三个遗传性抗凝血酶缺陷症家系临床表型和基因型分析

Molecular analysis of the genotypes and phenotypes in three pedigrees with inherited antithrombin deficiency

目的探讨遗传性抗凝血酶（AT）缺陷症先证者及其家系成员的AT活性（AT：A）、AT抗原含量（AT：Ag）及基因型在引起遗传性AT缺陷症发病的分子机制。方法收集3例AT缺陷症先证者及其家系成员血浆标本，采用发色底物法和免疫比浊法分别检测先证者及其家系成员血浆AT：A和AT：Ag，提取外周血基因组DNA。用PCR法扩增AT基因的全部7个外显子及侧翼序列，通过直接测序分析异常的突变基因。利用蛋白免疫印迹法分析血浆中AT含量的变化，用凝血酶生成试验检测患者体内的凝血状态。结果3例先证者AT：A和AT：Ag均降低到正常值的50％左右，分别在AT基因外显子4区的第7386位核苷酸发生杂合性G〉C突变，导致W（Trp）225C（Cys）错义突变；2号外显子区第2591位核苷酸发生C〉G突变，导致S（Ser）36X（stop）无义突变；5号外显子区第9819位核苷酸发生C〉T杂合突变，导致R（Arg）359X（stop）无义突变。部分家系成员表型和基因型检测结果与先证者一致。蛋白免疫印迹结果显示先证者及其家系成员血浆中的AT含量较正常混合血浆明显减少，凝血酶生成实验显示先证者体内呈现明显的高凝状态。结论Ⅰ型遗传性AT缺陷症中，W225C、S36X、R359X引起血浆中AT含量下降，是导致遗传胜AT缺陷症的分子致病机制。

Objective To investigate the clinical phenotype and genotype in three probands with antithrombin （AT） deficiency and their families, and to identify the molecular mechanism of AT deficiency. Methods Chromogenic substrate method and immunoturbidimetry assay was used to detect the plasma levels of AT： A and AT： Ag,respectively. Genomic DNA was extracted from the peripheral blood. All 7 exons and the flanking sequences were amplified by PCR, and the abnormal mutant genes were analyzed by direct sequencing. Western blot was used to detect the AT levels and thrombin generation tests were used to detect coagulation status. Results The plasma levels of AT： A and AT： Ag of the three probands declined by 50%. G7386C （Trp225Cys） mutation in exon 4, C2591G（Ser36stop） in exon 2 and C9819T（Arg359stop） in exon 5 were characterized in the three probands and they could result in W（Trp）225C（Cys） missense mutation, S （Ser） 36X （stop） nonsense mutation and R （Arg） 359X （stop） nonsense mutation respectively, The testing results of phenotype and genotype from some of their family members showed consistent with results from the probands. Western blot results indicated that the levels of PC： Ag were lower compared with the normal pooled plasma. The hypercoagulative status was present in the probands using thrombin generation tests. Conclusions Type I hereditary AT deficiency was found in these three families. The 3 heterozygous mutations, W225C,S36X and R359X are genetic defects of hereditary AT deficiency. W225C and S36X have not been described before.