LT-RPA: An Isothermal DNA Amplification Approach for Improved Microsatellite Genotyping and Microsatellite Instability Detection.

Microsatellites are short tandem repeats of one to six nucleotides that are highly polymorphic and extensively used as genetic markers in numerous biomedical applications, including the detection of microsatellite instability (MSI) in cancer. The standard analytical method for microsatellite analysis relies on PCR amplification followed by capillary electrophoresis or, more recently, next-generation sequencing (NGS). However, their amplification during PCR generates undesirable frameshift products known as stutter peaks caused by polymerase slippage, complicating data analysis and interpretation, while very few alternative methods for microsatellite amplification have been developed to reduce the formation of these artifacts. In this context, the recently developed low-temperature recombinase polymerase amplification (LT-RPA) is an isothermal DNA amplification method at low temperature (32 °C) that drastically reduces and sometimes completely abolishes the formation of stutter peaks. LT-RPA greatly simplifies the genotyping of microsatellites and improves the detection of MSI in cancer. In this chapter, we describe in detail all the experimental steps necessary for the development of LT-RPA simplex and multiplex assays for microsatellite genotyping and MSI detection, including the design, optimization, and validation of the assays combined with capillary electrophoresis or NGS.

A high-throughput real-time PCR tissue-of-origin test to distinguish blood from lymphoblastoid cell line DNA for (epi)genomic studies.

Lymphoblastoid cell lines (LCLs) derive from blood infected in vitro by Epstein-Barr virus and were used in several genetic, transcriptomic and epigenomic studies. Although few changes were shown between LCL and blood genotypes (SNPs) validating their use in genetics, more were highlighted for other genomic features and/or in their transcriptome and epigenome. This could render them less appropriate for these studies, notably when blood DNA could still be available. Here we developed a simple, high-throughput and cost-effective real-time PCR approach allowing to distinguish blood from LCL DNA samples based on the presence of EBV relative load and rearranged T-cell receptors γ and ß. Our approach was able to achieve 98.5% sensitivity and 100% specificity on DNA of known origin (458 blood and 316 LCL DNA). It was further applied to 1957 DNA samples from the CEPH Aging cohort comprising DNA of uncertain origin, identifying 784 blood and 1016 LCL DNA. A subset of these DNA was further analyzed with an epigenetic clock indicating that DNA extracted from blood should be preferred to LCL for DNA methylation-based age prediction analysis. Our approach could thereby be a powerful tool to ascertain the origin of DNA in old collections prior to (epi)genomic studies.

A Genome Wide Association Study on plasma FV levels identified PLXDC2 as a new modifier of the coagulation process.

BACKGROUND: Factor V (FV) is a circulating protein primarily synthesized in the liver, and mainly present in plasma. It is a major component of the coagulation process. OBJECTIVE: To detect novel genetic loci participating to the regulation of FV plasma levels. METHODS: We conducted the first Genome Wide Association Study on FV plasma levels in a sample of 510 individuals and replicated the main findings in an independent sample of 1156 individuals. RESULTS: In addition to genetic variations at the F5 locus, we identified novel associations at the PLXDC2 locus, with the lead PLXDC2 rs927826 polymorphism explaining ~3.7% (P = 7.5 × 10-15 in the combined discovery and replication samples) of the variability of FV plasma levels. In silico transcriptomic analyses in various cell types confirmed that PLXDC2 expression is positively correlated to F5 expression. SiRNA experiments in human hepatocellular carcinoma cell line confirmed the role of PLXDC2 in modulating factor F5 gene expression, and revealed further influences on F2 and F10 expressions. CONCLUSION: Our study identified PLXDC2 as a new molecular player of the coagulation process.

Endothelial Epas1 Deficiency Is Sufficient To Promote Parietal Epithelial Cell Activation and FSGS in Experimental Hypertension.

FSGS, the most common primary glomerular disorder causing ESRD, is a complex disease that is only partially understood. Progressive sclerosis is a hallmark of FSGS, and genetic tracing studies have shown that parietal epithelial cells participate in the formation of sclerotic lesions. The loss of podocytes triggers a focal activation of parietal epithelial cells, which subsequently form cellular adhesions with the capillary tuft. However, in the absence of intrinsic podocyte alterations, the origin of the pathogenic signal that triggers parietal epithelial cell recruitment remains elusive. In this study, investigation of the role of the endothelial PAS domain-containing protein 1 (EPAS1), a regulatory α subunit of the hypoxia-inducible factor complex, during angiotensin II-induced hypertensive nephropathy provided novel insights into FSGS pathogenesis in the absence of a primary podocyte abnormality. We infused angiotensin II into endothelial-selective Epas1 knockout mice and their littermate controls. Although the groups presented with identical high BP, endothelial-specific Epas1 gene deletion accentuated albuminuria with severe podocyte lesions and recruitment of pathogenic parietal glomerular epithelial cells. These lesions and dysfunction of the glomerular filtration barrier were associated with FSGS in endothelial Epas1-deficient mice only. These results indicate that endothelial EPAS1 has a global protective role during glomerular hypertensive injuries without influencing the hypertensive effect of angiotensin II. Furthermore, these findings provide proof of principle that endothelial-derived signaling can trigger FSGS and illustrate the potential importance of the EPAS1 endothelial transcription factor in secondary FSGS.