Abstract
Objective: The mechanism of immunoglobulin A nephropathy (IgAN) remains unclear. Genetic factors may be associated with the risk of IgAN. This study aims to identify the possible association of M268T (rs699) in the Angiotensinogen (AGT) gene and A1166C (rs5186) in the Angiotensin II receptor type 1 (ATR1) gene with IgAN risk. Methods: Study subjects included 351 patients with IgAN and 310 controls from the Chinese population. The tag SNPs (tSNPs) were genotyped by Sequenom MassARRAY RS1000. Statistical analysis of the association between tSNPs and IgAN was performed using the χ2 test and SNPStats software. Results: The AGT (M268T) genotypes were distributed in IgAN as CC 61.9%, CT 34.8%, and TT 3.2%, while in controls CC 64.1%, CT 31.3%, and TT 4.6%. Distribution of ATR1 (A1166C) was AA 87.7%, CA 12.3%, and CC 0%, while in controls AA 87.2%, CA 12%, and CC 0.8%. We further analyzed tSNPs under different inheritance models and found that there were no significant differences in the genotypes and allele frequencies of rs699 and rs5186 between two groups (p > 0.05). We also analyzed tSNPs based on the rate of pressure, proteinuria and Lee's classification, and no significant differences were found in the models (p > 0.05). Conclusion: rs699 in the AGT gene and rs5186 in the ATR1 gene were not associated with the risk and clinical outcomes of IgAN.
Introduction
I
Although the mechanism of IgAN remains unclear, gene polymorphisms in the renin-angiotensin system have been suggested as possible modulators in the progression of IgAN toward ESRD (Narita et al., 2003; Huang et al., 2010). However, the conclusions are inconsistent in different regions and races. For example, the ACE D allele is associated with IgAN risk in Asians, but not in Caucasian population (Qin et al., 2011; Mao et al., 2014). A previous study on association between polymorphisms of the genes Angiotensinogen (AGT) and Angiotensin II receptor type 1 (ATR1) with progression of IgAN revealed that the patients with one or both copies of the AGT T268 alleles had a faster rate of decline in their renal function than patients homozygous for the M268 allele (Pei et al., 1997). However, related studies did not find any association between IgAN and AGT or ATR1 in Japanese or Chinese (Huang et al., 2010; Teranishi et al., 2015).
The aim of this study is to further identify the possible association of M268T AGT (rs699) and A1166C ATR1 (rs5186) with the susceptibility of IgAN.
Materials and Methods
Study population
This study includes 351 patients with histologically proven IgAN and 310 healthy controls. All subjects were admitted to the First and Second Affiliated Hospital of Xi'an Jiaotong University from March 2009 to April 2014 and only people from the Chinese population were enrolled in this study. Informed consent was obtained from all subjects included. Clinical parameters and demographic features of control and patients were recorded. Specifically, we collected the data of gender, age, blood pressure, serum albumin level, serum creatinine level, serum IgA level, 24-h urine protein, Lee's classification, and so on. Following the approval of institutional ethics board, we collected morning fasting blood samples (5 mL) from patients and controls in vacutainer tubes containing acid citrate dextrose.
Genotyping methods
Genomic DNA was isolated from whole-blood samples using the GoldMag-Mini DNA Purification Kit (GoldMag Co. Ltd., Xi'an City, China). Sequenom MassARRAY Assay Design 3.0 software was used to design a multiplexed single-nucleotide polymorphism (SNP) MassEXTENDED assay (Gabriel et al., 2009). The tag SNP primer sequences of rs699 used for this study were 5′-ACGTTGGATGTGGACGTAGGTGTTGAAAGC-3′ (for forward) and 5′-ACGTTGGATGGATTGACAGGTTCATGCAGG-3′ (for reverse); and of rs5186 were 5′-ACGTTGGATGGAAAAGTCGGTTCAGTCCAC-3′ (for forward) and 5′-ACGTTGGATGATTCCTCTGCAGCACTTCAC-3′ (for reverse). Subsequently, SNP genotyping was performed by Sequenom MassARRAY RS1000 following the standard protocol recommended by the manufacturer. Finally, data were managed using Sequenom Typer 4.0 Software (Sequenom, San Diego, CA) (Thomas et al., 2007; Gabriel et al., 2009).
Statistical analysis
We used Microsoft Excel and SPSS 16.0 statistical package (SPSS, Chicago, IL) for statistical analysis. All p-values reported in this study were two-tailed and p < 0.05 was used as threshold of statistical significance. χ2 test was used to calculate the difference of each tSNP allele distribution between cases and controls (Adamec, 1964). We tested the genetic association between genotype and IgAN risk using SNPStats software from http://bioinfo.iconcologia.net/snpstats/start.htm (Solé et al., 2006). Odds ratios and 95% confidence intervals were calculated using unconditional logistic-regression analysis adjusted by age and sex (Bland and Altman, 2000).
Results
Patients characteristics
The clinical characteristics of the patients and controls are listed in Table 1. There were no significant differences in age and sex between the patients and control groups. To investigate the relationship with hypertension, proteinuria, and Lee's classification, the patients were divided into hypertensive and nonhypertensive groups, 24-h urine protein <3.5 or ≥3.5 g/24-h groups, and Lee's classification: I + II + III and IV + V groups.
p-Values were calculated from two-sided χ2 test.
p-Values were calculated by Student t-tests.
IgAN, immunoglobulin A nephropathy.
The association between genotype frequencies and IgAN risk
The genotype distribution of rs699 and rs5186 in the IgAN group and control group (Table 2). All of the tested tSNPs are in Hardy-Weinberg equilibrium in the controls of this study. The frequencies of T and C alleles at rs699 of AGT and C and A alleles at rs5186 were similar in IgAN patients and healthy donors (p = 0.85 and 0.60). We found that the distribution of genotypes for both AGT gene rs699 and ATR1 gene rs5186 was similar between the study patients and control subjects (p > 0.05).
p < 0.05 indicates statistical significance.
CI, confidence interval; OR, odds ratio.
The association between genotype frequencies and clinical characteristics
We further examined whether the distributions of the genotypes differed between subgroups segregated on the basis of urine protein, blood pressure, and Lee's classification. As shown in Table 3, we found that neither the rs699 polymorphism of the AGT gene nor the rs5186 polymorphism of the ATR1 gene were associated with pressure, proteinuria, or Lee's classification in our patients. For rs699, no evidence of the association was observed in the urine protein, blood pressure, and Lee's classification adjusted by age and sex among the models (p > 0.05). Moreover, genotypes of rs5186 examined in subgroups based on different levels of urine protein, blood pressure, and Lee's classification are similar in our study (p > 0.05).
p < 0.05 indicates statistical significance.
Model—Dominant: B/B versus others; Recessive: A/A versus others; Overdominant: B/B-A/A versus B/A.
A, the minor allele; B, the major allele.
Discussion
IgAN, a mesangial proliferative glomerulonephritis, accounts for about 40% of all biospsied primary glomerulonephritis in China and is a major cause of ESRD (Gong et al., 2002). Many gene polymorphisms have been reported to be associated with the development and/or progression of IgAN. A study in Caucasians with IgAN suggested that polymorphisms at the AGT gene loci are important markers for predicting progression to chronic renal failure (Pei et al., 1997), while some other studies in Japanese and Chinese found neither ATR1 (rs5186) nor AGT (rs699) predicted the IgAN progression of IgAN patients recently (Huang et al., 2010; Teranishi et al., 2015).
In this case-control study with a total of 351 IgAN cases and 310 controls, we investigated the associations of M268T (rs699) in AGT gene and A1166C (rs5186) in ATR1 gene with the risk and clinical characteristics of IgAN in a Chinese population. We found no significant association between the polymorphisms in AGT (rs699) and ATR1 (rs5186) and IgAN in the Chinese population. This concurred with the results of a previous study, which showed that the rs699 and rs5186 polymorphisms were not associated with IgAN or with its progression to ESRD in Chinese population (Huang et al., 2010). Since the occurrence of the CC genotype in rs5186 is very low in Chinese people, it seems unlikely to appear as a factor determining the susceptibility to IgAN and its progression.
Several limitations cannot be ignored in the present study. First, all participants were limited to Chinese who lived in Shaanxi Province or nearby, they may not be representative of the population in different regions of China. Second, the sample size (351 IgAN patients and 310 control subjects) was not relatively large enough.
In conclusion, our study suggested that rs699 in AGT gene and rs5186 in ATR1 gene were not associated with the risk and clinical outcomes such as hypertension, proteinuria, and Lee's classification in IgAN patients in Chinese. However, further studies with more samples from a broader population are required.
Footnotes
Acknowledgments
This work was financially supported by the National Nature Science Foundation of China (No. 81470968 and No. 81100530); the Science and Technology project of Shaanxi Province (2014K11-02-04-05).
Author Disclosure Statement
No competing financial interests exist.