两个遗传性蛋白C缺陷症家系临床表型和基因型变化的研究

Analysis of phenotype and genotype in two Chinese pedigrees with hereditary protein C deficiency

目的对两个遗传性蛋白C（PC）缺陷症家系进行临床表型和基因突变检测。方法血浆蛋白C活性（PC：A）和抗原（PC：Ag）分别用发色底物法和ELISA法测定，蛋白S活性（PS：A）和抗凝血酶活性（AT：A）用发色底物法测定。用PCR法对先证者PC基因的9个外显子及其侧翼、内含子序列进行扩增，PCR产物纯化后直接测序，检测其基因突变。仅对先证者家系成员基因突变部位的外显子及其侧翼序列进行PCR扩增和测序。突变位点经限制性内切酶酶切分析或直接测序证实。结果先证者1（Ⅱ7）的PC：A和PC：Ag分别为1．2％和0。基因测序显示，先证者1在PC基因外显子5存在C3135G杂合错义突变，致C（TGC）64W（TGG），同时在外显子7存在T6128G杂合错义突变，致F（TTC）139V（GTC）。家系成员中，先证者的父亲（Ⅰ4）和女儿（Ⅲ3）存在T6128G杂合突变，先证者的舅舅（Ⅱ）存在C3135G杂合突变，先证者丈夫（Ⅱ8）存在外显子76161-6163或6164～6166AAG（K150或K151）杂合缺失，而其女儿（Ⅲ3）亦有此突变。先证者2（Ⅲ1）的PC：A和PC：Ag分别为50．3％、1．9mg／L，基因测序显示其存在K150或K151杂合缺失，该突变遗传自其父亲。2个家系中所有成员在PC基因启动子区中存在-1654C／T、-1641A／G、-1476A／T多态性，先证者2为CC／GG／TT纯合型。限制性内切酶PSp5Ⅱ酶切分析显示T6168G不是多态性。所有成员的PS：A和AT：A均在正常范围。结论复合杂合性PC基因突变（C64W和F139V）是导致先证者1遗传性Ⅰ型PC缺陷症的原因，杂合性Lys150或151缺失突变和PC基因启动子区CC／GG／TI纯合多态性是致先证者2遗传性Ⅰ型PC缺陷症的原因。C64W为国际首次报道，F139V、K150或151缺失突变为国内首次报道。

Objective To identify the phenotype and gene mutation in two Chinese pedigrees with hereditary protein C deficiency. Methods The plasma level of protein C activity （PC： A）, protein C antigen （PC： Ag）, protein S activity （PS： A）, and antithrombin activity （AT： A） of the probands and their family members were detected using chromogenic assay and ELISA, respectively. All of the nine exons and intron-exon boundaries of protein C gene were amplified by PCR and analyzed by direct sequencing of the probands. Restriction enzyme site analysis was used to confirm the mutation. Results The plasma PC： A and PC： Ag for proband 1 was 1.2% and 0, respectively. Compound heterozygous mutations, C（TGC）64W （TGG） and F（TTC）139V（GTC）, were identified in her, the former being inherited from the maternal side and the later the paternal side. Further genetic analysis showed that her husband （Ⅱ8） had the heterozygous deletion mutation （K150 or 151 Del） in exon 7, her daughter had the same heterozygous deletion mutation and a F139V. The plasma PC： A and PC： Ag for proband 2 was 50.3% and 1.9 mg/L, respectively. He had the heterozygous Lys150 or Lys151 deletion mutation, which was inherited from his father. Polymorphisms of C/T at position - 1654, A/G at - 1641, and A/T at - 1476A/T in the promoter region of protein C were confirmed in all members of the two pedigrees, of which, proband 2 had homozygous CC/GG/Tr. The F139V mutation was confirmed by restriction enzyme site analysis and polymorphism for this mutation was excluded. PS： A and AT： A were in normal range for all members. Conclusion Compound heterozygous mutation C64W and F139V of protein C gene lead to type Ⅰ hereditary protein C deficiency for proband 1. K150 or 151 deletion mutation and polymorphyism of CC/GG/Tr might lead to type Ⅰ hereditary protein C deficiency for proband 2. C64W is a novel mutation for protein C gene. F139V and K150 or 151 deletion mutation are reported for the first time in China.