背景:苯丙酮尿症(PKU)是由PAH变异引起的常染色体隐性遗传先天性代谢紊乱。以前,在Sanger测序和多重连接依赖性探针扩增后,约5%的PKU患者仍未确诊.迄今为止,据报道,在100多个疾病相关基因中,有越来越多的致病性深层内含子变异.
方法:在本研究中,我们对无明确基因诊断的PKU患者进行了PAH的全长测序,以研究PAH的深层内含子变异.
结果:我们确定了五个深层内含子变体(c.1199502A>T,c.1065+241C>A,c.706+368T>C,c.706+531>C,和c.706+608A>C)。其中,c.1199502A>T变异在中国PKU中发现频率高,可能是热点PAH变异。c.706+531T>C和c.706+608A>C是扩展PAH的深内含子变体谱的两个新变体。
结论:深度内含子变异致病性分析可进一步提高PKU患者的基因诊断。计算机预测和小基因分析是研究深层内含子变异的功能和影响的有力方法。全长基因扩增后的靶向测序是一种经济有效的检测小片段基因深层内含子变异的工具。
Phenylketonuria (PKU) is an autosomal recessive congenital metabolic disorder caused by PAH variants. Previously, approximately 5% of PKU patients remained undiagnosed after Sanger sequencing and multiplex ligation-dependent probe amplification. To date, increasing numbers of pathogenic deep intronic variants have been reported in more than 100 disease-associated genes.
In this study, we performed full-length sequencing of PAH to investigate the deep intronic variants in PAH of PKU patients without definite genetic diagnosis.
We identified five deep intronic variants (c.1199+502A>T, c.1065+241C>A, c.706+368T>C, c.706+531>C, and c.706+608A>C). Of these, the c.1199+502A>T variant was found at high frequency and may be a hotspot PAH variant in Chinese PKU. c.706+531T>C and c.706+608A>C are two novel variants that extend the deep intronic variant spectrum of PAH.
Deep intronic variant pathogenicity analysis can further improve the genetic diagnosis of PKU patients. In silico prediction and minigene analysis are powerful approaches for studying the functions and effects of deep intronic variants. Targeted sequencing after full-length gene amplification is an economical and effective tool for the detection of deep intron variation in genes with small fragments.