Deep pockets are not necessarily a good thing in membranous nephropathy: evidence for a modifier allele

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recipients on 3 continents. Am J Transplant. 2018;18:1914–1923. 9. Kidney Disease: Improving Global Outcomes (KDIGO) Transplant Work Group. KDIGO clinical practice guideline for the care of kidney transplant recipients. Am J Transplant. 2009;9(suppl 3):S1–S155.

Deep pockets are not necessarily a good thing in membranous nephropathy: evidence for a modifier allele Laurence H. Beck Jr.1 and David J. Salant1 Risk (or susceptibility) alleles for primary membranous nephropathy exist within the DQ and DR loci of the human leukocyte antigen (HLA) region of chromosome 6. The discussed study identifies a novel allele, HLA DRB1*1502, in a Han Chinese cohort that acts as a modifier allele by associating not with the phenotype of membranous nephropathy, but rather with the severity of disease. This commentary addresses the potential biologic aspects of these new data. Kidney International (2018) 94, 855–857; https://doi.org/10.1016/j.kint.2018.07.010 Copyright ª 2018, International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

see clinical investigation on page 974

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rimary (or idiopathic) membranous nephropathy (PMN) is an organ-specific autoimmune disease mediated by the humoral aspect of the adaptive immune system. Autoantibodies target 1 or more epitopes within a protein (phospholipase A2 receptor [PLA2R] or THSD7A) expressed on the basal surface of podocytes to form antigen-antibody immune complexes, which ultimately lead to the activation of the complement system, subsequent podocyte injury, loss of slit diaphragms, and loss of massive amounts of protein into the urine. As is the case with many autoimmune diseases, a genetic association with the

1 Department of Medicine, Renal Section, Boston University School of Medicine and Boston Medical Center, Boston, Massachusetts, USA

Correspondence: Laurence H. Beck, Jr., Renal Section, Department of Medicine, Boston University School of Medicine and Boston Medical Center, 650 Albany Street, Room 504, Boston, Massachusetts 02118, USA. E-mail: [email protected] Kidney International (2018) 94, 849–860

human leukocyte antigen (HLA) region of chromosome 6 has been identified. The association of PMN with specific class II HLA serotypes or alleles has long been known. This association took a leap forward with the publication of a genome-wide association study by Stanescu et al. that showed, in a relatively small cohort of Europeans with PMN, a very significant signal within an intronic region of HLA-DQA1 that associated with disease.1 Importantly, this risk allele showed a strong genetic interaction with a risk allele within the PLA2R1 gene locus, suggesting a potential interaction at a biological level. Subsequent studies have confirmed this association in additional cohorts, typically limited to Asian or Caucasian ethnicity (reviewed in Mladkova and Kiryluk2 and Beck3), and have also identified other class II alleles, primarily in the DRB locus, that may better explain the association. Class II HLA molecules, expressed by B cells and other antigen presenting

cells, are encoded by HLA-D gene products (DR, DQ, and DP). Two gene products from each locus, an alpha (A) and a beta (B) chain, assemble to form the mature, antigen-presenting molecule. Although both chains contribute to the peptide-binding groove, most of the polymorphisms that govern peptide-binding interactions within the groove are within the beta chain. The incredible diversity at these gene loci and the molecular specificity defined by the particular amino acids lining the peptide-binding groove allow any one individual a restricted repertoire of peptides that can be successfully presented to a T cell bearing the appropriate T cell receptor. At a species level, however, the ability to respond to a known or emerging microbe is nearly infinite, ensuring the survival of the species as a whole. When the same diversity in HLA alleles is considered in the context of autoimmunity and presentation of intrinsic (self) antigens to the immune system, it is not surprising that there would be individual or familial susceptibility to certain autoimmune diseases that manifest as a complex (non-Mendelian) genetic trait. In contrast to the studies of patients with European ancestry, recent work in Han Chinese cohorts of PMN has shown that specific alleles within the DRB locus represent highly significant susceptibility alleles for PMN in this population.4,5 Cui et al. have implicated DRB1*1501 and DRB1*0301 as major independent risk alleles for PMN associated with circulating anti-PLA2R antibodies,4 whereas Le et al. have shown the additional and independent importance of DRB3*0202,5 a second DRB allele present in many individuals that was not assayed in the former study. Cui et al., using predictive algorithms, identified peptide sequences from PLA2R that would best fit into the binding groove of DRB1*1501. Notably, these sequences were located in the cysteine-rich, C-type lectin-like domain 1, and C-type lectin-like domain 7 domains of PLA2R, which are exactly the domains in which humoral (B-cell) epitopes have been shown to exist.6 The authors speculate that the genetic 855

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T-cell

Figure 1 | A potential mechanism to relate risk and modifier human leukocyte antigen (HLA) alleles to disease phenotype and expression. The biologic effects of risk alleles (HLA DRB1*1501 and *0301) and modifier alleles (HLA DRB1*1502) on the overall phenotype and expression of primary, phospholipase A2 receptor (PLA2R)–associated membranous nephropathy are speculative and are represented in this schematic as an “immunologic black box.” Specific risk alleles at the HLA locus may allow antigen-presenting cells (APC), under the proper environmental cues, to effectively present linear peptides of PLA2R to helper T cells, that then drive the proliferation of B cells leading to the production of antibodies to distinct epitopes of PLA2R, such as the immunodominant epitope within the N-terminal cysteine-rich region (CysR) or to more C-terminal epitopes such as the first or seventh (not shown) C-type lectin-like domain. The autoantibodies generated by these processes allow formation of antibody-antigen complexes beneath the podocyte, leading to the overall phenotype of membranous nephropathy (MN). Wang and colleagues have demonstrated a modifier allele, also in the HLA locus and differing from HLA DRB1*1501 by only 1 amino acid residue, that is not a risk allele per se, but rather modifies the phenotype by associating with decreased estimated glomerular filtration rate and possibly higher levels of anti-PLA2R. This modifier HLA class II protein may present additional peptides from PLA2R, or from other antigens, leading to more severe tissue injury and causing further anti-PLA2R either directly or through increased tissue injury and a vicious cycle of antigen presentation. MHC, major histocompatibility complex.

susceptibility to PMN is conferred by these particular HLA class II molecules by allowing presentation of 1 or more PLA2R epitopes to T helper cells, which then promote the proliferation of B cells and production of anti-PLA2R antibodies. The additional DRB3*0202 risk allele identified by Le et al. shares a haplotype with DRB1*0301; however, they did not examine whether specific PLA2R peptides might be presented by this additional DRB allele. Because both of these studies were conducted in Han Chinese cohorts, the implications for Caucasian or other ethnic groups of PMN are unclear. The current study by Wang et al.7 (2018) published in this issue of Kidney International adds a new dimension to the field. As noted above, this group had previously shown that HLA DRB1*1501, as well as the DRB1*0301 allele, are associated with PMN (although only 67% of the cohort was definitely shown to have PLA2R856

associated disease, so the significance of their results might have been diluted by the inclusion of an unknown fraction of patients with non-PLA2R–associated membranous nephropathy).4 These risk alleles associate with the overall phenotype of PMN, meaning that they are statistically more frequent in the disease cohort than in healthy controls. In the current analysis, using the constellation of HLA alleles identified within their larger cohort, they next ask, using a similar association analysis, whether any of these individual HLA alleles associate with clinical manifestations of the general membranous nephropathy phenotype: for example, age at presentation, gender, degree of proteinuria, anti-PLA2R level, and importantly, estimated glomerular filtration rate at presentation or last follow-up. The authors perform statistical association analyses with 93 HLA alleles and 8 clinical parameters and find that there are several alleles that are

significantly associated with 1 or more clinical parameters by univariate analysis. One of these, DRB1*1502 (found at 2.9% frequency in both PMN and healthy controls), is associated with estimated glomerular filtration rate at baseline and last follow-up in the PMN cohort. Because the allele is found at the same frequency in both PMN and controls, it is not a risk allele for PMN per se, but rather modifies the phenotype (“modifier” allele). It should be noted that, although DRB1*1502 was associated with progression to end-stage renal disease in univariate analysis, the effect disappeared on multivariate analysis, where only the absence of remission remained significantly associated. The authors also ruled out an effect of coinheritance of the DRB1*1502 with other susceptibility alleles, because there was low linkage disequilibrium between this proposed modifier allele and the known susceptibility alleles. There were gene-gene Kidney International (2018) 94, 849–860

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interactions such that individuals carrying both DRB1*1502 and 0301 had increased risk for higher anti-PLA2R levels, whereas having both DRB1*1502 and 1501 did not. Intriguingly, the DRB1*0301 allele, which itself was independently associated with higher antiPLA2R levels, was not associated with adverse renal function or outcome, suggesting that higher autoantibody levels are not necessarily responsible for the worsened renal outcomes. It is important to understand the difference between a genetic locus/allele being associated with a particular phenotype (in this case, primary membranous nephropathy) versus with the expression or variability of that phenotype. Within the overall phenotype of PMN, patients will differ in age of onset, proteinuria at time of biopsy, anti-PLA2R level, and renal function. What determines such variability may have a genetic basis but not necessarily confer susceptibility to PMN. For example, if an allele associates with one of these clinical parameters it could function as a “modifier” allele and alter the age of onset or severity of PMN without altering the probability that an individual will get the disease. The interested reader can find a thorough discussion of the modifier gene subject in an excellent review of the topic,8 also referenced by Wang et al. HLA DRB1*1501 (risk allele) and HLA DRB1*1502 (modifier allele) differ by a single amino acid (V86G), located within the peptide-binding groove. Molecular modeling predicts that the DRB1*1502 allele creates a deeper pocket that would accommodate a longer amino acid side chain of the antigenic peptide. The authors again use predictive algorithms to predict which peptides from human PLA2R would bind to DRB1*1502 with the highest affinity, and identify 4 potential linear sequences within the first, third, and fourth C-type lectin-like domains, raising the possibility that this allele

Kidney International (2018) 94, 849–860

modifies the disease phenotype by facilitating epitope spreading. In contrast to monogenic diseases in which the penetrance or expressivity of a single gene variant, for example, a mutation in PKD1, may be modified by 1 or more interacting genes to affect the age of onset or severity of disease in different family members harboring the same gene mutation, the effect of modifying genes in complex conditions like autoimmunity is more difficult to comprehend. In the context of PMN, if *1502 is indeed a modifier gene, the question arises as to what gene(s) it is modifying and what would be the unmodified phenotype of the target gene. At this point, the suggestion that the observed associations of HLA DRB1*1502, for example, a difference in anti-PLA2R levels and worsened kidney function, are due to presentation of additional PLA2R epitopes is merely a speculation. Although it presents an attractive hypothesis, it is also conceivable that this particular class II molecule furthers injury by presenting a distinct antigen from the glomerulus or tubulointerstitium, and that the increased anti-PLA2R levels are instead due to further tissue damage via these additional autoantibodies (see Murtas et al.9) and enhanced presentation of PLA2R via the previously identified HLA susceptibility alleles (Figure 1). Because only 15 patients carried the DRB1*1502 allele, larger studies are necessary to confirm the modifier effect of this allele in the Chinese population and to look for similar effects in Caucasians, in whom the association of HLA-DQA1*0501-DRB1*0301 haplotype has a stronger risk association with PMN than does the DRB1*1501 allele. It would also be of immense interest to study the effect of risk and modifier HLAs on the presence of epitope spreading beyond the immunodominant epitope within the cysteine-rich region. Studies using recombinant tetramers of the specific class II HLA proteins in question to bind and

stimulate patient-derived T cells could address whether patients possess T cells that are stimulated in the presence of PLA2R peptide, which would support biological plausibility. Wang et al. are to be credited with adding another piece to the as yet incomplete genetic puzzle that will eventually explain the remarkably strong interaction between PLA2R1 and class II HLA alleles, both at the susceptibility and modifier level. Although sequencing and genotyping of PLA2R1 have been quite unrevealing thus far, it seems that the answers may well come from more precise investigations addressing PLA2R B and T cell epitope mapping and class II HLA binding. DISCLOSURE

LHB and DJS are co-inventors on the patent “Diagnostics for membranous nephropathy.” REFERENCES 1. Stanescu HC, Arcos-Burgos M, Medlar A, et al. Risk HLA-DQA1 and PLA2R1 alleles in idiopathic membranous nephropathy. N Engl J Med. 2011;364:616–626. 2. Mladkova N, Kiryluk K. Genetic complexities of the HLA region and idiopathic membranous nephropathy. J Am Soc Nephrol. 2017;28: 1331–1334. 3. Beck LH. PLA2R and THSD7A: disparate paths to the same disease? J Am Soc Nephrol. 2017;28:2579–2589. 4. Cui Z, Xie LJ, Chen FJ, et al. MHC class II risk alleles and amino acid residues in idiopathic membranous nephropathy. J Am Soc Nephrol. 2017;28:1651–1664. 5. Le WB, Shi JS, Zhang T, et al. HLA-DRB1*15:01 and HLA-DRB3*02:02 in PLA2R-related membranous nephropathy. J Am Soc Nephrol. 2017;28:1642–1650. 6. Seitz-Polski B, Dolla G, Payre C, et al. Epitope spreading of autoantibody response to PLA2R associates with poor prognosis in membranous nephropathy. J Am Soc Nephrol. 2016;27:1517–1533. 7. Wang H-y, Cui Z, Xie L-j, et al. HLA class II alleles differing by a single amino acid associate with clinical phenotype and outcome in patients with primary membranous nephropathy. Kidney Int. 2018;94:974–982. 8. Riordan JD, Nadeau JH. From peas to disease: modifier genes, network resilience, and the genetics of health. Am J Hum Genet. 2017;101: 177–191. 9. Murtas C, Bruschi M, Candiano G, et al. Coexistence of different circulating anti-podocyte antibodies in membranous nephropathy. Clin J Am Soc Nephrol. 2012;7:1394–1400.

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