Original Article
Screening for Carriers of Tay-Sachs Disease among Ashkenazi Jews — A Comparison of DNA-Based and Enzyme-Based Tests
N Engl J Med 1990; 323:6-12July 5, 1990
- Abstract
- Abstract
Background and Methods.
The prevention of Tay-Sachs disease (GM2 gangliosidosis, type 1) depends on the identification of carriers of the gene for this autosomal recessive disorder. We compared the enzyme-based test widely used in screening for Tay-Sachs disease with a test based on analysis of DNA. We developed methods to detect the three mutations in the HEXA gene that occur with high frequency among Ashkenazi Jews: two mutations cause infantile Tay-Sachs disease, and the third causes the adult-onset form of the disease. DNA segments containing these mutation sites were amplified with the polymerase chain reaction and analyzed for the presence of the mutations.
Results.
Among 62 Ashkenazi obligate carriers of Tay-Sachs disease, the three specific mutations accounted for all but one of the mutant alleles (98 percent). In 216 Ashkenazi carriers identified by the enzyme test, DNA analysis showed that 177 (82 percent) had one of the identified mutations. Of the 177, 79 percent had the exon 11 insertion mutation, 18 percent had the intron 12 splice-junction mutation, and 3 percent had the less severe exon 7 mutation associated with adult-onset disease. The results of the enzyme tests in the 39 subjects (18 percent) who were defined as carriers but in whom DNA analysis did not identify a mutant allele were probably false positive (although there remains some possibility of unidentified mutations). In addition, of 152 persons defined as noncarriers by the enzyme-based test, 1 was identified as a carrier by DNA analysis (i.e., a false negative enzyme-test result).
Conclusions.
The increased specificity and predictive value of the DNA-based test make it a useful adjunct to the diagnostic tests currently used to screen for carriers of Tay-Sachs disease. Although some false positive results may be desirable on an enzyme-based test that is used in screening, the DNA test allows precise definition of the carrier state for the known mutations. (N Engl J Med 1990; 323:6–12.)
Media in This Article
Figure 1
Strategies Used to Detect the Three HEXA Mutations and Corresponding DNA Photographs.Figure 2
Hexosaminidase A Activity in Normal Subjects and Carriers of Tay-Sachs Disease, According to DNA Analysis.Article Activity
- Article
Tay-sachs disease (GM2 gangliosidosis, type 1, reviewed in references 1 and 2) is an autosomal recessive lysosomal-storage disorder that results from a deficiency of the α subunit of hexosaminidase A.3 , 4 In the absence of hexosaminidase A, GM2 ganglioside cannot be hydrolyzed and therefore accumulates primarily in neuronal tissues. This results in progressive neurologic degeneration, the severity of which appears to correlate with the level of residual hexosaminidase A activity.1 Patients with infantile Tay-Sachs disease die before the age of five, whereas patients with the juvenile and adult forms have a delayed onset.
Hexosaminidase occurs predominantly in two forms, hexosaminidase A and hexosaminidase B.5 Hexosaminidase A is made up of one α and one β subunit, encoded by the HEXA and HEXB genes,6 7 8 respectively. Hexosaminidase B is made up of two β subunits.9 Mutations of the α subunit affect hexosaminidase A activity, which can be detected in the serum and tissues of patients and carriers.3 , 10 Because of the high incidence of Tay-Sachs disease in the Ashkenazi Jewish population,11 worldwide screening programs were established.12
Hexosaminidase A activity can be measured with the use of an artificial, fluorogenic substrate. Since hexosaminidase B is thermostable and hexosaminidase A is heat labile, the difference in total hexosaminidase activity before and after heat denaturation is used to calculate the percentage of hexosaminidase A in the sample.13 Many factors,10 , 14 15 16 notably pregnancy10 , 17 , 18 and the presence of oral contraceptives,19 cause an elevation in total activity of heat-stable hexosaminidase in serum.20 The decrease in the apparent percentage of hexosaminidase A activity in serum can make the test an unreliable indicator of the carrier state.19 In these instances, leukocyte hexosaminidase is assayed to identify true carriers.14 , 17 , 19
The frequency of carriers of the gene for Tay-Sachs disease in the Ashkenazi population in the United States has been estimated by enzyme assay to be 1 in 27,27 accounting for an incidence of infants affected at birth that is 100 times higher (1 in 3600) than in other populations.22 The number of such infants born to Ashkenazi Jews has been reduced by 90 percent since the introduction of carrier-screening programs.1
The recent elucidation of the molecular basis of three HEXA mutations2 in Ashkenazi Jews introduced the possibility of DNA-based screening for carriers of Tay-Sachs disease in this population. The first mutation identified was a splice-junction mutation at the 5′ end of intron 12.23 24 25 Unexpectedly, it accounted for only 20 to 30 percent of the cases of infantile Tay-Sachs disease among Ashkenazi Jews, despite the hypothesis that the high incidence of the disease in this population was due to a single-founder effect.26 A second mutation, a 4-bp (base-pair) insertion in exon 11 that accounted for approximately 70 percent of the cases of infantile Tay-Sachs disease in Ashkenazi Jews, was subsequently identified.27 The third of these mutations, responsible for adult-onset GM2 gangliosidosis, was a G-to-A nucleotide substitution in exon 7 that reduced hexosaminidase A activity.28 , 29
In the present study, obligate carriers of Tay-Sachs disease were examined to determine whether the major disease-causing alleles in the Ashkenazi population had been identified. The DNA analysis was extended to Ashkenazi subjects identified as normal (noncarriers) or as carriers by enzyme analysis, and the results of the enzyme and DNA analyses were compared.
Methods
DNA Sources
Fibroblasta from cell lines established from patients with Tay-Sachs disease (GM02968, GM00515A, and GM00502B) were obtained from the Human Genetic Mutant Cell Repository (Camden, N.J.). Fibroblasts or leukocytes from other patients and obligate carriers of infantile Tay-Sachs disease or adult-onset GM2 gangliosidosis were supplied by the Boston and Toronto Tay-Sachs disease prevention programs. These centers also provided leukocyte pellets and sonicates that had been enzyniatically tested in their programs.
Sample Selection
Leukocyte pellets and sonicates from the Toronto center came from part of the samples from normal subjects and from all the samples from carriers who had been evaluated from 1987 to 1989. The samples supplied by the Boston center had been obtained only from obligate carriers, enzyniatically identified carriers, and patients with the disease. Any biologic parent of a fetus or child with any form of Tay-Sachs disease was considered an obligate carrier. Three of the obligate carriers were parents of an affected fetus in whom the diagnosis of Tay-Sachs disease was confirmed by two methods. Random carriers and noncarriers included any persons of Ashkenazi Jewish ancestry (i.e., descended from Jews from Central or Eastern Europe). A sample was excluded if the person was not of Ashkenazi descent or was a blood relative of an enzymatically identified or obligate carrier. DNA analysis was done without knowledge of the results of enzyme analysis. The allele frequencies of samples from the Boston and Toronto centers were compared by means of a chi-square test of a two-by-three contingency table, with two degrees of freedom.
Assay of Hexosaminidase A Activity
Serum hexosaminidase levels were determined with a manual method in Boston and an automated method in Toronto.17 , 30 Hexosaminidase was measured in leukocytes if the subject was known to be pregnant, was taking oral contraceptives, or had a serum level of the enzyme in the range of values that were inconclusive or indicated carrier status. Consequently, whereas enzyme levels in 48 percent of the samples from normal subjects were measured in serum, the levels in most of the samples from carriers were measured in leukocytes. In this report, serum values are differentiated from leukocyte values only if they alter the mean of the results of enzyme analysis.
Leukocytes were prepared from whole blood collected in tubes containing heparin by dextran precipitation17 or a modified dextran procedure.19 Leukocyte hexosaminidase A activity was determined in both centers with the artificial substrate 4-methyl-unibelliferyl-β-D-N-acetylglucosaminide and an assay based on thermal differentiation.17 Statistical analysis of the results of enzyme testing in the normal and carrier (control) groups was performed as described by Kaback et al.17 to establish the corresponding reference ranges; each range was unique to each center.
The Toronto and Boston centers are accredited by the International Tay-Sachs Prevention Program, administered through the California Testing Program, to maintain quality-control standards for screening for Tay-Sachs disease.
DNA Preparation
DNA was prepared from fibroblasts or leukocyte pellets obtained by dextran precipitation.31 Approximately one third of the leukocyte pellet, dispersed in water, or 100 μl of leukocyte sonicate was used for the preparation of DNA. The extracted DNA was resuspended in 50 μl of water, and 2 to 3 μl (equivalent to 100 to 200 μl of whole blood) was used for each amplification reaction. In general, the extraction of the DNA gave more consistent amplifications.
Amplification of Genomic DNA by Polymerase Chain Reaction
Target sequences were amplified from 0.5 to 1.0 μg of genomic DNA or directly from leukocyte pellets or lysates prepared according to the modified dextran procedure.32 Amplification reactions were conducted in a 50-μl volume containing 100 pmol of each oligonucleotide primer; 1.5 mmol each of deoxyadenosine triphosphate, deoxycytidine triphosphate, deoxyguanosine triphosphate, and deoxythymidine triphosphate per liter; and 1.25 to 2.5 units of recombinant Taq polymerase (Amplitaq; Cetus, Emeryville, Calif.) in 10 mM TRIS-hydrochloride (pH 8.3), 50 mM potassium chloride, 1.5 mM magnesium chloride, and 0.01 percent gelatin as recommended by the manufacturer. Amplification was performed for 30 cycles in a Perkin–Elmer–Cetus DNA Thermal Cycler; each cycle consisted of 30 seconds of denaturation at 94°C, 30 seconds of annealing at 55°C (or at 60°C for the oligonucleotide-primer pair used to amplify exon 7), and 90 seconds of extension at 72°C. Precautions were taken to avoid contamination of the polymerase chain reactions by DNA and included negative controls. Gel electrophoresis of the samples used in the reactions was chosen for the analysis because direct visualization would allow a more detailed assessment of the quality control. The mutations we studied, however, have also been analyzed by dot-blot assay.24 , 27
Sample aliquots (5 μl for undigested DNA and 10 μl for digested DNA) for analysis were taken from the amplified-product reaction tube, and 2 U of an appropriate restriction enzyme — HaeIII, DdeI (both enzymes from New England Biolabs, Beverly, Mass.), or EcoRII (Bethesda Research Laboratories, Gaithersburg, Md.) —was added. After the samples were incubated for two hours at 37°C, they were analyzed by electrophoresis on an 8 percent polyacrylamide gel, followed by staining with ethidium bromide and visualization with ultraviolet fluorescence. The strategies for amplification and analysis are shown in Figure 1Figure 1
Strategies Used to Detect the Three HEXA Mutations and Corresponding DNA Photographs..Results
Strategies of DNA Analysis
Samples were analyzed for the three known HEXA mutations among Ashkenazi Jews by means of DNA amplification with the polymerase chain reaction and restriction-enzyme digestion followed by polyacrylamide-gel electrophoresis (Fig. 1). The mutations could be detected because each creates a characteristic new band in the restriction-fragment pattern of the amplified product (Fig. 1D).
The most common of the mutations, a 4-bp insertion in exon 11 (Fig. 1A), was identified in both heterozygous states (Fig. 1D, lanes 3 and 4) and homozygous states (lanes 5 and 6) by digestion of the amplified product with the restriction enzyme HaeIII. In the presence of the 4-bp insertion, a 47-bp band was seen rather than a 43-bp band. When the product was separated by electrophoresis on polyacrylamide but not agarose gels, the undigested amplified product of heterozygotes always contained two extra bands that migrated more slowly (Fig. 1D). These bands were heteroduplexes resulting from annealing of DNA strands from the amplified normal and mutant alleles.33 When the heterologous products anneal, the 4-bp insertion forms a bubble that slows the migration of the duplex in polyacrylamide gels. Since either strand of the amplified mutant product can anneal with its counterpart in the normal amplified product, two types of heteroduplexes are formed that migrate at different rates.34 The heteroduplexes provide a simple diagnostic test for carriers of the 4-bp insertion mutation.
The strategy for detecting the intron 12 splice-junction mutation (Fig. 1B) has been described previously.23 The G-to-C base substitution at the 5′ end of intron 12 created a new DdeI site, producing two fragments (85 and 35 bp) from the 120-bp DdeI fragment (Fig. 1D, lanes 7 to 10).
Detection of the exon 7 mutation associated with adult-onset disease was based on the loss of an EcoRII site due to a G-to-A nucleotide substitution at the 3′ end of exon 7 (strategy shown in Fig. 1C). The loss of the EcoRII site creates a 52-bp fragment from the 44-bp and 8-bp fragments present in the normal allele (lanes 11 to 14). The 52-bp fragment appears more intense than the 44-bp fragment, as previously noted.29
DNA Analysis of Obligate Carriers
Samples from patients with Tay-Sachs disease and obligate carriers (see Methods) were analyzed to determine the proportion of the disease alleles in Ashkenazi Jews that could be accounted for by the three mutations. The DNA samples studied were obtained from 42 Ashkenazi obligate carriers, 11 Ashkenazi patients (with an equivalent of 22 mutant alleles), 6 non-Ashkenazi obligate carriers, and 6 non-Ashkenazi patients (with an equivalent of 12 mutant alleles). Of the alleles from Ashkenazi subjects examined, 61 of 62 (98 percent) were identified as having one of the three known mutations, whereas of the 20 alleles from non-Ashkenazi subjects, 4 (20 percent) had the insertion mutation. A single Ashkenazi patient (cell line GM00515A) had the insertion mutation in combination with an unidentified mutation.27 Thus, only 1 of 62 (2 percent) alleles of obligate Ashkenazi subjects had an unidentified mutation (Table 1Table 1
DNA Analysis of Obligate Carriers of Tay-Sachs Disease.). Because the frequency of the exon 7 mutation would be biased by the large number of obligate alleles from patients with adult-onset disease (n = 8), only the frequency of the two mutations identified as causing infantile disease was calculated for the Ashkenazi group. Of these two mutations, the insertion mutation accounted for 85 percent of the alleles and the splice-junction mutation for 15 percent. The intron 12 splice-junction mutation was never found in the homozygous state or in combination with the exon 7 mutation.DNA Analysis of Samples Previously Tested by Enzyme Assay
Analysis of Normal (Noncarrier) Samples
A pool of samples classified as normal according to the enzyme assay were evaluated with the DNA test to determine the false negative rate. The average (±SD) hexosaminidase A level of 152 enzymatically normal samples (both serum and leukocyte samples, obtained from the Toronto center) was 66±5 percent (Fig. 2Figure 2
Hexosaminidase A Activity in Normal Subjects and Carriers of Tay-Sachs Disease, According to DNA Analysis.A). A survey of their leukocyte DNA for the two alleles common in infantile disease revealed one sample that contained the insertion mutation (1 of 152, or 0.7 percent). (Two leukocyte samples from the subject had been stored at the time of the original enzyme test; both were retested, and the results indicated carrier status on DNA analysis and normal status on the enzyme test.) When an additional sample from the subject with the insertion mutation was tested, the results indicated carrier status on both enzyme analysis (46 percent hexosaminidase A level in leukocytes) and DNA analysis. The only identifiable difference between the two samples was that the first sample was obtained while the subject was pregnant, but the second was obtained after the pregnancy.Analysis of Carrier Samples
Samples from 216 subjects identified as carriers by enzyme testing (Boston and Toronto centers) were analyzed for the three mutations. The samples were first tested for the two mutations common in infantile disease; samples with neither mutation were then tested for the less common exon 7 mutation (Table 2Table 2
DNA Analysis of All Carriers and Normal Subjects.). Of the 216 carriers, 140 had the exon 11 insertion mutation, 32 had the intron 12 splice-junction mutation, and 5 had the less severe exon 7 mutation. The remaining 39 subjects (18 percent) appeared to be normal on DNA analysis. Thus, the known mutations in a population of random Ashkenazi Jewish carriers had the following frequencies: insertion mutation, 79 percent; splice-junction mutation, 18 percent; and exon 7 mutation, 3 percent. The frequencies observed at the two centers did not differ significantly (P≥0.5), although the false positive rates differed slightly (Fig. 2).The hexosaminidase A values of the subjects with false positive results did not differ appreciably from those of subjects confirmed as carriers by DNA analysis. In Toronto, the carriers had an average hexosaminidase A level of 42±5 percent, as compared with 46±3 percent in subjects found to have false positive results. Similarly, in Boston, the carriers had an average hexosaminidase A level of 46±5 percent, as compared with 52±6 percent in the subjects with false positive results. The differences between the two centers reflect a slight difference in their reference ranges.
Discussion
Our DNA analyses of obligate carriers of Tay-Sachs disease identified all the common mutations known to cause the disease in the Ashkenazi population. The three known mutations accounted for 98 percent (61 of 62) of the mutant alleles in the group of Ashkenazi obligate carriers. The frequency of identified mutations increases to 99 percent (93 of 94) of the alleles of all patients and carriers, or to 99 percent (78 of 79) of the alleles of all patients with infantile disease, if other obligate carriers described in the literature27 28 29 are included. These mutations were not restricted to the Ashkenazi population, since the insertion mutation was found in four non-Ashkenazi subjects, and the exon 7 mutation in a non-Ashkenazi (described as Assyrian) patient.29
Greenberg and Kaback have estimated that mutations other than the mutation (or mutations) common among Ashkenazi patients with infantile disease would have a frequency of 4.5X104 (2 to 3 percent of carriers of Tay-Sachs disease).35 Since the exon 7 mutation associated with adult-onset disease had a frequency of 3 percent in our analysis of carriers, HEXA mutations identified in other populations are unlikely to account for a large portion of the variant alleles in Ashkenazi Jews.36 37 38 The one mutant allele among the alleles of Ashkenazi obligate subjects that was not identified must be a variant with a low frequency.
Programs to prevent Tay-Sachs disease are designed to distinguish carriers from noncarriers by enzymatic-screening assays. Until now, the only way to assess false negative results on enzyme analysis has been to look for affected offspring of subjects classified as noncarriers by the enzyme assay. However, since the chance that a misclassified carrier will have a child with Tay-Sachs disease is low (effectively 1 in 120), false negative results are unlikely to be identified in this way. Although our study revealed a false negative result in 1 of 152 Ashkenazi noncarriers, the result of enzyme testing was confirmed on repeated assay.
An apparent false positive rate of 18 percent was found among 216 Ashkenazi subjects in whom values on the enzyme assay fell in the range for carriers. Possible explanations for the false positive results include (1) unidentified mutations, with or without implications for disease, that cause an actual or apparent reduction in hexosaminidase A activity (e.g., a reduction detected as a result of the thermal-denaturation step), (2) variation in hexosaminidase levels that is due to unidentified biologic factors, and (3) expected statistical variation and inconsistencies in the enzyme assay. As new cases of Tay-Sachs disease and benign alleles are identified, it will be possible to differentiate between the biochemical and biologic factors influencing the enzymatic test. In the interim, it will be important to determine whether other centers have high false positive rates, especially when different methods are used to differentiate hexosaminidase isozymes.
These findings show that the DNA test is more specific than the currently used enzyme test. Although the sensitivity of the two tests is similar, the positive predictive value of the DNA test is superior to that of the enzyme test. The simplicity of the DNA test will allow it to be performed in small centers without the need to maintain the rigorous quality control required for the enzyme assay. Laboratories establishing these procedures should not underestimate the necessity to take precautions to avoid external DNA contamination.39 The DNA test will be a useful supplement in prenatal testing if the mutations present in the parents have been identified, since it can predict both the genetic status of the child and the severity of the disease if the child is affected. We recommend the implementation of DNA testing in conjunction with enzyme screening for Tay-Sachs disease in Ashkenazi Jews, recognizing that variant mutations will continue to be identified and means for their detection incorporated into the DNA test.
Supported by a grant (MA-10432) from the Medical Research Council of Canada. Dr. Triggs-Raine is a postdoctoral fellow supported by the Medical Research Council of Canada, and Dr. Feigenbaum is a postdoctoral fellow supported by the Hospital for Sick Children.
We are indebted to Dr. Michael Kaback for critical discussions, to Andrea Cajolet and Irene Warren for technical assistance, and to Charles Troxel for the preparation of oligonucleotide primers.
Source Information
From The Research Institute (B.L.T.-R., A.S.J.F., M.-A.S., D.J.M., R.A.G.) and the Department of Pediatrics (J.T.R.C), Hospital for Sick Children, Toronto; the E.K. Shriver Center, Waltham, Mass., and Harvard Medical School, Boston (M.N., E.H.K.); and the Department of Biochemistry and Molecular Biology. University of Florida, Gainesville (S.M.S.). Address reprint requests to Dr. Gravel at MeGill-Montreal Children's Hospital Research Institute, 2300 Tupper St., Montreal, PQ H3H 1P3, Canada.
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Raelia Lew, Leslie Burnett, Anné Proos. (2011) Tay-Sachs disease preconception screening in Australia: self-knowledge of being an Ashkenazi Jew predicts carrier state better than does ancestral origin, although there is an increased risk for c.1421 + 1G > C mutation in individuals with South African heritage. Journal of Community Genetics 2:4, 201-209
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NOH JIN PARK, CRAIG MORGAN, RAJESH SHARMA, YUANYIN LI, RAYNAH M. LOBO, JOY B. REDMAN, DENISE SALAZAR, WEIMIN SUN, JULIE A. NEIDICH, CHARLES M. STROM. (2010) Improving Accuracy of Tay Sachs Carrier Screening of the Non-Jewish Population: Analysis of 34 Carriers and Six Late-Onset Patients With HEXA Enzyme and DNA Sequence Analysis. Pediatric Research 67:2, 217-220
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Soon Min Lee, Min Jung Lee, Joon Soo Lee, Heung Dong Kim, Jin Sung Lee, Jinna Kim, Seung Koo Lee, Young Mock Lee. (2008) Newly observed thalamic involvement and mutations of the HEXA gene in a Korean patient with juvenile GM2 gangliosidosis. Metabolic Brain Disease 23:3, 235-242
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