Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Progres de la biologie et avenir des productions animales

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In vitro studies of the long-range regulation of FOXL2 expression by epigenome editing

It has been shown that Forkhead boxL2 gene (FOXL2) is a master gene for female gonadal development. Firstly, it is involved in ovarian determination[1] and/or maintenance[2], and secondly it is necessary for ovarian follicle growth[3]. Interestingly, long-range deletions affecting FOXL2 expression have been reported in different species. In goats, a natural mutation called PIS for “Polled Intersex Syndrome” is responsible for female-to-male sex-reversal in homozygous XX PIS-/- mutant animals. This mutation corresponds to a 11.7kb homozygous deletion[4] lying at around 300 kb upstream of FOXL2 gene[5,6]. The PIS mutation is responsible for FOXL2 expression silencing and the sex-reversal[1]. In humans, various heterozygous long-range deletions have been shown to be responsible for decreasing FOXL2 expression, resulting in Blepharophimosis Ptosis Epicanthus inversus Syndrome[7]. To date, the Smallest Region of deletion Overlap (SRO) described in human patients, is a 7.4 kb region, encompassing the long non-coding RNA PISRT1 that was shown, in the goat species, to be closely regulated with FOXL2. All this data suggest that this distant region located at around 300 kb of FOXL2 may contain crucial regulatory elements for its expression. Analyzes of the epigenetic state of this region in human ovary (ChIP-Seq, ENCODE data), or of its counterpart in mouse ovary (ChIP-qPCR, personal data) revealed H3K27ac enrichment, which is an epigenetic mark preferentially observed on active gene enhancer or promoter. In order to prove that this region contains crucial enhancer(s), but also to demonstrate that long-range epigenetic modifications are able to control FOXL2 expression, we developed a program of epigenome editing. The CRISPR/dCas9 technology has been previously deflected to induce epigenetic modifications at specific loci[8]. Thus we designed several guide RNAs, coupled to a CRISPR/dCas9-p300 which is capable to acetylate H3K27. In a cellular model, different regions will be targeted, focusing on the most conserved elements of the SRO counterpart

Multicentre external quality control evaluating universal 16S polymerase chain reaction (PCR) in the diagnosis of bone and joint infections

International audienceObjectives:During a multicentrer French Study performed to assess the contribution of 16S PCR in the diagnosis of prosthesis osteoarticular infections, 300 patients were included from December 2009 to April 2012. An external quality control (QC) was considered essential due to the diversity of molecular equipment for each laboratory. Three sets were held, for each including 4 bacterial DNA extracts (E) and 4 crushed osteoarticular deep samples.Methods:Extraction: 0,2 ml of pretreated S (PK, 37 °C, 3h) with elution in 0.1 ml. Four laboratories used Qiagen manual extraction and 3 others used automated extraction 1 MagnaPur Roche, 1 Easy Mag, BioMérieux and 1 iPrep, Invitrogen. Real time 16S PCR with SybrGreen was performed with degenerate primers amplifying 658pb followed by sequencing. In the 7 centers, PCR thermocyclers used were 2 MX 3000p Agilent, 1 Roche Light Cycler, 1 Abi 7900 and 1 Applied Step one plus, 1 Smartcycler Cepheid,1 Biorad Chrono 4 and for PCR premix, Takara premix exTaq, Applied, Promega and Biorad were used.Results: 168 QC were sent and 160 responses were analyzed (1 laboratory did not participate in the first QC series). Expected results were obtained in 97.5% for Extracts and 95% for Samples. Sensitivity and Specificity were 100 and 90% for E and 93.3 and 100% for S. Ct standard deviations (SD) for E were from 1 to 9 while SD was 2 to 7 for S. For centers using the same premix, the results were closer, SD 0.5 to 1.5 (3 Ct gap max). For S, no influence of extraction system was observed.Conclusion:If extraction system had no influence, premix seems to be the most important factor influencing the value of threshold. This QC demonstrates the possibility to obtain good and homogeneous results by using the same 16S PCR in laboratories with different equipments for molecular bone and joint infection diagnosis

Evaluation of 16S rRNA gene PCR sensitivity and specificity for diagnosis of prosthetic joint infection: a prospective multicenter cross-sectional study.

International audienceThere is no standard method for the diagnosis of prosthetic joint infection (PJI). The contribution of 16S rRNA gene PCR sequencing on a routine basis remains to be defined. We performed a prospective multicenter study to assess the contributions of 16S rRNA gene assays in PJI diagnosis. Over a 2-year period, all patients suspected to have PJIs and a few uninfected patients undergoing primary arthroplasty (control group) were included. Five perioperative samples per patient were collected for culture and 16S rRNA gene PCR sequencing and one for histological examination. Three multicenter quality control assays were performed with both DNA extracts and crushed samples. The diagnosis of PJI was based on clinical, bacteriological, and histological criteria, according to Infectious Diseases Society of America guidelines. A molecular diagnosis was modeled on the bacteriological criterion (≥ 1 positive sample for strict pathogens and ≥ 2 for commensal skin flora). Molecular data were analyzed according to the diagnosis of PJI. Between December 2010 and March 2012, 264 suspected cases of PJI and 35 control cases were included. PJI was confirmed in 215/264 suspected cases, 192 (89%) with a bacteriological criterion. The PJIs were monomicrobial (163 cases [85%]; staphylococci, n = 108; streptococci, n = 22; Gram-negative bacilli, n = 16; anaerobes, n = 13; others, n = 4) or polymicrobial (29 cases [15%]). The molecular diagnosis was positive in 151/215 confirmed cases of PJI (143 cases with bacteriological PJI documentation and 8 treated cases without bacteriological documentation) and in 2/49 cases without confirmed PJI (sensitivity, 73.3%; specificity, 95.5%). The 16S rRNA gene PCR assay showed a lack of sensitivity in the diagnosis of PJI on a multicenter routine basis

Transgenic short-QT syndrome 1 rabbits mimic the human disease phenotype with QT/action potential duration shortening in the atria and ventricles and increased ventricular tachycardia/ventricular fibrillation inducibility

International audienceShort-QT syndrome 1 (SQT1) is an inherited channelopathy with accelerated repolarization due to gain-of-function in HERG/IKr. Patients develop atrial fibrillation, ventricular tachycardia (VT), and sudden cardiac death with pronounced inter-individual variability in phenotype. We generated and characterized transgenic SQT1 rabbits and investigated electrical remodelling. Transgenic rabbits were generated by oocyte-microinjection of β-myosin-heavy-chain-promoter-KCNH2/HERG-N588K constructs. Short-QT syndrome 1 and wild type (WT) littermates were subjected to in vivo ECG, electrophysiological studies, magnetic resonance imaging, and ex vivo action potential (AP) measurements. Electrical remodelling was assessed using patch clamp, real-time PCR, and western blot. We generated three SQT1 founders. QT interval was shorter and QT/RR slope was shallower in SQT1 than in WT (QT, 147.8 ± 2 ms vs. 166.4 ± 3, P < 0.0001). Atrial and ventricular refractoriness and AP duration were shortened in SQT1 (vAPD90, 118.6 ± 5 ms vs. 154.4 ± 2, P < 0.0001). Ventricular tachycardia/fibrillation (VT/VF) inducibility was increased in SQT1. Systolic function was unaltered but diastolic relaxation was enhanced in SQT1. IKr-steady was increased with impaired inactivation in SQT1, while IKr-tail was reduced. Quinidine prolonged/normalized QT and action potential duration (APD) in SQT1 rabbits by reducing IKr. Diverse electrical remodelling was observed: in SQT1, IK1 was decreased-partially reversing the phenotype-while a small increase in IKs may partly contribute to an accentuation of the phenotype. Short-QT syndrome 1 rabbits mimic the human disease phenotype on all levels with shortened QT/APD and increased VT/VF-inducibility and show similar beneficial responses to quinidine, indicating their value for elucidation of arrhythmogenic mechanisms and identification of novel anti-arrhythmic strategies