CLINICAL RESEARCH www.jasn.org Rare Variants in the Complement Factor H–Related Protein 5 Gene Contribute to Genetic Susceptibility to IgA Nephropathy †‡ †‡ †‡ †‡ †‡ Ya-Ling Zhai,* § Si-Jun Meng,* § Li Zhu,* § Su-Fang Shi,* § Su-Xia Wang,* § †‡ †‡ †‡ †‡ †‡ Li-Jun Liu,* § Ji-Cheng Lv,* § Feng Yu,* § Ming-Hui Zhao,* § and Hong Zhang* § *Renal Division, Department of Medicine, Peking University First Hospital, Beijing, China; †Institute of Nephrology, Peking University, Beijing, China; ‡Key Laboratory of Renal Disease, Ministry of Health of China, Beijing, China; and §Key Laboratory of Chronic Kidney Disease Prevention and Treatment (Peking University), Ministry of Education, Beijing, China ABSTRACT A recent genome–wide association study of IgA nephropathy (IgAN) identified 1q32, which contains multiple complement regulatory genes, including the complement factor H (CFH) gene and the comple- ment factor H–related (CFHRs) genes, as an IgAN susceptibility locus. Abnormal complement activation caused by a mutation in CFHR5 was shown to cause CFHR5 nephropathy, which shares many character- istics with IgAN. To explore the genetic effect of variants in CFHR5 on IgAN susceptibility, we recruited 500 patients with IgAN and 576 healthy controls for genetic analysis. We sequenced all exons and their intronic flanking regions as well as the untranslated regions of CFHR5 and compared the frequencies of identified variants using the sequence kernel association test. We identified 32 variants in CFHR5, includ- ing 28 rare and four common variants. The distribution of rare variants in CFHR5 in patients with IgAN differed significantly from that in controls (P=0.002). Among the rare variants, in silico programs predicted nine as potential functional variants, which we then assessed in functional assays. Compared with wild-type CFHR5, three recombinant CFHR5 proteins, CFHR5-M (c.508G.A/p.Val170Met), CFHR5-S (c.533A.G/p. Asn178Ser), and CFHR5-D (c.822A.T/p.Glu274Asp), showed significantly higher C3b binding capacity (CFHR5-M: 109.67%63.54%; P=0.02; CFHR5-S: 174.27%69.78%; P,0.001; CFHR5-D: 127.25%61.75%; P,0.001), whereas another recombinant CFHR5 (c.776T.A/p.Leu259Termination) showed less C3b bind- ing (56.89%60.57%; P,0.001). Our study found that rare variants in CFHR5 may contribute to the genetic susceptibility to IgAN, which suggests that CFHR5 is an IgAN susceptibility gene. J Am Soc Nephrol 27: 2894–2905, 2016. doi: 10.1681/ASN.2015010012 IgA nephropathy (IgAN) is the most common pri- IgAN. A recent genome–wide association study mary GN worldwide.1 Recently, important discov- (GWAS)7 and a GWAS replication study8 identified eries have been made regarding the pathogenesis of and confirmed 1q32 as an IgAN susceptibility IgAN,2–5 such as the aberrant glycosylation of IgA1 and the presence of antiglycan antibodies in circu- lation as well as the role of transferrin receptors in Received January 5, 2015. Accepted December 20, 2015. glomeruli. However, there are still many unknown Y.-L.Z. and S.-J.M. contributed equally to this work. aspects in IgAN. Although complement activation Published online ahead of print. Publication date available at has been widely accepted for a long time to occur in www.jasn.org. IgAN,6 the exact pathways and regulatory mecha- Correspondence: Dr. Li Zhu, Renal Division, Department of nisms are still unclear. Medicine, Peking University First Hospital, Peking University In- Meanwhile, from racial and ethnic variations in stitute of Nephrology, Beijing 100034, China. Email: funnyzhuli@ disease incidence to familial aggregation of IgAN, bjmu.edu.cn increasing evidence implied a genetic effect on Copyright © 2016 by the American Society of Nephrology 2894 ISSN : 1046-6673/2709-2894 JAmSocNephrol27: 2894–2905, 2016 www.jasn.org CLINICAL RESEARCH locus, which suggested the involvement of genetic abnormal- Their proteinuria and eGFR levels were 1.42 (0.81–2.66) g/d ities in the complement factor H (CFH)and/orthecomplement and 84.19627.03 ml/min per 1.73 m2, respectively. In total, factor H–related (CFHRs) genes in IgAN. This region contains 13.60% (68 of 500) had hypertension. Regarding pathologic several complement regulatory genes, CFH-CFHRs. CFHRs, features, 47.60% of patients with IgAN had a strong intensity which include CFHR3, CFHR1, CFHR4, CFHR2,andCFHR5, of C3 deposition (3+ or 4+). Haas 1–5 were found in 8.60%, share high sequence homology with CFH. Accordingly, they 0.80%, 45.20%, 33.20%, and 12.20% of patients, respectively. have similar structures, which are built on a motif of distinct All patients were regularly followed for at least 1 year, with a functional domains called short consensus repeats (SCRs), mean follow-up time of 56.26 months. Patients’ treatment and and similar functions in complement regulation,9 although follow-up were conducted at an IgAN-specific clinic in Peking their precise biologic roles are not completely identical. University First Hospital and consistent with the Kidney Recently, a novel disease named CFHR5 nephropathy was Disease Improving Global Outcomes guideline.14 During identified after genetic analysis of a pedigree of Cypriot ances- follow-up, 16.80% (84 patients) of these patients reached a try with primary GN.10 Patients with CFHR5 nephropathy composited end point (ESRD, 50% eGFR decline, or death). presented with many characteristics, including young age The clinical, laboratory, and pathologic characteristics of the onset, tendency in men, repeated episodes of synpharyngitic recruited patients were similar to those of previously reported hematuria, mesangial matrix expansion, increased glomerular IgAN cohorts.15 cellularity, glomerular staining for C3, mesangial electron– dense deposits, and high recurrence rate after renal transplan- Identification of Genetic Variants in CFHR5 tation, which shared many similarities with IgAN.11 Patients All exons in the CFHR5 gene were amplified and screened for with CFHR5 nephropathy bear the same heterozygous inter- genetic variants in recruited individuals. In total, 32 variants nal duplication of the CFHR5 gene, which is likely to be the were identified (Figure 1, Table 1), including 10 in exons, 18 in cause of their glomerular lesions. introns, two in promoter, and two in 39 untranslated region The CFHR5 gene is located at 1q31.3 and spans 42 kb of the (UTR). Of 10 exonic variants, eight were nonsynonymous human genome. Its encoded product is a 62-kD protein coding (including one nonsynonymous stop gain and nonsy- named CFHR5. The CFHR5 gene has 10 exons encoded in nonymous stop loss), and two were synonymous changes. nine SCRs, in which SCR3–7andSCR8–9 showed a high ho- Among 32 variants, 16 were found in the dbSNP database, mology with SCR10–14 and SCR19–20 in CFH, respectively. whereas the other 16 variants were novel. CFHR5 is the most recently discovered CFHR protein, and it is also the one that showed the most similarity with CFH among Genetic Association Analyses of CFHR5 with IgAN the five CFHRs.12 As with CFH and other CFHRs, CFHR5 is Among the identified 32 variants, four were common variants produced in the liver, circulates in plasma, and functions as a (c.58+17T.A, c.431–80G.A, c.434G.A, and c.1147+24T. complement regulatory protein. Until today, the precise G; minor allele frequency [MAF] .0.01 in controls), whereas mechanism for CFHR5 in complement regulation is still un- the other 28 were rare. We only observed individuals with known.13 homozygous minor alleles in two common variants (c.58+ However, with respect to IgAN, the disease presents 17T.A and c.1147+24T.G) and not in the other two com- complement activation in glomeruli and has clinical features mon variants (c.431–80G.A [MAF=0.013] and c.434G.A similar to CFHR5 nephropathy, but whether some patients [MAF=0.013]) and 28 rare variants. Of 28 rare variants, 14 with IgAN have genetic changes in the CFHR5 gene and were identified only in patients with IgAN (who exhibited a whether these variants contribute to IgAN susceptibility are total of 22 rare variants), nine were identified only in healthy still unknown. In this study, using a large population of pa- controls (with a total of 23 rare variants), and the other five tients with IgAN, we performed an intensive genetic analysis variants were identified in both patients (23 total rare variants) to explore the genetic effect of variants in the CFHR5 gene on and controls (21 total rare variants). IgAN susceptibility. Because analysis of rare variants requires statistical methods that are fundamentally different than testing commonvariants, we applied Sequence Kernel Association Test (SKAT), which RESULTS can evaluate not only rare variants’ effect but also, the cumu- lative effect of rare and common variants. The results of SKAT Baseline Clinical, Laboratory, and Pathologic showed that the combined effect of rare and common variants Characteristics in CFHR5 had a significant association with IgAN (P=0.03) In total, 1076 individuals were recruited, including 500 patients (Table 2). Furthermore, we investigated common and rare with IgAN (258 men and 242 women) and 576 healthy controls variants separately. Rare variants in CFHR5 presented a sig- (310 men and 266 women). The average ages for patients with nificant association with IgAN (P=0.002) (Table 2), but com- IgAN and controls were 34.23611.09 and 33.5067.66 years mon variants did not (P=0.92) (Table 2). old, respectively. For patients with IgAN, detailed clinical, lab- Regarding the four common variants, we also performed oratory, and pathologic characteristics were also available. single variant–level analysis. In accordance with the results J Am Soc Nephrol 27: 2894–2905, 2016 CFHR5 in IgAN 2895 CLINICAL RESEARCH www.jasn.org c.1513+126A.G in three patients; c.254– 66A.T in two patients; and the following 11 variants, c.-350G.T, c.332A.C, c.430+ 112T.C, c.776T.A, c.790+2T.C, c.790+47A.G, c.1148–121G.A, c.1330+ 78A.G, c.1708T.C, c.1710*3C.G, and c.1710*168G.T, in one patient each).