Human Phenylalanine Hydroxylase Gene Research Articles

Analysis of exon 7 of the human phenylalanine hydroxylase gene: a mutation hot spot?

Complete sequence analysis of 194 human phenylalanine hydroxylase genes from PKU patients originating from West Germany and Bulgaria revealed 13 different mutations within exon 7 of the gene. Four of these mutations (T238P: ACT-->CCT; L242F:CTC-->TTC; R252G:CGG-->GGG; and 1043 delta 11: nt 1043-nt 1053 deleted) have so far not been described in the literature. Including these new mutations at least 21 different gene lesions and one sequence polymorphism exist for exon 7. Despite this large number unbiased calculation of the mutation frequency/exon size ratio does not provide conclusive evidence that exon 7 is a hot spot for disease causing mutations. Extensive screening during our experiments also failed to demonstrate the existence of excessive polymorphism in this part of the gene. It might therefore be speculated that the functional importance of the highly conserved exon 7 sequence accounts for the clustering of observed mutations which result in clinically manifest PKU. In addition we report our experience in regard to the resolution capacity of denaturing gradient gel electrophoresis (DGGE), a nonradioactive technique for the rapid screening of unknown mutations in exon 7.

Application of natural and amplification created restriction sites for the diagnosis of PKU mutations

PCR amplification, either conventional, or as site directed mutagenesis using primers with mismatched 3'-ends, followed by restriction endonuclease digestion, provides rapid, non-isotope assays of known mutations in the human phenylalanine hydroxylase gene. Such assays were shown to have the potential to detect all of the 18 presently reported phenylketonuria mutations. The practical applicability of this approach was demonstrated for eight mutations in Norwegian phenylketonuria patients, among them the most common ones.

4965190 Methods for the identification of mutations in the human phenylalanine hydroxylase gene using DNA probes: Savio L Woo, Anthony G Dilella assigned to Howard Hughes Medical Institute

4965190 Methods for the identification of mutations in the human phenylalanine hydroxylase gene using DNA probes: Savio L Woo, Anthony G Dilella assigned to Howard Hughes Medical Institute

4965190 Methods for the identification of mutations in the human phenylalanine hydroxylase gene using DNA probes: Savio L Woo, Anthony G Dilella assigned to Howard Hughes Medical Institute

4965190 Methods for the identification of mutations in the human phenylalanine hydroxylase gene using DNA probes: Savio L Woo, Anthony G Dilella assigned to Howard Hughes Medical Institute

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 nucelotide 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 iesion. 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.

Phenylketonuria: detection of a frequent haplotype 4 allele mutation.

By sequence analysis of 94 phenylketonuria (PKU) alleles using polymerase chain reaction (PCR) based techniques, we identified a G to A transition in exon 5 of the human phenylalanine hydroxylase gene. This base substitution predicts an Arg158----Glu158 amino acid exchange and is strongly associated with the mutant haplotype 4 PKU allele.

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.

Polymorphic DNA haplotypes at the human phenylalanine hydroxylase locus and their relationship with phenylketonuria.

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 appears 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 utility of the PAH haplotypes for prenatal diagnosis and detection of PKU carriers is established.

GT to AT transition at a splice donor site causes skipping of the preceding exon in phenylketonuria.

Classical Phenylketonuria (PKU) is an autosomal recessive human genetic disorder caused by a deficiency of hepatic phenylalanine hydroxylase (PAH). We isolated several mutant PAH cDNA clones from a PKU carrier individual and showed that they contained an internal 116 base pair deletion, corresponding precisely to exon 12 of the human chromosomal PAH gene. The deletion causes the synthesis of a truncated protein lacking the C-terminal 52 amino acids. Gene transfer and expression studies using the mutant PAH cDNA indicated that the deletion abolishes PAH activity in the cell as a result of protein instability. To determine the molecular basis of the deletion, the mutant chromosomal PAH gene was isolated from this individual and shown to contain a GT-- greater than AT substitution at the 5' splice donor site of intron 12. Thus, the consequence of the splice donor site mutation in the human liver is the skipping of the preceding exon during RNA splicing.

Molecular basis of phenylketonuria and recombinant DNA strategies for its therapy.

Mutations in the human phenylalanine hydroxylase gene associated with two prevalent mutant alleles have been identified and shown to be in linkage disequilibrium with the corresponding mutant restriction fragment length polymorphism haplotypes. These results suggest the possibility of carrier detection in the population without a prior family history of phenylketonuria (PKU). Furthermore, recombinant retroviruses containing the full-length human phenylalanine hydroxylase cDNA have been constructed and used to transduce functional enzymatic activity into cultured hepatoma cells. Together with the recent success in retroviral infection of primary mouse hepatocytes, it will be possible to use the mouse model to investigate somatic gene therapy for PKU.

P-Chlorophenylalanine does not inhibit production of recombinant human phenylalanine hydroxylase in NIH3T3 cells or E. coli

P-chlorophenylalanine is an irreversible inhibitor of rat phenylalanine hydroxylase in vivo and in rat hepatoma cells and is frequently administered to rodents to create an animal model for phenylketonuria. We investigated the effect of p-chlorophenylalanine on production of human phenylalanine hydroxylase in human hepatoma cells and cells transformed with the recombinant human phenylalanine hydroxylase gene. P-chlorophenylalanine inhibited production of the human enzyme in human hepatoma cells and transformed mouse hepatoma cells but had no effect on the production of the enzyme in transformed NIH3T3 cells or in E. coli. Thus, phenylalanine hydroxylase inhibition does not result from a simple interaction between the drug and enzyme.

Tight linkage between a splicing mutation and a specific DNA haplotype in phenylketonuria.

The first phenylketonuria mutation identified in the human phenylalanine hydroxylase gene is a single base substitution (GT----AT) in the canonical 5'-splice donor site of intron 12. Direct hybridization analysis using specific oligonucleotide probes demonstrates that the mutation is tightly associated with a specific restriction fragment-length polymorphism haplotype among mutant alleles. The splicing mutation is the most prevalent phenylketonuria allele among Caucasians, and the results suggest the possibility of detecting carriers of the genetic trait who have no family history of phenylketonuria.

Molecular genetics of PKU.

AbstractThis review summarizes the isolation of rat phenylalanine hydroxylase mRNA and its use in the synthesis of its cDNA. As rat cDNA cross‐hybridized with human phenylalanine hydroxylase mRNA, the rat cDNA probe was used to screen a human liver cDNA library. A partial length cDNA human phenylalanine hydroxylase probe was obtained which showed restriction fragment length polymorphism (RFLP) with 3 restriction enzymes and was successfully used to trace the transmission of the mutant gene in PKU families with one or more affected children. Recently the partial‐length cDNA probe has been used to isolate a full‐length cDNA probe for human phenylalanine hydroxylase. Gene transfer experiments with the full‐length cDNA have led to expression of human phenylalanine hydroxylase in eukaryotic cultured cells and in recombinant bacteria which normally do not express phenylalanine hydroxylase activity. The full‐length cDNA of human phenylalanine hydroxylase has been sequenced, uncovering the nucleic acid sequence of the exons of the human phenylalanine hydroxylase gene, as well as the most likely amino acid structure of the human phenylalanine hydroxylase enzyme. The full‐length cDNA probe has 10 identifiable binding sites for restriction enzymes that show RFLP. These additional RFLPs have enabled haplotype analyses of the normal and mutant phenylalanine hydroxylase genes in PKU families. Haplotype analyses in Danish PKU families revealed 12 different haplotypes. However, of 132 chromosomes analysed from 66 obligate heterozygotes, 59 out of 66 PKU genes were associated with only 4 haplotypes. Cosmid cloning and preliminary characterization of the human phenylalanine hydroxylase gene have identified 13 exons distributed across a gene that is more than 190 kb in length. In β‐thalassaemia, distinct mutations in the β‐globin locus are associated with specific RFLP haplotypes within a given population. As in thalassaemia such an association forms a strategy for cloning and sequence characterization of mutant phenylalanine hydroxylase genes derived from each haplotype. If the PKU genes in the Danish population are the result of mutiple mutations which occurred on chromosomes of the most common haplotypes, the same strategy is potentially applicable for the molecular characterization of the various types of phenylalanine hydroxylase deficiency.

Molecular structure and polymorphic map of the human phenylalanine hydroxylase gene

Human phenylalanine hydroxylase is a liver-specific enzyme that catalyzes the conversion of phenylalanine to tyrosine. Absence of enzymatic activity results in phenylketonuria, a genetic disorder that causes development of severe mental retardation in untreated children. In this paper we report the cloning and structure of the normal human phenylalanine hydroxylase gene, which was isolated in four overlapping cosmid clones that span more than 125 kilobases (kb) of the genetic locus. The peptide coding region of the gene is about 90 kb in length and contains 13 exons, with intron sizes ranging from 1 to 23 kb. Exons at the 3' half of the gene are compact, whereas those at the 5' half are separated by large introns. The human phenylalanine hydroxylase gene codes for a mature messenger RNA of approximately 2.4 kb, and its noncoding to coding DNA ratio is one of the highest among eukaryotic genes characterized to date. The map positions of nine polymorphic restriction sites identified within the locus were established by restriction enzyme mapping of the cloned gene fragments. Two clusters of polymorphic sites were demonstrated: (1) BglII, PvuII(a), and PvuII(b) at the 5' end of the gene and (2) EcoRI, XmnI, MspI(a), MspI(b), EcoRV, and HindIII at the 3' end. The polymorphic site distribution within this gene is a useful tool for prenatal diagnosis and carrier detection of the genetic disorder, while knowledge of normal gene structure is a prerequisite for future characterization of mutant alleles.

Retroviral-mediated gene transfer of human phenylalanine hydroxylase into NIH 3T3 and hepatoma cells.

Phenylketonuria (PKU) is caused by deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH). A full-length human PAH cDNA sequence has been inserted into pzip-neoSV(X), which is a retroviral vector containing the bacterial neo gene. The recombinant has been transfected into psi 2 cells, which provide synthesis of the retroviral capsid. Recombinant virus was detected in the culture medium of the transfected psi 2 cells, which is capable of transmitting the human PAH gene into mouse NIH 3T3 cells by infection leading to stable incorporation of the recombinant provirus. Infected cells express PAH mRNA, immunoreactive PAH protein, and exhibit pterin-dependent phenylalanine hydroxylase activity. The recombinant virus is also capable of infecting a mouse hepatoma cell line that does not normally synthesize PAH. PAH activity is present in the cellular extracts and the entire hydroxylation system is reconstituted in the hepatoma cells infected with the recombinant viruses. Thus, recombinant viruses containing human PAH cDNA provide a means for introducing functional PAH into mammalian cells of hepatic origin and can potentially be introduced into whole animals as a model for somatic gene therapy for PKU.

PRENATAL DIAGNOSIS OF CLASSIC PHENYLKETONURIA BY DNA ANALYSIS

Prenatal diagnosis of classic phenylketonuria (PKU) was performed in two at-risk families by means of a cloned human phenylalanine hydroxylase gene probe which was used to analyse DNA isolated from cultured amniotic fluid cells. The diagnoses of a PKU fetus in one family and a heterozygous fetus in another family were confirmed after birth. The prenatal diagnosis procedure by DNA analysis can be confidently applied to 90% of caucasian families with previously affected children.

Cloned human phenylalanine hydroxylase gene allows prenatal diagnosis and carrier detection of classical phenylketonuria.

The human gene for the hepatic enzyme phenylalanine hydroxylase has been cloned and used to analyse the phenylalanine hydroxylase locus in the human genome. The detection of polymorphisms in this locus by several restriction enzymes has allowed feasibility studies of prenatal diagnosis of classical phenylketonuria and identification of carriers of the trait. Results indicate that these services could be provided for up to 75% of all families with phenylketonuric children in the general Caucasian population.