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.     

Two mutations within the coding sequence of the phenylalanine hydroxylase gene

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

Identification of a missense phenylketonuria mutation at codon 408 in Chinese

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

Geographical distribution gradients of the major PKU mutations and the linked haplotypes

Summary Analysis of 81 phenylketonuria families from Bulgaria, Lithuania and eastern Germany demonstrated a high frequency of haplotype 2 and the associated Arg⁴⁰⁸ Trp⁴⁰⁸ substitution. Haplotype 3 and the splicing mutation in intron 12 are rare or absent in the groups studies. Pooling the data on European populations suggests a Balto-Slavic origin of the defect in codon 408 of the phenylalanine hydroxylase gene and a geographical gradient in the distribution of both major PKU mutations which may contribute to the higher incidence of classic PKU in northern Europeans.     

CpG hotspot causes second mutation in codon 408 of the phenylalanine hydroxylase gene

Summary A new mutation has been identified in exon 12 of the gene encoding phenylalanine hydroxylase at codon 408. The single base change from guanine to adenine changes the amino acid arginine to glutamine; thus, the mutation is defined as R408Q. This codon is the site of a mutation known to causes phenylketonuria. Both these mutations are located at the same CpG site.     

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

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

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.     

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

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

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.     

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.     

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.     

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).     

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.     

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.     

苯丙酮尿症分子遗传学研究进展

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

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

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

Novel PKU mutation on haplotype 2 in French-Canadians.

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