Genetic distance as an alternative to physical distance for definition of gene units in association studies.

BACKGROUND: Some association studies, as the implemented in VEGAS, ALIGATOR, i-GSEA4GWAS, GSA-SNP and other software tools, use genes as the unit of analysis. These genes include the coding sequence plus flanking sequences. Polymorphisms in the flanking sequences are of interest because they involve cis-regulatory elements or they inform on untyped genetic variants trough linkage disequilibrium. Gene extensions have customarily been defined as ±50 Kb. This approach is not fully satisfactory because genetic relationships between neighbouring sequences are a function of genetic distances, which are only poorly replaced by physical distances. RESULTS: Standardized recombination rates (SRR) from the deCODE recombination map were used as units of genetic distances. We searched for a SRR producing flanking sequences near the ±50 Kb offset that has been common in previous studies. A SRR≥2 was selected because it led to gene extensions with median length=45.3 Kb and the simplicity of an integer value. As expected, boundaries of the genes defined with the ±50 Kb and with the SRR≥2 rules were rarely concordant. The impact of these differences was illustrated with the interpretation of top association signals from two large studies including many hits and their detailed analysis based in different criteria. The definition based in genetic distance was more concordant with the results of these studies than the based in physical distance. In the analysis of 18 top disease associated loci form the first study, the SRR≥2 genes led to a fully concordant interpretation in 17 loci; the ±50 Kb genes only in 6. Interpretation of the 43 putative functional genes of the second study based in the SRR≥2 definition only missed 4 of the genes, whereas the based in the ±50 Kb definition missed 10 genes. CONCLUSIONS: A gene definition based on genetic distance led to results more concordant with expert detailed analyses than the commonly used based in physical distance. The genome coordinates for each gene are provided to maintain a simple use of the new definitions.

Association of anti-citrullinated vimentin and anti-citrullinated α-enolase antibodies with subsets of rheumatoid arthritis.

OBJECTIVE: To determine whether the anti-citrullinated vimentin peptide 60-75 (anti-Cit-vimentin) and the immunodominant anti-citrullinated α-enolase peptide 1 (anti-CEP-1) antibodies are associated with subsets of patients with rheumatoid arthritis (RA) independently of the associations between anti-cyclic citrullinated peptide (anti-CCP) antibodies and clinical features of RA. METHODS: The 3 antibody types were quantified by enzyme-linked immunosorbent assay (ELISA) in serum samples from 521 patients with RA and 173 healthy controls of Spanish ancestry. Genotypes for HLA-DRB1 alleles and rs2476601 in PTPN22 were available for these patients and controls plus an additional 106 healthy controls. A combined analysis of the 3 antibodies was conducted using stratified contingency tables and logistic regression models. RESULTS: A differential, particularly strong, and independent association was observed between the presence of anti-Cit-vimentin antibodies and the presence of shared epitope (SE) alleles, specifically in patients carrying 2 SE alleles, and between the presence of anti- Cit-vimentin antibodies and the prevalence of joint erosion. Associations were observed between anti-CEP-1 positivity and the presence of HLA-DRB1 and PTPN22 risk alleles and their additive interaction. These associations were not accounted for by the anti-CCP status. CONCLUSION: Our results indicate that the 2 antibodies against citrullinated peptides analyzed in this study add specific information beyond that obtained with the anti-CCP status. They define subgroups of patients with RA in which genetic factors have different weight and there is an observed difference in the prevalence of erosions.

Particular association of clinical and genetic features with autoimmunity to citrullinated α-enolase in rheumatoid arthritis.

OBJECTIVE: To confirm that the presence of anti-citrullinated α-enolase peptide 1 (anti-CEP-1) antibodies identifies a subgroup of patients with rheumatoid arthritis (RA). METHODS: DNA and serum samples were obtained from 451 patients with RA and 279 healthy control subjects, all of whom were of Spanish ancestry. Antibodies to cyclic citrullinated peptide (CCP) and CEP-1 were measured by enzyme-linked immunosorbent assay. HLA-DRB1 and the R620W single-nucleotide polymorphism of PTPN22 were genotyped. RESULTS: Anti-CEP-1 and anti-CCP antibodies were observed in 26.8% and 71.2% of the patients with RA, respectively. Most of the patients (86.6%) with anti-CEP-1 antibodies also had anti-CCP antibodies. Erosive arthritis, rheumatoid factor (RF) positivity, and the presence of the HLA shared epitope (especially the DRB1*04 alleles) were disproportionately associated with the group of patients with both antibodies. In addition, evidence of a significant interaction between the shared epitope and the risk allele of PTPN22 was observed only in these patients. In contrast, the association with these clinical and genetic features was weaker in patients with anti-CCP antibodies but lacking anti-CEP-1 antibodies. These results were obtained in patients in whom the prevalence of RA risk factors differed from that in other previously studied patients. CONCLUSION: We observed that autoimmunity against citrullinated α-enolase may identify a subset of patients with a higher frequency of joint erosions and RF positivity. In addition, we confirmed the disproportionately large effect of the susceptibility alleles of HLA-DRB1 and their interaction with PTPN22 in this subset of patients. These results extend, confirm, and generalize the evidence supporting the specificity of the anti-CEP-1 antibody-positive subgroup of patients with RA among anti-CCP antibody-positive patients with RA.

Obesity-related genetic determinants of stroke.

As obesity, circulating lipids and other vascular/metabolic factors influence the risk of stroke, we examined if genetic variants associated with these conditions are related to risk of stroke using a case-control study in Galicia, Spain. A selection of 200 single-nucleotide polymorphisms previously found to be related to obesity, body mass index, circulating lipids, type 2 diabetes, heart failure, obesity-related cancer and cerebral infarction were genotyped in 465 patients diagnosed with stroke and 480 population-based controls. An unsupervised Lasso regression procedure was carried out for single-nucleotide polymorphism selection based on their potential effect on stroke according to obesity. Selected genotypes were further analysed through multivariate logistic regression to study their association with risk of stroke. Using unsupervised selection procedures, nine single-nucleotide polymorphisms were found to be related to risk of stroke overall and after stratification by obesity. From these, rs10761731, rs2479409 and rs6511720 in obese subjects [odds ratio (95% confidence interval) = 0.61 (0.39-0.95) (P = 0.027); 0.54 (0.35-0.84) (P = 0.006) and 0.42 (0.22-0.80) (P = 0.0075), respectively], and rs865686 in non-obese subjects [odds ratio (95% confidence interval) = 0.67 (0.48-0.94) (P = 0.019)], were independently associated with risk of stroke after multivariate logistic regression procedures. The associations between the three single-nucleotide polymorphisms found to be associated with stroke risk in obese subjects were more pronounced among females; for rs10761731, odds ratios among obese males and females were 1.07 (0.58-1.97) (P = 0.84), and 0.31 (0.14-0.69) (P = 0.0018), respectively; for rs2479409, odd ratios were 0.66 (0.34-1.27) (P = 0.21), and 0.49 (0.24-0.99) (P = 0.04), for obese males and females, respectively; the stroke-rs6511720 association was also slightly more pronounced among obese females, odds ratios were 0.33 (0.13-0.87) (P = 0.022), and 0.28 (0.09-0.85) (P = 0.02) for obese males and females, respectively. The rs865686-stroke association was more pronounced among non-obese males [odds ratios = 0.61 (0.39-0.96) (P = 0.029) and 0.72 (0.42-1.22) (P = 0.21), for non-obese males and females, respectively]. A combined genetic score of variants rs10761731, rs2479409 and rs6511720 was highly predictive of stroke risk among obese subjects (P = 2.04 × 10-5), particularly among females (P = 4.28 × 10-6). In summary, single-nucleotide polymorphisms rs1076173, rs2479409 and rs6511720 were found to independently increase the risk of stroke in obese subjects after adjustment for established risk factors. A combined score with the three genomic variants was an independent predictor of risk of stroke among obese subjects in our population.

LIPG endothelial lipase and breast cancer risk by subtypes.

Experimental data showed that endothelial lipase (LIPG) is a crucial player in breast cancer. However, very limited data exists on the role of LIPG on the risk of breast cancer in humans. We examined the LIPG-breast cancer association within our population-based case-control study from Galicia, Spain, BREOGAN (BREast Oncology GAlicia Network). Plasma LIPG and/or OxLDL were measured on 114 breast cancer cases and 82 controls from our case-control study, and were included in the present study. The risk of breast cancer increased with increasing levels of LIPG (multivariable OR for the highest category (95% CI) 2.52 (1.11-5.81), P-trend = 0.037). The LIPG-breast cancer association was restricted to Pre-menopausal breast cancer (Multivariable OR for the highest LIPG category (95% CI) 4.76 (0.94-28.77), P-trend = 0.06, and 1.79 (0.61-5.29), P-trend = 0.372, for Pre-menopausal and Post-menopausal breast cancer, respectively). The LIPG-breast cancer association was restricted to Luminal A breast cancers (Multivariable OR for the highest LIPG category (95% CI) 3.70 (1.42-10.16), P-trend = 0.015, and 2.05 (0.63-7.22), P-trend = 0.311, for Luminal A and non-Luminal A breast cancers, respectively). Subset analysis only based on HER2 receptor indicated that the LIPG-breast cancer relationship was restricted to HER2-negative breast cancers (Multivariable OR for the highest LIPG category (95% CI) 4.39 (1.70-12.03), P-trend = 0.012, and 1.10 (0.28-4.32), P-trend = 0.745, for HER2-negative and HER2-positive tumors, respectively). The LIPG-breast cancer association was restricted to women with high total cholesterol levels (Multivariable OR for the highest LIPG category (95% CI) 6.30 (2.13-20.05), P-trend = 0.018, and 0.65 (0.11-3.28), P-trend = 0.786, among women with high and low cholesterol levels, respectively). The LIPG-breast cancer association was also restricted to non-postpartum breast cancer (Multivariable OR for the highest LIPG category (95% CI) 3.83 (1.37-11.39), P-trend = 0.003, and 2.35 (0.16-63.65), P-trend = 0.396, for non-postpartum and postpartum breast cancer, respectively), although we lacked precision. The LIPG-breast cancer association was more pronounced among grades II and III than grade I breast cancers (Multivariable ORs for the highest category of LIPG (95% CI) 2.73 (1.02-7.69), P-trend = 0.057, and 1.90 (0.61-6.21), P-trend = 0.170, for grades II and III, and grade I breast cancers, respectively). No association was detected for OxLDL levels and breast cancer (Multivariable OR for the highest versus the lowest category (95% CI) 1.56 (0.56-4.32), P-trend = 0.457).

Gene-based analysis of ADHD using PASCAL: a biological insight into the novel associated genes.

BACKGROUND: Attention-Deficit Hyperactivity Disorder (ADHD) is a complex neurodevelopmental disorder (NDD) which may significantly impact on the affected individual's life. ADHD is acknowledged to have a high heritability component (70-80%). Recently, a meta-analysis of GWAS (Genome Wide Association Studies) has demonstrated the association of several independent loci. Our main aim here, is to apply PASCAL (pathway scoring algorithm), a new gene-based analysis (GBA) method, to the summary statistics obtained in this meta-analysis. PASCAL will take into account the linkage disequilibrium (LD) across genomic regions in a different way than the most commonly employed GBA methods (MAGMA or VEGAS (Versatile Gene-based Association Study)). In addition to PASCAL analysis a gene network and an enrichment analysis for KEGG and GO terms were carried out. Moreover, GENE2FUNC tool was employed to create gene expression heatmaps and to carry out a (DEG) (Differentially Expressed Gene) analysis using GTEX v7 and BrainSpan data. RESULTS: PASCAL results have revealed the association of new loci with ADHD and it has also highlighted other genes previously reported by MAGMA analysis. PASCAL was able to discover new associations at a gene level for ADHD: FEZF1 (p-value: 2.2 × 10- 7) and FEZF1-AS1 (p-value: 4.58 × 10- 7). In addition, PASCAL has been able to highlight association of other genes that share the same LD block with some previously reported ADHD susceptibility genes. Gene network analysis has revealed several interactors with the associated ADHD genes and different GO and KEGG terms have been associated. In addition, GENE2FUNC has demonstrated the existence of several up and down regulated expression clusters when the associated genes and their interactors were considered. CONCLUSIONS: PASCAL has been revealed as an efficient tool to extract additional information from previous GWAS using their summary statistics. This study has identified novel ADHD associated genes that were not previously reported when other GBA methods were employed. Moreover, a biological insight into the biological function of the ADHD associated genes across brain regions and neurodevelopmental stages is provided.

Novel Gene-Based Analysis of ASD GWAS: Insight Into the Biological Role of Associated Genes.

Background: Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by its significant social impact and high heritability. The latest meta-analysis of ASD GWAS (genome-wide association studies) has revealed the association of several SNPs that were replicated in additional sets of independent samples. However, summary statistics from GWAS can be used to perform a gene-based analysis (GBA). GBA allows to combine all genetic information across the gene to create a single statistic (p-value for each gene). Thus, PASCAL (Pathway scoring algorithm), a novel GBA tool, has been applied to the summary statistics from the latest meta-analysis of ASD. GBA approach (testing the gene as a unit) provides an advantage to perform an accurate insight into the biological ASD mechanisms. Therefore, a gene-network analysis and an enrichment analysis for KEGG and GO terms were carried out. GENE2FUNC was used to create gene expression heatmaps and to carry out differential expression analysis (DEA) across GTEx v7 tissues and Brainspan data. dbMDEGA was employed to perform a DEG analysis between ASD and brain control samples for the associated genes and interactors. Results: PASCAL has identified the following loci associated with ASD: XRN2, NKX2-4, PLK1S1, KCNN2, NKX2-2, CRHR1-IT1, C8orf74 and LOC644172. While some of these genes were previously reported by MAGMA (XRN2, PLK1S1, and KCNN2), PASCAL has been useful to highlight additional genes. The biological characterization of the ASD-associated genes and their interactors have demonstrated the association of several GO and KEGG terms. Moreover, DEA analysis has revealed several up- and down-regulated clusters. In addition, many of the ASD-associated genes and their interactors have shown association with ASD expression datasets. Conclusions: This study identifies several associations at a gene level in ASD. Most of them were previously reported by MAGMA. This fact proves that PASCAL is an efficient GBA tool to extract additional information from previous GWAS. In addition, this study has characterized for the first time the biological role of the ASD-associated genes across brain regions, neurodevelopmental stages, and ASD gene-expression datasets.

Reference genes for normalization of gene expression studies in human osteoarthritic articular cartilage.

BACKGROUND: Assessment of gene expression is an important component of osteoarthritis (OA) research, greatly improved by the development of quantitative real-time PCR (qPCR). This technique requires normalization for precise results, yet no suitable reference genes have been identified in human articular cartilage. We have examined ten well-known reference genes to determine the most adequate for this application. RESULTS: Analyses of expression stability in cartilage from 10 patients with hip OA, 8 patients with knee OA and 10 controls without OA were done with classical statistical tests and the software programs geNorm and NormFinder. Results from the three methods of analysis were broadly concordant. Some of the commonly used reference genes, GAPDH, ACTB and 18S RNA, performed poorly in our analysis. In contrast, the rarely used TBP, RPL13A and B2M genes were the best. It was necessary to use together several of these three genes to obtain the best results. The specific combination depended, to some extent, on the type of samples being compared. CONCLUSION: Our results provide a satisfactory set of previously unused reference genes for qPCR in hip and knee OA This confirms the need to evaluate the suitability of reference genes in every tissue and experimental situation before starting the quantitative assessment of gene expression by qPCR.

Expressed sequence tags (ESTs) from immune tissues of turbot (Scophthalmus maximus) challenged with pathogens.

BACKGROUND: The turbot (Scophthalmus maximus; Scophthalmidae; Pleuronectiformes) is a flatfish species of great relevance for marine aquaculture in Europe. In contrast to other cultured flatfish, very few genomic resources are available in this species. Aeromonas salmonicida and Philasterides dicentrarchi are two pathogens that affect turbot culture causing serious economic losses to the turbot industry. Little is known about the molecular mechanisms for disease resistance and host-pathogen interactions in this species. In this work, thousands of ESTs for functional genomic studies and potential markers linked to ESTs for mapping (microsatellites and single nucleotide polymorphisms (SNPs)) are provided. This information enabled us to obtain a preliminary view of regulated genes in response to these pathogens and it constitutes the basis for subsequent and more accurate microarray analysis. RESULTS: A total of 12584 cDNAs partially sequenced from three different cDNA libraries of turbot (Scophthalmus maximus) infected with Aeromonas salmonicida, Philasterides dicentrarchi and from healthy fish were analyzed. Three immune-relevant tissues (liver, spleen and head kidney) were sampled at several time points in the infection process for library construction. The sequences were processed into 9256 high-quality sequences, which constituted the source for the turbot EST database. Clustering and assembly of these sequences, revealed 3482 different putative transcripts, 1073 contigs and 2409 singletons. BLAST searches with public databases detected significant similarity (e-value < or = 1e-5) in 1766 (50.7%) sequences and 816 of them (23.4%) could be functionally annotated. Two hundred three of these genes (24.9%), encoding for defence/immune-related proteins, were mostly identified for the first time in turbot. Some ESTs showed significant differences in the number of transcripts when comparing the three libraries, suggesting regulation in response to these pathogens. A total of 191 microsatellites, with 104 having sufficient flanking sequences for primer design, and 1158 putative SNPs were identified from these EST resources in turbot. CONCLUSION: A collection of 9256 high-quality ESTs was generated representing 3482 unique turbot sequences. A large proportion of defence/immune-related genes were identified, many of them regulated in response to specific pathogens. Putative microsatellites and SNPs were identified. These genome resources constitute the basis to develop a microarray for functional genomics studies and marker validation for genetic linkage and QTL analysis in turbot.

Assessment of global DNA methylation in peripheral blood cell subpopulations of early rheumatoid arthritis before and after methotrexate.

INTRODUCTION: DNA methylation is an epigenetic mechanism regulating gene expression that has been insufficiently studied in the blood of rheumatoid arthritis (RA) patients, as only T cells and total peripheral blood mononuclear cells (PBMCs) from patients with established RA have been studied and with conflicting results. METHOD: Five major blood cell subpopulations: T, B and NK cells, monocytes, and polymorphonuclear leukocytes, were isolated from 19 early RA patients and 17 healthy controls. Patient samples were taken before and 1 month after the start of treatment with methotrexate (MTX). Analysis included DNA methylation with high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry-selected reaction monitoring (HPLC-ESI-MS/MS-SRM) and expression levels of seven methylation-specific enzymes by quantitative polymerase chain reaction (qPCR). RESULTS: Disease-modifying anti-rheumatic drug (DMARD)-naïve early RA patients showed global DNA hypomethylation in T cells and monocytes, together with a lower expression of DNA methyltrasnferase 1 (DNMT1), the maintenance DNA methyltransferase, which was also decreased in B cells. Furthermore, significantly increased expression of ten-eleven translocation1 (TET1), TET2 and TET3, enzymes involved in demethylation, was found in monocytes and of TET2 in T cells. There was also modest decreased expression of DNMT3A in B cells and of growth arrest and DNA-damage-inducible protein 45A (GADD45A) in T and B cells. Treatment with MTX reverted hypomethylation in T cells and monocytes, which were no longer different from controls, and increased global methylation in B cells. In addition, DNMT1 and DNMT3A showed a trend to reversion of their decreased expression. CONCLUSIONS: Our results confirm global DNA hypomethylation in patients with RA with specificity for some blood cell subpopulations and their reversal with methotrexate treatment. These changes are accompanied by parallel changes in the levels of enzymes involved in methylation, suggesting the possibility of regulation at this level.

Brief report: lack of replication of an association between anti-citrullinated fibrinogen and subclinical atherosclerosis in patients with rheumatoid arthritis.

OBJECTIVE: Results of a recent study suggested that the excess cardiovascular (CV) risk observed in patients with rheumatoid arthritis (RA) could be partially explained by the presence of immune complexes of antibodies against citrullinated proteins that locally promote and perpetuate inflammation and progression of atherosclerotic plaques. The present study was undertaken to attempt to replicate one of the observations supporting this hypothesis, i.e., association between anti-citrullinated fibrinogen (anti-Cit-fibrinogen) positivity and subclinical atherosclerosis. METHODS: Three surrogate markers of atherosclerosis were assessed in 124 RA patients with no previous history of CV events: carotid intima-media thickness (CIMT) assessed by carotid ultrasonography, carotid plaques assessed by carotid ultrasonography, and Coronary Artery Calcification Score (CACS) determined by multidetector computed tomography (CT) scanning. We analyzed the relationship of these 3 subclinical atherosclerosis markers to the presence and levels of autoantibodies, including anti-Cit-fibrinogen, anti-cyclic citrullinated peptide 2 (anti-CCP-2), and rheumatoid factor (RF). RESULTS: Carotid plaques and CIMT >0.90 mm were present in 69.4% and 15.3%, of the patients, respectively, and the CACS was moderate or high in 21.0%. None of these surrogate markers of atherosclerosis showed a significant association with positivity for or the level of anti-Cit-fibrinogen antibodies (either against the whole protein [present in 33.9% of the patients] or against an immunodominant peptide [present in 23.4%]), anti-CCP-2 (present in 60.7%), or RF (present in 58.1%) in this series of patients with RA. CONCLUSION: Our results do not support the notion that there is a relationship between anti-Cit-fibrinogen antibodies and subclinical atherosclerosis in RA, thus calling into question the claim that these antibodies have a role in the increased risk of CV disease observed in patients with RA.

Lack of replication of interactions between polymorphisms in rheumatoid arthritis susceptibility: case-control study.

INTRODUCTION: Approximately 100 loci have been definitively associated with rheumatoid arthritis (RA) susceptibility. However, they explain only a fraction of RA heritability. Interactions between polymorphisms could explain part of the remaining heritability. Multiple interactions have been reported, but only the shared epitope (SE) × protein tyrosine phosphatase nonreceptor type 22 (PTPN22) interaction has been replicated convincingly. Two recent studies deserve attention because of their quality, including their replication in a second sample collection. In one of them, researchers identified interactions between PTPN22 and seven single-nucleotide polymorphisms (SNPs). The other showed interactions between the SE and the null genotype of glutathione S-transferase Mu 1 (GSTM1) in the anti-cyclic citrullinated peptide-positive (anti-CCP+) patients. In the present study, we aimed to replicate association with RA susceptibility of interactions described in these two high-quality studies. METHODS: A total of 1,744 patients with RA and 1,650 healthy controls of Spanish ancestry were studied. Polymorphisms were genotyped by single-base extension. SE genotypes of 736 patients were available from previous studies. Interaction analysis was done using multiple methods, including those originally reported and the most powerful methods described. RESULTS: Genotypes of one of the SNPs (rs4695888) failed quality control tests. The call rate for the other eight polymorphisms was 99.9%. The frequencies of the polymorphisms were similar in RA patients and controls, except for PTPN22 SNP. None of the interactions between PTPN22 SNPs and the six SNPs that met quality control tests was replicated as a significant interaction term--the originally reported finding--or with any of the other methods. Nor was the interaction between GSTM1 and the SE replicated as a departure from additivity in anti-CCP+ patients or with any of the other methods. CONCLUSIONS: None of the interactions tested were replicated in spite of sufficient power and assessment with different assays. These negative results indicate that whether interactions are significant contributors to RA susceptibility remains unknown and that strict standards need to be applied to claim that an interaction exists.

Lack of replication of higher genetic risk load in men than in women with systemic lupus erythematosus.

INTRODUCTION: We aimed to replicate a recent study which showed higher genetic risk load at 15 loci in men than in women with systemic lupus erythematosus (SLE). This difference was very significant, and it was interpreted as indicating that men require more genetic susceptibility than women to develop SLE. METHODS: Nineteen SLE-associated loci (thirteen of which are shared with the previous study) were analyzed in 1,457 SLE patients and 1,728 healthy controls of European ancestry. Genetic risk load was calculated as sex-specific sum genetic risk scores (GRS(s)). RESULTS: Our results did not replicate those of the previous study at either the level of individual loci or the global level of GRS(s). GRS(s) were larger in women than in men (4.20 ± 1.07 in women vs. 3.27 ± 0.98 in men). This very significant difference (P < 10(-16)) was more dependent on the six new loci not included in the previous study (59% of the difference) than on the thirteen loci that are shared (the remaining 41%). However, the 13 shared loci also showed a higher genetic risk load in women than in men in our study (P = 6.6 × 10(-7)), suggesting that heterogeneity of participants, in addition to different loci, contributed to the opposite results. CONCLUSION: Our results show the lack of a clear trend toward higher genetic risk in one of the sexes for the analyzed SLE loci. They also highlight several limitations of assessments of genetic risk load, including the possibility of ascertainment bias with loci discovered in studies that have included mainly women.

Assessment of osteoarthritis candidate genes in a meta-analysis of nine genome-wide association studies.

OBJECTIVE: To assess candidate genes for association with osteoarthritis (OA) and identify promising genetic factors and, secondarily, to assess the candidate gene approach in OA. METHODS: A total of 199 candidate genes for association with OA were identified using Human Genome Epidemiology (HuGE) Navigator. All of their single-nucleotide polymorphisms (SNPs) with an allele frequency of >5% were assessed by fixed-effects meta-analysis of 9 genome-wide association studies (GWAS) that included 5,636 patients with knee OA and 16,972 control subjects and 4,349 patients with hip OA and 17,836 control subjects of European ancestry. An additional 5,921 individuals were genotyped for significantly associated SNPs in the meta-analysis. After correction for the number of independent tests, P values less than 1.58 × 10(-5) were considered significant. RESULTS: SNPs at only 2 of the 199 candidate genes (COL11A1 and VEGF) were associated with OA in the meta-analysis. Two SNPs in COL11A1 showed association with hip OA in the combined analysis: rs4907986 (P = 1.29 × 10(-5) , odds ratio [OR] 1.12, 95% confidence interval [95% CI] 1.06-1.17) and rs1241164 (P = 1.47 × 10(-5) , OR 0.82, 95% CI 0.74-0.89). The sex-stratified analysis also showed association of COL11A1 SNP rs4908291 in women (P = 1.29 × 10(-5) , OR 0.87, 95% CI 0.82-0.92); this SNP showed linkage disequilibrium with rs4907986. A single SNP of VEGF, rs833058, showed association with hip OA in men (P = 1.35 × 10(-5) , OR 0.85, 95% CI 0.79-0.91). After additional samples were genotyped, association at one of the COL11A1 signals was reinforced, whereas association at VEGF was slightly weakened. CONCLUSION: Two candidate genes, COL11A1 and VEGF, were significantly associated with OA in this focused meta-analysis. The remaining candidate genes were not associated.

Identification of three new cis-regulatory IRF5 polymorphisms: in vitro studies.

BACKGROUND: Polymorphisms in the interferon regulatory factor 5 (IRF5) gene are associated with susceptibility to systemic lupus erythematosus, rheumatoid arthritis and other diseases through independent risk and protective haplotypes. Several functional polymorphisms are already known, but they do not account for the protective haplotypes that are tagged by the minor allele of rs729302. METHODS: Polymorphisms in linkage disequilibrium (LD) with rs729302 or particularly associated with IRF5 expression were selected for functional screening, which involved electrophoretic mobility shift assays (EMSAs) and reporter gene assays. RESULTS: A total of 54 single-nucleotide polymorphisms in the 5' region of IRF5 were genotyped. Twenty-four of them were selected for functional screening because of their high LD with rs729302 or protective haplotypes. In addition, two polymorphisms were selected for their prominent association with IRF5 expression. Seven of these twenty-six polymorphisms showed reproducible allele differences in EMSA. The seven were subsequently analyzed in gene reporter assays, and three of them showed significant differences between their two alleles: rs729302, rs13245639 and rs11269962. Haplotypes including the cis-regulatory polymorphisms correlated very well with IRF5 mRNA expression in an analysis based on previous data. CONCLUSION: We have found that three polymorphisms in LD with the protective haplotypes of IRF5 have differential allele effects in EMSA and in reporter gene assays. Identification of these cis-regulatory polymorphisms will allow more accurate analysis of transcriptional regulation of IRF5 expression, more powerful genetic association studies and deeper insight into the role of IRF5 in disease susceptibility.

Nesfatin-1 in human and murine cardiomyocytes: synthesis, secretion, and mobilization of GLUT-4.

Nesfatin-1, a satiety-inducing peptide identified in hypothalamic regions that regulate energy balance, is an integral regulator of energy homeostasis and a putative glucose-dependent insulin coadjuvant. We investigated its production by human cardiomyocytes and its effects on glucose uptake, in the main cardiac glucose transporter GLUT-4 and in intracellular signaling. Quantitative RT-PCR, Western blots, confocal immunofluorescence microscopy, and ELISA of human and murine cardiomyocytes and/or cardiac tissue showed that cardiomyocytes can synthesize and secrete nesfatin-1. Confocal microscopy of cultured cardiomyocytes after GLUT-4 labeling showed that nesfatin-1 mobilizes this glucose transporter to cell peripherals. The rate of 2-deoxy-D-[(3)H]glucose incorporation demonstrated that nesfatin-1 induces glucose uptake by HL-1 cells and cultured cardiomyocytes. Nesfatin-1 induced dose- and time-dependent increases in the phosphorylation of ERK1/2, AKT, and AS160. In murine and human cardiac tissue, nesfatin-1 levels varied with diet and coronary health. In conclusion, human and murine cardiomyocytes can synthesize and secrete nesfatin-1, which is able to induce glucose uptake and the mobilization of the glucose transporter GLUT-4 in these cells. Nesfatin-1 cardiac levels are regulated by diet and coronary health.

Genetic risk load and age at symptom onset of knee osteoarthritis.

To test whether a higher genetic risk load for knee osteoarthritis (OA) is associated with an earlier age at symptom onset. Six polymorphisms in GDF5, PTGS2, 7q22 locus, DVWA, DIO3, and ASPN that have been associated with knee OA were analyzed in 255 patients that had undergone total knee replacement (TKR) because of primary OA and in 457 healthy controls. We looked for association between the number of risk alleles in each patient and his age at symptom onset with linear regression and t-tests between the upper and lower quartiles. There was not even a weak trend in the direction of a younger age at symptom onset in the patients carrying more risk alleles. Patients in the upper quartile of age at symptom onset (67.0 ± 2.8 years) carried the same number of OA risk alleles (5.4 ± 1.4 vs. 5.3 ± 1.0) than patients in the lower quartile (44.6 ± 5.5 years). We did not find any evidence in support of the hypothesis of an earlier knee OA symptom onset associated with higher genetic risk load as determined by the six loci. This result suggests that old age and genetic risk act as independent factors in the pathogenesis of OA. It also indicates that designing OA genetic studies with patients selected for early symptom onset will not provide any substantial power gain.

Increased expression of fatty-acid and calcium metabolism genes in failing human heart.

BACKGROUND: Heart failure (HF) involves alterations in metabolism, but little is known about cardiomyopathy-(CM)-specific or diabetes-independent alterations in gene expression of proteins involved in fatty-acid (FA) uptake and oxidation or in calcium-(Ca(2+))-handling in the human heart. METHODS: RT-qPCR was used to quantify mRNA expression and immunoblotting to confirm protein expression in left-ventricular myocardium from patients with HF (n = 36) without diabetes mellitus of ischaemic (ICM, n = 16) or dilated (DCM, n = 20) cardiomyopathy aetiology, and non-diseased donors (CTL, n = 6). RESULTS: Significant increases in mRNA of genes regulating FA uptake (CD36) and intracellular transport (Heart-FA-Binding Protein (HFABP)) were observed in HF patients vs CTL. Significance was maintained in DCM and confirmed at protein level, but not in ICM. mRNA was higher in DCM than ICM for peroxisome-proliferator-activated-receptor-alpha (PPARA), PPAR-gamma coactivator-1-alpha (PGC1A) and CD36, and confirmed at the protein level for PPARA and CD36. Transcript and protein expression of Ca(2+)-handling genes (Two-Pore-Channel 1 (TPCN1), Two-Pore-Channel 2 (TPCN2), and Inositol 1,4,5-triphosphate Receptor type-1 (IP3R1)) increased in HF patients relative to CTL. Increases remained significant for TPCN2 in all groups but for TPCN1 only in DCM. There were correlations between FA metabolism and Ca(2+)-handling genes expression. In ICM there were six correlations, all distinct from those found in CTL. In DCM there were also six (all also different from those found in CTL): three were common to and three distinct from ICM. CONCLUSION: DCM-specific increases were found in expression of several genes that regulate FA metabolism, which might help in the design of aetiology-specific metabolic therapies in HF. Ca(2+)-handling genes TPCN1 and TPCN2 also showed increased expression in HF, while HF- and CM-specific positive correlations were found among several FA and Ca(2+)-handling genes.

Further evidence of subphenotype association with systemic lupus erythematosus susceptibility loci: a European cases only study.

INTRODUCTION: Systemic Lupus Erythematosus (SLE) shows a spectrum of clinical manifestations that complicate its diagnosis, treatment and research. This variability is likely related with environmental exposures and genetic factors among which known SLE susceptibility loci are prime candidates. The first published analyses seem to indicate that this is the case for some of them, but results are still inconclusive and we aimed to further explore this question. METHODS: European SLE patients, 1444, recruited at 17 centres from 10 countries were analyzed. Genotypes for 26 SLE associated SNPs were compared between patients with and without each of 11 clinical features: ten of the American College of Rheumatology (ACR) classification criteria (except ANAs) and age of disease onset. These analyses were adjusted for centre of recruitment, top ancestry informative markers, gender and time of follow-up. Overlap of samples with previous studies was excluded for assessing replication. RESULTS: THERE WERE THREE NEW ASSOCIATIONS: the SNPs in XKR6 and in FAM167A-BLK were associated with lupus nephritis (OR=0.76 and 1.30, P(corr) =0.007 and 0.03, respectively) and the SNP of MECP2, which is in chromosome X, with earlier age of disease onset in men. The previously reported association of STAT4 with early age of disease onset was replicated. Some other results were suggestive of the presence of additional associations. Together, the association signals provided support to some previous findings and to the characterization of lupus nephritis, autoantibodies and age of disease onset as the clinical features more associated with SLE loci. CONCLUSION: Some of the SLE loci shape the disease phenotype in addition to increase susceptibility to SLE. This influence is more prominent for some clinical features than for others. However, results are only partially consistent between studies and subphenotype specific GWAS are needed to unravel their genetic component.