10例遗传性凝血因子Ⅶ缺陷症分子发病机制与临床特性分析

Molecular genetics and clinical features in 10 patients with hereditary F Ⅶ deficiency

目的 探讨 10例遗传性凝血因子Ⅶ (coagulationfactorⅦ ,FⅦ )缺陷症基因突变类型与临床特性。方法 检测凝血指标以明确诊断 ;用DNA直接测序法对先证者及其家庭成员FⅦ基因的全部外显子及其侧翼、5’和 3’非翻译区进行分析 ,寻找基因突变 ;将含插入或缺失突变序列克隆入pMD18 TTA克隆载体中 ,对所得两条染色体相应序列分别测序 ,以确定突变在染色体上的分布。应用限制性内切酶对先证者及家系成员相应基因片段进行酶切分析 ,无酶切位点改变的基因片段用等位基因特异的PCR(ASPCR)方法 ,证实测序所发现的突变。结果 在 10例遗传性凝血因子Ⅶ缺陷症患者中发现 8种类型的基因突变 ,其中 6 390T→C(Phe4 0Cys) ,94 82G→T(Arg15 2Leu) ,和 114 87 9delC 3种突变为国际首次报道 ;6种突变发生在催化区 ;除一种缺失突变外 ,其余均为点突变 ;所有的基因突变都来自先证者的父亲和 (或 )母亲。Thr35 9Met和Arg30 4Trp突变分别在 4个及 2个无亲缘关系的家系中重复出现。 2例Thr35 9Met纯合突变 (FⅦ :C分别为 2 %和 3% )及 1例Arg15 2Leu、114 87 9delC及Arg30 4Trp复合杂合突变 (FⅦ :C为 1% )临床表型为重型 ;2例双重杂合突变 (His348Gln和Thr35 9Met,Agr30 4Trp和Arg30 4Gln)临床表型分别为中型和无症状

Objective To investigate the mutations and polymorphisms of the FⅦ gene and the clinical features in 10 patients with hereditary FⅦ deficiency.Methods The diagnosis was validated by coagulant parameters. FⅦ gene mutations were analysed in the probands and their family members with amplified all exons, exon-intron boundaries and the 3',5'untranslated domains by PCR and sequenced directly. Some PCR fragments linkaged into pMD18-T vector were sequenced for characterizing the allele mutations of insertions or deletions, The PCR fragments contained the mutations were cleaved by the relative restriction enzymes or allele specific PCR(ASPCR) was amplified for no endonuclease sites being created or abolished to confirm the mutations detected by sequencing.Results 8 different mutations were identified in 10 unrelated probands and three(Phe40Cys, Arg152Leu, and 11487-9delC)of them were novel mutations. Most of the mutations located in protease domain. All of them were point-mutations exception for one deletion mutation and transmitted from the parents. Some mutations were repeated in unrelated pedigrees, The Thr359Met mutation was reoccurred in four unrelated families while the Arg304Trp mutation was determintered in two unrelated probands , The relationship between the genetic deficiencies and hemorrhagic disorder were characterized. The homozygosity of the Thr359Met in two probands with FⅦ:C activity 2% and 3%, respectively, were related to severe clinical symptoms; The proband with double heterozygous lesions of Arg152Leu and one single nucleotide deletion (C) at position 11487-9,combined with Arg304Trp in exon 8 with FⅦ activity 1% suffered severe clinical bleeding tendency. Two probands with double heterozygous mutations (His348Gln and Thr359Met, Agr304Trp and Arg304Gln) with FⅦ activities of 3.4% and 10% characterized moderate and asymptom. the heterozygous mutations(Phe40Cys combined with Arg353Gln heterozygous polymorphism, Thr359Met, Arg152Gln, -55C→T) with FⅦ:C 1%～5.5% showed moderate or mild clinical symptoms. In addition,the proband with three polymorphisms with FⅦ:C 1% revealed the mild hemorrhage feature.Conclusion Eight types of FⅦ gene mutations were identified and three of them were novel mutations in 10 unrelated probands with hereditary FⅦ deficiency. There were hotspot that causing factor Ⅶ deficiency in Chinese Han race, the genotypes were not related with the clinical phenotypes in hereditary coagulation factor Ⅶ deficiency.