Population-based genetic screening for reproductive counseling: the Tay-Sachs disease model.

UNLABELLED: Since 1970, more than 1.4 million individuals worldwide have been screened voluntarily to determine if they are carriers of the mutant gene for Tay-Sachs Disease (TSD). Employing both enzymatic and molecular methods (for optimal sensitivity and specificity) more than 1400 couples have been identified to be at-risk for TSD in their offspring, i.e., both parents heterozygotes. Through prenatal testing of more than 3200 pregnancies, births of over 600 infants with this uniformly fatal neurodegenerative disease have been prevented. In the United States and Canada, the incidence of TSD in the Jewish population has been reduced by more than 90%. More that 100 mutations in the hexosaminidase A gene (the TSD locus) have been identified to date. Some are associated with later onset or more chronic forms of neuronal storage disease. Two mutations cause a carrier-like "pseudo-deficiency" when enzymatic testing is used (false positives). A number of practical, social, and ethical complexities have been identified in this prototypic population-based effort. Educational and counseling components must be provided both before and after screening. Issues of privacy and confidentiality of test results must be addressed. In certain cultures insurability and employment may be involved. The public perception of the biomedical community as advocates for wide-scale testing and screening may be interpreted, in some systems, as conflicts of interest on the part of entrepreneurial scientists, clinicians, and institutions. CONCLUSION: Many new opportunities for population-based screening will be evident in this era of genome-related discovery. Accordingly, some of the experiences with Tay-Sachs disease prevention may be instructive.

Evaluation of mental retardation: recommendations of a Consensus Conference: American College of Medical Genetics.

A Consensus Conference utilizing available literature and expert opinion sponsored by the American College of Medical Genetics in October 1995 evaluated the rational approach to the individual with mental retardation. Although no uniform protocol replaces individual clinician judgement, the consensus recommendations were as follows: 1. The individual with mental retardation, the family, and medical care providers benefit from a focused clinical and laboratory evaluation aimed at establishing causation and in providing counseling, prognosis, recurrence risks, and guidelines for management. 2. Essential elements of the evaluation include a three-generation pedigree: pre-, peri-, and post-natal history, complete physical examination focused on the presence of minor anomalies, neurologic examination, and assessment of the behavioral phenotype. 3. Selective laboratory testing should, in most patients, include a banded karyotype. Fragile X testing should be strongly considered in both males and females with unexplained mental retardation, especially in the presence of a positive family history, a consistent physical and behavioral phenotype and absence of major structural abnormalities. Metabolic testing should be initialed in the presence of suggestive clinical and physical findings. Neuroimaging should be considered in patients without a known diagnosis especially in the presence of neurologic symptoms, cranial contour abnormalities, microcephaly, or macrocephaly. In most situations MRI is the testing modality of choice. 4. Sequential evaluation of the patient, occasionally over several years, is often necessary for diagnosis, allowing for delineation of the physical and behavioral phenotype, a logical approach to ancillary testing and appropriate prognostic and reproductive counseling.

Novel mutations and DNA-based screening in non-Jewish carriers of Tay-Sachs disease.

We have evaluated the feasibility of using PCR-based mutation screening for non-Jewish enzyme-defined carriers identified through Tay-Sachs disease-prevention programs. Although Tay-Sachs mutations are rare in the general population, non-Jewish individuals may be screened as spouses of Jewish carriers or as relatives of probands. In order to define a panel of alleles that might account for the majority of mutations in non-Jewish carriers, we investigated 26 independent alleles from 20 obligate carriers and 3 affected individuals. Eighteen alleles were represented by 12 previously identified mutations, 7 that were newly identified, and 1 that remains unidentified. We then investigated 46 enzyme-defined carrier alleles: 19 were pseudodeficiency alleles, and five mutations accounted for 15 other alleles. An eighth new mutation was detected among enzyme-defined carriers. Eleven alleles remain unidentified, despite the testing for 23 alleles. Some may represent false positives for the enzyme test. Our results indicate that predominant mutations, other than the two pseudodeficiency alleles (739C-->T and 745C-->T) and one disease allele (IVS9+1G-->A), do not occur in the general population. This suggests that it is not possible to define a collection of mutations that could identify an overwhelming majority of the alleles in non-Jews who may require Tay-Sachs carrier screening. We conclude that determination of carrier status by DNA analysis alone is inefficient because of the large proportion of rare alleles. Notwithstanding the possibility of false positives inherent to enzyme screening, this method remains an essential component of carrier screening in non-Jews. DNA screening can be best used as an adjunct to enzyme testing to exclude known HEXA pseudodeficiency alleles, the IVS9+1G-->A disease allele, and other mutations relevant to the subject's genetic heritage.

The carrier frequency of the BRCA1 185delAG mutation is approximately 1 percent in Ashkenazi Jewish individuals.

Since BRCA1, the first major gene responsible for inherited breast cancer, was cloned, more than 50 unique mutations have been detected in the germline of individuals with breast and ovarian cancer. In high-risk pedigrees, female carriers of BRCA1 mutations have an 80-90% lifetime risk of breast cancer, and a 40-50% risk of ovarian cancer. However, the mutation stats of individuals unselected for breast or ovarian cancer has not been determined, and it is not known whether mutations in such individuals confer the same risk of cancer as in individuals from the high-risk families studied so far. Following the finding of a 185delAG frameshift mutation in several Ashkenazi Jewish breast/ovarian families, we have determined the frequency of this mutation in 858 Ashkenazim seeking genetic testing for conditions unrelated to cancer, and in 815 reference individuals not selected for ethnic origin. We observed the 185delAG mutation in 0.9% of Ashkenazim (95% confidence limit, 0.4-1.8%) and in none of the reference samples. Our results suggest that one in a hundred women of Ashkenazi descent may be at especially high risk of developing breast and/or ovarian cancer.

Perspectives in genetic screening. Principles and implications.

Recent advances in genetic identification and characterization of a number or hereditary disorders have led to increased possibilities for genetic testing and screening. The context and methods of screening are important given that identification of otherwise healthy persons as being presymptomatic or at increased risk for genetic diseases may have serious consequences for their future lifestyle, employment, and insurability. This article examines general principles for genetic screening, including goals, delivery issues, and professional and lay responses to screening and counseling, and recommends areas in which social psychological research on screening is needed.

A second mutation associated with apparent beta-hexosaminidase A pseudodeficiency: identification and frequency estimation.

Deficient activity of beta-hexosaminidase A (Hex A), resulting from mutations in the HEXA gene, typically causes Tay-Sachs disease. However, healthy individuals lacking Hex A activity against synthetic substrates (i.e., individuals who are pseudodeficient) have been described. Recently, an apparently benign C739-to-T (Arg247Trp) mutation was found among individuals with Hex A levels indistinguishable from those of carriers of Tay-Sachs disease. This allele, when in compound heterozygosity with a second "disease-causing" allele, results in Hex A pseudodeficiency. We examined the HEXA gene of a healthy 42-year-old who was Hex A deficient but did not have the C739-to-T mutation. The HEXA exons were PCR amplified, and the products were analyzed for mutations by using restriction-enzyme digestion or single-strand gel electrophoresis. A G805-to-A (Gly269Ser) mutation associated with adult-onset GM2 gangliosidosis was found on one chromosome. A new mutation, C745-to-T (Arg249Trp), was identified on the second chromosome. This mutation was detected in an additional 4/63 (6%) non-Jewish and 0/218 Ashkenazi Jewish enzyme-defined carriers. Although the Arg249Trp change may result in a late-onset form of GM2 gangliosidosis, any phenotype must be very mild. This new mutation and the benign C739-to-T mutation together account for approximately 38% of non-Jewish enzyme-defined carriers. Because carriers of the C739-to-T and C745-to-T mutations cannot be differentiated from carriers of disease-causing alleles by using the classical biochemical screening approaches, DNA-based analyses for these mutations should be offered for non-Jewish enzyme-defined heterozygotes, before definitive counseling is provided.

Further investigation of the HEXA gene intron 9 donor splice site mutation frequently found in non-Jewish Tay-Sachs disease patients from the British Isles.

In a previous study we found that a Tay-Sachs disease (TSD) causing mutation in the intron 9 donor splice site of the HEXA gene occurs at high frequency in non-Jewish patients and carriers from the British Isles. It was found more frequently in subjects of Irish, Scottish, and Welsh origin compared with English origin (63% and 31% respectively). We have now tested, in a blind study, 26 American TSD carriers and 28 non-carriers who have British ancestry for the intron 9 splice site mutation. Six of the carriers and none of the controls were positive for the mutation. All six had Irish ancestry, compared with nine of the 20 other (intron 9 mutation negative) TSD carriers (p < 0.05). These results confirm the previously found high frequency of the intron 9 mutation in non-Jewish TSD families of British Isles, particularly Irish, origin, and reinforce the need to screen such families for this mutation.

A pseudodeficiency allele common in non-Jewish Tay-Sachs carriers: implications for carrier screening.

Deficiency of beta-hexosaminidase A (Hex A) activity typically results in Tay-Sachs disease. However, healthy subjects found to be deficient in Hex A activity (i.e., pseudodeficient) by means of in vitro biochemical tests have been described. We analyzed the HEXA gene of one pseudodeficient subject and identified both a C739-to-T substitution that changes Arg247----Trp on one allele and a previously identified Tay-Sachs disease mutation on the second allele. Six additional pseudodeficient subjects were found to have the C739-to-T mutation. This allele accounted for 32% (20/62) of non-Jewish enzyme-defined Tay-Sachs disease carriers but for none of 36 Jewish enzyme-defined carriers who did not have one of three known mutations common to this group. The C739-to-T allele, together with a "true" Tay-Sachs disease allele, causes Hex A pseudodeficiency. Given both the large proportion of non-Jewish carriers with this allele and that standard biochemical screening cannot differentiate between heterozygotes for the C739-to-T mutations and Tay-Sachs disease carriers, DNA testing for this mutation in at-risk couples is essential. This could prevent unnecessary or incorrect prenatal diagnoses.

Ganglioside GM2 levels in human melanoma cells: inverse correlation with lysosomal beta-hexosaminidase A activity.

GM2 is usually significantly elevated in human melanoma cells. The lysosomal hydrolase beta-hexosaminidase A (Hex A), is an isoenzyme required for terminal GalNAc hydrolysis from intact GM2 to GM3. The objective of the present studies is to determine if the elevated levels of gangliosides, particularly GM2, correlate with the activity of Hex A in cultured melanoma cells. The Hex A activity in 13 melanoma cell lines ranged from 26%-88.3% There was a inverse correlation between GM2 nmole/g and Hex A activity (r = -0.48; p less than 0.003). An inverse correlation (p less than 0.03) occurred between GD2 nmole/g and Hex A activity. There was an inverse correlation (r = -0.53; p less than 0.05 and r = -0.51; p less than 0.05, respectively) between the ratio of substrate/product (GM2/GM3) and (GD2/GD3) and Hex A activity. These results indicate that the level of GM2 in melanoma is inversely correlated with the level of activity of Hex A.

A mutation common in non-Jewish Tay-Sachs disease: frequency and RNA studies.

Tay-Sachs disease (TSD) is an autosomal recessive genetic disorder resulting from mutation of the HEXA gene encoding the alpha-subunit of the lysosomal enzyme, beta-N-acetylhexosaminidase A (Hex A). We have discovered that a Tay-Sachs mutation, IVS-9 + 1 G-->A, first detected by Akli et al. (Genomics 11:124-134, 1991), is a common disease allele in non-Jewish Caucasians (10/58 alleles examined). A PCR-based diagnostic test, which detects an NlaIII site generated by the mutation, revealed a frequency among enzyme-defined carriers of 9/64 (14%). Most of those carrying the allele trace their origins to the United Kingdom, Ireland, or Western Europe. It was not identified among 12 Black American TSD alleles or in any of 18 Ashkenazi Jewish, enzyme-defined carriers who did not carry any of the mutations common to this population. No normally spliced RNA was detected in PCR products generated from reverse transcription of RNA carrying the IVS-9 mutation. Instead, the low levels of mRNA from this allele were comprised of aberrant species resulting from the use of either of two cryptic donor sites, one truncating exon 9 and the other within IVS-9, spliced to exon 10. Numerous additional splice products were detected, most involving skipping of one or more surrounding exons. Together with a recently identified allele responsible for Hex A pseudodeficiency (Triggs-Raine et al. Am J Hum Genet, 1992), these two alleles accounted for almost 50% (29/64) of TSD or carrier alleles ascertained by enzyme screening tests in non-Jewish Caucasians.

Frequency of three Hex A mutant alleles among Jewish and non-Jewish carriers identified in a Tay-Sachs screening program.

Mutations in the HEX A gene, encoding the alpha-subunit of beta-hexosaminidase A (Hex A), are the cause of Tay-Sachs disease as well as of juvenile, chronic, and adult GM2 gangliosidoses. We have examined the distribution of three mutations--a 4-nucleotide insertion in exon 11, a G----C transversion at a 5' splice site in intron 12, and a 269Gly----Ser amino acid substitution in exon 7--among individuals enzymatically diagnosed as carriers of Hex A deficiency. Mutation analysis included polymerase chain reaction (PCR) amplification of the relevant regions of genomic DNA, followed by allele-specific oligonucleotide hybridization; another test for heterozygosity of the exon 11 insertion was based on the formation of heteroduplex PCR fragments of low electrophoretic mobility. The percentage distribution of the exon 11, intron 12, exon 7, and unidentified mutant alleles was 73:15:4:8 among 156 Jewish carriers of Hex A deficiency and 16:0:3:81 among 51 non-Jewish carriers. Regardless of the mutation, the ancestral origin of the Jewish carriers was primarily eastern and (somewhat less often) central Europe, whereas for the non-Jewish carriers it was western Europe. Because a twelfth of the Jewish carriers and four-fifths of the non-Jewish carriers of Hex A deficiency had mutant alleles other than the three common ones tested, enzyme-based tests cannot be replaced by DNA-based tests at the present time. However, DNA-based tests for two-carrier couples could identify those at risk for the chronic/adult GM2 gangliosidoses rather than for infantile Tay-Sachs disease.

Juvenile GM2 gangliosidosis caused by substitution of histidine for arginine at position 499 or 504 of the alpha-subunit of beta-hexosaminidase.

Juvenile GM2 gangliosidosis is a rare neurodegenerative disorder closely related to Tay-Sachs disease but of later onset and more protracted course. The biochemical defect lies in the alpha-subunit of the lysosomal enzyme beta-hexosaminidase. Cultured fibroblasts derived from patient A synthesized an alpha-subunit which could acquire mannose 6-phosphate and be secreted, but which failed to associate with the beta-subunit to form the enzymatically active heterodimer. By contrast, fibroblasts from patient B synthesized an alpha-subunit that was retained in the endoplasmic reticulum. To identify the molecular basis of the disorder, RNA from fibroblasts of these two patients was reverse-transcribed, and the cDNA encoding the alpha-subunit of beta-hexosaminidase was amplified by the polymerase chain reaction (PCR) in four overlapping fragments. The PCR fragments were subcloned and shown by sequence analysis to contain a G to A transition corresponding to substitution of histidine for arginine at position 504 in the case of patient A and at position 499 in the case of patient B. The mutations were confirmed by hybridization of allele-specific oligonucleotides to PCR-amplified fragments of DNA corresponding to exon 13 of the alpha-subunit gene. The Arg504----His mutation was found on both alleles of patient A as well as of another unrelated patient; the homozygosity of this mutant allele is attributable to consanguinity in the two families. The Arg499----His mutation was found in patient B in compound heterozygosity with a common infantile Tay-Sachs allele. There is additional heterogeneity in juvenile GM2 gangliosidosis, as neither mutation was found in the DNA of a fourth patient. The Arg----His mutations at positions 499 and 504 are located at CpG dinucleotides, which are known to be mutagenic "hot spots."

Molecular basis of adult-onset and chronic GM2 gangliosidoses in patients of Ashkenazi Jewish origin: substitution of serine for glycine at position 269 of the alpha-subunit of beta-hexosaminidase.

Chronic and adult-onset GM2 gangliosidoses are neurological disorders caused by marked deficiency of the A isoenzyme of beta-hexosaminidase; they occur in the Ashkenazi Jewish population, though less frequently than classic (infantile) Tay-Sachs disease. Earlier biosynthetic studies had identified a defective alpha-subunit that failed to associate with the beta-subunit. We have now found a guanosine to adenosine transition at the 3' end of exon 7, which causes substitution of serine for glycine at position 269 of the alpha-subunit [designated 269 (Gly----Ser) substitution]. An RNase protection assay was used to localize the mutation to a segment of mRNA from fibroblasts of a patient with the adult-onset disorder. That segment of mRNA (after reverse transcription) and a corresponding segment of genomic DNA were amplified by the polymerase chain reaction and sequenced by the dideoxy method. The sequence analysis, together with an assay based on the loss of a ScrFI restriction site, showed that the patient was a compound heterozygote who had inherited the 269 (Gly----Ser) mutation from his father and an allelic null mutation from his mother. The 269 (Gly----Ser) mutation, in compound heterozygosity with a presumed null allele, was also found in fetal fibroblasts with an association-defective phenotype and in cells from five patients with chronic GM2 gangliosidosis. It was not found in beta-hexosaminidase A-deficient cells obtained from patients with infantile Tay-Sachs disease nor in cells from individuals who do not have beta-hexosaminidase A deficiency. However, there must be additional mutations with similar consequences, since the 269 (Gly----Ser) substitution was not present in fibroblasts from two patients with juvenile GM2 gangliosidosis even though these had an association-defective alpha-subunit.