Next Generation Sequencing studies generate a large quantity of genetic data in a relatively cost and time efficient manner and provide an unprecedented opportunity to identify candidate causative variants that lead to disease phenotypes. A challenge to these studies is the generation of sequencing artifacts by current technologies. To identify and characterize the properties that distinguish false positive variants from true variants, we sequenced a child and both parents (one trio) using DNA isolated from three sources (blood, buccal cells, and saliva). The trio strategy allowed us to identify variants in the proband that could not have been inherited from the parents (Mendelian errors) and would most likely indicate sequencing artifacts. Quality control measurements were examined and three measurements were found to identify the greatest number of Mendelian errors. These included read depth, genotype quality score, and alternate allele ratio. Filtering the variants on these measurements removed ~95% of the Mendelian errors while retaining 80% of the called variants. These filters were applied independently. After filtering, the concordance between identical samples isolated from different sources was 99.99% as compared to 87% before filtering. This high concordance suggests that different sources of DNA can be used in trio studies without affecting the ability to identify causative polymorphisms. To facilitate analysis of next generation sequencing data, we developed the Cincinnati Analytical Suite for Sequencing Informatics (CASSI) to store sequencing files, metadata (eg. relatedness information), file versioning, data filtering, variant annotation, and identify candidate causative polymorphisms that follow either de novo, rare recessive homozygous or compound heterozygous inheritance models. We conclude the data cleaning process improves the signal to noise ratio in terms of variants and facilitates the identification of candidate disease causative polymorphisms.
whole exome sequencing; variant filtering; next-generation sequencing; disease causative polymorphisms; Mendelian errors; Mendel errors; CASSI
There are limited reports of the use of whole exome sequencing (WES) as a clinical diagnostic tool. Moreover, there are no reports addressing the cost burden associated with genetic tests performed prior to WES.
We demonstrate the performance characteristics of WES in a pediatric setting by describing our patient cohort, calculating the diagnostic yield, and detailing the patients for whom clinical management was altered. Moreover, we examined the potential cost-effectiveness of WES by examining the cost burden of diagnostic workups.
To determine the clinical utility of our hospital’s clinical WES, we performed a retrospective review of the first 40 cases. We utilized dual bioinformatics analyses pipelines based on commercially available software and in-house tools.
Of the first 40 clinical cases, we identified genetic defects in 12 (30%) patients, of which 47% of the mutations were previously unreported in the literature. Among the 12 patients with positive findings, seven have autosomal dominant disease and five have autosomal recessive disease. Ninety percent of the cohort opted to receive secondary findings and of those, secondary medical actionable results were returned in three cases. Among these positive cases, there are a number of novel mutations that are being reported here. The diagnostic workup included a significant number of genetic tests with microarray and single-gene sequencing being the most popular tests. Significantly, genetic diagnosis from WES led to altered patient medical management in positive cases.
We demonstrate the clinical utility of WES by establishing the clinical diagnostic rate and its impact on medical management in a large pediatric center. The cost-effectiveness of WES was demonstrated by ending the diagnostic odyssey in positive cases. Also, in some cases it may be most cost-effective to directly perform WES. WES provides a unique glimpse into the complexity of genetic disorders.
whole exome sequencing; next generation sequencing; diagnosis; children; clinical utility; pediatrics
Recent reports have associated NCF2, encoding a core component of the multi-protein NADPH oxidase (NADPHO), with systemic lupus erythematosus (SLE) susceptibility in individuals of European ancestry. To identify ethnicity-specific and -robust variants within NCF2, we assessed 145 SNPs in and around the NCF2 gene in 5325 cases and 21 866 controls of European-American (EA), African-American (AA), Hispanic (HS) and Korean (KR) ancestry. Subsequent imputation, conditional, haplotype and bioinformatic analyses identified seven potentially functional SLE-predisposing variants. Association with non-synonymous rs17849502, previously reported in EA, was detected in EA, HS and AA (PEA = 1.01 × 10−54, PHS = 3.68 × 10−10, PAA = 0.03); synonymous rs17849501 was similarly significant. These SNPs were monomorphic in KR. Novel associations were detected with coding variants at rs35937854 in AA (PAA = 1.49 × 10−9), and rs13306575 in HS and KR (PHS = 7.04 × 10−7, PKR = 3.30 × 10−3). In KR, a 3-SNP haplotype was significantly associated (P = 4.20 × 10−7), implying that SLE predisposing variants were tagged. Significant SNP–SNP interaction (P = 0.02) was detected between rs13306575 and rs17849502 in HS, and a dramatically increased risk (OR = 6.55) with a risk allele at each locus. Molecular modeling predicts that these non-synonymous mutations could disrupt NADPHO complex assembly. The risk allele of rs17849501, located in a conserved transcriptional regulatory region, increased reporter gene activity, suggesting in vivo enhancer function. Our results not only establish allelic heterogeneity within NCF2 associated with SLE, but also emphasize the utility of multi-ethnic cohorts to identify predisposing variants explaining additional phenotypic variance (‘missing heritability’) of complex diseases like SLE.
Macrophage activation syndrome (MAS), a life-threatening complication of systemic Juvenile Idiopathic Arthritis (SJIA), resembles Familial Hemophagocytic Lymphohistiocytosis (FHLH), a constellation of autosomal recessive immune disorders resulting from deficiency in cytolytic pathway proteins. We hypothesized that MAS predisposition in SJIA could be attributed to rare gene sequence variants affecting the cytotolytic pathway.
Whole exome sequencing (WES) was used in 14 SJIA/MAS patients and their parents to identify protein altering SNPs/indels in the known HLH-associated genes. To discover new candidate genes, the entire WES data were filtered to identify protein altering, rare recessive homozygous, compound heterozygous, and de novo variants with the potential to affect the cytolytic pathway.
Heterozygous protein-altering rare variants in the known genes (LYST, MUNC13-4, and STXBP2) were found in 5 of 14 SJIA/MAS patients (35.7%). This was in contrast to only 4 variants in 4 of 29 (13,7%) SJIA patients without MAS. Homozygosity and compound heterozygosity analysis applied to the entire WES data in SJIAMAS, revealed 3 recessive pairs in 3 genes, and 76 compound heterozygotes in 75 genes. We also identified 22 heterozygous rare protein altering variants that occurred in at least two patients. Many of the identified genes encode proteins with a role in actin and microtubule reorganization and vesicle-mediated transport. “Cellular assembly and organization” was the top cellular function category based on Ingenuity Pathways Analysis (p<3.10E-05).
WES performed in SJIA/MAS patients identified rare protein altering variants in the known HLH associated genes as well as new candidate genes.
Genome wide association studies have identified variants in PXK that confer risk for humoral autoimmune diseases, including systemic lupus erythematosus (SLE or lupus), rheumatoid arthritis and more recently systemic sclerosis. While PXK is involved in trafficking of epidermal growth factor Receptor (EGFR) in COS-7 cells, mechanisms linking PXK to lupus pathophysiology have remained undefined. In an effort to uncover the mechanism at this locus that increases lupus-risk, we undertook a fine-mapping analysis in a large multi-ancestral study of lupus patients and controls. We define a large (257kb) common haplotype marking a single causal variant that confers lupus risk detected only in European ancestral populations and spans the promoter through the 3′ UTR of PXK. The strongest association was found at rs6445972 with P < 4.62 × 10−10, OR 0.81 (0.75–0.86). Using stepwise logistic regression analysis, we demonstrate that one signal drives the genetic association in the region. Bayesian analysis confirms our results, identifying a 95% credible set consisting of 172 variants spanning 202 kb. Functionally, we found that PXK operates on the B-cell antigen receptor (BCR); we confirmed that PXK influenced the rate of BCR internalization. Furthermore, we demonstrate that individuals carrying the risk haplotype exhibited a decreased rate of BCR internalization, a process known to impact B cell survival and cell fate. Taken together, these data define a new candidate mechanism for the genetic association of variants around PXK with lupus risk and highlight the regulation of intracellular trafficking as a genetically regulated pathway mediating human autoimmunity.
lupus; PXK; fine-mapping; B cells; BCR
Sjögren’s syndrome is a common autoimmune disease (~0.7% of European Americans) typically presenting as keratoconjunctivitis sicca and xerostomia. In addition to strong association within the HLA region at 6p21 (Pmeta=7.65×10−114), we establish associations with IRF5-TNPO3 (Pmeta=2.73×10−19), STAT4 (Pmeta=6.80×10−15), IL12A (Pmeta =1.17×10−10), FAM167A-BLK (Pmeta=4.97×10−10), DDX6-CXCR5 (Pmeta=1.10×10−8), and TNIP1 (Pmeta=3.30×10−8). Suggestive associations with Pmeta<5×10−5 were observed with 29 regions including TNFAIP3, PTTG1, PRDM1, DGKQ, FCGR2A, IRAK1BP1, ITSN2, and PHIP amongst others. These results highlight the importance of genes involved in both innate and adaptive immunity in Sjögren’s syndrome.
Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by autoantibody production and altered type I interferon expression. Genetic surveys and genome-wide association studies have identified more than 30 SLE susceptibility genes. One of these genes, TNIP1, encodes the ABIN1 protein. ABIN1 functions in the immune system by restricting the NF-κB signaling. In order to better understand the genetic factors that influence association with SLE in genes that regulate the NF-κB pathway, we analyzed a dense set of genetic markers spanning TNIP1 and TAX1BP1, as well as the TNIP1 homolog, TNIP2, in case-control sets of diverse ethnic origins.
We fine-mapped TNIP1, TNIP2, and TAX1BP1 in a total of 8372 SLE cases and 7492 healthy controls from European-ancestry, African-American, Hispanic, East Asian, and African-American Gullah populations. Levels of TNIP1 mRNA and ABIN1 protein were analyzed using quantitative RT-PCR and Western blotting, respectively, in EBV-transformed human B cell lines.
We found significant associations between genetic variants within TNIP1 and SLE but not in TNIP2 or TAX1BP1. After resequencing and imputation, we identified two independent risk haplotypes within TNIP1 in individuals of European-ancestry that were also present in African-American and Hispanic populations. These risk haplotypes produced lower levels of TNIP1 mRNA and ABIN1 protein suggesting they harbor hypomorphic functional variants that influence susceptibility to SLE by restricting ABIN1 expression.
Our results confirmed the association signals between SLE and TNIP1 variants in multiple populations and provide new insight into the mechanism by which TNIP1 variants may contribute to SLE pathogenesis.
Amerindian-Europeans, Asians and African-Americans have an excess morbidity from SLE and higher prevalence of lupus nephritis than Caucasians. The aim of this study was to analyze the relationship between genetic ancestry and socio-demographic characteristics and clinical features in a large cohort of Amerindian-European SLE patients.
A total of 2116 SLE patients of Amerindian-European origin and 4001 SLE patients of European descent with clinical data were used in the study. Genotyping of 253 continental ancestry informative markers was performed on the Illumina platform. The STRUCTURE and ADMIXTURE software were used to determine genetic ancestry of each individual. Correlation between ancestry and socio-demographic and clinical data were analyzed using logistic regression.
The average Amerindian genetic ancestry of 2116 SLE patients was 40.7%. There was an increased risk of having renal involvement (P<0.0001, OR= 3.50 95%CI 2.63-4.63) and an early age of onset with the presence of Amerindian genetic ancestry (P<0.0001). Amerindian ancestry protected against photosensitivity (P<0.0001, OR= 0.58 95%CI 0.44-0.76), oral ulcers (P<0.0001, OR= 0.55 95%CI 0.42-0.72), and serositis (P<0.0001, OR= 0.56 95%CI 0.41-0.75) after adjustment by age, gender and age of onset. However, gender and age of onset had stronger effects on malar rash, discoid rash, arthritis and neurological involvement than genetic ancestry.
In general, genetic Amerindian ancestry correlates with lower socio-demographic status and increases the risk for developing renal involvement and SLE at an earlier age of onset.
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.
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.
Protein tyrosine phosphatase non-receptor type 22 (PTPN22) is a negative regulator of T-cell activation associated with several autoimmune diseases, including systemic lupus erythematosus (SLE). Missense rs2476601 is associated with SLE in individuals with European ancestry. Since the rs2476601 risk allele frequency differs dramatically across ethnicities, we assessed robustness of PTPN22 association with SLE and its clinical sub-phenotypes across four ethnically diverse populations. Ten SNPs were genotyped in 8220 SLE cases and 7369 controls from in European-Americans (EA), African-Americans (AA), Asians (AS), and Hispanics (HS). We performed imputation-based association followed by conditional analysis to identify independent associations. Significantly associated SNPs were tested for association with SLE clinical sub-phenotypes, including autoantibody profiles. Multiple testing was accounted for by using false discovery rate. We successfully imputed and tested allelic association for 107 SNPs within the PTPN22 region and detected evidence of ethnic-specific associations from EA and HS. In EA, the strongest association was at rs2476601 (P = 4.7×10−9, OR = 1.40 (95% CI = 1.25–1.56)). Independent association with rs1217414 was also observed in EA, and both SNPs are correlated with increased European ancestry. For HS imputed intronic SNP, rs3765598, predicted to be a cis-eQTL, was associated (P = 0.007, OR = 0.79 and 95% CI = 0.67–0.94). No significant associations were observed in AA or AS. Case-only analysis using lupus-related clinical criteria revealed differences between EA SLE patients positive for moderate to high titers of IgG anti-cardiolipin (aCL IgG >20) versus negative aCL IgG at rs2476601 (P = 0.012, OR = 1.65). Association was reinforced when these cases were compared to controls (P = 2.7×10−5, OR = 2.11). Our results validate that rs2476601 is the most significantly associated SNP in individuals with European ancestry. Additionally, rs1217414 and rs3765598 may be associated with SLE. Further studies are required to confirm the involvement of rs2476601 with aCL IgG.
Little is known about the genetic etiology of systemic lupus erythematosus (SLE) in individuals of African ancestry, despite its higher prevalence and greater disease severity. Overproduction of nitric oxide (NO) and reactive oxygen species are implicated in the pathogenesis and severity of SLE, making NO synthases and other reactive intermediate related genes biological candidates for disease susceptibility. This study analyzed variation in reactive intermediate genes for association with SLE in two populations with African ancestry.
A total of 244 SNPs from 53 regions were analyzed in non-Gullah African Americans (AA; 1432 cases and 1687 controls) and the genetically more homogeneous Gullah of the Sea Islands of South Carolina (133 cases and 112 controls) and. Single-marker, haplotype, and two-locus interaction tests were computed for these populations.
The glutathione reductase gene GSR (rs2253409, P=0.0014, OR [95% CI]=1.26 [1.09–1.44]) was the most significant single-SNP association in AA. In the Gullah, the NADH dehydrogenase NDUFS4 (rs381575, P=0.0065, OR [95%CI]=2.10 [1.23–3.59]) and nitric oxide synthase gene NOS1 (rs561712, P=0.0072, OR [95%CI]=0.62 [0.44–0.88]) were most strongly associated with SLE. When both populations were analyzed together, GSR remained the most significant effect (rs2253409, P=0.00072, OR [95%CI]=1.26 [1.10–1.44]). Haplotype and two-locus interaction analyses also uncovered different loci in each population.
These results suggest distinct patterns of association with SLE in African-derived populations; specific loci may be more strongly associated within select population groups.
systemic lupus erythematosus; African Americans; genetic association studies; oxygen compounds; single nucleotide polymorphism
We previously established an 80 kb haplotype upstream of TNFSF4 as a susceptibility locus in the autoimmune disease SLE. SLE-associated alleles at this locus are associated with inflammatory disorders, including atherosclerosis and ischaemic stroke. In Europeans, the TNFSF4 causal variants have remained elusive due to strong linkage disequilibrium exhibited by alleles spanning the region. Using a trans-ancestral approach to fine-map the locus, utilising 17,900 SLE and control subjects including Amerindian/Hispanics (1348 cases, 717 controls), African-Americans (AA) (1529, 2048) and better powered cohorts of Europeans and East Asians, we find strong association of risk alleles in all ethnicities; the AA association replicates in African-American Gullah (152,122). The best evidence of association comes from two adjacent markers: rs2205960-T (P = 1.71×10−34, OR = 1.43[1.26–1.60]) and rs1234317-T (P = 1.16×10−28, OR = 1.38[1.24–1.54]). Inference of fine-scale recombination rates for all populations tested finds the 80 kb risk and non-risk haplotypes in all except African-Americans. In this population the decay of recombination equates to an 11 kb risk haplotype, anchored in the 5′ region proximal to TNFSF4 and tagged by rs2205960-T after 1000 Genomes phase 1 (v3) imputation. Conditional regression analyses delineate the 5′ risk signal to rs2205960-T and the independent non-risk signal to rs1234314-C. Our case-only and SLE-control cohorts demonstrate robust association of rs2205960-T with autoantibody production. The rs2205960-T is predicted to form part of a decameric motif which binds NF-κBp65 with increased affinity compared to rs2205960-G. ChIP-seq data also indicate NF-κB interaction with the DNA sequence at this position in LCL cells. Our research suggests association of rs2205960-T with SLE across multiple groups and an independent non-risk signal at rs1234314-C. rs2205960-T is associated with autoantibody production and lymphopenia. Our data confirm a global signal at TNFSF4 and a role for the expressed product at multiple stages of lymphocyte dysregulation during SLE pathogenesis. We confirm the validity of trans-ancestral mapping in a complex trait.
Systemic lupus erythematosus (SLE/lupus) is a complex disease in which the body's immune cells cause inflammation in one or more systems to cause the associated morbidity. Hormones, the environment and genes are all causal contributors to SLE and over the past several years the genetic component of SLE has been firmly established. Several genes which are regulators of the immune system are associated with disease risk. We have established one of these, the tumour-necrosis family superfamily member 4 (TNFSF4) gene, as a lupus susceptibility gene in Northern Europeans. A major obstacle in pinpointing the marker(s) at TNFSF4 which best explain the risk of SLE has been the strong correlation (linkage disequilibrium, LD) between adjacent markers across the TNFSF4 region in this population. To address this, we have typed polymorphisms in several populations in addition to the European groups. The mixed ancestry of these populations gives a different LD pattern than that found in Europeans, presenting a method of pinpointing the section of the TNFSF4 region which results in SLE susceptibility. The Non-European populations have allowed identification of a polymorphism likely to regulate expression of TNFSF4 to increase susceptibility to SLE.
Systemic lupus erythematosus (SLE) is a common systemic autoimmune disease with complex etiology but strong clustering in families (λS = ~30). We performed a genome-wide association scan using 317,501 SNPs in 720 women of European ancestry with SLE and in 2,337 controls, and we genotyped consistently associated SNPs in two additional independent sample sets totaling 1,846 affected women and 1,825 controls. Aside from the expected strong association between SLE and the HLA region on chromosome 6p21 and the previously confirmed non-HLA locus IRF5 on chromosome 7q32, we found evidence of association with replication (1.1 × 10−7 < Poverall < 1.6 × 10−23; odds ratio 0.82–1.62)in four regions: 16p11.2 (ITGAM), 11p15.5 (KIAA1542), 3p14.3 (PXK) and 1q25.1 (rs10798269). We also found evidence for association (P < 1 × 10−5) at FCGR2A, PTPN22 and STAT4, regions previously associated with SLE and other autoimmune diseases, as well as at ≥9 other loci (P < 2 × 10−7). Our results show that numerous genes, some with known immune-related functions, predispose to SLE.
Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease with significant immune system aberrations resulting from complex heritable genetics as well as environmental factors. TRAF6 is a candidate gene for SLE, which has a major role in several signaling pathways that are important for immunity and organ development.
Fifteen single-nucleotide polymorphisms (SNPs), across TRAF6 were evaluated in 7,490 SLE and 6,780 control subjects from different ancestries. Population-based case-control association analyses and meta-analyses were performed. P values, false discovery rate q values, and odds ratios with 95% confidence intervals were calculated.
Evidence of associations in multiple SNPs was detected. The best overall p values were obtained for SNPs rs5030437 and rs4755453 (p=7.85×10−5 and p=4.73×10−5, respectively) without significant heterogeneity among populations (p=0.67 and p=0.50 in Q-statistic). In addition, rs540386 previously reported to be associated with RA was found to be in LD with these two SNPs (r2= 0.95) and demonstrated evidence of association with SLE in the same direction (meta-analysis p=9.15×10−4, OR=0.89, 95%CI=0.83–0.95). Thrombocytopenia improved the overall results in different populations (meta-analysis p=1.99×10−6, OR=0.57, 95%CI=0.45–0.72, for rs5030470). Finally evidence of family based association in 34 African-American pedigrees with the presence of thrombocytopenia were detected in one available SNP rs5030437 with Z score magnitude of 2.28 (p=0.02) under a dominant model.
Our data indicate the presence of association of TRAF6 with SLE in agreement with the previous report of association with RA. These data provide further support for the involvement of TRAF6 in the pathogenesis of autoimmunity.
TRAF6; polymorphism; systemic lupus erythematosus
To investigate and refine the relationships among systemic lupus erythematosus (SLE) and related autoantibodies, interferon-α (IFN-α), and various ancestral backgrounds.
We investigated quantitatively defined genetic ancestry through principal component analysis in place of self-reported ancestry.
African ancestry was found to be associated with presence of anti-RNP antibody (p = 0.0026), and anti-RNP was correlated with high levels of IFN-α (p = 2.8 × 10−5).
Our data support a model in which African ancestry increases the likelihood of SLE-associated autoantibody formation, which subsequently results in higher levels of serum IFN-α.
SYSTEMIC LUPUS ERYTHEMATOSUS; AUTOANTIBODIES; INTERFERONS; GENETICS
About 90% of patients with systemic lupus erythematosus (SLE) are female. We hypothesize that the number of X chromosomes, not sex, is a determinate of risk of SLE. Number of X chromosomes was determined by single nucleotide typing and then confirmed by karyotype or fluorescent in situ hybridization in a large group of men with SLE. Presence of an sry gene was assessed by rtPCR. We calculated 96% confidence intervals using the Adjusted Wald method, and used Bayes’ theorem to estimate the prevalence of SLE among 47,XXY and 46,XX men. Among 316 men with SLE, 7 had 47,XXY and 1 had 46,XX. The rate of Klinefelter’s syndrome (47,XXY) was statistically different from that found in control men and from the known prevalence in the population. The 46,XX man had an sry gene, which encodes the testes determining factor, on an X chromosome as a result of an abnormal crossover during meiosis. In the case of 46,XX, 1 of 316 was statistically different from the known population prevalence of 1 in 20,000 live male births. A previously reported 46,XX man with SLE had a different molecular mechanism in which there were no common gene copy number abnormalities with our patient. Thus, men with SLE are enriched for conditions with additional X chromosomes. Especially since 46,XX men are generally normal males, except for infertility, these data suggest the number of X chromosomes, not phenotypic sex, is responsible for the sex bias of SLE.
Systemic lupus erythematosus; Klinefelter’s syndrome; male 46; XX; female bias; X chromosome
Systemic lupus erythematosus (SLE) is a sexually dimorphic autoimmune disease which is more common in women, but affected men often experience a more severe disease. The genetic basis of sexual dimorphism in SLE is not clearly defined. A study was undertaken to examine sex-specific genetic effects among SLE susceptibility loci.
A total of 18 autosomal genetic susceptibility loci for SLE were genotyped in a large set of patients with SLE and controls of European descent, consisting of 5932 female and 1495 male samples. Sex-specific genetic association analyses were performed. The sex–gene interaction was further validated using parametric and nonparametric methods. Aggregate differences in sex-specific genetic risk were examined by calculating a cumulative genetic risk score for SLE in each individual and comparing the average genetic risk between male and female patients.
A significantly higher cumulative genetic risk for SLE was observed in men than in women. (P = 4.52×10−8) A significant sex–gene interaction was seen primarily in the human leucocyte antigen (HLA) region but also in IRF5, whereby men with SLE possess a significantly higher frequency of risk alleles than women. The genetic effect observed in KIAA1542 is specific to women with SLE and does not seem to have a role in men.
The data indicate that men require a higher cumulative genetic load than women to develop SLE. These observations suggest that sex bias in autoimmunity could be influenced by autosomal genetic susceptibility loci.
Cardiac manifestations of neonatal lupus, comprising atrioventricular conduction defects and cardiomyopathy, occur in fetuses exposed to anti-Ro/SSA antibodies, and carry substantial mortality. There is strong evidence of a genetic contribution to the risk. This study was undertaken to evaluate single-nucleotide polymorphisms (SNPs) for associations with cardiac neonatal lupus.
Children of European ancestry with cardiac neonatal lupus (n = 116) were genotyped using the Illumina 370K SNP platform and merged with 3,351 controls. Odds ratios (ORs) and 95% confidence intervals (95% CIs) for association with cardiac neonatal lupus were determined.
The 17 most significant associations with cardiac neonatal lupus were found in the HLA region. The region near the MICB gene showed the strongest variant (rs3099844; Pdom = 4.52 × 10−10, OR 3.34 [95% CI 2.29–4.89]), followed by a missense variant within C6orf10 (rs7775397; Pdom = 1.35 × 10−9, OR 3.30), which lies between NOTCH4 and BTNL2, and several SNPs near the tumor necrosis factor α gene, including rs2857595 (Padd = 1.96 × 10−9, OR 2.37), rs2230365 (Padd = 1.00 × 10−3, OR 0.46), and rs3128982 (Padd = 6.40 × 10−6, OR 1.86). Outside the HLA region, an association was detected at 21q22, upstream of the transcription regulator ets-related isoform 1 (rs743446; P = 5.45 × 10−6, OR 2.40). HLA notwithstanding, no individual locus previously implicated in autoimmune diseases achieved genome-wide significance.
These results suggest that variation near genes related to inflammatory and apoptotic responses may promote cardiac injury initiated by passively acquired autoantibodies.
High serum interferon α (IFNα) activity is a heritable risk factor for systemic lupus erythematosus (SLE). Auto-antibodies found in SLE form immune complexes which can stimulate IFNα production by activating endosomal Toll-like receptors and interferon regulatory factors (IRFs), including IRF5. Genetic variation in IRF5 is associated with SLE susceptibility; however, it is unclear how IRF5 functional genetic elements contribute to human disease.
1034 patients with SLE and 989 controls of European ancestry, 555 patients with SLE and 679 controls of African–American ancestry, and 73 patients with SLE of South African ancestry were genotyped at IRF5 polymorphisms, which define major haplotypes. Serum IFNα activity was measured using a functional assay.
In European ancestry subjects, anti-double-stranded DNA (dsDNA) and anti-Ro antibodies were each associated with different haplotypes characterised by a different combination of functional genetic elements (OR > 2.56, p >003C; 1.9×10−14 for both). These IRF5 haplotype-auto-antibody associations strongly predicted higher serum IFNα in patients with SLE and explained > 70% of the genetic risk of SLE due to IRF5. In African–American patients with SLE a similar relationship between serology and IFNα was observed, although the previously described European ancestry-risk haplotype was present at admixture proportions in African–American subjects and absent in African patients with SLE.
The authors define a novel risk haplotype of IRF5 that is associated with anti-dsDNA antibodies and show that risk of SLE due to IRF5 genotype is largely dependent upon particular auto-antibodies. This suggests that auto-antibodies are directly pathogenic in human SLE, resulting in increased IFNα in cooperation with particular combinations of IRF5 functional genetic elements.
SLE is a systemic autoimmune disorder affecting multiple organ systems including the skin, musculoskeletal, renal and haematopoietic systems. Humoral autoimmunity is a hallmark of SLE, and patients frequently have circulating auto-antibodies directed against dsDNA, as well as RNA binding proteins (RBP). Anti-RBP autoantibodies include antibodies which recognize Ro, La, Smith (anti-Sm), and ribonucleoprotein (anti-nRNP), collectively referred to as anti-retinol-binding protein). Anti-retinol-binding protein and anti-dsDNA auto-antibodies are rare in the healthy population.1 These auto-antibodies can be present in sera for years preceding the onset of clinical SLE illness2 and are likely pathogenic in SLE.34
The Xq28 region containing IRAK1 and MECP2 has been identified as a risk locus for systemic lupus erythematosus (SLE) in previous genetic association studies. However, due to the strong linkage disequilibrium between IRAK1 and MECP2, it remains unclear which gene is affected by the underlying causal variant(s) conferring risk of SLE.
We fine-mapped ≥136 SNPs in a ~227kb region on Xq28, containing IRAK1, MECP2 and 7 adjacent genes (L1CAM, AVPR2, ARHGAP4, NAA10, RENBP, HCFC1 and TMEM187), for association with SLE in 15,783 case-control subjects derived from 4 different ancestral groups.
Multiple SNPs showed strong association with SLE in European Americans, Asians and Hispanics at P<5×10−8 with consistent association in subjects with African ancestry. Of these, 6 SNPs located in the TMEM187-IRAK1-MECP2 region captured the underlying causal variant(s) residing in a common risk haplotype shared by all 4 ancestral groups. Among them, rs1059702 best explained the Xq28 association signals in conditional testings and exhibited the strongest P value in trans-ancestral meta-analysis (Pmeta=1.3×10−27, OR=1.43), and thus was considered to be the most-likely causal variant. The risk allele of rs1059702 results in the amino acid substitution S196F in IRAK1 and had previously been shown to increase NF-κB activity in vitro. We also found that the homozygous risk genotype of rs1059702 was associated with lower mRNA levels of MECP2, but not IRAK1, in SLE patients (P=0.0012) and healthy controls (P=0.0064).
These data suggest contributions of both IRAK1 and MECP2 to SLE susceptibility.
Systemic Lupus Erythematosus; Gene Polymorphism; Xq28; IRAK1; MECP2
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.
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.
Systemic lupus erythematosus (SLE) is an inflammatory autoimmune disease with a strong genetic component. African-Americans (AA) are at increased risk of SLE, but the genetic basis of this risk is largely unknown. To identify causal variants in SLE loci in AA, we performed admixture mapping followed by fine mapping in AA and European-Americans (EA). Through genome-wide admixture mapping in AA, we identified a strong SLE susceptibility locus at 2q22–24 (LOD = 6.28), and the admixture signal is associated with the European ancestry (ancestry risk ratio ∼1.5). Large-scale genotypic analysis on 19,726 individuals of African and European ancestry revealed three independently associated variants in the IFIH1 gene: an intronic variant, rs13023380 [Pmeta = 5.20×10−14; odds ratio, 95% confidence interval = 0.82 (0.78–0.87)], and two missense variants, rs1990760 (Ala946Thr) [Pmeta = 3.08×10−7; 0.88 (0.84–0.93)] and rs10930046 (Arg460His) [Pdom = 1.16×10−8; 0.70 (0.62–0.79)]. Both missense variants produced dramatic phenotypic changes in apoptosis and inflammation-related gene expression. We experimentally validated function of the intronic SNP by DNA electrophoresis, protein identification, and in vitro protein binding assays. DNA carrying the intronic risk allele rs13023380 showed reduced binding efficiency to a cellular protein complex including nucleolin and lupus autoantigen Ku70/80, and showed reduced transcriptional activity in vivo. Thus, in SLE patients, genetic susceptibility could create a biochemical imbalance that dysregulates nucleolin, Ku70/80, or other nucleic acid regulatory proteins. This could promote antibody hypermutation and auto-antibody generation, further destabilizing the cellular network. Together with molecular modeling, our results establish a distinct role for IFIH1 in apoptosis, inflammation, and autoantibody production, and explain the molecular basis of these three risk alleles for SLE pathogenesis.
African-Americans (AA) are at increased risk of systemic lupus erythematosus (SLE), but the genetic basis of this risk increase is largely unknown. We used admixture mapping to localize disease-causing genetic variants that differ in frequency across populations. This approach is advantageous for localizing susceptibility genes in recently admixed populations like AA. Our genome-wide admixture scan identified seven admixture signals, and we followed the best signal at 2q22–24 with fine-mapping, imputation-based association analysis and experimental validation. We identified two independent coding variants and a non-coding variant within the IFIH1 gene associated with SLE. Together with molecular modeling, our results establish a distinct role for IFIH1 in apoptosis, inflammation, and autoantibody production, and explain the molecular basis of these three risk alleles for SLE pathogenesis.
Several confirmed genetic susceptibility loci for lupus have been described. To date, no clear evidence for genetic epistasis is established in lupus. We test for gene-gene interactions in a number of known lupus susceptibility loci.
Eighteen SNPs tagging independent and confirmed lupus susceptibility loci were genotyped in a set of 4,248 lupus patients and 3,818 normal healthy controls of European descent. Epistasis was tested using a 2-step approach utilizing both parametric and non-parametric methods. The false discovery rate (FDR) method was used to correct for multiple testing.
We detected and confirmed gene-gene interactions between the HLA region and CTLA4, IRF5, and ITGAM, and between PDCD1 and IL21 in lupus patients. The most significant interaction detected by parametric analysis was between rs3131379 in the HLA region and rs231775 in CTLA4 (Interaction odds ratio=1.19, z-score= 3.95, P= 7.8×10−5 (FDR≤0.05), PMDR= 5.9×10−45). Importantly, our data suggest that in lupus patients the presence of the HLA lupus-risk alleles in rs1270942 and rs3131379 increases the odds of also carrying the lupus-risk allele in IRF5 (rs2070197) by 17% and 16%, respectively (P= 0.0028 and 0.0047).
We provide evidence for gene-gene epistasis in systemic lupus erythematosus. These findings support a role for genetic interaction contributing to the complexity of lupus heritability.