PROPOSAL FOR A

GENETICS RESEARCH CENTER

  Submitted to the

National Institute of

General Medical Sciences

June 1, 1973

School of Medicine
Stanford University


      DEPARTMENT OF
HEALTM, EDUCATION. AND WELFARE
   PUBLIC HEALTH SERVICE

                        Farm Approved
SECTION I                 Eudgm Burcw No. 6BA02C9
I               LEAVE BLANK
   TYPE   PROGRAM         NUMBER

GRANT APPLICATION

      I
REVIEW GROUP

kOUNCIL (Morth. Ynrr/

FORMERLY


DATE RECEIVED

                                                 I
TO BE COMPLETED BY PRINCIPAL INVESTIGATOR f/fwns 1 rhmu@ 78nd 16.4)
(1. TITLE OF PROPOSAL (Do not oxcad S3 t~@wdtW8p#~d
      Genetics Research Center

Lederberg, Joshua
28. TITLE OF POSITION

  Professor and Chairman
2C. MAI LING ADORESS IStrwt. City, SUh, Zip we)

              12/31/78
4. TOYAL DIRECT COSTS RE-  5. DIRECT COSTS REQUESTED
FTiE;ED FOR PERIOD IN    FOR FIRST 12-MONTH PERIOD

$2,659,305         $537,800
.   FQRMANrrrSlfElSr (Sn /5rn/ctions)

Department of Genetics
Stanford University School of Medicine    Department of Genetics and

Stanford, California 94305          Department of Pediatrics
                        Stanford University School of Medicine
                        Stanford, California 94305

  N/A
7 . Rrwch lnvolvirq Hums Subjuts fSm ItU~fucDpnS)            & lnvomiom IRwmw8l Applreim8 Only - Sm fttstnu204

A.0 NO 6.0 YES Avprwsd:                 A.0 NO B.0 YES - Not prwiously mponrd
~~~~~-~cnding~cviw6-29-;;)      D*t*         C. DYES - Pwiowty reoorted
TO BE COMPLETED BY RESPONSIBLE AOMINISTRATIVE AUTHORITY hlPS8 thmuph 13and 1581
& APPLICANT ORGANltATIONtSt /Swa IrrrtnutiMs)        11. TYPE OF ORGANlZATlON (Chsk aoplicabh ireml
                          OFEDERAL c] STATE 0 LOCAL m OTW3f IS=ifvJ
Stanford University                    Primtp- non-nrofl -+
Stanford, California 94305          12. NAME, TITLE, ADDRESS, AND TELEPHONE NLlh!EfR OF
  IRS No. 94-1156365                   OFFICIAL IN BUSINESS OFFICE WHO SHOULD ALSO BE
Congressional District No. 17             NOTIFIED IF AN AWARD IS MADE

                          K.D. Creighton

Deputy Vice President for Business d Finance
Stanford University

IQ NAME.TITLE. AN0 TELEPHONE NUMBER OF OFFlClALlSI   , Stanford, California 94305
lCNlNG FOR APPLltANT ORGANIZATIONISI              T.lcphan~Nurn~(415)3~1-3330 :<7,?51
                             i3 IOEN~
                                        NAL
Kathleen C. Butler                 FOR INSTITUTIONAL GRANT PURPOSES iSea lnsrrucrio~~
Sponsored Projects Officer            01 School of Medicine

C/O Sponsored Projects Office             14.  ENTITY NUYBER (Fomcrly PHS Account Nunbcr)
  ToIaphonrNumbw (sI& 5) 32-0 Eyt. ?883_f    458210
IL CERTIFICATION AND ACCEPTANCE Wo, thm undmrrlgne crrriti tht th* nrumena husin uu trur and Compkta to th. bat 0' our
kmwlaJp 8nd p m to 8fIy QInt mv.rdmd, tha obll@pn bD c&ply v&h Publk Hwlth Swvlca tonm o nd conditions in 8ff=t n the time of th*

  SIGNATURES
IS-urn rquird on

A, SI,GNATLjRE OF PERSON NAMED IN ITEM 2A   (See also page i)DATE     107'1
     .   :, ,-  ..z..--   : .* " 1.. ., -`- q           MAY 30 IJIJ

IN ITEM IO                      UATC
  /                   5f3Of73
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DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE
         PUBLIC HEALTH SERVICE
    RESEARCH OBJECTlVES

       LEAVE BLANK
PROJECT NUMBER

NAME AND ADDRESS OF APPLICANT ORGANIZATION
Stanford University, Stanford, California 94305

NAME. SOCIAL SECURITY NUMBER. OFFICIAL TITLE, AND DEPARTMENT OF ALL PROFESSIONAL PERSONNEL ENGAGED ON

PROJkT, BEGINNING WITH PRlNCl-PAL INVESTlGATbR
Joshua Lederberg, Professor and Chairman,
Howard Cann, Associate Professor of Pedia
Norman Kretchmer, Professor of Pediatrics
Leonard Herzenberg, Professor of Genetics
Luca Cavalli-Sforza, Professor of Genetic

Luigi Luzzatti, Professor ofA
  Department of Pediatrics   ~~1
Clifford R. Earnett, Associ   e
  Department of Pediatrics ;)tp

TITLE OF PROJECT

Co&unity and Preventive Medicine,

GENETICS RESEARCH CENTER

USE THIS SPACE TO ABSTRACT YOUR PROPOSED RESEARCH. OUTLINE OBJECTIVES AND METHODS. UNDERSCORE THE KEY WORDS
(NOT TO EXCEED 10) IN YOUR ABSTRACI-.

.A comprehensive program of basic and clinical research, uniting the efforts of
the Department of Genetics a& .Paatz$cs, will apply advances in analytical

.P. . .

instrumentation to problems of genetic and disease in man.

Program areas include:
Genetic errors of metabolism identified by computer-managed gas chromatography
and mass spectrometry, applied to urine, blood and amniotic fluid;
Detection of fetal cells in maternal circulation;
Expression of genetic markers in fetal cells, linkage studies; antenatal diagnosis;
Polymorphisms involving specific binding to plasma proteins;
Psychological impact of genetic disease and counseling practices.

* Kenneth Tsuboi, Sr. Scientist, Department of Pediatrics
Alan Duffield, Research Associate, Department of Geneti
Wilfried Pereira, Jr., Research Associate, Department o

PHS-398
RN. 3.70

PAGE 2


   The anaersigned agrees to accept responsibility for the
scientific dud technical condwt of this project and for
provision oE required progress reports if a grant is awarded as
the result Bf this application.

MAY 3 0 1973
---------w-----

    Date

   4-J       ,!
~~~&.A&-..A~L~~
4         Y-r----
Joshua Lederberg, '--.'
Principal Investigator

i


TABLE OF CONTFNTS

I.   introduction and Program Summary  .*.*................  P-l

Comments on Organization  . . . . . . . . . . . . . . . . . . . . . . . .  P-3

The Stanford Environment . . . . . . . . . . . . . . . . . . . . . . . .  P-6

Clinical Faci 1 i ties  . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  P-11

Research Program Out1 i ne  . . . . . . . . . . . . . . . . . . . . . . . .  P-16

il.  Screening and Characterization of inborn Errors of
   Metabolism by Gas Chromatography/Mass Spectrometry
   Analysis of Body Fluids
   (Drs. Lederberg, Kretchmer, Cann, and nuffield) .,... . P-17

a-76,~ Budget and Explanation  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  P-78

  iii. Maternal Blood Stream - Ar.other Source of Fetal
+dg    Tissue for Pre-Natal Diagnosis of Genetic
       Disorders (Drs. Herzenberg and Cann)   . . . . . . . . . . . . . . . .  P-85

      Budget and Explanation  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  P-97

   IV.  Polymorphic Genetic Markers in. Amniotic Fluid
      (Drs. Cann and Tsuboi)   . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  p-100

      Budget and Explanation  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  P-119


   v.   A Search for Genetic Polymorphisms and Variances
     Of Specific Binding Proteins in Blood
      (Dr. Cavall i-Sforza)   . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  P-122

Budget and Explanation  . . . . . . . . . . . . . . . . . . . ..*........  P-139

vi.  The impact of Genetic Counseling Practices on
   Family Decisions and Behavior
  (Drs. Barnett, Cann, and Luzzatti) . . . . . . . . . . . . . . . . . . P-133

   Budget and Explanation  ..............................  P-145

VII. Overall Budgets  .....................................  P-148

     Program Director's Office Budget  ................  P-143

     Aggregate Genetics Research Center Budget  .......  P-152

VIII. Concluding Remarks (Dr. Lederberg)   . . . . . . . . . . . . . . . . .  P-157


TABLE OF CONTENTS (cont.)

IX.  Professional Personnel   ..............................  P-160

      Key Personnel and Backgrounds  ...................  P-161

      Relevant Research Support Summary for
     Departments of Genetics and Pediatrics  ..........  P-165

      individual Biographies  ..........................  P-169


SECTION I

Introduction and Program Summary


INTRODUCTION

   The Genetics Research Center that would be enabled by this grant will te a new
level of cooperative involvement of the Departments of Genetics and Pediatrics of
the Stanford University School of Medicine in research in medical genetics and
the application of such research to clinical aspects of medical genetics. We are
requesting support for an interrelated set of basic and applied research projects
involving members of both departments. Besides their importance as basic science,
these investigations relate to genetic counselling, pathogenesis and diagnosis of
genetic disorders and the mechanisms of human adaptation in genetic variation.
11-l preparing this application, the two departments,  labelled as basic science
and clinical science respectively, have already enhanced the coordination of their
activities for the benefit of patients with genetic disorders and their famiiies.

   Since 1959, the inception of the School of Medicine on the Stanford University
campus, Genetics and Pediatrics each have been deeply involved in various aspects
of genetic research. A number of ad hoc cooperative efforts have also evolved.
Both departments are together teaching medical genetics to medical students. In-
formal communications in training graduate students and postdoctoral fellohs have
been excellent. Research projects undertaken in one department have involved
consultation or collaboration with various members of the other department,  and
clinical problems have stimulated basic research activities in both departments,

   In accordance with its clinical mission Pediatrics has focused on patients
with genetic disorders, diagnosis, pathogenesis, and therapy; and the couns2llis~
of such patients and their families. Pediatrics has likewise emphasized perinatal
biology, featuring a program of detection and management of high risk pregnancies
in which the potential for morbidity or mortality of mother, fetus and/or newborn
infant is high.

   Genetics is well known for its basic research programs in molecular biology,
population genetics, and immuriogenetics, whose potential for clinical applications
have long been recognized by members o+f 'both departments. As knowledge in molecular
biology has increased and the necessary technology has permitted applications
to the study of man and his disorders, it has become increasingly evident that
relevant research efforts of both departments should converge on problems connected
with clinical care. Both departments have begun to cooperatively design and carry
out research applicable to genetic medtcine and to plan for the application of the
findings to the care of patients encountered on the various clinical services of
the Department of Pediatrics.

   The underlying theme that unifies the range of specific studies outlined below    :
is genetic polymorphism in man.  The pediatrician views this as the source of
genetic disease; the basic scientist as an expression of gene mutation and evolu-
tionary pressures.  These are roles shared within as well as among individual in- !/

a vestigators.  To these challenges are brought a combination of clinical insights,  1
experience with several aspects of basic genetics, and new analytical technologies    3
-- the application of instruments like the mass-spectrometer, the computer and the
cell sorter.

  Besides the specific research projects to be funded under the Center grant,
we work in an extensive context of genetic and related  research -- and indispensable
aspect of our own environment, and a set of activities to which the Center organiza-
tion may also bring a new focus for developments that should advance both basic
scientific knowledge and its application to human problems.           P-2


COHtlBNTS ON ORGANIZATXON

   The Stanford Genetics Research Center is organized as a
cooperation of the Departments of Genetics and Pediatrics. Thus,
it follows, rather than conflicts with existing departmental
authority. This minimizes the need for new, formal arrangements
that right be essential in other circumstances. 1t does reflect a
deep-seated intention on the part of both departments to improve
the application of nev analytical methodology and of basic
genetic science to clinical problems, and to relate clinical
studies to enhancing knowledge of genetic polymorphism in man.
The length of the following comments on organizational detail may
lead one to believe that we are xore interested in management
than in scientific substance.

   Exactly the opposite is the case, but we cannot afford the
consequences of misunderstanding about how we vi11 work together.
Obviously, as at any other university, the actual character of
our @@organixationN has a larger compass and bears little
resemblance to the forxal hierarchy of imputed power, Research is
actually done in individual laboratories under the supervision of
an autonomoas faculty member.  Best HdirectionM must be in the
form of intellectual stimulation and criticism, and in the
selection of key people for roles of autonomous responsibility.

   Professor Joshua Lederbetg, Chairman of the Department of
Genetics, vi11 serve as Principal Investigator and Director of
the overall Genetics Center Proyraa. Dr. Howard Cann, Associate
Professor of Pediatrics, will serve as Associate Program Director
with special responsibility for clinical research related to the
Center's activities. (If this application is approved, and offers
a funding basis for the step, it is anticipate! that Dr. Cann
will receive a joint appointment in the Genetics Department as
well.)

   The Director will have executive responsibility for the
administration of the program, including the formulation of
extended an3 new projects, their budgets, and reports to the
NIGHS of progress under the grant, and internal functions of
coordination,  information and criticism.

   The internal adainistration of the program is facilitated by
the Director's position as Chairman of the Genetics Department,
and by the enthusiastic support and participation of Dr. Irving
Schulman, Chairma'n of Pediatrics,  and of other senior members of
both departrents. In practice,  the Director will be advised by,
and vi11 delegate subproject responsibility to, these colleagues
in accordance with their special skills and interests.

   The Director will also invite an informal visiting
committee, principally from among his colleagues involved in
similar lines of work at other West Coast institutions, to visit
Stanford annually and to advise him of advantageous directions of


-2-

policy. This group may also help to identify new opportunities
for inter-institutional cooperation and coordination, especially
to further our ,eaphasis on building complementary rather than
competitive capabilities. These annual visits may also
advantageously coincide with a local or regional conference on
research progress concerning genetic polymorphism and disease.

  The Director must play both an attractive and a critical
role. That is he nust encourage his colleagues to invest in the
effort needed to orient their activities towards the conson goals
of the Center's research. At various times this attraction may be
enhanced by the fiscal support of the Center's budget; on the
other hand, it is hard to predict what the level of that support
will be, an;3 the level of discretion that will be delegated to
the Director. Realistically speaking, then, his role is more
inspirational than directive, at least uith respect to the
tenured fellow-members of the faculty. If the Center's budget is
approved, aad develops some de facto continuity, he may have
greater formal authority -- but always with accountability to
outside revieu groups and to the independent authority of other
professors.  The moral and intellectual influence that the
Director can exert will obvjously be greatest in relation to
people in the same or closely co-functioning departments who
already relate in many other ways. His main positive role will be
to help bring in new ide&s -- his own and others -- into the
progress of the Centeres research.

   Conversely, he has responsibilities for negative functions
-- especially to discourage the growth or even survival of
projects that are inherently unsound, or become obsolete. Within
a close-knit institution there are often serious personal and
political obstacles to the exercise of objective scientific
quality judgments. These limitations are both aggravated and
compensated for by the limited discretion usually given to a
director, i.e., the fact that final decisions about project items
are generally made by peer review groups.

   All of these problems would be much uorse if the Director
had the responsibility of coordinating every genetics-relevant
activity in the school. He would have neither the budget nor the
political mandate to do this well. In fact, the present proposal
encompasses a serious and soul-searching effort to initiate a
major Center program at a practically achievable and useful level
of integration. It will also be the basis for less formal but
equally important co-operations with other departments, whose
policies and programs can be influenced but not directed in the
exercise of our responsibilities.

Provision for Succession of Directorship.

  Day to day deputization will be handled ad hoc in the same
fashion as the duties o-f the departmental chairman. The question
of possible succession, in the event of incapacity or transfer of
the Director, poses a more difficult question. The Associate
Director will serve pro-tea, in concert with the (acting)


-3-

chairaan of each department; and a new director will be proposed
by the Dean of the school after consultation with the visiting
committee and with the participating faculty of the departments.

   The Associate Director's role will be to maintain contacts
with other clinical departments for patient referals to Center
projects, and to assure a high standard of clinical
responsibility in all Center research activities that deal with
patients. Stanford has long experience with Clinical Research
Centers -- e.g. in medicine, in cancer therapy, in cardiovascular
disease, and in premature infant research. (These facilities
afford further research bed opportunities for protocols involving
intensive study of selected cases without burdening the present
proposal uith inappropriate ia-patient care charges). In
addition, they have led to the institutionalization of formal
procedures for the ethical revieu of research involving human
subjects. Dr. Cann vi11 be responsible for defending the present
proposal before that revieu conmittee, and for assuring that all
further research under this grant is properly submitted and
reviewed according to established procedures.

  An Application for renewal of a Genetics Research Training
Grant uas approved by Council for the.period 1974-1979. This
action was superseded, however, by the executive decision to wind
down such programs and we are nou operating on a decreasing
budget to cover only the previously enrolled trainees.

  That training grant reneual had, for the first time,
provided for clinical research in genetics as part of our
program. Lacking these funds, it is, of course, imperative that
the research activities of the Center also double as training
opportunities for graduate research assistants and postdoctoral
fellows (research associates.) We vi11 raake every effort to fit
each fellou into employment in the support roles of the Center.
We cannot pretend, however, to be able to support the training
functions at the level previously assumed without a compensatory
expansion of the funding for these research programs. This is not
reflected in the budgets submitted at this time.

  The department faculties having agreed to the programs of
the Center, there is much to.do in substance, but little in
organizational process,  to coordinate our activities and insure
meeting the Center's objectives. We are already in frequent,
almost daily, comvtunication in the pursuit of our other duties
and socially. These contacts ace promoted by the fact that our
laboratories and offices ace immediately adjacent in the nedical
Center Duilding.

   The Genetics Center Budget is intended to fund programs of
new substance and scope or those for which other support (e.g.
NASA) is no longer available. Lt goes into new territory beyond
many on-going research projects which are identified in the
material on each affiliated investigator. However, the
coordinated funding and administration of the Center allows more
careful planning and resource-sharing than would be possible for


-4-

a disparate set of individual proposals.

   We intend .to minimize the indefinite prolongation of
autonomous projects merely to keep a research group intact.
Instead we vi.11 promote an innovative search for new
opportunities ana on-going mutaal scrutiny of projects.

   For the continuity of a program as complex as the Center, we
are requesting an award for 5 years.  Yet it is obvious that many
unforeseen obstacles and opportunities will arise in that
interval to warrant changes of tactical direction and evolution
of major strategies. The responsibility of the Director to manage
this progression of emphasis is his most important executive
role. It vi.11 be discussed with the visiting committee and
reported to BIGHS from ,year to year, to assure the consistency of
his decisions with the approved mandate of the Center grant.

    This is not an assertion of unwarranted latitude. To the
contrary, the political structure of a university ensures, if
anything, a great deal of conservatism in changes of direction
and support for individual programs, once approved.

   The present proposU will also be a model for further
developments to be initiated uith the Departments of Gyn-Ob.,
fledicine, Psychiatry and .Dermatology -- to mention only those
with which we have had tangible discussions. For various reasons,
potential projects uith them are not yet ripe for formal
subnissious;  these will be t&e subject of future requests,
depending in part on the climate for funding expansion of genetic
research.  Heanuhile, we have working arrangements to assure their
awareness of our activities and the provision of patient material
for mutual advantage.

   A brief description of the role of each participant in this
prograa and of his research and/or clinical interests are
presented here:

THE STANFORD ENVIRONW3NT

   Besides the Frograa Director (Professor Lederberg) and
Associate Director (Professor Cann) the faculty and professional
personnel associated with the Center, their roles and
biographical detail are spelled out in Section IX.

   In addition,  we have included biographical sketches of a
number of key members of the environment of the Department of
Pediatrics and Genetics, although their research projects dre not
included in funding under the Center at this time.

   Within one or two years after the activation of this
prograa. we anticipate the direct participation of one or more
colleagues from the Department of Obstetrics and Gynecology. A
search for a chairman of this department is presently under Way,
and we expect that this individual will reaeu active research

P-L


-5-

here in fetal physiology and fetal monitoring. Ue look forward to
interacting in this program with our obstetrical colleagues in
various projects pertaining to antenatal detection of genetic
disorders and selective abortion.

   Ye hare also consulted uith the chairmen of the Departments
of Dediciae and of Dermatology,  and have encountered great
interest in the development of the Center, and assurances of
cooperation in the referal of patients who would be pertinent to
our screening technologies. Their position is best witness8d by
the attached correspondence. At some future date, we may expect
to formulate more specific proposals, as a consequence of
recruiting now under way.

  The Department of Psychiatry will be chaired by Albert
Stunkard (now at University of Pennsylvania) beginning September
1973. He is personally well known to the Director (who served on
the search committee for this appointsent, which ua,s chaired by
Professor Schulman) and aany other participants and we have
strong assurances that the Psychiatry Department's interest in
genetic etiologies of psychiatric disease will continue under his
leadership.  Dr. Stunkard has also voiced his concern that
psychiatrists have not hitherto been more directly involved in
problems of genetic counseling, like those reflected in Dr.
Barnett@s proposal hem; and we are looking forward to closer
cooperation on such issues after his assumption of his duties.
For some time,  Professor Cavalli-Sforza of the Genetics
Department has been cooperating with Professor Darchas of
Psychiatry on polymorphisms of biogenic amine metabolism.

   Genetic counseling, per se, is a responsibility mainly of
the Pediatrics Department -- diseases of adults only occasionally
present serious problems of reproductive policy of the farily.
Genetic disease, of course, presents itself to all of the medical
and surgical specialities, In recent years, these departments
have not seen any requirement for a special organization to deal
with genetic aspects of internal rmedicine or surgery -- and
indeed these are fully and competently integrated into their
overall teaching and practice. Rhen special problems do arise,
there are no iapediaents to consultation with pediatricians and
with basic geneticists. Plany, nembers of those departments are
already involved in immunogenetic and other genetic research.


DATE: May 25, 1973

To :    Joshua Lederberg, Ph.D.

FROM :   Daniel D. Fe&man, M.D. (Chairman, Department of Medicine)

SUBJECT:     Genetics Research Center

Dear Josh:

  I am not really clear&out your timetable for submission of the
proposal for a Genetics Research Center, but I thought I would make
explicit several areas of potential involvement of the Department of
Medicine.

  1. Pharmacogenetics -  We have a slot in Clinical Pharmacology
for a new Assistant Professor.  Stan Cohen and I have discussed finding
someone with a particular interest in the area of pharmacogenetics, but
at the present time, no particular individual has been chosen.

2. Endocrinology -

A. Hormonal control of gene expression. We will have at
least one new person in the Endocrine Division in the next
year and have placed as our first interest finding someone
working in the area of the effective hormone on the expres-
sion of genetic information.
link to a genetic center.   This would provide an obvious

  3. Early detection of hereditary endocrine disease. There are
at least 80 endocrinopathies which show Mendelian segregation and a
number more which probably have a major genetic control. Early de-
tection of assymptomatic patients can be achieved by suitable pro-
vocative testing.  This opens the way to family studies in which
a) the hereditary mechanism can be defined, b) preventive or therapeutic
intervention can be tried before irreversible damage has occurred, and
C) the effects that intervention can be compared to the natural history
of the disorder.  The latter approach has important application to the
management of "patients" discovered by multiphasic screening techniques.
  4. Imunogenetics -  This area of course is strongly represented in
our Department and we could, I believe, contribute significantly to the
activities of the genetic center in this area.

  I should have liked to provide more specific information, but until
particular people have been identified, it would be difficult to do so.
I hope this is of some help --
laboration.           1 am certainly anxious for an active col-

Sincerely,
d?!f

3-f


CLINIC FOFj Ctt[LDf$!`i WPTK ~SO~I.fWS AND OTHEFj ~NH.&kT/-j~bE S,KI;N DISEASES

                      Directors:

             Eugene M. Farber, M.D.
      Profe'ssor and Chairman, Department of Dermatology

              Alvin H. Jacobs, M.D.
         Professor of Dermatology and Pediatrics


In the clinics of the Department of Dermatology there are about 12,000

total patient visits annually,of these approximately 3,000 are children seen

in the Pediatric Dermatology Clinic,  Over half of these children have either
                                                                           c
psoriasis or atopic dermatitis, 60th conditions with important heritable

factors in their etiology.  In addition many patients are seen with the less

common genodermatoses, such as, the vat lous types of ichthyosis, epidermolysis

bullosa, and the many types of heritable neurocutaneous disorders. At the

present time a special clinic is being established for the care and study

of children with psoriasis and other inheritable skin diseases.  The purpose

of this clinic is not only to offer more complete care for these types of

patients, but to study the genetic aspects of these conditions and offer

genetic councelling to the patients and their families.

  The Department of Dermatology 6lready has an unmatched reservoir of
material for genetic study in its Psoriasis Life History Research files; a

data-bank of approximately 8000 patient histories. Not only is this computerized

information a source of information .on the epidemiology of psoriasis, but

has served as the basis for several published follow-up studies on the genetics

of psoriasis including family and twin studies.

  At present several genetic studies are under way. For example, Dr. Farber

and associates are studying the association between H-LA antigens and psoriasis.

Drs. Jacobs and Chan are studying and developing techniques for accurate counting

of melanocytes in pigmented and depigmented macules in order to predict the

development of certain genodermatoses, such as neurofibromatosis, tuberous


                                          P-9


-2-

sclerosis etc.

  Studies are also under way to properly classify the various genetic
types of ichthyosis and in cooperatton with Dr. Howard Cann we are planning

to investigate the x-linked blood type

ichthyosis.

in the families with x-linked

It is hoped that tn the future, w ith the vast resources availab le

in Genetics, Pediatrics, and Dermatology at Stanford, an investigation

could be launched into the genetlc aspects of atoptc dermatitis, one of

the commonest skin problems of childhood,


I  CLINICAL FkXLITIES
                      -

   The Stanford Yedical Center, located midway between the cities of Scn
Francisco and SalI Jose on the campus of Stanford University, co;,siats of tk.2
School of Xedicine , the University !lospital and the Stanford Clinics. Staforrl
is the only university hospital in the ikgional liedical Program (XZ?) .Arca I11
and functions as a tertiary care center.  R`iP Area III consists of eleven coti.i:ie-j
in mid California consisting of 2.6 million people, There are 60 acute h@%pital
facilities, and 4000 physicians practice in RX ? Area III. Approximately $0 PArcent
of patients admitted to Stanford University Hospital live in this area.Xdi'cticnall)`,
patients for the clinics and university hospital are also dra5.n from other areas i:,
and out of California. ElP Area III and its:population form a base from wbic:l is
drawn numerous patients with diseases resulting from major genes or chromosomal
abnormalities or with a significant genetic component. bloat of these patients are
seen primarily on one of t!le services of the Department of Pediatrics or arc
referred for diagnostic evaluation and/or genetic counsclling frcm other clinical
services at the medical center. Clinical teaching services at other hospitals;:
affiliated with the Department of Fediatrics add to the source of patients k.ith
medical genetic problems.

  As the Genetics Center activities evolve and research developrdnts become
available for clinical application, we anticipate that more patients t.511 be
referred to this medical canter for genetic counsclling and evaluation and
management of genetic disease. The number of patients seen annuall;: by ttc combii:zi
clinical facilities of the ikpartment of Pediatrics for genetic disorders 5s
estimated to be about 200. Ve predict that this number will double during t;r-r: cctirs;i
of the Genetics Center grant.

  Other clinical divisions of the Medical Center, of course, also see man;:
adult patients with genetic disorders. iiistorically, Genetics has had weaker tiss
with them than with Tediatrics, although several joint projects connect ::it:?
Medi tine (H. !!cDevitt) and vsith Psychiatry (J. Sarchas; S. Kessler). T:?e ap:cint:?nr
(effective February 1) of a "genetic endocrinoiogist", Dr. Daniel Federman, as net;
head of Medicine should remove administrative obstacles to extensions of Center-
related programs.

  Patients with genetic disorders are seen in the fo_llowing pediatric sub-
specialty clinics at the Stanford Xedical Center.

*The Children's hospital at Stanford and the pediatric services at the Santa
Clara County Hospital (San Jcse) and a,t the Kaiser-Permanente Medical Center
in Santa Clara.

PHS 2495.1 1245

P-N


Confinuot~- n page

Genetic Counselling Clinic (Director, Dr. Howard Cann, Associate Professor
                 of Pediatrics)

  Approximately fifty patients and their families are referred annually for
genetic counselling . These include patients from outside of the Stanford ?!cdical
Center and from various clinical services at the Piedical Center, About jO% of
fami.lies seen are counselled for disorders determined by major genes at singie
loci. The remaining 50% of patients represent more complex counselling problems,
e.g. possible sporadic cases and phenocopies, multifactorial threshold traits,
undiagnosed familial disorders, and excessive exposure to radiation. Counselling
i.ncludes confirmation of the diagnosis of the disorder and identification of
heterozygotes among asymptomatic relatives at risk (autosomal recessive and
X-linked recessive disorders). Families are always screened for the possibility
of applying ante-natal diagnostic techniques. The motivation for and expectations
from genetic counselling are explored by Dr. Cann and, for selected families,
by a social worker, in order to plan for effective communication with each ianily.

Birth Defects Clinics (Director, Dr. Luigi Luzzatti, Professor of Pediatrics)

    Approximately 100 new patients are seen in the Birth Defects Clinics
annually for diagnostic evaluaticn and comprehensive and interdisciplinary
management of congenital defects. There are about 400 return visits per annun
in the clinics. In addition, about 50 newborn infants are seen annually for
evaluation of congsnital defects in tlie Stanford Hospital Surseries. >lost ChiiLren
seen have multiple congenital dt2fects of unclear etiology, about 507; of thzse
are scrt?eneci for c1lronosonal aSnormalities, aSout 25X are patients with dlsor<srs
determined by major genes at single loci,  and about one third are seen for
structural malfori~atiorsw;lich ars multifactorially inherited threshold traits.
This is the clinic where most of tile patients with mucopolysaccharidoses,
mucoi ipidoses , simply inherited disorders of bone, Down's syndrome and other
chromosomal abnor;Jalities, cleft lip and/or palate and congenital defects or
neural tube development are seen. Where appropriate, genetic counselling is
provided to families of patients enrolled in the Birth Defects Clinics. In
addition about 50 families are referred each year specifically for genetic
counselling . ti average of about 100 karyotype analyses are done annually in thz
Cytogenetics Laboratory in proband s with congenital defects and/or in family mer?hrs
as indicated. To date diagnostic amniocentesis has been performed at 12-16 \:eeks
gestation on approximately 25 women referred to the clinic for either advanced
maternal age or a documented family history for an X linked gene.

Pediatric Hematology Clinic (Clinic Director, Dr. Herbert C. Schwartz,
                  Professor of Pediatrics)

  In this clinic approximately 100 ne;q patients are seen annually for various
disorders of blood. Between five and ten percent of these are children with
inherited disorders -- sickle cell anemia and other hemoglobinopathies,
thalassemia, cougenital spherocytosis, glucose-G-phosphate dehydrogenase
deficiency and other instances of congenital non-spherocytic hemolytic anemia
and hcmolytic disease of the newborn (feto-maternal blood group incomPatibility).
In this clinic workup of patients with genetic disorders includes idcntificati?n
of hetcrozygotes and honozygotcs among first degree relatives of probands and
subscqucnt genetic counselling.

s-.-v                     -.---- --ww- -v-l-   4-m --_.. . . . .


Continua!lsn page

       D?prx:~; c: !T-., .`;..
                                     .--.
.

The Stanford Nurseries (Director, Dr. Philip Sunshine, Associate Professor
               of Pediatrics)

   The newborn nurseries of the Stanford Xedical Center include facilities
for uncomplicated births, an intensive care nursery and the Premature Infant
Research Center. The research and clinical emphasis of these newborn units is on
perinatal biology of the fetus, newlyborn infant and the mother. This emphasis
has led to the development of a program dealing with high risk pregnancies, in
which mother, infant or both are at high risk for morbidity and mortality. T,Fil2n-
ever appropriate the fetus is monitored and such monitoring includes diagnostic
amniocentesis for evaluation of fetal maturity, for fetonaternal Rh incoc~sribiliiy
and early antenatal detection of genetic disordrrs. A study conducted in Cal.iiornia
in 1971 by the Bureau of Yaternal and Child H&alth indicated that the Stec_`ord
Medical Center is the only facility in RXP Area III with an intensive car2 .lur.scr>
(based on fairly ridgid criteria, e.g.  full time neonatologists and basic irlfant
monitoring equipment available at all times), and this is evident by referr&ls of   I

fetuses and their mothers and newborn infants. Approsimat2ly 700 infants arr
admitted per year to either the intensive care nursery of the Premature Rosearc
Center at Stanford and about 30;; of these are born at other facilitks. +proxlzatc3>
551 of newborn infants receiving intensive care at Stanford are ill because ::?y -
are hetero-ygous or homozygous for a major mutant gene,  because of a chromosJna1   i
abnormality or because of a malformation with a significant genetic contribution.
We anticipate a major expansion of pcrinatal activities involving detecticn and  I
management of high risk coth2rs and fetuses with the appointm&nt in the czar     !
future of a Chairman of the Department of Obstetrics and Gynecology. The scar&       j
is presently underway, and one of the criteria for selection is comT,stsnoe in the
areas of fetal monitoring and evaluation and fetal and maternal physiologic Ict2r-  i
actions.                               I

Pediatric Neurology Clinic (Clinic Director, Dr. Judith Koehler, Assistant Professor I
                of Pediatrics and N2urology)

  Approximately 500-600 patients with various neurologic disorders are se2n
annually in this clinic. Xany of these patients `nave genetically det2rmined or
inherited disease, e.g. learning disorders, familial myopathies, spinoc2r2bellar
degeneration, familial spastic paraparesis and other metabolic or degen2racive
disorders of the central and peripheral nervous system.  The Division of PedlatrLc  I
Neurology has special interest in clinical treatments of and research into rile bzs%z .
mechanisms of epilepsy. Some patients w!t.. h seizure disorders have heredofsmilLa1   i
determinants underlying their basic seizure problems, Additionally, response to
medication and drug metabolism may be genetically determined. The Divisicn of   I
Pediatric Neurology also has special clinical and research interest in neuro-
muscular disease, much of which is hereditofamilial. Specifically for this purpose,
a neuromuscular clinic is being established in conjunction with the Yuscuiar
Dystrophy Association. This clinic will see 200-300 patients, mostly children,
annually. A diagnostic neuromuscular histochemistry laboratory is already in   I
function to process nerve and muscle biopsies from these patients. Xctivz ressarzl;   I
in neuromuscular disease is being carried out by several workers in the   2n;!rt;"-?::::  ,F_
which incorporates ultrastructurz and basic physiologic studies.    3 :iSurOi cfgy ,
                  I
                                   !

    t
  I
      --2


h ,-a


Hemophilia Program (Clinic Director, Dr. John Gribble)

   The Hemophilia Program provides diagnostic evaluation and comprehensive
management for patients with disorders of blood coagulation. Approximately 109
new patients are seen annually; these patients represent the clinical spectrum of
disorders of blood coagulation, the most frequent being hemophilia A, heno?hilia !3
and van Willebrand's disease. The families of all patients with inherited disorders
are provided with genetic counselling. A comprehensive care clinic is located
at the Children's Hospital at Stanford in order to provide continuing care for
patients with hemophilia and includes prophylactic treatment of patients with
hemophilia A with Factor VIII preparations.

Pediatric Metabolic and Endocrine Clinic (Clinic Director, Dr. R.O. Christiansen,
                           Assistant Professor of Pediatrics)

   Seventy to eighty new patients are seen annually in this clinic which
logs about 703 patient visits each year. Xany of the patients may be classified
as inborn metabolic errors and these include phenylketonuria, galactosemia,
the various syndromes associated with adrenal hyperplasia, alcaptc>uria, the
various glycogenoses and defects in thyroxine oiosynthesis. Diet therapy of
patients with phenylketonuria and galactosenia is an ongoing activity of thzse
clinics. Furthermore, a number of patients with hypophosphatemia (X-linked)
are enrolled in this clinic. Genetic counselling is provided for the families of
all patients with hereditary metabolic and endocrine diseases, and this includes
attempts to designate heterozygotes among unaffected siblings of probands with
inborn errors of metabolism.

Growth and Development Clinic (Director, Dr. Norman Kretchmer, Professor of
                   Pediatrics)

  This clinic deals with genetic and environmental problems in infants and
children which affect their growth and development. Usually children are referred
to this clinic because of failure to grow or abnormally slow growth.
                                                   Thus children

with hereditary conditions which result in significant growth:failure in the
phenotype (e.g. isolated growth hormone deficiency, various aminoacidurias and
various chondrodystrophies) comprise a proportion of this clinic's patient
population. Approximately 150 new patients are seen annually.


Conlinual;-..n page

  The Children's Hospital at Stanford, located approximately l/2 mile from
the Medical Center on the Stanford campus, is affiliated with the Department of
Pediatrics for teaching and postdoctoral clinical and researc!] trainin?.
There are three clinical services relevant to a medical genetics program at thi;
hospital: 1) Cystic Fibrosis Service (Director, Dr. Birt Harvey, Clinicai
Associate Professor of Pediatrics), 2) Clinical Immunology Service (Director,
Dr. Vincent Narinkovich, Assistant Professor of Pediatrics) and 3) Pediatric
Oncology Service (Director, Dr. Jordan Wilbur,  Clinical Associate Professor of
Pediatrics). These services have in-patient and out-patient facilities at tile
Children's Hospital at Stanford. Here patients are evaluated, diagnosed and
treated for cystic fibrosis, hereditary immune defects and hereditary malignant
tumors (e. g. retinoblastoma). Genetic counselling is provided by the staff of thsst
services.

I   Research Facilities

   Research facilities in the Departments of Genetics and Pediatrics available
to this Genetics Center Program include some 15,000 square feet of laboratc*ri2s,
research support areas and offices.  The research and administrative Ireas for each
department are contiguous on the third floor (the Joseph P. Kennedy Jr.
Laboratories for Nolecular Xedicine) of the Joseph D. Grant Building of tiln
School of Medicine. In addition the Instrumentation Research Laboratory (D?;artr2nt
of Genetics), in the basement of this Suilding,conprises about 7,030 square feet
of offices, laboratories, and shops. Thus biochemical laboratories, fully devcicy\ed
tissue culture areas, heavy equipment areas,  cold and warm rooms and an electron
microscope facility are available for this program. A cell separator, a gas
chromatograph, a mass spectrometer and an electron microscope lead the long list
of equipment which is available for ths research projects to be carried out in
this program. Other equipment also includes centrifuges, scintillation counters,
lyophilizers, spectrophotometers, incubators, microscopes and coulter counters.
Rather good computer facilities are available.


Continuation page

THE RESEARCH PROGRAM

  The Center program consists of research projects which have direct relevance
to medical genetics. Some of these projects will require blood, urine, amniotic
fluid and cells from patients and others will require the patients themselves.
In none of these research projects are we ready to test methods for use in the
actual clinical situation; rather, we are developing methodology. The general
areas of research are as follows:

I.   Screening and Characterization of Inborn Errors of Metabolism by Gas
  Chromatography/Mass Spectrometry Analysis of Body Fluids.
  (Drs. Lederberg, Kretchmer, Cann and Duffield).

II.  Maternal Blood Stream - Another Source of Fetal Tissue for Pre-Natal
   Diagnosis of Genetic Disorders.
  (Drs. Herzenberg and Cann).

III. Polymorphic Genetic Markers in Amniotic Fluid.
   (Drs. Cann and Tsuboi).

IV.  A Search for Genetic Polymorphisms and Variances of Specific Binding
   Proteins in Blood.
   (Dr. Cavalli-Sforza).

V.  The Impact of Genetic Counseling Practices on Family Decisions and Behavior.
   (Drs. Bamett, Cann and Luzzatti).

The arrangement is also reflected in the budget presentation.

I
PHS-398
Rev. 249

Peg*


SECTION II

Screening and Characterization of Inborn Errors of
Metabolism by Gas Chromatography/Mass Spectrometry
        Analysis of Body Fluids

Drs. Lederberg, Kretchmer, Cann, and Duffield


Screening and Characterization of Inborn Errors of
Retabolism with Conpaterized Gas Chromatography
          And Bass Spec troaetry

Dr. J. Lederberg, Principal Investigator
Drs.  1. Rretchmer, 8. Cann, and A. Duffield,
       Associate Investigators

A.   IYTRODUCTIOB

A.1 Objectives

   The objectives of this work are to develop the uses of
gas-1iquiU chromatography (GC) and @ass spectrometry (ES)
instrurentation, under computer management, for the screening,
diagnosis (pre and postnatal), and study of inborn errors of
metabolism. The efficacy of these analytical tools has been
demonstrated uhen applied to limited populations of urine samples
in the research laboratory environment. Ue propose to enlarge the
clinical investigative applications of GCfRS technology and to
deaonstrate its utility for o ore econorical and routine diagnosis
and screening of disease.

   Specific goals include the application of GC/HS analysis
capabilities to larger and more diversified populations to
establisb better defined norms, deviations, and control
parameters necessary to relate GC/BS analysis results to
identifiable disease states. In order to ease the problems in
analyzing the prodigious amounts of information expected in this
research, we will augment the existing GC/BS data handling system
to provide for increased throughput and automation.

   Two other on-going or penbiag research projects relate to
the present application,  each with distinctive aims:

1. DBUDRAL (RIB: BE-00612; Principal Investigator, R. A.
Peigembaaa) is concerned uitb the advancement of artificial
iate$ligence (computer softuare) techniques for the automated
interpretation .of sass spectrometry data. These prograwx
attempt to emulate human reasoning processes in constructing
explanations for mass spectra fron basic principles.

2. SIJRBX (RIE: RR-00785 pending; Principal Investigator, J.
Lederberg) is a conprehensive resource grant to establish a
national facility for developing applications of artificial
intelligence in medicine. Our own use of this facility will
include the integration of DEYDRAL software with BIGR
RESOLDTIOR mass spectroietry instrumentation. Genetic
screening can be made even more sophisticated by using these
techniques for the corroboration of the structures of newly
discerned metabolites. Rowever,  the present program can also
operate stand-alone, if necessary, using low resolution RS on
line with the GC,


-2-

A.2 Background and Rationale

   The Instrumentation Research Laboratory was established in
the Genetics Department under NASA auspices in 1961. Its task was
to define and improve nicroanalytical methods for the detection
of living processes that might be useful for the biological
exploration of the planets. !iany of the concepts that we explored
have been embodied in NASA's planetary mission plans. However, we
have not undertaken to design and build harduare for such
missions, Instead, we have served as experienced advisors to the
experiment teams responsrble for scientific studies on the
Mariner and Viking Mars programs.  Our work on GC/HS is one of
several lines of instrumentation effort.

   Our original mandate from NASA included generous
encouragement to seek health-related applications as a spin-off
of the development work they were supporting. However, they have
not been able to support the full fledged extension of
space-related technology to genetic disease research per se. The
present application also comes at a time when overall funding for
basic research by NASA is declining rapidly and sag disappear
within the next year. We have already begun to reduce our W/MS
laboratory staff in response t3 these cutbacks. It is therefore
appropriate that we seek NIN support to help maintain this
existing laboratory to apply its capabilities to problems of
characterizing genetic metabolic disease.

   Our focus on mass spectroaetrp (ref 3c) originally stemmed
from the exquisite sensitivity,  speed, and specificity of this
technique for the identification of organic molecules. We have
had some experience with instruments (like the Bendix
Time-of-Plight Hass Spectrometer) which can generate a complete
low resolution mass spectrum in 100 microseconds and whose
sensitivity is limited by the statistics of the number of ionized
fragments, and by the data handling problems of averaging
repetitive spectra emerging at a rate of 10,000 frames per
second. Ue have also been led to look at the computational
challenges of fiS from another standpoint - namely the
aechanization of the scientific thinking that is entailed by the
interpretation of a mass spectrum. This task has been the focus
of the research in "artificial intelligence" of the DENDRAL
project.

   The application of these instruments to genetic research
requires another dimension, namely the separation of complex
mixtures, e.g., from body fluUs, into individual components.
These are then available for identification by the mass
spectrometer. Gas chromatography has proven to be a useful
companion to the mass spectrometer - the output gas stream can be
fed directly to the inlet of the spectrometer and much of the
carrier gas (helium) selectively deviated by a semi-permeable
membrane. (Automated, continuous flow into a mass spectrometer of
other chroaatographic streams, e.g., high pressure liquid
chromatography, is a speculation that may eventually materialize

.-.
i., ,'


-3-

to great advantage but is not yet available for applications like
ours).

  For some time then we have been developing the means to
integrate GC vith MS under computer management. The present
project represents the systematic application of these skills to
the recognition and identification of metabolic variations,
viewed both as genetic polymorphisms and as clinical problems of
genetic disease in man.

  The sample populations compr-ise mainly healthy (control) and
problematical newborns already under intensive study in the
Stanford Pediatrics Department.  Other samples will be furnished
by collaborative arrangements with physicians elsewhere and vith
other Departments at Stanford (e.g., Medicine, Psychiatry,
Dermatology, etc.). These inputs will have been prescreened for
conditions likely to relate to possible genetic etiologies or
otherwise to exercise the analytical utility of GC/MS screening.

The Techniques of nS and GC

  The technique of sass spectrometry gives information about
the structure of a molecular species by measuring the
characteristic mass abundance pattern of fragments resulting from
ionizing the parent molecule,
by electron bombardment.    Ionization is usually accomplished
               The compound under analysis must have a
measurable vapor pressure at about 200 degrees C. (This
temperature and a pressure of 0.01 microbars are the normal
operating conditions of a GC-coupled mass spectrometer ion
source). The ionizing electron beam (70 eV energy) removes one
electron from some of the molecules of the sample vapor to yield
excited positive molecalar ions:

II + e ----> El (+) + 2e

         The molecular ion, a(+), is generally unstable (especially
     if at a high energy of excitation) and may decompose within a few
     microseconds to yield a series of positively charged fragment
        ions. Each fragment ion can in turn decompose to ions of lesser
ion where they af-6 sepdrated           -     - ^    spectrometer into the analyzer reg
ratio.  In sector instruments              according to their mass-to-charge
in quadrupole instruments by m             this is done by a magnetic field,
ght instruments by dn                  electric field, and in time-of-fli
Y- The mass spectrum of an                 adjustable ion detection time dela
a table of positive ions of                organic compound thus consists of
olecular structure (for                    different Basses and abundances. n
teroatoms) determines the                   instance number and location of he
pture subsequent to ionization,              frequency uith which bonds will ru
rt,  a characteristic mass                thereby producing, for the most pa
rical isomers may shou subtle              spectrum for each compound. Geoaet
e mass spectra owing to the              differences vithin their respectiv


-4-

influence of the geometry of neighboring groups. Optical
enantioaers yield identical spectra.

   Although the technique of mass spectrometrp was extensively
used by petroleum chemists from the 1940's, it was not widely
utilized in organic chemistry until the late 1950's. The first
extensive monograph on biochemical applications of mass
spectroaetry has just appeared ("Biochemical Applications of Mass
Spectrometry, " edited by G, R. Wailer, Wiley-Interscience, New
York, 1972).  Our colleagues and close collaborators in the
Stanford Chemistry Department,  led by Professor Carl Djerassi,
have been among the pioneers in the development of nS for natural
product chemistry, especially as applied to steroids (4 books and
in excess of 200 papers on various aspects of the theory and
application of MS have been published by Prof.. Djerassi's group
since 1961).  During tbe 1960's,  mass spectrometry was applied to
many different types of organic compounds. The accumulation of
these reference mass spectra was necessary to establish
fragmentation rules for the interpretation of unknown mass
spectra. The experienced mass spectroscopist becomes adept at
recognizing the mass spectral signatures of those types of
compounds with which he works but he cannot encompass within his
memory all the relevant information contained in the literature.
In addition,  many reference spectra determined by mass
spectrometry laboratories have not been published. To overcome
this problem, libraries of mass spectra are being compiled for
computer storage and retrieval so that they will eventually be
available for matching by computer against the mass spectra of
unknown compounds.  Progress is now being made toward compiling
libraries of mass spectra relevant to general metabolic studies.
These will match the accumulation already available for special
classes of organic molecules and for some drugs whose spectra are
important for emergency toxicological analyses (ref 2).

   Instrumentation advances in mass spectrometry during the
past decade like improved sensitivity, direct coupling with GC
and the use of computers for the routine recording and
presentation of mass spectra, a11 facilitate the large scale
application of mass spectronetcy to biomedical problems.

   Body fiuids and other materials encountered in biomedical
research are complex mixtures, For example, urine is known to
contain several hundred organic compounds at levels exceeding on
the order of 1 nanogram per milliliter. The gas chromatograph is
indispensable for the separation of such mixtures into discrete
components.  Yith mediua resolution instruments, the mass
spectrometer can be scanned repeatedly once every 2-4 seconds.
The gas chronatographic separation of a urine mixture may require
40-50 minutes:  the result is the accumulation of over 700 mass
spectra per analysis. The simplest way to identify these mass
spectra is to search a library of known compounds. Even if the
mass spectrum of a test compound does not reside in the library,
the best match found may be a related compound. This can
facilitate the manual interpretation based on the chemist's
knowledge of and guesses about the rules of frageentation. The


-5-

problem of computerizing the identification of compounds whose
mass spectra are not in a library is addressed by the DENDRAL
project. Computer programs have been developed to interpret mass
spectra of unknown coapounds from first principles (i.e., to
emulate the reasoning processes of organic chemists).

   Prequently compounds of biochemical interest occur in small
amounts in biological fluids. (By definition, many frontier
problems concern compounds at the limit of easy detection by
existing techniques). Thus,  the effectiveness of GC/HS as a
detector of biological materials is directly related to its
sensitivity. Current systems routinely operate vith sensitivities
such that mixture components with as little as 50-300 nanograms
of material can be measured. This limitation is imposed by the
following instrument-related factors. In order to record an
interpretable mass spectrum, a low resolution mass spectrometer
must have input to its source en the order of S nanograms of
material per second. Since a gas chromatographic peak lasts for
approximately 5 to 30 seconds, in GC/HS operation some 25-150
nanograms per GC peak are required for mass spectral analysis.
Inherent in the gas chroaatographic column and in the
semi-permeable membrane separator (used to preferentially remove
the helium carrier gas from the effluent stream) are losses of up
to 50% which increase the input sample requirements in a
practical sense to about 50-300 nanograms of material per GC peak
analysis.

   Another limiting factor in the application of the GC/RS
technique to biological extracts concerns the volatility of the
material to be assayed.  before the system can detect many
non-volatile components (e.g., carbohydrates, amino acids, etc.),
they must be converted into volatile derivatives uhich will pass
through the gas chroeatograph at a maxinutP oven temperature of
300 degrees C.  Above this temperature, column bleed from the gas
chromatographic phase will tend to enter the ion source and
complicate the recorded spectra.  Thus the GC/YIS technique is
restricted to those organic compounds which can be converted to
volatile derivatives and is not, in general, applicable to
inorganic compounds. A recent report (ref I) describes the
analysis by GC/RS of ketose diphosphates (as their trimeth.ylsilyl
(TtiS) derivatives) uhile aldose diphosphates (also as their TMS
derivatives) proved to be too unstable to analyze. It is safe to
assert, however, based on our uun experience and the literature,
that a broad spectrum of organic compounds of biological
significance will be amenable to analysis by GC/HS methods.

   There is another mode of operation of a Gc/HS system which
enables greater sensitivities to be attained for the yuantitation
of KNOWN aetabolites, If the mass spectrum and the gas
chromatographic retention time of the compound to be quantitdted
are known, the mass spectrometer can be used as a specific
detecting system for this compound.  This technique is called mass
fragmentography. Under these experimental conditions, the mass
spectrometer is not scanned over the ENTIRti mass range but is
directed to measure one or two SPECIFIC masses known to be


-6-

characteristic of the compound(s) being quantitated.
Consequently, there is an appreciable increase in sensitivity
since the mass spectrometer samples only the significant datd
points and can integrate the signal longer.

   In this mode, existing GC/HS instrumentation matches the new
fluorescent reagents for aaines (reported to detect approximately
10 picomoles). It also embodies the specificity of the Bass
spectrum at individual mass numbers. At greater cost and
cumbersomeness, the W can be extended to a quantum-counting
range of sensitivity. These methods are therefore likely to be
complementary to the special purpose methods, like
flnoresciretry, which are often cheaper and more efficient for
well defined classes of compounds. On the other hand, the history
of pesticide analysis shows how the GC can also be made
ultrasensitive at the cost of some loss of specificity.

   Using deuterated analogs as standards for the test compound,
qnantitation can also be achieved at sub-nanogram levels. We have
recently exploited certain characteristics of the quadrupole mass
spectrometer and its data system to develop a method for the
qnantitation of ten amino acids in soil extracts (ref 3a, copy
attached) and subsequently for the amino acid content of
biological fluids (ref 3b). This represents an advance in the
technique of mass fragmentography since the sector mass
spectrometers used up to this time, have been severely limited in
the number of ions and the mass range they could monitor for any
one experiaent. Our technique of quadrupole mass frayaentography
was used for the quantitation of the amino acids in the urine of
a patient with suspected branch chain amino aciduria. The results
are discussed later (see nethods of Procedure and figure 5).

  The overall management of the system and the reduction and
selective presentation of the large volume of data emanating front
the analytical instruments is an important task of the control
computer. Our experience in instruaentation comprises a good deal
of computational software embracing real time instrument
aanagenent,  automated data reductxon, and artificial intelligence
(ref 4, appended to this application), It also requires
considerable effort in electronic and vacuum technology for the
instrumentation hardware, and a coherent system approach for the
overall integration of these components.

   Present GC/HS systems are designed mainly for laboratory
research, incorporating great analysis flexibility but the
ability to handle only a small nunber of samples. Such systems
are not practical for large volume screening, but they can be
adapted for the pilot studies contemplated here. A properly
designed automated spstem could reduce these costs by as much as
an order of magnitude,  as would be essential for cost-effective
applications to general health care.

   The routine screening of normal and abnormal body
aetabolites,  including drugs, in human body fluids (ref S) is
currently the object of several research programs. Various


-7-

non-specific methods, including thin layer (ref 6,-I), ion
exchange (ref S,lO), liquid (ref 9), and gas chromatoyraphy (ref
11-14 and 17t), are used primarily with the goal of separating d
large number of unnamed constituent materials. Using these
techniques, compound widentifizatlon8* is made by a comparison or
the migration, under identical conditions, of the unknown spot
with reference compounds. This approach can lead to erroneous
identifications, however. This point is illustrated by a recent
article (15) which describes the use of mass spectrometry in the
identification of a case of isovaleric acideaia. Previously, the
same patient was diagnosed as having butyric dnd hexanoic
acidemia on the basis of chromatographic evidence alone. This
type of error is especially important when analyzing a @@newtr
(previously undescribed) metabolic error uhere rigorous
identification of relevant metabolites in various body fluids and
tissues is essential.

   For positive identification using mass spectrometry, the
separated components must be transferred from the chromatographic
medium to the mass spectrometer. The unknoun spot can be leached
from paper or thin layer chrowatograms, or in the case of liquiti
chromatography, the solvent removed, and a mass spectrum recorded
on the residual material. Trapping the various effluent
components Erom a gas chromatograph, with subsequent introduction
into the mass spectrometer, has been used. This approach requires
considerable time and is inefficient when applied to complex
separations.  It has been superseded by the direct coupling of the
gas chroeatograph and mass spectrometer.

   Gas chromatography is unquestionably the most convenient
separation technique to couple to the mass spectrowater because
the carrier \jas can be removed efficiently and easily as the
analysis proceeds. For recent examples of the use of the GC/MS
technique for the analysis of body fluids see refs 15ld. dased
on the work cited, as well as our own on-going programs, the
ability of the GC/FiS technique for the analysis of body fluids is
well established. We have drawn upon the published literature in
helping to design our experimental protocols (ref 18).

   As mentioned earlier, urine dnd other body fluids (e.g.,
serum,  amniotic fluid, etc.) are complex mi.xtures. The separation
(by gas chromatography) dud subsequent identification (by mass
spectroaetry) of these components can be a difficult task. To
simplifg the separation problem, the body fluid under analysis is
chemically separated into a number of fractions which permit
analysis for acids, am.ino acids, and carbohydrates present in
free or in conjugated form. Drugs and hormones, as well as their
metabolites, will also appear in the chromatographic seyardtions.
The gas chramatographic analysis of each class of compounds;
presents a metabolic profile. Abnormal profiles (ContaininLJ
either novel peaks or peaks deviating from their expected
amplitudes) are then assayed by mass spectrometry. The mass
spectra recorded during the elntion of each gas chromdtogf~~phic
peak then serve to identify tha constituents present in that
peak.


-a-

  The importance of this diagnostic technology may be
illustrated by a family seen for a fatal inherited disease at the
Stanford fledical Center.  Three male infants have been born to
this couple and each of the offspring died in a very similar
manner by 10 days of age,  The first infant was not seen at
Stanford, but the second infant was transferred from another
hospital,  in extremis. Aside from the striking family history,
there were no diagnostic claes,  and the second child was dead
within 6 hours of arrival. During the period of observation,
blood and urine were collected from the proband, and analysis of
the latter by gas chromatography (at another institution)
revealed an excess of erotic acid. This diagnostic clue, coupled
with the clinical picture of relatively rapid delsise after
protein (breast milk) feedings were well established, resulted in
a diagnosis of a hereditary deficiency of hepatic ornithine
transcarbaaylase, with resulting hyperaamonemia (ref 19). 'This
diagnosis was eventually confirmed when the third mdle infant of
this couple behaved in a manner identical to that of his two
deceased brothers and, despite attempts at therapeutic
intervention which included low protein feedings, he developed
documented hyperamconemia and died in this medical center. Study
of the liver revealed the complete absence of ornithine
transcarbasylase.  It must be stressed that clinical observations
of the first two infants and post mortem examinations were not
helpful diagnostically,  and it was the chemical identification of
erotic acid which led to the understanding of the familial
pattern of neonatal mortality in this family.

   Ilost medical centers have access to amino acid analyzers in
order to screen patients for metabolic abnormalities of the
principal amino acids, but unless a special research interest
exists, other inborn errors of metabolism cannot easily be
studied. At this institution the tiC/EIS system provides us the
opportunity to detect a uide variety of inborn errors which show
accumulation of amino acids, fatty acids, and many other
metabolites in urine, blood, amniotic fluid, and other biOlOJiCdl
fluids and tissues.

  Another application of GC/tiS pertains to pre-natal diagnosis
of hereditary inborn errors of metabolism, Fetal urine
contributes to the amniotic fluid by the twelfth week of
gestation and it should provide information which is
diagnostically relevant to the fetus (ref LO). Surprisingly
little is known about the origin, fate, and components of normal
amniotic fluid, although information is accumulating because of
interest in pre-natal diagnosis  and evaluation of the fetus (ret
21). The fetal cells of the amniotic fluid, cultivated IN VITRO,
are used routinely for pre-natal diagnosis of ,Jenetic disorders
(ref 22). The fluid itself has been used infrequently for
diagnostic purposes, although there is increasing evidence for
the utility of direct assay of this component of amniotic tluir!
for pre-natal diagnosis of heritable metabolic errors. Rahoney et
al.  (ref 23) have recently reported the first successrul
pre-natal diagnosis of methylmalonic aciduria (confirmed by stu*:y
of the fetus and performed in time to elect abortion). Their


-9-

observations suggest that at 12 weeks of gestation, methylmalonic
acid is undetectable in the amniotic fluid surrounding a normal
fetus and, probably, a fetus who is heterozygous for the
(recessive] gene (ref 24). Goo3man. et al. (ref 25) have
successfullp diagnosed argininosuccinic aciduria in a 16 week
fetus. This involved detection of argininosuccinic acid in the
amniotic fluid plus enzymatic  studies on cultured amniotic fluid
cells. Normally this compound is not detected in amniotic fluid
at 16 ueeks of gestation. Thera are reports of other conditions
being diagnosed in the fetus by direct asstiy of amniotic fluid
(ref 26) as uell.

   The accuracy of diagnostic procedures probiny amniotic rluid
for soluble constituents has been guestioncd because of the
possibility of contamination with maternal blood and because of
lack of information on the normal state at various times of
gestation.  If the fluid component could be used for pre-natal
diagnostic purposes, the phenotype of the fetus could he detected
relatively rapidly as compared to the time required to culture
sufficient amniotic cells.  Hereditary diseases which are
potentially amenable to diagnosis by analysis of the soluble
constituents of amniotic fluid are those in which the
accumulating metabolite is not cleared by the placenta but is
expected to appear in fetal uri-ne. Defects in epithelial
transport, e.g., cystinuria an3 Hartnup disease, are examples of
such conditions. However,  it is clear that this class of
metabolic errors is not the only one which might be detectable by
direct assay of amniotic fluid.  The examples provided above
suggest. that fetuses with overflow type metabolic errors may also
be detected.

   The development of diagnostic and screening techniques
suitable for various inborn errors of metabolism will require a
suitable computer based methodology for screening a large
selected sample of subjects with the subsequent resolution of
data into classifications describing normal states and ranges as
well as specific correlations of lX/HS analysis abnormalities
uith disease states. With a modest augmentation of existing
instrumentation facilities, we can accomplish these analytical
tasks on increasing nuabers of patients.

B.   SPECIFIC AIM

a) We plan to use GC/NS to screen urine and pldsma from
normal individuals ok various ages, including premature and
newborn infants,  in order to establish adequate control clatd
and to anderstand variations encountered.

b) We plan to use GC/MS in  th o diagnosis of inherited
metabolic abnormalities an3 in the detection and study of
previously unrecognized metabolic disorders.


-Y-

observations suggest that at 12 weeks of gestation, methylmalonic
acid is undetectable in the amuiotic fluid surroundiny a normal
fetus and, probably, a fetus who is heterosygous for the
(recessive) gene (ref 24). Goodaaa, et al. (ref 25) have
successfully diagnosed argininosucciaic aciduria in a 16 week
fetus. This involved detection of aryininosuccinic acid in the
amniotic fluid plus enzymatic studies on cultured amniotic fluid
cells. Nocrally this compound is not detected in amniotic fluid
at 16 weeks of gestation. Thera are reports of other conditions
being diagnosed in the fetus by direct assttp of amniotic fluid
(ref 26) as well.

   The accuracy of diagnostic procedures probing amniotic fluid
for soluble constituents has been questioned because of the
possibility of contaaination with maternal blood and because of
lack of information on the normal state at various times of
gestation. If the fluid component could be used for yre-natal
diagnostic purposes, the phenotype of the fetus could be detected
relatively rapidly as compared to the time required to culture
sufficient arniotic cells. Hereditary diseases uhich are
potentially amenable to diagnosis by analysis of the soluble
constituents of amniotic fluid are those in which the
accumulating netabolite is not cleared by the placenta but is
expected to appear i.n fetal urine. Defects in epithelial
transport, e.g., cystinuria an3 Hartnup disease, are examples of
such conditions. Hoverer,  it is clear that this class of
metabolic errors is not the only one which might be detectable by
direct assay of amniotic fluid.  The examples provided above
suggest that fetuses with overflow type metabolic errors may also
be detected.

   The dewelopment of diagnostic and screening technigues
suitable for various inborn errors of metabolism will require a
suitable computer basea methodology for screening a large
selected sample of subjects with the subsequent resolution of
data into classifications describing normal states and ranges as
well as specific correlations of GC/NS analysis abnormalities
with disease states. With a modest augmentation of existing
instrumentation facilities, ue can accomplish these analytical
tasks on increasing numbers of patients.

B.   SPECXPIC rras

a) Ye plan to use GC/HS to screen urine ana plasma from
normal individuals of various ages, including premature and
newborn infants,  in order to establish adequate control datn
and to understand variations encountered.

b) Ho plan to use GC/HS in  th o diagnosis of inherited
metabolic abnormalities aad in the detection and study of
previously unrecognized metabolic disorder:;.


c) Ye plan to use GC/MS to study normal variation of
clinically significant aetabolites in amniotic fluid and then
to apply these techniques to pre-natal diagnosis of
hereditary inborn errors of metabolism.

d) In support of the above goals, we plan to augment our
existing GC/PlS system and to more fully automate it for
screening larger numbers of patients through improved
computer management. This would fit with current concepts ot
regionalization of diagnostic facilities for genetic
disorders and would'& appropriate for a Genetics Research
Center.


-ll-

c.   METHODS OF PROCEDURE

   We use the following procadures to fractionate mine, blood,
and amniotic fluid in preparation for their analysis by GC/fiS. In
the case of plasma, the protein is first precipitated (by the
addition of ethanol) and the supernatant liquid dried IN YACUO
and processed as for urine.  Amniotic fluid is treated in the same
way as urine.
          URINE (pH =  1, internal standards added)
     I
             I
           1 ethyl acetate extraction
             I
      --------------------------------

    I                         I
organic phase            aqueous phase
(free'acids)                     L
                       --------------------------------
    A  (carboh:drates)         I           I
                      (amino acids)      I  HC1 hydrolysis
                  c                  B           I  and ethyl acetate
                 1   extraction

---------------------------------

       I                            I
  organic phase              aqueous phase
(hydrolyz:d acids)       (aeino'&cids)
        D                                 E

   The experimental procedurs used for uorkiug with a fluid
sample is as follows. To an aliquot (2.5 ml.) of fluid is added
6N hydrochloric acid until the pH is 1. Two internal standards,
n-tetracosane and 2-amino octanoic acid are then added. Ethyl
acetate extraction isolates the free acids (fraction A) which are
then methglated and analyzed by W/HS.  An aliquot of the aqueous
phase (G.5 al.) is concentrate3 to dryness, reacted with
n-butanol/hydrochloric acid followed by methylene chloride
containing trifluoroacetic anhydride.  This procedure derivatizes
any amino acids (or uater soluble amines) which are then
subjected to GC/BS analysis (fraction B). Another aliquot (0.5
ml) of the aqueous phase can ba derivatized (T8lS) for the
detection of carbohydrates (Fraction C).

   Concentrated hydrochloric acid (0.15 ml) is added to the
urine (1.5 al) after ethyl acetate extraction dnd the mixture
hydrolyzed for 4 hours under raflux.  Ethyl acetate extraction
then separates the hydrolyzed acid fraction (D) which is
methglated and analyzed by GC/MS.  A portion of the aqueous phasca
(0.5 ml), from hydrolysis of the urine, is concentrated to


-12-

dryness and derivatized and analyzed for amino acids (Fraction
?o o

   The above scheme represents a general method for preparing
body fluids for analysis. The conditions required for gas
chromatoyraphic separation vary with different classes of
compounds.  For instance,  using our method for chromatographing
derivatized free acids, amino acids, and carbohydrates, steroids
will not be detected.  A different gas chromatography column and
derivatization procedure would be required. Large molecular
weight compounds (e.g.,  tripeptides and mucopolysaccharide
fragments) cannot be analyzed by this system without their
degradation to smaller unit molecules.

   A suitable computer data system can significantly ease the
tedious burden of analyzing the large amounts of data emanating
from GC/fiS instrumentation. The computer assists in instrument
set-up and calibration,  data collection and filing, and data
reduction dnd analysis.  We have developed considerable experience
with such data systems,  both for low and high resolution
instruments,  since our fxrst design uas described in 1966 (ref
27, copy attached).  Our most recent efforts have focussed on
facilitating user interaction uith the data system and on
automating various aspects of data analysis (ref 3~). 'The
research proposed in this application entails the analysis of
increased numbers of body fluid samples, beyond the capacity of
the present system.  We therefore propose to augment the current
system to handle the larger sample volume and at the same time to
extend the capabilities of the system to reduce X/AS data.

   Our present system is built around the NLH-subsidized ACME
tine-shared computer facility (Iflh 360/50) with the real tine
data interface being through an Itrtl 1800 computer. These machines
vi11 disappear at the termination of the ACHE grant in July 1973
and will be replaced by a fee-for-service IBM 370/158 system with
more limited real time support capabilities. With the transition
implied by the machine changeover, we have reevaluated the
approach to be taken in implementing our GC/HS supFort needs
relative to the new requirements presently proposed, the
projected costs of alternative approaches, and the evolving
computer technology.  Mini-computer capabilities have advanced
significantl.y sinct? the previous decision was made in 1965 to
implement the existing system on the ACPiE .facility.

   After examining the options of implementing a data system on
the new time-shared facility or on a stand-alone mini system, we
feel that a mini-system is the more advantageous for our needs.
Such a system is the cheaper of the two based on projected costs
and is more responsive to real time needs in view of a smaller
set of demands on the mini-machine. There are limitations
inherent in mini-machines, however,  in terms of memory size and
software/language support.  The large central system (370/158 or
SUMEX if approved) will dct as a backup for the mini-system in
the relatively small num1;er of instances where these
considerations are important in low resolution GC/hS

I, -
/-- ,... `I


-13-

applications,

   In preparation for the transition this July, we are
iaplementiug a minimal data system on a PDP-11/20 computer. This
machine is well suited for "front endUi data collection and filing
activities as vell as simple analysis tasks. It cannot
simultaneously support data acquisition and the more
sophisticated analysis activities conte0plated in this proJran,
however, because of capacity limitations. In addition, it lacks
the arithmetic speed desirable for these analysis procedures.
Since the proposed research program will entail a heavy duty
cycle of data collection throughout the day to analyze the
increased number of body fluid samples,  data analysis and program
development would have ,to take place during off hours. We propose
to solve this problem by acquiring a second machine to support
the PDP-1 l/20. This machine,  tentatively selected as a PDP-11/43,
would provide for data analysis and program development support
and would be well suited to anticipated data analysis functions
because of its speed and extended arithmetic capabilities. We
have also considered a PDF-11/40 machine but feel the PDP-II/45
to be a better choice because of the relatively small cost
differential (approximately $10,000) in return for a factor of
2-3 in performance. The proposed system configuration is shovn in
Pigure 1, Our choice of Digital Equipment Corporation (DEC)
maChiAeS is based on oar existing hardvare and software expertise
with this equipment. Our existing high resolution PlS system
utilizes a similar PDP-ll/20 machine as the data acquisition
computer a5 vell.

   The PDP-11/45 vould have 24K words of memory, floating point
hardware and a programmable clock.  The memory size is based on
projected needs for PORTPAN-based analysis programs. The two disk
drives (an existing fixed head drive,  262K vords and a proposed
moving head drive,  1.2fi words) provide needed space for system
software, programs (source and object riles), and for spectral
data.  It should be noted that a single GC/rSS run contains some
700 spectra, each containing 500 12-bit spectral amplitude
measurements. Each such file therefore requires 350,000 words
(uncompressed) so that our proposed disk space would be quickly
consumed by several such files in residence. We can increase the
effective space available by compressing the data files to
eliminate insignificant measurements.  Note that this requites at
least one data analysis pass over the full file, however. we
propose to augment this disk capacity in the second year by
adding an additional drive.

   In general, data files vi11 be stored on luagnetic tape. Both
DEC tape and industry compatible tape drives are pravided. The
former is required for system maintenance and is ideal for
relatively small private program files, data files, etc. The
industr,y compatible tape provides for large volume storage of K~LV
data during data acquisition and for archival storage.

   This augmented data system will allow increased
sophistication in the analysis of GC/MS runs and in prescreenin\j


-14-

GC profiles.  In process.iny body fluid samples to establish Norma;
and to investigate specific clinical abnormalities, full flS
analyses of the GC effluent will be required. This involves
extracting from the approxieataly 700 spectra collected during
each run, the 100 or so representing the components of the body
fluid sample for identification. The raw spectra are contaminated
with background *gcolu~n bleed" and some are composited with
adjacent constituent spectra unresolved by the GC,

   We have begun to develop a solution to the problem of
effectively increasing the resolution of the GC by computer
analysis of the data.  These programs will allow us to
autoaaticaly locate the body fluid constituent spectra and remove
the distortions caused by background and poor resolution. These
"cleaned-up" spectra can then be analyzed by library search
techniques 3r first principles as necessary. By using a
disk-oriented matrix transposition algorithm developed for image
processing applications, we can rotate the entire array of 700
spectra by 500 mass samples for each run,  Tn this way ve can gain
convenient access to the "mass chroeatograan form of the data.
This form of the data,  displayed at a few selected masses, is
used in mass fragsentography described elsewhere in this
proposal. fYass chrosatograas have the important property of
displaying much higher resolution in localizing GC effluent
constituents. The automatic analysis techniques currently being
developed for mass fragnentograms can be extended to this more
general case.  Thus by transposing the raw data to the mass
ChrOQatOgCam domain, we can systematically analyze these data for
baselines, peak positions, and correct amplitudes thereby
deriving idealized mass spectra for the constituent materials.
These spectra are free from background contamination and
influences of adjacent unresolved GC peaks.

    The results of this work zan also lead to reliable
prescreening analyses of GC traces alone by having available a
detailed list of expecteu GC effluent positions and amplitudes
for the particular body fluid fraction under consideration. By
dynamically determining peak shape parameters for detected GC
singlet peaks,  interpretation of more complex peaks can be made
to determine if unexpected constituents or abnormal amounts of
expected constituents are present. If so, d more thorough K/M
analysis can be trade. This typz ot prescreening is valuable in
processing body fluid samples which may or may not be abnormal
and saves by committing the expensive mass spectrometer
instrumentation to analyzing only suspect samples. This, of
course,  assumes that norms have been previously established by
processing body fluid samples in detail from a large population
of normal subjects.

   The problem of spectrum identification is addressed by using
rapid library search techniques for the identification of
previously encountered compoun3s. Those not in the library will
be identified by the manual interpretation of the spectrum usinq
other information as available, These results will be
incorporated into the library to extend its domain of usefulness.

P-3/


-1%

This progressive compilation of a library of bioaedically
relevant compounds will speed the throughput of the system. .This
library w-ill be freely shared with other investigators.
Eventually the extension of the library domain will be assisted
by adapting the computer programs under development in the
DENDRAL project (NIB RR-UO612). These seek to emulate the
reasoning processes organic chemists employ in interpreting
spectra using fragmentation rules and other knowledge to infer
the correct molecular structure from among those possible.

   The most appropriate target material for this developmental
effort is surely the metabolic output of NORBAL subjects under
controlled conditions of diet and other intakes. The eventual
application of this kind of analytical methodology to the
diagnosis of disease obviously depends on the establishment of
normal baselines, and much experience already tells us hou
important the influence of nutrient and medication intake can be
in influencing the composition of urine, body fluids, and breath
(ref 18).  Among the most attractive subjects for such a baseline
investigation are newborn infants already under close scrutiny in
the Premature Research Center and the newborn nurseries of the
Department of Pediatrics at this institution. Such patients are,
for valid medical reasons, under a degree of dietary control
difficult to match under any other circumstance. Many other
features of their physiological condition are being carefully
monitored for other purposes as well. The examination of their
urine and other effluents is therefore accompanied by the most
economical context of other information and requires the least
disturbance of these subjects.

   Two obvious factors which could profoundly influence the
excretion of eetabolites detected by GC/#S are maturity and diet.
Be have already initiated a program for serial screening of
urinary metabolite excretion in premature infants of various
gestational ages and a determination of changes in the pattern of
excretion of various netabolites as a function of age following
birth. A synopsis of this research is presented later in this
section. These studies are being performed on infants admitted to
the Center for Premature Infants and the Intensive Care Nursery
at Stanford,  a source of some 500 premature infants per year, In
addition, in conjunction uith an independent study on the effects
of both quality and quantity of oral protein intake on the
incidence and pathogenesis of late metabolic acidosis of
prematurity,  ue plan to measure the urinary excretion patterns of
various metabolites and thereby partially assess the effect of
diet on this screening method.

   We shall use the analyses on blood and urine specimens from
normal individuals in the final development of rapid, automated
identification of compounds described by mass spectrometry. ay
compiling records o-f the gas chromatographrc profiles dnd
associated computer-identified constituentx of the body fluids of
normal individuals,  we will establish the statistical norms and
expected variations for the component and levels present.
Quantitation is achieved by introducing internal stdnddrds into


-lb

the body fluid fractions prior to analysis. We will seek to
minimize or at least stabilize the variations in measurement
results by assessing the effects of various aspects of diet and
medication. lornal blood and urine specimeos will also allou us
to test the system's operational capacity for rapid and accurate
aetabolite identification.

  Given our abilitg to identify various constituents of urine
and plasma and to understand normal variation, we shall apply the
GC/hS system to patholo$y, making use of patients with already
identified aetabolic defects for control purposes. The aain
application uill, of course, be diagnostic and patients with
suggestive clinical manifestations, such as psychomotor
retardation and progressive neurologic disease, as uell as
suggestive pedigrees (e.y. affected offspring of consanguineous
parents or multiplex sibships) vi11 be investigated. Actually, we
have been studying limited numbers of such patients already.
These patients are seen relatively frequently at any university
hospital, and their presence in the various in-patient and
out-patient services of the Stanford Department of Pediatrics is
well documented.

  The GC/M system will be invaluable in diagnosing various
inborn errors of metabolism, especially those involviog excretion
of various fatty and other organic acids. Some of these are
isovaleric acidemia (ref IS), methylaalonic acidemia (ref 28),
the recently reported inherited disorder of isoleucine metabolism
causing accumulation of alpha-aethylacetoacetic acid and
alpha-aethyl-beta-hydroxybutyric acid ,(ref 29), lactic acideaia
(ref 20), Refsum's disease (a defect in the oxidation of phytanic
acid - ref 31), erotic dciduris (ref 32), dnd as illustrated
above, ornithine transcarbamylase deficiency (ref 19). lie
recognize the potential of this methodology for defining
previously undescribed (*nevcc) inborn errors of metabolism (e.g.,
ref 15).

  In considering the strategy for applying tiC/MS techniques to
problems of screening and characterizing inborn errors of
metabolism, particular liaitations in our present system in terms
of detection capabilities or throughput capacity must be
accounted for. For example, our present system is somewhat
limited for screening and diagnosing those conditions whose
phenotypes include the accumulation of amino acids in urine
and/or blood. He can detect all of the naturally occurring alpha
amino acids except hoaocystine, cystine, and tryptophan. In
addition, the derivatization procedure being used, converts
asparagioe and glutamine respectively to aspartic acid and
glutamic acid. Thus the presence and quantity of asparagine and
glutamine cannot be: separately measured from that of aspartic
acid and glutanic dcid by current procedures. This situation
could be remedied by the use of a different GC column in order to
detect homocystine, cystina, and tryptophan. By using an
additional derivatization procedure, asparagine and ylutamine
could be measured. This approach, while rigorous in its ability
to detect and quantitate amino acids, complicates the higher


-1'7-

volume screening throughput. In effect, it would be necessary to
install and operate an additional gas chromatograph just for this
purpose because,the temperature programming conditions for the
new column differ from those required for the column presently
used for acid and carbohydrate fractions. Ye are auare that an
amino acid analyzer, separate from the GC/nS system, offers a
practical solution to larger szale screening for metabolic errors
involving the accumulation of amino acids. This approach also has
the advantage of providing increased assurance that the clinical
analysis of amino acid accumulations can be performed at times
when the GC/LIS system may be tied-up, under repair, or undergoing
engineering change. Such an analyzer, using ion exchange
chromatography, does not require derivatization of the specimen
and can detect all of the naturally occurrinq amino acids. We
therefore propose to acquire an amino acid analyzer to assist in
the screening of the increased number of samples expected from
our work at Stanford and from collaborating researchers
elsewhere. In selected cases where the amino acid analyzer alone
cannot provide sufficient resolution and quantitative accuracy in
characterizing amino acid accumulations, the more lengthy,
augmented X/MS approach vi11 be available.

   Another area in which our existing equipment would limit
throughput is in the capacity 3f the single low resolution mdss
spectometer.  The present GC/MS system can process 1-2 specimens
per day (including all five derivatized fractions). This
throughput has sufficed for small sample research applications
but we anticipate that this load will at least be tripled when we
are actively studying normal variations and are screening
patients for metabolic errors. We could, of course, eliminate the
bottleneck by buying more mass spectrometers. I'his would be quite
expensive. An alternative approach appears feasible.

   By passing the samples submitted for screening through a
preliminary GC profile analysis (without HS), we feel it will be
possible to identify those deviating from established normal
linits and warranting a more rigorous GC/HS analysis. This will
allow store efficient use to be made of the HS instrument time.
For abnormal samples, the deviation, under IIS analysis, may be
readily identified by library search techniques or may require
more lengthy analysis procedures. These could require different
chemical derivatization techniques or additiondl information such
as high resolution mass spectrometry,  This additional analysis
could consume days or veeks of effort.  Thus it is important to
make efficient use of these analysis capabilities. The number of:
samples eliminated in GC prescreening depends, of course, on the
nature of the samples submitted. Prescreening will be less useful
for the processing of samples which have been carefully selected
as clinically suspect than on a general samFling of "normal"
individuals.  It is in this latter case that we see a benefit to
developing the prescreening capability. Fat: this purpose, we
propose to add to our laboratory a 4 column gas ChrOmdtOgrdph.

   The Department of Pediatrics has affiliations with the
Kaiser-Pernanente bediCd1 Center in Santa Clara, the Santa Clar<l

/ . . = . ,.
  . I


-18-

County 5ospita1, and the Children's Hospital at Stanford. These
clinical units will fannel samples (urine, blood, amniotic fluict,
etc.) from suspect patients into the laboratory of tttis program.
Ye are already receiving samples from various physicians in
California (e.g., Presno - see below) and from Dr. Jose H.
Garcia-Castro, 5edical Geneticist of the University of Puerto
Rico Hedical School (see below also).  Arrangements are in process
to obtain samples from the newborn nurseries of the Los Angeles
County Efospital. The proposed augaentation of our instrumentation
with an amino acid analyzer and an additional gas chromatogrdph
will assist in being able to handle this anticipated specimen
load.  with the experience afforded by our augmented screening and
diagnostic facilities, we will have the capabilities to develop
into a regional center for the screening and diagnosis of various
metabolic errors.

   As an example of the application of these methods to
biomedical problems,  we can use some recent studies we have
undertaken on the urine of a patient suffering from acute
lymphoblastic leukemia. The gas chromatographic profile (figure
2) of the amino acid fraction of his urine showed the presence of
an abnormal peak (A). The mass spectral analysis of this
chromatographic peak (Figure 3) identified this component as
beta-amino ISObutyric acid (BAIB) from a comparison with a
literature spectrum of authentic material (ref 32a). The
literature on human urinary 5AX5 excretion is extensive and it
has been reviewed in reference 33 . Avapara (ref 34) noted
increased urinary 5AI5 excretion from patients receiving nitrogen
o ustard therapy while Bubini (ref 35) reported high levels of
BAI5 excretion in people exposed to excessive radiation levels.
Wright and Fink (ref 36) found elevated urinary BAIB levels from
mongoloid children and non-mongoloid mental defectives but these
conclusions uere subsequently disputed (ref 37). Harris in
England (ref 35) and Calchi-lovata in Ita1.y (ref 39) found that
6-10X of a sample population had elevated urine concentrations of
BAIB while DeGrouchy and Sutton (ref 40) in a study of families
of oriental extraction, and Yani, et al. (ref 41) with Japanese
families, found about 40% of their subjects excreted elevated
levels of BAIB. Gartler concluded that the variation underlying
BAI5 excretion by Nev York families (ref 42), Apache Indians and
Black Caribs of British Bonduras (ref 35) was due to genetic
difference at one locus. Later studies showed that thymine was
probably a precursor of BAIB in man (ref 44).

   At least two authors (refs 33 and 41) have questioned the
accuracy of the methods used for the detection and quantitation
of 5AI5 in urine. The early uock (refs 32 and 51), including the
first isolation of BAIB from urine (ref 38). was completed using
paper chromatography and reaction with ninhydrin to develop the
appropriate spot for detection and quantitation. As suggested
(refs 25 ana 34) other compounds in urine migrate with
characteristics similar to BAID thereby introducing an element of
imprecision into the published quantitation results. In those
instances where BAIB is present in only d small amount, or for
that matter in large amounts, it is alwaps possible that another


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metabolite could be present within the confines of the "BAIB
spot", Subsequent methods employed for BAIB quantitation include
thin layer chromatography (ref 47 ) and paper electrophoresis
(ref 48). These methods must also be considered as non-specific
for the estimation of BAIB in urine. Our own procedure, must be
considered far superior to these other techniques for both the
detection and quantitation of 6AX8 in urine because it combines
the quantitative aspects of gas chromatography with the positive
identification of a mass spectrum. We have observed several
instances in which the gas chromatographic peak associated vith
BAIB vas shown by mass syectrometry to contain other components.
Using the technique of mass fragmentography (refs 3a aud 3b), ue
can quantitate for BAIB under these conditions. In this
technique, a characteristic fragment ion from the mdss spectrum
of BAIB (figure 3, a/e.=153 or 182, for example) is summed over
the total ion chroaatogram to give an area corresponding to the
amount of BAIB present. Using an internal standard and a
characteristic ion from its mass spectrum and knowing the
ionization efficiency of both BAIB and the internal standard,
qnantitation can be achieved.  This vork is almost finished and a
paper describing a convenient way to quantitate BAXB in urine
vi11 be prepared in the near future.

   We hate examined urine samples from 18 patients suffering
from leukemia and have observe3 BAIB in a total of five
individuals. The patient whose urine was used to record the
chromatogram shown in figure 2 vas excreting 1.2 grams of BAIB
per day and following drug therapy this compound could no longer
be detected nor vas it detected in a urine sample collected 3
man ths later. Avapara (ref 34) claimed that all leukemic patients
excrete varring amounts of BAILS which was not detected in normal
urine.  Rillrann, et al.  (ref 49) noted that two out of three
patients vith chronic granulocytic leukemia produced high levels
of BAXB but following drug therapy, the level returned to normal.
Lee, et al. (ref 50) examined the urine of 33 patients vith
leukemia receiving specific drug therapy and failed to observe
BAIB excretion in any of these subjects. Our ovn observations, in
agreement vith the literature (refs 45, 49, and 50), suggest that
during the disease cycle, leukemic patients may excrete large
a8ounts of BAXB but this ceases following chemotherapy. Schreier
(ref 51) studied the urinary excretion of BAIB in leukemic
subjects and noted extrenely variable results with "massive
amounts@* of BAIB present in the terminal stages of the disease.

   As mentioned earlier in this section, we started pilot GC/HS
studies of biological fluids by investigating the urinary
metabolic output of premature children. So far ue hdVe studied
over 80 urine specimens from a total of 11 premature or "ma11
for gestational age" infants. Of this population, six infants
vere closely studied for periods of betveen six dnd eight veeks
beginning at day 3 of life.  Five of the six babies shoved
clinical symptoms of late metabolic acidosis (ref 52) and all
five shoved the same abnormal metabolic profile of their acidic
fractions.  All five children excreted excessive amounts of
p-hydroxyphenyllactic acid (I),  together with smaller guantities

P-3&


of p-hydroryphenylpyrnvic acid (II), and p-hydroxyphenylacetic
acid (IIX) (see figure YA and the structures shown below). After
reaching a maximum, the daily excretion of these abnormal
metabolites diminished until they were almost completely absent
when the child's pH and weight gain had returned to normal values
(see figure 4-B). The sixth infant showed no clinical symptoms of
acidosis and excreted only minute amounts of I, II, and III
during the observation period. None of the six subjects showed
any increased level of urinary tyrosine excretion. A blood
sample, from a patient who excreted large urinary amounts of I,
II, and IfI, showed no detectable quantity of these acids.

  1         z: I         z
The heavy urinary content of I and II by premature infants

ingesting a relatively high protein diet (COY'S milk) was
described in 1941 (ref 53).  Classical chemical isolation
procedures were used for the identification of I and II and
quantitation was achieved by two calorimetric assays. The
quantity of I was determined from the difference between the two
coloriaetric assays, assuming that no p-hydroxyphenylacetic acid
(III) uas present. Our finding of appreciable amounts of II? in
the urine of these premature infants suggests that the
quantitation values given previously (ref 53) for the excretion
of (I) are incorrect. Figure 4 illustrates the gas
chromatographic trace from the analysis of the acid fraction (as
their methyl esters) present in the urine of one premature infant
while acidotlc (A) and after recovery (B). The identification of
the compounds on Figure 4 follow from their mass spectral
signatures uhile quantitation uas achieved from measuring the
area under the gas chromatographic peaks using tetracosane as an
internal standard.

  During the past six months we have commenced a preliminary
investigatron of the use of GC/M for analyzing body flu.ids from
both normal patients and those whose clinical history suggested
they had a high risk of suffering from a metabolic disorder. The
first two cases reflect some of the results we have obtained
while the third describes the preliminary work undertaken with
amniotic fluid from normal pregnancies being terminated for
medico-social reasons.


-2'1-

  The first case involves a deceased child, whose parents were
first cousins and who was suspected of having a branched chain
amino aciduria 'as indicated by thin layer chromatography at
another institution (ref. 54). Preliminary screening by GC/HS of
the urine which was sent to us shoved no abnormal levels of
leucine, iseleucine or valine, nor of their respective alpha-keto
acids. In order to verify this point ue analyzed the dalino acid
fraction using our recently developed mass fragoentogrdphy
technique (refs. 2, 3). A copy of the coaputer output for this
analysis is reproduced as Figure 5. Clearly there are no elevated
quantities of any of the branched chain amino dcids, which we
know ve can detect, and the cause of this child's death remains
unknoun at this time. It is possible that this child shoved
intermittent excretion of branched chain amino acids (54). This
urine vas sent to us by Dr. Jose #. Garcia-Castro, Puerto Rico
(see note on Collaborative Arrangements).

   Recently a child died 33 hours after birth in Fresno,
California, vith the classical signs of hypophosphatasia (ref
55). This genetic defect is marked by high phosphoethdnoldmine
(PEA) 'concentrations in urine of affected homozygotes and
unaffected heterozygotes, After derivatization (in this instance
the TM ethers of the water soluble carbohydrate fraction were
prepared) ue vere able to detect by GC/?lS large concentrations of
ethanolamine and phosphoric acid but not PEA itself. The
derivatization procedure we used aost likely hydrolyzed PPA (ref
56) . We uere able to quantitate for this compound in the infant's
urine using an amino acid analyzer, and PEA excretion uas
extremely high (over'2CO times normal values for infants)
confirming the diagnosis.
child% parents,       Bert we examined urine samples from the
          presumed heterozygotes, by GC/HS and by the
amino acid analyzer. Again, no PEA uas detected by the former
method although the presence of ethanolamine and phosphoric acid
was demonstrated. we determined the following excretion levels of
PEA by amin:, acid analyzer:

Newborn infant:  94 micromoles per 100 ml. (Normal O-21-0.33)
Father:      269 micronoles per 24 hours (normal 17-99)
tlother:       32 micromoles per 24 hours (normal 17-99)

  It is of interest that in this family the affected infant
and his unaffected father both show subnormal serum alkaline
phosphatase activity. The mother,  who aid not excrete increased
amounts of PYA,
in her serum.  was found to have normal activity of this enzyme
        The following table summarizes the serum
phosphatase activity measureaents:

Rewborn infant:   0.2 units* (normal 2.8-6.7)
Father:        0.7 units (normal 0.8-2.3)
Mother:        3.4 units (normal 0.8-2.3)

    P - 1 unit is that phosphatase activity which will liberate
1 millimole of p-nitrophenol per hour per liter of serum)


-22-

   These findings are quite consistent with hypophosphatasia.
The heterozygous unaffected parents of children with
hypophosphatasia say or may not show biocheaical evidence of the
carrier state.

   The analysis of the metabolic content of amniotic fluid has
been pursued by many -investigators with the aim of identifying
metabolic defects I% UTEHO. Historically the first studies on
amniotic fluid utilized paper shromatography (refs 57, 58, 59).
Amino acid quantitatioa in amniotic fluid was attempted by a
visual coaparison of paper chrosatographic spots with standard
spots (refs 60, 61) and by spectrophotowetrically measuring the
intensity of spots eluted from paper chromatoyrams.  (ref 62). Ion
exchange chromatography was applied (refs 20, 21) for the
analysis of the free amino acids in amniotic fluid while gas
chromatography (refs 63, 64) and GC/HS (ref 65) were used for the
determination of the free acid content of amniotic fluid. On the
basis of their results, Levy and flontag suggested (ref 21) that
amniotic fluid arises from at least 3 sources: maternal blood,
fetal blood,  and fetal urine.

   Our own endeavors in the field of amniotic fluid analysis
has involved a study to determine those constituents found in
normal amniotic fluid. Ten specimens have been analyzed, All of
these samples were obtained at Stanford, most at 14-16 weeks of
gestation for chromosoae studies of the fetus, usually because of
increased maternal age. Following separation by ten trifugation
and removal of the cells for culture, the amniotic fluid was
again ceatrifuged (2900 rpm, International Clinical Centrifuye,
for 10 minutes) and the supernatant was stored at -20 degrees C
until analyzed by GC/klS. To date we have identified the following
acids in amniotic fluid: citric, ayristic, palaitic, stearic,
oleic and esters of phthalic acid (probably leached from the
plastic containers used to store the specimens). The amniotic
fluid from one patient contained salicylic acid (aspirin
ingestion) and from another caffeine (tea or coffee ingestion).
The following amino acids have been identified in these "normal"
amniotic fluid samples: alanine, threonine, serine, glycine,
valine, i-leucine, leucine, proline, methionine, aspartic acid,
phenylalaniae, tyrosine, glutamic acid and lysine. In view of the
current interest in the pre-natal diagnosis of
methylmalonicaciduria,  we can state that methylmalonic acid was
not detected in the acidic fraction of any of the "normal"
amniotic fluid specimens.  We are continuing these baseline
studies of normal amniotic fluid in order to eventually identify
abnormal accuaulations or metabolites in the at-risk pregnancy.
Variables wh.ich we will consider include gestational age,
diagnosis and status of the fetus,  maternal plasma concentration
of the relevant substances and maternal plasma concentrations of
hormones and medications and their metabolites. We are presently
planning to investigate variation in amniotic fluid
pregnanetriol,  170ketosteroids and some of the specific androgens
which are elevated in the adrenogenital syndrome (ref bb).


-23-

   At this time we are not prepared to predict that GC/HS
analysis of amniotic fluid alone will provide the accurate
diagnostic information about the fetus required for a decision
for selective abortion. Heterozygous fetuses and contamination of
the amniotic fluid with maternal blood (that result of the
amniocentesis procedure) raise possibilities for false positive
diagnoses which may lead to ab;rrtion of unaffected fetuses if
cultured amniotic fluid cells are not also studied. however,
studies relating quantity of the pertinent metabolite in amniotic
fluid to enzyme activity in the cultured cells should be helpful
in providing information for discriminating between heterozpgous
and hoaozygoos fetuses by GC/l_S. l;n other words, we will monitor
at-risk pregnancies with GC/HS and with biochemical studies of
cultured cells until we are confident we can predict the
hoaozygons state from GC/HS analysis alone, Maternal blood
contamination will be monitored by gross and microscopic
examination of amniotic fluid samples, and the behavior of
contaminated fluid on GC/HS analysis will be anticipated by
studying a series of amniotic fluid samples to which various
amounts of blood from a heterozygous individual (The mother in
the high risk pregnancy is usually a heterozygote) have been
added.

D.   SXGNIPICA%CE

  An accurate diagnosis is especially important for genetic
counseling purposes. The diagnosis allovs reference to published
data on the mode of inheritance and, thus, expresses the
recurrence risk. Furthermore, accurate diagnosis of the
accumulated metabolite provides insight into the biochemical
pathogenesis and into therapeutic approaches to the control of
hereditary inborn errors of metabolism. The GC/HS system, with
its potential for automated and rapid identification of many
metabolites, provides the diagnostic accuracy necessary for a
clinical program. This system also provides the methodology for
detecting previously unrecognized inherited metabolic errors.

  The methodology developed by this project will decrease the
time required for antenatal diagnosis of certain metabolic
disorders. The elapsed time until diagnosis is important because
legal, psychological and, perhaps, obstetrical considerations
have set a deadline of 20 weeks of gestation for selective
abortion. This deadline is sosetimes not set when amniotic cells
obtained for diagnostic purposes fail to divide sufficiently
rapidly in culture to provide adequate material for biochemical
testing.

  The study of system designs for automated GC/HS systems in
the clinical environment will pave the way for a prototype system
which will make more routinely available these poverful
analytical tools. Such tools will be important in the inevitable
regionalization of facilities for the screening, diagnosis and
study of hereditar,y inborn errors of metabolism,

P-JYC


-24-

E.   FACILITIES AVAILABLE

    We vi11 derive much of the clinically significant material
for analysis from patients in the Premature Research Center and
the Clinical Research Center of the Department of Peuiatrics.
Analyses will be performed in part on existing GC and tlS
equipment in the Department of Genetics. We now have a two column
Varian Aerograph 2100 gas chromatograph used for specimen
prescreening and an older, less effective Varian Aerograph 1200
gas chroeatograph connected to a Yinnigan 1015 quadrupole mass
spectrometer. We have access to a Varian-flAP 711 high resolution
GC/M system to assist in the identification of compounds not
readily identified by lou resolution spectrometry alone. Also
available to the project are the electronics facilities and
software experience of the Instrumentation Research Laboratory of
the Department of Genetics.  Assuming the approval of the SOHEX
resource proposal,  we will have access to large scale coaputing
support for the later applications of artificial intelligence to
this research from the SUKEX PDP-10 machine,

F.   COLLABORATIVE ARRANGEHENTS

   This project involves an interdisciplinary collaboration
between Drs,  J, Lederberg (Principal Investigator) and A.
Duffield (Associate Investigator) of the Department of Genetics,
Drs.  N. Kretchner and tl. Cann (Associate Investigators) of the
Department or Pediatrics,  and the Instrumentation. Research
Laboratory (including Dr. E. C. Levinthal and Rr. T. C.
Hindfleisch), Dr. Jose M. Garcia-Castro, University of Puerto
Bico School.of Hedicine has agreed to send us samples (urine and
blood) fror selected patients.  We will also receive samples from
collaborators at the Kaiser-Peraanente bedical Center in Santa
Clara, the Santa Clara Valley Medical Center, the Children's
Hospital at Stanford, and the Los Angeles County Hospital,

   This arrangement is a prototype of efforts to organize d
systematic network of support to physicians at outlying centers
for the mutual benefit of better care for their patiertts, and
providing pre-screened, high-yield material for scientific study.
Before tbis is formalized, ue uish to build up practical
experience uith collaborations where personal understanding
allows good communication about respective needs and flexibility
in meeting urgent requirements.


-25-

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43) Gartler, S. B., Pirschein, I. L., and Kraus, R. S., @*An
Investigation into the Genetics and Racial Variation of BAIE
Excretion," Am. J. Huaan Genetics, 9, Paye 200 (1957).

44)  Gactler, S. fi., "A Betabolic Investigation of Urinary RAIU
Excretion in Man, Arch. Biochem. 80, 400 (19SY).

45) Pink, K., Henderson, R. B., and Fink, R. ai., "BAIB: A
Possible Factor in Pyrimidine fietabolism,ti Proc. Sot. Exptl.
Biol. Bed., 78, Page 135 (1951).

46) Crumpler, H.R., Dent, C.E., tiarris, H., and Westall, R.G.,
"BAIB, A Xew Amino Acid Obtained from Human Urine," ddture, lb7,
Page 307, (1951).


-29-

47) Goedde, H.W. and Brnnschede, H., 18frAIB: A TLC Plethod for the
Quantitative Estimation in Elnman Urine," Clin. Chim Acta, 11,
page 485, (1965).

48)  Pessas, P.R., Koniavitis, A., and Zeis, P.R., J. Clin,
Path., 22, Page 154, (1969).

49) Killmann, S.A., Rubini, J.R., Cronkite, B.P., and Bond,
V.P., *Urinary Excretion of Beta-Amino Isobutyric Acid (BAIBA)
in Leukemia and Polycytheaia Vera," Acta Haemat., 25, Page til,
(1961).

50) Lee, K.B., Reill, D-W., and Bridges, J.B., "Urinary Amino
Acid Excretion in Subjects with Leukemia," 3. Clin. Path., 20,
Page 70, (196'7).

51) Schreiec, "The 'Behavior of Amino Acids in Body Fluids During
Development and Growth: Physiology and Pathology in Amino Acid
Pools," J.T. Holden, Editor, Klsevier, blew York, Paye 2'76,
(1962).

52) Ranlov, P. and Siygaard-Aadersen, 0.. "Late Betabolic
Acidosis in Premature Infants;  Prevalence and Significancc,n
Acta Ped. Stand., SU, Page 531, (1965).

53) Leviae, S.Z., Bacyles, E., and Gordon. H.H., nA Defect in
the Metabolism of Tyrosine and Phenylalanine in Premature
Infants, I: Identification and Assay of Intermediary Products."
J. Clin. Invest., 20, Page 199, (1941).

54) Dancis,  J. and Levitz, M,  "Abnormalities of Branched-Chain
Amino acid Betabolisr:  The Metabolic Basis of Inherited
Disease ,I) Stanbury, J-t)., Wpngaarden, J-B., and Predrickson,
D.S..  Editors, McGraw-Hill, Raw York, 3rd Edition, Page 426.

55) Bartter, F.C., *Hypophosphatasia: The Retabolic Basis of
Inherited Disease," Stanbury, J.b., Wyngaarden, J. B., and
Predrickson, D.S., Editors, BcGcau-Rill, Neu York, 3rd Edition,
Page 1295.

56)  Rasmussen, K., WPhosphocylethanolamine and
Rypophosphatasia, Danish fled. Bull,, 15 (Suppl. II), 1 (196d).

57) Bevis, D.C.A., %omyositi~n of Liquor Amnii in Haemolytic
Disease of lewborn," Lancet, ii, Page 443, (1950).

58) Deflarca, C., WAminoacidie Chetoacidi Presenti nel Liquid0
Axniotico,N G. Biochem., 3, Page 296, (1954).

59) Thomas, J.A. and Thiery, J.P., "The Use of Deproteinized and
Purified Amniotic Fluid. Culture and Prolonged Conservation of
the Tissues; Bicrobiological Cultures," Bull. Sot. Chim. Biol.,
35, Page 841, (1953).


-3O-

60) Orlandi, C., Torsello, ft.!!., and Bottiqlioni, P., *'Analisi
Qualitativa e Dosaggio Semiquantitativo degli Aminoacidi
Contenuti nel Liquid0 Amniotico,** Attual, Ostet. Ginec., 4, Pacje
871,  (1958) *

61) Wirtschafter, Z.L.,  "Free Amino Acids in Human Amniotic
Fluid, Fetal and Maternal Serum,*' Amer. J. Obutet, Gynec., `76,
Page 1219, (1958).

62) Sassi, D., *#Sulla Presenzs degli Aminoacidi nel tiquido
Amniotico," Honogr, Ostet, Ginec., 33, Page 683, (1962),

63) Warren, C., Allen, J.T., and Holton, J.8.6 "Assessment of
Foetal Lung Maturity by Amniotic Fluid Fatty Acid AnaLps&,"
Clin. Chin, Acta, 44, Page 457, (1973).

64) Reiner,  E. and Hicks, J.J., "Pyrolysis-GLC Studies on
Amniotic Fluid: A Prospective nethod for Confirming Inborn
Errors of Fetuses," Chromatographia, S, Page 529, (1972),

65) Hagenfeldt, L. and Kagenfeldt, K.,  "Gas Chromatographic-Mass
Spectroaetric Analysis of Organic Acids in Amniotic Fluid,"
Cllin. Chia. Acta, 42, Page 219, (1972).

66)   Neu, ff. I., "Antenatal Diagnosis of the Adrenogeaital
Syndrome," Lancet, 1, 83 (1970).


                   L        I      I

Existing Equipmentm

FIGURE 1.  PROPOSED DATA SYSTEM HARDWARE CONFIGURATION


FIGURE 3


80

60

40

20

r53

40 60   `6'0  100 120 140 160 180 200 220 240 260

FIGURE 3

Malrs spectrum of P -amino isobutflic acid


         I-









.









                                         FIGUW 4
                             GAS CIIROMATO(;fiAPiI OF URINARY ACID PROPILE
                             A. PATIKIT ACIDOTIC (Z-17-73)
                                   I\" !`A'I'T)`yr' vfq."; 1, ( 2,. 1 (l-71 1


l.fIf!
n.52
3.51
f-l.23
Q.4P
-!I.51
').7c,
2.7')
n.74
I , :: 1
3 "I,

Areas

A r-as

Arr-as

Areas

Arwis

hrfvs

Areas

Arcas

At-pas

Art-as

Areas

11349.2/  21382.5

l&LX/ 109h7.3

6272.4/  6054.7


39;. 21  I;!i53.7


R35,1/  67621.3


7cn3.1/-330es.r

116?.7/ 2712.q

3293.4/  31f?4.7


2920.1/  5971.8


22El.E/  5751.0


lcZE3.1;/  19353.8

PATIC! =   0.5308 Locattan Error - 9

?????? ?? o 0.1771 Locatifh rrrar =`-2

F4T

PAT

PAT

PAT

I

1.0277

0.107G

0.1235

-0.2238

0.4270

        FIGURE 5
ANALYSIS OF 12 AMINO ACIDS IN URINE
   USING MASS FRAGMXNTOGRAPHY

Location Erroi = 2



Lccntfnn Error - -1



loc?tinn Frrnr = 2


Locatfon Crror = 2


LocatIon Error = 0



Location Error - 0

Lor?&an 'rror = -2


Locattnn rrror = 0


Loci+tlan Frror - -1


THE SIMULTANEOUS QUANTITATIPN OF TEN AMINO ACIDS IN SOIL EXTRACTS
       BY MA& FRAGMENTOGRAPHY

W. E. Pereira, Y. Hoyano, W. E. Reynolds, R. E. Summons and A. M. Duffield

    Department of Genetics, Stanford University Medical Center

               Stanford, California

Running Title:  Mass Fragmentography of Amino Acids

Address for Proofs: Dr. A. M. Duffield
            Department of Genetics
           Stanford University School of Medicine
            Stanford, California 94305

Received

P- 5-z


  The analysis of amino acids from terrestrial and extraterrestrial
sources is becoming increasingly, important (l-5) ,  The need for a specific,
                                    I

sensitive and rapid method of quantitation is desirable.  The methods
currentiy employed for amino acid analysis involve ion exchange procedures
(6,7) or gas chromatography (8-10). These techniques, although of

immense value, are limited by their non-specificity for the absolute

identification of any substance responsible for a gas chromatographic

peak.

  In the present communication we report an absolute, unambiguous

method for the positive identification rnd quantitation of ten amino acids

present in soil extracts using GLC-mass fragmentography. In mass

fragmentography the mass spectrometer is used only to detect certain

preselected ions known to be characteristic for each compound being
quantitated, and the internal standard.  The technique of mass fragmentography
                                           . .

using sector mass spectrometers is usually restricted to the simultaneous

monitoring of up to three integer mass values (11, 12), although with

one instrument five ions were used

spectrometer up to eight ions have

analog signals monitored (14). We

(13) o  Using a.quadrupole mass

been selected and their respective

now wish to report the modification

of the gas

previously

conqo~ of

chromatography-quadrupole mass spectrometer-computer system

described (15) for the simultaneous monitoring under computer
                          ., . . .                                               ,

          the ion currents from 2.5
  cc\nje br*w-- WIICS C Ld 750 i- con+tst 2 r;sc.l;Eetdcnteger mass _ values. -pjcsc via us cc-
                                ;yL  UVIIlCbIL 4or n`$l &8y--io+j ;I:;*(j SC'
P*r.tr'XIf required this number could be increased by suitable alteration of the

computer control programs.  Specifically we wish to report the application
                                .

of thii system to the quanti'tation of ten of the amino acids present in soil
                           . ___. --. _-
                                   .,
extracts .


Reagents:  A deuterated amino acid mixture was supplied by

Merck Laboratory Chemicals (New Jersey). 1.25N HCl in n-butanol,

25% (v/v) trifluoroacetic anhydride in methylene chloride and Tabsor

column packing were obtained from Regis Chemical Co., Illinois. A

standard amino acid solution was purchased from Pierce Chemical Co.,

Illinois.

Equipment:  A Varian model 1200 gas chromatograph was coupled

by an all glass membrane separator (16) to a Finnigan 1015 Quadrupole

mass spe-trometer which was interfaced to the ACME computer system of

the Stanford University Medical School (15). CLC separations were

conducted using a 6 foot by 4 mm. (I.D.) coiled glass column packed

with Tabsorb (Regis Chemical CO.).  The flow rate of the carrier gas

(helium) was 60 ml/minute.

. .

The uniqueness of the mass spectrometer instrumentation lies in the
                                                                        -_-

modified computer software (program) used.  The hardware is the system
previously described (15) and as:umes an operating cycle of:

(a) transmission of a control number, N, from the computer to an

interface controller which sets the quadrupole mass analyser to

  a particular mas-s point in the m/e continuum.
                                        --
(b) an integration of the ion.signal for a pre-set period, T

  (integration time = 8 milliseconds in our work), and
(c) computer reading of the integration value with a twelve bit A + D

      conversion.
     .                         * '
For the recording of normal mass spectra N is selected such that successive


cycles result in m/e values of 1,2, . . ...750. At the beginning of each
                   --

day the instrument is calibrated using a reference compound. Idiosyncracies

of the IBM 360,/50 to IBM 1800 computer data paths dictate that the mass
                                     s

values be buffered into groups of 250.%

   For normal g.c.-m.s. procedures the operator is allowed to select

a mass range of 1 to n x 250 (n = 1,2, or 3 buffers).  For mass fragmento-
graphy n is set to zero and instead,a "precision collect" buffer of 250
control-data acquisition cycles is employed.  The operator must then enter
the pre-selected m/e values he wishes to scan. When the precision collect
                   --

buffer is constructed, 10 cycles are allocated to each m/e value selected.
                                                               --

The first of the 10 cycles sets N to K -4.
                                           In   The returned integrated ion

measurement is discarded; this cycle serves only to slew the quadrupole
electronics from anywhere in the m/e continuum to the mass region of
                                      --

InterFst. The additional 9.cycles are used with N = N,-4....Nm.....Nm+4.

The returned values represent a set of readings about the m/e value of
                                                                  --

Interest + 0.5 amu.  The center three points are then smoothed with a

five point qudratic function (17).  The highest value of these three

smoothed points is then selected,as the precision collect value.  Thus
                                      ,

small drifts in calibration are corrected and a signal average obtained.
Finally ,i the abbreviated "spectrum" of 25 precision intensities for each

m/e are filed on disc.
a-

  Such a "spectrum" is recorded every 2 seconds and a summation of all

the ion intensities is used to cou$truct the ion chromatogram shown in Fig.

2.  Inditidual ion chromatograms can also be constructed if required

(Fig. 3).  A threshhold is established from the ion currents before and

aftet each gas chromatographic peak and a computer program performs

integration of the ion currents under each peak.


Procedure

  1 g of sieved, air-dried soil (Stanford University garden soil)

was refluxed with 6N HCl (10 ml) for 20 hrs.  The mixture was filtered
and the residue washed kth 1N HCl (5 ml).  The combined filtrate

and washings were extracted with chloroform (4 x 10 ml) and the

aqueous phase evaporated to dryness.  The residue is dissolved in

water (5 ml) and passed through a column of "Ion Retardation Resin"

AG 11-A8 (So-100 mesh, 1 x 21 cm).  The amino acids were eluted with
water (50 ml) and the eluate evaporated in vacua to dryness. The
residue is dissolved in water (5 ml) and placed on a column of

p-57


cation exchange resin (AC SOW-X12, SO-100 mesh, 1 x 21 cm) and washed

with water (50 ml) to remove neutral and anion contaminants.  The amino
acids were eluted with.4N NH4?H (80 ml) and the eluate evaporated to

dryness. The residue was disiolved in water and made up to a volume of

4 ml.  A portion of this solution (1 ml) was used for the amino acid

analysis using an amino acid ,analyser.  To another 2 ml of the processed

solution was added 2 ml of the deuterated amino acid standard solution

(100 mg in 100 ml of O.lN HCl) and the mixture evaporated to dryness.

The residue was refluxed with 1.2 N HCl in n-butanol (1 ml) for 30 min.

and evaporated to dryness in vacua.  To the residue trifluoroacetic

anhydride in methylene chloride (0.7: ml) was added and refluxed for 10

min.  The solution was evaporated to dryness at room temperature and the

residue dissolved in ethyl acetate (100 ~1). An aliquot (1 ~1) was
injected into the injector port of the gas chromatograph and the oven

kept at 100" for 1 min. when it was programmed at 4"/min. to 220'.

  To each of 4 tubes containing 2 ml of the deuterated amino acid
standard solution (100 mg in 100 ml of O.lN HCl) was added 150, 200,
250 and 300 ~1 respectively of a standard amino acid solution (2.5 ,,moles

                                                             -e
of each amino acid per ml).  The solutions were mixed and evaporated 'to

dryness.  Each residue was derivatized by the above method and an
aliquot of each (1 l.J) injected into the gas chromatograph which

was operated under the conditions described above.  This procedure

                                     . . . .

was used to construct a standard curve for the quantitation of each
am&o acid.  A typical standard curve is shown (Figure 1) for glutamic.

acid.

p- 5-z


RESULTS

The N-TFA, 0-n-butyl derivative was chosen for the derivatization
                              s

of amino acids for two reasons.. Firstly, these derivatives have
                 14

excellent glc separation characteristics @$j and secondly the selected
                                    .

characteristic fragment ions of the deuterated and non-deuterated

derivatives do not interfere with each other, nor with other a-amino

acids.  Table I records the individual ions monitored for quantitation

in the mass spectra of each of the deuterated and non-deuterated amino

acids.  Thezomput~tegrates-the-intensity of the deuterated and

Be-ratio-of-their-respective-peak-areas7

Our results of a typical soil analysis are compared with those

from an amino acid analyser in Table II. The higher value obtained

with lysine by the amino acid analyser is due to a ninhydrin positive

substance in soil interfering with the quantitation of lysine. In

this respect mass fragmentography is superior to the amino acid analyser

in that using a mass spectrometer as detector only characteristic

pre-selected ions are detected and quantitated and any impurity present'

under the same gas chromatographic peak is not measured*  A summation of

20 such characteristic ions was plotted as an ion chromatogram of a

derivatized soil sample and is shown in Fig. 2.

Preliminary experiments showed that when the deuterated amino

acid mixture was added directly to the soil sample extensive hydrogen-

deuterik exchange occurred during acid hydrolysis of the soil extract.

The removal of the isotopic label was catalysed by the hot mineral acid

in presence of excess mineral used in the soil hydrolysis step. Fox


and collaborators have reported (4) a similar finding concerning the
                                                                           _ -

decomposition of amino acids in soil upon direct acid hydrolysis. In

the present work the deuterated amino acid mixture was added just before

derivatization (i.e. after hydrolytic extraction of the soil) in order

to avoid this problem.  However, in cases where it is necessary to

quantitate the free amino acid content of complex mixtures, such as

In serum or urine samples, the deuterated amino acid mixture may be added

sample before processing without any deleterious

Although only ten amino acids present in soil were quantitated

the method can be extended to all the normal amino acids found in

protein.  The deuterated analogs of arginine, histidine, serine,

threonine and tyrosine are commercially available.  Appropriate

deuterated analogs of methionine, tryptophane, cysteine and cystine

would have to be chemically synthesized from the appropriate precursors.

In these instances at least two deuterium atoms should be incorporated

in non-exchangeable positions so that for the characteristic ion

chosen the P + 2 peak is separate from the 13 C isotope contribution

of the unlabeled amino acid.  Furthermore,  the deuterium substitution

need not be quantitative (>90%) provided the same characteristic ion

of that deuterated analog is used for the construction of a standard

curve such as Figure 1.
   -Lx


  Instrument analysis time is approximately one hour and with our

system we have been able to achieve accurate quantitation with samples
containing as little as 10 nano'grams of an amino acid,

SUMMARY

  A specific and sensitive method for the identification and

simultaneous quantitation by mass fragmentography of ten of the amino

acids present in soil has been developed.  The technique uses a computer

driven quadrupole mass spectrometer and a commercial preparation of

deuterated amino acids is used as internal standards for purposes of

quantitation.  The results obtained are comparable with those from an

amino acid analyser.  In the quadrupole mass spectrometer-computer

system used up to 25 pre-selected ions may be monitored sequentially.

This allows a maximum of 12 different amino acids (one specific ion
in each of the undeuterated and deuterated amino acid spectra) to be

quantitated.  The method is relatively rapid (analysis time of

approximately one hour) and is capable of the quantitation of nanogram

quantities of amino acids.

. . . . .

ACXNOWLEDGMEXTS

This research was funded by the Planetology Program Office, Office

of Space Science, NASA Headquarters under grant NGR-05-020-004.


REFERENCES

1.

2.


3.

4.

5.

6.

7.

8.

9.

10.

Il.

12.

'13.

14.

POCKLINGTON, R., Anal. Biochem. 45, 403 (1972).

TAYLOR, R. and DAVIES, M. G., Anal. Biochem. 51, 180 (1973).
                                                   -

HALPERN, B., WESTLEY, J. W., LEVINTHAL, E. C. and LEDERBERG, J.,

Life Sciences and Space Research - North Holland, Amsterdam, 239 (1967).

HARE, P. E., HARADA, K. and FOX, S. W., Proc. Apollo 11 Lunar

Science Conf. 2, 1799 (1970).
               =

KVENVOLDEN, K., LAWLESS, J. G., PERING, K., PETERSON, E., FLORES, J.,

PONNAMPERUMA, C., KAPLAN, I. R. and MOORE, C., Nature, 228, 923 (1970).
                                                               Z

SPACKMAN, D. H., STEIN, W. H. and MOORE, S., Anal. Chem. 3&, 1190 (1958).

HAMiLTON, P. B., Anal. Chem. 35, 2055 (1963).

GEHRKE, C. W., KUO, K. and ZUMWALT, R. W., J. Chromatog. 57,
                                                                 =

193 (1971).

GEHRKE, C. W., ZUMWALT, R. W. and WALL, L. L., J. Chromatog. 37,

398 (1968).                         . . .
                                     ._
JijNSSON, J., EYEM, J. and SJGQUIST, J., Anal. Biochem. 51, 204 (1973).
                                                               -

HAMMA!, C. G., HOLMSTEDT, B. and RYHAGE, R., Anal. Biochem. 2, 532

(1968).                                                 _-

HAMMAR, C. G. and HESSLING, R., Anal. Chem. 43, 298 (1971).
                                     --  -

SNEDDEN, W., PARKER, R. B. and WATTS, R. E., Int. Conf. on Mass
                                                   --
Spectrometry Brussels, 31&. i 4 Sept. 1970, Vol. 5, Institute
                          -

of Petroleum, London, and Elsevier, Amsterdam, 1971. p. 742.
                                . .

KNIGHT, J. B., Finnigin Spectra, &, No. 1 (1971).

.lS.. REYNOLDS, W.: E.; BACON;& A;, BRIDGES, J.. C., COBURN;T. C.,.

HALPEPN, B.,
.

LEDERBERG, J., LEVINTHAL, E. C., STEED, E. and TUCKER,

R. B., Anal.

        .`, .
Chem. 42, 1122 (1970).
      =


  16.  HAWES, J. E., MALLABY, R. and WILLIAMS, V. P., 2. Chromatog. Sci.
   2, 690 (1969).
(%H. GEHRKE, C. W. and STALLING, i. L., Separation g. 2, 101 (1967).
1q.6  PEREIRA, W. E., BACON, V. A., HOYANO, Y., SUMMONS, R. and DUFFIELD,
   A. k., Clin. Biochem. (In Press).


Table I.  CHARACTERISTIC FRAGMENT IONS SELECTED FOR MASS FRAGMENTOGRAPHY OF UNDEUTERATED AND-DEUTERATED
  .

N-TFA-0-IJ-BUTYL-AMINOrACIDS.

Amino
Acids i

Fragment Ion

VAL


GLY


ILEU


LEU

PRO

PHE


ASP


GLU


LYS

CH3CH=kICOCF3 (m/e 140)      CD3CD(NH2)COOH
I-C3H7CH=zHCOCF3 (m/e 168)     I-C3D7CD(NH2)COOH
CH2&COCF3 (m/e 126)        NH2CD2COOH
C2H5CH(CH3)CH=zHCOCF3 (m/e 182)   C2D5CD(CD3)CD(NH2)COOH
i-C3H7~2CH=kXOCF3 (m/e 182)    I-C3D7CD2CD(NU2)COOH

kCOCF3 (m/e 166)

C6H5CH=CHCODHl + (m/e 148)
BuOOCCH2CH&XOCF3 (m/.e 240)
HOOCCH2CH2CH=kKOCF3 (m/e 198)
CX2-CHCH2CH2CH-&COCF3 (m/e 180)

Deuterated Amino Acids

D

C6D5CD2CD(NH2)COOH
HOOCCD2CD(NH2)COOH
HOOCCD2CD2CD(NH2)COOH

Fragment Ion

CD3CD&COCF3 (m/e. 144)
                       . .
I-C3D7CD=hCOCF3 (m/e 176)
CD2=iHCOCF3 (m/e li8)
C2D5CD(CD3)CD=zHCOCf+3 x+/e 1%)
i-C3D7CD2CD=hCOCF~~~(m/e 192)

D     D
K2 hi -COG3 (m/e 173)
DD ; D

C6D5CD=CDC001q+ (m/0.155)
                      ,
BuOOCCD2CD=hCOCF3 (m/e 243)
HOOCCD2CD2CD=$HCOCFj, (m/e 203)
CD2-CDCD2CD2CD&O~~3 (m/e 188)


LEGENDS TO FIGURES

Fig. 1. Standard curve for the quantitation of Glutamic acid.

Fig. 2: Typical ion chromatogram of soil amino acids.
Ftg.3. fv\',sr Fc=p e-2          Q+ub& -i--*u-   O- f



                                .


Table II.  ANALYSIS OF AMINO ACIDS IN SOIL (pg/g SOIL)

Mass Fragmentography

Amino Acid

Ala

Val

GUY

Ileu


LeU


Pro


Phe

Amino Acid Analysis

    206.5

    148.3

    215.4

     95.4

    154.2

    143.4


     80.3


    218.3

    227.0

    129.7

Bl


198.7


151.0

ASP

Glu

LYS

S96.8

100.4

152.1

141.4


80.5


217.1

217.2

115.3

#2

202.7


150.5


201.6

100.2

149.7


142.8


80.7


219.8

215.6

113.5

83


198.3

149.9


201.3


92.3


154.2

141.2

80.0


219.9

214.1

114.9

P-Lb


Fig. 1.

GLU
- 4dded
D5^GLU -


                             3
                     J

3           E         i, I


IIC. 3


A Lomputer Uperated Mass Spectrometer System

W. E. Reynolds, V. A. Bacon, J. C. Bridges, T. C. Cohurn, Berthold Halpern,
Joshua Lederbcrg, E. C. Levinthal, Ernest Steed, and R. B. Tucker
Department o$ Genetics, Stan$ord University School of Medicine, Stanford, Calif. 94305

An integer resolution mass spectrometer-computer
system has been developed in which the computer
controls the "scan" of a mass spectrometer. In this
system, the computer queries the user for operating
parameters which are then translated into control
functions which operate the mass analyzer. The
spectral information acquired from the mass spec-
trometer is made available to the chemist within min-
utes in an on-line graphic system.
processing of GLC effluent are given. Examples of the

THE USE OF htAss SPECTROMETRY has been hampered by the
lagging development of a fast and convenient method of re-
ducing the spectral output of the mass spectrometer (MS)
to numerical data.  Usually the operator must convert a MS
chart recording, which is an analog plot of intensity cs. time,
to a digitized plot of intensity cs. mass number.  Because of
instrument instabilities, wide range of signal amplitudes,
large amounts of data, and other operational ditficulties,
(I; 2), it is often dificult and time-consuming to establish all
the correct mass peak identifications. One aid is to use a
reference compound (3) tither prior to the run or as an in-
ternal standard with the unknown sample.  By counting from
known mass peaks, unknown spectral peaks can bc idcntificd.
However, the processing of data by this tczhnique is still a
formidable task and it may take several days to accumulate
all the information from a gas chromatograph-mass spec-
trometer (GLC-hIS) run.

Several workers have demonstrated MS-computer systems
in which the computer monitors and records digital data
from a MS. In most of these applications the mass spcc-
trometer has operated indepcndcntly oi the computer, scannmg
in some time dcpcndcnt mode. mcnsuring ion intensities at
all points within the range of (500 to 5000 samples per second)
and afterward performs the computations required to reduce
the large amounts of digital data to useful information
(4-7). Much instrument time and sampling effort is ex-
pended in the intervals between integer peak positions where
there is little or no information.  One system that improved

(1) K. Biemann, "Mass Spectrometry." McGraw-Hill, New York,
1962, p 10.

(2) J. Lederberg, E. Levinthal, and Staff. "Cytochemical Studies
of Planetary Microorganisms Explorarlons in Exobiology,"
IRL Report No. 105-1, Instrumenration Research Laboratory,
Department of Genetics, Stanford University School of Medi-
tine, April 1966 to October 1966. NASA Accession No. N66-
34195. -
(3) J. H. Beynon. "Mass Spcctrometry and Its Applications to
Organic Chemistry," Elsevicr Publishers. Amsterdam, 1960,
p 44.

(4) R. A. Hitcs an K. Biemann. ANAL. CHEhq., 39,965 (1967).
(5) ILGd., 40, 1217 (196s).
(6) R. A. Hitcs, S. Markey, R. C. Murphy, and K. Biemnnn, 16th
Ann. CO/I./. ,lfrw Spcc/rarwrr.v Aliid To,Cs, ASTM E-I J,
Pittsburgh, Pa., hla>, 196s.
(7) R. B. Tucker, "A hlass Spcctromater Data Acquisition and
Analysis System."  IRL Report No. IOG?, Instrumcntntion
Research Laboratory. Depnrtmcnt of Gcncticb. St:tniorcl Um-
versity School of Medicine, NASA Acc:ss~on No. NOS-25743,
1968.

upon this latter ineficiency used step switches to step the
scan from position to position (8).

We now describe a MS-computer system, suitable for
routine laboratory use, in which the computer controls the
operation of a quadrupole mass spectrometer (9, 10). In
this system the "scan" is calibrated by relating known mass
positions of a reference compound to a computer g~ncxtcd
control voltage (V,).  R, is gencratcd as the result of a number
N, serit from the computer to a Digital-to-Analog (D-to-A)
converter in a MS-computer interface. The parameters of
this V,, or the N for each integer mass position, arc de-
termined by a computer program and stored in memory.
The subsequent use of this information allows the computcr-
directed MS output to be recorded directly as mass,chnrgz
(nz/r) cs. intensity.  On request, this data is then made al,ail-
able to the operator in an on-line system.
The use of this computer-.MS interaction, combined with
the decision-making ability of the opcra+?r, permits n sig-
nificant saving in data processing costs.  Furthermore, a
much larger duty cycle of analyzer "on peak" time is obtain-
able, resulting in the detection of more ions for a given rnxs
position than is possible in conventional time based scanning.
The new MS-computer system has at least thrcs unique
features. There is a hardware control intcrfncc to connt':t
the MS intimately with the computer; there is an improv:d
etlicicncy of information acquisition from spectral psahs that
are limited in ion production rates; and there is a uscr-
oriented control and data presentation system that csnc.xls
the foregoing details from the operator, but prssen:s 1i7c gxr
with prompt and concise data which include normalirsd mass
spectral plots.

The described system has evolved through three mass
spectrometers, three computers, and two basic computer pro-
grams (I I, 12).  The later systems have greater range. scnsi-
tivity, and convenience, but they all have a common concept.
Therefore the description that follows will be concrprunl
rather than specific to any one configuration.

(8) H. L. Friedman, H. W. Goldstein. and G. A. Griffith. "\lnjs
Spectrometric Thermal Annllsis of Polymrr DCCOnlpOhiIiJn
Products," 15th d~vr. CU/I~: .\fcrss S~cctronre!r.v rlllrc*ti 7'0,~jcs.
ASTM E-Id, Denver, Colo., hfay 1967.
(9) W. E. Reynolds, "A Small Computer Approach to Lou
Resolution hlnss Spectrometry." Pacitic Conference on Chc:l:ls:r>
and Spectroscopy, Anaheim. Cnlif.. November 1967.
(IO) W. E. Reynolds. T. B. Coburn. J. Bridges. and R. Tuc`ker.
"A Computer Operated ,Ilass Spectrometer S~stcm." IRL
Report No. 1062. lnstrumcnration Research Laboratorv [)c-
partment of Genetics, Stanford Univcwty School of \lc::c!zc.
NASA Accession No. N68-l ! S69. Nov. 1967.
(11) W. E. Reynolds. R. B. Tucker, R. A. Stillman. and J. C.
Bridges, "hlass Spcctromcrers In a Time Shxcd C:xn:~u!~r
Environment,"  I7 t h rlrur. Co,rjI iIl:,ss Sprcrror,~c*tr~ .~li!;,x
Topics, ASThl E-14. 1969.

(I?) J. Lectcrberg. E. Lcventh:ll. and StatT. "C! tochmxnl Stt~d~c; ot
Planetary hlicroorgcanisms Esplorshons in t\;olxoioc~ ." II;L
Report No. 1076. Instrumcntntion Rcscarch Labornror). I):-
partmcnt of Gcnctizs. Stnnf'urd C'mvcrsity School of X1:, I;,!::.
October 1967 to r\prll IYM.  Appcndiu A IS a ~LI!~~I~IICLI IC;':.IIL
of the abovc "hlass Spcctrometcrs in 2 Tuw Shurcd t. II\ iron-
menl," NASA Accession No. N6S-ZYS-IG.

   Reprinted from ANALYTICAL CHEMISTRY, Vol. 42. Papc 1112, Scptentbcr 1970
Copyright 1970 by the Amcricm Chemical Society and rcprintcd by permission of the copyright owner

P-7

1 ,


The present system is operating with a Finnigan 1015
quadrupole MS and a Varian Aerograph GOOD chromato-
graph.  The same computer programs and a similar interface
were also operated successfully with a Bendix Time-of-Flight
(T-o-F) MS (13) and an EAI qundrupole (14) MS. In all
cases the GLC-MS, the teletypew/riter, and the digital plotter
were situated in a wet chemical laboratory.

A schematic diagram of the GLC-MS combination is
shown in Figure 1. The emuent from the gas chromato-
graph, equipped with a flame ionization detector, first passes
through a variable splitter that diverts between Ii3 and *I?
of the flow through a Biemnnn separator (15) and into the
MS.  A solenoid-actuated valve in this line helps to keep the
large initial solvent peak from entering the MS system. A
reference gas reservoir containing a fluorine compound at a
vapor pressure of approximately 3 X IO--' Torr is also in-
corporated in the system and is connected to the MS by
another solenoid valve. The computer, tin the interfacing
electronics, has direct control of gas valves, and can valve in
or shut off the reference gas whenever it is needed for the
calibration routine.

These valves were constructed in our shops in such a way
that the back side is open to the vacuum system when the
valve is closed.  This avoids the common pressure burst when
conventional valves are opened to a vacuum.

The right side of Figure 1 illustrates the major components
and functions of the interface.  This computer-MS interface
was built in our Instrumentation Research Laboratory (16)
and contains all the electronics not normally supplied with a
standard configuration IMS or computer.  Al1 of thcl operating
parameters of the hlS are, or may be. controlled by a digital
word (binary number) sent from the computer.  The principal
control is ricl fhe "N" register to the D-to-A converter.  The
analog signal, V,, from the D-to-A sets and holds the mass
analyzer to pass ions of a predetermined ))I e.  Alternately
the digital output may be coded to operate auxiliary control
functions, such as actuate valves. set amplifier gains, the low
speed multiplexer, or enable the digital plotter.

STlEV'3 ONll33NN03 UOHS
      1

The characteristic method of controlling the nl:e passband
and taking measurements while the mass analyzer dwells
upon a m/e value converts what is normally measured as a
time dependent parameter, to a stationary signal. This
statistically stationary property of the signal enables the em-
ployment of full integration to enhance signal to noise.  Both
the electrometer and the integrator are standard commercial
FET operational amplifiers of the 550.00 class. The time
allowed for integration and the operation of the integrator
reset are controlled by signals (numbers) from the computer
to the "T" register.  The output of the integrator is sampled,
held, and read ciu the Analog-to-Digital (A-to-D) converter.
Auxiliary signal sensing is provided by the low speed
multiplexer. This is useful to determine the automatic
settings for self calibration,or may be used to record tempcra-
ture, pressure, etc. These sense functions, plus some valve

(13) D. B. Harrington and R. S. Gohlkc. "High Resolution Time of
Flight Mass Spectrometers." 10th .Irw. Coqj: ,Iltrss Sp~wrum-
erry A//id Topics, ASTM E-14. New Orlcnns. La.. 1962.
(14) W. I'& H. I'. Keinhard, and U. van Znhn, Z. Plrys., 152,
143 (1958).

Figure 1. The GLC-AIS instrumentation and the electronics
interface to enable computer systems integration

control and checkout functions, are controlled by the "C'
register.

(IS) J. T. Watson and K. Bicmnnn. ANAL. CIIEM., 37, WI (1965).
(16) W. E. Kqnolds, J. C. Hric!gcs. R. B. Tucker, and T. U. Co-
burn, "Computer Control of Xfass AnalyLcrs," 16th .&III. Co\!/:
Mass Sprclrowe~ry d//M Topics. ASTXl E-14, Pittsburgh. Pa..
1968.

      Thcrr are no manual opcrntor control functions in :lny o'
      the above steps.  The control is nccompli>hcd iIt tl~ I<!:[> 11
     writer keyboard. This keeps the system llcsiblc ant! IILI,L,.
     it indcpendcnt of the idiosyncrasies of indi\,idual con~put~ I
                                   P-1'
ANALYTICAL CHEMISTRY, VOL. 42, NO. 11, SEPTEMBER 1970 . 1123


    COMPUTER DIALOGUE
  (The rcser's respome is underlined)
OPTION = collect
EXPERIMENT; = test
               -

T=g

MODE = single

PLOT, TYPE, or FILE = plot
                   -

BY MASS, AMP. or ENTIRE = entire

FROM MASS = 1
g,;g n

PLOT, TYPE, or FILE = 1
T=I
EXPExIMENT,+' = MS26x15
              --~
CONTINUATION = yes
T=6            -
MODE = continuous

# OF SPECTRA = 130
FILED IN POSITION 930 to 1070
T=/
EXPE`i%IMENT; = /
OPTION = sum -
EXPn = MS26x15
      _~---
FROM SPECTRU,M = 9-U)
TO = 1020          -
FROMXSS = ilo

TO MASS = 500
PLOT = yes -

EXP,+ =, 1
OPTION = plot
EXPERIMEtii+ = XlS26x15
              _- -
FRO&M SPECTRU.M = 957
TO = 957           -

&OM%iASS = 1
TO MASS = 340 -
          -

interrupt lines and/or individual computer characteristics.
This straightforward system definition makes the software
design much like conventional computer programming rather
than encouraging intricate techniques highly dependent upon
the specific hardware.
Thus the system is not oriented specifically to any given
computer.  It has operated on an early model LINC (17) com-
puter with 2K words of 12 bits. memory, and on a time-shared,
locally programmed, IBM 360;50, butTered with an IBhl 1800
(18). In all cases the computer was somewhat remote,
separated by some 500 ft of cable from the rest of the in-
strumentation.  The system is very economical of computer
resources.  Most of todak's small general purpose computers
would be able to operate the described functions if it were
desired to avoid time-shared computer dependency. Some




(17) R. W. Stacy and B. Waxman. "Computers in Biomedical Re-
search." Vol. II. Academic Press. New York. N. Y.. 1965. DD
35-66..                                     . .

(18) W. J. Sanders, G. Breitbard. G. Wledcrhold. er (I/.. "An Ad-
vanced Computer for hlcdicnl Research." Full Joiut Cortrpwr
Colt/. Proc., ACM, Anaheim, Calif., 1967, p 497.

COhlMEm

The user reauests the data collection chase.
A catch-all name, "test". is given; the spectrum
will be used simply for a systems check.

An integration time of 35 milliseconds per
peak is requested.

Only one spectrum will be taken (single mode).
The data is acquired after this answer.
The user can plot, type. or file the data col-
lected; here a plot is requested.
The user can plot selected masses, the
highest intensities, or the entire
spectrum within requested limits.
The user indicates the limits.

A QUICK plot omits annotation, etc.
Note that "y" and "n" mean "yes" and "no".
The user completed the checkout and now
wishes to proceed with the experiment.
The "/" is used to backup through the
conversation.

An existing experiment name is given here.
The user confirms that the spectra are
to be added to the eslsting experiment file.

The user requests that spectra be collected
continuously until 130 are taken.

Data collection is complete.

The user then wishes to sum the elements of
each spectrum to produce a "total ion"
plot, analogous to a GLC trace.

Mass position 40 to 500 are summed for each
of the collected spectra.

The "total ion" curve is now plotted.
(Figures 6 and 7 are illustrative of this
&tiple dialogue.)

Guided by the total ion plot. the user
will plot interesting m3ss spectra.
Only the spectrum filed in position 957
is chosen.

The plot (Figure 17) is drawn and normalized
to the base peak, nlje = 31.

Figure 2.  An example of the user-computer dialogue during operation

sort of magnetic storage for object code programs and data
storage is most desirable. DEC-t)-pe tapes ha;-c be:n uzd
on the LINC system and disc packs on the IB\l s\-stem.



        THE SOFIV.IRE STRUCTURE

The objectives of the software are to operate and conrrol
the MS, acquire data from the MS, process and prcsznr rhis
data in a manner useful to the chemist, and provide ctrtn:n
control and information to aid in maintaining and ser\-l:ing
the instrument.
With the program loaded into the computer, the Csfr
requests any one of several functions (see Table I) by t!Fing
the name of that function. The computer responds \x::h a
series of prompts (see Figure 2) to elicit user m;lcrocomm:!ntis.
The computer then generates the detailed conrrol funz!ons
to perform the assigned task.  At the comp:<tion of the :.:,i.
requests are made for new parameters.  By striking the si.tlh
("/"), the user can "backup" through anj' conversntion to
correct errors or to go to a JilTercnt function.  This con\.::>.!-
tion technique makes the system both tlesiblrt an;l r<ason.l>l)
self-instructing.

1124  .  ANALYTICAL CHEMISTRY, VOL. 42, NO. 11, SEPTEMBER 1970                        P- 72


      Table I.  A List of Program Options
(1) CALIBRATE: Creates an accurate N Table. The .V "urn-
hers which correspond to the peaks in the reference gas are
used as the end points of a piecewise linear interpolation pro-
cedure for calculating a complete .V Table.
(2) COLLECT: Is the primary data collection step. It is here
that the 750 N Table values are sent to the MS and the 750
m/e intensities rccordcd. This operation can be repeated at
five-second intervals as the data are tiled on disk under an e.y-
periment name.
(3) TYPE: Allows the user to print out spectral data by indicating
what spectra in a given file are to be reviewed.  The user can
request that the amplitudes of particular rtr/e positions be
typed ; that a given number of the highest amplitudes be
typed, or that a consecutive number ot' them over a given range
be typed.
(4) PLOT:  Enables the user to have bar graphs produced by the
computer controlled digitai plotter. The amplitudes to be
plotted can be selected with the same flexibility as described in
  TYPE.
(5) SUM:  Produces a plot of the total ion current over a series
of gathered spectra.  All responses of a spectrum are summed
to produce one datum point on the plot. This plot corre-
spondsclosely with the GLC output when runrung with theGLC.
(6) TRACE: Produces a record of a spectrum similar to the
normal chart recording output. The analyzer is sampled at
all N values (about 10 per amu) over a given range and the
result is plotted as a "broken line." (Used for system check
out)

(7) MONITOR: Provides for inspecting the peak profiles by
sampling the ,$>ctrum around a given or position.  The
gathered dara are then typed out.  (Normally used for system
service or service log)
(8) DISPLAY:  Enables the user to display a given mass position
(or N numbcrj in the center of the console oscilloscope.  (Used
in the adjustment of the mass spectrometer)
(9) GAS:  Allows the user to remot+ turn the reference gas on or
  off.  This is helpful when operating the system from a remote
position.

n 31      69
:! 1'"     1212
         .0600

2060
.7600

&
2507
.260I

.il.
   169     :
   3327 1
D    1.300 #

PRES  Jr lo'7/orr     T FACTOR    40

rlICA '  40,&U.      123351    L/he
           I

IONV   20 r&,;r's      691218    dafe

          Figure 3.  A monitor plot indication of instrument scr\iccability

The example of a user-computer conversation gi*:cn in
Figure 2 represents the day-to-day conlput-r-r~sc:!r~her
dialogue given to direct the system's olxrarion. D:?per
level programming may be done at the terminal to rcdctine
these functions or to add new modes. AddiIionnl b!`;tcm
development may be done by the chemist-user, or his program-
mer in a manner typical of general purpcse ccrnptit<r soft-
ware. In normal daily practice, the user first rcqucsts the
calibrate function and then proceeds to data acquisition,
analysis, and presentation. Usually the calibrntion is done
once every four hours.

It is this calibration subsection of the prcgrnm rhnt nssigns
to each integer mass position a value Nwhich when wnt to the
D-to-A converter in the interface, will set the mass analyzer
to pass that particular species oinz.`e.  During this caiibration
phase a reference compound (perfluorotributylnmine. i-C-13),
is introduced into the ,&IS. The calibrarion proxdure in
addition to determining the N values, makes dltn ?.~~ailnble
that will aid the operator in making qua1itstii.e judgments
about the stability, sensitivity, and resolution oi thz \fS.
-41~0 a service or maintenance reccrd plot is n!x~l~ble (xc
Figure 3), that, at least indirectly, shows these and other im-
portant instrument conditions.  Figure 3 is acttxiiy 12 trxsd
segments of a complete spectra, each segnent co\ erin_r a szan
of about 4 amu and each taken at a diffsrcnt int<gratlon time
(gain).  The m/e value, its position. and a parnmetrrr indicat-
ing the gain is automatically printed be!ow each peak.  The
date and time is printed by the computer, but at prssent thz
operator must insert the sample pressure and icnimion pa-
rameters.  The file of these plots represents an exc?!lent rec-
ord of the instrument's serviceability,  The czlibrntion is
automatic and its use less complicated than ths dsscrlption.
It takes about 5 minutes, after which the refcrcnce gss is
pumped out of the system.  In the IBM 360-1600 system, the
time is used to compile the main program.

L+x
    264
. .
&I    5381  :;
    .se00 2.

I

I

P-

7;

ANALYTICAL CHEMISTRY, VOL. 42, NO. 11, SEPTEMBER 1970 . 1125


Total spectral data acquisition time depends upon the
number of m/e positions measured and the integration time
allowed per position.  It may be calculated:
    Spectrum acquisition time = P X (T, + 2")    (1)
where P is the number of m/e positions to be measured (they do
llot have to becontiguous or sequentiai), TI is a transition time
(2 msec in our system), and T is the integration time per peak
(nominally 6 to 17 msec, but we have usefully used 1 to 1000
msec).

Normally data are collected at each integer m/e position I
through 750.  The 750 N values arc sent through the D-to-A
converter to the MS and the 750 responses (a full spectrum)
are recorded by the computer cia the A-to-D converter.  This
process can be repeated approximately every 5 to 10 seconds
for an arbitrary number of times.  The spectra thus gathered
are stored by the computer on magnetic disks or tapes.  Pro-
gram changes may be made lo measure any subset of the 750
nr/e positions and thus achieve faster repetitive spectra.  Con-
versely more measurements may be made at any specific
peak position, a technique which may be used for accurate
isotopic ratio measurements.

Since many spectra are taken and stored during a GLC run
or a solid probe experiment, the user requires fast methods to
evaluate the data. The more useful data abstracting pro-
grams we use are:
`THE MATRIX SEALH. The user specifies which group of
spectra, what range of mass values in each spectra, and how
many large peaks he wants abstracted from each spectrum.
An abstract of these highest peaks is then typed out and in
many cases this abstract contains useful chemical information
or at least indicates the spectrum of interest.
THE TI~IE PRUEXATION (PLOT).  This is a computer drawn
plot of certain peak intensities or a sum of all peak intensities
ofeach spectrum (total ion current) plotted against time.  The
latter gives a good reproduction of the GLC curve and also
indexes the spectra of interest (19).

NOR~~ALIZED SPECTRUXI PLOTS. Conventional bar graphs
of mass US. time, normalized and annotated, are routinely
available.

AU data outputs are in the laboratory and are available
immediately after data acquisition.  All spectra are filed and
may be recalled at a later time or date and reprocessed in any
way desired.

Involved programs of these magnitudes are specifically
dependent upon the language of a given computer. The
logic may be easily transferred. but in general the specific pro-
gram may not.  We have about 4 man-years of programming
invested in this system.

THEORY OF OPERATION

During spectrum data acquisition, the computer directs the
mass analyzer to a program-selected mass position and reads
the output intensity of the MS. The mass analyzer is not
swept in a conventional sense.  As indicated in Figure 1, it is
controlled by a voltage (V,) such that

           m/e = WC)           (2)

where m/e is the mass/charge ratio and f(V,) is a monotonic
function characterized by the MS.  For every Mi, (M = ~!e),
to be passed by the mass analyzer, the computer has (according
to the prior run calibration program) a digital number N,

(19) R. A. Hires and K. Biemann, "Advances in Mnss Spectrometry
IV," Elsevier Publishers, Amsterdam, 1965, p 37.

which is transformed by the D-to-A converter to the voltage
VW

The determination of these values, Nr. is accomplished b)
the calibration program.  The value of IV for 12 keq ptxks of
the reference compound are known to approximately 1 amu
from prior calibrations.  The actual ,V value for the ccntrold
of each of these peaks is then determined by detailed examinn-
tion of the rll/e continuum in each of these areas.  Sutficlcnt
detail is obtained by designing the D-to-A resolution to be
10 or more values per peak width.
After determining these exact 1 2 Iv VaIues. linear interpola-
tion. superimposed uF:on the analyticai function, 111'e = jiVCj,
is used to expand the list of 12 experimentally determined
values to a full tabie of 750 entries. (The analytical func-
tion of tnie to control voltage is linear for the quad-
rupole and parabolic, m/e = k(Vcz), in the case of the T-o-F
MS.)

Thus the procedure to measure the intensity at any .Cli is as
fol1ows:  a. The number NI which corresponds to the se-
lected rll/e ratio (1Mi) is loaded from the computer into the D-
to-A converter. This sets the control voltage, 5/,, to the
mass analyzer. The output of the mass spectrometer is
proportional to the quantity of ions, IV,, passed from the
sample.

6. An analog circuit, reset and released by the computer,
integrates the output of the MS.

c. Several milliseconds after the integrator is released. (the
choice of integration time was initially supplied by the user
upon program request), the computer samples the output of
the integrator by means of an A-to-D converter.  This digital
value is stored as the intensity of Mi.

Steps (a) through (c) are repeated to acquire a complete
spectrum.

The fundamental restraint upon this system is the drift of
the function 1~ = j(V,) following calibration.  Our experience
with the Finnigan 1015 afld a Bendix T-o-F instrument and
our interface, is that this drift causes an error in ;V of less than
1,`) the value from one Nentry to the next in a l-hour period.
This is sufficiently small to allow an unambiguous mass idrnti-
fication.

Table II contains comparisons of signal-to-noise ratios and
the following defined figures of merit.  The comparisons are
made between the described control system. a linear scan m
time, a parabolic scan in time such as the T-o-F, and the ex-
ponential time scan characteristic of magnetic instruments.

Uniform conditions are used to give realistic values for
comparison; it is assumed that in each case the peak jha?es are
uniform if scanned in time, and that they are gaussian. and
that the resolution is commensurate with the 10% va!lcy (5 ",
points on a single peak side) definition (3).  In order to give
typical comparison figures. it is further assumed that a spt'c-
trum will be taken from mass 50 to 500 in 4500 milliseconds.

The first column in Table II is the time the mass snnlqzer
is on or about the mass position.  In the case of the computer
control system, the 4500 milliseconds is divided equally into
450 periods of 10 milliseconds each. Two milliseconds nre
allowed for each transition, and the mass analyzer will dwsll
on the peak position for 8 milliseconds.  In the CCISC` of a con-
ventional linear scan, the analyzer will enter a peak nren and
leave it 10 milliseconds I;lter.  By the 10% vnllc!~ con\`c`niLzn.
this means the time from the beginning 5 x level to the end 5 "g
level of a single peak.  However for the parabolic C:ISC (the
T-o-F) it bill be found th;u the resolution of the mstriimcnt
will have to be set for the uork case, peaks 499 and YlO.  It
will be found that there is 6.6 milliseconds between thcbc pc'ak

P- 74'

1126  o ANALYTICAL CHEMISTRY. VOL. 42, NO. 11, SEPTEMBER 1970                                     . ,


Type of scan
Control and
integrate
Linear scan
m = kf
Parabolic scan
m = kta
Exponential scan
~ m=m.ekl

Table II. A Comparison of Attributes Affecting Signal-to-Soise Efficiencies
Typical Operation Condition:  Scan from nr/e 50 to 500 in 4500 Xlilliseconds
  Time on,
  or between                  Ions detected:
  5 % points,      Time constant    (Peak intensities     Effective noise
of a peek, msec   of amplifiers, msec   of n ions/m@    bandwidth, Hz

Figure of merit:
1000,`~ X de:ccte;l
ions,`bandv.tdth

8          VA         8.011            40          200

10            2            5.1 n           80           64

6.6           1.3          3.4 n          120           28

3.9          0.8           2.0 n          200            10

centers, and the theoretical 5% heights must be at the mid-
point. Since all peaks are similar, all peaks of the scan will be
just 6.6 milliseconds from 5% point to 5% point. During
34% of the scan time, the analyzer is not in the area of any
peak position at all; but is mostly between peaks in the low
mass range. The case of the exponential scans is similar.
In this case it will be found that the peaks are 3.9 milliseconds
wide and 61% of the time no information can get through the
instrument.

The next column indicates the time constant (7) of the ampli-
fier channel appropriate to the scan pammctcrs.  The control
`system uses a full nitegrator, so the entry is not applicable.  In
the conventional scanning system, the time constant is usually
chosen as large as skewing permits to integrate signal and
discriminate against noise. The relationship between 7 and
the 3-db bandwidth (jhB) of an amplilier is simply r =
I/(2&). If 7 is chosen to be large, peak skewing and
broadening as illustrated in Figure 3 will occur.  If T is chosen
small, the bandwidth with its attendant noise is excessive and
there is little integration of the signal.

This is the dilemma always faced by the user of linear ampli-
fier circuits:  the desire to limit amplifier bandpass to smooth
the signal, as opposed to the need for a wide bandpass to pass

the signal without distortion. Since the purpose here is to
compare our described amplifier and integrator system with
conventional linear amplifiers, a T of 0.2 is assumed for the
conventional case.  This T is still large enough to cause deg-
radation of resolution in the conventional output signal (25
to 35% depending upon the definition used). It is felt that
this choice represents a fairly typical operational psrametsr.
The assumption of a rigorous lower value would rcsalt in an
unnecessary, and perhaps unrealistic, comparison ndiantngz
for the described control and inteprate signal skstcm.

The column "Effective Noise Bandwidth" is the !I:. for the
time dependent scans. However an equivalent 3-cib band-
width is not as well defined for the integrator.  It can be
shown that for an integration interval. T, (8 miliiseconds in
this example) an .& may be determined such that a linear
amplifier of bandwidth I;, would pass the same amount of
"white" noise as the integrator.  The actual bandpxs oi an
integrator is a sin(.y)`x type function.
The white noise power passed by either qstem may be es-
pressed as an integration of the white noise model, r'*`, (29)

(20) W. B. Davenport, Jr., and W. L. Root, "Random Signals and
Noise," McGraw-Hill, New York, 1958, p 88.

OUTPUT OF A UNITY     r-v-l

AMPLIFIER

TIME CONSTANTS

T

T

i  \?t 2=0.15 XT

T milliseconds

V'
In

R&=2

          1.35 T

Figure 4. Peak broadening and skewing effect of narrow bandr$idth amplifiers         p- 7<
                                                                          

                ANALYTICAL CHEMISTRY, VOL. 42, NO. 11, SEPTEMBER 1970  o  1127


J

oLc CM#tT RECDRO OF TFA - OK'EPTIOE METHYL ESTERS

IM k c.37 "Ill.

A WPUTER PLOT OF THE SUMMED PEAKS 00N.9 DF EACM SPECTRW

Figure 5.  The above trace is a G1.C flame ionization chart of
an cxpcrimcnt.  IlcIow is a total ion plot, crcatcd, and plotted
by lhc compufcr, that indcres the indi&duai spectra

OPTIOI!  -?TVPE

FROM t!ASS
70 -?750  -`II!
WI'RER~?~
SPECTPUM  955
MASS 31    b2    13
~MPL las    22    22
SPECTRUfl  956
MASS 31    45    56
A#PL 117    25    25

iPECTRi?I 957.

wss 11  ::  ::    69
AY,PL 124             57
SPECTRUI.1  959

MASS 31  ::  ::    69   `70
ACPl 11C             36    31
SPECTRUtl  9 59
PASS 31    41    56    69    70
AWPL 116    26    24    26    27
SPECTPW  960
PASS 31  ::  :E    46
AHPL 11s             23  i':

69   139   166   167
50    25    37    27
::  139 13  166 64  lb7 44
::  't:  166 102  167 76

72   139   166   167
Jb    12    60    b9

96   139  'ii   167
25    29         19

70       166   167
24  I: J6 10

Figure 6. The priucipal peak listing from the matrix search

times the circuit transfer function over the frequency range in
w. The first shown below (on the lelt side of the equation)
represents the white noise retained by a full integrator during
a time interval T. The second hprd'ses the white noise
passed by a simple amplifier of bandpass,/;, = I/(2 *r,J.
S,`~~r~u~d,~7do-~-/r'I +;Ur,Ql*d,,J (3)

A solution for rca may be made by numerical methods:

r., -
* 0.496 T

Using this value, it is found in the example that /., = 40
HZ.

A 1x4 analytical benct'it of the system is the rflicicncy of
detccting ions.  In any hlS with elcclronic ion dctcction, an
ion is dot&cd or not dctccted and there is no "5 % dctcction."

There is a 5 % probability an ion generated in the SOUTCC ~11
pass the analyzer and hit the detector. Or more accurn\&, i!
when a peak is "ccntcrcd," n ions rcgistcr upon the tlctcclor
each millisecond, it may be expected that at the npprol:ri::tc
low side of the peak, $20 ions will register each milli<ccond.
The control method is always "ccntcrcd" upon the peak, htlnce
the total expected ions will be T X II ions. Housvcr, in
scanning a gaussian peak, it can be shown that for an cquiv-
alent time, from the first 5 % point through the maximum and
out to the last 5 % point, the total cxpcctcd ions will by' :lprrox-
irnately 0.51 X T X II, where now Tis the lime from 5 "; point
to 5xpoinl.  Thcsc numbers of ions expected to register upon
the detector are tabulated as "Ions Detected" in Table II.
Many small, but important, peaks will have an n of 1 lo 10
ions per millisecond under ihe operating conditions imposed
by some modern research requirements.

A figure of merit for comparing the systems may be dclincd:

           1000
Figweof Merit = - X  Detected Ions
            Bandwidth (Hz)    (3
               n

This is the final tabulation in Table II.  Our use of the syhtcm
has verified these results, that introducing computer control 10
the quadrupolc or T-o-F MS can enhance the useful scnsilivir:J
by a factor of 3 to 8.  The grcatcr convenience olfcrcd to the
user is not at the cxpcnsc of performance, but oks these
intrinsic advantages.

One other practical benefit is possible by reviewing the
attributes of resolution. The idealized gaussian pt:~k con-
sidered here, which has a width commensurate with the IO z
valley resolulion criteria, has a contribution of only O.OG:;
at the neighboring integer mass posirion.  Since in the control
method, only Ihc cross contribution at the integer positions is
harmful for some cxpetiments, the resolution msy bc: dc-
graded until a O.S% or 1% cross contribution is obiavrd.
If the physics of thr: particular instrument arl exploited. II \<.!!I
be found that this will greatly incrensc the portion of gcncr-
ated ions that will be passed by the analyzer.

Some consideration should be given here to the soflivvnrc and
ils interaction with the user and the instrument.  A mode of
conversation has been programmed into the system ro prompt
the user for necessary parameters, and then expand upon
these parameters to conduct the necessary detailed opcrafion
of the instrument.  In retrospect we find that we hn\,e very
closely followed the concept of R. J. Spinard (?I).  X skiizl
problem occurs with the presentation of information. Lvhich is
often too voluminous for complete display. To o\`r`r:ome
this, the bulk of the information is held in the cornpurer. 2nd
is made available by similar conversational techniques.

It should be realized that the present software is in ;in cm-
bryonic state of drvclopmcnt compared to its eventual potcn-
tial. The system of computer, interface, and klS may be
defined in terms of computer syntax to a systems devclop<r in a
manner comparable to the definition of a specific data pro<css-
ing task to a system programmrr.  There is an enormous po-
tential in the implr`mcntcd synt`ax of this instrumcnt:lGon
system for the chemist who may have special requirements 2nd
who understands fuljy the principles of the augmented instru-
ment. The user can then program, or have progrnrnmcd,
efflcicnt solutions KO m`any of his instrumentation problem:.

The fenturcs that do allow the foregoing bcnclits. do rLL*nl-
selves impose certain limitations. The control sys[cm docj
not normally n`turn any information about doubly ioniwi odd

--
(21) R. J. Spinard, Sricvtcc*, 15s. 3797, 55 (1967).

1128  o ANALYTICAL CHEMISTRY, VOL. 42, NO. 11, SEPTEMBER 1970

--


P- 7c


CG          I
                3
  ct&- CHZ
I I
Y / CH-C==O+

`I'
c-o
I          h-ocn,1+ hit
                       593       324

20     40     60     80     100    120    140    160   lE0  200    220    240    260    280    300    320    343

`IFA DIPEPTIDE flfTHYL ESTERS
nS26 15
fILE 957

Figure 7.  The on-line, computer plotted spectrum

mass values (peaks at 0.5 integer position), wide metastable
peaks, or the value of mass defects.  It also might be expected
that mass defect deviations, cspocinlly at high m;e values,
would cause difirulty.  In practice there have not been any
problems attributed to this latter case, the high I~:E defect.
The resolution and other operational factors have masked this
inherent difficulty.  If opcrntional USC should be hampcrcd by
this problem, we have proposed techniques of concurrent
calibration on the unknown peaks themselves.

For example, the major peaks in the unknown compound,
identified to their nominal intcgcr value (Icss mass defects),
would be used to calibrate the N table.  After a programmed
computer decision to explore a peak area, similar to those
illustrated in Figure 3, the computer would direct the "scan" to
that area.  Data acquisition time would be about 100 msec for
10 data points over the peak profile.  Ccntroid identification
and recalibration might be nnothcr 100 msec.  If this were
repeated for 8 or 10 key peaks, total recalibration would be
in the order of 2 sec.  This technique could also be emp!oyed
to verify the linearity above the last reference peak (614 with
our system) or below the first.  (We have paid little attention
to m/e below 12 or 18.)

As a final minimal operating mode, the scan could be pro-
grammed to simulate any present mode of time-based scan and
data collection, with comparable attributes of performance.
As an example, doubly ionized masses or metastable peaks
might be identified and measured. Of course the MS used
must have sufficient resolution and/or sensitivity, also the
total spectrum acquisition time would be increased. Such
changes of operational mode would be by program only;
hence they could be established or removed in milliseconds
with no hardware or switch changes of any nature.

RESULTS

In a typical experiment a mixture of TFA-dipeptide methyl
esters (22) was injected into the GLC-MS system. and 130
complete mass spectra were collected and recorded by the
computer.  Eighty-ooc of the individu:tl spectra were summed
to obtain the total ion plot (Figure 5) (IY).  This may be com-
pared with th:: GLC flame ionization chart record on the top.
The mass spcctrnl output from peak "C." for example, in the
        ----.-

(27) B. 1lalpcrn and J. W. U.estlcy, Bioclrm~. Bioplrys. Rrs.
ConmIr/~l., 19, 361 (1965).

region of scans 955 to 960, was then abstracted by the TYPE
routine and the results are shown in Figure 6. This data
shows that Peak C is homogeneous and that a satisfactory
normalized plot can be obtained from scan No. 957.  Figure 7
is the plot as produced in the laborato,y.  The background
in the mass spectrum is primarily due to the GLC column
bleed.  Only the chemical notation was added by hand.
The taking of spectra, in this computer-compatibltt form.

opens the way to many types of further processing:  searching
through each stored spectrum to pick out the amplitude at a
specified mass position and plot this data to show the variation
of the ion current of a specific )?I e with time; check the horno-
gencity of each GLC peak; resolve the mass spectra of simple
mixtures into those of the individual components; and sub-
tract background signal from a mass spectrum.

The system is also well suited to the recording of spectra
from solid samples introduced directly into the ion source.
Because of the fast scanning speed and the recording of mrlny
spectra, there is no need to establish a constant vapor pressure
in the mass spectrometer and any contamination or decom-
position of the sample is readily detected.

- COSCLUSION

It is feasible to build a limited purpose computer and a
quadrupole MS as one unit, forming a compact inztrumcnt
with features similar to the system presented in this rcpcrr.
In addition, some of the software could be embodied in hard-
ware circuits. Such units would be more costly than rho~
using commcrcinl gencml purpose computers, but could bz
uniquely well adapted to space or other special physiCa\ cn-
vironments.  Alternatively, the system can be interfaced vvith
a larger time-shared computer.

RECEIVED for review March 23, 1970. Accepted June 15,
1970.  This research was aided by grants from the r\ia!lon;ll
Institutes of Health, Public Health Service, XX.1 1X37-31
and FR-0031 l-01.  The LINC computer was provided under
NH-1 grant FR-00151-01.  The v\.ork is a part of a yro:r::;;l o:
instrumentation rcseiuch oriented toward nuroni;tt~Li /l;.irl<-
tary experiments, supported by the National ;\cron:\u\i:s an,i
Space .\dministration.  rcscnrch grant NGR-05 `12 l-0114.
Principal investigator. Joshua Lsdcrbcrg; progr:un Liirc`;tL)r.

Elliott C. Lcvinthnl.

ANALYTICAL CHEMISTRY, VOL. 42, NO. 11, SEPTEMBER 1970 o 1129


PRIVILEGED COMMUNICATION                SECTION II     SUBSTITUTE THIS PACE FOR DETAILED BUDGET Pk5E

                                                     PERIOD COVERED           GRANT NUMBER
          SUBSTITUTE
IETAILED BUDGET FOR FIRST 12-MONTH PERIOD] EROkA
                                           ..-...           Ill-Ii         1
C                                         I    l/1/7

1. PERSONNEL (List e/l personnel engaged on omiect)        l-             AMOUNT REQUESTED (Omit cents)

1 ,

TITLE OF POSITION

4

TIME OR
EFFORT
X/HRS.

r4 At.4 E (Lest. first. initial)

,ederberg, J.

:retchmer, N.
Iann, H.
)uffield,A.
'ereira, W.
iummons, R.
tindfleisch, T.
Cucker, R.
leizades, N.
steed, E.
'earson, D.
Jegmann, A.
)pen - Genetics
)pen  - Genetics
(continued)

Principal Investigator or
Program Director
Prof. of Peds.
Assoc.Prof. of Peds
Research Associate
Research Associate
Post.Doc. Fellow
Research Associate
Computer Programmer
Research Engineer
Research Engineer
Elect. Tech.
Senior Res. Assist.
Grad. Res. Assist.
Grad. Res. Assist.

I     TOTAL

20


10
10
100
100
100
20
75
33
33
40
10
50
50

SCREENING FOR INBORN ERRORS
  OF METABOLISM

:









        I





        1

TOTAL

CONSULTANT COSTS (Include Fees and Travel)

-

s -

EQUIPMENT (Itemize)
             see attached list

$ 110,400*

SUPPLIES

See attached list

s 16,200

  STAFF
  TRAVEL   O.OOMESTIC 2 East Coast (lOOO), 1 Mid-west (3OO), 1 West Coast
See rn8tNctions) b FORE,GN                                  (200)

, PATIENT COSTS (Separate Inpatient md Outpatient)

S   1,500

S

S

. ALTERATIONS AND RENOVATIONS
Relocate equip., power, etc. for GC/MS Data System

s 1,800

. OTHER EXPENSES (Itemize per inrtructions)
Mini-computer maintenance            $6,000
Freight on capital equipment          $ 500
Office supplies, telephone, repro.,
postage, publication costs, etc.        $2,600

$ 9,100

         10. TRAINEE EXPENSES (See Instructions)

                   PREDOCTORAL

FOR . a S~,pENoS POSTDOCTORAL
                 OTHER (Specify)
TRAINING             DEPENDENCY ALLOWANCE

                                 I
GRANTS

        b. TUITION AND FEES
ONLY
        c. TRAINEE TRAVE'. (Describe)

Subtotal - Items 1 thru 8



No. Proposed           s

No. Proposed           S
No. Proposed           S

                   S
TOTAL STIPEND EXPENSES

*









c

$296,208

S

S

11.                               Subtotal - Trainee Exptnsas                 c

12. TOTAL DIRECT COST (Add Subtotals. Itsma 9 and II, end enter on Page 0                             0  5296,208

$ 157,208

Substltut. Budget Fog. 5-72
  . LL. PIIrr snrt . '


continued

1. PERSONNEL

Wyche, M.
Boswell, M.
Jamtgaard, R.
Allen, M.
Murray, R.
Harlow, W.
Open - Genetics

    TITLE


Lab. Tech.
Lab. Tech.
Administrator
Secretary - Genetics
Secretary - Peds.
Machinist
Lab. Glasswasher

3. EQUIPMENT

4. Column Gas Chromatograph
Mini-Computer System:
PDP-11/45 (8k memory)
Memory (16k)
Floating Point Hdw.
Prog. Clock
Bootstrap Loader
  Industry Tape
Disk System
  Lab Interface Units
  Interproc. Comm.

Amino Acid Analyzer                        32,500*
Event Counter                             2,700*
Digital Voltmeter                                    900*

4. SUPPLIES

Chemicals,glassware, and lab apparatus

GC supplies (columns, phases, etc.)               1,100
Dry ice and liquid nitrogen                          500
Data recording media (GC/MS, Calcomp, etc.)          1,800
Mini-computer supplies (start-up 6 continuing)        1,600
Mass spectrometer repair parts & supplies           2,300
Electronic parts & supplies                    1,900
Amino Acid Analyzer supplies                   2,000

20,700
5,200
5,200
  600
  600
10,500
11,700
4,600
  800

59,900

%; TIME

100
100
15
25
25
10
25

$14,400*

59,900*

Total Equipment    $110,400*

$5,000

Total Supplies     $16,200

P-79


`-a  TION II -PRIVILEGED COMMUNICATION     SCREENING FOR INBORN METABOLIC ERRORS
-

EUDi;ET ESTIMATES FOR ALL YEARS OF SUPPORT REQUESTED FROM PUBLIC HEALTH SERVICE
               DIRECT COSTS ONLY (Omit Cents)

DESCRIPTION

1ST PERIOD
(SAME AS DE.
TAILED BUDGET

ADDITIONAL YEARS SUPPt RT REQUESTED (This application only)
2ND YEAR    3RD YEAR  4TH YEAR 1 5Tt-i YEAR 1 6THYEAR i 7TH YEAR

157,208  168,338  215,892 )
  230,758 1 246,632 1

PERSONNEL
COSTS

CONSULTANT COSTS
(Include fees, travel, etc.)

EQUIPMENT         110.400*

22,500*

17,600*

5,300*   6,000*

21,400

SUPPLIES

16,900

20,200

16,200

1,800

1,900

ALTERATIONS AND       1,800

RENOVATIONS

12,700 I -I-~
      13,500

OTHER EXPENSES

11,500

12,000

9,100

TOTAL DIRECT COSTS    296,208  221,038  267,592 1
      272,058 1 290,932 1

TOTAL FOR ENTIRE PROPOSED PROJECT PERIOD (Enter on Page 1, Item 41  -  ' 1,347,828

REMARKS: Justify all costs for the first year for which the need may not be obvious. For future years, justify equipment costs, as well as any
significant increases in any other category. if a recurring annual increase in personnel costs is requested, give percentage. (Use continuation
pege if needed)

Budget Explanation attached

RF!. 3-w                                                                           I   --


BIJDGRT EXPLANATION

   The early phases of the present study have been funde.i by
NASA but that agency is rapidly severing its involvement in
biological projects. It has already severely reduced its fiscal
support of our program in anticipation of total withdrawal uithiri
one or a feu years. We have started to reduce laboratory stdrf in
response to those cutbacks and will not be able to maintain this
laboratory uithout new funding (like the present proposal). since
we cannot predict the level of funding we will receive for each
of these proposals, we simply note the overlap and will negotiate
a suitable joint effort for system development and operation,
depending on funds available.

   The follouing is a detailed explanation of the budget for
this subprogram. This budget covers only the iudicated segment,
with an indicated allocation for the time o.f a number of
participants who also appear in other segwnts, These allocatiotls
are integrated into the overall proposal budget summarized later
(see Table of Contents for location).

   It should be noted that support for Professor Lederberg's
time is shown at 20%. This includes not only his participation in
this SUbprOjeCt but also his role in overall program direction as
Principal Investigator.

   Salaries are increased at a rate of 6% per year to cover
merit and cost of living increases.  Staff benefits are applied
based on the following University projections: 175, g/73-8/74;
18.3%. 9/74-&i/75; 19.3X, 9/7%8/7b; 20.3%. g/76-0/77; 21.3X,
9/77-a/70; and 22.3%. Y/78-0/79.

YEAR 81  (Retabolic Screening only)

PERSOYMLL: Support is requested for 10% of Professor Cann in
Pediatrics (Professor Kretchmer will devote 10% of his effort
without grant salary support) for clinical and diagnostic
inputs t:, the program. Dss. Duffield, Periera, Summons, and
MS,  Uyche are responsible for the derivdtization and chemical
analysis of urine samples including GC/HS operations. Support
for an operator (Hs.  tioswell) for the prescreening amino acid
analyzer is included. Mr. Steed uill maintain the glassvork
aspects of the GC/HS system.  Hessers Nindfleisch and Tucker
will be responsible for system design and software
implementation respectively and Hr. Veizades, supported by hr.
Pearson,  will design the electronics. Ms. Wegmann operates the
K-High Resolution Rass Spectrometer in the Department of
Chemistry and will be available to run high resolution sass
spectra on critically important samples. Other part-time
personnel (machinist, secretaries,  and administrative support)
act in necessary supporting roles including assisting with
liaison work with collaborating physicians at Stanford and
elsewhere,


CAPITAL E(IUIPMENT:  We request a PDP-II/45 mini-computer system
as an extension of the data system for our low resolution ma-;s
spectrometer.  This system will initially have 24K uords of
memory and use a 1.2M word disk system .for monitor, program,
and spectral data storage.  Removable data storage for raw datd
and archival spectrum rccorainy is provided by the requested
industry compatible maynetiz tapes. The terminal, loader,
clock, instrument interface hardware, etc. complete the
system. Funds to maintain the computer system under
manufacturer contract are included under ggOFBER.~@ A detailed
computer equipment list is included in the "First 12 month
budget detail.18

We also request a 4 column gas chromatograph and an amino acid
analyzer which will increase our urine prescreening capacity
thereby alleviating the bottleneck at the mass spectrometer.
The latter instrument is particularly pertinent to the larger
scale prescreening of samples from the Kaiser Hospital, the
Santa Clara Valley Medical Center,  the Children's Hospital at
Stanford,  and the LA County Hospital. The amino acid analyzer
will also allov reliable continued access to screening
facilities for routine clinical studies during times when the
GC/bS may be tied up for more sophisticated studies, or may be
aout3 for maintenance, repair, or re-engineering. The
reliability offered by this back-up will help resolve the
problem faced by any clinician when he attempts to investigate
new techniques while committed to offering the highest
standard of care, Once an analytical technique is coupled to
patient care, it cannot ethically be disengaged - a priority
that pats great strains on any system also intended for
further research. In the present context, once a metabolite
has been identified,  it may be efficient to monitor its level,
in repetitive samples,  by techniques like the amino acid
analyzer.

We have access to both low resolution and high resolution mass
spectrocnaters (physically located in the Genetics and
Chemistry Departments respectively) and do not request
augmentation of this costly hardware for the present proposal,

SUPl?LIES:  Supplies are requested for sample preparation and
equipment operation. These include chemicals, glassware,
laboratory apparatus, CC supplies, amino acid analyzer
supplies, dry ice and liquid. nitrogen, recorder paper for the
GC/flS instruments and Calcontp plotters, and computer operation
supplies.  In addition, we request supplies and parts for the
maintenance (by our own hardware personnel) of the GC/Ms
system and electronic equipment as well as for the
implementation of necessary data system interface hardware.

TRAVEL:  Travel costs are estimated at round trip expenses for
two professional meetings on the east coast and 1 trip c?dch to
the mid-west and west coast.

/. . .


-3-

ALTERATIOYS AID REYOVATIOYS:  We will need to modify the
electrical service and relocate some equipment in order to
install the new data system and gas chronatograph and to allou
for future equipment ue are requesting. This estimate is based
on a similar modification just made in another laboratory.

OTHER EXPRRSES: Office supplies, telephone service, postage,
publication costs, etc. are requested based on operating
experience in our laboratory. Computer maintenance and
equipment freight are estimated for the proposed data system
augmentations.

YEAR #2

PERSORKEL:  Personnel are unchanged in year 2.

CAPITAL EQUIP1IENT:  As the data system adaptation reaches
completion after year 1, we request additional machine
features to facilitate operational use simultaneously for GC
prescreening and for tEi analyses. These include a block of
fast memory (1K words) to speed up the machine, an additional
disk drive for larger volume on-line storage requirements and
a high speed display dewice for system status and processing
result feedback. Appropriate equipment maintenance increases
are included for the expanded data system.

SUPPLIES, TRAVEL,  ANI) OTRER: These items are increased by 6%
to retlcct inflation and the needs of increased throughput of
urine screening.

YEAH t3

PERSObllYEL: Ye anticipate an increased volume of samples to be
processed by the third year as our baseline studies progress
and more extensive clinical screening is undertaken. This
implies the need for additional support both at the clinical
and laboratory level.  For this reason UC have added 50% time
of an Assistant Professor of Pediatrics and 100X time for a
laboratory technician for sample preparation and equipaaent
operation to the budget.

CAPITAL EQUIPRENT:  An additional prescreening GC is requested
to allou larger population analyses along with the appropriate
computer interface equipment.

SUPPLIES, TRAVEL, AND OTHER: The budget for supplies and
equipment maintenance are increased consistent with personnel
and equipment additions. Also provision is made for inflation
and longer daily operation.

YEAR 14

PERSOEiKEL:  Personnel remain unchanged in year 4.


-4-

CAPITAL EyUIPtlENT: Ue request a system for the vacuum
distillation of aqueous samples for specimen preparation and
derivatization as well as aiscellaneous test equipment which
will require replacing.

SUPPLIES, TRAVEL, AND OTHER: Increases are requested to cover
expected inflation.

CAPITAL EQUIPtiENT:  A small provision is made for replacement
test equipment.

SUPPLIES,  TRAVEL, AND OTHER:  Increases similar to year 4 ar2
requested.


SECTION III

Maternal Blood Stream - Another Source of Fetal
Tissue for Pre-Natal Diagnosis of Genetic
               Disorders

Drs. Herzenberg and Cann


The ffaternal Bloodstream -  Another Source of Petal Tissue tor
       Prenatal Diagnosis of Genetic Disorders

DC,  L. A. tierzenberg,  Principal Investigator
    Dr.  H. Cann,  Aasocrate Investigator

A.   INTRODUCTION

A, 1  Objectives

   We propose to study methods of labelling, detecting and
isolating fetal lymphocytes which pass into the maternal
circulation during gestation. Further data on the time, extent
and conditions of occurrence of these cells during normal human
pregnancy could lead to non-invasive methods for prenatal
diagnosis of genetic disease.

A.2 Background and Rationale

   Fetal lymphocytes have been reported to be present in the
mother's blood in the majority of pregnancies; they may be
present in the maternal circulation in all pregnancies. The
frequencies of fetal cells found, that is the percentage of fetal
lymphocytes in the pregnant uoman@s blood stream, have ranged
from 0.05% to as high as 1.5% in two studies. in one, by
Walknouska,  Conte and Grumbach (l),  the presence of 5 small
acrocentric chromosomes was.sought in lymphocytes obtained from
10 milliliters of maternal bloBd, stimulated by
phytohemagglutinin (PHA) and examined at aetaphase. Prom one to
three metaphases out of many hundreds, suggested a male fetus in
21 pregnancies. This diagnosis was checked at birth and 19 males
were correctly predicted.  Three ot these mothers were studled at
20 weeks or less of gestation, In the second study (2),
quinacrine fluorescent-staining Y bodies were looked for in
interphase lymphocytes obtained from blood (20 milliliter
specimens) Df women in the second and third trimesters of
pregnancy. Here eleven male infants were predicted and seven wele
verified at birth.  Since the method used by Ualknowska et al. is
relatively insensitive,  we suggest that fetal lymphocytes dre
present in the maternal bloodstream in frequencies of about O.lk
or more in all pregnancies at least from 14 weeks of gestation
on.

   Fetal lymphocytes possess HL-A antigens perhaps as early ils
12 weeks of gestation (3). The enormous amount of polymorphism
noted in Caucasian populations (4) is such that most individuals
will be hetecozygous at one or both HL-A sub-loci and the
likelihood of mates differing at one or both sub-loci is high.
These facts suggest that fetal lymphocytes may be separated fcon:
the maternal bloodstream by taking advantage of the HL-A
differences existing between parents, A cytotoxic antibody
directed toward the maternal HL-A antigen which the fetal
lymphocytes do not carry, will,  in the presence of complement,
lyse the maternal leukocytes spacing those of the fetus. OK,

1". ;`*


-2-

indirect labeling of the fetal lymphocytes coated with antibodies
to the paternal HL-A type with fluorescein conjugated anti-human
globulin will render these fluorescent in a sea of
non-fluorescing maternal cells.

   The fluorescent cell-separator perfected by engineers
working under the direction of one of us (Herzenberq) can
separate fluorescent from non-fluorescent cells with high
efficiency, viability and yield (Figure 1; also reference 5). It
can detect fluorescent-labeled cells present in frequencies lover
than lO**(-5) and can separate and thus enrich by a factor of
10+*3.  15 model experiments with human erythrocytes, Rh+ cells
stained with anti-D and fluorescein-conjugated anti-human
immunoqlobulins were detected about one time in two at a
frequency of one positive cell per lo**5 Rh negative cells.
Post-partoa blood samples of Rh- aothers who just gave birth to
an Rh+ child were found to hava from l/163,000 to l/5,000 Hh+
cells indicating fetal blood leakage, probably at birth, of from
20 to b80 microliters (6). Thus, it should be quite feasible to
enrich fetal lymphocytes from the reported frequencies of
lO**(-3) to close to purity after the fetal cells are stained by
imnunofluorescence for paternal HL-A antigens.

   We recognize the potential of the cell separator- to separate
fetal red blood cells from the aaternal bloodstream on the basis
of heritable erythrocyte antigens.  Such methodology could lead to
the developaent of prenatal diagnostic tests for sickle cell
anemia, possibly thalassemia, and other hereditary disorders of
the erythrocyte. We have decided, however, to emphasize isolation
of fetal lymphocytes in this research project because of the
reports (1,2) suggesting that these cells are detectably present
in the rraternal bloodstream in most, if not all, pregnancies. The
literature suggests that fetal red blood cells are detectable in
the maternal circulation in 5-10X of pregnancies at 20 weeks of
gestation or less (7,8). Yhether the application of the cell
separator t5 detection and separation of fetal red blood cells
will improve these figures, is a Question we have been
discussing. Ye believe that aore data on the frequency of mothers
showing fetal red blood cells in their circulations and the
frequency of the cells themselves at various times in pregnancy
are needed, and the cell separator could be applied to this
research question. At present,  our estimates of the amount of
fetal red blood cells ue can isolate,  based on the performance of
the cell separator and on the amount of fetal blood reported in
the maternal circulation, indicate that our yield will be too low
(perhaps 160 times too low) to permit diagnostic studies based on
incorporation of radioactive label into globin synthesized by
fetal cells (9,lO). For these reasons too, we dre persuaded to
work with fetal lymphocytes. However,  since we developed and us~li
the methodology to detect Rh- fetal cells in post pat-turn Hh+
maternal blood specimens (6), we are planning preliminary
experiments designed to answer questions ds to dmount, frequency,
and requirenants, raised above.  We are collabordting with Dr.
Herbert Schwdrtz,  Professor of Pediatrics and Chief of thz
2ediatric Uematoloyy Service at Stanford, in this Freliminlry


-3-

work. Dr. Schwartz will review the morphology of and determine
whether globin synthesis can ba measured in cells separated from
the maternal circulation. Our preliminary work will be aimed at
ways to increase gield and/or decrease amount of fetal blooll
required for prenatal detection tests. At this time, we arc! not
asking for budgetary support in this area, although we may,
should preliminary work show promise.

   The remainder of this proposal will deal only with the
separation of fetal lymphocytes from the aaternal circulatic.1.

D.   SPEC.I.FfC AIFJS

   We propose to use heritable surface antigens as a basis for
separating fetal lymphocytes from the maternal bloodstream during
gestation. These separated cells will be used to diagnose various
genetic abnormalities in the fetus.

C.  METHODS Of PROCEDUHR

C. 1 Iamunoselection of L,ymphocytes

    (a) POSITIVE IHHUROSELECTION WITH THE: FLUORESCENT CELL
SEPARATOR. Specific fluorescent staining for a variety of HL-A
antigens will be developed. In most cases, we will use the
indirect procedure, i.e. anti HL-A whole serum followed, after
washing,  by fluorescein or rhodamine conjugated anti-human
immunoglobin (P or B anti-Ig).  In pilot studies for this project,
we have already obtained staining sufficiently bright to achievr
excellent cell separation with a polyspecific and two specific
HL-A antisera-  In order to decrease non-specific staining (of
lymphocytes lacking the antigens to which the antibodies are
directed) ue have had to remove aggregated proteins of antiserum
by centrifugation at 1U0,OOO g for 2-3 hours.

   Lymphocytes are prepared by ficoll-Isoyaque isoppcnic
centrifugation (11) followed by filtration through nylon wool to
remove monocytes and many'R-lymphocytes. Since these latter bavc?
surface immonoglobins,  they stain with the fluorescent reagents,
although much more dimly than the HL-A stained cells. ciith thest,
virtually i;ure lymphocyte population, of which greater than 90%
are thymus-derived (T) lymphocytes, we have obtained bright
speckled fluorescence by staining at 0 degrees C. Uhen we have
passed positive and negative (stained for d particular HL-A type)
cells separately through the cell separator, the fluorescence
intensity distribution has shown two clear populations with very
little overLap (Figure 2). Thus, good separation can be obtained.

   Initially, reconstruction experiments will be periormctd
using various mixtures of cells positive and negative for dn HL-A
antigen or antigens.  In addition to numerical evidence (from the
fluorescence intensity distribution) for successful separation,
we will use male and female cells so that sex chromatin,
karyotypiny and quinacrine staining for Y bodies (2) cdn bt- usell
as confirmatory markers.


-4-

   In isolations from. maternal blood, antibodies directed to
paternal and not maternal antigens will be used followed by P
anti-Ig.

   (b) UECATIVE I!JfiUNOSELECTION UITH COIYYLEMEYT MtDXATED LYSIa.
As an alternate procedure to or a step preliminary to fluorescent
cell separation,  (maternal) cells with particular HL-A antigens
will be killed by incubation with antiserum and complement.
Antisera directed against maternal antigens not shared by the
father will be used uhen possible.  With separate aliquots of
lymphocytes,  antisera negative for one or the other set of
paternal antigens will be used.

C.2 Fetal Lymphocyte antigens as Alternates to HL-A for
  Immunoselection with the Fluorescent Cell Separator

   Several fetal antigens have been described (12) but, so far,
none of lymphocytes.  If an antigen could be found to be present
on early fetal lymphocytes but absent, or in greatly diminished
amount, on adult lymphocytes, it would be considerably more
convenient to use than HL-A antigens.  There would be no problem
of typing the father and having to select for each of the
paternal haplotypes,

   We vi11 attempt to find a fetal antigen by direct
immunization of other species with lymphocytes from aborted
fetuses. Anr antisera obtained will be absorbed with adult cells,
including lymphocytes, to render it specific. Of course, YZ
cannot predict in advance if this will succeed. Another means to
look for fetal lymphocyte antigens is to use antisera raised
against leukenias. Tumors often have fetal or embryonic antigens;
whether leukemias do is unknown,  ke will test for them with fetal
and normal adult lymphocytes,

C. 3 Mitogens Preferentially Stimulating Fetal Cells.

   It is possible that some mitogens will be relatively more
stimulatory to fetal than adult lymphocytes. If so, this would
decrease the importance of maternal contamination of separated
fetal cells.  We will screen all the available lectins including
PHA, PWfl (pukeweed mitogon), concanavalin A, wheat germ
agqlutinin, etc. for mitogenic activity with cells from aborted
fetuses of several ages and cells from adults. One precedent for
differential aitogenesis is PHA which stimulates fetal mouse
thymocytes 1Q times more than adult mouse lymphocytes (13).
Evidence of differential response to PHA and PWY by lymphoid
cells of huaan newborns (cord blood) and adults suggests that
this approach may be feasible (14).

C.4 Lymphocyte Culture and Karyotyping.

   Separated lymphocytes will be grown in micro-culture plate:<
with phytohenagglutinin (PHA) for 48 hours, colceaid added and
chromosome spreads made 16-20 hours later. In preliminary
experiments we have obtained high percentages of mcttaphases with


-5-

50,000 cells cultured in 250 aicroliters of eedium. Prior
staining uith anti RL-A does not seriously decrease (less than
2-fold) the oitotic index.  We have been successful in obtaininy
high quality chromosoae spreads by standard techniques with these
small numbers of cells in micro-cultures. We believe we could
culture (io 10 microliters medium) and spread as few as 1,OO;j
lymphocytes. However,  that is yet to be tried.

   To check our ability to separate, grow and karyotype fetal
lymphocytes, we will predict the sex of the fetuses and compare
with the sex observed at birth.

C.5 Cell Separations

   Fluorescent cells will be separated in the fluorescent cell
sorter we have built (see Figure 1). Cells suspended in medium
pass in single file in a stream past 2 laser light sources and 2
photodetectors,  One laser and detector observes cell iluorescence
and the other light scatter which is a measure of cell volume.
when a cell uith the desired fluorescence level and of the right
volume is detected, a charge is applied to the stream with a
delay of about 100 microseconds.  The stream is broken into 40,000
uniform droplets per second downstream of the observing point,
Charges are applied when droplets containiny the desired cells
are just being formed so they become charged, Phey are then
deflected by constantly charged deflecting plates into collecting
tubes.

   The number of cells processed per unit time is generally
inversely related to the enrichment obtained. with a frequency of
desired cells of lG**(-3),  lo**4 cells per second can be
processed to obtain about 100% purity of wanted cells. A yield of
at least 20,000 desired cells per hour should be obtained. This
is certain1.y enough to culture and karyotype. If the frequency of
fetal lymphocytes in the maternal bloodstream is lo-fold lower,
i.e.  13** (-4),  then only 2,000 cells per hour would be obtained.
This is Frobably a marginal number for culture. Furthermore,
there would probably be prohibitable losses uith so few cells.
However, there is no need to seek high purity. When 1ookin:j for
male karyotypes, or later for gross chromosome abnormalities,
only a feu successtully spread cells of the selected type are
needed.  If we predict 5X purity,  we need to screen about 1dO
cells to be quite certain of seeing a few eetaphases from the
selected cells. Thus, preliminary analysis of maternal samples
will be used to determine the amount of cells to be put through
the cell sorter.

   with the lo**{-4) frequency, which is at least 13 times
lower than the published estimates,  2 x 10**7 lymphocytes from
maternal blood will be processed per hour. This approximately
represents the number in 10 milliliters of blood. E'or a routine,
non-objectionable procedure, wa feel this (or perhaps 20
milliters) is about the limiting amount of blood which could be
used. Thus,  for screening purposes,  the minimum fetal lymphocytt\
frequency is probably slightly less than lO**(-4). We will


-6-

establish what the real frequencies are in a reasonable number of
pregnant uomen at various stages of gestation, If tha published
values are correct, we will not be limited by fetal cell
frequency. (Par high risk pregnancies, it may be that even with a
frequency of less than IO**{-4) , it would be preferable to do the
antenatal cptogenetic analysis with SO-100 milliliters of blood
rather than by amnioceutesis) .

C.6 Strategy (and Summary)

   (a) IIL-A specific staining by immunofluorescence will be
developed. This is presently in progress.

   (b) Preliminary enrichment by cytoxicity with HA,-A antisera
and complement will be assessed.

   (c) Separation from artifically mixed populations will be
carried oat using sex markers to confirm.

   (d) Fetal lymphoc,yte frequencies will be determined at
various stages of gestation.

(e) Petal lymphocyte antigens will be sought.

(f) Selective fetal mitogens will be sought.

   (9) If the frequency in late first trimester or early second
trimester pregnancies is about IO**(-li), separations, culture and
karyotyping will be done. Accuracy of prenatal sex prediction
will be used to confirm separations.

    Time Table : Development of the method with artificial
mixtures and maternal blood will take between one and two years.
Assessment of frequency of fetal lymphocytes in maternal blood TV.
a function of gestational time will take about six months.

   Subsequent steps involve Developing enough reagents for a
signif icant trial at detecting chromosome dbnormalities using
high risk pregnancies,  This stage will also require setting up a
cooperative effort involving patients at Stanford and other
nearby institutions (e.g. the University of California Medical
Center, San Yrancisco; Dr. C.J. Epstein). This will involve
comparing results from the cell separator technique with those
from amniocentesis. We anticipate applying for supplementary
funds dfter 3 years if tkir; pilot project is successful.

D. SIGNIPICAYCE

   A routine method of antenatal diagnosis using lb-20
milliliters of maternal blood would permit mass screening for
genetic disorders, especially those involvinrj chromosomal
aberrations.  The methods ue propose could result in dVOidinq the
use of aani3centesis even in high-risk pregnancies (A;nnioc.?ntesis
would be dif&icult to apilly for mass Screening).


-7-

   The cost of separation an3 karyotypiny could be brought into
the range of a few dollars per test with the development of
semi-automated micro-cultures and computer assisted karyotyping
(Dr. C.J. Epstein, personal communication). The cost of the
0.5-11 of congenital chromosome abnormalities among births is flir
higher than the cost of such screening. It is impossible to
assign a uniformly acceptable cost in total human terms, but the
actual costs to the family and/or society must be in excess of
100,OOG dollars for each infant with Down's syndrome born.
Screening to detect an extra chromosome 21 would yield 1 case in
600 at a cost of probably from l,OOO-10,OGG dollars, Thus, the
cost would likely be justified even for only this one condition.

   Further uork could permit biochemical and particularly
cytocheaical tests (15) for inherited disease to be performed on
the separated fetal lymphocytes either before or after culture.

    Ye wish to stress that the  use of maternal blood rather than
amniotic fluid samples provides a non-invasive method of
antenatal diagnosis. This methodology can eliminate the risks to
fetus and mother which are encountered in amniocentesis.


-a-

NEPERENCBS

1. Walknouska, J., Conte, F-A,, Grumbach, H.R., Lancet 1, 1119,
1969.

2, Schroeder, J., De la Chapelle, A., Blood 39, 153, 1972.

3. Payne, El.,  Blood 19, 411, 1962.

4. Bach, P.R., Science 168, 1170, 1970.

5. Rulett,  H.R. et al.  Science 166, 747, I969.

6. Jan, U., Herzenberg, L.A. in preparation.

7, Cohen, P. et al. Blood 23, 621, 1964

8. Clayton, E.H. Jr., Peldhaus, U.D., Whitacre, P.E., Obstet.
Gynecol. 23, 915, 1964.

9. Hollenberg, H.D., Kaback, H.H,, Kazazian, H.H. Jr., Science
174, 698, 1971,

10. Kan,  II.Y. et al.,  New England J. tied. 287, 1, 1972.

11. Borjua, A., Scandinav. J, Clin. Lab. Invest. 21, Suppl. 97,
1968.

12. Anderson, N.G., Coggin, J-ii. Jr., Cole, E., Holleman, J.W.
(eds.) EBBR~ONIC AND PETAL ANTIGENS IN CANCER,

Vol. 2, Oak Ridge National Lab., Tennessee, 1972. tlSAEC Heport
CONF- 7202G8.

13. Nosier, D.E., Nature New Riology 242, 184, 1973.

14. tlontgomery, J.R., Hason, E.O., South, H-A., Pediat, Res. 7,
365, 1973.

15. Galjaard, H, Jahodova, l!l., Nierrijer, tI.F., Brux, aed. 52,
109,  1972.

P- ;??


-9-

TITLES OF FIGUHES

Figure 1.  Diagram of fluorescent cell separator.

   Figure 2.  Demonstration of almost complete separation of
IiL-A stained and unstained cells.


- `RESSUAIZED
k-z--?-l r3 l4JTER FLOW
- ---
  - - -     RESERVOIR
- -em
I I
  - --
- ---
  - - -
-- --

FLUORESCENCE
EXCITATION

CHARGING PULSE

HELIUM NEON                          FLUORESCENCE    HELIUM NEON
  LASER          0            PHOTODETECTOR    SCATTERED LIGHT

                        0                      DETECTOR
                 0
                       0

    :
    :
-0 I-
    00
  -2KV  iii    +2Kv
    3
         I .o

CELL SORTER

SIMPLIFIED BLOCK
    DIAGRAM

NON- FLUORESCENT
    CELLS

UNWANTED CELLS


Frequency Distribution

HL-A2 positive
"V Lymphocytes

Weak anti HL-A2

6 Normal Human Serum

Stkong anti HLaA2
       .e

CHRNNEL NUMBER

Fluorescence Intensity


RIVILEGED COMMUNICATION              SECTION II     SUBSTITUTE THIS PAGE FOR DETAILED BUDGET PACF
                                                                           GRANT NUMBER
          SUBSTITUTE                           PERIOD COVERED
                                          FROM              THROUGH
IETAILED BUDGET FOR FIRST 12.MONTH PERIOD l/1/74       12/31/74

Herzenberg, Leonard
Cann, H.
Open - Pediatrics

Hulett, H.
Waters, Ruth
Sakaguchi, S.
*en - Genetics

Meyering, P.

TITLE OF POSITION

I

PERSONNEL (List all personnel en&e&d on muiect)

Principal Investigator or
Program Director
Assoc. Prof. Peds.

Research Associate

Research Associate

Research Assistant

Research Assistant

Engineering Tech.
Secretary

TIME OR
EFFORT         AMOUNT REQUESTED (Omit cents)

   7./HA.5.
t-i                               TOTAL


  10%

10%

100%

20%

100%

50%

50%

10%

 FETAL CELLS IN MATERNAL

     CIRCULATION

: '. '.







.
.:'.:. .'
   .,             .
. . I


           r

-

.-CONSULTANT COSTS [Include Fees and Trsvcl)

. EQUIPMENT (Itemize)         Carbon Dioxide Incubator   $2,500
                    Laminar Flow Hood        1,000
                Fluorescence Microscope    9,000


8 SUPPLIES
Tissue culture supplies, (filters, trays, etc.), dishes, media,
pipets, sera, etc.

STAFF     0. DOMESTIC
TRAVEL .        1 East Coast meeting

5     500

& c InrtNcflons) b. FOREIGN                                                                 L

. PATIENT COSTS (Separate Inpatient md Outpatient)

s

. ALTERATIONS AN0 RENOVATIONS

s

. OTHER EXPENSES (Itemize per inatmctionsJ
Office supplies, telephone, repro., postage, publication costs, etc.

.

Subtotal - Items 1 thw 8                c  s 75,782

I I

        10. TRAINEE EXPENSES (See Instructfans)

                   PREDOCTORAL

  FOR    a. STIPENDS POSTDOCTORAL
                 OTHER (Specify)
TRAINING             DEPENDENCY ALLOWANCE

GRANTS

        b. TUITION AND FEES
ONLY ,
        c. TRAINEE TRAVEL IDe.crlbe)

No. Proposed           s

No. Proposed           S
No. Proposed           s

                   I
TOTAL STIPEND EXPENSES -

11.

Subtotal - Trainee Expenses                 c -I

i2. TOTAL DIRECT COST (Add Subtotals. Items 9 and 11, and enter on Pale 0

c

I

s 54,782

s 12,500 *

s   7,000

s   1,000

5

-
s

s

s

' 75,782 1

Substitut. Budget Page 5-72

P-97


SECTION II -PRIVILEGED COMMUNICATION         FETAL CELLS IN MATERNAL CIRCULATION
  BUDGET ESTIMATES FOR ALL YEARS OF SUPPORT REQUESTED FROM PUBLIC HEALTH SERVICE

CONSULTANT COSTS
(Include fees, travel, etc.)

EQUIPMENT

12,500*

SUPPLIES          7,000

TRAVEL

PATIENT COSTS

ALTERATIONS AND
RENOVATIONS

OTHER EXPENSES

1,000

1 7'j ,782
TOTAL DIRECT COSTS          67,859   72,404 1 77,221 1 82,529 1

DIRECT COSTS ONLY (Omit Cents)

lDDlTlONAL YEARS SUPPORT REQUESTED (Thisapplication only1
2ND YEAR    3RD YEAR    4TH YEAR    5TH YEAR    6TH YEAR    7TH YEAR


58,659   62,704  67,021   71,629









       7,900   8,400    8,900

7,500

600

1,100

600     600      700

-





1,200   1,200    1,300

TOTAL FOR ENTIRE PROPOSED PROJECT PERIOD (Enter on Page 1, Item 41  -   s  375,795

REMARKS: Justify all costs for the first year for which the need may not be obvious For future years, justify equipment costs, as well as any
significant increases in any other category. If a recurring annual increase in personnel casts is requested, give percentage. /Use continuation
pege if needed. )

Budget explanation attached.


BUDGET EXPLANATION

   The budget covers 10% each of Professor Herzenberg's and
Professoc Cann's tiae in relation to this subproject. A full tiEe
Besearch Associate is required to obtain, prepare, and
fluorescent label the lyaphocytes for separation and then to
culture and Perform cytogenetic studies on the separated cells.
The part time (20%) Research Associate is to be responsible for
the actual separations. Tuo Research Assistants, one part-time
(SO%) for cytogenetic studies and omkfull-time for serological
studies, as well as a part-time (50%) cell separator operator art!
required to assist in these procedures.  Support is also budgetted
for 10% of Professor Berzenberg's secretary.

   Salaries are increased at a rate of 6X per year to cover
merit and cost of living increases.  staff benefits are applied
based on the following University projections: 17%,9/73-e/74;
18.3X, 9/74-a/75;  19.3%, 9/75-8/76; 20.32, g/76-8/77; 21.3X,
g/77-8/78; and 22.3%. Y/78-8/79.

   The lauinar flow hood is essential for aseptic culturing and
protection of personnel from possible infectious agents in human
blood. The carbon dioxide incubator is required for culturing
lymphocytes,  The fluorescent microscope will be used to detect
interphase fluorescent Y bodies and the Y chromosome at
metaphase. In the later stages of this study, the fluorescence
microscope vi11 be used to stu3y the karyotypes of lymphocytes of
fetuses with chroaosomal aberrations.

   The budget for this project represents the MINIMUh ADDED
COSTS to DC.  Herzenberg's and Dr. Cann's other programs (cell
separator project,  in particular) required for this work to be
performed.


SECTION IV

Polymorphic Genetic Markers in Amniotic Fluid

Drs.  Cann and Tsuboi


Polymorphic Genetic Rarkers in Amniotic fluid

  Dr.  EI. Cann, Principal Investigator
Dr. K. A, Tsuboi, bssociate Investigator

A.  INTRODUCTION

A.1 Objective

   We propose to study amniotic fluid (AP) and cultured
amniotic fluid cells for the expression and variation in the
expression of various polymorphic genes. As the loci of those
polymorphic genetic markers which are expressed in BP are shown
to be closely linked to loci of genes determining disease, we
will use the specific syntenic relationship for prenatal
detection of the disease in the fetus.

A.2 BACRGROUibiD AND BAT'IONALE

   Surprisingly little is knovn about the origin of AF, ancl it
appears that various sources contribute to the 981s water and 2%
solids making up this biological fluid of the fetal environment
??? o  Fetal urine, amniotic epitheliuo, cells of the fetal
respiratory tract and, perhaps,  the umbilical cord are thought to
contribute at different times during gestation (2). The fetal
cells suspended in the AY probably represent exfoliation from the
skin, umbilical cord, urinary tract, oropharyngeal uucosa and
amnion (3).  Erythrocytes which are seen following the majority of
amniocentesis procedures are almost certainly maternal in origin.
The soluble and cellular constituents of AP provide the
opportunity to study a saaple of the genome of the unborn fetus,
especially genetic systelas of antigens and enzyates. Detection of
certain of the latter in cultured BP cells, of course, forms the
basis for prenatal diagnosis of some of the hereditary metabolic
errors (4).  It is possible to study some polymorphic genetic
systems in the fetus froa AF analysis (5,6,7), although a
systematic search for expression of human polymorphisms in AF has
not been undertaken. The emphasis on prenatal diagnosis of
hereditary disorders has, understandably, focussed on detection
in Al? of enzymes whose deficiencies cause disease, and these
genetic markers are usually idiomorphs (8). The variation among
individuals iaplied by polymorphic genetic systems adds a
dimension to the research design vhich allows certain areas of
genetic significance to be explored. Coaparision of expressions
in the fetus of differing alleles at the same locus can be trade,
The investigator can pursue the effects of feto-maternal
incompatibility on expressions of various alleles.

   It is clear that a significant scientific advance has been
encountered in the successful exploitation of alsniotic fluid for
the prenatal diagnosis of genetic disorders, The ability to
isolate cells of fetal origin from AP and successfully culture
them is the basis for monitoring "high risk*@ preganancies to
detect fetuses with various chromososal aberrations and some

P-/O/


-2-

inborn errors of aetabolisn (4). The technique of prenatal
diagnosis removes the uncomfortable uncertainty from genetic
counseling and, coupled with selective abortion of affected
fetuses, joins the armamentariua of preventive medicine. As with
most desirable techniques, there are limitations. Considerable
time may elapse between amniocentesis and diagnosis in order to
obtain sufficient nuabers of cultured AF cells required for d
particular test, usually biochemical in nature. Sufficient cells
must be available for testing by (approximately) 20 weeks of
gestation, i.e. the deadline for performing a therapeutic
abortion. Uith reference to this limitation, the development of
methodology uhich accurately tests the AF, or AF cells, at once
is desirable. Another and more fundamental limitation presently
confronting prenatal diagnostic activities arises from the
inability to detect phenotypes at the cellular level. For some
diseases, although the biochemical phenotype is known, absence of
the normal protein from AP cells prevents their detection in the
fetus; sickle cell anemia, hes>philia, phenylketonuria and
ornithine transcarbaaylase are some of these prenatal diagnostic
*Dorphansa. For other hereditary diseases, sufficient information
on the underlying biochemical 3efect is not available, and
numerous autosonal recessive,  autosomal dominant and X-linked
pathologic characters can be cited for this category. Cystic
fibrosis (although this disorder may soon be liberated from this
category), X-linked ichthyosis,  neurofibromatosis and
huntington*s Chorea are just a few. while we can optimistically
look forward to increasing progress in the elucidation of basic
mechanisms of these hereditary disorders, a waiting period before
application to prenatal diagnosis of these conditions is not
necessarily implied. In other Yards, even though a biochemical or
other suitable cellular phenotype may not be available, it may
still be possible to diagnose with considerable accuracy some of
these conditions in the fetus. Genetic linkage may offer this
possibility.

  Human linkage (syntenic) groups are relevant to prenatal
diagnosis of inherited disorders because the detection of the
phenotype of a genetic marker which is closely linked to the
locus of an allele determining disease provides the possibility
of genotyping the fetus. This is already possible for myotonic
dystrophy, a disease expressed in heterozygotes for an allele at
a locus which is linked to the ABli secretor locus, the
recombination fraction being 0.04 (9). ABh substances are
secreted early in gestation by the fetus into amniotic fluid,
forming the basis of determining its secretor status (5). Thus,
if the coupling phase is known for an individual who is
heterozygous at both loci and married to a non-secretor,
detection of the secretor status of the fetus will predict
presence or absence of the allele determining mpotonic dystrophy.
The wagnituae of error in this prediction, 8X, is slightly
greater than the recombination fraction (9).

  The genetic map of man is growing. More and more liakeci loci
are being found, autosoaal linkage groups containing more thdn
two loci are recognized and loci are being assigned to visible


-3-

autosonal chromosomes (IO). The majority of the newer linkage
groups involve polymorphic biochemical or serological markers
rather than inherited disease markers. This is understandable
because ascertainment of infrequent diseases in families is
by-passed and accumulation of data is facilitated. Furthermore,
linkage analysis by in vitro, Sendai virus mediated,
interspecific somatic cell hybridization (11) does not require
polymorphic gene markers so long as differences between
interspecific allozyaes or other homologous markers can be
detected. Still, for prenatal diagnosis, spnteny involviny a
locus uith a disease determining allele is essential.

   At present, a small number of autosomal syntenic groups
involving clinically significant loci are known. The loci of five
autososal dDainant disorders have been shown by family studies to
be linked to polymorphic loci (revieued in reference 10):

,,c. , (' =


MAIN LOCUS

TEST LOCUS   DISTAPCE     95% PROBABILITY         COFMENTS
          CENTIHORGAIS LKHITS OP I'lAP DISTANCE
                      CENTIMORGANS

Congenital total    Duffy          0            o-17            Probably syntenic
nuclear cataract                                             with the two loci for
                                               pancreatic auylase.
                                              Assigned to
                                                             chromosoae #l,

Elliptocytosis       Bh            3            2-7

Nail-Patella

b    Sclerotylosis
2
e    Hyotonic
    Dystrophy

ABO
hdenylate
Kinase

13

0

HkiS            4

Secretor        4

8-21


O-6


0.3-19

Syntenic with loci
f Or 6-phDSphDglUCO-
natedehydrogenase E
phOSphDglUCDmUtaSe
(first locus), and
peptidase C.   If these
loci are correctly
assigned to
chromosome #l, they are
syntenic with loci for
the Duffy system, con-
genital total nuclear
cataract and pancreatic
amylase.
Linkage studies with
Rh have detected
genetic hetero-
geneity for ellipto-
cytosis.


-s-

The biochemical basis of almost all autosomal dominant disorders,
including those listed above, is unknown, denying us a rational
fOUndatiD8k for prenatal diagnostic testing. Genetic linkage
affords a potentially useful strategy for antenatal detection of
autosomal dominant disorders. This also applies at present to
some clinically significant loci on the human X chromosome:

P-/c 5


MAIN LOCUS

TEST LOCUS DISTANCE
           CENTIHORGAI'IS

Hemophilia A    Glucose-ti-
(Factor VIII   phosphate
deficiency)    dehldroyenase      4

Lchthyosis

Ocular Albinism

XY           18

xg           18

95% PRUBABILITY         COLIBEITS
LIHIT OF HAP DISTANCE
CENTIWORGANS

O-16

Polymorphism at
the test locus
in black,
tlediterranean E
some Asiatic
populations

ll-31(*)

7-34(*)

(references 12,13 and 14, respectively)
(+I  - 90% probability limits


-7-

With respect to the above autosonal syntenic groups it should be
pointed out that the ABU types of secretor + fetuses and secretor
types have been detected from AF (5). and that Gphosyhogluconate
dehpdrogenase (6-PGD), of the Rh syntenic group, has been
detected ia cultured AP cells (7).  The electrophoretic phenotype
of glucose-6-phosphate dehydrogenase (G-6-PD) has also been
detected in cultivated AP cells (7). Thus, the potential exists
for diagnosing the nail patella syndrome, myotonic dystrophy,
hemophilia A and perhaps elliptocytosis in fetuses at risk by
virtue of slntenic relationships.

   It is clear that, at present, there are limitations in the
use of syatenic groups for prenatal diagnosis of inherited
disorders. The method will be useful only for informative
families, i.e. those with a doubly heterozygoos (at test and main
loci) parent for autosomal dominant traits, a doubly heterozygous
mother for X-linked recessive characters and both parents doubly
heterozygous for autosomal recessive disorders. lleiotic crossing
over between the marker (test) and main loci vi11 always provide
the possibility of error in predicting the fetal genotype, and
the magnitude of this error is provided by the recombination
frequency.  The smaller the distance between these loci, the lover
this error vi11 be. Still this error will be, in general, much
less than the 50% "error" that nou attends genetic counseling
based on detecting a male fetus at risk for an X-linked disorder.

   Another limitation of utilizing linkage information for
prenatal diagnosis is the small number of syntenic groups
involving clinically significant loci. At present, there is good
reason to be optimistic about mapping the human chromosomes,
Huddle has indicated that each of the chromosomes wiJ.1 have a
known linkage assigament within "the next several years" (15).
Family studies and interspecific somatic cell hybridization are
providing data for new s,yntenic groups,  assignment to recognized
groups and to visible chromosomes, and these in vivo and in vitro
techniques for linkage analysis are also confirming each other's
findings (e.g. reference 16). Ye should inject a slightly less
optimistic coraent concerning the application of genetic linkaye
groups detected by somatic cell hybridization to prenatal
diagnosis of inherited disorders. The genetic markers used in
these studies with interspecific hybrids are usually enzymes
vhich are not necessarily polymorphic characters, as classified
by electroyhoresis. This map bn especially true for autosomal
recessive genes determining various metabolic errors. For
prenatal detection of autosomal recessive disorders, both carrier
parents must also be heterozygous,  preferably for codominant
alleles,  at the linked test locus, an unlikely expectation if the
test locus Ls not polymorphic. Thus, this restricts the number of
marker loci and the frequency of couples at risk which are
informative for application of linkage for the prenatal
diagnostic test. The situation is only slightly improved for
X-linked loci. because of the paucity of X-linked polymorphic
systems (Xg,  Xm and in some populations, glucose-6-phosphate
dehydrogenaso;  color blindness is a recessive trait).


-B-

   Another problem with linkage data collected from analysis of
somatic cell hybrids resides in the inability to estimate the
frequency of recombination betueen test and main loci. For
purposes of prenatal detection,  the recombination frequency is dn
estimate of the error in the prediction of the fetal genotype and
phenotype. The limitation in observing polymorphism at a number
of the (enzyme) loci uhich will be linked by this technique
suggests that for some linkages, confirming family studies will
not be forthcoming because informative families will be rarely
encountered.

   Despite these reservations,  the more linkages determined
between loci and between loci and identifiable chromosomes, the
more likely this knowledge can be applied to prenatal diagnosis
of various inherited disorders. Certainly we can expect
assignment of polymorphic loci to linkage groups and visible
chromosomes, and detection of polymorphism in a number of
presently monomorphic or idioaorphic systems is more than a
reasonable prediction, The amount of genetic variability in terms
of polymorphism, has surpassed estimates of a mere decade ago,
and,  indeed, it is not unreasonable to consider the possibility
that almost all loci are polymorphic in man (17). Thus, we can
expect assignment of a number of clinically significant loci to
syntenic groups.

   A ,final limitation to the use of synteny for prenatal
diagnosis stems from the relatively small amount of information
available on the expression of polymorphic genetic markers in AF.
Should additional, clinically relevant, syntenic groups be
detected,  ue still must be able to test for the expression of the
marker loci in AF in order fo infer the fetal genotypes. In
addition to providing information pertaining to the biology of
the fetus and BP, a systematic study of polyaorphisas in AP can
provide the information necessary to use syntenic relationships
for the prenatal diagnosis of inherited disorders.

B. SPECIFIC AIHS

   1. We propose to seek in AF and cultured AF cells the
expression of known polymorphisms.  Among the polyaorphisrs to be
studied will be erythrocyte antigenic systems, the hL-A system
and systems of various intracellular enzymes,

   2. Ye vi11 delineate variation in expression of allelic
markers in AF and attempt to understand the basis of the
variation.

   3, We will delineate expression of polymorphic markers in Af
in terms of gestational age.

    4.  Me will compare the expression of polymorphic markers in
AP with their expression in other tissues (e.g. blood, cultured
fibroblasts, etc.).


-9-

    5. As ue come to understand the variation encountered in the
expression of polymorphic markars in AF and the limitations of
the tests ue use, we shall begin to employ or help others employ
those markers involved in a syntenic relationship with clinically
significant loci for prenatal detection of affected fetuses.

C. HETHODS AND PROCEDURE

1. AP Samples

   AP samples for these studies are being obtained from
appropriate patients by staff physicians with full-time or
clinical faculty appointments to the Department of Obstetrics and
Gynecology. These physicians provide lo-15 AF samples each year
for prenatal diagnosis of chromosonal disorders (primarily
trisomy 21);  these specimens are usually obtained at 12-16
gestational ueeks.  Ye expect the numbers of Al? samples sent for
prenatal diagnosis will gradually increase as more obstetricians
take advantage of the service provided by the Cptogenetics
Laboratory at Stanford. One problem in this source of Al? samples
which concerns us pertains to maternal age. Host of the samples
obtained for prenatal diagnosis are taken from women over 35
years of age. The effect of maternal age on expression of
polymorphic markers in AP is a variable about which there is no
inf ormatioa.

   The obstetricians have been cooperative in providing us with
AF samples collected at the time of therapeutic abortion; these
specimens are usually obtained at 18-20 weeks of gestation. About
2-3 therapeutic abortions are performed each week at Stanford
University Hospital.

2. Cultivation of AF CELiS

    We shall work with cultivated AF cells in order to assure
ourselves that we are working uith only fetal cells. After
amniocentesis AP usually contains a mixture of maternal blood
cells (probably resulting from the procedure) and fetal cells.
Although it is possible to remove most of the maternal
erythrocytes by lysis (I&),  other nucleated cells from maternal
blood may remain.  Cultivation results in dilution of maternal
blood cells by the replicating fetal cells.

   The use of polymorphic markers and of markers of fetal sex
will enable us to decide on the origin of the cells growing in
culture (fetal or maternal), Obviously cultured cells carrying a
fluorescent interphase Y marker (19) or showing no sex chroadtin
(Barr) bodies are of fetal origin. For cells which do show
evidence of the XX karyotgpe, ue vi11 compare tha phenotypes of
the polymorphic markers we employ with the phenotypes in the
mother and father.  In most instances these precautionary
procedures vi11 help us avoid mistaking maternal for fetal cells
in AF.


-lO-

   We have been cultivating AF cells for the past year. Our
culture technique is similar to that used by other investigators
in the field.  Gsually 10 ml of Af (more, if obtained before
instilling a hypertonic saline solution for therapeutic abortion)
are centrifuged at 100 g for T-10 minutes. The supernatant Ah' is
removed and saved (see below) and the sedimented cells are
suspended in 0.5-1.0 ml of fetal calf serum. We carefully place
drops of the serum-cell suspension on the surface of a small
plastic Petri dish or on a cover slip in a Petri dish and then
incubate the cells at 37 degrees C in a 5% CO;! and air
environment.  After 6-18 hours, during which time cells have
attached to the surface of the dish (or coverslip), we add tissue
culture medium (FlO-Grand Island Uioloyical Co,) with 30% fetal
calf serum. This medium is changed every other day. with this
procedure ve can see cell growth within one week. There are
sufficient cells in a dish or on a coverslip for the first
subculture (0.05% trppsinsolution) within 2-3 ueeks. After the
first subculture,  we find that we can perform the second
subculture uithin a ueek.  At this time the cultivated BP cells
are groving well.

   The cel.ls with which ue are dealing at this time usually are
epithelioid cells.  It is our experience that these cultivated AY
epithelioid cells will grow vell for about 4-S subculture
passages after which their grouth will cease. Hithin these limits
we have been able to cultivate large qnantities of these
epithelioid cells in roller culture bottles. This will be
important for the study of polymorphic enzyme markers in
cultivated AP cells.

   Less frequently we have noted fibroblasts growing in
cultures. These cells grou for longer periods than the
epithelioid cells. For instance,  one AE' fibroblast culture which
we are propagating and using for various investigations (7B2B) is
now in its 14th subculture passage. We know this line is of fetal
origin because it does not shou Barr bodies and possesses the XY
karyotype; this is a diploid culture.  It is our impression that
more time is required for fibroblastic growth to be evident
initially than for epithclioid cell replication. We will use
either morphological type for our studies of cultured BP cells,
although for purposes of prenatal diagnosis epithelioid cells may
be more relevant.

   Aliquots of replicating cells from each AF culture are
frozen ia 10X DHSO (lo**6 cells per ml per vial) and storect in
liquid nitrogen for future studies with live cells. Aliquots of
lo**7 cells vhich have been washed,  sedimented and drained of
supernatant liquid are stored in liquid nitrogen for future
electrophoretic and enzpme activity studies.

3. The Supernatant AP Fluid

   After AP cells are separated from the supernatant AF by
centrifugation, we again centrifuge the latter (2,900 r.p.m. fee
10 minutes in an angle head International Clinical Centrifuge) to

p-//t


-1 I-

remove any remaining cells or large pieces of cell debris. The
supernatant AP is then transferred to another container and
stored at -20 degrees C.  This fraction of A.F is used for ABE
hemagglutination inhibition studies and will be used for studies
on fetal Lewis substances (6).

4. Controls for Expression of Polymorphic lYarkers

   Whenever possible blood samples will be obtained on both
parents of the fetus under study. These samples will be typed for
the various polymorphic markers being studied in AF. In addition
umbilical card blood specimens will be collected at birth of
those fetuses whose AP was studied and who were not aborted.
These specimens will also be typed for the various polymorphisms
which were studied in the AF. In those instances in uhich fetuses
are aborted,  ve shall atteapt to use appropriate material from
the abortuses to type for polymorphisms. The collections of the
various specimens just listed are meant to provide us with
material which will serve as controls for the observations we
make on Al?,  Working uith polymorphic systems permits us to
predict the phenotype of the fetus,  provided we know maternal and
biological paternal phenotypes.  A high frequency of discordance
between observed (from BP) and expected phenotypes will signal us
to examine our testing procedures,  to consider that changes are
occurring in cell culture or that interesting variations in
interallelic expression are occurring. The direct controls of
studies of expression of polyrorphisms in AF vi11 be the results
of testing cord bloods or aborted material.

5. The A&O Secretor and Lewis Polymorphisms

   Fetuses which are heterozygous or homozygons for the gene
which determines secretion of ABH substances (Se) secrete these
soluble blood group antigens which appear in AP (5,6). The
secretor type of the fetus and the ABO type of secretor + fetuses
can be detected by studying the AF in early gestation free of
cells, for hemagglutination inhibition of appropriate detector
systems.  Inhibition by AP of A,B, or H hemagglutinins indicates
that one (or tuo) of these blood group substances have been
secreted by the fetus.  In this case the fetus is secretor t, and
his ABO type is determined directly by specific inhibition of the
A,B or ti heragglutinia. If the AF does not inhibit agglutination
the fetus is a non-secretor and the ABO type cannot be
determined.  About 25% of Caucasian fetuses will be non-secretors
(20) o

   Wet are already determining in our laboratory secretor status
and ABO types of ABH secretor + fetuses. Al? is serially diluted
for nine or ten doubling dilutions in three series of tubes. In
one series of tubes the AF dilutions (including undiluted AF) due
incubated at room temperatures for 1 hour with an egual volume of
single donor,  non-commercial, anti-A hemagglutinin. In the second
series of tubes tht? AF dilutions are irlcubated with single donor,
non-commercial, anti-B and in the third with anti-h (Ulex
europaeus), A single dilution of each hemagglutinin is used


throughout the determination, this being determined from the
anti-A or anti-B titer of the serun; we do not dilute the anti-H
reagent which has a very low titer (commercial preparations or
our own preparation).  After the incubation of A8 and
heeagglutinins, ue add A, B and R erythrocytes to the first,
second and third series of tubes, respectively. Following
thorough mixing of cells and reagents (and AP), the tubes are
centrifuged and are then observed for gross hemagglutination. As
a control for each AF determination, agglutination inhibition is
carried out in a similar manner using salivas containing A, B and
H substance.

   As of the date of preparation of this application, we hdVe
standardized (for our laboratory) the conditions of the
hemagglutination inhibition procedure (e.g. titer of
hemagglutinins, volumes of reagents, incubation times,
centrifugation tines and speeds) and tested five amniotic fluids:

YBEK OF GISTATION  PBTAL TYPE   BECIPBOCAL BEDAGGLUTINATION
                   ABO   SECRETOR   IRHIBITTOB TITER II AF
                                    A    B     H

19  weeks            B           +         0    64   0
17  l/2 weeks       ?                 0     u   0
16  weeks           A           +         8     0    0
14  weeks                           +        16
16  weeks                           +         0

We believe that we vi.11 be able to distinguish between Al and AZ
secretor fetuses on the basis of inhibition of anti-R; A2
individuals secrete nore H substance than do Al individuals (20).
We are continuing to type AF specimens for ABH henagglutination
inhibition and to compare the inhibition titer with that of the
saliva controls. He plan to check the ABO types of secretor +
fetuses by typing cord blood specimens and ABH secretor status
with saliva collected in the newborn period.

   In a manner analogous to the methodology just described,
secretion of Lewis a, Le(a), and Lewis b, Le(b), substances will
be studied (6). Non-secretor fetuses do secrete Le(a) substance.

   We are planning to decrease the amount of serological
reagents and AF used in the ABH and Le typing procedures. He vi.11
attempt to adapt these hemagglutination inhibition tdsts to
tissue typing piates (21) which require only 1 nicroliter each of
AP dilution, antiserum and erythrocytes. These plates can be
"centrifuged n on a serological rotator and hemagglutination
abserved microscopically. Direct reacting (complete) antibodies
must be used in this nicrotechnique. Fortunately, ABH
hemagglutinins and some dntibo3ies to Le(a) and Le(b) do not
require anti-human globulin (Coomb's reagent) to agglutinate
erythrocytcs,

6, Other Polymorphic Systems of Erythrocyte Antigens


-13-

   Recently, the expression of the blood group P has been
detected on cultured cells and interspecific cell hybrids by
complement fixation (22). If this finding can be confirmed, a
technique might be available for detecting the expression of
various blood group polyaorphisas on cultured AF cells. ABO, Rh,
HtlS,  Duffy, P and Xg specificities will be sought on cultured Al
cells by complement fixation. Other serological methods which we
will explore for the detection of these substances on AF cells
include absorption of specific antisera and mixed agglutination.
Fuchs et al.  (23) used the latter technique to detect ABO types
of uncultured AP cells.

   Although there is no indication whatsoever of secretion of
blood group antigens other than ABH and Le substances, (except
for Sd(a), see reference 201,  we shall examine AF for
hemagglutinin inhibition activity for the above mentioned
erythrocyte antigens.

7, The EL-A System

   Ue have been able to type for HL-A antigens cells in the
fifth passage of the fibroblastic euploid culture (7R2R) derived
from AP obtained at 19 l/2 weeks of gestation. Cultured AP cells
grown to confluence in a plastic tissue culture flask (75 square
centimeter growing area) were released from the surface of the
flask with 3 ml of 0.05% trypsin solution. The cells were exposed
to trypsin for approximately five minutes, They were washed and
resuspended in medium PI0 without fetal calf serum; the cell.
concentration of this suspension was lo**6 cells per ml, Yh:,se
cells mete prepared and typed for HL-A antigens in Dr, Rose
Payne's laboratory, Department of Hedicine, Stanford, as follows:
Aliquots of the cell suspension were incubated with fluorasr:ein
diacetate (PIiA 2 micrograms in O-1 ml tissue culture medium; added
to each P ml aliquot of cell suspension) for 30 minutes an6 the
cells were separated from the FDA by centrifugation. Examination
of the FDA-treated,  cultured AF ccl Is under the fluorescertc:,?
microscope revealed masses of fluorescent cells. Aliquots of
l,OOO-2,900 of these cells were then added to approximately 60
HL-A antisera, representing 25 antigenic specificities, on tissue
plates and incubated in the dark at room temperature for 30
minutes. Rabbit serum, absorbed with cultured human fibrohhasts,
was used as a source of complement; an excess of complement uas
added to each well of the tissue typing plate containing cells
and antibodies. Following an incubation of 1-4 hours at room
temperature in the dark,  the cells were observed with a
fluorescence microscope for evidence of cytotoxicity. Cells which
remained fluorescent failed to react with antibodies and thus did
not carry the HL-A antigen which the antibodies detect. Peu+r or
absent fluorescent cell& indicated cytotoxicity mediated by the
antigens recognized by the antibodies with which the cells had
been mixed. Cytotoxicity was graded as 4+ when no fluorescent
cells were seen, 3+ few fluorescent cells, 2+ more fluorescent
cells and lt no decrease in fluorescent cells. This method has
been used by Dr. Payne and her associates to detect HL-A antigens
on cultured cells (24).

P-113


-14-

  Typing of the 7B2B cells by this method revealed HL-A first
series antigens, I&-A9 and R29, and the second series antigen
RL-A3. Ue ace especially confident of the detection of HL-A9;
most of the anti HL-A9 reagents in Dr. Payne's sublist of
antisera reacted with these cells.  Although more than one of the
antisera for W29 and for HL-A7 reacted with the 7B28 cells, there
were some uhich did not. Pe are about to type this AF cell
culture again, now at passage 14. We are especially interested in
the gain or loss of BL-A specificities, especially involving Y29
and HL-All. The genetic structure of the HL-A spstea ( 2 closely
linked loci with a lou frequency of recombination between them,
reference 25) serves as a control for our typing results. If we
are correctly detecting the HL-A phenotype of the fetus, we
should find no aore than two first series and two second series
antigens on the cultured AF cells. Unfortunately, this fetus was
aborted (by hypertonic saline injection), so that we cannot
colapare the UL-A antigenic type of the cultured BP cells with
that of (aborted) fetal tissue or of lymphocytes in cord blood.

   He look forward to studying variation in expression of HL-A
antigens in cultured AF cells. The genetic structure of the
system will permit us to look for variation within sub-series as
well as betueen series (i.e. do the antigens of one sub-series
tend to be expressed more frequently in cultured AF cells?)
Furthermore, we will study the evolution of expression of RL-A
antigens on AP cells throughout the life of the cell culture as
well as at different weeks of gestation. Finally, shifts in
expression of cross-reacting antigens (26) will be sought.

8. Polymorphic Enzyme Karkers of Cultured AF Cells

   The demonstration of the electrophoretic phenotypes of
G-6-PD and 6-PGD in cultured AP cells by Eadler (7) raises the
possibility of detecting other polymorphic enzyme markers. It is
not clear from ladler's report that he considered polymorphism
for G-b-PD as the source of the variation he observed in the
cultured cells frotu different AF samples. blor is it clear that he
studied sufficient saaples to note variation for 6-PGD in
cultured AF cells which he reported as failing to show variation.
Three to eight percent of Caucasian individuals shov variant
6-PGD electcophoretic phenotypas (27). Harris and Hopkinson (28)
have listed some 20 loci determining enzyme structure which have
been found bg electrophoretic studies to be polymorphic in
Europeans,  and G-6-PD can be added to this list for Black
populations. Of these enzyme polymorphisms ue can test extracts
of cultured amniotic fluid cells for seven by starch gel
electrophocesis: phosphoqlucomatase locus 1 (PGRl),
phosphoglucoautase locus 3 (PGM3), adenylate kinase (AK),
adenosine deaminase (ADA), 6-PGD, hexokinase and G-6-PD.
Furthermore, we believe we will be able to develop procedures for
five additional systems: Peptidase A, C and D, glutamate-pyruvate
transaainase (soluble - SGPT) .and galactose-l-phosphate uridyl
transferase (gal-I-P-tfase).


-lS-

   Cultured AF cells will be tested for total enzyme activity
(per unit weight of cell protein) and for the electrophoretic
phenotypes of the systems listed above (many of the procedures
which we will use are provided in references in 28). Quantitative
determinations and qualitative observations will be made
throughout the lifetime of the same AP cell cultures and on
cultivated cells from AF specimens taken at different times in
early gestation.  We will attempt to ascertain all common
phenotypes of each polgmorphisa. Over a three year period we will
probably have had the opportunity to encounter the expressions of
comu~oa alleles for most if not all of these phenotypes,

    Ye shall use standard cell cultures uith reference
phenotypes to control our electrophoretic procedures. These
cultures will be obtained from cell culture repositories (e.g.
The klaemalian Genetic Wutant Cell Repository, Institute for
Hedical Besearch) or from biopsies (after informed consent
procedures) from individuals who have been typed for various
enzyme electrophoretic phenotypes.

9. Applications to Prenatal Diagnosis

   As this study progresses we will become familiar with the
behavior and the limits of variation of expression of various
polymorphic markers in Au.  This information will serve as a base
for using syntenic relationships for prenatal diagnosis. WC
envision a cooperative effort uith a number of other centers
involved in genetic counseling and prenatal diagnostic activities
in finding families who are at risk for having affected fetuses
and to which the methodology discussed herein can be applied for
prenatal diagnosis of those fetuses. Such families will be typed
for the test marker in order t3 determine that one or both (for
autosomal -recessive traits) parents are heterozygons and to work
out the coupling phase.  We will then seek the expression of the
syntenic marker in the AF from those informative and suitable
pregnancies. Certainly, familias uith hereditary disorders seen
at Stanford vi11 be screened for the possibility of applying the
methodology presented in this application, but we believe we will
need access to a larger number of families to apply this prevatal
diagnostic technique.

D. SIGNIFICANCE

   1. This project will enable us to infer information about
well knoun polymorphic markers in the developing fetus. This
includes the development of the expression of these markers in
early antenatal life, The project gives us the opportunity to
find fetal forms of these markers.  By understanding differential
expression of polymorphic alleles in fetal life, we may be able
to make inferences about the nature of selective forces which mRay
be acting on these polpmorphisms.

    2. This project could ultimately result in expanding the
number of hereditary conditions a&enable to prenatal diagnostic
techniques.  This is especially true for autosomal dominant


-16-

disorders for which little information on basic biochemical
mechanisms is available to fashion appropriate techniques for
prenatal detection of the fetal phenotype.


-17-

REFE BENCES

1. Bosnes, R.W., Clin. Obstet, Gynec. 9:440, 1966.

2. Plentl, B.A., Clin. Obstet. Gynec. 9:427, 1966.

3. Huisjes, H.J., Amer. J. Obstet. Gynec. 106:1222, 1970.

4. Nadler, H-L., Adv. Hum, Genet. 3:1, 1972.

5. Harper, P. et al., J. fled, Genet. &:438, 1971.

6. Arcilla, K.B., Sturgeon, P., Pediat. Nes.  6:853, 1972.

7. Nadler, H.L., Biochem. Genet. 2:119, 1968.

8. Horton, R.E., Amer. J. Phys, Anthrop. 28: 191, 1968.

9. Renuick, J.H. et al., J. Med. Genet. 8:407, 1971.

IO, Renuick, J.H., Ann. Rev. Genet. 5:81, 1971.

11. Ruddle, P.H., Adv. Hue. Genet. 3:173, 1972.

12, Bayer, S-R., Graham, J.B., Amer. J. Hum. Genet. 17:320,
1965.

13. Adam, A. et al,, Lancet i:877, 1966.

14. Fialkou, P.J., Giblett, E.R., Hotulsky, A.G., Amer. J. Hum.
Genet.  19:63, 1967.

15. Ruddle, F.H., Birth Defects: Original Article Series 3:188,
1973.

16. Westerveld, A., Kahn, P.R., Nature 236:30, 19'12.

17. Cavalli-Sforza, L.L., Amer. J. Hue. Genet. 25382, 1973.

18, Lee, C.L.Y., Gregson, N.H., iJalker, S., Lancet ii:31b, 197$i.

19. Pearson, P.L., Bobrou, H,, Vosa, C.G., Nature 226:78, 1970.

20, Race, R.R., Sanger, R.,  Blood Groups in tian (5th ea.),
Blackwell, i)xtford, 1968.

21. Bodmec, W.F., Tripp, M., Bodmer, J.G., Uistocompatibility
Testing, Runksgaard, Copenhagen, 1967.

22. Felloos, !I. et al., 4th Internat. Congress Hum. Genet.,
Abstracts,  Excerpta Redica Intern. Congr. Ser. No. 233, page bb,
1971.


-It)-

23. Fuchs, F. et al., Lancet i:996, 1956.

24. Brautbar, C., Payne, R., Hayflick, L., Exper. Cell Res.
75:31, 1972.

25. Bodaer, W.F., Bodaer, J-G., Tripp, R!., Histocompatibility
Testing, tinnksgaard, Copenhagen, 1970.

26. Mittal, K.K., Terasaki, P.I., Tissue Antigens 2:94, 1971.

27. Giblett, E.R., Genetic )larkers in Blood, Davis,
Philadelphia, 1969.

28. Harris, f-f., Hopkinson, D.A., Ann. Hum. Genet., London 36:Y,
1972.


EGED COMMUNICATION                 SECTlClN It

SUBSTlfUTE TWIS PAtF FOR DFTAILFO Allnt.ET Pdr.1

,__. _----- --                            ---..-.. . .      --- -... -. - . . ..- _.-- -.. --.-.--- _--__, _",

          SUBSTITUTE                          PERIOD COVERED           GRANT NUMBER

                                        FROM
)ETAILED BUDGET FOR FIRST 12.MONTH PERIOD                   THROUGH
                        l/1/74       12/31/74

PERSONNEL (List all perso~el endaged 0" project)                TIME OR
                                                 EFFORT        AMOUNT RECUESTEO fOrnit cents)
   NAME (Lad. first, initial)          TITLE OF POSITION       A/HRS.            I     tOTAL

Cann, H.             Principal Investigator or
                    Program Director        20%
Tsuboi, K.           Senior Scientist    20%    PoL'fbfORPHIC GENETIC MARKBRS
                            '..' ,,
                                                           :     . .
Van West, B.      Research Assistant 100% '- '."
Makk, G.         Research Assistant 100% ./ "

Open - Pediatrics

Cusumano, M.
Murray, R.

                                
Research Assistant 50% ;.I i, :
Laboratory Diener    50%     
                       .I.;       . . .

Secretary         25x  : .j: f .:
                           :.:    

                   1. ,' I:..:
                            6'.
                             . . . : : . .
                             I
                   ',::.;.

TOTAL     R ' 52,330

, CONSULTANT COSTS (Include Feoa and Travel)


. EQUIPMENT (Itemize)
Eppendorf Microfuge and accessories   $500
Buchler Power Supply (constant voltage/current) $600

* SUPPLIES
Chemicals, antisera, tissue culture supplies, glassware, etc.

s

S  1,100 *


5,000

S

.
  STAFF     a. DDMESTIC  1 East Coast Meeting
TRAVEL t
sea xn8tNsrions~ b. FORE, GN

. PATIENT COSTS (Separate Inpatienf md Outprtlenf)

S   500

S



S

. ALTERATIONS AND RENOVATIONS                                                         S
. OTHER EXPENSES (Xlwni;e per Inrt~crlon~
Office supplies, telephone, repro., postage, publication costs, etc.$400
Central computer usage and terminal rental                $1600



                                                                           S  2,000


.                                         Subtotal - Items 1 thru 8         c ,I 60,930

        10. TRAINEE EXPENSES f&e Instnrctions)

                  PREDOCTORAL              No. Proposed           s

FOR . a STIPENDS P0ST00CT0RAL              No. Proposed           5
                 OTHER (Specify)              No. Proposed           S
TRAINING            DEPENDENCV ALLOWANCE                            S
                                .
GRANTS                              TOTAL STIPEND EXPENSES

        b. TUITION AND FEES
ONLY ,
        e. TRAINEE TRAVE', (Deari&)

         11.                             Subtotal - Trainee Exponrer

Ii. TOTAL DIRECT COST (Add Subrorala, Irema 9 uld II. and enter on Pale 1)

c s


    S

    S


+S

* s 60,930

h . . .

S&rtitutp Budget Pop. %72
c-m c--, PUC 20s --A 0oC uoo.1

f--/l ;r

GPO 933.:i'


SECTION II -PRIVILEGED COMMUNICATION

POLYMORPHIC GENETIC MARKERS

BUDGET ESTIMATES FOR ALL YEARS OF SUPPORT REQUESTED FROM PUBLIC HEALTH SERVICE
               DIRECT COSTS ONLY (Omit Cents)

   DESCRIPTION



PERSONNEL
COSTS

ST PERIOD
(SAME AS DE.  ADDITIONAL YEARS SUPPORT REQUESTED (Thisapplication on/y/
TAILED BUDGET)  2ND YEAR    3RD YEAR    4TH YEAR    5TH YEAR    6TH YEAR    7TH YEAR


52,330   56,035   59,897  64,022   68,424

CONSULTANT COSTS
(Include fees, travel, etc.)

EOUIPMENT         1,100*



SUPPLIES          5,000    5,300    5,700   6,000    6,400




            DOMESTIC     500      600      600     600      700
TRAVEL
            FOREIGN

PATIENT COSTS

ALTERATIONS AND
RENOVATIONS

OTHER EXPENSES       2,000    2,100    2,300   2,500    2,600

TOTAL DIRECT COSTS    60,930   64,035   68,497  73,122   78,124

TOTAL FOR ENTIRE PROPOSED PROJECT PERIOD (Enteron Pege 1, Item 41  -   s  344,708

I

REMARKS: Justify all costs for the first year for which the need may nor be obvious For future years. justify equipment costs, JS well JS any
significant increases in any other CJtJgOfy. If e recurring annual increase in personnel coets is requested, give percentage (Use conbnuarion
page if needed. I

Budge't explanation attached.


BUDGET EXPLAYATIOI'

  Professor Cam's and Dr. Tsuboi's time are each budgetted dt
20% in support of this project.  The project also requires two
full time and one part time research assistants. The part time
research assistant will assist in typing cultured cells from
amniotic fluid for W-A antigens. One full time assistant (firs
Van West) will assist in the development of serologic tests on
amniotic fluid and amniotic fluid cells. The other research
assistant (6, tlakk) will assist in culturing amniotic fluid cells
and in performing electrophoresis on cultured cell extracts. The
laboratory diener uill provide the needed support for cell
culture work ( washing, sterilization of equipment, etc) and also
for general laboratory uork. We are also requesting 25% of
secretarial time to support the activities of both professionals
in this project.

  Salaries are increased at a rate of 6% per year to cover
merit and cost of living increases. Staff benefits are applied
based on the following University projections: 17X, g/73-8/74;
18.3%. 9/n-e/75;  19.3%. g/75-8/76; 20.3%. 9/76-a/77; 21.3X,
9/77-a/78; and 22.31, Y/78-8/79.

  The microfuge will be use3 to centrifuge small dBOUntS of
cultured cells. The power pack will be used for electrophoresis.
Mecessarp supplies are budgetted to support laboratory work
including chemicals, glassware, antisera, etc.

  We are requesting $100 per month for computer time and data
file storage and $400 per year for our share of the rental of a
computer terminal. Access to c3aputation is necessary for
cataloging of cultured cells from AF specimens, automatic filing
and storage of test results on various cell cultures and amniotic
fluid samples, filing and storage of data on phenotypes of
parents, rela,ting parental data to fetal data, monitoring lists
of expected dates of delivery of fetuses ue have studied and data
analysis.

P-/l/


SECTION V

A Search for Genetic Polymorphisms and Variances
   Of Specific Binding Proteins in Blood

Dr. Cavalli-Sforza


Genetic Polyaorphisns and Variants
of Specific Binding Proteins in Blood

Dr. L. L. Cavalli-Sforza, Principal Investigator

A.  IRTRODUCTIOR

A.1 Objectire

   To search for neu medically significant genetic variants and
genetic polymorphisms of specific binding proteins in blood.

A. 2 Background and Bationale

   The recognition of yenetil" differences by electrophoretlc
studies of proteins is by now a widely applied procedure, nethods
employed for detectiny proteins after electrophoresis range from
unspecific Protein staining to the identification of specific
proteins by a great variety of techniques. Aarris and Hopkinson
(1972) have recently summarized the evidence collected in
electrophoretic studies of specific enzymes in Caucasians. Out of
7 enzymes analyzed, about l/3 were found to be polymorphic.
Certain other categories of enzymes may be subject to a lower
rate of polymorphism (Oaenn, Cohen and Rotnlsky, 1971).

   One method of labeling a variety of proteins is already
establ,ished but has not been tested systematically for its
capacity to identify polymorphisms or rare variants. It consxts
of adding to serum (or other biological fluids) a radioactive
ligand molecule and following its binding to a specific protein
(or proteins) by electrophoresis followed by autoradiography.
This method was used (Giblett, Rickman and Smithies, 1959) to
amplify the classical notion that transferrin binds iron and that
electroyhorntic variants of this molecule retain the capacity to
bind iron. The physiological function of this protein is fairly
well known and its important role in the organism uell
ascertained {for details see Giblett, 1969). Polymorphism is well
documented and many rare variants are also known. Other proteins
known to specifically bind metals (e.g. copper: ceruloplasmin,
see Giblett for details) and other substances (e.g. thyroxine;
see Heinonen et al., fialkow et al., and Penfold et al.) have
also been studied.

   The sugyestion advanced in the present application is to
test systematically samples of human blood for proteins binding
specific substances, taking advantage of the fact that many
physiologically important ligands are available in a radioactive
form. Two aims could be thus obtained: 1) increase the present
uealth of polymorphisms, a very desirable aim (see
Cavalli-Sforza, 1973);  2) a functional basis for each specific
polymorphism or rare variant conld be sought, given that the
nature of the substance would usually suggest possible advantages
or handicaps of the variants.

p -/`;z :


-2-

B. SPECIFIC AIdS

   (1) Search for polymorphisms in proteins binding specific
substances available in radioactive form. This may enable us to
look for a large number of new polymorphisms, allowing us to
considerably increase the genetic map and to find new linkages.

   (2) In all cases in which polymorphisms are .detected, test
for developmental behavior of the proteins in question, and for
the chemical specificity of the ligand.

   (3) In cases of toxicity or idiosyncratic drug reaction,
test for variation or absence of proteins binding the specific
poison or drug.

C.  KETLiODS AllD PROCRDURE

  The experimental methodology has already been developed on
the basis of experience accumulated in another research line. In
the analysis of platelet proteins binding certain
neurotransmftters (in particular, serotonin and norepinephrine) a
method of electrophoresis followed by autoradiography was set op.
In order to test the method for its capacity to reveal serum
proteins binding specific substances, it was applied to
lead-binding proteins. A short description of the method follows:
Pb(210) at an appropriate concentration is incubated at 37
degrees C.  for 30 minutes with human serum. 0.1 ml of the
incubated serum is electrophoresed on acrylanide gel. Bromophenol
blue is used as a tracking dye and electrophoresis stopped when
the dye is at the bottom of the column. (The procedure can be
used both on columns and on s13b gels). At the end of
electrophotesis, the gel column is cut longitudinally and the
flat surface applied on an X-ray Kodak film for 4-10 days in
conditions guaranteeing close adhesion and maximum efficiency of
arrival to the plate of the electrons emitted by the decaying
radioactive atoms. A diagram of a developed autoradiogram is
shovn in Figure 1.

   Four different individuals are shown. There is considerable
binding of l?b to albumin which forms the large thick band.
Albumin is known to have a high affinity for many substances. In
addition, there are other lighter bands of Pb-binding proteins in
the globulin region. Some of these correspond to thin protein
bands visible by staining. Tuo proteins in the beta globulin
region have been comstantlp found in all individuals tested so
far. In the gel at the extreme left, two bands are visible near
the origin (the top of the figure) while there is only one band
at the same location in the other three individuals. The double
band may be due to heterozygosis but so far only one individual
with a double band has been found on the limited number of
individuals tested. It is planned to continue the testing on at
least a hundred normal individuals, to establish if a
polymorphism really exists,  to test the families of individuals
showing electrophoretic differences, and to test all cases of
plumbism which may come to our attention by the cooperation of


-3-

the hospital staff. Some of these individuals may be
idiosyncratically sensitive to lead; or, alternatively, may show
physiological responses to lead intoxication. Other disease
states suspected of affecting plasma protein distributions will
also be examined.

    The extension of this uork to substances other than lead is
the subject of the present grant application, The substances to
be tested for binding to serum proteins can be many, the main
limitation being that the organic substances must be labelled
with C(14) or otherwise produce beta radiation of similar energy.
Pb emits also gaana-radiation, so that exposed plates must be
shielded; this, however,  is no serious liaitation since contact
of the experimenter with the radioactive material is very short
and his or her exposure controlled by the supervision ordinarily
carried out in the laboratory (film badges, routine checks of
benches and equipment). Results with h(3) labelled material have
so far been unsatisfactory due to the short range of the weak
electrons eritted in E(3) decay. C(14) labelled material is
eatirely satisfactory, as shoun by the previous experience with
platelets.

    A shortened list of the substances to be tested includes the
following:

(1) Elements which hava radioactive isotopes suitable for
the test.

(2) Amino acids, peptides

(3) Uucleotides, nucleosides

(0) Sugars

(5) Lipids

(6) tiormones

(7) Vitamins

W Qruw, including antibiotics, addiction drugs,
pesticides, poisons, etc.

   The analysis should be carried out initially on a sample of
at least one hundred random iniividuals. Over 100 substances can
be tested, but each test requires only 0.1 ml of serum and thus
20-30 ml of blood obtained from an individual will be enough for
all tests. On the assumption that one or more proteins binding
specifically a substance are found for most of these substances,
this experiment should give a good sample of prospective
polynorphisrs from which the frequency of polymorphism and
average heterozygosity for each can be computed and compared with
that observed for enzymes (see Introduction).


-Y-

  Substances found to hdVe polymorphic binding proteins cdn
then be subject to the following series of observations:

  1) Tests on families scored for other markers uhicb have
already been collected in other laboratories. Prospective
collaborations are being considered. It is expected (but should
be first tested) that in serum stored in freezers the specific
binding activity is stable. The existence of a number of projects
in which blood samples have been collected from families,
examined and stored makes it easier and more efficient to test on
such material inheritance of the protein differences (i.e.
segregation analysis) and linkage of the corresponding genes to
standard markers. Several such collections of samples are already
available.

  2) Ye plan to examine newborn infants born at Stanford
Hospital of matings in which the mother is honozygous for d
polymorphic protein of the type described, and the father
heterozygous (or hosozygous for another allele). The paternal
protein would be searched in cord blood and if not present, the
child uould be followed further to establish the age of
appearance of the paternal protein. This would give us a chance
to seek regulatory genes for the developmental pattern of these
proteins.  P3r instance, we will seek variation among individuals
of age of appearance of the protein and analyze the variation
with family studies.

   3) For every specific substance, patients with diseases that
may be explained by a variation or absence of a binding protein,
the specific substance should be examined.       .

D, SIGNIPICAYCE

  It is difficult to anticipate the total number of proteins
that can be identified by this procedure, hut existing
information would suggest that it can be as high as several
hundred. The method suggested then supplies a very economical
procedure for testing a great number of potential polymorphisms.
The frequency of polymorphic genes is one of the quantities which
is of interest to estimate for comparison with the existing
enzyme data. This result has obvious evolutionary significance in
view of the present discussion on neutrality of polymorphic
genes. If the proportion is the same as is known to be among
enzymes, then this investigation may generate enough markers to
more than double the existing genetic map of man, with all
consequent advantages of increased precision in genetic
counseling and research.

  The interest offered by such new polymorphisms would be
greatly enhanced by the possibility of detecting variation for
regulatory genes in the manner explained before. This is one of
the most d.ifficult fields in human general genetics today, the
development of which may be most fruitful.


-4-

Substances found to have polymorphic binding proteins can

then be subject to the following series of observations:

   1) Tests on families scored for other markers uhicb have
already been collected in other laboratories. Prospective
collaborations are being considered. It is expected (but should
be first tested) that in serum stored in freezers the specific
binding activity is stable. The existence of a number of projects
in which blood samples have been collected from families,
examined an3 stored makes it easier and more efficient to test on
such material inheritance of the protein differences (i.e.
segregation analysis) and linkage of the corresponding genes to
standard markers.  Several such collections of samples are already
available.

   2) Ye plan to examine newborn infants born at Stanford
Hospital of matings in uhich the mother is homozygous for a
polymorphic protein of the type described, and the father
heterozygous (or homozygous for another allele). The paternal
protein uould be searched in cord blood and if not present, the
child uould be followed further: to establish the age of
appearance of the paternal protein.  This uould give us a chance
to seek regulatory genes for the developaental pattern of these
proteins.  F9r instance, we will seek variation among individuals
of age of appearance of the protein and analyze the variation
with family studies.

   3) For every specific substance,  patients with diseases that
ray be explained by a variation or absence of a binding protein,
the specific substance should be examined.       .

D. SfGhIFXCIYCE

   It is difficult to anticipate the total number of proteins
that can be identified b,y this procedure, but existing
information uould suggest that it can be as high as several
hundred. The method suggested then supplies a very economical
procedure for testing a great number of potential polymorphisms.
The frequency of polymorphic genes is one of the guantities uhich
is of interest to estimate for comparison with the existing
enzyme data. This result has obvious evolutionary significance in
view of the present discussion on neutrality of polymorphic
genes. If the proportion is tha same as is known to be among
enzymes, then this investigation may generate enough markers to
more than double the existing genetic map of man. with all
consequent advantages of increased precision in genetic
counseling and research.

   The interest offered by such new polymorphisms would be
greatly enhanced by the possibility of detecting variation for
regulatory genes in the manner explained before. This is one of
the most difficult fields in human general genetics today, the
development of which may be most fruitful.

P-126


-5-

   Finally, each and every one of the proteins thus detected
and identified may offer unique possibilities of further research
and therapeutic developments. Taking again the model of
transferrin,  there is one well known case of congenital absence
of this protein which was lethal (Reilmeyer et al., 1961). III
similar cases, substitutional therapy by transfusion or plasma
infusions may prove life saving. Several dangerous rare drug
idiosyncrasies are kooun to exist, e.g. to chloraaphenicol.
Should they prove to be connected to the lack of a specific
binding protein, transfusion or plasma infusions may again prove
useful or at least these,patients could be identified before
becoming the victims of the administration of a drug potentially
lethal for them. Cases of vitamin or hormone resistance might
find similarly an unexpected explanation and therapeutic benefit,


-6-

REFEREYC ES

Caralli-Sfnrza, L.L. 1973, Some current problems of human
population genetics. Am. J. Hum. Gen. 25:82-104.

Giblet, E.R.  1969. Genetic Markers in Huaan Blood. Blackwell
Scientific Publications, Oxford.

Giblett, E-B., Bickran, C.G. and Smithies, 0. 1959. Serum
transferrins.  Rature 183:1589.

Harris, II. and Hopkinson, D.A.  1372. Average heterozygosity per
locus in man:  an estimate based on the incidence of enzyae
polymorphisms. Ann. Hua. Genet. 36:9-20.

Omenn, G.S., Cohen, P-T, and Hotulsky, A-G. 1971. Genetic
variation in glycolytic enzymes in human brain (abstr.).
Excerpta Bedica Int. Cong. Ser. Yo. 233:135.

Xeinonen, 0-P.. Lanberg, B.A., Virtaao, J, 19'10. Inherited
decrease of the binding capacity of thyroxine-binding globulin
(TBG). Acta Endocrinologica 64:171-180.

Pialkow, P.J., Giblett, E.R. nnd Husa, B. 1970. Increased serum
thyroxine-binding globulin capacity: inheritance and linkage
relationships. J. Clin. Endo. E hetab. 30:66-75.

Penfold, J.L., Kneebone, G.B., Welby, tl., Oldfield, B-K, 1971.
Growth retardation and thyroxine-binding globulin deficiency.
Arch. Dis.  in Childhood 46:115-117.


I
j .:
,
I
i
I
!


        ~"".,.."I..~-. I",.                   *Lb I I",. II      avua 1 I I" I L I r1,a I ClVL r "R YL I mmLLY ""VVC t 13-v:

         SUBSTITUTE                          PERIOD COVERED           GRANT NUMBER
IETAILED BUDGET FOR FIRST 12.MONTH PERIOD -O;,l,,4        THROUGH
                                12131174

PERSONNEL (List all personnel en9eged on project)
   NAME (Last, first, initfal)          TITLE OF POSITION

CavalUrSforza, L.       Principal Investigator or
                    Program Director

TIME OR
EFFORT       AMOUNT REQUESTED (Omit cents)

%/HRS.           I     TOTAL
                    ,      >.;

10%           .'.

open - Genetics         Research Associate  100% ';;.~PCLYMCRPRISMSS OF.SPRCIFIC
                                                        ,.:
Open - Genetics         Research Tech.    100% .'.j,' :  ~smimc PRCTRINS
                                                       : +;,,:.
                                                       ..:I ,..: j :,.,.   :.:          . .
                                                               .:.
                                                          . .    I.
                                                       1: i.     .:
                                       "".S :;:.: .   .., ;   . . '..   .:.::,   . . .::.,
                                                                           .: 1
                                                                        
                                                                  . . .                     
                                                       ; .:: .: :,.., . .      .I...      ::..    ,. ::. I
                                                                           ..:
                                                        . . .:.:....
                                                        " ::..               /
                                                       .1..::               : .."
                                                         .:::          :. :.  :.
                                                                           . .
                                                         .j            "'.
                                                                 :
                                                       :... :':'            
                                                       :.:    /
                                                          .;.:       . . .            
                                                        '.' :..       '..'. .,    :

                                                       .::
                                                       :. :               .
                                                                           .   :

TOTAL          *s  30,489

CONSULTANT COSTS (Include Fees and iravel)


EQUIPMENT (Itemize)   Constant current power supply   $1,000
          Slab gel electrophoresis          300
          Column acrylamide gel electrophor. 200
              Destainer                      200
SUPPLIES
Radioactive tracers       $5,000
Chemicals, glassware, lab app. 1,000
Expendable lab supplies,     1,500 (photographic sunplies. etc.1

I








S    1.700*






s   7.500

  STAFF     0. DOMESTIC  2 East coast meetings
TRAVEL .
:ee rn*tJuctfons) b. FORE,GN
PATIENT COSTS (Separate Inpetient md Outpatient)
Venapuncture for blood samples
ALTERATIONS AND RENOVATIONS


OTHER EXPENSES (Itemize per in&uctiond

S   1,000

S



S     500


S

Office supplies, telephone, repro., postage, publications costs, etc. 1000
Central computer usage                                   1000



                                                                           S   2,000

                                       Subtotal - Items 1 thru 8         * s 43,189

       10. TRAINEE EXPENSES (See Instnrctions)

                  PREDOCTORAL              No. Proposed           s

FOR . o sT,pENDS POSTDOCTORAL              No. Proposed           S

                OTHER (Specify)             No. Proposed           5
rRAlNlNG            DEPENDENCY ALLOWANCE                            S

GRANTS                          .          TOTAL JTIPEND EXPENSES - S

        b. TUITION AND FEES                                                         S
ONLY .
       C. TRAINEE TRAVE'- (Dercrlbe)                                                   S


         11.                              Subtotal - Trainee Erpmses                *S

2. TOTAL DIRECT COST (Add Subtotala, Items 9 md 11, and enter on Pa&?= 1)           c ' 43,189

,. _.__ ,a. .I    - --                                                         n r.-

kP~rmm rruagor rag* ~--II
For Forms PHJ 398 and PHS 2499-l

r-/Y v        GPO 930.731


SECTION 11- PRlVlLEGED COMMUNICATION       POLYMORPHISMS OF SPECIFIC BINDING PROTEINS

I     BUDGET ESTIMATES FOR ALL YEARS OF SUPPORT REQUESTEO FROM PUBLIC HEALTH SERVICE
                   DIRECT COSTS ONLY (Omit Cents)

CONSULTANT COSTS
(Include fees, travel, etc.)

EQUIPMENT

I  1,700* I    500*

SUPPLIES

I I
7,500    8,000 8,600 1 9,000 1 9,600 1

           DOMESTIC   1,000    1,100
TRAVEL
            FOREIGN

PATIENT COSTS  I I
                  500      500

ALTERATIONS AND
RENOVATIONS

OTHER EXPENSES  I  2,000 I  2,300

TOTAL DIRECT COSTS 1 43,189 1 45,048.

TOTAL FOR ENTIRE PROPOSED PROJECT PERIOD (Enter{

REMARKS: Justify all costs for the first year for which the nea
sbnifimnt increak in any other category. If a recurring annual
page if needed.)

Budget explanation attached

YEARS SUPPORT REQUESTED (Thisapplication only)
3RDYEAR 1  4THYEAR 1  5TH YEAR 1  6THYEAR  1  7Tt-i YEAR

34,898  37,301   39,867

500*

       I                      I
1,200   1,200    1,300

(,-I---

-2,400 / 2,400 1 2,700 )

may not be obvious For future years, justify equipment costs, as well as any
ncrease in personnel costs is requested, give percentage.  (Use continuation

I
PHS398
Rw. &70

           L
P-/.2/


BUDGET EXPLAHATION

   10% of Professor Cavalli-Sforza's time along with a
full-time Research Associate and a full-time Research Technician
are budgetted in support of this project. The Research Associate,
a biochemical geneticist, will be responsible for the
electrophoretic analysis of plasma proteins and will be assisted
by the Research Technician.

   Salaries are increased at a rate of 6% per year to cover
aerit and cost of living increases. Staff benefits are applied
based on the following University projections: 17X, Y/73-8/74;
18.3%. g/74-0/75; 19.3X, g/75-8/76;  20.31, 9/76-8/77; 21.3X,
g/77-8/78; and 22.3X, g/78-8/79,

   The budget includes slab qel and column gel electroyhoresis
equipment, and associated pouer supply, etc., as well as
supporting supplies. These supplies include radioactive tracers,
chemicals and laboratory apparatus, glassware, and expendable
supplies such as photographic plates, etc.

   Travel funds are requested for attending two professional
meetings on the east coast.

   Patient costs covering venepuncture to obtain blood samples,
are estimated at $500 per year.

P-/3;?


SECTION VI

The Impact of Genetic Counseling Practices on
    Family Decisions and Behavior

Drs. Barnett, Cann, and Luzzatti


The Impact of Genetic Counseling Practices
   on Paaily Decisions and Behavior

    Dr. C.R. Barnett, Principal Investigator
Drs,  R. Cann and L, Lozzstti,  Associate Investigators

A. INTRODDCTION

A.1 Objectives

   The overall objective of this study is to provide systematic
answers to some of the basic, unanswered questions in the
practice of genetic counseling.  (1) Yhat is the impact of genetic
counseling, that is, do families not receiving genetic coanseling
make decisions different from those who do? (2) What is the
difference in counseling effectiveness betueen a physician
trained in genetic counseling and a social worker trained in
genetics? (3) What is the difference in effectiveness between a
counselor who is directive in his counseling and one who
maintains a neutral stance? (4) Uhat is the relationship between
the structure and content of a genetic counseling session and the
pre-counseling training and attitude of the counselor'? (5) What
is the difference in effectiveness between a counselor who
receives social and psychological information about the family
before counseling and one uho 3oes not have such information? (6)
What are the expectations of farilies seeking counseling and how
do they use the information they obtain in making decisions?

A.2 Background

   A recent revieu of the social aspects of human genetics (1)
and an editorial on genetic counseling in the Neu England Journal
of Medicine (2) have consisted largely of lists of questions
regarding genetic counseling for uhich there are as yet no
answers. While much has been learned over the years regarding the
genetic basis for many diseases, their mode of inheritance and
the probability of occurrence in a given population, little
research attention has been paid to how this information is
transmitted to patients and the use they make of it. Typical of
the state of the art and the still prevailing emphasis on
agenetic prognosis", rather than "genetic counseling", is a
recent textbook on genetic counseling (3) which devotes only 3 of
its 355 pages to the counselor-patient relationship.

   The major issues in the field may be subsumed under three
basic questions:  1) What is or should be the impact of
counseling ? 2) What should be the counselor*s role? 3) Who should
do the counseling?

   The first question is most difficult to answer at this time
since there are little data available on the impact of
counseling.  A few studies have looked at impact by measuring the:
number of children families have had post-counseling, or by
learning of their post-counseling decision to practice or change

p-134


-2-

their aethois of contraception.  With regard to the findings from
such studies,  Hecht and Holmes (2) have noted: @*What is the
objective of genetic counseling ? If it is to lessen the chance of
subsequent affected sibs being born, the available data are
discouraging .I) One of the major problems with studies which have
reported somewhat favorable results (4) is that they have not
utilized control groups. The only study that used a control group
(made up of faailies uith children affected with a non-genetic,
chronic condition) reported that SO% of the control group decided
to limit the size of their families, in the absence of any
genetic counseling to do so (5).  Studies to measure the effects
of counseling have also used a number of other outcome criteria,
such as knouledge of probability or risk and information about
hereditary transmission of traits that was retained by the
family. There are two major deficiencies in these studies, First,
with one exception (6) none of the studies have made an
assessment of knowledge before the counseling took place. Indeed,
in some cases, the follow-up of knowledge retained by the
families was as long as 4 to 10 years after counseling (4,7),
uith no control or assessment of the effect of other sources of
information on the families.

   A second deficiency with the studies which have tested
post-counseling risk and genetic knowledge of families, is that
there is no indication, from the point of view of families, that
biological knowledge and inforaation regarding risk is used by
them in making decisions about reproduction, The actual
decision-making process in the family has remained an unopened
Rblack boxa.

   The second question regarding the counselor*s role involves
sharp differences of opinion on two issues: the question of
whether the counselor should be neutral or directive; and whether
the counseling should be narrowly focused or broadly
comprehensive.  The traditional neutral stance is most often
associated with the focused role prescribed for the counselor:
*It can be argued that a counselor's job is simply to
estimate.... risk as uell as possible and try to ensure that this
is understood.  This is, of course, true. It is entirely a matter
for parents to decide whether to avoid having further children,
or to seek sterilization or termination of pregnancyn (3). Yhen a
genetic counselor feels called upon to violate this principle of
neutrality,  he makes a point of explaining the deviation, as does
Carter (4),  in order to reassure low risk parents. There are two
untested assumptions in the argument presented by experts on both
sides of this controversy. The first assumption is that a neutral
counselor will not communicate unconsciously by his tone of
voice, mode of presentation or non-verbal cues, his true feelings
about what decision a family should make. Secondly, it is assumed
that presentation of his feelings of "what he would do if he were
in their shoes" will have a marked effect on the faiPily*s
decision. These assumptions can and should be tested, since
findings nay suggest that the argument for either position is
irrelevant to the outcome of counseling.

P-135


-3-

   The issue of whether genetic counseling should be narrowly
focused or broadly comprehensive has been linked to the question
of who should do the counseling. Thus those who have taken the
position that genetic counseling should be considered part of
family guidance, have argued that the family physician can best
play this role (8). Those vho argue that the priaary purpose of
genetic counseling is to answer questions the patient may have
about risk, feel that counseling should be left to the clinical
geneticist.  Franz Kallman (9),  uho favors a comprehensive
approach has phrased the question most realistically by
suggesting the type of training needed by the counselor: "There
can be no qaestion .,.-that the constructive management of genetic
family problems requires either geneticists uho are experienced
in counseling techniques or family guidance workers who have
adequate training in genetics*'.

   The issue of whether to take a narrow or a brodd approach to
counseling could be settled if information were available
regarding what problems they may acquire as a result of the
counseling. At present, genetic counseling has been not subject
to the same types of analysis that have been brought to bear on
the physician-patient relationship in other situations (lo),
Thus, what happens during genetic counseling has been described
only in anecdotal form (1).

   The question of whether the geneticist, the family
counselor, the family physician or any other type of professional
or lay couaselor can best meet the needs of the family seeking
genetic counseling can be determined by systematic evaluation of
what these people actually do in counseling and what impact they
have on the families. Uhen new roles have been established in
other areas of care delivery,  the behavior and effectiveness of
people taking the neu roles have been evaluated (11). There is no
reason uhy the same approach cannot be taken with regard to this
issue in genetic counseling,

A.3 Rationale

   Genetic counseling involves at least two parties, the
counselor and the family, and both parties to the event must be
studied, as well as the event (counseling) itself to determine
the effectiveness of genetic counseling under varying conditions.

   The counselor vi11 bring to counseling his expectations
about the nature of the coanselin9 situation, and a predetermined
view of the risk and burden a defect may represent for the
family. He may decide, beforehand, to communicate an optimistic,
pessimistic or neutral point of view to the family. His upublicH
position map vary from his "privately'* held view. Ke may, if he
is supplies uith additional information about the family, (their
state of knowledge, their values regarding having children,
differences between husband an3 uife on basic issues, other
decisions they are in the procsss of making, and their
expectations regarding counseling), tailor the information and
counseling he provides to the specific needs of the family.


-4-

   The family,  as noted above, may have expectations regarding
counseling that are at variance with those of the counselor. They
also come to counseling with a state of knowledge about the risk
of having a child with a defect, the burden it represents, the
genetic and biological principles underlying the defect and the
basis fot computing risk. Further, in their own fanily
decision-making experience they may make great or little use of
probabilities in coning to decisions. The fattily also comes with
a set of values or attitudes regarding what they want as
individuals and as members of a family unit, and these values
also determine the kind of information they seek and how they use
the information. Families will vary even with regard to the
number of sources of information they use, so that for some, the
genetic counselor may be the principal source, while for others,
the genetic counselor may be one among many.

   The counseling session represents an interaction between
these two parties and no matter what the prior expectations on
either side, the event say differ from what the two parties
believe will happen, and after the event, what they think
happened. Thus, the event itself aust be studied and compared
both witb prior expectations and with post-counseling
recollections. Did the counselor consciously or unconsciously
break his stance of neutrality,  and uas it noted by the family?
Yas the family so immersed in absorbing the information about
burden and prognosis that they recollected little about the risk
information given by the counselor?

   The expectations of both parties in genetic counseling
provides two measures of effectiveness of counseling, rather than
the single measure (goals of the counselor) which has been used
up to nou. Information obtained prior to counseling about the
values, knouledge, and decision states of the family, as well as
their expectations may enable the counselor to satisfy both his
and the family's expectations.

   An ideal design for answering the basic questions regarding
genetic counseling should satisfy the canons of experimental
design even though the issues are basically behavioral and
social. The study would be prospective in that it measures the
status of the parties before the counseling takes place and then
measures changes following counseling, against the pre-counseling
base (6). It should randomly assign counselees to varying types
of counselors (such as a physician or a social worker), and to
counselors uho have di-fferent types of information available to
them about the family before counseling. Finally, control groups
should be utilized to control for both the effects of the
research contacts on the family,  ds vell as a control group which
does not receive genetic counseling, but say also make decisions
about having children.

B. SPECIFIC AIHS

   1. To test the hypothesis that genetic counseling can be
done at least as effectively by a social worker with some

?-i37


-5-

training in clinical genetics as by an fl,D. trained in clinical
genetics.

   2. To test the hypothesis that genetic counselors, even when
holding consciously to the principle of "neutrality," will
divulge their ntrueM feelings to their counselees.

   3. To test the hypothesis that counselors who are informed
prior to counseling regarding the values, knouledge, decision
status and counseling expectations of the counselees will be more
effective than counselors who are not so informed.

    4, To develop measures for determining the effectiveness of
genetic counseling which utilize the goals of the counselees, as
veil as the objectives of the counselors.

   5. To learn how families utilize information provided in
genetic counseling (such as risk and burden) in reaching
decisions about child bearing.

C. STUD? DESIGB

   Four experimental groups and 3 control groups will be
established in order to test the significance of the major
variables in the study. all four of the experimental groups will
be subject to the following procedures.

   1. 48-72 hours, pre-counseling. Family receives
pre-counseling interview by 2 members of research team and fills
out inventory instruments to assess their values relating to
child-bearing, faeily relationships and life expectations; their
knowledge of probabilities, genetics and the disease or condition
about which they are seeking counseling; the family decisions
they have recently made or are in the process of making; and
their expectations regarding the counseling they are to receive.

   2. 24-48 hours pre-counseling. Genetic counselor writes a
summary of his understanding of the case; his expectations
regarding the session;  the position he expects to take with the
family (neutral, optimistic, pessimistic), and his personal
feelings about the decision the family ought to make.

   .3. Family receives genetic counseling. The entire intorviev
is audio-taped for analysis of the structure and content of the
interaction.

   4. 24-48 hours post-counseling. Summary and evaluation of
the counseling session by the genetic counselor including his
prediction about the decision the family vi11 make and
differences between his expectations recorded at point 112 and
what actually occurred during the counseling at point 83.

   5. 48-72 hours post-counseling.  Interview and administration
of instruments to the family, similar to point Xl. Probes on:
their view of the counseling sassion; what they learned; were

P-138


-6-

expectations net; what position did they feel the counselor took.

  6. 1 month post-counseling interview with family.
Information obtained as at tl; probes on other information
obtained by family sources of information, new experiences which
have led to value changes and decisions.

  7, 6 month post-counseling interview with family.
Information obtained as in 16.

    8. 1 pear post-counseling interview with family.
Information obtained as in #6,

  The 4 experimental groups will vary according to vhether
they receive counseling by an H.D. trained in 8edical genetics or
by a social worker trained in genetic counseling. They vi11 also
vary according to whether the counselor receives or does not
receive information about the family obtained from the
pre-counseling contact (point 1c1, above). The families in all 4
of the experimental groups defined belov will be subject to the
procedures outlined above (#l-R).

  Families seeking or referred for genetic counseling will be
assigned randonly to one of the folloving treatment groups:

Group E-l,

Group B-2,

Group E-3.

Groay B-4.

Receives counseling from M.D. trained in medical
genetics,  Counselor receives no information
obtained from pm-counseling interview.

Receives counseling from M.D. trained in medical
genetics,  Counselor receives information aboat the
family obtained in pre-counseling research
interview.

Receives counseling from social worker trained in
genetic counseling.  Counselor receives no
information obtained from pre-counseling research
interview.

Receives counseling from social vorker trained in
genetic counseling.  Counselor receives information
about the family obtained in pre-counseling
research interview.

  It has been our experience with other longitudinal studies
(12) that oaltiple interviews with families in order to obtain
research data actually provide considerable psychological and
social support for the family. In the case of the proposed study,
it could ev8n influence the decision made by the family by
helping them to focus on the problems they face and to make more
explicit the alternatives they may have. In order to control for
the effects of the interviews and instruments on the decisions
that may be made by the faeilias, the following 2 control 'droups
will be established by random assignment of families:


-7-

Group C-l.  Pamily does not receive pre-counseling research
        interview (#I above).  Receives counseling from H. D.
       trained in,medical genetics (as does Group R-1).
       Counselor completes pre- and post-counseling
       summary (points t2 and Y4). Family does not receive
       post-counseling follow-up (points #S,tb, and t7)
       until 1 year post-counseling (point #8).

Group C-2.  Family does not receive pre-counseling research
        interview (#l above). Receives counseling from
       social uorker trained in genetic counseling (as
       does Group E-3). Counselor completes pre- and
      post-counseling summary (points 12 and BY). Family
      does not receive post-counseling follow-up (points
       15, f6, and t7) until 1 year post-counseling (point
        t8) .

  A third control group (C-3) will consist of parents who have
a child with a chronic, non-genetic condition and who have not
received genetic counseling. This group vi11 provide an overall
control on the effect of genetic counseling on family decisions,
particularly with regard to knowledge and limitation of family
skze, Like control groups C-l and C-2 they vi11 be interviewed
one year after receiving information from a physician (in this
case, inforration about the diagnosis and prognosis for their
child).

MTRAYCE CRITERIA FOB THE STUDY

  For families in the 4 experiwental groups and families in
control groaps 1 and 2:

1. Family must seek or be referred for and receive genetic
counseling at Stanford University Medical Center.

2. Family must be intact, i.e. there must be a couple in an
already-established marriage or common-law relationship.

3. Family must be willing to participate in the number of
sessions involved for data collection. Counseling costs
and transportation for research interviews will be borne
by the project to encourage participation.

  Paorilies in control group 3 will meet the same criteria,
except that they vi11 have a child with a non-genetic, chronic
condition diagnosed at Stanfor University fledical Center or the
Children's Efospital at stanforj.

  The purpose of the entrance criteria is to control for some
of the background variables which must be considered in data
analysis. Patients receiving genetic counseling outside of the
medical center must be presumed to be a population with somewhat
different characteristics than the population seen at the medical
center, and the counseling they receive must also be assumed to
be somevhat different. A population outside of the aedical center


-0-

could be studied only b,y increasing the size of the study
population by 100%. Use of a medical center population, combined
with the requirement that families be intact, vi11 serve to
provide a population uith some homogeneity with regard to income,
education, occupation and family situation (13). This
requirement, for example, rules out from the study couples
seeking genetic counseling before marriage, unmarried teen-age
mothers, etc. Hhile the impact of counseling on these groups is
deserving of study, given the umber of variables in the study,
control of some of the population characteristics is necessary.
These criteria will also allow for random assignment of families
to treatment and control groups thus obviating the difficulties
and possible bias of selective matching.

IISTRUMITS ABD SCHEDULIW

   The first year of the study will be devoted to the
developnent and validation of the instruments to be utilized, the
training of personnel to do the coding of the transcripts of the
counseling and interview sessions, and a pilot test of the study
design. Approximately 50 families will be utilized during the
first year. Wring the second and third year of study,
approxisately 125 families will be taken in and followed each
year. The 4th year will be devoted to continued one year
post-counseling follow-up of the families and data analysis, The
5th year vi11 be exclusively data analysis and write-up of the
study.

  Among the instruments to be developed are those to assess
the attitudes, decision state, knowledge and expectations of
families relevant to genetic counseling. These are the
instruments to be utilized at point 81 in the study design and at
future follow-up points. These instruments will be pre-tested
with a variety of patients to determine their ability to
distinguish significant differences among families, their ease of
administration and numerical szoring. Pace validity vi.11 be
determined through use of standard pre-test procedures (14).
Particular attention will be given to the development of
instruments which vi11 deternine the ability of the families to
apply probability figures to every-day life situations.

   During the development period, genetic counseling sessions
will be tape recorded and a scoring system developed for analysis
of the sessions. Coders, who will have no knowledge of the
pre-counseling data obtained from the families or the counselors,
will apply the scoring system.  Using an adaptation of the
interaction methods developed by Bales (15), both the structure
and the content of the sessions will be analyzed. These data will
be tested against the pre-counseling data obtained from both tht!
counselors and families and against the recall, post-counseling,
of counselors and families.

   Pre-coded and pre- and post-counseling forms to be used by
the counselors will be developed,  Counselors will record their
understanding of the case, the stance they propose to take and


-9-

their own personal feelings about the decision the family should
make. The standardized post-counseling report vi11 include their
evaluation of the session, any changes from the pre-counseling
stance and their estimate of the decision the family might make
as a result of the counseling.  The expectations of the counselor
regarding the session vi11 be compared with the pre-counseling
expectations elicited independently from the couple. Similarly,
the post-counseling summary from the counselor vi.11 be compared
with the post-counseling wiev of the session obtained from the
counselees.

   Post-counseliny interviews will also be conducted with the
families (points 115-e in the study design). Some of the same
pre-counseling instruments vi11 be used along with a standard
intervieu format combining general and specific probe questions
similar in form to the type developed by the study director for a
study of family response to the birth of premature infants (12,
13). Included in the post-counseling interviews vi11 be questions
to elicit family reactions to the counseling, their assessment of
the point of view taken by the counselor, decisions they may have
reached and the reasons for making the decisions they have
arrived at. On the basis of our previous family studies, the
husband and uife will be interviewed separately to prevent
contamination of the decision-making process by forcing consensus
or facilitating husband-wife communication. Since the
pre-counseling assessment will also be obtained independently,
one form of data analysis will be to see to what extent the
values and information of the husband and wife coincide after
counseling,

   The timing of data collection for the post-counseling
period, beyond the first post-counseling interviews, is not
rigidly established. One purpose of the first year of
developmental work is to determine the best timing that will take
us closest to the point at which families do make decisions.

LIflITATfOffS

   There may be some loss of subjects to follow-up, but this
uill be minimized by paying transportation and counseling costs.
The number of families who refuse to participate in the study
will be kept to a miaimum through the same devices, but
background data will be obtainad in any case to see whether
refusing families differ in important respects from the study
population.

   The findings of the study will not apply, of course, to
couples who seek pre-martial counseling, to individuals who do
not constitate a faai1.y unit, and to those who do not seek or are
referred foe counseling. Further, it is anticipated that because
of the nature of the entrance criteria, the population Will have
fairly homogeneous middle class ChardCteriStiCS (as definer! by
income, occupation and education).


   A number of genetic coonsaling studies have attempted to
determine the relationship between the decisions families make
about reproduction and the risk and burden they face, As noted
previously,  the meaning of risk from the fnsilycs point of view
has not been determined. Further, there appear to be significant
differences among counselors regarding the nature of the burden
for the same disease. Therefore,  we have not chosen to classify
families on the basis of risk and burden before assigning them to
the experimental or control groups. Risk and burden will be
analytic variables in the study and random assiynlaent of families
should provide an appropriate mix of these variables in each
group,

SIGNIPXCAilCE

   The study will provide the first systematic test of the
significant questions relating to the practice and inpact of
genetic counseling. The study is unique in the experimental
nature of the design. The instruments to be developed in the
course of the study should be useful to counselors in guiding
their practices and in evaluation of their effectiveness.
Conceptually, the study places genetic counseling within the
general framework of family decisions, so t.hat the effect of
variables other than counseling on decision-making can be
assessed.


-11-

REPEHEPiCES

1. Sorenson, J.R.: Social Aspects of Applied Human Genetics.
Social Science Frontiers, No.3,  Hussell Sage poundation, N.Y.,
1971.

2.  Hecht, F., Holaes, L.B.: New England J. fied. 237:464, 1972.

3. Stevenson, A.C., Davidson, B.C.: Genetic Counseling. J.P.
Lipincott Co., Phila., 1970.

4, Carter, C.O., tvans, K.A., Fraser Roberts, J.A., Buck, A.R.:
Lancet 1:281, 1971.

5.  Leonard, C.O., Chase, G.A., Childs, B.: Neu England J. bed.
2873433, 1972.

6.  Reiss, J-A., henashe, V.: J. Peds. 80:655, 1972.

7. Carter, C.O.: Proc. of the Third Xntl. Congress of Human
Genetics (J. Crow E J. Reel, eds.), Baltimore, 1966, p. 97.

8.  Gordon, H.:  JARA 21.7:1215, 1971.

9. Kallmann, F-J.: Genetics S the Epidemiology of Chronic
Diseases (J. Neel, LI. Shau G W. Schull, eds.). Public health
Service Publication 1163. Washington, b.C., 1965.

10.  FreeaDn, B., legrete, V-F., Davis, Pl., Korsch, B.H.:
Pediatric Research 53298, 1971.

11. Adair, J., Deuschle, K., Rabin, D.: The People's Health:
Medicine and Anthropology in a Navajo Community.
Appleton-Century-Crofts, N.Y., 1'370.

12. Barnett, C.R., Leiderman, P.H., Grobstein, R., Klaus, R.:
Pediatrics 45:197, 1970.

13. Seashore, R., Leifer, A., Barn&t, C., Leiderman. P.ki.,
Williams, J.: Personality and Social Psychology (in press).

14. Festinper, L., Katz, D.:  Research Methods in thz Behavioral
Sciences. Bolt,  Rinehart E Winston, N.Y., 1953.

15. Bales, B.F.: Interaction Process Analysis: A tlethod for the
Study of small Groups. Addison h'esley, Reading, Mass., 1950.

P-/`/r/


                                            "b.b 1 ,"I. I,      ~"YJIIIVIL IrllJr N"L rvn YLI~IiL'J~`J"`J=I rr.

          SUBSTITUTE                           PERIOD COVERED           GRANT NUMBER
DETAILED BUDGET FOR FIRST 12.MONTH PERIOD FAo;,l,74         THROUGH
                                  12/31/74
---
. PERSONNEL (Lisf ail perso~ef enfagcd on project)                 TIME OR
                                                     EFFORT       AMOUNT REQUESTED (Omit cenrs)
     NAME (Last. first, initial)           TITLE OF POSITION       wnus.           I     TOTAL

                    Principal Investigator or                                  ~

Barnett, C.            Program Director      20% . .

Cann, H.,          Assoc. Prof./Peds.     10%  IMPACT OP'GBNBTIC COUNSELIXG
Luzzatti, L.          Professor of Peds.    10%      PRACTICES
Open               Research Assoc.-Stat   20% 1
Open-Pediatrics        Res. Assoc.-Sot. wk.  50% ::
Open-Pediatrics        Interviewer       50% '.
Open-Pediatrics        Interviewer       25% .' :.
Open-Pediatrics        Statistical Clerk    ,50x
Open-Pediatrics        Data Coder         '65% '
Open-Pediatrics       Typist         100% .;:









                                                            I                              ".-e--*,---






:. CONSULTANT COSTS (Include Fees and Travel)                                                      _ "_-.I_. ..!-



                                                                           . -I~-

         Tape Recorder



                                                                           -...-_.---_---







                                                                           -.-.- -._


                                                                       ---


                                                                          ---   ----.-. .-_--~-








_I_--
1. OTHER EXPENSES (itemize per inatmctions)

Office supplies, telephone, repro., postage, publicatti costs, etc. 1200
Central computer usage       \-









          a. STIPENDS









kkstituh Budast Paae S-72


SECTION II -PRIVILEGED COMMUNICATION         IMPACT OF GENETIC COUNSELING PRACTICES

    BUDGET ESTIMATES FOR ALL YEARS OF SUPPORT REQUESTED FROM PUBLIC HEALTH SERVICE
                    DIRECT COSTS ONLY (Omit Cents)

   DESCRIPTION



PERSONNEL
COSTS

1ST PER100
ISAME AS DE.  ADDITIONAL YEARSSUPPORT REQUESTED lThisapp/ication only1
TAILEO BUDGET)


47,377

SUPPLIES

            DOMESTIC
TRAVEL              500      600      600     600      700

            FOREIGN

PATIENT COSTS        2,500





RENOVATIONS ---I--   5,000    5,000



ALTERATIONS AND

OTHER EXPENSES       2,400    2,600    2,800   3,000    3,200




TOTAL DIRECT COSTS    53,277   58,932   62,629   61,564   65,850



TOTAL FOR ENTIRE PROPOSED PROJECT PERIOD (Enter on Page 1. item 4) _  s 302,252

REMARKS: Justify all costs for the first year for which the need may nor be obvious For future years, justify equipment costs, as well as any

significant increases in any other category. If a recurring annual increase in personnel casts is requested, give percentage.  (Use continuation
page if needed)

Budget explanation attached


BUDGET SXPLAhATIOU

     Barnett has a 9 month academic appointment supported
joint!;.by the Department of Pediatrics and the Department of
Anthropology, His project salary 1s computed on the basis of 1Oi
time during the 9 month academic year and 70% time during July
and August for an average of approximately 20% during each year.

   The social worker (50% time),  a research associate (23%
time), two interviewers (50% and 25% time respectively), a
statistical clerk (50% time), a data coder (65% time) and a
typist (100% time) are require3 for the project. Two interviewers
are required because husband and wife will be seen separately.
The research associate is a biomathematician experienced in
design ot and data analysis for behaviorial research projects.
Computer time will be used for data analysis.

   Salaries are increased at a rate of 6X per year to cover
8erit and cost of living increases. Staff benefits are applied
based on the following University projections: 17X, Y/73-8/74;
18.3%. 9/74-a/75; 19.3X, 9/75-8/7b; 20.3%. 9/76-h/77; 21.3X,
g/77-8/78; and 22.3%. g/78-8/79.

  The tape recorder vi11 be used by the typist to transcribe
tape recordings of genetic counseling sessions and pre- and
post-counseling interviews in the project on qenetic counseling
impacts on the family.

  To insure that ve obtain adequate patient material for the
project on genetic counseling, ue propose to waive the fee for
this service to any participating family and therefore include
these costs in our budget. The cost of genetic counseling to d
family is $50. This does not include laboratory tests and
amniocentesis. We anticipate doubling the number of patients in
the second and third years of the project. Xn years 4 and 5 no
new patients will be studied although follou-up interviews will
be carried out for those studied in year 3.

p- i'r 7


SECTION VII




Overall Budgets


DIRECTOR'S OFFICE BUDGET EXPLANATION

   Salary support for the Program Director (Profassor
Lederhery) has been included entirely under the subproject budget
for Screening and Characterizatiou of Inborn Errors of Hetaboli~m
Using GC/HS.  The 2UX of his time budgetted there, includes
support for his role in overall program direction as well ds his
direct involvement in that research project, This 20% allocation
has not been subdivided between that budget and the present
Program Director's Office budget.  Such a suballocation would be
difficult to make realistically since the apportionment of
Professor Lederberg's time vi11 vary from time to time, depending
on program needs.

   This budget does include support for 30% of the Program
Director's secretary. She will support the Director in overall
program management as veil as in liaison vork with the Visiting
Committee and in implementing the planned annual symposium on
aspects of genetic disease. An important responsibility of the
Director is maintaining current awareness of the relevant
literature which spaas a number of fields. Us. Aedse will spend
considerable time in assisting at this task with the help of
modern information services an?L devices. She will also undertake
to disseminate notices to the appropriate colldboratiny
investigators.

    ns. ilease's salary is increased at a rate of 6% per year to
cover merit and cost of living increases. Staff benefits are
applied based on the folloving University projections: 17%,
9/7 3-8/74; -16.3X, Y/74-8/75; 19.3%, Y/75-8/76; 20.3%` Y/76-8/77;
21.3%, Y/77-8/78; and 22.3%. Y/78-8/79.

   Secretarial support for the individual Principal
Investigators is provided in those respective subproject budgets.

   The bulget also covers estimated expenses for Visiting
Committee honoraria and travel and expenses related to the
planned annual symposia.


1, , PERSONNEL (Lisf all personnel enlale  m omiect)                TIME OR
                                                - EFFORT        AMOUNT REQUESTED fOrnit cen,s)
     NAME (Lest, lirsl. hiHal)          TITLE OF POSITION      OIHRS.           I     TOTaiL

                      Principal Investigator or
                       Program Director

1 Redse, R.

Program Director's   30
                             :.  PROGRAM DIRECTOR'S OFFICE
Secretary                    '.           
                             :. i

2 , CONSULTANT COSTS (Incfude Fees and TravelJ


3

I . SUPPLIES

5 .   STAFF     Q. DOMESTIC
  TRAVEL
I. Sea rnducrfond b FORE,GN

6 . PATIENT COSTS (Sepsrs~e InpsUenl md Oulpalienl)

7 . ALTERATIONS ANO RENOVATIONS                                                        6 -.

6 . OTHER EXPENSES (ILemire per insOucUona)

  Visiting Committee honoraria and travel and expenses for annual
  symposium.   Communications:  information services (e.g., abstracts,
   ASCA; MEDLINE).                                    $5,000

9                                          Subtotal - Items 1 thru 8               t  $8,414

         10. TRAINEE EXPENSES (See Ins,mctions)

                   PREOOCTORAL              No. Proposed           s                            -
  FOR   o. ST,PENDS POSTDOCTORAL              No. Proposed           s
                 OTHER (Specrfy)              No. Proposed           s
TRAINING            DEPENDENCY ALLOWANCE                            s

GRANTS                          .,            TOTAL STIPEND EXPENSES -  s


                                                                           s
  ONLY   b. TUITION AND FEES

        c. TRAINEE TRAVEL (Describe)                                                  s


          11.                              Subtotal - Trainee Expenses                c  s
I

1

I& TOTAL DIRECT COST (Add Sublolals. Ilsma 9 and 11, and enter on Page I)

c

$8,414

s

~PRIVILEGED COMMUNICATION              SECTION It     SUBSTITUTE THIS PAGE FOR DETAILED BUDGET PAGE
.
           SUBSTITUTE                          PERIOD COVERED           GRANT NUMBER
DETAILED BUDGET FOR FIRST 12.MONTH PERIOD FRoM             THROUGH

:;j.; :.,.:>.
:.:.
.J' :.. .:..

..:.:..   (     

..:. .
. . . .
..::
:.   . . .   


,.

s -

s -

s -



s -

Substltut. Budget Page S-72
For Forms PHS 398 and PtlS 2499-1

P cio

GPO 933.751


SECTION II -PRIVILEGED COMMUNICATION

PROGRAM DIRECTOR'S OFFICE

BUDGET ESTIMATES FOR ALL

DESCRIPTION

1ST PERIOD
ISAME AS DE-
TAILED BUDGET)

PERSONNEL
COSTS



CONSULTANT COSTS
(Include fees, travel, etc.1

3,414

EOUIPMENT   I -

SUPPLIES

           DOMESTIC
TRAVEL
            FOREIGN

PATIENT COSTS

ALTERATIONS AND
RENOVATIONS

OTHER EXPENSES      5,000

TOTAL DIRECT COSTS    8,414

TOTAL FOR ENTIRE PROPOSED PROJECT I

REMARKS: Justify all costs for the first year fc
significant increases in any other category. If a I
pege if needed.)

fEARS OF SUPPORT REQUESTED FROM PUBLIC HEALTH SERVICE
DIRECT COSTS ONLY (Omit Cents)

ADDITIONAL YEARS SUPPORT REQUESTED llhis application only)

2ND YEAR    3RD YEAR    4TH YEAR    5Tti YEAR    6TH YEAR    7TH YEAR
                     L                      I

3,656
3,656    3,908
      3,908   4,177
            4,177    4,464
                  4,464

ZNDYEAR 1 3RD YEAR t 4TH YEAR 1 5Tti YEAR 1 6TH YEAR 1 7TH YEAR

5,300
5,300    5,700
       5,700   6,000
            6,000    6,400
                  6,400


8,956
8,956    9,608
      9,608   -0,177
            -0,177    10,864
                  10,864

               I
ERIOD (Enter on Page f, Item 4) -   s   48,019

ERIOD (Enter on Page f, Item 4) - I  s   48,019

I

r which the need may not be obvious. For future years, justify equipment costs, as well as any
Cuffing annual increase in personnel caHs is requested. give percentage.  Use continuation

Budget explanation attached

MS-398
Rev. 3-70                                                     p-/y/


BGGREGATP BUDGET EYPLAUATIOR

  The following budget is the aggregate of the five individual
subprogram budgets and the Program Director's Office budget.
Individual budget items such as personnel appearing in more than
one budget and supply items like Vhemicals, glassware, etc."
have been combined to show only one such item each in this
overall budget.

  In considerinq the percentages of personnel time committed
to this project, it should be noted that.facnlty personnel, as
distinct from staff, also receive a portion of their salary from
the institution. This institutional salary support assists in
covering fasulty efforts along research lines overlapping those
in the present application. Therefore the faculty time
percentages reflect contractual lower bounds on faculty
commitment to this program,

  Salaries are uniforrlp increased at a rate of 6% per year to
cover expected merit and cost of living increases. Staff benefits
are applied based on the following University projections: 17%,
9/J3-0/74; 18.3%, Y/74-&/75; 19.35, g/75-8/76; 20.3%. 9/J&0/77;
21.3%. 9/77-b/78; 22.3X. 9/30-B/19.

   Budget items other than %quipmentH and "Patient Costs" are
in general increased by 6% per year to cover inflation, except in
specific instances noted in the individual budgets.


n,. ,LL"LY                                   JELIIUN II


DETAILED BUDGET FOR







Cann, H.
Kretchmer, N.
Herzenberg, L.              of Genetics
Cavalli-Sforza, L.           of Genetics      - :AkGREGATE PROGRAM BUDGET

Bamett, C.
Luzzatti, L.
Tsuboi, K.              nior Scientist
Duffield, A.             search Associate
Pereira, W.             search Associate
Rindfleisch, T.          search Associate
Hulett, H.              search Associate
Open - Genetics          search Associate                                I

Open - Pediatrics       Research Associate








. CONSULTANT COSTS (Include Fees and Travel)









                   see attached









                 emtzs per matruct



                   see attached

                                              s 21,500

                                          Subtotal - Items 1 thru 8          . s 537,800

        IO. TRAINEE EXPENSES (See instructions)

                    PREDOCTORAL               No. Proposed           s

  FOR    Q. STIPENDS  POSTDOCTORAL             No. Proposed           s
                 0 TH ER (Specity)              No. Proposed         - s
rRAlNlNG            DEPENDENCY ALLOWANCE                             s

GRANTS                               TOTAL STIPEND EXPENSES            c s

        b. TUITION AND FEES
ONLY                                                                           S

        C. TRAINEE TRAVEL (Describ.,)                                                     S


          11.                              Subtotal - Trainee Expenses                 *s

2. TOTAL DIRECT COST (Add Subtotala, Item= 9 and II. and enter on Page I)                 t s 537,800

Substltuto Budget Peg. S-72                                     P-63
For Forms PM 398 and PHS 2499-1                                                                   GPO 930.751


continued

1. PERSONNEL

Open
Open - Pediatrics
Veizades, N.
Steed, E.
Tucker, R.
Wegman, A.
Waters, R.
Sakaguchi, S.
Van West, B.
Makk, G.
Open - Pediatrics
Summons, R.
Open -  Genetics
Open - Genetics
Pearson, D.
Wyche, M.
Boswell, M.
Open - Genetics
Open- Genetics
Open - Pediatrics
Open - Pediatrics
Open - Pediatrics
Open - Pediatrics
Jamtgaard, R.
Redse, R.
Allen, M.
Murray, R.
Meyering, P.
Open - Pediatrics
Harlow, W.
Cusumano, M.
Open - Genetics

TITLE

Research Assoc.-Stat.            20
Research Assoc. - Sot. Worker     50
Research Engineer               33
Research Engineer              33
Computer Programmer             75
Senior Res. Assist.              10
Research Assist.               100
Research Assist.                50
Research Assist.                100
Research Assist.               100
Research Assist.                50
Post.Doc. Fellow               100
Grad. Res. Assist.              50
Grad. Res. Assist.              50
Electronics Tech.               40
Lab. Tech.                    100
Lab. Tech.                    100
Engin. Tech.                   50
Research Tech.                  100
Interviewer                   50
Interviewer                    25
Statist. Clerk                  50
Data Coder                    65
Administrator                  15
Secretary-Prog.Dir.             30
Secretary - IRL                25
Secretary - Peds.                50
Secretary - Gen.                10
Typist                       100
Machinist                      10
Laboratory Diener               50
Lab Glasswasher                25

X TIME


continued

3. EQUIPMENT

4 Column Gas Chromatograph                $14,400*
Mini-computer System & Inst. Interface          59,900*
Amino Acid Analyzer                      32,500*
Event Counter                         2,700*
Digital Voltmeter                                900*
Carbon Dioxide Incubator                   2,500*
Laminar Flow Hood                        1,000*
Fluorescence Microscope                    9 ,ooo*
Eppendorf Microfuge                              500*
Buchler Power Supply                             600*
Constant Current Power Supply                1,000*
Slab Gel Electrophoresis                          300*
Column Acrylamide Gel Electrophoresis                200*
Destainer                                     200*
Tape Recorder                                  500*

Total Equipment $126,200*

4.  SUPPLIES

Chemicals, glassware & lab. apparatus
GC Supplies (Columns, Phases, etc.)
Dry ice & liquid nitrogen
Data recording media (GC/MS, Calcomp, etc.)
Mini-computer supplies (start-up 6, continuing)
Mass spectrometer repair parts & supplies
Electronic parts & supplies
Amino acid analyzer supplies
Radioactive tracers
Expendable supplies (photo. plates, etc.)

$18,000
1,100
   500
1,800
1,600
2,300
1,900
2,000
5,000
1,500

Total Supplies   $35,700

8.   OTHER

Visiting committee honoraria & expenses for
annual symposium
Office supplies, telephone, repor.,postage,
publication costs, etc.
Mini-computer maintenance
Freight on capital equipment
Central computer usage & terminal rental

$5,000

6,200
6,000
  500
3,800

Total Other    $21,500


II -

AGGREGATE BUDGET

.",I",. I, -, lll"lLL"LV Y"I.II.I"I.IVr.lIV..
   BUDGET ESTIMATES FOR ALL YEARS OF SUPPORT REQUESTED FROM PUBLIC HEALTH SERVICE
                  DIRECT COSTS ONLY (Omit Cents)

   OESCRIPTION



PERSONNEL
COSTS



CONSULTANT COSTS
llnclude fees, travel, etc.)

1ST PERIOD
ISAME AS DE-  ADDITIONAL YEARS SUPPORT REQUESTED (Thisapplication only1
TAILED BUOGET)   2ND YEAR    3RD YEAR    47-H YEAR    5TH YEAR    6TH YEAR    7TH YEAR

$345,600 370,068 431,528  461,243  492,966

EOUIPMENT        126,200*  23,000* 18,100*   5,300*   6,000*



SUPPLIES          35,700   37,700  42,400   44,800   47,700



            DOMESTIC   4,000    4,700   4,900    4,900    5,400
TRAVEL
            FOREIGN

             3,000    5,500   5,500      500      500
PATIENT COSTS

ALTERATIONS AN0
RENOVATIONS        1,800



OTHER EXPENSES      21,500   24,900   26,400   27,800   29,700

TOTAL DIRECT COSTS    537,800  465,868 528,828  544,543   582,266



TOTAL FOR ENTIRE PROPOSED PROJECT PERIOD (Enteron Page 1, item 4) -   t   2,659,305

REMARKS: Justify ail costs for the first year for which the need may nor be obvious For future years, justify equipmenr costs, as well as any
significant increases in any other category. If e recurring annual increase in personnel msts is requested, give percentage.  (Use continuation
page if needed, I

See individual subprojects for budget explanations


SECTION VIII

Concluding Remarks

Dr. Lederberg

p-k-7


Continuation page

CONCLUDING REMARKS      (Joshua Lederberg)

  The overt justification for the proposed Center is, of course, the collective
value of the well-defined research projects that will make up its day to day work.
However, its most important utility in the long run may be a new institutional
arrangement that will generate now unforeseen lines of investigation. Molecular
biology as a basic science has leaped far beyond its practical application to
human problems. The more intimate intellectual association of gifted scientists
and clinicians envisaged here, the development of an active interface among workers
who spend much of their time still either in basic laboratory work or in the clinic,
is the only possible way of advancing these applications.

   Speaking more personally, I can point to a number of anticipations from my
own research career that might have been explored more aggressively had I then
pressed them in an interdisciplinary context. My early work (with Tatum, Zinder
and others) on genetic recombination and on viral transduction in bacteria was
foreseen years ago as laying a groundwork for the development of a genetics of
somatic cells and of means of importing reparative genetic information ("genetic
engineering"). Subsequently, I tried to stress that the new molecular genetics
would overtake other lines of concern about genetic impairment, and improvement,
and that ethical issues would loom as large as technological opportunLties in
public policy debates. While public reactions to these potentialities are in some
respect overdrawn, geneticists obviously must inculcate and exhibit great sensitivit!
to the ethical issues of genetic intervention and how they are perceived by the
public (1).

  Although much of my own laboratory research might go under the heading of
genetic engineering of bacteria (2) I have been persuaded that prenatal diagnosis
represents the most important practical avenue of genetic therapy or rather pre-
emption of disease. For this reason, the application of automated technologies
(growing in large measure out of NASA-supported work) is emphasized in this
application.

  What is more difficult to foresee is the intensive development of the whole
repertoire of tools of molecular genetics. Somatic cell genetics is well launched;
but we will surely also have to learn how to use specific DNA replication,
hybridization, transcription, and translation, i.e. the full range of gene action
vitro,
in     to solve pressing diagnostic problems.

  Laboratory research shares a place with other interests. I can summarize these
under the heading of a concern for the overall process of science, and its
application for human benefit. These meta-scientific interests have been expressed
in the public arena, as in efforts to enhance public understanding of science and
provision for its support. More recently,  I have been more involved in efforts to
enhance the infrastructure of science, with work on new instrumentation coupled
with feeble steps towards the use of computers for scientific "intelligence" (3).

  The Genetics Research Center is a plan to bring all of these themes to a focus
'on an area most likely to advance genetic knowledge -- my fundamental scientific
base -- and to generate tangible health benefits for the mutual advantage of both
disciplines, and to satisfy the public's motives for continued investment in the
health sciences.

PM-398
Rev. 2-69

Page


Continuation page

Bibliography

1.  Lederberg, J.: in Challenging Biological Problems, Oxford LJ. Press, N.Y. 1972,
    p. 7 (J.A. Behnke, ed.) "Biological Innovation and Genetic Intervention".

2.  Sgaramella, V.: Proc. Nat. Acad. Sci. 69:3389, 1972.
   "Enzymic Oligomerization of BacterGphage P22 DNA and of Linear
      Simian Virus 40 DNA."

3.  Lederberg, J.: in Biochemical Applications of Mass Spectrometry, John Wiley
    & Sons, N.Y. 1972, p. 193 (G.R. Waller, ed.) "Use of a Computer to
     Identify Unknown Compounds: The Automation of Scientific Inference."

HS-398
BY. 2.49

Page

0Po : ,889 0 - 350-360
  P-/Q


SECTION IX

Professional Personnel

P-/60


KEY PERSONNEL AND BIOCRAPBICAL MATERIAL

Joshua Lederberg, Ph.D. - Principal Investigator, Professor and
Chairman, Department of Genetics.

Professor Lederberg will serve as the Principal Investigator
for the overall program, and for part 1, automated screening
methods (SC/RS) for inborn errors of metabolism. In addition,
Professor Lederbery aaintains active research programs in
genetic recombination in bacteria; mechanisms of D#A
replication and reunion;  interactions of environmental mutagens
with DYA;  and computer emulation of human cognitive processes.

Reward Cann, M.D. - Associata Program Director, Associate
Professor of Pediatrics.

Dr. Cann vi11 assuae responsibility for coordinating laboratory
research with clinical applications within the program. This
will involve screening of patients for their suitability of
participation in the relevant reseach projects, obtaining
appropriate patients for projects, informing patients about the
research projects and obtaining their consent for
participation, insuring that proper evaluation, care and
counseling are provided to patients and ianilies involved in
these projects and planning for the application of the research
findings to patieat care.  Dr. Cann will be directly involved
with research projects on genetic markers in amniotic fluid,
separation of fetal cells from the maternal circulation, the
impact of genetic counseling on family decisions and screening
and detection of inborn errors of metabolisn. Dr. Cann's
research activities pertain to human polymorphisms and to
somatic cell genetics and his clinical work involves him with
hereditary illness and genetic counseling. He has set up and
directs a laboratory for typing for various polymorphisms of
erythrocgte antigens and enzymes and serum proteins. He has
initiated an investigative program for applying somatic cell
hybridization to prenatal diagnosis of hereditary disorders.

L.L. Cavalli-Sforza, H.D. - Professor of Genetics

'The investigations of Professor Cavalli-Sforza have long
contributed and continue to contribute important information on
human population genetics.  He has pioneered in applying
demography to studies of population and genetic structure of
man. Particularly pertinent to this program is his interest and
work on variation among populations in the frequency of
inherited disorders and their determining genes,

Professor Gaoalli-Sforza als3 directs a laboratory program
oriented toward the detection of specific binding proteins in

P-/6/


-2-

blood. The detection of genetic polymorphisms of these proteins
will be carried out by Professor Cavalli-Sforza as a research
activity of this Genetics Center.

Norman Itcetchmer, H-D., Ph.D. - Professor of Pediatrics.

Professor Sretchmec will participate in the research project
dealing with screening and detection of inborn errors of
metabolism.  De is well known for his research and clinical uork
in various inborn errors of metabolism. One of Professor
Kretchmer's main research interests presently deals uith
lactase intolerance in man. His research program also involves
the developmental biochemistry of the urea cycle and the
pyriaidine biosynthetic pathuay in eukaryote cells and tissues.
He has recently relinquished the chairmanship of the faculty
committee for teaching the human biology curriculum at Stanford
University.  Prom 3959-3969 Professor Kretchner was Chairman of
the Department of Pediatrics,

Leonard Herzenberg, Ph.D. - Professor of Genetics.

Professor Heczenberg directs an active research program in
immunogenetics with special emphasis on genetic and structural
studies of mouse immunoglobulins and on the mechanism and
control of antibody synthesis. The development under his
direction of instrumentation for separating cells by means of
fluorescent sensing has provided potential methodology for
separating fetal cells from the maternal bloodstream. Professor
Herzenberg vi11 participate in the project which will apply
this methodology to antenatal diagnosis of genetic disorders.

Clifford Barnett, Ph.D. - Professor of Anthropology and
Associate Professor of Pediatrics.

Professor Barnett has worked in medical anthropology for 10
years. His research activities have dealt with cultural and
genetic aspects of congenital dislocation of the hips,
fertility and birth spacing of partially acculturated
Guatenalan Indians,  and mother and infant interactions in
reference to premature infants. He has designed and will direct
the research project on the impact of genetic counseling on
family decisions and behavior.

Alan Duffield, Ph.D.  - Research Associate in Genetics.

Dr. Duffield is an organic chemist who has carried out
fundamental studies in mass spectrometry of organic compounds.
He is presently directing research involving the development of
a multicomponent, automated system for analysis of biolorjic
substances. He shall apply this system, which involves d gas
liquid chromatograph, a mass spectrometer and computer


-3-

facilities to screening, detecting, and studying inborn
metabolic errors in various body fluids and tissues.

Kenneth Tsuboi, Ph.D. - Senior Scientist, Department of
Pediatrics.

Dr. Tsuboi*s research interests have included the physiological
chemistry and enzymology of erythrocyte interaediate metabolism
and more recently, the biochemical genetics of various
erythrocyte enzymes. He is also working oti the enzyaological
characteristics of various priaate cell culture lines. In this
prograa he vi11 participate in the project on linkage and
prenatal diagnosis of inherited disorders. In particular, he
will uork in the detection of polyaorphic enzyme markers in
cultured amniotic fluid cells.

Luigi Luzzatti, M.D.  - Professor of Pediatrics.

Professor Luzzatti is the director of the clinical cytogenetics
laboratory and of the Birth Defects Clinics in the Department
of Pediatrics. His clinical activities which are pertinent to
this program, include comprehensive care for patients (and
their families) with birth defects, genetic counseling and
screening amniotic fluid samples {usually from women over 35
years of age) for chroaosomal abnormalities. He will
participate in the project which investigates the impact of
genetic counseling on family decisions.

Herbert Schwartz, tl,D. - Professor of Pediatrics.

Professor Schwartz's research interests in hemoglobin
synthesis, structures and function in health and disease, and
their application to the prenatal detection of hereditary
disorders involving hemoglobin, e.g. sickle cell anemia crnd
thalassemia, will be incorporated into the program on exploring
the maternal bloodstream for fetal cells. Dr. Schwartz will
COlldbOCdte with Drs. Herzenherg and Cann in preliminary
studies of fetal erythrocvtes separated from maternal blood.
Dr. Schwartz directs the hematology service of the Department
of Pediatrics.

   Other members of the Departaent of Genetics who will not
participate directly in this program but with whom ve interact
daily are Dr. Eric Shooter (Professor) dnd Dr. A.T. cidnesan
(Associate Professor).  Frofessor Shooter directs research into
the structure and mechanism of action of the nerve growth factor
protein. Dr. Ganesan is investigating the genetic control of
chromosome replication in B. subtilis. the chemical basis of DEiA
replication, the mechanism of genetic recombination during Dt~b
mediated transformation and the role of the nuclear membrane in
chromosome replication in mammalian cells.


-4-

   The clinical and research interests of Professor Irving
Schulman, Chaiman of the Department of Pediatrics, are devoted
to various defects of coagulation,  including inherited disorders.
Other members of the Departaent of Pediatrics whose clinical and
research activities relate to this prograta are Dr. Phillip
Sunshine (Associate Professor of Pediatrics) and Dr. John Johnson
(Assistant Professor of Pediatrics), Director and Associate
Director of the Revborn and Premature Infant Nurseries,
respectively.  Dr. Sunshine and Dr.  Johnson are together
investigating the metabolic consequences of neonatal ornithine
transcarbamylase deficiency and detection of heterozygotes for
the gene which determines this disorder. The research activities
of Dr.  Merton Bernfield,  Associate Professor of Pediatrics, are
designed to assess the aorphogenetic behavior and cell surface
properties of human cells and to utilize these assessments in
studies of cells derived from individuals with birth defects, Dr.
John Gribble, Assistant Qrofessor of Pediatrics, is a pediatric
hematologist whose research activities have dealt with in vitro
biosynthesis of hetaoglobin and biochemical and physiological
aspects of pinniped hemoglobins. Dr. Gribble's clinic&
activities involve evaluation and treatment of patients with
hereditary disorders of blood coagulation, including genetic
counseling ot their faailies. The clinical and research
activities of Dr.  R.O. Christiansen (Assistant Professor of
Pediatrics and Director of the Pediatric Pletabolic and Endocrine
Service) an3 Dr. Judith Koehler (Assistant Professor of
Pediatrics and Director of Pediatric Neurology) are quite
relevant to the Genetics Center program.  Even though the research
activities of all of these individuals will not be supported by
this grant,  we are including bibliographic sketches on each of
them because of their relevance  to the overall Genetics Center
program.

   Within one or-tro years after the activation of this program
we anticipate the ditect participation of one or @ore colleagues
from the Department of Obstetrics and Gynecology. A search for
chairnan of this department is presently under uay, and ue expect
that this individual will bring competence to Stanford in fetal
physiology and fetal monitoring.  We look forward to interacting
in this program with our obstetrical colleagues in various
projects pertaining to antenatal detection of genetic disorders
and selective abortion.


Principal Investigator:
(unless otherwise noted)

Research Support Summary for Departments of Genetics and Pediatrics
           Relevant to Genetics Research Center

Program Director: J. Lederberg
Associate Program Director: H. Cann

HOWARD CANN
Associate Professor of Pediatrics
Department of Pediatrics

Grant Title                Current Year

Total Award     Grant Term   Budgeted
                       % time

1) NIH:HL-15008

Pre-Natal Detection of Sickle Cell Anemia $ 38,717

LUCA CAVALLI-SFORZA
Professor of Genetics
Department of Genetics

1) AEC:AT-04-3-326    Mutation Rates and Mutational Loads in     32,000
                   Man

2) NIH:NS-10711

Genetic Study of Metabolism of Neural
Transmitters

65,000

3) NIHiGM-20467

Gene Diffusion, Natural Selection and
Drift in Man

47,587

ADAYAPALAM GANESAN
Associate Professor of Genetics
Department of Genetics

1) NIH:GM50,199

Development Award-Research Career Program   21,433
Chromosome Replication, Recombination and
Cell Division

2) NIH:GM14108

DNA Synthesis and Genetic Recombination

34,902

$118,000      g/72-8/75

32,000

10/72-g/73      10%
(Renewal pending)

207,000      g/72-8/75      20%

147,351      5/73-4/76       20%

Support recommended for 2 additional
years ending 12/31/75, the amounts to
be determined annually.

211,996      6/72-5177       50%


LEONARD HERZENBERG
Professor of Genetics
Department of Genetics

1) NIH:GM-17367


2) NIH:AI-08917

3) NIH:CA-04681

4) NIH:HD-01287

Automated Cell Sorting - Clinical and
Biological Uses

Genetics of Immunoglobins

Genetic Studies of Mammalian Cells

Fetal-Maternal Immunological Inter-
actions

JOHN D. JOHNSON
Assistant Professor
Department of Pediatrics

1) United Cerebral   Developmental Aspects of Heme
  Palsy           Protein Catabolism

NORMAN KRETCHMER
Professor of Pediatrics
Department of Pediatrics

1) NIH:RR-00081

Clinical Research Center for Premature
Infants

2) NIH:HD-02147     Biochemical Studies of Development

3) National Found.
  CRBS-252

Growth and Differentiation of the
Placenta

4) NIH:HD-CC047

Human Development and Pediatrics Train-
ing Grant

5) NllH:HD-00391
b
*;
P

Regulatim of Enzyme Action During
Development

160,802

585,977

l/73-12/75      15%

52,774        246,386      5/69-4/ 74      20%

80,994        424,981      9172-8177       30%

38,198        222,897      5/73-4178       10%

28,772

418,532

206,593

23,293

90,588

42,289

53,322

7/71-6/73

1,974,008

10/69-9/74       5%
(Renewal pending)

1,309,278

6/66-5/74       15%
(Renewal pending)

23,293      7172-16174      15%

448,936      7170-6 J74       10%

183,822

g/68-8/73       15%
(Renewal pending)


NORMAN KRETCHMER (continued)

5) NIH:CAl4917

Pyrimidine Synthesis and Cellular
Proliferation in Colon

7) Educational Found.  Epidemiology, Etiology, and
   of America      Physiology of Diarrhea in the
                 American Indian

JOSHUA LEDERBERG
Professor and Chairman
Department of Genetics

42,689

45,156

1) NASA:NGR-05-020   Cytochemical Studies of Planetary      180,000
              Micro-organisms

2) NIH:AI-05160

Genetics of Bacteria

60,000

3) NIH:RR-00612

Resource Related Research - Computers    194,408
and Chemistry (E. Feigenbaum, Principal
Investigator; J. Lederberg and C. Djerassi,
Co-Investigators)

4) NIH:RR-00785

Stanford University Medical
Experimental Computer Facility
(SUMEX)

765,573

5) NIH:GM0295      Training Grant in Genetics

i43,964

GILDA LOEW
Research Associate
Department of Genetics

1) NSF:GB17980

Quantum Chemical Investigations
of Heme Proteins and Ferredoxins

2 2) NIH:DAOO770
w

Quantum Chemical  Szudfec ef Opiate
Narcotics

130,737      6/73/5/76       20%

92,915

7/73-6/75        5%
(Renewal pending)

3,800,OOO

g/60-8/73
(Future support
dubious)

11%

280,000

g/68-8/73       15%
(Renewal pending)

6i5,OOO

5/71-4174        0%
(Renewal pending)

4,246,621

8/73-7J78
(pending)

20%

755,550      7/69-6J74       15%

34,799

34,799

2/72-l/74       50%
(Renewal pending)

57,557

184 * 139

g/73-8/76
(Pending)

50%


ERIC M. SHOOTER
Professor of Genetics
Department of Genetics

1) NIH:NS04270

Molecular Neurobiology - Proteins
in the Nervous System

2) NSF:GB31982

Structure and Mechanism of Action
of the Nerve Growth Factor

KENNETH TSUBOI
Senior Scientist
Department of Pediatrics

1) NIH: Contract-    Biochemical Parameters of Primate
  Div. of Biol.     Cell Cultures
   Standards

84,558        404,979      12/70-11/75     40%

60,000

60,000

l/72-12/73      20%

23,598

47,196

6/72-5 J74


                                                 ROT. j. lo*:?+g'r:
                                                                           . ,
SECTION II - PRI\`ILZ.;ED CO'.".:`J:!!CATIO'J                                  ppnt+Y.`.' -1 ,!-..,A a. --
                        BIOGF:RPl-IICAL SKETCH

      lGivc the following information for allprcfcGcw~l personnel listed on page 3, beginning with the Principal /nvA-tigator.
               Use continwtion p.?gc' a~:d fol!o;v rhe same general format for each pe,son.j                      ---
NAME                                 TITLE                         BIRTHDATE IYo., Dab/, Yr.)
Clifford R. Barnett         Associate Professor of Pediatrics
                       Professor of Anthropologv       8/17/29

PLACE OF BIRTH (City, State, Country]

---
FCiESENT NATIONALITY I/f non-U.S cifizen,
in6'icate kind of visa end expiration date)

SEX

New York, New York                U.S. Citizen                m Male   l-J Fenlol;:
              EDUCAl ION (Begin with baccalaureate training and include postdoctoral)

INSTITUTION AND LOCATION               DEGREE        YEA?             SCIENTIFIC
                                                CONFERRED             FIELD

The City College, New York, New York         B.S.S.
Cornell University, Ithaca, New York         M.A.
Cornell University, Ithaca, New York        Ph.D.

1950     Anthropology-Psychol$;:
1951     Anthropology
1960     Anthropology

HONORS

     Executive Board, Society for Applied Anthropology 1967-70, Vice-President, 1971-72,
President 1972-73, Past President 1973-74; Executive Board, Society for Medical
Anthropology 1971-73; Chairman, Committee on Ethics, American Anthropological Association,

-.-

Cultural Anthropology
RESEARCH SUPPORT I.% instructions)

I    Investigator

Research Grant: Genetic Studies in the Lake Atitlan Basin, Guatemala, GM 15593; $13,133
for the current year; $300,000 for the six year period 1967 - 1973;
National Institutes of Health. Howard Pi. Cann, M.D. - principal investigator.

RESEARCH kr:D/OR PROFESSIONAL EXPERIEF!CE IStar:im v/;thpresentposition, list traininqandexperiencerelcvant toarea Sfprojjcct Lasta:;
Or mat representative publications  Do not exceed 3 pages for each individL:al. )

1964-    Associate to Full Professor, Anthropology; Associate Professor, Pediatrics, Stan. :Jr,;1
1964-1970 Assistant Director to Acting Director, Program in Medicine and the Behavioral
      Sciences, Stanford University.

1962-1964 Research Associate to Associate Project Director, Navajo-Cornell Field Health
      Research Project, Dept. of Public Health, Cornell Univ. Xedical College.
1961-1963 Resident Anthropologist, Navajo-Cornell Field Health. Research Project at ?!z-q Fzrzs,
       Arizona; Resident in ?rofessional Practice, Russell Sage Foundation.
1955-1961 Senior Research Associate to Team Chairman, Foreign Areas Studies Division of Specid
     Oper.ations Research Office of the American University, Washington, D.C.

Publications (selected)

"Untreated Congenital Hip Disease: A Study of the Epidemiology, Natural History and Social
Aspects of the Disease in a Navajo Population". With D. Rabin, W. Arnold, R. Freiberger and
  G. Brooks.  Supplement, @erican Journal of Public Health 55, 2. 1965.
"Genetics of Diego Blood Groups in Guatemalan Indians: Use of Antiserums to Diego a and Diego b
Antigens". With Howard M:Cann and Betty Van West. Science, 162:1391, 1968.
"Neonatal Separation: The Maternal Side of Interactional Deprivation". With P.H. Leiderman,
R. Grobstein, M. Klaus.  Pediatrics 45:197, 1970.
                                           -

(publications continued)

                                                                           ---
RI-IS-398
Rev. 370                                         P-q


Publications (continued)

"Child Spacing in a Highland Guatemala Community". With Jean Jackson and
  Howard M. Cann.  In Culture and Population: A Collection of Current
Studies, Polgar, (Ed.).  Monograph 9, Carolina Population Center,
Chapel Hill, 1971. pp. 139-148.
"Health Care Experiment at Many Farms". With W. McDermott and K. Deuschle.
Science 175:4017, January 7, 1972, pp. 23-31.
"The Effects of Denial of Early Mother-Infant Interaction on Natemal
  Self-Confidence".  With M. Seashore, A. Leifer and P. Leiderman.  Child
Development 43:1203, 1972.
                 -
"Effects of Mother-Infant Separation on Maternal Attachment Behavior".
With A. Leifer, P. Leiderman and J. Williams.  In Press, Personality
  and Social Psychology.


       . .   . .*<. a._\"-" "C.
-                        4I\I"`-.I ICS,l
             --
                        _*      .    BIOGRAPi-!ICAL S1:E-l ~4
      (Give the folkwing infurmSiou fgr al/prof~~;rion;rlp~rsonn~/ listed on pqe 3, bsinning with the Princ!~al I,qvc<rigJto;.
               use CL~fltiI~~JofiOf~ I).xJ~' end follow the s?me genertil format for each person.)
iih$$                                                                           A--_
                                      TITLE                       SlRTHGI,l~E I.??cI, LX,;/, Vr.!
Herton R. Bernfield          Assok!ate Professor        April 9, 193S

PLhCE OF UIRTH (City, State, Country)          PRESENT NATIONALITY f/f non-L/S citizen,    SEX
Chicago, Illinois               ind'ca c kind of visa and expiration date)
                   ti. &.A.
                     .                                          m,,ble   El F- E,WZl!C
                EDUCkTiOirl (Begin with baccalaurca te training and include postdoctoral1
      `INSTITUTION AND LOCdTlON               DEGREE        YEA9             scIEr4nFic
                                                        CONFERFlED            FIrI.
                                                                           -_.e -. ".~.I____

University of Illinois, Urbana, Illinois     B.S.      1959       Eedicinc
Graduate Division, University of Illinois,             
  Chicago, Illinois                 M.S.       .1961      Biochemistry
College of ,le
      W dicine, 1Jniv. of Illinois,Chi. 1i.D,       1961

;----
HONORS                                               _L--                __ ly -__ -.-.----
Alpha ticga Alpha, 1959                 Ross Award for Pediatric Research, 197:
Borden Undergraduate Kesearch.kwatd in Medicine
C,V, Ibsby Company Research Award
I- --._-.._-.---_
hMJOl3 I<fISfIAliCW It415 HEST

         :~x~oneclease and Oligor~bucocleotide Synthesis, Seytcr~ber 1, 1370 to
August 30, 1975. Current year $26,816. Total $140, 381.
EIIH XX) 06763 Extracellular hlateria1.s .- end Embryonic O,,& Forrktion, Piay 1, 1372 -
April 30, 1977.  Current year $45,401, Total $203, 648.
National Foundation-March of Dimes  R-73-146 Morphogenetic Behavior of 'Human Cells
July 1, 1973 - June 30, 1934. Total $40,000.

--------_                           --        -r        -        l___--_l_ _-..-. ___
FiESEAtICtl I:!`~~l/Ot? Fi?O!:CSSIO~\14L. t:XFEHltl:CE (St-rt/nT t;~jthpr~~,~ntposi~;on, /irt I,rr:
                                               ininqandexp-Crience re:evant to arcs' of ;rti,+:t. LI;;
or most rcprrscnlativcpvLt~cattions.  DJ not exceed S p.ycs for fact, individ:lal.)
`1970 -                      -5
        Associate Professor, Departnqnc of Pediatrics, Stanford University Stilccl :

19vo ,:      Wdicixx; Stanford, California                                    .

        Co-Director, Xedical Scib?ntist Training Program, Stanford Universi.ty Sc?xo:
      Eledicinc; Stnnford, cz.1.ifcIrni.a. - -
1969~197-O    Associate Director, X~~dj.c.aJ. Scientist 'i'rrining Program, Stanford. Unjversir;

1967 -     School of Kcdicjne; Stanford, Czli.fcrnia ..
         Director,  Birth Defects !T-2.SP.arCii Center ',  Associate 'Director, d
                                                             y\;.r ty, -G > f c.- f:
                                                                           t.
        Clltnic, Dcpartmeili: of Yediatri.cs, Stanford University ScliGol of 1.;ediclx;
        Stanford, California
1967-1970   Assistact Professor, Department of Pediatrics, Stanford Ur;iversity StkxP,.
        p!edicine; Stanford, California
J 96-i-1367    Chief ksident, T~e~~arixir,~~t of Pcdbtrics, Stanford Medixal Center, Sta:;f>:-
f96S-1966    Research Invcsjgaxr,  Nstional 1Ixtitut.e of Child llcalth and 1k:;l:x Dr~;.~~:z:
        with Dr. Cliffo?:d Grcbstcin, i~i the D~p2rttncrit of fiiology, University 0;
19634965    California, San Diego; La Jolla, California
        Research Associate, btional Heart Institute, with Dr. brshall Kire?bcr:;:;
       Bethesda, 'Xarylznc?
1962-1963    Assistilnt Resident, 1'!e~?:~rtx.:rznt cf Padiatrlcs, Xew York ~losp~tril-Corni~3.!. I'..
         Center; Ilcw York, 33: i'urk
1961-1962    Rotating Internship, Research anh Education Hospitals, Unj.versify of I1El:
       Chicago, Illinois

                                                         P-171
i7iTxx--                                -u___--" . .                  -o--e- --_-.-_ . . . -. - .

llev. 3-m                            51


BioBrhical. Sketch of Dr. Merton Bernfield
--

1'8;~ Two, continuation of page one

PLblications
-... -- ---

Bernfield, M.R. and Nirenberg, M.W.:  RUA Codewords and Protein Synthesis.
   Iv,  The nuclcotide sequences of multiple codewords for phcnylaianine,
   serine, leucine, and proline.  Science 147, 479-484, 1965.

Bernfield, M.R. and Rottmzn, F.M.:  Ribonuclcase and Oligoribonucleotide
   Synthesis.   III.  Oligonucleotide synthesis with 5'-substituted
  uridine 2', 3'-cyclic phosphates,  J. Biol. Chem. 242 4134-4143,
                                                              ..-'
   1967.

Haenpaa, P.R., and Eernfield, N.R.:  Quantitative Variation in Serine Trcnsfc;l
   Ribonuclcic Acid durir;SEstrogen-Induced Phosphoprotein Synthesis in
   Rooster Liver.  Biochemistry g, 4926-4935, 1969.

Bernfield', K. R. :  Collagen Synthesis during Epitheliomesenchymal Inter-
   .actions.  Develop. Biol. 22, 213-231, 1970.

Eernfield, >I.R., and Vessells, N.K.:  Intra: and Extracellular Control of
   Epithelial Korphogenesis.  Develop. Biol. Supplentnt 4-, 195-249,
   1970.

Bernfield, M.R., Banerjee, S.D., and Cohn, R.H.: Dependence of ,Salivnry
  Epithelial 1lorphology and Eranching Morphogencsis upon Acid Huco-
  polysaccharide:Protein (Proteoglycan) at the Epithelial Surface.
   J. Cell. Biol., 2, 674-689, 1972.

52

P-/7&


-_.____ ------ ----- - ----------_-_-_  _ .__ ._-__ -.-. -_--.
NAME                                                                           -:. -
                                            -.I                                  

Howard M. Cann, M.D.
PLACE OF BIRTH (City, State, Country]

`Associate Professor of Pediatri s March 3i, 12;:
PRESENT NATIONALITY (If non-US citizen,     SEX
indicj te kind of visa and expiration da tel

Chicago, Illinois

           U.S. Citizen       I   D hlzie   0 Female
EDUCATION (Zqin cvi:h baccalaurcste training and include poztdoarorall

INSTITUTION AND LOCATION

I    DEGREE  I     YEAR
              CONFERRED I

SCIENTIFIC
  FIELD

University of Colorado, Colorado           B.A.

1950

Chemistry, Gun Laude
(General Studies)

University of Colorado School of Medicine, M.D.
Colorado.

  1954

                                           .                                                            ,
HoNDRS 1971 - 1972 National Institutes of Health Special Research Fellowship, Genetics
          Laboratory, Department of Biochemistry, University of Oxford, Englad.
    1966 - 1971 Scholar in Academia Medicine, The John and Mary R. Markle Foundation.
                                                                         I
MAJOR RESEARCH INTEREST            ROLE IN PROPOSED PROJECT k-M "`I -
                                                                           b   +---+

Human Somatic Cell Genetics

ASSOCIATE INVESTIGATOR

RESEARCH SUPPORT (See instructions)
Research Grant:  `Genetic Studies in the Lake Atitlan Basin, Guatemala, GM 15593;
$28,260. for the current year;  $300,000. for the six year period 1967 - 1973;
35% effort; National Institutes of Health.
National Institutes of Health Special Research Fello\Iship for sabbatical leave at the
University of Oxford (Professor-Walter F. Bodmer), Genetic Control of ?ucan Transa?2x:?:i.:
Antigens, l-F03, HD51401-01; $12,371. for one year, September 1, 1971 - August 31, 1'172,-
100% effort.

RESEARCH AND,`GR PROFESSIONAL EXPERIENCE /S:ar:irrg v.:ithp;esentposition, /i:t trainingandexperiancer~;rvant toarc's cf,?rl;,-!l; i.;: c :
Orfmxt representative publrcationr  Do not ex cecd 3 pqes for each ino. didual.)

September 1, 1971 -

1970 -

1964 - 1970

1963 -` 1964

1962 - 1963

1960 - 1962

1957 - 1960

1956 - 1957

1955 - 195G

1954 - 1955

August 31, 1972 National Institutes of Health Special Research
Fellowship, Gene tics Laboratory , Department of 3iochenistry,
..H+?gr,si ty of Osf ord , England.
Associate Professor, Department-of ,Pediafrics ti,Stanford Un,iv.ergity
School of Medicine.                                                      .W' )

Assistant Professor of Pediatrics, Stanford University School of
Medicine

Instructor, Department of Pediatrics, Stanford University School cf
                                                    .
Medicine.

National Science .Foundation Senior Postdoctoral Fellow, institute
of Genetics, University of Pavia, Pavia, Italy.
Postdoctoral Fellosr, Department of Genetics, Stanford University
School of Medicine, Palo Alto, California.
United States Pub1j.c Health Service, Washington, D.C., Director,
National Clearinghouse for Poison Control Centers. Draft
Classif ication: 4A.

Assistant Resident and Senior Resident in Pediatrics, Stanford
University Hospital, San Francisco, California.
Assistant Resident in Pediatrics, Cniversity of Colorado >Iedica1
Center, Denver, Colorado.
Rotating Intern at San Francisco Hospital, University of California
Service, San Francisco, California.

RHS-393
Rev. 3-m

Page 17

--

b/7.3


'Committees and Consultant

1968 -      Committee on Drugs, American Academy of Pediatrics.
1965 - 1968   Genetic Consultant, Hereditary Defects Unit, California State
         Department of Public Health.
1965 -     Genetic Consultant, Congenital Malformations Branch, Epideniolcgy
        Section, Dental Health Center, U.S. Public Health Service.

Societies and Organizations
American Academy of Pediatrics
American Association of Poison Control Centers
American Federation for Clinical ,Research
American Public Health Association, Epidemiology Section
American Society of Human Genetics
Western Society for Pediatric Research

Specialty Board Certification

19tio        American Board of Pediatrics

State and National Certification
L__-

Diplomate of National Board of Medical Examiners
Licensed to practice medicine in California and Colorado.
                                                      _

BIBLIOGR!?HY

1.  Cann, H.Ff. and Herzenberg, L.A.:  In Vitro Studies of Mammalian Somatic Cell
                                             --
   Variation. I.  Detection of H-2 i?henotype in Cultured Mouse Cell Lines, J.

2.  Cann, H.hf. and Herzenberg, L.A.:  In Vitro Studies of Xaznalian Somatic Call
                                             --
   Variation. II.  Isoimmune Cytotoxicity !*Jith a Cultured Xouse Lycphoma and
  Selection of'Resistant Variants.'. J;,, Expi-.Med. .2..=:267,- i963...:. . . ~... ., ,-,. ,_,. :'

3.            Segregation Analysis o) Juvenile Diabetes   I
- Berrai, I. and Cann, H.M.:
  J. Med. Genet. 2:8, 1965.                     Xellitus, /
                                    I

4.   Cann, H.M.:  Computer Analysis in Human Genetics: *Segregation Anal::sis and    1
  Demographic Genetics, Ann. New York Acad. Sci. 126:728,  1965.

5.   Barrai, I., Cann, H.M., Cavalli-Sforza, L.L. and DeNicola, P.:  The Effect of 4
  Parental Age on Rates of Mutation for Hemophilia and Evidence for Differing
  Mutation Rates for Hemophilia A and B, American J. Hum. Genet. g:175, 1968.

6.  Cann, H.M. and Cavalli-Sforza, L.L.:  Effects of Grandparental and Parental
  Age, Birth Order and Geographic Variation on the Sex Ratio of Liveborn and
  Stillborn Infants, American J. Hum. &net., 20:381, 19G8.
                                                        -

7.   Cann, H.Ef. : Principlks of HLnan Inl-eritanca, in Human Genetics, Birth D~fectd,
  Original Article Series, The National Foundation, Vol. IV, November, 1958.

Qo9e 18

?-/7f/

            __ -
*u.s.cPo. 1910 - l/J-cqi


8.

9.





10.



11.







12.



13.



14.

BIBLIOGRAPHY (Continued)

Cann, H.&l., Van West, B., and .3arnctt, C.R.:  Genetics of Diego Blood Grox?s in.
Guatemalan Indians:  Use of Antiserums to Dia and Dib Antigens, Science, 162:
1391, 1968.

Greenstein, R.M., Harris, D.J., Luzzatti, L.L. and Cann, H.M.:  Cytogenetic
Analysis of a Boy with the XXXY Syndrome: Origin of the X-Chromosomes;
Pediatrics, 45:677, 1970.
             -

Dungy, C.I., Aptekar, R.G. and Cann, H.M.:  Herediatry Hydrometrocolijos with
Polydactyly in Infancy.  Pediatrics, 47:138, 1971.

Barnett, C.R., Jackson, J. ind Cann, H.M.:  Childspacing in a Highland
Guatemala Community, in Culture and Population:  A Collection of Current
Studies, Polgar, S. (ea.), Carolina Population Center, Univ. North Carolina,
Chapel Hill, 1971.

Watson, W. and Cann, H.M.:  Genetic Counseling in Dermatology. Pediat. Clin.
N. Ame,rica 5:757, 1971.

Watson, W., Cann, H.M., Farber, E. and Nall, I.,.: The Genetics of Psoriasis.
Arch. Dermatol., In Press.                   -- -

Bodmer, W.F., Bodmer, J.G., Coukell, A.,.Cann, H.M. and Van Widest, B.:   SO"2
Further Data on the Joint Segregation of HL-A and Haptoglobin. Ann. Hum. Gene:.
(London), In Press.

Book Review

Carter, C.O.:  Human Heredity, Cytogenetics 1:55, 1963.

HONORS (Continued from Page 17)
1964       Society for Pediatric Research
1963 - 1968   Research Career Development Award, National Institutes of Health,
           U.S. Public Health Service
1962  - 1963  National Science Foundation Senior Postdoctoral Fellowship.


CURRICULUM VITAE

NAME:  L. L. Cavalli-Sforza

BIRTHDATE:  January 25, 1922

NATIOXALITY:  Xtalian

Married, four children

ACADEXIC HISTORY:

  University of Pavia, Italy

  Cambridge University, U.K.

  Universities of Pavia and Parma, Itily

EMPLOYXENT RECORD:

   1943-44

Intern of Genetics Department, University of Pavia
(then located at Istituto Italian0 di Idrobilogia, fc.rSar-ia,
Italy)

1944-45

1945-47

Intern and resident at Vatbania Hospital, Italy

Assistant in research, Istituto, Sieroterapico Xilanese,
Milan

1948

Scholarship in genetic statistics, John Innes Horticultural
Institution, London (U.K.)

1948-50

Research Fellow and Assistant in Research, Genetics De-
partment, Cambridge University (U.K.)

1950-57

Director of Research in Microbiology, Istituto Siero-
terapico Mlanese (Milan, Italy)

1951-54

Lecturer in Genetics and in Statistics, Science Faculty
(part-time), University of Parma and L'niversity of Pavia,
Italy

1958-62

1962-70

Professor of Genetics, University of Parma, Italy
Profeisor of Genetics, and Director of Istituto di Genetica,
University of Pavia (Italy)

1970-       Professor of Genetics, Stanford University, Palo Alto,
Present        California

TITLE:  Professor of Genetics

PLACE OF BIRTH: Genoa, Italy


SEX:  Male

M.D., 1944
iA .,  1950

Libera Docenza, 1952 (!Ecrobiolog:;1

           1952 (Genetics)

           1960 (Statistics)

P-/76


Curriculum Vitae - 2
L. L. Cavalli-Sforza

Earlier Sojourns in the United States:

1954

Rockefeller Fellow (six months) at Genetics Department,
University of Wisconsin, Madison, Wise.

1958       Research Associate tit University of Wisconsin

1960



1962

Visiting Professor at Genetics Department, Stanford
University (three months)
           *
Visiting Professor at Genetics Department and Statistics
Department, Stanford University (one month)

1964-65

Visiting Professor at Department of Biology, Harvard
University (three months)

1968-69

VisitTng Professor at Genetics Department, Stanford
University (one year)

Expeditions to Africa in the Fall-Winter periods of 1965-66, 1966-67, 1967-
          68, 1968-69, 1969-70, 1970-71, conducting research in the
            following countries:  Central African Republic, CaEerocn,
            Zaire and Ethiopia

ACADEMIC HONORS:

President, Biometric Society, 1967-68
Vice President, International Congress of Genetics, Tokyo,
1968

Foreign Honorary Member of American Academy of Arts and
Sciences, 1969 '
Royal Anthropological Institute of Great Britain and Ireland,
Huxley Award in Anthropology, 1972

MILITARY SERVICE:   Medical Officer in the Italian Army, 1947-48

MAJOR RESEARCH INTERESTS:  Bacterial' genetics; presently, human population
            genetics and allied fields.

RESEARCH ACTIVITY:

  Apart from early work in the fields of immunology and Drosophila popu-
lation genetics, research activity during the first fifteen years was concen-
trated mostly on bacterial genetics.  Among results of the analysis of sex
and recombination in E. coli strains:  the finding of the first coli mutant
with a high recombination frequency (Hfr), of Hfr linkage with chromosome
markers, and of the infective transmission of mating capacity (F episomes).
Among results on the analysis of drug resistance in bacteria: a quantitative

p-177


Curriculum Vitae - 3
L. L. Cavalli-Sforza

technique for sib selection, demonstrating the spontaneous origin of drug
resistant mutants; the analysis by crossing of polygenic inheritance for chlor-
amphenicol resistance; streptomycin resistance as a genetic modifier.  Some of
this,work was in collaboration with the Lederbergs.

  From 1954, activity was concentrated increasingly on human population
genetics -- the analysis of a population in the Parma Valley showed that
microgeographic variation can largely be explained by drift alone.   This
required the development of special techniques, including the computer simu-
lation of human populations, methods for the reconstruction of differentiation
of racial groups, and the characterization of selective and random causes of
variation.  Several archives, from consanguinity records to parish books, have
been the subject of study to extract genetic information. A full description
of the history of consanguinity in Italy has resulted.  Demographic analysis
of pari,h book records by computer is still in progress.

  An analysis, during six winters, of an African Pygmy population with the
help of a large team of collaborators allowed to accumulate information on the
population structure of hunters-gatherers, to detect new genetic types, to
account for the physiology of 10:~ stature in Pygmies and other problems of this
population.  Yost recent work has centered on the impact of technolcgical
developments and cultural change, essentially the domestication of plants and
animals, on human biological evolution.

AUTHOR OF BOOKS:

"The Genetics of Human Populations" in collaboration with W. Bodmer (now
Professor of Genetics at Oxford, U.K.), and a short handbook of biostatistics.

/3-/7F


100.  A. ?lORONI, 1960. Sources, reliability and usefulness of consanguincity

data with special reference to Catholic records.  International Sy:;n-
osium of Demography and Genetics, Geneva.

101.  L.L. CAVALLI-SFOEZA, 1960.  Indagine'speciale sulla consanguineita dei
     matrimoni.  Istituto Centrale di Statistica, Roma.

102.  A. FlORONI, 1960.  Analisi metodologica de1 rilievo della consanguineita.
     Parte Prima - Legislazione e materiale religioso e civile italiano per
   il rilievo della consanguineita.
     fast. 3: 199-247.         Folia Hereditaria et Patholcgica, 2,

103.  L.L. CAVALLI-SFORZA, 1961.  Lczioni di metodologia statistica per
      ricercatori - 11 numero di osscrvazioni necessaris in esperinenti
    semplici e l'efficienza dell'esperimento.  Istituto di Calcolo
   della Probabilita, Istituto di Statistis, 4: Articolo Terzo.
                                                          -
                                              1

104.  L.L. CAVALLI-SFORZX, 1961.  Un netodo per la stima della frequenza di
    ,nutazione dell'uomo; risultati preliminari.  Atti X.G.I., r1: 151-162.
                                                                           -

105.  L.L. CAVALLI-SFORZA, J.A. ROPER, G. SERXO!ITI, S.I. ALIKIXXAS, D.X.
   HOPKOOD and J.F. STAL'FFER, 1YGl. Panel discussion: microbial genetics
    and its application to fermentations.  Sci. Repts., 1st Super. San%l-.a,
    1: 441-483.

106.  L.L. CAVALLI-SFORZA, 1961.  Biologia e genetica.  L'Xteneo Parr.ens;l:   -.,.y-
                                                                           --' -\-:-IT,
     fast. 6: l-20.

107.  PI. ?fAINX-RDI, I. BAR?&, L.L. CXVALLI-SFORZX, 1962.  The distribution of t:le
     number of collateral relatives.  Xtti X.G.I.,  VII: 123-130.


108.  L`I>. CAVALLI-SFORZX, 1960.  !,fetodi e problemi nella analisi degli eff2tt.i
    biologici delle radiazioni sull'uomo. VII Basser.na I~.tsrnazi~n~l~
                                                                   -
    Elettronica e Sucleare, ;\tti Ufficiali de1 Congresso Scitnrific?.  %=a.
                                                              --

109.  L.L. CAVALLI-SFORZA, 1962.  Studying human evolution by census and computer.
    New Scientist, 13: 92-93.

110.  I. BARRAI, L.L. CAVALLI-SFORZ.! and A. XORONI, 1962.  Frequencies of pedi-
    grees of consanguineous marriages and mating structure of the populaticn.
    Ann. Hum. Genet., 25: 347.
                             -

111.  L.L.CAVALLI-SFORZA, 1961.  I fattori letali nell'Uom0. Prim0 Simposio
    di Statistica !ledica, Roma, 11-12 giugno 1961.

112.  L.L. CAVALLI-SFORZA, 1962.  Risposta ad alcune critiche sul neodamrinisco.
    Rendiconti della Classe di Scienze Fisiche, ?fatematic!le e ?jaturali;
    Serie VIII, Vol. SXXI, fasc.1.

113.  L.L. CAVALLI-SFORZA, 1962'.  Indagine speciale su alcune caratteristiche
    genetiche della popolazione italiana.  Note e Relazioni N. 17, Istltuto
     Centrale di Statistica.


114.  L.L. CAVALLI-SFORZA, 1963. Sir Ronald Fisher. Patti A.G.I., VIII: l-8.

115.  G. MODIANO, A.S. BENERECETTI-SANTACHIAPA, F. GONANO, A. CAPALDO e
   L.L. CAVALLI-SFORZA, 1963.  Analisis della popolazione leccese per
   gruppi sanguigni G6PD, aptoglobine, e transferrine. Atti A.G.I., S:
     64-79.

116.  L.L. CAVALLI-SFORZA, 1963.  Nuclear threat and human genetics.  Exposure
    of man to radiation in nuclear warfare.  Elsevier Publishing Company,
     Amsterdam.

117.  L.L. CAVALLI-SFORZA, 1963..  The distribution of. migration distances,
    models and application to genetics.  Entretiens de Monaco en Science
    Humaines, Preniere Session, 24-29 Xai, 1962.  Human Displacements,
     139-15s.

118.  E.M. LEDERBERG, L.L. CAVALLI-SFOBZA and J. LEDERBERG, 1964.  Interaction
    of streptomycin and a suppressor for galactose fermentation in E. coli
     P-12.  P.N.X.S.,  51: 678-682.
                            -

119.  I. BARRAI, L.L. CXVALLI-SFORZA and >I. YAINAPDI, 1964. Testing a model
     of dominant inheritance for metric traits in man.  Heredity, 19: 651-668.
                                                                           -

120.  A.W.F. EDIJARDS and L.L. CXYXLLI-SFORZA, 1964.  Reconstruction of c-solution-
     ary trees.  Svstematic Association Publication X.6, ?henetic and rl:..-1c-
   Benetic Classification, 67-56.

121.  L.L. CAVALLI-SFOBZA and A.!d.F. EX~ARDS, 1964. Analysis of human evolution.
    Genetics Todav (Proceelinos of the XI International Congress of Genetics,
     The Hague, Netherlands, September, 1963. ;/oL.;r: y&z-7&w

122.  L.L. CAVALLI-SFORZX, I. BARRAI and A.W.F. EDb!ARDS, 1964. Analysis of
    huinan evolution under random genetic drift. Cold Soring Harbor
   Symposia on Quantitative Biolc<y, 24: 9-20.
                                               -
                                                             /-
123.  I. BARRAI, L.L. CAVALLI-SFORZX and A. MORONI, 1965. Record linkage from
    parish books.  >!athematics and Comouter Science in Siologv and Y?edicine,
    Medical Research Council, 51-60.

124.  A.W.F. EDXARDS and L.L. CAVALEI-SFORZA, 1965. A method for cluster analysis.
    Biometrics, 21: 362-375.
                      -

125.  G. MODIANO, A.S. BENERECETTI-SANTACHIAR4, F. GONANO, G. ZEI, A. CAPALDO and
    L.L. CAVALLI-SFORZA, 1965.  An analysis of ABO, XX, Rh, Hp, Tf and
   G6PD types in a sample from the human population of the Lecce Province.
    Ann. Hum. Genet., London, 29: 19-31.
                                      -

126.  L.L. CAVALLI-SFORZA, 1965.  Interesse genetic0 dei matrimoni consanguinei.
    Atti III Corso di Acgiornamento Professionale "Ereditarieta nella
    Patologia e nella Clinica" dell'ordine dei Xedici di Rona e Provincia,
      95-101.


127.  CAVALLI-SFORZA, L.L. and A.W.F. EDWARDS, 1965.  Estimation procedures
    for evolutionary branching processes. Bulletin of the Internaticnel
    Statistical Institute, Proceedings of the 35th Session, Beograd.

128.  CAVALLI-SFORZA, L.L., 1966.  Genetic drift in popolazioni umane. Atti
     A.G.I.,  11: 3-50.
        --

129.  CAVALLI-SFORZA, L.L., 1966.  Population structure and human evolution.
     Proceedings, Royal Societv of London, 194: 362-379.

130.  CAVALLI-SFORZA, L.L., :4: Kimura and I. Barrai, 1966. The probability
    of consanguineous marriages.  Genetics, 54: 37-6Q.
                                                      -
                                  *

130.  DE NICOLA, P., I. Barrai, H.M. Cann and L.L. Cavalli-Sforza, 1966.
bis    Mutation rate in hemophilia A and B, and effect of parental age.
    Proc. 3rd Congr. Wld. Fed. Hemophilia, Paris 1965; Eibl. haenat.,
     fast. 26: 91-93, Sew York.        r
                                             I

131.  C.LVALLI-SFORZA, L;L., 1967. Human populations, Heritage from Xendei.
                                                                           --
     Ed. Alexander Brink.  University of Wisconsin Press, >iadison, 309-331.

132.  CAVALLI-SFORZA, L.L. and A.W.F. Edwards, 1967. Phylogenetic analysis.
    Models and estimation procedures.  Am. J. Human Genetics, 19: 233- 257;
                                                                           -
     and Evolution, 21: 550-570.
                         -

133.  CAVALLI-SFORZA, L.L., 1967.  Some old and new data on the genetics
    of human populations.  T'ne Alabama Journal of :,!edical Sciences, 2:
     376-381.

134.  CAVALLI-SFORZA, L.L., 1967.  Da ?lendel alla Genetica Umana ?Ioderna.
    "Cosi Comincio la Genetica", Acta Geneticae ?!edicae 2: Gemel-
    lologiae, Roma, 103-130.

135.  RIMOIN, D.L., T.J. Xerimee, D. Rabinowitz, V.X. XcKusick and L.L.
    CAVALLI-SFORZA, 1967.  Growth hormone in African Pygmies.  The
    Lancet, 523-526.

136.  CAVALLI-SFORZA, L.L. and G. Zei, 1967.  Experiments with an artificial
    population.  Proc. III Intern. Congr. Hum. Genet., Johns Hopkins
    Press, Baltimore, 473-478.

137.  CAVALLI-SFORZA, L.L., 1968.  Introduzione all'Impiego dei Calcolatori
    in Genetica Umana. Quaderni de "La Ricerca Scientifica," 47: 13-16.
                                                                           -

138.  BODMER, W. and L.L. CAVALLI-SFORZA, 1968. A migration matrix model
   for the study of random genetic drift. Genetics, 59: 565-592.

139.. BARRAI, I., H.M. Cann, L.L. CAVALLI-SFORZA, P. De Nicola, 1968.
    The effect of parental age on rates of mutation for hemophilia,
    and evidence for differing mutation rates for hemophilia A and B.
    Amer. J. Hum. Genet., 20: 175-196.
                                  -


140.  CAVALLI-SFORZA, L.L. L.A. Zonta, F. NUSSO, L. Bernini, W.W.W. De
     Jong, P. Meera Khan, A.K. Ray, L.N. Went, M. Siniscalco, L.E.
    Nijenhuis, E. Van Loghem and G. Nodinao, 1969. Slitdies on
    African Pygmies. I.  A pilot.investigation of Babinga Pygmies
    in the Central African Republic (with an analysis of genetic
   distances). F+.J~-/u.&~;L/: 34-2 -279,

141.  PAOLUCCI, A.M., M.A. Spadoni,. V. Pennetti and L.L. CAVALLI-SFORZX,
     1968.  Elevated serum phenylalanine-tyrosine ratios in African
    Pygmy and non-Pygmy subjects.  Unpublished.

142.  RIVA, S., I. Barrai,+ L.L. CAVALLI-SFORZA and A. Falaschi, 1969.
    Dependence on the buoyant density of single-stranded DNA on
     base composition. J. Yol. Biol., 45: 367.
                                                - .

143.  SANTACHIAII-BESERECETTI, A.S., G. Modiano e L.L. CAVALLI-SFORZX,
     1968.  Studio de Alcuni Pol!imorfismi Enzimatici dei Globuli
     Rossi nei Pigmei Babinga. Atti A.G.E., 12: 99-101.
                                                      -

144.  CAVALLI-SFORZA; L.L., 1968.  Teaching of biometry in secondary
      schools.  Biometrics, 24(3): 736-740.
                                  -

145.  CAVALLI-SFORZA, L.L., 1968.  Recherches Genetiques sur les Pygnees
     Babingas de la Republique Centrafricaine. Cahiers de La I.:abol-e,
    5(l): 19-25.

146.  CANN, H.?l. and L.L. CXVXLLI-SFCRZA, 1968. Effects of grandp&rental
    and parental age, birth order, and geographic variation on the

      sex ratio of live-born and stillborn infants.
     Genet., ZO(4): 391-391.
                  -

147,  CAVALLI-SFORZA, L.L., 1968. Research on African
    national Biological Programme.  Biology of Xan

.tier. 3. Hum.

Pygmies.   Inter-
in Africa,

Warsaw meeting, 1968.  N.78 Yaterialv I Prace Antrooologiczne
Wroclaw (Poland), 1970.                     --

148.  CAVALLI-SFORZX, L.L., 1968. Population structure. Proceedinss
    of the Conference on Computer Xnnlications in Genetics, Honolulu,
      Hawaii.                   8

149.  CAVALLI-SFORZA, L.L., 1969. Human diversity. Proc. XII Intern.
    Congr. Genet., Tokyo, Japan, 1968, 2: 405-416.

150.  RIMOIN, D.L., T.J. Merimee, D. Rabinowitz, L.L. CXVXLLI-SFORZh and
    V.A. McKusick, 1969.  Peripheral subresponsiveness to human growth
    hormone in the African Pygmies. New England Journal of ?!edicine,
     281: 1383-1388.  (December, 1969).

151.  CAVALLI-SFORZA,, L.L., 1968.  Studi sulla struttura genetica di una
    popolazione italiana.  Le Scicnze, L(4): 7-19.


152.  RIIIOIN, D.L., T.J. Merimee, D. Rabinowitz, L.L. CAVALLI-SFORZX

and V.A. McKusick, 1969.  Genetic aspects of isolated growth
hormone deficiency. Proc. Int. Symp. on Grcwth Hormone, !4ilan,
11-13 September, 1968, 418-432.

153.  MERIXEE, T.J., D.L; Rimoin, D. Rabinowitz, L.L. CAVALLI-SFORZA and
     V.A. McKusick, 1969.  Metabolic studies in the African Pygmy.
    Unpublished.
154.  PAOLUCCI, A.!4., M.A. Spadoni, V. Pennetti and L.L. CXVALLI-SFORZX,
     1969.  Serum free amino acid pattern in a Babinga Pygmy adult
    population. Amer. J. Clin.'Sutrition, 22(12): 1652-1659.
                                                     -
155.  NALCOLN, L.A., P.B. Booth -.nd L.L. CAVALLI-SFORZA, 1971.   Inter-
    marriage patterns and blood group frequencies in the Bundi
    people of the New Guinea highlands. Human Biology, 43(2).
                                                                    -
156.  BAR&X, I., L.L. CAVALLI-SFORZA and A. Yoroni, 1969.  The prediction
    of consanguineous marriages.  Japan Journal of Genetics, G<(l):
                                                                          -
     230-233.

157; CAVALLI-SFORZA, L.L., 1969.  Genetic drift in an Italian population.
    Scientific American, 221(2): 30-37.

158.  EDWARDS, A.W.F. and L.L. CXVALLI-SFORZA, 1969. Affinity as revealed

    by differences in gene frequencies. Unpublished.

159.  BARRAI, I., L.L. CAVALLI-SF&A and A. Xoroni, 1969. Demography
                                                       --_
    and genealogy. I. Family reconstitution by computer. World

Conference on Records and Genealogical Seminar, Salt Lake City,
Utah, 5-8 August, 1969..

P-/P3


2i.

160.  CAVALLI-SFORZA, L. L;, 1969.  Present trends in basic biomedical research.

C.I.O.~LS, Table Ronde organisee avcc l'assistance de 1'O.X.S. et de l'LiXESC0,

Geneve, S-10 Octobre 1969.

161.  CAVALLI-SFORZA, L. L., F. COXTERIO and A. NORON. 1970. Valutazione de1
   carico genetic0 in relazione a matrimoni fra consanguinei.  Societa Italiana
   di Ostetricia e Ginecologia. 54' Congress0 Nazionale, Elano, 16-19
                                      *
    Settembre 1970.

162.  BODKER, W., and L. L. CAVALLI-SFORZX, 1970.  Intelligence and Race.   Scizntif.'-=.
                                                                           --
   American, 223: 19-29.                   

163. CAVALLI-SFORZX, L. L., 1970. Proble'ms and prospects of genetic analysis of
   inteliigence at the intra and interracial level. Disadvantsned Child. 3:
   111-123. Ed., J. Hellcuth, Brunner >!azel, Inc., Xe:J York.

164.  CAVALLI-SFOUA, L. L., 1971.  Sisiiarities and dissimilarities of socieculzxrz1

   and biological evolution.  In Xbth.xzntics in t:?e ~~rch~eolc~icsl 2-2 I-`.lr:rl::l
                                                                           ---.

    Sciences.  Eds. F. R. Hodson, D. G. Kendall, and P. Tautu.  Ediaburzh ~~<-.-~r~ 1:;:
   Press, Edinburgh. 535-541.

165. KIDD, I;. K. and L. L. CXVX,LI-SFOXX, 1971. Sumber of characters esamir.zi

    and error in reconstructicn of evclutionary trees.  In vht;~~~.~tf=S ;- -.-z
                                                                           -.. -..-

   Archaeoloqical and 'Ilsccrical Scf~~ces. Eds. F. R. Hodscn, D. G. Kendall,

    and P. Tautu.  T-,dizhurgh Cniv2rsi.E:; ?ress, Edinburgh. 335-346.
166.  sKoLsIcK, ?f. H., A. XORO?z, C. CAhYISZS and L. L.-hYhLLI-SFOXZ-4, 1?71.
   The reconstruction of genealogies zrom parish books. In >!ethemstics I::


-167.

168.

169.

170.

171.

172.

173.





174.





175.



176.







177.

22.

CAVALLI-SFORZA, L, L., and W. BODXER. 1971.  THE GEXETICS OF HLXU POPiTLXTIO:;S.
W. H. Freeman and Company, San Francisco.
A?DEEM, A. , and L. .L. CAVALLI-SFORZA.  1971.  Measuring the rate of spread
of early farming in Europe. Man, 4-+l&w &b?4- L ?f
AYMER?44~~, A. J ., and L. L. CAVALLI-SFORZA, 1971. A population model for the
diffusion of early farming in Europe.  Proceedings of the Research Sc>inar in
Archaeology and Related Subjects, Sheffield, Dec. 13-17, 1971. In press.
KIDD, K. K., P. ASTOLFI, L.' L. CAVALLI-SFORZA, 1971.  Error in the reconstruc-
tion of evolutionary trees: In GEXETIC DISTUCE, Ed., J. F. Crow,
in press.

CAVALLI-SFORZA, L. L., 1971. Pygmies, an example of hunters-gatherers,
and genetic consequences for man of ldomestication of plants and animals.
IV Int. Conq. !r?uman Genetics, Paris, Proceedines.  In press.
RINOIS, D. L., T. J. XkRI?IEE, and L. L. CAVALLI-SFORZA, 1971.  Growth h0rzor.e

unresponsiveness in the African Pygmies.  IV Int. Conpr. lhzan Genetics, Par<?.
Proceedinrs, in press.
SKOLXCI;, Y. H:, A. XOROSI, and L. L. CAVALLI-SFORZA, 1971.  The population
structure of Parea Valley, Italy.  IV Tnt. Conz. Hnman Ganetics, Paris,
Proceedings.  T+yed3A&ze&?~

BODXER W F
       ? o  ., and L. L. CAVXLLI-SFORZA, 1971. Variation in fitness 2r-k r,ole-

cular evolution.  Proceedings of the Sixth Berkelev STTnosium an :!athezz:Iczl
                                                ;;; L/ys,   -;'_r'
'Statistics and Probability, Conference on Evolution, X3ri1, 1971.,X"k-+&>.
CAVALLI-SFORZA, L. L., 1971.  I1 &co genetic0 nell'uoirio. Atti A. G. I.,
16:

ZEI, G., A. XORONI, and L. L. CAVALLI-SFORZA. 1971. Age of consanguineous
marriages.  In "Genetique et Populations.  Hommage a Jean Sutter." Institut
National d'Etudes Demographiquei. Travaux et Documents. Cahier n060. Presses
Universitaires de France. 147-153.

KIDD, K. K., and L. L. CAVALLI-SFORZA, 1971. An analysis of the genetics of
schizophrenia.  IV Int. Congr. Human Genetics, Paris, ?roCeedinyS. h


-,  Sde-G&.


178.

179.

180.

181.

182.

183.

BOD>IER, W., and L. L. CAVALLI-SFORZX, 1971. Nigration matrices. IV
Int. Congress Human Genet., Paris, Proceedings.  In press.


CAVALLI-SFORZA, L. L., and K. K. KIDD, 1972. Considerations on genetic

models of schizophrenia.  Xeurosciences Research Program Bulletin. r:5:
pws./&Z+a@,/~74

CAVALLI-SFORZA, L. L., 197.2.  Book review (David Rosenberg) in Psychiatry.

SGARMELLA-ZOSTA, L., and L. CAVALLI-SFORZA, 1972. A method of the

detection of a denic cline.  Proceedings, Workshop on Population
Structure, Hawaii.  In press.


CAVALLI-SFORZX, L. L.; and !I. W. FELDXAS, 1972. Models for cultural

inheritance. I.:  Group mean and within group variation. Theoretic?1
Population Biolozv.  In press.


CAVALLI-SFOPJX, L. L., and C. CASNISGS, 1972.  Human Pcnulation Strcct*ire
                                                                           2

Advances in Human Genetics.   Ed. K. Hirschhorn.

184..  CAVALLI-SFORZX, L. L., 1972. Phylogenetic analysis. In Histoconpa:ibilL::*

Testing, Nunksgaard, Copenhagen, 1972.

185.  CAVALLI-SFORZA, L. L., 1972. Elements of human genetics. An Addison-
    Wesley Xodule, Addison-Wesley, Reading, >!ass.  In press.

                                         .

186.  CAVALLI-SFO.RZA, L. L., 1972.   Evolution.  In Enciclopedia de1 Sovecentro.
  Fd. V. dappelletti.  In press.

187.  CAVALLI-SFORZA, L. L., 1972.  Some current problems of human population
    genetics.  Am. J. Hum. Gen.  In press.

                               :*
188.  CAVALLI-SFORZA, L.L. and N'. Yasuda, 1972. The evolution of surnames. For
    submission to Theoretical Fonulation Biology.


--
pLj\CE OF` 8111 iti lo:;<, S!src; Courr!ryj
S$n Francisco, California U.S.A.

     INSTlTUi-ION AN0 LOCATION               OEGREE


Stanford university                       B.A.
Stanford University School of Medicine   M.D.

  YEAR             SCIE::TIFIC
COfdFEflFiED             FIELO

1958      Basic Medical Science:
1961      Medicine

MAJOR AESE23CH INrEfiEST

flOLE ItJ PROPOSC-0 PROJECT

Endocrine Bioshemistry

RESEA.HCH SL;i~?O:iT ISA2 ;f/,:fIuc!,o:ls~
1. Support from September 1969 to September 1972:HD02147 (principal investigator: Norman
Kretchmer, M.D.,Ph.D.) Program Grant in Human Development, from which I was allocated
$25,000 per year, which supported ongoing projects herein described.
2. Current.Research Support: HDU6&68-01, Biochemical Studies of Male Sexual Maturation,
from 9/l/72 to 8/31/75. Budget for 9/1/72,to 8/31/73 is $26,342.
3. Patient care costs for ongoing projects are derived from RR-70, General Clinica.
Research Centers Branch, and RR-00-81-11, Premature Infant Clinic Research Centers
Branch, both of NIH.

RESEARCH AxC.Oi? P3OiE2SIOX;IL E XPE ZIEiJCE (StJrtrr:g v,,tn pfescnt position, list trainhp andexpcricncc fcfCvJftt :O area Of prO,'?C:  LL: d.
ormost rcprcsenratwc puohc~tions.~ 00 not cxcccd 2 pqcs for each individual.)
Assistant Professor of Pediatrics, Division of Metabolism, Stanford Medical School,
    "1969-present                                                          .
Post-doctoral Fellow, Department of Biochemistry & Molecular--Biology, Cornell University,
    Ithaca, New York, 1967-1969 .'.

Post"doctora1 Fellow in Metabolism, Department of Pediatrics, Stanford Medical School, 1967
Chief Resident in Pediatrics,Sta.nford Medical School, 1966
Post-doctoral Fellow in Metabolism, Department of Pediatrics, Stanford Medical School,

       1965-1966                                             -.
Resident in Pediatrics, Stanford Medical School, 1964-1965
Pediatrician, USAH, La Chapelle Saint Mesmin, France, 1962-1964
Intern, University of Utah Medical School, Salt Lake City, Utah, 1961-1962

Societies:  American Association of University'Professors
        American Association for the Advancement of Science
       Society for Pediatric Research
       Western Society for Pediatric Research
       The Endocrine Society

Society for Developmental Biology

Licensure:  National Poard of Medical Examiners, 1952
        State of California, 1964
       Diplomate, American Board of Pediatrics, 1967
'Awards:  Special Fellotiship, American Cancer Society, 1967-1969

P-/87


Publications:

1.


2.


3.

4.

5 `

6.

7.


8.

9.


!O.


Ll.




L2.




b3.

                                                                L#c,>:,.: `. ., .?' -. -
Loyter, Aor R.O. Christiansen, and E. Racker. Respiratory control in s*&tito-
chondrial particles and Ca++ transport. Biochem.Biophys,?.es,Com?, 29:450, 1957.
Christiansen, R.O., L.A. Page, and R.E. Greenberg. Glycogen storagein a
hepatoma: Dephosphophosphorylase kinase defect. Pediatrics 42:694, 1968.
Christiansen, R.O., A. Loyter, and E. Racker. Effect of anions on oxidztive
phosphorylation in submitochondrial particles. Biochem.Biophys.Acta lEg:207,
1969.
Loyter, A.; R.O. Christiansen, H.. Steensland, J. Saltzgaber, and E. Racker.
Energy-linked ion translocation in submitochondrial particles. I. ~a++
accumulation in submitochondrial particles. J.Biol.Cnem. 242:4422, 1969.
Christiansen, R.O., A. Loyter, H. Steensland, J. Saltzgaber, and E. Racker.
Energy-linked ion translocation in submitochondrial particles.  II. Prnrnrties
                                                                           -v-d-
of submitochondrial particles capable-of Ca++ translocation. J.Biol.Chea. 244:
                                                                           --
4428, 1969.
Loyter, A., C. Burstein, R.O. Christiansen, and E. Racker. The polarity of the
mitochondrial membrane, Proceedings of the Bari Symaosium, B.B.A. Library,
Adriatica Editrice Bari, p. 235, 1970.
Greenberg, R.E., and R.O. Christiansen. The critically ill child. XV.
Hypoglycemia. Pediatrics 46:915, 1970.
                --
Loyter, A., R.O. Christiazen, and E.  Racker. The two sides of the mitochon,d:ial
membrane in B. Chance (ed.), Johnson Foundation Symposia: "Probes of structx-2
and function of macromolecules and membranes", p. 407, Acader:c Press, :;mr
York, 1971.
Mann, E. and R.O. Christiansen. Adenosine 3',5'-monophosphate phosphodiesteraser
Multiple molecular forms. Science 173:540, 1971.
Mann, E.  and R.O. Christiansen. "GGylate kinase in man: Multiple molecular
forms", Human Heredity 22:X-27, 1972.
Johnson, J.D., X0. Chrztiansen, and N. Rretchmer. "Lactose synthetase in
mammary gland of the California sea lion". Biochem.Bioz:hys.Res.Csx?.,
47:393-397, 1972.
Enn, E., M. Desautel and R.O. Christiansen. Highly specific testicular
adenosine 3' ,5'-monophosphate phosphodiesterase associated with sexual
maturation, Endocrinolcgy 91:716-720, 1972.
Johnson, J.D., Hansen, R.CR Albritton, W.L., Werthemann, V. and R.O.
Christiansen. Hypoplasia of the anterior pituitary and neonatal hypoglycemia.,
J.Ped., in press.

Abstracts:

1: Christiansen, R.O., L.A. Page, and R.E. Greenberg. Analysis of glycogen
  storage in hepatoma: dephosphophosphorylase kinase defect. J.Pcdiat. g:?'83,
   1966.
2.  Christiansen, R.O., and R.E. Greenberg. Hypoglycemia due to diminished hePatic
  gluconeogenesis. Proc.Xest.Soc.Pediat.Rcs. 14:79, 1967..
3;  ChriStianSen, R.O.,and R.E. Greenberg. Reguihon of gluconeogenic enzymes in
  the newborn period. Soc.Pediat.Rcs.Abs. 37:158, 1967.
                                    --- I
4.  Greenberg,R.E., R.O. Christiansen, R.X. Rcmcn, A.?. iCohr;na, and G. Re~~~nn.
  Physiologic mechanisms in infantile hypoglycemia. lediat.Rcs. 1:292, 1?67.
                                                              ---
5.  Christiansen, R.O., and A. Loyter. Respiratory control and calcium binding in
  submitochondrial particles. Fcd.Proc. 27:527, 1968.
6.  DiJeso, F., R.O. Christiansen, H. Steensland, and A. Loyter, Localizatio:] of
  inner mitochondrial meniorane components. Fed.Proc. 2G:663, 1369.
7.  Christiansen, R.O., pf.J.W. Fan, D.A. Eclenky, and Lx. Page. Effect of


chlorpropamide in                                 p;:: - - .?i , .
          central and nephrogenic.diabetes insipidus. Amer.Iedi,z:..C:::z.;
Soc.Pediat.Res.Abs., p. 192, 197Q.                                                   ---

Christiansen, R.O., J.D. Johnson, J. Kellema, and G. Reaven. Severe prolonged
neonatal hypoglycemia, nodular hyperplasia of the pancreas and hypcrinsulinism.
Amer.Pediat.Soc.&Soc.Pediat.Res.Abs., p. 193, 1971.
Christiansen, R.O., Z. Monn and M. Desautel. Highly specific isozyne of cyclic
nucleotide phosphodiesterase associated with sexual maturation. Cli.n.Res., 20:
252, 1972..                                                                           -

Hansen, R., J. Johnson, W. Albritton, R.O. Christiansen and V. Werthemann.
Absent anterior pituitary and neonatal hypoglycemia, Clin.Res., 20:254, 1972.
                                                                         -
Christiansen, R.O. and W. Desautel. Highly specific testicular isozyce of cyclic
nucleotide phosphodieterase associated wtih sexual maturation., Ped.Res.
c&349, 1972.
Christiansen, R.0 ., E. Monn and M. Desautel.'Highly specific isozyme of

phosphodiesterase associated with male sexual maturation. Abstracts, IV int.
Cong.Endo., 4~536, 1972.


       lNSTtTUTlON A?:i) LOCATION

University oI e Uestern Australia

University of Western Xustrnlia

     SCIELJTIFIC


Orcanic C:-,e=istry .
Organic Che=si",:y

MAJOH RiSiAFcCH iN:EiiEST             A0L.r I:4 ?FiO?CSED i'iO;f CT

Applicatiocs of tzss s?ectrc=etr/ to   Organic Chesist/zass s;ectroscor?st
B~olory/ c,d =~~r.c:c'l =ZCo'~le-s
                    ..- - e... .-   "Zb...
5ESEAFICH SU??Oa i iSr* `nstrcCLonsJ
     N/A

PUBLTCATIO::S SIKCE 1971

,I ILlUl ,II,L11 t:,Lti,b.. ..s . ..- Inrerpreto!isn of b',sss CZCc!:::.

I .   R.W.G. Milne, Ed., John \Viicy ono kns,

        1.                     o ? ????oo????*??? ??? ?????
         . .,* .  An Application of Arti"-.-
          Mass Spcctromct? ,
          New York, 1971, ps, &;i- -

i~~~~,,d       By B . G 3:Jchoncn, A, M. Dufficld and A. V. Robertson

`m. 5 70                                                        P-150


2.

3.

6.

7.

8.

9.






10.





11,







12,

Mass Spectromeiri in Structural a,r.l Stereochemical Hrsble~s. CCI'I. Sc=--c-
                                                                           ---a 1
of Hydantoins.11.  Electron In?sct Induced Fracectaticn of sme S*>stirzrei
Hydantoins.

Org. Mass Spectr. , 5_, 551 (1971)
By R. A. Corral, 0. 0. Orazi, A. M. Duffield and C. Djerassi

Electron Impact Induced Hydrogen Scradling in Cyclohexanol and Isczeric
Methylcyclohexanols.

Org. Mass Spectr'., 2, 383 (1971)
By R. H. Shapiro, S. P. Levine and A. M. Duffield

Derivatives of 2-Biphenylcarboxylic Acid.
  Rev. Roumain. Chen., 16, 1095 (1971)
  By A. T. Balaban and A.!4. Duffield

Alkaloide aus Evonymus europaea L.
  Helv. Chin. Acta, 54, 2144 (1971)
  By A. Kldsek, T. Rxchstein, A. M. Duffield and F. Santa@
                              I

Studies cn Indian Medicinal Plarts. XXVIII. Sesquiterpene Lactones cf

Enhy<r> Fluctczns Lou-. Strccturc. of Enhidrin, Fluctuanin and Flu~~'~=~;
                                                                           c ,,,,n ,
  Tetrahedrcn, 23 32SS (13'7.1).
  By E. Ali, P.3: Ghosh Dastiizr, S. C. Pakrashi, L. J. Durham
   ar,d A. L1. Cuffield

The Electrsn Impact Prcmoted Frazmentaticn of Aurone Epoxiies.
   Or-z.  Mass  SZr'C?
  By B. A. Brady, _. 6, 139 (lc72j
        VI r. G'Sullivan.and A. M. Duffield

The Detezzicaticn of Cy clshex~~lazine in Aqueous Solutions cf Scdi*:n Cyclz.t:e
by Electron Ca;ture Zas Ch~zarcgrzphy.

Anal. Letters, 5, 331 (17-71)
.By !!. D. Solman, ii. E.  ?erei-a and A. ti. Duffield
   .

Computer Zeccznition of Ilctastable Ions. Nineteenth Anr.l;al Ccnference cr:
Mass Spectrcmetry, ;stlanra, 1971, 2. 63            =,
By A. $1. Duffield, :l. E. Reynolds,.D. A. Anderzon, R. A. Stillzn, u'r.
   and C. E. Carroll

Spectrometrie de E&se. VI.  Frasentation de Dimethyl-2,2-dicxolanes-1,3-
Insatures.

Org. Mass Spectr., 5, 1409 (1971)
By J. Kossanyi, J. Chuche and A. M. Duffield

Chlorprccazlne !.:et&olism in Sheep. II. In vitro Ketzbolisn and Preparaticz
of 3H-7-Hydroxychicrprcnlzine. _        --.

  Journees D'Qressolcgie, 12  333 cl9711
  By L. G. Brooks, Iy.  A. Hold., I. S. Forrest, V. A. Bacon,
   A, M. Duffield and M. D. Solcmon

        .                .
Mass Spectronetry in S&c&al and Stereocheaical Problems.  CCXVII.

Electron Impact sromoted Frac--?
                ,.,,,ntaticn of 0-Yethyl Oximes of Some
a&Unsatwa+ed Ketcnes and Ijethyl Substituted Cyclohexanones.
  'Canadia;; J. Chem., 50, 2776 (1972)
  By Y. M. Sheikh, R.?. Licdtke, A. M', Duffield`afid t. `Djerassi


ADAYAPhLA?l T. GXSESXN

Born:      Madras State, India. May 15, 1932.
Citizenship:  U. S. Citizen
Married Ann K. Cook, Ph.D. (Stanford University, Department of Genetics),
       Research Associate, Department of Biology, Stanford University.

Eddcation:   1947-1951 Annamalai University, Madras State, India. Physics,
               Chemistry, English.
              B. S. (1951) Botany.

1951-1953 Annamalai University, Madras State, India.
      M. A. (1953) Plant Physiology and Genetics

1959-1963 Stanford University, Department of Genetics, Palo Alto,
      California ("laticnal Institutes of Health Trainee under
      Professor Joshua Lederberg).

Ph. D. .(1963) Genetics. Thesis title: Phyeical and
Biological Studies on Transforming D:SX from B. subtilis.

Profession,1 Experience:

Research Fellow (1953-55), Department of Biochemistry, Indian Institute of
        Science, Bangalore, India.  Awarded Institute Fellowship.
Research Associate (1955-57), 2otar.y Department, Indian Agricultural Research
         Institute, Xe:.r Delhi, India.  Responsible for plant tissue cu1turs,
        teaching of genetics course (laboratory and lectures).
Fellowship (1957-59). ATlarded by Rask @rsted Foundation of Denmark for study at:

        Department of Physiology, Carlsberg Laboratory, Copenhagen,
          Denmark.  Worked under Professor 0. !:'inge, F.S.S. (fermentation
        genetics and some aspects of cytology).
        Department of Physiological Chemistry, Carlsberg Laboratory,
        Copenhagen. Xorked under i?rofessor Halter (physioiogy of sporu-
        lation in yeasts)..
        Department of Gei?etics, University of Copenhagen. Seurospora
        genetics and methods.

Research Associate (1963-65), Department of Genetics, Stanford University, ?alc
        Alto, California.  Xssistant Professor (1965-70); Xssociate
        Professor (1970-present).

Special Fields of Research Interest: D>?A Replication and Recombination Mechanisms
           in B. subtills;
      Repair of DX  in vitro Synthesis of Biologically Active DXX.
                                  --
                 1Iolecules and its genetics.  Phage DSX Biosynthesis.
        Cytogenetics of Eukaryotic Cells.

Recent Publications:

Studies on in vitro Replication of g, subtilis DNA. Cold Spring Harbor Syap.
   Quant. Biol. 33:45 (1968).
_ .
ATP Dependent Synthesis of Biologically Active DNA by Azide Poisoned Bacteria.
   Proc. Natl. .'.zad. Scie, 6831236 (1971).


DNA Synthesis in Bacteriophage SPO-1 Infected Bacillus subtilis. J, Virology
   9:263 (19721, with C. 0. Yehle.                                     .
A Deoxyribonucleic Acid Polymerase I deficient Xutant of B. subtilis.  J. Biol.
  Chem. 247:5867 (19721, with P. J. Laipis.                       C'

In vitro Repair of X-irradiated DNA Extrac'ted from a Polymerase I Deficient
   Bacillus subtilis. Proc. Natl. Acad. Sci.(i+~~~ber$-9?2), with P. J. Laipis.
                                                        I .,T : t
In vitro DSA Synthesis and Function of DNA Polymerasds&i6 Bacillus subtills
--
  in "DKA Synthesis in vitro".' Ed. R..D. \!ells and R. B. Inman, University
   Park Press, Baltimore, Maryland (1972), with C. 0. Yehle and P. J. Laipis.

Source of Funds:  NIH, General Pledical Sciences
         NIH Research Career Development Award (G?l-50199)

Title of Project: DNA Replication and Recombination in Bacillus subtilis
Principle Investigator: A. T. Ganesan

Grant Number:  GM 14108, G?l-50199 (AwardTthrough November 1975)

P-133


SECTION II - PRiVILEC-ED CO'ZX!~IFJIC,~TIC)N                                       nil. j. -.:-. : :q
                                             co?;c,Te-: pl ? rt, . .
                    BIOGI7APHiCAL SKETCH

     (Give the fOhv~~rtg fnformarion for allprof~:;,;riop.?/ p,?r~o,,~c~ listed on o z;e 3, bqinning w.th tho Principal Invcst;g3ror,
            Use COfJfifWafiOn P3tfs Jfld follo:v the ume general former for each person.)
NAME                               TITLE '
                                              . '                    BIRTHDATE (20 GJY Y/ I
                                                                           .., I.

Terence John Gribble          Assistant Professor      April 6, 1937 .
PLACE OF BIRTH ICity, Stare, Country)         PRESENT NATIONALITY f/f non-US
                       fndica te kind of visa and cxpira tion date) citizen,    SEX

Cardiff, Wales                 U.S.A.            CT h4a!e
            EDUCAl ION Iikgin vitlr bscch/eurtare training and ir~/ude portdoctora/)          r3 Ftira!c!


        INSTITUTION AND LOCATION              DEGREE        YEAR           SCIENTIFIC
                                                   CONFERRED           FIELD      -

University of the South, Scwanee, Tenn.   B.S.        1959
Stanford University, Stanford, California XD. _            Chemistry
                                 1964

HONORS

Sigma Pi Sigtm,,Physics Uonor Society (1958); Phi Beta Kappa (1959); Alpha @mea Alpha;
Faber Pediatric,kard (1964); Eorden Student Research Award (1964); Dernhan Senior
Fellow (1970). SOCIETIES: Western Sot. for Pcdiat. Resaarch (1968):Pzer. See. EeL-.atolc
MAJOR RESEARCH INi ERESI                   ROLE IN PROPOSED PROJECT

*Pediatric Hematology - Hemoglobin

Investigator

Dernham Senior Fellow, Amricsn Cancer Society, July 1, 1970 - June 30, 1973
Current year $20,690. Total $60,989.'                 = e     -_ --

RESEARCH AtdD;gR PHOFES%l'OS,\L EXPERIEt~!CE (S:~:i;l~~ ~,,;r;lhprc"cc,~t'po:if;on, /;sr tr~i~inoand~~pericnccrc/er,cnt toarc o:crckecf LJT
ormost reprcscfmrivc publ;c,?tion~.  DO 83 t FxcPCd J" ,E;eS for uch ino'ivid::si.)
1969 to present - Assistant Professor of Pediatrics, Stanford University
1969       - Chief Residc~t in Pcdiatr.ics, Stanford University
1968       - Research Fcllor~~ in Fcdiatric Rezatology, Stanford University .
1966 --1968   - U.S. Public Health-Service (NIH) Rational Eeart Irktitute, Eethezda,
           Naryland,
Publi&ions (Selected)

Walters, T.R., Gribble, T.J., and Sch::artz, U.C.: Heme S$nthesis in Norm1 and Lcukezi
  Leukocytes.  Xaturc 197:,,1213, 1963.                       5

Gribblc, 'T.J. and Scht?artz, 1I.C.:  Effect of Yrotoporphyrin on Hemoglobin Synthesis,
  Biochcn. Riophys. Acts 1@3:333, 1965.

Gribblc, T.J., Cornstock, T.J., and Udcnfrimd, S.:  Collagen Chain Pm-nation 2-d Per\::
Proline Uydroxylati.on in PIonolaycr Tissue Cultures of L-929 Fibroblasts. Arch.'
  Biochcm. Biophys. 129:3OS, 1569.
                         --

Cornstock, J.P., Gribblc, T.J,, aild Udenfricnd, S.:  Further Studies on the Act?\-atioil
  of Collagen Prol.kc i!yci,jrox~l.st:i.on in Cultures of L.-929 Pitrob3asts.  rQ: c :i f.>\' (J r .I
   Biochcx. cr.d Eiq~I:ys* 1.37:!.!..5, 1!?20.
                             -we

Lincoln, D.R., Edmunds, D.J., Gribblc, T.J.', and Schvart-z, H.C.: Studies on the
   Ucmoglobins of Pinniped.  Ill pre!;s, Jclnunry 1373, Clood.


                                                         pj-:r. J, 1:-. ._ :
                                           Q#y+~;t ;f 1:: ' :" .-;
                      wai w-c . . . --i .,-. I ;-;5 -1.;  :..c,:'  :e 2.7-i j.%.`.-,, ";,-ra: `3.. *jc,y;.T,-;,,:.,                   -. -- ____ -.
NANE                                TITLE                       BIRTHDATE ia;:=., 2ji, `:.j
Leonard A. Herzenberg         Professor of Genetics        Nov. 5, 1931

PLACE OF BIRTH (City, Stare, COUnw/          PRESENT NATIONALITY l/f n0n-U.S c/r;zen,    SEX
                          indicate kind of visa and expiration date)
Brooklyn, New York            . U.S.               XHhlzle c Fr?jl:
                 EDUCATION (Begin with bacca/aurea :e training dnd include pcrstdx:orJ/j

         INSTITUTION AND LOCATION          OiGREE        YEAR             SClEIvTlFlC
                                                    CONFERRED            FIELD

Brooklyn College, Brooklyn, Net:/ York       A.B.
California Institute of Technology
Pasadena, Calif.                    Ph.D.
Pasteur Institue, Paris, Postdoctoral Fellos

1952   Biology, Chemistry

1955   Biochemistry, Irxunclc;,
1957

HONORS
Phi Ekta Kappa, Sigma Xi

Distinguished Alumnus blard, Brooklyn College, 1970
Genetics Study Section, Eational Institues of Health
MAJOR RES'ARCH.PJTEREST                 FiOLE I." PR.O?GSED PROJECT
Immunogenetics, somatic cell genetics    Prlnclpal Investigator

RESEARCH SUPPORT (See instmcdons)

I

N.I.H. G:+17367, Automated Cell--Sorting-Clinical & Biological Uses, S160,&02 current
year direct costs, total funds for nro.ject S585,977 (l/1/73-12/31/75).
N.I.tl/ AI-02917, Genetics of !mmuno9loSulins, 552,774 current year direct costs,
total funds for project 5246,386 (5/l/69-4/31/74).
N.I.H. CA 04681, Genetic Studies xizn ;!amma~ian Cellsj-SE0,993, current year cirect
costs, total funds for project 5424,981 (9/l/72-8/31/77)
N.I.H. HD 01287, Fetal-maternal Immunological interactions. S35,GOO current year dirzit
costs, total funds for project $101,572 (5/l/70-4/30/73)

RESEARCH AS3 OR ?=CFESSiCNAL EXPEF'.lE:<CE is;zr5,Ty ivith pr+;ent posrcjon, ii,-r rra~`li-q and ex;en%ie relevant t3 are3 c? ;*;, it:  2.;: 2 :
OrmartN3rrsent3~vrpuo:icat~on~  Do not exceed 3 fages ior each ifz /idual.)
1969-present Stanford University School of Medicine, Stanford, California, Professor s?
Genetics                                   
1964-1969 Stanford University School of Kedicine, Stanford?`California, ASSO. ?XfG;:r
of Genetics
1959-1964 Stanford University School of Medicine, Stanford: California, Assist?nt ?r:r`~::
of Genetics

1957:1959 l'!ational Institutes of Health, Bethesda, Maryland. Officer, US?% (Dr. :-:a~-:;
Eagle)

1955-1957 Pasteur Institute, Paris, France (Prof. Jacques Konod, American Cancer Sccie:/
Postdoctoral Fellow)

1952-1955 Ph.D. California Institute of Technology,,Pasadena, California (Ptajcr: 3icc~:-
istry, Prof. H.K. Mitchell; Ilinor: Immunology, Prof. R.D. Owen)
1948-1952 A.B. Brooklyn College, Piew York


Leonard A, HerzenL?rs, 7:;,:,
bublication List

8.





2.




3.



4,





5.






6.



7.





8.




9,

Mitchell, H. K. and Herzenbcrg, L.A.
Enzymatic degradation of cytochromz C
Colowick, S.P. and Kaplan, II. 0. (cds.)
Methods of Enzymology 60:167-69, Academic Press, M.Y., 1955.
                       -
Mitchell, H. K. and Herzenberg, L.A. .'
Zone electrophoresis on sponge rubber
Analytical Chemistry 29-:1229, 1957.

Herzenberg, L.A. and Herzenberg, L.A;
Adapt6tion tc lactose
Nutrition Reviews 17:65-67, 1959.
                    -

Herzenberg , L. A.

Studies on the induction of beta-galactosidase in a cryptic strain
of escherichia coli

Biochimica et Biophysics Acta 31:525-38, 1959.
                                  -     f

Herzenberg, L.A.

Discussion of genetics of somatic cells (Bar Harbor, Maine, Sei,t. .l960)
Burdette, \I. J. (ed) .
Methodolosy in flarl;malian Genetics, p. 462-63, Holden-Day, Inc.,
San Francisco, 1963.

Herzenberg, L.A. and Roosa, R.A.
Nutritional requirements for grokrth of a mouse lymphoma in cell culture.
Experimental Cell Research 2:430-38, 1960.

Herzenberg, L.A.

Isolation and identification of derivatives formed in the cow-se of
intracellular accuxlation of thio?alsctosides by escherichia coli.
Archives of Biochemistry and Biophysics 93:314-15, 1961.
                                             -

Cann, H.M. and Herzenberg, L.A.
Detection and localization of H-2 antigen in cells groxn in culture.
American Journal of.Diseases of Children 102:477, 1961. (Abstract)

Herzenberg, L.A.

Chemical and serological characterization of purified H-2 antigens.
"Mechanisma of Trxwnological Tolerance" Proceedings of a Conference
of the Czechoslovak Academy of Science, Prague (Nov. 1961) p. 495-99,
1962.

`10 c  Herzenberg, L.A. and Herzenberg, L.A.
  Association of H-2 antigens with the cell membrane fraction of
'.    mouse liver.
  Proceedings of the Nationql Academy of Sciences 47:762-67, 1961.
                                                             -.

.


12.  Herzenberg, L. A.
  Histoconlpatibility antigens and tissu? transplantation.
  American College of Physicians, Annual kieeting, April 1962.

13.  Herzenbcrg, L. A.
  A genetic and iLnunoloqic approach to the purification of an
  histocompatibility antigen.
  Leone, C. A. (ed.)

The Effects of Ionizing Radiations on Immune Processes, Gordon and
Breach Science Publishers, ftl. Y., 1962.

15.  Cann, H. Il. and,Herzenberg, L. A.
  In vitro section for isoantiqenic variants of msnimalian somatic
   cells.

American Journal of Diseases of Children, p. 533, 1962. (Abstract)

16 &
17.  Herzenberg, L. A.
   Part I.  Steps to:lard a genetics of somatic cells in culture.
   Part II.  Naternal isoimmunizationr as a result of breeding in the
    mouse.
$l;nal of Cellular.and Comparative Physiology, Suppl. 1, f;o:145-57,
           .

18.  Roosa, R. A., Bradley, T. R., Lakr, L, \J. and Herzenberg, L. A.
  Characterization of resistance to amethopterin, 8-azaguanine and
  several fluorinated pyrimidines in the murine lyaphocytic neoplasm,
   p. 388.

Journal of Cellular and Conparatjve Physiology, 60:109-26, 1962.
                                                      -

19.  Wunderlich, 3. R. and Herzenberg, L. A.
  A second gamma globulin isoantigen (allotype) in the mouse.
  Genetics 47:995, 1962 (Abstract).
                -

20.  Cann, H. M. and Herzenberg, L.A.
   In vitro studies of m xxalian sonatic cell variation I.  Detection
  of H-2 pheontype in cultured mouse cell lines.
  Journal of Experimzntal F;edicine, 117:259-65, 1963.

21.  Cann, H. t4. and Herzenberg, L. A.
  In vitro studies of mammalian somatic cell variation. II. Isoimmune
  cytotoxicity with a cultured mouse lymphoma and selection of resistant
   variants.

  Journal of Experimental Medicine , 117:267-84, 1963.

22.  Mishell, R. I., Herzenberg, L. A. and Herzenberg, L.A.
   Leukocyte agglutination in mice, Detection of H-2 and non H-2
   jsoantigens.

Journal of Inxwnology, 90:628-33, 1963.
                          -

23.  Herzenberg, L. A. Tachibana, D. K., Herzenberg, L.A. and Rosenberg,
   L.T.
  A $ene locus concerned with hemolytic complement in mus musculus.
   Genetics 48:711-15, 1963.
                 -

P-197


24.

25.

26.





72,




28,





29.

31.  Herzenberg, L.A.

32.





33.

34.

Vunderlich, J. and Herzenberg, L.A.
Genetics of a gamma globulin isoantigen (allotype) in the mouse.
Proceedings of the National Academy of Sciences 49:592-98, 1963.
                                                      -

Herzenberg, L. A., .Rosenberg, P. and Herzenberg, L. A.
Gamma globulin isoantigens (allo:'Lypes) in the mouse.
Genetics 483892, 1963. (Abstract).
          -

Herzenberg, L.A., Kishell, R. I. and Herzenberq, L.A.
Gamma-globulin isoantigens (allotypes) in the house mouse.
Proc. of the XI.International Congress of Genetics, The Hague,
The Netherlands, Sept. 1963. _1:136. (Abstract).

Erickson, R. P. Herzenberg, L.A. and Goor, R.
Partial immune elimination of homologous red blood cells in mice.
Transplantation &:175-82, 1964.

Herzenberg, L. A. and Cole, L. J. (
Presence of donor specific gamma-globulins in sera of allogeneic
mouse radiation chimeras.
Nature 202:352-53, 1964.

Erickson, R. P., Tachibana, D.K. Herzenberg, L. A. and Rosenberg,
L. T.
A single gene controlling hemolytic complement and a serum antigen in
the mouse.
Journal of Immunology 92:611-15, 1964.
                        -

Paper-master, B. II. and Herzenberg, L.A.
In vitro selection of an isoantigenic variant from a cultured mouse
lymphoma heterozygous at the H-2 locus.
Genetics =:274

Study of the H-2 locus in murine cell cultures
Krooth, R. S. (ed..)
Somatic Cell Genetics, p. 140-166, University of Michigan Press,
Ann Arbor, 1964.

Herzenberg, L. A.

A chromosome region for gamma2A and beta2A globulin h chain
isoantigens in the mouse.
Cold Spring Harbor Sypm. Quant. Biol. c:455-64, 1964.

Herzenberg, L. A., blamer, FL. L., and Herzenberg, L.A.
Immunoglobulin isoantigcns (allotypes) in the mouse,
I. Genetics and cross-reactions of the 7s gamma2A lsoantigens
controlled by alleles at the 19-l locus.
Journal of Experimental Medicine 121:415-38, 1965.

blamer, N. L. and Herzenberg, L. A., Cole, L.A. and Davis, G!. E. ,Jr.
Dissociation of skin homograft tolerance and donor type gamma globulin
synthesis in allogeneic mouse radiation chimaeras.
Nature 205:1077-79, 1965.

                                          P-/98


35.

36,

37.

38.

39.

40.

41.

42.

43.






44.

Papennas ter, B. bl. and Herzenberg, Leonard A, 1966. Isolation and
characterization of an isoantigenic variant from a heterozggous
mouse lymphoma in culture.  Journal of Cell PhysiGlOgy 67-:407-20, 1966,
tterzenberg, L. A.;1966, tl-2 and immunoglobulin isoantigens (allotypes)
iq somatic cell genetics.  (Antigenic variation of sc:;latic cells and
genetics cytoa ,,enetic aspects of cell antigenicity-introduction), p. 353-
65.  In Jan Klein, Marta Vojtiskova and Valdimire Zeleny (Genetic Variations
in SoKtic Cells, Proc. Syzp. f~lutational Process.
1965. Academia (Czechoslovakian publisher).    Praha, August 9-11,

Herzenberg, L. A. and Herzenberg, L.A. 1966..
Suppression of a $G-Globulin allotype in mice by anti-allotype anti-
bodies, pp. 227-32.  In Jan Klein, Rarta Vojtiskova and Vladimir-e
Zeleny (ed.), Genetic-variation in Somatic Cells, Proc. Symp. tiutational
Process, Praha, August 9-11, 1965.  Academia (Czechoslovakian publisher)

Uarner, Noel L., Herzenberg, L. A., and Goldstein, Gideon, 1966.
Immunoqlo!)ulin isoantigens (allotypes) in the mouse. II Allotypic
analysis of three yG myeloma Troteins from (UZB X BALB/c)F hybrids
and of normal yG qloiulins
           2 -           .  Journal of Experimental Medicine 123:707-721,
1966.

Warner, floe1 L., tlerzcnberg, Leonard A., 1966.  Immunoglobulin isoantigens
(allotypcs) in the mouse. III.  Detection of allotypic antigens with
heterol ogous antisera.  Journal of Immunology =:525-31, 1966.

Herzenberg, Leonard A, and !;farner, r!oel L., 1967. Genetic control of
mouse imwnoglobulins, p. In B.  Cinader (ed.), Regulation of the
Antibody Response, Chapter-KV.  C. C. Thcmas, Springfield, Illinois,
1967.

Tyan, Marvin L,, Cole, Leonard J., and Herzenberg, Leonard A., 1967.
Fetal liver cells:  A source of thqus-dependent specific immunoglcbulin
production in radiation chimeras.  Proceedings of the Society of
Experimental Biology and t<edicine 124:1161-63, 1367.

Klein, Jan, Martinkova, Jitka, and Herzenberg, Leonard A., 1967.
Analysis of the histo-compatibility-2 (H-2) locus of fIZ6 mice.
Transplantation 5:1335-37, 1967.

Klein, Jan and Herzenberg, Leonard A., 1967.
Cbngenic mouse strains with different immunoglobulin allotypes. I. Breeding
scheme, histocompatibility tests and kinetics of yGZA globulin production
by transferred cells for C3H. S. 1~1. and its congenlc partner U!B/5.
Transplantation 5-:1484-1495, 1967.
Melton, John, Gfalker, Samuel R.; Sharp, Gordon C, Herzenbera, Leonard A.,
Wistar, Richard, and Creger, William P., 1968. flacroglobulinemia with bone
destruction.  Difficulty of distinguishing between macroglobulinemia and
myel oma.

American Journal of f!edi tine 4J:280-,88, 1968.

P-199


45. Uarner, Noel L., and Herzenberg, Leonard A., 1967
  Imwnoglobulin isoantigens (allotypes) in the mouse. IV.
  Allotypic specificities common to two distinct immunoglobulin
   classes.
  Journal of Immunology 99: 675-678, 1967.
46. Minna, John D., Iierson, G. Mi'chacl, and Herzenberg, Leonard A., 1967.
  Identification of a gene locus for YG  irnmrrnoglobulin H chains and its
  linkage to the H chain chronlosow reg on in the mouse.
                 1
Proceedings of the National Academy of Sciences 58: 1880194,.1967.
                                                            -
47. Tyan M. L., Cole, L. J., and Hew&berg, Leonard A., 1967.
   Fetal liver cells:  A source of speci'fic immtino~lobulin production
  in radiation chimeras, pp. 87-89.  In J. Dausset, 3. Hanibruger,
  G. Mathe(Eds.) Advance in transplaxation, Proceedings First Inter-
  national Congress Transplantation Society, Paris, June 27-30, 1967.
  (Munksgaard, Publisher).

48. Herzenberg, Lconore A., Herzenberg, Leonard A., Goodlin, Robert C.,
  and Rivera, Edna C., 1967. Immunoglobulin synthesis in mice:
  Suppression by anti-alloiype antibody.
  Journal of Experimental Kedicine 126: 701, 1967.

49, !M;enberg, Leonard A., Minna, John D., and Herzenberg, Leonore A.,
The ihronoso?e region for iwunoglobulin heavy chains in the mouse:
  Allelic elcctrophoretic nobility differences and sllotype suppression.
  Cold Spring Harbor Symposia on Quantitative Biology 32: 181-86, 1967.
                                                                -

50. Herzenberg, Leonard A., McDevitt, Hugh O., Herzenberg, Leonore A.,
   1968.

Genetics of antibodies.
Annual Review of Genetics, 1968.

51. Tyan, N.L. and Herzenberg, L.A., 1968.
  Ontogeny of the Kouse Ili;:lune System. 11. Immunoglobulin-producing
   Cells.

Journal of Inmunoiogy, 101:: 446.450, 1968.

52. Tyan, Marvin L., HcDevitt, Hugh O., and Herzenberg, Leonard A., 1968.
  Genetic control of the antibody response to a synthetic polypeptide:
  transfer of response with spleen cells or lymphoid precursors.
  Abstract and paper submitted to 2nd International Transplantation
  Congress, Ne:.l York, Sept. 1968.

53. Herzenberg, Leonard A., and Tyan, tjarvin, 1968.
  Genetics of antibody formation:  role of the thymus in the evolution
  of the immune response.  12th International Congress of Genetics,
  Tokyo, Japan, August 19-28, 1968.

54. Tyan, Marvin L. and.Herzenbe?g, Leonard A., 1968.
  Irnmunoglobulin production by embryonic tissues: thymus independent.
  Proceeding of Society for Experirllental Biology and Kedicine, 128:
  952-954, 1958.


55. Moods, Roy and Herzenberg, Leonard A.
  Specificities common to mouse IgG and IgA molecules.
  (In preparation).

56. Lanzerotti, Richard I!. and Horzenberg, Leonard A.
  Population of antibodies recognizing distinct allotypic specificities
  in mouse im?unoglobulin. V.          
(In preparation).

57.' Herzenberg, Leonard A., Pernis, B., and Eelus, A.S.
  A second locus controlling rabbit heavy chain allotypcs on the
  Fd fragment of a second-class of immunoglobulin.
  (In preparation).

58. Herzenberg, Leonard A., Carbonara, A.;Tosi, R., and Pernis, B.
  Comparison of a locus allotypic specificities.in IgG, IgA, and
  Igt4 in the rabbit.


       .t:.t--a:., = _... -..;p*. -; _c ,,rj `; ,s..  ,..`,  **`l-s*",., !,:r**,l c,m :..;`a 3. 3r.:#nr,,?. *,:- **. 2a.r.: .y.: -:--. L -I r-,,r:rS
                                                                           "
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                                                                           a-
NAh:E                                  TiTLE                                       c .--
                                                           is~h Tn.,- a c I.`.`&, r.,,, : :. ,

  H. Russell Hulett               Research Associate        Nay 5,,1920   t
                                                     i
PLACE OF UIl\Tti IC~tv. StJtL*, CourwyJ          PIICXNT NA';`lOt~ALlTY 111 nun-U.S crtrrvn,    Ski4
                              indicale kind of vu md orptralion daloJ
Ncspclem, Washington             U.S.             .-I
                                                                           -I
                                                              ?r_, 5.iw   hFr--r,r
                                                                           _ ,    I
                 E OGCA iIGt; (liqin with l~,*rcal.wrcJ:c tf.unrng nnd lncllrth oo:ldClurail

         INSTlTUTlON AN0 LOCATION            bEGFlEE 1     YEAR  I.    scli:*TJilc
                                                CONFEFIREC)            FIELD  1

Oregon State College, Corvallis, Ore.     B.S. *    1941
Oregon State College, Corvallis, Ore.   . M.S.       1942                     _ h.

Stanford University, Stanford, Cal.    . .Ph.D.     1964    Chemistry


HONORS   Zla;ncersnlp 1l-l:
Phi Lambda Upsilon                                                                           /
                               .
  Pi Mu Upsilon                                      /
                                                                J'                                       ;
  Sigma Xi
`i,lAJOI\ RESEAHCH INTERNIST                  IHOLE IN PROPOSE0 Pf?OXCT

  Biomedical

RESEAHCH SUWOR 1

                         I
instrumentation, origin of l:.fe. ' Instrumentation developmentt investigat.
                      of soeclfic cell seoaraticzs. e*::!*.:.zzfz-
1sc~rnrlrJitronll      . .

.

N/A

.                             . . .

1966 - present:  Research Associate, Stanford University
1964 - 1965: Associate Professor, Head,Department of Chemistry, College of Xotre .Da-.
1965 - 1966: Dqartment Head, It& Corporation           .
1959 - 196::  Research Director, Advanced Technology Labs
1957 * 1959:  Slibsystem ?tanagcr, Lo&heed
1952 - 1957: Eleccrcnics Dcpartmcnt Enad, Detroit Controls
19X'- 1952:  Electronics Department ticad, Santa Barbara Research
1946 - 1951:  Electronic Engineer, Hug;hes Aircraft
1942 - 1946:. U. S. Army                 .

                       
Publications: (selected -- since 1965)

Hulett, H. R. and H. Si Loring.  "Reactions of metals with tobacco mosaic virus ."
    Federation Proceedings. 23: 1943 (1965)
  Hulett, H. R. and H. S. Lorinz "Effect of particle length distribu:ion on infec-
   tivity of tobacco mosaic virus." Virology 25:418 (1965)
Hulett, H. R.  "Turbulence limitations in photographic resolution of planet
     'surfaces."  J. Opt. Sac. Am. 5721335 (1967)
Ehrlich, P. and H. R. Hulctt. "Lixng on capital." :kw Scientist%:426 (1968)
  Hulett, H. 3. "Linltations on prcbiolnzicnl synthesis." J. Thcor. 5~01. 2:>~-7? (:'
Hulet:, II. i:., Bonncr, :I. A., E.~rr,?tt, J. and L. A. Uerzcnbcrg. "Ccl1 sor;ir.;:
   automated scpnration of mammalian cells as a function of intracellular
    fluorcsccncfz:"   Science 166:  747-749 (1969)
                                        _---__
A rcl B
A:::'&                             me                      P-20&


- .__ _`.._m._
__ -_ _..._. - .--..-a.  a.- -

Hulett, H. R.  "Optima world population." Bioscience., g:160 (1970)
Hulett, II. Ii.  "Non-cnzynatic hydrolysis of adcnosine phosphates." K'attire
  225:1248 (1970                                       
HuleK H.-R., Coukcll, A., and W. Bodmcr. 'Tissue typing instrumcnta:ion using
  the fluorochtonatic cytotoxicity assay.'  Transplantation lo:135 (1970).
Hulett, H. R, and L. A. Herzenberg: "Approaches to prcscrecniG with
special consideration of flow systems'."  Acta Cytologica (in press)
Hulett, H. R. and L. A. Herzenberg, W. Bonner and R. L. Wolf. "Rapid cell
   sorter -- a new tool for cell study with clinical appliations."
  Laboratory Investigations 22:501 (19.70).
Hulett, H. R.  "Shock.synthesirof amino acids in simulated primitive atmosphere."
  Science 17091000 (1970). Letter to the Editor.

SzSen patents on electro-optical devices and instrumentation
Publications after 1970:                                                                  ,
Merrill, .I. T., N. 'Veizades, H. R. Hulett, P. L. Wolf and L. A. Herzenberg.
  "An Improved Cell Volume Analyzer" Rev. Sci. Instr. 42, No. 8, 1157-1163 (1971).
Bonner, W. A., H. R. Hulett, R. G. Sweet, and L. A. Herzenberg.       .
  "Fluorescent Cell Sorting,, Rev. Sci. 'Instr. 43, 404 (1972).
Hulett, H. R., R. G. Sweet, L. A. Heryenberg.  "Development and Application of
  Rapid Cell Sorter,,, to-be presented at O.R.N.L. symposium on Advanced
  Analytical Nethods for the Clinical Laboratory, Xar. 15, 1973 (and later
  published in Clinical Chemistry.)
Hulett, H. R.  "Formaldehyde and Ammonia as Precursors to Prebiotic Amino Acids.
  Science 174, 1038, 1971.


John D. Johnson

PIACE Of BIRTH ICity, Sate, Country/
Palo Alto, California

      .
Assistant Professor       September 14, 1935

PHESENT NATIONALITY (If nonUS cirizcn,    SEX
indicate kind of visa end expiration datel
U.S.A.                        .     ah!ale n Fen-i!-

EDUCATION (fie@ v,,ifh hxcalaurate trainmg and include postdocrorai)

          INSTITUTION AND LOCATION             DEGREE        YEAR            SCIENTIFIC
                                                     CONFERRED          FlELq

Wabash College, Crawfordsville, Ind.       B;A.       1960     _2oology'~~
e Stanford Medical School, Stznford, Calif. M.D.    .. 1965       Medicine
Johns Hopkins Hospital, Baltimore, Md.            1965-67      Pediatrics,2 yea
: Stanford Medical School, Stanford, Calif.          I     1970         Post-Doctorel F;-
                                          lSf?~5~~fE~~~
HONORS
Phi Beta Kappa, Alpha Omega Alpha, Borden Award for Undergraduate Research (Stanford
Medical School)

MAJOR RESEARCH INTEREST            .-       /ROLE IN PRGPOSED ?ROJECT

Developmental Biochemistry,
bilirubin metabolism
RESEARCH SiJ?PORT~ irlstructionsl

Investigator

United Cerebral Palsy Associations, II-X. Grant R-245-71,
Developmental Aspects of Mene Protein' Catabolism      - -.
Current $28,772. July 1, 1971 to June 30, 1973 Totif $53,322.

RESEARCH AIC3jGfI Ph3FESS;GNitL EXfEfilENCE (St.?fi;f,l ?::I -h pr~=cnt ~O:jt;o,), list trG;rlhT sndcxperialce retevent 10 2red Of ;W:Xf
                                  L;., i t
ormost rcprcsentatire publicirtions.  Do nor cxcrcd 3 pqes fsr c:ch ir;dividulual.J
.a 1971 to pre'sent - Assistant Professor of Pediatrics, Stanford University
1971 to present - Assistant Director of !<et;born Service, Stanford University
1970      - Post-Doctoral Fell.ow, lkpcrtmcnt of Pediatrics, St?nford Universlt:?
1967-- 1969    - Research Associatc,.NICHD
1960 - 1963   - Pre-Doctoral Fellow, Department of Pediatrics, Stanford University
         'i
Public.ations

Johnson, J.D., Hurwktz, R., and Kretchmer, N.:  Utilization of fat and glycerol for
glycogenesis by the neonatal rat. J. Nutrition, E, 299, 1971.
-Johnson, J.D., Jant, B.A., b*Lfm.?n, S., and Sokoloff, L.:  Effect of ionic strength
  on the RNA pol~,...--~-
          *-ln**lse activities of isolated nuclei and nucleoli of rat liver.
  Arch. Biochem. Bfqhys.,

Johnson, J.D., Christiansen, R.C!. , and Krct-chr;cr, N.: Lactose synthctasc in mnzxry
gland of the California Se2 Lion. Biochcm. Biophys. Rcs. COIT~. 47, 393, 1972.

Johnson, J.D., Albrittdn, W.L., and Sunshine, P.:  Hyperammoncmin accompanying parcntcrc-
  nutrition in newborn infnnts.  .T. Pc;dS.;itrics 81 154, 1972.
                                                       -.-'
Johnson, J.D.:  NeiJt.r;:l !rc~erQ.-,iZ~nlf,c';o.ri;~,?s, fi fro3 the srx+ll intcstinc of the rs>'Jl'i.
  Submitted for publication to Giochim. Xiophys. Acta.

                                                .*s+--- -c-w.----              -..---.-- . . .-m.-..
.-.-.-em____                         --I --.e.-
RHS.:4~ir;          . .                                       P-;rw
:: P i :: :m


                                                                           .-.. -.
SECTIOI"J It - PRIVIl.FGED CO:::I1VNICATION                           Cpe,<--.?-a ! q-.- . . .-
                                                                           
                       BIOGRAPHICAL SKETCH

      (Give the following information for all prv/rssion,-I personnel lis:ed on p?yc 3, &inning v;ith the Principal Invcstig&ar.
                Use continuation p2g7w and follow the wme general forma: for each penonl
NAME                                 TITLE  Assistant Professor of     BIRTHDATE /MC., Lby, Yr.)

  Judith P. Koehler, M.D.         Neurology and Pediatrics     April 27, 1939

PUCE OF BIHTH ICjry, State, Country)           PRESENT NATIONALITY I/f non-U.S. citizen,    SEX
  New York, New York             indicate kind of visa and expiration date)

                              U.S. Citizen                 0 Male   a Fcmzk
                 EDUCATION Begin with baccalaurca te training and include postdoctoral)

            INSTITUTION AND LOCATION


Mount Holyoke College -.
Dartmouth Medical School
University of Pennsylvania

DEGREE        YEAR             SCIENTIF:C
            CONFERRED            FIELD '


A.B.       1960
B.M.S. .     1962
M.D.       1966

American Academy of Neurology (Junior Member)

MAJOR RESEARCH INTEREST

   Pediatr&* Neurology

RESEARCH SUPPORT (Set instrucrions)

ROLE IN PROPOSED PROJECT

--

RESEARCH AND,`3R PROFESSIONAL EXPEFtlENCE (Starting with p,.esantposition, list trainicqandexperience relevant toarea of projzc  List ,r.i
or most rcprasenta tive publications.  Do not exceed 3 pages for each individual.)

1966-1967 Intern, Mixed Pediatrics, Xontefiore Hospital and Medical Center, Bronx, N.Y.
1967-1968 Junior Resident, Neurology, Albert Einstein College Hospital and Bronx ?funicipal Eos
1968-1970 Senior Resident, Neurology, Albert Einstein College Hosp. and Bronx >!unicipal
      Hospital Center, New York.
1969-1971 Postdoctoral Fellow, Anatomy, College of Physicians and Surgeons, Columbia C'niv., X.
1971-1972 Fellow in Pediatric Neurology, Columbia-Presbyterian Medical Center, S.Y.
1972-    Assistant Professor of Neurology and Pediatrics, Stanford Medical Center.
                                                                         \

Publications (selected).

Koehler, J.P., Lovelace, R.E., Spiro, A.J.: Basement membrane and capillary endothelial altsra-
tions in hypokalemic periodic paralysis: an electron microscopic study. (Abstract) Present--c
at the American Association of Neuropathologists, 47th Annual Meeting, June 1971.
Koehler, J.P., Spiro, A.J., Lovelace, R.E.: I. Thyrotoxic periodic paralysis: light and electrr
microscopic observations of nerve and muscle. (In preparation).
Koehler, J.P. and Chuzorian, A.: Tissue culture of medulloblastoma cells from spinal fluid, a
useful diagnostic technique. (In preparation).
Spiro, A.J., Koehler, J.P., Taylor, J.M.: Oculopharyngeal dystrophy: ultrastructural and his:*>-
  chemical observations of skeletal muscle.  (Abstract) Presented at the American Associatiz-
of Neuropathologists, June 1972,
Koehler, J.P., Duffy, P.E., Carter, S. (1973) Selective type I fiber atrophy in childhood
neuropathy: light and electron microscopic observations of peripheral nerve, Schwann ccllj.
neuromuscular junction, and muscle.  (In preparation).


    (Ghe the foh'owing information for s:iprofzsion~/ perscnnrl Irsrcd on pqs 3, brj!nrriry ?ji!h t.`w Principal Inrzr;,-3rof. .-.I ..
               Use conrifwatinn pq3j- ~4 ff2lcw th* 2rne genefit format fcr wctj parson.1
NAME
  Norman Kretchmer        TITLE,' Harold K. Faber professor IClaTHpATE (a!!~, Cdl', Yr.l-
                           of Pediatrics    I January 20,-1923

PUCE OF ljlHTH (Ciry, State, Cowtfq4                                                                    -.
                           PRESENT NAIIOt2ALITY f/f non-U,S cir;ien,    SEX           .,..
                            indicate kind of visa ad expiration date)
New York, N.Y.                      U.S.A.                 Q M3Ie   0 Fr~:le
               EDWXTIDN (&yin v.?th Dsccckurtute rrainh? aqd incluti ros;doctoq[j

         INSTITUTION AND LOCATION   :

Cornell University
University of Ninnesota
University of Xinnesota           .

Downstate >lcdical Center, State Univ. of

DEGREE
B.S.
M.S.
Ph.D:

YEAR -+ -    SCIENiIFlC
CONFERRED        FIELD
                   
1944    Animal Phycidlc;?
1945    Physiological C!;e;:.
1947     Physiological Cheer..

m& Commonvealth*Fund Fellow, 1952,57,65             1957
                             President, Int. Organ. Stuc!y i;.uzan i>cy:r!is
  . .Mead Johnson Award, 1958;         ment, 1969; President, Soc.Ped,Res., Y$:c:c.
                                                                           A-/. s_
  . Borden Award, 1969              Council, Amer. Sot. Clin. Invest., 1064-~j:

MAJOR RESEARCH VJlEfiEST



C~ESEARCH n?i'DRi (kr ins:rccrjn,T.c)

  Alumni Hedallicn SlXS, 1969.
        --
ROLE IN PROWSED PKOJECT

Investigator

RR 81 - Clinical Research Center for Premature Infants, 10/l/63-9/30174           -- .1
    Amount current year: 418,532 - Total amount: 2,021.300-. (5%;) renewal pence.,
HD 02147 - Eiochemical Studies of Dcvelopcent, 6/l/66-5/30/73,
    Amount current year: 206,593 - Total amount: 1,309,278- (15X) rexc$.-al pendil:z.
CRBS 252 - sational,Foundation - Cellular and Xolecular Determinants cf X~r~:ic;~nc,ii~
    Amount current year: $27,273 - Total amount: 54,546 (7/l/71-6/30/73)- ilS::>
HD 00049 -'Human Development and Pediatrics Training Grant* 7/l/70-6/30/74,           .
    Amountrurrent year: $$9,538 - Total amount: 448,936.- (1X)renewal pzr.@ing    .
                   (Please see continuation on attached sheet)                -
RESf1.f.:C!i b?.!D,:>R izI:3PiS.5ib:;i;L                                 :!,r *
Of most rcprcxnr2tLe prh5rrcr!icrB.5   ~i:I'El~! E;`:CE'(Str;i;t~~ ,:*/IA F:CSZT~ positio;t , --md!~t,c am c*pcri'en:e fc:ev~nr ta 8x3 c/~:rr,:.-;;  L;.;: I
                Un flcf wccrd3 ps;lcs ior cxh iwYlrid23f.)
1971 to present -  Harold El. Faber Professor of Pediatrics
1969.-.1972   .-. Chairman, .Progran.in Ruman Biology (Eaccalaureate Prog., Stanford i'r:-.i
1959 - 1969     - Professor and Executive kad, Pediatrics
1953 - 1959    - Assistant then Associate Professor, Dept. of Peds, Cornell L`Liv.
       --.         New York l!ospital
1950 -:1935    - Lecturer, Dcpartmcnt of Biology, Brooklyn College
1948 - 1952    - Research hssxiate, Dept. of Pathol., State Univ. of Xew Ycrk
1947 - 1948    - Assistant Professor, Departments of Pathology and Biocheris:rg, L'niv.
             of Vermont,.
From 1952     - Various cl+nical appointments from Intern to Pediatrician-in-Chief

Publications (Six relevant publications are listed from a total of 101)

Rretchmer, N.:  Lactose and lactase: .A historical perspective. Castrcenteroiogy 61:&O-,
           1971.

Levine, R.L., Hoogenraad, N.J., and Kretchmcr, N,: Regulation of activity of cerb z:yl-
      phosphate synthctase from mouse spleen.  Biochemistry 10:3694, 1971.
Lebenthal, E., Sunshi.ne, P,, and Kretchner, N,:  Dffect of carbo@rate and
      ccrticostcrc~jds on ac';j.vi:y oi 3.-~~lUCOSLiG2SC!S in ir=ssr:ine Of the

.        infant rat ,  J, Clin, Invest. 51.:1244, 1972.
                                               -4
Mretchmcr, D., Ransomc-Ruti, O., llur*Jitz, R., Dungy, C., and Xlakija, W.:
      Intestinal absorption of J.actosc *iii Kigerian ethnic groups.
     The Lancct 2:392, 1971.                             p-2Ob
ic {CT+<. p                   --..I--        _A_---                     ---f*.-.. __.- -
               . _                                                            : -- --7

--


Biographical Sketch of Dr. Norm& Kretchmer

Page Two

Research Support, continued

. HII 00391  i Regulation of enzyme'actlon durlng'development,
         9/l/68-8/31/73               *
      'Amount current year: 42,289 - total amount': 183,822 (15X)

Publications, continued

Welchsel, M.E., Jr., Moogenraad, N.J., Levine, R.L., and Kretchmer, N.
    Pyrlmidine biosynthesis during development of rat cerebellum
   Pediatric Res. 6:682, 1972.

Johnson, J.D., Christiansen, R.O., and Kretcber, N. Lactose synthetase
   in mammary gland of the California sea lion. Biochem. Biophys.
   Res. Comun. 47:393, 1972.

Note:  Renewal of HD-02147 is pending.  This present renewal application,
   HII 00391, in part overlaps with some material include4 in ID-02147.


a .I,, _I.  ,,-r....I.b"."  ~~I~I..."...~0..~..                                                 ( =----= -- -_   .--=- .,-,
                                                                           -----..
                           BIOGRAPHICAL SKETCH                           APPLICATION NUMiEY (Ir.,, r l~~e,k/

   (Give tha followiT infarmatton for o och KEY professional staff member, whether or not solorY is requested. Begin witti tha
   Program Director.)

JAMf (Last. jint, i~likiUlJ                   fl"fProfessor of Pediatrics and    BIRlHDATE I IJo.. 1L1.t. j-r.,
                         Community & Preventive Medicine I  916114
  Luigl Luzzatti, M.D.                                                        SEX

`LACE OF BIRTH (City, Slrtre. Cornrr~)                 PRESENT NATIONALITY (If aorr-L'.S. ririzm, indic&r      ?I3 MALE   0 FEMALE
                                 ha sprbof J                              SOC. SEC. NO.
  Rome, Italy                          U.S. Citizen           570-42-7539

lElATlONSHlP TO PROPOSED PROGRAIA

  Investigator


                       .
                     EDUCAUON f Begin -wir/, b.ar&wratc truiving and inchdr pnrtdxtorul)


                        ORGANIZATION AND LOCATION                           DEGREE      YEAR
                                                                           CONFERRED       DISCIPLINE


    University of Minnesota, Mpls, Minn.               M.S.  1942
    University of Minnesota, Npls, Minn.               M.D.  1943


                 .









40NORS






MJOR RESARCH OR PROFESSIONAL INTE IS1 (If apphbk)

   Congenital Defects and Cytogenetics

IS1 RECEM RELEVAM PUBLICATIONS
Miles, C.P., Luzzatti, L., Starey, S. and Peterson, C.D.: A male pseudo-
  hermaphrodite with a probable XO/AXY mosaiclsm. Lancet, Sept. 1, 1962, 455.
Luzzattl, L.:  Failure to thrive - a diagnostic approach.  Postgrad. Med.
   35:270-8, March 1964.
Peterson, C.D. and Luzzatti, L.: The role of chromosome translocatlon in the
   recurrence risk of Down's syndrome.  Pediat. 35:463-9,-March 1965.
Welchsel, XE., Jr., and Luzzatti, L.: Trisomy 17-18 syndrome with congenital extrahe?
   blliary atresia and congenital amputation of the left foot. J. Ped. 67:32G-7, .-\uz. '
`WFESSIONAL AND/OR RESEARCH EXPERIENCE (S&r: with pr~mt poritim und list recmt sigu&mt r.~$wrimsr rrlrumt lo pwww~) (publications  con: .
1972-   Professor of Pediatrics & Community and Preventive Medicine
1967-   Director, Birth Defects Center, Stanford Univ. School of Xedicine
1963-   Director, Cytogenetic Lab., Stanford Univ. School of Medicine
1957-72 Associate Prof. of Pediatrics and Preventive Yedicine, Stanford Univ. Sch. of ?
1955-67 Director, Pediatric Outpatient Dept., Stanford Univ. School of Medicine
1954-57 Assistant Prof. of Pediatrics and Preventive hledicine, Stanford Univ. Sch. of 1.
1953-54 Director of Cerebral Palsy Training Program, Children's Hospital of
       San Francisco









                                                                           . .


                                                    P-2Db

PHS 2499-l                                     PAGE
trv. s-70                                                        U.8. OOt+ENNMSNT PRINTING OFPICE : 1070 OF - 166-466


Publications(continued)

Craig, A., and Luzzatti, L.: Translocation in De Lange's syndrome? Lancet 2:445-6,
August 1965.

Craig, A., and Luzzatti L.: Translocation in trisomy D syndrome. A case of
probable D/18 translocation.  J. Pediat. 70:264-g, Feb. 1967.
Greenstein, R.M., Harris, D.J., Luzzatti, L. and Cann, H,.M.: Cytogenetic
analysis of a boy with the XXXY syndrome: origin of the X-chromosomes.
  Pediatrics, 45:677-686, April 1970.
Knight, L., Sakaguchi, S., and Luzzatti, L.: Unusual mechanism of transmission
of a chromosome translocation from mother to offspring.  American J. Dis.
  of Children 121:162-167. Feb. 1971.

Knight, L., and Luzzatti, L.: Replication patterns of X & Y chromosomes in
partially synchronized human lymphocyte cultures. Chromosoma, Vol. 40,
153-166, 1973.


NAME                                TITLE                     GIFITHD;\TE /::I. ;J,; i'r./

  Wilfred E. PSPZI?A
PV\CE OF BIRTH fGry, Sirte. Counrryl

  Research Associate        June 23 193.5
PfiESEF;T P'AT,.`;' * 1
        . " .C.L ITY l/i xc-US c/rizen.    SEX
indicate kind of viz and exsirdrlon dare)

INSTITUTION AND LOCATION    I    DEGREE        YEa9  I       SCIESTIFIC
                                          CONFERRED            FIELD

' Madras Medical College, EIadras, India   B. Pharm
  Saugar Univ, 2. .
          ?f2dhva Pradesh,, India   14. Pharm
  U.C. Med. Center, San Francisco, Calif Ph.D.


HONORS

1960
1902
1968

Pharmaceutical Che~ist~~~
Pharm. Chern L Cher. of :;a%
harm:Chen b Phzrrscolo-

.

MAJOR RESEi,RCH lNTE.i;EST
Identificetion of !(stabolites t drug
metabolites in Bzological fluids
RESEARCH SiJiPORT (See:nsmcoonsl

ROLr IN PROPOSED PROJECT


      Organic chemist

RESEARCH A:;o,oa tT:C-cS3iOXAC EXiE?,E?:ZE rS:3mr.~ .-.trn orzen. . ~ouo'on, jjs: pj,c,rrq and expene.xe rQ?fdnt :O ared Of Ur:;t'."  i:ir j.'

       e-t3rIve ;~:.'~:3::
oi~ggY~97 0        or!  Go no: exceed 3 c3;es for ex.7 mcivraust.1
           Fcst 3cctoral Pellcv, Zep5, of Genetics Stanford University :.!ed. School
1970 - present Resezreh kscciate s.ze institution              e
During these fox years i have been in-solved wizh Feptide syxtkeiis, azino acid esz~~sis
and synthetic orgzic chemistry.  I hel; ed develop rethods for the segarzzicn of
diasterioisocers ky gzs ck.rc~2to~~~~y zzd kzve been involved with t.*.e r0r;tir.e t;se of
`gas chrozsto~~a3~9 :zss ssectrczetry for the identification of *xir.zrj- -,etsbolizes i:: xrxi
and pathological ~7ir.e and serum sezrples.  t?y a??lications of ~255 spectrozezrg k27t
included the Ceveclyez 0:' ~12~s fra,zeatoGgagky for the detemicztion of tke amino aci?
Contents of soil and *zxx serum.   2~2 present project involves tkc screening of xrice
fkom leukenic aatiezts for 2bnorz21 cetabolites and to investigate the metabolic fate of
anti-leukemic chemotherapeutic agents in the body,

PUBLICATICX

1, Transesterification with an Anion-exchange Resin;
W, Pereira, V. Close, W. Pattcn and B. Halpern,
J. Org. Chem. &:2032 (1959).

.

2. Alcoholysis of the ,. verrifield-type Peptide-polymer Bond with an Anion

Exchange Resin;

W, Pcrcira, V. A. Close, E. Jellum, W. Patton and 9. Xalpern,
Australian J. of Chem. =:1337 (1969).


3r  The Action of Xitrosyl CbLoriCe cn Fhenylalanifie Pegtides;
  W, Patton, 2, Jell=, D, Zitecki, W. Pcreira and B, Halpern,
.Australian J. of Chen. 22:2709 (1969).
               -
4, Abnomal Circular Dichroisn of #.-Amino Acid Esters;
J, Cyzermn Craie and $1. Z. Pereira,
Tet. Let. M-:1563 (1970).

5, The Use of (+)-2,220Trifluoro-LPhenglethylhydrazine in the Optical
  Analysis of Asymetric %eto=les by Gas Chrorzatography;
  W. E, Pereira, Is!. Solocon acd B. Ealpern,
Australian J. of Chez,&:1103 (1971).

6, The Kicrosoral Ozq-genation of Ethyl Benzene. Isotopic, Stereocherical,
  snd Induction Studies; *

R, E, Kc3!&on, H. PI. Sullivan, J. Cjr;leman Craig and W. E. Pereira,
Arch, Eiochen. Biophys, 132:575 (1969).

7. The Steric Analysis of Aligha tic Azines with 'P-do Asyxzetric Centers
  by Gas-liquid ChrosatoGra,..
                 -by of Ciastereoiscceric At&es.
  W. E, Pereira aad 3. Ralpern,
  Australian J. Ches. 25:667 (lgn).

8, Optical Rotatory Dispersion and Absolute Configuration -XVII.
  &-Alkylphesrlacetic Acids;
  J, Cyizexan Crai<, V. Z. ?ereira, B. Halpern and J. W. Vestley,
Tetrahedroa 2:X73 (1971).

9. The Optical Rotary Dispersion and Circ ilar Dichroisn of &-Amino and    _
  A-Hydroxy Acids;

J, Cy-,er=an Crztg, a-,i W. 6. Per&a
Tetrahedron sj: $57 (1970) .

10,' The Detemination of Cycl,,.,.
                *&arJlaziice in Aqueous Solutions of Sodium
  Cyclari3te by Ziectron-capture Gas Chrocatography;
  M, D. SO~OLO~, 3. 2. Pereira ar.d A. X. Duffield, __
  AntLL Let. 4:301 (1971).


u.

12,

13.

14.

15.

16.

17.
.




18.





19e




20.

Chlorination Studies. I. The Reaction of Aqueous
vith Cytosine; lCsn

Hypochlorous Acid

W. Patton, V, S~bx, A. M. Duffield, B. Halpern, Y. Hoyano, W, Pereira
and J, Lederberg,
Biochen. Biophys. Res. Cormun. %:880 (1972).

The Use of R-(+)-l-Phenylethylisocyanate in the Optical Analysis of
Asymmetric Secondary Alcohols by Gas Chroztography;
W. Pereira, V. A. Bacon, W. Patton, B, Halpera, and G, E, Pollock,
Anal. Let, 2:23 (1970).

A Rapid and Quantitative Gas Chronatographic Analysis for Phenylalanine
in Serum;
B, Halpern, W. E. Pereira, M. D. Solomon and E. Steed,
Anal, Biochea, 39:1.56 (1971).

Electron-i&pact ?ro;noted Fraq entation of Alkyl-X-(1.Phezjjlethyl)-
CarbaLzates .of Primary, Seccr,dary 2nd Tertiary Alcohols;
W, E, Pereira, B. Ralpern, b!. D. SoloTon and A. M. Duffield,
Org. blass Spectrozetry 2:157 (197?).

Peptide Sequerxing by L&f Resolution Xass Spectronetry;
V, Bacon, E. Jell=, il. ?ztton, ii. Pereira aad 3. iialpern,
Biochen. Eiophys. 3es. Cozr,;~?. x:873 (1969).

A Gas Liquid Chrozstographic Xethed for the Detemination of Phenylalasise
in Serum;
E, Jellun, V. A, Close, V. Patton, W, Pereira and B, !Mpern,
Anal, Biochea. 2:227 (1959).

Quantitative Deterzication of Riologically Important Thiols acd
Disulfides by Gzs 3iy;id C1:roza:o;rzp?~;
E, Jellun, 'rl, ?atton, V. A. Zacon, 'ri, 3. Pereira and B, Halpern,
Anal. Biochea. z:339 (1~69).                           e
                                          .              .    M
A Study of the Zlectron Icpact-,, --c=:oted Fragmentation of Prczazine
Sulfofide a& pro-szi,?e Usiz,- Specifically Deucerat-ed h.&ogUeS;
M, D. Soloxn, R. S~xs, 'ri, 3ereira and A. 51. Duffield,
Australian J. Chen. (1973, in press).

The Detemination of PInenylalenirie in Serum by Mass Frag=ectogapkJ;
WI Pereira, V. A. Sacon, Y. Eoyano, R. Surxons ard A. 14. Duffield,
Clin, Biochen. (In press).

Chlorination Studies II.  The Reaction of Aqueous HyFocblorous Acid
vith A-Amino Acids and Dipeptides;
W, E, Pereira, Y. Eoyano, R. Su==ons, V. A, Bacon and A, fi. Duffield,
Biochen, et Biophys. Acta (In press),


r                          026)

L SECTION II -PRIVILEGED COMMUNICATION   BIOGRAPHICAL SKETCH

       (G&e the fol~owhtg infomution for aN professional personnel listad on page 3. boginnlng wfth rho Principel Invatl~tor.
                Use continuation pages and follow the mm gene& fmet for eech penon)
NAME                                  TITLE                       WRTHQATE (Ma. Day, Yr.)

Thomas C. Rindfleisch             Research Associate     12-10-41
PLACE Of Elf? TH (City, Stete, Country)          PRESENT NATIONALITY (If non-US citizen,    SEX
                             indiceh khd of vita end o xpimtlon &to)
Oshkosh, Wisconsin, USA              USA              I3 MaI0   0 Femrlr
                 EDUCATION (Begin with becceleuroete treining end include postdoctor8lj

         INSTITUTION AN0 LOCATION             DEGREE        YEAR            SCIENTIFIC
                                                     CONFERRED        FIELD
Purdue University, Lafayette, Ind.        B.S        1962     Physics               .

California Institute of Technology,       M.S       1965     Physics
Pasadena, CA                         Ph.D     Thesis to bc completed. All
                                        course work and examinations
                                   completed.
HONORS
Purdue University, Graduated wlth.Hlghest Honors, Sigma
Xi.

MA@R RESEARCH INTEREST
Space sciences, computer science and
image processing

ROLE IN PROPOSED PROJECT

Technics1 Support

RESEARCH ANDDR PROFESSIONAL EXPERIENCE (Staning rvithpr~ntposidon,~ondoxpe~ecrr~w~nt toana ofprojac Listrtl
W mai nprwenmdve publiationr  Do not uce& 3 es for mch indfvidrul.)
19710Present Stanford University Medical School, Department of Genetics,
         Stanford, CA.
        Research Associate - Mass Spectrometry, Instrumentation research.
1962-1971   Jet Propulsion Laboratory, California Institute of Technology,
         Pasadena, CA.
         Relevant Experience:
           1969-1971:  Supervisor of Image Processing Development and
                Applications Group.
          1968-1969:  Mariner Mars 1969 Cognizant Engineer for Image
                Processing
         1962-1968: Engineer - design and Implement image processing
               computer software.

1. Rindfleisch, T. and Willingham, D., "A Figure of Merit Measuring Picture
  Resolution," JPL Technical Report 32-666, September 1, 1965.

2. Rindfleisch, T. and Willingham, D., "A Figure of Merit Measuring Picture
  Resolution," Advances in Electronics and Electron Physics, Volume 22A,
  Photo-Electronic Image Devices, Academic Prese, 1966.                     ,


Thomas C. Rindfleisch
PUBLICATIONS (cont'd)

3.

4.

5,

6.

7.




a.




9.

Rindfleisch, T., "A Photometric Method for Deriving Lunar Topographic
Information," JPL Technical Report 32-786, September 15, 1965.

Rindfleisch, T., 'Photometric Method for Lunar Topography,' Photo-
grammetric Engineering, March 1966.
                  .                                                      * .
Rindfleisch, T., "Generalizations and Limitations of Photoclinometry,"
JPL Space Science Summary Volume III, 1967.

,

Rindfleisch, T., "The Digital Removal of Noise from Imagery,' JPL Space
Science Summary 37-62 Volume III, 1970.

Rindfleisch, T., 'Digital Image Processing for the Rectification of
Television Camera Distortions," Astronomical Use of Television-Type
Image Sensors, NASA Special Publication SP-256, 1971.

Rindflpiach, T., Dunrie, J., Frieden, H., Stromberg, W., and Rulz, R.,
"Digital Processing of the Mariner 6 and 7 Pictures," Journal of
Geophysical Research, Volume 76, Number 2, January 1971.

Rindfleisch, T., "Digital Image Processing," To be published, IEEE
Special Issue, July 1972. '


(Give the following informs don for all prof~ssio,rajperson~el liskd on ,oage 3, bginnbg with the Principal I~~vcsti~~lcr.
        USC continuation pqcs and foilov/ the Simc yencrel formet for each parson.)

PRESENT NATIONALITY I/f non-US ritizen,
indicate kind of visa end expiration date)

New York City, New York    I   U.S.A.        I  n VI+
                                                                 J, . ,.. . .  g Fcmr;lc
                                                                           _
              ED lJ CA TI ON (Eq!n v;irh b~cc~:aur~~tc trdinirq and include pas tdoc toral)

INSTITUTION AND LOCATION       ,  I    DEGREE        YEA9             SCIENTIFIC
                                            COiJFERRFD            FIELD

New York University, Kcw York                    .
New York University College of Medicine,    B.A.        1942
New York                             M.D.        1945

Phi Zeta Kappa, Alpha Omega Alpha
Wead Johnson Aorard for Pediatric Research, 1960
President, Society for Pediatric Research, 1966
            _ ----.
MP..IOF( OE:;!ll,t<Cti Iid~iEEEST                    `icOLE IN PROPOSED PROJECT
Coagulation physiology, Hemorrhagic dis-
eases

See next'pagc

"ESEA~~C~ AND!;J~ PHOFESSIO:<AL EXPEfilENC: (s(art;fip ~i:hpr~gmtposilion, list trainiruandexpelicncerefevant toareaofUc&t lk:aT:
or most representative publications.  Do not exceed 3 pages for each individual.)
Professor and Chairman, Dept. of Pediatrics, Stanford University- 1972
Professor a;ld Head, Dept. of Pediatrics, University of Illinois - 1961 - 1972
Profes.sor of Pediatrics, Northwestern Uiliversity - 1956-1961
Associate Professor of Pediatrics, Cornell University - 1956-1956
Assistant I'rofessor of Pediatrics, Cornell University - 1952-1956
USI'DS Post-Doctoral FelloF: in Pediatrics, Cornell University - 1950-1952
Resident in Pediatrics, Bellevue Uospital -1948-1950
Military Service - 1946-194s

Intern, Queens General Hospital, Kew York City - 1945-1946


Privileged Communication - Irving Schulman, El. .D.

       .
Biographical Sketch (continued)

Research Support

HD-00568-12

Hemost&sis and Hcmorrhogic Disease.in Children

Principal Investigator 20X time
Project period       l/1/70 - 12/31/72
Total Funds        (Direct) $122,993
Current Year '   ,(Direct) $ 15,814 l/1/72 - S/31/72
            University of 'Illinois

HD-07300-01

-------- $27,426 6/l/72 - 8131172
Stanford University

TI-AH205344

Training Grant in Pediatric Hematology

Program Director      20% time
Period          7/l/67 - 6/30/72
Total Funds        $263,465
Current Year        $ 54,458
*Training grant renewed at University of Illinois for
five years effccttvc 7/l/72 ($332,600)- application
for new traininS grant in Pediatric Hematology has been
submitted from Stanford University,


Privileged Cozunication - Irving Schulman, FI. D.

Representative Bibliography
Irving Schulman, ?I.D.
   (Past 5 Years)

1.  Abildgaard, C.F., Corrigan, J.J., Seeler, R.A., Simone, J.V., and
   Schulnan, I.: Meningoccemia associated with intravascular coagulation.
Pediatrics 40:78, 1968
                  -

2.  Johnson, C.A., Abildgaard; C.F., and Schulman, ,I.: Coagulation studies
  in children with cyanotic congenital heart disease. Lancet 2_:660, 196s

3.  Seelcr, R.A., Forman, E.N., Bolger, J.F., Abildgaard, C.F., and
   Schulman, I.: Induction of intravascular coagulation and renal cortical
  necrosis in rabbits by simultaneous injection of thorotrast and endotoxin.
   Brit. J.  Haemat. 16:501, 1969.
                          -
4. -
   Forman, R.N., Abildgaard, C.F., Bolger, J.F., Johnson, C.A., and Schulinzn,I.:
   Generalized Shwartzman reaction: Role of the granulocyte in intravascular
  coagulation and renal cortical .lccrosis. Bltit. J. Haemat. ij:507, 1969.
                                                                       -

5.  Hods, G.R., For-man, E.f?., Johnson, C.A., Seeler, R.A., Abildgeard, C.F.,
   and Schulman, I.: Administration of single doses of hEF (Factor VX11)
  concentrates in the treatment of hemophilic hcmarthroscs. Pediatrics
o 43:26, 1969.
     -

6.  Honig, G.'R., Abildgaard, C. F., Forman, E.N., Gotoff, S.P., LinZley, A.,
   and Schulman, I.: Some properties of the anticoagulant factor of ased
   pooled plasma.  Thrombosis et Diathesis Haemorrhagic 22:151, 1969.
                                                                 -

7.  Schulmsn, I., bbildgaard, C. F., Johnson, C. A. and Lindley, A.: Irnr~~-
  suppressive therapy in the management of acquired inhibitors of Factor TIII
   in.hemophilia.  Hemophilia snd ;Ie!w Hemorrhc~ic States  (Erickhous, editor),
   New York, The University of North Carolina Press, 1970, p.164.

8.  Johnson, C.A., Abildgaard, C.F., and Schulnan, -1.: Functional studies of

.   young versus old platelets in a patient with chronic thrombocytopenia.
   Blood 37:163, 1971.
             -                                          .x.
                      -. . .                                    \

9.  Corby, D.G., Zirbel, C.L., and Schulman, I.: Thrombasthenia. Am. J. Dis.
   Child. 121:40,  1971.

10.  Corby, D.G., Zirbel, C.L., Gibson,X.S., and Schulnan,I.: Effects of
   antenatal drug administration on platelet function of newborn infants.
   J. Ped. 79:307, 1971.

11.   Corby, D.G., and Schulman, 1,: Effect of bilirubin on unwashed platelets
   in experimental hyperbilirubinemia. J.Ped. (in press).


    fGke t/h? foh~~ing hfO~~3rion for z4prcf~+oa~I pcrso.~cl l&cd on p;)?e 3, beginn;rg v,jth the PriAci>aI /n~s: L-r:g,:
             U:e conthua?iu:l pqcc ii!d f.?\lO;Y fhe ShW? gencfJ/ fofn:;lt for e&l per;on.)
NAME                                                                           --.--_---_
                                   TITLE '                      BIFtTiiDA't E I,:`?., ,-.

Herbert C. Schwartz              Professor of Pediatrics     May 8, j-926
PLACE OF BIRTH (city, Smte, Country)                                                               --
                              PRESENT NATlOlJALlTY Cf con-U.S
                          indicate kind of vim and cxpirztion date/ citizen,     55x

New Haven, Connecticut             U.S.A.                   f?J 5.';!-?

               EDUCATION licp:`n with bxcil~vrc.~tc trJainin~andinc/u~cpo:cd~:torJ//

         INSTITUTION AKD LOCATION              DEGREE         YEAR
                                                     CONFERfiED
Alma College, Alma, :*!ichigan

Illinois Institute of Technology, Chicago
Yale University, New Haven, Connecticut    A.B. -      1948     Psychology-C!:::.:.
State University of New York, Brooklyn     M.D.       1952
.

HCNORS                                                                           - _ -
John and Mary Markle Scholar in Acade mic Meeicine (1962); Visiting &of. State yr:i..-
Netherlands (19G7); SOCIETIES:  Society for Pediatric Research (1961); &.ericz.n s:z.
Clinical Invesgigation (1962); American Pediatric Society (1967), etc.

MAJOR FiESEARCH If47 EHEST                                                                       -.
                                       ROLE IN PROPOSED PROJECT

Hemoglobin Structure and Synthesis       Investigator

U. S.P.H.S. Grant P.01 X4 12467-10, ForrAtion of Hemoglobin and Other HezproteLnc
June 1, 1968, to Play 31, 1973.  Current year $28,866.- Total (Years 6-10) SiSj,TSJ.

                          .
RESEARCH kl<&`Zi? PFOFESSIOSAL EV?EEIE>~CE                                                        --
                        fSI~rti,-~ !:?th prmmr posirion, ks t I rain I:?? end exocricnce r2;e.v .? t to c .-r'~ c :. :` _ -7 . .
. OInlOSt Cpk5enlJi;VC publ~w;ionr.  00 ncr cxcezd 3 ;.ye s ror P3ilI ind:`viSYa/. )
1968 - 1971 Professor of Pediatrics, Stanford University
1969 - 1971 Ch.cirTan, &.pnrtcxtt cf E'eGiatrics, Stanford University
1963 - 1968 Associate Proiesscr cf Pediatrics, Stanford Unversity
1960 -.1963  Assistant i?rofcssor'of.Pc<istrics, Stanfcrd University
1958.: -1960 Research Ii-structor in >:cdLci.nc, University of Utah
1957 - 1358 Research Fellow in Ciochaxistry, University of Utah
1955 - 1957 Research Fcllotr in hfedicine ( Hematoiogy ), `Cnivcrsity of Utah

Publications (Selected from a total of 19.)
                                 .

Schwartz, H.C. , Cert\,right, G.E., Smith, E.L., and Wintrohe, M.M.:  Studies on th? :
  thesis of Hcxc from Iron and Protcporp?iyrin, Blood 14:486, 1959.
                                                            -
Hill, R.L., S>z.nson, R.T., and Scheartz, H.C.:  Characterization of a Chcxical. i-t:.::
  in Hemoglobin C.  J. Biol. Chcm. 2?5:3l.S2, 1960.
Gribble, T.J., and Schwartz, H.C.:  Effect of Protoporphyrin on Hemoglobin Sy:::::,::
  them. Biophys. Acts.  103:333, 1965.
Dnllun, P.R. and Sch~zrtz, 1i.C.:  :i:,.oy,?o'~ir. and C srOCl:!:C-:C C Cci:OS~sc dl!l-ilt~ ::~ . ...1
   DCiiCiCtlCy ir; the !;at.  J. Clin, invust. 4<:1631, 1365.
                                                  -
FIalters, T.R. , \.!el.I.at~d, F,H. , Gribble., T.J. ,a and Sch~xrtz, 1I.C. :  Bj.osynth~t7~ :` '.
  LCUkCTTl!.C J8Clll:OC~tCSs CZllCClY ?.0:117# 1367.
                                    ---
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~.,S~::i:-;~,~-c'crd-..(~tl-!:~:c~~-,s ,-- Jriu~ls.r:y-,l9-%3).. .__. _-_--__.__--a- ,,-,.,~-,-__-..,~.:_: 
   . rrv


CURRICULUM   VITAE

Eric M. Shooter

Born:    April 18, 1924  Mansfield, Nottingham, England.

Married:  Elaine Arnold (Born Dec. 22, 1924) Newhall, Burton-on-Trent.
Children:  Annette (Born Nov. 18, 1956) Redhill, Surrey, England.
Permanent Address:     Department of Genetics       -

1942-45

  1942

  1943

  1945

  1945-46
nd 1946-49

1949

1950

1949-50

1950-53

1953-63

1961-62

1963-68

1964

Senior Scientist in charge of Biochemistry, Brewing Industry
Research Foundation, Nutfield (Proteins and enzymes of barley
and other brewing materials).
Lecturer in Biochemistry, Department of Biochemistry, University
College, London with Professor Ernest Baldwin.  (Molecular
biology of normal and abnormal haemoglobins; protein-ion inter-
actions of ribonuclease).                     :

U.S.P.H.S. International Fellow, Department of Biochemistry,
Stanford University School of Medicine, with Professor R. L.
Baldwin (Replication of DNA).
Associate Professor of Genetics, Stanford University School of
Medicine (Molecular Neurobiology).  Head of Neurobiology Group,
Lt. Joseph P. Kennedy, Jr. Laboratories for Molecular Medicine.
D.Sc. University of London (awarded for distinguished work in
the field of Biochemistry).

1968     Professor of Genetics, Stanford University School of Medicine.

Stanford University School of Medicine
Stanford, California 94305

Natural Sciences Tripos (Part If in Chemistry)
University of Cambridge

Exhibitioner of Gonville & Caius.College, Cambridge
Minor Scholar of same.

B.A. (Cantab.)

:

Research under Professor Sir 'Eric Rideal in the Department of
&lloid Science, Cambridge and the Davy Faraday Laboratory of
the Royal Institution, London (Proteins of the ground nut).
M.A. (Cantab.)

Ph.D. (Cantab.)

Postdoctoral Fellowship with Dr. J. W. Williams, Department of
Chemistry, University of Wisconsin, *Madison, and partly with
Dr. D. E. Green, Enzyme Institute, University of Wisconsin.
(Enzymes of the electron transport system).

1968-present  Professor of Genetics and Biochemistry, Stanford University
      School of Medicine.


69. Varon, S., Nomura, J. and E. M. Shooter, 1968. Reversible dissociation of
  the mouse nerve growth factor protein into different subunits.  Biochemistry
  1, 1296-1303.

70. Shooter, E. M., Smith, A. P., and S. Varon, 1968. Heterogeneity of the nerve
  growth factor protein and its subunits. 'Fed. Proc. 27, 464.
                                                                -

71.  Herschkowitz, N., McKhann, G. M., Saxena, S. and E. M. Shooter, 1968.     '..
  Characterization of sulfatide-containing lipoproteins in rat brain. J.
  Neurochem. l5, 1181-1183.

72.  Smith, A. P., Varon, S. and E. M. Shooter,'1968.  Multiple forms of the nerve
  growth factor protein and its subunits. Biochemistry L, 3259-3268.

73. Greene, L. A., Shooter, E. M. and S. V&on,' 1968. Enzymatic activities of
  mouse nerve growth factor and its subunits.  P.N.A.S. 60, 1383-1388.

74. McKhann, G. M. and E. M. Shooter, 1969.  Genetics of Seizure Susceptibility
  & Basic Mechanisms of the Epilepsies, H. H. Jasper, A. A. Ward, Jr., and
  A. Pope, Ed., Boston, Little, Brown and Company, Chapter 24.

75. Herschkowitz, N., McKhann, G. M., Saxena, S., Shooter, E. M. and R. Herndon
   1969.  Synthesis of sulfatide-containing lipoproteins in rat brain. J.
  Neurochem. l6, 1049-1057.

76. Goodall, P. T. and E. M. Shooter, 1969.  Changes in Heme Environment due to
  subunit interaction in hemoglobin.  J. Mol. Biol. 39, 675-678.

77.  Smith, A. P., Varon, S. and E. M. Shooter, 1969. Equilibria of the Nerve
  Growth Factor proteins and their subunits. Fed. Proc. 28, 897.

78. Greene, L. A., Shooter, E. M. and Silvio Varon, 1969. Subunit interaction
  and enzymatic activity of mouse 7s Nerve Growth Factor.  Biochemistry 8,
   3735-3741.

79. Shooter, E. M., Smith, A. P., Greene, L. A. and S. Varon, 1969. Aspects
  of the dissociation equilibria between 7s NGF and its subunits.  Abstracts
  of Second International Meeting of the International Society for Neurochemistry,
  p. 366.

80. Waehneldt, T. V., Grossfeld, R. M. and E. M. Shooter, 1969. The solubili-
  zation and electrophoretic analysis of membrane proteins from mouse brain.
  Abstracts of Second International Meeting of the International Society for
  Neurochemistry, p. 411.


81'.  Swfth, A. P., Greene, L. A., Fisk, H, R,, Varon, S. and E. M. Shwter,
   1969.  Subunit equilibria of the 7s Nerve Growth Factor protein,  Bit?-
  chemistry 8, 4918-4926.

8& Shooter, E. M, and S. Yawns 1974,  Macromolecular aspects of the Nerve
  Gmwtk Factor proteins a Protefn MetaB&isar of the Nemw $ystem, A.
  Lajtha, Ed. q New York, N,Y. $ Plenum Pww, 41%438,-


SECTION II - FPIVILEGED CC:!!.`U"JIC:iTlOtJ                                              CI ,.,-. ,- ( .) - .
                                                                           -- ---
                     BIOGRAPHICAL S!:ETCH

(Give the fofio&ng information for ~IIprofcss.kn~l F?rsonnd lislcd or) pqc 3, bYinning v.5'th the Prin?ipaf Inv=t:gzlor.

N&E
Philip Sunshine

Use continuarion p.?:cs and IO:/OW th? same general format for each person,)
             TITLE *                      BIRTHDATE !:.!a, Cd/, Yc.1
                       .
           'As'sociate Professor        June 16, 1930

PLACE OF BIRTH Kiry, Sfafc, Country)          PRESENT NkTlOrdALITY (If noa-U,S CitiZFn,    SEX
                            indicate kind of visa and expiration date)
Denver, Colorado .               American                 gQ F.%lls   g FPT>:~
                EDUCATION (Sqin rvith bzcczlaornfc twining and kcludo pcstdsctoral)

INSTITUTION AND LOCdTlON             DEGREE        YEAR            SClElJTlFlC
                                          CONFERRED             FIELD

University of Colorado, Eouldcr, Cola,     B.A.    1952      Pre-Medicine
. .

. University of Colorado,. Denver, Colo. M.D.*

'1955

Medicine

.

HONORS

Alpha Omega Alpha               Council Western Society for Pediatrics
Ross Award for Pediatric Research 1970    Research, 1972

MAJOR RESEAfiCH I;,!TE!?EST                     ROLE IN PROf'OSEU PfiOJECT
Developmental Gastroeneerology and
   Nutriticn                    Investigator

RESEARCH SUPPORT (Se jnsrrv;tjonsj

RR 81 - Clinical Research Center for Premature Infants, lo/%9 - g/30/74
    Amt. Current Year: $385,882 - Total Account:- $2,3&0,59$ (Xorman Kretcker -
    Principal Investigator)                                             .

RESEARCH AND,QR PROFESSIONAL E.XPERi EIJCE (Starting r;ith pruent po:ition, list rr,~~nin~~andexpcience rc!&ant 13 arca nf ,?ro,+st
                         L..:-
`Of most rr?prcscnt.&c pub//cations.  00 not pxcced 3 pqes for each indrviduaI,I

   .         -
Associate Professor of Pediatrics   1968~present    Stanford Univ. Sc!;ool of licr`lcl::c
Director, Cmter for Frerxature Infants 1968- present Stanford Vniv. Scksl of :-Ictlic1-.:
Instructor through Assist. Professor 1963-1968      Stanford Univ. School of :!sdicir%
Fellow in Pediatrics          . .  -'-X961-1963      Stanford Univ. Sch:c)ol of :I;3ici::.z
Resideqt in Pediatrics           1959-1961     Stanford Univ. School of tlz.diclz;~

Publications (Selected)

         .                      .
Sunshine,. P. and Kretchmr, N.:  Studies of mall intestine during developezt. III.
  Infantile diarrhea associated with intolerance to disaccharides. Pediatrics 22:.
   38,' 1964.

Sunshine, P. and Kretchmcr, N.:  Absence of intestinal disaccharidases in two species
     of se2 lions.  Science 144:SSO,  1964.
Herbst, J.J., Hurwitz, R., Sunshine, P. and Krctchmer, N,:  Effect of colchicir:e on
     intestinal disaccharidascs:  Correlation with biochcnical aspects of ceJ.luler
      rcneval.  J. Clin, Invest. 19:532, 1970.
                                        -
Sunshinc, P., Ilcrbst, J.J., !:oldwsi:y: 0. md Krctchxr, !3.: hdqtztion of t!.,?
   gastrointesr.inal tract to extrautcrJ.nc iife. Ann. N.Y. Acad. Sci. lik:iG-7'?,
    1971. ,                                   *

Sunshine, P., ct al.:  1Ipperaemoncmia due to a dcfcct in hcpatic ornithine t!-:rrr-.cnr? '.

--
n,ls.~;~r  Pediatrics z:lOO, 1972,  -- -...-- ---s-L_
    1 ,         . . -                                 -pz5i- - -. , . .


Tsuboi, Kenneth K.              Senior'Scicntist
                       -   t-                  February 7, 1922
I'LI.CC CF BIRl H (City, Stole, Country)         PRESENT NATIOtJALITY (If rmn4.1.S~ citizen,
                            hdicu te kind of visa and expiration date)

Okayama, Japan
           ---

INSTITUTION AND LOCATION

St , Thomas College, St. Paul, Minnesota
University of Xinncsota, Xinn. , Plinil.
. University of Xinnesota, Ninn., Minn.     d

                   .

tlOI:OHS

DEGREE

B.S.       1944
F1.S.       1946
Ph.D.      1948

P ,ch.fc ~?,,sfdo:to:

  YEAR
CONFEftfcED

- Established Investigator;
     .             American lieart Association; 1960 - 1964.

                                               .

SCIENTIFIC
  FIELD

Chem,
Biochem.
Biochem.

MAJOR RESEAHCtf I!:TIil<ESl'                        .
Bioch&%stry, Enzymology, Yucleotides,     ROLE IN C'ROPOSLD PROJECT

Xuscle Proteins                 Investigator
                   --                                                                   --
RESEARCI: IlJPfQRT (500 ir.structions)
Contract A:?ard:  Division of Biologic Standards, N.I.H., Biochemical Parameters of
Primate Cell Cultures, June 1, 1972 to ?lay 31, 1974.
$47,196.                          Current year $23,598.  Total
                                                                  _
                                                               - _
                                                                -

Ml 03978 Yetaiwlism of I:uman Erythrocyte, June 1, 3.967 -a >iay 31, 1972.
Current $12,500. Total $102,880.

RESkAfXtl AtiD/OR PHOf`ESSlO!:I..L EXf C'itEt:Cc _ (Sftir!ir~.? with prrsrnt position. fist trnininb and cxpcricnco rolcvcnt tz (IICG ol prc;rc?.
Lirt nil 0' rr8.3:t tc,zrar.nt.~tive p;r.l,licoticns. (Ijo net c*csrS                   ----.i
1966 to prcsc:nt                    3 p;;c s for cat.? indiviiu;z:.)
        - Senior Scientist, Biochemistry in Pediatrics
1460 - 1966    - Associate Professor, Biochzxistry in Pediatrics, Stanford ?!edical Schoo:
1957 - 1960   - Assistant Yroicssor, Siochcmistry in Pediatrics, Cornell _ Xedical '(:::ool
1951 - 1957   - Researcil Associate, Ziocixxiistry,
1948 - 1951                       Columbia University ?ledicai Sc:;col
        - Research Associate, Oncology, University of Kansas Xedicrl Sc!;ool
Publicati&x (Five relevant publications are listed frcm a total of 48).
Tsuboi, K.K., Greenberg, R.E., and Kretchner, I!.:  CarbohydGhte PIetnbolisn, ix Bi.olo:ic,-1
Baais of Pediatric Practice, R.E. Cooke, cd., XcGraw-Hill (1968).            ----.A--.

    ----

Tsuboi, X.K., Fukuncga, K., and Petricciani, J.C.:
  Properties of Eryt!lrocyte Uridine         Purification and Specific Kinetic
                   Diphosphste Glucose Pyrophosphorylase.  J.;l.lol. Chxn.
  2U, 3.008 (1969).
Tsuboi, K.K., i'ukunaga, K.:  Relationship of solute permeability to crythrocytc gl~colys::.
Biochem. Biophps. Ac.ta, w9(, 215 (197Oj.

Uchino, 3. and Tsuboi, K.K.:  Actin Accumulation in Developing Rat Muscle. Amer. J.
Physiology, 2l9, 154 (1970).

Tsuboi, K.K., Fukunn:a, I;., and Chervenka, C.H.:  Phosphoglucose Isonwrase from Iluxn
  Erythroc:;tc. Preparation and Propertxes.  J. dial. &cm., 2!!6, 7SSG (1971).
                                                                      --

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