Mouse Models of Human Phenylketonuria

Phenylketonuria (PKU) results from a deficiency in phenylalanine hydroxylase, the enzyme catalyzing the conversion of phenylalanine (PHE) to tyrosine. Although this inborn error of metabolism was among the first in humans to be understood biochemically and genetically, little is known of the mechanism(s) involved in the pathology of PKU. We have combined mouse germline mutagenesis with screens for hyperphenylalaninemia to isolate three mutants deficient in phenylalanine hydroxylase (PAH) activity and cross-reactive protein. Two of these have reduced PAH mRNA and display characteristics of untreated human PKU patients. A low PHE diet partially reverses these abnormalities. Our success in using high frequency random germline point mutagenesis to obtain appropriate disease models illustrates how such mutagenesis can complement the emergent power of targeted mutagenesis in the mouse. The mutants now can be used as models in studying both maternal PKU and somatic gene therapy.     

A 3-base pair in-frame deletion of the phenylalanine hydroxylase gene results in a kinetic variant of phenylketonuria.

Phenylketonuria (PKU) is an autosomal recessive disease due to deficiency of a hepatic enzyme, phenylalanine hydroxylase (PAH). The absence of PAH activity results in typical PKU while persistence of a residual enzyme activity gives rise to variant forms of the disease. We report here a 3-base pair in-frame deletion of the PAH gene (delta 194) in a mild variant, with markedly reduced affinity of the enzyme for phenylalanine (Km = 160 nM), and we provide functional evidence for responsibility of the deletion in the mutant phenotype. Since the deletion was located in the third exon of the gene, which presents no homology with other hydroxylases, we suggest that exon 3 is involved in the specificity of the enzyme for phenylalanine. Finally, since none of the 98 PKU patients tested were found to carry this particular deletion, our study suggests that this molecular event probably occurred recently on the background of a haplotype 2 gene in Portugal.     

Molecular basis for nonphenylketonuria hyperphenylalaninemia.

Nonphenylketonuria hyperphenylalaninemia (non-PKU HPA) is defined as phenylalanine hydroxylase (PAH) deficiency with blood phenylalanine levels below 600 mumol/liter (i.e., within the therapeutic range) on a normal dietary intake. Haplotype analysis at the PAH locus was performed in 17 Danish families with non-PKU HPA, revealing compound heterozygosity in all individuals. By allele-specific oligonucleotide (ASO) probing for common PKU mutations we found 12 of 17 non-PKU HPA children with a PKU allele on one chromosome. To identify molecular lesions in the second allele, individual exons were amplified by polymerase chain reaction and screened for mutations by single-strand conformation polymorphism. Two new missense mutations were identified. Three children had inherited a G-to-A transition at codon 415 in exon 12 of the PAH gene, resulting in the substitution of asparagine for aspartate, whereas one child possessed an A-to-G transition at codon 306 in exon 9, causing the replacement of an isoleucine by a valine in the enzyme. It is further demonstrated that the identified mutations have less impact on the heterozygote's ability to hydroxylate phenylalanine to tyrosine compared to the parents carrying a PKU mutation. The combined effect on PAH activity explains the non-PKU HPA phenotype of the child. The present observations that PKU mutations in combination with other mutations result in the non-PKU HPA phenotype and that particular mutation-restriction fragment length polymorphism haplotype combinations are associated with this phenotype offer the possibility of distinguishing PKU patients from non-PKU individuals by means of molecular analysis of the hyperphenylalaninemic neonate and, consequently, of determining whether a newborn child requires dietary treatment.     

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.     

DNA haplotype analyses of patients with hyperphenylalaninemia.

Linkage analysis of phenylketonurics has shown a strong association between the DNA haplotype at the phenylalanine hydroxylase (PAH) locus and phenylketonuria (PKU). Similarly, a genetic linkage between less severe forms of hyperphenylalaninemia (HPA) and the PAH locus has been suggested. In the present study we analyzed this linkage in more detail. Haplotypes at the PAH locus were determined for 19 individuals with moderately elevated plasma phenylalanine and normal urinary neopterin/biopterin ratios. Fourteen of these individuals had plasma phenylalanine levels of 4-10 mg/dl (mild HPA), and the other five had plasma phenylalanine levels of 10-19 mg/dl (atypical PKU). Thirteen of the 15 HPA families consisted of an affected child and at least one other sibling. Elevated plasma phenylalanine was seen to genetically segregate with specific PAH alleles in each family. Summation of the LOD scores for both categories of moderate plasma phenylalanine elevation gave a maximum value of 3.556 at theta = 0. At theta = 0 this gives a probability of linkage between the PAH locus and the locus for moderate phenylalanine elevations that is approximately 3,600:1. None of the alleles segregating with either mild HPA or atypical PKU were of haplotype 2 or 3, and 13/20 were of types 1 or 4. This is in agreement with the most deleterious mutations being on haplotypes 2 and 3 and with the less severe mutations being on haplotypes 1 and 4. chi 2 Analyses indicated no statistically significant correlation between HPA and a particular haplotype or restriction-enzyme site.     

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.     

Rapid single-base mismatch detection in genotyping for phenylketonuria

Phenylketonuria (PKU) is a metabolic disorder that results from a deficiency of hepatic phenylalanine hydroxylase (PAH). Identification of the PKU genotype is useful for predicting clinical PKU phenotype. More than 400 mutations resulting in PAH deficiency have been reported worldwide. We used a genedetecting instrument to identify the nine prevalent Japanese mutations in the PAH gene among 31 PKU patients as a preliminary study. This instrument can automatically detect mutations through the use of allele-specific oligonucleotide (ASO) capture probes, and gave results comparable to those of sequencing studies. Each country has uniquely prevalent and specific mutations causing PKU, and less than 50 types of such mutations are generally present in each country. Early genotyping of PKU makes it possible to identify the phenotype and select the optimal therapy for the disease. For early genotyping, the instrumental method described here shortens the time required for genotyping based on mRNA and/or genomic DNA of PKU parents.     

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.     

The phenylketonuria locus: current knowledge about alleles and mutations of the phenylalanine hydroxylase gene in various populations

Summary The hyperphenylalaninemic disorders of classic phenylketonuria (PKU), mild phenylketonuria, and hyperphenylalaninemia (HPA), result from a deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH) or its cofactor (tetrahydrobiopterin). Use of the complementary DNA of this enzyme has allowed the establishment of a restriction fragment length polymorphism (RFLP) haplotype-analysis system. This haplotype analysis system provides the means for determination of mutant PAH alleles in most affected families and is the basis for mutational analysis of the PKU locus. This review is focused on two major areas of current PKU research: (1) the use of DNA haplotype analysis in the study of the population genetics of PAH deficiency, and (2) the study of genotypes, and their various combinations, as a means of explaining and predicting the phenotypic variability observed for the disorders of PAH deficiency.     

Simultaneous measurement of phenylalanine and tyrosine in phenylketonuric plasma and dried blood by high-performance liquid chromatography

Phenylketonuria (PKU) is a disorder characterized by an interruption in the conversion of phenylalanine to tyrosine, a reaction catalyzed by phenylalanine hydroxylase (PAH). Animal models of PKU used in this study were induced by daily subcutaneous injections of pups with alpha-methylphenylalanine plus phenylalanine in utero and postnatally from day 4 to day 14. Dry blood and plasma were utilized to measure phenylalanine concentration in PKU rats. The results indicated that the concentration of phenylalanine is higher and more stable in plasma than dry blood. Precolumn derivatization of dried blood and plasma free amino acids were conducted with phenylisothiocyanate (PITC). The phenylthiocarbamyl (PTC) derivatives were separated on a reversed-phase C-18 column (15 cm x 4.6 mm). A gradient high-performance liquid chromatography method with two eluents, 0.1 M sodium acetate buffer and 100% acetonitrile was developed to facilitate the separation of nine amino acids within 11 min. Tyrosine and phenylalanine eluted the column at 5.4 and 9.4 min, respectively. This method provides a quick and reliable technique for neonatal screening.     

Molecular characterization of Thai patients with phenylalanine hydroxylase deficiency and in vitro functional study of two novel PAH variants

Molecular Biology Reports - Phenylketonuria (PKU) is an autosomal recessive amino acid metabolism disorder caused by variants in the gene encoding phenylalanine hydroxylase (PAH; EC1.14.16.1). This...     

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.     

Mutations in the regulatory domain of phenylalanine hydroxylase and response to tetrahydrobiopterin

Tetrahydrobiopterin (BH4) is a co-factor that enhances the activity of other enzymes, and this co-factor level is found to be affected in phenylketonuria (PKU), an amino acid metabolism disorder. The present study was aimed at understanding the effect of BH4 on mutations in the regulatory domain of phenylalanine hydroxylase (PAH). Among 14 patients, 5 patients were classical PKU, 3 were atypical PKU, and 6 were mild PKU. All of these patients had at least one mutation in the regulatory domain. Patients were given 10 mg/kg BH4, and the response of blood phenylalanine (Phe) levels was monitored following treatment. The level of blood Phe decreased after BH4 treatment in all of the patients. These studies suggest that mutations in the regulatory domain also responded to BH4 even if the patient had classical PKU.     

中国北方人苯丙氨酸羟化酶基因外显子7内新突变的鉴定

应用ＰＣＲ－单链构象多态性分析及ＤＮＡ直接测序,对４５例中国北方苯丙酮尿症（ＰＫＵ）患者苯丙氨酸羟化酶（ＰＡＨ）基因外显子７内突变进行了鉴定。共检出６种错义突变及一种静止突变：Ｒ２４３Ｑ．Ｒ４１Ｈ,Ｇ２４７Ｖ．Ｌ２４９Ｈ．Ｐ２５４Ｉ．Ｇ２５７Ｖ和Ｖ２４５Ｖ。经与国际ＰＡＨ基因突变数据库比较,确认Ｇ２５７Ｖ．Ｐ２５４Ｉ和Ｌ２４９Ｈ为国际上首次发现的突变。结果揭示,中国人与其他种族及中国北方与南方人群ＰＡＨ突变特点不同。明确了中国北方人群中ＰＡＨ基因外显子７基因突变分布,有助于提高ＰＫＵ的基因诊断率,对基因的起源、进化研究有参考价值     

Human Phenylalanine Hydroxylase Mutations and Hyperphenylalaninemia Phenotypes: A Metanalysis of Genotype-Phenotype Correlations

We analyzed correlations between mutant genotypes at the human phenylalanine hydroxylase locus (gene symbol PAH) and the corresponding hyperphenylalaninemia (HPA) phenotypes (notably, phenylketonuria [OMIM 261600]). We used reports, both published and in the PAH Mutation Analysis Consortium Database, on 365 patients harboring 73 different PAH mutations in 161 different genotypes. HPA phenotypes were classified as phenylketonuria (PKU), variant PKU, and non-PKU HPA. By analysis both of homoallelic mutant genotypes and of "functionally hemizygous" heteroallelic genotypes, we characterized the phenotypic effect of 48 of the 73 different, largely missense mutations. Among those with consistent in vivo expression, 24 caused PKU, 3 caused variant PKU, and 10 caused non-PKU HPA. However, 11 mutations were inconsistent in their effect: 9 appeared in two different phenotype classes, and 2 (I65T and Y414C) appeared in all three classes. Seven mutations were inconsistent in phenotypic effect when in vitro (unit-protein) expression was compared with the corresponding in vivo phenotype (an emergent property). We conclude that the majority of PAH mutations confer a consistent phenotype and that this is concordant with their effects, when known, predicted from in vitro expression analysis. However, significant inconsistencies, both between in vitro and in vivo phenotypes and between different individuals with similar PAH genotypes, reveal that the HPA-phenotype is more complex than that predicted by Mendelian inheritance of alleles at the PAH locus.     

Preliminary mutation analysis in the phenylanaline hydroxylase gene in Greek PKU and HPA patients

The presence of nine mutations in the phenylalanine hydroxlase (PAH) gene, previously described in phenylketonuria (PKU) patients of other Mediterranean and European populations, was assessed in 47 Greek PKU and 3 hyperphenylalaninaemia (HPA) patients. Of the nine mutations investigated, only five were detected, characterizing 31 % of the PKU alleles in our patients.     

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.