Genetic variation in human carboxylesterase CES1 confers resistance to hepatic steatosis

Obesity often leads non-alcoholic fatty liver disease, insulin resistance and hyperlipidemia. Expression of carboxylesterase CES1 is positively correlated with increased lipid storage and plasma lipid concentration. Here we investigated structural and metabolic consequences of a single nucleotide polymorphism in CES1 gene that results in p.Gly143Glu amino acid substitution. We generated a humanized mouse model expressing CES1^WT (control), CES1^G143E and catalytically dead CES1^S221A (negative control) in the liver in the absence of endogenous expression of the mouse orthologous gene. We show that the CES1^G143E variant exhibits only 20% of the wild-type lipolytic activity. High-fat diet fed mice expressing CES1^G143E had reduced liver and plasma triacylglycerol levels. The mechanism by which decreased CES1 activity exerts this hypolipidemic phenotype was determined to include decreased very-low density lipoprotein secretion, decreased expression of hepatic lipogenic genes and increased fatty acid oxidation as determined by increased plasma ketone bodies and hepatic mitochondrial electron transport chain protein abundance. We conclude that attenuation of human CES1 activity provides a beneficial effect on hepatic lipid metabolism. These studies also suggest that CES1 is a potential therapeutic target for non-alcoholic fatty liver disease management.     

The same amino acid substitution in orthologous esterases confers organophosphate resistance on the house fly and a blowfly.

Organophosphate (OP) insecticide resistance in certain strains of Musca domestica is associated with reduction in the carboxylesterase activity of a particular esterase isozyme. This has been attributed to a 'mutant ali-esterase hypothesis', which invokes a structural mutation to an ali-esterase resulting in the loss of its carboxylesterase activity but acquisition of OP hydrolase activity. It has been shown that the mutation in Lucilia cuprina is a Gly137-->Asp substitution in the active site of an esterase encoded by the Lc alpha E7 gene (Newcomb, R.D., Campbell, P.M., Ollis, D.L., Cheah, E., Russell, R.J., Oakeshott, J.G., 1997. A single amino acid substitution converts a carboxylesterase to an organophosphate hydrolase and confers insecticide resistance on a blowfly. Proc. Natl. Acad. Sci. USA 94, 7464-7468). We now report the cloning and characterisation of the orthologous M. domestica Md alpha E7 gene, including the sequencing of cDNAs from the OP resistant Rutgers and OP susceptible sbo and WHO strains. The Md alpha E7 gene has the same intron structure as Lc alpha E7 and encodes a protein with 76% amino acid identity to Lc alpha E7. Comparisons between susceptible and resistance alleles show resistance in M. domestica is associated with the same Gly137-->Asp mutation as in L. cuprina. Bacterial expression of the Rutgers allele shows its product has OP hydrolase activity. The data indicate identical catalytic mechanisms have evolved in orthologous Md alpha E7 and Lc alpha E7 molecules to endow diazinon-type resistance on the two species of higher Diptera.     

Molecular basis of ESD*5 and ESD*7 and haplotype analysis with new polymorphisms in introns

The two polymorphic alleles of esterase D (ESD), ESD*5 and ESD*7, are specific to Europeans and Asians, respectively. In this study the molecular basis was characterized: ESD*5, arising from ESD*1, has a G to A transition, resulting in Gly257(GGT) --> Asp(GAT); and ESD*7, originating from ESD*2, has an A to G transition, resulting in Asp231(GAT) --> Gly(GGT). Glycine is also involved in the common ESD*1/ESD*2 polymorphism [Gly190(GGA) --> Glu(GAA)]. Haplotype analysis using a few novel intragenic polymorphisms showed strong associations among polymorphic sites, suggesting that recombination has been less frequent in the human ESD gene, although it spans about 25 kb from exon 1 to exon 10. A marked difference was observed in the distribution of haplotype frequencies between Germans and Japanese.     

Novel three missense mutations observed in Von Hippel-Lindau gene in a patient reported with renal cell carcinoma

Von Hippel-Lindau (VHL) disease is an autosomal dominant hereditary cancer syndrome that predisposes to the development of a variety of benign and malignant tumors, especially cerebellar hemangioblastomas, retinal angiomas and clear-cell renal cell carcinomas (RCC). We have identified of VHL gene using immunohistochemistry in a patient who was diagnosed for RCC. In order to understand the involvement of mutation in the VHL gene exon 1 was amplified and sequenced (accession number: JX 401534). The sequence analysis revealed the presence of novel missense mutations c.194 C>T, c.239 G>A, c.278 G>A, c.319 C>G, c. 337 C > G leading to the following variations p.Ala 65 Val, p.Gly 80 Asp, p.Gly 93 Glu, p.Gln 107 Glu, p.Gln 113 Glu in the protein.     

Spontaneous missense mutations in the rplX gene for ribosomal protein L24 from Escherichia coli.

Temperature-resistant pseudorevertants of the temperature-sensitive Escherichia coli mutant KNS19, harboring a mutation in rplX, the gene for ribosomal protein L24, were isolated, cloned, and sequenced. The codon GAC for the amino acid Asp in the temperature-sensitive mutant corresponding to position 84 in the protein chain mutated either back to the wild type (Gly) or to codons for the amino acids Tyr and Glu. Furthermore, rplX genes from two other mutants with an altered protein L24 were cloned and sequenced. The mutations were localized at position 56 (Gly to Asp) and at position 62 (Glu to Lys) in the rplX gene. The latter two mutants lacked a conditional lethal phenotype. The results suggest that the amino acid Gly at positions 56 and 84 in the protein might be involved in loop formations.     

Reverse genetic screen for loss‐of‐function mutations uncovers a frameshifting deletion in the melanophilin gene accountable for a distinctive coat color in Belgian Blue cattle

In the course of a reverse genetic screen in the Belgian Blue cattle breed, we uncovered a 10‐bp deletion (c.87_96del) in the first coding exon of the melanophilin gene (MLPH), which introduces a premature stop codon (p.Glu32Aspfs*1) in the same exon, truncating 94% of the protein. Recessive damaging mutations in the MLPH gene are well known to cause skin, hair, coat or plumage color dilution phenotypes in numerous species, including human, mice, dog, cat, mink, rabbit, chicken and quail. Large‐scale array genotyping undertaken to identify p.Glu32Aspfs*1 homozygous mutant animals revealed a mutation frequency of 5% in the breed and allowed for the identification of 10 homozygous mutants. As expression of a colored coat requires at least one wild‐type allele at the co‐dominant Roan locus encoded by the KIT ligand gene (KITLG), homozygous mutants for p.Ala227Asp corresponding with the missense mutation were excluded. The six remaining colored calves displayed a distinctive dilution phenotype as anticipated. This new coat color was named ‘cool gray’. It is the first damaging mutation in the MLPH gene described in cattle and extends the already long list of species with diluted color due to recessive mutations in MLPH and broadens the color palette of gray in this breed.     

Isolation and characterization of phi X174 mutants carrying lethal missense mutations in gene G.

A previously constructed Escherichia coli transformant carrying a functional copy of bacteriophage phi X174 gene G on a plasmid, p phi XG, was used to isolate gene G mutants carrying temperature sensitive and lethal missense mutations. Two of the mutations have been characterized by sequencing: one carries a G --> A transition at residue 2821 producing a Gly --> Ser change in codon 143 of the G spike protein; the other carries an A --> G transition at residue 2678 producing Glu --> Gly change in codon 95. Sequencing DNA from 2 other mutants carrying lethal mutations that are rescued with p phi XG did not reveal any changes in the coding sequence. The lesion is believed to be in the intercistronic region between genes F and G. The adsorption kinetics for these mutants appear to be normal. Their burst size is about 25% that of wild type phi X174 on the host carrying p phi XG. These results along with previous results from the senior author's laboratory demonstrate that p phi XG can be used to rescue any gene G mutant of phi X174 regardless of the nature of the mutation involved.     

Moderately severe hemophilia A resulting from Glu----Gly substitution in exon 7 of the factor VIII gene.

To define the molecular basis of a TaqI site alteration in the factor VIII gene of a patient with moderately severe hemophilia A, we used a combination of genomic amplification followed by direct sequencing and oligonucleotide hybridization, to demonstrate an A-to-G substitution in exon 7 (codon 291) of this gene. This mutation generates a Gly in place of Glu at amino acid 272 of the mature factor VIII protein. The mutation arose de novo in a germ cell of the patient's mother.     

Identification of a Novel Synonymous Mutation in the Human β -Ureidopropionase Gene UPB1 Affecting Pre-mRNA Splicing

β-Ureidopropionase is the third enzyme of the pyrimidine degradation pathway and it catalyzes the conversion of N-carbamyl-β-alanine and N-carbamyl-β-aminoisobutyric acid to β-alanine and β-aminoisobutyric acid, respectively, and ammonia and CO₂. To date, only 16 genetically confirmed patients with a complete ß-ureidopropionase deficiency have been reported. Here, we report the clinical, biochemical, and molecular analysis of a newly identified patient with β-ureidopropionase deficiency. Mutation analysis of the UPB1 gene showed that the patient was compound heterozygous for a novel synonymous mutation c.93C >T (p.Gly31Gly) in exon 1 and a previously described missense mutation c.977G >A (p.Arg326Gln) in exon 9. The in silico predicted effect of the synonymous mutation p.Gly31Gly on pre-mRNA splicing was investigated using a minigene approach. Wild-type and the mutated minigene constructs, containing the entire exon 1, intron 1, and exon 2 of UPB1, yielded different splicing products after expression in HEK293 cells. The c.93C >T (p.Gly31Gly) mutation resulted in altered pre-mRNA splicing of the UPB1 minigene construct and a deletion of the last 13 nucleotides of exon 1. This deletion (r.92_104delGCAAGGAACTCAG) results in a frame shift and the generation of a premature stop codon (p.Lys32SerfsX31). Using a minigene approach, we have thus identified the first synonymous mutation in the UPB1 gene, creating a cryptic splice-donor site affecting pre-mRNA splicing.     

A frameshift mutation results in a truncated alpha 1-antitrypsin that is retained within the rough endoplasmic reticulum

The major physiological role of the serine protease inhibitor alpha 1-antitrypsin (alpha 1-AT) is to protect elastic fibers in the lung from excessive hydrolysis by neutrophil elastase. Genetic deficiency of alpha 1-AT predisposes individuals toward the development of emphysema. We have cloned and characterized a mutant alpha 1-AT gene from an individual exhibiting a total absence of immunoreactive alpha 1-AT in serum. Nucleotide sequence analysis of this "null" allele has demonstrated a TC dinucleotide deletion within the codon for Leu318 in exon IV. This frame-shift mutation results in the generation of a premature termination codon at residue 334, which is upstream of the active inhibitory site. To determine the biochemical basis of the null phenotype, the mutant and normal genes were transferred into mouse hepatoma cells for expression analysis. Pulse-chase experiments demonstrated that the mutant gene is expressed into a truncated protein of 45 kDa, which is retained within the rough endoplasmic reticulum. The complete lack of secretion of the truncated protein is consistent with the absence of immunoreactive alpha 1-AT in the patient's serum. In addition, a G to A transition was identified in exon II of the mutant gene, changing the codon for Arg101 to His101. Finally, an A to C transversion was identified in exon V changing the codon for Glu376 to Asp376. Since the latter conservative amino acid substitution has previously been identified in the common PiM2 variant, the frame-shift mutation might have occurred on a PiM2 background chromosome. Using the birthplace of this index case, this mutant alpha 1-AT allele has been designated "nullHong Kong."     

Identification of catalytically essential residues in Escherichia coli esterase by site-directed mutagenesis.

Escherichia coli esterase (EcE) is a member of the hormone-sensitive lipase family. We have analyzed the roles of the conserved residues in this enzyme (His103, Glu128, Gly163, Asp164, Ser165, Gly167, Asp262, Asp266 and His292) by site-directed mutagenesis. Among them, Gly163, Asp164, Ser165, and Gly167 are the components of a G-D/E-S-A-G motif. We showed that Ser165, Asp262, and His292 are the active-site residues of the enzyme. We also showed that none of the other residues, except for Asp164, is critical for the enzymatic activity. The mutation of Asp164 to Ala dramatically reduced the catalytic efficiency of the enzyme by the factor of 10(4) without seriously affecting the substrate binding. This residue is probably structurally important to make the conformation of the active-site functional.     

KRAS mutations in colorectal cancer from Tunisia: relationships with clinicopathologic variables and data on TP53 mutations and microsatellite instability

Mutations in KRAS gene are among the critical transforming alterations occurring during CRC tumorigenesis. Here we screened 51 primary CRC tumors from Tunisia for mutations in KRAS (codons 12 and 13) using PCR-direct sequencing. Our aim was to analyze tumor mutation frequencies and spectra in Tunisian patients with CRC. KRAS status and mutation site/type were than correlated with familial and clinicopathologic variables and data on TP53 mutations and nuclear protein accumulation and microsatellite instability (MSI). A KRAS somatic mutation has been detected in the CRC tumor of 31.5 % (16/51) of the patients. 81.2 % had a single mutation at codon 12 and 23 % had a single mutation at codon 13. The most common single mutation (50 %) was a G>A transition in codon 12 (c.35G>A; p.Gly12Asp). 81.25 % of the KRAS mutations were transitions and 23 % were transversions. All the mutations in codon 13 were a c.38G>A transition, whereas both G>A transitions and G>T and G>C transversions were found in codon 12. The mutation spectrum was different between MSS and MSI-H tumors and more varied mutations have been detected in MSS tumors. Some amino acid changes were detected only in MSS tumors, i.e. p.Gly12Ser, p.Gly12Cys and p.Gly12Ala. Whereas, the KRAS mutation p.Gly13Asp have been detected only in MSI-H. 43.75 % of the patients harboured combined mutations in KRAS and TP53 genes and the tumor of 71.42 % of them showed TP53 overexpression. In conclusion, the frequency and types of KRAS mutations were as reported for non-Tunisian patients. However, no significant associations have been detected between KRAS mutations and clinicopathologic variables and MSI in Tunisian patients as previously reported.     

Characterization and genetic analysis of bovine αs1-casein I variant

The aim of this study was to identify the molecular genetic origin underlying the I variant of α_s1-casein and to develop a DNA-based test for this polymorphism as a tool for genetic analyses independent of milk sample testing. All coding exons and flanking regions of the α _{s 1} -casein gene were sequenced in DNA samples from cattle of known α _{s 1} -casein genotypes ( BI , CI , II , CC ), determined by isoelectric focusing of milk samples. A nucleotide substitution (A>T) in exon 11 (g.19836A>T) leads to the exchange of Glu with Asp at amino acid position 84 of the mature protein (p.Glu84Asp) and perfectly co-segregated with the presence of the α_s1-casein I variant in the milk of the analysed animals. Genotyping of a total of 680 DNA samples from 31 Bos taurus and Bos indicus cattle breeds and from Bos grunniens , Bison bison and Bison bonasus by restriction fragment length polymorphism analysis revealed the occurrence of Asp at position 84 at low frequencies in Bos taurus and Bos indicus breeds and established its origin from the α _s1-casein C variant (p.Glu192Gly). Ten different intragenic haplotypes in the gene region from intron 8 to intron 12 were observed by sequencing, of which two occurred in Bison bison and one in Bison bonasus only. Using available casein gene complex information, an association of Asp at position 84 to β-casein A ² and κ-casein B was shown in the Bos indicus breed Banyo Gudali . Taken together, we can postulate that the α _s1-casein variant I is caused by a non-synonymous nucleotide substitution in exon 11 of the gene and that it originated within Bos indicus and spread to Bos taurus subsequently.     

Novel mutations in the SLC12A3 gene causing Gitelman's syndrome in Swedes.

PURPOSE: Gitelman's syndrome (GS) is an inherited autosomal recessive disorder due to loss of function mutations in the SLC12A3 gene encoding the Na-Cl co-transporter (NCCT), the target of thiazide diuretics. The defective function of the NCCT, which normally is expressed in the apical membrane of the distal convolute tubule in the kidney, leads to mild hypotension, hypokalemia, hyperreninemic hyperaldosteronism, mild metabolic alkalosis, hypomagnesemia and hypocalciuria. Up to now, more than 100 mutations of the SLC12A3 gene have been described in GS patients. METHODS: We have collected 30 patients from Sweden with a clinical diagnosis of GS and undertaken a mutation screening by SSCP and successive sequencing of the 26 exons and intronic boundaries. Both mutations were identified in most (n = 28, 93%) and at least one mutation was identified in all patients. RESULTS: We found 22 different mutations evenly distributed throughout the gene, 11 of which have not been described previously. The new variants include 8 missense mutations (Glu68Lys, His69Asn, Argl45His, Vall53Met, Gly230Asp, Gly342Ala, Val677Leu and Gly867Ser), 1 insertion (c.834_835insG on exon 6) and 2 splice-site mutations (c.2667 + lT>G substitution in splicing donor site after exon 22, c.1569-1G>A substitution in the splicing acceptor site before exon 13). CONCLUSION: In Swedish patients with the clinical features of GS, disease-causing mutations in the SLC12A3 gene were identified in most patients. The spectrum of GS mutations is wide making full mutation screening of the SLC12A3 gene necessary to confirm the diagnosis.     

Identification of mutations in exons 1 through 8 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene.

Five different mutations have been identified in the gene causing cystic fibrosis (CF) through sequencing regions encompassing exons 1-8, including the 5' untranslated leader. Two of these apparent mutations are missense mutations, one in exon 3 (Gly to Glu at position 85; G85E) and another in exon 5 (Gly to Arg at 178; G178R), both causing significant changes in the corresponding amino acids in the encoded protein--cystic fibrosis transmembrane conductance regulator (CFTR). Two others affect the highly conserved RNA splice junction flanking the 3' end of exons 4 and 5 (621 + 1G----T, 711 + 1G----T), resulting in a probable splicing defect. The last mutation is a single-basepair deletion in exon 4, causing a frameshift. These five mutations account for the 9 of 31 non-delta F508 CF chromosomes in our Canadian CF family collection and they are not found in any of the normal chromosomes. Three of the mutations, 621 + 1G----T, 711 + 1G----T, and G85E, are found in the French-Canadian population, with 621 + 1G----T being the most abundant (5/7). There are two other sequence variations in the CFTR gene; one of them (129G----C) is located 4 nucleotides upstream of the proposed translation initiation codon and, although present only on CF chromosomes, it is not clear whether it is a disease-causing mutation; the other (R75Q) is most likely a sequence variation within the coding region.     

人肝细胞癌中抑癌基因PTEN/MMAC1/TEP1的突变分析

PTEN／MMAC1／TEP1(PTEN)是新近分离到的抑癌基因,在多种肿瘤中存在突变。我们检测了34例人肝细胞癌中PTEN基因第5外显子和第8外显子的突变。采用聚合酶链方法以内含子引物扩增第5和第8外显子,继之以单链构象多态性和测序技术分析PTEN基因突变。有4例肝细胞癌SSCP显示异常条带并经测序证实存在突变。2例发生于第4内含子,突变位点相同;另两例发生于第8外显子,其中1例碱基颠换导致PTEN蛋白产物304位半胱氨酸突变为甘氨酸。     

人肝细胞癌中抑癌基因PTEN／MMAC1／TEP1的突变分析

PTEN/MMAC1/TEP1 is a tumor suppressor gene. Its mutation has been found in several different types of human cancers. 34 primary human hepatocellular carcinomas have been examined for mutations in exon 5 and exon 8 of the PTEN gene. Exon 5 and exon 8 were amplified by polymerase chain reaction (PCR) with intronic primers and subjected to single strand conformation polymorphism (SSCP) analysis. SSCPs were found in 4 of the 34 hepatocellular carcinomas analyzed. Direct sequencing of the PCR products identified single base-pair substitutions in the four tumor DNA samples, two in intron 4 and two in exon 8. One of the base-pair substitution in exon 8 is a missense mutation, which changed codon 304 of PTEN protein from Cys to Gly. These data suggest that PTEN may be involved in the carcinogenesis and development of hepatocellular carcinoma.     

Deleterious effect of the Qo inhibitor compound resistance-conferring mutation G143A in the intron-containing cytochrome b gene and mechanisms for bypassing it

The mutation G143A in the inhibitor binding site of cytochrome b confers a high level of resistance to fungicides targeting the bc(1) complex. The mutation, reported in many plant-pathogenic fungi, has not evolved in fungi that harbor an intron immediately after the codon for G143 in the cytochrome b gene, intron bi2. Using Saccharomyces cerevisiae as a model organism, we show here that a codon change from GGT to GCT, which replaces glycine 143 with alanine, hinders the splicing of bi2 by altering the exon/intron structure needed for efficient intron excision. This lowers the levels of cytochrome b and respiratory growth. We then investigated possible bypass mechanisms that would restore the respiratory fitness of a resistant mutant. Secondary mutations in the mitochondrial genome were found, including a point mutation in bi2 restoring the correct exon/intron structure and the deletion of intron bi2. We also found that overexpression of nuclear genes MRS2 and MRS3, encoding mitochondrial metal ion carriers, partially restores the respiratory growth of the G143A mutant. Interestingly, the MRS3 gene from the plant-pathogenic fungus Botrytis cinerea, overexpressed in an S. cerevisiae G143A mutant, had a similar compensatory effect. These bypass mechanisms identified in yeast could potentially arise in pathogenic fungi.     

Characterisation of the alpha-1-antitrypsin M3 gene,a normal variant

Summary By sequence analysis of the complete proteincoding region of the human alpha-1-antitrypsin gene using polymerase chain reaction techniques, we have characterised one of the normal variants, M3. We have identified a single point mutation between M1 Va1²¹³ and M3 at codon position 376 which is a GAA(Glu) to GAC(Asp) transversion.