The PKU mutation S349P causes complete loss of catalytic activity in the recombinant phenylalanine hydroxylase enzyme

The mutation S349P in exon 10 of the phenylalanine hydroxylase (PAH) gene was identified in one Norwegian and one Polish phenylketonuria (PKU) allele on a haplotype 1.7 background. This missense mutation in PAH codon 349 is a T to C transition in cDNA position 1267. This mutation has been reported both on haplotype 1 and 4, suggesting recurrent mutation. In two different expression systems, the pET and the pMAL systems of Escherichia coli, it was shown that the S349P mutation, introduced by site directed mutagenesis, results in complete loss of enzymatic activity. Thus, protein instability alone does not seem to be the direct cause of the lack of activity of this PKU mutation as previously reported.We have identified mutations in the PAH gene of 118 PKU patients in Norway. To obtain information about how the different mutations affect the catalytic properties of the PAH enzyme we have used two prokaryotic expression systems.We detected the mutation S349P (Forrest et al. 1991) in one Norwegian patient and one of Polish ancestry. This mutation has previously been reported on haplotype 4 in North-African Jews (Weinstein et al. 1993), and on haplotype 1 in French-Canadians (John et al. 1992) and in Danes (Guldberg et al. 1993a). Here we present gene expression data showing that the recombinant mutant enzyme has no measurable residual catalytic activity.     

Missense mutations associated with RFLP haplotypes 1 and 4 of the human phenylalanine hydroxylase gene.

We report missense mutations associated with haplotype 1 and haplotype 4 alleles of the human phenylalanine hydroxylase (PAH) gene. Individual exon-containing regions were amplified by polymerase chain reaction from genomic DNA of a PKU patient who was a haplotype 1/4 compound heterozygote. The amplified DNA fragments were subcloned into M13 for sequence analysis. Missense mutations were observed in exons 5 and 7, resulting in the substitution of Arg by Gln at residues 158 and 261 of the enzyme, respectively. Expression analysis in heterozygous mammalian cells after site-directed mutagenesis demonstrated that the Arg158-to-Gln158 mutation is a PKU mutation, whereas the Arg261-to-Gln261 mutation is apparently silent in the assay system. Hybridization analysis using allele-specific oligonucleotide probes demonstrated that the Arg158-to-Gln158 mutation is present in two of six mutant haplotype 4 alleles among the Swiss and constitutes about 40% of all mutant haplotype 4 alleles in the European population. The mutation is not present in normal alleles or in any mutant alleles of other haplotypes. The results provide conclusive evidence that there is linkage disequilibrium between mutation and haplotype in the PAH gene and that multiple mutations have occurred in the PAH gene of a prevalent haplotype among Caucasians.     

Identification of three novel PKU mutations among Chinese: evidence for recombination or recurrent mutation at the PAH locus.

Three novel mutations have been identified in the phenylalanine hydroxylase (PAH) genes of Chinese classical phenylketonuria (PKU) patients. Two of these substitutions (W326X and Y356X) result in the generation of a premature stop codon, while the third (IVS-7nt2) alters an invariant dinucleotide splicing signal. These mutations together account for about 10% of all PKU alleles in the Chinese population. The W326X mutation is associated with PAH RFLP haplotype 4, the most common haplotype in Orientals, while the IVS-7nt2 mutation occurs once on a haplotype 7 chromosome. The Y356X mutation is associated with multiple haplotypes, possibly due to crossover, gene conversion, or recurrent mutation.     

A frameshift mutation in exon 2 of the phenylalanine hydroxylase gene linked to RFLP haplotype 1

Summary A deletion of a single base in codon 55 (exon 2) of the phenylalanine hydroxylase (PAH) gene has been identified by direct DNA sequencing of 94 phenyl-ketonuria (PKU) chromosomes. This mutation alters the reading frame so that a stop signal (TAA) is generated in codon 60 of the PAH gene. Haplotype analysis revealed that all PKU alleles showing the codon 55 frameshift mutation exhibited haplotype 1. In our panel of DNA probes 13% of all mutant haplotype 1 alleles carry this particular mutation. Patients who were compound heterozygotes for this deletion and R408W in exon 12, or the splice mutation in intron 12, were affected by severe PKU. Thus, the clinical data provide additional evidence that haplotype 1 PKU alleles carry molecular defects which confer a null phenotype. In addition, we were able to show that the newly detected mutation occurs on alleles of different ethnic background.     

Molecular genetics of phenylketonuria in Orientals: linkage disequilibrium between a termination mutation and haplotype 4 of the phenylalanine hydroxylase gene.

Phenylketonuria (PKU) is a common metabolic disorder among Chinese, with a prevalence of about 1 in 16,500 births. This frequency is very similar to that among Caucasians. Individual exons of the phenylalanine hydroxylase (PAH) gene with flanking introns were amplified by polymerase chain reaction and cloned into M13 for sequence analysis. An Arg111-to-Ter111 mutation has been identified in exon 3 of the PAH gene in a Chinese PKU patient. The mutation is in linkage disequilibrium with the mutant haplotype 4 alleles which are the most prevalent haplotype among the Orientals. The mutation accounts for about 10% of the Chinese PKU alleles and is absent from the Caucasians, demonstrating that independent mutational events have occurred in the PAH locus after racial divergence.     

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     

A termination mutant prevalent in Norwegian haplotype 7 phenylketonuria genes.

RFLPs in the phenylalanine hydroxylase (PAH) gene locus were determined in 47 Norwegian nuclear families that had at least one child with phenylketonuria (PKU). The PKU haplotype distribution differed somewhat from that of other European populations. Mutant haplotype 7 is relatively rare in other populations but constituted 20% of all mutant haplotypes in Norway. In 14 of the 17 mutant haplotypes 7, a previously unreported deletion of the BamHI restriction site in exon 7 of the PAH gene was observed. The abrogation of the BamHI site was shown to be due to a G-to-T transversion, changing Gly 272 to Ter 272 in exon 7 of the gene, thus directly identifying the PKU mutation. Unlike the families of the other PKU patients, the families with this mutation clustered along the southeastern coast of Norway, suggesting a founder effect for this mutation.     

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.     

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.     

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 .     

Haplotype distribution and mutations at the PAH locus in Croatia

Summary Restriction fragment length polymorphism (RFLP) haplotypes and mutations at the phenylalanine hydroxylase (PAH) locus have been studied in 25 unrelated families from Croatia. The results of RFLP analysis demonstrated that 80% of the mutant alleles were associated with three haplotypes (1, 2 and 4). Eight mutations were detected on the background of six mutant haplotypes, comprising 68% of phenylketonuria (PKU) alleles in Croatia. The mutation in codon 408 was most frequent, as was the haplotype 2 allele with which it was associated. These data are in accordance with formerly published population genetic analyses at the PAH locus, and with studies revealing the molecular basis of the phenotypic heterogeneity of PKU. The codon 281 mutation was more frequent in Croatia than previously observed in other populations.     

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.     

Molecular genetics of phenylketonuria in Mediterranean countries: a mutation associated with partial phenylalanine hydroxylase deficiency.

We report the characterization of a mutation in the phenylalanine hydroxylase (PAH) gene associated with partial residual activity of the enzyme. This point mutation (280glu----lys) was found by sequencing a mutant cDNA clone derived from a needle biopsy of the liver in a child with variant form of phenylketonuria. There is a strict concordance between homozygosity for the mutation and this particular phenotype. The (280glu----lys) mutation is linked to an original and rare RFLP haplotype at the PAH locus found in south Europe and North Africa. So far, this genotype-haplotype association is both inclusive and exclusive. Thirty-three PAH-deficient patients were screened for the mutation by using polymerase chain-reaction amplification of their genomic DNA extracted from Guthrie cards. Since a large number of patients can be screened for a particular mutation by using Guthrie cards, the possibility arises of using these samples collected by national newborn screening centers for prospective and retrospective detection of other mutations in the human genome.     

Identification of a novel phenylketonuria (PKU) mutation in the Chinese: further evidence for multiple origins of PKU in Asia

A novel mutation has been identified in the human phenylalanine hydroxylase (PAH) gene of a Chinese patient with classical phenylketonuria (PKU). It is a single base transition of G to A at the last base in intron 4 of the gene, which abolishes the 3'-acceptor site of the intron. Population screening indicates that this mutation constitutes about 8% of all PKU chromosomes in Chinese but is absent in Japanese and Caucasian PKU patients. It is prevalent in southern China but rare in northern China, providing additional evidence that there were multiple founding populations of PKU in east Asia.     

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.     

CpG dinucleotides are mutation hot spots in phenylketonuria

The coding region of the phenylalanine hydroxylase (PAH) gene contains 22 CpG dinucleotides, including five doublets in the seventh exon of the gene. We hypothesized that CpG doublets could represent mutation hot spots in PAH deficiencies and we carried out the systematic sequence analysis of exon 7 in 20 unrelated PAH-deficient kindreds of Mediterranean ancestry. This procedure resulted in the detection of two novel missense mutations whose location and nature (CG to CA and CG to TG) were consistent with the accidental deamination of a 5-methylcytosine in a CpG doublet (codon 261arg----gln and codon 252arg----trp). Moreover, the codon 261 mutation was found to be associated with mutant restriction fragment length polymorphism (RFLP) haplotype 1, the most frequent mutant RFLP haplotype at the PAH locus in the studies reported thus far. However, since the mutation was detected in only 36% of haplotype 1 mutant alleles, it appears that this haplotype at the PAH locus is genotypically heterogeneous in Mediterranean countries.     

PAH 399 GTA(Val)→GTT(Val), a new silent mutation found in the Chinese

Summary A silent mutation or sequence polymorphism, an A to T substitution at codon 399 in exon 11 of the phenylalanine hydroxylase (PAH) gene has been identified by DNA sequence analysis in the Chinese. The frequencies of this new mutation in normal and abnormal (phenylketonuria; PKU) genes are 0.005 and 0.09, respectively, based on the analyses of 100 apparently normal individuals and 39 PKU patients, as demonstrated by DNA amplification with polymerase chain reaction (PCR) and oligonucleotide hybridization methods. The results suggest that there is linkage disequilibrium between this polymorphism and PKU mutations in the PAH gene; approximately 10% of defect PAH alleles in the Chinese population may be identified with this sequence polymorphic marker.     

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.     

Functional Characterization of Novel Phenylalanine Hydroxylase p.Gln226Lys Mutation Revealed Its Non-responsiveness to Tetrahydrobiopterin Treatment in Hepatoma Cellular Model

Treatment with tetrahydrobiopterin (BH4) is the latest therapeutic option approved for patients with phenylketonuria (PKU)—one of the most frequent inborn metabolic diseases. PKU or phenylalanine hydroxylase (PAH) deficiency is caused by mutations in the PAH gene. Given that some PAH mutations are responsive to BH4 treatment while others are non-responsive, for every novel mutation that is discovered it is essential to confirm its pathogenic effect and to assess its responsiveness to a BH4 treatment in vitro, before the drug is administered to patients. We found a c.676C>A (p.Gln226Lys) mutation in the PAH gene in two unrelated patients with PKU. The corresponding aberrant protein has never been functionally characterized in vitro and its response to BH4 treatment is unknown. Computational analyses proposed that glutamine at position 226 is an important, evolutionary conserved amino acid while the substitution with lysine probably disturbs tertiary protein structure and impacts posttranslational PAH modifications. Using hepatoma cellular model, we demonstrated that the amount of mutant p.Gln226Lys PAH detected by Western blot was only 1.2% in comparison to wild-type PAH. The addition of sepiapterin, intracellular precursor of BH4, did not increase PAH protein yield thus marking p.Gln226Lys as BH4-non-responsive mutation. Therefore, computational, experimental, and clinical data were all in accordance showing that p.Gln226Lys is a severe pathogenic PAH mutation. Its non-responsiveness to BH4 treatment in hepatoma cellular model should be considered when deciding treatment options for PKU patients carrying this mutation. Consequently, our study will facilitate clinical genetic practice, particularly genotype-based stratification of PKU treatment.