Isolation of subtelomeric DNA sequences labelling sheep and goat chromosome ends

Two techniques that make it possible to isolate telomere DNA are presented, using sheep as an example. The first technique is based upon the screening of a sheep BAC library with PCR amplified DNA segments preserved from high-power laser beam irradiation. Twenty-three BACs hybridising to 13 subtelomeric regions in sheep and goats were obtained (out of 27 in the sheep complement), of which 13 recognised more than one region, telomeric or not. Twenty-three microsatellites were isolated from these BACs and 22 were genetically mapped on the sheep international genetic map, always consistently with the cytogenetical localisation in 17 cases out of 22. These results are discussed. The second technique is based upon the selective cloning of subtelomeric enriched DNA. Preliminary results were obtained by this approach

A Missense Mutation in PPP1R15B Causes a Syndrome Including Diabetes, Short Stature, and Microcephaly.

Dysregulated endoplasmic reticulum stress and phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) are associated with pancreatic β-cell failure and diabetes. Here, we report the first homozygous mutation in the PPP1R15B gene (also known as constitutive repressor of eIF2α phosphorylation [CReP]) encoding the regulatory subunit of an eIF2α-specific phosphatase in two siblings affected by a novel syndrome of diabetes of youth with short stature, intellectual disability, and microcephaly. The R658C mutation in PPP1R15B affects a conserved amino acid within the domain important for protein phosphatase 1 (PP1) binding. The R658C mutation decreases PP1 binding and eIF2α dephosphorylation and results in β-cell apoptosis. Our findings support the concept that dysregulated eIF2α phosphorylation, whether decreased by mutation of the kinase (EIF2AK3) in Wolcott-Rallison syndrome or increased by mutation of the phosphatase (PPP1R15B), is deleterious to β-cells and other secretory tissues, resulting in diabetes associated with multisystem abnormalities.This work was supported by the European Union 7th Framework Programme (project BetaBat), the Actions de Recherche Concertées de la Communauté Française, and Fonds National de la Recherche Scientifique (FNRS), Belgium, and by grants from the Agence Nationale pour la Recherche (ANR-09-GENO-021), the European Foundation for the Study of Diabetes/JDRF/Novo Nordisk, the Assistance Publique-Hôpitaux de Paris Programme Hospitalier de Recherche Clinique (DIAGENE), the GIS Maladies Rares, and the Wellcome Trust (084812/Z/08/Z). A.T.H. is a Wellcome Trust and National Institute for Health Research senior investigator, and D.R. is a Wellcome Trust Principal Research Fellow. B.A. was supported by an European Molecular Biology Organization Short-Term Fellowship and an FNRS-FRIA fellowship. M.I.-E. is a scientific collaborator of the FNRS. M.D. was supported by a doctoral fellowship from the Ministère de l’Education Nationale, de l’Enseignement Supérieur et de la Recherche, France.This is the author accepted manuscript. The final version is available from the American Diabetes Association via http://dx.doi.org/10.2337/db15-047

Rare coding variants in PLCG2, ABI3, and TREM2 implicate microglial-mediated innate immunity in Alzheimer's disease

We identified rare coding variants associated with Alzheimer’s disease (AD) in a 3-stage case-control study of 85,133 subjects. In stage 1, 34,174 samples were genotyped using a whole-exome microarray. In stage 2, we tested associated variants (P<1×10-4) in 35,962 independent samples using de novo genotyping and imputed genotypes. In stage 3, an additional 14,997 samples were used to test the most significant stage 2 associations (P<5×10-8) using imputed genotypes. We observed 3 novel genome-wide significant (GWS) AD associated non-synonymous variants; a protective variant in PLCG2 (rs72824905/p.P522R, P=5.38×10-10, OR=0.68, MAFcases=0.0059, MAFcontrols=0.0093), a risk variant in ABI3 (rs616338/p.S209F, P=4.56×10-10, OR=1.43, MAFcases=0.011, MAFcontrols=0.008), and a novel GWS variant in TREM2 (rs143332484/p.R62H, P=1.55×10-14, OR=1.67, MAFcases=0.0143, MAFcontrols=0.0089), a known AD susceptibility gene. These protein-coding changes are in genes highly expressed in microglia and highlight an immune-related protein-protein interaction network enriched for previously identified AD risk genes. These genetic findings provide additional evidence that the microglia-mediated innate immune response contributes directly to AD development

Single cell transcriptome sequencing of stimulated and frozen human peripheral blood mononuclear cells

Abstract Peripheral blood mononuclear cells (PBMCs) are blood cells that are a critical part of the immune system used to fight off infection, defending our bodies from harmful pathogens. In biomedical research, PBMCs are commonly used to study global immune response to disease outbreak and progression, pathogen infections, for vaccine development and a multitude of other clinical applications. Over the past few years, the revolution in single-cell RNA sequencing (scRNA-seq) has enabled an unbiased quantification of gene expression in thousands of individual cells, which provides a more efficient tool to decipher the immune system in human diseases. In this work, we generate scRNA-seq data from human PBMCs at high sequencing depth (>100,000 reads/cell) for more than 30,000 cells, in resting, stimulated, fresh and frozen conditions. The data generated can be used for benchmarking batch correction and data integration methods, and to study the effect of freezing-thawing cycles on the quality of immune cell populations and their transcriptomic profiles

Identification of biological pathways specific to phases preceding rheumatoid arthritis development through gene expression profiling

The etiopathogenesis of rheumatoid arthritis is partially understood; however, it is believed to result from a multi-step process. The immune onset followed by pre-clinical phases will eventually lead to the development of symptomatic disease. We aim at identifying differentially expressed genes in order to highlight pathways involved in the pre-clinical stages of rheumatoid arthritis development. The study population consisted of first-degree relatives of patients with rheumatoid arthritis, known to have an increased risk of developing disease as compared to the general population. Whole transcriptome analysis was performed in four groups: asymptomatic without autoantibodies or symptoms associated with possible rheumatoid arthritis (controls); having either clinically suspect arthralgias, undifferentiated arthritis or autoimmunity associated with RA (pre-clinical stages of RA: Pcs-RA); having subsequently developed classifiable RA (pre-RA); and early untreated rheumatoid arthritis patients (RA). Differentially expressed genes were determined, and enrichment analysis was performed. Functional enrichment analysis revealed 31 pathways significantly enriched in differentially expressed genes for Pcs-RA, pre-RA and RA compared to the controls. Osteoclast pathway is among the seven pathways specific for RA. In Pcs-RA and in pre-RA, several enriched pathways include TP53 gene connections, such as P53 and Wnt signalling pathways. Analysis of whole transcriptome for phenotypes related to rheumatoid arthritis allows highlighting which pathways are requested in the pre-clinical stages of disease development. After validation in replication studies, molecules belonging to some of these pathways could be used to identify new specific biomarkers for individuals with impending rheumatoid arthritis

Systematic analysis of TruSeq, SMARTer and SMARTer Ultra-Low RNA-seq kits for standard, low and ultra-low quantity samples.

International audienceHigh-throughput RNA-sequencing has become the gold standard method for whole-transcriptome gene expression analysis, and is widely used in numerous applications to study cell and tissue transcriptomes. It is also being increasingly used in a number of clinical applications, including expression profiling for diagnostics and alternative transcript detection. However, despite its many advantages, RNA sequencing can be challenging in some situations, for instance in cases of low input amounts or degraded RNA samples. Several protocols have been proposed to overcome these challenges, and many are available as commercial kits. In this study, we systematically test three recent commercial technologies for RNA-seq library preparation (TruSeq, SMARTer and SMARTer Ultra-Low) on human biological reference materials, using standard (1 mg), low (100 ng and 10 ng) and ultra-low (<1 ng) input amounts, and for mRNA and total RNA, stranded and unstranded. The results are analyzed using read quality and alignment metrics, gene detection and differential gene expression metrics. Overall, we show that the TruSeq kit performs well with an input amount of 100 ng, while the SMARTer kit shows decreased performance for inputs of 100 and 10 ng, and the SMARTer Ultra-Low kit performs relatively well for input amounts <1 ng. All the results are discussed in detail, and we provide guidelines for biologists for the selection of an RNA-seq library preparation kit

Isolation of subtelomeric DNA sequences labelling sheep and goat chromosome ends

Two techniques that make it possible to isolate telomere DNA are presented, using sheep as an example. The first technique is based upon the screening of a sheep BAC library with PCR amplified DNA segments preserved from high-power laser beam irradiation. Twenty-three BACs hybridising to 13 subtelomeric regions in sheep and goats were obtained (out of 27 in the sheep complement), of which 13 recognised more than one region, telomeric or not. Twenty-three microsatellites were isolated from these BACs and 22 were genetically mapped on the sheep international genetic map, always consistently with the cytogenetical localisation in 17 cases out of 22. These results are discussed. The second technique is based upon the selective cloning of subtelomeric enriched DNA. Preliminary results were obtained by this approach.Isolement de séquences d'ADN subtélomériques chez les ruminants. Deux techniques permettant d'isoler l'ADN télomérique sont présentées, le mouton servant d'exemple. La première technique est basée sur le criblage d'une banque de BACs ovins avec des segments d'ADN amplifiés par PCR préservés de l'irradiation par un laser à forte puissance. Vingt trois BACs s'hybridant à 13 régions sub-télomériques (parmi les 27 du génome ovin) ont été obtenus, dont treize reconnaissent plus qu'une région unique, télomérique ou non. Vingt-trois microsatellites ont été isolés à partir des BACs et 22 cartographiés génétiquement sur la carte ovine internationale, de façon cohérente avec la localisation cytogénétique dans 17 cas sur 22. Ces résultats sont discutés. La seconde technique est basée sur le clonage sélectif d'ADN enrichi en régions sub-télomériques. Des résultats préliminaires ont été obtenus par cette approche

Genetic investigation of four meiotic genes in women with premature ovarian failure

International audienceOBJECTIVE: The goal of this study was to determine whether mutations of meiotic genes, such as disrupted meiotic cDNA (DMC1), MutS homolog (MSH4), MSH5, and S. cerevisiae homolog (SPO11), were associated with premature ovarian failure (POF). DESIGN: Case-control study. METHODS: Blood sampling, karyotype, hormonal dosage, ultrasound, and ovarian biopsy were carried out on most patients. However, the main outcome measure was the sequencing of genomic DNA from peripheral blood samples of 41 women with POF and 36 fertile women (controls). RESULTS: A single heterozygous missense mutation, substitution of a cytosine residue with thymidine in exon 2 of MSH5, was found in two Caucasian women in whom POF developed at 18 and 36 years of age. This mutation resulted in replacement of a non-polar amino acid (proline) with a polar amino acid (serine) at position 29 (P29S). Neither 36 control women nor 39 other patients with POF possessed this genetic perturbation. Another POF patient of African origin showed a homozygous nucleotide change in the tenth of DMC1 gene that led to an alteration of the amino acid composition of the protein (M200V). CONCLUSIONS: The symptoms of infertility observed in the DMC1 homozygote mutation carrier and in both patients with a heterozygous substitution in exon 2 of the MSH5 gene provide indirect evidence of the role of genes involved in meiotic recombination in the regulation of ovarian function. MSH5 and DMC1 mutations may be one explanation for POF, albeit uncommon

Genotyping on blood and buccal cells using loop-mediated isothermal amplification in healthy humans

International audienceGenetic variations contribute to phenotypic individual vulnerabilities to sleep debt, particularly for five single nucleotide polymorphisms (SNPs). Loop-mediated isothermal amplification and melting curve analysis (LAMP-MC) is a recently developed method to characterize SNPs. The aim of present study was to evaluate the LAMP-MC method on blood and buccal cells for detection of five SNPs of interest in healthy humans. We first analyzed signals obtained from LAMP-MC method on 42 samples. Then we compared the results with those of referent TaqMan method. The LAMP-MC method produced specific melting curves for the five SNPs. A high concordance of genotyping results was observed between the two methods for rs5751876_ADORA2A, rs1800629_TNF-α, rs73598374_ADA and rs228697_PER3 in blood and saliva (Cohen’s kappa coefficient >0.80). A good agreement ( = 0.61) was observed for rs4680_COMT in blood only. LAMP-MC is a simple and reliable method to study genetic influences on health, sleep debt-related performance impairments and countermeasures