Systemic and intrinsic functions of ATRX in glial cell fate and CNS myelination in male mice.

Myelin, an extension of the oligodendrocyte plasma membrane, wraps around axons to facilitate nerve conduction. Myelination is compromised in ATR-X intellectual disability syndrome patients, but the causes are unknown. We show that loss of ATRX leads to myelination deficits in male mice that are partially rectified upon systemic thyroxine administration. Targeted ATRX inactivation in either neurons or oligodendrocyte progenitor cells (OPCs) reveals OPC-intrinsic effects on myelination. OPCs lacking ATRX fail to differentiate along the oligodendrocyte lineage and acquire a more plastic state that favors astrocytic differentiation in vitro and in vivo. ATRX chromatin occupancy in OPCs greatly overlaps with that of the chromatin remodelers CHD7 and CHD8 as well as H3K27Ac, a mark of active enhancers. Overall, our data indicate that ATRX regulates the onset of myelination systemically via thyroxine, and by promoting OPC differentiation and suppressing astrogliogenesis. These functions of ATRX identified in mice could explain white matter pathogenesis observed in ATR-X syndrome patients.

Face mask and protective eyewear-associated headache among healthcare workers during the COVID-19 pandemic.

OBJECTIVE: The objective of this study was to investigate the prevalence, clinical features, and factors related to personal protective-associated headaches. METHODS: We conducted a cross-sectional study among healthcare workers using an online questionnaire. RESULTS: We surveyed 305 participants. The N95 face-mask was the most used device by 93%. Of 305 respondents, 206 experienced headaches while wearing protective equipment; 36.06% suffered from a headache disorder before the pandemic. The prevalence of de novo headache was 39.01%. Gender, age, or exposure to coronavirus disease were not determining factors to develop headache. Headache intensity was higher in front-line healthcare workers and was correlated (r = 0.728) with the time wearing protective equipment. The more days per month the participants wore personal protective equipment the shorter the time to headache onset after donning equipment. CONCLUSION: Our study confirms the relationship between frequent and prolonged use of protection devices with headaches and reaffirms the implication of external pressure as a primary mechanism.

OBJETIVO: Investigar la prevalencia, las características clínicas y los factores relacionados con las cefaleas asociadas al equipo de protección personal. MÉTODOS: Realizamos un estudio transversal entre trabajadores de la salud por medio de un cuestionario en línea. RESULTADOS: Encuestamos a 305 participantes. La mascarilla N95 fue el dispositivo más utilizado opor 93%. Del total de encuestados, 206 experimentaron cefalea mientras usaban el equipo de protección; el 36.06% padecía algun trastorno cefalálgico antes del inicio de la pandemia. La prevalencia de cefalea de novo fue del 39.01%. El género, la edad o la exposición a la enfermedad por coronavirus no fueron factores determinantes para desarrollar cefalea. La cefalea fue de mayor intensidad en los trabajadores de primera línea y se correlacionó (r = 0.728) con el tiempo que se uso el equipo de protección personal. Mientras más días por mes los participantes usaron el equipo de protección personal menor fue el tiempo de inicio de la cefalea tras la colocación del equipo cada vez. CONCLUSIONES: Nuestro estudio confirma la asociación del uso frecuente y prolongado de dispositivos de protección con el desarrollo de cefalea y reafirma la implicación de la compresión externa como mecanismo primario.

Karyon: a computational framework for the diagnosis of hybrids, aneuploids, and other nonstandard architectures in genome assemblies.

BACKGROUND: Recent technological developments have made genome sequencing and assembly highly accessible and widely used. However, the presence in sequenced organisms of certain genomic features such as high heterozygosity, polyploidy, aneuploidy, heterokaryosis, or extreme compositional biases can challenge current standard assembly procedures and result in highly fragmented assemblies. Hence, we hypothesized that genome databases must contain a nonnegligible fraction of low-quality assemblies that result from such type of intrinsic genomic factors. FINDINGS: Here we present Karyon, a Python-based toolkit that uses raw sequencing data and de novo genome assembly to assess several parameters and generate informative plots to assist in the identification of nonchanonical genomic traits. Karyon includes automated de novo genome assembly and variant calling pipelines. We tested Karyon by diagnosing 35 highly fragmented publicly available assemblies from 19 different Mucorales (Fungi) species. CONCLUSIONS: Our results show that 10 (28.57%) of the assemblies presented signs of unusual genomic configurations, suggesting that these are common, at least for some lineages within the Fungi.

Selective isolation of mouse glial nuclei optimized for reliable downstream omics analyses.

BACKGROUND: Isolation of cell types of interest from the brain for molecular applications presents several challenges, including cellular damage during tissue dissociation or enrichment procedures, and low cell number in the tissue in some cases. Techniques have been developed to enrich distinct cell populations using immunopanning or fluorescence activated cell/nuclei sorting. However, these techniques often involve fixation, immunolabeling and DNA staining steps, which could potentially influence downstream omics applications. NEW METHOD: Taking advantage of readily available genetically modified mice with fluorescent-tagged nuclei, we describe a technique for the purification of cell-type specific brain nuclei, optimized to decrease sample preparation time and to limit potential artefacts for downstream omics applications. We demonstrate the applicability of this approach for the purification of glial cell nuclei and show that the resulting cell-type specific nuclei obtained can be used effectively for omics applications, including ATAC-seq and RNA-seq. RESULTS: We demonstrate excellent enrichment of fluorescently-tagged glial nuclei, yielding high quality RNA and chromatin. We identify several critical steps during nuclei isolation that help limit nuclei rupture and clumping, including quick homogenization, dilution before filtration and loosening of the pellet before resuspension, thus improving yield. Sorting of fluorescent nuclei can be achieved without fixation, antibody labelling, or DAPI staining, reducing potential artifactual results in RNA-seq and ATAC-seq analyses. We show that reproducible glial cell type-specific profiles can be obtained in transcriptomic and chromatin accessibility assays using this rapid protocol. COMPARISON WITH EXISTING METHODS: Our method allows for rapid enrichment of glial nuclei populations from the mouse brain with minimal processing steps, while still providing high quality RNA and chromatin required for reliable omics analyses. CONCLUSIONS: We provide a reproducible method to obtain nucleic material from glial cells in the mouse brain with a quick and limited sample preparation.

Massive gene presence-absence variation shapes an open pan-genome in the Mediterranean mussel.

BACKGROUND: The Mediterranean mussel Mytilus galloprovincialis is an ecologically and economically relevant edible marine bivalve, highly invasive and resilient to biotic and abiotic stressors causing recurrent massive mortalities in other bivalves. Although these traits have been recently linked with the maintenance of a high genetic variation within natural populations, the factors underlying the evolutionary success of this species remain unclear. RESULTS: Here, after the assembly of a 1.28-Gb reference genome and the resequencing of 14 individuals from two independent populations, we reveal a complex pan-genomic architecture in M. galloprovincialis, with a core set of 45,000 genes plus a strikingly high number of dispensable genes (20,000) subject to presence-absence variation, which may be entirely missing in several individuals. We show that dispensable genes are associated with hemizygous genomic regions affected by structural variants, which overall account for nearly 580 Mb of DNA sequence not included in the reference genome assembly. As such, this is the first study to report the widespread occurrence of gene presence-absence variation at a whole-genome scale in the animal kingdom. CONCLUSIONS: Dispensable genes usually belong to young and recently expanded gene families enriched in survival functions, which might be the key to explain the resilience and invasiveness of this species. This unique pan-genome architecture is characterized by dispensable genes in accessory genomic regions that exceed by orders of magnitude those observed in other metazoans, including humans, and closely mirror the open pan-genomes found in prokaryotes and in a few non-metazoan eukaryotes.

An estimate of the number of white sharks Carcharodon carcharias interacting with ecotourism in Guadalupe Island.

The aim of the present study is to provide an estimate of the number of white sharks Carcharodon carcharias that seasonally interact with ecotourism boats in Guadalupe Island using Schnabel's mark-recapture method and 6316 records of white sharks during 2012-2014. The results of the estimation highlight an abundance of 78 white sharks 95% C.I. (62.1, 105.6) interacting with ecotourism. The regulations regarding the number of tourists, boats and the monitoring of white sharks should be assessed to improve management decisions regarding the conservation and sustainable use of this threatened species.

Pulmonary Neuroendocrine Tumor Presenting as a Left Pleural Effusion.

The presence of a symptomatic and recurrent unilateral pleural effusion should alert physicians to consider thoracentesis with mindful use of biomarkers not only for therapeutic purposes, but also for diagnosis of both benign and malignant etiologies.

Fungal evolution: cellular, genomic and metabolic complexity.

The question of how phenotypic and genomic complexity are inter-related and how they are shaped through evolution is a central question in biology that historically has been approached from the perspective of animals and plants. In recent years, however, fungi have emerged as a promising alternative system to address such questions. Key to their ecological success, fungi present a broad and diverse range of phenotypic traits. Fungal cells can adopt many different shapes, often within a single species, providing them with great adaptive potential. Fungal cellular organizations span from unicellular forms to complex, macroscopic multicellularity, with multiple transitions to higher or lower levels of cellular complexity occurring throughout the evolutionary history of fungi. Similarly, fungal genomes are very diverse in their architecture. Deep changes in genome organization can occur very quickly, and these phenomena are known to mediate rapid adaptations to environmental changes. Finally, the biochemical complexity of fungi is huge, particularly with regard to their secondary metabolites, chemical products that mediate many aspects of fungal biology, including ecological interactions. Herein, we explore how the interplay of these cellular, genomic and metabolic traits mediates the emergence of complex phenotypes, and how this complexity is shaped throughout the evolutionary history of Fungi.

Fungal evolution: diversity, taxonomy and phylogeny of the Fungi.

The fungal kingdom comprises a hyperdiverse clade of heterotrophic eukaryotes characterized by the presence of a chitinous cell wall, the loss of phagotrophic capabilities and cell organizations that range from completely unicellular monopolar organisms to highly complex syncitial filaments that may form macroscopic structures. Fungi emerged as a 'Third Kingdom', embracing organisms that were outside the classical dichotomy of animals versus vegetals. The taxonomy of this group has a turbulent history that is only now starting to be settled with the advent of genomics and phylogenomics. We here review the current status of the phylogeny and taxonomy of fungi, providing an overview of the main defined groups. Based on current knowledge, nine phylum-level clades can be defined: Opisthosporidia, Chytridiomycota, Neocallimastigomycota, Blastocladiomycota, Zoopagomycota, Mucoromycota, Glomeromycota, Basidiomycota and Ascomycota. For each group, we discuss their main traits and their diversity, focusing on the evolutionary relationships among the main fungal clades. We also explore the diversity and phylogeny of several groups of uncertain affinities and the main phylogenetic and taxonomical controversies and hypotheses in the field.

Fungal evolution: major ecological adaptations and evolutionary transitions.

Fungi are a highly diverse group of heterotrophic eukaryotes characterized by the absence of phagotrophy and the presence of a chitinous cell wall. While unicellular fungi are far from rare, part of the evolutionary success of the group resides in their ability to grow indefinitely as a cylindrical multinucleated cell (hypha). Armed with these morphological traits and with an extremely high metabolical diversity, fungi have conquered numerous ecological niches and have shaped a whole world of interactions with other living organisms. Herein we survey the main evolutionary and ecological processes that have guided fungal diversity. We will first review the ecology and evolution of the zoosporic lineages and the process of terrestrialization, as one of the major evolutionary transitions in this kingdom. Several plausible scenarios have been proposed for fungal terrestralization and we here propose a new scenario, which considers icy environments as a transitory niche between water and emerged land. We then focus on exploring the main ecological relationships of Fungi with other organisms (other fungi, protozoans, animals and plants), as well as the origin of adaptations to certain specialized ecological niches within the group (lichens, black fungi and yeasts). Throughout this review we use an evolutionary and comparative-genomics perspective to understand fungal ecological diversity. Finally, we highlight the importance of genome-enabled inferences to envision plausible narratives and scenarios for important transitions.

aaquetzalli is required for epithelial cell polarity and neural tissue formation in Drosophila.

Morphogenetic movements during embryogenesis require dynamic changes in epithelial cell polarity and cytoskeletal reorganization. Such changes involve, among others, rearrangements of cell-cell contacts and protein traffic. In Drosophila melanogaster, neuroblast delamination during early neurogenesis is a well-characterized process requiring a polarized neuroepithelium, regulated by the Notch signaling pathway. Maintenance of epithelial cell polarity ensues proper Notch pathway activation during neurogenesis. We characterize here aaquetzalli (aqz), a gene whose mutations affect cell polarity and nervous system specification. The aqz locus encodes a protein that harbors a domain with significant homology to a proline-rich conserved domain of nuclear receptor co-activators. aqz expression occurs at all stages of the fly life cycle, and is dynamic. aqz mutants are lethal, showing a disruption of cell polarity during embryonic ventral neuroepithelium differentiation resulting in loss of epithelial integrity and mislocalization of membrane proteins (shown by mislocalization of Crumbs, DE-Cadherin, and Delta). As a consequence, aqz mutant embryos with compromised apical-basal cell polarity develop spotty changes of neuronal and epithelial numbers of cells.

Genome Sequence of the Brown Rot Fungal Pathogen Monilinia laxa.

Monilinia laxa (phylum Ascomycota) is a plant pathogen responsible for the brown rot blossom blight disease in stone fruit trees of the Rosaceae family, such as apricots. We report here the genome sequence of strain 8L of this species, which was assembled into 618 scaffolds, having a total size of 40.799 Mb and encoding 9,567 unique protein-coding genes.

Partial characterization of chayotextle starch-based films added with ascorbic acid encapsulated in resistant starch.

Chayotextle starch was modified by subjecting it to a dual treatment with acid and heating-cooling cycles. This caused a decrease in the content of amylose, which showed values of 30.22%, 4.80%, 3.27% and 3.57% for native chayotextle starch (NCS), starch modified by acid hydrolysis (CMS), and CMS with one (CMS1AC) and three autoclave cycles (CMS3AC), respectively. The percentage of crystallinity showed an increase of 36.9%-62% for NCS and CMS3AC. The highest content of resistant starch (RS) was observed in CMS3AC (37.05%). The microcapsules were made with CMS3AC due to its higher RS content; the total content of ascorbic acid of the microcapsules was 82.3%. The addition of different concentrations of CMS3AC microcapsules (0%, 2.5%, 6.255% and 12.5%) to chayotextle starch-based films (CSF) increased their tensile strength and elastic modulus. The content of ascorbic acid and RS in CSF was ranged from 0% to 59.4% and from 4.84% to 37.05% in the control film and in the film mixed with CMS3AC microcapsules, respectively. Water vapor permeability (WVP) values decreased with increasing concentrations of microcapsules in the films. Microscopy observations showed that higher concentrations of microcapsules caused agglomerations due their poor distribution in the matrix of the films.

Evolutionary genomics of yeast pathogens in the Saccharomycotina.

Saccharomycotina comprises a diverse group of yeasts that includes numerous species of industrial or clinical relevance. Opportunistic pathogens within this clade are often assigned to the genus Candida but belong to phylogenetically distant lineages that also comprise non-pathogenic species. This indicates that the ability to infect humans has evolved independently several times among Saccharomycotina. Although the mechanisms of infection of the main groups of Candida pathogens are starting to be unveiled, we still lack sufficient understanding of the evolutionary paths that led to a virulent phenotype in each of the pathogenic lineages. Deciphering what genomic changes underlie the evolutionary emergence of a virulence trait will not only aid the discovery of novel virulence mechanisms but it will also provide valuable information to understand how new pathogens emerge, and what clades may pose a future danger. Here we review recent comparative genomics efforts that have revealed possible evolutionary paths to pathogenesis in different lineages, focusing on the main three agents of candidiasis worldwide: Candida albicans, C. parapsilosis and C. glabrata We will discuss what genomic traits may facilitate the emergence of virulence, and focus on two different genome evolution mechanisms able to generate drastic phenotypic changes and which have been associated to the emergence of virulence: gene family expansion and interspecies hybridization.

Chitosan enhances parasitism of Meloidogyne javanica eggs by the nematophagous fungus Pochonia chlamydosporia.

Pochonia chlamydosporia (Pc), a nematophagous fungus and root endophyte, uses appressoria and extracellular enzymes, principally proteases, to infect the eggs of plant parasitic nematodes (PPN). Unlike other fungi, Pc is resistant to chitosan, a deacetylated form of chitin, used in agriculture as a biopesticide to control plant pathogens. In the present work, we show that chitosan increases Meloidogyne javanica egg parasitism by P. chlamydosporia. Using antibodies specific to the Pc enzymes VCP1 (a subtilisin), and SCP1 (a serine carboxypeptidase), we demonstrate chitosan elicitation of the fungal proteases during the parasitic process. Chitosan increases VCP1 immuno-labelling in the cell wall of Pc conidia, hyphal tips of germinating spores, and in appressoria on infected M. javanica eggs. These results support the role of proteases in egg parasitism by the fungus and their activation by chitosan. Phylogenetic analysis of the Pc genome reveals a large diversity of subtilisins (S8) and serine carboxypeptidases (S10). The VCP1 group in the S8 tree shows evidence of gene duplication indicating recent adaptations to nutrient sources. Our results demonstrate that chitosan enhances Pc infectivity of nematode eggs through increased proteolytic activities and appressoria formation and might be used to improve the efficacy of M. javanica biocontrol.

Widespread Inter- and Intra-Domain Horizontal Gene Transfer of d-Amino Acid Metabolism Enzymes in Eukaryotes.

Analysis of the growing number of available fully-sequenced genomes has shown that Horizontal Gene Transfer (HGT) in eukaryotes is more common than previously thought. It has been proposed that genes with certain functions may be more prone to HGT than others, but we still have a very poor understanding of the selective forces driving eukaryotic HGT. Recent work uncovered that d-amino acid racemases have been commonly transferred from bacteria to fungi, but their role in the receiving organisms is currently unknown. Here, we set out to assess whether d-amino acid racemases are commonly transferred to and between eukaryotic groups. For this we performed a global survey that used a novel automated phylogeny-based HGT-detection algorithm (Abaccus). Our results revealed that at least 7.0% of the total eukaryotic racemase repertoire is the result of inter- or intra-domain HGT. These transfers are significantly enriched in plant-associated fungi. For these, we hypothesize a possible role for the acquired racemases allowing to exploit minoritary nitrogen sources in plant biomass, a nitrogen-poor environment. Finally, we performed experiments on a transferred aspartate-glutamate racemase in the fungal human pathogen Candida glabrata, which however revealed no obvious biological role.

Complicaciones de la canalización venosa central con técnica ecoguiada y convencional en el servicio de urgencias del Hospital de San José, Bogotá, Colombia / Complications of the central venous catheter with ecoguide and conventional technique in emergency department of Hospital de San Jose, Bogota, Colombia

Introducción: la utilización de la ultrasonografía como técnica de elección para la colocación de catéteres venosos centrales durante situaciones de emergencia aumenta la seguridad del paciente al presentar baja frecuencia de complicaciones, menor exposición a irradiación e incremento de la calidad de atención. Objetivo: comparar las complicaciones mecánicas de la colocación de catéter venoso central por la técnica guiada por ultrasonografía y la convencional; realizada por residentesde la especialización en Medicina de Urgencias del Hospital de San José (Bogotá,Colombia). Materiales y métodos: estudio descriptivo de dos cohortes en pacientes mayores de 15 años que ingresaron al servicio de urgencias del Hospital de San José, con indicación de colocación de catéter venoso central por técnica convencional entre 2012 y 2013 (cohorte no expuesta) y técnica ecoguiada en 2014 (cohorte expuesta). Como desenlace se observó la presencia de complicacionesen cualquiera de los dos grupos. Resultados: se incluyeron 73 casos, 38 (52,1%) con colocación del catéter venoso central por la técnica ecoguiada. Se presentaron nueve (12,3%) complicaciones, ocho (88,9%) de ellas con la técnica convencional. La mediante del tiempo del procedimiento fue 20 minutos (rango intercuatílico: 15-20 minutos) con la técnica convencional y 10 minutos (rango intercuatílico: 7-10 minutos) con la técnica ecoguiada; esta última con colocación del catéter en un único intento en el 78,9% de los casos. Conclusión: se observó un menor número de complicaciones de tipo mecánico cuando con la técnica ecoguiadarespecto a la técnica convencional en los pacientes críticos con indicación de canulación venosa central.

Introduction: the use of ultrasound as a technique of choice for central venous catheter placement during emergencies increases patient safety due to low frequencyof complications, reduction of exposure to radiation, and increasing quality of care. Objective: To compare the mechanical complications of central venous catheterplacement under ultrasound guided and conventional technique by residents of emergency medicine specialty at the Hospital de San Jose (Bogota, Colombia). Material and methods: Descriptive study of two cohorts, which include patients over 15 years old, admitted to the emergency department of Hospital de San Jose with indication of central venous catheter placement by conventional technique between2012 and 2013 (unexposed cohort) and ultrasound-guided technique in 2014 (exposed cohort). As outcome was observed the presence of complications in both groups. Results: A total of 73 cases were included, 38 (52.1%) of them with central venous catheter placement by ultrasound-guided technique. A total of nine complications(12.3%) were presented, eight (88.9%) of them with the conventional technique.The median of time procedure was of 20 minutes (interquartile range: 15-20 minute) with the conventional technique and 10 minutes (interquartile range: 7-10 minutes) with ecoguide technique. In the ultrasound-guided technique, the catheter placement was achieved in an only attempt in 78.9% of cases. Conclusion: Fewer mechanical complications were observed with ultrasound guided technique respect conventional technique in critically ill patients who have indication of central venous cannulation.

A contrast between DEMATEL-ANP and ANP methods for six sigma project selection: a case study in healthcare industry.

BACKGROUND: The project selection process is a crucial step for healthcare organizations at the moment of implementing six sigma programs in both administrative and caring processes. However, six-sigma project selection is often defined as a decision making process with interaction and feedback between criteria; so that it is necessary to explore different methods to help healthcare companies to determine the Six-sigma projects that provide the maximum benefits. This paper describes the application of both ANP (Analytic Network process) and DEMATEL (Decision Making trial and evaluation laboratory)-ANP in a public medical centre to establish the most suitable six sigma project and finally, these methods were compared to evaluate their performance in the decision making process. METHODS: ANP and DEMATEL-ANP were used to evaluate 6 six sigma project alternatives under an evaluation model composed by 3 strategies, 4 criteria and 15 sub-criteria. Judgement matrixes were completed by the six sigma team whose participants worked in different departments of the medical centre. RESULTS: The improving of care opportunity in obstetric outpatients was elected as the most suitable six sigma project with a score of 0,117 as contribution to the organization goals. DEMATEL-ANP performed better at decision making process since it reduced the error probability due to interactions and feedback. CONCLUSIONS: ANP and DEMATEL-ANP effectively supported six sigma project selection processes, helping to create a complete framework that guarantees the prioritization of projects that provide maximum benefits to healthcare organizations. As DEMATEL- ANP performed better, it should be used by practitioners involved in decisions related to the implementation of six sigma programs in healthcare sector accompanied by the adequate identification of the evaluation criteria that support the decision making model. Thus, this comparative study contributes to choosing more effective approaches in this field. Suggestions of further work are also proposed so that these methods can be applied more adequate in six sigma project selection processes in healthcare.

Influence of the LILRA3 Deletion on Multiple Sclerosis Risk: Original Data and Meta-Analysis.

BACKGROUND: Multiple sclerosis (MS) is a neurodegenerative, autoimmune disease of the central nervous system. Genome-wide association studies (GWAS) have identified over hundred polymorphisms with modest individual effects in MS susceptibility and they have confirmed the main individual effect of the Major Histocompatibility Complex. Additional risk loci with immunologically relevant genes were found significantly overrepresented. Nonetheless, it is accepted that most of the genetic architecture underlying susceptibility to the disease remains to be defined. Candidate association studies of the leukocyte immunoglobulin-like receptor LILRA3 gene in MS have been repeatedly reported with inconsistent results. OBJECTIVES: In an attempt to shed some light on these controversial findings, a combined analysis was performed including the previously published datasets and three newly genotyped cohorts. Both wild-type and deleted LILRA3 alleles were discriminated in a single-tube PCR amplification and the resulting products were visualized by their different electrophoretic mobilities. RESULTS AND CONCLUSION: Overall, this meta-analysis involved 3200 MS patients and 3069 matched healthy controls and it did not evidence significant association of the LILRA3 deletion [carriers of LILRA3 deletion: p = 0.25, OR (95% CI) = 1.07 (0.95-1.19)], even after stratification by gender and the HLA-DRB1*15:01 risk allele.

MANBA, CXCR5, SOX8, RPS6KB1 and ZBTB46 are genetic risk loci for multiple sclerosis.

A recent genome-wide association study reported five loci for which there was strong, but sub-genome-wide significant evidence for association with multiple sclerosis risk. The aim of this study was to evaluate the role of these potential risk loci in a large and independent data set of ≈ 20,000 subjects. We tested five single nucleotide polymorphisms rs228614 (MANBA), rs630923 (CXCR5), rs2744148 (SOX8), rs180515 (RPS6KB1), and rs6062314 (ZBTB46) for association with multiple sclerosis risk in a total of 8499 cases with multiple sclerosis, 8765 unrelated control subjects and 958 trios of European descent. In addition, we assessed the overall evidence for association by combining these newly generated data with the results from the original genome-wide association study by meta-analysis. All five tested single nucleotide polymorphisms showed consistent and statistically significant evidence for association with multiple sclerosis in our validation data sets (rs228614: odds ratio = 0.91, P = 2.4 × 10(-6); rs630923: odds ratio = 0.89, P = 1.2 × 10(-4); rs2744148: odds ratio = 1.14, P = 1.8 × 10(-6); rs180515: odds ratio = 1.12, P = 5.2 × 10(-7); rs6062314: odds ratio = 0.90, P = 4.3 × 10(-3)). Combining our data with results from the previous genome-wide association study by meta-analysis, the evidence for association was strengthened further, surpassing the threshold for genome-wide significance (P < 5 × 10(-8)) in each case. Our study provides compelling evidence that these five loci are genuine multiple sclerosis susceptibility loci. These results may eventually lead to a better understanding of the underlying disease pathophysiology.