Genome-wide association study of multiple sclerosis confirms a novel locus at 5p13.1.

Multiple Sclerosis (MS) is the most common progressive and disabling neurological condition affecting young adults in the world today. From a genetic point of view, MS is a complex disorder resulting from the combination of genetic and non-genetic factors. We aimed to identify previously unidentified loci conducting a new GWAS of Multiple Sclerosis (MS) in a sample of 296 MS cases and 801 controls from the Spanish population. Meta-analysis of our data in combination with previous GWAS was done. A total of 17 GWAS-significant SNPs, corresponding to three different loci were identified:HLA, IL2RA, and 5p13.1. All three have been previously reported as GWAS-significant. We confirmed our observation in 5p13.1 for rs9292777 using two additional independent Spanish samples to make a total of 4912 MS cases and 7498 controls (ORpooled = 0.84; 95%CI: 0.80-0.89; p = 1.36 × 10-9). This SNP differs from the one reported within this locus in a recent GWAS. Although it is unclear whether both signals are tapping the same genetic association, it seems clear that this locus plays an important role in the pathogenesis of MS.

Polymorphism analysis of the CTLA-4 gene in paracoccidioidomycosis patients

The CTLA-4 protein is expressed in activated T cells and plays an essential role in the immune response through its regulatory effect on T cell activation. Polymorphisms of the CTLA-4 gene have been correlated with autoimmune, neoplastic and infectious illnesses. This work aimed to verify possible associations between single nucleotide polymorphisms (SNPs) in CTLA-4, -318C/T in the promoter and +49A/G in exon 1 and paracoccidioidomycosis (PCM) caused by Paracoccidioides brasiliensis. For this purpose, 66 chronic form PCM patients and 76 healthy controls had their allele, genotype and haplotype frequencies determined. The genetic admixture structure of the patients and controls was evaluated to eliminate ancestral bias. The comparison of frequencies indicated no significant differences between patients and controls that could link the SNPs to PCM. Groups were admixture matched with no difference observed in population ancestry inference, indicating that the absence of association between CTLA-4 polymorphisms and PCM could not be attributed to ancestral bias. This study showed that there was no association between the CTLA-4 SNPs -318 and +49 and the resistance or susceptibility to PCM.

Sources of pre-analytical variations in yield of DNA extracted from blood samples: analysis of 50,000 DNA samples in EPIC.

The European Prospective Investigation into Cancer and nutrition (EPIC) is a long-term, multi-centric prospective study in Europe investigating the relationships between cancer and nutrition. This study has served as a basis for a number of Genome-Wide Association Studies (GWAS) and other types of genetic analyses. Over a period of 5 years, 52,256 EPIC DNA samples have been extracted using an automated DNA extraction platform. Here we have evaluated the pre-analytical factors affecting DNA yield, including anthropometric, epidemiological and technical factors such as center of subject recruitment, age, gender, body-mass index, disease case or control status, tobacco consumption, number of aliquots of buffy coat used for DNA extraction, extraction machine or procedure, DNA quantification method, degree of haemolysis and variations in the timing of sample processing. We show that the largest significant variations in DNA yield were observed with degree of haemolysis and with center of subject recruitment. Age, gender, body-mass index, cancer case or control status and tobacco consumption also significantly impacted DNA yield. Feedback from laboratories which have analyzed DNA with different SNP genotyping technologies demonstrate that the vast majority of samples (approximately 88%) performed adequately in different types of assays. To our knowledge this study is the largest to date to evaluate the sources of pre-analytical variations in DNA extracted from peripheral leucocytes. The results provide a strong evidence-based rationale for standardized recommendations on blood collection and processing protocols for large-scale genetic studies.

Mitochondrial haplogroups and polymorphisms reveal no association with sporadic prostate cancer in a southern European population.

BACKGROUND It is known that mitochondria play an important role in certain cancers (prostate, renal, breast, or colorectal) and coronary disease. These organelles play an essential role in apoptosis and the production of reactive oxygen species; in addition, mtDNA also reveals the history of populations and ancient human migration. All these events and variations in the mitochondrial genome are thought to cause some cancers, including prostate cancer, and also help us to group individuals into common origin groups. The aim of the present study is to analyze the different haplogroups and variations in the sequence in the mitochondrial genome of a southern European population consisting of subjects affected (n = 239) and non-affected (n = 150) by sporadic prostate cancer. METHODOLOGY AND PRINCIPAL FINDINGS Using primer extension analysis and DNA sequencing, we identified the nine major European haplogroups and CR polymorphisms. The frequencies of the haplogroups did not differ between patients and control cohorts, whereas the CR polymorphism T16356C was significantly higher in patients with PC compared to the controls (p = 0.029). PSA, staging, and Gleason score were associated with none of the nine major European haplogroups. The CR polymorphisms G16129A (p = 0.007) and T16224C (p = 0.022) were significantly associated with Gleason score, whereas T16311C (p = 0.046) was linked with T-stage. CONCLUSIONS AND SIGNIFICANCE Our results do not suggest that mtDNA haplogroups could be involved in sporadic prostate cancer etiology and pathogenesis as previous studies performed in middle Europe population. Although some significant associations have been obtained in studying CR polymorphisms, further studies should be performed to validate these results.

Discovery and fine mapping of serum protein loci through transethnic meta-analysis.

Many disorders are associated with altered serum protein concentrations, including malnutrition, cancer, and cardiovascular, kidney, and inflammatory diseases. Although these protein concentrations are highly heritable, relatively little is known about their underlying genetic determinants. Through transethnic meta-analysis of European-ancestry and Japanese genome-wide association studies, we identified six loci at genome-wide significance (p < 5 × 10(-8)) for serum albumin (HPN-SCN1B, GCKR-FNDC4, SERPINF2-WDR81, TNFRSF11A-ZCCHC2, FRMD5-WDR76, and RPS11-FCGRT, in up to 53,190 European-ancestry and 9,380 Japanese individuals) and three loci for total protein (TNFRS13B, 6q21.3, and ELL2, in up to 25,539 European-ancestry and 10,168 Japanese individuals). We observed little evidence of heterogeneity in allelic effects at these loci between groups of European and Japanese ancestry but obtained substantial improvements in the resolution of fine mapping of potential causal variants by leveraging transethnic differences in the distribution of linkage disequilibrium. We demonstrated a functional role for the most strongly associated serum albumin locus, HPN, for which Hpn knockout mice manifest low plasma albumin concentrations. Other loci associated with serum albumin harbor genes related to ribosome function, protein translation, and proteasomal degradation, whereas those associated with serum total protein include genes related to immune function. Our results highlight the advantages of transethnic meta-analysis for the discovery and fine mapping of complex trait loci and have provided initial insights into the underlying genetic architecture of serum protein concentrations and their association with human disease.

DRB1*03:01 haplotypes: differential contribution to multiple sclerosis risk and specific association with the presence of intrathecal IgM bands.

BACKGROUND Multiple sclerosis (MS) is a multifactorial disease with a genetic basis. The strongest associations with the disease lie in the Human Leukocyte Antigen (HLA) region. However, except for the DRB1*15:01 allele, the main risk factor associated to MS so far, no consistent effect has been described for any other variant. One example is HLA-DRB1*03:01, with a heterogeneous effect across populations and studies. We postulate that those discrepancies could be due to differences in the diverse haplotypes bearing that allele. Thus, we aimed at studying the association of DRB1*03:01 with MS susceptibility considering this allele globally and stratified by haplotypes. We also evaluated the association with the presence of oligoclonal IgM bands against myelin lipids (OCMB) in cerebrospinal fluid. METHODS Genotyping of HLA-B, -DRB1 and -DQA1 was performed in 1068 MS patients and 624 ethnically matched healthy controls. One hundred and thirty-nine MS patients were classified according to the presence (M+, 58 patients)/absence (M-, 81 patients) of OCMB. Comparisons between groups (MS patients vs. controls and M+ vs. M-) were performed with the chi-square test or the Fisher exact test. RESULTS Association of DRB1*03:01 with MS susceptibility was observed but with different haplotypic contribution, being the ancestral haplotype (AH) 18.2 the one causing the highest risk. Comparisons between M+, M- and controls showed that the AH 18.2 was affecting only M+ individuals, conferring a risk similar to that caused by DRB1*15:01. CONCLUSIONS The diverse DRB1*03:01-containing haplotypes contribute with different risk to MS susceptibility. The AH 18.2 causes the highest risk and affects only to individuals showing OCMB.

A multicenter study confirms CD226 gene association with systemic sclerosis-related pulmonary fibrosis.

INTRODUCTION CD226 genetic variants have been associated with a number of autoimmune diseases and recently with systemic sclerosis (SSc). The aim of this study was to test the influence of CD226 loci in SSc susceptibility, clinical phenotypes and autoantibody status in a large multicenter European population. METHODS A total of seven European populations of Caucasian ancestry were included, comprising 2,131 patients with SSc and 3,966 healthy controls. Three CD226 single nucleotide polymorphisms (SNPs), rs763361, rs3479968 and rs727088, were genotyped using Taqman 5'allelic discrimination assays. RESULTS Pooled analyses showed no evidence of association of the three SNPs, neither with the global disease nor with the analyzed subphenotypes. However, haplotype block analysis revealed a significant association for the TCG haplotype (SNP order: rs763361, rs34794968, rs727088) with lung fibrosis positive patients (PBonf = 3.18E-02 OR 1.27 (1.05 to 1.54)). CONCLUSION Our data suggest that the tested genetic variants do not individually influence SSc susceptibility but a CD226 three-variant haplotype is related with genetic predisposition to SSc-related pulmonary fibrosis.

Polymorphisms in DNA-Repair Genes in a Cohort of Prostate Cancer Patients from Different Areas in Spain: Heterogeneity between Populations as a Confounding Factor in Association Studies.

BACKGROUND Differences in the distribution of genotypes between individuals of the same ethnicity are an important confounder factor commonly undervalued in typical association studies conducted in radiogenomics. OBJECTIVE To evaluate the genotypic distribution of SNPs in a wide set of Spanish prostate cancer patients for determine the homogeneity of the population and to disclose potential bias. DESIGN SETTING AND PARTICIPANTS A total of 601 prostate cancer patients from Andalusia, Basque Country, Canary and Catalonia were genotyped for 10 SNPs located in 6 different genes associated to DNA repair: XRCC1 (rs25487, rs25489, rs1799782), ERCC2 (rs13181), ERCC1 (rs11615), LIG4 (rs1805388, rs1805386), ATM (rs17503908, rs1800057) and P53 (rs1042522). The SNP genotyping was made in a Biotrove OpenArray® NT Cycler. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Comparisons of genotypic and allelic frequencies among populations, as well as haplotype analyses were determined using the web-based environment SNPator. Principal component analysis was made using the SnpMatrix and XSnpMatrix classes and methods implemented as an R package. Non-supervised hierarchical cluster of SNP was made using MultiExperiment Viewer. RESULTS AND LIMITATIONS We observed that genotype distribution of 4 out 10 SNPs was statistically different among the studied populations, showing the greatest differences between Andalusia and Catalonia. These observations were confirmed in cluster analysis, principal component analysis and in the differential distribution of haplotypes among the populations. Because tumor characteristics have not been taken into account, it is possible that some polymorphisms may influence tumor characteristics in the same way that it may pose a risk factor for other disease characteristics. CONCLUSION Differences in distribution of genotypes within different populations of the same ethnicity could be an important confounding factor responsible for the lack of validation of SNPs associated with radiation-induced toxicity, especially when extensive meta-analysis with subjects from different countries are carried out.

Extended genetic analysis of Brazilian isolates of Bacillus cereus and Bacillus thuringiensis

Multiple locus sequence typing (MLST) was undertaken to extend the genetic characterization of 29 isolates of Bacillus cereus and Bacillus thuringiensis previously characterized in terms of presence/absence of sequences encoding virulence factors and via variable number tandem repeat (VNTR). Additional analysis involved polymerase chain reaction for the presence of sequences (be, cytK, inA, pag, lef, cya and cap), encoding putative virulence factors, not investigated in the earlier study. MLST analysis ascribed novel and unique sequence types to each of the isolates. A phylogenetic tree was constructed from a single sequence of 2,838 bp of concatenated loci sequences. The strains were not monophyletic by analysis of any specific housekeeping gene or virulence characteristic. No clear association in relation to source of isolation or to genotypic profile based on the presence or absence of putative virulence genes could be identified. Comparison of VNTR profiling with MLST data suggested a correlation between these two methods of genetic analysis. In common with the majority of previous studies, MLST was unable to provide clarification of the basis for pathogenicity among members of the B. cereus complex. Nevertheless, our application of MLST served to reinforce the notion that B. cereus and B. thuringiensis should be considered as the same species.

Fine mapping and functional analysis of the multiple sclerosis risk gene CD6.

CD6 has recently been identified and validated as risk gene for multiple sclerosis (MS), based on the association of a single nucleotide polymorphism (SNP), rs17824933, located in intron 1. CD6 is a cell surface scavenger receptor involved in T-cell activation and proliferation, as well as in thymocyte differentiation. In this study, we performed a haptag SNP screen of the CD6 gene locus using a total of thirteen tagging SNPs, of which three were non-synonymous SNPs, and replicated the recently reported GWAS SNP rs650258 in a Spanish-Basque collection of 814 controls and 823 cases. Validation of the six most strongly associated SNPs was performed in an independent collection of 2265 MS patients and 2600 healthy controls. We identified association of haplotypes composed of two non-synonymous SNPs [rs11230563 (R225W) and rs2074225 (A257V)] in the 2(nd) SRCR domain with susceptibility to MS (P max(T) permutation = 1×10(-4)). The effect of these haplotypes on CD6 surface expression and cytokine secretion was also tested. The analysis showed significantly different CD6 expression patterns in the distinct cell subsets, i.e. - CD4(+) naïve cells, P = 0.0001; CD8(+) naïve cells, P<0.0001; CD4(+) and CD8(+) central memory cells, P = 0.01 and 0.05, respectively; and natural killer T (NKT) cells, P = 0.02; with the protective haplotype (RA) showing higher expression of CD6. However, no significant changes were observed in natural killer (NK) cells, effector memory and terminally differentiated effector memory T cells. Our findings reveal that this new MS-associated CD6 risk haplotype significantly modifies expression of CD6 on CD4(+) and CD8(+) T cells.

Virulence profiles of bacteremic extended-spectrum β-lactamase-producing Escherichia coli: association with epidemiological and clinical features.

There is scarce data about the importance of phylogroups and virulence factors (VF) in bloodstream infections (BSI) caused by extended-spectrum β-lactamase-producing Escherichia coli (ESBLEC). A prospective multicenter Spanish cohort including 191 cases of BSI due to ESBLEC was studied. Phylogroups and 25 VF genes were investigated by PCR. ESBLEC were classified into clusters according to their virulence profiles. The association of phylogropus, VF, and clusters with epidemiological features were studied using multivariate analysis. Overall, 57.6%, 26.7%, and 15.7% of isolates belonged to A/B1, D and B2 phylogroups, respectively. By multivariate analysis (adjusted OR [95% CI]), virulence cluster C2 was independently associated with urinary tract source (5.05 [0.96-25.48]); cluster C4 with sources other than urinary of biliary tract (2.89 [1.05-7.93]), and cluster C5 with BSI in non-predisposed patients (2.80 [0.99-7.93]). Isolates producing CTX-M-9 group ESBLs and from phylogroup D predominated among cluster C2 and C5, while CTX-M-1 group of ESBL and phylogroup B2 predominantes among C4 isolates. These results suggest that host factors and previous antimicrobial use were more important than phylogroup or specific VF in the occurrence of BSI due to ESBLEC. However, some associations between virulence clusters and some specific epidemiological features were found.

The association between dietary energy density and type 2 diabetes in Europe: results from the EPIC-InterAct Study

BACKGROUND Observational studies implicate higher dietary energy density (DED) as a potential risk factor for weight gain and obesity. It has been hypothesized that DED may also be associated with risk of type 2 diabetes (T2D), but limited evidence exists. Therefore, we investigated the association between DED and risk of T2D in a large prospective study with heterogeneity of dietary intake. METHODOLOGY/PRINCIPAL FINDINGS A case-cohort study was nested within the European Prospective Investigation into Cancer (EPIC) study of 340,234 participants contributing 3.99 million person years of follow-up, identifying 12,403 incident diabetes cases and a random subcohort of 16,835 individuals from 8 European countries. DED was calculated as energy (kcal) from foods (except beverages) divided by the weight (gram) of foods estimated from dietary questionnaires. Prentice-weighted Cox proportional hazard regression models were fitted by country. Risk estimates were pooled by random effects meta-analysis and heterogeneity was evaluated. Estimated mean (sd) DED was 1.5 (0.3) kcal/g among cases and subcohort members, varying across countries (range 1.4-1.7 kcal/g). After adjustment for age, sex, smoking, physical activity, alcohol intake, energy intake from beverages and misreporting of dietary intake, no association was observed between DED and T2D (HR 1.02 (95% CI: 0.93-1.13), which was consistent across countries (I(2) = 2.9%). CONCLUSIONS/SIGNIFICANCE In this large European case-cohort study no association between DED of solid and semi-solid foods and risk of T2D was observed. However, despite the fact that there currently is no conclusive evidence for an association between DED and T2DM risk, choosing low energy dense foods should be promoted as they support current WHO recommendations to prevent chronic diseases.

Meta-analysis of studies analyzing the role of human papillomavirus in the development of bladder carcinoma

PURPOSE We aimed to ascertain the degree of association between bladder cancer and human papillomavirus (HPV) infection. MATERIALS AND METHODS We performed a meta-analysis of observational studies with cases and controls with publication dates up to January 2011. The PubMed electronic database was searched by using the key words "bladder cancer and virus." Twenty-one articles were selected that met the required methodological criteria. We implemented an internal quality control system to verify the selected search method. We analyzed the pooled effect of all the studies and also analyzed the techniques used as follows: 1) studies with DNA-based techniques, among which we found studies with polymerase chain reaction (PCR)-based techniques and 2) studies with non-PCR-based techniques, and studies with non-DNA-based techniques. RESULTS Taking into account the 21 studies that were included in the meta-analysis, we obtained a heterogeneity chi-squared value of Q(exp)=26.45 (p=0.383). The pooled odds ratio (OR) was 2.13 (95% confidence interval [CI], 1.54 to 2.95), which points to a significant effect between HPV and bladder cancer. Twenty studies assessed the presence of DNA. The overall effect showed a significant relationship between virus presence and bladder cancer, with a pooled OR of 2.19 (95% CI, 1.40 to 3.43). Of the other six studies, four examined the virus's capsid antigen and two detected antibodies in serum by Western blot. The estimated pooled OR in this group was 2.11 (95% CI, 1.27 to 3.51), which confirmed the relationship between the presence of virus and cancer. CONCLUSIONS The pooled OR value showed a moderate relationship between viral infection and bladder tumors.

Sensitivity of genome-wide-association signals to phenotyping strategy: the PROP-TAS2R38 taste association as a benchmark.

Natural genetic variation can have a pronounced influence on human taste perception, which in turn may influence food preference and dietary choice. Genome-wide association studies represent a powerful tool to understand this influence. To help optimize the design of future genome-wide-association studies on human taste perception we have used the well-known TAS2R38-PROP association as a tool to determine the relative power and efficiency of different phenotyping and data-analysis strategies. The results show that the choice of both data collection and data processing schemes can have a very substantial impact on the power to detect genotypic variation that affects chemosensory perception. Based on these results we provide practical guidelines for the design of future GWAS studies on chemosensory phenotypes. Moreover, in addition to the TAS2R38 gene past studies have implicated a number of other genetic loci to affect taste sensitivity to PROP and the related bitter compound PTC. None of these other locations showed genome-wide significant associations in our study. To facilitate further, target-gene driven, studies on PROP taste perception we provide the genome-wide list of p-values for all SNPs genotyped in the current study.

Obesity and survival in operable breast cancer patients treated with adjuvant anthracyclines and taxanes according to pathological subtypes: a pooled analysis.

INTRODUCTION Obesity is an unfavorable prognostic factor in breast cancer (BC) patients regardless of menopausal status and treatment received. However, the association between obesity and survival outcome by pathological subtype requires further clarification. METHODS We performed a retrospective analysis including 5,683 operable BC patients enrolled in four randomized clinical trials (GEICAM/9906, GEICAM/9805, GEICAM/2003-02, and BCIRG 001) evaluating anthracyclines and taxanes as adjuvant treatments. Our primary aim was to assess the prognostic effect of body mass index (BMI) on disease recurrence, breast cancer mortality (BCM), and overall mortality (OM). A secondary aim was to detect differences of such prognostic effects by subtype. RESULTS Multivariate survival analyses adjusting for age, tumor size, nodal status, menopausal status, surgery type, histological grade, hormone receptor status, human epidermal growth factor receptor 2 (HER2) status, chemotherapy regimen, and under-treatment showed that obese patients (BMI 30.0 to 34.9) had similar prognoses to that of patients with a BMI < 25 (reference group) in terms of recurrence (Hazard Ratio [HR] = 1.08, 95% Confidence Interval [CI] = 0.90 to 1.30), BCM (HR = 1.02, 0.81 to 1.29), and OM (HR = 0.97, 0.78 to 1.19). Patients with severe obesity (BMI ≥ 35) had a significantly increased risk of recurrence (HR = 1.26, 1.00 to 1.59, P = 0.048), BCM (HR = 1.32, 1.00 to 1.74, P = 0.050), and OM (HR = 1.35, 1.06 to 1.71, P = 0.016) compared to our reference group. The prognostic effect of severe obesity did not vary by subtype. CONCLUSIONS Severely obese patients treated with anthracyclines and taxanes present a worse prognosis regarding recurrence, BCM, and OM than patients with BMI < 25. The magnitude of the harmful effect of BMI on survival-related outcomes was similar across subtypes.