一例遗传性蛋白C缺陷症家系的实验诊断

Analysis of one Chinese pedigree with hereditary protein C deficiency

目的对一个蛋白C(PC)缺陷家系进行临床表型及基因突变检测,初步探讨突变位点的作用机制。方法采用发色底物法和ELISA双抗体夹心法分别检测先证者和家系成员的血浆PC活性和PC抗原含量。用PCR法对先证者PROC基因9个外显子及其侧翼序列进行扩增,PCR产物纯化后测序,检测基因突变;对于家系成员,仅检测先证者发现基因突变部位的外显子及其侧翼序列。通过对突变前后蛋白质的构象分析,初步推测突变位点的分子机制。结果先证者的PC活性和PC抗原含量分别为38%和33.6%。基因测序显示先证者的PROC基因外显子7区同时存在1个杂合错义突变[c.541T>G(p.Phe181Val)]和1个无义突变[c.595C>T(p.Arg199*)]。前者引起181位苯丙氨酸(Phe)变为缬氨酸(Val),后者使199位精氨酸(Arg)突变为终止密码子。其父亲和儿子的PC活性分别为49%和42.2%,PC抗原含量分别为90%和97.4%,存在一个相同的杂合无义突变c.595C>T。其母亲的PC活性为84.4%,PC抗原含量为100%,外显子7区在位点c.541T>G存在杂合错义突变。所有成员蛋白S活性和AT活性均在正常范围。结论通过表型检测、家系调查和基因诊断,明确先证者为遗传性PC缺陷症,先证者的2个突变分别来自父亲和母亲,是导致先证者患PC缺陷症的原因,突变位点c.595C>T可能是导致患者PC活性下降的主要原因。

Obiective To identify the phenotype and gene deficiency, and explore the action mechanism of mutant site. mutation in one Chinese pedigree with hereditary protein C Methods The plasma levels of protein C activity and protein C antigen of the propositus and family members were detected using chromogenic assay and ELISA respectively. All of the nine exons and intron-exon boundaries of PROC gene were amplified by PCR and analyzed by direct sequencing for the propositus. To the family members, only the exons with mutations were amplified and analyzed. The molecular mechanism of mutant site was explored through conformational analysis of protein before and after mutation. Results The plasma levels of protein C activity and protein C antigen of the propositus were 38% and 33.6% respectively. Gene sequencing revealed that there existed a heterozygous missense mutation [ c. 541T 〉 G （ p. Phel81Val） ] and a nonsense mutation [ c. 595C 〉 T （p. Arg199 ＊ ） ] on exon 7 of PROC gene of the propositus. The former caused the transformation from Phe to Val on site 181, and the latter led to the mutation from Arg to stop codon on site 199. The plasma levels of protein C activity of the father and son were 49% and 42.2% respectively, and those of protein C antigen were 90% and 97.4% respectively, and both had the same heterozygous nonsense mutation c. 595C 〉 T. The plasma level of protein C activity of the mother was 84.4%, that of protein C antigen was 100%, and there was missense mutation on site c. 541T 〉 G in exon 7. The levels of protein S： A and AT： A were in the normal range in all the members. Conclusion Inherited protein C deficiency is indicated by phenotype diagnosis and gene analysis for the propositus. Two mutations of the propositus are inherited from father and mother respectively, which may be the cause for protein C deficiency, and c. 595C 〉 T mutation may be the main cause of decrease in protein C activity.Objective