The major defect in Ashkenazi Jews with Tay-Sachs disease is an insertion in the gene for the alpha-chain of beta-hexosaminidase.

Abstract

The Ashkenazi Jewish population is enriched for carriers of a fatal form of Tay-Sachs disease, an inherited disorder caused by mutations in the alpha-chain of the lysosomal enzyme, beta-hexosaminidase A. Until recently it was presumed that Tay-Sachs patients from this ethnic isolate harbored the same alpha-chain mutation. This was disproved by identification of a splice junction defect in the alpha-chain of an Ashkenazi patient which could be found in only 20-30% of the Ashkenazi carriers tested. In this study we have isolated the alpha-chain gene from an Ashkenazi Jewish patient, GM515, with classic Tay-Sachs disease who was negative for the splice junction defect. Sequence analysis of the promoter region, exon and splice junctions regions, and polyadenylation signal area revealed a 4-base pair insertion in exon 11. This mutation introduces a premature termination signal in exon 11 which results in a deficiency of mRNA in Ashkenazi patients. A dot blot assay was developed to screen patients and heterozygote carriers for the insertion mutation. The lesion was found in approximately 70% of the carriers tested, thereby distinguishing it as the major defect underlying Tay-Sachs disease in the Ashkenazi Jewish population.

Full text

Page 1

Communication
Vol. 263, No. 35, Issue of December 15, pp. 18587-18589,1988
THE JOURNAL
OF BIOLOGICAL
CHEMISTRY
Printed in U.S.A.
The Major Defect in Ashkenazi
Jews with Tay-Sachs Disease Is
an Insertion in the Gene for the
at-Chain of &Hexosaminidase*
(Received for publication, September 16, 1988)
Rachel MyerowitzS and F. Christine Costigan
From the Laboratory of Biochemistry and Metabolism,
National Institute of Diabetes and Digestive and Kidney
Diseases, National Institutes of Health, Bethesda,
Maryland 20892
The Ashkenazi Jewish population is enriched for
carriers of a fatal form of Tay-Sachs disease, an inher-
ited disorder caused by mutations in the a-chain of the
lysosomal enzyme, @-hexosaminidase A. Until recently
it was presumed that Tay-Sachs patients from this
ethnic isolate harbored the same a-chain mutation.
This was disproved by identification of a splice junc-
tion defect in the a-chain of an
which could be found in only 20-30% of the Ashkenazi
carriers tested. In this study we have isolated the
a-chain gene from an Ashkenazi Jewish patient,
GM515, with classic Tay-Sachs disease who was neg-
ative for the splice junction defect. Sequence analysis
of the promoter region, exon and splice junctions re-
gions, and polyadenylation signal area revealed a 4-
base pair insertion in exon 11. This mutation intro-
duces a premature termination signal in exon 11 which
results in a deficiency of mRNA in Ashkenazi patients.
A dot blot assay was developed to screen patients and
heterozygote carriers for the insertion mutation. The
lesion was found in approximately 70% of the carriers
tested, thereby distinguishing it as the major defect
underlying Tay-Sachs disease in the Ashkenazi Jewish
population.
Ashkenazi patient
Mutations in the a-chain
(0-N-acetyl-D-hexosaminidase,
zyme composed of two polypeptides (a, @), result in Tay-
Sachs disease, an inherited disorder displaying clinical and
biochemical variation (1, 2). The disease is inherited in an
autosomal recessive manner. Heterozygote carriers harbor one
a-chain mutant allele and are asymptomatic,
in whom both a-chain alleles are defective display neurode-
generation due to accumulation of undegraded gangliosides
(1). Patients with a severe type of Tay-Sachs disease called
the “classic” form show clinical
months of age, and death follows in early childhood. Ashke-
nazi Jews are carriers for this classic form 1 0 times more
frequently than the general population. Until recently it
presumed that a single defect, probably arising via one foun-
of the A form of @-hexosaminidase
EC 3.2.15.2), a lysosomal en-
while individuals
symptoms as early as
6
was
* This work was supported in part by March of Dimes Birth Defects
Foundation Grant 1-1071. The costs of publication of this article
were defrayed in part by the payment of page charges. This article
must therefore be hereby marked “advertisement” in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
$To whom correspondence should be addressed: Bldg. 10, Rm.
9N114, National Institutes of Health, Bethesda, MD 20892.
der, underlies classic Tay-Sachs disease in this ethnic group
(1). This was disproved by discovery of a splice junction defect
(3-5) in the a-chain gene of approximately 20-30% of the
Ashkenazi carriers tested (3, 4). This report describes a 4-
base pair insertion in exon 11 of the a-chain gene of an
Ashkenazi patient. Since 70% of the carriers tested are posi-
tive for this mutation, it appears to be the
underlying classic Tay-Sachs disease in the Ashkenazi Jewish
population.
major defect
EXPERIMENTAL PROCEDURES
DNA Sources
Fibroblast cultures GM2968, GM3051, GM3052, GM515,
IMR9O were obtained from the National Institute of General Medical
Sciences Human Genetic Mutant Cell Repository (Camden, NJ).
Conditions for culture were as described (6). Blood samples of Ash-
kenazi patients with classic Tay-Sachs disease and of their parents
were obtained from Lyndon Badal of Kingsbrook Jewish Medical
Center, Brooklyn, NY. Leukocyte pellets from Ashkenazi Jewish
obligate heterozygote carriers and normal Ashkenazi Jews were kindly
provided by Eugene Grebner (Thomas Jefferson University, Phila-
delphia, PA).
and
Construction of Genomic Library and Isolation and Characterization
of Clones
Genomic DNA was isolated from cultured fibroblasts of an Ash-
kenazi Jewish patient with classic Tay-Sachs disease, GM515, and
used to construct a library in vector XEMBL3 (Promega Biotec) as
described (7). Recombinant clones were screened with a cDNA clone
that encodes the entire a-chain for &hexosaminidase, ppHa-5 (8).
The probe was labeled with [a-32P]dCTP (6000 Ci/mmol, Du Pont-
New England Nuclear) by the random primer method (9). Positive
clones were purified and assigned to their appropriate location on the
a-chain gene map (10) by analyses as previously reported (7). a-
Chain inserts or portions thereof were transferred into pTZ-18 vectors
by standard methods (11), and plasmids so generated were purified
as described (12, 13).
Amplification of Specific Genomic DNA Sequences
Genomic DNA isolated from either cultured cells (ll), whole blood,
or leukocyte pellets (14) was used (1 pg) as the initial template in the
polymerase chain reaction (15) to amplify a segment of exon 11
encompassing the insertion defect. The reaction was carried out for
27 cycles utilizing two 23-base primers, 5”GTGTGGCGAGAGGA-
TATTCCAGT-3’ and 5’-TTCAAATGCCAGGGGTTCCACTA-3’
(Midland Certified Reagent Co.) and TaqI polymerase along with a
DNA amplification kit (Gene Amp, Perkin-Elmer Cetus) as described
by the manufacturer with the exception that the annealing step was
performed at 55 “C. Following amplification, one-tenth of each sample
was electrophoresed in a low-melting agarose (4% Nu-Sieve, FMC
Corp.) and stained with ethidium bromide to assay for reaction
specificity and yield.
DNA Sequencing
Plasmids-Plasmid DNA was used as template in the sequencing
reaction (16) with 35S-substituted dATP (500 Ci/mmol) and either
avian myeloblastosis virus reverse transcriptase (Promega Biotec) or
sequenase (United States Biochemicals) as described by the manu-
facturer. a-Chain exon-specific oligomers (18 or 19 bases, prepared
by the Midland Certified Reagent Co.) were utilized as primers
without purification.
Amplified Genomic DNA-Amplified DNA was freed of deoxynu-
cleotides and salts by spin dialysis of the sample in a Centricon 30
(Amicon Corp.) microconcentrator as described (17). Direct sequenc-
ing of the sample was performed utilizing a sequencing primer, 5’-
GTGAACTATATGAAGGAGC-3’ end-labeled with [y”P] (6000 Ci/
18587

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18588
Ashkenazi Tay-Sachs Insertion Mutant
mmol), and avian myeloblastosis virus reverse transcriptase by the
method of Saiki et al. (18)
Dot Blot Assay for a-Chain Insertion Defect
One-twentieth of each amplified DNA sample was applied in
duplicate to a Biotrans nylon membrane (0.2 pm, ICN), denatured,
and fixed to the membrane according to the dot blot protocol supplied
by the manufacturer. One set of samples was hybridized to mutant
probe (5'-GAACCGTATATCTATCCTA-3')
plementary to that found in mutant exon 11. The other set of samples
was hybridized to normal probe (5"GAACCGTATATCCTATGGC-
3' ) with a sequence complementary to that found in normal exon 11.
Both probes were end-labeled to a specific activity of 5 X lo8 cpmlpg
with [y3'P]ATP (6000 Ci/mmol). The blots were washed as described
(3) and exposed to x-ray film at -70 "C in the presence of a Cronex
intensifying screen.
with a sequence com-
RESULTS AND DISCUSSION
Although previous studies (3, 4) did not exclude the possi-
bility that a multiplicity of defects underlies classic Tay-Sachs
disease in the Ashkenazi Jewish population, we began our
search by assuming that in addition to the splice junction
defect only one other lesion was present in this ethnic group.
The mutant a-chain gene of an Ashkenazi Jewish patient,
GM515, was selected for isolation and study because it was
negative for the splice junction mutation (3) and therefore
presumably homozygous for the second unknown defect. A
genomic library was constructed in XEMBL3 with DNA iso-
lated from fibroblast cultures of GM515 and screened for the
mutant a-chain gene by using a-chain cDNA, &Ha-5, as
probe (8). Clones spanning the entire 40-kilobase a-chain
mutant gene were isolated. GM515 as well as fibroblasts from
other Ashkenazi patients was previously shown to have an
intact a-chain gene by Southern analysis (19, 20) and a
deficiency of a-chain mRNA by Northern analysis (20-22).
Therefore we hypothesized that a subtle defect affecting tran-
scription or RNA processing would be likely and sequenced
the promoter region, all of the 14 exons including the splice
junction regions, and the polyadenylation signal area. Com-
parison of these sequences to those of the corresponding
region in the normal a-chain gene revealed only one differ-
ence, a 4-base pair insertion (5'-TATC-3') in exon 1 1 (Fig.
1) identical in sequence to the four bases preceding it. The
mutation causes a shift in the reading frame which results in
a nonsense mutation 9 base pairs from the insertion (Fig. 1).
We developed an assay to screen for the 4-base pair inser-
tion in other Ashkenazi Jewish patients and carriers. A 113-
base pair region of exon 11 encompassing the insertion is
amplified from genomic DNA
chain reaction (15). A fraction of the amplified product is
applied in duplicate to a nylon membrane; one set of samples
is hybridized to an oligonucleotide probe with a sequence
specific for normal exon 11, the other set to a probe with a
sequence specific for mutant exon 11. DNA samples from 20
normal Ashkenazi controls were negative for the insertion
mutation. Fourteen of the twenty obligate heterozygote car-
riers screened were positive for this defect. These insertion-
positive carriers were negative for the splice junction muta-
tion. This result was anticipated because a carrier can have
only one mutant allele. Similarly, those carriers that were
negative for the insertion were positive for the splice junction
mutation. A family which had tested negative for the splice
junction lesion (3) was evaluated for the insertion defect.
Both alleles of the affected child, R.B., harbor the insertion
defect while the father, B.B., and the mother, T.B., both have
one normal allele and one bearing the insertion (Fig. 2). In a
family where the affected child, GM2968, demonstrated the
splice junction mutation in only one allele inherited from the
samples by the polymerase
430
ASPI; NORMAL
CLONE
429jT
Pro /C
AGCT
C
A
GIStop T
MUTANT
CLONE
GACT
FIG. 1. Nucleotide sequence analysis of normal and mutant
a-chain genes in the region of exon 11 containing the inser-
tion. The normal a-chain gene was cloned from a human liver library
as described (10). The mutant gene was cloned from DNA of an
Ashkenazi Jewish patient, GM515, as described in the text. The
sequencing reaction was performed using plasmid DNA as a template
and an exon 11-specific 19-base oligomer, ATTCAGCCAGACA-
CAATC, as the primer. The insertion is marked with asterisks, and
the nucleotide sequences determined from the films are listed beside
the autoradiograms.
FIG. 2. Assay for the exon 11 insertion defect in various
echain genotypes. Genomic DNA samples were assayed for the
insertion defect as described under "Experimental Procedures": IMR
90 (normal), S.W. (Ashkenazi normal), S.S. (Ashkenazi obligate
heterozygote carrier), R.B. (Ashkenazi with classic Tay-Sachs), B.B.
(Ashkenazi obligate heterozygote, father of R.B.), T.B. (Ashkenazi
obligate heterozygote, mother of R.B.), GM2968 (Ashkenazi with
classic Tay-Sachs), GM3052 (Ashkenazi obligate heterozygote,
mother of GM2968), GM3051 (Ashkenazi obligate heterozygote, fa-
ther of GM2968), GM5I.5 (Ashkenazi with classic Tay-Sachs). Ex-
posure was for 2 h.
father, GM3051, we predicted the presence of an unknown
mutation in the other allele which must have been inherited
from the mother, GM3052 (3). Indeed, the child and the
mother bear the insertion defect in one allele. Both alleles
from the father and one each from mother and child exhibit
normal exon 11 (Fig. 2). This dot blot assay is simple and
rapid and may be useful clinically in
splice junction assay for heterozygote screening when the
enzymatic test is indeterminant.
Examination of GM515, the cell strain of the Ashkenazi
Tay-Sachs patient orginally used to clone the mutant gene,
gave a surprising result. Because it was negative for the splice
junction defect, we presumed that it would be homozygous for
the insertion. But our assay shows that only one allele in
GM515 bears the insertion (Fig. 2). Since GM515 is an
affected patient and must have two mutant a-chain alleles,
we predict the presence of a third a-chain
patient, different from the splice junction and insertion de-
fects. The third mutation must be present in very low fre-
quency in the Ashkenazi Jewish population or is a private
conjunction with the
defect in this

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Ashkenazi Tay-Sachs Insertion Mutant
18589
ASHKENAZI
NORMAL
/f
q
G
G
G
T
A
T
C
C
G
t
normal
allele
C
C
C
G
G
T
f ]
C
T
A
T
T
A
T
A
T
G
C
t
mutant
allele
FIG. 3 . Direct sequencing of polymerase chain reaction-am-
plified DNA samples. A region of exon 11 in the a-chain of target
DNA samples was amplified and sequenced as described under “Ex-
perimental Procedures.” The left panel is the sequence of an area of
exon 11 found in the a-chain of a normal Ashkenazi Jew, D.B. The
middle panel is that of an Ashkenazi Jewish patient, R.B., homozygous
for the exon 11 insertion defect. The right panel is that of an obligate
heterozygote carrier for this mutation, T.B., mother of R.B. The
nucleotide sequences determined from the films are listed beside the
autoradiogram. The insertion is bracketed. Sequencing lanes in each
panel are from left to right, A, G, C, T.
family mutation. The parents of GM515 were unavailable for
testing.
The region of exon 11 that had been amplified in the
genomic DNA samples was sequenced directly. As expected,
the sequence found in the amplified sample of an Ashkenazi
Jew with noncarrier status is normal (Fig. 3), whereas that
observed in an Ashkenazi patient with classic Tay-Sachs,
R.B. (homozygous for the a-chain exon 11 defect by the dot
blot assay (Fig. 2)), does ineeed contain the 4-base pair
insertion (Fig. 3). In an obligate heterozygote carrier, B.B.
(the father of R.B.), both normal and mutant alleles are
equally amplified by the polymerase chain reaction so that
extension from sequencing primer occurs on both alleles. Up
until the insertion defect the sequencing ladders of the two
alleles are identical (Fig. 3). At the point of insertion the
sequences diverge and result in a superimposition of the
sequencing ladders derived from the normal and mutant al-
leles (Fig. 3).
Because the insertion introduces a premature termination
signal, one would have expected to find a truncated a-chain
polypeptide (10 kDa shorter than normal) in cultured cells of
Ashkenazi patients. In fact, no a-chain polypeptide can be
observed by immunological techniques in GM515 or other
Ashkenazi patient fibroblasts (23). Rather, the insertion re-
sults in a deficiency of a-chain mRNA (19). The thalassemias
provide similar examples of nonsense mutations associated
with mRNA deficiency (24). The mechanism by which a
premature termination codon within an exon affects mRNA
levels is unknown but may involve mRNA stability, intranu-
clear mRNA metabolism (25), or mRNA processing.
The etiology of the high gene frequency of classic Tay-
Sachs disease in the Ashkenazi population has been the
subject of controversy, Le. the founder effect uersus selective
advantage (26, 27). Identification of the major mutant a-
chain allele (-70%) and suggestion of a very low-frequency
allele in this report, along with the description of the splice
junction allele, appear to support the
theory.
selective advantage
Acknowledgments-We thank the patients at Long Island Jewish
Hospital, Brooklyn, NY, and their families for donating blood and
Lyndon Badal for facilitating transport of the samples to our labo-
ratory. We thank Eugene Grebner for providing leukocyte samples of
carriers and normals and thank Richard L. Proia for providing a
normal a-chain genomic clone. We are grateful to April Robbins and
William B. Jakoby for carefully reading this manuscript.
REFERENCES
1 . O’Brien, J. S. (1983) in The Metabolic Basis of Inherited Disease
(Stanbury, J. B., Wyngaarden, J. B., Fredrickson, D. S., Gold-
stein, J. L., and Brown, M. s., eds) pp. 945-969, McGraw-Hill
Book Co., New York
2 . Sandhoff, K., and Conzelmann, E. (1984) Neuropediatrics Suppl.
15.85-92
3 . Myerowitz, R. (1988) Proc. Natl. Acad. Sei. U. S. A. 85, 3955-
3959
4 . Arpaia, E., Dumbrille-Ross, A., Maler, T., Neote, K., Tropak, M.,
Troxel, C., Stirling, J. L., Pith, J. S., Bapat, B., Lamhonwah,
A. M., Mahuran, D. J., Schuster, S. M., Clarke, J. T. R.,
Lowden, J. A., and Gravel, R. A. (1988) Nature 333,85-86
5 . Ohno, K., and Suzuki, K. (1988) Bwchem. Biophys. Res. Commun.
153,463-469
6. Sando, G. N., and Neufeld, E. F. (1977) Cell 12,619-627
7 . Myerowitz R., and Hogikyan, N. D. (1987) J. Bwl. Chem. 262,
15396-15399
8 . Myerowitz, R., Piekan, R., Neufeld, E. F., Shows, T. B., and
Suzuki, K. (1985) Proc. Natl. Acad. Sei. U. S. A. 82,7830-7834
9. Feinberg, A. P., and Vogelstein, B. (1983) Anal. Biochem. 132,
6-13
10. Proia, R. L., and Soravia, E. (1987) J. Bwl. Chem. 262, 5677-
5681
11. Maniatis, T . , Fritsch, E. F., and Sambrook, J. (1982) Molecular
Cloniw: A Laboratory Manual. Cold Spring Harbor Laboratory,
Cold Spring Harbor,-NY
12. Birnboim, H. C., and Doly, J. (1979) Nucleic Acids Res. 7,1513-
1523
”
..
13. Garger, S. J., Griffith, 0. M., and Grill, L. K. (1983) Biochern.
Biophys. Res. Commun. 117,835-842
14. Bell, G. I., Karam, J. H., and Rutter, W. J. (1981) Proc. Natl.
Acad. Sei. U. S. A. 78,5759-5763
15. Mullis, K. B., and Faloona, F. A. (1987) Methods Enzymol. 155,
335-350
16. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl.
Acad. Sci. U. S. A. 74,5463-5467
17. Wirschnik, L . A., Higuchi, R. G., Stoneking, M., Erlich, H. A.,
Arnheim, N., and Wilson, A. C. (1987) Nucleic Acids Res. 15,
529-541
18. Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharfe, S. J., Higuchi,
R., Horn, G. T., Mullis, K. B., and Erlich, H. A. (1988) Science
239,487-491
19. Myerowitz, R., and Hogikyan, N. D. (1986) Science 232, 1646-
1648
20. Korneluk, R. G., Mahuran, D. J., Neote, K., Klavins, M. H.,
O’Dowd, B. F., Tropak, M., Willard, H. F., Anderson, M. J.,
Lowden, J. A., and Gravel, R. A. (1986) J. Biol. Chem. 261,
8407-8413
21. Myerowitz, R., and Proia, R. L . (1984) Proc. Natl. Acad. Sci.
U. S. A. 81,5394-5398
22. Paw, B. H., and Neufeld, E. F. (1988) J. Biol. Chem. 263,3012-
3015
23. Hasilik, A., and Neufeld, E. F. (1980) J. Biol. Chem. 255,4937-
4945
24. Orkin, S. H., and Kazarian, H. H. (1984) Annu. Rev. Genet. 18,
131-171
25. Baserga, S. J., and Benz, E. J., Jr. (1988) Proc. Natl. Acad. Sci.
U. S. A. 85, 2056-2060
26. Chase, G. A. (1977) Prog. Clin. Biol. Res. 18,107-110
27. Myrianthopoulos, N. C., and Melnick, M. (1977) Prog. Clin. Bwl.
Res. 18,95-106