Fourth Annual DNA Grantees' Workshop
Tuesday, June 24, 2003
MORNING SESSION
Research Update Briefings: Tools for Tomorrow
Anthony J. Tambasco, Moderator
Biography
MR. TAMBASCO: Good morning. My name is Tony Tambasco. I'm the director of the Mansfield Police Laboratory in Ohio.
We have a wonderful panel up here this morning. As Carl pointed out yesterday, all the bios are in the back of the book, so we're just going to skip right to the good stuff.
Our session is basically on research tools for tomorrow.
Gene Polymorphisms and Human Pigmentation
Murray H. Brilliant
Biography
MR. TAMBASCO: Our first speaker this morning, Dr. Murray Brilliant, earned his Ph.D. in molecular, cellular, and developmental biology from the University of Colorado. Dr. Brilliant is a Lindholm professor of genetics with the Department of Pediatrics, College of Medicine, at the University of Arizona. He's going to speak to us about gene polymorphism and human pigmentation. Dr. Brilliant.
DR. BRILLIANT: Thank you. Good morning.
My work has to do with the genetics of pigmentation. It dawned on me that this could be very useful with forensic applications. So hopefully you'll see what I mean. Brilliant: Slide 1
Before I start, I'd just like to acknowledge the people who are doing this work. Some students in my laboratory are doing the subject recruitment and DNA analysis, and then many of my colleagues at the University of Arizona are helping with the data input and analysis. I'll show you some of those results today. Brilliant: Slide 2
Our goals are to collect 1,000 samples from people who ethnically represent the U.S. population and to determine the polymorphisms in genes that are known to affect pigmentation, that is, pigment variations in the eyes, hair, and skin. We're trying to understand how the genes involved in pigmentation interact with each other and how they determine ultimately what our constitutive pigmentation is. Brilliant: Slide 3
People come in many different flavors, and these flavors are encoded by our genes. Brilliant: Slide 4
There are very powerful selective pressures on human pigmentation. This has to do with the fact that our skin needs ultraviolet light to make vitamin D, which is very important for calcium and phosphate metabolism. People who have a deficiency in calcium and phosphate have, among other problems, a reduction in fertility. So this is a very powerful selective pressure. Brilliant: Slide 5
Melanin, the pigment in our skin, for example, is an important protective agent against sunburn and skin cancer. That's why, in general, people who live in sun-exposed areas or who evolved in sun-exposed areas have darker pigmentation than those who live very far north, say, in northern climates.
In the 1970s, there was an estimate done on the number of genes that might affect skin pigmentation. This was a kind of politically incorrect and scientifically flawed study, but it did give us some idea of the number of genes that might be involved in skin pigmentation. Brilliant: Slide 6
Most of these curves correspond to different gene pair variations that one might see in a population, such as the African-American population, which on average has an admixture of about 20 percent non-African derived genes. From this data, it was estimated that four genes are responsible for the major pigmentation variation that is found, say, among African Americans.
Skin pigmentation arises in the pigment cells or melanocytes. These pigment cells have long projections that go into the skin and manufacture melanin in organelles. Brilliant: Slide 7
Organelles are called melanosomes. They act as kind of a factory where melanin pigment is made. Melanin pigment, which act as a motor that moves pigment granules along the dendritic processes of the melanocytes, get taken up in the epithelial cells of the epidermis. They actually form a little umbrella around the nucleus of the epidermal cells to shield it from ultraviolet light.
If we look at the skin of different individuals of different races, we would see that amounts of melanin and, in particular, the size and packaging of melanosomes vary considerably among different individuals. Brilliant: Slide 8
This is an example of some important genes in pigmentation that, at least medically, lead to forms of albinism: Type 1 albinism or oculocutaneous albinism type 1 (OCA1) is the result of deficiencies in the tyrosinase enzyme involved in making melanin. This is not the most common form of albinism, but it's the one that most people recognize as albinism. The P gene is responsible for the most common form of albinism called OCA2 (oculocutaneous albinism type 2). I'll talk about that in a few minutes. OCA3 (oculocutaneous albinism type 3), which is mostly recognized in Africa, is another form of albinism. Although it may or may not be a true form, it does affect pigmentation. OCA4 (oculocutaneous albinism type 4) is a relatively recent form of albinism that our group has identified in a gene called MATP (membrane associated transporter protein). So these are some of the genes that are involved in albinism, but we're also interested in some other ones. Brilliant: Slide 9
The most common form of albinism is OCA2. Another gene, MC1R (melanocortin receptor 1), has already been shown to be involved in a red hair variation, and there's a gene that interacts with MC1R called the agouti signaling protein. One polymorphism of that has been linked to pigmentation changes in human beings. Then there are a bunch of other genes that I won't go into at this time. Brilliant: Slide 10
In regards to melanosomes, a protein called the tyrosinase enzyme goes through the membrane of the melanosome and converts the tyrosine amino acid to the melanin polymer, which is the pigment in human beings. The OCA3 gene product is thought to help stabilize tyrosinase.
In addition, there's an energy-dependent proton pump that pumps protons inside the organelle. Counter-ions and ions come in via this P transporter which is involved in OCA2. Together, these two regulate the pH (potential of hydrogen) of the melanosome. If the pH is regulated at different times or to different extents, variations in pigmentation can occur. The MATP (or OCA4) protein is also a transporter and may be involved in releasing some protons. Again, this is a dynamic process and it varies within melanosomes of the eyes, skin, and hair. It's the interaction of these proteins and how they regulate the action of tyrosinase that's responsible for our constitutive pigmentation. Brilliant: Slide 11
Indonesian populations are among the populations that I study. In one case, there was a girl with a pigmentation typical of what one sees in Indonesia, whose first cousin carries a null mutation for the P gene, and she has brown hair, which is unusual in this population. Brilliant: Slide 12
So already we can tell that just the action of the P gene could be involved in pigment variation. Brilliant: Slide 13
So far, we've recruited about 750 people, mostly students at the University of Arizona, between 18 and 40 years of age. We don't want people as old as I am with gray hair or those who have dyed their hair during the past 3 months, which is kind of difficult on a college campus these days. Involving a lot of students has been quite helpful: We've gotten television coverage and all sorts of things for this project, so we haven't had a problem recruiting students. We pay them $20 for participating in the study. Brilliant: Slide 14
Because the ethnic distribution of our subjects underrepresents African Americans, we plan to recruit African-American subjects at other universities. I think we're going to be okay, at least as far as representation of the U.S. population. Brilliant: Slide 15
The subjects complete a questionnaire. They write down their hair color and talk about whether or not they're tanned or things like that, and we also collect data from their driver's licenses, such as hair and eye color. We collect a buccal cell sample, from which we extract DNA and determine the polymorphisms that I'll show you in a moment. We collect hair samples that are sent to our colleagues in Japan for chemical analysis of the last centimeter of growth. We also measure reflectance of skin pigmentation using a reflectometer. Brilliant: Slide 16
Dermatologists classify six types of skin: Type I is the lightest skin (i.e., skin that doesn't tan and always burns), and type VI is the darkest skin. But this classification is kind of subjective. Brilliant: Slide 17
Color is seen (or perceived) by us by the light that gets reflected. We use a reflectometer to measure color. This portable machine is one that's used in paper manufacturing, for example, to make sure that the paper is the right color and such. It can also be used to measure human pigmentation. We generally use the upper part of the arm. It's a place that typically does not get much sun and rarely presents trouble for measuring. Brilliant: Slides 18 and 19
An output of 61 is almost average for human pigmentation. Brilliant: Slide 20
Eye color is difficult to measure because eyes are not a uniform color. I always thought it was rather subjective. However, we've compared eye color using a chart that's available from companies that make artificial eyes. The chart is used to match the eye colors, and we can record fairly accurately what the eye colors are. Brilliant: Slides 21–23
Hair color also can be rather subjective. The hairs that we collect are subjected to chemical analysis.
Human pigmentation actually involves two different forms of melanin: yellow-red melanin and brown-black melanin. Most of the spectrum of human hair color is represented in these two forms, and we can measure them chemically. Brilliant: Slides 24–27
Looking at a coding polymorphism in the P gene or the OCA2 gene, we can compare various hair and eye colors. Brilliant: Slide 28
However, for this polymorphism, which is another coding polymorphism, we do have some significant data. Medium-brown hair has a relatively high chi-square value. However, the degrees of freedom of this particular analysis are quite high, such that this is just borderline significant. Hazel eyes, for example, have a very high chi-square value. It's a very significant bit of data, such that we can correlate the presence of this particular polymorphism with a high incidence of hazel eyes. Brilliant: Slide 29
The black hair/very dark eyes polymorphism is overrepresented and has a significant chi-square value. However, this particular polymorphism may reflect ethnicity of this population. This polymorphism is quite common among East Asians. The high correlation with hazel eyes, appears to be independent of ethnicity. Brilliant: Slide 30
What we're trying to do is to come up with a series of measurements of specific polymorphisms that will give us a high probability of predicting what an individual looks like just from their DNA sample. I suspect that given a specific DNA pattern/polymorphic pattern, we could come up with data with a 60-percent probability that an individual has hazel eyes, dark-brown hair, and a medium complexion.
I hope that this will be useful for the forensic community and that's why we're proceeding with this. Thank you very much for your attention.