青岛市苯丙氨酸羟化酶缺乏症患儿基因突变分析

目的探讨青岛市苯丙氨酸羟化酶(phenylalanine hydroxylase,PAH)缺乏症患儿的基因突变特点,为青岛市PAH缺乏症的产前诊断、治疗提供科学参考依据。方法对经青岛市新生儿疾病筛查确诊的44例PAH缺乏症患儿,应用第二代高通量测序及多重连接酶探针依赖扩增(multi-ligase probe dependent amplification,MLPA)技术进行基因分析,检测患儿基因突变位点,应用Sanger测序对其父母的PAH基因相应突变位点进行检测并验证。根据患儿血苯丙氨酸浓度,分为经典型苯丙酮尿症、轻度苯丙酮尿症和轻度高苯丙氨酸血症。结果①44例PAH缺乏症患儿PAH基因中均检测到2个突变位点,其中2例为纯合突变,纯合突变的频率为4.6%,所有突变在患儿父母相应突变位点处均能检测到。②44例PAH缺乏症患儿共检测到突变36种,其中c.728G>A突变频率最高(15.9%,14/88),其次是c.1068C>A(10.2%,9/88),再次为c.158G>A(9.1%,8/88)。③21例经典型苯丙酮尿症患儿PAH基因突变19种,其中c.1068C>A突变频率最高(21.4%,9/42),其次是c.728G>A(19.0%,8/42)。10例轻度苯丙酮尿症患儿PAH基因突变14种,其中c.721C>T/722delG突变频率最高(15.0%,3/20),其次为c.1197A>T、c.1301C>A、c.721C>T、c.728G>A(均为10.0%,2/20)。13例轻度高苯丙氨酸血症患儿PAH基因突变17种,其中c.158G>A突变频率最高(26.9%,7/26),其次为c.728G>A(15.4%,4/26)。结论青岛市PAH缺乏症患儿PAH基因突变以复合杂合突变为主,具有明显热点突变(c.728G>A、c.1068C>A、c.158G>A),经典型苯丙酮尿症患儿以c.1068C>A、c.728G>A为主,轻度苯丙酮尿症患儿以c.721C>T/722delG为主,轻度高苯丙氨酸血症患儿以c.158G>A为主。本研究明确了青岛市PAH缺乏症患儿基因的突变类型与特点,为深入开展PAH缺乏症的诊断以及进一步的基因治疗奠定了基础。

Study on mutations of gene in children with phenylalanine hydroxylase deficiency in Qingdao

Objective To explore the mutation characteristics of phenylalanine hydroxylase(PAH)gene in children with PAH deficiency in Qingdao,and provide scientific reference for prenatal diagnosis and treatment.Methods Genetic analysis was performed on 44 children with PAH deficiency detected by neonatal screening in Qingdao.The second generation high-throughput sequencing and multi-ligase probe dependent amplification(MLPA)technique were used for gene analysis to detect the mutation sites in children,and Sanger sequencing was used to detect and verify the corresponding mutation sites of their parents.According to the concentration of phenylalanine in children's blood,PAH deficiency is divided into phenylketonuria(PKU),mild PKU and mild hyperphenylalanine(HPA).Results①There were two mutation sites in each case.Two of the 44 children with PAH deficiency had homozygous mutations in the PAH gene and the frequency of homozygous mutations was 4.6%.All mutations were detected in their parents'corresponding mutation sites.②A total of 36 mutations were found in 44 patients with PAH deficiency.The mutation frequency of c.728G>A was the highest(15.9%,14/88),and that of c.1068C>A and c.158G>A was 10.2%(9/88)and 9.1%(8/88).③There were 19 PAH gene mutations in 21 children with PKU.The mutation frequency of c.1068C>A was the highest(21.4%,9/42),and that of c.728G>A was 19.0%(8/42).There were 14 PAH gene mutations in 10 children with mild PKU.The mutation frequency of c.721C>T/722delG was the highest(15.0%,3/20),and that of c.1197A>T,c.1301C>A,c.721C>T,and c.728G>A was 10.0%(2/20).There were 17 PAH gene mutations in 13 children with mild HPA.The mutation frequency of c.158G>A was the highest(26.9%,7/26),and that of c.728G>A was 15.4%(4/26).Conclusions The mutation of PAH gene in children with PAH deficiency in Qingdao is mainly heterozygous mutation and has obvious hot spot mutations(c.728G>A,c.1068C>A,c.158G>A).Children with PKU were mainly c.1068C>A,c.728G>A,while children with mild PKU were mainly c.721C>T/722delG,and children with mild HPA were mainly c.158G>A.The study of PAH gene mutations lays the foundation for the in-depth development of PAH deficiency genetic diagnosis,prenatal diagnosis and further gene therapy.