H H Kazazian

Molecular characterization of seven beta-thalassemia mutations in Asian Indians.

To characterize systematically the mutations which produce beta‐thalassemia in Asian Indians, we first determined the DNA polymorphism haplotype in the beta‐globin gene cluster of 44 beta‐thalassemia chromosomes in the ethnic group. Nine different haplotypes were observed. Upon molecular cloning and partial DNA sequencing of one beta‐gene from each of eight haplotypes and two from the ninth, seven different mutations were found. None of these have been identified in Mediterranean patients, even among the five haplotypes which appeared identical in the two groups. Asian Indian mutations included one nonsense and three frameshift mutations, one deletion affecting an acceptor splice site, and two mutations affecting a donor splice site. The correlation of a specific mutation with a specific haplotype was high but not invariant. Two mutations were associated with more than one haplotype but, in each instance, the mutation spread to a new haplotype could be explained most simply by recombination 5′ to the beta‐globin gene. In addition, four mutations, one reported here and three others previously reported, have been observed on two chromosome backgrounds that are identical except for the status of a polymorphic HinfI site 5′ to the beta gene. This HinfI site does not show significant linkage disequilibrium with markers both 5′ and 3′ to it, suggesting that it lies within a region of relative sequence randomization.

Thalassemia due to a mutation in the cleavage-polyadenylation signal of the human beta-globin gene.

A beta‐globin gene cloned from a person with beta‐thalassemia contained a T‐‐‐‐C substitution within the conserved sequence AATAAA that forms a portion of the recognition signal for endonucleolytic cleavage and polyadenylation of primary mRNA transcripts. By Northern blot analysis a novel beta‐globin RNA species, 1500 nucleotides in length, was detected in erythroid RNA. Nuclease protection studies of erythroid RNA, as well as RNA generated upon transient expression of the cloned mutant gene in HeLa cells, located the 3′ terminus of this novel, polyadenylated RNA 900 nucleotides downstream of the normal poly(A) addition site, within 15 nucleotides of the first AATAAA in the 3′‐flanking region of the beta‐globin gene. These findings define the in vivo terminus of an elongated RNA and establish that human beta‐globin transcription may extend at least 900 nucleotides 3′ of the normal polyadenylation site.

Improved detection of the sickle mutation by DNA analysis: application to prenatal diagnosis.

DETECTION of sickle hemoglobin in the human fetus was first accomplished nearly 10 years ago.1 , 2 This marked the beginning of a technology for prenatal diagnosis of the hemoglobinopathies. When m...

Direct detection of the common Mediterranean beta-thalassemia gene with synthetic DNA probes. An alternative approach for prenatal diagnosis.

The most common form of beta-thalassemia among Mediterraneans results from a single nucleotide substitution within the first intervening sequence (IVS-1) of the beta-globin gene. This particular mutation is not detectable in uncloned DNA by restriction enzyme analysis. Using synthetic DNA of 19-nucleotides in length corresponding to the normal and mutant IVS-1 sequences as probes, we have developed a direct assay for this gene defect. Under carefully controlled experimental conditions these synthetic probes detect only their homologous sequences in restriction digests of both cloned and uncloned DNA samples. The method is sufficiently sensitive to establish the genotype of individuals with respect to this defect using approximately 20 micrograms total DNA. This assay provides an alternative to fetal blood and DNA linkage analysis for the prenatal diagnosis of this variety of beta-thalassemia, particularly among Greek families where it is especially common.

Linkage analysis of neurofibromatosis.

Linkage analysis of neurofibromatosis was performed using genes on chromosomes 1, 8, 11, and 12. No linkage was found between NF and C-myc, AT 3, IGF-1, PTH, and gamma globin genes. Evidence for linkage was found between C-ets 1, on the long arm of chromosome 11 and NF in two families with a lod score of 1.88 at theta = 0. More families are being studied to confirm this linkage.