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.     

Protein stability and in vivo concentration of missense mutations in phenylalanine hydroxylase

A previous computational analysis of missense mutations linked to monogenic disease found a high proportion of missense mutations affect protein stability, rather than other aspects of protein structure and function. The purpose of this study is to relate the presence of such stability damaging missense mutations to the levels of a particular protein present under "in vivo" like conditions, and to test the reliability of the computational methods. Experimental data on a set of missense mutations of the enzyme phenylalanine hydroxylase (PAH) associated with the monogenic disease phenylketonuria (PKU) have been compared with the expected in vivo impact on protein function, obtained using SNPs3D, an in silico analysis package. A high proportion of the PAH mutations are predicted to be destabilizing. The overall agreement between predicted stability impact and experimental evidence for lower protein levels is in accordance with the estimated error rates of the methods. For these mutations, destabilization of protein three-dimensional structure is the major molecular mechanism leading to PKU, and results in a substantial reduction of in vivo PAH protein concentration. Although of limited scale, the results support the view that destabilization is the most common mechanism by which missense mutations cause monogenic disease. In turn, this conclusion suggests the general therapeutic strategy of developing drugs targeted at restoring wild type stability.     

Mutational spectrum of phenylalanine hydroxylase deficiency in the population resident in Catalonia: genotype-phenotype correlation

Hyperphenylalaninemia (HPA) is a group of diseases characterized by the persistent elevation of phenylalanine levels in tissues and biological fluids. It is an autosomal recessive disorder affecting 1 in 10,000 individuals in Caucasian populations and about 1 in 6,600 in Catalonia. We report the mutational spectrum of phenylalanine hydroxylase deficiency in the population living in Catalonia and the genotype-phenotype correlation. The molecular study was performed in 383 samples corresponding to 115 patients from 99 unrelated families and 268 relatives. We have characterized 90% of the mutant alleles; there were 57 different mutations, 49 of which have previously been described, 8 being novel mutations and two being large deletions. The 57 mutations detected corresponded to: five nonsense, seven frameshift, and eight splice defects, the remainder being missense mutations. These mutations cause 72 different genotypes in the 83 families characterized, confirming the mutational heterogeneity of phenylketonuria (PKU) in the Mediterranean population. According to our biochemical classification, our HPA population is composed of 40 PKU (35%), 36 variant PKU (31%), and 39 non-PKU HPA (34%). Mutations such as IVS 10, A403 V, and E390G correlated as expected with the phenotype and the predicted residual activity in vitro. However, in four cases (165 T, V388 M, R261Q, and Y414 C), the observed metabolic phenotype was not consistent with the predicted genotypic effect. The identification of the mutations in the PAH gene and the genotype-phenotype correlation should facilitate the evaluation of metabolic phenotypes, diagnosis, implementation of optimal dietary therapy, and determination of prognosis in the patients and genetic counselling for the patient's relatives.     

A European multicenter study of phenylalanine hydroxylase deficiency: classification of 105 mutations and a general system for genotype-based prediction of metabolic phenotype.

Phenylketonuria (PKU) and mild hyperphenylalaninemia (MHP) are allelic disorders caused by mutations in the gene encoding phenylalanine hydroxylase (PAH). Previous studies have suggested that the highly variable metabolic phenotypes of PAH deficiency correlate with PAH genotypes. We identified both causative mutations in 686 patients from seven European centers. On the basis of the phenotypic characteristics of 297 functionally hemizygous patients, 105 of the mutations were assigned to one of four arbitrary phenotype categories. We proposed and tested a simple model for correlation between genotype and phenotypic outcome. The observed phenotype matched the predicted phenotype in 79% of the cases, and in only 5 of 184 patients was the observed phenotype more than one category away from that expected. Among the seven contributing centers, the proportion of patients for whom the observed phenotype did not match the predicted phenotype was 4%-23% (P<.0001), suggesting that differences in methods used for mutation detection or phenotype classification may account for a considerable proportion of genotype-phenotype inconsistencies. Our data indicate that the PAH-mutation genotype is the main determinant of metabolic phenotype in most patients with PAH deficiency. In the present study, the classification of 105 PAH mutations may allow the prediction of the biochemical phenotype in >10,000 genotypes, which may be useful for the management of hyperphenylalaninemia in newborns.     

E-cadherin destabilization accounts for the pathogenicity of missense mutations in hereditary diffuse gastric cancer

E-cadherin is critical for the maintenance of tissue architecture due to its role in cell-cell adhesion. E-cadherin mutations are the genetic cause of Hereditary Diffuse Gastric Cancer (HDGC) and missense mutations represent a clinical burden, due to the uncertainty of their pathogenic role. In vitro and in vivo, most mutations lead to loss-of-function, although the causal factor is unknown for the majority. We hypothesized that destabilization could account for the pathogenicity of E-cadherin missense mutations in HDGC, and tested our hypothesis using in silico and in vitro tools. FoldX algorithm was used to calculate the impact of each mutation in E-cadherin native-state stability, and the analysis was complemented with evolutionary conservation, by SIFT. Interestingly, HDGC patients harbouring germline E-cadherin destabilizing mutants present a younger age at diagnosis or death, suggesting that the loss of native-state stability of E-cadherin accounts for the disease phenotype. To elucidate the biological relevance of E-cadherin destabilization in HDGC, we investigated a group of newly identified HDGC-associated mutations (E185V, S232C and L583R), of which L583R is predicted to be destabilizing. We show that this mutation is not functional in vitro, exhibits shorter half-life and is unable to mature, due to premature proteasome-dependent degradation, a phenotype reverted by stabilization with the artificial mutation L583I (structurally tolerated). Herein we report E-cadherin structural models suitable to predict the impact of the majority of cancer-associated missense mutations and we show that E-cadherin destabilization leads to loss-of-function in vitro and increased pathogenicity in vivo.     

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.     

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.     

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.     

Missense mutations prevalent in Orientals with phenylketonuria: molecular characterization and clinical implications

Two missense mutations in the phenylalanine hydroxylase (PAH) genes of Orientals with phenylketonuria (PKU) have been identified. A G-to-A transition in exon 7 of the gene results in the substitution of Gln243 for Arg243 (R243Q) and accounts for 18% of all PKU chromosomes among Chinese. An A-to-G transition in exon 6 of the gene results in the substitution of Cys204 for Tyr204 (Y204C) and identifies about 13 and 5% of all PKU chromosomes in the Chinese and Japanese populations, respectively. The R243Q construct produced less than 10% of normal PAH activity in in vitro expression analysis in a eukaryotic cell system, and patients homozygous for this substitution exhibit a severe clinical phenotype. These results are consistent with previous findings in this expression system. The Y204C construct, however, produced near normal levels of PAH enzyme activity and immunoreactivity in this in vitro expression system. Because this substitution is present only on PKU chromosomes, it is a valuable marker for identifying the corresponding mutant allele for carrier screening of PKU. With the characterization of these two substitutions, about 60% of PKU alleles in China can now be identified. The continuing search for additional PKU mutations will permit effective carrier screening and prenatal gene diagnosis of PKU in East Asia.     

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.     

Mutation spectrum of phenylketonuria in Syrian population: Genotype–phenotype correlation

Characterization of the molecular basis of phenylketonuria (PKU) in Syria has been accomplished through the analysis of 78 unrelated chromosomes from 39 Syrian patients with PKU. Phenylalanine hydroxylase (PAH) gene mutations have been analyzed by using molecular detection methods based on the restriction fragment length polymorphism (RFLP), artificial constructed restriction sites (ACRS) PCR and direct DNA sequencing. 56.4% of the patients had cPKU. A mutation detection rate of 79.49% was achieved and sixteen different mutations were found: missense 56.25%, splice site 37.5%, and frameshift 6.25%. The predominant mutation in this population sample was p.R261Q G>A, p.F55>Lfs and p.R243Q G>A. No mutation in six PKU patients was observed. In 57.9% of patient genotypes, the metabolic phenotype could be predicted. The identification of the mutations in the PAH gene and the genotype–phenotype correlation should facilitate the evaluation of metabolic phenotypes, diagnosis, implementation of optimal dietary therapy, and determination of prognosis in the patients and genetic counseling for the patient's relatives.     

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.     

Structure‐energy‐based predictions and network modelling of RASopathy and cancer missense mutations

The Ras/MAPK syndromes (‘RASopathies’) are a class of developmental disorders caused by germline mutations in 15 genes encoding proteins of the Ras/mitogen‐activated protein kinase (MAPK) pathway frequently involved in cancer. Little is known about the molecular mechanisms underlying the differences in mutations of the same protein causing either cancer or RASopathies. Here, we shed light on 956 RASopathy and cancer missense mutations by combining protein network data with mutational analyses based on 3D structures. Using the protein design algorithm FoldX, we predict that most of the missense mutations with destabilising energies are in structural regions that control the activation of proteins, and only a few are predicted to compromise protein folding. We find a trend that energy changes are higher for cancer compared to RASopathy mutations. Through network modelling, we show that partly compensatory mutations in RASopathies result in only minor downstream pathway deregulation. In summary, we suggest that quantitative rather than qualitative network differences determine the phenotypic outcome of RASopathy compared to cancer mutations.     

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     

Analysis of disease-linked rhodopsin mutations based on structure, function, and protein stability calculations

Retinitis pigmentosa (RP) refers to a heterogeneous group of inherited diseases that result in progressive retinal degeneration, characterized by visual field constriction and night blindness. A total of 103 mutations in rhodopsin are linked to RP to date, and the phenotypes range from severe to asymptomatic. To study the relation between phenotype and rhodopsin stability in disease mutants, we used a structure-based approach. For 12 of the mutants located at the protein-lipid interphase, we used the von Heijne water-membrane transfer scale, and we find that 9 of the mutations could affect membrane insertion. For 91 mutants, we used the protein design algorithm FoldX. The 3 asymptomatic mutations had no significant reduced stability, 2 were unsuitable for FoldX analysis since the structure was incorrect in this region, 63 mutations had a significant change in protein stability (> 1.6 kcal/mol), and 23 mutations had energy change values under the prediction error threshold (< 1.6 kcal/mol). Out of these 23, the disease-causing effect could be explained by the involvement in other functions (e.g., glycosylation motifs, the interface with arrestin and transducin, and the cilia-binding motif) for 19 mutants. The remaining 4 mutants were probably incorrectly associated with RP or have functionalities not discovered yet. For destabilizing mutations where clinical data were available, we found a highly significant correlation between FoldX energy changes and the average age of night blindness and between FoldX energy changes and daytime vision loss onset. Our detailed structural, functional, and energetic analysis provides a complete picture of the rhodopsin mutations and can guide mutation-specific therapies.     

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 .     

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.