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.     

甘肃地区苯丙酮尿症患者苯丙氨酸羟化酶基因突变分析

.2%),IVS4-1G>A(5.2%),较常见的突变有Y356X(3.4%),R111X(3.4%),INS7+2T>A(3.4%).结论 甘肃地区PAH基因突变与中国其他地区相似,但部分突变基因比例存在明显的差异.     

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。     

中国北方地区苯丙氨酸羟化酶基因的突变构成

目的了解中国人苯丙氨酸羟化酶（phenylalanine hydroxylase,PAH）基因的突变构成。方法应用PCR单链构象多态结合序列分析检测230例苯丙酮尿症患儿PAH基因全部外显子及其两侧内含子。结果（1）在460个PAH等位基因中检测出75种不同的突变基因,总检测率达94.6%（435/460）,其中3种突变基因（S251-R252〉SfsX89、Y387D和A389G）尚未见到报道。常见的突变基因为：R243Q（21.7%）、EX6-96A〉G（10.2%）、R111X（8.3%）、R413P（6.5%）和Y356X（6.1%）。较常见的突变基因为：V399V（4.1%）、IVS4-1G〉A（3.5%）、IVS7＋2T〉A（2.2%）和R241C（2.2%）。大部分突变集中在第3、5、6、7、11和12外显子及其两侧内含子区域。（2）检测出10种多态性位点,突变率高的4个位点IVS3-22C〉T（56.7%）、IVS10＋97G〉A（75.9%）、Q232Q（89.0%）和V245V（81.9%）,提示PAH基因cDNA序列存在人种差异。结论中国人PAH基因的突变构成与欧洲人群完全不同,与亚洲其它人群有频率的差异。     

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。     

新疆回族中苯丙酮尿症基因的突变鉴定

目的探讨新疆回族中苯丙氨酸羟化酶(PAH)基因的突变特点。方法采用PCR产物直接测序的方法,测定7例回族苯丙酮尿症(PKU)患者PAH基因全部外显子及其启动子区域序列。结果在14个回族PAH等位基因中,共检出突变基因10个,包括错义突变5种、无义突变1种、剪切位点突变1种、缺失突变1种,其中的R243Q和R176X为种纯合子突变,这些突变分布于六个外显子,均系在回族中首次发现。检出的突变基因中包含2种新的突变基因类型,即R155H和A300S基因突变,通过查阅国内外文献、查询PAH专业网站及www.pahdb.mcgill.ca国际PAH数据库,证实是国内未见报道的新突变。结论新疆回族PKU患者中PAH基因突变具有明显的异质性特点,我们的研究丰富了PAH基因突变研究的内容,是我国PKU研究中民族资料的重要补充。     

云南苯丙氨酸羟化酶基因点突变及外显子5内新突变Y166X(C→G)的鉴定

目的研究云南苯丙氨酸羟化酶基因点突变分布概况,以提高该地区苯丙酮尿症（ＰＫＵ）的基因诊断率。方法应用ＰＣＲ－ＡＳＯ探针斑点杂交,ＰＣＲ－ＳＳＣＰ、ＡＳＰＣＲ和ＤＮＡ直接测序等技术,对１３／１４名云南籍ＰＫＵ患儿的苯丙氨酸羟化酶（ＰＡＨ）基因外显子４、５、６、７、１０、１１和１２进行了鉴定分析。结果共检出５种错义突变：Ｒ２４３Ｑ（５／２６）、Ｙ２０４Ｃ（３／２８）、Ｒ４１３Ｐ（２／２８）、Ｔ４１８Ｐ（１／２８）及Ｇ２４７Ｖ（１／２６）,３种无义突变Ｙ１６６Ｘ（Ｃ→Ｇ）（２／２６）、Ｗ３２６Ｘ（１／２８）及Ｙ３５６Ｘ（２／２６）和１种静止突变Ｖ３９９Ｖ（２／２６）。经检索国际ＰＡＨ基因突变数据库,其中Ｙ１６６Ｘ（Ｃ→Ｇ）为首次发现的新突变。结论云南藉ＰＫＵ与中国北方人群有相类似的最常见的ＰＡＨ基因突变类型（Ｒ２４３Ｑ、Ｙ２０４Ｃ、Ｙ３５６Ｘ、Ｖ３９９Ｖ和Ｒ４１３Ｐ）,但与南方人群ＰＡＨ基因突变特点则不同。该发现有助于提高云南ＰＫＵ的基因诊断率,并对我国ＰＡＨ基因突变不同地区及人群的分布、起源研究有参考价值。     

高分辨率熔解曲线分析技术检测苯丙酮尿症患者的苯丙氨酸羟化酶基因突变

目的 应用高分辨率熔解曲线（HRM）分析技术检测苯丙酮尿症患者苯丙氨酸羟化酶基因突变.方法 利用PCR扩增13例苯丙酮尿症（PKU）患者的苯丙氨酸羟化酶（PAH）基因第6、7、11及12外显子,然后对PCR产物进行HRM分析,通过DNA测序对HRM结果进行验证.结果 在13例PKU患者的26个PAH等位基因中共检测出5种不同突变基因,总检出率为38.5％.常见的突变类型是R243Q和A434D.检测出两种多态性位点为Q232Q和V245V.HRM分析结果与测序结果完全一致.结论 HRM技术具有简单、快速、易操作、准确等优点,可用于PKU患者PAH基因突变筛查.     

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.     

四氢生物蝶呤反应性的苯丙酮尿症研究进展

本文综述对四氢生物蝶呤（BH4）反应性的苯丙氨酸羟化酶（PAH）基因缺陷的苯丙酮尿症（PKU）的研究进展。分析BH4反应性的PAH基因突变，探讨这一现象潜在的发生机制，介绍一种新的临床分类法，并对PKU的BH4替代治疗前景进行展望。     

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.     

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.     

Silent mutations in the phenylalanine hydroxylase gene as an aid to the diagnosis of phenylketonuria.

Direct sequencing of the phenylalanine hydroxylase (PAH) gene indicated the existence of silent mutations in codons 232, 245, and 385, linked to specific RFLP haplotypes in several Caucasian populations, namely Germans, Bulgarians, Italians, Turks, and Lithuanians. All three mutations create a new restriction site and can be easily detected on PCR amplified DNA. The usefulness of the silent mutations for diagnostic purposes depends on the haplotype distribution in the target population. The combined analysis of these markers and one or two PKU mutations forms a simple panel of diagnostic tests with full informativeness in a large proportion of PKU families, which helps to avoid the problems of genetic heterogeneity and of prenatal genomic Southern blot analysis.     

Haplotype analysis of the phenylalanine hydroxylase gene in Turkish phenylketonuria families

We have estimated the haplotype distribution of mutant and normal phenylalanine hydroxylase (PAH) alleles for 17 Turkish phenylketonuria (PKU) families: 20 normal and 27 mutated PAH alleles could be identified. Of the latter, the most prevalent were associated with haplotype 6 (29.6%), 1 (18.5%) and 36 (11.1%), while the normal alleles were preferentially associated with haplotype 1 (20%). Of the 19 different haplotypes observed, 5 have not been described previously. The haplotype distribution differed significantly from that of the Northern European population. Two of the eight polymorphic sites were in association with PKU. No deletions of exon sequences were found in the families analysed.     

应用等位基因特异性扩增检测经典型苯丙酮尿症PAH基因第6外显子基因突变

目的建立快速简便、特异灵敏的鉴定PAH基因第6外显子c.611A〉G突变热点的基因诊断方法。方法针对突变频率较高的PAH基因第6外显子的突变热点c.611A〉G,设计等位基因特异性扩增（amplification refractory mutationsystem,ARMS）的特异引物,对山西省已经临床确诊的70例经典型PKU患儿、c.611A〉G突变患儿的家长及50例正常儿童进行第6外显子扩增,扩增产物测序验证。结果在受检的山西省患儿中,PAH基因第6外显子c.611A〉G突变位点ARMS检测结果和测序结果完全相符。结论 ARMS技术操作简便,重复性和稳定性好,可作为PAH基因热点突变的快速灵敏检测方法。     

Molecular basis of phenylketonuria and related hyperphenylalaninemias: mutations and polymorphisms in the human phenylalanine hydroxylase gene.

Mutations in the human phenylalanine hydroxylase gene producing phenylketonuria or hyperphenylalaninemia have now been identified in many patients from various ethnic groups. These mutations all exhibit a high degree of association with specific restriction fragment-length polymorphism haplotypes at the PAH locus. About 50 of these mutations are single-base substitutions, including six nonsense mutations and eight splicing mutations, with the remainder being missense mutations. One splicing mutation results in a 3 amino acid in-frame insertion. Two or 3 large deletions, 2 single codon deletions, and 2 single base deletions have been found. Twelve of the missense mutations apparently result from the methylation and subsequent deamination of highly mutagenic CpG dinucleotides. Recurrent mutation has been observed at several of these sites, producing associations with different haplotypes in different populations. About half of all missense mutations have been examined by in vitro expression analysis, and a significant correlation has been observed between residual PAH activity and disease phenotype. Since continuing advances in molecular methodologies have dramatically accelerated the rate in which new mutations are being identified and characterized, this register of mutations will be updated periodically.