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1.  Multiple Lupus Associated ITGAM Variants Alter Mac-1 Function on Neutrophils 
Arthritis and rheumatism  2013;65(11):2907-2916.
Multiple studies have demonstrated that single-nucleotide polymorphisms (SNPs) in the ITGAM locus (including the non-synonymous SNPs rs1143679, rs1143678, rs1143683) are associated with SLE. ITGAM encodes the protein CD11b, a subunit of the β2 integrin Mac-1. The purpose of this study was to determine the effects of ITGAM genetic variation on the biological functions of neutrophil Mac-1.
Neutrophils from ITGAM genotyped and sequenced healthy donors were isolated for functional studies. The phagocytic capacity of neutrophil ITGAM variants was probed with complement coated erythrocytes, serum treated zymosan, heat treated zymosan and IgG coated erythrocytes. The adhesion capacity of ITGAM variants, in adhering to either purified intercellular adhesion molecule 1 or tumor necrosis factor α-stimulated endothelial cells was assessed in a flow chamber. Expression levels of total CD11b and activation of CD11b were assessed by flow cytometry.
Mac-1–mediated neutrophil phagocytosis, determined in cultures with 2 different complement-coated particles, was significantly reduced in individuals with nonsynonymous variant alleles of ITGAM. This reduction in phagocytosis was related to variation at either rs1143679 (in the β-propeller region) or rs1143678/rs1143683 (highly linked SNPs in the cytoplasmic/calf-1 regions). Phagocytosis mediated by Fcγ receptors was also significantly reduced in donors with variant ITGAM alleles. Similarly, firm adhesion of neutrophils was significantly reduced in individuals with variant ITGAM alleles. These functional alterations were not attributable to differences in total receptor expression or activation.
The nonsynonymous ITGAM variants rs1143679 and rs1143678/rs113683 contribute to altered Mac-1 function on neutrophils. These results underscore the need to consider multiple nonsynonymous SNPs when assessing the functional consequences of ITGAM variation on immune cell processes and the risk of SLE.
PMCID: PMC3969028  PMID: 23918739
2.  Evaluation of imputation-based association in and around the integrin-α-M (ITGAM) gene and replication of robust association between a non-synonymous functional variant within ITGAM and systemic lupus erythematosus (SLE) 
Human Molecular Genetics  2009;18(6):1171-1180.
We recently identified a novel non-synonymous variant, rs1143679, at exon 3 of the ITGAM gene associated with systemic lupus erythematosus (SLE) susceptibility in European-Americans (EAs) and African-Americans. Using genome-wide association approach, three other studies also independently reported an association between SLE susceptibility and ITGAM or ITGAM-ITGAX region. The primary objectives of this study are to assess whether single or multiple causal variants from the same gene or any nearby gene(s) are involved in SLE susceptibility and to confirm a robust ITGAM association across nine independent data sets (n = 8211). First, we confirmed our previously reported association of rs1143679 (risk allele ‘A’) with SLE in EAs (P = 1.0 × 10−8) and Hispanic-Americans (P = 2.9 × 10−5). Secondly, using a comprehensive imputation-based association test, we found that ITGAM is one of the major non-human leukocyte antigen susceptibility genes for SLE, and the strongest association for EA is the same coding variant rs1143679 (log10Bayes factor=20, P = 6.17 × 10−24). Thirdly, we determined the robustness of rs1143679 association with SLE across three additional case–control samples, including UK (P = 6.2 × 10−8), Colombian (P = 3.6 × 10−7), Mexican (P = 0.002), as well as two independent sets of trios from UK (PTDT = 1.4 × 10−5) and Mexico (PTDT = 0.015). A meta-analysis combing all independent data sets greatly reinforces the association (Pmeta = 7.1 × 10−50, odds ratio = 1.83, 95% confidence interval = 1.69–1.98, n = 10 046). However, this ITGAM association was not observed in the Korean or Japanese samples, in which rs1143679 is monomorphic for the non-risk allele (G). Taken together along with our earlier findings, these results demonstrate that the coding variant, rs1143679, best explains the ITGAM-SLE association, especially in European- and African-derived populations, but not in Asian populations.
PMCID: PMC2649018  PMID: 19129174
3.  Combined protein- and nucleic acid-level effects of rs1143679 (R77H), a lupus-predisposing variant within ITGAM 
Human Molecular Genetics  2014;23(15):4161-4176.
Integrin alpha M (ITGAM; CD11b) is a component of the macrophage-1 antigen complex, which mediates leukocyte adhesion, migration and phagocytosis as part of the immune system. We previously identified a missense polymorphism, rs1143679 (R77H), strongly associated with systemic lupus erythematosus (SLE). However, the molecular mechanisms of this variant are incompletely understood. A meta-analysis of published and novel data on 28 439 individuals with European, African, Hispanic and Asian ancestries reinforces genetic association between rs1143679 and SLE [Pmeta = 3.60 × 10−90, odds ratio (OR) = 1.76]. Since rs1143679 is in the most active region of chromatin regulation and transcription factor binding in ITGAM, we quantitated ITGAM RNA and surface protein levels in monocytes from patients with each rs1143679 genotype. We observed that transcript levels significantly decreased for the risk allele (‘A’) relative to the non-risk allele (‘G’), in a dose-dependent fashion: (‘AA’ < ‘AG’ < ‘GG’). CD11b protein levels in patients' monocytes were directly correlated with RNA levels. Strikingly, heterozygous individuals express much lower (average 10- to 15-fold reduction) amounts of the ‘A’ transcript than ‘G’ transcript. We found that the non-risk sequence surrounding rs1143679 exhibits transcriptional enhancer activity in vivo and binds to Ku70/80, NFKB1 and EBF1 in vitro, functions that are significantly reduced with the risk allele. Mutant CD11b protein shows significantly reduced binding to fibrinogen and vitronectin, relative to non-risk, both in purified protein and in cellular models. This two-pronged contribution (nucleic acid- and protein-level) of the rs1143679 risk allele to decreasing ITGAM activity provides insight into the molecular mechanisms of its potent association with SLE.
PMCID: PMC4082363  PMID: 24608226
4.  Association Study of ITGAM, ITGAX, and CD58 Autoimmune Risk Loci in Systemic Sclerosis: Results from 2 Large European Caucasian Cohorts 
The Journal of Rheumatology  2011;38(6):1033-1038.
Accumulating evidence shows that shared autoimmunity is critical for the pathogenesis of many autoimmune diseases. Systemic sclerosis (SSc) belongs to the connective tissue disorders, and recent data have highlighted strong associations with autoimmunity genes shared with other autoimmune diseases. To determine whether novel risk loci associated with systemic lupus erythematosus or multiple sclerosis may confer susceptibility to SSc, we tested single-nucleotide polymorphisms (SNP) from ITGAM, ITGAX, and CD58 for associations.
SNP harboring associations with autoimmune diseases, ITGAM rs9937837, ITGAX rs11574637, and CD58 rs12044852, were genotyped in 2 independent cohorts of European Caucasian ancestry: 1031 SSc patients and 1014 controls from France and 1038 SSc patients and 691 controls from the USA, providing a combined study population of 3774 individuals. ITGAM rs1143679 was additionally genotyped in the French cohort.
The 4 polymorphisms were in Hardy-Weinberg equilibrium in the 2 control populations, and allelic frequencies were similar to those expected in European Caucasian populations. Allelic and genotypic frequencies for these 3 SNP were found to be statistically similar in SSc patients and controls. Subphenotype analyses for subgroups having diffuse cutaneous subtype disease, specific autoantibodies, or fibrosing alveolitis did not reveal any difference between SSc patients and controls.
These results obtained through 2 large cohorts of SSc patients of European Caucasian ancestry do not support the implication of ITGAM, ITGAX, and CD58 genes in the genetic susceptibility of SSc, although they were recently identified as autoimmune disease risk genes.
PMCID: PMC3404507  PMID: 21362770
5.  Evaluation of genetic association between an ITGAM non-synonymous SNP (rs1143679) and multiple autoimmune diseases 
Autoimmunity Reviews  2011;11(4):276-280.
Many autoimmune diseases (ADs) share similar underlying pathology and have a tendency to cluster within families, supporting the involvement of shared susceptibility genes. To date, most of the genetic variants associated with systemic lupus erythematosus (SLE) susceptibility also show association with others ADs. ITGAM and its associated ‘predisposing’ variant (rs1143679, Arg77His), predicted to alter the tertiary structures of the ligand-binding domain of ITGAM, may play a key role for SLE pathogenesis. The aim of this study is to examine whether the ITGAM variant is also associated with other ADs. We evaluated case-control association between rs1143679 and ADs (N=18,457) including primary Sjögren’s syndrome, systemic sclerosis, multiple sclerosis, rheumatoid arthritis, juvenile idiopathic arthritis, celiac disease, and type-1 diabetes. We also performed meta-analyses using our data in addition to available published data. Although the risk allele ‘A’ is relatively more frequent among cases for each disease, it was not significantly associated with any other ADs tested in this study. However, the meta-analysis for systemic sclerosis was associated with rs1143679 (pmeta=0.008). In summary, this study explored the role of ITGAM in general autoimmunity in seven non-lupus ADs, and only found association for systemic sclerosis when our results were combined with published results. Thus ITGAM may not be a general autoimmunity gene but this variant may be specifically associated with SLE and systemic sclerosis.
PMCID: PMC3224188  PMID: 21840425
ITGAM; autoimmune diseases; genetic susceptibility
6.  Polymorphisms of the ITGAM Gene Confer Higher Risk of Discoid Cutaneous Than of Systemic Lupus Erythematosus 
PLoS ONE  2010;5(12):e14212.
Lupus erythematosus (LE) is a heterogeneous disease ranging from mainly skin-restricted manifestations (discoid LE [DLE] and subacute cutaneous LE) to a progressive multisystem disease (systemic LE [SLE]). Genetic association studies have recently identified several strong susceptibility genes for SLE, including integrin alpha M (ITGAM), also known as CD11b, whereas the genetic background of DLE is less clear.
Principal Findings
To specifically investigate whether ITGAM is a susceptibility gene not only for SLE, but also for cutaneous DLE, we genotyped 177 patients with DLE, 85 patients with sporadic SLE, 190 index cases from SLE families and 395 population control individuals from Finland for nine genetic markers at the ITGAM locus. SLE patients were further subdivided by the presence or absence of discoid rash and renal involvement. In addition, 235 Finnish and Swedish patients positive for Ro/SSA-autoantibodies were included in a subphenotype analysis. Analysis of the ITGAM coding variant rs1143679 showed highly significant association to DLE in patients without signs of systemic disease (P-value  = 4.73×10−11, OR  = 3.20, 95% CI  = 2.23–4.57). Significant association was also detected to SLE patients (P-value  = 8.29×10−6, OR  = 2.14, 95% CI  = 1.52–3.00), and even stronger association was found when stratifying SLE patients by presence of discoid rash (P-value  = 3.59×10−8, OR  = 3.76, 95% CI  = 2.29–6.18).
We propose ITGAM as a novel susceptibility gene for cutaneous DLE. The risk effect is independent of systemic involvement and has an even stronger genetic influence on the risk of DLE than of SLE.
PMCID: PMC2996302  PMID: 21151989
7.  ITGAM is associated with disease susceptibility and renal nephritis of systemic lupus erythematosus in Hong Kong Chinese and Thai 
Human Molecular Genetics  2009;18(11):2063-2070.
ITGAM was recently found to be associated with systemic lupus erythematosus (SLE) in populations of not only European ancestry, but also in Hispanic- and African-Americans, Mexicans and Colombians. The risk alleles in the gene, however, were found to be monomorphic in two Asian populations examined: Japanese and Korean. In this study, using a collection of 910 SLE patients and 2360 controls from Chinese living in Hong Kong, analyzed by both genome-wide association and direct sequencing, we confirmed the association of the same risk alleles in ITGAM with the disease. These findings were further replicated in the Thai population with 278 patients and 383 ethnicity- and geography-matched controls. Subphenotype stratification analyses showed significantly more involvement of the gene in patients with renal nephritis and neurological disorders. Although our results support a pivotal role by rs1143679 (R77H) in disease association, our data also suggests an additional contribution from rs1143683, another non-synonymous polymorphism in this gene (A858V). Therefore, despite the low-allele frequencies of the risk alleles of the gene in our two Asian populations, ITGAM was confirmed to be a risk factor related to disease susceptibility and probably severe manifestations of SLE.
PMCID: PMC2678927  PMID: 19286673
8.  Genetically Determined Amerindian Ancestry Correlates with Increased Frequency of Risk Alleles for Systemic Lupus Erythematosus 
Arthritis and rheumatism  2010;62(12):3722-3729.
To analyze if genetically determined Amerindian ancestry predicts the increased presence of risk alleles of known susceptibility genes for systemic lupus erythematosus.
Single nucleotide polymorphisms within 16 confirmed genetic susceptibility loci for SLE were genotyped in a set of 804 Mestizo lupus patients and 667 Mestizo normal healthy controls. In addition, 347 admixture informative markers were genotyped. Individual ancestry proportions were determined using STRUCTURE. Association analysis was performed using PLINK, and correlation of the presence of risk alleles with ancestry was done using linear regression.
A meta-analysis of the genetic association of the 16 SNPs across populations showed that TNFSF4, STAT4, PDCD1, ITGAM, and IRF5 were associated with lupus in a Hispanic-Mestizo cohort enriched for European and Amerindian ancestry. In addition, two SNPs within the MHC region, previously associated in a genome-wide association study in Europeans, were also associated in Mestizos. Using linear regression we predict an average increase of 2.34 risk alleles when comparing a lupus patient with 100% Amerindian ancestry to an SLE patient with 0% American Indian Ancestry (p<0.0001). SLE patients with 43% more Amerindian ancestry are predicted to carry one additional risk allele.
Amerindian ancestry increased the number of risk alleles for lupus.
PMCID: PMC3078084  PMID: 20848568
9.  Resequencing the susceptibility gene, ITGAM, identifies two functionally deleterious rare variants in systemic lupus erythematosus cases 
Arthritis Research & Therapy  2014;16(3):R114.
The majority of the genetic variance of systemic lupus erythematosus (SLE) remains unexplained by the common disease-common variant hypothesis. Rare variants, which are not detectable by genome-wide association studies because of their low frequencies, are predicted to explain part of this ”missing heritability.” However, recent studies identifying rare variants within known disease-susceptibility loci have failed to show genetic associations because of their extremely low frequencies, leading to the questioning of the contribution of rare variants to disease susceptibility. A common (minor allele frequency = 17.4% in cases) nonsynonymous coding variant rs1143679 (R77H) in ITGAM (CD11b), which forms half of the heterodimeric integrin receptor, complement receptor 3 (CR3), is robustly associated with SLE and has been shown to impair CR3-mediated phagocytosis.
We resequenced ITGAM in 73 SLE cases and identified two previously unidentified, case-specific nonsynonymous variants, F941V and G1145S. Both variants were genotyped in 2,107 and 949 additional SLE cases, respectively, to estimate their frequencies in a disease population. An in vitro model was used to assess the impact of F941V and G1145S, together with two nonsynonymous ITGAM polymorphisms, A858V (rs1143683) and M441T (rs11861251), on CR3-mediated phagocytosis. A paired two-tailed t test was used to compare the phagocytic capabilities of each variant with that of wild-type CR3.
Both rare variants, F941V and G1145S, significantly impair CR3-mediated phagocytosis in an in vitro model (61% reduction, P = 0.006; 26% reduction, P = 0.0232). However, neither of the common variants, M441T and A858V, had an effect on phagocytosis. Neither rare variant was observed again in the genotyping of additional SLE cases, suggesting that there frequencies are extremely low.
Our results add further evidence to the functional importance of ITGAM in SLE pathogenesis through impaired phagocytosis. Additionally, this study provides a new example of the identification of rare variants in common-allele-associated loci, which, because of their extremely low frequencies, are not statistically associated. However, the demonstration of their functional effects adds support to their contribution to disease risk, and questions the current notion of dismissing the contribution of very rare variants on purely statistical analyses.
PMCID: PMC4060450  PMID: 24886912
10.  Serological and Genetic Evidence for Altered Complement System Functionality in Systemic Lupus Erythematosus: Findings of the GAPAID Consortium 
PLoS ONE  2016;11(3):e0150685.
Systemic lupus erythematosus is a chronic autoimmune disease with multifactorial ethiopathogenesis. The complement system is involved in both the early and late stages of disease development and organ damage. To better understand autoantibody mediated complement consumption we examined ex vivo immune complex formation on autoantigen arrays. We recruited patients with SLE (n = 211), with other systemic autoimmune diseases (n = 65) and non-autoimmune control subjects (n = 149). Standard clinical and laboratory data were collected and serum complement levels were determined. The genotype of SNP rs1143679 in the ITGAM gene was also determined. Ex vivo formation of immune complexes, with respect to IgM, IgG, complement C4 and C3 binding, was examined using a functional immunoassay on autoantigen microarray comprising nucleic acids, proteins and lipids. Complement consumption of nucleic acids increased upon binding of IgM and IgG even when serum complement levels were decreased due to consumption in SLE patients. A negative correlation between serum complement levels and ex vivo complement deposition on nucleic acid autoantigens is demonstrated. On the contrary, complement deposition on tested protein and lipid autoantigens showed positive correlation with C4 levels. Genetic analysis revealed that the non-synonymous variant rs1143679 in complement receptor type 3 is associated with an increased production of anti-dsDNA IgG antibodies. Notwithstanding, homozygous carriers of the previously reported susceptible allele (AA) had lower levels of dsDNA specific IgM among SLE patients. Both the non-synonymous variant rs1143679 and the high ratio of nucleic acid specific IgG/IgM were associated with multiple organ involvement. In summary, secondary complement deficiency in SLE does not impair opsonization of nucleic-acid-containing autoantigens but does affect other antigens and potentially other complement dependent processes. Dysfunction of the receptor recognizing complement opsonized immune complexes promotes the development of class-switched autoantibodies targeting nucleic acids.
PMCID: PMC4780778  PMID: 26950932
11.  Risk Alleles for Systemic Lupus Erythematosus in a Large Case-Control Collection and Associations with Clinical Subphenotypes 
PLoS Genetics  2011;7(2):e1001311.
Systemic lupus erythematosus (SLE) is a genetically complex disease with heterogeneous clinical manifestations. Recent studies have greatly expanded the number of established SLE risk alleles, but the distribution of multiple risk alleles in cases versus controls and their relationship to subphenotypes have not been studied. We studied 22 SLE susceptibility polymorphisms with previous genome-wide evidence of association (p<5×10−8) in 1919 SLE cases from 9 independent Caucasian SLE case series and 4813 independent controls. The mean number of risk alleles in cases was 15.1 (SD 3.1) while the mean in controls was 13.1 (SD 2.8), with trend p = 4×10−128. We defined a genetic risk score (GRS) for SLE as the number of risk alleles with each weighted by the SLE risk odds ratio (OR). The OR for high-low GRS tertiles, adjusted for intra-European ancestry, sex, and parent study, was 4.4 (95% CI 3.8–5.1). We studied associations of individual SNPs and the GRS with clinical manifestations for the cases: age at diagnosis, the 11 American College of Rheumatology classification criteria, and double-stranded DNA antibody (anti-dsDNA) production. Six subphenotypes were significantly associated with the GRS, most notably anti-dsDNA (ORhigh-low = 2.36, p = 9e−9), the immunologic criterion (ORhigh-low = 2.23, p = 3e−7), and age at diagnosis (ORhigh-low = 1.45, p = 0.0060). Finally, we developed a subphenotype-specific GRS (sub-GRS) for each phenotype with more power to detect cumulative genetic associations. The sub-GRS was more strongly associated than any single SNP effect for 5 subphenotypes (the above plus hematologic disorder and oral ulcers), while single loci are more significantly associated with renal disease (HLA-DRB1, OR = 1.37, 95% CI 1.14–1.64) and arthritis (ITGAM, OR = 0.72, 95% CI 0.59–0.88). We did not observe significant associations for other subphenotypes, for individual loci or the sub-GRS. Thus our analysis categorizes SLE subphenotypes into three groups: those having cumulative, single, and no known genetic association with respect to the currently established SLE risk loci.
Author Summary
Systemic lupus erythematosus is a chronic disabling autoimmune disease, most commonly striking women in their thirties or forties. It can cause a wide variety of clinical manifestations, including kidney disease, arthritis, and skin disorders. Prognosis varies greatly depending on these clinical features, with kidney disease and related characteristics leading to greater morbidity and mortality. It is also complex genetically; while lupus runs in families, genes increase one's risk for lupus but do not fully determine the outcome. The interactions of multiple genes and/or interactions between genes and environmental factors may cause lupus, but the causes and disease pathways of this very heterogeneous disease are not well understood. By examining relationships between the presence of multiple lupus risk genes, lupus susceptibility, and clinical manifestations, we hope to better understand how lupus is triggered and by what biological pathways it progresses. We show in this work that certain clinical manifestations of lupus are highly associated with cumulative genetic variations, i.e. multiple risk alleles, while others are associated with a single variation or none at all.
PMCID: PMC3040652  PMID: 21379322
12.  Combined Effects of ICAM-1 Single-Nucleotide Polymorphisms and Environmental Carcinogens on Oral Cancer Susceptibility and Clinicopathologic Development 
PLoS ONE  2013;8(9):e72940.
In Taiwan, oral cancer has causally been associated with environmental carcinogens. Intercellular adhesion molecule (ICAM)-1, a cell adhesion molecule with a key role in inflammation and immunosurveillance, was implicated in carcinogenesis by facilitating instability in the tumor environment. The current study explored the combined effect of ICAM-1 gene polymorphisms and exposure to environmental carcinogens on the susceptibility of developing oral squamous cell carcinoma (OSCC) and the clinicopathological characteristics of the tumors.
Methodology and Principal Findings
Four single-nucleotide polymorphisms (SNPs) of the ICAM-1 gene from 595 patients with oral cancer and 561 non-cancer controls were analyzed by a real-time PCR. We found that the ICAM-1 rs5498 polymorphism and the TAGG or TACG haplotype of 4 ICAM-1 SNPs (rs3093030, rs5491, rs281432, and rs5498) combined were associated with oral-cancer susceptibility. Among 727 smokers, ICAM-1 polymorphisms carriers with the betel-nut chewing habit had a 27.49–36.23-fold greater risk of having oral cancer compared to ICAM-1 wild-type (WT) carriers without the betel-nut chewing habit. Among 549 betel-nut chewers, ICAM-1 polymorphisms carriers who smoked had a 9.93–14.27-fold greater risk of having oral cancer compared to those who carried the WT but did not smoke. Finally, patients with oral cancer who had at least 1 T allele of ICAM-1 rs5491 or 1 G allele of rs281432 were at lower risk of developing an advanced clinical stage (III/IV) (p<0.05), compared to those patients with AA or CC homozygotes.
Our results suggest that the ICAM-1 rs5498 SNP and either of 2 haplotypes of 4 SNPs combined have potential predictive significance in oral carcinogenesis. Gene-environment interactions of ICAM-1 polymorphisms, smoking, and betel-nut chewing might alter oral-cancer susceptibility. ICAM-1 rs5491 and rs281432 may be applied as factors to predict the clinical stage in OSCC patients.
PMCID: PMC3771971  PMID: 24069166
13.  Rare variants at 16p11.2 are associated with common variable immunodeficiency 
Common variable immunodeficiency (CVID) is characterized clinically by inadequate quantity and quality of serum immunoglobulins with increased susceptibility to infections resulting in significant morbidity and mortality. Only a few genes have been uncovered and the genetic background of CVID remains elusive to date for the majority of patients.
To seek novel associations of genes and genetic variants with CVID.
We performed association analyses in a discovery cohort of 164 CVID cases and 19,542 healthy control subjects genotyped on the Immuno BeadChip from Illumina (iCHIP); replication of findings were examined in an independent cohort of 135 CVID cases and 2,066 healthy control subjects, followed by meta-analysis.
We identified 11 SNPs at the 16p11.2 locus associated with CVID at genome-wide significant level in the discovery cohort. The most significant SNP, rs929867 (p = 6.21×10−9) is in the gene FUS (fused-in-sarcoma) with four other SNPs mapping to ITGAM (integrin CD11b). Results were confirmed in our replication cohort. Conditional association analysis suggests a single association signal at the 16p11.2 locus. A strong trend of association was also seen by 38 SNPs (p < 5×10−5) in the MHC region, supporting that this is a genuine CVID locus. Interestingly we found that 80% of patients with the rare ITGAM variants have reduced counts of switched-memory B-cells.
We report a novel association of CVID with rare variants at the FUS/ITGAM (CD11b) locus on 16p11.2. The association signal is enriched for promoter/enhancer markers in the ITGAM gene. ITGAM encodes the integrin CD11b, a part of complement receptor 3 (CR3/Mac-1), a novel candidate gene implicated here for the first time in the pathogenesis of CVID.
PMCID: PMC4461447  PMID: 25678086
immunodeficiency; immunogenetics; genome-wide association study; ITGAM; rare variants
14.  European Genetic Ancestry is Associated with a Decreased Risk of Lupus Nephritis 
Arthritis and rheumatism  2012;64(10):10.1002/art.34567.
African Americans, East Asians, and Hispanics with systemic lupus erythematosus (SLE) are more likely to develop renal disease than SLE patients of European descent. We investigated whether European genetic ancestry protects against the development of lupus nephritis and explored genetic and socioeconomic factors that might explain this effect.
This was a cross-sectional study of 1906 adults with SLE. Participants were genotyped for 126 single nucleotide polymorphisms (SNPs) informative for ancestry. A subset of participants was also genotyped for 80 SNPs in 14 candidate genes for renal disease in SLE. We used logistic regression to test the association between European ancestry and renal disease. Analyses adjusted for continental ancestries, socioeconomic status, and candidate genes.
Participants (n=1906) had on average 62.4% European, 15.8% African, 11.5% East Asian, 6.5% Amerindian, and 3.8% South Asian ancestry. Among participants, 34% (n=656) had renal disease. A 10% increase in European ancestry was associated with a 15% reduction in the odds of having renal disease after adjustment for disease duration and sex (OR 0.85, 95% CI 0.82-0.87, p=1.9 × 10−30). Adjusting for other genetic ancestries, measures of socioeconomic status, or SNPs in genes most associated with renal disease (IRF5 (rs4728142), BLK (rs2736340), STAT4 (rs3024912), ITGAM (rs9937837) and HLA-DRB1*0301 and DRB1*1501, p<0.05) did not substantively alter this relationship.
European ancestry is protective against the development of renal disease in SLE, an effect independent of other genetic ancestries, common risk alleles, and socioeconomic status.
PMCID: PMC3865923  PMID: 23023776
15.  Differential Genetic Associations for Systemic Lupus Erythematosus Based on Anti–dsDNA Autoantibody Production 
PLoS Genetics  2011;7(3):e1001323.
Systemic lupus erythematosus (SLE) is a clinically heterogeneous, systemic autoimmune disease characterized by autoantibody formation. Previously published genome-wide association studies (GWAS) have investigated SLE as a single phenotype. Therefore, we conducted a GWAS to identify genetic factors associated with anti–dsDNA autoantibody production, a SLE–related autoantibody with diagnostic and clinical importance. Using two independent datasets, over 400,000 single nucleotide polymorphisms (SNPs) were studied in a total of 1,717 SLE cases and 4,813 healthy controls. Anti–dsDNA autoantibody positive (anti–dsDNA +, n = 811) and anti–dsDNA autoantibody negative (anti–dsDNA –, n = 906) SLE cases were compared to healthy controls and to each other to identify SNPs associated specifically with these SLE subtypes. SNPs in the previously identified SLE susceptibility loci STAT4, IRF5, ITGAM, and the major histocompatibility complex were strongly associated with anti–dsDNA + SLE. Far fewer and weaker associations were observed for anti–dsDNA – SLE. For example, rs7574865 in STAT4 had an OR for anti–dsDNA + SLE of 1.77 (95% CI 1.57–1.99, p = 2.0E-20) compared to an OR for anti–dsDNA – SLE of 1.26 (95% CI 1.12–1.41, p = 2.4E-04), with pheterogeneity<0.0005. SNPs in the SLE susceptibility loci BANK1, KIAA1542, and UBE2L3 showed evidence of association with anti–dsDNA + SLE and were not associated with anti–dsDNA – SLE. In conclusion, we identified differential genetic associations with SLE based on anti–dsDNA autoantibody production. Many previously identified SLE susceptibility loci may confer disease risk through their role in autoantibody production and be more accurately described as autoantibody propensity loci. Lack of strong SNP associations may suggest that other types of genetic variation or non-genetic factors such as environmental exposures have a greater impact on susceptibility to anti–dsDNA – SLE.
Author Summary
Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that can involve virtually any organ system. SLE patients produce antibodies that bind to their own cells and proteins (autoantibodies) which can cause irreversible organ damage. One particular SLE–related autoantibody directed at double-stranded DNA (anti–dsDNA) is associated with kidney involvement and more severe disease. Previous genome-wide association studies (GWAS) in SLE have studied SLE itself, not particular SLE manifestations. Therefore, we conducted this GWAS of anti–dsDNA autoantibody production to identify genetic associations with this clinically important autoantibody. We found that many previously identified SLE–associated genes are more strongly associated with anti–dsDNA autoantibody production than SLE itself, and they may be more accurately described as autoantibody propensity genes. No strong genetic associations were observed for SLE patients who do not produce anti–dsDNA autoantibodies, suggesting that other factors may have more influence in developing this type of SLE. Further investigation of these autoantibody propensity genes may lead to greater insight into the causes of autoantibody production and organ damage in SLE.
PMCID: PMC3048371  PMID: 21408207
16.  A Comprehensive Analysis of Shared Loci between Systemic Lupus Erythematosus (SLE) and Sixteen Autoimmune Diseases Reveals Limited Genetic Overlap 
PLoS Genetics  2011;7(12):e1002406.
In spite of the well-known clustering of multiple autoimmune disorders in families, analyses of specific shared genes and polymorphisms between systemic lupus erythematosus (SLE) and other autoimmune diseases (ADs) have been limited. Therefore, we comprehensively tested autoimmune variants for association with SLE, aiming to identify pleiotropic genetic associations between these diseases. We compiled a list of 446 non–Major Histocompatibility Complex (MHC) variants identified in genome-wide association studies (GWAS) of populations of European ancestry across 17 ADs. We then tested these variants in our combined Caucasian SLE cohorts of 1,500 cases and 5,706 controls. We tested a subset of these polymorphisms in an independent Caucasian replication cohort of 2,085 SLE cases and 2,854 controls, allowing the computation of a meta-analysis between all cohorts. We have uncovered novel shared SLE loci that passed multiple comparisons adjustment, including the VTCN1 (rs12046117, P = 2.02×10−06) region. We observed that the loci shared among the most ADs include IL23R, OLIG3/TNFAIP3, and IL2RA. Given the lack of a universal autoimmune risk locus outside of the MHC and variable specificities for different diseases, our data suggests partial pleiotropy among ADs. Hierarchical clustering of ADs suggested that the most genetically related ADs appear to be type 1 diabetes with rheumatoid arthritis and Crohn's disease with ulcerative colitis. These findings support a relatively distinct genetic susceptibility for SLE. For many of the shared GWAS autoimmune loci, we found no evidence for association with SLE, including IL23R. Also, several established SLE loci are apparently not associated with other ADs, including the ITGAM-ITGAX and TNFSF4 regions. This study represents the most comprehensive evaluation of shared autoimmune loci to date, supports a relatively distinct non–MHC genetic susceptibility for SLE, provides further evidence for previously and newly identified shared genes in SLE, and highlights the value of studies of potentially pleiotropic genes in autoimmune diseases.
Author Summary
It is well known that multiple autoimmune disorders cluster in families. However, all of the genetic variants that explain this clustering have not been discovered, and the specific genetic variants shared between systemic lupus erythematosus (SLE) and other autoimmune diseases (ADs) are not known. In order to better understand the genetic factors that explain this predisposition to autoimmunity, we performed a comprehensive evaluation of shared autoimmune genetic variants. First we considered results from 17 ADs and compiled a list with 446 significant genetic variants from these studies. We identified some genetic variants extensively shared between ADs, as well as the ADs that share the most variants. The genetic overlap between SLE and other ADs was modest. Next we tested how important all the 446 genetic variants were in our collection with a minimum of 1,500 SLE patients. Among the most significant variants in SLE, the majority had already been identified in previous studies, but we also discovered variants in two important immune genes. In summary, our data identified diseases with common genetic risk factors and novel SLE effects, and this supports a relatively distinct genetic susceptibility for SLE. This study helps delineate the genetic architecture of ADs.
PMCID: PMC3234215  PMID: 22174698
17.  Admixture in Hispanic-Americans: Its impact on ITGAM association and implications for admixture mapping in SLE 
Genes and immunity  2009;10(5):539-545.
Systemic Lupus Erythematosus (SLE) disproportionately affects minorities, such as Hispanic-Americans. Prevalence of SLE is 3–5 times higher in Hispanic Americans (HA) than European derived populations, and have more active disease at the time of diagnosis, with more serious organ system involvement. HA is an admixed population, it is possible that there is an effect of admixture on the relative risk of disease. This admixture can create substantial increase of linkage disequilibrium (LD) in both magnitude and range, which can provide a unique opportunity for admixture mapping. Main objectives of this study are to (a) estimate hidden population structure in HA individuals; (b) estimate individual ancestry proportions and its impact on SLE risk; (c) assess impact of admixture on ITGAM association, a recently identified SLE susceptibility gene; and (d) estimate power of admixture mapping in HA. Our dataset contained 1,125 individuals, of whom 884 (657 SLE cases and 227 controls) were self classified as HA. Using 107 unlinked ancestry informative markers (AIMs) we estimated hidden population structure and individual ancestry in HA. Out of 5,671 possible pair-wise LD, 54% were statistically significant, indicating recent population admixture. The best fitted model for HA was a four population model with average ancestry of European (48%), American-Indian (40%), African (8%) and a fourth population (4%) with unknown ancestry. We also identified significant higher risk associated with American-Indian ancestry (OR=4.84, P=0.0001, 95%CI=2.14—10.95) on overall SLE. We showed that ITGAM is associated as a risk factor for SLE (OR= 2.06, P=8.74×10−5, 95%CI=1.44–2.97). This association is not affected by population substructure or admixture. We have demonstrated that HA have great potential and are an 3 appropriate population for admixture mapping. As expected, the case-only design is more powerful than case-control design, for any given admixture proportion or ancestry risk ratio.
PMCID: PMC2714406  PMID: 19387459
SLE; Association; Hispanics; Admixture mapping; Hispanic-American; Population structure
18.  Phenotypic associations of genetic susceptibility loci in systemic lupus erythematosus 
Annals of the rheumatic diseases  2011;70(10):1752-1757.
Systemic lupus erythematosus is a clinically heterogeneous autoimmune disease. A number of genetic loci that increase lupus susceptibility have been established. This study examines if these genetic loci also contribute to the clinical heterogeneity in lupus.
Materials and methods
4001 European-derived, 1547 Hispanic, 1590 African-American and 1191 Asian lupus patients were genotyped for 16 confirmed lupus susceptibility loci. Ancestry informative markers were genotyped to calculate and adjust for admixture. The association between the risk allele in each locus was determined and compared in patients with and without the various clinical manifestations included in the ACR criteria.
Renal disorder was significantly correlated with the lupus risk allele in ITGAM (p=5.0×10−6, OR 1.25, 95% CI 1.12 to 1.35) and in TNFSF4 (p=0.0013, OR 1.14, 95% CI 1.07 to 1.25). Other significant findings include the association between risk alleles in FCGR2A and malar rash (p=0.0031, OR 1.11, 95% CI 1.17 to 1.33), ITGAM and discoid rash (p=0.0020, OR 1.20, 95% CI 1.06 to 1.33), STAT4 and protection from oral ulcers (p=0.0027, OR 0.89, 95% CI 0.83 to 0.96) and IL21 and haematological disorder (p=0.0027, OR 1.13, 95% CI 1.04 to 1.22). All these associations are significant with a false discovery rate of <0.05 and pass the significance threshold using Bonferroni correction for multiple testing.
Significant associations were found between lupus clinical manifestations and the FCGR2A, ITGAM, STAT4, TNSF4 and IL21 genes. The findings suggest that genetic profiling might be a useful tool to predict disease manifestations in lupus patients in the future.
PMCID: PMC3232181  PMID: 21719445
19.  Study of the common genetic background for rheumatoid arthritis and systemic lupus erythematosus 
Annals of the Rheumatic Diseases  2010;70(3):463-468.
Evidence is beginning to emerge that there may be susceptibility loci for rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) that are common to both diseases.
To investigate single nucleotide polymorphisms that have been reported to be associated with SLE in a UK cohort of patients with RA and controls.
3962 patients with RA and 9275 controls were included in the study. Eleven SNPs mapping to confirmed SLE loci were investigated. These mapped to the TNFSF4, BANK1, TNIP1, PTTG1, UHRF1BP1, ATG5, JAZF1, BLK, KIAA1542, ITGAM and UBE2L3 loci. Genotype frequencies were compared between patients with RA and controls using the trend test.
The SNPs mapping to the BLK and UBE2L3 loci showed significant evidence for association with RA. Two other SNPs, mapping to ATG5 and KIAA1542, showed nominal evidence for association with RA (p=0.02 and p=0.02, respectively) but these were not significant after applying a Bonferroni correction. Additionally, a significant global enrichment in carriage of SLE alleles in patients with RA compared with controls (p=9.1×10−7) was found. Meta-analysis of this and previous studies confirmed the association of the BLK and UBE2L3 gene with RA at genome-wide significance levels (p<5×10−8). Together, the authors estimate that the SLE and RA overlapping loci, excluding HLA-DRB1 alleles, identified so far explain ∼5.8% of the genetic susceptibility to RA as a whole.
The findings confirm the association of the BLK and UBE2L3 loci with RA, thus adding to the list of loci showing overlap between RA and SLE.
PMCID: PMC3033530  PMID: 21068098
20.  ICAM1 and VCAM1 polymorphisms, coronary artery calcium, and circulating levels of soluble ICAM-1: The Multi-Ethnic Study of Atherosclerosis (MESA) 
Atherosclerosis  2008;201(2):339-344.
Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) may be important contributors to the development and progression of atherosclerosis. Using a stratified random sample of 2,880 participants of the Multi-Ethnic Study of Atherosclerosis we investigated the relationship of 12 ICAM1 and 17 VCAM1 SNPs and coronary artery calcium (CAC) and ICAM1 SNPs and circulating levels of soluble ICAM-1 (sICAM-1). There were no ICAM1 or VCAM1 SNPs significantly associated with CAC in any of the four race/ethnic groups. In a subset of 1,451 subjects with sICAM-1 measurements, we observed a significant association with rs5491 in all four race/ethnic groups corroborating previous research that has shown that the T-allele of rs5491 interferes with the monoclonal antibody used to measure sICAM-1 in this study. After excluding all rs5491 T-allele carriers, several ICAM1 SNPs were significantly associated with sICAM-1 levels; rs5496 in African Americans, rs5498 and rs3093030 in European Americans, and rs1799969 in Hispanics. Our results identified ICAM1 polymorphisms that were significantly associated with sICAM-1 level but not CAC, a subclinical marker of atherosclerosis.
PMCID: PMC2615541  PMID: 18420209
coronary artery calcium; intercellular adhesion molecule-1 (ICAM-1); vascular adhesion molecule-1 (VCAM-1); soluble intercellular adhesion molecule-1 (sICAM-1); gene; single nucleotide polymorphism (SNP); haplotypes
21.  Association of STAT4 Polymorphism with Severe Renal Insufficiency in Lupus Nephritis 
PLoS ONE  2013;8(12):e84450.
Lupus nephritis is a cause of significant morbidity in systemic lupus erythematosus (SLE) and its genetic background has not been completely clarified. The aim of this investigation was to analyze single nucleotide polymorphisms (SNPs) for association with lupus nephritis, its severe form proliferative nephritis and renal outcome, in two Swedish cohorts. Cohort I (n = 567 SLE cases, n =  512 controls) was previously genotyped for 5676 SNPs and cohort II (n = 145 SLE cases, n = 619 controls) was genotyped for SNPs in STAT4, IRF5, TNIP1 and BLK.
Case-control and case-only association analyses for patients with lupus nephritis, proliferative nephritis and severe renal insufficiency were performed. In the case-control analysis of cohort I, four highly linked SNPs in STAT4 were associated with lupus nephritis with genome wide significance with p = 3.7×10−9, OR 2.20 for the best SNP rs11889341. Strong signals of association between IRF5 and an HLA-DR3 SNP marker were also detected in the lupus nephritis case versus healthy control analysis (p <0.0001). An additional six genes showed an association with lupus nephritis with p <0.001 (PMS2, TNIP1, CARD11, ITGAM, BLK and IRAK1). In the case-only meta-analysis of the two cohorts, the STAT4 SNP rs7582694 was associated with severe renal insufficiency with p  = 1.6×10−3 and OR 2.22. We conclude that genetic variations in STAT4 predispose to lupus nephritis and a worse outcome with severe renal insufficiency.
PMCID: PMC3873995  PMID: 24386384
22.  Impact of Intercellular Adhesion Molecule-1 Genetic Polymorphisms on Coronary Artery Disease Susceptibility in Taiwanese Subjects 
The principal pathogenesis of coronary artery disease (CAD) is coronary artery atherosclerosis, a chronic inflammatory disease of the vessel walls of the coronary artery. Intercellular adhesion molecule-1 (ICAM-1) displays an important role in the development of the inflammation reaction and atherosclerosis. Few studies report the association of ICAM-1 genetic polymorphisms with CAD in Taiwanese subjects. Therefore, we conducted a study to associate the single nucleotide polymorphisms (SNPs) of ICAM-1, rs5491, rs5498, rs281432 and rs3093030 with CAD. Five hundred and twenty-five male and female subjects, who received elective coronary angiography in Taiwan Chung Shan Medical University Hospital, were recruited to determine four ICAM-1 SNPs by real time-polymerase chain reaction and genotyping. The relationships among ICAM-1 SNPs, haplotypes, demographic and characteristics and CAD were analyzed. This study showed that rs281432 (C8823G) was the only ICAM-1 SNP which affect the development of CAD. Multivariate analysis revealed that ICAM-1 SNP rs281432 CC/CG [p=0.016; odds ratio (OR): 2.56, 95% confidence interval (CI): 1.19-5.56], male gender (p=0.018; OR: 1.66, 95% CI: 1.09-2.51), aspirin use in the past 7 days (p=0.001; OR: 2.05, 95% CI: 1.33-3.14), hypertension (p<0.001; OR: 2.15, 95% CI: 1.42-3.25), serum cardiac troponin I elevation (p<0.001; OR: 2.14, 95% CI: 1.47-3.24) and severe angina in recent 24 hours (p=0.001; OR: 1.97, 95% CI: 1.31- 2.95) increase the risk of CAD. In conclusion, ICAM-1 SNP rs281432 is an independent factor to predict the development of CAD. ICAM-1 SNP rs281432 homozygotic mutant GG can reduce the susceptibility to the CAD in Taiwanese subjects.
PMCID: PMC4466516  PMID: 26078712
Coronary artery disease; genetic polymorphism; haplotype; intercellular adhesion molecule-1; rs281432; Taiwanese.
23.  Large-scale genomic studies reveal central role of ABO in sP-selectin and sICAM-1 levels 
Human Molecular Genetics  2010;19(9):1863-1872.
P-selectin and intercellular adhesion molecule-1 (ICAM-1) participate in inflammatory processes by promoting adhesion of leukocytes to vascular wall endothelium. Their soluble levels have been associated with adverse cardiovascular events. To identify loci affecting soluble levels of P-selectin (sP-selectin) and ICAM-1 (sICAM-1), we performed a genome-wide association study in a sample of 4115 (sP-selectin) and 9813 (sICAM-1) individuals of European ancestry as a part of The Cohorts for Heart and Aging Research in Genome Epidemiology consortium. The most significant SNP association for sP-selectin was within the SELP gene (rs6136, P = 4.05 × 10−61) and for sICAM-1 levels within the ICAM-1 gene (rs3093030, P = 3.53 × 10−23). Both sP-selectin and sICAM-1 were associated with ABO gene variants (rs579459, P = 1.86 × 10−41 and rs649129, P = 1.22 × 10−15, respectively) and in both cases the observed associations could be accounted for by the A1 allele of the ABO blood group. The absence of an association between ABO blood group and platelet-bound P-selectin levels in an independent subsample (N = 1088) from the ARIC study, suggests that the ABO blood group may influence cleavage of the P-selectin protein from the cell surface or clearance from the circulation, rather than its production and cellular presentation. These results provide new insights into adhesion molecule biology.
PMCID: PMC2850624  PMID: 20167578
24.  The complement receptor 3 (CD11b/CD18) agonist Leukadherin‐1 suppresses human innate inflammatory signalling 
Complement receptor 3 (CR3, CD11b/CD18) is a multi‐functional receptor expressed predominantly on myeloid and natural killer (NK) cells. The R77H variant of CD11b, encoded by the ITGAM rs1143679 polymorphism, is associated robustly with development of the autoimmune disease systemic lupus erythematosus (SLE) and impairs CR3 function, including its regulatory role on monocyte immune signalling. The role of CR3 in NK cell function is unknown. Leukadherin‐1 is a specific small‐molecule CR3 agonist that has shown therapeutic promise in animal models of vascular injury and inflammation. We show that Leukadherin‐1 pretreatment reduces secretion of interferon (IFN)‐γ, tumour necrosis factor (TNF) and macrophage inflammatory protein (MIP)‐1β by monokine‐stimulated NK cells. It was associated with a reduction in phosphorylated signal transducer and activator of transcription (pSTAT)‐5 following interleukin (IL)‐12 + IL‐15 stimulation (P < 0·02) and increased IL‐10 secretion following IL‐12 + IL‐18 stimulation (P < 0·001). Leukadherin‐1 pretreatment also reduces secretion of IL‐1β, IL‐6 and TNF by Toll‐like receptor (TLR)‐2 and TLR‐7/8‐stimulated monocytes (P < 0·01 for all). The R77H variant did not affect NK cell response to Leukadherin‐1 using ex‐vivo cells from homozygous donors; nor did the variant influence CR3 expression by these cell types, in contrast to a recent report. These data extend our understanding of CR3 biology by demonstrating that activation potently modifies innate immune inflammatory signalling, including a previously undocumented role in NK cell function. We discuss the potential relevance of this to the pathogenesis of SLE. Leukadherin‐1 appears to mediate its anti‐inflammatory effect irrespective of the SLE‐risk genotype of CR3, providing further evidence to support its evaluation of Leukadherin‐1 as a potential therapeutic for autoimmune disease.
PMCID: PMC4991522  PMID: 27118513
complement receptor 3; innate immunity; Leukadherin‐1; NK cells; systemic lupus erythematosus
25.  The complement receptor 3 (CD11b/CD18) agonist Leukadherin‐1 suppresses human innate inflammatory signalling 
Complement receptor 3 (CR3, CD11b/CD18) is a multi‐functional receptor expressed predominantly on myeloid and natural killer (NK) cells. The R77H variant of CD11b, encoded by the ITGAM rs1143679 polymorphism, is associated robustly with development of the autoimmune disease systemic lupus erythematosus (SLE) and impairs CR3 function, including its regulatory role on monocyte immune signalling. The role of CR3 in NK cell function is unknown. Leukadherin‐1 is a specific small‐molecule CR3 agonist that has shown therapeutic promise in animal models of vascular injury and inflammation. We show that Leukadherin‐1 pretreatment reduces secretion of interferon (IFN)‐γ, tumour necrosis factor (TNF) and macrophage inflammatory protein (MIP)‐1β by monokine‐stimulated NK cells. It was associated with a reduction in phosphorylated signal transducer and activator of transcription (pSTAT)‐5 following interleukin (IL)‐12 + IL‐15 stimulation (P < 0·02) and increased IL‐10 secretion following IL‐12 + IL‐18 stimulation (P < 0·001). Leukadherin‐1 pretreatment also reduces secretion of IL‐1β, IL‐6 and TNF by Toll‐like receptor (TLR)‐2 and TLR‐7/8‐stimulated monocytes (P < 0·01 for all). The R77H variant did not affect NK cell response to Leukadherin‐1 using ex‐vivo cells from homozygous donors; nor did the variant influence CR3 expression by these cell types, in contrast to a recent report. These data extend our understanding of CR3 biology by demonstrating that activation potently modifies innate immune inflammatory signalling, including a previously undocumented role in NK cell function. We discuss the potential relevance of this to the pathogenesis of SLE. Leukadherin‐1 appears to mediate its anti‐inflammatory effect irrespective of the SLE‐risk genotype of CR3, providing further evidence to support its evaluation of Leukadherin‐1 as a potential therapeutic for autoimmune disease.
PMCID: PMC4991522  PMID: 27118513
complement receptor 3; innate immunity; Leukadherin‐1; NK cells; systemic lupus erythematosus

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