The Genetic Architecture of Iris Colour and

Surface Feature Variation in Populations of

Diverse Ancestry

By

Melissa Edwards

A thesis submitted in conformity with the requirements

for the degree of Doctor of Philosophy
Department of Anthropology
University of Toronto

© Copyright by Melissa Edwards 2016

 ProQuest Number: 10138105

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 The Genetic Architecture of Iris Colour and Surface Feature
Variation in Populations of Diverse Ancestry
Melissa Edwards

Doctor of Philosophy

Department of Anthropology
University of Toronto

2016

Abstract

Research focused on iris colour and surface features has begun to play an increasingly

important role in the fields of anthropology, forensics and public health. Although both eye

colour and, to a much lesser extent, iris features have been studied in populations of European

ancestry, very few research groups have attempted to investigate these traits in regions outside of

Europe. This may be partly due to difficulties associated with obtaining a quantitative

measurement of eye colour and a lack of standardized methodology. For my doctoral thesis, I

developed a novel method of characterizing iris colour and feature variation in diverse

populations. I then applied this method to a sample of individuals of East Asian, European and

South Asian ancestry. I was primarily interested in looking at the phenotypic distribution and

genetic basis of these traits across all three sample sets. I found that: 1/ Quantitative methods

provide an ideal means of characterizing iris colour in populations of diverse ancestry; 2/ The

phenotypic distributions of iris colour and surface features are different in East Asian, European

and South Asian populations; 3/ HERC2 rs12913832 controls iris pigmentation variation in both

European and South Asian populations. However, it affects eye colour differently in both

regions; 4/ The variants responsible for controlling iris pigmentation in Europe are different from

ii
those responsible for controlling iris pigmentation in East Asia; 5/ SEMA3A rs10235789 is

significantly associated with Fuchs’ crypts in East Asian, European and South Asian populations.

Ultimately, my thesis is the first research work to compare iris pigmentation and structure

variation in populations of diverse ancestry. The methodology that I have developed will provide

other researchers with a new framework that they can use to approach the study of iris colour and

feature diversity.

iii
 Table of Contents
Abstract .......................................................................................................................................... ii
Table of Contents ......................................................................................................................... iv
List of Tables ............................................................................................................................... vii
List of Figures ............................................................................................................................. viii
Forward ........................................................................................................................................ ix
Chapter 1: .....................................................................................................................................1
Introduction
1.1 The Evolution of Global Iris Colour Variation ......................................................................2
1.2 Anatomy of the Human Iris ....................................................................................................3
1.2.1 Iris Structure ...................................................................................................................3
1.2.2 Iris Pigmentation ............................................................................................................8
1.3 Factors that Influence Iris Pigmentation Variation ................................................................9
1.4 Genetic Basis of Iris Pigmentation .......................................................................................11
1.5 Genetic Basis of Iris Surface Features .................................................................................14
1.6 Limitations in our Understanding of Iris Colour and Texture Variation .............................16
1.6.1 Characterizing Iris Colour and Surface Feature Variation ...........................................16
1.6.2 Characterizing Central Heterochromia .........................................................................17
1.6.3 Population Variation .....................................................................................................18
1.7 Rationale and Study Goals ...................................................................................................20
1.8 References ............................................................................................................................22
Chapter 2: ...................................................................................................................................28
Quantitative Measures of Iris Colour Using High Resolution Photographs
2.1 Abstract ................................................................................................................................29
2.2 Introduction ..........................................................................................................................30
2.3 Materials and Methods .........................................................................................................31
2.3.1 Recruitment ...................................................................................................................31
2.3.2 Genotyping ....................................................................................................................32
2.3.3 Acquisition and Processing of Iris Photographs ............................................................32
2.3.4 Acquisition of Colour Measurements ............................................................................33
2.3.5 Statistical Analysis ........................................................................................................34
2.4 Results ..................................................................................................................................34

iv
 2.5 Discussion ............................................................................................................................36
2.6 Tables ...................................................................................................................................42
2.7 Figures ..................................................................................................................................44
2.8 References ............................................................................................................................47
Chapter 3: ...................................................................................................................................49
Iris Pigmentation as a Quantitative Trait: Variation in Populations of European, East Asian and
South Asian Ancestry and Association with Candidate Gene Polymorphisms
3.1 Abstract ................................................................................................................................50
3.2 Introduction ..........................................................................................................................51
3.3 Materials and Methods .........................................................................................................54
3.3.1 Sample Collection .........................................................................................................54
3.3.2 Acquisition of Photographs ..........................................................................................54
3.3.3 Acquisition of Iris Colour .............................................................................................55
3.3.4 Marker Selection and Genotyping ................................................................................57
3.3.5 Statistical Analysis ........................................................................................................57
3.4 Results ..................................................................................................................................59
3.5 Discussion ............................................................................................................................62
3.5.1 Iris Colour in European Populations .............................................................................63
3.5.2 Iris Colour in South Asian Populations ........................................................................65
3.5.3 Iris Colour in East Asian Populations ..........................................................................67
3.5.4 Central Heterochromia ..................................................................................................69
3.5.5 A Global View of the Genetic Basis of Iris Pigmentation ............................................70
3.6 Tables ...................................................................................................................................73
3.7 Figures ..................................................................................................................................86
3.8 References ............................................................................................................................89
Chapter 4: ...................................................................................................................................94
Analysis of Iris Surface Features in Populations of Diverse Ancestry
4.1 Abstract ................................................................................................................................95
4.2 Introduction ..........................................................................................................................96
4.3 Materials and Methods .........................................................................................................98
4.3.1 Participant Recruitment .................................................................................................98
4.3.2 Genotyping ..................................................................................................................98
4.3.3 Acquisition and Processing of Iris Photographs ...........................................................99

v
 4.3.4 Iris Analysis ...................................................................................................................99
4.3.5 Characterization of Iris Structure ..............................................................................101
4.3.6 Statistical Analysis .....................................................................................................102
4.4 Results ................................................................................................................................103
4.5 Discussion ..........................................................................................................................106
4.5.1 Fuchs’ Crypts...............................................................................................................106
4.5.2 Pigment Spots .............................................................................................................108
4.5.3 Contraction Furrows ...................................................................................................109
4.5.4 Wolfflin Nodules .........................................................................................................111
4.5.5 Conjunctival Melanosis ...............................................................................................112
4.5.6 Correlations of Iris Features ........................................................................................113
4.5.7 Future Research ...........................................................................................................113
4.6 Tables .................................................................................................................................116
4.7 Figures ................................................................................................................................120
4.8 References ..........................................................................................................................129
Chapter 5: .................................................................................................................................132
Concluding Remarks
5.1 Introduction ........................................................................................................................133
5.2 Summary of Findings .........................................................................................................133
5.2.1 Quantifying Iris Pigmentation .....................................................................................133
5.2.2 Characterizing Iris Surface Features ...........................................................................134
5.2.3 Phenotypic Distribution of Iris Colour and Central Heterochromia ...........................135
5.2.4 Phenotypic Distribution of Iris Surface Features ........................................................136
5.2.5 Genetic Basis of Iris Colour ........................................................................................136
5.2.6 Genetic Basis of Iris Surface Features.........................................................................138
5.3 Research Limitations ..........................................................................................................139
5.4 Conclusion ..........................................................................................................................140
5.5 References ..........................................................................................................................142
Copyright Acknowledgements ................................................................................................144

vi
 List of Tables

Table 2-1 General Descriptive Statistics........................................................................................42

Table 2-2 General Linear Model Analysis Results ........................................................................43
Table 3-1 Results of the Inter- and Intra-Rater Reliability Analysis .............................................73
Table 3-2 Summary Statistics for All 1448 Participants Included in the Study ............................74
Table 3-3 Results of the Meta-Analysis in the European Sample (A) Before and (B) After
Conditioning for the Effects of HERC2 rs12913832 .....................................................................75
Table 3-4 Results of the Meta-Analysis in the South Asian Sample (A) Before and (B) After
Conditioning for the Effects of HERC2 rs12913832 .....................................................................79
Table 3-5 Results of the Meta-Analysis in the East Asian Sample (A) Before and (B) After
Conditioning for the Effects of OCA2 rs1800414 ........................................................................83
Table 3-6 Average, Minimum and Maximum ∆E Stratified by HERC2 rs12913832 genotype ...85
Table 4-1 General Descriptive Statistics for the East Asian, European and South Asian Irises .116
Table 4-2 Results of the Genetic Ordinal Regression ..................................................................117
Table 4-3 Iris Surface Feature Frequencies .................................................................................118
Table 4-4 Distribution of Iris Surface Features ...........................................................................119

vii
 List of Figures

Figure 1-1 Surface Features of the Humans Iris ..............................................................................5

Figure 1-2 Cross-Section of the Human Iris ....................................................................................6
Figure 2-1 Colour Extraction Methodology and Iris Colour Coordinates .....................................44
Figure 2-2 Distribution of Irises in CIELAB Colour Space ..........................................................45
Figure 2-3 Visualization of Central Heterochromia ......................................................................46
Figure 3-1 Distribution of Irises Across the b* and a* (A), a* and L* (B), and b* and L* (C),
Coordinates of CIE 1976 L*a*b* (CIELAB) Colour Space for the Second Camera Body ..........86
Figure 3-2 Sample Iris Wedges with Corresponding Colour Space Values ..................................88
Figure 4-1 Five Surface Features Commonly Found in the Human Iris .....................................120
Figure 4-2 Boundaries of the Iris .................................................................................................121
Figure 4-3 Iris Quadrants .............................................................................................................122
Figure 4-4 Pigment Spot Categories ............................................................................................123
Figure 4-5 Contraction Furrow Categories ..................................................................................124
Figure 4-6 Wolfflin Nodule Categories .......................................................................................125
Figure 4-7 Crypt Categories.........................................................................................................126
Figure 4-8 Self-Described Iris Colour, as Defined by the Fitzpatrick Phototype Scale ..............127
Figure 4-9 Conjunctival Melanosis Categories............................................................................128

viii
 Forward
This thesis consists of five chapters that explore the global distribution and genetic basis of iris
colour and surface features in populations of East Asian, European and South Asian ancestry.

Chapter 1 provides a general introduction to the structure and colour of the human eye. It
provides important information about the evolution of iris colour, the anatomy of the iris, the
process of melanogenesis and the genetic basis of human eye colour and surface features. This
chapter also addresses some of the limitations that are currently present in this field.

Chapter 2 introduces a quantitative method for measuring iris colour variation that was
developed using a pilot sample of 205 volunteers of East Asian, European and South Asian
ancestry. It also looks at the association between HERC2 rs12913832 and quantitative measures
of eye colour in the European and South Asian groups. This work has been previously published
in The American Journal of Physical Anthropology (Edwards et al., 2012)

Chapter 3 improves the quantitative method for measuring iris colour that was introduced in
Chapter 2. It also looks at the association between 14 putative pigmentation markers and eye
colour variation in 1,465 individuals of East Asian, European and South Asian descent. This
work has been previously published in Pigment Cell and Melanoma Research (Edwards et al,
2015)

Chapter 4 looks at the global distribution of four surface features (Fuchs’ crypts, contraction
furrows, Wolfflin nodules and pigment spots) in the human eye. It also investigates the genetic
basis of these traits. This work has been previously published in Royal Society Open Science
(Edwards et al., 2016)

Lastly, Chapter 5 summarizes the general conclusions of the preceding articles and highlights
some persisting limitations in this field.

ix
Chapter 1:
Introduction

1
 2

1.1 The Evolution of Global Iris Colour Variation

The iris is a complex structure that displays extensive colour variation in human
populations. Some eyes even show heterochromia, where multiple colours are found in the same
(central or sectoral heterochromia) or different (binocular heterochromia) eyes. The full spectrum
of iris colour variation is largely restricted to European populations, although moderate diversity
can also be observed in North Africa, the Middle East and parts of South Asia. Throughout the
rest of the world, however, iris colour is primarily limited to varying shades of brown. However,
even these browns can show extensive diversity and range from light reddish-yellows to dark
brownish blacks.
The evolution of light eye colour does not appear to be a simple by-product of genetic
drift or other random genetic forces. Rather, this trait appears to have been subjected to strong
selection pressures. This is supported by the exceptionally high-frequency of blue eyes in some
European populations, and the fact that the genetic marker (HERC2 rs12913832) which controls
the difference between blue and brown eye colour shows strong signals of positive selection in
both modern and ancient sample sets (Eiberg et al., 2008; Mathieson et al., 2015; Nakagome et
al., 2015; Wilde et al., 2014). It has thus far been very difficult to date the origin of blue eye-
colour. It was initially believed that blue eyes first evolved 6,000-10,000 years ago when humans
moved into the Baltic Sea region of Europe (Eiberg et al., 2008). However, this estimate has
since lengthened considerably. One recent study suggested that it is unlikely that selection on
HERC2 rs12913832 began any more recently than the spread of agriculture 12,800 years ago
(Nakagome et al., 2015). A second study, which looked at 230 ancient Eurasian skeletal samples,
found that the blue eye colour variant had a frequency of 100% in European Hunter Gatherers
dating from 7,500-8,200 years ago (Mathieson et al., 2015). In addition, the distribution HERC2
rs12913832 within other skeletal groups mirrored the regional distribution of this marker today.
These findings suggest that selection on blue eyes occurred prior to this time period.
The actual selective forces that drove the evolution of light eye colour in European
populations are still poorly understood. Even within Europe, eye colour shows a unique
distribution where the frequency of blue eyes increases with latitude (Donnelly et al., 2011).
Several research groups have suggested that blue eye colour may have initially evolved as a
product of frequency-dependent sexual selection. When humans penetrated the steppe-tundra,
they encountered harsh climates (Frost, 2006, 2014). Food was primarily acquired by hunting
 3

over long distances and this in turn led to a high male mortality rate and a low-operational sex
ratio. It is possible that light eye colour provided females with a rare colour advantage that
helped them procure mates. Once this trait became established in European populations, it
continued to be favored as an indicator of group membership (Wilde et al., 2014). It may have
also allowed males to better ascertain paternity confidence. This hypothesis is supported by an
excess of HERC2 rs12913832 homozygotes in both modern and ancient populations (Wilde et
al., 2014). Alternatively, iris colour may not have been selected for at all. Rather, light eyes may
have hitchhiked along with some other trait that is controlled by the same genes (Donnelly et al.,
2011; Wilde et al., 2014). HERC2 rs12913832 is a pleiotropic marker that has also been
associated with skin pigmentation, hair colour and tanning ability (Cook et al., 2008; Han et al.,
2008; Nan et al., 2009a). In addition, it has been found to interact with a number of other genes
that play a role in the pigmentation pathway (Branicki et al., 2009; Pospiech et al., 2011). Thus,
iris colour variation may simply be the product of selection on other traits, such as skin and/or
hair colour. Some researchers have even pointed towards behavioural selection pressures. Most
notably, Sturm and Larsson (2009) suggested that blue eyes may help mitigate some of the
effects of seasonal affective disorder. It is unlikely that any one hypothesis can fully explain the
global diversity that is observed in eye colour today. Rather, the evolution of iris pigmentation
variation is likely the product of many complex interacting environmental factors (Mathieson et
al., 2015; Wilde et al., 2014).

1.2 Anatomy of the Human Iris

1.2.1 Iris Structure
The embryogenesis of the iris is a long and complicated process (see review in Oyster,
1999). The optical vessels first appear during the fourth embryonic week. By the sixth week, a
structure known as the pupillary membrane grows in front of the lens. The different components
and layers of the iris then begin to differentiate underneath this membrane. At around eight
months, the centre of the pupillary membrane is absorbed, forming the pupil. The tissue that
remains after resorption resembles a small and jagged circular ring on the surface of the iris. This
ring, which is known as the collarette, divides the iris into two different regions: the pupillary
 4

zone and the ciliary zone (Figure 1-1). The white region surrounding the iris is known as the
sclera.
The resulting iris is comprised of five distinct layers (Eagle, 1988; Oyster, 1999) [Figure
1-2]. The posterior-most layer is called the iris pigment epithelium (IPE). This layer is primarily
protective and blocks the retina from absorbing excess light (Wilkerson et al., 1996). Attached to
the IPE are two muscle layers known as the sphincter muscle and the dilator muscle (Eagle,
1988). These muscles assist in the contraction and dilation of the pupil. Lastly, the two anterior-
most layers are the stromal layer and the anterior border layer (Eagle, 1988). Both of these layers
contain an assortment of melanocytes, collagen fibres and fibroblasts. However, the anterior
border layer is much more densely packed then the stromal layer (Eagle, 1988). The majority of
iris colour and structure variation can be attributed to differences in the colour and composition
of these two layers (Baranoski and Lam, 2007; Mackey et al., 2011; Rennie, 2012; Sturm and
Larsson, 2009). The texture of the iris surface also shows substantial variation. This is largely
due to the presence of several surface features that can affect the organization and overall
stability of the eye. These features include Fuchs’ crypts, contraction furrows, Wolfflin nodules
and pigment spots (Figure 1-1).
Fuchs’ crypts are diamond-shaped lacunae in the anterior border and anterior stromal
layers of the iris (Purtscher, 1965). They first appear at around 6 months of age and resemble
deep pits covering the iris surface (Larsson and Pedersen, 2004). Although there are several
different types of lacunae, Fuchs’ crypts are notable because they vary widely in size, are found
in the ciliary region and most often originate at the collarette (Larsson and Pedersen, 2004;
Purtscher, 1965; Sidhartha et al., 2014a). The developmental factors that control the formation of
Fuchs’ crypts are not well-understood. However, it has been suggested that they may be a
phylogenetic defect (Purtscher, 1965). Due to the decreasing importance of the anterior layers of
the iris over the course of evolutionary history, these layers may have become rudimentary
tissues in the anthropoid apes. As a result, they are more prone to acquiring Fuchs’ crypts and
other forms of hypoplasia. Interestingly, the extent of Fuchs’ crypts in the iris appears to be
correlated with age (Larsson and Pedersen, 2004). This suggests that secondary crypts may also
form due to the pushing and pulling of the pupil over time. However, both primary and
secondary crypts are ultimately a product of the overall stability and strength of the anterior
layers (Purtscher, 1965).
 5

Figure 1-1 Surface Features of the Human Iris

This figure shows the surface of a typical human iris. The iris can be divided into two zones that are
separated by the collarette (shown in white). The pupillary zone is the region adjacent to the pupil and the
ciliary zone encompasses the rest of the iris. There are a number of textural elements that can be observed
on the iris surface. Fuchs’ crypts are diamond-shaped lacunae. Wolfflin nodules are small bundles of
atrophied collagen. Contraction furrows are discontinuous rings that extend around the outer edge of the
ciliary zone. Pigment spots are small accumulations of melanin.
 6

Figure 1-2 Cross-Section of the Human Iris

The human iris is composed of five distinct layers. The posterior-most layer is known as the iris pigment
epithelium (IPE). This is a melanin-dense layer that does not vary greatly between individuals. Attached
to the IPE are two muscle layers, known as the sphincter muscle and the dilator muscle. The anterior-most
layers are the stromal layer and the anterior border layer. The majority of variation in iris pigmentation
and surface texture between individuals can be attributed to differences in these two layers.
 7

Contraction furrows are thin rings that extend around the outer border of the iris. They
resemble ‘wrinkles’ on the surface of the eye and are formed by the contraction and dilation of
the pupil (Eagle, 1988; Larsson et al., 2011). Although contraction furrows can extend around
the entire iris, each individual furrow is rarely longer than a quadrant in length (Eagle, 1988).
Consequently, furrows often look like a series of discontinuous and staggered lines. Very little is
known about how contraction furrows affect the overall stability and organization of the iris.
However, it is interesting to note that more extensive contraction furrows have been associated
with a thicker peripheral iris (Sidhartha et al., 2014a).
Wolfflin nodules are round, whitish-yellow collagen deposits that are distributed along
the peripheral border of the iris (Donaldson, 1961; Sturm and Larsson, 2009; Williams, 1981).
They are formed from the remnants of atrophied collagen fibres in the anterior border and
stromal layers. Wolfflin nodules are strongly associated with iris colour. This is exemplified
particularly well in one study, which found that Wolfflin nodules had an incidence of 35% in
blue-eyed individuals and only 13% in brown-eyed individuals (Donaldson, 1961). Although it
was initially believed that Wolfflin nodules are not present in darker irises, they are actually just
obscured by pigment in the anterior border layer (Falls, 1970). Wolfflin nodules have received
much attention over the years due to their apparent similarity to an iris structure known as
Brushfield spots. Brushfield spots are found in over 80% of children who have been diagnosed
with Down’s syndrome (Donaldson, 1961; Falls, 1970; Kim et al., 2002; Williams, 1981). They
closely resemble Wolfflin nodules in appearance. However, they tend to be slightly smaller and
more variable in shape. In addition, whereas Wolfflin nodules are located near the periphery of
the iris, Brushfield spots are distributed around the mid-zone area.
Many irises show small, discrete areas of brown pigmentation on the iris surface. These
areas are known as pigment spots. There are two different types of pigment spots that are
commonly found in human eyes: nevi and freckles (Larsson et al., 2011; Rennie, 2012; Sturm
and Larsson, 2009). Nevi have a prevalence of 4-6% in individuals of European ancestry
(Harbour et al., 2004; Schwab et al., 2015). They are nodular in shape and distort the underlying
stromal layer. In contrast, freckles have a prevalence of 48-70% in European populations and
have no effect on the stromal architecture (Harbour et al., 2004; Reese, 1944). Both types of
pigment spots first appear around the age of six, and gradually increase in frequency over time
(Larsson and Pedersen, 2004). Although nevi and freckles affect the stromal layer of the iris in
 8

different ways, they are very difficult to distinguish topographically. Consequently, throughout
this work I will use the term ‘pigment spot’ to refer to both nevi and freckles. Interestingly,
despite their structural differences, nevi and freckles appear to have a similar physiological
origin (Harbour et al., 2004). This is because individuals with nevi are also much more likely to
have freckles.

1.2.2 Iris Pigmentation

Eyes become pigmented due to the presence of a polymer known as melanin. In humans,
there are two types of melanin that contribute to normal colour variation: eumelanin and
pheomelanin. Eumelanin is a dark brownish-black pigment that is found in high quantities in
dark skin, hair and eyes (Hu et al., 2009; Prota et al., 1998; Wakamatsu et al., 2008). In contrast,
pheomelanin is a reddish-yellow pigment that is found in higher ratios in light skin, hair and
eyes. Apart from their colour, there are a number of biochemical properties that differentiate
eumelanin and pheomelanin (Menon et al., 1987; Samokhvalov et al., 2005). Eumelanin is
photoprotective and an antioxidant. In contrast, pheomelanin is photoreactive and oxidative. This
means that eumelanin is more protective against the damaging effects of ultraviolet radiation.
Melanin is synthesized in specialized organelles known as melanosomes, which are
located in cells called melanocytes (Eagle, 1988). Melanocytes and melanosomes are both
distributed throughout the three non-muscle layers of the iris. However, they take a different
form in each of the layers. Melanosomes in the iris pigment epithelium are large, spherical and
made up primarily of eumelanin (Imesch et al., 1996; Peles et al., 2009; Prota et al., 1998).
Interestingly, the amount of melanin in the iris pigment epithelium appears to show little
variation between individuals, irrespective of eye colour (Imesch et al., 1996). In contrast, the
melanosomes in the anterior and stromal layers are much smaller and ovoid-shaped (Eagle, 1988;
Peles et al., 2009).
The synthesis of pheomelanin and eumelanin both begin with the hydroxylation of L-
tyrosine to L-dopa, and the oxidation of L-dopa to L-dopaquinone (reviewed in Sturm and
Frudakis, 2004). These steps are catalyzed by the enzyme tyrosinase (TYR). After these initial
steps, L-dopaquinone can either be converted into cyclodopa (in the case of eumelanin
production) or cysteinyldopa (in the case of pheomelanin production) depending on which of two
divergent molecular pathways is activated. A membrane bound receptor, known as MC1R, is the
 9

primary control switch between pheomelanin and eumelanin synthesis. When MC1R binds to
agonist α-MSH (the alpha-melanocyte stimulating hormone), cAMP levels inside the
melanosome increase and eumelanin production is stimulated. If MC1R binds to an antagonist
known as the agouti signalling protein (ASIP), or if α-MSH is unavailable, pheomelanin
synthesis begins. It has been suggested that pheomelanin is created preferentially over eumelanin
when the correct compounds are available (Peles et al., 2009). Consequently, most melanin takes
the form of a bipolymer with an eumelanic coat covering a pheomelanic core.
Apart from the proteins and genes that are directly involved in the melanogenesis
pathway there are many other genes and gene-gene interactions that play a role in the synthesis
of melanin (Parra, 2007; Sturm and Frudakis, 2004). A large number of membrane transporters
(i.e. OCA2, MATP/SLC25A4, and SLC24A4) are required to maintain appropriate ionic
conditions and transport substrates into the melanosomes. TYRP1 is needed to stabilize the TYR
protein and maintain melanosome structure and transcription factors (i.e. IRF4, MITF) regulate
the expression of the different components in the pathway.

1.3 Factors that Influence Iris Pigmentation Variation

The primary factor controlling iris pigmentation diversity is the amount and type of
melanin in the iris. Multiple studies have found that darker coloured eyes have much higher
concentrations of melanin than lighter coloured eyes (Hu et al., 2009; Prota et al., 1998; Wielgus
and Sarna, 2005). The eumelanin to pheomelanin ratio also plays a role. Most eyes appear to be
primarily eumelanic in nature and contain only trace amounts of pheomelanin. In fact,
pheomelanin shows roughly similar concentrations in both blue and brown eyes (Wielgus and
Sarna, 2005). Green eyes, however, are unique in that they contain higher concentrations of
pheomelanin and very low concentrations of eumelanin (Prota et al., 1998).
Although much of iris pigmentation can be explained by the melanin composition of the
iris, there are other physiological, biochemical and demographic processes that must also be
considered. This is particularly noticeable when looking at intermediate irises, which show
melanin concentrations that do not appear to correlate very strongly with colour (Prota et al.,
1998; Wielgus and Sarna, 2005). Other contributing factors may include the structure of the
 10

anterior border and stromal layers, the distribution of melanosomes in the iris, the presence of
iris surface features, and life stage.
Interestingly, light blue eyes may contain as much as 40% more melanin than dark blue
eyes (Wielgus and Sarna, 2005). There are several explanations for this finding. Blue eye colour
is primarily the product of light entering the melanin-poor stromal layer of the eye and bouncing
against collagen fibres (Mackey et al., 2011; Rennie, 2012; Sturm and Larsson, 2009). Thus, the
thickness of the stroma, as well as the ordering of the collagen fibres, may influence the intensity
of blue that is reflected back. The distribution of melanosomes across the anterior layer of the iris
may also play some role (Baranoski and Lam, 2007). Computer simulations have found that
brown eyes tend to be darker when most of the melanin is present in the anterior border layer (as
opposed to the stromal layer). In contrast, blue eyes tend to be darker when most of the melanin
is present in the stromal layer. Thus, it is very important to consider the overall structure of the
iris when investigating eye colour variation.
The presence or absence of surface features also influences iris pigmentation diversity.
Many individuals show substantial colour variation within the eye. The most common form of
this is central heterochromia, where the pupillary zone is different in colour than the ciliary zone
(Bond, 1913; Gladstone, 1969; Rennie, 2012). Central heterochromia can greatly influence
perceived eye colour. When I was collecting data for this thesis, a number of participants who
believed they had green eyes actually had blue eyes with a brown ring surrounding the pupil. The
genetic and physiological factors that control central heterochromia are still largely unknown.
However, this condition may result from differences in the thickness of the ciliary and pupillary
zones (Imesch et al., 1996). Alternatively, it may be controlled by genetic variants that determine
where pigment is placed in the iris (Larsson et al., 2011). Although central heterochromia may be
the most visible iris surface feature, the presence of Fuchs’ crypts, pigment spots and the position
of the collarette have also been found to influence perceived eye colour (Mackey et al., 2011).
Lastly, iris colour has been tentatively associated with demographic factors, like age and
sex. A study that followed monozygotic and dizygotic twins from childhood to adulthood noted
that 17% of twins showed a change in iris colour with age (Bito et al., 1997). Before the age of
six, eye colour was most likely to darken. After the age of six, however, dark eyes tended to
become lighter and light eyes tended to become darker. These changes appear to be genetic, as
the eye colour of monozygotic twins was more likely to change in the same direction.
 11

Interestingly, eye colour changes are also seen in older adults (Bito et al., 1997; Taylor, 1967).
However, in this age group, eyes tends to become lighter. This may be the product of age-related
melanin degeneration. There is some evidence that sex may also play a role in iris colour
variation. Two recent research studies using Italian and Spanish sample sets found that males
were more likely to have blue eyes and females were more likely to have brown/intermediate
eyes (Martinez-Cadenas et al., 2013; Pietroni et al., 2014). However, this finding was not
replicated in Danish or Swedish samples (Pietroni et al., 2014). Consequently, the association
between eye colour and sex may be largely restricted to Mediterranean populations.

1.4 Genetic Basis of Iris Pigmentation

Human eye colour has a very high (~98%) heritability (Larsson and Pedersen, 2004).
This means that most of the diversity observed in this trait can be explained by variation in the
human genome. At present, a large number of polymorphisms have been identified in European
populations that are believed to play some role in determining eye colour.
Remarkably, the vast majority of iris pigmentation diversity has been attributed to a
single locus known as rs12913832 (Eiberg et al., 2008; Kayser et al., 2008; Sturm et al., 2008).
This marker, which is located in intron 86 of the gene HERC2, is believed to be responsible for
determining the difference between brown and blue eyes. This variant can carry either an
adenine (A) or guanine (G) allele. The ‘A’ allele is present at very high frequencies in
populations of non-European ancestry, and has been associated with brown iris colour. In
contrast, the ‘G’ allele is found at very high frequencies in European populations and has been
associated with blue iris colour. It has traditionally been believed that this marker operates in a
dominant/recessive manner; individuals who are homozygous for the ‘G’ allele have blue eyes,
and individuals who are heterozygous or homozygous for the ‘A’ allele have brown eyes. One
research group found that individuals who had the genotype G/G only had a 1% probability of
having brown eyes (Sturm et al., 2008). Likewise, individuals who had the genotype A/A had an
80% probability of having brown eyes. A similar study found that 98.3% of individuals with blue
eyes were homozygous for the derived allele (Mengel-From et al., 2010).
HERC2 rs12913832 is located in a highly conserved region of the genome that contains
binding sites for many important regulatory proteins (Sturm et al., 2008; Visser et al., 2012). The
 12

marker itself falls in the middle of a binding site for the Helicase-Like Transcription Factor
(HLTF), which remodels the chromatin structure around genes. As HERC2 does not appear to
play any direct role in the pigmentation pathway, it is unlikely that the gene itself is affected by
the mutation. Rather, HERC2 rs12913832 may influence the regulation of the nearby
pigmentation gene, OCA2. When the ancestral ‘A’ allele is present, HLTF is able to bind to this
region and begin unwinding the chromatin structure. This exposes sites for other regulatory
proteins that promote the transcription of OCA2. In contrast, when the derived allele is present,
the binding site for HLTF is disrupted. As a result, fewer regulatory proteins are able to access
this region and OCA2 transcription is greatly reduced. This hypothesis has been supported by a
recent research study that observed cross-linking between HERC2 rs12913832 and OCA2
(Visser et al., 2012).
Although HERC2 rs12913832 may play the primary role in controlling the difference
between blue and brown iris colour, the lack of complete concordance between HERC2 and iris
pigmentation suggests that many other markers may also be involved. Thus far, a number of
variants that are believed to play a more subtle role in iris colour diversity have been identified in
European populations. A review of these markers can be found below:

I. ASIP. This gene contains a polymorphism known as rs6058017 that results in the
exchange of a ‘G’ allele for an ‘A’ allele in the 3’ UTR of exon 4. It has been suggested
that this variant may have a negative impact on overall mRNA stability, causing the
transcript to degrade prematurely (Kanetsky et al., 2002). The role that this marker plays
in iris pigmentation is still under heavy debate. A few early pigmentation studies found
that the derived ‘G’ allele was significantly associated with darker skin, eyes and hair
(Bonilla et al., 2005; Kanetsky et al., 2002). However, these results have not been
consistently replicated (Frudakis et al., 2003; Liu et al., 2010).
II. IRF4. The marker rs12203592 has been associated with iris pigmentation variation in
multiple studies (Han et al., 2008; Liu et al., 2010). The ancestral cytosine (C) allele
appears to be associated with darker skin, hair and eye colour. Not much is known about
how this marker functions. However, the derived thymine (T) allele has a very high
frequency in Irish populations. This has led some researchers to suggest that this variant
may play a role in generating the set of pigmentation characteristics (i.e. red hair, green
 13

eyes, light skin) that is commonly observed in this region of the world (Walsh et al.,
2012).
III. OCA2. Although many variants have been identified in OCA2, there is only one marker
that has been consistently associated with iris pigmentation variation. OCA2 rs1800407 is
a non-synonymous mutation that results in the replacement of an arginine by a glutamine
at position 419 of the protein. This marker is associated with the penetrance of
hazel/green eye colour when HERC2 rs12913832 is either heterozygous or homozygous
for the ancestral ‘A’ allele (Mengel-From et al., 2010; Sturm et al., 2008). Remarkably,
although OCA2 rs1800407 has a very low frequency in all human populations, it has a
relatively strong effect on iris colour. This marker is presently the second strongest
predictor of iris pigmentation diversity in European populations (Walsh et al., 2012).
IV. SLC24A4. The marker rs12896399 has been associated with iris pigmentation variation
in multiple research studies. One study found that this variant was associated with the
difference between blue and green eye colour (Sulem et al., 2007). A second found it to
be positively associated with the hue and saturation of the iris (Liu et al., 2010). Although
not much is known about rs12896399, this marker may fall in the middle of a
transcription factor binding site (Hart et al., 2013). Interestingly, a recent study noted that
this marker may also be associated with central heterochromia (Larsson et al., 2011).
Therefore, rs16891982 may actually function by determining where pigment is placed in
the eye.
V. SLC24A5. The marker rs1426654 is presently believed to be one of the principal
polymorphisms responsible for the lightening of skin pigmentation in populations of
European ancestry (Lamason et al., 2005). This marker codes for a non-synonymous
mutation that results in the substitution of a threonine for an alanine at position 111 of the
protein. The derived allele has become largely fixed in European populations. Therefore,
it is difficult to test the association between this variant and iris colour. However, recent
studies in an admixed Cape Verdean population and an Italian population suggest that
rs1426654 may contribute extensively to eye colour diversity (Beleza et al., 2013;
Pietroni et al., 2014).
VI. SLC45A2. A marker in this gene known as rs16891982 is believed to have played a large
role in evolution of light skin pigmentation in European populations (Graf et al., 2005).
 14

This is a non-synonymous polymorphism that results in the substitution of a leucine for a

phenylalanine at position 374 of the protein. The derived allele has been strongly
associated with light eye and hair colour in multiple pigmentation studies (Graf et al.,
2005; Liu et al., 2010).
VII. TYR. Two variants in this gene, rs1393350 and rs1126809, have been associated with iris
pigmentation variation in multiple pigmentation studies (Liu et al., 2010; Sulem et al.,
2007). Both of these markers are located very close together in the genome and are in
strong linkage disequilibrium. Therefore, it is likely that they have the same effect on eye
colour variation. TYR rs1126809 is most likely the causative variant, as it is a non-
synonymous mutation that results in the replacement of an arginine by a glutamine at
position 402 of the protein (Sulem et al., 2007). Interestingly, although neither of these
variants show any signals of positive selection, they are largely restricted to European
populations.
VIII. TYRP1. A variant in this gene, known as rs1408799, has been significantly associated
with iris colour variation in several research studies. One study found that this marker
was associated with the difference between blue and non-blue eyes (Frudakis et al.,
2003). A second found that it played a role in controlling the hue and saturation of the iris
(Liu et al., 2010). TYRP1 rs1408799 shows strong signals of positive selection in
European populations (Myles et al., 2006). However, its function is still largely unknown.
IX. Other Genes. A recent genome-wide association study on quantitative iris colour
variation identified several new markers that may contribute to eye colour diversity (Liu
et al., 2010). These include rs7277820 in DSCR9, rs3768056 in LYST, and rs9894429 in
NPLOC4. However, these markers have not yet been replicated in any additional studies.
Interestingly, none of these genes appear to be directly involved in the pigmentation
pathway. Rather, they may play some role in regulating the overall structure and function
of the iris.

1.5 Genetic Basis of Iris Surface Features

The heritability of Fuchs’ crypts (66%), contraction furrows (78%), Wolfflin nodules
(78%) and pigment spots (58%) is moderately high (Larsson and Pedersen, 2004). However, at
 15

present, the genetic basis of these traits is still very poorly understood. Thus far, only one
research group has attempted to identify the variants in the human genome that are directly
associated with iris surface feature diversity (Larsson et al., 2011; Sturm and Larsson, 2009). It
appears that the markers that determine iris structure may be very different from those that
determine eye colour (Larsson et al., 2011). Whereas most of the genes associated with iris
pigmentation code for proteins directly involved in the pathway of melanogenesis, the markers
that have been associated with iris surface features are primarily responsible for the
embryogenesis and development of the eye.
A variant known as rs10235789 in the gene SEMA3A has been the most strongly
associated with Fuchs’ crypts (Larsson et al., 2011). This marker, which was identified in an
Australian population of European ancestry, appears to explain approximately 1.5% of the
overall variation in this trait. It has been suggested that the derived ‘C’ allele may negatively
affect the overall stability of the anterior layers of the iris, leading to an increased frequency of
crypts. The variant rs3739070 in TRAF3IP1 has been strongly associated with contraction
furrows (Larsson et al., 2011). This variant, which explains about 1.7% of the variation in
furrows, may be linked to the overall thickness and density of the iris. Lastly, the polymorphism
rs11630290 in HERC1 has been associated with the presence of pigment spots (Larsson et al.,
2011). Although the function of HERC1 is largely unknown, it may play some role in membrane
transport.
It has long been suggested that the Downs Syndrome Critical Region (DSCR) of the
genome may hold the genetic variants responsible for Wolfflin nodules (Sturm and Larsson,
2009). This is largely because of the perceived similarity between Wolfflin nodules and
Brushfield spots. Interestingly, in a recent genome-wide association study on iris colour
variation, a variant known as rs7277820 in DSCR9 was significantly associated with multiple iris
colour characteristics (Liu et al., 2010). It was suggested that this variant may modulate iris
pigmentation by controlling the presence and extension of Wolfflin nodules in the eye. However,
the relationship between DSCR9 rs7277820 and Wolfflin nodules has not yet been tested.
At present, the genetic basis of iris surface features is still very poorly understood. The
genetic markers that have been identified thus far explain a very small percentage of the overall
variation in in these traits. Given the moderate heritability of all four iris features, there are
probably many as of yet unidentified markers involved.
 16

1.6 Limitations in our Understanding of Iris Colour and Texture

Variation
Although both iris colour and structure variation have begun to receive much attention
over the past decade, there are still many gaps and limitations in our understanding of these
traits. The most significant of these limitations stem from difficulties associated with obtaining
quantitative measurements of iris colour, the complexities of characterizing central
heterochromia, and the limited population groups that have traditionally been studied.

1.6.1 Characterizing Iris Colour and Surface Feature Variation

Skin colour and hair colour have conventionally been measured using a technique known
as reflectance spectroscopy (reviewed in Shriver and Parra, 2000). Spectroscopy involves
shining light at a particular wavelength on the skin or hair, and measuring the amount of light
that is reflected back. Due to the nature of the eye, spectroscopy cannot be used on the iris.
Consequently, early iris pigmentation studies chose to characterize iris colour using sets of
discrete categories (i.e. blue, green, brown) [Frudakis et al., 2003; Graf et al., 2005; Mengel-
From et al., 2009; Rebbeck et al., 2002; Sulem et al., 2007]. Such categorical classification
methods have severe limitations. Iris colour is an innately quantitative trait. Although discrete
categories may capture gross differences in iris colour (i.e. blue versus brown), they are unable to
capture the full range of eye colour variation. In addition, there has been no formal consensus on
how many categories should be used. Some research studies use as few as three categories, while
others use as many as eight. Thus, it is not surprising that the inter- and intra-observer reliability
of categorical methods is not very high (Frudakis et al., 2003; Seddon et al., 1990).
Over the past decade, a number of research groups have begun to develop quantitative
methods of measuring iris colour. Liu et al. (2010) designed an automated system to extract hue
and saturation values from photographs of the iris. Saturation was used to represent the amount
of melanin in the eye, and hue was used to represent the type. Beleza et al. (2013) extracted RGB
values from digital photographs of the eye. They used the term ‘T-index’ to describe the
distribution of irises across RGB colour space. Most recently, Andersen et al. (2013)
automatically extracted a Pixel Index of the Eye (PIE) score from photographs of the eye. This
score, which ranges from -1 to 1, is calculated by comparing the number of pixels labelled blue
 17

with the number of pixels labelled brown. Such quantitative methods offer numerous advantages.
For one, they are able to pick up much more subtle variation in pigmentation variation. As a
result, they have been very successful at identifying several of the genetic variants that play a
smaller role in controlling iris colour variation. In addition, they allow researchers to approach
the study of iris colour in populations with more homogenously coloured eyes.
The distribution of iris surface features is also remarkably complex. Fuchs’ crypts vary
considerably in number, shape and size. Some eyes show many clear and well-demarcated
crypts, whereas others show indistinct and sparsely distributed crypts. It is also not uncommon
for an iris to be completely devoid of crypts, or only show small, bulbous-shaped crypts scattered
around the collarette. Pigment spots can be either large or small. They differ widely in colour and
can range from light yellowish-browns to dark brownish-blacks. Some pigment spots show clear
boundaries, whereas others are not very well-defined. Contraction furrows may extend around
the entire iris, or be restricted to particular quadrants of the eye. Some eyes show numerous strata
of furrows, whereas others have only one or two. Wolfflin nodules differ between individuals in
both number, size and colour. Some irises show large, nodules whereas others eyes show
numerous, small nodules. In some eyes they are a pale white, whereas in other eyes they appear
orange or yellow.
Due to the extensive diversity inherent in iris surface features, it has traditionally been
very difficult to characterize these traits. Consequently, most research groups have used
categorical schema that attempt to capture the most important properties of surface feature
variation. However, there has been very little standardization across these methods. Research
studies have used anywhere between three and seven categories to characterize iris texture
(Larsson et al., 2011; Quillen et al., 2011; Sidhartha et al., 2014a). What this means is that the
results obtained from different groups are not comparable. Accordingly, the overall frequency
and distribution of these traits is still poorly understood.

1.6.2 Characterizing Central Heterochromia

Central heterochromia has thus far been one of the most poorly studied areas of iris
research. Thus, it is not surprising that we currently know very little about the genetic basis and
global distribution of this trait.
 18

Early categorical methods tended to group irises that showed evidence of heterochromia
into a broad ‘intermediate’ category (Graf et al., 2005; Rebbeck et al., 2002; Sulem et al., 2007).
When heterochromia was studied on its own, it was characterized using a ‘presence’ or ‘absence’
schema (Larsson et al., 2011). However, this greatly simplifies the overall variation present in
heterochromatic irises. Some irises with heterochromia show a gross colour difference between
the ciliary and pupillary zones, whereas others show a much more subtle difference. Just as iris
pigmentation variation is quantitative, so is heterochromatic variation. Current quantitative
methods have not improved the study of central heterochromia. Although such methods are
capable of picking up subtle colour differences, they tend to use a single ‘average’ value to
capture eye colour. This means that an iris that is uniformly green might give the same average
colour value as a blue iris with a heterochromatic ring.
The difficulties associated with measuring central heterochromia in the iris have had a
significant impact on our overall understanding of iris colour variation. At present, a number of
forensic groups have developed algorithms that can be used to predict eye colour from small
samples of DNA (Hart et al., 2013; Spichenok et al., 2011; Walsh et al., 2012). The most well-
known of these algorithms is perhaps IrisPlex, which uses six SNPs (HERC2 rs12913832,
SLC45A2 rs16891982, IRF4 rs12203592, OCA2 rs1800407, SLC24A4 rs12896399, TYR
rs1393350) to differentiate between blue, brown or intermediate/green eye colour (Walsh et al.,
2012). Although IrisPlex has been found to work well in populations with a high frequency of
blue or brown eyes, it does poorly in populations with a high frequency of intermediate eyes. In a
sample of 803 individuals of mixed ancestry from New York City, IrisPlex showed 209
inconclusive results and 135 errors (Pneuman et al., 2012). It is clear we need to develop more
effective methods of characterizing central heterochromia.

1.6.3 Population Variation

One of the biggest gaps in our current understanding of iris colour variation is the lack of
attention that has been devoted to populations of non-European ancestry. Although brown eye
colour may predominate in regions outside of Northern Europe, there is still substantial variation
within these browns. In fact, brown iris colour can range from light reddish-yellows to dark
brownish-blacks. The lack of research being conducted outside of European populations may be
largely due to the categorical methods that have been traditionally use to characterize iris colour.
 19

As such methods can only pick up gross differences in eye colour, they are largely ineffective in
populations with more homogenously coloured irises. However, even with the advent of new
quantitative methodologies, there has been a noticeable lack of research being performed in
populations of non-European ancestry.
This dearth of research is problematic for a number of reasons. Firstly, there may be
many genetic variants associated with iris pigmentation variation that cannot be identified in
European populations. Such variants may either be fixed in Europe, or not present at all. In
addition, as the sample size of brown eyes is moderately low in Northern Europe, it may be more
difficult to detect variants that play a role in controlling brown iris colour. The lack of iris
pigmentation research outside of Europe also means that we have a very poor understanding of
the distribution of iris colour in these regions. Iris colour in non-European populations is
typically described using the blanket term ‘brown’. However, is it possible that the shade and
intensity of these browns may differ from one population to the next? Is the brown iris colour
found in South Asian populations the same as the brown iris colour found in East Asian
populations? Are the genetic variants that control brown iris colour variation the same in all
populations? These are questions that we are still unable to answer. Finally, it is very difficult to
trace the evolutionary history of a trait when we only know the genetic basis of that trait in one
particular region of the world. At the moment, the evolutionary factors that drove the evolution
of light eye colour are poorly understood. Looking at the distribution of genetic variants
associated with iris pigmentation in other populations may provide some insights into the
evolution of this trait.
In contrast to iris colour research, there have been some attempts to understand iris
structure diversity in different populations. At present, there have been extensive studies
performed in East Asian, European, Portuguese, Brazilian and Cape Verdean populations
(Larsson and Pedersen, 2004; Larsson et al., 2011; Quillen et al., 2011; Sidhartha et al., 2014a,
2014a). However, all of these studies have limited their work to a particular continent. As each
research group categorized iris structure variation differently, we are still not able to compare the
phenotypic distribution of these traits in different continental regions of the world. There is some
evidence that the distribution of the textural features of the iris may be population-specific (Qiu
et al., 2005; Quillen et al., 2011). However, until different ancestral groups are compared using
 20

the same methodology, we will not be able to fully understand the global distribution of these
traits.

1.7 Rationale and Study Goals

Developing a better understanding of iris colour and surface features will contribute
greatly to the fields of forensics, anthropology and public health. At present, many forensics
groups are developing pigmentation predictor algorithms. The primary purpose of these
algorithms is to allow crime scene investigators to piece together the hair, skin and eye colour of
unidentified individuals from small samples of their DNA. IrisPlex, which was discussed briefly
in the previous section, is perhaps the most well-known of these systems (Walsh et al., 2012).
However, other algorithms that rely on different sets of genetic variants have also been proposed
(Hart et al., 2013; Spichenok et al., 2011). The use of iris colour predictor equations has far-
reaching effects outside of the field of forensics. Anthropologists and molecular biologists are
now able to piece together the pigmentation characteristics of early humans and their ancestors.
For example, genetic variants in the MC1R gene have been used to show that Neanderthals most
likely had red hair and pale skin (Lalueza-Fox et al., 2007). Likewise, HERC2 rs12913832, the
polymorphism that controls the difference between blue and brown eyes, has been used to
predict the iris colour of ancient skeletal remains (Keyser et al., 2009; Olalde et al., 2014). Such
research will ultimately provide us with important information about the evolution and early
migratory history of human populations.
Apart from the anthropological and forensic benefits, there is some evidence that both iris
colour and structure may have an influence on individual health and well-being. Many of the
markers associated with iris colour variation have also been associated with an increased risk of
basal cell carcinoma, squamous cell carcinoma, and/or melanoma (Gudbjartsson et al., 2008;
Nan et al., 2009b). Understanding how these markers function may provide important
information about the distribution and prevalence of such carcinomas in different global
populations. Similarly, there appears to be an association between several ocular disorders and
eye colour. Within populations, for instance, individuals with lighter coloured irises are more
likely to develop age-related macular degeneration (AMD) than individuals with darker coloured
irises (Frank et al., 2000; Wakamatsu et al., 2008). The surface features found on the iris may
 21

also serve as important risk indicators of a number of diseases and disorders. Two recent studies
in a Malaysian population living in Singapore found that increasing grades of Fuchs’ crypts are
associated with a thinner iris and a wider angle closure and more extended contraction furrows
are associated with a thicker peripheral iris (Sidhartha et al., 2014a, 2014b). As iris thickness and
angle closure are both linked to a number of ocular disorders, the use of photographs to predict
these traits may represent a cost-effective way of evaluating overall iris structure.
It is clear that iris colour and structure have the potential to contribute greatly to a number
of different fields. However, we cannot fully use these traits until we overcome some of the
numerous limitations in these fields. How do we measure iris colour quantitatively? How do we
take central heterochromia into account when using such quantitative methods? What categories
best characterize iris structure across global populations? To what extent do the genes involved
in iris colour and texture variation overlap in different populations? Do the surface features on
the iris show the same frequencies in populations of different ancestry? These are questions that
need to be answered before we can begin to fully understand the evolutionary events that led to
the iris colour variation that we see in the world today. With that in mind, my research has six
specific goals:

1) To develop a quantitative method of measuring iris colour from high-resolution

photographs
2) To develop a quantitative method of investigating central heterochromia
3) To develop a categorical method of characterizing Fuchs’ crypts, contraction furrows,
Wolfflin nodules, and pigment spots from high-resolution photographs of the iris
4) To look at the phenotypic distribution of both iris colour and surface features in
populations of East Asian, European and South Asian ancestry
5) To look at the association between putative genetic markers and iris colour and texture
variation in populations of East Asian, European and South Asian ancestry

In summary, this research will look at the distribution and genetic basis of iris colour and
surface feature variation in three different populations. It will also provide other researchers
with a methodology that they can use to look at iris colour and structure in their own sample
sets. This will have many benefits for the fields of forensics, anthropology and public health.
 22

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 23

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 Chapter 2:
Quantitative Measures of Iris Colour Using High
Resolution Photographs

Author Contributions: E.J. Parra and I designed this project. A. Gozdzik carried out the
participant recruitment and data collection. J. Miles created the macro that automatically cropped
a square from each iris photograph. I prepared the samples for genotyping with help from K.
Ross. I prepared the iris photographs, analysed the data, developed the methodology and wrote
the manuscript with help from E. J Parra.

This Chapter Has Been Previously Published As: Edwards, M., Gozdzik, A., Ross, K., Miles
J., and Parra, E.J. (2012) Technical note: quantitative measures of iris colour using high
resolution photographs. Am. J. Phys. Anthro. 147, 141-149.

Acknowledgements: The authors thank all the individuals who participated in the study. Jon
Miles is the owner of Miles Research, a company that designs and builds specialized iris
cameras.

28
 29

2.1 Abstract
Our understanding of the genetic architecture of iris colour is still limited. This is partly
related to difficulties associated with obtaining quantitative measurements of eye colour. Here
we introduce a new automated method for measuring iris colour using high resolution
photographs. This method extracts colour measurements in the CIE 1976 L*a*b* (CIELAB)
colour space from a 256 by 256 pixel square sampled from the 9:00 meridian of the iris. Colour
is defined across three dimensions: L* (the lightness coordinate), a* (the red-green coordinate),
and b* (the blue-yellow coordinate). We applied this method to a sample of individuals of
diverse ancestry (East Asian, European and South Asian) that was genotyped for the HERC2
rs12913832 polymorphism, which is strongly associated with blue eye colour. We identified
substantial variation in the CIELAB colour space, not only in the European sample, but also in
the East Asian and South Asian samples. As expected, rs12913832 was significantly associated
with quantitative iris colour measurements in subjects of European ancestry. However, this SNP
was also strongly associated with iris colour in the South Asian sample, although there were no
participants with blue irides in this sample. The usefulness of this method is not restricted only to
the study of iris pigmentation. High-resolution pictures of the iris will also make it possible to
study the genetic variation involved in iris textural patterns, which show substantial heritability
in human populations.
 30

2.2 Introduction
Eye colour is determined by the type of melanin present and the density and distribution
of melanosomes located within the melanocytes of the iris stroma (Sturm and Larsson, 2009).
Iris pigmentation exhibits a variable global distribution. In most populations, eye colour is
primarily limited to varying shades of brown. However, individuals of European, and to a lesser
extent, North African, Middle Eastern, Central Asian, and South Asian ancestry, express a wide
range of colours that include shades of brown, green, and blue. In recent years, the use of linkage
analyses and genome-wide association studies has led to the identification of several of the key
genes associated with iris colour variation (Frudakis et al., 2003; Duffy et al., 2007; Sulem et al.,
2007; Eiberg et al., 2008; Kayser et al., 2008; Sturm et al., 2008; Branicki et al., 2009; Mengel-
From et al., 2009; Liu et al., 2010). However, most of the research efforts have focused on
European populations, and there have been very few studies exploring the phenotypic variation
and genetic basis of iris pigmentation in populations of non-European ancestry.
One of the major challenges for unraveling the genetic architecture of iris pigmentation is
obtaining a quantitative measurement of eye colour. Traditional methods of measuring skin and
hair pigmentation, which are based on reflectometry, cannot be used on the iris. Consequently,
the majority of studies investigating iris pigmentation variation have used a limited number of
discrete categories to characterize eye colour (for a recent review about iris colour classification,
see Mackey et al., 2011). Although such discrete classification methods have successfully
identified some of the major genes associated with iris colour, they also have a number of
weaknesses. For one, they are subjective and have limited inter- and intra-observer reliability
(Seddon et al., 1990; Frudakis et al., 2003). Additionally, they are unable to account for the
extensive quantitative variation that is inherent in iris pigmentation.
Recently, a number of research groups have developed quantitative methods for
measuring iris colour. Melgosa et al. (2000) used a spectroradiometer to obtain a measurement of
the combined pupil and iris in the Commission Internationale de L'Eclairage L*a*b* (CIELAB)
colour space. German et al. (1998) studied drug response in irides by manually extracting a
number of colour measurements from photographs taken of the human eye. These measurements
included parameters in the XYZ and CIELAB colour spaces. Frudakis (2008) extracted RGB and
luminosity measurements from iris photographs and condensed this information into a single ‘iris
melanin index.’ Most recently, Liu et al. (2010) isolated hue and saturation values from iris
 31

photographs; hue was used to represent the type of melanin, while saturation was used to
represent the amount of melanin in the iris. The development of such quantitative methods is
creating new opportunities to study the genetics of iris pigmentation variation in a much more
objective way.
In this article, we introduce a new automated method to quantify iris colour. This method
extracts colour measurements in the CIELAB colour space from high-resolution photographs of
the iris. CIELAB is an international standardized colour system that was designed to
approximate human colour vision (CIE, 1986). The CIELAB colour space provides quantitative
measurements across three different dimensions: a brightness dimension (L*), a green/red
dimension (a*), and a blue/yellow dimension (b*). The CIELAB space has advantages over other
colour systems because it is perceptually uniform: the distance between two colour points closely
corresponds to perceptual differences in human vision. Additionally, the CIELAB is a metric
space that factors in the colour temperature of the illuminant, also known as the white balance of
the source illumination. Colour data from an RGB colour space does not include the illuminant
temperature; this factor is however included in the conversion from RGB to CIELAB. The
output of the xenon flash as used in photography is approximated as D55—that is, Daylight at
5,500 degrees Kelvin.
We applied this new method to a sample of individuals of diverse ancestry (East Asia,
Europe, and South Asia) that was genotyped for the HERC2 rs12913832 polymorphism, which
shows a strong association with blue eye colour (Eiberg et al., 2008; Sturm et al., 2008).

2.3 Materials and Methods

2.3.1 Recruitment
Study participants were recruited at the Molecular Anthropology Laboratory of the
University of Toronto Mississauga (UTM) between 2007 and 2009. The majority of participants
were UTM students and staff who responded to online and print advertisements that were
distributed throughout the University of Toronto community. To assess geographic ancestry,
each participant was asked to complete a questionnaire that inquired about the participant's
parental and grandparental places of birth and native languages. For example, individuals who
stated that their ancestors were from China, Japan, and Korea, were grouped as East Asian, while
 32

those who reported ancestors from India and Pakistan were grouped as South Asian. Individuals
who reported being of multiple ancestries were placed into a subgroup designated as ‘Other,’ and
were not included in the analysis. In total, 205 subjects were included in the study. Of these, 66
were of East Asian ancestry, 72 were of European ancestry, and 67 were of South Asian
ancestry.
This study was approved by the University of Toronto Health Sciences Research and
Ethics Board. All participants provided written informed consent.

2.3.2 Genotyping
We selected the rs12913832 polymorphism in intron 86 of the HERC2 gene for
genotyping. The derived ‘G’ allele of this marker has been strongly associated with blue iris
colour in populations of European ancestry (Eiberg et al., 2008; Sturm et al., 2008). A blood
sample was collected from each participant in a 4-mL EDTA tube, and DNA was extracted from
the blood samples using the E.Z.N.A. Blood DNA Midi Kit (Omega Bio-Tek, United States).
The samples were then sent to Kbioscience (United States) for genotyping. Kbioscience uses a
genotyping method that combines allele-specific PCR with detection by a Fluorescence
Resonance Energy Transfer (FRET) system. To evaluate genotyping quality, blind duplicates
were included in the genotyping plates. The concordance rate for the genotype calls of the
duplicated samples was 100%.

2.3.3 Acquisition and Processing of Iris Photographs

We took a photograph of each subject's left iris using the Miles Research Professional Iris
camera (Miles Research, United States). This camera consists of a Fujifilm Finepix S3 Pro 12-
megapixel DSLR mounted on a Nikkor 105-mm macro lens. A coaxial biometric illuminator was
used to deliver a constant and uniform source of light to each iris at 5,500 K (D55 illuminant).
Camera performance in terms of colour recording fidelity was verified using the 24-patch
GretagMacbeth Colour Checker and Imatest Master software (http://www.imatest.com). All
photographs were taken using an aperture of f19 and exposure sensitivity (ISO) of 200. The
shutter speed was set to 1/60 second and all pictures were recorded in 12-megapixel JPEG
format.
 33

Upon examination of the photographs used for the analysis of iris colour there was
evidence of underexposure. As a corrective measure, an exposure normalization procedure was
applied to all 205 images using a colour-neutral brightness operator, the screen-mode blending of
each image with itself. This exposure compensation filter shifts the colours away from black and
white so as to improve discrimination of small differences in colour. The effect is analogous to
using two slide projectors illuminating the same transparency image, aligned on a screen, to
create a brighter result. Mathematically, the screen blend lightening filter (blending an eight-bits-
per-channel image with itself) is expressed as: c′ = 255 – ((255 − c0)2/255), where c0 represents
each R, G, B channel value as an eight-bit integer from 0 to 255, and c′ is the filter result value.

2.3.4 Acquisition of Colour Measurements

A single RGB measurement was extracted from each iris image using a multistep
process: 1/ A macro was created in Adobe Photoshop CS5 that cropped each image to the
middle-third of the image height, centred on the pupil; 2/ A second macro was developed that
cropped out the regions of the image containing the sclera; 3/ A final macro was used to crop the
sample down to a 256 by 256 pixel square at the ciliary zone of the iris (mid-periphery) in the
9:00 meridian of each sample; and 4/ The program ImageJ (National Institutes of Health, United
States) was used to extract RGB data from each of the cropped image samples. We used the
‘average RGB’ of the 256 by 256 pixel square to characterize the iris colour.
The RGB values were transformed into CIE 1976 L*a*b* (CIELAB) colour space. The
CIELAB colour space is a universal standardized system that uses three dimensions to represent
colour. The first dimension, L*, is used to represent brightness and can have values that range
from 0 to 100, where 0 is black and 100 is white. The a* and b* dimensions correspond to
differences in colour, with negative values of a* indicating green and positive values of a*
indicating red, and negative values of b* indicating blue and positive values of b* indicating
yellow. To transform the RGB values into the CIELAB colour space, the RGB measurements
were first converted into XYZ values using standard equations found at
http://www.easyrgb.com/index.php?X=MATH&H=02#text2. The XYZ values were then
transformed into L*, a*, and b* measurements using standard equations found at
http://www.easyrgb.com/index.php?X=MATH&H=07#text7. As the coaxial biometric
 34

illuminator delivered light to each iris at a temperature of 5,500 K, the colour space conversion
was based on the D55 standard illuminant. The observer was set at 2°.

2.3.5 Statistical Analysis

Deviations from Hardy–Weinberg proportions in each population were evaluated using
the Hardy–Weinberg exact test (see http://ihg2.helmholtz-muenchen.de/cgi-bin/hw/hwa1.pl).
We used general linear model (GLM) analyses to test the association between the rs12913832
genotypes and L*, a*, b* values in the European and South Asian samples (in the East Asian
sample, 64 out of 65 individuals were homozygous for the ancestral ‘A’ allele, so due to the lack
of polymorphism in this sample, we did not carry out the statistical analysis). The tests were
done with the statistical program SPSS (version 17.0, SPSS, 2008, United States). Associations
were considered significant when P ≤ 0.05.

2.4 Results
Figure 2-1 shows a graphical representation summarizing the procedure that was used to
obtain measurements in the CIELAB colour space from each iris, with a representative sample
including two blue, one green, and three brown irides. Figure 2-2 depicts the measurements
obtained for the full sample. There is a substantial dispersion of the L*, a*, and b* values in the
three-dimensional space. As expected, the dispersion is more pronounced in the European
sample, which includes eye colours that ranged from light blue to dark brown. In the European
sample, the L* values ranged from 17.63 to 67.08, the a* values from −6.56 to 25.52, and the b*
values from −14.34 to 37.62. There was also considerable variation in the East Asian and South
Asian samples. In the East Asian sample, which is comprised primarily of dark brown irides, the
L* values fluctuated from 10.96 to 40.92, the a* values from −2.67 to 24.42, and the b* values
from 5.73 to 11.12. In the South Asian sample, which includes a broader representation of shades
of brown than the East Asian sample, the L* (ranging from 10.18 to 44.31) and a* (ranging from
−2.86 to 25.42) values showed a distribution that is broadly similar to that observed in the East
Asian sample. However, there was considerably more dispersion in the b* axis (0.32–31.36).
When examining the CIELAB coordinates considering broad categorical classifications of iris
colour, blue irides tended to have very high L* values and negative a* and b* values. Green
 35

irides usually mapped between brown and blue irides, and had a high L* value, a b* value that
hovered around 0, and a negative a* value. In contrast, brown irides tended to have low L*
values and positive a* and b* values. Irides with lighter shades of brown had higher L*, a*, and
b* values than irides with darker shades of brown (see Figure 2-1B). Overall, it is evident that
classifications that use broad categories such as ‘brown,’ ‘intermediate,’ or ‘blue’ do not
satisfactorily capture iris pigmentation variation. We also observed that in many individuals,
particularly those of European ancestry, the pigmentation of the central pupillary zone is darker
than the pigmentation of the peripheral ciliary zone (central heterochromia).
To test the usefulness of our method to quantify iris colour, we evaluated the effect of the
HERC2 rs12913832 polymorphism on the L*, a*, and b* values. This single nucleotide
polymorphism (SNP) has been associated with iris pigmentation in previous studies (Eiberg et
al., 2008; Sturm et al., 2008; Liu et al., 2010). Table 2-1 shows the genotype and allele
frequencies in the East Asian, European, and South Asian samples. There were no significant
deviations from Hardy–Weinberg proportions in any of the samples (Table 2-1). The frequency
of the derived ‘G’ allele, which has been associated with blue iris colour in previous studies, was
71% in the European sample and 13% in the South Asian sample. In the East Asian sample, 64
out of 65 individuals were homozygous for the ancestral ‘A’ allele, so we eliminated this sample
from the statistical analysis. We employed an unconstrained genetic model, which estimates
separately the effects of the homozygotes for the derived ‘G’ allele and the heterozygotes, using
the homozygotes for the ancestral ‘A’ allele as the reference genotype. The results of these tests
for the European and South Asian sample are depicted in Table 2-2. In the European sample,
rs12913832 was significantly associated with the L*, a*, and b* (P < 0.001) dimensions of
colour space. In the lightness dimension, the estimates of the regression coefficient (beta)
indicated that L* increased by 12.37 U in AG heterozygotes and by 23.27 U in GG
homozygotes, with respect to AA homozygotes. In the green/red dimension, a* decreased by
7.19 U in AG heterozygotes and by 23.16 U in GG homozygotes, with respect to AA
homozygotes. In contrast, in the blue/yellow dimension, b* decreased dramatically (22.58 U) in
the GG homozygotes, but was very similar for the AG heterozygotes and the AA homozygotes.
Therefore, rs12913832 seems to fit a dominant model for the blue/yellow dimension (b*) of the
CIELAB colour space, with the ancestral ‘A’ allele dominant over the recessive ‘G’ allele, and a
codominant model for the green/red (a*) and lightness (L*) dimensions. In the South Asian
 36

sample, rs12913832 was also significantly associated with the L*, a*, and b* dimensions of the
CIELAB colour space (P < 0.001). In this sample we only observed one homozygote for the
derived ‘G’ allele, so it was not possible to explore in detail the effects of the three rs12913832
genotypes on the colour coordinates. However, it is interesting to note that in the South Asian
sample, heterozygotes for the AG allele showed higher L*, a*, and b* values than homozygotes
for the ancestral ‘A’ allele, indicating that, on average, they have lighter shades of brown (see
Figure 2-1B).
To verify the results, we ran the same tests on a smaller collection of irides that were
photographed 1 year later, using the same camera, but with a slightly different shutter speed.
This collection included photographs of 27 East Asian, 37 European, and 25 South Asian
participants and all of the major eye colour phenotypes (blue, brown, green) were represented.
The results are very consistent with those described above. In the European sample, the
rs12913832 marker was strongly associated (P < 0.001) with the three dimensions of the
CIELAB colour space, and the parameters of the unconstrained model are overly similar to those
of the larger sample. L* increased by 17.45 U in AG heterozygotes and by 24.79 U in GG
homozygotes, a* decreased by 11.24 U in AG heterozygotes and by 20.80 U in GG
homozygotes, and b* decreased by 20.45 U in GG homozygotes with respect to AA
homozygotes, but there were very small differences between AA homozygotes and AG
heterozygotes in the b* dimension. In the South Asian sample, rs12913932 was significantly
associated with the L* (P < 0.001) and b* (P = 0.021) dimensions, and was not significant for the
a* dimension (P = 0.369). Again, heterozygote AG individuals showed, on average, higher L*,
a*, and b* values than homozygote AA individuals.

2.5 Discussion
We introduce a new method to measure iris colour, using a series of crops from high-
resolution photographs to obtain a quantitative estimate of colour in the three coordinates of the
CIELAB colour space. The CIELAB colour space is a universal colour system that was designed
to approximate human vision (CIE, 1986). Iris colour variation is reported across three different
planes: a brightness plane (L*) and two colour planes (a* and b*). When we plotted our set of
205 human irides in CIELAB colour space, most of the points were distributed throughout the
 37

three-dimensional space in a fairly continuous manner. This confirms that iris pigmentation, like
skin and hair colour, is a quantitative trait. This is evident in the three population groups studied
in our analysis, which included two samples (East Asians and South Asians) that were comprised
primarily of individuals with ‘brown’ irides. Treating eye colour as a categorical trait in studies
aimed at characterizing the genetic basis of iris pigmentation disregards a substantial amount of
the existing variation and reduces the statistical power to identify main effects and gene–gene
interactions.
To validate our method, we tested the association between the HERC2 rs12913832
polymorphism and our CIELAB colour space measurements in the European and South Asian
samples. This particular marker is found in a highly conserved region of intron 86 of the HERC2
gene and has been strongly associated with blue/brown iris colour in previous studies in
populations of European ancestry (Eiberg et al., 2008; Sturm et al., 2008). Although the exact
mechanisms of action of this polymorphism have not yet been fully determined, it is purported to
play a role in the regulation of OCA2. Eiberg et al. (2008) suggested that rs12913832 falls within
an OCA2 silencer complex that may be disturbed or stabilized depending on which of the two
rs12913832 alleles is present (Eiberg et al., 2008). In contrast, Sturm et al. (2008) have argued
that this particular polymorphism may be a binding site for the regulatory protein HLTF (Sturm
et al., 2008). Under this alternative model, when the ancestral ‘A’ allele is present, HLTF binds
to this region and promotes the transcription of OCA2. When the derived ‘G’ allele is present,
however, HLTF cannot properly bind to the DNA and OCA2 transcription is reduced. Studies in
human melanocyte strains have shown that the presence of the rs12913832 ancestral ‘A’ allele is
associated with increased levels of OCA2 transcript compared to the derived ‘G’ allele (Cook et
al., 2009).
The frequencies for the derived ‘G’ allele that we observed in our sample (East Asians
<1%, Europeans = 71%, and South Asians = 13%) are in overall agreement with previously
reported data. For example, the frequency of this allele in the Human Genome Diversity Project
(HGDP) East Asian samples was <1%, for the European samples 51%, and for Central-South
Asian samples, 15% (http://spsmart.cesga.es/).
As expected, rs12913832 was significantly associated with the L*, a*, and b* planes of
the CIELAB colour space in our European sample. The use of quantitative measures, instead of
categorical variables, highlights some interesting aspects of the effect of rs12913832 on iris
 38

colour. For the b* (blue/yellow) dimension of the CIELAB colour space, rs12913832 fits a
dominant model of inheritance quite well, with the ancestral ‘A’ allele dominant over the G-
derived allele. Homozygous GG individuals have lower b* values (corresponding to the blue
regions of the CIELAB space) than heterozygous AG or homozygous GG individuals, which
show similar b* values. However, the L* and a* dimensions appear to fit a codominant model,
with AG heterozygotes showing intermediate values between AA and GG homozygotes AA.
Traditionally, eye colour has been described as a trait that fits a dominant model of inheritance,
with ‘brown’ dominant over ‘blue,’ although there have been many reports demonstrating
inconsistencies with a dominant model of inheritance (Frudakis, 2008; Sturm and Larsson,
2009). Focusing specifically on the rs12913832 polymorphism, Eiberg et al. (2008) showed that
in a sample of ∼200 Danish individuals this polymorphism was perfectly associated with
blue/brown colour, strongly supporting a model in which blue eye colour is caused by
homozygosity of the rs12913832 ‘G’ allele. However, Sturm et al. (2008) showed that the
association of the rs12912832 GG genotype with blue eye colour is not perfect: some individuals
with this genotype had brown eyes, and some heterozygous AG individuals blue eyes. These two
studies used categorical classifications of iris colour based on self-report (Eiberg et al., 2008), or
the rating of a trained observer with cross-validation with the individual's self-report (Sturm et
al., 2008). In our sample, we also see clear evidence that, although strongly associated with blue
eye colour, rs12913832 does not fit a simple dominant model of inheritance. Some homozygous
GG individuals and heterozygous AG individuals occupy positions in the CIELAB space that do
not overlap with the coordinates observed for most of the GG or AG genotypes. We also
observed that, contrary to the expectations of a simple dominant model, the AG heterozygotes
show L* and a* values that are intermediate between those characteristic of the AA and GG
genotypes. This suggests that AG heterozygotes have intermediate levels of OCA2 expression,
and this is reflected as perceptible differences in the L* and a* dimensions of the colour space.
When we performed the same analysis on the South Asian sample, rs12913832 was also
significantly associated with the L*, a*, and b* dimensions of colour space. As our South Asian
sample is comprised almost entirely of irides that would have been defined as ‘brown’ using a
categorical iris classification system, this shows that rs12913832 also plays a role in modulating
subtle gradations in brown eye colour. In particular, AG heterozygotes showed higher L*, a*,
 39

and b* values (e.g., lighter shades of brown) than AA homozygotes. Again, this strongly
suggests that rs12913832 does not fit a dominant model of inheritance.
We repeated the analysis in an independent sample measured one year later, and we
obtained similar results. However, the parameter values (beta coefficients) obtained in this
replication sample should be considered with caution due to the small sample size (e.g., in the
European sample, there were only three homozygotes for the ancestral allele).
It is worth noting that, in our combined iris samples, we found two individuals of South
Asian ancestry that had two copies of the rs12913832 derived ‘G’ allele and brown or
heterochromatic irides. Additionally, we observed that, whereas AG heterozygotes show
significantly lower a* values than homozygotes for the ancestral ‘A’ allele in the European
sample, in the South Asian sample AG heterozygotes have significantly higher a* values than
AA homozygotes. These findings, as well as information from other studies described above,
clearly indicate that eye colour is a polygenic trait with a complex genetic architecture, where the
effect of some of the major loci may be modified by other polymorphisms (gene–gene
interaction). A recent study in a large sample of European descent has shown that interactions
between the genes HERC2 and OCA2, HERC2, and SLC24A5 and HERC2 and TYRP1 play a
role in the determination of eye colour (Pospiech et al., 2011). Similarly, Liu et al. (2010)
described that interactions of several polymorphisms in pigmentation genes are responsible for
some of the variation of hue and saturation observed in their iris sample.
In this study, we automatically extracted a 256 by 256 pixel square from the iris to
estimate the average colour coordinates in the CIELAB colour space. As shown above, this
method is an improvement over the categorical classifications conventionally used to study the
genetics of eye colour. However, this method does not fully capture the complexity of iris colour.
Central heterochromia is a common feature observed in human irides. In individuals with central
heterochromia, the pupillary zone of the iris has a darker colour than the peripheral ciliary zone.
Alternative methods of quantifying iris colour based on high-resolution photographs can be
employed to specifically study central heterochromia. For example, a measure describing colour
(e.g., hue angle, saturation, or just wavelength) can be obtained in a radial section of the iris,
making it possible to compare the colour of the pupillary and ciliary zones of the iris. Figure 2-3
illustrates such a method using high-resolution photographs of individuals with and without
central heterochromia. In our sample, central heterochromia was present in the three populations
 40

examined. As well, it was found in irides of all of the major colour groups, although it tends to
be most noticeable in individuals with lighter coloured eyes.
Finally, apart from central heterochromia, there are a number of other iridial structures
that can be studied using high-resolution photographs, such as nevi (hyper-pigmented spots),
Wolfflin nodules (pale nodules encircling the iris that are composed primarily of collagen tissue),
Fuchs' crypts (pit-like depressions near the collarette and in the ciliary zone of the iris), and
contraction furrows (circular and radial folds due to iris contraction or dilation) [Larsson and
Pedersen, 2004; Sturm and Larsson, 2009]. These traits show a substantial heritability in human
populations (between 58 and 78%), but the genetic basis of these and other iris patterns remains
largely unexplored (Larsson et al., 2003). Using a genome-wide association (GWA) strategy,
Larsson et al. (2011) reported that variants within the gene SEMA3A are significantly associated
with crypt frequency, polymorphisms within the gene TRAF3IP1 with contraction furrows, and
variants near the pigmentation gene SLC24A4 with the presence of a peripupillary pigmentary
ring. Interestingly, the genes SEMA3A and TRAFIP1 are involved in pathways that control
neurogenesis, neural migration, and synaptogenesis, and this led Larsson et al. (2011) to suggest
that genes involved in normal neuronal pattern development may also influence iris structures.
In conclusion, we present a method to measure iris colour quantitatively using high-
resolution photographs obtained with uniform illumination and show that, using this approach in
individuals of diverse ancestry, it is possible to gain interesting insights about the role of the
HERC2 rs12913832 polymorphism in iris pigmentation. There are still important gaps in our
understanding of the genetic architecture of iris pigmentation. The application of quantitative
methods for measuring eye colour opens up new avenues in pigmentation research. As these
methods are able to distinguish between subtle colour gradations, it will be possible to increase
the statistical power to identify main effects and gene interactions, and to study the genetics of
iris pigmentation in populations in which traditional categorical systems are of limited use.
Additionally, such methods will provide a means of unraveling the genetic architecture of other
traits, such as central heterochromia, and a number of iris patterns that show substantial variation
in human populations. Advancing our current knowledge of the genetic basis of iris colour and
structure is important from multiple perspectives. For example, from a forensic perspective it
will be useful to reliably predict iris colour and structure based on DNA information. From an
anthropological and evolutionary perspective, it will allow researchers to gain new insights about
 41

the major evolutionary events associated with the current distribution of eye colour variation,
which is quite different from the geographical distribution of skin pigmentation. Of interest is to
which extent natural or sexual selection, or the action of demographic factors (e.g., genetic drift,
range expansions) shaped the current distribution of eye colour. Finally, from the developmental
and physiological perspectives, discovering the genes associated with iris colour and structure
will improve our understanding of the pathways that determine these traits.
 42

2.6 Tables
Table 2-1 General Descriptive Statistics
Observed and expected genotype frequencies, allele frequencies, and the Hardy–Weinberg exact test for
HERC2 rs12913832 in the East Asian, European, and South Asian samples

Observed and Expected Genotype Hardy-Weinberg Exact

Population Allele Frequencies
Frequencies Test (p)
A:A = 64 (64.00)
A = 99%
East Asian A:G = 1 (0.99) 1.000
G = 1%
G:G = 0 (0.00)
A:A = 9 (6.00)
A = 29%
European A:G = 23 (28.99) 0.087
G = 71%
G:G = 38 (35.00)
A:A = 50 (50.09)
A = 87%
South Asian A:G = 15 (14.81) 1.000
G = 13%
G:G = 1 (1.09)
 43

Table 2-2 General Linear Model Analysis Results

Results of the general linear model analysis for the brightness dimension (L*), the green/red dimension
(a*), and the blue/yellow dimension (b*) of the CIELAB colour space in the European and South Asian
samples

Population Colour Space Dimension p-value Genotype Beta p-value

AA - -
L* 0.000 AG 12.368 0.000
GG 23.267 0.000
AA - -
European a* 0.000 AG -7.187 0.003
GG -23.158 0.000
AA - -
b* 0.000 AG 1.989 0.585
GG -22.584 0.000
AA - -
L* 0.000
AG 8.358 0.000
AA - -
South Asian a* 0.000
AG 2.701 0.037
AA - -
b* 0.000
AG 6.788 0.000
 44

2.4 Figures
Figure 2-1 Colour Extraction Methodology and Iris Colour Coordinates
(A) An illustration of the sample 256 by 256 pixel square that was isolated from the 9:00 meridian of six
irides of varying colour (two blues, one green, three browns). The average RGB of this square was
determined, and transformed into L*, a*, and b* values in the CIELAB colour space. (B) The coordinates
of overall iris colour of six exemplar samples in the CIELAB colour space. Each iris is graphically
represented by the 256 by 256 pixel square sample region that was used to evaluate iris colour and its
colour space coordinates (L*, a*, b*).
 45

Figure 2-2 Distribution of Irises in CIELAB Colour Space

(A) The distribution of the full sample of irides of participants of East Asian (red circles), South Asian
(green triangles), and European (blue squares) ancestry in the three coordinates of CIELAB colour space:
L* (the lightness coordinate), a* (the red-green coordinate), and b* (the blue-yellow coordinate). (B) The
coordinates of the same irides for a* and b*, the chromatic components of the CIELAB colour space.
 46

Figure 2-3 Visualization of Central Heterochromia

One method of visualizing central heterochromia. The iris photographs of two heterochromatic and one
homogenous irides were reduced to a width of 125 pixels. The program ImageJ (National Institutes of
Health, United States) was used to extract a horizontal band (left to right) from the 9:00 meridian of each
iris in RGB space and the RGB values were transformed into saturation values using standard equations
found at http://www.easyrgb.com/index.php?X=MATH&H=18#text18. Irides with substantial
heterochromia showed sharp changes in saturation across the width of the iris.
 47

2.7 References
Branicki, W., Brudnik, U., and Wojas-Pelc, A. (2009). Interactions between HERC2, OCA2 and
MC1R may influence human pigmentation phenotype. Ann. Hum. Genet. 73, 160–170.
CIE. (1986). CIE publication no. 15.2—colorimetry. Austria: Central Bureau of the Commission
Internationale de L'Eclairage.
Cook, A.L., Chen, W., Thurber, A.E., Smit, D.J., Smith, A.G., Bladen, T.G., Brown, D.L.,
Duffy, D.L., Pastorino, L., Bianchi-Scarra, G., et al. (2008). Analysis of cultured human
melanocytes based on polymorphisms within the SLC45A2/MATP, SLC24A5/NCKX5, and
OCA2/P loci. J Invest Dermatol 129, 392–405.
Duffy, D.L., Montgomery, G.W., Chen, W., Zhao, Z.Z., Le, L., James, M.R., Hayward, N.K.,
Martin, N.G., and Sturm, R.A. (2007). A three–single-nucleotide polymorphism haplotype in
intron 1 of OCA2 explains most human eye-color variation. Am. J. Hum. Genet. 80, 241–252.
Eiberg, H., Troelsen, J., Nielsen, M., Mikkelsen, A., Mengel-From, J., Kjaer, K.W., and Hansen,
L. (2008). Blue eye color in humans may be caused by a perfectly associated founder mutation in
a regulatory element located within the HERC2 gene inhibiting OCA2 expression. Hum. Genet.
123, 177–187.
Frudakis, T. (2010). Molecular photofitting: predicting ancestry and phenotype using DNA
(Elsevier).
Frudakis, T., Thomas, M., Gaskin, Z., Venkateswarlu, K., Chandra, K.S., Ginjupalli, S., Gunturi,
S., Natrajan, S., Ponnuswamy, V.K., and Ponnuswamy, K.N. (2003). Sequences associated with
human iris pigmentation. Genetics 165, 2071–2083.
German, E.J., Hurst, M.A., Wood, D., and Gilchrist, J. (1998). A novel system for the objective
classification of iris colour and its correlation with response to 1% tropicamide. Ophthalmic
Physiol. Opt. 18, 103–110.
Kayser, M., Liu, F., Janssens, A.C.J.W., Rivadeneira, F., Lao, O., Duijn, K. van, Vermeulen, M.,
Arp, P., Jhamai, M.M., Ijcken, W.F.J. van, et al. (2008). Three genome-wide association studies
and a linkage analysis identify HERC2 as a human iris color gene. Am. J. Hum. Genet. 82, 411–
423.
Larsson, M., and Pedersen, N. (2004). Genetic correlations among texture characteristics of the
human iris. Mol Vis 10, 821–831.
Larsson, M., Pedersen, N.L., and Stattin, H. (2003). Importance of genetic effects for
characteristics of the human iris. Twin Res. Hum. Genet. 6, 192–200.
Larsson, M., Duffy, D.L., Zhu, G., Liu, J.Z., Macgregor, S., McRae, A.F., Wright, M.J., Sturm,
R.A., Mackey, D.A., Montgomery, G.W., et al. (2011). GWAS findings for human iris patterns:
associations with variants in genes that influence normal neuronal pattern development. Am. J.
Hum. Genet. 89, 334–343.
 48

Liu, F., Wollstein, A., Hysi, P.G., Ankra-Badu, G.A., Spector, T.D., Park, D., Zhu, G., Larsson,
M., Duffy, D.L., Montgomery, G.W., et al. (2010). Digital quantification of human eye color
highlights genetic association of three new loci. PLoS Genet 6, e1000934.
Mackey, D.A., Wilkinson, C.H., Kearns, L.S., and Hewitt, A.W. (2011). Classification of iris
colour: review and refinement of a classification schema. Clin. Experiment. Ophthalmol. 39,
462–471.
Melgosa, M., Rivas, M.J., Gómez, L., and Hita, E. (2000). Towards a colorimetric
characterization of the human iris. Ophthalmic Physiol. Opt. 20, 252–260.
Mengel-From, J., Wong, T.H., Morling, N., Rees, J.L., and Jackson, I.J. (2009). Genetic
determinants of hair and eye colours in the Scottish and Danish populations. BMC Genet. 10, 1–
13.
Pospiech, E., Draus-Barini, J., Kupiec, T., Wojas-Pelc, A., and Branicki, W. (2011). Gene-gene
interactions contribute to eye colour variation in humans. J Hum Genet 56, 447–455.
Seddon, J.M., Sahagian, C.R., Glynn, R.J., Sperduto, R., Gragoudas, E., Group, E.D.C.-C.S., and
others (1990). Evaluation of an iris color classification system. Invest Ophthalmol Vis Sci 31,
1592–1598.
Sturm, R.A., and Larsson, M. (2009). Genetics of human iris colour and patterns. Pigment Cell
Melanoma Res. 22, 544–562.
Sturm, R.A., Duffy, D.L., Zhao, Z.Z., Leite, F.P.N., Stark, M.S., Hayward, N.K., Martin, N.G.,
and Montgomery, G.W. (2008). A single SNP in an evolutionary conserved region within Intron
86 of the HERC2 gene determines human blue-brown eye color. Am. J. Hum. Genet. 82, 424–
431.
Sulem, P., Gudbjartsson, D.F., Stacey, S.N., Helgason, A., Rafnar, T., Magnusson, K.P.,
Manolescu, A., Karason, A., Palsson, A., Thorleifsson, G., et al. (2007). Genetic determinants of
hair, eye and skin pigmentation in Europeans. Nat Genet 39, 1443–1452.
 Chapter 3:
Iris Pigmentation as a Quantitative Trait: Variation
in Populations of European, East Asian and South
Asian Ancestry and Association with Candidate
Gene Polymorphisms

Author Contributions: M. Edwards participated in the design of the study, collected the data,
conducted the data analysis and drafted the manuscript. D. Cha developed the iris colour
program and assisted with data analysis. S. Krithika, M. Johnson and G. Cook assisted with data
collection and molecular laboratory work. E.J. Parra conceived the study, participated in the
design of the study, coordinated the study and helped draft the manuscript.

This Chapter Has Been Previously Published As: Edwards, M., Cha, D., S. Krithika.,
Johnson, M., Cook, G., Parra, E.J. (2015) Iris pigmentation as a quantitative trait: variation in
populations of European, East Asian and South Asian ancestry and association with candidate
gene polymorphisms. Pigment Cell Melanoma Res.

Acknowledgements: We would like to thank all the individuals who participated in this study.
ME was funded by a 3-yr Natural Sciences and Engineering Research Council (NSERC) CGSD
award and an Ontario Graduate Scholarship (OGS). EJP was funded by an NSERC Discovery
Grant.

49
 50

3.1 Abstract
In this study, we present a new quantitative method to measure iris colour based on high
resolution photographs. We applied this method to analyse iris colour variation in a sample of
individuals of East Asian, European and South Asian ancestry. We show that measuring iris
colour using the coordinates of the CIELAB colour space uncovers a significant amount of
variation that is not captured using conventional categorical classifications, such as ‘brown’,
‘blue’ or ‘green’. We tested the association of a selected panel of polymorphisms with iris colour
in each population group. Six markers showed significant associations with iris colour in the
European sample, three in the South Asian sample and two in the East Asian sample. We also
observed that the marker HERC2 rs12913832, which is the main determinant of ‘blue’ vs.
‘brown’ iris colour in European populations, is also significantly associated with central
heterochromia in the European sample.
 51

3.2 Introduction
Iris colour in humans is a complex trait that is primarily the product of differences in the
structure and organization of the eye. The typical iris consists of five layers: the iris pigment
epithelium (IPE), the sphincter and dilator muscles, the stromal layer (SL) and the anterior
border layer (ABL) (Eagle, 1988; Oyster, 1999; Sturm and Larsson, 2009). The IPE is derived
from the neuroectoderm and contains many large, densely-packed spherical melanosomes
(Eagle, 1988; Prota et al., 1998; Wilkerson et al., 1996). The amount and composition of melanin
in this layer is similar in all healthy humans as the primary purpose of the IPE is to protect the
retina and absorb excess light (Wilkerson et al., 1996). As a result, variation in this layer does
not contribute significantly to normal iris pigmentation variation. In contrast, the melanosomes in
the SL and ABL, which originate from the neural crest, are smaller, ovoid-shaped and differ
widely between individuals (Imesch et al., 1996; Prota et al., 1998; Sturm and Larsson, 2009). It
is presently believed that the majority of iris pigmentation variation can be directly linked to the
amount and type of melanin found in these two layers, with darker eyes having more melanin
and a higher eumelanin/pheomelanin ratio than lighter eyes (Peles et al., 2009; Wakamatsu et al.,
2008; Wielgus and Sarna, 2005). However, there are a number of other factors that can also
influence iris colour, including the depth of the SL, the organization of the extracellular
components in the SL and ABL, the thickness and curvature of the cornea, the presence of
structures (e.g. pigment spots, Wolfflin nodules) in the iris and the amount of melanin found in
the SL relative to the ABL (Baranoski and Lam, 2007; Mackey et al., 2011; Prota et al., 1998;
Wielgus and Sarna, 2005).
Iris colour shows a very distinct global distribution. Across Europe, and to a lesser degree
North Africa, the Middle East, and Central Asia, irises show extensive pigmentation variation
and range from light blues to greens to browns (Donnelly et al., 2012; Eiberg et al., 2008). Many
irises in these regions also show central heterochromia, where there is a band of colour around
the pupil that differs from the rest of the eye. Throughout the rest of the world, however, iris
colour appears to be much more homogenous and is primarily limited to varying shades of
brown. At present, the genetic basis of iris colour has been extensively studied in populations of
European ancestry. The majority of variation between blue and brown eye colour has been
attributed to the marker HERC2 rs12913832, which is located in a highly conserved region of the
genome that is believed to regulate the transcription of the nearby pigmentation gene, OCA2
 52

(Eiberg et al., 2008; Kayser et al., 2008; Sturm et al., 2008). It has been suggested that this
polymorphism is located in the middle of a helicase-like transcription factor (HLTF) binding site
(Sturm et al., 2008; Visser et al., 2012). When the ancestral ‘A’ allele is present, HLTF (a
chromatin remodelling protein) is able to recognize the sequence and begin unwinding the
chromatin. This exposes sequences for other regulatory proteins and promotes the synthesis of
OCA2. When the derived ‘G’ allele is present, however, there is reduced recruitment of HLTF
and OCA2 transcription is limited. Although HERC2 rs12913832 may be the primary
determinant of global iris colour variation, a number of other markers have also been identified
that have a more subtle effect on iris pigmentation. These include polymorphisms in OCA2,
SLC45A2, SLC24A4, TYRP1 and IRF4 (Graf et al., 2005; Rebbeck et al., 2002; Sturm et al.,
2008; Sulem et al., 2007; Walsh et al., 2011). The identification of markers associated with iris
colour in European populations has allowed several forensic science groups to develop
algorithms that are capable of predicting iris colour from DNA samples obtained at crime scenes
and from other unidentified persons (Ruiz et al., 2014; Spichenok et al., 2011; Walsh et al.,
2011).
Despite our growing understanding of the genetic basis of iris pigmentation in European
populations, very few groups have attempted to look at eye colour in populations of non-
European ancestry. Although brown eye colour does dominate in regions outside of Europe,
there is much variation within these browns and they can range from light reddish-yellows to
dark brownish-blacks. In recent years, a number of methods have been developed that allow
researchers to characterize iris colour quantitatively. Liu et al. (2010) obtained hue and saturation
values from iris photographs, with hue being used to represent the type of melanin and saturation
being used to represent the amount of melanin in the iris. Andersen et al. (2013) developed a PIE
(Pixel Index of the Eye) score that was computed using the number of pixels labelled brown and
the number of pixels labelled blue in photographs of the iris. Recently, we extracted a colour
measurement in CIE 1976 L*a*b* (CIELAB) colour space from a 256 by 256 pixel square
isolated using high-resolution photographs of the iris (Edwards et al., 2012). These methods go
beyond the traditional categorical classification systems that have been used in the past (i.e.
‘green’ ‘blue’ ‘brown’) and provide a means of studying iris colour variation in populations with
more homogenously coloured irises. However, thus far, they have not yet been broadly applied
to global populations.
 53

Using quantitative methods to study the genetic basis of iris colour in populations of non-
European ancestry would greatly contribute to our overall understanding of global iris colour
variation. At present, we have a limited understanding of the markers responsible for
intermediate iris colours. This is reflected in the fact that most eye colour prediction algorithms
perform poorly and inconsistently when looking at intermediately coloured eyes (Dembinski and
Picard, 2014; Pneuman et al., 2012; Yun et al., 2014). In addition, identifying novel variants
associated with iris colour variation in populations of non-European ancestry would provide
valuable information about the genetic basis of skin and hair colour in these regions, given the
known pleiotropic effects of some genetic markers on pigmentary traits. Studying iris colour may
also be of biomedical interest. Eye colour has been found to be associated with a number of
ocular disorders (Mitchell et al., 1998; Wakamatsu et al., 2008). The most well-known of these is
perhaps age-related macular degeneration (AMD). This disease, which primarily affects older
adults, results from damage to the retina and can lead to vision loss and blindness. Age-related
macular degeneration has been strongly linked to population-specific genetic effects (Frank et
al., 2000). However, within populations eye colour may be one of the determinants of AMD. In
European samples, it has been found that individuals with blue eyes have a higher risk of
developing AMD (Frank et al., 2000; Mitchell et al., 1998). Similarly, the incidence of primary
angle-closure glaucoma in East Asian populations may be associated with variation in brown iris
colour. A recent study found that individuals with dark brown eyes in a Malaysian population
living in Singapore had a narrower angle closure than individuals with light brown eyes
(Sidhartha et al., 2014). Thus, it is not only necessary to investigate the genes responsible for the
difference between blue, brown and intermediate phenotypes in European populations, but also
to look at the genes that modulate brown iris pigmentation in other regions as well.
In this study, we have three primary goals: 1/ To improve our quantitative method of
measuring iris colour in order to better capture iris colour variation in populations of diverse
ancestry; 2/ To look at the association between putative pigmentation markers and iris colour in a
sample of European, East Asian, and South Asian ancestry; 3/ To look at the association between
pigmentation markers and central heterochromia within the European and South Asian samples.
 54

3.3 Materials and Methods

3.3.1 Sample Collection
Between 2012 and 2014, 1465 healthy volunteers of East Asian, European and South
Asian ancestry volunteered for a research study on human pigmentation variation. All
participants ranged between 18 and 35 years of age and were recruited using online and print
advertisements directed towards the University of Toronto student community. A personal
questionnaire was administered to each participant to determine their age, sex, self-described eye
colour and whether or not they had been diagnosed with any pigmentation-related diseases or
disorders.
Biogeographical ancestry was determined using information from the personal
questionnaire, which inquired about the ancestry, place of birth and first language of each
participant’s maternal and paternal grandparents. Individuals who stated that all of their
grandparents originated in China, Japan, Korea or Taiwan were categorized as East Asian, and
individuals who stated that all of their grandparents originated in Pakistan, India, Bangladesh or
Sri Lanka were categorized as South Asian. Individuals were categorized as European if all of
their grandparents originated in any country in Europe, other than Turkey. Admixed individuals
who had grandparents from two different regions (i.e. East Asia and Europe) were excluded from
the analysis. When information about the grandparents was not known, the self-described
ancestry of both parents was used to assess biogeographical ancestry. In addition to the 1465
participants that were included in this paper, 308 additional volunteers were recruited and
excluded because they could not be categorized as East Asian, European or South Asian using
this criteria.
This study was approved by the University of Toronto Research and Ethics Board
(Protocol Reference #27015), and all participants were required to provide written informed
consent.

3.3.2 Acquisition of Photographs

A photograph of each participant’s right eye was taken using a Miles Research
Professional Iris Camera (Miles Research, United States). This camera consists of a Fujifilm
Finepix S3 Pro DSLR 12-megapixel camera body attached to a 105-mm Nikkor lens. A
 55

biometric coaxial cable was used to deliver light to the iris at a constant light temperature to
maintain colour and brightness fidelity and reduce the impact of ambient light. The camera body
needed to be replaced after the first 552 participants and a camera body with an identical make
and model was acquired. We could not adequately assess if the photographs taken with the first
and second camera bodies were identical as we did not have a large enough sample of
individuals whose eyes had been photographed using both cameras. Therefore, we split our
sample into two groups. All photographs were taken with an ISO of 200, a shutter speed of
1/125” and an aperture of f19.
Photographs were initially acquired in RAW format and later converted to JPEG format
using Adobe Camera Raw in Adobe Photoshop CS5 (Adobe Systems Incorporated, United
States). They were resized from 3043 x 2036 pixels to 1200 x 803 pixels to optimize the
processing of iris colour. The white balance was set to flash, the contrast and blacks levels were
set to zero, and all other camera defaults were preserved for each conversion.

3.3.3 Acquisition of Iris Colour

One of the authors (D.C.) designed an eleven step Web-based application to characterize
iris structure and acquire iris colour from photographs of the eye. The web application can be
accessed at http://iris.davidcha.ca/. Accounts can be set up for interested users by request. Only
the first six steps are necessary to obtain a measurement of iris colour. In the first step, the user
must note whether or not the iris is obstructed to an extent that could affect colour and structure
measurements. In steps 2-6, the user is asked to identify the approximate centre point of the iris,
the approximate centre point of the pupil and then draw a best fit circle around the scleral,
pupillary and collarette boundaries.
After the scleral, pupillary and collarette boundaries are defined, the application is able to
automatically extract a measurement of average eye colour. We decided to estimate iris colour
from a 60˚ angle wedge taken from the left side of the iris. This region was chosen because it
was least likely to be obscured by eyelids, eyelashes or reflections in our sample. To isolate this
wedge, the program defines a start point located at the centre of the iris and moves to a point on
the scleral boundary located 240˚ clockwise from the top of the iris. It then follows an arc that
stretches for 60˚ until it reaches the point that is 300˚ from the top of the iris. This point is
connected to the centre of the iris, which demarcates an isolated wedge. These steps are repeated
 56

for the pupillary boundary and the collarette boundary so that the pupil can be completely
excised from the wedge and the pupillary and ciliary zones can be separated. Two images are
then saved in PNG format: the portion of the wedge that represents the ciliary zone and the
portion of the wedge that represents the pupillary zone (Figure 3-2). After processing all iris
photographs, we manually scanned the wedges for any evidence of obstruction or incorrect
cropping.
To obtain a measurement of average iris colour, the application counts all of the pixels
located in the wedge (consisting of both the ciliary and pupillary zones) and determines a Red,
Green and Blue (RGB) value for each individual pixel. The average RGB value of the entire
wedge is calculated by adding up the R, G and B values for each pixel, and then dividing this
value by the total number of counted pixels. We chose to describe iris colour in CIE 1976
L*a*b* (CIELAB) colour space (McLaren, 1976). CIELAB is a colour system that was designed
to characterize colour across three different coordinates. The L* coordinate represents the
lightness dimension and ranges from 0 to 100, with 0 being black and 100 being white. The a*
and b* coordinates represent variation in colour, with negative values of a* indicating green and
positive values of a* indicating red, and negative values of b* indicating blue and positive values
of b* indicating yellow. To convert our RGB measurements into CIELAB colour space, the
application first transformed the RGB coordinates into XYZ coordinates and then into L*, a* and
b* coordinates using the equations provided by EasyRGB (Logicol Colour Technology, United
States). For each conversion, the illuminant was set to D55 and the observer was set to 2 degrees.
These are standard conversion settings when using flash photography. This process ultimately
provides an average colour estimate for ciliary zone, the pupillary zone and the entire iris in
CIELAB colour space.
We were also interested in quantifying the total amount of central heterochromia in the iris.
To do this, we used CIEDE2000 (ΔE), a colour metric that looks at the difference between two
colours in CIELAB colour space (McLaren, 1976). The Web application used the equations
provided by EasyRGB (Logicol Colour Technology, United States) to calculate the difference
between the CIELAB values in the pupillary and ciliary zones.
After acquiring estimates of iris colour from all 1465 irises, the Web application was used to
output an Excel spreadsheet which contained the average colour of the entire iris, the average
colour of the ciliary zone, and the average colour of the pupillary zone in RGB and CIELAB
 57

colour space and the ΔE between the ciliary and pupillary zones. The spreadsheet also produced
information on how many pixels were counted in each eye during the calculation of iris colour.
All iris analyses were carried out by M.E.

3.3.4 Marker Selection and Genotyping

A 2-mL saliva sample was obtained from each participant using the Oragene˖DNA (OG-
500) collection kit (DNA Genotek, Canada). All participants were instructed not to eat, drink or
smoke for at least 30 min prior to obtaining the sample to ensure maximal sample purity. DNA
was isolated from each sample using the protocol provided by DNA Genotek and eluted in 500
mL of TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) Buffer.
We selected fourteen markers for genotyping that have either previously been associated
with iris colour in European populations or been associated with some other pigmentation
phenotype (i.e. skin colour) in East or South Asia (Eaton et al., 2015; Edwards et al., 2012;
Eiberg et al., 2008; Graf et al., 2005; Kayser et al., 2008; Liu et al., 2010; Rebbeck et al., 2002;
Sturm et al., 2008; Walsh et al., 2011). These markers consisted of HERC2 rs12913832, OCA2
rs1800407, SLC24A4 rs12896399, SLC45A2 rs16891982, SLC24A5 rs1426654, TYR rs1393350,
IRF4 rs12203592, DSCR9 rs7277820, TYRP1 rs1408799, NPLOC4 rs9894429, LYST rs3768056,
ASIP rs6058017, OCA2 rs1800414 and OCA2 rs74653330. Markers were only included for a
population if the minor allele frequency (MAF) exceeded 0.05. The exception to this was OCA2
rs74653330, which has a low MAF (<0.04) in East Asia, but has been found to have a strong
effect on skin pigmentation variation (Eaton et al., 2015).
All DNA samples were sent to LGC Genomics (United States) for genotyping. LGC
Genomics uses a KASP based genotyping method that combines allele-specific amplification
with FRET (fluorescent resonance energy transfer) technology. Twenty-nine samples were sent
as blind duplicates and 14 samples were sent as blanks to validate the quality of the genotyping
results. The concordance rate for both blind duplicates and blanks was 100%.

3.3.5 Statistical Analysis

Unless otherwise noted, all statistical analyses were carried out using PLINK
(http://pngu.mgh.harvard.edu/~purcell/plink/), a genome analysis program designed to look at
the association between genotype and phenotype data. We carried out the following analyses: 1/
 58

A test of deviations from Hardy-Weinberg proportions for each marker included in the East
Asian, European and South Asian sample. 2/ A linkage disequilibrium test to determine the
amount of linkage between the markers. 3/ An exploratory analysis to look at the association
between age and sex and the four colour space measurements (L*, a*, b* and ∆E). As no
significant associations were noted between iris colour and age or sex, these variables were not
included in the downstream statistical analyses. 4/ A linear regression analysis to determine the
effect of each SNP on the L*, a* and b* dimensions of CIELAB colour space. In each
population, we only looked at SNPs that reached a MAF of at least 0.05 (with the exception of
OCA2 rs74653330 in the East Asian sample). For each SNP, we used a genotypic model, which
reports a P-value and beta coefficient independently for the minor allele homozygote and the
heterozygote, with respect to the major allele homozygote. We also explored whether there was
evidence for deviations from an additive model of inheritance for any of the markers. Deviations
were considered significant if the statistic reporting deviation from additivity (DOMDEV)
yielded a P-value of < 0.05 for both cameras. Each population was divided into two groups
(representing the first camera body and the second camera body) and the regression was carried
out individually on each group to account for differences in colour and brightness between the
two cameras. 5/ A linear regression analysis in the European and South Asian samples
conditioning on the effect of HERC2 rs12913832 and a linear regression analysis in the East
Asian sample conditioning on the effect of OCA2 1800414. Again, this analysis was performed
separately on each camera body. 6/ A meta-analysis of the linear regression results from both
camera bodies. This meta-analysis was carried out both before and after conditioning on HERC2
rs12913832 in the European and South Asian samples and OCA2 rs1800414 in the East Asian
sample. For each marker, we reported the P-value of the association, the effect size (beta) and the
P-value for Cochran's Q statistic (a measure of the heterogeneity between the two groups used in
the meta-analysis). After Bonferroni's correction for multiple comparisons, associations were
significant in the East Asian population if P < 0.00714, in the European population if P <
0.00454 and in the South Asian population if P < 0.00500. 7/ A linear regression analysis in the
European and South Asian samples looking at the association between the colour difference
between the pupillary and ciliary zones (∆E) and the putative pigmentation SNPs. This analysis
was carried out separately on each camera body. 8/ A meta-analysis on the linear regression
results for ∆E from each camera body. The P-value of the association, the effect size (beta) and
 59

the P-value for Cochran's Q statistic were reported for each marker. 9/ Four months after the
initial iris colour classification, intra-rater reliability measurements were carried out by M.E and
inter-rater reliability measurements were carried out by D.C on 40 random irises. The intraclass
correlation coefficient between the original and repeated iris colour measurements was
calculated in IBM Statistics SPSS (version 20.0, SPSS Incorporated, United States) using a two-
way random model with absolute agreement for the L*, a*, b* and ∆E measurements.

3.4 Results
In total 474 East Asians, 624 Europeans and 367 South Asians were included in the
study. Six East Asians, four Europeans and four South Asians were unable to remove their
contact lenses for the photograph and were excluded from the analysis. Two participants of
South Asian descent were removed from the study because they had an obstructed iris wedge. No
iris obstruction could be observed in any of the other participants. A final participant of South
Asian descent was excluded because the participant reported having a form of albinism that
affected ocular pigmentation.
Inter- and intra-rater reliability measurements were excellent for all four colour space
measurements (Table 3-1). Substantial dispersion across CIE 1976 L*a*b* (CIELAB) colour
space could be seen in all three populations (Figure 3-1). Blue eyes tend to show high L* values
and negative a* and b* values, while brown eyes have low L* values and high a* and b* values.
Due to the arc shape in which irises are distributed across CIELAB colour space, dark brown
eyes have a lower L*, a* and b* values than lighter brown eyes (Figure 3-1). Green eyes tend to
have a* values that are intermediate between blue and brown irises. The greatest amount of iris
colour variation was found in the European sample. However, there was also extensive variation
in the South and East Asian groups. The average, minimum and maximum L*, a*, b* and ∆E
measurements for all 1448 participants is presented in Table 3-2. There is a noticeable difference
between the two camera bodies in the L* dimension, confirming that the bodies have some
variation in brightness. As a* and b* are colour dimensions that were designed to be independent
from brightness, it is not surprising that there is less variation in these coordinates (McLaren,
1976). However, all three dimensions appear to show somewhat higher values in photographs
taken with the first camera compared to those taken with the second camera.
 60

In each population, we looked at the association between markers purported to play a role
in pigmentation variation and the L*, a* and b* coordinates. In the European and South Asian
populations, the association between each of these markers and ∆E was also examined. With the
exception of OCA2 rs74653330 in the East Asian sample, we only looked at polymorphisms that
had a minor allele frequency of at least 0.05. For the European population, this included HERC2
rs12913832, OCA2 rs1800407, SLC24A4 rs12896399, SLC45A2 rs16891982, TYR rs1393350,
IRF4 rs12203592, DSCR9 rs7277820, TYRP1 rs1408799, NPLOC4 rs9894429, LYST rs3768056
and ASIP rs6058017. In the East Asian population, this included markers SLC24A4 rs12896399,
DSCR9 rs7277820, NPLOC4 rs9894429, LYST rs3768056, ASIP rs6058017, OCA2 rs1800414
and OCA2 rs74653330. Lastly, in the South Asian population this included markers HERC2
rs12913832, SLC24A4 rs12896399, SLC45A2 rs16891982, SLC24A5 rs1426654, TYR
rs1393350, IRF4 rs1126809, DSCR9 rs7277820, TYRP1 rs1408799, NPLOC4 rs9894429, LYST
rs3768056 and ASIP rs6058017. Linkage disequilibrium was low (r2 < 0.1) between all pairs of
markers located in the OCA2/HERC2 region. Most markers showed no deviations from Hardy-
Weinberg equilibrium. However, DSCR9 rs7277820 showed minor deviations in the European
(P = 0.0169) and South Asian (P = 0.0473) samples and SLC24A5 rs1426654 showed major
deviations (P = 1.199 x 10-5) in the South Asian sample. Twenty-five individuals (5 East Asians,
10 Europeans and 10 South Asians) were missing genotypes for more than 20% of the
polymorphisms and were excluded from the genetic analysis.
As the two camera bodies appeared to show some variation in the L*, a* and b*
dimensions, we chose to carry out a meta-analysis on the two data sets. A linear association
analysis was first performed in each population to look at the association between each SNP and
the L*, a* and b* values for each camera-body. In the European and South Asian populations, an
additional analysis was carried out for the ∆E measurement. The meta-analysis function in
PLINK, using a fixed effects model, was then used to determine the combined significance and
effect size in each population.
The results of the meta-analysis in the European population can be found in Table 3-3A.
HERC2 rs12913832 was strongly associated with the L*, a* and b* dimensions of colour space,
with the ancestral ‘A’ allele decreasing the L* and increasing the a* and b* coordinates. For this
marker, the a* and b* dimensions showed significant deviations from additivity, indicating that
these two colour dimensions are best modelled using a dominant/recessive mode of inheritance
 61

(e.g. the ancestral ‘A’ allele is dominant over the derived ‘G’ allele). In contrast, the L*
coordinate showed no significant deviations from additivity. SLC24A4 rs12896399, SLC45A2
rs16891982 and IRF4 rs12203592 were significantly associated with the a* and b* dimensions.
For all three markers, the derived alleles decreased both coordinates. Although we had no
individuals who were homozygous for the derived genotype in our sample, OCA2 rs1800407 was
significantly associated with an increase in the b* dimensions of colour space. Apart from
HERC2 rs12913832, none of the markers showed any significant deviations from an additive
mode of inheritance.
As HERC2 rs12913832 is known to be the primary variant responsible for controlling the
difference between blue and brown eye colour, we ran the analysis again after conditioning for
this marker (Table 3-3B). Most polymorphisms showed similar effects after conditioning.
Interestingly, although OCA2 rs1800407 was no longer associated with the b* dimension of
colour space, it was now associated with the L* and a* dimensions, with the derived allele
increasing the L* coordinate and decreasing the a* coordinate. Lastly TYR rs1393350 was now
associated with the L* dimension of colour space, with the derived ‘A’ allele decreasing this
coordinate.
In the South Asian sample, HERC2 rs12913832 was strongly associated with the L*, a*
and b* dimensions of colour space (Table 3-4A). The derived ‘G’ allele was associated with an
increase in the L* and b* dimensions, and a decrease in the a* dimension. HERC2 rs12913832
showed significant deviations from additivity for all three dimensions of colour space indicating
that it is best modelled using a dominant/recessive mode of inheritance. SLC24A5 rs1426654 was
strongly associated with the L*, a* and b* dimensions of colour space, and each copy of the
ancestral allele was responsible for a decrease in all three coordinates. The only other marker
associated with iris colour in the South Asian sample was LYST rs3768056, with the derived ‘G’
allele increasing both the a* and b* coordinates. Conditioning on HERC2 rs12913832 in this
population had very little effect on both significance levels and effect size (Table 3-4B).
In the East Asian population, only OCA2 rs1800414 and OCA2 rs74653330 were
associated with iris colour variation (Table 3-5A). OCA2 rs1800414 had a significant effect on
the L*, a* and b* dimensions of colour space, with each copy of the ancestral allele decreasing
all three coordinates. Although no participants were homozygous for the derived allele at OCA2
rs74653330, having one copy significantly increased both the a* and b* coordinates. After
 62

conditioning for OCA2 rs1800414, the effect of OCA2 rs74653330 became stronger, and this
marker was now associated with all three dimensions of colour space (Table 3-5B). For this
marker, the derived ‘A’ allele increases the L*, a* and b* coordinates. Neither marker showed
any significant deviations from an additive mode of inheritance.
The only marker that was associated with ∆E in the European sample was HERC2
rs12913832. This marker had an additive effect, with each copy of the ancestral allele decreasing
the colour difference between the ciliary and pupillary zones (Table 3-3A). In the South Asian
sample, both HERC2 rs12913832 and SLC24A5 rs1426654 were associated with ∆E. HERC2
rs12913832 showed significant deviations from additivity, suggesting that it is best modelled
using a dominant/recessive mode of inheritance. In contrast, SLC24A5 did not show any
deviation from additivity. After conditioning for the effects of HERC2 rs12913832, SLC24A5
rs1426654 was no longer associated with ∆E.

3.5 Discussion
In this study, we present an improved method for measuring iris colour in diverse
populations and looked at the association between 14 SNPs purported to play a role in global
pigmentation variation and eye colour in a sample of East Asian, European and South Asian
ancestry. CIE 1976 L*a*b* (CIELAB) colour space is an ideal system for measuring iris colour.
This colour space was designed to capture perceptible differences in colour variation, which is
important when looking at visible human traits that are typically distinguished by colour
difference (McLaren, 1976). Each unit change in CIELAB colour space is visible to at least 50%
of observers (Kuehni and Marcus, 1979). CIELAB also characterizes colour across three
coordinates that closely parallel visual differences in eye colour. The L* dimension represents a
brightness dimension and ranges from 0 to 100, with 0 being black and 100 being white. The a*
and b* dimensions represent variation in colour, with negative values of a* indicating green and
positive values of a* indicating red, and negative values of b* indicating blue and positive values
of b* indicating yellow. CIELAB also has a colour metric, ∆E that can be used to quantify the
difference between two points in colour space (McLaren, 1976). This allows us to investigate
colour variation between different regions of the iris. Unlike previous studies, we selected a
wedge to represent iris colour instead of the entire iris. We made this decision because the left
 63

quadrant of the iris was least likely to be obstructed in our sample. In addition, if we chose to
use the entire iris but crop out regions of obstruction, such as eyelashes and eyelids, it would bias
the colour of the iris towards the pupillary region. Although several automated methods have
been developed to facilitate the isolation of the iris from photographs of the eye (Liu et al., 2010;
Pietroni et al., 2014), we chose to manually define the boundaries of the iris. This allowed us to
separate the eye into different regions, and look at the difference in colour between the ciliary
and pupillary zones.
Contrary to previous studies, we did not find any association between iris colour and age
or sex in the East Asian, European, or South Asian sample populations. Iris colour has been
tentatively associated with sex in a small number of European populations (Pietroni et al., 2014).
It has been suggested that this association may be highly population-specific. As we looked at a
broad range of biogeographical ancestries across Europe and had a small number of male
participants, it is not surprising that this association was absent in our sample. Similarly, the lack
of an association between age and eye colour is not surprising, as we sampled from a relatively
narrow age range compared to other studies that included participants that ranged from children
to seniors (Bito et al., 1997).

3.5.1 Iris Colour in European Populations

The European sample showed the greatest variation and spread across CIELAB colour
space. This is to be expected, given that eye colour in this population ranges from blues to greens
to browns. Interestingly, very few individuals of European descent fell into the region of the
colour space occupied by the darkest brown irises. Instead, when individuals of European
ancestry had brown eyes, they tended to be lighter. This suggests that there may be fixed genetic
markers in this population that modulate the intensity of brown iris pigmentation.
In recent years, a number of iris colour prediction models have been developed by
forensic groups (Ruiz et al., 2014; Spichenok et al., 2011; Walsh et al., 2011, 2013). These
models predict the iris colour of an individual based on their genotype at a set of established iris
pigmentation markers. The best-known of these is perhaps IrisPlex, a forensically validated
genetic assay and prediction model that uses 6 iris colour polymorphisms (HERC2 rs12913832,
OCA2 rs1800407, SLC24A4 rs129896399, SLC45A2 rs16891982, TYR rs1393350, IRF4
rs12203592) to estimate blue, brown and intermediate eye colour from unknown DNA samples
 64

(Walsh et al., 2011, 2013). A second system suggested that the inclusion of additional markers
from the HERC2-OCA2 region could improve the prediction of intermediate iris colour
phenotypes (Ruiz et al., 2014). A third system, known as 7-Plex was designed to estimate both
skin and iris colour (Spichenok et al., 2011). This system uses three SNPs (HERC2 rs12913832,
SLC45A2 rs16891982, IRF4 rs12203592) associated with iris and skin colour, three SNPs
associated with genetic ancestry (MC1R rs885479, ASIP rs6119471 and OCA2 rs1545397) and
one SNP associated with skin colour alone (SLC24A5 rs1426654) to predict brown, not brown,
not blue and green eye colour. Apart from the markers that are currently incorporated into iris
prediction models, several studies have identified additional polymorphisms that may have an
effect on iris colour. TYRP1 rs1408799 has been associated with the difference between ‘blue
and not blue’ eyes in a Northern European population and ASIP rs6058017 may play some role
in modulating brown iris colour (Kanetsky et al., 2002; Sulem et al., 2008). In addition, three
new potential iris colour predictors (DSCR9 rs7277820, NPLOC4 rs9894429, LYST rs3768056)
were recently identified in a genome-wide association study (Liu et al., 2010). However, as of
yet, none of these markers have been incorporated into any forensic algorithms.
In our sample, HERC2 rs1291832 had the strongest effect on eye colour across all three
dimensions of colour space. HERC2 rs12913832 has been traditionally characterized as having a
dominant/recessive mode of inheritance, where heterozygotes and homozygotes for the ancestral
allele have brown or intermediate eyes and homozygotes for the derived allele have blue eyes.
This was largely reflected in our sample, as both the a* and b* dimensions of colour space
showed significant deviations from additivity. In contrast, the effect of HERC2 rs12913832 on
the L* coordinate was largely additive, with one copy of the ancestral allele increasing the
brightness in the eye by 7.143 units and two copies increasing it by 12.586 units. This suggests
that the inheritance of this polymorphism in European populations is more complex than
traditionally modelled. Although this marker may control the difference between blue and brown
eye colour, it also has more subtle effects on the overall lightness of the iris. The additive nature
of HERC2 rs12913832 is supported by recent functional studies (Cook et al., 2009). Cultured
melanocyte strains that were homozygous for the ancestral allele had more melanin content than
homozygotes for the derived allele, while heterozygotes had intermediate amounts. Thus, it
appears that HERC2 rs12913832 may have both dominant and additive effects on iris colour
variation, depending on which dimensions of colour space are being studied.
 65

In addition to HERC2 rs12913832, our study supports previous research showing that
OCA2 rs1800407, SLC45A2 rs16891982, SLC24A4 rs12896399, IRF4 rs12203592 and TYR
rs1393350 are the primary determinants of iris colour in European populations (Eiberg et al.,
2008; Kayser et al., 2008; Liu et al., 2010; Sturm et al., 2008; Walsh et al., 2011). All of these
markers were significantly associated with iris colour in at least one dimension either before or
after conditioning for HERC2 rs12913832. We were not able to identify an association between
iris colour variation and any of the other markers. However, after conditioning for HERC2
rs12913832, having two copies of the derived allele at TYRP1 rs1408799 showed a borderline
significant association with the L* (P = 0.054, β = -1.629) and a* (P = 0.007, β = 3.091)
dimensions of colour space and having one copy of the ancestral allele at ASIP rs6058017
showed a borderline significant association (P = 0.007, β = 2.942) with the b* dimension. It is
possible that our sample size was too small to pick up these associations as significant.

3.5.2 Iris Colour in South Asian Populations

The South Asian sample showed considerably less diversity across CIELAB colour space
than the European sample, and the vast majority of participants in this sample had eyes that
would be traditionally described as brown. However, there were still a small number of
participants that fell into the intermediate region of colour space.
At present, very little research has been devoted to the study of iris colour in populations
of South Asian ancestry. HERC2 rs12913832 is present at low frequencies in South Asia
(Edwards et al., 2012). In this population, this polymorphism appears to modulate variation in
brown iris colour. In a recent study on brown-eyed individuals of South Asian ancestry, one copy
of the derived ‘G’ allele was found to significantly increase the L*, a* and b* dimensions of
CIELAB colour space (Edwards et al., 2012). However, there was only one homozygote for the
derived allele in that study. Apart from HERC2 rs12913832, the role that other iris colour
markers play in South Asia has not yet been tested. Both OCA2 rs1800407 and IRF4 rs12203592
have very low minor allele frequencies (MAF) in this population and are unlikely to contribute to
normal pigmentation variation.
In our South Asian sample, we were able to replicate the association between HERC2
rs12913832 and iris colour variation. This marker showed significant deviations from additivity
across all three dimensions of colour space. Having two copies of the derived allele increased L*
 66

values by 17.870 and decreased a* values by 10.856 relative to the ancestral homozygotes. These
effects are similar to what was observed in the European sample. However, there is a clear
difference in the effect of HERC2 rs12913832 in the b* dimension of colour space in Europeans
and South Asians. In Europeans, the derived homozygote is associated with a very strong
reduction in the b* values with respect to the ancestral homozygote (~20 units). In contrast, in
the South Asian sample, the derived homozygote has no significant effect on b* values. In fact,
in this sample, derived homozygotes have slightly higher b* values than the ancestral
homozygote. In our South Asian sample, there were only 7 individuals homozygous for the
derived HERC2 rs12913832 allele. These individuals had an average b* value between 8 and 9
(depending on the camera), indicative of intermediate colour irises, in contrast to the average b*
values observed in individuals homozygous for the derived allele in Europe, which is around -7,
within the blue region of the colour space. Additionally, the South Asian individuals
homozygous for the ancestral allele have much lower b* values (average 7-8, depending on the
camera) than the European ancestral homozygotes (average 13-15, depending on the camera).
This is primarily due to the fact that the South Asian individuals have darker brown irises than
the Europeans, and dark brown colours have lower b* values than light brown colours. The
difference in the effect of the HERC2 rs12913832 polymorphism in Europe versus South Asia
strongly suggests that there are other polymorphisms modifying the effect of this marker in both
populations. Our hypothesis is that the effect of HERC2 rs12913832 may be modified by other
variants that are common in Europe, but not in South Asia. Exploring this would require much
larger samples including substantial numbers of derived homozygotes in South Asia, to explore
potential interactions of HERC2 rs12913832 with other pigmentation variants.
The only other marker that had a strong effect on iris pigmentation in the South Asian
sample was SLC24A5 rs1426654. SLC24A5 rs1426654 is a non-synonymous polymorphism
(A111T) that results in the substitution of a guanine to an adenine in exon three of SLC24A5.
This marker shows strong signals of positive selection in Europe and South Asia, and has been
associated with skin pigmentation variation in South Asians and also in admixed individuals of
African-European descent (Lamason et al., 2005; Stokowski et al., 2007). Functional analyses
have found that homozygotes for the ancestral ‘G’ allele have 2.2 fold higher melanin content
and 1.7 higher TYR activity than homozygotes for the derived ‘A’ allele (Cook et al., 2009).
This is not the first study to suggest that this marker may play some role in iris pigmentation
 67

variation. Beleza et al. (2013) found that this marker was strongly associated with iris colour in
an admixed population of African-European ancestry. In our study, SLC24A5 rs1426654 was one
of the main determinants of iris colour in South Asians. This marker showed an additive mode of
inheritance for all three dimensions of colour space, with one copy of the ancestral allele
decreasing the L*, a* and b* values by 2.337, 2.118 and 3.512 respectively and a second copy
decreasing the values by 3.673, 3.905 and 5.882 relative to the homozygous-derived genotype.
This suggests that the ancestral ‘G’ allele is responsible for darkening brown iris colour in this
population. It is important to note that in our South Asian sample, SLC24A5 rs1426654 showed
significant deviations from Hardy-Weinberg proportions. This is not surprising as there is well-
documented population stratification in South Asia and this marker is under strong global
selection (Izagirre et al., 2006; McEvoy et al., 2006; Stokowski et al., 2007). Although it is
possible that the association between this marker and iris colour is a secondary effect resulting
from population stratification, it is not likely. SLC24A5 rs1426654 has well-established
functional effects on the synthesis of melanin and was the only marker that showed major
deviations from Hardy-Weinberg proportions in this group.
Although SLC24A5 rs1426654 and HERC2 rs12913832 were the primary determinants of
iris colour in the South Asian sample, LYST rs3768056 also showed a much smaller association
with the a* and b* dimensions of CIELAB colour space, with the derived ‘G’ allele increasing
both coordinates. The association between LYST rs3768056 and iris pigmentation in the South
Asian population is interesting, as this is one of the new putative pigmentation markers that was
suggested for the European population (Liu et al., 2010). This marker may be a stronger
predictor of iris colour in populations of non-European ancestry. In addition, having one copy of
the derived ‘T’ allele at TYRP1 rs1408799 showed a borderline significant effect on the a* and
b* dimensions of colour space, with the derived ‘C’ allele increasing both coordinates.

3.5.3 Iris Colour in East Asian Populations

Iris colour showed the most limited distribution in the East Asian sample. Blue iris colour
was completely absent in this group, and only a small number of eyes had an intermediate or
green phenotype. The majority of irises fell into the region of colour space associated with
various shades of brown. Average iris colour was comparable to the South Asian sample.
 68

Very little is known about pigmentation phenotypes in East Asia. Light skin pigmentation
appears to have evolved independently in Europe and East Asia, and there is very little overlap in
the markers responsible for pigmentation diversity between these two populations (Eaton et al.,
2015; Edwards et al., 2010; McEvoy et al., 2006; Norton et al., 2007). The majority of
polymorphisms associated with light skin pigmentation in Europe and South Asia are absent in
East Asia. In addition, the only putative iris pigmentation markers that have a MAF allele
frequency greater than 0.05 are SLC24A4 rs129896399, DSCR9 rs7277820, NPLOC4 rs9894429,
LYST 3768056 and ASIP rs6058017. Thus, the genetic basis of iris pigmentation in East Asian
populations is likely very different than in other groups.
The strongest determinants of iris pigmentation diversity in our sample were OCA2
rs1800414 and OCA2 rs74653330. OCA2 rs1800414 had an additive effect on all three
dimensions of CIELAB colour space. One copy of the ancestral ‘A’ allele decreased the value of
L*, a* and b* by 1.460, 1.318 and 1.757 respectively, and two copies of the ancestral ‘A’ allele
decreased the value by 2.793, 2.831 and 4.024. This suggests that the ancestral allele for this
marker plays a role in darkening iris colour. Although we did not have any participants who
were homozygous for the derived ‘A’ allele for OCA2 rs74653330, having one copy of the
derived allele increased both the a* and b* values. After conditioning for OCA2 rs1800414, the
effect of OCA2 rs74653330 became even stronger, with heterozygotes having L*, a* and b*
values that were 2.1509, 2.6713, 3.9180 higher respectively than the homozygous ancestral
genotype.
OCA2 rs1800414 is a non-synonymous polymorphism (His615Arg) that is present in
very high frequencies in East Asia (Donnelly et al., 2012; Yuasa et al., 2007). The derived ‘G’
allele is strongly associated with lower skin melanin levels, with each copy of the ‘G’ allele
decreasing the skin melanin index by approximately 0.9 units (Edwards et al., 2010). OCA2
rs74653330 is another non-synonymous (Ala481Thr) polymorphism that is largely restricted to
East Asia (Eaton et al., 2015). The derived ‘T’ allele has a low frequency in this region. Each
copy of the derived ‘T’ allele has been found to reduce the skin melanin index by approximately
1.9 units (Eaton et al., 2015). The OCA2 gene appears to be under strong selective pressure in
East Asia (Donnelly et al., 2012; Eaton et al., 2015; Edwards et al., 2010; Lao et al., 2007).
Given the role that the derived allele plays in lightening skin pigmentation for both of these
markers, it is likely that their influence on iris colour is only secondary.
 69

3.5.4 Central Heterochromia

Central heterochromia is a trait that is prevalent in populations with lighter coloured
irises (Larsson et al., 2011). It usually takes the form of a blue/green iris with a ring of darker
pigment around the pupil. When central heterochromia is present in the iris, the darker colour is
typically restricted to the pupillary zone of the iris, although it occasionally extends into the
ciliary zone. To characterize the magnitude of central heterochromia in the iris, we looked at the
difference in iris colour between the ciliary and pupillary zones. We used the ∆E metric to
quantify this trait, as it was designed to capture perceptible differences in colour across CIELAB
colour space (Kuehni and Marcus, 1979).
In our sample, the degree of central heterochromia was largely population-dependent,
with the European group showing the highest prevalence of this trait (Table 3-2). There was
some heterochromia in the South Asian sample, however most irises had very low ∆E values.
The East Asian group showed the least colour difference. As heterochromia was largely absent in
our East Asian sample, we only looked at the association between the putative pigmentation
markers and heterochromia in the European and South Asian samples.
The only polymorphism that was associated with central heterochromia in the European
sample was HERC2 rs12913832. The effect of this marker appeared to be additive, with one
copy of the ancestral allele decreasing the colour difference by 1.220, and two copies of the
ancestral allele decreasing the colour difference by 3.828 relative to the homozygous derived
genotype. HERC2 rs12913832 was also associated with central heterochromia in the South
Asian population. However, in this group, it appears to be better modelled using a
dominant/recessive mode of inheritance, with one copy of the derived allele increasing the colour
difference by 1.305 and two copies increasing the colour difference by 7.520 relative to the
homozygous ancestral genotype. It is interesting to note that heterozygotes for this marker in
the European and South Asian population show very different amounts of colour difference
(Table 3-6). In the European sample, the average ∆E ranged between 6.668 and 8.128 when
HERC2 rs12913832 is in the heterozygous state. In the South Asian sample, however, the
average ∆E only ranged between 3.634 and 3.722. In contrast, when this marker is found in the
derived homozygous state both groups showed closer amounts of colour difference. It is well
known that HERC2 rs12913832 can have an effect on intermediate phenotypes. (Spichenok et
al., 2011; Walsh et al., 2011, 2012). In particular, individuals who are heterozygous for this
 70

marker commonly have iris colours that range from blues, to greens to browns. However, it is
likely that there are other polymorphisms interacting with HERC2 rs12913832 to produce
variation in central heterochromia. This is particularly evident when looking at the average ∆E
in heterozygotes for this marker in the European and South Asian groups. Although
heterozygotes in the European sample show substantial amounts of central heterochromia, this is
not the case in the South Asian sample. Therefore, there are likely other polymorphisms
modulating the effect of this marker in both of these groups.
Larsson et al. (2011) identified an association between SLC24A4 rs12896399 and
heterochromatic variation in a genome-wide association study performed in a sample of
European ancestry living in Australia. However, we were not able to replicate this finding in our
study. This may be because they looked at both the extent and spread of central heterochromia,
and we only looked at colour difference. Interestingly, we did find a borderline significant effect
between ∆E and TYR rs1393350 after conditioning for HERC2 rs12913832 in the European
sample, with the derived ‘A’ allele increasing the amount of heterochromia in the eye. TYR
rs1393350 has been previously associated with the difference between blue and green eyes
(Sulem et al., 2008). Likewise, in the South Asian sample, SLC24A5 rs1426654 was associated
with ∆E before conditioning for HERC2 rs12913832. However, after conditioning, this marker
was no longer significant.
There are a number of structural differences between the pupillary and ciliary zones,
including the thickness and opacity of these regions (Mackey et al., 2011; Oyster, 1999). It is
possible that markers associated with the structure of the iris may also be determinants of central
heterochromia.

3.5.5 A Global View of the Genetic Basis of Iris Pigmentation

Although the evolution and genetic basis of iris colour in European populations has been
well-studied over the past decade, very few research groups have attempted to explore the
genetic basis of iris pigmentation variation in populations of non-European ancestry. Although
European populations may show the greatest diversity in eye colour phenotypes, iris colour
variation extends far past the difference between blue and brown. This is especially evident
when looking at the distribution of iris pigmentation in East and South Asian samples across CIE
1976 L*a*b (CIELAB) colour space. Although the majority of the irises in these populations
 71

would traditionally be described as brown, these browns showed much diversity and ranged from
very light to very dark in colour. Thus, studying iris colour in populations with more
homogenously coloured irises will allow us to approach the study of iris colour from a number of
new directions and better understand the global diversity in this trait.
It is interesting to note that the markers associated with iris colour variation in all three
groups have largely been associated with other pigmentary traits. SLC45A2 rs16891982,
SLC24A5 rs1426654, OCA2 rs1800414 and OCA2 rs7465330 are responsible for major
differences in global skin pigmentation diversity (Eaton et al., 2015; Edwards et al., 2010; Graf
et al., 2005; Lamason et al., 2005; Stokowski et al., 2007). In addition, SLC24A4 rs12896399 and
HERC2 rs12913832 have been associated with hair colour variation and IRF4 rs12203592 has
been associated with both hair and skin pigmentation (Han et al., 2008; Sulem et al., 2007). TYR
rs1126809, which is in strong linkage disequilibrium with TYR rs1393350, has also been
associated with both skin and hair colour (Nan et al., 2009; Sulem et al., 2008). Thus, it is very
likely that many of the iris pigmentation markers were selected for because of the effect that they
had on other pigmentary characteristics, and not for their effect on iris colour variation. HERC2
rs12913832, which shows evidence of strong positive selection in regions where blue eyes
dominate, may be the exception (Donnelly et al., 2012).
It is also interesting to note that the markers which modulate iris colour variation in each
region appear to be very different. The six markers commonly used as predictors in European
populations were all significant in our European sample. However, only HERC2 rs12913832 was
associated with eye colour in the South Asian sample and none of the markers were associated
with eye colour in East Asia. Rather, the markers that were significantly associated with iris
colour variation in the East and South Asian populations were largely polymorphisms that played
a role in skin and hair pigmentation variation in those regions. These include SLC24A5
rs1426654 in the South Asian sample, and OCA2 rs1800414 and OCA2 rs7465330 in the East
Asian sample.
Continuing to develop a better understanding of the global distribution of iris colour
variation will have a number of advantages. At present, the markers associated with intermediate
eye colours are largely unknown. As a result, forensic eye colour predictor models have been
found to perform poorly when applied to populations with a large proportion of green or
intermediate eyes (Dembinski and Picard, 2014; Pneuman et al., 2012; Yun et al., 2014). As the
 72

genetic basis of iris colour appears to be highly population-specific, it is critical to study eye
colour in populations outside of Europe to get a better understanding of the genetic architecture
of this trait. Determining the variants responsible for variation in brown iris colour may allow
future iris predictor systems to distinguish between dark and light brown eyes in populations
with more homogenous irises. This would broaden their use beyond European populations.
Lastly, given that the markers associated with iris pigmentation appear to be strongly associated
with skin and hair pigmentation variation, identifying novel variants associated with iris colour
variation in populations of non-European ancestry may provide valuable information about the
genetic basis of skin and hair colour in these regions.
The development of quantitative methods of measuring iris colour has opened up many
doors in pigmentation research. However, these methods have not yet been widely applied to
populations of non-European ancestry. In this study, we present a new method to estimate iris
colour and heterochromia based on high-resolution photographs. This method has been
implemented in a Web application that can be accessed at http://iris.davidcha.ca/. Accounts can
be set up for interested users by request. Using this quantitative approach, we identified one
novel variant associated with iris colour in a South Asian sample and two novel variants in an
East Asian sample. We suggest that future research should apply such quantitative methods to
other global populations, such as African-American and Hispanic groups. In addition, as the
genetic basis of central heterochromia and intermediate irises continues to be poorly understood,
we suggest that using quantitative methods that allow researchers to divide the iris into separate
regions may provide a new approach for studying these traits.
 73

3.6 Tables

Table 3-1 Results of the Intra- and Inter-Rater Reliability Analysis

We report the intracorrelation coefficient for a two-way mixed model for both the intra-rater and inter-
rater measurements.

Intra-Rater Intracorrelation Coefficient Inter-Rater Intracorrelation Coefficient

L* 0.999 0.999
a* 0.999 0.997
b* 0.997 0.992
∆E 0.985 0.985
 74

Table 3-2 Summary Statistics for All 1448 Participants Included in the Study
We show the sex, average, minimum and maximum age, L*, a*, b* and ∆E values of all 1448 participants included in the analysis (excluded
individuals have been omitted from this table) for each of the two camera bodies. Participants are divided both by broad ancestry (East Asian,
European and South Asian) and regional ancestry. European participants are divided into regions using the United Nations geoscheme for Europe
(http://unstats.un.org/unsd/methods/m49/m49regin.htm). East Asian and South Asian participants are divided by country of origin.
Regional Camera Total Average Age Males Average L* Average a* Average b* Average ∆E
Ancestry Body Number (Min, Max) (Females) (Min, Max) (Min, Max) (Min, Max) (Min, Max)
1 87 21 (18,34) 26 (61) 26.914 (19.525,36.683) 13.674 (8.832,20.391) 8.158 (0.982,17.891) 2.476 (0.638,4.748)
China
2 274 22 (18,35) 78 (196) 22.182 (14.239,36.669) 12.9 (7.299,20.010) 7.289 (0.3,18.203) 2.109 (0.063,5.705)
1 2 21 (19,23) 1 (1) 27.372 (24.384,30.36) 14.311 (13.202,15.419) 7.329 (6.544,8.115) 1.762 (1.605,1.918)
Japan
2 6 24 (18,29) 1 (5) 21.846 (15.43,26.468) 13.563 (9.845,15.087) 8.714 (5.543,11.411) 2.006 (0.657,2.788)
East 1 21 24 (18,29) 10 (11) 27.055 (22.65,38.532) 13.732 (8.399,17.410) 8.728 (0.932,16.177) 2.251 (0.399,6.519)
Korea
Asian 2 52 22 (18,31) 24 (28) 21.034 (15.814,28.17) 12.56 (6.594,18.147) 7.136 (0.098,18.372) 2.203 (0.523,5.199)
1 2 21 (20,22) 1 (1) 27.08 (23.85,30.31) 12.524 (10.539,14.509) 6.531 (3.354,9.708) 2.607 (2.046,3.168)
Taiwan
2 7 20 (19,23) 2 (5) 19.798 (16.502,23.522) 10.961 (7.709,13.119) 4.635 (1.36,6.767) 1.881 (1.329,2.868)
1 4 21 (19,25) 0 (4) 27.43 (24.298,30.477) 13.437 (10.063,16.724) 7.911 (3.51,12.166) 2.992 (2.311,3.861)
Other
2 13 21 (18,26) 5 (8) 20.44 (14.685,26.547) 11.949 (7.921,16.724) 6 (-0.664,11.902) 1.999 (0.748,3.203)
Eastern 1 53 21 (18,35) 24 (29) 45.002 (30.834,62.23) 1.055 (-8.444,18.733) 2.252 (-15.797,20.44) 9.277 (1.114,16.684)
Europe 2 67 23 (18,35) 25 (42) 38.229 (18.899,53.004) 1.663 (-7.734,19.962) 1.795 (-17.65,21.611) 7.671 (0.683,16.153)
Northern 1 33 23 (19,35) 11 (22) 45.279 (30.857,57.16) 0.43 (-8.225,16.027) 1.828 (-18.61,21.473) 9.129 (3.508,17.378)
Europe 2 74 24 (18,35) 33 (41) 37.97 (25.269,49.584) -1.5 (-6.961,17.543) -3.406 (-15.832,22.095) 6.832 (1.702,14.721)
Southern 1 47 21 (18,27) 16 (31) 38.821 (26.084,55.444) 10.894 (-6.601,22.009) 12.512 (-14.18,22.418) 6.451 (0.796,14.674)
European
Europe 2 52 22 (18,35) 22 (30) 33.813 (21.294,52.467) 8.942 (-6.713,21.318) 10.045 (-12.828,19.776) 6.4 (0.657,15.402)
Western 1 3 20 (18,23) 0 (3) 47.903 (41.213,52.606) -3.663 (-6.977,-1.149) 2.576 (-1.76,4.923) 12.418 (10.731,13.312)
Europe 2 5 24 (19,32) 2 (3) 35.911 (28.341,43.317) 4.705 (-5.882,13.278) 5.376 (-17.356,13.636) 6.456 (1.515,10.712)
1 99 22 (18,31) 36 (63) 44.622 (27.588,61.609) 1.611 (-8.035,21.032) 1.911 (-18.957,24.087) 8.447 (1.343,19.264)
Other
2 187 23 (18,35) 76 (110) 38.191 (19.674,52.872) 0.436 (-7.315,19.87) 0.328 (-18.475,22.067) 7.239 (0.509,16.921)
1 5 24 (18,33) 1 (4) 27.792 (22.245,30.599) 12.977 (7.632,15.868) 6.099 (0.481,9.061) 3.257 (2.339,4.588)
Bangladesh
2 12 21 (18,32) 5 (7) 25.107 (19.351,31.789) 13.419 (11.086,16.25) 8.679 (3.468,17.146) 2.663 (0.416,8.207)
1 93 21 (18,32) 32 (61) 29.56 (19.918,50.209) 13.81 (-0.912,22.671) 8.564 (-1.732,20.147) 2.807 (0.908,10.391)
India
2 88 21 (18,35) 38 (50) 25.105 (15.533,37.93) 14.16 (6.26,21.256) 8.134 (-1.427,19.367) 2.117 (0.156,7.782)
South 1 48 20 (18,24) 15 (33) 30.401 (21.266,50.995) 14.665 (0.429,25.925) 9.595 (-1.2,26.796) 3.204 (0.451,13.486)
Pakistan
Asian 2 36 20 (18,31) 9 (27) 27.53 (18.311,48.769) 14.028 (1.643,19.802) 9.148 (-1.955,18.111) 2.99 (0.561,11.616)
1 23 20 (18,21) 5 (18) 27.698 (18.31,42.544) 12.049 (7.101,18.796) 6.001 (-0.198,15.982) 2.343 (0.247,7.118)
Sri Lanka
2 26 20 (18,26) 5 (21) 23.467 (16.704,40.457) 11.5 (1.746,21.599) 4.772 (-1.493,17.479) 2.317 (0.926,9.633)
1 19 20 (18,23) 6 (13) 32.627 (25.853,47.392) 13.747 (-2.94,18.521) 9.735 (2.136,21.973) 3.437 (0.378,10.773)
Other
2 10 20 (18,27) 2 (8) 24.451 (18.644,30.629) 14.169 (10.034,19.247) 8.212 (3.967,16.983) 2.577 (1.003,5.219)
 75

Table 3-3 Results of the Meta-Analysis in the European Sample (A) Before and (B) After
Conditioning for the Effects of HERC2 rs12913832
We report the P-value of the association, the effect size (beta) and the P-value for Cochran's Q statistic for
each additional copy of the minor allele relative to the reference genotype. After the Bonferroni correction
for multiple comparisons, associations were significant if P < 0.00454. Reference genotypes marked with
an asterisk (*) represent the homozygous ancestral state. Significant associations are bolded and
italicized.

Reference Colour Significance Heterogeneity

Gene Marker Genotype Beta
Genotype Metric (P) (P)
(A)
GA 3.907 x 10-54 -7.143 0.041
L*
AA 1.023 x 10-56 -12.586 0.023
GA 4.641 x 10-216 12.971 0.031
a*
AA 5.203 x 10-137 17.985 0.039
HERC2 rs12913832 GG
GA 1.872 x 10-223 18.343 0.071
b*
AA 1.863 x 10-96 20.659 0.271
GA 1.098 x 10-05 -1.236 0.443
∆E
AA 3.667 x 10-15 -3.846 0.088
GA 0.796 0.221 0.252
L*
AA - - -
GA 0.150 1.542 0.748
a*
AA - - -
OCA2 rs1800407 GG*
GA 0.003 4.297 0.887
b*
AA - - -
GA 0.852 -0.080 0.431
∆E
AA - - -
GT 0.066 1.121 0.556
L*
TT 0.325 0.789 0.648
GT 0.004 -2.202 0.759
a*
TT 0.080 -1.759 0.596
SLC24A4 rs12896399 GG*
GT 7.191 x 10-04 -3.446 0.470
b*
TT 1.758 x 10-04 -5.018 0.707
GT 0.949 -0.019 0.718
∆E
TT 0.071 -0.725 0.060
GC 0.059 -1.610 0.085
L*
CC 0.230 -3.818 0.026
GC 9.655 x 10-07 5.139 0.170
a*
CC 0.050 7.714 0.047
SLC45A2 rs16891982 GG
GC 2.069 x 10-06 6.740 0.666
b*
CC 0.244 6.100 0.116
GC 0.150 -0.621 0.659
∆E
CC 0.209 -2.039 0.422
GA 0.543 -0.349 0.022
L*
AA 0.029 2.604 0.077
GA 0.443 -0.556 0.356
a*
AA 0.261 -1.694 0.725
TYR rs1393350 GG*
GA 0.077 -1.721 0.308
b*
AA 0.261 -2.258 0.883
GA 0.896 -0.038 0.247
∆E
AA 0.019 1.404 0.026
 76

Reference Colour Significance Heterogeneity

Gene Marker Genotype Beta
Genotype Metric (P) (P)
CT 0.206 0.802 0.791
L*
TT 0.033 3.381 0.921
CT 0.008 -2.077 0.473
a*
TT 0.003 -5.917 0.464
IRF4 rs12203592 CC*
CT 0.013 -2.624 0.167
b*
TT 1.072 x 10-04 -10.058 0.523
CT 0.041 0.652 0.967
∆E
TT 0.704 -0.305 0.634
CT 0.790 -0.156 0.238
L*
TT 0.041 -1.857 0.409
CT 0.245 0.858 0.425
a*
TT 0.007 3.091 0.890
TYRP1 rs1408799 CC
CT 0.248 1.151 0.739
b*
TT 0.083 2.669 0.447
CT 0.154 -0.420 0.381
∆E
TT 0.096 -0.763 0.134
CT 0.109 -1.035 0.305
L*
TT 0.686 -0.321 0.359
CT 0.890 0.112 0.623
a*
TT 0.117 -1.564 0.518
NPLOC4 rs9894429 CC*
CT 0.845 -0.213 0.252
b*
TT 0.040 -2.755 0.549
CT 0.489 0.225 0.245
∆E
TT 0.167 0.554 0.530
AG 0.151 -0.842 0.762
L*
GG 0.438 1.010 0.015
AG 0.097 1.226 0.354
a*
GG 0.814 0.387 0.142
LYST rs3768056 AA*
AG 0.056 1.890 0.594
b*
GG 0.904 -0.267 0.095
AG 0.165 -0.408 0.462
∆E
GG 0.875 -0.103 0.006
AG 0.185 -0.906 0.296
L*
GG 0.670 -0.951 0.391
AG 0.047 1.695 0.448
a*
GG 0.393 2.396 0.480
ASIP rs6058017 AA
AG 0.012 2.887 0.536
b*
GG 0.309 3.789 0.248
AG 0.391 -0.294 0.023
∆E
GG 0.065 2.079 0.551
(B)
GA 1.767 x 10-07 3.552 0.498
L*
AA - - -
GA 2.203 x 10-09 -3.835 0.872
a*
AA - - -
OCA2 rs1800407 GG*
GA 0.013 -2.389 0.624
b*
AA - - -
GA 0.082 0.736 0.772
∆E
AA - - -
 77

Reference Colour Significance Heterogeneity

Gene Marker Genotype Beta
Genotype Metric (P) (P)
GT 0.111 0.909 0.200
L*
TT 0.457 0.555 0.520
GT 0.007 -1.902 0.311
a*
TT 0.115 -1.453 0.464
SLC24A4 rs12896399 GG*
GT 0.001 -3.205 0.201
b*
TT 2.134 x 10-04 -4.725 0.617
GT 0.691 -0.118 0.439
∆E
TT 0.039 -0.803 0.039
GC 0.687 -0.327 0.176
L*
CC 0.876 -0.458 0.000
GC 2.278 x 10-04 3.675 0.340
a*
CC 0.258 4.105 0.001
SLC45A2 rs16891982 GG
GC 9.493 x 10-05 5.458 0.968
b*
CC 0.602 2.625 0.017
GC 0.647 -0.197 0.946
∆E
CC 0.542 -0.961 0.106
GA 0.907 -0.062 0.005
L*
AA 0.004 3.185 0.109
GA 0.214 -0.828 0.192
a*
AA 0.086 -2.367 0.999
TYR rs1393350 GG*
GA 0.034 -1.974 0.201
b*
AA 0.127 -2.914 0.876
GA 0.845 0.055 0.158
∆E
AA 0.005 1.614 0.033
CT 0.332 0.575 0.862
L*
TT 0.027 3.220 0.688
CT 0.016 -1.745 0.561
a*
TT 0.001 -5.809 0.617
IRF4 rs12203592 CC*
CT 0.024 -2.282 0.651
b*
TT 5.917 x 10-05 -9.916 0.997
CT 0.058 0.588 0.951
∆E
TT 0.628 -0.375 0.487
CT 0.839 -0.111 0.506
L*
TT 0.054 -1.629 0.363
CT 0.254 0.776 0.820
a*
TT 0.008 2.811 0.864
TYRP1 rs1408799 CC
CT 0.289 1.011 0.892
b*
TT 0.106 2.372 0.405
CT 0.149 -0.415 0.613
∆E
TT 0.124 -0.684 0.111
CT 0.124 -0.925 0.179
L*
TT 0.698 -0.287 0.291
CT 0.844 -0.147 0.795
a*
TT 0.081 -1.608 0.465
NPLOC4 rs9894429 CC*
CT 0.637 -0.494 0.349
b*
TT 0.029 -2.808 0.539
CT 0.399 0.267 0.194
∆E
TT 0.155 0.553 0.477
 78

Reference Colour Significance Heterogeneity

Gene Marker Genotype Beta
Genotype Metric (P) (P)
AG 0.166 -0.755 0.909
L*
GG 0.589 0.655 0.006
AG 0.098 1.122 0.395
a*
GG 0.636 0.712 0.094
LYST rs3768056 AA*
AG 0.061 1.773 0.631
b*
GG 0.985 0.040 0.071
AG 0.198 -0.367 0.328
∆E
GG 0.718 -0.228 0.004
AG 0.143 -0.923 0.298
L*
GG 0.964 0.093 0.404
AG 0.027 1.737 0.479
a*
GG 0.682 1.053 0.528
ASIP rs6058017 AA
AG 0.007 2.942 0.589
b*
GG 0.491 2.444 0.277
AG 0.380 -0.290 0.020
∆E
GG 0.024 2.444 0.560
 79

Table 3-4 Results of the Meta-Analysis in the South Asian Sample (A) Before and (B) After
Conditioning for the Effects of HERC2 rs12913832
We report the P-value of the association, the effect size (beta) and the P-value for Cochran's Q statistic for
each additional copy of the minor allele relative to the reference genotype. After the Bonferroni correction
for multiple comparisons, associations were significant if P < 0.00500. Reference genotypes marked with
an asterisk (*) represent the homozygous ancestral state. Significant associations are bolded and
italicized.

Reference Colour Significance Heterogeneity

Gene Marker Genotype Beta
Genotype Metric (P) (P)
(A)
AG 5.042 x 10-14 4.652 0.530
L*
GG 7.535 x 10-26 17.867 0.879
AG 7.571 x 10-04 1.601 0.885
a*
GG 9.703 x 10-17 -10.856 0.897
HERC2 rs12913832 AA*
AG 7.073 x 10-11 4.165 0.669
b*
GG 0.390 1.510 0.770
AG 8.583 x 10-10 1.305 0.074
∆E
GG 6.443 x 10-38 7.520 0.916
AG 8.901 x 10-04 -2.337 0.948
L*
GG 0.002 -3.673 0.689
AG 1.400 x 10-05 -2.118 0.710
a*
GG 1.538 x 10-06 -3.905 0.956
SLC24A5 rs1426654 AA
AG 6.703 x 10-09 -3.512 0.789
b*
GG 5.271 x 10-09 -5.881 0.921
AG 0.002 -0.775 0.527
∆E
GG 0.261 -0.476 0.379
GT 0.807 0.147 0.414
L*
TT 0.793 -0.299 0.871
GT 0.147 -0.615 0.133
a*
TT 0.343 -0.763 0.186
SLC24A4 GG*
rs12896399 GT 0.172 -0.741 0.200
b*
TT 0.523 -0.657 0.470
GT 0.325 0.210 0.874
∆E
TT 0.210 0.507 0.542
CG 0.943 -0.050 0.094
L*
GG 0.607 -1.597 0.427
CG 0.409 -0.408 0.225
a*
GG 0.701 0.849 0.431
SLC45A2 rs16891982 CC*
CG 0.964 -0.028 0.114
b*
GG 0.398 2.385 0.365
CG 0.609 0.127 0.759
∆E
GG 0.964 -0.050 0.639
GA 0.534 0.510 0.321
L*
AA 0.907 -0.280 0.241
GA 0.715 -0.213 0.807
a*
AA 0.437 1.315 0.121
TYR rs1393350 GG*
GA 0.752 0.233 0.135
b*
AA 0.572 1.217 0.170
GA 0.448 0.222 0.174
∆E
AA 0.317 -0.850 0.962
 80

Reference Colour Significance Heterogeneity

Gene Marker Genotype Beta
Genotype Metric (P) (P)
TC 0.116 0.946 0.170
L*
CC 0.795 0.252 0.857
TC 0.009 1.106 0.226
a*
CC 0.014 1.674 0.280
TYRP1 rs1408799 TT*
TC 0.024 1.224 0.606
b*
CC 0.007 2.366 0.671
TC 0.109 -0.344 0.027
∆E
CC 0.509 -0.229 0.940
TC 0.615 -0.315 0.382
L*
CC 0.802 -0.213 0.891
TC 0.297 -0.464 0.412
a*
CC 0.636 -0.284 0.877
NPLOC4 rs9894429 TT
TC 0.148 -0.819 0.696
b*
CC 0.163 -1.068 0.717
TC 0.198 0.283 0.097
∆E
CC 0.839 -0.061 0.359
AG 0.091 1.012 0.933
L*
GG 0.292 1.145 0.603
AG 0.002 1.308 0.475
a*
GG 0.046 1.544 0.168
LYST rs3768056 AA*
AG 0.002 1.696 0.808
b*
GG 0.064 1.836 0.241
AG 0.730 -0.074 0.655
∆E
GG 0.331 -0.380 0.630
GA 0.961 -0.031 0.844
L*
AA 0.628 -0.450 0.174
GA 0.321 0.441 0.228
a*
AA 0.276 -0.714 0.114
DSCR9 rs7277820 GG*
GA 0.356 0.524 0.271
b*
AA 0.507 -0.556 0.050
GA 0.311 -0.228 0.907
∆E
AA 0.556 -0.195 0.988
AG 0.204 0.797 0.895
L*
GG 0.763 -0.356 0.655
AG 0.472 0.318 0.027
a*
GG 0.661 -0.367 0.523
ASIP rs6058017 AA
AG 0.248 0.655 0.171
b*
GG 0.733 0.366 0.505
AG 0.406 0.185 0.168
∆E
GG 0.750 0.134 0.349
(B)
AG 0.003 -1.918 0.979
L*
GG 0.002 -3.255 0.637
AG 1.651 x 10-08 -2.461 0.759
a*
GG 4.900 x 10-09 -4.233 0.997
SLC24A5 rs1426654 AA
AG 5.237 x 10-09 -3.560 0.823
b*
GG 4.738 x 10-09 -5.922 0.902
AG 0.007 -0.586 0.499
∆E
GG 0.417 -0.292 0.283
 81

Reference Colour Significance Heterogeneity

Gene Marker Genotype Beta
Genotype Metric (P) (P)
GT 0.896 0.071 0.093
L*
TT 0.450 -0.778 0.329
GT 0.157 -0.554 0.337
a*
TT 0.547 -0.447 0.471
SLC24A4 GG*
rs12896399 GT 0.167 -0.757 0.196
b*
TT 0.501 -0.699 0.461
GT 0.362 0.166 0.261
∆E
TT 0.399 0.291 0.782
CG 0.386 -0.542 0.019
L*
GG 0.633 -1.320 0.381
CG 0.766 -0.135 0.328
a*
GG 0.744 0.662 0.389
SLC45A2 rs16891982 CC*
CG 0.903 -0.078 0.097
b*
GG 0.404 2.365 0.370
CG 0.781 -0.059 0.357
∆E
GG 0.957 0.051 0.572
GA 0.492 0.507 0.568
L*
AA 0.965 0.094 0.198
GA 0.653 -0.240 0.474
a*
AA 0.494 1.059 0.089
TYR rs1393350 GG*
GA 0.788 0.200 0.151
b*
AA 0.577 1.204 0.174
GA 0.358 0.228 0.347
∆E
AA 0.331 -0.701 0.939
TC 0.025 1.215 0.382
L*
CC 0.407 0.719 0.729
TC 0.021 0.905 0.405
a*
CC 0.032 1.343 0.269
TYRP1 rs1408799 TT*
TC 0.028 1.208 0.680
b*
CC 0.007 2.362 0.643
TC 0.225 -0.224 0.069
∆E
CC 0.926 -0.027 0.945
TC 0.601 -0.296 0.682
L*
CC 0.864 -0.130 0.751
TC 0.290 -0.432 0.630
a*
CC 0.532 -0.343 0.986
NPLOC4 rs9894429 TT
TC 0.177 -0.771 0.623
b*
CC 0.165 -1.066 0.715
TC 0.135 0.280 0.205
∆E
CC 0.930 -0.022 0.383
AG 0.041 1.093 0.828
L*
GG 0.369 0.870 0.231
AG 0.001 1.299 0.319
a*
GG 0.014 1.732 0.325
LYST rs3768056 AA*
AG 0.002 1.744 0.743
b*
GG 0.069 1.808 0.227
AG 0.813 -0.042 0.713
∆E
GG 0.137 -0.486 0.848
 82

Reference Colour Significance Heterogeneity

Gene Marker Genotype Beta
Genotype Metric (P) (P)
GA 0.948 -0.037 0.658
L*
AA 0.264 -0.932 0.062
GA 0.287 0.437 0.484
a*
AA 0.485 -0.421 0.173
DSCR9 rs7277820 GG*
GA 0.378 0.506 0.273
b*
AA 0.469 -0.611 0.056
GA 0.204 -0.242 0.337
∆E
AA 0.144 -0.41 0.635
AG 0.168 0.778 0.307
L*
GG 0.429 -0.840 0.882
AG 0.476 0.291 0.089
a*
GG 0.884 -0.112 0.871
ASIP rs6058017 AA
AG 0.278 0.621 0.145
b*
GG 0.790 0.288 0.549
AG 0.315 0.191 0.580
∆E
GG 0.885 -0.052 0.759
 83

Table 3-5 Results of the Meta-Analysis in the East Asian Sample (A) Before and (B) After
Conditioning for the Effects of OCA2 rs1800414
We report the P-value of the association, the effect size (beta) and the P-value for Cochran's Q statistic for
each additional copy of the minor allele relative to the reference genotype. After the Bonferroni correction
for multiple comparisons, associations were significant if P < 0.00714. Reference genotypes marked with
an asterisk (*) represent the homozygous ancestral state. Significant associations are bolded and
italicized.

Reference Colour Significance Heterogeneity

Gene Marker Genotype Beta
Genotype Metric (P) (P)
(A)
GA 1.434 x 10-05 -1.460 0.503
L*
AA 5.875 x 10-09 -2.793 0.902
GA 5.187 x 10-08 -1.319 0.493
OCA2 rs1800414 GG a*
AA 1.704 x 10-16 -2.831 0.926
GA 1.137 x 10-07 -1.757 0.510
b*
AA 1.308 x 10-17 -4.024 0.783
GA 0.069 1.201 0.177
L*
AA - - -
GA 4.310 x 10-04 1.700 0.153
OCA2 rs74653330 GG* a*
AA - - -
GA 1.141 x 10-04 2.556 0.063
b*
AA - - -
GT 0.273 -0.375 0.432
L*
TT 0.902 -0.067 0.267
GT 0.088 -0.429 0.263
SLC24A4 GG* a*
rs12896399 TT 0.161 -0.562 0.190
GT 0.027 -0.761 0.129
b*
TT 0.018 -1.294 0.057
GA 0.186 0.477 0.296
L*
AA 0.251 0.521 0.499
GA 0.140 0.395 0.984
DSCR9 rs7277820 GG* a*
AA 0.087 0.577 0.468
GA 0.042 0.745 0.734
b*
AA 0.024 1.041 0.609
TC 0.634 -0.161 0.506
L*
CC 0.844 0.170 0.089
TC 0.782 -0.069 0.648
NPLOC4 rs9894429 TT a*
CC 0.990 0.008 0.154
TC 0.442 -0.265 0.739
b*
CC 0.590 -0.474 0.337
AG 0.306 0.359 0.323
L*
GG 0.379 0.879 0.001
AG 0.168 0.364 0.471
LYST rs3768056 AA* a*
GG 0.703 0.288 0.748
AG 0.161 0.505 0.388
b*
GG 0.397 0.873 0.032
 84

Reference Colour Significance Heterogeneity

Gene Marker Genotype Beta
Genotype Metric (P) (P)
AG 0.338 0.332 0.796
L*
GG 0.434 0.582 0.081
AG 0.670 0.109 0.754
ASIP rs6058017 AA a*
GG 0.725 0.195 0.093
AG 0.921 0.035 0.682
b*
GG 0.707 0.283 0.003
(B)
GA 8.781 x 10-04 2.151 0.298
L*
AA - - -
GA 3.071 x 10-09 2.671 0.310
OCA2 rs74653330 GG* a*
AA - - -
GA 1.847 x 10-10 3.918 0.143
b*
AA - - -
GT 0.304 -0.347 0.315
L*
TT 0.984 -0.011 0.373
GT 0.085 -0.420 0.138
SLC24A4 GG* a*
rs12896399 TT 0.189 -0.509 0.229
GT 0.023 -0.754 0.052
b*
TT 0.021 -1.213 0.112
GA 0.275 0.388 0.321
L*
AA 0.495 0.306 0.468
GA 0.255 0.294 0.888
DSCR9 rs7277820 GG* a*
AA 0.282 0.352 0.416
GA 0.090 0.599 0.808
b*
AA 0.107 0.718 0.567
TC 0.712 -0.123 0.274
L*
CC 0.796 0.220 0.132
TC 0.812 -0.058 0.259
NPLOC4 rs9894429 TT a*
CC 0.908 0.071 0.255
TC 0.451 -0.250 0.726
b*
CC 0.645 -0.388 0.525
AG 0.293 0.363 0.298
L*
GG 0.445 0.749 0.002
AG 0.146 0.369 0.410
LYST rs3768056 AA* a*
GG 0.819 0.167 0.814
AG 0.138 0.510 0.324
b*
GG 0.474 0.706 0.032
AG 0.491 0.235 0.802
L*
GG 0.480 0.519 0.117
AG 0.971 0.009 0.751
ASIP rs6058017 AA a*
GG 0.816 0.124 0.157
AG 0.744 -0.110 0.681
b*
GG 0.805 0.178 0.006
 85

Table 3-6 Average, Minimum and Maximum ∆E Stratified by HERC2 rs12913832 Genotype
We report the average, minimum and maximum ∆E values for the three possible HERC2 rs12913832
genotypes for both the European and South Asian populations. A * is used to represent the homozygous
ancestral state. The East Asian sample was monomorphic for this marker, and was not included in the
table. Note the substantial difference in average ∆E between the heterozygous European and South Asian
samples.

HERC2 genotype Camera Average ∆E Minimum ∆E Maximum ∆E

Camera 1 4.806 0.823 11.503
AA*
Camera 2 4.578 0.509 9.936
Camera 1 8.128 1.394 16.527
European AG
Camera 2 6.668 0.683 16.153
Camera 1 9.669 1.515 19.264
GG
Camera 2 7.751 1.605 16.921
Camera 1 2.589 0.247 9.433
AA
Camera 2 2.015 0.156 5.655

South Camera 1 3.634 0.957 10.391

AG
Asian Camera 2 3.722 0.511 9.918
Camera 1 10.043 5.328 13.486
GG
Camera 2 9.598 7.545 11.616
 86

3.7 Figures

Figure 3-1 The Distribution of Irises Across the b* and a* (A), a* and L* (B), and b* and L* (C),
Coordinates of CIE 1976 L*a*b* (CIELAB) Colour Space for the Second Camera Body
East Asian participants are represented by triangles, European participants are represented by squares, and
South Asian participants are represented by circles. The colour inside of each shape is equivalent to the
average iris colour of the participant's wedge in CIELAB colour space. Ample dispersion across colour
space can be observed in all three populations. Both cameras show very similar distributions across colour
space. However, the average L*, a* and b* values from the first camera are higher than those from the
second camera.
87
 88

Figure 3-2 Sample Iris Wedges with Corresponding Colour Space Values
A wedge with a 60-degree angle is cropped from the left side of the iris based on the scleral and pupillary
boundaries that the user manually defines during iris categorization. To obtain a measurement of average
iris colour, the application determines the red, green and blue (RGB) value of each pixel located in the
wedge and divides the sum of the RGB values by the total number of counted pixels. The RGB values are
then transformed into CIE 1976 L*a*b* (CIELAB) colour space using an illuminant of D55 and an
observer angle of 2 degrees. The Web application also divides the iris into a ciliary and pupillary zone
based on the user-defined collarette. In the image below, we show the pupillary and ciliary wedges for six
irises of differing colour. The L*, a*, b* and ∆E values are written to the right of each iris.
 89

3.8 References
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Nielsen, M.M., Sørensen, E., Ullum, H., Hansen, T., et al. (2013). Genetic analyses of the human
eye colours using a novel objective method for eye colour classification. Forensic Sci. Int. Genet.
7, 508–515.
Baranoski, G.V.G., and Lam, M.W.Y. (2007). Qualitative assessment of undetectable melanin
distribution in lightly pigmented irides. J. Biomed. Opt. 12, 1–3.
Beleza, S., Johnson, N.A., Candille, S.I., Absher, D.M., Coram, M.A., Lopes, J., Campos, J.,
Araújo, I.I., Anderson, T.M., and Vilhjálmsson, B.J. (2013). Genetic architecture of skin and eye
color in an African-European admixed population. PLoS Genet 9, e1003372.
Bito, L.Z., Matheny, A., Cruickshanks, K.J., Nondahl, D.M., and Carino, O.B. (1997). Eye color
changes past early childhood. The Louisville Twin Study. Arch. Ophthalmol. 115, 659–663.
Cook, A.L., Chen, W., Thurber, A.E., Smit, D.J., Smith, A.G., Bladen, T.G., Brown, D.L.,
Duffy, D.L., Pastorino, L., and Bianchi-Scarra, G. (2009). Analysis of cultured human
melanocytes based on polymorphisms within the SLC45A2/MATP, SLC24A5/NCKX5, and
OCA2/P loci. J. Invest. Dermatol. 129, 392–405.
Dembinski, G.M., and Picard, C.J. (2014). Evaluation of the IrisPlex DNA-based eye color
prediction assay in a United States population. Forensic Sci. Int. Genet. 9, 111–117.
Donnelly, M.P., Paschou, P., Grigorenko, E., Gurwitz, D., Barta, C., Lu, R.-B., Zhukova, O.V.,
Kim, J.-J., Siniscalco, M., and New, M. (2012). A global view of the OCA2-HERC2 region and
pigmentation. Hum. Genet. 131, 683–696.
Eagle, R.C. (1988). Iris pigmentation and pigmented lesions: an ultrastructural study. Trans. Am.
Ophthalmol. Soc. 86, 581–687.
Eaton, K., Edwards, M., Krithika, S., Cook, G., Norton, H., and Parra, E.J. (2015). Association
study confirms the role of two OCA2 polymorphisms in normal skin pigmentation variation in
East Asian populations. Am. J. Hum. Biol. 520–525.
Edwards, M., Bigham, A., Tan, J., Li, S., Gozdzik, A., Ross, K., Jin, L., and Parra, E.J. (2010).
Association of the OCA2 polymorphism His615Arg with melanin content in east Asian
populations: further evidence of convergent evolution of skin pigmentation. PLoS Genet 6,
e1000867.
Edwards, M., Gozdzik, A., Ross, K., Miles, J., and Parra, E.J. (2012). Technical note:
quantitative measures of iris color using high resolution photographs. Am. J. Phys. Anthropol.
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 Chapter 4:
Analysis of Iris Surface Features in Populations of
Diverse Ancestry

Author Contributions: M. Edwards participated in the design of the study, collected the data, conducted
the data analysis and drafted the manuscript. D. Cha developed the iris structure program and assisted
with data analysis. S. Krithika and M. Johnson assisted with data collection and molecular laboratory
work. E.J. Parra conceived the study, participated in the design of the study, coordinated the study and
helped draft the manuscript.

This Chapter Has Been Previously Published As: Edwards, M., Cha, D., S. Krithika., Johnson, M.,
Parra, E.J. (2016) Analysis of iris surface features in populations of diverse ancestry. R. Soc. Open Sci. 3,
150424.

94
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4.1 Abstract
There are many textural elements that can be found in the human eye, including Fuchs’
crypts, Wolfflin nodules, pigment spots, contraction furrows and conjunctival melanosis.
Although iris surface features have been well-studied in populations of European ancestry, the
worldwide distribution of these traits is poorly understood. In this paper, we develop a new
method of characterizing iris features from photographs of the iris. We then apply this method to
a diverse sample of East Asian, European and South Asian ancestry. All five iris features showed
significant differences in frequency between the three populations, indicating that iris features
are largely population dependent. Although none of the features were correlated with each other
in the East and South Asian groups, Fuchs’ crypts were significantly correlated with contraction
furrows and pigment spots and contraction furrows were significantly associated with pigment
spots in the European group. The genetic marker SEMA3A rs10235789 was significantly
associated with Fuchs’ crypt grade in the European, East Asian and South Asian samples and a
borderline association between TRAF3IP1 rs3739070 and contraction furrow grade was found in
the European sample. The study of iris surface features in diverse populations may provide
valuable information of forensic, biomedical, and ophthalmological interest
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4.2 Introduction
The human iris is a complex tissue consisting of many different regions and strata. A
healthy human eye typically has five different layers. The posterior-most layer is called the iris
pigment epithelium (IPE). This layer is tightly packed with cuboidal melanin-rich melanocytes in
all healthy individuals and does not contribute significantly to variation in iris colour or structure
(Eagle, 1988; Peles et al., 2009; Wilkerson et al., 1996). Just above the IPE are two muscle
layers, known as the sphincter muscle and the dilator muscle (Eagle, 1988). The two layers that
are responsible for most of the variation between individuals are the anterior border layer and the
stromal layer. The anterior border layer is made up primarily of fibroblasts and melanocytes
(Eagle, 1988; Imesch et al., 1996; Oyster, 1999). By contrast, the stroma is a loose mesh of
collagen fibres, melanocytes, fibroblasts and clump cells. The surface of the eye can also be
divided into two regions: the pupillary zone and ciliary zone (Oyster, 1999). These regions are
bounded by a ring of tissue known as the collarette, which is a product of the reabsorption of the
pupillary membrane during development. There are differences in thickness between these two
zones, which leads to variation in colour and structure (Oyster, 1999).
There are many textural elements that can be found in the healthy human eye. These
include Fuchs’ crypts, Wolfflin nodules, pigment spots, contraction furrows and conjunctival
melanosis (Figure 4-1). Fuchs’ crypts are diamond-shaped lacunae in the anterior border layer of
the iris, which first arise during the reabsorption of the pupillary membrane (Purtscher, 1965).
Wolfflin nodules are small bundles of collagen that accumulate along the outer edge of the iris
(Donaldson, 1961; Williams, 1981). Pigment spots are small regions of hyper-pigmentation in
the anterior-border layer. They may be superficial (freckles) or distort the underlying stromal
layer (nevi) [Eagle, 1988; Harbour et al., 2004; Rennie, 2012]. Lastly, contraction furrows are
folds that fall in rings around the outer edge of the iris (Eagle, 1988). They are believed to be the
product of the dilation and the contraction of the pupil. Some irises may also show conjunctival
melanosis, which is pigment spotting that can be found on the sclera surrounding the iris
(Damato and Coupland, 2008). This tends to be more common in populations with darker irises.
Considerable research has been devoted to iris pigmentation variation (Eiberg et al.,
2008; Liu et al., 2010; Sturm et al., 2008). However, very few studies have attempted to look at
global variation in iris surface features. Although the functional consequences of these features
remain largely unknown, they have become a topic of significant forensic, biomedical, and
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ophthalmological interest. From a forensics perspective, a number of markers have been

identified over the past 10 years that are capable of predicting pigmentation characteristics, such
as hair, skin and iris colour, from crime scene DNA samples (Walsh et al., 2011). It has been
suggested that some of the iris surface features, such as Wolfflin nodules and pigment spots, may
have an influence on the perception of overall iris colour (Liu et al., 2010; Mackey et al., 2011).
Therefore, a better understanding of the genetic basis of iris surface features may lead to
improved eye colour predictor algorithms. Photographs of the iris may also represent a cost-
effective alternative to more expensive ophthalmological procedures. Sidhartha et al. (2014a,
2014b) recently characterized contraction furrow and Fuchs’ crypt grade in a Malaysian
population living in Singapore. They found that both features were associated with iris thickness
and/or the degree of iris angle closure. Thus, it may be possible to predict patient’s disease risk
from iridial surface features. Lastly, although very little is known about the pathology of iris
texture, there is some evidence that these features may influence individual health and well-
being. In particular, nevi and pigment spots may be a risk marker for uveal melanoma (Holly et
al., 1990; Horn et al., 1994).
As the textural elements in the iris have the potential to be of interest to many different
disciplines, it has become imperative to develop a better understanding of the worldwide
frequency and genetic basis of these traits. At present, iris features have been primarily studied in
populations of European ancestry (Larsson and Pedersen, 2004; Larsson et al., 2011; Sturm and
Larsson, 2009). Very few studies have focused on non-European populations, and these studies
have indicated that there are differences in the distribution of iris features between major
population groups (Qiu et al., 2005; Quillen et al., 2011). When iris texture was examined in
Portuguese, Cape Verdean, and Brazilian populations, for example, increasing European
biogeographical ancestry was significantly associated with a greater number of pigment spots,
Fuchs’ crypts, and contraction furrows (Quillen et al., 2011).
This paper has four primary goals: 1/ To develop a method for describing iris surface
features in populations of diverse ancestry; 2/ To characterize global differences in iris features
across European, East Asian and South Asian populations; 3/ To look at correlations between the
structural elements within each of the populations; 4/ To look at the association between genetic
markers that have been associated with iris surface features in European populations and our iris
feature measurements.
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4.3 Materials and Methods

4.3.1 Participant Recruitment
Between 2012 and 2014, 1773 healthy volunteers of East Asian, European and South
Asian ancestry were recruited at the University of Toronto to participate in a study looking at
global pigmentation diversity. All participants ranged in age between 18 and 35 years, and were
recruited using online and print advertisements that targeted members of the University of
Toronto student community. A personal questionnaire asking about each participant’s maternal
and paternal grandparents was administered in order to assess geographical ancestry. Individuals
who stated that all of their grandparents came from Japan, Korea, China or Taiwan were
categorized as East Asian, and individuals who stated that their grandparents originated in
Bangladesh, Pakistan, Sri Lanka or India were categorized as South Asian. Participants were
defined as European if their grandparents came from any region in Europe, other than Turkey.
Only individuals who stated that all relatives came from the same general geographical region
(i.e. East Asia) were included in the analysis. Admixed participants who came from two different
regions (i.e. East Asian and Europe) were eliminated. When information about the grandparents
was unknown, the self-reported ancestry of both parents was used to assess geographical
ancestry. In total, our study included 623 participants of European ancestry, 475 participants of
East Asian ancestry and 367 participants of South Asian ancestry. The remaining 308
participants were excluded from the analysis. In addition to determining ancestry, the personal
questionnaire was also used to ensure that each participant was healthy, and had not been
previously diagnosed with any ocular pigmentation-related disorders. Lastly, participants were
asked to provide a self-assessment of their iris colour using the Fitzpatrick Phototype Scale
(Fitzpatrick, 1975).

4.3.2 Genotyping
A 2-mL saliva sample was taken from each participant using the Oragene˖DNA (OG-
500) collection kit (DNA Genotek, Canada). All participants were instructed not to eat, drink or
smoke for at least 30 min prior to their appointment in order to ensure maximal sample purity.
DNA was then isolated from each sample using the protocol provided by the manufacturer.
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We selected four markers for genotyping that have either been directly associated with, or
are purported to be associated with, iris texture in European populations (Larsson et al., 2011;
Liu et al., 2010). These include TRAF3IP1 rs3739070 (contraction furrows), SEMA3A
rs10235789 (crypts), DSCR9 rs7277820 (Wolfflin nodules) and HERC1 rs11630290 (pigment
spots).
All DNA samples were sent to LGC Genomics (United States) for genotyping. LGC
Genomics uses a KASP based genotyping method that combines allele-specific amplification
with FRET (fluorescent resonance energy transfer) technology. Twenty-nine samples were
included as blind duplicates and 14 samples were included as blanks in order to check the quality
of the genotyping results. The concordance rate for both blind duplicates and blanks was 100%.

4.3.3 Acquisition and Processing of Iris Photographs

A photograph of each participant’s right iris was acquired using a Miles Research
Professional Iris Camera (Miles Research, United States). This camera consists of a FujiFilm
Finepix S3 Pro 12-Megapixel DSLR mounted on a 105-mm Nikkor macro lens. All photographs
were taken in RAW format with an aperture of f19, a shutter speed of 1/125” and an ISO of 200.
A biometric coaxial cable was used to deliver light to the iris at a constant temperature in order
to maintain colour and brightness fidelity and reduce the impact of ambient light. A Krypton
2.33 V light bulb was used as a focusing light, which allowed participants’ pupils to become
adjusted to a standardized light source. A chin rest and camera mount was used to ensure that
each photograph was taken from a standard distance and angle.
Photographs were converted to JPEG format and resized from 3043 by 2036 to 1200 by
803 pixels using Adobe Camera Raw in Adobe Photoshop CS5 (Adobe Systems Incorporated,
United States). The white balance was set to flash, the contrast and blacks levels were set to zero,
and the default values were maintained for all remaining settings.

4.3.4 Iris Analysis

One of the authors (D.C.) designed a web-based application in order to accurately and
reliably characterize iris texture. The web application can be accessed at http://iris.davidcha.ca/.
Accounts can be set up for interested users by request. This program consists of 11 steps, which
take approximately 2 min to complete for each iris. In the first step, the user is required to note
 100

whether or not the iris is significantly obstructed by eyelids, eyelashes or reflections (to the
extent that any of the five structures of interest cannot be accurately characterized). Steps 2 to 6
have the user identify the approximate centre point of the iris, the approximate centre point of the
pupil, and then draw a best fit circle around the scleral boundary, the collarette, and the pupillary
boundary. This allows the program to separate the iris into a ciliary zone and pupillary zone
(Figure 4-2), and divide the iris into four different quadrants based on the centre of the iris
(Figure 4-3).
In Step 7, the user must click on all of the pigment spots present in the iris. The iris is
magnified by 1.5× times for this step, in order to more easily distinguish between pigment spots
and other iridial textural elements. For each iris, the program records the number of pigment
spots, as well as the quadrant in which each pigment spot is found.
In step 8, the user notes the presence and extension of contraction furrows. A rotating
line, fixed at the centre point of the iris, is used to help the user determine whether or not the
furrows cover more than 180˚ of the iris. In order to facilitate the identification of furrows, the
iris is magnified by 1.5× for this step. The user also notes in which quadrants, if any, the furrows
are found.
Wolfflin nodules are characterized in a manner similar to contraction furrows. In step 9, a
rotating line is fixed at the centre point of the iris, and the user must determine whether nodules
are present, and if so, whether or not they extend around more than 180˚ of the iris. As with
contraction furrows, the user must also note in which quadrants the Wolfflin nodules can be
found.
In step 10, the user clicks on the outermost edge of all of the crypts found in the iris.
Crypts that originate from the collarette are characterized as either ‘small’ or ‘large’. Large
crypts are defined as those that extend from the collarette into more than 50% of the ciliary zone.
Small crypts are defined as those that do not extend into more than 50% of the ciliary zone. This
is calculated automatically by the program. The program also records the quadrant in which each
crypt is found. Crypts that do not originate from the collarette are manually defined as small. The
iris is magnified by 1.5× for this step in order to increase the visibility of crypts in darker irises.
For the last step, the user must note whether or not there are any pigment rings or
pigment spotting on the visible scleral region of the eye. The iris is obscured for this step, and
only the sclera is visible.
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After analysis of all 1465 irises, the program was used to output an Excel spreadsheet
which contained the category for each of the five surface features, the quadrants in which these
features were found and the diameter of the iris in pixels. Detailed information about the position
of each of the crypts and pigment spots identified could also be retrieved. All initial iris
categorizations were carried out by M.E.

4.3.5 Characterization of Iris Structure

We developed iris feature categories that could capture variation in diverse populations
with different frequencies of iris colour. These were primarily based on categorization systems
that had been developed in prior studies (Larsson and Pedersen, 2004; Larsson et al., 2011;
Sidhartha et al., 2014a, 2014b; Sturm and Larsson, 2009). However, they were modified to
account for the pigmentation diversity present in our sample set. Pigment spots were initially
measured using 4 grades: 1, no pigment spots; 2, between one and two pigment spots; 3, between
three and five pigment spots; 4, more than five pigment spots. However, grades 3 and 4 were
later collapsed into a single grade to account for the lower frequency of pigment spots in the East
and South Asian groups compared to the European group (Figure 4-4). As it is not possible to
reliably differentiate between pigment spots and nevi in photographs, we chose pigment spot
categories that attempted to capture the overall magnitude of spotting in the iris. Both contraction
furrows and Wolfflin nodules were measured using a grading system that took into account the
extension of these structures around the iris. Contraction furrows were measured across three
grades: 1, no contraction furrows; 2, contraction furrows that extend less than 180° around the
iris and 3, contraction furrows that extend more than 180° around the iris (Figure 4-5). Wolfflin
nodules were also measured using three grades: 1, no Wolfflin nodules; 2, Wolfflin nodules that
extend less than 180° around the iris and 3, Wolfflin nodules that extend more than 180° around
the iris (Figure 4-6). Crypts were measured across a four grade system that attempted to capture
the size and overall grade of crypts in the iris: 1, no crypts; 2, only small crypts centred around
the collarette; 3, at least one large crypt located in fewer than three quadrants of the iris and 4, at
least three large crypts located in three or more quadrants of the iris (Figure 4-7). Self-described
iris colour was measured using the categories developed for the Fitzpatrick Phototype Scale
(Figure 4-8) [Fitzpatrick, 1975]. These categories emphasize the intensity, rather than the shade,
of the eye: 1, light blue, green or grey; 2, blue, green or grey; 3, hazel or light brown; 4, dark
 102

brown and 5, brownish black. Lastly, conjunctival melanosis was characterized using a presence
or absence schema (Figure 4-9).

4.3.6 Statistical Analysis

Unless otherwise noted, statistical analyses were carried out using IBM Statistics SPSS
(v. 20.0, SPSS Incorporated, United States). Correlations between the ordinal iris feature
categories and iris colour were tested using Goodman and Kruskal’s gamma statistic, a measure
of rank correlation typically used for ordinal traits. We report both the G-value and p-value for
each of the correlations. Surface features were considered to be significantly correlated with each
other or with iris colour if p<0.05. The relationships between iris feature and sex, age and iris
width (the diameter of the best fit circle around the outer border of the iris) were examined using
ordinal regression. The assumptions of proportional odds and goodness of fit were tested.
Differences in iris feature frequency between the three sample sets were tested using the chi-
square test. For features that were significantly different, additional pairwise comparisons
between populations were conducted using an independent samples t-test with a Bonferonni
correction for multiple comparisons. After correction, differences between samples were
significant if p<0.0167. Differences in the width of the iris between the three populations were
tested using a one-way ANOVA. Normality was tested using Q–Q plots and the Levene Statistic
was checked before running the analysis.
Associations between the four polymorphisms of interest and the iris feature categories
were evaluated using ordinal regression in each population, including other relevant covariates in
the analyses (e.g. other iris structures, iris width or sex). The assumptions of proportional odds
and goodness of fit were tested before running the analysis. Genotype deviations from Hardy–
Weinberg proportions were evaluated using the Court Lab Calculator (Court, 2008).
Four months after the initial iris classification, intra-rater reliability was assessed on 40
irises by M.E. and inter-rater reliability was assessed on 40 irises by D.C. using the linear
weighted kappa statistic. This was done using the web application provided by GraphPad
software (GraphPad, United States).
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4.4 Results
A total of 475 East Asian, 623 European and 367 South Asian irises were evaluated using
the iris structure program (Table 4-1). Of these, 14 irises (eight East Asian, four European and
two South Asian) were judged to be too obscured or blurry to accurately characterize, and were
excluded from the analysis. One additional participant of South Asian ancestry with a
pigmentation-related ocular disorder (albinism) was also removed from the study. None of the
remaining participants reported ocular disorders. However, as medical history was self-reported,
it is possible that additional participants with ocular disorders may have been incorporated into
the study. The intra- and inter-rater reliability was assessed using the kappa statistic (for
additional information, see Material and Methods section) [Viera and Garrett, 2005]. The intra-
and inter-rater reliabilities were good for all five structures (intra-rater kappa values; crypt
κ=0.881, furrow κ=0.835, nodule κ=0.884, pigment spot κ=0.889, melanosis κ=0.827; inter-rater
kappa values; crypt κ=0.809, furrow κ=0.857, nodule κ=1.000, pigment spot κ=0.898, melanosis
κ=0.867).
The width of the iris was significantly different across the three sample sets (F=200.161,
p<0.001), with East Asians having the smallest iris widths (mean=376.72 pixels), followed by
South Asians (mean=384.92) and then Europeans (mean=394.45 pixels).
Within the European sample set, there was a significant negative correlation between the
grade of Fuchs’ crypts and the extension of contraction furrows (G=−0.474, p<0.001) and a
significant, but weaker, negative correlation between the grade of Fuchs’ crypts and the number
of pigment spots (G=−0.238, p<0.001). There was also a significant, positive correlation between
the extension of furrows and the number of pigment spots (G=0.218, p=0.007). No significant
correlations between the five iridial structures were found in either the South or East Asian
sample sets. In the European sample set, self-reported darker iris colour showed a significant
positive correlation with the extension of contraction furrows (G=0.461, p<0.001) and a
significant negative correlation with Wolfflin nodules (G=−0.409, p<0.001). In the South Asian
sample set, in contrast with what was observed in the European sample set, darker iris colour
showed a significant negative correlation with the extension of contraction furrows (G=−0.233,
p=0.041).
Sex was significantly associated with crypt grade in the East Asian (Nagelkerke
R2=0.013, p=0.019), European (Nagelkerke R2=0.022, p<0.001) and South Asian (Nagelkerke
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R2=0.037, p<0.001) samples. In all three groups, males had a significantly higher crypt grade
than females (East Asian male/female OR=1.54, European male/female OR=1.72, South Asian
male/female OR=2.07). Sex was not associated with any of the other four iridial structures. Iris
diameter was significantly associated with a higher number of pigment spots in the European
(Nagelkerke R2=0.012, p=0.009) and South Asian sample sets (Nagelkerke R2=0.018, p=0.041).
Iris diameter was also significantly associated with more extended Wolfflin nodules in
Europeans (Nagelkerke R2=0.021, p=0.001). No associations were found between age and iris
structure in any of the three groups.
All five structures examined showed significant differences in frequency between the
three sample sets (Fuchs’ crypts: χ2=67.388, p<0.001; contraction furrows: χ2=186.819, p<0.001;
pigment spots: χ2=260.587, p<0.001; Wolfflin nodules: χ2=350.627, p<0.001; conjunctival
melanosis: χ2=273.177, p<0.001). Additional pairwise independent sample T-tests with a
Bonferonni correction were run in order to determine the source of these differences. Europeans
had a significantly higher grade of Fuchs’ crypts (T=8.333, p<0.001), more extended contraction
furrows (T=10.802, p<0.001), more pigment spots (T=14.686, p<0.001) and more extended
Wolfflin nodules (T=18.028, p<0.001) than individuals of East Asian ancestry and a significantly
higher grade of crypts (T=2.961, p=0.003), greater number of pigment spots (T=15.571, p<0.000)
and more extended Wolfflin nodules (T=16.345, p<0.001) than individuals of South Asian
ancestry. The South Asian and East Asian samples had a higher proportion of individuals with
conjunctival melanosis than the European sample (South Asian: T=15.954, p<0.001; East Asian:
T=6.674, p<0.001). There was no significant difference in contraction furrow extension between
the European and South Asian groups (T=−0.306, p=0.759). Individuals of South Asian ancestry
had a significantly higher grade of crypts (T=4.231, p<0.001), more extended contraction
furrows (T=10.474, p<0.001) and a higher frequency of conjunctival melanosis (T=6.674,
p<0.001) than individuals of East Asian ancestry. However, there was no significant difference
in the number of pigment spots (T=−1.527, p=0.127) and the presence of Wolfflin nodules
(T=2.372, p=0.018) between these two groups. Self-described iris colour was significantly
different (χ2=892.674, p<0.001) across all three samples. Individuals of East Asian ancestry had
significantly darker eyes than individuals of South Asian (T=4.327, p<0.001) and European
(T=40.776, p<0.001) ancestry and individuals of South Asian ancestry had significantly darker
eyes (T=32.724, p<0.001) than individuals of European ancestry.
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Genotype proportions were in agreement with the expected Hardy–Weinberg proportions,

with the exception of moderate deviations observed for DSCR9 rs7277820 in the European
(χ2=5.373, p=0.020) and South Asian (χ2=3.930, p=0.047) sample sets and HERC1 rs11630290
in the South Asian sample set (χ2=5.224, p=0.022). We looked at the association between
SEMA3A rs10235789 and Fuchs’ crypts, TRAF3IP1 rs3739070 and contraction furrows, DSCR9
rs7277820 and Wolfflin nodules, and HERC1 rs11630290 and pigment spots. In the statistical
tests, we included as covariates the variables that were significantly associated with each iris
structure in our exploratory analyses (e.g. other iris structures, iris width, self-reported iris colour
and sex).
Fuchs’ crypt grade was significantly associated with SEMA3A rs10235789 in all three
sample sets (Table 4-2). In the European sample set, having one copy of the derived ‘C’ allele
significantly increased the odds of having a higher grade of crypts by 1.507 (p=0.023) and
having two copies of the derived ‘C’ allele significantly increased the odds of having a higher
grade of crypts by 2.203 (p<0.001). In the South Asian sample, having one copy of the derived
‘C’ allele significantly increased the odds of having a higher grade of crypts by 2.206 (p<0.001)
and having two copies of the derived ‘C’ allele significantly increased the odds of having a
higher grade of crypts by 2.721 (p=0.011). In the East Asian sample set, having one copy of the
derived ‘C’ allele significantly increased the odds of having a higher grade of crypts by 1.679
(p=0.018). As there were fewer than five individuals with two copies of the derived ‘C’ allele,
they were eliminated from this analysis.
The association between contraction furrow grade and TRAF3IP1 rs3739070 was
borderline significant in the European group but not in the East and South Asian sample sets
(Table 4-2). Having two copies of the derived ‘A’ allele increased the odds of having more
extended contraction furrows by 6.385 (p=0.051). However, having only one copy of the ‘A’
allele did not significantly (p=0.333) increase the odds. Both pigment spot grade and Wolfflin
nodule grade were not significantly associated with their respective markers in any of the three
groups examined.
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4.5 Discussion
The use of a program to characterize iris surface features has a number of advantages over
traditional qualitative assessments of iris photographs. For one, it allows for the storage and
retrieval of a substantial amount of information that would not be easy to obtain using
descriptive methods. This includes the size of the iris, the position in which each pigment spot
and crypt is found, and the distribution of traits across the different quadrants of the iris. In
addition, the program displays irises in a random order to limit bias, can handle large amounts of
data and is relatively fast. It is also simple to set up replicate users for intra- and inter-reliability
estimates. The inter- and intra-rater reliability estimates were both good for all five iris structures
examined, which underscores the ability of the program to return consistent and repeatable
results.
The iris structure categories that we developed were designed to account for the extensive iris
texture variation that is inherent in global populations. Previous attempts to study iris structure
have primarily been targeted at single, more homogenous populations (Larsson and Pedersen,
2004; Larsson et al., 2011; Sidhartha et al., 2014a, 2014b; Sturm and Larsson, 2009). In this
paper, we have attempted to expand on these methods and develop categories that can capture
iris structure variation in global populations with different iris characteristics.

4.5.1 Fuchs’ Crypts

Substantial amounts of hypoplasia can develop in the iris following the absorption of the
pupillary membrane. The optic vessels first appear around the fourth week of fetal life, and the
anterior layer begins to grow in front of the lens around the second embryonic month (Eagle,
1988; Eriksson et al., 1965; Oyster, 1999). This ultimately forms the iridopupillary membrane,
which covers the entire iris. By the sixth month, this membrane begins to atrophy and is
gradually reabsorbed until the pupil is completely free of mesodermal tissue. It has been
suggested that the anterior layer that remains is a rudimentary tissue that has no major function in
humans (Purtscher, 1965). As a result, there is a sparsity of tissue in this layer. Most irises have a
moderate amount of hypoplasia around the collarette. However, in some eyes, this hypoplasia
can extend widely into the ciliary zone. This leads to the formation of diamond-shaped lacunae
known as Fuchs’ crypts. These lacunae are primarily believed to be a phylogenetic defect that
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results from the decreasing importance of the anterior layer over evolutionary history (Purtscher,
1965). However, secondary lacunae can also gradually form due to the pushing and pulling
effects of the pupil on the anterior layer when it dilates and contracts. Thus, it is not surprising
that age has been associated with a higher number of crypts in prior studies (Larsson and
Pedersen, 2004). For Fuchs’ crypts, we attempted to develop a categorical system that could
capture the total amount of hypoplasia in the iris. Irises that contained very little hypoplasia, or
hypoplasia centred solely along the collarette, were placed into the first two categories. Irises that
had a greater amount of hypoplasia that extended into at least 50% of the ciliary zone were
placed in the latter two categories.
In all three sample sets, the largest number of participants fell into the second category (small
crypts around the collarette in at least three quadrants of the iris). However, individuals of
European descent had a greater probability of having a higher crypt grade than individuals of
South Asian descent, and individuals of South Asian ancestry had a greater probability of having
a higher crypt grade than individuals of East Asian descent (Table 4-3). Among East Asians,
only 29.8% of participants had crypts that extended into the ciliary zone, compared with 52.9%
of Europeans and 43.4% of South Asians. There may be several explanations for this finding.
Individuals of East Asian ancestry have significantly darker irises than those of South Asian
ancestry or European ancestry. It is possible that the presence of greater amounts of melanin in
the anterior stromal layer may make the iris less prone to the development of both primary and
secondary crypts. However, this is unlikely as the frequency of crypts appears to be independent
of iris colour within populations (Larsson and Pedersen, 2004). Iris width may be another
explanation, as we noted that individuals of East Asian ancestry had significantly smaller irises
than those of South Asian and European ancestry. However, we were unable to find an
association between iris width and crypt grade within populations. As the frequency of Fuchs’
crypts is believed to be associated with the overall stability of the anterior border layer, it is
possible that there are other genetic and developmental population differences that affect the
stability of this layer.
In all three groups, sex was weakly associated with Fuchs’ crypts. Most notably, males
consistently had greater odds of having a higher crypt grade than females. We were not able to
replicate an association between Fuchs’ crypts and age (Larsson and Pedersen, 2004). However,
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this was most likely because of the relatively narrow age range used in our study, compared with
prior studies where participants ranged in age from children to seniors.
SEMA3A rs10235789 is one marker that appears to be associated with variation in Fuchs’
crypts (Larsson et al., 2011). This marker, which is believed to play a role in the initial
development of the pupillary membrane, has previously been associated with approximately
1.5% of the variation in Fuchs’ crypts in an Australian sample of European ancestry. When we
looked at the association between SEMA3A rs10235789 and Fuchs’ crypt grade in our European,
East Asian, and South Asian samples, we found significant associations in all three groups. The
effects of SEMA3A rs10235789 appear to be additive, with two copies of the derived ‘C’ allele
having a greater effect than one copy. The geographical distribution of the SEMA3A rs10235789
polymorphism may help to explain some of the differences observed in the frequency of crypts
in the three groups. The derived ‘C’ allele is found at a very high frequency in our European
sample (0.48), while it is found at much lower frequencies in the East Asian (0.08) and South
Asian (0.28) groups. Therefore, we would expect crypts to be found at a lower frequency in the
latter groups. Given the role of SEMA3A rs10235789 in the absorption of the pupillary
membrane, it is possible that there are population differences in the stability of the anterior
border layer after pupil reabsorption in the different population groups.

4.5.2 Pigment Spots

Pigment spots are discrete areas of dark pigmentation that are found on the anterior border
layer of the iris (Rennie, 2012). There are many different types of spots, the most common of
which are iris freckles and nevi. Both variants of pigment spots are similar topographically, but
are very different ultrastructurally (Eagle, 1988; Harbour et al., 2004). Freckles, which range
from light to dark in colour, are found in 50-60% of healthy adults (Reese, 1944; Rennie, 2012).
They lie flat on the surface of the iris, and do not distort the iris stroma. By contrast nevi are
found only in 4-6% of adults, and look like well-demarcated nodular lesions (Rennie, 2012).
They tend to be more common on the lower half of the iris and may increase in size over time
(Horn et al., 1994). Unlike freckles, nevi do distort the underlying stromal layer. Although older
individuals have a predisposition for both types of pigment spots, they may be found in all ages
(Reese, 1944; Sturm and Larsson, 2009).
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Pigment spotting was significantly more common in individuals of European ancestry than in
individuals of East or South Asian ancestry (Table 4-3). Over half of our European sample
(57.9%) showed some degree of iridial spotting, compared with only 22.1% of East Asians and
16.7% of South Asians. This finding is not surprising. As pigment spots are discrete regions of
brown or black pigment, we would expect to see fewer spots in populations with a predisposition
for darker iris colours given that regions of hyper-melanin can be difficult to distinguish in
darker eyes. Interestingly, in all three groups, pigment spots were most likely to be found in the
lower temporal quadrant of the iris (Table 4-4). Several prior studies have noted that pigment
spots preferentially appear on the lower half of the iris (Schwab et al., 2014). This is because sun
exposure appears to be a risk factor for the development of iridial spots, and the lower half of the
iris is least protected by the eyelids. However, we only noted this preference on the lower
temporal quadrant, not on the lower nasal quadrant. In fact, the second most common quadrant
for pigment spotting was the upper temporal quadrant. It is possible that the upper temporal
quadrant may be less protected by the eyelids than the lower nasal quadrant, leading to this
discrepancy.
We did not find a correlation between pigment spotting and sex or age in any of the three
groups. However, we did note an association between iris width and pigment spot grade in the
European and South Asian sample sets. Given the well-established link between sun exposure
and iris pigment spots, it may simply be the case that a wider iris has more contact with the sun
than a smaller iris. We were also not able to identify an association between HERC1 rs11630290
and pigment spots in any of the three samples. This is not surprising, as HERC1 rs11630290 was
the weakest association reported in prior studies. It is possible that our sample size was too small
to replicate this association.

4.5.3 Contraction Furrows

Contraction furrows, which are produced by the contraction and dilation of the pupil, are
deep depressions that lie around the outer periphery of the iris (Eagle, 1988; Reese, 1944).
Although each contraction furrow rarely extends more than an iris quadrant in length, there are
typically many furrows staggered around the eye. It has been suggested that the overall thickness
and density of the iris play an important role in their formation and overall appearance (Larsson
and Pedersen, 2004; Larsson et al., 2011). Although prior studies have used several different
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systems for describing contraction furrows, we were primarily interested in looking at their
overall extension around the iris. This is because furrow extension is easier to quantify than
furrow depth or number of furrows when looking at irises of varying shades.
In all three samples, there were very few individuals with the lowest grade of contraction
furrows (15.2% of East Asians, 6.6% of Europeans, 3.8% of South Asians). Individuals of South
Asian and European ancestry had similar distributions of contraction furrow grade (Table 4-3).
However, participants of East Asian ancestry had a significantly lower furrow grade than either
South Asians or Europeans. In several prior studies, contraction furrow grade has been
associated with both darker irises and a thicker peripheral iris within populations (Larsson and
Pedersen, 2004; Quillen et al., 2011; Sidhartha et al., 2014a). Thus, it is unexpected to see an
overall lower grade of contraction furrows in the East Asian sample, given that this group has the
darkest self-described eye colour and the fact that East Asian individuals have also been found to
have a thicker peripheral iris than individuals of European ancestry (Lee et al., 2013). This
suggests that some factor other than iris colour or iris depth is responsible for generating
differences in contraction furrow grade between populations. One explanation may be iris width
and iris area. There is some evidence that a higher overall iris area (irrespective of iris depth)
may be associated with a higher grade of contraction furrows (Sidhartha et al., 2014a). In our
sample, individuals of East Asian ancestry had a significantly smaller iris width than individuals
of European or South Asian ancestry. Likewise, prior studies have found that individuals of East
Asian ancestry have an overall smaller iris area than individuals of other population groups
(Albert et al., 2003). Although, we were unable to find a significant association between
contraction furrow grade and iris width in any of the population groups, this association was
close to the established significance value of p<0.05 in the East Asian (p=0.096) and European
(p=0.073) groups.
TRAF3IP1 rs3739070 has been associated with approximately 1.7% of the variation in
contraction furrows in Australian individuals of European ancestry (Larsson et al., 2011). It has
been suggested that this marker may play a role in determining the overall thickness and density
of the iris. We were able to detect a borderline significant association (p=0.051) between
TRAF3IP1 rs3739070 and contraction furrows in our European sample. The derived allele
appears to have a recessive effect, with one copy of the derived allele not increasing the odds of
having more extended contraction furrows. We estimated that having two copies of the derived
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‘A’ allele increases the odds by 6.385. However, it is important to note that we only had five
individuals who were homozygous for the ancestral allele. In both East and South Asians, the
ancestral TRAF3IP1 rs3739070 allele also has a very low frequency. No participants of South
Asian descent, and only one individual of East Asian descent, were homozygous for the ancestral
‘A’ allele. In addition, the frequency of heterozygotes was also very low in both populations.
Thus, it is not surprising that we were unable to find an association between TRAF3IP1 and
furrow grade in either group. It is likely that markers other than TRAF3IP1 rs3739070 play a role
in the development of contraction furrows in populations of non-European ancestry. East Asian
populations may be an ideal group for studying the genetic basis of contraction furrows. Unlike
the European and South Asian samples, where there is little trait variation and the vast majority
of individuals have highly extended furrows, participants of East Asian ancestry were more
likely to fall into the lower furrow grades.

4.5.4 Wolfflin Nodules

Wolfflin nodules are small (0.1-0.2 mm) circular lesions that are distributed uniformly along
the outer border of the ciliary zone (Sturm and Larsson, 2009; Williams, 1981). They primarily
comprise of atrophied collagen from the anterior border and stromal layers. Wolfflin nodules are
highly associated with iris colour, and are much more common in light-eyed individuals than in
dark-eyed individuals. For example, in one study of 123 brown-eyed East Asian children,
Wolfflin nodules were absent from all participants (Kim et al., 2002). Much attention has been
devoted to Wolfflin nodules during the past 50 years due to their structural similarity to
Brushfield spots, an iris feature found in 85-90% of Down’s syndrome patients (Skeller and
Øster, 1951; Williams, 1981). Although the genetic basis of Brushfield spots remains largely
unknown, they closely resemble Wolfflin nodules and also appear to be restricted to individuals
with light-coloured eyes. However, they tend to be larger, less uniform, and located closer to the
mid-zone of the iris.
We decided to characterize Wolfflin nodules based on their overall extension around the iris.
Individuals who had Wolfflin nodules that extended more than 180° around the iris were put into
the highest grade, and individuals who had Wolfflin nodules that extended less than 180° were
placed in the second grade. Individuals with no discernable Wolfflin nodules were placed in the
lowest grade. Interestingly, very few individuals fell into the second category and participants
 112

were more likely to have no Wolfflin nodules, or Wolfflin nodules that extended around the
entire iris (Table 4-3).
Unsurprisingly, Wolfflin nodules were significantly more common in the European sample,
with 25.8% of this group having Wolfflin nodules that extended more than 180˚ around the iris,
compared to 0% of East Asians and 0.8% of South Asians. Given the strong association between
Wolfflin nodules and iris colour, we would expect to see fewer Wolfflin nodules in the primarily
brown-eyed East and South Asian populations. It has been suggested that both Wolfflin nodules
and Brushfield spots are not actually absent in brown irises, but merely obscured by the
abundance of melanin particles in the anterior border layer (Falls, 1970). Therefore, it is possible
that methods other than colour photography may be necessary to accurately characterize Wolfflin
nodules in populations of diverse ancestry. Interestingly, we did find an association between
larger iris width and more extended Wolfflin nodules in the European population. As Wolfflin
nodules are composed of atrophied collagen from the stromal layer, it is possible that larger irises
may be more prone to the accumulation of nodules in the anterior stromal layer.
In a recent genome-wide association study on iris colour in a population of Danish ancestry,
DSCR9 rs7277820 was found to be associated with iris colour variation (Liu et al., 2010). It was
suggested that this marker may actually be correlated with Wolfflin nodules, due to its presence
in the Downs’ Syndrome Critical Region. When we looked at the association between DSCR9
rs7277820 and Wolfflin nodules in the European sample, we were not able to find a significant
relationship.

4.5.5 Conjunctival Melanosis

Finally, the last trait that we looked at was conjunctival melanosis. In some irises, there
are areas of pigment spotting on the scleral region surrounding the iris (Damato and Coupland,
2008). These spots can comprise either of discrete regions, or rings surrounding the iris.
Conjunctival melanosis has not yet been widely explored. However, it does appear to be found
more commonly in populations with darker irises. This was largely reflected in our sample. Here,
almost half of our South Asian participants showed some form of conjunctival melanosis,
compared to only 23.3% of East Asians and 2.1% of Europeans (Table 4-3). It is important to
note, however, that these results may be an underestimate given that the upper and lower
portions of the sclera were not fully visible in our sample. Interestingly, the presence of
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conjunctival melanosis does not appear to be entirely linked to iris colour, as the distribution of
this trait is very different among the East and South Asian participants.

4.5.6 Correlations of Iris Features

Correlations between iris features and sex, age and iris width were examined in all three
population groups. Within the European population we were able to replicate a number of the
correlations that have been identified in prior studies. These include: 1/ A higher grade of crypts
is correlated with less extended contraction furrows (Larsson and Pedersen, 2004); 2/ A higher
grade of crypts is correlated with fewer pigment spots (Quillen et al., 2011); 3/ More extended
contraction furrows are correlated with a greater amount of pigment spotting (Larsson and
Pedersen, 2004). We were not initially able to replicate the association between crypt grade and
Wolfflin nodule grade (Larsson and Pedersen, 2004). However, we were interested in
determining if this was just because of the higher frequency of brown-eyed Europeans in our
sample, in comparison with the previous studies reporting the correlation. When we restricted
our sample to individuals who self-described their iris colour as either ‘light blue, green or grey’
or ‘blue, green or grey’, a higher grade of crypts (p<0.044, G=0.142) and more extended
contraction furrows (p=0.001, G=0.343) were both correlated with more extended Wolfflin
nodules. This emphasizes the effects that iris colour can have on the study of global Wolfflin
nodule variation. In contrast with the significant correlations observed in the European sample,
none of the five traits were significantly correlated in either the East or South Asian populations.
There are several potential explanations for this: 1/ Iris colour may be obscuring some of the
associations; 2/ There may be population differences responsible for the lack of correlations; or
3/ Our ability to identify significant correlations in the East Asian and South Asian samples was
hampered due to smaller sample sizes.

4.5.7 Future Research

Several prior studies have suggested that the distribution of iris features may be population-
dependent (Qiu et al., 2005; Quillen et al., 2011). In this paper, we looked at the global
distribution of Fuchs’ crypts, Wolfflin nodules, contraction furrows, pigment spots and
conjunctival melanosis in participants of East Asian, European and South Asian ancestry. We
found that all five traits showed significant differences in frequency across the three groups. We
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also showed that SEMA3A rs10235789 is significantly associated with crypts not only in the
European sample, but also in the East Asian and South Asian samples. By contrast, TRAF3IP1
rs3739070 is only correlated with contraction furrows in Europeans. Lastly, we were able to
replicate all of the iris feature correlations that had been identified in prior studies in our
European sample. However, we were not able to identify any correlations in the East and South
Asian groups. Future research will be necessary to explore the frequency of these features in
other populations, such as African and Hispanic groups.
There are still many gaps in our understanding of iris surface features. At present, very little
is known about the genetic basis and global distribution of Wolfflin nodules. This is primarily
because it is difficult to study this trait in populations that are primarily composed of individuals
with brown eyes. While it was initially suggested that these collagen bundles were absent in
darker irises, it has since been established that they are most likely masked when there are large
amounts of melanin particles in the anterior stromal layer. At present, the only way to accurately
characterize Wolfflin nodules is through magnification (Falls, 1970). Pigment spots are similarly
very difficult to study. Most notably, we do not have a way of confidently differentiating
between pigment freckles and nevi from photographs of the iris. Nevi and freckles both appear to
have a different relationship with the development of ocular disorders such as uveal melanoma
(Holly et al., 1990; Horn et al., 1994). Therefore, it would be extremely useful to develop ways
of distinguishing these features in photographs.
We suggest that infrared photography may provide a potential solution to these issues. As
infrared light is neither absorbed nor reflected by melanin, it may provide a way of visualizing
traits, such as Wolfflin nodules, that lie below the melanin granules in the anterior border layer.
In addition, as the primary difference between nevi and freckles is whether or not the underlying
stromal layer is distorted, infrared photography may provide a means of identifying the spots that
have an effect on this layer. The field of iris recognition already relies heavily on the use of
infrared photography. However, it also amalgamates all of the structures in the iris into a single
code (Acharya and Kasprzycki, 2010). Looking at infrared photographs from a qualitative
perspective may provide valuable information about the distribution of both pigment spots and
Wolfflin nodules in global populations.
Iris features are beginning to play an increasingly important role in many different fields.
However, there are still many gaps in our knowledge of these traits. Future research will be
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necessary in order to determine the functional differences in these traits in global populations, as
well as the effects that these traits may have on population-specific ocular diseases and disorders.
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4.6 Tables

Table 4-1 General Descriptive Statistics for the East Asian, European and South Asian Irises

Population Irises Included Females Males Average Age Average Iris Width
East Asian 467 320 147 21.61 376.72
European 619 376 243 22.65 394.45
South Asian 364 246 118 20.67 384.92
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Table 4-2 Results of the Genetic Ordinal Regression.

Significance values and odds ratios for each genotype are presented for each population. Genotypes
labelled with an ‘a’ refer to the ancestral genotype.

Polymorphism Population Genotypes Significance Odd's Ratios

CC - -
East Asian CT 0.018 1.679
TT a a
CC < 0.001 2.203
rs10235789
European CT 0.023 1.507
(Fuchs' Crypts)
TT a a
CC 0.011 2.721
South Asian CT < 0.001 2.206
TT a a
AA - -
East Asian CA 0.956 0.980
CC a a
AA 0.051 6.385
rs3739070
European CA 0.333 2.601
(Contraction Furrows)
CC a a
AA - -
South Asian CA 0.168 0.360
CC a a
TT - -
East Asian CT 0.173 0.241
CC a a
TT 0.187 1.685
rs11630290
European CT 0.998 1.000
(Pigment Spots)
CC a a
TT - -
South Asian CT 0.136 0.536
CC a a
AA 0.375 0.758
rs7277820
European GA 0.345 0.794
(Wolfflin Nodules)
GG a a
 118

Table 4-3 Iris Surface Feature Frequencies

The percentage (and number) of individuals with each category of Fuchs’ crypts, contraction furrows
pigment spots, Wolfflin nodules, melanosis and iris colour in all three populations.

Trait Category East Asian European South Asian

0 20.3% (95) 9.2% (57) 15.1% (55)

Fuchs’ 1 49.9% (233) 38.0% (235) 41.5% (151)

Crypts 2 19.7% (92) 30.9% (191) 25.0% (91)
3 10.1% (47) 22.0% (136) 18.4% (67)
0 15.2% (71) 6.6% (41) 3.8% (14)
Contraction
1 37.7% (176) 11.1% (69) 15.4% (56)
Furrows
2 47.1% (220) 82.2% (509) 80.8% (294)
0 77.9% (364) 42.1% (260) 83.2% (303)
Pigment
1 20.6% (96) 38.4% (238) 14.8% (54)
Spots
2 1.5% (7) 19.5% (121) 1.9% (7)
0 100% (467) 62.7% (388) 98.6% (359)
Wolfflin
1 0% (0) 12.0% (74) 0.5% (2)
Nodules
2 0% (0) 25.4% (157) 0.8% (3)
0 76.7% (358) 97.9% (606) 54.9% (200)
Melanosis
1 23.3% (109) 2.1% (13) 45.1% (164)
0 0% (0) 11.8% (73) 0% (0)
1 0% (0) 45.1% (279) 1.6% (6)
Eye
2 4.5% (21) 23.1% (143) 8.2% (30)
Colour
3 45.4% (212) 19.1% (118) 51.9% (189)
4 50.1% (234) 1.0% (6) 38.2% (139)
 119

Table 4-4 Distribution of Iris Surface Features

The percentage (and number) of irises with large crypts, contraction furrows, pigment spots, and Wolfflin
nodules in each of the four quadrants of the iris

Trait Population Quadrant 1 Quadrant 2 Quadrant 3 Quadrant 4

East Asian 10.9% (51) 15.8% (74) 14.3% (67) 18.4% (86)
Large Crypts European 31.5% (195) 35.5% (220) 25.8% (160) 29.2% (181)
South Asian 23.6% (86) 29.9% (109) 22.5% (82) 22.3% (81)
East Asian 26.8% (125) 70.4% (329) 82.4% (385) 61.0% (285)
Furrows European 82.1% (508) 84.8% (525) 89.2% (552) 84.3% (522)
South Asian 70.6% (257) 91.2% (332) 95.1% (346) 84.1% (306)
East Asian 2.8% (13) 5.1% (24) 10.3% (48) 8.1% (38)
Pigment Spots European 17.3% (107) 18.6% (115) 31.7% (196) 31.2% (193)
South Asian 2.7% (10) 4.9% (18) 6.3% (23) 6.6% (24)
East Asian 0% (0) 0%(0) 0% (0) 0% (0)
Nodules European 23.7% (147) 28.6% (177) 34.9% (216) 31.2% (193)
South Asian 0.8% (3) 1.1% (4) 1.4% (5) 1.4% (5)
 120

4.7 Figures

Figure 4-1 Five Surface Features Commonly Found in the Human Iris
The arrows are pointing to the features in images b, c and e. Fuchs’ crypts (a) are lacunae in the anterior
border of the iris which arise during resorption of the pupillary membrane. They may be either large or
small and closely resemble windows. Four sample crypts are outlined in the image below. Wolfflin
nodules (b) are small bundles of collagen that are the consequence of atrophy in the stromal layer of the
iris. Pigment spots (c) are discrete areas of pigmentation that can be observed on the surface of the iris.
Spots that distort the stromal layer are referred to as nevi and spots that do not distort the stromal layer are
referred to as freckles. Contraction furrows (d) are rings that extend around the outer border of the iris.
They closely resemble wrinkles and are the product of the contraction and dilation of the pupil. Furrows
are typically discontinuous and staggered across the iris. In the image below, the black line follows the
path of the furrows around the eye. Conjunctival melanosis (e) is spotting that can be observed on the
scleral region surrounding the iris. It is usually benign, and is found more commonly in some ancestries
than in others.
 121

Figure 4-2 Boundaries of the Iris

In steps 2-6 of the web-based application, the user draws a best fit circle around the pupillary ruff (purple
circle), collarette (blue circle) and iris (red circle). This allows the program to identify the pupillary zone
(bounded by the blue and purple circles) and the ciliary zone (bounded by the blue and red circles)
 122

Figure 4-3 Iris Quadrants

The program divides the iris into four different quadrants based on the user-defined centre of the iris.
Quadrant 1 is the upper nasal quadrant, quadrant 2 is the lower nasal quadrant, quadrant 3 is the lower
temporal quadrant, and quadrant 4 is the upper temporal quadrant.
 123

Figure 4-4 Pigment Spot Categories

Category 1: no pigment spots. Category 2: between one and two pigment spots. Category 3: more than two pigment spots. Any well-demarcated
lesion that ranged in colour from tan to dark brown was considered a pigment spot. The black arrows illustrate example pigment spots.
 124

Figure 4-5 Contraction Furrow Categories

Category 1: no contraction furrows. Category 2: contraction furrows that extend less than 180° around the iris. Category 3: contraction furrows
that extend more than 180° around the iris. The black lines follow the extension of contraction furrows around the iris.
 125

Figure 4-6 Wolfflin Nodule Categories

Category 1: no Wolfflin nodules. Category 2: Wolfflin nodules that extend less than 180° around the iris. Category 3: Wolfflin nodules that extend
more than 180° around the iris. Wolfflin nodules were defined as well-demarcated lesions that were white to orange in colour. The black arrows
are pointing at example Wolfflin nodules.
 126

Figure 4-7 Crypt Categories

Category 1: no crypts. Category 2: only small crypts centred around the collarette. Category 3: at least
one large crypt located in fewer than three quadrants of the iris. Category 4: at least three large crypts
located in three or more quadrants of the iris. Large crypts are defined as those that extend from the
collarette into more than 50% of the ciliary zone. The web application used the scleral and iris boundaries
to automatically distinguish between large crypts and small crypts. Examples of crypts are illustrated in
the images below. Not all crypts are labelled in each image. Crypts that would be defined as large are
bounded in green and crypts that would be defined as small are bounded in yellow.
 127

Figure 4-8 Self-Described Iris Colour, as Defined by the Fitzpatrick Phototype Scale
The Fitzpatrick scale characterizes iris colour across five categories: 1/ light blue, green or grey, 2/ blue, green or grey, 3/ hazel or light brown, 4/
dark brown, and 5/ brownish black. The photographs below represent a self-described example from each of the five categories.
 128

Figure 4-9 Conjunctival Melanosis Categories

Conjunctival melanosis (black arrows) was measured using a presence or absence schema. Any iris that showed spotting on the sclera was
characterized as having conjunctival melanosis. This spotting typically took the form of a ring around the iris, or as isolated spots on the sclera.
 129

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Falls, H.F. (1970). Ocular changes in mongolism. Ann. N. Y. Acad. Sci. 171, 627–636.
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Larsson, M., Duffy, D.L., Zhu, G., Liu, J.Z., Macgregor, S., McRae, A.F., Wright, M.J., Sturm,
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of the iris. Eye 26, 29–50.
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A.M., Langmann, G., and Richtig, E. (2014). New insights into oculodermal nevogenesis and
proposal for a new iris nevus classification. Br. J. Ophthalmol. 99, 644-649.
Sidhartha, E., Gupta, P., Liao, J., Tham, Y.-C., Cheung, C.Y., He, M., Wong, T.Y., Aung, T.,
and Cheng, C.-Y. (2014a). Assessment of iris surface features and their relationship with iris
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Sidhartha, E., Nongpiur, M.E., Cheung, C.Y., He, M., Wong, T.Y., Aung, T., and Cheng, C.-Y.
(2014b). Relationship between iris surface features and angle width in Asian eyes. Invest.
Ophthalmol. Vis. Sci. 55, 8144–8148.
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and Montgomery, G.W. (2008). A single SNP in an evolutionary conserved region within intron
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 Chapter 5:
Concluding Remarks

132
 133

5.1 Introduction
The overall goal of my research was to investigate the global distribution and genetic
basis of iris colour and structure in populations of diverse ancestry. In order to accomplish this, I
acquired high-resolution iris photographs from participants of East Asian, European and South
Asian ancestry who lived in the Greater Toronto Area. For my first paper (Chapter 2), I used a
pilot sample of 205 participants in order to develop my methodology. For my second and third
papers (Chapters 3-4), I carried out a full research study on 1,465 participants. I was specifically
interested in answering the following research questions: 1/ How can I quantify iris colour and
central heterochromia in populations of diverse ancestry? 2/ How can I characterize iris surface
features in populations of diverse ancestry? 3/ How does the phenotypic distribution of iris
colour and surface features differ in populations of East Asian, European and South Asian
ancestry? 4/ What genetic variants are responsible for iris colour and structure variation in
European, East Asian and South Asian populations?

5.2 Summary of Findings

5.2.1 Quantifying Iris Pigmentation
Obtaining a measurement of iris colour has traditionally been a very difficult endeavor.
Categorical methods of characterizing iris colour are unable to account for the extensive
continuous variation observed in this trait. In contrast, quantitative methods ignore regions of
heterochromia in the eye. A major aim of my research was to develop a quantitative method of
measuring eye colour that could also account for central heterochromia. In the first study of my
thesis (Chapter 2), I attempted to extract iris colour from a 256 by 256 pixel square in the left
quadrant of the eye. A collaborator created a macro in Adobe Photoshop CS5 that carried out this
process automatically. The average colour measurement of this square was then recorded in CIE
1976 L*a*b* (CIELAB) colour space. CIELAB is a universal colour system that closely
approximates human vision (McLaren, 1976). This means that most people should be able to
detect a one unit change in colour space (Kuehni and Marcus, 1979). This is very advantageous
for a trait like eye colour, which has traditionally been graded visually.
Although this initial quantitative method was quite successful in capturing phenotypic
variation, it had considerable limitations. Other research groups correctly noted that this method
 134

isolated only a very small portion of the iris (Andersen et al., 2013). This means that if a
pigmentation abnormality, such as a large pigment spot, is found in that 256 by 256 pixel square,
the overall colour of that iris might be grossly misrepresented. In addition, this method provided
no easy way to isolate or study central heterochromia.
For my second study (Chapter 3), I refined my iris colour quantification methodology to
account for these limitations. A web application was designed by a second collaborator that was
able to extract a 60˚ wedge from the left quadrant of the iris. Although this new method is not
automatic, it delivers a much deeper and comprehensive estimation of iris colour. As the user is
required to define the scleral, pupillary and collarette boundaries, the program is able to obtain
individual colour measurements from both the pupillary and ciliary zones. This means that the
amount of central heterochromia in the iris, as well as the overall colour of the iris, can be
quantified. I continued to use CIELAB colour space to quantify iris colour, as this system proved
to be very effective in the early stages of my research. This new methodology is powerful,
accurate and allows researchers to approach iris pigmentation variation from an entirely new
direction.

5.2.2 Characterizing Iris Surface Features

Although diversity in iris surface features has been studied within several continental
population groups, there has not yet been an attempt to look at variation in these traits across
different continental groups. As a result, most of the methods that have been used to
characterize iris structure were designed with a single sample set in mind. In the third research
study of my thesis (Chapter 4), I attempted to develop a method of grading iris surface features
that could be applied to diverse populations. At present, the most effective way of
characterizing iris structure is categorically. Both Wolfflin nodules and contraction furrows
were graded based on their presence and extension around the iris. Fuchs’ crypts were graded
based on the amount and extent of hypoplasia in the iris. Finally, pigment spots were graded
based on their overall count in the eye. The same web application that was used to characterize
iris colour was modified to also describe iris texture. This web application consists of eleven
steps that have the user manually note the presence and extent of each of the surface features in
the iris. The program then automatically assigns a grade for each of the features. Interestingly,
while investigating iris texture variation I noticed that many eyes showed spots or rings of
 135

pigment on the sclera. This condition, which is known as conjunctival melanosis, has not yet
been widely characterized. Therefore, I chose to also incorporate the ‘presence or absence’ of
this trait into the web application.
This categorical method of measuring iris surface features shows consistent and reliable
results. In addition, the frequency of each of the traits described in my study mirrors previously
reported frequencies (Donaldson, 1961; Harbour et al., 2004; Sidhartha et al., 2014a, 2014b). A
standardized method of measuring iris surface features in diverse populations should allow
researchers to better compare the distribution of these traits across different ancestral groups.

5.2.3 Phenotypic Distribution of Iris Colour and Central Heterochromia

It is well-established that the greatest range of iris pigmentation diversity is found in
populations of European ancestry. However, very little is known about the distribution of iris
colour variation in populations outside of Europe. In the first and second papers of my thesis
(Chapters 2 and 3), I investigated the distribution of this trait in East Asian, European and
South Asian populations.
Unsurprisingly, the European sample showed the greatest range of colour variation across
CIELAB colour space. However, extensive diversity could also be observed in the South and
East Asian samples, with the South Asian sample showing somewhat more dispersion than the
East Asian sample. The distribution of all three populations across CIELAB colour space
confirms that eye colour is a continuous trait that needs to be approached quantitatively.
Interestingly, brown eyes in the European sample appear to be much lighter than brown eyes in
the East and South Asian samples. This suggests that there may be fixed population markers
that control the intensity of brown eye colour in these different groups.
The distribution of central heterochromia also showed clear population differences. The
European sample displayed the greatest amounts of central heterochromia, followed by the
South Asian and East Asian samples. Given the low colour difference between the pupillary
and ciliary zones in populations outside of Europe, central heterochromia appears to be a trait
found almost exclusively in lighter coloured irises. Populations with darker irises do not show
very much variation between the different regions of the eye.
 136

5.2.4 Phenotypic Distribution of Iris Surface Features

In the third paper of my thesis (Chapter 4), I looked at the phenotypic distribution of
Fuchs’ crypts, contraction furrows, Wolfflin nodules and pigment spots in populations of East
Asian, European and South Asian ancestry. Interestingly, the global distribution of all four iris
features appears to be largely dependent on ancestry. Fuchs’ crypts, pigment spots and Wolfflin
nodules had a significantly higher grade in the European sample compared to the other two
groups. Only contraction furrows showed an equally high grade in both the European and South
Asian samples. In turn, the South Asian sample had a higher grade of Fuchs’ crypts and
contraction furrows than the East Asian sample. However, both of these groups showed a
similar degree of pigment spotting and Wolfflin nodules. The population-specific differences in
pigment spots and Wolfflin nodules are most likely explained by differing eye colour
frequencies. As both of these traits are more prominent in lighter irises, it makes sense that they
would be most common in individuals of European ancestry. However, it is unlikely that the
global differences in contraction furrow and crypt grade can be attributed to iris colour alone.
Fuchs’ crypts were not associated with iris colour in any of the three groups. Similarly, the
darkly pigmented East Asian population showed the lowest grade of contraction furrows.
However, within the European population, contraction furrow extension was positively
associated with darker irises.
These results suggest that the distribution of iris surface features is population dependent.
This may help explain why so many ocular diseases and disorders show ancestry-dependent
distributions.

5.2.5 Genetic Basis of Iris Colour

At present, the genetic basis of iris pigmentation is fairly well understood in populations
of European ancestry. The majority of variation between blue and brown eye colour can be
attributed a to a single marker, rs12913832, located in intron 86 of the gene HERC2 (Eiberg et
al., 2008; Kayser et al., 2008; Sturm et al., 2008). However, other polymorphisms in OCA2,
SLC24A4, SLC45A2, TYR, IRF4, TYRP1, ASIP, DSCR9, NPLOC4 and LYST may play a more
subtle role as well (Beleza et al., 2013; Frudakis et al., 2003; Kanetsky et al., 2002; Liu et al.,
2010; Sulem et al., 2007). At present, very few studies have attempted to examine iris colour
diversity in populations of non-European ancestry.
 137

In my first and second publications (Chapters 2 & 3), I looked at the association between
putative iris pigmentation markers and eye colour in participants of East Asian, European and
South Asian ancestry. My first publication consisted of a small-scale pilot study that looked
specifically at HERC rs12913832. My second publication included HERC rs12913832 and 13
additional markers. In both studies, HERC rs12913832 was significantly associated with iris
pigmentation variation in the European and South Asian sample sets. However, this marker had a
very different effect in each population. This is exemplified particularly well in the second
publication by individuals who were homozygous for the derived allele. European homozygotes
had b* values that fell into the ‘blue’ region of colour space. In contrast, South Asian
homozygotes had b* values that fell into the ‘green/intermediate’ region of colour space. This
suggests that HERC2 rs12913832 does not just code directly for the difference between blue and
brown eye colour. Rather, there must be other variants modifying its effect that are different in
both populations. In the European sample, five other markers were also associated with iris
pigmentation diversity: OCA2 rs1800407, SLC45A2 rs16891982, SLC24A4 rs12896399, IRF4
rs12203592 and TYR rs1393350.
Apart from HERC2 rs12913832, eye colour in the South Asian sample set was largely
determined by SLC24A5 rs1426654. This marker had an additive effect on iris colour, with each
copy of the derived allele producing lighter brown eyes. SLC24A5 rs1426654 is an interesting
polymorphism because it is one of the principal markers associated with the lightening of skin
pigmentation in European populations (Lamason et al., 2005). It has also been found to
contribute substantially to skin pigmentation diversity in South Asia (Stokowski et al., 2007).
Thus, it is unlikely that it was selected for due to its effect on iris pigmentation. A second
marker, LYST rs3768056, was also associated with eye colour in the South Asian sample.
However, it had a much smaller effect.
The lone determinants of eye colour in the East Asian sample were OCA2 rs1800414 and
OCA2 rs74653330. The derived allele at both markers was associated with lighter iris
pigmentation. Although not much is known about pigmentation variation in East Asia, both
markers show a strong association with skin pigmentation in this region, and it is believed that
both of these markers may have played a role in the evolution of light skin pigmentation in East
Asia (Eaton et al., 2015; Edwards et al., 2010). Thus, their association with iris colour is likely
secondary to their effect on skin colour.
 138

My first and second research papers ultimately showed that the genetic basis of iris
pigmentation is, at least to some extent, population-specific. With the exception of HERC2
rs12913832, the markers associated with iris pigmentation variation were different in all three
groups. In the South Asian sample, this may be largely related to allele frequency differences.
The derived allele frequency at SLC24A4 rs12896399 (European: 0.402, South Asian: 0.278),
SLC45A2 rs16891982 (European: 0.931, South Asian: 0.1134) and TYR rs1393350 (European:
0.259, South Asian: 0.083) is much lower in the South Asian group compared to the European
group. Thus, it is possible that the sample size in the South Asian group was simply too small to
pick up these associations. In contrast, the markers controlling iris colour in the East Asian
sample appear to be entirely independent from those observed in the European and South Asian
samples. HERC2 rs12913832, OCA2 rs1800407, SLC45A2 rs16891982, IRF4 rs12203592 and
TYR rs1393350 are all either entirely absent or found at negligible (< 0.010) frequencies in the
East Asian sample. It is interesting to note that the genes that determine iris pigmentation
diversity in all three regions are largely those that have been associated with other pigmentary
characteristics, such as skin colour. This may provide some support to the hypothesis arguing
that these markers were initially selected for due to the influence that they had on other
pigmentary phenotypes.

5.2.6 Genetic Basis of Iris Surface Features

In the third paper of my thesis (Chapter 4), I looked at the association between Fuchs’
crypts, contraction furrows, Wolfflin nodules and pigment spots and a set of genetic variants that
had previously been identified in an Australian population of European ancestry (Larsson et al.,
2011). Fuchs’ crypts were significantly associated with SEMA3A rs10235789 in the East Asian,
European and South Asian samples. In all three groups, each additional copy of the derived allele
was associated with a higher grade of crypts. I was also able to detect a borderline association
between TRAF3IP1 rs3739070 and contraction furrow grade in the European sample. However,
these results were not replicated in the East or South Asian groups. The ancestral TRAF3IP1
allele has a very low frequency in all three groups. However, it was found at a higher frequency
in the European group (0.08) compared to the East (0.04) and South Asian (0.03) groups. It is
possible that a larger sample size would be necessary to pick up this association.
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Although HERC1 rs10235789 has been tentatively associated with the number of nevi
in the eye, I was unable to detect a relationship between this marker and pigment spots in my
sample set. Likewise, DSCR9 rs7277820 was not associated with Wolfflin nodules in any of the
three ancestral groups.
Even though iris surface features show a moderately high heritability, SEMA3A
rs10235789 and TRAF3IP1 rs3739070 explain very little of the total variation in Fuchs’ crypts
and contraction furrows. Given that all four iris features showed very different distributions in
my three study populations, it will be necessary to investigate the genetic basis of these traits
using denser panels of genetic markers, and a diverse representation of populations in order to
identify other contributing markers.

5.3 Research Limitations

The findings published in my three research papers represent an important contribution to
the fields of iris structure and colour research. However, there are several limitations which must
be addressed. These limitations include standardization across different sample sets, the number
of populations studied, and difficulties associated with characterizing colour-dependent iris
features
The web application that was developed for this thesis represents a novel approach to the
study of iris colour and structure variation. Unlike other methods, which generate an average
measurement of eye colour and ignore other features like heterochromia, this application allows
users to obtain a separate colour measurement from the ciliary and pupillary zones. In addition,
because the web application does not require the eye to be a certain size or located in a particular
place in the photograph, it can be applied to a broad set of iris photographs. Despite these
advantages, this method still does not allow for the comparison of iris photographs taken from
different instruments and cameras. This is largely because different instruments do not have the
same exposure and colour settings. The next step in iris pigmentation research will be to find a
way to standardize the ways in which photographs of the eye are taken across different research
groups. Perhaps one solution would be to place a colour card in each photograph. This colour
card could then be used to standardize colour and brightness variation in different sample sets.
However, implementing such a method would be challenging. An alternative tactic to combine
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the results of different studies would be to use meta-analytic approaches in which a global effect
can be calculated based on the effect and variance of the individual studies.
Another limitation of my research was the number of populations that I chose to study.
The papers presented in this thesis are the first to look at iris colour in East and South Asian
populations. They are also the first to compare iris features across different continental groups.
However, there are many populations that still need to be studied in order to fully understand the
global diversity inherent in these traits. The Greater Toronto Area was an ideal setting for the
recruitment of East Asian, European and South Asian volunteers. However, the pilot study
indicated that I would not be able to recruit a sufficient number of individuals of African,
Hispanic and Middle Eastern ancestry. Future research will be needed in order to obtain a larger
sample size with a better ancestry representation.
Lastly, we need to develop better ways of measuring colour-dependent iris features.
Although high-resolution photographs of the iris do a wonderful job of capturing Fuchs’ crypts
and contraction furrows, the same cannot be said for Wolfflin nodules and pigment spots.
Wolfflin nodules are often impossible to see in darker coloured eyes (Falls, 1970). This means
that their overall frequency is greatly underestimated in populations where brown eyes
predominate. Likewise, ‘pigment spots’ is a category that is used to encompass two types of
pigment aberrations: nevi and freckles. Both of these structures affect the iris in different ways.
Although pigment spots lay flat on the anterior border layer, nevi ultimately distort the
underlying stromal layer (Eagle, 1988; Harbour et al., 2004). It is presently not possible to
distinguish between these two types of spots using photographs of the iris. I believe that the use
of near infrared photography may be a solution to both of these problems. Infrared light is
neither absorbed nor reflected by melanin. Therefore, its use may allow for the visualization of
structures that lie below the iris surface. In the case of pigment spots, it may increase the
visibility of nevi and decrease the visibility of pigment spots due to their differing effects on the
stromal layer.

5.4 Conclusions

Ultimately, this thesis has made the following findings:

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1) Quantitative methods of measuring eye colour represent an ideal means of investigating iris
colour variation in populations of diverse ancestry. Quantitative methods can also be used to
successfully quantify heterochromatic variation in the iris.
2) The phenotypic distribution of iris colour shows large population differences. Individuals of
European ancestry show the greatest amounts of colour diversity. However, substantial
variation can also be observed in East and South Asia. Central heterochromia is largely
restricted to lighter coloured irises.
3) Fuchs’ crypts, contraction furrows, Wolfflin nodules and pigment spots show very different
distributions in populations of East Asian, European and South Asian ancestry.
4) The marker HERC2 rs12913832 is a major determinant of iris colour variation in individuals
of both European and South Asian ancestry. However, it has different effects in each group.
5) The markers associated with iris pigmentation variation in East and South Asia are, to some
extent, different from the markers associated with iris pigmentation variation in Europe.
OCA2 rs1800414 and OCA2 rs74653330 are the primary determinants of iris colour in the
East Asian sample. HERC2 rs12913832, SLC24A5 rs1426654 and LYST rs3768056 are the
primary determinants in the South Asian sample.
6) Many of the markers associated with iris pigmentation variation in all three groups have been
associated with other pigmentary characteristics, like skin and hair colour.
7) SEMA3A rs10235789 shows an association with Fuchs’ crypt grade in the East Asian,
European and South Asian samples. TRAF3IP1 rs3739070 shows a borderline association in
the European sample.

Iris colour and structure research has the potential to contribute greatly to the fields of
anthropology, forensics, molecular biology and public health. In this thesis, I have shown that
both eye colour and surface features are highly complex traits that show considerable inter-
population variation. Thus, it is important that research groups branch out and begin to consider
the genetic basis and global distribution of both traits in populations outside of Europe. Although
the study of these topics is still very much ongoing, this thesis has provided a framework which
can be used by other researchers in the same field.
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Copyright Acknowledgements

Chapter 1 has been previously published as:

Edwards, M., Gozdzik, A., Ross, K., Miles J., and Parra, E.J. (2012) Technical note: quantitative
measures of iris colour using high resolution photographs. Am. J. Phys. Anthro. 147, 141-149.

Permission has been acquired from John Wiley and Sons for publication in this thesis (License
Number: 3802541113357)

Chapter 2 has been previously published as:

Edwards, M., Cha, D., S. Krithika., Johnson, M., Cook, G., Parra, E.J. (2015) Iris pigmentation
as a quantitative trait: variation in populations of European, East Asian and South Asian ancestry
and association with candidate gene polymorphisms. Pigment Cell Melanoma Res.

This article was published as open access. No copyright permissions were required.

Chapter 3 has been previously published as:

Edwards, M., Cha, D., S. Krithika., Johnson, M., Parra, E.J. (2016) Analysis of iris surface
features in populations of diverse ancestry. R. Soc. Open Sci. 3, 150424.

This article was published as open access. No copyright permissions were required.