Am. J. Hum. Genet. 51:371-377, 1992

Identification and Rapid Detection of Three Tay-Sachs Mutations in the Moroccan Jewish Population Liat Drucker,*,t Richard L.

Proia,$ and Ruth Navon*

*Human Genetics Department, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv, Tel-Aviv; tMolecular Genetics Unit, Sapir Medical Center, Kfar-Sava, Israel; and $Genetics and Biochemistry Branch, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, Bethesda

Summary Infantile Tay-Sachs disease (TSD) is caused by mutations in the HEXA gene that result in the complete absence of P-hexosaminidase A activity. It is well known that an elevated frequency of TSD mutations exists among Ashkenazi Jews. More recently it has become apparent that elevated carrier frequencies for TSD also occur in several other ethnic groups, including Moroccan Jews, a subgroup of Sephardic Jews. Elsewhere we reported an in-frame deletion of one of the two adjacent phenylalanine codons at position 304 or 305 (AF304/305) in one HEXA allele of a Moroccan Jewish TSD patient and in three obligate carriers from six unrelated Moroccan Jewish families. We have now identified two additional mutations within exon 5 of the HEXA gene that account for the remaining TSD alleles in the patient and carriers. One of the mutations is a novel C-to-G transversion, resulting in a replacement of Tyr180 by a stop codon. The other mutation is a G-to-A transition resulting in an Arg170-to-Gln substitution. This mutation is at a CpG site in a Japanese infant with Tay-Sachs disease and was described elsewhere. Analysis of nine obligate carriers from seven unrelated families showed that four harbor the AF3041305 mutation, two the Arg170-->Gln mutation, and one the Tyr'80 Stop mutation. We also have developed rapid, nonradioactive assays for the detection of each mutation, which should be helpful for carrier screening. Introduction

Tay-Sachs disease (TSD), an infantile form of GM2 gangliosidosis (reviewed in Sandhoff et al. 1989), is very heterogeneous at the molecular level, with over 30 allelic mutations reported to date (Akli et al. 1991; Gravel et al. 1991; Mules et al. 1991; Neufeld 1991; Triggs-Raine et al. 1991). The mutations result in a complete lack of 1-hexosaminidase A, a dimer composed of a and 13 subunits. The disease had long been considered to be an Ashkenazi Jewish affliction because of a carrier frequency (1 / 31) 10 times higher than that in the general population. Recently, it has become apparent that the frequency of the disease is also elevated among other ethnic groups-e.g., a

Received January 29, 1992; revision received March 23, 1992. Address for correspondence and reprints: Dr. Ruth Navon, Molecular Genetics Unit, Sapir Medical Center, Kfar-Sava 44281, Israel. This material is in the public domain, and no copyright is claimed.

French-Canadian population in eastern Canada (Andermann et al. 1977), a Pennsylvania Dutch semiisolate (Kelly et al. 1975), a French-Acadian (Cajun) population (McDowell et al. 1991), and Moroccan Jews (Vecht et al. 1983). We have been particularly interested in identifying HEXA mutations in Jewish populations in order to facilitate TSD screening and to gain insight into the origin and expansion of mutations in this ethnic group. In our earlier study, we had shown that neither of the Ashkenazi Jewish infantile TSD mutations-the 4-bp insertion in exon 11 (Myerowitz and Costigan 1988) and the G-to-C transversion in the donor splice site of intron 12 (Arpaia et al. 1988; Myerowitz 1988; Ohno and Suzuki 1988)-was found in six Moroccan Jewish obligate TSD carriers (Navon and Proia 1991). It became apparent, therefore, that the mutations causing TSD in the Moroccan Jews and Ashkenazi Jews were different. Analysis of a Moroccan Jewish TSD patient revealed an in-frame deletion (AF304/305) of one of the two adjacent phenylalanine codons that 371

Drucker et al.

372 are present at positions 304 and 305 in the a-subunit sequence. The AF3041305 mutation impaired the subunit assembly of 1-hexosaminidase A, resulting in an absence of enzyme activity. Analysis of obligate carriers from six unrelated Moroccan Jewish families showed that three harbored the AF3041305 mutation. The aims of the present study are to identify the other mutations that cause TSD in the Moroccan Jewish population and to develop assays for their rapid identification. Material and Methods Tay-Sachs Patient and Carriers

The Moroccan Jewish TSD patient was diagnosed by the age of 8 mo. A total deficiency of IB-hexosaminidase A was found in her serum leukocytes and skin fibroblasts. In our previous study (Navon and Proia 1991), the patient was found to carry the AF304/305 mutation in one of her HEXA alleles. Eight of the Moroccan obligate carriers from six unrelated families were also studied previously (Navon and Proia 1991). An additional, unrelated carrier also has been found to harbor the AF304/305 mutation (J. Grinsphun and R. Navon, unpublished data). Reagents Restriction enzymes were purchased from New England Biolabs. The Gene Amp kit was from Perkin Elmer Cetus. [32P]ATP (3,000 Ci/mmol) and Gene Screen Plus were from New England Nuclear. The Sequenase version 2.0 kit was from United States Bio-

chemical Corporation. PCR Amplification DNA was isolated from blood samples, according to standard procedures (Maniatis et al. 1982). PCR amplification for the different exons was performed

using the Gene Amp kit from Perkin Elmer Cetus. The sequences of the primers for the amplification of exon 5 were 5'TGCTCCATCACCCTAGAACTCTTA3' and 5'ATCTCCCTGTGCCCCCATAGTAA3'. The sequences of the primers for the amplification of exon 8 for analysis of the AF304/305 mutation were as described by Navon and Proia (1991). Initial denaturation was at 940C for 10 min, followed by 30 cycles of denaturation at 940C for 30 s, annealing at 591C for 30 s, and extension at 720C for 90 s. Direct Sequencing of PCR Products Amplified DNA was freed from the primers and salts by using a Centricon 100 (Amicon) according to

the manufacturer's procedure. Direct sequencing was performed utilizing a 32P end-labeled primer 5'ATCTCCCTGTGCCCCCATAGTAA3'. Sequencing reactions were performed using the Sequenase (version 2) kit. Allele-specific Oligonucleotide Hybridization

One twentieth of each amplified DNA sample was dot blotted in duplicate onto GeneScreen Plus membranes. The following 19 mers were used for detection ofthe Arg'70- Stop mutation: normal probe, 5'TTCCTCACCGGGGCTTGCT3'; and mutant probe, 5'TTCCTCACCAGGGCTTGCT3'. The hybridization conditions were as described elsewhere (Navon and Proia 1989). For the detection of the Tyrl80-*Stop mutation the following 18 mers were used: normal probe, 5'CTCGCCATTACCTGCCACT3'; and mutant probe, 5'CTCGCCATTAGCTGCCACT3'. Washing conditions were performed according to the procedure described by Navon and Proia (1989), except that the final wash was for 10 min at 60'C. Heteroduplex and Restriction Enzyme Assays for Detection of Moroccan Mutations

For detection of the AF3041305 mutation, PCR-amplified fragments of exon 8 that were prepared as described above were electrophoresed on a 10% polyacrylamide gel in 1 x TBE buffer for 40 min at 180 V. For detection of the Tyrl80-Stop and Arg170- Gln mutations, PCR amplification of exon 5 was performed as above, and one-fifth of the amplified PCR fragments was digested overnight at 370C, with 5-10 units of the appropriate restriction enzyme. The digested and undigested fragments were applied to 8% polyacrylamide gels and electrophoresed in 1 x TBE buffer for 30 min at 180 V. After electrophoresis, the DNA bands were visualized by staining with ethidium bromide. Results

We had previously described a Moroccan Jewish TSD patient who carried the AF3041305 mutation in one HEXA allele and an unidentified mutation in the other allele which caused a deficiency of HEXA mRNA (Navon and Proia 1991). Direct sequence analysis of amplified exons from genomic DNA of the compound heterozygous patient revealed a novel mutation in exon 5 (fig. 1). The mutation, a C540-to-G transversion, results in the replacement of tyrosine (TAC) by a termination codon (TAG) at position 180 of the a-polypeptide. The presence of both the normal C and

Tay-Sachs Mutations in Moroccan Jews

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Analysis of Tyr'80-Stop (C540-BEG) mutation by Figure 2 allele-specific oligonucleotides. Genomic DNA from a normal control, the Moroccan TSD patient, her parents, and six other Moroccan obligate carriers for TSD was amplified in the region of exon 5. The amplified DNA samples were hybridized with 32P-labeled allele-specific oligonucleotide probes, one corresponding to the mutation (C540) and the other corresponding to the normal sequence (C540).

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to contain the mutation, cleavage with AluI, and examination of the products by electrophoresis on a polyacrylamide gel. AluI digestion of normal control DNA yielded a 205-bp fragment, whereas digestion of DNA from the TSD patient and her mother, both of

Nucleotide sequence in exon 5 of the HEXA gene Figure I from the Moroccan TSD patient showing the Tyr80-Stop (Cs50-OG) mutation. Genomic DNA from the patient and from a normal control were amplified by PCR in the region of exon 5 and were sequenced directly. The relevant portion of the sequencing gel is shown.

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Identification and rapid detection of three Tay-Sachs mutations in the Moroccan Jewish population.

Infantile Tay-Sachs disease (TSD) is caused by mutations in the HEXA gene that result in the complete absence of beta-hexosaminidase A activity. It is...
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