NAT2 Gene Polymorphisms in Turkish Patients with Psoriasis Vulgaris

Psoriasis is a common, chronic, and autoimmune skin disease. Factors that play a role in etiopathogenesis of psoriasis include internal factors such as genetic susceptibility and immunological factors and external factors such as stress, infection, trauma, drug, and environmental compounds. N-acetyltransferase 2 (NAT2) is a xenobiotic enzyme that is involved in the metabolism of drugs, environmental toxins, and carcinogens. In this study, we aimed to demonstrate whether the variations in the NAT2 gene lead to a predisposition to psoriasis by affecting the enzyme's ability to metabolize drugs and environmental components or not. Three polymorphisms (rs1799929, rs1799930, and rs1799931) in NAT2 gene were genotyped and compared by real-time PCR method in 260 psoriasis vulgaris patients and 200 healthy controls. There was no difference in the genotype distributions and allele frequencies of polymorphisms between psoriasis vulgaris patients and controls. When the effects of polymorphisms on the clinical features of the disease, such as onset age and severity, are assessed, it has been found that rs1799930 and rs1799929 are, respectively, associated with early onset age and severity of the disease. In conclusion, rs1799929, rs1799930, and rs1799931 polymorphisms of the NAT-2 gene do not appear to be a risk factor for the development of psoriasis. Conversely, they may have an effect on either more severe or early onset cases of the disease.


Introduction
Psoriasis is a common, chronic, and recurrent autoimmune inflammatory skin disease that can also affect nails and joints [1]. It is characterized by erythematous, scaly, sharply demarcated papules and plaques on skin and scalp [2]. It can be seen in all age groups without gender discrimination [1]. The prevalence of the disease is between 1% and 3% worldwide, varying among different ethnic populations and geographical regions [3]. Psoriasis has four major clinical phenotypes, which are distinguished by morphological characteristics of the associated lesions: (i) psoriasis vulgaris, (ii) guttate psoriasis, (iii) pustular psoriasis, and (iv) erythrodermic psoriasis. The most common one of these clinical phenotypes is psoriasis vulgaris, accounting for 90% of all cases, and is also known as plaque psoriasis [4]. In this phenotype, the lesions are dry, sharply demarcated, oval/circular plaques and can be localized all over the body, mostly affecting the knees, elbows, lumbosacral area, intergluteal cleft, and scalp [4,5]. Although the etiopathogenesis of psoriasis is not fully understood, it is known that the disease manifests as a result of biochemical, immunological, and vascular system abnormalities in the presence of a genetic predisposition [6,7]. Mutual interactions of T-cells which play a central role in pathogenetic mechanism, other immune-related cells, skin cells, and proinflammatory cytokines and chemokines cause histopathologic changes observed in psoriatic skin [8]. These histopathological changes bring out the clinical picture of the disease, which involves hyperproliferation and aberrant differentiation of keratinocytes, capillary dilatation, angiogenesis, and leukocytes infiltration into dermis [8][9][10]. The reported number of cellular and molecular factors contributing to its etiopathogenesis is increasing day by day [11][12][13][14][15][16][17][18]. Psoriasis is associated with several comorbidities, such as inflammatory bowel diseases [19,20], cardiovascular syndrome [21,22], metabolic syndrome [22][23][24], depression [25], and cancer [26,27]. The disease leads to a serious reduction in patient's life quality, due to its link with social stigmatization, pain, discomfort, physical disability, aesthetic concerns about physical appearance, and psychological distress [28,29]. Now, it is accepted that environmental factors such as trauma, infections, exposure to ultraviolet (UV) radiation, drugs, stress, and smoking/alcohol consumption play an effective role in emergence and development of psoriasis [30][31][32][33].
As in any other autoimmune disease, the pathogenesis of psoriasis requires three puzzle components to come together: genetic predisposition, environmental factors, and immune regulation disturbance [34][35][36]. Studies have shown that environmental factors such as trauma, infections, intestinal microbiota, exposure to UV radiation, drugs, stress, and dietary components can be the largest component of this puzzle [37,38]. In recent years, many studies have been published on polymorphisms in genes which code xenobiotic metabolizing enzymes, in order to better understand the role of environmental factors in the pathogenesis of diseases [39][40][41][42][43][44][45][46][47][48][49]. One of the enzymes involved in xenobiotic metabolism is N-acetyl transferase II (NAT2). It performs detoxification of xenobiotics, which have the structure of aromatic amines, hydrazine, sulphonamides, and aliphatic amines, by acetylation reactions. These xenobiotics include environmental and dietary compounds, carcinogens, and drugs used to treat a variety of diseases, particularly tuberculosis, AIDS-related complex diseases, psoriatic arthritis, and hypertension [50][51][52]. NAT2 enzyme is encoded by intronless NAT2 gene that is located at 8p22. NAT2 gene is highly polymorphic and so far 106 NAT2 variants have been identified (http://nat.mbg.duth.gr). NAT2 variants are often in the form of single nucleotide polymorphisms (SNP) and alter the catalytic activity or stability of the enzyme, leading to amino acid substitution [53,54]. These alterations in gene and protein products determine the acetylation phenotypes that can be observed individually as "rapid" and "slow" acetylator phenotypes [55]. NAT2 acetylation polymorphisms are also very important in pharmacological, toxicological, and clinical aspects, since some of these polymorphisms may lead to adverse drug reactions or decreased drug effectiveness and even to increased susceptibility to certain diseases via decreasing the enzyme's acetylation capacity [51,52].
There are a number of studies showing that NAT2 gene polymorphisms lead to increased susceptibility to various diseases, primarily cancer [40,[56][57][58][59][60][61] and autoimmune disorders [47,50,[62][63][64]. However, to the best of our knowledge, there are only two studies on the association of NAT2 gene variants with psoriasis vulgaris [65,66]. Only in one of these studies [65] the association of NAT2 polymorphisms with age of the disease onset has been investigated. In our study, however, we simultaneously assessed whether NAT2 polymorphisms were associated with the age of disease onset and the severity of the disease. The starting point of our study is the two fundamental principles about psoriasis and NAT2: (1) psoriasis pathogenesis can be triggered by drugs and various other environmental factors. (2) NAT2 enzyme is responsible for detoxification of various drugs and environmental compounds. In that case, can NAT2 gene acetylation polymorphisms contribute to the pathogenesis of the disease by facilitating the factors that may trigger psoriasis? With this hypothesis, our goals were (i) to investigate whether the NAT2 gene polymorphisms are related to development of psoriasis vulgaris (ii) and to detect whether the NAT2 gene polymorphisms have an impact on the clinical features of psoriasis vulgaris such as age of onset and severity.
In the present study, we conducted a research on 481C>T (rs1799929), 590G>A (rs1799930), and 857G>A (rs1799931) in the NAT2 gene. We also confirmed that the acetylator phenotypes are induced by these three SNPs at "http://nat.mbg.duth.gr": rs1799929 is responsible for the rapid acetylator phenotype while rs1799930 and rs1799931 are responsible for the slow acetylator phenotype. We chose these SNPs, as they are among the most commonly used SNPs for inferring NAT2 acetylator phenotype [64], and rs1799930 and rs1799931 especially are defined to be the cause of 95% of the low enzymatic activity alleles [67].

Subjects. Two hundred and sixty (260) unrelated Turkish psoriasis vulgaris patients and two hundred (200) unrelated
Turkish healthy controls were included in the study. Psoriasis vulgaris patients (142 females/118 males; mean age±SD: 39.83± 24.52) were recruited from the Dermatology Clinic. The patients with other chronic and autoimmune disorders or cancer were excluded from the study. The control subjects (99 females/101 males; mean age±SD: 38.94±18.80) were healthy individuals who had been examined for psoriasis, cancer, autoimmune symptoms, and family history. The patients and controls were matched according to their gender and age. Severity of psoriasis was assessed via Psoriasis Area and Severity Index (PASI), ranging from 0 (no disease) to 72, with higher scores indicating the severity of disease [68]. To determine the level of association of NAT-2 gene polymorphisms with clinical features of psoriasis vulgaris, the patients were divided into two groups according to the severity of disease (PASI<12 group and PASI≥12 group) and then assigned into two groups according to the onset of disease (early onset group: <40 age; late onset group: ≥40 age) ( Table 1). This study was conducted in accordance with the Declaration of Helsinki principles and was approved by the Meram Medical Faculty Ethics Committee (No: 2011/204). Informed consent was obtained from all participants before beginning of the study.

2.2.
Genotyping. Blood specimens from psoriasis vulgaris patients and controls were drawn into EDTA-containing tubes and were kept at 4 ∘ C until the blood collection from all subjects was completed. Genomic DNA from peripheral blood leukocytes of all patients and controls was extracted with the High Pure PCR Template Preparation Kit (Roche Molecular Diagnostics, Manheim, Germany) according to the manufacturer's instructions. Genotyping of rs1799929 (481C>T), rs1799930 (590G>A), and rs1799931 (857G>A) was performed by using the LightCycler-NAT2 mutation detection kit by real-time PCR with the LightCycler 480 instrument (Roche Molecular Diagnostic, Manheim, Germany). Reaction mixture for real-time PCR comprised 10.4-14.4 l distilled water, 1.6 l MgCl 2 (25 mM), 10 pmol of each primer, 4 pmol of each probe, and 2 l genomic DNA (50-200 ng/ml). The cycling conditions were as follows: an initial denaturation step at 95 ∘ C for 10 min followed by 45 cycles for 10 s at 95 ∘ C, 10 s at 62 ∘ C, and 15 s at 72 ∘ C. Wild type, heterozygote, and polymorphic genotypes were detected by specific melting temperature (Tm) of amplicons.

Statistical Analysis.
The expected genotype values for testing Hardy-Weinberg equilibrium (HWE) were calculated from allele frequencies. Deviations from observed genotype values were determined by 2 -test. The SNPStats program was used to test the relationship between genotype and disease according to different inheritance models. Haploview version 4.2 was also applied to construct haplotype structures and to determine the relationship between haplotype and disease.  into subgroups according to onset age of psoriasis vulgaris (early onset group: <40 age; late onset group: ≥40 age) and severity of psoriasis vulgaris (PASI<12 group; PASI≥12 group) ( Figure 1).

Haplotype Analysis.
Linkage disequilibrium (LD) map of SNPs was genotyped using Haploview ( Figure 2). rs1799929 and rs1799930 were in strong LD and form a haplotype block. rs1799931 located outside of this block was not in LD with rs1799929 and rs1799930. Haplotype analysis was performed  using all 3 SNPs (rs1799929, rs1799930, and rs1799931) and 2 SNPs (rs1799929 and rs1799930) at the same block. Four haplotypes were detected in patients and controls among which the frequencies did not differ significantly (Table 4).

Association of NAT2 Gene Polymorphisms with Clinical
Characteristics of Psoriasis Vulgaris. Firstly, NAT2 polymorphisms were evaluated for having an effect on the onset age of psoriasis vulgaris. The comparisons of genotypes with the onset age of psoriasis vulgaris under the genetic models were presented in Table 5. We observed an increased risk of early onset age of psoriasis vulgaris in rs1799930 according to the codominant model (OR=1.75, 95% CI=0.65-4.73, and p=0.046, for the AA genotype) and to the overdominant model (OR=0.44, 95% CI=0.21-0.93, and p=0.026, for the GA genotype).
The distribution of acetylator phenotypes in early onset age and late onset age groups was shown in Table 6. There was no significant difference in the distribution of the slow acetylator and fast acetylator phenotypes between the early onset and late onset age groups.
Haplotype analysis results were presented in Table 7. None of the haplotypes had a significant relationship with the onset age of psoriasis vulgaris.
The distribution of acetylator phenotypes in PASI≥12 and PASI<12 groups is given in Table 9. Although the frequencies of TT/GG/GG-fast acetylator, TT/GA/GA-slow acetylator,    and CT/GA/GA-slow acetylator phenotypes were found to be significantly higher in the PASI≥12 group (p=0.04, p=0.04, and p=0.03, respectively), total fast and total slow acetylator phenotype frequencies did not differ significantly between the PASI≥12 and PASI<12 groups. In haplotype analysis, no significant difference in haplotype distribution was detected in the PASI≥12 group compared to the PASI<12 group (Table 10).
The NAT2 gene, which encodes the NAT2 enzyme, is a highly polymorphic gene. The polymorphisms of the NAT2 gene have major pharmacogenetic considerations as they lead to adverse drug reactions and individual differences in drug response [51,52]. NAT2 polymorphisms have also clinical importance, due to the fact that these polymorphisms lead to susceptibility to many diseases such as autoimmune diseases and cancer, for which the etiopathogenesis is affected by environmental factors [40,47,50,[56][57][58][59][60][61][62][63][64]. Indeed, psoriasis is one of these diseases with an etiopathogenesis affected by environmental factors.
In the present study, we investigated whether the most common three polymorphisms (481C>T, 590G>A, and 857G>A), effective in the acetylation phenotype of the NAT2 gene, have any effect on the development and clinical features of psoriasis vulgaris, i.e., the most common clinical type of psoriasis. The study was performed with 260 patients (118 males/142 females) and 200 controls (101 males/99 females). Although there was no statistically significant difference in sex ratios between the two groups, the proportion of females (54.62%) was slightly higher than that of males (45.38%). Despite various studies reporting that the incidence of psoriasis is higher either in females or males [33], there is a theoretical consensus on no gender discrimination in psoriasis incidence [6]. In a study on the incidence of psoriasis in Turkey, the prevalence of the disease is given as 1.2% in females and as 1.0% in males [69]. In another study, the female/male ratio in Turkish psoriasis patients has been reported as 200/129 [70]. In our study, it is possible to explain the excess of women in the group of psoriasis as follows: psoriatic women are more often referred to clinic than psoriatic men, due to their physical appearance/aesthetic concerns.
In our study, the genotype and allele frequencies of three polymorphisms of the NAT2 gene (481C>T, 590G>A, and 857G>A) were compared between psoriasis vulgaris patients and controls. The genotypes and allele distributions of three polymorphism did not manifest a statistically significant difference between patients and controls, according to genetic models we applied. No significant association was observed between acetylator phenotypes and susceptibility to psoriasis vulgaris either. To our knowledge, there are only two studies on the relationship between psoriasis and NAT2 polymorphisms in the literature. In one of these studies, Reich et al. [65] studied seven NAT2 polymorphisms, including 481C>T, 590G>A, and 807G>A, in 151 psoriasis patients and 123 controls. They found no significant differences in the distribution of NAT2 polymorphisms between psoriasis patients and controls. In another study, Kozhekbaeva et al. [66] studied NAT2 282C>T, 341T>C, 481C>T, 590G>A, 803A>G, and 857G>A polymorphisms in 180 psoriasis patients and 99 controls, with the findings that suggest 341T>C, 481C>T,  and 803A>G polymorphisms and some lifestyles could be considered as risk factors of psoriasis development.
There is a great deal of research on the possible association of NAT2 gene polymorphisms with other autoimmune diseases. The results of studies on inflammatory bowel disease (IBD) are contradictory. Baranska et al. [63] evaluated 481C>T, 803A>G, 590G> A, and 857G>A polymorphisms in IBD and found that only the 857A allele was correlated with IBD. Similarly, in another study on IBD patient group, it was reported that NAT2 * 6A (282C>T, 590G>A) haplotype had correlation with neither of ulcerative colitis and Crohn's disease, with a higher frequency of NAT2 * 7B (282C>T, 857G>A) haplotype found only for Crohn's disease [71]. Chen et al. [72] have studied the same polymorphisms as those in our study, with consistent results. The researchers found that 481C>T, 590G>A, and 857G>A polymorphisms did not count as a susceptibility factor for inflammatory bowel disease but that slow acetylator genotypes were significantly associated with dose-related adverse effects of sulfasalazine in the treatment of inflammatory bowel disease.
The more frequent occurrence of drug-induced systemic lupus erythematosus (SLE) in individuals with slow acetylator genotypes has led to hypothesis that xenobiotics accumulate and convert into reactive molecules due to defect in acetylation to stimulate immune system and T-cells in favor autoimmunity [73]. However, in idiopathic SLE, the role of the acetylator genotype and phenotypes on susceptibility to disease is controversial. The study on the association of NAT2 polymorphisms with SLE overall shows that 481C>T, 590G>A, and 857G>A polymorphisms and slow acetylator genotypes do not form a risk factor for SLE [74]. But Lima dos Santos et al. [64] reported that only 857G>A polymorphism among 481C>T, 590G>A, 857G>A, and 191G>A polymorphisms was associated with SLE.
In a study conducted with a very small systemic sclerosis patient population (n=39), the distributions of 481C>T, 590G>A, 803A>G, and 857G>A polymorphisms have been compared between patients and controls and no significant difference was found [75].
NAT2 acetylation polymorphisms have also been studied in rheumatoid arthritis (RA). In a study evaluating NAT2 * 4, NAT2 * 5, NAT2 * 6, and NAT2 * 7 genotypes, it has been suggested that NAT2 slow acetylator genotypes might lead to RA susceptibility [62]. Oqal et al. [76] also have studied the same polymorphisms and found that only the NAT2 * 5/7 genotype correlates with RA, while other slower acetylator genotypes are not a risk factor for RA.
In the case-control study conducted by Tamer et al. it was suggested that NAT2 * 5A and NAT2 * 6A slow acetylator genotypes were significantly associated with Behçet's disease [47].
In the current study, we also aimed to assess whether the NAT2 polymorphisms have any effect on the clinical characteristics of psoriasis (such as the age of the disease onset and the severity of the disease). We compared the distribution of genotype and allele frequencies of polymorphisms between "early onset age" and "late onset age" and between "PASI<12" and "PASI≥12" groups. In the analyses, we found a statistically significant correlation between the 590G>A polymorphism and the age of the disease onset according to the codominant (p=0.046) and overdominant (p=0.026) inheritance patterns; however the two other polymorphisms did not correlate with age of onset. It is not surprising that we found a relationship between 590G>A polymorphism and age of the disease onset according to codominant and overdominant inheritance patterns. As a matter of fact, the comparisons were made according to the 4 inheritance patterns and allele frequencies. The alleles frequencies did not differ between the two groups while a correlation was found according to the two of inheritance patterns. Given the statistically significant difference between two groups according to only two inheritance patterns, suggesting 590G> A polymorphism to be a distinct risk factor for the early age onset of psoriasis would not be realistic.
In the analysis of polymorphisms-disease severity relations, we only found the frequency of CT+TT genotypes (p=0.026) and minor allele (T allele) frequencies of 481C>T polymorphism significantly higher in PASI≥12 group compared to PASI<12 group. Other polymorphisms did not correlate with the severity of the disease. Concordantly, 481C>T polymorphism being associated with the severity of the disease is not surprising, according to only the dominant inheritance pattern. However, the fact that the frequency of the minor allele (T) was higher in the PASI≥12 group than in the PASI<12 group suggests that 481C>T polymorphism may have a precautionary effect on the severity of the disease. The association of NAT2 polymorphisms with the clinical features of psoriasis has only been analyzed by Reich et al. [65]. The researchers evaluated the association of NAT2 polymorphisms with age of the disease onset. Their data suggest a relationship between early onset age (<40 age) and fast acetylator phenotype. When we consider the acetylator phenotypes in our study, we found no relationship between the acetylator phenotypes and the age of the disease onset. When we evaluated the relationship between the severity of the disease and acetylator phenotypes, TT/GG/GG-fast acetylators (p=0.04), TT/GA/GA-slow acetylators (p=0.04), and CT/GA/GA-slow acetylators (p=0.03) were all statistically significantly higher in PASI≥12 group compared to the PASI <12 group (order of genotypes is rs1799929/rs1799930/rs1799931).
Here, the question might come to mind: Could the relationship between certain SNPs and age of onset/severity of the disease be due to linkage disequilibrium between these alleles and functional polymorphisms at distant sites? However, to the best of our knowledge, none of the polymorphisms directly associated with psoriasis are found on chromosome 8p [77,78]. We, therefore, think that the relationship between certain SNPs and the clinical features of the psoriasis vulgaris is directly related to the NAT2 itself, rather than to linkage disequilibrium between these alleles and functional polymorphisms.
Our study has some limitations: (i) We evaluated the polymorphisms of gene encoding NAT2 enzyme that work in the detoxification of environmental compounds and drugs in psoriasis patients, but we could not question or categorize lifestyle, nutritional habits, drug use, smoking-alcohol habits, and environmental/occupational exposures of patients and controls. (ii) It was not possible for us to analyze all known polymorphisms of NAT2 gene. These two issues make it difficult to assess a disease affected by the gene-gene and/or geneenvironment interactions during etiopathogenesis, such as psoriasis.
NAT2 is extensively polymorphic, and the distribution of polymorphic alleles varies widely between populations and ethnic groups. Table 11 shows the distribution of allele frequencies of the NAT2 polymorphisms rs1799929, rs1799930, and rs1799931 in healthy individuals from different ethnic groups. The allele distributions of rs1799929, rs1799930, and rs1799931 in our study population are close to the respective distribution of Indian [84] and Mexican [82], British [95] and Polish [92], and Egyptian [79] and Polish [92] populations.
In a study carried out by Tamer and colleagues [94] for the Turkish population living in Mersin (a city in South Anatolia), polymorphic allele frequencies have been reported as 0.34 for rs1799929, 0.23 for rs1799930, and 0.23 for rs1799931. Our study was conducted on the other hand in Konya (in central Anatolian city) and the results we found were similar to Tamer and colleagues' study population only in terms of rs1799929 allele frequencies. The distributions of rs1799930 and rs1799931 allele frequencies differ between the two populations. Presumably these differences stem from the ethnic differences of the populations in the two major cities of Anatolia. This suggests that the NAT2 gene is so polymorphic that it can cause serious differences in the allele frequency even between populations of two neighboring cities in the same geographical region.

Conclusions
According to the data of this study, NAT2 gene 481C>T, 590G>A, and 807G>A polymorphisms do not appear to be effective in the emergence of psoriasis vulgaris disease. However, further studies should consider the possibility that 590G> A polymorphism may have an effect on the age of the disease onset and 481C> T polymorphism may have an effect on the severity of the disease. As a result, the NAT2 is a very remarkable gene due to its high polymorphism rates and the crucial role of the enzyme it encodes in several detoxification events. This high polymorphism rate and variation variability also necessitate working with large populations and as many relevant individuals/cases as possible in studies. NAT2 will continue to be a suspicious gene in the pathogenesis of many