应用高分辨熔解曲线技术检测经典型苯丙酮尿症患者PAH基因第7外显子基因突变

目的 建立操作简便、快速,使用成本低,结果准确的鉴定PAH基因第7外显子c.728G>A突变热点的基因诊断方法.方法 应用高分辨熔解曲线(high resolution melting,HRM)技术,对山西省临床确诊的88例经典型苯丙酮尿症患儿第7外显子c.728G>A突变热点进行检测,并将检测结果进行测序验证.结果 受检患儿PAH基因第7外显子c.728G>A突变位点HRM技术检测结果和测序结果完全相符.结论 高分辨熔解曲线技术操作简便、快速,使用成本低,结果准确,可作为PAH基因第7外显子热点突变的快速、灵敏检测方法.     

应用DNA PCR法扩增检测苯丙酮尿症基因突变

DNA体外扩增是近年来分子遗传学与分子生物学发展起来的高效DNA分析技术．多聚酶链反应(Polymerase Chain Reaction，PCR)是在一个简单的试管体系中，利用人工合成的与特异突变点两侧DNA序列互补的两寡核苷酸引物，通过DNA变性、退火、引物延冲反应这样数十个循环后，目的区域的DNA可扩增百万倍，从而提高了DNA分析的灵敏度，     

16例苯丙酮尿症PAH基因Exon7突变的检测

应用选择突变扩增系统方法对１６例苯丙酮尿症患儿ＰＡＨ基因Ｅｘｏｎ７区域的多聚酶链反应扩增产物进行了突变位点的检测。结果在３２个突变位点中Ｒ２４３Ｑ和Ｒ２６１Ｑ突变位点的检出率分别为４６．８８％和９．３８％，二者占整个突变位点的５６．２５％。表明ＰＡＨ基因Ｅｘｏｎ７突变在中国苯丙酮尿症患者中具有很高的发生频率。     

经典型苯丙酮尿症基因全长外显子的突变检测和分析

目的探讨中国PKU患者PAH基因突变特征。方法运用PCR-SSCP及PCR-DNA直接测序检测40例经典型PKU患者和30例正常对照的PAH基因。结果在PAH基因上8个外显子共发现11种突变和3种多态,其中R243Q和Y204C为两个高频突变位点,突变率分别为27.5%和10%,280 insT、M276K、M276R、IVS10nt+32T→A、IVS4nt+46C→T、H290R是首次发现的新突变。结论中国PKU患者基因外显子突变是以两个突变热点和罕见突变并存为特征。     

16例经典型苯丙酮尿症的产前基因诊断

应用聚合酶链反应－等位基因特异的寡核苷酸斑点杂交、聚合酶链反应－短串联重复序列对１６例经典型苯丙酮尿症（ＰＫＵ）作家系连锁分析，以及应用聚合酶链反应－单链构型多态性，对其进行产前诊断。其中１０例已获验证，结果与产前诊断相符。     

山西省经典型苯丙酮尿症患者苯丙氨酸羟化酶基因突变研究

目的 探讨山西省经典型苯丙酮尿症(phenylketonuria,PKU)患者苯丙氨酸羟化酶(phenylalanine hydroxylase,PAH)基因第3、6、7、11和12外显子的突变特征.方法 通过测序及序列比对的方法对山西省59例经典型PKU患者和100名正常儿童PAH基因进行序列分析,以确定其突变位点、性质和突变频率.结果 通过序列分析,发现在患儿和正常儿童中均出现Q232Q(CAA→CAG)、V245V(GTG→GTA)和L385L(CTG→CTC)3种单核苷酸多态性位点(single nucleotide polymorphism,SNP),其中患儿cDNA 696位点的SNP发生率高达96.2%,正常儿童的SNP发生率为97.0%;患儿cDNA 735位点的SNP发生率为76.1%,正常儿童的SNP发生率为77.3%;患儿cDNA1155位点的SNP发生率仅为7.6%,正常儿童SNP发生率为8.3%.正常儿童的其它序列与GenBank中序列比较的无差异.在患儿的基因序列中还发现了16种共计72个突变基因,占全部PAH突变基因的61.0%.第3外显子发现3种突变R111X、H64＞TfsX9和S70 del,突变频率分别为5.1%、0.8%、0.8%;第6外显子仅发现1种突变EX6-96A＞G,突变频率达10.2%;第7外显子中R243Q的突变频率最高,占12.7%,其次是Ivs7+2T＞A,占5.1%,T278I占2.5%,G247V、R252Q、L255S、R261Q、E280K均占0.8%;第11外显子中,Y356 X占5.9%,V399V占5.1%;第12外显子中,R413P占5.9%,A434D占2.5%.在16种突变中,有9种错义突变、3种剪接位点突变、2种无义突变及2种缺失,其中,H64＞TfsX9为本次研究新发现.结论 明确了山西省经典型PKU患者PAH基因第3、6、7、11和12外显子的突变种类和分布等特征,EX6-96A＞G、R243Q可能属于山西人群中PAH基因突变的热点.     

16例苯丙酮尿症的PAH基因Exon7突变的检测

应用选择突变扩增系统方法对１６例苯丙酮尿症患儿ＰＡＨ基因Ｅｘｏｎ７区域的多聚酶链反应扩增产物进行了突变位点的检测，结果在３２个突变位点中Ｒ２４３Ｑ和Ｒ２６１Ｑ突变位点的检出率分别为４６．８８％和９．３８％，二者占整个突变位点的５６．２５％，表明ＰＡＨ基因Ｅｘｏｎ７突变在中国苯丙酮尿症患者中具有很高的发生频率。     

经典型苯丙酮尿症苯丙氨酸羟化酶基因的新突变鉴定

目的研究经典型苯丙酮尿症(phenylketonuria, PKU)基因突变.方法应用聚合酶链反应,单链构象多态分析和DNA直接测序等技术,对内蒙古地区32个PKU家系苯丙氨酸羟化酶(phenylalanine hydroxylase, PAH)基因第3～12外显子进行了鉴定分析. 结果检出14种PAH基因点突变R243Q (12/64)、Y356X(6/64)、Y204C(5/64)、R261Q(2/64)、Y161S(2/64)、R252Q(1/64)、R111X(2/64)、D282G(1/64)、S303P(1/64)、G239D(1/64)、R413P(1/64)、IVS7nt+2(2/64)、IVS4nt+3(1/64)、IVS9nt+34(2/64),经检索国际PAH基因突变数据统计库(截至到2004年7月),确认IVS4nt+3(G>C)、IVS9nt+34(G>A)为国际首次发现的新突变,S303P(T>C) 、D282G(A>G)为国内首次报道的新突变.结论内蒙古人群苯丙氨酸羟化酶基因存在突变的多样性,R243Q、Y356X、Y204C是PAH基因的突变热点.     

非经典型苯丙酮尿症的基因突变检测

为探讨非经典型苯丙酮尿症的基因突变特征，应用高效液相层折法技术、反转录－聚合酶链反应和ｃＤＮＡ序列分析等方法，对２例６－丙酮酰－四氢生物喋呤合成酶（６－ｐｙｒｕｖｏｙｌｔｅｔｒａｈｙｄｒｏｂｉｏｐｔｅｒｉｎｓｙｎｔｈａｓｅ，ＰＴＰＳ）缺陷症患儿及其家系进行了基因分析。其结果发现２个基因突变位点，分别为第１５５Ａ→Ｇ突变和第２５９Ｃ→Ｔ突变。为早期诊断ＰＫＵ患儿提供了依据。     

苯丙酮尿症（PKU）研究又进一步

上海交通大学医学院附属新华医院，上海市儿科医学研究所顾学范教授自1981年在国内率先开展新生儿PKU筛查，25年来筛查100多万患儿。对全国来诊的1100多名患儿进行了诊断治疗。基因诊断35例。他们又在国内首次建立了可检测包括PKU在内的34种疾病的串联质谱遗传性代谢病检测技术平台，获得新生儿遗传性代谢病的患病率为1：5263的数据。     

A comprehensive study of phenylalanine hydroxylase gene mutations in the Iranian phenylketonuria patients

Phenylketonuria (PKU) is a metabolic disorder caused by mutations in the phenylalanine hydroxylase (PAH) gene. After thalassemia, PKU is considered as the most common autosomal recessive diseases in the Iranian population. Therefore, an efficient diagnostic strategy is required to identify disease-causing mutations in this population. Following our first report in 2003, here we presented a comprehensive study on the mutation spectrum of the PAH gene in the Iranian population. This study was performed on 280 unrelated chromosomes from 140 Iranian patients with classic PKU. All 13 exons as well as exon-intron boundaries of the PAH gene were analyzed by direct DNA sequencing. Thirty four different mutations were identified by a mutation detection rate of 100%. IVS10-11G > A, p.P281L, R261Q, p.F39del and IVS11+1G > C were the most prevalent mutations with frequencies of 26.07%, 19.3%, 12.86%, 6.07 and 3.93%, respectively. All other mutations represented a relative frequency less than 3.5%. The data from this study provided a comprehensive spectrum of the PAH gene mutations which can facilitate carrier detection and prenatal diagnosis of PKU disease in the Iranian population.     

Ten novel mutations in the phenylalanine hydroxylase gene (PAH) observed in Brazilian patients with phenylketonuria

In the present study we report on the identification of ten novel mutations in the phenylalanine hydroxylase (PAH) gene of Brazilian patients with phenylketonuria (PKU): IVS5-54A>G, IVS6+17G>T, E205A, F240S, K274E, I318T, L321L, C357G, IVS11+17G>A and S411X. These mutations were detected during the characterization of the PAH genotypes of 115 patients with PKU from the southeast region of Brazil. The results obtained confirm the high heterogeneity of the PAH gene and provide information about the distribution of PKU mutations in the Brazilian population.     

Mutations of the phenylalanine hydroxylase (PAH) gene in Brazilian patients with phenylketonuria

In the present study, 115 Brazilian families with phenylketonuria (PKU), mainly from the Southeast of the country, were studied using three laboratory methods (DGGE, SSCP, and sequencing). All 13 exons of the PAH gene were analyzed, including the splicing sites and the promoter region. We identified 50 distinct mutations and characterized 91% of the mutant alleles. The five most prevalent mutations of the 50 mutations identified (50% of the PKU alleles) were IVS10nt-11G-->A (17.4%), followed by R261Q (12.2%), V388M (9.1%), R252W (6.5%), and R270K (4.8%). The other mutations were rare. The mutation spectrum included 10 novel mutations (IVS5nt-54A-->G, IVS6nt17G-->T, E205A, F240S, K274E, I318T, L321L, C357G, IVS11nt17G-->A, and S411X). To characterize the origin and distribution of the PAH alleles we determined the association between the detected mutations and the PCR/RFLP haplotypes and VNTR alleles located on the PAH gene. For those patients whose mutant alleles were detected, we calculated the correlation with pretreatment phenylalanine levels, thus establishing a genotype/phenotype correlation. The present results confirm the marked heterogeneity observed at the PAH locus and contribute to the understanding of the distribution and frequency of PKU mutations in the Brazilian population.     

云南经典PKU基因外显子4,10和12突变的检测

目的：探讨云南经典ＰＫＵ的基因突变特征。方法：应用ＰＣＲˉＳＣＰ和ＰＣＲ－循环测序技术对云南１３个ＰＫＵ家系１４名患儿的ＰＡＨ基因外显子４、１０和１２进行了检测。结果：Ｒ４１３Ｐ、Ｗ３２６Ｘ的突变频率分别是７１４％、３５７％；Ａ／Ｃ杂合频率是１０７１％，同时检测到外显子４的３种异常带型。结论：云南ＰＫＵ患者的基因突变不同于北方人群，Ｐ４１３Ｐ、Ａ／Ｃ杂合率约为北方人群突变的１／２；Ｗ３２６Ｘ则高于北方人群；外显子４的突变也可能高于北方人群。     

Splicing of phenylalanine hydroxylase (PAH) exon 11 is vulnerable: molecular pathology of mutations in PAH exon 11

In about 20-30% of phenylketonuria (PKU) patients, phenylalanine (Phe) levels can be controlled by cofactor 6R-tetrahydrobiopterin (BH(4)) administration. The phenylalanine hydroxylase (PAH) genotype has a predictive value concerning BH(4)-response and therefore a correct assessment of the mutation molecular pathology is important. Mutations that disturb the splicing of exons (e.g. interplay between splice site strength and regulatory sequences like exon splicing enhancers (ESEs)/exon splicing silencers (ESSs)) may cause different severity of PKU. In this study, we identified PAH exon 11 as a vulnerable exon and used patient derived lymphoblast cell lines and PAH minigenes to study the molecular defect that impacted pre-mRNA processing. We showed that the c.1144T>C and c.1066-3C>T mutations cause exon 11 skipping, while the c.1139C>T mutation is neutral or slightly beneficial. The c.1144T>C mutation resides in a putative splicing enhancer motif and binding by splicing factors SF2/ASF, SRp20 and SRp40 is disturbed. Additional mutations in potential splicing factor binding sites contributed to elucidate the pathogenesis of mutations in PAH exon 11. We suggest that PAH exon 11 is vulnerable due to a weak 3' splice site and that this makes exon 11 inclusion dependent on an ESE spanning position c.1144. Importantly, this implies that other mutations in exon 11 may affect splicing, since splicing is often determined by a fine balance between several positive and negative splicing regulatory elements distributed throughout the exon. Finally, we identified a pseudoexon in intron 11, which would have pathogenic consequences if activated by mutations or improved splicing conditions. Exonic mutations that disrupt splicing are unlikely to facilitate response to BH(4) and may lead to inconsistent genotype-phenotype correlations. Therefore, recognizing such mutations enhances our ability to predict the BH(4)-response.     

127例PKU患者PAH基因第12外显子点突变及其频率研究

目的　了解中国人苯丙酮尿症 ( phenylketonuria,PKU)患者的苯丙氨酸羟化酶( phenylalanine hydroxylase,PAH)基因第 12外显子点突变种类和频率。方法　应用单链构象多态性( single strand conformation polymorphism,SSCP)、变性梯度凝胶电泳 ( denaturing gradient gelelectrophoresis,DGGE)、DNA测序分析了 12 7例 PKU患者的 PAH基因第 12外显子点突变种类及频率。结果　 DNA测序分析显示 10例患者存在 R4 13P、S4 11X、R4 0 8W、R4 0 8Q 4种杂合突变 ,其突变频率分别为 2 .76 %、0 .39%、0 .39%、0 .39% ,S4 11X突变为中国人中首次报道。 SSCP分析仅发现 2例 R4 13P杂合突变 ,DGGE分析显示 10例出现 3种类型的异常电泳带型。R4 13P突变在南北方人之间、在经典型 PKU和高苯丙氨酸血症之间的分布差异无显著性。结论　 DGGE对 PAH基因第 12外显子点突变检出率明显高于 SSCP。 DGGE结合 DNA测序是明确 PAH基因第 12外显子点突变种类和频率较好的方法。 R4 13P突变在南北方人中分布无明显差异