DNASE1 VNTR Polymorphism and SLE

Deoxyribonuclease I (DNASE1) polymorphism and the risk of systemic lupus erythematosus in Iranian population

  • Milad Mohammadoo-Khorasani, Saeedeh Salimi, Maryam Moossavi, Mahnaz Sandoughi, Zahra Zakeri, Maryam Khoddamian

Abstract

Background: Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease. The etiology of SLE is multi-factorial and both genetic and environmental factors play role. In SLE patients a reduction of DNASE1 activity is observed. The aim of this study was to investigate the association of DNASE1 VNTR polymorphism and the risk of SLE in South East of Iran.

Material and method: In this case-control study 163 SLE patients and 180 healthy controls that have no close family relationship with SLE patients were chosen. Genotyping was performed by polymerase chain reaction (PCR).

Results: Results of genotypic frequency investigation indicates that 3/6 genotype frequency in patients affected with SLE was more than healthy controls (OR=1.6[95%CI, 1.173 to 2.183]). Moreover, 3/4 and 4/6 genotype frequencies in healthy cohorts was further in comparison with patient cohorts (OR=0.024 [95%CI, 0.004 to 0.123]) and (OR=0.375[95%CI, 0.224 to 0.629]). Therefore, it is considered as a protective factor against SLE.

Discussion: Findings of this study manifested that 3/6 genotype of DNASE1gene in patients affected with SLE was significantly more than healthy controls, thus it can be regarded as a risk factor. While, 3/4 and 4/6 genotypes were significantly higher in healthy controls.

Key words: systemic lupus erythematosus, polymorphism, DNASE1

Introduction

Systemic lupus erythematosus (SLE) is a chronic multisystem autoimmune disorder with unknown etiology [1]. SLE features comprises of altered immune tolerance mechanisms, hyper-activation of T and B cells, reduced ability of immune complexes and auto-antibody production which gives rise to inflammation and ultimately tissue injury such as renal, skin, joints, blood, cardiovascular, pulmonary, neurologic and immunologic involvements [2]. Of particular interest, although SLE is seen throughout the world, it is more prevalence in some countries and also in some ethnics within a country. Moreover, the disease courses and its manifestations vary in various ethnics [3]. Diverse factors including genetic and environmental play role in affecting to SLE. Smoking, occupational exposure to silica, childhood infections and sun exposure are some of environmental factors which lead to SLE. Genetic basis of SLE is very complicated to predict the genes which are involved in SLE susceptibility. It is estimated that more than 100 genes can contribute to SLE [4]. Deoxyribonuclase I (DNASE1) as one of the susceptible genes has been implicated in a number of immune disorders and is a remarkable candidate gene for SEL. DNASE1 is an endonuclease enzyme that facilitates degradation of the double stranded DNA in apoptosis pathway and thus suppression of anti-DNA autoimmunity [5-7]. DNASE1 is a calcium and magnesium-dependent enzyme that can be found in fluids such as serum and urine in large amount [8]. Napirei et al. showed that reduction of DNASE1activity in animal model resulted in SLE-like manifestations [6]. Notably, a reduction of DNASE1 activity in patients with SLE has been observed which is probably due to the inhibition of lymphocytes expression against nucleoproteins complex. These findings suggested that the reduction or lack of DNASE1 activity is an important factor in the early onset of systemic lupus erythematosus [9]. DNASE1 gene is located on the long arm of chromosome 16 which is containing 9exons. Recent researches have shown the presence of a novel 56-bp variable number of tandem repeat (VNTR) polymorphism in intron 4 at DNase I. Genetic variation in DNASE1 seems to have correlation with SLE [10]. The purpose of this study was to highlight the association of DNASE1 gene VNTR polymorphism in susceptibility to systemic lupus erythematosus in south east of Iran.

Material and Methods

Patients and sample collection

We evaluated a cohort of 163(150 females and 13 males) SLE patients including 81 Baluch ethnics and 82 Fars ethnics who referred to rheumatology clinic of Zahedan city since 2011 to 2013. Patients confirmed SLE according to ACR 1998 criteria (American Rheumatology Association)[11]. Healthy cohorts were 180 (166 females and 14 males) age and sex matched which include 94 Baluch ethnics and 84 Fars ethnics with negative ANA and systemic diseases. They also have no family relation with SLE patients. All participants provided written informed consent according to the Declaration of Helsinki and all samples collection approved by ethics committee of Zahedan University of Medical Sciences.

Genomic DNA extraction and genotyping

Each participant of this study donated 2 mL of whole blood which collected with EDTA as anti-coagulant and kept frozen at -20EsC until use. Salting out method [12]was used to extract DNA from the whole blood of samples from patients and controls. Polymerase chain reaction (PCR) technique was carried out for genotyping. Forward and reverse primers are as follows; forward: 5`-GGACCTTTTGTTTCTTCAA-3`,reverse: 5`-ACCGCAGACACCTGGTCA-3`. PCR was performed in a 25 ?l final volume contained 25 pmol of each primers, 0.1 mmol of dNTP (Fermentas, Lithuania), 0.5 ?g of genomic DNA, 1.5 mmol/L of MgCl2, 2.5 ?l of 10X PCR buffer and 1.5 unit of Taq DNA polymerase (Fermentas, Lithuania), according to the following protocol: initial denaturation at 95?C for 6min; 30 cycles of denaturation at 95?C for 30 s, ,annealing for 30s at 60?C and extension at 72?C for 1min; and final extension at 72?C for 7 minutes. PCR products were separated by electrophoresis on a 2.5% agarose gel and visualized by ethidium bromide staining. Fragments size of 2 to 6 alleles appeared as follows respectively: 469, 525, 581, 637 and 693 bps.

Statistical analysis

Data was analyzed using the statistical software SPSS 18 (SPSS, Chicago, IL). The differences between groups were analyzed by independent sample t – test, Chi square test. The genotypes frequencies were compared between SLE patients and controls by ?2 test and Fisher’s exact test. The odds ratio (OR) and 95% confidence intervals (CI) were also estimated. Values of p<0.05 were considered statistically significant.

Results

In current study, 163 SLE patients with a mean age 32.6 ±8.6 years (150females and 13males) were genotyped for VNTR polymorphism in DNASE1 gene. Control experiments were also performed in the same way, with a mean age 32.1±11.7 years (166females and 14males). Demographic data of SLE patients and control group are shown in table 1.

Table 1. Demographic characteristics of SLE and healthy cohorts

Parameter SLE

N=163

Controls

N=180

p-Value ?2
Age (yr) 32.6±8.6 32.1±11.7 0.68 0.04
Sex (male/female) 13/150 14/166 0.6 0.04
Race N (%)

Persian

Balouch

82 (50)

81 (50)

86 (48)

94 (52)

0.36 0.27

Among 6 probable alleles for DNASE1 gene, we only observed 4 alleles. Allelic and genotypic frequency distributions of the HumDNAse1 VNTR are shown in table 2. Current findings indicate that 3/4 and 4/6 genotypes can be regarded as potent protective factors against SLE disease (OR=0.024 [95%CI, 0.004 to 0.123]) and (OR=0.375[95%CI, 0.224 to 0.629]). Moreover, 3/6 genotype considered as a risk factor so that, the individuals who have this genotype seem susceptible to SLE (OR=1.6 [95%CI, 1.173 to 2.183]). Frequency of allele 5 in SLE patients is significantly more than healthy controls that suggested individuals with this allele, than individuals without allele 5 may predispose to SLE (OR=1.715[95%CI, 1.131to 2.604]).

Table II. Genotypic and allelic frequencies of DNASE1 polymorphisms in SLE patients and controls

Genotype SLE patients

Number=163

Healthy controls

Number=180

P value Odds ratio
3/3 (9.2%) 15 9(5%) Ref=1
3/4 2(1.2%) 50(27.8%) 0.0001 0.024(0.004-0.123)
3/5 33(20.2%) 48(26.7%) 0.050 0.412(0.161-1.05)
3/6 17(10.4%) 0 0.004 1.6(1.173-2.183)
4/4 38(23.3%) 25(13.9%) 0.527 1.096(0.416-2.88)
4/5 55 (33.7%) 27(15%) 0.427 1.222(0.474-3.144)
4/6 0 21(11.6%) 0.0001 0.375(0.224-0.629)
5/6 (2%) 3 0 0.279 1.6(1.173-2.183)
Allele
3 82 116 Ref=1
4 133 148 0.117 0.787(0.545-1.136)
5 91 75 0.007 1.715(1.131-2.604)
6 20 21 0.243 0.742(0.378-1.457)

Discussion

Systemic lupus erythematosus (SLE) is a prototype and systemic autoimmune disorder that is characterized by presence of self-antibody, complement involvement, and tissue injury [13]. The precise etiology of SLE is not known; however, involving environmental and genetic factors independently and most probably synergistically [14]. Severity, risk factor, and clinical manifestations of this disease can alter by ethnic, region, and gender with a high prevalence in women [15]. Genetic factors presumably play a remarkable role in susceptibility to SLE [16]. DNASE1 is a candidate endonuclease that breakdown double-stranded (ds) DNA and producing oligonucleotides with 5?-phosphate and 3?-hydroxyl termini [17, 18]. Its enzymatic activity is around 7.5 PH, and concentrations of Ca 2+, Mg2+, or Mn2+ are required [8]. Low serum DNase1 activity observed in SLE patients which can be responsible for clearance of post-apoptotic products actually DNA, and consequently, suppression of anti-DNA autoimmunity which could be an important pathogenesis of SLE [19] .The failure of this mechanism could be due to the ini¬‚uence of variants within DNASE1 gene. Recent research has shown the presence of a novel 56-bp variable number of tandem repeat (VNTR) polymorphism in intron 4 at DNaseI. Genetic variation in DNASE1 seems to have correlation to SLE susceptibility [10, 20]. In 1995 Yasuda et al. [10] showed that serum and urine levels of DNASE1 in SLE patients is low. Moreover, Macanovic et.al, reported a reduction in DNAse1 protein level in mice affected with SLE [21]. Several studies have indicated that VNTR analysis can improve our understanding of the factors involved in different diseases [22, 23]. In this study 6/3 genotype frequency was significantly higher in SLE patients than in healthy control which implies the possible role of this genotype in SLE susceptibility. Furthermore, 3/4 and 4/6 genotype frequencies were remarkably high in patients group indicated the protective role of this genotypes against the disease. In addition, frequency of allele 5 is higher in patients than controls. This difference was statistically significant which reveals the possible role of this allele in predisposing to SLE. In 2004, Yasuda et al. [24] conducted a study on Japanese and German populations and showed that allele frequencies of 2 and 6 are very rare in these populations which are similar to our findings. Panneer et.al, reported +2373 A>G polymorphism on DNAse1 gene can associate with SLE disorder [25]. In similar study which was carried by AlFadhli et al.[23] they showed that 3/5 and 4/5 genotypes were considerably high in patients affected with SLE that is in contrast with findings of this study. Nevertheless, we see a rise in 4/5 genotype frequency but it is not statistically significant. In Kuwait populations [23], the 3/3 and 4/4 genotypes considered as protective factors that are in opposite to our findings. The data obtained from this study can be used as a marker in predicting SLE susceptibility. Therefore, it is suggested to provide a larger sample sizes and also other races for precise research.