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1.  Selective Involvement of the Amygdala in Systemic Lupus Erythematosus 
PLoS Medicine  2006;3(12):e499.
Background
Antibodies specifically affect the amygdala in a mouse model of systemic lupus erythematosus (SLE). The aim of our study was to investigate whether there is also specific involvement of the amygdala in human SLE.
Methods and Findings
We analyzed a group of 37 patients with neuropsychiatric SLE (NP-SLE), 21 patients with SLE, and a group of 12 healthy control participants with diffusion weighted imaging (DWI). In addition, in a subset of eight patients, plasma was available to determine their anti-NMDAR antibody status. From the structural magnetic resonance imaging data, the amygdala and the hippocampus were segmented, as well as the white and gray matter, and the apparent diffusion coefficient (ADC) was retrieved. ADC values between controls, patients with SLE, and patients with NP-SLE were tested using analysis of variance with post-hoc Bonferroni correction. No differences were found in the gray or white matter segments. The average ADC in the amygdala of patients with NP-SLE and SLE (940 × 10−6 mm2/s; p = 0.006 and 949 × 10−6 mm2/s; p = 0.019, respectively) was lower than in healthy control participants (1152 × 10−6 mm2/s). Mann-Whitney analysis revealed that the average ADC in the amygdala of patients with anti-NMDAR antibodies (n = 4; 802 × 10−6 mm2/s) was lower (p = 0.029) than the average ADC of patients without anti-NMDAR antibodies (n = 4; 979 × 10−6 mm2/s) and also lower (p = 0.001) than in healthy control participants.
Conclusions
This is the first study to our knowledge to observe damage in the amygdala in patients with SLE. Patients with SLE with anti-NMDAR antibodies had more severe damage in the amygdala compared to SLE patients without anti-NMDAR antibodies.
Patients with SLE who also had antibodies against the NMDA receptor had more severe damage in the amygdala as compared with patients with SLE without these antibodies.
Editors' Summary
Background.
The human body is continually attacked by viruses, bacteria, fungi, and parasites, but the immune system usually prevents these pathogens from causing disease. To be effective, the immune system has to respond rapidly to foreign antigens (bits of proteins that are unique to the pathogen) but ignore self-antigens. In autoimmune diseases, this ability to discriminate between self and nonself fails for unknown reasons, and the immune system begins to destroy human tissues. In the chronic autoimmune disease systemic lupus erythematosus (SLE or lupus), the immune system attacks the skin, joints, nervous system, and many other organs. Patients with SLE make numerous “autoantibodies” (antibodies are molecules made by the immune system that recognize and attack antigens; autoantibodies attack self-antigens). These autoantibodies start the attack on the body; then other parts of the immune system join in, causing inflammation and forming deposits of immune cells, both of which damage tissues. Common symptoms of SLE include skin rashes and arthritis, but some patients develop NP-SLE, a form of SLE that includes neuropsychiatric symptoms such as amnesia, dementia, mood disorders, strokes, and seizures. There is no cure for SLE, but mild cases are controlled with ibuprofen and other non-steroidal anti-inflammatory drugs; severe cases are kept in check with corticosteroids and other powerful immunosuppressants.
Why Was This Study Done?
In most of the tissues affected by SLE, the damage done by autoantibodies and immune cells can be seen when the tissues are examined with a microscope. But there is little microscopic damage visible in the brains of patients with NP-SLE. More generally, it is unclear how or even whether the immune system affects mental functions and emotion. In this study, researchers used magnetic resonance imaging (MRI) to investigate whether there are any structural changes in the brains of patients with NP-SLE that could explain their neuropsychiatric symptoms. They have also examined whether any changes in the brain can be linked to the presence of autoantibodies that recognize a protein called the NMDA receptor (anti-NMDAR antibodies) that is present on brain cells.
What Did the Researchers Do and Find?
The researchers used an MRI technique called diffusion weighted imaging to examine the brains of several patients with NP-SLE or SLE and the brains of several healthy individuals. Using this technique, it is possible to quantify the amount of structural damage in different regions of the brain. The researchers found no differences in most areas of the brain between the two groups of patients and the healthy controls. However, there were clear signs of damage in the amygdala (the part of the brain that regulates emotions and triggers responses to danger) in the patients with SLE or NP-SLE when compared to the control individuals. The researchers also found that the damage was more severe in the patients who had anti-NMDAR autoantibodies than in those that did not have these autoantibodies.
What Do These Findings Mean?
These findings suggest that autoantibodies produced by patients with SLE specifically damage the amygdala, a discovery that helps to explain some of the neuropsychiatric symptoms of this condition. Previous work has shown that the treatment of mice with anti-NMDAR antibodies and epinephrine, a stress hormone that causes leaks in the blood-brain barrier (antibodies can't usually get into the brain because of this barrier), results in damage to the amygdala and a deficient response to dangerous stimuli. The researchers suggest that a similar series of events might happen in SLE—patients often mention that a period of major stress precedes the development of symptoms. To provide stronger evidence for such a scenario, a detailed study of how stress relates to neuropsychiatric symptoms is needed. The damage to the amygdala (and the lack of damage elsewhere in the brain) and the possible association between brain damage and anti-NMDAR antibodies seen in this small study also need to be confirmed in more patients. Nevertheless, these findings provide an intriguing glimpse into the interplay between the immune system and the brain and into how stress might lead to physical damage in the brain.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030499.
MedlinePlus encyclopedia pages on autoimmunity and on systemic lupus erythematosus
US National Institute of Arthritis and Musculoskeletal and Skin Diseases booklet for patients with SLE
American College of Rheumatology information for patients on SLE
NHS Direct Online Health Encyclopedia pages on SLE
The Lupus Foundation of America information and support for patients with SLE
doi:10.1371/journal.pmed.0030499
PMCID: PMC1702559  PMID: 17177602
2.  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.
doi:10.1371/journal.pgen.1002406
PMCID: PMC3234215  PMID: 22174698
3.  MicroRNA-3148 Modulates Allelic Expression of Toll-Like Receptor 7 Variant Associated with Systemic Lupus Erythematosus 
PLoS Genetics  2013;9(2):e1003336.
We previously reported that the G allele of rs3853839 at 3′untranslated region (UTR) of Toll-like receptor 7 (TLR7) was associated with elevated transcript expression and increased risk for systemic lupus erythematosus (SLE) in 9,274 Eastern Asians [P = 6.5×10−10, odds ratio (OR) (95%CI) = 1.27 (1.17–1.36)]. Here, we conducted trans-ancestral fine-mapping in 13,339 subjects including European Americans, African Americans, and Amerindian/Hispanics and confirmed rs3853839 as the only variant within the TLR7-TLR8 region exhibiting consistent and independent association with SLE (Pmeta = 7.5×10−11, OR = 1.24 [1.18–1.34]). The risk G allele was associated with significantly increased levels of TLR7 mRNA and protein in peripheral blood mononuclear cells (PBMCs) and elevated luciferase activity of reporter gene in transfected cells. TLR7 3′UTR sequence bearing the non-risk C allele of rs3853839 matches a predicted binding site of microRNA-3148 (miR-3148), suggesting that this microRNA may regulate TLR7 expression. Indeed, miR-3148 levels were inversely correlated with TLR7 transcript levels in PBMCs from SLE patients and controls (R2 = 0.255, P = 0.001). Overexpression of miR-3148 in HEK-293 cells led to significant dose-dependent decrease in luciferase activity for construct driven by TLR7 3′UTR segment bearing the C allele (P = 0.0003). Compared with the G-allele construct, the C-allele construct showed greater than two-fold reduction of luciferase activity in the presence of miR-3148. Reduced modulation by miR-3148 conferred slower degradation of the risk G-allele containing TLR7 transcripts, resulting in elevated levels of gene products. These data establish rs3853839 of TLR7 as a shared risk variant of SLE in 22,613 subjects of Asian, EA, AA, and Amerindian/Hispanic ancestries (Pmeta = 2.0×10−19, OR = 1.25 [1.20–1.32]), which confers allelic effect on transcript turnover via differential binding to the epigenetic factor miR-3148.
Author Summary
Systemic lupus erythematosus (SLE) is a debilitating autoimmune disease contributed to by excessive innate immune activation involving toll-like receptors (TLRs, particularly TLR7/8/9) and type I interferon (IFN) signaling pathways. TLR7 responds against RNA–containing nuclear antigens and activates IFN-α pathway, playing a pivotal role in the development of SLE. While a genomic duplication of Tlr7 promotes lupus-like disease in the Y-linked autoimmune accelerator (Yaa) murine model, the lack of common copy number variations at TLR7 in humans led us to identify a functional single nucleotide polymorphism (SNP), rs3853839 at 3′ UTR of the TLR7 gene, associated with SLE susceptibility in Eastern Asians. In this study, we fine-mapped the TLR7-TLR8 region and confirmed rs3853839 exhibiting the strongest association with SLE in European Americans, African Americans, and Amerindian/Hispanics. Individuals carrying the risk G allele of rs3853839 exhibited increased TLR7 expression at the both mRNA and protein level and decreased transcript degradation. MicroRNA-3148 (miR-3148) downregulated the expression of non-risk allele (C) containing transcripts preferentially, suggesting a likely mechanism for increased TLR7 levels in risk-allele carriers. This trans-ancestral mapping provides evidence for the global association with SLE risk at rs3853839, which resides in a microRNA–gene regulatory site affecting TLR7 expression.
doi:10.1371/journal.pgen.1003336
PMCID: PMC3585142  PMID: 23468661
4.  Elevated Serum Levels of Interferon-Regulated Chemokines Are Biomarkers for Active Human Systemic Lupus Erythematosus 
PLoS Medicine  2006;3(12):e491.
Background
Systemic lupus erythematosus (SLE) is a serious systemic autoimmune disorder that affects multiple organ systems and is characterized by unpredictable flares of disease. Recent evidence indicates a role for type I interferon (IFN) in SLE pathogenesis; however, the downstream effects of IFN pathway activation are not well understood. Here we test the hypothesis that type I IFN-regulated proteins are present in the serum of SLE patients and correlate with disease activity.
Methods and Findings
We performed a comprehensive survey of the serologic proteome in human SLE and identified dysregulated levels of 30 cytokines, chemokines, growth factors, and soluble receptors. Particularly striking was the highly coordinated up-regulation of 12 inflammatory and/or homeostatic chemokines, molecules that direct the movement of leukocytes in the body. Most of the identified chemokines were inducible by type I IFN, and their levels correlated strongly with clinical and laboratory measures of disease activity.
Conclusions
These data suggest that severely disrupted chemokine gradients may contribute to the systemic autoimmunity observed in human SLE. Furthermore, the levels of serum chemokines may serve as convenient biomarkers for disease activity in lupus.
A comprehensive survey of the serologic proteome in human SLE suggests that severely disrupted chemokine gradients may contribute to the systemic autoimmunity observed.
Editors' Summary
Background.
The term “lupus,” meaning wolf in Latin, is often used as an abbreviation for the disease systemic lupus erythematosus (SLE). The name may have been given because some people with SLE have a rash that slightly resembles a wolf's face. The condition affects around 50 to 100 people per 100,000, and is much more common in women than men. SLE is a complicated disease that comes about when antibodies inappropriately attack the body's own connective tissues, although it is not known why this happens. Symptoms vary between different people; the disease may get better and then worse, without explanation; and can affect many different organs including the skin, joints, kidneys, blood cells, and brain and nervous system. SLE is difficult for doctors to diagnose. Although the disease cannot be cured, patients who are diagnosed with SLE can be treated for their symptoms, and the right management can slow progress of the disease. One area of SLE research focuses on finding “molecular markers” (e.g., proteins or other compounds) that could be tested for in the blood. Researchers hope this would help doctors to more accurately diagnose SLE initially, and then also help to track progress in a patient's condition.
Why Was This Study Done?
“Gene expression” is a term meaning the process by which a gene's DNA sequence is converted into the structures and functions of a cell. These investigators had found in previous studies that certain genes were more “highly expressed” in the blood cells of patients with SLE. Some of these genes were already known to be regulated by interferons (a group of proteins, produced by certain blood cells, that are important in helping to defend against viral infections). The investigators performing this study wanted to understand more clearly the role of interferon in SLE and to see whether the genes that are more highly expressed in patients with SLE go on to produce higher levels of protein, which might then provide useful markers for monitoring the condition.
What Did the Researchers Do and Find?
This research project was a “case-control” study, in which the researchers compared the levels of certain proteins in the blood of people who had SLE with the levels in people who did not have the condition. Thirty people were recruited as cases, from a group of patients with SLE who have been under evaluation at Johns Hopkins School of Medicine since 1987. Fifteen controls were recruited from a group of healthy people of similar age and sex as the patients with SLE; everyone involved in the study gave their consent to take part. Blood samples were taken from each individual, and the serum (liquid component of blood) was separated out. The serum levels of 160 different blood proteins were then measured. When comparing levels of blood proteins between the groups, the researchers found that 30 specific proteins were present at higher or lower levels in the SLE-affected patients. Many of these proteins are cytokines, which are regulated by interferons and are involved in the process of “signaling” within the immune system. A few proteins were found at lower levels. Levels of the interferon-regulated proteins were, on average, seen at higher levels in people whose condition was more severe.
What Do These Findings Mean?
These results suggest that patients with SLE are likely to have a very different pattern of regulation of certain proteins within the blood, particularly the proteins involved in signaling within the immune system. The authors propose that these proteins may be involved in the progression of the disease. There is also the possibility that some of these proteins may prove useful in diagnostic tests, or in tests for monitoring how the disease progresses. However, before any such tests could be used in clinical practice, they would need to be further developed and then thoroughly tested in clinical trials.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030491
Patient information from the UK National Health Service on systemic lupus erythematosus
Patient handout from the US National Institutes of Health
MedlinePLUS encyclopedia entry on lupus
Information on lupus from the UK Arthritis Research Campaign
doi:10.1371/journal.pmed.0030491
PMCID: PMC1702557  PMID: 17177599
5.  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.
doi:10.1371/journal.pgen.1001323
PMCID: PMC3048371  PMID: 21408207
6.  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.
doi:10.1371/journal.pgen.1001311
PMCID: PMC3040652  PMID: 21379322
7.  Identification of Two Independent Risk Factors for Lupus within the MHC in United Kingdom Families 
PLoS Genetics  2007;3(11):e192.
The association of the major histocompatibility complex (MHC) with SLE is well established yet the causal variants arising from this region remain to be identified, largely due to inadequate study design and the strong linkage disequilibrium demonstrated by genes across this locus. The majority of studies thus far have identified strong association with classical class II alleles, in particular HLA-DRB1*0301 and HLA-DRB1*1501. Additional associations have been reported with class III alleles; specifically, complement C4 null alleles and a tumor necrosis factor promoter SNP (TNF-308G/A). However, the relative effects of these class II and class III variants have not been determined. We have thus used a family-based approach to map association signals across the MHC class II and class III regions in a cohort of 314 complete United Kingdom Caucasian SLE trios by typing tagging SNPs together with classical typing of the HLA-DRB1 locus. Using TDT and conditional regression analyses, we have demonstrated the presence of two distinct and independent association signals in SLE: HLA-DRB1*0301 (nominal p = 4.9 × 10−8, permuted p < 0.0001, OR = 2.3) and the T allele of SNP rs419788 (nominal p = 4.3 × 10−8, permuted p < 0.0001, OR = 2.0) in intron 6 of the class III region gene SKIV2L. Assessment of genotypic risk demonstrates a likely dominant model of inheritance for HLA-DRB1*0301, while rs419788-T confers susceptibility in an additive manner. Furthermore, by comparing transmitted and untransmitted parental chromosomes, we have delimited our class II signal to a 180 kb region encompassing the alleles HLA-DRB1*0301-HLA-DQA1*0501-HLA-DQB1*0201 alone. Our class III signal importantly excludes independent association at the TNF promoter polymorphism, TNF-308G/A, in our SLE cohort and provides a potentially novel locus for future genetic and functional studies.
Author Summary
Systemic lupus erythematosus (SLE/lupus) is a complex autoimmune disease in which the body's immune system attacks its own tissues, causing inflammation in a variety of different organs such as the skin, joints, and kidneys. The cause of lupus is not known, but genes play a significant role in the predisposition to disease. The major histocompatibility complex (MHC) on Chromosome 6 contains at least 100 different genes that affect the immune system, including the genes with the strongest effect on lupus susceptibility. Despite the importance of the MHC in SLE, the identity of the actual genes in the MHC region that cause SLE has remained elusive. In the present study, we used the latest set of genetic markers present at the MHC in lupus families to identify the actual genes that affect the disease. To our knowledge, we have shown for the first time that two separate groups of genes are involved in SLE. One group of genes alters how the immune system may inappropriately target its own tissues in the disease. How the second set of genes predisposes to SLE is the subject of ongoing study.
doi:10.1371/journal.pgen.0030192
PMCID: PMC2065882  PMID: 17997607
8.  Variation in the ICAM1–ICAM4–ICAM5 locus is associated with systemic lupus erythematosus susceptibility in multiple ancestries 
Annals of the rheumatic diseases  2012;71(11):1809-1814.
Objective
Systemic lupus erythematosus (SLE; OMIM 152700) is a chronic autoimmune disease for which the aetiology includes genetic and environmental factors. ITGAM, integrin αΜ (complement component 3 receptor 3 subunit) encoding a ligand for intracellular adhesion molecule (ICAM) proteins, is an established SLE susceptibility locus. This study aimed to evaluate the independent and joint effects of genetic variations in the genes that encode ITGAM and ICAM.
Methods
The authors examined several markers in the ICAM1–ICAM4–ICAM5 locus on chromosome 19p13 and the single ITGAM polymorphism (rs1143679) using a large-scale case–control study of 17 481 unrelated participants from four ancestry populations. The single marker association and gene–gene interaction were analysed for each ancestry, and a meta-analysis across the four ancestries was performed.
Results
The A-allele of ICAM1–ICAM4–ICAM5 rs3093030, associated with elevated plasma levels of soluble ICAM1, and the A-allele of ITGAM rs1143679 showed the strongest association with increased SLE susceptibility in each of the ancestry populations and the trans-ancestry meta-analysis (ORmeta=1.16, 95% CI 1.11 to 1.22; p=4.88×10−10 and ORmeta=1.67, 95% CI 1.55 to 1.79; p=3.32×10−46, respectively). The effect of the ICAM single-nucleotide polymorphisms (SNPs) was independent of the effect of the ITGAM SNP rs1143679, and carriers of both ICAM rs3093030-AA and ITGAM rs1143679-AA had an OR of 4.08 compared with those with no risk allele in either SNP (95% CI 2.09 to 7.98; p=3.91×10−5).
Conclusion
These findings are the first to suggest that an ICAM–integrin-mediated pathway contributes to susceptibility to SLE.
doi:10.1136/annrheumdis-2011-201110
PMCID: PMC3466387  PMID: 22523428
9.  Recent insights into the genetic basis of systemic lupus erythematosus 
Genes and immunity  2009;10(5):373-379.
Genetic variation was first shown to be part of the cause of systemic lupus erythematosus (SLE or lupus) in the 1970s with associations in the human leukocyte antigen (HLA) region. Almost four decades later, and with the help of increasingly powerful genetic approaches, more than 25 genes are now known to contribute to the mechanisms that predispose individuals to lupus. Over half of these loci have been discovered in the past two years, underscoring the extraordinary success of recent genome-wide association approaches in SLE. The now well established genetic risk factors include alleles in the MHC region (multiple genes), IRF5, ITGAM, STAT4, BLK, BANK1, PDCD1, PTPN22, TNFSF4, TNFAIP3, SPP1, ATG5, XKR6, PXK, some of the Fcγ receptors, and deficiencies in several complement components, including C1q, C4, and C2. As reviewed here, many of these genes fall into key pathways that are consistent with previous studies implicating immune complexes, host immune signal transduction, and interferon pathways in the pathogenesis of SLE. Other genetic loci have no known function or apparent immunological role and have the potential to reveal novel disease mechanisms. Certainly, as our understanding of the genetic etiology of SLE continues to mature, important new opportunities will emerge for developing more targeted and effective diagnostic and clinical management tools for this complex autoimmune disease.
doi:10.1038/gene.2009.39
PMCID: PMC3144759  PMID: 19440199
10.  Association of Genetic Variants in Complement Factor H and Factor H-Related Genes with Systemic Lupus Erythematosus Susceptibility 
PLoS Genetics  2011;7(5):e1002079.
Systemic lupus erythematosus (SLE), a complex polygenic autoimmune disease, is associated with increased complement activation. Variants of genes encoding complement regulator factor H (CFH) and five CFH-related proteins (CFHR1-CFHR5) within the chromosome 1q32 locus linked to SLE, have been associated with multiple human diseases and may contribute to dysregulated complement activation predisposing to SLE. We assessed 60 SNPs covering the CFH-CFHRs region for association with SLE in 15,864 case-control subjects derived from four ethnic groups. Significant allelic associations with SLE were detected in European Americans (EA) and African Americans (AA), which could be attributed to an intronic CFH SNP (rs6677604, in intron 11, Pmeta = 6.6×10−8, OR = 1.18) and an intergenic SNP between CFHR1 and CFHR4 (rs16840639, Pmeta = 2.9×10−7, OR = 1.17) rather than to previously identified disease-associated CFH exonic SNPs, including I62V, Y402H, A474A, and D936E. In addition, allelic association of rs6677604 with SLE was subsequently confirmed in Asians (AS). Haplotype analysis revealed that the underlying causal variant, tagged by rs6677604 and rs16840639, was localized to a ∼146 kb block extending from intron 9 of CFH to downstream of CFHR1. Within this block, the deletion of CFHR3 and CFHR1 (CFHR3-1Δ), a likely causal variant measured using multiplex ligation-dependent probe amplification, was tagged by rs6677604 in EA and AS and rs16840639 in AA, respectively. Deduced from genotypic associations of tag SNPs in EA, AA, and AS, homozygous deletion of CFHR3-1Δ (Pmeta = 3.2×10−7, OR = 1.47) conferred a higher risk of SLE than heterozygous deletion (Pmeta = 3.5×10−4, OR = 1.14). These results suggested that the CFHR3-1Δ deletion within the SLE-associated block, but not the previously described exonic SNPs of CFH, might contribute to the development of SLE in EA, AA, and AS, providing new insights into the role of complement regulators in the pathogenesis of SLE.
Author Summary
Systemic lupus erythematosus (SLE) is a complex autoimmune disease, associated with increased complement activation. Previous studies have provided evidence for the presence of SLE susceptibility gene(s) in the chromosome 1q31-32 locus. Within 1q32, genes encoding complement regulator factor H (CFH) and five CFH-related proteins (CFHR1-CFHR5) may contribute to the development of SLE, because genetic variants of these genes impair complement regulation and predispose to various human diseases. In this study, we tested association of genetic variants in the region containing CFH and CFHRs with SLE. We identified genetic variants predisposing to SLE in European American, African American, and Asian populations, which might be attributed to the deletion of CFHR3 and CFHR1 genes but not previously identified disease-associated exonic variants of CFH. This study provides the first evidence for consistent association between CFH/CFHRs and SLE across multi-ancestral SLE datasets, providing new insights into the role of complement regulators in the pathogenesis of SLE.
doi:10.1371/journal.pgen.1002079
PMCID: PMC3102741  PMID: 21637784
11.  A Functional Variant in MicroRNA-146a Promoter Modulates Its Expression and Confers Disease Risk for Systemic Lupus Erythematosus 
PLoS Genetics  2011;7(6):e1002128.
Systemic lupus erythematosus (SLE) is a complex autoimmune disease with a strong genetic predisposition, characterized by an upregulated type I interferon pathway. MicroRNAs are important regulators of immune homeostasis, and aberrant microRNA expression has been demonstrated in patients with autoimmune diseases. We recently identified miR-146a as a negative regulator of the interferon pathway and linked the abnormal activation of this pathway to the underexpression of miR-146a in SLE patients. To explore why the expression of miR-146a is reduced in SLE patients, we conducted short parallel sequencing of potentially regulatory regions of miR-146a and identified a novel genetic variant (rs57095329) in the promoter region exhibiting evidence for association with SLE that was replicated independently in 7,182 Asians (Pmeta = 2.74×10−8, odds ratio = 1.29 [1.18–1.40]). The risk-associated G allele was linked to reduced expression of miR-146a in the peripheral blood leukocytes of the controls. Combined functional assays showed that the risk-associated G allele reduced the protein-binding affinity and activity of the promoter compared with those of the promoter containing the protective A allele. Transcription factor Ets-1, encoded by the lupus-susceptibility gene ETS1, identified in recent genome-wide association studies, binds near this variant. The manipulation of Ets-1 levels strongly affected miR-146a promoter activity in vitro; and the knockdown of Ets-1, mimicking its reduced expression in SLE, directly impaired the induction of miR-146a. We also observed additive effects of the risk alleles of miR-146a and ETS1. Our data identified and confirmed an association between a functional promoter variant of miR-146a and SLE. This risk allele had decreased binding to transcription factor Ets-1, contributing to reduced levels of miR-146a in SLE patients.
Author Summary
Genome-wide association studies have identified quite a number of susceptibility loci associated with complex diseases such as systemic lupus erythematosus (SLE). However, for most of them, the intrinsic link between genetic variation and disease mechanism is not fully understood. SLE is characterized by a significantly upregulated type I interferon (IFN) pathway, and we have previously reported that underexpression of a microRNA, miR-146a, contributes to alterations in the type I IFN pathway in lupus patients. Here we identified a novel genetic variant in the promoter region of miR-146a that is directly related to reduced expression of miR-146a and is associated with SLE susceptibility. The risk allele of this variant confers weaker binding affinity for Ets-1, which is a transcription factor encoded by a lupus susceptibility gene found in recent GWAS. These findings suggest that reduced expression of Ets-1 and its reduced binding affinity to the miR-146a promoter both may contribute to low levels of this microRNA in SLE patients, which may contribute to the upregulated type I IFN pathway in these patients. To our knowledge, this is also the first piece of evidence showing association between a genetic variant in a promoter region of a miRNA gene and a human disease.
doi:10.1371/journal.pgen.1002128
PMCID: PMC3128113  PMID: 21738483
12.  Resequencing the susceptibility gene, ITGAM, identifies two functionally deleterious rare variants in systemic lupus erythematosus cases 
Arthritis Research & Therapy  2014;16(3):R114.
Introduction
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1186/ar4566
PMCID: PMC4060450  PMID: 24886912
13.  Specificity of the STAT4 Genetic Association for Severe Disease Manifestations of Systemic Lupus Erythematosus 
PLoS Genetics  2008;4(5):e1000084.
Systemic lupus erythematosus (SLE) is a genetically complex disease with heterogeneous clinical manifestations. A polymorphism in the STAT4 gene has recently been established as a risk factor for SLE, but the relationship with specific SLE subphenotypes has not been studied. We studied 137 SNPs in the STAT4 region genotyped in 4 independent SLE case series (total n = 1398) and 2560 healthy controls, along with clinical data for the cases. Using conditional testing, we confirmed the most significant STAT4 haplotype for SLE risk. We then studied a SNP marking this haplotype for association with specific SLE subphenotypes, including autoantibody production, nephritis, arthritis, mucocutaneous manifestations, and age at diagnosis. To prevent possible type-I errors from population stratification, we reanalyzed the data using a subset of subjects determined to be most homogeneous based on principal components analysis of genome-wide data. We confirmed that four SNPs in very high LD (r2 = 0.94 to 0.99) were most strongly associated with SLE, and there was no compelling evidence for additional SLE risk loci in the STAT4 region. SNP rs7574865 marking this haplotype had a minor allele frequency (MAF) = 31.1% in SLE cases compared with 22.5% in controls (OR = 1.56, p = 10−16). This SNP was more strongly associated with SLE characterized by double-stranded DNA autoantibodies (MAF = 35.1%, OR = 1.86, p<10−19), nephritis (MAF = 34.3%, OR = 1.80, p<10−11), and age at diagnosis<30 years (MAF = 33.8%, OR = 1.77, p<10−13). An association with severe nephritis was even more striking (MAF = 39.2%, OR = 2.35, p<10−4 in the homogeneous subset of subjects). In contrast, STAT4 was less strongly associated with oral ulcers, a manifestation associated with milder disease. We conclude that this common polymorphism of STAT4 contributes to the phenotypic heterogeneity of SLE, predisposing specifically to more severe disease.
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. It is thought that 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 subtypes of lupus and specific genes, we hope to better understand how lupus is triggered and by what biological pathways it progresses. We show in this work that the STAT4 gene, very recently identified as a lupus risk gene, predisposes specifically to severe manifestations of lupus, including kidney disease.
doi:10.1371/journal.pgen.1000084
PMCID: PMC2377340  PMID: 18516230
14.  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.
doi:10.1093/hmg/ddp007
PMCID: PMC2649018  PMID: 19129174
15.  Emerging Roles for the Interferon-Inducible p200-Family Proteins in Sex Bias in Systemic Lupus Erythematosus 
Systemic lupus erythematosus (SLE) is a complex autoimmune disease involving multiple organs. The disease is characterized by the production of pathogenic autoantibodies to DNA and certain nuclear antigens, chronic inflammation, and immune dysregulation. Genetic studies involving SLE patients and mouse models have indicated that multiple lupus susceptible genes contribute to the disease phenotype. Notably, the development of SLE in patients and in certain mouse models exhibits a strong sex bias. In addition, several lines of evidence indicates that activation of interferon-α (IFN-α) signaling in immune cells and alterations in the expression of certain immunomodulatory cytokines contribute to lupus pathogenesis. Studies have implicated factors, such as the X chromosomal gene dosage effect and the sex hormones, in gender bias in SLE. However, the molecular mechanisms remain unclear. Additionally, it remains unclear whether these factors influence the “IFN-signature,” which is associated with SLE. In this regard, a mutually positive regulatory feedback loop between IFNs and estrogen receptor-α (ERα) has been identified in immune cells. Moreover, studies indicate that the expression of certain IFN-inducible p200-family proteins that act as innate immune sensors for cytosolic DNA is differentially regulated by sex hormones. In this review, we discuss how the modulation of the expression of the p200-family proteins in immune cells by sex hormones and IFNs contributes to sex bias in SLE. An improved understanding of the regulation and roles of the p200-family proteins in immune cells is critical to understand lupus pathogenesis as well as response (or the lack of it) to various therapies.
doi:10.1089/jir.2011.0073
PMCID: PMC3234491  PMID: 21902548
16.  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.
doi:10.1016/j.autrev.2011.07.007
PMCID: PMC3224188  PMID: 21840425
ITGAM; autoimmune diseases; genetic susceptibility
17.  Polymorphisms of the ITGAM Gene Confer Higher Risk of Discoid Cutaneous Than of Systemic Lupus Erythematosus 
PLoS ONE  2010;5(12):e14212.
Background
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).
Significance
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.
doi:10.1371/journal.pone.0014212
PMCID: PMC2996302  PMID: 21151989
18.  Identification of MAMDC1 as a Candidate Susceptibility Gene for Systemic Lupus Erythematosus (SLE) 
PLoS ONE  2009;4(12):e8037.
Background
Systemic lupus erythematosus (SLE) is a complex autoimmune disorder with multiple susceptibility genes. We have previously reported suggestive linkage to the chromosomal region 14q21-q23 in Finnish SLE families.
Principal Findings
Genetic fine mapping of this region in the same family material, together with a large collection of parent affected trios from UK and two independent case-control cohorts from Finland and Sweden, indicated that a novel uncharacterized gene, MAMDC1 (MAM domain containing glycosylphosphatidylinositol anchor 2, also known as MDGA2, MIM 611128), represents a putative susceptibility gene for SLE. In a combined analysis of the whole dataset, significant evidence of association was detected for the MAMDC1 intronic single nucleotide polymorphisms (SNP) rs961616 (P –value = 0.001, Odds Ratio (OR) = 1.292, 95% CI 1.103–1.513) and rs2297926 (P –value = 0.003, OR = 1.349, 95% CI 1.109–1.640). By Northern blot, real-time PCR (qRT-PCR) and immunohistochemical (IHC) analyses, we show that MAMDC1 is expressed in several tissues and cell types, and that the corresponding mRNA is up-regulated by the pro-inflammatory cytokines tumour necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) in THP-1 monocytes. Based on its homology to known proteins with similar structure, MAMDC1 appears to be a novel member of the adhesion molecules of the immunoglobulin superfamily (IgCAM), which is involved in cell adhesion, migration, and recruitment to inflammatory sites. Remarkably, some IgCAMs have been shown to interact with ITGAM, the product of another SLE susceptibility gene recently discovered in two independent genome wide association (GWA) scans.
Significance
Further studies focused on MAMDC1 and other molecules involved in these pathways might thus provide new insight into the pathogenesis of SLE.
doi:10.1371/journal.pone.0008037
PMCID: PMC2785483  PMID: 19997561
19.  Distinct roles for complement in glomerulonephritis and atherosclerosis revealed in mice with a combination of lupus and hyperlipidemia 
Arthritis and Rheumatism  2012;64(8):2707-2718.
Objective Although the accelerating effect of systemic lupus erythematosus (SLE) on atherosclerosis is well established, the underlying mechanisms are unknown. The aim of this study was to explore the hypothesis that lupus autoimmunity modulates the effect of hypercholesterolemia in driving arterial pathologic development.
>Methods Low-density lipoprotein receptor–deficient (Ldlr−/−) mice were crossed with B6.129-Sle16 (Sle16)–congenic autoimmune mice to obtain Sle16. Ldlr−/− mice, which were compared with Ldlr−/− and Sle16 control mice. All mice were fed either a low-fat or high-fat diet. Groups of mice were compared, by strain and by diet group, for features of accelerated atherosclerosis and autoimmunity.
Results Presence of the Sle16 locus significantly increased the extent of atherosclerosis in Ldlr−/− mice. Circulating C3 levels were significantly reduced in Sle16.Ldlr−/− mice compared to Ldlr−/− control mice and this was paralleled by a marked reduction in arterial lesion C3 deposition despite similar levels of IgG deposition between the groups. Increased numbers of apoptotic cells in plaques were observed in the high-fat–fed Sle16.Ldlr−/− mice, consistent with the observed defective clearance of cellular debris. After receiving the high-fat diet, Sle16.Ldlr−/− mice developed glomerulonephritis and displayed enhanced glomerular C3 deposition.
Conclusion These results indicate that accelerated atherosclerosis and renal inflammation in SLE are closely linked via immune complex formation and systemic complement depletion. However, whereas hyperlipidemia will enhance renal immune complex–mediated complement activation and the development of nephritis, accelerated atherosclerosis is, instead, related to complement depletion and a reduction in the uptake of apoptotic/necrotic debris. These results suggest that aggressive treatment of hyperlipidemia in patients with SLE may reduce the occurrence of lupus nephritis, as well as diminish the risk of accelerated atherosclerosis.
doi:10.1002/art.34451
PMCID: PMC3607248  PMID: 22392450
20.  Copy number variants in genetic susceptibility and severity of systemic lupus erythematosus 
Cytogenetic and Genome Research  2009;123(1-4):142-147.
Systemic lupus erythematosus (SLE) is a systemic autoimmune disorder characterized by the presence of auto-antibodies to nuclear antigens, immune complex deposition, and subsequent tissue destruction. Early studies in twins suggested that SLE has, at least in part, a genetic basis, and a role for class II alleles in the major histocompatibility complex has been known for over 30 years. Through both linkage studies and candidate gene studies, numerous additional genetic risk factors have been identified. The recent publication of two SNP-based genome-wide association studies (GWAS) has resulted in the confirmation of a number of previously identified genetic risk loci and has identified new previously unappreciated loci conferring risk for development of SLE. A role for gene copy number variation (CNV) in SLE has also been appreciated through studies of the complement component 4 (C4) loci and more recent work in the IgG Fc receptor loci. The availability of large SNP-based GWAS datasets will undoubtedly lead to the genome-wide analysis and identification of copy number variants related to genetic susceptibility for development of SLE. We review current studies of CNV in SLE susceptibility that include reports of association between SLE and CNV in C4, IgG Fc receptors, TLR7, and CCL3L1.
doi:10.1159/000184701
PMCID: PMC2826785  PMID: 19287148
21.  Association of NCF2, IKZF1, IRF8, IFIH1, and TYK2 with Systemic Lupus Erythematosus 
PLoS Genetics  2011;7(10):e1002341.
Systemic lupus erythematosus (SLE) is a complex trait characterised by the production of a range of auto-antibodies and a diverse set of clinical phenotypes. Currently, ∼8% of the genetic contribution to SLE in Europeans is known, following publication of several moderate-sized genome-wide (GW) association studies, which identified loci with a strong effect (OR>1.3). In order to identify additional genes contributing to SLE susceptibility, we conducted a replication study in a UK dataset (870 cases, 5,551 controls) of 23 variants that showed moderate-risk for lupus in previous studies. Association analysis in the UK dataset and subsequent meta-analysis with the published data identified five SLE susceptibility genes reaching genome-wide levels of significance (Pcomb<5×10−8): NCF2 (Pcomb = 2.87×10−11), IKZF1 (Pcomb = 2.33×10−9), IRF8 (Pcomb = 1.24×10−8), IFIH1 (Pcomb = 1.63×10−8), and TYK2 (Pcomb = 3.88×10−8). Each of the five new loci identified here can be mapped into interferon signalling pathways, which are known to play a key role in the pathogenesis of SLE. These results increase the number of established susceptibility genes for lupus to ∼30 and validate the importance of using large datasets to confirm associations of loci which moderately increase the risk for disease.
Author Summary
Genome-wide association studies have revolutionised our ability to identify common susceptibility alleles for systemic lupus erythematosus (SLE). In complex diseases such as SLE, where many different genes make a modest contribution to disease susceptibility, it is necessary to perform large-scale association studies to combine results from several datasets, to have sufficient power to identify highly significant novel loci (P<5×10−8). Using a large SLE collection of 870 UK SLE cases and 5,551 UK unaffected individuals, we firstly replicated ten moderate-risk alleles (P<0.05) from a US–Swedish study of 3,273 SLE cases and 12,188 healthy controls. Combining our results with the US-Swedish data identified five new loci, which crossed the level for genome-wide significance: NCF2 (neutrophil cytosolic factor 2), IKZF1 (Ikaros family zinc-finger 1), IRF8 (interferon regulatory factor 8), IFIH1 (interferon-induced helicase C domain-containing protein 1), and TYK2 (tyrosine kinase 2). Each of these five genes regulates a different aspect of the immune response and contributes to the production of type-I and type-II interferons. Although further studies will be required to identify the causal alleles within these loci, the confirmation of five new susceptibility genes for lupus makes a significant step forward in our understanding of the genetic contribution to SLE.
doi:10.1371/journal.pgen.1002341
PMCID: PMC3203198  PMID: 22046141
22.  Amelioration of Lupus Nephritis by Serum Amyloid P Component Gene Therapy with Distinct Mechanisms Varied from Different Stage of the Disease 
PLoS ONE  2011;6(7):e22659.
Background
Our previous study revealed that administration of syngeneic female BALB/c mice with excessive self activated lymphocyte-derived DNA (ALD-DNA) could induce systemic lupus erythematosus (SLE) disease, indicating that overload of self-DNA might exceed normal clearance ability and comprise the major source of autoantigens in lupus mice. Serum amyloid P component (SAP), an acute-phase serum protein with binding reactivity to DNA in mice, was proved to promote the clearance of free DNA and prevent mice against self-antigen induced autoimmune response. It is reasonable to hypothesize that SAP treatment might contribute to alleviation of SLE disease, whereas its role in ALD-DNA-induced lupus nephritis is not fully understood.
Methodology/Principal Findings
The ratios of SAP to DNA significantly decreased and were negatively correlated with the titers of anti-dsDNA antibodies in ALD-DNA-induced lupus mice, indicating SAP was relatively insufficient in lupus mice. Herein a pcDNA3-SAP plasmid (pSAP) was genetically constructed and intramuscularly injected into BALB/c mice. It was found that SAP protein purified from the serum of pSAP-treated mice bound efficiently to ALD-DNA and inhibited ALD-DNA-mediated innate immune response in vitro. Treatment of ALD-DNA-induced lupus mice with pSAP in the early stage of SLE disease with the onset of proteinuria reversed lupus nephritis via decreasing anti-dsDNA autoantibody production and immune complex (IC) deposition. Further administration of pSAP in the late stage of SLE disease that had established lupus nephritis alleviated proteinuria and ameliorated lupus nephritis. This therapeutic effect of SAP was not only attributable to the decreased levels of anti-dsDNA autoantibodies, but also associated with the decreased infiltration of lymphocytes and the reduced production of inflammatory markers.
Conclusion/Significance
These results suggest that SAP administration could effectively alleviated lupus nephritis via modulating anti-dsDNA antibody production and the inflammation followed IC deposition, and SAP-based intervening strategy may provide new approaches for treating SLE disease.
doi:10.1371/journal.pone.0022659
PMCID: PMC3143173  PMID: 21799927
23.  Immunopathogenesis of environmentally induced lupus in mice. 
Environmental Health Perspectives  1999;107(Suppl 5):723-727.
Systemic lupus erythematosus (SLE) is a systemic autoimmune syndrome defined by clinical and serologic features, including arthritis, glomerulonephritis, and certain autoantibodies such as anti-nuclear ribonucleoprotein (nRNP)/Smith antigen (Sm), DNA, and ribosomal P. Although lupus is considered primarily a genetic disorder, we recently demonstrated the induction of a syndrome strikingly similar to spontaneous lupus in many nonautoimmune strains of mice exposed to the isoprenoid alkane pristane (2,6,10,14-tetramethylpentadecane), a component of mineral oil. Intraperitoneal injection of pristane leads to the formation of lipogranulomas consisting of phagocytic cells that have engulfed the oil and collections of lymphocytes. Subsequently, pristane-treated BALB/c and SJL mice develop autoantibodies characteristic of SLE, including anti-nRNP/Sm, antiribosomal P, anti-Su, antichromatin, anti-single-stranded DNA, and anti-double-stranded DNA. This is accompanied by a severe glomerulonephritis with immune complex deposition, mesangial or mesangiocapillary proliferation, and proteinuria. All inbred mice examined appear to be susceptible to this novel form of chemically induced lupus. Pristane-induced lupus is the only inducible model of autoimmunity associated with the clinical syndrome as well as with the characteristic serologic abnormalities of SLE. Defining the immunopathogenesis of pristane-induced lupus in mice may provide insight into the causes of spontaneous (idiopathic) lupus and also may lead to information concerning possible risks associated with the ingestion or inhalation of mineral oil and exposure to hydrocarbons in the environment.
Images
PMCID: PMC1566261  PMID: 10502537
24.  Mutations in Complement Regulatory Proteins Predispose to Preeclampsia: A Genetic Analysis of the PROMISSE Cohort 
PLoS Medicine  2011;8(3):e1001013.
Jane Salmon and colleagues studied 250 pregnant patients with SLE and/or antiphospholipid antibodies and found an association of risk variants in complement regulatory proteins in patients who developed preeclampsia, as well as in preeclampsia patients lacking autoimmune disease.
Background
Pregnancy in women with systemic lupus erythematosus (SLE) or antiphospholipid antibodies (APL Ab)—autoimmune conditions characterized by complement-mediated injury—is associated with increased risk of preeclampsia and miscarriage. Our previous studies in mice indicate that complement activation targeted to the placenta drives angiogenic imbalance and placental insufficiency.
Methods and Findings
We use PROMISSE, a prospective study of 250 pregnant patients with SLE and/or APL Ab, to test the hypothesis in humans that impaired capacity to limit complement activation predisposes to preeclampsia. We sequenced genes encoding three complement regulatory proteins—membrane cofactor protein (MCP), complement factor I (CFI), and complement factor H (CFH)—in 40 patients who had preeclampsia and found heterozygous mutations in seven (18%). Five of these patients had risk variants in MCP or CFI that were previously identified in atypical hemolytic uremic syndrome, a disease characterized by endothelial damage. One had a novel mutation in MCP that impairs regulation of C4b. These findings constitute, to our knowledge, the first genetic defects associated with preeclampsia in SLE and/or APL Ab. We confirmed the association of hypomorphic variants of MCP and CFI in a cohort of non-autoimmune preeclampsia patients in which five of 59 were heterozygous for mutations.
Conclusion
The presence of risk variants in complement regulatory proteins in patients with SLE and/or APL Ab who develop preeclampsia, as well as in preeclampsia patients lacking autoimmune disease, links complement activation to disease pathogenesis and suggests new targets for treatment of this important public health problem.
Study Registration
ClinicalTrials.gov NCT00198068
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Most pregnancies culminate in the birth of a healthy baby but, sadly, about a quarter of women lose their babies during pregnancy. A common pregnancy-related medical problem that threatens the life of both baby and mother is preeclampsia. Mild and severe preeclampsia affects up to 10% and 1%–2% of pregnancies, respectively. Preeclampsia occurs because of a problem with the function of the placenta, the organ that transfers nutrients and oxygen from mother to baby and removes waste products from the baby. Although preeclampsia begins early in pregnancy, it is diagnosed by the onset of high blood pressure (hypertension) and the appearance of protein in the urine (proteinuria) after 20 weeks of pregnancy. Other warning signs include headaches and swelling of the hands and face. The only cure for preeclampsia is delivery, and labor is usually induced early to prevent eclampsia (seizures), stroke, liver and kidney failure, and breathing and blood vessel problems developing in the mother. Although delivery before 37 weeks of pregnancy is not generally recommended, in cases of preeclampsia it may be too dangerous for both the baby and the mother to allow the pregnancy to continue. Unfortunately when severe preeclampsia occurs in the second trimester, babies weighing only 500 grams may be delivered and they may not survive.
Why Was This Study Done?
Because the exact cause of preeclampsia is unknown, it is difficult to develop treatments for the condition or to find ways to prevent it. Many experts think that immune system problems—in particular, perturbations in complement activation—may be involved in preeclampsia. The complement system is a set of blood proteins that attacks invading bacteria and viruses. The activation of complement proteins is usually tightly regulated (overactivation of the complement system causes tissue damage) and, because preeclampsia may run in families, one hypothesis is that mutations (genetic changes) in complement regulatory proteins might predispose women to preeclampsia. In this study, the researchers test this hypothesis by sequencing genes encoding complement regulatory proteins in pregnant women with the autoimmune diseases systemic lupus erythematosus (SLE) and/or antiphospholipid antibodies (APL Ab) who developed preeclampsia. In autoimmune diseases, the immune system attacks healthy human cells instead of harmful invaders. Both SLE and APL Ab are characterized by complement-mediated tissue injury and are associated with an increased risk of preeclampsia and miscarriage.
What Did the Researchers Do and Find?
Two hundred fifty women with SLE and/or APL Ab were enrolled into the PROMISSE study (a multi-center observational study to identify predictors of pregnancy outcome in women with SLE and/or APL Ab) when they were 12 weeks pregnant and followed through pregnancy. Thirty patients developed preeclampsia during the study and ten more had had preeclampsia during a previous pregnancy. The researchers sequenced the genes for complement regulatory proteins: membrane cofactor protein (MCP), factor I, and factor H in these 40 patients. Seven women (18%) had mutations in one copy of one of these genes (there are two copies of most genes in human cells). Five mutations were alterations in MCP or factor I that are gene variants that increase the risk of hemolytic uremic syndrome, a disease characterized by blood vessel damage. The sixth mutation was a new MCP mutation that impaired MCP's ability to regulate complement component C4b. The final mutation was a factor H mutation that did not have any obvious functional effect. No mutations in complement regulatory proteins were found in 34 matched participants in PROMISSE without preeclampsia but, among a group of non-autoimmune women who developed preeclampsia during pregnancy, 10% had mutations in MCP or factor I.
What Do These Findings Mean?
These findings identify MCP and factor I mutations as genetic defects associated with preeclampsia in pregnant women with SLE and/or APL Ab. Importantly, they also reveal an association between similar mutations and preeclampsia in women without any underlying autoimmune disease. Taken together with evidence from previous animal experiments, these findings suggest that dysregulation of complement activation is involved in the development of preeclampsia. Although further studies are needed to confirm and extend these findings, these results suggest that proteins involved in the regulation of complement activation could be new targets for the treatment of preeclampsia and raise the possibility that tests could be developed to identify women at risk of developing preeclampsia.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001013.
Tommy's, a UK charity that funds scientific research into the causes and prevention of miscarriage, premature birth, and stillbirth, has information on preeclampsia
The March of Dimes Foundation, a nonprofit organization for pregnancy and baby health, has information on preeclampsia
The UK National Health Services Choices website also has information about preeclampsia
Wikipedia has pages on the complement system, on autoimmune disease, and on preeclampsia (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
More information on the PROMISSE study is available
doi:10.1371/journal.pmed.1001013
PMCID: PMC3062534  PMID: 21445332
25.  Preferential Binding to Elk-1 by SLE-Associated IL10 Risk Allele Upregulates IL10 Expression 
PLoS Genetics  2013;9(10):e1003870.
Immunoregulatory cytokine interleukin-10 (IL-10) is elevated in sera from patients with systemic lupus erythematosus (SLE) correlating with disease activity. The established association of IL10 with SLE and other autoimmune diseases led us to fine map causal variant(s) and to explore underlying mechanisms. We assessed 19 tag SNPs, covering the IL10 gene cluster including IL19, IL20 and IL24, for association with SLE in 15,533 case and control subjects from four ancestries. The previously reported IL10 variant, rs3024505 located at 1 kb downstream of IL10, exhibited the strongest association signal and was confirmed for association with SLE in European American (EA) (P = 2.7×10−8, OR = 1.30), but not in non-EA ancestries. SNP imputation conducted in EA dataset identified three additional SLE-associated SNPs tagged by rs3024505 (rs3122605, rs3024493 and rs3024495 located at 9.2 kb upstream, intron 3 and 4 of IL10, respectively), and SLE-risk alleles of these SNPs were dose-dependently associated with elevated levels of IL10 mRNA in PBMCs and circulating IL-10 protein in SLE patients and controls. Using nuclear extracts of peripheral blood cells from SLE patients for electrophoretic mobility shift assays, we identified specific binding of transcription factor Elk-1 to oligodeoxynucleotides containing the risk (G) allele of rs3122605, suggesting rs3122605 as the most likely causal variant regulating IL10 expression. Elk-1 is known to be activated by phosphorylation and nuclear localization to induce transcription. Of interest, phosphorylated Elk-1 (p-Elk-1) detected only in nuclear extracts of SLE PBMCs appeared to increase with disease activity. Co-expression levels of p-Elk-1 and IL-10 were elevated in SLE T, B cells and monocytes, associated with increased disease activity in SLE B cells, and were best downregulated by ERK inhibitor. Taken together, our data suggest that preferential binding of activated Elk-1 to the IL10 rs3122605-G allele upregulates IL10 expression and confers increased risk for SLE in European Americans.
Author Summary
Systemic lupus erythematosus (SLE), a debilitating autoimmune disease characterized by the production of pathogenic autoantibodies, has a strong genetic basis. Variants of the IL10 gene, which encodes cytokine interleukin-10 (IL-10) with known function of promoting B cell hyperactivity and autoantibody production, are associated with SLE and other autoimmune diseases, and serum IL-10 levels are elevated in SLE patients correlating with increased disease activity. In this study, to discover SLE-predisposing causal variant(s), we assessed variants within the genomic region containing IL10 and its gene family member IL19, IL20 and IL24 for association with SLE in case and control subjects from diverse ancestries. We identified SLE-associated SNP rs3122605 located at 9.2 kb upstream of IL10 as the most likely causal variant in subjects of European ancestry. The SLE-risk allele of rs3122605 was dose-dependently associated with elevated IL10 expression at both mRNA and protein levels in peripheral blood samples from SLE patients and controls, which could be explained, at least in part, by its preferential binding to Elk-1, a transcription factor activated in B cells during active disease of SLE patients. Elk-1-mediated IL-10 overexpression could be downregulated by inhibiting activation of mitogen-activated protein kinases, suggesting a potential therapeutic target for SLE.
doi:10.1371/journal.pgen.1003870
PMCID: PMC3794920  PMID: 24130510

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