Mutant NDUFV2 subunit of mitochondrial complex I causes early onset hypertrophic cardiomyopathy and encephalopathy

Respiratory chain complex I deficiencies represent a genetically heterogeneous group of diseases resulting from mutations in either mitochondrial or nuclear DNA. Combination of denaturing high performance liquid chromatography and sequence analysis allowed us to show that a 4-bp deletion in intron 2 (IVS2+5_+8delGTAA) of the NDUFV2 gene (encoding NADH dehydrogenase ubiquinone flavoprotein 2) causes complex I deficiency and early onset hypertrophic cardiomyopathy with trunk hypotonia in three affected sibs of a consanguineous family. The homozygous mutation altering the consensus splice-donor site of exon 2 resulted in 70% decreased NDUFV2 protein and complex I deficiency. While mutation in a number of genes encoding complex I subunits essentially result in neurological symptoms, this first mutation in NDUFV2 is strikingly associated with cardiomyopathy, as previously observed in the unique case of NDFUS2 mutations.     

Different respiratory-defective phenotypes of Neurospora crassa and Saccharomyces cerevisiae after inactivation of the gene encoding the mitochondrial acyl carrier protein

The nuclear genes (acp-1, ACP 1) encoding the mitochondrial acyl carrier protein were disrupted in Neurospora crassa and Saccharomyces cerevisiae. In N. crassa acp-1 is a peripheral subunit of the respiratory NADH: ubiquinone oxidoreductase (complex I). S. cerevisiae lacks complex I and its ACP1 appears to be located in the mitochondrial matrix. The loss of acp-1 in N. crassa causes two biochemical lesions. Firstly, the peripheral part of complex I is not assembled, and the membrane part is not properly assembled. The respiratory ubiquinol: cytochrome c oxidose (complex IV) are made in normal amounts. Secondly, the lysophospholipid content of mitochondrial membranes is increased four-fold. In S. cerevisiae, the loss of aCP1 leads to a pleiotropic respiratory deficient phenotype.     

Catalog of 258 single-nucleotide polymorphisms (SNPs) in genes encoding three organic anion transporters, three organic anion-transporting polypeptides, and three NADH:ubiquinone oxidoreductase flavoproteins

We present here a series of high-density maps of single-nucleotide polymorphisms (SNPs) detected in genes encoding three organic-anion transporters, three organic anion-transporting polypeptides, and three nicotinamide adenine dinucleotide, reduced:ubiquinone oxidoreductase flavoproteins. A total of 258 SNPs were identified among these nine genes through systematic screening of DNA from 48 Japanese individuals: 17 in 5′ flanking regions, three in 5′ untranslated regions, 13 in coding regions, 211 in introns, six in 3′ untranslated regions, and 8 in 3′ flanking regions. By comparing our data with SNPs deposited in the dbSNP database in the National Center for Biotechnology Information, we determined that 236 (91.5%) were novel. In addition, 46 genetic variations of other types were discovered within these loci. These high-resolution maps will serve as a useful resource for analyzing potential associations between variations in these nine genes and differences in human susceptibilities to common diseases or response to drug therapies. Received: August 15, 2001 / Accepted: August 23, 2001     

Mutations in genes encoding polycomb repressive complex 2 subunits cause Weaver syndrome

Weaver syndrome (WS) is a rare congenital overgrowth disorder caused by heterozygous mutations in EZH2 (enhancer of zeste homolog 2) or EED (embryonic ectoderm development). EZH2 and EED are core components of the polycomb repressive complex 2 (PRC2), which possesses histone methyltransferase activity and catalyzes trimethylation of histone H3 at lysine 27. Here, we analyzed eight probands with clinically suspected WS by whole‐exome sequencing and identified three mutations: a 25.4‐kb deletion partially involving EZH2 and CUL1 (individual 1), a missense mutation (c.707G>C, p.Arg236Thr) in EED (individual 2), and a missense mutation (c.1829A>T, p.Glu610Val) in SUZ12 (suppressor of zeste 12 homolog) (individual 3) inherited from her father (individual 4) with a mosaic mutation. SUZ12 is another component of PRC2 and germline mutations in SUZ12 have not been previously reported in humans. In vitro functional analyses demonstrated that the identified EED and SUZ12 missense mutations cause decreased trimethylation of lysine 27 of histone H3. These data indicate that loss‐of‐function mutations of PRC2 components are an important cause of WS.     

Catalog of 434 single-nucleotide polymorphisms (SNPs) in genes of the alcohol dehydrogenase, glutathione S-transferase, and nicotinamide adenine dinucleotide, reduced (NADH) ubiquinone oxidoreductase families

An approach based on development of a large archive of single-nucleotide polymorphisms (SNPs) throughout the human genome is expected to facilitate large-scale studies to identify genes associated with drug efficacy and side effects, or susceptibility to common diseases. We have already described collections of SNPs present among various genes encoding drug-metabolizing enzymes. Here we report SNPs for such enzymes at additional loci, including 8 alcohol dehydrogenases, 12 glutathione S-transferases, and 18 belonging to the NADH-ubiquinone oxidoreductase family. Among DNA samples from 48 Japanese volunteers, we identified a total of 434 SNPs at these 38 loci: 27 within coding elements, 52 in 5′ flanking regions, five in 5′ untranslated regions, 293 in introns, 20 in 3′ untranslated regions, and 37 in 3′ flanking regions. The ratio of transitions to transversions was approximately 2.1 to 1. Among the 27 coding SNPs, 13 were nonsynonymous changes that resulted in amino acid substitutions. Our collection of SNPs derived from this study should prove useful for investigations designed to detect associations between genetic variations and common diseases or responsiveness to drug therapy. Received: March 15, 2001 / Accepted: April 6, 2001     

Sequence analysis of aMolluscum contagiosum virus DNA region which includes the gene encoding protein kinase 2 and other genes with unique organization

The nucleotide sequence of a near left-terminal region from the genome ofMolluscum contagiosum virus subtype I (MCVI) was determined. This region was contained within three adjacentBamHI fragments, designated L (2.4 kilobases (kb)), M (1.8 kb), and N (1.6 kb).BamHI cleavage of MCVI DNA produced another 1.6-kb fragment (N), which had been mapped 30–50 kb from the L,M region. The MCVI restriction fragments were cloned and end-sequenced. The N fragment that maps at the L,M region was identified by the polymerase chain reaction, using primers devised from the sequence of each fragment. The results from this analysis led to establish the relative position of these fragments within the MCVI genome. The analysis of 3.6 kb of DNA sequence revealed the presence of ten open reading frames (ORFs). Comparison of the amino acid sequence of these ORFs to the amino acid sequence of vaccinia virus (VAC) proteins revealed that two complete MCVI ORFs, termed N1L and L1L, showed high degree of homology with VAC F9 and F10 genes, respectively. The F10 gene encodes a 52-kDa serine/threonine protein kinase (protein kinase 2), an essential protein involved in virus morphogenesis. The MCVI homologue (L1L) encoded a putative polypeptide of 443 aa, with a calculated molecular mass of 53 kDa, and 60.5/30.2% sequence identity/similarity to VAC F10. The MCV N1L (213 aa, 24 kDa) showed 42.6/40.6% amino acid sequence identity/similarity to VAC F9, a gene of unknown function encoding a 24-kDa protein with a hydrophobic C-terminal domain, which was conserved in MCVI. The genomic arrangement of MCVI N1L and L1L was equivalent to that of the vaccinia and variola virus homologues. However, the ORFs contained within MCVI fragment M (leftward) showed no homology, neither similarity in genetic organization, to the genes encoded by the corresponding regions of vaccinia and variola viruses.The contribution to this paper by Antonia Martin-Gallardo and Marta Moratilla is equal, and the order of authorship is arbitrary.     

Characterization of genes encoding members of the nuclear hormone receptor superfamily from Onchocerca volvulus

Several lines of evidence suggest that molting in parasitic nematodes is controlled through the action of steroid molting hormones, or ecdysones. In other organisms, the central mediator of steroid hormone action is the hormone receptor. These receptor molecules are members of a superfamily of proteins called the nuclear hormone receptor family. Using an oligonucleotide derived from the amino-acid sequence of the Drosophila melanogaster ecdysone receptor, genes encoding homologues of the nuclear hormone receptor family were identified in the genome of the human filarial parasite Onchocerca volvulus. The O. volvulus genome contains at least three genes that encode putative members of the nuclear hormone receptor superfamily. A complete cDNA for one of these genes, designated OvNHR-1, has been isolated and characterized. The OvNHR-1 cDNA was 2378 bp in length, and contained a single open reading frame of 1104 bp. The open reading frame encoded a peptide with all of the features characteristic of a member of the nuclear hormone receptor superfamily of proteins. OVNHR-1 appeared to be encoded by a single-copy gene. Expression of the mRNA corresponding to OvNHR-1 was developmentally regulated, with maximal expression occurring during early embryogenesis. The polypeptide encoded by the OvNHR-1 open reading frame is antigenic in a minority of individuals exposed to O. volvulus.     

The 49 K subunit of NADH: ubiquinone reductase (complex I) from Neurospora crassa mitochondria: primary structure of the gene and the protein

Summary The primary structure of the 49 K subunit of the respiratory chain NADH:ubiquinone reductase (complex I) from Neurospora crassa was determined by sequencing cDNA, genomic DNA and the N-terminus of the mature protein. The sequence lengths correlate to a molecular mass of 54002 daltons for the preprotein and 49239 daltons for the mature protein. The presequence consists of 42 amino acids of typical composition for sequences which target nuclear-encoded proteins into mitochondria. The mature protein consists of 436 amino acids and shows 64% similarity to a 49 K subunit of bovine heart NADH:ubiquinone reductase and 33% to a predicted translation product of an open reading frame in the chloroplast DNAs of Marchantia polymorpha and Nicotiana tabacum. Evidence for an iron-sulfur cluster in the subunit is discussed.     

Catalog of 300 SNPs in 23 genes encoding G-protein coupled receptors

We previously published a series of detailed maps of single nucleotide polymorphisms (SNPs) in the genomic regions of 209 gene loci encoding drug metabolizing enzymes, transporters, receptors, and other potential drug targets. In addition to the maps reported earlier, we provide here high-resolution SNP maps of 23 genes encoding G-protein coupled receptors in the Japanese population. A total of 300 SNPs were identified through screening of these loci; 83 in four adenosine receptor family genes, 45 in three adrenergic receptor family genes, 22 in three EDG receptor family genes, 29 in three melanocortin receptor family genes, 22 in two somatostatin receptor family genes, 21 in five anonymous G protein-coupled receptor family genes, and 78 in the others (AVPR1B, OXTR, and TNFRSF1A). We also discovered a total of 33 genetic variations of other types. Of the 300 SNPs, 132 (44%) appeared to be novel on the basis of comparisons with the dbSNP database of the National Center for Biotechnology Information (US) or with previous publications. The maps constructed in this study will serve as an additional resource for studies of complex genetic diseases and drug-response phenotypes to be mapped by linkage-disequilibrium association analyses.     

Mutated NDUFS6 is the cause of fatal neonatal lactic acidemia in Caucasus Jews

NADH:ubiquinone oxidoreductase (complex I; EC 1.6.5.3), the largest respiratory chain complex is composed of 45 proteins and is located at the mitochondrial inner membrane. Defects in complex I are associated with energy generation disorders, of which the most severe is congenital lactic acidosis. We report on four infants from two unrelated families of Jewish Caucasus origin with fatal neonatal lactic acidemia due to isolated complex I deficiency. Whole genome homozygosity mapping, identified a 2.6 Mb region of identical haplotype in the affected babies. Sequence analysis of the nuclear gene encoding for the NDUFS6 mitochondrial complex I subunit located within this region identified the c.344G>A homozygous mutation resulting in substitution of a highly evolutionary conserved cysteine residue by tyrosine. This is the second report of NDUFS6 mutation in humans. Both reports describe three diverse homozygous mutations with variable consequential NDUFS6 protein defects that result in similar phenotype. Our study further emphasizes that NDUFS6 sequence should be analyzed in patients presenting with lethal neonatal lactic acidemia due to isolated complex I deficiency.     

Cloning and analysis of the nuclear gene MRP-S9 encoding mitochondrial ribosomal protein S9 of Saccharomyces cerevisiae

The Saccharomyces cerevisiae nuclear gene MRP-S9 was identified as part of the European effort in sequencing chromosome II. MRP-S9 encodes for a hydrophilic and basic protein of 278 amino acids with a molecular mass of 32 kDa. The C-terminal part (aa 153–278) of the MRP-S9 protein exhibits significant sequence similarity to members of the eubacterial and chloroplast S9 ribosomal-protein family. Cells disrupted in the chromosomal copy of MRP-S9 were unable to respire and displayed a characteristic phenotype of mutants with defects in mitochondrial protein synthesis as indicated by a loss of cytochrome c oxidase activity. Additionally, no activities of the gluconeogenetic enzymes, fructose-1,6-bisphosphatase and phosphoenolpyruvate carboxykinase, could be observed under conditions of glucose de-repression. The respiration-deficient phenotype could not be restored by transformation of the disruption strain with a wild-type copy of MRP-S9, indicating that MRP-S9 disruption led to rho^- or rho^o cells. Sequence similarities of MRP-S9 to other members of the ribosomal S9-protein family and the phenotype of disrupted cells are consistent with an essential role of MRP-S9 is assembly and/or function of the 30s subunit of yeast mitochondrial ribosomes.     

人凋亡相关半胱氨酸肽酶4基因在川崎病中的表达及与核转录因子-κB信号通路的相关性

目的 探讨人凋亡相关半胱氨酸肽酶4基因(Caspase4,Casp4)在川崎病中的表达及与核转录因子-κB(nuclear factor-kappa B,NF-κB)信号通路的相关性.方法 收集上海市第六人民医院东院儿科2012年1月至2015年1月的15例川崎病患者血液样本然后提取单核细胞,采用mRNA定量检测血液中单核细胞Casp4及NF-κB信号通路相关因子表达情况,同时用酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)和免疫蛋白印迹方法检测Casp4蛋白及NF-κB信号通路相关因子表达情况,并对结果进行生物统计学的分析.结果 mRNA定量、ELISA和及免疫蛋白印迹方法结果表明川崎病患儿血液CASP4基因表达与健康对照组相比显著升高,NF-κB信号通路相关因子表达也发生变化.数据分析证明川崎病患儿血液中NF-κB p65和Casp4基因mRNA表达情况存在线性关系.结论 婴幼儿在受到环境因素和遗传因素影响后,NF-κB信号通路激活,NF-κB被释放进入细胞核内,引起Casp4基因变化,CASP4基因在川崎病中的高表达及与NF-κB信号通路相关.     

Sequence of the canine major histocompatibility complex region containing non-classical class I genes

Abstract:  We have sequenced a segment of 150,102 nucleotides of canine major histocompatibility complex (MHC) DNA, corresponding to the junction of the class I and class III regions. The distal portion contained five class III genes including two tumor necrosis factor genes and the proximal portion contained five genes or pseudogenes belonging to the class I region. The order of the class III region genes was conserved as in the porcine and human MHC regions. The order of the class Ib loci from the proximal side outwards was DLA-53, DLA-12a, DLA-64, stress-induced phosphoprotein-1, followed by DLA-12. Only DLA-64 and DLA-12 display an overall predicted protein sequence compatible with the expression of membrane-anchored glycoproteins. The other class 1b loci do not appear to be functional by sequence analysis. In all, these 10 genes spanned 24% of the total sequence. The remaining 76% comprised of a number of non-coding and repetitive DNA elements including long interspersed nuclear element (LINE) fragments, short interspersed nuclear elements (SINE), and microsatellites.     

Rapid screening for nuclear genes mutations in isolated respiratory chain complex I defects

Complex I or reduced nicotinamide adenine dinucleotide (NADH): ubiquinone oxydoreductase deficiency is the most common cause of respiratory chain defects. Molecular bases of complex I deficiencies are rarely identified because of the dual genetic origin of this multi-enzymatic complex (nuclear DNA and mitochondrial DNA) and the lack of phenotype–genotype correlation. We used a rapid method to screen patients with isolated complex I deficiencies for nuclear genes mutations by Surveyor nuclease digestion of cDNAs. Eight complex I nuclear genes, among the most frequently mutated (NDUFS1, NDUFS2, NDUFS3, NDUFS4, NDUFS7, NDUFS8, NDUFV1 and NDUFV2), were studied in 22 cDNA fragments spanning their coding sequences in 8 patients with a biochemically proved complex I deficiency. Single nucleotide polymorphisms and missense mutations were detected in 18.7% of the cDNA fragments by Surveyor nuclease treatment. Molecular defects were detected in 3 patients. Surveyor nuclease screening is a reliable method for genotyping nuclear complex I deficiencies, easy to interpret, and limits the number of sequence reactions. Its use will enhance the possibility of prenatal diagnosis and help us for a better understanding of complex I molecular defects.     

Identification of functionally important amino acid residues in the mitochondria targeting sequence of hepatitis B virus X protein

Chronic hepatitis B virus (HBV) infection has been strongly associated with hepatocellular carcinoma (HCC) and the X protein (HBx) is thought to mediate the cellular changes associated with carcinogenesis. Recently, isolation of the hepatitis B virus integrants from HCC tissue by others have established the fact that the X gene is often truncated at its C-terminus. Expression of the GFP fusion proteins of HBx and its truncation mutants with a GFP tag in human liver cell-lines in this study revealed that the C-terminus of HBx is indispensable for its specific localization in the mitochondria. A crucial region of seven amino acids at the C-terminus has been mapped out in which the cysteine residue at position 115 serves as the most important residue for the subcellular localization. When cysteine 115 of HBx is mutated to alanine the mitochondria targeting property of HBx is abrogated.     

一株多重耐药肺炎克雷伯菌KF3的质粒研究

目的 通过对肺炎克雷伯菌KF3质粒DNA全序列测定,从基因组水平研究质粒DNA的结构、功能基因和与宿主菌耐药相关性.方法 碱裂解法提取质粒DNA,构建质粒DNA文库并测序.采用Phred/Phrap/Consed软件包进行序列拼接,Glimmer软件预测开放阅读框架(ORF)及功能分析.结果 构建包含3个质粒DNA的pUC18文库和Fosmid文库,测序获得3个质粒全序列.功能注释分析发现3个质粒均为可接合转移质粒,编码大量耐药相关基因.结论 肺炎克雷伯菌KF3的3个质粒都是可接合转移质粒,将耐药基因在细菌间进行水平转移,造成了耐药菌的播散.