Alteration in P-glycoprotein Functionality Affects Intrabrain Distribution of Quinidine More Than Brain Entry—A Study in Rats Subjected to Status Epilepticus by Kainate

This study aimed to investigate the use of quinidine microdialysis to study potential changes in brain P-glycoprotein functionality after induction of status epilepticus (SE) by kainate. Rats were infused with 10 or 20 mg/kg quinidine over 30 min or 4 h. Plasma, brain extracellular fluid (brain ECF), and end-of-experiment total brain concentrations of quinidine were determined during 7 h after the start of the infusion. Effect of pretreatment with tariquidar (15 mg/kg, administered 30 min before the start of the quinidine infusion) on the brain distribution of quinidine was assessed. This approach was repeated in kainate-treated rats. Quinidine kinetics were analyzed with population modeling (NONMEM). The quinidine microdialysis assay clearly revealed differences in brain distribution upon changes in P-glycoprotein functionality by pre-administration of tariquidar, which resulted in a 7.2-fold increase in brain ECF and a 40-fold increase in total brain quinidine concentration. After kainate treatment alone, however, no difference in quinidine transport across the blood–brain barrier was found, but kainate-treated rats tended to have a lower total brain concentration but a higher brain ECF concentration of quinidine than saline-treated rats. This study did not provide evidence for the hypothesis that P-glycoprotein function at the blood–brain barrier is altered at 1 week after SE induction, but rather suggests that P-glycoprotein function might be altered at the brain parenchymal level

Multiplexed SNP Typing of Ancient DNA Clarifies the Origin of Andaman mtDNA Haplogroups amongst South Asian Tribal Populations

The issue of errors in genetic data sets is of growing concern, particularly in population genetics where whole genome mtDNA sequence data is coming under increased scrutiny. Multiplexed PCR reactions, combined with SNP typing, are currently under-exploited in this context, but have the potential to genotype whole populations rapidly and accurately, significantly reducing the amount of errors appearing in published data sets. To show the sensitivity of this technique for screening mtDNA genomic sequence data, 20 historic samples of the enigmatic Andaman Islanders and 12 modern samples from three Indian tribal populations (Chenchu, Lambadi and Lodha) were genotyped for 20 coding region sites after provisional haplogroup assignment with control region sequences. The genotype data from the historic samples significantly revise the topologies for the Andaman M31 and M32 mtDNA lineages by rectifying conflicts in published data sets. The new Indian data extend the distribution of the M31a lineage to South Asia, challenging previous interpretations of mtDNA phylogeography. This genetic connection between the ancestors of the Andamanese and South Asian tribal groups ∼30 kya has important implications for the debate concerning migration routes and settlement patterns of humans leaving Africa during the late Pleistocene, and indicates the need for more detailed genotyping strategies. The methodology serves as a low-cost, high-throughput model for the production and authentication of data from modern or ancient DNA, and demonstrates the value of museum collections as important records of human genetic diversity

A high density linkage map of the bovine genome

<p>Abstract</p> <p>Background</p> <p>Recent technological advances have made it possible to efficiently genotype large numbers of single nucleotide polymorphisms (SNPs) in livestock species, allowing the production of high-density linkage maps. Such maps can be used for quality control of other SNPs and for fine mapping of quantitative trait loci (QTL) via linkage disequilibrium (LD).</p> <p>Results</p> <p>A high-density bovine linkage map was constructed using three types of markers. The genotypic information was obtained from 294 microsatellites, three milk protein haplotypes and 6769 SNPs. The map was constructed by combining genetic (linkage) and physical information in an iterative mapping process. Markers were mapped to 3,155 unique positions; the 6,924 autosomal markers were mapped to 3,078 unique positions and the 123 non-pseudoautosomal and 19 pseudoautosomal sex chromosome markers were mapped to 62 and 15 unique positions, respectively. The linkage map had a total length of 3,249 cM. For the autosomes the average genetic distance between adjacent markers was 0.449 cM, the genetic distance between unique map positions was 1.01 cM and the average genetic distance (cM) per Mb was 1.25.</p> <p>Conclusion</p> <p>There is a high concordance between the order of the SNPs in our linkage map and their physical positions on the most recent bovine genome sequence assembly (Btau 4.0). The linkage maps provide support for fine mapping projects and LD studies in bovine populations. Additionally, the linkage map may help to resolve positions of unassigned portions of the bovine genome.</p

Genetic risk and a primary role for cell-mediated immune mechanisms in multiple sclerosis.

Multiple sclerosis is a common disease of the central nervous system in which the interplay between inflammatory and neurodegenerative processes typically results in intermittent neurological disturbance followed by progressive accumulation of disability. Epidemiological studies have shown that genetic factors are primarily responsible for the substantially increased frequency of the disease seen in the relatives of affected individuals, and systematic attempts to identify linkage in multiplex families have confirmed that variation within the major histocompatibility complex (MHC) exerts the greatest individual effect on risk. Modestly powered genome-wide association studies (GWAS) have enabled more than 20 additional risk loci to be identified and have shown that multiple variants exerting modest individual effects have a key role in disease susceptibility. Most of the genetic architecture underlying susceptibility to the disease remains to be defined and is anticipated to require the analysis of sample sizes that are beyond the numbers currently available to individual research groups. In a collaborative GWAS involving 9,772 cases of European descent collected by 23 research groups working in 15 different countries, we have replicated almost all of the previously suggested associations and identified at least a further 29 novel susceptibility loci. Within the MHC we have refined the identity of the HLA-DRB1 risk alleles and confirmed that variation in the HLA-A gene underlies the independent protective effect attributable to the class I region. Immunologically relevant genes are significantly overrepresented among those mapping close to the identified loci and particularly implicate T-helper-cell differentiation in the pathogenesis of multiple sclerosis

Transcriptome and genome sequencing uncovers functional variation in humans

Summary Genome sequencing projects are discovering millions of genetic variants in humans, and interpretation of their functional effects is essential for understanding the genetic basis of variation in human traits. Here we report sequencing and deep analysis of mRNA and miRNA from lymphoblastoid cell lines of 462 individuals from the 1000 Genomes Project – the first uniformly processed RNA-seq data from multiple human populations with high-quality genome sequences. We discovered extremely widespread genetic variation affecting regulation of the majority of genes, with transcript structure and expression level variation being equally common but genetically largely independent. Our characterization of causal regulatory variation sheds light on cellular mechanisms of regulatory and loss-of-function variation, and allowed us to infer putative causal variants for dozens of disease-associated loci. Altogether, this study provides a deep understanding of the cellular mechanisms of transcriptome variation and of the landscape of functional variants in the human genome

ATM variants and cancer risk in breast cancer patients from Southern Finland

BACKGROUND: Individuals heterozygous for germline ATM mutations have been reported to have an increased risk for breast cancer but the role for ATM genetic variants for breast cancer risk has remained unclear. Recently, a common ATM variant, ATMivs38 -8T>C in cis with the ATMex39 5557G>A (D1853N) variant, was suggested to associate with bilateral breast cancer among familial breast cancer patients from Northern Finland. We have here evaluated the 5557G>A and ivs38-8T>C variants in an extensive case-control association analysis. We also aimed to investigate whether there are other ATM mutations or variants contributing to breast cancer risk in our population. METHODS: Two common ATM variants, 5557G>A and ivs38-8T>C, previously suggested to associate with bilateral breast cancer, were genotyped in an extensive set of 786 familial and 884 unselected breast cancer cases as well as 708 healthy controls. We also screened the entire coding region and exon-intron boundaries of the ATM gene in 47 familial breast cancer patients and constructed haplotypes of the patients. The identified variants were also evaluated for increased breast cancer risk among additional breast cancer cases and controls. RESULTS: Neither of the two common variants, 5557G>A and ivs38-8T>C, nor any haplotype containing them, was significantly associated with breast cancer risk, bilateral breast cancer or multiple primary cancers in any of the patient groups or subgoups. Three rare missense alterations and one intronic change were each found in only one patient of over 250 familial patients studied and not among controls. The fourth missense alteration studied further was found with closely similar frequencies in over 600 familial cases and controls. CONCLUSION: Altogether, our results suggest very minor effect, if any, of ATM genetic variants on familial breast cancer in Southern Finland. Our results do not support association of the 5557G>A or ivs38-8T>C variant with increased breast cancer risk or with bilateral breast cancer

In Vitro vs In Silico Detected SNPs for the Development of a Genotyping Array: What Can We Learn from a Non-Model Species?

Background: There is considerable interest in the high-throughput discovery and genotyping of single nucleotide polymorphisms (SNPs) to accelerate genetic mapping and enable association studies. This study provides an assessment of EST-derived and resequencing-derived SNP quality in maritime pine (Pinus pinaster Ait.), a conifer characterized by a huge genome size (~23.8 Gb/C). [br/] Methodology/Principal Findings: A 384-SNPs GoldenGate genotyping array was built from i/ 184 SNPs originally detected in a set of 40 re-sequenced candidate genes (in vitro SNPs), chosen on the basis of functionality scores, presence of neighboring polymorphisms, minor allele frequencies and linkage disequilibrium and ii/ 200 SNPs screened from ESTs (in silico SNPs) selected based on the number of ESTs used for SNP detection, the SNP minor allele frequency and the quality of SNP flanking sequences. The global success rate of the assay was 66.9%, and a conversion rate (considering only polymorphic SNPs) of 51% was achieved. In vitro SNPs showed significantly higher genotyping-success and conversion rates than in silico SNPs (+11.5% and +18.5%, respectively). The reproducibility was 100%, and the genotyping error rate very low (0.54%, dropping down to 0.06% when removing four SNPs showing elevated error rates). [br/] Conclusions/Significance: This study demonstrates that ESTs provide a resource for SNP identification in non-model species, which do not require any additional bench work and little bio-informatics analysis. However, the time and cost benefits of in silico SNPs are counterbalanced by a lower conversion rate than in vitro SNPs. This drawback is acceptable for population-based experiments, but could be dramatic in experiments involving samples from narrow genetic backgrounds. In addition, we showed that both the visual inspection of genotyping clusters and the estimation of a per SNP error rate should help identify markers that are not suitable to the GoldenGate technology in species characterized by a large and complex genome

Development and implementation of a highly-multiplexed SNP array for genetic mapping in maritime pine and comparative mapping with loblolly pine

<p>Abstract</p> <p>Background</p> <p>Single nucleotide polymorphisms (SNPs) are the most abundant source of genetic variation among individuals of a species. New genotyping technologies allow examining hundreds to thousands of SNPs in a single reaction for a wide range of applications such as genetic diversity analysis, linkage mapping, fine QTL mapping, association studies, marker-assisted or genome-wide selection. In this paper, we evaluated the potential of highly-multiplexed SNP genotyping for genetic mapping in maritime pine (<it>Pinus pinaster </it>Ait.), the main conifer used for commercial plantation in southwestern Europe.</p> <p>Results</p> <p>We designed a custom GoldenGate assay for 1,536 SNPs detected through the resequencing of gene fragments (707 <it>in vitro </it>SNPs/Indels) and from Sanger-derived Expressed Sequenced Tags assembled into a unigene set (829 <it>in silico </it>SNPs/Indels). Offspring from three-generation outbred (G2) and inbred (F2) pedigrees were genotyped. The success rate of the assay was 63.6% and 74.8% for <it>in silico </it>and <it>in vitro </it>SNPs, respectively. A genotyping error rate of 0.4% was further estimated from segregating data of SNPs belonging to the same gene. Overall, 394 SNPs were available for mapping. A total of 287 SNPs were integrated with previously mapped markers in the G2 parental maps, while 179 SNPs were localized on the map generated from the analysis of the F2 progeny. Based on 98 markers segregating in both pedigrees, we were able to generate a consensus map comprising 357 SNPs from 292 different loci. Finally, the analysis of sequence homology between mapped markers and their orthologs in a <it>Pinus taeda </it>linkage map, made it possible to align the 12 linkage groups of both species.</p> <p>Conclusions</p> <p>Our results show that the GoldenGate assay can be used successfully for high-throughput SNP genotyping in maritime pine, a conifer species that has a genome seven times the size of the human genome. This SNP-array will be extended thanks to recent sequencing effort using new generation sequencing technologies and will include SNPs from comparative orthologous sequences that were identified in the present study, providing a wider collection of anchor points for comparative genomics among the conifers.</p

Genetic association analyses implicate aberrant regulation of innate and adaptive immunity genes in the pathogenesis of systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a genetically complex autoimmune disease characterized by loss of immune tolerance to nuclear and cell surface antigens. Previous genome-wide association studies (GWAS) had modest sample sizes, reducing their scope and reliability. Our study comprised 7,219 cases and 15,991 controls of European ancestry, constituting a new GWAS, a meta-analysis with a published GWAS and a replication study. We have mapped 43 susceptibility loci, including ten new associations. Assisted by dense genome coverage, imputation provided evidence for missense variants underpinning associations in eight genes. Other likely causal genes were established by examining associated alleles for cis-acting eQTL effects in a range of ex vivo immune cells. We found an over-representation (n = 16) of transcription factors among SLE susceptibility genes. This finding supports the view that aberrantly regulated gene expression networks in multiple cell types in both the innate and adaptive immune response contribute to the risk of developing SLE

Оценка качества образования на основе компетентностного подхода

В работе представлен практический опыт оценки качества образования в новом формате компетентностного подход