Alternative splicing of transcripts encoding the alpha- and beta-subunits of mouse glucosidase II in T lymphocytes.
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Glucosidase II is a processing enzyme of the endoplasmic reticulum that functions to hydrolyze two glucose residues in immature N -linked oligosaccharides attached to newly synthesized polypeptides. We previously reported the cDNA cloning of the alpha- and beta-subunits of mouse glucosidase II from T cells following copurification of these proteins with the highly glycosylated transmembrane protein-tyrosine phosphatase CD45. Subsequent examination of additional cDNA clones, coupled with partial genomic DNA sequencing, has revealed that both subunits are encoded by gene products that undergo alternative splicing in T lymphocytes. The catalytic alpha-subunit possesses two variably expressed segments, box Alpha1, consisting of 22 amino acids located proximal to the amino-terminus, and box Alpha2, composed of 9 amino acids situated between the amino-terminus and the putative catalytic site in the central region of the molecule. Box Beta1, a variably expressed 7 amino acid segment in the beta-subunit of glucosidase II, is located immediately downstream of an acidic stretch near the carboxyl-terminus. Screening of reverse transcribed RNA by polymerase chain reaction confirms the variable inclusion of each of these segments in transcripts obtained from a panel of T-lymphocyte cell lines. Thus, distinct isoforms of glucosidase II exist that may perform specialized functions. (+info)
Sequence heterogeneity within three different regions of the hepatitis G virus genome.
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Two sets of primers derived from the 5'-terminal region and the NS5 region of the hepatitis G virus (HGV) genome were used to amplify PCR fragments from serum specimens obtained from different parts of the world. All PCR fragments from the 5'-terminal region (5'-PCR, n = 56) and from the NS5 region (NS5-PCR, n = 85) were sequenced and compared to corresponding published HGV sequences. The range of nucleotide sequence similarity varied from 74 and 78% to 100% for 5'-PCR and NS5-PCR fragments, respectively. Additionally, five overlapping PCR fragments comprising an approximately 2.0-kb structural region of the HGV genome were sequenced from each of five sera obtained from three United States residents. These sequences were compared to 20 published sequences comprising the same region of the HGV genome. Nucleotide and deduced amino acid sequences obtained from different individuals were homologous from 82.9 to 93. 6% and from 90.4 to 99.0%, respectively. Sequences obtained from follow-up specimens were almost identical. Comparative analysis of deduced amino acid sequences of the HGV structural proteins and hepatitis C virus (HCV) structural proteins combined with an analysis of predicted secondary structures and hydrophobic profiles allowed prediction of processing sites within the HGV structural proteins. A phylogenetic sequence analysis performed on the 2.0-kb structural region supports the existence of three previously identified HGV genetic groups. However, phylogenetic analysis performed on only small DNA fragments yielded inconsistent genetic grouping and failed to confirm the existence of genetic groups. Thus, in contrast to HCV where almost any region can be used for genotyping, only large or carefully selected genome fragments can be used to identify consistent HGV genetic groups. (+info)
Analysis of chromosome 1q42.2-43 in 152 families with high risk of prostate cancer.
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One hundred fifty-two families with prostate cancer were analyzed for linkage to markers spanning a 20-cM region of 1q42.2-43, the location of a putative prostate cancer-susceptibility locus (PCAP). No significant evidence for linkage was found, by use of both parametric and nonparametric tests, in our total data set, which included 522 genotyped affected men. Rejection of linkage may reflect locus heterogeneity or the confounding effects of sporadic disease in older-onset cases; therefore, pedigrees were stratified into homogeneous subsets based on mean age at diagnosis of prostate cancer and number of affected men. Analyses of these subsets also detected no significant evidence for linkage, although LOD scores were positive at higher recombination fractions, which is consistent with the presence of a small proportion of families with linkage. The most suggestive evidence of linkage was in families with at least five affected men (nonparametric linkage score of 1.2; P=.1). If heterogeneity is assumed, an estimated 4%-9% of these 152 families may show linkage in this region. We conclude that the putative PCAP locus does not account for a large proportion of these families with prostate cancer, although the linkage of a small subset is compatible with these data. (+info)
Genetic linkage of IgA deficiency to the major histocompatibility complex: evidence for allele segregation distortion, parent-of-origin penetrance differences, and the role of anti-IgA antibodies in disease predisposition.
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Immunoglobulin A (IgA) deficiency (IgAD) is characterized by a defect of terminal lymphocyte differentiation, leading to a lack of IgA in serum and mucosal secretions. Familial clustering, variable population prevalence in different ethnic groups, and a predominant inheritance pattern suggest a strong genetic predisposition to IgAD. The genetic susceptibility to IgAD is shared with a less prevalent, but more profound, defect called "common variable immunodeficiency" (CVID). Here we show an increased allele sharing at 6p21 in affected members of 83 multiplex IgAD/CVID pedigrees and demonstrate, using transmission/diseqilibrium tests, family-based associations indicating the presence of a predisposing locus, designated "IGAD1," in the proximal part of the major histocompatibility complex (MHC). The recurrence risk of IgAD was found to depend on the sex of parents transmitting the defect: affected mothers were more likely to produce offspring with IgAD than were affected fathers. Carrier mothers but not carrier fathers transmitted IGAD1 alleles more frequently to the affected offspring than would be expected under random segregation. The differential parent-of-origin penetrance is proposed to reflect a maternal effect mediated by the production of anti-IgA antibodies tentatively linked to IGAD1. This is supported by higher frequency of anti-IgA-positive females transmitting the disorder to children, in comparison with female IgAD nontransmitters, and by linkage data in the former group. Such pathogenic mechanisms may be shared by other MHC-linked complex traits associated with the production of specific autoantibodies, parental effects, and a particular MHC haplotype. (+info)
ERGIC-53 gene structure and mutation analysis in 19 combined factors V and VIII deficiency families.
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Combined factors V and VIII deficiency is an autosomal recessive bleeding disorder associated with plasma levels of coagulation factors V and VIII approximately 5% to 30% of normal. The disease gene was recently identified as the endoplasmic reticulum-Golgi intermediate compartment protein ERGIC-53 by positional cloning, with the detection of two founder mutations in 10 Jewish families. To identify mutations in additional families, the structure of the ERGIC-53 gene was determined by genomic polymerase chain reaction (PCR) and sequence analysis of bacterial artificial chromosome clones containing the ERGIC-53 gene. Nineteen additional families were analyzed by direct sequence analysis of the entire coding region and the intron/exon junctions. Seven novel mutations were identified in 10 families, with one additional family found to harbor one of the two previously described mutations. All of the identified mutations would be predicted to result in complete absence of functional ERGIC-53 protein. In 8 of 19 families, no mutation was identified. Genotyping data indicate that at least two of these families are not linked to the ERGIC-53 locus. Taken together, these results suggest that a significant subset of combined factors V and VIII deficiency is due to mutation in one or more additional genes. (+info)
Thrombophilia as a multigenic disease.
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BACKGROUND AND OBJECTIVE: Venous thrombosis is a common disease annually affecting 1 in 1000 individuals. The multifactorial nature of the disease is illustrated by the frequent identification of one or more predisposing genetic and/or environmental risk factors in thrombosis patients. Most of the genetic defects known today affect the function of the natural anticoagulant pathways and in particular the protein C system. This presentation focuses on the importance of the genetic factors in the pathogenesis of inherited thrombophilia with particular emphasis on those defects which affect the protein C system. INFORMATION SOURCES: Published results in articles covered by the Medline database have been integrated with our original studies in the field of thrombophilia. STATE OF THE ART AND PERSPECTIVES: The risk of venous thrombosis is increased when the hemostatic balance between pro- and anti-coagulant forces is shifted in favor of coagulation. When this is caused by an inherited defect, the resulting hypercoagulable state is a lifelong risk factor for thrombosis. Resistance to activated protein C (APC resistance) is the most common inherited hypercoagulable state found to be associated with venous thrombosis. It is caused by a single point mutation in the factor V (FV) gene, which predicts the substitution of Arg506 with a Gln. Arg506 is one of three APC-cleavage sites and the mutation results in the loss of this APC-cleavage site. The mutation is only found in Caucasians but the prevalence of the mutant FV allele (FV:Q506) varies between countries. It is found to be highly prevalent (up to 15%) in Scandinavian populations, in areas with high incidence of thrombosis. FV:Q506 is associated with a 5-10-fold increased risk of thrombosis and is found in 20-60% of Caucasian patients with thrombosis. The second most common inherited risk factor for thrombosis is a point mutation (G20210A) in the 3' untranslated region of the prothrombin gene. This mutation is present in approximately 2% of healthy individuals and in 6-7% of thrombosis patients, suggesting it to be a mild risk factor of thrombosis. Other less common genetic risk factors for thrombosis are the deficiencies of natural anticoagulant proteins such as antithrombin, protein C or protein S. Such defects are present in less than 1% of healthy individuals and together they account for 5-10% of genetic defects found in patients with venous thrombosis. Owing to the high prevalence of inherited APC resistance (FV:Q506) and of the G20210A mutation in the prothrombin gene, combinations of genetic defects are relatively common in the general population. As each genetic defect is an independent risk factor for thrombosis, individuals with multiple defects have a highly increased risk of thrombosis. As a consequence, multiple defects are often found in patients with thrombosis. (+info)
Sanfilippo type B syndrome (mucopolysaccharidosis III B): allelic heterogeneity corresponds to the wide spectrum of clinical phenotypes.
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Sanfilippo B syndrome (mucopolysaccharidosis IIIB, MPS IIIB) is caused by a deficiency of alpha-N-acetylglucosaminidase, a lysosomal enzyme involved in the degradation of heparan sulphate. Accumulation of the substrate in lysosomes leads to degeneration of the central nervous system with progressive dementia often combined with hyperactivity and aggressive behaviour. Age of onset and rate of progression vary considerably, whilst diagnosis is often delayed due to the absence of the pronounced skeletal changes observed in other mucopolysaccharidoses. Cloning of the gene and cDNA encoding alpha-N-acetylglucosaminidase enabled a study of the molecular basis of this syndrome. We were able to identify 31 mutations, 25 of them novel, and two polymorphisms in the 40 patients mostly of Australasian and Dutch origin included in this study. The observed allellic heterogeneity reflects the wide spectrum of clinical phenotypes reported for MPS IIIB patients. The majority of changes are missense mutations; also four nonsense and nine frameshift mutations caused by insertions or deletions were identified. Only five mutations were found in more than one patient and the observed frequencies are well below those observed for the common mutations in MPS IIIA. R643C and R297X each account for around 20% of MPS IIIB alleles in the Dutch patient group, whilst R297X, P521L, R565W and R626X each have a frequency of about 6% in Australasian patients. R643C seems to be a Dutch MPS IIIB allele and clearly confers the attenuated phenotype. One region of the gene shows a higher concentration of mutations, probably reflecting the instability of this area which contains a direct repeat. Several arginine residues seem to be 'hot-spots' for mutations, being affected by two or three individual base pair exchanges. (+info)
Heterogeneity of T-cell receptor usage in experimental autoimmune neuritis in the Lewis rat.
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In experimental autoimmune neuritis (EAN), T-cell receptor (TCR) variable (V)-region gene usage by neuritogenic T cells has been reported to be clonally restricted at the RNA level. This study was designed to verify TCR usage by neuritogenic T cells at the protein level. We generated two monoclonal antibodies (mAbs) 7H4 and 8G8 specific for a Vbeta4/Valpha11 associated idiotype expressed by the majority of neuritogenic cells of P2-specific T-cell lines. The remaining neuritogenic P2-specific T cells either exhibited a dominant usage of the TCR Vbeta13 chain recognized by the recently generated mAbs 17D5 and 18B1 or showed diverse Vbeta usage. Treatment of adoptive-transfer (AT)-EAN or of EAN actively induced with the neuritogenic P2 peptide by mAbs 7H4 and 8G8 led to a partial, but significant, reduction of clinical disease. Treatment with Vbeta13-specific mAb 17D5 had no clear effect on active EAN. Our data show that at least three different TCR are used by P2-specific pathogenic T cells in EAN, an animal model for human inflammatory neuropathies. (+info)