Multiple abnormal beta-hexosaminidase alpha chain mRNAs in a compound-heterozygous Ashkenazi Jewish patient with Tay-Sachs disease

Abnormal beta-hexosaminidase alpha chain cDNA clones were isolated from fibroblasts of an Ashkenazi Jewish patient with Tay-Sachs disease. Four abnormal cDNA clones were sequenced in their entirety. We showed previously that three of these mRNAs retained intron 12 with a mutation from G to C at the 5' donor site and that the patient was heterozygous with respect to this splicing defect (Ohno, K., and Suzuki, K., (1988) Biochem. Biophys. Res. Commun. 153, 463-469). One clone retained, in addition to intron 12, intron 13, which was truncated and polyadenylated due to a polyadenylation signal within intron 13. The fourth clone did not contain intron 12 and was missing exon 12. Some of these abnormal mRNAs were also missing one or more of upstream exons. The regions of exon 12-intron 12 and of upstream exons were evaluated in a total of 30 clones, including those completely sequenced, by restriction mapping and Southern analysis with appropriate probes. Of the 25 cDNA clones that included the exon 12-intron 12 region, 11 contained the exon 12-intron 12 sequence with the junctional transversion, and 11 were missing both exon 12 and intron 12. Among the 12 clones that included the region of exon 3-exon 9, 7 were missing one or more of upstream exons. Three clones gave results expected of normal cDNA in the region of exons 12 and 13. One of the three, furthermore, was 3.6-kilobases long and contained the completely normal beta-hexosaminidase alpha chain mRNA sequence on the 3' side and an abnormal 1.7-kilobase segment at the 5' end. These findings suggest that the splicing defect results in either retention of intron 12 or skipping of exon 12 in approximately equal proportions and that remote upstream exons are also frequently excised out. The three clones that were normal in the exon 12-intron 12 region could have derived from the other yet-to-be-characterized mutant allele. However, we were unable to obtain firm evidence that the abnormal upstream sequence is directly related to Tay-Sachs disease.     

The presence of two different infantile Tay-Sachs disease mutations in a Cajun population.

A study was undertaken to characterize the mutation(s) responsible for Tay-Sachs disease (TSD) in a Cajun population in southwest Louisiana and to identify the origins of these mutations. Eleven of 12 infantile TSD alleles examined in six families had the beta-hexosaminidase A (Hex A) alpha-subunit exon 11 insertion mutation that is present in approximately 70% of Ashkenazi Jewish TSD heterozygotes. The mutation in the remaining allele was a single-base transition in the donor splice site of the alpha-subunit intron 9. To determine the origins of these two mutations in the Cajun population, the TSD carrier status was enzymatically determined for 90 members of four of the six families, and extensive pedigrees were constructed for all carriers. A single ancestral couple from France was found to be common to most of the carriers of the exon 11 insertion. Pedigree data suggest that this mutation has been in the Cajun population since its founding over 2 centuries ago and that it may be widely distributed within the population. In contrast, the intron 9 mutation apparently was introduced within the last century and probably is limited to a few Louisiana families.     

Sequence of DNA flanking the exons of the HEXA gene, and identification of mutations in Tay-Sachs disease.

The rapid identification of mutations causing Tay-Sachs disease requires the capacity to readily screen the regions of the HEXA gene most likely to be affected by mutation. We have sequenced the portions of the introns flanking each of the 14 HEXA exons in order to specify oligonucleotide primers for the PCR-dependent amplification of each exon and splice-junction sequence. The amplified products were analyzed, by electrophoresis in nondenaturing polyacrylamide gels, for the presence of either heteroduplexes, derived from the annealing of normal and mutant DNA strands, or single-strand conformational polymorphisms (SSCP), derived from the renaturation of single-stranded DNA. Five novel mutations from Tay-Sachs disease patients were detected: a 5-bp deletion of TCTCC in IVS-9; a 2-bp deletion of TG in exon 5; G78 to A, giving a stop codon in exon 1; G533 to T in exon 5, producing the third amino acid substitution detected at this site; and G to C at position 1 of IVS-2, expected to produce abnormal splicing. In addition, two mutations, (G1496 to A in exon 13 and a 4-bp insertion in exon 11) that have previously been reported were identified.     

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.     

Seven novel Tay-Sachs mutations detected by chemical mismatch cleavage of PCR-amplified cDNA fragments.

Total RNA was isolated from cultured fibroblasts from 12 unrelated patients with Tay-Sachs disease, an autosomal recessive disorder due to beta-hexosaminidase A deficiency. beta-Hexosaminidase mRNA was amplified by cDNA-PCR in four overlapping segments spanning the entire coding sequence. In two patients, abnormal size cDNA-PCR fragments in which exons were removed resulted from splicing mutations that were characterized at the genomic DNA level: both were G to A transitions, at the first position of intron 2 and at the fifth position of intron 4. Five other mutations have been identified by cDNA-PCR chemical mismatch analysis and direct sequencing of an amplified fragment containing the mismatch site. One missense mutation alters the codon for Ser210 to Phe in exon 6 and the other one alters the codon for Arg504 to Cys in exon 13. A 3-bp deletion results in the deletion of a phenylalanine residue in exon 8. Two nonsense mutations in exon 3 (Arg137 to stop) and in exon 11 (Arg393 to stop) are associated with a marked decrease of mRNA abundance, probably because they result in mRNA instability. Three of the six single base mutations involve the conversion of a CpG dinucleotide in the sense strand to TpG. These results demonstrate the extreme molecular heterogeneity of mutations causing Tay-Sachs disease. The procedure described in this paper allows the rapid detection of any type of mutation, except those impairing the promoter function. Applicable even to patients with splicing or nonsense mutations and very low mRNA abundance, it has therefore a potentially broad application in human genetics, for both diagnostic and fundamental purposes.     

A gel electrophoretic assay for detecting the insertion defect in Ashkenazi Jewish carriers of Tay-Sachs disease

A simple, rapid, nonradioactive assay for detecting the 4-bp insertion defect found in the beta-hexosaminidase alpha-chain gene of 70% of the Ashkenazi Jewish carriers of Tay-Sachs disease is described. In this assay, DNA derived from such carriers serves as a template for the polymerase chain reaction. Following amplification of a 159-bp fragment of exon 11 inclusive of the insertion, a portion of the product is subjected to electrophoresis in a 4% NuSieve agarose minigel. Visualization of the DNA with ethidium bromide demonstrates that heterozygote carriers for the defect display two distinct bands. In contrast, DNA from carriers of the splice junction defect, a mutation found in 30% of the Ashkenazi Jewish carriers of Tay-Sachs disease, displays only one band.     

Insertional inactivation of the WT1 gene in tumour cells from a patient with WAGR syndrome

The WT1 gene was analysed using DNA from a Wilms' tumour derived from a patient with the WAGR syndrome using single strand conformation polymorphism analysis and polymerase chain reaction sequencing. A 14-bp insertion was found in the intron part of the splice donor site of exon 7 and was a tandem duplication of an upstream exon sequence. This mutation would be expected to disrupt the correct processing of the WT1 mRNA and is predicted to result in a non-functional protein. This observation further supports the role of WT1 in Wilms' tumorigenesis in patients with constitutional 11p13 deletions.     

Nature and frequency of mutations in the argininosuccinate synthetase gene that cause classical citrullinemia

Citrullinemia is an autosomal recessive disorder caused by a genetic deficiency of argininosuccinate synthetase (ASS). So far 20 mutations in ASS mRNA have been identified in human classical citrullinemia, including 14 single base changes causing missense mutations in the coding sequence of the enzyme, 4 mutations associated with an absence of exons 5, 6, 7, or 13 in mRNA, 1 mutation with a deletion of the first 7 bases in exon 16 (which is caused by abnormal splicing), and 1 mutation with an insertion of 37 bases between the exon 15 and 16 regions in mRNA. In order to identify the abnormality in the ASS gene causing the exon 7 and 13 deletion mutations and the 37-base insertion mutation between exons 15 and 16 in mRNA, and to establish a DNA diagnostic test, we isolated and sequenced the genomic DNA surrounding each exon. The absence of exon 7 or 13 in ASS mRNA resulted from abnormal splicing caused by a single base change in the intron region: IVS-6^–2 (a transition of A to G at the second nucleotide position within the 3 splice cleavage site of intron 6) and IVS-13⁺⁵ (a transition of G to A at the fifth nucleotide position within the 5 splice cleavage site of intron 13), respectively. The IVS-6^–2 mutation resulted in the creation of an MspI restriction site. DNA diagnostic analysis of 33 Japanese alleles with classical citrullinemia showed that 19 alleles had the IVS-6^–2 mutation (over 50% of the mutated alleles in Japanese patients). It was thus confirmed that one mutation is predominant in Japan. This differs from the situation in the USA where there is far greater heterogeneity. The insertion mutation in mRNA on the other hand resulted from abnormal splicing caused by a 13-bp deletion at the splice-junction between exon 15 and intron 15. The deletion had a short direct repeat (CTCAGG) at the breakpoint junction and presumably resulted from slipped mispairing.     

Human ferrochelatase: a novel mutation in patients with erythropoietic protoporphyria and an isoform caused by alternative splicing

Erythropoietic protoporphyria (EPP), attributable to deficiency of ferrochelatase activity (FECH), is characterised mainly by cutaneous photosensitivity. To define the molecular defect in two EPP-affected siblings and their parents in a Swiss family, ferrochelatase cDNA was amplified by the polymerase chain reaction (PCR) and subjected to sequence analysis. A 5-bp deletion (T580G584) was identified on one allele of the ferrochelatase gene in both patients and their mother. Screening of the mutation among family members by RsaI digestion of PCR-amplified genomic DNA revealed autosomal dominant inheritance associated with abnormal protoporphyrin concentration and enzyme activity. We also isolated ferrochelatase cDNAs containing a 18-bp insertion (part of the intron 2 sequence) between exons 2 and 3; this corresponded to six extra amino acids (YESNIR) inserted between Arg-65 and Lys-66 of the known ferrochelatase. This isoform was identified initially in mRNAs derived from both alleles of the ferrochelatase gene in one patient. Its existence was confirmed in six additional EPP patients, in five out of seven controls, and in four different cell lines (fibroblast, muscle, hepatoma and myelogenous leukaemia). This isoform, roughly 20% of the total ferrochelatase mRNA, was generated through splicing at a second donor site in intron 2 and its presence was not linked to EPP.     

Biallelic discrimination assays for the three common Ashkenazi Jewish mutations and a common non-Jewish mutation, in Tay-Sachs disease, using fluorogenic TaqMan probes

We have developed rapid semiautomated fluorogenic TaqMan assays for the three common Jewish mutations that occur in Tay-Sachs disease, the TATC 4-bp insertion in exon 11 (1,278insTATC), the IVS 12 + 1G --> C, splice site mutation in intron 12 (1421 + 1 G --> C), and the G --> A change at the 3' end of exon 7 (G269S), as well as for a non-Jewish mutation, IVS9 + I G --> A, believed to be prevalent in patients of Celtic descent. The TaqMan assays are designed to run on the ABI SDS 7700 sequence detection system, using allele-specific probes that carry a reporter dye at the 5' end and a quencher dye at the 3' end. Using a 96-well format, all four assays can be performed simultaneously on the same plate, with real-time fluorescence detection or just an end-point plate read. DNA samples from 78 patients identified as carriers by biochemical screening and genotyped by conventional techniques were used to assess the accuracy and efficiency of the probes in allelic discrimination assays. There were no discrepancies noted between previously assigned genotypes and the results obtained by application of this methodology.     

A novel point mutation in an acceptor splice site of intron 32 (IVS32 A–12→G) but no exon 3 mutations in the glycogen debranching enzyme gene in a homozygous patient with glycogen storage disease type IIIb

Genetic deficiency of the glycogen-debranching enzyme (debrancher) causes glycogen storage disease type III (GSD III), which is divided into two subtypes: IIIa and IIIb. In GSD IIIb, glycogen accumulates only in the liver, whereas both liver and muscles are involved in GSD IIIa. The molecular basis for the differences between the two subtypes has not been fully elucidated. Recently, mutations in exon 3 of the debrancher gene were reported to be specifically associated with GSD IIIb. However, we describe a homozygous GSD IIIb patient without mutations in exon 3. Analysis of the patient’s debrancher cDNA revealed an 11-bp insertion in the normal sequence. An A to G transition at position –12 upstream of the 3′ splice site of intron 32 (IVS 32 A^–12→G) was identified in the patient’s debrancher gene. No mutations were found in exon 3. Mutational analysis of the family showed the patient to be homozygous for this novel mutation as well as three polymorphic markers. Furthermore, the mother was heterozygous and the parents were first cousins. The acceptor splice site mutation created a new 3′ splice site and resulted in insertion of an 11-bp intron sequence between exon 32 and exon 33 in the patient’s debrancher mRNA. The predicted mutant enzyme was truncated by 112 amino acids as a result of premature termination. These findings suggested that a novel IVS 32 A^–12→G mutation caused GSD IIIb in this patient. Received: 1 August 1997 / Accepted: 22 September 1997     

Molecular relationships between alcohol dehydrogenase null-activity alleles from natural populations of Drosophila melanogaster.

Alcohol dehydrogenase null-activity alleles extracted from a number of natural populations of Drosophila melanogaster in Tasmania were shown to be molecularly similar by probing, with an oligonucleotide specific to an inserted region in intron 2 of the gene, genomic DNA amplified by the polymerase chain reaction. This insertion had previously been shown to be the cause of the loss of activity in one of the null alleles whose DNA sequence was known. Three Adh null alleles from mainland populations did not contain the insertion. Two of these null alleles, extracted from the Coffs Harbour population in different years, were cloned, and their DNA sequences showed that they were identical and that both had a 438-bp deletion which removed most of exon 2. The third null allele, identified in a sample of flies from Chateau Tahbilk, was shown by 4-bp restriction-endonuclease mapping to contain a 320-bp insertion in intron 1, although this may not be the cause of the loss of activity. The data show that at least three different Adh null alleles have been found in Australian populations and that at least two have been maintained as heterozygotes over a period of years.     

Molecular analysis of five independent Japanese mutant genes responsible for hypoxanthine guanine phosphoribosyltransferase (HPRT) deficiency

Five independent mutations in the hypoxanthine guanine phosphoribosyltransferase (HPRT) gene were identified in a partially HPRT deficient patient with gout and in four Lesch-Nyhan patients. Using the polymerase chain reaction (PCR) technique coupled with direct sequencing, the nucleotide sequences of the entire HPRT coding region amplified from the cDNA and also of each exon amplified form the genomic DNA were analyzed. Three independent point mutations in the coding region were detected in the partially HPRT deficient patient (Case 1) and in two Lesch-Nyhan patients (Case 2 and 3), resulting in single amino acid substitutions. The family study of Case 3, utilizing a PvuII restriction site created in the mutant gene, indicated that the mother was a heterozygote, and a sister and a fetal brother had inherited the normal HPRT gene from the mother. In two other mutants causing Lesch-Nyhan syndrome, a portion of the HPRT gene was deleted, and RNA splicing was missing in both mutants. A 4-bp deletion at the 5 end of exon 4 resulted in formation of three different types of abnormal mRNA (Case 4). The other mutant (Case 5) produced abnormal mRNA including 26bp of intron 8 instead of the deleted 58bp at the 5 end of exon 9, because of a 74-bp deletion from intron 8 to exon 9.     

The search for south European cystic fibrosis mutations: identification of two new mutations, four variants, and intronic sequences

The major mutation in the cystic fibrosis (CF) gene is a 3-bp deletion (delta F508) in exon 10. About 50% of the CF chromosomes in Southern Europe carry this mutation, while other previously described mutations account for less than 4%. To identify other common mutations in CF patients from the Mediterranean area, we have sequenced, exon by exon, 16 chromosomes that did not show the delta F508 deletion from a selected panel of eight unrelated CF patients. We describe here one missense and one nonsense mutation, and four sequence polymorphisms. We have also found two previously reported mutations in three chromosomes. Overall, these mutations may account for about 20% of CF alleles in the Italian and Spanish populations. No other mutations were detected in 10 out of 16 CF chromosomes after analyzing about 90% of the coding region of the CF gene, and 39 out of 54 intron/exon boundaries. Therefore, about 26% of CF mutations remain to be identified. In addition we provide the intron/exon boundary sequences for exons 4 to 9. These results together with previously reported linkage data suggest that in the Mediterranean populations further mutations may lie in the promoter region, or in intron sequences not yet analyzed.     

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

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.