Meta-analysis of two Chinese populations identifies an autoimmune disease risk allele in 22q11.21 as associated with systemic lupus erythematosus

Introduction Systemic lupus erythematosus (SLE) is a heterogeneous disease with a diverse spectrum of clinical symptoms, ranging from skin rash to end-organ damage. 22q11.21 has been identified as a susceptibility region for several autoimmune diseases, including SLE. However, detailed information for SLE association and the underlying functional mechanism(s) is still lacking. Methods Through meta-analysis of two genome-wide association studies (GWAS) on Han Chinese populations, comprising a total of 1,659 cases and 3,398 controls matched geographically, we closely examined the 22q11.21 region, especially on the reported single-nucleotide polymorphisms (SNPs) associated with different autoimmune diseases and their relationships. We further replicated the most significant associations of SNPs with SLE using 2,612 cases and 2,323 controls of Asian ancestry. Results All reported SNPs in the 22q11.21 region with different autoimmune diseases were examined using the two GWAS data and meta-analysis results, and supportive evidence of association with SLE was found (meta-analysis: P_meta ≤ 7.27E-05), which might require further investigation. SNP rs2298428 was identified as the most significant SNP associated with SLE in this region (P_meta =2.70E-09). It showed independent effects through both stepwise and conditional logistic regression, and there is no evidence of other independent association signals for SLE in this region. The association of rs2298428 was further replicated in three cohorts from Hong Kong, Anhui and Thailand comprising a total of 2,612 cases and 2,323 controls (joint analysis of GWAS and replication result: P_all =1.31E-11, odds ratio =1.23). SNP rs2298428 was shown to be an expression quantitative locus for UBE2L3 gene in different cell types, with the risk allele (T) being correlated with higher expression of UBE2L3. This is consistent with earlier reports on higher expression of UBE2L3 in patients with SLE. Conclusions Association with distinct autoimmune diseases highlights the significance of this region in autoreactive responses and potentially shared functional mechanisms in these diseases. Electronic supplementary material The online version of this article (doi:10.1186/s13075-015-0577-6) contains supplementary material, which is available to authorized users.


Introduction
Systemic lupus erythematosus (SLE) is an autoimmune disease with an unclear etiology. It usually presents with a diverse spectrum of clinical manifestation spanning from malar rash to kidney injury. Genetic factors explain about 50% to 60% of the disease etiology [1]. The concordance rate for SLE is much higher in monozygotic (25% to 70%) than in dizygotic (2% to 9%) twins [2,3], indicating the importance of genetic contributions.
In the present study, we examined the association for SLE in the 22q11.21 region and further replicated the association of SNP rs2298428 in a total of 4,271 cases and 5,721 controls of Asian ancestry. To that end, our results confirmed rs2298428 as the most significant SNP associated with SLE in this region. Meanwhile, the risk allele of this SNP is highly correlated with higher expression of UBE2L3 in different cell lines.

Study participants
The samples included in the present study were collected from Hong Kong and Anhui, China, and from Bangkok, Thailand (Additional file 1). All the cases fulfilled the revised criteria of the

Genotyping
The GWAS on the Hong Kong and Anhui cohorts were conducted using the Illumina Human610-Quad BeadChip array (Illumina, San Diego, CA, USA), as previously reported (HK_GWAS and AH_GWAS). Further replication of the candidate SNPs was performed by using the Taq-Man genotyping method (Life Technologies, Carlsbad, CA, USA) with the remaining samples from the Hong Kong cohort that were not included in the discovery stage (HK_REP); samples collected from Bangkok, Thailand (TH_REP); and samples from an independent Anhui cohort (AH_REP). Genotyping concordance between Illumina Human610-Quad BeadChip and TaqMan SNP genotyping method was also checked on randomly selected samples, and the two methods showed complete concordance.

Association analysis
We used inverse variance method for the meta-analysis installed in METAL [24]. Joint analysis of association was conducted using the Cochran-Mantel-Haenszel (CMH) test, taking into account the effect of SNP differences between cohorts. The homogeneity of the effect size between different cohorts and different stages of the study was evaluated by using the Breslow-Day test (P_het in Table 1), both installed in PLINK 1.07.
Stepwise logistic regression was performed using IBM SPSS 16.0 software (IBM, Armonk, NY, USA). Tests of independent contributions toward disease associations for SNPs in a single locus were done using logistic regression, adjusting for the effect of a specific SNP in the same locus, while also taking into account differences among cohorts. SNPTEST v2.2.0 was used to perform the logistic regression tests in this study [25]. Linkage disequilibrium (LD) patterns and values were obtained using Haploview [26].

Imputation and meta-analysis of two genome-wide association studies on Han Chinese populations from Hong Kong and Anhui
First, imputation was performed using IMPUTE2 [27] on two GWAS on Han Chinese populations. Association analysis was performed using SNPTEST v2.2.0, taking the genotype uncertainty into account. Meta-analysis was performed using METAL [24] with the inverse variancebased model. We examined the meta-analysis results in 22q11.21 and observed a total of 4,834 SNPs in this 1.9-Mb region, from 20,220,110 to 22,131,990 bp (GRCh37/hg19). On the basis of meta-analysis P-values (P_meta), 121 SNPs showed suggestive associations (P_meta <0.0001), aggregating in a 187-kb region (Additional file 2). SNP rs2298428 showed the most significant P-value (P_meta =2.70E-09). Of the 96 SNPs, 25 SNPs had P_meta-values reaching genome-wide significance (5E-08), including SNP rs2298428. The other 24 SNPs all had high LD with rs2298428 (r 2 > 0.9).
Linkage disequilibrium pattern of single-nucleotide polymorphisms in 22q11.21 associated with immune-related diseases For the purpose of finding potentially shared susceptible variants and/or causal variants between SLE and other immune-related diseases, we focused on SNP rs2298428, which showed the most significant P-values for SLE in our meta-analysis results, and other reported SNPs in this region that showed association with SLE (rs5754217, rs463426 and rs131654) [14,18], CD and PS (rs181359) [22], and IBD (rs2266959). SNP rs2298428 was also reported to be associated with other immune-related diseases, including SSc [19], CeD and RA [21]. As shown in Table 2, all six SNPs reported for different diseases showed strong evidence of association with SLE in Asian populations (2.70E-09 ≤ − ≤ 7.27E-05). The LD patterns of these six SNPs are shown in Figure 1, based on different populations from HapMap data including Han Chinese, Beijing, population and Utah residents with ancestry from northern and western Europe (CHB and CEU, respectively), Hong Kong (HK) and Anhui (AH). The LD patterns in CHB, HK and AH populations were similar. In these three Chinese populations, all but rs463426 showed moderate to high LD between each other (r 2 > 0.5). We also compared the LD patterns between the Chinese populations and CEU ( Figure 1). In Caucasians, SNPs rs463426 and rs131654 showed minimal LD with all the other SNPs (r 2 < 0.2). The other four SNPs showed high LD with each other (r 2 > 0.95).
Independence test on the single-nucleotide polymorphisms associated with immune-related diseases A stepwise logistic regression analysis was performed to test the independence of these SNPs ( Table 3). The method begins with an empty model, to which the variables were added one at a time. The analysis showed that SNP rs2298428 exhibited the strongest, and the only, significant association with SLE. Further addition of any other SNPs involved did not show significant improvement of the model, which is partially explained by the high LD among most of these SNPs. However, the SNPs with moderate LD did not show significant improvement in the model, demonstrating a lack of evidence of further independent signals of association for SLE in this region.
Conditional logistic regression analysis was performed to investigate the independent effects among these replicated SNPs with immune-related diseases (Table 4). SNP rs2298428 remained significant when the effect of any other SNPs was accounted for, except for SNP rs2266959. More intuitively, the association P-values of all SNPs before and after the effect of rs2298428 was adjusted for are shown in Figure 2.

Replication on single-nucleotide polymorphism rs2298428
Replication for SNP rs2298428 was performed by using a TaqMan SNP genotyping method (Life Technologies) on the Hong Kong cohort independent from Hong Kong samples genotyped in the GWAS stage; samples collected from Bangkok, Thailand; and samples from Anhui, China, which were independent from the Anhui GWAS cohort. As shown in Table 5, replications in different cohorts showed consistent results as those from the discovery stage (Table 1). The joint analysis of association, taking into account the effect of SNP differences among cohorts from Hong Kong, Anhui and Thailand from both discovery stage and replication stage, was conducted using the Cochran-Mantel-Haenszel test. SNP rs2298428 showed stronger evidence of association with SLE (P =1.31E-11), and testing of between-population heterogeneity of odds ratios by the Breslow-Day test did not show significant differences among the cohorts (P_het =0.26).

Expression quantitative trait loci in this region
Expression quantitative trait loci (eQTL) associations between SNP rs2298428 and UBE2L3 and other genes in this region were closely examined. Two datasets, from Stranger et al. [28] and Fairfax et al. [29], were investigated. In the first study, the researchers examined the correlation of SNPs to gene expression using the lymphoblastoid cell lines (LCLs) of 726 individuals in 8 cohorts from the HapMap3 project. In the second study, the investigators assessed the correlation of SNPs to gene expression in a cell-specific manner using 288 paired, purified primary monocytes and B cells from Caucasians. As shown in Table 5, the genotype of SNP rs2298428 correlated with expression of UBE2L3 in five different populations. (The other three cohorts did not show significant correlation, possibly due to the low frequency of the alternative allele, thus lower power.) The genotypes of the SNP also significantly correlated with the expression of UBE2L3 in B cells and monocytes. The results consistently demonstrated that the risk allele T from rs2298428 is correlated with higher expression of UBE2L3.
The gene expression pattern of UBE2L3 was also examined using a publicly available database, NextBio [30] (Additional file 3). Five independent studies reported increased expression of UBE2L3 from patients with lupus compared with healthy controls in different cell lines. In addition, UBE2L3 was found to have increased expression in a number of other autoimmune diseases, including CD, T1D, SS, PS and PA, using the same  database, indicating that UBE2L3 might be the key player in disease association of this region.

Discussion
In this study, through meta-analysis of two existing GWAS on Han Chinese populations with a total number of 1,659 cases and 3,398 controls matched geographically, we identified SNP rs2298428 as the SNP with the highest association with SLE in the 22q11.21 region (P_meta =2.70E-09). The association of rs2298428 was further supported by replication in three cohorts from Hong Kong, Anhui and Thailand, and the results  improved by two orders of magnitude after joint analysis from the discovery stage and the replication stage (P_all =1.31E-11, odds ratio =1.23). Many GWAS hits were aggregated in this region for different autoimmune diseases, and here we tried to find out whether all the reported SNPs (six SNPs included) were linked to the same causal variant or whether they were derived from independent signals. All of the SNPs showed strong evidence of association with SLE in the current investigation with P_meta <7.27E-05. Stepwise logistic regression and conditional logistic regression were performed to examine the independence of these SNPs. The results supported the notion that rs2298428 exhibited the strongest association with SLE. According to the LD pattern, SNP rs463426 is relatively independent from the other five SNPs (Figure 1). However, we were unable to find evidence of independence based on the presently reported results (Figure 2). This might be due to the fact that the exploration of independently contributing variants from this region is based mainly on the meta-analysis data, which might not have enough power to detect multiple independent signals. The five SNPs reported for different autoimmune diseases are located in the same LD block in Chinese population, and their association may be derived from the same casual variant. These cross-phenotype associations in this region highlighted the shared genetic involvement in autoimmune diseases.
In addition, to identify how this region might influence susceptibility to SLE and other autoimmune diseases, we investigated the potential biological function of the gene. eQTL analysis is an important approach in detecting functional mechanisms underlying association by testing whether identified variants may lead to variations in mRNA expression of nearby genes. Using publicly available eQTL datasets, the SNPs in the 22q11.21 region were analyzed. All the results pointed to increased expression of UBE2L3 as the mechanism for association with SLE. UBE2L3 encodes a member of the E2 ubiquitinconjugating enzyme family. This enzyme was demonstrated to participate in the ubiquitination of p53, c-Fos and the nuclear factor κB precursor p105 in vitro [31,32]. There is also evidence showing the interaction between UBE2L3 and RNF125 [33]. RNF125 is reported as a negative regulator of type I interferon (IFN) signaling. It is well known that patients with SLE have elevated serum levels of type I IFN [34] and that these increased levels correlate with disease activity and severity [35]. Among numerous immunologic alterations present in patients with lupus, the type I IFN system is thought to play a pivotal role in pathogenesis [36][37][38], which points to a possible role of UBE2L3. However, the exact mechanism of UBE2L3 is still not fully understood.

Conclusions
Focusing on the SNPs in 22q11.21 region with strong evidence of being associated with SLE in previous work, we have identified one more novel susceptibility variant showing the most significant genetic contribution for SLE via meta-analysis and further replication in independent cohorts. The putative susceptibility gene, UBE2L3, is suggested to be related to the type I IFN signaling pathway in SLE pathogenesis. Our findings may shed light on the shared biological mechanisms between different diseases with immunological components.

Additional files
Additional file 1: Detailed information of the sample included in this study.

Competing interests
The authors declare that they have no competing interests.
Authors' contributions YLL conceived and designed the experiments and revised the manuscript. WY designed the experiments and revised the manuscript. YZ performed the experiments, analyzed the data and drafted the manuscript. JY and JZ performed the experiments and helped to revise the manuscript. YFW analyzed the data and drafted the manuscript. LZ performed the GWAS data quality control and participated in drafting the manuscript. JJS analyzed the