Spectrum and methods of detection of mutations in a phenylalanine hydroxylase gene from patients with phenylketonuria from the Novosibirsk region]

Phenylketonuria (PKU) is a widespread autosome recessive hereditary disease caused by a deficiency of the liver enzyme phenylalanine hydroxylase, which results in the distortion of phenylalanine metabolism and accumulation of toxic metabolites. The knowledge of molecular bases of PKU is of a high social importance as it enables phenotypic correction of the disease in the case of its early diagnostics. This disease is known to be associated with mutations in the phenylalanine hydroxylase gene, the distribution and mutation spectrum having pronounced ethnic and regional features. We studied the spectrum of mutations in the phenylalanine hydroxylase gene in a group of patients with PKU from the Novosibirsk region to reveal 10 missense point mutations, 1 mutation in the splice donor site, and 1 microdeletion. For these mutations, most widely distributed in the region, we used straightforward detection methods based on the restriction fragment length polymorphism (RFLP), artificial constructed restriction sites (ACRS) PCR, and denaturing gradient gel electrophoresis (DGGE).     

苯丙酮尿症分子遗传学研究进展

苯丙酮尿症是由于苯丙氨酸羟化酶基因突变引起的常染色体隐性遗传病。文章综述了苯丙酮尿症中的苯丙氨酸羟化酶基因的定位、结构、突变、调控以及突变基因的体外表达和苯丙氨酸羟化酶的三维结构特点等分子遗传学进展,阐述了苯丙氨酸羟化酶基因的突变对苯丙氨酸羟化酶的体外表达及其三维结构的影响, 以及部分基因型与表型相关的分子机制。 Abstract: Phenylketonuria(PKU) is one kinds of autusomal recessive disease caused by phenylalanine hydroxylase(PAH) gene mutation. This article reviews the recent molecular heredity progress on the phenylalanine hydroxylase gene’s orientation、structureand gene mutation and gene regulation. At same time, mutation gene in vitro expression and the character of 3D structure of PAH in PKU are involved. In this paper, also discussed the inflence of vitro expression and 3D protein structure by gene mutations and the molecular mechanism of the relationship between genotype and phenotype in PKU patient.     

Phenylketonuria in Iranian population: a study in institutions for mentally retarded in Isfahan

Phenylalanine hydroxylase (PAH) deficiency is caused by mutations in the PAH gene (12q22-q24) resulting in a primary deficiency of the PAH enzyme activity, intolerance to the dietary intake of phenylalanine (Phe) and production of the phenylketonuria (PKU) disease. To date there have been no reports on the molecular analysis of PKU in Iranian population. In this study, the states of the PKU disease in terms of prevalence and mutation spectrum among patients reside in the institutions for mentally retarded in Isfahan was investigated. In the first step, 611 out of 1541 patients with PKU phenotype or severe mental retardation were screened for the PKU disease using the Guthrie bacterial inhibition assay (GBIA) followed by HPLC. Among the patients screened 34 (5.56%) were found positive with abnormal serum Phe of above 7mg/dl. In the next step, the presence of 18 common mutations of the PAH gene in 26 of the patients with classical PKU (serum Phe above 20mg/dl) was investigated, using the polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). Of the 52 independent mutant alleles that were analyzed, 34 (65.38%) were genotyped showing 8 mutations as follows: R252W (15.38%), Q232Q (13.46%), R261Q (7.69%), delL364 (7.69%), IVS10-11g>a (5.77%), L333F (5.77%), V245V (5.77%) and S67P (3.85%). The results from this study may serve as a reference to analyze the PKU mutations in other part of Iran, and to establish diagnostic tests for carrier detection and prenatal diagnosis of the PKU disease in Iranian population.     

Polymorphic DNA haplotypes at the human phenylalanine hydroxylase locus and their relationship with phenylketonuria

Summary Eight polymorphic restriction enzyme sites at the phenylalanine hydroxylase (PAH) locus were analyzed from the parental chromosomes in 33 Danish nuclear families with at least one phenylketonuric (PKU) child. Determination of haplotypes of 66 normal chromosomes and 66 chromosomes bearing mutant allele (S) demonstrated that there are at least two haplotypes which occur predominantly on PKU chromosomes and rarely otherwise. Overall, the relative frequencies of the various haplotypes are significantly different on PKU-and normal-allele bearing chromosomes, even though there is no predominantly occurring unique haplotype which can characterize the PKU chromosomes. In addition, no significant association (linkage disequilibrium) between any single polymorphic site and the mutant allele (s) was observed. The results suggest that either the phenylketonuric mutation was very ancient so that the polymorphic sites and the mutation have reached linkage equilibrium or the mutant allele (s) are the results of multiple mutations in the phenylalanine hydroxylase gene in man. Furthermore, a crude relationship between standardized linkage disequilibria and physical map distances of the polymorphic sites indicates that there is no apparent recombination hot-spot in the human phenylalanine hydroxylase gene, since the recombination rate within the locus apears to be uniform and likely to be occurring at a rate similar to that within the HLA gene cluster. The limitations of this later analysis are discussed in view of the sampling errors of disequilibrium measure used, and the potential untility of the PAH haplotypes for prenatal diagnosis and detection of PKU carriers is established.     

PKU in Slovakia: mutation screening and haplotype analysis

The restriction fragment length polymorphism haplotypes and seven common mutations in the phenylalanine hydroxylase gene were analysed in 49 unrelated Slovak phenylketonuria (PKU) families of Caucasian origin. The predominant mutation in this population sample is R408W, with a frequency of 45.9%. In addition, four other mutations have been identified at relatively high frequencies: IVS12nt1, 10.2%; R158Q, 7.1%; R261Q, 7.1%; R252W, 2.0%. The mutation-haplotype associations correspond to those described in other European populations. The high proportion of mutations (72.4%) amenable to simple rapid detection based on the polymerase chain reaction provides a good basis for direct DNA-diagnosis of PKU in the Slovak population.     

Phenylketonuria mutations in Germany

We report the spectrum of mutations and associated modified haplotypes in patients with phenylketonuria living in Germany. A total of 546 independent alleles was investigated, including 411 of German and 65 of Turkish descent. Mutations were identified for 535 PKU alleles (98%) and there were 91 different mutations. The most common mutation was R408W on 22% of alleles. Two mutations, IVS12+1G→A and IVS10–11G→A accounted for just under 10% of alleles, whereas the remaining mutations were found at relative frequencies of 6% or less; 43 mutations were observed once only. IVS10–11G→A was the most common mutation (38% of alleles) in the subgroup of patients of Turkish descent. Modified haplotypes were determined from the analysis of four silent mutations, three diallelic restriction fragment length polymorphisms, a variable number of tandem repeats minisatellite and a short tandem repeat microsatellite in the phenylalanine hydroxylase gene, showing that a considerable proportion of mutations must have recurred in independent founders; other mutations may have changed chromosomal haplotype backgrounds by gene conversion. The spectrum of PKU mutations in Germany reflects the history of a heterogenous Central European population living at the crossroads of migration throughout the centuries. Received: 11 January 1999 / Accepted: 11 March 1999     

The PKU locus in man is on chromosome 12

Classical phenylketonuria (PKU) is a typical example of inborn errors in metabolism and is characterized by a complete lack of the hepatic enzyme phenylalanine hydroxylase, which normally converts phenylalanine to tyrosine. The genetic disorder causes impairment of postnatal brain development, resulting in severe mental retardation in untreated children. The disease is transmitted as an autosomal recessive trait and has a collective prevalence of about one in 10,000 among Caucasians, so that 2% of the population are carriers of the PKU trait. We have recently reported the cloning of human phenylalanine hydroxylase cDNA and that the human chromosomal phenylalanine hydroxylase gene is encoded by a unique DNA sequence. Using the human phenylalanine hydroxylase cDNA clone to analyze a clonal human/mouse hybrid cell panel by Southern hybridization, the phenylalanine hydroxylase gene has been assigned to human chromosome 12. Since the hypothesis that classical PKU is caused by structural mutations in the phenylalanine hydroxylase gene itself rather than through some transregulatory mechanisms has recently been confirmed by gene mapping, the PKU locus in man is determined to be on chromosome 12.     

苯丙氨酸羟化酶（PAH）基因外显子7及其两侧内含子的突变研究

为探讨中国苯丙酮尿症（PKU）人群中苯丙氨酸羟化酶（PAH）基因外显子7的突变特征,对147例PKU患儿的294个PAH基因外显子7以及两侧部分内含子序列,应用PCR－单链构象多态性（SSCP）分析及基因序列分析的方法进行了筛查和确定。共发现13种突变基因：G239D、R241C、R241fs、R243Q、G247S、G247V、R252Q、L255S、R261Q、M276K、E280G、P281L、Ivs7+2T>A,其中7 种突变基因在中国PKU人群首次发现：G239D 、R241fs 、G247S 、E280G、L255S、R261Q、P281L,前4种在国际上尚未见到报道,并已提交到国际PAH突变数据库（www.pahdb.mcgill.ca）。突变基因的总频率为30.61%（90 /294）。突变涉及了错义、缺失、移码和剪接位点4种突变类型。结果明确了PAH基因外显子7的突变种类和分布等特征,表明外显子7是中国人PAH基因突变的热点区域。 Abstract: To study mutation in exon 7 of the gene for the phenylalanine hydroxylase（PAH）, the mutations in exon 7 and flanking sequence of PAH gene were detected by means of SSCP analysis and DNA sequencing, in 147 unrelated Chinese children with phynelketonuria and their parents. Thirteen different mutations, including 11 missense, 1 deletion and 1 splice mutation, were revealed in 90/294 mutant alleles (30.61%). The prevalent mutations were R243Q (22.8%) and Ivs7nt2t->a (2.38%). Seven novel mutations were identified: G239D, R241fsdelG, G247S, E280G, L255S, R261Q, P281L. These new mutations have not been described in Chinese PKU population and the first 4 mutants have not been reported and thus been submitted to www.pahdb,mcgill.ca. The missense was the most common type. The deletion and frameshift mutations were detected for the first time in Chinese PKU population. This study showed the mutation characteristics and their distribution in exon 7 of PAH gene and proved that the exon 7 was the hot region of PAH gene mutation in Chinese PKU population .     

Mutation spectrum of the <Emphasis Type="Italic">PAH</Emphasis> gene in phenylketonuria patients in the Karachay-Cherkess Republic (Russia)

A comprehensive population and medical-genetic study was carried out in ten districts and two cities in the Karachay-Cherkess Republic (Russia). As a result, 57 patients with phenylketonuria were revealed. PAH gene genotypes for 40 probands and their diseased and healthy relatives were determined. The mutation spectrum of the PAH gene in the Karachay-Cherkess Republic was investigated. The major mutation in this region is R261X with allelic frequency of 68.4%. We elaborated a convenient system for detection of six PAH gene mutations common in the Karachay-Cherkess Republic, with the total information content of the system being 89.9%. As a result of processing the clinical data, association of the diet and phenylalanine levels in the blood was verified. Genophenotypic analysis confirms the association of the residual activity of phenylalanine hydroxylase and the severity of the disease. It is shown that common mutation R261X is severe and that patients who are homozygous for this mutation have classical phenylketonuria (PKU).     

Phenylalanine hydroxylase deficiency caused by a single base substitution in an exon of the human phenylalanine hydroxylase gene

A novel restriction fragment length polymorphism in the phenylalanine hydroxylase (PAH) locus generated by the restriction endonuclease MspI was observed in a German phenylketonuria (PKU) patient. Molecular cloning and DNA sequence analyses revealed that the MspI polymorphism was created by a T to C transition in exon 9 of the human PAH gene, which also resulted in the conversion of a leucine codon to a proline codon. The effect of the amino acid substitution was investigated by creating a corresponding mutation in a full-length human PAH cDNA by site-directed mutagenesis followed by expression analysis in cultured mammalian cells. Results demonstrate that the mutation in the gene causes the synthesis of an unstable protein in the cell corresponding to a CRM- phenotype. Together with the other mutations recently reported in the PAH gene, the data support previous biochemical and clinical observations that PKU is a heterogeneous disorder at the gene level.     

Phenylketonuria mutation in Chinese haplotype 44 identical with haplotype 2 mutation in northern-European Caucasians

Summary DNA amplification with the polymerase chain reaction was employed to identify the phenylketonuria (PKU) mutation in Chinese PKU families. The amplified DNA was hybridized with oligonucleotides corresponding to the two most common mutant alleles, i.e., mutations associated with PKU haplotype 2 and 3 among Caucasians of northern-European ancestry. The results of analysis demonstrate that the mutation in Chinese haplotype 44 is a single-base substitution corresponding to the mutation associated with haplotype 2 in Caucasians, whereas the mutations of the phenylalanine hydroxylase gene in haplotypes 4, 7, 11 and 28 among Chinese do not correlate with either of the two mutations identified in northern-European Caucasians.     

Phenylalanine hydroxylase gene: novel missense mutation in exon 7 causing severe phenylketonuria.

By direct sequence analysis of 94 mutant phenylalanine hydroxylase alleles using polymerase chain reaction-based techniques, we identified a C to T transition in exon 7 of the human phenylalanine hydroxylase gene that is associated with RFLP haplotypes 1 and 4. A leucine for proline substitution at position 281 can be predicted from the nucleotide sequence of the mutant codon. Expression analysis in cultured mammalian cells after site-directed mutagenesis proved that the base substitution is a disease causing gene lesion. Dot-blot hybridization analysis using allele-specific oligonucleotides revealed that 25% of all mutant haplotype 1 alleles in the German population bear this mutation. In addition, this mutation could be detected on one mutant haplotype 4 allele. The fact that this mutation is associated with only 25% of all mutant haplotype 1 alleles suggests that multiple mutations may be associated with this haplotype. The occurrence of several different mutations would be in agreement with the clinical heterogeneity observed in the group of patients whose PKU alleles belong to haplotype 1.     

Recurrent mutation in the human phenylalanine hydroxylase gene.

We report the identification of a missense mutation of Glu280 to Lys280 in the phenylalanine hydroxylase (PAH) gene of a phenylketonuria (PKU) patient in Denmark. The mutation is associated with haplotype 1 of the PAH gene in this population. This mutation has previously been found in North Africa, where it is in linkage disequilibrium with haplotype 38. While it is conceivable that this mutation could have been transferred from one haplotype background to another by a double crossover or gene conversion event, the fact that the mutation is exclusively associated with the two different haplotypes in the two distinct populations supports the hypothesis that these two PKU alleles are the result of recurrent mutations in the human PAH gene. Furthermore, since the site of mutation involves a CpG dinucleotide, they may represent hot spots for mutation in the human PAH locus.     

Novel PKU mutation on haplotype 2 in French-Canadians.

We analyzed DNA from nine French-Canadian probands from eastern Quebec province; all had hyperphenylalaninemia (phenylketonuria [PKU] or non-PKU forms) caused by mutations at the phenylalanine hydroxylase locus. Analysis of RFLP haplotypes and mutations revealed a novel mutation, an A-to-G transition (met----val) in codon 1 (the translation-initiation codon). It occurred on 5 of the 18 mutant chromosomes and was associated each time with haplotype 2. A proband homozygous for this mutation had the PKU phenotype. In other probands, the codon 1 mutation was inherited once with the splice junction mutation in exon 12 (on haplotype 3), conferring PKU, and was inherited twice with a mutation on haplotype 1, conferring PKU in one proband and non-PKU hyperphenylalaninemia in the other. The other five probands carried mutations, conferring PKU, on the following haplotype combinations: 1/3 (twice), 1/9, 3/4, and 1/1. The mutations on haplotypes 1, 4, and 9 are not yet characterized. This preliminary study reveals a novel PKU mutation and considerable genetic heterogeneity at the phenylalanine hydroxylase locus in French-Canadians.     

Compound heterozygosity in nonphenylketonuria hyperphenylalanemia: the contribution of mutations for classical phenylketonuria

Hyperphenylalaninemia (HPA) results from defective hydroxylation of phenylalanine in the liver, in most cases because of defective phenylalanine hydroxylase. HPA is highly variable, ranging from moderate elevation of plasma phenylalanine with no clinical consequences to a severe disease, classical phenylketonuria (PKU). Non-PKU HPA was found in excess of PKU in Israel, while the opposite is true in Europe. To study the genetic basis of non-PKU HPA, we performed haplotype analysis at the phenylalanine hydroxylase locus in 27 families with non-PKU HPA. All individuals with this condition were compound heterozygotes. In six of these families, in which both PKU and non-PKU HPA were segregating, haplotype analysis showed that non-PKU HPA resulted from compound heterozygosity for a PKU mutation and a second mutation, with milder effect, which is probably expressed only when it interacts with the severe mutation. The involvement of PKU mutations in non-PKU HPA was further demonstrated in Jewish Yemenite families with non-PKU HPA, in which the individuals with this condition were carriers of the single PKU allele which exists in this community. In addition, two previously known PKU point mutations (R261Q and R408W) were found in individuals with non-PKU HPA. These mutations are associated, in our population, with the same haplotypes as those with which it is associated in Europe. Based on the above-mentioned genetic model for non-PKU HPA, successful prenatal diagnosis of this condition was performed in one family.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two mutations within the coding sequence of the phenylalanine hydroxylase gene

Summary Two previously unidentified mutations at the phenylalanine hydroxylase locus were found during a study of the relationship between genotype and phenotype in phenylketonuria and hyperphenylalaninemia. One mutation eliminates the BamHI site in exon 7 and the other eliminates the HindIII site in exon 11 of the phenylalanine hydroxylase gene. They were suspected because of deviating restriction fragment patterns and confirmed by amplification, via the polymerase chain reaction, of exon 7 and exon 11, respectively, followed by digestion with the appropriate restriction enzyme. Direct sequencing of amplified mutant exon 7 revealed a G/C to T/A transversion at the first base of codon 272, substituting a GGA glycine codon for a UGA stop codon. Direct sequencing of amplified mutant exon 11 revealed a deletion of codon 364, a CTT leucine codon. The exon 7 mutation can be expected to result in a truncated protein and the exon 11 mutation in the elimination of an amino acid in the catalytic region of the enzyme. A patient who is a compound heterozygote for these two mutations has classical phenylketonuria. It is concluded that each of the two mutations leads to a profound loss of enzymatic activity. The segregation of these mutations with disease alleles in 4 and 2 families, respectively, supports the hypothesis that multiple mutations at the phenylalanine hydroxylase locus explain the variable phenylalanine tolerance in patients with phenylalanine hydroxylase deficiency.     

Identification of a missense phenylketonuria mutation at codon 408 in Chinese

Summary A single base transition of G to A at codon 408 of the phenylalanine hydroxylase gene is identified. This missense mutation results in the substitution of Arg⁴⁰⁸ for Gln⁴⁰⁸ (R408Q) and accounts for about 5% of phenylketonuria (PKU) chromosomes among Chinese. This mutation is in linkage disequilibrium with restriction fragment length polymorphism haplotype 4. In addition, another mutation (R408W), at the same codon and prevalent on haplotype 2 PKU chromosomes in Caucasians, is identified in a PKU allele of haplotype 41. Previously, this mutation has been observed on a haplotype 44 background in Chinese PKU patients.     

中国北方人苯丙氨酸羟化酶基因外显子7内新突变的鉴定

应用ＰＣＲ－单链构象多态性分析及ＤＮＡ直接测序,对４５例中国北方苯丙酮尿症（ＰＫＵ）患者苯丙氨酸羟化酶（ＰＡＨ）基因外显子７内突变进行了鉴定。共检出６种错义突变及一种静止突变：Ｒ２４３Ｑ．Ｒ４１Ｈ,Ｇ２４７Ｖ．Ｌ２４９Ｈ．Ｐ２５４Ｉ．Ｇ２５７Ｖ和Ｖ２４５Ｖ。经与国际ＰＡＨ基因突变数据库比较,确认Ｇ２５７Ｖ．Ｐ２５４Ｉ和Ｌ２４９Ｈ为国际上首次发现的突变。结果揭示,中国人与其他种族及中国北方与南方人群ＰＡＨ突变特点不同。明确了中国北方人群中ＰＡＨ基因外显子７基因突变分布,有助于提高ＰＫＵ的基因诊断率,对基因的起源、进化研究有参考价值     

Haplotype distribution and molecular defects at the phenylalanine hydroxylase locus in Italy

Summary In order to investigate the molecular basis of phenylketonuria (PKU) in Italy, we characterized the RFLP haplotypes at the phenylalanine hydroxylase gene in 38 unrelated Italian PKU families. The distribution of haplotypes associated with PKU alleles differs from that of other European populations. In particular, haplotypes 1 and 6 are present in 39.7% and 17.6% of the PKU chromosomes, whereas the frequencies of haplotypes 2 and 3 are 5.9% and 2.9%, respectively. The characterization of PKU mutations using the polymerase chain reaction and allele-specific oligonucleotides shows that 1 out of 2 haplotypes 3 carries the splicing mutation and that 2 out of 4 haplotypes 2 carry the missense mutation associated with these haplotypes in North European populations. Our results indicate that the two molecular defects most frequent in Northern Europe represent a minority of PKU mutations in Italy.     

Two novel PAH gene mutations detected in Italian phenylketonuric patients

We report the identification by denaturing gradient gel electrophoresis and sequence analysis of two new phenylalanine hydroxylase (PAH) gene mutations (IVS4nt-2 and N207S) in single chromosomes of two unrelated Italian phenylketonuric (PKU) patients. Interestingly, mutation Y204C, found on the second mutant allele of family F1, has been previously detected in Chinese patients. Haplotype analysis showed that the latter mutation is linked to the same haplotype (H4) in both Chinese and Italian patients, suggesting a common origin. In vivo assessment of mutation severity indicates that N207S is associated with classic PKU. The identification of these two new mutations further extends the remarkable heterogeneity of the PAH locus in the Italian population. Received: 23 May 1996