Objectives

Altered signaling in B-cells is a predominant feature of systemic lupus erythematosus (SLE). The genes BANK1 and BLK were recently described as associated with SLE. BANK1 codes for a B-cell-specific cytoplasmic protein involved in B-cell receptor signaling and BLK codes for an Src tyrosine kinase with important roles in B-cell development. To characterize the role of BANK1 and BLK in SLE, we performed a genetic interaction analysis hypothesizing that genetic interactions could reveal functional pathways relevant to disease pathogenesis.

Methods

We Used the method GPAT16 to analyze the gene-gene interactions of BANK1 and BLK. Confocal microscopy was used to investigate co-localization, and immunoprecipitation was used to verify the physical interaction of BANK1 and BLK.

Results

Epistatic interactions between BANK1 and BLK polymorphisms associated with SLE were observed in a discovery set of 279 patients and 515 controls from Northern Europe. A meta-analysis with 4399 European individuals confirmed the genetic interactions between BANK1 and BLK.

As BANK1 was identified as a binding partner of the Src tyrosine kinase LYN, we tested the possibility that BANK1 and BLK could also show a protein-protein interaction. We demonstrated co-immunoprecipitation and co-localization of BLK and BANK1. In a Daudi cell line and primary naïve B-cells the endogenous binding was enhanced upon B-cell receptor stimulation using anti-IgM antibodies.

Conclusions

Here, we show a genetic interaction between BANK1 and BLK, and demonstrate that these molecules interact physically. Our results have important consequences for the understanding of SLE and other autoimmune diseases and identify a potential new signaling pathway.

Osteopontin is a proinflammatory molecule, modulating TH1 and TH17 responses. Several reports suggest its involvement in multiple sclerosis (MS) pathogenesis. We previously reported that OPN gene variations at the 3′ end are a predisposing factor for MS development and evolution. In this paper, we extended our analysis to a gene variation at the 5′ end on the −156G > GG single nucleotide polymorphism (SNP) and replicated our previous findings at the 3′ end on the +1239A > C SNP. We found that only +1239A > C SNP displayed a statistically significant association with MS development, but both +1239A > C and −156G > GG had an influence on MS progression, since patients homozygous for both +1239A and −156GG alleles displayed slower progression of disability and slower switch to secondary progression than those carrying +1239C and/or −156G and those homozygous for +1239A only. Moreover, patients homozygous for +1239A also displayed a significantly lower relapse rate than those carrying +1239C, which is in line with the established role of OPN in MS relapses.

Background

Iron involvement/imbalance is strongly suspected in multiple sclerosis (MS) etiopathogenesis, but its role is quite debated. Iron deposits encircle the veins in brain MS lesions, increasing local metal concentrations in brain parenchyma as documented by magnetic resonance imaging and histochemical studies. Conversely, systemic iron overload is not always observed. We explored the role of common single nucleotide polymorphisms (SNPs) in the main iron homeostasis genes in MS patients.

Methods

By the pyrosequencing technique, we investigated 414 MS cases [Relapsing-remitting (RR), n=273; Progressive, n=141, of which: Secondary (SP), n=103 and Primary (PP), n=38], and 414 matched healthy controls. Five SNPs in 4 genes were assessed: hemochromatosis (HFE: C282Y, H63D), ferroportin (FPN1: -8CG), hepcidin (HEPC: -582AG), and transferrin (TF: P570S).

Results

The FPN1-8GG genotype was overrepresented in the whole MS population (OR=4.38; 95%CI, 1.89-10.1; P<0.0001) and a similar risk was found among patients with progressive forms. Conversely, the HEPC -582GG genotype was overrepresented only in progressive forms (OR=2.53; 95%CI, 1.34-4.78; P=0.006) so that SP and PP versus RR yielded significant outputs (P=0.009). For almost all SNPs, MS disability score (EDSS), severity score (MSSS), as well as progression index (PI) showed a significant increase when comparing homozygotes versus individuals carrying other genotypes: HEPC -582GG (EDSS, 4.24±2.87 vs 2.78±2.1; P=0.003; MSSS, 5.6±3.06 vs 3.79±2.6; P=0.001); FPN1-8GG (PI, 1.11±2.01 vs 0.6±1.31; P=0.01; MSSS, 5.08±2.98 vs 3.85±2.8; P=0.01); HFE 63DD (PI, 1.63±2.6 vs 0.6±0.86; P=0.009). Finally, HEPC -582G-carriers had a significantly higher chance to switch into the progressive form (HR=3.55; 1.83-6.84; log-rank P=0.00006).

Conclusions

Polymorphisms in the genes coding for iron binding and transporting proteins, in the presence of local iron overload, might be responsible for suboptimal iron handling. This might account for the significant variability peculiar to MS phenotypes, particularly affecting MS risk and progression paving the way for personalized pharmacogenetic applications in the clinical practice.

Objectives

To perform fine mapping of the autoimmunity susceptibility gene BLK and identify functional variants involved in SLE.

Methods

Genotyping of 1163 European SLE patients and 1482 controls and imputation were performed covering the BLK gene with 158 SNPs. Logistic regression analysis was done using PLINK and conditional analyses using GENABEL’s test score. Transfections of BLK constructs on HEK293 cells containing the novel mutation or the wild-type form were made to analyse their effect on protein half-life using a protein stability assay, cycloheximide and Western blot. CHiP-qPCR for NFkB binding.

Results

Fine mapping of BLK identified two independent genetic effects with functional consequences: one represented by two tightly linked associated haplotype blocks significantly enriched for NFκB-binding sites and numerous putative regulatory variants whose risk alleles correlated with low BLK mRNA levels. Binding of NFkBp50 and p65 to an associated 1.2Kb haplotype segment was confirmed. A second independent genetic effect was represented by an Ala71Thr, low-frequency missense substitution with an OR = 2.31 (95% c.i. 1.38–3.86). The 71Thr decreased BLK protein half-life.

Conclusions

Our results show that rare and common regulatory variants in BLK are involved in disease susceptibility and both, albeit independently lead to reduced levels of BLK protein.

Multiple sclerosis (OMIM 126200) is a common disease of the central nervous system in which the interplay between inflammatory and neurodegenerative processes typically results in intermittent neurological disturbance followed by progressive accumulation of disability.1 Epidemiological studies have shown that genetic factors are primarily responsible for the substantially increased frequency of the disease seen in the relatives of affected individuals;2,3 and systematic attempts to identify linkage in multiplex families have confirmed that variation within the Major Histocompatibility Complex (MHC) exerts the greatest individual effect on risk.4 Modestly powered Genome-Wide Association Studies (GWAS)5-10 have enabled more than 20 additional risk loci to be identified and have shown that multiple variants exerting modest individual effects play a key role in disease susceptibility.11 Most of the genetic architecture underlying susceptibility to the disease remains to be defined and is anticipated to require the analysis of sample sizes that are beyond the numbers currently available to individual research groups. In a collaborative GWAS involving 9772 cases of European descent collected by 23 research groups working in 15 different countries, we have replicated almost all of the previously suggested associations and identified at least a further 29 novel susceptibility loci. Within the MHC we have refined the identity of the DRB1 risk alleles and confirmed that variation in the HLA-A gene underlies the independent protective effect attributable to the Class I region. Immunologically relevant genes are significantly over-represented amongst those mapping close to the identified loci and particularly implicate T helper cell differentiation in the pathogenesis of multiple sclerosis.

Background

neuropeptide S (NPS) and its receptor NPSR1 act along the hypothalamic-pituitary-adrenal axis to modulate anxiety, fear responses, nociception and inflammation. The importance of the NPS-NPSR1 signaling pathway is highlighted by the observation that, in humans, NPSR1 polymorphism associates with asthma, inflammatory bowel disease, rheumatoid arthritis, panic disorders, and intermediate phenotypes of functional gastrointestinal disorders. Because of the genetic complexity at the NPSR1 locus, however, true causative variations remain to be identified, together with their specific effects on receptor expression or function. To gain insight into the mechanisms leading to NPSR1 disease-predisposing effects, we performed a thorough functional characterization of all NPSR1 promoter and coding SNPs commonly occurring in Caucasians (minor allele frequency >0.02).

Principal Findings

we identified one promoter SNP (rs2530547 [−103]) that significantly affects luciferase expression in gene reporter assays and NPSR1 mRNA levels in human leukocytes. We also detected quantitative differences in NPS-induced genome-wide transcriptional profiles and CRE-dependent luciferase activities associated with three NPSR1 non-synonymous SNPs (rs324981 [Ile107Asn], rs34705969 [Cys197Phe], rs727162 [Arg241Ser]), with a coding variant exhibiting a loss-of-function phenotype (197Phe). Potential mechanistic explanations were sought with molecular modelling and bioinformatics, and a pilot study of 2230 IBD cases and controls provided initial support to the hypothesis that different cis-combinations of these functional SNPs variably affect disease risk.

Significance

these findings represent a first step to decipher NPSR1 locus complexity and its impact on several human conditions NPS antagonists have been recently described, and our results are of potential pharmacogenetic relevance.

Systemic Lupus Erythematosus (SLE) is an autoimmune disease with a very varied spectrum of clinical manifestations that could be partly determined by genetic factors. We aimed to determine the relationship between prevalence of 11 clinical features and age of disease onset with European population genetic substructure. Data from 1413 patients of European ancestry recruited in nine countries was tested for association with genotypes of top ancestry informative markers. This analysis was done with logistic regression between phenotypes and genotypes or principal components extracted from them. We used a genetic additive model and adjusted for gender and disease duration. Three clinical features showed association with ancestry informative markers: autoantibody production defined as immunologic disorder (P = 6.8×10−4), oral ulcers (P = 6.9×10−4) and photosensitivity (P = 0.002). Immunologic disorder was associated with genotypes more common in Southern European ancestries, whereas the opposite trend was observed for photosensitivity. Oral ulcers were specifically more common in patients of Spanish and Portuguese self-reported ancestry. These results should be taken into account in future research and suggest new hypotheses and possible underlying mechanisms to be investigated. A first hypothesis linking photosensitivity with variation in skin pigmentation is suggested.

Several single nucleotide polymorphism (SNP) genome-wide association studies (GWAS) have been completed in multiple sclerosis. Follow-up studies of the variants with the most promising rankings, especially when supplemented by informed candidate gene selection, have proven to be extremely successful. We report here the results of a multi-stage replication analysis of the putatively associated SNPs identified in the Wellcome Trust Case-Control Consortium non-synonymous SNP screen. In total the replication sample consisted of 3444 patients and 2595 controls. A combined analysis of the nsSNP screen and replication data provides evidence implicating a novel additional locus, rs3748816 in MMEL1 (OR=1.16, p=3.54×10−6) in multiple sclerosis susceptibility.

Studies suggest that different autoimmune diseases share a common genetic background, in particular, an overlap between multiple sclerosis (MS) and type 1 diabetes (T1D) susceptibility loci has been established. A recent study found that four rare SNPs in the IFIH1 (interferon induced with helicase C domain 1) were significantly associated with T1D.

To establish if these SNPs were also involved in MS susceptibility, we chose to examine the nonsynonymous SNP rs35667974/Ile923Val which displayed the strongest effect in T1D and was also shown to lead to a loss of IFIH1 function in an in vitro study. We have performed the first association study to test if this rare variant is involved in MS susceptibility in a very large sample consisting of 3037 MS patients and 10657 healthy controls recruited from Italy and the UK. This study has 99% power to demonstrate an association at the 5% level with this rare variant. Our analysis shows that the nsSNP rs35667974/Ile923Val does not have a role in susceptibility to MS.

Background

Albeit several studies pointed out the pivotal role that CD4+T cells have in Multiple Sclerosis, the CD8+ T cells involvement in the pathology is still in its early phases of investigation. Proteasome degradation is the key step in the production of MHC class I-restricted epitopes and therefore its activity could be an important element in the activation and regulation of autoreactive CD8+ T cells in Multiple Sclerosis.

Methodology/Principal Findings

Immunoproteasomes and PA28-αβ regulator are present in MS affected brain area and accumulated in plaques. They are expressed in cell types supposed to be involved in MS development such as neurons, endothelial cells, oligodendrocytes, macrophages/macroglia and lymphocytes. Furthermore, in a genetic study on 1262 Italian MS cases and 845 controls we observed that HLA-A*02+ female subjects carrying the immunoproteasome LMP2 codon 60HH variant have a reduced risk to develop MS. Accordingly, immunoproteasomes carrying the LMP2 60H allele produce in vitro a lower amount of the HLA-A*0201 restricted immunodominant epitope MBP111–119.

Conclusion/Significance

The immunoproteasome LMP2 60HH variant reduces the risk to develop MS amongst Italian HLA-A*02+ females. We propose that such an effect is mediated by the altered proteasome-dependent production of a specific MBP epitope presented on the MHC class I. Our observations thereby support the hypothesis of an involvement of immunoproteasome in the MS pathogenesis.

A gain-of-function R620W polymorphism in the PTPN22 gene, encoding the lymphoid tyrosine phosphatase LYP, has recently emerged as an important risk factor for human autoimmunity. Here we report that another missense substitution (R263Q) within the catalytic domain of LYP leads to reduced phosphatase activity. High-resolution structural analysis revealed the molecular basis for this loss of function. Furthermore, the Q263 variant conferred protection against human systemic lupus erythematosus, reinforcing the proposal that inhibition of LYP activity could be beneficial in human autoimmunity.

Introduction

We aimed to replicate association of newly identified systemic lupus erythematosus (SLE) loci.

Methods

We selected the most associated SNP in 10 SLE loci. These 10 SNPs were analysed in 1,579 patients with SLE and 1,726 controls of European origin by single-base extension. Comparison of allele frequencies between cases and controls was done with the Mantel–Haenszel approach to account for heterogeneity between sample collections.

Results

A previously controversial association with a SNP in the TYK2 gene was replicated (odds ratio (OR) = 0.79, P = 2.5 × 10-5), as well as association with the X chromosome MECP2 gene (OR = 1.26, P = 0.00085 in women), which had only been reported in a single study, and association with four other loci, 1q25.1 (OR = 0.81, P = 0.0001), PXK (OR = 1.19, P = 0.0038), BANK1 (OR = 0.83, P = 0.006) and KIAA1542 (OR = 0.84, P = 0.001), which have been identified in a genome-wide association study, but not found in any other study. All these replications showed the same disease-associated allele as originally reported. No association was found with the LY9 SNP, which had been reported in a single study.

Conclusions

Our results confirm nine SLE loci. For six of them, TYK2, MECP2, 1q25.1, PXK, BANK1 and KIAA1542, this replication is important. The other three loci, ITGAM, STAT4 and C8orf13-BLK, were already clearly confirmed. Our results also suggest that MECP2 association has no influence in the sex bias of SLE, contrary to what has been proposed. In addition, none of the other associations seems important in this respect.

Immune-mediated nephritis contributes to disease in systemic lupus erythematosus, Goodpasture syndrome (caused by antibodies specific for glomerular basement membrane [anti-GBM antibodies]), and spontaneous lupus nephritis. Inbred mouse strains differ in susceptibility to anti-GBM antibody–induced and spontaneous lupus nephritis. This study sought to clarify the genetic and molecular factors that may be responsible for enhanced immune-mediated renal disease in these models. When the kidneys of 3 mouse strains sensitive to anti-GBM antibody–induced nephritis were compared with those of 2 control strains using microarray analysis, one-fifth of the underexpressed genes belonged to the kallikrein gene family, which encodes serine esterases. Mouse strains that upregulated renal and urinary kallikreins exhibited less evidence of disease. Antagonizing the kallikrein pathway augmented disease, while agonists dampened the severity of anti-GBM antibody–induced nephritis. In addition, nephritis-sensitive mouse strains had kallikrein haplotypes that were distinct from those of control strains, including several regulatory polymorphisms, some of which were associated with functional consequences. Indeed, increased susceptibility to anti-GBM antibody–induced nephritis and spontaneous lupus nephritis was achieved by breeding mice with a genetic interval harboring the kallikrein genes onto a disease-resistant background. Finally, both human SLE and spontaneous lupus nephritis were found to be associated with kallikrein genes, particularly KLK1 and the KLK3 promoter, when DNA SNPs from independent cohorts of SLE patients and controls were compared. Collectively, these studies suggest that kallikreins are protective disease-associated genes in anti-GBM antibody–induced nephritis and lupus.

Objectives

To perform fine mapping of the autoimmunity susceptibility gene BLK and identify functional variants involved in systemic lupus erythematosus (SLE).

Methods

Genotyping of 1163 European SLE patients and 1482 controls and imputation were performed covering the BLK gene with 158 single-nucleotide polymorphisms. Logistic regression analysis was done using PLINK and conditional analyses using GENABEL's test score. Transfections of BLK constructs on HEK293 cells containing the novel mutation or the wild type form were analysed for their effect on protein half-life using a protein stability assay, cycloheximide and western blot. CHiP-qPCR for detection of nuclear factor κ B (NFkB) binding.

Results

Fine mapping of BLK identified two independent genetic effects with functional consequences: one represented by two tightly linked associated haplotype blocks significantly enriched for NFκB-binding sites and numerous putative regulatory variants whose risk alleles correlated with low BLK mRNA levels. Binding of NFkBp50 and p65 to an associated 1.2 Kb haplotype segment was confirmed. A second independent genetic effect was represented by an Ala71Thr, low-frequency missense substitution with an OR=2.31 (95% CI 1.38 to 3.86). The 71Thr decreased BLK protein half-life.

Conclusions

These results show that rare and common regulatory variants in BLK are involved in disease susceptibility and both, albeit independently, lead to reduced levels of BLK protein.