Article Association of Systemic Lupus Erythematosus Susceptibility Genes

Article

Association of Systemic Lupus Erythematosus Susceptibility Genes with IgA Nephropathy in a Chinese Cohort

Xu-Jie Zhou, Fa-Juan Cheng, Li Zhu, Ji-Cheng Lv, Yuan-Yuan Qi, Ping Hou, and Hong Zhang

Abstract Background and objectives One hypothesis states that IgA nephropathy (IgAN) is a syndrome with an Renal Division, Peking University First autoimmune component. Recent studies strongly support the notion of shared genetics between immune-related Hospital; Peking diseases. This study investigated single-nucleotide polymorphisms (SNPs) reported to be associated with University Institute of systemic lupus erythematosus (SLE) in a Chinese cohort of patients with IgAN and in controls. Nephrology; Key Laboratory of Renal Disease, Ministry of Design, setting, participants, & measurements This study investigated whether SNP markers that had been Health of China; and reported to be associated with SLE were also associated with IgAN in a Chinese population. The study cohort Key Laboratory of consisted of 1194 patients with IgAN and 902 controls enrolled in Peking University First Hospital from 1997 to Chronic Kidney 2008. Disease Prevention and Treatment (Peking University), Ministry , 3 25 Results Ninety-six SNPs mapping to 60 SLE loci with reported P values 1 10 were investigated. CFH of Education, Beijing, 2 2 2 2 2 (P=8.41310 6), HLA-DRA (P=4.91310 6), HLA-DRB1 (P=9.46310 9), PXK (P=3.62310 4), BLK (P=9.32310 3), People’s Republic of and UBE2L3 (P=4.0731023) were identiﬁed as shared genes between IgAN and SLE. All associations reported China herein were corroborated by associations at neighboring SNPs. Many of the alleles that are risk alleles for SLE are protective alleles for IgAN. By analyses of two open independent expression quantitative trait loci (eQTL) Correspondence: databases, correlations between genotypes and corresponding gene expression were observed (P,0.05 in mul- Dr. Hong Zhang, Renal Division, Peking tiple populations), suggesting a cis-eQTL effect. From gene-expression databases, differential expressions of these University First genes were observed in IgAN. Additive interactions between PXK rs6445961and HLA-DRA rs9501626 Hospital, Peking (P=1.5131022), as well as multiplicative interactions between CFH rs6677604 and HLA-DRB1 rs9271366 University Institute of (P=1.7731022), and between HLA-DRA rs9501626 and HLA-DRB1 rs9271366 (P=3.2331022) were observed. Nephrology, No. 8 Xi Shi Ku Street, Xi Disease risk decreased with accumulation of protective alleles. Network analyses highlighted four pathways: Cheng District, Beijing MHC class II antigen presentation, complement regulation, signaling by the B-cell receptor, and ubiquitin/ 100034, People’s proteasome-dependent degradation. Republic of China. Email: hongzh@bjmu. Conclusion From this “systems genetics” perspective, these data provide important clues for future studies on edu.cn pleiotropy in IgAN and lupus nephritis. Clin J Am Soc Nephrol 9: 788–797, 2014. doi: 10.2215/CJN.01860213

Introduction identified loci collectively explain ,10% of the genetic Over the past two decades, considerable progress has risk for IgAN, highlighting the fact that much of the been made in unraveling the complex pathogenesis of heritable basis for IgAN has yet to be identified. IgA nephropathy (IgAN). However, the exact patho- Previously, we reported that genetic factors have an genesis remains poorly determined. Current data sug- appreciable influence on the production of under- gest that genetic factors combined with environmental galactosylated IgA1 and that GWAS data strongly factors lead to increased synthesis of aberrantly implicate new clues as to the pathogenesis of IgAN galactosylated IgA1, formation of glycan-specificanti- (7,8,11–15). We also reported on the overlap between bodies to IgG and IgA, and mesangio-podocytic-tubular several autoimmune diseases: systemic lupus erythema- cross-talk in the occurrence and development of the tosus (SLE), rheumatoid arthritis, ANCA-associated disease (1–6). Whether IgAN should be termed an “au- small vasculitis, and anti–glomerular basement toimmune disease” is controversial. However, recent membrane disease (16–25). Thus, we hypothesized genome-wide association studies (GWAS) strongly in- that refinement of GWAS data or identification of dicate that many of its associated loci also affect other IgAN susceptibility genes could be underpinned by autoimmune and infectious diseases (5,7–9), further investigation of the genetic variants reported to be supporting the notion of shared genetics of immune- associated with other immune-related diseases. Iden- related diseases (10). Recent estimates suggest that the tification of novel IgAN genes and shared genetic

pathways could improve understanding of common ge- Statistical Analyses netic mechanisms and eventually the development of im- Only SNPs meeting the quality-control criteria of ,1% proved methods of diagnosis, prognosis, and targeted overall missing data as well as consistency with Hardy– therapies. Weinberg equilibrium genotype frequency expectations SLE is an autoimmune disease. Lupus nephritis is charac- (P,0.05) were included. As reported previously, after ad- terized by multiple immune complexes depositing in the justment for population substructure, the inflation factor kidney, including IgA molecules. IgAN is an immune using all SNPs was l=1.02, indicating a minimal effect of complex–mediated GN defined by the predominant IgA residual population structure. Thus, no further genomic molecule that deposits in the kidney. A recent study showed control corrections were applied. the pathogenicity of anti-glycan antibodies in IgAN, which Genotype frequencies between IgAN cases and controls suggested that IgAN is a type of autoimmune disease (26). A were compared using the chi-squared trend test implemented new theory suggests that most types of GN are primarily in PLINK software to determine whether individual SLE autoimmune diseases. Certain pathogenic similarities be- susceptibility loci were also associated with IgAN. Genetic tweenautoimmunediseases(e.g., greater prevalence among models were defined relative to the minor allele. To reduce the Asians than Europeans, chronic course, renal involvement, risk of false-positive findings, all positive associations were circulating immune complexes, complement activation, mor- checked further by associations at neighboring SNPs. phologic similarities, certain pathways being involved in To test for additive interactions, the methods were taken ESRD) prompted us to investigate the overlap in genetic using a 232 factorial design to calculate the attributable susceptibility between SLE and IgAN. Well established co- proportion due to interaction, the relative excess risk due occurrences of SLE with IgAN suggest common etiologic to interaction, and the synergy index (20,43). P values factors (27–29). Little progress has been made regarding ,0.05 for attributable portion due to interaction were con- the identification of genetic factors specific to lupus nephri- sidered to be indicators of additive interactions. Ninety- tis, but a genetic cause in SLE has been substantiated. More five percent confidence intervals (95% CIs) were calculated than 40 genes have been robustly associated with SLE. using the delta method (44). Multiplicative interaction was We investigated whether single-nucleotide polymorphism assessed by adding an interaction variable (SNP3SNP) to (SNP) markers that had been reported to be associated with the regression models. P,0.05 was considered to be evi- SLE were also associated with IgAN in a Chinese population. dence for multiplicative interactions. Analyses of carriage of SLE alleles in patients with IgAN were carried out to determine whether there was an overall Materials and Methods enrichment of SLE susceptibility variants in IgAN cases. The protocol of this study complied with the Declaration Analyses were also undertaken to determine whether com- of Helsinki. The protocol was approved by the Ethics Com- bining those risk alleles conferred a higher risk of disease. mittee of Peking University First Hospital (Beijing, China). Written informed consent was obtained from each patient. Analyses of Bioinformatics To explore whether the identified SNPs had expression Study Population quantitative trait loci (eQTLs) effects, Genevar software The samples used in the present study have been described was used to determine associations between sequence previously. Briefly, exclusion of duplicates and first-degree variation and gene expression (http://www.sanger.ac. relatives yielded 1194 IgAN cases and 902 healthy controls uk/resources/software/genevar). The sequence variation recruited in the Renal Division of Peking University First and gene-expression profiling data were from lymphoblas- Hospital from 1997 to 2008 (8). All the cases were con- toid cell lines of 726 HapMap3 individuals. Another global firmed by renal biopsy, and all the controls were healthy map of the effects of polymorphism on gene expression in blood donors without indicators of renal disease. Quality 400 children from families recruited through a proband control was undertaken as described (8). Unexpected re- with asthma was also investigated to associate gene ex- latedness was excluded with a PLINK pi-hat cutoff of pression on the basis of imputed genotypes (45). 0.125. We included men and women of Northern Chinese The differential expressions of suspected IgAN candidate Han ancestry. genes were compared with those of healthy controls using publically available data from the ArrayExpress Archive Selection and Genotyping of SNPs database (http://www.ebi.ac.uk/arrayexpress/) using “IgA We systemically examined data from GWAS, as well as nephropathy” as the search term. Three experiments (E- large-scale replications conducted in SLE genetics through GEOD-37460, E-GEOD-35489, and E-GEOD-14795) involv- December 1, 2012. The reported SNPs associated with SLE ing comparatively large samples were included in the current in the GWAS context with a P value ,131025 were analysis. The former two experiments took kidney biopsy selected for analysis (30–42). The reported risk variants samples and the latter experiment took whole-blood samples for SLE using data from the Catalog of Published for gene-expression analyses. The normalized data available Genome-Wide Association Studies from the National on the public databases were tested as reported previously. Human Genome Research Institute (http://www.genome. To integrate data in biologic networks, Cytoscape soft- gov/gwastudies) were also checked. Finally, a panel of 96 ware (which allows visualization of data in the context of SNPs representative of 60 genes or loci was selected networks) was applied (46). Cytoscape is widely used (Supplemental Table 1). Genotyping was undertaken using open-source software for the analyses of bimolecular in- the Illumina Human 610-Quad BeadChip, which involved teraction networks. MiMI integrates data on 119,880 mol- 498,322 SNPs with a mean call rate of 0.9992. ecules, 330,153 interactions, and 579 complexes from 790 Clinical Journal of the American Society of Nephrology

multiple, well known protein-interaction databases. An MiMI plugin, version 3.1.1, installed within Cytoscape 2.8.3, was used to determine the genetic interactions in po- sitional/functional networks. Direct query of genes and 1.19 (41) 1.18 (35) 1.20 (31) 1.86 (32) 1.26 (36) 1.42 (32) SLE Risk Allele OR their nearest neighbors from all data resources was done, (Reference) and no further modiﬁcations were made. 5 3 3 5 10 2 dence interval; SLE, 2 2 2 2 2 2 ﬁ 10 10 10 10 10 10 3 3 3 3 3 3

Results 0.06 1.28 (34) Values

Analyses of SLE Risk Alleles in IgAN Show Suggestive IgAN P Genotype Protective Alleles 5.47 2.96

Among the selected 96 SNPs, 10 SNPs of the lupus risk 2 2 2 alleles in the region of HLA, CFH (suggested to be a tag- 2 2 2 10 D 10 10 3 ging SNP for CFHR1, 3 ), PXK, BLK, UBE2L3,andLYST 3 3 0.180.070.12 3.15 0.81 9.00 0.05 2.60 3.40 0.070.15 0.04 0.08 1.45 (34) 0.06 1.35 (36) 1.20 (36) showed evidence for association at an allele-type level Values P Recessive (P,0.05) (Table 1). Of note, the associations between al- 1.80 4.88 leles in the HLA region, CFH, and IgAN were the top signals in our previous reports on GWAS. Interestingly, in 5b 3 3 6b 10b 2 2 2 2 2 2 2 2 2 2 2 2 2

comparing odds ratio (OR) values for these alleles in SLE 10 10 10 10 10 10 10 10 10 3 3 3 3 3 3 3 3 3 and IgAN, all the directions of association were opposite Values P thoseobservedintheSLEstudies,exceptforUBE2L3. Dominant Control allele frequencies were similar to those reported in SLE GWAS data. Thus, these findings suggested that a the SLE risk alleles may be protective for susceptibility to IgAN. However, only SNPs in CFH and HLA regions could retain statistically significant evidence for association (Table 1). Although nonsignificant after applying a Bonferroni correction, PXK, BLK, UBE2L3,andLYST re-

mained interesting candidates for further investigation. 0.55 (0.42 to 0.72)0.81 (0.67 to 0.98)0.79 (0.68 to 3.37 0.92)0.66 (0.55 to 6.31 0.79)0.63 (0.53 to 3.78 0.75)0.83 (0.72 to 4.68 0.96)0.83 (0.72 to 4.37 0.95)0.86 (0.75 to 3.28 0.99)1.15 (1.02 to 2.35 1.30)1.16 (1.02 to 7.78 1.31) 2.36 0.17 2.38 Allele OR (95% CI) by SLE Risk Allele

Analyses of Neighboring SNPs Support Disease Effects 6 2 3 6 8 2 3 2 2 2 2 2 2 2 2 2 2 2 2 2 To reduce the chance of false-positive ﬁndings using a 10 10 10 10 10 10 10 10 10 10 3 3 3 3 3 3 3 3 3 single marker by chance, all the positive associations were 3 Values

checked further by analyzing the neighboring SNPs. Multiple P significant association signals were observed (Figure 1). In Trend Test CFH, HLA-DRA, and BLK, the top signals were from the SNPs selected. In HLA-DRA, rs2027856 (protective allele T; 2 P=4.91310 6; OR, 0.66; 95% CI, 0.55 to 0.79) also showed the same significance compared with rs9501626 in associa- 2 4.10/7.26 8.41 Control (%) 11.39/16.2612.60/18.65 4.91 6.96 47.32/43.74 2.11 MAF Case/ tion with IgAN (r value between rs2027856 and rs9501626 cance after multiple correction. is 1.00). In HLA-DRB1, rs9270984 (protective allele G; OR, fi

0.63; 95% CI, 0.53 to 0.74) and rs9271055 (protective allele T; T A G A /T 12.87/10.71 3.31 /T 26.59/23.23 1.23 /A 26.63/23.12 9.32 /C 29.94/26.94 3.33 /G 46.48/49.94 2.63 OR, 0.63; 95% CI, 0.53 to 0.74) showed the same most sig- /G 23.79/19.79 2.01 C C T T T G Allele 29 2 Minor nificance with P=9.46310 (r =1.00 between rs9270984 and Major/ rs9271055; r2=0.84 between rs9270984 and rs9271366). HLA- DRB1 rs9270984 and rs9271055 showed no better fit 2 (P=3.1310 9 for both) in association at the genotype level 2 than that of rs9271366 (P=3.40310 10) as well as their in- rs6677604 G/ rs131654 rs2254546 rs9782955 rs6445975 rs9501626rs9271366 C/ rs7812879 A/ rs2736340 complete information in eQTL analyses; rs9271366 was still rs5754217 G/ selected as tag SNPs in further analyses. In PXK, rs6445961 (r2=0.92 between rs6445961 and rs6445975) showed the most 24 significant association, with P=3.62310 (protective allele regions could retain statistical signi A; OR, 0.77; 95% CI, 0.66 to 0.89). In UBE2L3, rs2298428 2 HLA CFH UBE2L3 BLK LYST PXK HLA-DRA HLA-DRB1 BLK BLK (r =0.95 between rs2298428 and rs5754217) showed the UBE2L3 23 most significant association, with P=4.07310 (risk allele and T; OR, 1.21; 95% CI, 1.06 to 1.37). These findings suggested that associations with PXK, BLK,andUBE2L3 may be true CFH associations because all the associations reported herein were corroborated by associations at neighboring SNPs. Nevertheless, the significances were too weak to meet the 1 194953541 8 11381089 13 234106500 66 58345217 8 32508322 32694832 8 11377591 11381382 Only SNPs in ORs were calculated on the basis of SLE risk alleles for comparison. Reported ORs were derived from the references listed. 22 20247190 threshold in multiple testing. However, the associations be- 22 20269675 systemic lupus erythematosus. Table 1. Association results for systemic lupus erythematosus risk variantsChr in IgA nephropathy Base Pair Locus SNP The reported SLE risk alleles are set in boldface.a Chr, chromosome; SNP, single-nucleotide polymorphism;b MAF, minor allele frequency; OR, odds ratio; 95% CI, 95% con tween SNPs within LYST and IgAN were not convincing. Clin J Am Soc Nephrol 9: 788–797, April, 2014 Shared Genetic Study in IgAN and SLE, Zhou et al. 791

Figure 1. | Regional plots of identified loci in Chinese patients with IgA nephropathy. Genotyped SNPs are plotted with their P values (as– log10[P values]) as a function of genomic position (Human Genome Build 18) within a region surrounding the reported systemic lupus erythematosus risk alleles. SNP, single-nucleotide polymorphism. eQTL Analyses Provides Functional Clues not available), all of the genes were differentially ex- We investigated whether the most associated SNPs were pressed from IgAN than those of controls, with elevated expression SNPs because they affected the abundance of a expressions of CFH, HLA-DRA,andHLA-DRB1 in renal protein or gene product by altering transcription. In lym- biopsy specimens as well as BLK and UBE2L3 in whole- phocyte cell lines from HapMap individuals, rs2298428 and blood samples (Table 3). Only HLA genes were signifi- rs9271366 were correlated consistently with UBE2L3 expres- cantly differentially expressed when subjected to multiple 2 sion (P=0.01–5.0310 5)andHLA-DRB1 expression testing, but only in renal biopsy specimens rather than in 2 2 (P=4.7310 13–3.9310 19), respectively, without population blood samples. restrictions (Table 2). This conclusion was confirmed by data from lymphoblastoid cell lines from 405 siblings in the Additive and Multiplicative Interaction Analyses Suggest fi United Kingdom (45). These ndings suggested that the as- Gene–Gene Interactions sociations between rs6677604 and CFH, rs2254546, and BLK Fifteen tests involving different combinations of six of the were more pronounced in Asian populations (although sim- most significantly associated SNPs (n3[n21])/2) within ilar trends between genotypes and gene expressions could their respective loci (PXK rs6445961, UBE2L3 rs2298428, be observed among different populations). For rs6445961 CFH rs6677604, HLA-DRB1 rs9271366, HLA-DRA fi and PXK, the correlation was marginally signi cant in white rs9271366, and BLK rs2254546) were conducted in the Chi- patients living in Utah and Han Chinese from Beijing, China. nese population. Supplemental Table 2 shows the results of However, different association patterns appeared to exist analyses for additive and multiplicative interactions be- between white and Chinese individuals. tween identified SNPs categorized by whether they had or did not have protective alleles. There was a modest additive Differential Gene-Expression Analyses Suggest Gene (but not multiplicative) gene–gene interaction between PXK Involvement in IgAN rs6445961 and HLA-DRA rs9501626, with the proportion of We ascertained whether the associated genes described risk due to an additive interaction of 2.86 (0.55–5.16), inter- 2 above were expressed differently in patients with IgAN action P=1.51310 2 for IgAN. Significant multiplicative and healthy controls. Except for PXK (for which data were interactions were observed between FH rs6677604 and 9 lnclJunlo h mrcnSceyo Nephrology of Society American the of Journal Clinical 792

Table 2. Correlation between genotypes of identiﬁed IgA nephropathy–associated single-nucleotide polymorphisms with gene expression in Epstein–Barr virus–transformed lymphoblastoid cell lines from an open database

HapMap 3 Unrelated Individuals (P Value) Children Siblings of SNP Gene CEU (n=165) CHB (n=137) JPT (n=113) YRI (n=203) British Descent (n=405) 2 rs6445961-A PXK 0.27 (4.10310 3)a 20.20 20.18 0.02 ND 0.07 0.10 0.83 2 2 2 rs2298428-C UBE2L3 20.28 (3.30310 3)a 20.28 (0.01)a 20.43 (5.00310 5)a – 20.390 (8.50310 5)a rs6677604-A CFH 0.12 (0.22) 0.02 (0.84) 0.26 (0.03)a 0.11 (0.26) – rs9501626-A HLA-DRA ––––– 2 2 2 2 rs9270984-G HLA-DRB1 0.59 (1.00310 11 )a 0.72 (1.30310 13 )a 0.68 (1.40310 12 )a 0.68 (4.90310 16 )a – 2 2 2 2 2 rs9271366-G HLA-DRB1 0.63 (4.70310 13 )a 0.74 (3.80310 15 )a 0.75 (3.10310 16 )a 0.73 (3.90310 19 )a 0.878 (4.00310 17 )a 2 2 rs2254546-G BLK 0.02 (0.82) 20.43 (8.20310 5)a 20.51 (1.10310 6)a 20.06 (0.57) ND

With the function of each increase of the risk allele, the table depicts the correlation between genotypes and gene expressions. Protective alleles were regarded as reference alleles in the correlation. Pearson correlation coefﬁcients are presented with P values in brackets. CEU, Caucasians living in Utah who were of northern and western European ancestries; CHB, Han Chinese from Beijing, China; JPT, Japanese in Tokyo, Japan; YRI, Yoruba in Ibadan, Nigeria; ND, no data could be derived from the database. aP,0.05.

Table 3. Differential candidate gene expressions in patients with IgA nephropathy compared with healthy controls from an open database

Samples

Renal Biopsies Candidate Gene Experiment E-GEOD-37460 Experiment E-GEOD-35489 Whole Blood: Experiment E-GEOD-14795

IgAN Controls IgAN Controls Controls P Value P Value IgAN (n=12) P Value (n=27) (n=27) (n=25) (n=6) (n=8) 2 CFH 9.4160.94 8.9560.64 4.09310 2a 5.7260.32 5.5160.14 0.14 96.90656.10 88.11661.04 0.74 2 2 HLA-DRA 11.5960.33 10.8960.54 6.56310 7a,b 9.4260.76 8.6260.27 2.56310 4a,b 8576.4362251.01 8638.2462355.87 0.95 2 2 HLA-DRB1 13.1060.26 12.5260.51 4.22310 6a,b 11.3160.65 10.4360.28 5.58310 5a,b 16661.5865086.23 15779.1063730.21 0.68 PXK –––––– – – – 2 BLK 4.9160.25 4.8260.17 0.14 4.4860.13 4.4460.13 0.53 372.316148.09 245.606104.07 3.75310 2a 2 2 UBE2L3 9.5860.18 9.6660.29 0.21 7.9460.13 7.7560.16 3.24310 3a 492.78694.12 362.576132.65 1.90310 2a

Data are the means6SD. IgAN, IgA nephropathy. aP,0.05. bP values remained signiﬁcant after multiple correction using Benjamini and Hochberg false-discovery rate methods. Clin J Am Soc Nephrol 9: 788–797, April, 2014 Shared Genetic Study in IgAN and SLE, Zhou et al. 793

2 HLA-DRB1 rs9271366 (P=1.77310 2), as well as for HLA- PXK, BLK and UBE2L3. Although some of the associations 2 DRA rs9501626 and HLA-DRB1 rs9271366 (P=3.23310 2). did not remain significant after the Bonferroni correction was applied, all the associations reported herein were cor- Analyses of Joint Effects Suggest Cumulative Effects on the roborated by associations at neighboring SNPs, suggesting Risk of Disease that they are true associations. To determine the cumulative effect of six SNPs, disease It is widely accepted that initial GWAS can detect just the risk was assessed according to the number of protective alleles greatest effects rather than all the susceptibility variants. they had. Individuals with more protective alleles seemed The ORs of all the novel variants were much weaker (0.8 or 2 to be less prone to IgAN (whole model P=5.96310 13) 1.2) than the ORs from variants within CFH and HLA (0.6), fi (Table 4). With each increase in the number of protective both of which were previously identi ed signals in GWAS. alleles, the disease risk decreased by approximately 7% The observation that the associated allele was the reverse of 2 (r2=–0.97; P=1.38310 3). The disease risk decreased up that reported previously for SLE was in accordance with a to seven-fold in individuals with eight or more protec- report stating that the protective alleles within MHC, 1q32 and tive alleles compared with those with fewer than 2. 22q12 regions for IgAN had been implicated as risk factors for other autoimmune disorders (8). Most of these associated loci showed the same tendency for disease susceptibility, so the Integrating Identifies Molecules in Cytoscape-Supported result is not likely to be a coincidence. The different association Network Involvement The six identified molecules showed physical interactions directions of the same alleles nevertheless supported the notion of pleiotropy (effect of a singlegeneonmultiplepheno- between genes or through their products/neighbors (Figure fl 2). The network was divided mainly into four modules, rep- types), quantitative genetics (combination of the in uences of resentative of pathways: MHC class II antigen presentation, multiple genes together with environmental variation result- complement regulation, signaling by the B-cell receptor ing in continuous distributions of phenotypes), and the human “diseasome” (the synthesis of all human genetic disorders (BCR), and ubiquitin/proteasome-dependent degradation. “ ” “ Several cellular interrelated genes have been suggested to [ disease phenome ]) and all human disease genes [ disease genome”]). Ideally, GWAS testing for identifying the common participate in the pathogenesis of IgAN as well as SLE: fl HLA, ITGAM, C3, CFI, FCGR, and PTEN (26,47–50). We or shared genetic in uences on SLE and IgAN in the same also checked the differential expression of those interrelated population should be carried out and is underway. genes: great enrichment of differences in gene expression GWAS have been used to identify multiple SNPs associ- between IgAN and healthy controls was observed (Supple- ated with disease risk, and attention has turned to explaining mental Table 3). Whole genome-wide expression data were the underlying molecular mechanisms of action (5). One hy- just from tens of samples, but C3 (it was linked with CFH), pothesis is that a proportion of the causal variants tagged by ITGAM (CFH), CD74 (HLA-DRB1), HLA-DMA (HLA-DRB1), these disease-associated markers may affect the abundance HLA-DMB HLA-DRA EGFR BLK SMAD7 UBE2L3 of a protein (or the relative abundance of its different iso- ( ), ( ), ( ), and fi fi PTEN (UBE2L3) still produced significant associations in the forms) by altering transcription. Ef cient identi cation of context of multiple testing. additional susceptibility loci with more modest effects might benefit from the integration of statistical evidence with some assessment of functional candidacy. Here, we investigated Discussion the positive correlations between identified SNPs and their In recent years, three GWASs in IgAN have been con- corresponding gene expression, especially for HLA-DRB1, ducted. They uncovered several susceptibility loci and UBE2L3,andBLK. The data further supported the candidacy greatly broadened our understanding of the genetic archi- of those genes as causal factors in IgAN. Data from Epstein– tecture of the susceptibility to IgAN (5,7–9). Among these Barr virus B cell–transformed lymphoblastoid cell lines three GWAS, we took part in two of them (7,8). As reported, should be more illustrative than data based on other cell all the identified associations within the regions of MHC, lines in IgAN, because gene expression and eQTLs can be 1q32, 8p23, 17p13, and 22q12 could be confirmed in our tissue-specific and because IgAN is a disease characterized cohort (7,8), which proved to be the cornerstone of credibil- by production of the nephritogesnic IgA1 molecule from B ity of the present study. The findings of the present study cells. Confirmation from a different gene-expression data- added to the loci showing associations with IgAN, as well as base strongly supported the probability of reliability (45). overlap between IgAN and SLE: CFH, HLA-DRA, HLA-DRB1, In addition, immortalized lymphoblasts that were clonal

Table 4. Joint effects of newly identiﬁed loci stratiﬁed by the number of protective alleles

Protective Alleles (n) Frequency in Cases/Controls (%/%) Odds Ratio (95% CI) P Value

#2 5.4/1.9 1.00 (Reference) 2 3 13.5/10.3 0.46 (0.25 to 0.83) 9.11310 3 2 4 25.7/19.5 0.46 (0.26 to 0.82) 6.68310 3 2 5 26.3/25.4 0.36 (0.21 to 0.64) 2.73310 4 2 6 19.0/21.4 0.31 (0.18 to 0.55) 3.06310 5 2 7 6.4/13.3 0.17 (0.09 to 0.31) 1.44310 9 2 $8 3.7/8.0 0.16 (0.08 to 0.31) 8.77310 9 794 Clinical Journal of the American Society of Nephrology

Figure 2. | A network plot of connections between identified loci in Chinese patients with IgA nephropathy. could more readily be studied without the environmental has proved difficult, and an optimal statistical approach is influences or transcriptome diversity found in mixed lym- not available, so combining several analytical methods may phocyte populations in vivo (51). Also, when differential be best for detecting epistatic interactions. Gene–gene inter- gene expressions in IgAN patients were checked, the ex- actions can be assessed with additive or multiplicative math- pression of all of those genes was upregulated in IgAN ematical models. We demonstrated significant additive and patients. However, the associations seemed to have tissue multiplicative interactions among the identified SNPs: that specific-characteristics because elevated expressions of CFH, is, additive interactions between PXK rs6445961and HLA- HLA-DRA and HLA-DRB1 seemed to be restricted to renal DRA rs9501626, as well as multiplicative interactions biopsies and BLK and UBE2L3 to whole-blood samples. between CFH rs6677604 and HLA-DRB1 rs9271366, and be- More widespread gene-expression analyses will be warran- tween HLA-DRA rs9501626 and HLA-DRB1 rs9271366. ted, especially in specific cell clones. It seemed that HLA- However, because of the moderate effects of these alleles DRA and HLA-DRB1 protective alleles corresponded to and a low incidence of IgAN (estimated incidence in the lower gene expressions, whereas PXK, BLK, and UBE2L3 general population, 25–50 cases per 100,000 individuals), protective alleles corresponded to higher gene expressions, our study remained underpowered to detect epitasis with which may indicate an abnormal balance between antigen oursamplesize(calculatedpower for epistasis was approxi- presentation and lymphocyte signaling. Nevertheless, future mately 0.1–0.2). In joint analyses, we observed that the disease studies linking alleles and differential gene expressions in risk decreased by about 7% with each increase in the alleles, specific tissues will be needed. In addition, rare variants, and it decreased up to 7-fold in individuals with eight or which may have a greater effect in conferring disease risk more protective alleles compared with those who have fewer and may contribute to a substantial fraction of heritability, than two. These results repeatedly supported the notion that will need further evaluations in future genetic studies in IgAN. the identified genes were the susceptibility genes for IgAN. Furthermore, to determine whether the identified genes One of the most compelling reasons for identifying the cause effects in a joint manner or epistatic fashion, we con- genetic underpinnings of common diseases is to generate ducted gene–gene interaction analyses as well as cumula- new hypotheses about the mechanisms and pathogenesis tive gene effect analysis. Investigating genetic interactions of disease (5). Hence, we checked further the newly Clin J Am Soc Nephrol 9: 788–797, April, 2014 Shared Genetic Study in IgAN and SLE, Zhou et al. 795

identified genes in a pathway-based manner. A molecular and ubiquitin/proteasome-dependent degradation) were network using a correlation structure was produced in highlighted. From the “systems genetics” perspective, our which all the identified genes were connected to each other data represent important clues for future studies on pleiotropy by intermediary genes, and four modules were highlighted. in IgA nephropathy and lupus nephritis. Great enrichment of differences in gene expression between IgAN and healthy controls was observed even though the Acknowledgments whole genome-wide expression data were just from tens of We thank our collaborators, Ali G. Gharavi and Krzyszt of Kiryluk samples. The pathways were MHC class II antigen presen- (Department of Medicine, Columbia University College of Physi- tation, complement regulation, signaling by the BCR, and cians and Surgeons, New York, NY), for kindly providing GWAS ubiquitin/proteasome-dependent degradation. The role of data and giving advice for the manuscript. We thank Sai-Nan Zhu MHC and complement in IgAN has been supported (Department of Biostatistics, Peking University First Hospital) for strongly by several observational studies. BLK encodes a ty- assistance with statistical analysis. We thank ELIXIGEN for their rosine kinase that is involved in the regulation of B-cell ac- editing assistance. We are grateful to the patients and their families tivation. B-cell signaling may have a key role in the for their participation in this study. pathogenesis of IgAN through elevation of IgA levels in This work was supported by grants from the Major State Basic serum, production of autoantibodies, antigen presentation Research Development Program of China (973 program, No. to T cells, and cytokine production (52). Also, B-cell deple- 2012CB517700),theNationalNaturalScienceFoundationofChina(No. tion has proved successful in the treatment of GN. UBE2L3 81200524), the Research Fund of Beijing Municipal Science and Tech- encodes a ubiquitin-conjugating enzyme involved in ubi- nology for the Outstanding Program (20121000110), the Foundation of quitin/proteasome-dependent degradation, which is Ministry of Education of China (20120001120008), and the Natural important in the cell cycle, cell differentiation, apoptosis, Science Fund of China to the Innovation Research Group (81021004). sodium-channel function, and modulation of inflamma- tory responses. The ubiquitin/proteasome pathway has Disclosures been suggested to be implicated in the development of None. multiple kidney diseases (53),andproteasomeinhibitors have been efficacious in some forms of renal disorders, such as lupus nephritis (54), renal ischemia-reperfusion References injury (55), and ANCA-induced GN (56). PXK encodes a 1. Floege J: The pathogenesis of IgA nephropathy: What is new and how does it change therapeutic approaches? Am J Kidney Dis 58: multimodular protein composed of a phox homology 992–1004, 2011 domain, a protein kinase–like domain, and a Wiskott- 2. Lai KN: Pathogenesis of IgA nephropathy. Nat Rev Nephrol 85: Aldrich syndrome protein homology 2 domain. The 275–283, 2012 gene product of PXK regulates the activity of Na-ATPase 3. Boyd JK, Cheung CK, Molyneux K, Feehally J, Barratt J: An update on the pathogenesis and treatment of IgA nephropathy. Kidney Int and K-ATPase ion transport, and is expressed in the kidney 81: 833–843, 2012 (57,58). Recent data suggest that PXK has a critical role in 4. Suzuki H, Kiryluk K, Novak J, Moldoveanu Z, Herr AB, Renfrow trafficking of the EGF receptor through modulation of ligand- MB, Wyatt RJ, Scolari F, Mestecky J, Gharavi AG, Julian BA: The induced ubiquitination of the receptor. Thus, the present study pathophysiology of IgA nephropathy. J Am Soc Nephrol 22: provided important clues for better elucidation of IgAN path- 1795–1803, 2011 5. Kiryluk K, Novak J, Gharavi AG: Pathogenesis of immunoglob- ophysiology in the future and possible therapy optimization. ulin A nephropathy: Recent insight from genetic studies. Annu Nevertheless, one must be cautious because most of the find- Rev Med 64: 339–356, 2013 ings from the initial GWA studies were association signals 6. Mestecky J, Raska M, Julian BA, Gharavi AG, Renfrow MB, rather than direct information about susceptibility genes. Moldoveanu Z, Novak L, Matousovic K, Novak J: IgA nephropathy: Molecular mechanisms of the disease. Annu Rev Pathol 8: The degree of shared genes between IgAN and SLE is 217–240, 2013 substantial, but is likely to still be an underestimate. First, 2 7. Yu XQ, Li M, Zhang H, Low HQ, Wei X, Wang JQ, Sun LD, we analyzed only associated loci at the P,1310 5 level, Sim KS, Li Y, Foo JN, Wang W, Li ZJ, Yin XY, Tang XQ, Fan L, Chen and the SNPs or genes meeting this criterion are increasing J, Li RS, Wan JX, Liu ZS, Lou TQ, Zhu L, Huang XJ, Zhang XJ, with enrollment of larger sample sizes. Second, because of Liu ZH, Liu JJ: A genome-wide association study in Han Chinese identifies multiple susceptibility loci for IgA nephropathy. Nat different linkage disequilibrium between variants in cases Genet 44: 178–182, 2012 and controls, a different variant in the same locus may be 8. Gharavi AG, Kiryluk K, Choi M, Li Y, Hou P, Xie J, Sanna-Cherchi responsible for disease risk in a second phenotype. Third, S, Men CJ, Julian BA, Wyatt RJ, Novak J, He JC, Wang H, Lv J, Zhu GWASs directly conducted in patients with lupus nephri- L, Wang W, Wang Z, Yasuno K, Gunel M, Mane S, Umlauf S, Tikhonova I, Beerman I, Savoldi S, Magistroni R, Ghiggeri GM, tis are still underway. For these reasons, the gene overlap Bodria M, Lugani F, Ravani P, Ponticelli C, Allegri L, Boscutti G, between the two diseases may be higher than that identi- Frasca G, Amore A, Peruzzi L, Coppo R, Izzi C, Viola BF, Prati E, fied in the present study. Salvadori M, Mignani R, Gesualdo L, Bertinetto F, Mesiano P, In conclusion, we identified CFH, HLA-DRA, HLA-DRB1, Amoroso A, Scolari F, Chen N, Zhang H, Lifton RP: Genome- PXK, BLK,andUBE2L3 as shared loci between IgAN and wide association study identifies susceptibility loci for IgA nephropathy. Nat Genet 43: 321–327, 2011 SLE. Many of the alleles that are risk alleles for SLE are 9. Feehally J, Farrall M, Boland A, Gale DP,Gut I, Heath S, Kumar A, protective alleles for IgAN. Genotypes were correlated Peden JF, Maxwell PH, Morris DL, Padmanabhan S, Vyse TJ, with the corresponding gene expression, suggesting a Zawadzka A, Rees AJ, Lathrop M, Ratcliffe PJ: HLA has strongest cis-eQTL effect. Positive gene–gene interactions were ob- association with IgA nephropathy in genome-wide analysis. JAm Soc Nephrol 21: 1791–1797, 2010 served, and disease risk decreased with accumulation of pro- 10. Zhernakova A, van Diemen CC, Wijmenga C: Detecting shared tective alleles. Four pathways (MHC class II antigen pathogenesis from the shared genetics of immune-related dis- presentation, complement regulation, signaling by the BCR, eases. Nat Rev Genet 10: 43–55, 2009 796 Clinical Journal of the American Society of Nephrology

11. Li GS, Zhu L, Zhang H, Lv JC, Ding JX, Zhao MH, Shen Y, Wang Edberg JC, Freedman BI, Gilkeson GS, Jacob CO, James JA, HY: Variants of the ST6GALNAC2 promoter influence tran- Kamen DL, Kimberly RP,Kim JH, Martin J, Merrill JT, Niewold TB, scriptional activity and contribute to genetic susceptibility to IgA Park SY, Petri MA, Pons-Estel BA, Ramsey-Goldman R, Reveille nephropathy. Hum Mutat 28: 950–957, 2007 JD, Scofield RH, Song YW, Stevens AM, Tsao BP,Vila LM, Vyse TJ, 12. Lin X, Ding J, Zhu L, Shi S, Jiang L, Zhao M, Zhang H: Aberrant Yu CY, Guthridge JM, Kaufman KM, Harley JB, Wakeland EK, galactosylation of IgA1 is involved in the genetic susceptibility of Langefeld CD, Gaffney PM, Montgomery CG, Moser KL; BIO- Chinese patients with IgA nephropathy. Nephrol Dial Transplant LUPUS Network; GENLES Network: Identification of IRF8, 24: 3372–3375, 2009 TMEM39A, and IKZF3-ZPBP2 as susceptibility loci for systemic 13. Zhu L, Tang W, Li G, Lv J, Ding J, YuL, Zhao M, Li Y,Zhang X, Shen lupus erythematosus in a large-scale multiracial replication Y, Zhang H, Wang H: Interaction between variants of two gly- study. Am J Hum Genet 90: 648–660, 2012 cosyltransferase genes in IgA nephropathy. Kidney Int 76: 190– 31. Ramos PS, Criswell LA, Moser KL, Comeau ME, Williams AH, 198, 2009 Pajewski NM, Chung SA, Graham RR, Zidovetzki R, Kelly JA, 14. Lv J, Xu D, Perkovic V, Ma X, Johnson DW, Woodward M, Levin Kaufman KM, Jacob CO, Vyse TJ, Tsao BP, Kimberly RP, Gaffney A, Zhang H, Wang H; TESTING Study Group: Corticosteroid PM, Alarcoń-Riquelme ME, Harley JB, Langefeld CD; In- therapy in IgA nephropathy. J Am Soc Nephrol 23: 1108–1116, ternational Consortium on the Genetics of Systemic Eryth- 2012 ematosus: A comprehensive analysis of shared loci between 15. Zhao N, Hou P, Lv J, Moldoveanu Z, Li Y, Kiryluk K, Gharavi AG, systemic lupus erythematosus (SLE) and sixteen autoimmune Novak J, Zhang H: The level of galactose-deficient IgA1 in the diseases reveals limited genetic overlap. PLoS Genet 7: sera of patients with IgA nephropathy is associated with disease e1002406, 2011 progression. Kidney Int 82: 790–796, 2012 32. Okada Y, Shimane K, Kochi Y, Tahira T, Suzuki A, Higasa K, 16. Chang DY, Luo H, Zhou XJ, Chen M, Zhao MH: Association of Takahashi A, Horita T, Atsumi T, Ishii T, Okamoto A, Fujio K, HLA genes with clinical outcomes of ANCA-associated vasculi- Hirakata M, Amano H, Kondo Y, Ito S, Takada K, Mimori A, Saito tis. Clin J Am Soc Nephrol 7: 1293–1299, 2012 K, Kamachi M, Kawaguchi Y, Ikari K, Mohammed OW, Matsuda 17. Luo H, Chen M, Cui Z, Yang R, Xu PC, Zhou XJ, Zhao MH: The K, Terao C, Ohmura K, Myouzen K, Hosono N, Tsunoda T, association of HLA-DQB1, -DQA1 and -DPB1 alleles with anti- Nishimoto N, Mimori T, Matsuda F, Tanaka Y, Sumida T, glomerular basement membrane (GBM) disease in Chinese pa- Yamanaka H, Takasaki Y,Koike T, Horiuchi T, Hayashi K, Kubo M, tients. BMC Nephrol 12: 21, 2011 Kamatani N, Yamada R, Nakamura Y, Yamamoto K: A genome- 18. Zhou XJ, Cheng FJ, Lv JC, Luo H, Yu F, Chen M, Zhao MH, Zhang wide association study identified AFF1 as a susceptibility H: Higher DEFB4 genomic copy number in SLE and ANCA- locus for systemic lupus eyrthematosus in Japanese. PLoS associated small vasculitis. Rheumatology (Oxford) 51: 992– Genet 8: e1002455, 2012 995, 2012 33. Yang W, Shen N, Ye DQ, Liu Q, Zhang Y, Qian XX, Hirankarn N, 19. Zhou XJ, Lu XL, Lv JC, Yang HZ, Qin LX, Zhao MH, Su Y, Li ZG, Ying D, Pan HF, Mok CC, Chan TM, Wong RW, Lee KW, Mok MY, Zhang H: Genetic association of PRDM1-ATG5 intergenic Wong SN, Leung AM, Li XP, Avihingsanon Y, Wong CM, Lee TL, region and autophagy with systemic lupus erythematosus in a Ho MH, Lee PP, Chang YK, Li PH, Li RJ, Zhang L, Wong WH, Chinese population. Ann Rheum Dis 70: 1330–1337, 2011 Ng IO, Lau CS, Sham PC, Lau YL; Asian Lupus Genetics Con- 20. Zhou XJ, Lu XL, Nath SK, Lv JC, Zhu SN, Yang HZ, Qin LX, Zhao sortium: Genome-wide association study in Asian populations MH, Su Y, Shen N, Li ZG, Zhang H; International Consortium on identifies variants in ETS1 and WDFY4 associated with sys- the Genetics of Systemic Lupus Erythematosus: Gene-gene in- temic lupus erythematosus. PLoS Genet 6: e1000841, 2010 teraction of BLK, TNFSF4, TRAF1, TNFAIP3, and REL in systemic 34. Han JW, Zheng HF, Cui Y, Sun LD, Ye DQ, Hu Z, Xu JH, Cai ZM, lupus erythematosus. Arthritis Rheum 64: 222–231, 2012 Huang W, Zhao GP, Xie HF, Fang H, Lu QJ, Xu JH, Li XP, 21. Zhou XJ, Lv JC, Bu DF, Yu L, Yang YR, Zhao J, Cui Z, Yang R, Zhao Pan YF, Deng DQ, Zeng FQ, Ye ZZ, Zhang XY, Wang QW, Hao F, MH, Zhang H: Copy number variation of FCGR3A rather than Ma L, Zuo XB, Zhou FS, Du WH, Cheng YL, Yang JQ, Shen SK, FCGR3B and FCGR2B is associated with susceptibility to anti- Li J, Sheng YJ, Zuo XX, Zhu WF, Gao F, Zhang PL, Guo Q, Li B, GBM disease. Int Immunol 22: 45–51, 2010 Gao M, Xiao FL, Quan C, Zhang C, Zhang Z, Zhu KJ, Li Y, Hu DY, 22. Zhou XJ, Lv JC, Yu L, Cui Z, Zhao J, Yang R, Han J, Hou P, Zhao Lu WS, Huang JL, Liu SX, Li H, Ren YQ, Wang ZX, Yang CJ, MH, Zhang H: FCGR2B gene polymorphism rather than Wang PG, Zhou WM, Lv YM, Zhang AP, Zhang SQ, Lin D, Li Y, FCGR2A, FCGR3A and FCGR3B is associated with anti-GBM Low HQ, Shen M, Zhai ZF, Wang Y, Zhang FY, Yang S, Liu JJ, disease in Chinese. Nephrol Dial Transplant 25: 97–101, 2010 Zhang XJ: Genome-wide association study in a Chinese Han 23. Zhou XJ, Lv JC, Zhao MH, Zhang H: Advances in the genetics of population identifies nine new susceptibility loci for systemic anti-glomerular basement membrane disease. Am J Nephrol 32: lupus erythematosus. Nat Genet 41: 1234–1237, 2009 482–490, 2010 35. Cunninghame Graham DS, Morris DL, Bhangale TR, Criswell LA, 24. Zhou XJ, Zhang H: Autophagy in immunity: Implications in eti- Syva¨nen AC, Ro¨nnblom L, Behrens TW, Graham RR, Vyse TJ: ology of autoimmune/autoinflammatory diseases. Autophagy 8: Association of NCF2, IKZF1, IRF8, IFIH1, and TYK2 with sys- 1286–1299, 2012 temic lupus erythematosus. PLoS Genet 7: e1002341, 2011 25. Lu X, Guo J, Zhou X, Li R, Liu X, Zhao Y, Zhu B, Liu X, Xu J, Zhu P, 36. Gateva V, Sandling JK, Hom G, Taylor KE, Chung SA, Sun X, Wu X, He J, Liu X, Zhang H, Li Z: Deletion of LCE3C_LCE3B is Ortmann W, Kosoy R, Ferreira RC, Nordmark G, Gunnarsson I, associated with rheumatoid arthritis and systemic lupus eryth- Svenungsson E, Padyukov L, Sturfelt G, Jo¨nsen A, Bengtsson AA, ematosus in the Chinese Han population. Ann Rheum Dis 70: Rantapaä¨-Dahlqvist S, Baechler EC, Brown EE, Alarcoń GS, 1648–1651, 2011 Edberg JC, Ramsey-Goldman R, McGwin G Jr, Reveille JD, Vila´ 26. Vernon KA, Cook HT: Complement in glomerular disease. Adv LM, Kimberly RP, Manzi S, Petri MA, Lee A, Gregersen PK, Seldin Chronic Kidney Dis 19: 84–92, 2012 MF, Ro¨nnblom L, Criswell LA, Syva¨nen AC, Behrens TW, Graham 27. Corrado A, Quarta L, Di Palma AM, Gesualdo L, Cantatore FP: RR: A large-scale replication study identifies TNIP1, PRDM1, IgA nephropathy in systemic lupus erythematosus. Clin Exp JAZF1, UHRF1BP1 and IL10 as risk loci for systemic lupus er- Rheumatol 25: 467–469, 2007 ythematosus. Nat Genet 41: 1228–1233, 2009 28. Horino T, Takao T, Terada Y: IgA nephropathy in a patient with 37. Hom G, Graham RR, Modrek B, Taylor KE, Ortmann W, Garnier systemic lupus erythematosus. Lupus 19: 650–654, 2010 S, Lee AT, Chung SA, Ferreira RC, Pant PV, Ballinger DG, Kosoy R, 29. Basile C, Semeraro A, Montanaro A, Giordano R, De Padova F, Demirci FY, Kamboh MI, Kao AH, Tian C, Gunnarsson I, Marangi AL, Ligorio VA, Santese D: IgA nephropathy in a patient Bengtsson AA, Rantapaä¨-Dahlqvist S, Petri M, Manzi S, Seldin with systemic lupus erythematosus. Nephrol Dial Transplant 13: MF, Ro¨nnblom L, Syva¨nen AC, Criswell LA, Gregersen PK, 1891–1892, 1998 Behrens TW: Association of systemic lupus erythematosus with 30. Lessard CJ, Adrianto I, Ice JA, Wiley GB, Kelly JA, Glenn SB, Adler C8orf13-BLK and ITGAM-ITGAX. NEnglJMed358: 900–909, AJ, Li H, Rasmussen A, Williams AH, Ziegler J, Comeau ME, 2008 Marion M, Wakeland BE, Liang C, Ramos PS, Grundahl KM, 38. Harley JB, Alarcoń-Riquelme ME, Criswell LA, Jacob CO, Gallant CJ, Alarcoń-Riquelme ME, Alarcoń GS, Anaya JM, Bae Kimberly RP, Moser KL, Tsao BP, Vyse TJ, Langefeld CD, Nath SK, SC, Boackle SA, Brown EE, Chang DM, Cho SK, Criswell LA, Guthridge JM, Cobb BL, Mirel DB, Marion MC, Williams AH, Clin J Am Soc Nephrol 9: 788–797, April, 2014 Shared Genetic Study in IgAN and SLE, Zhou et al. 797

Divers J, Wang W, Frank SG, Namjou B, Gabriel SB, Lee AT, biological networks and gene expression data using Cytoscape. Gregersen PK, Behrens TW, Taylor KE, Fernando M, Zidovetzki R, Nat Protoc 2: 2366–2382, 2007 Gaffney PM, Edberg JC, Rioux JD, Ojwang JO, James JA, Merrill 47. Cox SN, Sallustio F, Serino G, Pontrelli P, Verrienti R, Pesce F, JT, Gilkeson GS, Seldin MF, Yin H, Baechler EC, Li QZ, Torres DD, Ancona N, Stifanelli P, Zaza G, Schena FP: Altered Wakeland EK, Bruner GR, Kaufman KM, Kelly JA; International modulation of WNT-beta-catenin and PI3K/Akt pathways in IgA Consortium for Systemic Lupus Erythematosus Genetics (SLEGEN): nephropathy. Kidney Int 78: 396–407, 2010 Genome-wide association scan in women with systemic lupus 48. Tanaka Y, Suzuki Y, Tsuge T, Kanamaru Y, Horikoshi S, Monteiro erythematosus identifies susceptibility variants in ITGAM, PXK, RC, Tomino Y: FcgammaRIIa-131R allele and FcgammaRIIIa- KIAA1542 and other loci. Nat Genet 40: 204–210, 2008 176V/V genotype are risk factors for progression of IgA ne- 39. Jacob CO, Zhu J, Armstrong DL, Yan M, Han J, Zhou XJ, Thomas phropathy. Nephrol Dial Transplant 20: 2439–2445, 2005 JA, Reiff A, Myones BL, Ojwang JO, Kaufman KM, Klein- 49. Zhou XJ, Lv JC, Qin LX, Yang HZ, Yu F, Zhao MH, Zhang H: Is Gitelman M, McCurdy D, Wagner-Weiner L, Silverman E, Ziegler FCGR2A a susceptibility gene to systemic lupus erythematosus in J, Kelly JA, Merrill JT, Harley JB, Ramsey-Goldman R, Vila LM, Chinese? Lupus 20: 1198–1202, 2011 Bae SC, Vyse TJ, Gilkeson GS, Gaffney PM, Moser KL, Langefeld 50. Zhou XJ, Cheng FJ, Qi YY, Zhao YF, Hou P, Zhu L, Lv JC, Zhang H: CD, Zidovetzki R, Mohan C: Identification of IRAK1 as a risk gene FCGR2B and FCRLB gene polymorphisms associated with IgA with critical role in the pathogenesis of systemic lupus eryth- nephropathy. PLoS ONE 8: e61208, 2013 ematosus. Proc Natl Acad Sci U S A 106: 6256–6261, 2009 51. Grundberg E, Adoue V, Kwan T, Ge B, Duan QL, Lam KC, Koka V, 40. Shen N, Fu Q, Deng Y, Qian X, Zhao J, Kaufman KM, Wu YL, Yu Kindmark A, Weiss ST, Tantisira K, Mallmin H, Raby BA, Nilsson CY, Tang Y, Chen JY, Yang W, Wong M, Kawasaki A, Tsuchiya N, O, Pastinen T: Global analysis of the impact of environmental Sumida T, Kawaguchi Y, Howe HS, Mok MY, Bang SY, Liu FL, perturbation on cis-regulation of gene expression. PLoS Genet 7: Chang DM, Takasaki Y, Hashimoto H, Harley JB, Guthridge JM, e1001279, 2011 Grossman JM, Cantor RM, Song YW, Bae SC, Chen S, Hahn BH, 52. McCarthy DD, Kujawa J, Wilson C, Papandile A, Poreci U, Lau YL, Tsao BP: Sex-specific association of X-linked Toll-like Porfilio EA, Ward L, Lawson MA, Macpherson AJ, McCoy KD, Pei receptor 7 (TLR7) with male systemic lupus erythematosus. Proc Y, Novak L, Lee JY, Julian BA, Novak J, Ranger A, Gommerman JL, Natl Acad Sci U S A 107: 15838–15843, 2010 Browning JL: Mice overexpressing BAFF develop a commensal 41. Zhao J, Wu H, Khosravi M, Cui H, Qian X, Kelly JA, Kaufman KM, flora-dependent, IgA-associated nephropathy. J Clin Invest 121: Langefeld CD, Williams AH, Comeau ME, Ziegler JT, Marion MC, 3991–4002, 2011 Adler A, Glenn SB, Alarcoń-Riquelme ME, Pons-Estel BA, 53. Fukasawa H: The role of the ubiquitin-proteasome system in Harley JB, Bae SC, Bang SY, Cho SK, Jacob CO, Vyse TJ, Niewold kidney diseases. Clin Exp Nephrol 16: 507–517, 2012 TB, Gaffney PM, Moser KL, Kimberly RP, Edberg JC, Brown EE, 54. Neubert K, Meister S, Moser K, Weisel F, Maseda D, Amann K, Alarcon GS, Petri MA, Ramsey-Goldman R, Vila´ LM, Reveille JD, Wiethe C, Winkler TH, Kalden JR, Manz RA, Voll RE: The pro- James JA, Gilkeson GS, Kamen DL, Freedman BI, Anaya JM, teasome inhibitor bortezomib depletes plasma cells and protects Merrill JT, Criswell LA, Scofield RH, Stevens AM, Guthridge JM, mice with lupus-like disease from nephritis. Nat Med 14: 748– Chang DM, Song YW, Park JA, Lee EY, Boackle SA, Grossman JM, 755, 2008 Hahn BH, Goodship TH, Cantor RM, Yu CY, Shen N, Tsao BP; 55. Huber JM, Tagwerker A, Heininger D, Mayer G, Rosenkranz AR, BIOLUPUS Network; GENLES Network: Association of genetic Eller K: The proteasome inhibitor bortezomib aggravates renal variants in complement factor H and factor H-related genes with ischemia-reperfusion injury. Am J Physiol Renal Physiol 297: systemic lupus erythematosus susceptibility. PLoS Genet 7: F451–F460, 2009 e1002079, 2011 56. Bontscho J, Schreiber A, Manz RA, Schneider W, Luft FC, Kettritz R: 42. Graham RR, Hom G, Ortmann W, Behrens TW: Review of recent Myeloperoxidase-specific plasma cell depletion by bortezomib genome-wide association scans in lupus. J Intern Med 265: 680– protects from anti-neutrophil cytoplasmic autoantibodies-induced 688, 2009 glomerulonephritis. J Am Soc Nephrol 22: 336–348, 2011 43. Karlson EW, Chang SC, Cui J, Chibnik LB, Fraser PA, De Vivo I, 57. Mao H, Ferguson TS, Cibulsky SM, Holmqvist M, Ding C, Fei H, Costenbader KH: Gene-environment interaction between HLA- Levitan IB: MONaKA, a novel modulator of the plasma mem- DRB1 shared epitope and heavy cigarette smoking in predicting brane Na,K-ATPase. J Neurosci 25: 7934–7943, 2005 incident rheumatoid arthritis. Ann Rheum Dis 69: 54–60, 2010 58. Zou X, Qiu G, Chen C, Wu M, Hu Y, Zheng H, Li X, Gu S, Ji C, 44. Hosmer DW, Lemeshow S: Confidence interval estimation of Mao Y: Expression pattern and subcellular localization of five interaction. Epidemiology 3: 452–456, 1992 splice isoforms of human PXK. Int J Mol Med 16: 701–707, 2005 45. Dixon AL, Liang L, Moffatt MF, Chen W, Heath S, Wong KC, Taylor J, Burnett E, Gut I, Farrall M, Lathrop GM, Abecasis GR, Received: February 13, 2013 Accepted: October 30, 2013 Cookson WO: A genome-wide association study of global gene expression. Nat Genet 39: 1202–1207, 2007 46. Cline MS, Smoot M, Cerami E, Kuchinsky A, Landys N, Workman Published online ahead of print. Publication date available at www. C, Christmas R, Avila-Campilo I, Creech M, Gross B, Hanspers K, cjasn.org. Isserlin R, Kelley R, Killcoyne S, Lotia S, Maere S, Morris J, Ono K, Pavlovic V, Pico AR, Vailaya A, Wang PL, Adler A, This article contains supplemental material online at http://cjasn. Conklin BR, Hood L, Kuiper M, Sander C, Schmulevich I, asnjournals.org/lookup/suppl/doi:10.2215/CJN.01860213/-/ Schwikowski B, Warner GJ, Ideker T, Bader GD: Integration of DCSupplemental.