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AVRF 2008 PROGRESS REPORT

Grant Title: “The Role of Genetics in Vitiligo Susceptibility”

Principal Investigator : Margaret R. Wallace, Ph.D.

Professor, Dept. of Molecular Genetics and Microbiology

Box 100266 , University of Florida College of Medicine

1600 S.W. Archer Road

Gainesville , FL 32610-0266

(352) 392-3055 (phone)

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Co-Principal Investigator : Wayne T. McCormack, Ph.D.

Associate Professor,

Dept. of Pathology, Immunology, & Laboratory Medicine

Box 100275 Health Science Center

University of Florida College of Medicine

Gainesville , FL 32610-0275

(352) 392-7413 (phone)

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Vitiligo Genetics Research at the University of Florida
College of Medicine

Introduction

Vitiligo is a pigment disorder of the skin, which is seen on patients as de-pigmented areas of the skin that may gradually enlarge.  Vitiligo is a common condition, affecting about 1% of all racial groups worldwide, and predisposes affected persons to skin damage by sunburn.  Vitiligo is also associated with an increased risk for other autoimmune diseases, such as thyroid conditions.  The current treatments for vitiligo are difficult, expensive, and often disappointing.  The cause of vitiligo is unknown, but is thought to involve both genetic and environmental factors.  Vitiligo sometimes runs in families, and one study found that 20% of the relatives of vitiligo patients also have vitiligo, with the highest risks for children and siblings of patients.  This suggests that some people are born with genes that make them more susceptible to developing vitiligo.  These genes may be important for the normal function of the pigment-producing cells in the skin (melanocytes) and/or in cells of the immune system (lymphocytes).  Environmental factors, such as traumatic skin injury, sunburn, or stress, appear to influence whether or not an individual with those “susceptibility genes” will develop vitiligo during their lifetime.

Genetic Studies

By identifying disease susceptibility genes, we can learn more about the cause of vitiligo.  Depending on which genes are involved, what their normal functions are, and what genetic changes are found in vitiligo patients, it might be possible to design new treatments based on understanding those genes.  Knowing which genes are important may also allow genetic testing for individuals that might be predisposed to having vitiligo, so that environmental risk factors can be more carefully avoided.  Theoretically, it is possible that future advances in gene therapy might also help susceptible or affected persons.

We believe that in addition to environmental factors that trigger vitiligo, some individuals are genetically susceptible to vitiligo, due to variations in multiple genes important for melanin biosynthesis, response to oxidative stress, and/or regulation of autoimmunity.  The goal of our research is therefore to test the hypothesis that vitiligo is caused in part by both autoimmune and autotoxic events in the epidermis due to genetic differences in genes involved in the regulation of the immune response, melanin production and oxidative stress.

We are using “case/control association” to try to identify additional vitiligo susceptibility genes.  Case/control studies compare the variations of potential vitiligo susceptibility genes that are present in patients compared to an unaffected control group.  A gene variant or change will be considered to be associated with vitiligo susceptibility when it is found more often in vitiligo patients than in controls.  Large numbers of association tests must be performed for the most reliable statistical results, and repeating the studies in different populations or ethnic groups is extremely useful to confirm real associations.  We are also using “family-based association,” which requires DNA samples from family members of the vitiligo patient.  Families with one or more affected children can be analyzed, and statistical tests can help determine whether a particular genetic change in a vitiligo susceptibility gene is passed from parents to affected children more frequently than expected due to random chance.  The advantages of family-based association studies include the use of families with any number of affected children, and the combining of information from multiple ethnic groups and/or geographical populations.

Previous Results

In 2002, we reported case-control and family-based association studies for the catalase gene (CAT) in vitiligo patients [Casp, C.B., J.X. She, & W.T. McCormack, 2002, Genetic association of the catalase gene (CAT) with vitiligo susceptibility, Pigment Cell Res. 15:62-66].  The CAT gene was selected as a candidate gene because the reduced catalase enzyme activity and accompanying accumulation of excess hydrogen peroxide observed in the entire epidermis of vitiligo patients.  One of three CAT genetic markers studied was found to be informative for genotypic analysis of Caucasian vitiligo patients and control subjects.  Both case/control and family-based genetic association studies of this genetic marker suggested possible association between the CAT gene and vitiligo susceptibility.  The observations that individuals who carried different gene variations (i.e., were heterozygous) are more frequent among vitiligo patients than controls and that the one variant is transmitted more frequently to patients than controls suggest that linked mutations in or near the CAT gene might contribute to a quantitative deficiency of catalase activity in the epidermis and the accumulation of excess hydrogen peroxide.  This evidence for the CAT gene as a vitiligo susceptibility gene in some vitiligo patients supports the epidermal oxidative stress model for vitiligo pathogenesis.

We also reported in 2002 that the LMP/TAP gene region is significantly associated with vitiligo [Casp, C.B., J.X. She & W.T. McCormack, 2003, Genes of the TAP/LMP cluster are associated with the human autoimmune disease vitiligo, Genes & Immunity 4:492-499].  These genes are located within the class II region of the major histocompatibility complex (MHC), which includes genes involved in antigen processing and presentation, and have been reported to be associated with several autoimmune diseases.  The LMP/TAP gene products are responsible for processing and transport of antigenic peptides for presentation to the immune system via MHC class I molecules.  Case/control analyses revealed genetic association of vitiligo in Caucasian patients with an early age of onset with the transporter associated with antigen processing-1 (TAP1) gene.  A family-based association method revealed biased transmission of specific alleles from heterozygous parents to affected offspring for the TAP1 gene, as well as for the closely linked LMP2 and LMP7 genes encoding subunits of the immunoproteasome.  No association with vitiligo was found for the MECL1 gene, which encodes a third immunoproteasome subunit and is unlinked to the MHC class II region.  These results suggested a possible role for the MHC class I antigen processing and/or presentation pathway in the anti-melanocyte autoimmune response involved in vitiligo pathogenesis.

Past AVRF Project (2002-2004)

The American Vitiligo Research Foundation funded a project at the University of Florida College of Medicine from 4/1/02 through 2/28/04 ($27,000) entitled “The Role of the CAT Gene in Vitiligo Susceptibility”.  The principal investigator for this study was Wayne T. McCormack, Ph.D.  This project built on previous results by Ph.D. candidate Courtney B. Casp, which demonstrated a genetic association between two candidate genes and vitiligo susceptibility. 

In the AVRF-funded study (2002-2004), gene expression studies were conducted for all of these candidate vitiligo susceptibility genes.  Catalase is an important antioxidant responsible for breaking down hydrogen peroxide.  Our study looked at expression of active catalase in purified vitiligo patient monocytes (white blood cells), and revealed a significantly decreased expression of catalase in the monocytes of vitiligo patients.  We observed no significant difference in the catalase activity of patient and control red blood cells.  We also looked at catalase messenger RNA (mRNA) expression in these same monocytes through semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR).  We found no significantly different expression of catalase mRNA in vitiligo patients compared with normal controls.  Taken together these results suggest that the decrease in vitiligo patient monocytes catalase enzyme activity is not a result of changes in mRNA expression or quantity.  These results support those discovered by Schallreuter and coworkers, who found mRNA expression extracted from keratinocytes and melanocytes from both patients and controls did not show any differences in expression, while catalase activity was decreased in the epidermis of vitiligo patients.  The question remains as to whether the deficiency of catalase enzyme activity in vitiligo patient skin is due to defects in the catalase enzyme itself, or is due substrate inhibition caused by a localized accumulation of hydrogen peroxide from other sources.  Nevertheless, we have observed functional changes in catalase enzyme activity in circulating monocytes of vitiligo patients, which helps to support the genetic association found through our case/control and family-based analyses.

Messenger RNA expression studies of LMP2, LMP7, TAP1 and TAP2 genes did not show alterations in expression of mRNA for any of these genes in monocytes derived from vitiligo patients and normal controls.  However, expression studies of MHC class I revealed decreased expression of MHC class I on monocytes from vitiligo patients, suggesting some alterations in the MCH class I presentation pathway.  The results of this AVRF-funded project demonstrate a possible role for genes involved in immune system regulation, as well as for genes involved in regulating oxidative stress in vitiligo susceptibility.  Thus, the etiology of vitiligo may rely on both autotoxic events in the melanocyte, allowing for increased oxidative stress in the epidermis and inappropriate autoimmune presentation of self-peptides to the immune system.

Current AVRF Project (2006-2008)

The AVRF is currently funding a project entitled “The Role of Genetics in Vitiligo Susceptibility” (Margaret R. Wallace, Ph.D., Principal Investigator) from 6/1/06 through 5/31/08 ($150,000).  Co-Investigators include Wayne T. McCormack, Ph.D., and Ph.D. candidate Deborah M. Herbstman.  The goal of these studies is to test the hypothesis that susceptibility to vitiligo is a complex genetic trait involving a number of genes.  Some of the genes we are investigating are known to be expressed in melanocytes, which may predispose them to self-destruction and/or target them for autoimmune destruction.  Other genes we are investigating are known to be involved in the immune system, which may contribute to the autoimmune response in vitiligo patients.  The long-term goals of this research include determining whether “at risk” individuals can be identified in families with a history of vitiligo, which might influence the choice of strategies for treatment or prevention of vitiligo, and whether novel therapies that might prevent or ameliorate vitiligo can be devised based on an understanding of vitiligo pathogenesis and genetic susceptibility.  In addition, these studies may contribute to an overall understanding of genetic susceptibility to autoimmune disease, given the possible association of vitiligo with other autoimmune endocrinopathies.