High-resolution hepatitis C virus subtyping using NS5B deep sequencing and phylogeny, an alternative to current methods

HepatitisCvirus(HCV)is classified into seven major genotypesand67 subtypes. Recent studies haveshownthat inHCVgenotype 1-infected patients, response rates to regimens containingdirect-acting antivirals(DAAs)are subtype dependent. Currently available genotypingmethods have limited subtyping accuracy.Wehave evaluated theperformanceof adeep-sequencing-basedHCVsubtyping assay, developed for the 454/GS-Junior platform, in comparisonwith thoseof two commercial assays (VersantHCVgenotype 2.0andAbbott Real-timeHCVGenotype II)andusingdirectNS5Bsequencing as a gold standard (direct sequencing), in 114 clinical specimenspreviously tested by first-generation hybridization assay (82 genotype 1and32 with uninterpretable results). Phylogenetic analysis of deep-sequencing reads matched subtype 1 callingbypopulation Sanger sequencing(69%1b,31%1a) in 81 specimensandidentified amixed-subtype infection (1b/3a/1a) in one sample. Similarly,amongthe 32previously indeterminate specimens, identical genotypeandsubtype results were obtained by directanddeep sequencing in all but four samples with dual infection. In contrast, both VersantHCVGenotype 2.0andAbbott Real-timeHCVGenotype II failed subtype 1 calling in 13 (16%) samples eachandwere unable to identify theHCVgenotype and/or subtype inmore than half of the nongenotype 1 samples.Weconcluded that deep sequencing ismore efficient forHCVsubtyping than currently available methodsandallows qualitative identificationofmixed infectionsandmay bemorehelpfulwith respect to informing treatment strategies withnewDAA-containing regimens across allHCVsubtypesThis study has been supported by CDTI (Centro para el Desarrollo Tecnológico Industrial), Spanish Ministry of Economics and Competitiveness (MINECO), IDI-20110115; MINECO projects SAF 2009-10403; and also by the Spanish Ministry of Health, Instituto de Salud Carlos III (FIS) projects PI10/01505, PI12/01893, and PI13/00456. CIBERehd is funded by the Instituto de Salud Carlos III, Madrid, Spain. Work at CBMSO was supported by grant MINECO-BFU2011-23604, FIPSE, and Fundación Ramón Areces. X. Forns received unrestricted grant support from Roche and has acted as advisor for MSD, Gilead, and Abbvie. M. Alvarez-Tejado, J. Gregori, and J. M. Muñoz work in Roche Diagnostic

Role of the von Hippel-Lindau tumor suppressor gene in the formation of beta1 integrin fibrillar adhesions

8 páginas, 7 figuras -- PAGS nros. 2929-2936The von Hippel-Lindau tumor suppressor gene (VHL) is absent or inactivated in the VHLcancer syndrome and in most sporadic renal cancers. VHL is requiredfor the assembly of a proper extracellular fibronectin matrix, although the exact mechanism remains unknown. In this report, we demonstrate that 786-O renal cancer cells are unable to organize an adequate matrix even in the presence of an excess of exogenous fibronectin. Because the formation of integrin fibrillar adhesions plays a pivotal role in the organization of extracellular fibronectin, we next examined the expression and subcellular distribution of integrins in VHL(−) cells and their wild-type VHL stably transfected counterparts. The levels of β1 and αv integrins were increased in VHL(−) cells when compared with VHL(+) transfectants. Early after plating, both VHL(+) and VHL(−) cells were capable of assembling classic “patch-like” αv focal contacts. As the culture advanced and cells became confluent, αv integrins partly relocated to the intercellular junctions in VHL(+) transfectants, which then developed large β1 fibrillar-type adhesions and anchored firmly to the substrate. In contrast, confluent VHL(−) cells were unable to assemble β1 fibrillar adhesions, and αv focal contacts remained unchanged at all stages of the culture. Exogenous activation of β1 integrins with either divalent cations or activating antibodies partly restored the capability of VHL(−) cells to assemble β1 fibrillar adhesions and fibronectin fibers. Finally, pulse-chase studies of metabolically labeled 786-O cells revealed that the maturation of the common β1-integrin chain was delayed in VHL(−) cells when compared with VHL(+) cells. Our results show that VHL is an important regulator of integrins and is essential for the formation of β1 fibrillar adhesions. These findings help to explain the abnormal extracellular matrix organization and increased motility of VHL(−) renal cancer cellsThis work was supported by Grants SAF2001/0215 and FEDER-2FD971870 from the Ministerio de Educación y Cultura. Miguel A. Esteban-Barragán, Miguel Álvarez-Tejado, and M. Dolores Gutiérrez were supported by fellowships from Comunidad Autónoma de MadridPeer reviewe

Reproducibility: Comparison of mutant abundance at each polymorphic site for a single amplicon replicated on three platforms, located in two laboratories when considering all CH>0.5%.

<p>Reproducibility: Comparison of mutant abundance at each polymorphic site for a single amplicon replicated on three platforms, located in two laboratories when considering all CH>0.5%.</p

PCR and UDPS errors.

<p>Experiment with clones. Point mutations and haplotypes found when cutting consensus point mutations (analysis by columns) or consensus haplotypes (analysis by rows) at 0.25% abundance (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083361#pone-0083361-t001" target="_blank"><b>Table 1a</b></a>) at a cut-off of 0.50% abundance (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083361#pone-0083361-t001" target="_blank"><b>Table 1b</b></a>). Err indicates the number of false segregating sites, and OK the number of true segregating sites.</p

Number of reads per sample and strand at each data treatment step, and haplotype distribution at a consensus haplotype abundance >0.5%.

<p>Number of reads per sample and strand at each data treatment step, and haplotype distribution at a consensus haplotype abundance >0.5%.</p

Reproducibility: Measures of quasispecies complexity for ten amplicons replicated on GS-FLX and GS-FLX+ located in two different laboratories.

<p>Data from CH>0.5%.</p><p>Hpl haplotypes, Eta number of mutations, S number of polymorphic sites, Mf mutation frequency, Sn normalized Shannon entropy, Pi nucleotide diversity.</p

UDPS data treatment workflow to obtain error-free HCV haplotypes as a sampling of the actual HCV viral quasispecies.

<p>UDPS data treatment workflow to obtain error-free HCV haplotypes as a sampling of the actual HCV viral quasispecies.</p

Reproducibility: Quasispecies complexity measures for a single amplicon replicated on three platforms, located in two laboratories.

<p>Hpl stand for number of haplotypes, Eta for number of mutations, S for number of polymorphic sites, Mf for mutation frequency, Sn for normalized Shannon entropy and Pi for nucleotide diversity.</p

267 Spanish Exomes Reveal Population-Specific Differences in Disease-Related Genetic Variation.

Recent results from large-scale genomic projects suggest that allele frequencies, which are highly relevant for medical purposes, differ considerably across different populations. The need for a detailed catalog of local variability motivated the whole-exome sequencing of 267 unrelated individuals, representative of the healthy Spanish population. Like in other studies, a considerable number of rare variants were found (almost one-third of the described variants). There were also relevant differences in allelic frequencies in polymorphic variants, including ∼10,000 polymorphisms private to the Spanish population. The allelic frequencies of variants conferring susceptibility to complex diseases (including cancer, schizophrenia, Alzheimer disease, type 2 diabetes, and other pathologies) were overall similar to those of other populations. However, the trend is the opposite for variants linked to Mendelian and rare diseases (including several retinal degenerative dystrophies and cardiomyopathies) that show marked frequency differences between populations. Interestingly, a correspondence between differences in allelic frequencies and disease prevalence was found, highlighting the relevance of frequency differences in disease risk. These differences are also observed in variants that disrupt known drug binding sites, suggesting an important role for local variability in population-specific drug resistances or adverse effects. We have made the Spanish population variant server web page that contains population frequency information for the complete list of 170,888 variant positions we found publicly available (http://spv.babelomics.org/), We show that it if fundamental to determine population-specific variant frequencies to distinguish real disease associations from population-specific polymorphisms