Showing papers by "Stylianos E. Antonarakis published in 1988"

Haemophilia A resulting from de novo insertion of L1 sequences represents a novel mechanism for mutation in man.

Abstract: L1 sequences are a human-specific family of long, interspersed, repetitive elements, present as approximately 10(5) copies dispersed throughout the genome. The full-length L1 sequence is 6.1 kilobases, but the majority of L1 elements are truncated at the 5' end, resulting in a fivefold higher copy number of 3' sequences. The nucleotide sequence of L1 elements includes an A-rich 3' end and two long open reading frames (orf-1 and orf-2), the second of which encodes a potential polypeptide having sequence homology with the reverse transcriptases. This structure suggests that L1 elements represent a class of non-viral retrotransposons. A number of L1 complementary DNAs, including a nearly full-length element, have been isolated from an undifferentiated teratocarcinoma cell line. We now report insertions of L1 elements into exon 14 of the factor VIII gene in two of 240 unrelated patients with haemophilia A. Both of these insertions (3.8 and 2.3 kilobases respectively) contain 3' portions of the L1 sequence, including the poly (A) tract, and create target site duplications of at least 12 and 13 nucleotides of the factor VIII gene. In addition, their 3'-trailer sequences following orf-2 are nearly identical to the consensus sequence of L1 cDNAs (ref. 6). These results indicate that certain L1 sequences in man can be dispersed, presumably by an RNA intermediate, and cause disease by insertional mutation.

Nonsense and missense mutations in hemophilia A: Estimate of the relative mutation rate at CG dinucleotides

Abstract: Hemophilia A is an X-linked disease of coagulation caused by deficiency of factor VIII. Using cloned cDNA and synthetic oligonucleotide probes, we have now screened 240 patients and found CG-to-TG transitions in an exon in nine. We have previously reported four of these patients; and here we report the remaining five, all of whom were severely affected. In one patient a TaqI site was lost in exon 23, and in the other four it was lost in exon 24. The novel exon 23 mutation is a CG-to-TG substitution at the codon for amino acid residue 2166, producing a nonsense codon in place of the normal codon for arginine. Similarly, the exon 24 mutations are also generated by CG-to-TG transitions, either on the sense strand producing nonsense mutations or on the antisense strand producing missense mutations (Arg to Gln) at position 2228. The novel missense mutations are the first such mutations observed in association with severe hemophilia A. These results provide further evidence that recurrent mutations are not uncommon in hemophilia A, and they also allow us to estimate that the extent of hypermutability of CG dinucleotides is 10-20 times greater than the average mutation rate for hemophilia A.

DNA polymorphism haplotypes of the human apolipoprotein APOA1-APOC3-APOA4 gene cluster.

Abstract: The genes coding for apolipoproteins A1, C3, and A4 (APOA1, APOC3, APOA4) are closely linked and tandemly organized within a 15-kilobase (kb) DNA segment on the long arm of human chromosome 11 The nucleotide variability of a 61-kb DNA segment containing these genes and their flanking sequences was studied by restriction analysis of a sample of 18 unrelated Northern Europeans using seven different genomic DNA probes Eleven restriction site polymorphisms located within this DNA segment were used for haplotype analysis of 129 Mediterranean and 67 American black chromosomes Estimation of the extent of nonrandom association between these polymorphisms indicated considerable linkage disequilibrium within the APOA1-APOC3-APOA4 gene cluster Several haplotypes arose by recombination, and the rate of recombination within this gene cluster was estimated to be at least 4 times greater than that expected based on uniform recombination The polymorphism information content of each of these polymorphisms, taken individually, ranges between 0053 and 0375, while that of their haplotypes ranges between 0858 and 0862 Therefore, DNA polymorphism haplotypes in the APOA1-APOC3-APOA4 gene cluster constitute a highly informative genetic marker on the long arm of human chromosome 11

Chromosomal localization and racial distribution of the polymorphic human dihydrofolate reductase pseudogene (DHFRP1).

Abstract: The human dihydrofolate reductase (DHFR) gene family comprises one functional gene and at least four intronless processed pseudogenes. The functional DHFR gene is on chromosome 5, and DHFRP4 is on chromosome 3. Using in situ hybridization, we have now localized the functional DHFR gene to the region q11.1-q13.3 on chromosome 5. By genomic DNA analysis of a panel of human X rodent somatic-cell hybrids, we determined the chromosomal assignment of the DHFRP1 pseudogene to chromosome 18 and that of the DHFRP2 pseudogene to chromosome 6. The DHFRP1 pseudogene exhibits a novel form of polymorphism in humans in that it is present in the DNA of some individuals and absent in that of others. We investigated the racial distribution of this pseudogene in five racial groups. The allelic frequency as defined by analysis of 180 chromosomes was found to be 94% in Mediterraneans, 77% in Asian Indians, 67% in Chinese, 57% in Southeast Asians, and 32% in American blacks. These data suggest that the transposition of this "perfect" pseudogene occurred prior to the inception of the human racial groups.

Moderately severe hemophilia A resulting from Glu→Gly substitution in exon 7 of the factor VIII gene

Abstract: To define the molecular basis of a TaqI site alteration in the factor VIII gene of a patient with moderately severe hemophilia A, we used a combination of genomic amplification followed by direct sequencing and oligonucleotide hybridization, to demonstrate an A-to-G substitution in exon 7 (codon 291) of this gene. This mutation generates a Gly in place of Glu at amino acid 272 of the mature factor VIII protein. The mutation arose de novo in a germ cell of the patient's mother.

The Molecular Genetics of Hemophilia A and B in Man

Abstract: Hemophilias are relatively common inherited disorders of blood coagulation arising from deficiency of two different clotting factors VIII and IX. Hemophilia A, or classic hemophilia, is associated with abnormality of factor VIII and affects about 1 in every 10,000 males; hemophilia B, or Christmas disease, is associated with abnormality of factor IX and affects about 1 in every 50,000 males (McKee, 1983). Both factors are involved in the middle phase of the intrinsic clotting cascade, which consists of several inactive proteases and cofactors that are serially activated in response to an initial stimulus. The end product of the cascade is the production of the insoluble fibrin from the soluble protein fibrinogen. Fibrin then forms a filamentous network and stabilizes the platelet plug. Factor IX is a serine protease, which after proteolytic cleavage by factor XIa becomes activated (IXa) and with the help of factor VIII:Ca, Ca2+, and phospholipid activates factor X. Factor VIII in its “activated” form VIII:Ca is actually a cofactor for the activation of factor X [see Jackson and Nemerson (1980) for review].

Molecular characterization of β‐globin gene mutations in patients with β‐thalassaemia intermedia in South China

Abstract: We have studied the spectrum of mutations producting beta-thalassaemia intermedia in South China. The methods of mutation detection include oligonucleotide analysis, polymerase chain reaction amplification of the beta-globin gene and direct genomic sequencing. The mutations have been identified in 22 beta-globin genes from the patients in 11 unrelated families. Seven different mutations have been identified and the A to G substitution in the TATA box of the beta-globin gene accounts for 42% of these mutant beta-globin genes. Most patients have a beta(+) thalassaemia and one copy of the TATA box mutation. In two patients with beta(0) thalassaemia intermedia the mild phenotype may be explained in one by the presence of the - + - + + 5' beta-globin gene cluster haplotype which contains the Xmn I site -158 nt to the G gamma-globin gene or in the other by the number of alpha-globin genes present.

Restriction endonuclease mapping of six novel deletions of the factor VIII gene in hemophilia A.

Abstract: Hemophilia A is an X-linked disease of blood coagulation caused by deficiency of factor VIII. Using cloned cDNA, genomic and synthetic oligonucleotide factor VIII probes, we have identified six novel partial gene deletions in patients with severe hemophilia A. We have previously reported six other deletions of the factor VIII gene. The number of gross molecular defects (deletions, insertions) in the factor VIII gene in our series of 240 patients is 17 (3 insertions and 2 complicated deletions will be described elsewhere). No association was observed between the size or location of the deletions and the presence of inhibitors to factor VIII. No deletion breakpoint "hotspots" have been identified by restriction analysis. The parental origin of several of the deletions was determined.

Accuracy and limitations of pulsed field gel electrophoresis in sizing partial deletions of the factor VIII gene

Abstract: Pulsed field gel electrophoresis (PFGE) has been recently used to separate DNA fragments ranging from 100 to 2000 kb in size. In order to assess the accuracy of the sizes of the DNA fragments and the resolving capability of this technique, we used PFGE combined with Southern blotting and probe hybridization techniques to determine the size and approximate location of four partial deletions of the factor VIII gene. This gene was chosen because of its large size (186 kb) and the availability of hemophiliac patients with well-characterized partial deletions. The sizes of three deletions estimated by PFGE (55 kb, 60 kb and 133 to 145 kb) were within 10% of the sizes calculated from conventional restriction analysis. Therefore, concatemers of lambda DNA and intact chromosomal DNA of Saccharomyces cerevisiae provide a relatively accurate system (within 10%) for sizing mammalian DNA fragments that are 100 to 550 kb in size. However, analysis of the fourth deletion (9 to 12 kb) revealed that it is difficult to detect changes in the size of mammalian DNA fragments of 8% or less using Southern blotting and PFGE.

Apolipoprotein B gene haplotypes. Association between Ag and DNA polymorphisms.

Abstract: Berg et al. (Clin Genet 1986;30:515-520) have reported that an Xba I DNA polymorphic site in exon 26 of the apolipoprotein (apo) B gene is associated both with the Ag(x/y) immunochemical polymorphism and with elevated serum lipoprotein levels. Ma et al. (Arteriosclerosis 1987;7:301-305) have reported that the same Xba I polymorphism is associated with a different immunochemical polymorphism, Ag(c/g). To extend and clarify these observations, we have determined the Ag and Xba I polymorphism for 106 individuals. We find that the Xba I restriction fragment length polymorphism is in linkage disequilibrium with both Ag(x/y) and Ag(c/g) loci; thus, all 31 Xba I(X1/X1) genotypes observed in this study are also Ag(y/y). All but one of 22 Xba I(X2/X2) genotypes are also Ag(g/g). For individuals homozygous at either two or three of these loci, it was possible to determine the haplotypes for 128 apo B alleles. Of the eight possible apo B haplotypes, only four were represented in this unambiguous subpopulation, although other minor haplotypes were present in the total population from which it was derived. The identification of major apo B haplotypes in human populations may simplify the search for significant correlations between certain apo B alleles and lipid levels and atherosclerosis.

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

Abstract: 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.

Two polymorphisms for the human hepatic lipase (HL) gene.

Abstract: SOURCE AND DESCRIPTION OF CLONE: A 1.65 kb ECORI cDNA insert corresponding to the full length hepatic lipase (HL) sequence isolated from a rat liver cDNA library (I). CHROMOSOMAL LOCALIZATION: Chromosome 15q21 by somatic cell and in situ hybridization (2).