本文選題：苯丙酮尿癥 + 苯丙氨酸羥化酶��； 參考：《石河子大學(xué)》2017年碩士論文

【摘要】：目的:(1)利用二代測序技術(shù)對新疆地區(qū)64名苯丙酮尿癥(PKU)患者的PAH、GCH1、PTS、QDPR、PCBD1五種基因進(jìn)行突變檢測,建立PKU突變基因高效準(zhǔn)確快速的檢測技術(shù),評估二代測序技術(shù)在苯丙酮尿癥基因診斷中的應(yīng)用價值,為苯丙酮尿癥的基因診斷和產(chǎn)前診斷提供技術(shù)支撐。(2)對新疆地區(qū)PKU患者進(jìn)行基因突變分析,掌握其基因的突變類型和分布頻率,確立本地區(qū)的PKU基因突變常見類型。方法:針對PAH、GCH1、PTS、QDPR、PCBD1五個基因設(shè)計引物,多重PCR擴(kuò)增獲取目標(biāo)序列,產(chǎn)物純化后加入標(biāo)簽序列富集產(chǎn)物,評估文庫質(zhì)量后使用Next Seq500 Desktop Sequencer二代測序平臺雙向測序驗證,并以Sanger測序技術(shù)驗證檢測結(jié)果。結(jié)果:成功建立高效、快速和方便的目標(biāo)區(qū)域二代測序分析體系;檢測64例PKU樣本,在128個PAH等位基因中檢測出124個突變基因,共55種突變類型,其中錯義突變41種,無義突變6種,剪切位點突變7種,缺失突變1種,以上結(jié)果均經(jīng)Sanger測序驗證;Guldberg AV系統(tǒng)分析顯示59.26%(16/27例)的PKU患者基因型與實際生化表型相一致,其中預(yù)測表型為經(jīng)典型PKU與實際生化表型的一致率為60%(3/5例),預(yù)測表型為輕度高苯丙氨酸血癥與實際生化表型的一致率為57.14%(12/21例)。結(jié)論:(1)利用二代測序技術(shù)對大樣品量的候選基因目的區(qū)域進(jìn)行富集測序和變異位點篩查,建立了高效、快速和方便的目標(biāo)區(qū)域二代測序分析體系,實現(xiàn)在單一反應(yīng)管中完成對PAH、GCH1、PTS、QDPR、PCBD1五個基因全部36個外顯子及其旁側(cè)內(nèi)含子區(qū)域基因突變分析;(2)對新疆地區(qū)64例PKU患者PAH、GCH1、PTS、QDPR、PCBD1五個基因進(jìn)行檢測,經(jīng)過嚴(yán)格過濾、篩選、定位,在128個等位基因中共檢測出124個PAH突變基因,突變檢出率為96.88%(124/128),確定了三種新發(fā)突變:c.673CT、c.1316-1GA、c.1200-2AC;(3)通過與國內(nèi)外其他地區(qū)PAH突變類型與頻率的比較分析,顯示新疆地區(qū)的遺傳基因不僅具有獨立、保守的特性,而且還存在著相互交叉、相互融合的特征,是研究基因突變多樣性、PAH基因異質(zhì)性以及人類起源、遷徙等的遺傳資源寶庫,同時也是對我國PKU研究中民族和地區(qū)資料的重要補充,為在本地區(qū)制定有針對性的PKU防治戰(zhàn)略提供了科學(xué)依據(jù)。
[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.
【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R589;R440

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