Yuan-yuan Qi

Brief Report: Identification of MTMR3 as a Novel Susceptibility Gene for Lupus Nephritis in Northern Han Chinese by Shared-Gene Analysis With IgA Nephropathy

Several novel susceptibility genes for systemic lupus erythematosus (SLE) and IgA nephropathy have been identified in recent genome‐wide association studies. Since both lupus nephritis and IgA nephropathy are autoimmune diseases of the kidney, they may share common disease mechanisms that overlap with genetic susceptibility. To test this hypothesis, we sought to identify genetic variants associated with IgA nephropathy in lupus nephritis.

FCGR2B and FCRLB Gene Polymorphisms Associated with IgA Nephropathy

Background IgA nephropathy (IgAN) is a complex syndrome characterized by deposition of IgA and IgA containing immune complexes (ICs) composed of IgG and complement C3 proteins in the mesangial area of glomeruli. The low-affinity receptors for the Fc region of IgG (FcγRs) are involved in autoantibody/immune complex-induced organ injury as well as ICs clearance. The aim of the study was to associate multiple polymorphisms within FCGR gene locus with IgAN in a large Chinese cohort. Patients and Methods 60 single nucleotide polymorphisms (SNPs) spanning a 400 kb range within FCGR gene locus were analyzed in 2100 DNA samples from patients with biopsy proven IgAN and healthy age- and sex-matched controls from the same population in Chinese. Results Among the 60 SNPs investigated, 15 gene polymorphisms within FCGR gene locus (25%) were associated with susceptibility to IgAN. The most significantly associated SNPs within individual genes were FCGR2B rs12118043 (p = 8.74*10−3, OR 0.76, 95% CI 0.62–0.93), and FCRLB rs4657093 (p = 2.28*10−3, OR 0.77, 95% CI 0.65–0.91). Both conditional analysis and linkage disequilibrium analysis suggested they were independent signals associated with IgAN. Associations between FCGR2B rs12118043 and proteinuria (p = 3.65×10−2) as well as gross hematuria (p = 4.53×10−2), between FCRLB rs4657093 and levels of serum creatinine (p = 2.67×10−2) as well as eGFR (p = 5.41*10−3) were also observed. Electronic cis-expression quantative trait loci analysis supported their possible functional significance, with protective genotypes correlating lower gene expressions. Conclusion Our data from genetic associations and expression associations revealed potentially pathogenic roles of Fc receptor gene polymorphisms in IgAN.

A Replication Study from Chinese Supports Association between Lupus-Risk Allele in TNFSF4 and Renal Disorder

A recent phenotypic association study of genetic susceptibility loci in SLE suggested that TNFSF4 gene might be useful to predict renal disorder in lupus patients. To replicate the association, two single-nucleotide polymorphisms (SNPs: rs2205960 and rs10489265) were genotyped in 814 SLE patients. Correlations between genotypes and TNFSF4 expression were determined. The stainings of TNFSF4 in renal biopsy specimens were checked by immunohistochemistry. The SNPs of TNFSF4 were associated with renal involvement in lupus patients from the Chinese population (P values for rs2205960 and rs10489265 were 0.014 and 0.005 in additive model, resp.). An association between risk genotypes and low C3 levels was also observed (P = 0.034). Functional prediction suggested that rs2205960 had a regulatory feature. The risk alleles seemingly correlated with lower TNFSF4 expression. Strong TNFSF4 expression was detected in lymph nodes and “apparently normal” paratumor renal biopsy but not in renal biopsies from lupus nephritis. In genome-wide expression data, TNFSF4 was also observed to be downregulated in LN in both glomeruli and tubulointerstitium from kidney biopsies. However, the associations were marginally significant. Our data firstly replicated the association of TNFSF4 with renal disorder in SLE patients in the Chinese population, which supported that TNFSF4 may act as a marker of lupus nephritis. The detailed mechanisms of its role in pathogenesis will still be further needed.

Cumulative Effects of Variants Identified by Genome-wide Association Studies in IgA Nephropathy

The effect of genetic markers associated with IgA nephropathy on risk of disease in sub-phenotype and progression is uncertain. Data from 2096 Chinese patients were used to create both un-weighted (uw) and weighted (w) genetic risk score (GRS). The association between GRS with disease susceptibility and clinical parameters were assessed. All nine selected single nucleotide polymorphisms (SNPs) were associated with susceptibility to IgAN. uwGRS and wGRS showed a similar fit in disease associations. With every 1-unit increase in the uwGRS, the disease risk increased by approximately 20%; whereas every one standard deviation increase in the wGRS, disease risk increased by approximately 40% ~ 60%. Association between rs3803800 and serum IgA was replicated, and risk groups in GRSs were associated with increased IgA/IgA1 levels. uwGRS9 ≥ 16 was an independent predictor for end stage renal disease (ESRD) in IgAN, with a relative risk of 2.52 (p = 6.68 × 10−3). In conclusion, we observed that GRSs comprising nine SNPs identified in a GWAS of IgAN were strongly associated with susceptibility to IgAN. The high risk GRS9 group had a high risk of ESRD in follow-up.

Rare Variants of ATG5 Are Likely to Be Associated With Chinese Patients With Systemic Lupus Erythematosus

AbstractRecently, common variants within or near ATG5, which is a key autophagy gene required for the formation of autophagosomes, have been identified as a candidate gene of systemic lupus erythematosus (SLE) by several genome-wide association studies. Moreover, elevated ATG5 expression was observed in SLE as well as other autoimmune diseases. However, no significant associations between variants within ATG5 and SLE were identified in several Chinese populations. The present study was conducted to further check the genetic role of ATG5 by associating both common and rare variants of ATG5 in Chinese patients with lupus nephritis (LN), a major phenotype with poor prognosis in SLE.To detect the association of common variants of ATG5 with LN, 7 tagging single nucleotide polymorphisms (SNPs) designed in immunochip and 4 SNPs reported to be associated with SLE were genotyped in 500 LN patients and 500 healthy controls. Furthermore, direct sequencing of exons and their flanking regions in 90 LN patients, 30 SLE patients, and 60 healthy controls were performed. Functional genomic annotation was performed by using public databases.None of the 11 tagging SNPs was observed to be associated with LN. By sequencing, 13 variants were identified, including 5 common SNPs, 7 not previously described, and 1 reported as rare variants (<1%) in the Single Nucleotide Polymorphism Database or the 1000 Genome project. None of the 5 common SNPs showed significant association between patients and controls, whereas increased frequencies of rare or novel variants were observed in patients compared with healthy controls, with 6/90 in LN patients, 2/30 in SLE patients, and 1/163 in healthy controls. Although these rare variants were observed to be located in the flanking regions of exons instead of missense mutations, patients carrying them tended to have severe clinical phenotype, and in silicon analysis suggested their regulatory effects.Increased frequencies of rare variants of ATG5 were identified in patients with LN and SLE compared with healthy controls, highlighting a likely important role of rare ATG5 variants in Chinese SLE patients.

Autophagy-related gene LRRK2 is likely a susceptibility gene for systemic lupus erythematosus in northern Han Chinese

Autophagy is associated with various immune diseases, including systemic lupus erythematosus (SLE). Seven variants within autophagy-related genes previously reported to show top association signals by genome-wide association studies in immune diseases were selected for analysis. Initially, 510 SLE patients (631 controls) were enrolled in the study. An additional independent cohort of 511 SLE patients (687 controls) was included for replication. Polymorphism rs2638272 in LRRK2 gene showed significant association with susceptibility to SLE (P = 1.14 × 10−2) within the initial patient population. This was independently replicated (second patient cohort), and was reinforced with combination (P = 2.82 × 10−3). By combining multiple layers of regulatory effects, rs1491941 in high linkage disequilibrium with rs2638272 (r2 = 0.99) was regarded to have the strongest function in LRRK2. The rs1491941 protective A-allele exhibited an increase of nuclear protein binding, and an increase in LRRK2 transcription compared with G-allele. Furthermore, we observed increased transcription levels of LRRK2 in peripheral blood mononuclear cells from SLE patients compared with controls. In conclusion, we have identified a novel genetic association between the autophagy-related LRRK2 gene and susceptibility to SLE. By integrating layers of functional data, we derived the beneficial effect of autophagy on the pathogenesis of SLE.

Association of Variants in CCR6 With Susceptibility to Lupus Nephritis in Chinese

Systemic lupus erythematosus (SLE) is a common autoimmune disease with a complex genetic etiology. In the last 2 years, exciting insights supporting the notion that the interleukin17 (IL-17)–producing Th 17 cell subset exerts proinflammatory activity in the pathogenesis of several autoimmune or allergic diseases, including asthma, atopic dermatitis, rheumatoid arthritis (RA), and SLE, have emerged (1,2). High production of IL-17 is observed in SLE patients, and several compounds targeting IL-17 have demonstrated marked clinical efficacy (1,2). Genetic studies have reproducibly demonstrated an association between susceptibility to RA and polymorphisms of the CCR6 gene, a surface marker for Th17 cells (3,4). However, to date no direct link between CCR6 and SLE has been found from genetic studies, although higher percentages of CCR61 T helper cells were observed in patients with SLE, and especially in those with lupus nephritis (LN), compared to controls (5,6). The current study was undertaken to investigate whether there are any associations between CCR6 polymorphisms and susceptibility to LN. The discovery cohort in this study comprised 500 LN patients (mean 6 SD age 32.0 6 11.5 years; 83.6% female) and 900 unrelated healthy individuals (age 31.5 6 8.4 years; 40.1% female). The replication cohort consisted of 626 LN patients (age 32.5 6 12.8 years; 84.0% female) and 1,932 healthy individuals (age 40.9 6 12.7 years; 47.7% female), as well as 1,063 SLE patients without evidence of renal involvement (age 36.6 6 13.4 years; 90.5% female) as an additional control group. All of the SLE patients met the American College of Rheumatology revised criteria for the disease (7). The study was approved by the medical ethics committee of Peking University, and all subjects provided informed consent. Two intronic single-nucleotide polymorphisms (SNPs), rs3093023 and rs3093024, known to be associated with RA with high association signals (3,4), were selected for study, and genotyping was conducted by TaqMan allele discrimination assay (Applied Biosystems) as previously described (8,9). Direct sequencing was performed in 45 samples that were randomly selected on the basis of rs3093024 genotype (15 subjects with the A/A genotype, 15 with the G/A genotype, and 15 with the G/G genotype) to determine whether rs3093024 could tag the recently identified functional CCR6DNP (a triallelic dinucleotide polymorphism of CCR6) (3). The power of the study was calculated using CaTS (http://www.sph.umich.edu/csg/abecasis/CaTS/). As rs3093023 and rs3093024 are in high linkage disequilibrium (LD) (r 5 0.98), indicating that statistical tests performed on each SNP are highly dependent, no multiple correction was applied. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated. Statistical analyses were performed with SPSS16.0 software. Functional annotations of variants were obtained from the HaploReg and regulomeDB databases. A total of 5,021 Chinese subjects were included as described above. The call rates for rs3093024 and rs3093023 were 99.82% and 98.94%, respectively. Both SNPs studied were in Hardy-Weinberg equilibrium in patients and controls (P . 0.05). With the expected frequency of the rs3093024 and rs3093023 minor allele (40%), the study had a power of 96– 99% to detect a 1.20-fold increased risk in the discovery and replication cohorts separately and combined. The frequency of the A allele of rs3093024 was significantly higher in patients with LN as compared to healthy controls both in the discovery cohort and in the replication cohort (P 5 1.32 3 10 and P 5 4.15 3 10, respectively) (Table 1).

Variants in CCR6 are associated with susceptibility to lupus nephritis in Chinese

Systemic lupus erythematosus (SLE) is a common autoimmune disease with a complex genetic etiology. In the last 2 years, exciting insights supporting the notion that the interleukin17 (IL-17)–producing Th 17 cell subset exerts proinflammatory activity in the pathogenesis of several autoimmune or allergic diseases, including asthma, atopic dermatitis, rheumatoid arthritis (RA), and SLE, have emerged (1,2). High production of IL-17 is observed in SLE patients, and several compounds targeting IL-17 have demonstrated marked clinical efficacy (1,2). Genetic studies have reproducibly demonstrated an association between susceptibility to RA and polymorphisms of the CCR6 gene, a surface marker for Th17 cells (3,4). However, to date no direct link between CCR6 and SLE has been found from genetic studies, although higher percentages of CCR61 T helper cells were observed in patients with SLE, and especially in those with lupus nephritis (LN), compared to controls (5,6). The current study was undertaken to investigate whether there are any associations between CCR6 polymorphisms and susceptibility to LN. The discovery cohort in this study comprised 500 LN patients (mean 6 SD age 32.0 6 11.5 years; 83.6% female) and 900 unrelated healthy individuals (age 31.5 6 8.4 years; 40.1% female). The replication cohort consisted of 626 LN patients (age 32.5 6 12.8 years; 84.0% female) and 1,932 healthy individuals (age 40.9 6 12.7 years; 47.7% female), as well as 1,063 SLE patients without evidence of renal involvement (age 36.6 6 13.4 years; 90.5% female) as an additional control group. All of the SLE patients met the American College of Rheumatology revised criteria for the disease (7). The study was approved by the medical ethics committee of Peking University, and all subjects provided informed consent. Two intronic single-nucleotide polymorphisms (SNPs), rs3093023 and rs3093024, known to be associated with RA with high association signals (3,4), were selected for study, and genotyping was conducted by TaqMan allele discrimination assay (Applied Biosystems) as previously described (8,9). Direct sequencing was performed in 45 samples that were randomly selected on the basis of rs3093024 genotype (15 subjects with the A/A genotype, 15 with the G/A genotype, and 15 with the G/G genotype) to determine whether rs3093024 could tag the recently identified functional CCR6DNP (a triallelic dinucleotide polymorphism of CCR6) (3). The power of the study was calculated using CaTS (http://www.sph.umich.edu/csg/abecasis/CaTS/). As rs3093023 and rs3093024 are in high linkage disequilibrium (LD) (r 5 0.98), indicating that statistical tests performed on each SNP are highly dependent, no multiple correction was applied. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated. Statistical analyses were performed with SPSS16.0 software. Functional annotations of variants were obtained from the HaploReg and regulomeDB databases. A total of 5,021 Chinese subjects were included as described above. The call rates for rs3093024 and rs3093023 were 99.82% and 98.94%, respectively. Both SNPs studied were in Hardy-Weinberg equilibrium in patients and controls (P . 0.05). With the expected frequency of the rs3093024 and rs3093023 minor allele (40%), the study had a power of 96– 99% to detect a 1.20-fold increased risk in the discovery and replication cohorts separately and combined. The frequency of the A allele of rs3093024 was significantly higher in patients with LN as compared to healthy controls both in the discovery cohort and in the replication cohort (P 5 1.32 3 10 and P 5 4.15 3 10, respectively) (Table 1).

Polymorphism rs3828903 within MICB Is Associated with Susceptibility to Systemic Lupus Erythematosus in a Northern Han Chinese Population

Objectives. The variant rs3828903 within MICB, a nonclassical MHC class I chain-related gene, was detected to contribute to systemic lupus erythematosus (SLE) in a Caucasian population. This study aimed to investigate the association in a northern Han Chinese population. Methods. We recruited 1077 SLE patients and 793 controls for analysis. rs3828903 was genotyped by TaqMan allele discrimination assay. Using the public databases, its functional annotations and gene differential expression analysis of MICB were evaluated. Results. Significant association between the allele G of rs3828903 and risk susceptibility to SLE was observed after adjusting for sex and age (P = 1.87 × 10−2). In silico analyses predicted a higher affinity to transcription factors for allele G (risk) and cis-expression quantitative trait loci (cis-eQTL) effects of rs3828903 in multiple tissues (P ranging from 2.79 × 10−6 to 6.27 × 10−38). Furthermore, higher mRNA expressions of MICB were observed in B cells, monocytes, and renal biopsies from SLE patients compared to controls. Conclusion. An association between rs3828903 and susceptibility to SLE has been detected in a Chinese population. This together with the functional annotations of rs3828903 converts MICB into a main candidate in the pathogenesis of SLE.

Detecting Genetic Associations between ATG5 and Lupus Nephritis by trans-eQTL

Objectives. Numerous loci were identified to perturb gene expression in trans. As elevated ATG5 expression was observed in systemic lupus erythematosus (SLE), the study was conducted to analyze the genome-wide genetic regulatory mechanisms associated with ATG5 expression in a Chinese population with lupus nephritis (LN). Methods. The online expression quantitative trait loci database was searched for trans-expression single nucleotide polymorphisms (trans-eSNPs) of ATG5. Tagging trans-eSNPs were genotyped by a custom-made genotyping chip in 280 patients and 199 controls. For positive findings, clinical information and bioinformation analyses were performed. Results. Four trans-eSNPs were observed to be associated with susceptibility to LN (P < 0.05), including ANKRD50 rs17008504, AGA rs2271100, PAK7 rs6056923, and TET2 rs1391441, while seven other trans-eSNPs showed marginal significant associations (0.05 < P < 0.1). Correlations between the trans-eSNPs and ATG5 expression and different expression levels of ATG5 in SLE patients and controls were validated, and their regulatory effects were annotated. However, no significant associations were observed between different genotypes of trans-eSNPs and severity or outcome of the patients. Conclusion. Using the new systemic genetics approach, we identified 10 loci associated with susceptibility to LN potentially, which may be complementary to future pathway based genetic studies.