Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Variant identification”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,459 records · Page 81Linked to original sources
PubMed1999

Preformulation study of the vaccine candidate P64k against Neisseria meningitidis.

We have previously isolated, cloned and expressed in Escherichia coli the lpdA gene coding for a high-molecular-mass protein (P64k) common to many meningococcal strains. P64k is an outer membrane lipoamide dehydrogenase that is highly immunogenic in animals. Here we describe a preformulation study of the recombinant protein as a vaccine candidate against Neisseria meningitidis, in which six variants containing the candidate were tested. Three assays were used to identify the most suitable variant for further evaluation: percentage of adsorption, identification of P64k by SDS/PAGE, and immunogenicity in mice. All the preformulation variants studied showed more than 98% of adsorption of P64k on the aluminium gel. After desorption, P64k was also identified by SDS/PAGE in the six preformulation variants. Seroconversion was attained in all groups analysed. On the basis of these results, the most effective variant consisted of 20 microg/ml P64k plus 0.5 mg/ml aluminium hydroxide.

Adjuvants, Immunologic↗
PubMed1998

Characterization of human and rat brain myristoyl-CoA:protein N-myristoyltransferase: evidence for an alternative splice variant of the enzyme.

Using 5'-rapid amplification of cDNA ends, we have identified an extended 5'-end of mRNA coding for human myristoyl-CoA:protein N-myristoyltransferase (NMT). PCR using primers based on this new 5'-sequence and reverse primers within the currently accepted coding sequence of the enzyme resulted in the identification of a novel splice variant of NMT. In vitro translation of these cDNAs resulted in the production of proteins with apparent molecular masses of 63 kDa and 48 kDa. Immunoprecipitation of NMT from human cell lines and immunoblotting of a range of rat tissues has identified proteins with molecular masses corresponding to those derived from these cDNAs, and provided evidence that their relative abundance differs among tissues. Our results provide evidence that this enzyme exists in different forms resulting from alternative splicing of the mRNA.

Acyltransferases↗
PubMed2002

SAF-2, a splice variant of SAF-1, acts as a negative regulator of transcription.

Serum amyloid A-activating factor-1 (SAF-1), a Cys(2)His(2)-type zinc finger transcription factor, regulates inflammation-induced expression of serum amyloid A protein that is linked to the pathogenesis of reactive amyloidosis, rheumatoid arthritis, and atherosclerosis. Here we report the identification of a novel splice variant, SAF-2, of the SAF family bearing strong sequence similarity to SAF-1. The N-terminal 426 amino acids of both SAF-1 and SAF-2 are identical containing two polyalanine tracts, one proline-rich domain, and six zinc fingers. However, the C terminus of SAF-2 containing two additional zinc fingers is different from SAF-1, which indicates the capability of different biochemical function. We show that SAF-2 interacts more avidly with the SAF-binding element, but its transactivation potential is much lower than SAF-1. Furthermore, co-expression of SAF-2 markedly suppresses SAF-1-regulated promoter function. Finally, we show that the level of SAF-2 protein is reduced during many inflammatory conditions, whereas the SAF-1 protein level remains unchanged. Together, these data suggest that the relative abundance of SAF-2 plays a critical role in the fine tuned regulation of inflammation-responsive genes that are controlled by SAF-1.

Base Sequence↗
PubMed2004

Non-sequencing molecular approaches to identify preS2-defective hepatitis B virus variants proved to be associated with severe liver diseases.

BACKGROUND/AIMS: PreS2-defective hepatitis B virus (HBV) variants may emerge during chronic HBV infection. These variants carry mutation(s) at the ATG-start-codon and/or in-frame deletion into the preS2 genomic region and are commonly detected by sequencing analyses. We evaluated the prevalence of these variants in a large series of chronic HBV infected patients through non-sequencing molecular approaches. METHODS: We examined HBV isolates from 110 HBV carriers: 15 were inactive carriers (IC); 50 had chronic hepatitis (CH); 25 were cirrhotics; 19 had hepatocellular carcinoma (HCC). The entire preS2 genomic region was amplified by PCR technique. The amplicons were processed: (A) through electrophoresis on acrylamide gel to reveal deleted genomes; (B) through electrophoresis on agarose gel after digestion by NlaIII enzyme that cuts the wild ATG-start-codon but not the mutated one. RESULTS: We detected preS2 variants in 56/110 cases (51%). In particular, we found preS2-defective mutants in 2/15 IC, 25/50 CH, 13/26 cirrhotics, and 16/19 HCC. The presence of these variants was thus significantly associated with active infection and liver disease (P<0.002). Moreover, among cases with liver disease preS2-mutants were more prevalent in HCC patients (P<0.02). CONCLUSIONS: Our non-sequencing molecular methods are sensitive and specific, and simplify the identification of all preS2 HBV variant forms. Infection by these variants is significantly associated with active infection and HCC.

Carcinoma, Hepatocellular↗
PubMed1993

A novel silent variant at codon 711 and a variant at codon 708 of the APP sequence detected in Spanish Alzheimer and control cases.

Pathogenic mutations have been identified in exons 16 and 17 of the beta-amyloid precursor protein (APP) gene in some cases of early onset Alzheimer's disease. Screening of these exons in a number of familial and sporadic cases of Alzheimer's disease in Spain, resulted in the identification of a novel silent variant at codon 711 whose relevance to the AD pathogenesis remains unclear. The 708 variant was also detected in one of normal controls.

Alzheimer Disease↗
PubMed1996

A new method of STR interpretation using inferential logic--development of a criminal intelligence database.

A short tandem repeat (STR) system consisting of seven multiplexed loci has recently been introduced in the UK to support a National strategy to create large DNA databases for criminal intelligence purposes. The process uses automated sequencers, employing dye-labelled primers. Identification of tetrameric loci such as HUMTH01 are straightforward. Sizing windows are estimated by running a series of control allelic ladders on several gels and 'unknown' samples are designated if they fall within a defined window. However, utilisation of complex STRs (eg. D21S11) characteristically have common variants which differ by just 2 bp. In addition, rare alleles are encountered which may differ by just 1 bp from a common variant. To assist with the identification of alleles, we have introduced a series of allelic ladders, so that direct comparisons with 'unknown' samples can be made on the same gel. To designate an allele, it should be within 0.5 bp of an allelic ladder marker. Not all alleles (in particular rare alleles) can be included within an allelic ladder, however their expected positions can be easily calculated by reference to existing alleles in the ladder. Measurement of band shift is also a useful diagnostic tool. A series of guidelines are described to enable reliable allelic identification. These guidelines can be converted into computer programmes which form the basis of an expert system.

Alleles↗
PubMed1984

Molecular cloning and analysis of a new variant of human T-cell leukemia virus (HTLV-ib) from an African patient with adult T-cell leukemia-lymphoma.

We report the identification and characterization of a new variant of HTLV-I in an African patient with adult T-cell leukemia/lymphoma (ATL). Proviral sequences were detected by Southern blot analysis in three T-cell lines established from this patient's peripheral blood lymphocytes (PBL) and lymph-node cells. We molecularly cloned and analyzed proviruses from two of these cells lines, one established by direct culture of PBL and one established by co-cultivation of PBL with cord-blood T cells. These two HTLV clones contained full-length proviruses which were identical to each other in 44 out of 44 restriction enzyme sites. They were closely related to, but distinct from, the prototype HTLV-I, having divergence in their envelope and 5' pX regions and therefore represented a new variant of HTLV-I. We designated it as HTLV-Ib. Despite the genomic differences, however, HTLV-Ib retained its tropism for OKT4+ lymphocytes as well as its ability to initiate and maintain transformation of these cells. The finding of a variant of HTLV-I in this African ATL patient, along with the results of recent seroepidemiological studies, extends to the African continent the prevalence of HTLV-I associated malignancy previously identified in the Caribbean and Japan.

Adult↗
PubMed1994

Biochemical characterization of bovine MHC DQ allelic variants by one-dimensional isoelectric focusing.

Previous studies on expressed bovine MHC class II polymorphism using one-dimensional isoelectric focusing (1D-IEF) allowed the identification of at least 12 allelic variants of the DRB3 gene. So far, only limited data have been available on the expression of other class II genes. The present study involved biochemical analysis of bovine MHC class II molecules using a set of monoclonal antibodies presupposed to be bovine DR and DQ reactive. After essential modification of the standard electrophoresis conditions used for 1D-IEF typing of bovine DR products, biochemical polymorphism was observed for non-DR molecules, revealing polymorphic sets of basic and acidic focusing bands. Because of the extensive DNA polymorphism described for bovine DQA and DQB genes, and the apparent similarity with the focusing pattern of human DQ products, these molecules were considered to be the bovine DQ homologues. The definition of the DQ-associated banding patterns was made possible by using two half-sib sire families. Four different DQA-like patterns and nine DQB-like patterns were detected. Segregation of the DQ types was supported by serological class I and class II typing. These results show that it is now possible to discriminate between expressed bovine DR and DQ polymorphism.

Alleles↗
PubMed2026

Double heterozygous germline pathogenic variants in patients referred to a tertiary cancer genetics clinic in Singapore.

BACKGROUND: The increasing use of multigene panel testing has led to a rise in the identification of double heterozygous (DH) pathogenic variants in cancer patients, although their frequency and clinical relevance remain poorly characterised, particularly in Asian populations. METHODS: We conducted a retrospective review of 5,178 patients referred to a tertiary cancer genetics clinic in Singapore. DH pathogenic variants were defined as the presence of two or more distinct pathogenic or likely pathogenic germline variants identified by multigene panel testing. Clinical, pathological, and family history data were reviewed and analysed using appropriate non-parametric and exact statistical methods. RESULTS: Among 2,802 index patients with available genetic test results, 593 (21.2%) carried at least one pathogenic/likely pathogenic variant. DH pathogenic variants were identified in 21 individuals (0.75%), of which 9 were patients with breast cancer, 10 with non-breast malignancies, and 2 were cancer-free. The frequency of multiple primary cancers was 26.3% in DH variant carriers, 23.3% in single variant carriers, and 16.5% in those with no pathogenic variants. The median ages of first cancer diagnosis were 47, 44, and 48 years. Several DH combinations involved moderate- or low-penetrance genes, and some clinically unsuspected variants were detected only through broad multigene testing. CONCLUSION: DH pathogenic variants represent a rare subgroup that is increasingly detected through multigene panel testing. Their phenotypic expression is variable, and the influence of additional pathogenic variants remains uncertain. Our findings emphasise the need for tailored genetic evaluation and further research to guide evidence-based management for this complex patient population.

Asia↗
PubMed2005

Basal cell carcinoma: from host response and polymorphic variants to tumour suppressor genes.

The molecular factors and events that characterize susceptibility and outcome in cutaneous basal cell carcinoma (BCC) have been the focus of much research interest. As a result, we are beginning to understand the complex relationships between exposure to ultraviolet radiation (UVR), host response and the resulting damage to key genes that characterize these tumours. In this review, we will focus on genetic factors that influence susceptibility and outcome. While the search for susceptibility genes has generally resulted in the identification of low penetrance allelic variants, studies on modifier genes influencing outcome variables such as tumour number, age of onset and tumour subtype have identified factors with higher potential impact. Here we will briefly describe some recent work on the genetic basis of the immune response to UVR, the effect of UVR on the generation of reactive oxygen species and their detoxification, and the role of onco- and tumour suppressor genes. Areas for further research are highlighted, together with a consideration of possible applications in clinical practice.

Basal Cell Carcinoma↗
PubMed2002

Exip, a new alternative splicing variant of p38 alpha, can induce an earlier onset of apoptosis in HeLa cells.

One of the major families of the mitogen-activated kinases (MAPK), p38, has been shown to transduce extracellular stress stimuli into cellular responses. Among them, p38 alpha is the best characterized isoform and many biological phenomena, especially in the inflammatory responses, were attributed to the specific inhibitor-sensitive isoforms, namely p38 alpha and p38 beta. However, the roles played by each member are still unclear. Here, we report the identification of a new splice variant of p38 alpha, Exip (for exon skip), by RT-PCR using mRNA derived from a renal tumor cell line, OS-RC-2. Exip is predicted to encode a 307-amino-acid protein and the absence of exons 10, 11, and 11' results in the shift of the reading frame at the exon 9-12 junction to produce a unique 53-amino-acid C-terminus. The expression of mRNA was barely observed in cultured cells tested, but substantial amounts of mRNA were detected in mouse tissues. Unlike p38 alpha, Exip lost a common docking domain well conserved in major MAPK families for their specific interactions with upstream kinases or downstream substrates. Even though Exip is not phosphorylated at conserved TGY motif by p38-activating treatments, such as an osmotic shock or coexpression with a constitutive active form of MKK6 in HeLa cells, Exip can induce an earlier onset of apoptosis in HeLa cells. These results indicate that Exip has unique properties as a member of p38 alpha and may play role(s) in the signal transduction pathway(s) different from those of p38 alpha.

3T3 Cells↗
PubMed2004

Hemoglobin C and hemoglobin O-Arab variants can be diagnosed using the Bio-Rad Variant II high-performance liquid chromatography system without further confirmatory tests.

CONTEXT: Current standards for laboratory accreditation from the College of American Pathologists state that when high-performance liquid chromatography (HPLC) is used as a screening test, all non-A, non-S abnormal hemoglobin (Hb) variants must be confirmed by an alternative method, including alkaline and acid electrophoresis. OBJECTIVE: To determine whether confirmation of Hb C and Hb O-Arab variants by an alternative method is required when using the Bio-Rad Variant II HPLC system. DESIGN: We reviewed 48 478 consecutive hemoglobin identification test results performed on the Bio-Rad Variant II HPLC system during the period November 15, 2000 to January 15, 2003. SETTING: Special Hematology Laboratory, Department of Pathology, Bellevue Hospital Center, New York, NY. MAIN OUTCOME MEASURES: The chromatogram patterns and retention times (RTs) for specimens containing Hb C and Hb O-Arab were analyzed. We compared the results by the HPLC method with those by the confirmatory tests (alkaline and acid electrophoresis) for both variants. RESULTS: We identified 3668 cases of abnormal hemoglobin variants, including 660 cases of Hb C trait (17%), 5 cases of Hb O-Arab trait (0.1%), and 1 case of Hb SO-Arab (0.03%). A unique pattern of separation on the chromatogram for Hb O-Arab was revealed, presenting as 2 distinct peaks in 2 different manufacturer-defined RT windows, namely, D and C. The chromatogram for Hb C did not show the D window in any of the reviewed cases. The RT in the C window (C-RT) revealed a statistically significant difference for Hb C and Hb O-Arab (5.18 +/- 0.01 minutes and 4.91 +/- 0.01 minutes, respectively; P <.001). CONCLUSION: According to our review, the identification of Hb C and Hb O-Arab is accurate using HPLC methodology, as performed by the Bio-Rad Variant II HPLC system. This method can be both confirmatory and diagnostic at the same time.

Blood Protein Electrophoresis↗
PubMed2004

Identification of a 67-amino-acid region of the Plasmodium falciparum variant surface antigen that binds chondroitin sulphate A and elicits antibodies reactive with the surface of placental isolates.

The complications of malaria in pregnancy are caused by the massive sequestration of parasitized erythrocytes (PE) in the placenta. Placental isolates of Plasmodium falciparum are unusual in that they do not bind the primary microvasculature receptor CD36 but instead bind chondroitin sulphate A (CSA). Pregnant mothers develop antibodies that recognize placental variants worldwide, suggesting that a vaccine against malaria in pregnancy is possible. Some members of the Duffy binding-like gamma (DBL-gamma) domain of the large and diverse P. falciparum erythrocyte membrane protein-1 (PfEMP-1) family, when expressed on Chinese hamster ovary (CHO) cells, bind CSA. To characterize better the molecular requirements for DBL-gamma adhesion to CSA, we determined the binding of various DBL-gamma domains. Most DBL-gamma did not bind CSA, and no conserved region was identified that strictly differentiated binders from non-binders. Structure-function analysis of the FCR3-CSA DBL-gamma domain localized the minimal CSA binding region to a 67-residue fragment. This region was partially conserved among some binding sequences. Serum from a rabbit immunized with the minimal domain reacted with CSA-binding parasite lines, but not with non-CSA-adherent PE lines that adhered to CD36 and other receptors. The identification of a minimal binding region from a highly variable cytoadherent family may have application for a vaccine against malaria in pregnancy.

Amino Acid Sequence↗
PubMed2002

Hypermodification and immune escape of an internally deleted middle-envelope (M) protein of frequent and predominant hepatitis B virus variants.

Naturally occurring deletions within the human hepatitis B virus (HBV) preS2 region have frequently been identified in patients with hepatocellular carcinoma (HCC), while chronic carriers without cirrhosis and HCC contain no detectable preS2 deletion variants. We have characterized two different preS2 internal deletion variants from two patients. In addition to several weak phenotypes, our study revealed three unexpected strong phenotypes: (1) a paradoxical "hypermodification" phenomenon was observed with significantly increased size heterogeneity and molecular weights of the secreted middle (M) envelope proteins containing a preS2 internal deletion. This phenomenon was observed in transient transfection with a human hepatoma Huh7 cell line as well as in stable transfection with a rodent hepatoma cell line 7777. (2) A significantly increased intracellular accumulation of all three envelope proteins (large, middle, and small) was detected by both Western blot analysis and immunofluorescence microscopy. (3) The middle envelope proteins with a preS2 internal deletion were not recognized in vitro by a putative neutralizing antiserum, suggesting that these variants can evade immune recognition in vivo. To our knowledge, this is the first identification and characterization of the M deletion variant protein in HBV natural infection.

Animals↗
PubMed2005

SMYD3-NY, a novel SMYD3 mRNA transcript variant, may have a role in human spermatogenesis.

Identification of genes specifically expressed in adult and fetal testis is important to further our understanding of testis development and function. In this study, a novel SMYD3 transcript variant, termed SMYD3-NY (GenBank Accession No. AY186742), was identified by hybridization of adult and fetal human testis cDNA probes with a human cDNA microarray. SMYD3-NY transcript was expressed at 2.3-fold higher levels in adult human testis than in fetal testis, with a low expression level in human spermatozoa. Bioinformatical analysis showed that SMYD3-NY protein has the SET domain that is involved in histone methyltransferase (HTMase) activity. Southern blotting showed that SMYD3-NY is distributed in several tissues, including testis. In summary, SMYD3-NY is a novel transcript variant of the SMYD3 gene, and SMYD3-NY protein may influence transcriptional regulation during spermatogenesis via HTMase activity.

Adult↗
PubMed1999

Two buffer PAGE system-based SSCP/HD analysis: a general protocol for rapid and sensitive mutation screening in cystic fibrosis and any other human genetic disease.

The large size of many disease genes and the multiplicity of mutations complicate the design of an adequate assay for the identification of disease-causing variants. One of the most successful methods for mutation detection is the single strand conformation polymorphism (SSCP) technique. By varying temperature, gel composition, ionic strength and additives, we optimised the sensitivity of SSCP for all 27 exons of the CFTR gene. Using simultaneously SSCP and heteroduplex (HD) analysis, a total of 80 known CF mutations (28 missense, 22 frameshift, 17 nonsense, 13 splicesite) and 20 polymorphisms was analysed resulting in a detection rate of 97.5% including the 24 most common mutations worldwide. The ability of this technique to detect mutations independent of their nature, frequency, and population specificity was confirmed by the identification of five novel mutations (420del9, 1199delG, R560S, A613T, T1299I) in Swiss CF patients, as well as by the detection of 41 different mutations in 198 patients experimentally analysed. We present a three-stage screening strategy allowing analysis of seven exons within 5 hours and analysis of the entire coding region within 1 week, including sequence analysis of the variants. Additionally, our protocol represents a general model for point mutation analysis in other genetic disorders and has already been successfully established for OTC deficiency, collagene deficiency, X-linked myotubular myopathy (XLMTM), Duchenne and Becker muscular dystrophy (DMD, BMD), Wilson disease (WD), Neurofibromatosis I and II, Charcot-Marie-Tooth disease, hereditary neuropathy with liability to pressure palsies, and defects in mitochondrial DNA. No other protocol published so far presents standard SSCP/HD conditions for mutation screening in different disease genes.

Base Sequence↗
PubMedDate not supplied

Identification and molecular characterization of five novel kallikrein gene 13 (KLK13; KLK-L4) splice variants: differential expression in the human testis and testicular cancer.

The kallikrein gene family is comprised of genes that have either established or potential applications in prostate and breast cancer diagnostics. New members of the human kallikrein gene family have been recently identified. By using the positional candidate gene approach, we were able to clone a novel human serine protease gene that maps to chromosome 19q13.3-q13.4, the location of the kallikrein gene family. We named this gene KLK-L4 (now also known as KLK13). Here, we describe the identification of five new KLK-L4 splice variants which are not expressed in any other tissue except the human testis. We have further established that these splice variants can be detected in normal testis but not in the adjacent matched testicular tumors. In addition, differential expression of the KLK-L4 gene was found in various histological types of testicular cancer. Our results suggest that the KLK-L4 gene is expressed in normal and cancerous testicular tissue; however, its five variants are all expressed in normal tissue but not in testicular tumors. The physiological relevance of these variants and the implications of their differential expression between cancerous and normal tissues are currently unknown.

Alternative Splicing↗
PubMed2000

DNA technology for the detection of common genetic variants that predispose to thrombophilia.

With the identification of common single locus point mutations as risk factors for thrombophilia, many DNA testing methodologies have been described for detecting these variations. Traditionally, functional or immunological testing methods have been used to investigate quantitative anticoagulant deficiencies. However, with the emergence of the genetic variations, factor V Leiden, prothrombin 20210 and, to a lesser extent, the methylene tetrahydrofolate reductase (MTHFR677) and factor V HR2 haplotype, traditional testing methodologies have proved to be less useful and instead DNA technology is more commonly employed in diagnostics. This review considers many of the DNA techniques that have proved to be useful in the detection of common genetic variants that predispose to thrombophilia. Techniques involving gel analysis are used to detect the presence or absence of restriction sites, electrophoretic mobility shifts, as in single strand conformation polymorphism or denaturing gradient gel electrophoresis, and product formation in allele-specific amplification. Such techniques may be sensitive, but are unwielding and often need to be validated objectively. In order to overcome some of the limitations of gel analysis, especially when dealing with larger sample numbers, many alternative detection formats, such as closed tube systems, microplates and microarrays (minisequencing, real-time polymerase chain reaction, and oligonucleotide ligation assays) have been developed. In addition, many of the emerging technologies take advantage of colourimetric or fluorescence detection (including energy transfer) that allows qualitative and quantitative interpretation of results. With the large variety of DNA technologies available, the choice of methodology will depend on several factors including cost and the need for speed, simplicity and robustness.

DNA↗