Assessment of use of microsatellite polymorphism analysis for improving spatial distribution tracking of echinococcus multilocularis

Alveolar echinococcosis (AE)--caused by the cestode Echinococcus multilocularis--is a severe zoonotic disease found in temperate and arctic regions of the northern hemisphere. Even though the transmission patterns observed in different geographical areas are heterogeneous, the nuclear and mitochondrial targets usually used for the genotyping of E. multilocularis have shown only a marked genetic homogeneity in this species. We used microsatellite sequences, because of their high typing resolution, to explore the genetic diversity of E. multilocularis. Four microsatellite targets (EmsJ, EmsK, and EmsB, which were designed in our laboratory, and NAK1, selected from the literature) were tested on a panel of 76 E. multilocularis samples (larval and adult stages) obtained from Alaska, Canada, Europe, and Asia. Genetic diversity for each target was assessed by size polymorphism analysis. With the EmsJ and EmsK targets, two alleles were found for each locus, yielding two and three genotypes, respectively, discriminating European isolates from the other groups. With NAK1, five alleles were found, yielding seven genotypes, including those specific to Tibetan and Alaskan isolates. The EmsB target, a tandem repeated multilocus microsatellite, found 17 alleles showing a complex pattern. Hierarchical clustering analyses were performed with the EmsB findings, and 29 genotypes were identified. Due to its higher genetic polymorphism, EmsB exhibited a higher discriminatory power than the other targets. The complex EmsB pattern was able to discriminate isolates on a regional and sectoral level, while avoiding overdistinction. EmsB will be used to assess the putative emergence of E. multilocularis in Europe.

G870A polymorphism of cyclin D1 gene influences cancer risk - From epidemiological study to mechanism analysis

Alternate splicing of the cyclin D1 gene gives rise to transcript a and b which encode two protein isoforms cyclin D1a and cyclin D1b. Through testing transcript a and transcript b in a series of human samples, we found that cyclin D1 transcript b is ubiquitously expressed as transcript a but in the lower abundance compared to transcript a. Epidemiological studies have reported that the cyclin D1 gene (CCND1) G870A polymorphism influences the risk for a variety of cancer. In this investigation, we examined the cyclin D1b levels in tumor samples with different genotypes and found that higher levels of cyclin D1b are expressed from the A allele than the G allele. Cyclin D1 is known as a cell cycle regulator facilitating the progression of the cell cycle from G1 to S phase in response to the mitogenic signals. It also interacts with several transcription factors and transcriptional coregulators to modulate their activities. It has been reported that cyclin D1a can substitute for estrogen to activate estrogen receptor α (ERα) mediated transcription and can induce the proliferation of estrogen responsive tissues. However the biological role of cyclin D1b in ERα transcriptional regulation has not been previously explored. In this study, we determined that cyclin D1b antagonizes the action of cyclin D1a on ERα mediated transcription. Cell proliferation assays provided the evidence that cyclin D1b negatively regulates estrogen responsive breast cancer cell growth. Taken together, our findings show that the CCND1 G870A polymorphism is correlated with increased levels of cyclin D1b and that cyclin D1b antagonizes the action of cyclin D1a on ERα mediated transcription providing evidence for the mechanism by which the CCND1 G870A polymorphism may be protective in certain types of breast cancer. ^

Utilización de marcadores morfológicos y moleculares AFLP en la identificación de germo plasma nativo y cultivado de Elymus scabrifolius (Poaceae)

Elymus scabrifolius es una gramínea perenne nativa de Sudamérica con gran potencial como recurso forrajero para ambientes con limitantes edáficas. En el presente trabajo se analizó la utilización de caracteres morfológicos y marcadores moleculares AFLP para la identificación genotípica de seis accesiones, un cultivar comercial y siete híbridos artificiales de esta especie. Ambos tipos de marcadores permitieron diferenciar a los materiales analizados en los respectivos dendrogramas, aunque las relaciones entre materiales variaron según el tipo de marcador. El Análisis de Componentes Principales permitió identificar las variables más relevantes para la diferenciación morfológica. Los híbridos se diferenciaron morfológicamente de ambos parentales, excepto un híbrido que se agrupó con su material paterno. Aunque en el análisis de los marcadores AFLP los híbridos se agruparon con uno de sus parentales, se pudo corroborar su origen híbrido mediante el registro de bandas paternas y polimórficas entre parentales. Se concluye que las metodologías empleadas para caracterizar los materiales analizados de E. scabrifolius serían de gran utilidad para el manejo eficiente de colecciones de germoplasma como así también para su utilización en programas de mejoramiento genético.

Half tetrad analysis in alfalfa using multiple restriction fragment length polymorphism markers.

A maximum likelihood approach of half tetrad analysis (HTA) based on multiple restriction fragment length polymorphism (RFLP) markers was developed. This procedure estimates the relative frequencies of 2n gametes produced by mechanisms genetically equivalent to first division restitution (FDR) or second division restitution and simultaneously locates the centromere within a linkage group of RFLP marker loci. The method was applied to the diploid alfalfa clone PG-F9 (2n = 2x = 16) previously selected because of its high frequency of 2n egg production. HTA was based on four RFLP loci for which PG-F9 was heterozygous with codominant alleles that were absent in the tetraploid tester. Models including three linked and one unlinked RFLP loci were developed and tested. Results of the HTA showed that PG-F9 produced 6% FDR and 94% second division restitution 2n eggs. Information from a marker locus belonging to one linkage group was used to more precisely locate the centromere on a different linkage group. HTA, together with previous cytological analysis, indicated that in PG-F9, FDR 2n eggs are likely produced by diplospory, a mechanism common among apomictic species. The occurrence of FDR 2n eggs in plant species and their importance for crop evolution and breeding is discussed together with the potential applicability of multilocus HTA in the study of reproductive mutants.

HLA-Cw allele analysis by PCR-restriction fragment length polymorphism: study of known and additional alleles.

We describe a technique for HLA-Cw genotyping by digestion of PCR-amplified genes with restriction endonucleases. Locus-specific primers selectively amplified HLA-Cw sequences from exon 2 in a single PCR that avoided coamplification of other classical and nonclassical class I genes. Amplified DNAs were digested with selected enzymes. Sixty-three homozygous cell lines from International Histocompatibility Workshop X and 113 unrelated individual cells were genotypes for HLA-Cw and compared with serology. The present protocol can distinguish 23 alleles corresponding to the known HLA-Cw sequences. Genotyping of serologically undetectable alleles (HLA-Cw Blank) and of heterozygous cells was made possible by using this method. Six additional HLA-Cw alleles were identified by unusual restriction patterns and confirmed by sequencing; this observation suggests the presence of another family of allele-sharing clusters in the HLA-B locus. This PCR-restriction endonuclease method provides a simple and convenient approach for HLA-Cw DNA typing, allowing the definition of serologically undetectable alleles, and will contribute to the evaluation of the biological role of the HLA-C locus.

Restriction fragment length polymorphism-coupled domain-directed differential display: a highly efficient technique for expression analysis of multigene families.

In this paper, a reverse-transcriptase PCR-based protocol suitable for efficient expression analysis of multigene families is presented. The method combines restriction fragment length polymorphism (RFLP) technology with a gene family-specific version of mRNA differential display and hence is called "RFLP-coupled domain-directed differential display. "With this method, expression of all members of a multigene family at many different developmental stages, in diverse tissues and even in different organisms, can be displayed on one gel. Moreover, bands of interest, representing gene family members, are directly accessible to sequence analysis, without the need for subcloning. The method thus enables a detailed, high-resolution expression analysis of known gene family members as well as the identification and characterization of new ones. Here the technique was used to analyze differential expression of MADS-box genes in male and female inflorescences of maize (Zea mays ssp. mays). Six different MADS-box genes could be identified, being either specifically expressed in the female sex or preferentially expressed in male or female inflorescences, respectively. Other possible applications of the method are discussed.

Genetic time-series analysis identifies a major QTL for in vivo alcohol metabolism not predicted by in vitro studies of structural protein polymorphism at the ADH1B or ADH1C loci

After ingestion of a standardized dose of ethanol, alcohol concentrations were assessed, over 3.5 hours from blood (six readings) and breath (10 readings) in a sample of 412 MZ and DZ twins who took part in an Alcohol Challenge Twin Study (ACTS). Nearly all participants were subsequently genotyped on two polymorphic SNPs in the ADH1B and ADH1C loci known to affect in vitro ADH activity. In the DZ pairs, 14 microsatellite markers covering a 20.5 cM region on chromosome 4 that includes the ADH gene family were assessed, Variation in the timed series of autocorrelated blood and breath alcohol readings was studied using a bivariate simplex design. The contribution of a quantitative trait locus (QTL) or QTL's linked to the ADH region was estimated via a mixture of likelihoods weighted by identity-by-descent probabilities. The effects of allelic substitution at the ADH1B and ADH1C loci were estimated in the means part of the model simultaneously with the effects sex and age. There was a major contribution to variance in alcohol metabolism due to a QTL which accounted for about 64% of the additive genetic covariation common to both blood and breath alcohol readings at the first time point. No effects of the ADH1B*47His or ADH1C*349Ile alleles on in vivo metabolism were observed, although these have been shown to have major effects in vitro. This implies that there is a major determinant of variation for in vivo alcohol metabolism in the ADH region that is not accounted for by these polymorphisms. Earlier analyses of these data suggested that alcohol metabolism is related to drinking behavior and imply that this QTL may be protective against alcohol dependence.

Meta-analysis of the population and phenotypic expression of CYP2C9/2C19 polymorphism on drug metabolism in different ethnicities

The term "pharmacogenetics" has been defined as the scientific study of inherited factors that affect the human drug response. Many pharmacogenetie studies have been published since 1995 and have focussed on the principal enzyme family involved in drug metabolism, the cytochrome P450 family, particularly cytochrome P4502C9 and 2C19. In order to investigate the pharmacogenetic aspect of pharmacotherapy, the relevant studies describing the association of pharmacogenetic factor(s) in drug responses must be retrieved from existing literature using a systematic review approach. In addition, the estimation of variant allele prevalence for the gene under study between different ethnic populations is important for pharmacogenetic studies. In this thesis, the prevalence of CYP2C9/2C19 alleles between different ethnicities has been estimated through meta-analysis and the population genetic principle. The clinical outcome of CYP2C9/2C19 allelic variation on the pharmacotherapy of epilepsy has been investigated; although many new antiepileptic drugs have been launched into the market, carbamazepine, phenobarbital and phenytoin are still the major agents in the pharmacotherapy of epilepsy. Therefore, phenytoin was chosen as a model AED and the effect of CYP2C9/2C19 genetic polymorphism on phenytoin metabolism was further examined.An estimation of the allele prevalence was undertaken for three CYP2C9/2C19 alleles respectively using a meta-analysis of studies that fit the Hardy-Weinberg equilibrium. The prevalence of CYP2C9*1 is approximately 81%, 96%, 97% and 94% in Caucasian, Chinese, Japanese, African populations respectively; the pooled prevalence of CYP2C19*1 is about 86%, 57%, 58% and 85% in these ethnic populations respectively. However, the studies of association between CYP2C9/2C19 polymorphism and phenytoin metabolism failed to achieve any qualitative or quantitative conclusion. Therefore, mephenytoin metabolism was examined as a probe drug for association between CYP2C19 polymorphism and mephenytoin metabolic ratio. Similarly, analysis of association between CYP2C9 polymorphism and warfarin dose requirement was undertaken.It was confirmed that subjects carrying two mutated CYP2C19 alleles have higher S/R mephenytoin ratio due to deficient CYP2C19 enzyme activity. The studies of warfarin and CYP2C9 polymorphism did not provide a conclusive result due to poor comparability between studies.The genetic polymorphism of drug metabolism enzymes has been studied extensively, however other genetic factors, such as multiple drug resistance genes (MDR) and genes encoding ion channels, which may contribute to variability in function of drug transporters and targets, require more attention in future pharmacogenetic studies of antiepileptic drugs.

Childhood asthma exacerbations and the Arg16 β2-receptor polymorphism : a meta-analysis stratified by treatment

Peer reviewed

Transforming Growth Factor-b1 polymorphism is not associated with chronic graft disease: evidence from a meta-analysis

Kidney transplantation has been recognised as the optimal treatment choice for most end stage renal disease patients and the increase of allograft survival rates is achieved through the refinement of novel immunosuppressive agents. Chronic Graft Disease (CGD) is a multifactorial process that likely includes a combination of immunological, apoptotic and inflammatory factors. The application of individualised immunosuppressive therapies will also depend on the identification of risk factors that can influence chronic disease. Despite being the subject of several independent studies, investigations of the relationship between transforming growth factor-b1 (TGF-b1) polymorphisms and kidney graft outcome continue to be plagued by contradictory conclusions.

AFLP-based genetic mapping of the " bud-flowering" trait in heather (Calluna vulgaris)

Background: Calluna vulgaris is one of the most important landscaping plants produced in Germany. Its enormous economic success is due to the prolonged flower attractiveness of mutants in flower morphology, the so-called bud-bloomers. In this study, we present the first genetic linkage map of C. vulgaris in which we mapped a locus of the economically highly desired trait " flower type" .Results: The map was constructed in JoinMap 4.1. using 535 AFLP markers from a single mapping population. A large fraction (40%) of markers showed distorted segregation. To test the effect of segregation distortion on linkage estimation, these markers were sorted regarding their segregation ratio and added in groups to the data set. The plausibility of group formation was evaluated by comparison of the " two-way pseudo-testcross" and the " integrated" mapping approach. Furthermore, regression mapping was compared to the multipoint-likelihood algorithm. The majority of maps constructed by different combinations of these methods consisted of eight linkage groups corresponding to the chromosome number of C. vulgaris.Conclusions: All maps confirmed the independent inheritance of the most important horticultural traits " flower type" , " flower colour" , and " leaf colour". An AFLP marker for the most important breeding target " flower type" was identified. The presented genetic map of C. vulgaris can now serve as a basis for further molecular marker selection and map-based cloning of the candidate gene encoding the unique flower architecture of C. vulgaris bud-bloomers. © 2013 Behrend et al.; licensee BioMed Central Ltd.

Relationship between IL-10 gene -819C/T polymorphism and the risk of inflammatory bowel disease: a meta-analysis.

Background: The -819C/T polymorphism in interleukin 10 (IL-10) gene has been reported to be associated with inflammatory bowel disease (IBD) ,but the previous results are conflicting. Materials and Methods: The present study aimed at investigating the association between this polymorphism and risk of IBD using a meta-analysis.PubMed,Web of Science,EMBASE,google scholar and China National Knowledge Infrastructure (CNKI) databases were systematically searched to identify relevant publications from their inception to April 2016.Pooled odds ratio (OR) with 95% confidence interval (CI) was calculated using fixed- or random-effects models. Results: A total of 7 case-control studies containing 1890 patients and 2929 controls were enrolled into this meta-analysis, and our results showed no association between IL-10 gene -819C/T polymorphism and IBD risk(TT vs. CC:OR=0.81,95%CI 0.64- 1.04;CT vs. CC:OR=0.92,95%CI 0.81-1.05; Dominant model: OR=0.90,95%CI 0.80-1.02; Recessive model: OR=0.84,95%CI 0.66-1.06). In a subgroup analysis by nationality, the -819C/T polymorphism was not associated with IBD in both Asians and Caucasians. In the subgroup analysis stratified by IBD type, significant association was found in Crohn’s disease(CD)(CT vs. CC:OR=0.68,95%CI 0.48-0.97). Conclusion: In summary, the present meta-analysis suggests that the IL-10 gene -819C/T polymorphism may be associated with CD risk.

Rs266882 polymorphism analysis in KLK3 gene by RFLPPCR in cases of prostate cancer

Poster presented at the From Basic Sciences to Clinical Research - First International Congress of CiiEM. Egas Moniz, Caparica, Portugal, 27-28 November 2015