Genetic diagnosis and molecular characterization of three novel variations in the phenylalanine hydroxylase gene from Chinese patients with phenylketonuria

Loss-of-function variants in the human phenylalanine hydroxylase (PAH) gene are the most common genetic causal factors for Phenylketonuria (PKU). Currently, a broad spectrum of variations is recognized in the human PAH gene. However, the molecular function and clinical significance of some novel PAH variants remain unclear. Here, we report on five PKU-affected families carrying three novel PAH variants, including one missense variant (PAH: c.271C>A (p.Leu91Met)) and two deletions (PAH: c.206_208delCTT (p.Ser70del) and PAH: c.541_544delGAGG (p.Glu181Lysfs*13)). These variations constitute different compound heterozygous genotypes with other known pathogenic variants such as PAH: c.721C>T (p.Arg241Cys), PAH: c.168+5G>C, and PAH: c.1238G>C (p.Arg413Pro), which probably led to the patients’ PKU etiopathology. qRT-PCR and immunoblotting showed that the protein levels of PAH (S70del) and PAH (E181Kfs*13) were significantly reduced compared with the wild-type control, although their transcript levels were not. Also, the enzyme activity of PAH (S70del) and PAH (E181Kfs*13) mutants was significantly decreased relative to the wild type (P < 0.001). PAH: c.271C>A (p.Leu91Met) had no significant effect on PAH mRNA and protein levels or enzyme activity. Collectively, our data demonstrate that the two deletions PAH: c.206_208delCTT and PAH: c.541_544delGAGG are clinically significant for pathogenicity. Our findings are anticipated to contribute to the advancement of prenatal diagnosis, population-based carrier screening, and genetic counseling for individuals affected by PKU, and is expected to help reduce the incidence of PKU and ameliorate the associated disease burden.

人类苯丙氨酸羟化酶（phenylalanine hydroxylase, PAH）基因的功能丧失性变异是苯丙酮尿症（Phenylketonuria, PKU）最常见的遗传致病因素。目前，人类PAH基因中已被鉴定出多种类型的变异。然而，部分新型PAH变异的分子功能与临床意义仍未明确。本研究报道了5个携带3种新型PAH变异的PKU受累家系，其中包含1种错义变异（PAH: c.271C>A (p.Leu91Met)）以及2种缺失变异（PAH: c.206_208delCTT (p.Ser70del)与PAH: c.541_544delGAGG (p.Glu181Lysfs*13)）。上述变异与PAH: c.721C>T (p.Arg241Cys)、PAH: c.168+5G>C及PAH: c.1238G>C (p.Arg413Pro)等已知致病变异构成不同的复合杂合基因型，推测其可能是患者发生PKU的致病机制。实时荧光定量PCR（quantitative real-time polymerase chain reaction, qRT-PCR）与免疫印迹实验结果显示，与野生型对照相比，PAH(S70del)与PAH(E181Kfs*13)的蛋白水平显著降低，但其转录水平未出现明显改变。同时，相较于野生型，PAH(S70del)与PAH(E181Kfs*13)突变体的酶活性显著下降（P < 0.001）。PAH: c.271C>A (p.Leu91Met)对PAH的mRNA水平、蛋白水平及酶活性均无显著影响。综上，本研究数据证实，PAH: c.206_208delCTT与PAH: c.541_544delGAGG这两种缺失变异具有明确的致病临床意义。本研究结果有望推动PKU患者的产前诊断、人群携带者筛查及遗传咨询工作的发展，并有助于降低PKU的发病率，减轻相关疾病负担。