MspI polymorphic site in intron 22 of the factor VIII gene in the Japanese population

Summary A novel MspI DNA polymorphic site has been found in intron 22 of the human factor VIII gene. This site is informative almost exclusively in the Japanese population (heterozygosity 0.45) and will be of considerable importance in carrier detection and prenatal diagnosis of hemophilia A in this population.     

Analysis of the AluI polymorphism in intron 1 of the human coagulation factor VIII gene: a new marker for the hemophilia A carrier detection

Frequencies of the CIT SNP alleles at position 2403 of the human coagulation factor VIII gene intron 1, containing the AluI restriction endonuclease recognition site, were examined. Genomic DNA samples for the analysis were obtained from the consulted women and their relatives from the families with hemophilia A. A total of 221 unrelated X chromosomes were studied. The two allelic variants were found with similar frequencies of T(Alu+), 0.53 and C(Alu-), 0.47. The heterozygosity index evaluated as equal to 0.50 was correlated with the experimental heterozygote number. The absence of a tight linkage between the AluI SNP and the widely used in the hemophilia A gene diagnostics HindIII polymorphism (CIT SNP at position 103 of intron 19) was demonstrated. Summarized informativity of these two markers for obligate carriers and for those detected in this study constituted 68% (32 out of 47). At the same time using one of the markers, only 40% (HindIII) and 51% (AluI) of the consulted women were informative. The new marker was used in 13 prenatal DNA diagnostics of hemophilia A. A new deletion polymorphism (del TGA, position 2281-2283 of intron 1) was described in close proximity of the AluI SNP with the frequency of about 0.05. among the five other SNP of the factor VIII gene examined (Bme 18I, intron 1; HpaII, intron 13; MnlI, exon 14; Bst4CI, exon 25; and MseI, exon 26) no effective diagnostic markers were found. Only the MnlI polymorphism could be recommended for limited usage.     

Mild hemophilia A associated with a cryptic donor splice site mutation in intron 4 of the factor VIII gene

Hemophilia A, an X-linked disease caused by deficiency of factor VIII, is characterized by variation in clinical severity and coagulation activity. This variation is though to reflect heterogeneity of mutations in the factor VIII gene. Here we describe a CG-to-CA mutation within a potential cryptic donor splice site in intron 4 of the factor VIII gene from a patient with mild disease. This mutation makes the cryptic sequence resemble more closely the consensus sequence for donor splice sites. We infer that the mutation activates the cryptic donor splice site, which in turn causes a defect in RNA processing.     

Nonhomologous recombination in the human genome: deletions in the human factor VIII gene

Four deletions in the human factor VIII gene have been characterized at the sequence level in patients with hemophilia A. Deletion JH 1 extends 57 kb from IVS 10 to IVS 18. Intron 13 and exon 14 are partially deleted in patients JH 7 and JH 37, with a loss of 3.2 and 2.4 kb of DNA, respectively. The 3' deletion breakpoint of the JH 21 event resides in intron 3 and extends 5' into intron 1, resulting in the loss of exons 2 and 3. Seven of the eight breakpoints sequenced (5' and 3' for each of the four deletions) occur in nonrepetitive sequence, while the 3' breakpoint of the JH 1 resides in an Alu repetitive element. All of the deletions are the result of nonhomologous recombination. The 5' and 3' breakpoints of JH 1, JH 7, and JH 37 share 2- to 3-bp homologies at the deletion junctions. In contrast, two nucleotides have been inserted at the JH 21 deletion junction. Short sequence homologies may facilitate end-joining reactions in nonhomologous recombination events.     

MCM3AP is transcribed from a promoter within an intron of the overlapping gene for GANP

MCM3 acetylase (MCM3AP) and germinal-centre associated nuclear protein (GANP) are transcribed from the same locus and are therefore confused in databases because the MCM3 acetylase DNA sequence is contained entirely within the much larger GANP sequence and the entire MCM3AP sequence is identical to the carboxy terminus of GANP. Thus, the MCM3AP and GANP genes are read in the same reading frame and MCM3AP is an N-terminally truncated region of GANP. However, we show here that MCM3AP and GANP are different proteins, occupying different locations in the cell and transcribed from different promoters. Intriguingly, a promoter for MCM3AP lies within an intron of GANP. This report is an interesting example in nature of two separate gene products from the same locus that perform two entirely different functions in the cell. Therefore, to avoid further confusion, they should now be referred to as separate but overlapping genes.     

A HindIII RFLP and a gene lesion in the coagulation factor VIII gene

Summary The presence and inheritance of restriction fragment length polymorphisms (RFLPs) and gene lesions in the coagulation factor VIII gene were investigated in 15 hemophilia families. An abnormal HindIII 2.6-kb band, previously detected in a severe hemophiliac, was observed in a not severely affected patient and also in the normal gene of a woman carrying a hemophilic gene in which the lesions was found. The TaqI site in exon 24 of this defective gene was removed by a C to T transition causing an amino acid change (ArgGln). Very low amounts of factor VIII activity and antigen were detected in the severely affected grandson. The presence of the HindIII 2.6-kb fragment in both normal and patholgoical genes indicates that a factor VIII RFLP without functional meaning was found. Its frequency, determined in 60 chromosomes, is 0.18. Double digestions enabled us to map the polymorphic site 3 to the exon 19.     

Combined analysis of the MspI and XbaI polymorphisms in intron 22 of the factor VIII gene for detection of hemophilia A in a Korean population

To determine the usefulness of MspI/int22h-1 (intron 22 homologous region-1) polymorphism of the factor VIII gene for molecular genetic diagnosis of hemophilia A in the Korean population, MspI/intron 22 and XbaI/intron 22 polymorphisms were analyzed in 101 unrelated Korean families with severe hemophilia A. The expected heterozygosity rates of MspI/int22h-1 and XbaI/int22h-1 polymorphisms were 49.5 and 43.6%, respectively; these polymorphisms were not in complete linkage disequilibrium. Combined analysis using both polymorphisms provided an informative rate of 66.3%. These results suggest that PCR analysis of the MspI/int22h-1 polymorphism of the factor VIII gene would be useful for carrier detection and prenatal diagnosis of hemophilia A in the Korean population.     

Plasma exchange and human factor VIII concentrate in managing haemophilia A with factor VIII inhibitors.

Plasma exchanges were combined with human factor VIII concentrate therapy in the treatment of major bleeding episodes in five patients with haemophilia A and factor VIII inhibitors. All patients had a good clinical response to combined treatment. Inhibitor levels showed satisfactory falls before rapid secondary increases of inhibitor levels took place. A sixth patient with von Willebrand''s disease and a factor VIII clotting activity inhibitor was successfully prepared for operation using plasma exchange. Postoperative haemostasis and healing were normal. In two patients the plasma exchanges were relatively more effective than the administered human factor VIII in reducing the levels of factor VIII inhibitor. Combined plasma exchange and human factor VIII treatment may offer a rapidly effective means of reducing factor VIII inhibitor levels in this group of patients, together with significant saving of costs.     

A nuclear factor which interacts with an AT-cluster in the first intron of rat alpha 2-macroglobulin gene

We have identified a nuclear protein which binds specifically to the first intron of rat alpha 2-macroglobulin gene. The protein became insoluble at low salt concentration retaining the binding specificity. Its molecular weight was estimated to be 22 kilodalton by a protein blotting procedure. The binding site of the protein determined by DNase I footprinting was an AT-strech which shared 80% homology with the cleavage consensus of Drosophila topoisomerase II.     

Physical mapping of the factor VIII gene proximal to two polymorphic DNA probes in human chromosome band Xq28: implications for factor VIII gene segregation analysis

Genomic DNA segments for the coagulation factor VIIIc gene (F8C), which exhibits only limited restriction length polymorphism, map to the proximal region of band Xq28 by somatic cell hybridization analysis and in situ hybridization. Using somatic cell hybrids, we have obtained data which place probes DX13 (used to detect locus DXS15) and St14 (used to detect DXS52) distal to F8C, within band Xq28. Previous studies have mapped the factor IX gene (F9) and probe 52A (used to detect DXS51) proximal to F8C, in Xq26----q27 and Xq27, respectively (Camerino et al., 1984; Drayna et al., 1984; Mattei et al., 1985). Thus, the relative order of genetic marker loci in the Xq27----qter region is most likely cen-F9-DXS51-F8C-(DXS15, DXS52)-Xqter. The collection of these molecular probes is thus potentially useful in three-factor crosses of factor VIII gene segregation.     

Characterization of recombinant human factor VIII

Recently, complete human factor VIII DNA clones have been obtained and subsequently expressed in baby hamster kidney cells (Wood, W. I., Capon, D. J., Simonsen, C. C., Eaton, D. L., Gitschier, J., Keyt, B., Seeburg, P. H., Smith, D. H., Hollingshead, P., Wion, K. L., Delwart, E., Tuddenham, E. G. D., Vehar, G. A., and Lawn, R. M. (1984) Nature 312, 330-337). The recombinant factor VIII (rVIII) protein secreted from these cells has now been purified allowing its structural analysis and comparison to plasma-derived factor VIII (pdVIII). Analysis of purified rVIII by sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that it consists of multiple polypeptides with relative mobilities (Mr) ranging from 80,000-210,000. The same pattern of polypeptides is also observed for pdVIII resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins associated with rVIII are recognized by pdVIII antibodies in a Western blot. When rVIII and pdVIII are subjected to isoelectric focusing they are resolved into a similar pattern of protein bands. Thrombin, factor Xa, and activated protein C, which modulate factor VIII activity by proteolysis, process rVIII in the same manner they do pdVIII. As is the case for pdVIII, thrombin activation of rVIII coagulant activity correlates with the generation of subunits with Mr of 73,000, 50,000 and 43,000. These subunits appear to form a metal-(perhaps Ca2+) linked complex. EDTA inactivates thrombin-activated rVIII and pdVIII, with the activity being regenerated after the addition of a molar excess of MnCl2. The results suggest that rVIII is structurally and functionally very similar to pdVIII.     

A new polymorphism in the factor VIII gene for prenatal diagnosis of hemophilia A.

A restriction fragment length polymorphism (RFLP) has been found in the gene for clotting factor VIII. Defects in this gene are the cause of hemophilia A. The DNA polymorphism affects an XbaI site in intron 22 of the gene. Two alleles occur in a frequency of 59 and 41 percent of the X chromosomes tested. Furthermore, about 25 percent of females who are homozygous for the previously reported BclI RFLP in the factor VIII gene are heterozygous for the XbaI polymorphism. This new RFLP thus represents a significant addition to available probes for the DNA-based prenatal diagnosis and carrier detection of this disease.