Three novel variants (p.G1u178Lys, p.Va1245Met, p.Ser250Phe) of the phenylalanine hydroxylase (PAH) gene impair protein expression and function in vitro
Three novel variants (p.G1u178Lys, p.Va1245Met, p.Ser250Phe) of the phenylalanine hydroxylase (PAH) gene impair protein expression and function in vitro
复制标题
苯丙氨酸羟化酶 (PAH) 基因的三种新变体（p.G1u178Lys、p.Va1245Met、p.Ser250Phe）在体外损害蛋白质表达和功能
作者：
Zong Y;Liu N;Ma S;Bai Y;Guan F;Kong X
苯丙酮尿症（PKU）是最常见的遗传代谢性疾病，是一种常染色体隐性遗传疾病，全球每年影响超过10，000名新生儿。它可以由苯丙氨酸羟化酶（PAH）基因中超过1000种不同的天然突变引起。我们分析了三种新的天然存在的PAH基因变体：p.Glu178Lys（c.532G>A），p.Val245Met（c.733G>A）和p.Ser250Phe（c.749C>T）。利用生物信息学软件预测突变体对PAH酶结构和功能的影响。产生表达相应PAH变体的载体用于在E中表达。大肠埃希菌在HEK 293 T细胞中表达。采用定量逆转录聚合酶链反应（RT-qPCR）检测三种PAH变异体的RNA表达。采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）、蛋白质印迹和酶联免疫吸附试验（ELISA）检测突变PAH蛋白水平。通过生物信息学分析，预测所有三种变体都是致病的。在HEK 293 T细胞中，三种PAH变异体的转录与野生型PAH基因相似。与此相反，突变PAH蛋白质的水平显着下降相比，野生型对照，在两个E。coli和HEK 293 T细胞。我们的研究结果表明，三个新的PAH基因变异体（p.Glu178Lys，p.Val245Met，p.Ser250Phe）在原核和真核细胞中损害PAH蛋白的表达和功能。
Phenylketonuria (PKU) is the most common inherited metabolic disease, an autosomal recessive disorder affecting >10,000 newborns each year globally. It can be caused by over 1000 different naturally occurring mutations in the phenylalanine hydroxylase (PAH) gene. We analyzed three novel naturally occurringPAHgene variants: p.Glu178Lys (c.532G>A), p.Val245Met (c.733G>A) and p.Ser250Phe (c.749C>T). The mutant effect on the PAH enzyme structure and function was predicted by bioinformatics software. Vectors expressing the correspondingPAHvariants were generated for expression inE. coliand in HEK293T cells. The RNA expression of the threePAHvariants was measured by quantitative reverse transcription polymerase chain reaction (RT-qPCR). The mutant PAH protein levels were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), western blot and enzyme-linked immunosorbent assay (ELISA). All three variants were predicted to be pathogenic by bioinformatics analysis. The transcription of the threePAHvariants was similar to the wild typePAHgene in HEK293T cells. In contrast, the levels of mutant PAH proteins decreased significantly compared to the wild type control, in bothE. coliand HEK293T cells. Our results indicate that the three novelPAHgene variants (p.Glu178Lys, p.Val245Met, p.Ser250Phe) impair PAH protein expression and function in prokaryotic and eukaryotic cells.