A refined, rapid and reproducible high resolution melt (HRM)-based method suitable for quantification of global LINE-1 repetitive element methylation

<p>Abstract</p> <p>Background</p> <p>The methylation of DNA is recognized as a key mechanism in the regulation of genomic stability and evidence for its role in the development of cancer is accumulating. LINE-1 methylation status represents a surrogate measure of genome-wide methylation.</p> <p>Findings</p> <p>Using high resolution melt (HRM) curve analysis technology, we have established an in-tube assay that is linear (r > 0.9986) with a high amplification efficiency (90-105%), capable of discriminating between partcipant samples with small differences in methylation, and suitable for quantifying a wide range of LINE-1 methylation levels (0-100%)--including the biologically relevant range of 50-90% expected in human DNA. We have optimized this procedure to perform using 2 μg of starting DNA and 2 ng of bisulfite-converted DNA for each PCR reaction. Intra- and inter-assay coefficients of variation were 1.44% and 0.49%, respectively, supporting the high reproducibility and precision of this approach.</p> <p>Conclusions</p> <p>In summary, this is a completely linear, quantitative HRM PCR method developed for the measurement of LINE-1 methylation. This cost-efficient, refined and reproducible assay can be performed using minimal amounts of starting DNA. These features make our assay suitable for high throughput analysis of multiple samples from large population-based studies.</p

Sex-specific effect of CPB2 Ala147Thr but not Thr325Ile variants on the risk of venous thrombosis: A comprehensive meta-analysis

International audienceBackground: Thrombin activatable fibrinolysis inhibitor (TAFI), encoded by the Carboxypeptidase B2 gene (CPB2), is an inhibitor of fibrinolysis and plays a role in the pathogenesis of venous thrombosis. Experimental findings support a functional role of genetic variants in CPB2, while epidemiological studies have been unable to confirm associations with risk of venous thrombosis. Sex-specific effects could underlie the observed inconsistent associations between CPB2 genetic variants and venous thrombosis.Methods: A comprehensive literature search was conducted for associations between Ala147Thr and Thr325Ile variants with venous thrombosis. Authors were contacted to provide sex-specific genotype counts from their studies. Combined and sex-specific random effects meta-analyses were used to estimate a pooled effect estimate for primary and secondary genetic models.Results: A total of 17 studies met the inclusion criteria. A sex-specific meta-analysis applying a dominant model supported a protective effect of Ala147Thr on venous thrombosis in females (OR = 0.81, 95%CI: 0.68,0.97; p = 0.018), but not in males (OR = 1.06, 95%CI:0.96–1.16; p = 0.263). The Thr325Ile did not show a sex-specific effect but showed variation in allele frequencies by geographic region. A subgroup analysis of studies in European countries showed decreased risk, with a recessive model (OR = 0.83, 95%CI:0.71–0.97, p = 0.021) for venous thrombosis.Conclusions: A comprehensive literature review, including unpublished data, provided greater statistical power for the analyses and decreased the likelihood of publication bias influencing the results. Sex-specific analyses explained apparent discrepancies across genetic studies of Ala147Thr and venous thrombosis. While, careful selection of genetic models based on population genetics, evolutionary and biological knowledge can increase power by decreasing the need to adjust for testing multiple models

The role of the Deki Reader™ in malaria diagnosis, treatment and reporting: findings from an Africare pilot project in Nigeria

Abstract Background The Deki Reader is a diagnostic device used with rapid diagnostic tests (RDTs) and linked to an online database for real-time uploads of patient information and results. This is in contrast to visual interpretation of malaria RDTs recorded on the District Health Information System (DHIS). This paper compares records for use of the Deki Reader with DHIS records of visual interpretation of RDTs. Results A total of 4063 patient encounters/tests were recorded on the Deki Reader database between June 1st and December 31st, 2016. These tests were for 2629 persons who presented with fever and had RDT done. In comparison, data from DHIS 2.0 for same period recorded 7201 persons presenting with fever. 2421 out of the 2629 persons (92.1%), received RDT using Deki Reader compared to 6535 out of 7201 persons (90.4%) recorded on DHIS (p = 0.04). From DHIS records, malaria positivity rate was 51.6% (3375 out of 6535 persons) compared to Deki Reader records of 23.6% (572 out of 2421 persons). The difference between these two rates was significant (p < 0.001). The odds ratio (95% CI) for the association between use of Deki Reader and having a positive malaria result was 0.29 (0.26–0.32). DHIS showed that 4008 persons received Artemisinin-based combination therapy (ACT) while 3989 persons tested positive with RDT or microscopy, compared to 691 out of 705 persons (98.0%) using Deki Reader. Finally, Deki Reader identified 618 processing and manufacturers errors with an error rate of 15.3%. Conclusion The Deki Reader is likely a useful tool for malaria diagnosis, treatment, and real-time data management. It potentially improves diagnostic quality, reduces wastage in ACT administration and improves data quality

Genome-wide investigation of DNA methylation marks associated with FV Leiden mutation.

In order to investigate whether DNA methylation marks could contribute to the incomplete penetrance of the FV Leiden mutation, a major genetic risk factor for venous thrombosis (VT), we measured genome-wide DNA methylation levels in peripheral blood samples of 98 VT patients carrying the mutation and 251 VT patients without the mutation using the dedicated Illumina HumanMethylation450 array. The genome-wide analysis of 388,120 CpG probes identified three sites mapping to the SLC19A2 locus whose DNA methylation levels differed significantly (p<3 10-8) between carriers and non-carriers. The three sites replicated (p<2 10-7) in an independent sample of 214 individuals from five large families ascertained on VT and FV Leiden mutation among which 53 were carriers and 161 were non-carriers of the mutation. In both studies, these three CpG sites were also associated (2.33 10-11<p<3.02 10-4) with biomarkers of the Protein C pathway known to be influenced by the FV Leiden mutation. A comprehensive linkage disequilibrium (LD) analysis of the whole locus revealed that the original associations were due to LD between the FV Leiden mutation and a block of single nucleotide polymorphisms (SNP) located in SLC19A2. After adjusting for this block of SNPs, the FV Leiden mutation was no longer associated with any CpG site (p>0.05). In conclusion, our work clearly illustrates some promises and pitfalls of DNA methylation investigations on peripheral blood DNA in large epidemiological cohorts. DNA methylation levels at SLC19A2 are influenced by SNPs in LD with FV Leiden, but these DNA methylation marks do not explain the incomplete penetrance of the FV Leiden mutation

Meta-analysis results of the <i>CPB2</i> Thr325Ile variant and risk of venous thrombosis in European study populations.

<p>The solid squares represent the ORs from the individual studies; horizontal lines represent corresponding CIs; the diamonds show the combined ORs.</p

Meta-analysis results of the <i>CPB2</i> Thr325Ile variant and risk of venous thrombosis using a recessive model.

<p>The solid squares represent the ORs from the individual studies; horizontal lines represent corresponding CIs; the diamonds show the combined ORs.</p

Distribution of Ala147Thr genotypes by sex among cases and non-cases in studies included in the meta-analysis.

<p>Distribution of Ala147Thr genotypes by sex among cases and non-cases in studies included in the meta-analysis.</p

Leveraging cell type specific regulatory regions to detect SNPs associated with tissue factor pathway inhibitor plasma levels

International audienceTissue factor pathway inhibitor (TFPI) regulates the formation of intravascular blood clots, which manifest clinically as ischemic heart disease, ischemic stroke, and venous thromboembolism (VTE). TFPI plasma levels are heritable, but the genetics underlying TFPI plasma level variability are poorly understood. Herein we report the first genome‐wide association scan (GWAS) of TFPI plasma levels, conducted in 251 individuals from five extended French‐Canadian Families ascertained on VTE. To improve discovery, we also applied a hypothesis‐driven (HD) GWAS approach that prioritized single nucleotide polymorphisms (SNPs) in (1) hemostasis pathway genes, and (2) vascular endothelial cell (EC) regulatory regions, which are among the highest expressers of TFPI. Our GWAS identified 131 SNPs with suggestive evidence of association (P‐value < 5 × 10 −8), but no SNPs reached the genome‐wide threshold for statistical significance. Hemostasis pathway genes were not enriched for TFPI plasma level associated SNPs (global hypothesis test P‐ value = 0.147), but EC regulatory regions contained more TFPI plasma level associated SNPs than expected by chance (global hypothesis test P‐value = 0.046). We therefore stratified our genome‐wide SNPs, prioritizing those in EC regulatory regions via stratified false discovery rate (sFDR) control, and reranked the SNPs by q‐value. The minimum q‐value was 0.27, and the top‐ranked SNPs did not show association evidence in the MARTHA replication sample of 1,033 unrelated VTE cases. Although this study did not result in new loci for TFPI, our work lays out a strategy to utilize epigenomic data in prioritization schemes for future GWAS studies

Meta-analysis of the association between Thr325Ile variant and risk of venous thrombosis in European study populations.

<p>Meta-analysis of the association between Thr325Ile variant and risk of venous thrombosis in European study populations.</p