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At least 217 records · Page 12Linked to original sources
PubMed1997

Sequence analysis of the Epstein-Barr virus (EBV) latent membrane protein-1 gene and promoter region: identification of four variants among wild-type EBV isolates.

Sequence variations in the Epstein-Barr virus (EBV) encoded latent membrane protein-1 (LMP-1) gene have been described in a Chinese nasopharyngeal carcinoma-derived isolate (CAO), and in viral isolates from various EBV-associated tumors. It has been suggested that these genetic changes, which include loss of a Xho I restriction site (position 169425) and a C-terminal 30-base pair (bp) deletion (position 168287-168256), define EBV genotypes associated with increased tumorigenicity or with disease among particular geographic populations. To determine the frequency of LMP-1 variations in European wild-type virus isolates, we sequenced the LMP-1 promoter and gene in EBV from lymphoblastoid cell lines from healthy carriers and patients without EBV-associated disease. Sequence changes were often present, and defined at least four main groups of viral isolates, which we designate Groups A through D. The widespread prevalence of LMP-1 sequence variations, particularly the Xho I polymorphism and the 30-bp deletion, indicate that they cannot be used as simple markers for oncogenic viruses related to particular forms of EBV-associated tumor. Several of the structural changes detected occur, however, at sites where they may affect transcription, translation, or function of LMP-1. Future in vitro studies should aim to establish the functional importance of variations at these sites.

Amino Acid Sequence↗
PubMed1997

[Identification of 5 variants of the hemoglobin beta chain using DNA analysis (amplification, sequencing and restriction enzymes)].

PURPOSE: To confirm the conventional techniques for studying structural haemoglobinopathies and to show off the simplicity and efficacy of new methods based on the study of DNA. PATIENTS AND METHODS: Peripheral blood samples of 17 patients with 5 haemoglobin variants detected by conventional and shown off by means of sequencing according to Sanger's method, plus PCR-RFLP, were studied. RESULTS: Five structural haemoglobin variants were found, which distributed as follows: 7 cases of Hb Complutense (beta 127 Gln-->Glu), 1 Hb D-Punjab (beta 121 Glu-->Gln), 3 Hb Hofu (beta 126 Val-->Glu), 3 Hb J-Baltimore (beta 16 Gly-->Asp) and 3 Hb San Diego (beta 109 Val-->Met). CONCLUSIONS: These results allow us to stress the simplicity and usefulness of DNA analysis (sequencing , amplification and enzymatic digestion) to identify haemoglobin variants as opposed to laborious analysis of the primary structure by means of HPLC peptide separation.

DNA Mutational Analysis↗
PubMed1996

Identification of a variant estrogen receptor lacking exon 4 and its coexpression with wild-type estrogen receptor in ovarian carcinomas.

By means of reverse transcription-PCR we have identified an alternatively spliced mRNA coding for a variant estrogen receptor (ER) that lacks exon 4 (ERDelta4) and is coexpressed with the wild-type ER mRNA in ovarian carcinomas. Furthermore, Western blot analysis revealed the expression of the ERDelta4 protein in normal as well as neoplastic ovarian tissues along with the wild-type ER, although the relative amounts of the wild-type ER and ERDelta4 proteins varied. The trans-activational properties of this variant were studied in ER-negative COS1 cell lines by cotransfection of the ERDelta4 expression vector and a reporter gene containing the estrogen response element. The ERDelta4 protein was not able to activate transcription of a reporter gene. However, it inhibited estrogen-dependent transcriptional activation in a dominant negative fashion when it was cotransfected with the wild-type ER and reporter plasmid. Because it has been shown that ERDelta4 is not able to bind to its response element, the observed inhibitory effect probably occurs through protein-protein interactions. Although several variants of the ER have been described from cancerous cells, none has been identified in ovarian tissues, and ERDelta4 is the only isoform detected in normal tissues. These results may have implications for understanding the physiological role of ERDelta4 in normal cells, because it may affect the function of the wild-type ER, depending on the level of the variant ER protein relative to that of the wild-type ER.

Alternative Splicing↗
PubMed2003

Identification of alternate splice variants for murine SmX5.

We report here the identification of three additional murine SmX5 alternative transcripts, designated SmX5a, b and c. The cDNA of SmX5a possesses intron 1 in its entirety, and SmX5b harbors a part of intron 2. The retained fragment of SmX5c starts at the same position as SmX5b, but the retention extends through the remaining portion of intron 2. The sequence of retained DNA at all the intron-exon boundaries conforms to the "GT-AG" rule. RT-PCR analysis indicates that the three isoforms are stable transcripts, all at low frequency when compared with SmX5. Expression analysis by RT-PCR indicates that SmX5 and its isoforms exist in murine brain, kidney, testis, thymus, liver, spleen and heart. The SmX5a isoform is predominantly expressed in the thymus, while SmX5b and c are expressed in all tissues examined. The presence of alternatively spliced SmX5 isoforms and tissue-specific variation in splicing patterns suggests that SmX5 expression is complex and that the regulation of all four SmX5 mRNA levels may be critical in maintaining proper pre-mRNA splicing machinery in different cell types.

Alternative Splicing↗
PubMedDate not supplied

Molecular and phylogenetic characterization of HIV variants in Italian primary HIV infections (PHI): identification of non-B subtype variants.

The distribution of Human Immunodeficiency Virus type 1 (HIV-1) clades is evaluated in primary HIV-1 infections (PHIs) occurring through sexual transmission in Lombardia, the Italian region with the highest prevalence/incidence of HIV-1 infections. The two primary inclusion parameters for enrollment were sexual transmission and < 1 year seroconversion. Thirty-four enrolled patients have been analysed so far at the molecular level, to characterize their infecting HIV-1 population. Two HIV-1 genomic regions with different rates of genetic variability, the hypervariable C2-V3 fragment of the env gene and the conserved 5' end of the gag p17, were amplified by Polymerase Chain Reaction (PCR) in peripheral blood mononuclear cells (PBMCs) and characterized by direct DNA sequence analysis. Pairwise nucleotide alignment and phylogenetic analyses show that, although with a high range of nucleotide variability, 32 out of the 34 HIV-1 isolates identified in this PHI cohort fall under the clade B genotype. The two remaining isolates, detected in a couple formed by a Nigerian woman and her Italian partner, consistently cluster with clade G standards in both sub-genomic regions. The amino acid sequences confirm this classification, showing clade-specific residues both in the V3 and p17 regions. These data suggest that the B clade is still prevalently associated with acute primary HIV-1 infections occurring in Italy through sexual transmission. However, the significant intra-clade variability and the identification of non-B clades strongly indicate the relevance of continuous molecular monitoring of the HIV-1 isolates circulating in Italy, for prognostic evaluations as well as preventive and therapeutic strategies.

Acute Disease↗
PubMed1984

Immunochemical identification of normal and variant hemoglobins after electrophoretic separation and transfer to nitrocellulose membranes.

A highly specific method for the conclusive identification of normal and variant human hemoglobins is described in this communication. The method employs the standard electrophoretic technique in combination with the immunoblot technique using monospecific antisera raised in rabbits. Various hemoglobins such as Hb, S, C, A2, and F were separated on an alkaline polyacrylamide gel or a cellulose-acetate membrane, and transferred to a nitrocellulose membrane and the individual hemoglobins were identified by the immunoblot procedure. With this method hemoglobins with similar or identical electrophoretic mobilities can be definitively identified with the use of monospecific antisera.

Electrophoresis, Cellulose Acetate↗
PubMed2006

[Identification strategy of RHCE gene variants at the National Blood Group Reference Laboratory: impact on transfusion safety].

The RH blood group is the most polymorphic and immunogenic blood group system. The RH locus is composed of 2 highly homologous genes: the RHD gene encoding the D polypeptide; and the RHCE gene, encoding C or c together with either E or e polypeptides. Numerous variants exist for both RHD and RHCE genes. Among them we were interested in the serological and molecular definition of numerous pre-published RHCE variants encountered in different populations. The identification of these variants is crucial to ensure the transfusion safety for patients expressing these variants. Here we propose a procedure to identify some of them and discuss the adequate transfusion strategy. This procedure has enable us to identify new variants to be presented at the symposium.

Blood Group Incompatibility↗
PubMed2000

Separation of hemoglobin variants with similar charge by capillary isoelectric focusing: value of isoelectric point for identification of common and uncommon hemoglobin variants.

Clinical assays for the primary evaluation of congenital hemoglobin (Hb) disorders must detect and identify a variety of Hb variants. We analyzed hemolysates containing Hb variants with similar charge to evaluate the diagnostic sensitivity and specificity of automated capillary isoelectric focusing (CIEF). Peak separation was observed for each variant in samples containing Hb S, D, and G. The calculated isoelectric points (pI) of these variants were significantly different such that each could be identified in a single run with pI as the sole criterion of identification. The pI of Hb C was significantly different from that of Hb E, C-Harlem, and O-Arab. Hb E, C-Harlem, and O-Arab had similar pI and were not readily differentiated. Hb Koln, M-Saskatoon, Aida, and S/Aida hybrid were readily separated from common Hb variants and detected by CIEF. We conclude that CIEF exhibits both diagnostic sensitivity and specificity, and that pI is an objective and specific criterion of Hb variant identification.

Child↗
PubMed1991

[Diagnosis of familial amyloid polyneuropathy--gene analysis with primer-directed enzymatic amplification of DNA, isolation of plasma variant prealbumin and immunohistochemical identification of tissue amyloid protein].

Type I familial amyloid polyneuropathy (FAP) is an autosomal dominant hereditary generalized amyloidosis characterized by polyneuropathy and autonomic nerve failure. The main component of the amyloid fibril protein in this disorder has been shown to be a variant prealbumin with a single substitution of a methionine residue for valine at position 30. In the present study we have investigated 19 patients with FAP aged 31 to 67 and an asymptomatic family member using gene analysis with primer-directed enzymatic amplification (PCR) of DNA, isolation of plasma variant prealbumin and immunohistochemical identification of tissue amyloid protein. All patients and a symptom-free boy in the affected family had the mutant prealbumin gene showing abnormal DNA fragments by treatment with restriction endonuclease Bal I, and plasma variant prealbumin was also detected in all of them by reverse-phase high performance liquid chromatography. Rectum biopsies obtained from 9 patients showed amyloid deposits which were specifically immunostained by anti-human prealbumin antiserum. However, an asymptomatic carrier at the age of 16 showed no rectal amyloid deposition. Recent studies of FAP have disclosed that the expression of type I FAP is closely associated with the gene mutation of prealbumin. Accordingly, a simple and rapid method to detect this gene abnormality using PCR technique is considered to be very useful for diagnosis of type I FAP and can also provide valuable information for the genetic counselling of the family members at risk.

Adolescent↗
PubMed2001

Identification of multiple gephyrin variants in different organs of the adult rat.

The neurotransmitter receptor anchoring protein gephyrin is encoded by a highly mosaic gene whose primary transcript is subject to extensive alternative splicing. Gephyrin mRNAs are widely expressed in various mammalian tissues, and gephyrin has been implicated in neuron-specific and general metabolic functions. Using a novel affinity isolation procedure, we report the identification of different gephyrin variants in various organs of the adult rat. In particular, polypeptides of 52, 56, 60, and 91 kDa were detected in addition to the previously characterized 93-kDa protein. Our results suggest tissue-specific functional differences between gephyrin variants.

Animals↗
PubMed1999

Characterization of transthyretin mutants from serum using immunoprecipitation, HPLC/electrospray ionization and matrix-assisted laser desorption/ionization mass spectrometry.

A mass spectrometry approach for the detection and identification of variants of the plasma protein transthyretin (TTR) is presented. The single amino acid substitutions found in TTR are closely associated with familial transthyretin amyloidosis (ATTR), a hereditary degenerative disease. A definitive diagnosis of ATTR relies on the detection and identification of TTR variants. The approach presented here is based on isolation of serum TTR using immunoprecipitation. The detection of the variant is achieved by mass measurement of the intact protein with electrospray ionization mass spectrometry (ESIMS). The liquid chromatography/ESIMS analysis of the tryptic digest of the protein followed by subsequent matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry and MALDI postsource decay of the relevant recovered chromatographic fraction containing the variant peptide allows the identification of unknown variants. The method was successfully tested using serum from ATTR patients with known variants (Val30-->Met and Val122-->Ile). A new TTR variant, Ser23-->Asn, was detected and identified using the above method where isoelectric focusing and restriction enzyme analysis failed to identify the nature of the variant.

Amino Acid Sequence↗
PubMed1996

Identification of a novel variant hepatocyte growth factor secreted by spleen-derived stromal cells.

Stromal cells can interact with parenchymal cells by secreting various cytokines to affect the growth, differentiation or movement of the latter. Here we report the identification and characterization of a novel variant hepatocyte growth factor (HGF) from the conditioned medium of stromal cells derived from mouse spleen. Compared to human HGF, it has much lower heparin-binding activity and lacks the beta-chain. Its molecular weight, 70 kDa, is very close to that of the alpha-chain of HGF. Human HGF homologue was not found in the conditioned medium. The conditioned medium of stromal cells, like recombinant HGF, could inhibit the growth of rat hepatoma cells. The inhibitory activity was presumably attributed to this novel HGF because the inhibitory activity, as the existence of this novel HGF, was confined to the identical fractions after heparin-column chromatography. Furthermore, this activity could be specifically abrogated by neutralizing anti-HGF antibodies.

Animals↗
PubMed1981

High performance liquid chromatography in the identification of human hemoglobin variants.

A description is given of the nature of column packings for reverse phase high performance liquid chromatography (HPLC) and of the equipment that is available for HPLC procedures. HPLC methods have been applied to the identification of abnormal hemoglobins. In most instances, the variant peptide(s) is easily detected on the chromatogram. The effect of differences in column packings and developer sequences is described; modifications in either provide many means for achieving desired separations. Thus, HPLC separates peptides well and is a rapid, sensitive method for studying hemoglobin. Although the emphasis has been on the examination of peptides of abnormal hemoglobins, the procedure can be extended to other aspects such as the separation of hemoglobin chains themselves.

Chromatography, High Pressure Liquid↗
PubMed1988

Variant translocations (9;11): identification of the critical genetic rearrangement.

The t(9;11)(p22;q23) is a recurring abnormality in acute nonlymphocytic leukemia. The analysis of complex 9;11 translocations will aid in the identification of the conserved chromosomal junction or the critical genetic alteration created by the rearrangement; however, variant translocations involving chromosomes #9 and #11 have not been reported. We have identified such variants in two patients who had acute myelomonocytic leukemia and acute monocytic leukemia, characterized by a t(9;11;18)(p22;q23;q12) and a t(9;11;13)(p22;q23;q34), respectively. The conserved junction resulting from these rearrangements is created by the translocation of chromosomal material from 9p to 11q.

Aged↗
PubMed2004

Comprehensive analysis of thiopurine S-methyltransferase phenotype-genotype correlation in a large population of German-Caucasians and identification of novel TPMT variants.

The thiopurine S-methyltransferase (TPMT) genetic polymorphism has a significant clinical impact on the toxicity of thiopurine drugs. It has been proposed that the identification of patients who are at high risk for developing toxicity on the basis of genotyping could be used to individualize drug treatment. In the present study, phenotype-genotype correlation of 1214 healthy blood donors was investigated to determine the accuracy of genotyping for correct prediction of different TPMT phenotypes. In addition, the influence of gender, age, nicotine and caffeine intake was examined. TPMT red blood cell activity was measured in all samples and genotype was determined for the TPMT alleles *2 and *3. Discordant cases between phenotype and genotype were systematically sequenced. A clearly defined trimodal frequency distribution of TPMT activity was found with 0.6% deficient, 9.9% intermediate and 89.5% normal to high methylators. The frequencies of the mutant alleles were 4.4% (*3A), 0.4% (*3C) and 0.2% (*2). All seven TPMT deficient subjects were homozygous or compound heterozygous carriers for these alleles. In 17 individuals with intermediate TPMT activity discordant to TPMT genotype, four novel variants were identified leading to amino acid changes (K119T, Q42E, R163H, G71R). Taking these new variants into consideration, the overall concordance rate between TPMT genetics and phenotypes was 98.4%. Specificity, sensitivity and the positive and negative predictive power of the genotyping test were estimated to be higher than 90%. Thus, the results of this study provide a solid basis to predict TPMT phenotype in a Northern European Caucasian population by molecular diagnostics.

Adolescent↗
PubMed1999

Identification of a novel variant of hepatitis E virus in Italy.

Hepatitis E infection is typically associated with areas in which hepatitis E virus (HEV) is endemic. Except for a few cases in Europe and in the United States, acute hepatitis E is usually associated with travel to endemic areas. We set out to determine the etiologic role of HEV in acute non-A-C hepatitis in Italy. The presence of HEV-RNA and antibody was determined in 218 patients diagnosed with acute viral non-A-C hepatitis. Acute hepatitis E infection was defined by the presence of HEV-RNA in sera and positivity for IgM anti-HEV and seroconversion to IgG anti-HEV. Acute hepatitis E was found in 10.1% of the patients with acute non-A-C, with 95.5% exhibiting a benign course. A more severe course was observed in a patient co-infected with HAV and HEV. Most cases were travelers to endemic areas, although 18.2% reported no travel. One patient was from a household with an infected patient. Sequence analyses of the polymerase chain reaction (PCR) product derived from a patient who never visited endemic areas, identified an isolate that is divergent significantly from all reported isolates of HEV (79.5-85.8% nucleotide identity). Evidence from this study suggests that HEV accounts for approximately 10% of acute non-A-C viral hepatitis in Italy, diagnosed generally in travelers returning from endemic areas. However, the identification of a new HEV variant in an individual who never indicated travel or contact with individuals associated with endemic areas, suggests that this virus may be native to Italy.

Adolescent↗
PubMed2004

Identification of a functional variant of estrogen receptor beta in an African population.

In this study, we identified five novel polymorphisms in the estrogen receptor beta (ERbeta) gene in an African population. Interestingly, two of these variants are expected to change the amino acid sequence of the ERbeta protein. These changes correspond to an isoleucine to valine substitution at amino acid position 3 (I3V) and a valine to glycine substitution at position 320 (V320G), respectively. The functional consequences of these amino acid substitutions were determined in different in vitro assays. The I3V mutation displayed no differences with regard to transcriptional activity in a reporter assay, as compared with the wild-type receptor. The V320G mutation, however, showed significantly decreased maximal transcriptional activity in a reporter assay, although its binding affinity for 17beta-estradiol was not affected. A pull-down assay indicated that the interaction of full-length TIF2 with hERbetaV320G was weaker than with hERbetawt. Moreover, surface plasmon resonance analysis revealed reduced interaction of the V320G ERbeta variant with the NR box I and II modules of TIF2. To our knowledge, this represents the first identification of a functional polymorphism in the ERbeta gene. This novel polymorphism provides a tool for human genetic studies of diseases in the African population.

Amino Acid Sequence↗
PubMed1992

Thyroxine-binding globulin variant (TBG-Kumamoto): identification of a point mutation and genotype analysis of its family.

Thyroxine-binding globulin (TBG) is the major thyroid hormone transport protein. Several inherited TBG variants resulting in partial or complete TBG deficiencies have been shown to be caused by either one or two nucleotide substitutions, or one nucleotide deletion in the coding regions of the TBG gene. In this report, a Japanese female patient (proband) with hyperthyroid state, whose lower TBG levels did not return to normal under the euthyroid state after treatment was examined. Genomic DNA samples from the proband with thyroxine-binding globulin deficiency (termed TBG-Kumamoto) and her family were subjected to the polymerase chain reaction, and the generated DNA fragments were sequenced. A single nucleotide substitution in the codon for the amino acid 363 of native TBG molecule (CCT to CTT) was found, resulting in the replacement of proline by leucine. It was revealed that the proband was a heterozygote and her father was a hemizygote. The mutation was confirmed by the allele-specific amplification of genomic DNAs from the proband and her father using oligonucleotide primers of normal or mutant residues at the 3' position in the polymerase chain reaction. These results indicate that the abnormality of TBG-Kumamoto is the consequence of this mutation. Genetically, this point mutation observed in TBG-Kumamoto might be classified as a new type of TBG deficiency.

Adult↗