Gene Polymorphisms And Functional Analysis Of Gsts,CAT Gene And Vitiligo Susceptibility

Vitiligo is a relatively common, acquired, and non-contagious depigmenting disorder of the skin characterized by a progressive, patchy loss of pigmentation from skin. Multi-factorial and overlapping pathogenetic mechanisms are proposed for the disease, but the cellular mechanisms leading to the loss of functioning epidermal melanocytes remain unclear. Many hypotheses have been proposed for the pathogenesis of depigmentation including autoimmune, biochemical, neural, self-destructive and genetic hypotheses. Oxidative stress has attracted much more attention in recent years, because numerous findings showed that it may be the triggering event of the melanocyte degeneration in vitiligo. Although reactive oxygen species(ROS) are constantly generated by mitochondria via enzyme complexes during normal cellular processes such as oxidative phosphorylation, excess amount can cause severe cell injury and even cell death.The unbalance between oxidative and antioxidant activities, such as accumulation of hydrogen peroxide (H2O2) and a low catalase (CAT) level/activity, may induce the destruction of melanocytes. A number of CAT gene single nucleotide polymorphisms (SNPs) and mutations have been associated with disease manifestations such as catalasemia/hypocatalasemia, hypertension, and type 2 diabetes mellitus in different races The polymorphism at catalase(CAT) gene may exert deleterious effects upon the expression or function of catalase, many were reported associated with disease manifestations such as catalasemia/hypocatalasemia, hypertension, and type 2 diabetes mellitus in different races. The polymorphisms in GSTs were correlated with increased susceptibility to diseases associated with oxidative stress, such as cancer, diabetes, asthma, and Parkinson's disease.Hence, we hypothesized that the foregoing common GSTs and the CAT polymorphisms were associated with the susceptibility to vitiligo in the Chinese population, and tested this hypothesis in our hospital-based case-control study of 749 vitiligo patients and 763 vitiligo-free control subjects. We further analyzed the function of a CAT polymorphism in promoter.MethodsWe detected genotypes of the 8 polymorphisms in the cases and control by the means of PCR-RFLP and multi-PCR, and analyzed the association between genotype distributions with vitiligo susceptibility. To investigate the function of positive associated polymorphism in CAT promoter region, we constructed recombinant luciferase reporter plasmids containing A or T allele at -89 locus, then transiently transfected HEK293 cells and pigment cells, and the relative activities were evaluated with the luciferase reporter system. We also observed the serum catalase activities in different genotype carriers.Results1. A significantly increased risk of vitiligo susceptibility was associated with the GSTT1 null type (adjusted OR 1.42, 95% CI 1.16-1.75), while the null type of GSTM1 genotype only showed a borderline increased vitiligo risk (P=0.059, adjusted OR 1.22, 95% CI 0.99-1.49). There was also significant association of vitiligo risk with GSTT1 /GSTM1 deleted types, and the trend test was significant (Ptrend <0.001). 2. The frequency of the alleles and genotypes in vitiligo patients did not show significant over-representation compared with the controls (P=0.103). For the GSTP1 Ala113Val, Val allele was rarely detected (only one Ala/Val genotype was present in 200 control subjects). For the GSTP1 Gly169As, Asp allele could not be detected in any of the control subjects or the vitiligo patients, so all individuals recruited in this study might be homozygous for the Gly allele.3. The CAT -89 variant T allele frequency was significantly higher among the cases than among the controls (P = 0.001).No evidence showed that CAT 389C>T was associated with the susceptibility to vitiligo. For the CAT 419C>T, the T allele could not be detected in any of the control subjects or the vitiligo patients, then all individuals recruited in this study might be homozygous for the C allele.4. CAT -89A>T and CAT 389C>T were in linkage disequilibrium (D' = 0.652, P < 0.001), suggesting that joint effect between the two SNPs exist, and the haplotypes containing -89T allele were associated with increased risk of vitiligo. The patients carrying the CAT -89AT genotype were more likely to carry the CAT 389 TT genotype than the controls, and patients carrying two risk genotypes of -89AT and 389TT had an OR of 3.00, suggesting an interaction between the CAT -89A>T and CAT 389C>T genotypes in intensifying risk of developing vitiligo5. Evaluated with the luciferase reporter system, the ?89A→T polymorphism affects the promoter regulation, and the promoter fragment containing T allele might be the Sp1-binding site.6. The serum catalase activity in vitiligo patients significantly decreased, compared with control individuals. There were no obvious different among different genotype carriers.Conclusion1. There was significant association of vitiligo risk with GSTT1 /GSTM1 gene. GSTP1 gene might not play a major role in the pathogenesis of vitiligo. 2. CAT -89A>T was associated with vitiligo, and had joint effect with 389C>T on vitiligo.3. CAT -89 A>T might alter the gene transcription activity by binding transcription factor Sp1.4. Further studies need to be done for in-depth molecule mechanism of antioxidase keeping oxidation-antioxidation balance.