Molecular basis of phenylketonuria and related hyperphenylalaninemias: mutations and polymorphisms in the human phenylalanine hydroxylase gene.

Mutations in the human phenylalanine hydroxylase gene producing phenylketonuria or hyperphenylalaninemia have now been identified in many patients from various ethnic groups. These mutations all exhibit a high degree of association with specific restriction fragment-length polymorphism haplotypes at the PAH locus. About 50 of these mutations are single-base substitutions, including six nonsense mutations and eight splicing mutations, with the remainder being missense mutations. One splicing mutation results in a 3 amino acid in-frame insertion. Two or 3 large deletions, 2 single codon deletions, and 2 single base deletions have been found. Twelve of the missense mutations apparently result from the methylation and subsequent deamination of highly mutagenic CpG dinucleotides. Recurrent mutation has been observed at several of these sites, producing associations with different haplotypes in different populations. About half of all missense mutations have been examined by in vitro expression analysis, and a significant correlation has been observed between residual PAH activity and disease phenotype. Since continuing advances in molecular methodologies have dramatically accelerated the rate in which new mutations are being identified and characterized, this register of mutations will be updated periodically.     

Mutation analysis of the phenylalanine hydroxylase gene and its clinical implications in two Japanese patients with non-phenylketonuria hyperphenylalaninemia

We describe a mutation analysis for the phenylalanine hydroxylase gene and the clinical outcome of two Japanese patients with non-phenylketonuria (PKU) hyperphenylalaninemia (serum phenylalanine level below 600 μmol/l under a free diet) detected by a mass-screening program. Single strand conformation polynorphism analysis and direct sequencing of their genomic DNAs revealed that non-PKU hyperphenylalaninemia resulted from compound heterozygosity for a mutation causing classical PKU and a mutation with a milder effect on phenylalanine hydroxylase activity. The mutations were R241C and R243Q in exon 7, and R413P in exon 12. The mutation genotypes of the two patients were R241C/R243Q and R241C/R413P. It has been demonstrated that homozygosity for the R243Q or R413P mutation is associated with a severe phenotype of PKU and low in vitro expression activity. In contrast, the R241C mutation has much less effect on phenylalanine hydroxylase activity. The metabolic consequence of each variant allele was confirmed by a phenylalanine loading test in the patients and their parents. The patients achieved normal results in all intellectual and neurologic tests. Brain magnetic resonance imaging revealed no abnormalities. The dietary restriction was continued until 10 years of age in order to maintain the serum phenylalanine level below 400 μmol/l. The genetic analysis to distinguish non-PKU hyperphenylalaninemia from classical PKU helps to determine the principles of dietary management in the early infantile period. Received: June 2, 1998 / Accepted: July 17, 1998     

High frequency of large genomic deletions in the PCCA gene causing propionic acidemia

Mutations in either the PCCA or PCCB genes are responsible for propionic acidemia (PA), one of the most frequent organic acidemias inherited in autosomal recessive fashion. Most of the mutations detected to date in both genes are missense. In the case of PCCA deficient patients, a high number of alleles remain uncharacterized, some of them suspected to carry an exonic deletion. We have now employed multiplex ligation probe amplification (MLPA) and long-PCR in some cases to screen for genomic rearrangements in the PCCA gene in 20 patients in whom standard mutation detection techniques had failed to complete genotype analysis. Eight different deletions were found, corresponding to a frequency of 21.3% of the total PCCA alleles genotyped at our center. Two of the exonic deletions were frequent, one involving exons 3–4 and another exon 23 although in the first case two different chromosomal breakpoints were identified. Absence of exons 3 and 4 which is also the consequence of the novel splicing mutation c.231 + 1g > c present in two patients, presumably results in an in-frame deletion covering 39 aminoacids, which was expressed in a eukaryotic system confirming its pathogenicity. This work describes for the first time the high frequency of large genomic deletions in the PCCA gene, which could be due to the characteristics of the PCCA gene structure and its abundance in intronic repetitive elements. Our data underscore the need of using gene dosage analysis to complement routine genetic analysis in PCCA patients.     

Molecular mechanisms underlying large genomic deletions in ornithine transcarbamylase (OTC) gene

Ornithine transcarbamylase deficiency (OTCD) is an X-linked urea cycle error causing hyperammonemia and orotic aciduria. Clinical diagnosis is generally confirmed by mutation detection. However, in ∼20% of the patients, no mutation is found by conventional mutation-searching strategies, which fail to detect deletions spanning at least a whole exon, large rearrangements, or mutations at non-coding regions. To detect large deletions or duplications, we have applied the multiplex ligation-dependent probe amplification (MLPA) methodology to three OTCD patients (two females and one male). MLPA revealed copy number alterations of OTC exons in all of them. The two females were found to be heterozygous for deletions of either exon 2 or exons 6–9, and the male was confirmed to lack all OTC exons. Females' characterization of the deletion breakpoints by long polymerase chain reaction and sequencing revealed the mutations c.78-3544_217-129del5921 and c.541-600_1005 + 1880del10862 corresponding to exon 2 and exon 6–9 deletions, respectively. Examination of the deletion-flanking regions suggests that exon 2 deletion probably resulted from replication slippage facilitated by a secondary structure formed by two inverted Alu repeats, whereas an Alu – Alu homologous recombination was probably responsible for the exon 6–9 deletion. This work contributes to the identification of novel disease-causing mutations in OTCD and increases the knowledge on possible mutational mechanisms generating deletions in OTC .     

A de novo phenylketonuria mutation: ATG (Met) to ATA (Ile) in the start codon of the phenylalanine hydroxylase gene.

We here describe the detection of a de novo mutation in the phenylalanine hydroxylase gene in a Norwegian phenylketonuria (PKU) patient. This novel mutation, M1I, disrupts the start codon of the gene by a G to A transition. The compound heterozygote genotype (IVS-12/M1I) of this patient predicts that no phenylalanine hydroxylase enzyme is formed, thus leading to a severe classical PKU. Determination of haplotypes and DNA fingerprint patterns indicates a paternal origin of the de novo mutation.     

In vivo regulation of phenylalanine hydroxylase in the genetic mutant hph-1 mouse model

The hph-1 mouse has low liver activity of GTP cyclohydrolase 1, the rate limiting enzyme in the biosynthesis of tetrahydrobiopterin (BH₄). BH₄ is the cofactor for phenylalanine hydroxylase (PAH) and in the early stages of life the hph-1 mouse is hyperphenylalaninemic. At approximately 15 days after birth the blood phenylalanine levels normalize. During this period the animals provide an in vivo model which can be used to study the regulatory effects of phenylalanine on PAH, and for related pediatric metabolic disease in humans; from birth to youth. We therefore, examined; liver PAH activity using BH₄ and 6-methyltetrahydropterin (6MPH₄) as cofactor; PAH total enzyme concentration by Western blotting using the PH8 antibody, and PAH state of phosphorylation using the PH7 antibody from 4 to 18 days after birth. The findings were compared to the wild type animals that are not hyperphenylalaninemic during this period. PAH (6MPH₄) activity and total protein (PH8 antibody) rose steadily in the hph-1 mice. In control mice, both activity and total protein fluctuated. The degree of phosphorylation of PAH in the mutants and the state of activation (as measured by the 6MPH₄/BH₄ activity ratio) increased as phenylalanine levels rose, and decreased when they fell. Similar patterns were not seen in the control animals. These studies provide in vivo evidence that phenylalanine concentration regulates the activity of PAH in the hph-1 mouse and that this acts via a mechanism that includes phosphorylation of the PAH molecule. The kinetic values (K_m and V_max) for mouse PAH are also reported.     

Identification of GLA gene deletions in Fabry patients by Multiplex Ligation-dependent Probe Amplification (MLPA)

Fabry disease is an under-recognized X-linked lysosomal disorder, due to α-galactosidase A deficiency. Most of the mutations in the GLA gene are detectable using genomic sequencing analysis. However, deletions of one or more exons or deletion encompassing the entire gene are undetectable, especially in heterozygous females. The Multiplex Ligation-dependent Probe Amplification (MLPA) is an efficient tool for discovering these rearrangements. In this study two novel different deletions were detected using MLPA assay on two Fabry patients, both resulted mutation negative by sequencing analysis. These data suggest that this screening should be systematically included in genetic testing surveys of patients with Fabry disease.     

应用等位基因特异性扩增检测经典型苯丙酮尿症PAH基因第6外显子基因突变

目的建立快速简便、特异灵敏的鉴定PAH基因第6外显子c.611A〉G突变热点的基因诊断方法。方法针对突变频率较高的PAH基因第6外显子的突变热点c.611A〉G,设计等位基因特异性扩增（amplification refractory mutationsystem,ARMS）的特异引物,对山西省已经临床确诊的70例经典型PKU患儿、c.611A〉G突变患儿的家长及50例正常儿童进行第6外显子扩增,扩增产物测序验证。结果在受检的山西省患儿中,PAH基因第6外显子c.611A〉G突变位点ARMS检测结果和测序结果完全相符。结论 ARMS技术操作简便,重复性和稳定性好,可作为PAH基因热点突变的快速灵敏检测方法。     

In vitro expression analysis of mutations in phenylalanine hydroxylase: Linking genotype to phenotype and structure to function

Mutations in the human phenylalanine hydroxylase gene (PAH) altering the expressed cDNA nucleotide sequence (GenBank U49897) can impair activity of the corresponding enzyme product (hepatic phenylalanine hydroxylase, PAH) and cause hyperphenylalaninemia (HPA), a metabolic phenotype for which the major disease form is phenylketonuria (PKU; OMIM 261600). In vitro expression analysis of inherited human mutations in eukaryotic, prokaryotic, and cell-free systems is informative about the mechanisms of mutation effects on enzymatic activity and their predicted effect on the metabolic phenotype. Corresponding analysis of site-directed mutations in rat Pah cDNA has assigned critical functional roles to individual amino acid residues within the best understood species of phenylalanine hydroxylase. Data on in vitro expression of 35 inherited human mutations and 22 created rat mutations are reviewed here. The core data are accessible at the PAH Mutation Analysis Consortium Web site ( http://www.mcgill.ca/pahdb ). Hum Mutat 11:4–17, 1998. © 1998 Wiley-Liss, Inc.     

Characterization of deletions at 9p affecting the candidate regions for sex reversal and deletion 9p syndrome by MLPA

The distal region on the short arm of chromosome 9 is of special interest for scientists interested in sex development as well as in the clinical phenotype of patients with the 9p deletion syndrome, characterized by mental retardation, trigonocephaly and other dysmorphic features. Specific genes responsible for different aspects of the phenotype have not been identified. Distal 9p deletions have also been reported in patients with 46,XY sex reversal, with or without 9p deletion syndrome. Within this region the strongest candidates for the gonadal dysgenesis phenotype are the DMRT genes; however, the genetic mechanism is not clear yet. Multiple ligation-dependent probe amplification represents a useful technique to evaluate submicroscopic interstitial or distal deletions that would help the definition of the minimal sex reversal region on 9p and could lead to the identification of gene(s) responsible of the 46,XY gonadal disorders of sex development (DSD). We designed a synthetic probe set that targets genes within the 9p23-9p24.3 region and analyzed a group of XY patients with impaired gonadal development. We characterized a deletion distal to the DMRT genes in a patient with isolated 46,XY gonadal DSD and narrowed down the breakpoint in a patient with a 46,XY del(9)(p23) karyotype with gonadal DSD and mild symptoms of 9p deletion syndrome. The results are compared with other patients described in the literature, and new aspects of sex reversal and the 9p deletion syndrome candidate regions are discussed.     

Prediction of multiple hypermutable codons in the human PAH gene: Codon 280 contains recurrent mutations in Quebec and other populations

The predicted mutability profile (MUTPRED) of the phenylalanine hydroxylase (PAH) gene shows that the 48 CpG sites (template and atemplate strands) are either empty of known mutations (7 sites), harbour “PKU” alleles involving CpG doublets (16 sites), or contain mutations that do not involve a C→ T or G→ A substitution in the doublet. These hypermutable sites harbour 32 different mutations in association with at least 66 different haplotypes and hyperphenylalaninemia. The E280K mutation in exon 7 of the PAH gene is a cause of phenylketonuria. It occurs on four different haplotypes in Europeans and on haplotypes 1 and 2 in Quebec. Whereas a single recombination event could explain the two haplotype associations in Quebec, the mutation does involve a CpG dinucleotide. By analyzing multiallelic markers 5′ (STR) and 3′ (VNTR) to the E280K allele on 12 mutant and 30 normal chromosomes, we conclude that recurrent mutation is the likely origin of E280K in Quebec. The PAH mutation databse shows that the allele accounts for 1.5% of PKU chromosomes worlwide. Hum Mutat 9:316–321, 1997. © 1997 Wiley-Liss, Inc.     

Alterations in protein aggregation and degradation due to mild and severe missense mutations (A104D,R157N) in the human phenylalanine hydroxylase gene (PAH)

Phenylalanine hydroxylase (PAH) catalyzes the conversion of phenylalanine to tyrosine; its activity is the major determinant of phenylalanine disposal. Mutations in the corresponding human gene (PAH), which encodes the human hepatic PAH enzyme, result in hyperphenylalaninemia; the resulting phenotypes can range in severity from mild forms of hyperphenylalaninemia with benign outcome to the severe form, phenylketonuria with impaired cognitive development. This paper describes the detailed characterization of two inherited recessive missense mutations in PAH, c.311C→A (A104D) and [c.470G→A;c.471A→C] (R157N), which are associated, respectively, in the homozygous or functionally hemizygous states, with mild and severe metabolic phenotypes. We used three different in vitro PAH expression systems (in Escherichia coli, cell-free rabbit reticulocyte lysates, and human embryonal kidney cells), as well as a unique assay for phenylalanine oxidation in vivo. In each system, we observed alterations of PAH function and physical properties, compared with wild-type enzyme, and differences in relative severity of effects between these two mutations. Pulse-chase experiments showed increased PAH degradation, probably related to observed aberrations in protein folding and altered oligomerization, as a basic mechanism underlying effects of these missense mutations. Hum Mutat 12:344–354, 1998.© 1998 Wiley-Liss, Inc.     

The neuronal nitric oxide synthase (nNOS) gene contributes to the regulation of tyrosine hydroxylase (TH) by cocaine

Recently, we demonstrated that intact nitric oxide (NO) signaling is essential for the development of cocaine behavioral sensitization in adulthood [M.A. Balda, K.L. Anderson, Y. Itzhak, Differential role of the nNOS gene in the development of behavioral sensitization to cocaine in adolescent and adult B6;129S mice, Psychopharmacology (Berl) 200 (2008) 509–519]. Given the requirement of dopamine (DA) transmission in cocaine-induced behavioral sensitization and the interactions between NO and DA systems, the present study investigated the role of the neuronal nitric oxide synthase (nNOS) gene and the effect of cocaine on the expression of tyrosine hydroxylase (TH)-immunoreactive (-ir) neurons. Adult (postnatal day 80) wild type (WT) and nNOS knockout (KO) mice received saline or a sensitizing regimen of cocaine (20 mg/kg) for 5 days. After 24 h, TH immunoreactivity was assessed in the ventral tegmental area (VTA) and the dorsal striatum (dST) using stereology and Western blotting, respectively. We report that (a) nNOS KO mice express lower levels of TH-ir neurons in the VTA compared to WT counterparts, (b) cocaine administration to WT mice significantly increased striatal TH expression, and (c) the same cocaine administration to nNOS KO mice significantly decreased striatal TH expression. Thus, the nitrergic system may contribute to cocaine-induced behavioral sensitization by regulating dopaminergic neurotransmission.     

Double deletions and missense mutations in the first nucleotide-binding fold of the ATP-binding cassette transporter A1 (<Emphasis Type="Italic">ABCA1</Emphasis>) gene in Japanese patients with Tangier disease

 Tangier disease (TD) is a rare autosomal recessive disease characterized by plasma high-density lipoprotein deficiency caused by an ATP-binding cassette transporter A1 (ABCA1) gene mutation. We describe three different mutations in Japanese patients with TD. The first patient was homozygous for double deletions of 1221 bp between intron 12 and 14 and 19.9 kb between intron 16 and 31. The breakpoint sequence analyses suggest that it is a simultaneous event caused by double-loop formation through multiple Alu. The second patient was homozygous for a novel mutation of A3198C in exon 19, resulting in Asn935His. The third patient was homozygous for A3199G of exon 19 that leads to Asn935Ser, which is the same mutation found in German and Spanish families. Both Asn mutations involved Walker A motif of the first nucleotide-binding fold. Received: January 24, 2002 / Accepted: March 11, 2002     

Analysis of the polymorphic (GT)n repeat at the dopamine β‐hydroxylase gene in Spanish patients affected by schizophrenia

The presence of a polymorphic (GT)_n repeat, a microsatellite repeat, at the human dopamine β‐hydroxylase (DBH) gene had been previously investigated in healthy people and in schizophrenic patients. The different DBH genotypes had been found to be associated to different DBH biochemical function, but no differences were found in the allelic and genotype frequencies between schizophrenic and control groups. To further clarify the potential involvement of the variation at the DBH gene in schizophrenia we have studied the DBH (GT)_n repeat in a sample of 47 Spanish schizophrenic patients, in their healthy relatives (n = 72), and in a control population (n = 74). We have been able to identify five different variants of the DBH gene (A1, A2, A3, A4, A5) in the different groups. Subsequent statistical analysis revealed that the genotypes as well as the allele frequencies did not differ significantly among schizophrenic patients and the control population. Interestingly, the allelic variant A2 and the genotype A4/A2 were significantly more frequent in schizophrenic patients as compared with their healthy relatives. However, the association of the A2 allele with schizophrenia was not supported by the haplotype relative risk analysis of transmitted versus nontransmitted alleles. Therefore, although it will be important to extend the present analysis in a larger sample of schizophrenic patients and controls, our results suggest that the (GT)_n does not seem to play a major role in the genetics of schizophrenia at least in this group of Spanish schizophrenic patients. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 96:88–92, 2000. © 2000 Wiley‐Liss, Inc.     

SPG35 contributes to the second common subtype of AR‐HSP in China: frequency analysis and functional characterization of FA2H gene mutations

Hereditary spastic paraplegias (HSPs) encompass a clinically and genetically heterogeneous group of neurodegenerative disorders. Recently, mutations in fatty acid 2‐hydroxylase gene (FA2H) have been identified responsible for HSPs type 35 (SPG35). This study aims to define the contribution of FA2H to Chinese autosomal recessive HSP (AR‐HSP) patients and provide insights into the enzymatic functions of the novel mutations. Direct sequencing of FA2H was conducted in 31 AR‐HSP families and 55 sporadic cases without SPG11, SPG15, SPG5 and SPG7 gene mutations. Enzymatic activity of the mutated proteins was further examined. Three novel mutations were found in two Chinese families, including two compound heterozygous mutations (c.388C>T/p.L130F and c.506+6C>G) and one homozygous mutation (c.230T>G/p.L77R). The c.506+6C>G splice‐site mutation led to the deletion of exon 3. Measurement of enzymatic functions revealed a significant reduction in the enzymatic activity of FA2H associated with p.L130F and p.L77R. Overall, our data widens the spectrum of the mutations on FA2H, and functional analyses indicate that these mutations severely impair the enzymatic activity of FA2H. Furthermore, frequency analysis shows that SPG35 is the second most common subtype of AR‐HSP in China.     

Papillary carcinoma of the thyroid: evidence for a role for hepatocyte growth factor (HGF) in promoting tumour angiogenesis

The pattern of vascularization of papillary carcinoma was investigated in tumour sections from 31 cases and in primary cultures from 12 cases. Tumour sections were immunostained for von Willebrand Factor (vWF) to visualize blood vessels; for endothelial-specific nitric-oxide-synthase (EC-NOS), as a marker of endothelial cell activation; and for Ki-67 to evaluate endothelial cell proliferation. It was found that endothelial cells lining venous vessels located in peritumoural fibrous tissue were intensely EC-NOS-positive and occasionally Ki-67-positive. Capillary vessels of tumour papillae were not stained for Ki-67 and were weakly EC-NOS-positive. Primary cultures of papillary carcinoma cells were used as a potential source of factors active on endothelial cells. It was found that thyroid tumour cells contain RNAs for angiopoietin, vascular endothelial growth factor (VEGF), and VEGF-C; moreover, they release large amounts of VEGF into culture supernatants and exert chemotactic activity in vitro for the endothelial cell line SIEC. The ability of papillary carcinoma cells to release angiogenic factors could be stimulated in vitro. Hepatocyte growth factor (HGF; 25 ng/ml) induced a 1.2- to 5-fold increase in the amount of VEGF released by tumour cells and a 1.2- to 4.2-fold increase in the amount of chemotactic activity present in culture supernatants. Met protein, the high affinity HGF-receptor, is overexpressed in a large proportion of cases of papillary carcinoma. These findings are consistent with the possibility that HGF-Met protein interaction is one of the molecular mechanisms promoting the vascularization of papillary carcinoma of the thyroid.     

Sequence analysis of the gene coding for glyceraldehyde-3-phosphate dehydrogenase (gpd) of Podospora anserina: use of homologous regulatory sequences to improve transformation efficiency

Summary The glyceraldehyde-3-phosphate dehydrogenase (gpd) gene of Podospora anserina has been isolated from a genomic library by heterologous hybridization with the corresponding gene of Curvularia lunata. The coding region consists of 1014 nucleotides and is interrupted by a single intron. The amino-acid sequence encoded by the gpd gene shows a high degree of sequence identity with the corresponding gene products of various fungi. Multiple alignments of all fungal GPD sequences so far available resulted in the construction of a phylogenetic tree. The evolutionary relationships of the various fungi belonging to different taxa will be discussed on the basis of these data. Sequence analysis of 1.9 kbp of the 5 non-coding region revealed the presence of typical fungal promoter elements. Utilizing different parts of the 5 regulatory sequence of the Podospora gpd gene, expression vectors containing a dominant selectable marker gene (hygromycin B phosphotransferase) have been constructed for the transformation of P. anserina protoplasts. The use of these homologous gpd regulatory sequences resulted in a significant increase in transformation efficiencies compared to those obtained with vectors in which the selectable marker gene is under the control of the corresponding heterologous promoter of Aspergillus nidulans.