Genetic and Prenatal Diagnostic Center, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China.
School of Life Science, Zhengzhou University, Zhengzhou, China.
Gene. 2018 Aug 20;668:135-139. doi: 10.1016/j.gene.2018.03.078. Epub 2018 Apr 10.
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 occurring PAH gene 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 corresponding PAH variants were generated for expression in E. coli and in HEK293T cells. The RNA expression of the three PAH variants 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 three PAH variants was similar to the wild type PAH gene in HEK293T cells. In contrast, the levels of mutant PAH proteins decreased significantly compared to the wild type control, in both E. coli and HEK293T cells. Our results indicate that the three novel PAH gene variants (p.Glu178Lys, p.Val245Met, p.Ser250Phe) impair PAH protein expression and function in prokaryotic and eukaryotic cells.
苯丙酮尿症（PKU）是最常见的遗传性代谢疾病，是一种常染色体隐性遗传病，全球每年影响超过 10000 名新生儿。它可能由苯丙氨酸羟化酶（PAH）基因中超过 1000 种不同的自然发生突变引起。我们分析了三种新的自然发生的 PAH 基因突变：p.Glu178Lys（c.532G>A）、p.Val245Met（c.733G>A）和 p.Ser250Phe（c.749C>T）。生物信息学软件预测了突变对 PAH 酶结构和功能的影响。为了在大肠杆菌和 HEK293T 细胞中表达，生成了表达相应 PAH 变体的载体。通过定量逆转录聚合酶链反应（RT-qPCR）测量三种 PAH 变体的 RNA 表达。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）、western blot 和酶联免疫吸附测定（ELISA）确定突变型 PAH 蛋白水平。生物信息学分析预测所有三种变体均为致病性。在 HEK293T 细胞中，三种 PAH 变体的转录与野生型 PAH 基因相似。相比之下，与野生型对照相比，突变型 PAH 蛋白水平在大肠杆菌和 HEK293T 细胞中均显著降低。我们的结果表明，三种新的 PAH 基因突变（p.Glu178Lys、p.Val245Met、p.Ser250Phe）会损害原核和真核细胞中 PAH 蛋白的表达和功能。
