Association of CYP2C9 Genetic Variants with Vitiligo.

BACKGROUND: Vitiligo is a depigmenting skin disorder in which genetic factors play an important role.
OBJECTIVE: To examine the association of CYP2C9 (*) 1/(*) 2/(*) 3 gene polymorphism with vitiligo.
METHODS: In this case controlled study, 95 Saudi patients with vitiligo (50 men and 45 women), with a mean age of 27.3 years, were analyzed. Patients were compared to 86 healthy controls from the same locality (76 men and 10 women), with a mean age of 20.1 years. In all participants, DNA was extracted and processed for characterization of 2C9 (*) 1/(*) 2/(*) 3 gene variants using real time-polymerase chain reaction.
RESULTS: Vitiligo patients have a significantly higher CYP2C9 (*) 3 allele carriage rate compared to controls (32.7% versus 4.7%, p=0.00, odds ratio=9.9, 95% confidence interval=3.3~29.6). On the other hand, frequencies of CYP2C9 (*) 2 genotypes and alleles did not show any significant difference between vitiligo cases and controls. When the frequencies of CYP2C9 genotypes were compared among subgroups of age, gender, family history, and disease patterns, the cases with positive consanguinity had significantly higher frequencies of homozygous genotypes than others (p=0.029).
CONCLUSION: CYP2C9 (*) 3 allele carriage is probably associated with vitiligo susceptibility.

INTRODUCTION

Vitiligo is an acquired skin depigmentation that affects all races but is far more disfiguring in blacks. The precise cause of vitiligo is unknown1. An autoimmune process targeting melanocytes is considered to mediate its pathogenesis. Consistent with this hypothesis histological studies have reported the absence of melanocytes in the affected skin2. In addition to cellular immunity, multiple autoantibodies against melanocyte antigens including various enzymes and other substances have been detected in the sera of some patients with vitiligo3,4. Since genetic factors appear to play a role, 20% to 30% of patients were reported with a positive family history of the disorder5,6. Nevertheless, many vitiligo patients have neither a family history of vitiligo nor a history of other autoimmune diseases6. Consequently, many other hypotheses have been proposed to explain the pathogenesis of this disorder, including an inadequate defense from the toxic effects of free radicals and exposure of industrial chemicals7,8. These effects were hypothesized to be controlled by the variable expression of cytochrome P450 (CYP or P450) genes that encode a superfamily of multi-functional monooxygenases, which comprise more than 6,000 individual enzymes9. CYPs play a major role in the metabolism of foreign lipophilic compounds, including drugs and chemical carcinogens, as well as endogenous compounds such as steroids, fat-soluble vitamins, fatty acids, and biogenic amines9. In addition, CYP expression and activity can be in uenced by various factors such as genetic variations, presence of inhibitors or inducers, and disease states with differential tissue-specific expression pattern including the skin10,11,12,13,14,15.

The polymorphisms of important CYP450 genes such as CYP2C9, CYP2C19, CYP2D6, and CYP2E1 have been studied extensively in a large number of populations and showed a significant heterogeneity in the frequency of different alleles/genotypes and consequently in the resulting metabolizer phenotypes. Cytochrome P/450/2C9 (CYP2C9) is primarily localized in the liver but can be expressed in other tissues like the skin. This enzyme belongs to the subfamily cytochrome 2C, which comprise CYP2C9 and 3 isoenzymes, 2C8, 2C18, and 2C1916. The CYP2C9 gene is polymorphic and within the inactive alleles 2C9, *3, *6, *15, and *25, only *2 and *3 occur more frequently in Caucasians. In 2C9 (rs 1799853) the amino acid arginine Arg 144 is replaced by Cys while in 2C9 (rs 1057910) Ile 359 is replaced by Leu17,18. Variations in CYP2C9 can be detected by real time polymerase chain reaction (PCR) using Taqman probes or probe-based melting curve analysis with the light cycler instrument17,18. This work aims to investigate the association of slow or mutant metabolizer variants of CYP2C9 gene; *2 and *3 with vitiligo among Saudi patients.

MATERIALS AND METHODS

This is a case controlled study on 95 Saudi vitiligo cases in addition to a control sample of 86 healthy unrelated subjects from the same locality. Cases included 50 men and 45 women with a mean age of 27.3±14.5 years and a median age of 23 years. They were recruited from the Outpatient Dermatology Clinics affiliated to Qassim University and King Saud University, Saudi Arabia from January to August 2012. Diagnosis of vitiligo was made by a consultant dermatologist. The detected vitiligo cases were of focal (22 cases), vulgaris (52 cases), acrofacial (20 cases), and universal (1 case) types. The clinical classification of Hercogová et al.19, was used for the categorization of focal vitiligo as localized lesions that are clinically and pathologically typical of vitiligo presenting in the form of one or more macules in one area, but not clearly in a segmental distribution.

Among these cases, 39 (41.1%) patients had positive family history of vitiligo, whereas 32 (33.7%) patients had positive parental consanguinity. Patients' data were compared with that of the control subjects, which comprised 76 men and 10 women with a mean age of 20.1±3.3 years. An informed consent was obtained from all participants and the study was approved by the Scientific and Ethical Committees of Qassim University, Saudi Arabia.

Blood samples were taken from all participants and DNA was isolated from the peripheral blood using a MagNA Pure LC instrument (LC DNA Isolation Kit LV; Roche Molecular Biochemicals, Mannheim, Germany).

Oligonucleotide primers and fluorescence-labeled hybridization probes were designed for amplification and sequence-specific detection of both CYP2C9 *2 and 2C9 *3 (TIB MolBiol, Berlin, Germany). The master mix used in the PCR reaction contained 2µl of a 10×mixture of LightCycler FastStart DNA master hybridization probes, 5 mM MgCl2 (final concentration), 1µM final concentration of primers, 0.075µM final concentration of specific primers, and 0.2µM final concentration of hybridization probes. Real time PCR was done using LightCycler instrument (Roche Diagnostics, Mannheim, Germany). The specificity of the amplified product was confirmed by corresponding melting curve analysis.

Statistical analysis

Statistical analysis was performed using the statistical software program SPSS version 13.0 (SPSS Inc., Chicago, IL, USA). Comparisons between cases and controls' genotype and allele frequencies were done using the chi-square test and odds ratio (with 95% confidence intervals). In addition, conformity to the Hardy Weinberg law of genetic equilibrium was tested among cases and controls using the chi square test through the assessment of the difference between the frequencies of the observed and the expected genotypes. A p-value <0.05 was considered statistically significant.

RESULTS

Normal controls showed 5 different genotypic variants including CYP2 C9 *1/*1, *1/*2, *1/*3, *2/*2, *2/*3, and *3/*3 with frequencies of 67.4%, 22.1%, 3.5%, 5.8%, 1.2%, and 0.0% respectively. Vitiligo patients had significantly higher CYP2C9 *3 allele carriage rate (both *1/*3, *2/*3, and *3/*3 genotypes) compared to controls (32.7% vs. 4.7%, p=0.00, odds ratio [OR]=9.9, 95% confidence interval [CI]=3.3~29.6). This was confirmed by the higher CYP2C9 *3 allele frequency among patients compared to controls (16.8% vs. 2.3%, OR=8.1, 95% CI=2.8~23.4, p=0.0; Table 1). Interestingly, statistical analysis of the vitiligo cases excluding the focal type that is liable for mistyping, according to a recent classification, has confirmed the previous results indicating a significantly higher CYP2C9 *3 allele frequency among cases compared to controls (17.12% vs. 2.3%, OR=8.4, 95% CI=2.8~24.8, p=0.0; data not shown). On the other hand, frequencies of CYP2C9 *2 genotypes and alleles did not show any significant difference between vitiligo cases and controls (p>0.05; Table 1). Conformity to the Hardy Weinberg Equilibrium (HWE) was noted in CYP2C9*3 locus variants among cases and controls, which did not show any significant difference between the expected and observed genotypes (p>0.05). However, CYP2C9*2 variants showed significant difference between expected and observed frequencies of polymorphic genotypes (p<0.001; Table 1).

Table 1

Frequency of CYP2C9 *1/*2/*3 polymorphism in vitiligo cases compared to controls

Values are presented as number (%) or median (range). HWE: Hardy Weinberg Equilibrium, OR: odds ratio, CI: confidence interval.

†Marks indicate significant difference, p<0.05.

Combined CYP2C9 *2 and *3 genotypic variants confirmed the higher frequency of the heterozygous mutant forms *2/*1 and *3/*1 genotypes among cases compared to controls (47.4% vs. 25.6%) and a lower frequency of *1/*1 (48.4% vs. 67.4%). Comparison of the frequencies of CYP2C9 normal, heterozygous, and homozygous mutant genotypes among subgroups of age, gender, family history, and disease patterns indicated insignificant difference. On the other hand, cases with positive consanguinity showed higher homozygous genotypes than others (p=0.029; Table 2).

Table 2

Frequency of CYP2C9 *2 and *3 alleles and genotypes in Saudi cases of vitiligo related to their demographic data and the pattern of the disease

Values are presented as number (%). †Mark indicates significant difference, p<0.05.

DISCUSSION

Cytochrome P450 2C9 has been suggested to be very similar to epoxide hydrolase (sEH), which hydrolyzes a wide variety of endogenous and exogenous epoxides that are believed to be formed by cytochrome P450 epoxygenases. Moreover, sEH was found in various tissues including the epithelial cells in the skin20. In active vitiligo patients, an increase in oxidative stress in the entire epidermal compartment has been demonstrated; in particular, the imbalance in catalase activity, reduced glutathione, and vitamin E levels was associated with hyperproduction of reactive oxygen species21,22,23. Oxidative DNA damage in vitiligo patients manifested by DNA breakage in mononuclear leukocytes was shown to be comparatively higher24. Since these factors might contribute to the susceptibility to vitiligo, we have undertaken this research to clarify the association of CYP2C9 gene polymorphism with vitiligo among Saudi patients.

Frequencies of CYP2C variants among normal Saudi subjects showed a relatively unique pattern in the form of high carrier rate of *2 allele (*1/*2, *2/*3, and *2/*2 genotypes), which was 29.1% of controls that was much higher than the carriage rate for the allele *3 (*1/*3, *2/*3, and *3/*3) which was 4.7%, thus resulting into allele frequencies of 17.44% and 2.33% for alleles *2 and *3, respectively. In this respect, we would note that normal Saudi subjects had a lower carriage rate and allele frequency of CYP 2C *3 when compared to Iranians and Caucasians, whose carriage rate was as high as 5% to 10%16,25,26, but relatively closer to that of the Asian Indians, Korean, Chinese, Hispanics, and African Americans whose carriage rate was 2% to 4%26,27,28,29,30.

Interestingly, the Saudi vitiligo patients showed a significantly higher frequencies of *3 allele carriage rate corresponding to 32.7% of cases with a significantly higher *3 allele frequency of 16.84% but with a lower *2 allele frequency (11.05%) that was statistically insignificant compared to controls. This suggests the potential association of CYP2C9 *3 with the susceptibility to vitiligo, in this particular population. However, it is not clear whether this susceptibility is due to a primary genetic predisposition or secondary to an error related to the metabolism of certain chemicals or other environmental agents. Since CYP2C9 is an oxidative metabolizer of exogenous drugs and toxins as well as endogenous polypeptide enzymes and hormones, we can speculate that the slow metabolizing genetic form of the *3 allele predisposes the patient skin to oxidative stress probably under the effect of inhibitory drugs or toxins to the natural enzymes involved in melanin synthesis. Furthermore, the oxidative stress hypothesis is supported by the higher hydrogen peroxide levels in vitiligo epidermis that was previously attributed to factors like catalase genetic polymorphisms, reduced glutathione peroxidase activity and increased levels of tetrahydrobiopterins (6BH4 and 7BH4), which are inhibitors of tyrosinase,and phenyl alanine hydroxylase enzymes31,32.

We propose an extensive analysis of all potential forms of exposure to chemicals, drugs, pollutants or radiations for all affected subjects probably using investigative techniques like HPLC. Genome wide association studies indicated that most vitiligo susceptibility loci (more than 20) encode immunoregulatory proteins or melanocyte components that likely mediate immune targeting and genetic relationships among vitiligo, malignant melanoma, and normal variation of eye, skin, and hair color33,34. These loci are distributed along diverse chromosomal locations including the chromosome 10q22-23 in areas nearby the area coding the CYP2C9 (10q24). A strong association with a single nucleotide polymorphism within the major histocompatibility complex region had been also identified in a recent genome-wide association study of generalized vitiligo35.

Since degradation of drugs in humans is driven by detoxification mechanisms whose efficiency is influenced by genetic mutations, Weise et al.36 studied the association between type 2 diabetes with mutations in prominent members of the CYP 450 2C9 isoenzyme family.

Probable genetic contribution to the occurrence of vitiligo among Saudi subjects is relatively higher in patients with positive family history (41.1%) and consanguinity (33.7%). This study, demonstrated that consanguinity apparently had a role into the appearance of homozygous genotypes although most of them were for the normal or wild type allele *1 with some mutant forms. On the other hand, the distribution of genetic variants of CYP2C9 were not affected by age, gender, family history or pattern of vitiligo among the patients. The gene frequencies related to the CYP 2C*3 allele were in conformity with the HWE. In contrast, gene frequencies related to allele *2 were not in accordance to the HWE that might be due to higher levels of consanguinity or due to the relatively small sized sample. So, this research probably needs to be investigated in a wider study by including other interactive haplotypes and genetic polymorphisms as suggested by other scientists37.

Interestingly, Saudi cases did not show any significant difference from the control subjects in terms of the frequency of their CYP2C9 *2 allelic variants. Similarly, Semiz et al.38 reported that no significant difference in allele frequencies for CYP2C9 *2, was demonstrated between diabetic and non-diabetic subjects. Recently, Kaur-Knudsen et al.39 reported the findings of large studies on the association between genetic variation in CYP1B1 and CYP2C9 and the risk of disease, and rebutted the hypotheses that these genetic variants?influenced the risk of tobacco-related cancer, female cancer (as cervical and endometrial cancers), chronic obstructive pulmonary disease, and ischemic vascular disease. Veenstra et al.18 found that genetic variation in CYP2C9 exons, rather than the promoter or other regulatory regions, is largely responsible for warfarin sensitivity associated with CYP2C9 variants in a European American population. Other studies have reported that CYP2C9 *3 genotype did not affect the required warfarin dose while it was associated with increased risk of bleeding when treated with routine dosage regimen during the initiation of treatment40.

In conclusion, this study provides presumptive evidence that CYP2C9 *3 is probably associated with the susceptibility to vitiligo among Saudi subjects regardless of the clinical pattern, gender, and presence of family history. Nonetheless, our study is limited in terms of the relatively small sample size, lack of protein studies or cell culture analyses to fully examine the underlying mechanism of vitiligo pathogenesis.