Human prealbumin and related methods and products

Recombinant human prealbumin and a method for producing such human prealbumin through recombinant DNA techniques including preparation and isolation of a cDNA sequence coding for human prealbumin, construction of a cloning vector and an expression vector containing said cDNA sequence, and expression of human prealbumin in a prokaryotic cell transformed by said expression vector are disclosed. The invention further relates to the use of human prealbumin cDNA in the diagnosis by hybridization methodologies of medical conditions with which variant forms of prealbumin are associated. Also disclosed is a method for diagnosing some Type I familial amyloid polyneuropathies by a restriction endonuclease assay with an enzyme which recognizes the nucleotide base sequence 5'-ATGCAT-3'.

FIELD OF THE INVENTION 
This invention relates to human prealbumin, to the production of human 
prealbumin by recombinant DNA techniques, including preparation and 
isolation of cDNA coding for human prealbumin and particularly to the use 
of human prealbumin cDNA in the diagnosis by hydridization methodologies 
of medical conditions in whichvariant forms of prealbumin have been 
implicated. The invention further relates to the expression of recombinant 
DNA to produce human prealbumin, to expression vectors useful for 
expressing human prealbumin, to microorganisms and cell lines transformed 
with said vectors, and to methods for producing human prealbumin by 
recombinant DNA techniques. 
BACKGROUND OF THE INVENTION 
The familial amyloid polyneuropathies (FAPs) are systemic amylid syndromes 
which are inherited in an autosomal dominant fashion (Glenner, G. G. et 
al., The Metabolic Basis of Inherited Disease pp 1308-1339, McGraw-Hill, 
New York 1978). They are characterized by the deposition of fibrils 
containing the plasma protein prealbumin (transthyretin) (Costam, P. P. et 
al., Proc. Natl. Acad. Sci., USA (1978) 75:4499; Benson, M. D., J. Clin. 
Invest. (1981); 67: 1035; and Skinner, M. et al., Biochem. Biophys. Res. 
Comm. (1981) 99: 1326.). Prealbumin is a 55 K Mr protein, composed of four 
identical subunits, which is involved in thyroid hormone and vitamin A 
transport (Blake, C.C.F., Proc. R. Soc. Lond. (1981) B211: 413.) Recently 
prealbumin isolated from the amyloid deposits of FAP patients has been 
sequenced and shown to have various single amino acid substitutions not 
found in the circulating prealbumin of normal individuals. One FAP 
associated prealbumin variant has a methionine for valine substitution at 
amino acid 30 and is found in kinships of Portuguese (Saraiva, M. J. M. et 
al., J. Clin. Invest (1984) 74: 104), Japanese (Tawara, S. et al., 
Biochem. Biophys. Res. Comm. (1983) 116: 880) and Swedish (Dwulet, et al., 
Proc. Natl. Acad. Sci., USA (1984) 81: 694; and Whitehead, A. S. et al., 
Mol. Biol. Med. (1985) Vol. 7 in press) ancestry type 1 (FAP). This 
prealbumin variant is present in the plasma of some kinships afflicted 
with type 1 FAP and it has been proposed that peptide mapping would 
provide a definitive diagnostic test for this form of the disease 
(Saraiva, M. J. M. et al., supra; Benson, M. D. et al., J. Clin. Invest. 
(1985) 75: 71.) Another type 1 FAP associated prealbumin variant has been 
identified by Whitehead et al., Mol. Biol. Med. supra. The variant 
prealbumin allele associated with type 2 FAP has a serine for isoleucine 
substitution at position 84 (Wallace et al., Clin. Res. (1985) 33: 592A). 
Variant prealbumin alleles have also been implicated in Alzheimers Disease 
(Shirahama, T. et al., Am. J. Pathol. (1982) 107: 41; A. S. Cohen and J. 
Sipe, NIH Grant No. P50-AG/NH05141 original submitted 3/19/84, revised 
11/07/84). 
There are three basic methods currently in use for detecting variant 
protein alleles: detecting the altered protein molecule itself; 
determining the nucleotide sequence of the coding regions of the variant 
gene; and detecting mutations that affect Southern blot hydridization 
patterns arising from either insertions or deletions in the gene or point 
mutations which either create or destroy restriction endonuclease 
recognition sequences. 
The method described by Benson et al. for detecting carriers of the Met 30 
human prealbumin variant falls into the first of the basic methods 
described. (Benson, M. D. et al., supra.) Since normal prealbumin has only 
one methionine (position 13), treatment with cyanogen bromide (CNBr), 
which cleaves only at methionines, results in two peptides. CNBr treatment 
of the Met 30 variant gives three peptides. The extra peptide is detected 
by high performance liquid chromatography (HPLC) or sequential Edman 
degradation. 
Nakazato et al. report a radioimmunossay for the Met 30 variant of human 
prealbumin based on a nonapeptide (position 22-30) of the prealbumin 
variant. (Nakagato, M. et al., The Lancet (1985) 1: 99). 
The second approach described above to determine the DNA structure of the 
mutant gene is a long and labor intensive process requiring cloning of 
normal and variant alleles. As such there is not much interest in this 
approach. 
In order to detect variant prealbumin alleles by the third approach 
described above, use of restriction endonuclease based assays, it is 
necessary to clone and sequence cDNA for the prealbumin variant of 
interest. Recent advances in biochemistry and in recombinant DNA 
technology have made it possible to clone DNA and to achieve the 
controlled synthesis of specific proteins using the technique of molecular 
cloning. Molecular cloning involves isolating and purifying a nucleotide 
sequence coding for a particular protein, inserting the sequence into a 
plasmid or other cloning vehicle and transferring the cloned gene to a 
suitable cell for amplification and/or expression of the protein. 
Maniatis, T. et al.--Molecular Cloning--A Laboratory Manual, Cold Spring 
Harbor Laboratory, Cold Spring Harbor, New York, 1982. The protein can 
then be isolated and purified by conventional techniques. 
Restriction endonucleases are enzymes, isolated primarily from prokaryotes, 
which recognize specific nucleotide sequences within a DNA molecule 
(Maniatis et al., supra, at pp 98-106). Generally, restriction 
endonucleases may be classified into three groups. Type-I and type-III 
enzymes carry methylase and an ATP-requiring cleavage activity in the same 
protein. Both types of this enzyme recognize unmethylated sequences in a 
substrate, but type-I enzymes cleave randomly, while type-III enzymes 
cleave at specific sites. Type-II restriction enzymes consist of a 
separate restriction endonuclease and modification methylase. A large 
number of type-II enzymes have been isolated (Roberts, R. J. Nuc. Acids 
Res. (1982) 10:R117), many of which are useful in molecular cloning. These 
enzymes cut DNA within or near a particular recognition sequence, which 
typically is from four to six nucleotides in length. In general, different 
restriction enzymes recognize different target nucleotide sequences. 
However, there are several enzymes isolated from different sources which 
recognize the same target sequences. These enzymes are known as 
isochizomers. Most restriction enzymes recognize either tetranucleotide, 
pentanucleotide or hexanucleotide sequences; see Roberts, R. J., Nuc. 
Acids Res. (1983) 11:R135. 
A restriction endonuclease has been employed in an assay for detecting the 
sickle cell allele (beta.sup.S gene) (Wilson et al., U.S. Pat. No. 
4,395,486). Wilson et al. analyzed amniotic fluid by isolating DNA 
therefrom, digesting the DNA with the restriction enzyme Dde I, which 
recognizes the nucleotide base sequence CTNAG, and separating the DNA 
fragments following cleavage. Using standard hybridization probe detection 
methods and a specific probe for the human beta-globin gene, the presence 
of an approximately 376 base pair fragment and the absence of an 
approximately 175 base pair fragment in the analyte was stated to be 
indicative of the presence of the sickle cell genotype. 
It would be advantageous to develop diagnostic tests for medical conditions 
associated with variant prealbumin alleles using hybridization based 
assays. Moreover, it is desirable to have a recombinant source of human 
prealbumin that can supply normal prealbumin protein substantially free of 
other proteins of human origin. Thus, there is much interest in cloning 
cDNA for human prealbumin in quantities and purity sufficient for use in 
clinical applications. 
SUMMARY OF THE INVENTION 
The present invention provides recombinant human prealbumin and a means or 
method for producing such prealbumin through recombinant DNA techniques 
including (1) preparation and isolation of cDNA sequences coding for 
prealbumin, (2) construction of expression vectors containing said cDNA 
sequences, and (3) expression of prealbumin in a prokaryotic cell 
transformed by said expression vector. The present invention also provides 
the use of human prealbumin cDNA in the diagnosis, by use of hybridization 
methodologies, of medical conditions in which variant forms of prealbumin 
have been implicated.

DETAILED DESCRIPTION OF THE INVENTION 
The following definitions are supplied in order to facilitate the 
understanding of this case. To the extent that the definitions vary from 
meaning circulating within the art, the definitions below are to control. 
Amplification means the process by which cells produce gene repeats within 
their chromosomal DNA. 
An enhancer is a nucleotide sequence that can potentiate the transcription 
of a gene independent of the position of the enhancer in relation ot the 
gene or the orientation of the sequence. 
A gene is a deoxyribonucleotide sequence coding for a given protein. For 
the purposes herein, a gene shall not include untranslated flanking 
regions such as RNA transcription initiation signals, polyadenylation 
addition sites, promoters or enhancers. 
Orientation refers to the order of nucleotides in a DNA sequence. An 
inverted orientation of a DNA sequence is one in which the 5-prime to 
3-prime order of the sequence in relation to another sequence is reversed 
when compared to a point of reference in the DNA from which the sequence 
was obtained. Such points of reference can include the direction of 
transcription of other specified DNA sequences in the source DNA or the 
origin of replication of replicable vectors containing the sequence. 
Transcription means the synthesis of RNA from a DNA template. 
Transformation means changing a cell's genotype by the cellular uptake of 
exogenous DNA. Transformation may be detected in some cases by an 
alteration in cell phenotype. Transformed cells are called transformants. 
Translation means the synthesis of a polypeptide from messenger RNA. 
As used herein the term "recombinant human prealbumin" refers to human 
prealbumin expressed in transferred cells. 
The patent and scientific literature is replete with processes useful for 
the production of recombinant products. Generally, these techniques 
involve the isolation or synthesis of a desired gene sequence, and the 
expression of that sequence in either a procaryotic or eucaryotic cell, 
using techniques well known to the skilled artisan. Once a given gene has 
been isolated, purified and inserted into a transfer vector (i.e., 
cloned), its availability in substantial quantity has typically been 
assured. The vector with the cloned gene is transferred to a suitable 
microorganism or cell line, for example, bacteria, yeast, mammalian cell 
lines such as COS (monkey kidney) and CHO (Chinese hamster ovary), insect 
cell lines, and the like, wherein the vector replicates as the 
microorganism or cell line proliferates and from which the vector can be 
isolated by conventional means. Thus there is provided a continuously 
renewable source of the gene for further manipulation, modification and 
transfer to other vectors or other loci within the same vector. 
Expression can be obtained by transferring the cloned gene, in proper 
orientation and reading frame, into an appropriate site in an expression 
vector such that translational read-through from a procaryotic or 
eucaryotic gene results in synthesis of a protein precursor comprising the 
amino acid sequence coded by the cloned gene including or linked to Met or 
an amino-terminal sequence from the procaryotic or eucaryotic gene. 
Vectors used for eukaryotic cell expression typically contain various 
elements such as enhancers, promoters, introns, polyadenylation sites, 
3-prime and 5-prime noncoding regions and translational activators. A 
variety of specific protein cleavage techniques may be used to cleave the 
protein leader, if produced, at a desired point so as to release the 
desired mature amino acid sequence, which can then be purified by 
conventional means. In some cases, the mature protein containing the 
desired amino acid sequence is produced without the need for specific 
cleavage techniques and may also be released from the cells into the 
extracellular growth medium. 
Prealbumin specific cDNA clones have been isolated from an adult human 
liver library. An oligonucleotide mixture (5' TTPy-CAPy-GAu-CaPy-GCX-GA 
3') based on the prealbumin amino acid sequence between residues 87 and 92 
was used to screen 40,000 recombinant colonies of an adult human liver 
cDNA library by established procedures (Woods, D. E. et al., Proc. Natl. 
Acad. Sci. USA (1982) 79: 5661). Approximately fifty positive clones were 
identified and eight were selected for further study. Nucleotide sequence 
analysis obtained by the dideoxy chain termination method (Sanger, F. et 
al., Proc. Natl. Acad. Sci. USA (1977) 74: 5463) and hybridization 
analysis confirmed that these clones were complementary to prealbumin. The 
largest clone, pTTR3 (approximately 600 bp), spans the codons specifying 
amino acids 30 and 31 and was used in subsequent experiments. 
Nsi1 digested DNA from normal individuals and type 1 FAP patients kown to 
have the methionine for valine substitution has been analyzed in Southern 
blot hybridization experiments and a variant pattern associated with the 
disease phenotype has been identified (Whitehead et al., supra). This 
variant pattern in which the nucleotide sequence specifying amino acid 
residues 30 and 31 is such that a methionine substituted for a valine at 
position 30 creates a new Nsi1 restriction endonuclease recognition site 
in the gene encoding the variant product. This variant pattern permits 
direct identification of individuals carrying this disease-associated 
prealbumin allele and will facilitate diagnosis prior to clinical 
presentation, genetic counselling and renatal screening procedures in some 
families with type 1 FAP. 
A number of FAP patients whose amyloid deposits contain a variant 
prealbumin molecule other than that with the methionine for valine 
substitution have been described above. Although the above analysis based 
on a new Nsi1 site is not applicable to FAP kinships whose disease is in 
association with other amino acid substitutions of prealbumin, the normal 
prealbumin clone, pTTR3, may be used in analysis of any variant prealbumin 
using appropriate restriction endonucleases. Prealbumin specific cDNA 
clones according to the present invention have been used to localize the 
prealbumin gene to human chromosome 18 (Whitehead et al., supra.). 
A human prealbumin specific cDNA clone, pTTR3, was used to analyze DNA 
samples from several patients with familial amyloid polyneuropathy. In 
classical type 1 FAP a variant prealbumin molecule with a methionine for 
valine substitution at position 30 has been implicated in the pathogenesis 
of this autosomal dominant condition. The codon for methionine is AUG, 
therefore the variant ellele would be expected to contain the nucleotide 
sequence ATGCAT specifying amino acids 30 and 31. ATGCAT is the 
recognition sequence for the restriction endonuclease Nsi1. In Southern 
blot hybridization experiments the prealbumin clone, pTTR3, detects Nsi1 
DNA fragments of 6.5 Kb and 3.1 Kb in normal individuals. In a family 
known to have the methionine for valine substitution (Family 1, infra) 2 
individuals with Type 1 FAP were shown to have novel bands of 4.9 Kb and 
1.6 Kb which are derived from cleavage of the additional Nsi1 site in the 
region of the variant allele corresponding to the 6.5 Kb band in the 
normal allele. Of seven offspring subjected to similar analysis 4 were 
shown to have the variant prealbumin DNA patterns indicative of the 
presence of the FAP-associated allele. All offspring were younger than the 
usual age of onset of Type 1 FAP. The above analysis, therefore, allows 
diagnosis of FAP in members of this family prior to clinical presentation 
and forms a basis for genetic counselling and prenatal diagnosis in this 
and other similar families. 
Prealbumin has been found associated with amyloid deposits in the 
peripheral nervous system (Costa et al., supra) and in the central nervous 
system in senile cerebral amyloid, Alzheimer's disease, (Shirahama, T. et 
al., supra) for which there is a familial form. In addition prealbumin 
amyloid fibrils occur in senile cardiac amyloidosis (Cornwall, G. G. et 
al., Immun. (1981) 44: 447.) and familial amyloid cardiomyopathy (Husby, 
G. et al. Clin. Exp. Immunol. (1985) 60: 207). There is no known function 
for prealbumin in the nervous system, however two carrier interactionshave 
been described for the circulating form. Prealbumin is involved with 
vitamin A (retinol) transport through its association with retinol binding 
protein and with transport of thyroid hormone through direct binding (RAZ, 
A. et al., J. Biol. Chem. (1969) 244: 3230.) The latter function may be 
compromised in familial euthyroid hyperthyroxinemia in which a variant 
prealbumin with an increased binding capacity for thyroxine has been 
described (Moses, A. C. et al., N. Engl. J. Med. (1982) 306: 966.) Clones 
for human prealbumin will therefore be of use in studying the genetic 
basis for the many autosomal dominant diseases, in addition to FAP, in 
which variant forms and activities of prealbumin are implicated. 
Since all types of FAP and Alzheimer's disease usually are not manifest 
until after the child bearing years methods according to the present 
invention of identifying carriers of the gene provide a basis for 
diagnosis before clinical presentation, genetic counseling and prenatal 
screening. 
In accord with the preferred embodiment of this invention, the vector, 
pKT218, utilized for the amplication of human prealbumin cDNA pTTR3 has 
been deposited and is available from the American Type Culture Collection 
as Deposit Number ATCC 53306. 
EXAMPLES 
Example I 
Patients and Methods 
FAP kinships: Family 1 (COhen, A. S. et al., in Glenner, G. G., et al., 
eds., Amyloid and Amyloidosis, pp 67-77, Excerpts Medica, Amsterdam, 1980) 
is of Swedish origin and has had a history of variably diagnosed 
neurological disorders for over 60 years. The amyloid involvement in this 
kinship is typical of type 1 FAP (Glenner et al., supra) and is 
characterized by severe sensory neuropathy beginning in the lower 
extremities. Patients exhibit some autonomic nervous system dysfunction, 
including incontinence, impotence, postural hypotension and nocturnal 
diarrhea. The age of onset is before 40 years and the most frequent cause 
of death is severe renal disease. Autopsy typically reveals extensive 
involvement of blood vessels throughout the body with particularly marked 
involvement of skin, nerves, gastrointestinal tract and kidney. The 
methionine for valine substitution in prealbumin was determined from 
amyloid fibrils isolated from thyroid tissue removed at autopsy from one 
individual in family 1. DNA samples from two maternal aunts diagnosed as 
having type 1 FAP and their offspring were analyzed. 
Family 2 (Libbey, C. A. et al., Am. J. Med. (1984) 76: 18) is of 
German/English ancestory and by clinical criteria is classified as type 1 
FAP. This kinship, however, differs in that onset is in the seventh decade 
and the clinical course is milder. Although histologic and 
immunohistochemical evidence has established that an amyloid fibril of 
prealbumin origin is present in diseased members of this family, sequence 
analysis on the isolated fibril has not been performed. DNA samples from 2 
affected individuals whose disease has been apparent for several years 
were analyzed. 
In addition one individual from a third kinship with classical type 1 FAP 
symptoms and age of onset (Skinner, M. and Cohen, A. S. unpublished) was 
also analyzed. 
Example II 
Amino Acid Sequence Analysis of Amyloid Fibrils 
A 14,000 M.sub.r protein was isolated from amyloid laden thyroid tissue of 
individual IV-1 (FIG. 1) who died at age 34 of malnutrition. The sequence 
of the first twenty amino acid residues had previously been reported to be 
identical to normal prealbumin (Skinner et al., supra). In this study, 
sequence analysis was continued through residue 42 with identification of 
amino acids by high performance liquid chromatography and thin layer 
chromatography. When compared with normal prealbumin the only difference 
was at position 30 where methionine residues were found in addition to 
valine indicating that the amyloid fibrils were composed of a mixture of 
the normal and variant forms of prealbumin. 
Example III 
Isolation of Prealbumin Specific cDNA Clones 
RNA was extracted from the liver of an organ donor who had sustained severe 
injuires in an automobile accident 36 hours prior to brain death (Woods et 
al., supra). Polyadenylated RNA-containing message for perealbumin was 
prepared and the enzyme reverse transcriptase was used to prepare a single 
stranded DNA complementary to the mRNAs. Formation of ds cDNA was 
catalyzed by DNA polymerase and the overhanging ends were removed by S1 
nuclease. A poly C tail was added to the ds cDNA using terminal 
deoxynucleotydyl transferase. Then the tailed cDNA was inserted into the 
plasmid pKT218 at the Pst I restriction enzyme recognition site to which 
had been added a poly G tail. Plasmid and cDNA were annealed and used to 
transform E. coli MC1061. 
Cultures of transformed bacteria were replica plated, a process by which 
duplicate copies of a master plate were grown, and used for filter colony 
hybridization (Grunstein et al., infra). After lysis of the colonies to 
denature DNA and baking of the single strands to nitrocellulose, the 
filters were hybridized with .sup.32 P prealbumin specific oligonucleotide 
probes while the master plate was kept as a source of positive colonies. 
A synthetic olignucleotide mixture comprising all 17-nucleotide DNA 
sequence that could code for the amino acid sequences of prealbumin 
between residues 87 and 92 was prepared by a solid-phase phosphodiester 
method using a library or dimer anions (Gait et al., Nuc. Acids Res. 
(1980) 8: 1081; Markham et al., Nuc. Acids Res. (1980) 8: 5193). To ensure 
the presence of the correct sequence, the mixtures contained 64 different 
oligonucleotides in order to cover the degrees of codon ambiguity for each 
region of 6 amino acids. 
To identify prealbumin specific cDNA clones, the oligonucleotide mixture 
was radio-labeled with .sup.32 P and used to screen 40,000 clones. 
Approximately 50 of them hybridized specifically with this mixture. The 
largest clone, pTTR3 (approximately 600 bp), spans the codons specifying 
amino acids 30 and 31. 
Example IV 
Expression of Human Prealbumin cDNA 
Plasmid DNA is isolated from cultures of pTTR3 by the cleared lysate method 
of Clewell, D. B. et al., Proc. Nat. Acad. Sci. (1969) 62: 1159. A 
concentrated suspension of bacteria is lyzed by the action of lysozyme and 
SDS. The lysate is cleared by ultracentrifugation to pellet the cell 
debris and chromosomal DNA. Protein is removed by phenol/chloroform 
extraction. DNA is precipitated with ethanol, and redissolved in buffered 
saline and the supercoiled plasmid is recovered by ultracentrifugation on 
cesium chloride gradients (Clewell et al., supra). The prealbumin specific 
cDNA contained in pTTR3 is excised by PstI digestion and purified by 
agarose gel electrophoresis. (Methods in Molecular Biology Vol. 2, ed. J. 
M. Walker, Humama Press, Clifton, N.J. 1984). This dC-tailed double 
stranded cDNA is hybridized to PstI - digested, dG-tailed pBR322 and the 
resulting ampicillin sensitive recombinant plasmids are used to transform 
E. coli RRI made competent by treatment with CaCl.sub.2 (Birch, H. E. et 
al., Biochem. Intl. (1983) 6:653; J. M. Walker, supra). 
In situ colony hybridization is carried out as described for pTTR3 in 
Example III above. The colonies which are positive to the pTTR3 cDNA 
insert are screened with anti-prealbumin antibodies purified from whole 
antiserum to prealbumin by affinity chromatography (Sipe, J. D. et al., J. 
Immunol. (19769 116: 1151). 
The E. coli expressing the pTTR3 cDNA insert, i.e. normal human prealbumin 
protein, are extracted with 7M guanidine hydrochloride (Lomedico, P. T., 
et al., (Nature (1984) 312: 458). Transformed E. coli are used as a 
control. The crude extract is concentrated by ammonium sulfate 
precipitation and is fractionated by gel filtration on Ultragel AcA54 (LKB 
Instruments, Inc., Gaithersburg, MD). The fractions containing prealbumin 
are pooled and applied to an anti-prealbumin immunosorbent column. The 
prealbumin is eluted with 4M KSCN. The presence of bacterial contaminants 
is assessed by chemical and functional (LPS) analysis of the control 
extract from nontransformed bacteria. 
Example V 
Somatic Cell Hybrids 
Somatic cell hybrids were derived from fusions of hypoxanthine 
phosphoribosyl transferase deficient mouse RAG cells or hamster E36 cells 
with white blood cells or fibroblasts from 3 unrelated human individuals 
and have been described previously (Mantzouranis, E. C. et al., J. Biol. 
Chem. (1985) 260: 7752; and Bruns, G. A. P. et al., Biochem. Genetics 
(1979) 17: 1031.) 
Example VI 
Isolation and Analysis of Human, Rodent and Somatic Cell Hybrid DNA 
High Molecular weight DNA from human peripheral blood lymphocytes, the 
rodent cell lines RAG and E36 and somatic cell hybrids was prepared 
(Gross-Bellard, M. et al., Eur. J. Bioch. (1977) 36: 32). Following 
digestions with the restriction endonucleases Sac1 or Nsi1 (New England 
Biolabs), DNA was size fractionated by egarose gel electrophoresis, alkali 
denatured and transferred to nitrocellulose filters (Southern, E. M., J. 
Mol. Biol. (1975) 98: 503.) The resulting Southern blots were hybridized 
overnight under standard conditions (Jeffreys, A. J. et al., Cell (1977) 
12: 429) with pTTR3 which had been radiolabeled by nick translation 
(Rigby, P. W. J. et al., J. Mol. Biol. (1977) 113: 237.) washed in 30 mM 
NaCl/3 mM Na citrate/0.1% SDS at 65.degree. C. for one hour, dried and 
exposed to Kodak XAR5 film. 
Example VII 
Analysis of DNA from FAP patients 
The nucleotide sequence of human prealbumin specifies the codons GUG and 
CAU for amino acid residues 30 and 31 (valine and histidine). In type 1 
FAP the valine at position 30 is replaced by a methionine. The codone 
specifying methionine is AUG, thus the substitution giving rise to the 
disease phenotype is probably derived from a single G to A mutation of the 
first nucleotide of the codon specifying amino acid 30. The coding 
sequence of the variant allele would therefore contain a novel ATGCAT Nsi1 
restriction endonuclease recognition site not present in the normal 
allele. DNA from the peripheral blood lymphocytes of 15 unrelated 
individuals was digested with Nsi1 and subjected to Southern blot analysis 
using radiolabeled pTTR3 as a hybridization probe. In all cases two bands 
of 6.5 Kb and 3.1 Kb were observed (data not shown), the intensity of each 
being approximately 1:1. DNA from the peripheral blood lymphocytes of 10 
members of a kinship (FIG. 1) with Type 1 FAP was subjected to similar 
analysis (FIG. 2). Amino acid sequencing of prealbumin deposited in the 
tissues of one member (IV1 deceased) of this kinship had previously 
established the presence of the methionine for valine substitution in the 
affected members of this family. Individuals III2 and III4 have had a 
classical type 1 FAP clinical course for several years. When digested with 
Nsi1 and hybridized with radiolabeled pTTR3 their DNA patterns (FIG. 2, 
tracks B and H) differ from those found in normal individuals; the ratio 
of the 6.5 Kb band to the 3.1 Kb band is approximately 1:2 and two new 
bands 4.9 Kb and 1.6 Kb are apparent. The relative intensity and sizes of 
these bands indicate that they are derived from a novel Nsi1 restriction 
site in the region of the variant prealbumin gene corresponding to the 6.5 
Kb fragment of the normal allele. The DNA patterns of the 7 offspring of 
individuals III2 and III4 (IV3-7; Tracks 3-7; and IV8,9;, Tracks 9 and 10) 
show that in 4 cases the variant allele has been transmitted to the next 
generation. These individuals are younger than the typical age of onset of 
type 1 FAP and their clinical assessment reveals no disease state as yet. 
Of interest is the analysis of DNA from two FAP patients of another kinship 
(FIG. 2, tracks 11 and 12). These individuals are from family 2 in which 
the symptoms presented during the course of the disease are similar, 
though not identical, to those of classical type 1 FAP patients. A 
significant difference is an age of onset of the disease towards the end 
of the sixth decade of life, about 20-30 years later than that of type 1 
FAP. Nsi1 digested DNA samples from these individuals shows a "normal" 
pattern detected by radiolabeled pTTR3, indicating that their disease is 
not a result of the prealbumin variant characterized by the methionine for 
valine substitution. Similar analysis of an unrelated FAP patient (FIG. 2, 
track 13) with classical type 1 symptoms and age of onset also reveals a 
"normal" pattern, thereby establishing the heterogeneity of the underlying 
biochemical defect in type 1 FAP. 
Example VIII 
Chromosomal Localization of the Human Prealbumin Gene 
Somatic cell hybrids between human cells and mouse RAG or hamster E36 cells 
which have a full complement of rodent chromosomes and a limited but 
varied human chromosome content were analyzed for the presence or absence 
of the human prealbumin gene. DNA was isolated from 125 unrelated 
individuals, digested with the restriction endonuclease SacI, and 
subjected to Southern blot analysis using radiolabeled pTTR3 as a 
hybridization probe. Prealbumin specific bands of 5.6 Kb and 1.7 Kb, which 
were distinct from the mouse RAG or hamster E36 SacI restriction 
fragments, were identified (data not shown). DNA from 24 somatic cell 
hybrid lines was analyzed similarly and assessed for the presence or 
absence of the human prealbumin specific 5.6 Kb and 1.7 Kb bands (FIG. 3). 
The presence or absence of these bands correlates perfectly only with the 
presence or absence of chromosome 18 (Table 1 below) thereby allowing 
assignment of the prealbumin gene to human chromosome 18. 
Table 1: Segregation of hybridization of the DNA probe pTTR3 to 
SacI-digested DNA from 24 somatic cell hybrids. Concordant: (++) probe 
hybridizes, chromosome present; (--) probe does not hybridize, chromosome 
not present. Discordant: (+-) probe hybridizes, chromosome not present; 
(-+) probe does not hybridize, chromosome present. Data are summarized 
from hybridization of pTTR3 to DNA from aromatic cell hybrids 
(Mantzourani's, E. C. et al., supra and Burns et al., supra.) (FIG. 3) 
previously analyzed from human chromosome content. *Clones with chromosome 
present, in less than 15% of metaphases, or in which a chromosome specific 
isoenzyme of DNA probe exhibited only a weak signal were excluded from 
analysis for that chromosome. Clones with a rearranged chromosome were 
likewise excluded from the analysis. 
TABLE 1 
______________________________________ 
HYBRIDATION OF TTR3 PROBE 
Human Concordant 
Discordant 
Total 
Chromosome 
++ -- +- -+ Concordant 
Discordant 
______________________________________ 
1* 8 7 3 3 15 6 
2* 6 10 5 0 16 5 
3* 7 5 5 6 12 11 
4* 5 5 7 6 10 13 
5 9 9 3 3 18 6 
6 9 8 3 4 17 7 
7 9 6 3 6 15 9 
8 5 7 7 5 12 12 
9* 3 7 8 4 10 12 
10 10 5 2 7 15 9 
11* 9 8 3 3 17 6 
12* 10 8 2 3 18 5 
13* 5 5 4 6 10 10 
14* 8 3 4 8 11 12 
15* 6 8 6 3 14 9 
16* 7 5 3 7 12 10 
17* 4 9 6 3 13 9 
18 12 12 0 0 24 0 
19* 9 2 1 9 11 10 
20* 9 7 3 4 16 7 
21* 7 4 5 7 11 12 
22* 8 3 4 8 11 12 
X 12 0 0 12 12 12 
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Additional advantages and modifications of the invention disclosed herein 
will occur to those persons skilled in the art. Accordingly, the invention 
in its broader aspects is not limited to the specific details or 
illustrated examples described herein. Therefore, all departures made from 
the detail are deemed to be within the scope of the invention as defined 
by the appended claims.