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.     

The molecular basis of phenylketonuria in Latvia

Characterization of the molecular basis of phenylketonuria (PKU) in Latvia has been accomplished through the analysis of 96 unrelated chromosomes from 50 Latvian PKU patients. Phenylalanine hydroxylase (PAH) gene mutations have been analyzed through a combined approach in which R158Q, R252W, R261Q, G272X, IVS10-11G>A and R408W mutations were first screened for by PCR or restriction generating PCR amplification of PAH gene exons 5, 7, 11 and 12 followed by digestion with the appropriate diagnostic enzyme. Subsequently 'broad range' denaturing gradient gel electrophoresis analysis of the 13 PAH gene exons has been used to study uncharacterized PKU chromosomes. A mutation detection rate of 98% was achieved. 12 different mutations were found, with the most frequent mutation, R408W, accounting for 76% of Latvian PKU alleles. Six mutations (R408W, E280K, R158Q, A104D, R261Q and P281L) represent 92% of PKU chromosomes. PAH VNTR and STR alleles have been also identified and minihaplotype associations with PKU mutations were also determined.     

Discordant phenylketonuria phenotypes in one family: the relationship between genotype and clinical outcome is a function of multiple effects.

Four members spanning three generations of one family have phenylketonuria of varying degrees of severity. Two first cousins were screened in the neonatal period and have had dietary phenylalanine restriction since diagnosis, the older patient having been classified as having more severe PKU and the younger one as having mild PKU. Their mutual grandfather and his older brother also have a significant hyperphenylalaninaemia and are of normal intelligence despite never having had restricted phenylalanine intake. Mutation analysis of the phenylalanine hydroxylase (PAH) gene has established that there are four different mutations, two in exon 2 (F39L and L48S) and two in exon 3 (R111X and S67P), which give rise to PKU in this family. In order to establish their relative severity, we screened the PKU populations of western Scotland and the south west of England for these mutations. The exon 3 mutations are rare; however, F39L is relatively common in Scotland and L48S in England. A comparison of diagnostic blood phenylalanine concentrations in subjects carrying L48S/null or F39L/null mutations with those carrying two null mutations suggest that these exon 2 mutations are less deleterious. Thus, in this family, the different biochemical phenotypes can be explained, in part, by different genotypes at the PAH locus but our results show that the relationship between genotype and clinical outcome is more complex and is a function of multiple effects.     

Phenylketonuria: variable phenotypic outcomes of the R261Q mutation and maternal PKU in the offspring of a healthy homozygote.

Phenylketonuria (PKU) and benign hyperphenylalaninaemia (HPA) result from a variety of mutations in the gene for the hepatic enzyme phenylalanine hydroxylase. PKU has been found in the Israeli population in two variants, classical and atypical. The two are clinically indistinguishable and require treatment with low phenylalanine diet to prevent mental retardation, but show differences in serum phenylalanine levels and in tolerance to this amino acid. Maternal PKU is a syndrome of congenital anomalies and mental retardation that appears in offspring of PKU mothers as a result of fetal exposure to the high phenylalanine level in the maternal blood. We studied a family in which two children with severe, classical PKU and their unaffected brother showed mild signs of maternal PKU. Their mother had no clinical signs of PKU, but the phenylalanine concentration in her serum reached a level that usually characterises PKU patients. This woman represents a rare phenotype, benign atypical PKU. Such 'hidden' PKU in women may lead to maternal PKU in the offspring, similar to overt PKU. Special attention should therefore be paid to women having children with any of the clinical hallmarks of maternal PKU, and to children born to women known to have benign HPA. The mother was also found to be homozygous for a missense mutation at the phenylalanine hydroxylase locus, R261Q, which does not abolish enzymatic activity completely. In two other families, homozygosity for this mutation resulted in atypical PKU in four children. This observation suggests that mutations that do not completely destroy phenylalanine hydroxylase activity may exhibit variable phenotypic expression which is unpredictable. Compound heterozygosity for R261Q and other mutations led in other patients either to classical PKU or to mild benign HPA.     

Response of patients with phenylketonuria in the US to tetrahydrobiopterin

Tetrahydrobiopterin (BH4) responsive forms of phenylketonuria (PKU) have been recognized since 1999. Subsequent studies have shown that patients with PKU, especially those with mild mutations, respond with lower blood phenylalanine (Phe) concentrations following oral administration of 6-R-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4). To determine the incidence of BH4 responding PKU patients in the United States and characterize their phenylalanine hydroxylase (PAH) mutations, a study was undertaken at UTMB in Galveston and the Children's Hospital of Los Angeles on 38 patients with PKU. Patients were screened by a single oral dose of BH4, 10 mg/kg and blood Phe and tyrosine were determined at 0, 4, 8, and 24 h. Twenty-two individuals (58%) responded with marked decrease in blood Phe (>30%) at 24h. Some of the patients that responded favourably were clinically described as having Classical PKU. Blood tyrosine concentrations did not change significantly. Twenty subjects with PKU, responsive and non-responsive to BH4, were enrolled in a second study to evaluate blood Phe response to ascending single doses of BH4 with 10, 20, and 40 mg/kg and to evaluate multiple daily doses, for 7 days each, with 10 and 20 mg/kg BH4. The 7-day trial showed a sustained decrease in blood Phe in 14 of 20 patients taking 20 mg/kg BH4 (70%). Of these 14 patients, 10 (71%) responded with a significant decrease in blood Phe following 10 mg/kg BH4 daily. To understand the mechanism of response to BH4, the kinetics and stability of mutant PAH were studied. We found that mutant PAH responds with increase in the residual enzyme activity following BH4 administration. The increase in activity is multi-factorial caused by increased stability, chaperone effect, and correction of the mutant Km. These studies indicate that BH4 can be of help to patients with PKU, including some considered to have Classical PKU. The PKU population in US is heterogeneous and mutations can be varied so mutations need to be characterized and response to BH4 tested. It is more likely that mutations with residual activity should respond to BH4, therefore the clinical definition of "Classical PKU" should be reconciled with the residual activity of PAH mutations.     

Low proportion of whole exon deletions causing phenylketonuria in Denmark and Germany

Phenylketonuria (PKU) is an autosomal recessive genetic disorder caused by mutations of the gene encoding phenylalanine hydroxylase (PAH). More than 500 different PAH mutations have been identified and about 90% of these are single base mutations. Although the identification rate of the PAH mutations is generally very high, some variants remain unidentified. A fraction of these mutations are the result of genomic deletions or duplications, which are not recognized with standard PCR-based methods. Here we present the results of exon deletion or duplication analysis in a total of 34 families, in which two mutations had not been identified using conventional diagnostic screening techniques. Using multiplex ligation-dependent probe amplification (MLPA), we found a deletion covering exon 1 and exon 2 (c.1-?_168+?del) in one patient, a deletion of exon 3 (c.169-?_352+?del) in four patients, and a deletion of exon 5 (c.442-?_509+?del) in two patients. A deletion was thus detected in about 20% (7/34) of the families tested. Out of a combined cohort of 570 independent PKU patients from Denmark and Germany, exon deletions were identified in a total of four patients. The estimated allelic frequency of exon deletions in PKU patients in these two populations is therefore below 0.5%.     

Molecular characterization of phenylketonuria and tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency in Japan

Phenylketonuria (PKU) is a heterogeneous metabolic disorder caused by a deficiency in hepatic phenylalanine hydroxylase (PAH). On the basis of phenotype/genotype correlations, determination of phenylketonuric genotype is important for classification of the clinical phenotype and treatment of PKU, including tetrahydrobiopterin therapy. We characterized the genotypes of 203 Japanese patients with PKU and hyperphenylalaninemia using the following systems: (1) denaturing high-performance liquid chromatography with a GC-clamped primer; (2) direct sequencing; and, (3) multiplex ligation-dependent probe amplification. Of 406 mutant alleles, 390 (96%) were genotyped; 65 mutations were identified, including 22 new mutations. R413P, R241C, IVS4-1g>a, R111X and R243Q were prevalent mutations. Mutations prevalent in the Japanese cohort are also common in Korean and Northern Chinese populations, suggesting same origin. The spectrum of prevalent mutations was not significantly different among six Japanese districts, indicating that Japan comprises a relatively homogeneous ethnic group. We classified the mutations by clinical phenotypes and in vivo PAH activity and estimated the mutations with potential tetrahydrobiopterin (BH(4)) responsiveness. The frequency of BH(4) responsiveness based on the genotype was 29.1% in Japanese PKU patients. A catalog of PKU genotypes would be useful for predicting clinical phenotype, deciding on the subsequent treatment of PKU including BH(4) therapy, and genetic counseling in East Asia.     

Mutational and phenotypical spectrum of phenylalanine hydroxylase deficiency in Denmark

We describe the genotypes of the complete cohort, from 1967 to 2014, of phenylketonuria (PKU) patients in Denmark, in total 376 patients. A total of 752 independent alleles were investigated. Mutations were identified on 744 PKU alleles (98.9%). In total, 82 different mutations were present in the cohort. The most frequent mutation c.1315+1G>A (IVS12+1G>A) was found on 25.80% of the 744 alleles. Other very frequent mutations were c.1222C>T (p.R408W) (16.93%) and c.1241A>G (p.Y414C) (11.15%). Among the identified mutations, five mutations; c.532G>A (p.E178K), c.730C>T (p.P244S), c.925G>A (p.A309T), c.1228T>A (p.F410I), and c.1199+4A>G (IVS11+4A>G) have not been reported previously. The metabolic phenotypes of PKU are classified into four categories; ‘classical PKU’, ‘moderate PKU’, ‘mild PKU’ and ‘mild hyperphenylalaninemia’. In this study, we assigned the phenotypic outcome of three of the five novel mutations and furthermore six not previously classified mutations to one of the four PKU categories.     

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.     

Classical phenylketonuria in Bulgaria: RFLP haplotypes and frequency of the major mutations.

RFLP haplotypes and common mutations in the phenylalanine hydroxylase gene have been studied in a group of 29 Bulgarian PKU families. Haplotype distribution differs from that in other European populations, with a predominance of haplotypes 2 and 6 and a total absence of haplotype 3. The amino acid substitution in codon 408 is the most frequent molecular defect. The splicing defect in intron 12 is not found in Bulgarian PKU patients. Testing for three mutations, reported to be common among haplotype 1 and 4 alleles, has shown that they occur less frequently in Bulgarian PKU patients. Screening with five pairs of allele specific oligonucleotides failed to show the mutation in 59% of the patients. These findings add to the evidence that PKU is heterogeneous and that significant interpopulation differences exist. At present, DNA data cannot be used as an aid in early clinical classification and prognosis of hyperphenylalaninaemia in Bulgaria.     

Frequency and distribution of phenylketonuric mutations in Orientals.

The frequency and distribution of eight mutations (R111X, IVS4nt-1, Y204C, R243Q, IVS7nt-2, W326X, Y356X, and R413P) in the phenylalanine hydroxylase gene of Orientals in Japan and Korea were examined by allele-specific oligonucleotide hybridization. The mutant alleles comprised 54 and 55% of the phenylketonuria (PKU) chromosomes examined in 36 patients in Japan and 10 patients in Korea, respectively. The spectrum of PKU mutations in Japan was similar to that in China, particularly in northern China, but different from that in Korea. The IVS4nt-1 mutation had a high frequency in Korea and southern China, due to the result of the founder effect and genetic drift. The R413P mutation, which may have originated in the regions surrounding the Baikal, expanded to northern China and Japan. We did not find Caucasian mutations in the Japanese or Korean PKU chromosomes. Thus, PKU mutations occurred after racial divergence between Caucasians and Mongoloids, and there were different founding populations for PKU in the two populations.     

Phenylketonuria mutations in Northern China

Mutation spectrum of phenylalanine hydroxylase (PAH) gene in patients with phenylketonuria (PKU) in Northern China is described with a discussion on genotype-phenotype correlation. By using PCR/SSCP and DNA sequencing, all exons of PAH gene in the 185 unrelated patients with PKU from Northern China were studied. A total of 70 different mutations, including 42 missense, 12 splice, 7 nonsense, 5 deletion, 3 insertion, and 1 silence/splice mutations, were detected in 349/370 mutant alleles (94.3%). Deletion, insertion, and frameshift mutations were found for the first time in China PKU patients. The mutations R243Q, EX6-96A>G, R111X, Y356X, and R413P were the prevalent mutations with relative frequencies of 22.2, 11.1, 8.7, 6.5, and 6.5%, respectively. Fifteen novel mutations were identified in this study: I38fsX19, IVS4+3G>C, Y154H, R157K, R157I, T200fsX6, Q267H, Q267E, F302fsX39, G346R, S349A, L367L, R400K, IVS12+4A>G, and IVS12+6T>A. Each of them occurs at very low frequency (0.3-1.1%). The mutation spectrum of PKU in Chinese is similar to other Asian populations but significantly different from European populations. Altogether, 70 different mutations are found in 109 genotypes distributed among 185 PKU patients. As shown by the analysis, the predicted residual activity found in the majority of PKU individuals match their in vivo phenotypes, though evidence is also found for both phenotypic inconsistencies among subjects with similar genotypes and discordance between the in vitro and in vivo effects of some mutant alleles. The study enables us to construct a national database in China serving as a valuable tool for genetic counseling and prognostic evaluation of future cases of PKU.     

Mutation and haplotype analysis of phenylalanine hydroxylase alleles in classical PKU patients from the Czech Republic: identification of four novel mutations.

Mutations, haplotypes, and other polymorphic markers in the phenylalanine hydroxylase (PAH) gene were analysed in 133 unrelated Czech families with classical phenylketonuria (PKU). Almost 95% of all mutant alleles were identified, using a combination of PCR and restriction analysis, denaturing gradient gel electrophoresis (DGGE), and sequencing. A total of 30 different mutations, 16 various RFLP/VNTR haplotypes, and four polymorphisms were detected on 266 independent mutant chromosomes. The most common molecular defect observed in the Czech population was R408W (54.9%). Each of the other 29 mutations was present in no more than 5% of alleles and 13 mutations were found in only one PKU allele each (0.4%). Four novel mutations G239A, R270fsdel5bp, A342P, and IVS11nt-8g-->a were identified. In 14 (5.1%) alleles, linked to four different RFLP/VNTR haplotypes, the sequence alterations still remain unknown. Our results confirm that PKU is a heterogeneous disorder at the molecular level. Since there is evidence for the gene flow coming from northern, western, and southern parts of Europe into our Slavic population, it is clear that human migration has been the most important factor in the spread of PKU alleles in Europe.     

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.     

Mutation screening of phenylketonuria in the Far East of Russia

We analyzed mutant genotypes at the human phenylalanine hydroxylase (PAH) locus among phenylketonuria (PKU) patients in the Far East of Russia. A total of 60 variant alleles from 30 PKU families were analyzed for prevalent Caucasian mutations and restriction fragment length polymorphism/variable number of tandem repeats (RFLP/VNTR) haplotypes. Seventy-eight percent of all variant alleles carried six mutations. The most prevalent mutation was R408W (63%), with a haplotype background of 2.3. It also showed a very high degree of homozygosity (43%). The other five mutations (R158Q, R261Q, R252W, R261X, and IVS12nt-1) accounted for 1.7%–6.7% of all PKU alleles, and a single haplotype was associated with each genotype, except for R261Q. The genetic structure of PKU patients in the Far East of Russia seems to be relatively homogeneous, compared with that in the other Slavic and Oriental populations of surrounding countries. Prediction of a clinical phenotype and carrier detection will be feasible using DNA tests. Received: June 30, 1999 / Accepted: August 10, 1999     

Two novel mutations in exon 11 of the PAH gene (V1163del TG and P362T) associated with classic phenylketonuira and mild phenylketonuria. Mutations in brief no. 143. Online

PKU is one of the commonest genetic disease in man, affecting 1/10,000 individuals. It presents a wide phenotypical spectrum, from classic PKU to moderate Hyperpheylalaninemia depending on the residual enzymatic activity. Two novel mutations 1163/1164delTG and P362T in exon 11 have been detected during the mutational screening of the PAH gene in 84 families. 1163/1164delTC can be confused with V388M if the mutational screening is performed with BsaAI restriction enzyme, this mutation in heterozigosis presents a moderate phenotype. P362T mutation in heterozigosis with V388M shows a classical PKU phenotype. We report here two new mutations in exon 11 of the PAH gene (GenBank U49897), V1163delTG and P362T (using cDNA sequence), detected during the analysis of 57 PKU and 36 HPA patients belonging to 84 unrelated families detected under a neonatal screening program performed in Catalonia.     

The molecular basis of phenylalanine hydroxylase deficiency in Croatia

We present the results of a comprehensive analysis of mutations, polymorphisms and haplotypes in the phenylalanine hydroxylase (PAH) gene in 39 Croatian families with phenylketonuria (PKU). A total of 21 disease-causing mutations was identified on 78 out of 79 independent chromosomes. The commonest mutation, R408W on haplotype 2 was found with a relative frequency of 37 %. P281L accounted for 11 %, R261Q and E390G each for 9 % of mutant chromosomes. There were three novel mutations: L249P (c.746T>C) in exon 7, IVS8+2T>C (c.912T>C) in intron 8, and F402L (c.1206T>G) in exon 12 of the PAH gene. Two known PKU mutations were found in cis on the same chromosome in one family, highlighting the need to perform full mutation scanning in recessive disease genes for molecular diagnosis even if two known mutations have been identified in a patient. This is the first comprehensive report on PKU mutations in southeastern Europe, adding to the growing bulk of molecular data for population genetic investigations.     

Molecular and phenotypic characteristics of patients with phenylketonuria in Serbia and Montenegro

Phenylketonuria (PKU) is the most common inborn error of amino acid metabolism in Caucasians. PKU is caused by mutations in the gene encoding phenylalanine hydroxylase (PAH) enzyme. Here, we report the spectrum and the frequency of mutations in the PAH gene and discuss genotype-phenotype correlation in 34 unrelated patients with PKU from Serbia and Montenegro. Using both polymerase chain reaction-restriction fragment length polymorphism and 'broad-range' denaturing-gradient gel electrophoresis/DNA sequencing analysis, 19 disease-causing mutations were identified, corresponding to mutation detection rate of 97%. The most frequent ones were L48S (21%), R408W (18%), P281L (9%), E390G (7%) and R261Q (6%), accounting for 60% of all mutant alleles. The genotype-phenotype correlation was studied in homozygous and functionally hemizygous patients. We found that the most frequent mutation, L48S, was exclusively associated with the classical (severe) PKU phenotype. The mutation E390G gave rise to mild PKU. For the mutation R261Q, patients had been recorded in two phenotype categories. Considering allele frequencies, PKU in Serbia and Montenegro is heterogeneous, reflecting numerous migrations over the Balkan Peninsula.     

Mutations in the phenylalanine hydroxylase gene identified in 95 patients with phenylketonuria using novel systems of mutation scanning and specific genotyping based upon thermal melt profiles

Phenylketonuria (PKU, MIM 261600; EC 1.14.16.1) results from mutations in the phenylalanine hydroxylase (PAH) gene. Newborn metabolic disease screening uses blood dried on filter paper (DBS) to prospectively identify candidate newborns affected with PKU via an elevated concentration of phenylalanine. However, it is then important to confirm the specific category of PKU since classical PKU requires a stringent diet while milder categories may not require diet and a very important BH4-responsive category may be treated with the PAH cofactor 6R-tetrahydrobiopterin (BH4). Since there is a close genotype-phenotype correlation in PKU, determining the PAH genotype can be extremely important for therapy as well as prognosis. A simple and rapid method of accurately determining the PAH genotype would be a valuable addition to the diagnosis of PKU. Described herein is a means to identify variants in the PAH gene using high-resolution melt profiling, which compares the thermal denaturation profile of a patient sample to that of a control. Regions where the patient and control samples produce a common profile were not further evaluated, while those regions where the patient profile deviates from the control were assessed by DNA sequencing. Additionally described is a scheme utilizing redundant analysis with melt profile controls and a novel multiplex genotyping assay to triage deviation owing to known polymorphisms. Two mutations were identified in 93 of the 95 patients assessed and in the remaining two patients a single mutation was identified. Melt profiling provided 99% sensitivity to identify sequence variants in the PAH gene.     

Phenylketonuria and hyperphenylalaninemia in eastern Germany: a characteristic molecular profile and 15 novel mutations

Phenylketonuria (PKU) is an important error of amino acid metabolism which results in most patients from phenylalanine hydroxylase (PAH) deficiency. PKU displays a marked genotypic heterogeneity both within and between different populations. The aim of this study was to establish the genotypic spectrum of PKU in eastern Germany, and to compare this to the distribution of mutations in western Germany. The study population included 302 patients in 290 families who were followed at treatment centers in Berlin, Leipzig and Jena. The study showed marked genotypic variability with a total of 75 mutations, including 15 that have so far not been described (eleven missense mutations, one splicing mutation, and three small deletions). One of these novel mutations, E183Q, occurred in cis to a R408W mutation. In the non-immigrant eastern German population, the frequency of R408W accounted for 40.1% of the PKU alleles. In the immigrant Turkish population of the former West Berlin, the most prevalent mutation was IVS10-11G>A (57%). There was a marked difference of the genotypic spectrum between the population studied here and the data reported from the western part of the country.