Superoxide Dismutase 1 (SOD 1) A251G Polymorphism

Introduction

Oxidative stress is accepted as one of the main factors involved in the development and progression of many diseases [1], [2]. Many studies have shown the relationship between the genetic polymorphism of SODs genes and multifactorial diseases such as cancers and age-related macular degeneration [3], [4], [5], [6], [7]. Superoxide dismutases are a class of enzymes that catalyze the dismutation of superoxide into oxygen and hydrogen peroxide. They play an important role in the antioxidant mechanism in almost all cells exposed to oxygen [8]. Three isoforms of SOD [cytoplasmic superoxide dismutase (SOD1), mitochondrial superoxide dismutase (SOD2) and extracellular superoxide dismutase (SOD3)] have been identified in mammals [9], [10], [11]. SOD1 accounts for approximately 85% of the total cellular SOD activity of the most mammalian cells [12].

The SOD1 gene is localized in the chromosome 21 (region 21q22) in humans. It contains four introns and five exons. Possible associations of single nucleotide polymorphisms in SOD1 gene in patients with diabetes mellitus, amyotrophic lateral sclerosis, encephalitis, and breast cancer have been reported [2], [13], [14]. A genetic polymorphism of A251G (rs2070424) is located in 3rd intron of the SOD1 gene. This polymorphism plays important roles as a risk factor of many diseases, including Alzheimer's disease [15], type 1 diabetic nephropathy [16], in age-related cataract [4], noise-induced hearing loss [17], myelomeningocele [18], antituberculosis drugs-induced hepatitis [19], ulcerative colitis [20] and gastric cancer [21].

This study is the first study of the Turkish population in the SOD1 A251G polymorphism. The aim of this study was to determine the frequencies of SOD1 A251G gene polymorphisms in a Turkish population. Here, we describe a polymerase chain reaction (PCR) based method for identifying single nucleotide polymorphism.

Materials and methods

Genotyping

SOD1 A251G in polymorphism in Turkish population was examined using a MspI-RFLP method. For amplifying this polymorphism, forward primer 5′-AGTACTGTCAACCACTAGCA-3′ and reverse primer 5′-CCAGTGTGCGGCCAATGATG-3′ were used. PCR reactions contained 1 μL (10 pmol/μL) of each primer, 1 μL dNTPs (1 mmol/L), 3μL of 25 mmol/L MgCI₂, 5 μL buffer, 1 Units of Taq DNA polymerase and 37.5 μL sterile deionized water. 50–100 ng DNA in a total volume of 50 μL. PCR conditions were 94 °C for 4 min, followed by 35 cycles of 94 °C for 50 s, 63 °C for 50 s, 72 °C for 50 s, and a final extension step at 72 °C for 7 min. Amplified products were digested with MspI (MBI Fermentas, Burlington, CA) at 37 °C for 5 h and analyzed with 2% agarose gels. Genotypes were determined for the polymorphism as 570 bp AA; two fragments of 369 and 201 bp GG; and three fragments of 570, 369, and 201 bp AG [4] (Figure 1).

Figure 1:

PCR-RFLP patterns of polymorphisms of SOD1 A251G, SOD1 PCR product (570 bp); 2, AA (570 bp); 3, AG (570, 369, 201 bp); 4, GG (369, 201) bp; Marker GeneRuler 100bp Plus DNA Ladder; 3000, 2000, 1500, 1200, 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100. It contains two reference bands (1000 and 500 bp) for easy orientation.

Results

In this study, SOD1 A251G single nucleotide polymorphism was determined in human subjects. The distribution of these polymorphisms in Turkish population was examined using a MspI-RFLP method. The principal characteristics of the study population are listed in Table 1. There were no significant differences for sex, age, and ethnicity when compared to the controls. SOD1 A251G, 494 (216 men and 278 women) Turkish individuals were genotyped for polymorphisms of SOD1 gene. The demographic characteristics of the study population are listed in Table 1. The mean age of the study population was 38.41±16.59 years (males, 39.81±17.10; females, 37.32±16.14). Allele and genotype frequencies of the subjects for SOD1 A251G polymorphism in the SOD1 gene are shown in Table 2. The distributions of the SOD1 A251G genotype were in accordance with the Hardy–Weinberg Equilibrium among the study population (p>0.05). The frequencies of AA, AG and GG genotypes were found to be % 86.2, % 13.4 and % 0.4 respectively in the controls. The frequency distributions of A and G alleles were found to be % 92.9 and % 7.1 respectively in the controls.

Discussion

Genetic variations in the antioxidant genes coding for the SOD, CAT, and GPX enzymes may lead to decreased or impaired regulation of their enzymatic activity and changed ROS detoxification. Hence, genetic variations among enzymes that protect the cell against ROS may modulate the disease risk [23]. Due to the high interaction opportunity of ROS with genetic material, polymorphisms in genes coding for antioxidant enzymes may play an important role for inter-individual differences in maintaining the human genome’s integrity. Genetic polymorphisms in SOD, CAT, and GPX enzymes have been suggested to involve in the tendency to cancer and other diseases [24], [25], [26]. SOD1 plays important roles in diseases like heart failure [27], cancer [28], diabetes [25], Down’s syndrome [29], and amyotrophic lateral sclerosis [30]. In fact, the first paper about Down’s syndrome was published in 1984 [31], and the first paper on SOD1 gene mutations associated with familial amyotrophic lateral sclerosis were described in 1993 [30]. These possible significant genetic variants related to the oxidative stress have already been studied extensively, including SNP A251G of the SOD1 gene. SOD1 A251G polymorphisms are the most common and important of antioxidant enzymes. Most of these polymorphisms result in changes either in the level or in the activity of this enzyme, which can lead to reduced protection against oxidative stress [23], [24], [25], [26]. Our study aimed to determine the allelic and genotypic frequency distribution of polymorphisms of A251G in the SOD1 gene and to compare findings with other ethnic groups. The frequencies of A and G alleles were 92.9% and 7.1% respectively, and the frequencies of the AA, AG and GG genotypes were 86.2%, 13.4%, and 0.4% respectively in the study population. The distribution of three genotypes fitted the Hardy–Weinberg equation (χ²=0.107, p=0.743) (Table 2). There is wide range variation of AA genotype frequency ranging from 23.4% [17] to as high as 88.3% [6] (Table 3). AA genotype frequency was found to be 86.2% in the present study in which the sample size was much larger than those of other studies performed in Turkish population which is very important for the making of more precise estimations in epidemiological studies in a such population.

SOD1 is an intracellular enzyme that primarily protects cells against cytosolic-generated superoxide [36]. SOD1 was found to have a widespread distribution in a variety of cells [37]. The expression of cytoplasmic SOD1 is stable and its activity is often considered as an internal control for SOD1 gene expression. SOD1 A251G polymorphisms result in changes in the levels or the activities of SOD1 enzyme, which can lead to reduced protection against oxidative stress [23], [24], [25]. In the present study, A allele frequency was found more than G allele frequency (χ²=60.5, p=0.001) (Table 2). This finding could suggest that SOD1 A251G polymorphism in Turkish population may pose a risk for some diseases such as cancer. Several studies have reported that the A allele of SOD1 contributed to a high risk of cancer in various populations [21].

In this study, we compared the distribution of three genotypes in different ethnic groups of the world in Table 3. Data presented here show that the Turkish population has the similar GG genotype frequency as seen in Chinese [3], Iranian [33], and Polish populations [15]. We detected higher GG genotype frequency than those found in American populations [6] and fairly lower frequencies than those found in Chinese [4], [17], Korean [32], Indian [35], Iranian [34] populations. Eventually, GG genotype frequency in populations of the eastern countries have been found higher (between 0.3 and 34.8%) than the western countries (between 0.0 and 0.82%) (Table 3). GG genotype frequency found in the present study was consistent with the above mentioned situation that the frequency value (0.4%) was between the values of eastern and western populations. Geographical situation confirms this result.

This study provides basic information about the allele and genotype frequency distributions of polymorphisms of A251G in the SOD1 gene studied. Our sample size was much larger than those of other studies, which is very important for the making of more precise estimations in epidemiological studies. The results of the present study, in conjunction with the results regarding SOD1 polymorphisms in a Turkish population, provide a framework for further studies concerning the role of this enzyme as a susceptibility of many diseases, including certain cancers.