二代测序技术在苯丙酮尿症分子诊断的应用研究

发布时间：2018-04-16 13:32

本文选题：苯丙酮尿症 + 苯丙氨酸羟化酶　；参考：《石河子大学》2017年硕士论文

【摘要】：目的:(1)利用二代测序技术对新疆地区64名苯丙酮尿症(PKU)患者的PAH、GCH1、PTS、QDPR、PCBD1五种基因进行突变检测,建立PKU突变基因高效准确快速的检测技术,评估二代测序技术在苯丙酮尿症基因诊断中的应用价值,为苯丙酮尿症的基因诊断和产前诊断提供技术支撑。(2)对新疆地区PKU患者进行基因突变分析,掌握其基因的突变类型和分布频率,确立本地区的PKU基因突变常见类型。方法:针对PAH、GCH1、PTS、QDPR、PCBD1五个基因设计引物,多重PCR扩增获取目标序列,产物纯化后加入标签序列富集产物,评估文库质量后使用Next Seq500 Desktop Sequencer二代测序平台双向测序验证,并以Sanger测序技术验证检测结果。结果:成功建立高效、快速和方便的目标区域二代测序分析体系;检测64例PKU样本,在128个PAH等位基因中检测出124个突变基因,共55种突变类型,其中错义突变41种,无义突变6种,剪切位点突变7种,缺失突变1种,以上结果均经Sanger测序验证;Guldberg AV系统分析显示59.26%(16/27例)的PKU患者基因型与实际生化表型相一致,其中预测表型为经典型PKU与实际生化表型的一致率为60%(3/5例),预测表型为轻度高苯丙氨酸血症与实际生化表型的一致率为57.14%(12/21例)。结论:(1)利用二代测序技术对大样品量的候选基因目的区域进行富集测序和变异位点筛查,建立了高效、快速和方便的目标区域二代测序分析体系,实现在单一反应管中完成对PAH、GCH1、PTS、QDPR、PCBD1五个基因全部36个外显子及其旁侧内含子区域基因突变分析;(2)对新疆地区64例PKU患者PAH、GCH1、PTS、QDPR、PCBD1五个基因进行检测,经过严格过滤、筛选、定位,在128个等位基因中共检测出124个PAH突变基因,突变检出率为96.88%(124/128),确定了三种新发突变:c.673CT、c.1316-1GA、c.1200-2AC;(3)通过与国内外其他地区PAH突变类型与频率的比较分析,显示新疆地区的遗传基因不仅具有独立、保守的特性,而且还存在着相互交叉、相互融合的特征,是研究基因突变多样性、PAH基因异质性以及人类起源、迁徙等的遗传资源宝库,同时也是对我国PKU研究中民族和地区资料的重要补充,为在本地区制定有针对性的PKU防治战略提供了科学依据。
[Abstract]:Objective: to detect the mutations of five genes of PKU gene in 64 patients with phenylketonuria (PKU) in Xinjiang by using the second generation sequencing technique, and to establish an efficient, accurate and rapid method for the detection of PKU mutation gene.To evaluate the application value of second generation sequencing technique in gene diagnosis of phenylketonuria, and to provide technical support for gene diagnosis and prenatal diagnosis of phenylketonuria.To know the mutation type and distribution frequency of its gene and to establish the common type of PKU gene mutation in our region.Methods: five primers were designed for the five genes of PAHN GCH1 PCR, the target sequences were amplified by multiple PCR amplification, the products were purified and the products were enriched with tagged sequences. The quality of the library was evaluated and verified by bidirectional sequencing using the Next Seq500 Desktop Sequencer second-generation sequencing platform.The results were verified by Sanger sequencing technique.Results: a highly efficient, rapid and convenient second generation sequencing analysis system for target region was successfully established, 64 PKU samples were detected, 124 mutation genes were detected in 128 PAH alleles, 55 mutation types were detected, including 41 missense mutations.There were 6 nonsense mutations, 7 split-site mutations and 1 deletion mutation. All of the above results were confirmed by Sanger sequencing. The results showed that the genotype of 59.26g / 16 / 27 patients with PKU was consistent with the actual biochemical phenotype.The coincidence rate between the predicted phenotypes of classical PKU and actual biochemical phenotypes was 60 / 3 / 5 and that of mild hyperphenylalaninemia and actual biochemical phenotypes was 57.14 / 12 / 21 respectively.ConclusionThe second generation sequencing technique was used to analyze the target regions of candidate genes in large amount of samples, and to establish an efficient, rapid and convenient analysis system for second generation sequencing of target regions.The mutation analysis of all 36 exons and their adjacent intron region of the five genes of PAHN GCH1 PTSPR1 PTSPR1 PCBD1 in a single reaction tube was carried out in 64 patients with PKU in Xinjiang. The five genes of PTDPR1 QDPR1 were screened, screened and located.A total of 124 PAH mutation genes were detected in 128 alleles. The detection rate of PAH mutation was 96.8884 / 1280.Three new mutations were identified as follows: 1: c.673CTU c.1316-1GAX c.1200-2AC3) by comparing with other regions at home and abroad, the type and frequency of PAH mutations were analyzed.The results show that the genetic genes in Xinjiang are not only independent and conserved, but also intersected and fused. It is a treasure house of genetic resources to study the heterogeneity of PAH gene and the origin and migration of human beings.At the same time, it is also an important supplement to the national and regional data in the study of PKU in China, which provides a scientific basis for the establishment of targeted PKU prevention and control strategy in our region.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R589;R440

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