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PubMed2003

B19 virus genome diversity: epidemiological and clinical correlations.

Genetic analysis of parvovirus B19 has been carried out mainly to establish a framework to track molecular epidemiology of the virus and to correlate sequence variability with different pathological and clinical manifestations of the virus. A good amount of information regarding B19 virus sequence variability is available, and presently there are about 400 sequence records deposited in the nucleotide database of NCBI. A few are almost complete genomic sequences, and these allow the construction of a global alignment framework. Many others are partial genomic sequences, limited to selected regions, and these allow comparison of a higher number of isolates from well-defined epidemiological settings and/or pathological conditions. Most studies showed that the genetic variability of B19 virus is low, that molecular epidemiology is possible only on a limited geographical and temporal setting, and that no clear correlations are present between genome sequence and distinctive pathological and clinical manifestations. More recently, several viral isolates have been identified that show remarkable sequence diversity with respect to reference sequences. The identification of variant isolates added to the knowledge of genetic diversity in this virus group and allowed the identification of three divergent genetic clusters, about 10% divergent from each other and still quite distinct from other parvoviruses, that can be thought of as different genotypes within the human erythrovirus group and that show clearly resolved phylogenetic relationship. These variant isolates pose interesting questions regarding the real extent of genetic variability in the human erythroviruses, the relevance of these viruses in terms of epidemiology and their possible implication in the pathogenesis of erythrovirus-related diseases.

Genes, Viral↗
PubMed2026

Mitochondrial DNA diversity in Ecuadorian populations: Recurrence of variant 16136 within haplogroup B2.

The identification of lineage-defining variants, frequently found in the coding region of mitochondrial DNA (mtDNA), is essential for refining haplogroup classification. Most mtDNA studies in South American populations have focused on the control region (CR), which has provided important insights into population structure and maternal lineage origins, although information needed for more robust phylogenetic resolution has been neglected. This study investigates the maternal genetic structure of Ecuadorian populations by combining CR and whole mitogenome analyses. Sequences from the mtDNA CR were obtained from 461 individuals (253 Mestizos and 208 Native Americans), while complete mitogenomes were sequenced for 127 individuals to improve phylogenetic resolution by identifying lineage-defining variants present in coding region. Most mtDNA haplogroups in the two population groups analyzed were of Native American origin (A2, B2, B4, C1, D1, D4), with significant differences in the distribution of specific lineages between them. Among Mestizos, African haplogroups (all within the L branches) and Eurasian haplogroups (H, K, R, U) were detected at low frequencies, whereas no African lineages were observed among Native Americans. The results obtained highlighted a heterogeneity within Ecuadorian populations that must be considered when developing mtDNA haplotype databases for forensic purposes. Whole mitogenome sequences enabled the identification of variants that refined haplogroup classifications, provided a more accurate reconstruction of the maternal genetic diversity, and improve the discrimination between Native American and Asian maternal lineages within haplogroup B4b.

Humans↗
PubMed2004

Cloning and identification of two novel splice variants of human PD-L2.

PD-L2, a newly identified member of B7 family, plays a role in down-regulating T cell responses. The common PD-L2 mRNA (type I) is the splicing product containing all 6 exons. We report here the identification of two human PD-L2 splice variants in activated leukocytes. One splice variant (type II) is generated through splicing out exon 3 encoding Ig constant-like domain; it retains all other regions without a frame shift. The other variant (type III) is created by splicing out exon 3 to an alternative acceptor site 5 bp downstream of the canonical acceptor site, leading to a frame shift. Consequently, the translated protein should be a soluble form. Furthermore, type I isoform is expressed on the plasma surface whereas type II isoform showed a pattern of intracellular membrane distribution in the transiently transfected K562 cells. In addition, the expression patterns of PD-L2 splice variants are variable in different individuals and distinct cellular status. These results suggest that PD-L2 expression may be controlled by posttranscriptional regulation through alternative splicing, and modulation of PD-L2 isoform expression may influence the outcome of immune response.

Alternative Splicing↗
PubMed1991

A study of schizophrenic outpatients with the Defense Mechanism Test.

Intrapsychic defensive strategies are allegedly expressed on the Defense Mechanism Test through the presentation, at increasing tachistoscopic exposure times, of a picture in which a central figure is threatened by a peripheral person. Several types of perceptual alterations of the stimulus configuration are coded as defenses in the subjective reports. A number of test variables have already been shown to differentiate significantly between nonclinical controls and nonpsychotic psychiatric patients. In the present study, after a review of the pertinent percept-genetic literature, nonpsychotic patients (n = 57) were compared with a group of schizophrenic outpatients in the active phase (n = 21). As predicted, significantly more schizophrenics than controls were coded for projection and regression and for certain variants of these signs. Four subcodings of repression, three of isolation, the sign of reaction formation, and several variants of identification differentiated in the same direction. Reports in which the central figure was too old and changed from a correct to an incorrect sex attribution were highly characteristic of the schizophrenic sample. No defensive variable was significantly linked with nonpsychotic pathology. Two variants, one of regression and one of identification, allowed a correct diagnostic placement for all schizophrenics and for 82.4% of the nonpsychotic patients. The findings, besides being a further clinical validation of the Defense Mechanism Test, provide a preliminary distinction between high- and low-level defenses. It is suggested that they are congruent with an hierarchical model which implies inclusive nonreflexive relationships between classes of mental disorders.

Adult↗
PubMed2004

Identification of two novel sequence variants affecting thiopurine methyltransferase enzyme activity.

The polymorphic enzyme thiopurine methyltransferase (TPMT) is involved in the methylation of thiopurines. On comparing the phenotype with the genotype in Swedish patients with inflammatory bowel disease and healthy individuals, we found two discordant cases with low TPMT enzyme activity (0.3 and 0.4 U/ml packed red blood cells (pRBC). Genotyping by pyrosequencing revealed that they carried the nucleotide substitutions 460G>A and 719A>G, giving two possible genotypes (TPMT*1/*3A or TPMT*3B/*3C). DNA sequencing of exon III to X was performed in the patients and their parents. We identified an A>G transition in the start codon (exon III, 1A>G, Met>Val, TPMT*14) in one of the patients and her father (6.3 U/ml pRBC). The mother in this family carried the 460G>A and 719A>G nucleotide substitutions (TPMT*1/*3A; 5.0 U/ml pRBC). In the second family, sequencing revealed a G>A transition in the acceptor splice site in intron VII/exon VIII (IVS7 -1G>A, TPMT*15) in the patient and his mother (6.9 U/ml pRBC). His father was genotyped as TPMT*1/*3A (6.0 U/ml pRBC). Hence, we report the identification of two novel sequence variants, present in highly conserved nucleotide positions of the human TPMT gene, resulting in a loss of enzyme activity.

Base Sequence↗
PubMed1992

The dermal-epidermal junction of human skin contains a novel laminin variant.

We report the identification of a novel laminin variant that appears to be unique to a subset of epithelial basement membranes. The variant contains two chains electrophoretically and immunologically identical to the B1 and B2 chains. Epitopes contained in the laminin A chain are absent from the molecule, and a 190-kD chain substitutes for the A chain. V8 protease analysis and Western blotting studies indicate that the variant 190-kD chain shows structural and immunological similarity to the 200-kD chain of kalinin. Rotary shadowing analysis indicates that the 190-kD chain contributes a large globular structure to the variant long arm, but lacks the short arm contributed to laminin by the A chain. The variant is produced by cultured skin explants, human keratinocytes and a squamous cell carcinoma line, and is present in human amniotic fluid. Polyclonal antibodies raised to kalinin, a recently characterized novel component of anchoring filaments, and mAb BM165 which recognizes a subunit of kalinin (Rousselle et al., 1991) cross react with the variant under nonreducing conditions. Immunohistological surveys of human tissues using the crossreacting antikalinin antiserum indicate that the distribution of this laminin variant is at least restricted to anchoring filament containing basement membranes. We propose the name K-laminin for this variant.

Basement Membrane↗
PubMed2005

Rapid identification of the coxsackievirus A24 variant by molecular serotyping in an outbreak of acute hemorrhagic conjunctivitis.

We evaluated the clinical applicability of a molecular serotyping method for determination of the cause of epidemic acute hemorrhagic conjunctivitis. Seventy conjunctival swab specimens from individuals involved in a nationwide acute hemorrhagic conjunctivitis outbreak were tested. Viral culture and a molecular biology-based assay were compared by directly using clinical specimens. On the one hand, virus culture was done to isolate the enteroviruses, and serotyping was done by a coxsackievirus A24 variant-specific PCR. On the other hand, the original clinical specimens were directly screened for enterovirus by reverse transcription (RT)-PCR with panenterovirus-specific primers. Enterovirus screening-positive specimens were subjected to RT-PCR for detection of the VP1 region of enterovirus, and the amplicons were sequenced. Molecular serotyping was done by calculating the pairwise identity scores for the sequences with the maximum identities to the sequences of known prototype enteroviruses. Thirty-two specimens (45.7%) were culture positive, whereas 37 specimens (52.8%) were screening PCR positive (P < 0.001). The VP1 regions were amplified from 21 of the 37 specimens (56.8%), and the products amplified from 9 specimens were appropriately sequenced. These nine sequences were homologous with the sequence of the coxsackievirus A24 variant. Molecular serotyping by direct use of clinical specimens without cell culture could be applied for the rapid identification of the causative agent of epidemic acute hemorrhagic conjunctivitis.

Base Sequence↗
PubMed1988

[Genetic variants of human albumin: structural characterization of allotypes used as references for electrophoretic classification].

Eight different types of genetic variants of albumin are observed in the French population. The analysis of electrophoretic patterns of sera containing these variants, performed a three different pHs (8.6, 5.0 and 6.9) after addition of a reference protein (transferrin), allows the identification each variant by a quantitative estimation of its relative mobilities. The accuracy and reproducibility of the technique make it a useful reference method, commonly employed for studying European variants. The samples used as references for five genetic variant types, proalbumins Christchurch and Lille, albumins Vanves, B and Reading, were subjected to sequence analysis to determine the nature and localization of their structural change. Together with the mutations of albumins Gent and Roma previously described, the data presented here make available seven reference specimens for which the structural changes are characterized out of the eight variants known to exist in France.

Amino Acid Sequence↗
PubMed2004

Identification of novel alternative splice variants of human constitutive androstane receptor and characterization of their expression in the liver.

Human constitutive androstane (or active) receptor (hCAR), a member of the nuclear receptor superfamily NR1I3, regulates the expression of several genes that are mainly involved in the metabolism of endogenous and xenobiotic compounds (e.g., CYP2B6, CYP3A4, and UGT1A1). We found four novel splice variants in the ligand-binding domain (LBD) of hCAR (NCBI reference sequence, NM_005122; designated SV0 herein). The variants designated SV1 and SV2 contained in-frame 12- and 15-base pair (bp) insertions, respectively. SV3 carried both of the insertions, and SV4 contained an in-frame 117-bp deletion. The insertion site of SV1 is located in the alpha6 helix of hCAR LBD, which makes up the ligand-binding cavity, and that of SV2 is located in the highly conserved loop between helices alpha8 and alpha9. SYBR Green real-time reverse transcription-polymerase chain reaction analysis of each splice variant revealed that the hepatic expression of SV2 was almost comparable with that of SV0 (approximately 40%), whereas other variants accounted for 6 to 10% of the total hCAR transcripts. In the reporter gene assays employing the phenobarbital-responsible enhancer module (PBREM) from CYP2B6 and UGT1A1 genes, the splice variants, except for SV1, were inactive, whereas SV1 transactivated the CYP2B6 PBREM but not the UGT1A1 PBREM reporter. A nuclear translocation assay in rat hepatocytes revealed that all the splice variants lack the responsiveness toward phenobarbital and 6-(4-chloropheny-l)imidazo[2,1-b][1,3]thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime (CITCO) in terms of the ligand-dependent nuclear translocation. Further characterization, such as the identification of specific ligands, will help elucidate physiological implication of these hCAR splice variants.

Alternative Splicing↗
PubMed1998

Qualitative and quantitative analysis of hemoglobin variants by capillary isoelectric focusing.

We developed two capillary isoelectric focusing (CIEF) assays, in narrow pH gradients, with the aim of routinely separating and quantitating normal and abnormal hemoglobins (Hbs): a one-step CIEF assay where residual electroosmotic flow mobilizes the proteins during focalization, and a two-step CIEF assay where focused Hbs are mobilized by low pressure by maintaining high-voltage. The resolution of 0.10 pH unit obtained with the one-step assay allowed the separation of the Hbs A, F, S and C; but Hb A2, which represents about 2-3% of whole Hb, could not be quantitated. The better resolution of 0.02 pH unit obtained with the two-step assay allowed the separation of some Hb variants of very close isoelectric points. The reproducibility of retention times was satisfactory (C.V.<5%). Moreover, in this configuration quantitation of Hb A2, Hb F and Hb S led to a standard deviation of less than 5%, allowing the diagnosis of thalassemias. The one-step assay could be useful only for the detection of abnormal variants, while the two-step assay could be applied to the routine analysis of Hbs, with quantitation of minor fractions and presumptive identification of variants.

Electrochemistry↗
PubMed1991

Infectious crystalline keratopathy. Role of nutritionally variant streptococci and other bacterial factors.

Infectious crystalline keratopathy (ICK) is a chronic corneal infection characterized by interlamellar plaques of gram-positive coccal bacteria in the absence of inflammatory cells. It generally occurs within a corneal graft. Viridans streptococci are usually isolated, but the clinical response to antibiotics is poor and disparate with the in vitro antimicrobial sensitivities. These features suggest the possibility of unusual bacterial factors in pathogenesis. Four cases caused by nutritionally variant viridans streptococci are described. The organisms were fully characterized. They have a rare nutritional requirement for pyridoxal and require defined culture conditions and specific identification. Nutritional variant streptococci (NVS) are principally described as causing endocarditis, another infection involving an avascular collagenous tissue, and exhibiting similar biologic behavior. Electronmicrographic evidence is also adduced that suggests the possible importance of intracorneal glycocalyx deposition. Such factors might explain the anomalous clinical characteristics of this condition.

Adult↗
PubMed2002

Integrated platform for detection of DNA sequence variants using capillary array electrophoresis.

We have developed a highly versatile platform that performs temperature gradient capillary electrophoresis (TGCE) for mutation/single-nucleotide polymorphism (SNP) detection, sequencing and mutation/SNP genotyping for identification of sequence variants on an automated 24-, 96- or 192-capillary array instrument. In the first mode, multiple DNA samples consisting of homoduplexes and heteroduplexes are separated by CE, during which a temperature gradient is applied that covers all possible temperatures of 50% melting equilibrium (Tms) for the samples. The differences in Tms result in separation of homoduplexes from heteroduplexes, thereby identifying the presence of DNA variants. The sequencing mode is then used to determine the exact location of the mutation/SNPs in the DNA variants. The first two modes allow the rapid identification of variants from the screening of a large number of samples. Only the variants need to be sequenced. The third mode utilizes multiplexed single-base extensions (SBEs) to survey mutations and SNPs at the known sites of DNA sequence. The TGCE approach combined with sequencing and SBE is fast and cost-effective for high-throughput mutation/SNP detection.

DNA↗
PubMed2003

Digital genotyping using molecular affinity and mass spectrometry.

The goal of DNA sequencing and genotyping is to efficiently generate accurate high-throughput digital genetic information that unambiguously identifies sources of genetic variation and clearly distinguishes heterozygous from homozygous variants. Recent advances in mass-spectrometry-based DNA sequencing and genotyping bode well for meeting these criteria. Pilot studies show that these recently developed approaches allow unambiguous multiplex detection of heterozygous variants and the identification of deletion and insertion variants.

Genotype↗
PubMedDate not supplied

Biochemical characteristics of four common molecular variants in glucose-6-phosphate dehydrogenase-deficient Chinese in Singapore.

Biochemical characteristics were determined for four common molecular variants of glucose-6-phosphate dehydrogenase (G6PD) deficiency and 10 non-deficient Chinese males in Singapore. The variants included one Mediterranean (nt563 C-->T), two Canton (G-->T at nt1376) and one each of Kaiping (1388 G-->A) and Chinese-5 (1024 C-->T) variants. Molecular identification was carried out by amplication of genomic DNA with specific oligonucleotide primers followed by digestion with restriction enzymes that recognize artificially created or naturally occurring restriction sites. All the variants had low enzyme activity in red cells (0.2-0.6 IU/g Hb). All but the Chinese-5 variant (nt1024) had a normal Km for NADP (7-10 microM). The Mediterranean variant had a high utilization of deamino-NADP (296%), followed by the Canton variant 1376 substitution (131%). The Km for glucose-6-phosphate was low in the Mediterranean and 1376 variant (18-40 microM) but high in the 1024 substitution (104 microM). Electrophoretic mobility in TEB buffer (pH 8.6) was slightly faster (103%) for the 1376 mutation while 100% for all the others. All but the 1024 substitution had increased analogue utilization for galactose-6-phosphate and 2-deoxy-glucose-6-phosphate (58 and 68% for the Mediterranean mutation and 14-23% for the 1376 and 1388 substitutions, respectively), and reduced heat stability.

China↗
PubMed2004

Identification of a novel splice variant of the human anti-apoptopsis gene survivin.

Survivin, a new member of the inhibitor of apoptosis protein (IAP) family, has been reported to be expressed in many cancers but not in differentiated normal tissues. In the present study, we describe the identification of a novel alternatively spliced survivin transcript, designated as survivin-3B. It comprises 5 exons including novel exon 3B derived from a 165-bp long portion of intron 3. Acquisition of a new in-frame TGA stop codon within the novel exon 3B results in an open reading frame (ORF) of 363 nucleotides, predicting a truncated 120 amino acid protein. Expression of survivin-3B was detected in human colon and gastric adenocarcinoma cell lines as well as mononuclear cells prepared from patients with myelodysplastic syndrome (MDS) and acute myelogenous leukemia (AML). Survivin-3B contains a single baculovirus IAP repeat (BIR), which is critical for apoptosis inhibition. However, it lacks a carboxyl-terminal coiled-coil domain, suggesting that survivin-3B may not be associated with G2/M phase. These data indicate that the function of survivin-3B may be different from that of regular survivin.

Alternative Splicing↗
PubMed2005

On the probability that a novel variant is a disease-causing mutation.

When a novel variant is found in a patient and not in a group of controls, it becomes a candidate for the disease-causing mutation in that patient. At present, no sampling theory exists for assessing the probability that the novel SNP might actually be a neutral variant. We have developed a population genetics-based method for calculating a P-value for a mutation-detection effort. Our method can be applied to a heterozygous patient, a homozygous patient, with or without inbreeding, or to a patient who is a compound heterozygote. Additionally, the method can be used to calculate the probability of finding a neutral variant at frequencies that differ between a group of patients and a group of controls, given some length of sequence examined. This method accounts for the multiple testing that is inherent in identification of variants through sequencing, to be used in subsequent case-control analyses. We show, for example, that for complete resequencing of 10 kb, the probability of finding a neutral variant in a patient and not in 50 controls is about 15%. Thus, discovery of a variant in a patient and not in a group of controls is, on its own, very weak evidence of involvement with disease.

Animals↗
PubMed1988

Colony hybridization to identify mammalian cells containing amplified, transfected, or expressed sequences.

The identification of variant cell clones has played an important role in the elucidation of various biochemical pathways. Such clones are typically selected by altering the medium or the growth conditions to give a selective advantage to the desired variant. The availability of cloned DNA probes has made it possible to identify bacterial colonies or viral plaques containing desired sequences, even in the absence of selection. We describe here a new method for the identification and isolation of mammalian cells containing any sequence for which a probe exists. Mammalian cell clones are first replicated onto nylon cloth. The original clones and the replicas are grown for a few days to enlarge the clones. All the clones on the original dish are transferred in situ to a filter and processed for hybridization. By altering the processing, either DNA or RNA sequences in the clones can be identified. When a clone of interest is located by hybridization, the cells can be isolated from the nylon replicas. We expect this method to be of value in the isolation of clones containing amplified, transfected, or mutated genes, as well as those exhibiting altered gene expression.

Animals↗
PubMed2006

Identification of wild type and variants of oestrogen receptors in polymorphonuclear and mononuclear leucocytes.

OBJECTIVE: Leucocytes play an important role in the pathogenesis of autoimmune and cardiovascular diseases. Clinical and epidemiological observations indicate that the sex steroid hormones, particularly oestrogens, may regulate leucocyte functions. The assumption that oestrogens have a direct effect on leucocytes has to be supported by identification of functional oestrogen receptors (ER) in leucocytes. This study aimed at investigating the presence of ER subtypes in different types of leucocytes isolated from peripheral blood of female and male donors. DESIGN AND PATIENTS: A total of nine men (age range 18-43 years) and nine women (age range 19-42 years) all healthy blood donors, were recruited for the study. The donors did not receive any medication or hormonal contraceptives for the last three months. Ten millilitres of peripheral blood was collected from each donor. Polymorphonuclear leucocytes (PMN) and peripheral blood mononuclear cells (PBMC) were purified by density gradient centrifugation. MEASUREMENTS: ERalpha and ERbeta mRNA expression was measured by real-time reverse transcriptase-polymerase chain reaction (RT-PCR), and ER proteins were analysed by Western blot in the PBMC and PMN leucocyte populations. In addition, expression profiles of ER variant isoforms were characterized by conventional PCR using the splice-targeted primer approach. RESULTS: Although we detected wild-type ERalpha and ERbeta mRNAs in PBMC but not in PMN cells, the ERalpha and ERbeta proteins were found in both cell types using Western blot. We observed that both ERalpha and ERbeta proteins differ in size between PMN and PBMC, suggesting that the two leucocyte populations contain diverse variant isoforms of ERalpha and ERbeta. RT-PCR analysis of exon-deleted ER splice variants revealed that PBMC express several exon-deleted variants of ERalpha and ERbeta, along with wild-type receptor, whereas the PMN cells only express exon-deleted variant isoforms and no wild-type ERalpha or ERbeta. CONCLUSIONS: Our study demonstrates the presence of ERalpha and ERbeta in PBMC and PMN cells from female and male donors. The ERalpha and ERbeta genes have complex transcriptional profiles, with many receptor variant isoforms being expressed. Considering the diversity of ER isoforms in leucocyte subtypes, we conclude that the expected effect of oestrogen would be highly cell type-specific. Further studies are needed to test the functional activity of ER isoforms and their relation to disease.

Adolescent↗