Genetic Complexities of the HLA Region and Idiopathic Membranous Nephropathy
Authors: Nikol Mladkova, Krzysztof Kiryluk
Journal: Journal of the American Society of Nephrology (2017)

The first human disease associations of the major histocompatibility (MHC) genes were reported over half a century ago, providing important insights into the genetic basis of autoimmunity. Since then, numerous association studies have examined the effect of specific MHC variants, also referred to as the human leukocyte antigen (HLA) alleles, on the risk of autoimmune conditions. These initial studies, however, were frequently flawed by low resolution of HLA typing, small cohort sizes, and spurious associations due to the extended linkage disequilibrium across this region. The modern era of genome-wide association studies (GWAS) provided robust validation of the pivotal role of this region in genetic susceptibility to many autoimmune and inflammatory disorders, including SLE, multiple sclerosis, inflammatory bowel disease, type 1 diabetes, rheumatoid arthritis, and many others. In the kidney field, GWAS provided strong evidence for HLA involvement in susceptibility to common forms of immune-mediated glomerular diseases, such as IgA nephropathy (IgAN)1–4 and membranous nephropathy (MN).5 Suggestive HLA signals have also been reported in recent studies of steroid-sensitive nephrotic syndrome6 and lupus nephritis.7 The described effects of HLA are consistently large, with per allele disease risk increasing from 50% to >400% above baseline, highlighting the critical role of HLA in the pathogenesis of these disorders. The HLA region resides on chromosome 6p21.3 and is among the most gene-dense portions of DNA, with gene products ranging from antigen-binding molecules and receptors to signaling factors. This region is also among the most polymorphic in humans, with 21 core HLA genes coding for proteins critical for the human immune response to infectious pathogens. The region can be subdivided into classic class I, II, and III and extended class I and II. Class I encompasses HLA-A, -B, and -C that function as presenters of peptides to cytotoxic T cells, and class II consists of HLA-DR and HLA-DQ molecules that present epitopes to CD4-positive T cells. The currently known number of unique HLA alleles surpasses 8000 and 2400 for class I and II, respectively, requiring a precise hierarchical nomenclature to discern a specific allele at a given locus: two-digit resolutions differentiate between allele groups, four-digit accuracy between specific alleles, and six- and eight-digit resolutions discern between exonic and intronic HLA variants, respectively. Owning to the complex structure and a high level of linkage disequilibrium in the HLA locus, genetic dissection of specific disease-causing alleles has been challenging. Most frequently, a complex pattern of associations is observed with multiple independent haplotypes conferring risk or protection, such as in type 1 diabetes or IgAN. Moreover, HLA alleles often have pleiotropic effects including opposed effects on the susceptibility to different conditions. For example, the same allele that increases the risk of IgAN may also increase the risk of type 1 diabetes but protect against SLE.4 Idiopathic MN provides another fascinating example of strong genetic effects at the HLA locus. First described as a distinct form of glomerular disease in 1957, MN and its pathogenesis has been the subject of intensive research efforts. Through a series of experiments and animal models, including Heymann nephritis, it has been established that MN represents a kidney-specific antigen-driven autoimmune disease. However, the identity of the human antigen had long been elusive. In 2009, the M-type phospholipase A2 receptor (PLA2R) had finally been identified as the key antigen in the pathogenesis of MN in humans.8 An additional antigen, thrombospondin type-1 domain-containing 7A (THSD7A), was subsequently identified, explaining a smaller fraction of patients with idiopathic MN who were negative for anti-PLA2R antibodies.9 The first report of genetic contributions of the HLA locus to the risk of MN was published in 1979.10 This report was followed by a series of additional HLA association studies, building the case for the involvement of this locus. The first and only GWAS for MN carried out in a relatively small cohorts of French, Dutch, and British patients resulted in an astonishing discovery: two disease association peaks with large effects dominated the Manhattan plot, one at the HLA locus and another at the PLA2R1 locus.5 The PLA2R1 locus may influence the immunogenicity of PLA2R molecule (the primary autoantigen in MN), whereas the production of anti-PLA2R autoantibodies may be further amplified in individuals with permissive MHC haplotypes. Moreover, a strong genetic interaction between these two loci was described. Similar genetic interactions between variants in antigen-presenting and antigen-encoding genes have been reported in only a handful of other autoimmune diseases, mainly in psoriasis11 and type 1 diabetes.12 Although the above GWAS findings have now been replicated in multiple cohorts,13–17 the loci have not yet been fine-mapped, and the exact mechanisms underlying these associations remain unknown. Notably, the original GWAS in Europeans lacked conditional analyses of the region or imputation of classical alleles, thus the precise number of independently associated haplotypes or the identity of specific causal variants had not been defined.18 Two studies in this issue of the Journal of the American Society of Nephrology provide novel insights into the role of specific HLA alleles in idiopathic MN. The first study by Cui et al.19 performed four-digit resolution typing of HLA-DRB1, DQA1, DQB1, and DPB1 genes followed by case-control association analysis in 261 patients and 599 healthy controls, and pointed to two classical alleles, DRB1*1501 and DRB1*0301, with highly significant independent effects on the risk of idiopathic MN among Han Chinese. The second study by Le et al. performed targeted high-throughput sequencing and four-digit resolution HLA analysis in 99 anti-PLA2R–positive MN cases and 100 healthy controls.20 This study confirmed the association of DRB1*1501 with anti-PLA2R–positive MN, and suggested DRB3*0202 as the second independent risk factor, subsequently replicating both of these associations in an independent cohort of 293 cases 285 controls. In the first study, the DRB1*1501 and DRB1*0301 alleles had large, independent, and genome-wide significant effects with allelic odds ratios of 4.65 and 3.96, respectively. These alleles were also significantly associated with circulating anti-PLA2R antibodies. Similar to single-nucleotide polymorphisms in the original GWAS, both HLA alleles exhibited statistical interaction with the PLA2R1 variant rs4664308. Notably, the homozygosity for risk alleles at the PLA2R1 locus combined with DRB1*1501 or DRB1*0301 positivity conferred up to 30-fold increased risk of kidney disease. This synergistic effect is suggestive of a physical interaction between DRβ1 molecules and the PLA2R epitope(s) during the process of antigen presentation. The analysis of individual amino acid substitutions within MHC proteins further narrowed down the search to the amino acid positions 13 and 71 within the MHC-DRβ1 chain. Both of these positions participate in the formation of the fourth peptide-binding pocket of MHC-DRβ1 chain. Furthermore, in silico modeling of PLA2R peptides presented by this pocket revealed several candidate epitopes that now require experimental validation. Interestingly, modeling sequence variants of PLA2R1 had little effect on T cell epitope prediction, suggesting that coding variants in PLA2R1 are less likely to alter the immunogenicity of its gene product. In comparison to the first study, Le et al. performed a more comprehensive analysis by targeted high-throughput sequencing of all HLA genes in the region, including class I and class II genes. Despite smaller sample size of the discovery cohort, additional power was gained by stratifying the analysis on the basis of the PLA2R antibody status. Significant associations of two independent alleles, DRB1*1501 and DRB3*0202, were detected in the discovery phase and replicated in additional case-control cohorts. Notably, the effects were significantly larger compared with the first study, with allelic odds ratios of 24.9 and 17.7, respectively. These larger effects are likely explained by differences in the ascertainment of cases: the first study included all cases of idiopathic MN but the second study used "PLA2R-related MN," defined strictly by anti-PLA2R seropositivity combined with positive glomerular PLA2R antigen staining. Notably, DRB3 alleles were not typed in the first study, but DRB3*0202 resides on the same haplotype as DRB1*0301, thus the results of both studies are technically in full agreement. In fact, the second study suggests that DRB3*0202 is the most likely culprit allele explaining the reported signal at DRB1*0301, considering its stronger statistical significance and the conditional analysis that removes any residual signal at DRB1*0301 after accounting for both DRB1*1501 and DRB3*0202. Interestingly, secondary genotype–phenotype correlation analyses also revealed that DRB1*1501 was strongly associated with an earlier age at kidney disease onset. Among PLA2R-positive MN cases, patients carrying the DRB1*1501 risk allele presented at a median of 35 years of age compared with the median age of 50 for all other patients. Although these studies present important novel findings, one needs to be mindful of several limitations, including the relatively small sample size of both studies, study cohorts restricted to Han Chinese, and analysis of HLA alleles performed without accounting for genetic rather than self-reported ancestry. Moreover, the study by Cui et al. appears to have missed an important association of DRB3*0202 by not accounting for the full genomic context of the HLA region. Follow-up studies will clearly require a large-scale comprehensive sequencing effort involving the entire locus, also including noncoding segments that may regulate HLA gene expression. Moreover, because the frequencies and effects of individual HLA alleles may vary greatly between populations, such studies would be ideally performed in cohorts of diverse ancestries, including representative cohorts of Asian, European, and African ancestry. In summary, although the above studies define two critical HLA haplotypes conveying high risk of MN in Han Chinese, it is not yet clear if these haplotypes are equally important in other populations, or if additional variants were missed because of power or coverage limitations. With respect to future studies, an adequately powered multiethnic GWAS combined with sequencing of the HLA region is still greatly needed for MN, not only to refine the HLA signal but also to potentially define additional loci outside of the HLA and PLA2R1 regions. Stratifying cases by PLA2R antibody status clearly reduces heterogeneity and can potentially enhance future discovery efforts. Moreover, it is essential to explore the precise nature of the interaction between PLA2R1 and HLA alleles across different ancestries, and systematically evaluate its role in the context of potential environmental disease triggers. Disclosures None.