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.     

中国人群苯丙氨酸羟化酶基因突变研究进展

苯丙氨酸羟化酶（ｐｈｅｎｙｌａｌａｎｉｎｅｈｙｄｒｏｘｙｌａｓｅ，ｐｈｅｎｙｌａｌａｎｉｎｅ４ｍｏｎｏｘｙｇｅｎｅｍａｓｅ：ＰＡＨ：ＥＣ１．１４．１６．１）是体内苯丙氨酸转化为酪氨酸的关键酶。ＰＡＨ由二个分子量约为５０，０００的亚单位组成。ＰＡＨ肽链合成障碍导致其活性降低或缺如，造成Ｐｈｅ羟化受阻，Ｐｈｅ在体内储积。体内过量的Ｐｈｅ引发出一系列临床症状，从轻度的高苯丙氨酸血症到典型的苯丙酮尿症（ｐｈｅｎｙｌｋｅｔｏｎｕｒｉａ，ＰＫＵ）［１］。一、ＰＡＨ基因分子学研究已证明ＰＡＨ基因位于１２ｑ２…     

河南地区苯丙酮尿症患者苯丙氨酸羟化酶基因突变研究

目的 了解河南地区苯丙酮尿症(phenylketonuria,PKU)患者苯丙氨酸羟化酶(phenylalanine hydroxylase,PAH)基因突变情况,以便为苯丙酮尿症产前诊断和遗传咨询提供理论依据.方法 应用PCR产物直接测序对47例PKU患者及其父母PAH基因第1～13外显子及其两侧内含子进行序列分析.结果 在94条染色体中共检测到了83个PAH基因突变位点,检出率为88.3%(83/94),共发现了25种突变,其中突变E79fX13、H271R和D415Y国内外未见报道,突变VS10-14C＞G为国内首次报道.河南地区PKU患者的PAH基因突变集中在第6、7和11外显子,常见的7种突变是p.R243Q(20.5%)、EX6-96A＞G(12.0%)、p.Y356X(9.6%)、VS4-1G＞A(9.6%)、p.R111X(8.4%)、p.V399V(8.4%)、p.R413P(7.2%).结论 河南地区PKU患者PAH基因突变与中国其他地区相似,通过PAH基因直接测序可对大部分的PKU家系进行产前诊断.

Abstract：

Objective To study the characteristics of the phenylalanine hydroxylase gene (PAH)mutations in patients with phenylketonuria (PKU) in Henan province, in order to provide basic information for genetic counseling and prenatal diagnosis. Methods Mutations of the PAH gene were detected in exons 1-13 with flanking introns of PAH gene by PCR and DNA sequencing in 47 families with PKU. Results A total of 25 different mutations were detected in 83 out of 94 PAH alleles (88. 3%). Among them,E79fX13, H271R and D415Y have not been reported previously. It was the first time that IVS10-14C＞Gmutation was reported in Chinese PKU population. The mutations p. R243Q, EX6-96A＞G, p. Y356X,IVS4-1G＞A, p. R111X, p. V399V and p. R413P, were the prevalent mutations with relative frequencies of 20. 5 %, 12.0%, 9.6%, 9. 6%, 8. 4%, 8. 4% and 7.2% respectively. Conclusion The mutations of the PAH gene in patients with classical phenylketonuria in Henan province were similar to that in other areas of China. Prenatal gene diagnosis for PKU by PAH gene sequencing is efficient for most PKU families.     

河南地区苯丙酮尿症患者苯丙氨酸羟化酶基因突变研究

Objective To study the characteristics of the phenylalanine hydroxylase gene (PAH)mutations in patients with phenylketonuria (PKU) in Henan province, in order to provide basic information for genetic counseling and prenatal diagnosis. Methods Mutations of the PAH gene were detected in exons 1-13 with flanking introns of PAH gene by PCR and DNA sequencing in 47 families with PKU. Results A total of 25 different mutations were detected in 83 out of 94 PAH alleles (88. 3%). Among them,E79fX13, H271R and D415Y have not been reported previously. It was the first time that IVS10-14C＞Gmutation was reported in Chinese PKU population. The mutations p. R243Q, EX6-96A＞G, p. Y356X,IVS4-1G＞A, p. R111X, p. V399V and p. R413P, were the prevalent mutations with relative frequencies of 20. 5 %, 12.0%, 9.6%, 9. 6%, 8. 4%, 8. 4% and 7.2% respectively. Conclusion The mutations of the PAH gene in patients with classical phenylketonuria in Henan province were similar to that in other areas of China. Prenatal gene diagnosis for PKU by PAH gene sequencing is efficient for most PKU families.     

河南地区苯丙酮尿症患者苯丙氨酸羟化酶基因突变研究

Objective To study the characteristics of the phenylalanine hydroxylase gene (PAH)mutations in patients with phenylketonuria (PKU) in Henan province, in order to provide basic information for genetic counseling and prenatal diagnosis. Methods Mutations of the PAH gene were detected in exons 1-13 with flanking introns of PAH gene by PCR and DNA sequencing in 47 families with PKU. Results A total of 25 different mutations were detected in 83 out of 94 PAH alleles (88. 3%). Among them,E79fX13, H271R and D415Y have not been reported previously. It was the first time that IVS10-14C＞Gmutation was reported in Chinese PKU population. The mutations p. R243Q, EX6-96A＞G, p. Y356X,IVS4-1G＞A, p. R111X, p. V399V and p. R413P, were the prevalent mutations with relative frequencies of 20. 5 %, 12.0%, 9.6%, 9. 6%, 8. 4%, 8. 4% and 7.2% respectively. Conclusion The mutations of the PAH gene in patients with classical phenylketonuria in Henan province were similar to that in other areas of China. Prenatal gene diagnosis for PKU by PAH gene sequencing is efficient for most PKU families.     

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.     

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.     

新疆地区苯丙氨酸羟化酶基因的突变研究

目的 了解新疆地区苯丙氨酸羟化酶(phenylalanine hydroxylase,PAH)基因的突变规律及特点.方法 应用聚合酶链反应一单链构象多态性分析及基因测序列方法 ,检测46例苯丙酮尿症患者PAH基因第3、5、6、7、11和12外显子及其两侧内含子序列.结果 在92个PAH等位基因中共检出20种不同的突变基因,总检出率为73.9%(68/92).其中常见基因突变R243Q、EX6-96A＞G、R111X、Y356X和V399V与我国北方地区基本类似.较常见基因突变F161S、L255S、P281L和R413P与国内其他地区相比差异较大.E280G和A434D为在国际上第2次检出;L255S、P281L、R261Q和165T为在国内第2次检出.新疆少数民族也发现了13种PAH基因突变,均系在本民族中首次报道,其突变基因的类型表现出鲜明的民族特色.结论 从对新疆地区PAH突变基因的研究结果 来看,该地的遗传基因不仅具有独立、保守的特性,而且还存在着相互交叉、相互融合的特征.     

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。     

苯丙氨酸羟化酶基因的六种新型突变

目的　检测中国苯丙酮尿症 ( phenylketonuria ,PKU )患者苯丙氨酸羟化酶 (phenylalaninehy droxylase ,PAH)基因新的突变位点。 方法　应用聚合酶链反应 单链构象多态性及DNA直接测序检测 40例经典型PKU患者和 3 0名正常对照的PAH基因。结果　在PAH基因上共发现 11种突变和 3种多态 ,3 0名正常对照者PAH基因未发现异常。结论　经与国际PAH基因突变数据库比较 ,确认M 2 76K、M 2 76R、2 80insT、IVS10nt 3 2T→A、IVS4nt 4 7C→T是国际上首次发现的突变 ,H 2 90R是中国PKU患者基因上首次发现的新型突变。     

天津及周边地区苯丙氨酸羟化酶基因突变谱和新突变分析

目的 分析天津地区人苯丙氨酸羟化酶(phenylalanine hydroxylase gene,PAH)基因突变谱,明确本地区该基因突变类型和特点,为遗传咨询和产前基因诊断提供科学依据.方法 用聚合酶链反应-单链构象多态、变性高效液相色谱法和DNA测序法对天津及河北等地99例已确诊各种类型苯丙酮尿症患儿基因组DNA进行PAH全基因13个外显子分析.结果 全基因共检出41种突变,含错义突变22种、无义突变7种、剪接位点突变9种和缺失突变3种.其中新突变6种(IVS3nt+1g→a、A165D、Q301X、G344D、P362L和R413G).198个PAH等位基因突变总检出率为93.94%.结论 天津及周边地区PAH基因突变谱广、遗传异质性高,一些常见突变的发生频率与以往报道的地域分布略不同.     

Eight new mutations of the phenylalanine hydroxylase gene in Italian patients with hyperphenylalaninemia

This report identifies eight new mutations of the phenylalanine hydroxylase gene detected in Italian patients with hyperphenylalaninemia. The trivial name of the mutations, predicted phenotypic effect, and population of origin (Italian region) are as follows: F55L (nonconservative change: classic, moderate, mild PKU ?; Sicily), IVS2nt-13 (splicing defect, classic PKU; Tuscany), I65N (nonconservative change classic, moderate, mild PKU ?; Sicily), H201Y (non-PKU HPA; Sicily), I269L (non-PKU HPA, or polymorphism; Sicily), IVS7nt3 (splicing defect or polymorphism; Sicily), I283N (classic PKU; Sicily), IVS12nt2 (splicing defect, classic PKU; Sicily and Apulia). In Sicily, the relative frequency of mutations F55L, I65N, H201Y, I269L, IVS7nt3, I283N, IVS12nt2 is < 1%. The seven new mutations identified in the Sicilian population increase the remarkable genetic heterogeneity typical of this population with an estimated homozygosity value at the PAH locus of 0.041. Hum Mutat 11:240–243, 1998. © 1998 Wiley-Liss, Inc.     

Mutational spectrum in German patients with phenylalanine hydroxylase deficiency

We report on the spectrum and frequency of mutations in the phenylalanine hydroxylase (PAH) gene in 226 German families with PAH deficiency, most of them from Southern Germany. A total of 88 mutations were identified in 428 out of 438 mutant PAH alleles including one novel stop mutation L293X (c.878T>A). In three families, two phenylketonuria (PKU) mutations were found in cis, and in one family a de novo mutation was observed. A comparison of the results from Southern Germany with those of other parts of Western Germany showed no obvious local mutation clustering. In addition we studied the genotypic spectrum of 39 Turkish families with PAH deficiency. Twenty-three mutations were identified in 73 out of 75 Turkish chromosomes including two novel mutations: E280A (c.839A>G) in Exon 7 and IVS10-7C>A (c.1066-7C>A) in Intron 10. A new polymorphism IVS4+47C/T (c.441+47C>T) was found in mutant and normal PAH alleles. Screening of 170 German and 150 Turkish individuals without family history of PAH deficiency revealed 10 and 12 heterozygotes, respectively, a higher frequency of carriers than expected. A novel mutations of uncertain functional relevance, R169H (c.506G>A) in Exon 5 was found in two Turkish heterozygotes. Most of the Turkish heterozygotes carried mild mutations, indicating that mild forms of PAH deficiency may be more common in that population than previously recognised.     

Gypsy phenylketonuria: A point mutation of the phenylalanine hydroxylase gene in Gypsy families from Slovakia

A direct mutational analysis of the phenylala nine hydroxylase gene (PAH) in Gypsy families with phenylketonuria (PKU) has not yet been presented. However, they obviously represent a group at high risk for this inherited disease. We analyzed the PAH loci of 65 Gypsies originating from Eastern Slovakia by a combination of PCR amplification, direct sequencing and ASO hybridization. These studies uncovered 10 “classical PKU” patients to be homozygous for a R252W (CGG-TGG) transition, and 29 heterozygous carriers of this mutation. Fifteen control Caucasoid PKU patients from the Czech and Slovak Republics were selected. In this group we detected R252W mutation in two subjects (6.67% of all mutant alleles). Both were compound heterozygous for two different mutations. Previous haplotype studies of Welsh Gypsies with PKU were uniformative in the determination of heterozygosity. ASO hybridization served us effectively for the consequent analyses in Gypsy PKU-related families and to identify the carriers among the unrelated subjects. © 1994 Wiley-Liss, Inc.     

Comparative diagnostic value of phenyla-lanine challenge and phenylalanine hydroxylase activity in phenylketonuria

Serum phenylalanine (phe) concentrations during and following phe challenges and liver phenylalanine hydroxylase (PH) activity were compared in 13 phenylketonuric (PKU) patients. These patients were separated into two groups: eight patients with no detectable PH activity (PH°) and five patients with residual PH activity (PH^-) ranging from 9 to 24% of the activity obtained in 10 non-PKU subjects. The rise in serum phe concentration during 3 days of oral loading did not differentiate the two groups. However, the difference in serum phe concentration of the PH° and PH^- groups reached statistical significance at 24 h postloading (p<0.01). We concluded that combined results from multiple measurements during the oral challenge, namely serum phe concentration after termination of loading, serum phe clearance rate, post-loading phe tolerance index and urinary metabolite excretion, make a better indicator for predicting residual PH activity for the majority of PKU subjects than peak phe concentrations during phe challenge.