Large-Scale Whole-Genome Sequencing Reveals the Genetic Architecture of Primary Membranoproliferative GN and C3 Glomerulopathy

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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 mutations 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

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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- v1.9. Haplotype association analysis was performed using pean ancestry, in Supplemental Appendix 1. PLINK v1.07.

Structural and Copy Number Variants Replication The occurrence of previously described rare structural variants HLA serotypes from the UK National Health Service Blood and copy number variants for PMG5–7,23,24 was examined by and Transplant (NHSBT) service were utilized as an indepen- manually inspecting all structural variants and copy number dent replication cohort. The analyzed cohort was a subset of variants involving the genes of relevance in unrelated PMG data from all White individuals listed for a kidney transplant individuals of all ethnicity. Subsequent analyses were re- in the UK within the past 25 years. HLA serotype data were stricted to the unrelated European cohort of cases and con- available for HLA-A, HLA-B, HLA-C, HLA-DR, and HLA-DQ. trols. A genome-wide comparison of the frequency of Only those serotypes observed at a frequency .0.05 in controls deletions per gene between PMG and controls was under- were analyzed for association (n=28). Chi-squared allelic tests taken, with P values calculated by permutation testing and logistic regression were performed using PLINK v1.9. (n=100,000). Serotypes were converted to molecular subtypes using the HLA Dictionary.29 Comparison with Previously Described PMG and aHUS Variants We examined the occurrence of common and rare variants RESULTS in C3, CD46, CFB, CFH, CFHR1, CFHR3, CFHR5, CFI, DGKE, and THBD previously observed in patients with aHUS, age-related The initial PMG cohort comprised 184 participants. After macular degeneration, C3G, or thrombotic microangiopathy, centralized biopsy review, 19 were excluded, most because as per the Database of Complement Gene Variants—a compi- the biopsy showed mesangial proliferative GN rather than lation of rare variant data from 3128 patients with aHUS and MPGN, leaving 165 (47.9% male). Histologic subtypes, com- 443 with C3G tested in six national reference laboratories plement abnormalities, and clinical features are summarized (http://www.complement-db.org)14 and a further study.11 in Table 1. Full clinical details for all individuals with C3G (C3GN and DDD) are provided in Supplemental Table 2. A Rare Variant Candidate Gene and Exome-wide Coding C3NeF was more likely to have been observed in those with Variant Burden Analysis DDD (Supplemental Table 2), and almost all of those with a Rare coding variants (gnomAD-Non-Finnish European C3NeF or DDD had exhibited a low C3 level on at least one [NFE] minor allele frequency [MAF] ,0.0001) of moderate occasion. Consistent with previous reports,30 transiently low or high impact were extracted. Per-gene rare variant burden serum C4 was documented in some patients with DDD and was enumerated as the proportion of individuals (cases versus C3GN, but this almost always normalized within weeks of controls) with at least one alternate allele in each gene with initial presentation. significance calculated using the exactCMC function in The total number of individuals in each BR-RD cohort is RVTESTS,25 which uses the Fisher exact test. Analyses were shown in Supplemental Table 1. Excluding PID, PAH, and also conducted filtering variants on the basis of their predicted SRNS, and control individuals with phenotypic codes compat- deleteriousness, using CADD scores.26 ible with aHUS or age-related macular degeneration (n=11), there were 10,250 individuals for use as controls. Of these, 6491 Common Variant Genome-Wide Association Study were unrelated and of European ancestry. All the cases were Common, high-quality variants (MAF$0.05 in gnomAD- genetically unrelated with the exception of one sibling NFE and BR-RD) were retained. Standard quality-control pair and the majority (n=146) were genetically classified procedures27 were undertaken (detailed in Supplemen- as of European ancestry (Supplemental Figure 2). After further tal Appendix 1). The final data set included 5,897,512 var- quality control measures, the final data set comprised 146 PMG iants with the call rate across the samples exceeding 0.999. cases and 6442 non-PMG controls. A genome-wide association study was undertaken with In the genes typically screened in patients with C3G and PLINK v1.9, assuming additive allele effects using logistic aHUS (namely C3, CD46, CFB, CFH, CFHR5, CFI, DGKE,and regression with the first five principal components as THBD), there was no enrichment of rare (gnomAD-NFE covariates. MAF ,0.0001) variants of moderate or high predicted impact in the PMG cohort (Figure 1). The number of individuals with HLA Imputation at least one such variant was 10 (6.8%; 95% confidence interval HLA genotyping was performed using BWAKIT/BWAMEM [95% CI], 3.5% to 12.6%) in the PMG cohort, compared with v0.7.15 (https://github.com/lh3/bwa/tree/master/bwakit) 381 (5.9%; 95% CI, 5.4% to 6.5%) in non-PMG controls and HLA-HD v1.2.0.128. Alleles with MAF,0.05 in controls (P=0.37, one-tailed Fisher exact test), consistent across were excluded. Logistic regression with the first five princi- each of the control cohorts (Supplemental Figure 3). Among pal components as covariates was performed using PLINK the PMG cohort, there was no difference in the candidate

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Table 1. Histologic categorization and clinical details Category Total, % C3NeF Detected (%) Low C3 (<0.68 g/L), % Low C4 (<0.18 g/L), % All participants 165 (100) 25 (36.2) 58 (69.0) 38 (45.2) IC-PMG 53 (46.5) 6 (24.0) 19 (63.3) 19 (63.3) C3GN 39 (34.2) 8 (33.3) 19 (67.9) 8 (28.6) DDD 22 (19.3) 10 (58.8) 16 (84.2) 9 (47.4) Pediatric onset 85 (51.5) 23 (40.4) 51 (77.2) 33 (50.0) Immunosuppression 75 (56.0) 18 (38.3) 42 (77.8) 27 (50.0) ESKD 42 (25.4) 5 (41.7) 6 (42.9) 1 (7.1) Renal transplant 30 (18.2) 4 (57.1) 4 (44.4) 0 (0.0) C3NeF detected 25 (36.2) — 23 (92.0) 11 (44.0) Ultrastructural and immunostaining data to allow sub-classification into IC-PMG, C3GN, or DDD were available for 114 participants. Results of clinically accredited C3NeF assays were available for 69 and serum C3 and C4 levels were available for 84 participants, but only a small number of those with ESKD. gene rare variant burden between the histologic subgroups signals, that were statistically significant after correction for C3GN, DDD, IC-PMG, and PMG unclassified, between those multiple testing (Bonferroni threshold for ten loci, P,0.005) with and without C3NeF, and those with low C3 (Supplemental (Table 2). The full association statistics for all 16 variants are Figure 3). The details of the 11 and 318 variants identified provided in Supplemental Table 6. There was no evidence of in the PMG participants and in non-PMG controls, respec- epistasis between the associated variants (P.0.05). tively, are provided in Supplemental Tables 3 and 4. Analyses Across the whole exome, there was no enrichment of rare were also performed imposing a variable CADD threshold variants with a moderate or high predicted impact per gene (none to $20) and control allele frequency (gnomAD-NFE in PMG (Supplemental Figure 5). The minimum P value 2 MAF ,0.0001 to ,0.01); however, in none of these permu- across the exome was 1.9310 4 as compared with the tations was there a significant difference between PMG and exome-wide significance threshold, correcting for 28,252 2 controls (Supplemental Figure 4). Furthermore, there was genes, of P,1.77310 6. The QQ plot showed no evidence no enrichment of rare variants previously classified as patho- of deviation from the null (Supplemental Figure 6). When fil- genic or likely pathogenic in the Database of Complement tering the data using a CADD threshold of $15, the minimum 2 Gene Variants, with one and 13 such variants identified in P value was also 1.9310 4. PMG and non-PMG individuals, respectively (Supplemental The only previously reported rare structural variant ob- Table 5). served in PMG cases was the 6.3 kbp CFHR5 tandem dupli- Sixteen previously described common complement gene cation (chr1:196950207–196956508) known to cause CFHR5 variants (gnomAD-NFE MAF $0.05) were identified (Supple- nephropathy,5 present in a single individual of Cypriot ances- mental Table 6). Computing pairwise linkage disequilibrium try. The common CFHR3-CFHR1 deletion was observed at demonstrated that these variants represented ten independent a similar frequency in European PMG cases and controls at signals at r2,0.8. Association analysis using logistic regression 0.164 and 0.201, respectively (Fisher exact test P=0.14), sim- including principal components as covariates identified four ilar to that in the UK population.31 Across all the candidate variants in the genes C3 and CFH, representing two independent genes, a total of 65 structural variants and copy number variants were identified, of which only one was seen in a PMG case: a 128.3 kbp heterozygous deletion involving exon 1 of 0.07 PMG CFH – Controls (chr1:196498350 196626665). Genome-wide, there was 0.06 no enrichment of deletions in PMG cases either in total or per 0.05 gene, after correcting for multiple testing by permutation analysis. 0.04 A common-variant genome-wide association study exam- 0.03 ining unrelated individuals of European ancestry identified Frequency fi P, 3 28 0.02 one locus achieving genome-wide signi cance ( 5 10 ) at 6p21.32 (Figure 2). The genomic inflation (l) was 1.017 0.01 (QQ plot in Supplemental Figure 7). Association statistics 28 0.00 for all variants achieving P,5310 are provided in Supple-

C3 CFB CFI CFH mental Table 7. At the 6p21.32 locus, the lead variant CD46 THBD DGKE 28 CFHR5 (rs35406322) was associated at P=3.29310 (odds ratio ﬁ Figure 1. Similar burden of rare variants with moderate or high [OR], 1.93; 95% CI, 1.53 to 2.44). Signi cance was maximal predicted impact in candidate genes comparing unrelated for variants within the gene C6orf10 (Figure 3). The control European PMG cases (n=146) with controls (n=6442). Vertical allele frequency of the lead variant (0.361) approximated to dotted lines indicate 95% CIs. that in gnomAD-NFE (0.376) and was consistent across all of

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Table 2. Association statistics comparing PMG with controls for four common variants in complement genes previously described at altered frequency in individuals with aHUS/MPGN Chr Position Ref Alt rsID Gene HGVSp Case Control gnomAD-NFE OR (95% CI) P Value 2 1 196654324 A C rs1061147 CFH p.A307A 0.534 0.620 0.617 0.71 (0.56 to 0.89) 3.5310 3 2 1 196659237 C T rs1061170 CFH p.H402Y 0.534 0.620 0.616 0.71 (0.56 to 0.89) 3.4310 3 2 19 6713262 G A rs1047286 C3 p.P314L 0.284 0.213 0.200 1.47 (1.13 to 1.90) 3.9310 3 2 19 6718387 G C rs2230199 C3 p.R102G 0.295 0.218 0.206 1.49 (1.16 to 1.93 2.1310 3 The two chromosome 19 variants and two chromosome 1 variants are in linkage disequilibrium (r2=0.844 and r2=0.999, respectively). Chr, chromosome; Ref, reference allele; Alt, alternate allele; rsID, dbSNP identifier; HGVSp, HGVS protein sequence change; gnomAD-NFE, allele frequency in non-Finish Europeans in the gnomAD database; Position, reference and alternate alleles are given with reference to Build 37 of the human genome. the BR-RD control cohorts (Supplemental Figure 8). There at a frequency of 0.233 compared with 0.122 in controls 2 was no statistically significant difference in the frequency of (P=1.21310 8; OR, 2.19; 95% CI, 1.66 to 2.89). The control the lead variants by PMG histologic subtype, C3NeF status, or frequency of this haplotype approximates to that observed in in those with low C3 (Supplemental Figure 8). Conditioning 1899 European American individuals (0.131).32 These analy- on the lead variant abrogated the signal. The second lead ses were repeated using HLA types imputed using HLA-HD, variant (rs3117135) is a known eQTL for multiple genes in yielding similar results (Supplemental Table 9). multiple tissue types at genome-wide significance, including HLA serotypes from the NHSBT were available from 338 HLA-DRB5, CYP21A1P, C4A,andNOTCH4 (Supplemental Table individuals with MPGN (both primary and secondary) and 8). There was no evidence of epistasis (P.0.05) between the 15,614 non-MPGN controls with renal failure of nonimmune 6p21.32 variants and the nominally associated common candidate or unknown cause, the largest groups of which were unknown complement gene variants. Testing dominant and recessive mod- (n=6836), polycystic kidney disease (n=4442), and pyelone- els for the lead variant showed weaker evidence of association, phritis/interstitial nephritis (n=1958). Using a Bonferroni 2 suggesting an additive genetic model best explains the association. threshold of P,1.8310 3 (n=28), three serotypes were sta- A second locus at 12q14.1 was not statistically significant at the tistically significantly associated with MPGN, with an OR of 2 genome-wide level (lead variant rs61938185, P=6.14310 8). approximately 1.4: DR17 (corresponding to DRB1*03:01/04), Fully imputed HLA genotypes at all six loci were available B8 (B*08), and DQ2 (DQB1*02:01/02/03/04/05) (Table 4). The for all 146 of the PMG cases and 6386 of the controls. A total of frequency of the most significant serotype, DR17, was approx- 39 HLA alleles were observed with a frequency .0.05. The imately consistent across each of the control cohorts, particularly 2 strongest association was with DQA1*05:01 at P=2.09310 8 those with larger sample sizes (Supplemental Figure 9), and (OR, 1.94; 95% CI, 1.54 to 2.45), followed by DRB1*03:01 equal to the frequency in 1043 UK blood cell donors.33 The and DQB1*02:01 (Table 3). Full association details for all al- significance of B8 and DQ2 was abrogated by conditioning leles tested are provided in Supplemental Table 9. The associ- on DR17. The DR17|DQ2 haplotype was observed in cases ation with both DQB1*02:01 and DRB1*03:01 was abrogated at a frequency of 0.175 compared with 0.129 in controls 2 by conditioning on DQA1*05:01. The DQA1*05:01| (P=4.55310 4; OR, 1.43; 95% CI, 1.17 to 1.75). DQB1*02:01|DRB1*03:01 haplotype was observed in cases

DISCUSSION 8 In this study we have examined the genetics of PMG using 6 whole-genome sequence data generated from a UK-wide collection of cases and a large number of genetic ancestry– ( p )

10 4 matched, non-PMG controls. The high prevalence of C3NeFs and reduced serum C3 levels, especially in patients with DDD –log and/or a C3NeF, is consistent with previous literature11,13 and 2 suggests that the cohort under study was comparable with previously reported PMG cohorts. Although we did observe rare,

0 protein-altering variants in the candidate genes (encoding 1 2 3 4 5 6 7 8 9

10 11 12 13 15 17 19 21 components of the complement alternative pathway and other Chromosome genes observed in the related disease aHUS), PMG cases were Figure 2. Genome-wide association study comparing unrelated not enriched for such variants, which occurred at a frequency European PMG cases (n=146) with controls (n=6442) at 5,897,512 of approximately 6% across all cohorts. Our study of 146 common variants identiﬁes a single locus surpassing the genome- European cases and 6442 European controls had .92% power 2 wide signiﬁcance threshold (P,5310 8, indicated by the hori- to detect a .15% burden of rare complement gene variants in zontal line) on chromosome six. PMG. We also observed association of PMG with common

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100 R 2 8 rs3117135 r ecombination rate 80

) 0.8 6 0.6 60

−value 0.4 (p 4 10 0.2 40 g ( cM/Mb −lo 2 20 ) 0

CFB TNXB PRRT1 C6orf10 HLA−DRA HLA−DRB6 HLA−DQB1 NELFE NOTCH4 HLA−DRB5 MIR1236 BTNL2 HLA−DQA2 SKIV2L

EGFL8 AGPAT1 MIR6721 RNF5 CYP21A1P RNF5P1 MIR6833

GPSM3

32 32.2 32.4 32.6 Position on chr6 (Mb)

Figure 3. LocusZoom plot for the chromosome 6 locus associated with PMG at genome-wide significanceshowsthesignaltoreside within the gene C6orf10 within the HLA complex. Variants are colored on the basis of their linkage disequilibrium (LD), using 1000 Genomes (November 2014) European data. As there were no reference LD data available for the lead marker, the second most 2 significant marker was used (chr6:32313531, rs3117135). A horizontal dotted line indicates P=5310 8. alleles of the candidate complement genes and, although not number than this, which suggests that any currently unrec- statistically significant at the genome-wide level, this is consis- ognized monogenic disorders (caused by coding mutations) tent with previous data and provides evidence that variation in are unlikely to account for a significant proportion of PMG in genes encoding components of the complement alternative the UK population. pathway affects susceptibility to PMG. Analysis of the frequency of common genetic variants Power calculation, which we performed before recruit- across the genome identified a single locus achieving genome- 2 ment to this study (using previously described methods)34 wide significance of P,5310 8. Numerous markers at the indicated that, using whole-exome analysis, 100 individuals HLA locus were strongly associated with PMG, and imputa- would have provided .80% power to detect association with tion identified an associated haplotype containing rare variants in a novel gene accounting for 20% of unex- DQA1*05:01, DQB1*02:01, and DRB1*03:01. This finding plained cases under a dominant model (power would was replicated in an independent cohort that included both be .95% under a recessive model). We recruited a greater primary and secondary MPGN, in which we observed association with the corresponding HLA serotypes DQ2 (DQB1*02:01) and DR17 (DRB1*03:01), which are associated with a number of Table 3. Association statistics comparing PMG and controls immune-mediated disorders. These genes encode components fi for the three most signi cant HLA alleles imputed using of the MHC class 2 molecule that are found on the surface of BWAKIT/BWAMEM antigen-presenting cells and are important in initiation of the Allele Case Control OR (95% CI) P Value adaptive immune response, including antibody production.35 2 DQA1*05:01 0.291 0.154 1.94 (1.54 to 2.45) 2.09310 8 This suggests that a key step in the pathogenesis of these disor- 27 DRB1*03:01 0.264 0.146 1.94 (1.51 to 2.50) 2.46310 ders is an aberrant adaptive immune response,36 which is con- 3 27 DQB1*02:01 0.264 0.144 1.81 (1.43 to 2.29) 7.69 10 sistent with the high frequency of autoantibodies (i.e.,C3NeFs)

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Table 4. Association statistics for the HLA serotypes Transplant (NHSBT) Service for kindly providing HLA serotype data. associated with MPGN in the NHSBT data after correcting for Genomic data from the NIHR BioResource Rare Diseases study has 23 multiple testing (P,1.8310 ) been deposited in the European Genome Archive under accession Serotype Case Control OR (95% CI) P Value number EGAS00001001012. 2 DR17 0.188 0.137 1.46 (1.20 to 1.78) 1.4310 4 Dr. Gale conceived and designed the study. Dr. Levine, Dr. Chan, 2 B8 0.186 0.140 1.42 (1.16 to 1.72) 4.7310 4 and Dr. Sadeghi-Alavijeh analyzed the data with the assistance of 2 DQ2 0.283 0.228 1.33 (1.12 to 1.58) 9.3310 4 Dr.Carss,Dr.Penkett,andDr.Tuna.Dr.Johnsondirectedthees- tablishment of the primary membranoproliferative GN cohort. Prof. Cook undertook the centralized biopsy review. Dr. Gale and in these patients. However, the possibility has not been excluded Dr. Levine drafted the manuscript. All other authors were responsible that the observed associations are mediated by one of the non- for recruitment of patients to the study and data acquisition. HLA genes spanned by this haplotype, including those encod- The members of the MPGN/DDD/C3 Glomerulopathy Rare ing complement components C2, Factor B, and C4. Variation Disease Group and the NIHR BioResource may be found in the in dose of C4A and C4B, which encode isotypes of C4 that Supplemental Material. preferentially bind antibody-protein or antibody-cell surface complexes, respectively,37 is known to affect serum C4 activity and has previously been implicated in SLE and schizophrenia.38,39 DISCLOSURES However, direct comparison of the copy number of each of these genes showed no significant differences between PMG cases and Dr. Carss reports personal fees from AstraZeneca, outside the submitted controls. Examining C4NeF levels and the relationship with the work. Dr. Gale reports personal fees from Alexion, personal fees from Novartis, outside the submitted work. Dr. Harris reports other from Admirx, other from observed variants may be informative. GlaxoSmithKline, other from Gyroscope Therapeutics, grants from RaPharma, We observed shared genetic risk factors in all the subgroups other from Roche, outside the submitted work. Dr. Marchbank reports other of PMG (IC-PMG, C3GN, and DDD), as well as those with from Gemini Therapeutics LTD, outside the submitted work; In addition, and without C3NeF, and those with low C3 (Supplemental Dr. Marchbank has a patent “Modified Complement Proteins and Uses ” Figure 5) in this study, implying shared underlying disease Thereof pending. Dr. Megy reports grants from National Institute for Health Research, during the conduct of the study. mechanisms. We did not observe any significant genetic differences between these subgroups and it is likely larger studies would be needed to identify such differences, if they exist. Tests FUNDING for the presence of C4 nephritic factors or autoantibodies against complement regulators (which have been reported in This work was funded by the National Institute for Health Research (NIHR; patients with PMG40) were not available for the whole cohort, grant numbers: RG65966, BH141504, and PD00400) and Kids Kidney so we are unable to determine the proportion of patients in Research. Dr. Gale is supported by a Medical Research Council Clinician Sci- entist Fellowship and St Peter’s Trust. Dr. Chan is supported by a Kidney whom an autoantibody was present; however, the HLA asso- Research UK Clinical Training Fellowship. Dr. Sadeghi-Alavijeh is supported ciation we observed is consistent with other reports in which by an NIHR clinical fellowship. multiple autoantibodies are detectable in cohorts of patients with PMG40 and previous observations showing an associa- SUPPLEMENTAL MATERIAL tion between MPGN/C3G and autoimmune disorders.16–19 Some of the variants and HLA alleles that we observed to be This article contains the following supplemental material associated with PMG have previously been associated with a online at http://jasn.asnjournals.org/lookup/suppl/doi:10.1681/ number of immune mediated diseases, including membranous ASN.2019040433/-/DCSupplemental. nephropathy,41 rheumatoid arthritis,42 myasthenia gravis,43 Supplemental Appendix 1. Consortia, supplemental methods, asthma,44 celiac disease,45 and type 1 diabetes mellitus,46,47 results, detailed legends for tables and references. potentially explaining the observed phenotypic association be- Supplemental Figure 1. Sample and analytic workflow. tween these different disorders. Together, these findings imply Supplemental Figure 2. Principal component analysis. that, rather than resulting from a primary genetic disorder of Supplemental Figure 3. Candidate gene rare variant cumulative complement alternative pathway regulation, in most cases burden by cohort and PMG subphenotype. PMG is actually an autoimmune disease. Supplemental Figure 4. Candidate gene rare variant cumulative burden with variable filtering (CADD and frequency). ACKNOWLEDGMENTS Supplemental Figure 5. Exome-wide rare variant burden analysis Manhattan plot. The authors gratefully acknowledge the participation of all National Supplemental Figure 6. Exome-wide rare variant burden analysis Institute for Health Research (NIHR) BioResource volunteers, and QQ plot. thank the NIHR Cambridge BioResource Centre and the NIHR Supplemental Figure 7. Genome-wide association study QQ plot. Newcastle Biomedical Research Centre for their contributions. Supplemental Figure 8. Allele frequency of chromosome 6 lead We also acknowledge the UK National Health Service Blood and variant by cohort and PMG subphenotype.

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AFFILIATIONS

1Department of Renal Medicine, University College London, London, United Kingdom; 2Renal Department, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, United Kingdom; 3Faculty of Medical Sciences, Translational & Clinical Research Institute, Newcastle University, Newcastle upon Tyne, United Kingdom; 4The National Renal Complement Therapeutics Centre, Royal Victoria Infirmary, Newcastle upon Tyne, United Kingdom; 5Centre for Inflammatory Disease, Department of Immunology and Inflammation, Imperial College London, London, United Kingdom; 6National Institute of Health Research BioResource, Cambridge University Hospitals, Cambridge, United Kingdom; 7Department of Haematology, University of Cambridge, Cambridge, United Kingdom; 8Children’s Renal and Urology Unit, Nottingham Children’s Hospital, Queen’s Medical Centre, Nottingham University Hospitals NHS Trust, Nottingham, United Kingdom; 9Department of Nephrology, Nottingham University Hospitals NHS Trust, Nottingham, United Kingdom; 10Department of Paediatric Nephrology, Great Ormond Street Hospital and University College London Great Ormond Street Institute of Child Health, NIHR Great Ormond Street Hospital Biomedical Research Centre, London, United Kingdom; 11Department of Paediatric Nephrology, Royal Hospital for Children, NHS Greater Glasgow and Clyde, Glasgow, United Kingdom; 12Renal Department, University Hospitals of North Midlands NHS Trust, Stoke-on-Trent, United Kingdom; 13Department of Paediatric Nephrology, Great North Children’s Hospital, Newcastle upon Tyne Hospitals NHS Foundation Trust, Newcastle upon Tyne, United Kingdom; and 14Rare Renal Disease Registry, UK Renal Registry, Bristol, United Kingdom

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