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At least 1,153 records · Page 64Linked to original sources
PubMed1998

Identification and characterization of human fructose or glucose taste variants with hypogeusia for one monosaccharide but not for the other.

Human psychophysical functions for sweetness are similar for sucrose and fructose, but different for glucose, and suggest different mechanisms for fructose and glucose. Drosophila behavioral and electrophysiological data are similar to the human data and indicate separate receptor cell mechanisms for the monosaccharides. Moreover, fructose 'nontasters' (NTs) and glucose NTs have been identified in two Drosophila species. Identification of human NTs would confirm separate mechanisms and could lead to identification of proteins in human sweet taste by molecular genetic techniques. To identify human NTs, we first obtained responses for sucrose, fructose and glucose from 20 subjects. They tasted seven concentrations of each sugar (2-128 mM), paired with water, and indicated the sweeter of each pair. Functions for recognition indices (RIs) (proportion of subjects recognizing the sugar as sweeter) were similar for sucrose and fructose and different for glucose; this result agrees with the previous studies and supports different mechanisms for the monosaccharides. At 128 mM, RIs for all three sugars were 1.0; this result is consistent with the monogeusia reported by Breslin et al. for concentrations higher than those tested here. Eleven rising-phase concentrations (10-35 mM fructose, 10-90 mM glucose) then were tested on 32 subjects. A statistically significant interaction indicated different regression slopes and supported different monosaccharide mechanisms. From these data, positive identification values (PIDs) (lowest concentration at which the sugar always was judged sweeter than the water) were determined for each subject. The fructose log(PID) and glucose log(PID) data were not well correlated; thus separate mechanisms were supported further. Next, NT traits were defined by log(PID)s > or = 2 SD above the mean for one sugar, while the PID for the other remained within 1 SD of the population mean log(PID). Ninety-two subjects were screened to identify 12 glucose NTs and four fructose NTs. Two glucose NTs and three average subjects were tested in six additional sessions. The NTs showed an experience-induced change: there was a statistically significant reduction of glucose PIDs, but not of fructose PIDs. No change occurred in PIDs of the average subjects for either sugar.

Animals↗
PubMed2005

The APOA1/C3/A4/A5 gene cluster, lipid metabolism and cardiovascular disease risk.

PURPOSE OF REVIEW: APOA1/C3/A4/A5 are key components modulating lipoprotein metabolism and cardiovascular disease risk. This review examines the evidence regarding linkage disequilibrium and haplotype structure within the A1/C3/A4/A5 cluster, and assesses its association with plasma lipids and cardiovascular disease risk. In addition, we use genomic information from several species to draw inferences about the location of functional variants within this cluster. RECENT FINDINGS: The close physical distance of these genes and the interrelated functions of these apolipoproteins have encumbered attempts to determine the role of individual variants on lipid metabolism. Therefore, current research aims to define linkage disequilibrium and haplotype structure within this cluster. Functional variants in regulatory regions are most interesting as they are potentially amenable to therapy. Comparative genomics can contribute to the identification of such functional variants. SUMMARY: Genetic variability at the APOA1/C3/A4/A5 cluster has been examined in relation to lipid metabolism and cardiovascular disease risk. However, the findings are inconsistent. This is partly due to the classic approach of studying single and mostly nonfunctional polymorphisms. Moreover, allelic expression may depend on the concurrent presence of environmental factors. Association studies using haplotypes should increase the power to detect true associations and interactions. We hypothesize that phenotypes observed in association with transcriptional regulatory variants can be readily modified by environmental factors. Therefore, studies focusing on regulatory variants may be more fruitful to locate/define future therapeutic targets.

Apolipoprotein A-I↗
PubMed1984

The detergent solubility properties of a malarial (Plasmodium knowlesi) variant antigen expressed on the surface of infected erythrocytes.

Four detergents have been compared for identification of the Plasmodium knowlesi variant antigen on infected erythrocytes by immunoprecipitation analysis. Erythrocytes infected with late trophozoite and schizont forms of cloned asexual parasites were labeled by lactoperoxidase-catalyzed radioiodination and extracted either with the anionic detergents sodium dodecyl sulfate (SDS) or cholate, the neutral detergent Triton X-100, or the zwitterion 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS). After addition of Triton X-100 to SDS and cholate extracts, parallel immunoprecipitations of the four extracts were performed using rhesus monkey antisera of defined agglutinability. Identical results were obtained with clone Pk1(A+), which has 125I-variant antigens of Mr 210,000 and 190,000, and with clone Pk1(B+)1+, which has variant antigens of Mr 200,000-205,000. SDS yielded maximal levels of immunoprecipitated 125I-variant antigens. Variant-specific immunoprecipitation was detected in some experiments with Triton X-100 and cholic acid but with significantly lower recovery than with SDS. CHAPS extraction did not yield the variant antigens on immunoprecipitation. The variant antigens could also be identified in Triton X-100-insoluble material by subsequent extraction with SDS, indicating that failure to recover these proteins in the Triton X-100-soluble fraction is due to failure of this detergent to extract the variant antigens rather than to degradation during extraction. We suggest that the 125I-variant antigens either have a structure that renders them intrinsically insoluble in Triton X-100, cholate, or CHAPS, or that they are associated in some way with host cell membrane components that also resist solubilization by these detergents.

Animals↗
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↗
PubMed2006

Distribution of optineurin sequence variations in an ethnically diverse population of low-tension glaucoma patients from the United States.

PURPOSE: Previous studies have suggested that Optineurin (OPTN) sequence variants contribute to low-tension glaucoma (LTG) in ethnically homogeneous populations. The purpose of this study is to evaluate the prevalence of OPTN sequence variants in an ethnically diverse population of LTG patients from the United States, and to describe the phenotype of patients with OPTN sequence variants preferentially found in LTG patients. METHODS: Genomic DNA purified from 67 LTG patients was screened for DNA sequence variants located in the exons and flanking introns of the OPTN gene using high-performance liquid chromatography analysis and direct genomic DNA sequencing. Eighty-six primary open-angle glaucoma probands and 100 control patients were also analyzed. RESULTS: Nine OPTN DNA sequence variants were identified in this patient population including the 2 previously identified heterozygous nonsynonymous single-nucleotide polymorphisms in exons 4 and 5. Four LTG patients with severe disease and positive family history of glaucoma, were found to have DNA sequence changes not found in primary open-angle glaucoma probands or control individuals including the previously reported E50K variation. CONCLUSIONS: The results of this study support the rare association of OPTN sequence variants with familial forms of LTG. The E50K mutation seems to be associated with a severe form of LTG, and although rare, the identification of this sequence variant in patients at risk may help direct appropriate therapy.

Adult↗
PubMed1992

Nosological considerations of the neurofibromatoses.

We are on the threshold of evaluating the NF1 and NF2 loci with respect to variant forms of the neurofibromatoses. Genetic mapping of NF1, gene cloning and characterization of its encoded product, neurofibromin, provides a framework for the evaluation of the variant forms of NF. This may also apply to NF2 variant forms in the near future. The mapping approach in evaluating variant forms of NF should begin with the rigorous clinical assessment of familial cases whereby the establishment of genetic linkage in families with overlap syndromes might determine if either NF1, NF2, or a separate locus is involved in the phenotype. Conditions mapping to the NF1 locus could then be screened for mutations in hopes of identifying the etiologies of the variant forms of NF. Mutation identification should provide a molecular-based classification scheme for the variant forms of NF, now tentatively divided into alternative and related forms. It is expected that the nosology of the neurofibromatoses will most certainly change as more is learned of the NF1 and NF2 loci.

Diagnosis, Differential↗
PubMed2003

Modulation of splicing events in histone deacetylase 3 by various extracellular and signal transduction pathways.

Within the context of the chromatin environment histone deacetylases are important transcriptional regulators. Three classes of human histone deacetylases have currently been identified on the basis of their similarity to yeast proteins. The class I enzymes contain four members: HDACs 1-3 and HDAC8. Of these, HDAC3 is known to generate transcript variants with altered amino-terminal regions. Here we describe the identification of a novel splice variant of HDAC3, in which exon 3 is alternatively spliced from the messenger RNA transcript. We show that this human HDAC3 splice transcript is upregulated by treatments with histone deacetylase inhibitors. We also demonstrate evidence of splicing events in murine HDAC3 as a response to various signals, including switching between splice transcript isoforms following treatments with kinase inhibitors or by osmotic shock. In contrast, such switching events were not observed in human cells. These results indicate that differential pathways in mouse and human may control the regulation of HDAC3, and that splice variants may play important roles in responding to exogenous stimuli that act via signal transduction pathways.

Alternative Splicing↗
PubMed1997

Capillary electrophoretic analysis of genetic variants of milk proteins from different species.

Polymorphism of bovine, ovine and caprine milk proteins was studied by CE. Identification of some rare bovine variants was carried out by isoelectric focussing (IEF) using PhastSystem. Genetic variants A and D of bovine alpha s2-casein, beta-casein variants A1, A2, A3, B and C and alpha s1-casein variants B and C were determined by CE. In addition, the different casein fractions including some genetic variants of ovine and caprine milk were identified by CE. In order to carry out this identification, collected fractions from a cation-exchange FPLC separation were injected by CE.

Animals↗
PubMed2006

Genomic fossils as a snapshot of the human transcriptome.

Processed pseudogenes (PPGs) are cDNA sequences that were generated through reverse transcription of mature, spliced mRNAs and have subsequently been reinserted at a new genomic location. These cDNA sequences are usually no longer transcribed and are considered "dead on arrival." Here we show that PPGs can be used to generate a map of the transcriptome. By analyzing thousands of human PPGs, we were able to discover hundreds of transcript variants so far unidentified. An experimental verification of a subset of these variants by RT-PCR indicates that most of them are still active in the human transcriptome. Furthermore, we demonstrate that PPGs can enable the identification of ancient splice variants that were expressed ancestrally but are now extinct. Our results show that the genome itself carries a "virtual cDNA library" that can readily be used to analyze both present and ancestral transcripts. Our approach can be applied to sequenced metazoan genomes to computationally annotate splicing variation even when expressed sequences are unavailable.

Alternative Splicing↗
PubMed2006

MLH3 mutation in endometrial cancer.

MLH3 is a recently described member of the DNA mismatch repair gene family. Based on its interaction with the MutL homologue MLH1, it was postulated that MLH3 might play a role in tumorigenesis. Germ line and somatic mutations in MLH3 have been identified in a small fraction of colorectal cancers, but the role of MLH3 in colorectal cancer tumorigenesis remains controversial. We investigated MLH3's role in endometrial tumorigenesis through analysis of tumor and germ line DNA from 57 endometrial cancer patients who were at increased risk for having inherited cancer susceptibility. Patients with known MSH2 or MSH6 mutations were excluded as well as those who had MLH1-methylated tumors. Sixteen different variants were identified by single-strand conformational variant analysis. Of the 12 missense changes identified, three were somatic mutations. One patient had a germ line missense variant and loss of heterozygosity (LOH) in her tumor specimen. There was no evidence of MLH3 promoter methylation based on combined bisulfite restriction analysis. The identification of inherited missense variants, somatic missense mutations (present in 3 of 57 tumors), and LOH in the tumor from a patient with a germ line missense change suggest a role for MLH3 in endometrial tumorigenesis.

Base Sequence↗
PubMed1993

Plasma cholinesterase: gene and variations.

The traditional tests that have been used for the past 30 yr to determine plasma cholinesterase phenotype--measurement of esterase activity with a variety of substrates, dibucaine inhibition, fluoride inhibition, and Ro2-0683 inhibition--are inadequate for identifying some variants of this enzyme and leave many cases of prolonged response to succinylcholine unexplained. The application of the techniques of molecular genetics has permitted precise identification of plasma cholinesterase variants and has resulted in the discovery of previously unrecognized variants. It is now possible, in cases of prolonged response to succinylcholine resulting from genetically determined alterations in plasma cholinesterase, to ascertain the nature of the mutations in the alleles, and from them to deduce the structural changes in the enzymes responsible for the impairment in drug metabolism.

Butyrylcholinesterase↗
PubMed2005

Multiple 5'-splice variants of the rat glutamate transporter-1.

In most brain areas, uptake of extracellular glutamate predominantly occurs through the glutamate transporter subtype, glutamate transporter-1 (GLT-1), which is enriched in astroglia. Here, we report the identification of five splice variants of the 5'-leader sequence of rat GLT-1 which contain varying numbers of upstream open reading frames and encode putative GLT-1 proteins with two distinct N-terminal modifications. We further demonstrate that the identified rat 5'-GLT-1 splice variants are expressed in a brain region-specific manner. Our findings point to potential influences of RNA splicing on glial glutamate transport in the intact and injured rat brain.

Alternative Splicing↗
PubMed2026

Exploring genomic regions regulating the liver transcriptome and energy homeostasis in pigs.

In pigs, energy homeostasis has an impact on meat quality and health. In a Duroc pig population, 30 quantitative trait locus (QTL) regions associated with fatty acid (FA) composition in adipose tissue, plasma, liver and muscle were previously identified. Mapping of expression quantitative trait locus (eQTL) regions will provide a molecular hypothesis for genotype-phenotype interactions and may allow the identification of shared causal variants, key to increasing our understanding of the genetic regulation of FA composition and energy homeostasis. However, gene expression is impacted by environmental factors, while individual-level allelic imbalance (AI) can be more reliable and can be surveyed via allelic-specific expression (ASE) analysis. Furthermore, treatment of ASE as a quantitative trait allows the identification of allele-specific expression quantitative trait loci (aseQTLs), which are variants whose heterozygosity is linked to the AI of a nearby single-nucleotide polymorphism (SNP), pointing to regulatory elements. In this study, liver was selected as a key metabolic hub with an important role in the regulation of energy homeostasis, and 310 liver RNA sequencing samples were analysed using a combination of (1) eQTL mapping, (2) ASE analysis, and (3) aseQTL mapping methods. A total of 2 188 eQTL regions were identified, mostly cis-eQTL regions (73.17%). ASE analysis reported 1 964 ASE SNPs, associated with 633 genes. Finally, aseQTL mapping reported 64 172 aseQTL, associated with the AI of 31 genes. Colocalisation analysis combined with ASE analysis showed that the expression of FADS1 and FADS2 genes is associated with the polyunsaturated FA composition in several tissues, where microRNA regulation may be present. Finally, in the DGAT2 gene, annotated in a QTL region associated with multiple FAs in adipose tissue, ASE revealed allelic imbalance in the 3' untranslated region (UTR) of this gene. Allelic differential expression can be caused by a 13-bp insertion affecting messenger RNA stability, previously described, exemplifying how allelic imbalance is caused by a post-transcriptional regulatory mechanism undetectable by eQTL mapping. Furthermore, aseQTLs were associated with this gene, linked to a previously identified copy number variant not yet associated with DGAT2 expression. These results demonstrate how ASE analysis and aseQTL mapping can complement eQTL mapping, as they resolved a complex region affected by allelic heterogeneity, a main confounding effect of QTL mapping. In conclusion, the combination of eQTL mapping, ASE analysis and aseQTL mapping allowed the characterisation of the regulation of liver gene expression, improving our understanding of the genetic determinism of energy homeostasis.

Allele-specific expression↗
PubMed2026

LDLR Variant Classification Through Activity-Normalized Prime Editing Screening.

BACKGROUND: Inherited variants in the LDL (low-density lipoprotein) receptor (LDLR) gene are the most common cause of familial hypercholesterolemia, significantly increasing coronary artery disease risk. Early identification of pathogenic LDLR variants enables prompt lipid-lowering therapy and cascade testing of at-risk relatives; however, most LDLR variants observed in the population have uncertain or absent clinical classifications, leaving many patients without actionable information. METHODS: We developed the first activity-normalized prime editing screening pipeline to measure the impact of 5184 LDLR coding variants on LDL-cholesterol (LDL-C) uptake. Each prime editing guide RNA is paired with a genotypic outcome reporter to correct for variable editing efficiency, overcoming a key limitation of previous pooled genome editing screens. A statistical framework further improves variant effect estimates by jointly analyzing all missense variants at each amino acid position. RESULTS: We show that prime editing of the reporter construct correlates with endogenous variant installation frequency, validating the activity normalization approach. The resulting scores capture a continuous spectrum of functional effects, robustly separate pathogenic versus benign ClinVar variants, and show concordance with LDL-C levels in UK Biobank participants. We calibrate functional evidence strengths to the ACMG/AMP variant interpretation framework, enabling integration into a clinical variant classification workflow. By combining functional, computational, population, and contextual evidence, 322 of 434 LDLR variants currently classified as variants of uncertain significance, conflicting, or absent from ClinVar appear to meet evidence thresholds for reclassification and can be prioritized for expert review, substantially expanding the pool of actionable variant classifications. The screen also reveals a cluster of gain-of-function variants in LDLR class A repeat 5, at least some of which enhance LDL-C uptake through increased apolipoprotein B interaction, with implications for therapeutic genome editing. Last, prime editing uniquely detects splice-altering coding variants missed by cDNA-based screens and pathogenicity predictors, revealing an advantage of endogenous variant installation. CONCLUSIONS: Altogether, activity-normalized prime editing provides a scalable framework for LDLR variant classification that substantially expands the proportion of variants with evidence for genetic diagnosis and reveals novel biology with therapeutic relevance.

CRISPR screening↗
PubMed2000

Quantitative and qualitative analysis of haemoglobin variants using capillary zone electrophoresis.

Capillary zone electrophoresis (CZE) has been introduced into the clinical chemistry laboratory because of its range of potential applications. In this paper, we evaluate an alkaline CZE method for the quantification of HbA2 and HbF and also assess the combination of the alkaline CZE method with an acid CZE method for the determination of haemoglobin variants in an automated fashion. Correlation of the HbA2% determined between the HbA2-CZE method (alkaline conditions) and the Helena Sickle Thal Quick Column method was good (r = 0.91). The correlation between the HbF% determined by the HbA2-CZE method and by the alkaline denaturation method was acceptable (r = 0.81). The HbA2-CZE method was able to identify a large number of haemoglobin variants. The variants HbC and HbE or HbS and HbD, however, had the same characteristics under alkaline conditions and could therefore not be discriminated from each other. The identification of these overlapping variants could be accomplished by the analysis of the blood specimens in combination with the HbA1c-CZE method (acid conditions). We conclude that the presented applications for capillary zone electrophoresis can be used for quantitative and qualitative analysis of haemoglobin variants.

Chromatography, Liquid↗
PubMed1988

A heritable variant of mouse liver ornithine aminotransferase (EC 2.6.1.13) induced by ethylnitrosourea.

A variant of ornithine aminotransferase (OAT, EC 2.6.1.13) has been detected in an offspring of a male mouse treated with ethylnitrosourea. The evidence presented to support the identification of the protein variant (ENU 2) as altered OAT includes (a) a corresponding 50% decrease in the abundance of a protein, located one charge unit basic to the variant, which comigrates on two-dimensional gel patterns with purified mouse liver OAT; (b) the binding of anti-rat-OAT antibody to the variant; (c) the increased abundance of the variant protein in the livers of mice fed a high protein diet (85% casein); and (d) purification of the variant through an OAT purification protocol.

Animals↗
PubMed2002

Galactosyltransferase I is a gene responsible for progeroid variant of Ehlers-Danlos syndrome: molecular cloning and identification of mutations.

A human cDNA encoding a novel galactosyltransferase was identified based on BLAST analysis of expressed sequence tags, and the cDNA clones were isolated, showing a type II membrane protein with 327 amino acids and 38% homology to the Caenorhabditis elegans sqv-3 gene involved in vulval invagination and oocyte development. This cDNA exhibited marked galactosyltransferase activity specific for p-nitrophenyl-beta-D-xylopyranoside, and also restored glycosaminoglycan (GAG) synthesis to galactosyltransferase I-deficient CHO mutant pgsB-761 cells. The enzyme product contained beta-1,4-linked galactosyl residues, indicating that the enzyme is galactosyltransferase I (UDP-D-galactose: D-xylose beta-1,4-D-galactosyltransferase; EC 2.4.1.133) involved in the synthesis of the GAG-protein linkage region of proteoglycans. Mutations of this gene were investigated in a case of Ehlers-Danlos syndrome (progeroid variant), since reduced activity of galactosyltransferase I had been reported in this disease by others. As expected, the patient gene contained two different mutations (A186D, L206P). The mutations showed, respectively, 10-50% and 0% of the enzyme activity compared with wild type, suggesting that galactosytransferase I (XGal-T1) is at least one of the genes responsible for Ehlers-Danlos syndrome (progeroid variant).

Amino Acid Sequence↗
PubMed2004

Inhibitors of hepatitis C virus NS3.4A protease. Part 3: P2 proline variants.

We recently described the identification of an optimized alpha-ketoamide warhead for our series of HCV NS3.4A inhibitors. We report herein a series of HCV protease inhibitors incorporating 3-alkyl-substituted prolines in P(2). These compounds show exceptional enzymatic and cellular potency given their relatively small size. The marked enhancement of activity of these 3-substituted proline derivatives relative to previously reported 4-hydroxyproline derivatives constitutes additional evidence for the importance of the S(2) binding pocket as the defining pharmacophore for inhibition of the NS3.4A enzyme.

Carrier Proteins↗