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Results for “variant calling”

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PubMed1976

GD (--) Aachen, a new variant of deficient glucose-6-phosphate dehydrogenase. Clinical, genetic, biochemical aspects.

A deficient G-6PD variant was discovered in 4 males of one family from northwestern Germany. Five generations of this family could be studied. The deficient G-6PD was a new variant, called "Gd (--) Aachen". Its main characteristics are the following: severe enzyme deficiency in erythrocytes (3% of normal), contrasting with an almost normal activity in leukocytes; normal molecular specific activity (i.e., normal ratio enzyme activity/cross-reacting material); slow mobility in starch gel electrophoresis (92-94% of normal); increased Michaelis constant for glucoes-6-phosphate (60-70 muM) and NADP+ (20-25 muM); decreased inhibition constant by NADPH with respect to NADP+ (7 muM); increased inhibition by ATP; normal utilization of the substrate analogues; slightly biphasic pH curve; thermal instability, and normal activation energy of the enzymatic reaction. The relationships between the hematologic disorders (severe and frequent hemolytic crises) and the unfavorable kinetic modifications are discussed.

Adult↗

PubMed1997

On the role of the low-affinity neurotrophin receptor p75LNTR in nerve growth factor induction of differentiation and AP 1 binding activity in PC12 cells.

Three clones of PC12 cells that differ with respect to their nerve growth factor (NGF) receptors were examined: wild-type PC12 cells that have both trkA and p75LNTR receptors; the MR-1 clone that possesses a normal trkA receptor and a truncated form of p75LNTR without the extracellular NGF-binding part; and a new PC12 variant, called v-clone, that is partly characterized here. The v-clone had no demonstrable binding to trkA, but displayed binding to p75LNTR as assessed by chemical crosslinking. NGF did not induce any change in the tyrosine phosphorylation of phosphatidy-3'-kinase in the v-clone. NGF induced neurite extension in wild-type cells, induced it more rapidly in mR-1, but not at all in v-clone cells. The v-clone lacked the b-form of protein kinase C, but transfection with this enzyme did not restore responsiveness to NGF. Neurite extension in response to staurosporine and basic fibroblast growth factor was equal in wild-type and v-clone cells. All three clones responded to forskolin, with the mR-1 clone the most responsive. NGF stimulated AP 1 binding activity in all clones. The response was transient in the MR-1 clone but prolonged in the wild-type and v-clone cells. In the wild-type and MR-1 clone cells, AP 1 binding activity was reduced by a tyrphostin analog, whereas in the v-clone cells it was inhibited by staurosporine. NGF increased inositol (1,4,5)-trisphosphate (InsP3) formation in all clones. In the wild-type and v-clone cells the InsP3 responses were followed by [Ca2+]i increases. It is concluded that although trkA is required for differentiation in response to NGF in PC12 cells, the concomitant stimulation, by NGF, of p75LNTR may affect phospholipase C and AP 1. This may be important for the reported ability of p75LNTR to modify the phenotypic changes induced in PC12 cells by NGF.

Animals↗

PubMed2000

Papillary glioneuronal tumour: clinicopathological and biochemical study of one case with 7-year follow up.

Among the mixed glioneuronal tumours, a new variant called papillary glioneuronal tumour has recently been delineated. A case occurring in a 23-year-old man is reported. The tumour was cystic with a mural nodule enhanced by gadolinium injection. It was located within the left parieto-occipital lobe. Surgical excision showed a greyish friable tumour with cystic areas. Histopathological examination revealed a pseudopapillary component comprising a single layer of regular cells, arranged around hyalinised vessels. These cells were immunoreactive with anti-glial fibrillary acidic protein and HNK1 antibodies. A neurocytoma-like component coexisted with round blind cells and focal fibrillary rosettes. These cells were immunostained by anti-neuron-specific enolase and anti-synaptophysin antibodies. Neither mitoses nor ganglioid cells were seen. HNK1, the three isoforms of NCAM, and the L1 adhesion molecule were detected by Western blot analysis. Ultrastructural study showed three different types of cells. The first contained gliofilaments, the second showed long processes with true synapses, and the third was poorly differentiated. However, all had identical nuclei and contained dense bodies. These findings suggest a common origin for the tumour cells derived from a bipotential neuroglial progenitor. As for other mature mixed neuroglial tumours, the prognosis is good. Our patient is free of disease 7 years after complete surgical treatment.

Adult↗

PubMed2026

A transcriptome-wide approach for rapid pathotype discrimination of Puccinia striiformis f. sp. tritici in north-western India.

Stripe rust of wheat caused by Puccinia striiformis f. sp. tritici (Pst) remains a major constraint to wheat production in India due to the rapid evolution and frequent emergence of virulent pathotypes. Rapid and reliable discrimination of Pst pathotypes is essential for effective resistance deployment and surveillance. In the present study, transcriptome-wide simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs) were exploited to develop and validate molecular markers for pathotype-specific detection of Pst pathotypes prevalent in North India (110S119, 238S119, 46S119, 110S84 and 78S84). Microsatellite mining from 6103 core orthologous clusters comprising 51,127 transcripts mined 14,634 SSR loci, from which 93 primer pairs were synthesized. However, only three SSR markers exhibited polymorphism indicating limited discrimination potential of expressed sequence-derived (EST) SSRs for pathotype differentiation. In contrast, SNP discovery through stringent variant calling and filtration yielded 186 pathotype-specific homokaryotic SNPs, of which 56 high-confidence loci were selected for Kompetitive Allele-Specific PCR (KASP) assay development. A total of 48 KASP markers were synthesized and 14 demonstrated clear pathotype- or cluster-specific polymorphism representing substantially higher resolution than SSR markers. The high SNP-to-KASP conversion efficiency (~ 95%) and reproducible fluorescence-based clustering emphasize the robustness of KASP assay. Comparative evaluation revealed that SNP-based KASP markers provide superior discriminatory capacity for closely related Pst pathotypes and represent a promising complementary molecular approach for rapid identification of predominant Indian Pst pathotypes. The validated marker panel developed in this study can complement conventional virulence phenotyping and field pathogenomics approaches for surveillance of currently known pathotypes, while continued refinement may accommodate future changes in pathogen populations.

India↗

PubMed2005

Specific detection and semi-quantitative analysis of TRPC4 protein expression by antibodies.

In mouse tissues two variants of the transient receptor potential (canonical) (TRPC) 4 protein are expressed: the "full-length" TRPC4 protein and a slightly smaller variant, called TRPC4Delta(761-864), which lacks 84 amino acid residues. Although the presence of mRNA encoding the TRPC4 protein in mammalian cells and the detection of the heterologously expressed TRPC4 protein by Western blot analysis have been reported, the unequivocal detection of endogenous TRPC4 proteins has proven difficult. In the present study we compared polyclonal antibodies for the detection of TRPC4 proteins in mouse tissues and monitored their specificity and reliability by analysing corresponding tissues from TRPC4-deficient mice. In addition we introduced a procedure that allows us to estimate the amount of TRPC4 protein expressed in a single cell. Using this technique it appears that the amount of TRPC4 protein expressed stably in HEK 293 cells is at least fourfold higher than the amount of TRPC4 protein expressed endogenously in the bovine adrenocortical cell line SBAC.

Animals↗

PubMed1996

A detailed mutational analysis of the VSG gene expression site promoter.

The African trypanosome Trypanosoma brucei is a protozoan parasite that causes the disease African sleeping sickness. The parasite avoids the host's immune response by the process of antigenic variation, or by sequentially expressing antigenically different cell-surface coat proteins. These proteins, called variant surface glycoproteins (VSGs), are expressed from a specific locus, the VSG gene expression site (ES). In an attempt to understand expression of VSG genes, we expanded on earlier investigations of the promoter that controls the large VSG gene expression site transcription unit. We studied VSG ES promoter function both in transient transfection assays, and after stable integration at a chromosomal locus. Analysis of closely spaced deletion mutants showed that the minimum VSG ES promoter fragment that gives full activity is extremely small, and mapped precisely to a fragment that contains no more than -67 bp 5' to the putative transcription initiation site. The promoter lacked an upstream control element, or UCE, an element found at the PARP promoter, and at most eukaryotic Pol I promoters. Furthermore, linker scanning mutagenesis demonstrated that the VSG ES promoter contains at least two essential regulatory elements, including sequences within the region -67/-60 and the region -35/-20, both numbered relative to the initiation site. An altered promoter with mutated nucleotides surrounding the transcription initiation site still directed wild-type levels of expression. In this study, the results were similar for both insect and bloodstream form trypanosomes, suggesting that the same basic machinery for expression from the VSG ES promoter is found in both stages of the parasite.

Animals↗

PubMed1991

Inhibition of antibody-dependent allergic autocytotoxicity in rheumatoid arthritis by OM-89.

Rheumatoid arthritis (RA) is a disease of multiple etiologies and clinical evidence suggests that a separate variant called "allergic arthritis" induced by food antigens could exist. A missing link in the confirmation of such an observation is a relative lack of a reliable in vitro assay which can confirm the in vivo oral ingestion challenge. Therefore, white blood cells (WBC) from 33 rheumatoid arthritis patients were separated and their disintegration was measured in the presence of specific IgE RAST positive sera and gluten-gliadin antigens. This assay was called the antibody-dependent allergic autocytotoxicity (ACT) test which represents an equivalent of an oral ingestion challenge with food antigens. Control WBC expressed 10-45% disintegration as compared to 75-95% in RA. Preincubation of WBC with OM-89 (immunomodulating fractions of Escherichia coli, OM Laboratories Ltd, Geneva, Switzerland) inhibited significantly antibody-dependent ACT in a dose-related manner (P less than 0.001) in our patients.

Adjuvants, Immunologic↗

PubMedDate not supplied

Fluorescent Y-chromosomes in hairs and blood stains.

Y-chromosome detection by way of fluorescence microscopy in biological materials has made sex determination possible in various areas of investigation. The present report describes the results of sex determination on hairs and blood stains. Significant differences were found between the Y-body count for female and male materials. In blind trials it was demonstrated that a reliable sex determination of hairs was possible for at least 27 weeks and of blood stains on cotton cloth and glass for 6 weeks. There were no false positive findings, but there was one male with a "female" blood smear count, who revealed an abnormally small fluorescent region on his Y-chromosome. The existence of such variants calls for caution when evaluating a low count.

Blood Stains↗

PubMedDate not supplied

Clinical applications and methodological developments of the RARE technique.

The RARE technique is an extremely useful tool for clinical diagnosis, since it delivers images comparable to those produced by X-ray myelography and X-ray urography. Contrary to these methods, RARE does not use the application of contrast agents. A low-flip-angle variant called FLARE (fast low angle refocused echo imaging) makes this technique accessible for high-field systems.

Humans↗

PubMed2026

Whole genome sequence data on Ethiopian key sorghum landraces and founder lines.

Sorghum (Sorghum bicolor (L.) Moench) is the fifth most important cereal globally. Its genetic diversity is key to improving yield stability, stress tolerance, and adaptation to different environments. Ethiopia is one of the centers for the crop's origin, diversity, and use in both human food and livestock feed. However, genomic data on Ethiopian sorghum remain limited, especially for landraces preferred by local farmers. This dataset consists of whole-genome sequencing data for 188 Ethiopian sorghum accessions, including founder lines and important landraces from major agroecological zones. Sequencing was performed using the Complete Genomics DNBSEQ-T7 platform, generating high-coverage whole-genome data (20 × coverage). On average, each accession produced 64.7 million reads. Reads were aligned to the Sorghum bicolor NCBIv3 reference genome and variants called using GATK HaplotypeCaller with joint genotyping (GATK v4.6.1.0). Hard-filtering followed GATK best-practice thresholds (QD <2.0, FS> 60.0, MQ <40.0, MQRankSum <-12.5, ReadPosRankSum <-8.0), retaining biallelic SNPs with mean depth 10-50×, missingness ≤20%, and MAF ≥0.05, yielding 6095,752 high-confidence SNPs across 185 accessions. Both the raw FASTQ files and processed VCF files are publicly available to support studies of sorghum genetic diversity, population structure, selection, and the genetic basis of important traits.

Adaptation↗

PubMed2004

Germ-line specific variants of components of the mitochondrial outer membrane import machinery in Drosophila.

A search of the Drosophila genome for genes encoding components of the mitochondrial translocase of outer membrane (TOM) complex revealed duplication of genes encoding homologues of Tom20 and Tom40. Tom20 and Tom40 were represented by two differentially expressed homologues in the Drosophila genome. While dtom20 and dtom40 appeared to be expressed ubiquitously, the second variants, called tomboy20 and tomboy40, were expressed only in the male germ-line. Transcripts for tomboy20 and tomboy40 were detected in primary spermatocytes as well as post-meiotic stages. Transcription of tomboy20 and tomboy40 in spermatocytes was not dependent on the transcription factor Cannonball, which is responsible for controlling expression of gene products exclusively required for post-meiotic germ cell differentiation. Epitope-tagging and transient expression of dTom20 and Tomboy40 in mammalian cell culture showed proper targeting to mitochondria.

Amino Acid Sequence↗

PubMed2005

The clinical importance of the nomenclature, evolution and taxonomy of human papillomaviruses.

Human papillomaviruses (HPVs) are formally described by isolation of their circular double-stranded DNA genomes and establishment and comparison of the nucleotide sequence of these genomes. Alternatives such as serological diagnosis and maintenance of HPVs in culture are neither clinically useful nor consistently feasible. Novel HPV isolates have traditionally been described as "types". The analysis of specific HPV types is of medical importance, because HPV types typically induce type-specific lesions, i.e. they may be specific for cutaneous or mucosal epithelia, or give rise to benign warts or malignant carcinomas. Recently, it was formally decided that papillomaviruses are a virus family separate from the polyomaviruses. Within the papillomavirus family, closely or remotely related types form species or genera. These formal agreements were important as they brought the taxonomy of papillomaviruses in line with that of other viruses, bacteria and higher organisms, although their impact on medical practice and terminology used in clinical studies is limited. Notably, however, HPV types that are closely related (i.e. form "species") are associated with similar lesions. Confusion of the terms "type" and "subtype" should be avoided, as the latter term refers to some specific but rare taxonomic assemblages. In contrast to many RNA viruses, HPV types evolve very slowly, and diverged since the origin of humans only by about 2%. These divergent isolates are called "variants". HPVs evolved together with humankind and Homo sapiens was never without HPVs, and consequently never without warts and cervical cancer. Variants of the same HPV type may have different pathogenicity and may account for part of the worldwide disparities in the occurrence of genital cancers.

Evolution, Molecular↗

PubMed2006

Dystrophin Dp71f associates with the beta1-integrin adhesion complex to modulate PC12 cell adhesion.

Dystrophin Dp71 is the main product of the Duchenne muscular dystrophy gene in the brain; however, its function is unknown. To study the role of Dp71 in neuronal cells, we previously generated by antisense treatment PC12 neuronal cell clones with decreased Dp71 expression (antisense-Dp71 cells). PC12 cells express two different splicing isoforms of Dp71, a cytoplasmic variant called Dp71f and a nuclear isoform called Dp71d. We previously reported that antisense-Dp71 cells display deficient adhesion to substrate and reduced immunostaining of beta1-integrin in the cell area contacting the substrate. In this study, we isolated additional antisense-Dp71 clones to analyze in detail the potential involvement of Dp71f isoform with the beta1-integrin adhesion system of PC12 cells. Immunofluorescence analyses as well as immunoprecipitation assays demonstrated that the PC12 cell beta1-integrin adhesion complex is composed of beta1-integrin, talin, paxillin, alpha-actinin, FAK and actin. In addition, our results showed that Dp71f associates with most of the beta1-integrin complex components (beta1-integrin, FAK, alpha-actinin, talin and actin). In the antisense-Dp71 cells, the deficiency of Dp71 provokes a significant reduction of the beta1-integrin adhesion complex and, consequently, the deficient adhesion of these cells to laminin. In vitro binding experiments confirmed the interaction of Dp71f with FAK and beta1-integrin. Our data indicate that Dp71f is a structural component of the beta1-integrin adhesion complex of PC12 cells that modulates PC12 cell adhesion by conferring proper complex assembly and/or maintenance.

Actinin↗

PubMed2006

Clinical characteristics of cortical multiple sclerosis.

There are several articles in this special issue in which authors eloquently describe neurobehavioural and cognitive complications of multiple sclerosis with relevant neuropsychological assessments and neuroimaging findings. However behavioural and cognitive presentation of multiple sclerosis remains poorly understood. Two years ago, we reported a series of patients with multiple sclerosis who presented with neurobehavioural symptoms and had neuropsychological deficits consistent with cortical dysfunction. Based on previous case reports, pathological studies of cerebral cortex in multiple sclerosis and advanced neuroimaging studies we suggested that neurobehavioural presentation of multiple sclerosis represents a new variant called "cortical multiple sclerosis". The condition is characterised by predominant or exclusive cortical pathology presenting with neurobehavioural symptoms, such as depression, amnesia or distinct cortical syndromes. Since the publication of our report, there has been further neuroimaging and neuropathological findings that further supported the above concept. In addition, observation of more patients with this condition helped us to formulate a logical approach in the detection of these patients. This article focuses on their clinical characteristics.

Behavioral Symptoms↗

PubMed2004

Detection of wild type and deleted latent membrane protein 1 (LMP1) of Epstein-Barr virus in clinical biopsy material.

The latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is one of two postulated viral oncogenic proteins. Sequence variations, and in particular a 30 base pair deletion variant called CAO, may define different disease populations. We developed a panel of rat monoclonal antibodies (MAb) specific for the non-wild type LMP1 and compared the presence of the antibody staining with LMP1 DNA sequence analysis on clinical samples of nasopharyngeal carcinoma, peripheral T-cell lymphoma (PTCL), Hodgkin's disease, lymphoblastoid cell lines (LCL) from normal volunteers, and patients with nasopharyngeal carcinoma. The results demonstrate specificity of the monoclonal cocktail for detecting the non-wild type LMP1 and the ability to sub-differentiate between the mediterranean type of LMP1 and the CAO-LMP1. Double immunofluorescence on paraffin material using the traditional CS1-4 monoclonal antibodies and the CAO-cocktail revealed no dual population of cells in the biopsy material from the Asian region.

Amino Acid Sequence↗

PubMed2026

The SMN locus in the T2T era: Structure, gene conversion, and clinical implications.

Long-read sequencing, paralog-aware variant calling, and telomere-to-telomere (T2T) human genome assemblies now enable the resolution of copy-, haplotype-, and nucleotide-level complexities in segmentally duplicated loci, which were previously inaccessible with short-read sequencing. In this review, we highlight how current technologies and analysis methods reveal extensive diversity in copy number (CN), structure, and gene conversion within the spinal muscular atrophy-associated survival motor neuron (SMN) locus. We summarize how understanding population-level structural variation could be translated into clinical practice, where a nucleotide-level view of the SMN locus may refine prognostic accuracy beyond SMN2 CN and explain variable treatment responses. Finally, we discuss how the approaches and methodologies required to study the SMN locus may be applied elsewhere, providing a scaffold to characterize other complex human genetic regions.

Humans↗

PubMed2006

Dissecting the sequence specific functions of alternative N-terminal isoforms of mouse bullous pemphigoid antigen 1.

Bullous pemphigoid antigen 1 (BPAG1) is a member of the plakin family of proteins that is involved in cross-linking the cytoskeletal elements and attaching them to cell junctions. BPAG1 null mice develop severe degeneration of sensory neurons that was attributed in part due to the absence of a splice variant called BPAG1a that harbors an actin-binding domain at the N-terminus. Additional alternative splicing also results in BPAG1a isoforms with different first exons, leading to three additional types of BPAG1a called isoforms 1, 2 and 3 (or BPAG1a1, BPAG1a2, and BPAG1a3). These unique N-terminal extensions of the BPAG1a isoforms are of variable length. In this study, we characterized these N-terminal isoforms and evaluated the influence of these unique N-terminal sequences to the actin-binding properties. The unique N-terminal region of isoform 1 is very short and was not expected to affect the property of the ABD that followed it. In contrast, transfection studies and mutagenesis analyses signified that the N-terminal sequences of isoform 2 had the ability to bundle actin filaments and the N-terminal region that contained isoform 3 showed cortical localization. Isoforms 1, 2 and 3 also displayed differential tissue expression profiles. Taken together, these data suggested that the unique N-terminal regions of these isoforms have different roles that may be tailored to meet tissue specific functions.

Actins↗

PubMed2005

eGFP reporter genes silence LCRbeta-globin transgene expression via CpG dinucleotides.

beta-Globin transgenes regulated by the locus control region (LCR) are dominantly silenced by linked bacterial reporter genes in transgenic mice. Enhanced green fluorescent protein (eGFP) from jellyfish is an alternative reporter used in retrovirus vectors to transfer LCRbeta-globin genes into bone marrow. We show here that the eGFP coding sequence silences LCRbeta-globin in transgenic mice, but the PGK promoter did not provoke such silencing. As eGFP contains 60 CpG dinucleotides, which are targets of DNA methylation, we synthesized a novel CpG-free variant called dmGFP. Its utility was demonstrated in MSCV retrovirus vectors transcriptionally controlled by the viral 5'LTR or internal PGK or EF1alpha promoter. Specific fluorescence was detected from eGFP, and at lower levels from dmGFP, in transduced mouse CFU-S and embryonic stem cells. While eGFP was rarely silenced in CFU-S, dmGFP was not silenced in these progenitors. Moreover, the dmGFP coding sequence did not silence LCRbeta-globin in transgenic mice, showing that the eGFP silencing mechanism acts primarily via CpG dinucleotides. However, LCRbeta-globin expression remained suboptimal, indicating that other silencing pathways recognize dmGFP in the absence of CpG dinucleotides. We conclude that dmGFP ameliorates silencing, but optimal LCRbeta-globin expression is obtained in the absence of nonmammalian reporters.

Animals↗