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.

Dominant thalassemia-like phenotypes associated with mutations in exon 3 of the beta-globin gene.

Mutations producing beta-thalassemia reach individual gene frequencies greater than .01 in malarial-endemic regions because beta-thalassemia trait individuals have increased genetic fitness over that of normal individuals. Exon 3 of the beta-globin gene has been relatively spared as a site of common beta-thalassemia mutations. Frameshifts caused by the loss of a single nucleotide and nonsense mutations produce beta-thalassemia trait when they occur in exons 1 and 2. In contrast, they usually produce chronic hemolytic anemia when present in exon 3. Certain missense mutations in exon 3 produce unstable globins and thalassemia intermedia with hemolysis in heterozygotes. Here we report two new mutations in exon 3 of the beta-globin gene. One is a single nucleotide deletion in codon 109 in a 78-year-old Lithuanian with chronic hemolytic anemia and features of thalassemia. It leads to an abnormal globin (beta Manhattan) that is elongated to 156 amino acids. The second is a CAG-CGG missense mutation at codon 127 that causes a Gln----Pro substitution (beta Houston) and a thalassemia intermedia with hemolysis in three generations of a British-American family. Although the clinical phenotypes of these two patients differed little, differences in globin-synthetic ratios were significant, presumably reflecting differences in the ability of each abnormal beta-globin to form alpha beta dimers. The paucity of high-frequency exon 3 mutations and their worldwide distribution is likely attributable to their phenotypic severity and loss of increased genetic fitness vis-a-vis malaria.

Molecular basis of hexosaminidase A deficiency and pseudodeficiency in the Berks County Pennsylvania Dutch.

Following the birth of two infants with Tay-Sachs disease (TSD), a non-Jewish, Pennsylvania Dutch kindred was screened for TSD carriers using the biochemical assay. A high frequency of individuals who appeared to be TSD heterozygotes was detected (Kelly et al., 1975). Clinical and biochemical evidence suggested that the increased carrier frequency was due to at least two altered alleles for the hexosaminidase A alpha-subunit. We now report two mutant alleles in this Pennsylvania Dutch kindred, and one polymorphism. One allele, reported originally in a French TSD patient (Akli et al., 1991), is a GT-->AT transition at the donor splice-site of intron 9. The second, a C-->T transition at nucleotide 739 (Arg247Trp), has been shown by Triggs-Raine et al. (1992) to be a clinically benign "pseudodeficient" allele associated with reduced enzyme activity against artificial substrate. Finally, a polymorphism [G-->A (759)], which leaves valine at codon 253 unchanged, is described.

A novel mutation in the invariant AG of the acceptor splice site of intron 4 of the beta-hexosaminidase alpha-subunit gene in two unrelated American black GM2-gangliosidosis (Tay-Sachs disease) patients.

Samples of genomic DNA from three unrelated American black infants having both biochemical and clinical features of classical infantile Tay-Sachs disease were sequenced following PCR amplification. A G----T transversion was observed in the AG acceptor splice site preceding exon 5 of the beta-hexosaminidase alpha-subunit gene in the first black family. This transversion changed the acceptor splice site from the consensus sequence, AG, to AT, thereby interfering with splicing at this intron 4/exon 5 junction. The proband was homozygous for this mutation; his mother and a brother are heterozygous. The same mutation was found in a second, apparently unrelated, black GM2-gangliosidosis patient. The second patient was a compound heterozygote, as only one allele carried this mutation. The mother and a brother in this second family are carriers for this mutation, while the father and a noncarrier sister are normal for this region of the gene. The third proband did not have this mutation; nor did the mother of a fourth black proband. Eight other independently ascertained non-black, non-Jewish, GM2-gangliosidosis families did not have this mutation. The observation of the same novel mutation in two unrelated black GM2-gangliosidosis patients indicates that the American black population has segregating within it at least one GM2-gangliosidosis mutation which may be specific to this population and not a result of migration.

Evolution of a genetic disease in an ethnic isolate: beta-thalassemia in the Jews of Kurdistan.

beta-Thalassemia is a hereditary disease caused by any of 90 different point mutations in the beta-globin gene. Specific populations generally carry a small number of mutations, the most common of which are those that are widely distributed regionally. The present study constitutes an extensive molecular characterization of this disease in a small, highly inbred ethnic group with a high incidence of beta-thalassemia--the Jews of Kurdistan. An unusual mutational diversity was observed. In 42 sibships 13 different mutations were identified, of which 3 are newly discovered: a C----A transversion at -88 to the cap site, a frameshift in codon 36/37, and an A----G transition in the polyadenylylation signal. Four of the mutations are unique to Kurdish Jews and have not been discovered in any other population. A fifth was found outside Kurdish Jews only in an Iranian from Khuzistan, a region bordering Kurdistan. Two-thirds of the mutant chromosomes carry the mutations unique to Kurdish Jews. We traced the origin of the mutations to specific geographic regions within Kurdistan. This information, supported by haplotype analysis, suggests that thalassemia in central Kurdistan (northern Iraq) has evolved primarily from multiple mutational events. In Turkish Kurdistan, the primary mechanism is genetic admixture with the local population. In Iranian Kurdistan, a founder effect appears to be partly responsible. We conclude that several evolutionary mechanisms contributed to the evolution of beta-thalassemia in this small ethnic isolate.

Homozygous beta-thalassemia without anemia.

A 37-year-old man of Guyanese origin was found to have homozygous beta-thalassemia without anemia. There were no physical stigmata of thalassemia. The hematocrit value was 41 to 45.8, the mean corpuscular volume was 61 fL, and the mean corpuscular hemoglobin was 18.9 pg. The HbF was 45% with a G gamma:A gamma ratio of 3:1. An acid elution preparation of the peripheral blood showed heterogeneous distribution of HbF, but all erythrocytes stained for fetal hemoglobin. The beta/alpha synthesis ratio in the peripheral blood was 0.25; the (beta + gamma)/alpha ratio was 0.55. Haplotype analysis revealed homozygosity for the -+-+ + + + pattern (Senegal, type IX) at seven polymorphic restriction sites within the beta-like gene complex. Digestion of DNA with Xmnl indicated that the -158 C-to-T transition was present in both beta-globin gene clusters. Oligomer hybridization analysis demonstrated homozygosity for the -29 A-to-G mutation in the beta-globin promoter region. Although this form of thalassemia can cause transfusion-requiring anemia, the high-HbF, high-G gamma phenotype associated with the linked +-+ + subhaplotype and -158 C-to-T substitution appears to have ameliorated the disease in this subject.

The spectrum of beta-thalassaemia mutations in Sicily.

To characterize beta-thalassaemia genes among the Sicilian population we have previously determined the DNA haplotypes in the beta-globin gene cluster of 99 beta-thal chromosomes. We found seven haplotypes, although 95 of 99 beta-thal chromosomes contained framework 1 and framework 3 beta genes. We have now determined the mutation in all 99 of these beta-thal genes by the use of oligonucleotide hybridization. PCR-amplification and direct genomic sequencing, and direct restriction analysis. Our results indicate that (1) the beta (0)-39 mutation is most frequent (35%); (2) beta(0)-39, IVS-1 nt 110 and IVS-1 nt 6 mutations account for 90% of beta-thal genes: (3) the IVS-1 nt 6 mutation is more frequent in thalassaemia intermedia (77%) than in Cooley's disease (34%): (4) the association between haplotypes and specific mutations is imperfect, but mutation spread has occurred within haplotypes containing the same beta-gene framework: (5) the beta(0)-39 and the IVS-1 nt 6 mutations, with a mutation spread to two major haplotypes, may be older than the IVS-1 nt 110 mutation: (6) these data make possible first-trimester prenatal diagnosis in many families (85%) in Sicily using only three pairs of oligonucleotides. In addition, a new mutation, a frameshift at codon 76 due to loss of a C residue, was found in a single beta-thal chromosome.

Beta-thalassemia in China: a systematic molecular characterization of beta-thalassemia mutations.

In order to initiate a program of prenatal diagnosis for the prevention of beta-thalassemia in China, we have begun systematic studies of the beta-thalassemia mutations among the Chinese. DNA polymorphisms in the beta-globin gene cluster were examined in 46 beta-thalassemia chromosomes. Six different haplotypes were observed. One beta-thalassemia gene associated with a new haplotype was cloned and sequenced. The mutation was a single base substitution (A----G) at position -29 within the highly conserved proximal promoter element (the "TATA" box). This mutation was not observed previously in the Chinese. The beta-thalassemia genes were further screened with oligonucleotide probes specific for all known mutations in the Chinese. Five mutations were identified and accounted for 35 beta-thalassemia alleles.

Characterization of beta-thalassaemia mutations using direct genomic sequencing of amplified single copy DNA.

Direct sequencing of specific regions of genomic DNA became feasible with the invention of the polymerase chain reaction (PCR) which permits amplification of specific regions of DNA. Recently, human mitochondrial DNA was amplified and directly sequenced. Using a thermostable DNA polymerase of T. aquaticus (Saiki, R.K. et al., manuscript in preparation) in the PCR, we have applied a combination of PCR and direct sequence analysis of the amplified product to a human single-copy gene. We studied the genomic DNA of five patients with beta-thalassaemia whose mutant alleles were uncharacterized, and found two previously undescribed mutations, along with three known alleles. One new allele is a frameshift at codons 106-107 and the other is an A-C transversion at the cap site (+1) of the beta-globin gene. This latter is the first natural mutation observed at the cap site and it occurs in a gene which is poorly expressed.

Molecular characterization of beta-thalassemia major and beta-thalassemia intermedia in China and Southeast Asia.

We have studied the spectrum of mutations producing beta-thalassemia (beta-thal) in South China and Southeast Asia in two groups of patients. In randomly selected patients with beta-thal major we characterized 78 beta-thal genes. In patients with beta-thal intermedia, 22 beta-thal genes were studied. The relevant mutation was characterized in all 78 genes of the first group, and 21 of 22 (96%) of mutant genes in the second group. Eight point mutations were found among the 100 genes studied. Of these eight alleles, four constituted 90% of the total. Prenatal diagnosis of beta-thalassemia in this region should be feasible by simplified techniques for direct detection of point mutations.

The spectrum of beta-thalassemia genes in China and Southeast Asia.

To make possible prenatal diagnosis of beta-thalassemia in China and Southeast Asia by direct detection of mutant beta-globin genes, we have determined the spectrum of mutations producing the disorder in this region of the world. Seventy-eight beta-thalassemia genes from Chinese and Southeast Asians were randomly obtained, and the relevant mutation was characterized in 76 (98%) of them. Seven different point mutations were found among the 78 genes studied. Of these seven beta-thalassemia alleles, two constitute 62%, and two others account for 29% of the total. Since only four alleles make up 91% of the mutant genes, prenatal diagnosis of beta-thalassemia in China and Southeast Asia should be feasible by simplified techniques for direct detection of point mutations.

Characterization of a spontaneous mutation to a beta-thalassemia allele.

We have studied a nuclear family containing a single child with severe beta-thalassemia intermedia, a Greek-Cypriot mother with hematological findings of beta-thalassemia trait, and a Polish father who is hematologically normal. Since both the child and her father were heterozygous for a DNA polymorphism within the beta-globin gene, it was possible to clone and sequence the beta-globin gene identical by descent from both the child and her father. A nonsense mutation in codon 121 (GAA----TAA) was found in the beta-globin gene of the child, while the same gene from her father lacked this mutation and was normal. This mutation has not been previously observed among over 200 beta-thalassemia genes characterized in Caucasians. Since the mutation eliminates an EcoRI site in the beta-globin gene, we could show that the mutation is not present in genomic DNA of the father. To rule out germinal mosaicism, sperm DNA of the father was also digested with EcoRI, and the mutant EcoRI fragment was not observed under conditions that would detect the mutation if it were present in at least 2% of sperm cells. Routine HLA and blood group testing supported stated paternity. In addition, studies with 17 DNA probes that detect multiple allele polymorphisms increased the probability of stated paternity to at least 10(8):1. These data provide evidence that the G----T change in codon 121 of the beta-globin gene in the child is the result of a spontaneous mutation that occurred during spermatogenesis in a paternal germ cell.

Use of oligonucleotide hybridization in the characterization of a beta zero-thalassemia gene (beta 37 TGG----TGA) in a Saudi Arabian family.

Analysis of restriction site polymorphisms in the beta-globin gene cluster of a Saudi Arabian female with beta zero-thalassemia demonstrated that both of her beta-globin genes were missing a nonpolymorphic AvaII site in exon 2. Examination of the normal nucleotide sequence surrounding this AvaII site revealed that either of two nucleotide substitutions, TGG----TAG or TGG----TGA, could produce a nonsense codon at codon 37 and eliminate the AvaII site. Consequently, two oligonucleotides (19-mers spanning codons 36 through 41 and containing either TAG or TGA at codon 37) were synthesized and hybridized against genomic DNA of the proband and her family. Specific hybridization with one of the oligomers demonstrated that the patient's beta o-thalassemia was the result of homozygosity for the TGG----TGA mutation at codon 37. In certain cases, oligonucleotide hybridization using genomic DNA may obviate the need for gene cloning and sequencing in the characterization of point mutations.

Evidence supporting a single origin of the beta(C)-globin gene in blacks.

In order to characterize the origin(s) of the beta C-globin gene in blacks, 25 chromosomes bearing this gene were characterized at eight polymorphic restriction sites within the beta-globin gene cluster. Twenty-two of the 25 chromosomes were identical at all sites and possessed a haplotype seen only infrequently among beta A-bearing chromosomes in black Americans. Two different haplotypes were observed among the three exceptional chromosomes. These haplotypes were identical to the most common beta C allele in the 3' end of the beta-globin gene cluster, but differed in the 5' region. Partial haplotype analysis on an additional 14 beta C alleles demonstrated complete association with the typical beta C-associated polymorphisms in the 3' region of the cluster. These data can be most easily explained by a single origin of the mutation followed by spread of the mutation to other haplotypes through meiotic recombination 5' to the beta-globin gene.