Eye colour: portals into pigmentation genes and ancestry
5:19 AMEye colour: portals into pigmentation genes and
ancestry
Richard A. Sturm1 and Tony N. Frudakis2
Institute for Molecular Bioscience, University of Queensland, Brisbane, Qld 4072, Australia
DNAPrint genomics, Incorporated, 900 Cocoanut Ave, Sarasota, FL 34236, USA
Several recent papers have tried to address the genetic determination of eye colour via microsatellite linkage, testing of pigmentation candidate gene polymorphisms
and the genome wide analysis of SNP markers that are informative for ancestry. These studies show that the OCA2 gene on chromosome 15 is the major determinant of brown and/or blue eye colour but also indicatethat other loci will be involved in the broad range ofhues seen in this trait in Europeans.
One of the first investigations into the concept of mendelian inheritance in humans was the consideration of eye colour. Iris colour exists on a continuum from the lightest shades of blue to the darkest of brown or black, although genetic studies have usually categorised: blue,
grey, green, yellow, hazel, light brown and dark brown (Figure 1a) in addition to the colour deficiencies apparent in those with oculocutaneous albinism. In 1907, the Davenports [1] outlined what is still commonly taught in schools today as a beginners guide to genetics that brown eye colour is always dominant to blue, with two blue-eyed parents always producing a blue-eyed child, never one with brown eyes. Unfortunately, as with many physica traits, this simplistic model does not convey the complexities of real life and the fact is that eye colour is inherited as a polygenic not as a monogenic trait. Although not common, two blue-eyed parents can produce children with brown eyes. The apparently non-mendelian examples of
iris colour transmission from parents to offspring, combined with the quantitative nature of iris pigmentation indicate that the inheritance of this apparently simple
trait as a dichotomous value must be reconsidered. The use of eye colour as a paradigm for ‘complete’ recessive and dominant gene action should be avoided in the teaching of
genetics to the layperson, which is often their first encounter with the science of human heredity. The phenotypes of eye, hair and skin colour [2] in addition to stature and facial features will always be observed to run in families but families need to know that these are
complex traits (i.e. conditioned by several genes) [3].
Physical basis of eye colour: melanocytes,
melanogenesis and ancestry The physical basis of eye colour is determined by the distribution and content of the melanocyte cells in the uveal tract of the eye (Box 1). The iris consists of several Corresponding author: Richard A. Sturm (R.Sturm@imb.uq.edu.au).
www.sciencedirect.com
layers: the anterior layer and its underlying stroma are the most important for the appearance of eye colour [4]. In the brown iris there is an abundance of melanocytes and melanosomes in the anterior layer and stroma, whereas in the blue iris these layers contain little melanin. As
light traverses these relatively melanin-free layers, the minute protein particles of the iris scatter the short blue
-A schematic representation of the eye ball from the front view shows the anatomical division of the sclera, the white connective tissue, the iris and the coloured disk surrounding the central black pupil (Figure I). In cross section view the cornea is seen as a transparent tissue above the
iris enabling light to enter through the pupil, which is then focused by the lens onto the retina. The iris comprises two tissue layers, the innermost consists of cuboidal, pigmented cells that are tightly fused and is known as the iris pigment epithelium (IPE), which is formed from the optic cup during development. The outermost layer is referred to as the anterior iridial stroma and is composed mainly of loosely arranged connective tissue, fibroblasts and melanocytes and are of the same embryological origin as dermal melanocytes, which arise and migrate from the neural crest. Apart from albino patients, who lack melanin pigment and have eyes that might appear pink as a result of the reflection of light from blood vessels, the IPE does not exert a major influence on the perceived eye colour of normal individuals because the melanin in this layer is distributed similarly in irides of different colour. Notably, it is the density and cellular composition of the iris stroma that must be considered as major factors in the colouration of the eye [5].
The melanocyte cells are aggregated in the anterior border layer of the iridial stroma, parallel to the surface of the eye, and store melanin pigment in a specialized organelle within their cytoplasm termed the melanosome. White light entering the iris can absorb or reflect a
spectrum of wavelengths giving rise to the three common iris colours, blue, green–hazel and brown, but it should be recognized that these broad classifications are simplistic and that there is actually a continuum in the range of eye colours seen in Europeans. The middle of the panel illustrates the intracellular distribution and content of the melanosome particles within the iridial melanocytes with the varied melanin pigment quantity, packaging and qualities giving the range of eye shades [6]. Although blue eyes have similar numbers of melanocyte cells they contain minimal pigment and few melanosomes; green hazel irides are the product of moderate pigment levels, melanin intensity and melanosome number and with brown irides are the result
of high melanin levels and melanosomal particle numbers. Each of these eye colours can occur with or without a darker pigmented iris peripupillary ring, represented to the right of the figure. Insufficient studies have been performed into the nature of the peripupillary ring; however, the possibility that the number of melanocytes, their melanin granule size, distribution or content can differ between ethnic groups has been recognized [26], and further ultrastructural investigations are needed to clarify this issue.
wavelengths to the surface, thus blue is a consequence of structure not of major differences in chemical composition. The number of melanocytes does not appear to differ between eye colours [5], but the melanin pigment quantity, packaging and quality does vary, giving a range of eye shades [6]. The common occurrence of lighter iris colours is found almost exclusively in Europeans (i.e. recent monophyletic, non-East Asian, non-Native American and non African lineages) and individuals of European admixture.The study of biogeographical ancestry admixture is becoming more popular and soon it might be possible to date the genesis of lighter irides; that is to distinguish whether lighter iris colours are exclusive to the continental European populations, as opposed to unadmixed Middle Eastern or Central and/or Southern Asian popu-
lations with whom they share some common ancestry.
There are two forms of melanin pigment particles produced during melanogenesis and both occur in the iris of the eye, the cutaneous and follicular (skin and hair) melanocyte cells (Box 2). However, unlike the skin and hair in which melanin is produced continuously and secreted, in the eye the melanosomes containing the pigment are retained and accumulate in the cytoplasm of
the melanocytes within the iris stroma. Eumelanin is a brown – black form of pigment that is responsible for dark colouration and is packaged in ovoid eumelanosomes, which are striated particles, whereas pheomelanin is a red –yellow pigment produced in granular immature
pheomelanosomes [7].
The study of mouse-coat colours and the comparative genomic analysis with other mammals, including humans, has provided enormous insight into the genetic basis of pigmentation [8,9]. Several loci are known to have major effects on pigmentation (Table 1) including the enzymes
that are involved in the catalytic formation of melanin [including tyrosinase (TYR), tyrosinase related proteins TYRP1 and dopachrome tautomerase (DCT)], the melano-somal proteins [P and membrane-associated transporter protein (MATP) encoded by the OCA2 and MATP genes, respectively] and the melanocortin-1 receptor (MC1R), which is involved in pheomelanin– eumelanin pigment switching of the melanocyte [7,9].
Box 2. Melanin pigment formation
Melanin is an inert light-absorbing biopolymer of no fixed size and of uncertain unit structure that is extraordinarily resistant to chemical degradation. Melanogenesis is based on the chemical reactions that take place within the melanosome beginning with tyrosine, dopa and cysteine that result in the formation of the eumelanin and pheomelanin pigments, through a bifurcated biosynthetic pathway [27]. When tyrosine is oxidised by the tyrosinase (TYR) enzyme, dopaquinone (DQ) is produced as an intermediate (Figure I). In the absence of cysteine, DQ undergoes intramolecular addition producing cyclodopa, with a redox exchange between cyclodopa and DQ giving rise to dopa and dopachrome. Dopa is a substrate that stimulates TYR to further increase the production of DQ and increase the rate of melanogenesis.
Dopachrome decomposes to give mostly 5,6-dihydroxyindole (DHI) with the catalytic action of dopachrome tautomerase (DCT) also producing 5,6-dihydroxyindole-2-carboxylic acid (DHICA). These compounds are further oxidised by the TYR and tyrosinase-related protein-1 (TYRP1) enzymes to produce the brown –black eumelanin.
In a separate pathway, DQ can be conjugated with cysteine to give 5-S-cysteinyldopa and to a lesser extent 2-S-cysteinyldopa. These cysteinyldopas are then oxidised to give benzothiazine intermediates that are incorporated into the red – yellow pheomelanin polymer.
Little is known about the chemical regulatory or catalytic processes that are involved in pheomelanogenesis, but it is thought that the addition of cysteine to DQ is a rapid process that continues as long as cysteine is made available within the melanosome. The oxidation of
cysteinyldopas and incorporation into pheomelanin is proposed to continue as long as the cysteinyldopas are present. Depletion of melanosomal cysteine and cysteinyldopas enables the eumelanogenic pathway to commence, with eumelanin then deposited upon the preformed pheomelanin. Therefore, each melanocyte has the capacity to produce both types of pigment, which are known as mixed melanogenesis. However, the ratios of the two forms of melanin can vary widely between individuals as seen in the different shades of eye, hair and skin colour [2].
Genetic linkage analysis for eye colour Early linkage studies for eye and hair colours were
performed using blood groups as markers and provided evidence of association of a green or blue eye colour locus [eye colour 1 (EYCL1), also known as GEY; OMIM 227240;
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db ¼ OMIM] www.sciencedirect.com
to the Lutheran-Secretor systems on chromosome 19p13.1 – 19q13.11 [10]. Another major locus for brown or blue eye colour [eye color 3 (EYCL3) also known as BEY2; OMIM 227220] and brown hair [hair color 3 (HCL); OMIM 601800] was found on chromosome 15q11 –15q21 using linkage analysis with DNA markers within this region in
Table 1. Human pigmentation-related genesa
families segregating for BEY2 [11], with the OCA2 gene recognized as a candidate within this region. In these studies, a three-point scale of blue – grey, green– hazel and brown eye colour was used. The same three categories have now been used in the first complete genome scan in
an attempt to map genes responsible for eye colour using microsatellites at a 5 – 10 cM level [12]. These studies were performed in a sample of 502 twin families and obtained a peak LOD score of 19.2 in a region on 15q that contains OCA2 gene (Figure 1b), which had already been implicated in brown or blue eye colour [11]. This peak has a long tail towards the telomere, suggesting that other eye colour quantitative trait loci (QTL) might lie there [interestingly, both the Myosin Va (MYO5A) and RAB27A proteins that are involved in melanosome trafficking are located in this region [7]). Zhu and colleagues estimate that 74% of variance of eye colour might be due to this single QTL peak and conclude that most variation in eye colour is due to the
OCA2 locus (encoding the P melanosomal protein) but that there will be modifiers at several other loci [12].
OCA2 and candidate pigmentation gene polymorphism for eye colour The human P-gene transcript encoded by the OCA2 locus consists of 24 exons and is . 345 kb [13]. The gene encodes an 838 amino acid open reading frame producing a 110 kD protein that contains 12 transmembrane spanning regions; it has been classified as an integral melanosomal
membrane protein. In mouse, the P-protein is encoded by the pink-eyed (p) dilute mouse coat-colour locus, and mutations in the orthologous human OCA2 result in type II albinism [14]. At least 35 apparently non-pathogenic variant alleles of OCA2 have been identified: 24 of which
are exonic and six of these result in amino acid changes (for more information, see the Albinism database www.cbc. umn.edu/tad/). Some of these polymorphisms have markedly different frequencies in different populations indicating the potential to explain difference in pigmenta-
tion phenotypes between ethnic groups. Using a candidate www.sciencedirect.com gene analysis approach in a sample of 629 individuals the Rebbeck group recently found two of these OCA2 coding- region variants, R305W and R419Q were associated with brown and green– hazel eyes, respectively [15]. These same polymorphisms were tested in the twin collection
described by Zhu et al. and each was confirmed as being associated with green and brown but not with blue eyes [16]. Another locus that has been tested for association for human pigmentation phenotypes is the agouti signalling protein gene (ASIP) [15]. A g8818A-G single nucleotide polymorphism (SNP) in the 30 untranslanted region of this gene was genotyped in 746 participants, and the G nucleotide allele was found to be significantly associated
with brown eyes [17].
Genome wide SNP analysis for eye colour
A recent paper by Frudakis et al. has taken a different approach at dissecting the genetic basis of eye colour using SNPs [18]. They used a hypothesis-driven SNP screen, focusing on pigmentation candidate genes and a hypothesis-free approach analogous to admixture map-
ping to screen a genome-wide set of Ancestry Informative SNP Markers (AIMs) [19]. AIMs are genetic loci showing alleles with large frequency differences between populations and can be used to estimate bio-geographical ancestry and admixture of an individual from founder
populations or subgroups (Figure 2).
The candidate gene portion of their study confirmed
some associations and introduced others. More than 335 SNPs within 13 known pigmentation genes were screened in 851 individuals of European descent. Individual SNPs and haplotypes significantly associated with eye colour were identified within the OCA2, TYR, TYRP1, DCT,
MATP and MYO5A loci. Alleles for several additional genes – ASIP, MC1R, pro-opiomelanocortin (POMC) and Silver homologue (SILV) – were associated at the haplo-
type level but not at the individual SNP level. Of the 335 SNPs in known pigmentation genes, only 61 were associated with iris pigmentation at the SNP level; most of
linkage disequilibrium with OCA2, suggesting these two genes might act independently to affect eye colour. After OCA2, the TYRP1 associations were the next strongest, followed by those for MATP, which were significant using any colour grouping scheme; this was the first indication that common variants for these genes explain extant human iris in addition to skin colour variation. It is debatable whether the weaker associations found in the other pigment genes are due to low allelic penetrance or are due to the sequences being informative for certain elements of cryptic population substructure that correlate with iris colours.
The hypothesis-free AIM screening produced interesting results for other regions. Linkage disequilibrium can extend for megabases in recently admixed populations and this can be useful for mapping loci that underlie common human traits [19– 22]. Frudakis et al. used AIMs in an
unconventional manner – their goal was to draw a connection between trait value (iris colour) and elements of cryptic population structure that are present within the European population (Figure 2c). AIMs from CYP2C8 and CYP2C9 located in 10q23 and 10q24, respectively, were
found to be associated with iris colours. Although neither of these genes is a pigment gene, both are located between two Hermansky-Pudlak syndrome (HPS) pigment genes [8,23] that were not tested in the candidate gene portion of the study, HPS1 (10q23.1–10q23.3) and HPS6 (10q24.32).
Interestingly, the linkage screen by Zhu et al. also showedmodestly elevated LOD scores for this region [12]. The use of AIMs in this way suggests that crude and cryptic population structure might be useful in developing sequence-based classification tools for complex anthropo-
metric and other human traits, such as iris colour. Iris patterns and change of eye colour
The human iris has many other characteristic patterns (Figure 1a) that are not measured through an assessment of eye colour and these will also be under strong genetic influence [24] but remain to be fully investigated. Forexample, although eye colour is assumed to be fixed for
adult life there can be changes as an individual ages or changes in disease states. Notably, there is a genetic component to the drug induced changes that can occur in iris pigmentation for the treatment of glaucoma [25].
References
1 Davenport, G.C. and Davenport, C.B. (1907) Heredity of eye-color in man. Science 26, 590 – 592
2 Sturm, R.A. et al. (1998) Human pigmentation genetics: the difference is only skin deep. BioEssays 20, 712 – 721
3 Badano, J.L. and Katsanis, N. (2002) Beyond Mendel: an evolving view of human genetic disease transmission. Nat. Rev. Genet. 3, 779 – 789
4 Boissy, R.E. (1998) Extracutaneous Melanocytes. In The Pigmentary System – Physiology and Pathophysiology (Nordlund, J.J. et al. eds),
pp. 59– 72, Oxford University Press
5 Imesch, P.D. et al. (1997) The color of the human eye: a review of morphologic correlates and of some conditions that affect iridial pigmentation. Surv. Ophthalmol 41 (Suppl. 2), S117 – S123
6 Prota, G. et al. (1998) Characterization of melanins in human irides and cultured uveal melanocytes from eyes of different colors. Exp. Eye Res. 67, 293– 299
7 Sturm, R.A. et al. (2001) Human pigmentation genes: identification, structure and consequences of polymorphic variation. Gene 277, 49 – 62
8 Bennett, D.C. and Lamoreux, M.L. (2003) The color loci of mice – a genetic century. Pigment Cell Res. 16, 333– 344
9 Rees, J.L. (2003) Genetics of hair and skin color. Annu. Rev. Genet. 37, 67 – 90
10 Eiberg, H. and Mohr, J. (1987) Major genes of eye color and hair color linked to LU and SE. Clin. Genet. 31, 186 – 191
11 Eiberg, H. and Mohr, J. (1996) Assignment of genes coding for brown eye colour (BEY2) and brown hair colour (HCL3) on chromosome 15q. Eur. J. Hum. Genet. 4, 237– 241
12 Zhu, G. et al. (2004) A genome scan for eye colour in 502 twin familes: most variation is due to a QTL on chromosome 15q. Twin Res. 7, 197 – 210
13 Lee, S.T. et al. (1995) Organization and sequence of the human P gene and identification of a new family of transport proteins. Genomics 26, 354 – 363
14 Brilliant, M.H. (2001) The mouse p (pink-eyed dilution) and human P genes, oculocutaneous albinism type 2 (OCA2), and melanosomal pH. Pigment Cell Res. 14, 86 – 93
15 Rebbeck, T.R. et al. (2002) P gene as an inherited biomarker of human eye color. Cancer Epidemiol. Biomarkers Prev. 11, 782 – 784
16 Duffy, D.L. et al. (2004) Interactive effects of MC1R and OCA2 on melanoma risk phenotypes. Hum. Mol. Genet. 13, 447– 461
17 Kanetsky, P.A. et al. (2002) A polymorphism in the agouti signaling protein gene is associated with human pigmentation. Am. J. Hum. Genet. 70, 770 – 775
18 Frudakis, T. et al. (2003) Sequences associated with human iris pigmentation. Genetics 165, 2071 – 2083
19 Shriver, M.D. et al. (2003) Skin pigmentation, biogeographical ancestry and admixture mapping. Hum. Genet. 112, 387 – 399
20 Chakraborty, R. and Weiss, K.M. (1988) Admixture as a tool for finding linked genes and detecting that difference from allelic association between loci. Proc. Natl. Acad. Sci. U. S. A. 85, 9119 – 9123
21 McKeigue, P.M. et al. (2000) Estimation of admixture and detection of linkage in admixed populations by a Bayesian approach: application to African-American populations. Ann. Hum. Genet. 64, 171– 186
22 McKeigue, P.M. (2000) Multipoint admixture mapping. Genet. Epidemiol. 19, 464 – 467
23 Swank, R.T. et al. (2000) Abnormal vesicular trafficking in mouse models of Hermansky-Pudlak syndrome. Pigment Cell Res. 13 (Suppl. 8), 59 – 67
24 Larsson, M. et al. (2003) Importance of genetic effects for character- istics of the human iris. Twin Res. 6, 192 – 200
25 Stjernschantz, J.W. et al. (2002) Mechanism and clinical significance of prostaglandin-induced iris pigmentation. Surv. Ophthalmol. 47 (Suppl. 1), S162 – S175
26 Albert, D.M. et al. (2003) Iris melanocyte numbers in Asian, African- American, and Caucasian irides. Trans. Am. Ophthalmol. Soc. 101, 217– 222
27 Ito, S. (2003) The IFPCS presidential lecture: a chemist’s view of melanogenesis. Pigment Cell Res. 16, 230– 236
Unexpected conserved non-coding DNA blocks in
mammals
Daniel J. Gaffney and Peter D. Keightley
Ashworth Laboratories, School of Biological Sciences, University of Edinburgh, West Mains Road, Edinburgh EH9 3JT,
United Kingdom
The significance of non-coding DNA is a longstanding riddle in the study of molecular evolution. Using a comparative genomics approach, Dermitzakis and colleagues have recently shown that at least some non coding sequence, frequently ignored as meaningless noise, might bear the signature of natural selection. If functional, it could mark a turning point in the way we
think about the evolution of the genome. Few genomic features are more puzzling than the vast
amounts of apparently functionless non-coding DNA thatmake up the greater proportion of human, mouse and many other eukaryotic genomes. However, although the view of non-coding sequence as genomic debris has been widespread, recent results by Dermitzakis and colleagues [1 – 3] offers a fascinating hint that a significant proportion can retain a function that, for the moment, remains amystery.
For much of the past 50 years, the functional genome has been viewed as one that codes for protein and, until recently, most evolutionary studies of DNA sequences have focused almost entirely on this translated fraction,Corresponding author: Daniel J. Gaffney (Daniel.Gaffney@ed.ac.uk).
www.sciencedirect.com
which we now think accounts for as little as 1 – 2% of both human and mouse DNA [4,5]. Many theories of the origin of non-coding DNA are founded on the perception that the bulk of such sequence is meaningless [6] and invoke random processes of accumulation of this ‘junk’, for
example, the action of ‘selfish’ self-replicating elements [7]. Whole genome sequencing has, to some extent, borne these views out. Approximately 40% of mouse and human genomes are composed of the repetitive signatures that characterize past insertion of such retroelements [4,5]. Indeed, , 20% of the entire mouse genome appears to have originated via the activity of a single class of element, the long interspered elements (LINEs) [5]. However, excluding repetitive DNA sequence still leaves enormous quantities of non-coding sequence that we know little about. One of the most intriguing suggestions arising from the compari- son of human and mouse genomes is that protein-coding sequences only account for approximately a fifth of the
total amount of each species’ genome that is subject to purifying selection [5]. The implication is that relatively large amounts of non-coding DNA are functional and it is clear, therefore, that the elucidation of potential functions (or otherwise) of non-coding DNA is a primary challenge in
evolutionary genomics.
777777777777777777777
777777777777777777777
ancestry
Richard A. Sturm1 and Tony N. Frudakis2
Institute for Molecular Bioscience, University of Queensland, Brisbane, Qld 4072, Australia
DNAPrint genomics, Incorporated, 900 Cocoanut Ave, Sarasota, FL 34236, USA
Several recent papers have tried to address the genetic determination of eye colour via microsatellite linkage, testing of pigmentation candidate gene polymorphisms
and the genome wide analysis of SNP markers that are informative for ancestry. These studies show that the OCA2 gene on chromosome 15 is the major determinant of brown and/or blue eye colour but also indicatethat other loci will be involved in the broad range ofhues seen in this trait in Europeans.
One of the first investigations into the concept of mendelian inheritance in humans was the consideration of eye colour. Iris colour exists on a continuum from the lightest shades of blue to the darkest of brown or black, although genetic studies have usually categorised: blue,
grey, green, yellow, hazel, light brown and dark brown (Figure 1a) in addition to the colour deficiencies apparent in those with oculocutaneous albinism. In 1907, the Davenports [1] outlined what is still commonly taught in schools today as a beginners guide to genetics that brown eye colour is always dominant to blue, with two blue-eyed parents always producing a blue-eyed child, never one with brown eyes. Unfortunately, as with many physica traits, this simplistic model does not convey the complexities of real life and the fact is that eye colour is inherited as a polygenic not as a monogenic trait. Although not common, two blue-eyed parents can produce children with brown eyes. The apparently non-mendelian examples of
iris colour transmission from parents to offspring, combined with the quantitative nature of iris pigmentation indicate that the inheritance of this apparently simple
trait as a dichotomous value must be reconsidered. The use of eye colour as a paradigm for ‘complete’ recessive and dominant gene action should be avoided in the teaching of
genetics to the layperson, which is often their first encounter with the science of human heredity. The phenotypes of eye, hair and skin colour [2] in addition to stature and facial features will always be observed to run in families but families need to know that these are
complex traits (i.e. conditioned by several genes) [3].
Physical basis of eye colour: melanocytes,
melanogenesis and ancestry The physical basis of eye colour is determined by the distribution and content of the melanocyte cells in the uveal tract of the eye (Box 1). The iris consists of several Corresponding author: Richard A. Sturm (R.Sturm@imb.uq.edu.au).
www.sciencedirect.com
layers: the anterior layer and its underlying stroma are the most important for the appearance of eye colour [4]. In the brown iris there is an abundance of melanocytes and melanosomes in the anterior layer and stroma, whereas in the blue iris these layers contain little melanin. As
light traverses these relatively melanin-free layers, the minute protein particles of the iris scatter the short blue
-A schematic representation of the eye ball from the front view shows the anatomical division of the sclera, the white connective tissue, the iris and the coloured disk surrounding the central black pupil (Figure I). In cross section view the cornea is seen as a transparent tissue above the
iris enabling light to enter through the pupil, which is then focused by the lens onto the retina. The iris comprises two tissue layers, the innermost consists of cuboidal, pigmented cells that are tightly fused and is known as the iris pigment epithelium (IPE), which is formed from the optic cup during development. The outermost layer is referred to as the anterior iridial stroma and is composed mainly of loosely arranged connective tissue, fibroblasts and melanocytes and are of the same embryological origin as dermal melanocytes, which arise and migrate from the neural crest. Apart from albino patients, who lack melanin pigment and have eyes that might appear pink as a result of the reflection of light from blood vessels, the IPE does not exert a major influence on the perceived eye colour of normal individuals because the melanin in this layer is distributed similarly in irides of different colour. Notably, it is the density and cellular composition of the iris stroma that must be considered as major factors in the colouration of the eye [5].
The melanocyte cells are aggregated in the anterior border layer of the iridial stroma, parallel to the surface of the eye, and store melanin pigment in a specialized organelle within their cytoplasm termed the melanosome. White light entering the iris can absorb or reflect a
spectrum of wavelengths giving rise to the three common iris colours, blue, green–hazel and brown, but it should be recognized that these broad classifications are simplistic and that there is actually a continuum in the range of eye colours seen in Europeans. The middle of the panel illustrates the intracellular distribution and content of the melanosome particles within the iridial melanocytes with the varied melanin pigment quantity, packaging and qualities giving the range of eye shades [6]. Although blue eyes have similar numbers of melanocyte cells they contain minimal pigment and few melanosomes; green hazel irides are the product of moderate pigment levels, melanin intensity and melanosome number and with brown irides are the result
of high melanin levels and melanosomal particle numbers. Each of these eye colours can occur with or without a darker pigmented iris peripupillary ring, represented to the right of the figure. Insufficient studies have been performed into the nature of the peripupillary ring; however, the possibility that the number of melanocytes, their melanin granule size, distribution or content can differ between ethnic groups has been recognized [26], and further ultrastructural investigations are needed to clarify this issue.
wavelengths to the surface, thus blue is a consequence of structure not of major differences in chemical composition. The number of melanocytes does not appear to differ between eye colours [5], but the melanin pigment quantity, packaging and quality does vary, giving a range of eye shades [6]. The common occurrence of lighter iris colours is found almost exclusively in Europeans (i.e. recent monophyletic, non-East Asian, non-Native American and non African lineages) and individuals of European admixture.The study of biogeographical ancestry admixture is becoming more popular and soon it might be possible to date the genesis of lighter irides; that is to distinguish whether lighter iris colours are exclusive to the continental European populations, as opposed to unadmixed Middle Eastern or Central and/or Southern Asian popu-
lations with whom they share some common ancestry.
There are two forms of melanin pigment particles produced during melanogenesis and both occur in the iris of the eye, the cutaneous and follicular (skin and hair) melanocyte cells (Box 2). However, unlike the skin and hair in which melanin is produced continuously and secreted, in the eye the melanosomes containing the pigment are retained and accumulate in the cytoplasm of
the melanocytes within the iris stroma. Eumelanin is a brown – black form of pigment that is responsible for dark colouration and is packaged in ovoid eumelanosomes, which are striated particles, whereas pheomelanin is a red –yellow pigment produced in granular immature
pheomelanosomes [7].
The study of mouse-coat colours and the comparative genomic analysis with other mammals, including humans, has provided enormous insight into the genetic basis of pigmentation [8,9]. Several loci are known to have major effects on pigmentation (Table 1) including the enzymes
that are involved in the catalytic formation of melanin [including tyrosinase (TYR), tyrosinase related proteins TYRP1 and dopachrome tautomerase (DCT)], the melano-somal proteins [P and membrane-associated transporter protein (MATP) encoded by the OCA2 and MATP genes, respectively] and the melanocortin-1 receptor (MC1R), which is involved in pheomelanin– eumelanin pigment switching of the melanocyte [7,9].
Box 2. Melanin pigment formation
Melanin is an inert light-absorbing biopolymer of no fixed size and of uncertain unit structure that is extraordinarily resistant to chemical degradation. Melanogenesis is based on the chemical reactions that take place within the melanosome beginning with tyrosine, dopa and cysteine that result in the formation of the eumelanin and pheomelanin pigments, through a bifurcated biosynthetic pathway [27]. When tyrosine is oxidised by the tyrosinase (TYR) enzyme, dopaquinone (DQ) is produced as an intermediate (Figure I). In the absence of cysteine, DQ undergoes intramolecular addition producing cyclodopa, with a redox exchange between cyclodopa and DQ giving rise to dopa and dopachrome. Dopa is a substrate that stimulates TYR to further increase the production of DQ and increase the rate of melanogenesis.
Dopachrome decomposes to give mostly 5,6-dihydroxyindole (DHI) with the catalytic action of dopachrome tautomerase (DCT) also producing 5,6-dihydroxyindole-2-carboxylic acid (DHICA). These compounds are further oxidised by the TYR and tyrosinase-related protein-1 (TYRP1) enzymes to produce the brown –black eumelanin.
In a separate pathway, DQ can be conjugated with cysteine to give 5-S-cysteinyldopa and to a lesser extent 2-S-cysteinyldopa. These cysteinyldopas are then oxidised to give benzothiazine intermediates that are incorporated into the red – yellow pheomelanin polymer.
Little is known about the chemical regulatory or catalytic processes that are involved in pheomelanogenesis, but it is thought that the addition of cysteine to DQ is a rapid process that continues as long as cysteine is made available within the melanosome. The oxidation of
cysteinyldopas and incorporation into pheomelanin is proposed to continue as long as the cysteinyldopas are present. Depletion of melanosomal cysteine and cysteinyldopas enables the eumelanogenic pathway to commence, with eumelanin then deposited upon the preformed pheomelanin. Therefore, each melanocyte has the capacity to produce both types of pigment, which are known as mixed melanogenesis. However, the ratios of the two forms of melanin can vary widely between individuals as seen in the different shades of eye, hair and skin colour [2].
Genetic linkage analysis for eye colour Early linkage studies for eye and hair colours were
performed using blood groups as markers and provided evidence of association of a green or blue eye colour locus [eye colour 1 (EYCL1), also known as GEY; OMIM 227240;
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db ¼ OMIM] www.sciencedirect.com
to the Lutheran-Secretor systems on chromosome 19p13.1 – 19q13.11 [10]. Another major locus for brown or blue eye colour [eye color 3 (EYCL3) also known as BEY2; OMIM 227220] and brown hair [hair color 3 (HCL); OMIM 601800] was found on chromosome 15q11 –15q21 using linkage analysis with DNA markers within this region in
Table 1. Human pigmentation-related genesa
families segregating for BEY2 [11], with the OCA2 gene recognized as a candidate within this region. In these studies, a three-point scale of blue – grey, green– hazel and brown eye colour was used. The same three categories have now been used in the first complete genome scan in
an attempt to map genes responsible for eye colour using microsatellites at a 5 – 10 cM level [12]. These studies were performed in a sample of 502 twin families and obtained a peak LOD score of 19.2 in a region on 15q that contains OCA2 gene (Figure 1b), which had already been implicated in brown or blue eye colour [11]. This peak has a long tail towards the telomere, suggesting that other eye colour quantitative trait loci (QTL) might lie there [interestingly, both the Myosin Va (MYO5A) and RAB27A proteins that are involved in melanosome trafficking are located in this region [7]). Zhu and colleagues estimate that 74% of variance of eye colour might be due to this single QTL peak and conclude that most variation in eye colour is due to the
OCA2 locus (encoding the P melanosomal protein) but that there will be modifiers at several other loci [12].
OCA2 and candidate pigmentation gene polymorphism for eye colour The human P-gene transcript encoded by the OCA2 locus consists of 24 exons and is . 345 kb [13]. The gene encodes an 838 amino acid open reading frame producing a 110 kD protein that contains 12 transmembrane spanning regions; it has been classified as an integral melanosomal
membrane protein. In mouse, the P-protein is encoded by the pink-eyed (p) dilute mouse coat-colour locus, and mutations in the orthologous human OCA2 result in type II albinism [14]. At least 35 apparently non-pathogenic variant alleles of OCA2 have been identified: 24 of which
are exonic and six of these result in amino acid changes (for more information, see the Albinism database www.cbc. umn.edu/tad/). Some of these polymorphisms have markedly different frequencies in different populations indicating the potential to explain difference in pigmenta-
tion phenotypes between ethnic groups. Using a candidate www.sciencedirect.com gene analysis approach in a sample of 629 individuals the Rebbeck group recently found two of these OCA2 coding- region variants, R305W and R419Q were associated with brown and green– hazel eyes, respectively [15]. These same polymorphisms were tested in the twin collection
described by Zhu et al. and each was confirmed as being associated with green and brown but not with blue eyes [16]. Another locus that has been tested for association for human pigmentation phenotypes is the agouti signalling protein gene (ASIP) [15]. A g8818A-G single nucleotide polymorphism (SNP) in the 30 untranslanted region of this gene was genotyped in 746 participants, and the G nucleotide allele was found to be significantly associated
with brown eyes [17].
Genome wide SNP analysis for eye colour
A recent paper by Frudakis et al. has taken a different approach at dissecting the genetic basis of eye colour using SNPs [18]. They used a hypothesis-driven SNP screen, focusing on pigmentation candidate genes and a hypothesis-free approach analogous to admixture map-
ping to screen a genome-wide set of Ancestry Informative SNP Markers (AIMs) [19]. AIMs are genetic loci showing alleles with large frequency differences between populations and can be used to estimate bio-geographical ancestry and admixture of an individual from founder
populations or subgroups (Figure 2).
The candidate gene portion of their study confirmed
some associations and introduced others. More than 335 SNPs within 13 known pigmentation genes were screened in 851 individuals of European descent. Individual SNPs and haplotypes significantly associated with eye colour were identified within the OCA2, TYR, TYRP1, DCT,
MATP and MYO5A loci. Alleles for several additional genes – ASIP, MC1R, pro-opiomelanocortin (POMC) and Silver homologue (SILV) – were associated at the haplo-
type level but not at the individual SNP level. Of the 335 SNPs in known pigmentation genes, only 61 were associated with iris pigmentation at the SNP level; most of
linkage disequilibrium with OCA2, suggesting these two genes might act independently to affect eye colour. After OCA2, the TYRP1 associations were the next strongest, followed by those for MATP, which were significant using any colour grouping scheme; this was the first indication that common variants for these genes explain extant human iris in addition to skin colour variation. It is debatable whether the weaker associations found in the other pigment genes are due to low allelic penetrance or are due to the sequences being informative for certain elements of cryptic population substructure that correlate with iris colours.
The hypothesis-free AIM screening produced interesting results for other regions. Linkage disequilibrium can extend for megabases in recently admixed populations and this can be useful for mapping loci that underlie common human traits [19– 22]. Frudakis et al. used AIMs in an
unconventional manner – their goal was to draw a connection between trait value (iris colour) and elements of cryptic population structure that are present within the European population (Figure 2c). AIMs from CYP2C8 and CYP2C9 located in 10q23 and 10q24, respectively, were
found to be associated with iris colours. Although neither of these genes is a pigment gene, both are located between two Hermansky-Pudlak syndrome (HPS) pigment genes [8,23] that were not tested in the candidate gene portion of the study, HPS1 (10q23.1–10q23.3) and HPS6 (10q24.32).
Interestingly, the linkage screen by Zhu et al. also showedmodestly elevated LOD scores for this region [12]. The use of AIMs in this way suggests that crude and cryptic population structure might be useful in developing sequence-based classification tools for complex anthropo-
metric and other human traits, such as iris colour. Iris patterns and change of eye colour
The human iris has many other characteristic patterns (Figure 1a) that are not measured through an assessment of eye colour and these will also be under strong genetic influence [24] but remain to be fully investigated. Forexample, although eye colour is assumed to be fixed for
adult life there can be changes as an individual ages or changes in disease states. Notably, there is a genetic component to the drug induced changes that can occur in iris pigmentation for the treatment of glaucoma [25].
References
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2 Sturm, R.A. et al. (1998) Human pigmentation genetics: the difference is only skin deep. BioEssays 20, 712 – 721
3 Badano, J.L. and Katsanis, N. (2002) Beyond Mendel: an evolving view of human genetic disease transmission. Nat. Rev. Genet. 3, 779 – 789
4 Boissy, R.E. (1998) Extracutaneous Melanocytes. In The Pigmentary System – Physiology and Pathophysiology (Nordlund, J.J. et al. eds),
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7 Sturm, R.A. et al. (2001) Human pigmentation genes: identification, structure and consequences of polymorphic variation. Gene 277, 49 – 62
8 Bennett, D.C. and Lamoreux, M.L. (2003) The color loci of mice – a genetic century. Pigment Cell Res. 16, 333– 344
9 Rees, J.L. (2003) Genetics of hair and skin color. Annu. Rev. Genet. 37, 67 – 90
10 Eiberg, H. and Mohr, J. (1987) Major genes of eye color and hair color linked to LU and SE. Clin. Genet. 31, 186 – 191
11 Eiberg, H. and Mohr, J. (1996) Assignment of genes coding for brown eye colour (BEY2) and brown hair colour (HCL3) on chromosome 15q. Eur. J. Hum. Genet. 4, 237– 241
12 Zhu, G. et al. (2004) A genome scan for eye colour in 502 twin familes: most variation is due to a QTL on chromosome 15q. Twin Res. 7, 197 – 210
13 Lee, S.T. et al. (1995) Organization and sequence of the human P gene and identification of a new family of transport proteins. Genomics 26, 354 – 363
14 Brilliant, M.H. (2001) The mouse p (pink-eyed dilution) and human P genes, oculocutaneous albinism type 2 (OCA2), and melanosomal pH. Pigment Cell Res. 14, 86 – 93
15 Rebbeck, T.R. et al. (2002) P gene as an inherited biomarker of human eye color. Cancer Epidemiol. Biomarkers Prev. 11, 782 – 784
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Unexpected conserved non-coding DNA blocks in
mammals
Daniel J. Gaffney and Peter D. Keightley
Ashworth Laboratories, School of Biological Sciences, University of Edinburgh, West Mains Road, Edinburgh EH9 3JT,
United Kingdom
The significance of non-coding DNA is a longstanding riddle in the study of molecular evolution. Using a comparative genomics approach, Dermitzakis and colleagues have recently shown that at least some non coding sequence, frequently ignored as meaningless noise, might bear the signature of natural selection. If functional, it could mark a turning point in the way we
think about the evolution of the genome. Few genomic features are more puzzling than the vast
amounts of apparently functionless non-coding DNA thatmake up the greater proportion of human, mouse and many other eukaryotic genomes. However, although the view of non-coding sequence as genomic debris has been widespread, recent results by Dermitzakis and colleagues [1 – 3] offers a fascinating hint that a significant proportion can retain a function that, for the moment, remains amystery.
For much of the past 50 years, the functional genome has been viewed as one that codes for protein and, until recently, most evolutionary studies of DNA sequences have focused almost entirely on this translated fraction,Corresponding author: Daniel J. Gaffney (Daniel.Gaffney@ed.ac.uk).
www.sciencedirect.com
which we now think accounts for as little as 1 – 2% of both human and mouse DNA [4,5]. Many theories of the origin of non-coding DNA are founded on the perception that the bulk of such sequence is meaningless [6] and invoke random processes of accumulation of this ‘junk’, for
example, the action of ‘selfish’ self-replicating elements [7]. Whole genome sequencing has, to some extent, borne these views out. Approximately 40% of mouse and human genomes are composed of the repetitive signatures that characterize past insertion of such retroelements [4,5]. Indeed, , 20% of the entire mouse genome appears to have originated via the activity of a single class of element, the long interspered elements (LINEs) [5]. However, excluding repetitive DNA sequence still leaves enormous quantities of non-coding sequence that we know little about. One of the most intriguing suggestions arising from the compari- son of human and mouse genomes is that protein-coding sequences only account for approximately a fifth of the
total amount of each species’ genome that is subject to purifying selection [5]. The implication is that relatively large amounts of non-coding DNA are functional and it is clear, therefore, that the elucidation of potential functions (or otherwise) of non-coding DNA is a primary challenge in
evolutionary genomics.
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