一个丙酮酸激酶缺乏症家系PKLR基因的突变分析及产前诊断

Analysis and prenatal diagnosis of PKLR gene mutations in a family with pyruvate kinase deficiency

目的对一个丙酮酸激酶缺乏症（pyruvatekinasedeficiency，PKD）家系进行致病基因突变分析及产前诊断。方法应用目标序列捕获和高通量测序技术对临床拟诊为PKD的患儿的PKLR基因外显子及其侧翼序列进行测序，采用SIFT及PolyPhen-2数据库对突变进行蛋白功能预测，在确定先证者致病基因型后，应用Sanger测序技术进行验证，同时检测其父母基因型，并对孕16周的高危胎儿抽取羊水进行产前诊断。结果患儿PKLR基因存在罕见的双重杂合突变C．661G〉A（Asp221Asn）以及C．1528C〉T（Arg510Ter），导致该基因的第221位氨基酸由天冬氨酸变化为天冬酰胺，第510位由精氨酸改变为终止密码子。Sanger测序验证了该双重突变的存在，先证者父母分别检出c．661G〉A（Asp221Asn）与c．1528C〉T（Arg5lOTer）突变。胎儿检出与先证者相同的致病突变。在终止妊娠后，对流产物进行突变位点分析，结果与产前诊断一致。结论PKLR基因c．661G〉A与C．1528C〉T复合突变是该PKD家系的分子发病机制。通过产前诊断可以有效阻止致病基因的传递，降低生育患儿的风险。

Objective To evaluate the feasibility of genetic and prenatal diagnosis for a family affected with pyruvate kinase deficiency （PKD）. Methods Targeted sequence capture and high-throughput sequencing technology was used to detect the exons and exon-intron boundaries of the PKLR gene in a clinically suspected PKD patient. Meanwhile, the genotype of the pedigree was validated by Sanger sequencing. Prenatal genetic diagnosis was performed by amniotic fluid sampling after genotype of the mother of the proband was determined. Results The proband was found to harbor double heterozygous mutations, c. 661 G〉A （Asp221Asn） and c. 1528C〉T （Arg510Ter）, which resulted in amino acid substitution Asp221Asn and Arg510Ter, Such mutations were confirmed by Sanger sequencing. The mother and father of the proband were detected to have respectively carried c. 1528C~ T （Arg510Ter） and c. 661G~〉A （Asp221Asn） mutation. The fetus was found to have carried the same mutations as the proband. Following selected abortion, analysis of fetal tissue was consistent with the result of prenatal diagnosis. Conclusion The compound mutations of c. 661 G〉A and c. 1528 C〉T of PKLR gene probably underlie the PKD in the family. Prenatal diagnosis of the mutations analysis can facilitate detection of affected fetus in time.