| Backgrounds: Human coagulation factor V is a single-chain glycoprotein that plays an important role in maintaining the hemostatic blance. It circulates in blood as an inactive procoagulant with a Mr of 330 kd and a structure consisting of 3 homologous A-type domains and 2 homologous C-type domains connected by d heavily glycosylated B domain in the order A1-A2-B-A3-C1-C2. Deficiency of FV, or parahemophilia, was first described in 1747 by Owren. It is a rare autosomal recessive bleeding disorder with an estimated frequency of one in one million. The phenotypic expression of FV deficiency is variable; heterozygotes are usually asymptomatic, whereas homozygous patients show mild, moderate, or severe bleeding symptom. Recently the complete nucleotide sequence of FV gene(GenBank accession number Z99572) has been published, it is mapped to chromosome 1q23 and spans 80 kilobasees. FV gene consists of 25 exons and the messenger RNA (mRNA) encodes aleader peptide of 28 amino acids and a mature protein of 2196 amino acids. Roughly, the heavy chain is encoded by exons 1 to 12 and the light chain by exons 14 to 25. The entire B domain is encoded by exon 13. Identifying the molecular basis underlying this disease will help to obtain more insight into the mechanisms involved in this variable clinical expression, and cure this disorder ultimately.Objective: First, to intruduce the diagnosis and treatment for these two patients, and to explore the substitution strategy for major surgery in congenital FV deficiency. The inherited trait of FV deficiency in these two families was also observed. Second, to identify the molecular mechanisms involved in these two patients with congenital FV deficiency. All mutations of FV gene in probands and their family numbers were confirmed by restriction enzyme analysis. Their occurrences were investigated in the control group. Methods: APTT(activated partial thromboplastin time), PT(prothrombin time), TT(thrombin time) and FV coagulant activity were determined on an C-1500 coagulation analyzer by using DADE Bchring kit . The level of the FV inhibitor was measured according to the Bethesda assay. Patient A in whom a large haematoma under right duramater was surgically excised by substitution with fresh-frozen plasma. Family screening was also done among relatives of these two patients. The primers used for polymerase chain reaction (PCR) and DNA sequence analysis of the 25 exons were designed in the intronic regions (GenBank accession number Z99572) to check the splicing junctions. Genomic DNA was isolated from peripheral mononucleate cells using DNAZOL. The PCR products were purified prior to DNA sequence analysis using DNA purification kit. Automated DNA sequencing reactions were carried out in forward and reverse direction on an ABI 377 DNA sequencer. If necessary, DNA sequence analysis was performed again using another PCR product. The exon 13 was cloned into PGEM-T Easy Vector prior to DNA sequencing. The mutations were identified as comparing sequence data to published ones in GeneBank . Finally, all mutations of FV gene in probands and their family numbers were confirmed by restriction enzyme analysis. Their occurence were investigated in the control group.Results:.The Patient A and patient B were diagnosed as homozygous congenital FV deficiency. The parents of patient A, mother of patient B and her son were all heterozygous. Patient A in whom a large haematoma under right duramater was successfully surgically excised by substitution with fresh-frozen plasma.This primary study identified 2 novel mutations associated with congenital FV deficiency in 2 patients and their family numbers: In patient A, we detected a single point mutation, a homozygous A to G substitution at position 3' splice site of intron 8 where was in the invariant AG dinucleotide. In patient B, a homozygous missense mutation C5729 A, Pro1880Gln was revealed。Conclution: These two mutations of FV gene are related to the pathogenesis of congenital FV deficiency .
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