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PubMed1997

Identification of GB virus C variants by phylogenetic analysis of 5'-untranslated and coding region sequences.

Phylogenetic analysis of 44 GB virus C (GBV-C) 5'-untranslated region (5'-UTR) sequences from 37 individuals suggested the presence of GBV-C genotypes (A. S. Muerhoff, J. N. Simons, T. P. Leary, J. C. Erker, M. L. Chalmers, T. J. Pilot-Matias, G. J. Dawson, S. M. Desai, and I. K. Mushahwar, J. Hepatol. 25:379-384, 1996) that correlated with geographic origin: type 1, 2a and 2b, and 3 isolates are found predominantly in West Africa, the United States and Europe, and Japan, respectively. We have extended our analysis to include 5'-UTR sequences from 129 globally distributed GBV-C isolates and sequences from the second envelope protein (E2) gene and nonstructural (NS) regions 3 and 5b from a subset of these isolates. Bootstrap analysis of a 157-nucleotide segment of the 5'-UTR from 129 sequences provided weak support for the existence of the four major groups of GBV-C isolates previously described, although phylogenetic analysis of a 374-nucleotide segment of the 5'-UTR from 83 isolates provided stronger support. Thus, the groups of GBV-C variants previously identified upon analysis of the entire 5'-UTR can be distinguished by analysis of the shorter, 374-nucleotide region from the 5'-UTR. In contrast, independent analysis of the E2, NS3, or NS5b region sequences does not identify groups of GBV-C variants that correlate with geographic origin. However, bootstrap analysis of these coding sequences, when linked to form colinear sequences, demonstrates that longer coding regions can produce GBV-C groupings that are similar to that determined from 5'-UTR sequence analysis. The inability to distinguish between GBV-C variants by using small segments of coding sequence suggests that the GBV-C genome is constrained. As a result of these constraints, there is a high degree of nucleotide and amino acid sequence conservation between isolates from widely separated geographic areas. Hence, substitutions at many nucleotide positions are not tolerated, so that substitutions at the positions which can change are saturated, thereby obscuring the evolutionary relationships.

Base Sequence↗

PubMed1984

Structurally and functionally modified forms of pp60v-src in Rous sarcoma virus-transformed cell lysates.

When analyzed from transformed cell lysates, pp60v-src, the product of the Rous sarcoma virus src gene, typically appears as a single polypeptide of 60,000 molecular weight, phosphorylated at two major sites, an amino-terminal region serine residue and carboxy-terminal region tyrosine residue. We describe here the identification of variant forms of pp60v-src present in transformed cell lysates that exhibited an altered electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels. This change in migration appeared to be the result of some alteration in the amino-terminal portion of the molecule and paralleled the appearance of extensive amino-terminal region tyrosine phosphorylation on the pp60v-src molecule. These structural modifications were further correlated with a dramatic increase in the protein kinase-specific activity of pp60v-src. The detection of these variant forms of pp60v-src depended on the prior treatment of the transformed cell cultures with vanadium ions or the inclusion in the cell disruption buffer of Mg2+ or ATP-Mg2+. The implications is that modified, highly active forms of the pp60v-src protein exist in transformed cells, but are transient and rapidly converted to stable forms, possibly by specific dephosphorylation. We suggest that amino-terminal region tyrosine phosphorylation of pp60v-src, presumably the result of autophosphorylation, serves to greatly enhance src protein enzymatic activity, but that much of the regulation of this transforming protein's function may involve a phosphotyrosyl protein phosphatase.

Animals↗

PubMed1999

Identification of a new transthyretin variant (Ile49) in familial amyloidotic polyneuropathy using electrospray ionization mass spectrometry and nonisotopic RNase cleavage assay.

Mutation of the transthyretin (TTR) plasma protein and gene in a Japanese patient with amyloid polyneuropathy was investigated by electrospray ionization mass spectrometry (ESI-MS) and nonisotopic RNase cleavage assay (NIRCA), respectively. ESI-MS analysis showed normal TTR peaks and additionally a variant TTR with 12-dalton-higher molecular weight than normal TTR. NIRCA suggested that the mutation existed near either the 5' or 3' end of exon 3. Direct DNA sequencing revealed both a normal ACC (threonine) and a variant ATC (isoleucine) at codon 49, which was located near the 5' end of exon 3. The molecular weight shift of this mutation was 12 D, consistent with the result of ESI-MS.

Aged↗

PubMed2006

Barriers to autopsy: Creutzfeldt-Jakob disease in New York state.

Surveillance of Creutzfeldt-Jakob disease (CJD) monitors trends and ensures timely identification of variant CJD and other emergent prion diseases. Brain tissue is needed to definitively diagnose these diseases. A survey of neurologists and pathologists in New York State was conducted to understand neurologists' and pathologists' views on autopsy and CJD. Neurologists reported using autopsy rarely or never. Over half of the pathologists worked in facilities that did not perform autopsies when CJD was suspected. Barriers to autopsy included family reluctance, infection control concerns, and local facilities unable to perform brain autopsy. More accurate, complete recognition of CJD and variant forms depends on physician awareness of the manifestations of CJD and its diagnosis, access to pathologists and facilities willing and able to perform brain biopsies and autopsies, and family acceptance of such procedures.

Attitude of Health Personnel↗

PubMed2006

Identification of a novel C5L2 variant (S323I) in a French Canadian family with familial combined hyperlipemia.

OBJECTIVE: A functional acylation stimulating protein (ASP) receptor, C5L2, has been recently identified in ASP-responsive cells. Impaired ASP-mediated triglyceride synthesis has previously been described in a subset of hyperapolipoprotein B/familial combined hyperlipidemia subjects. METHODS AND RESULTS: DNA sequencing of C5L2 coding region in 61 unrelated probands identified a heterozygous variant (G968-->T) in 1 subject, resulting in Ser323-->Ile substitution in the carboxyl terminal region. This variant was not detected in 2176 additional chromosomes by restriction fragment length polymorphism or fluorescence polarization genotyping. Eight family members of the proband were identified with one altered (+/-)C5L2 allele. Nine other family members had the wild-type (+/+)C5L2 sequence. The abnormal allele was associated with increased plasma triglyceride, plasma cholesterol, low-density lipoprotein (LDL) cholesterol, apolipoprotein B and ASP. Of 23 subjects tested in cell-based ASP bioactivity assays, those with C5L2(+/-) variant (n = 2) had a 50% reduction in ASP-stimulated triglyceride synthesis, glucose transport and marked reduction in maximal binding (B(max)). By contrast, a C5L2(+/+) family member responded normally, as did hyperapolipoprotein B normal ASP subjects compared with C5L2(+/+) controls (n = 6). CONCLUSIONS: The S323I variant may alter C5L2 function and might be one molecular basis contributing to familial combined hyperlipidemia.

Adult↗

PubMed2005

Cellular response to gene expression profiles of different hepatitis C virus core proteins in the Huh-7 cell line with microarray analysis.

In the current investigation, we constructed recombinants of expression of HCV genotype 1b, 2a, and 4d whole core proteins and established a human hepatoma (Huh-7) cell line which expressed different core proteins constitutively. In the Affymetrix human gene chip, the HG-U133 A and B were employed for identification of variant core protein gene expression in the Huh-7 cell line. In data analysis, we applied a threshold that eliminated all genes that were not increased or decreased by at least a 3-fold change in a comparison between transfected cells and control cells. All of these genes were annotated by using NetAffx analysis through the Affymetrix website and categorized on the basis of their biological processes. The microarray analysis result suggested that the gene expression profiles caused by three kinds of core proteins were mainly shown in metabolism, signal transduction, protease activity, immune responses, etc. and that some pathogenesis/oncogenesis, apoptosis, or anti-apoptosis gene expression were up/down-regulated simultaneously in the Huh-7 cell line. In conclusion, the gene expression profiles of variant core proteins were implicated in HCV replication, pathogenesis, or oncogenesis in the Huh-7 cell line, which is useful for our understanding of HCV variant core protein biological function and its pathogenic mechanism.

Carcinoma, Hepatocellular↗

PubMed1982

Identification and measurement of molecular variants of cholecystokinin in duodenal mucosa and plasma. Diminished concentrations in patients with celiac disease.

The amount and type of cholecystokinin (CCK) in duodenal extracts and plasma of celiac patients and normal subjects was studied by radioimmunoassay and gel filtration. In both groups there were similar patterns of molecular forms in extracts of duodenal biopsies, but concentrations in celiac disease were significantly depressed. In boiling water extracts of duodenal mucosa from both groups a factor with the properties of the COOH-terminal octapeptide of cholecystokinin predominated, but there were also significant amounts of a larger molecular weight form. In acid extracts of mucosa a factor with the properties of the 33 or 39 residue form was identified in amounts that were approximately 25% those of CCK8; there were also similar amounts of an acid-soluble form that had an apparent molecular weight higher than CCK39. Plasma immunoreactive cholecystokinin was studied after concentration by immunoaffinity adsorption and fractionation by gel filtration. In normal subjects fasting CCK-like immunoreactivity was less than 0.8 pmol/liter, and after a light breakfast increased to 2.0 +/- 0.7 (range 1.0 to 4.8) pmol/liter; CCK8-like activity accounted for all the increased immunoreactivity. In five of six celiac patients the concentrations of both fasting and postprandial CCK-like immunoreactivity in plasma were undetectable (less than 0.8 pmol/liter). We conclude that diminished production and release of CCK could account for the impaired pancreatic and gall bladder responses to intraluminal stimuli in celiac disease.

Adult↗

PubMed1983

Identification of a new structural variant of human apolipoprotein E, E2(Lys146 leads to Gln), in a type III hyperlipoproteinemic subject with the E3/2 phenotype.

A type III hyperlipoproteinemic subject having the apolipoprotein E (apo E) phenotype E3/2 was identified. From isoelectric focusing experiments in conjunction with cysteamine treatment (a method that measures cysteine content in apo E), the E2 isoform of this subject was determined to have only one cysteine residue, in contrast to all previously studied E2 apoproteins, which had two cysteines. This single cysteine was shown to be at residue 112, the same site at which it occurs in apo E3. From amino acid and sequence analyses, it was determined that this apo E2 differed from apo E3 by the occurrence of glutamine rather than lysine at residue 146. When phospholipid X protein recombinants of the subject's isolated E3 and E2 isoforms were tested for their ability to bind to the human fibroblast apo-B,E receptor, it was found that the E3 bound normally (compared with an apo E3 control) but that the E2 had defective binding (approximately 40% of normal). Although they contained E3 as well as E2, the beta-very low density lipoproteins (beta-VLDL) from this subject were very similar in character to the beta-VLDL from an E2/2 type III hyperlipoproteinemic subject; similar subfractions could be obtained from each subject and were shown to have a similar ability to stimulate cholesteryl ester accumulation in mouse peritoneal macrophages. The new apo E2 variant has also been detected in a second type III hyperlipoproteinemic subject.

Aged↗

PubMed1985

Identification of carriers of a variant plasma prealbumin (transthyretin) associated with familial amyloidotic polyneuropathy type I.

A method is described for detecting carriers of a variant plasma prealbumin that is associated with familial amyloidotic polyneuropathy (FAP) type I. It is based on the finding of an extra methionine in the variant prealbumin, at position 30 from the amino terminals. Since normal prealbumin has only one methionine (position 13), treatment with cyanogen bromide (CNBr), which cleaves only at methionines, results in two peptides. CNBr treatment of the variant prealbumin gives three peptides. The extra can then be detected in two ways: by HPLC using a reverse phase C18 column, and by sequential Edman degradation. Each method can detect as little as 1% variant prealbumin in isolated plasma prealbumin, and therefore, can identify carriers of the gene for the variant protein. Since FAP type I usually is not manifest until after the childbearing years, this method to identify carriers of the gene offers a new approach for genetic counseling of families with this disease. To date, kindreds with hereditary amyloidosis that could benefit from these studies include those with FAP type I of Swedish, Japanese, and Portuguese origins.

Adult↗

PubMed1981

Empathy: its nature and limitations.

Empathy is usually regarded as an irreducible inborn capacity, operative from birth, for knowing the inner experience of another person without necessarily perceiving cues from that person about his thoughts or feelings. Merging of the type characteristic of early infant-mother symbiosis has often been considered the origin and basic component of empathy. However, merging is an illusory experience which cannot function as an active mechanism in the perceptual process, and the psychological structures needed for certain kinds of empathy do not commence development until eighteen months of age. The mechanism of empathy has also been ascribed to vaguely defined variants of identification. This is not a settled issue, but the idea is not compatible with a recent rigorous effort to define identification. The author offers a different theory of empathy, according to which empathy is a capacity that evolves with neuropsychological maturation and interpersonal interactions in the course of individual development. Empathy depends on sensory perception of behavioral cues from the object about his inner state. The empathizer compares these behavioral cues with one or more kinds of referent in this own mind which could be expressed by similar behavior. He then infers that the inner experience of the object qualitatively matches that associated with his referent. Limitations in the accuracy and scope of empathy are threefold: patients may limit or distort the expression of behavioral cues about their state of mind; referents available in the mind of the empathizer may be inadequate; and the inferential process is inherently uncertain. As a result, knowledge of another person's thoughts and feelings which can be acquired through empathy is limited. The theoretical understanding of empathy offered in this paper implies ways for improving empathic accuracy, especially by means of applying two or more kinds of referents to the same set of perceived cues.

Cues↗

PubMed2002

Cloning and characterization of the mouse Mcoln1 gene reveals an alternatively spliced transcript not seen in humans.

BACKGROUND: Mucolipidosis type IV (MLIV) is an autosomal recessive lysosomal storage disorder characterized by severe neurologic and ophthalmologic abnormalities. Recently the MLIV gene, MCOLN1, has been identified as a new member of the transient receptor potential (TRP) cation channel superfamily. Here we report the cloning and characterization of the mouse homologue, Mcoln1, and report a novel splice variant that is not seen in humans. RESULTS: The human and mouse genes display a high degree of synteny. Mcoln1 shows 91% amino acid and 86% nucleotide identity to MCOLN1. Also, Mcoln1 maps to chromosome 8 and contains an open reading frame of 580 amino acids, with a transcript length of approximately 2 kb encoded by 14 exons, similar to its human counterpart. The transcript that results from murine specific alternative splicing encodes a 611 amino acid protein that differs at the c-terminus. CONCLUSIONS: Mcoln1 is highly similar to MCOLN1, especially in the transmembrane domains and ion pore region. Also, the late endosomal/lysosomal targeting signal is conserved, supporting the hypothesis that the protein is localized to these vesicle membranes. To date, there are very few reports describing species-specific splice variants. While identification of Mcoln1 is crucial to the development of mouse models for MLIV, the fact that there are two transcripts in mice suggests an additional or alternate function of the gene that may complicate phenotypic assessment.

Journal Article↗

PubMed2006

Identification of novel functional sequence variants in the gene for peptidase inhibitor 3.

BACKGROUND: Peptidase inhibitor 3 (PI3) inhibits neutrophil elastase and proteinase-3, and has a potential role in skin and lung diseases as well as in cancer. Genome-wide expression profiling of chorioamniotic membranes revealed decreased expression of PI3 in women with preterm premature rupture of membranes. To elucidate the molecular mechanisms contributing to the decreased expression in amniotic membranes, the PI3 gene was searched for sequence variations and the functional significance of the identified promoter variants was studied. METHODS: Single nucleotide polymorphisms (SNPs) were identified by direct sequencing of PCR products spanning a region from 1,173 bp upstream to 1,266 bp downstream of the translation start site. Fourteen SNPs were genotyped from 112 and nine SNPs from 24 unrelated individuals. Putative transcription factor binding sites as detected by in silico search were verified by electrophoretic mobility shift assay (EMSA) using nuclear extract from Hela and amnion cell nuclear extract. Deviation from Hardy-Weinberg equilibrium (HWE) was tested by chi2 goodness-of-fit test. Haplotypes were estimated using expectation maximization (EM) algorithm. RESULTS: Twenty-three sequence variations were identified by direct sequencing of polymerase chain reaction (PCR) products covering 2,439 nt of the PI3 gene (-1,173 nt of promoter sequences and all three exons). Analysis of 112 unrelated individuals showed that 20 variants had minor allele frequencies (MAF) ranging from 0.02 to 0.46 representing "true polymorphisms", while three had MAF < or = 0.01. Eleven variants were in the promoter region; several putative transcription factor binding sites were found at these sites by database searches. Differential binding of transcription factors was demonstrated at two polymorphic sites by electrophoretic mobility shift assays, both in amniotic and HeLa cell nuclear extracts. Differential binding of the transcription factor GATA1 at -689C>G site was confirmed by a supershift. CONCLUSION: The promoter sequences of PI3 have a high degree of variability. Functional promoter variants provide a possible mechanism for explaining the differences in PI3 mRNA expression levels in the chorioamniotic membranes, and are also likely to be useful in elucidating the role of PI3 in other diseases.

Binding Sites↗

PubMed2024

Identification of a novel intronic variant in COL4A2 gene associated with fetal severe cerebral encephalomalacia and subdural hemorrhage.

BACKGROUND: Genetic variants in COL4A2 are less common than those of COL4A1 and their fetal clinical phenotype has not been well described to date. We present a fetus from China with an intronic variant in COL4A2 associated with a prenatal diagnosis of severe cerebral encephalomalacia and subdural hemorrhage. METHODS: Whole exome sequencing (WES) was applied to screen potential genetic causes. Bioinformatic analysis was performed to predict the pathogenicity of the variant. In in vitro experiment, the minigene assays were performed to assess the variant's effect. RESULTS: In this proband, we observed ventriculomegaly, subdural hemorrhage, and extensive encephalomalacia that initially suggested cerebral hypoxic-ischemic and/or hemorrhagic lesions. WES identified a de novo heterozygous variant c.549 + 5G > A in COL4A2 gene. This novel variant leads to the skipping of exon 8, which induces the loss of 24 native amino acids, resulting in a shortened COL4A2 protein (p.Pro161_Gly184del). CONCLUSION: Our study demonstrated that c.549 + 5G > A in COL4A2 gene is a disease-causing variant by aberrant splicing. This finding enriches the variant spectrum of COL4A2 gene, which not only improves the understanding of the fetal neurological disorders associated with hypoxic-ischemic and hemorrhagic lesions from a clinical perspective but also provides guidance on genetic diagnosis and counseling.

Female↗

PubMed2001

Identification of an apolipoprotein(e) variant associated with type III hyperlipoproteinaemia in an indigenous Australian.

As a result of testing for lipid and apolipoprotein(e) (apo E) phenotype status of an indigenous Australian community, an apo E variant associated with type III hyperlipoproteinaemia has been identified. Apo E phenotype was determined by analysis of VLDL by isoelectric focusing, and genotype on DNA amplified by polymerase chain reaction, using two different restriction enzyme isotyping assays. Phenotypes and genotypes were discordant in samples from two subjects and an abnormal-sized restriction fragment was also observed in their genotyping gel patterns. DNA sequencing studies revealed this was due to a single nucleotide deletion, 3817delC, at amino acid 136 on apo E. This resulted in a new reading frame and the premature termination of the apo E protein due to a stop codon (TGA) at nucleotide 4105. The variant apo E null allele showed a recessive mode of inheritance and, in combination with the E2 allele, resulted in the type III hyperlipoproteinaemic phenotype but when inherited with the E4 allele had no marked effect on plasma lipids.

Adolescent↗

PubMed2005

Genome-wide associations of gene expression variation in humans.

The exploration of quantitative variation in human populations has become one of the major priorities for medical genetics. The successful identification of variants that contribute to complex traits is highly dependent on reliable assays and genetic maps. We have performed a genome-wide quantitative trait analysis of 630 genes in 60 unrelated Utah residents with ancestry from Northern and Western Europe using the publicly available phase I data of the International HapMap project. The genes are located in regions of the human genome with elevated functional annotation and disease interest including the ENCODE regions spanning 1% of the genome, Chromosome 21 and Chromosome 20q12-13.2. We apply three different methods of multiple test correction, including Bonferroni, false discovery rate, and permutations. For the 374 expressed genes, we find many regions with statistically significant association of single nucleotide polymorphisms (SNPs) with expression variation in lymphoblastoid cell lines after correcting for multiple tests. Based on our analyses, the signal proximal (cis-) to the genes of interest is more abundant and more stable than distal and trans across statistical methodologies. Our results suggest that regulatory polymorphism is widespread in the human genome and show that the 5-kb (phase I) HapMap has sufficient density to enable linkage disequilibrium mapping in humans. Such studies will significantly enhance our ability to annotate the non-coding part of the genome and interpret functional variation. In addition, we demonstrate that the HapMap cell lines themselves may serve as a useful resource for quantitative measurements at the cellular level.

Chromosome Mapping↗

PubMed2005

Identification of two further splice variants of GABABR1 characterizes the conserved micro-exon 4 as a hot spot for regulated splicing in the rat brain.

Inhibitory neurotransmission in the mammalian brain is principally mediated by gamma-aminobutyric acid (GABA) acting through different subtypes of cell membrane GABA receptor (GABAR). The expression of one GABAR gene, GABABR1, is distinguished by the expression of multiple splice variants that encode different isoforms of the receptor. In the present study, we have identified two novel GABABR1 variants, GABABR1h (R1h) and GABABR1i (R1i), which appear to arise from alternative splicing of the GABABR1 gene. The expression of R1h and R1i is differentially regulated in brain and peripheral tissues, but expression is not altered in the brain of a genetic model of absence epilepsy (GAERS rat [genetic absence epilepsy rat from Strasbourg]). Both the R1h and R1i variants exhibit a novel 80-bp insert downstream of exon 4 that is flanked by consensus splice sites, and both encode C-terminal-truncated proteins. The new insight into the family of GABABR1 variants gained from this study identifies exon 4 as a preferred locus, or hot spot for regulated splicing in the GABABR1 gene. This finding correlates with the micro-exonic nature of exon 4 (21 bp). Bioinformatic analysis of micro-exon 4 and its flanking pre-mRNA sequences has revealed multiple, potentially competitive, exonic splicing enhancers that provide a mechanistic basis for the preponderance of alternative splicing events at this locus. Conservation of GABABR1 micro-exon 4 across species suggests a conserved functional role, facilitating either N-terminal protein production or post-transcriptional gene regulation through regulated splicing coupled to transcript decay.

Alternative Splicing↗

PubMed2001

GABA(C) receptors: a molecular view.

In the central nervous system inhibitory neurotransmission is primarily achieved through activation of receptors for gamma-aminobutyric acid (GABA). Three types of GABA receptors have been identified on the basis of their pharmacological and electrophysiological properties. The predominant type, termed GABA(A), and a recently identified GABA(C) type, form ligand-gated chloride channels, whereas GABA(B) receptors activate separate cation channels via G proteins. Based on their homology to nicotinic acetylcholine receptors, GABA(C) receptors are believed to be oligomeric protein complexes composed of five subunits in a pentameric arrangement. To date up to five different GABA(C) receptors subunits have been identified in various species. Recent studies have shed new light on the biological characteristics of GABA(C) receptors, including the chromosomal localization of its subunit genes and resulting links to deseases, the cloning of new splice variants, the identification of GABA(C) receptor-associated proteins, the identification of domains involved in subunit assembly, and finally structure/function studies examining functional consequences of introduced mutations. This review summarizes recent data in view of the molecular structure of GABA(C) receptors and presents new insights into the biological function of this protein in the retina.

Animals↗

PubMed1987

Mockery through caricature: a variant of introjection utilized by a masochistic woman.

This case study of a woman with moral masochism focuses upon her characteristic defenses. Particular attention is paid to her utilization of a variant of identification. This patient's mother was a strict, harsh disciplinarian who insisted upon total compliance as a condition for her approval and love. This roused considerable anger and resentment in this patient but also intense anxiety, lest she lose her mother's love. To deal with this, she adopted an ingenious strategy. She established a veneer of pseudocompliance, following her mother's dictates to the letter. She incorporated many of her severe attitudes. She took these to an extreme, in effect caricaturing her. Through this process of exaggerating these bits of maternal behavior she made herself miserable, i.e., she became anhedonic, anorectic, and anorgasmic except via painful anal intercourse. This was a most useful compromise formation that enabled her to appear as her mother's ideal child and, therefore, retain her love. At the same time, by achieving a state of misery through following her mother's orders, she exposed her as ridiculous, and thus covertly discharged considerable aggression.

Adult↗