Characterization of mutations at the mouse phenylalanine hydroxylase locus

By using a three-dimensional computed tomography (CT) scanner, we compared the anatomic features of the pelvis of three fetuses of same gestational age, one with a normal pelvis representing the reference model, one with classic bladder exstrophy, and one with cloacal exstrophy. The tomography slices were selected at the same levels for each case. Three angles expressing external opening of the pelvis were defined. Comparing normal and abnormal pelvises allowed definition of three criteria for the correction of the malformation: (a) the sum of the differential angles gives the amplitude of the correction needed; (b) a supraacetabular osteotomy appears to allow best closure of the pelvic ring; (c) only three slices of a CT scan are needed, which cannot be harmful, especially for neonates. Therefore, we believe that a CT scan of the pelvis should be performed whenever an osteotomy is planned in the surgical reconstruction of bladder and cloacal exstrophy.     

P53 tumour-suppressor gene mutations are mainly localised on exon 7 in human primary and metastatic prostate cancer

Mutations in the p53 tumour-suppressor gene are among the most common genetic alterations in human cancers. In the present study we analysed the mutations in the p53 tumor-suppressor gene in 25 primary and 20 metastatic human prostate cancer specimens. DNA extracted from the paraffin-embedded sections was amplified by hot-start polymerase chain reaction, and p53 gene mutations in the conserved mid-region (exons 4-9) were examined using single-strand conformation polymorphism (SSCP) analysis and immunohistochemistry. In the present study, we used a novel hot-start PCR-SSCP technique using DNA Taq polymerase antibody, which eliminates primer-dimers and non-specific products. Because of this new technique, the results of PCR-SSCP showed very high resolution. Polymerase chain reaction products were sequenced directly for point mutations for the p53 gene. Mutations were found in 2 out of 25 primary prostate cancers (8%) and 4 out of 20 metastatic cancers (20%). Mutations were observed exclusively in exon 7 and not in exons 4, 5, 6, 8 or 9. Nuclear accumulation of p53 protein, determined by immunohistochemistry, correlated with the degree of metastasis in prostatic cancer.     

Mutation to phenylalanine of tyrosine 371 in tyrosine hydroxylase increases the affinity for phenylalanine

The aromatic amino acid hydroxylases tyrosine and phenylalanine hydroxylase both contain non-heme iron, utilize oxygen and tetrahydrobiopterin, and are tetramers of identical subunits. The catalytic domains of these enzymes are homologous, and recent X-ray crystallographic analyses show the active sites of the two enzymes are very similar. The hydroxyl oxygens of tyrosine 371 in tyrosine hydroxylase and of tyrosine 325 of phenylalanine hydroxylase are 5 and 4.5 A, respectively, away from the active site iron in the enzymes. To determine whether this residue has a role in the catalytic mechanism as previously suggested [Erlandsen, H., et al. (1997) Nat. Struct. Biol. 4, 995-1000], tyrosine 371 of tyrosine hydroxylase was altered to phenylalanine by site-directed mutagenesis. The Y371F protein was fully active in tyrosine hydroxylation, eliminating an essential mechanistic role for this residue. There was no change in the product distribution seen with phenylalanine or 4-methylphenylalanine as a substrate, suggesting that the reactivity of the hydroxylating intermediate was unaffected. However, the KM value for phenylalanine was decreased 10-fold in the mutant protein. These results are interpreted as an indication of greater conformational flexibility in the active site of the mutant protein.     

The STR252-IVS10nt546-VNTR7 phenylalanine hydroxylase minihaplotype in five Mediterranean samples

IVS10nt546 (IVS10nt-11g-->a) is the most common molecular defect of the phenylalanine hydroxylase gene causing phenylketonuria in Mediterranean populations. Previous studies have proposed various and alternative hypotheses concerning the geographical origin and pattern of diffusion of this mutation in this area. In this study, this issue was re-examined on a large sample (149) of "Mediterranean" IVS10nt546 mutant alleles analysed with multiallelic intragenic polymorphisms. The analysis of intragenic microsatellite (STR) and minisatellite (VNTR) polymorphisms shows allelic heterogeneity of the IVS10nt546 mutation. Eight STR and three VNTR alleles were found in association with the splicing defect. Of the ten detected STR-VNTR combinations ("minihaplotypes"), we identified a predominant allelic association (VNTR7-STR252) embedded in a RFLP-haplotype 6 background, which seems to correspond to the ancestral gene originating in the Turkey-Israel area. Analysis of both absolute and relative gene frequencies of the STR252-IVS10nt546-VNTR7 minihaplotypes, shows statistically significant (P < 0.02) variations and may suggest gene flow from Turkey and/or Israel to Italy and Spain. The associated migratory events need not be unique in time (and people) but seem to suggest they may be traced back to the expansion of the Neolithic culture and people, thus allowing dating of the origin of this mutation to at least 5000-10000 years ago. Alternative hypotheses are discussed to explain, in light of the available historical and pre-historical evidence, the pattern of diffusion of the IVS10nt546 mutation in the Mediterranean basin.     

The role of phenylalanine in structure-function relationships of phenylalanine hydroxylase revealed by radiation target analysis

The activity of rat liver phenylalanine hydroxylase (PAH; phenylalanine 4-monooxygenase, EC 1.14.16.1) is regulated by interaction with its substrate, phenylalanine, and its coenzyme, BH4 [tetrahydrobiopterin (6R-dihydroxypropyl-L-erythro-5,6,7,8-tetrahydropterin)]. The structural changes accompanying these interactions have been studied by radiation target analysis. PAH purified from rat liver was incubated with 2 mM phenylalanine to achieve complete activation of the enzyme. Frozen samples were irradiated with various doses of high energy electrons; samples were subsequently thawed, and several surviving properties of the enzyme were determined. Each parameter decreased as a single exponential function of radiation dose. Radiation target analysis of enzymatic activity yielded a dimeric target size. Similar radiation effects on subunit monomers and on tetrameric structure were observed. Together with results from unactivated enzyme, these data show that phenylalanine increases the interactions between the subunits in a dimer and weakens the interactions between dimers in a tetramer. These alterations prevent the natural cofactor, a tetrahydrobiopterin, from exerting a negative effect on activity.     

High frequency of two mutations in codon 778 in exon 8 of the ATP7B gene in Taiwanese families with Wilson disease

The gene for Wilson disease (WD) has been cloned as a P type copper transporter gene (ATP7B). To elucidate the possible genetic mechanism for the diversity of clinical manifestations, we characterised 22 Taiwanese families with WD by microsatellite haplotyping of close DNA markers D13S314-D13S301-D13S316. We also screened for mutations of codon 778 in the transmembrane region. There were at least 15 haplotypes within eight broad subgroups observed among 44 WD chromosomes. Among the 22 unrelated patients, we found that six patients (27%) carried a codon 778 mutation. Nucleotide sequence analysis showed there were two different mutations: the previously reported Arg778Leu mutation in four patients and Arg778Gln, a new mutation, in two patients. The two different mutations of the same codon occurred in two distinct microsatellite haplotypes.     

APC gene messenger RNA: novel isoforms that lack exon 7

The APC gene at human chromosome 5q21 is responsible for familial adenomatous polyposis coli. Furthermore, sporadic cancers of not only colon but also other digestive organs often contain mutations in the APC gene. A dominant mouse mutation Min that was generated by chemical mutagenesis and causes polyposis in the digestive tract is in the mouse homologue of the human APC gene. The APC mRNA is generated from 15 exons. Two mRNA isoforms were reported which are produced by alternative splicing in the 9th exon. Here, we report novel mRNA isoforms that lack the 7th exon in both mouse and human cells.     

Novel COL7A1 mutations in dystrophic forms of epidermolysis bullosa

OBJECTIVE: To characterize the use of the esophageal tracheal combitube (ETC) in trauma patients who fail orotracheal rapid sequence intubation (RSI). DESIGN: Prospective protocol design and retrospective chart review. MATERIALS AND METHODS: Flight nurses were trained in the use of the ETC by mannequin simulation, videotape review, and didactic sessions. ETC insertion was attempted after failure of two or more attempts at orotracheal RSI. Over a 12-month period, 12 patients had ETC insertion, and 10 cases qualified for review. Injuries, number of failed orotracheal RSI attempts, definitive airway, initial arterial blood gas results, and outcome were recorded. RESULTS: ETC insertion was successful in all 10 patients in whom it was attempted. Definitive airway control was achieved by conversion to orotracheal intubation in seven patients, emergency department cricothyroidotomy in one patient, and operative room tracheostomy in two patients. No patient died because of failure to control the airway. Seven patients requiring ETC had mandible fractures. CONCLUSION: ETC insertion is an effective method of airway control in trauma patients who fail orotracheal RSI. It may be particularly useful in the patient with maxillofacial trauma and offers a practical alternative to surgical cricothyroidotomy in difficult airway situations.     

Mutation of the phenylalanine hydroxylase gene in the population of central Bohemia. Relation to the clinical picture of phenylketonuria

BACKGROUND: Phenylketonuria (PKU) is an autosomal recessive, disease, heterogeneous at the molecular level, caused by mutations in the gene of phenylalanine hydroxylase (PAH). The objective of the present work was to identify mutations and their frequency in the Central Bohemian and Prague population in relation to the clinical phenotype. METHODS AND RESULTS: The authors analyzed a group of 33 patients from 32 unrelated families. The phenotypic manifestations were classified as non-PKU hyperphenylalaninaemia (non-PKU-HPA), mild and classical PKU. Sixty-six mutant alleles of the PAH gene were analyzed by means of the polymerase chain reaction on a Perkin Elmer (480) apparatus and on PHC Techne. A total of eight mutations linked with five haplotypes were detected. R408W mutation linked with 2.4 haplotype was detected on 53% of mutant alleles. No type of mutation was detected by hitherto published procedures in 27% of mutant alleles. CONCLUSIONS: The finding on the distribution and frequency of mutations indicate a genotypic homogeneity of the PKU population in the Central Bohemian area and Prague and are consistent with hitherto published data from the Czech Republic. The revealed data can be used in prenatal and postnatal DNA diagnosis and genotype classification of PKU.     

Association study of structural mutations of the tyrosine hydroxylase gene with schizophrenia and Parkinson''s disease

Although pulmonary metastases are typical of chondrosarcoma, only 2 patients with intermediate grade tumours have been reported with bone metastases and without pulmonary involvement. We report one patient with an intermediate grade chondrosarcoma which metastasised to the lumbar spine following surgical resection of a locally recurrent tumour. The local recurrence and the metastases were resected and she is alive and well after 20 months.     

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

Although the importance of the father for the development of the child is well known and although an increased presence of the father in the family system is often demanded, processes of active shutting out the father occur. The deterioration of relation between father and child is not seen as a result of pathological male personality, but rather as a result of family or couple system. The special situation for counsellors of such cases is discussed.     

Structure of tetrameric human phenylalanine hydroxylase and its implications for phenylketonuria

Phenylalanine hydroxylase (PheOH) catalyzes the conversion of L-phenylalanine to L-tyrosine, the rate-limiting step in the oxidative degradation of phenylalanine. Mutations in the human PheOH gene cause phenylketonuria, a common autosomal recessive metabolic disorder that in untreated patients often results in varying degrees of mental retardation. We have determined the crystal structure of human PheOH (residues 118-452). The enzyme crystallizes as a tetramer with each monomer consisting of a catalytic and a tetramerization domain. The tetramerization domain is characterized by the presence of a domain swapping arm that interacts with the other monomers forming an antiparallel coiled-coil. The structure is the first report of a tetrameric PheOH and displays an overall architecture similar to that of the functionally related tyrosine hydroxylase. In contrast to the tyrosine hydroxylase tetramer structure, a very pronounced asymmetry is observed in the phenylalanine hydroxylase, caused by the occurrence of two alternate conformations in the hinge region that leads to the coiled-coil helix. Examination of the mutations causing PKU shows that some of the most frequent mutations are located at the interface of the catalytic and tetramerization domains. Their effects on the structural and cellular stability of the enzyme are discussed.     

Deletion of twenty seven nucleotides within exon 11 of the band 3 gene identified in ovalocytosis in Lombok Island, Indonesia

This study reports the molecular characterization of ovalocytosis in Lombok Island, Indonesia. The analysis of genomic DNA by polymerase chain reaction shows that all 21 ovalocytotic individuals have two amplified products of different size from a region encompassing exon 11 of the band 3 gene. The sequence of the larger product matched perfectly with that of normal individuals. In the sequence of the smaller product, 27 nucleotides within exon 11 were deleted. The heterozygous presence of the deletion identified in other parts of Southeast Asia was confirmed in patients with ovalocytosis in an isolated island of eastern Indonesia.     

Crystallization and preliminary diffraction analysis of a truncated homodimer of human phenylalanine hydroxylase

A recombinant truncated form (delta1-102/delta428-452) of the non-heme iron-dependent metalloenzyme human phenylalanine hydroxylase (hPAH, phenylalanine 4-monooxygenase; EC 1.14.16.1) was expressed in E. coli, purified to homogeneity as a homodimer (70 kDa) and crystallized using the hanging drop vapour diffusion method. The crystals are orthorhombic, space group C222 with cell dimensions of a = 66.6 A, b = 108.4 A, c = 125.7 A. The calculated packing parameter (Vm) is 3.24 A3/Da with four 2-fold symmetric dimers (or eight momomers) in the unit cell. Data have been collected to 2.0 A resolution.     

Familial adenomatous polyposis coli in the Czech population. I. Detection of an additional 3 mutations out of a total of 7 in exon 15 of the APC gene

BACKGROUND: Familial adenomatous polyposis (FAP) is an autosomal dominant inherited disease characterized by multiple adenomatous polyps in the colon which progress to carcinoma. FAP is caused by germ-line mutation of the tumor-suppressor adenomatous polyposis coli (APC) gene, the structure and coding sequence of which have been known from 1991. The diagnosis of FAP has classically been based on the detection of multiple colorectal adenomas, often after carcinoma development. Presymptomatic genetic testing for the presence of an allele carrying the FAP mutation is now possible using a variety of techniques. METHODS AND RESULTS: The present paper is the first part of an analysis of 37 different Czech families with 83 members affected by FAP. Our goal is to identify the mutation characteristic for each family for early diagnosis of FAP. We screened clinically manifest representatives of nine families for mutations in exon 15 of the APC gene. First, we searched for the mutation hot spots (codons 1061 and 1309, respectively) and later for the entire exon 15. Denatured gradient gel electrophoresis (DGGE) of amplified regions ov exon 15 has been used to identify DNA sequence variations followed by sequencing verification. In seven patients, seven different mutations in exon 15 of APC gene, four deletion mutants (5-base deletions in codons 1061 and 1309, 1-base deletion in codons 759 and 7-base deletion combined with a 2-base insertion in codon 712), one insertion mutation (1-base/A/insertion in codon 1554) and two point mutations (C to T and C to A substitutions in codons 737 and 935, respectively, in both cases leading to formation of stop codons) have been found. CONCLUSIONS: From seven different mutations found, 4 mutations have been previously described (mutations in codons 935, 1061, 1309 and 1554), 3 mutations in the APC gene are described here for the first time, namely the mutations in codons 712, 759 and 767.     

Mutation spectrum and phenylalanine hydroxylase RFLP/VNTR background in 44 Romanian phenylketonuric alleles

PURPOSE: Simultaneous fluorescence in situ hybridization (FISH) was used in a preimplantation genetic diagnosis program to determine which embryos were normal for chromosomes X, Y, 13, 18, and 21. METHODS: Single blastomeres were disrupted and attached to glass slides using acetic acid and ethanol. Using a ratio mixture of chromosome enumeration DNA probes in combination with locus-specific identifier DNA probes, FISH was performed for the identification of chromosomes X, Y, 13, 18, and 21. RESULTS: Fourteen couples enrolled in IVF produced 134 embryos for biopsy. Blastomeres subjected to five-color FISH revealed that 22% of embryos were normal for chromosomes X, Y, 13, 18, and 21. In addition, 52% were abnormal and no results could be detected for 25%. Twelve couples underwent embryo transfer, two couples did not receive embryos due to lack of any normal embryos, and three couples became pregnant. CONCLUSIONS: The simultaneous detection of five-color FISH is a feasible method to detect aneuploidy in preimplantation embryos from women of advanced maternal age.