山西省经典型苯丙酮尿症患者PAH基因的突变研究

发布时间：2018-06-25 14:37

本文选题：PKU + PAH　；参考：《山西医科大学》2012年硕士论文

【摘要】：背景苯丙酮尿症是可治疗的少数遗传病之一,我国PKU的发病率为1/11572,而我省2004年—2009年的新生儿的发病率为1/3425,远远高于全国的平均水平,早期诊断是决定苯丙酮尿症预后的关键。目前国内外对苯丙酮尿症患儿的诊断方法耗时长且繁琐,而且仅适用于已经出生的婴儿,对产前诊断无能为力。尤其对已生育过苯丙酮尿症患儿的家庭,再次怀孕生育苯丙酮尿症患儿的概率为25%,盲目生育导致悲剧发生的可能性极大。因此,通过对山西地区苯丙酮尿症患者中苯丙氨酸羟化酶基因的热点突变位点及分布特征、基因突变的发生频率的研究,建立山西地区苯丙酮尿症苯丙氨酸羟化酶基因的基因资料库,为山西地区苯丙酮尿症患者产前诊断、治疗、遗传咨询、优生优育奠定基础有重要意义。目的研究山西省苯丙酮尿症患儿PAH基因的突变,并制定出适合我省PKU患者孕前、产前诊断的最佳诊断筛查方案,为我省PAH基因突变提供有效的分子图谱数据,并为研究突变发生的分子机制,基因诊断、产前诊断、新药开发提供重要的参考。方法 1、把PAH基因3、6、7、11、12外显子同时进行体外扩增和测序,然后与正常PAH基因序列进行比对,通过统计学处理,从而得到我省的PKU患者PAH基因的全部突变位点信息； 2、收集山西省各地区2007年5月-2011年5月经临床确诊的经典型PKU患儿97例,正常儿童158例,采用ARMS(等位基因特异性扩增)方法检测经典型苯丙酮尿症患者常见的热点突变位点(c.611AG)； 3、首次采用HRM(高分辨熔解曲线)方法检测经典型苯丙酮尿症患者常见的热点突变位点(c.728GA)。结果 1、在59例患者和100名正常儿童的PAH基因第3、6、7、11、12外显子序列研究中发现,在患儿和正常儿童中均出现了3种同义突变,还发现4种突变类型,16个突变位点,其中H64TfsX9为本次研究新发现,属首次报道,并得出Y204C和R243Q属于山西地区的突变热点； 2、采用ARMS扩增,在70例PKU患儿中检测出10例存在PAH第6外显子热点突变c.611AG,并对其患儿父母进行检测,经测序验证,ARMS检测结果和测序结果完全相符； 3、根据PCR-HRM方法测定,在88例PKU患儿中检测出20例PAH基因第7外显子热点突变c.728GA,其中野生型68例、纯合突变2例和杂合突变18例。经测序验证,PCR-HRM产物的测序结果与第7外显子PCR产物测序结果完全相同。结论 1、山西地区PKU患儿PAH基因的突变研究,为我省PKU患儿PAH基因突变提供有效的分子图谱数据,并为丰富我国PKU资料库做出贡献； 2、ARMS方法具有重复性好、特异性和准确率均较高的特点,为开展PAH基因的研究提供了简便、快速、准确的基因分析方法； 3、HRM方法分析能够有效区分不同SNP位点与不同基因型。为开展PAH基因的研究提供了简便、快速、准确的基因分析方法,也是一种适合临床PKU筛查和治疗检测的好方法。
[Abstract]:Background phenylketonuria is one of the few treatable hereditary diseases. The incidence of PKU in China is 1 / 11572, while the incidence rate of newborns in our province from 2004 to 2009 is 1 / 3425, which is much higher than the national average. Early diagnosis is the key to the prognosis of phenylketonuria. At present, the diagnostic methods of phenylketonuria in China and abroad are time-consuming and tedious, and can only be applied to infants who have already been born, and can not be used for prenatal diagnosis. Especially for families who have already given birth to children with phenylketonuria, the probability of having children with phenylketonuria again is 25. The possibility of blind birth leading to tragedy is very great. Therefore, the hot spot and distribution of phenylalanine hydroxylase gene and the frequency of phenylalanine hydroxylase gene mutation were studied in patients with phenylketonuria in Shanxi area. To establish the gene database of phenylalanine hydroxylase gene in phenylketonuria in Shanxi province is of great significance for the prenatal diagnosis, treatment, genetic counseling and eugenics of phenylketonuria patients in Shanxi area. Objective to study the mutation of PAH gene in children with phenylketonuria in Shanxi province, and to establish the best screening program for prenatal diagnosis of PKU patients before pregnancy, and to provide effective molecular map data for the mutation of PAH gene in Shanxi province. It provides important reference for studying molecular mechanism of mutation, gene diagnosis, prenatal diagnosis and new drug development. Methods 1. The exons of PAH gene were amplified and sequenced simultaneously in vitro, and then compared with the normal PAH gene sequence. The total mutation site information of PAH gene in PKU patients in our province was obtained by statistical analysis. 2. From May 2007 to May 2011, 97 cases of classical PKU and 158 cases of normal children were collected. Allele specific amplification (ARMS) was used to detect common hot spot mutation sites (c. 611 AG) in patients with classical phenylketonuria. 3. HRM (high resolution melting curve) was used to detect the common hot spot mutation sites (c. 728GA) in patients with classical phenylketonuria for the first time. Results 1. In 59 patients and 100 normal children, 3 synonymous mutations, 4 mutation types and 16 mutation sites were found in exon 3 of PAH gene. H64TfsX9 is the new discovery of this study, which belongs to the first report, and concludes that Y204C and R243Q belong to the hot spots of mutation in Shanxi region. Ten out of 70 children with PKU were found to have a hot spot mutation c.611AGin exon 6 of PAH, and their parents were detected. The results of ARMS and sequencing were confirmed by sequencing. 3, the results were determined by PCR-HRM. In 88 children with PKU, 20 cases of hot spot mutation in exon 7 of PAH gene c.728GA were detected, including 68 cases of wild type, 2 cases of homozygous mutation and 18 cases of heterozygous mutation. The sequencing results of PCR-HRM products were identical to those of exon 7 PCR products. Conclusion 1. The study on the mutation of PAH gene in children with PKU in Shanxi province provides effective molecular map data for the mutation of PAH gene in children with PKU and contributes to enrich the PKU database in China, 2ARMS method has good reproducibility. The specificity and accuracy are high, which provides a simple, rapid and accurate gene analysis method for the study of PAH gene, and can effectively distinguish different SNP sites from different genotypes by 3H HRM method. It provides a simple, rapid and accurate gene analysis method for the study of PAH gene. It is also a good method for clinical PKU screening and therapy.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R725.8

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