一个杂合缺失变异致遗传性蛋白C缺陷症家系的表型和基因型分析  

Phenotypic and genotypic analysis of a pedigree affected with hereditary protein C deficiency due to heterozygous deletional mutation of PROC gene

作　　者：刘斯奇 余芳 罗莎莎 李小龙[1] 金艳慧[1] 杨丽红[1] 周星星[1] 张海月 王明山[1] Liu Siqi;Yu Fang;Luo Shasha;Li Xiaolong;Jin Yanhui;Yang Lihong;Zhou Xingxing;Wang Mingshan;Zhang Haiyue(Department of Clinical Laboratory,the First Affiliated Hospital of Wenzhou Medical University,Zhejiang 325000,China)

机构地区：[1]温州医科大学附属第一医院医学检验中心,浙江325015

出　　处：《中华医学遗传学杂志》2020年第10期1108-1112,共5页Chinese Journal of Medical Genetics

基　　金：浙江省科技厅公益技术研究计划(LGF18H080003)。

摘　　要：目的分析1个遗传性蛋白C(protein C,PC)缺陷症家系的表型和基因变异。方法对先证者及家系成员(共4代11人)采用发色底物法检测血浆蛋白C活性(protein C activity,PC:A),酶联免疫吸附法检测蛋白C抗原(protein C antigen,PC:Ag)含量。PCR法对先证者蛋白C基因(protein C,PROC)的9个外显子及侧翼序列进行扩增,PCR产物纯化后进行直接测序;对发现的疑似变异用克隆测序予以验证,并对家系其他成员相同变异位点进行检测。分别用生物信息学软件Mutation Taster和ClustalX-2.1-win分析变异的致病性和保守性;用Swiss-PdbViewer软件分析蛋白质三维模型和变异氨基酸之间的相互作用。结果先证者PC:A和PC:Ag分别降至46%和50%,其奶奶、大姑、表姐和弟弟的PC:A和PC:Ag也有不同程度的下降,为正常参考值的50%左右。基因分析显示,上述5名成员PROC第9外显子均存在c.1212-1212delG(p.Met364Trp fsX15)杂合缺失变异,该缺失变异导致第364位的甲硫氨酸(Met)变成色氨酸(Trp)后框移了15个氨基酸,并在第378位产生提前的终止密码子,最后形成截短蛋白。MutationTaster评分结果为1.000,预示该杂合缺失变异为致病变异;保守性分析显示15个框移的氨基酸在9种同源物种中均位于保守区域;蛋白质模型分析显示该缺失变异破坏了PC的催化结构域,从而影响PC的功能。结论先证者PROC第9外显子存在c.1212-1212delG(p.Met364Trp fsX15)杂合缺失变异,可能是导致该家系PC:A和PC:Ag减少的主要原因。Objective To analyze the phenotype and genetic variants of a pedigree affected with hereditary protein C(PC)deficiency.Methods The protein C activity(PC:A)of the proband and her family members(a four-generation pedigree including 11 individuals)were tested by chromogenic substrate method,and the protein C antigen(PC:Ag)was detected with an enzyme-linked immunosorbent assay(ELISA).The 9 exons and flanking sequences of the protein C(PROC)gene were amplified by PCR and directly sequenced.Suspected mutation was validated by clone sequencing and in other members of the family.MutationTaster and ClustalX-2.1-win was used to analyze the pathogenicity and conservation of the mutation site,respectively.Three-dimentional protein model and amino acids interaction were analyzed with Swiss-PdbViewer software.Results The PC:A and PC:Ag of the proband were decreased to 46%(reference range:70%-130%)and 50%(referencerange:70%-140%),respectively.Her grandmother,aunt,cousin and younger brother also showed declined PC:A and PC:Ag by approximately 50%.Genetic analysis revealed that the above individuals have all carried a deletional mutation c.1212-1212delG(p.Met364TrpfsX15)in exon 9 of the PROC gene which can cause replacement of Methionine at position 364 by Tryptophan and alteration of 15 downstream amino acids,and produce a premature stop codon at position 378.The score of MutationTaster was 1.000,indicating that the variant is pathogenic.Conservative analysis showed that the 15 altered amino acids are located in a conserved region across nine homologous species.Protein model analysis showed that the mutation has disrupted a catalytic domain of protein C thereby affected its function.Conclusion The heterozygous c.1212-1212delG deletional mutation in exon 9 of the PROC gene,which was unreported previously,probably accounts for the decrease of PC:A and PC:Ag in this pedigree.

关 键 词：遗传性蛋白C缺陷症 基因变异 框移 截短蛋白 

分 类 号：R596[医药卫生—内科学] R440[医药卫生—临床医学]