本文選題：PKU + PAH�。� 參考：《山西醫(yī)科大學(xué)》2012年碩士論文

【摘要】：背景 苯丙酮尿癥是可治療的少數(shù)遺傳病之一,我國PKU的發(fā)病率為1/11572,而我省2004年—2009年的新生兒的發(fā)病率為1/3425,遠(yuǎn)遠(yuǎn)高于全國的平均水平,早期診斷是決定苯丙酮尿癥預(yù)后的關(guān)鍵。目前國內(nèi)外對(duì)苯丙酮尿癥患兒的診斷方法耗時(shí)長且繁瑣,而且僅適用于已經(jīng)出生的嬰兒,對(duì)產(chǎn)前診斷無能為力。尤其對(duì)已生育過苯丙酮尿癥患兒的家庭,再次懷孕生育苯丙酮尿癥患兒的概率為25%,盲目生育導(dǎo)致悲劇發(fā)生的可能性極大。因此,通過對(duì)山西地區(qū)苯丙酮尿癥患者中苯丙氨酸羥化酶基因的熱點(diǎn)突變位點(diǎn)及分布特征、基因突變的發(fā)生頻率的研究,建立山西地區(qū)苯丙酮尿癥苯丙氨酸羥化酶基因的基因資料庫,為山西地區(qū)苯丙酮尿癥患者產(chǎn)前診斷、治療、遺傳咨詢、優(yōu)生優(yōu)育奠定基礎(chǔ)有重要意義。 目的 研究山西省苯丙酮尿癥患兒PAH基因的突變,并制定出適合我省PKU患者孕前、產(chǎn)前診斷的最佳診斷篩查方案,為我省PAH基因突變提供有效的分子圖譜數(shù)據(jù),并為研究突變發(fā)生的分子機(jī)制,基因診斷、產(chǎn)前診斷、新藥開發(fā)提供重要的參考。 方法 1、把PAH基因3、6、7、11、12外顯子同時(shí)進(jìn)行體外擴(kuò)增和測(cè)序,然后與正常PAH基因序列進(jìn)行比對(duì),通過統(tǒng)計(jì)學(xué)處理,從而得到我省的PKU患者PAH基因的全部突變位點(diǎn)信息； 2、收集山西省各地區(qū)2007年5月-2011年5月經(jīng)臨床確診的經(jīng)典型PKU患兒97例,正常兒童158例,采用ARMS(等位基因特異性擴(kuò)增)方法檢測(cè)經(jīng)典型苯丙酮尿癥患者常見的熱點(diǎn)突變位點(diǎn)(c.611AG)； 3、首次采用HRM(高分辨熔解曲線)方法檢測(cè)經(jīng)典型苯丙酮尿癥患者常見的熱點(diǎn)突變位點(diǎn)(c.728GA)。 結(jié)果 1、在59例患者和100名正常兒童的PAH基因第3、6、7、11、12外顯子序列研究中發(fā)現(xiàn),在患兒和正常兒童中均出現(xiàn)了3種同義突變,還發(fā)現(xiàn)4種突變類型,16個(gè)突變位點(diǎn),其中H64TfsX9為本次研究新發(fā)現(xiàn),屬首次報(bào)道,并得出Y204C和R243Q屬于山西地區(qū)的突變熱點(diǎn)； 2、采用ARMS擴(kuò)增,在70例PKU患兒中檢測(cè)出10例存在PAH第6外顯子熱點(diǎn)突變c.611AG,并對(duì)其患兒父母進(jìn)行檢測(cè),經(jīng)測(cè)序驗(yàn)證,ARMS檢測(cè)結(jié)果和測(cè)序結(jié)果完全相符； 3、根據(jù)PCR-HRM方法測(cè)定,在88例PKU患兒中檢測(cè)出20例PAH基因第7外顯子熱點(diǎn)突變c.728GA,其中野生型68例、純合突變2例和雜合突變18例。經(jīng)測(cè)序驗(yàn)證,PCR-HRM產(chǎn)物的測(cè)序結(jié)果與第7外顯子PCR產(chǎn)物測(cè)序結(jié)果完全相同。 結(jié)論 1、山西地區(qū)PKU患兒PAH基因的突變研究,為我省PKU患兒PAH基因突變提供有效的分子圖譜數(shù)據(jù),并為豐富我國PKU資料庫做出貢獻(xiàn)； 2、ARMS方法具有重復(fù)性好、特異性和準(zhǔn)確率均較高的特點(diǎn),為開展PAH基因的研究提供了簡(jiǎn)便、快速、準(zhǔn)確的基因分析方法； 3、HRM方法分析能夠有效區(qū)分不同SNP位點(diǎn)與不同基因型。為開展PAH基因的研究提供了簡(jiǎn)便、快速、準(zhǔn)確的基因分析方法,也是一種適合臨床PKU篩查和治療檢測(cè)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.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R725.8

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