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Vitiligo Scientific Study

The Genetic Basis of Vitiligo

Vitiligo is a complex disease in which autoimmune destruction of epidermal melanocytes results inpatches of depigmented white skin. Vitiligo has an estimated prevalence of about 0.2e2% in different populations and approximately 0.4% in the European derived white (EUR) population. The fraction of disease risk attributable to genetic variation, termed heritability, is high, with estimates from family studies in EUR of 0.75e0.83 and from SNP based studies estimated at 0.78. About 70% of genetic risk comes from common genetic variants and about 30% from rare genetic variants. Through candidate gene, genomewide linkage, and genomewide association studies, over 50 vitiligo susceptibility loci have been discovered. These have been combined into a vitiligo polygenic risk score, which has allowed various aspects of vitiligo genetic architecture in the EUR population to be better understood. Vitiligo has thus proved to be a particularly tractable model for investigation
of complex disease genetic architecture. Here, we summarize progress to date including dissection of heritability, discovery of vitiligo susceptibility loci through candidate gene, genomewide linkage, and genomewide association studies, relationships to other autoimmune diseases, polygenic architecture of vitiligo risk, vitiligo triggering, and disease onset, and provide suggestions for future directions.

INTRODUCTION

Vitiligo is a common disease in which autoimmune destruction of epidermal melanocytes results in patches of depigmented white skin. Pigment loss is generally progressive, and until very recently, there were no good treatments. Vitiligo is a complex disease, involving both genetic and environmental components. From the genetic standpoint, vitiligo behaves as a typical polygenic condition, each specific genetic factor making a relatively small individual contribution. Nevertheless, vitiligo polygenicity is relatively low and heritability relatively high compared with those of most other complex traits. As a result, the genetic architecture of vitiligo has been easier to discover and understand than that of most other complex traits (Roberts et al., 2020b). Thus, vitiligo provides a particularly tractable model for investigation of complex disease genetic architecture and provides important lessons for predictive, personalized medicine of complex diseases.

Historical Context

Because of its visually dramatic phenotype, vitiligo was recognized relatively early, probably thousands of years ago (Krauss, 2018), with the first known formal medical description in 1765 (Le Cat, 1765). The fundamental pathological defect in vitiligo was defined about a hundred years later when Kaposi (1879) observed a lack of pigmented cells in the involved lesions. Another key observation came with Addison’s (1855) initial description of Addison’s disease, 2 of his 12 cases additionally having vitiligo as well as pernicious (Addisonian) anemia (Figure 1). DeMowbray (1965) suggested that such co-occurring diseases were autoimmune and, furthermore, involved a shared genetic predisposition. The first specific consideration of vitiligo genetics came in 1950 when Stüttgen (1950) and Teindel (1950) simultaneously described multiplex families having multiple relatives affected with vitiligo. Indeed, Stüttgen’s (1950) suggestion that co-occurrence of vitiligo and autoimmune thyroid disease in his family might reflect simultaneous segregation of both dominant and recessive factors constitutes one of the earliest specific hypotheses of complex inheritance.

In the 1960s, efforts began attempting to identify genes that underlie genetic susceptibility to vitiligo (Roberts and Spritz, 2018Spritz and Andersen, 2017). The earliest studies analyzed polymorphic blood proteins, followed subsequently by candidate gene association studies, targeted genetic linkage analyses, genome-wide linkage analyses, and finally GWAS. The GWAS approach has been by far the most successful, thus far defining 50 confirmed loci that are associated with vitiligo susceptibility in the European-derived white (EUR) population and several others in other populations, especially in Han Chinese (CHN). For many of these loci, the corresponding genes have now been identified and, in many cases, also the specific causal DNA sequence variants. This has led to a deep understanding of vitiligo pathobiological pathways and relationship to environmental triggers and has suggested potential new approaches to vitiligo treatment.

Vitiligo Epidemiology and Heritability

Vitiligo has an estimated prevalence of about 0.2–2% in different populations (Zhang et al., 2016), approximately 0.4% in the EUR population. Although about 91% of cases are sporadic (Roberts et al., 2019), the frequency of vitiligo is considerably elevated in probands’ close relatives, about 7% in first-degree relatives (Alkhateeb et al., 2003). Overall, about 8% of patients report at least one affected relative, with a non-Mendelian pattern of recurrence characteristic of complex, polygenic inheritance (Laberge et al., 2005).

The fraction of disease risk attributable to genetic variation is termed heritability (h2), with the remainder attributable to the environment. Heritability sets a ceiling on the importance of genes in disease causation and provides a key benchmark of progress toward the completeness of disease gene discovery. Classical approaches to estimating heritability analyze disease recurrence rates in multiplex families or twins (Tenesa and Haley, 2013); here, we term such family-based heritability h2FAM. The recent availability of very large genetic reference panels from multiple human populations (McCarthy et al., 2016), coupled with techniques for deep imputation of millions of untyped common and rare variants (Das et al., 2018), has enabled the estimation of overall genomic similarity among unrelated, singleton (simplex) cases versus controls (Yang et al., 2011Yang et al., 2015); here, we term such DNA-based estimates of heritability h2SNP.

For vitiligo, estimates of h2FAM and h2SNP are remarkably congruent. Estimates of vitiligo h2FAM from small studies range from about 0.50 to 0.80 in different populations (Arcos-Burgos et al., 2002Das et al., 1985Hafez et al., 1983Zhang et al., 2004) and from a large-scale study in the EUR population was recently estimated as 0.75–0.83, depending on the specific types of relatives studied (Roberts et al., 2020a). These estimates of vitiligo h2FAM are all much higher than those of many other complex diseases, for which h2FAM estimates are typically in the range of 0.3–0.5 (Polderman et al., 2015).

In parallel, vitiligo h2SNP was recently estimated as 0.78 in the EUR population (Roberts et al., 2020a), virtually identical to the estimates of h2FAM in this population. Thus, for vitiligo, essentially all heritability estimated by classical family-based methods can be captured by array-based genotyping and deep imputation of common and rare genomic variation; there is no missing heritability (Manolio et al., 2009). With the progress of imputation and sequencing methods that account for both common genetic variants (risk allele frequency ≥ 0.01), such as those detected by typical GWAS, and rarer genetic variants (risk allele frequency < 0.01), this is also proving to be true for other complex traits (Yang et al., 2015; Wainschtein et al., 20191).

Furthermore, the vitiligo h2SNP estimate can be partitioned into the fraction attributable to common genetic variants versus rarer genetic variants (Roberts et al., 2020a). As shown in Figure 2, in the EUR population, common genetic variants account for about 70% of vitiligo h2SNP, whereas rarer genetic variants in the aggregate account for about 30%. Specific identification of such rare vitiligo susceptibility alleles will likely require family studies or GWAS with very large sample sizes that may be difficult to attain for vitiligo.

Figure 2Partitioning of vitiligo risk. Heritability studies (Roberts et al., 2020a) enable total vitiligo risk to be partitioned into an environmental component (∼20%) and a genetic component (∼80%). Moreover, total vitiligo genetic risk can be further partitioned in a ∼70% fraction attributable to common genetic variants (MAF ≥ 0.01), such as those detected by GWAS, and a ∼30% fraction that represents rare genetic variants (MAF < 0.01). MAF, minor allele frequency.

Together, these findings show that vitiligo heritability is quite high; about 80% of vitiligo risk is genetically based, with the remaining 20% attributable to environmental factors (Figure 2). Furthermore, of the genetic risk component, about 70% (56% of total risk) comes from common genetic variants and about 30% (24% of total risk) comes from rare variants, with no remaining missing heritability. Similar patterns have recently been observed for other complex human traits (Hartman et al., 20192).

Identification of Vitiligo Susceptibility Genes

Candidate gene studies

The first attempts to identify specific genes that underlie vitiligo susceptibility were candidate gene association studies. Unfortunately, this study design has proven highly prone to false-positive results because of population stratification artifacts, incomplete correction for multiple testing, and publication bias of positive results (Hirschhorn et al., 2002). Accordingly, candidate gene studies are no longer generally accepted as appropriate for de novo identification of susceptibility genes for complex traits. Nevertheless, this approach successfully provided the first indications of involvement in vitiligo susceptibility of two immune-related candidate genes, CTLA4 (Blomhoff et al., 2005Kemp et al., 1999) and PTPN22 (Cantón et al., 2005), both subsequently confirmed by GWAS results.

Genome-wide linkage studies

Genome-wide mapping methods, including both genome-wide linkage studies and GWAS, are not subject to the problems inherent in candidate gene studies. Genome-wide linkage studies search for genomic regions that cosegregate along with the occurrence of disease among affected relatives in multiplex families. Genetic linkage studies are difficult to accomplish, have relatively modest statistical power and low genetic resolution, and are best suited to detecting uncommon causal variants that exert relatively large effects on disease risk. A total of seven putative vitiligo susceptibility loci were detected by genome-wide linkage analysis (Table 1). Of these, five have been associated with a proposed underlying causal gene: FOXD3 (Alkhateeb et al., 2005), NLRP1 (Jin et al., 2007), PDGFRA (Xu et al., 2010), HLA (Yang et al., 2018), and XBP1 (Ren et al., 2009).

Table 1. Vitiligo Susceptibility Loci Detected by Genome-wide Linkage Studies

Chromosome LOD Score Proposed Gene Population References
1p31.3-p32.2 5.59 FOXD3 EUR Alkhateeb et al., 2005Spritz et al., 2004
4q12-q21 4.01 PDGFRA CHN Chen et al., 2005Xu et al., 2010
6p21-p22 3.73 HLA CHN Liang et al., 2007Yang et al., 2018
7q21.11 3.73 Unknown EUR Spritz et al., (2004)
8p12 3.36 Unknown EUR Spritz et al., (2004)
17p13.3 3.07 NLRP1 EUR Jin et al., 2007Nath et al., 2001Spritz et al., 2004
22q12 3.26 XBP1 CHN Liang et al., 2007Ren et al., 2009

Abbreviations: EUR, European-derived white population; CHN, Han Chinese population; LOD, logarithm of the odds.

GWAS

GWASs typically search for differential genetic associations in singleton cases versus unrelated controls, interrogating hundreds of thousands or millions of DNA polymorphisms across the genome (Altshuler et al., 2008). Furthermore, the genotypes of millions of additional polymorphisms can be imputed using various genome-wide reference panels (Yang et al., 2011Yang et al., 2015). Unlike candidate gene association studies, GWAS can adequately control for population stratification artifacts, apply appropriate multiple-testing correction, and further require independent replication; thus, GWAS results have been largely reproducible across multiple studies. Over the past decade, GWASs have become the gold standard for the primary discovery of genes involved in complex traits, providing deep insights into the corresponding underlying biology (Visscher et al., 2017). At least five GWASs have been reported for vitiligo; three in the EUR population (Jin et al., 2016Jin et al., 2012Jin et al., 2010a), one in CHN (Quan et al., 2010Tang et al., 2013), a small GWAS in Japanese (Jin et al., 2015), as well as a small partial GWAS in subjects from the Indian subcontinent (Birlea et al., 2013) based on the Immunochip (Cortes and Brown, 2011). For reasons that are unclear, the greatest yield has come from the GWAS of vitiligo in EUR, in which 50 loci have thus far been identified that contribute to vitiligo risk (Table 2). Several additional loci have been discovered in CHN (Table 2). A number of vitiligo susceptibility loci appear to be shared across populations, although others may not be, suggesting that the general pathobiology of vitiligo is likely similar across different populations and efficacy of vitiligo treatments might thus transcend ethnic populations.

Table 2. Vitiligo Susceptibility Loci Detected by GWAS

Chromosome Proposed Gene Population OR References
1p36.23 RERE EUR 1.21 Jin et al., (2010a)
1p13.2 PTPN22 EUR, IND, AR 1.38 Jin et al., (2010a)
1q24.3 FASLG EUR, CHN 1.32 Jin et al., (2016)
1q31.3-q32.1 PTPRC EUR 0.83 Jin et al., (2016)
2p16.1 PPP4R3B EUR 1.51 Jin et al., (2016)
2q13 BCL2L11 EUR 1.15 Jin et al., (2016)
2q24.2 IFIH1 EUR 0.77 Jin et al., (2012)
2q33.2 CTLA4 EUR 1.18 Jin et al., (2016)
2q37.3 FARP2 – STK25 EUR 0.80 Jin et al., (2016)
3p24.3 UBE2E2 EUR 0.87 Jin et al., (2016)
3p13 FOXP1 EUR 0.80 Jin et al., (2010b)
3q13.33 CD80 EUR 1.31 Jin et al., (2012)
3q28 LPP EUR 1.32 Jin et al., (2010a)
3q29 FBXO45 – NRROS EUR, CHN 0.87 Jin et al., (2016)
4q24 PPP3CA EUR 0.87 Jin et al., (2016)
6p25.3 IRF4 EUR 0.75 Jin et al., (2016)
6p25.2 SERPINB9 EUR 0.79 Jin et al., (2016)
6p22.1 MHC class I
HLA-A1 EUR, JPN, CHN 1.53 Hayashi et al., 2016Jin et al., 2015Jin et al., 2010a
HLA-A/HLA- B/HLA-C CHN 1.90 Quan et al., (2010)
MHC class II
HLA-DRB1- HLA-DQA12 EUR 1.77 Cavalli et al., 2016Jin et al., 2016Jin et al., 2010a
HLA-DQA1 – HLADQB13 EUR 8.1 Jin et al., (2019)
HLA-DQB14 CHN 1.79 Yang et al., (2018)
BTNL2 – HLA-DRA IND 1.67 Birlea et al., (2013)
6q15 BACH2 EUR 1.27 Jin et al., (2012)
6q27 RNASET2 – FGFR1OP – CCR6 EUR, CHN 0.79 Jin et al., 2010bQuan et al., 2010
7p14.3 CPVL EUR 1.84 Jin et al., (2010b)
8q24.21 PVT1 EUR 1.17 Ben et al., (2018)
8q24.22 SLA EUR 1.19 Jin et al., (2012)
9q33.3 NEK6 EUR 1.15 Jin et al., (2016)
10p15.1 IL2RA EUR 0.77 Jin et al., (2010a)
10q21.2 ARID5B EUR 1.18 Jin et al., (2016)
10q22.1 SLC29A3 – CDH23 CHN 0.88 Tang et al., (2013)
10q22.3 ZMIZ1 CHN 1.18 Quan et al., 2010Sun et al., 2014
10q25.3 CASP7 EUR 0.82 Jin et al., (2012)
11p13 CD44 EUR 1.23 Jin et al., (2012)
11q13.1 PPP1R14B – PLCB3 – BAD – GPR137 –KCNK4 – TEX40 – ESRRA – TRMT112 – PRDX5 EUR 0.87 Jin et al., (2016)
11q14.3 TYR EUR 0.67 Jin et al., (2010a)
11q21 Gene desert EUR 1.34 Jin et al., (2010a)
11q23.3 DDX6-CXCR5 CHN 1.22 Tang et al., (2013)
12q13.2 IKZF45 EUR, CHN 1.33 Jin et al., 2010bTang et al., 2013
12q24.12 SH2B3 EUR 0.79 Jin et al., (2012)
13q14.11 TNFSF11 EUR 1.17 Jin et al., (2016)
14q12 GZMB EUR, CHN 1.25 Jin et al., (2010a)
15q12-q13.1 OCA2 – HERC2 EUR 1.37 Jin et al., (2012)
16q24.3 MC1R EUR 0.71 Jin et al., (2012)
17q21.2 KAT2A-HSPB9-RAB5C EUR 1.21 Jin et al., (2016)
18q21.33 TNFRSF11A EUR 1.21 Jin et al., (2016)
19p13.3 TICAM1 EUR 1.21 Jin et al., (2016)
19q13.33 SCAF1-IRF3-BCL2L12 EUR 0.84 Jin et al., (2016)
20q11.22 RALY – ASIP EUR 0.61 Jin et al., (2016)
20q13.13 PTPN1 EUR 1.15 Jin et al., (2016)
21q22.3 UBASH3A EUR 1.35 Jin et al., (2010a)
22q12.3 C1QTNF6 EUR 1.32 Jin et al., (2010a)
22q13.2 ZC3H7B – TEF EUR 0.79 Jin et al., (2012)
Xp21.3-p21.2 IL1RAPL1 EUR 1.77 Jin et al., (2016)
Xp11.23 CCDC22-FOXP3-GAGE EUR, CHN, IND 0.86 Jin et al., (2016)

Abbreviations: AR, Arab; CHN, Han Chinese; EUR, European-derived white; IND, Indian subcontinent; JPN, Japanese population; MHC, major histocompatibility complex.

For associations observed in multiple populations, the first-listed was determined by GWAS and the others by non-GWAS methods.

1

All three populations have major vitiligo association with HLA-A∗02:01.

2

Intergenic SNP rs9271597-rs9271600-rs9271601 haplotype associated with vitiligo susceptibility.

3

Intergenic SNP rs145954018 associated with early-onset vitiligo.

4

Principal association is with HLA-DQB1∗02:02.

5

Tang et al. (2013) interpreted this association as possibly representing PMEL.

As shown in Figure 3, the majority of the loci that have been associated with vitiligo encode genes involved in immunoregulation, apoptosis, and melanocyte biology. For a number of these genes, the causal genetic variants have been discovered, providing important insights into biological mechanisms that underlie vitiligo risk attributable to these genes, Furthermore, together, the corresponding proteins define a functional network that suggests a general pathobiological pathway of melanocyte damage, antigen processing and presentation, immune cell activation, and melanocyte targeting and apoptosis (Spritz and Andersen, 2017).

Figure 3Functional interaction network of proteins encoded by candidate genes associated with vitiligo susceptibility. Unsupervised functional interaction network analysis was performed using STRING, version 11.0 (Szklarczyk et al., 2019) with default settings. Primary nodes were proteins encoded by all the confirmed vitiligo susceptibility loci (Tables 1 and 2). Nodes that shared no edges with any other nodes were then excluded. Edge colors are from STRING: teal, interactions from curated databases; purple, experimentally determined interactions; dark green, gene neighborhood; red, gene fusions; dark blue, gene co-occurrence; light green, text mining; black, coexpression; lavender, protein homology.

Relationship to Other Autoimmune Diseases

Epidemiological studies have shown that the frequencies of several other autoimmune diseases are elevated in patients with vitiligo. In the EUR population, these principally include autoimmune thyroid disease (principally Hashimoto thyroiditis), rheumatoid arthritis, adult-onset type 1 diabetespernicious anemia, Addison’s disease, and systemic lupus erythematosus (Alkhateeb et al., 2003Cunliffe et al., 1968Laberge et al., 2005). Epidemiological studies of autoimmune diseases in vitiligo cases from other populations have shown generally similar associations, although with perhaps some differences (Alissa et al., 2011Ayanlowo et al., 2009Chen et al., 2015Gopal et al., 2007Liu et al., 2005Narita et al., 2011Silva de Castro et al., 2012Zhang et al., 2009) Likewise, these same autoimmune diseases occur at an elevated frequency in vitiligo probands’ first-degree relatives, suggesting that these autoimmune disease associations likely reflect at least partially shared genetic underpinnings (Alkhateeb et al., 2003).

This hypothesis has been completely borne out by genetic studies of vitiligo-associated genes and loci in other autoimmune diseases. Genetic associations of various major histocompatibility complex (MHC) genes and HLA alleles are of course well-established with many different autoimmune diseases. Of the 47 non-MHC loci that have been associated with vitiligo in the EUR population, six appear to involve genes that are specifically relevant to melanocytes and, thus, would not be expected to play roles in autoimmune diseases other than vitiligo. Of the remainder, at least 19 have also been genetically associated with at least one of the other autoimmune diseases that are epidemiologically associated with vitiligo (Figure 4). Many of these shared genetic associations appear to involve the same associated marker alleles and, thus, may potentially represent the same underlying causal variant. Thus, the hypothesis that shared epidemiological associations among various autoimmune diseases to some extent reflect shared underlying genetic predisposition has been fully confirmed.

Figure 4Shared genetic associations among vitiligo-associated autoimmune diseases. In addition to various HLA alleles, at least 19 of the 47 non-MHC loci that have been associated with vitiligo in the EUR population have also been associated with at least one of the autoimmune diseases that are epidemiologically associated with vitiligo (autoimmune thyroid disease, adult-onset type 1 diabetes mellitusrheumatoid arthritis, pernicious anemia, systemic lupus erythematosus, and Addison’s disease), in many cases involving the same associated marker alleles. Red, immune-related; blue, apoptosis-related; white, function unknown. Note that not all diseases have been studied to similar extents, and so for some diseases, relatively few genetic associations are yet known. EUR, European-derived white population; MHC, major histocompatibility complex.

Polygenic Architecture of Vitiligo Risk

As noted above, about 8% of vitiligo cases report at least one affected relative, with a pattern suggestive of non-Mendelian, complex inheritance (Laberge et al., 2005). Alkhateeb et al. (2003) found that the frequency of vitiligo in probands’ first-degree relatives was about 5–7%, depending on the ethnic population studied.

Roberts et al. (2019) combined data for the most significant SNPs from the 48 autosomal vitiligo GWAS loci—all relatively common—to create a vitiligo polygenic risk score, which they then used as a tool to probe various aspects of vitiligo genetic architecture in the EUR population. Comparing the top 1% of the cases with the rest, this risk score yielded an OR of 8.79—far higher than the risk scores for most other complex diseases (Khera et al., 2018)—owing at least in part to higher ORs of the vitiligo-associated variants than those of most complex diseases. Roberts et al. (2019) found that the positive predictive value of that vitiligo risk score was 71%, again better than polygenic risk scores for most other complex diseases (Wald and Old, 2019). Together, these results indicate that vitiligo is polygenic, although less so than many other complex diseases, and, as noted above, common genetic variants account for a high fraction (about 70%) of vitiligo heritability (Roberts et al., 2020a).

Roberts et al. (2019) also showed a major role for polygenicity in families with multiple affected relatives. They found that the vitiligo risk score in cases from such multiplex families was higher than that in singleton cases, with the risk score generally proportional to the number of affected relatives, and that this high polygenic risk was disproportionately transmitted to those affected relatives. Even in a family with a known high-risk rare variant (in FOXD3), the polygenic risk from common vitiligo variants was exceedingly high. Thus, whereas rare, high-penetrance variants undoubtedly play a role in some multiplex vitiligo families, even in such families, much of the genetic risk comes from over-representation of the same common risk variants involved in singleton vitiligo cases. This suggests that the application of a polygenic risk score might prove clinically useful for risk prediction in such families. Furthermore, the same genetic risk variants and corresponding biological pathways are involved in both simplex and multiplex vitiligo cases, suggesting that the same treatments will generally be applicable to both types of cases.

Vitiligo Triggering and Disease Onset

For autoimmune diseases such as vitiligo, case versus control genetic studies identify inherited risk factors that together increase the likelihood of loss of tolerance in response to an environmental trigger. Whereas no vitiligo environmental triggers are known with certainty, the Köbner phenomenon (Köbner, 1877) is particularly frequent in vitiligo (van Geel et al., 2011), suggesting a major role for skin damage in disease triggering.

Spritz et al. (2004) have studied the vitiligo age of onset in the EUR population as an indirect proxy for disease triggering. Jin et al. (2011) initially showed that vitiligo age of onset is genetically associated with the MHC class II region. These investigators (Jin et al., 2019) later showed that the distribution of vitiligo age of onset is bimodal and that early-onset vitiligo is genetically associated with a specific extreme-risk MHC class II haplotype (OR = 8.1) containing an enhancer variant that upregulates the expression of HLA-DQB1 mRNA and HLA-DQ protein on peripheral blood monocytes and dendritic cells. Elevated HLA class II protein expression on such professional antigen-presenting cells might thus enhance the response to triggering antigens, facilitating loss of tolerance by autoreactive T cells.

In addition to a genetic component underlying vitiligo triggering and onset, Jin et al. (2020) also searched for evidence for an environmental component by analyzing the long-term trends in vitiligo age of onset. Studying EUR vitiligo cases from North America and Europe, these investigators observed a dramatic shift over the period 1970–2004 from the mean onset of about 15 years of age in 1970 to over 30 years of age in 2004 (Figure 5). This change was unrelated to the extreme-risk MHC class II haplotype. Moreover, the pattern of change appeared generally similar among EUR vitiligo cases from both North America and Europe. Together, these findings suggest that exposure to one or more important vitiligo environmental triggers became reduced or delayed over this period in these populations. Whereas the cause of this seemingly beneficial change is not known, important clues might be gleaned by comparison with analogous data from other world populations.

Figure 5Mean age at vitiligo onset in 4,406 EUR cases from North America and Europe by calendar year of onset, 1951–2013. Circles denote means, and vertical bars delimit 95% CIs. Black segments show regression lines for time periods of 1951–1969, 1970–2004, and 2005–2013. Reprinted from Jin et al. (2020) with permission. CI confidence interval; EUR, European-derived white population.

Perspectives and Future Directions

The past two decades have seen extraordinary progress in deciphering the genetic basis of vitiligo risk, both in general terms and in the identification of specific genes and genetic variants that underlie risk. This progress has provided a profound understanding of the pathobiology of vitiligo and its relationship to other autoimmune diseases. Vitiligo behaves as a complex polygenic disease. For the average vitiligo case, about 20% of risk comes from the environment, about 56% from various common genetic variants, and about 24% from a large diversity of rare variants. The common genetic variants associated with vitiligo risk, principally involved in immunoregulation, apoptosis, and melanocyte biology, can be combined in a vitiligo polygenic genetic risk score that has impressive positive predictive value compared with those for other complex diseases.

Nevertheless, many important questions remain. It is clear that the fundamental genetic architecture of vitiligo risk is generally polygenic and that vitiligo polygenic risk is generally additive at the macro level. However, it is not yet known whether polygenic vitiligo involves just one basic pathobiological process or there are multiple vitiligo biological endotypes, defined by different genes and pathways, with risk additive within each endotype. This is an important distinction, because the former would suggest that effective vitiligo treatments would be biologically generic, whereas the latter would suggest that different treatments might be needed for different vitiligo biological endotypes.

A related question is whether the general genetic architecture and pathobiology of vitiligo are similar in different world populations. Many vitiligo susceptibility loci and perhaps some corresponding causal genetic variants appear to be shared in patients from the EUR, CHN, and perhaps other populations. That would suggest that vitiligo in these populations involves shared pathobiological pathways. Nevertheless, other vitiligo susceptibility loci appear to be population-specific. That would suggest the possibility that some cases of vitiligo in one or another population might involve some aspects of differing underlying biology.

At least in the EUR population, about 70% of vitiligo genetic risk derives from common genetic variants. The remaining 30% of vitiligo genetic risk derives from a large diversity of rare genetic variants. As of now, only a single rare, highly penetrant vitiligo susceptibility variant, in FOXD3, has been identified, although it is likely that rare and uncommon variants in MC1R and IFIH1 also play roles. To identify additional specific rare vitiligo risk variants will likely require either association studies with much larger sample sizes or family-based studies to track the cosegregation of rare genomic variants along with genetic risk.

As of now, no large-scale genetic studies have yet addressed segmental vitiligo. The prevalence of associated autoimmune disorders does not appear to be elevated in segmental vitiligo (Iacovelli et al., 2005), suggesting that segmental vitiligo might not represent a fundamentally autoimmune process. Nevertheless, at least some susceptibility genes might be shared between segmental and nonsegmental vitiligo. Taïeb et al. (2008) speculated that segmental vitiligo might reflect some form of cutaneous somatic mosaicism. Might evidence of that be observed by single-cell DNA sequencing or other modern omics approaches?

Perhaps most important, all genetic studies of vitiligo to date have compared the genetic basis of case status with that of noncase status, either in large patient populations or within families. Fundamentally, such studies address the underlying biological determinants of disease occurrence; in the case of vitiligo, autoimmune triggering. Whereas that topic is of key importance, these biological determinants may not be the same as those that influence vitiligo clinical course once autoimmune triggering has occurred, which of course might have the greatest relevance to vitiligo treatment.

Finally, whereas there has thus been great progress toward discovering the genetic components of vitiligo susceptibility, as of now, no common environmental triggers for vitiligo have been identified with certainty. Such knowledge would provide an even deeper understanding of vitiligo pathobiology and furthermore, might provide opportunities to ameliorate vitiligo risk. Unfortunately, whereas we have a rigorous global systematic scientific method to identify genetic risk factors for disease, we have yet to develop an analogous global systematic approach to identify specific environmental risk factors.

Conflict of Interest

The authors state no conflict of interest.

Acknowledgement

We thank the thousands of patients with vitiligo, their family members, and normal control individuals around the world who participated in our studies of vitiligo genetics, as well as our many medical and scientific collaborators.

Richard A. Spritz1,2 and Stephanie A. Santorico

Source: 

Translational Research in Vitiligo

Erica L. Katz and  John E. Harris

Vitiligo is a disease of the skin characterized by the appearance of white spots. Significant progress has been made in understanding vitiligo pathogenesis over the past 30 years, but only through perseverance, collaboration, and open-minded discussion. Early hypotheses considered roles for innervation, microvascular anomalies, oxidative stress, defects in melanocyte adhesion, autoimmunity, somatic mosaicism, and genetics. Because theories about pathogenesis drive experimental design, focus, and even therapeutic approach, it is important to consider their impact on our current understanding about vitiligo. Animal models allow researchers to perform mechanistic studies, and the development of improved patient sample collection methods provides a platform for translational studies in vitiligo that can also be applied to understand other autoimmune diseases that are more difficult to study in human samples. Here we discuss the history of vitiligo translational research, recent advances, and their implications for new treatment approaches.

Introduction: Early Research in Vitiligo

Focused research in vitiligo has surged over the past 30 years. This is revealed by the exponential increase in scientific papers published with vitiligo in the title (Figure 1) and significant progress in developing new targeted treatment approaches. However, the first documentation of vitiligo and its treatment exists from 3,500 years ago. These ancient texts describe the appearance of vitiligo, emphasize the social stigma associated with the disease, and list treatment approaches used at the time. Vitiligo patients still experience the social stigmas documented in ancient Indian and Buddhist writings, including the restriction of marriage (1). Most of the treatments noted in these ancient texts, including the application of elephant stool or ingestion of cobra bones, are not currently utilized. However, the basis of one remedy is still used today. According to the Atharvaveda, an ancient Indian medical text written in ~1400 B.C., patients with vitiligo were instructed to chew on black seeds from the Bavachi (Psoralea corylifolia) plant, which was later determined to be a source of psoralen. They then sat in the afternoon sun until they began to sweat profusely, which led to blistering sunburns of their exposed skin. While this treatment is unacceptable by today’s standards, it would have likely been effective, as evidenced by its continued use in ancient times and its later rediscovery in the mid 1900’s as topical or oral psoralen derivatives in combination with UVA light treatment, currently known as (PUVA) (2).

Figure 1

www.frontiersin.orgFIGURE 1 Graph showing the exponential increase of publications available on Pubmed with titles including vitiligo published over time.

Vitiligo has been discussed in modern medical literature for over one hundred years. These early records were primarily case studies characterizing clinical observations and their correlation with other diseases. Vitiligo was described as white patchy depigmentation of the skin beginning as small macules that gradually increased and coalesced into larger patches over time, frequently sparing the hair pigment (3). Authors noted that patients with vitiligo exhibited a higher incidence of other autoimmune diseases, such as alopecia areata, type 1 diabetes, and Addison’s Disease (4). The documented therapeutic approaches for treating vitiligo included arsenic, sulfur, mercury, antimony, and sulphuric acid, which are thankfully not used any longer due to limited efficacy and the potential for severe side effects (5). Sulfuric acid was used to burn the skin at borders of the lesions to make them less distinct, and therefore less obvious to the observer. The tolerance of these toxic approaches highlights the motivation of patients to treat their disease.

It was not until the 1940s when more in-depth research studies were performed. Haxthausen in 1947 reported that grafting vitiligo lesional skin onto non-lesional sites led to varying degrees of repigmentation of the lesional skin, while transplanting nonlesional skin onto a lesional area led to the graft depigmentation. He hypothesized that the local environment, which might be determined by the nervous system, influenced the pigmentation state of the graft (6). Comel, in 1948, reported similar results with pedicled and tubed flaps. The lesional flaps repigmented, while the nonlesional flaps attached to lesional skin depigmented (7). Conversely, Spencer and Tolmach in 1951 reported that graft transplantation between lesional and nonlesional skin did not lead to the repigmentation of lesional grafts or depigmentation of nonlesional grafts, but reported extension of depigmentation around the lesional skin graft (8). A possible reason for the conflicting results is the different methodologies used for the skin grafts. Haxthausen used thin split thickness grafts, referred to as a Thiersch graft, while Spencer and Tolmach used full thickness skin grafts. Although these were small case studies that showed mixed results, they suggested that the environment in which the graft is transplanted is an important factor. It wasn’t until 1964 when Behl applied skin grafts as a large scale treatment option for vitiligo (9), utilizing the Thiersch grafting technique similar to the Haxthausen study and limiting the variability in repigmentation and relapse by restricting the procedure to only those with stable disease.

Pathogenesis of Vitiligo

There are a number of hypotheses offered to address the pathogenesis of vitiligo. These hypotheses consider roles for innervation, microvascular anomalies, melanocyte degeneration from oxidative stress, defects in melanocyte adhesion, autoimmunity, somatic mosaicism, and genetic influences (11015) (Figures 2A–G). Some have strong evidence to support them, while others are relatively unsupported but continue to be discussed. We will outline these hypotheses and the evidence offered to support them below.

Figure 2

www.frontiersin.orgFIGURE 2 Overview of pathogenesis for vitiligo. (A) Neuronal involvement – neurons within the skin release neuropeptides like catecholamines, which act on melanocytes and lead to depigmentation. (B) Microvascular theory – vitiligo lesions have increased blood flow during segmental vitiligo, which allows for increased infiltration of lymphocytes that results in autoimmune attack of melanocytes. (C) Somatic mosaicism – depigmentation develops because a somatic mutation in melanocytes leads to genetically distinct populations that are susceptible to autoimmune attack. (D) Melanocyte adhesion – friction or oxidative stress in melanocytes or keratinocytes leads to melanocyte loss because of reduced adhesion to the skin. (E) Degenerative theory – depigmentation occurs because of intrinsic melanocyte defects, such as increased susceptibility to environmental stressors and dysregulation of reactive oxidative species (ROS). (F) Autoimmunity theory – autoreactive immune cells attack and kill melanocytes, ultimately leading to depigmentation. (G) Genetics – underlies all pathways leading to vitiligo. Genetic studies most clearly implicate autoimmunity, but also melanocyte contributions. Neuronal and microvascular theories are least supported, represented by (A, B) being grayed. Figure created in BioRender.com.

Neural Theory

The hypothesis that innervation influences depigmentation, often labeled the “neural hypothesis”, is primarily based on a few largely unsupported observations: 1) Segmental vitiligo is unilateral, and therefore mistakenly labeled as dermatomal; however, disease is rarely limited to a single dermatome, and often crosses many dermatomes (111618). 2) Lerner hypothesized that vitiligo is caused by the increase of neuropeptides released by neurons and that this decreases melanocyte melanin production (19). Catecholamines have also been reported to be elevated in the urine of vitiligo patients, leading some to assume this came from dysfunctional neurons (2022). However, others dispute these results and note catecholamines are also produced by melanocytes (2324). Thus, these studies do not implicate nerves in disease pathogenesis. 3) There are case reports of vitiligo preferentially affecting limbs that had been traumatically denervated, but the opposite is also true, that denervated limbs have been reported to repigment or be spared from vitiligo altogether (192526). In most cases, denervation does not affect vitiligo. Thus, these discordant case reports are more likely chance observations than actual indications of disease pathogenesis. 4) Sweating and vasoconstriction have been reported to be altered in lesions (2728). However, many reports are contradictory. Some reports claim an increase in cholinergic influence in all vitiligo lesions, while others report this observation occurs in segmental lesions but not bilateral lesions. Data on the level of sympathetic tone is also conflicting, as increased sympathetic tone has been observed by some groups while others observed decreased sympathetic tone (2734). Since these studies are largely inconclusive, it is premature to suggest they provide any insight into vitiligo pathogenesis. 5) Stress or trauma appears to exacerbate vitiligo (19). This is often thought to be due to neurohormonal mechanisms, however there is currently no evidence to support neuronal involvement. Many autoimmune conditions are exacerbated by stress, an observation that has not implicated neurons in their pathogenesis. 6) Animal studies reveal neural control of pigmentation, particularly in fish (3335). However, there is no evidence for this in mammals, especially humans (36). Thus, we suggest future discussions defer mention of the “neural hypothesis”, at least until there is solid evidence available to support it.

Microvascular Theory

The microvascular theory suggests there is an increased blood flow into lesional skin during segmental vitiligo. The use of iontophoresis and laser Doppler flowmetry demonstrated up to triple the blood flow in lesional skin of segmental vitiligo patients compared to healthy, but not within nonsegmental lesional skin (37). The unilateral presentation was proposed to be from the activation and expansion of the autoreactive T cells in the regional lymph nodes that then exit into the blood circulation. An increased blood flow may then escalate the migration of lymphocytes, specifically melanocyte-specific cytotoxic T cells because of their expression of cutaneous homing receptors, to the lesion for the destruction of melanocytes (133840). However, a contradictory observation is that when vitiligo develops in melanoma patients as the result of a vaccine injected for the expansion of melanocyte-specific T cells, disease is typically reported to develop in a nonsegmental pattern. If the microvascular theory were true, then it would be expected to develop in a segmental pattern originating in the region of the injection site (12). Furthermore, the microvascular theory is often grouped closely with the neural theory because it has been proposed that damaged sympathetic nerves leads to the increased blood flow due to the vasodilation of the skin blood vessels (41). The lack of real evidence for this theory supports discarding it until further studies provide support.

Degenerative Theory

The degenerative theory focused on the idea that there is an intrinsic defect within melanocytes that leads to their loss within the skin, thereby causing depigmentation. This theory arose from observations that melanocytes from vitiligo patients are difficult to culture and proliferate much slower than melanocytes from healthy individuals (42). Additionally, dysregulation of reactive oxidative species (ROS) and increased susceptibility to chemicals that induce ROS have been reported in melanocytes from patients with vitiligo (4344). Catalase is an enzyme that converts hydrogen peroxide, a source of ROS, into water, and expression of this enzyme is reportedly reduced in vitiligo lesional skin (45). Attempts to mitigate oxidative stress in melanocytes using a topical psuedocatalase cream does not seem to have an impact on disease, but use in combinational treatments with calcium and UVB light treatments have been reported to result in effective repigmentation (4647). However, Gilhar et al. demonstrated that melanocytes are functional within lesions because human vitiligo skin grafted onto nude mice fully repigmented and the number of melanocytes within the graft increased (48). Therefore, although there is clear evidence indicating melanocytes are abnormal in lesional skin, the idea that vitiligo exclusively develops from intrinsic melanocyte degeneration is unlikely because of the disputing studies supporting the fact that melanocyte defects are not sufficient to cause disease.

Melanocyte Adhesion Theory

The melanocyte adhesion theory suggests that melanocytes lose or have decreased adhesion to the skin and are therefore easily eliminated or otherwise lost during oxidative stress or mechanical pressure, such as friction from clothing (49). Some studies report a decreased expression of the adhesion molecule E-cadherin on melanocytes, while another reported no significant differences in expression of adhesion molecules between healthy, nonlesional, and lesional skin but elevated expression of an “anti-adhesion molecule” tenascin in vitiligo lesional and nonlesional skin, which might lead to the loss of melanocytes within the skin (5051). More in vivo data is needed to determine the role of melanocyte adhesion defects during melanocyte loss, such as whether adhesion molecules knocked out in melanocytes in mouse models lead to increased disease severity. Keratinocytes may also play a role in the loss of melanocytes. Keratinocytes and melanocytes comprise a “epidermal-melanin unit” and maintain homeostasis in the epidermis through paracrine signaling of growth and survival factors (52). Studies report that keratinocytes within vitiligo perilesional and lesional skin have dysregulation of oxidative stress (52). For example, keratinocytes have mitochondrial abnormalities evident from increased ROS production, lower production of antioxidants, and expression of apoptotic proteins (5354). Keratinocytes clearly have a role in pathogenesis, as their production of CXCL9 and CXCL10 recruits autoreactive CD8+ T cells to the skin (55). Therefore, it is conceivable that intrinsic defects within keratinocytes may play contribute to abnormalities in melanocyte adhesion during vitiligo pathogenesis. The current available data is intriguing and may support a role in vitiligo pathogenesis. The loss of melanocyte adhesion to keratinocytes might be a cause or effect of autoimmune inflammation. For example, it may trigger autoimmunity by a large release of autoantigens, or intrinsic defects in handling stress or autoimmune attack itself might lead to reduced adhesion (56).

Autoimmunity Theory

The autoimmunity theory offered that self-reactive immune cells attack melanocytes, which then leads to their death and loss from the skin, resulting in depigmentation. This was one of the earliest theories, since initial case studies noted the coincident onset of vitiligo with other autoimmune diseases, thus implicating autoimmunity by association. In the late 1970s, a study revealed the presence of autoantibodies against melanocytes only in vitiligo patients and not in healthy controls using immunofluorescence complement fixation tests (57). This supported the notion that autoimmunity played a functional role in disease because it indicated activation of adaptive immunity specifically against melanocytes. Later studies revealed that autoantibodies could lead to the destruction of melanocytes in vivo by grafting human skin onto nude mice and subsequently injecting purified IgG antibodies from vitiligo patients (58). This resulted in a decrease of melanocytes in the grafts on mice injected with vitiligo patient IgG compared to healthy patient control IgG (58). It was later determined that autoantibodies are likely not the main drivers of disease because autoantibody levels do not correlate with disease severity in vitiligo patients and autoantibodies are equally distributed throughout the body, rather than concentrated in lesions (59).

While autoantibodies indicated an autoimmune response against melanocytes in vitiligo, it became apparent that melanocyte damage results from another mechanism. Early immunohistochemical (IHC) studies noted the decrease of melanocytes within lesional skin and the presence of immune cells in the upper dermis and epidermis (3360). It was then reported that CD8+ T cells infiltrate the skin and are positioned within close proximity to dying melanocytes (3961). Van den Boorn et al. determined that the immune system, and specifically CD8+ T cells, were both necessary and sufficient for melanocyte destruction in the skin of vitiligo patients by incubating T cells isolated from perilesional skin with nonlesional skin explants from the same donor, which resulted in melanocyte death (62). Furthermore, autoimmunity is supported by genome-wide association (GWA) studies that revealed genes associated with vitiligo risk are predominately immune-related, including genes associated with both adaptive and innate immune responses (63).

Melanocytes during vitiligo appear to be abnormal at baseline, producing heat shock proteins to help manage cellular stress, which activate the unfolded protein stress response. These abnormalities may lead to the release of damage-associated molecular patterns (DAMPs), which signal through pattern recognition receptors (PRRs) like TLR2, TLR4, and NLRP inflammasomes (64). Vitiligo-inducing chemicals like phenols initiate the unfolded protein response (UPR) in melanocytes, which results in the production of IL-6 and IL-8 (65), two cytokines that promote immune cell recruitment. Additional studies report infiltration of vitiligo lesional skin by innate immune cells, including inflammatory dendritic cells, macrophages, and natural killer (NK) cells (396667). Yu et al. reported an increase of NK cells in vitiligo nonlesional skin, suggesting a role in the onset of disease (67). These studies suggest that melanocytes may initiate the innate immune response through the recruitment of innate immune cells from inflammatory signals and the release of DAMPs that are able to signal through pathogen recognition receptors (PRRs) on these cells. Additional studies will be important to further support these observations and enhance our understanding how the innate immune system bridges melanocyte stress and adaptive autoimmunity in vitiligo.

Genetics

Genetics clearly influences the risk of developing vitiligo, although should not be described as a theory in itself. Indeed, genetic variation influences all pathways in the body, including immunology, melanocyte stress and adhesion, etc. Several epidemiological and familial studies addressing the incidence of vitiligo report that vitiligo affects 1%–2% of the overall population and that there is a higher risk for people who have first-degree relatives (FDR) with disease (6871). Many of these studies were limited due to the low number of participants, although a recent study by Kim et al. incorporated a large number of individuals in a Korean population, providing significant power in their analysis. Their study calculated incidence risk ratio (IRR) for FDRs and found that siblings of vitiligo patients have a higher incidence of disease, with a larger increase if the sibling is a twin (697172). Unlike previous studies, Kim et al. also reported a higher IRR for offspring that have a mother with vitiligo compared to a father (71). Although this increased risk could be from shared environmental risks, another possibility is the inheritance of mitochondrial DNA that is only obtainable maternally. Overall, homogeneity of the study population provided strong, powered data about vitiligo heritability, but more studies will be needed to determine whether these observations can be applied to other ethnicities and mixed populations.

Recent GWA studies revealed small nucleotide polymorphisms (SNPs) in many immune-related and melanocyte specific genes (63). The identification of melanocyte-specific genes (OCA2-HERC2, PMEL) through GWAS support a role for melanocyte involvement, while genes involving stress and apoptosis (SOD2, GSTP1, RERE, XPB1) support a role for melanocyte stress as well (687374). The autoimmunity theory is validated by GWA study hits, as well with the melanocyte-specific genes are often autoantigens (PMEL) and immune-related genes (FOXP3, GMZB, XPB1). While these SNPs have provided significant insight, functional genomic studies are needed to understand the full relationship SNPs have on disease pathogenesis. However, it is difficult to identify gene target of SNPs because most occur in noncoding regions that likely regulate gene transcription, which may be present at a significant distance from their target genes. Recent three-dimensional (3D) genome analysis studies have reported that SNPs can influence genes that are linearly distant because of higher-order chromatin structures allow interactions of distant genome regions (7577).

While genetics clearly plays a role in vitiligo, environmental exposures, and stochastic events factor into pathogenesis as well. This is indicated by the fact that coincident rates for identical twins to develop vitiligo is only 23%–26%, rather than the expected 100% if pathogenesis was only influenced by genetics (6971). Chemicals in permanent hair dyes and cosmetics, as well as chemicals like 4-TBP and monobenyzl ether of hydroquinone, induce and exacerbate vitiligo (7880). This is likely through the induction of cellular stress pathways within melanocytes when they attempt to convert the chemical into melanin, as the chemicals are very similar in structure to the amino acid tyrosine, the building block of melanin synthesis. Additionally, stochastic events within the immune system may result in the develop of vitiligo because T cell receptors and antibodies are generated through random recombination of existing genes. Thus, a single panel of genetic factors as found in identical twins could result in very different immune responses through stochastic events. Therefore, a combination of genetic predisposition, environmental exposures, and stochastic events contribute to the risk of developing vitiligo.

Segmental Vitiligo

While vitiligo is typically bilateral and symmetric, segmental vitiligo is a subtype of the disease that presents as unilateral depigmentation that does not cross the midline. Historically, the segmental subtype has been misnamed “dermatomal vitiligo” because its unilateral presentation was reminiscent of shingles, which affects the skin in a distribution overlying a single sensory nerve, which also localizes unilaterally. However, segmental vitiligo rarely, if ever, follows a single dermatome, but frequently involves multiple dermatomes and even runs perpendicular to dermatomes, indicating that this disease is not related to single nerves (12). Because of its unilateral nature and dermatomal misnomer, many believed that segmental vitiligo may have a separate and distinct pathogenesis, hypothesizing that nerves might play a role in this subtype of disease. However, more recent studies have shed light on mechanisms that lead to segmental vitiligo, including strong evidence that it is also driven by autoimmunity (4062).

In fact, there may be an important role for somatic mosaicism in the pathogenesis of segmental vitiligo. Somatic mosaicism occurs when there are two genetically distinct populations of cells arising from a mutation that appears within the zygote during development. A post-zygotic mutation occurring in an embryonic melanocyte can be passed on to daughter cells that then migrate ventrally from the neural crest (81). The somatic mosaicism theory suggests that a somatic mutation creates susceptible melanocytes by inducing melanocyte stress or increasing the expression of melanocyte specific markers (12), which then become the targets for autoimmune attack that is limited to the mutant population of cells (82). The ventral migration of these melanocytes during development without crossing the midline may lead to the unilateral distribution of disease, characteristic of segmental vitiligo (11). Future studies will be important to test this hypothesis directly.

Hypotheses Influence Research Questions and Data Interpretation

Hypotheses that address the pathogenesis of disease influence experimental design, as well as data interpretation, which can be either beneficial or detrimental to progress in understanding disease, as this natural bias can focus investigation leading to accelerated discovery, or negatively influence data interpretation within a narrow framework. This has been done in some respect with the “neural hypothesis”. As discussed above, the characteristic unilateral nature of segmental vitiligo for years was misinterpreted as supportive of neural involvement, despite the fact that lesions did not follow specific dermatome patterns at all (11). In addition, the presence of elevated catecholamines in the blood and urine of vitiligo patients were interpreted to indicate support for the neural hypothesis, suggesting that these catecholamines emanated from dysfunctional nerves (21). An alternative interpretation proposed that the increased catecholamines were a product of melanocyte destruction. This interpretation led to studies that observed that melanocytes also produce catecholamines, thus providing new insight about melanocyte biology during disease and further refutes the neural hypothesis (2324). Discussions and new interpretations when exploring both proposed hypotheses about the presence of catecholamines and nerve mapping helped drive focus to more promising pathogenesis ideas.

Another example highlighting different data interpretations driving experimental approaches is the observation that immune cells infiltrate vitiligo lesions. Immunohistochemical studies revealed infiltrating immune cells within the upper dermis and epidermis of vitiligo lesions (3360). It was then identified these immune infiltrates were predominantly CD8+ T cells and macrophages, which were positioned within close proximity to dying melanocytes (3961). Some interpreted these immune cells as simply “cleaning up” after melanocyte degeneration, which led to exploration of the role of macrophage clearance of debris in promoting repigmentation (83). Alternatively, researchers hypothesized immune infiltration into the epidermis supported a direct autoimmune attack against melanocytes. This led to additional characterization of the infiltrating T cells, which reported a higher frequency of melanocyte specific CD8+ T cells in the circulation of vitiligo patients expressing the skin homing receptor cutaneous leukocyte-associated antigen (CLA) (388485). Further studies on T cells isolated directly from lesional skin revealed that cytotoxic CD8+ T cells selectively kill melanocytes, further validating the autoimmune theory (62).

In addition, increased production of heat shock proteins (HSPs) was observed in melanocytes from patients with vitiligo. One interpretation of this data in the context of the degenerative theory is that the elevated HSPs are simply a result of increased melanocyte stress from intrinsic defects in regulating ROS production. This led to the exploration of melanocyte ROS and HSP production during normal cellular processes and under environmental stressors (658688). Another interpretation is that the increased production of HSPs by melanocytes acts as danger signals that induce inflammation, leading to autoimmunity. With this in mind, studies indicate that HSP70i is elevated in human melanocytes during chemical-induced vitiligo, has the capacity to activate skin dendritic cells, and is required for the induction of vitiligo in mouse models (6689).

In fact, both possibilities may be true. Melanocyte intrinsic defects regulating ROS production may play in a role in activating autoimmunity. The interplay of endoplasmic reticulum (ER) stress and ROS production have been implicated in the development of multiple autoimmune diseases, including vitiligo (9092). Melanocytes within vitiligo skin show signs of oxidative and ER stress, including elevated ROS, a dilated ER, as well as gene expression consistent with UPR activation, including X-box protein 1 (XBP1), a transcription factor that is needed to produce cytokines involved in innate immune cell recruitment (6587). Vitiligo-inducing phenols induce expression of XBP1 in melanocytes (93), providing a feasible connection between melanocyte oxidative and ER stress to the immune response. Furthermore, these stress pathways within the melanocytes lead to the production of HSPs to help mitigate this stress, which are then secreted from the melanocyte or released during cell death to signal through TLRs and other PRRs to activate the immune response. Exploring multiple hypotheses, such as melanocyte stress, innate immune activation, and adaptive autoimmunity through complementary approaches will give a better understanding of the complex cell interactions and mechanisms at play during vitiligo pathogenesis.

Addressing Existing Hypotheses in Future Research

To further explore existing hypotheses and to better understand how they interface to cause vitiligo, genetic and functional analyses comparing lesional, nonlesional, and healthy skin will be needed. Tracing mutations within melanocyte lineages will interrogate somatic mosaicism development and progression. Performing single cell RNA sequencing and functional genomics techniques like Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) can guide functional studies addressing the mutations found in lineage tracing to determine their direct role on melanocyte homeostasis and function.

The results of genetic studies have launched many experiments. Since many GWAS hits are in both melanocyte and immune related genes, the identified genomic risk alleles have guided studies that address both the degenerative and autoimmunity theories. Studies to dissect intrinsic pathways within melanocytes have provided insight about stress responses, ROS production, metabolism, and adhesion. These insights have led to new questions that have yet to be answered, such as understanding the interface between melanocytes and inflammation. For example, future studies may dissect how melanocyte stress and inflammation influence each other during disease initiation and progression. Stressed melanocytes may induce local inflammation that then initiates and perpetuates autoimmunity in vitiligo. Inflammation could be also a stressor to melanocytes, and since melanocytes are defective in vitiligo this stress may weaken the melanocytes further, exacerbating autoimmune attack.

Additionally, the interplay of inflammation and melanocyte adhesion should be pursued. Skin inflammation could be reducing melanocyte adhesion or melanocytes that lose adhesion could be triggering inflammation and breaking tolerance. One reason for these unanswered questions is that cell interactions are not as easily studied in cell culture because multiple cell types interact within the tissue within a 3-dimensional (3D) environment. 3D skin models and animal models could be useful tools for exploring these questions. These models will also be helpful in working to define the complex, multicellular interactions that characterize autoimmunity within the skin. Unanswered questions include the mechanism of autoimmune melanocyte killing, tolerance mechanisms for suppression or prevention of disease, and whether other immune cells besides autoreactive CD8+ T cells contribute to melanocyte death.

These questions will be elucidated by creatively utilizing developing technologies and tools. Interest in understanding these interactions has supported the advancement and utilization of translational research tools such as flow cytometry, cell sorting, and live in vivo imaging. As these technologies develop, opportunities to explore these cellular interactions will expand.

Translational Tools for Vitiligo Research

Rubio et al. defined translational research as the merging of basic, clinical, and population research, which continuously build on each other (94). The multi-directionality of translational research is advantageous because it aligns mechanistic studies in a model system with clinical observations and studies on human tissue. Thus, translational research leverages the best of both basic and clinical approaches, understanding the mechanism of disease while maintaining physiologic relevance (Figure 3).

Figure 3

www.frontiersin.orgFIGURE 3 Translational tools to study vitiligo. Vitiligo is uniquely positioned for translational studies because of ready access to the skin and multiple collection techniques for human skin samples such as shave, punch, and suction blister biopsies. These biopsies can then be used for the identification and characterization of cell populations on the single cell level with technologies like flow cytometry and single cell RNA sequencing. Animal models are available for studying initiating factors and adaptive immune responses, as well as cell culture techniques like 3D skin models that have been useful for elucidating mechanisms during vitiligo pathogenesis. ( Figure created in BioRender.com.)

Vitiligo is uniquely suited to translational research because of its high prevalence in the population, opportunity for visual evaluation of disease progression, and ready access to the target tissue. Procedures for obtaining patient skin biopsies are less invasive than in other organs, such as the intestines where endoscopic procedures are required or pancreas and brain where samples can only be safely obtained postmortem. Tissue samples are critically important for understanding human disease pathogenesis, because other sources of patient tissue such as the blood is at best a diluted sample of disease-causing factors in the tissues, and at worst may be specifically depleted of these factors.

Traditional methods used to sample the skin include punch or shave biopsies, which yield small pieces of solid tissue for analysis. These biopsies can be analyzed using histology, immunohistochemistry, and immunofluorescence to determine protein expression, which enables positional information that indicates spacial relationships among cells. Tissue can be homogenized for RNA or protein quantification; however, this eliminates information on individual cells or their position within tissues. Finally, solid tissue can be enzymatically digested to yield single cells for single cell analyses such as flow cytometry, RNA sequencing, and others. Disadvantages to these approaches include pain, need for anesthesia, scarring of the sampled site, and requirement for sutures in some cases. In addition, immune cells infiltrating vitiligo lesions can be difficult to isolate, since they are frequently present in a perilesional location, somewhere beyond the lesional border. This is because these cells migrate through the skin outward from the lesion center while melanocyte loss requires epidermal turnover to observe clinically, so that the true lesion edge is located beyond the visible border. Thus, it can be very difficult to determine where the biopsy should be taken in order to “catch” migratory immune cells.

The use of suction blistering on human volunteers was first described by Kiistala and Mustakallio in 1964 for the purpose of separating the epidermis and dermis (95). Originally, suction blister roofs had been used for obtaining cells for culture, quantifying bacteria on the epidermis, and studying epidermal permeability and biochemical properties (9698). The time blisters took to form on various skin disease states compared to healthy could also be used to categorize skin diseases and their severity, especially blistering diseases like pemphigus vulgaris (99). Blister fluid was utilized for to analyze immune cells in the skin, such as cell recruitment during allergic inflammation because an influx of immune cells into the blister occurred over multiple days after induction of the blister (100). Suction blistering later was used for epidermal grafting, using cells from both the blister roofs and fluid, to treat stable vitiligo lesions (101). More recently, this technique has been used in other contexts to identify cell populations, protein levels of soluble receptors, and secreted growth factors and cytokines (102104).

The suction blistering approach is less invasive, not painful, does not typically scar, and does not require anesthesia or sutures. It is accomplished through application of negative pressure by a suction chamber, often with the addition of gentle heating (99). While a single punch biopsy collects 4-6 mm of skin, suction blistering can yield many more total cells because the interstitial skin fluid that forms the blister (up to 10mm) is drawn from an even larger area and each subject can contribute multiple blisters, we frequently acquire up to 10 per subject. We optimized this technique to sample the involved and uninvolved skin of subjects with vitiligo as well as healthy controls, and found that the extracted blister fluid contains multiple cell types that can be characterized by flow cytometry, as well as proteins that can be measured with ELISA (104). Thus, it is an efficient technique to isolate migratory immune cells from vitiligo lesions as well as soluble proteins from interstitial fluid. However, blisters do not preserve the architecture of the skin, and thus positional information is lost. In addition, it may preferentially sample the epidermis and upper dermis, potentially missing cells (particularly structural cells like fibroblasts and endothelial cells) and fluid from the deeper dermis (105106). Since vitiligo consists of melanocyte destruction at the basal epidermis, the blistering approach is well-suited for translational studies in vitiligo.

Once single cells have been obtained from skin through suction blistering or enzymatic digestion of solid tissue biopsies, several techniques can be used for analysis of the cells (Table 1). Flow cytometry has enabled the identification and characterization of cell phenotypes on the protein level in vitiligo patients. It does not only distinguish the number of cells that are expressing a marker of interest but can also measure the amount of protein expressed by each cell as mean fluorescence intensity (MFI). In addition, since vitiligo has known autoantigens, the use of tetramers and pentamers have been valuable tools for the detection and characterization of circulating autoreactive cells in peripheral blood and skin. Flow cytometry has limitations though, as many transcription factors and intracellular proteins are difficult to detect due to the lack of available antibodies or sufficient permeabilization methods, and some receptors are quickly internalized so protein expression by flow cytometry may not always be an accurate assessment.

Table 1

www.frontiersin.orgTABLE 1 Table comparing the advantages and disadvantages of translational tools available to study vitiligo.

Single cell RNA sequencing can provide valuable insights at the single cell level (107). This approach enables researchers to identify unknown immune cell interactions through signaling networks with ligand/receptor pairing, unlike flow cytometry, which detects a small set of previously selected target proteins. It also can use gene expression within individual cells to discover new cell subtypes, which is not possible through bulk tissue gene expression analysis. However, not all changes on the RNA transcript level are reflected at the protein level. Further regulation can occur post-transcriptionally and post-translationally, which can affect whether a protein is available or functional. Therefore, validation studies must be done to determine whether RNA expression changes are reflected at the protein level.

Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) is a technique to assess genome-wide chromatin accessibility by probing open chromatin with hyperactive transposases that are pre-loaded with sequencing adaptors (108). This technique can be performed in bulk sequencing and on single cells (109). If done at the single cell level, cell specific transcription factors, binding sites and changes in cell-to-cell DNA accessibility variability within different disease states can be explored (110). These epigenetic approaches could be applied to understanding vitiligo pathogenesis, as already described for Systemic Lupus Erythematosus (SLE) (111). However, open chromatin does not always lead to higher expression of genes or their expression as proteins, and thus further studies are required to validate findings at this level, similar to RNA sequencing.

Interstitial fluid, which cannot be obtained using conventional biopsy approaches, can be used to analyze soluble factors that might contribute to disease pathogenesis. For example, we used ELISA to determine that CXCL9 was a sensitive and specific biomarker of disease activity in vitiligo lesions (104). Metabolomics has been performed on interstitial skin fluid, which could be used to understand changes in metabolic processes within nonlesional and lesional skin (112). While soluble factors in fluid cannot be measured at the single cell level, when combined with single cell techniques like scRNA-seq, pathways can be inferred, and new hypotheses developed. These tools will provide new insight into vitiligo pathogenesis and potentially fast track the discovery of new pathways and mechanisms involved in vitiligo, in hopes of developing better and durable therapeutic options. All these techniques would provide a wealth of new and exciting insights into vitiligo pathogenesis. While current techniques using suction blister fluid lack spatial information, emerging techniques seek to examine this, including spatial transcriptomics like Slide-seq and RNA seqFISH+, as well as spatial mass spectrometry imaging (113114).

All biopsy techniques allow for the isolation of cells for culture. While healthy melanocytes can be cultured, melanocytes from vitiligo lesions were difficult to establish in vitro because they had a long lag time for growth and could not be passaged (42115). In the early 1990’s, cell culture techniques improved to enable the culture of both healthy and vitiligo melanocytes by the addition of growth factors, such as fibroblast growth factor (bFGF), phorbol 12-myristate 13-acetate (PMA), and bovine pituitary extract (BPE) (115116). This has supported investigation of melanocyte biology and autoimmune killing in co-culture experiments. There are limitations to what can studied in 2D cell culture however, and so the future development of re-creating the skin environment in vitro with 3D skin cultures may help further dissect mechanisms of autoimmunity from more physiologically relevant interactions (117119). This has been used by Van den Boorn et al., who cultured T cells isolated from punch biopsies with additional biopsies from normal skin from the same patient (62). Boukhedouni et al. also used 3D skin model to study the effect of TNFα on melanocyte adhesions (56).

Translational research covers a vast range of experimental designs. While many only consider experiments containing human samples and clinical studies to be translational, others consider animal models that parallel human disease to be translational work as well. It is easy to recognize the direct impact for patients when working with patient samples, but these studies tend to lack mechanistic insights in this context, which basic research is better positioned to address. Therefore, the development and use of animal models has helped to add mechanistic understanding to clinical observations. Animal models of vitiligo have been extensively reviewed by Essien and Harris (120). Models of vitiligo include both spontaneous and induced systems, and spontaneous models include Smyth line chickens, Sinclair swine, Gray-allele horses, and a variety of mouse models (121125). These are useful for studying disease initiation but can be costly to maintain and may be more difficult to study because of limited reagents available that are compatible with these species. Inducible models are helpful to study disease progression after onset and include the mouse model developed by Harris et al. that parallels human disease by developing patches of skin depigmentation without involving hair, and thus has the potential to repigment with treatment. Like humans, this model is mediated by CD8+ T cells and exhibits IFN-γ driven disease with a similar Type 1 cytokine signature (126128). Animal models are most useful for focused studies that probe specific questions for which the model is optimized to answer. For example, questions about disease initiation, including genetic influences, should be studied in spontaneous models, models designed for this purpose, or directly in humans, since many induced models bypass this step of pathogenesis.

Lessons Learned from Translational Research

The long history of studying vitiligo using translational methods has enabled us to develop a more complete picture about disease pathogenesis, which has been more difficult to achieve in diseases that target less-accessible tissues. Translational studies have been great assets for understanding the mechanisms of disease, which can then promote the development of promising new treatments (Figure 4). For instance, patient biopsies revealed that there is an influx of melanocyte specific CD8+ T cells into lesional skin, which produce IFNγ and IFNγ-induced genes, and express NKG2D (3962127129131). One study reported type I IFN expression within lesions using CXCL9 and MxA as surrogate markers induced by IFNs, however these can be induced by IFNγ as well. Mouse studies do not support a role for type I IFNs in vitiligo, as IFNα receptor (IFNaR) knockout mice still develop vitiligo to the same extent (132). Thus, while IFNγ clearly plays an important role in vitiligo pathogenesis, it is still unclear whether other IFNs contribute to disease.

Figure 4

www.frontiersin.orgFIGURE 4 Vitiligo pathogenesis revealed through translational research. A simple overview of the current understanding of vitiligo pathogenesis. Autoreactive CD8+ T cells are recruited to the skin by CXCL10 produced by keratinocytes and kill melanocytes. These cells convert to resident memory T cells, which maintain vitiligo and require IL-15 signaling in the skin. JAKi and IL15i, outlined in red, may be effective treatments for vitiligo and are currently being tested in clinical trials. Some questions that remain include how melanocyte abnormalities initiate autoimmunity, the mechanism by which CD8+ T cells kill melanocytes, how Tregs suppress disease, and how changes in metabolism effect disease activity. Existing and developing translational tools will help to answer these and other questions. Figure created in BioRender.com.

Infiltrating CD8+ T cells express CXCR3 at high levels within lesions, and expression of CXCR3 ligands are characteristic of vitiligo lesions (104127133134). Supporting data using mouse models reveal that CXCR3 and CXCR3 ligands are expressed within lesional vitiligo skin in mouse models, and it is required for recruitment of T cells during disease progression and maintenance (127135136). Mouse models also made it possible to determine that keratinocytes respond to IFNγ production from T cells and secrete CXCL10 to recruit melanocyte-reactive CD8+ T cells to the skin (55). These discoveries provided the rationale to test JAK inhibitors, which block IFNγ signaling, as treatments for vitiligo and eventually led to a successful Phase 2 clinical trial to test topical ruxolitinib as a novel treatment for the disease (137143).

However, conventional treatments (144145) and even JAK inhibitors (137139) do not appear to provide durable responses following treatment, as disease relapses within previous lesions once they are stopped. Translational studies revealed that autoreactive CD8+ T cells form tissue resident memory cells (Trm) in vitiligo lesional skin (133146147). Mouse models confirmed the presence of Trm in vitiligo lesions and revealed that they are responsible for maintenance and relapse of disease after stopping treatment (146148150). Additional studies determined that melanocyte-specific autoreactive Trm require IL-15, and that targeting IL-15 signaling eliminates Trm from the skin and results in durable repigmentation in mice (146). IL-15 expression is elevated in vitiligo lesional skin and may be induced by oxidative stress in keratinocytes (151). Therefore, targeting IL-15 may provide a durable treatment option for vitiligo, and clinical trials to test this hypothesis are imminent (152).

As mentioned above, increased expression of HSP70i has been reported in vitiligo lesions, and activates skin dendritic cells that could prime autoreactive T cells against melanocytes (89). A mouse model required HSP70i for vitiligo initiation and when inhibited, disease is prevented (6689). Additionally, melanocytes produce HSP70 in response to oxidative stress, which may provide a link between cellular stress in melanocytes and inflammation that initiates autoimmunity in vitiligo (92153). Therefore, melanocyte stress could be mitigated by targeting this pathway and thus provide a new opportunity for treatment.

Regazzetti et al. reported a decreased expression of WNT signaling components in melanocytes when exposed to oxidative stress. The authors went on to demonstrate that treatment with a chemical WNT agonist can promote melanoblast differentiation in vitiligo skin, suggesting that activating WNT signaling in melanocytes might promote their regeneration (154). While that study did not show whether this differentiation fully progresses to a mature functional melanocyte, Yamada et al. reported that WNT signaling is critical in melanocyte stem cell differentiation and that WNTs are secreted predominantly by keratinocytes and melanocytes (154155). Furthermore, WNT signaling regulates E-cadherin, an important adhesion molecule for melanocytes, through the availability of β-catenin (156). Wagner et al. reported that there is a reduction of E-cadherin expression on melanocytes in vitiligo skin (51). Together these studies suggest that a decrease in WNT signaling might impair melanocyte adhesion. Additionally, Boukhedouni et al. reported that the type 1 cytokines IFNγ and TNFα impair melanocyte adhesion from the epidermis by inhibiting melanocyte E-cadherin expression and inducing keratinocyte release of protease MMP-9 (56). MMP-9 cleaves E-cadherin complexes, which results in melanocyte detachment from the epidermis. This study also reported that when type 1 cytokines were inhibited with JAK inhibitors, or when MMP-9 activity was inhibited, fewer melanocytes detached (56). Therefore, anti-inflammatory treatments like JAK inhibitors may also stabilize melanocytes and potentially could become more durable if used in conjunction with WNT agonists or inhibitors of detachment to promote melanocyte regeneration and maintenance.

Some have suggested that activating immune checkpoint inhibitors, which are receptors expressed on the surface of T cells that negatively regulate their activity, might be an effective therapeutic target for vitiligo (157158). Blocking cutaneous lymphocyte antigen-4 (CTLA-4) and program cell death protein 1 (PD-1) for melanoma treatment induce and exacerbate vitiligo, presumably by removing negative regulation that prevents autoimmune activation of these cells (159162). Decreased CTLA-4 mRNA expression has been reported in whole blood of vitiligo patients, and PD-1 expression on CD8+ T cells is positively correlated with disease activity, suggesting increasing activation (163164). PD-L1 inhibits cytokine production and is associated with maintaining peripheral tolerance (165166). Miao et al. reported that the injection of a PD-L1 fusion protein into a mouse model of vitiligo led to reduced disease severity and an increased Treg to effector T cell ratio (167). However, PD-1 is also a marker for T cell exhaustion and its upregulation was reported on regulatory T cells in the peripheral blood of vitiligo patients (168). Theoretically, PD-L1 may negatively regulate Tregs similarly to effector T cells, however neutralization of PD-L1 in vitro led to an expansion of human Tregs isolated from chronically infected HCV patients (169). Targeting these or other negative stimulatory molecules may limit the autoreactive CD8+ T cell activity, which may allow for Tregs to recover to reinstate tolerance, but further investigation is needed.

There have been recent advancements in Treg-based therapeutics. Tregs appear to actively prevent vitiligo in healthy subjects and may be impaired in those with the disease (170). Many report possible defects in Tregs in vitiligo patients, although they do not necessarily agree whether the defects are in total Treg number, ability to migrate to the skin, or suppressive function (168171176). Expansion of Tregs and adoptive transfer of these cells into patients have promising results in mitigating some autoimmune diseases and transplantation (177178). Selective expansion of antigen-specific Tregs reportedly controls inflammation in mouse studies to a greater extent than administration of polyclonal Tregs (179181). Chimeric antigen receptor (CAR)-Tregs are being developed as treatment options for autoimmunity and GVHD. CAR-Tregs allow for a more stable, antigen-specific Treg population that can be engineered to migrate to the site of inflammation, however more improvements are needed as off-target immunosuppression can result in susceptibility to infections and cancer development (177182183). Chatterjee et al. reported that adoptively transferred Tregs into a spontaneous vitiligo mouse model reduced disease onset. In addition, treating these mice with rapamycin limited disease progression and increased the number of circulating Tregs (184). Further investigation will be important to optimize these therapies and reduce risks for the treatment of vitiligo.

Using Vitiligo to Understand Organ-Specific Autoimmunity

Progress made through translational research in vitiligo may facilitate progress in other diseases that are more difficult to study because they are less common and/or because target tissues are less accessible for translational research. Diseases in which CD8+ T cells target a single cell type resulting in loss of that cell type likely share a similar pathogenesis with vitiligo (92). Examples include type 1 diabetes (T1D) within the pancreas, Hashimoto’s thyroiditis in the thyroid, Addison’s disease in the adrenal cortex, and multiple sclerosis (MS) in the brain. In support of this hypothesis, GWA studies reveal common risk SNPs (92185188). In addition, studies have implicated CD8+ T cells in Hashimoto’s thyroiditis, MS, and Addison’s Disease, similar to vitiligo (189192), as well as an important role for the IFN-γ chemokine axis during pathogenesis. IFN-γ and CXCL10 are expressed in human islets to recruit lymphocytes in T1D, autoreactive cells produce IFN-γ in Addison’s Disease, and IFN-γ level positively correlates with disease severity in Hashimoto’s thyroiditis (193196). IFN-γ is also involved in MS, although its role is still not fully understood (197). Furthermore, targeting IL-15 in a NOD mouse model reduced disease severity, suggesting IL-15 may play a role in T1D pathogenesis (198). Hence, vitiligo pathogenesis can be used as a guide for these diseases to launch more targeted investigations, as well as repurposing treatments. Narrowing the potential areas for investigation can help accelerate the understanding of pathogenesis for these autoimmune diseases.

Conclusion

Vitiligo is an organ-specific autoimmune disease that results in white patchy skin depigmentation. Since the beginning of modern vitiligo research over 40 years ago, translational work has been at the forefront of vitiligo studies because of the simple, non-invasive collection of patient skin samples. These observations have led to clinical studies and a successful clinical trial that are likely to change how we manage patients in the clinic. In addition to sample collection, the discussion, collaboration, and open-minded consideration among researchers have made vitiligo one of the best understood autoimmune diseases. Independent, parallel studies addressing both the degenerative and autoimmune theories of pathogenesis have led to a broader understanding of vitiligo pathogenesis by preventing narrow of experimental focus. This has not only helped our understanding of vitiligo but has also provided a valuable platform to study organ-specific autoimmunity in general. The knowledge gained about vitiligo may be beneficial for those who study similar autoimmune diseases that do not have ready access to tissues for translational studies. Our understanding for vitiligo and other autoimmune disease will improve as more discussions, collaborations, and technology continue to advance. These advancements will be continuously applied toward new therapeutic development to help patients.

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COVID‐19 and autoimmune phenomena: Vitiligo after Astrazeneca vaccine

by Irene López Riquelme,corresponding author 1 María Dolores Fernández Ballesteros, 1 Ana Serrano Ordoñez, 1 and Daniel Jesús Godoy Díaz

Aktas et al. published an interesting case of vitiligo after COVID‐19 vaccination.Recently, similar cases of vitiligo have been reported after,as well as other cutaneous reactions after COVID‐19 vaccination (both mRNA and ChAdOx1 vaccines) such as immune thrombocytopenic purpura and psoriatic flare‐up.Even though no cutaneous adverse events were encountered in Phase 3 studies of mRNA vaccines, the chronology of the symptoms is very suggestive. We would like to report another case of vitiligo 3 days after COVID‐19 vaccine and to comment about possible theories that have been suggested to explain autoimmune phenomena and vitiligo following COVID 19 infection or vaccination.

A 60 year‐old woman presented to the Dermatology department with amelanotic macules and patches in her face and arms that have appeared in the last 3 weeks. Three days before the onset of the symptoms, she had received the first dose of Astrazeneca vaccine against COVID‐19 (ChAdOx1/AZD1222).

On physical examination, circumscribed depigmented patches were observed, affecting both cheeks (Figure 1), forehead and between the eyebrows, as well as both arms. The lesions became more clearly visible when examined under Wood’s lamp (Figure 2).

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Well‐circumscribed depigmented patches in both cheeks, forehead and between eyebrows

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Depigmented patches were more noticeable when examined under Wood’s lamp

Blood test including thyrotropin was normal. Antithyroid peroxidase and antithyroglobulin antibodies were negative. With the diagnosis of vitiligo, she initiated a treatment with tacrolimus ointment 0.1% twice a day.

Vitiligo is an autoimmune disease in which there is a progressive depigmentation due to the loss of melanocytes in epidermis. In cell stress conditions, oxygen free radicals cause cell damage. In this inflammatory microenvironment, specific neoantigens are generated by melanocytes, like HSP70i, HMGB1 and S100B, leading to activation of innate and adaptive immune response. Eventually, dendritic cells present antigens to T lymphocytes, leading to destruction of melanocytes. Various studies have proven the central role that HSP70i plays in the pathogenesis of vitiligo, since its overexpression is associated with greater activation of dendritic cells, and therefore an increased lymphocytic infiltration in depigmented areas.

A relation between COVID‐19 and autoimmune phenomena has been reported. The most accepted theory considers that molecular mimicry between antigenic epitopes of the virus and certain human proteins, such as heat shock proteins (HSP60 y 90),could be the origin. In fact, these proteins have been associated with several autoimmune diseases triggered by COVID‐19 infection, like Guillain‐Barré syndrome, autoimmune bullous diseasesand some forms of vasculitis. Additionally, cross‐reactivity between SARS‐CoV‐2 and other tissular human proteins has been demonstrated, especially transglutaminase 2 and 3, ENA, myelin basic protein and even S100B.These findings suggest that a similar mechanism could be the cause of autoimmune diseases triggered by covid‐19 vaccination. However, IgG antibodies generated against SARS‐CoV‐2 have not been shown to be able to recognize and react against heat shock proteins.

Another possible mechanism suggested by Abdullah et al.posits that COVID‐19 infection would stimulate dendritic cells to produce massive amounts of IFN‐I, which also plays an important role in the pathogenesis of vitiligo. In the case of psoriatic flare‐up reactions, it has been suggested that the mRNA vaccines may cause a significant increase in IL‐6 production and recruitment of Th17 cells,which not only participate in the pathogenesis of psoriasis, but also in many other autoimmune diseases.

In conclusion, we report another case of vitiligo after COVID‐19 vaccine in a patient with unremarkable medical history. Although mechanism remains unclear, given the suggestive time sequence following vaccination and the increasing number of cases reported, we believe these hypothesis deserve further investigation

 

 

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FDA Approves New Vitiligo Treatment, Ruxolitinib (Opzelura) The JAK inhibitor cream is the first medication that can restore pigment in people with this autoimmune disease.

By Becky Upham July 22, 2022

On July 18, the U.S. Food and Drug Administration (FDA) approved ruxolitinib (Opzelura) cream 1.5 percent as a treatment for the most common form of vitiligo, according to a statement by Incyte, the manufacturer of the drug.

Vitiligo is a chronic autoimmune condition that causes patches of skin to lose pigment and turn milky white. The most prevalent form is nonsegmental (also known as generalized) vitiligo, in which white patches appear symmetrically on both sides of the body, such as on both hands or both knees, often covering large areas.

Ruxolitinib is the first medication that can restore pigment in patients with nonsegmental vitiligo. The FDA approved Incyte’s ruxolitinib cream for adults and children ages 12 and up.

“This approval is monumental,” says Daniel Gutierrez, MD, assistant professor of dermatology at NYU Grossman School of Medicine and dermatologist at NYU Langone Health in New York City, who was not involved in the drug development. “With Opzelura, we will have an FDA-approved pharmaceutical treatment option that can actually bring back color in patients who have vitiligo,” says Dr. Gutierrez.

He adds that prior to ruxolitinib, the only FDA-approved medication for vitiligo was monobenzyl ether of hydroquinone, a topical drug that removes pigment from skin to even out tones.

What Is Vitiligo?

Researchers estimate that between 1.9 and 2.8 million adults in the United States have vitiligo, with perhaps 40 percent of adults with vitiligo going undiagnosed.

Vitiligo causes immune cells to destroy melanocytes, the skin cells that produce pigment, according to the National Institute of Arthritis and Musculoskeletal and Skin Diseases. “This makes vitiligo much more noticeable in patients of color — people whose skin is much more richly pigmented — because there is going to be much more of a contrast between the unaffected skin and the skin affected by the vitiligo,” says Gutierrez.

Vitiligo can occur at any age, but most people experience the initial symptoms before age 30.

About 50 Percent of People Using Ruxolitinib Had Significant Repigmentation After One Year

Ruxolitinib belongs to a class of drugs called Janus kinase (JAK) inhibitors. While doctors prescribe oral JAK inhibitors for diseases such as rheumatoid arthritis, ruxolitinib is the only topical JAK inhibitor approved in the United States.

The FDA previously approved ruxolitinib for mild to moderate atopic dermatitis (eczema), in the fall of 2021.

JAK inhibitors work by decreasing the activity of the immune system, blocking certain enzymes that cause inflammation.

Patients using ruxolitinib apply the cream twice daily to the affected areas, covering up to 10 percent of their body’s surface area. It may take 24 weeks or more for people with vitiligo to see satisfactory results, according to Incyte.

The FDA based its approval on data from a clinical trial program that compared ruxolitinib to a placebo cream in more than 600 people (age 12 and older) with nonsegmental vitiligo. Investigators used the Vitiligo Area Scoring Index (VASI), a tool used to gauge disease severity and to measure improvements in face and body repigmentation.

In the two trials, by week 24 approximately 30 percent of people treated with ruxolitinib experienced significant improvements (at least 75 percent) as measured by VASI, which was the goal of the study. At one year, about 50 percent of those using the medication achieved that level of repigmentation.

“People using Opzelura had much more improvement in their vitiligo — very meaningful — compared to the placebo,” says Gutierrez.

The most common side effects seen in the trials were application-site acne, redness and itchiness, pharynx and nasal cavity inflammation, headache, urinary tract infection, and fever.

Ruxolitinib Comes With a Black Box Warning

The FDA added a black box warning to ruxolitinib, based on data showing that people taking oral JAK inhibitors faced a small increased risk of serious infections, major heart issues, clotting (thrombosis), cancer, and even death.

“However, in the clinical trials for people using ruxolitinib as a topical cream, the concentrations of the drug found in the blood were observed to be much lower compared to people who take ruxolitinib orally,” says Gutierrez. The same risks were not observed in the ruxolitinib trials, but the FDA is taking a “better safe than sorry” approach by including a warning on the box, he adds.

A conversation with your healthcare provider is the best way to determine whether the benefits of ruxolitinib outweigh the potential risks, as well as the need for any baseline and/or ongoing monitoring.

Patients Can Use Ruxolitinib on Their Face

Although dermatologists sometimes prescribe topical steroids off-label for vitiligo, there are risks when applying these medications to the face — the area where loss of pigment can impact appearance the most, says Gutierrez.

When used on the face, topical steroids can cause an acne-like rash that can persist for many months, called perioral dermatitis. Plus, “they can cause atrophy or dispigmentation, meaning you can have skin color changes. They can also thin the skin, cause stretch marks, and cause the growth of small blood vessels in the area,” Gutierrez says.

Ruxolitinib does not pose these risks, notes Gutierrez.

FDA Approval Means Better Access to Vitiligo Treatment

The FDA’s approval of ruxolitinib will definitely improve access to the drug by validating it as medically necessary. “Because vitiligo just creates a color change in the skin — there’s no itching or dermatitis under normal circumstances — sometimes it’s considered a cosmetic condition, meaning it’s not medically necessary to treat,” Gutierrez says. As a result, some insurers have declined to cover vitiligo treatments, according to the Vitiligo Research Foundation.

“However, this condition can dramatically impact how a patient sees themselves and how they present to the world. Vitiligo can cause significant psychological distress and negatively impact quality of life,” says Gutierrez.

“Vitiligo disproportionately impacts patients of color,” he adds. “This approval is an important step in improving a health disparity that does exist, and hopefully there will be more treatment options for vitiligo in the pipeline.”

How Much Will Ruxolitinib Cost?

The current Wholesale Acquisition Cost pricing is $1,950 for a 60 gram tube of Opzelura, according to Gabriella Greig, a spokesperson for Incyte. The actual cost to the consumer will vary depending on insurance coverage and how much of the cream is required for treatment.

“Incyte is committed to working with insurance providers in the U.S. to ensure eligible patients who can benefit from Incyte’s products have access to them,” says Greig. The company offers a copay savings card on its website for people with commercial insurance.

Source

Image Source: Demetr White/Stocksy; Canva

Factors affecting quality of life in patients with vitiligo Part 2

Patients and methods

Participants and settings

A nationwide questionnaire-based study was conducted in 21 hospitals and clinics in Korea from July 2015 to June 2016. All patients aged ≥ 20 years diagnosed with vitiligo by dermatologists, and who provided written informed consent prior to the survey were enrolled. We restricted the participants to adult patients, because different questionnaires should be applied to children and adults. We explored demographic characteristics and vitiligo phenotypes and determined Skindex-29 scores. All patients first completed the questionnaires in paper-and-pencil format, and dermatologists then confirmed the clinical profiles after interviewing and examining the patients. The study protocol was designed in accordance with the Declaration of Helsinki and was approved by the institutional review board of each hospital.

Demographic characteristics

Demographic characteristics recorded included age, sex, marital status (single or married) and educational background (elementary school graduate, middle school graduate, high school graduate or college graduate).

Vitiligo phenotypes

The vitiligo phenotypes included subtype (segmental or nonsegmental), disease duration (< 1, 1–4, 5–9 or ≥ 10 years), affected body surface area (BSA; < 05, 05–09, 1–4, 5–9, 10–19 or ≥ 20%), involvement of visible body parts (yes or no) and the particular body parts involved (face and neck, scalp, upper extremities, lower extremities, trunk and genital area).

The Skindex-29 questionnaire

QoL was assessed using the Korean version of Skindex-29. This instrument is employed extensively to measure the effects of skin disease on a patient’s life;8,9 the Korean version was cross-culturally adapted by Ahn et al.10 The semantic equivalence of all back-translated items has been confirmed,10 and the Korean version has been validated by several previous studies.11–13 The questionnaire contains 29 items exploring the influence of skin disease on daily life using a five-point scale: 0 (never), 1 (rarely), 2 (sometimes), 3 (often) and 4 (all the time). The responses were transformed into linear scores varying from 0 (no effect) to 100 (effect always experienced). Each item belongs to one of three domains (symptoms, functioning and emotions); the scores of the three domains were calculated as the mean score of the items included in each domain.

The major items affecting patients with vitiligo

The proportions of patients affected by each item (sometimes, often and all the time) were calculated. The items of most concern were identified based on the answers.

Quality-of-life impairment

The outcomes of the study were mild or severe impairment of QoL, as determined by each domain (symptoms, functioning and emotions) of Skindex-29. Mild and severe QoL impairments were defined using the cut-off scores suggested by Prinsen et al.:14,15 ≥ 39 (mild) and ≥ 52 (severe) for symptoms, ≥ 21 (mild) and ≥ 37 (severe) for functioning, and ≥ 24 (mild) and ≥ 39 (severe) for emotions.

Statistical analyses

Absolute and percentage frequencies were determined for categorical variables, and position (mean and median) and scattering (SD, range) were described for continuous variables. Univariate and multivariate logistic regression analyses were sequentially performed to identify the factors independently associated with QoL impairment in each domain of Skindex29. All analyses were performed using R 3.3.1 (R Foundation for Statistical Computing, Vienna, Austria).

Results

In total, 1123 patients with vitiligo were recruited from 21 hospitals and clinics. Of the participants, 609 (542%) were male and 514 female (458%), with a mean age of 498  152 years (range 20–84). The median duration of disease was 30 years (range 0–60). The detailed clinical characteristics of the enrolled patients are summarized in Table 1.

The results of Skindex-29

The Skindex-29 items of most concern to patients with vitiligo were as follows: no. 13: I worry that my skin condition may get worse (746% of patients); no. 3: I worry that my skin condition may be serious (629%); no. 22: My skin condition is a problem for the people I love (556%); and no. 6: My skin condition makes me feel depressed (535%) (Table 2).

Impaired quality of life and associated factors in terms of the symptoms domain

Of the patients with vitiligo, 146% (164 of 1123) had mild and 52% (58 of 1123) had severe QoL impairment in terms of the symptoms domain (Fig. 1). On multivariate logistic

Table 1 The clinical characteristics of the 1123 patients with vitiligo included in the present study

 

 

 

 

 

 

regression modelling, the involvement of visible body parts (OR 153) and a larger affected BSA (compared with < 05%; 5–9%: OR 278, 10–19%: OR 284 and ≥ 20%: OR 469) were associated with mild symptom impairment (Table 3). A larger affected BSA was also associated with severe symptom impairment (compared with < 05%; ≥ 20%: OR 810).

Impaired quality of life and associated factors in terms of the functioning domain

Of the patients with vitiligo, 488% (548 of 1123) had mild and 282% (317 of 1123) had severe QoL impairment in terms of the functioning domain. The factors associated with

Table 2 The Skindex-29 items that affected > 25% of patients with vitiligo

Affected

Items (in order of frequency)                        patients     Domain

13. I worry that my skin condition may          746%         Emotions get worse

3. I worry that my skin condition may            629%         Emotions be serious

22. My skin condition is a problem for          556%                Functioning the people I love

6. My skin condition makes me feel               535%         Emotions depressed

9. I worry about getting scars from my           498%         Emotions skin condition

12. I am ashamed of my skin condition        497%       Emotions

28. I am annoyed by my skin condition        476%       Emotions

5. My skin condition affects my social           450%                Functioning life

15. I am angry about my skin condition        448%       Emotions

24. My skin is sensitive                                 433%          Symptoms

4. My skin condition makes it hard to            427%                Functioning work or do hobbies

21. I am embarrassed by my skin 410%         Emotions condition

19. My skin is irritated                                  379%          Symptoms

23. I am frustrated by my skin condition      356%       Emotions

20. My skin condition affects my 354%         Functioning interactions with others

11. My skin condition affects how close       328%       Functioning

I can be with those I love

17. My skin condition makes showing           305%                Functioning affection difficult

26. I am humiliated by my skin    296%         Emotions condition

10. My skin itches                                         291%          Symptoms

14. I tend to do things by myself  275%         Functioning because of my skin condition

30. My skin condition makes me tired          264%       Functioning

8. I tend to stay at home because of my          263%                Functioning skin condition

 

Source

 

Anxiety and depression in pediatric patients with vitiligo and alopecia areata and their parents: A cross-sectional controlled study Part 1

Department of Dermatology, University of

Health Sciences, Sultan Abdulhamid Han

Training and Research Hospital, Istanbul,

Turkey

Correspondence

Sevil Savaş Erdoğan, Department of

Dermatology, Sultan Abdulhamid Han Training and Research Hospital, Tıbbiye Street., 34668, Usküdar, Istanbul, Turkey. Email: doktorsevilsavas@gmail.com

2232  |  © 2020 Wiley Periodicals LLC                                                       wileyonlinelibrary.com/journal/jocd       J Cosmet Dermatol. 2021;20:2232–2239.

 

1       | INTRODUCTION

Anxiety and depressive disorders are the most common psychopathologies in children and adolescents.1 Alopecia areata (AA) is a common hair disorder characterized by a sudden-onset non-cicatricial but has a great negative effect on cosmetic appearance. Several studies have estimated the prevalence of AA worldwide to be 1%-2%, and in the majority, the first patch presents before the age of 20 years.2 Vitiligo is characterized by the partial or complete absence of melanocytes that cause the development of white macules or patches in various parts of the body.3,4 The worldwide prevalence of vitiligo is estimated to be 0.5%-2%, and approximately 50% of patients experience vitiligo before the age of 20 years.5,6 Since their visibility, chronic and disfiguring nature, and lack of curative therapy, AA and vitiligo may have high psychosocial effects on patients. AA and vitiligo are associated with various psychiatric comorbidities. In different studies, high anxiety and depressive disorder rates have been reported in children and adolescents with AA.7-11 Studies have shown that vitiligo patients suffer more from depression and anxiety.12-14 However, there are only a limited number of studies investigating the relationship between vitiligo and psychiatric state in children and adolescents.15-17

Skin diseases have a negative effect on family life,18-20 and the care of a child with AA or vitiligo may be associated with higher anxiety and depression in their parents than that of healthy children. Unlike other studies, in the current work, children and adolescents with AA and vitiligo and their parents were compared with each other and also with the healthy control group. Therefore, we aimed to identify psychiatric findings in children and adolescent groups with AA and vitiligo and to evaluate the levels of anxiety and depression in their parents. We also aimed to investigate the quality of life (QoL) of patients, and the impact of the disease on their parents.

2       | MATERIAL AND METHOD

2.1      | Study design and subjects

This is a prospective cross-sectional study conducted in our dermatology outpatient clinic between October 2018 and December 2019. The study was approved by the institutional ethical committee and carried out in accordance with the principles of the Declaration of Helsinki. Patients, controls, and their parents, who agreed to participate in the study, provided informed consent.

The study included children and adolescents aged seven to 17 years and their parents. Of the patients, 31 had AA and 29 had vitiligo. In addition, 30 age- and gender-matched healthy controls without any dermatological disease and their parents with a similar education level to that of the patients’ parents were included as a reference group. The following exclusion criteria were used for both the patient and control groups: systemic treatment (systemic steroid, cyclosporin and/or neuropsychiatric drugs) within the last three months prior to the study and any other chronic disease.

2.2      | Demographic variables and clinical severity

Demographic data (patient age and gender, and parental age and education level) and medical information (disease duration, disease severity, and presence of stressful life events within the past year from the onset of disease) were noted in separate forms for the patients. In addition, through a clinical examination, the Severity of Alopecia Tool (SALT) score21 was calculated for the patients with AA, and the Vitiligo Area Severity Index (VASI)22 for those with vitiligo.

The questionnaires explained below were administered in a single interview. The patients and controls completed the Revised Child Anxiety and Depression Scales-Child version (RCADS-C), and their parents completed the parent version (RCADS-P) to support the results of the child version. The parents also completed the Beck Anxiety Inventory (BAI) and Beck Depression Inventory (BDI). Lastly, the Children’s Dermatology Life Quality Index (CDLQI) scale was completed by the patients, and the Dermatological Family Impact Scale (DeFIS) by their parents.

2.3      | Scales on psychological parameters and QoL

CDLQI is a four-point Likert scale that evaluates the state of the patients and can be used in children aged four to 17 years. Higher scores indicate a greater level of deterioration in QoL. In this study, we used the Turkish version of this scale, for which validity and reliability studies have been previously conducted.23

RCADS-C and RCADS-P are self-report questionnaires, each containing 47 items designed to assess DSM-IV depression and anxiety disorders in children and adolescents. Response options are based on four-point Likert scales. Both versions have six subscales [separation anxiety disorder (SAD), social phobia (SP), obsessive-compulsive disorder (OCD), panic disorder (PD), generalized anxiety disorder (GAD), and major depressive disorder (MDD)] and provide a total anxiety score (sum of the score in five anxiety scales) and a total internalizing score (sum of the six scores in subscales).24 In this study, we used the Turkish versions of these questionnaires. The validity and reliability studies of the Turkish versions have been previously conducted.25,26

DeFIS is a 15-item, five-choice response scale to investigate how the family QoL of patients with chronic dermatosis has been affected within the last month and produces a score of 0-4. Higher scores indicate greater impairment in QoL. The scale was developed for the Turkish society by Turan et al, and the validity and reliability studies have been undertaken by the same authors.27

BAI is a 21-item self-report scale that was used to measure parents’ anxiety levels in this study. The items include symptoms of anxiety and higher total scores indicating greater anxiety levels. BAI was previously adapted to the Turkish society, and the Turkish version of the scale was in the current study.28

BDI is a 21-item self-report scale that was used to measure the level of depression in parents. There are four statements for each symptom, and a higher total score indicates a higher level of depression. The validity and reliability studies of the Turkish adaptation of the 1979 version of BDI have been undertaken.29,30

2.4      | Statistical analysis

As the statistical method, descriptive analyses (frequency distributions, percentage, mean, and standard deviation) were used. For the analysis of continuous data, conformance to normal distribution was checked using the Kolmogorov-Smirnov test. For the data that were normally distributed, the comparison of more than two groups was undertaken with ANOVA and the post hoc Bonferroni test and the comparison of two groups using the t-test.

In the absence of normal distribution, Kruskal-Wallis and MannWhitney U tests were performed, the chi-square test was used for discrete data, and Fisher’s exact test was conducted depending on the data compatibility. Relationships were investigated by the

TABLE 1 Characteristics of patients and controls

Spearman rho correlation coefficient. The results were evaluated at the 95% confidence interval and P < .05 significance level.

3       | RESULTS

3.1                | Demographic and clinical characteristics of the study groups

Thirty-one patients with AA, 29 patients with vitiligo, and 30 age- and gender-matched controls were included in the study. The mean (± standard deviation) ages of the in the AA, vitiligo, and control groups were 12.5 ± 3.6, 13 ± 3, and 12.6 ± 3.1 years, respectively. There was no statistically significant difference between the patient and control groups in terms of age, gender, number of siblings, parental education level, and parental marital status (Table 1). All patients with AA had the patchy type, and their mean SALT score was 6.3 ± 13.1. In the vitiligo group, the type of disease was vulgaris in 51.7% (n = 15), focal in 44.8% (n = 13), and segmental in 3.4% (n = 1). The mean VASI score of the vitiligo group was 3.6 ± 7.4. The mean disease duration was 6.5 ± 15.5 (range, 1-84) months in the AA group and 31.4 ± 35.5 (range, 2-132) months in the vitiligo group.

The difference between the AA, vitiligo, and control groups in terms of the presence of stressful life events was statistically

 

Groups

Alopecia areata

Vitiligo Controls P-value
Age, mean ± SD 12.54 ± 3.56 13 ± 3.03 12.6 ± 3.06 .85
Sex Female, n (%) 14 (45.16) 13(44.82) 14(46.66) .98
Male, n (%) 17 (54.83) 16(55.17) 16(53.33)
Mother’s educational level Illiterate, n (%) 1 (3.2) 3(10.3) 0 .56
Primary school, n (%) 12 (38.7) 9(31) 12(40)
Middle School, n (%) 5 (16.1) 5(17.2) 8(26.7)
High school, n (%) 9 (29) 6(20.7) 7(23.3)
University, n (%) 4 (12.9) 6(20.7) 3(10)
Father’s educational level Illiterate, n (%) 11 (35.5) 6(20.7) 13(43.3) .6
Primary school, n (%) 5 (16.1) 6(20.7) 4(13.3)
Middle School, n (%) 8 (25.8) 11(37.9) 9(30)
High school, n (%) 7 (22.6) 5(17.2) 4(13.3)
University, n (%) 0 1(3.4) 0

Parental marital status                                         Married, n (%)                                 27 (87.1)                                   25(86.2)                       30(100)                                                                    .11

Separated, n (%)                            4 (12.9)                                        4(13.8)                                                                                                               0

Number of siblings 1, n (%) 4 (12.9) 3(10.3) 4(13.3) .56
2, n (%) 12 (38.7) 17(58.6) 18(60)
3, n (%) 10 (32.3) 7(24.1) 6(20)
4, n (%) 3 (9.7) 2(6.9) 2(6.1)
5, n (%) 2 (6.5) 0 0

TABLE 2 Comparison of groups’ psychological test scores and presence of stressful life event

Groups P-value
                                             Alopecia areata                       Vitiligo                                          Controls AA-Vi                         AA-C                         Vi-C

Stressful life event, n (%)                        19 (61.3)                                      25 (86.2)                                      9 (30)                                                                               .041                                               .014                                         <.0001

RCADS-Child, mean ± SD

Separation Anxiety

55.61 ± 12.94 50.21 ± 9.6 48.83 ± 9.9 .116 .026 .291
Generalized Anxiety 48.61 ± 11.37 45.14 ± 9.09 42.5 ± 7.41 .195 .021 .261
Panic 49.65 ± 12.3 47.31 ± 10.9 44.67 ± 8.17 .454 .177 .470
Social Phobia 46.19 ± 12.48 43.66 ± 8.72 39.93 ± 8.34 .625 .050 .069
Obsessive-Compulsive 48 ± 12.58 48.03 ± 11.56 46.93 ± 8.89 .935 .874 .952
Depression 49.68 ± 12.21 47.38 ± 14.3 41.07 ± 7.55 .314 .006 .162
Total Anxiety 49.03 ± 12.96 45.69 ± 10.5 42.43 ± 7.88 .287 .057 .295
Total 49.13 ± 12.9 45.86 ± 11.63 41.47 ± 7.95 .245 .010 .261

RCADS-Parent, mean ± SD

Separation Anxiety 57.55 ± 15.3 54.38 ± 9.8 51.03 ± 8.43 .700 .167 .181
Generalized Anxiety 54.81 ± 9.64 52.9 ± 11.55 51.6 ± 10.8 .415 .133 .660
Panic 55.06 ± 12.64 54.24 ± 11.82 48.57 ± 9.9 .795 .014 .030
Social Phobia 49.26 ± 12.09 49.45 ± 10.91 45.83 ± 8.47 .900 .291 .199
Obsessive-Compulsive 57.39 ± 11.18 55.86 ± 11.17 54.97 ± 11.18 .573 .422 .558
Depression 58.74 ± 14..95 56.24 ± 14.15 47.37 ± 8.23 .529 .001 .014
Total Anxiety 55.06 ± 10.92 53.9 ± 12.39 50.2 ± 10.65 .569 .041 .316
Total 56.32 ± 11.1 54.72 ± 12.24 49.43 ± 10.22 .428 .012 .074
Parents’ BAI, mean ± SD 11.71 ± 8.78 11.45 ± 10.21 8.97 ± 8.71 .667 .169 .235
Parents’ BDI, mean ± SD 10 ± 6.72 9.83 ± 5.79 7.53 ± 5.78 .906 .110 .093

Abbreviations: AA, Alopecia Areata; Vi, Vitiligo; C, Controls; BAI, Beck Anxiety Inventory; BDI, Beck Depression Inventory; RCADS-Child, Revised Child Anxiety and Depression Scales, Child Version; RCADS-Parent, Revised Child Anxiety and Depression Scales, Parent Version.

 

significant (P < .0001). Furthermore, this parameter was statistically significantly higher in the vitiligo group compared to the AA and control groups (P = .014 and P < .0001, respectively) (Table 2).