The Role of hOGG1 C1245G Polymorphism in the Susceptibility to Lupus Nephritis and Modulation of the Plasma 8-OHdG in Patients with Systemic Lupus Erythematosus †

We investigated whether the C1245G polymorphism of human 8-oxoguanine glycosylase 1 (hOGG1) gene confers the susceptibility to systemic lupus erythematosus (SLE) occurrence of lupus nephritis and affects the plasma level of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in patients with SLE. A total of 45 healthy controls and 85 SLE patients were recruited. The C1245G polymorphism of the hOGG1 gene was determined by direct sequencing. The frequency of occurrence of the hOGG1 1245 GG genotype in SLE patients was 31.8% (27/85), which is lower than that of healthy controls of 53.3% (24/45). Thirty-three (33/85, 38.8%) SLE patients developed lupus nephritis. Significantly, SLE patients harboring the hOGG1 1245 GG genotype had a higher incidence to develop lupus nephritis than did those harboring the hOGG1 1245 CC or CG genotype (15/27, 55.6% vs.18/58, 31.0%, p = 0.031). Divided into subgroups, SLE patients harboring the hOGG1 1245 GG genotype had the highest plasma levels of 8-OHdG among patients with all genotypes, with regard to the coexistence of lupus nephritis (p = 0.020, ANOVA), including those with nephritis harboring the hOGG1 1245 CC or CG genotypes (p = 0.037), those without nephritis harboring the hOGG1 1245 GG genotype (p = 0.050), and those without nephritis harboring the hOGG1 1245 CC or CG genotype (p = 0.054). We conclude that the C1245G polymorphism of hOGG1 may be one of the factors that confer the susceptibility to lupus nephritis and modulate the plasma level of 8-OHdG in patients with SLE.


Introduction
Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by the generation of arrays of pathogenic auto-antibodies that cause cascades of organ damages [1]. Similar to several human degenerative diseases, elevated reactive oxygen species (ROS) and ROS-triggered oxidative damages have been demonstrated to be involved in the pathogenesis of SLE [2][3][4][5]. Theoretically, ROS can attack the intracellular molecules randomly, e.g., protein, lipid, RNA and DNA, and thereby cause cellular dysfunction. Among these oxidative DNA damages, the formation of 8-hydroxy-2'-deoxyguanosine , an oxidative damage product of 2'-deoxyguanine (dG), is the most common one [6]. Normally, the dG is matched with 2'-deoxycytosine (dC) during DNA replication. However, if the 8-OHdG is not repaired efficiently and accumulated in the affected tissues, it would pair wrongly with 2'-deoxyadenine (dA) rather than dC, and cause a G→C to T→A transversion [7]. As a result, if the affected gene harbors 8-OHdG, functional impairment may subsequently develop.
In human cells, 8-OHdG is mainly repaired by human 8-oxoguanine glycosylase 1 (hOGG1) through the base excision repair mechanism [8]. There have been three hOGG1 genotypes identified because of a C to G shifting at base-pair (bp) 1245 (C1245G) in exon 7 of the hOGG1 gene. Such a shifting causes a serine (Ser) to cysteine (Cys) substitution at codon 326 during translation. This phenomenon is referred to as hOGG1 C1245G polymorphism. Considering the DNA repair efficiency, homozygous hOGG1 1245 CC genotype has the highest DNA repair activity followed by the heterozygous hOGG1 1245 CG genotype and then the homozygous hOGG1 1245 GG genotype in order [9]. This discrepancy in hOGG1 repair activity implies that hOGG1 1245 GG genotype with lower efficiency in DNA repair might be related to a higher level of 8-OHdG in human tissues [10], and may contribute to the development of several human degenerative diseases, including type 2 diabetes mellitus [11], Huntington's disease [12], chronic obstructive pulmonary disease [13], Graves' ophthalmopathy [14], and higher susceptibility to cancer formation, including lung cancer [15] and esophageal squamous cell carcinoma [16]. However, the role of hOGG1 C1245G polymorphism in the pathogenesis of SLE has remained obscure.
Lupus nephritis is a serious clinical manifestation of SLE. Studies revealed that excessive ROS production and oxidative stress may exacerbate inflammation, dampen antioxidant capacity and lead to tissue damage and the formation of lupus nephritis in SLE patients [3,17]. Furthermore, some authors also reported that hOGG1 1245 GG genotype is related to higher 8-OHdG content in patients with end-stage renal disease (ESRD) [18,19]. In one of our previous studies, we demonstrated that SLE patients had higher plasma levels of 8-OHdG than did healthy controls [20]. In light of these findings, we aimed to appraise (1) whether the hOGG1 1245 GG genotype contributes to a higher rate of SLE development and higher plasma level of 8-OHdG; (2) whether the hOGG1 1245 GG genotype confers a higher incidence to the development of nephritis in SLE patients; and (3) whether the hOGG1 1245 GG genotype modulates the plasma level of 8-OHdG in SLE patients with or without nephritis.

Distributions of hOGG1 C1245G Polymorphism in Healthy Controls and SLE Patients
The genotypes of hOGG1 C1245G polymorphisms are illustrated in Figure 1. As revealed by direct sequencing, among the 45 healthy controls, 3 (6.7%) had the CC, 18 (40.0%) had the CG and 24 (53.3%) had the GG genotype, respectively. On the contrary, among the 85 SLE patients, 14 (16.5%) had the CC genotype, 44 (51.8%) had the CG genotype and 27 (31.8%) had the GG genotype, respectively. For the subsequent comparative analysis, CC genotype plus CG genotype were gathered as one group in this study ( Table 1).
As shown in Table 1, among the 85 SLE patients, 27 (31.8%) harbored the GG genotype, and the rate was significantly lower than the 24 (53.3%) of the 45 healthy controls (p = 0.017). For further comparisons, healthy controls harboring the CC or CG genotypes were designated as Group A, healthy controls harboring the GG genotype were designated as Group B, SLE patients harboring the CC or CG genotypes were designated as Group C and SLE patients harboring the GG genotype were designated as Group D, respectively.

Figure 1.
Illustrations are the results of direct sequencing of the DNA segment harboring the hOGG1 gene. Green, red, blue and black colors represent A, T, C and G, respectively. systemic lupus erythematosus (SLE) patient S4 harbored a single C peak at bp 1245 (homozygous C/C allele) and belonged to the homozygous CC genotype. SLE patient S6 harbored a single peak of G at bp 1245 (homozygous G/G allele) and belonged to the homozygous human 8-oxoguanine glycosylase 1 (hOGG1) GG genotype. SLE patient S5 harbored concurrent C and G peaks at bp1245 (heterozygous C/G allele) and thus belonged to the heterozygous CG genotype.  Error bar = 95% C.I. We have demonstrated that SLE patients had a higher plasma level of 8-OHdG than did the healthy controls [20]. Since 8-OHdG is repaired by hOGG1 in human tissues, the possible influence of hOGG1 1245 GG genotype on SLE susceptibility or the elevation of 8-OHdG level in SLE patients deserves evaluation. In our study cohort, the percentage of hOGG1 1245 GG genotype in SLE patients was 31.8%, which was lower (p = 0.017) than that in healthy controls (24/45, 53.3%). On the contrary, the percentage of hOGG1 CC genotype in healthy control was 6.7% (3/45), which was lower than in SLE patients (16.5%, 14/85). As reported in the published data, hOGG1 1245 GG genotype frequency in SLE patients is variable, higher in normal Taiwanese (20.5%) population [10] and Japanese populations (13.7%) [21], similar to normal Chinese population (31.4%) [18], and lower in another normal Taiwanese cohorts (37.6% [22] and 42.0% [19]). There is actually a wide range of frequency in the occurrence of hOGG1 1245 GG genotype in the normal Taiwanese population. Furthermore, the percentage of CC genotype in our healthy cohort was 6.7%, which was lower than those reported previously [10,18,19,21,22]. This might suggest that our healthy cohort had a lower repair activity for oxidative damage to DNA, but it was not that low to lead to a significant elevation of plasma levels of 8-OHdG compared with those found in SLE patients. Our results have indicated that hOGG1 1245 GG genotype does not predispose to SLE and is not the prominent contributor to a higher plasma 8-OHdG levels in SLE patients. However, if SLE patients harbor the hOGG1 1245 GG genotype (Group D), they might exhibit diminished capacity to ameliorate oxidative stress and resulted in higher plasma levels of 8-OHdG (Table 2 and Figure 2). Alternatively, there might be other contributing factors not yet found that also resulted in higher 8-OHdG. Although the CC genotype was previously reported as a wild type, it was mainly found in the European/American populations. The distribution variations of the hOGG1 gene polymorphisms might be dependent on ethnic group. Thus, the CC, CG or GG genotype cannot be regarded as the wild type of the hOGG1 gene.

Distributions of hOGG1 C1245G Polymorphisms in SLE Patients with or without Lupus Nephritis
As shown in Table 3, the 27 SLE patients who harbored the GG genotype were more susceptible to the development of lupus nephritis compared with the other 58 SLE patients who harbored the CC or CG genotypes (15/27, 55.6% vs.18/58, 31.0%, p = 0.031). Thus, for further comparisons, the 85 SLE patients were divided into 4 groups according to the clinical presentations of lupus nephritis and hOGG1 C1245G polymorphism. These include SLE patients without nephritis and with the CC/CG genotypes (Group I), SLE patients without nephritis and with the GG genotype (Group II), SLE patients with nephritis and with the CC/CG genotypes (Group III), and SLE patients with nephritis and with the GG genotype (Group IV).

Plasma Levels of 8-OHdG in SLE Patients as Related to the Clinical Presentations of Lupus Nephritis and hOGG1 C1245G Polymorphism
In the patients who harbored the CC or CG genotypes (Group I and Group III), there was no significant difference in their plasma levels of 8-OHdG (0.224 ± 0.064 vs. 0.201 ± 0.046 ng/mL, p = 0.165). In those who harbored the GG genotype (Group II and Group IV), Group IV had higher plasma levels of 8-OHdG than Group II (0.280 ± 0.145 vs. 0.197 ± 0.040 ng/mL, p = 0.050). In the 33 SLE patients with nephritis (Group III and Group IV), Group IV had higher plasma levels of 8-OHdG than did Group III (0.280 ± 0.145 vs. 0.201 ± 0.046 ng/mL, p = 0.037). However, such a difference was not observed between Group I and II in the 52 SLE patients without nephritis (0.197 ± 0.040 vs. 0.224 ± 0.064 ng/mL, p = 0.164). Furthermore, SLE patients with nephritis who harbored the GG genotype (Group IV) had higher plasma levels of 8-OHdG than SLE patients without nephritis who harbored the CC or CG genotypes (Group I) (0.280 ± 0.145 vs. 0.224 ± 0.064 ng/mL, p = 0.054). Group IV SLE patients with nephritis and with the GG genotype had higher levels of plasma 8-OHdG than did patients in the other groups (p = 0.020, ANOVA) ( Table 4 and Figure 3). Table 4. Plasma levels of 8-OHdG in SLE patients with or without nephritis as related to the hOGG1 C1245G polymorphisms. M, mean, SD, standard deviation; * compared between SLE patients without nephritis who harbored the CC or CG genotypes (Group I) and SLE patients with nephritis who harbored the CC or CG genotypes (Group III), and between SLE patients without nephritis who harbored the GG genotype (Group II) and SLE patients with nephritis who harbored the GG genotype (Group IV); ** compared between SLE patients without nephritis who harbored the GG genotype and those harboring the CC or CG genotypes (Group II and Group I), and in SLE patients with nephritis who harbored the GG genotype and those harboring the CC or CG genotype (Group IV and Group III); *** compared between SLE patients with nephritis who harbored the GG genotype and SLE patients without nephritis who harbored the CC or CG genotypes (Group IV and Group I); **** compared among all groups (ANOVA).
It was reported that hOGG1 1245 GG genotype is related to the development of several human degenerative diseases, including type 2 diabetes mellitus [11], Huntington's disease [12], chronic obstructive pulmonary disease [13], chronic renal failure [23] and Graves' ophthalmopathy [14]. Furthermore, the hOGG1 1245 GG genotype is associated with multiple vessel involvement in patients with coronary artery disease [22], and with the progression of IgA nephropathy [23]. However, the role of hOGG1C1245G polymorphism in lupus nephritis remains unclear. Interestingly, we demonstrated that in our SLE cohort, the 27 patients harboring the GG genotype had a significantly higher rate (15/27, 55.6%) of nephritis than the other 58 patients harboring the CC or CG genotypes (18/58, 31.0%, p = 0.031, Table 3). Several previous studies have shown that the GG genotype is related to the higher 8-OHdG concentration in patients with ESRD [18,19]. Thus, it is important to clarify whether the hOGG1 1245 GG genotype in patients with lupus nephritis confers a higher oxidative damage to DNA compared with SLE patients without nephritis. As shown in Table 4 and Figure 3, SLE patients with nephritis and with the GG genotype (Group IV) had a higher plasma level of 8-OHdG than did other groups of SLE patients (p = 0.020, ANOVA). Therefore, we suspect that the SLE patients harboring hOGG1 1245 GG genotype tend to have a higher risk of developing nephritis and accumulating more oxidative damage to DNA in affected tissues.

Recruitment of SLE Patients and Healthy Controls
With fulfillment of the American College of Rheumatology (ACR) criteria for the classification of SLE [24], we collected a total of 85 SLE patients (male/female = 12/73) with a mean age of 44.6 years from Outpatient Clinic of the Division of Allergy, Immunology and Rheumatology, Taipei Veterans General Hospital. 45 age-matched healthy controls (male/female = 7/38) with a mean age of 42.6 years served as the controls. We included the same cohort, their demographic data and plasma levels of 8-OHdG, which had been reported previously for other research purposes [20,25]. Among the 85 SLE patients, 33 (38.8%) were confirmed to have lupus nephritis according to the ACR classification criteria [24,25]. Approval from the Institutional Review Board of Taipei Veterans General Hospital was obtained to conduct this study.

Blood Sample Collection, Plasma Separation and Leukocyte DNA Preparation
About 10 mL of venous blood was drawn in a specific tube (VACUETTE ® , Greiner Bio-one, Monroe, NC, USA) that contained EDTA. After centrifugation at 3000× g for 10 min at 4 °C, the plasma and leukocyte-enriched buffy coat were collected separately. The plasma was then subject to the determination of 8-OHdG. After addition with the erythrocyte (RBC) lysis buffer (Bioman, Taiwan), leukocyte DNA was extracted by standard phenol-chloroform procedures and kept at −20 °C until use [26].

Determination of 8-OHdG in Plasma by ELISA
Plasma 8-OHdG was detected by using ELISA kit (High Sensitive 8-OHdG Check ELISA, Japan Institute for the Control of Aging, Nikken SEIL Co., Ltd., Fukuroi, Shizuoka, Japan) according to the manufacturer's instruction [20,27]. Each reaction was done in duplicate and the mean value was used for data presentation.

Analysis of hOGG1 C1245G Polymorphisms
The hOGG1 C1245G polymorphisms were detected by direct genome sequencing. Briefly, 1 μL (10 ng/μL) of the leukocyte DNA was amplified in a 50 μL PCR reaction that containing 25 μL of RBC SensiZyme ® Hotstart Taq Premix (RBC Bioscience, Taipei, Taiwan), 22 μL of PCR grade H2O, 1 μL of each primer (F: 5'-ACTGTCACTAGTCTCACCAG-3' and R: 5'-GGAAGGTGCTTGGGGAAT-3') to generate a 200-bp product [16]. PCR conditions were set as: 95 °C for 10 min followed by 40 cycles of amplification at 95 °C for 30 s, 58 °C for 30 s and 72 °C for 60 s, and a final extension at 72 °C for 7 min. After gel electrophoresis to confirm the DNA band of interest, 30 μL of the PCR-amplified product was subjected to direct sequencing (MB Mission Biotech, Taipei, Taiwan). During sequencing, single C peak at bp1245 indicates a homozygous CC genotype, single G peak indicates a homozygous GG genotype, and concurrent C plus G peaks indicates the heterozygous CG genotype [16,21].

Statistical Analysis
The plasma 8-OHdG concentrations are presented as the mean ± standard deviation (M ± SD). The continuous variables between two groups or among three or more groups were compared using the Student's t-test, Mann-Whitney U-test, ANOVA (analysis of variance) or Kruskal-Wallis test when appropriate. A difference was considered significant when a p-value was less than 0.05.

Conclusions
In conclusion, we have provided evidence that hOGG1 C1245G polymorphism might be one of the multiple factors that confer a higher susceptibility to lupus nephritis and modulate the plasma level of 8-OHdG in patients with SLE.