American Vitiligo Research Foundation
Grant Report - 2002

The Role of the Catalase Gene in Vitiligo Susceptibility

           Vitiligo is a pigment disorder of the skin that results in areas of depigmentation that may gradually enlarge.  Vitiligo is common, affecting about 1% of all racial groups worldwide.  The average age of onset is about 20 years of age, so half of all vitiligo patients develop the condition during childhood or adolescence.  Vitiligo may predispose affected persons to skin damage by sunburn, and may be associated with an increased risk for some other autoimmune diseases, such as thyroid conditions.  Most current standard 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 the disease, 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).  However, 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.  In some patients disease progression appears to involve inappropriate activation of an immune response against the patient’s own melanocytes, therefore vitiligo is considered to be an autoimmune disease.

            By identifying 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 developing 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. 

 With the help of hundreds of vitiligo patients and family members, our vitiligo research at the University of Florida College of Medicine has focused on the possible genetic basis for being predisposed to having vitiligo.  We have collected vitiligo family DNA samples and performed “genetic association” studies of candidate genes, which are genes that might be involved in causing vitiligo based on their important normal functions in the pigment-producing melanocytes or in regulation of the immune system.  In many cases there were reports in the research literature suggesting some change in the expression or function of the candidate gene in vitiligo patients.  Variations in the gene sequences that have no known effect on gene function were used as “markers” to examine how common gene variants are in vitiligo patients vs. people who do not have vitiligo.  The theory was that if a particular genetic marker is more common in vitiligo patients, then that marker may be “linked” to another genetic change (mutation) that does impact gene function in some way, and therefore contributes to the disease process.  These studies found no association between vitiligo and about two dozen candidate susceptibility genes.  However, one of the candidate genes for which evidence was found for genetic association was the CAT gene, which codes for the enzyme catalase. 

 The enzyme catalase is important for breaking down hydrogen peroxide, a normal byproduct of many biochemical processes in the human body, including pigment production in the skin.  If hydrogen peroxide is not broken down by catalase (into oxygen and water), it can cause damage to cells and contribute to disease processes.  CAT was originally selected as a candidate gene because of reports from Dr. Karin Schallreuter’s research team that hydrogen peroxide levels are abnormally high in the entire skin of vitiligo patients and that catalase enzyme activity is depressed.  A deficiency in catalase enzyme activity in vitiligo patient skin is the biochemical basis for pseudocatalase treatment.

 One of the three genetic markers we studied for the human CAT gene suggested possible association between the CAT gene and vitiligo susceptibility in Caucasian patients.  We all have two copies of our genes, one inherited from each of our parents.  Interestingly, vitiligo patients tended to have only one copy of the gene variant (geneticists call them “heterozygous”), which suggested a possible mechanism for decreased enzyme activity.  In order to be functional, the catalase enzyme has four enzyme molecules put together in a complex.  Our hypothesis is that “mixed molecules”, that is, catalase complexes made up of some “mutant” subunits and some “normal” subunits, might have lower enzyme activity, resulting in higher hydrogen peroxide levels in the skin and therefore contributing to the cause of vitiligo. 

 In order to test this hypothesis, two experiments were planned in our 2002 AVRF grant project.  The first experiment has been completed, and the second experiment is still in progress.  A third experiment, part of our 2003 AVRF grant project, will also be briefly discussed. 

1.  Mutations in the CAT gene of vitiligo patients.  DNA samples were screened for mutations in the CAT gene of vitiligo patients.  Using a technique called polymerase chain reaction (PCR), specific regions of the CAT gene were amplified so the genetic code for the catalase gene could be determined.  Several previously unknown gene variations were discovered, however, none of the genetic changes discovered so far appear to be able to affect catalase enzyme activity.

2.  Deficiency of catalase enzyme activity due to heterozygous CAT mutations.  A second approach to directly test our hypothesis was to compare the catalase enzyme activities of cells artificially engineered to be heterozygous for the CAT gene mutations.  Molecular models of catalase enzyme structure were examined to identify amino acids within the catalase enzyme subunits that could be altered in a way that might affect enzyme activity in mixed molecules.  A copy of the human catalase gene cloned in bacteria cells was altered by site-directed mutagenesis to change four specific amino acids.  All of the mutations have confirmed by checking the gene sequences.  In the remainder of the experiment, these genes will be introduced into human cells grown in culture, and catalase enzyme levels will be measured to determine whether the presence of mixed molecules results in decreased catalase enzyme activity.

            3.  Altered CAT gene expression in patient immune system cells.  The catalase enzyme is expressed by many cell types, including some cells in the immune system.  Because of the involvement of inflammatory responses in the skin of some vitiligo patients, we are examining the expression of the CAT gene in vitiligo patient white blood cells.  A variation of the PCR technique is being used to measure gene expression for CAT and several other genes in patient white blood cells (monocytes) grown in cell culture under conditions that cause inflammation.  In addition, the levels of catalase enzyme activity expressed under these conditions by monocytes of vitiligo patients and persons without vitiligo are being measured for direct comparison.

In summary, our AVRF-funded research seeks to confirm a role of the CAT gene in vitiligo susceptibility.  Gene sequences were compared for vitiligo patients and control subjects, but no obvious mutations affecting gene function were identified in vitiligo patients.  Cells grown in the laboratory are being used to investigate whether specific genetic changes artificially introduced into the catalase gene will influence levels of catalase enzyme activity as predicted by our previous genetic data.  Finally, patient cells are being studied to determine whether catalase enzyme activity is altered under inflammatory conditions, as seen in the skin of some vitiligo patients.  The ultimate goal of our research is to increase our understanding of what causes vitiligo in terms of susceptibility, disease onset, and progression.  These studies may allow the future identification of “at risk” individuals in families with a history of vitiligo, and might be the basis for developments in treating or preventing vitiligo.