Understanding Genetics of Human Eye Color

Human Eye Color

• In 1907, Charles and Gertrude Davenport outlined what is still commonly taught in schools today:– Brown eye color is always dominant to blue;– Two blue-eyed parents always produce a blue-eyed

child, never one with brown eyes.

• Unfortunately, as with many phenotypes, this simple model does not explain the complexities of real life.

• Eye color is inherited as a polygenic not as a monogenic trait.

Iris colors: blue, grey, green, yellow, hazel, light brown and dark brown

http://www.biosci.ohio-state.edu/~pfuerst/courses/eeobmg640/reading1eyecolor.pdf

Human eye colorcomes from melanocytes: cells which make the brown pigment melanin.

Melanocytes are cells making melanin and melanosomes are bodies in the cytoplasm storing melanin. In the eye, melanin is not made continuously like in skin and hair.

The peripupillary ring is shown on the right and isn’t understood genetically yet.

http://www.biosci.ohio-state.edu/~pfuerst/courses/eeobmg640/reading1eyecolor.pdf

Downside to Being Blonde:

• Individuals with lower melanin pigments in skin, hair, and eye color who are exposed to more environmental UV light are at higher risk for developing both melanoma and nonmelanoma skin cancer (NMSC)

Evolution of Human Skin Color Differences

• There are several hypotheses– Dark skin blocks out the sun and protects the body's folate

reserves. Low folate is correlated with spina bifida and other problems.

– Dark skin protects against cancer- but not usually until past age of reproduction.

– Fair skin produces adequate amounts of vitamin D during the long winter months.

– Women are generally 1-3% lighter than men because they have to balance their need for vitamin D which is aided by the sun against their need for folic acid which is destroyed by the sun. Women accept a higher folate loss as opposed to men in exchange for getting the D they need to reproduce.

http://magma.nationalgeographic.com/ngm/0211/feature2/online_extra.html

Predicted map of skin color based on UV light protection models

What is the full name of the OCA2 gene?

• The official name of this gene is “oculocutaneous albinism II (pink-eye dilution homolog, mouse).”

• OCA2 is the gene's official symbol. • The OCA2 gene is located on the long (q) arm of

chromosome 15 between positions 11.2 and 12.• More precisely, the OCA2 gene is located from

base pair 25,673,627 to base pair 26,018,060 on chromosome 15.

• http://ghr.nlm.nih.gov/gene=oca2

What is the normal function of the OCA2 gene?

• The OCA2 gene (formerly called the P gene) provides instructions for making a protein called the P protein.

• This protein is located in melanocytes, which are specialized cells that produce a pigment called melanin.

• Melanin is the substance that gives skin, hair, and eyes their color.

• Melanin is also found in the light-sensitive tissue at the back of the eye (the retina), where it plays a role in normal vision.

MC1R Gene is also Important

• The MC1R gene provides instructions for making a protein called the melanocortin 1 receptor.

• This receptor plays an important role in normal pigmentation. The receptor is primarily located on the surface of melanocytes, specialized cells that produce melanin.

• Melanin is the pigment that gives skin, hair, and eyes their color. Melanin is also found in the light-sensitive tissue at the back of the eye (the retina), where it plays a role in normal vision.

• http://ghr.nlm.nih.gov/gene=mc1r

Melanin

• Melanocytes make two forms of melanin, eumelanin and pheomelanin.

• The relative amounts of these two pigments help determine the color of a person's hair and skin.

• People who produce mostly eumelanin tend to have brown or black hair and dark skin that tans easily. Eumelanin also protects skin from damage caused by ultraviolet (UV) radiation in sunlight.

• People who produce mostly pheomelanin tend to have red or blond hair, freckles, and light-colored skin that tans poorly.

Biosynthesis of Eumelanin and Pheomelanin

Eumelanin is darker pigment in brown hair. Pheomelanin is lighter pigment in red hair

What MC1R Does

• The melanocortin 1 receptor controls which type of melanin is produced by melanocytes.

• When the receptor is activated, it triggers a series of chemical reactions inside melanocytes that stimulate these cells to make eumelanin. If the receptor is not activated or is blocked, melanocytes make pheomelanin instead of eumelanin.

What MC1R Does

• Polymorphisms in the MC1R gene are associated with normal differences in skin and hair color.

• Certain genetic variations () are most common in people with red hair, fair skin, freckles, and an increased sensitivity to sun exposure.

• These MC1R polymorphisms reduce the ability of the melanocortin 1 receptor to stimulate eumelanin production, causing melanocytes to make mostly pheomelanin.

Oculocutaneous Albinism

• Certain genetic changes in the MC1R gene modify the appearance of people with oculocutaneous albinism type 2. This form of albinism, which is caused by mutations in the OCA2 gene, is characterized by fair hair, light-colored eyes, creamy white skin, and vision problems.

• People with genetic changes in both OCA2 and MC1R genes have many of the usual features of oculocutaneous albinism type 2; but, they often have red hair instead of the usual yellow, blond, or light brown hair seen with this condition.

Ancient DNA with MC1R polymorphisms purified from the bones of two Neanderthals suggests that at least some of them had red

hair and pale skin

Carles Lalueza-Fox, Holger Römpler, Michael Hofreiter et al., A melanocortin 1 receptor allele suggests varying pigmentation among Neanderthals. Science, October 25, 2007

MC1R and OCA2 are Unlinked

• The MC1R gene is located on the long (q) arm of chromosome 16 at position 24.3.

• More precisely, the MC1R gene is located from base pair 88,512,526 to base pair 88,514,885 on chromosome 16.

• This is an example of pleiotropic interaction between genes and their alleles.

The Paper We Read

A Three–Single-Nucleotide Polymorphism Haplotype in Intron 1 of OCA2 Explains Most Human Eye-Color Variation

David L.  Duffy, Grant W.  Montgomery, Wei  Chen, Zhen Zhen  Zhao, Lien  Le, Michael R.  James, Nicholas K.  Hayward, Nicholas G.  Martin, and Richard A.  Sturm

• American Journal of Human Genetics. 2007 February; 80(2): 241–252.

• http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1785344

What is the normal function of the OCA2 gene?

• Although the exact function of the P protein is unknown, it is essential for normal pigmentation and is likely involved in the production of melanin.

• Within melanocytes, the P protein may transport molecules into and out of structures called melanosomes (where melanin is produced).

• Researchers believe that this protein may also help regulate the relative acidity (pH) of melanosomes.

• Tight control of pH is necessary for most biological reactions to proceed properly.

What is the normal function of the OCA2 gene?

• The distribution of melanin with the melanocytes of the uveal tract of the eye is the physical basis of eye color and brown irides have up to 70% higher concentrations than do those of other colors.

• Age-related changes in eye color do occur, but eye color becomes stable by age 6 years.

• Recently, eye color has been accepted as being a polygenic trait, with multiple genes contributing to the expressivity of eye color.

Subjects

• “Adolescent twins and their siblings were recruited for an investigation of genetic and environmental factors contributing to the development of pigmented nevi and were also phenotyped for pigment traits, including skin, hair, and eye color. The pigmentation characteristics of the twins were examined on up to three occasions, at ages 12, 14, and 16 years.”

• Subjects were 95% Northern European origin and from South Queensland in Australia.

Distribution of Pigmentation Phenotypes among Genotyped Study Participants

No. (%) of Subjects with Phenotype

Eye color: Females Males AllBlue/gray 617 (45.5) 613 (47.7) 1314 (46.1)

Green/hazel 405 (29.8) 333 (25.9) 789 (27.7)

Brown 335 (24.7) 340 (26.4) 749 (26.3)

Total 1,357 (100) 1,286 (100) 2,852 (100)

From: Duffy et al. Am J Hum Genet. 2007 February; 80(2): 241–252.

Distribution of Pigmentation Phenotypes among Genotyped Study Participants

No. (%) of Subjects with Phenotype Hair color:

Red/auburn 81 (6.0) 46 (3.6) 146 (5.1)

Fair/blond 210 (15.5) 158 (12.3) 377 (13.2)

Light brown 464 (34.2) 445 (34.6) 974 (34.2)

Dark brown 570 (42.0) 574 (44.7) 1234 (43.3)

Black 32 (2.4) 62 (4.8) 120 (4.2)

Total 1,357 (100) 1,285 (100) 2,851 (100)

From: Duffy et al. Am J Hum Genet. 2007 February; 80(2): 241–252.

Distribution of Pigmentation Phenotypes among Genotyped Study Participants

No. (%) of Subjects with Phenotype

Skin Color: Females Males AllFair/pale 547 (41.8) 512 (40.8) 1,099 (40.4)

Medium 630 (48.1) 580 (46.3) 1,295 (47.6)

Olive/dark 132 (10.1) 162 (12.9) 324 (11.9)

Total 1,309 (100) 1,254 (100) 2,718 (100)

From: Duffy et al. Am J Hum Genet. 2007 February; 80(2): 241–252.

Distribution of Pigmentation Phenotypes among Genotyped Study Participants

No. (%) of Subjects with Phenotype

Skin Color: Females Males AllFair/pale 547 (41.8) 512 (40.8) 1,099 (40.4)

Medium 630 (48.1) 580 (46.3) 1,295 (47.6)

Olive/dark 132 (10.1) 162 (12.9) 324 (11.9)

Total 1,309 (100) 1,254 (100) 2,718 (100)

From: Duffy et al. Am J Hum Genet. 2007 February; 80(2): 241–252.

DNA Sampling

• “We analyzed genomic DNA samples from 40 individuals: 9 with red/auburn hair, 10 each with fair/blond and light brown hair, and 11 with dark brown hair (representing 23 blue/gray, 9 green/hazel, and 8 brown eye colors). PCRs were conducted for each OCA2 coding-region exon (amplimers and size products for each OCA2 coding-region exon are available on request) that encompassed the six reported amino acids changing alleles Ala257Asp, Arg305Trp, Arg419Gln, Leu440Phe, His615Arg, and Ile722Thr.”

• “PCR products were denatured and analyzed on a Transgenomic Wave System (model 2100D), for mutation detection, by use of the Navigator Software package. Samples with a mismatch in the analyzed PCR fragment were sequenced from new PCR products through use of ABI BigDye Terminator version 3.1 chemistry and were separated on a capillary-based genetic analyzer (Applied Biosystems). Sequence chromatograms were analyzed using the Sequencher program (Gene Codes).”

Transgenomic Wave System

• Denaturing high performance liquid chromatography (DHLCP)

• Normal and test or mutant type alleles are amplified separately, mixed, heated and then cooled to form homoduplexes and heteroduplexes (if a mutation is present).

• http://www.roswellpark.org/Research/Shared_Resources/Gene_Expression_Resource/TransgenomicWaveSystem

Sequence Mutants to Confirm

OCA2 SNP Genotyping

• A combination of exonic and intronic SNPs and 40 SNPs were selected from the HapMap database as suitable for haplotype coverage of the OCA2 locus. They genotyped all their samples and did a linkage analysis with the traits they were measuring for eye color etc.

• This is the data summarized in Tables 2 and 3 of the paper. Subset of Table 3 is next.

Location and dbSNP Number A1a A2b No. Typed LRTSc Pc

Intron 1:

rs7495174 .94 T .06 C 3,066 274.81 1.02×10−61

rs6497268 .83 G .17 T 3,057 434.66 1.57×10−96

rs11855019 .87 T .13 C 3,023 239.76 4.45×10−54

rs4778137 .71 G .29 C 2,923 21.14 4.27×10−6

Intron 2:

rs7179994 .86 T .14 C 3,033 55.73 8.32×10−14

rs1448481 .91 A .09 G 3,030 4.09 .04

rs1597196 .82 C .18 A 3,067 74.09 7.46×10−18

rs1375164 .78 G .22 A 3,057 94.11 2.98×10−22

rs1448485 .86 C .14 A 3,030 82.75 9.31×10−20

rs7176759 1.00 G .00 C 3,066 6.05 .014

a Allele frequency associated with blue/gray eye color in this study.bAllele frequency associated with nonblue eye color in this study.cLikelihood-ratio test (MENDEL 6.01) for association between SNP and blue eye color.

Likelihood Ratio Tests

• You have two hypotheses H0 and H1 and you want to distinguish between them.

• For each hypothesis and the observed data assume there is a probability that the data observed would have arisen if Ho were true.

• Call that probability l 0 and an analogous probability l 1 that the data would have arisen if H1 were true.

• The ratio of l 1/l 0 is called the likelihood ratio (L) and it amounts to the odds that H1 indeed is correct as opposed to H0.

Advantages to the Likelihood Ratio Test

• It follows a natural probability model which leads naturally to the so called LOD score employed in pedigree analysis.

• The LOD score which is based on Log10 can easily be converted to a G score and vice versa.

• More complex tests of this type can be developed which allow the G score to be partitioned into different components or sources of variation much as can often be done with analysis of variance.

• http://staff.jccc.net/PDECELL/bio205/likelihood.html

Results

• “The human P-gene transcript encoded by the OCA2 locus is divided into 24 exons, covering >345 kb; 23 of these exons span the 836-aa coding region, with exon 1 representing exclusively a noncoding 5′ UTR. The translational initiation codon is located in exon 2, and exon 24 includes the termination codon plus the 3′ UTR. A possible alternative spliced region, previously referred to as “exon 19” and containing an in-frame stop codon, was neither included in the analysis nor used in our exon or P-protein numbering system.”

Results

• “At least 42 apparently nonpathogenic variant alleles of the OCA2 gene have been identified in the literature, 22 of which are exonic; of these, 6 result in amino acid changes (see the Albinism Database). Some of these polymorphisms have markedly different frequencies in different populations, which indicates the potential to explain differences in pigmentation phenotypes among ethnic groups.”

• “DNA analysis of 40 individuals in our study identified 10 coding-region SNPs and 10 flanking intronic-region SNPs that confirmed the presence and allele frequencies of several of the polymorphisms that had already been described in the literature for the white population.”

• “A total of 3,839 subjects were genotyped, with the final 58 SNPs used for statistical analyses”

Linkage Disequilibrium Heat Plot: Bright colors have higher LD (r2)

The surprise- noncoding regions were linked to Blue eye color.

Coding Region SNPs weren’t linked to Blue Eye Color

• Of the two common OCA2 coding-region changing SNPs identified elsewhere as modifying the association of green/hazel or brown eye color, the Arg305Trp rs1800401 change did not show significant association in this expanded study (P=.84), although the Arg419Gln rs1800407 polymorphism was strongly associated with nonblue eye colors (P=4.96×10-10).

Some SNPs were good at predicting Eye Color in Regression

Results

• “Given the facts that the three OCA2 intron 1 SNPs were most strongly associated with blue/nonblue eye color and were grouped together into a single haplotype block, the frequencies of the eight possible haplotype combinations present in the twin population were deduced using the program MENDEL.”

Results

• One major haplotype TGT (haplotype 1 in table 4) was predicted, representing 78.4% of alleles, with four minor haplotypes as TTT (haplotype 2 in table 4) at 7.9%, CTC (haplotype 8 in table 4) at 6.4%, with TGC (haplotype 3 in table 4) and TTC (haplotype 4 in table 4) each at 3.4%. The three other haplotypes—CGT (haplotype 5 in table 4), CTT (haplotype 6 in table 4), and CGC (haplotype 7 in table 4)—were considered rare in the twin collection, all falling well below 1%.

Results

• The coding-region polymorphisms were predicted to occur as 305Arg-419Arg (haplotype 9 in table 4) at 87.2% of alleles, 305Trp-419Arg (haplotype 10 in table 4) at 5%, and 305Arg-419Gln (11) at 7.7%; the 305Trp-419Gln haplotype was not found in our sample.

• These data are consistent with the TGT haplotype 1 acting as a highly penetrant recessive blue-eye-color allele. The TTC haplotype 4 is also associated with blue eyes, and the remaining haplotypes were dominant green- or brown-eye-color alleles.

• In other words, they could sequence just these SNPs and get a good fit with their eye color predictions.

Results

• “For hair and skin color, the 1/1 diplotype had the highest frequency (40.2%) in subjects with light brown hair and was also enriched (92.6%) for fair/pale and medium skin types (table 5).

• Each of the diplotypes 1/2 to 1/5 had higher frequencies in subjects with dark brown hair and medium skin types, again consistent with TGT haplotype 1 acting as a recessive modifier associated with lighter pigmentary phenotypes.”

Eye Color and Freckles

Intron 1 Haplotype Frequencies in HapMap

Haplotype Number Nucleotides African East Asian European

Twin Collection

1 TGT .074 .121 .825 .784

2 TTT .173 .102 .075 .079

3 TGC .285 .018 .042 .034

4 TTC .313 .051 … .034

5 CGT .020 .014 … .003

6 CTT … … … .001

7 CGC … … … .000

8 CTC .135 .690 .042 .064

Coding Region Haplotype Frequencies in HapMap

Haplotype Number 305/419

African African

Pacific Rim

Native American-Hispanic White

Twin Collection

9 RR 1.0 .96 .89 .84 .872

10 WR … .04 .07 .06 .050

11 RQ … … .04 .10 .077

Discussion

• “The recessive action of the TGT haplotype is likely to explain effects of loss or gain of alleles at this locus on the pigmentation changes associated with Prader-Willi and Angelman syndromes noted earlier.

• Nevertheless, the modeling of eye-color inheritance by use of a single locus determining eye color is insufficient to explain the range of eye color phenotypes, and the description of other loci influencing the appearance of green eye color (GEY/EYCL1 [MIM 227240]) has been noted.”

Why the Intron SNPs?

• The position of the three major diagnostic SNPs for eye color located at the 5′ end of the OCA2 gene near proximal regulatory regions immediately suggests that transcriptional regulation may be important in the action of the TGT haplotype 1. It is not apparent that the three SNPs in themselves play a direct role in expression of the OCA2 transcript levels…. Thus, although unlikely to be responsible for regulation of the OCA2 gene in themselves, they may be in tight linkage with regulatory elements that are affected by other changes.

Unstable RNA?

• “Another human locus that has been tested for association with pigmentary traits is the agouti signaling protein gene (ASIP). A g8818A/G SNP in the 3′ UTR of this gene has been reported to be associated with brown eye color and dark hair and is thought to destabilize the ASIP mRNA, which leads to premature degradation of the transcript.

• Quantification of the ASIP transcripts in melanocytes genotyped for this SNP did show decreases in levels of the ASIP mRNA, which suggests that expression of human pigmentary genes at the level of transcription occurs in genes other than OCA2.”

Conclusion

• Polymorphism of gene-regulatory regions is likely to be one of the major contributors to phenotypic variation between and within human populations.

• Our data demonstrate that variation in the OCA2 gene 5′ region explains most human eye-color variation, but the molecular basis of the association with the blue/brown eye color phenotype remains to be elucidated.