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At least 739 records · Page 41Linked to original sources
PubMed2004

Identification of genetic variants in the neuronal form of tryptophan hydroxylase (TPH2).

OBJECTIVE: We screened the complete protein coding sequence of the newly identified neuronal form of tryptophan hydroxylase (TPH2) for genetic variants. METHODS: Genomic DNA samples from 24 African-Americans and 24 Caucasian-Americans in the Coriell human variation collection were screened by denaturing high-performance liquid chromatography followed by sequencing. RESULTS: We identified a number of genetic variants in both the coding and exon-flanking intronic sequences. Only one variant was identified that predicts a structural change in the TPH2 protein, and this was seen in only one out of 96 chromosomes. CONCLUSIONS: The gene for TPH2 contains a number of polymorphisms that might serve as useful markers for association analyses of complex behavioral phenotypes or as actual risk factors. Structural polymorphisms are extremely rare in TPH2 and cannot therefore act as substantial risk factors for behavioral disorders in African-American and Caucasian populations.

Black or African American↗
PubMed1998

Identification and differential regional expression of KOR-3/ORL-1 gene splice variants in mouse brain.

KOR-3, also known as ORL-1, is a member of the opioid receptor family, encoding the murine receptor for orphanin FQ/nociceptin. In the current studies we have identified five different splice variants of KOR-3 in mouse brain, three of which have not been previously reported. In addition to variants with a 15 bp deletion at the 3'-end of the first coding exon (KOR-3d) and an 81 bp insertion between the second and third coding exons (KOR-3e), three new variants with insertions of 34 (KOR-3a), 98 (KOR-3b), and 139 bp (KOR-3c) between the first and second coding exons have been obtained. The expression of the three variants in mouse brain varies markedly among brain regions with a distribution which is quite distinct from KOR-3 itself. Of greatest interest was the presence of high levels of KOR-3a in the striatum, a region with no demonstrable KOR-3, and in the cortex. KOR-3c was seen in the periaqueductal gray and hypothalamus, regions where KOR-3 predominated. The brainstem had similar levels of KOR-3, KOR-3a, and KOR-3d. In contrast, KOR-3d was most prominent in the cerebellum. KOR-3b levels were very low throughout.

Alternative Splicing↗
PubMed2002

CABYR, a novel calcium-binding tyrosine phosphorylation-regulated fibrous sheath protein involved in capacitation.

To reach fertilization competence, sperm undergo an incompletely understood series of morphological and molecular maturational processes, termed capacitation, involving, among other processes, protein tyrosine phosphorylation and increased intracellular calcium. Hyperactivated motility and an ability to undergo the acrosome reaction serve as physiological end points to assess successful capacitation. We report here that acidic (pI 4.0) 86-kDa isoforms of a novel, polymorphic, testis-specific protein, designated calcium-binding tyrosine phosphorylation-regulated protein (CABYR), were tyrosine phosphorylated during in vitro capacitation and bound (45)Ca on 2D gels. Acidic 86-kDa calcium-binding forms of CABYR increased during in vitro capacitation, and calcium binding to these acidic forms was abolished by dephosphorylation with alkaline phosphatase. Six variants of CABYR containing two coding regions (CR-A and CR-B) were cloned from human testis cDNA libraries, including five variants with alternative splice deletions. A motif homologous to the RII dimerization domain of PK-A was present in the N-terminus of CR-A in four CABYR variants. A single putative EF handlike motif was noted in CR-A at aas 197-209, while seven potential tyrosine phosphorylation-like sites were noted in CR-A and four in CR-B. Pro-X-X-Pro (PXXP) modules were identified in the N- and C-termini of CR-A and CR-B. CABYR localizes to the principal piece of the human sperm flagellum in association with the fibrous sheath and is the first demonstration of a sperm protein that gains calcium-binding capacity when phosphorylated during capacitation.

Amino Acid Sequence↗
PubMed2005

Genomic characterization of KIR2DL4 in families and unrelated individuals reveals extensive diversity in exon and intron sequences including a common frameshift variation occurring in several alleles.

The KIR2DL4 gene including a portion of exon 1 through exon 9 was sequenced from two families and eight cell lines from the International Histocompatibility Workshop (IHWS). Two known alleles and eight variants were detected. Overall, there were five synonymous and three non-synonymous changes when the variants were compared to the coding sequences of the most closely related known alleles plus a common frameshift change in five of the variant alleles. Alignment of the new variants with all known alleles showed that the regions encoding the extracellular region and the cytoplasmic tail were the most polymorphic. Two non-synonymous changes, P146H and L161V, occurred in an extracellular immunoglobulin-like domain. Five of the eight variants had a single adenine deletion in the exon encoding the transmembrane region, potentially resulting in a truncated protein lacking the cytoplasmic tail. The distribution of the deletion variant among many KIR2DL4 alleles may explain the high frequency of this variation in the population. Four of the eight consanguineous IHWS cell lines were found to be heterozygous for KIR2DL4 carrying two alleles that differed from one another by a few nucleotide substitutions. Analysis of intron sequences in the families revealed the nature and distribution of interspersed repeat elements which comprise 46% of the KIR2DL4 nucleotide sequence and consist of 12 elements including six SINEs (13.73% of the total length), one LINE (12.41%), and five LTR elements (19.51%). The results revealed the presence of extensive diversity in the KIR2DL4 gene. This is the first extensive report providing both exon and intron data in related individuals.

Alleles↗
PubMedDate not supplied

[Molecular genetic analysis of a cold-adapted donor attenuation strain for a pediatric liver influenza vaccine, of recombinants based on it and of isolates from vaccinated children].

The recombination test demonstrated that in the genome of a highly attenuated cold-adapted variant of influenza A/Leningrad/134/47/57 virus five genes coding for nonglycosylated proteins (1, 2, 5, 7, 8) carried ts mutations. Hybridization of this variant with epidemically important strains of influenza H1N1 and H3N2 viruses produced recombinants which inherited hemagglutinin and neuraminidase from the epidemic strains and 5 (H1N1) and 6 (H3N2) genes coding for nonglycosylated proteins from the cold-adapted variant. The genomes of both recombinants were found to contain five genes coding for nonglycosylated proteins which carried ts mutations. High genetic stability of cold-adapted recombinant vaccine strains was established in immunized children.

Adaptation, Physiological↗
PubMed2006

Association of enzyme and transporter genotypes with the pharmacokinetics of imatinib.

OBJECTIVE: Our objective was to explore the relationships between imatinib pharmacokinetics and 9 allelic variants in 7 genes coding for adenosine triphosphate-binding cassette transporters (ABCB1 and ABCG2) and enzymes (cytochrome P450 [CYP] 2C9, CYP2C19, CYP2D6, CYP3A4, and CYP3A5) of putative relevance for imatinib. METHODS: Imatinib transport in vitro was studied by use of human embryonic kidney 293 cells transfected with wild-type ABCG2 and an ABCG2 Q141K clone. Steady-state pharmacokinetics of imatinib was obtained in 82 patients with gastrointestinal stromal tumors treated with oral imatinib at doses ranging from 100 to 1000 mg/d. Genotyping was carried out via direct sequencing or restriction fragment length polymorphism-based techniques. RESULTS: Human embryonic kidney 293 cells transfected with ABCG2 Q141K exhibited greater drug accumulation in vitro in comparison with cells expressing wild-type ABCG2 (P = .028). However, pharmacokinetic parameters of imatinib in vivo were not statistically significantly different in 16 patients who were heterozygous for ABCG2 421C>A compared with 66 patients carrying the wild-type sequence (P = .479). The apparent oral clearance of imatinib was potentially reduced in individuals with at least 1 CYP2D6*4 allele (median, 7.78 versus 10.6 L/h; P = .0695). Pharmacokinetic parameters were not related to any of the other multiple-variant genotypes (P >or= .230), possibly because of the low allele frequencies. CONCLUSIONS: This study indicates that common genetic variants in the evaluated genes have only a limited impact on the pharmacokinetics of imatinib. Further investigation is required to quantitatively assess the clinical significance of homozygous variant ABCG2 and CYP2D6 genotypes in patients treated with imatinib.

ATP-Binding Cassette Transporters↗
PubMed2004

Characterization of genomic organization of the adenosine A2A receptor gene by molecular and bioinformatics analyses.

The adenosine A(2A) receptor (A(2A)R) is abundantly expressed in brain and emerging as an important therapeutic target for Parkinson's disease and potentially other neuropsychiatric disorders. To understand the molecular mechanisms of A(2A)R gene expression, we have characterized the genomic organization of the mouse and human A(2A)R genes by molecular and bioinformatic analyses. Three new exons (m1A, m1B and m1C) encoding the 5' untranslated regions (5'-UTRs) of mouse A(2A)R mRNA were identified by rapid amplification of 5' cDNA end (5' RACE), RT-PCR analysis and genome sequence analyses. Similar bioinformatics analysis also suggested six variants of the non-coding "exon 1" (h1A, h1B, h1C, h1D, h1E and h1F) in the human A(2A)R gene, which were confirmed by RT-PCR analysis, while three of the human exon 1 variants (h1D, h1E and h1F) were likewise verified by 5' oligonucleotide capping analysis suggesting multiple transcription start sites. Importantly, RT-PCR and quantitative PCR analysis demonstrated that the A(2A)R transcripts with different exon 1 variants displayed tissue-specific expression patterns. For instance, the mouse exon m1A mRNA was detected only in brain (specifically striatum) and the human exon h1D mRNA in lymphoreticular system. Furthermore, the determination of the three new transcription start sites of human A(2A)R gene by 5' oligonucleotide capping and bioinformatics analyses led to the identification of three corresponding promoter regions which contain several important cis elements, providing additional target for further molecular dissection of A(2A)R gene expression. Finally, our analysis indicates that A(2A)R mRNA and a novel transcript partially overlapping with the 3' exon h3, but in opposite orientation to the A(2A)R gene, could conceivably form duplexes to mutually regulate transcript expression. Thus, combined molecular and bioinformatics analyses revealed a new A(2A)R genomic structure, with conserved coding exons 2 and 3 and divergent, tissue-specific exon 1 variants encoding for 5'-UTR. This raises the possibility of generating multiple tissue-specific A(2A)R mRNA species by alternative promoters with varying regulatory susceptibility.

Animals↗
PubMed2002

Significance of genetic information in risk assessment and individual classification using silicosis as a case model.

Over the last decade the role of genetic data in epidemiological research has expanded considerably. We recently published a case-control study that evaluated the interaction between silica exposure and minor variants in the genes coding for interleukin-1alpha (IL-1alpha), interleukin-1 receptor antagonist (IL-1RA) and tumor necrosis factor alpha (TNFalpha) as risk factors associated with silicosis, a fibrotic lung disease. In contrast, this report uses data generated from these studies to illustrate the utility of genetic information for the purposes of risk assessment and clinical prediction. Specifically, this study will address how, given a known exposure, genetic information affects the characterization of risk groups. Relative operating characteristic (ROC) curves were then used to determine the impact of genetic information on individual classification. Logistic regression modeling procedures were used to estimate the predicted probability of developing silicosis. This probability was then used to construct predicted risk deciles, first for a model with occupational exposure only and then for a model containing occupational exposure and genetic main effects and interactions. Results indicate that the exposure-only model effectively captures an increasing relationship between predicted risk deciles and prevalence of observed silicosis cases. Individuals comprising the highest risk decile were almost four times as likely to have silicosis as opposed to the lowest risk decile. The addition of genetic data, however, substantially improved characterization of risk categories; the proportion of cases in the highest risk decile was almost eight times that in the lowest risk decile. However, the ROC curve and classification analysis demonstrated that the addition of genetic main effects and interactions did not significantly impact on prediction of the individual's case status. These results indicate that genetic information plays a valuable role in effectively characterizing risk groups and mechanisms of disease operating in a substantial proportion of the population. However, in the case of fibrotic lung disease caused by silica exposure, information about the presence or absence of the minor variants of IL-1alpha, IL-1RA and TNFalpha is unlikely to be a useful tool for individual classification.

Aged↗
PubMed1992

Molecular abnormality of a Japanese glucose-6-phosphate dehydrogenase variant (G6PD Tokyo) associated with hereditary non-spherocytic hemolytic anemia.

The entire coding sequence of a Japanese class 1 variant (G6PD Tokyo) was amplified by the polymerase chain reaction from genomic DNA. Nucleotide analysis by a direct sequencing technique revealed a unique nucleotide substitution (1246 G to A) in exon 10, which predicts a Glu to Lys substitution at the 416th amino acid. This is another member of a conspicuous mutation cluster surrounding the putative NADP-binding domain.

Anemia, Hemolytic↗
PubMed2005

Mitochondrial DNA as a cancer biomarker.

As part of a national effort to identify biomarkers for the early detection of cancer, we developed a rapid and high-throughput sequencing protocol for the detection of sequence variants in mitochondrial DNA. Here, we describe the development and implementation of this protocol for clinical samples. Heteroplasmic and homoplasmic sequence variants occur in the mitochondrial genome in patient tumors. We identified these changes by sequencing mitochondrial DNA obtained from tumors and blood from the same individual. We confirmed previously identified primary lung tumor changes and extended these findings in a small patient cohort. Eight sequence variants were identified in stage I to stage IV tumor samples. Two of the sequence variants identified (22%) were found in the D-loop region, which accounts for 6.8% of the mitochondrial genome. The other sequence variants were distributed throughout the coding region. In the forensic community, the sequence variations used for identification are localized to the D-loop region because this region appears to have a higher rate of mutation. However, in lung tumors the majority of sequence variation occurred in the coding region. Hence, incomplete mitochondrial genome sequencing, designed to scan discrete portions of the genome, misses potentially important sequence variants associated with cancer or other diseases.

Adult↗
PubMed2002

Strains of JC virus in Amerind-speakers of North America (Salish) and South America (Guaraní), Na-Dene-speakers of New Mexico (Navajo), and modern Japanese suggest links through an ancestral Asian population.

Previously we showed that strains of human polyoma virus JC among the Navajo in New Mexico, speakers of an Athapaskan language in the Na-Dene language phylum, and among the Salish people in Montana, speakers of a language of the Salishan group in the Amerind family, were mainly of a northeast Asian genotype found in Japan (type 2A). We now report partial VP1-gene, regulatory region, and complete genome sequences of JC virus (JCV) from the Guaraní Indians of Argentina. The Tupí-Guaraní language represents the Equatorial branch of the Amerind language family proposed by Greenberg ([1987] Language in the Americas, Stanford: Stanford University Press). The partial VP1 gene sequences of the Guaraní revealed several variants of strains found in northeast Asia (Japan), as did the Salish. In contrast, the strains in the Navajo largely conformed to the prototype type 2A sequence (MY). Phylogenetic reconstruction with both the neighbor-joining and maximum parsimony methods utilized three complete Guaraní JCV genome sequences, three genomes from the Salish people, and 27 other complete JCV genomes, including three from the Navajo and three from Japan. Both trees showed that all type 2A JCV strains from the North and South Americans are closely related phylogenetically to strains in present-day Japan. However, variant sites in the coding regions, the T-antigen intron, and the regulatory region link the type 2A strains in Amerind groups (Guaraní and Salish), but differentiate them from those in a Na-Dene-speaking (Navajo) population. The data suggest separation from a population ancestral to modern Japanese, followed by a second division within the ancestral group that led to Amerind- and Na-Dene-speaking groups. The data cannot, however, localize the latter split to the Asian mainland (two migrations) or to North America (one migration).

Adult↗
PubMed2004

The human FOXL2 mutation database.

Blepharophimosis-ptosis-epicanthus inversus syndrome (BPES; MIM# 110100) is an autosomal dominant genetic condition in which an eyelid malformation is associated (type I) or not associated (type II) with premature ovarian failure (POF). In 2001, mutations in the FOXL2 gene, encoding a forkhead transcription factor, were shown to cause both BPES type I and II. Since then, a number of reports have appeared that describe intragenic FOXL2 mutations in BPES patients. In addition, a few FOXL2 variants have been reported in isolated POF patients and XX males. Previously, our group has described a large number of FOXL2 mutations, thereby demonstrating the existence of two mutational hotspots in FOXL2, intra- and interfamilial phenotypic variability in BPES families, and genotype-phenotype correlations for a number of mutations in BPES patients. Here we describe a locus-specific Human FOXL2 Mutation Database (http://medgen.ugent.be/foxl2/), created using the MuStaR software. Our database contains general information about the FOXL2 gene, as well as details about 135 intragenic mutations and variants of FOXL2, obtained from published papers, abstracts of meetings, and from unpublished data produced by our group. Not included in the current version of the database are variants residing outside the coding region of FOXL2 and molecular cytogenetic rearrangements of the FOXL2 locus. The Human FOXL2 Mutation Database was created to provide a unique publicly available online resource of information about human FOXL2 mutations/variants associated with BPES and POF. It allows remote users to submit new mutations to the database and to query the database using a web form. It will facilitate evaluation of the pathogenicity of a particular mutation, as it contains data about disease-causing mutations and polymorphisms in BPES and isolated POF patients, and a link to the known FOXL2 orthologs. Moreover, it will allow us to establish more accurate genotype-phenotype correlations, since clinical information is contained in the database.

Alleles↗
PubMed1987

Polarized secretion of an ectopic protein in Drosophila salivary glands in vivo.

Epithelial cells can secrete specific proteins in a polarized manner, either from the apical or basolateral surface. Intracellular protein sorting which results in polarized secretion has previously been studied using epithelial tissue culture cells. We describe here the use of Drosophila larval salivary glands for the study of polarized secretion by epithelia in vivo, and address whether an ectopically synthesized secretory protein can be sorted and targeted to the correct cell surface for secretion. Larval cuticle proteins (LCPs) and salivary gland secretion (Sgs) proteins of Drosophila melanogaster are apically secreted proteins that are produced respectively by the epidermis and salivary glands. We have transformed Drosophila with a hybrid gene consisting of the sgs-4 promoter sequence and the coding sequence for a variant (LCP-f2) of LCP-2. We have found that transgenic late third instar larvae produce LCP-f2 only in the salivary glands and that LCP-f2 is properly secreted in vivo in a polarized manner from only the apical surface of the cells into the gland lumen. The results indicate that apical secretion does not depend on a tissue-specific targeting signal contained within the protein.

Animals↗
PubMed2001

Identification of GABA(B) receptor in rat testis and sperm.

gamma-Aminobutyric acid (GABA) can mimic and potentiate the action of progesterone in initiating the acrosome reaction (AR) of mammalian sperm, indicating that sperm contain receptors for GABA. This contention was validated by identifying the receptor (R) subtype, GABA(A)R, in mammalian sperm. In the present study a second subtype, GABA(B)R, was identified in rat testis and sperm. Total RNAs of rat testis and sperm were prepared and used as template to synthesize the respective cDNAs by the RT-PCR method. Two splice variants of the cDNA coding GABA(B)R1 (GABA(B)R1a and GABA(B)R1c) and GABA(B)R2 were identified. Extracts of rat testis, spermatogenic cells and sperm contained two proteins with estimated molecular sizes of 130 and 100 kDa, corresponding to GABA(B)R1a and GABA(B)R1c/lb, respectively, determined by Western blot using polyclonal anti-GABA(B)R1 antibody. By an indirect immunofluorescence technique, GABA(B)R1 was located on the head of rat sperm. The present finding is the first direct demonstration that mammalian sperm contain GABA(B)R.

Alternative Splicing↗
PubMed1996

Guided evolution of enzymes with new substrate specificities.

A gene library was constructed coding for all possible variants of two amino acids (101, 102) in a solvent-exposed surface return loop (alpha E-beta D) of Bacillus stearothermophilus L-lactate dehydrogenase (bsLDH). All but one of 38 enzyme variants examined were thermally stable and had native-like hydrodynamic properties. In this sample, there was no bias detected in either the DNA or amino acid sequences encoded. We argue that the alpha E-beta D surface loop sequence is unimportant for protein folding or stability and can be fully varied to select enzymes with new substrate specificities. The selection of NAD-dependent dehydrogenases with specificity for: malate, phenyllactate, hydroxyisocaproate and 4-phenyl-2-hydroxy-butanoate from two bsLDH libraries is described. This required a highly discriminatory screen for 2-hydroxy acid dehydrogenase activity to select enzymes which, in the absence of the natural allosteric activator fructose-1,6-bisphosphate (FBP), maintained high temperature stability and catalytic activity without substrate inhibition. In general the amino acid residues at positions 101 and 102 which determined substrate specificity were as expected from hydrophobic and ionic complementarity to the substrate. For example, a bsLDH variant with Asn101Va1102 is as efficient with phenylpyruvate as is the wild-type enzyme (Asn101Gln102) with pyruvate. Using molecular modelling, the valine at position 102 can be fitted into the active site without significant structural distortion caused by the aromatic side-chain of the substrate. Similarly, nine out of ten malate dehydrogenases (MDHs) selected had an arginine residue at position 102 to complement the negatively charged carboxyl group in oxaloacetate. One, Arg101Arg102 (Kcat/Kmoxaloacetate = 1.6 x 10(6) M-1 S-1) is 25% more active than the previous best synthetic MDH. There were surprises: present understanding would not have predicted the oxaloacetate transforming activity of Ser101Leu102 or the phenylpyruvate activity of Pro101Lys102. The former is about one-third as efficient as the best malate dehydrogenase selected, whilst the latter had about one-seventh of the best phenylpyruvate dehydrogenase activity.

Amino Acid Sequence↗
PubMed1995

A Tsp509I variant in exon 13 of the neurofibromatosis type 1 (NF1) gene allows the identification of both alleles at the mRNA level.

We describe here a new sequence variant occurring in the coding region of the neurofibromatosis (NF1) gene (exon 13). This exonic polymorphism can be directly investigated by simple restriction enzyme digestion of RT-PCR (reverse transcription-polymerase chain reaction) products, making it a powerful tool for examining allele-specific mRNA expression levels.

Alleles↗
PubMed1994

Drosophila melanogaster acetylcholinesterase: identification and expression of two mutations responsible for cold- and heat-sensitive phenotypes.

AceIJ29 and AceIJ40 are cold- and heat-sensitive variants of the gene coding for acetylcholinesterase in Drosophila melanogaster. In the homozygous condition, these mutations are lethal when animals are raised at restrictive temperatures, i.e., below 23 degrees C for AceIJ29 or above 25 degrees C for AceIJ40. The coding regions of the gene in these mutants were sequenced and mutations changing Ser374 to Phe in AceIJ29 and Pro75 to Leu in AceIJ40 were found. Acetylcholinesterases bearing these mutations were expressed in Xenopus oocytes and we found that these mutations decrease the secretion rate of the protein most probably by affecting its folding. This phenomenon is exacerbated at restrictive temperatures decreasing the amount of secreted acetylcholinesterase below the lethality threshold. In parallel, the substitution of the conserved Asp248 by an Asn residue completely inhibits the activity of the enzyme and its secretion, preventing the correct folding of the protein in a non-conditional manner.

Acetylcholinesterase↗