systemic lupus erythematosus ( sle ) ( omim 152700 ) is a debilitating autoimmune disease which affects multiple organs and is characterized by a loss of tolerance to self - antigens , inflammation , and dysregulated immune responses , resulting in significant morbidity and mortality . although many new loci which contribute to the pathogenesis of sle have been identified by genome wide association studies ( gwas ) and other association studies , they collectively do not explain all the risk contributed by heritable factors , indicating that other , as - of - yet unidentified genes are likely to be involv1ed . the power of gwas is in their agnostic approach , which does not require prior knowledge of any of the genetics or cellular mechanisms underlying a phenotypic trait . this comes at the cost of testing a large number of single nucleotide polymorphisms ( snps ) , thereby increasing the multiple - testing correction , and ignoring ( often decades of ) prior research into a phenotypic trait which may increase the power of a genetic study . in the present study we report the discovery of 13 novel genes outside of the human leukocyte antigen ( hla ) region ( 4 family - wise error rate ( fwer ) significant ) and confirmation of 4 genes ( 4 fwer significant ) which were reported as significant in previously published studies ( table 1 ) . furthermore , we have imputed all associated regions using the largest panels publicly available and used bioinformatics tools to test for deleterious effects of non - synonymous variants . imputation can be used to deconvolute multiple signals in regions which have complex interdependent signals , such as the hla region . imputation ( hla*imp ) has also been used in this study to assign samples to hla alleles and then to hla serotypes , connecting modern gwas based association results to earlier genetic and serotypic association results . many snps are associated with a disease because they are in linkage disequilibrium ( ld ) with another snp which is the primary signal in that region . to disambiguate between independent and dependent signals , we used conditional regression analysis and lasso regularization to eliminate signals whose association was due to correlation with another primary signal . this enabled us to identify the minimum number of signals required to explain the association results present in every associated region , an approach pioneered by raychaudhuri et al . , and adapted here for sle . this study has also assigned hla alleles and serotypes to each subject and has refined the association signals within the hla and other regions . we detected 430 significant snps ( false discovery rate ( fdr )  0.05 ) in 160 genes . 405 snps were in the hla region ( supplemental table s1 and s3 ) , and 25 snps were in 17 genes in other regions ( table 1 , supplemental table s3 ) . an additional 7 genes were found to be consistent with previously published results , although they did not meet the non - hla fdr level of this study ( supplemental table s2 ) . figure 1 shows a manhattan plot of the genome wide association data showing the p values of all tested snps . as this figure shows , because of these results , coupled with the well - known association of the hla region with sle and the extensive ld in the hla region , we have separated the analysis and discussion of hla genes from non - hla genes . 25 significant snps within 16 regions corresponding to 17 genes were detected in non - hla regions . of these genes , 13 have not been previously associated with sle ( previous association columns in table 1 ) . two of the 17 genes are transcription factors ( ehf and med1 ) , and two are involved in nfb signaling ( rassf2 and rnf114 ) . two genes are involved in antigen presentation ( bin1 and sec61 g ) , and one in cell adhesion / tissue remodeling cntn6 . all of the significant genes which have mrna expression information available are expressed in at least one relevant immune cell type ( figure s2 ) . the presence of multiple significant snps in a region can be due to ld or multiple independent signals . to distinguish between these possibilities , we performed multiple regression analysis on the significant snps in each of the 4 regions which contained multiple significant snps . we found snps in 1 gene which were dependent on other genes or interdependent on each other where significance of either gene could be explained by the other gene ( table 1  dependent on  column ) . in the region including and surrounding irf5 and tnpo3 ( 7q32.1 ) there are 8 significant snps ( figure 2 panel a , supplemental table s3 ) . after accounting for rs10488631 in tnpo3 ( figure 2 panel b ) and rs4728142 in irf5 ( figure 2 panel c ) , the next most - significant snp is rs1665105 , whose p value ( 3.2910 ) is greater than the irf5/tnpo3 region multiple - family fdr ( 2.2210 ) , and furthermore was not found to be significant in this study . this suggests that there are at least two causal variants in the irf5 and tnpo3 region , and that irf5 and tnpo3 are independently associated with sle . in the 15q14 region , the significant snp in fam98b , rs11073328 , accounts for the original significance of the snp in tyro3 , rs12259 ( pmult = 2.6110 , pmult = 8.3010 in the multiple regression , respectively ) this indicates that the association of tyro3 is due to fam98b ; there is evidence for only one signal in the 15q14 region . the final two regions which have multiple snps are sec61 g and ehf ; each of these regions has two significantly associated snps , and in both regions , the significantly associated snps are in complete ld with each other . thus , there is only evidence for a single signal in sec61 g and another single signal in ehf . imputation was performed on all 16 non - hla regions comprising over 2.9 million snps . 840 additional significant snps were found in these regions , 3 of these snps were non - synonymous . however , bioinformatic analyses of these non - synonymous snps using polyphen , sift , and provean   do not predict deleterious effects as a consequence of these mutations . thus , it is unlikely that we have identified the causal mutations responsible for the association of these regions with sle , and deep sequencing or other follow up studies of these regions are required . we have previously established that rs17849502 in ncf2 ( 1q25 ) is a causal mutation associated with sle which produces a two - fold reduction in fc receptor - mediated vav1 nadph oxidase response . in this region there is are multiple significant and suggestive ( fdr  0.1 ) snps by genotyping and imputation in each of edem3 , lamc1 , and ncf2 genes as shown in figure 3 . multiple regression indicates that the signals in edem3 and lamc1 are independent of each other ( data not shown ) . to determine if the significant association of edem3 and the suggestive association of lamc1 was due to the presence of rs17849502 in ncf2 , we examined the imputation results in this region . while rs17849502 was present in the imputation panels , it is rare , so the ability of the imputation panels to accurately impute this snp is greatly diminished , leading to its non - significance upon imputation ( impute v2 reports an metric of 0.05 for this snp ; accurately imputed snps should have an  info however , rs17849502 and rs12565776 are in strong linkage disequilibrium ( d = 0.96 , figure 3 panel b ) , so it remains possible that the significance of rs12565776 is due to rs1789502 . the hla region has by far the largest number and most highly significant snps , reaching a minimum p value of 7.7610 ( fdr=2.5610 ) ( figure 1 , supplemental table s1 ) . the 6p21 - 22 region , which includes the classical hla genes , contains many alleles which are in linkage disequilibrium with each other . thus , many of the significant results seen could be due to a smaller number of primary signals . to determine the minimum number of signals capable of accounting for the significance seen in the 6p21 - 22 region we performed a step - wise multiple regression analysis , accounting for the most significant snp in the model at each step , and repeating until no additional terms had p values less than the p value corresponding to an fdr of 0.05 in this study . as shown in figure 4 , the most significant snp ( from 405 significant snps out of 4,748 genotyped snps in this region ) was rs558702 ( p = 7.76  10 ) ( figure 4 panel a ) . after accounting for rs558702 , the most significant unaccounted snp is rs9275572 ( p = 1.94  10 , figure 4 panel b ) . subsequently , after accounting for both of these snps , rs2764208 was the most significant snp unaccounted for ( p = 3.6110 , figure 4 panel c ) . finally , rs10946940 was most significant snp after accounting for all three of these snps ( p = 2.3710 , figure 4 panel d ) . these four snps accounted for the vast majority of the significant snps seen in the 6p21 - 22 region , although 6 additional weak signals remained ( figure 4 panel e ) . using lasso regularization   across all significant snps in this region , both rs558702 and rs9275572 were the components in the model with the largest coefficients , further indicating their importance ( data not shown ) . thus , two signals in the classical hla are able to account for the vast majority of the significant associations seen in hla , and two additional signals in znf184 and snrpc explain most significance seen in the periphery of the hla region ( table 2 and figure 4 ) . the other signal is in snrpc which is involved in the formation of the spliceosome which is often a target of autoantibodies in sle . thus , the majority of the association of classical hla with sle in this study can be explained by two primary signals . to reconcile our snp findings with the established hla allele and serotype association with sle , we imputed hla alleles using hla*imp [ 1214 ] which assigned an hla haplotype to each sample on the basis of genotyped snps in the hla region . the alleles of hla - a , b , c , dpb1 , dqa1 , dqb1 , and drb1 were imputed at four digit accuracy and hla - drb3 , drb4 , and drb5 were imputed at two digit accuracy . notably , hla - dqa1 * 05:01 and hladqb1 * 02:01 , which have previously been shown to be significantly associated with sle and are part of the dr17/dq2 extended haplotype , were found to be highly associated in this study . we did not detect any significant associations with any of the alleles of hla - a , c , dpb1 , drb4 , or drb5 . finally , multiple regression with hla - dqb1 and hla - drb1 indicated that hla - dqb1 and hla - drb1 have an independent association with sle , and the association of one is not merely due to linkage with the other ( table s5 ) . furthermore , the association of hla - b is largely explained by hla - drb1 , but not hla - dqb1 ( table s5 ) . a regression analysis with the significant alleles of hla - dqb1 and hla - drb1 with the two significant primary snps signals in classical hla ( rs558702 and rs9275572 ) indicates that hla - dqb1 and hla - drb1 are strongly correlated with these snps . the snps outside of the hla region ( rs2764208 and rs10946940 ) were not accounted for by hla - dqb1 or hla - drb1 , indicating that their association is independent of the classical hla signal . dq7 ( broad antigen dq3 ) had a protective effect , whereas dr17 and b8 were associated with disease . we did not see any significant associations with the hla class i a or c antigens , nor did we see any significant associations with alleles of the extended dr / dq haplotype . 25 significant snps within 16 regions corresponding to 17 genes were detected in non - hla regions . of these genes , 13 have not been previously associated with sle ( previous association columns in table 1 ) . two of the 17 genes are transcription factors ( ehf and med1 ) , and two are involved in nfb signaling ( rassf2 and rnf114 ) . two genes are involved in antigen presentation ( bin1 and sec61 g ) , and one in cell adhesion / tissue remodeling cntn6 . all of the significant genes which have mrna expression information available are expressed in at least one relevant immune cell type ( figure s2 ) . the presence of multiple significant snps in a region can be due to ld or multiple independent signals . to distinguish between these possibilities , we performed multiple regression analysis on the significant snps in each of the 4 regions which contained multiple significant snps . we found snps in 1 gene which were dependent on other genes or interdependent on each other where significance of either gene could be explained by the other gene ( table 1  dependent on  column ) . in the region including and surrounding irf5 and tnpo3 ( 7q32.1 ) there are 8 significant snps ( figure 2 panel a , supplemental table s3 ) . however , after accounting for rs10488631 in tnpo3 ( figure 2 panel b ) and rs4728142 in irf5 ( figure 2 panel c ) , the next most - significant snp is rs1665105 , whose p value ( 3.2910 ) is greater than the irf5/tnpo3 region multiple - family fdr ( 2.2210 ) , and furthermore was not found to be significant in this study . this suggests that there are at least two causal variants in the irf5 and tnpo3 region , and that irf5 and tnpo3 are independently associated with sle . in the 15q14 region , the significant snp in fam98b , rs11073328 , accounts for the original significance of the snp in tyro3 , rs12259 ( pmult = 2.6110 , pmult = 8.3010 in the multiple regression , respectively ) this indicates that the association of tyro3 is due to fam98b ; there is evidence for only one signal in the 15q14 region . the final two regions which have multiple snps are sec61 g and ehf ; each of these regions has two significantly associated snps , and in both regions , the significantly associated snps are in complete ld with each other . thus , there is only evidence for a single signal in sec61 g and another single signal in ehf . imputation was performed on all 16 non - hla regions comprising over 2.9 million snps . 840 additional significant snps were found in these regions , 3 of these snps were non - synonymous . however , bioinformatic analyses of these non - synonymous snps using polyphen , sift , and provean   do not predict deleterious effects as a consequence of these mutations . thus , it is unlikely that we have identified the causal mutations responsible for the association of these regions with sle , and deep sequencing or other follow up studies of these regions are required . we have previously established that rs17849502 in ncf2 ( 1q25 ) is a causal mutation associated with sle which produces a two - fold reduction in fc receptor - mediated vav1 nadph oxidase response . in this region there is are multiple significant and suggestive ( fdr  0.1 ) snps by genotyping and imputation in each of edem3 , lamc1 , and ncf2 genes as shown in figure 3 . multiple regression indicates that the signals in edem3 and lamc1 are independent of each other ( data not shown ) . to determine if the significant association of edem3 and the suggestive association of lamc1 was due to the presence of rs17849502 in ncf2 , we examined the imputation results in this region . while rs17849502 was present in the imputation panels , it is rare , so the ability of the imputation panels to accurately impute this snp is greatly diminished , leading to its non - significance upon imputation ( impute v2 reports an  info metric of 0.05 for this snp ; accurately imputed snps should have an  info however , rs17849502 and rs12565776 are in strong linkage disequilibrium ( d = 0.96 , figure 3 panel b ) , so it remains possible that the significance of rs12565776 is due to rs1789502 . the hla region has by far the largest number and most highly significant snps , reaching a minimum p value of 7.7610 ( fdr=2.5610 ) ( figure 1 , supplemental table s1 ) . the 6p21 - 22 region , which includes the classical hla genes , contains many alleles which are in linkage disequilibrium with each other . thus , many of the significant results seen could be due to a smaller number of primary signals . to determine the minimum number of signals capable of accounting for the significance seen in the 6p21 - 22 region we performed a step - wise multiple regression analysis , accounting for the most significant snp in the model at each step , and repeating until no additional terms had p values less than the p value corresponding to an fdr of 0.05 in this study . as shown in figure 4 , the most significant snp ( from 405 significant snps out of 4,748 genotyped snps in this region ) was rs558702 ( p = 7.76  10 ) ( figure 4 panel a ) . after accounting for rs558702 , the most significant unaccounted snp is rs9275572 ( p = 1.94  10 , figure 4 panel b ) . subsequently , after accounting for both of these snps , rs2764208 was the most significant snp unaccounted for ( p = 3.6110 , figure 4 panel c ) . finally , rs10946940 was most significant snp after accounting for all three of these snps ( p = 2.3710 , figure 4 panel d ) . these four snps accounted for the vast majority of the significant snps seen in the 6p21 - 22 region , although 6 additional weak signals remained ( figure 4 panel e ) . using lasso regularization   across all significant snps in this region , both rs558702 and rs9275572 were the components in the model with the largest coefficients , further indicating their importance ( data not shown ) . thus , two signals in the classical hla are able to account for the vast majority of the significant associations seen in hla , and two additional signals in znf184 and snrpc explain most significance seen in the periphery of the hla region ( table 2 and figure 4 ) . the other signal is in snrpc which is involved in the formation of the spliceosome which is often a target of autoantibodies in sle . thus , the majority of the association of classical hla with sle in this study can be explained by two primary signals . to reconcile our snp findings with the established hla allele and serotype association with sle , we imputed hla alleles using hla*imp [ 1214 ] which assigned an hla haplotype to each sample on the basis of genotyped snps in the hla region . the alleles of hla - a , b , c , dpb1 , dqa1 , dqb1 , and drb1 were imputed at four digit accuracy and hla - drb3 , drb4 , and drb5 were imputed at two digit accuracy . notably , hla - dqa1 * 05:01 and hladqb1 * 02:01 , which have previously been shown to be significantly associated with sle and are part of the dr17/dq2 extended haplotype , were found to be highly associated in this study . we did not detect any significant associations with any of the alleles of hla - a , c , dpb1 , drb4 , or drb5 . finally , multiple regression with hla - dqb1 and hla - drb1 indicated that hla - dqb1 and hla - drb1 have an independent association with sle , and the association of one is not merely due to linkage with the other ( table s5 ) . furthermore , the association of hla - b is largely explained by hla - drb1 , but not hla - dqb1 ( table s5 ) . a regression analysis with the significant alleles of hla - dqb1 and hla - drb1 with the two significant primary snps signals in classical hla ( rs558702 and rs9275572 ) indicates that hla - dqb1 and hla - drb1 are strongly correlated with these snps . the snps outside of the hla region ( rs2764208 and rs10946940 ) were not accounted for by hla - dqb1 or hla - drb1 , indicating that their association is independent of the classical hla signal . dq7 ( broad antigen dq3 ) had a protective effect , whereas dr17 and b8 were associated with disease . we did not see any significant associations with the hla class i a or c antigens , nor did we see any significant associations with alleles of the extended dr / dq haplotype . in the present study we report the discovery of novel non - hla genes not previously associated with sle and the confirmation of genes which were reported as significant in previously published studies ( table 1 ) . the novel discoveries were made in part due to the increase in statistical power of the study through the use of a fdr multiple testing correction instead of a fwer multiple testing correction . it is therefore expected that genes involved in regulation of various immune cells and their functions would be an important part of the genetic susceptibility architecture of sle . indeed , among the novel genes associated with sle susceptibility found in the present study , two genes code for transcription factors ( ehf and med1 ) , two are components of the nfb pathway ( rassf2 and rnf114 ) , two are involved in antigen presentation ( bin1 and sec61 g ) , and one is involved in adhesion and endothelial migration ( cntn6 ) . some of the proteins encoded by these susceptibility genes have been previously considered to be involved in the pathogenesis of the disease , but this is the first study to demonstrate that they are actually associated with genetic susceptibility to sle . the ets family of transcription factors and especially ets-1 , have been previously associated with sle . here we identified ehf , another member of the ets family as associated with sle . ehf is particularly important in the differentiation of dendritic cells ( dc ) , an important antigen presenting cell and producer of cytokines , including type i ifns that are essential for disease pathogenesis . interestingly , the cytokine tgfa , specifically involved in dc differentiation , is suggestively associated with sle ( p = 2.62  10 , fdr = 1.6010 ) . furthermore , mafb , a repressor of ets - mediated transcription , is also suggestively associated with sle ( p = 9.8810 , fdr = 8.8310 ) . the ets pathway exemplifies the recurring motif of genes at multiple steps of a disease - relevant pathway being found to be associated with that disease . the transcription factor med1 is essential for the intrathymic development of inkt cells , a subgroup of immune cells found at abnormal levels in sle subjects . components of the nfb pathway , the master transcriptional regulator of inflammation , have been associated with increased sle susceptibility ( irak1 , tnfaip3 , ube2l3 , prkcb ) . the present study adds two new genes involved in nfb signaling ( rassf2 and rnf114 ) , highlighting the importance of this pathway for sle genetic susceptibility and pathogenesis . rnf114 , an ubiquitin binding protein , regulates a positive feedback loop that enhances dsrna - induced production of type i ifn through the activation of the irf3 and nfb transcription factors , previously shown to be associated with the immune - mediated skin disease psoriasis . genetic variants related to adhesion and endothelial migration of the various immune cell types have been associated with sle susceptibility ( itgam , icam1 , selp)[1 , 20 ] . in the present study we have identified an additional gene involved in cell adhesion and tissue remodeling ( cntn6 ) . although the primary auto - antigens driving sle pathogenesis are quite elusive , it is clear that abnormal antigen processing and presentation play an important part . in the current study we identified two new susceptibility genes involved in antigen processing and presentation ( bin1 and sec61 g ) . classical mhc class i and class ii genes on the surface of antigen presenting cells are responsible for antigen presentation to the t cell receptor on t lymphocytes . mhc loci were the first reported genetic association with sle and remain the strongest . understanding the genetic risk of this region is therefore critical to the understanding of the pathogenesis of the disease however , the region is extremely gene - dense with long range ld and hundreds of immunologically active genes , which makes identification of the true causal loci very difficult . following the approach of raychaudhuri et al . using conditional logistic regression , we showed that two signals inside of the classical hla and two additional in the periphery can account for the vast majority of the significant snps in the entire hla region . we confirm the findings of morris et al . by showing the involvement of hla - drb1 * 03:01 and the involvement of snps in addition to hla alleles . we extend their findings by showing the association of sle with serotypes , confirm the number of primary signals using lasso regularization , and provide evidence for the requirement of hla - dqb1 * 02:01 to explain association of hla with sle . our use of hla imputation has also enabled us to deconvolute the large number of associated snps in the hla region and reconcile these findings with decades - old work showing the association of serotypes and hla alleles with sle . the new sle - associated genes and genetic regions discovered in this study expand our understanding of sle and provide new putative targets for diagnostics and treatment of this important autoimmune disease . this study was approved by the irb of university of southern california school of medicine , and by the irb of the institutions participating in the study , namely , university of california los angeles , university of california san francisco , oklahoma medical research foundation , university of oklahoma health sciences center , university of alabama at birmingham , cincinnati children s hospital medical center , and the irb of king s college , london . 735 unrelated self - identified females of european ancestry with sle ( satisfying the revised criterion for sle from the american college of rheumatology [ 22 , 23 ] ) were collected ( along with 480 unrelated female controls ) by slegen members . in addition , 2057 female controls were obtained from the illumina icontroldb resource giving 2537 total controls . genotyping was performed using the illumina infinium humanhap300 genotyping bead chip as previously described . in order to avoid including samples with genetic background not representative of the overall study , a principle component analysis ( pca ) analysis was performed which identified 109 non - representative samples which were removed from further analysis . to eliminate duplicate and cryptic relationships , a set of 500 snps with minor allele frequency ( maf )  0.4 were selected . 98 sets of duplicates ( three cases , two slegen controls , 93 illumina controls ) which matched at 99.9% of the snps and 44 sets of related samples ( 16 cases , three slegen controls and 25 illumina controls ) had all but one duplicate / related removed . in order to address population structure which can be a source of confounders [ 25 , 26 ] , we performed pca on a subset of snps which are informative for ancestry ( ancestry informative markers ( aims ) ) . in figure s1 , a pca biplot of the samples collected for this study is shown . the vast majority of samples are present in a homogeneous grouping , with no clear delineations between case and control samples . a prominent exception to this are the samples in orange which are found in the lower left of panel a and lower right of panel b. these 109 samples ( 10 cases , 6 slegen controls , and 93 illumina controls ) are not representative of the ancestral background of the other samples , and therefore have been excluded from further analysis . to control for any remaining population structure , the same first three pca rotation axes were used as cofactors in the association analysis . after correcting for these three factors and eliminating the outlier samples , the remaining dispersion of cases and controls is even and symmetrical ( figure s1 panels c and d ) . the significance of association was calculated using logistic regression with sle status as the dependent variable and the additive dosage of the snp and the three first pca axes as independent variables with custom routines written in r . p values obtained were corrected for multiple testing by estimating the fdr using the benjamini and hochberg method . since the vast majority of significant snps are located in the hla region , calculating the fdr study - wide would result in an under - estimation of the fdr in the non - hla region . thus , the fdr was calculated separately for the hla regions and non hla regions . because each of these regions contains significant results imputation on regions containing significantly associated snps was performed using impute ( v2.3.0 )   with the 1k genomes   and hapmap   panels , covering over 3.1 million snps . significance of association of imputed snps was calculated in the same manner as for genotyped snps , with multiple - testing corrections being applied within the region instead of study - wide . multiple logistic regression was performed by adding the most significant snp in a region as covariates to the logistic regression model , and repeating the regression on the remaining snps in the region . this procedure was repeated until no snps had a multiple - testing corrected fdr less than the per - family fdr cutoff of 2.6810 ( q = 0.05 , m = 298 , r = 16 as defined in   using the ( bh  q , bh  rq / m ) procedure ) . the best guess haplotype was used in logistic regression with three pca axes as was done for genotyped values . hla haplotypes were assigned to serotypes using the wmda directory release 3.12.0 , and logistic regression with three pca axes was performed on the resultant serotypes .
in a genome wide association study ( gwas ) of individuals of european ancestry afflicted with systemic lupus erythematosus ( sle ) the extensive utilization of imputation , stepwise multiple regression , lasso regularization , and increasing study power by utilizing false discovery rate ( fdr ) instead of a bonferroni multiple test correction enabled us to identify 13 novel non - human leukocyte antigen ( hla ) genes and confirmed the association of 4 genes previously reported to be associated . 
 novel genes associated with sle susceptibility included two transcription factors ( ehf , and med1 ) , two components of the nfb pathway ( rassf2 and rnf114 ) , one gene involved in adhesion and endothelial migration ( cntn6 ) , and two genes involved in antigen presentation ( bin1 and sec61 g ) . 
 in addition , the strongly significant association of multiple single nucleotide polymorphisms ( snps ) in the hla region was assigned to hla alleles and serotypes and deconvoluted into four primary signals . 
 the novel sle - associated genes point to new directions for both the diagnosis and treatment of this debilitating autoimmune disease .