Polymorphisms of the GCLC Gene Are Novel Genetic Markers for Susceptibility to Psoriasis Associated with Alcohol Abuse and Cigarette Smoking

The aim of this pilot study was to investigate whether single nucleotide polymorphisms (SNP) in the gene encoding the catalytic subunit of glutamate cysteine ligase (GCLC) are associated with the risk and clinical features of psoriasis. A total of 944 unrelated individuals, including 474 patients with a diagnosis of psoriasis and 470 healthy controls, were recruited for the study. Six common SNPs in the GCLC gene were genotyped using the MassArray-4 system. Polymorphisms rs648595 (OR = 0.56, 95% CI 0.35–0.90; Pperm = 0.017) and rs2397147 (OR = 0.54, 95% CI 0.30–0.98; Pperm = 0.05) were associated with susceptibility to psoriasis in males. In the male group, diplotype rs2397147-C/C × rs17883901-G/G was associated with a decreased risk of psoriasis (FDR-adjusted p = 0.014), whereas diplotype rs6933870-G/G × rs17883901-G/G (FDR-adjusted p = 0.045) showed an association with an increased disease risk in females. The joint effects of SNPs with tobacco smoking (rs648595 and rs17883901) and alcohol abuse (rs648595 and rs542914) on psoriasis risk were observed (Pperm ≤ 0.05). We also found multiple sex-independent associations between GCLC gene polymorphisms and various clinical features such as earlier disease onset, the psoriatic triad, and specific localizations of skin lesions. The present study is the first to show that polymorphisms of the GCLC gene are significantly associated with the risk of psoriasis and related to its clinical features.


Introduction
Psoriasis is a chronic immune-inflammatory-mediated dermatosis characterized by thickened, scaly erythema or plaques [1,2]. Psoriasis is recognized by the World Health Organization as a serious non-communicable disease [3]. Clinical variants of the disease include psoriasis vulgaris, arthritis, and pustular and erythrodermic types; however, psoriasis vulgaris is the most common form, accounting for about 90% of cases and affecting 3% of Caucasians [4]. A study by Kubanov and co-workers demonstrated a substantial disease burden on psoriasis patients in Russia [5].
The etiology and pathogenesis of psoriasis remain mysteries, making the disease's management more challenging [6]. Psoriasis is characterized by sustained inflammation, which results in uncontrolled keratinocyte proliferation and defective differentiation [7]. Psoriatic inflammation is caused and maintained by disruptions in innate and adaptive cutaneous immune responses [6,8], which coexist with autoinflammatory perpetuation or T-cell-driven autoimmune reactions [7]. The overlap of autoimmune and autoinflammatory mechanisms in the pathogenesis of psoriasis has led to the development of biological therapy for the disease. However, despite the fact that targeted therapies focusing on the inhibition of cytokines such as IL-23 and IL-17 showed high clinical efficacy, psoriasis remains an incurable disease [7].
Psoriasis is known as a complex multifactorial disease for which development is determined by the interaction between genetic, environmental, and epigenetic factors [9][10][11]. Linkage analysis, an effective method to identify the chromosomal location of disease genes, has discovered nine separate genomic regions known as psoriasis susceptibility regions (PSORS1-9) comprising many genetic variants, a part of which has been fine-mapped as disease-linked loci [11,12]. Progress in the development of high-throughput genotyping technologies enabled the implementation of genome-wide association studies (GWAS), a research approach in which large case-control cohorts are genotyped for tens of thousands of single nucleotide polymorphisms (SNPs) across the genome [11]. According to the GWAS catalog (https://www.ebi.ac.uk/gwas/home, accessed on 29 April 2023), 57 GWASs have been conducted so far to unravel the genetic background of psoriasis in different populations around the world, and 946 SNPs have been identified as loci associated with disease susceptibility or severity and those influencing the efficacy of anti-psoriatic therapy. Nevertheless, despite considerable genetic research and achievements, the etiology of psoriasis and its primary molecular mechanisms remain elusive.
It has been argued that the increased production of reactive oxygen species (ROS) and a decreased antioxidant defense leading to the activation of oxidative stress are involved in the pathogenesis of psoriasis and influence disease duration and severity [13][14][15][16]. Despite the fact that the important role of oxidative stress in the etiopathogenesis of psoriasis remains undisputable after decades of research, a limited number of studies have been undertaken so far to assess whether genetic variation in antioxidant defense enzymes contributes to psoriasis susceptibility. A larger portion of the studies looked for the link between psoriasis risk and genetic polymorphisms of glutathione-S-transferases [17][18][19][20], enzymes catalyzing the conjugation of reduced glutathione (GSH) to xenobiotic compounds for their detoxification.
Glutathione is a low-molecular-weight thiol, a tripeptide consisting of glutamate, cysteine, and glycine, which plays a major role in maintaining intracellular redox balance and antioxidant defense [21]. It is involved in many crucial biological functions, such as xenobiotic detoxification, maintaining mitochondrial function, the modulation of cell proliferation, wound healing, and the inhibition of apoptosis [21,22]. Furthermore, glutathione is utilized as a cofactor by glutathione peroxidases and glutathione S-transferases for the glutathionylation of selected proteins and toxic substance conjugation. GSH is also required for the maturation of cytosolic iron-sulfur proteins, which are essential for cell viability and involved in the maintenance of DNA metabolism, genome integrity, protein translation, and other critical biological functions [22,23]. It is important to note that glutathione is involved in the skin metabolic clearance system [24], protects DNA and mitochondria from oxidative damage, and ensures the survival of keratinocytes in normal and wounded skin [25]. Glutathione deficiency is well known to be associated with an increased susceptibility to oxidative stress, a pathological condition implicated in the pathogenesis of psoriasis [26], and, therefore, we can suggest that oxidative stress may be responsible for the modulation of inflammatory and autoimmune mechanisms underlying the diseases [27,28]. Despite the obvious importance of glutathione in skin metabolism, existing research data in psoriasis on the roles of genes encoding enzymes involved in glutathione metabolism, primarily glutamate cysteine ligase, an enzyme catalyzing the initial rate-limiting step of GSH biosynthesis [29], are surprisingly absent. We propose that genetic polymorphisms of glutamate cysteine ligase may explain inter-individual differences in glutathione biosynthesis and influence the risk of psoriasis, making SNPs attractive markers for testing disease susceptibility. Therefore, the purpose of our pilot study was to investigate whether common polymorphisms at the gene encoding the catalytic subunit of glutamate cysteine ligase (GCLC) are associated with the risk and clinical features of psoriasis.

Study Participants and Clinical Examination
Informed consent was signed by all subjects involved in this study. The protocol of the present study was approved by the Ethical Review Committee of Kursk State Medical University (protocol No. 8, 13.11.2017). A total of 944 unrelated individuals of European descent (predominantly Russians), including 474 patients with a diagnosis of psoriasis and 470 healthy controls, were used for this study. The enrollment of patients with psoriasis was conducted in Medvenka Central District Hospital (Kursk region), the Center for Medical Examinations and Prevention (Kursk), and Kursk Regional Multidisciplinary Clinical Hospital in a period between September 2018 and December 2021. The control group of subjects without chronic diseases was recruited from our previous studies [30][31][32]. The diagnosis of psoriasis was verified by qualified dermatologists based on the typical clinical picture of skin rashes and their localization [6]. The study included patients with classic plaque psoriasis; palmoplantar, seborrheic, and scalp psoriasis; the von Zumbusch type of generalized pustular psoriasis; inverse psoriasis; guttate psoriasis; and erythrodermic psoriasis, as well as psoriasis comorbidities such as psoriatic arthritis and onychodystrophy [33]. The Psoriasis Area and Severity Index (PASI) was used for the clinical assessment of the severity of the course of psoriasis [34]. Enrolled patients did not suffer from chronic infectious diseases, including HIV and hepatitis, and did not have severe chronic conditions that manifested before psoriasis. Patients who were receiving biologic therapy at the time of the recruitment and pregnant women were not included in the study. Study participants completed a validated doctor-administered questionnaire [35] to assess risk factors for psoriasis, such as cigarette smoking [36] and alcohol consumption [37]. Information on smoking status (ever/never) was available from all psoriatic patients and healthy subjects. Data on alcohol intake were available from all patients with psoriasis and only 220 individuals from the control group. Alcohol intake habits were assessed by the number of drinks consumed per week, as described previously [38,39]. Briefly, according to the reported frequency of alcohol intake, study individuals were categorized into two groups: (1) subjects who consumed alcohol 1 to 2 days a month or less and (2) those drinking alcohol 1 or more days a week. The second group was considered alcohol abusers.

Genetic Analysis
Venous blood samples were collected from the cubital vein of study subjects into EDTA-coated tubes and immediately frozen and stored at −20 • C until processed. Total DNA was purified by the standard phenol/chloroform extraction and ethanol precipitation. Genotyping of the SNPs was performed with the MALDI-TOF mass spectrometry iPLEX platform on the MassArray-4 system (Agena Bioscience, Inc., San Diego, CA, USA). Primer sequences used for genotyping are available upon request. To guarantee quality

Statistical Analysis
Statistical power was estimated using the GAS power calculator (https://csg.sph. umich.edu/abecasis/gas_power_calculator/, accessed on 21 May 2022). It has been estimated that we could detect a genotype relative risk (GRR) of 1.30-1.45 with 82-98% power in the overall analysis (474 cases and 470 controls) and a GRR of 1.40-1.5 with 76-83% power in the analysis of groups stratified by sex/risk factors at α = 0.05. Fisher's exact test was used to assess the distribution of genotype frequencies according to the Hardy-Weinberg equilibrium (HWE). Allele and genotype frequencies in the study groups and their associations with the risk of psoriasis were analyzed using the PLINK software v.1.9 [40]. Logistic regression analysis was used to evaluate the associations of GCLC gene polymorphisms with the risk of psoriasis and binary clinical phenotypes. The crude odds ratio (OR) and 95 percent confidence intervals (95% CI) were calculated to assess SNP-phenotype associations phenotype associations. Associations of SNPs with continuous phenotypes were evaluated with linear regression analysis, with estimation of differences in mean between genotypes and 95% CI using the SNPstats software (https://www.snpstats.net/start.htm, accessed on 12 April 2023). For SNP-disease associations, allelic, recessive, dominant, and log-additive genetic models were evaluated. Haplotype analysis and visualization of the haplotypic structure of the GCLC gene were performed by the Haploview software, v.4.2 [41]. p-values (P perm ) for allele/genotype/haplotype associations were estimated via adaptive permutations using PLINK and Haploview. Gene-environment interactions were analyzed in groups stratified by risk factors such as cigarette smoking and alcohol abuse. Replication of associations between GCLC gene polymorphisms and psoriasis was performed using the Gene ATLAS database of the UK Biobank (http://geneatlas.roslin.ed.ac.uk (accessed on 17 January 2023)). Associations of pairwise genotype combinations (diplotypes) with the risk of psoriasis were estimated by the chi-squared test and adjusted for multiple comparisons by the false discovery rate (FDR) procedure (False Discovery Rate Online Calculator, https://tools.carbocation.com/FDR, accessed on 9 April 2023).

Baseline and Clinical Characteristics of the Study Patients
The baseline and clinical characteristics of the study patients are listed in Table 1. The group of patients with psoriasis was matched to the control group for sex (p = 0.30). The psoriasis patients were more than ten years younger than the healthy subjects. The duration of psoriasis was 10 (4-21) years. The mean age of disease onset was 27  years old. The number of smokers in each group was about equal. However, the number of subjects abusing alcohol in the patient group was seven times higher than in the control group (p < 0.0001). The psoriatic triad was diagnosed in 54.4% of patients. Most often, psoriatic rashes in patients were observed in the upper (80.0%) and lower (57.4%) extremities, the head (47.9%), and the trunk (33.08%), which is typical for psoriasis.

Association of GCLC Gene Polymorphisms with the Risk of Psoriasis
Genotype frequencies for five polymorphisms of the GCLC gene satisfied the Hardy-Weinberg equilibrium in both cases and controls. Only one SNP, rs17883901, showed a deviation from the HWE in both groups (p = 0.001). We analyzed associations between the GCLC gene polymorphisms and the risk of psoriasis in entire groups and groups stratified by sex. Table 2 shows a summary of associations between GCLC gene polymorphisms and psoriasis risk in the entire and sex-stratified groups. Allelic, additive, dominant, and recessive genetic models of SNP-disease associations were evaluated, and p-values (P perm ) were assessed using adaptive permutation tests. The most significant Pperm was considered to be the selected genetic model of SNP-disease associations.   The genotype and allele frequencies of the GCLC gene in healthy controls and patients with psoriasis, along with the most significant P perm of the SNP-disease associations, are reported in Table 3. As can be seen from Table 3, none of the polymorphisms showed an association with the risk of psoriasis as analyzed in the entire group of patients. However, the sex-stratified analysis detected that SNPs rs648595 (OR = 0.56, 95% CI 0.35-0.90; P perm = 0.017, recessive model) and rs2397147 (OR = 0.54, 95% CI 0.30-0.98; P perm = 0.05, recessive model) of the GCLC gene were associated with susceptibility to psoriasis in males. None of the polymorphisms was significantly associated with the risk of psoriasis in females.

Joint Effects of GCLC Gene Polymorphisms on the Risk of Psoriasis
The joint effects of GCLC gene polymorphisms on psoriasis risk were evaluated via haplotype and diplotype analyses. The GCLC haplotypes and their association with psoriasis risk in the entire and sex-stratified groups are shown in Table 4. Four common haplotypes of GCLC (H1-H4) with a frequency of more than 5% were identified in the study groups. The rare haplotype H12, with a frequency of 1%, was detected only in females. Figure 1 shows the linkage disequilibrium plot of the GCLC gene generated by the Haploview software. The polymorphism rs17883901 was not linked to any of the other studied SNPs in the GCLC gene. As can be seen from Table 4, none of the haplotypes was meaningfully associated with the risk of psoriasis, both in the entire and sex-stratified groups (P perm > 0.05). Table 4. Haplotypes of the GCLC gene and their association with psoriasis risk in the entire and sex-stratified groups.

Patients with Psoriasis Healthy Controls
Chi Square p-Value rs524553 rs542914 rs648595 rs6933870 rs2397147 rs17883901 Entire groups Males  The results of the diplotype analysis are shown in Table 5. In the entire group, genotype combinations such as rs542914-C/C × rs648595-G/T (FDR-adjusted p = 0.03) and rs648595-G/G × rs6933870-C/G (FDR-adjusted p = 0.016) of GCLC showed associations with an increased and decreased risk of psoriasis, respectively. In the male group, diplotype rs2397147-C/C × rs17883901-G/G was associated with a decreased risk of psoriasis (FDR-adjusted p = 0.014), whereas diplotype rs6933870-G/G × rs17883901-G/G (FDR-adjusted p = 0.045) showed an association with an increased disease risk in females. The remaining six diplotypes associated with disease risk in males did not reach statis- The results of the diplotype analysis are shown in Table 5. In the entire group, genotype combinations such as rs542914-C/C × rs648595-G/T (FDR-adjusted p = 0.03) and rs648595-G/G × rs6933870-C/G (FDR-adjusted p = 0.016) of GCLC showed associations with an increased and decreased risk of psoriasis, respectively. In the male group, diplotype rs2397147-C/C × rs17883901-G/G was associated with a decreased risk of psoriasis (FDRadjusted p = 0.014), whereas diplotype rs6933870-G/G × rs17883901-G/G (FDR-adjusted p = 0.045) showed an association with an increased disease risk in females. The remaining six diplotypes associated with disease risk in males did not reach statistical significance after adjusting for multiple tests.

Gene-Environment Interactions and Psoriasis Risk
Since psoriasis is a multifactorial disease, it appears important to investigate the joint influence of environmental risk factors and gene polymorphisms on disease development. Two risk factors, such as cigarette smoking and alcohol abuse, were used for the analysis of gene-environment interactions in psoriasis. Table 6 shows a summary of associations between GCLC gene polymorphisms and psoriasis risk in groups stratified by cigarette smoking and alcohol abuse habits. We found that SNP rs648595 is associated with the risk of psoriasis in cigarette smokers (OR = 0.55, 95% CI 0.31-0.99; P perm = 0.049, recessive model), whereas no association of this polymorphism was seen in non-smokers (OR = 0.88, 95% CI 0.59-1.31; P perm = 0.52, recessive model). In contrast, SNP rs17883901 showed an association with the risk of psoriasis in non-smokers (OR = 0.22, 95% CI 0.02-1.97; p = 0.14; P perm = 0.002, recessive model), whereas no association with this variant was observed in smoker subjects (OR = 0.89, 95% CI 0.11-5.90; p = 0.84; P perm = 0.99, recessive model). Notably, polymorphisms rs542914 (OR = 0.57, 95% CI 0.36-0.90; P perm = 0.015, recessive model) and rs648595 (OR = 0.60, 95% CI 0.39-0.92; P perm = 0.03, recessive model) of GCLC were associated with a decreased risk of psoriasis in non-drinkers of alcohol.
However, no protective effects of these SNPs against the risk of psoriasis were identified in alcohol abusers (p > 0.05).

Replication of Associations between GCLC Gene Polymorphisms and Psoriasis Risk in a Population of UK Biobank
It is stated that replication helps ensure that a genotype-phenotype relationship discovered in an original study represents a credible association and is not a chance finding or an artifact due to uncontrolled biases [42,43]. Therefore, we performed a replication analysis of associations between the studied GCLC gene polymorphisms and psoriasis susceptibility in two large populations from the UK Biobank. Table 7 shows the results of replication analysis to confirm associations between the studied polymorphisms of the GCLC gene and psoriasis risk in a population of the UK Biobank. It has been revealed that two SNPs of GCLC, rs6933870 (p = 0.063) and rs2397147 (p = 0.057), showed a clear tendency in their association with the risk of psoriasis in one of the UK cohorts. Formally, we cannot conclude that the GCLC gene polymorphisms we studied have been replicated in an independent population. The non-replication of SNP-disease associations might be in part explained by inter-population genetic differences, and this issue has been proposed to be readily resolved by the use of a gene-based approach rather than either an SNPbased or a haplotype-based approach [42,44]. Pursuing this proposal, we performed an association analysis of psoriasis with all SNPs of the GCLC gene genotyped in the UK Biobank cohorts. As a result (Table 8), 75 and 21 SNPs of the GCLC gene in the first and second UK Biobank cohorts, respectively, have been found to be associated with the risk of psoriasis at a p-value ≤ 0.05. Two polymorphisms of GCLC, rs547541077 (p = 0.004) and rs7764361 (p = 0.039), were associated with psoriasis risk in both cohorts.

Discussion
Since the skin is frequently exposed to environmental insults such as ultravio radiation, exposure to toxic chemicals, or mechanical injury causing oxidative or c cal stress, one of the principal physiologic roles of the skin is as a robust barrier ag xenobiotics and free radicals for their metabolic elimination and detoxification [25,4 For promoting these functions, human skin possesses a significant potential for ph

Discussion
Since the skin is frequently exposed to environmental insults such as ultraviolet irradiation, exposure to toxic chemicals, or mechanical injury causing oxidative or chemical stress, one of the principal physiologic roles of the skin is as a robust barrier against xenobiotics and free radicals for their metabolic elimination and detoxification [25,45,46]. For promoting these functions, human skin possesses a significant potential for phase II metabolism via multiple reactions including glutathione conjugation [45], and, therefore, the cytoprotective effects of GSH are likely to be of importance in this tissue. Experimental studies by Telorack and co-workers [25] have revealed that knockout mice with keratinocyte-specific deficiency in glutamate cysteine ligase showed a strong reduction in the viability of cell culture in vitro and in the skin in vivo. Furthermore, the authors observed that keratinocytes in glutathione-deficient mice died from apoptosis, ferroptosis, and necroptosis, and the increased cell death was attributed to increased levels of reactive oxygen and nitrogen species, causing DNA and mitochondrial damage [25]. This important research demonstrates the epidermis's exceptional antioxidant capability (especially with glutathione), which ensures skin integrity and effective wound healing. A deficiency of skin glutathione may contribute to psoriasis development. Genetic polymorphisms of glutamate cysteine ligase that are correlated with a decrease in GCLC mRNA and protein expression, enzyme activity, and GSH content [47][48][49][50][51] represent attractive markers for studying the molecular mechanisms of psoriasis. Polymorphisms of the GCLC gene have been found to be associated with the risk of cardiometabolic diseases such as coronary artery disease [52,53], ischemic stroke [54], type 1 [55,56] and type 2 [51] diabetes mellitus, polycystic ovary syndrome [57], and nonalcoholic fatty liver disease [58], as well as other multifactorial disorders such as bronchial asthma [59], pulmonary tuberculosis [60], and colorectal cancer [61]. However, no studies have been designed so far to investigate the role of GCLC gene polymorphisms in psoriasis susceptibility.
The present study is the first to show that polymorphisms of the GCLC gene are significantly associated with the risk of psoriasis and related to its clinical features. Two SNPs, rs648595 and rs2397147, were found to be associated with a decreased risk of psoriasis in males, suggesting sexual dimorphism in the relationship between the gene variation and susceptibility to psoriasis. Sexual dimorphism was also seen in associations between GCLC diplotypes and disease risk: rs2397147-C/C × rs17883901-G/G was associated with a decreased risk of psoriasis in males, whereas diplotype rs6933870-G/G × rs17883901-G/G showed an association with an increased disease risk in females. These findings were not surprising because gender differences in psoriasis risk and severity have become a discussable issue among dermatologists in the last few years [62,63].
Notably, sexual dimorphism has also been demonstrated in some genetic association studies on skin disease such as atopic dermatitis [64]. Environmental risk factors such as cigarette smoking and alcohol abuse may explain the mechanisms by which sexual dimorphism determines susceptibility to psoriasis [36,37,65]. We investigated the effect of GCLC gene polymorphisms on psoriasis risk depending on these environmental risk factors in the studied population. We found that polymorphism rs648595 is associated with the risk of psoriasis in cigarette smokers exclusively. Another SNP of GCLC, rs17883901, was associated with the risk of psoriasis only in non-smokers. Furthermore, polymorphisms rs542914 and rs648595 were found to be associated with a decreased risk of psoriasis in non-drinkers of alcohol, whereas no protective effects of these SNPs against disease risk were seen in subjects who were alcohol abusers.
The present study revealed sex-independent associations between GCLC gene polymorphisms and some clinical features such as the psoriatic triad, earlier onset, and more frequent flare-ups of disease, as well as localizations of psoriatic lesions. The last finding suggests that there are area-specific genetic effects of the studied polymorphisms of the GCLC gene that may be attributed to inter-individual differences in gene expression and, therefore, rates in glutathione biosynthesis by the skin from different body areas, as was demonstrated with regard to the rate of glutathione conjugation in different organs [66]. It is also known that the levels of glutathione may vary in sun-exposed and sun-protected areas [67], suggesting that UV exposure may impact glutathione biosynthesis in the skin.
The replication analysis in the UK Biobank cohorts showed a non-significant but clear association between rs6933870 and rs2397147 and psoriasis risk, suggesting that inter-population genetic differences may explain the non-replication of SNP-disease relationships. When we analyzed the associations between psoriasis and all the SNPs of the GCLC gene genotyped in the same cohorts, more than 70 polymorphisms were associated with disease risk, meaning that different SNPs may contribute to disease susceptibility in different ethnicities.
The functional annotation of some polymorphisms of the GCLC gene was performed in our previous study [54]. In particular, we found that allele rs648595G (this SNP showed the most significant association with psoriasis) is associated with a decreased expression of GCLC in blood, non-sun-exposed suprapubic skin, and sun-exposed lower leg skin. This SNP has regulatory potential and is located in transcription factor (TF)-binding or DNase hypersensitivity sites [54]. As predicted by HaploReg v4.2 tools, the rs648595 polymorphism is located within the TF-binding site for transcription factor AP-1 (activator protein 1), which is known to control gene expression in response to various stimuli such as cytokines, growth hormones, stress, and infections [68]. In the liver, SNP rs648595 is enriched with enhancer (H3K4me1 and H3K27ac) and promotor (H3K4me3 and H3K9ac) histone marks that regulate the transcriptional activity. In particular, H3K4me1 is a dynamic modification that was specifically found to mark both active and primed enhancers [69]. Enhancers bearing the H3K4me1 mark were found to be poised for activation in response to external stimuli [70]. H3K4me3 was found to promote rapid gene activation [71]. H3K9ac co-occurs highly with H3K14ac and H3K4me3 histone marks associated with active gene promoters [72]. Taken together, the epigenetic data clearly show that the polymorphism rs648595 of the GCLC gene represents an important genetic variant capable of activating gene expression in the liver.
Our findings of gene-environment interactions indicate that risk factors such as cigarette smoking and alcohol abuse can modify the associations between GCLC gene polymorphisms and the risk of psoriasis (Table 6). It is known that reduced glutathione plays an important role in ethanol detoxification, and acute ethanol administration was found to deplete GSH in the liver and other organs [73]. The leveling of the protective effects of the rs648595 and rs542914 polymorphisms in chronic alcohol abusers appears to be explained by the fact that persistent ethanol intake may diminish the endogenous pool of glutathione [74,75]. Meanwhile, an in vitro study by Kimura and co-workers [76] has revealed that primary human hepatocytes treated with 100 and 200 mM of ethanol showed the induction of GCLC gene expression via the activation of the NF-κB pathway. Tobacco smoking is also well known to deplete glutathione [77][78][79]. Thus, our study supports the causative roles of tobacco smoking and alcohol abuse in the development of psoriasis, and the negative effects of these environmental factors eliminate the protective role of polymorphisms of the GCLC gene against disease risk.
Sexual dimorphism in the discovered associations of GCLC gene polymorphisms with psoriasis risk is apparently attributed to differences in environmental exposures (i.e., smoking and alcohol abuse) between sexes. Considering an important role of oxidative stress in the pathogenesis of psoriasis [13][14][15][16], the mechanisms by which glutathione exerts protective effects against disease risk can be explained by the key role of glutathione in detoxifying ROS and environmental toxicants, penetrating and generating in the skin. However, the role of glutathione in psoriasis pathogenesis is not limited to protecting skin from oxidative damage. GSH is also involved in the regulation of cell proliferation, wound healing, and the inhibition of apoptotic pathways [21,22]. Furthermore, glutathione plays an important role in the regulation of the immune system and inflammation, two faces of the same biological coin [80]. Glutathione possesses a wide range of effects on the immune system, either activating or suppressing the immune response to control inflammation. In particular, reduced glutathione is required for the control of innate and adaptive immunological processes such as T-lymphocyte proliferation, the phagocytic activity of polymorphonuclear neutrophils, and dendritic cell functions, as well as antigen presentation by antigen-presenting cells [80][81][82]. Changes in glutathione concentrations may be critical in many autoimmune disease disorders, including psoriasis [83]. In particular, glutathione may suppress the immune reaction in mice with allergic contact dermatitis [84], inhibit the production of inflammatory cytokines, and maintain the adequate production of interferon-gamma by dendritic cells [80].
Our study has several limitations. Since our study was the first to investigate the contribution of GCLC gene polymorphisms to psoriasis risk in relatively small groups of patients, further studies in populations with a larger sample size are required to replicate the observed associations. The relatively small number of subjects in the study groups did not allow the analysis of the joint effects of GCLC gene polymorphisms and environmental risk factors (smoking and alcohol abuse) separately in males and females to obtain estimates of sex-specific gene-environment interactions contributing to psoriasis susceptibility. Since the studied polymorphisms of the GCLC gene are located in noncoding regions, their phenotypic effects should be interpreted with caution because no investigations were conducted to assess gene expression in skin biopsies from study patients. Further genetic association studies are recommended to follow the gene-based approach to look for the link between psoriasis and a wider spectrum of polymorphisms in the GCLC gene. Following this approach, nevertheless, it should be taken into account that SNPs might be characterized by weak or moderate phenotypic effects that cannot be reproduced in independent populations given their genetic heterogeneity in minor allele frequencies and linkage disequilibrium between the loci [85,86]. Importantly, some studies have recently reported genetic differences in glutathione metabolism between races or ethnicities [47,87].

Conclusions
The present study demonstrated, for the first time, that polymorphisms in the gene encoding the catalytic subunit of glutamate cysteine ligase represent novel genetic markers for susceptibility to psoriasis. The phenotypic effects of GCLC polymorphisms on psoriasis risk are modified by tobacco smoking and alcohol abuse, which are known environmental factors that increase disease risk. The GCLC gene may contribute to the pathogenesis of psoriasis via the diminished biosynthesis of glutathione in both the liver and skin, where GSH regulates a plethora of cellular processes such as redox homeostasis, the detoxification of xenobiotics, innate and adaptive immune functions, inflammation, cell proliferation and differentiation, and apoptosis. A better understanding of the relationship between GCLC gene polymorphisms and glutathione biosynthesis, as well as the molecular mechanisms by which this gene contributes to psoriasis, will open new scientifically based options for disease therapy and prevention targeting for glutathione metabolism. In particular, the use of L-cysteine and glycine as food supplements to restore the endogenous glutathione pool in patients with psoriasis is supported by our study results. Thus, this approach has potential in dermatological practice as a means of adjuvant therapy for psoriasis and the prevention of disease progression. Furthermore, pharmacogenetic and precision medicine approaches [88,89] would make it possible to subclassify patient groups based on environmental risk factors (e.g., cigarette smoking and alcohol abuse) and clinically significant genetic variants affecting glutathione metabolism, thus personalizing and improving the treatment and prevention of psoriasis.