Transcript Understanding Eye Color

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 color
comes 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 lightcolored 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
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, lightcolored 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.fc
gi?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:
Blue/gray
Females
617 (45.5)
Males
613 (47.7)
All
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:
Fair/pale
Females
547 (41.8)
Males
512 (40.8)
All
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:
Fair/pale
Females
547 (41.8)
Males
512 (40.8)
All
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_Re
sources/Gene_Expression_Resource/Transgen
omicWaveSystem
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
Intron 1:
A1a
A2b
No. Typed
LRTSc
Pc
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
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
Intron 2:
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 305Trp419Gln 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
1
Nucleotides
TGT
African
.074
East Asian
.121
European
.825
Twin
Collection
.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
305/419
RR
African
African
1.0
Pacific
Rim
.96
Native
AmericanHispanic
.89
10
WR
…
.04
.07
.06
.050
11
RQ
…
…
.04
.10
.077
Haplotype
Number
9
White
.84
Twin
Collection
.872
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.