Identification of Yeast and Human 5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAr) Transporters

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Improved V3 genotyping with duplicate PCR amplification for determining HIV-1 tropism.

International audienceOBJECTIVES: To determine whether genotyping of HIV-1 by duplicate PCR amplification of the region encoding the V3 loop is more sensitive than single PCR for detecting CXCR4-using viruses. PATIENTS AND METHODS: The V3 genotypes of the HIV-1 infecting 152 patients enrolled in the multicentre GenoTropism ANRS study were determined by all the participating laboratories using a single PCR and V3 bulk sequencing. In parallel, one laboratory determined the V3 genotype using duplicate PCR and bulk sequencing of pooled amplicons. HIV tropism was predicted with the geno2pheno10 algorithm. The phenotypes of all samples were determined with the Trofile assay and the Toulouse tropism test (TTT) recombinant virus assay. RESULTS: Geno2pheno10 was 56.8% sensitive and 75.9% specific when compared with the Trofile assay for detecting CXCR4-using viruses after a single PCR. Duplicate amplification and bulk sequencing of the pooled PCR amplicons increased the sensitivity to 68.2% and specificity to 79.6%. Geno2pheno10 was 64.1% sensitive and 77.0% specific when compared with the TTT assay for detecting CXCR4-using viruses after a single PCR. Duplicate amplification and sequencing of the pooled PCR amplicons increased sensitivity to 76.9% and specificity to 80.5%. CONCLUSIONS: The genotypic determination of HIV-1 tropism can be improved by duplicate amplifications and sequencing the pooled PCR products. This is a good compromise between improved sensitivity and reasonable cost for the genotype-based determination of tropism

Exome sequencing and pathogenicity-network analysis of five French families implicate mTOR signalling and autophagy in familial sarcoidosis

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Whole exome sequencing in three families segregating a pediatric case of sarcoidosis

International audienceAbstractBackgroundSarcoidosis (OMIM 181000) is a multi-systemic granulomatous disorder of unknown origin. Despite multiple genome-wide association (GWAS) studies, no major pathogenic pathways have been identified to date. To find out relevant sarcoidosis predisposing genes, we searched for de novo and recessive mutations in 3 young probands with sarcoidosis and their healthy parents using a whole-exome sequencing (WES) methodology.MethodsFrom the SARCFAM project based on a national network collecting familial cases of sarcoidosis, we selected three families (trios) in which a child, despite healthy parents, develop the disease before age 15 yr. Each trio was genotyped by WES (Illumina HiSEQ 2500) and we selected the gene variants segregating as 1) new mutations only occurring in affected children and 2) as recessive traits transmitted from each parents. The identified coding variants were compared between the three families. Allelic frequencies and in silico functional results were analyzed using ExAC, SIFT and Polyphenv2 databases. The clinical and genetic studies were registered by the ClinicalTrials.gov - Protocol Registration and Results System (PRS) (https://clinicaltrials.gov) receipt under the reference NCT02829853 and has been approved by the ethical committee (CPP LYON SUD EST – 2 – REF IRB 00009118 – September 21, 2016).ResultsWe identified 37 genes sharing coding variants occurring either as recessive mutations in at least 2 trios or de novo mutations in one of the three affected children. The genes were classified according to their potential roles in immunity related pathways: 9 to autophagy and intracellular trafficking, 6 to G-proteins regulation, 4 to T-cell activation, 4 to cell cycle and immune synapse, 2 to innate immunity. Ten of the 37 genes were studied in a bibliographic way to evaluate the functional link with sarcoidosis.ConclusionsWhole exome analysis of case-parent trios is useful for the identification of genes predisposing to complex genetic diseases as sarcoidosis. Our data identified 37 genes that could be putatively linked to a pediatric form of sarcoidosis in three trios. Our in-depth focus on 10 of these 37 genes may suggest that the formation of the characteristic lesion in sarcoidosis, granuloma, results from combined deficits in autophagy and intracellular trafficking (ex: Sec16A, AP5B1 and RREB1), G-proteins regulation (ex: OBSCN, CTTND2 and DNAH11), T-cell activation (ex: IDO2, IGSF3), mitosis and/or immune synapse (ex: SPICE1 and KNL1). The significance of these findings needs to be confirmed by functional tests on selected gene variants

Enhancing the learning of evolutionary anthropology skills by combining student‐active teaching with actual and virtual immersion of Master's students in fieldwork, laboratory practice, and dissemination

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Additional file 1: of Whole exome sequencing in three families segregating a pediatric case of sarcoidosis

Table S1. Recessive variants found in at least two affected children of different trios. Possibly pathogenic recessive variants (polymorphisms) found by whole-exome -sequencing in at least two affected children of the trios (T). Chr., chromosome; SNP, single nucleotide polymorphism; QUAL., a quality parameter measuring the probability p that the observation of the variant is due to chance (for ex: QUAL = n, p = 1/n). As detailed in the text, Alamut® Visual integrates missense variant pathogenicity prediction tools and in silico study of variants’ effect on RNA splicing, allowing the assessment of their potential impact on splice junctions and splicing regulatory sequences. Alamut® Visual helped us also to exclude well known mutations identified in recessive diseases for those genes which have been related to known genetic diseases (as shown in Table 3). (DOCX 23 kb

Additional file 4: of Whole exome sequencing in three families segregating a pediatric case of sarcoidosis

Figure S1. CADD scoring of prioritized variants versus other variants in the selected genes. (PDF 71 kb