Polymorphisms of CARD9 Gene Predict Disease Progression and Renal Survival of Immunoglobulin A Nephropathy

Immunoglobulin A nephropathy (IgAN) is one of the most common primary glomerulonephritis throughout the world, which is characterized by the deposition of IgA-containing immune complexes in the mesangial area of glomeruli and histopathological lesions of mesangial cell proliferation [1, 3]. Approximately 20–40% of IgAN patients could progress to end-stage renal disease within 20 years [4], which results in a significant clinical and financial burden on global public health. Although the exact etiology of IgAN remains unclear, it is suggested that both environmental and genetic factors contribute to the susceptibility and disease progression [5, 6].

Up to now, there have been five large genome-wide association studies (GWASs) [7, 11] and a genome-wide meta-analysis [12] for IgAN, which reported dozens of susceptibility loci for IgAN and advanced our understanding of disease pathogenesis. The susceptibility loci discovered by GWASs are mainly involved in adaptive immunity, the complement system, mucosal immunity, and the regulation of mucosal IgA production [4]. Notably, CARD9 (caspase recruitment domain family member 9) was identified as a susceptibility gene for IgAN [10], which is a pro-inflammatory molecule and an adapter protein that promotes activation of the NF-κB pathway in the innate immune system. Therefore, this gene provided the genetic evidence for the involvement of NF-κB pathway in the pathogenesis of IgAN [13]. Interestingly, genetic variations in CARD9 are also associated with a variety of traits, such as fungal infection [14], inflammation, and autoimmune disorders [15].

Taken together, it is speculated that CARD9 may play an essential role in IgAN. However, until now, there has been no systematic investigation about the role of CARD9 in disease progression and prognosis of IgAN. Thus, we attempted to demonstrate whether single nucleotide polymorphisms (SNPs) within CARD9 correlate with clinicopathological features or the prognosis of IgAN in an independent Chinese Han cohort in current study.

As previously described [16], 986 IgAN patients were initially enrolled from the First Affiliated Hospital of Sun Yat-Sen University in this study. Patients with Henoch-Schönlein purpura nephritis, secondary IgAN, systemic lupus erythematosus, chronic liver diseases, other autoimmune diseases, or under immunosuppressive treatments before renal biopsy were excluded.

Demographic and clinical details were collected from patients with IgAN at diagnosis, including serum IgA, IgM, C3, albumin (Alb), 24-h urine protein (UPRO) levels, blood pressure, stages of chronic kidney disease (CKD), history of gross hematuria, history of upper respiratory infections such as pharyngitis, tonsillitis, and rhinitis, or other types of infections (Epstein-Barr virus infection, gingivitis, measles, and herpes virus infection), the estimated glomerular filtration rate (eGFR), and renal histopathological classification. The eGFR was evaluated using the simplified MDRD formula for Chinese patients, and stages of CKD were classified based on the eGFR intervals according to the 2021 KDIGO practice guidelines [17]. The renal histopathological classification was evaluated using the updated Oxford MEST-C system [18, 19], which consists of five predictive lesions as follows: mesangial hypercellularity (M), endocapillary hypercellularity (E), segmental sclerosis (S), tubular atrophy/interstitial fibrosis (T), and crescents (C). We also collected the data on the proportion of global glomerulosclerosis, segmental sclerosis, and crescent lesions.

Additionally, we gathered data from all participants with or without hypertension (systolic blood pressure ≥140 mm Hg and/or diastolic blood pressure ≥90 mm Hg), hyperuricemia (blood uric acid >420 µmol/L for males and >360 µmol/L for females), and hyperlipidemia (serum cholesterol ≥5.18 mmol/L and/or triglycerides ≥1.70 mmol/L and/or high-density lipoprotein cholesterol <1.04 mmol/L and/or low-density lipoprotein cholesterol ≥3.37 mmol/L) [20]. Follow-up data of 718 patients after kidney biopsy were available with a median time of 26.94 months. All the participants were stratified into different groups according to clinical characteristics, including Alb levels (<30 g/d or ≥30 g/d), serum IgA levels (<3.5 g/L or ≥3.5 g/L), 24-h UPRO excretion (<1.0 g/24 h, 1.0–3.5 g/24 h, or >3.5 g/24 h), the presence of renal dysfunction (defined as eGFR <60 mL/min/1.73 m²), proportion of crescent lesions (<5%, 5–10%, 10–25%, or ≥25%) [21], and serum C3 levels with a cut-off value of 0.79 (<0.79 g/L or ≥0.79 g/L).

DNA was extracted from 4 mL EDTA-treated peripheral blood samples using a commercial DNA extraction Kit (Qiagen, Hilden, Germany). SNP genotype information (Release 27 phase I + II + III, CHB populations) was retrieved from the HapMap database (https://ftp.ncbi.nlm.nih.gov/hapmap/), including 2 kb upstream and downstream of the CARD9 gene. In total, seven SNPs (rs10870077, rs10747047, rs3812552, rs9411205, rs10781533, rs3829109, and rs3812555) were selected based on the criteria of r² ≥0.80 and minor allele frequency ≥0.05 using HaploView (version 4.2) software [22]. Additionally, we selected the SNP rs10870149, which has been reported to be associated with ankylosing spondylitis (AS) through the GWAS. Ultimately, a total of eight SNPs were successfully genotyped through the Sequenom MassARRAY platform (Sequenom, San Diego, USA) for the participants.

Functional annotation of the candidate SNPs and their proxy SNPs was performed by the HaploReg database (version 4.1, http://compbio.mit.edu/HaploReg). The RegulomeDB (version 2.0.3, http://www.regulomedb.org/) and rVarBase (version 2.0, http://rv.psych.ac.cn/) databases were used to investigate the regulatory properties of the SNPs. Expression quantitative trait locus (eQTL) effects for SNPs were investigated by querying the eQTLGen Consortium data (https://eqtlgen.org/, a blood eQTL meta-analysis performed in 31,684 individuals) [23], the Genotype-Tissue Expression (GTEx) project [24] (https://www.gtexportal.org/home/), and the Human Kidney eQTL Atlas (http://www.susztaklab.com/) [25].

Categorical variables were expressed as frequencies and percentages. Quantitative variables were presented as medians and interquartile ranges due to their skewed distributions. For genotypic-phenotypic analyses, odds ratio (OR) or β and 95% confidence interval (95% CI) were provided for the evaluation of relevance between different polymorphisms. Linear regression analyses were applied to reveal relationships between continuous/multiple categorical variables and CARD9 SNPs. All the analyses were adjusted by age and gender under the additive genetic model. Logistic regression analyses were applied to reveal relationships between binary variables and CARD9 SNPs under the same conditions. The Kaplan-Meier method and Cox proportional hazard models with hazard ratios (HRs) and 95% CIs were applied to analyze the associations between genetic variants and renal survival. In the multivariate Cox model, age, gender, hypertension, eGFR, and 24-h UPRO were set as confounding variables. Notably, 11 patients were excluded for the absence of hypertension or 24-h URPO values, and 707 patients were finally enrolled in the multivariate Cox model.

Statistical significance was defined at p ≤ 0.05, and the step-up false discovery control (FDR)-corrected significance level was set at p’ ≤ 0.05 [26]. The associations between candidate SNPs and IgAN were implemented by PLINK (version 1.90) [27]. Statistical analyses and figure preparation were accomplished with R (version 4.1.3).

All 986 cases were eligible after quality control (SNP genotyping rate >95%). The SNP rs3812555 was not successfully designed, and the rs3829109 was eliminated due to the minor allele frequency (<0.05). Consequently, six candidate SNPs were included in the following association analysis. The general information of all six candidate SNPs is summarized in online supplementary Table 1 (for all online suppl. material, see https://doi.org/10.1159/000530262). The detailed baseline demographics and clinicopathological features of the 986 eligible IgAN patients are summarized in Table 1.

The six candidate SNPs were studied for associations with clinical phenotypes by using different regression methods (Table 2). We checked the normality of all quantitative phenotypes, and natural log transformation was performed for some continuous variables, including the IgA, IgM, C3, eGFR, Alb, 24-h UPRO, and Scr measurements.

Patients with the rs10870149-A and rs10870077-G alleles tended to present with milder proteinuria (p = 0.041 and β = −0.049; p = 0.022 and β = −0.056, respectively). The G allele carriers of rs3812552 were more likely to exhibit higher levels of C3 (p = 0.018 and β = 0.014). Importantly, the T allele of SNP rs10747047 showed associations with decreased eGFR (p = 0.005 and β = −0.078), increased Scr (p = 0.005 and β = 0.067), and CKD (p = 0.010 and β = 0.246). These associations remained significant even after FDR correction (p’ ≤ 0.05). The T allele of SNP rs10747047 was also associated with hyperuricemia (p = 0.021 and OR = 1.430). In addition, the rs9411205-C allele was nominally correlated with earlier disease age onset (p = 0.050 and β = −0.425). We also investigated the relationships between CARD9 SNPs and history of infections. However, we did not observe any significant associations.

Further stratification analyses of several vital predictive parameters for IgAN (24-h UPRO, proportion of crescent lesions, eGFR, C3, Alb, and serum IgA) are shown in Table 3. Serum C3 levels at the time of biopsy were significantly higher (≥0.79) in patients with the C allele of rs9411205 than in those with the T allele (p = 0.029 and OR = 1.323).

Subsequently, we investigated the relationships between six candidate SNPs and the pathological patterns among patients (Table 4; online suppl. Table 2). The results indicated that patients with the T allele of rs10747047 showed a higher risk of endocapillary hypercellularity lesions (p = 0.004 and OR = 1.763), reaching the FDR-corrected significance threshold (p’ = 0.022). The T allele of rs10747047 was also associated with a higher proportion of global glomerulosclerosis (p = 0.023 and β = 4.657, online suppl. Table 2). Patients with the C allele of the rs10781533 tended to have reduced occurrence of segmental glomerulosclerosis lesions (p’ = 0.005 and OR = 0.719, Table 4) and reduced proportion of segmental glomerulosclerosis lesions (p’ = 0.024 and β = −0.929, online suppl. Table 2) in contrast to the T allele. Similar trends were also observed in patients with the A allele of rs10870149, G allele of rs3812552, and G allele of rs10870077 in regard to both segmental glomerulosclerosis lesions (p’ = 0.015 and OR = 0.755; p’ = 0.021 and OR = 0.702; p = 0.041 and OR = 0.810, respectively, Table 4) and the proportion of segmental glomerulosclerosis lesions (p’ = 0.023 and β = −1.057; p’ = 0.023 and β = −1.526; p’ = 0.024 and β = −0.952, respectively, online suppl. Table 2). Additionally, the C allele of rs9411205 was negatively associated with the segmental glomerulosclerosis proportion (p’ = 0.024 and β = −0.942, online suppl. Table 2). The associations were statistically robust and surpassed the threshold of FDR-corrected significance (p’ ≤ 0.05).

A total of 718 of the 986 patients had long-term retrospective progression data, and 105 patients (15.25%) experienced end-stage renal disease or doubled Scr after a median follow-up period of 26.94 (17.02–39.42) months. The results of both univariate and multivariate Cox models in the cohort are shown in Table 5.

Through multivariate Cox regression analysis, the presence of the CC genotype of rs10747047 was correlated with poor renal outcomes (TT vs. CC: HR = 0.138, 95% CI = 0.022–0.871, p = 0.035, Table 5, online suppl. Fig. 1a). Additionally, we detected a strong independent association between the GG genotype of rs10870077 and renal survival among 707 patients with available data (GG vs. CC: HR = 0.321, 95% CI = 0.123–0.836, p = 0.020, Table 5, online suppl. Fig. 1b). However, through Kaplan-Meier curve analyses, the difference in cumulative renal survival among 718 patients of three genotypes for all six SNPs was not statistically significant (online suppl. Table 3; Fig. 2, log-rank p > 0.05).

SNP functional annotation based on different databases is shown in online supplementary Tables 4–7. Through the eQTL analysis, we found that all candidate SNPs could regulate CARD9 expression levels in whole blood (online suppl. Table 4). Additionally, rs10747047, rs10781533, and rs10870149 were closely associated with transcriptional regulation through the RegulomeDB database (online suppl. Table 5), which was in accordance with the eQTL results. In addition, we found that candidate SNPs rs10781533, rs10870077, and rs10870149 could also regulate CARD9 gene expression levels in other tissues, including the lung, cultured fibroblast cells, testis, and visceral omental adipose tissues (online suppl. Table 4). However, we did not find any related eQTLs in the kidney through the GTEx portal and the Human Kidney eQTL Atlas (online suppl. Table 4).

CARD9 has been implicated in autoimmune and inflammatory-related diseases during the past decades [15] and has been identified as a novel susceptibility gene for IgAN [10]. But few publications have been reported to investigate the links between CARD9 variants and the progression and prognosis of disease. To our knowledge, this is the first study that shows CARD9 gene polymorphisms are associated with the progression and prognosis of IgAN in the Chinese Han population.

Evidence has indicated that several clinical and pathological features could predict increased disease severity for IgAN, including the level of 24-h UPRO, hypertension, renal function at biopsy, and the MEST-C scores [28, 32]. In addition, a lower plasma level of C3 is correlated with disease progression [33]. In this study, we systematically reported genotypic associations between the CARD9 gene and the abovementioned clinicopathological phenotypes and prognosis in a Chinese Han population. Briefly, IgAN patients carrying the T allele of rs10747047 are inclined to suffer from severe renal dysfunction, presenting with increased Scr levels and decreased eGFR levels. Moreover, the T allele of rs10747047 predisposes patients to more severe endocapillary hypercellularity lesions and advanced CKD. Compared with noncarriers, carriers with rs10870077-C and rs10870149-G alleles tend to develop heavy 24-h UPRO excretion and more segmental sclerosis lesions, indicating that both the C allele of rs10870077 and the G allele of rs10870149 promote the progression of IgAN. The Cox regression results show that patients with rs10870077-CC genotypes are observed to have decreased renal survival after adjustments for multiple confounders, further suggesting that IgAN patients carrying the C allele of rs10870077 are more likely to develop renal failure and receive poor prognosis. Therefore, we propose that the rs10870077-C allele is a risk indicator of renal function and disease progression. Moreover, the cumulative burden of the risk C allele from rs9411205 causes earlier disease onset. This implies that early onset of IgAN may have a stronger genetic determination, which is in line with previous studies [10, 13]. Further studies need to be performed to support our hypothesis. In general, these findings indicate the involvement of CARD9 gene variants in IgAN progression and prognosis.

In our study, we also investigated the associations between candidate SNPs and history of infections for IgAN patients and found no robust associations, which may be due to the relatively small sample size. However, we do not deny the true relationships between CARD9 and infections and we will gather more clinical data regarding infections to explore the potential associations in a larger sample size. CARD9 polymorphisms are reported to confer susceptibility to several human diseases, especially infectious diseases and autoimmune disorders [15]. The association between the CARD9 rs10870077 SNP and inflammatory bowel diseases (IBD) was studied in a prior work, and this SNP was identified as a predisposing variant exhibiting an increased risk of IBD [34]. Recently, this SNP was also investigated as a potential functional genetic candidate factor for five different chronic inflammatory diseases, including AS, ulcerative colitis, and Crohn’s disease [35]. However, another study reported that associations between rs10870077 and susceptibility to IBD cannot be replicated in the Chinese Han population [36]. The SNP rs10870077 presumably regulates gene expression levels in the whole blood (online suppl. Table 4), and we therefore propose that rs10870077 has a cis functional effect on CARD9 signaling via modification of CARD9 expression. In addition, the SNP rs10870149 showed a nominal association with AS in a small GWAS [37], and the haplotype CGCCA containing rs9411205 is reported to be a protective factor against BD [38]. Moreover, SNPs rs10747047 and rs10781533 are predicted to play a regulatory role and are defined as cis-eQTLs after functional annotation (online suppl. Table 4, 6). These findings suggest that rs10747047 and rs10781533 may participate in the development of IgAN through the cis-transcriptional regulation of CARD9 gene expression in whole blood.

Collectively, these findings further suggest that CARD9 also plays a crucial role in promoting disease progression and affecting renal survival. CARD9 is shown to play a generalized role in mucosal immunity and be a vital molecule for activating the NF-κB pathway in prior studies [10]. It is also correlated with gastrointestinal tract disorders such as ulcerative colitis and Crohn’s disease and can mediate intestinal epithelial injury in mice [39, 40]. In this way, it may be assumed that polymorphisms of CARD9 have a regulatory function on CARD9 expression in whole blood, subsequently activating the NF-κB pathway, generating pro-inflammatory factors, and finally inhibiting intestinal repair, initiating mucosal immunity, and inducing IgAN.

Taken together, our findings indicate that variations within CARD9 are independently related to prognosis and can be optimized as potential biomarkers for clinicopathological manifestations that contribute to predicting the deterioration and progression of IgAN, such as Scr, eGFR, endocapillary hypercellularity lesions, and segmental sclerosis lesions. The exact functions of the genetic variations within CARD9 are not fully understood, but it may be assumed that they are involved in the NF-κB pathway and mucosal immunity. Our study provides insights into risk stratification for patients according to genotyping, which may be a significant future component of IgAN therapy and prognosis evaluation. However, patients with acute kidney injury were not excluded on admission, and there may be a potential selection bias of subjects. Therefore, further studies with stringent inclusion/exclusion criteria and larger sample size should be conducted to verify our results.

The polymorphisms of CARD9 are associated with disease severity and progression for IgAN in a Chinese Han population.

We appreciated all participants and staff for their efforts and contributions to this study.

This study was approved by the Research Ethics Board of First Affiliated Hospital of Sun Yat-Sen University and was conducted in accordance with the Helsinki Declaration (No. 2016215). Written informed consent was also obtained from all the participants simultaneously.

The authors have no conflicts of interest to declare.

This work was supported by the National Key Research and Development Project, China (No. 2016YFC0906100), National Natural Science Foundation of China (No. 81770661; No. 81920108008; No. 82170714), Guangdong-Hong Kong Joint Laboratory on Immunological and Genetic Kidney Diseases (2019B121205005), Guangdong Provincial Key Laboratory of Nephrology (No. 2020B1212060028), NHC Key Laboratory of Clinical Nephrology (Sun Yat-Sen University), Guangdong Provincial Program of Science and Technology (No. 2017A050503003), Young and Middle-aged Talents Program of The First Affiliated Hospital (Sun Yat-Sen University), and Guangdong Basic and Applied Basic Research Foundation (No. 2021A1515111054).

Ming Li and Dianchun Shi designed the study and were responsible for the project management. Chunhong He, Dianchun Shi, Lin Guo, and Zhong Zhong collected the clinical data of patients. Chunhong He analyzed the data. Chunhong He and Dianchun Shi wrote the article. Ming Li, Dianchun Shi, and Xueqing Yu conducted critical revision of the article. All authors reviewed and approved the final manuscript.

All data generated or analyzed during this study are included in this article and its online supplementary materials. Further inquiries can be directed to the corresponding author.

Chunhong He and Dianchun Shi both contributed equally to this work.