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PubMed2001

Identification of genetic variants in the human thromboxane synthase gene (CYP5A1).

Thromboxane synthase (CYP5A1) catalyzes the conversion of prostaglandin H2 to thromboxane A2, a potent mediator of platelet aggregation, vasoconstriction and bronchoconstriction. It has been implicated in the patho-physiological process of a variety of diseases, such as atherosclerosis, myocardial infarction, stroke and asthma. On the basis of the hypothesis that variations of the CYP5A1 gene may play an important role in human diseases, we performed a screening for variations in the human CYP5A1 gene sequence. We examined genomic DNA from 200 individuals, for mutations in the promoter region, the protein encoding sequences and the 3'-untranslated region of the CYP5A1. Eleven polymorphisms have been identified in the CYP5A1 gene including eight missense mutations R61H, D161E, N246S, L357V, Q417E, E450K, T451N and R466Q. This is the first report of genetic variants in the human CYP5A1 altering the protein sequence. The effect of these variants on the metabolic activity of CYP5A1 remains to be further evaluated.

Alleles↗
PubMed2003

Identification of HepG2 variant cell lines by short tandem repeat (STR) analysis.

In the past years, in our laboratory, several cell lines have been generated starting from a human liver (H7). Some of them have been used successfully in studies of the infection with and propagation of Hepatitis B and Hepatitis C viruses. Recently, several lines of evidence indicated that the origin of these cell lines was uncertain. Therefore, we now have determined the genetic characteristics of these cell lines in comparison to HepG2 cells received from ATCC and to HepG2 isolates grown at other laboratories. Quadruplex fluorescent short tandem repeat (STR) typing and karyotyping were performed. In addition, some biochemical characteristics of selected clones were studied. Genetically, all H7-derived cell lines were identical to HepG2 cells. However, some liver-specific functions varied between the different sub-cloned lines. The H7-derived cell lines that were generated proved to be sub-cloned lines of HepG2. The problem of cross-contamination during cloning of cell lines appears to be not uncommon. We found that two out of six HepG2 isolates obtained from other laboratories were not derived from the same individual as the original HepG2 cells. Therefore, STR typing should be applied as a rapid and sensitive technique to determine and monitor the origin of cell lines and to safeguard against contamination.

Albumins↗
PubMed2002

Identification of a variant associated with adult-type hypolactasia.

Adult-type hypolactasia, also known as lactase non-persistence (lactose intolerance), is a common autosomal recessive condition resulting from the physiological decline in activity of the lactase-phlorizin hydrolase (LPH) in intestinal cells after weaning. LPH hydrolyzes lactose into glucose and galactose. Sequence analyses of the coding and promoter regions of LCT, the gene encoding LPH, has revealed no DNA variations correlating with lactase non-persistence. An associated haplotype spanning LCT, as well as a distinct difference in the transcript levels of 'non-persistence' and 'persistence' alleles in heterozygotes, suggest that a cis-acting element contributes to the lactase non-persistence phenotype. Using linkage disequilibrium (LD) and haplotype analysis of nine extended Finnish families, we restricted the locus to a 47-kb interval on 2q21. Sequence analysis of the complete region and subsequent association analyses revealed that a DNA variant, C/T-13910, roughly 14 kb upstream from the LCT locus, completely associates with biochemically verified lactase non-persistence in Finnish families and a sample set of 236 individuals from four different populations. A second variant, G/A-22018, 8 kb telomeric to C/T-13910, is also associated with the trait in 229 of 236 cases. Prevalence of the C/T-13910 variant in 1,047 DNA samples is consistent with the reported prevalence of adult-type hypolactasia in four different populations. That the variant (C/T-13910) occurs in distantly related populations indicates that it is very old.

Adult↗
PubMed1999

Identification of four variants in the tryptophan hydroxylase promoter and association to behavior.

One of the most replicated findings in biological psychiatry is the observation of lower 5-hydroxyindoleacetic acid concentrations, the major metabolite of serotonin, in the brain and cerebrospinal fluid of subjects with impulsive aggression. Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in the synthesis of serotonin, however functional variants have not been reported from the coding sequence of this gene. Therefore, we screened the human TPH promoter (TPH-P) for genetic variants which could modulate TPH gene transcription. The TPH-P (2093 nucleotides) was screened for sequence variation by SSCP analysis of 260 individuals from Finnish, Italian, American Caucasian, and American Indian populations. Four common polymorphisms were identified: -7180T>G, -7065C>T, -6526A>G, and -5806G>T (designated as nucleotides upstream of the translation start site). In the Finns, the four polymorphisms had a minor allele frequency of 0.40 and in this population linkage disequilibrium between the four loci was complete. In the other populations the minor allele frequencies ranged from 0.40 to 0.45. TPH -6526A>G genotype was determined in 167 unrelated Finnish offenders and 153 controls previously studied for the TPH IVS7+779C>A polymorphism. A significant association was observed between -6526A>G and suicidality in the offenders. TPH -6526A>G and the previously reported intron seven polymorphism, TPH IVS7+779C>A, exhibited a normalised linkage disequilibrium of 0.89 in Finns. Normalized linkage disequilibrium was reduced in other populations, being 0.49 and 0.21 in Italians and American Indians, respectively. In conclusion, four TPH-P variants were identified which can be used for haplotype-based analysis to localize functional TPH alleles influencing behavior.

Adult↗
PubMed2000

Identification of DNA variants in the SNAP-25 gene and linkage study of these polymorphisms and attention-deficit hyperactivity disorder.

The gene for the synaptic vesicle docking fusion protein, synaptosomal-associated protein of 25 kDa (SNAP-25), has been implicated in the etiology of attention-deficit hyperactivity disorder (ADHD) based on the mouse mutant strain coloboma. This neutron-irradiation induced mouse strain is hemizygous for the deletion of the SNAP-25 gene and displays spontaneous hyperactivity that is responsive to dextroamphetamine. Because of these characteristics, this strain has been suggested to be a mouse model for ADHD. We identified using single stranded conformational polymorphism analysis (SSCP) four DNA sequence variants in the 3' untranslated region of the human SNAP-25 gene. We searched for polymorphisms in the 3' untranslated region because the intron/exon structure of this gene has not yet been determined. We tested for linkage of this gene and ADHD using two of the identified polymorphisms that change a restriction enzyme recognition site. We examined the transmission of the alleles of each of these polymorphisms and the haplotypes of both polymorphisms using the transmission disequilibrium test in a sample of 97 small nuclear families consisting of a proband with ADHD, their parents, and affected siblings. We observed biased transmission of the haplotypes of the alleles of these two polymorphisms. Our findings are suggestive of a role of this gene in ADHD.

Adolescent↗
PubMed2001

Identification of sequence variants and analysis of the role of the glycogen synthase kinase 3 beta gene and promoter in late onset Alzheimer's disease.

Alzheimer's disease (AD) is a disorder characterised by a progressive deterioration in memory and other cognitive functions. Glycogen synthase kinase 3 beta (GSK3 beta) phosphorylates the microtubule associated protein tau at sites that are aberrantly phosphorylated in AD. GSK3 beta binds to presenilin 1 and plays a role in wnt and insulin signalling cascades, both of which have been proposed to be linked to AD. Moreover GSK3 beta activity may be altered in AD brain. These observations suggest a central role for GSK3 beta in AD and led us to investigate GSK3 beta as a candidate gene for AD. We sought to identify sequence variations in the gene and its promoter, as these could have an effect on activity and expression leading to abnormal function. Sequencing over 3000 bp of the GSK3 beta putative promoter revealed there to be five sequence variations, two of which were common (>10%). However on further examination none of these, either alone or in synergy, had any association with late onset AD. Stratification of the data by APOE epsilon 4 status also produced no significant association. Sequencing of the GSK3 beta coding region revealed no variations. This would suggest that the aberrant phosphorylation of tau by GSK3 beta in AD is not due to sequence variations in the gene or its promoter.

Age of Onset↗
PubMed1986

Species variants of cathepsin L and their immunological identification.

Cathepsin L variants purified from sheep and ox liver are shown to have similar catalytic properties to those from rat, rabbit and man with regard to activity against the substrate benzyloxycarbonyl-Phe-Arg-7-(4-methyl)coumarylamide and inhibition by benzyloxycarbonyl-Phe-Phe-diazomethane, thus identifying cathepsin L in these species for the first time. All five variants of cathepsin L are shown to be immunologically related by their interaction with antibodies raised to the human enzyme. Sheep liver was found to yield more enzyme than any other species, suggesting that this tissue is a good source of cathepsin L. Cathepsin S, a closely related enzyme, could not be detected in livers of any of these species.

Animals↗
PubMed2001

Identification of functionally variant MDR1 alleles among European Americans and African Americans.

MDR1 (P-glycoprotein) is an important factor in the disposition of many drugs, and the involved processes often exhibit considerable interindividual variability that may be genetically determined. Single-strand conformational polymorphism analysis and direct sequencing of exonic MDR1 deoxyribonucleic acid from 37 healthy European American and 23 healthy African American subjects identified 10 single nucleotide polymorphisms (SNPs), including 6 nonsynonymous variants, occurring in various allelic combinations. Population frequencies of the 15 identified alleles varied according to racial background. Two synonymous SNPs (C1236T in exon 12 and C3435T in exon 26) and a nonsynonymous SNP (G2677T, Ala893Ser) in exon 21 were found to be linked (MDR1*2 ) and occurred in 62% of European Americans and 13% of African Americans. In vitro expression of MDR1 encoding Ala893 (MDR1*1 ) or a site-directed Ser893 mutation (MDR1*2 ) indicated enhanced efflux of digoxin by cells expressing the MDR1-Ser893 variant. In vivo functional relevance of this SNP was assessed with the known P-glycoprotein drug substrate fexofenadine as a probe of the transporter's activity. In humans, MDR1*1 and MDR1*2 variants were associated with differences in fexofenadine levels, consistent with the in vitro data, with the area under the plasma level-time curve being almost 40% greater in the *1/*1 genotype compared with the *2/*2 and the *1/*2 heterozygotes having an intermediate value, suggesting enhanced in vivo P-glycoprotein activity among subjects with the MDR1*2 allele. Thus allelic variation in MDR1 is more common than previously recognized and involves multiple SNPs whose allelic frequencies vary between populations, and some of these SNPs are associated with altered P-glycoprotein function.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1981

Identification of a variant of mucolipidosis III (pseudo-Hurler polydystrophy): a catalytically active N-acetylglucosaminylphosphotransferase that fails to phosphorylate lysosomal enzymes.

Fibroblasts from patients with I-cell disease (mucolipidosis II) or with pseudo Hurler polydystrophy (mucolipidosis III) are markedly deficient in UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase. As a consequence, the common phosphomannosyl recognition marker of acid hydrolases is not regenerated, and these enzymes are not targeted to lysosomes. We have developed a sensitive assay for the transferase that uses alpha-methyl mannoside as an acceptor, and this has allowed us to distinguish between fibroblasts from these two types of patients. The enzyme activity is less in the former than in the latter (less than 0.4-2.0 pmol/mg per hr vs 2.9-39.4). This may provide an explanation for the difference in clinical severity between the two syndromes, However, in two siblings with pseudo Hurler polydystrophy (GM 3392), the enzyme activity was normal when assayed by using alpha-methyl mannoside as acceptor whereas it was low when assayed with endogenous glycoprotein acceptors or with human placental beta-hexosaminidase A. The apparent Km values of the mutant enzyme toward alpha-methyl mannoside, high-mannose oligosaccharides, and UDP-GlcNAc were not different from those of the normal enzyme. Mixing experiments demonstrated that the mutant fibroblasts contained endogenous acceptors and were free of inhibitors. We conclude that the N-acetylglucosaminylphosphotransferase in the mutant fibroblasts has normal catalytic activity but is defective in the ability to recognize lysosomal enzymes as specific substrates for phosphorylation. This variant form of pseudo Hurler polydystrophy demonstrates the biological importance of this recognition mechanism in the generation of the phosphomannosyl marker.

Glucosyltransferases↗
PubMed1983

Antigenic variation of Plasmodium knowlesi malaria: identification of the variant antigen on infected erythrocytes.

Erythrocytes infected with mature asexual stages of Plasmodium knowlesi express a new surface antigen such that rhesus monkey antisera specifically agglutinate these cells. Cloned parasites can express different antigenic variants of this antigen. The variant antigen has been identified by comparison of the surface membrane antigens of a clone and of an antigenic variant of that clone of different agglutination phenotype. After lactoperoxidase labeling, 125I-labeled proteins of Mrs 210,000 and 190,000 with one clone and of Mrs 205,000 with the antigenic variant were only immunoprecipitated by antisera containing homologous anti-variant antibody. Antigens of the same Mr from each clone were labeled by [35S]methionine incorporation during parasite growth and also specifically immunoprecipitated only by agglutinating antisera. Therefore, the variant antigens are malarial proteins rather than modified host proteins.

Animals↗
PubMed1986

Characterization of somatomedins from human fetal brain: identification of a variant form of insulin-like growth factor I.

A fetal form of somatomedin (insulin-like growth factor) that crossreacts in the fetal brain radioreceptor assay has been proposed to exist in humans. Using this assay to monitor activity during purification, we have isolated a variant form of insulin-like growth factor I (IGF-I) from human fetal brain tissue. The variant IGF-I showed potent crossreaction in the fetal brain radioreceptor assay and stimulated DNA synthesis in fetal brain cells in vitro. Structural analysis revealed the variant IGF-I to have a truncated NH2-terminal region compared to IGF-I isolated from serum. An additional peptide, which displayed less potent crossreaction in the fetal brain radioreceptor assay, was also isolated from the human fetal brain. Partial amino acid sequence analysis revealed identity to insulin-like growth factor II.

Amino Acid Sequence↗
PubMed2002

RasGRP4, a new mast cell-restricted Ras guanine nucleotide-releasing protein with calcium- and diacylglycerol-binding motifs. Identification of defective variants of this signaling protein in asthma, mastocytosis, and mast cell leukemia patients and demonstration of the importance of RasGRP4 in mast cell development and function.

A cDNA was isolated from interleukin 3-developed, mouse bone marrow-derived mast cells (MCs) that contained an insert (designated mRasGRP4) that had not been identified in any species at the gene, mRNA, or protein level. By using a homology-based cloning approach, the approximately 2.6-kb hRasGRP4 transcript was also isolated from the mononuclear progenitors residing in the peripheral blood of normal individuals. This transcript information was then used to locate the RasGRP4 gene in the mouse and human genomes, to deduce its exon/intron organization, and then to identify 10 single nucleotide polymorphisms in the human gene that result in 5 amino acid differences. The >15-kb hRasGRP4 gene consists of 18 exons and resides on a region of chromosome 19q13.1 that had not been sequenced by the Human Genome Project. Human and mouse MCs and their progenitors selectively express RasGRP4, and this new intracellular protein contains all of the domains present in the RasGRP family of guanine nucleotide exchange factors even though it is <50% identical to its closest homolog. Recombinant RasGRP4 can activate H-Ras in a cation-dependent manner. Transfection experiments also suggest that RasGRP4 is a diacylglycerol/phorbol ester receptor. Transcript analysis of an asthma patient, a mastocytosis patient, and the HMC-1 cell line derived from a MC leukemia patient revealed the presence of substantial amounts of non-functional forms of hRasGRP4 due to an inability to remove intron 5 in the precursor transcript. Because only abnormal forms of hRasGRP4 were identified in the HMC-1 cell line, this immature MC progenitor was used to address the function of RasGRP4 in MCs. HMC-1 leukemia cells differentiated and underwent granule maturation when induced to express a normal form of RasGRP4. Thus, RasGRP4 plays an important role in the final stages of MC development.

Amino Acid Sequence↗
PubMed2002

Identification of a variant antioxidant response element in the promoter of the human glutamate-cysteine ligase modifier subunit gene. Revision of the ARE consensus sequence.

Constitutive and inducible expression of the gene encoding the modulator subunit of human glutamate-cysteine ligase (GCLM) is regulated by either of two regions of the promoter; an antioxidant response element (ARE) at -302:-291 and a 44-bp fragment (-346:-303) upstream of the ARE. This second region includes a consensus AP-1 site previously considered responsible for the enhancer activity of the upstream fragment. Deletion of a 165-bp fragment (-348:-183) including the ARE and upstream 44-bp fragment totally ablated t-butyl hydroquinone (tBHQ) inducibility of a GCLM promoter/luciferase transgene. Mutation analyses confirmed that both the ARE and the -346:-303 fragment could support induction following tBHQ exposure but demonstrated that induction in the latter case did not involve the AP-1 site at -341:-335. A region sharing significant homology with the consensus ARE sequence except for a single nucleotide mismatch at -330 (5'-TTACnnnGCA-3' versus 5'-TGACnnnGCA-3') was identified at the 5'-end of the 44-bp fragment immediately adjacent to the AP-1 site. A G in this position has been considered an invariant requirement of functional ARE sequences. Mutation of T(-330) to A (a substitution known to ablate ARE function) or C eliminated basal and inducible expression. Substitution of a G at -330 enhanced basal expression relative to the wild-type sequence, but induction following tBHQ exposure was comparable, indicating that either sequence (5'-TTACnnnGCA-3' versus 5'-TGACnnnGCA-3') may function as an ARE, although the former sequence is less effective at directing basal expression. This possibility was confirmed by similar mutational analyses of the core sequence of hNQO1, a prototypic ARE. Electrophoretic mobility shift competition assays revealed that the 5'-TTACnnnGCA-3' sequence could compete with the hNQO1 ARE for protein binding but was less effective than a similar probe containing the 5'-TGACnnnGCA-3' motif. Probes including the T(-330)A or T(-330)C mutations were ineffective. These results reveal that the GCLM promoter includes two functional AREs, one having a variant sequence. The results indicate that the consensus ARE sequence should be revised to 5'-RTKAYnnnGCR-3'.

Amino Acid Sequence↗
PubMed2004

Identification of splicing variants of Rapostlin, a novel RND2 effector that interacts with neural Wiskott-Aldrich syndrome protein and induces neurite branching.

Rho family GTPases regulate neuronal morphology. Rnd subfamily is a new branch of Rho family GTPases. Of these GTPases, Rnd2 is specifically expressed in brain. We recently identified Rapostlin as a novel effector of Rnd2. Rapostlin induces neurite branching in response to Rnd2 in PC12 cells. During the cloning of Rapostlin, we have found two mainly expressed splicing variants of Rapostlin (renamed as RapostlinL), RapostlinM and RapostlinS, lacking 29 residues and 61 residues within the unique insert region at the center, respectively, and three minor variants, RapostlinLd, RapostlinMd, and RapostlinSd, each with the identical five-amino acid deletion from RapostlinL, RapostlinM, and RapostlinS, respectively. RapostlinL is predominantly expressed in brain, whereas RapostlinS is expressed ubiquitously. In a dot-blot assay, all splicing variants bind to Rnd2 in a GTP-dependent manner. However, RapostlinM and RapostlinS induce less neurite branching when coexpressed with Rnd2 in PC12 cells, indicating that the insert region is important for the branching activity of RapostlinL. All splicing variants bind to N-WASP in vitro and in vivo through the SH3 domain at the carboxyl terminus, and the SH3 domain is essential for branching activity of RapostlinL. In immunoprecipitation experiments, Rnd2 reduces RapostlinL-N-WASP interaction, whereas it has little effect on the interaction of RapostlinM or RapostlinS with N-WASP. Therefore, we found that functionally different splicing variants of Rapostlin have different responses to Rnd2 in association with N-WASP.

Alternative Splicing↗