A capillary electrophoresis-based variant hotspot genotyping method for rapid and reliable analysis of the phenylalanine hydroxylase gene in the Chinese Han population

Hyperphenylalaninemia (HPA) is a common autosomal recessive disorder of phenylalanine metabolism, mainly caused by the deficiency of phenylalanine hydroxylase gene (PAH). A simple, fast, and accurate assay to achieve early diagnosis for children with HPA is required.In the present study, we established a SNaPshot-based assay that allows the simultaneous genotyping of 96 hotspot variants in the PAH gene. First, 18 Chinese HPA patients were analyzed by next generation sequencing (NGS) and SNaPshot in parallel. Then, the SNaPshot assay was performed to analyze the mutational spectrum of the PAH in 4,276 individuals in Eastern China.A total of 36 variants in the PAH gene were successfully identified by NGS, while the SNaPshot assay identified 34 PAH variants in these patients. Thus, the SNaPshot assay achieved the sensitivity and specificity of 91.6% and 100.0%, respectively. Furthermore, the carrier rate was approximately 1 in 58 (1.73%) in 4,276 individuals, and c.728G > A was the most common variant.In summary, SNaPshot can accurately and rapidly detect PAH gene variants at a comparable performance and lower cost as compared with NGS. Our results suggest that SNaPshot may serve as a promising approach for a routine genetic test in clinical practice.