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当前位置:首页 > 医疗器械资讯 > 学术论文 > 苯丙酮尿症患者32例家系基因突变研究 1

苯丙酮尿症患者32例家系基因突变研究 1

文章来源:中华实用儿科临床杂志发布日期:2017-01-17浏览次数:228

【摘要】 目的检测苯丙酮尿症( PKU)患儿及其父母苯丙氨酸羟化酶(PAH)基因突变谱,并探讨PAH基 因突变与临床严重程度的相关性,为本地区PKU患儿的早期诊断及遗传咨询提供基础数据。方法使用高通 量测序技术对来自江苏省无锡和宿迁的32例PKU患儿及其父母的PAH基因13个外显子及其附近内含子区域进行测序分析。结果32例PKU共检测出61个突变位点,33种突变基因,突变检出率为95. 31%( 61/64个)。本地区PKU患儿常见致病突变位点为c.721C>T、c.1068C>A、c.611A>G、c.1197A>T、c.728G>A、 c.331C>T和c.442-1G>A,其突变频率均在5%以上。报道1个新的突变基因位点c.699C>G和3个汉族人口中新的突变基因位点c.265C>T、c.722G>A和c.1194A>G。Guldberg AV系统分析显示38. 0010( 8/21例)的PKU患者基因型与实际生化表型相一致,其中预测表型为中重度的与实际生化表型的一致率为92.3%(12/13例),轻度的与实际生化表型的一致率为50.0%(4/8例)。

结论江苏地区PKU患儿的PAH基因突变集中在外显子7上,其中频率高的基因位点是c.721C>T,并发现1个新的基因突变位点c.699C>G; PKU患者基因型与生化表型之间有一定的相关性。

 【关键词】 苯丙酮尿症;苯丙氨酸羟化酶;基因突变
Gene mutations in 32 family with phenylketonuria Chen Yafen* ,Jiang Xinye, Chen, Zhonghai,Jia Haitao, Pei Jingjing,Qiu Yali,Wu Zhijun,Wang Jing. *Department of Children, Health Care, Wuxi Maternal and Child Health HosP pital,Wuxi 214002 ,Jiangsu Province , China Correspon,din,g author : Jiang Xinye,Email :fyjxy2110@163. com
[Abstract] ob[x]jective By detecting the mutations spectrum of phenylalanine hydroxylase( PAH) gene in phenylketonuria( PKU) patients and their parents. The researchers analyzed the gene mutation features and high - frequency mutations and determined the relationship between the genotype and the phenotype,which would provide a theoretical basis for the early diagnosis and genetic consultation of PKU children in the region. Methods In this study,13 exons and their flanking introns of the PAH gene in 32 PKU patients and their parents from Wuxi and Suqian in Jiangsu province were sequenced by using the next - generation sequencing( NGS) technology. Results Sixty -one mutant sites and 32 mutant genes were detected in 32 PKU patients,and the mutation detection rate was 95. 31% (61/64cases). The variants at c. 721C > T,c. 1068C > A,c. 611A > G,c. 1197A > T,c. 728G > A,c. 331C > T and c. 442 1G >A were common mutations in the region with mutation frequency over 5a/o. What's more,4 novel variants of c. 699C > G,c.265C >T,c.722G > A and c.1194A > G were found. Of those,c. 699C > G was not recorded in the PAH variant databa[x]se and HGMD databa[x]se and. c.265C > T,c. 722G > A,and c. 1194A > G were first reported in the Chinese population. Genotype - accurate biochemical phenotype correlation by using the Guldberg AV system revealed consistency rate of 38.00/o ( 8/21 cases) , which the consistency rate between accurate biochemical phenotype and predictive phenotype of moderate to severe genotype was 92. 3% (12/13 cases) ,and mild genotype was 50.0% (4/8 cases).
Conclusions The PAH gene variants of PKU patients in Jiangsu province are distributed mainly in exons 7,of which the highest frequency gene mutation is c.721 c > T. Moreover,one novel variant c. 699C > G was reported for the first time. The PKU children inherit the PAH mutation gene mainly from both parents. There are definite correlation between the genotypes and phenotypes.
[Key words] Phenylketonuria;Phenylalanine hydroxylase;Mutations
苯丙酮尿症(PKU)是一种常见的常染色体隐性遗传代谢病,中国人群中发病率为1/14000[1],由编码肝苯丙氨酸羟化酶(PAH)基因的缺陷引起,主要的临床表现是高苯丙氨酸血症( HPA)和不可逆的进行性的智力障碍,部分患者还合并癫痫。本研究检测了PKU患儿及其父母PAH基因突变谱,分析了本地区PKU患者基因突变类型及高频突变位点,并探讨了PAH基因突 变率与临床严重度的相关性,为本地区PKU的早期诊断及遗传咨询提供了依据。

 1资料与方法

1.1一般资料选择来自无锡市妇幼保健院及宿迁妇
幼保健院确诊的PKU患儿及其父母,共32个家系。男18例,女14例;年龄2—10岁。采集受检者外周血5—10 mL,对其PAH基因突变位点进行测序。患儿诊治符合卫生部《PKU和先天性甲状腺功能减低症诊治技术规
范》[2]及“HPA的诊治共识”[3]标准。患儿监护人均签署知情同意书,所做研究获医院医学伦理委员会批准。

1.2临床分型  根据其治疗前血苯丙氨酸水平分为临床表型,经典型PKU:血苯丙氨酸(PHE)水平≥l200μmol/L,中度PKU:血PHE>600~1200μmol/L,轻度 PKU:血PHE> 360~600μmol/L,轻度HPA:血PHE>120~360 μmol/L[4-5]。

 1.3实验方法
1. 3.1基因组DNA  提取利用全基因组提取试剂盒 ( QIAGEN,USA)提取患儿及其父母外周血的全基因组 DNA。对PAH基因13个外显子及其附近内含子区域进行测序,对应的特异性引物根据文献[6]报道合成。

1. 3.2 PCR反应采用  ABI GeneAmp9700型扩增仪进行PCR反应。将PCR扩增产物制备成多样本混合池 ( DNA Pooling),给产物添加不同接头(Adapter),定量浓度达到文库制备要求后,上机检测。

1. 3.3基因测序  研究中将采用2种检测方法寻找导致PKU发病的PAH基因单核甘酸多态性(SNP)位点:一种为基于MassArray芯片技术的质谱检测,另外一种为采用高通量测序技术的测序检测。对测序结果进行图像识别( ba[x]se calling),通过与参考基因组序列比对,结合国际PAH基因数据库(PAHdb)信息,寻找目标区域的SNPs并注释,汇总得到需要的PAH致病SNP位点、序列长度( Reads)、Reads数量和测序深度等。采用美国Illumina公司的HiSeq2000测序仪进行样本测序。 样品纯化及序列分析由深圳华大基因研究院技术人员完成。

1.4基因型与表型关系分析  参考Guldberg任意值 (AV)分析法预测患儿病情轻重程度[7],将各种突变类 型分组,每组突变设定一个AV:设经典型PKU AV =1,中度PKU AV =2