Identification of three novel PKU mutations among Chinese: evidence for recombination or recurrent mutation at the PAH locus.

Three novel mutations have been identified in the phenylalanine hydroxylase (PAH) genes of Chinese classical phenylketonuria (PKU) patients. Two of these substitutions (W326X and Y356X) result in the generation of a premature stop codon, while the third (IVS-7nt2) alters an invariant dinucleotide splicing signal. These mutations together account for about 10% of all PKU alleles in the Chinese population. The W326X mutation is associated with PAH RFLP haplotype 4, the most common haplotype in Orientals, while the IVS-7nt2 mutation occurs once on a haplotype 7 chromosome. The Y356X mutation is associated with multiple haplotypes, possibly due to crossover, gene conversion, or recurrent mutation.     

Identification of a novel phenylketonuria (PKU) mutation in the Chinese: further evidence for multiple origins of PKU in Asia

A novel mutation has been identified in the human phenylalanine hydroxylase (PAH) gene of a Chinese patient with classical phenylketonuria (PKU). It is a single base transition of G to A at the last base in intron 4 of the gene, which abolishes the 3'-acceptor site of the intron. Population screening indicates that this mutation constitutes about 8% of all PKU chromosomes in Chinese but is absent in Japanese and Caucasian PKU patients. It is prevalent in southern China but rare in northern China, providing additional evidence that there were multiple founding populations of PKU in east Asia.     

Identification and expression of eight novel mutations among non-Jewish patients with Canavan disease.

Canavan disease is inherited as an autosomal recessive trait that is caused by the deficiency of aspartoacylase (ASPA). The majority of patients with Canavan disease are from an Ashkenazi Jewish background. Mutations in ASPA that lead to loss of enzymatic activity have been identified, and E285A and Y231X are the two predominant mutations that account for 97% of the mutant chromosomes in Ashkenazi Jewish patients. The current study was aimed at finding the molecular basis of Canavan disease in 25 independent patients of non-Jewish background. Eight novel and three previously characterized mutations accounted for 80% (40/50) of mutant chromosomes. The A305E missense mutation accounted for 48% (24/50) of mutant chromosomes in patients of western European descent, while the two predominant Jewish mutations each accounted for a single mutant chromosome. The eight novel mutations identified included 1- and 4-bp deletions (32 deltaT and 876 deltaAGAA, respectively) and I16T, G27R, D114E, G123E, C152Y, and R168C missense mutations. The homozygous 32 deltaT deletion was identified in the only known patient of African-American origin with Canavan disease. The heterozygosity for 876 deltaAGAA mutation was identified in three independent patients from England. Six single-base changes leading to missense mutations were identified in patients from Turkey (D114E, R168C), The Netherlands (I16T), Germany (G27R), Ireland (C152Y), and Canada (G123E). A PCR-based protocol is described that was used to introduce mutations in wild-type cDNA. In vitro expression of mutant cDNA clones demonstrated that all of these mutations led to a deficiency of ASPA and should therefore result in Canavan disease.     

Structural impact of three Parkinsonism-associated missense mutations on human DJ-1

A number of missense mutations in the oxidative stress response protein DJ-1 are implicated in rare forms of familial Parkinsonism. The best-characterized Parkinsonian DJ-1 missense mutation, L166P, disrupts homodimerization and results in a poorly folded protein. The molecular basis by which the other Parkinsonism-associated mutations disrupt the function of DJ-1, however, is incompletely understood. In this study we show that three different Parkinsonism-associated DJ-1 missense mutations (A104T, E163K, and M26I) reduce the thermal stability of DJ-1 in solution by subtly perturbing the structure of DJ-1 without causing major folding defects or loss of dimerization. Atomic resolution X-ray crystallography shows that the A104T substitution introduces water and a discretely disordered residue into the core of the protein, E163K disrupts a key salt bridge with R145, and M26I causes packing defects in the core of the dimer. The deleterious effect of each Parkinsonism-associated mutation on DJ-1 is dissected by analysis of engineered substitutions (M26L, A104V, and E163K/R145E) that partially alleviate each of the defects introduced by the A104T, E163K and M26I mutations. In total, our results suggest that the protective function of DJ-1 can be compromised by diverse perturbations in its structural integrity, particularly near the junctions of secondary structural elements.     

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.     

DNA sequence analysis of three missense mutations affecting colicin E3 bactericidal activity

Summary
We have determined the nucleotide sequence changes caused by three missense mutations leading to the production of inactive colicin E3 proteins. The ceaC1 mutation, affecting the transiocation of colicin E3 through bacterial membranes, is caused by a serine to phenylalanine change at position 37 within the glycine-rich region at the N-terminal part of colicin E3. This confirms previous results suggesting a role for this domain in colicin uptake by sensitive cells. The ceaC2 and ceaC3 mutations, abolishing colicin E3 RNase activity, affect the C-terminal enzymatic domain of the molecule, in the ceaC2 mutant, serine at position 529 was converted to leucine. The ceaC3 mutation replaced a glycine residue at position 524 with an aspartic acid residue. The two mutations ceaC2 and ceaC3 yieid information on the amino acid residues involved in the RNase activity of colicin E3.     

Identification of two novel mutations in OCTN2 of three patients with systemic carnitine deficiency

Systemic carnitine deficiency is a potentially lethal, autosomal recessive disorder characterized by cardiomyopathy, myopathy, recurrent episodes of hypoketotic hypoglycemia, hyperammonemia, and failure to thrive. This form of carnitine deficiency is caused by a defect in the active cellular uptake of carnitine, and the gene encoding the high affinity carnitine transporter OCTN2 has recently been shown to be mutated in patients suffering from this disorder. Here, we report the underlying molecular defect in three unrelated patients. Two patients were homozygous for the same missense mutation 632A→G, which changes the tyrosine at amino acid position 211 into a cysteine (Y211C). The third patient was homozygous for a nonsense mutation, 844C→T, which converts the arginine at amino acid position 282 into a stop codon (R282X). Reintroduction of wild-type OCTN2 cDNA into fibroblasts of the three patients by transient transfection restored the cellular carnitine uptake, confirming that mutations in OCTN2 are the cause of systemic carnitine deficiency. Electronic Publication     

DNA sequence analysis of three missense mutations affecting colicin E3 bactericidal activity

We have determined the nucleotide sequence changes caused by three missense mutations leading to the production of inactive colicin E3 proteins. The ceaC1 mutation, affecting the translocation of colicin E3 through bacterial membranes, is caused by a serine to phenylalanine change at position 37 within the glycine-rich region at the N-terminal part of colicin E3. This confirms previous results suggesting a role for this domain in colicin uptake by sensitive cells. The ceaC2 and ceaC3 mutations, abolishing colicin E3 RNase activity, affect the C-terminal enzymatic domain of the molecule. In the ceaC2 mutant, serine at position 529 was converted to leucine. The ceaC3 mutation replaced a glycine residue at position 524 with an aspartic acid residue. The two mutations ceaC2 and ceaC3 yield information on the amino acid residues involved in the RNase activity of colicin E3.     

Identification of three novel mutations in the COL2A1 gene in four unrelated Chinese families with spondyloepiphyseal dysplasia congenita

Introduction

Spondyloepiphyseal dysplasia congenita (SEDC) is an autosomal dominant skeletal dysplasia characterized by short stature, abnormal epiphyses, and flattened vertebral bodies. The condition occurs through a mutation in the COL2A1 gene that encodes the type II procollagen alpha1 chain (proalpha1 (II)).

Method and Results

We investigated nine affected individuals from four unrelated Chinese families with SEDC. We screened for COL2A1 gene mutations, and identified found four missense mutations (G447A, G456A, R789C and G1152D). The G447A, G456A and G1152D mutations are novel and the R789C mutation has been reported previously in several other studies with a strikingly similar phenotype.

Conclusions

Our study extends the mutation spectrum of SEDC and is helpful in early molecular diagnoses of SEDC.     

Distribution of three alpha-chain beta-hexosaminidase A mutations among Tay-Sachs carriers.

DNA from 176 carriers of the Tay-Sachs gene was tested for the presence of the three mutations most commonly found among Ashkenazi Jews: the so-called insertion, splice junction, and adult mutations. Among 148 Ashkenazi Jews tested, 108 had the insertion mutation, 26 had the splice junction mutation, five had the adult mutation, and nine had none of the three. Among 28 non-Jewish carriers tested, most of whom were obligate carriers, four had the insertion mutation, one had the adult mutation, and the remaining 23 had none of the three.     

三株新型发酵产氢菌生物学特征鉴定与比较

比较了三株发酵产氢细菌的形态和生理生化特征,分析了它们的16S rDNA序列系统发育关系,并测定了它们的产氢量和液相发酵产物。认为它们同属于一类新的发酵产氢菌属,其液相产物以乙醇和乙酸为主,在一定的培养条件下可获得较高产氢量。此外,对该类细菌的某些共同的生物学特征也作了描述。     

Identification of three novel mutations in the CFTR gene using temperature-optimized non-radioactive conditions for SSCP analysis

Optimal temperature conditions for the detection of 28 known mutations on 15 exons of the human cystic fibrosis transmembrane conductance regulator gene by single strand conformation polymorphism analysis using the Diagen TGGE Apparatus were established. This procedure was applied to the detection of unknown mutations in 58 non-deltaF₅₀₈ chromosomes. Three novel mutations,-471del3 (5 flanking region), 3171insC (exon 17a) and 4700(T)_8/9 (3 non-translated region) of the CFTR gene were found. Mutation 3171insC occured in conjunction with the delta F₅₀₈ mutation on the other allele of a child presenting with severe pathology. Mutation -471 del3 has so far only been found in one healthy individual and her father, and 4700(T)_8/9 is a DNA sequence polymorphism.     

Functional consequences of PRODH missense mutations

PRODH maps to 22q11 in the region deleted in the velocardiofacial syndrome/DiGeorge syndrome (VCFS/DGS) and encodes proline oxidase (POX), a mitochondrial inner-membrane enzyme that catalyzes the first step in the proline degradation pathway. At least 16 PRODH missense mutations have been identified in studies of type I hyperprolinemia (HPI) and schizophrenia, 10 of which are present at polymorphic frequencies. The functional consequences of these missense mutations have been inferred by evolutionary conservation, but none have been tested directly. Here, we report the effects of these mutations on POX activity. We find that four alleles (R185Q, L289M, A455S, and A472T) result in mild (<30%), six (Q19P, A167V, R185W, D426N, V427M, and R431H) in moderate (30%-70%), and five (P406L, L441P, R453C, T466M, and Q521E) in severe (>70%) reduction in POX activity, whereas one (Q521R) increases POX activity. The POX encoded by one severe allele (T466M) shows in vitro responsiveness to high cofactor (flavin adenine dinucleotide) concentrations. Although there is limited information on plasma proline levels in individuals of known PRODH genotype, extant data suggest that severe hyperprolinemia (>800 microM) occurs in individuals with large deletions and/or PRODH missense mutations with the most-severe effect on function (L441P and R453C), whereas modest hyperprolinemia (300-500 microM) is associated with PRODH alleles with a moderate reduction in activity. Interestingly, three of the four alleles associated with or found in schizophrenia (V427M, L441P, and R453C) resulted in severe reduction of POX activity and hyperprolinemia. These observations plus the high degree of polymorphism at the PRODH locus are consistent with the hypothesis that reduction in POX function is a risk factor for schizophrenia.     

Two novel SRY missense mutations reducing DNA binding identified in XY females and their mosaic fathers.

Two novel mutations in the sex-determining gene SRY were identified by screening DNA from 30 sex-reversed XY females by using the SSCP assay. Both point mutations lead to an amino acid substitution in the DNA-binding high-mobility-group domain of the SRY protein. The first mutation, changing a serine at position 91 to glycine, was found in a sporadic case. The second mutation, leading to replacement of a highly conserved proline at position 125 with leucine, is shared by three members of the same family, two sisters and a half sister having the same father. The mutant SRY proteins showed reduced DNA-binding ability in a gel-shift assay. Analysis of lymphocyte DNA from the respective fathers revealed that they carry both the wild-type and the mutant version of the SRY gene. The fact that both fathers transmitted the mutant SRY copy to their offspring implies that they are mosaic for the SRY gene in testis as well as in blood, as a result of a mutation during early embryonic development.     

Identification of three novel mutations in the PIG-A gene in paroxysmal nocturnal haemoglobinuria (PNH) patients

Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired haemolytic disorder caused by the absence of glycosyl phosphatidylinositol (GPI)-anchored surface proteins resulting from a defect in one step of GPI-anchor biosynthesis. Recent analysis has shown that mutations at the PIG-A (phosphatidylinositoglycan-class A) gene are responsible for GPI-anchor deficiency in all PNH patients. In the current study, we describe three new mutations of the PIG-A gene in Italian patients with PNH. The analysis has been performed by RNA/single-strand conformation polymorphism using genomic DNA purified from nucleated peripheral blood cells. An abnormal pattern of migration of polymerase chain reaction amplified fragments containing exons 2 and 5 was observed. Sequencing analysis led to the identification of three mutations: a transversion C-to-A creating a stop codon (Y98X), an A insertion at position 460 (460insA), and a C deletion (1114delC). All the mutations cause a premature termination of the translation of the PIG-A protein.     

Identification of a missense phenylketonuria mutation at codon 408 in Chinese

Summary A single base transition of G to A at codon 408 of the phenylalanine hydroxylase gene is identified. This missense mutation results in the substitution of Arg⁴⁰⁸ for Gln⁴⁰⁸ (R408Q) and accounts for about 5% of phenylketonuria (PKU) chromosomes among Chinese. This mutation is in linkage disequilibrium with restriction fragment length polymorphism haplotype 4. In addition, another mutation (R408W), at the same codon and prevalent on haplotype 2 PKU chromosomes in Caucasians, is identified in a PKU allele of haplotype 41. Previously, this mutation has been observed on a haplotype 44 background in Chinese PKU patients.     

Identification of three novel mutations in the dihydropyrimidine dehydrogenase gene associated with altered pre-mRNA splicing or protein function

Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of the pyrimidine bases uracil and thymine, as well as of the widely used chemotherapeutic drug 5-fluorouracil (5FU). Analysis of the DPD gene ( DPYD ) in two patients presenting with complete DPD deficiency and the parents of an affected child showed the presence of three novel mutations, including one splice site mutation IVS11 + 1G-->T and the missense mutations 731A-->C (E244V) and 1651G-->A (A551T). The G-->T mutation in the invariant GT splice donor site flanking exon 11 (IVS11 + 1G-->T) created a cryptic splice site within exon 11. As a consequence, a 141-bp fragment encoding the aminoacid residues 400-446 of the primary sequence of the DPD protein was missing in the mature DPD mRNA. Analysis of the crystal structure of pig DPD suggested that the E244V mutation might interfere with the electron flow between NADPH and the pyrimidine binding site of DPD. The A551T point mutation might prevent binding of the prosthetic group FMN and affect folding of the DPD protein. The identification of these novel mutations in DPYD will allow the identification of patients with an increased risk of developing severe 5FU-associated toxicity.