Mutation in the structure of exon 7 of the phenylalanine hydroxylase in phenylketonuria patients from the Novosibirsk area

The spectrum and frequency of mutations of exon 7 of the gene for phenylalanine hydroxylase (PAH) were studied in 34 phenylketonuria (PKU) patients living in Novosibirsk oblast. The five most prevalent mutations constituted 17.64% of defective alleles: R243Q (1.47%), R252W (1.47%), R261Q (5.88%), E280K (1.47%), and P281L (7.35%). A neutral polymorphic locus V245V was found within exon 7.     

Analysis of mutations in the phenylalanine hydroxylase gene in Ukrainian families at high risk for phenylketonuria

The data on analysis of phenylalanine hydroxilase (PAH) gene mutations in 39 phenylketonuria (PKU) families from Ukraine are presented. Obtained results indicate that the most common mutation observed in the Ukrainian population is R408W mutation (66.6%). Besides two minor mutations R158Q (2.6%) and Y414C (1.25%) were revealed.     

Rapid mutation screening of phenylketonuria by polymerase chain reaction-linked restriction enzyme assay and direct sequence of the phenylalanine hydroxylase gene: clinical application in northern Japan and northern China

We describe a simple and technically feasible method for mutation screening of the phenylalanine hydroxylase (PAH) gene and its application to Japanese and Chinese patients with hyperphenylalaninemia. The strategy is based on the identification of a nucleotide substitution by restriction enzyme analysis, coupled with PCR and direct sequencing of exon 7 of the PAH gene. Because the detection of various mutations can proceed simultaneously using the same technique, it is quite rapid and reproducible, making it possible to perform effective molecular diagnosis and carrier screening in most laboratories. Using this procedure, we found that the most common molecular defects were R413P in Hokkaido, Japan (35 %) and R243Q in Heilongjiang, China (50%). R111X, IVS4nt-1, and five mutations in exon 7 (R241C, R243Q, R252W, A259T, and S273P) accounted for 55% of phenylketonuria (PKU) alleles in Hokkaido. In Heilongjiang, the R111X, Y356X, and R408W mutations accounted for 35% of PKU alleles. Clinically, homozygotes or compound heterozygotes of null alleles, which express nonfunctional enzyme activity, were all associated with classic PKU. On the other hand, patients heterozygous for the R241C allele had a benign phenotype of mild hyperphenylalaninemia. The DNA diagnosis in early infancy can predict various PKU phenotypes, and can prove useful in decision-making concerning dietary therapy.     

Analysis of mutations and VNTR-polymorphism in the phenylalanine hydroxylase gene

The data on 5 PAH gene mutations analysis are presented. The most common mutation observed in Ukrainian population was determined to be R408W (66.6%). As well two minor mutations R158Q (2.5%) and Y414C (1.25%) were identified. The allelic variation of the VNTR-polymorphism in 470 healthy volunteers and 39 PKU-patients were analysed. 7 allelic variants and 15 haplotypes were found. The linkage disequilibrium was displayed between mutation R408W and VNTR-haplotypes 03. An advantages of molecular genetic analysis of mutations and VNTR-polymorphism for diagnosis of PKU in Ukraine are discussed.     

Associations between mutations and a VNTR in the human phenylalanine hydroxylase gene.

The HindIII RFLP in the human phenylalanine hydroxylase (PAH) gene is caused by the presence of an AT-rich (70%) minisatellite region. This region contains various multiple of 30-bp tandem repeats and is located 3 kb downstream of the final exon of the gene. PCR-mediated amplification of this region from haplotyped PAH chromosomes indicates that the previously reported 4.0-kb HindIII allele contains three of these repeats, while the 4.4-kb HindIII allele contains 12 of these repeats. The 4.2-kb HindIII fragment can contain six, seven, eight, or nine copies of this repeat. These variations permit more detailed analysis of mutant haplotypes 1, 5, 6, and, possibly, others. Kindred analysis in phenylketonuria families demonstrates Mendelian segregation of these VNTR alleles, as well as associations between these alleles and certain PAH mutations. The R261Q mutation, associated with haplotype 1, is associated almost exclusively with an allele containing eight repeats; the R408W mutation, when occurring on a haplotype 1 background, may also be associated with the eight-repeat VNTR allele. Other PAH mutations associated with haplotype 1, R252W and P281L, do not appear to segregate with specific VNTR alleles. The IVS-10 mutation, when associated with haplotype 6, is associated exclusively with an allele containing seven repeats. The combined use of this VNTR system and the existing RFLP haplotype system will increase the performance of prenatal diagnostic tests based on haplotype analysis. In addition, this VNTR may prove useful in studies concerning the origins and distributions of PAH mutations in different human populations.     

The importance of arginine mutation for the evolutionary structure and function of phenylalanine hydroxylase gene

Phenylalanine hydroxylase (PAH) gene mutations were investigated in 23 (46 alleles) unrelated phenylketonuria (PKU) patients in Cukurova region. First, all exons of PAH gene were screened by denaturing high performance liquid chromatography (DHPLC), and then, the suspicious samples were analyzed by direct sequencing technique. Consequently, the following results were obtained: IVS10-11g-->a splicing mutation in 27/46 (58.7%), R261Q mutation in 7/46 (15.2%) and E178G, R243X, R243Q, P281L, Y386C, R408W mutations, each found in the frequency of 2/46 (4.3%). In many countries, Arginine mutations have the highest frequency among PAH gene mutations in PKU patients. Although, CpG dinucleotids are effective in mutations resulting in arginine changes, this finding originated from the studies on the causes of mutations rather than the studies on the importance of arginine amino acid. In our analyses, we have detected that a majority of mutations causing a change in arginine and other amino acids concentrated in exon 7 comprising the catalytic domain (residues 143-410) of PAH gene. Several studies has emphasized the role of arginine amino acid; with the following outcomes; arginine repetition is significant for RNA binding proteins, and for histon proteins in eukaryotic gene expression, and also arginine repetition occurring in the structure of signal recognition particle's (SRPs) as a consequence of post-translational processes is very important in terms of gene expression. Therefore, the role of arginine amino acid in PAH gene is rather remarkable in that it shows the role of amino acids in the protein/RNA interaction that has started in the evolutionary process and is still preserved and maintained in the motif formation of active domain structure due to its strong binding properties. Thus, such properties imply that both arginine amino acid and exon 7 is of great significance with regards to the structure and function of the PheOH enzyme.     

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 .     

Multiple origins for phenylketonuria in Europe

Phenylketonuria (PKU), a disorder of amino acid metabolism prevalent among Caucasians and other ethnic groups, is caused primarily by a deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH). PKU is a highly heterogeneous disorder, with more than 60 molecular lesions identified in the PAH gene. The haplotype associations, relative frequencies, and distributions of five prevalent PAH mutations (R158Q, R261Q, IVS10nt546, R408W, and IVS12n1) were established in a comprehensive European sample population and subsequently were examined to determine the potential roles of several genetic mechanisms in explaining the present distribution of the major PKU alleles. Each of these five mutations was strongly associated with only one of the more than 70 chromosomal haplotypes defined by eight RFLPs in or near the PAH gene. These findings suggest that each of these mutations arose through a single founding event that occurred within time periods ranging from several hundred to several thousand years ago. From the significant differences observed in the relative frequencies and distributions of these five alleles throughout Europe, four of these putative founding events could be localized to specific ethnic subgroups. Together, these data suggest that there were multiple, geographically and ethnically distinct origins for PKU within the European population.     

Phenylketonuria mutations and their relation to RFLP haplotypes at the PAH locus in Czech PKU families

A detailed study of the mutant phenylalanine hydroxylase (PAH) gene from the eastern part of the Czech Republic (Moravia) is reported. A total of 190 mutant alleles from 95 phenylketonuria (PKU) families were analyzed for 21 prevalent Caucasian mutations and restriction fragment length polymorphism /variable number of tandem repeats (RFLP/VNTR) haplotypes. Eighty per cent of all mutant alleles were found to carry 11 mutations. The most common molecular defect was the mutation R408W (55.3%), with a very high degree of homozygosity (34.6%). Each of four other mutations (R158Q, R243X, G272X, IVS12nt1) accounted for more than 3% of PKU alleles. Rarely present were mutations IVS10nt546 (2.6%), R252W (2.6%), L48S (2.1%), R261Q (1.6%), Y414C (1.0%) and I65T (0.5%). Mutations that have been predominantly described in southern Europe (IVS7nt1, A259V, Y277D, R241H, T278N) were not detected. A total of 14 different mutant haplotypes were observed. Three unusual genotype-haplotype associations were identified (R158Q on haplotypes 2.3 and 7.8 and R252W on haplotype 69.3). There was a strong association between the mutation R408W and haplotype 2.3 (54.7%). Heterogeneity was found at mutations R408W (haplotypes 2.3 and 5.9), R158Q (haplotypes 4.3, 2.3 and 7.8) and IVS10nt546 (haplotypes 6.7 and 34.7). The molecular basis of PKU in the Moravian area appears to be relatively homogeneous in comparison with other southern and western European populations, thus providing a good starting point for prenatal diagnosis and early clinical classification.     

Single-strand conformation polymorphism for detection of mutations and base substitutions in phenylketonuria.

In the past few years, more than 20 different mutations have been reported in hyperphenylalaninemias. In southwestern Europe and Mediterranean countries, however, the mutant genotypes reported account for only a fraction (27%) of all mutant alleles at the phenylalanine hydroxylase (PAH) locus, and most of the mutations causing the disease remain unknown. In order to develop a strategy for rapid detection of mutation-containing exons, we applied the single-strand conformation-polymorphism (SSCP) technique to exons 3, 5, 7, and 12 of the PAH gene. We observed five abnormal patterns of migration in mutant PAH genes, and we consistently found base substitutions in the corresponding exons, with no false-positive results. By this procedure, two novel putative mutations were detected in the seventh exon of the PAH gene, (A259V and Y277D) and we were able to demonstrate that the delta I94, R158Q, R408W, and E280K mutations were easily detectable by the SSCP technique. This procedure is therefore of particular interest for rapid detection of mutation-containing exons and for determination of further genotype-phenotype correlations in hyperphenylalaninemias.     

Molecular analysis of 16 Turkish families with DHPR deficiency using denaturing gradient gel electrophoresis (DGGE)

Dihydropteridine reductase (DHPR) catalyses the conversion of quinonoid dihydrobiopterin (qBH2) to tetrahydrobiopterin (BH4), which serves as the obligatory cofactor for the aromatic amino acid hydroxylases. DHPR deficiency, caused by mutations in the QDPR gene, results in hyperphenylalaninemia and deficiency of various neurotransmitters in the central nervous system, with severe neurological symptoms as a consequence. We have studied, at the clinical and molecular levels, 17 patients belonging to 16 Turkish families with DHPR deficiency. The patients were detected at neonatal screening for hyperphenylalaninemia or upon the development of neurological symptoms. To identify the disease causing molecular defects, we developed a sensitive screening method that rapidly scans the entire open reading frame and all splice sites of the QDPR gene. This method combines PCR amplification and "GC-clamping" of each of the seven exonic regions of QDPR, resolution of mutations by denaturing gradient gel electrophoresis (DGGE), and identification of mutations by direct sequence analysis. A total of ten different mutations were identified, of which three are known (G23D, Y150C, R221X) and the remaining are novel (G17R, G18D, W35fs, Q66R, W90X, S97fs and G149R). Six of these mutations are missense variants, two are nonsense mutations, and two are frameshift mutations. All patients had homoallelic genotypes, which allowed the establishment of genotype-phenotype associations. Our findings suggest that DGGE is a fast and efficient method for detection of mutations in the QDPR gene, which may be useful for confirmatory DNA-based diagnosis, genetic counselling and prenatal diagnosis in DHPR deficiency.     

Mutations of the phenylalanine hydroxylase gene in patients with phenylketonuria in Shanxi,China

The variation in mutations in exons 3, 6, 7, 11 and 12 of the phenylalanine hydroxylase (PAH) gene was investigated in 59 children with phenylketonuria (PKU) and 100 normal children. Three single nucleotide polymorphisms were detected by sequence analysis. The mutational frequencies of cDNA 696, cDNA 735 and cDNA 1155 in patients were 96.2%, 76.1% and 7.6%, respectively, whereas in healthy children the corresponding frequencies were 97.0%, 77.3% and 8.3%. In addition, 81 mutations accounted for 61.0% of the mutant alleles. R111X, H64 > TfsX9 and S70 del accounted for 5.1%, 0.8% and 0.8% mutation of alleles in exon 3, whereas EX6-96A > G accounted for 10.2% mutation of alleles in exon 6. R243Q had the highest incidence in exon 7 (12.7%), followed by Ivs7 + 2 T > A (5.1%) and T278I (2.5%). G247V, R252Q, L255S, R261Q and E280K accounted for 0.8% while Y356X and V399V accounted for 5.9% and 5.1%, respectively, in exon 11. R413P and A434D accounted for 5.9% and 2.5%, respectively, in exon 12. Seventy-two variant alleles accounted for the 16 mutations observed here. The mutation characteristics and distributions demonstrated that EX6-96A > G and R243Q were the hot regions for mutations in the PAH gene in Shanxi patients with PKU.     

Recurrent mutation, gene conversion, or recombination at the human phenylalanine hydroxylase locus: evidence in French-Canadians and a catalog of mutations.

The codon 408 mutation (CGG----TGG, Arg----Trp) in exon 12 of the phenylalanine hydroxylase (PAH) gene occurs on haplotype 1 in French-Canadians; elsewhere this mutation (R408W) occurs on haplotype 2. A CpG dinucleotide is involved. The finding is compatible with a recurrent mutation, gene conversion, or a single recombination between haplotypes 2 and 1. A tabulation of 20 known mutations at the PAH locus reveals three instances of putative recurrent mutation.     

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.     

Phenylketonuria missense mutations in the Mediterranean.

Two missense mutations have been identified in the phenylalanine hydroxylase (PAH) genes of an Italian phenylketonuria (PKU) patient. Both mutations occurred in exon 7 of the PAH gene, resulting in the substitution of Trp for Arg at amino acid 252 (R252W) and of Leu for Pro (P281L) at amino acid 281 of the protein. Expression vectors containing either the normal human PAH cDNA or mutant cDNAs were constructed and transfected into cultured mammalian cells. Extracts from cells transfected with either mutant construct showed negligible enzyme activity and undetectable levels of immunoreactive PAH protein as compared to the normal construct. These results are compatible with the severe classical PKU phenotype observed in this patient. Population genetic studies in the Italian population revealed that both the R252W and the P281L mutations are in linkage disequilibrium with mutant restriction fragment length polymorphism (RFLP) haplotype 1, which is the most prevalent RFLP haplotype in this population. The R252W mutation is present in 10% and the P281L mutation is present in 20% of haplotype 1 mutant chromosomes. These mutations are both very rare among other European populations, suggesting a Mediterranean origin for these mutant chromosomes.     

RFLP haplotyping and mutation analysis of the phenylalanine hydroxylase gene in Dutch phenylketonuria families

Restriction fragment length polymorphism haplotyping of mutated and normal phenylalanine hydroxylase (PAH) alleles in 49 Dutch phenylketonuria (PKU) families was performed. All mutant PAH chromosomes identified by haplotyping (n = 98) were screened for eight of the most predominant mutations. Compound heterozygosity was proven in 40 kindreds. Homozygosity was found for the IVS12nt1 mutation in 5 families, and for the R158Q and IVS10nt546 mutations in one family each. All patients from these families suffer from severe PKU, providing additional proof that these mutations are deleterious for the PAH gene. Genotypical heterogeneity was evident for mutant haplotype 1 (n = 27) carrying the mutations R261Q (n = 12), E280K (n = 4), P281L (n = 1) and unknown (n = 10), and likewise for mutant haplotype 4 (n = 30) carrying the mutations R158Q (n = 13), Y414C (n = 1) and unknown (n = 16). Mutant haplotype 3 (n = 20), in tight association with mutation IVS12nt1, appeared to be in strong linkage disequilibrium (LDE) with its normal counterpart allele (n = 4). Mutant haplotype 6 (n = 4), in tight association with the IVS10nt546 mutation, showed moderate LDE with its counterpart allele (n = I). The distribution of the mutant PAH haplotypes 1, 3 and 4 among the Dutch PKU population resembles that in other Northern and Western European countries, but it is striking that mutant haplotype 2 and its associated mutation R408W is nearly absent in The Netherlands, in strong contrast to its neighbouring countries.     

Molecular Genetics of Phenylketonuria in Inhabitants of Novosibirsk Region

Phenylketonuria is a wide-spread autosomal-recessive hereditary disease due to a deficient activity of the enzyme phenylalanine hydroxylase (EC 1.14.16.1). A decrease of the enzyme activity results from mutations in structure of the phenylalanine hydroxylase gene, whose incidence has pronounced regional and ethnic peculiarities. We have carried out a search for mutations in structure of exons of the phenylalanine hydroxylase gene in the group of 34 phenylketonuric patients, inhabitants of the Novosibirsk region, and evaluated frequencies of the alleles in comparison with other populations. The performed study has shown that the spread of mutant alleles in Siberia seems to be affected by gene flows from Eastern Europe (mutations R408W and R252W) and, to a lesser degree, from Scandinavia (mutations IVS12ntl and Y414C), Western (mutations E280K, R158Q, and R261Q) and Southern Europe (P281L). Alleles have been revealed also characteristic of Southeast Asia (R243Q) and Turkey (R261Q).     

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     

Association between minihaplotypes and mutations at the PAH locus in Polish hyperphenylalaninemic patients

Hereditary hyperphenylalaninemia (HPA) is a disorder of amino acid metabolism and results from an insufficiency of hepatic phenylalanine hydroxylase (PAH). HPA phenotypes form a spectrum ranging from classical phenylketonuria (PKU) to mild hyperphenylalaninemia (MHP). The phenotypic diversity reflects heterogeneity at the molecular level, and more than 320 different mutations in the PAH gene are known to date. The association of 3 mutations (R408W, IVS10 and A403V) common in different European populations with a variable number tandem repeat (VNTR) and short tandem repeat sites (minihaplotype) in the PAH gene was examined in a group of Polish PKU and MHP patients. Additionally, minihaplotypes were established for another 16 mutations. The presented data support the hypothesis that the R408W/VNTR3/STR238 allele originated among pre-Indo-Europeans on the territory in present-day Lithuania and Belarus. Mutation IVS10nt-11g-->a (IVS10) is strongly associated with VNTR7/STR250 minihaplotype and is possibly of Mediterranean origin.