Molecular characterization of phenylketonuria in Japanese patients

We characterized phenylalanine hydroxylase (PAH) genotypes of Japanese patients with phenylketonuria (PKU) and hyperphenylalaninemia (HPA). PKU and HPA mutations in 41 Japanese patients were identified by denaturing gradient gel electrophoresis and direct sequencing, followed by restriction fragment length polymorphism analysis to find a large deletion involving exons 5 and 6. Of 82 mutant alleles, 76 (92%) were genotyped showing 21 mutations. The major mutations were R413P (30.5%), R243Q (7.3%), R241 C (7.3%), IVS4nt-1 (7.3%), T278I (7.3%), E6nt-96A→g (6.1%), Y356X (4.9%), R111X (3.7%), and 442–706delE5/6 (2.4%). Eight new mutations (L52 S, delS70, S70P, Y77X, IVS3nt-1, A132 V, W187 C, and C265Y) and a polymorphism of IVS10nt-14 were detected. In vitro PAH activities of mutant PAH cDNA constructs were determined by a COS cell expression system. Six mutations, viz., R408Q, L52 S, R241 C, S70P, V388 M, and R243Q, had 55%, 27%, 25%, 20%, 16% and 10% of the in vitro PAH activity of normal constructs, respectively. The mean pretreatment phenylalanine concentration (0.83±0.21 mmol/l) of patients carrying the R408Q, R241 C, or L52 S mutation and a null mutation was significantly lower (P<0.0005) than that (1.99±0.65 mmol/l) of patients with both alleles carrying mutations associated with a severe genotype. Simple linear regression analysis showed a correlation between pretreatment phenylalanine concentrations and predicted PAH activity in 29 Japanese PKU patients (y=31.9–1.03x, r=0.59, P<0.0001). Genotype determination is useful in the prediction of biochemical and clinical phenotypes in PKU and can be of particular help in managing patients with this disorder. Received: 24 July 1998 / Accepted: 12 September 1998     

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.     

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.     

Identification of Mutations in Phenylalanine Hydroxylase Gene Using an Automated Genetic Analyzer

Mutations were studied in phenylalanine hydroxylase gene of phenylketonuria patients from Kemerovo oblast and Altaiskii krai (15 and 2 families, respectively). The following mutations were identified in exons of this gene: R408W, R261Q, R243Q, R158Q, Y414C, Y386C, P281L, Y168H, R68S (lead to amino acid substitutions), R243X (leads to stop codon formation), and three splice site mutations (IVS12nt1g a, IVS2nt-13t g, IVS7nt 1g a).     

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.     

Identification of mutation of the phenylalanine hydroxylase gene using an automated DNA sequencer

Mutations were studied in phenylalanine hydroxylase gene of phenylketonuria patients from Kemerovo oblast and Altaiskii krai (15 and 2 families, respectively). The following mutations were identified in exons of this gene: R408W, R261Q, R243Q, Y414C, Y386C, P281L, Y168H, R68S (lead to amino acid substitutions), R243X (leads to stop codon formation), and three splice site mutations (IVS12nt 1g-->a, IVS2nt-13t-->g, IVS7nt 1g-->a).     

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.     

Frequencies of phenylalanine hydroxylase mutations I65T, R252W, R261Q, R261X, IVS10nt11, V388M, R408W, Y414C, and IVS12nt1 in Minas Gerais, Brazil

In order to determine the phenylketonuria (PKU) mutation spectrum in the population of Minas Gerais State, Brazil, 78 unrelated PKU patients found by the neonatal screening program from 1993 to 2003 were tested for nine phenylalanine hydroxylase mutations. These mutations were selected due to their high frequencies in other Brazilian populations and in Portugal, where the largest contingent of the Caucasian component of the Brazilian population originated from. The most frequent mutations were V388M (21%), R261Q (16%), IVS10nt11 (13.4%), I65T (5.7%), and R252W (5%). The frequencies of the other four mutations (R261X, R408W, Y414C, and IVS12nt1) did not reach 2%. By testing these nine mutations, we were able to identify 64% of the PKU alleles in our sample. V388M frequency was higher than in any other known population and almost three times larger than that observed in Portugal, probably reflecting genetic drift. The mutation profile, as well as the relative frequency of the different mutations, suggest that the Minas Gerais population more closely resembles that of Portugal than do the other Brazilian populations that have already been tested.     

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.     

Analysis of phenylalanine hydroxylase gene mutations in phenylketonuria patients from Kemerovo oblast and the Sakha Republic

This paper presents the results of a molecular genetic study on the phenylalanine hydroxylase (PAH) gene among phenylketonuria (PKU) patients and their family members residing in Kemerovo oblast and the Sakha Republic. To reveal the PAH gene mutations, the researchers applied exon amplification and a direct determination of their nucleotide sequences. The study has revealed both well-known mutations (R158Q, R252W, R261Q, P281L, IVS10 ? 11G > A, R408W, and IVS12 + 1G > A) and some rarely encountered ones (IVS2 + 5G > A, R155H, Y168H, W187R, E221-D222 > Efs, A342T, Y386C, and IVS11 + 1G > C). Some of the studied populations with a mixed ethnic ancestry have been shown to demonstrate a wider spectrum of their PKU-associated alleles.     

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.     

Phenylketonuria in Spain: RFLP haplotypes and linked mutations

In order to investigate the molecular basis of phenylketonuria (PKU) in Spain, we analyzed the restriction fragment length polymorphism (RFLP) haplotypes and common mutations in the phenylalanine hydroxylase (PAH) gene in 32 unrelated Spanish PKU families. The distribution of RFLP haplotypes differs from that of northern Europe. Mutant haplotypes 2 and 3 were completely absent in our sample. Approximately 65% of the mutant alleles are confined to three RFLP haplotypes, namely haplotypes 1, 6 and 9, also frequently found in other Mediterranean populations. We screened for previously described PKU mutations using the polymerase chain reaction and allele-specific oligonucleotides, and found IVS10,165T, E280K and P281L as the major mutations, representing 41% of the PKU alleles. Other mutations found were Y414C, and a new one, P244L. Mutations R408W and IVS12, prevalent in northern Europe, as well as others present in southern European populations (R252W, R261Q, L249F) were not detected in our sample. Our results reveal the genetic heterogeneity present in the Spanish PKU population, which shows similarities to others of Mediterranean origin.     

Phenylalanine hydroxylase deficiency in the Slovak population: Genotype–phenotype correlations and genotype-based predictions of BH4-responsiveness

We investigated the mutation spectrum of the phenylalanine hydroxylase gene (PAH) in a cohort of patients from 135 Slovak PKU families. Mutational screening of the known coding region, including conventional intron splice sites, was performed using high-resolution melting analysis, with subsequent sequencing analysis of the samples showing deviated melting profiles compared to control samples. The PAH gene was also screened for deletions and duplications using MLPA analysis. Forty-eight different disease causing mutations were identified in our patient group, including 30 missense, 8 splicing, 7 nonsense, 2 large deletions and 1 small deletion with frameshift; giving a detection rate of 97.6%. The most prevalent mutation was the p.R408W, occurring in 47% of all alleles, which concurs with results from neighboring and other Slavic countries. Other frequent mutations were: p.R158Q (5.3%), IVS12 + 1G>A (5.3%), p.R252W (5.1%), p.R261Q (3.9%) and p.A403V (3.6%). We also identified three novel missense mutations: p.F233I, p.R270I, p.F331S and one novel variant: c.− 30A>T in the proximal part of the PAH gene promoter. A spectrum of 84 different genotypes was observed and a genotype based predictions of BH4-responsiveness were assessed. Among all genotypes, 36 were predicted to be BH4-responsive represented by 51 PKU families. In addition, genotype–phenotype correlations were performed.     

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.     

Mutation spectrum of the PAH gene in the PKU patients from Khorasan Razavi province of Iran

Background

Characterization of the molecular basis of phenylketonuria (PKU) in North-east of Iran has been accomplished through the analysis of 62 unrelated chromosomes from 31 Iranian PKU patients.

Methods

Phenylalanine hydroxylase (PAH) gene mutations have been analyzed by direct DNA sequencing exons 6, 7, 10 and 11.

Results

A mutation detection rate of 74% was achieved. Eleven different mutations were found, with the most frequent mutation, IVS10-11G > A, accounting for 19% of Khorasan-Razavi PKU alleles. Ten mutations (R176X, E280K, IVS11 + 1G > C, S231P, Q383X, R243X, I224T, E390G, R252W and P281L) represent the rest PKU chromosomes. One novel mutation, Q383X in the homozygote form was identified which is located in the catalytic domain (residues143–410).

Conclusion

With this high detection rate of mutations in North-east of Iran, new strategy for carrier testing could be DNA sequencing of these four exons. The other exons and boundaries will be studied only when either one or no mutations are detected in the initial screen.     

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).     

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)     

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.     

PKU mutations R408Q and F299C in Norway: Haplotype associations,geographic distributions and phenotype characteristics

Summary Details are given concerning the phenylketonuria (PKU) mutations R408Q and F299C. Both mutations were identified among 47 PKU patients, derived from the Norwegian PKU registry. A novel PKU mutation (R408Q) was identified, by single-strand conformation polymorphism analysis, on six out of eight mutant haplotype 12 chromosomes and on none of the other PKU chromosomes. The F299C mutation occurred exclusively on mutant haplotype 8, and was the only mutation associated with this haplotype (on six chromosomes). One patient homozygous for each mutation was found. The patient homozygous for F299C manifested severe PKU, whereas the R408Q homozygote exhibited a mild PKU variant. Pedigree analysis of these families has not, so far, revealed consanguinity. Information on the place of birth of the relevant grandparents of the PKU patients with these mutations suggests that each of these mutations in Norway has originated from a common gene source.     

Mutational spectrum of phenylalanine hydroxylase deficiency in the population resident in Catalonia: genotype-phenotype correlation

Hyperphenylalaninemia (HPA) is a group of diseases characterized by the persistent elevation of phenylalanine levels in tissues and biological fluids. It is an autosomal recessive disorder affecting 1 in 10,000 individuals in Caucasian populations and about 1 in 6,600 in Catalonia. We report the mutational spectrum of phenylalanine hydroxylase deficiency in the population living in Catalonia and the genotype-phenotype correlation. The molecular study was performed in 383 samples corresponding to 115 patients from 99 unrelated families and 268 relatives. We have characterized 90% of the mutant alleles; there were 57 different mutations, 49 of which have previously been described, 8 being novel mutations and two being large deletions. The 57 mutations detected corresponded to: five nonsense, seven frameshift, and eight splice defects, the remainder being missense mutations. These mutations cause 72 different genotypes in the 83 families characterized, confirming the mutational heterogeneity of phenylketonuria (PKU) in the Mediterranean population. According to our biochemical classification, our HPA population is composed of 40 PKU (35%), 36 variant PKU (31%), and 39 non-PKU HPA (34%). Mutations such as IVS 10, A403 V, and E390G correlated as expected with the phenotype and the predicted residual activity in vitro. However, in four cases (165 T, V388 M, R261Q, and Y414 C), the observed metabolic phenotype was not consistent with the predicted genotypic effect. The identification of the mutations in the PAH gene and the genotype-phenotype correlation should facilitate the evaluation of metabolic phenotypes, diagnosis, implementation of optimal dietary therapy, and determination of prognosis in the patients and genetic counselling for the patient's relatives.