两个遗传性蛋白C缺陷症家系表型与基因突变分析

Analysis of phenotypes and genetic mutations in two pedigrees affected with hereditary protein C deficiency

目的对两个遗传性蛋白C（proteinC，PC）缺陷症家系进行临床表型和基因突变检测，建立蛋白模型，探讨其分子发病机制。方法分别应用发色底物法和酶联免疫吸附法检测两个家系先证者和家系成员的血浆蛋白C活性（protein Cactivity，PC：A）和蛋白C抗原（protein Cantigen，PC：Ag）。用PCR法对先证者蛋白C（protein C，PROC）基因9个外显子及侧翼序列进行扩增，PCR产物纯化后用Sanger测序法进行基因突变检测；对家系其他成员相应基因突变位点进行扩增和测序。用生物信息学软件PolyPhen-2预测突变位点的危害程度；用ClustalX软件分析突变位点的保守性；用Swiss-PdbViewer软件对突变基因进行蛋白模型和氨基酸相互作用分析。结果家系1先证者PC：A为30％，PC：Ag为35％，其父亲、母亲及姑姑的PC：A均略低于正常参考值范围；先证者的PROC第7外显子存在c．565C〉T杂合错义突变（p．Arg147Trp），并在第5外显子存在c．383G〉A杂合错义突变（p．Gly86Asp）；其父亲和姑姑均携带c．565C〉T杂合突变，母亲携带c．383G〉A杂合突变。家系2先证者和儿子的PC：A分别为50％、64％；测序结果显示两人的PROC第7外显子均存在c．565C〉T杂合突变。生物信息学软件PolyPhen-2分析显示p．Arg147Trp为良性突变，p．Gly86Asp为有害突变。ClustalX分析显示p．Argl47Trp在同源物种间不保守，而p．Giy86Asp则高度保守。突变蛋白模型分析显示p．Arg147Trp突变产生的Trpl47芳香环破坏了Argl47和Lys146、Lys151的两个氢键，并与Arg178之间产生分子碰撞。p．Gly86Asp突变后Asp86侧链延长，和Gln90侧链之间产生分子碰撞力。基因突变导致的氢键破坏和碰撞作用改变了氨基酸的空间构型，使突变蛋白稳定性下降或分泌障碍。结论两个家系先证者均为遗传性PC缺陷症，杂合突变p．Arg147Trp和p．Gly86Asp是导致患者PC活性下降的主要原因。其中p．Gly86Asp为未报道过的新突变。

Objective To explore the pathogenesis of protein C deficiency in two pedigrees through mutation detection and model analysis. Methods Chromogenic substrate method and enzyme linked immunosorbent assay （ELISA） were used to determine the plasma protein C activity （PC ： A） and protein C antigen （PC ： Ag） in the two probands and their family members. All of the 9 exons and intron-exon boundaries of the PROC gene were amplified by PCR and analyzed with Sanger sequencing after purification. Corresponding mutate sites of the family members were also amplified and sequenced. The PolyPhen-2 software was used to analyze the perniciousness of the mutations and Clustal X was to analyze the conservatism. The protein model and amino acids interaction of the mutations were analyzed by Swiss- PdbViewer software. Results The PC ： A and PC , Ag of proband 1 was 30% and 35 %, while PC ： A of his father, mother and aunt were all slightly under the reference range. Two heterozygous missense mutations were found in exons 7 and 5 of the PROC gene, namely c. 565 C〉T （p. Arg147Trp） and c. 383 G 〉A （p. Gly86Asp）. His father and aunt were carriers for c. 565 C〉T, while his mother had carried c. 383 GI〉A. The PC ： A of proband 2 and his son were 50% and 64%, respectively. And they were both positive for p. Arg147Trp. Analysis of PolyPhen-2 indicated that p. Arg147Trp was benign, while p. Gly86Asp was damaging. Clustal X analysis indicated that the p. Arg147Trp was non-conservative, while the p. Gly86Asp was highly conservative. Modeling for the mutant proteins revealed that the simple aromatic ring of Trp147 in p. Arg147Trp destroyed the two hydrogen bonds between Arg147-Lys146 and Arg147-Lys151, and steric hindranted with Arg178. The side chain of Asp86 extended and generated steric clash with Gln90 with the occurrence of p. Gly86Asp. The change of hydrogen bonds and steric effects has altered the spatial configuration of amino acids, which led to unstable mutate proteins and interfered with the secretion. Conclusion Both probands had hereditary protein C deficiencies, for which their parents were all carriers. The heterozygous mutations p. Arg147Trp and p. Gly86Asp were the main cause for PC ： A activity decrease. Among these, p. Gly86Asp was discovered for the first time.