Mohmmad Shoab Mansuri

Vitiligo: interplay between oxidative stress and immune system

Vitiligo is a multifactorial polygenic disorder with a complex pathogenesis, linked with both genetic and non‐genetic factors. The precise modus operandi for vitiligo pathogenesis has remained elusive. Theories regarding loss of melanocytes are based on autoimmune, cytotoxic, oxidant–antioxidant and neural mechanisms. Reactive oxygen species (ROS) in excess have been documented in active vitiligo skin. Numerous proteins in addition to tyrosinase are affected. It is possible that oxidative stress is one among the main principal causes of vitiligo. However, there also exists ample evidence for altered immunological processes in vitiligo, particularly in chronic and progressive conditions. Both innate and adaptive arms of the immune system appear to be involved as a primary event or as a secondary promotive consequence. There is speculation on the interplay, if any, between ROS and the immune system in the pathogenesis of vitiligo. The article focuses on the scientific evidences linking oxidative stress and immune system to vitiligo pathogenesis giving credence to a convergent terminal pathway of oxidative stress–autoimmunity‐mediated melanocyte loss.

Interleukin-4 genetic variants correlate with its transcript and protein levels in patients with vitiligo

Background  Vitiligo is an acquired pigmentary disorder resulting from loss of melanocytes. Interleukin (IL)‐4 has been shown to stimulate B‐cell proliferation, to regulate immunoglobulin class switching (IgG1 and IgE) and to promote T‐cell development. Polymorphisms in the IL4 gene are known to increase its expression, thereby implicating its role in vitiligo susceptibility.

Role of oxidative stress and autoimmunity in onset and progression of vitiligo

Vitiligo is an acquired depigmentation disorder characterized by the loss of functional melanocytes from the epidermis. Two major theories of vitiligo pathogenesis include autoimmunity and oxidative stress‐mediated toxicity in melanocytes. The present study aimed to evaluate both the hypotheses in vitiligo patients and to investigate their role in the disease onset and progression. Antimelanocyte antibody levels and lipid peroxidation (LPO) levels were evaluated in 427 patients and 440 controls; antithyroid peroxidase (TPO) antibody levels were estimated in 102 patients and 72 controls. Patients showed a significant increase in LPO and antimelanocyte antibody levels compared to controls. Antimelanocyte antibody and LPO levels were higher in active vitiligo compared to stable. Only 9.8% of patients showed the presence of anti‐TPO antibodies in their circulation. Oxidative stress may be the initial triggering event to precipitate vitiligo in Gujarat population, which is exacerbated by contributing autoimmune factors together with oxidative stress.

Association of generalized vitiligo with MHC class II loci in patients from the Indian subcontinent.

TO THE EDITOR Generalized vitiligo is a disease in which patches of depigmented skin and overlying hair result from autoimmune destruction of melanocytes in involved regions (Spritz, 2012). Clinic-based studies cite high prevalence of vitiligo in India, up to 8.8% (e.g. Handa and Kaur, 1999), though population-based surveys report much lower prevalence, 0.46% in Calcutta (Das et al., 1985) and 1.79% in South Gujarat (Mehta et al., 1973). Vitiligo is a distressing cosmetic problem in individuals of dark skin phototypes, due to striking contrast between lesions and unaffected skin. This may explain the reported high prevalence of vitiligo in India and negative impact on perceived quality of life in this population (Parsad et al., 2003). Indeed, vitiligo has long been recognized in India (Singh et al., 1974), the specific use of ultraviolet light treatment was pioneered in India (Menon, 1945), and some of the earliest genetic studies of vitiligo were carried out there: of ABO blood groups, α1-antitrypsin, and haptoglobin, and subsequent candidate gene studies, including GCH1, ACE, CAT, CTLA4, GPX1, IL4, MBL2, and PTPN22, most yielding negative or conflicting results. Recently, Singh et al. (2012) tested genetic association of vitiligo in Indian patients with HLA–A, -B, -C in the MHC class I region and HLA-DRB1 in the class II region, identifying primary genetic association with HLA-DRB1* 07:01. Here, we describe a more comprehensive genetic association study of generalized vitiligo on the Indian subcontinent, utilizing the Immunochip® (Cortes and Brown, 2011) to screen 196,524 SNPs in 128 loci previously implicated in autoimmune and inflammatory diseases, including 9441 SNPs spanning the extended major histocompatibility complex (MHC) on chromosome 6p. Our results suggest there are at least two independent association signals in the MHC class II region, one located upstream of HLA-DRA and the other located between HLA-DRB1 and HLA-DQA1, generally similar to what we previously found in a genomewide association study of vitiligo in European-derived whites (EUR) (Jin et al., 2010). Our initial study group consisted of 255 patients with generalized vitiligo and 377 unrelated non-vitiligo controls of Indian subcontinent (Pakistan, India, Sri Lanka, Bangladesh) derivation. After quality control procedures, data for 120,724 remaining SNPs from 251 remaining cases were compared to those from 349 remaining controls. Suggestive association signals were considered as clusters of nearby SNPs with trend P-values <10−5. The International Immunochip Consortium has agreed on a genomewide significance criterion of P<5 × 10−8 for studies utilizing the Immunochip (Cortes and Brown, 2011). As shown in Figure 1a and Supplementary Table S1, the only highly suggestive association signals were in the MHC class II gene region (Figure 1b), from rs3134942 (chr6:32168770) to rs2856674 (chr6:32659644), spanning the upstream part of NOTCH4 through HLA-DQB1. The principal region of association encompassed c6orf10--BTNL2--HLA-DRA--HLA-DRB5--HLA-DRB1--HLA-DQA1 (Figure 1b), with extensive LD through this region in this population (Figure 1c). One SNP, rs482044, located towards the centromeric end of the region, between HLA-DRB1 and HLA-DQA1, achieved genomewide significance (G allele; P=1.94 × 10−8, OR=1.93; Table 1), remaining significant (P = 4.86 × 10−8) even after correction for the observed genomic inflation factor λ = 1.06. Figure 1 Immunochip association results for generalized vitiligo Table 1 MHC class II region SNPs genotyped in Immunochip screening and in replication studies To determine which SNPs in the MHC class II region represent primary association with vitiligo versus are signals secondary to LD, we applied a backward regression procedure, comparing a model including the seven most significant MHC class II SNPs to alternative models in which each SNP was removed one by one. This analysis suggested that this region contains two independent associated loci, one represented by rs482044-G (located between HLA-DRB1 and HLA-DQA1) and the other represented by rs3129859-C (located 6680 nt upstream of HLA-DRA). Forward regression analysis of these two SNPs showed that the model composed of rs3129859 was significantly (P=4.4 × 10−5) improved by adding rs482044, and that the model composed of rs482044 was significantly improved (P=6.0 × 10−4) by adding rs3129859. In contrast to our previous findings in EUR (Jin et al., 2010), we observed no apparent association of vitiligo with SNPs in the MHC class I region in this Indian-Pakistani population (Figures 1a and 1b). Furthermore, considering loci represented on the Immunochip that have been reported to be associated with vitiligo in previous candidate gene studies from India, no SNPs in the ACE (3 SNPs), CTLA4 (505 SNPs), or IL4 (103 SNPs) gene regions showed even nominal association in the present study. To confirm association of generalized vitiligo with MHC class II region SNPs in the Indian subcontinent, we carried out a replication study of rs3129859 and rs482044, as well as the third most significant Immunochip SNP, rs3096691 (located just upstream of NOTCH4) (Fig. 1b). These three SNPs were genotyped in 685 unrelated generalized vitiligo cases and 774 unrelated controls from Gujarat state, India. All three were in Hardy-Weinberg equilibrium in the controls, and all three achieved at least nominal significance in the replication study (Table 1). Most significant association in the replication study was observed for rs3129859-C (P=9.48 × 10−9), with no significant heterogeneity of OR between the two studies (PBreslow-Day=1.15 × 10−1). Cochran-Mantel-Haenszel meta-analysis of the rs3129859 data from the Immunochip screen and replication study likewise yielded strongest overall association (P=4.30 × 10−14, OR=1.67; 95% C.I. 1.46–1.91). Association was also confirmed in the replication study for rs482044 (P= 1.11 × 10−4), with only nominal association for rs3096691 (P=2.32 × 10−2), although both of these SNPs exhibited heterogeneity of OR. Both rs482044 (P=1.58 × 10−2) and rs3129859 (P = 1.20 × 10−6) remained significant when each was conditioned on the other. Overall, our findings thus generally confirm association of vitiligo with at least two independent loci in the MHC class II region. In a previous genomewide-association study of generalized vitiligo in EUR subjects, we found that both vitiligo susceptibility (Jin et al., 2010) and age of onset (Jin et al., 2011) are likewise associated with at least two independent loci in the MHC class II region. To assess whether the MHC class II loci observed in the Indian subcontinent and EUR populations might correspond ancestrally, we carried out trans-ethnic meta-analysis using MANTRA (Morris, 2011), which indicated that the MHC association signal represented by rs482044 in the Indian subcontinent population apparently corresponds to the MHC signal represented by rs532098 in EUR (Jin et al., 2010) (Table S2). In contrast, rs3129859 is not significantly associated with vitiligo in EUR (Jin et al., 2010), and correspondence between the association signals upstream of HLA-DRA observed in both populations remains uncertain. Our findings thus highlight both similarity and differences of vitiligo MHC genetic associations in subjects from different major world populations. On the Indian subcontinent, this study and that of Singh et al. (2012) support association of vitiligo with loci in the MHC class II region, but show no primary association in the MHC class I region. Similarly, in the EUR population, vitiligo is also associated with multiple signals in the MHC class II region, at least one of which, between HLA-DRB1 and HLA-DQA1, appear to correspond to one in the Indian subcontinent population. However, in the EUR population vitiligo shows primary association with HLA-A in the distal class I region (Jin et al., 2010); specifically, HLA-A*02:01 (Jin et al., 2011). In addition, studies in Chinese show principal MHC association in the class III region (Quan et al., 2010) and in the proximal class I region, between HLA-B and HLA-C (Liu et al., 2012). Together, these similarities and differences of principal MHC genetic associations with generalized vitiligo among different populations may in part underlie differing prevalence of this autoimmune disease in different groups around the world.

Association of Neuropeptide Y ( NPY ), Interleukin-1B ( IL1B ) Genetic Variants and Correlation of IL1B Transcript Levels with Vitiligo Susceptibility

Background Vitiligo is a depigmenting disorder resulting from loss of functional melanocytes in the skin. NPY plays an important role in induction of immune response by acting on a variety of immune cells. NPY synthesis and release is governed by IL1B. Moreover, genetic variability in IL1B is reported to be associated with elevated NPY levels. Objectives Aim of the present study was to explore NPY promoter −399T/C (rs16147) and exon2 +1128T/C (rs16139) polymorphisms as well as IL1B promoter −511C/T (rs16944) polymorphism and to correlate IL1B transcript levels with vitiligo. Methods PCR-RFLP method was used to genotype NPY -399T/C SNP in 454 patients and 1226 controls; +1128T/C SNP in 575 patients and 1279 controls and IL1B −511C/T SNP in 448 patients and 785 controls from Gujarat. IL1B transcript levels in blood were also assessed in 105 controls and 95 patients using real-time PCR. Results Genotype and allele frequencies for NPY −399T/C, +1128T/C and IL1B −511C/T SNPs differed significantly (p<0.0001, p<0.0001; p = 0.0161, p = 0.0035 and p<0.0001, p<0.0001) between patients and controls. ‘TC’ haplotype containing minor alleles of NPY polymorphisms was significantly higher in patients and increased the risk of vitiligo by 2.3 fold (p<0.0001). Transcript levels of IL1B were significantly higher, in patients compared to controls (p = 0.0029), in patients with active than stable vitiligo (p = 0.015), also in female patients than male patients (p = 0.026). Genotype-phenotype correlation showed moderate association of IL1B -511C/T polymorphism with higher IL1B transcript levels. Trend analysis revealed significant difference between patients and controls for IL1B transcript levels with respect to different genotypes. Conclusion Our results suggest that NPY −399T/C, +1128T/C and IL1B −511C/T polymorphisms are associated with vitiligo and IL1B −511C/T SNP influences its transcript levels leading to increased risk for vitiligo in Gujarat population. Up-regulation of IL1B transcript in patients advocates its possible role in autoimmune pathogenesis of vitiligo.

Association of NLRP1 genetic variants and mRNA overexpression with generalized vitiligo and disease activity in a Gujarat population

It has been suggested that NLRP1 is involved in susceptibility to a wide range of autoimmune diseases including generalized vitiligo (GV). Genetic polymorphisms in the gene encoding NLRP1 (previously known as NALP1) have previously been shown to be associated with GV and there is speculation about their involvement in the regulation of NLRP1 expression.

Tumor necrosis factor B (TNFB) genetic variants and its increased expression are associated with vitiligo susceptibility.

Genetic polymorphisms in TNFB are involved in the regulation of its expression and are found to be associated with various autoimmune diseases. The aim of the present study was to determine whether TNFB +252A/G (rs909253) and exon 3 C/A (rs1041981) polymorphisms are associated with vitiligo susceptibility, and expression of TNFB and ICAM1 affects the disease onset and progression. We have earlier reported the role of TNFA in autoimmune pathogenesis of vitiligo, and we now show the involvement of TNFB in vitiligo pathogenesis. The two polymorphisms investigated in the TNFB were in strong linkage disequilibrium and significantly associated with vitiligo. TNFB and ICAM1 transcripts were significantly increased in patients compared to controls. Active vitiligo patients showed significant increase in TNFB transcripts compared to stable vitiligo. The genotype-phenotype analysis revealed that TNFB expression levels were higher in patients with GG and AA genotypes as compared to controls. Patients with the early age of onset and female patients showed higher TNFB and ICAM1 expression. Overall, our findings suggest that the increased TNFB transcript levels in vitiligo patients could result, at least in part, from variations at the genetic level which in turn leads to increased ICAM1 expression. For the first time, we show that TNFB +252A/G and exon 3 C/A polymorphisms are associated with vitiligo susceptibility and influence the TNFB and ICAM1 expression. Moreover, the study also emphasizes influence of TNFB and ICAM1 on the disease progression, onset and gender bias for developing vitiligo.

MicroRNA profiling reveals differentially expressed microRNA signatures from the skin of patients with nonsegmental vitiligo

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Association of Cytotoxic T-Lymphocyte Antigen 4 (CTLA4) and Thyroglobulin (TG) Genetic Variants with Autoimmune Hypothyroidism

Autoimmune hypothyroidism is known to be caused by immune responses related to the thyroid gland and its immunological feature includes presence of autoimmune antibodies. Therefore the aim was to analyze presence of anti-TPO antibodies in hypothyroidism patients in Gujarat. Cytotoxic T-Lymphocyte Antigen 4 (CTLA4) is one of the susceptibility genes for various autoimmune diseases. Hence, exon1 +49A/G and 3’UTR CT60A/G single nucleotide polymorphisms (SNPs) in CTLA4 and its mRNA expression levels were investigated in autoimmune hypothyroidism patients. Thyroglobulin (TG) is known to be associated with autoimmune thyroid disorders and thus exon 33 (E33) SNP in TG was investigated. We analyzed the presence of anti-TPO antibodies in the plasma samples of 84 hypothyroidism patients and 62 controls by ELISA. PCR-RFLP technique was used for genotyping of polymorphisms. sCTLA4 and flCTLA4 mRNA expression levels were assessed by real time PCR. 59.52% of hypothyroid patients had anti-TPO antibodies in their circulation. The genotype and allele frequencies differed significantly for +49A/G (p = 0.0004 for +49AG, p = 0.0019 for +49GG & p = 0.0004 for allele), CT60 (p = 0.0110 for CT60AG, p = 0.0005 for CT60GG & p<0.0001 for allele) and TG E33 (p = 0.0003 for E33TC p<0.0001 for E33CC& p<0.0001 for allele) SNPs between patients and controls. Patients had significantly decreased mRNA levels of both sCTLA4 (p = 0.0017) and flCTLA4 (p<0.0001) compared to controls. +49A/G and CT60 polymorphisms of CTLA4 were in moderate linkage disequilibrium. Logistic regression analysis indicated significant association of CT49A/G, CT60A/G and TG exon 33 polymorphisms with susceptibility to autoimmune hypothyroidism when adjusted for age and gender. Our results suggest +49A/G and CT60 polymorphism of CTLA4 and E33 polymorphism of TG may be genetic risk factors for autoimmune hypothyroidism susceptibility and down regulation of both forms of CTLA4 advocates the crucial role of CTLA4 in pathogenesis of autoimmune hypothyroidism.

miRNA signatures and transcriptional regulation of their target genes in vitiligo.

BACKGROUND miRNAs are small non-coding RNA molecules that post-transcriptionally regulate gene expression. We have earlier reported the skin miRNA expression profiling in patients with non-segmental vitiligo. OBJECTIVE In the present study, we show the expression of previously identified skin miRNAs signatures in blood and their target genes in whole blood and PBMCs as well as skin micro-environment of vitiligo patients and controls. METHODS miRNA expression profiling in whole blood was performed using customized TaqMan® Low Density Array. We predicted the potential targets of differentially expressed miRNAs and investigated their expression levels in skin, whole blood and PBMCs from patients and controls using Real-time PCR. RESULTS Our results showed miR-1, miR-184, miR-328, miR-383 and miR-577 hold similar pattern of expression as of skin, suggesting their potent eminence for being putative markers for vitiligo. In silico target prediction revealed miR-1 targets EDN1, G6PD, HSP60, HSP70, SERP1, SIRT1 & TYR; miR-184 targets EZR & LAMP1; miR-328 targets IL1B, POLH & TRPM1; miR-383 targets EDN1 & TYRP1; and miR-577 targets PTPN22 & TYRP1 which were corroborated by our validation study. CONCLUSIONS In conclusion, the present study for the first time provides new insights into the crucial role of miRNA regulated gene network involved in oxidative stress, autoimmunity and ER stress mediated pathogenesis of vitiligo.