Takuya Nishimura

A deletion involving intron 13 and exon 14 of factor VIII gene in a haemophiliac with anti-factor VIII antibody

SummaryA deletion mutation in the factor VIII gene of a severe haemophiliac patient was found along with a high level of factor VIII inhibitor in the blood plasma among seventy Japanese haemophilia A patients. The 6 kbp long deletion involved a region from somewhere between PstI and SstI sites at nucleotide positions 2659 and 2991 of exon 14 and intron 13, respectively (nucleotide positions were defined as in Wood et al., 1984).

Heterogeneity of factor IX BM difference of cleavage sites by factor XIa and Ca2+in factor IX Kashihara, Factor IX Nagoya and Factor IX Niigata

Abnormal factor IX was isolated from the plasma of a patient with hemophilia B Kashihara and two patients with hemophilia BM. The F.IX was purified to homogeneity by using monoclonal anti-F.IX-Sephrose, heparin-Sepharose and DEAE-Sephadex A-50 affinity chromatography successively. The isolated proteins have the same molecular weight and the same mobility on crossed immunoelectrophoresis as normal F.IX. The limited proteolysis of purified proteins was induced by F.XIa/Ca2+ or by RVV-X/Ca2+. A time course study showed that F.IX Nagoya seemed to be cleaved by neither F.XIa nor RVV-X, F.IX Kashihara was cleaved partially by F.XIa but not by RVV-X, and that F.IX Niigata was cleaved completely at the rate similar to normal F.IX, though the resultant product of F.IX Niigata did not show any F.IXa activity. These results favored the view that hemophilia B+ or BM is a heterogeneous disorder.

NONSENSE MUTATION IN FACTOR VIii GENE OF A SEVERE HAEMOPHILIAC PATIENT WITH ANTI-FACTOR VIII ANTIBODY

SummaryA nonsense mutation was found in exon 23 of the factor VIII gene of a haemophiliac patient with anti-factor VIII antibody. Genomic DNA of lymphocyte cells from the patient analyzed by Southern blot analysis with various segments of factor VIII cDNA revealed that the TaqI site in exon 23 was erased in the patient gene. The 0.3 kbp nucleotide sequence of the exon 23 was cloned and sequenced, and the substitution of nonsense (TGA) codon for the arginine (CGA) codon was found to be the possible cause of the factor VIII deficiency.

RFLPs of factor IX gene in Japanese haemophilia B families and gene deletion in two high-responder-inhibitor patients

SummaryThe factor IX genes in four Japanese families with haemophilia B were analysed for the restriction fragment length polymorphisms (RFLPs) of TaqI, XmnI and DdeI, using subcloned intragenic DNA fragments as probes (probes VIII and XIII). The factor IX genes in 12 patients with haemophilia B and three high-responder-inhibitor cases showed no size difference using a cDNA probe (cVII) when restricted by TaqI, EcoRI and HindIII. Complete gene deletions were observed in two other high-responder-inhibitor cases.

Carrier detection in Japanese haemophilia a families using factor VIII gene probe (F8A) and the gene-linked ST 14-1 probe

SummaryCarriers of haemophilia A were detected in 20 Japanese families with using the factor VIII gene probe (F8A) and the gene-linked ST 14-1 probe. Polymorphism by the use of the BclI and F8A probe detected the smaller allele (0.9 kb) in 86% and the larger allele (1.2 kb) in 14% of the 65 normal X chromosomes. The frequency of BclI polymorphism was 30% in 30 normal fameles. In six out of 20 haemophilia A families, the haemophilia gene was identified by BclI allele polymorphism. The use of TaqI and the ST 14-1 probe detected 11 carriers in 20 haemophilia A families. In 14 families (70%), either the BclI polymorphism in the factor VIII locus or TaqI polymorphisms in the ST 14 locus was useful for carrier detection.

First trimester prenatal diagnosis of haemophilia a using factor VIII gene probe

SummaryAccurate first-trimester prenatal diagnosis was achieved in a Japanese haemophilia A family by the use of a restriction fragment length polymorphism (RFLP) located within the F.VIII gene. Since the pregnant womans heterozygosity for BclI polymorphism in F.VIII/intron 18 (F8A) probe was informative, chorionic villus sampling (CVS) was performed at 9 weeks of gestation. Restriction analysis showed that the fetus was heterozygous for the BclI site and had received a normal paternal X chromosome (0.9 kb) and a normal maternal X (1.2 kb). Therefore, we concluded that the fetus was a non-carrier female. Pregnancy went to term and woman gave birth to an apparently healthy female. At one week after birth a coagulation study confirmed that the newborn infant is not a carrier. The first-trimester prenatal diagnosis of haemophilia A is possible by CVS due to a RFLP in the F.VIII gene.