Vitiligo belongs to a family of cutaneous diseases, characterized by a lack of pigmentation in the skin.1 This disease affects between 0.1% and 2% of the world’s population; however, its incidence varies considerably among the populations or ethnic groups analyzed and is estimated to be 0.14% in Russia, between 1% and 2.5% in the United States and Japan,2 and high in Mexico (4%) and India (8.8%).1,3

The causes of vitiligo are complex and not yet fully understood. Several hypotheses and theories have been developed to explain the depigmentation and melanocyte destruction observed in this condition; however, these theories do not explain the full spectrum of this disease.4 Thus, the clinical data, embryonic origin of the melanocytes, association with autoimmune disorders, and presence of relatives affected with this disease, among other factors, should be considered, suggesting that vitiligo results from a combination of environmental, autoimmune and genetic factors, and several theories have been proposed.5,6

Studies worldwide have explored genes that are potentially involved in the development of vitiligo and other autoimmune diseases using gene expression analysis studies, candidate gene association studies, genetic linkage studies, and genomic association studies (GWAS).7Gene expression studies performed on skin biopsies obtained from patients with vitiligo have enabled the analyses of changes in the expression patterns of several genes associated with immunomodulation,7 melanogenesis, and regulation of the development and survival of melanocytes.8-11

To date, no fully effective treatment for vitiligo has been identified. Techniques promoting repigmentation have been used, such as narrow-band ultraviolet B light (nb-UVB), which is currently considered the treatment of choice for vitiligo.12 This therapy uses a light spectrum of 311nm, presenting a penetrance to the basal layer of the epidermis, which induces local immunosuppression and stimulates the proliferation of melanocytes in the skin and in the external epithelial root sheath of the hair follicle, thereby stimulating melanogenesis and the production of melanocyte-stimulating hormone (MSH).12-14

Currently, there are no studies analyzing the changes in gene expression in the skin of subjects in response to the treatment of vitiligo, particularly the application of nb-UVB treatment. Using the currently available molecular tools, including reverse transcription polymerase chain reaction (RT-PCR) as well as transcript identification and gene expression quantification through RNA sequencing (RNA-seq) analysis, we can efficiently analyze the expression profiles of multiple genes present in cells and tissues under pathological and non-pathological human conditions, among other conditions.15-18 In particular, RNA-seq using next-generation sequencing (NGS) also facilitates a more accurate measurement of the levels of transcript expression, even the presence of isoforms in the analyzed samples.19 TruSeq RNA Targeted sequencing can effectively detect changes in patterns of gene expression by simultaneously analyzing a large variety of selected targets with custom designs, using smaller sample amounts, shorter processing times and lower costs than other technologies (qPCR and Microarray).20,21 This technology has previously been applied in the identification of expression profiles in other dermatological diseases, such as psoriasis and lupus erythematosus, suggesting that TruSeq RNA Targeted sequencing could be a useful tool for vitiligo.22-24 However, the discrimination power of this method has not been explored in vitiligo.

In a previous study conducted by our research group on skin biopsies obtained from a smaller number of untreated vitiligo patients and healthy controls, we detected changes in the expression of the genes involved in a wide variety of biological processes, including signaling, apoptosis, oxidative stress control, cell survival and pigmentation (unpublished data). Using the information from the gene expression profiles and the addition of other genetic targets identified by the previously mentioned methods, valuable information concerning gene expression patterns, clinical classification, and the effectiveness of the treatment could be obtained using RNA-seq.

Thus, the objective of the present study was to explore the use of RNA-seq technology for the identification of changes in the expression profiles of selected genes in skin biopsies obtained from vitiligo patients before and after treatment with nb-UVB; the present study used NGS TruSeq Targeted RNA Expression from Illumina.

In this research, a group of genes involved in skin pigmentation, apoptosis and cell survival, oxidative stress and signal transduction were selected to identify changes in the expression patterns of these genes in the skin of vitiligo patients undergoing treatment with nb-UVB phototherapy. Until recently, the relationship between the skin expression patterns of a group of genetic markers participating in the main routes involved in vitiligo, patients affected by this condition and responses to experienced standard treatments had not been documented.

For this purpose, nb-UVB phototherapy was applied for 6 months, which is considered the standard treatment for vitiligo vulgaris because of its effectiveness and safety.12

Evaluation by 2 dermatologists revealed that, consistent with patients undergoing treatment, 42 of the 45 subjects included in the expression analysis showed some degree of response to treatment. The three subjects who did not present repigmentation after treatment presented active vitiligo (6.67%). Kumar et al. (2009) and Chen et al. (2005)43,44 reported similar results, showing a high percentage of responses to this type of treatment. However, the latter study did not describe the type of vitiligo or disease activity presented by patients who showed no response to treatment.

TruSeq RNA Targeted sequencing, unlike other methodologies for gene expression analysis, offers the following advantages: i) effective detection of changes in the “transcriptome” or patterns of gene expression through the simultaneous analysis of a variety of targets, ii) reduced processing and analysis times, iii) the use of small sample sizes to maximize analyses in subjects affected by this condition compared to the patterns present in the genes of healthy subjects, and iv) affordable costs.20,21 Due to the limited biological sample size, the numerous genetic targets to be analyzed and the need for economic resources for its development, this methodology is presented as the best option for analysis, even over RT-qCR.

Using RNAseq technology for the analysis of gene expression, we observed differences in the expression patterns between the analyzed samples. The heat maps generated from the expression results obtained through the massive sequencing of Illumina TruSeq RNA (Figures 1 and 2) revealed similar expression patterns between the affected skin that repigmented after treatment and the unaffected skin of patients biopsied at the beginning of the study. Similar expression patterns were observed in the skin of vitiligo patients before treatment and affected skin that did not repigment after 6 months of phototherapy. Comparison of the different types of skin before and after treatment revealed the reduced expression of MLANA, DCT and TYRP1 genes, all of which are involved in skin pigmentation in non-pigmented skin compared to unaffected skin and repigmented skin after treatment. These results are consistent with those described in the PhD thesis (2012) of Salinas45 and results published by Regazzetti et al. (2015), who validated a group of genes using real-time PCR, reporting low levels of MLANA, DCT and TYRP1 expression in affected skin (vitiligo lesions, compared to perilesional skin and non-affected – pigmented - patients).30

Statistical analysis of the expression patterns obtained from the skin samples before and after treatment revealed alterations in the expression of genes related to skin pigmentation, apoptosis, cell survival, oxidative stress and signal transduction mechanisms.

Reduced gene expression patterns in non-pigmented skin were observed in 5 of the 7 genes included in this study, corresponding to MLANA, DCT and TYRP1, as previously mentioned, and MC1R and MC4R. These last two genes correspond to melanocortin receptors 1 and 4, which together with POMC form part of the hypothalamic-hypophyseal-adrenal axis in the skin, which acts as a coordinator and enforcer of stress responses.8

Kingo et al. (2007) published the results of an expression analysis performed on genes participating in the melanocortin system in skin biopsies obtained from non-pigmented lesions and pigmented (normal) skin from patients with vitiligo. These results were compared to the expression patterns of skin samples from healthy subjects, demonstrating that the expression of proopiomelanocortin and its receptors is reduced in the affected skin compared to the unaffected skin of patients with vitiligo and controls. Recently, Nagui et al. (2015) reported the reduced expression of MC1R and POMC genes in affected skin obtained from a vitiligo lesion after analyzing only MC1R, MC4R and POMC.11

In contrast, Kingo et al.8,9 analyzed the expression profiles of the genes involved in pigmentation in biopsies of affected and unaffected skin of vitiligo patients, compared to the expression levels observed in skin of normal control subjects, without immediate treatment, and our results correspond to the expression profile of a group of genes participating in 5 cell signaling pathways, including pigmentation, in biopsies from the affected and unaffected skin of patients obtained before and after nb-UVB treatment.

When analyzing the total RNA from each of the biopsies obtained from the patients, changes in the expression profiles were observed in most of the selected genes. In pigmentation, the genes that presented differential expression profiles before and after treatment were DCT, MC1R, MC4R, MLANA and TYRP1. Differences were detected after comparing the expression profiles of skin biopsies with pigment (non-affected skin and affected skin that repigmented after treatment) vs. non-pigmented biopsies (biopsies of vitiligo skin and biopsies of affected skin that did not repigment after treatment). In addition, there were no statistically significant differences between the biopsies with pigment, obtained before and after phototherapy application. The same phenomenon was observed among biopsies without pigment, indicating that, once the treatment was applied, the expression profile of the skin underwent changes favoring the manifestation of the final pigmented phenotype. In skin that recovered pigmentation, the expression patterns were similar or slightly higher in genes related to pigment.

The pigmentation gene expression results obtained in the present study reaffirm those of Kingo et al. (2007, 2008) on biopsies before treatment8,9 and are consistent with those of Nagui et al. (2015) in the pigment routes.11However, we excluded the participation of POMC in this process because no statistically significant difference in the expression profile of the different biopsies analyzed was observed.

Two of the eleven analyzed genes involved in apoptosis presented statistically significant differences (p <0.05): cysteine protease 3 (CASP3, which is processed by caspases 8, 9 and 10 and participates in the execution of the signaling cascade of cellular apoptosis, activating caspases 6, 7 and 9) and tumor necrosis factor receptor family member 1A gene (TNFRS1A) (which induces cell death signaling through caspase 8). In both cases, these genes are slightly over expressed in affected vitiligo skin, indicating the induction of an extrinsic apoptoticpathway. Previously, Kumar R et al. (2011) reported an increase in CASP3 levels in melanocyte cultures obtained from samples of unstable vitiligo using cell cultures and immune fluorescence techniques.46

For the TNFRS1A gene, no alterations in the expression levels in the different skin types of vitiligo patients have been reported.

However, two of the four selected genes related to cell survival showed some pattern of differential expression between the skin types (p <0.05). Among these genes, the CDC5L gene, involved in cell cycle control, and the MITF gene, which controls the expression of genes related to melanin synthesis and melanocyte survival, showed higher expression patterns for pigmented tissues (not affected and repigmented) compared to affected vitiligo skin and skin that did not repigment after treatment. Kingo et al. (2008) published a similar observation for the MITF gene using real-time PCR expression analysis. In their study, MITF showed higher expression patterns in skin from healthy controls and the unaffected skin of vitiligo patients compared to skin affected with vitiligo lesions.9A decrease in the expression pattern of the CDC5L gene was observed in skin that repigmented after treatment compared to affected vitiligo and unaffected skin before treatment. However, the participation of this gene in vitiligo has not yet been described.

For the genes involved in responses to oxidative stress, the GPD1 gene, involved in the oxidation-reduction metabolism of the cell (redox),38 was over expressed in tissue that repigmented after treatment. The TXN gene, involved in the inhibition of oxidative stress and activation of caspase 3, is over expressed in vitiligo and non-repigmented tissues, suggesting potential activation mechanisms in response to apoptosis and oxidative stress generated through mechanisms of cell death.

To date, only changes in the levels of TXN protein in the skin of vitiligo patients,39 and their relationship to the control of reactive oxygen species production and UV damage have been reported.47 The involvement of the GPD1 gene in the control of oxidative stress in vitiligo has not yet been explored.

Finally, the involvement of the WNT pathway in melanocyte differentiation and the development of vitiligo has recently been described.30 However, in our study, only statistically significant differences in the expression patterns observed for the CSNK1G3 gene were seen. The product of this gene is a serine/threonine kinase that phosphorylates a number of proteins involved in the WNT pathway, participating in important cellular processes, such as DNA repair, cell division, nucleus-cytoplasmic shuttling of transcription factors, and transport through the membrane. As shown in table 1, an increase in the levels of vitiligo-affected skin expression may reflect, among other processes, the activation of DNA repair mechanisms in damaged melanocyte/keratinocyte cells. However, a decrease in the expression of this gene was observed in affected skin that did not repigment after treatment, which may account for a decrease in the cellular mechanisms underlying melanocyte differentiation and DNA repair in the damaged cells of skin affected with vitiligo.

The nb-UVB phototherapy produced significant changes in the expression patterns of the genes analyzed, primarily reflecting repigmentation and cellular apoptosis control.

Expression analysis revealed significant differences in the expression patterns of the DCT, MLANA and TYRP1 genes in pigmented skin (unaffected and affected then repigmented after treatment) compared to affected skin (vitiligo and non-pigmented skin after treatment). The statistical analysis of the expression patterns obtained using TruSeq Targeted RNA Expression technology from skin samples obtained before and after treatment showed changes in the expression profiles in selected genes in response to nb-UVB phototherapy, and some of these changes were comparable with those previously reported by other research groups, particularly in genes related to skin pigmentation.

In addition, the identification of novel targets genes, including TNFRS1A, GPD1 and CSNK1G3, implicated in apoptosis, oxidative stress control and signal transduction, suggests that these genes play a central role in many aspects of vitiligo development.

We would like to acknowledge MD. Osvaldo Vázquez-Martínez, MD. Alejandra Villarreal-Martínez and MD. Verónica Garza-Rodríguez for their assistance in patient selection and screening, skin biopsy obtaining, application of phototherapy treatment and data analysis. We would also like to thank Dr. Juan José Vila-ta-Corell and Dr. José Luis Alfonso-Sánchez for their unconditional support in the study and in data analysis, and PhD Eduardo de la Rosa-Moreno for his contributions in study design, gene selection, TruSeq RNA sample preparation, sequencing and data analysis. We would also like to thank all those who participated in this study for contributing to the proofreading the manuscript. ❑