云南各民族PAH基因突变谱及中国人群PAH基因新突变体外表达功能鉴定研究

Phenylketonuria (PKU) is a disease caused by mutation of phenylalanine hydroxylase (PAH) gene. Previous studies proved the wide variability in the common mutations between ethnic groups and geographical areas, and the PAH genotypes is correlation with variable metabolic phenotypes. Yunnan is a province of China whose ethnic groups takes 38% of its whole population, but mutation spectrum of PAH gene of Yunnan people is kept unknown by now. We want to continue our previous study on PAH gene mutation, by collecting an enlarged sample size to more than 100 cases of PKU patients and to employ multiples ligation-dependent probe amplification (MLPA) as an additional method to traditional sequencing of 13 exons of PAH gene, aiming to detect large deletion and duplication, and finally to get a better mutation spectrum of PAH gene of the Yunnan population. PKU is a genetic disease with great allelic heterogeneity, but numerous novel mutations in Chinese people have been reported without the functional analysis. There are no more than four documents about the genotype-phenotype analysis of and only of missence of PAH gene founded in Chinese PKU patients, with most of novel mutations, including missence, splicing mutation, and deletion /insertion, still untested. We want to build in vitro expression systems of novel missense, splicing mutation, and deletion /insertion previously reported in Yunnan people, and then also other mutations founded in Chinese people in other provinces, and to explore the genotype-phenotype correlation through comparison of protein levels and residual enzyme activities．We will clone the PAH cDNA into an expression plasmid, then mutation was introduced into the PAH sequence by gene modification technology. The expression plasmids were individually transfected into COS-1 cells by the calcium phosphate method. The quantities of proteins and residual enzyme activities of the mutants were assessed with the wild-type PAH gene as reference. This research may help us to understand the mechanism of mutants on PAH gene that causing PKU.

苯丙氨酸羟化酶(PAH)基因突变导致苯丙酮尿症，突变型呈现地域和人种特异性，不同缺陷导致的临床表型差异较大。云南人口38%为少数民族，但PAH基因突变谱未知，本课题拟扩大前期研究样本至100例以上，采用基因测序和MLPA技术检测PAH基因突变、缺失和重复，获取云南各民族PAH基因突变谱数据。另外，关于PAH基因突变表型研究，国内仅有少量PAH基因错义突变的表达研究报道，剪接突变、插入/缺失突变理论推断会导致酶活性的缺失，但体外表达研究较少。近年来国内研究发现了大量中国人PAH基因突变新类型，但许多都未经基因表达实验验证，这些数据应用临床可能带来医疗隐患。我们拟构建PAH-cDNA的SV40质粒载体，并通过突变诱导技术构建PAH基因突变体，建立错义突变、剪接突变、插入/缺失突变的基因突变体真核表达体系，结合生物信息学技术，对尚未证实的PAH基因新突变进行表型研究，揭示PAH基因突变致病机制。

苯丙氨酸羟化酶(PAH)基因缺陷导致苯丙酮尿症，不同缺陷导致的临床表型差异较大，且呈现地域和人种特异性。.本课题收集了云南地区195例PKU，民族覆盖汉族、回族、彝族、白族、纳西族、佤族、普米族、傈僳族等，完成了PAH基因检测，第一次获得了较大样本的云南人群PAH基因突变谱，发现云南人群中PAH基因突变75种，频率最高的分别为c.728G>A（p.R243Q）、c.331C>T（p.R111X）、c.1068C>A（p.Y356X）、c.1238G>C（p.R413P）和c.442-1G>A（IVS4-1G>A）。其主要特点有：云南地区PAH基因突变总体与中国北方人群相似；但云南人群，特别是云南少数民族人群具有一些特定的PAH基因突变类型，比如c.[59A>C;60G>C] (p.Q20P)，该基因突变类型仅仅在云南大理的2例患者中检出，该突变为云南人群的特有突变之一。其次，本团队的前期研究发现了云南人群中存在的c.[59A>C;60G>C] (Q20P), c.690-691insG (S231fxX51)和c.1119-1120insT (I374fxX20)三种突变为新的致病突变类型。本课题通过CRISPR/Cas9基因编辑的方法构建了3种苯丙氨酸羟化酶基因突变的细胞模型，并以突变细胞模型为材料鉴定了PAH 基因突变后的表型变化，首次检测了三种新突变的残留酶活性，分别为53.7%、22.8%和20.3%，该细胞模型的检测结果和临床病例观察的结果一致，结合了生物信息学技术，对未证实的PAH基因新突变进行表型研究，揭示PAH基因突变致病机制。另外，为27例云南地区苯丙酮尿症家庭提供了产前基因诊断服务，未出现误诊漏诊等情况。另外，本项目创新性地采用TaqMan SNP基因分型分析技术，设计了一套PAH基因连锁的特异性TaqMan MGB探针，可以用于PKU家系产前基因诊断中的连锁分析，丰富了PAH产前诊断技术体系。