BASIC RESEARCH www.jasn.org Large-Scale Whole-Genome Sequencing Reveals the Genetic Architecture of Primary Membranoproliferative GN and C3 Glomerulopathy Adam P. Levine,1 Melanie M.Y. Chan,1 Omid Sadeghi-Alavijeh,1 Edwin K.S. Wong,2,3,4 H. Terence Cook,5 Sofie Ashford,6 Keren Carss,6,7 Martin T. Christian,8 Matthew Hall,9 Claire Louise Harris ,3 Paul McAlinden,2 Kevin J. Marchbank ,3,4 Stephen D. Marks,10 Heather Maxwell,11 Karyn Megy,6,7 Christopher J. Penkett,6,7 Monika Mozere,1 Kathleen E. Stirrups,6,7 Salih Tuna,6,7 Julie Wessels,12 Deborah Whitehorn,6,7 MPGN/DDD/C3 Glomerulopathy Rare Disease Group,14 NIHR BioResource,6 Sally A. Johnson,3,4,13 and Daniel P. Gale 1 Due to the number of contributing authors, the affiliations are listed at the end of this article. ABSTRACT Background Primary membranoproliferative GN, including complement 3 (C3) glomerulopathy, is a rare, untreatable kidney disease characterized by glomerular complement deposition. Complement gene mu- tations can cause familial C3 glomerulopathy, and studies have reported rare variants in complement genes in nonfamilial primary membranoproliferative GN. Methods We analyzed whole-genome sequence data from 165 primary membranoproliferative GN cases and 10,250 individuals without the condition (controls) as part of the National Institutes of Health Research BioResource–Rare Diseases Study. We examined copy number, rare, and common variants. Results Our analysis included 146 primary membranoproliferative GN cases and 6442 controls who were unrelated and of European ancestry. We observed no significant enrichment of rare variants in candidate genes (genes encoding components of the complement alternative pathway and other genes associated with the related disease atypical hemolytic uremic syndrome; 6.8% in cases versus 5.9% in controls) or exome-wide. However, a significant common variant locus was identified at 6p21.32 (rs35406322) 2 (P=3.29310 8; odds ratio [OR], 1.93; 95% confidence interval [95% CI], 1.53 to 2.44), overlapping the HLA locus. Imputation of HLA types mapped this signal to a haplotype incorporating DQA1*05:01, 2 DQB1*02:01, and DRB1*03:01 (P=1.21310 8; OR, 2.19; 95% CI, 1.66 to 2.89). This finding was replicated by analysis of HLA serotypes in 338 individuals with membranoproliferative GN and 15,614 individuals with nonimmune renal failure. Conclusions We found that HLA type, but not rare complement gene variation, is associated with primary membranoproliferative GN. These findings challenge the paradigm of complement gene mutations typically causing primary membranoproliferative GN and implicate an underlying autoimmune mechanism in most cases. JASN 31: 365–373, 2020. doi: https://doi.org/10.1681/ASN.2019040433 Membranoproliferative GN (MPGN) refers to inflam- Received April 30, 2019. Accepted November 3, 2019. matory kidney disease in which there is increased Published online ahead of print. Publication date available at glomerular mesangial matrix and cellularity, thicken- www.jasn.org. ing of the capillary walls, and deposition of immuno- Correspondence: Dr. Daniel P. Gale, UCL Department of Renal globulins (Igs) and/or complement. Such appearances Medicine, Royal Free Hospital, Rowland Hill Street, London NW3 can be seen when the immune system is chronically 2PF, United Kingdom. Email: [email protected] activated; the term primary membranoproliferative Copyright © 2020 by the American Society of Nephrology JASN 31: 365–373, 2020 ISSN : 1046-6673/3102-365 365 BASIC RESEARCH www.jasn.org GN (PMG) refers to those cases in which an underlying infec- Significance Statement tious, neoplastic, or autoimmune disorder is not identified. PMG is divided into immune complex primary membranopro- A minority of cases of primary membranoproliferative GN are fa- liferative GN (IC-PMG), where there is positive immunostain- milial, caused by mutations in complement genes, and nonfamilial ing for Igs and complement, and complement 3 glomerulopathy cases have also been reported to harbor such mutations. To char- acterize the genetic factors contributing to this disease, the authors (C3G), where complement 3 (C3) is the predominant immu- analyzed whole-genome data from 165 cases of primary mem- noprotein deposited. C3G is subdivided by electron microscopic branoproliferative GN and 10,250 control individuals, including 146 appearances into C3 glomerulonephritis (C3GN) and dense cases and 6442 controls who were unrelated and of European an- deposit disease (DDD), in which there is characteristic dense cestry. Although they observed no significant enrichment of rare transformation of the glomerular basement membrane.1 variants in complement genes or exome-wide among cases com- – pared with controls, they found that the HLA locus was strongly PMG is rare, with incidence estimated at 3 5 per million associated with primary membranoproliferative GN, a finding rep- – population.2 4 In most cases the cause is not known but famil- licated in an independent cohort. These findings imply that in most ial C3G has been linked to genomic rearrangements in the cases, primary membranoproliferative GN is driven by autoimmu- Complement Factor H Related genes (CFHR1–5),5–8 biallelic nity rather than an underlying monogenic disorder of complement loss of function variants of Complement Factor H (CFH),9 and regulation. an activating mutation of C3.10 In addition, studies of nonfamilial cases of PMG have identified rare variants in these and other whole-genome sequencing has been undertaken on 13,342 indi- complement genes (previously associated with atypical hemolytic viduals: 12,525 across 16 rare disease domains and 817 apparently uremic syndrome; aHUS) in up to 40% of patients.11–14 These healthy individuals (see Supplemental Table 1). Given the poten- findings, together with the almost invariable presence of C3 in the tial for a shared genetic cause with PMG, cohorts with diseases glomerulus, have implicated complement alternative pathway with a known immunologic basis (pulmonary artery hyperten- activation as a key causal mechanism and testing for comple- sion [PAH] and primary immunodeficiencies [PID]) and steroid- ment gene mutations is currently recommended in C3G, espe- resistant nephrotic syndrome (SRNS) were excluded. Clinical cially where living related renal transplantation is considered.15 phenotypic data for all participants was encoded using Human However, the current paradigm, in which the disease is fre- Phenotype Ontology,21 SNOMED CT, and ORPHANET codes. quently assumed to result from a rare genetic defect of com- Among those without PMG, three participants with the pheno- plement regulation, seems incompatible with the following types microangiopathic hemolytic anemia, thrombocytopenia observations: first, the disease is usually not familial; second, a and acute kidney injury, or SNOMED CT or ORPHANET C3 nephritic factor (C3NeF), an autoantibody that activates the codes compatible with hemolytic uremic syndrome, were identi- complement alternative pathway in the blood, is detectable in a fied and excluded from the control cohort, as were eight partic- substantial proportion of patients, including those in whom a ipants with evidence of retinal drusen or macular degeneration. rare variant in a complement gene is identified11; and third, there A summary of the analytic workflow, number of samples is a recognized association of MPGN with other autoimmune analyzed, and main findings is provided in Supplemental diseases16–18 including a very substantially increased rate of Figure 1. type 1 diabetes mellitus in relatives of patients with DDD.19 Here, we use whole-genome sequencing to investigate the PMG Cohort role of genetic variation in the causation of PMG in the United Recruitment of patients with PMG was undertaken from Kingdom (UK) population, and resolve all three of these ten British pediatric (64 patients) and 18 adult centers anomalous observations: although rare genetic variation in the (120 patients, of whom 21 had pediatric onset of disease). a priori candidate genes was not enriched in PMG (or the subset Patients with histologically confirmed MPGN either with or with C3G), there is a strong association with common varia- without immune-complex deposition (IC-PMG or C3G, re- tion at the HLA locus, explaining the phenotypic association spectively) in the absence of a known or suspected underlying with established autoimmune diseases and implicating auto- systemic cause22 were considered eligible. No genetic prescreen- immunity as the key causal mechanism. ing was applied. Clinical data were extracted from the UK Rare Renal Disease Registry (http://rarerenal.org/radar-registry). Where available, kidney biopsies were reviewed centrally to METHODS confirm the histologic diagnosis and to classify as IC-PMG, C3GN, or DDD. Serum C3NeF and C3 and C4 levels were Abbreviated Methods Follow measured using standard, clinically validated assays. Detailed methods are provided in Supplemental Appendix 1. Whole-Genome Sequencing: Data Generation, Variant National Institute for Health Research BioResource Calling, Annotation, Relatedness, and Ancestry Rare Diseases Study The methods used for data generation and variant calling have This study is a part of the National Institute for Health Re- been previously described20 and are further detailed, along search BioResource Rare Diseases study (BR-RD),20 in which with information on quality control, variant annotation, and 366 JASN JASN 31: 365–373, 2020 www.jasn.org BASIC RESEARCH the identification of a subset of unrelated individuals of Euro-