Genome-wide pathway analysis identifies VEGF pathway association with oral ulceration in systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a disabling multisystem rheumatic disease with substantial epidemiological variation [1]. SLE is characterized by the dysregulation of the immune system and high phenotypical diversity [2]. This phenotypical heterogeneity includes a wide range of clinical manifestations that are exemplified by the current use of multiple clinical phenotypes as criteria to diagnose the disease [3]. So far, however, little is known about the causes of this phenotypic variation. Understanding the molecular mechanisms associated with the pathogenesis of SLE phenotypes could therefore be of high relevance to develop more efficient therapeutic approaches and preventive strategies.

SLE is characterized by a strong genetic component, with a sibling recurrence rate (λ_s) of 8–29 and estimated heritability of approximately 66% [4]. To date, nine genome-wide association studies (GWAS) of SLE risk have been performed in European and Asian populations [5]. Together these studies have led to the identification of >40 loci associated with SLE susceptibility. Despite this extraordinary success, there is still a lack of understanding of the genetic variation that is relevant for the development of specific phenotypes within the disease. There is evidence, however, that the main clinical phenotypes in SLE aggregate in families [6], suggesting a genetic basis underlying disease heterogeneity.

To date, only a few candidate gene studies have been performed to uncover the genetics of clinical heterogeneity in SLE [7]. These studies have identified immunity-related genes associated with clinically relevant SLE phenotypes [8]. From these, the most significant associations have been detected between renal disorder and genetic variation in the ITGAM and STAT4 genes, which have been also associated with discoid rash and oral ulceration, respectively [9, 10]. Other significant findings include the association between renal disorder and TNFSF4, malar rash and FCGR2A and hematological disorder and variation in the IL21 gene. So far, however, the genetic component for most SLE phenotypes has been only partially explained. Therefore, the analysis of genetic variation at a genome-wide scale is needed to identify additional variation associated with SLE clinical heterogeneity.

Complex traits like disease risk or clinical phenotypes have been shown to be caused by multiple genes of small effect size [11]. The identification of these small-effect genes is currently one of the major challenges in the characterization of the genetic background for disease phenotypes [12]. Importantly, single-marker GWAS do not allow the identification of genetic variants with small effect sizes, unless extremely large sample sizes are used [13]. In this common type of GWAS, a large number of markers are tested for association and, consequently, stringent significance thresholds are applied, which makes the identification of small-effect variants very difficult [14]. In addition, single-marker GWAS ignore the joint contribution of multiple genes that act coordinately in the same biological process [15]. The characterization of the genetic basis of many complex traits will therefore require the development of new powerful methods that are able to leverage biological knowledge and efficiently integrate the evidence from multiple loci with moderate to small effect sizes.

Recently, novel statistical methodologies that are able to test genetic risk associations at the pathway level have been developed [16]. Pathway-based approaches test whether sets of functionally related genes are jointly associated with a particular phenotype [17]. This methodology strongly reduces the number of association tests and, therefore, it can substantially increase the power to identify new genetic variation compared to single-marker GWAS [18]. The genome-wide pathway analysis (GWPA) has been recently used to characterize the genetic basis of several complex diseases like cancer [19]. Very recently, using the GWPA approach we have identified new genetic variation associated with psoriasis, an autoimmune disease of the skin [20]. This result confirms the utility of GWPA in the study of the genetics of autoimmune diseases.

To gain a better understanding of the genetic basis underlying phenotype heterogeneity in SLE we have performed, for the first time, a GWAS of clinical phenotypes using the GWPA approach. In this study we have analyzed a discovery cohort of 482 SLE patients of European ancestry to determine the association between 798 reference biological pathways and the main clinical phenotypes of SLE represented that are used as diagnosis criteria. Using an independent cohort of 425 SLE patients from the same ancestry, we have then performed a validation study of the most significant genetic pathways. Based on these results, we have performed an in silico validation analysis to evaluate the functional impact of drugs commonly used to treat the associated phenotype. Our findings provide new insights into the biological mechanisms associated with clinical phenotypes of SLE.

One of the major challenges in the pathogenesis of SLE is to understand the biological mechanisms responsible for its phenotypic heterogeneity. Although single-marker GWAS have successfully identified a large number of genetic variants associated with SLE risk, the genetic basis of SLE phenotypes has so far been analyzed only in a few candidate gene studies. In order to identify new genetic variation, we have here performed the first GWAS on SLE phenotypes using a pathway-based approach. Using a discovery cohort of individuals with European ancestry and a validation cohort with the same ancestry, we have identified and validated the association between VEGF genetic pathway and oral ulcers, a common manifestation of SLE. The results of this study provide new insights into the genetic basis and the biological mechanisms associated with the clinical heterogeneity of SLE.

The VEGF pathway is a network of genes that are involved in the transduction of different intracellular signals and act coordinately to modulate inflammatory and angiogenic processes [35, 36]. The dysregulation of angiogenesis has been described as an important biological mechanism in the pathogenesis of SLE [37]. In particular, there is growing evidence that angiogenesis is also involved in the development of skin manifestations in SLE patients [38, 39]. The serum levels of VEGFA protein itself have been suggested as a useful marker for disease activity monitoring in SLE patients [40]. Importantly, the serum levels of VEGFA have also been found to be significantly elevated in SLE patients with cutaneous manifestations [41]. Despite this clinical evidence, the VEGF pathway had not been previously associated with SLE at the genetic level. Our study provides the first evidence of a genetic association between the VEGF pathway and oral ulceration in SLE.

Oral ulcers are frequently chronic mucocutaneous lesions that affect up to 54% of patients with SLE [42, 43]. This clinical manifestation is characterized by high angiogenic activity and a loss of the epithelial and connective tissue in the oral mucosa [44, 45]. Accordingly, anti-angiogenic therapies like thalidomide have been suggested to promote oral ulcer healing and to control ulcer recurrence [46, 47]. From a clinical perspective, oral ulceration has been associated with an increase in the disease activity and a worse prognosis in SLE [48, 49]. The early detection of oral ulcers is therefore highly valuable as it contributes to an earlier diagnosis of SLE and, consequently, to faster initiation of treatment.

The genetic association identified in this study is consistent with previous evidence from other ulcer-related diseases like Behçet disease (BD), recurrent aphtosus ulceration (RAU) and gastroduodenal ulcers. BD is an inflammatory disorder characterized by an extremely high frequency of oral ulcers (>95%) [50]. Clinical evidence suggests that VEGF cytokine could be directly implicated in the formation of oral ulcers in BD [51]. In RAU, the most common oral mucosal disease, the salivary levels of VEGF have been also associated with oral ulceration [52]. Finally, genetic variation in the VEGF gene has been also associated with the risk of developing gastroduodenal ulcers [53]. Evidence from these studies implicates angiogenesis in the pathophysiological development of oral ulcers, at both the genetic and at the functional level. Accordingly, the genes in the VEGF pathway are strong candidates for susceptibility in diseases with a high prevalence of oral ulcers like BD or RAU. Future studies aimed at testing the association between VEGF pathway genes and these diseases are therefore warranted.

Topical steroid and non-steroid immunotherapies have been successfully used for the treatment of the cutaneous manifestations in SLE. However, topical immunotherapies are not exempt from side effects, including skin atrophy or telangiectasias with corticosteroids and the exacerbation of the inflammatory processes with non-steroid immunotherapies like imiquimod [32]. In this study, we have demonstrated that topical steroid and non-steroid immunomodulators significantly affect the expression of the VEGF pathway genes. The results of this in silico analysis indicate that the VEGF genetic pathway could be a key mediator of the benefits of topical immunotherapy to reduce oral ulceration. Therefore, our findings suggest that the VEGF pathway is a relevant source of new drug targets for oral ulceration that could allow more specific treatment while reducing the undesirable side effects of current therapies. In order to confirm the VEGF pathway as new source for drug discovery in the treatment of SLE oral ulceration, further prospective studies using oral ulcer samples from SLE patients are clearly needed.

In the present study, we have also identified and validated the association between SLE risk locus PTPN22 and the production of antinuclear antibodies and with the presence of a hematologic disorder. It is the first time that this coding SNP (rs2476601) has been associated with the development of hematologic disease in SLE. A previous study reported a non-significant trend for association between PTPN22 and antinuclear antibody positivity [54]. The results from our study provide the evidence to confirm the association between this susceptibility gene and the production of a common autoantibody in SLE. Also, the previously identified association between genetic variation in the TNFSF4 gene and renal disorder was replicated in the discovery cohort [9]. Conversely, the reported association between renal disorder and ITGAM and STAT4 genes was not replicated. The renal disorder phenotype encompasses different clinical manifestations (e.g. persistent proteinuria or cellular casts) and, therefore, differences in frequencies in any of these sub-phenotypes could have prevented the replication. Additional studies performing specific analysis of association for each renal disease subtype could help to further refine this genetic association. Oral ulceration was also previously found to be associated with variation in STAT4. This association was not replicated in the present single-marker analysis. The lack of replication could be explained by the comparatively smaller sample size of our study cohorts and by the small effect size reported for the association (OR ~ 1.12). For example, in the European cohort, STAT4 association was not statistically significant despite analyzing >4,000 individuals [9]. Finally, after combining both patient cohorts, we also found seven other SLE risk loci - IL10, IKZF2, MHC class III, UHRF1BP1, ETS1-FLI1, SH2B3 and SLC15A4 - to be nominally associated with different phenotypes. These also represent new genetic associations with SLE heterogeneity. Further studies using independent cohorts of phenotypically well-characterized SLE patients like the present one are needed to corroborate these associations.

GWPA represents a new and powerful approach to identify genetic variation associated with complex phenotypes. However, this study has limitations. First, the sample size of the discovery and replication cohorts is moderate compared to recent case-control GWAS and this could have led to missed pathway associations with SLE clinical phenotypes. Second, the statistical power to detect significant associations is lower for those clinical phenotypes with more extreme frequencies (e.g. 7% of SLE patients have a neurologic disorder). Therefore, larger cohorts of well-characterized SLE patients will be needed to identify additional genetic variation associated with clinical phenotypes. Finally, the GWPA methodology also has intrinsic limitations, mainly related to the current knowledge of biological processes and subsequent definition of the pathways [55]. For many human genes, the functional annotation is far from complete, which precludes their mapping to reference biological pathways. Also, genomic variation located far from the transcribed region itself could be relevant for the regulation of the gene expression. Conversely, variants within genes could be influencing the activity of other distant genes or genes in other chromosomes (i.e. trans-regulation). With the increase in knowledge of genomic regulation [56], the mapping of SNPs to their functionally related genes will clearly improve. Consequently, the integration of this knowledge into GWPA will likely increase the power of this approach to identify new pathways associated with human diseases.

In conclusion, we have performed, for the first time, a genome-wide association analysis to identify genetic risk factors for the main phenotypes in SLE. To do this, we have used a pathway-based analysis approach. Using this approach, we have identified and validated the association between the VEGF genetic pathway and the presence of oral ulcers in SLE. These findings show the existence of a genetic basis underlying SLE heterogeneity that is independent from the genetic component associated with disease risk. The results of the present study could contribute to the development of more efficient therapies to treat cutaneous manifestations of SLE in the near future.