MHC associations with clinical and autoantibody manifestations in European SLE.

Systemic lupus erythematosus (SLE) is a clinically heterogeneous disease affecting multiple organ systems and characterized by autoantibody formation to nuclear components. Although genetic variation within the major histocompatibility complex (MHC) is associated with SLE, its role in the development of clinical manifestations and autoantibody production is not well defined. We conducted a meta-analysis of four independent European SLE case collections for associations between SLE sub-phenotypes and MHC single-nucleotide polymorphism genotypes, human leukocyte antigen (HLA) alleles and variant HLA amino acids. Of the 11 American College of Rheumatology criteria and 7 autoantibody sub-phenotypes examined, anti-Ro/SSA and anti-La/SSB antibody subsets exhibited the highest number and most statistically significant associations. HLA-DRB1*03:01 was significantly associated with both sub-phenotypes. We found evidence of associations independent of MHC class II variants in the anti-Ro subset alone. Conditional analyses showed that anti-Ro and anti-La subsets are independently associated with HLA-DRB1*0301, and that the HLA-DRB1*03:01 association with SLE is largely but not completely driven by the association of this allele with these sub-phenotypes. Our results provide strong evidence for a multilevel risk model for HLA-DRB1*03:01 in SLE, where the association with anti-Ro and anti-La antibody-positive SLE is much stronger than SLE without these autoantibodies.

High-resolution haplotype structure in the human genome.

Linkage disequilibrium (LD) analysis is traditionally based on individual genetic markers and often yields an erratic, non-monotonic picture, because the power to detect allelic associations depends on specific properties of each marker, such as frequency and population history. Ideally, LD analysis should be based directly on the underlying haplotype structure of the human genome, but this structure has remained poorly understood. Here we report a high-resolution analysis of the haplotype structure across 500 kilobases on chromosome 5q31 using 103 single-nucleotide polymorphisms (SNPs) in a European-derived population. The results show a picture of discrete haplotype blocks (of tens to hundreds of kilobases), each with limited diversity punctuated by apparent sites of recombination. In addition, we develop an analytical model for LD mapping based on such haplotype blocks. If our observed structure is general (and published data suggest that it may be), it offers a coherent framework for creating a haplotype map of the human genome.

A susceptibility locus for asthma-related traits on chromosome 7 revealed by genome-wide scan in a founder population.

The genetics of asthma and atopy have been difficult to determine because these diseases are genetically heterogeneous and modified by environment. The pedigrees in our study (n=86) originate in eastern central Finland (Kainuu province). According to census records, this region had only 200 households (2,000 inhabitants) in the mid sixteenth to mid seventeenth centuries. The current population of 100,000 represents the expansion of these founders within the past 400 years. Because this population is relatively homogeneous, we hypothesized that the molecular genetic mechanisms underlying asthma might also have reduced heterogeneity and therefore be easier to dissect than in mixed populations. A recent twin family study supported a strong genetic component for asthma in Finland. We carried out a genome-wide scan for susceptibility loci in asthma in the Kainuu subpopulation. We identified two regions of suggestive linkage and studied them further with higher-density mapping. We obtained evidence for linkage in a 20-cM region of chromosome 7p14-p15 for three phenotypes: asthma, a high level of immunoglobulin E (IgE; atopy) and the combination of the phenotypes. The strongest linkage was seen for high serum IgE (non-parametric linkage (NPL) score 3.9, P=0.0001), exceeding the threshold for genome-wide significance based on simulations. We also observed linkage between this locus and asthma or atopy in two independent data sets.

Genetic variation in the 5q31 cytokine gene cluster confers susceptibility to Crohn disease.

Linkage disequilibrium (LD) mapping provides a powerful method for fine-structure localization of rare disease genes, but has not yet been widely applied to common disease. We sought to design a systematic approach for LD mapping and apply it to the localization of a gene (IBD5) conferring susceptibility to Crohn disease. The key issues are: (i) to detect a significant LD signal (ii) to rigorously bound the critical region and (iii) to identify the causal genetic variant within this region. We previously mapped the IBD5 locus to a large region spanning 18 cM of chromosome 5q31 (P<10(-4)). Using dense genetic maps of microsatellite markers and single-nucleotide polymorphisms (SNPs) across the entire region, we found strong evidence of LD. We bound the region to a common haplotype spanning 250 kb that shows strong association with the disease (P< 2 x 10(-7)) and contains the cytokine gene cluster. This finding provides overwhelming evidence that a specific common haplotype of the cytokine region in 5q31 confers susceptibility to Crohn disease. However, genetic evidence alone is not sufficient to identify the causal mutation within this region, as strong LD across the region results in multiple SNPs having equivalent genetic evidence-each consistent with the expected properties of the IBD5 locus. These results have important implications for Crohn disease in particular and LD mapping in general.

A targeted association study in systemic lupus erythematosus identifies multiple susceptibility alleles.

Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease. Multiple genetic and environmental factors contribute to the pathogenesis of this disease. Recent genome-wide association studies have added substantially to the number of genes associated with SLE. To replicate some of these susceptibility loci, single-nucleotide polymorphisms reported to be associated to SLE were evaluated in a cohort of 245 well-phenotyped Canadian SLE trios. Our results replicate previously reported associations to alleles of interferon regulatory factor 5 (IRF5), major histocompatibility complex (MHC), tumor necrosis factor (ligand) superfamily member 4 (TNFSF4), Kell blood group complex subunit-related family member 6 (XKR6), B-cell scaffold protein with ankyrin repeats 1 (BANK1), protein tyrosine phosphatase non-receptor type 22 (PTPN22), ubiquitin-conjugating enzyme E2L 3 (UBE2L3) and islet cell autoantigen 1 (ICA1). We also identify putative associations to cytotoxic T-lymphocyte-associated protein 4 (CTLA4), a gene associated with several autoimmune disorders, and ERBB3, a locus on 12q13 that was previously reported to be associated with type 1 diabetes. This study confirms the existence of multiple genetic risk factors for SLE, and supports the notion that some risk factors for SLE are shared with other inflammatory disorders.

Genetic variants in the region harbouring IL2/IL21 associated with ulcerative colitis.

OBJECTIVES: Genetic susceptibility is known to play a large part in the predisposition to the inflammatory bowel diseases (IBDs) known as Crohn's disease (CD) and ulcerative colitis (UC). The IL2/IL21 locus on 4q27 is known to be a common risk locus for inflammatory disease (shown in coeliac disease, type 1 diabetes, rheumatoid arthritis, systemic lupus erythematosus and psoriasis), while the roles that interleukin 2 (IL2) and IL21 play in the immune response also make them attractive candidates for IBD. The objective of this study was to test for association between the IL2/IL21 locus and the IBDs. METHODS: The four single nucleotide polymorphisms (SNPs) in the IL2/IL21 locus most associated with coeliac disease were genotyped in 1590 subjects with IBD and 929 controls from The Netherlands, and then replicated in a North American cohort (2387 cases and 1266 controls) and an Italian cohort (805 cases and 421 controls), yielding a total of 4782 cases (3194 UC, 1588 CD) and 2616 controls. Allelic association testing and a pooled analysis using a Cochran-Mantel-Haenszel test were performed. RESULTS: All four SNPs were strongly associated with UC in all three cohorts and reached genome-wide significance in the pooled analysis (rs13151961 p = 1.35 x 10(-10), rs13119723 p = 8.60 x 10(-8), rs6840978 p = 3.0 7x 10(-8), rs6822844 p = 2.77 x 10(-9)). A moderate association with CD was also found in the pooled analysis (p value range 0.0016-9.86 x 10(-5)). CONCLUSIONS: A strong association for the IL2/IL21 locus with UC was found, which also confirms it as a general susceptibility locus for inflammatory disease.

Association of LY9 in UK and Canadian SLE families.

Systemic lupus erythematosus (SLE) is a complex disease trait of unknown aetiology. Genome-wide linkage studies in human SLE identified several linkage regions, including one at 1q23, which contains multiple susceptibility genes, including the members of the signalling lymphocyte activation molecule (SLAM) locus. In mice there is a syntenic linkage region, Sle1. The SLAM genes are functionally related cell-surface receptors, which regulate signal transduction of cells in the immune system. Family-based association study in UK and Canadian SLE families identified variants in the promoter and coding region of SLAMF7 and LY9 contributing to SLE disease susceptibility. The strongest association was from rs509749, in exon 8 of LY9 (P=0.00209). rs509749 encodes a Val/Met nonsynonymous change in amino acid 602 in the cytoplasmic domain of LY9. In the parents and affected individuals from the Canadian SLE families, the risk allele of rs509049 skews the T-cell population by increasing the number of CD8+ memory T cells, while decreasing the proportion of CD4+ naïve T cells and activated T cells. Since rs509749 lies within the consensus binding site for SAP/SH2D1a, which influences downstream signalling events from LY9, the mechanism for increased CD8+ memory T cells may include differential binding SAP/SH2D1a to the cytoplasmic domain of LY9.

MAST3: a novel IBD risk factor that modulates TLR4 signaling.

Inflammatory bowel disease (IBD) is a chronic disorder caused by multiple factors in a genetically susceptible host. Significant advances in the study of genetic susceptibility have highlighted the importance of the innate immune system in this disease. We previously completed a genome-wide linkage study and found a significant locus (IBD6) on chromosome 19p. We were interested in identifying the causal variant in IBD6. We performed a two-stage association mapping study. In stage 1, 1530 single-nucleotide polymorphisms (SNPs) were selected from the HapMap database and genotyped in 761 patients with IBD. Among the SNPs that passed the threshold for replication, 26 were successfully genotyped in 754 additional patients (stage 2). One intronic variant, rs273506, located in the microtubule-associated serine/threonine-protein kinase gene-3 (MAST3), was found to be associated in both stages (pooled P=1.8 x 10(-4)). We identified four MAST3 coding variants, including a non-synonymous SNP rs8108738, correlated to rs273506 and associated with IBD. To test whether MAST3 was expressed in cells of interest, we performed expression assays, which showed abundant expression of MAST3 in antigen-presenting cells and in lymphocytes. The knockdown of MAST3 specifically decreased Toll-like receptor-4-dependent NF-kappaB activity. Our findings are additional proofs of the pivotal role played by modulators of NF-kappaB activity in IBD pathogenesis.

An SNP linkage scan identifies significant Crohn's disease loci on chromosomes 13q13.3 and, in Jewish families, on 1p35.2 and 3q29.

Inflammatory bowel disease (IBD) is a complex genetic disorder of two major phenotypes, Crohn's disease (CD) and ulcerative colitis (UC), with increased risk in Ashkenazi Jews. Twelve genome-wide linkage screens have identified multiple loci, but these screens have been of modest size and have used low-density microsatellite markers. We, therefore, performed a high-density single-nucleotide polymorphism (SNP) genome-wide linkage study of 993 IBD multiply affected pedigrees (25% Jewish ancestry) that contained 1709 IBD-affected relative pairs, including 919 CD-CD pairs and 312 UC-UC pairs. We identified a significant novel CD locus on chromosome 13p13.3 (peak logarithm of the odds (LOD) score=3.98) in all pedigrees, significant linkage evidence on chromosomes 1p35.1 (peak LOD score=3.5) and 3q29 (peak LOD score=3.19) in Jewish CD pedigrees, and suggestive loci for Jewish IBD on chromosome 10q22 (peak LOD score=2.57) and Jewish UC on chromosome 2q24 (peak LOD score=2.69). Nominal or greater linkage evidence was present for most previously designated IBD loci (IBD1-9), notably, IBD1 for CD families at chromosome 16q12.1 (peak LOD score=4.86) and IBD6 in non-Jewish UC families at chromosome 19p12 (peak LOD score=2.67). This study demonstrates the ability of high information content adequately powered SNP genome-wide linkage studies to identify loci not observed in multiple microsatellite-based studies in smaller cohorts.

Anti-DNA and anti-platelet specificities of SLE-derived autoantibodies: evidence for CDR2H mutations and CDR3H motifs.

Although polyreactivity appears to be a characteristic feature of natural autoantibodies, polyreactive anti-DNA autoantibodies can be derived both from patients with autoimmune disease and from normal individuals. It is unclear whether these autoantibodies differ depending on their origin, but previous studies from our laboratory have suggested that polyreactive systemic lupus erythematosus (SLE)-derived platelet-binding anti-DNA autoantibodies have more restricted antigen reactivity and greater functional activity than normal-derived polyreactive autoantibodies. The objective of the present study was to characterize the VH and VL region sequences of 10 human hybridoma anti-DNA autoantibodies derived from peripheral blood lymphocytes of different origins [SLE, rheumatoid arthritis (RA), or normal] to determine whether there are structural differences between these autoantibodies. We show that although some unmutated germline structures (VH and VL) are represented, these are not restricted to anti-DNA autoantibodies from normal individuals and that two normal-derived anti-DNA antibodies showed quite extensively mutated VH genes. However, these mutations, unlike those found in the CDR2H of several of the SLE-derived antibodies, did not appear to be antigen-selected. Three different amino acid motifs, putatively involved in antigen binding specificity, were observed in the CDR3H segments of some of the autoantibodies. One was the previously described YYGSG motif, which was found in a normal-derived anti-DNA autoantibody, while two new potential motifs were observed only in SLE-derived platelet-binding anti-DNA autoantibodies. These data suggest that antigenic and functional differences between SLE-derived and normal-derived platelet-binding anti-DNA autoantibodies may be due to antigen-selected mutations in the CDR2H and specific amino acid motifs in the CDR3H.

Restricted variable region gene usage and possible rheumatoid factor relationship among human monoclonal antibodies specific for the AD-1 epitope on cytomegalovirus glycoprotein B.

The nucleotide sequences of the variable region genes encoding five different human, high affinity antibodies, specific for the major neutralization determinant (AD-1) expressed by human cytomegalovirus glycoprotein B (gp58/116), have been determined. Three of the five heavy chain variable regions belonged to the small VHV-family, although they combined with a diverse set of light chains (V kappa IIIb, V lambda II and V lambda III). The other two antibodies belonged to VH-families III and IV. One of the VHV-family genes most likely originated from a previously unreported germline gene or allele, since it carries a nine nucleotide insert in framework 1. In addition, V lambda-genes showed variable homology (77-95%) to known germline sequences, while V kappa-genes showed high homology (approximately 98%) with their proposed germline origin. Despite the close homology of the V kappa IIIb-gene used to express one of the antibodies with its corresponding germline gene, the protein did not strongly express some idiotypes associated with this light chain family. There is, thus, no direct relation between the expression of these crossreactive idiotypes and the use of even modestly mutated light chains belonging to this V kappa-family, which has been implicated in the development of anti-idiotypic networks possibly inducing autoantibodies, such as rheumatoid factors.

Structural characteristics of four human hybridoma antibodies specific for the pp65 protein of the human cytomegalovirus and their relationship to human rheumatoid factors.

Four human hybridoma antibodies directed against the human cytomegalovirus (CMV) were characterized with respect to their immunoglobulin gene usage and expression of rheumatoid factor (RF) associated idiotypes and variable region epitopes. The aims of these experiments were: (1) to characterize the immunoglobulin gene usage of four antibodies directed against a single protein of a human pathogen; and (2) to examine how this humoral response may be linked to the production of RFs, autoantibodies found in the majority of patients with rheumatoid arthritis (RA). All four anti-CMV antibodies were of the gamma heavy chain isotype and were specific for the immunodominant 65 kDa viral matrix phosphoprotein (pp65). The four anti-pp65 antibodies expressed different light (L) and heavy (H) chain variable region gene combinations. These were: VkIII/VH3, V lambda 1/VH3, V lambda 1/VH4 and V lambda 3/VH3, respectively for the HCV-2, HCV-3, HCV-63 and HCV-65 hybridoma cell lines. Although none had RF activity, each of these antibodies expressed a unique set of RF-associated determinants, implying different three-dimensional configurations of the variable regions of these antibodies. The HCV-2 antibody, however, had the most extensive similarities to human RFs since it not only expressed the greatest number of RF-associated determinants but also had a protein sequence that was very homologous to RFs of the "Po" idiotypic family. Furthermore, predicted germline gene usage by anti-CMV antibodies and RFs suggest that some are encoded by identical or similar genes and that the different specificities are achieved by somatic mutations in the L and H chain complementarity determining regions (CDRs) and genetic diversity in the H chain CDR3.

Integrating risk factors: HLA-DRB1*1501 and Epstein-Barr virus in multiple sclerosis.

BACKGROUND: Individuals with high levels of antibodies to the Epstein-Barr virus nuclear antigen 1 (EBNA-1) have an increased risk of developing multiple sclerosis (MS), but this association could be confounded by genetic susceptibility. METHODS: We conducted a nested case-control study including 148 women with MS (18 with blood collected before disease onset) and 296 age-matched healthy women to determine whether the human leukocyte antigen (HLA) DRB1*1501 allele (DR15) and anti-Epstein-Barr virus (anti-EBV) antibody titers are independent risk factors for MS. RESULTS: The association between anti-EBNA-1 antibody titers and MS risk was not affected by adjustment for DR15 and was similar in DR15-positive and DR15-negative women. The relative risk of MS among DR15-positive women with elevated (>1:320) anti-EBNA-1 titers was ninefold higher than that of DR15-negative women with low (<1:80) anti-EBNA-1 titers. CONCLUSIONS: Anti-Epstein-Barr virus nuclear antigen 1 (anti-EBNA-1) antibody titers are a risk factor for multiple sclerosis (MS), independently from the DR15 allele. Carriers of the DR15 allele with elevated anti-EBNA-1 antibody titers may have a markedly increased risk of MS.

Gene-centric association mapping of chromosome 3p implicates MST1 in IBD pathogenesis.

Association mapping and candidate gene studies within inflammatory bowel diseases (IBD) linkage regions, as well as genome-wide association studies in Crohn's disease (CD) have led to the discovery of multiple risk genes, but these explain only a fraction of the genetic susceptibility observed in IBD. We have thus been pursuing a region on chromosome 3p21-22 showing linkage to CD and ulcerative colitis (UC) using a gene-centric association mapping approach. We identified 12 functional candidate genes by searching for literature cocitations with relevant keywords and for gene expression patterns consistent with immune/intestinal function. We then performed an association study composed of a screening phase, where tagging single nucleotide polymorphisms (SNPs) were evaluated in 1,020 IBD patients, and an independent replication phase in 745 IBD patients. These analyses identified and replicated significant association with IBD for four SNPs within a 1.2 Mb linkage disequilibrium region. We then identified a non-synonymous coding variant (rs3197999, R689C) in the macrophage-stimulating 1 (MST1) gene (P-value 3.62 x 10(-6)) that accounts for the association signal, and shows association with both CD and UC. MST1 encodes macrophage-stimulating protein (MSP), a protein regulating the innate immune responses to bacterial ligands. R689C is predicted to interfere with MSP binding to its receptor, suggesting a role for this gene in the pathogenesis of IBD.

The role of the Toll receptor pathway in susceptibility to inflammatory bowel diseases.

The intestinal flora has long been thought to play a role either in initiating or in exacerbating the inflammatory bowel diseases (IBD). Host defenses, such as those mediated by the Toll-like receptors (TLR), are critical to the host/pathogen interaction and have been implicated in IBD pathophysiology. To explore the association of genetic variation in TLR pathways with susceptibility to IBD, we performed a replication study and pooled analyses of the putative IBD risk alleles in NFKB1 and TLR4, and we performed a haplotype-based screen for association to IBD in the TLR genes and a selection of their adaptor and signaling molecules. Our genotyping of 1539 cases of IBD and pooled analysis of 4805 cases of IBD validates the published association of a TLR4 allele with risk of IBD (odds ratio (OR): 1.30, 95% confidence interval (CI): 1.15-1.48; P=0.00017) and Crohn's disease (OR: 1.33, 95% CI: 1.16-1.54; P=0.000035) but not ulcerative colitis. We also describe novel suggestive evidence that TIRAP (OR: 1.16, 95% CI: 1.04-1.30; P=0.007) has a modest effect on risk of IBD. Our analysis, therefore, offers additional evidence that the TLR4 pathway - in this case, TLR4 and its signaling molecule TIRAP - plays a role in susceptibility to IBD.

The role of inflammatory bowel disease susceptibility loci in multiple sclerosis and systemic lupus erythematosus.

To date, three loci have been validated to confer susceptibility to inflammatory bowel disease (IBD): the CARD15/NOD2 gene, the discs large homolog 5 gene (DLG5), and the IBD5 locus on 5q31 (IBD5). We have explored the possibility that these loci may also be associated with susceptibility to two other chronic inflammatory diseases, multiple sclerosis (MS) and systemic lupus erythematosus (SLE). As the CARD15 risk alleles had previously been assessed in our collection of 496 MS trios, we focused our efforts on the DLG5 risk allele and the IBD5(risk) haplotype (IBD5(risk)) for MS. While there is no evidence of association within our MS sample with either of these polymorphisms, screening of 1027 subjects with SLE suggests that IBD5(risk) may have a modest contribution to disease risk in the subset of SLE subjects without lupus nephritis. In addition, a pooled analysis of existing published and unpublished data in 1305 cases of SLE genotyped for the CARD15 risk alleles suggests that only the CARD15(908R) IBD risk allele may have a strong effect on risk of SLE. Our data, therefore, suggest that both the CARD15 gene and the IBD5 locus may have a role as general susceptibility loci for certain common, genetically complex inflammatory diseases.

Quantitative founder-effect analysis of French Canadian families identifies specific loci contributing to metabolic phenotypes of hypertension.

The Saguenay-Lac St-Jean population of Quebec is relatively isolated and has genealogical records dating to the 17th-century French founders. In 120 extended families with at least one sib pair affected with early-onset hypertension and/or dyslipidemia, we analyzed the genetic determinants of hypertension and related cardiovascular and metabolic conditions. Variance-components linkage analysis revealed 46 loci after 100,000 permutations. The most prominent clusters of overlapping quantitative-trait loci were on chromosomes 1 and 3, a finding supported by principal-components and bivariate analyses. These genetic determinants were further tested by classifying families by use of LOD score density analysis for each measured phenotype at every 5 cM. Our study showed the founder effect over several generations and classes of living individuals. This quantitative genealogical approach supports the notion of the ancestral causality of traits uniquely present and inherited in distinct family classes. With the founder effect, traits determined within population subsets are measurably and quantitatively transmitted through generational lineage, with a precise component contributing to phenotypic variance. These methods should accelerate the uncovering of causal haplotypes in complex diseases such as hypertension and metabolic syndrome.

Immunoglobulin gene usage in the human anti-pathogen response.

The human antibody response to foreign pathogens is generated to a relatively small number of target surface proteins and carbohydrates that nonetheless have an extensive array of epitopes. The study of human monoclonal antibodies to different pathogens shows that there are a diversity of mechanisms used to generate a sufficient repertoire of antibodies to combat the invading pathogens. Although many different immunoglobulin gene elements are used to construct the anti-pathogen response, some elements are used more often than would be expected if all elements were used randomly. For example, the immune response to Haemophilus influenzae polysaccharide appears to be quite narrow, being restricted primarily to a specific heavy-chain gene, 3-15, and a lambda light-chain family II member, 4A. In contrast, for the immune response to cytomegalovirus proteins, a wider group of gene elements is needed. It is also surprising that despite an investigator bias for IgG- rather than IgM-secreting immortal B cells (because of their high affinity and neutralizing abilities), 26% of light chains and 13% of heavy chains showed a very low level of somatic mutation, equivalent to an IgM molecule that has not undergone affinity maturation. Although some highly mutated IgG molecules are present in the anti-pathogen response, most of the monoclonal antibodies specific for viruses or bacteria have a level of somatic hypermutation similar to that of the adult IgM repertoire. A number of studies have shown that there are similarities in the antibody responses to pathogens and to self (autoantibodies).(ABSTRACT TRUNCATED AT 250 WORDS)

A human rheumatoid factor C304 shares VH and VL gene usage with antibodies specific for ubiquitous human viral pathogens.

Analysis of the variable region gene sequences of a human hybridoma rheumatoid factor (RF), derived from a patient with rheumatoid arthritis (RA), revealed the expression of genes from the V lambda I and VH3 families. Specifically, the C304 RF had rearranged the DPL8/Humlv1042 and VH26 germline VL and VH genes, respectively. This gene usage has been observed in the rearrangement of human anti-viral antibodies specific for the herpes group of viruses. This overlap between the autoimmune and anti-viral antibody gene repertoires suggests a possible structural relationship between the immune response directed against ubiquitous pathogens and the induction of RF production.