Genetic Determinants of IgA Nephropathy: Eastern Perspective

Genetic Determinants of IgA Nephropathy: Eastern Perspective Ming Li, MD, PhD,*,† and Xue-Qing Yu, MD, PhD*,†,‡ Summary: IgA nephropathy (IgAN) is one of the most common primary glomerulonephritides throughout the world and a major cause of end-stage renal disease among the East Asian population. It is widely considered that genetic factors play an important role in the pathogenesis of IgAN. This article summarizes the recent achievements in the genetic studies of IgAN, focusing mainly on studies performed in East Asia, from the early association studies of candidate genes and family based designs, to the recent genome-wide association studies. There have been five large genome-wide association studies performed that have identified multiple susceptibility loci for IgAN, especially some novel loci identified in the Chinese population. Genes within these loci have provided important insights into the potential biological mechanisms and pathways that influence genetic risk to IgAN. In susceptibility loci/genes, the study of genetic interaction and structural variants (such as copy number variation) was conducted to identify more variants associated with IgAN and disease progression. Genetic studies of IgAN from East Asia have made great achievements over the years. Most susceptibility loci discovered to date encode genes involved in the response to mucosal pathogens, suggesting that an intestinal-immune network for IgA production may be involved in the pathogenesis of IgAN. Although genetic studies of the complex diseases are challenging, for future genetic studies in IgAN, new genetic techniques and methods of analysis, especially next-generation sequencing, need to be applied to push the genetic studies forward. Semin Nephrol 38:455
460 Ó 2018 Elsevier Inc. All rights reserved. Keywords: IgA nephropathy (IgAN), genome-wide association study (GWAS), common variants, rare variants, copy number variants (CNV)

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gA nephropathy (IgAN) is the most common form of glomerulonephritis worldwide since described by Berger in 1968.1 The diagnosis of IgAN is based on a renal biopsy that shows deposition of IgA-containing immune complexes in the mesangial area of glomeruli and histopathologic lesions such as mesangial cell proliferation and accumulation of extracellular matrix.2 It leads to progressive loss of kidney function, and between 20% and 40% of cases will progress to end-stage renal disease within 20 years of disease onset.3-6 There is substantial variation in the prevalence of IgA nephropathy globally, with the highest frequency in some Asian populations (40%-50%), moderate frequency in the European population (20%-30%), and the lowest frequency in the African population (<5%), and there was a clear west-to-east gradient of ascending prevalence.7-10 However, whether this geographic variation may be influenced by differences in policies and techniques for performing renal biopsies is debatable.

Nonetheless, the difference in the prevalence of IgAN between ethnic groups, together with evidence of familial clustering11-13 and renal abnormalities among relatives of cases, strongly suggest the presence of a substantial genetic contribution to IgAN.

*Department of Nephrology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China. y Key Laboratory of Nephrology, National Health Commission (NHC) and Guangdong Province, Guangzhou, Guangdong, China. z Guangdong Medical University, Zhanjiang, Guangdong, China. Financial disclosure and conflict of interest statements: none. Address reprint requests to Xue-Qing Yu, MD, PhD, Department of Nephrology, The First Affiliated Hospital, Sun Yat-sen University, Key Laboratory of Nephrology, National Health Commission (NHC), Guangzhou, Guangdong, 510080 China. E-mail: [email protected] 0270-9295/ - see front matter © 2018 Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.semnephrol.2018.05.015

Both linkage and association studies have been performed to identify genetic risk factors for IgAN. The first application of a genome-wide linkage study of familial IgAN identified a significant linkage peak on chromosome 6q22-23,14 under an autosomal-dominant mode of inheritance with incomplete penetrance. Another two linkage studies of IgAN families reported additional suggestive peaks at 4q26-31 and 17q12-22.12,15 However, in exploring the causative genes at these loci, no disease genes were identified, which partly may be owing to the genetic heterogeneity and called for distinction based on genetic/biomarker data.

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TAGEDH1GENETIC STUDY OF IgAN IN THE EAST Because of the variable prevalence of IgAN among different ethnicities and high familial aggregation of IgAN, a genetic factor is considered an important part of the disease. To identify susceptibility genes of IgAN, family based analysis and population-based analysis were used, and to find common risk genes in large populations, several genome-wide association studies (GWAS) have been performed since 2011.

TAGEDH1LINKAGE STUDY AND CANDIDATE-GENE ASSOCIATION STUDYTAGEDN

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Candidate gene association studies were conducted more widely in East Asia during the past 3 decades compared with linkage studies. Many encoding proteins involved in adaptive and innate immunity, glycosylation of IgA1, and the renin-angiotensin system have been investigated,16 including the HLA molecules of HLADQ and HLA-DR alleles.17-19 For the family based association studies, using a “transmission disequilibrium test”20 design in a Chinese population, the Megsin gene was shown to confer susceptibility and disease progression of IgAN.21-22 However, most of these studies were limited by small sample sizes, insufficient methodologies, and lack of validation in independent samples.

TAGEDH1GENE-GENE INTERACTION STUDY IN IgAN Because the effect of a single gene is relatively weak in a polygenic disorder, studies focusing on combining the effect of genes on disease were performed. To detect gene-gene interaction, 24 candidate genes associated with the pathogenesis of IgAN were selected and analyzed using the multifactor dimensionality reduction method. The interaction between C1GALT1-330G/T and IL5RA31t197A/G was discovered to affect the pathogenesis of IgAN,23 and the combination of P-selectin2441A/G and CD14-159C/T was associated with macroscopic hematuria in IgAN patients. In addition, the interaction of TGF-b1 509T/C, P-selectin-2441A/G, and MCP-1 2518A/G had an integrated effect on crescent formation.24 Another study on the interaction between two key susceptibility genes, C1GALT1 and ST6GALNAC2, was analyzed and showed that these two genes may have an additive effect on IgAN predisposition and disease progression.25

TAGEDH1GWAS IN IgAN GWAS have identified common variants within several loci (chromosomal regions) associated with the risk of developing IgAN. A GWAS was conducted in a British cohort composed of 431 cases and 4,980 public controls and identified a significant association at the major histocompatibility complex (MHC) region.26 The second GWAS was performed in 3,144 cases and 2,822 controls which involved Han Chinese in discovery and follow-up validation in Chinese and European cohorts, in which five susceptibility loci for IgAN were identified. These included three distinct loci in the MHC region as well as the 1q32 (CFH/CFHR) locus and the 22q12 (HORMAD2) locus.27 In 2014, a large GWAS by Kiryluk et al28 analyzed 7,658 cases and 12,954 controls, of which 3,685 cases and 2,682 controls were East Asians and the rest were Europeans. The study identified six novel genome-wide significant associations, four in ITGAM-ITGAX, VAV3, and CARD9, and two new independent signals at HLADQB1 and DEFA. A further study also implicated that

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most of these loci are associated with the risk of inflammatory bowel disease or maintenance of the intestinal epithelial barrier and response to mucosal pathogens. Because of the difference in prevalence and genetic heterogeneity, a GWAS of a Chinese population was performed in our group since 2011. We performed a two-stage GWAS study of IgAN in Han Chinese, with 1,434 IgAN patients and 4,270 controls in the discovery phase, and follow-up evaluation of the top 61 SNPs in an additional 2,703 cases and 3,464 controls. We identified associations at 17p13 and 8p23 that implicated the genes encoding tumor necrosis factor (TNFSF13) and a-defensin (DEFA) as susceptibility genes. In addition, we found multiple associations in the MHC region. We also found that rs660895 was associated with clinical subtypes of IgAN, proteinuria, and serum IgA levels. Our findings show that IgAN is associated with variants near genes involved in innate immunity and inflammation.29 By adding more samples in the discovery and validation stage, and using the imputation analysis for deep mining the GWAS data, we conducted an extended GWAS, analyzing a total of 8,313 cases and 19,680 controls from the Han Chinese population across four stages.30 In this large study of IgAN, we discovered novel associations at ST6GAL1 on 3q27.3, ACCS on 11p11.2, and ODF1-KLF10 on 8q22.3, validated the recently reported association at ITGAX-ITGAM (16p11.2), and moderately replicated the reported associations at VAV3 (1p13) and CARD9 (9q34). Most of these loci, including two of our newly discovered ones (ST6GAL1 and UBR5), implicate genes involved in innate immunity and IgA production, in particular mucosal immunity in the gut. Several loci are shared with a variety of other autoimmune diseases. In addition, variants at several loci including ST6GAL1, ACCS, and ITGAX showed a trend of increasing risk allele frequencies from the African, European, to Asian samples of the HapMap project, suggesting that they may contribute cumulatively to geographic differences in genetic susceptibility and thus disease prevalence of IgAN. We estimate that these novel association signals explain approximately 1.7% of the disease variance and 5.5% of the variance in combination with previously published loci. Collectively, these GWAS have identified 11 new loci (Table 1), providing initial insight into the genetic architecture of IgAN.

TAGEDH1COPY NUMBER VARIATION IN IgAN Structural variants, including copy number variant (CNV), reversion, and deletion/insertion, are the important contributors to human genome variation, which have been considered to be involved in the pathogenesis of many common diseases. Although multiple susceptibility variants have been identified by GWAS in IgAN, these loci typically have

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Table 1. New IgAN Susceptibility Loci Discovered in GWAS Locus

SNP

Effect Allele

6p21

rs9275596

C

rs1883414 rs9357155

Gene

Type of SNP

Population

0.60

HLA-DRB1, HLA-DQA1/B1

Noncoding

Han Chinese and European

T A

0.83 0.75

HLA-DPA1/B1/B2 TAP2, TAP1, PSMB8, PSMB9

rs2523946 rs660895 rs1794275 rs6677604 rs2738048 rs2738058 rs9314614 rs12716641 rs10086568

C G A A G T C T A

1.21 1.34 1.30 0.68 0.79 1.23 1.13 1.15 1.16

HLA-A HLA-DRB1 HLA-DQA/B CFHR1-CFHR3

17p13.1

rs3803800 rs4227

A G

1.21 1.23

TNFSF13

Coding

Han Chinese

22q12

rs12537 rs2412971

T A

0.78 0.75

MTMR3 HORMAD2

Noncoding

Han Chinese and European

3q27.3 8q22.3 11p11.2

rs7634389 rs2033562 rs2074038

C C T

1.13 1.13 1.14

ST6GAl1 ODF1-KLF10 ACCS

Noncoding Noncoding Noncoding

Han Chinese

16p11.2

rs7190997 rs11150612 rs11574637 rs17019602 rs4077515

C A T G T

1.22 1.18 1.32 1.17 1.16

ITGAX-ITGAM

Noncoding

Han Chinese and European

VAV3 CARD9

Noncoding coding

1q32 8p23

1p13 9q34

Effect Size, Odds Ratio

Noncoding

DEFA Noncoding

relatively small effects, which may account for approximately 10% heritability. Current studies have indicated that structural variants, such as CNVs, may play important roles in IgAN. CNVs can convey phenotypes by gene dosage, gene interruption, gene fusion, position effects, unmasking of recessive alleles or functional polymorphism, and potential transvection effects.31,32 We recently reported the important role of DEFA1A3 gene copy number variations in the development of IgAN in a large Chinese cohort.33 Low copy numbers of the DEFA1A3 gene, especially the 4 base pair deletion (TATC) variant within the second intron of the DEFA1A3 gene, showed strong associations with increased risks for IgAN and also showed significant associations with renal dysfunction in patients with IgAN. Cumulatively, the DEFA1A3 CNV locus could explain up to 4.96% of phenotypic variance. Another study from the European IgAN Consortium reported that IgAN patients with CKD carried a low copy number of the TLR9 gene. And the TLR9 gene expression correlated significantly with the loss aberration.34 As a major source of genetic variation, the role of CNVs in the pathogenesis of IgAN should be investigated further.

TAGEDH1GENETIC INFLUENCES ON MUCOSAL IMMUNITY FOR IgAN The potential link between IgAN and the mucosa has been investigated since the 1970s. It is commonly shown

that mucosal immunity may play a part in the pathogenesis of IgAN because hematuria is one typical clinical feature and gross hematuria frequently coincides with mucosal infections.35 For many years, attention has been focused on the possibility of modulating the mucosal immune system by tonsillectomy.36,37 Some data also suggested an association between IgAN and gastrointestinal diseases in which the mucosa plays a role in a small number of patients.38-40 For these reasons, the concept of a mucosa-kidney axis was proposed. In the past decade, analyses that showed abnormalities in the O-glycosylation of the IgA1 molecule have advanced our understanding of the pathogenesis of IgAN. In IgAN, galactose-deficient IgA1 (Gd-IgA1) is significantly more abundant than in healthy controls.41 It is interesting that dysregulated systemic responses to mucosally acquired antigens are responsible for the increased circulating levels of IgA1. These studies are likely to imply that a defect in mucosal IgA production may be one of the sources of pathogenic IgA1 because the majority of polymeric IgA1 originated from mucosal inductive sites. Next, Gd-IgA1 as an autoantigen resulted in the production of IgA or IgG autoantibodies and the formation of immune complexes that deposit in the kidney and cause tissue injury.42,43 Recently published GWASs can provide some evidence to support these notions.26-30 Our previous two GWASs in Han Chinese cohorts identified some

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Figure 1. A proposed pathway in IgAN focused on the synthesis and deposition of IgA1. Genetic and epigenetic factors can have an effect on the production of galactose-deficient IgA1. Some of the events during an infection or exposure to an alimentary component can send signals via cells in the mucosa such as T cells, dendritic cells (DCs), and B cells, which then result in differentiation of IgA1-secreting plasma cells, leading to increased production of galactose-deficient IgA1. As a result of misplaced synthesis of mucosal-type IgA occurring in the bone marrow, Gd-IgA1 can reach the circulation and facilitate mesangial IgA deposition. Nevertheless, the presence of these IgA1 alone cannot cause IgA nephropathy. In a genetically predisposed individual, glycan-specific IgG can recognize the Gd-IgA1 molecule to form the immune complex, which can deposit in mesangial area. Deposition of glomerular immune complex can activate the local production of cytokines and growth factors, eventually leading to complement activation. In this model, there are many of potential susceptibility genes (in red) that may influence the progression of IgA1.

Genetics of IgAN from the East

significant signals that implicated TNFSF13, DEFA, ITGAX-ITGAM, and FCRL3 as susceptibility genes. After dysregulated response to mucosal antigen exposure to bacterial, viral, or alimentary component, the immune system can send signals via cells such as T cells, dendritic cells, and B cells, and eventually lead to the production of IgA1. Gd-IgA1 production possibly is influenced by variants in cytokine genes such as LIF, OSM, ITGAX-ITGAM, or TNFSF13, which regulate mucosal IgA production and class switching. However, Gd-IgA1 alone is insufficient to form an immune complex. In a susceptible individual, the MHC-II alleles can mediate the synthesis of glycan-specific IgG, which can recognize the galactose-deficient IgA1 molecules to form immune complexes. Identified susceptibility genes such as DEFA, TNFSF13, FCRL3, and others may have an effect on enhancing inflammatory signals and lead to increased production of antiglycan antibodies.44-46 Finally, the deposition of glomerular immune complexes can lead to complement activation. The alternative complement pathway, influenced by variants in the CFH and ITGAX-ITGAM genes, can cause tissue injury and regulate the risk of renal inflammation locally (Fig. 1). The available genetic data provide some support for the hypothesis that functional modifications in the mucosal immune system are a key factor in the pathogenesis of IgAN. Nevertheless, this hypothesis has little confirmatory evidence in vitro or in vivo from any other studies. Additional work is needed to verify the effects of these loci on the synthesis and O-glycosylation of IgA1.

TAGEDH1CONCLUSIONS AND FUTURE DIRECTIONSTAGEDN Genetic studies of IgAN have provided some evidence of the pathogenesis and identified multiple novel molecular candidate genes for disease, especially after five GWAS studies were performed in different populations. However, the susceptibility loci discovered to date explain only a small fraction of the genetic risk, suggesting there may be additional genetic and environmental risk factors involved in the pathogenesis of IgAN. Because the susceptibility loci identified in the GWAS of IgAN only suggested that there may be some risk genes within this region, fine mapping of risk loci is necessary, which can identify causal genes and potentially show additional independent risk alleles that can explain a large proportion of disease variance. GWAS mainly discovered the common variants with moderate effects, which can explain only a small fraction of disease heritability, and it is hard to identify rare and low-frequency variants. Nowadays, rare variants are considered to have a large effect on the complex disease because such variants may implicate the consequences of the gain or loss of function for organismal physiology and potentially could inform about the therapeutic effects of the gene products. In addition, structural

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variants were considered to be involved in the pathogenesis of the complex disease. With reduction in the cost of next-generation sequencing and improvement in the statistical methods of analyzing sequencing results, it is possible to continue mining the genome for new loci, by increasing sample size and therefore statistical power to detect variants with smaller effects, as well as to evaluate the roles of low-frequency/rare variants and structural variants. More importantly, we need to identify genetic factors that influence clinical phenotypes and the risk of progression to end-stage renal disease. This will require the integration of the genomic profile with other omics profiles (eg, transcriptomics, metabolomics, and immunomics) in patients with long-term clinical follow-up evaluation to better understand the factors underlying interindividual variability, not only in disease susceptibility, but also in the long-term prognosis and health care requirements.

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