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目的:探讨红细胞丙酮酸激酶缺乏症(pyruvate kinase deficiency,PKD)的分子发病机制。方法:应用目标序列捕获和高通量二代测序技术(next-generation sequencing,NGS)对临床拟诊PKD患儿的PKLR基因12个外显子及其侧翼序列进行测序,采用SIFT及PolyPhen-2数据库预测突变对蛋白质功能的影响,在确定患者致病基因型后,应用Sanger测序技术对此基因型进行验证。结果:NGS结果显示,患儿PKLR基因存在罕见的双重杂合突变:第5外显子661G>A(Asp221Asn)和第10外显子1528C>T(Arg510Ter),导致该基因的第221位氨基酸由天冬氨酸突变为天冬酰胺,第510位氨基酸由精氨酸突变为终止密码子,使PKLR基因氨基酸链编码提前终止;Sanger测序技术进一步验证了该双重突变的存在。检索相关文献及数据库显示,这两种突变在人群中的发生率极低,蛋白质功能预测均显示为有害。结论:PKLR基因661 G>A与1528 C>T双重杂合突变是该PKD患儿的分子发病机制,PKD患者同时存在上述复合突变的报道在国内外尚属首次。
Objective: To investigate the molecular pathogenesis of erythrocyte pyruvate kinase deficiency (PKD). METHODS: Twelve exons of PKLR gene and their flanking sequences were sequenced by targeted sequencing and high-throughput second generation sequencing (NGS). SIFT and PolyPhen-2 The database predicts the effect of the mutation on the function of the protein. Sanger sequencing was used to validate the genotype after determining the patient’s pathogenic genotype. Results: The results of NGS showed that there was a rare double heterozygous mutation in PKLR gene in children: exon 5 661G> A (Asp221Asn) and exon 10 1528C> T (Arg510Ter), leading to the 221th amino acid From aspartate to asparagine, the amino acid at position 510 was changed from arginine to stop codon, and the amino acid chain of PKLR gene was prematurely terminated. Sanger sequencing further validated the existence of this double mutation. Retrieval of relevant literature and databases shows that these two mutations are extremely low in the population and protein function predictions are shown to be deleterious. CONCLUSION: The double heterozygous mutation of PKLR gene 661 G> A and 1528 C> T is the molecular pathogenesis in children with PKD. The coexistence of the above compound mutations in PKD patients is the first time in China and abroad.