Technologies for Detecting Heritable

Mutations in Human Beings

September 1986

NTIS order #PB87-140158
Recommended Citation:

U.S. Congress, Office of Technology Assessment, Technologies for Detecting Heritable

Mutations in Human Beings, OTA-H-298 (Washington, DC: U.S. Government Printing

Office, September 1986).

Library of Congress Catalog Card Number 86-600523

For sale by the Superintendent of Documents

U.S. Government Printing Office, Washington, DC 20402
Foreword

Ensuring the health of future generations of children is of obvious importance to

American society. Heritable mutations, permanent changes in the genetic material that

can be passed on to succeeding generations, are the cause of a large but currently unquantifiable share of embryonic and fetal loss, disease, disability, and early death in

the United States today. The methods now available to study heritable mutations,

however, offer relatively little information about the kinds of mutations that can occur, their frequency, or their causes. Recent advances in molecular genetics have opened

the door to new and innovative technologies that may offer a great deal more information about DNA. It may soon be possible to characterize mutations precisely, to measure their frequency, and perhaps also to associate particular mutations with exposures

to specific mutagenic influences. While some of the new technologies are still on the

drawing board, they are developing quickly and several of them may become available

for wide-scale use in the next 5 to 10 years.

The Senate Committee on Veterans’ Affairs, the House Committee on Science and

Technology, and the House Committee on Energy and Commerce requested that OTA

assess the available information about current and proposed means for detecting heritable

mutations and on the likelihood and potential impact of such technological advances.

These committees have wrestled with the problems of determining whether past exposures

to potential mutagens have affected the health of Americans, and of framing reasonable public health laws, given current knowledge and technologies. This report summarizes OTA’s findings as they relate to these issues.

An advisory panel, chaired by Arno G. Motulsky, provided guidance and assistance during the assessment. The OTA Health Program Advisory Committee, OTA

staff, and scientific and policy experts from the private sector, academia, and the Federal Government provided information during the assessment and reviewed drafts of

the report. We thank all who assisted us. As with all OTA reports, the content of the

assessment is the sole responsibility of OTA and does not necessarily constitute the consensus or endorsement of the advisory panel or the Technology Assessment Board. Key

OTA staff involved in the assessment were Michael Gough, Julie Ostrowsky, and Hellen

Gelband,

JOHN H. GIBBONS

Director

I l l
Advisory Panel for Technologies for Detecting

Heritable Mutations in Human Beings

Arno G. Motulsky, Panel Chair

Center for Inherited Diseases

University of Washington School of Medicine

Richard J. Albertini

Department of Medicine

University of Vermont

College of Medicine

Michael S. Baram

School of Medicine and Public Health

Boston University

Charles R. Cantor

Department of Human Genetics and

Development

Columbia University College of Physicians

and Surgeons

Dale Hattis

Center for Policy Alternatives

Massachusetts Institute of Technology

Ernest B. Hook

Bureau of Maternal and Child Health

New York State Department of Health

Alfred G. Knudson, Jr.

Institute for Cancer Research

Fox Chase Cancer Center

Nan M. Laird

Department of Biological Statistics

Harvard University

School of Public Health

Mortimer L. Mendelssohn

Lawrence Livermore National Laboratory

Jeffrey H. Miller

Department of Biology

University of California at Los Angeles

James V. Neel

Department of Human Genetics

University of Michigan School of Medicine

Norton Nelson

Department of Environmental Medicine

New York University School of Medicine

Mark L. Pearson

E.I. du Pent de Nemours & Co.

Richard K. Riegelman

Department of Medicine

George Washington University School of

Medicine

Liane B. Russell

Oak Ridge National Laboratory

Richard B. Setlow

Brookhaven National Laboratory

William J. Schull

The University of Texas Health Science Center

William G. Thilly

Department of Nutrition and Food Science

Massachusetts Institute of Technology

Richard M. Myers, Special Consultant

University of California at San Francisco

NOTE: OTA appreciates and is grateful for the valuable assistance and thoughtful critiques provided by the advisory panel members. The

panel does not, however, necessarily approve, disapprove, or endorse this report. OTA assumes full responsibility for the report

and the accuracy of its contents.

iv
OTA Project Staff—Technologies for Detecting

Heritable Mutations in Human Beings

Roger C. Herdman, Assistant Director, OTA

Health and Life Sciences Division

Clyde J. Behney, Health Program Manager

Michael Gough, Project Director

Julia T. Ostrowsky, Analyst

Hellen Gelband, Analyst

Other Contributing Staff

Cheryl M. Corsaro, Analystl

Virginia Cwalina, Administrative Assistant

Carol Ann Guntow, Secretary/Word Processor Specialist

Diann G. Hohenthaner, Word Processor/P. C. Specialist

Eric Passaglia, Senior Distribution Specialist

Contractors

Elbert Branscomb, Lawrence Livermore National Laboratory

Neal Cariello, Massachusetts Institute of Technology

Leonard Lerman, Genetics Institute

Harvey Mohrenweiser, University of Michigan Medical School

Richard M. Myers, University of California at San Francisco

Janice A. Nicklas, University of Vermont School of Medicine

Maynard Olson, Washington University Medical School

Cassandra Smith, Columbia University College of Physicians and Surgeons

L.H.T. Van der Ploeg, Columbia University College of Physicians and Surgeons

Diane K. Wagener, Washington, DC

‘On detail from the National Institutes of Healthr April-June 1985.
Contents

1.

2.

3.

4.

5.

6.

7.

8.

9.

Summary and Options. . . . . . . . . . . . .

Genetic Inheritance and Mutations . .

Kinds and Rates of Human Heritable

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

New Technologies for Detecting Heritable Mutations . . . . . . . . . . . . . . . . . . . . . . . . . .

New Methods for Measuring Somatic Mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Laboratory Determination of Heritable Mutation Rates . . . . . . . . . . . . . . . . . . . . . . . .

Extrapolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3

23

37

55

73

81

91

Mutation Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..................103

Mutagens: Regulatory Considerations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .......113

Appendixes Page

A. Federal Spending formulation Research . . . . . . . . . . ...........................121

B. Acknowledgments and Health Program Advisory Committee . .................123

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. ....129

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .139
Glossary of Acronyms and Terms

Glossary of Acronyms

A

AEC

ATSDR

BEIR

c

CERCLA

DNA

DOE

EBV

EDB

EPA

ENU

EtO

FDA

FRC

G

HbA

HbM

hprt

HPRT

HTT

ICPEMC

ICRP

mRNA

MSHA

NCRP

NIH

NRC

NRC

NTP

OSHA

OTA

PBL

PFGE

PKU

R

—Adenine

—Atomic Energy Commission

—Agency for Toxic Substances and Disease Registry (Centers for Disease Control)

–Committee on the Biological Effects of

Ionizing Radiations (NRC)

—Cytosine

—Comprehensive Environmental Response,

Compensation, and Liability Act

(“Superfund”)

—Deoxyribonucleic Acid

—Department of Energy

—Epstein-Barr Virus

—Ethylene dibromide

—Environmental Protection Agency

—Ethylnitrosourea

—Ethylene oxide

–Food and Drug Administration (DHHS)

—Federal Radiation Council

—Guanine

—Hemoglobin A

—Hemoglobin M

—Hypoxanthineguanine phosphoribosyl

transferase (gene)

—Hypoxanthineguanine phosphoribosyl

transferase (enzyme)

—Heritable Translocation Test

—International Commission for Protection

Against Environmental Mutagens and

Carcinogens

—International Commission on Radiological Protection

–Messenger RNA

—Mine Safety and Health Administration

(Department of Labor)

—National Council on Radiation Protection and Measurements

—National Institutes of Health

—Nuclear Regulatory Commission

—National Research Council (National

Academy of Sciences)

–National Toxicology Program (DHHS)

–Occupational Safety and Health Administration (Department of Labor)

–Office of Technology Assessment (U.S.

Congress)

–Peripheral Blood Lymphocyte

—Pulsed Field Gel Electrophoresis

—Phenylketonuria

—Roentgen

RFLP —Restriction Fragment Length Polymorphism

RNA —Ribonucleic Acid

SCE —Sister-chromatid Exchange

SLT —Specific Locus Test

T —Thymine

T G

r —Thioguanine resistant

TSCA —Toxic Substances Control Act

UNSCEAR—United Nations Scientific Committee on

the Effects of Atomic Radiation

2DDGGE —Two Dimensional Denaturing Gel Electrophoresis

2DPAGE —Two Dimensional Polyacrylamide Gel

Electrophoresis

6TG —6Thioguanine

6TGr —6Thioguanine resistant

Glossary of Terms

Achondroplasia: A disease marked by a defect in the

formation of cartilage at the ends of long bones (femur, humerus) that leads to a type of dwarfism.

There are a number of hereditary forms, the most

common of which is autosomal dominant.

Allele: An alternative form of a gene, or a group of

functionally related genes, located at the corresponding site on a homologous chromosome. Each

allele is inherited separately from each parent.

Alleles can be dominant, recessive, or co-dominant

for a particular trait.

Alpha thalassemia: A genetic defect caused by an

alteration in a portion of the gene coding for the

alpha globin molecule. The result is an insufficient

number of alpha globin molecules and a deficiency

of adult hemoglobin.

Amino acid: One of a group of 20 molecules that bind

together in various sequences to form all protein

molecules. The specific sequence of amino acids

determines the structure and function of a protein.

Amniocentesis: A procedure that involves withdrawing a sampIe (usually 2 to 8 miIliIiters) of the amniotic

fluid surrounding the fetus in utero. This fluid contains cells shed by the developing fetus. The cells

can be grown in cell culture and analyzed either biochemically or cytogenetically to detect a variety of

genetic abnormalities in the fetus, including genetic

diseases such as Down syndrome and Tay Sachs

disease.

Autoradiography: A technique for identifying radioactively-labeled molecules or fragments of molecules, useful for analyzing DNA for the presence

of mutations.

vii
Autosome: A chromosome not involved in sex-determination. In a complete set of human chromosomes, there are 44 autosomes (22 pairs of homologous

chromosomes) and a pair of sex-determining chromosomes.

Base pair (of nucleic adds): A pair of hydrogen-bonded

nitrogenous bases (one purine and one pyrimidine)

that join the two strands of the DNA double helix.

Adenine pairs with thymine, and cytosine pairs with

guanine.

Beta globin: A constituent of hemoglobin, which is a

molecule consisting of four globin subunits and a

heme group (two alpha and two beta globins).

Carcinogen: A chemical or physical agent that causes

cancer.

Cell: The smallest membrane-bound protoplasmic

body, consisting of a nucleus and its surrounding

cytoplasm, capable of independent reproduction.

Cell culture: Growth in the laboratory of cells isolated

from multicellular organisms. Each culture is usually

of one cell type (e.g., lymphocytes, fibroblasts,

etc.).

Cell line: A sample of cells, having undergone the process of adaptation to artificial laboratory cultivation,

that is now capable of sustaining continuous, longterm growth in culture.

Chromosome: A threadlike structure that carries

genetic information arranged in a linear sequence.

It consists of a complex of nucleic acids and proteins.

Chromosome abnormalities: A group of pathological

conditions associated with abnormalities in the

number or structure (e.g., insertions, deletions,

rearrangements) of chromosomes.

Chromosome banding: The chemical process of staining chromosomes to identify each pair of homologous chromosomes. Staining by various techniques

produces patterns of light and dark bands (visible

under the microscope) that are characteristic of each

chromosome pair. Chromosome banding is particularly useful in detecting structural chromosome abnormalities such as inversions and deletions.

Codon: A sequence of 3 adjacent nucleotides in mRNA

that specifies 1 of the 20 amino acids or the initiation or termination of the peptide chain. The linear sequence of codons determines the order in

which amino acids are added to a polypeptide chain

during translation.

Complementary DNA (cDNA): Single-stranded DNA

that is synthesized from a messenger RNA template.

It is often used as a probe to help locate a specific

gene in an organism.

Congenital: Refers to a condition that is present at

birth.

Congenital abnormality: Any abnormality, genetic or

nongenetic, that is present at birth.

Cytoplasm: The contents of a cell exclusive of the nucleus. It consists of an aqueous solution and the

organelles suspended in it and is the site of most

chemical activities of the cell, including protein

synthesis.

Denaturation: The separation of double-stranded

DNA into its complementary, separate strands by

treatment with chemicals or heat. The narrow range

of temperature or chemical concentration at which

DNA denaturation occurs is characteristic of the

nucleotide sequence of the particular molecule.

Diploid: The chromosome state in which each

homologous chromosome is present in pairs. All

human somatic cells are diploid (i.e., they have 46

chromosomes), whereas reproductive cells, with 23

chromosomes, are haploid.

DNA: Deoxyribonucleic acid. The nucleic acid in chromosomes that contains the genetic information. The

molecule is double-stranded, with an external “backbone” formed by a chain of alternating phosphate

and sugar (deoxyribose) units and an internal ladderlike structure formed by nucleotide base-pairs held

together by hydrogen bonds. The nucleotide base

pairs consist of the bases adenine (A), cytosine (C),

guanine (G), and thymine (T), whose structures are

such that A can hydrogen bond with T, and C with

G. The sequence of each individual strand can be

deduced by knowing that of its partner. This complementarily is the key to the information-transmitting capabilities of DNA. (See also nucleotide

and gene. )

DNA probe: A segment of complementary DNA that

is used to detect the presence of a particular nucleotide base sequence.

DNA sequence: The order of nucleotide bases in DNA.

Dominant: A term used to refer to a genetic trait that

is expressed in an individual who is heterozygous

for a particular gene. Compare recessive.

Down syndrome: A genetic abnormality characterized

by mental retardation, congenital heart defects,

immune system abnormalities, various morphological abnormalities and a reduced life expectancy.

Down syndrome is caused by either an extra copy

of chromosome 21 (called trisomy 21) or by two

copies of chromosome 21 and another chromosome

21 translocated to a different chromosome (usually

to chromosome 14). The latter results in “translocation Down syndrome.” Trisomy 21 has been

shown to increase in frequency with advanced

maternal age.

Electrophoresis: A technique used to separate molecules (such as DNA fragments or proteins) from a

mixture of similar molecules. By passing an electric

current through a medium containing the mixture,

each type of molecule travels through the medium

at a rate that corresponds to its electric charge and

size. Separation is based on differences in net electrical charge and in size or arrangement of the

molecule.

Vlli
Enzyme: A protein that catalyzes a chemical reaction

without being permanently altered or consumed by

the reaction, so that it can be used repeatedly.

Enzyme deficiency variants: Abnormal proteins characterized by reduction or elimination of enzymatic

activity. These changes may be caused by the absence of a gene product, or the presence of proteins

that are non-functional or abnormally unstable.

Epidemiologic studies: Studies concerned with the relationships of various factors determining the frequency and distribution of diseases in a human population.

Escherichia coli (E. coli): A species of rod-shaped, gram

negative bacteria that inhabit the normal intestinal

tract of vertebrates. Many nonpathogenic strains

of E. coli are hosts in recombinant DNA technologies.

Eukaryote: Cells or organisms with membrane-bound,

structurally discrete nuclei and well-developed cell

organelles. Eukaryotes include all organisms except

viruses, bacteria, and blue-green algae. Compare

prokaryote.

Exons: DNA sequences that determine the sequence

of amino acids in proteins. Exons are separated on

the DNA by introns, or intervening sequences, that

are transcribed and later removed, or spliced out,

during the production of mature messenger RNA.

Gametes: Mature male or female reproductive cells

(germ cells: sperm or ova) with a haploid chromosome content (23 chromosomes in humans).

Gene: A linear sequence of nucleotides in DNA that

is needed to synthesize a protein and/or regulate

cell functions. A mutation in one or more of the

nucleotides in a gene may lead to abnormalities in

the structure of the gene product or in the amount

of gene product synthesized.

Genetic code: The sequence of nucleotide triplets along

the DNA that determines the sequence of amino

acids in protein synthesis. This code is common to

nearly all living organisms.

Genome: A term used to refer to all the genetic material carried by a single gamete.

Genotype: The genetic constitution of an organism,

as distinguished from its physical appearance (its

phenotype). For example, an individual may have

a heterozygous genotype for eye color consisting

of an allele for brown eyes (which is dominant) and

an allele for blue eyes (which is recessive) or a

homozygous genotype, with two alleles (both dominant) for brown eyes. In either case, the phenotype

is the same: brown eyes.

Germinal mutations: Mutations in the DNA of reproductive cells—egg or sperm. Germinal mutations

can be transmitted to the offspring only if one of

those particular germ cells is involved in fertilization.

Germ line: The tissue or cell line that produces gametes

and is used for reproductive purposes, as opposed

to tissue or cell line from somatic cells. Also known

as “germinal tissue. ”

Haploid: Half of the full set of genetic material, or one

chromosome of each homologous pair. Gametes

have a haploid set of DNA. Fertilization of an ovum

by a sperm produces a diploid number of chromosomes in the zygote.

Hemoglobin: The oxygen-carrying molecule found in

red blood cells. Adult hemoglobin is a protein

composed of a single heme group bound to two

alpha globin chains and two beta globin chains.

Hemoglobinopathies: A collection of hereditary disorders of hemoglobin structure and/or function.

Examples are thalassemia and sickle cell anemia.

Hemophilia: A genetic disorder distinguished by a

deficiency of one or more coagulation factors—e.g.,

Factor VIII (hemophilia A) or Factor IX (hemophilia

B). The underlying mutations are located on the X

chromosome. Hemophilia occurs most often in

males who have only one X chromosome, and is

transmitted to offspring by asymptomatic females,

who have two X chromosomes, one of which carries

the hemophilia mutation.

Heritable mutation: A mutation that is passed from

parent to offspring and was present in a germ cell

of one of the parents.

Heteroduplex: A double-stranded nucleic acid molecule composed of individual strands of different

origin, e.g., a parent’s DNA strand hybridized to

a child’s DNA strand, or RNA hybridized to DNA.

Heterozygote: An individual who has two different

alleles of any one particular gene, e.g., an individual who has one copy of the gene for thalassemia

at the locus for beta globin is heterozygous for this

trait.

Homoduplex: A double-stranded nucleic acid molecule composed of two strands of the same origin,

e.g., genomic DNA isolated from human ceils

(DNA hybridized with DNA from the same individual).

Homozygote: An individual with the same allele at

both genes responsible for a particular trait.

Homozygous: Having identical alleles at a given locus.

Compare heterozygous.

Hybridization: The process of joining two singlestrands of RNA or DNA together so that they

become a double-stranded molecule. For hybridization to occur, the two strands must be nearly or

perfectly complementary in the sequence of the

nucleotide base pairs.

Induced mutation: A change in the structure of DNA

In

or the number of chromosomes, caused by exposure

of the DNA to a mutagenic agent.

vitro: Literally, “in glass, ” pertaining to a biological process or reaction taking place in an artificial

environment, usually a laboratory. Sometimes used

to include the growth of cells from multicellular

organisms under cell culture conditions.

ix
In vivo: Literally, “in the living, ” pertaining to a biological process or reaction taking place in a living

cell or organism.

Karyotype: A photomicrograph of an individual’s

chromosomes arranged in a standard format,

showing the number, size, and shape of each chromosome.

Kilobase: A unit of measurement for the length of nucleic acids along a chromosome. One kilobase

(abbreviated kb) consists of 1,000 nucleotide bases.

Genes can be several kilobases in length.

Lambda: A bacterial virus that infects E. coli, producing many copies of itself within the bacterium.

Lesch-Nyhan syndrome: An X-linked recessive disorder characterized by compulsive self-mutilation

and other mental and behavioral abnormalities. It

is caused by a defect in the gene that produces a

particular enzyme (hypoxanthine-guanine phosphoribosyl transferase) important in metabolism.

The causal relationship to the behavioral aspects of

the disorder is not yet understood.

Locus: The position of a gene or of a group of

functionally related genes on a chromosome.

Locus Test: A measurement of genes examined in a

study of mutant proteins, based on the number of

proteins examined, on the number of gene loci represented by each such protein, and on the number

of individual samples obtained.

Lymphocytes: Specialized white blood cells involved

in the body’s immune response. B-lymphocytes

originate in the bone marrow and when stimulated

by an antigen produce circulating antibodies (humoral immunity). T-lymphocytes are produced in

the bone marrow and mature in the thymus gland

and engage in a type of defense that does not depend

directly on antibody attack (cell-mediated immunity).

Marfan’s syndrome: A genetic disorder characterized

by abnormally long fingers and toes and by abnormalities of the eye lenses and heart. (Abraham

Lincoln is thought by some to have suffered from

this disease). Also called arachnodactyly (“spider

fingeredness”).

Meiosis: The process of cell division in reproductive

cells that reduces the number of chromosomes to

half of the full set so that each parent contributes

half of the genetic material to each offspring. When

the egg and sperm fuse at conception, the full set

of chromosomes (a total of 46 chromosomes in

humans) is restored in the offspring.

Mendelian: A term used to refer to a trait that follows

Mendel’s laws of inheritance and is controlled by

a single gene, and which therefore shows a simple

pattern of inheritance (dominant or recessive). So

named because traits of this sort were first recognized by Gregor Mendel, the Austrian monk whose

early research laid the basis for modern genetics.

Mendel’s laws include the Law of Segregation,

which describes how each pair of alleles separates

into different gametes and the Law of Independent

Assortment, which describes how different alleles

are assorted independently of the other alleles in

gametes and how the subsequent pairing of male

and female gametes occurs randomly.

Messenger RNA (mRNA): Ribonucleic acid that serves

as the template for protein synthesis. It is produced

by transcribing a DNA sequence into a complementary RNA sequence. Messenger RNA molecules

carry the instructions for assembling proteins on the

ribosomes.

Mitosis: The process of division involving DNA replication, and resulting in two daughter cells with

the same number of chromosomes and cytoplasmic

material as the parent cell.

Multifactorial traits: Traits that are not determined

solely by a single gene. Multifactorial, or polygenic,

traits (e.g., height) have variable phenotypic effects

that depend for their expression on the interaction

of many genes and environmental influences.

Mutagen: A chemical or physical agent that causes

changes in the structure of DNA.

Mutation rate: The number of mutations per unit of

DNA (e.g., per gene, per nucleotide, per genome,

etc. ) occurring per unit of time (usually per generation).

Mutations: Changes in the composition of DNA, generally divided according to size into “gene mutations” (changes within a single gene, such as nucleotide substitutions) and “chromosome mutations”

(affecting larger portions of the chromosome, or the

loss or addition of an entire chromosome). A “heritable mutation” is a mutation that is passed from

parent to offspring and therefore was present in the

germ cell of one of the parents. See also induced

mutation and spontaneous mutation.

Nucleic acids: Macromolecules composed of sequences

of nucleotides that carry genetic information, There

are two kinds of nucleic acids: DNA, containing the

sugar deoxyribose and RNA, containing the sugar

ribose.

Nucleotide: A subunit of DNA or RNA, consisting of

a nitrogenous base (adenine, guanine, thymine,

cytosine, or uridine), a phosphate molecule, and a

sugar molecule (deoxyribose in DNA or ribose in

RNA). The linkage of thousands of these subunits

forms the DNA or RNA molecule.

Nucleus: The intracellular structure of eukaryotes that

contains DNA.

Oligonucleotide: A polymer made up of a few (generally fewer than 10 or 20) nucleotides; a short sequence of DNA or RNA.

Peptide: A compound consisting of two or more amino

acids linked together, formed through a chemical

process that produces one molecule of water for

each joining of one amino acid to another. Peptides

are the building blocks of proteins.

x
Phenotype: The appearance of an individual or the

observable properties of an organism that result

from the interaction of genes and the environment.

PhenyIketonuria (PKU): An autosomal recessive genetic disorder of amino acid metabolism, caused by

the inability to metabolize phenylalanine to tyrosine. The resulting accumulation of phenylalanine

and derived products causes mental retardation,

which can be avoided by dietary restriction of

phenylalanine beginning soon after birth.

Polymorphism: A single gene trait (e.g., a red blood

cell surface antigen) that exists in two or more alternative forms (such as types A, B, AB, and O blood).

Genetic variants are considered polymorphisms if

their frequency exceeds 1 percent each, but are considered “rare mutations” if they are found in less

than 1 percent of the population.

Polypeptide: A sequence of amino acids joined in a

chain.

Probe: A molecule that has been tagged or labeled in

some way with a tracer substance (a radioactive

isotope or specific dye-absorbing compound) that

is used to locate or identify a specific gene or gene

product. For example, a radioactive mRNA probe

for a DNA gene, or a monoclinal antibody probe

for a specific protein. See also DNA probes.

Prokaryote: A cell or organism lacking membranebound, structurally discrete nuclei. Compare

eukaryote.

Protein: A molecule composed of hundreds of linked

amino acids in a specific sequence, which is, in turn,

determined by the sequence of nucleotides in DNA.

Proteins are required for the structure, function,

and regulation of cells, tissues, and organs in the

body.

Recessive: A genetic trait that is manifested phenotypically only when both alleles for the trait are

present at a locus. E.g., sickle cell anemia is manifest only when both copies of the gene for beta

globin contain the betas mutation, whereas when

individuals have only one copy of the betas gene,

they do not develop the disease, but have sicklecell trait, a generally benign condition. X-linked

traits act as if they were recessive in females and

dominant in males. Compare dominant.

Recombinant DNA (rDNA) technology: Techniques

involving the incorporation of DNA fragments,

generated with the use of restriction enzymes, into

a suitable host organism’s DNA (a vector). The host

is then grown in culture to produce clones with

multiple copies of the incorporated DNA fragment.

The clones containing this particular DNA fragment

can then be selected and harvested.

Replication: The synthesis of new DNA from existing

DNA.

Restriction enzyme: An enzyme that has the ability to

recognize a specific nucleotide sequence in a nucleic

acid (ranging from 4 to 12 base pairs in length) and

cut or cleave the nucleic acid at that sequence. They

are termed “restriction” enzymes because, occurring

naturally in bacteria, they recognize foreign nucleic

acid (e. g., the DNA of a bacterial virus as it begins

to infect and destroy its host) and destroy it, thus

restricting the ability of the virus to prey upon

certain potential host strains. Over 400 different restriction enzymes are known, recognizing a great

variety of different nucleotide base sequences. Use

of restriction enzymes has made possible the cutting

and splicing together of nucleic acid within and

between different organisms and species.

Restriction Fragment Length Polymorphism (RFLP):

Fragments of DNA that may vary in length from

individual to individual, depending on the presence

of polymorphisms or rare mutations.

Ribosome: A cellular organelle that is the site of

protein synthesis, the process of reading the

instructions in mRNA and using it as the guide to

constructing the specified protein.

RNA (ribonucleic acid): The nucleic acid found mainly

in the nucleus and ribosomes and is involved in the

control of cellular activities. There are several

classes of RNA that serve different purposes, including messenger RNA (mRNA), transfer RNA (tRNA),

and ribosomal RNA (rRNA). The functions of various kinds of RNA include “translating” selected

gene sequences coded in the cell’s DNA, and transferring that information outside of the nucleus to

structures that then synthesize the proteins indicated

by the RNA vehicle.

RNaseA: An enzyme that cleaves RNA where certain

sequences of nucleotide bases occur. RNaseA has

been used to cut fragments of RNA/DNA heteroduplexes where particular mismatches in the heteroduplexes occur.

Sentinel phenotypes: A group of autosomal dominant

or X-linked conditions that can occur sporadically.

They are thought to result from new germinal

mutations in the parents’ reproductive cells.

Sex chromosomes: The X and the Y chromosomes, 2

of the 46 chromosomes in human cells, that determine the sex of the individual. Females have two

X chromosomes, while males have one X and one

Y chromosome.

Sickle-cell disease: A genetic disorder of hemoglobin

function caused by the presence of an abnormal

beta globin chain. Patients with sickle-cell disease

have red blood cells that tend to deform into a

sickle-like shape. The specific defect is caused by

an abnormal gene, resulting in the replacement of

the usual amino acid, glutamic acid, with valine,

in the sixth amino acid position in the beta-hemoglobin molecule. This increases its propensity to

crystallize, thus rupturing the red blood cell and

causing the cells to lodge in small blood vessels.

Also called “sickle-cell anemia.” See sickle-cezl trait.

Sickle-cell trait: The generally benign condition shown

xi
by individuals carrying the variant betas gene as

well as the normal betaa gene. Such individuals are

heterozygous for the sickle-cell gene, and are healthy

(i.e., are usually asymptomatic), but two heterozygous parents have a 25 percent risk with each

pregnancy of having a child with homozygous

sickle-cell disease.

Single gene disorder: A genetic disease caused by a single gene and showing a simple pattern of inheritance (e.g., dominant or recessive, autosomal or XIinked). Also called “Mendelian disorder.”

Somatic: A term used to refer to body tissues, as opposed to reproductive (germinal) tissues.

Somatic cell: Any cell in the body except reproductive cells or their precursors.

Spontaneous mutation: In the absence of any known

causative agent, a change in the structure of DNA

or in the number of chromosomes. Also called a

“background” mutation. Also see mutations.

Stem cells: Undifferentiated cells in the bone marrow

that have the ability to replicate and to differentiate into blood cells.

Tay-Sachs disease: An autosomal recessive genetic disease resulting in developmental retardation, paralysis, dementia and blindness, usually fatal in early

childhood. The defective gene codes for hexosaminidase A, an enzyme that is involved in certain chemical pathways in the brain. Symptoms are caused

by an accumulation of cerebral gangliosides, fatty

acid and sugar molecules found in the brain and

nervous tissue. The gene is found in highest frequency among Jews of Eastern European origin.

Teratogen: A physical or chemical agent (e.g., thalidomide, radiation, alcohol, etc. ) that can cause congenital abnormalities as a result of exposure in

utero.

Tetramer: A complex molecule consisting of four major portions joined together (e.g., hemoglobin, in

which two alpha chains and two beta chains are

joined to a central heme group. )

Thalassemia: A group of autosomal recessive genetic

disorders characterized by abnormalities in synthesis of the globin polypeptides of hemoglobin. The

two most common forms are alpha-thalassemia and

beta-thalassemia, disorders of the alpha- and betaglobin polypeptides, respectively, which cause imbalances in the production of the globins and lead

to an overall deficiency of adult hemoglobin. The

thalassemias are most common in people of Mediterranean, Middle Eastern, and Asian descent.

T-lymphocytes: See Lymphocytes,

Transcription: The synthesis of messenger RNA (mRNA)

on a DNA template. The resulting RNA sequence

is complementary to the DNA sequence.

Transfer RNA (tRNA): Specialized RNA molecules

that function to bring specific amino acids to ribosomes that translate messenger RNA (mRNA) into

proteins.

Translation: The process in which the genetic code

contained in the nucleotide base sequence of messenger RNA directs the synthesis of a specific order of amino acids to produce a protein.

Trisomy: The presence of an extra chromosome, resulting in three homologous chromosomes instead

of two, e.g., Down syndrome can result from

Trisomy 21, or the presence of an extra chromosome number 21 in each body cell.

tRNA: See transfer RNA.

X-linked mutation: A mutation that occurs in a region

of the X-chromosome.

Zygote: A fertilized egg that results from the fusion

of sperm and egg.

xii
Chapter 1

Summary and Options
Contents

Introduction .

Request for

. . .

the

. . . . . . . . . . . . . . . . . . . . . . . .

Assessment . . . . . . . . . . . . .

Scope of the Report.. . . . . . . . . . . . . . . . . . .

Background ... ..~.. . . . . . . . . ........+ . . . .

Kinds and Effects of Mutations in Human

Current Methods for Studying Mutations . . .

Animal Studies . . . . . . . . . . . . . . . . . . . . . . . .

Studies in Human Beings . . . . . . . . . . . . . . .

. . . . . .

. . . . . .

. . . . . .

. . . . . .

Beings

. . . . . .

. . . . . .

. . . . . .

New Technologies for Detecting Human Mutations

Page

. . . . . . . . . . . . . . . . . . . . . . . . . . 3

. . . . . . . . . . . . . . . . . . . . . . . .,, 4

. . . . . . . . . . . . . . . . . . . . . . . . . . 4

. . . . . . . . . . . . . . . . . . . . . . . . . . 5

. . . . . . . . . . . . . . . . . . . . . . . . . . 5

. . . . . . . . . . . . . . . . . . . . . . . . . . 6

. . . . . . . . . . . . . . . . . . . . . . . . . . 6

. . . . . . . . . . . . . . . . . . . . . . . . . . 7

. . . . . . . . . . . . . . . . . . . . . . . . . . 10

Detection and Measurement of Heritable Mutations. . . . . . . . . . . . . . . . . . . . . .. .11

Detection and Measurement of Somatic Cell Mutations . . . . . . . . . . . . . . . . . . . . 13

Use of New Technologies in Research and Public Policy. . . . . . . . . . . . . . . . . . . . . .14

Feasibility and Validity Testing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .14

Epidemiologic Activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .......14

Extrapolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .......14

Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Federal Spending formulation Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Development of New Technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...16

Feasibility and Validity Testing . . .

Field Studies . . . . . . . .

Integration

Figure No.

. . . . . . . . . . .

and Human

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

FIGURE

Page

1. Organizational Hierarchy of DNA, the Carrier of Genetic Information

in Human Cells.... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Chapter 1

Summary and Options

INTRODUCTION

Mutations, lasting changes in the genetic information carried in the deoxyribonucleic acid

(DNA) of cells, can cause severe diseases and disabilities, none of which is curable and relatively

few of which can be treated effectively. Such

genetic diseases represent a significant fraction of

chronic disease and mortality in infancy and childhood; they generally impose heavy burdens expressed in premature mortality, morbidity, infertility, and physical and mental handicap. Some

of the most common of the 3,000 or more different disorders known to result from mutations include Down syndrome, Duchenne muscular dystrophy, and hemophilia. In addition, mutations

have been associated with increased susceptibilities to certain chronic diseases, including some

forms of diabetes, heart disease, and cancer. Most

mutations that are expressed as genetic disease already exist in the population and are carried from

generation to generation. A smaller proportion

of mutations arises anew, “sporadically,” in each

generation, and the specific causes of these mutations are unknown.

The public and the government have expressed

concern about the possibility that environmental

exposures are contributing to or increasing the frequency of mutations. Mutations are among the

chronic health effects singled out in the Toxic Substances Control Act of 1976 (TSCA) and in the

Comprehensive Environmental Response, Compensation, and Liability Act of 1980 (CERCLA or

“Superfund”). In those laws, Congress specified

public protection from exposures that can cause

mutations. The other major environmental statutes contain language broad enough to include

protection from mutagens.

Unfortunately, little is currently known about

the kinds and rates of mutations that occur in

human beings. Available methods to study such

mutations are inadequate to provide sufficient information for evaluating mutagenic risks.

Much of our knowledge of genetic risks to human health from exposures to environmental

agents has been derived from the study of the effects of mutagens on experimental animals. These

experiments are useful in manipulating various

aspects of the mutagenic process, for example, to

examine how mutagens act on DNA and to study

effects of varying doses and rates of exposure to

mutagenic agents administered either singly or in

combination. Experimentation with animals is essential for assessing potential hazards of new

chemical and physical agents before human populations have been exposed to them. However,

at present, technical problems in detecting and

measuring mutations limit animal experiments as

they limit human studies, so the results from animal experiments, using current methods, detect

only a small proportion of the kinds and numbers of mutations that can occur.

Recent advances in molecular biology have led

to the development of new technologies for examining DNA that may provide insight into the kinds

and rates of mutations that occur in human beings. This report assesses these developments and

discusses their potential for predicting risks of mutation from particular exposures.

At present, these new technologies propose reasonable and verifiable ways of detecting heritable mutations in human DNA and proteins, but

none is efficient enough to be used on a large scale.

However, there is considerable optimism in the

scientific community that these new technologies

can provide, for the first time, the means to obtain basic knowledge about the primary causes

of mutation and the means to assess the kinds and

rates of mutations that occur in human beings.

Data derived from studies in human beings,

along with verifiable methods to extrapolate from

corresponding animal data, will permit a more informed assessment of the medical and biological

3
4 ● Technologies for Detecting Heritable Mutations in Human Beings

consequences of mutagenic exposures. At present,

without such comparative data, it is difficult to

know whether general extrapolations from animal data would lead to underestimates or overestimates of the genetic risks for humans. Continuing to rely on inadequate data may incur both

human and financial costs, since conclusions

drawn from this information contribute to decisions about acceptable levels of exposure and the

level of society’s resources that are devoted to providing protection from such exposures.

A combination of factors—concern that environmental exposures may be contributing to human mutations, questions about the fundamental nature of mutations, and increasing knowledge

of the structure and function of DNA—increase

the likelihood that new technologies will be developed and field tested. However, studies using

these technologies may be expensive and will

probably require the collaboration of a large number of scientists; their continued development, pilot testing, and large-scale application may require

sufficient interest and financial support outside

the scientific community. With such support, and

with continued development of the techniques,

some of these techniques could be ready for largescale use in the next 5 to 10 years.

Congressional interest in supporting basic research on human mutations and in the continued

development of these technologies is necessary if

the regulatory agencies are eventually to have the

tools to evaluate risks from most occupational or

environmental exposures. The current lack of information on kinds and rates of human mutations

is largely due to the inadequacy of present methods to study heritable mutations. Efforts to comply with the agencies’ mandates to protect people from mutagens may be impeded unless basic

knowledge of causes, kinds, and rates of human

mutations is obtained.

Request for the Assessment

This assessment was requested by the Senate

Committee on Veterans’ Affairs, the House Committee on Science and Technology, and the House

Committee on Energy and Commerce. Interest in

the assessment was also expressed by the Senate

Committee on Public Works and the Environment, the Senate Committee on Labor and Human Resources, and the House Committee on Veterans’ Affairs.

These committees have wrestled with problems

of determining whether past exposures to potential mutagens have affected the health of veterans

and civilians and of framing reasonable public

health laws that can be implemented, given current knowledge and technologies. OTA was asked

to assess the available information about current

means for detecting mutations as they relate to

these issues and on the likelihood and potential

impact of technological developments.

Scope of the Report

This chapter summarizes current knowledge

about the kinds and rates of human mutations and

the methods that have been used to detect heritable mutations in human beings and in experimental animals. New technologies assessed in this

report for detecting and measuring human heritable mutations are briefly described. Methods for

measuring human somatic mutations are discussed

as tools for evaluating the risks of heritable mutations. The final section of this chapter presents

options for congressional action.

Chapter 2 provides background information

about human genetics and DNA, and discusses

the types of mutations that can occur and their

potential health effects. Chapter 3 reviews the

literature on current methods for studying mutations and summarizes current knowledge about

the frequency of heritable mutations in human

populations.

The new technologies for examining human

DNA for heritable mutations are described in

chapter 4, followed by descriptions of new somatic mutation tests in chapter 5.

Chapter 6 summarizes data from experimental

animals on spontaneous and induced mutations,

and discusses the possible use of such data for

identifying human mutagens and determining

their potency. Chapter 7 focuses on the problems

of extrapolating from the results of animal experiments to human risks.

Chapter 8 discusses epidemiologic considerations in the application of the new technologies,
Ch. l—Summary and Options 5

such as validation of the new methods and selec- A summary of current Federal expenditures in

tion of at-risk populations to study. Chapter 9 dis- putation research and potential costs of studies

cusses Federal involvement in protecting against to detect mutations using the new technologies is

genetic risks and the regulatory mechanisms avail- presented in appendix A.

able to control exposures to mutagenic agents.

BACKGROUND

Kinds and Effects

in Human Beings

of Mutations

Mutations can occur “spontaneously,” that is,

in the apparent absence of any unusual stimuli,

or they can be “induced” by particular agents. It

is likely that many or most “spontaneous” mutations are caused by external forces, possibly including ionizing radiation, ultraviolet radiation,

viruses, and certain chemicals, but the appropriate links have not been made. Some mutagens

present around us may also be necessary for sustaining life, for example, oxygen, components of

our food, and some of the body’s own metabolizes. Experiments in animals have shown that

many substances present in agricultural, industrial, and pharmaceutical chemicals in use today

are mutagenic in some test systems. Which of

these cause mutations in human beings is still a

matter of speculation. Precise causes for essentially all mutations that have been identified in

human beings are unknown.

At present, more than 3000 different genetic

diseases have been identified, including disorders

resulting from mutations in DNA, and disorders

resulting from the interaction of genetic and environmental components. Approximately 10 in

1,000 liveborn infants are born with a single gene

disorder and an additional 6 in 1,000 liveborn infants are born with a major chromosome abnormality. It is estimated that approximately 80 percent of the single gene disorders are the direct

result of mutations that occurred in germ cells of

distant ancestors and were passed along to succeeding generations. The remaining 20 percent of

these cases (0.2 percent of all livebirths) and the

majority of chromosome abnormalities are believed to be due to sporadic mutations in the reproductive cells of one of the parents of the infant. An additional 10 in 1,000 liveborn infants

manifest a serious genetic disease sometime after

birth, and a far higher proportion of newborns

will show indirect effects of one or several parental

or ancestral mutations in later life as, for example, in increased susceptibilities to some forms of

heart disease, diabetes, or cancer.

Mutations are changes in the composition of

the genetic material, DNA (see fig. 1), and are generally divided according to size into gene mutations and chromosome mutations. Gene mutations refer to changes within a single gene, for

example, substitutions of single component nucleotides, or small losses or additions of genetic

material in expressed or nonexpressed regions of

the gene. Chromosome mutations affect larger

portions of the chromosome (e.g., structural rearrangements of genetic material in the chromosomes) or result in the loss or addition of an entire chromosome. Since DNA directs the synthesis

and regulation of molecules in the body, either

group of mutations can influence a wide range of

biological and physiological functions, including

reproduction, longevity, intelligence, and physical development. Individual differences in susceptibility to disease may result from the effects of

one gene, several genes, or combinations of genes

and environmental factors.

Depending on the nature and location of the

mutations and on the function of the genes in

which they occur, mutations may, in theory, be

beneficial, neutral, or harmful to the individual.l

The kinds and effects of known mutational events

range from single nucleotide substitutions (the

smallest unit of change in DNA) with no clinically observable effects, to single nucleotide subICurrent theory maintains that most newly arising mutations in

regions of DNA that directly determine the structure and regulation of proteins are more likely to be detrimental than beneficial.

Little is known of effects of mutations in other regions of DNA.
6 . Technologies for Detecting Heritable Mutations in Human Beings

Figure I.–Organizational Hierarchy of DNA, the

Carrier of Genetic Information in Human Cells

Nucleotides

A GC T

AAA CGC GAC CGA

-ACGAAAATCCGCGCTTCAGATACCTTA –

SOURCE: Office of Technology Assessment.

Adenine, guanine,

cytosine, and

thymine, the basic

building blocks of

DNA.

Codons

Nucleotides arranged in a triplet code,

each corresponding

to an amino acid

(components of proteins) or to a

regulatory signal.

Genes

Functional units of

DNA needed to synthesize proteins or

regulate cell function.

Chromosome

Thousands of genes

arranged in a linear

sequence, consisting of a complex of

DNA and proteins.

Genome

The complete set of

genetic information;

each human reproductive cell contains

23 chromosomes,

and all other cells in

the body contain a

full set of 46

chromosomes.

stitutions resulting in severe diseases; from major

structural and numerical chromosome abnormalities (the largest observed unit of change in DNA)

leading to various abnormalities and impairments,

to those resulting in embryonic, fetal, or neonatal death.

A child’s entire genetic endowment comes from

the DNA of two single reproductive cells (or gametes), one egg and one sperm, from his parents.2

A mutation occurring in the DNA of either of

these germ cells, a germinal mutation, is passed

on to the child, who is born with a “new” heritaMe mutation. Mutations in germ cells that are not

involved in fertilization are not passed on to the

offspring. If a mutation arises in the DNA of the

parents’ nonreproductive cells (collectively termed

somatic cells), such somatic mutations are not

transferred to the reproductive cells. Somatic mutations may, however, affect the parents’ health,

and indirectly, their ability to bear a healthy child.

‘Each of the gametes (the male spermatozoan and the female

ovum) is the product of a series of developmental stages of reproductive (or germ) cells.

CURRENT METHODS FOR STUDYING MUTATIONS

Current empirical methods to study mutations

in human beings focus on physiological and biochemical effects of mutations because, until recently, it was not possible to examine changes in

DNA directly. Each of the current methods detects only a limited portion of the spectrum of

mutational changes. These methods have been

used to derive estimates of baseline frequencies

of some kinds of human mutations.

Animal Studies

Much of our knowledge about how substances

interact with DNA and how they may cause mutations is derived from studies with experimental

animals. In addition, some estimates of human

mutation rates have been derived, by extrapolation, from animal studies. Beginning soon after

World War II and still continuing, spontaneous

and induced gene mutation rates have been studied in laboratory mice. The rate of spontaneous

heritable gene mutations, as detected in these experiments, is roughly two to eight mutations per

1 million genes per generation of mice. Radiation

and approximately two-thirds of about 20 chemicals3 tested so far increase the frequency of de-

—3 —

0n the basis of animal experiments using the specific locus test

and the heritable translocation test, in which they were found to

be mutagenic in all germ cell stages, these chemicals are strongly

suspected to be mutagenic in humans (154). They included both common environmental agents and chemicals available in the laboratory that are not normally found in the environment.
Ch. l—Summary and Options . 7

tectable heritable gene mutations in these experimental mice. Generally, the chemical substances

that induce heritable mutations in mature and

maturing germ cells also induce somatic mutations, and vice versa. However, substances that

induce somatic mutations do not necessarily induce heritable mutations in immature germ cells

(stem cells). Unlike mature germ cells or somatic

cells, these germinal stem cells may have efficient

systems for repair of mutational or premutational

damage. There is, in fact, indirect evidence from

dose-response and dose-rate experiments that germinal stem cells have good repair systems.

It may be useful to quantify relationships between somatic and germinal cells with regard to

mutagenic potency of different chemicals, and to

study mutations in equivalent sets of genes in both

types of cells. Animal experimentation is useful

for determining the feasibility of using human somatic mutation rates for predicting the risk of human heritable mutations. This work in animals

may demonstrate whether it is possible to extrapolate from the occurrence of somatic to that of

heritable mutations at all, and may help determine whether it is possible to generalize from animal data to human beings. In addition, animal

studies on heritable mutations are useful not only

for determining whether a given agent is mutagenic but also for more general explorations of

the factors that may influence the occurrence of

mutations.

Studies in Human Beings

Spontaneous heritable mutations in human beings have been studied by examining: 1) the incidence of certain genetic diseases (“sentinel phenotypes”), 2) gross changes in chromosome structure

or number, and 3) changes in the structure or

function of blood proteins. Epidemiologic studies

of specific populations, in particular, the survivors

of atomic bombs in Japan, provide some information about induced heritable mutations in human beings.

Sentinel Phenotypes

The classic method for identifying human heritable mutations is the empirical observation of infants and children for the presence of certain rare

genetic diseases known as sentinel phenotypes. Examples include achondroplasia (dwarfism), aniridia,

and some childhood cancers, such as retinoblastoma and Wilms’ tumor. By recording the occurrence of sentinel phenotypes as a proportion of

the total number of livebirths in a defined population over time, the frequency of each disorder

(and of its corresponding mutation) can be estimated (165).

The characteristic of sentinel phenotypes that

is most useful for mutation studies is that these

conditions are “sporadic” in most or all cases; they

almost always result from a new germinal mutation in one of the parents of the afflicted individual.4 Each different sentinel phenotype is thought

to result from a different, single, mutant gene, although precise genetic information to confirm the

single gene hypothesis is lacking in most cases.

Despite the distinctive characteristics of sentinel

phenotypes, the relevance of existing data on the

frequency of the various sentinel phenotypes to

the study of kinds and rates of human mutations

is limited by a lack of knowledge of the genetic

bases of the phenotypes, and by the small fraction of DNA that accounts for these phenotypes.

Of the several thousand known genetic diseases,

only 40 are thought to satisfy the criteria for inclusion as “sentinel phenotypes. ” Roughly 40

genes are involved in the 40 sentinel phenotypes,

among a total of an estimated 50,000 to 100,000

expressed genes in an individual’s DNA.

Sentinel phenotypes are severely debilitating

conditions that require accurate diagnosis and

long-term medical care. However, practical difficulties arise in gathering and maintaining data

on the incidence of sentinel phenotypes for the

purpose of tracking mutation rates. Infants with

sentinel phenotypes are rare, numbering approximately 1 in 10,000 to 1 in 10 million Iiveborn

infants, depending on the particular disease. Consequently, a huge number of infants must be observed in order to find even a few infants with

sentinel phenotypes. Diagnosis of individual phenotypes is complicated by the genetic heterogeneity

of these disorders, so that highly trained specialists in various pediatric subdiscipline, which are

‘Various tests are done to exclude nonsporadic cases, which could

result from X-linked recessive inheritance, mistaken parentage, and

the occurrence of other genetic or nongenetic conditions that mimic

the appearance of sentinel phenotypes.
8 ● Technologies for Detecting Heritable Mutations in Human Beings

few in number, would be needed to make these

observations. Millions of consecutive newborn infants would have to be monitored thoroughly and

accurately for many years, and registries, much

larger than those currently in use, would be

needed to collect and maintain the necessary data.

Chromosome Abnormalities

Another method for identifying a certain class

of human heritable mutations is the examination

of chromosomes under a light microscope for the

presence of chromosome abnormalities (“cytogenetic analysis”). Normal human DNA is organized into 46 chromosomes (44 autosomes and 2

sex chromosomes), distinguishable by size, proportional shape, and staining pattern, or “banding.” Chromosome abnormalities are defined as

either numerical (extra or missing whole chromosome[s]) or structural (deletions, insertions, translocations, inversions, etc., of sections of chromosomes). In total, chromosome abnormalities are

estimated to occur in at least 5 percent of all human conceptions. The majority of such conceptuses are spontaneously aborted, but the few that

survive comprise approximately 0.6 percent of all

liveborn infants. The incidence of Down syndrome is 1 in 650 at birth, making it the most common chromosomal disorder in newborns.

With the most advanced chromosome staining

methods currently available, approximately 1,000

bands are distinguishable in one set of human

chromosomes, although far fewer bands are produced with routine methods. Mutations in DNA

sometimes cause a change in the banding pattern,

particularly if such mutations involve large sections of a chromosome. With routine banding

methods, there may be several hundred genes

present in each visible band, and with higher resolution banding methods, a single band may contain about 100 genes. However, smaller mutations, from single nucleotide changes within genes

on up to some deletions and insertions of entire

genes, generally are not visible by any banding

method.

Measurement of Mutant Proteins

Most of the available information on rates of

spontaneous human mutations has been derived

from studies of sentinel phenotypes and chromosome abnormalities, but data on different kinds

of mutations in humans is now emerging from

studies of mutant proteins. In general, mutant

proteins are more precise indicators of genetic

damage than are clinical and cytogenetic observations. Certain mutations in genes that determine

the structure of proteins alter the chemical characteristics of the proteins, causing them to behave

differently in separation and purification procedures. These differences suggest that a mutation

has occurred because proteins are constructed

according to blueprints in DNA, and changes in

DNA can lead to the production of altered proteins. If the protein under study is an enzyme, a

mutation within the gene that codes for it can

alter, diminish, or eliminate the enzyme’s biochemical activity.

Operationally, mutant proteins are identified

by taking samples of blood from each member

of a “triad,” including both parents and the child.

Proteins are extracted from blood components,

and the proteins are separated by electrophoresis. Putative mutations are identified when a protein from a child behaves differently from the corresponding protein from both parents.

One-Dimensional Separation of Proteins.—The

technique most commonly used to study mutant

proteins is electrophoresis, a method of separating proteins on the basis of their electrical charges.

The term “electrophoretic variant,” or “electromorph, ” is used to describe a protein that behaves

differently in electrophoresis from the corresponding protein found in the parents.

Although one-dimensional electrophoretic analysis of proteins is well established and can be

improved by including functional assays for additional enzymes, it is limited to detecting: 1) mutations that do not eliminate the functional ability of the proteins, 2) nucleotide substitutions only

in coding regions of genes for the proteins examined, and 3) only those nucleotide substitutions

that alter the electrical charge on these proteins;

such substitutions are thought to account for

about one-third of all nucleotide substitutions in

coding regions, which, in turn, account for a fraction of all the kinds of mutations that can occur.

Electrophoresis does not detect many other types

of mutations, including small duplications, rearrangements, or mutations that result in the
Ch. l—Summary and Options ● 9

absence of gene products; these mutations are

thought to constitute the majority of the mutations induced by certain mutagens, including radiation. Moreover, electrophoresis does not detect mutations that occur anywhere outside the

coding regions of a certain set of genes, including mutations in other coding genes and in nonexpressed regions of the DNA.

Data from several studies using one-dimensional electrophoresis have been used to estimate

the rate at which mutations produce electrophoretic variants, and from this estimate, to infer the

total rate of amino acid substitutions in proteins,

and the corresponding mutation rate per nucleotide in human DNA.

Two-Dimensional Separation of Proteins.—An

extension of one-dimensional electrophoresis involves separation of proteins in a second dimension. With two-dimensional electrophoresis, about

300 proteins from each person can be separated

and examined, compared with about 100 proteins

per sample that can be separated in one-dimensional electrophoresis. Further improvements may

be possible with the use of computer algorithms

to assist in interpreting the complex two-dimensional gels. This technique is currently feasible,

and although it detects the same types of mutations as one-dimensional electrophoresis, it can

examine more proteins per sample.

Epidemiologic Studies

An extensive body of data from experimental

animals demonstrates that exposure to radiation

and to certain chemicals can induce mutations in

mammalian germ cells. In humans, exposure to

ionizing radiation is known to cause somatic mutations, and it is suspected to enhance the probability of heritable mutations. To date, however,

the available methods have provided no direct evidence for the induction (by chemicals or by radiation) of mutations in human germ cells.

The single largest population studied for induced mutations is the group of survivors of the

atomic bombs detonated in Hiroshima and Nagasaki in 1945. Many survivors of the bombs received doses of radiation that could have caused

germinal mutations; in experiments with mutation induction by radiation in mammals, similar

kinds and doses of radiation were sufficient to

cause observable mutations in offspring. Therefore, it was assumed that germinal mutations

could have been induced in people exposed to the

radiation from the blasts.

Medical examinations of the survivors soon after the blasts revealed the immediate effects of

whole body irradiation: loss of hair; reduction in

bone marrow activity; and reduction in circulating white blood cells, associated with a reduction

in the body’s resistance to infection. Among those

who recovered from the immediate effects of the

radiation, there was a significant excess of cancer deaths later in life. Certain types of leukemia

were the first cancers to appear in excess, but continuing followup has revealed later increases in

other cancers, such as multiple myeloma and

cancers of the breast, thyroid, colon, esophagus,

stomach, lung, ovaries, and possibly of the spinal cord and nerves (24,55).

Exposure of pregnant women to radiation was

found to be associated with an increased incidence

in their liveborn infants of small head circumference, mental retardation, and an increased incidence of childhood cancers. The critical time for

fetal brain damage from radiation exposure was

identified as the period of 8 to 15 weeks of gestation (99).

Analysis of the chromosomes prepared from

peripheral blood lymphocytes of survivors exposed to the radiation has indicated an excess of

chromosome aberrations (7). Certain types of

chromosome aberrations (mainly balanced structural rearrangements, such as reciprocal translocations and inversions) have been found to persist in circulating lymphocytes long after exposure

to radiation, whereas other types of chromosome

aberrations (e.g., unbalanced rearrangements) in

lymphocytes declined in number soon after exposure. Overall, the frequency of chromosomally

aberrant cells in the survivors’ blood was found

to be proportional to the estimated dose of radiation received at the time of the bombing. It has

not yet known whether these somatic mutations

are correlated with specific cancers or other diseases in the survivors.

Survivors’ children who were conceived after

the acute radiation exposure were examined to
10 . Technologies for Detecting Heritable Mutations in Human Beings

study mutagenic effects on the parents’ reproductive cells. Using various methods available from

1945 to the present, survivors’ offspring were studied for “untoward pregnancy outcomes, ”5 for certain chromosome abnormalities, or most recently,

for abnormal blood proteins. The offspring of parents exposed to atomic radiation were compared

with the offspring of parents who were beyond

the zone of radiation (greater than 2,500 meters

from the hypocenter at the time of the bombings).

Observation and analysis of some 70,000 offspring has revealed no statistically significant excess in the incidence of stillbirths, congenital malformations, neonatal deaths, or chromosome

abnormalities. These findings suggest that the frequency of radiation-induced germinal mutations

that led to certain gross abnormalities in newborn

infants was not high enough to be detectable in

a population of that size and genetic heterogeneity. However, they do not rule out the possibility of other manifestations of genetic damage

in these children, or of latent expressions of such

damage, since the methods used to study this population could examine only a small subset of DNA

and only a limited number of genetic endpoints.

Analysis of the children’s blood proteins for

electrophoretic variants was later done to detect

recessive mutations, that is, mutations that are

not expressed as disease (unless present in both

5These were defined as major congenital defects and/or stillbirths

and/or death in the survivors’ offspring during the first postnatal

week. These abnormalities can be caused by exposure to radiation,

as well as to other environmental agents, and by socioeconomic

factors.

copies of a particular gene). This analysis, begun

in 1976, found few mutations in either the exposed

or control groups, making interpretation problematic. While the results indicate no significant excess of mutant proteins in the children of exposed

parents, they do not exclude the possibility that

an excess exists undetected. Unfortunately, electrophoresis is inefficient at detecting deletions, one

of the most likely types of radiation-induced mutations. Overall, these findings do not rule out

the possibility of genetic damage to the offspring

of survivors of the atomic bombs, but they put

upper limits of the frequency of occurrence of certain types of mutations that the current methods

are able to detect.

Taking these findings at face value, and cognizant of an enormous body of data on the genetic

effects of radiation on experimental animals, the

investigators suggest that the dose of radiation

necessary to double the human mutation rate (the

“doubling dose”) was between 139 and 258 rem,6

but they caution that there is a possibility of large

error attached to that estimate since genes other

than the ones sampled may demonstrate different sensitivity to radiation, and some types of mutations may be repaired more efficiently than

others. Their estimate of the doubling dose, if correct, however, indicates that man could be considerably less sensitive to radiation than laboratory mice.

6A rem (Roentgen-Equivalent-Man) is a measure of absorbed radiation dose. For comparison, a chest X-ray exposes an individual

to about 0.1 rem.

NEW TECHNOLOGIES FOR DETECTING HUMAN MUTATIONS

Recent advances in molecular biology have led

to the development of techniques that allow direct examination of DNA for evidence of mutations. These methods can examine large regions

of human DNA without requiring detailed knowledge of the genes contained in those regions and

without preparing genetic probes for particular

sequences. Unlike current methods—observing

sentinel phenotypes, chromosome abnormalities,

and electrophoretic variants—that are limited to

detecting a small fraction of all kinds and numbers of mutations, the new techniques have the

potential for detecting a wide, unselected spectrum

of mutations across the DNA. These techniques

are examples of the state-of-the-art in molecular

genetics and they are now promising to provide

the basis for better approaches to studying mutagenesis.
Ch. I—Summary and Options ● 1 1

Applying recent developments in molecular biology to the problem of detecting sporadic mutations, the new techniques described in this report

propose reasonable and verifiable ways of examining human DNA for alterations in sequence and

structure. These developments include the ability to clone specific genes, to cut up DNA into

predictable fragments, to hybridize complementary DNA strands, to detect less-than-perfect

hybridizations due to single base pair changes, and

to separate large fragments of human DNA. Some

of these methods detect similar types of mutations

and some complement each other by detecting different types. Some of these may merit further development and, eventually, pilot testing. Several

new technologies, representing different approaches,

are discussed below.

None of these new techniques has been applied

to large human populations or to experimental

animals and it is not known how well they will

perform. At present, none of these techniques is

approaching the efficiency needed for examining

the kinds and rates of mutations in a population

or for determining whether mutation rates are increasing. With technical improvements in efficiency, some of the techniques, or derivatives of

them, could be available in the next 5 to 10 years

for large-scale use. Since these technologies provide new information about DNA, the health implications of any newfound mutations may not

be immediately known. Additional research and

methods would be needed to examine biological

and physiological implications of the identified

mutations for the populations studied and for their

descendants.

Detection and Measurement of

Heritable Mutations

Restriction Fragment Length Polymorphisms

Restriction endonucleases are enzymes, isolated

from bacteria, that can be used experimentally to

cleave isolated DNA molecules into fragments at

specific sites in the DNA sequence that they recognize. If any of these sites have been altered by

mutation, the resulting pattern of fragment sizes

would also be altered. Restriction enzymes can

be used to detect mutations that either: 1) create

a new restriction site, 2) eliminate an old one, or

3) change the distance between existing restriction

sites. (These mutations may include single nucleotide substitutions, or multiple nucleotide deletions

or insertions. )

To use this method to detect mutations, DNA

would be treated with a set of restriction endonucleases and the resulting DNA fragments separated by electrophoresis and examined for differences between those present in the child’s DNA

and those in either parent’s DNA. Restriction site

analysis does not allow examination of every nucleotide. However, the use of a set of combined

restriction enzymes increases the number of restriction sites identified, allowing a larger portion

of the DNA to be examined, including both expressed and nonexpressed regions.

Genomic Sequencing

The most straightforward approach to looking

for mutations is by determining the sequence of

every nucleotide in a child’s genome and then

comparing this with the DNA sequences of the

child’s parents. To determine its sequence, human

DNA is cut with restriction endonucleases into

fragments, and then each fragment is analyzed for

its sequence of nucleotides. Genomic sequencing

would detect mutations regardless of where they

occur— in regions that code for specific proteins

and regulatory functions as well as in regions

without known functions—and is therefore potentially very informative. While it is technically possible at present, sequencing is currently feasible

only for very limited sections of DNA, such as

the length of DNA comprising only a few genes.

Because of current technical inefficiencies, it would

be an enormous task, involving many laboratories, a large number of scientists, and at least

several decades to sequence even one entire genome, the complete set of DNA in an individual’s

germ cell. At present, it is not feasible to use

genomic sequencing to examine several peoples’

genomes for mutations, although sequencing can

be used in conjunction with other techniques to

examine small sections of DNA.

One-Dimensional Denaturing Gradient

Gel Electrophoresis

A modification of the standard electrophoretic

gel procedure, denaturing gel electrophoresis al-
12 ● Technologies for Detecting Heritable Mutations in Human Beings

lows DNA fragments to be separated not only on

the basis of size, but also on the basis of sequence

of nucleotides. Double-stranded DNA separates

(“denatures”) into its constituent strands when it

is heated or when it is exposed to denaturing

chemicals. A gradient of increasing strength of

such chemicals can be produced in an electrophoretic gel so that DNA samples will travel in

the direction of the electric current, separate by

size, and begin to dissociate as they reach their

particular critical concentration of denaturing

chemical.

Every unique strand of DNA dissociates at a

different concentration of denaturant. In fact, a

sequence difference of only one nucleotide between two otherwise identical strands of doublestranded DNA is enough to cause the strands to

dissociate at different concentrations of denaturant chemical, and to stop traveling at different

locations in the gel. Using this technique, the parents’ and child’s DNA would be cut into fragments

with restriction enzymes, dissociated into single

stranded DNA, and reannealed with radioactively

labeled probe DNA. The resulting heteroduplex

fragments would then be separated on the basis

of their DNA composition in a denaturing gradient gel. Mismatches between the sequences of

probe and child’s DNA and not between probe

and parents’ DNA would appear as different

banding patterns on the gel. Again, a comparison between the banding pattern of parents’ and

child’s DNA analyzed in this way may identify

a wide range of mutations in all DNA regions.

Two-Dimensional Denaturing Gradient

Gel Electrophoresis

Another approach to detecting mutations is a

technique whereby sizing and denaturing gels are

used to differentiate among DNA sequences common to parents’ and child’s DNA, polymorphisms

in either parent’s DNA which are transmitted to

the child, and any new mutations in the child’s

DNA. Like the two-dimensional polyacrylamide

gel procedure for protein separation described

earlier, this approach compares locations of spots

on a gel (in this case, DNA spots) for evidence

of new mutations. In this method, various combinations of parents’ and child’s DNA are produced and compared on the basis of the denaturant concentration at which they dissociate.

Parents’ and child’s DNAs are compared to each

other, rather than to relatively small probes. This

approach, which would allow detection of mutations in the complete genome, would detect differences (“mismatches”) between a child’s DNA and

his or her parents’ DNA. Such mismatches would

represent various types of mutations in the nucleotide sequence in expressed and nonexpressed DNA

regions.

DNA-RNA Heteroduplex Analysis

This technique hinges on the production of

DNA bound to complementary strands of ribonucleic acid (RNA) or “DNA-RNA heteroduplexes,”

and on the use of specific enzymes, such as

“RNaseA,” that cleave the DNA at particular sequences where the DNA and RNA are not perfectly bound at every nucleotide. This is similar

to the use of restriction enzymes, which cleave

DNA at particular normal sequences, except that

RNaseA cleaves the RNA strand in RNA/DNA

hybrid molecules where there are mismatched

nucleotides, indicating mutations. The resulting

fragments are then separated electrophoretically

to detect differences between parents’ and child’s

DNA. The efficiency of this approach depends on

the number of different-mismatches that can be

recognized and cleaved. This method would detect nucleotide substitutions over a large portion

of the DNA.

Subtractive Hybridization

Detecting mutations would be much easier if

it were possible to ignore the majority of DNA

sequences that are the same in parents and child

and, instead, focus only on the few sequences that

may be different. Subtractive hybridization is an

idea for selecting and characterizing sequences in

a child’s DNA that are different from either parent’s DNA.

First, the double-stranded DNA of both parents is cut into fragments with restriction enzymes,

dissociated into single-stranded DNA, and then

mixed together with a set of single-stranded reference DNA sequences. These reference sequences

represent all possible sequences of 18 nucleotides

(analogous to a dictionary of 18-letter words using
Ch. l—Summary and Options . 13

only 4 different letters. ) Each reference sequence

binds to its complementary sequence in the parental DNA and can be removed from the mixture.

Any reference sequences left over, not bound to

parental DNA, represent sequences not found in

the parents’ DNA. If the child’s DNA binds with

any of these left-over sequences, such hybrids

would indicate that the child’s DNA contains

different sequences from those in the parents’

DNA. These hybrids could be separated and analyzed for mutations.

This approach is the least well developed of all

the ones discussed in this report, and its feasibility is unknown. If it does prove feasible, however, this approach would identify short sequences

containing mutations in any part of the DNA, allowing further detailed study (e.g., by DNA sequencing) of the kinds of mutations that may

occur.

Pulsed Field Gel Electrophoresis

If human DNA were short and simple, it could

be cutup with restriction enzymes and separated

electrophoretically into discrete bands, each representing a particular segment of the total DNA.

However, human DNA is so long that when restriction-digested DNA is electrophoretically separated, the resulting fragments of the whole set

of DNA form a continuous smear of bands. Even

if the DNA could be cut into 100 or 200 fragments,

the pieces would be too big to pass individually

through the pores of a standard electrophoretic

gel. A new technique, pulsed field gel electrophoresis is being developed to separate large fragments of human DNA and to examine such fragments for evidence of mutations. The procedure

may detect submicroscopic chromosome mutations, including rearrangements, deletions, breaks,

and transpositions. At the present time, however,

the method cannot handle whole human chromosomes, though it works well with fragments of

human chromosomes and with smaller whole

chromosomes from lower organisms. This technique may be useful in detecting chromosome mutations that are intermediate in size between major rearrangements (observable by cytogenetic

methods) and single base pair changes, potentially

a large proportion of all possible mutations.

Detection and Measurement of

Somatic Cell Mutations

The methods for detecting heritable mutations

rely on comparing the DNA of parents with the

DNA of their children to infer the kinds and rates

of mutations that previously occurred in parents’

reproductive cells. While this information is valuable in learning about heritable mutations, it

may come months or years after the mutations

have actually occurred, and this temporal separation of events makes it difficult to draw associations between mutations and their causes. Tests

to detect somatic mutations maybe useful in signaling the probability of heritable mutations. Such

tests may be useful in relating exposures to specific mutagens with particular genetic events in

the cell, and they may help to identify individuals and populations at high risk for mutations.7

It is thought that the mutation process is fundamentally similar in germinal and somatic cells.

If this is true, then it may be possible to predict

the risk of germinal and heritable mutations on

the basis of measurements of somatic mutations,

which are inferred from the frequency of mutant

cells. Several investigators are currently working

on methods to relate the frequency of mutant cells

to the number and kinds of underlying mutations.

Several new techniques for detecting and measuring somatic mutations are described in this

report. Mutant somatic cells may occur during

growth and development and may appear as

rarely as one in a million normal cells. Detection

of somatic mutants requires methods for scanning

through a million or so nonmutant cells to find

a single variant cell. Two general approaches are

used: 1) screening, which uses high-speed machinery to look at the total population of cells and

either count or sort out the variants; and 2) selection, in which a population of cells is cultured in

the laboratory under conditions that permit the

growth of variant cells and that restrict the growth

of the majority of cells that are nonvariants. The

7Even without knowing the exact relationship between rates and

kinds of somatic and heritable mutations, it is reasonable to predict that people with high somatic mutation rates might beat higher

risk for heritable mutations, either because of a particular environmental exposure or a genetic susceptibility to mutations.
14 Technologies for Detecting Heritable Mutations in Human Beings

new DNA technologies could be used to characterize the mutations in any such somatic variants

found.

Different mutagens have been shown to produce distinguishable types of mutations (“mutational spectra”) in human cells grown in culture.

Determining mutational spectra may be useful in

associating specific mutational changes in somatic

cells with particular mutagenic agents to which

individuals may be exposed. Such information

could be useful in understanding the causes of

USE OF NEW TECHNOLOGIES IN

Feasibility and Validity Testing

The new technologies now range from ideas on

paper to being in various stages of laboratory development, but none is yet ready for use in the

field. A critical step before a technology is used

in an investigation of a population thought to be

at risk for mutations is that the technology be

tested for validity and feasibility. To assure that

a technology is a “valid” method, that is, that it

detects the types of mutations that it theoretically

is capable of detecting, and to characterize the degree to which it gives the “right” answer, it will

be necessary to test a technology in a series of validation studies. A first step might be to test the

technologies against pieces of DNA with known

mutations, and at a later stage, in offspring of animals exposed to known mutagens. Feasibility testing will be required to make sure the technology

can be efficiently scaled up for analyzing large

numbers of samples.

Epidemiologic Activities

If the value of new technologies for detecting

mutations is to be realized, it will be as tools for

determining rates and patterns of mutations in epidemiologic studies of human beings. Once a technology has successfully passed through validation

and feasibility tests, it will become a candidate

for use in three major types of epidemiologic

activities: surveillance, monitoring, and ad hoc

studies.

mutational changes as well as in monitoring atrisk populations.

Data on kinds and rates of somatic mutations

may provide a monitor for exposure to mutagens

and carcinogens, and are relevant to the study of

carcinogenesis and of aging. However, measurements of somatic mutation rates per se have little direct applicability to intergenerational (or

transmitted) effects, without corresponding information on heritable mutations.

RESEARCH AND PUBLIC POLICY

Surveillance is a routine activity whose aim, in

the context of this report, would be to measure

the “baseline” rate of mutations in a defined population over the course of time and to facilitate

rapid recognition of changes in those rates. Monitoring consists of observations over time in a population thought to be at increased risk of, in this

case, heritable mutations, because of exposure to

a known or suspected mutagen, for the purpose

of helping the specific at-risk population in whatever way is possible. People living around hazardous waste disposal sites have been monitored

for endpoints other than mutations, e.g., cancer

and birth defects, and they would be likely candidates for mutation monitoring when technologies become available to do so efficiently. Ad

hoc studies of a variety of designs are carried out

to test hypotheses about suspected causes of mutations. Ideally, the results of ad hoc studies can

be generalized to populations other than those

specifically studied.

Extrapolation

Making predictions from observations of cause

and effect in one system to probable effects in

another is one form of extrapolation. The process involves a set of assumptions in moving from

one system to another. The practical importance

of extrapolation for mutagenicity is, ideally, to

be able to predict mutagenic effects on human beings from the response in laboratory animals or

lower test systems. The ability to extrapolate to
Ch. l—Summary and Options . 15

human responses addresses one of the major goals

of public health protection, the ability to identify substances harmful to human beings before

anyone is exposed, thereby providing a rational

basis for controlling exposure.

Extrapolation can be qualitative or quantitative. Qualitative, also called biologic, extrapolation involves predicting the direction of a result,

for example, if a chemical causes mutations in a

laboratory test, can we also expect mutations in

human beings? Quantitative extrapolation involves translating a quantitative result in an animal test into a quantitative estimate of mutagenic

risk in humans. Going a step further in extrapolation, can an estimate of mutagenic damage be

translated into a measure of genetic disease?

A number of theoretical models for extrapolating mutagenic effects have been proposed, based

on various parallel relationships. For instance, it

might be true that if the relationship between somatic and heritable effects in animals were known

after exposure to a specific mutagen, and if one

could measure a somatic effect in human beings

who had been exposed to the same substance, a

heritable effect in human beings could be predicted, assuming the relationship between somatic

and heritable effects is parallel in animals and human beings. Because of the paucity of data, particularly from human studies, it has been impossible to validate such an extrapolation model. The

new technologies should allow a major increase

in the database which, in turn, should allow researchers to more fully explore relationships

among various types of test results.

Regulation

Congress has mandated public protection from

mutagens in certain environmental health laws

(e.g., TSCA and CERCLA), and other laws provide mandates broad enough to empower agencies to take action against mutagens in virtually

any appropriate situation. Except for radiation,

however, very little regulatory evaluation has

taken place on the subject of heritable mutations.

This is directly related to the lack of sensitive

methods to detect heritable mutations in human

beings, and the related difficulty in extrapolating

from results in nonhuman test systems to probable human responses.

The Environmental Protection Agency (EPA)

has recently issued “Guidelines for Mutagenicity

Risk Assessment. ” EPA’s approach is relatively

simple and pragmatic. It requires only the types

of information that can be acquired with current

technologies, but allows for information from new

technologies, as they become available. The

guidelines require evidence of: 1) mutagenic activity from any of a variety of test systems, and 2)

chemical interactions of the mutagen in the mammalian gonad. Using a “weight-of-evidence” determination, the evidence is classified as “sufficient, ” “suggestive, ” or “limited” for predicting

mutagenic effects in human beings.

Although chemicals have not generally been

regulated as mutagens, it is probable that exposures to mutagens have been reduced by regulations for carcinogenicity. Strong evidence supports the idea that a first stage in many cancers

involves mutation in a somatic cell, and one of

the most widely used screening tests for potential carcinogens, the Ames test, is actually a test

of mutagenicity. The extent to which people are

protected against heritable mutations if their cancer risk from a specific agent is minimized is at

present unknown. The new technologies should

greatly improve our ability to make that judgment.

Federal Spending for

Mutation Research

OTA queried Federal research and regulatory

agencies about their support of research directed

at understanding human mutations. For fiscal year

1985, they reported about $14.3 million spent on

development or applications of methods for detecting and/or counting human somatic or heritable mutations. An estimated $207 million was

spent in the broader category of related genetic

research.
16 ● Technologies for Detecting Heritable Mutations in Human Beings

OPTIONS

Research related to the new technologies described in this report has the dual aims of increasing the knowledge base in human genetics about

the causes and effects of mutations, and producing information that could be used to estimate

mutagenic risks for the purpose of protecting public health. The pace and direction of research

toward developing these methods and the quality and efficiency of preliminary testing of methods could be influenced by congressional and executive branch actions and priorities. Integration

of research in different test systems and progress

in developing extrapolation models also can be

influenced by actions now and in the near future.

Continued progress in the development and application of new technologies will depend not only

on support of the individuals and laboratories

directly involved in this research, but also on

work in other areas. Although not directly addressed in this assessment, support of research in

medical, human, mammalian, and molecular

genetics will be essential to a full understanding

of the mutation process.

The options that follow are grouped in four sections: 1) options to influence the development of

new technologies, 2) options to address various

aspects of feasibility and validity testing, 3) options concerning the use of new technologies in

field studies, and 4) options to encourage coordination of research and validation of extrapolation

models.

Development of New Technologies

The Department of Energy (DOE), several

agencies of the Department of Health and Human

Services, and EPA currently provide funding to

researchers in independent and government laboratories for various research and development

activities pertaining to human mutation research

and the development of new technologies. Each

agency, appropriately, proceeds down a slightly

different path. OTA estimates that a total of about

$14.3 million was spent on human mutation research in fiscal year 1985. Progress in this research

could be speeded up by increased funding, though

it is difficult to quantify an expected gain. It is

clear also that less money spent on human mutation research will slow progress in laboratories already engaged in this research, and could deter

other scientists from pursuing research in this

field.

Option I: Congress could assure that funding

levels for human mutation research and closely

related studies do not decline without the responsible agencies assessing the impact of funding cuts on research progress. This requirement

could be expressed in appropriation, authorization, or oversight activities.

Of the several funding agencies, DOE has taken

the lead in funding much of the research on new

methods for mutation detection described in this

report, and researchers at some of DOE’s National

Laboratories are among the leaders in the research. DOE also is the agency responsible for

funding U.S. participation in the Radiation Effects

Research Foundation, the joint United StatesJapan body that continues to study the health of

Japanese atomic bomb survivors.

Option Z: A lead agency for research related to

detecting and characterizing mutations could

be designated. The lead agency would be responsible for tracking the development of new

technologies, facilitating the interchange of information among scientists developing the technologies and those in related fields, and encouraging and facilitating coordinated studies

involving different subdiscipline. The lead

agency also would keep Congress informed

about activities in this area. DOE may be the

logical choice for a lead agency.

The types of activities that a lead agency might

engage in are described below. These activities are

important whether or not a lead agency is designated, and Congress should consider directly encouraging them if there is no lead agency.

Tracking the Development of

New Technologies

All the new technologies require improvements

in efficiency before they become useful tools for

studying human beings. As research proceeds,

some techniques will develop more quickly than
Ch. l—Summary and Options . 17

others, some will be dropped, and some may

change in character, altering the kinds of mutations they can detect. The lead agency would be

responsible for keeping track of these developments.

It would be useful if the “tracking” responsibility could lead to actions on the part of the lead

agency that would promote the rapid and efficient

development of the technologies. At some point,

the technologies will be ready for feasibility testing and eventually, field testing. It would help researchers to know the stages of development of

various methods, and it could help agencies make

decisions about funding studies using certain

methods. In addition, the lead agency could assist researchers by anticipating needs that will be

common to all research programs and by encouraging efficient use of resources.

Facilitating Information Exchange

Among Researchers

In 1984, DOE organized and funded a meeting

that brought together for the first time many of

the researchers involved in laboratory-based mutation research. This meeting is acknowledged

among those who attended as a milestone for information exchange and the generation of new

ideas concerning detection of heritable mutations.

In fact, the ideas for some of the new methods

described in this report were born at that meeting. There is a continuing need for this type of

information exchange.

Keeping Congress Informed

Congress has already directed regulatory agencies to reduce exposures to environmental agents

that may cause mutations. As part of its oversight

of both regulatory and research activities, Congress could benefit from up-to-date information

about the development of various technologies.

The lead agency could report in a specified manner, e.g., a brief annual report describing the level

of current research, its goals, results of completed

or ongoing work, and expected near-term developments. This information could also be the basis for informing the public about mutation research. This activity will continue to be valuable

to Congress through later phases of development

and application of new technologies.

Feasibility and Validity Testing

After technologies pass through a phase of development to improve their efficiency and to work

out technical details, a period of feasibility and

validity testing will be necessary before a technology can rationally be used as a tool in a largescale study of heritable mutations in human beings. There are some ways to make validity testing an efficient process. As an example, a “DNA

library, ” a collection of known DNA sequences,

particularly sequences carrying known mutations,

could be established and maintained by one laboratory, which could make DNA sequences available to all researchers developing mutation detection technologies. This material could be used to

determine whether a particular technology detects

those mutations that it is designed to detect, analogous to testing chemical procedures and equipment against known chemical “standards. ” At a

more advanced stage, new technologies might also

be tested in animal experiments with a selection

of mutagens known to cause specific types of mutations. If a lead agency is designated, some of

these options would logically be among that

agency’s responsibilities. If there is no lead agency,

these functions could still be encouraged by Congress through oversight activities.

Banking Biological Samples

Biological samples, especially blood samples,

are often collected during the course of medical

examinations for people thought to be exposed

to environmental or occupational agents. Implementation of a plan to bank those samples would

facilitate human mutation studies and other related research.

Examining stored samples is not nearly so disruptive as collecting samples from a population.

The very act of specifically collecting samples for

a study of mutations would raise expectations that

definitive information about risks would be forthcoming. Examining stored samples would avoid

that human cost and could, at the same time, provide a realistic test of new technologies before they

are applied to people who are anxious about the

effects of environmental exposures on their genes.

Although stored samples offer many advantages, preparing human samples for storage and
18 ● Technologies for Detecting Heritable Mutations in Human Beings

maintaining the stored samples is a significant

task. Currently the high cost of storing samples

inhibits establishment of sample banks. There has

been little research directly aimed at improved

methods for storing biological samples.

Option 3: Congress could encourage banking of

biological material obtained from at-risk individuals and their spouses and offspring, with

the objective of studying somatic and heritable mutations from these stored samples before

technologies are used directly in a study of an

exposed population. A centralized samples

bank available to all researchers would be the

most efficient means of establishing a repository of sufficient size and scope for validity

testing.

Option 4: Congress could encourage the appropriate agencies (through the lead agency, if one

is designated) to investigate the potential for improvng technologies for storing biological samples. Where possible, funding should be encouraged for such improvements. This option

is relevant to a broad spectrum of human health

research, and collaboration among disciplines

should be encouraged for determining the specific storage needs of different lines of research.

Field Studies

Sometime in the next 5 to 10 years, it is likely

that one or more of the technologies for studying human mutations will be brought to the point

of readiness for epidemiologic studies of human

beings thought to be at risk for mutations. Such

a study could be initiated by an independent scientist or a group of scientists involved in the development of a new technology who have identified an at-risk population; a new technology or

technologies could be used in an environmental

health investigation triggered by the discovery of

a potentially at-risk population; or Congress could

mandate a study of mutations in a specific population. Depending on the way a study is initiated,

different agencies will be drawn in, and different

funding mechanisms will come into play.

Should a study be initiated by one or a group

of investigators who submit a grant proposal for

funding, the current system for review of research

awards is probably appropriate for making scientific judgments about whether a method is ready

for large-scale testing. From that point on, however, there are significant differences between a

proposal to use a new technology in a human population and most other proposals. The major differences are size and money. Application of any

promising technology will require that state-ofthe-art methods in molecular biology be expanded

from small-scale laboratory use to large-scale examination of blood samples collected from hundreds or thousands of people.

A study such as that described above would require a significant commitment of funds over a

period of years, and could account for a large percentage of a granting agency’s funds. The amount

of money necessary can only be estimated, but

it could amount to tens of millions of dollars, not

a large amount in government spending, but large

in comparison to most biomedical research grants,

which usually range from less than $100,000 to

about $500,000 annually. The necessary commitment of funds from any single agency, considering current spending for this type of research, is

likely to be impossible, no matter how worthwhile

the proposed study.

Option 5: Congress could consider providing specific add-on funding to finance an investigatorinitiated realistic test of a promising method to

study human heritable mutations.

A study mandated by Congress or an agencyinitiated study using new technologies will not

necessarily undergo the rigorous review and criticism that a proposal submitted to a granting

agency would. Congress already has some experience in mandating epidemiologic studies. Concerns about cancer and birth defects convinced

Congress to mandate studies of military veterans

who were exposed to Agent Orange or who participated in atomic bomb tests. In addition, Congress has vested the Public Health Service with

the authority to carry out a wide range of health

investigations of exposures from toxic waste

dumps through “Superfund.” The role of the mandated “Agency for Toxic Substances and Disease

Registry” (ATSDR) is to “effectuate and implement the health related authorities” of Superfund.

ATSDR, located within the Centers for Disease

Control, is a logical place for new technologies

to be used as exposed populations are identified

through other provisions of Superfund.
Ch. l—Surmmary and Options ● 19

An unusual array of health problems or an apparent excess of disease among people living

around toxic waste sites could trigger a study by

ATSDR. Such problems could also prompt local

residents to press Congress for studies to determine whether, among other things, the disorders

had been caused by mutations, and whether these

mutations could have resulted from chemicals in

the waste site.

Some scientific societies, such as the American

Society of Human Genetics, could be asked to

participate in a review of the feasibility of detecting possible health effects from environmental exposures. If the study is determined to be feasible,

Congress and the public can be reassured that the

study’s findings are likely to be useful in decisionmaking. Alternatively, a decision that no study

is currently feasible would underline the importance of developing and testing new methods. Reviewing the feasibility of a study may be perceived

as delaying an investigation unnecessarily. However, performing a study that has little or no likelihood of answering questions about exposure and

mutations has marginal value at best and would

not serve the people who requested it.

Option 6: If Congress mandates a study of heritable mutations in an at-risk population using the

new technologies, the mandate should include

a feasibility assessment by a panel of experts

before the study begins.

Option 7: Congress could require agencies to plan

for a rigorous outside review by a panel of experts of any agency-initiated study using a new

technology, before such a study can proceed.

Integration of Animal and

Human Studies

Much of our current ability to estimate the effects of various external agents on human beings

is derived from animal studies. In the future, animals will continue to be used to test for mutagenicity and, ideally, to predict effects in human beings. Right now, the available methods and body

of data provide an inadequate basis for making

predictions from animal results to human effects.

The new technologies described in this report for

detecting heritable and somatic mutations can be

applied in both animal systems and in human beings. This information and information from currently available animal systems could be used in

an integrated fashion to study relationships between somatic, germinal, and heritable mutations,

and to pursue the development of extrapolation

models for predicting effects in human beings.

The kind of integrated research necessary will

not occur spontaneously if the current pattern

holds. There are few researchers engaged in studies

that integrate comparable information from different systems for the purpose of elucidating relationships among such systems. Improvements in

extrapolation from animal to human data and

from somatic to heritable mutations await efforts

to encourage and coordinate the appropriate research. Single laboratories or centers are unlikely

to be able to perform all the different tests necessary to develop and test extrapolation models,

making coordination among laboratories essential.

The National Toxicology Program (NTP) is the

center of Federal mutation research using experimental animals, funding research grants and contracts nationally and internationally. The National

Center for Toxicological Research, a laboratory

of the Food and Drug Administration that is part

of NTP, has facilities and experience to carry out

large-scale animal tests. EPA also has a genetic

toxicology program, and it is actively pursuing

development and application of methods for

studying human somatic mutations. DOE’s National Laboratories have directed or carried out

the majority of large-scale animal studies of mutation rates and mechanisms, and this experience,

as well as the advanced technology that these laboratories have developed for sorting and studying chromosomes and cells, will be useful in improving methods for extrapolation. These three

agencies could encourage the development of

methods for linking information from animal

studies to somatic and heritable mutation risks in

humans.

Option 8: Congress could encourage studies of somatic, germinal, and heritable mutations in experimental animals using both currently available and new technologies. Further, research

funding agencies should encourage animal

studies directed at identifying the mechanisms

of mutagenesis and elucidating relationships between mutagenic potencies in animals and in

human beings.
Chapter 2

Genetic Inheritance

and Mutations
Contents

Page

Function and Organization of DNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .......23

Protein Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Inheritance of Genetic Traits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .......27

Kinds of Mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

Gene Mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

Chromosome Mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .......29

Health Effects of Mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .......30

Early Mortality and Morbidity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .......31

Diseases During Adult Life . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .......31

Effects of Somatic Mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .......31

Persistence of New Mutations in the Population . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

FIGURES

Figure No. Page

2. The Structure of DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

3. Replication of DNA... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

4. Diagrammatic Representation of Protein Synthesis According to the

Genetic Instructions in DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

5. The Genetic Code . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

6. Chromosomal Translocation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Chapter 2

Genetic Inheritance and Mutations

“Like begets like” is an expression that describes

repeated observations in families. Except in rare

instances, most children are normal and generally resemble their biological parents, brothers,

and sisters. In these cases, the genetic information has been passed intact from the parents to

their children.

This report is about the instances when something goes wrong, when a genetic mistake is made;

a mutation in the DNA of the parents’ reproductive cells, both of whom are physiologically norFUNCTION AND ORGANIZATION

Deoxyribonucleic acid, DNA, is the carrier and

transmitter of genetic information. Its functions

require that it is relatively stable and that it is able

to produce identical copies of itself. Certain

agents, such as some forms of radiation, viruses,

and certain chemicals, alter DNA’s ability to

maintain these characteristics. Mutations, changes

in the composition of DNA, can occur as a result

of these agents acting on DNA or on the systems

in place to repair DNA damage.

Human DNA can be thought of as an immense

encyclopedia of genetic information; each person’s

DNA contains a unique compilation of roughly

3 billion nucleotides in each of its two chains, the

exact sequence shared by no other person except

an identical twin. The genetic information encoded in DNA is contained in the nucleus of cells,

and is packaged in units, or chromosomes, that

consist of long twisted double strands of DNA surrounded by a complex of proteins. Each chromosome contains thousands of genes, the functional

units of DNA, which are “read,” or transcribed,

by the cell so that genetic information can be used

to make proteins or to regulate cell functions. It

is thought that only a small proportion, approximately 1 to 10 percent, of human DNA is translated into specific proteins. Functions of the nontranslated majority of DNA are largely unknown.

There are 23 pairs of chromosomes in all nucleated

real, is passed to a child who may or may not appear normal, depending on the nature and severity

of the mutation. All types of mutations, from the

benign to the severe, are considered in this report.

As an introduction to human genetics for nonspecialists, this chapter summarizes some basic information about normal functions of DNA, the

various kinds of mutations that can occur, and

health effects of mutations. More complete information can be found in a general reference such

as Vogel and Motulsky (165).

OF DNA

human cells except human reproductive cells

(germ cells). The latter contain one of each pair

of chromosomes, since two germ cells (one egg

and one sperm) fuse at conception and re-create

a full set of DNA in the offspring.

DNA is composed of two chains of nucleotides

bound together in a double helical structure (fig.

2). The backbones of the two chains, formed by

sugar (deoxyribose) and phosphate molecules, are

held to each other by hydrogen-bonded nitrogenous bases: adenine, thymine, guanine, and cytosine, abbreviated A, T, G, and C, respectively.

Each of the chains’ units, or nucleotides, consist

of a sugar, phosphate, and nitrogenous base. The

linear sequence of nucleotides repeated in various combinations thousands of times along the

DNA determines the particular genetic instructions for the production of proteins and for the

regulation of cell functions.

A change in even a single nucleotide, e.g., substituting G for T, among the 3 billion nucleotides

in the DNA sequence, constitutes one type of mutation. Depending on where it occurs, such a mutation could be sufficient to cause significant

pathological changes and dysfunction, or it could

cause no impairment at all. In thalassemia, for

example, a genetic disorder of hemoglobin synthesis, approximately 40 different “spelling er23
24 . Technologies for Detecting Heritable Mutations in Human Beings

Figure 2.—The Structure of DNA

Adenlne Guanlne 0

0

-0’

3

5

0 .

~

2

2a, The pairing of the four nitrogenous bases of DNA: o

Adenine (A) pairs with Thymine (T)

Guanine (G) pairs with Cytosine (C)

o

7

-O”p

Y

d

2 H2

,o–

‘ 0

; H2

,o– \

CH2

o -

‘ 0

; H2

)

sugar-phosphate

backbone

2b. The four bases form the four letters in the alphabet of

the genetic code. The sequence of the bases along the

sugar-phosphate backbone encodes the genetic information.

e pairs

phosphate

one

A schematic diagram of the DNA double helix. A three-dimensional representation of the DNA double helix.

2c. The DbJA molecule is a double helix composed of two chains. The sugar-phosphate backbones twist around the outside, with the paired bases on the inside serving to hold the chains together.

SOURCE: Office of Technology Assessment.
Ch. 2—Genetic Inheritance and Mutations ● 2 5

rors, ” or (single or multiple) nucleotide changes,

have been identified in a particular gene that specifies the production of globin, an essential constituent of hemoglobin. In complex diseases such as

heart disease, susceptibility is thought to be influenced by the interaction of environmental factors such as diet, and genetic factors such as mutations in one or several genes.

During DNA replication, each chain is used as

a template to synthesize copies of the original

DNA (fig. 3). New mutations are transmissible

to daughter cells. To replicate, the two chains of

the DNA double helix separate between nucleotides, and each is copied by a series of enzymes

that insert a complementary nitrogenous base opposite each base in the original strand, creating

two identical copies of the original one. As a result of specific pairing between nitrogenous bases,

each chain builds its complementary strand using

the single chain as a guide.1

Protein Synthesis

The normal functioning of the human body—

including the digestion of food; the production

of muscle, hair, bone, and skin; and the functioning of the brain and nervous system—is dependent on a well-ordered series of chemical reactions. Proteins, especially the enzymes, promote

the chemical reactions on which these processes

are based. Each enzyme has a specific function;

it recognizes a chemical, attaches to it, reacts with

it, alters it, and leaves, ready to promote the same

reaction again with another chemical.

To synthesize a protein, the genetic information contained in one or more genes along the

DNA is transcribed to small pieces of ribonucleic

acid (RNA), which are faithful replicas of one

strand of DNA (see fig. 4). Each strand of RNA

moves out of the nucleus to the cytoplasm of the

cell, where it serves as a template for protein

synthesis. Proteins are composed of hundreds of

linked subunits called amino acids. At the ribosomes, RNA is used to gather and link amino

acids in a specific sequence and length according

to the design specified in the DNA to form the

different proteins.

‘The molecular structure of the nitrogenous bases in DNA requires

that A pairs only with T (or with U in RNA) and G pairs only with C.

Figure 3.–Replication of DNA

Old Old

Old New New Old

When DNA replicates, the original strands unwind and

serve as templates for the building of new complementary

strands. The daughter molecules are exact copies of the

parent, with each having one of the parent strands.

SOURCE: Office of Technology Assessment

Genetic information in DNA is organized in a

triplet code, or sequence of three nucleotides. Each

triplet specifies a complementary codon in “messenger RNA” (mRNA) which, in turn, specifies

a particular amino acid. The sequence of such

triplets in a gene ultimately determines the amino

acid sequence of the corresponding protein.

Codons exist for each of the 20 amino acids that

make up the myriad of different proteins in the

body. Additional codons exist to signal “start” and
26 Technologies for Detecting Heritable Mutations in Human Beings

Figure 4.—Diagrammatic Representation of Protein Synthesis According to the Genetic Instructions in DNA

1. TRANSCRIPTION

3’ DNA 5’

AA AT TCTCGCACACCCTTC G

5’ 1

Nuclear ~ ~ ~

RNA

UUU A AGAGCGUGUGGGAAG C

I

Remove introns,

2. RNA PROCESSING

process ends

5’ 3,

U u u G A G G U G A A G

Transfer

to I Nucleus

Nuclear membrane

Mature

messenger

RNA

(mRNA)

5, i 3’

UUUGAGGUGAA G

3. TRANSLATION mRNA 5’ I

transfer RNA

.

. / ”

T

Ribosome

Protein polypeptide chain

composed of linked amino acids

SOURCE: Adapted from A.E, H, Emery, An /nfroduction fO Recombinant DNA (Chichester: John Wiley & Sons, 1984)
Ch. 2—Genetic Inheritance and Mutations • 27

Figure 5.—The Genetic Code

Codon Amino Acid Codon Amino Acid Codon Amino Acid Codon Amino Acid

Uuu Each codon, or triplet of nucleotidesPhenylalanine Ucu Serine UAU Tyrosine UGU Cysteine in RNA, codes for an amino acid (AA). UUC Phenylalanine UCC Serine UAC Tyrosine UGC Cysteine Twenty different amino acids are proUUA Leucine UCA Serine UAA atop UGA stop

UUG Leucine UCG Serine UAG

duced from a total of 64 different RNA

stop UGG Tryptophan codons, but some amino acids are

Cuu Leucine Ccu Proline CAU Histidine CGU Arginine specified by more than one codon (e.g.,

CuC Leucine ccc Proline CAC Histidine CGC Arglnine phenylalanine is specified by UUU and

CUA

by UUC). In addition, one codon (AUG)

Leucine CCA Proline CAA Glutamine CGA Arginine

CUG Leucine CCG Proline CAG GIutamine CGG Arginine specifies the ‘(start” of a protein, and 3

codons (UAA, UAG, and UGA) specify

AUU Isoleucine ACU Threonine AAU Asparagine AGU Serine termination of a protein. Mutations in

the nucleotide sequence can change

AUC isoleucine ACC Threonlne AAC Asparagine AGC Serine the resulting protein structure if the

AUA Isoleucine ACA Threonine AAA Lysine AGA Arginine

AUG Methionine ACG Threonine AAG Lysine AGG Arginine

mutation alters the amino acid specified by a triplet codon or if it alters the

(start) reading frame by deleting or adding a

GUU

nucleotide.

Valine GCU Alanine GAU Aspartic acid GGU Glycine

GUC Valine GCC Alanine GAC Aspartic acid GGC Glycine U - uracil (thymine) A - adenine

GUA Valine GCA Alanine GAA Glutamic acid GGA Glyclne C - cytoslne G. guanine

GUG Valine GCG Alanine GAG Glutamic acid GGG Glycine

SOURCE Off Ice of Technology Assessment and National Institute of General Medical Sciences

“stop” in the construction of a polypeptide (protein) chain. The triplet code provides more information, however, than is needed for 20 amino

acids; all except 2 of the amino acids are specified by more than one codon (see fig. 5).

Inheritance of Genetic Traits

A child’s entire genetic endowment comes from

the DNA in a single sperm and egg that fuse at

conception. The fertilized egg then divides, becoming a multicellular embryo, and the cells

differentiate into specialized tissues and organs

during embryonic development. In terms of

genetic material, there are two general types of

cells: somatic cells and germinal cells. Those that

have a full set of DNA at some time in their development and do not participate in the transmission of genetic material to future generations are

somatic cells, which include all cells in the body

except the reproductive cells. Germ cells include

the gametes (egg and sperm, each of which contains half of the total set of DNA) and the cell

types from which the gametes arise.

A normal, full set of human DNA in somatic

cells consists of 46 chromosomes, or 23 pairs of

homologous chromosomes: 22 pairs of “autosomes” and 1 pair of sex-determining chromosomes. Females have 22 pairs of autosomes and

two X chromosomes, while males have 22 pairs

of autosomes and an X and a Y chromosome. Reproductive cells, with half the normal set of DNA,

are produced by reduction division, or meiosis,

from sex-cell progenitor cells in the female ovary

or male testis. These progenitor cells are set aside

early in fetal development and are destined to be

the only source of germ cells. Each ovum or sperm

has 23 single, unpaired chromosomes: ova have

22 autosomes and one X chromosome; sperm

have 22 autosomes and one X or one Y chromosome.

Most genes are present in two copies, one on

each member of a homologous pair of chromosomes. For instance, if gene A is found on chromosome 1 it will be present at the same location

or locus on both copies of chromosome 1. The

exact nature of the DNA sequence of gene A may

differ between the two chromosomes, and the

word allele, classified as either normal or mutant,

is used to refer to different forms of the same gene.

Many different mutant alleles are possible, each

one defined by a particular change in the DNA

sequence. A mutation that is not expressed when

a normal gene is present at the same locus on the

sister chromosome is called recessive and a mutation that is expressed even in the presence of

a normal gene is called dominant.
28 . Technologies for Detecting Heritable Mutations in Human Beings

A Normal Karyotype of 46 Human Chromosomes

7 8 9 )1 12

13

Photo credit: Gail Stettt?n, Johns Hopkins Hospital

Karotypes typically show all the chromosomes of a single cell, and are used mainly to identify abnormalities in the number

or structure of chromosomes. Chromosomes can be isolated from any nucleated cell type in the body, but are most

often isolated from white blood cells derived from a sample of venous blood or from amniotic fluid cells obtained at

amniocentesis. The cells are stimulated to grow and divide in the laboratory, yielding a large number of cells which

contain a sufficient amount of DNA to isolate and examine. The cells are then blocked in their dividing phase by the

addition of a chemical, such as colcemid, to their growth media. After harvesting the cells, they are treated with a hypotonic

solution to cause them to swell, and then with a chemical fixative to maintain this fragile, swollen state. When small

amounts of the cell suspension are carefully dropped onto microscope slides, the cells break open, spreading out their

chromosomes. To visualize the chromosomes and to distinguish the different homologous pairs, the chromosomes are

stained to produce a characteristic banding pattern for each of the 23 pairs of homologous chromosomes. The chromosomes

can then be photographed under the microscope, cut out from the final print, and numbered and arranged in order of

decreasing size as shown in this karyotype.
Ch. 2—Genetic Inheritance and Mutations ● 2 9

KINDS OF MUTATIONS

Mutations are changes in the composition of

DNA. They may or may not be manifested in outward appearances. Some mutations, depending

on where they occur in the DNA, have no apparent effects at all, and some cause changes that are

detectable, but are without obvious impairment.

Still other mutations lead to profound effects on

human health and behavior: stillbirths, neonatal

and early childhood deaths, various diseases,

severe physical impairments, and mental deficiencies.

If mutations occur in genes that determine the

structure or regulation of essential proteins, the

results are often detrimental to health. Since most

DNA in human cells is not expressed or used in

any known way, mutations in these regions may

or may not be clinically apparent, although it is

possible that subtle physiological variations may

occur. Interactions among several such mutations

may result in unusual responses to environmental

stimuli or they may influence susceptibility to

various chronic diseases.

Mutations can be generally divided according

to size into two groups: gene mutations and chromosome mutations. Chromosome mutations include numerical and structural abnormalities.

Gene Mutations

Gene mutations occur within or across a single gene, resulting from the substitution of one

nucleotide for another or from the rearrangements

(e.g., deletions) within the gene, or they may

duplicate or delete the entire gene. The mutation

responsible for sickle cell anemia, for example,

is a point mutation: a single nucleotide in the gene

coding for globin, a constituent of hemoglobin,

is substituted for another nucleotide, resulting in

the substitution of one amino acid (glutamic acid)

for another (valine) at a certain position in the

globin chain. One of the mutations responsible

for a form of alpha-thalassemia, another form of

anemia, involves the deletion of an entire alphaglobin gene, resulting in the absence of alphaglobin synthesis.

Since proteins are produced from the instructions in genes, a mutation in a gene that codes

for a specific protein may affect the structure, regulation, or synthesis of the protein, A mutation

that results in a different codon may or may not

change the resulting protein, since different

codons can code for the same amino acid. Alternatively, such a mutation may result in the substitution of one amino acid for another, and possibly change the charge distribution and structure

of the protein. Another mutation in the same gene

may result in gross reduction or complete loss of

activity of the resulting protein. Detailed analysis of the mutations underlying thalassemia disease has shown that mutations occurring in the

bordering areas, not within the translated parts

of the gene, can also impair gene function (171).

Chromosome Mutations

Major changes affecting more than one gene are

called chromosome mutations. These involve the

loss, addition, or displacement of major parts of

a chromosome or chromosomes. They are often

large enough to be visible under the light microscope as a change in the shape, size, or staining

pattern (“banding”) of a chromosome. Major

chromosome abnormalities are often lethal or result in conditions associated with reduced lifespan

and infertility.

Structural Abnormalities

Structural mutations in the chromosomes

change the arrangement of sets of genes along the

chromosome. Sections of one chromosome, including many genes, may break and reattach to

another chromosome (e.g., in a balanced translocation, shown in fig. 6) or another location on

the same chromosome. Alternatively, sections of

chromosomes can be deleted, inserted, duplicated,

inverted.

Numerical Abnormalities

Numerical chromosomal mutations increase or

decrease the number of whole chromosomes,
30 ● Technologies for Detecting Heritable Mutations in Human Beings

Figure 6. —Chromosomal Translocation

Normal Translocation

chromosome 8 chromosome 8q –

I

Sites of

breakage

SOURCE: Office of Technology Assessment

HEALTH EFFECTS OF MUTATIONS

In Western countries, genetic diseases are more

visible now than in the earlier part of this century. Advances in medical care, public health

measures, and living conditions have contributed

to a gradual decline in the contribution of environmental factors to certain types of diseases,

particularly, infectious diseases and nutritional

deficiencies (32). Genetic disorders as a group now

represent a significant fraction of chronic diseases

and mortality in infancy and childhood, although

each disorder is individually rare. Such disorders

generally impose heavy burdens, expressed in

mortality, morbidity, infertility, and physical and

mental handicap. Approximately 90 percent of

them become clinically apparent before puberty.

The incidence of genetic disease is not known

precisely, but it is estimated that genetic disorders

are manifested at birth in approximately 1 percent of liveborn infants, and are thought to account for about 7’ percent of stillbirths and

neonatal deaths and for about 8.5 percent of childhood deaths. Nearly 10 percent of admissions to

pediatric hospitals in North America are reported

for genetic causes. The majority of these genetic

diseases lack effective treatment. A small, but increasing number are avoidable through prenatal

diagnosis and selective abortion (21,32,40).

without changing the structure of the individual

chromosomes. Errors in the production of the

germ cells can lead to abnormal numbers of chromosomes in the offspring. For example, Down

syndrome can be caused by the presence of an extra chromosome (Trisomy 21); three copies of

chromosome 21 are present while all other chromosomes are present in normal pairs, giving a total of 47 chromosomes in each cell. A similar, but

opposite, error in production of the germ cells results in the lack of one chromosome. Turner syndrome (Monosomy X), in which affected females

have 44 autosomes and only one sex chromosome,

an X chromosome, results in abnormal development of the ovaries and in sterility. The effects

of these mutations may result not from alteration

in the nature of the gene products but from the

abnormal amount of gene products present.

In most of these cases, the particular genetic disorder is passed along in families, or the asymptomatic carrier state for the disease is passed along

for many generations, before it maybe expressed

in a child with the disorder. In some of these cases,

however, the disorder may suddenly appear in a

child when no relatives have had the disorder. Not

uncommonly, such an event can be explained by

mistaken parentage or by one of the parents showing mild or almost unnoticeable signs of the disorder, so that this parent has a disease-causing

gene that can be passed on to his or her children.

However, the sudden, unexpected appearance of

a new condition in a family can also result from

a new mutation in the reproductive cells of the

parents. Examples of conditions that result from

new mutations in a large percentage of cases include Duchenne muscular dystrophy, osteogenesis

imperfect (a condition resulting in brittle bones),

and achondroplasia (dwarfism).

The exact causes of new heritable mutations are

unknown. It is thought that the risk of some new

heritable mutations increases with increasing age

of the father. Certain rare disorders due to mutations occur at as much as four times the average rate when fathers are 40 years of age or older

at the time of conception of their children. Such
Ch. 2—Genetic Inheritance and Mutations . 31

disorders include achondroplasia, Apert’s syndrome (a disorder with skull malformations and

fusion of bones in the hands), and Marfan’s syndrome (a complex syndrome including increased

height). Advanced maternal age is a risk factor

for some newly arising chromosome abnormalities, including Down syndrome.

Very small deletions of a particular chromosome have been found in children having certain

cancers such as Wilm’s tumor (chromosome number 11) and retinoblastoma (chromosome number 13). Some chromosome mutations are compatible with normal life, and others may be

associated with increased morbidity and reproductive problems. For example, when a section

of a chromosome breaks off and reattaches to

another chromosome, both copies of all of the

genes may still be present, but they are located

abnormally. If no genetic material has been lost

in the move, this type of rearrangement is called

a balanced translocation. However, a gene that

was previously “turned off” may, in its new location, be suddenly “turned on”; this explanation

has been invoked to explain how cancer genes can

be activated. Furthermore, in the production of

germ cells, an abnormal complement of DNA

could result, so that a parent with a balanced

translocation may have offspring with unbalanced

chromosomes, an outcome that usually has deleterious consequences for the child.

Early Mortality and Morbidity

Numerical and structural chromosome abnormalities have been detected in as many as 50 to

60 percent of spontaneous abortions, 5 to 6 percent of perinatal deaths, and 0.6 percent of liveborn infants (43). Fetuses that survive to term with

such abnormalities are apparently only a small

percentage of the number of fetuses with chromosome abnormalities that have been conceived.

Abnormalities such as trisomy 13 or trisomy 18

are among the more common disorders found in

the liveborn group, whereas many numerical and

structural abnormalties, most of which are not

found in surviving infants, have been found in

spontaneous abortuses.

Some of the numerical chromosome abnormalities, such as trisomy 13 or trisomy 18, can be

compatible with a full-term pregnancy but may

cause severe handicap in infancy and childhood

and may be associated with a reduced lifespan.

The health effects of structural chromosome aberrations (involving only parts of chromosomes) depend on the size and location of the aberration.

However, features shared by many of these conditions include low birthweight; mental retardation; and abnormalities of the face, hands, feet,

legs, arms, and internal organs, often necessitating medical care throughout an individual’s life.

Diseases During Adult Life

Some genetic disorders, although present in an

individual’s DNA since his conception, do not

manifest themselves until adult life. Huntington

disease, a degenerative neurological disorder characterized by irregular movements of the limbs and

facial muscles, mental deterioration, and death

usually within 20 years of its first physiological

appearance, is an example of a genetic disorder

with adult onset.

Many disorders that develop during adulthood

and that tend to cluster in families do not have

classical patterns of genetic inheritance. Several

genes may be involved in the development of these

“multifactorial diseases, ” and environmental factors may also influence the manifestation and

severity of these disorders. Susceptibilities to conditions such as diabetes mellitus, hypertension,

ischemic heart disease, peptic ulcer, schizophrenia, bipolar affective (manic depressive) disorder,

and cancer are thought to have genetic as well as

environmental components.

Effects of Somatic Mutations

Mutations that occur in somatic cells may affect the individual, but they are not passed on to

future generations. Somatic mutations may play

a role in the development of some malignant

tumors by removing normal inhibition to cell

growth and regulation, producing cells with a

selective growth advantage. A wide range of experiments in animals and observations in humans

indicate that somatic mutation may be one step

in the development of certain types of cancer, in-
32 ● Technologies for Detecting Heritable Mutations in Human Beings

eluding leukemia and cancers of the breast and disease xeroderma pigmentosum, can also lead to

thyroid. Mutations that cause deficiencies in the cancer following exposure to a mutagenic agent.

normal DNA repair system, such as in the genetic

PERSISTENCE OF NEW MUTATIONS IN THE POPULATION

The “spontaneous mutation rate” represents the

sum of “natural” error arising during the course

of life and reproduction, and external influences,

both natural and manmade. The mutation rates

that have prevailed throughout recent human history may be in a state of relative equilibrium, although accurate information is lacking. Suppose,

though, that mutagens are suddenly introduced

heavily into the environment, and that there is

an increase in mutation rates. It is impossible to

accurately predict the number of people born with

genetic diseases in future generations that will result from that increase, but some predictions can

be made based on what is known about the effects of different types of mutations on subsequent

generations. Two cases are considered below: 1)

the effect of a doubling of the spontaneous mutation rate in one generation, and 2) the effect of

a permanent doubling of the spontaneous mutation rate.

Assume that a population was exposed to a

“pulse” of a mutagenic agent, such as radiation

from an atomic bomb or from chemical agents

in a toxic spill, and that the exposure caused a

doubling of the heritable mutation rate. Overall,

the number of extra cases of disease associated

with mutation in the first generation after the

pulse (the generation in which the effect would

be greatest), will be comparatively small. One estimate (from a 1977 United Nations Scientific Committee on the Effects of Atomic Radiation report)

is that 10.5 out of every 100 liveborn infants has

some kind of genetic disorder (88). Almost all of

those cases are from mutations already present

in the gene pool, and not from new mutations.

The National Research Council (NRC) estimates

that one mutational pulse would add an extra 0.66

cases, so instead of 10.5 per 100, 11.16 cases

would occur. The effects on subsequent generations would be smaller, but even after hundreds

of generations, there might be some small increment of the mutational load as a result of the

pulse. To understand this phenomenon, it is useful to consider different kinds of mutations separately.

Chromosome mutations would be the shortest

lived in the population, because in most cases they

result in sterility, so they would not be passed on

to the next generation. They would probably be

eliminated completely after about 1.25 generations.

Dominant and X-linked (sex-linked) mutations

are the next shortest lived. Many of these, because

they often cause such severe disease, interfere with

a normal lifespan and reproduction. After about

four or five generations, on average, they would

no longer be transmitted to future generations.

New recessive mutations will probably have the

greatest chance of being maintained in the population. Virtually none would be eliminated in the

first generation, because a single individual will

have only one copy of the gene, and two would

be needed to cause overt disease. Recessive mutations may take generations to surface as over

disease. There is much less certainty about the ultimate fate of recessive mutations than about dominant, X-linked, or chromosome mutations.

Many “common” or multifactorial diseases are

partially influenced by genetic traits carried in

families or arising anew. These include congenital malformations, and such common diseases as

heart disease, diabetes mellitus, bipolar affective

disorder, immunological disorders, and cancer.

Since it is difficult to estimate the proportion of

disease that is currently influenced by mutations,

it is also difficult to estimate how long such mutations induced from a pulse of a mutagenic agent

would persist in the population. However, the

NRC report (88) estimates that these mutations

would persist for an average of 10 generations.

These estimates suggest a surprisingly small increase over background rates of genetic disease
Ch. 2—Genetic /inheritance and Mutations Ž 33

in the first few generations after intense exposure

to mutagenic agents, which would decline slowly

over many generations. The NRC report (88) suggests that about half of the total effect would occur in the first six generations. The most severe

mutations would be more easily observable as

dominant genetic diseases. These would be eliminated within one or two generations since they

interfere with survival and fertility. Mutations

with a less severe initial impact would persist in

the population for a longer period of time.

A permanent doubling of the current mutation

rate would, however, after perhaps hundreds of

generations, lead to a new equilibrium, with the

incidence of genetic disease at about twice its current level. One of the biggest unknowns about

predicting future effects of mutations is the impact of an accumulation of recessive mutations

in the population. There is also little information

to bring to bear on the question of the impact of

an increase in mutations to the prevalence of common diseases.
Chapter 3

Kinds and Rates of Human

Heritable Mutations
Page

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....37

Surveillance of Sentinel Phenotypes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

Mutation Rates Estimated From Phenotypic Data . . . . . . . . . . . . . . . . . . . .......39

Limitations of the Phenotype Approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..40

Cytogenetic Analysis of Chromosome Abnormalities . . . . . . . . . . . . . . . . . . .......40

Mutation Rates Derived From Chromosome Abnormalities . . . . . . . . . . .......42

Limitations of Cytogenetic Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .......42

Detection of Blood Protein Variants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .....44

One-Dimensional Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....44

Quantitative and Kinetic Studies of Enzyme Deficiency Variants . . . . . . . . . . ...45

Molecular Analysis of Hemoglobin M and Unstable Hemoglobins . . . . .......46

Studies of Populations Exposed to Atomic Radiation. . . . . . . . . . . . . . . . . ... .48

Early Epidemiologic Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...49

Cytogenetic Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .......49

Biochemical Analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .......49

Mutation Studies in Populations Exposed to Radiation

in the Marshall Islands . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .......50

TABLES

Table No. Page

I. Candidate Sentinel Phenotypes . . . . . + . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

2. Prevalence of Chromosome Abnormalities at Birth . . . . . . . . . . . . . . . . . . . . . . . . 43

3. Rates of Mutation Leading to HbM and Unstable Hb Disorders . . . . . . . . . . . . 48

4. Results in Studies To Detect Heritable Mutations by One-Dimensional

Electrophoresis in Human Populations Not Known To Beat High Risk

for Germinal Mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

5. Spontaneous Mutation Rates of Genetic Abnormalities in

Human Populations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Chapter 3

Kinds and Rates of Human

Heritable Mutations

INTRODUCTION

The genetic code specified in DNA directs the

transcription of RNA, which is translated into

polypeptide chains; these chains, in turn, are assembled into proteins, which are the body’s tools

for the regulation of physiological, biochemical,

and behavioral functioning. Mutations, transmissible alterations in DNA, can change the messages, causing alterations in RNA molecules and

proteins. Some of these changes can lead to subtle abnormalities, diseases, and disabilities.

Environmental factors and specific interactions

between environmental and genetic factors are

thought to account for the majority of diseases

or susceptibilities to disease. Mutations alone are

thought to account for only a fraction of the current burden of disease. However, there is a group

of disorders for which new mutations are the primary causes. Among these “genetic” diseases is

a group of rare clinical conditions, “sentinel phenotypes, ” that occur sporadically in each generation; children with sentinel phenotypes are usually born to parents who do not have the same

disease, indicating a new mutation that arose in

a germ cell of one of the parents,

There are several other indicators of new mutations in human beings that do not rely on detecting diseases. The most widely used methods

have been cytogenetic analysis of chromosomes

and biochemical analysis of proteins.

The structure and number of chromosomes visible by light microscopy has been used to detect

a certain class of heritable mutations. Gross chromosome abnormalities, often associated with high

fetal and neonatal death rates, are identifiable

with increasing precision by cytogenetic analysis. A large portion of these chromosome abnormalities arise anew each generation,

Studies of mutationally altered proteins have

been done on a limited basis. The proteins most

accessible for analysis are those circulating in

blood; other body proteins are not routinely obtained for analysis. The method available for

detecting abnormal proteins is electrophoresis.

Heritable mutations have been detected by identifying electrophoretic variants of blood proteins.

In addition, a combination of approaches has been

used to estimate the mutation rate per nucleotide

in genes coding for globin polypeptides, constituents of hemoglobin. These approaches to identifying mutations are discussed in this chapter.

The term “mutation rates” for particular types

of mutations in specific regions of DNA is used

frequently in this report. Mutation rates for different kinds of mutations, or corresponding to different overt effects of mutations, can be expressed

as mutations per locus, per gene, per nucleotide,

and per gamete. All of these indicate a specific

type of mutation occurring per generation, reflecting mutations arising anew from one generation

to the next. This chapter summarizes current

knowledge about such mutation rates as they have

been measured thus far; it is not yet known

whether these rates apply also to other populations, to other gene regions, or to other types of

mutations than the ones examined.

Rather than looking directly at germinal mutations—mutations in egg and sperm DNA—the

methods described in this report focus on heritable mutations in liveborn offspring, a subset of

all germinal mutations.l A larger subset of germinal mutations includes new mutations observed

in offspring before birth. In recent years, cytogenetic techniques have been used to identify

chromosome abnormalities in spontaneous abortuses. These investigations have shown that the

frequency of new mutations in spontaneous aborlAt present it is not feasible to examine egg or sperm DNA for

genetic damage, although some information on major chromosome

abnormalities can be obtained by cytogenetic examination of sperm.

37
38 . Technologies for Detecting Heritable Mutations in Human Beings

tuses is much higher than in liveborn infants; the agents. Nevertheless, the most lasting medical and

observed incidence of new mutations at birth only social consequences of new heritable mutations

partially represents the “true” incidence of such are derived from those mutations that are comheritable mutations in all conceptuses, and may patible with life at least through infancy and

give an incomplete picture of the potential risk childhood.

from environmental exposures to mutagenic

SURVEILLANCE OF SENTINEL PHENOTYPES

The United Nations Scientific Committee on the

Effects of Atomic Radiation (144) and the Committee on the Biological Effects of Ionizing Radiations (87) estimate that 1 percent of liveborn infants carry a gene for an autosomal dominant

disease; in 20 percent of these cases (0.2 percent

of livebirths) their disease is due to a new, or “sporadic, ” mutation that arose in the reproductive

cells of one of their parents, Observing and recording the birth of infants with such sporadic

conditions is the classical approach to identifying heritable mutations that lead to serious disease (36). 2

A subset of the group of autosomal dominant

and X-linked conditions, indicator conditions

known as “sentinel phenotypes,” maybe particularly useful in identifying heritable mutations.

Sentinel phenotypes are clinical disorders that occur sporadically, probably as the result of a single mutant gene. They are manifested at birth or

within the first months of life, and usually require

long-term, multidisciplinary medical care (83). In

addition, sentinel phenotypes are associated with

low fertility, so their appearance suggests mutations that have been passed not from affected parents to offspring, but instead, have been passed

from unaffected parents to offspring as a result

of a newly arising mutation in a germ cell of one

of the parents of that offspring. Affected infants

would have this mutation in the DNA of all their

somatic cells and 50 percent of their germ cells.

‘Rates of a wide variety of new heritable mutations leading to

severe diseases have also been estimated by an “indirect method. ”

This method is a theoretical approach, based on the loss of diseasecausing genes from the population (due to reduced reproductive capabilities of individuals with these genes) and on the sporadic frequency of the genes in the population. The mutation rate necessary

to maintain such genes in the population, despite their recurrent loss,

is inferred from these observations. The types of populations and

conditions suitable for this method are limited, and its precision is

unknown (91,165).

Mulvihill and Czeizel (83) compiled a list,

shown in table 1, of 41 genetic disorders (36 dominant and 5 X-linked) that satisfy the above criteria. Each of these phenotypes is individuall y

rare, some of them occurring in only one in a million liveborn infants.

Since children with sentinel phenotypes have

serious health problems associated with their conditions, they may first come to the attention of

the family practitioner or pediatrician, and then

be referred to subspecialists for a complete diagnosis. Clinical expertise in dysmorphology,3 medical genetics, pediatric ophthalmology, and oncology may be needed to diagnose accurately the

small numbers of children with sentinel phenotypes present among the vast majority of unaffected children and among children with phenotypically similar, but nongenetic conditions (called

“phenocopies”) (71,83,165).

Reliable incidence data are available only for

13 dominant and 5 X-linked sentinel phenotypes

(166); only those that are manifested in infancy,

rather than in childhood or adolescence, Q are systematically recorded. Diagnostic records of sentinel phenotypes may be available in populationbased registries of childhood cancers, birth defects, and genetic diseases, with initial entries

based on birth certificates and later entries added

as the conditions are diagnosed during infancy.

After investigators rule out the possibility of a discrepancy between stated and biological parentage by genetic testing (166), sporadic cases of a

sentinel phenotype are accepted as evidence for

‘Physicians who specialize in diagnosing syndromes characterized by unusual physical form and stucture, for example, congenital malformations.

‘lSuch disorders include achondroplasia, acrocephalosyndactyly,

osteogenesis imperfect, retinoblastoma, and Wilms’ tumor (66).
Ch. 3—Kinds and Rates of Human Heritable Mutations • 39

Table 1 .—Candidate Sentinel Phenotypes’

Inheritancea

Phenotypes identifiable at birth:

Achondroplasia . . . . . . . . . . . . . . . . . . . . . . . . . .

Cataract, bilateral, isolated . . . . . . . . . . . . . . . .

Ptosis, congenital, hereditary . . . . . . . . . . . . . .

Osteogenesis Imperfect type I . . . . . . . . . . . .

Oral-facial-digital (Gorlin-Psaume) syndrome

type I. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Incontinentia pigmenti, Bloch-Sulzberger

syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Split hand and foot, bilateral atypical . . . . . . .

Aniridia, isolated . . . . . . . . . . . . . . . . . . . . . . . . .

Crouzon craniofacial dysostosis . . . . . . . . . . .

Holt-Oram (heart-hand) syndrome . . . . . . . . . .

Van der Woude syndrome (cleft lip and/or

palate with mucous cysts of lower lip). . . .

Contractual arachnodactyly . . . . . . . . . . . . . . .

Acrocephalosyndactyly type 1, Alpert’s

syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Moebius syndrome, congenital facial

diplegia. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Nail-patella syndrome . . . . . . . . . . . . . . . . . . . .

Oculodentodigital dysplasia

(ODD syndrome) . . . . . . . . . . . . . . . . . . . . . . .

Polysyndactyly, postaxial . . . . . . . . . . . . . . . . .

Treacher Collins syndrome, mandibulofacial

dysostosis . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Cleidocranial dysplasia . . . . . . . . . . . . . . . . . . .

Thanatophoric dwarfism . . . . . . . . . . . . . . . . . .

EEC (ectrodactyly, ectodermal dysplasia,

cleft lip and palate) syndrome . . . . . . . . . . .

Whistling face (Freeman-Sheldon)

syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Acrocephalosyndactyly type V, Pfeiffer

syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Spondyloepiphyseal dysplasia congenita. . . .

Phenotypes not identifiable at birth:

Amelogenesis imperfect . . . . . . . . . . . . . . . . .

Exostosis, multiple . . . . . . . . . . . . . . . . . . . . . . .

Marfan syndrome . . . . . . . . . . . . . . . . . . . . . . . .

Myotonic dystrophy . . . . . . . . . . . . . . . . . . . . . .

Neurofibromatosis . . . . . . . . . . . . . . . . . . . . . . .

Polycystic renal disease . . . . . . . . . . . . . . . . . .

Polyposis coli and Gardner syndrome

Retinoblastoma, hereditary . . . . . . . . . . . . . . . .

Tuberous sclerosis . . . . . . . . . . . . . . . . . . . . . . .

von Hippel-Lindau syndrome . . . . . . . . . . . . . .

Waardenburg syndrome. . . . . . . . . . . . . . . . . . .

Wiedemann-Beckwith (EMG) syndrome . . . . .

Wilms’ tumor, hereditary . . . . . . . . . . . . . . . . . .

Muscular dystrophy, Duchenne type . . . . . . . .

Hemophilia A. . . . . . . . . . . . . . . . . . . . . . . . . . . .

Hemophilia B. . . . . . . . . . . . . . . . . . . . . . . . . . . .

AD

AD

AD

AD

XD

XD

AD

AD

AD

AD

AD

AD

AD

AD

AD

AD

AD

AD

AD

AD

AD

AD

AD

AD

AD

AD

AD

AD

AD

AD

AD

AD

AD

AD

AD

AD

AD

XR

XR

XR

aAD refers to autosomal dominant inheritance, XD refers to X-linked dominant

inheritance, and XR refers to X-1inked recessive inheritance.

SOURCE: J.J Mulvihill and A. Czeizel, “Perspectives in Mutations Epidemiology

6: A 1963 View of Sentinel Phenotypes,” Mutat. Res. 123:345-361, 1963.

a new germinal mutation originating in a germ

cell of one of the parents (65).

In general, recording the incidence of sentinel

phenotypes is not useful for monitoring a small

population exposed to a suspected mutagen. Surveillance of sentinel phenotypes in large populations is potentially more useful for estimating the

background mutation rates leading to dominant

genetic disorders. Given the rarity of the individual sentinel phenotypes, the most reliable data for

estimating mutation rates would be derived from

the largest target populations with, for example,

international cooperation among study centers

(62,66).

Mutation Rates Estimated From

Phenotypic Data

Data are available on the incidence of various

sentinel phenotypes from different time periods

in particular regions worldwide, though representation is incomplete. Crow and Denniston (22)

summarized the most current data; these data

have not been significantly updated since the

1940s and 1950s. The frequencies for any given

phenotype are generally consistent between

studies and the frequencies of occurrence of the

different phenotypes vary over a thousandfold

range, from 1 in 10,000 to 1 in 10 million depending on the particular disorder. The arithmetic

mean of the rates for the different disorders is approximately 2 mutations per 100,000 genes per

generation (17,89,166). This average rate is often

cited as the “classical” rate of heritable mutations.

‘Mutation rates corresponding to sentinel phenotypes are expressed

as the frequenc-y of mutations per gene. It assumes one gene per disorder and one disorder per person and it is designated “per generation” because the mutations are detected in offspring of individuals

whose germ celk have incurred mutation. In general, the mutation

rate is defined as the incidence of a sporadic case of some indicator

(e.g., a sentinel phenotype or a protein variant) divided by two times

the total number of cases examined (e.g., newborn infants or protein determinations). The two in the denominator is to express the

mutation rate, by convention, as the rate “per fertilized germ cell, ”

instead of “per concepts,” which is made from two germ cells (162).
40 • Technologies for Detecting Heritable Mutations in Human Beings

Limitations of the Phenotype

Approach

Several sources of error may affect estimates

of heritable mutation rates based on the incidence

of sentinel phenotypes. The most important one

results from the choice of phenotypes studied:

phenotypes that occur in the population most

often are the ones most likely to be recorded.

Phenotypes that are so severe as to cause prenatal death, or so mild as to be indistinguishable

from the normal variety of phenotypic traits, are

difficult or impossible to include in these population surveys. It is not known how large a bias

this selection occasions. In addition, the mutation

rates corresponding to clinically recognizable

phenotypes may not be representative of mutation rates for other traits.

Other problems contributing to an over- or underestimated mutation rate may include: 1) incomplete ascertainment among certain populations of livebom infants; 2) mistaken identification

of parentage, a significant issue since the number of cases of mispaternity may be high enough

to exceed the number of true sporadic cases of sentinel phenotypes; and 3) the occurrence of phenocopies (phenotypically similar nongenetic causes

of the same condition) and genocopies (recessive

forms of the same phenotype inherited from unaffected carrier parents). In addition, particular

phenotypes do not necessarily correspond to a

unique, single gene (163); often, mutations in any

one of several genes can lead to the same phenotype. This is not likely to influence the rate estimate significantly unless a large number of different mutant genes are involved, in which case the

rate of mutation of the particular phenotype

would be the sum of the rates of mutation at each

of the alternative genes.

Surveillance for sentinel phenotypes is an important part of assessing the clinical impact of new

mutations. The number of sentinel phenotypes

available to study is limited, however, so that surveillance of these phenotypes illuminates only a

fraction, albeit an important one, of this overall

impact.

Individually, the sentinel phenotypes are quite

rare. Keeping track of the frequencies of the different phenotypes over periods of time would require

large-scale surveillance of huge numbers of infants

over many years. A large team of medical specialists and a well-organized database would be

needed to obtain complete and accurate ascertainment (65). Unfortunately, the practical difficulties in screening huge populations of newborns,

diagnosing these rare disorders, and organizing

and maintaining the data, make it difficult, though

not impossible, to generate reliable data. The first

such effort is currently being organized by the

European Collaborative Study (62).

CYTOGENETIC ANALYSIS OF CHROMOSOME ABNORMALITIES

The adverse effects of chromosome abnormalities on human health are well established and

provide sufficient medical reasons for screening

infants for them. Data on the frequency of chromosome abnormalities is also useful in assessing

the impact of detectable new chromosome mutations on the current generation.

Chromosome abnormalities are defined as

either numerical (extra or missing whole chromosomes) or structural (deletions, insertions, translocations, inversions, etc., of sections of chromosomes). In liveborn infants, the presence of an

extra or missing sex chromosome is often associated with physical, behavioral, and intellectual

impairment. The presence of an extra autosome

is even more detrimental: it is usually associated

with severe mental and physical retardation and

often with premature death (39,119).

There is no doubt that fetuses with recognized

numerical chromosome aberrations are at a much

higher risk for aborting spontaneously than fetuses without such defects. Such chromosome abnormalities contribute to a large proportion of

spontaneous abortions and stillbirths, and in liveborn survivors may also contribute to repeated

early abortions and fertility problems in adult-
Ch. 3—Kinds and Rates of Human Heritable Mutations • 41

hood (169). The significance to human health of

structural rearrangements of the chromosomes is

less well defined, but such defects have been associated with mental retardation, physical malformations, and a variety of malignant diseases (43).

In addition, carriers of balanced translocations

may produce offspring with unbalanced complements of DNA; these offspring may die in utero

and may account for a high spontaneous abortion rate among such carrier parents.

It is believed that at least 5 percent of all recognized human conceptions have a chromosome

abnormality, although this is a crude estimate because it is not corrected for factors which could

influence the occurrence of such abnormalities

(such as maternal age) or for factors which could

affect the rate of embryonic or fetal death in some

of the disorders (43). Numerical and structural

chromosome abnormalities have been detected in

so to 60 percent of recognized spontaneous abortions (data summarized in ref. 12), 5 to 6 percent

of perinatal deaths (ibid.), and 0.6 percent of liveborn infants (42).

Chromosome abnormalities are useful for measuring heritable mutation rates since they are usually identified as new mutations, either because

the defect causes virtual infertility or sterility (as

in the case of numerical chromosome abnormalities) and therefore could not have been inherited

from a parent with the same defect, or because

the parents of an individual with a chromosome

abnormality (e.g., a structural abnormality) have

been shown not to carry the same mutation. Numerical chromosome abnormalities and some structural abnormalities are detectable with cytogenetic

methods using current chromosome staining and

banding techniques.

In general, little is known about the genetic

mechanisms of either numerical or structural rearrangements. They appear to result from complex

processes during gametogenesis in which various

defects may lead to the same or different outcomes

(18,119). Consequently, frequencies of the various cytogenetic abnormalities may correspond to

a combined rate of several different mutational

events or to a single mutational event.

Individuals with chromosome abnormalities

can be identified on the basis of karyotypes (see

ch. 2). However, since the vast majority of conceptuses with chromosome abnormalities such as

trisomies and unbalanced structural rearrangements are spontaneously aborted, many before

a pregnancy is recognized, the frequency of these

mutations at birth is known to represent only a

small fraction of the presumed frequency of these

mutations at conception.

Data on the frequency of chromosome abnormalities in fetuses at the time of amniocentesis

(generally done at 16 to 18 weeks gestation) provides information not available in newborn

screening. In addition, recent progress in cytogenetic analysis of chorionic villi cells makes it

possible to identify chromosome abnormalities in

fetuses at 8 to 10 weeks gestation (116). This technique provides even earlier information on possible chromosome abnormalities, before fetuses

with certain abnormalities (particularly trisomies

and unbalanced structural rearrangements) would

have aborted spontaneously. The analysis of fetal cells obtained at amniocentesis has provided

valuable data on the frequency of chromosome

abnormalities in the second trimester of pregnancy

(12,45,159.) However, estimates of mutation rates

from those measurements may be inflated unless

certain biases are corrected. Women who elect

amniocentesis—generally older mothers, couples

who have had previous reproductive problems,

and/or couples who may have been exposed to

mutagenic agents (101) —are at increased risk for

chromosome abnormalities in their offspring.

Cytogenetic surveys of consecutive liveborn infants have provided the most extensive data on

chromosome mutation rates, although it is known

that these data underestimate the “true” rate of

new chromosome mutations. It should also be

noted that the prevalence of sentinel phenotypes

and biochemical variants in liveborn infants may

also underestimate their “true” rate at conception,

but little is known about selection against these

mutations in the developing fetus (e. g., molecular repair of the defect, or spontaneous abortion

of the fetus), in contrast to the documented evidence for selection against chromosome mutations

in the fetus (135. ) However, the frequency at birth

is probably the best indicator of events with the

highest cost in terms of the social and economic

burden of these diseases.
42 . Technologies for Detecting Heritable Mutations in Human Beings

Mutation Rates Derived From

Chromosome Abnormalities

At least 10 major research efforts worldwide

have attempted to determine the prevalence of

chromosome abnormalities in liveborn infants. It

is possible to estimate the rate of new mutations

underlying these defects, but difficult to compare

with rates of sentinel phenotypes, since chromosome mutations, by definition, are multigenic

changes and sentinel phenotypes correspond to

single gene mutations.

The combined data from studies surveying a

total of 67,014 newborn infants for various

periods between 1974 and 1980 in the United

States, Canada, Scotland, Denmark, the U. S. S. R.,

and Japan (120) are shown in table 2. Two of the

studies used newer banding techniques that identify more abnormalities than conventional staining methods used in the remaining eight studies.

For conditions that are associated with virtual

infertility, complete sterility, or prereproductive

death, it is often assumed that each case is the result of a new mutation (unless there is evidence

for gonadal mosaicism6

). Such conditions include

the autosomal trisomies (e.g., Down syndrome)

and the sex chromosome aneuploidies (e.g.,

Turner’s syndrome).

For these defects, the apparent mutation rate

observed in liveborn infants is calculated as the

number of individuals identified with a given abnormality divided by the total number of individuals examined. This mutation rate is expressed

as the number of mutations per generation. For

conditions which could be the result of either new

mutations or inherited mutations, such as the various balanced and unbalanced structural rearrangements, it is necessary to karyotype the parents of

infants found with these chromosome abnormalities, and to exclude inherited mutations from calculations of the de novo mutation rate. For these

6Mosaicism refers to the presence of two genetically different cell

populations in the same individual. This can result from a mutation during one of the earliest divisions in the zygote, a few days

after conception, that led to the proliferation of a large number of

cells that were derived from the original cell bearing the mutation.

conditions, the observed mutation rate is determined using the following formula:

Number of patients X Proportion who are likely

Mutation + with abnormality to have de novo mutations7

rate Total number examined

The data show that the frequencies of chromosome abnormalities in liveborn infants range from

2 in 100,000 newborns per generation for inversions to 121 in 100,000 newborns per generation

for Trisomy 21 (Down syndrome), the most common newly arising numerical chromosome abnormality found at birth. Overall, the numerical

abnormalities are much more frequent than the

structural abnormalities. However, the rates for

balanced structural rearrangements may be low

in these data, since only two of the surveys used

more sensitive banding methods which are needed to identify these more subtle aberrations.

Limitations of Cytogenetic Analysis

Screening for chromosome mutations in newborn infants gives only a partial picture of their

incidence. The prevalence of a particular genetic

mutation at birth can be thought of as the result

of the incidence of the mutation at conception interacting with the probability of survival of conceptuses with the mutation. Fetal survival, in turn,

depends on individual fetal and maternal attributes, as well as on the interaction between the

two (136). Some mutations in the fetus may affect fetal survival, whereas other mutations may

have no effect on survival. Major chromosome

aberrations, in particular, numerical abnormalities and most unbalanced structural rearrangements, are often incompatible with fetal survival

and are associated with an increased risk of prenatal loss (45).

Balanced chromosome rearrangements, however, may not lead to excessive prenatal loss. For

this reason, the balanced structural rearrangements may be a useful indicator of new germinal

mutations observed in liveborn offspring. Since

some of these infants identified as having a bal-

“Such proportions are based on empirical data for each type of

chromosome aberration. In this case, data from Jacobs, 1981 were

used and are shown in table z in parentheses in the third column.
Ch. 3—Kinds and Rates of Human Heritable Mutations “ 43

Table 2.— Prevalence of Chromosome Abnormalities at Birth

Number New chromosome

Total of new abnormalities per 100,000

Chromosome abnormality population mutants a

newborns per generation

Numerical anomalies:

Autosomal trisomies:b

Trisomy 13 . . . . . . . . . . . . . . . . . . . . .

Trisomy 18 . . . . . . . . . . . . . . . . . . . . .

Trisomy 21 . . . . . . . . . . . . . . . . . . . . .

Male sex chromosome anomalies:

47,XYY . . . . . . . . . . . . . . . . . . . . . . . .

47,XXY . . . . . . . . . . . . . . . . . . . . . . . .

Female sex chromosome anomalies:

45,x . . . ., , . . . . . . ... , . . . . . ., ., . .

47, XXX . . . . . . . . . . . . . . . . . . . . . . . .

Balanced structural rearrangements:

Robertsonian translocations:c

D/Dd . . . . . . . . . . . . . . . . . . . . . . . . . .

D/G . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . 67,014

. . . . . . . . . . . . . . . . . . 67,014

. . . . . . . . . . . . . . . . . . 67,014

. . . . . . . . . . . . . . . . . . 43,048 males

. . . . . . . . . . . . . . . . . . 43,048 males

. . . . . . . . . . . . . . . . . . 23,966 females

. . . . . . . . . . . . . . . . . . 23,966 females

. . . . . . . . . . . . . . . . . . 67,014

. . . . . . . . . . . . . . . . . . 67,014

3

8

81

43

42

2

24

48(2/29) = 3.3

14(2/1 1) =2.5

5

12

121

100

98

8

100

5

4

Reciprocal translocations and insertions. . .............67,014 60(13/43) =18 27

Inversions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67,014 12(1/8)= 1.5 2

Unbalanced structural rearrangements:

Translocations, inversions, and deletions . .............67.014 37(7/16) =16 , 24

asee discussion in text for proportion of new mutants among all those identified with Structural r0arran9ementS.

bTrisomies refer t. conditions in which there are 47 chromosomes (instead of the normal 46 chromosomes or 23 pairs of homologous chromosomes, indicated as

46,XX for females or 46,XY for males) because of the presence of an extra copy of one chromosome; three copies of this particular chromosome would be present

instead of the normal two. Autosorna/ trisomies indicate an extra autosome, which includes any chromosome except one of the sex chromosomes

Prearrangements in which the long arms of two chromosomes fuse, resulting in one “hybrid” chromosome and the IOSS of the short WTls Of each chromosome,

dD and G refer to groups of chromosome numbers 13-15 and 21 ’22, resPectivf31Y

SOURCE: K Sankaranarayanan, Genetic Effecfs of /orrizina Radiafiorr in kfu/tice//u/ar Eukarvofes and the Assessment of Genetic Radiafion Hazards in Man (Amsterdam Elsewer Blomedlcal Press, 1982), pp 161-i64

anced chromosomal mutation may have inherited

the relatively subtle abnormality from one of their

parents, it is necessary to determine whether the

mutation observed in the child is actually a new

germinal mutation or not.

Although it is not understood how bands observable under the microscope correspond to the

structure and composition of DNA, mutations in

DNA can cause a visible change in the banding

pattern, particularly if such mutations involve a

large section of a chromosome. Increased resolution and sensitivity of cytogenetic techniques, specifically the “high-resolution” banding methods

(72), makes it possible to detect smaller and more

subtle aberrations than was previously possible.

Currently, routine cytogenetic methods can produce 200 to 300 bands (containing several hundred genes in each band) in a complete set of

DNA, and the high-resolution methods can distinguish as many as 1,000 bands (containing about

100 or fewer genes per band) (59). With more

smaller bands distinguishable, fewer genes are

present per band, and a change in one gene has

a greater likelihood of showing up as a change

in the banding pattern.

At present, data available for large populations

rely on only the routine staining and banding

methods. Like the observations of sentinel phenotypes in newborn infants, cytogenetic methods

focus on a subset of mutations, albeit those with

serious clinical consequences. Even though chromosome abnormalities are not as rare as sentinel

phenotypes, these two groups of abnormalities together probably account for only a small fraction

of heritable mutations.

Mutations detectable by the methods discussed

in the remainder of this chapter and in the following chapter do not necessarily have adverse

clinical consequences. Their detection broadens

the spectrum of identifiable mutations by including those that may not have an immediate adverse

effect on the individual, and also offers more precise information about the nature of the genetic

changes.
44 • Technologies for Detecting Heritable Mutations in Human Beings

—

DETECTION OF BLOOD PROTEIN VARIANTS

In a single-gene, dominant genetic disease, if

the causative mutant gene occupies one allele of

the pair of genes and the normal form of the gene

occupies the other allele, the one mutant gene is

dominant over the normal gene and is sufficient

to cause overt disease. Dominant diseases are said

to be expressed in individuals who have a single

dose, or are heterozygous, for the disease-causing

gene. Autosomal recessive diseases, however, are

clinically apparent only when the mutant gene is

present at both alleles, or in the homozygous state;

a single dose, the heterozygous state, can, for instance, destroy the activity of an enzyme or produce a defective gene product, but the corresponding normal gene supplies a sufficient amount of

normal gene product to prevent overt clinical

symptoms. It is difficult to infer the frequency of

recessive genes by population studies of phenotypes since individuals with one copy of the recessive mutant gene are phenotypically indistinguishable from individuals with only normal genes for

the particular protein. Biochemical means of identifying of heterozygotes, who are phenotypically

normal, is one way of identifying new mutations

that are expressed as recessive traits.

Biochemical studies on asymptomatic individuals with protein variants (indicative of single

gene, recessive mutations) can be useful in expressing the cumulative, “invisible” effect of an increased mutation rate. Any protein in the body

could be studied, but since peripheral blood is easily accessible, proteins from blood are most commonly used for these studies. Three major techniques for the detection and characterization of

variant proteins have been used in studies of human

1.

2.

3.

populations:

electrophoresis of blood proteins,

quantitative and kinetic studies of enzyme

deficiency variants from erythrocytes, and

molecular analysis of Hemoglobin M (HbM)

and unstable hemoglobins.

One-Dimensional Electrophoresis

Electrophoresis separates proteins according to

net molecular charge. A mixture of proteins is applied to a starch or acrylamide gel and exposed

to an electric current. Each protein moves according to its own electric charge and separates from

proteins with different charges. The subsequent

addition to the gel of stains that bind to the proteins makes it possible to visualize the location

of the proteins. Electrophoresis can be used to detect variant proteins in individuals and in their

parents. The identification in an offspring of a variant protein that is absent from either parent is

evidence for a new mutation. As in the other approaches, it is necessary to exclude the possibility of a discrepancy between stated and biological parentage so that only new, not inherited,

mutations are considered.

Electrophoretic variants are detectable because

a mutation results in a change in the net charge

of a protein. On a theoretical basis it is expected

that approximately one-third of amino acid substitutions produce a change in molecular charge

of a protein, and there is also evidence that electrophoresis can detect changes in a molecule’s configuration due to a nucleotide substitution. All in

all, it is estimated that electrophoresis probably

detects about 50 percent of nucleotide substitutions in expressed regions of coding genes (96).

The protein variants detected by electrophoresis

are usually not associated with clinically recognizable problems. However, this technique has an

important advantage for mutation research over

the phenotypic approach; it measures changes at

a level closer to the level of the mutational events

and therefore may provide more accurate estimates of mutation rates. It also reflects more subtle genetic changes, broadening the spectrum of

detectable genetic endpoints.

Mutation Rates Derived From Electrophoresis

Several large-scale studies to detect protein variants in human populations have been reported

(excluding studies in Japanese atomic bomb survivors, which are described below). Neel, et al.

(94), and Mohrenweiser (76) devised a large-scale

pilot program to study mutation rates using placental cord blood obtained from newborn infants

in Ann Arbor, Michigan (approximately 3,500

samples). They found no new heritable mutations
Ch. 3—Kinds and Rates of Human Heritable Mutations ● 4 5

in 36 different proteins in a total of 218,376 locus tests in Caucasian and 18,900 such tests in

black infants.g Harris and colleagues (38) summarized data from studies in the United Kingdom in

which 43 different loci coding for blood proteins

were analyzed, and no new mutations were found

in 133,478 locus tests.9 Altland and colleagues (5)

examined blood from filter paper submitted originally for screening for phenylketonuria in approximately 25,000 newborn infants in West Germany.

They identified one putative new mutation among

five different hemoglobin proteins in approximately 225,000 locus tests.

Feasibility of Electrophoresis

The availability of human blood for analysis

of variant proteins makes electrophoresis feasible for studies of large populations. In addition,

it permits comparisons of the frequency of occurrence of variant proteins between humans and

other species, since comparable proteins are usually associated with homologous gene sequences

(93). Perhaps the most important advantage of

this approach is that it permits the study of a defined set of genes expressing functional proteins

and offers more precise information on the number of genes involved in the biochemical observations made and on the effect of mutation on

some proteins (90).

However, electrophoretic analysis can provide

information only on mutations in genes that are

transcribed into RNA and subsequently translated

into proteins, and only for mutations that alter

the protein’s net molecular charge or configuration. It cannot provide information on mutations

in nontranscribed regions of the DNA, which

comprise the larger portion of the genome (165).

Since the baseline frequency of new protein variants is still poorly defined, it is difficult to estimate the size and scope of a population study necessary to detect an increase over a period of time

or to detect differences in rates between two

populations. One-dimensional electrophoresis is

limited by the number of proteins that can be

examined for variants, approximately the same

number of different phenotypes included in the

list of sentinel phenotypes. However, one-dimensional electrophoresis in a large population is a

more straightforward approach to studying new

heritable mutations than is population surveillance for sentinel phenotypes.

Vogel and Altland (164) estimated that analysis of two populations of 10 million individuals

each would be needed to have a 95-percent chance

of detecting a 10-percent increase in spontaneous

mutation rates. This calculation is based on a

theoretical rate of 1 mutation per 1 million genes

per generation and 50 loci tested per individual

in the study. The population size could be reduced

if the proteins that were examined mutated more

frequently or if detection of only large increases

in the mutation rate (e.g., 50 percent) were acceptable, or if many more proteins could be examined from each individual tested.

Quantitative and Kinetic Studies of

Enzyme Deficiency Variants

Mutations that result in variant proteins with

greatly reduced or no biological activity, when

present in the heterozygous state, are not readily

detectable in electrophoretic studies. Such “enzyme deficiency variants, ” or “nulls,”10 can be

identified through automated biochemical tests

that measure enzyme activity in red blood cells.

These studies increase the detectable range of

mutational events.

Loss of function of a protein may result from:

● the absence of a gene product,

● the presence of a protein which is nonfunctional catalytically, or

● abnormally unstable enzymes (75).

The types of mutations causing these abnormalities include chromosome rearrangement or loss,

deletions, frameshift mutations, and nucleotide

*The number of locus tests takes into account the number of proteins examined, the number of gene loci represented by each such

protein, and the number of individual samples obtained.

9

The number of individuals tested varies depending on the enzyme examined, but for some of the enzyme tests, more than 10,000

individuals were tested.

l’JThese mutations are characterized by the presence of an enzy -

matically inactive protein, or by an absence of a protein. These can

be caused by gene deletions, nucleotide substitutions in critical locations in the gene, mutations in the “start” and “stop” codons for

the protein, or even by nucleotide substitutions at splicing junctions

for the introns.
46 . Technologies for Detecting Heritable Mutations in Human Beings

substitutions in coding or noncoding regions of

DNA. The range of genetic events underlying the

production of enzyme deficiency variants is

thought to be larger than for electrophoretic mobility variants, since mutations in noncoding regions (e.g., intervening sequences, flanking regions, etc. ) as well as in coding regions could be

detected (74).

Four studies have provided data on the frequency of enzyme deficiency variants. The frequencies of such variants have been measured in:

1) placental cord blood samples from approximately 2,500 individuals in Ann Arbor, Michigan (74,75); 2) blood samples obtained from participants of the studies in Japan coordinated by

the Radiation Effects Research Foundation (122);

3) blood samples obtained from 3,OOO hospitalized

individuals in West Germany (3o); and 4) blood

samples obtained from Amerindians of South and

Central America (78). No new mutations were

found in any of these studies. However, rare but

not new enzyme deficiency mutations that have

been inherited over many generations have been

measured at an overall frequency of 2 variants

per 1,000 determinations in a total of approximately 110,000 locus tests. If new mutations

producing enzyme deficiency variants occur, as

predicted, no more than twice as often as new mutations leading to electrophoretic variants, the lack

of new mutations in these 110,000 locus tests is

not inconsistent with electrophoretic data.

Molecular Analysis of Hemoglobin M

and Unstable Hemoglobins

Stamatoyannopoulos and Nute (131) calculated

mutation rates for certain nucleotides in the globin

genes, which, when mutated, produce autosomal

dominant disorders expressed as chronic hemolytic anemia or methemoglobinemic cyanosis.

Their data provided the first direct measurements

of heritable mutation rates for particular nucleotides in human DNA. Although these data are

limited to the specific characteristics of the globin

genes, they suggest the kind of information which

may be possible to obtain for other genes as the

genetic basis for various single gene disorders is

discovered. Even if the mutation rates derived

from these studies of the globin genes are not representative of rates of mutation at other loci, the

hemoglobin system described below provides a

model for combining epidemiologic, clinical, and

molecular information to estimate heritable mutation rates in human populations.

Structure and Function of Hemoglobin

Hemoglobin molecules, which exist in a number of slightly different forms, are the universal

carriers of oxygen in the blood from the lungs to

all cells of the human body. Different mixtures

of hemoglobins are present at all stages of development from embryonic to adult, although a particular type of hemoglobin tends to predominate

at any one stage. In addition, hundreds of abnormal hemoglobins have been identified, ranging in

clinical severity from benign through serious

hematologic disorders. Human hemoglobin is a

protein consisting of four separate globin polypeptide chains and four iron-containing heme

groups. In adults, the predominant form of hemoglobin is hemoglobin A (HbA), composed of

two alpha-globin chains and two beta-globin

chains each joined to a heme group.

Hemoglobin’s primary function of binding, carrying, and releasing oxygen is closely regulated

by several factors, including the maintenance of

a precise three-dimensional conformation of the

hemoglobin molecule, the composition and balance of globin chains, and the distribution of ionic

charges in and around the molecule. Mutations

which result in amino acid substitutions at particular sites in the globin polypeptide chain can

markedly alter these specific functional properties of the hemoglobin molecule.

Two types of abnormal hemoglobin, HbM and

unstable hemoglobin, usually occur sporadically

as a result of a new mutation and are expressed

as dominant mutations.11 Stamatoyannopoulos

and Nute (131) described the genetic changes leading to these particular abnormal hemoglobins and

their incidence in human populations. From this

11A heterozygous individual, having one mutant globin gene and

one normal allele, would have clinically recognizable symptoms of

the disease. This is in contrast to other, more common hemoglobin

diseases, such as sickle cell anemia and thalassemia, where the heterozygote is asymptomatic and the disease occurs only when an individual is homozygous for the variant gene.
Ch. 3—Kinds and Rates of Human Heritable Mutations ● 47

information, they were able to calculate the rate

of mutation leading to these abnormal hemoglobins.

Unstable Hemoglobins

Various mutations in the globin genes can lead

to an altered sequence of amino acids in the globin

chains and to a major change in the three-dimensional structure of the hemoglobin molecule.

Often these changes create a less stable hemoglobin molecule than its normal counterpart. “Unstable hemoglobin” can result in the hemoglobin

precipitating within the red cell, leading to membrane damage and premature destruction of the

red cells by the liver and spleen (165).

The degree of clinical severity of the unstable

hemoglobin depends in part on the site at which

the amino acid replacement occurs and on the

properties of the amino acid that is substituted

(37). However, manifestations of the disorder

vary from mild hemoglobin instability which may

not be clinically significant, to severe hemoglobin instability, which causes chronic hemolytic

anemia.

Unstable hemoglobin is diagnosed by isolating

and characterizing the abnormal hemoglobin, and

by determining its molecular stability. Since the

amino acid sequence of normal hemoglobin is

known, and the sequences for some abnormal

hemoglobins have been determined, the genetic

mutation underlying these unstable globins have

been identified.

Hemoglobin M—The Methemoglobinemias

A blue, cyanotic appearance is the chief clinical manifestation of methemoglobinemia, caused

by HbM. Cyanosis occurs whenever a significant

proportion of the hemoglobin in the circulating

red blood cells is not carrying oxygen, and in this

case it is because of an inability of the hemoglobin to bind and carry oxygen (37).

Five different mutations in the globin gene

which lead to the production of HbM have been

identified, All of these are expressed as dominant

phenotypes. The common characteristic of these

mutations is that each results in substitution of

an amino acid in the globin peptide where the

heme groups are attached. (These mutations are

specific substitutions of histidine for tyrosine on

the alpha-globin chain [at positions 58 and 87] or

on the beta-globin chain [at positions 63 and 92],

or by substitution of valine for glutamic acid on

the beta-globin chain [at position 67]. ) By altering the site of attachment of heme on the globin

chains, these mutations change the three-dimensional organization of the entire hemoglobin molecule and interfere with its ability to combine with

oxygen (100).

Measurement of Mutation Rates in the

Globin Genes

Using published data on the number of cases

of hemoglobin disorders from 10 countries (134),

Stamatoyannopoulos and Nute (131) determined

the number of children with HbM and unstable

hemoglobin among an estimated number of livebirths in the defined populations. They recorded

a total of 55 cases of de novo hemoglobin mutants, each of which derived from substitutions

of single nucleotides in the coding portions of the

globin genes: 40 children with unstable hemoglobin (all beta chain mutations) and 15 with HbM

(10 beta chain and 5 alpha chain).12 The requisite paternity data, also from published information on these mutations, had been collected on

19 of the 55 cases which were used to calculate

mutation rates (131). Their data are shown in

table 3.

The mutation rates derived from these data are

expressed in mutational events per alpha- or betagene nucleotide per generation, and range from

5.9 X 10-9 to 19X 10-9 for alpha- and beta-gene

nucleotides, respectively. The rate for all betagene variants together is 7.4X 10-9 per nucleotide per generation. These estimates correspond

to mutation rates per globin chain gene per generation of 2.6X 10-6 to 8.3 X 10-6. The rate per

gene is obtained by multiplying the rate per nu-

‘

2 The number of alpha chain mutations detected was less than

expected, given the existence of two alpha-globin genes per betaglobin gene in an individual. A possible reason for this may be that

alpha-chain defects would produce physiological abnormalities at

an earlier stage of fetal development than would beta-chain abnormalities, since the alpha gene is activated much earlier, and may

be causing a greater loss of fetuses early in gestation.
48 ● Technologies for Detecting Heritable Mutations in Human Beings

—

Table 3.–Rates of Mutation Leading to HbM

and Unstable Hb Disorders

Mutation rate/

nucleotide/ Mutation rate/

Type of disorder generation globin geneb

Unstable Hbs . . .

Alpha

M variants . 4.2x 10-6/423 nucleotides

Beta

M variants . . 18.9x 10-9 8.3 x 10-6/438 nucleotides

Ail beta-chain

variants . . . . . . 7.4 x 10-9 3.2x 10-6/438 nucleotides

aThe mutation rate per nucleotide is based on the rate of appearance Of the Particular disorder (number of mutants observed divided by 2 multiplied by the number of births in the generation(s) Included). This is the average mutation rate

for the particular disorder expressed per globin gene affected per generation.

To calculate the mutation rate per nucleotide per generation, StamatoyannopouIos and Nute took the frequency of each mutant divided by 2 (for two beta genes

in a genorne) multiplied by 3 (since a given nucleotide can be substituted by

any one of three other nucleotides), then divided that product by the number

of different substituted nucleotides actually observed In their data.

bT’he estimated mutation rate per alpha-globin and beta-globin gene was cakuIated as the mutation rate per nucleotide multiplied by the number of nucleotides constituting the coding portion of the corresponding gene (423 in the

alpha-globin gene and 438 in the beta-globin gene).

SOURCE: G. Stamatoyannopoulos and P.E. Nute, “De Novo Mutations Producing Unstable Hbs or Hbs M. 11: Direct Estimates of Minimum Nucleotide Mutation Rates in Man,” Hum, Genef. 60:181-88, 1982.

cleotide by the number of nucleotides in the coding portion of the gene.

orders. The diagnosis of these hemoglobin disorders requires structural and functional analyses of the aberrant proteins, and not all cases

would be identified initially by their phenotypic

abnormalities; some degree of underascertainment

is inevitable in these data. This is particularly true

in the data for the unstable hemoglobins, where

clinical manifestations of the genotype range from

mild to severe. The more severe cases are likely

to be identified and reported in the literature.

Overall, the order of magnitude of the estimates

for gene mutation rates, 1 mutation in 1 million

genes per generation, is consistent with that of the

estimates of the spontaneous heritable mutation

rate made by Neel and colleagues in the Japanese

populations that comprised the control group in

their ongoing study of the genetic effects of the

atomic bombs (see below). This suggests that even

though the rates derived from these data are based

on mutations observed in only alpha- and betaglobin genes, they are not very different from mutation rates in other expressed genes.

The rates calculated from these data are likely

to be minimum rates corresponding to these disSTUDIES OF POPULATIONS EXPOSED TO ATOMIC RADIATION

The first demonstration that ionizing radiation

could induce genetic mutation is usually credited

to Muller (81) for his work with Drosophila, and

since then, many scientists have examined the nature and frequency of induced mutations in various species of animals, including whole mammals

(120). There is an extensive body of experimental

data that suggests that exposure to radiation and

to certain chemicals can induce mutations in mammalian germ cells. In humans, exposure to ionizing radiation is known to cause somatic mutation,

and is suspected to enhance the probability of germinal mutation (165). To date, however, the

available methods have provided no documented

evidence for the induction (by chemicals or by radiation) of mutations in human germ cells.

The single largest population at risk for induced

heritable mutations is the group of survivors of

the atomic bombs dropped on Hiroshima and

Nagasaki in 1945. Survivors of these bombs are

estimated to have received doses of radiation considered to be “biologically effective”; in experiments with mutation induction by acute exposure

to radiation in mammals, similar kinds and doses

of radiation were sufficient to cause phenotypically apparent mutations in offspring (and presumably other nonexpressed mutations as well).

On the basis of this kind of experimental data,

the assumption was made that germinal mutations

could have been induced in people exposed to the

radiation.

Efforts to detect genetic consequences of the

atomic bombs detonated in Hiroshima and

Nagasaki have been in progress continuously since

1946, sponsored by the Atomic Bomb Casualty

Commission and by its successor in 1977, the Radiation Effects Research Foundation. These studies

have attempted to determine whether germinal

mutations are expressed as heritable mutations in

the survivors’ offspring, particularly as “untoward
Ch. 3—Kinds and Rates of Human Heritable Mutations ● 4 9

pregnancy outcomes,” as single gene disorders,

as chromosome abnormalities, or most recently,

as abnormal blood proteins.

Early Epidemiologic Studies

The earliest clinical studies, as well as the continuing mortality surveillance study, sought to

identify a variety of health problems in the offspring of exposed atomic bomb survivors. These

studies consisted of epidemiologic surveys of

70,082 newborn infants born in Hiroshima and

Nagasaki between 1948 and 1953, including about

38,000 infants for which at least one parent had

been proximally exposed to the radiation (defined

as being within 2,OOO meters of the hypocenter,

while distally exposed was defined as being greater

than 2,500 meters from the hypocenter or not

exposed at all). Stillbirths, infant mortality, birthweight, congenital abnormalities, childhood mortality, and sex ratio were examined. An “untoward pregnancy outcome” was defined as a

stillbirth, a major congenital defect, death during the first postnatal week of life, or a combination of these events. The investigators found no

statistically significant, radiation-related increase

for any “untoward pregnancy outcomes” (123).

One reason for this finding may be that these

health problems each have some genetic basis, but

that all are influenced by a variety of nongenetic

factors as well, some of which may have obscured

an effect of genetic mutation. In addition, a continuing study (the F1 Mortality study) of mortality among children born between 1946 and 1980

has not demonstrated a relationship between radiation exposure of the parents and mortality in

the offspring (56).

Cytogenetic Studies

Since 1967, Awa and colleagues have screened

the children of survivors of the atomic bombs for

cytogenetic abnormalities to determine whether

exposure of the parents’ gametes to the radiation

caused a higher frequency of chromosome abnormalities in the offspring. After first reporting a

higher frequency of sex-chromosome abnormalities among 2,885 children of exposed parents

compared with 1,090 children of the control group

of parents, Awa (6) concluded that a mutagenic

effect of the radiation was not demonstrated in

these data. The total frequency of chromosomally

abnormal children of exposed parents (0.62 percent) was higher than in the control group (0.28

percent), although the difference was not statistically significant, and was similar to the frequency of such abnormalities observed in several

studies of unselected, consecutive newborn populations (120).

Results of their more recent analysis of the data

showed that 0.52 percent of the offspring of exposed parents were determined to have an abnormal chromosome constitution, compared to 0.49

percent of the offspring of the control group of

parents, which suggests that there is no significant difference between the two groups of offspring in frequency of autosomal balanced and

unbalanced rearrangements and of sex-chromosome numerical abnormalities (8). This study did

not provide information on chromosome abnormalities which are associated with high mortality rates, such as most of the unbalanced structural rearrangements and numerical chromosome

abnormalities involving the autosomes.

Biochemical Analyses

Beginning in 1976, one-dimensional electrophoresis was used to look for mutations affecting proteins found in red blood cells and blood plasma.

Rare variants of these proteins, differing by a single constituent amino acid, are taken to represent

mutations which occurred in the germ cells of one

of the parents if the variant is detected in a child

and is absent from both parents. Classification as

a “probable mutation” is made after inquiry into

possible errors of diagnosis and of stated parentage (124).

Neel and colleagues (93,96,121) analyzed 28 enzyme and serum proteins from blood samples obtained from children who were identified in the

F1 Mortality study and who were born between

1959 and 1975 to survivors of the atomic bombs.

Over 10,000 children were examined in each

group of exposed and unexposed parents. These

children were also participants in the cytogenetic

studies, and the biochemical analyses made use

of the same blood samples that were used for the
50 ● Technologies for Detecting Heritable Mutations in Human Beings

chromosome analyses. To date, two probable mutations have been identified among offspring of

proximally exposed parents (estimated to have received an average conjoint gonadal exposure of

approximately 600mSv [60 rem13]) in 419,66610-

cus tests, while 3 new mutations have been found

among 539,170 locus tests of unexposed individuals. Although the number of new mutants is

small, a crude mutation rate can be calculated

from these data. The rate of mutation in the exposed group is approximately 5 mutations in 1

million genes, and the rate in the unexposed group

is 6 mutations in 1 million genes per generation.

Such rates have wide margins of error and are not

statistically different (96).

In 1979, the tests of electrophoretic variants

were expanded to include assays for reduced activlsThe doses of radiation that the survivors are estimated to have

received are currently being re-evaluated in light of new information concerning the quantity and quality of radiation released by

the two bombs (125). The revised estimate of exposure will probably be lower than the current estimate of 60 rem.

CONCLUSIONS

This chapter has described several currently feasible approaches for determining rates of heritable mutations. Each of the methods has its particular problems. The genetic defects underlying

the sentinel phenotypes are largely unknown.

These phenotypes may result from mutations

within genes at one or several genes, and may be

complicated by the presence of phenotypically

similar but nongenetic traits, all serving to bias

estimates of their frequency upwards and to create a wide margin of error. Even with a collection of sentinel phenotypes that can be reliably

identified in newborn infants, population surveys

of a sufficient size are difficult to perform. To

detect a small or moderate increase of sentinel

phenotypes, consecutive newborns of entire national populations would have to be monitored

thoroughly for many years, perhaps as part of an

ongoing surveillance program for birth defects and

childhood cancers.

ity variants of certain red cell enzymes .14 The presence of such variants suggests a deletion in the

DNA, producing “null” mutations which would

not be identified by one-dimensional electrophoresis. No such variants were found among 11,852

locus tests of 10 erythrocyte enzymes in children

of exposed and unexposed parents (122).

Mutation Studies in Populations

Exposed to Radiation in

the Marshall Islands

Neel and his colleagues (93) reported results of

electrophoretic studies of blood proteins from offspring of parents exposed to radioactive fallout

from the Bravo thermonuclear test explosion at

Bikini in the Marshall Islands in 1954. No variant proteins indicating new mutations were found

in 1,897 locus tests of 25 proteins measured in children of unexposed parents, and in 1,835 such tests

in children of exposed parents.

‘—14An enz—yme level three standard deviations below the mean

and/or less than or equal to 66 percent of normal was defined as

a deficiency variant.

The routine staining and banding of chromosomes is the most straightforward of the current

methods, but they allow only major chromosomal

changes to be identified. More advanced banding techniques, which are not performed routinely, allow smaller chromosome abnormalities

to be detected in some cases.

Among the current methods described here, the

detection of electrophoretic protein variants

comes closest to mirroring precise changes in

DNA. However, one-dimensional electrophoresis is limited by the relatively small number of proteins it can examine and by the fact that it does

not readily detect the absence of gene products,

caused by deletions or by specific nucleotide substitutions. Detection of enzyme deficiency variants, although still in early stages of development,

could complement electrophoresis by detecting

loss-of-activity variants. In addition, large pop-
Ch. 3—Kinds and Rates of Human Heritable Mutations . 51

ulation surveys are required to generate statistically significant results, and many more paternity

tests are likely to be needed as a result of the biochemical approach compared to the phenotypic

and cytogenetic methods. (Since the protein variants are not associated with reduced fertility, as

are most of the sentinel phenotypes and chromosome abnormalities, only a fraction of the protein variants will be new mutations. )

An interesting example of an approach for detecting mutations in a single gene or in a group

of genes is provided by the analysis of mutations

underlying unstable hemoglobins and HbM.

Analyses of other accessible and well-studied

genes may help to generate better estimates of mutation rates per nucleotide and per gene on the

human genome, although it is not known how

representative any one gene is of the remainder

of the DNA. Moreover, characterizing the mutations at the molecular level provides important

information, especially where specific mutagens

are suspected to have caused the mutations.

The different approaches have led to various

estimates of heritable mutation rates in human beings (see table 4). Sentinel phenotypes, representing the occurrence of a single dominant mutant

gene, are detected in about 2 individuals, on average, per 100,000 examined per generation. Individual types of chromosome abnormalities are up

to 50 times more frequent than the average frequency of sentinel phenotypes. Analyses of the

mutant hemoglobins studied by Stamatoyannopoulos and Nute (131) give estimated spontaneous mutation rates of about three mutations per

1 million genes per generation. The rate measured

in one-dimensional electrophoresis of blood proteins is also about three mutations per 1 million

genes per generation, a figure based on the examination of over 1 million locus tests involving

30 to 40 different proteins.

The different approaches currently available for

detecting heritable mutations (listed in table 5) in

human beings generate data that are generally not

comparable. Each set of data reflects the nature

of the approach itself, not necessarily the nature

of the different genes examined or their spontaneous mutation rates. It is particularly difficult to

compare directly the results of studies of mutaTable 4.—Results of Studies To Detect Heritable

Mutations by One-Dimensional Electrophoresis

in Human Populations Not Known To Be

at High Risk for Germinal Mutations

Population Locus tests Mutations

United Statesa

: Caucasian . . . . . . 218,376 0

Black . . . . . . . . . . 18,900 0

United Kingdomb . . . . . . . . . . . . . . 133,478 0

West Germanyc . . . . . . . . . . . . . . . 225,000 1

Central and South Americad . . . . 118,475 0

Japan d . . . . . . . . . . . . . . . . . . . . . . . 539,170 3

Marshall Islandse . . . . . . . . . . . . . . 1,897 0

Total . . . . . . . . . . . . . . . . . . . . . . . 1,255,296 4

Overall “spontaneous” mutation rate = 4 mutat!ons/1 ,255,298 locus tests IS equwalent to approximately 3 mutations/l million genes per generation

SOURCES

aJ, Ij, Neel, H,W, Mollreniveiser, and M H Meisler, “Rate Of Spontaneous Muta.

tion at Human Loci Encoding Protein Structure,” Proc Nat/ Acad SCI USA

77(10):603743041, IWO; H W Mohrenweiser, 1986, unpublished data, cited In Neel,

et al., 1986.

bH. Harris, D.A Hopklnson, and E B Robson, “The Incidence of Rare Alleles De.

termining Electrophoretlc Variants” Data on 43 Enzyme LOCI In Man, ” Ann Hum

Genet. Lend. 37 ”237-253, 1974,

cK, Altland, M Kaempfer, M Forssbohm, and W. Werner, “Monitoring for Chmg.

Ing Mutation Rates Using Blood Samples Submitted for PKU Screen ing, ” F/u.

man Genetics, Part A: The Unfo/ding Genome (New York Alan R Liss, 1982),

pp 277-287.

dJ,V, Neel, c, Satoh, K Gorlkl, et al , “The Rate With Which Spontaneous Mutation Alters the Elect rophoretic Mobility of Polypeptides, ” Proc Naf Acad SCI

USA 83389-393, 1986, J V Neel, unpublished data.

eJ ,V Neel, H Mohrenweiser, S Hanash, et al., “Biochemlcai Approaches TO Monitoring Human Populations for Germinal Mutation Rates 1. Elect rophoresis, ”

Utilization of Marnma/ian Specific Locus Stud/es In Hazard Eva/uatfon and Es.

timation of Genet/c f%sk, F J deSerres and W Sheridan (eds ) (New York Pie.

num Press, 1983), pp 71-93

Table 5.-Spontaneous Mutation Rates of Genetic

Abnormalities in Human Populations

Mutations/

Genetic anomaly generation References

Sentinel phenotypes 2 mutations

per 100,000

individuals

Chromosome 2 to 121 mutations

abnormalities per 100,000

individuals

Electrophoretic 3 mutations per

protein variants million genes

Analysis of unstable 3 mutations per

hemoglobins million globin

genes

(166)

Table 3-2

Table 3-4; (96)

Table 3-3; (131)

SOURCE: Off Ice of Technology Assessment

tion rates based on clinical syndromes with biochemical studies. Measurements from each of the

current methods can be used to generate a separate mutation rate and, for the time being, those

rates can be viewed as baselines for the frequency

of various kinds of human mutations and as reference points for some of the most severe effects

of heritable mutations.
52 ● Technologies for Detecting Heritable Mutations in Human Beings

—

The different approaches currently available reflect the various forms that mutations and their

consequences take. Each of the current approaches

to studying mutations is generally limited to particular kinds of mutations or to a particular set

of outcomes. The cytogenetic and biochemical approaches discussed are limited to detecting a small

subset of mutations that occur in human DNA.

Identification of sentinel phenotypes and chromosome abnormalities limits the observations to a

particular set of outcomes, mostly in the severe

end of the spectrum. The available data are based

on mutations observed in a small fraction of the

genes, about 40 to sO, out of a total of about

50,000 to 100,000 in the human genome.

Aspects of the current methods that limit their

scope also make them inefficient. The proportion

of all mutations that these methods can identify

is relatively small and the limited number of suitable genetic endpoints (acceptable sentinel phenotypes, chromosome abnormalities, and protein

variants) makes it difficult to sample more than

a few mutational events per individual. As a result, huge populations of “exposed” and “unexposed” people would be needed to find out

whether the exposure resulted in excess heritable

mutations (71). Not surprisingly, perhaps, there

is no documented evidence to date for a meaningful difference in rates for any type of mutation

between two populations (or in the same population at different times).

Sentinel phenotypes and cytogenetic analyses

provide a highly selective approach to some of

the worst possible consequences of new mutations. However, they do not permit more than

a partial view of the broader consequences of mutations in human beings. Detailed molecular,

physiological, and clinical information on the

kinds and rates of new mutations will eventually

be needed to assess the immediate or potential impact of new mutations.
Chapter 4

New Technologies for

Detecting Heritable Mutations
Contents

Page

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

Short Descriptions of the New Technologies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

Two-Dimensional Polyacrylamide Gel Electrophoresis . . . . . . . . . . . . . . . . . . . . . 56

DNA Sequencing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

Restriction Fragment Length Polymorphisms. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

One-Dimensional Denaturing Gradient Gel Electrophoresis . . . . . . . . . . . . . . . . . 59

Two-Dimensional Denaturing Gradient Gel Electrophoresis. . . . . . . . . . . . . . . . . 61

Ribonuclease Cleavage of Mismatches in RNA/DNA Heteroduplexes . . . . . . . . 64

Subtractive Hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

Pulsed Field Gel Electrophoresis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

TABLE

Table No. Page

6. Current and New Methods for Detecting Human Heritable Mutations . . . . . . . 55

FIGURES

Figure No. Page

7. Production of Restriction Fragment Length Polymorphisms Using

Restriction Enzymes and Separation of Different DNA Fragment

Sizes by Agarose Gel Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

8. Restriction Fragment Length Polymorphism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

9. One-Dimensional Denaturing Gradient Gel Electrophoresis. . . . . . . . . . . . . . . . 62

10. Two-Dimensional Denaturing Gradient Gel Electrophoresis . . . . . . . . . . . . . . . 63

11. Ribonuclease Cleavage of Mismatches in RNA/DNA Heteroduplexes. . . . . . . 65

12. Subtractive Hybridization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Chapter 4

New Technologies for

Detecting Heritable Mutations

INTRODUCTION

New developments in molecular biology have

opened the way for direct examination of large

portions of human DNA to detect kinds and rates

of mutations. Until recently, it has been possible

only to infer the occurrence of genetic damage

from indirect, limited, and often imprecise observations (see ch. 3). In principle, these new technologies would allow samples of DNA from people exposed to potential mutagens to be examined

for precise genetic damage.

Little is known about the nature and frequency

in humans of the majority of mutational events,

since previous information was available only on

DNA sequences that could be selected on the basis of gene expression. Until recently, human mutation was studied in fine detail only in particular genes (e.g., hprt and globin genes) that could

be fished out of genomic DNA by using some

gene-specific probe, or in genes whose products

(mRNA, proteins, etc. ) could be isolated and

examined.

In this chapter, new methods are described for

examining proteins and for examining large regions of genomic DNA (see table 6), regardless

of their function or location in the genome and

without devising selection methods for specific

known genes. These new technologies are currently under development or being considered by

the scientific community. Most of them were explored as potentially feasible technologies at a

workshop cosponsored by the International Commission for Protection Against Environmental

Mutagens and Carcinogens (ICPEMC) and the

U.S. Department of Energy, in December 1984.

Table 6.—Current and New Methods for Detecting

Human Heritable Mutations

Indicators of mutation Method of detection

Current methods:

Sporadic genetic Sentinel phenotypes

disease

Numerical or structural Cytogenetic analysis

chromosome

abnormal i ties

Variant blood proteins 1 D-electrophoresis

Quantitative enzyme

2D-electrophoresis

New methods:

Altered DNA sequences DNA sequencing

analysis

Restriction fragment length

polymorphisms

1 D-denaturing gel

electrophoresis

2D-denaturing gel

electrophoresis

Ribonuclease cleavage

Subtractive hybridization

Pulsed field gel electrophoresis

SOURCE Office of Technology Assessment

Several different approaches are used in these

new technologies. Two-dimensional electrophoresis of proteins follows from the techniques of

one-dimensional electrophoresis and incorporates

new methods of analyzing protein spots on the

gel. In several of the DNA methods, restriction

enzymes are used to fragment genomic DNA before sizing and sequence analyses are performed.

Other technologies hinge on the production of hybrid DNA molecules, or heteroduplexes, and on

gradient denaturing gels that are used to identify

heteroduplexes with base pair changes. Another

method devises a way of separating larger fragments of DNA than were previously possible to

separate. These new methods are discussed below,

55
56 . Technologies for Detecting Heritab/e Mutations in Human Beings

SHORT DESCRIPTIONS OF THE NEW TECHNOLOGIES

Two-Dimensional Polyacrylamide Gel

Electrophoresis

A development following from the experience

with one-dimensional electrophoresis for protein

variants (see ch. 3) is two-dimensional polyacrylamide gel electrophoresis, “2-D PAGE, ” the only

technique described in this chapter that is currently in use in studies of human beings. With this

technique, proteins are separated first by isoelectric focusing on the basis of their molecular

charge, and then by electrophoresis on the basis

of their molecular weight. From 500 to 1,000 proteins may be visible from a single sample on one

gel, and about 100 of these may be sufficiently

clear to detect rare variants in a child that are not

present in the parents (92).

To search for new mutations in proteins using

2-D PAGE, blood samples are taken from child/

mother/father trios. The program that has had

most experience with this technique for mutation

research, the University of Michigan Medical

School, collects cord blood from newborns and

parental blood samples at the time of the child’s

birth. Each blood sample is fractionated into six

parts: platelets, plasma, nonpolymorphonuclear

cells, polymorphonuclear cells, erythrocyte (red

blood cell) membranes, and erythrocyte cytosol

(the contents of the cell) (95). Each fraction is

treated and analyzed separately.

Separation in the first dimension is by the

charge of the protein molecules, qualitatively similar to the separation using one-dimensional electrophoresis. Each sample is run on a long, thin

(1 to 2 mm in diameter) cylinder of polyacrylamide in a hollow glass tube using a technique

called isoelectric focusing. The separation time can

be speeded up or slowed down depending on the

voltage applied. After the first separation, the gel,

which looks like a clear noodle, is “extruded” from

the tube.

In the second separation, the “noodle” is laid

across the top of a gel about 7 inches square, that

is held between two glass or plastic plates. A current is passed through the gel, and the protein

molecules migrate down through the gel at a speed

that is proportional to their size. A final step

renders the proteins visible by autoradiography

(for proteins that can be isotonically labeled) or

by stains such as Coomassie Brilliant Blue and

silver- or nickel-based stains.

The trained eye or a computer program detects

patterns of spots on the gel, though most of the

stained areas are too crowded or indistinct for

drawing conclusions about specific proteins. Only

spots in relatively clear areas and of a sufficient

intensity are chosen for scoring. The analysis entails comparing the parent/child trios for differences. No two gels are run in precisely the same

way, so it is often difficult to superimpose a set

of three gels over one another and to look for

differences. While protein spots bear the same

relationships to each other on different gels, they

routinely appear at slightly different coordinates.

For each type of sample mentioned above, a

standard constellation of protein spots is scored

for genetic variation. A mutant protein can appear on the gel as a spot for which there is no corresponding spot on parental gels. The gels can be

interpreted visually, which is the traditional

method, and currently the most accurate, but

computer-assisted interpretation is desirable. Two

steps in analysis lend themselves to computerization. First, and now routinely done, the protein

spots can be “read” by computer for location and

intensity. The second step is computerized comparison of different gels. Two approaches for

comparison are being developed in different laboratories, each of which will probably be useful

on a “production scale” in the near future (92).

Eventually, computers will be relied on to screen

gels, and to eliminate from further consideration

the overwhelming majority of parent/child trios

with no variants.

In comparison to one-dimensional protein separation, a relatively small number of parent/child

trios have been analyzed with 2-D PAGE, and no

heritable mutations have been found. However,

the results of these analyses have revealed a wide

array of largely unknown proteins, and distinct

differences in the spectrum of proteins from different cell types. The range of electrophoretic vari-
Ch. 4—New Technologies for Detecting Heritable Mutations ● 5 7

ants of a number of these proteins have been characterized in plasma (104) and in erythrocyte lysate

(105). In plasma, the variation is substantially

higher than has been reported in other blood

fractions.

The advantage of 2-D PAGE is that it can be

done now without a major developmental breakthrough and without a great initial cost, though

the running expenses are significant. It may be

some years before any of the DNA analytic methods catch up to protein analysis in practical application. The future of protein analysis for detecting mutations after DNA methods become

routine is unclear, however.

There are technical limitations of the 2-D PAGE

technique. First, the technique itself is technically

demanding. Second, the number of proteins that

can be scored on each gel is limited by the degree

of separation possible on plates that are of a practical size.

As with most of the other techniques, only a

certain spectrum of mutations is detectable with

2-D PAGE. Chromosome rearrangements in general are not revealed through electrophoretic variants. This technique has its greatest strength in

detecting point mutations and larger deletions in

the DNA that change the size or charge of a protein molecule. Point mutations that do not cause

such changes, and small insertions or deletions,

are less likely to be detectable with this method.

Currently, null mutations cannot be detected reliably, and this is probably the greatest disadvantage

of 2-D PAGE. Overall, 2-D PAGE should detect

an estimated 25 to 35 percent of all spontaneous

gene mutations. Several aspects of 2-D PAGE of

blood proteins, in addition to computerized comparisons of gels, are amenable to improvement,

and at least one approach is being developed to

improve detection of null mutations (92).

The identity of most of the proteins that can

be visualized on two-dimensional gels is unknown, Although it has not yet been done, the

technology exists to recover proteins from gels,

purify them, and then determine their amino acid

sequences (92). Once amino acid sequences are

worked out, the nucleotide sequences that code

for them can be deduced and nucleic acid probes

can be assembled to correspond to the proteincoding nucleotide sequences. The probes could

theoretically be used to locate the corresponding

DNA within the human genome, thereby determining exactly where in the genome the coding

sequence lies.

DNA Sequencing

Conceptually, the most thorough and straightforward analysis of the genome would consist of

lining up the chromosomes and determining the

nucleotide sequence from end to end on each one.

Once the sequence was known, it could be stored

in computer memory.

The advantage of determining the complete

genomic sequence would be that any nucleotide

sequence that was identified in any laboratory

could immediately be mapped to its chromosomal

location. As nucleotide sequences for various

parts of the genome were obtained in different laboratories, both common polymorphisms and rare

mutations could be identified by making comparisons between the total genomic sequence and partial sequences. Furthermore, the method would

detect mutations and polymorphisms no n-tatter

where they occur—in introns, exons, and repetitive sequences.

Walter Gilbert, Fred Sanger, and their colleagues developed the theoretical basis for the

techniques that makes it possible to consider sequencing the genome (67,118) and in 1984, George Church and Walter Gilbert described a

method for sequencing any part of the genome

(2o), although the method could be applied to sequencing the entire genome. The genomic DNA

would be cut into pieces small enough to be handled in the laboratory, and then chemical methods would be used to determine the nucleotide sequence within each fragment. The methods are

well understood, are being applied in dozens of

laboratories around the world, and are a standard exercise in the education of molecular biology graduate students (33). Through February

‘Polymorphisms are “common” mutations that are present in the

population at a relatively high frequency. Several alternative versions of the same basic gene sequence, all equally functional, can

co-exist. New mutations are defined as occurring in less than 1 percent of the population, excluding the more common polymorphic

variants.
58 . Technologies for Defecting Heritable Mutations in Human Beings

—

1985, the methods have been used to examine discrete and defined segments of DNA from organisms with large genomes and complete genomes

from smaller organisms, sequencing a total of

4,045,305 base pairs (150)—roughly 1/800 of the

human genome.

Practically, we are far away from being able

to sequence the entire genome, not because the

techniques are unavailable but because they are

expensive and slow when applied to a task of the

magnitude of sequencing 3 billion nucleotides. A

meeting during the summer of 1985 considered

this enormous task and estimated the cost at about

$1 per nucleotide, or $3 billion overall. The effort can also be estimated in terms of technicianyears, and that comes to about 200 technicianyears to sequence 100 million nucleotides, which

would be expected to yield about one new mutation.

Although it is conceptually satisfying, the enormous resources necessary to sequence the entire

genome make it unlikely that the task will be undertaken soon. However, genomic sequencing

technology is still progressing, and further automation of the process may increase its efficiency.

Continued encouragement of those efforts and

provision of sufficient resources to develop techniques may make it possible to sequence the entire human genome in a reasonable time. When

it is done, it will probably be undertaken as part

of an effort to provide more general information

about human genetics, rather than to identify human mutations. If the genome is sequenced, for

whatever reason, it will be a great boon to mutation studies by providing a reference for all techniques that involve sequencing or the use of restriction enzymes.

Restriction Fragment Length

Polymorphisms

A method that offers a less exhaustive survey

than genomic sequencing, but which is potentially

more efficient, involves the use of restriction enzymes and gel electrophoresis to detect nucleotide substitutions or small deletions in the DNA

(98).

Genomic DNA is isolated from white blood

cells and treated with restriction enzymes, chemicals that recognize specific sequences in the DNA

and “cut” the DNA wherever such sequences occur (see fig. 7). (The number of fragments produced is determined by the frequency of occurrence of the particular enzyme recognition site in

the DNA sequence. ) Restriction site analysis does

not, in practice, examine every nucleotide. However, the use of a set of combined restriction enzymes increases the number of restriction sites

identified, allowing examination of a larger portion of the DNA, including both expressed and

nonexpressed regions.

After the DNA is fragmented (see fig. 8), there

are two alternative ways of proceeding, one using

a gene cloning method, and the other using a noncloning, direct method.

Cloning is the process of “growing” human

DNA in other organisms; it reduces the amount

of DNA needed—in most cases corresponding to

the amount of blood that must be drawn—but it

increases the number of laboratory manipulations.

Additionally, it introduces some uncertainty

about the possibility that DNA changes will occur during cloning. This can be checked, but requires diligence.

In the gene cloning method, the human DNA

fragments are incorporated into the genetic material of a virus called “lambda. ” Lambda reproduces in the bacterium E.coli, and large quantities of viral DNA containing the human segments

are produced. Following isolation of the cloned

human DNA, each clone is treated with a set of

restriction enzymes that cut it into smaller fragments. These collections of fragments are then

separated by gel electrophoresis, producing distinct bands visible by gel staining methods. A

visual comparison of the location of the bands derived from parental DNA and from children’s

DNA shows whether a mutation has occurred in

a site for a restriction enzyme. The presence of

a band in the child’s DNA that is not present in

the parents’ DNA, or vice versa, indicates a

nucleotide substitution or deletion restriction enzyme recognition site and suggests that a heritable mutation has occurred.
Ch. 4—New Technologies for Detecting Heritable Mutations ● 5 9

Figure 7.— Production of Restriction Fragment Length Polymorphisms Using Restriction Enzymes and

Separation of Different DNA Fragment Sizes by Agarose Gel Electrophoresis

Agarose sizing gel

electrophoresis

DNA I

DNA II

I

t t t

I

4 fragments result: c, b, d, and a

,

t t

Base change leads to

loss of restriction

enzyme site and

creates 3 fragments c, e, and a

t

= restriction enzyme site of cleavage

SOURCE: Off Ice of Technology Assessment

An alternative method bypasses the lambda

cloning procedure, and analyzes DNA fragments

directly. Fragments from the entire human genome are spread out on an electrophoretic gel,

forming smears of indistinguishable bands. In order to visualize the location of particular segments

of DNA and to evaluate their position relative to

the equivalent band in the reference sample, the

naturally double-stranded DNA is dissociated into

single-stranded DNA while still in the gel and allowed to reassociate with small pieces of specific

genes which are added to the gel and which are

radioactive (probes). Those original DNA sequences that pair with the probes are now tagged.

The DNA is then transferred to a flexible plastic

membrane conserving the spatial arrangement of

the DNA (a procedure known as Southern blotting) and autoradiographed; bands are detected

where the tagged sequences are present. As with

the cloning method above, the appearance of a

t

(-)

Larger

fragments

I

Direction

of

current

Smaller

fragments

(+)

DNA I DNA II

Positions of different

fragment sizes after

treatment with

restriction enzyme

new band or the disappearance of an old band

suggests a nucleotide substitution or deletion in

a recognition site of one of the restriction

enzymes.

One= Dimensional Denaturing Gradient

Gel Eiectrophoresis

A modification of the standard electrophoretic

gel procedure, proposed by Fischer and Lerman

(34), allows DNA to be separated not only on the

basis of size, but also on the basis of sequence

of nucleotides even if differences do not occur at

restriction enzyme recognition sites. Doublestranded DNA dissociates into single-stranded

DNA when it is heated or when it is exposed to

denaturing chemicals (e.g., formamide or urea).

A gradient of increasing strength of such chemicals can be produced in a gel so that DNA samples will travel in the direction of the electric cur-
.

60 • Technologies for Detecting Heritable Mutations in Human Beings

1

Figure 8.— Restriction Fragment Length

Mother’s DNA Father’s DNA

Polymorphism

Child’s DNA

L /

GENE CLONING METHOD / \

DIRECT (NONCLONING) METHOD

Viral

DNA

Fragments

of

viral

DNA

o3A ( - )

Electric

current

\

Separate fragments

restriction enzymes, cut DNA into double-stranded fragments of

2A Clone each fragment into the bacterial virus lambda, infect into E.co/i, grown in petri dishes, and allow the virus to

replicate within the bacteria. Isolate large quantities of the viral DNA, which now contains segments of human DNA, and

cleave it with restriction enzymes.

o3A Separate DNA fragments using agarose gel electrophoresis. Visualize bands corresponding to different sizes of

fragments using fluorescent staining. Fragments found in the child’s DNA samples and not in the parents’ DNA may contain heritable mutations. These bands can be removed and the DNA analyzed for specific mutations.

Direct (noncloning) Method:

o2B Apply samples of DNA fragments to an electrophoretic gel, producing smears of indistinguishable bands.

o3B Dissociate DNA fragments into single strands and incubate with radioactive, single-stranded, 32P-labeled DNA probes for

specific human genes. The probes hybridize with complementary sequences in the DNA and label their position in the gel.

Visualize the position of bands using autoradiography. Changes in the position of bands in the child’s DNA compared to

the parents’ DNA suggest possible new mutations. Bands can be isolated and DNA analyzed for sequence differences.

SOURCE: Office of Technology Assessment
Ch. 4—New Technologies for Detecting Heritable Mutations ● 6 1

rent, separating by size, and will also begin to

dissociate as they reach their particular critical

concentration of denaturing chemical. Dissociation causes the molecule to split into constituent

parts, or unravel, and get stuck in the pores of

the gel (84).

Every unique strand of DNA dissociates at a

unique concentration of denaturant. In fact, the

difference of only one nucleotide between two

otherwise identical strands of double-stranded

DNA of 250 nucleotides in length is enough to

cause the strands to dissociate at different concentrations of denaturant chemical, and to stop

traveling at different locations in the gel. Again,

a comparison between the banding pattern of parents’ and child’s DNA analyzed in this way may

identify a wide range of mutations in all regions

of the DNA.

In this method, total genomic DNA is isolated

from individuals and is heated to form singlestranded DNA (see fig. 9). It is then mixed with

radioactive, single-stranded DNA probes that correspond to a small portion of the genome, Following incubation so that double-stranded hybrid

molecules (“heteroduplexes”) will form, they are

treated with restriction enzymes and eIectrophoresed

in a denaturing gradient gel. The presence of a

single mismatch between a nucleotide in the “normal” probe and the corresponding segment from

a person bearing a mutation makes the heteroduplex slightly more sensitive to denaturing

chemicals so that such a molecule will stop traveling in the gel before the point at which a perfectly paired heteroduplex will stop. The gel is

dried and exposed to X-ray film, and the resulting autoradiogram is examined visually for differences in banding patterns between parents’ and

child’s DNA.

Two-Dimensional Denaturing Gradient

Gel Electrophoresis

Leonard Lerman has proposed a technique

whereby a two-dimensional separation of parents’

and child’s DNA is used to differentiate among

DNA sequences common to all three members,

polymorphisms in either parent which are transmitted to the child, and new mutations in the

child’s DNA (57). Like the two-dimensional poly -

acrylamide gel procedure for protein separation

described above, two-dimensional denaturing

gradient gel electrophoresis (2 DGGE) compares

locations of spots on a gel (in this case, DNA

spots) representing various types of mutations in

the nucleotide sequence in expressed and nonexpressed regions of the DNA. This method separates DNA fragments on the basis of differences

in base composition (or sequence), after separating them on the basis of differences in length.

2DGGE detects differences between DNA heteroduplexes by responses of their structure to gradual changes in denaturant concentration in the gel.

Genomic DNA from the parents in one sample

and from the parents and children in another

sample would first be treated with restriction enzymes and separated by size on separate agarose

gels in a single lane (see fig. 10). The gel strips

are then laid across the top of two denaturant gradient gels and the DNA is electrophoresed through

the increasing gradient of denaturant. Each gel is

then cut into horizontal slices, each of which contains pieces of the original homoduplex DNA of

all sizes, but including only those pieces that dissociated at the same denaturing concentration

(“isomelting” groups). These gel slices containing

homoduplexes of parents’ DNA or of parents’ and

child’s DNA are physically removed (or collected

on removable membranes) heated, and allowed

to reassociate as heteroduplex molecules (see fig.

lo).

In order to differentiate between common areas

of the DNA among parents and children, another

gradient denaturing gel is used to denature the

mixtures of double-stranded, isomelting DNA of

the parents (M1/Mz + F1/Fz) and another of the

parents’ DNA mixed with the child’s (M,/Mz2 +

F1/F2 +C1/Cz). Various double stranded combinations of parents’ and child’s DNA are produced

(e.g., M2/Cl, F1,/C2, etc.). The two sets of mixtures are compared in the final two gels.

Sequences that pair up perfectly represent common sequences to all three (level “i” in fig. 10).

At the bottom, a narrow region, dense with heteroduplex fragments, represents the largest fraction

of the sample—those that are perfectly matched

between base pairs and therefore are located at

the same denaturant concentration as the original homoduplexes contained in the narrow slice
62 . Technologies for Detecting Heritable Mutations in Human Beings —

Figure 9.—One-Dimensional Denaturing Gradient Gel Electrophoresis

Mother’s DNA Father’s DNA Child’s DNA

o1

/- I

\

I

i I 1

Treat with 1

/

1

I restriction

\

/

1

I enzymes

/

1 ~

&

,

I

i ‘, I

Treat with /’ \

I \ restriction

Dissociate and

reanneal with

32P-DNA probe

Dissociate and

reanneal with

32P-DNA probe

o2 Dissociate and

reanneal with

‘2P-DNA probe

I

Separate fragments

o3

Probe Father Mother Child

( - )

( + )

Increasing

concentration

of denaturant

Iisolate genomic DNA from white blood cells. Using restriction enzymes, cut DNA into (double-stranded) fragments of

- various lengths.

o2 Dissociate double-stranded DNA fragments into single strands, and reanneal in the presence of radioactive 32P-labeled,

single-stranded DNA probes. Heteroduplexes form between probe and sample DNA even if the base sequences are not

perfectly complementary; if mutations are present, some of these heteroduplexes will contain mismatches.

o3 Separate heteroduplex fragments in a denaturing gradient gel. Fragments with mismatches denature in a lower

concentration of denaturant than fragments that are perfectly complementary. Visualize the position of the fragments

with autoradiography. Fragments containing the child’s DNA that denature sooner than the parents’ fragments can be

analyzed for new mutations.

SOURCE: Office of Technology Assessment.
Ch. 4—New Technologies for Detecting Heritable Mutations • 63

01

02

03

Figure 10.—Two-Dimensional Denaturing Gradient Gel Electrophoresis

Mother’s DNA Father’s DNA Child’s DNA

o5

06

Increasing

o7 concentration

of denaturant

Increasing

concentration

of denaturant

(1)lsolate genomic DNA from white blood cells. Using restriction enzymes, cut DNA into double-stranded fragments Of

- various lengths.

o2 Mix mother’s and father’s DNA fragments (M1

/M, + F,/F,) in one set, and mix parents’ and child’s DNA fragments

(M,/M, + F,/F, + Cl

/C,) in another set.

o3 Separate fragments by length in an agarose sizing gel. Cut out a lengthwise strip of the gel, containing a range of fragment

sizes, and lay the strip across the top of a denaturing gradient gel.

o4 Separate fragments along an increasing concentration of denaturant. Cut out a slice of the gel at a narrow interval of the

temperature gradient, or collect a similar interval on a removable membrane.

o5 While maintaining spatial arrangement of fragments in the narrow slice, dissociate the double-stranded fragments into

single strands, and reanneal them to form heteroduplexes (e. g., M2

/C1, F,/CZ, F2/M,, etc.) of various combinations of strands

of the original homoduplexes (M,/Mz

, F1/Fz, and Cl/CZ).

o6 Lay the thin strip of heteroduplex fragments arranged by size across the top of a new gradient gel, and separate in the

direction of an increasing concentration of denaturants.

o7 Comparing the two final gels, the DNA spots are grouped in three distinct regions (see text for explanation). Analyze spots

in the highest region of the gel for DNA fragments with possible new mutations.

SOURCE: Office of Technology Assessment.
64 c Technologies for Detecting Heritable Mutations in Human Beings

removed from the gel or collected in the membrane.

Polymorphisms that are present in one parent

or the other show up as spots that denature earlier

than the perfectly matched DNAs (level “ii”). In

the middle region of the gel, a diffuse distribution of fragments represents heteroduplexes with

single mismatches between the two strands, causing them to dissociate earlier in the gel (at a lower

denaturant concentration, occurring above the

perfectly matched heteroduplexes). This region

would contain inherited polymorphisms as well

as new mutations, but the region would be too

dense with DNA spots to distinguish individual

spots.

Molecules that contain more than one polymorphism per fragment denature even earlier (level

“iii”) since multiple mismatches in a fragment have

an additive effect on the fragments stability in the

denaturing gradient. The highest area in the gel,

where heteroduplexes dissociate in the lowest

denaturant concentration, is where fragments with

more than one mismatch would be located. These

mismatches could be new mutations or inherited

polymorphisms. Since the number of such fragments in the sample is likely to be small, this region would show discrete, nonoverlapping spots;

DNA spots in this third level of the gel can be

removed from the gel and analyzed to identify

possible new heritable mutations. By comparing

the two final gels, new spots should be apparent

to a trained eye. These would represent single

base pair changes or very small deletions and rearrangements in all regions of the DNA.

In theory, it should be possible to cut many

strips of isomelting regions out of the first gel and

derive an analysis of each melting region by a series of second gels. By comparing the child’s DNA

to his or her parents’ DNA, this approach allows

for large portions of the genome to be examined

simultaneously.

Ribonuclease Cleavage of Mismatches

in RNA/DNA Heteroduplexes

A technically simpler approach to detecting mutations in cloned and genomic DNA has been proposed by Richard Myers (85,86). This technique

uses an enzyme, ribonuclease A (RNaseA), that

cleaves double-stranded RNA/ DNA heteroduplexes where a specific mismatch of base pairs occurs. RNaseA cleaves the RNA/ DNA molecule

where a cytosine (C) in RNA occurs opposite to

adenine (A) in DNA. (C normally pairs with G

[guanine] and A normally pairs with T [thymidine]. ) The idea behind this method is similar to

that of restriction enzymes, each of which cleaves

DNA at specific normal sequences, except that

RNaseA cleaves RNA/DNA hybrid molecules

where mismatches occur. The efficiency of this

approach depends on the number of different mismatches that can be recognized and cleaved. The

greater the number of enzymes that can be found

to cleave different mismatches, the more types of

mutations can be detected. This method should

detect nucleotide substitutions over a large portion of the DNA.

With this method, genomic DNA is first isolated and mixed with radioactively labeled RNA

probes (see fig. 11). The mixture is heated so that

the strands dissociate and then reassociate randomly with complementary strands. When RNA

molecules bind with DNA molecules of homologous sequences, the result is one type of “heteroduplex.” “Homologous” means that the sequence

is close enough between the two molecules that

they will form a stable double-stranded molecule.

The presence of a single base pair mismatch does

not prevent double-stranded heteroduplexes from

forming, however.

The heteroduplexes are treated with RNaseA

and separated according to size on a standard

agarose gel. Perfectly paired molecules will be unaffected by RNaseA and will form bands on the

gel that correspond to their original size. However, imperfectly paired molecules will be cut by

the enzyme at the site of the C:A mismatch, and

two separate fragments will result, showing up

as two separate bands on the gel. For analysis,

parents’ and child’s DNA are treated and electrophoresed separately, and the gels compared for

different patterns by autoradiography to reveal

the occurrence of any possible mismatches. Currently this method is applicable only for (RNA)C:

A(DNA) mismatches, which represent 1 of 12 possible types of mismatches between these heteroduplexes.
Ch. 4—New Technologies for Detecting Heritable Mutations ● 6 5

Figure 11 .—Ribonuclease Cleavage of Mismatches in RNA/DNA Heteroduplexes

o1 Mother’s DNA Father’s DNA Child’s DNA

Probe

o3

mismatch

cut into 2 pieces)

o4 ( - )

Electric

current

i

(+)

-

—

—

—
66 • Technologies for Detecting Heritable Mutations in Human Beings

Subtractive Hybridization

Detecting mutations would be much easier if

it were possible to ignore the millions of nucleotide sequences that are identical in both parents

and child and, instead, focus only on the relatively

few sequences that are different. Currently, the

best estimate for the frequency of human mutations is about 1 in 100 million base pairs, or about

30 per genome, so it is necessary to examine 3 billion base pairs from each of the parents and a child

and find those 30 that differ. George Church had

conceived of a method to find sequences in a

child’s DNA that are not present in either parent’s

DNA (25).

While there is no experience with Church’s proposed method, and therefore, no information

about its feasibility, it is a promising idea. The

basis of Church’s method is to view the human

genome as a group of unique nucleotide sequences. In this context, “unique sequence” means

a sequence that may occur only once in the entire genome. To explain what a unique sequence

is, consider the shortest and longest sequence in

the genome, a single nucleotide and the entire genome. A single nucleotide is not unique; A, T,

G, and C occur repeatedly throughout the genome. At the other extreme, the entire genome

is unique. Somewhere in between those extremes

is a length of nucleotides that is long enough to

be unique without being too long to handle experimentally.

It turns out that a sequence of 18 nucleotides

is long enough to be unique; on statistical

grounds, any sequence of that length or greater

that occurs once in the genome is not expected

to occur a second time. Therefore, if every possible sequence of 18 nucleotides, or “18-mer,” is

synthesized, this collection of 18-mers will include

sequences that together represent every possible

sequence of 18 nucleotides of DNA (there are 41

8

or 70 billion possibilities). Some proportion of

these are actually present in human sequences,

some are not. It is feasible to make the entire collection of 70 billion different 18-mers, and a single synthesis provides sufficient 18-mers for many

experiments.

To look for mutations, DNA from parents and

child is isolated and cut with restriction enzymes

into relatively small lengths of 40 to 200 base pairs

and dissociated into single strands (see fig. 12).

This parental DNA alone is mixed with the collection of 18-mers under conditions that permit

the formation of perfect hybrids: each nucleotide

sequence of an 18-mer hybridizes exactly with

each corresponding genomic sequence. The conditions are also such that an 18-mer that interacts with a genomic sequence complementary to

just 17 of the 18 nucleotides will not form a stable hybrid. When the hybridization reaction is

complete, the 18-mers that did not find a perfect

match with any parental DNA sequences are left

behind as single-stranded molecules, unchanged

by their participation in the hybridization reaction. Because they are single stranded, they can

be separated from all the other 18-mers that are

hybridized to parental DNA. This unbound fraction of the mixture is retained, while the fragments

of parental DNA that bind to the 18-mers are discarded.

This pool of single-stranded 18-mers which did

not bind to any parental DNA sequence is then

mixed with similarly prepared DNA from the

child, and hybridized under the same conditions.

This time, however, the molecules of interest are

the sequences that bind to the child’s DNA (and

not previously to the parents’ DNA). Any 18-mer

that hybridizes perfectly with a sequence in the

child’s DNA will identify a sequence that is present

in the child’s DNA and absent from the parents’

DNA. Such sequences may contain new mutations present in the child’s DNA. It should then

be possible to isolate and characterize this hybrid

formed between the 18-mer and child’s DNA, so

that the mutant sequence can be determined. If

mutations occur at a frequency of 1 per 100 million nucleotides, and if this technique works perfectly, it could detect 30 to 40 nucleotide sequences

in the child’s DNA that are not present in the parents’ DNA.

This approach is the least well developed of all

the ones discussed in this report, and its feasibility is unknown. If it does prove feasible, this approach could identify short sequences containing

mutations in any part of the DNA, allowing further detailed study (e.g., by DNA sequencing) of

the kinds of mutations that are occurring.
Ch. 4—New Technologies for Detecting Heritable Mutations • 67

Figure 12.—Subtractive Hybridization

Father’s DNA Child’s DNA

o2

Child’s DNA fragments

/

Separate and

discard bound

fragments

o3

04

05

Discard

unbound

fragments

o1 Isolate genomic DNA from parents’ and child’s white blood cells; mix parents’ DNA samples together, while keeping

child’s DNA separate. Cleave DNA into fragments with restriction enzymes.

o2 Dissociate parents’ DNA fragments into single strands, and reanneal with a set of unique single-stranded 18mer

sequences. 18mer sequences complementary to sequences in the parents’ DNA will form double-stranded, or bound,

fragments. The remainder, the unbound fraction, will remain single stranded.

o3 Separate the bound fraction from the unbound fraction. The bound fraction is discarded.

o4 Unbound 18mers for which no parental complement exists are allowed to hybridize with the child’s DNA fragments under

the same conditions of dissociation and reannealing. Any fragments of the child’s DNA that hybridize with unbound

fraction of 18mers represent sequences present in the child’s DNA that are not present in either parents’ DNA.

o5 Isolate these heteroduplexes and analyze them for specific new mutations.

SOURCE Off Ice of Technology Assessment
68 . Technologies for Detecting Heritable Mutations in Human Beings

Pulsed Field Gel Electrophoresis

If human DNA were short and simple, it could

be cutup with restriction enzymes and separated

electrophoretically into discrete bands, each representing a particular segment of the total DNA.

However, human DNA is so long that electrophoretically separated fragments make a continuous

smear of bands. If the DNA were cut in only a

few places to produce only 100 or 200 bands, the

pieces would be too big to pass individually

through the pores of a standard electrophoretic

gel. A new technique, pulsed field gel electrophoresis (PFGE) is being developed to allow separation of fragments of human DNA larger than can

be separated with other electrophoretic techniques

and to examine such fragments for evidence of

mutations. In theory, the procedure will detect

submicroscopic chromosome mutations, including rearrangements, deletions, breaks, and transpositions. At present, the method cannot handle

intact human chromosomes although it works

very well with smaller chromosomes from lower

organisms and with small fragments of human

chromosomes (157,158).

The standard process for isolating genomic

DNA randomly breaks the long molecules into

pieces. In the PFGE procedure, genomic DNA is

treated so that random breaks are avoided. Whole

cells are suspended in liquid agarose and after the

agarose solidifies into a gel, enzymes are added

to degrade proteins and RNA. This apparently

minimizes random damage to the high molecular weight DNA. DNA is cut into exact and predictable fragments by addition of a “rare cutter, ”

a restriction enzyme (or group of enzymes) that

cuts DNA consisting of 6 billion nucleotides, into

CONCLUSIONS

In a short period of time, enormous advances

have been made in the ability to “read” the genetic

material and to understand what it means. The

techniques described in this chapter are examples

of state-of-the-art molecular genetics. Some of the

components of these techniques were originally

developed for other purposes in genetic research,

while others were designed specifically for detectonly 3,OOO pieces averaging 2 million nucleotides

each (about 1/60 of an average chromosome)

(126).

Ten different fragments are each labeled with

a radioactive probe, so that each lane in the gel

will have 10 visible bands, the most that can be

analyzed per lane. The fragments are then separated on an agarose gel in which the electrical field

is applied alternately (“pulsed”) in perpendicular

directions for 24 to 72 hours. The pulse time is

adjusted, according to the particular size of the

fragments, to maximize separation of the fragments. The optimal pulse time is one in which the

fragments are constantly untangling and reorienting themselves. Autoradiography is the final step

in the procedure. Each of the 10 fragments bound

to a labeled probe appears as a visible band on

the gel on exposure to X-rays; chromosomal mutations would appear as a shift in the position of

the fragment containing the mutation.

Recent evidence suggests that fairly large pieces

of human chromosomes can be separated, although most of the experience with the technique

is limited to certain lower organisms such as yeasts

and unicellular parasites. Results of experiments

with DNA derived from these organisms, which

have much smaller genomes than humans, suggest that chromosome mutations would be apparent if they were at least 5 percent as large as the

fragment itself. The procedure would have to be

modified for human chromosomes due to their

larger size and complexity (127). This technique

may be useful in detecting chromosome mutations

that are intermediate in size between major rearrangements (observable by cytogenetic methods)

and single base pair changes.

ing new heritable mutations. They all are successes

in the sense that they propose reasonable and verifiable ways of examining human DNA for alterations in the nucleotide sequence. However, none

of the DNA-based techniques is approaching the

efficiency that would be needed for any of them

to be appropriate for field use. As these technologies develop and information is shared among
Ch. 4—New Technologies for Detecting Heritable Mutations ● 6 9

investigators, it may be possible to design combinations of methods that complement each other

and that select for those samples in which there

is a greater likelihood of finding new mutations.

In general, despite impressive achievements that

have been made, the technology remains too unwieldy for large-scale applications. Olson (98)

likens the current situation in mutation research

to the status of computer technology in the late

1950s, The spectacular improvement in computers

came not from scale-up of 1950s technology but

from development of the integrated circuit. He

suggests that big improvements in technology will

provide the appropriate methods; doing more of

what we have already done is unlikely to be

sufficient.

Analyzing complete sets of human DNA from

large numbers of people using these methods

would, at present, require an enormous commitment of resources. In considering the wisdom of

devoting major resources to such research, Olson

(98) emphasizes that the program’s ultimate goal

would have to be far more ambitious than the

detection of a few bona-fide examples of new mutations. A program large enough to have a legitimate impact on public policy about environmental exposures would have to be much larger

than a 5 or 10 person laboratory and be several

orders of magnitude more efficient than existing

restriction fragment length polymorphism technology.

One of the outstanding characteristics of current research in molecular genetics is the rapid

pace of new developments and ideas; assuming

this continues or accelerates, we can count on better methods to replace the proposed ones described in this report. The better ones should be

able to examine at least the major portions of the

genome and be practical in studying populations

at higher risk for heritable mutations. They should

also be able to detect a wide, if not the entire,

spectrum of mutational events.

Until the next generation of techniques is developed, the best course for the next few years

may be to maintain a balance between the support of current methods and the support of basic

research which is the source of better ones. Maintaining a balance implies that resource-intensive

efforts should not pull unwarranted amounts of

resources away from developing new techniques,

although no special scientific effort seems to be

needed to encourage this highly innovative and

rapidly paced area of research.
Chapter 5

New Methods for Measuring

Somatic Mutations
Contents

Page

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

Detection of Somatic Mutations in White Blood Cells . . . . . . . . . . . . . . . . . . . . . . . 74

Selection for HPRT Mutants in T-Lymphocytes. . . . . . . . . . . . . . . . . . . . . . . . . . . 74

Mutational Spectra From Human T-Lymphocyte HPRT Mutants,.. . . . . . . . . . 75

The hprt Gene. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

Results of Studies of hprt- T-lymphocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

Detection of Somatic Mutations in Red Blood Cells . . . . . . . . . . . . . . . . . . . . . . . . . 76

Glycophorin Somatic Cell Mutation Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76

Hemoglobin Somatic Cell Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78

TABLE

Table No. Page

7. Frequencies of hprt- T-Lymphocytes in Human Beings . . . . . . . . . . . . . . . . . . . . 76
Chapter 5

New Methods for Measuring

Somatic Mutations

INTRODUCTION

All the techniques for learning about heritable

mutations described in chapter 4 rely on comparing DNA or proteins of parents with DNA or proteins of their children to infer the kinds and rates

of mutations that occurred in parental germ cells.

While that information is of great value in learning about heritable mutations, it comes well after the mutations have actually occurred. Heritable mutation tests are not useful for determining

rapidly that an individual or a population has

been exposed to a mutagen, so that people who

have been exposed can be appropriately monitored and others, who have not been exposed, can

be warned and spared exposure to that same

mutagen. For this reason, it is desirable to develop

tests for somatic cell mutations that might allow

inferences to be made about possible heritable effects of some mutagenic exposure. Instead of providing an exhaustive catalog of somatic cell mutation assays, this chapter provides examples of

developing technologies that may soon be available to measure somatic mutations in human populations.

It is not known whether the rates and kinds of

somatic mutations in blood or any other nongerminal tissue have any predictable relationship

with rates and kinds of germinal mutations; it is

only with further development and testing of

those methods, together with methods for detecting germinal mutations, that such relationships

might be elucidated. However, even without

knowing the precise relationships between somatic

and germinal mutations, somatic tests may provide an early warning that people are being exposed to environmental mutagens. Furthermore,

identifying and characterizing somatic mutants

will provide important guidance for reducing exposures to mutagens and carcinogens, and is relevant to the study of aging and of carcinogenesis.

Exposure to mutagens may increase the risk of

developing some kinds of cancer. In addition, increases in the frequencies of second cancers occurring years after treatment of a first cancer with

radiation therapy have been noted (see e.g., ref.

11). In a study of survivors of cervical cancer, the

risk of developing a second cancer was found to

be highest for organs close to where radiation

therapy was delivered, strongly suggesting that

the development of second cancers maybe related

to the mutagenic effects of radiation (11).

Measurement of somatic mutants offers the possibility of identifying differences in somatic mutation rates over time within individuals, among

different individuals, and among different populations. Such studies can be used to examine possible associations between exposures to particular environmental or occupational agents and the

frequency of mutations. Ultimately, they maybe

used to identify individuals or populations who

are exposed to mutagens. This information could

be useful to the regulatory agencies in identifying genotoxic compounds and their potencies.

To be useful for studying human populations,

somatic tests must be based on reasonably accessible cells (e.g., blood cells) and on recognizable

markers for mutation (e. g., variant proteins or

enzymes). The most easily sampled human tissue

is blood, and thus far, all the somatic tests that

have been developed for studying mutations have

used red and white blood cells. Somatic cell

mutation assays focus on two types of cellular

changes that result from mutations: 1) alterations

in phenotypic properties of cells, such as changes

in cells’ resistance to drugs added to their growth

media; l and 2) alteration in a gene product, such

‘Studying drug resistance in humans is inherently limited to studying single copy genes, such as genes on the X chromosome (including the hprt gene) or dominant selectable markers.

73
74 . Technologies for Detecting Heritable Mutations in Human Beings

as a protein. The first type of change has been bers of mostly identical normal cells of a specific

most extensively studied using the mutation as- type. Although somatic cell mutation assays desay based on 6-thioguanine (6-TG) resistance, and tect phenotypic events that represent, as closely

the second type has been studied using two types as possible, actual mutations in DNA, the data

of proteins found in circulating red blood cells. are expressed as numbers of mutants, rather than

Instead of examining DNA directly, current and in numbers of mutations. Various analytic techdeveloping somatic tests detect rare mutant cells niques have been used to estimate the mutation

based on marker phenotypes among large num- frequency from the observed mutant frequency (97).

DETECTION OF SOMATIC MUTATIONS IN WHITE BLOOD CELLS

Albertini and his colleagues (3) and Morley and

his colleagues (79) have developed assays using

human peripheral blood lymphocytes to detect

mutations that result in a phenotypic change in

these cells. The assays are based on the ability of

mutant T-lymphocytes, which are present in the

peripheral blood at the time the sample is drawn,

to replicate DNA and to grow under conditions

that do not allow growth of normal cells. The mutant cells are identified by their resistance to

6-TG, a drug that is normally toxic to human

T-lymphocytes.

Normal cells produce an enzyme, hypoxanthineguanine phosphoribosyl transferase (HPRT), that

metabolizes 6-TG to a toxic chemical, killing the

cells. Mutations in the hprt gene (the gene that

directs the production of HPRT), can result in cells

that do not produce HPRT. The mutation from

the normal gene (hprt +

) to the mutant gene

(hprt-) makes the cell resistant to 6-TG (6-TG’)

because they cannot metabolize 6-TG into its toxic

form.

This particular selective system has been shown

to meet all the necessary criteria for a valid genetic

assay: 1) the 6-TGr mutants are genetic variants

that “breed true”: whether grown with or without 6-TG, they remain resistant to 6-TG when

challenged with the drug (3,79); 2) exposure of

laboratory cultures of T-lymphocytes to known

mutagenic agents such as X-rays or ultraviolet radiation increases the frequency of 6-TGr mutants,

indicating an increase in mutation in these cells

(17,160); 3) there is a demonstrable change in the

gene product—a deficiency in the enzyme HPRT;

and 4> there is a demonstrable change in the DNA

sequence of the hprt gene—a direct demonstration that the hprt gene has mutated (4,143).

Selection for HPRT Mutants in

T= Lymphocytes

Nicklas (97) described two methods for identifying mutant T-lymphocytes in human blood

samples. The first is the autoradiographic assay.

After blood is drawn, the T-lymphocytes are purified and frozen. On thawing, the cells are split

into two cultures, both of which are treated with

phytohemagglutinin, a chemical that stimulates

DNA synthesis. One of the cultures is also treated

with 6-TG. The other culture is an untreated

control.

A label, radioactive thymidine, is added to both

cultures, and all cells capable of DNA replication

and growth incorporate the labeled thymidine into

their DNA. In the the control culture not exposed

to 6-TG, essentially all the cells divide and incorporate thymidine, In the culture exposed to 6-TG,

normal cells are killed and therefore do not incorporate the radioactive thymidine into their

DNA. The cells from both cultures are then fixed

on microscope slides, covered with an X-ray film

or emulsion, and then developed using standard

radiographic techniques. Cells that have incorporated radioactive thymidine emit radiation, causing a black, exposed spot on the developed film.

The developed films are then viewed under a

microscope. This technique is quite sensitive;

radioactive cells can be counted at a rate of 2 per

10,000 to 1 per 10 million cells. The procedure

can also be automated to make it relatively effi-
Ch. 5—New Methods for Measuring Somatic Mutations ● 7 5

cient for identifying rare mutants. Dividing the

fraction of radioactive cells in the 6-TG-exposed

culture by the fraction in the culture not exposed

to the drug produces an estimate of the frequency

of 6-TG-resistant mutants.

The second method, called the clonal assay,

selects for cells that are resistant to 6-TG. White

blood cells are dispensed in small test tubes or

wells in a small plastic device resembling a tiny

muffin tin, each test tube or well containing 6-

TG in the growth medium. Only the mutant 6-

TG-resistant cells grow and divide, forming visible colonies within 10 to 14 days. These colonies

can then be analyzed further by isolating their

DNA and analyzing it for mutations. The clonal

assay allows characterization of mutations found

in these cells, whereas the autoradiographic

method permits only determination of their frequency.

Mutational Spectra From Human

T= Lymphocyte HPRT Mutants

Cariello (16) described a technique for analysis

of the hprt gene that may correlate mutational

patterns with particular mutagenic exposures. In

this method, all of the mutant hprt- T-lymphocytes in a small blood sample are isolated and

grown in culture for 2 weeks to produce enough

DNA for analysis. DNA of these clones of different hprt- mutants is then examined by first cutting it into fragments with restriction enzymes,

adding a group of different DNA probes and separating DNA bound to the probes by gradient gel

electrophoresis. DNA with different mutations

will bind to different probes. The pattern of mutations in the original blood sample appears as

the pattern of DNA probe positions on a gel.

Different mutagens are known to produce clearly

different patterns of mutation in human as well

as bacterial cells, so the aim of this technique is

to identify the probable causes of mutations in

human blood cell samples by using these patterns,

or “mutational spectra” (139). This approach includes studies of the pattern of spontaneous mutation in human T-lymphocyte samples as well

as studies of the mutational spectra produced by

potentially mutagenic chemicals to which human

beings may be exposed. The goal of this approach

is to “fingerprint” for the presence of a particular

mutagen in an individual’s life history.

The hprt Gene

One of the advantages of studying the hprt gene

is that much has been learned about it from studies

of a particular genetic disease. Heritable mutations

in the hprt gene located on the X chromosome lead

to Lesch-Nyhan syndrome, a severely debilitating disorder characterized by mental retardation

and self-mutilation. The hprt gene has been isolated from blood samples taken from patients with

Lesch-Nyhan syndrome, and the mutations in the

hprt gene have been studied. Comparisons have

been made between the types of mutations present

in cells of Lesch-Nyhan patients and those present

in rare, mutant hprt- T-lymphocytes selected

from blood samples of people without the disease.

DNA from 5 of 28 Lesch-Nyhan patients were

found to have major hprt gene alterations—

deletions of DNA, amplification of DNA, and detectable changes in DNA sequence (172). Studies

of mutant T-lymphocytes selected from normal

blood samples have shown similar gene alterations

in several cases (4,143). These results show that

the hprt- T-lymphocyte assay detects spontaneous mutations in the hprt gene in somatic cells

that are qualitatively similar to clinically apparent heritable mutations in humans, and also demonstrates that the clonal assay in T-lymphocytes

detects true mutations, not phenotypic changes

that mimic mutations.

Results of Studies of

hprt - T-lymphocytes

A number of studies have measured frequencies of T-lymphocytes resistant to 6-TG in normal individuals. Average mutant frequencies for

the groups of people tested fall within a fifteenfold range, between about 1 and 15 mutations per

million cells (table 7). However, mutant frequencies for different individuals vary widely. For instance, Albertini reports a range of frequencies

from 0.4 to 42 mutants per million T-lymphocytes

from 23 individuals, representing a hundredfold

range in somatic mutant cell frequencies among

a small number of people (2). This wide range in
76 ● Technologies for Detecting Heritable Mutations in Human Beings

Table 7.—Frequencies of hprt - T-Lymphocytes

in Human Beings

mutant frequencies may be due to different ages

and other characteristics of the T-lymphocyte

donors. Two investigators have shown that there

is a linear increase of hprt- mutants in T-lymphocytes with increasing age (141,160). Donor

characteristics, such as smoking behavior, have

been shown to increase somatic mutant frequency;

genetic variation in sensitivity to mutagens may

also exist among individuals.

The degree to which the observed frequency of

mutants reflects the actual frequency of somatic

mutations in this assay is not fully understood.

Descendants of the original mutant cells increase

the observed frequency, so growth of mutants in

vivo could falsely lead to a conclusion of higher

mutation rates than is actually the case, unless the

data are corrected for this problem. In addition,

hprt- cells maybe shorter-lived than hprt+ cells.

Before the technique can be used to estimate possible increases in somatic mutation rates from

environmental exposures, the wide range of background rates of these mutants has to be investigated, including discrimination between true genetic events and any possible phenocopies. While

imprecision of the assay may contribute to this

wide range of mutant frequencies, such a range

is not unexpected for genetically and geographically diverse human populations.

DETECTION OF SOMATIC MUTATIONS IN RED BLOOD CELLS

Unlike white blood cells, mature red blood cells

have no nuclei and contain no DNA. They cannot be grown in the laboratory since they do not

have the capacity to replicate. Red blood cells can,

however, be used to detect mutations expressed

in altered proteins on their surface. Red blood cells

are also easily obtained from a blood sample.

There are about 1 billion red blood cells per milliliter of whole blood. Branscomb (13) described

two methods to detect variant red cells, using

high-speed automated microscopy and flow cytometry to analyze and sort hundreds of red blood

cells per second to detect rare red cell mutants.

These two methods are described below.

Glycophorin Somatic Cell

Mutation Assay

The glycophorin assay detects gene-loss mutations expressed by the absence of cellular proteins

corresponding to those genes. Such mutations can

be detected where two or more variants of a particular protein normally exist, where the genes for

the alternative forms are said to be codominant

(both variants are expressed when a gene for each

is present), and where different variants are expressed in the same cell. A mutation causing the

loss of function of the gene for one of the variant

forms results in a cell that expresses only the other

form of the protein (9,10,5o).

One such protein is glycophorin A, a glycoprotein present on the surface of red blood cells. Two

variants of glycophorin A normally exist, called

“M” and “N,” which differ from each other by

2 out of a total of 131 amino acids. The M and

N serotypes have no known biological function,

and each allele functions independently of the

other. The M and N variants of glycophorin A

can be labeled independently using monoclinal

antibodies carrying different fluorescing molecules. As a result of the labeling, the M variant

appears green and the N appears red when viewed

under a fluorescent microscope. A flow cytometer, a machine through which cells are passed at
Ch. 5—New Methods for Measuring Somatic Mutations ● 7 7

very high speed, can sort and count the number

of cells that are green, containing only the M variant, and the number that are red, containing only

the N variant.

Individuals whose red cells display both variants of glycophorin are heterozygous for the M

and N alleles (i. e., they inherited an M gene from

one parent and an N gene from the other). As used

currently, this assay detects gene loss mutations

at glycophorin genes in individuals who are heterozygous for the M and N variants. Red blood

cells from heterozygotes show both red and green

fluorescence; if a mutation inactivates one of the

glycophorin A alleles, only the other variant

would be present on the cell surface, hence only

one color would appear. Single-color cells are recognized and distinguished from the majority of

double-colored cells, and are counted as somatic

mutant cells.

The glycophorin A assay is currently being used

at the Lawrence Livermore Laboratory to study

mutations in two populations: cancer patients’

blood cells examined before and after treatment

with known doses of chemotherapeutic drugs, and

Japanese atomic bomb survivors whose dose of

radiation can be estimated (69).

Hemoglobin Somatic Cell Assay

The frequency of particular hemoglobin variants that exist at low frequencies in red blood cells

of normal individuals can be measured. Cells containing these variants presumably arise in bone

marrow stem cells, the precursors of red blood

cells. Methods have been developed using monoclonal antibodies, prepared for each type of variant, to label particular mutant hemoglobin molecules. This method detects small mutations, such

as single nucleotide changes resulting in amino

acid substitutions, frame shift mutations, etc., in

the gene for beta-globin, a constituent of hemoglobin.

Investigators at the University of Washington

(132,133) and at Lawrence Livermore National

Laboratory (50) have developed an approach to

detect hemoglobin mutations in red blood cells.

In this technique, an antibody to the mutant hemoglobin is bound to a chemical called a fluorochrome, which is then mixed with red cells in suspension. The fluorochrome emits fluorescent light

of a specific color when light of a particular wavelength is shone on it. The cells are screened under

a microscope that allows the investigator to direct light of a particular wavelength on the sample. If any of the cells contain the mutant hemoglobin bound to fluorescing antibody, they can

be viewed and counted.

Stamatoyannopoulos and his colleagues have

used this technique on blood samples from 15 individuals using antibodies specific for Hemoglobin S (the mutant form of hemoglobin present in

sickle cell anemia) and Hemoglobin C. An average of 50 million cells per individual was screened,

and an average mutant frequency of 1.1 in 10 million cells per subject was obtained, with a range

of 4 in 100 million to 3 in 10 million. These investigators also screened the red blood cells of 10

individuals who had been exposed to known

mutagens—X-rays or mutagenic drugs used to

treat cancer. The frequencies of mutant hemoglobins obtained from these samples were within

the range found for normal individuals.

This technique is labor-intensive because it relies on visual screening of the cells: it takes one

technician nearly 1 month to screen 100 million

red cells. The investigators have since collaborated

with Dr. Mendelssohn and his associates at the

Lawrence Livermore Laboratory to use their automated fluorescence-activated cell sorter to screen

the cells. Although some technical difficulties were

encountered with this instrument, results obtained

on red cells from six normal individuals (on

screening 500 million cells per individual) yielded

an average mutant frequency of 1 in 10 million,

about the same as the average frequency determined from visually counted cells.

With an array of different mutant hemoglobins

to use for production of antibodies, a variety of

mutations can be detected in hemoglobins found

in rare cells present in normal individuals: single

nucleotide substitutions, deletions, frame-shift

mutations, or virtually any mutation that causes

the production of mutant hemoglobins.

There are currently two technical problems with

this technique. First, a significant number of false

positives are generated. Better methods of fixa-
78 . Technologies for Detecting Heritable Mutations in Human Beings

tion and antibody staining of the hemoglobins are reported that with present capabilities using flow

necessary, along with verification of each vari- sorting, 88 antibody-labeled red cells can be idenant found. Second, the method is not yet fully tified in a mixture that contains a known quanautomated, but research in several laboratories tit y of 100 such labeled red cells and 1 billion unis progressing toward full automation. Significant labeled red cells, a relatively good retrieval rate.

progress has already been made; Mendelson (68)

CONCLUSIONS

The methods to detect and quantify somatic

mutations suggest some additional approaches for

studying the nature and mechanisms of mutation

in human beings. Somatic tests offer the possibility of drawing associations between exposures

to specific mutagens and particular genetic events

in the cell, and they may help to identify individuals and populations at high risk for mutations.

They may lead to understanding the relationships

between the frequency and nature of somatic and

germinal mutations, and if those relationships can

be identified, somatic tests may contribute significantly to understanding why human heritable

mutations occur and may help predict their future occurrence. At present, however, it is not

possible to directly extrapolate from mutations

in somatic cells to risks of heritable mutations in

offspring.

Animal studies can be used to measure quantitatively the correlations between exposure to

mutagens and rates of somatic mutation, as well

as to investigate possible associations between somatic and heritable mutations. Coordinated tests

of somatic and germinal mutations in experimental animals may help guide such research in

humans. Such data, combined with data on human somatic mutation rates, may suggest specific

risks of heritable mutations in human populations.

In this regard, the tests for somatic mutation

described in this report appear promising. None

of these methods is currently ready for application to human population studies, however, even

though many of the technical details have been

worked out. Since the assays do not detect DNA

mutations directly, it is particularly important to

verify the results from these methods by isolating variant cells and confirming that the altered

proteins do indeed reflect primary changes in the

genes coding for these proteins. It is also necessary, although more difficult, to determine how

often these methods miss the mutations they are

designed to detect,

In general, the most useful somatic cell mutation assay may be one that detects a spectrum of

mutations (single amino acid substitutions to large

deletions and translocations) and that can detect

mutations in different cell types. Different tissues

may demonstrate different frequencies of somatic

mutants, and equally as important, some types

of mutant cells may have longer lifespans in the

body than others. Consideration of such characteristics could be useful in choosing the most

appropriate assay for particular circumstances.

Short-lived mutants could be useful in studying

the effects of short-term exposures to potential

mutagens, while longer lived mutants could be

better indicators of mutagenic effects of long-term,

chronic exposure to environmental mutagens.

The genetic analysis of white blood cell DNA

offers the possibility of identifying genetic “fingerprints, ” or particular patterns of mutations induced by specific mutagenic agents. Such patterns

are an essential first step toward understanding

the molecular mechanisms of genetic change in

human DNA,
Chapter 6

Laboratory Determination of

Heritable Mutation Rates
Introduction , . . . . . . . . . . . . . . . . . ., . :. ..,..... . . . . . . . . . . . . . . . . . . . . . . . . . . .

Rodent Tests for Heritable Mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

The Dominant Lethal Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

The Heritable Translocation Test . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Specific Locus Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Other Studies in Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Factors That Influence Induced Mutation Rates in Mice . . . . . . . . . . . . . . . . . . . . . .

Effects in Males . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Effects in Females . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Estimating Effects in Human Beings From Animal Data . . . . . . . . . . . . . . . . . . . . . .

81

81

81

82

82

83

84

84

85

86

TABLE

Table No. page

8. Spontaneous Mutation Rates in Mice. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

FIGURE

Figure No. Page

13. Spermatogonial Mutation Rates in Mice After Low-Dose Rate and

High-Dose Rate Radiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Chapter 6

Laboratory Determination of

Heritable Mutation Rates

INTRODUCTION

The genetic information of all organisms is encoded in DNA (with the exception of some viruses

that depend on RNA), and mutations have been

found in all species that have been examined. The

biological similarity of DNA structure and function in all species has allowed scientists to study

mutation rates and mechanisms of mutagenesis

in several species for over half a century. In addition to these efforts in basic research, in recent

years the biotechnology industry has provided

impetus, including financial incentives, for sophisticated inquiry into mutations and mutagenesis

in bacteria and yeast. The industry’s interest is

in using mutagenesis to benefit from some of the

particularly useful characteristics of certain microorganisms, such as the ability of some bacteria

to digest oil, and the fermentation capabilities of

various yeasts.

Another reason for studying mutations in lower

organisms is to identify agents that cause mutations in their DNA, agents that might act similarly on human DNA. For instance, concern about

human risks from the genetic effects of ionizing

radiation led to the funding of extensive mouse

studies, which have provided quantitative data

on factors that influence the yield of various types

of heritable mutations. In the case of chemicals,

however, concern about cancer, not about heritable mutations, has been the primary motivation

for developing short-term laboratory test systems

to detect mutations. In the last decade, as more

and more evidence incriminates somatic mutation

as an early event in the development of cancer,

the properties of mutagenicity and carcinogenicity have been conjoined, making identification of

mutagens an important part of efforts to identify

cancer-causing agents. Many of the tests for mutagens measure a chemical’s capacity to alter the

DNA of bacteria or yeast (145). While such a

short-term test in a lower organism may distinguish between agents that can and cannot change

DNA, it provides no information for making

quantitative estimates of mutagenic potency in humans nor does it show that the chemical can necessarily reach animal or human gonads in an active form to cause heritable mutations (108).

Whole animal tests specifically designed to detect

germ-cell or heritable mutations provide some information of that type.

RODENT TESTS FOR HERITABLE MUTATIONS

At least 15 tests and variations on them are The Dominant Lethal Test available for the detection and measurement of

heritable mutations induced in mammalian germ The dominant lethal test detects mutations that

cells. Lower organisms, notably Drosophila, are cause the death of rodent embryos. The usual prothe subjects of similar tests, but most of the in- cedure in this test is to treat male mice with a

formation about mutation rates comes from mice. suspected mutagen and then mate them with unThe three most widely used rodent tests are de- treated females. If the treatment causes a mutascribed below (58,108). tion in a male germ cell that is lethal at some stage

81
82 . Technologies for Detecting Heritable Mutations in Human Beings

in the development of the embryo (after fertilization), the lethal events are detectable by dissecting female uteri a few days after implantation of

embryos (implantation occurs about nine days after fertilization). If there are more dead embryos

than expected (compared with matings of unexposed mice) and/or more implantation sites than

expected in the uterus where there is no longer

an embryo, the effect is assumed to be the result

of a mutation in the male germ cell. Chromosomal

studies in mice have shown that almost all “dominant lethal” events are associated with major

chromosome abnormalities.

Human analogs of the postimplantation fetal

losses counted in dominant lethal tests are spontaneous abortions (miscarriages). Fifty to 60 percent of spontaneous abortions are associated with

numerical and structural chromosome abnormalities, the same basic types of abnormalities detected by dominant lethal tests (12). These similarities between mice and men, and the general

pragmatic acceptance of higher animals as predictors for human risk, underlie the idea that agents

that are positive in a mouse dominant lethal test

may also cause spontaneous abortions or chromosome abnormalities in liveborn infants.

The Heritable Translocation Test

The heritable translocation test detects breakage

and rejoining of fragments from different chromosomes. When a translocation involves no loss

of genetic information (a “balanced translocation”), often it has no adverse effect on the functioning of the carrier individual. The unusual

chromosomes produced by the breaking and rejoining are present in all cells, and they can pair

with each other and divide and function like normal chromosomes. However, such translocations

cause reproductive problems because the germ

cells may have incomplete sets of genetic material and may lead to unbalanced chromosome abnormalities in offspring, often resulting in death

during embryonic or fetal development. Heritable translocations are thus detectable in mice because they result in reduced litter size in the second generation after exposure to the suspect agent,

or in sterility in the first generation. In addition,

cytogenetic studies can detect abnormal chromosomes and can be used to study the association

between reduced fertility and chromosome abnormalities.

Specific Locus Tests

Since 1927, when Herman Muller first demonstrated that radiation causes mutations in fruit flies

(8 1 ), there has been concern about possible genetic

effects of radiation in human beings. Following

World War II, in anticipation of widespread use

of nuclear power, the Atomic Energy Commission

(AEC) contracted for research on the mutagenic

effects of radiation in mice to provide an experimental underpinning for estimating effects in

human beings, Specific locus tests that detect mutations in a few specific genes in mice were developed for that purpose. This work and use of

specific locus tests to measure chemically-induced

mutations have provided the animal data most

useful for estimating human risk.

Through research supported by the AEC, William L. Russell (109) bred a strain of “tester” mice

that differs from wild-type mice in 7 morphological features—coat colors, patterns of pigmentation, eye color, and ear shape—that are recessive traits. Each of the features is thought to be

controlled by a different genetic locus. Tester mice

are homozygous for the recessive alleles of these

genes, while wild-type mice are homozygous for

the dominant alleles. When the tester strain is

mated to a strain of wild-type mice, the offspring

should all be heterozygous at the seven test loci,

and therefore they should appear wild-type because the the wild-type alleles are associated with

the dominant phenotype. However, if a germinal

mutation occurs in a gene for one of the seven

loci in the wild-type strain, causing the dominant

allele not to be expressed, the offspring from that

mutation will have a phenotype characteristic of

the tester strain.

In a specific locus test, the wild-type mouse is

exposed to radiation or to a known or suspected

chemical mutagen and then mated to the tester

strain. In most cases, only male wild-type mice

are exposed, and the tester strain mice are female.

The reason for this is largely a practical one: one

exposed male can sire a large number of progeny
Ch. 6—Laboratory Determination of Heritable Mutation Rates . 83

when mated to many females, but an exposed female can have only one litter at a time. In addition, the information so far available suggests that

male germ cells are more sensitive to mutagens

than are female germ cells. After mating exposed

wild-type and tester mice, the offspring are examined for the easily seen morphological features

related to the seven loci. Since the morphologic

variants are readily identified, large numbers of

offspring can be examined quickly by an investigator. The frequency of mutant offspring of exposed parents is compared with offspring of unexposed parents. The importance of the ease of

recognizing mutants is underscored by the low frequency at which mutations occur spontaneously

—as few as 50 mutations per 1 million offspring

of unexposed mice. (Because mutations can be detected at any of the seven loci, examination of one

mouse provides information about one observation at each of the seven loci. )

Estimates for the rate of spontaneous mutations

in mice based on specific locus test results are summarized in table 8. These data are based on examining almost 1 million progeny of exposed mice,

or about 7 million loci. Though the reliability of

the estimate is limited by the small number of mutations seen, the calculated rate of between two

and eight mutations per 1 million genes is not inconsistent with the human rate estimated by onedimensional electrophoresis of blood proteins (see

ch. 3).

Although the “average” rate of mutations is a

useful touchstone, it is used in the full knowledge

that the average includes very different rates at

the seven loci. For instance, there is a 35-fold

difference in radiation-induced mutation rates between the most and least sensitive of these particular seven loci. The choice of a different set of

loci for examination might yield quite different

results.

Other Studies in Animals

There is a family of animal experiments called

dominant mutation tests, which use the appearance of dominant features, such as skeletal

anomalies or cataracts, to detect mutations (108).

Many of the endpoints used resemble known human genetic disorders, and the results of these tests

have been used to estimate human genetic risk.

In addition to the types of studies described

above, some of the techniques used to study human beings have been applied to experimental animals. Electrophoresis of blood proteins and quantitative enzyme assays in human beings, described

in chapter 3, have been used to study corresponding proteins in mice. The electrophoretic approach

has been used in studies of mutagenized mice at

the National Insitute of Environmental Health Sciences (51,52,53,63) and also at the Institute for

Genetics, Neuherberg, Germany (19,19a). A small

effort in electrophoretic analysis has also been

conducted at the Oak Ridge National Laboratory

(107). Similar studies have been reported in Drosophila (80,103,140,161).

Mutagenized mice have been tested for enzyme

deficiency variants using quantitative enzyme

assay techniques similar to those used to study

human populations (19a,73). Enzyme assays have

also been used in mouse and Drosophila experiments that involve selective mating, similar to the

specific locus test strategy. By selecting parental

strains that have electrophoretically distinct alleles at a locus, enzyme deficiency variants can

be detected in offspring by the absence of one

activity staining band that, in the absence of a

Table 8.—Spontaneous Mutation Rates in Micea

Number of Number of

Sex mutants progeny a Mutations/locus b Reference

Female ., . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 or 8c

204,639 2.1 or 5.6 x 10-8 (1 10)

Male ... , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 727,319 7.6 X 1 0

- 6

(1 13)

aDala about spontaneous mutat!on rates were collected from animals used as controls in experiments to determine the effects of radiation or chemical agents on

males and females separately, There are more progeny from males because more experiments have been done in males

bA rate of I x IO-6 means that, on average, a mutation occurs once In a million loci

cThe choice of three or eight mutants exemplifies a problem tn counting mutations Three I itters from female mice had mutant Off Spring TWO I itters had one mutant

each, the third had SIX mutants, all of which may have resulted from a s!ngle mutation The result is three probable mutations and eight mutant of fspr!ng

SOURCE Office of Technology Assessment
84 • Technologies for Detecting Heritable Mutations in Human Beings

mutation, would be inherited from one of the par- in the mouse derived from these experimental data

ents (51,52,103,161). is in the same range as the estimate of the sponData from animal experiments using electro- taneous rate derived from studies in human bephoretic and quantitative enzyme assays are lim- ings (77), recognizing, however, that these estiited, but at this stage a general, cautious conclu- mates are based on small numbers and may be

heavily influenced by chance fluctuations. sion can be drawn. The background mutation rate

FACTORS THAT INFLUENCE INDUCED MUTATION RATES IN MICE

When radiation or a chemical is tested for mutagenicity, the experiment does not necessarily

produce definitive answers to the questions: “Is

this agent mutagenic’?” And “if so, how mutagenic

is it?” The answers differ depending on several

factors, two important ones being whether the

animal is male or female and the stage of development of the exposed germ cell. There is ample

evidence from radiation studies that male and female germ cells differ in their sensitivity to mutagens. In males, germ cells appear to be more likely

to sustain a mutation the further along the developmental path they are. Overall, female germ cells

appear to be more resistant to mutagens than are

male germ cells.

Effects in Males

The male testis, from puberty on, contains a

mixture of germ cells at different developmental

stages, including spermatogonial stem cells, differentiating spermatogonia, and intermediate

stage cells, the spermatocytes and spermatids,

which are maturing into spermatozoa. The mechanisms of sperm production are qualitatively very

similar in mice and men, differing largely in timing: in mice, sperm production from spermatogonia to spermatozoa takes 35 days; in men, it takes

74 days (60). When a male is irradiated, germ cells

at all stages of development are exposed. By varying the times of mating, the effects of radiation

on different stages can be examined. For instance,

if male mice are irradiated and mated immediately, any mutants that appear in the progeny

must result from mutations that were caused in

fully mature sperm. In a mating two weeks after

exposure, eggs are fertilized by spermatozoa that

were spermatids at the time of irradiation. If seven

or more weeks are allowed to pass, sperm in the

ejaculate would have been irradiated at the spermatogonial stem-cell stage. Experimental evidences

shows that cells at each stage have varying sensitivities to induction of mutations by radiation, the

later, postspermatogonial stages being most sensitive.

While spermatogonial stem cells are more resistant to the effects of mutagens than are germ cells

in later stages of development, mutations in stem

cells are of greater concern. Stem cells persist

throughout the lives of males, constantly giving

rise to new generations of sperm cells. A mutation in stem cell DNA results in every cell that

buds off from the stem cell bearing a mutation.

The lifespan of later germ-cell stages constitutes

only a small fraction of the total reproductive

lifespan (especially in humans). Thus, in the case

of acute exposure to these later germ-cell stages,

only conceptions occurring during a short postexposure interval are at risk. For these reasons, the

effects of radiation and some chemicals have been

especially closely studied in spermatogonial stem

cells in animals.

The most common way for people to be exposed to ionizing radiation is through acute exposure to medical X-rays, or long-term, low-level

exposure to natural background radiation. Certain groups are exposed occupationally (e.g., uranium miners, nuclear powerplant workers, some

medical personnel); and some may have special

low-level environmental exposures from living

near nuclear installations, nuclear waste sites, uranium mine tailings, or areas of high natural radon concentrations.

in mice, exposure of males to X-rays or other

forms of ionizing radiation at low dose rates

causes a measurable increase in the number of

mutations, while such exposures do not measura-
Ch. 6—Laboratory Determination of Heritable Mutation Rates ● 85

bly increase mutation rates in females. Russell

and Kelly (113) counted the numbers of mutants

caused by varying doses of radiation in male mice.

They find no evidence for a dose so low that it

is not mutagenic, but the number of mutations

decreases with lower and lower doses (see fig. 13).

The data in the figure also show the differing results of exposure at high and low dose rates. For

example, the number of mutations caused by a

total dose of 300 roentgens (R), I administered at

a dose rate of between 72 and 90 R per minute

is about 80 X 10-6 per locus; the same total dose

administered at dose rates of 0.08 R per minute

or lower causes fewer mutations, about 30 X

10-’ per locus. These data may be interpreted

by saying that low dose rates allow time for repair of some of the damage caused by the radiation. (For further discussion of dose and dose-rate

relationships, see ch. 7.)

Effects in Females

Female mammals differ from males in the biology of germinal stem cells. Whereas male spermatogonial stem cells divide constantly during

adulthood, females are born with their total complement of egg precursor cells. Shortly after birth,

all cells destined to become eggs (ova) are present

in the ovaries as “arrested oocytes. ” Exposure of

arrested oocytes to radiation has caused no increase in mutation rates in mice (110). During the

six weeks before ovulation an arrested oocyte matures, undergoing meiosis and, in a mature state,

travels from the ovary to the uterus. Irradiation

during this maturation period does increase mutation rates in female germ cells.

Two different explanations can be advanced for

the failure to recover mutations from irradiated

arrested oocytes. First, it might be that arrested

oocyte DNA is somehow protected from the mutagenic effects of radiation, perhaps by having

very efficient repair mechanisms that correct

radiation-induced mutations. On the other hand,

in mice, arrested oocytes are very sensitive to the

killing effects of radiation (27). These lethal effects could also account for the absence of mutagenic effects in female mice. It is argued that since

1A measure of radiation. In human terms, a chest X-ray examination produces a gonadal radiation dose of less than 0.0005 R (148).

Figure 13.—Spermatogonial Mutation Rates in Mice

After Low-Dose Rate and High-Dose Rate Radiation

24

20

16

12

8

4

r)

100 300 500 700 900

Dose (Roentgens)

Straight lines fitted to experimental results from a number

of specific-locus tests involving single doses of radiation.

Line A: Radiation delivered at low dose rates (0.8

Roentgens/minute and lower). Line B: Radiation delivered

at high dose rates (72 to 90 Roentgens/minute). The figure

shows that a dose of radiation delivered at a faster rate

causes more mutations per locus than the same total dose

of radiation delivered at a slower rate. (90°10 confidence

limits are shown.)

SOURCE: W. L. Russell and E. M. Kelly, “Mutation Frequencies in Male Mice

and the Estimation of Genetic Hazards of Radiation in Men, ”

Proceedings of the National Academy of Sciences (U. S.) 79:542-544,

19s2.

radiation kills oocytes very efficiently, oocytes

would either die before they were mutated or they

would die with mutations that would never be expressed.

The extreme sensitivity to the killing effects of

radiation in mouse arrested oocytes is not common to all animals. In the squirrel monkey, high

sensitivity is limited to oocytes irradiated during

fetal life. Arrested oocytes in other monkey species are no more sensitive to lethal effects of radiation than are other cells. Similarly, human

oocytes, at least those that are irradiated after
86 ● Technologies for Detecting Heritable Mutations in Human Beings

birth, appear resistant to the lethal effects of radiation.

The differing sensitivities of oocytes of different species to lethal effects of ionizing radiation

complicate efforts to extrapolate from mutation

rates in female mice to the expected rates in human females (28). In particular, radiation may result in more mutations in human females than

would be predicted from female mice because human arrested oocytes are less likely to be killed

by radiation. That might allow a human oocyte

to survive a mutagenic dose of radiation and to

be fertilized.

Using experimental data from female mice, Russell (1977) made several calculations relating effects of radiation in mice to potential effects in

human females. In each calculation, he made the

“worst case” assumption that all stages of female

germ cells were as sensitive to the mutagenic effects of radiation as are the most sensitive stages—

maturing and mature oocytes-in mice. Because

there are different methods of handling the data,

he made four different calculations. Three produced estimates indicating that radiation would

not increase the mutation rate in human arrested

oocytes. The fourth estimated that human arrested oocytes would be somewhat less than half

as sensitive (from 17 to 44 percent as sensitive)

to radiation as are human spermatogonia.

ESTIMATING EFFECTS IN HUMAN BEINGS FROM ANIMAL DATA

Most extrapolations from animal data to human mutagenic risks are based on studies in male

mice because: 1) there are more data for male mice

than for female mice, 2) there appear to be fewer

biological differences in germ cell development between males of the two species than there are between females (see above), and 3) male germ cells

appear to be more sensitive to radiation than female germ cells and may provide a more sensitive

indicator of genetic risk.

The dose-response curves in figure 13 are derived from experiments on male mice and they

provide information for estimating human risks.

The absence of a detectable threshold for genetic

effects in male mice suggests that humans are at

some increased risk for mutations at any level of

radiation exposure. This finding increases the importance of making quantitative estimates of the

effects of radiation; since risk does not go to zero

except at zero dose, what is it at levels of human

exposure? The “doubling dose, ” which is the dose

of radiation that induces an additional number

of mutations equal to the number that occur spontaneously (resulting from all mutagenic influences,

known and unknown), can be derived from the

information in figure 13. The doubling dose differs

depending on the radiation dose rate. The doubling dose in male mice for high dose rates is about

40 R; for low dose rates, about 110 R. This is of

practical importance for human beings, since

some exposures are at high dose rates—e.g. the

populations of Nagasaki and Hiroshima and people receiving radiation therapy for some types of

cancer—but most population exposure to radiation is delivered at a low dose rate—e.g., people

who live near radioactive mines. About three

times as many mutations are caused by radiation

delivered at a high dose rate as opposed to an

equal total radiation dose delivered at a low dose

rate (114).

In chapter 3, the effects on mutation rates of

acute irradiation of the populations at Nagasaki

and Hiroshima are discussed. The exact doubling

dose for those populations is still being debated

because of uncertainties about the total dose and

because of insensitivity of the methods used to

detect mutations. Using the best information

available about those populations, however, humans appear to be less sensitive than mice to high

dose rate exposures.

Current radiation exposures of United States

citizens average about 200 millirem (mrem) per

year. On a population basis, this exposure is generally divided about equally between high dose

rate medical exposures and low dose rate exposures to natural sources of radiation. These exposures do not approach the doubling dose for
Ch. 6—Laboratory Determination of Heritable Mutation Rates • 87

mutations. If the average rate of human mutations

is now on the order of one mutation per 1 million genes per generation, we can estimate the effect of a hypothetical increase in human radiation exposure to twice the current level. If

exposures increased to 400 mrem and if that exposure were as mutagenic as the high dose rate

delivered to mice, it would increase the average

human rate to 1.005 mutations per 1 million genes

per generation. Such an increase would be undetectable by any current method.

Animal studies have proven useful for learning about the various relationships of dose, particularly for radiation, and germ-cell and heritable mutation rates. They are limited, however,

in providing information directly applicable to human beings. First and most important, human beings differ from animals in ways that we know

about and in more ways that are not understood.

Second, the endpoints measured in animal tests

do not generally correspond directly to endpoints

measurable in human beings under nonexperimental conditions. Third, the available animal

tests, like the currently available methods for

studying human beings, detect mutations arising

in specific, selected, loci which may not be representative of other loci. Evidence from Russell’s

specific locus test, cited above, shows a 35-fold

difference between the least sensitive and most

sensitive of the loci tested. A fourth consideration, again similar to the limitations of studies in

humans, is that each test does not detect more

than a fraction of the mutations that can occur,

and the exact nature of the mutations that are detectable cannot be characterized with these

methods.

When the new technologies discussed in this report are further developed, they presumably will

be applied to experimental animals as well as to

human beings. Studies that examine corresponding genes with the same techniques in

animals may provide some of the

comparisons that now are lacking.

humans and

interspecies
Chapter 7

Extrapolation
Contents

Page

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

The Aims of Extrapolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Extrapolation Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Attempts at Qualitative Extrapolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Attempts at Quantitative Extrapolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Dose-Response Relationships . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Generalizations About Dose-Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Limits of Extrapolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

91

91

93

94

97

97

99

99

TABLE

Table No. Page

9. Weighting of Test Results for Presumption of Germ-Line Mutagenicity . . . . . . 95

FIGURES

Figure No. Page

14. Biological Test Systems for Studying Mutations . . . . . . . . . . . . . . . . . . . . . . . . . 92

15. An Example of a-Parallelogram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Chapter 7

Extrapolation

INTRODUCTION

Currently, predictions of possible risk of heritable mutations in human beings are based on inferences, or “extrapolating” results, of mutagenicity

tests in other organisms or in laboratory cell cultures. One of the key problems in genetic extrapolation is that, while there is no shortage of mutagenicity tests using a variety of organisms and cell

types, researchers have just begun the painstaking work of drawing out relationships among results of different tests, and of eventually validating models for extrapolating to heritable effects

in human beings (see fig. 14). This chapter reviews

the basic constructs that have been devised for

framing genetic extrapolations and then presents

some efforts that have been made to carry out specific extrapolations.

The Aims of Extrapolation

For many years, the emphasis in mutagenicity

testing has been on developing individual tests and

learning about their properties. Test development

has not been targeted exclusively toward developing predictive models for heritable mutations

in human beings and in fact the quest for tests that

could be used to predict carcinogenicity through

somatic mutation has predominated. This being

the case, the test systems vary tremendously and

include, for example, tests in bacteria, insects, animal somatic and germ cells in culture, whole mammals, and human somatic cells. Efforts to relate

results from one test system to another or from

various tests to human beings, even on a qualitative level, have been relatively recent and have

not progressed very far to date. Bridges (15) summarized this situation:

Despite the extremely large number of screening tests that have been performed, remarkably

little rational thought has been devoted to the

use that should be made of the results of such

tests. Mutagenicity tests in lower organismsl are

“’Lower organisms” refers to bacteria, yeast, Drosophila, etc.,

and not to whole mammals.

essentially screening tests warning of a possible

human hazard. They do not give any quantitative indication of the level of risk to man, not

so much because of differences in the organization of DNA in man and lower organisms, but

because of the complex metabolic capabilities of

man which may greatly enhance or diminish the

mutagenic effectiveness of an agent.

In recent years, scientists who use different experimental approaches to study mutations have

begun to discuss methods to correlate results from

various test systems. The first consideration is

whether results from one system are qualitatively

predictive of results in another, i.e., are appropriate endpoints being considered and do the results agree in whether they are positive or negative. This is called “biologic extrapolation. ” The

second type of extrapolation is quantitative, which

deals with the relationship between the quantitative response in a test to a quantitative estimate

of the likely effect in human beings.

Examples of questions posed in extrapolation

are the following: If experiments demonstrate that

a single exposure to a high dose of a chemical induces heritable mutations in mice, what would

be the result of a single exposure to a lower realistic dose or of a long-term exposure to a lower dose

of that chemical in humans? How do results of

mutagenesis experiments on rodent somatic cells

in a test tube cell culture (e. g., Chinese hamster

ovary cells “in vitro”) relate to the issue of mutation rates inhuman somatic cells, or mutation rates

in human germ cells? What are appropriate dose

conversions between various experimental systems

and human beings? If a worker in a chemical plant

has a tenfold increase in mutation frequencies in

his or her somatic cells, what are the long-term

health implications? Is that worker at increased

risk of having a child with a new mutation? These

types of questions illustrate the complex issues that

arise in using results from one system to make predictions of risks to human health.

91
—

92 ● Technologies for Detecting Heritable Mutations in Human Beings

Figure 14.-BiologicaI Test Systems for Studying Mutations

Mammalian L

o

w

e

r

m

a

m

m

n

i

s

m

s

Test systems for studying mutations, arrayed according to biological similarity to the endpoint of interest—heritable mutations in human beings. Some test systems can be used to

measure a single endpoint; more than one type of endpoint (e.g., both chromosomal and gene

mutations) can be measured in other systems. Extrapolation from any system to human germ

cells in vivo involves many, mostly untested, assumptions.

SOURCE: Office of Technology Assessment,
Ch. 7—Extrapolation ● 9 3

In translating results from one system to another

(using similar genetic endpoints), a number of separate extrapolations may have to be made: 1) from

species to species; 2) from experimental doses to

actual environmental doses; 3) from one cell type

to another; 4) from in vitro to in vivo physiological conditions; and 5) the biggest and most uncertain leap, from estimates of mutation frequencies to estimates of genetic disease in humans. The

EXTRAPOLATION MODELS

Several researchers have begun developing models for extrapolating from one test system to

another (14,15,46,49,61,64,106, 129,130,138). one

of the key features common to all the extrapolation models developed is that a result from a single test system would not be used alone to predict

a result in another test system. Instead, results from

several related test systems are correlated and used

together.

Several of the methods described are extensions

or rearrangements of the first extrapolation method developed by Sobels (1290)—the parallelogram

(130):

The underlying principle is to obtain information on genetic damage that is hard to measure

directly, for example mutation in mouse germ

cells, by comparison with endpoints that can be

determined experimentally, e.g., alkylation per

nucleotide in mammalian cells in vitro and in

mouse germ cells, and mutation induction in

mammalian cells in vitro.

Sobels’ parallelogram is illustrated in figure 15.

It is relatively easy to determine the mutation frequency in mouse somatic cells (a quantity called

“A”) upon exposure to a particular chemical mutagen in culture. With certain types of chemicals.

(alkylating agents), it is also possible to derive a

measure of the interaction of the mutagen with

the DNA of those cells, which is quantified as

“alkylations per nucleotide” (a quantity called

“A”). Alkylations per nucleotide can also be measured in mouse germ cells after exposure to the same

mutagen (B’). The relationship of these different

values is then used to calculate the expected mutation frequency in mouse germ cells (B) on exkind of information that would give the biggest

boost to the ability to predict effects in humans

with information from other test systems is knowing exactly what kinds of mutations (e.g., point

mutations, chromosomal rearrangements, etc. )

each of the tests detects. The new technologies discussed in this report may provide this type of information.

Figure 15.—An Example of a Parallelogram

A ’

A

B ’

B

Estimated mutations

(Note that the same test system is represented in the vertical

dimension and the same genetic endpoint in the horizontal

dimension.)

SOURCE: F.H, Sobels, “The Parallelogram: An Indirect Approach for the Assess.

ment of Genetic Risks from Chemical Mutagens, ” pp. 323-327 in K.C.

Born et al. (eds.), Progress in Mutation Research, vol. 3 (Amsterdam:

Elsevier, 1982).

posure to the same mutagen. A major, untested

assumption is that the ratio of A to A‘ is proportional to the ratio of B to B‘, i.e., A/A = B/B

If that is true, it is then a matter of simple algebra

to predict the mutation frequency in mouse germ

cells (B) by solving the equation for B, which is

the only unknown quantity.

A similar parallelogram can be used to extrapolate results from mouse germ cells to human germ

cells. Mutation frequencies are measured in somatic cells of both humans and mice. Germ-cell

mutation frequencies are measured in the mouse

and compared to somatic-cell mutation frequencies in the mouse. Assuming that the ratio of germcell to somatic-cell mutation frequencies is the

same in mice and human beings, germ-cell mutation frequencies in human beings can be predicted

from the measured human somatic-cell mutation

frequencies (64).

Streisinger (138) has proposed a more complex

extrapolation method using two sequential paral-
94 • Technologies for Detecting Heritable Mutations in Human Beings

lelograms. In the first parallelogram, a measure

of chemical interaction with human germ cell DNA

is estimated from measurements of the effects of

the chemical in human and animal (e.g., monkey)

somatic cells, and a measure of interaction of the

same chemical with germ-cell DNA in the same

animal. In the second parallelogram, the ratio of

a measure of chemical interaction with mouse

germ-cell DNA to mouse germ-cell mutation rates

(from the specific locus test) is used to predict the

human germ-cell mutation rate using the estimated

value of chemical interaction with human germcell DNA from the first parallelogram. This construct embodies several untested assumptions.

Bridges (1980) also developed a more complex

extrapolation model based on the original parallelogram model of Sobels. Based on his approach,

Bridges outlined the types of results needed to fill

information gaps, ultimately to assess the impact

of mutagens at the: 1) molecular; 2) cellular; and

3) whole organism levels in both animals and man.

He suggested studies to determine: 1) the presence

of an effective dose of mutagen at the molecular

level by measuring the concentration of mutagen

in the gonads or blood or the extent of reaction

with DNA; 2) whether there appears to be a relationship between the presence of the mutagen and

a biological response at the cellular level by measuring somatic mutation frequencies or chromosomal changes in lymphocytes; and 3) whether

there is an effect at the whole organism level by

measuring the frequency of heritable genetic

defects, congenital malformations, or fetal loss.

The values Bridges specifies are obtainable in

animal systems. To obtain such values for man,

Bridges suggests that use be made of certain otherwise normal human populations that are exposed

to large doses of mutagens. Examples of such populations are patients treated for diseases, such as

cancer, with drugs that are known to be mutagenic, and certain occupational cohorts in which

there are known excesses of cancer (1,155). Simultaneous studies using the same mutagens could

be carried out in experimental mice to determine

the relative sensitivities of mouse and man to these

mutagens.

Parallelogram models are attractive for their

simplicity and inherent logic. They are appropriate starting points for exploring relationships

among test results when sufficient data become

available to do so. However, the assumptions embodied in parallelogram models—consistent, predictable relationships among various cell types,

translatable among species—are almost entirely

untested. The great differences among species

make it unlikely that these parallelogram models

will survive validation studies intact. While they

may continue to be useful research tools for posing logical questions, they may or may not prove

practical for predicting risks of heritable mutations in human beings.

ATTEMPTS AT QUALITATIVE EXTRAPOLATION

L. Russell and colleagues (106) compared the

results of mutagenicity tests carried out in a variety of systems other than whole mammals with

results from specific locus tests (SLTs) and heritable translocation tests (HTTs) in mice (see ch. 6

for descriptions of these two tests). The purpose

of the comparison was to find out how well results of the nongerm-cell tests corresponded to the

qualitative results (positive or negative) of the two

germ-cell tests. The analysis was limited by the

relatively small number of chemicals that have

been tested in either the SLT or the HLT. About

35 chemicals have been tested in one or both of

the germ-cell assays, out of a total of about 2,000

chemicals for which some test results are available from any system.2

The comparison tests were grouped into 18 categories and the categories given relative weights

according to their biological relationship to one

of the germ-cell tests. The categories and their

weights are given in table 9. The lowest weighted

category includes tests using prokaryotes, such

as bacteria, directly treated by the suspected mutagen. Higher scores signify moving toward higher

‘These test systems are not the focus of this report and are not

described here in detail. Descriptions of these tests can be found

in (88).
Ch. 7—Extrapolation ● 9 5

Table 9.—Weighting of Test Results for Presumption of Germ-Line Mutagenicity

Exposure Exposure

not within within

mammalian mammalian

body Weight body Weight Germ cells Weight

Prokaryotes, all endpoints SAL 1 BFT 2

W P– HMA

Lower eukaryotes, all endpoints YEA

YEP 2

ASP

NEU

Higher eukaryotes, chromosome aberrations PYC 3

Higher eukaryotes, gene mutations PGM 3

Mammals, genetically nondefined endpoints SC1

SC2 4

UDH

UDP

Mammals, chromosome aberrations CYC 5

Mammals, gene mutations CHO

V79 5

SC3

SC4

DAN

DHT

6

MNT 7 DLT

CYE

}

CY9

CYB 10 CYO

CY5

CY8

8

8

4

8

15

MST 10 SPF 15

L51

NOTE’ In general, the weights increase from top to bottom and from left to right in the table, From top to bottom, the tests progress from lower to higher organisms

and from more general endpoints to endpoints of direct relevance to human heritable mutations. From left to right, the categories progress from in vitro tests

In both somatic and germ cells, to in vivo germ-cell tests

SOURCE L.B Russell, C S Aaron, F de Serres, et al , “Evaluation of Mutagenicity Assays for Purposes of Genetic Risk Assessment,” Mutation Research 134:143-157, 1984

mammals, toward germ cells, and toward treatment with the chemical in a whole mammal.

A single composite score was calculated for each

chemical tested, adding together scores from each

category in which there were test results. There

is only one score per category regardless of the

number of tests. Positive results are scored as positive numbers; negative results as negative numbers, e.g., an in-vitro somatic-cell chromosome

aberration test with a positive result yields a score

of +5, one with a negative result, a score of —5.

Russell and colleagues found that nearly all

chemicals that tested positive in either or both the

SLT and HTT had high composite scores from

other tests. A number of chemicals with negative

SLT and HTT results also had high, positive composite scores, representing “false positives” in the

comparison tests.

Similar analyses looked separately at the SLT

and HTT and the comparison tests that specifically detect gene mutations or chromosome aberrations, respectively. The results are similar to

those matching the results in all comparison tests

against both mammalian germ-cell assays: high

scores for most chemicals positive in the germcell tests, and a number of false positives.
96•Technologies for Detecting Heritable Mutations in Human Beings

In an additional analysis, the comparison tests

are ranked according to how well each predicts

the results of the two germ-cell tests. In general,

the tests in higher numbered categories in the

earlier analyses, i.e., those that are closer biologically to whole mammal germ-cell tests, had better correlations with the SLT and HTT. For the

SLT, the best predictors overall were the mouse

spot test, unscheduled DNA synthesis in mouse

testis, and the micronucleus test. For the HTT,

unscheduled DNA synthesis in testis, the dominant-lethal test, and one lower ranked test, sisterchromatid exchange in cultured animal cells, were

the strongest predictors.

While it appears that the results of some of the

comparison tests correlate relatively well with the

mammalian germ-cell tests, in fact, not one of these

correlations reaches conventional statistical significance, meaning that the tests do not predict

reliability better than chance. The lack of significant results is due, in large part, to the small number of comparisons for many tests, and in part

because of the process for selecting chemicals for

SLT and HTT. From a practical, public policy

standpoint, this is an important finding. The lack

of statistically significant results does not mean

that these comparisons are without value. The

study provides a status report on the quality and

quantity of existing data.

Since the two whole mammal tests (the SLT and

HTT) are relatively expensive and time-consuming, they are usually reserved for testing chemicals highly suspected of causing heritable mutations. The suspicion is based on results of other

tests, specifically the comparison tests examined

in Russell and colleagues’ analysis. It is hardly surprising, therefore, that the comparison test results

are largely positive for chemicals eventually tested

in the mammalian germ-cell assays. Russell and

colleagues took the preponderance of positive results into account in their analyses.

Many chemicals have been tested in mutagenicity assays because, for reasons of chemical

structure or other properties, there is a high likelihood that they will be mutagenic. While these

chemicals have proved useful as laboratory tools,

they are not necessarily useful for drawing conclusions about what people are actually exposed

to. W. Russell (111), using the same database used

by L. Russell and colleagues (106), looked exclusively at the 11 “environmental chemicals” (those

found in the home or workplace) that have been

tested in the SLT and examined the results. All

11 are positive in the Drosophila sex-linked lethal

test, the 10 that have been tested are positive in

mammalian somatic-cell tests, and there are a variety of positive results in other test systems. None

of the 11, each of which was tested at very high

doses, is positive in the SLT, suggesting no increase

in mutations in spermatogonia (the pre-meiotic

male germ-cell stage) although several have positive results in tests of later sperm developmental

stages.

What conclusions can be drawn about the validity of qualitative extrapolation from various

mutagenicity tests to a risk of heritable mutations

in human beings? The available data give no direct

information about mutagenic effects of chemicals

on human germ cells in vivo. Application to humans rests on a series of assumptions about the

response of human germ cells in relation to responses in other types of cells and in other species.

The analysis of the these test results does allow

some generalizations about biologic extrapolation

and about the nature of the available test database. On the first point, there is evidence suggesting that chemicals that test positive in some comparison tests for gene mutations or chromosomal

aberrations have a likelihood of being positive in

the SLT or HTT, respectively, but are not invariably so. To date, chemicals testing negative in the

comparison tests have not produced clear positives in whole mammal germ-cell tests, but the

database supporting this comparison is limited.

Biologically, it seems unlikely that chemicals that

are consistently negative in comparison tests

would, in fact, be germ-cell mutagens in whole

animals. But it is unlikely also that very many

chemicals with negative results in one or two tests

will have been tested in enough systems to allow

an empirical test of that hypothesis.

Both L. Russell’s and W. Russell’s analyses described above suggest that a number of chemicals

that test positive in the simpler comparison tests

will be not be shown to cause mutations in spermatogonial germ cells. At present, however, it is

impossible to know based on comparison test results, which chemicals are true human germ-cell

mutagens and which are not.
Ch. 7—Extrapolation . 97

A major limitation of the database is that nearly The new technologies described in this report,

all tests have been in exposed male animals. Two which could be used to provide information on

strong animal mutagens—radiation and a chemi- the types of mutations detected by the various

cal agent, ethylnitrosourea—do not appear to tests, may improve our ability to apply the recause heritable mutations in the immature germ suits of simpler tests to predicting human risk.

cells of female mice, but the data for females are

not sufficient to draw firm conclusions.

ATTEMPTS AT QUANTITATIVE EXTRAPOLATION

Not surprisingly, quantitative extrapolation is

even less advanced than is qualitative extrapolation. However, there are some data bearing on

quantitative relationships that may eventually be

useful in predicting effects in human beings. First,

there is some information about dose-response

relationships. Second, some preliminary attempts

are being made to determine relationships among

certain corners of the parallelogram models described earlier in this chapter. One such effort is

described below.

In an ongoing effort, a group of investigators

is using a parallelogram approach to evaluate the

effects of gamma radiation, cyclophosphamide (a

medical drug) and benzo[a]pyrene (an environmental chemical) on mouse and human cells by

assaying chromosomal aberrations and sister chromatid exchanges in mitogen-stimulated peripheral

blood lymphocytes (PBLs). While these investigators have as yet little data, a paper presenting

some preliminary results (170) lays out the rigorous procedures necessary for proper extrapolation of results from different studies of just one

substance to predictions in untested systems. For

instance, in the case of radiation, the authors relate an experimental result in irradiated cultured

human PBLs to reports from the literature of the

same chromosomal endpoint (dicentrics) in PBLs

from patients who have received therapeutic radiation. Thus far, these authors have not presented

any conclusions about the relationships they are

studying.

Dose= Response Relationships

Extrapolation from high to low dose, and from

high to low dose rate, requires knowledge of doseresponse relationships. For heritable gene mutations, adequate data on dose-response relationships is limited to the effects of ionizing radiation

and one chemical, ethylnitrosourea (ENU), in male

mice. The available information comes from results of SLTs in mice, most of the work being done

in a small number of laboratories in different parts

of the world. Radiation and ENU have also been

assayed in many short-term in vivo and in vitro

tests in human somatic cells, so some generalizations may be drawn about the nature of doseresponse relationships for these two agents. In both

cases, there are independent effects of dose rate

and of total dose. This means that a fixed dose

may cause a different rate of mutations depending

on the intensity of the dose, i.e., a more protracted

administration may result in fewer mutations than

if the total dose is administered at one time.

Radiation

Specific-Locus Test. —In a series of experiments

from the mid-195@ to the present, a range of radiation doses, delivered at a range of dose rates,

has been tested in male mice. Results are available for both spermatogonial and postspermatogonial stages. In spermatids and spermatozoa

(postspermatogonial stages), the dose-response

relationship is generally linear, and there is no effect of dose rate. This means that approximately

the same mutation rate results from a short, highdose exposure and from a chronic, low-dose exposure when the same total dose is given.

In spermatogonia, the early, pre-meiotic stage,

for equal total exposure, radiation given at high

dose rates causes more mutations per unit of dose

than radiation at lower dose rates. At a high dose

rate of 90 Roentgens per minute (R/rein) or intermediate dose rate of 8 R/rein (work cited in 111),
98 ● Technologies for Detecting Heritable Mutations in Human Beings

the mutation rate decreases with decreasing dose

faster than would be predicted by a linear relationship. At dose rates of 0.8 R/rein or below,

however, the rate of mutations per unit dose appears to be constant, and without a threshold. At

low dose rates, the dose-response relationship is

linear, and above about 0.8 R/rein, the mutation

rate per unit of dose rises more quickly than would

be predicted by a linear relationship. Above a total dose of between 600 and 1,000 rem, the mutation rate begins to decline rapidly.

Investigators using the SLT offer an explanation for the observed dose-response patterns (111,

114,115). They postulate that the difference in

dose-rate response between spermatogonia and

postspermatogonial stages is a function of an active repair mechanism in metabolically active spermatogonial cells, which does not function at later

stages. In the earlier, spermatogonial stages, a

larger percentage of changes can be repaired before the spermatogonia complete meiosis when exposure is at a lower dose rate than is the case with

an acute, high-dose-rate exposure. In post-spermatogonial stages when capacity for repair is low,

the total radiation dose, irrespective of dose rate,

is the determinant of the mutation rate.

Heritable Translocation Test.—Generoso and

co-workers (135) have investigated heritable translocations induced by high-dose rate (96 R/rein)

irradiation of spermatogonial stem cells of mice.

They report a linear dose-response relation between O and 600 R of total irradiation, and repetition of doses in this range gave additive effects

up to 2,OOO R. From these data, the expected increase in heritable translocations at high-dose-rates

is calculated to be about 0.00004 per R.

Cytogenetics.— Waters and colleagues (170) describe a dose-response relationship for gamma radiation after both in vitro and in vivo irradiation,

using as an endpoint a certain type of chromosomal mutation, dicentrics, in PBLs. The in vivo

dose-response, which was derived from reports

in the literature, is linear at lower dose rates, and

quadratic at higher dose rates, meaning that the

increase in the number of dicentrics rises faster

than it would if the relation continued to be linear. The quadratic component may be explained

by an interaction of mutational events causing

some dicentrics, and the interactions being increasingly more likely as the dose rate increases.

Ethylnitrosourea (ENU)

Specific Locus Test. —As is the case with radiation, both total dose of ENU and dose-rate independently affect the mutation rate in mouse spermatogonia. Experimental data indicate that the

response at doses below 100 mg/kg of body weight

is “infralinear,” meaning that as the dose is lowered,

the mutation rate drops faster than would be predicted by a linear relationship. At doses between

100 and 400 mg/kg, the response appears to be

linear (4 I). No threshold was detected over the

range of doses tested, but the possibility of a

threshold at a dose lower than 25 mg/kg (the lowest single dose tested) is not excluded by the data.

In an experiment examining mutational responses at different dose rates, the mutation rate

was measured in mice that were given 10 weekly

doses of 10 mg/kg of ENU, and compared with

the mutation rate for a single dose of 100 mg/kg

of ENU. The mutation rate for the fractionated

dose was only about 15 percent of the rate for a

single dose (112).

Russell (111) notes that, in light of information

indicating that ENU reaches germ cells in doses

proportional to injected amounts (see next section),

these results cannot be explained by differences

in metabolic processes. He interprets the infralinear

portion of the dose-response curve and lower mutation rate that follows dose fractionation to be

the result of effective mutational repair systems

in spermatogonia, the same reasoning as in the

case of radiation.

Unscheduled DNA Synthesis in Mouse Spermatids.—Carricarte and Sega (cited in 111) found the

dose-response of “unscheduled DNA synthesis”

in mouse spermatids to be linear over the range

from 10 to 100 mg/kg, the same range over which

W. Russell (111) found an infralinear relationship

in the SLT. Sega (cited in 111) also measured “adduct formation” after injections of ENU, and found

a linear response in the range from 5 to 100 mg/kg.

One conclusion from these observations is that

chemical interaction with DNA may not always

be directly related to the rate of mutation, and
Ch. 7—Extrapolation . 99

this presents a major difficulty

parallelogram, described earlier in

Sister-Chromatid Exchange and

for Sobels’

this chapter.

Thioguanine

Resistance.–Jones and colleagues (54) investigated

the dose-response relationship for two somaticcell endpoints in whole mice exposed to ENU. The

frequency of sister-chromatid exchange (SCE) and

the frequency of thioguanine resistant (TGr) cells

were measured in white blood cells in the spleen.

Corresponding endpoints exist in human beings,

which makes them potentially useful for extrapolating to human responses. The investigators measured both the response over time at several dose

levels, and the response to a range of doses at several points in time after exposure.

Linear relationships were discovered between

dose and reponse for both SCEs and TGr cells,

but the timing of these responses was quite different. For all dose levels, the highest SCE levels were

measured on the first day after exposure, and SCE

LIMITS OF EXTRAPOLATION

The main limitation to validating extrapolation

models is a virtual lack of data to complete the

parallelogram models. Until there is enough empirical information to determine whether qualitative and quantitative generalizations can be made,

it is impossible to know how useful extrapolation

could be. At present, qualitative extrapolation is

at least a tentative guide for identification of mutagenic agents.

Even when data are available, many questions

bedevil the use of experimental animal data to extrapolate risks to humans. In particular, little is

known about the comparability of species with

respect to activation, detoxification, and tissue distribution of specific chemicals, as well as other

interspecies differences in metabolism. In addition,

mutational responses of germ cells at different

stages of development can differ for different types

of mutagens. Many of these gaps in information

could be filled by studies that are now technically

feasible.

levels decreased back to baseline after about 70

days, whereas the TGr response rose linearly

over time to a peak after about 80 days for each

dose level.

Generalizations About Dose-Response

While dose-response relationships cannot be

generally and simply described some generalizations can be made. It appears that mutations in

somatic cells are more predictable from the various experimental test systems available than are

mutations germ-cell. Repair of mutations in germ

cells may explain this difference. Both a better

understanding of the mechanisms of mutation and

repair at the molecular level, and empirical comparisons of test data on more substances will contribute to a better understanding of dose-response

relationships. Once again, the new technologies

may contribute to this understanding.

In using test results from somatic cell systems,

there are differences in the sensitivities of various

types of somatic cells and germ cells to different

mutagens. Even within a single cell type, different gene loci can respond very differently to a specific mutagen. In making comparisons, for instance, between somatic and germ cells, if the same

locus (or set of loci) cannot be tested, any differences in outcome cannot necessarily be attributed

solely to a difference in cell type. These are a few

of the many uncertainties in extrapolations from

somatic to germ cells.

An organizational problem that affects extrapolation in a practical way is that collaborations

have been rare among researchers working with

different systems, and are particularly rare among

scientists working in different laboratories. In addition, the test systems are not necessarily standardized among laboratories working with the same

systems, so results from different laboratories cannot always be combined or directly compared.
.

100 • Technologies for Detecting Heritable Mutations in Human Beings

Even relatively simple aspects of experimental procedures, for instance, the conditions under which

cells or organisms are exposed to radiation or

chemicals, vary enough among laboratories to render results incomparable to varying degrees.

Overall, the development of methods to extrapolate from different experimental test systems to

the risk of heritable mutations in human beings

is still in its infancy. While the various parallelogram models are appealing because of their potential usefulness, some of the data already available

suggest that they may never broadly applicable.

Both qualitative and quantitative improvements

in the database of experimental results will be necessary before the usefulness of extrapolation can

be reasonably assessed.
Chapter 8

Mutation Epidemiology
Contents

Page

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........................103

Validation of Measurement Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...104

Surveillance and Disease Registries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .......104

Monitoring and Exposure Registries . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........106

Ad Hoc Epidemiologic Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..107

Population to Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. ...108

Radiation-Exposed Groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............108

Cancer Patients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......................108

Other Populations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .....................109

Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .............................110
Chapter 8

Mutation Epidemiology

INTRODUCTION

This report is about biochemical and genetic

techniques for studying heritable mutations. These

new techniques will ultimately be used to study

people who are suspected of being at high risk for

excess heritable mutations. The types of epidemiologic activities in which mutation detection techniques may eventually be used are: surveillance,

monitoring, and ad hoc studies. Before any technique can be used for those purposes, a series of

validation studies will be needed, calling for different populations that are appropriate for study

at different stages of development of the technologies.

Surveillance is a routine activity whose aim, in

the context of this report, would be to measure

the “baseline” rate of mutations in a defined population over the course of time, and to facilitate

rapid recognition of changes in these rates. Following the distinction made by Hook and Cross

(44), the term monitoring is reserved for observations over time in populations thought to be

at increased risk for heritable mutations because

of a known or suspected exposure to a known or

suspected mutagen, for the purpose of helping the

specific population in whatever way possible. Ad

hoc studies of a variety of designs are carried out

to test hypotheses about suspected causes of mutations.

The different aims of surveillance, monitoring,

and ad hoc studies require that different criteria

be applied for deciding when and whether to carry

out one or more of those activities. Surveillance

and monitoring are not designed as hypothesistesting activities, though they may be sources for

hypothesis development.

The reasons a population is chosen for surveillance may be largely opportunistic. It is unlikely

that an entirely new system of data collection

would be put in place for mutation surveillance.

It is more probable that mutation surveillance

would be added to an existing program that is

established for another purpose, for instance,

birth defects surveillance. The population covered

must be large enough to generate reliable rates for

mutational events that may be relatively rare, but

there is no fixed requirement for size. Of the three

types of activities, surveillance generally would

involve the smallest effort and resource expenditure per individual, but because large numbers of

people would be routinely subject to the surveillance test, the total cost could be large. There is,

therefore, a great need to consider the costs and

benefits of such a program before embarking on

one, and for choosing the detection technique accordingly. The threshold for initiating mutation

surveillance would be relatively high. Information about trends sometimes can be obtained by

means other than full-scale surveillance, and some

such studies for that purpose have been done.

The main reason for instituting a mutation

monitoring program is concern about the potential effects of a mutagenic exposure in a specific

group of people. If there is enough concern, there

undoubtedly will be a greater expenditure of resources and effort per person than is the case for

surveillance, meaning that more extensive contact with the population and testing would be

justified. Given today’s knowledge, it is hard to

conceive of a situation in which there would be

a concern only about heritable mutations, so any

mutation monitoring effort would most likely be

part of a larger program. The obvious concurrent

concerns for exposures thought to be mutagenic

are cancer and birth defects. Strict criteria based

on tests of the statistical power to detect certain

levels of effects are not appropriate for monitored

populations, since the concern is about that particular group of people. Any finding is of interest in that situation. The information maybe used

incidentally to calculate upper limits of risk which

could be generalized to other populations with

similar exposures. The most important consideration in making a decision to monitor is that there

is reasonable evidence suggesting that the population may be at a substantially increased risk.

103
104 ● Technologies for Detecting Heritable Mutations in Human Beings

This decision may well be influenced by political

pressures to act, but ideally there should be a recognition that the best scientific judgment either

does or does not support a monitoring effort.

The purpose of ad hoc studies is to test hypotheses about exposures and effects. This is the one

place where it is imperative that studies be designed to achieve a high probability of detecting

an effect if it is present. Such studies are valuable

not only for the sake of the populations involved

in the studies, but for their generalizability to

other populations. Results of these studies can

form the basis for public health actions, if levels

of risk can be established.

VALIDATION OF MEASUREMENT TECHNIQUES

For the most part, the new technologies described in this report have not been “validated. ”

They are new, and have not been applied to large

numbers of people. Surveillance, monitoring, and

ad hoc studies all require tools—in this case techniques for measuring mutations—with an acceptable

degree of validity. That is, the tests must measure what they are designed to measure, within

some definable limits. Generally, it is not possible

to gather reliable information about a population

and concurrently gather validating information

about a technique used to measure outcome, unless another technique, with known validity, and

known relationship to the new technique, is also

applied in the study. Even though that is technically feasible, it is probably not an efficient way

to gather validating data.

A first step in the validation process would be

to use laboratory-prepared samples with known

DNA sequences to confirm that the types of mutations that should theoretically be detected with

a new technique actually are reliably detected. Beyond that stage, the need to move to clinical samples can be met using stored blood from individuals studied previously for other reasons. These

stored samples need not be from parent/child

triads initially, but triads will be needed at a later

stage.

A number of research organizations are storing samples that would be appropriate for studies

of mutations using new DNA techniques. The National Cancer Institute for example, is storing

blood samples from cancer patients who have

been treated with drugs and radiation, and the

Radiation Effects Research Foundation has stored

blood from Japanese citizens who were exposed

to atomic bombs. In both of these cases, DNA

is stored according to an established technique

that uses Epstein-Barr virus to transform lymphocytes, thereby “immortalizing” the cells so they

can be grown indefinitely. The transformed cells

can be frozen for the long term in liquid nitrogen. Both sample preparation and long-term storage costs are substantial. With currently available

technologies, it is unlikely that large numbers of

samples will be stored. This limits the number and

variety of samples available for validation studies,

and also for later studies of people exposed to potential mutagens.

SURVEILLANCE AND DISEASE REGISTRIES

The methods and aims of disease surveillance track the incidence of cancer. Although the first

have developed based on experience in infectious national cancer surveillance system, which covdisease control. Reporting of vital statistics, in ers about 12 percent of the population, was put

particular births and deaths for calculating birth in place as recently as 1972, New York State inand death rates, is also a form of surveillance. Sur- stituted a reporting system for cancer cases in

veillance of noninfectious diseases is a relatively 1940, and Connecticut followed the next year.

recent development, with roots in the desire to There are now dozens of cancer surveillance sys-
Ch. 8—Mutation Epidemiology • 105

terns operating around the country and internationally, covering a range of populations from

counties to whole countries (145).

Information about individual patients in cancer surveillance systems forms the basis for “cancer registries. ” The routine output of registries

consists of cancer rates by sex, age, and race

(where applicable) for each cancer site. Registries

also are an important source for researchers investigating hypotheses about cancer causation. In

this sense, cancer surveillance, with information

about individuals recorded on registry forms, is

similar to mortality statistics, with information

about individuals recorded on death certificates.

It should come as no surprise that there are no

“heritable mutation surveillance systems” now in

place. There are, in various places around the

world, registries of birth defects, which include

records of at least some sentinel phenotypes. Beyond that, as this report shows, there are at

present no techniques for detecting mutations that

are suitable for use in a large-scale population surveillance program. Because developments have

been so rapid, however, there maybe one or several good candidate techniques within 5 or 10

years.

Surveillance traditionally has involved reporting, to a central place, information already collected by some segment of the health care system

for reasons directly related to the health of individuals. Even for infectious diseases, only cases

that come to the attention of physicians are reported. Active “case-finding” in the population

is not a usual feature of surveillance. For chronic

disease, the same is true. Cancers diagnosed by

physicians are entered into registries. Case-finding

programs, such as breast cancer screening programs, are instituted on the basis of their effectiveness in identifying cases early in the course

of disease, for the benefit of the individual with

the disease.

Infants are examined for birth defects because

of the potential impact on the children and their

parents’ lives, and not mainly for the purpose of

computing the rates of birth defects in a population. Testing programs for newborns, including

biochemical tests for metabolic diseases (not necessarily a result of a mutation), also have been

instituted because of their importance to the health

of the individual. Nearly all States now require

testing newborns for phenylketonuria (PKU), and

some require additional tests. For instance, New

York requires testing for PKU, sickle-cell anemia,

and congenital hypothyroidism, which are moderately rare, and also for very rare conditions including maple syrup urine disease, homocystinuria, histidinemia, galactosemia, and adenosine

deaminase deficiency (102). The tests do not impose an added burden on the newborn, since all

are carried out using the same blood sample.

Cytogenetic techniques have not been used for

population-based surveillance of chromosome abnormalities, but some large hospital-born series

of newborns have been tested (102). Most of the

recorded cases of chromosome abnormalities found

in this way might eventually have been detected

later in life because of health and reproductive

problems, but some others might otherwise have

gone undetected.

There is no formula for deciding whether to institute a surveillance program, but there are characteristics of the disease, of the population, and

of the particular test to be used that contribute

to the decision: 1) the seriousness of the disease

(if the measured endpoint is known to be associated with a disease); 2) the ability to alter its clinical course after diagnosis; 3) the prevalence of

the disease in the population; 4) the reliability and

validity of the test; 5) the acceptability of the test

to the population; 6) the cost of the screening program; and 7) the cost of not screening (i.e., the

cost of treatment and social support). It is worth

considering these factors in thinking about screening and surveillance for heritable mutations.

The idea of surveillance for heritable mutations

represents a departure from the traditional applications of surveillance. It appears to be the case

that most heritable mutations are not related to

disease over the course of an individual’s lifetime,

and no predictions useful to the individual can

currently be made about the effect of a heritable

mutation in the absence of recognizable disease,

beyond those that are associated with sentinel

phenotypes and major chromosome abnormalities. As mutation detection techniques become

more and more sensitive, in fact, a greater per-
106 Ž Technologies for Detecting Heritable Mutations in Human Beings

centage of the mutations detected may not be related to a known effect on health.

Heritable mutation surveillance beyond reporting sentinel phenotypes will require more than just

a reporting of events already detected. It will require imposing a test burden on a population for

the sole purpose of collecting information about

mutations that may never affect an individual’s

life. This argues against instituting surveillance.

A reason in favor of surveillance is that it is clear

that increased mutation rates will be looked for

in special populations, those being monitored because of worries that they have been exposed to

a mutagen. Surveillance systems can provide a

range of estimates of “baseline” or “background”

rates, even though they may be from different

populations. In a more general sense, one of the

original aims of surveillance is relevant: to substantiate long-term trends and patterns in health

events and to detect changes that may be addressed by public health action.

MONITORING AND EXPOSURE REGISTRIES

Monitoring is the “long-range observation of

individuals who are at presumptive high risk for

adverse outcomes because of specific life events, ”

(44) in particular, exposures to suspected mutagens. The event may be catastrophic, such as exposure at the time of detonation of an atomic

bomb, or a chemical plant explosion. Or the

“event” may be long term, such as an occupational

or an environmental exposure. There are about

two dozen populations around the world currently monitored for long-term health effects, and

some of those programs include various studies

of heritable mutations.

The most extensive population monitoring, including monitoring for mutations, is of the Japanese residents of Hiroshima and Nagasaki, many

of whom were exposed to substantial amounts of

radiation during World War II when atomic

bombs were detonated in those cities. The population around a chemical plant that exploded near

Seveso, Italy in 1976, releasing several pounds of

dioxin, is the subject of health monitoring activities, including monitoring for birth defects. The

people exposed to methyl isocyanate in Bhopal,

India, will undoubtedly be followed for years to

come. Because these groups were exposed, and

because it is conceivable that something could be

done to alleviate health problems if they are detected early, or if warning signals are picked up,

they are being monitored; the scientific knowledge gained as a result is a secondary benefit.

The most prominent examples of chronic exposures are from occupational activities and toxic

chemicals in the environment. The populations

exposed to hazards often are not geographically

determined, but may be a collection of workers

from around the country. Workers exposed to

radiation in the nuclear power industry are an example of this. There are several “exposure registries” in existence worldwide, though none specifically because of a perceived increased risk of

mutations. One such registry has the names of all

workers who were exposed to dioxin during the

manufacture of various chemicals in this country. There also is an international dioxin registry, with names of workers from all around the

world. The registry does not, however, have information about the health status of those workers. A similar registry for workers exposed to

beryllium exists in this country. A report prepared

for the Nuclear Regulatory Commission in 1980

recommended that a registry be started for workers exposed to low-level ionizing radiation in certain types of workplaces, because of a possible

increased cancer risk (29). These registries could

be used for monitoring and as a potential population to include in ad hoc studies.
Ch. 8—Mutation Epidemiology ● 107

AD HOC EPIDEMIOLOGIC STUDIES

Surveillance, monitoring, medical case reports,

and laboratory research can all lead to hypotheses about possible causes of heritable mutations.

An investigator wishing to test a hypothesis must

find suitable subjects to study, in contrast to a

monitoring activity, where the existence of the exposed population is the reason for acting. A study

should be undertaken only if there is a good chance

of answering the question of interest. Disease and

exposure registries are common sources of individuals to study, depending on the question.

A cohort design will probably prove the most

useful approach for studies of heritable mutations,

though case-control studies of sentinel phenotypes

may also prove valuable. A cohort study involves

identifying a group of individuals, some exposed

to the suspected mutagen and some not exposed.

The health outcomes, i.e., the presence or absence

of mutations in offspring, of the two sub-cohorts

are compared. A higher rate of mutations in the

exposed group would signify an “association” between the exposure and heritable mutations. Statistical tests are applied to the results to estimate

the likelihood of the result occurring if in fact there

was no real difference in mutation rates between

the two groups.

In a case-control design, a group of “cases,” individuals with conditions of interest, e.g., sentinel

phenotypes, is compared to a group of individuals who do not have the condition of interest,

but who are otherwise similar demographically.

The cases and controls are compared according

to their past histories of exposures or other characteristics that might be associated with the mutation and an assessment made as to whether their

histories differ in important ways.

The important question for all studies is not just

whether the exposure is “associated with” mutations, but whether it causes them. That is a difficult if nearly impossible judgment to make in most

instances, but there are some generally accepted

guidelines for evaluating the likelihood of an association being causal based on epidemiologic evidence. These are:

ent times and in different populations, and

in studies of different designs.

2. Strength: The size of the effect of an exposure

is the measure of strength of association.

This is usually measured as an estimate of

relative risk (a ratio of the rate of mutations

in an exposed group to the rate in an unexposed group). The presence of a dose-response relationship, that is, the size of the

effect changes in a logical way with the level

of exposure and in at least some cases, with

the dose rate.

3. Specificity: Specificity refers to the degree

to which the exposure is associated exclusively with the outcome of interest, in this

case a mutation, and the degree to which a

mutation is associated exclusively with the

exposure. The concept of specificity derives

from study of infectious disease and is relevant to the study of mutations (and chronic

diseases generally) only in special cases, for

example, a specific mutation that almost

never occurs in the general population but

appears to be exclusively related to a particular exposure. While a highly specific relationship can provide positive evidence for

a causal relationship, a lesser degree of specificity does not necessarily argue strongly

against causality.

4. Temporal Relationship: The exposure must

occur before the effect. In the case of heritable mutations, the picture is more complicated. See chapter 6 for a discussion of the

timing of exposure for males and females for

a plausible effect on germ cells.

5. Coherence: All available information from

medical and biological science, and from epidemiologic observations and studies, fits together in a way that supports the hypothesis. The greater the variety of information,

and types of study designs, the stronger the

finding of coherence.

These criteria are quite stringent, and even in

the best of cases, often cannot be met, but they

are useful as standards.

1. Consistency: The association is observed in

studies by different investigators, at differ-
108 . Technologies for Detecting Heritable Mutations in Human Beings

POPULATIONS TO STUDY

There are elements in the environment that

damage human health under certain conditions

of exposure. Biologic, chemical, and physical

agents cause acute and chronic diseases in humans. At present, there are no exposures unequivocally known to cause heritable mutations

in human beings. A combination of factors, including the rather insensitive methods for detecting heritable mutations that have been available,

and the possibility that human germ cells may not

be very susceptible to some mutagens, probably

contribute to this situation. As a consequence, investigators looking for the effects of mutagens

must do so in people who have been highly exposed to agents that are very likely to be mutagenic in germ cells. There are not very many large

groups of people fitting that description, a fact

that many might find surprising.

Radiation= Exposed Groups

Radiation causes heritable mutations in laboratory mice and is the most likely potential germcell mutagen to which large numbers of human

beings have been exposed, either intentionally or

accidentally. The largest population with a known

high radiation exposure, the Japanese atomic

bomb survivors, continue to be followed for effects on cancer incidence, birth outcomes, and

heritable mutations. Heritable mutations have

been studied by clinical observations, cytogenetic

techniques, one-dimensional electrophoresis of

blood proteins, and more recently with the most

sensitive technique of two-dimensional gel electrophoresis of blood proteins (see ch. 3).

A report was prepared in 1980, under contract

to the Nuclear Regulatory Commission (NRC),

that evaluated opportunities for studying the

health effects of low-level ionizing radiation (29).

The report is focused on cancer, but the evaluation methods apply equally to studying mutations. The authors initially identified 100 candidate populations. About 30 remained after two

broad criteria were applied: 1) that there be data

identifying exposed individuals, and 2) that there

were at least 10,000 people in a single population

group or one comprising several similar groups.

Those 30 populations were evaluated further, and

recommendations made that if additional studies

were to be undertaken, three occupational groups

and one group with environmental exposure held

out the greatest promise of yielding a reliable result. Even the best of these, however, has a relatively low power: less than a 50 percent chance

of finding an excess of cancer if it exists. In general, this level of power would be unacceptable

in an epidemiologic study. Although political considerations might influence a decision to go ahead

with a study, they do nothing to increase the

power of the method.

The power figures for these studies refer to cancer detection, and the probability of detecting

heritable mutations is undoubtedly far lower,

making it unlikely at best that anything could be

learned about radiation and heritable mutations

by studying any of these groups with currentlyavailable methods.

The report to the NRC contained one other recommendation, that a registry for radiation workers be initiated. The registry would maintain

information about radiation doses and some information about other exposures. This recommendation has not been acted on. There are examples of radiation-exposure registries, but these are

mainly for people acutely exposed accidentally,

and not for the more usual long-term chronic exposures of workers.

Cancer Patients

Treatment for many cancers includes chemotherapy with cytotoxic drugs, some of which are

carcinogenic in laboratory animals and mutagenic

in vitro, and treatment with high doses of radiation. There is a growing body of evidence that

cancer patients are at a severalfold increased risk

of developing second cancers, and some of these

second cancers may be attributable to treatment

of the first cancer with drugs and radiation (see,

e.g., ref. 149). Cancer is mostly a disease of old

age, but certain cancers have their peak incidence

in younger people. Hodgkin’s disease, for instance, occurs with greatest frequency in young
Ch. 8—Mutation Epidemiology . 109

men. Childhood leukemias, some brain cancers,

and tumors with strong genetic components, e.g.

Wilms’ tumor, retinoblastoma, and neuroblastoma,

occur in the first few years of life. As treatment

for these early cancers has improved over the last

two decades, larger numbers of people are surviving, and it is these survivors who are at an increased risk of a second cancer, and possibly of

heritable mutations.

Results from four studies of the offspring of

childhood cancer survivors, and nine studies of

offspring of adult cancer patients have been published as of mid-1985. Several other studies are

in progress (82).

The combined published studies represent more

than 700 cancer patients (both male and female)

and more than 1,500 pregnancies, about 1,200 of

which resulted in live births. Four percent of the

liveborn babies had major birth defects, which is

similar to the incidence in the general population.

Two of the liveborn children had cancer. One had

a hereditary bilateral retinoblastoma, as his father had. The other, the daughter of a brain cancer survivor, had acute myelocytic leukemia. One

child had a condition that could have been the

result of a new mutation, the Marfan syndrome,

which fits the definition of a sentinel phenotype.

Several other children had defects that might have

had genetic components, but none of these represented sentinel phenotypes.

The largest study of offspring of childhood cancer survivors, including about 2,300 individuals

from five population-based cancer registries, is

nearing completion. Preliminary results indicate

no increased risk of cancer in offspring compared

with a control group, but the analysis is not yet

final (82). Another long-term followup study,

with more than 3,3oo cases enrolled to date, is

under way in the United Kingdom. No results are

yet available from that study (82).

The findings of a large international cooperative study of second tumors in children treated

for cancers are provocative (142). Overall, 12 percent of children who survive at least 2 years after a first cancer develop a second cancer sometime during the 25 years following the first cancer.

Most of the patients in the study were treated with

high-dose radiation therapy. The risk of second

cancers was highest among children with cancers

known to be strongly genetically influenced. In

that group, there may well be a genetic defect that

predisposes to mutations, e.g., a faulty repair

mechanism, which could also be related to a

higher risk of heritable mutations in that group.

Cancer registries are the most numerous registries of any type, and cohorts of treated patients

and patients with second tumors are relatively

easy to identify, compared with identifying other

populations potentially exposed to mutagens.

These groups should be considered when studies

of heritable mutations using the new technologies

become feasible.

Other Populations

A study of birth outcomes in people who had

attempted suicide by self-poisoning in Hungary

is an example of opportunistic use of available

information (23), A cohort of about 1,300 individuals who took large doses of drugs in suicide

attempts has been studied since 1976. Early on,

the investigators looked for short-term effects on

somatic cells, using cytogenetic and biochemical

testing. Long-term followup of birth outcomes examined spontaneous abortions, ectopic pregnancies, stillbirths, low birthweight, and congenital

anomalies. The study suffered a large loss of followup of study subjects, but in those evaluated,

no important excesses in any of these endpoints

were discovered.
110 . Technologies for Detecting Heritable Mutations in Human Beings

CONCLUSIONS

A very important question is answered by sim- greatly increase the power to identify mutations

ply observing birth outcomes in people thought in studies such as those described above, adding

to be at high risk, namely whether those individ- another dimension to knowledge about the relauals are at risk of having children with serious tionship between exposure to mutagens, the presdiseases and disabilities. The new mutation de- ence of detectable mutations in DNA, and the extection technologies discussed in this report may istence of observable health effects.
Chapter 9

Mutagens: Regulatory

Considerations
Contents

Page

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .....................113

Federal Involvement in Protecting Against Genetic Risk..... . ................113

Radiation Protection. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...............113

Agents Other Than Radiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........114

Regulatory Issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... ...115

A Regulatory Definition of a Germ-Cell Mutagen . . . . . . . . . . . . . . . ... ... ...115

Quantitative Extrapolation . . . . . . . . . . . . . . . . . . . ..........................117

Mutagenicity and Carcinogenicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..,117

New Methods for Measuring Mutation Rates and Their Potential Effects

on Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......118
Chapter 9

Mutagens: Regulatory Considerations

INTRODUCTION

It is clear from the information gathered in this

report that we are some years distant from being

able to muster convincing direct evidence of any

but very large increases in the rates of heritable

mutations in human beings. The ability to predict from experimental data which agents are

likely to increase the mutation rate if human beings were exposed has not been put to the test.

Even for the most likely mutagens, the ability to

make quantitative extrapolations is relatively undeveloped, Nonetheless, it is reasonable and prudent to accept that the environment may contain

human germ-cell mutagens and that, to the extent possible, human beings should be protected

from them at levels that might cause heritable mutations.

Ionizing radiation was recognized as a cause of

heritable mutations in fruitflies in the 1920s, and

the Federal Government has since made efforts

to protect workers and the public from excessive

radiation exposure. Widespread concern about the

mutagenic potential of chemicals is more recent,

an issue brought into focus by the environmental

movement that took shape in the late 1960s. While

the potential cancer-causing properties of manmade chemicals have been the driving force behind environmental health laws, two more recent

laws specifically mention mutation as an endpoint

against which the public should be protected.

About a dozen other statutes include language

broad enough to charge the Federal Government

with the responsibility to protect against heritable mutations. Evidence from current methods for

measuring mutation rates suggests that basing regulation of environmental agents on carcinogenicity will likely assure protection against heritable

mutations, but new, more sensitive detection technologies, such as those discussed in this report,

may necessitate a reexamination of that conclusion.

FEDERAL INVOLVEMENT IN PROTECTING AGAINST GENETIC RISK

Radiation Protection

In 1928, the newly created International X-Ray

and Radium Protection Commission was charged

by the Second International Congress on Radiology with developing recommendations for protection against radiation (156). The following

year, the Advisory Committee on X-Ray and Radium Protection was formed to represent the U.S.

viewpoint to the international commission. These

two bodies were the forerunners of the current

International Commission on Radiological Protection (ICRP) and the National Council on Radiation Protection and Measurements (NCRP),

the latter chartered by the U.S. Congress in 1964.

The ICRP and NCRP have, since their first recommendations in the 1930s based their acceptable radiation exposure limits on both heritable

and somatic effects. The limits recommended have

been lowered over the years, reflecting increased

knowledge about radiation effects, and particularly about the effects on the population of low

levels of radiation.

Neither the ICRP nor NCRP recommendations

have the force of law, but by and large, they have

formed the basis for the radiation protection limits

adopted by U.S. regulatory agencies. The first

Federal entity officially charged with providing

the agencies with guidance for developing radiation protection standards was the Federal Radiation Council (FRC), established in 1959. In 1960,

FRC issued recommendations for both occupational exposure and exposure of members of the

public, which drew on ICRP and NCRP work

(156). Over the years, the National Academy of

Sciences Committee on the Biological Effects of

Ionizing Radiation and the United Nations Sci113
114 ● Technologies for Detecting Heritable Mutations in Human Beings

entific Committee on the Effects of Atomic Radiation have also been influential in providing analyses that undergird exposure limits.

The National Environmental Protection Act of

1970 transferred the responsibilities of FRC to the

new Environmental Protection Agency (EPA).

EPA administers several environmental health

statutes under which that Agency is responsible

for setting standards for radiation exposure in specific conditions. Under its broader responsibilities, EPA has provided guidance for exposure

from diagnostic X-rays, which is the regulatory

responsibility of the Federal Food and Drug

Administration (FDA), and for exposure of uranium miners, who are the responsibility of the

Mine Safety and Health Administration (MSHA).

Agencies other than EPA with responsibility for

some aspects of radiation protection include: the

Nuclear Regulatory Commission, the Department

of Energy, the Department of Defense, FDA,

MSHA, the Occupational Safety and Health

Administration (OSHA), and the Department of

Transportation. The States have responsibilities

as well. Each entity, depending on its specific

charge, is required to protect workers, the public, or both in accordance with the guidance provided by EPA.

The basic occupational and population exposure guidelines have not been revised since

1960. In 1981, EPA proposed new occupational

guidelines (153), in line with 1977 ICRP recommendations (47), but these have not been made

final, and they do not represent a change in total

acceptable dose from the earlier guidelines. They

do, however, place less of the emphasis on mutagenesis, and relatively more on somatic effects

than do the 1960 guidelines. The ICRP includes

in its risk estimates only genetic effects occurring

in the first two generations after exposure. That

probably accounts for roughly half of the total

genetic effect, whatever its size.

The current occupational exposure limit is 5 rem

total body dose per year, with not more than 3

rem total body dose from occupational exposure

in any one quarter of the year, and a more detailed

breakdown for different groups of organs. The

1977 ICRP recommendations abandon the specifications by organ, and use a weighted wholebody dose.

Today, almost all radiation exposures of U.S.

workers are well below the regulated limits,

though there are exceptions. The quantitative

limits set by the ICRP and NCRP and adopted

by Federal groups, are accompanied by the

“ALARA principle’ ’—that radiation exposures

should be “as low as reasonably achievable. ”

Exposure to the public are to be limited to below 25 millirem (mrem) to the whole body, 75

mrem to the thyroid, and 25 mrem to any other

organ. These levels mainly affect the regulation

of radionuclides in air and the disposal of radioactive waste. The numbers come from ICRP and

NCRP recommendations, and are based on consideration of both genetic and somatic effects.

Agents Other Than Radiation

Congress formally recognized the need to protect against chemical mutagens in the Toxic Substances Control Act of 1976 (TSCA), and again

in 1980 in the Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA,

or “Superfund”). These two laws are administered

by EPA, as are other statutes that include broad

mandates to protect the public from environmental hazards. Other laws designed to protect

citizens from external agents under which chemical mutagens could be regulated include the Federal Food, Drug, and Cosmetics Act, administered

by FDA; the Occupational Safety and Health Act,

administered by OSHA; the Consumer Product

Safety Act, administered by the Consumer Product Safety Commission; and the Atomic Energy

Act, through which the Nuclear Regulatory Commission is empowered to protect certain workers

from radiation hazards.

Although it is almost certain that chemicals that

might cause heritable mutations have been regulated, no regulations have been written or standards set for these agents because of that property.

In a few cases, the mutagenic potential of chemicals has been considered by regulatory agencies,

but carcinogenic properties have driven standardsetting.
Ch. 9—Mutagens: Regulatory Considerations . 115

OSHA has included thorough reviews of mutagenicity data in notices of regulatory actions for

two of the best-publicized chemical hazards of the

1980s: ethylene oxide (EtO) and ethylene dibromide (EDB). Tests for heritable mutagenicity in

Drosophila and experimental mammals have

yielded positive results in at least some systems

for both of these chemicals. While results of

mutagenicity tests are included in the Federal Register notices for these chemicals, quantitative extrapolations for both the final EtO standard (152),

and the proposed rulemaking for EDB (151), are

based on protecting against carcinogenicity.

REGULATORY ISSUES

Currently there does not appear to be a scientific basis for the specifics of regulatory action

against mutagens. The following questions face

regulators and the scientific community involved

in mutation research:

1.

2.

3.

4.

What is an appropriate regulatory definition

of a probable human germ-cell mutagen and

how does that definition relate to what is

known about mutagens from epidemiologic

studies and experimental studies?

Is it possible to derive quantitative estimates

of the risk of heritable mutations in humans

from experimental evidence in animals or

from somatic-cell mutation tests in human

beings? If it is not, what kinds of information are necessary before such extrapolation

is possible?

How likely is it that a substance will require

a more stringent standard as a mutagen than

it will as a carcinogen or for other toxic

effects?

How will information from the new technologies for detecting heritable mutations that

are described in this assessment change our

perception of the kinds of “adverse effects”

against which regulation should be directed?

The discussion in the remainder of this chapter addresses these questions.

EPA has recognized germ-cell mutagenicity as

a class of adverse effects, particularly in its responsibilities under the Federal Insecticide, Fungicide,

and Rodenticide Act, under which it, in addition

to OSHA, has acted to regulate exposures to EtO

and EDB. Unique among the regulatory agencies,

EPA’s Reproductive Effects Assessment Group (in

the Office of Research and Development) has prepared guidelines for mutagenicity testing, which

are described later in this chapter.

A Regulatory Definition of

a Germ-Cell Mutagen

Strategies for regulating mutagens to protect

public health cannot today rely on data from current studies of heritable mutations in human beings. Just as is the case in regulating carcinogens,

a regulatory definition must serve as a substitute,

particularly for making judgments about the potential risks of new substances.

Certain lessons can be learned from experience

in regulating carcinogens. (For a discussion of the

issues surrounding carcinogen regulation, see

145. ) The most convincing evidence for carcinogenicity, from well-conducted epidemiologic studies, is that human beings have developed cancer

after exposure to a given agent. If an increase in

genetic disease could be convincingly shown to

be related to a specific agent, there would certainly

be no problem in acting against that agent. The

spirit of the regulatory laws, however, embody

the concept of taking protective action before people are harmed. In the absence of direct evidence

from human beings, regulators must rely on indirect evidence from a variety of experimental test

systems which will never be absolutely predictive

of effects in human beings. A regulatory definition of a mutagen will be pragmatic and rely on
116 . Technologies for Detecting Heritable Mutations in Human Beings

information that it is possible to collect, and on

a number of untested assumptions.

EPA is the first U.S. regulatory agency to have

proposed “Guidelines for Mutagenicity Risk Assessment” (154). The guidelines require evidence

of: 1) mutagenic activity and 2) chemical interactions in the mammalian gonad. The decision to

regulate is to be based on a “weight-of-evidence”

determination. EPA has proposed no formal

method for quantitative extrapolation by which

acceptable exposure levels could be set.

According to the EPA guidelines, evidence for

mutagenic activity may come from tests that detect point mutations and structural or numerical

chromosome aberrations. Structural aberrations

include deficiencies, duplications, inversions, and

translocations. In the absence of evidence of

heritable mutations in human beings, evidence

from a variety of experimental test systems may

be invoked. For mutagens that cause point mutations, whole animals tests (e.g., the mouse

specific-locus test) provide the highest degree of

evidence, but these tests are relatively more expensive than short-term tests, and there is a

limited capacity for laboratories to perform them.

Other tests for point mutations include those in

bacteria, eukaryotic micro-organisms, higher

plants, insects, and mammalian somatic cells.

Structural chromosome aberrations can be detected either in somatic or germ cells in different

assays. The organisms used include higher plants,

insects, fish, birds, and several species of mammals. Mutagens that cause numerical changes in

chromosomes may be missed by the tests that

directly measure DNA damage. Tests specifically

directed at detecting changes in chromosome number are not as well developed as are those for point

mutations or structural changes in chromosomes.

Tests are in various stages of development in

fungi, Drosophila, mammalian cells in culture,

and intact mammals, including mammalian germcells tests.

Results from tests that measure endpoints other

than mutagenicity directly may also be used in

judging the potential mutagenicity of a substance.

DNA damage, unscheduled DNA synthesis in

mammalian somatic and germ cells, mitotic recombination and gene conversion in yeast, and

sister chromatid exchange in mammalian somatic

and germ cells are cited by EPA as tests that provide evidence known to be correlated with mutagenicity, though they measure other genetic

events.

Evidence from various kinds of mutagenicity

tests is weighted with regard to the relationship

of the test to human germ-cell mutation. Greater

weight will be given to results from tests in: 1)

germ cells over somatic cells, 2) mammalian cells

over submammalian cells, and 3) eukaryotic cells

over prokaryotic cells.

EPA lists two classes of evidence for chemical

interactions in the mammalian gonad: sufficient

and suggestive. Sufficient evidence is from studies

in whole mammals that demonstrate, for example, unscheduled DNA synthesis, sister chromatid

exchange, or chromosomal aberrations in germ

cells. Adverse effects on the gonads or on reproductive outcomes after exposure, which are consistent with the substance reaching the gonads but

which do not indicate direct interaction with

DNA, are considered as providing suggestive

evidence.

The final step in EPA’s mutagenicity risk assessment is the weight-of-evidence determination,

which classifies the evidence for potential germcell mutagenicity as “sufficient,” “suggestive,” or

“limited.” In this step, results of tests plus any information about effects in human beings is evaluated. Sufficient evidence consists of a positive

mammalian germ-cell test. In addition, positive

responses in at least two different test systems,

at least one of which is in mammalian cells, and

evidence of germ-cell interaction, together constitute sufficient evidence. Evidence of lesser quantity and/or quality of both mutagenic response

and germ-cell activity constitute suggestive evidence. Limited evidence consists of positive results in either mutagenicity assays or tests for

chemical interactions in the gonad, but not both.

EPArS guidelines became final in September

1985. Currently and for the foreseeable future, the

greatest value of EPA’s guidelines is the recognition of germ-cell mutagenicity as a legitimate endpoint to consider in assessing the potential adverse

effects of substances in the environment.
Ch. 9—Mutagens: Regulatory Considerations • 117

Quantitative Extrapolation

If the levels of risk from suspected human germcell mutagens is to be estimated in the absence of

direct evidence of harm in human beings, data

from experimental systems must be used in a

“quantitative extrapolation. ” The experimental

systems are basically those mentioned in the EPA

guidelines discussed in the previous section, and

those discussed elsewhere in this report. The three

categories of tests are: 1) whole animal heritable

mutation studies; 2) animal somatic-cell mutation

studies, either in vivo or in vitro; and 3) human

somatic-cell mutation studies, either in vivo or in

vitro. Unfortunately, the kind of information (i.e.,

measures of human mutations) that would link

results from these three categories of tests to human heritable mutations is scanty. It is encouraging, however, that using tests available now, such

information can be generated at least for some

substances. For EtO and EDB, for instance, mutagenicity data are available in both somatic and

germ-cell systems in animals, and some somatic

cell (cytogenetic) data are available from human

beings exposed at known occupational levels.

Obtaining more information to fill in the arrows of the extrapolation “parallelograms” presented in chapter 7 of this report should be a high

priority for regulators. In fact, EPA’s Reproductive Effects Assessment Group has collaborated

with other groups in the Federal Government to

fund such studies (168). Without the kind of information that would come from coordinated

studies in several test systems, there is little chance

of writing a successful regulation that limits exposure to a specific level (short of a complete ban

for an agent acknowledged to be unacceptably

risky at any level). Given the experience with carcinogens, a regulation that states an exposure level

without adequate experimental evidence and theory behind it will not survive a court challenge,

which, in the United States today, appears to be

the final test of a regulation.

Even with good experimental data, some of the

same problems that plague extrapolating from animals to humans to determine acceptable exposure

levels for carcinogens are certain to hinder quantitative extrapolation for estimating levels of

mutagenic risk at specific levels of exposure. In

carcinogen extrapolation, there still are unresolved

controversies about the appropriate conversion

factors between species and about the shape of

dose-response curves. The latter is important because most animal bioassays test extremely high

doses in relation to the animals’ body weights,

while humans are generally exposed at lower

levels over longer periods of time. Though the details of extrapolation for mutagenicity differ from

those for carcinogenicity, the problems will undoubtedly be similar. Right now, there is not

enough information about the relationships between results in various test systems to address

intelligently the practical problems of actually performing extrapolations.

Given the appropriate information, it may become possible to carry out quantitative extrapolations for mutagenicity. When the time comes,

the regulatory agencies will need to require that

the appropriate tests are done, either by manufacturers or by the Federal Government. The testing requirement may take various forms, which

may vary by statute.

Mutagenicity and Carcinogenicity

As mentioned previously, while the regulatory

apparatus exists for acting against human germcell mutagens, in fact no regulations based on

germ-cell mutagenesis exist except for radiation

exposure limits. There are several reasons for this.

First, there are no proven human germ-cell mutagens, and only a limited number of presumptive

human mutagens known from animal tests. Second, it has often been thought that regulations

based on demonstrated carcinogenicity would

automatically protect against mutagenicity as

well. In fact, however, this may not always be

true. Voytek (167) reported a preliminary assessment indicating that the risk of heritable genetic

disease in the first generation after exposure to

EDB was greater than the lifetime risk of cancer

in the exposed individuals, based on an extrapolation from animal data.

It is widely held that a somatic mutation is a

necessary step in the development of cancer.

Many substances that are mutagenic in bacteria,

in cells in culture, and in Drosophila also are carcinogenic in laboratory rats, mice, or both. Short-
118 . Technologies for Detecting Heritable Mutations in Human Beings

term tests based on mutagenicity in these lower

organisms are therefore used as screens for carcinogenicity. The most widely used screening test

for carcinogenicity is the Ames test, which measures mutagenicity in strains of the bacterium

Salmonella. Under some statutes (e.g., TSCA),

negative results in short-term tests, meaning that

the substance is not mutagenic in those systems,

can obviate the need for a long-term bioassay, a

savings of up to $1 million to a manufacturer

(145). Positive results in short-term tests, meaning that the substance is mutagenic in these systems, have not been accepted as a basis for regulation under any statute, but they have probably

halted the development of new chemicals. Manufacturers know that positive mutagenicity tests

may trigger the requirement for a long-term bioassay, which in a high proportion of cases will

turn out positive. The product, whatever it is,

might never get to market. Rather than risk a financial loss, many manufacturers simply will not

proceed with that product.

New Methods for Measuring Mutation

Rates and Their Potential Effects on

Regulation

The new techniques for detecting heritable mutations and the somatic-cell techniques that eventually may be used to predict germ-cell mutagenesis will lead to the consideration of effects that

are increasingly removed from measurable or even

hypothetical adverse health effects in humans.

Mutagenicity endpoints may be detected that

could be more sensitive than those currently used

to predict carcinogenicity. In the regulatory context, judgments will have to be made, most likely

in the absence of certain knowledge of effects,

about the appropriate actions to be triggered by

demonstrations of various kinds of changes in

DNA, detectable by various analytic methods,

that can be convincingly linked to specific exposures. From a public health standpoint, it is

most appropriate to act under the assumption that

mutations of any kind are deleterious, and that

environmental agents at levels that cause any

reliably detected changes in the DNA should be

subject to available regulatory controls. This does

not get around the problem of defining “safe” or

“acceptable” exposure levels, however. Animal experiments will be needed to explore the quantitative relation between subtle changes detectable

anywhere in DNA and the levels of adverse effects that might be observed in the animal.

At present, testing for safety is a significant part

of the research and development investment in

new products, whether they are chemicals, drugs,

or food additives; determining the risks of substances already in the environment is a significant

task for the Federal Government. If they become

available, new mutagenicity testing technologies

using experimental animals could either impose

significant new testing requirements in addition

to those already in place, or could replace some

expensive and not entirely reliable tests for carcinogens. Regardless of the methods used to detect mutations, the relation between specific types

of changes in DNA and health effects will have

to be studied experimentally to shed light on the

meaning of a “positive result. ” Until this knowledge is available, the ability to detect an effect

without knowing the likelihood of any health consequences for human beings will remain a thorny

public policy question which scientists, regulators,

and politicians must address.
Appendixes
Appendix A

Federal Spending for Mutation Research

A quick review of the origins of biotechnology illustrates the difficulty of identifying the specific areas

of research that would further our knowledge of human mutations, and, in turn, the problems of estimating the current amount of Federal expenditures for such

research.

In 1968 and 1969, Hamilton O. Smith, a microbiologist at Johns Hopkins University, had no thought of

studying human heritable mutations. No venture capital firm was looking for biotechnology companies to

invest in, and had it been, it would have been disappointed. No such company existed,

Smith was studying how some bacteria can take up

molecules of DNA from their growth medium and

recombine it into their genetic material. He knew that

the bacteria would not take up DNA from other species of bacteria or from viruses, and he added some

viral DNA to the experiment expecting that it would

remain inert and intact while the bacterial DNA underwent recombination. Instead, the viral DNA was

rapidly degraded. Looking at that result, Smith hypothesized that a bacterial enzyme(s) could discriminate between foreign DNA (from the virus), which it

degraded and self DNA (from the same kind of bacteria), which it left alone. He purified the enzyme and

showed that it recognized particular sequences of

nucleotides on the viral DNA and cut it at those sites

(128).

These experiments, which identified and characterized the first known restriction enzyme, opened the

door to the discovery of hundreds of others. The use

of restriction enzymes, allowing precise cutting of

DNA and precise joining of DNA pieces from different parts of an organism’s genome as well as joining

of pieces from different organisms, led to fundamental advances in the basic approach of research in

molecular biology and genetics (146,147). Accordingly, these enzymes are basic ingredients of the new

technologies for studying human heritable mutations

described in this report,

However, someone in 1969 who had tried to identify research projects that would contribute to our understanding of human mutation rates would almost

certainly not have included Smith’s. His was basic research directed at understanding the mechanism of

recombination in an organism far removed from humans. This OTA report provides additional examples

of the difficulty of estimating how much money is being spent on research that may be important to understanding human mutations.

Current Federal Expenditures for

Mutation Research

OTA queried Federal research and regulatory agencies about their support of research directed at understanding human mutations. Questions were asked

about the amount of money spent specifically on human mutation research and the amount spent on “related” areas of biological research. The first category

was tightly defined—the research had to focus on development or applications of methods for detecting

and/or counting human somatic or heritable mutations

in vivo. The category of “related” research was much

broader, including studies examining genetic contributions to such common diseases as diabetes and arthritis, studies of mutagenesis and repair mechanisms,

development of instruments for measuring cellular effects of various kinds, and tests for mutagenic activity in lower organisms as well as in cultured human

cells.

The expenditures shown in table A-1 must be treated

with some reservation about their accuracy, the total

of $14.3 million estimated as the amount spent on human mutation research being more precise than the approximately $207 million estimated for related genetic

research. The $14.3 million may be an overestimate—

Table A-1 .—Federal Expenditures for Research

Related to Human Mutations

Human Related

mutation genetic

Federal aqency research research

Department of Energy $ 6,220,000a $ 30,123,000

Department of Health and Human

Services:

Centers for Disease Control 277,000 1,387.000

National Institutes of Health, 7,244,000b156,959,000C

Food and Drug Administration 1,200,000

Environmental Protection Agency 606,000 1,189,000

National Science Foundation 0 16,336,000

Tota l 14,347,000 207,194,000

aThl~ includes $1 ,e,50 000 (or half of the total $3 700,000 at current exchange rates) spent annually for the U S contribution to the Radlatlon Effects Research Foundation the group that coor.

dmates ongoing genetic studies of the survwors of the Nagasaki and Hlroshlma bombings and

thetr offspr!ng

bThls total was obtained by inspecting grant Mles and reading grant abstracts to ldefltlfy thOSe

that focused on human mutahon research

cThls figure IS fhe budget for the (%netlcs Program m the National lnStltW? Of General Medcal

Sctences It does not include all genetics research at NIH some of which goes on m other Institutes

SOURCE Off Ice of Technology Assessment

721
122 . Technologies for Detecting Heritable Mutations in Human Beings

some projects studying clinical effects of mutations

may provide no information about detection—and the

$207 million for related genetic research is probably

an underestimate, since much genetic research is supported by parts of the National Institutes of Health

(NIH) not queried. For comparison, the total NIH

budget for the fiscal year 1985 was about $4 billion.

Most of the NIH funding for mutation research is directed at identifying mutations in at-risk families

(where specific tests for specific mutations are appropriate), whereas DOE supports a search for more general methods to detect mutations.

Successes of the research efforts in human mutation

research and related genetic research will probably

generate a requirement for additional funding in the

near future. It will be a costly venture to take any new

technique (such as those described inch. 4) and apply

it to studies of populations sufficiently large to provide a good possibility of yielding information about

human mutations. For instance, the ongoing monitoring of the Nagasaki and Hiroshima populations using

clinical examinations and protein analyses (see ch. 3)

costs about $3.7 million annually to the U.S. and Japanese governments together (48).
Appendix B

Acknowledgments and

Health Program Advisory Committee

HEALTH PROGRAM ADVISORY COMMITTEE

Sidney S. Lee, Chair

President, Milbank Memorial Fund

H. David Banta

Project Director

STG Project on Future Health Technology

The Netherlands

Rashi Fein

Professor

Department of Social Medicine and Health Policy

Harvard Medical School

Harvey Fineberg

Dean

School of Public Health

Harvard University

Patricia King

Professor

Georgetown Law Center

Joyce C. Lashof

Dean

School of Public Health

University of California, Berkeley

Alexander Leaf

Professor of Medicine

Harvard Medical School

Massachusetts General Hospital

Frederick Mosteller

Professor and Chair

Department of Health Policy and Management

School of Public Health

Harvard University

Norton Nelson

Professor

Department of Environmental Medicine

New York University Medical School

Robert Oseasohn

Associate Dean

School of Public Health

University of Texas

Nora Piore

Senior Fellow and Advisor to the President

United Hospital Fund of New York

Dorothy Rice

Regents Lecturer

Department of Social and Behavioral Sciences

School of Nursing

University of California, San Francisco

Richard Riegelman

Associate Professor

George Washington University

School of Medicine

Walter Robb

Vice President & General Manager

Medical Systems Operations

General Electric

Milwaukee, WI

Frederick C. Robbins

University Professor

Department of Epidemiology and Biostatistics

School of Medicine

Case Western Reserve University

Cleveland, OH

Frank E. Samuel, Jr.

President

Health Industry Manufacturers’ Association

Rosemary Stevens

Professor

Department of History

and Sociology of Science

University of Pennsylvania

123
124 ● Technologies for Detecting Heritable Mutations in Human Beings

ACKNOWLEDGMENTS

OTA would like to thank the members of the advisory panel and the contractors who provided information

for this assessment. In addition, OTA acknowledges the following individuals for their assistance in reviewing

drafts or supplying information.

F. Alan Andersen

U.S. Food and Drug Administration

Benjamin J. Barnhart

U.S. Department of Energy

Fred Bergmann

National Institute of General Medical Sciences

David Botstein

Massachusetts Institute of Technology

Noel Bouck

Northwestern University School of Medicine

Michele Calos

Stanford University School of Medicine

L.L. Cavalli-Sforza

Stanford University School of Medicine

George Church

University of California at San Francisco

Mary Ann Danello

U.S. Food and Drug Administration

Amiram Daniel

Health Industry Manufacturers Association

Frederick J. DeSerres

National Institute of Environmental Health Sciences

Robert Dixon

U.S. Environmental Protection Agency

William Fitzsimmons

National Institute of General Medical Sciences

W. Gary Flamm

U.S. Food and Drug Administration

Joseph Fraumeni

National Cancer Institute

Helen Gee

National Institutes of Health

Margaret Gordon

National Institutes of Health

Ronald Hart

National Center for Toxicological Research

John Heddle

Ludwig Institute for Cancer Research (Canada)

Richard Hill

U.S. Environmental Protection Agency

Abraham Hsie

Oak Ridge National Laboratory

Seymour Jablon

National Research Council

Irene Jones

Lawrence Livermore National Laboratory

Moin Khoury

Johns Hopkins University

Norman Mansfield

National Institutes of Health

Karen Messing

University of Quebec at Montreal

Alexander Morley

Flinders Medical Center (Australia)

John J. Mulvihill

National Cancer Institute

DeLill Nasser

National Science Foundation

Neal S. Nelson

U.S. Environmental Protection Agency

Per Oftedal

University of Oslo

Doug Ohlendorf

E.I. du Pent de Nemours & Co.

Dilys Parry

National Cancer Institute

Claude Pickelsimer

Centers for Disease Control

William L. Russell

Oak Ridge National Laboratory

F. Raymond Salemme

E.I. du Pent de Nemours & Co.

Michael M. Simpson

Congressional Research Service

David A. Smith

U.S. Department of Energy
App. B—Acknowledgments and Health Program Advisory Committee • 125

Gail Stetten Larry R. Valcovic

Johns Hopkins Hospital U.S. Environmental

J. Timothy Stout Peter E. Voytek

Baylor College of Medicine U.S. Environmental

Gary Strauss

U.S. Environmental Protection Agency

Protection

Protection

Agency

Agency
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Index
Index

Achondroplasia (dwarfism), 7, 30, 31

Ad hoc studies, epidemiologic, 14, 103, 104, 107

Adenine (A), 23, 24, 27, 64, 66

Adenosine deaminase deficiency, 105

Agarose gel electrophoresis, 59, 60, 61, 63, 68

Agency for Toxic Substances and Disease Registry

(ATSDR), 18-19

Agent Orange, 18

Alanine, 27

ALARA principle, 114

Alkylating agents, 93-94

Alleles, 27, 44, 77, 82

Alpha-globin chain, 47

Alpha-thalassemia, 29

American Society of Human Genetics, 19

Ames test, 15, 118

Amino acids, 9, 25-27, 29, 44, 46, 47, 49, 57, 58,

78; see also specific amino acids

Amniocentesis, 41

Animals, mutation studies in, 3-5, 6-7, 19, 48, 78,

81-87, 91-100, 117-118

Aniridia, 7

Antibodies, in somatic mutation studies, 77-78

Apert’s syndrome, 31

Arginine, 27

Asparagine, 27

Aspartic acid, 27

Atomic Bomb Casualty Commission, 48

Atomic bomb survivors, effects of radiation on,

9-10, 32, 48-50, 77, 86, 106, 108

Atomic Energy Act, 114

Atomic Energy Commission (AEC), 82, 114

Autoradiographic assay, in somatic mutation

studies, 74-75

Autoradiography, 56, 59, 61, 64, 65, 68, 74-75

Autosomes, 27, 38, 42, 44, 46, 49

Bacteria, 81, 91, 92, 94, 116, 117, 118

Benzo[a]pyrene, 97

Beta-globin chain, 47, 77

Biochemical analyses, 37, 44-48, 49-50, 51, 52

Biologic extrapolation, see Extrapolation,

qualitative

Biological effects of ionizing radiation, 113

Biological samples, banking of, 17-18

Blood cells, somatic mutation studies on, 73-78

Blood proteins

detection of variants in, 44-48, 49-50

electrophoretic analysis of, 8-9, 10, 37, 44-46,

50, 51, 83

molecular analysis of, 46-48

see also, Enzymes; Hemoglobin proteins

Cancer, 3, 7, 9, 15, 31-32, 38, 50, 73, 77, 104-105,

108-109, 117

Cancer registries, 105, 109

Case-control studies, in epidemiologic testing, 107

Cells, see Blood cells; Chorionic villi cells; Germ

cells; Red blood cells; Somatic cells; Stem

cells; TGr cells; White blood cells

Centers for Disease Control, 18

Chemicals

denaturing, 12, 59, 61

effects on DNA, 23, 59, 81

effects on germinal mutations, 7, 9

effects on heritable mutations, 6-7

effects on somatic mutations, 7, 74-78

in extrapolation models, 93-97, 100

as mutagenic agents, 6-7, 9, 32, 48, 91-97, 106

“standards” for, 17

Chemotherapy, 108

Children, genetic diseases in, 7-8, 30-32, 37, 38-40,

47-48, 49/ 50, 109, 110

Chorionic villi cells, 41

Chromosomes

abnormal at birth, 41-43

banding methods, 8, 28, 41, 42, 43, 50

biochemical analyses of, 49-5o

cytogenetic analysis of, 37, 40-43, 49, 52, 55, 98

DNA and, 23-25, 27-28, 41, 43

health effects of abnormal, 40-43, 49

mutations in, 5, 6, 8, 9, 13, 29-30, 31, 32,

40-43, 49, 51, 68, 82, 92, 95

numerical abnormalities, 8, 29-30, 31, 41, 49,

116

pulsed field gel electrophoresis and, 68

staining methods, 8, 28, 41, 42, 43, 50

structural abnormalities, 8, 29, 31, 41, 49

studies in mice, 82

surveillance of abnormal, 105

translation of, 30, 82

Chronic hemolytic anemia, 46, 47

Clonal assay, in somatic mutation studies, 75

Cloning, 58-59, 60, 64-65, 75

Codons, 6, 25-27, 29

Cohort studies, in epidemiologic testing, 107, 109

Comprehensive Environmental Response,

Compensation, and Liability Act of 1980

(CERCLA), 3, 15, 114

Congenital hypothyroidism, 105

Consumer Product Safety Act, 114

Consumer Product Safety Commission, 114

Cyanosis, 47

Cyclophosphamide, 97

Cysteine, 27

139
140 ● Technologies for Detecting Heritable Mutations in Human Beings

Cytogenetic analysis, of chromosome

abnormalities, 37, 40-43, 49, 52, 55, 98

Cytosine (C), 23, 24, 27, 64, 66

Cytotoxic drugs, 108

Deoxyribonucleic acid (DNA)

chromosomes and, 23-25, 27-28, 41, 43

current methods for studying, 6-10

denaturing of, 11-12, 59-64

double helix, 24, 25, 27

double-stranded, 12-13, 23, 59-68

EPA guidelines and, 116

extrapolation studies and, 91-1OO

Federal expenditures on research, 121-122

function and organization of, 23-28

genetic nature of, 3, 4, 5-6

genomic, 6, 11, 55, 57-68

heteroduplexes, 12, 55, 61, 62, 63, 64-65, 67

homoduplexes, 61, 63, 67

hybridization, 66-67

nontranscribed, 45

nucleotide chains in, 23-25

organizational hierarchy of, 6

replication of, 23-25, 74

sequencing, 23-25, 55, 57-58, 60, 61-64, 66-67,

104

single-stranded, 12-13, 59-68

structure of, 23-25, 81

use of new technologies in examining, 10-14, 17,

55-69, 74-76

Department of Defense, 114

Department of Energy (DOE), 16, 17, 19, 55, 114,

122

Department of Transportation, 114

Diabetes, 3

Dioxin, 106

Disease registries, 104-106, 107

Diseases

dominant, 44

environmental factors in, 31

genetic, 3, 5, 7-8, 9, 30-33, 37, 38-40, 42, 44-45,

47-48, 49, 50, 93, 94, 103-110, 108-109, 110,

115

“multifactorial, ” 31

recessive, 44

see also Sentinel phenotypes and specific diseases

DNA-RNA heteroduplex analysis, 12, 64-65

Dominant lethal test, 81-82

Dominant mutation tests, 83

Dose-response relationships, in quantitative

extrapolation studies, 97-99

Down syndrome, 3, 8, 30, 31, 42

Drosophila, 48, 81, 82, 96, 116, 117

Duchenne muscular dystrophy, 3, 30

Electrophoresis, 8-9, 10, 11-12, 13, 37, 44-46, 49,

50, 51, 55, 68, 83, 84

see also Gel electrophoresis and specific types of

gel electrophoresis

Embryos, in rodent tests, 81-82

Endonucleases, restriction, use in detecting

mutations, 11

Environmental Protection Agency (EPA), 15, 16,

19, 114, 115, 116, 117

Enzymes

biochemical analyses of, 49-5o

HPRT, 74

kinetic studies of, deficiency variants, 44, 45-46

quantitative studies of deficiency variants, 45-46,

83, 84

restriction, 11, 13, 58-64, 66, 67, 68, 75

RNaseA, 12

role in protein synthesis, 25-27

somatic mutation studies on, 73

Epidemiologic studies, 9-1o, 14, 18, 49, 103-11o

Epstein-Barr virus, 104

Escherichia coli, 58, 60

Ethylene dibromide (EDB), 115, 117

Ethylene oxide (EtO), 115, 117

Ethylnitrosourea (ENU), in quantitative

extrapolation studies, 97, 98-99

Eukaryotes, 92, 94, 95, 116

Exposure registries, 106, 107

Extrapolation

aims of, 91-92

from animal to human data, 86-87

defined, 14-15

limits of, 99-100

qualitative, 15, 91, 94-97, 99-100

quantitative, 15, 91, 97-100, 115, 117

theoretical models for, 15, 93-94

Federal Food, Drug, and Cosmetics Act, 114

Federal Insecticide, Fungicide, and Rodenticide

Act, 115

Federal Radiation Council (FRC), 113, 114

Fetuses, chromosome abnormalities in, 40, 42, 82

Fluorochrome, 77

Food and Drug Administration (FDA), 19, 114

Fruit flies, see Drosophila

Fungi, 92

Galactosemia, 105

Gamma radiation, 97

Gel electrophoresis, 11-12, 13, 55, 56, 58, 59, 65

see also Agarose gel electrophoresis; Onedimensional denaturing gradient gel

electrophoresis; Pulsed field gel

electrophoresis; Two-dimensional denaturing
Index • 141

gradient gel electrophoresis; Two-dimensional

polyacrylamide gel electrophoresis

Genes

dominant, 27, 44, 51

globin, 29, 46, 47-48, 55

hprt, 55, 74-76

mutations in, 5, 6-7, 29, 38-40, 42-43, 44, 47-48,

50, 51, 55, 57-68, 74-76, 92, 95

recessive, 27, 44

Genomes and genomic sequencing, 6, 11, 57-58,

66-67, 68

Germ cells, 6, 7, 9, 13, 27, 30, 31, 37, 38, 39, 48,

49, 80-84, 91-100, 108, 115-116, 117; see also

Germinal mutations

Germinal mutations

in cells, 7

defined, 6

electrophoretic studies on, 51

new, 37-38

radiation-induced, 9-10, 48-49

relationship to somatic mutations, 73

see also Mutations

Glossary of Acronyms and Terms, vii-xii

Glutamic acid, 26, 27, 29

Glutamine, 27

Glycine, 27

Glycophorin A, 76-77

Glycophorin assay, in somatic mutation studies,

76-77

Guanine (G), 23, 24, 27, 64, 66

“Guidelines for Mutagenicity Risk Assessment, ”

116

Health effects

of chromosome abnormalities, 40-43

of mutations, 4, 30-32, 108-109

see also Children; Diseases; Humans; Infants

Heart disease, 3, 25, 32

Hemoglobin A (HbA), 46

Hemoglobin assays, in somatic mutation studies,

77-78

Hemoglobin C, 77

Hemoglobin M (HbM), molecular analysis of, 44,

46-48, 51

Hemoglobin S, 77

Hemoglobins, unstable, molecular analysis of, 44,

46-48, 51

Hemophilia, 3

Heritable mutations

chemicals and, 6-7, 9, 32, 48, 91-97

defined, 6

detection and measurement of, 11-13

epidemiologic studies in, 9-10, 14, 18, 49,

103-110

estimating effects in humans from animal data,

86-87

extrapolation of data to predict risk in humans,

91-100

factors that influence induced rates in mice,

84-86

health effects of, 30-32, 108-109

induced by chemicals, 6-7

kinds and rates of, in humans, 32-33, 37-52

laboratory studies on, 81-87

new technologies and, 3, 10-14, 55-69

rodent tests for, 81-84

spontaneous, studies in humans, 7-IO

see also Mutations

Heritable translocation tests (HTTs), 82, 94-96, 98

Heteroduplexes, 12, 55, 61, 62, 63, 64-65, 67

Heterozygotes, 44, 45, 77, 82

Histidine, 27, 47

Histidinemia, 105

Hodgkin’s disease, 108

Homocystinuria, 105

Homoduplexes, 61, 63, 67

Homologous sequences, 64

Homozygotes, 44, 82

hprt, 55, 74-76

Humans

animal studies and, 84-87

epidemiologic studies in, 9-10, 14, 18, 49,

103-110

Federal expenditures on mutation research in,

121-122

frequencies of hprt – T-lymphocytes in, 76

health effects of mutations in, 30-32, 108-109

kinds and effects of mutations in, 4, 5-6, 29-32

kinds and rates of heritable mutations in, 32-33,

37-52

mutagenic risk predictions by extrapolation,

91-100

mutation studies in, 3-4, 7-14, 19, 44-52, 55-69,

73-78

new technologies for detecting mutations in,

10-14, 55-69, 73-78

persistence of new mutations in, 32-33, 37

sentinel phenotypes in 7-8, 37, 38-40, 45, 50, 51,

52, 55, 105, 106, 109

spontaneous mutation rates in, 32-33, 51

see also Children; Diseases; Infants

Huntington disease, 31

Hypoxanthineguanine phosphoribosyl transferase

(HPRT), 74

Infants, genetic diseases in, 7-8, 30-32, 37, 38-40,

42, 44-45, 49, 50, 109

Insects, 91, 92, 116
142 . Technologies for Detecting Heritable Mutations in Human Beings

International Commission for Protection Against

Environmental Mutagens and Carcinogens

(ICPEMC), 55

International Commission on Radiological

Protection (ICRP), 113, 114

International dioxin registry, 106

Isoleucine, 27

Karyotypes, 28, 41, 42

Kinetic studies, of enzyme deficiency variants,

45-46

Laboratory studies, of heritable mutation rates,

81-87; see also Animals

Lambda (virus), 59, 60

Leucine, 27

Leukemia, 9, 32, 109

Lysine, 26, 27

Maple syrup urine disease, 105

Marfan’s syndrome, 31, 109

Measurement techniques, validation of, 104

Messenger RNA (mRNA), 25, 26

Methemoglobinemia, 47

Methemoglobinemic cyanosis, 46

Methionine, 27

Methyl isocyanate, 106

Mice

effects of radiation on, 82-86, 97

in extrapolation tests, 93, 96

factors affecting mutations in, 84-86

heritable mutation tests in, 82-84

mutation effects in females, 85-86

mutation effects in males, 84-85

spermatogonial studies in, 84-85, 97-98

unscheduled DNA synthesis in spermatids of,

97-98

Mine Safety and Health Administration (MSHA),

114

Monitoring, in epidemiologic testing, 14, 103-104,

106, 107

Mortality and morbidity, 31, 41, 49, 82

Mouse spot test, 96

Mutagenicity assays, 91-1oo; see also Mutations

and specific tests for mutagenicity

Mutants, in somatic mutation studies, 74-78; see

also Mutations

Mutations

animal studies, 3-5, 6-7, 19, 48, 78, 81-87,

117-118

at birth, 41

in chromosomes, 5, 6, 7, 8, 9, 13, 29-30, 31, 32,

40-43, 49, 51, 68, 82, 92

at conception, 41

current methods for studying, 6-10, 37-52

dominant, 32, 38, 44

environmental factors in, 3, 4, 37, 73, 96, 106,

108-109

Federal expenditures on research, 15, 18, 117,

121-122

in genes, 5, 6-7, 29, 38-40, 42-43, 44, 47-48, 50,

51, 55, 57-68, 74-76, 92, 95

genetic inheritance and, 23-33

germinal, see Germinal mutations

health effects of 4, 30-32, 108-109

heritable, see Heritable mutations

human studies, 3-4, 7-14, 19, 44-52, 55-69,

73-78, 103-110

kinds of, and effects in humans, 4, 5-6, 29-32,

55

new, persistence of in humans, 32-33, 37, 41

new technologies for detecting in humans, 10-19,

55-69

options for research on, 16-19

in proteins, 3, 8-9, 25-27, 29, 44-48, 49, 56-57

recessive, 32, 33

regulatory considerations, 15, 17, 113-118

somatic, see Somatic mutations

spontaneous, 5, 7, 32-33, 51, 83

test methods for research in, 14-16

X-linked (sex-linked), 32, 37, 38

National Cancer Institute, 104

National Center for Toxicological Research, 19

National Council on Radiation Protection and

Measurements (NCRP), 113, 114

National Environmental Protection Act of 1970,

114

National Institutes of Health (NIH), 122

National Research Council (NRC), 32-33

National Toxicology Program (NTP), 19

Neuroblastoma, 109

Nuclear Regulatory Commission (NRC), 106, 108,

114

Nucleotides and nucleotide sequencing, 5-6, 8, 11,

12, 23-25, 29, 44, 46, 47, 57-58, 59, 61, 66,

68, 93

Occupational Safety and Health Act, 114, 115

Occupational Safety and Health Administration,

114, 115

Office of Technology Assessment (OTA), 4, 15,

121

One-dimensional denaturing gradient gel

electrophoresis, 55, 59-61, 63

Oocytes, in mice, 85-86

Osteogenesis imperfect, 30
Index . 143

Parallelogram, as extrapolation model, 93, 97, 117

Peripheral blood lymphocytes (PBLs), 97

PFGE, see Pulsed field gel electrophoresis

Phenotypic data

limitations of, 40, 45

mutation rates estimated from, 39-40

surveillance of, 38-40

see also Sentinel phenotypes

Phenylalanine, 26, 27

Phenylketonuria (PKU), 45, 105

Polyacrylamide, use in protein separation, 5.5,

56-57

Polymorphisms, 58-59, 64

Polypeptide chain, 26, 37, 46; see also Proteins

Prokaryotes, 94, 95

Proline, 27

Proteins

biochemical analysis of, 37, 44-48, 49-50, 51, 52

DNA and, 23-27, 45, 46, 50, 57

mutations in, 3, 8-9, 25-27, 29, 44-48, 49, 56-57

one-dimensional separation of, 8-9, 44-45, 49, 56

synthesis of, 25-27

variant, mutation studies on, 8-9, 44-50, 55,

56-57, 73-74

two-dimensional separation of, 9, 56-57

see also Amino acids; Blood proteins; Enzymes;

Hemoglobins, Nucleotides and nucleotide

sequencing

Public Health Service, 18

Pulsed field gel electrophoresis (PFGE), 13, 55, 68

Qualitative studies, 15, 91, 94-97, 99-100

Quantitative studies, 15, 45-46, 55, 91, 97-100,

115, 117

Radiation

effects on atomic bomb survivors, 9-10, 32,

48-50, 77, 86, 106, 108

effects on DNA, 23

effects on fruit flies, 82

effects on human germ cells, 9

effects on mice, 82-86, 97

effects on pregnant women, 9

effects on U.S. citizens, 86-87, 106

in extrapolation studies, 97-1oo

in Marshall Islands populations test, 50

in somatic mutation studies, 74

mutagenic effects of, 73

U.S. Government efforts to protect humans

from, 113-114

Radiation Effects Research Foundation, 16, 48, 104

Radioactive thymidine, 74

Red blood cells, somatic mutation studies on, 73,

76-78

Regulatory considerations

definition of germ-cell mutagen, 115-117

mutagenicity and carcinogenicity, 117-118

new methods for measuring mutation rates, 118

protection against agents other than radiation,

114

quantitative extrapolation, 117

radiation protection, 113-114

role of U.S. Government, 15, 17, 113-118

Research and public policy, use of new

technologies in, 14-16

Restriction fragment length polymorphisms

(RFLPs), 55, 58-59, 60

Retinoblastoma, 7, 31, 109

Ribonuclease A (RNaseA), 64, 65

Ribonuclease cleavage of mismatches in

RNA/DNA heteroduplexes, 12, 55, 64-65

Ribonucleic acid (RNA)

heteroduplexes, 55, 64-65

role in protein synthesis, 26, 45

Risk assessment, EPA guidelines for, 116

RNA, transcribed, 45

RNA/DNA heteroduplexes, ribonuclease cleavage

of mismatches in, 12, 55, 64-65

Rodents, heritable mutation tests in, 81-84

Salmonella, 118

Sentinel phenotypes, 7-8, 37, 38-40, 45, 50, 51, 52,

55, 105, 106, 109; see also Phenotypic data

Serine, 27

Sickle-cell anemia, 105

Sister-chromatid exchange (SCE), 99, 116

6-TG, use in somatic mutation studies, 74-75

Somatic cel]s, 7, 13-14, 27, 31-32, 73, 91-100, 115,

116, 117; see also Somatic mutations

Somatic mutations

defined, 6

detection and measurement of, 4, 13-14

effects of, 31-32

new methods for measuring in humans, 73-78

see also Mutations

Specific locus tests (SLTs), 50, 82-83, 85, 94-96,

97-98, 116

Spermatogonial studies in mice, 84-85, 97-98

Spontaneous mutation rates, 5, 7, 32-33, 51, 83

Squirrel monkeys, 85

Stem cells, in mice, 84, 85

Subtractive hybridization, 12, 55, 66-67

“Superfund,” 18, 114

Surveillance, in epidemiologic testing, 14, 103,

104-106, 107

T-lymphocytes, 74-76

Technologies, new
144 . Technologies for Detecting Heritable Mutations in Human Beings

for detecting mutations in humans, 10-19,

development of, and research on, 16-19

and heritable mutations, 3, 10-14, 55-69

55-69

use in research and

Tester mice, 82-83

Thioguanine resistant

Threonine, 27

public policy, 14-16

(TGr

) cells, 99

Thymine (T), 23, 24, 27, 64, 66

Toxic Control Substances Act of 1976 (TSCA), 3,

15, 114, 118

Transfer RNA (tRNA), 26

Tryptophan, 27

Turner’s syndrome, 30, 42

Two-dimensional denaturing gradient gel

electrophoresis, 55, 61-64

Two-dimensional polyacrylamide gel

electrophoresis, 56-57

2DGGE, see Two-dimensional denaturing gradient

gel electrophoresis

2-D PAGE, see Two-dimensional polyacrylamide

gel electrophoresis

Tyrosine, 27, 47

United Nations Scientific

of Atomic Radiation

Committee on the Effects

(UNSCEAR), 113-114

U.S. Congress, 4, 15, 16-19, 113; see also U.S.

Government

U.S. Government

efforts to protect humans from radiation,

113-114

expenditures on mutation research, 15, 18, 117,

121-122

regulation by, 15, 17, 113-118

role in mutation research, 4, 5, 15, 16-19,

113-118

see also U.S. Congress and specific Federal

agencies

Validation of measurement techniques in

epidemiologic testing, 104

Valine, 26, 27, 29

Viruses, effects on DNA, 23

White blood cells, somatic mutation studies on,

73, 74-76

Whole mammal tests, see Specific locus tests;

Heritable translocation tests

Wild-type mice, 82-83

Wilms’ tumor, 7, 31, 109

Xeroderma pigmentosum, 32

Yeast, 81, 92, 116

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