一个遗传性蛋白C缺陷症家系表型与基因突变分析

目的：对一个遗传性蛋白C（PC）缺陷症家系进行实验室表型检测和基因突变分析，探讨其分子发病机制。方法：对先证者及其家系成员（共3代6人）进行血浆蛋白C活性（PC:A）、蛋白C抗原（PC:Ag）含量及其他相关凝血指标检测。采用DNA直接测序法分析先证者蛋白C基因（PROC）9个外显子及侧翼序列，发现突变位点，再对其家系成员进行该位点的突变检测。用ClustalX-2.1-win软件分析氨基酸突变位点的保守性；用PolyPhen-2在线生物信息学软件分析突变对蛋白质功能的危害程度；用Swiss-PdbViewer软件和PIC程序进行蛋白模型分析。结果：先证者、其儿子和二姐的血浆PC:A与PC:Ag均平行下降，介于39%～58%。这3人的PROC基因第9外显子携带c.997G＞A杂合错义突变（p.Ala291Thr）。生物信息学软件分析提示：p.Ala291Thr为有害突变；Ala291在同源物种间不高度保守；蛋白模型分析显示：p.Ala291Thr突变导致Thr291与Pro327之间新增一氢键，改变了氨基酸的空间构型，使PC的稳定性下降。结论：该先证者PROC基因第9外显子存在c.997G＞A杂合错义突变，导致p.Ala291Thr；p.Ala291Thr为未报道过的新突变，是该家系遗传性PC缺陷症的主要原因。

Phenotypic and genetic analysis of a pedigree with inherited protein C deficiency

Objective: To ascertain the mutant gene of a family with hereditary protein C (PC) deficiency and to explore the molecular pathogenesis. Methods: The plasma protein C activity (PC:A), protein C antigen (PC:Ag) and other coagulation index of the family members were measured. For the proband, potential mutations in exons, flanking introns and 5’, 3’untranslated regions of PROC gene were screened by direct DNA sequencing. The Mutational site was further detected in the other family members. The ClustalX-2.1-win software and the online bioinformatics tool PolyPhen-2 were used to checke the conservatism and the possible impact of the mutation respectively. Structural analysis of the mutational site was processed with software Swiss-PdbViewer and PIC program. Results: The proband, her son and sister had reduced level of PC:A and PC:Ag among 39%~58%. Gene sequencing revealed that three people carried a heterozygous mutation c.997G>A in exon 9 of PROC gene resulting in a substitution of Alanine 291 by Threonine (p.Ala291Thr). According to the results of bioinformatics software analysis, p.Ala291Thr was “probably damaging” and Ala291 was not highly conserved. Model analyzing illuminated that replacement of Ala291 with Thr291 led to additional hydrogen bonding between Thr291-Pro327, affecting the normal spatial conformation and stability of protein. Conclusion: The proband carried a heterozygous mutation c.997G>A in exon 9 resulting in p.Ala291Thr which is a novel mutation. The p.Ala291Thr mutation could potentially account for the reduced activity of PC in this pedigree.