Membrane Associated Transporter Protein Gene (SLC45A2) and the Genetic Basis of Normal

Human Pigmentation Variation

Justin GrafBachelor of Applied Science (Honours)

Institute of Health and Biomedical InnovationCRC for Diagnostics, School of Life ScienceQueensland University of Technology

Brisbane, Qld, Australia

A thesis submitted for the degree of Doctor of Philosophy of theQueensland University of Technology, 2008

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Abstract

This work is concerned with the genetic basis of normal human pigmentation variation.

Specifically, the role of polymorphisms within the solute carrier family 45 member 2

(SLC45A2 or membrane associated transporter protein; MATP) gene were investigated

with respect to variation in hair, skin and eye colour ― both between and within

populations. SLC45A2 is an important regulator of melanin production and mutations in

the gene underly the most recently identified form of oculocutaneous albinism. There is

evidence to suggest that non-synonymous polymorphisms in SLC45A2 are associated

with normal pigmentation variation between populations. Therefore, the underlying

hypothesis of this thesis is that polymorphisms in SLC45A2 will alter the function or

regulation of the protein, thereby altering the important role it plays in melanogenesis

and providing a mechanism for normal pigmentation variation.

In order to investigate the role that SLC45A2 polymorphisms play in human

pigmentation variation, a DNA database was established which collected pigmentation

phenotypic information and blood samples of more than 700 individuals. This database

was used as the foundation for two association studies outlined in this thesis, the first of

which involved genotyping two previously-described non-synonymous polymorphisms,

p.Glu272Lys and p.Phe374Leu, in four different population groups. For both

polymorphisms, allele frequencies were significantly different between population

groups and the 272Lys and 374Leu alleles were strongly associated with black hair,

brown eyes and olive skin colour in Caucasians. This was the first report to show that

SLC45A2 polymorphisms were associated with normal human intra-population

pigmentation variation.

The second association study involved genotyping several SLC45A2 promoter

polymorphisms to determine if they also played a role in pigmentation variation. Firstly,

the transcription start site (TSS), and hence putative proximal promoter region, was

identified using 5' RNA ligase mediated rapid amplification of cDNA ends (RLM-

RACE). Two alternate TSSs were identified and the putative promoter region was

screened for novel polymorphisms using denaturing high performance liquid

chromatography (dHPLC). A novel duplication (c.–1176_–1174dupAAT) was

identified along with other previously described single nucleotide polymorphisms (c.–

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1721C>G and c.–1169G>A). Strong linkage disequilibrium ensured that all three

polymorphisms were associated with skin colour such that the –1721G, +dup and –

1169A alleles were associated with olive skin in Caucasians. No linkage disequilibrium

was observed between the promoter and coding region polymorphisms, suggesting

independent effects. The association analyses were complemented with functional data,

showing that the –1721G, +dup and –1169A alleles significantly decreased SLC45A2

transcriptional activity. Based on in silico bioinformatic analysis that showed these

alleles remove a microphthalmia-associated transcription factor (MITF) binding site,

and that MITF is a known regulator of SLC45A2(Baxter and Pavan, 2002; Du and

Fisher, 2002), it was postulated that SLC45A2 promoter polymorphisms could

contribute to the regulation of pigmentation by altering MITF binding affinity.

Further characterisation of the SLC45A2 promoter was carried out using luciferase

reporter assays to determine the transcriptional activity of different regions of the

promoter. Five constructs were designed of increasing length and their promoter activity

evaluated. Constitutive promoter activity was observed within the first ~200 bp and

promoter activity increased as the construct size increased. The functional impact of the

–1721G, +dup and –1169A alleles, which removed a MITF consensus binding site,

were assessed using electrophoretic mobility shift assays (EMSA) and expression

analysis of genotyped melanoblast and melanocyte cell lines. EMSA results confirmed

that the promoter polymorphisms affected DNA-protein binding. Interestingly,

however, the protein/s involved were not MITF, or at least MITF was not the protein

directly binding to the DNA. In an effort to more thoroughly characterise the functional

consequences of SLC45A2 promoter polymorphisms, the mRNA expression levels of

SLC45A2 and MITF were determined in melanocyte/melanoblast cell lines. Based on

SLC45A2’s role in processing and trafficking TYRP1 from the trans-Golgi network to

stage 2 melanosmes, the mRNA expression of TYRP1 was also investigated. Expression

results suggested a coordinated expression of pigmentation genes.

This thesis has substantially contributed to the field of pigmentation by showing that

SLC45A2 polymorphisms not only show allele frequency differences between

population groups, but also contribute to normal pigmentation variation within a

Caucasian population. In addition, promoter polymorphisms have been shown to have

functional consequences for SLC45A2 transcription and the expression of other

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pigmentation genes. Combined, the data presented in this work supports the notion that

SLC45A2 is an important contributor to normal pigmentation variation and should be

the target of further research to elucidate its role in determining pigmentation

phenotypes. Understanding SLC45A2’s function may lead to the development of

therapeutic interventions for oculocutaneous albinism and other disorders of

pigmentation. It may also help in our understanding of skin cancer susceptibility and

evolutionary adaptation to different UV environments, and contribute to the forensic

application of pigmentation phenotype prediction.

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List of keywords

Membrane associated transporter protein (MATP), Solute carrier family 45 member 2

(SLC45A2), antigen in melanoma (AIM1), underwhite, single nucleotide polymorphism

(SNP), normal human pigmentation variation, promoter, hair, skin and eye colour,

genotyping, association study.

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List of publications and conference abstracts

The following is a list of publications, manuscripts prepared for submission and

conference abstracts that have been derived from the work performed for this thesis:

Journal publications

Graf J, Hodgson R, van Daal A. 2005. Single nucleotide polymorphisms in the MATP

gene are associated with normal human pigmentation variation. Human Mutation

Mar;25(3):278-84.

Graf J, Voisey J, Hughes I, van Daal A. 2007. Promoter polymorphisms in theMATP

(SLC45A2) gene are associated with normal human skin color variation. Human

Mutation Jul;28(7):710-7.

Graf J, Voisey J, Hughes I. 2008. Functional characterisation of polymorphic variation

in the MATP (SLC45A2) gene promoter. Pigment Cell and Melanoma Research.

(Prepared for submission)

Conference abstracts

Graf J, Hodgson R, van Daal A. 2005. MATP and the genetic basis of human

pigmentation. ASMR QLD Postgraduate Medical Research Student Conference,

Brisbane, Queensland.

Graf J, Hodgson R, van Daal A. 2005. MATP and the genetic basis of normal human

pigmentation variation. 19th International Pigment Cell Conference, Reston, Virginia,

USA.

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Table of contentsAbstract iiiList of keywords viList of publications and conference abstracts viiJournal publications viiConference abstracts viiTable of contents viiiList of abbreviations xiDeclaration xvDedication and acknowledgements xvi

Chapter 1 – Introduction 11.1 A description of the research problem investigated 11.2 Overall objectives 31.3 Specific aims 31.4 An account of scientific progress linking the research papers 41.5 References 8

Chapter 2 – Literature review 112.1 Fundamentals of human pigmentation 112.1.1 Melanin biosynthesis 112.1.2 Melanosome biogenesis 12

2.2 Sites of pigmentation 132.2.1 Skin pigmentation 132.2.2 Hair pigmentation 142.2.3 Eye pigmentation 16

2.3 Evidence for a genetic contribution to human pigmentation variation 19

2.4 The evolution of normal pigmentation variation 202.4.1 Geographical variation of human skin colour 202.4.2 Evidence for positive selection of human pigmentation variation 23

2.5 Genetics of human pigmentation dysfunction 282.5.1 Oculocutaneous albinism (OCA) 292.5.1.1 OCA Type I and Tyrosinase (TYR) 292.5.1.2 OCA Type II and OCA2 (P) 312.5.1.3 OCA Type III and Tyrosinase-related protein I (TYRP1) 332.5.1.4 OCA Type IV and solute carrier family 45 member 2 (SLC45A2) 36

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2.6 Genetics of normal pigmentation variation 402.6.1 Albinism genes involved in normal pigmentation variation 412.6.1.1 Tyrosinase (TYR) 412.6.1.2 OCA2 (P) 422.6.1.3 Tyrosinase-related protein 1 (TYRP1) 452.6.1.4 Solute carrier family 45 member 2 (SLC45A2) 462.6.2 Other genes involved in normal pigmentation variation 482.6.2.1 Melanocortin 1 receptor (MC1R) 482.6.2.2 Agouti signalling protein (ASIP) 532.6.2.3 SLC24A5 (Golden) 55

2.7 Genetic inference of human pigmentation 57

2.8 Transcriptional regulation of human pigmentation 592.8.1 Microphthalmia-associated transcription factor (MITF) 592.8.2 Transcriptional targets of MITF 59

2.9 Summary and relevance to experimental program 63

2.10 References 65

Chapter 3 87Single nucleotide polymorphisms in the MATP gene are associated with normal human pigmentation variationStatement of joint authorship 88

Chapter 4 97Promoter polymorphisms in the MATP (SLC45A2) gene are associated with normal human skin color variationStatement of joint authorship 98

Chapter 5 109Functional characterisation of polymorphic variation in the SLC45A2 (MATP) gene promoterStatement of joint authorship 110

Summary 112Introduction 113Results 115Discussion 117Methods 122References 127Tables 130Figure legends 131Figures 133

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Chapter 6 – General discussion 1376.1 Introduction 1376.2 Experimental considerations and limitations 1386.3 Principal outcomes, significance and applications of this research 1416.4 Future research 1486.5 Final conclusions 151

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List of abbreviations

ABI Applied Biosystems

ACTH Adrenocorticotropic hormone

AGRF Australian Genome Research Facility

Aiapy Intracisternal A-particle yellow

AIM1 Antigen in human melanoma

AIMs Ancestry informative markers

Aiy Intermediate yellow

ANOVA Analysis of variance

ASIP Agouti-signalling protein

AS-PCR Allele-specific polymerase chain reaction

Asy Sienna yellow

Avy Viable yellow

Ay Lethal yellow

α-MSH α-melanocyte stimulating hormone

BCA Bicinchoninic acid

b-HLH-Zip Basic helix-loop-helix leucine zipper

BOCA Brown oculocutaneous albinism

BSA Bovine serum albumin

C Iris colour score

cAMP Cyclic adenosine monophosphate

cDNA Complementary DNA

CEPH Centre d'Etude du Polymorphisme Humain

CHB Han Chinese in Beijing

CHS Chediak-Higashi syndrome

CIP Calf intestinal phosphatase

CLR Composite likelihood ratio

DCT Dopachrome tautomerase

DHI 5, 6-dihydroxyindole

DHICA 5, 6-dihydroxyindole-2-carboxylic acid

dHPLC Denaturing high performance liquid chromatography

dNTP Deoxynucleotide triphosphate

DOPA 3, 4 dihydroxyphenylanine

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DTT Dithiothreitol

EDTA Ethylenediaminetetraacetic acid

EHH Extended haplotype heterozygosity

EMSA Electrophoretic mobility shift assay

ER Endoplasmic reticulum

FBS Fetal bovine serum

Fst Fixation index or F statistic

GPCR G-protein coupled receptor

GS Griscelli syndrome

HAND2 Heart and neural crest derivatives expressed 2

Hap Haplotype

Hox Homeobox

HPS Hermansky-Pudlak syndrome

hr Hours

HWE Hardy-Weinberg equilibrium

IPCC International Pigment Cell Conference

JPT Japanese in Tokyo

kb Kilo-bases

kDa Kilo-Daltons

LD Linkage disequilibrium

LINE Long interspersed nuclear element

MATP Membrane associated transporter protein

MC1R Melanocortin 1 receptor

min Minutes

MITF Microphthalmia-associated transcription factor

MSE Melanocyte-specific element

MSH Melanocyte-stimulating hormone

NCBI National Centre for Biotechnology Information

NCKX Potassium-dependent sodium/calcium exchangers

Ngn 1/3 Neurogenin 1 and 3

nm Nanometres

OCA Oculocutaneous albinism

OMIM Online Mendelian Inheritance in Man

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OR Odds ratio

p Pink-eyed dilution

Pax Paired box

PCR Polymerase chain reaction

POMC Pro-opiomelanocortin

raly Heterogeneous nuclear ribonucleo-protein associated with lethal yellow

RFLP Restriction fragment length polymorphism

RHC Red hair colour

RLM-RACE RNA ligase mediated rapid amplification of cDNA ends

RNA Ribonucleic acid

ROCA Rufous oculocutaneous albinism

RPMI Roswell Park Memorial Institute

sec Seconds

SINE Short interspersed nuclear element

SLC24A5 Solute carrier family 24 member 5

SLC45A2 Solute carrier family 45 member 2

SNP Single nucleotide polymorphism

TAP Tobacco acid pyrophosphatase

TBE Tris borate ethylenediaminetetraacetic acid

Tbx2 Brachyury-related transcription factor

TDE Tyrosinase distal element

TEAA Triethylammonium acetate

TF Transcription factor

TFE3 Transcription factor binding to IGHM enhancer 3

TFEB Transcription factor EB

TFEC Transcription factor EC

TPE Tyrosinase proximal element

Tris Trishydroxymethylaminomethane

TSS Transcription start site

TYR Tyrosinase

TYRP1 Tyrosinase-related protein 1

TYRP2 Tyrosinase-related protein 2

TYRP3 Tyrosinase-related protein 3

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USF Upstream regulatory factor

UTR Untranslated region

UVR Ultraviolet radiation

uw Underwhite

uwd Underwhite dense

uwdbr Underwhite dominant brown

V Volts

WS Waardenburg syndrome

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Declaration

The work contained in this thesis has not previously been submitted for a degree or

diploma at this or any other higher education institution. To the best of my knowledge

and belief, the thesis contains no material previously published or written by any other

person(s) except where due reference is made.

Signed…………………………

Justin Graf

Date…………………………..

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Dedication and acknowledgements

This thesis is dedicated to my parents, my wife and our future family

Like many things in life, this thesis would not have been possible had certain events not

occurred. On reflection, I would probably not have undertaken this thesis had it not

been for the real life examples of where a PhD can take you. I refer to my good friends,

Dr Fabien Knab and Dr Christian Angst, whom I first met at Roche Products in Sydney,

Australia, while working for the company as part of QUT’s Industrial Internship

program in 2000. They provided the initial inspiration and drive to complete my PhD

and for that I am forever thankful.

Throughout my PhD I have faced several challenges, none more so emotionally

challenging than changing supervisors midway through my candidature. It is times such

as these that having a good mentor will either make or break a PhD. I am indebted to

my current principal supervisor, Dr Ian Hughes, for taking on the mentorship and

supervision of my PhD. Ian, you provided immeasurable support when I needed it most

and not only provided critical and thoughtful analysis of my work, direction and useful

scientific discussions, but you also provided friendship and respect for a fellow

scientist. You continually drove me to be a better scientist and you challenged me to

think more broadly. Even while I have been in Canberra, you have continued to provide

the support and guidance any student would be envious of. It has been an absolute

pleasure working with you and I hope we can keep in contact in the years to come.

Thanks so much, Ian. My associate supervisor, Dr Joanne Voisey, you were always

available to offer your expert knowledge of the pigmentation field and I thank you for

that.

To the friends I have made while being at QUT, particularly during honours and PhD, it

has been a long, tough, but most of all fun journey. Thanks to Alex Stephens and Chris

Swagell for improving my golf game and all the fun we had mucking around in the lab

and anywhere else we saw fit. To others in the CRC, Anna Coussens, Levi Carroll, Shea

Carter, Iman Muharam, Erin Price, Andy Pow and Naomi Knoblauch, thanks for all the

fun times. The memories of CRC retreats will be with me forever. I must also thank

other members of IHBI who have either made lab time more enjoyable or helped me

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with lab techniques: John Lai, Steve Meyers, Pete Cunningham, Steven Bell, Chris

Barker, Mark Turner, Tegan Harris, Jess van Haeften, and YuPei Tan. Thanks also to all

the SPAM boys who made life outside of the PhD memorable as well.

As any PhD student knows, a scholarship is a wonderful thing and it must end at some

point. For this reason, I must also acknowledge several sources of financial support

throughout my PhD. Firstly to the Cooperative Research Centre for Diagnostics, which

initially provided my scholarship, and supported my research throughout my PhD.

Towards the end, I specifically thank Prof. Peter Timms for providing financial support

of laboratory activities. Thanks must also go to the Department of Primary Industries

and Fisheries, who provided necessary part-time work after my scholarship ended. I

also thank my colleagues at the Australian Institute of Health and Welfare in Canberra,

Ms Tracy Dixon and Dr Kuldeep Bhatia, who have supported my efforts to complete

my PhD while working full time and have generously allowed me to take time out to

complete my thesis.

To my parents, you have always encouraged me to pursue my education, you supported

me while I did uni, and were there whenever I needed a hand. You taught me discipline,

persistence, and working hard for what ever I wanted. For this, I thank you and I hope

this achievement makes you eternally proud.

The completion of this thesis would not have been possible without the support, love

and friendship of my beautiful wife, Angelique. Thank you for putting up with the

seven days a week in the lab, when I was tired, cranky and not pleasant to be around.

Thanks just for being there, every step of the way. When we look back at these years, in

our alpine cabin somewhere in Switzerland, I know the hard work will have been worth

it.

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– Chapter 1 –

INTRODUCTION1.1 A description of the research problem investigated

Normal human pigmentation variation exists between different ethnogeographic

population groups as well as within them. It is believed that environmental factors have

led to pigmentation phenotypes being favoured in different parts of the world ― largely

correlated with latitude. Although exposure to ultraviolet radiation (UVR) can influence

pigmentation phenotype, constitutive pigmentation is highly heritable, with genetics

playing a major role in determining pigmentation phenotype (Brauer and Chopra, 1978;

Clark et al., 1981; Bito et al., 1997; Zhu et al., 2004). Evidence for a genetic

contribution to human pigmentation variation was first provided early in the 20th

century when Charles Davenport and his wife Gertrude published several papers on the

heredity of eye colour, hair colour and skin pigmentation (Davenport and Davenport,

1907; Davenport and Davenport, 1909; Davenport and Davenport, 1910). Since then,

over 120 genes have been suggested to contribute to human pigmentation, because they

have been implicated in the processes of melanin production, melanosome biogenesis

and transport, and pigment cell development (Oetting and Bennett, 2003). However, of

these 120 genes, very few have been extensively studied in order to explain the

contribution of genetic variation to normal human skin, hair and eye colour phenotypes.

Human pigmentation largely depends on the size, number and distribution of

melanosomes as well as the type of melanin within them (Hearing, 1999). A failure to

synthesise melanin results in the condition known as oculocutaneous albinism (OCA),

which is characterised by hypopigmentation in the skin, hair and eyes. Mutations in four

genes (tyrosinase, tyrosinase-related protein 1, oculocutaneous albinism type 2, and

solute carrier family 45 member 2) have been found to result in OCA (Reviewed in

Tomita and Suzuki, 2004). The most recently identified gene causing OCA is solute

carrier family 45 member 2 (SLC45A2), also known as antigen in melanoma 1 (AIM-1)

and membrane associated transporter protein (MATP) (Newton et al., 2001). Studying

pigmentation dysfunction is a valuable tool in understanding how pigmentation genes

affect normal pigmentation variation.

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The exact role of SLC45A2 in human pigmentation remains essentially unknown.

Initially, comparative protein sequence analysis identified similarities between

SLC45A2 and plant sucrose/proton symporters (Fukamachi et al., 2001). Although there

are no known sucrose transporters in mammals, it was considered feasible that

SLC45A2 could co-transport a sugar molecule with a proton (Fukamachi et al., 2001;

Newton et al., 2001). Slc45a2 mutations in the medaka fish have shown to impair the

differentiation of melanophores (fish melanocytes) and cause deterioration of

melanogenesis within melanosomes (Hirose and Matsumoto, 1993). Based on the above

evidence, Fukumachi et al. (2001) proposed that SLC45A2 might be a component of the

melanosomal membrane. The putative function of SLC45A2 as a proton transporter has

outlined a possible relationship with the oculocutaneous albinism type 2 (OCA2)

protein, which has been suggested to be an anion transporter in the melanosome

membrane (Puri et al., 2000). The murine OCA2 protein is thought to play a role in

regulating melanosomal pH, a key factor that contributes to the activity of tyrosinase

(Puri et al., 2000; Fuller et al., 2001; Chen et al., 2002). If both proteins are localised to

the melanosomal membrane as suggested above, they may act in conjunction to regulate

the pH of the melanosome (Newton et al., 2001). Currently, SLC45A2 is not believed to

play a specific enzymatic role in melanogenesis but is important for the correct

processing and trafficking of Tyrosinase, TYRP1 and DCT (TYRP2) and therefore

affects melanogenesis indirectly (Costin et al., 2003). Costin and colleagues (2003)

concluded that the function of SLC45A2 involves the sorting of these three

melanogenic proteins from the trans-Golgi network to Stage II melanosomes. To date,

this study has provided the best evidence for the role of SLC45A2 in human

pigmentation. For a more detailed discussion of the role of SLC45A2 in melanogenesis,

see Chapter 2.

If, and how, non-pathogenic mutations in SLC45A2 affect normal human pigmentation

variation is unclear, although some insight has been gained through researching

mutations in murine slc45a2. For example, studying the phenotypically most severe

mutation of the underwhite allele series in mice (Figure 9, Chapter 2) has provided the

best evidence for the role of SLC45A2 in human pigmentation (Costin et al., 2003). In

human SLC45A2, two non-synonymous polymorphisms have been genotyped in

differently pigmented population groups and shown to exhibit a distinctive distribution

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of allele frequencies (Nakayama et al., 2002). Nakayama’s 2002 report provided the

first evidence that SLC45A2 could be involved in determining normal pigmentation

variation between populations. However, no evidence existed that supported a role for

these polymorphisms, or other variants, in determining pigmentation variation within a

population. Identifying polymorphisms that play an important role in determining intra-

population pigmentation will have important implications for studies of human

evolution, the inference of pigmentation phenotype, susceptibility to UVR-induced

cancer, and therapeutics for altering pigmentation phenotype.

1.2 Overall objectives

The overall object of this work was to characterise non-pathogenic polymorphisms in

the solute carrier family 45 member 2 gene and determine their relationship to normal

human pigmentation variation and the function of SLC45A2. It was first postulated that

two non-synonymous SLC45A2 polymorphisms might alter the function or efficiency of

the SLC45A2 protein and therefore be associated with normal human pigmentation

variation. A large sample of unrelated individuals belonging to several different

population groups was used to test this hypothesis. We also sought to determine if the

promoter region of SLC45A2 was also involved in normal pigmentation variation. To do

this, the proximal promoter was partially characterised, novel polymorphisms were

sought, and several polymorphisms were genotyped in a large unrelated sample pool.

The final objective of this thesis was to determine the functional impact of the promoter

variants.

1.3 Specific aims

The specific aims of this study were:

1.To genotype two previously identified non-synonymous coding polymorphisms,

p.Glu272Lys and p.Phe374Leu, in SLC45A2 and determine if they are associated

with normal pigmentation variation.

2.To locate the transcription start site of SLC45A2 and to screen the proximal

SLC45A2 promoter region for both known and novel polymorphisms.

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3.To genotype a large sample of people from within a Caucasian population and other

ethnogeographic population groups for SLC45A2 promoter polymorphisms, and

determine if they are associated with normal pigmentation variation.

4.To characterise the SLC45A2 promoter by

a.using in silico bioinformatic software tools, and

b.performing luciferase reporter assays to identify functionally relevant regions of

the SLC45A2 promoter.

5.To determine if SLC45A2 promoter polymorphisms affect the transcriptional

activity of the gene using luciferase activities.

6.To assess if the two tightly linked SLC45A2 promoter polymorphisms, c.–1176_–

1174dupAAT and c.–1169G>A, affect DNA-protein binding.

7.To ascertain if SLC45A2 promoter genotype is associated with the mRNA

expression of SLC45A2 itself, or other pigmentation genes such as MITF, TYRP1

and ASIP.

1.4 An account of scientific progress linking the research papers

This thesis is presented in the format of “PhD Thesis by Submitted Manuscript”,

according to the guidelines of Queensland University of Technology. Chapter 2

provides a detailed literature review, while the subsequent three chapters represent

manuscripts that have been published or submitted for publication to international peer

reviewed journals.

Chapter 2 – Literature review

This chapter provides a brief introduction to the fundamentals of human pigmentation,

providing some detail on the process of melanogenesis and the major sites of

pigmentation variation. To put pigmentation in an evolutionary and historical context, a

critical review is given of the evidence for the role that selection has played in shaping

human pigmentation variation. The remainder of the literature review focuses on the

molecular genetics of human pigmentation variation. The molecular genetics and

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pathogenesis of oculocutaneous albinism is discussed with particular reference to what

can be learned about normal pigmentation by understanding pigmentation dysfunction.

Given that the mouse has been an important model organism for studying pigmentation,

murine pigmentation studies are included where human data does not provide sufficient

insight. The involvement of the OCA genes, along with other important pigmentation

genes, is also critically reviewed with reference to their involvement in normal

pigmentation variation. In summary, this literature review highlights the limited

evidence for the role that SLC45A2 polymorphisms play in normal human pigmentation

variation, as well as providing relevant background information to put the experimental

results of this thesis in to context.

Chapter 3 - Single nucleotide polymorphisms in the MATP gene are associated with

normal human pigmentation variation (2005) Human Mutation 25:278-284.

In 2001, mutations in the SLC45A2 gene were shown to underlie the fourth and most

recently identified type of oculocutaneous albinism (OCA4) (Newton et al., 2001).

OCA4 is characterised by generalised hypopigmentation. Therefore, it is reasonable to

postulate that the SLC45A2 gene plays a significant role in determining pigmentation

phenotype. A preliminary study that investigated two SLC45A2 non-pathogenic coding

region polymorphisms (p.Glu272Lys and p.Phe374Leu) reported a distinctive

distribution of alleles between several differently pigmented populations (Nakayama et

al., 2002). This was the first evidence that SLC45A2 could be involved in determining

normal pigmentation variation between populations. In this chapter, to confirm and

extend this result, p.Glu272Lys and p.Phe374Leu were genotyped in 608 samples from

four different populations groups (456 Caucasians, 31 Asians, 70 African-Americans,

and 51 Australian Aborigines). Tests of association were performed for allele frequency

differences and normal pigmentation variation between populations and within the

Caucasian population. Results indicated that the allele frequencies of both

polymorphisms were significantly different between population groups and the variant

alleles (272Lys and 374Leu) were strongly associated with black hair, brown eyes and

olive skin colour in Caucasians. This was the first report to show that SLC45A2

polymorphisms were associated with normal human pigmentation variation within a

population. To further understand the role of SLC45A2 polymorphisms in normal

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pigmentation variation, an investigation of the SLC45A2 putative promoter was

undertaken.

Chapter 4 – Promoter polymorphisms in the MATP (SLC45A2) gene are associated

with normal human skin color variation (2007) Human Mutation 28:710-717.

Following the encouraging results of Chapter 3, the proximal promoter region of

SLC45A2 was targeted, to determine if polymorphisms in this region might also be

associated with normal pigmentation variation. It has been suggested that complex

traits, of which pigmentation is included, are more often influenced by non-coding

regulatory polymorphisms rather than coding region polymorphisms (Mackay, 2001;

Korstanje and Paigen, 2002). It follows that if a promoter polymorphism is functional, it

is more likely to exhibit large differences in allele frequencies because it has been under

greater selective pressure. First, however, the promoter region was characterised by

initially defining the transcription start site (TSS). A putative transcription start site for

human SLC45A2 had previously been postulated at position –61 bp based on 5' RACE

results from mouse mRNA (Fukamachi et al., 2001). The work presented in Chapter 4

defines two potential alternate TSSs at –78 bp and –111 bp of SLC45A2. Following this,

dHPLC was used to screen the region encompassing –1287 to +145 in 95 DNA samples

of varying pigmentation phenotypes. One novel (c.–1176_–1174dupAAT) and two

previously described polymorphisms (c.–1721C>G and c.–1169G>A) were identified in

our sample pool. These three promoter polymorphisms were genotyped in a total of 700

volunteers from five different population groups (529 Caucasians, 38 Asians, 46

African Americans, 47 Australian Aborigines, and 40 Spanish Basques) and tested for

association with normal pigmentation variation. All three polymorphisms were

associated with skin colour such that the –1721G, +dup and –1169A alleles were more

common in olive-skinned Caucasian individuals. It was hypothesised that one or more

of the promoter polymorphisms affected pigmentation phenotype by altering

transcriptional activity of SLC45A2. Luciferase assays showed that transcriptional

activity was indeed altered. Based on in silico bioinformatic analysis, and that the

microphthalmia-associated transcription factor (MITF) is a known regulator of

SLC45A2(Baxter and Pavan, 2002; Du and Fisher, 2002), it was postulated that

SLC45A2 promoter polymorphisms could contribute to the regulation of pigmentation

by altering MITF binding affinity.

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Chapter 5 - Functional characterisation of polymorphic variation in the SLC45A2

(MATP) gene promoter (Pigment Cell and Melanoma Research – submitted).

In Chapter 4, it was shown that SLC45A2 transcriptional activity was altered by

promoter polymorphisms. Based on bioinformatic analyses, it was suggested that this

may have been due to altered MITF binding. In Chapter 5, this possibility was

investigated and further characterisation of the SLC45A2 promoter occurred by

designing various promoter constructs and expressing them in MM96 melanoma cells.

Five constructs were designed of increasing length and their promoter activity

evaluated. In addition, bioinformatic analysis was used to identify potential functional

elements. Constitutive promoter activity was observed within the first ~200 bp and

promoter activity increased as the construct size increased. Electrophoretic mobility

shift assays (EMSA) were used to investigate the functional consequences of the –dup

and –1169G alleles, showing that DNA-protein binding was altered. While this was not

an unexpected result it was interesting that the protein/s involved were not MITF, or at

least MITF was not the protein directly binding to the DNA. In an effort to more

thoroughly characterise the functional consequences of SLC45A2 promoter

polymorphisms, the mRNA expression levels of SLC45A2, TYRP1 and MITF were

determined in melanocyte/melanoblast cell lines that were genotyped for c.–1721C>G,

c.–1176_–1174dupAAT and c.–1169G>A. Allele-specific expression was observed for

TYRP1, suggesting a coordinated expression of pigmentation genes. The findings

summarised above, and what they mean to our understanding of human pigmentation,

are discussed in the general discussion (Chapter 6), along with some of the relevant

experimental considerations and limitations, future experiments, and possible

applications of this work.

8

1.5 References

Baxter, L.L. and Pavan, W.J. (2002). The oculocutaneous albinism type IV gene Matp is a new marker of pigment cell precursors during mouse embryonic development. Mech Dev. 116(1-2):209-12.

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(5):659-63.

Brauer, G. and Chopra, V.P. (1978). Estimation of the heritability of hair and eye color. Anthropol Anz. 36(2):109-20.

Chen, K., Manga, P. and Orlow, S.J. (2002). Pink-eyed dilution protein controls the processing of tyrosinase. Mol Biol Cell. 13(6):1953-64.

Clark, P., Stark, A.E., Walsh, R.J., Jardine, R. and Martin, N.G. (1981). A twin study of skin reflectance. Ann Hum Biol. 8(6):529-41.

Costin, G.E., Valencia, J.C., Vieira, W.D., Lamoreux, M.L. and Hearing, V.J. (2003). Tyrosinase processing and intracellular trafficking is disrupted in mouse primary melanocytes carrying the underwhite (uw) mutation. A model for oculocutaneous albinism (OCA) type 4. J Cell Sci. 116(Pt 15):3203-12.

Davenport, G. and Davenport, C. (1907). Heredity of eye-color in man. Science. 26(670):590-592.

Davenport, G. and Davenport, C. (1909). Heredity of hair color in man. The American Naturalist. 43(508):193-211.

Davenport, G. and Davenport, C. (1910). Heredity of skin pigmentation in man. The American Naturalist. 44(527):641-672.

Du, J. and Fisher, D.E. (2002). Identification of Aim-1 as the underwhite mouse mutant and its transcriptional regulation by MITF. J Biol Chem. 277(1):402-6.

Fukamachi, S., Shimada, A. and Shima, A. (2001). Mutations in the gene encoding B, a novel transporter protein, reduce melanin content in medaka. Nat Genet. 28(4):381-5.

Fuller, B.B., Spaulding, D.T. and Smith, D.R. (2001). Regulation of the catalytic activity of preexisting tyrosinase in black and Caucasian human melanocyte cell cultures. Exp Cell Res. 262(2):197-208.

Hearing, V.J. (1999). Biochemical control of melanogenesis and melanosomal organization. J Investig Dermatol Symp Proc. 4(1):24-8.

Hirose, E. and Matsumoto, J. (1993). Deficiency of the gene B impairs differentiation of melanophores in the medaka fish, Oryzias latipes: fine structure studies. Pigment Cell Res. 6(1):45-51.

Korstanje, R. and Paigen, B. (2002). From QTL to gene: the harvest begins. Nat Genet. 31(3):235-6.

Mackay, T.F. (2001). Quantitative trait loci in Drosophila. Nat Rev Genet. 2(1):11-20.Nakayama, K., Fukamachi, S., Kimura, H., Koda, Y., Soemantri, A. and Ishida, T.

(2002). Distinctive distribution of AIM1 polymorphism among major human populations with different skin color. J Hum Genet. 47(2):92-4.

Newton, J.M., Cohen-Barak, O., Hagiwara, N., Gardner, J.M., Davisson, M.T., King, R.A. and Brilliant, M.H. (2001). Mutations in the human orthologue of the mouse underwhite gene (uw) underlie a new form of oculocutaneous albinism, OCA4. Am J Hum Genet. 69(5):981-8.

Oetting, W. and Bennett, D. (2003). Albinism Database - International Albinism Center at the University of Minnesota.

9

Puri, N., Gardner, J.M. and Brilliant, M.H. (2000). Aberrant pH of melanosomes in pink-eyed dilution (p) mutant melanocytes. J Invest Dermatol. 115(4):607-13.

Tomita, Y. and Suzuki, T. (2004). Genetics of pigmentary disorders. Am J Med Genet C Semin Med Genet. 131C(1):75-81.

Zhu, G., Evans, D.M., Duffy, D.L., Montgomery, G.W., Medland, S.E., Gillespie, N.A., Ewen, K.R., Jewell, M., Liew, Y.W., Hayward, N.K.et al. (2004). A genome scan for eye color in 502 twin families: most variation is due to a QTL on chromosome 15q. Twin Res. 7(2):197-210.

10

11

– Chapter 2 –

LITERATURE REVIEW

2.1 Fundamentals of human pigmentation

Some of the most striking and variable traits in humans are pigmentation of the hair,

skin and eyes. In each case this pigmentation is primarily due to one chemically inert

and stable visual pigment known as melanin. Melanin is a polymer of indolequinone or

dihydroxyindole carboxylic acid, the exact structure of which are still being elucidated

(Kaxiras et al., 2006). Melanin biosynthesis takes place in specialised organelles called

melanosomes where the amino acid tyrosine is utilised to synthesise two different forms

of melanin, eumelanin and pheomelanin (Seiji et al., 1963a). Human pigmentation of

the hair and skin depends on the size, number and distribution of melanosomes as well

as the type of melanin within them (Hearing, 1999). A failure to synthesise melanin

results in the condition known as albinism. Over 120 genes are likely involved in human

pigmentation, relating to the processes of melanin production, melanosome biogenesis

and transport, and pigment cell development (Oetting and Bennett, 2003). A

combination of environmental influences, genetic regulation, and epigenetic factors

define human pigmentation as a complex physical trait.

2.1.1 Melanin biosynthesis

Melanin is synthesised from tyrosine as two forms, black/brown eumelanin and

yellow/red pheomelanin. Melanin biosynthesis takes place in specialised organelles of

dendritic melanocytes called melanosomes (Seiji et al., 1963b). Tyrosinase catalyses the

first two steps of the eumelanin and pheomelanin enzymatic pathways by converting

tyrosine to 3, 4 dihydroxyphenylanine (DOPA) and DOPA to DOPAquinone (Figure 1)

(Reviewed in Sturm et al., 2001). In the presence of thiols such as cysteine, glutathione,

and thioredoxin, melanin synthesis can be directed towards production of pheomelanin

through the metabolites of 5-S-cysteinylDOPA and benzothiazine intermediates

(Reviewed in Sulaimon and Kitchell, 2003). Pheomelanogenesis is also favoured when

tyrosinase activity is low, regardless of cysteine availability (Ozeki et al., 1997). In the

absence of thiols, eumelanin production is favoured. DOPAquinone undergoes

12

cyclisation to leucodopachrome and DOPAchrome which inturn utilises the enzyme

dopachrome tautomerase or tyrosinase-related protein 2 (DCT or TYRP2) to form 5, 6-

dihydroxyindole-2-carboxylic acid (DHICA). Spontaneous decarboxylation of DHICA

can also occur forming 5, 6-dihydroxyindole (DHI). Oxidative polymerisation of

DHICA to eumelanin is catalysed by tyrosinase-related protein 1 (TYRP1) whilst

tyrosinase is again utilised to catalyse the formation of the more darkly coloured DHI-

melanin (Reviewed in Hearing, 2005). Most melanin pigments are present as complex

mixtures or copolymers of eumelanin and pheomelanin (Ito and Wakamatsu, 2003).

Figure 1. Biosynthetic pathway of eumelanin and pheomelanin. Source:Wakamatsu and Ito (2002)

2.1.2 Melanosome biogenesis

Melanocytes are pigment-forming cells that contain specialised intracellular organelles

called melanosomes. Melanosomes are derived from late stage endosomes from the

endoplasmic reticulum (ER)/Golgi network and are involved in the synthesis and

storage of melanin (Kushimoto et al., 2001; Raposo et al., 2001). Their maturation can

13

be classified into four morphologically distinct stages (Figure 2). Stage I melanosomes,

often referred to as premelanosomes as they do not yet produce melanin, are common to

eumelanogenesis and pheomelanogenesis (Reviewed in Sturm et al., 2001).

Transformation into elongated, fibrillar organelles that now contain tyrosinase and other

enzymes for the first time, characterise Stage II eumelanosomes. Compared to Stage I

melanosomes in which irregular fibrous structures exist, Stage II melanosomes contain

regular parallel fibres which can be easily visualised using electron microscopy.

Melanin is synthesised and deposited evenly on the fibrous structures forming Stage III

melanosomes. When the melanosomes are essentially filled with melanin and little or no

structure is visible they are classified as Stage IV melanosomes.

Figure 2. Ultrastructure level from stages I-IV of melanosomes. Source:Hearing (2005)

Melanosomes differ in appearance and structure according to the type of melanin within

them. Eumelanosomes are large, ellipsoidal organelles with a highly structured internal

glycoprotein matrix. Pheomelanosomes are smaller and spherical (Reviewed in Sturm et

al., 2001).

2.2 Sites of pigmentation

2.2.1 Skin pigmentation

Skin melanocytes reside in the basal layer of the epidermis and have dendritic

projections that infiltrate surrounding keratinocytes (Figure 3A). Melanosomes are

transported along the dendrites as they mature before being transported into

keratinocytes (Figure 3A) (Boissy, 1988). In humans, one epidermal melanocyte can

make contact with approximately 30–40 keratinocytes (Eisinger and Marko, 1982). All

individuals have, essentially, similar numbers of melanocytes but the number and type

of melanosomes differs markedly (Reviewed in Barsh, 2003), as does the rate of

transfer to keratinocytes (Rees, 2003), all of which determine skin colour. As seen in

14

Figure 3B, darker-skinned individuals such as those from Africa, have keratinocytes

that contain larger eumelanosomes. Lighter pigmented populations such as Caucasians

(Europeans) and Asians generally have more pheomelanosomes, which are clustered in

small membrane-bound packages (Figure 3B) (Reviewed in Szabo et al., 1969; Sturm et

al., 1998). Variation in skin pigmentation also occurs in response to ultraviolet radiation

(UVR) exposure (Friedmann and Gilchrest, 1987). This “tanning” response occurs as a

result of a slight increase in melanocyte number and greater increase in transfer rate of

melanosomes to the keratinocytes and occurs to a greater extent in people with dark

skin (Tadokoro et al., 2005).

A B

Figure 3. Distribution of melanosomes to surrounding keratinocytes. A) The

dendrites of the melanocyte allow eumelanin- and pheomelanin-containing

melanosomes to be transferred to surrounding keratinocytes. B) Interpopulation skin

colour variation is determined by the type of melanin as well as the distribution of

melanosomes within surrounding keratinocytes.Source: A) http://www.pg.com/science/skincare/Skin_tws_16.htm, B) Sturm et al., (1998)

2.2.2 Hair pigmentation

The hair is an appendage to the epidermis with its origin residing in the dermis of the

skin: the layer below the epidermis (Figure 4). Hair growth occurs in the follicle and has

15

several morphologically and histologically defined sub-phases. A mature follicle

undergoes phases of growth (anagen), regression (catagen), rest (telogen) and shedding

(exogen) (Reviewed in Stenn and Paus, 2001). The duration of the cycle depends on the

site of the hair on the body and can take as long as five years in the human scalp

(Reviewed in Trotter, 1924; Saitoh et al., 1970; Stenn and Paus, 2001). Melanogenesis

only occurs during anagen with 90% of hair follicles in anagen at any one time. During

anagen, melanin production is carried out by follicle melanocytes which reside in the

lower, wider part of the hair follicle known as the hair bulb. Although follicle

melanocytes migrate from the epidermis they differ from their epidermal counterparts

by being larger, more dendritic and by producing larger melanosomes (Bell, 1967).

Melanogenically active hair bulb melanocytes (follicular melanocytes) transfer their

melanosomes to surrounding pre-cortical keratinocytes, which in turn form the cortex of

the hair shaft (Reviewed in Slominski et al., 2005). There is approximately one hair

bulb melanocyte for every five keratinocytes in the hair bulb (Tobin and Paus, 2001).

Cortical kertinocytes gradually harden and then die to form a mature hair shaft (Ryder,

1973; Wood and Bladon, 1985).

Figure 4. Hair shaft and the determination of hair colour. Epidermal melanocytes

migrate from the epidermis of the skin to the hair bulb where they transfer their

melanosomes to pre-cortical keratinocytes which then form the cortex of the hair shaft.Source: Modified fromSlominski et al., (2005)

16

2.2.3 Eye pigmentation

Eye, or more specifically iris, colour in normal individuals is observed in a continuous

spectrum from light blue through green to dark brown (Figure 5). Three main types of

pigment-containing cells can be found in the iris: pigmented epithelial cells, iridial

melanocytes and the clump cells of Koganei (Wilkerson et al., 1996; Stjernschantz et

al., 2002) (Figure 6). Pigmented epithelial cells are located in two layers at the posterior

surface and essentially contain eumelanin. Cells of the posterior iris epithelial layer are

more densely concentrated with melanin granules than those of the anterior iris

epithelium. Iridial melanocytes are located in the collagenous stroma, which lies

anterior to the two epithelial layers. They contain both eumelanin and pheomelanin and

the ratio of these two melanins varies in different iris colours (Prota et al., 1998). The

anterior border layer of the iris also contains melanocytes, which are orientated parallel

to the surface of the iris. Along with iridial melanocytes in the stroma, melanocytes in

the anterior border layer are the most important determinants of eye colour variation

(Eagle, 1988; Wilkerson et al., 1996; Imesch et al., 1997). Clump cells, which have

been suggested to be macrophage-like, acquire melanosomes through phagocytosis.

They are primarily located in the stroma at the pupillary border and in the periphery of

the iris (Wobmann and Fine, 1972; Zinn et al., 1973; Freddo, 1996).

Figure 5. Examples of iris colour variation. A range of iris colours ranging from blue,

grey, green, hazel, light brown to dark brown are shown. The peripupillary ring can be

seen in some examples and is characterised by a eumelanic ring surrounding the pupil.Source:Sturm and Frudakis (2004)

17

Both the pigmented epithelial cells and clump cells play a minor role in iris colour

variation. Epithelial cells contain a similar amount and distribution of melanin in

different iris colours whilst clump cells are only present in small numbers, and therefore

are very minor contributors to eye colour variation (Wilkerson et al., 1996). Iridial

melanocytes, including those in the anterior border layer, are believed to be the major

determinant of eye colour variation. Unlike melanocytes in the hair and skin, stromal

melanocytes do not transfer their melanosomes to surrounding cells but rather retain and

accumulate them in their cytoplasm (Reviewed in Sturm and Frudakis, 2004). The

number of melanocytes in the iris do not differ between different eye colours, but

increased numbers and increased size of melanosomes are observed as iris colour

darkens from green to dark brown (Eagle, 1988; Imesch et al., 1996; Wilkerson et al.,

1996; Prota et al., 1998). However, Imesch et al. (1996) did not observe a significant

difference in average melanosome size across colour groups. Blue iris colour results

mainly from the structure and density of the stroma of the eye whereby short

wavelength light is reflected and backscattered from the stroma (Wilkerson et al., 1996;

Imesch et al., 1997). Blue irides contain little pigment in the iridial melanosomes of the

stroma but the iris pigment epithelium still contains pigmented melanosomes. Melanin

quantity and melanosome numbers are increased in green eyes compared to blue eyes

and contain mostly pheomelanin. Brown eyes have the highest level of pigmentation

with densely packed melanocytes and large amounts of mostly eumelanin (Prota et al.,

1998). The presence or absence of a more darkly pigmented peripupillary ring in the iris

can also be observed (Figure 5). Eye colour has been shown to change with age. For

most people, eye colour will stabilise by the age of six although approximately 10–15%

of Caucasians will experience eye colour change throughout adolescence and adulthood

(Bito et al., 1997).

Melanogenesis was previously thought to occur only in ocular melanocytes during

foetal development and in the early years of life. Currently, it is believed that

melanogenesis can take place at low but detectable levels in both iridial melanocytes

and iris pigment epithelium throughout adulthood (Smith-Thomas et al., 1996;

Lindquist et al., 1998; Grierson et al., 2001). Much of the evidence for this has been

brought about through studying the effect a prostaglandin glaucoma drug, known as

latanoprost, on iris pigmentation changes (Reviewed in Stjernschantz et al., 2002).

18

Figure 6. Iris Anatomy. A)Whole eye showing pigmentation of the iris. B) Surface

portion of the iris showing clump cells and radial folds. C) Cross section of human iris

showing melanocytes, posterior iris pigment epithelium and clump cells.Source: http://eyecareug.com/anatomy_eye.htm

A

B

C

19

2.3 Evidence for a genetic contribution to human pigmentation

variation

The environment undoubtedly plays an important role in determining the levels of non-

constitutive pigmentation, particularly for hair and skin colour. The effects of UVR-

induced bleaching of hair colour and UVR-induced melanin production on exposed skin

can have a significant effect on pigmentation phenotype. However, constitutive

pigmentation is highly heritable, with genetics playing a major role in determining

pigmentation phenotype (Sturm et al., 1998; Rees, 2003), response to UVR exposure

(Miyamura et al., 2007) and susceptibility to cancer (Palmer et al., 2000), among others.

Evidence for a genetic contribution to human pigmentation variation arose early in the

20th century when Charles Davenport and his wife Gertrude published several papers on

the heredity of eye colour, hair colour and skin pigmentation (Davenport and

Davenport, 1907; Davenport and Davenport, 1909; Davenport and Davenport, 1910).

More recently, the heritability of eye colour was estimated to be 0.80, skin colour 0.83

(Clark et al., 1981), and hair colour to be 0.61 (Brauer and Chopra, 1978). Other studies

have assessed the contribution of genetic vs. non-genetic factors on pigmentation

variation by examining the phenotypes of monozygotic twins. For example, twin studies

have shown that one genetic loci contributes 74% of the variation of eye colour (Zhu et

al., 2004).

The Davenports used self-report questionnaire data on family pigmentation

characteristics and applied Mendelian principles to the inheritance of these traits.

Unfortunately, like other complex traits, pigmentation does not neatly fit Mendelian

inheritance and the Davenports first suggested a polygenic mode of inheritance. Later,

the number of genes responsible for pigmentation variation grew from two gene pairs,

as suggested by the Davenports, to 3–6 pairs (Reviewed in Kittles, 1995; Sturm et al.,

1998). It is now generally accepted that over 120 different loci contribute to

pigmentation variation but there are several major determinants and many modifier

genes that play a role in human pigmentation variation (Bennett and Lamoreux, 2003).

20

2.4 The evolution of normal pigmentation variation

2.4.1 Geographical variation of human skin colour

Normal pigmentation variation exists between and within indigenous populations, with

the most visually striking being that of skin colour. Skin colour variation has been the

subject of intrigue for centuries and has been used to define human races (Jablonski and

Chaplin, 2000; Millington and Levell, 2007). Quantitative measures of normal skin

colour variation have shown that variation is strongly correlated with latitude (Figure 7).

Darker pigmentation is observed in populations near the equator while lighter

pigmentation is observed with increasing distance away from the equator (Roberts and

Kahlon, 1976; Roberts, 1977; Tasa et al., 1985). In addition, hemispheric differences in

normal skin colour exist such that skin colour is darker in the southern hemisphere

(Relethford, 1997).

Figure 7. Global map of human skin colour variation.Source:O'neill (2007)

Normal human pigmentation variation also exists between sexes within a population.

Although this difference is relatively small compared to variation between populations,

females have lighter constitutive pigmentation than males (Byard and Lees, 1981). Of

course, this may be due to variations in hormone levels but it may also be the result of

selective mating (Aoki, 2002). In a study of 51 “ethnically” diverse populations, males

and females have shown preference for lighter than average skin colour when choosing

21

a sexual partner (van den Berghe and Frost, 1986). This research also noted a sexual

asymmetry, whereby a lighter than average skin colour is preferred more in females

than in males. Sexual selection theory has also been suggested to predict the latitudinal

variation in skin colour (Aoki, 2002).

It is interesting to note that unlike the majority of human genetic diversity and some

other quantitative traits such as craniometrics, skin pigmentation varies greatly between

populations, rather than within populations (Lewontin, 1972; Relethford, 2002). It is

estimated that 88% of total variation for skin colour exists between populations, with

only 3% existing between and 9% existing within local populations (Relethford, 2002).

This is not surprising considering that skin pigmentation has undergone intense natural

selection due to the skin being a major interface with the environment (Parra et al.,

2004).

Ultraviolet radiation stimulates changes in human skin pigmentation levels within

individuals and is also believed to be one of the most important environmental factors

that has driven natural selection of pigmentation between populations (Friedmann and

Gilchrest, 1987; Ortonne, 1990; Tadokoro et al., 2005). However, the causal pathway

by which UVR may have affected skin pigmentation is still debated. The mechanism by

which UVR leads to cancer has been well established (Melnikova and Ananthaswamy,

2005), and therefore some propose that darker skin, which has increased levels of

melanin, has been selected for in areas of high UVR by providing protection against

sunburn and cancer (Agar and Young, 2005; Izagirre et al., 2006). It can do this by

acting as an optical filter attenuating UVR by scattering. In addition, melanin acts as a

scavenger of damaging free radical generated by UVR (Schwabe et al., 1989; Kollias et

al., 1991; Bustamante et al., 1993; Rozanowska et al., 1999). Further evidence for

UVR-based selection can be found when comparing genetic variation in the

melanocortin 1 receptor (MC1R; discussed in section 2.6.2.1) gene between

populations. In Europeans, variant alleles (R151C, R160W, D294H) in MC1R are

associated with an increased risk of UVR-induced cancer by contributing to fair skin

colour (Valverde et al., 1995; Palmer et al., 2000). Usually, African populations show

greater genetic variation than do Europeans (Shriver et al., 1997), but for MC1R, the

African populations show much less variation than Europeans. This indicates that

MC1R is undergoing strong genetic selection. Any genetic variation in MC1R that

22

results in lighter skin pigmentation would be evolutionarily deleterious to populations in

areas of high UVR and would be selected against (Harding et al., 2000). Thus, these

results suggest that the increased number of heterozygous MC1R loci in European

populations represents a relaxation of natural selection rather than events indicative of

positive selection (Rana et al., 1999).

Increased melanin is also thought to protect against UVR-induced photolysis of folate

which is an essential nutrient for DNA biosynthesis and foetal development (Branda

and Eaton, 1978; Bower and Stanley, 1989; Jablonski and Chaplin, 2000). Folate is

essential to ensure correct neural tube development (Bower and Stanley, 1989).

Insufficient folate can result in serious deformities in newborn children, such as for

spina bifida, and in the worst case, it can result in embryonic lethality (Pritchard et al.,

1970; Tamura and Picciano, 2006). In addition, folate deficiency has been shown to

result in spermatogenic arrest in mice and rats and is therefore believed to be important

for male fertility (Mathur et al., 1977; Cosentino et al., 1990). The maintenance and

regulation of folate levels are therefore important for reproductive success. Thus,

protection of folate degradation, through changes in epidermal melanin, could be an

underlying mechanism that has driven the natural selection of skin colour in areas with

different UVR levels (Jablonski and Chaplin, 2000).

Positive selection for lighter skin colour is more controversial, with most discussion

centred on the “vitamin D hypothesis”. This hypothesis argues that as the selective force

maintaining dark skin colour weakens away from the equator, lighter skin colour is

selected for to ensure adequate photoproduction of vitamin D in order for humans to

inhabit non-tropical regions (Loomis, 1967). Robins (1991) has made strong arguments

against vitamin D-related depigmentation, arguing that early Homo would have had

sufficient exposure to UVR during spring and summer to enable adequate storage of

vitamin D in fat and muscle during periods of low UVR (Mawer et al., 1972; Robins,

1991). Further, Robins (1991) adds that vitamin D deficiencies are a consequence of

urbanisation, industrialisation and over-population, which early Homo would not have

been exposed to. However, the ability to store vitamin D depends on pre-existing stores

of the vitamin (Mawer et al., 1972). Therefore, for adequate storage, a previously

sufficient high intake or endogenous production of vitamin D would be required.

Jablonski and Chaplin (2000) refute the arguments of Robins (1991) by providing

23

evidence of geographical zones representing the different potential for vitamin D

synthesis and data showing a strong association between UVR levels and skin colour

reflectance of indigenous populations.

2.4.2 Evidence for positive selection of human pigmentation variation

Positive selection is the process whereby beneficial traits become more frequent in a

population. Beneficial traits are made possible due to an increased accumulation of

advantageous genetic mutations, usually relating to the ability to survive and reproduce.

When alleles undergo positive selection, they leave distinct and identifiable patterns of

genetic variation. These patterns can be identified by comparing the type and degree of

genetic variation, to the neutral model of evolution. This model states, “the great

majority of evolutionary changes at the molecular (DNA) level are caused not by

Darwinian selection but by random fixation of selectively neutral or nearly neutral

mutants” (Kimura, 1986). This process is also known as “genetic drift”. However, the

occurrence of confounding population demographic events such as population

bottlenecks or expansions can make identifying true positive selection difficult (Sabeti

et al., 2006).

With the advent of whole-genome sequencing and the generation of large sets of

genome-wide genetic variation data, such as those from the International Haplotype

Map Project (The International HapMap Consortium, 2005), Single Nucleotide

Polymorphism (SNP) consortium (Altshuler et al., 2000) and Perlegen Sciences (Hinds

et al., 2005), it is possible to detect positive selection on a genome-wide level. The rates

of non-synonymous mutations can be compared with synonymous mutations

(presumably neutral) between species, in order to detect regions that have an increased

frequency due to beneficial function-altering mutations. Some data sets, such as the

HapMap data set, were established primarily for use in association studies and therefore

are biased towards SNPs with higher levels of heterozygosity (Clark et al., 2005). Some

methods used to detect positive selection are sensitive to this sort of SNP ascertainment

bias and should be used with some caution.

When an allele increases within a population, nearby mutations (including neutral and

even deleterious mutations) also increase due to the effects of linkage disequilibrium.

This is known as “hitchhiking” and results in a “selective sweep” of alleles in the

24

vicinity of the beneficial allele (Smith and Haigh, 1974). If alleles reach fixation or

100% frequency, a complete selective sweep is said to have occurred. A reduction in

genetic diversity is commonly detected using statistical tests such as the Tajima’s D

(Tajima, 1989) and Fu an Li’s D (Fu and Li, 1993). When non-ancestral derived alleles

arise due to new mutations, a selective sweep can also hitchhike these mutations to high

frequency. Many non-ancestral alleles will not reach fixation as they are simply

hitchhiking with the beneficial allele. Regions of high frequency derived alleles can be

detected and compared with ancestral alleles to detect positive selection. The Fay and

Wu’s H statistic is commonly used for this purpose (Fay and Wu, 2000). Selective

sweeps of recent mutations can cause long haplotypes to arise before recombination has

had sufficient time to break them down. The age of haplotypes and evidence of

selection can be determined by measuring the frequency of alleles at increasing distance

from the primary locus (Sabeti et al., 2002). Higher levels of haplotype homozygosity,

indicative of positive selection, are expected to extend much further than that expected

under a neutral model (Voight et al., 2006). The extended haplotype heterozygosity

(EHH) test is useful to detect this.

Another commonly used method to detect positive selection involves comparing

differences in allele frequencies between populations, such as those between Europeans

and Africans. Allele frequency differences may arise due to environmental or cultural

pressures such that the populations are reproductively isolated. The fixation index (F

statistic or Fst) can detect positive selection by measuring the level of heterozygosity, or

more specifically the reduction in homozygosity, and comparing them to the expected

values determined by Hardy-Weinberg equilibrium.

Regional, population-specific, signatures of selection can play an important role in

“signposting” potentially functionally relevant genes for pigmentation variation and

other phenotypic traits (Ronald and Akey, 2005; McEvoy et al., 2006; Sabeti et al.,

2006). By investigating the frequency of genetic variants in admixed populations,

evidence that the variant actually does affect pigmentation can be provided. Known as

“admixture mapping”, Norton et al. (2007) define it as ‘a test for linkage in the presence

of population stratification which makes it possible to test specifically for functionally

important variations between two particular ancestral populations that differ for the

phenotype of interest’ (Norton et al., 2007). A good example was shown for TYR and

25

oculocutaneous albinism type II (OCA2) gene when comparing European American,

African American and Caribbean American populations (Shriver et al., 2003). TYR and

OCA2 were shown to have a significant impact on the difference between European and

admixed African population, in terms of skin colour. Further examples of the usefulness

of admixture mapping have been shown for solute carrier family 24 member 5

(SLC24A5) (Lamason et al., 2005) and solute carrier family 45 member 2 (SLC45A2)

gene (Norton, 2005). However, admixture mapping is not suitable for detecting within-

population pigmentation variation. Shriver et al. (2003) also investigated MC1R and

showed no evidence of this gene in determining between population pigmentation

differences, despite its well-established role in determining red hair colour and pale

skin.

It was assumed that light skin colour evolved due to the relaxation of natural selection

because UVR levels decrease away from the equator, decreasing the necessity for

darkly pigmented skin. Recently, genetic evidence of positive selection for light skin

pigmentation in “out of Africa” populations has been identified in pigmentation

candidate genes. For a good review see McEvoy et al. (2006). The discovery of a new

gene, SLC24A5 (discussed in section 2.6.2.3), and ensuing research has shown it to be

one of the major genes involved in the skin colour differences between Europeans and

non-Europeans (Lamason et al., 2005). Large allele frequency differences for a non-

synonymous polymorphism (A111T) in SLC24A5, between European and African

populations, provided initial evidence that this gene was a target of natural selection.

This was further supported by a striking reduction in heterozygosity within a 150 kb

region surrounding the gene in European populations, indicating a selective sweep had

occurred in this region (Lamason et al., 2005). Subsequent investigations by others have

confirmed the positive selection of pigmentation in European populations using several

measures (Izagirre et al., 2006; McEvoy et al., 2006; Voight et al., 2006; Norton et al.,

2007).

Evidence of recent positive selection has also been provided for another pigmentation

gene, SLC45A2, by observing reduced heterozygosity in a 7.55 kb region surrounding

the F374L polymorphism in a European population (Soejima et al., 2006). The F374L

polymorphism has previously been shown to exhibit distinctive allele frequencies in

different populations (Nakayama et al., 2002; Yuasa et al., 2004). By comparing the

26

frequency of non-synonymous and synonymous polymorphisms in five different

populations with that of the ancestral chimpanzee sequence, Soejima et al. (2006) did

not show any significant differences. However, when applying four different measures

of nucleotide diversity (or heterozygosity), a significant deviation from selective

neutrality was observed for the European population. Further, the composite likelihood

ratio (CLR) test was used to show a reduction in variation, presumably caused by a

hitchhiking event, indicating positive selection has occurred in Europeans in the region

surrounding the F374L polymorphism. The possible bias caused by the effects of

demography was ruled out by applying the goodness of fit (GOF) test.

In a screen of 81 loci, another study provided weak evidence of positive selection for

SLC45A2(Izagirre et al., 2006). Phylogenetic analysis revealed that there was no

evidence of positive selection for SLC45A2, rather it was classified as “neutral”. Using

merged SNP data from Perlegen, HapMap and Applera, multiple-test-corrected Fstanalysis showed a significant difference between Caucasians and East Asians for

SLC45A2. When using the Perlegen and HapMap data sets individually, Myles et al.

(2007) confirmed the Fst differences between Europeans/Caucasians and East

Asians/Chinese. In addition, Myles et al. (2006) observed Fst differences between their

European and African populations. A possible reason why Izagirre et al. (2006) did not

identify the same difference is due to the pooling of African allele data from the

Perlegen, HapMap and Applera data sets. The “African” populations used by Izagirre et

al. (2006) contained data from African Americans and other African populations such as

the Yoruba of Nigeria. The frequencies of some alleles in these two populations are

known to differ considerably, thus explaining the differences between these two reports

(Myles et al., 2007). Using HapMap genetic data and extended haplotype

heterozygosity (EHH)-based analyses (Sabeti et al., 2002), Izagirre et al. (2006) and

Myles et al. (2007), observed different results. Using DNA sequence of 200 kb either

side of SLC45A2, Izagirre et al. (2006) failed to achieve high EHH values for SLC45A2

in any population. In contrast, Myles et al (2007) observed high EHH values for their

European population, when analysing smaller surrounding regions (100 kb total) and

using a slightly modified EHH-based test of positive selection (Myles et al., 2007).

Strong evidence of positive selection for SLC45A2 in European populations was

recently provided (McEvoy et al., 2006; Norton et al., 2007). Using the Fst method to

27

detect inter-population divergence, Norton et al. (2007) used Affymetrix SNP arrays to

analyse genetic data from six different populations (West African, Island Melanesian,

South Asian, Native American, East Asian and European). Significant Fst differences

were observed between the European population and all other populations analysed.

The same study utilised HapMap data and three different indicators of positive selection

to further show the involvement of SLC45A2 in the evolution of light skin in Europeans.

Lao et al. (2007) employed a 6-step hierarchical approach to identify pigmentation

genes that could explain human skin colour variation between populations. One step

included using SNP data from Perlegen to identify unusual patterns of genetic variation

between populations. As expected, genetic variation in SLC45A2 was shown to

significantly differ between the three major continental populations used in the Perlegen

data set (Lao et al., 2007). Included in this analysis were measures of haplotype

heterozygosity, which can identify evidence of positive selection due to the effects of a

selective sweep. Lao et al. (2007) and Izagirre et al. (2006) used the EHH test to detect

positive selection and both observed no evidence of positive selection for SLC45A2.

However, others (Soejima et al., 2006; Myles et al., 2007) have identified positive

selection for SLC45A2 using haplotype-based methods. Lao et al. (2007) suggest that

the EHH test does not provide sufficient power to detect positive selection when the

frequency of the core haplotype is high, as is the case for SLC45A2. It is also possible

that slight variations in EHH-based methods can determine partial selective sweeps

which the original EHH method cannot (Voight et al., 2006). Lao et al. (2007) also

acknowledged that they identified signatures of positive selection in East Asians,

whereas Izagirre et al. (2006) did not. To explain this, they cite possible SNP

ascertainment bias for SNPs with higher major allele frequencies, thereby affecting the

result of Fst analysis. Further, they also criticise the use of the Fst pairwise method,

which can create a bias towards identifying differences between certain populations.

It is clear from the evidence presented above that many factors influence the detection

of positive selection. The choice of genetic data, the type of statistical method, the

strength of selection and the thresholds of significance will determine if a gene is a

candidate for positive selection. Whilst the studies discussed above support that skin

pigmentation has not evolved neutrally, but has evolved through positive selection and

convergently in European and Asian populations, they differ in the specific genes

28

involved. However, sufficient evidence has been provided for the role of SLC45A2 in

the positive selection of pigmentation. Evidence suggests that SLC45A2 is involved in

determining pigmentation difference between major continental populations as well as

within a population. Genetic changes in SLC45A2 have likely been a major driving

force for the evolution of light skin pigmentation in European populations. Therefore,

functional polymorphisms in SLC45A2 are likely to contribute to the major differences

between geographical populations and the more subtle variation within Europeans.

2.5 Genetics of human pigmentation dysfunction

Although the current study is focussed on the genetic basis of normal human

pigmentation variation, genes known to cause abnormal pigmentation are likely

candidates to play important roles in normal pigmentation variation. This is thought to

be possible, in part, through allelic strength variation in pigmentary-dysfunction loci

(Rees, 2003; Sturm and Frudakis, 2004). Using a comparative genomics approach,

model organisms such as the mouse, has allowed the in-depth investigation of

pigmentation dysfunction. In turn, much of our knowledge in relation to human

pigmentary disorders and normal human pigmentation has been derived from these

investigations.

Tomita and Suzuki (2004) formalised the classification of pigmentation disorders into

the following types: 1) disorders of melanoblast migration in the embryo from the

neural crest to the skin e.g. Piebaldism and Waardenburg Syndrome types 1–4 (WS1–

4); 2) disorders of melanosome formation in the melanocyte e.g. Hermansky-Pudlak

Syndrome types 1–7 (HPS1–7) and Chediak-Higashi Syndrome 1 (CHS1); 3) disorders

of melanin synthesis in melanosomes e.g. Oculocutaneous Albinism types 1–4 (OCA1–

4); and 4) disorders of mature melanosome transfer to the tips of the dendrites e.g.

Griscelli Syndrome types 1–3 (GS1–3) (Tomita and Suzuki, 2004). It is likely and

indeed our hypothesis, that other, non-pathogenic mutations or polymorphisms within

these genes have a more subtle effect on pigmentation and are able to contribute to the

normal variation seen in human populations. In this section, the genes involved in the

more common congenital hypopigmentary disorders relating to dysfunction of

melanogenesis, such as oculocutaneous albinism, will be reviewed with an emphasis on

the affects of polymorphic variation. The other pigmentary disorders have been

excluded from this literature review based on their rarity and their high degree of non-

29

pigmentary co-morbidity. Later in this literature review, some of these same genes and

their associated polymorphic variation will be discussed with reference to their role in

normal pigmentation.

2.5.1 Oculocutaneous albinism (OCA)

OCA is clinically and genetically heterogenous often making diagnosis complicated.

Four types of OCA are recognised based on the expression of mutations in four

different genes, TYR, P, TYRP1 and SLC45A2. Mutations in these genes prevent the

correct sorting and trafficking of their respective proteins, which ultimately prevents the

maturation of melanosomes and the production of melanin (Figure 8). In this section,

each form of OCA (types 1–4) will be discussed in relation to the causative gene’s role

in melanogenesis, the impact of mutations on its function, and how this provides an

insight into normal human pigmentation.

2.5.1.1 OCA Type I and Tyrosinase (TYR)

Tyrosinase has been coined the “key player” in melanogenesis as it is the first enzyme

in the pathway of melanin production (Figure 1). Tyrosinase catalyses the hydroxylation

of tyrosine to 3, 4 dihydroxyphenylalanine (DOPA) as well as the subsequent DOPA to

DOPAquinone oxidation step (Reviewed in Spritz, 1994). There are at least 189

mutations in TYR that cause oculocutaneous albinism type 1 (OCA1) (Online Mendelian

Inheritance in Man, OMIM: #203100) which is characterised by hypopigmentation of

the skin, hair, and eyes. Of the 189 TYR mutations attributable to OCA1, 148 are

missense or nonsense mutations, 23 are small deletions and the remainder are other

types of mutations (Ray et al., 2007). In OCA1, the mutant tyrosinase protein is not

transported to the melanosomes as it is retained in the endoplasmic reticulum (Figure 8)

(Halaban et al., 2000; Toyofuku et al., 2001a; Toyofuku et al., 2001b). The prevalence

of OCA1 is approximately 1 in 40,000 in most populations and accounts for

approximately 40% of OCA worldwide (King et al., 2000; Newton et al., 2001). Two

clinically distinct types of OCA1 exist depending on the effect of the TYR mutations

(Oetting et al., 1998a). The classical OCA1 phenotype, termed OCA1A (OMIM:

#203100) or Tyrosinase-negative, is caused by mutations that completely inactivate the

tyrosinase enzyme (King and Witkop, 1977; Claudy and Ortonne, 1982; Tomita et al.,

1989; Giebel et al., 1990; Oetting and King, 1992). A lack of tyrosinase activity leads to

a complete lack of melanin synthesis, an inability to tan, as well as reduced visual

30

acuity. However, some TYR mutations lead to a reduction in TYR activity (Tripathi et

al., 1992; Toyofuku et al., 2001a) allowing the accumulation of minimal to moderate

levels of cutaneous and ocular melanin. This condition is termed OCA1B (OMIM:

#606952). The ocular abnormalities associated with OCA1 are common to both

OCA1A and OCA1B and the severity of reduced visual acuity is associated with the

degree of hypopigmentation (Summers and King, 1994; Summers, 1996).

Figure 8. Melanosomal protein trafficking and types of OCA. Mutations in TYR, P,

TYRP1 and SLC45A2 are involved in the aberrant processing of melanosomal proteins

from the endoplasmic reticulum (ER). In OCA1 and OCA3, tyrosinase (TYR) is

disrupted at the level of the ER and at the post-Golgi level for OCA2. In OCA4, the

normal routing of Tyr, Tyrp1 and Dct (Tyrp2) is disrupted causing abnormal secretion

out of the cell leading to a hypopigmented phenotype.Source:Costin et al., (2003)

Tyrosinase belongs to a family of closely related, melanocyte-specific proteins with two

other known members, namely tyrosinase-related proteins 1 and 2 (TYRP1, TYRP2 or

DCT). Tyrosinase is believed to exist in a multienzyme complex that acts to regulate the

activity of other genes. Termed the “melanogenic complex”, the high molecular weight

complex consists of TYR, TYRP1, DCT (TYRP2) and OCA2 (P) (Orlow et al., 1994;

Lamoreux et al., 1995). Although this hypothesis remains unconfirmed in humans,

increasing evidence suggests that these proteins are highly dependant upon each other in

31

the mouse (Chen et al., 2002), and therefore may act similarly in humans. The

tyrosinase gene is located on chromosome 11q14–21 and spans over 118 kb with five

exons and four introns. Exons range in size from 1,321 bp for exon 1 to 148 bp for exon

3 and introns range in size from 10 kb to over 30 kb (Kwon et al., 1987; Barton et al.,

1988; Giebel et al., 1991a; Ponnazhagan et al., 1994). The gene transcript is 2,384 bp

long and encodes a 529 amino acid enzyme. The tyrosinase enzyme contains an 18

amino acid signal peptide, two copper binding regions which serve as the active sites, a

transmembrane region at the carboxyl end, and an epidermal growth factor-like motif

(Reviewed in Oetting, 2000). Tyrosinase is a copper-containing enzyme that requires

ATP7A, a copper-transporting P-type ATPase, for its activation (Petris et al., 2000).

2.5.1.2 OCA Type II and OCA2 (P)

The human OCA2 gene is the homologue of the murine pink-eyed dilution gene (p)

(Ramsay et al., 1992; Rinchik et al., 1993). It spans 250–600 kb, encodes 25 exons (one

is non-coding) and has been mapped to chromosome 15q11.2–12 (Ramsay et al., 1992;

Rinchik et al., 1993; Lee et al., 1995). Much of the characterisation of this gene has

been done in the mouse homologue of OCA2 (referred to as‘p’). Both the human and

mouse proteins are predicted to have 12 transmembrane domains spanning the

melanosomal membrane, although their function is still debated (Gardner et al., 1992;

Rinchik et al., 1993; Rosemblat et al., 1994). Several functions have been proposed for

the p protein, including a possible role as a tyrosine transporter (Sidman et al., 1965;

Rosemblat et al., 1998). However, studies have shown that this is unlikely since no

significant difference in the rate of tyrosine uptake between wild-type and p-null

melanocytes was observed (Gahl et al., 1995; Potterf et al., 1998).

OCA type 2 (or OCA2) (OMIM: +203200) is a Tyrosinase-positive form of albinism in

which mutations in the causative gene indirectly affect the function of melanogenic

enzymes. OCA2 is characterised by hypopigmentation of the skin, hair, and eyes with

pigmentation darkening with increased age (Nordlund et al., 1998). People with OCA2

have a potentially fully functional Tyrosinase enzyme, but its activity is reduced as a

consequence of OCA2 mutations, which ultimately causes a reduction in melanogenesis.

OCA2 is the most prevalent form of oculocutaneous albinism with an estimated

worldwide prevalence of 1 per 20,000 which accounts for approximately half of all

known OCA (Newton et al., 2001; Oetting et al., 2005). The prevalence of OCA2 in the

32

European American population is 1 per 36,000, although the African American

population has a higher prevalence of 1 per 10–15,000 (Witkop, 1989; Lee et al., 1994).

OCA2 is more common in indigenous African populations with a prevalence of 1 per

3,900 in South Africans (Kromberg and Jenkins, 1982), 1 per 1,400 in Tanzanians

(Spritz et al., 1995), 1 per 1,100 in the Ibo of Nigeria (Okoro, 1975) and 1 per 2,833 in

Zimbabweans (Kagore and Lund, 1995). A similarly high prevalence of OCA2 has been

identified in a Tonga population in Zimbabwe (1 per 1000) (Lund et al., 1997). The

prevalence of OCA2 has also been reported in a Native American Indian Navajo

population (between 1 per 1,500 and 1 per 2,000) (Yi et al., 2003) and other

indigenous populations in North and South America (1 per 28 to 1 per 6,500)

(Reviewed in Woolf, 2005). A much lower prevalence is reported in Japan where OCA2

is responsible for about 8% of oculocutaneous albinism (Suzuki et al., 2003).

The clinical manifestations of OCA2 gene mutations are broad, however, two main

phenotypes of OCA2 are associated with mutations in the OCA2 gene. The more

extreme phenotype is characterised by yellow to orange hair, white skin and blue-hazel

eyes in Caucasians, Africans, and African Americans (King et al., 2000). A milder

phenotype resulting from mutations in the OCA2 gene is seen in Africans and African

Americans and is known as Brown OCA (BOCA). Affected BOCA individuals have

cream to light tan skin, beige to light brown hair, and blue-green to brown irides

(Witkop et al., 1972; King et al., 1978; King et al., 1980; King et al., 1985; Manga et

al., 2001). A subclinical form of OCA2 has been described in a Japanese patient who

had a heterozygous mutation (A481T) common in the normally pigmented Japanese

population (Kawai et al., 2005). The mild phenotype (very pale skin but brown hair and

eyes) resulted in the patient not being diagnosed with OCA2 until being severely

sunburned and referred to a specialist.

A 2.7 kb deletion in OCA2 has been shown to account for the majority of OCA2 cases

seen in South Africa, Tanzania, Cameroon, Zimbabwe and other regions of sub-Saharan

Africa (Durham-Pierre et al., 1994; Spritz et al., 1995; Stevens et al., 1995; Lund et al.,

1997; Puri et al., 1997). The same deletion accounts for 25–50% of mutant OCA2

alleles in African-Americans (Durham-Pierre et al., 1996). However, the 2.7 kb deletion

is rare in German albino patients and is usually present only due to African ancestry.

Instead, a large number of different OCA2 gene mutations are responsible for OCA2,

33

which represents about 22% of albinism cases in Germany (Passmore et al., 1999). A

Navajo-specific, 122.5 kb deletion in OCA2, results in OCA2 in indigenous Navajo

Americans but not in other native American populations (Yi et al., 2003). Other

mutations, occurring at low frequencies, have been found in the OCA2 gene that result

in OCA2 (Spritz et al., 1997; Oetting et al., 1998b; Kerr et al., 2000; Kato et al., 2003;

Yi et al., 2003; Oetting et al., 2005). It is likely that more, currently unidentified

mutations, exist in intronic or promoter regions of the OCA2 gene (Kerr et al., 2000).

Unlike mutations in other pigmentation genes, missense mutations in OCA2 do not

seem to cluster in any specific region, although most are confined to regions coding for

the 12 transmembrane domains and the carboxy half of the protein. Mutations in the

melanocortin 1 receptor (MC1R) gene have shown to modify the classic OCA2

phenotype. Instead of the characteristic yellow/orange hair of OCA2, MC1R mutations

were found to modify hair colour to red in OCA2 affected individuals from a variety of

ethnic backgrounds (King et al., 2003). This was the first example of epistasis in human

OCA.

2.5.1.3 OCA Type III and Tyrosinase-related protein I (TYRP1)

A rare form of oculocutaneous albinism, OCA3 (OMIM #203290), is caused by

mutations in the Tyrosinase-related protein I (TYRP1) gene. The precise role of TYRP1

in melanogenesis has been a topic of debate for some time and evidence for a variety of

catalytic functions have been noted in the literature. Much of what is known about the

role of this gene in melanogenesis has come from studies involving the mouse

homologue of TYRP1, Tyrp1. Despite numerous roles proposed for the function of

Tyrp1 (Jackson, 1988; Halaban and Moellmann, 1990; Urabe et al., 1993; Winder et al.,

1994b), it is now generally accepted that Tyrp1 acts as a 5,6-dihydroxyindole-2-

carboxylic acid (DHICA) oxidase within the murine eumelanin biosynthetic pathway

(Figure 1) (Jimenez-Cervantes et al., 1994; Kobayashi et al., 1994a; Kobayashi et al.,

1994b). Based on a high degree of homology between murine and human TYRP1

proteins (approximately 93%), a similar function was proposed for human TYRP1

(Jimenez et al., 1991; Jimenez-Cervantes et al., 1993; Manga et al., 2000). However,

Boissy et al. (1998) did not identify significant DHICA oxidase activity of human

TYRP1 above control levels (Boissy et al., 1998). Their results showed no difference in

DHICA oxidase activity between a melanocyte cell line containing TYRP1 and a cell

line that completely lacked TYRP1 expression. Further investigation of the role of Tyrp1

34

revealed it may also act as a tyrosine hydroxylase (tyrosinase activity) (Jimenez et al.,

1991; Boissy et al., 1996), but at a reduced level compared to tyrosinase. An earlier

study contradicted these findings showing that Tyrp1 protein did not have detectable

levels of tyrosinase activity (Muller et al., 1988).

Tyrosinase, Tyrp1, and Dct (Tyrp2) exist in a multimeric complex known as the

melanogenic complex (Orlow et al., 1994; Winder et al., 1994a). Tyrp1 has been shown

to play an important role in stabilising Tyr within this complex, which may indirectly

affect melanogenesis (Kobayashi et al., 1994b; Kobayashi et al., 1998; Manga et al.,

2000). Kobayashi et al. (1998) showed that Tyr was degraded more quickly in

melanocyte cells with mutant Tyrp1 than in melanocytes that had a wild-type Tyrp1.

This study concluded that the principal function of Tyrp1 may be to stabilise Tyr in

melanocytes and therefore may play a key role in regulation of basal melanin

production. Therefore, due to the decreased stability of human TYR, TYRP1 may play a

more active and essential role in regulation of human pigmentation (Kobayashi et al.,

1998). It was shown that mutations in Tyrp1 affect the retention and maturation of Tyr

from the endoplasmic reticulum, thereby slowing its transport to the melanosome

(Figure 8) (Toyofuku et al., 2001b).

OCA3 was initially diagnosed in an African-American person exhibiting the phenotype

of Brown OCA, who had a single base pair deletion in exon six of TYRP1(Boissy et al.,

1996). The deletion of an adenosine (A) base at codon 368 caused a premature stop

codon at codon 384 severely truncating the 537-residue TYRP1 protein and

consequently deregulated the activity of tyrosinase (Boissy et al., 1996). However, it

was later suggested that the brown OCA phenotype identified by Boissy et al. (1996)

was actually attributable to the 2.7 kb deletion mutation in the OCA2 gene and

background mutations or mixed ancestry were responsible for the BOCA phenotype

(Manga et al., 2001). This phenomenon highlights the heterogenous nature of OCA and

indeed pigmentation in humans.

A type of albinism in African populations, previously known as Rufous OCA (ROCA),

has been attributed to mutations in TYRP1(Manga et al., 1997). ROCA is clinically

diagnosed in African people with red-bronze skin colour, ginger-red hair, and blue or

brown irises. The 368delA mutation mentioned above as well as another nonsense

35

mutation (S166X) have been identified in a South African black population with OCA3

(Manga et al., 1997). The frequency of OCA3 (ROCA) in this population was 1 per

8,500.

OCA3 was thought to exist only in African populations. However, the first Caucasian

OCA3 mutation (R321Q) was inconspicuously reported by personal communication in

a review of TYRP1 and OCA3 (Sarangarajan and Boissy, 2001). This review article

reported unpublished data, which identified three OCA individuals with a R321Q

mutation in TYRP1, who had no other mutations in either TYR or OCA2. No further

mention of this mutation has been made in the literature. As not all albinism genes were

screened, it is possible that the OCA phenotype was due to mutations in SLC45A2

which underlies the fourth type of OCA. It has been proposed that OCA3 does exist in

Caucasian populations, but may simply not be getting diagnosed because the degree of

hypopigmentation is within the normal range of pigmentation variation (Tomita and

Suzuki, 2004). Since then, a non-African mutation (R373X nonsense mutation) in a

Pakistani family with OCA has been published (Forshew et al., 2005). This study also

confirmed the milder phenotype of TYRP1 mutations. Recently, a German patient with

OCA was identified with two TYRP1 mutations (R356E and L36X) (Rooryck et al.,

2006).

Attempts to clone the tyrosinase gene resulted in the identification of TYRP1 which is

the human homologue of the murine brown locus (Shibahara et al., 1986; Jackson,

1988; Cohen et al., 1990). Human TYRP1 is located on chromosome 9p23 (Murty et al.,

1992) and encodes eight exons spanning 24 kb with the first exon being non-coding

(Box et al., 1998). TYRP1 protein is a type 1 membrane glycoprotein comprised of 537

amino acids with a molecular weight of 75 kDa (Halaban and Moellmann, 1990;

Vijayasaradhi and Houghton, 1991). TYRP1 protein shows conserved regions of

homology with TYR including two copper-binding regions, two cysteine-rich domains,

a transmembrane domain, a signal peptide sequence, and 6 glycosylation sites (Hearing

and Jimenez, 1989).

Regulation of TYRP1 expression is complex with many known positive and negative

transcriptional regulators identified upstream of the transcription initiation site

(Lowings et al., 1992). Central to the transcription of TYRP1 is binding of the

36

microphthalmia-associated transcription factor (MITF), a basic helix-loop-helix

transcription factor, to the M-box transcription element in the TYRP1 promoter (Aksan

and Goding, 1998; Goding, 2000). Expression can be down regulated by negative

regulatory factors, even in the presence of MITF (Fang and Setaluri, 1999). Two other

melanocyte-specific elements (MSE), MSEu and MSEi, also regulate TYRP1 expression

in a negative manner due to the binding of the brachyury-related transcription factor

(Tbx2). Binding of Pax3 to these elements promotes up-regulation of TYRP1(Carreira

et al., 1998; Galibert et al., 1999).

2.5.1.4 OCA Type IV and solute carrier family 45 member 2 (SLC45A2)

OCA4 (OMIM #606574) is the most recent form of OCA to be identified and was first

reported in a patient of Turkish descent with the causal mutation determined to be in the

membrane associated transporter protein (MATP) gene (Newton et al., 2001). This

gene was first known as antigen in melanoma (AIM1) before being named MATP and

undergoing another name change to solute carrier family 45 member 2 (SLC45A2).

Currently, SLC45A2 is not believed to play a specific enzymatic role in melanogenesis

but is important for the correct processing and trafficking of TYR, TYRP1 and DCT

(TYRP2) and therefore affects melanogenesis indirectly (Costin et al., 2003). The

function of SLC45A2 will be discussed later in this section.

Hypopigmented mice have provided a valuable tool in attempting to understand the role

pigmentation genes play in humans. Much of the research on OCA4 has used a mouse

model to understand the role that SLC45A2 plays in human albinism and human

pigmentation variation. Mutations in murine Slc45a2, previously named the underwhite

(uw) locus, lead to generalised hypopigmentation of the eyes and fur by significantly

reducing or abolishing tyrosinase activity and melanin production (Sweet et al., 1998;

Lehman et al., 2000). A range of phenotypes is observed in the mouse based on the

presence of different Slc45a2 mutations as well as the genetic background of the mouse.

Four major alleles have been described, although only three are currently available from

Jackson mouse laboratories. The first mutant allele, simply known as underwhite (uw),

was identified in the 1960s (Dickie, 1964). The homozygote of the uw allele is

characterised by a white coat colour and an age-dependent accumulation of

pigmentation in the eyes resulting in a dark reddish colour by adulthood (Figure 9)

(Sweet et al., 1998). The uw allele is the most phenotypically severe of the underwhite

37

allele series. It is a 7 bp deletion in exon 3 which causes a 43 amino acid frameshift and

a premature stop at codon 308 (Du and Fisher, 2002). The underwhite dense (uwd) allele

is a T to C SNP in exon 6, which results in a serine to proline substitution in the tenth

transmembrane domain of the protein. The uwd/ uwd mouse has light eyes at birth but by

adulthood the eyes are a dark ruby colour. On a nonagouti background the coat colour is

dark grey (Figure 9). The adult homozygous underwhite-intense mouse has a slightly

darker eye (not visible in Figure 9) and coat colour than homozygous underwhite mice

(Figure 9). Unfortunately, this mouse mutant was lost and the causal mutation has not

been identified (Sweet et al., 1998). The semidominant Matp allele, dominant brown

(uwDbr), is caused by a G to A polymorphism in exon 2 resulting in a missense mutation

(p.Asp153Asn) that alters the fourth transmembrane domain (Sweet et al., 1998; Du and

Fisher, 2002). This same mutation results in OCA4 in humans and is also responsible

for the cream coat colour in the horse (Mariat et al., 2003). On a nonagouti background,

homozygous uwDbr mice are light beige and heterozygotes are dark brown and eye

colour again is absent at birth but darkens with age (Figure 9) (Cook and Davisson,

1993).

Figure 9. Coat colours of underwhite mutant mice.From left to right: +/ uwd,

uwd/uwd, uwDbr/uwDbr, +/uwDbr, uw/uw, two uwderwhite-intense.Source:Sweet et al., (1998)

OCA4 in humans is rare. Since the first report of OCA4 in a person of Turkish descent

in 2001 (Newton et al., 2001), OCA4 has been reported in a German population

(Rundshagen et al., 2004), Japanese populations (Inagaki et al., 2004; Inagaki et al.,

2005; Inagaki et al., 2006), a Korean population (Suzuki et al., 2005) and Indian

38

populations (Sengupta et al., 2007). So far, at least 28 pathological mutations have been

identified in SLC45A2(Newton et al., 2001; Inagaki et al., 2004; Rundshagen et al.,

2004; Suzuki et al., 2005; Inagaki et al., 2006; Sengupta et al., 2007). OCA4 represents

the most common form of tyrosinase-positive OCA in Japan, with approximately 24%

of all albinism in Japan due to SLC45A2 mutations. The clinical phenotype of OCA4 in

these populations shows a high degree of heterogeneity. Phenotypes range from white

hair, blue irides, and presence of nystagmus to brown/black hair, brown irides, and no

nystagmus (Inagaki et al., 2004). Some individuals will accumulate pigmentation with

age whilst others remain completely hypopigmented throughout life. OCA4 may be

misdiagnosed as the clinical presentation is within the phenotypic range of OCA2

(Newton et al., 2001).

Figure 10. Predicted membrane topology of the human SLC45A2 protein.Using

several programs, such as MacVector, TMHMM (v. 2.0) and TopPred 2, the ~58 kD

human SLC45A2 protein was predicted to span the melanosomal membrane 12 times. Source: Newton et al., (2001)

SLC45A2 is located on chromosome 5p and has 7 exons spanning 40 kb. The predicted

protein shares 82% identity with the mouse Slc45a2 protein and is predicted to have 12

transmembrane domains (Newton et al., 2001) (Figure 10). Of the 24 known

pathological mutations, 16 are missense mutations and are located within or nearby to

the predicted transmembrane domains. Two transcripts have been identified by

39

Northern blot, 1.7 kb and 2.8 kb respectively, with the latter transcript most likely

resulting from an alternate splicing event (Harada et al., 2001). The 530 amino acid

human SLC45A2 protein is expressed in numerous melanoma and melanocyte cell lines

(F002-S mel, 1300 mel, 888 mel, 1199 mel, 1383 mel, 1280 mel, 1376 mel and 8F0366

melanocyte) but not at significant levels in 15 different normal tissues investigated

(Harada et al., 2001).

The exact role of SLC45A2 in human pigmentation remains largely unknown. Initially,

comparative protein sequence analysis identified similarities between SLC45A2 and

plant sucrose/proton symporters (Fukamachi et al., 2001). Although there are no known

sucrose transporters in mammals, it was considered feasible that SLC45A2 could co-

transport a sugar molecule with a proton (Fukamachi et al., 2001; Newton et al., 2001).

Slc45a2 mutations in the medaka fish have shown to manifest within the melanosome

(Hirose and Matsumoto, 1993) and therefore Fukumachi et al. (2001) proposed that

SLC45A2 might be a component of the melanosomal membrane. The possible function

of SLC45A2 as a proton transporter has outlined a possible relationship with the OCA2

protein, which has been suggested as an anion transporter in the melanosome membrane

(Puri et al., 2000). If SLC45A2 is similarly localised to the melanosomal membrane as

suggested above, it may act in conjunction with OCA2 protein to regulate the pH of the

melanosome (Newton et al., 2001). The importance of pH in the regulation of

melanogenesis is described briefly in sections 2.6.1.1 and 2.6.1.2.

Study of the mouse model of OCA4 has shown that Slc45a2 plays a crucial role in the

processing and intracellular trafficking of melanosomal proteins (Costin et al., 2003). In

this study, Costin and her colleagues established primary melanocyte cultures from

wild-type mice and mice carrying the uw/uw mutation. After centrifugation of media

from both cell lines, medium from uw/uw cells showed pigmented vesicles were

released in to the medium. Although these vesicles contained Tyrosinase, Tyrp1 and

Dct (Tyrp2), some of these proteins were retained in the melanocyte, which explains the

gradual accumulation of pigmentation associated with OCA4. Tyrosinase activity and

stability in uw/uw melanocyte extracts was significantly lower than in wild-type

melanocyte extracts and total melanin was negligible in uw/uw melanocytes. However,

through the use of metabolic labelling and Western-blotting, tyrosinase was transcribed

and translated without defect in both cell lines. Costin et al. (2003) used

40

immunohistochemical staining and confocal microscopy to investigate the subcellular

localisation of Tyrosinase, Tyrp1 and Dct (Tyrp2) melanogenic proteins in the

underwhite mutant melanocytes. The melanogenic proteins were shown to reach the

endoplasmic reticulum but were not delivered to the melanosomal compartment

suggesting a defect later in the secretory pathway. Costin et al. (2003) concluded that

the function of SLC45A2 involves the sorting of these three melanogenic proteins from

the trans-Golgi network to Stage II melanosomes (Figure 8). To date, this study has

provided the best evidence for the role of SLC45A2 in human pigmentation.

2.6 Genetics of normal pigmentation variation

When endeavouring to identify genes involved in normal pigmentation variation, there

are two obvious approaches. As discussed in section 2.5 above, investigating those

genes known to be involved in pathological pigment changes in both humans and model

organisms such as the mouse or fish, would be logical. Another good starting point,

given the noted inter-population variation in human pigmentation, is to identify genes

that show large allele frequency differences between populations which differ

substantially in average pigmentation levels. Other genes have received a degree of

research attention due to their link with an increased risk of developing skin or other

cancers (Bliss et al., 1995; Valverde et al., 1996; Box et al., 2001; Sturm, 2002; Sturm

et al., 2003a; Debniak et al., 2006). Investigating these genes has substantially added to

our knowledge of the genetic basis of normal pigmentation variation as well as their

role in cancer.

Over 120 loci have been implicated in normal pigmentation variation, which have been

identified via the above-mentioned rationales (Bennett and Lamoreux, 2003).

Considering this large number of contributing genes, the following review focuses only

on the major contributors or genes that have been studied more extensively, along with

the “albinism” genes. These genes will be discussed with particular reference to the

results of genetic association studies and the functional consequence of non-pathogenic

mutations.

41

2.6.1 Albinism genes involved in normal pigmentation variation

2.6.1.1 Tyrosinase (TYR)

Tyrosinase is the rate-limiting enzyme in melanogenesis and has therefore been

investigated to explain the dramatic difference in skin colour observed between

different populations (Iozumi et al., 1993; Alaluf et al., 2003). To explain the

differences in melanin content of hair and skin, several studies have investigated the

levels of tyrosinase activity, synthesis and mRNA levels. Tyrosinase activity levels can

be as much as 10-fold higher in melanocytes derived from dark skin, than in those from

lightly pigmented Caucasian melanocytes (Iozumi et al., 1993). Although

transcriptional regulation of tyrosinase is important for its expression, tyrosinase-

specific mRNA levels do not correlate with melanin content or tyrosinase activity

(Naeyaert et al., 1991). Variations in tyrosinase abundance have been shown to account

for differences in tyrosinase activity (Halaban et al., 1983; Iwata et al., 1990; Burchill et

al., 1991; Abdel-Malek et al., 1993). However, other studies have shown that variations

in tyrosinase activity are not due to enzyme abundance (Fuller et al., 1993; Iozumi et al.,

1993), suggesting a role for post-translational mechanisms.

An alternative explanation for ethnic variation has been proposed that involves

regulation of TYR activity through the regulation of melanosomal pH (Ancans et al.,

2001; Fuller et al., 2001; Watabe et al., 2004). Data exists that supports a model of high

pH in Caucasian melanosomes resulting in an almost inactive TYR and a neutral pH in

Negroid melanosomes where TYR is fully active (Fuller et al., 2001). Optimum pH

levels for tyrosinase activity in both Negroid and Caucasian melanocyte cell lines, was

determined to be above seven (Fuller et al., 2001).

The TYR gene is highly polymorphic and has been suggested to explain pigmentation

differences between populations. However, the majority of the 534 polymorphisms

recorded in the database are associated with OCA1 and relatively few are non-

pathogenic. The non-pathogenic polymorphisms, such as Y192S (rs1042602) and

R402Q (rs1126809), have allele frequency information for several different population

groups. Early studies suggested that amino acid and DNA sequences for tyrosinase were

identical in black and Caucasian skin types (Giebel et al., 1991b; Spritz et al., 1991;

Fuller et al., 2001), further supporting the role of post-translational control. However,

42

the availability of NCBI SNP database data for African and Caucasian populations

show that this is not the case. Small-scale genotyping studies showed that the

aforementioned non-pathogenic polymorphisms were present in all but Asian

populations (Giebel and Spritz, 1990; Tripathi et al., 1991; Johnston et al., 1992). The

NCBI SNP database confirms this and also shows that the Y192S and R402Q

polymorphisms are present at low frequency in African populations. The Y192S has

since been shown to have a substantial effect on the skin pigmentation differences

between populations of European and West African ancestry (Shriver et al., 2003).

Polymorphisms in TYR have been shown to be associated with intra-population skin

pigmentation. Specifically, the Y192S polymorphism is associated with skin colour in

African-American and African-Caribbean populations, even after adjustment for

admixture proportions(Hoggart et al., 2003). A recent study has reported an association

between Y192S and normal pigmentation variation within a population group

(Stokowski et al., 2007). Using a genomewide association approach, this study showed

that the Y192S polymorphism is associated with skin colour in a South Asian

population. Although non-pathogenic TYR polymorphisms are present in high frequency

in European populations, no reports of intra-population pigmentation variation

attributable to Y192S have been reported.

2.6.1.2 OCA2 (P)

Mutations in the OCA2 gene (also known as the P gene) underlie the most common

form of albinism (Oetting et al., 1998b) and is thought to affect melanogenesis by

regulating intramelanosomal pH levels. A model for OCA2 protein function has been

proposed based on the role that the murine OCA2 protein (known as p) plays in

regulating pH in the melanosome (Brilliant, 2001). The p protein is thought to play a

role in the alkalisation of melanosomal pH creating an environment for optimal

tyrosinase activity based on the observation that when p is absent the pH of

melanosomes drops thereby inactivating tyrosinase and inhibiting melanogenesis (Fuller

et al., 2001; Chen et al., 2002). Contrary to this are the results seen by Puri et al. (2000)

in which the p protein was shown to play a key role in the acidification of melanosomes.

This study showed a near absence of acidic melanosomes in p mutant melanocytes as

compared with wild-type melanocytes (Puri et al., 2000). The acidic nature of

melanosomes has also been seen in another study in which the pH of wild-type

43

melanosomes was determined to be 3.0–4.6 (Bhatnagar et al., 1993). In light of this

information it seems more likely, based on the recent findings that OCA2 gene

mutations decrease the likelihood of having lighter coloured eyes (Rebbeck et al.,

2002), that the pH of human melanosomes is acidic. Therefore, mutations in OCA2

would result in a more alkaline environment, which is more conducive to optimum TYR

activity. The p protein is also known to affect the trafficking of melanosomal proteins

(Manga, 2001) and recent studies have shown that the trafficking of Tyr and Tyrp1 are

altered in p-null melanocytes (Chen et al., 2002).

The human OCA2 gene is large (25 exons spanning 350–600 kb) and over 1,400

polymorphisms have been registered in the NCBI SNP database for this gene. Of these,

18 are non-synonymous, 17 are synonymous and the majority are intronic. At least 52

OCA2 gene mutations lead to OCA2 (Oetting and Bennett, 2003; Hongyi et al., 2007)

while a further 42 non-pathogenic mutations, 22 of which are exonic, have been

reported in the literature (Oetting and Bennett, 2003; Duffy et al., 2007). The OCA2

gene is a good candidate to explain normal pigmentation variation (Sturm and Frudakis,

2004) based on genetic studies which reveal allele frequency differences between

populations with different pigmentation characteristics (Shriver et al., 2003). OCA2

gene variants are believed to account for approximately 15% of skin pigmentation

variation between populations, based on measurements of skin reflectance (Brilliant et

al., 2005). One early study (Lee et al., 1995) characterised 28 non-pathogenic

polymorphisms in Caucasian, African American, indigenous African, Asian, Middle

Eastern, and Indo-Pakistani samples. This study observed genetic variation between the

population groups for several synonymous and non-synonymous polymorphisms. In

particular, an arginine to tryptophan amino acid substitution at position 305 (R305W;

rs1800401) showed significantly different frequencies such that the W coding allele was

seen at a rate of 0.83 in the Caucasian population compared with 0.10 in black skinned

people (Lee et al., 1995). Another OCA2 gene polymorphism (A355A; rs1800404) has

also showed a dramatic difference in genotype frequency between a Nigerian (Ibadan)

African population (0.115) and four European populations (0.746; German, Spanish,

English, Irish) (Shriver et al., 2003). This SNP has been included in a panel of SNPs,

which can enable the inference of ancestry.

44

Epistatic interactions between the OCA2 gene and other pigmentation genes can affect

within-population pigmentation variation. An OCA2 polymorphism, located 15 bp

upstream of the acceptor splice site in intron 13 (IVS13–15), was shown to interact with

a melanocortin 1 receptor polymorphism (V92M). Using a gene-gene interaction

model, the combination of genotypes for these two SNPs could explain some of the

inter-individual variation in skin pigmentation within a Tibetan population (Lee et al.,

1995; Akey et al., 2001). However, this study did not provide any functional evidence

to explain the observed statistical interaction, but proposed that the physical interaction

may reside within the intracellular signalling pathway.

The OCA2 gene is also a strong candidate for determining human hair and eye colour

(Frudakis et al., 2003; Zhu et al., 2004; Brilliant et al., 2005; Frudakis et al., 2007;

Branicki et al., 2008). It is co-located within a genomic region in which the putative eye

and hair colour pigmentation genes, brown eye colour 2 (BEY2) and hair colour 3

(HCL3), were initially identified through linkage studies (Eiberg and Mohr, 1996). The

mouse OCA2 was shown to be critical for melanosome biogenesis in ocular

melanocytes and for maintaining the levels of Tyr, Tyrp1 and Dct (Tyrp2) proteins in

the eye (Orlow and Brilliant, 1999). Later, two human polymorphisms (R305W and

R419Q; rs1800407) were shown to be associated with human eye colour variation in a

sample of 629 Caucasians (Rebbeck et al., 2002). Specifically, individuals were less

likely to have blue or grey eyes if they had either of these OCA2 gene variants or in

combination (305W and/or 419Q). The same study also suggested that these OCA2 gene

variants were associated with hair colour; however, the study did not have sufficient

statistical power to draw any convincing conclusions. Further, the R305W

polymorphism along with 12 other OCA2 gene SNPs, have been shown to be strongly

associated with iris pigmentation in a second study (Frudakis et al., 2003). These OCA2

gene SNPs were by far the most significant of the 754 pigmentation and non-

pigmentation gene SNPs genotyped by Frudakis et al. (2003). A recent investigation of

the R305W and R419Q polymorphisms has provided further evidence for their role in

determining eye colour (Branicki et al., 2008). This study used 390 unrelated

individuals of European (polish) ancestry, showing that the R419Q polymorphism was

associated with eye colour in the blue vs non-blue test, and contributes about 4% to eye

colour variation. Specifically, the 419Q allele was most strongly associated with

green/hazel eye colour. This study also showed that the 305W allele was associated

45

with hazel/brown eye colour, supporting results from previous studies (Rebbeck et al.,

2002; Frudakis et al., 2003; Jannot et al., 2005). However, when a Bonferroni

adjustment was applied for multiple testing, significance was no longer obtained for the

305W association.

Data from a recent study highlighted the importance of OCA2 in the determination of

eye colour (Duffy et al., 2007). The complete screen of all 24 coding exons along with

the genotyping of 58 previously-known exonic and tagging OCA2 gene SNPs in 3839

samples revealed the strongest associations were for three intronic SNPs, which

explained 74% of the variation in eye colour (Duffy et al., 2007). Another recent OCA2

gene study has confirmed it as the major predictor of human iris colour (Frudakis et al.,

2007). Using a shotgun-style association scan, 33 SNPs (one non-synonymous, two

synonymous, three 3' untranslated region (UTR) and 27 intronic SNPs), assembled in

multilocus haplotypes (diplotypes), were identified for the prediction of iris colour.

Unlike most previous studies of eye colour, this study used digital photography and in-

silico spectrophotometry to quantify the eumelanin content of each iris, thus providing a

more objective measure of eye colour. A measure of iris colour concordance between

samples sharing the same OCA2 diplotype was used to assess classification accuracy. In

a sample of 1072 samples a 96.3% rate of concordance was achieved.

2.6.1.3 Tyrosinase-related protein 1 (TYRP1)

Although there is evidence that the expression of TYRP1 varies in the skin of people

from different population groups (Maeda et al., 1997; Alaluf et al., 2003; Tadokoro et

al., 2005), very little evidence of polymorphic variation in TYRP1 has been provided to

explain this (Lao et al., 2007). Only small allele frequency differences are noted in the

NCBI SNP database between different population groups and none have been published

in the literature in relation to pigmentation variation.

One of the first attempts to investigate the role of TYRP1 in normal human pigmentation

variation used an RNase mismatch method to screen for polymorphisms in 100

Caucasian people (Box et al., 1998). Only two polymorphisms, a non-synonymous

polymorphism (R87R) and an intron 7 deletion (IVS7–18delA), were detected and these

were tested for an association with hair colour. No associations were identified when

each polymorphism was considered individually. However, when both polymorphisms

46

were considered together, and hair phenotype was grouped into red or non-red

(presumably to increase the power of the analysis), a significant number of IVS7–

18delA homozygotes were observed in the non-red hair colour group. In an analysis of

TYRP1 polymorphisms and eye colour, several haplotypes of TYRP1 polymorphisms

have been associated with blue and brown eye colour (Frudakis et al., 2003). The small

number of associations between TYRP1 polymorphisms and human pigmentation

phenotype, relative to other pigmentation candidate genes, suggest that TYRP1 is not a

major contributor to normal pigmentation variation.

2.6.1.4 Solute carrier family 45 member 2 (SLC45A2)

Non-pathogenic mutations in SLC45A2 have been investigated in the hope of explaining

normal human pigmentation variation as well as population and ancestry inference. Two

polymorphisms, c.814G>A (p.Glu272Lys; NCBI dbSNP rs26722) and c.1122C>G

(p.Phe374Leu; NCBI dbSNP rs16891982), are prime candidates as they show distinct

population frequency differences (Nakayama et al., 2002) (Table 1). A study

investigating these polymorphisms in white South African, Ghanaian, Japanese, and

New Guinea Islander populations showed that the Phe374 allele was only present in the

white South African population and not in the three other, more darkly pigmented

populations (Nakayama et al., 2002). The 272Lys allele was only identified in the

Japanese and New Guinea Islander populations. A subsequent study showed that the

Phe374 allele was not present in a second Japanese population and that a German

population showed similar allele frequencies to that seen in the white South African

population used by Nakayama et al. (2002) (Yuasa et al., 2004). Recently, Yuasa et al.

(2006) continued their work on the p.Phe374Leu polymorphism by genotyping 1649

unrelated individuals from 13 populations of mixed European and Asian ancestry

(Eurasian), and one African population. Again, the Phe374 allele was seen at much

higher frequencies in the more lightly pigmented European populations such as

Germans, Italians and French (Yuasa et al., 2006; Zuhlke et al., 2007). Lower

frequencies of Phe374 were observed in Turkish, Indian, and Asian populations whilst

the Phe374 allele was not detected in an African, Chinese, and another Japanese

population. Yuasa et al. (2006) concluded that the Phe374 allele might be an important

factor in hypopigmentation of Caucasian populations. Other analyses of the

p.Phe374Leu polymorphism in different population groups further highlights the

usefulness of this SNP as an ancestry informative marker and for explaining normal

47

pigmentation variation between population groups (Norton et al., 2007; Soejima and

Koda, 2007).

Table 1. Summary of allele frequencies of p.Glu272Lys and p.Phe374Leu

ALLELE FREQUENCYREFERENCE POPULATION

n Phe374 374Leu Glu272 272Lys

Nakayama et al., (2002) White South African 54 0.890 0.110 1 0African - Ghanaian 50 0 1 1 0Japanese 49 0 1 - presentNew Guinea Islanders 52 0 1 - present

Yuasa et al., (2004) German 93 0.962 0.038 0.968 0.032Japanese 103 0 1 0.617 0.383

Soejima et al., (2006) Chinese 80 0 1 0.500 0.500Sinhalese 54 0 1 0.700 0.300European 101 0.94 0.06 0.970 0.030Xhosans 102 0 1 1 0African - Ghanaian 121 0 1 0.950 0.050

Yuasa et al., (2006) German 241 0.965 0.035 - -French 98 0.893 0.107 - -Italian 97 0.851 0.149 - -Turkish 200 0.615 0.385 - -Indian 51 0.147 0.853 - -Bangladeshi 118 0.059 0.941 - -Khala 173 0.113 0.887 - -Buryat 143 0.115 0.885 - -Chinese – Han 89 0.028 0.972 - -Chinese – Han 119 0 1 - -Chinese – Han 111 0.005 0.995 - -Japanese 87 0 1 - -Indonesian 105 0.005 0.995 - -African 17 0 1 - -

Unpublished data (available at http://www.mostgene.org/Brilliant.pdf) which were

presented at the 19th International Pigment Cell Conference (IPCC) in 2005, implicates

p.Phe374Leu and other polymorphisms in skin, hair, and eye colour variation (Brilliant

et al., 2005). In this study, about 800 individuals from a diverse range of population

groups were genotyped for 48 polymorphisms located in 17 different genes. The genetic

variance of SLC45A2 was shown to account for 32.1% of variance in skin reflectance,

57.8% of variance in total melanin content of hair, 25.3% of eumelanin/pheomelanin

ratio variance in hair, and 5.7% of variance in eye colour.

48

To date, little evidence is available showing that SLC45A2 variants are responsible for

normal pigmentation variation within a population. One unpublished example,

presented on a poster at the 19th IPCC in 2005 by researchers from the multinational

company Unilever, showed that the Phe374 allele is more common in lighter skin types

in Southern Asian populations from India, Bangladesh, Pakistan and Sri Lanka (Ginger

et al., 2005). These results have not, subsequently, been published in a peer-reviewed

journal. At the September 2006 meeting of the European Society of Pigment Cell

Research, the same researchers confirmed the importance of this polymorphism in

South Asian skin colour variation using whole genome SNP association (Fereday et al.,

2006). The only published study to provide any evidence that SLC45A2 polymorphisms

are involved in Caucasian pigmentation variation was carried out by Frudakis et al.

(2003). This study showed that two intronic SNPs (rs35391 and rs40132) and one

missense polymorphism (rs276722; p.Glu272Lys) were associated with iris colour in a

Caucasian population (Frudakis et al., 2003).

Unfortunately, no data currently exists which sheds any light on the functional

consequence of SLC45A2 polymorphisms in normal pigmentation. Further

characterisation of the p.Phe374Leu polymorphism and SLC45A2 polymorphisms in

other regions of the gene, will provide further insights into the role that this important

pigmentation gene has in determining normal pigmentation phenotypes.

2.6.2 Other genes involved in normal pigmentation variation

2.6.2.1 Melanocortin 1 receptor (MC1R)

The melanocortin 1 receptor gene is highly polymorphic (Rana et al., 1999; Harding et

al., 2000) and is the most characterised of all pigmentation genes with respect to normal

pigmentation. The scope of this literature review does not allow for a comprehensive

review of the extensive research conducted into MC1R. Therefore, this section will

briefly discuss the function of the MC1R and highlight some of the more common and

functionally relevant polymorphisms relating to normal pigmentation variation.

Specifically, the alleles involved in the red hair colour (RHC) phenotype (i.e. red hair

and fair skin) will be discussed in more detail. A number of good reports have

summarised the role of MC1R in human pigmentation (Rees, 2003), the highly

49

polymorphic nature of MC1R(Makova and Norton, 2005; Gerstenblith et al., 2007) and

the functional consequence of MC1R polymorphisms (Garcia-Borron et al., 2005;

Beaumont et al., 2007).

The melanocortin signalling family consists of five receptors, MC1R – MC5R,

representing a family of G-protein coupled receptors (GPCRs) that contain seven

transmembrane domains. The name ‘melanocortin’ refers to the group of ligands that

the receptors bind. These structurally-related ligand peptides are all derived from the

common precursor, pro-opiomelanocortin (POMC) and include adrenocorticotropic

hormone (ACTH), and α-, β- and γ-melanocyte-stimulating hormones (MSHs) (Cone et

al., 1996; Adan and Gispen, 1997; Rees and Healy, 1997; Slominski et al., 2000).

MC1R is a cell-surface receptor which is primarily expressed on melanocytes and

melanoma cells but is also expressed on many other types of cells and tissues, including

keratinocytes, fibroblasts, testis, pituitary, endothelial cells, osteoclasts and others

(Reviewed in Chhajlani, 1996; Garcia-Borron et al., 2005; Roberts et al., 2006). MC1R

preferentially binds α-MSH and ACTH, with β-MSH and γ-MSH showing less affinity

respectively (Reviewed in Gantz and Fong, 2003). Upon binding of α-MSH (or ACTH,

β-MSH) to MC1R, the α subunit of the G protein activates adenylate cyclase which in

turn increases the production of cyclic adenosine monophosphate (cAMP). The elevated

cAMP levels stimulate increased tyrosinase expression and activity which begins

eumelanogenesis (Chhajlani and Wikberg, 1992; Mountjoy et al., 1992; Abdel-Malek et

al., 1995; Busca and Ballotti, 2000). However, when agouti signalling protein (ASIP)

binds to MC1R, a pigment-type switch occurs from eumelanin to pheomelanin

production (Figure 11) (discussed in section 2.6.2.2).

Individuals with red hair and fair skin are known to have increased levels of

pheomelanin and a reduced ability to produce eumelanin (Thody et al., 1991). Based on

the pigment-type switch role that MC1R has been found to play in other mammals,

Valverde et al. (1995) sequenced the MC1R coding region in a sample of 135 British

and Irish Caucasians, in the hope of identifying polymorphisms responsible for

pheomelanin production and therefore the RHC phenotype. Indeed, several different

MC1R variants were identified and shown to be more common in persons with red hair

50

Figure 11. Pigment-type switching through the MC1R. Eumelanin production

requires the binding of α-MSH to the MC1R. When agouti (ASIP) is expressed it

antagonises this binding causing a pigment-type switch from eumelanin to pheomelanin

production. Agouti requires an accessory receptor, attractin (Atrn), for its antagonistic

action.Source: Adapted from He et al., (2001)

and fair skin (Valverde et al., 1995). Other workers have subsequently investigated such

MC1R association and it soon became apparent that whilst MC1R was highly

polymorphic with several polymorphisms being noticeably more common in, and more

significantly associated with, the RHC phenotype (Box et al., 1997; Smith et al., 1998;

Flanagan et al., 2000; Palmer et al., 2000; Bastiaens et al., 2001; Box et al., 2001; Healy

et al., 2001; Kennedy et al., 2001). Hence, variant alleles found to be strongly

associated with RHC phenotype have been designated ‘R’, and weakly associated RHC

alleles have been designated ‘r’ (Figure 12) (Sturm et al., 2003b).

The variant alleles of the R polymorphisms (D84E, R151C, R160W and D294H) show

odds ratios ranging from 50.5 to 118.3 for red hair colour and 3.2 to 12.5 for fair/pale

skin, relative to the wild-type allele (Sturm et al., 2003b; Duffy et al., 2004). In

addition, the rare R142H variant has also been designated as an R allele by some (Rees,

2004; Wong and Rees, 2005). At least one variant R allele is present in 93% of red-

haired individuals and three polymorphisms (R151C, R160W and D294H) account for

>60% of all cases of red hair colour (Healy et al., 2001; Sturm et al., 2003a; Duffy et

al., 2004). The r alleles (V60L, V92M and R163Q) show odds ratios from 2.4 to 6.4 for

red hair and 1.7 to 2.3 for fair/pale skin (Sturm et al., 2003a; Duffy et al., 2004).

51

Figure 12. Structure of the human MC1R protein. ‘R’ and ‘r’ RHC polymorphisms

are highlighted in red.Source:Garcia-Borron et al., (2005) adaptation of Ringholm et al., (2004)

A number of studies have investigated the functional consequence of the RHC alleles. It

is generally accepted that the R variant alleles represent diminished function alleles. The

degree of impaired function varies between them although none are considered to be

complete loss of function alleles (Newton et al., 2005). An early study, however,

reported that the R151C polymorphism produced a completely non-functional receptor

(Frandberg et al., 1998). Although binding of α-MSH was not inhibited by the R151C

polymorphism, the mutant receptor did not stimulate production of cAMP (Frandberg et

al., 1998). As amino acid 151 is located in the second intracellular loop of MC1R, it is

believed to disrupt the coupling of MC1R to the G-protein, thereby inhibiting cAMP

production. Similar findings to those obtained for R151C have been found for other R

alleles (D84E, R142H, R160W and D294H). However, these studies reported that these

mutant receptors retained some low-level residual signalling to stimulate cAMP

production (Schioth et al., 1999; Scott et al., 2002). In fact, the R160W mutant receptor

and, in contrast to the earlier study by Frandberg et al. (1998), the R151C mutant

receptor were later shown to retain considerable signalling capacity by demonstrating

52

significant agonist-mediated (α-MSH and NDP-MSH) increases in cAMP production

(Newton et al., 2005). Interestingly, the D294H polymorphism showed a marked

impairment of MC1R function compared to the R151C and R160W polymorphisms,

highlighting that the RHC alleles are not functionally equivalent. These findings

prompted the hypothesis that reduced levels and differing localisation of cell surface

MC1 receptors could underlie the reduced signalling observed by MC1R variants

(Beaumont et al., 2005). Indeed, three of the R alleles (D84E, R151C and R160W)

showed a marked reduction in cell surface expression on melanocytes which most likely

occurs due to aberrant processing with intracellular retention (Beaumont et al., 2005;

Sanchez-Laorden et al., 2006). The ability ofMC1R polymorphisms (R151C, R160W

and D294H) to rescue the recessive yellow phenotype of Mc1re homozygous transgenic

mice (i.e. restore the more darkly pigmented wild-type agouti hair banding pattern of

the lightly pigmented recessive yellow mouse), by increasing eumelanin production,

provided further evidence for the functional impact of the RHC alleles (Healy et al.,

2001). In this study, the transgenic mice showed agouti-banding patterns after rescue,

indicating that the agouti protein antagonised the variant receptors.

The weaker r alleles (V60L, V92M and R163Q) have also been shown to result in

diminished function of MC1R. This has been characterised either by observing a

lowering of agonist binding affinity or a reduction in cAMP production. The V60L

polymorphism was investigated along with the R alleles and showed significantly lower

cAMP levels after α-MSH stimulation (Schioth et al., 1999). Although clearly different

from the wild type allele, the V60L allele showed some residual signalling consistent

with its designation as a weaker r allele. The V92M allele was initially shown to result

in a 5-fold decrease in α-MSH affinity (Xu et al., 1996). These results were confirmed

by showing that the V92M allele had a 100-fold reduced affinity for α-MSH compared

to the wild-type receptor and consequently a significantly lower cAMP response

(Ringholm et al., 2004; Nakayama et al., 2006). However, other workers showed that

the V92M allele was able to activate adenylate cyclase, (and therefore increase cAMP)

in a dose-dependent manner, comparable to the wild type receptor (Koppula et al.,

1997; Scott et al., 2002). Unlike other RHC alleles, V92M did not show cell surface

expression level changes (Beaumont et al., 2005).

53

Present at very high frequencies in East and Southeast Asian populations, the R163Q

variant allele is only weakly associated with the RHC phenotype (OR= 2.4 for red hair

and 2.0 for fair skin) (Rana et al., 1999). The R163Q variant allele has been shown to

have a 20-fold reduced affinity for α-MSH compared to the wild type and a small

reduction in cell surface expression level (Ringholm et al., 2004; Beaumont et al.,

2005). No significant decreases in cAMP production by the R163Q allele receptor have

been reported (Ringholm et al., 2004; Nakayama et al., 2006).

2.6.2.2 Agouti signalling protein (ASIP)

In humans, the role of agouti signalling protein (ASIP) is not well understood.

However, the role of the murine analogue, agouti (officially named a or nonagouti), has

been relatively well characterised. As mentioned above, agouti is intimately involved

with the function of the mouse melanocortin 1 receptor. Expression of agouti causes a

pigment type switch from black/brown eumelanin to red/yellow pheomelanin by

antagonising the Mc1r (Figure 11) (Lu et al., 1994). Another protein, attractin (Atrn), is

a downstream regulator of ASIP and acts as a low affinity accessory receptor for agouti

(He et al., 2001). As well as antagonising Mc1r, agouti also down regulates other

pigmentation genes (Tyr, Tyrp1 and Dct) to inhibit eumelanin synthesis (Sakai et al.,

1997). In wild-type agouti mice, agouti is expressed between days 4–6 of the hair

growth cycle leading to the development of a subapical yellow band on individual hairs

creating a brindle-coloured coat (Sakurai et al., 1975).

Five dominant agouti mutations, intracisternal A-particle yellow (Aiapy), intermediate

yellow (Aiy), viable yellow (Avy), sienna yellow (Asy) and lethal yellow (Ay), lead to the

ectopic and ubiquitous expression of agouti in the mouse (Lyon et al., 1985; Duhl et al.,

1994; Michaud et al., 1994b; Yen et al., 1994; Siracusa et al., 1995). For example, the

Ay mutation is a 120–170kb deletion spanning the entire coding region of the

ubiquitously expressed heterogeneous nuclearribonucleo-protein associated with lethal

yellow (raly) gene, leading to agouti being under the direct control of the raly promoter.

In the homozygous Ay state the mutation results in embryonic lethality, while in the

heterozygous state it results in yellow fur, obesity, hyperphagia, increased linear growth

and non insulin-dependant diabetes (Michaud et al., 1994a). The ubiquitous expression

of agouti means that the mc1 receptor is continually antagonised and can produce the

lighter pigment pheomelanin, thus resulting in the yellow fur phenotype. On the other

54

hand, recessive agouti mutations are associated with increased eumelanin and decreased

pheomelanin production (Bultman et al., 1994; Hustad et al., 1995). Mutations in the

co-receptor for agouti, attractin, have been shown to suppress the pigment type switch

caused by agouti expression (Miller et al., 1997; Gunn et al., 1999; Nagle et al., 1999).

Human ASIP, is located on chromosome 20q11.2–q12 and encodes a 132 amino acid

protein (Kwon et al., 1994; Wilson et al., 1995). Although ASIP mutations in humans do

not show the characteristic subapical hair pattern seen in wild-type agouti mice, ASIP is

believed to play a role in human pigmentation. ASIP acts in a similar manner to agouti

by down regulating genes (TYR, TYRP1) that control eumelanin synthesis (Suzuki et al.,

1997). The ability of ASIP to inhibit human MC1R also supports a role for ASIP

function in human pigmentation (Yang et al., 1997). Further, human ASIP also acts to

reduce eumelanin production in the presence of α-MSH but also acts independently of

α-MSH to reduce production of both pigments (Hunt and Thody, 1995; Graham et al.,

1997).

Due to the effects of agouti mutations on mouse coat colour, polymorphisms in ASIP

have been investigated to explain human pigmentation variation. Polymorphism screens

of the ASIP coding region have failed to identify any coding region polymorphisms

(Norman et al., 1999; Voisey et al., 2001; Kanetsky et al., 2002), although three are now

listed in the NCBI dbSNP database. Voisey et al. (2001) first identified a polymorphism

in the 3' UTR of ASIP. Kanetsky et al. (2002) confirmed this polymorphism and

designated it, g.8818A>G. Voisey et al. (2001) showed that the g.8818A>G SNP was

present in different population groups, namely, African American, Asian and European.

Later, the frequency of the g.8818A>G SNP was determined in European, East Asian,

African American, and a West African population (Zeigler-Johnson et al., 2004). Using

a much larger sample set than previous studies, Zeigler-Johnson et al. (2004) showed

that Americans of European heritage had the lowest (0.12) 8818G frequency and West

Africans had the highest (0.80).

The g.8818A>G SNP is also responsible for within population pigmentation variation.

The first published example showed that the 8818G allele was significantly associated

with dark hair and brown eyes in Caucasians (Kanetsky et al., 2002). Later, Voisey et

al. (2006) confirmed this association with dark hair colour in a sample of 271 unrelated

55

Australian Caucasians but noted no association with eye colour (Voisey et al., 2006).

Interestingly, the 8818G allele is also associated with pigmentation variation in another

population group, African Americans (Bonilla et al., 2005). Using skin reflectometry

measurements of 234 African American individuals, the 8818G allele was associated

with a higher melanin index (M-index) in female African Americans. A function for this

polymorphism was first postulated by Kanetsky et al (2002), who suggested that the

8818G allele could cause mRNA instability. This instability would lead to premature

degradation of the transcript, decreasing the levels of ASIP and preventing it from

inhibiting α-MSH mediated activation of MC1R and consequently favouring eumelanin

synthesis. Voisey et al. (2006) measured ASIP mRNA concentrations and showed them

to be significantly lower in 8818G homozygous cells thereby supporting the proposed

effect of this polymorphism (Voisey et al., 2006).

The g.8818A>G SNP was investigated along with other ASIP SNPs for associations

with iris colour (Frudakis et al., 2003). This study could not confirm the previous

association of this SNP and eye colour, observed by Kanetsky et al. (2002). However, a

haplotype of several polymorphisms was found to be associated with pigmentation.

Specifically, the ACA haplotype of three ASIP polymorphisms (rs242987, rs2424984

and rs819135) was positively associated with hazel eye colour (Frudakis et al., 2003).

This haplotype was only observed 13 times in a sample size of 851 and this low

frequency may be the reason for the observed association. If indeed the association is

true, this result may reflect the power of haplotype association analysis for the

discovery of polymorphisms associated with pigmentation phenotypes.

2.6.2.3 SLC24A5 (Golden)

In a recent exciting finding, a new human gene has been identified as a major

determinant of human skin colour (Lamason et al., 2005). The human orthologue of the

mouse golden gene (also known as slc24a5 or nckx5), solute carrier family 24 member

5 (SLC24A5), was identified using positional cloning and a morpholino knockdown

approach in the zebrafish. The golden mutation (golb1) was the first recessive mutation

to be studied in zebrafish and results in hypopigmentation of skin melanophores

(zebrafish melanocyte equivalents) and retinal pigment epithelium (Streisinger et al.,

1981). Phenotypically, the golden zebrafish is most distinctly characterised by

lightening of the stripes on the side of its body (Figure 13). Lamason et al. (2005)

56

showed that the golden phenotype could be rescued by injection of human SLC24A5

mRNA into golden embryos, successfully highlighting the conservation of function

across vertebrate evolution.

Figure 13. Phenotype of golden mutation in Zebrafish. A) Wild-type zebrafish. B)

Golden zebrafish with reduced melanin in horizontal stripes. Insets show melanin

content of melanophores.Source:Lamason et al., (2005)

The human SLC24A5 gene belongs to a family of potassium-dependent sodium/calcium

exchangers (NCKX). It is located on chromosome 15q21.1 and encodes a 500 amino

acid protein. Lamason et al. (2005) interrogated the International Haplotype Map (The

International HapMap Consortium, 2005) and identified only one coding region, non-

synonymous SNP in SLC24A5. The rs1426654 SNP results in an alanine to threonine

substitution at amino acid 111 (A111T) and shows distinct allele frequencies in

different populations. The 111T allele is present at 100% in the HapMap European

population, whereas the ancestral allele, A111, is present at 93–100% in African,

Indigenous American and East Asian populations (Lamason et al., 2005). The

contribution of this polymorphism to skin pigmentation was investigated using two

admixed populations, African American and African Caribbean. The results suggested

that allelic differences in SLC24A5 were responsible for between 25–38% of the

variation in skin melanin index between Europeans and Africans. This data, along with

the finding that the genomic region surrounding SLC24A5 shows a reduction in

heterozygosity in European samples, strongly suggests evolutionary pressure has been

57

exerted on the 111T allele. Recent studies (Izagirre et al., 2006; Norton et al., 2007) also

support the finding of strong selective pressure in European populations which

contributes to the belief that light skin is not simply the result of loss or relaxation of

environmental pressure but is at least in part due to positive directional selection

(discussed at length in section 2.4.2).

The first polymorphism study to specifically target SLC24A5 determined the allele

frequencies of A111T in 570 samples from seven different populations with varying

pigmentation phenotypes (Soejima and Koda, 2007). This study confirmed the findings

of previous studies (Smith et al., 2004; Lamason et al., 2005) in which the European

population predominantly had the 111T allele. The authors concluded that the F374L

polymorphism in SLC45A2 (also investigated in the same study) more clearly

differentiates populations with specific pigmentation characteristics (such as those

populations from Sri Lanka and Europe) than the A111T polymorphism in SLC24A5.

There is currently no data that implicates the SLC24A5 gene with intra-population

pigmentation variation. In the coming years it will be interesting to see whether

polymorphisms in this gene are important contributors to pigmentation variation within

Europeans/Caucasians.

2.7 Genetic inference of human pigmentation

One of the major applications of identifying polymorphisms that are associated with

pigmentation characteristics is to develop a genetic test that can infer human

pigmentation phenotype. Probably the best examples of these practical applications are

the ancestry and forensic products and services offered by DNAprint Genomics

(www.dnaprint.com). Founded by Dr Tony Frudakis, this company has patented the use

of many pigmentation gene SNPs for ancestry and physical trait inference. To do this,

they have identified hundreds of polymorphisms in pigmentation genes (including all of

those mentioned above except SLC24A5) and non-pigmentation genes, and genotyped

them in a range of well-phenotyped populations. They used single SNP, haplotype and

diplotype (pairs of haplotypes) associations in a nested statistical approach

(classification tree solution), taking into account genetic differences due to ancestry, to

predict pigmentation phenotype. The development of these methods did not involve

58

investigating the function of the SNPs, but rather was only concerned with identifying

SNPs that were statistically associated with a pigmentation trait.

DNAprint has combined several of their ancestry and physical trait inference products

into its DNAWitness™ 2.5 forensic testing service. This service can infer ancestry by

determining the percentage genetic make-up of four possible groups, Sub-Saharan

African, Native American, East Asian and European. If required another test can

distinguish ancestry from North Western European, South Eastern European, Middle

Eastern and South Asian populations. Further inference of specific European ancestry

can be achieved using the Euro-DNA™ 2.0 to distinguish between South Eastern

European,Iberian,Basque, ContinentalEuropean and NorthEasternEuropean. In

addition, the service includes using the RETINOME™ assay to infer eye colour, which

is the first genetic test developed to predict a complex human trait. Unpublished

validation experiments have shown that RETINOME™ is accurate 92% of the time.

Early variations of the DNAwitness™ 2.5 service were put to good use in a well-

documented criminal case. The test was used to apprehend an African American man,

Derrick Todd Lee, an alleged serial killer in Louisiana, USA. Faulty eyewitness

accounts led police to pursue a man of Caucasian appearance, however, the DNAprint

tests showed that the suspect was 85% Sub-Saharan African and 15% Native American.

After police refocussed their investigation, Derrick Todd Lee was arrested and later

convicted of his alleged crimes.

Of course, physical trait inference from DNA will be complicated by the ability of

individuals to change their physical appearance. For example, a hair colour inference

product similar to RETINOME™ would most likely be of little use to crime scene

investigators because the perpetrator can easily change their hair colour. Nonetheless,

such a product will contribute to creating an overall picture of the alleged perpetrator,

which should be helpful. Now that DNAPrint Genomics have created the platform for

phenotype inference, it is only a matter of time before more physical traits can be

inferred in this manner.

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2.8 Transcriptional regulation of human pigmentation

2.8.1 Microphthalmia-associated transcription factor (MITF)

The MITF gene has been coined the ‘master regulator’ of human pigmentation as it

regulates the expression of several key enzymes (TYR, TYRP1 and TYRP2) involved

in melanogenesis (Reviewed in Levy et al., 2006). Mutations in MITF cause

Waardenburg Syndrome (WS) type IIA (OMIM: #193510) (Hughes et al., 1994;

Tassabehji et al., 1994) and Tietz syndrome (OMIM: #103500) (Tietz, 1963; Amiel et

al., 1998; Smith et al., 2000). WS type IIA is a heterogenous condition characterised by

partial and isolated hypopigmentation, heterochromia, deafness and gastrointestinal

problems. Tietz syndrome is characterised by more generalised hypopigmentation with

the ability to accumulate pigment over time, and bilateral, profound deafness (Smith et

al., 2000).

MITF is located on chromosome 3p14.1–p12.3 and has nine exons which encode a 419

amino acid protein (Tachibana et al., 1994). MITF belongs to a family of basic helix-

loop-helix leucine zipper (b-HLH-Zip) transcription factors known as the MiT family.

Other members include transcription factor EB (TFEB), transcription factor binding to

IGHM enhancer 3 (TFE3) and transcription factor EC (TFEC) (Hemesath et al., 1994).

Nine different MITF isoforms have been identified in humans (MITF-A, B, C, D, E, H,

J, M and MC), all of which have different promoter-exon one units (Hodgkinson et al.,

1993; Steingrimsson et al., 1994; Tassabehji et al., 1994; Fuse et al., 1996; Amae et al.,

1998; Fuse et al., 1999; Udono et al., 2000; Oboki et al., 2002; Takeda et al., 2002;

Takemoto et al., 2002; Hershey and Fisher, 2005). The nine isoforms have exons two to

eight in common, which encode the functional domains of the protein (two

transactivation domains, a b-HLH-Zip domain and various phosphorylation consensus

sequences) (Hershey and Fisher, 2005; Levy et al., 2006). MITF is expressed in heart,

mast cells and osteoclast precursors, however, some isoforms show tissue-specific

expression, such as MITF-M, which shows highly specific expression in melanocytes

and melanomas (Hodgkinson et al., 1993; Steingrimsson et al., 1994; Fuse et al., 1996).

2.8.2 Transcriptional targets of MITF

Like other DNA-binding proteins, MITF requires the b-HLH structure to bind to DNA

(Fisher et al., 1991; Kadesch, 1993). It does so in either a homodimeric or heterodimeric

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fashion with other members of the MiT transcription factor family (Hemesath et al.,

1994). MITF binds to a highly conserved E-box motif (underlined) within an 11 bp M-

box (5'-AGTCANNTGCT-3') (Shibahara et al., 1991; Lowings et al., 1992). Several

key pigmentation genes have E-box or M-box elements within their promoter sequences

and are known transcriptional targets of MITF. These genes include, TYR, TYRP1, DCT

(TYRP2), and SLC45A2(Bentley et al., 1994; Yasumoto et al., 1997; Bertolotto et al.,

1998b).

A 20 bp element (5'-GGAGATCATGTGATGACTTC-3'), located between –1861 and –

1842 of the TYR promoter, is responsible for the pigment cell-specific gene expression

of TYR(Shibata et al., 1992; Yasumoto et al., 1994). Termed the TYR distal element

(TDE), the TDE contains a CATGTG motif located in the centre of the 20 bp element

(underlined above) (Yasumoto et al., 1994). There are three additional copies of the

highly conserved CATGTG motif located at –1972 to –1967, –104 to –99, and –12 to –

7 (Shibata et al., 1992; Bentley et al., 1994; Yasumoto et al., 1994). The –1972 to –1967

element is not believed to be essential for pigment cell-specific expression (Shibata et

al., 1992). However, a –104 to –99 element was shown to be more important than the –

12 to –7 initiator E-box element for pigment cell-specific expression of TYR. The –104

to –99 E-box forms part of a larger element known as the TYR proximal element (TPE:

–112 to –93) (Yasumoto et al., 1994). The TPE functions as a weak cell-specific

enhancer of TYR expression.

Coexpression of MITF cDNA and a TDE construct in a luciferase reporter assay system

showed that MITF transactivates the TYR promoter directly through the TDE

(Yasumoto et al., 1994). However, gel mobility shift assays failed to confirm the

binding of MITF to the TDE. The authors proposed that certain post-translational

modifications may be required for MITF to perform its transcriptional regulatory

function or that the gel mobility shift assays used were unable to detect MITF-TDE

complexes (Yasumoto et al., 1994). They also suggested that MITF might form a dimer

with another transcription factor such as upstream regulatory factor (USF) or TFE3,

which in turn functions to direct the pigment cell-specific transcription of TYR.

Subsequent work by the same authors showed that MITF does actually bind to the

CATGTG motifs in the TYR promoter (Yasumoto et al., 1997). The successful approach

61

used GST-MITF fusion protein, rather than nuclear extracts, and synthetic TDE as a

probe in gel mobility shift assays.

Luciferase reporter assays also suggest that overexpression of MITF cDNA

transactivates the TYRP1 promoter, but to a lesser extent than that for TYR(Yasumoto et

al., 1997). The region between –133 and +82, which contains an M-box, is responsible

for the preferential expression of TYRP1 in pigment cells by MITF. When using a

TYRP1 M-box oligonucleotide as a competitor in a gel shift assay, with GST-MITF

fusion protein and TDE as a probe, Yasumoto et al. (1997) observed significant

inhibition of the binding reaction, suggesting that MITF binds the TYRP1 M-box.

Two upstream regulatory regions direct melanocyte-specific gene expression of

TYRP2, a 32 bp element containing a E-box CAATTG motif (located between –447

and –416) and the M-box-containing (CATGTG) proximal region (–268 to –56)

(Yokoyama et al., 1994). MITF overexpression in luciferase assays failed to detect the

transactivation of the TYRP2 gene promoter by MITF when using constructs containing

either of these elements (Yasumoto et al., 1997). Interestingly, others have shown a 20-

fold transactivation of the TYRP2 promoter by murine microphthalmia coexpression,

but this could be explained by the specific behaviours of the murine protein (Bertolotto

et al., 1998b). A TYRP2 oligonucleotide, containing the 32 bp element, was used in

competition binding reactions and failed to inhibit the binding of GST-MITF fusion

protein to a TDE oligonucleotide. This result indirectly indicated that the 32 bp element

is not a target of MITF. However, an oligonucleotide spanning the TYRP2 proximal

region, which contains the CATGTG M-box motif, did inhibit binding of GST-MITF to

the TDE oligonucleotide, indicating that MITF binds to the TYRP2 proximal M-box

(Yasumoto et al., 1997). Another study supports this result by showing that murine

microphthalmia binds to the human TYRP2 proximal M-box (Bertolotto et al., 1998b).

The results of the competition binding reactions performed by Yasumoto et al. (1997)

has highlighted the significance of the CATGTG motif for MITF binding. The

functional significance of the M-box was determined by using mutated M-box

constructs in luciferase assays. Such experiments showed that the M-box is essential for

transcription in pigment cells but that MITF does not significantly transactivate the

TYRP2 promoter in its un-mutated form (Yasumoto et al., 1997).

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Whilst the nature of the specific M-box or E-box sequences are an important factor in

determining the degree of affinity that MITF has for a promoter region, surrounding

nucleotides also play a significant role. A single nucleotide immediately upstream of the

CANNTG motif is essential for murine microphthalmia binding (Aksan and Goding,

1998). Using in vitro binding assays and an in vivo yeast one-hybrid assay, the presence

or absence of the 5′ flanking T nucleotide was shown to be critical for microphthalmia

binding to DNA. Other pigmentation genes, such as the OCA2 gene, also have promoter

CATGTG motifs and were also expected to be regulated by MITF. Based on the

absence of the 5′ flanking T nucleotide from OCA2 gene E-box motifs, Aksan and

Goding (1998) successfully predicted and showed through competition binding assays,

that microphthalmia would not bind to OCA2 gene E-box sequences (Aksan and

Goding, 1998).

The relevance of MITF binding to the promoter of pigmentation genes can be explained

with reference to cAMP and melanogenesis. Upon binding of α-MSH to MC1R, an

increase in cAMP levels stimulates melanogenesis. Using mouse tyrosinase promoter

constructs in binding reactions, Bertolotto et al. (1996) showed that cAMP elevating

agents (such as forskolin) increase microphthalmia expression thereby stimulating the

binding of murine microphthalmia to M-box and E-box motifs, which in turn stimulates

the expression of tyrosinase and melanin synthesis (Bertolotto et al., 1996; Bertolotto et

al., 1998a). Bertolotto et al. (1998) later showed that this was also the case for the

murine TYRP1 promoter and human TYRP2 promoter, when using murine

microphthalmia (Bertolotto et al., 1998b). This indicates that these three melanogenic

enzymes share similar regulatory mechanisms by which cAMP can regulate their

expression.

MITF has also been implicated in the transcriptional regulation of SLC45A2(Baxter and

Pavan, 2002; Du and Fisher, 2002). Direct transcriptional regulation through MITF

usually involves binding to consensus E-box and M-box sequences, as noted above for

TYR, TYRP1 and TYRP2 (Reviewed in Levy et al., 2006). However, data suggest that

MITF does not bind recognised E-box binding sites in the proximal promoter of

SLC45A2(Du and Fisher, 2002). By measuring mRNA levels in human SKMEL5

melanoma cells and human primary melanocytes that were infected with adenovirus

over-expressing MITF, Du and Fisher (2002) observed upregulation of SLC45A2. A

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bioinformatic analysis of the SLC45A2 promoter revealed the presence of 55 E-boxes in

a 1.2 kb repetitive region which appears to be unique within the human genome.

Despite the large number of consensus E-box motifs, chromatin immunoprecipitation

assays failed to measure an interaction between MITF and two regions at each end of

the repeat. Further, luciferase reporter assays using these same regions did not show

significant responsiveness to MITF overexpression (Du and Fisher, 2002). Taken

together, these results suggest that MITF does not regulate SLC45A2 expression via

direct interactions but rather it acts via an indirect mechanism or at an alternate location.

Considering that the only evidence, which supports the regulation of SLC45A2 by

MITF, was via measuring endogenous mRNA levels, it is also possible that mRNA

stability is a causal factor.

2.9 Summary and relevance to experimental program

Constitutive human pigmentation variation is primarily determined by the size, number

and distribution of melanin-containing melanosomes. The most distinguishable sites of

pigmentation are the skin, hair and eyes. In rare cases, mutations in genes that are

crucial for melanin production occur, resulting in an under production of melanin and a

hypopigmented phenotype (oculocutaneous albinism). However, more subtle

pigmentation variation exists within populations. To explain this subtle variation, some

investigators have identified associations between non-pathogenic polymorphisms and

normal human pigmentation variation. The most well characterised being the effect of

melanocortin 1 receptor gene variants in determining the red hair colour (RHC)

phenotype.

In this study, a recently identified “albinism” gene, known as the solute carrier family

45 member 2 (SLC45A2) gene, was chosen for further investigation (Newton et al.,

2001). We hypothesise that non-pathogenic polymorphisms in SLC45A2 contribute to

normal human pigmentation variation, both between and within populations. Little is

known about the role of SLC45A2 in human pigmentation, however, by studying the

effects of the major pathogenic mutation in SLC45A2, one study has provided the best

evidence for its role in melanogenesis (Costin et al., 2003). For this reason, a review of

the genetics of human pigmentation dysfunction was included, specifically focussing on

disorders of melanin synthesis in melanosomes. In addition, the role of “albinism”

genes in determining normal pigmentation variation has been extensively reviewed.

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Other key genes for which mutations do not cause albinism, but are strongly associated

with normal pigmentation variation, have also been extensively reviewed.

This review has highlighted the need for more information regarding the genetic basis

of normal human pigmentation variation, particularly within populations. Therefore,

this thesis has attempted to address this need by investigating the role of SLC45A2

polymorphisms in determining between and within population pigmentation variation.

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2.10 References

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– Chapter 3 –

Single nucleotide polymorphisms in the MATP gene are

associated with normal human pigmentation variation

Justin Graf, Richard Hodgson and Angela van Daal

Human Mutation 2005 Mar;25(3):278-84.

88

Statement of joint authorship

In the case of this chapter:

Single nucleotide polymorphisms in the MATP gene are associated with normal

human pigmentation variation.

Human Mutation 2005 Mar;25(3):278-84.

The authors listed below have certified* that:

1.they meet the criteria for authorship in that they have participated in the conception,

execution, or interpretation, of at least that part of the publication in their field of

expertise;

2.they take public responsibility for their part of the publication, except for the

responsible author who accepts overall responsibility for the publication;

3.there are no other authors of the publication according to these criteria;

4.potential conflicts of interest have been disclosed to (a) granting bodies, (b) the

editor or publisher of journals or other publications, and (c) the head of the

responsible academic unit, and

5.they agree to the use of the publication in the student’s thesis and its publication on

the Australasian Digital Thesis database consistent with any limitations set by

publisher requirements.

Contributor Statement of contribution*

Justin Graf

(candidate)

Involved in the conception and design of the study, extraction of

DNA from blood samples, genotyping the majority of samples,

collection of samples, interpretation and analysis of all data and

composed the manuscript.

Richard Hodgson* Involved in the conception and design of the study, performed

some genotyping and critically reviewed a portion of the

manuscript.

Angela van Daal* Involved in the conception and design of the study, collection of

samples, assisted with the interpretation and analysis of data and

critically reviewed the manuscript.

89

Principal Supervisor Confirmation

I have sighted email or other correspondence providing certification from all co-authors.

_____________________ ____________________ ____________________

Name Signature Date

90

97

– Chapter 4 –

Promoter polymorphisms in the MATP (SLC45A2)

gene are associated with normal human skin color

variation

Justin Graf, Joanne Voisey, Ian Hughes and Angela van Daal

Human Mutation 2007 Jul;28(7):710-7.

98

Statement of joint authorship

In the case of this chapter:

Promoter polymorphisms in the MATP (SLC45A2) gene are associated with

normal human skin color variation.

Human Mutation 2007 Jul;28(7):710-7.

The authors listed below have certified* that:

1.they meet the criteria for authorship in that they have participated in the conception,

execution, or interpretation, of at least that part of the publication in their field of

expertise;

2.they take public responsibility for their part of the publication, except for the

responsible author who accepts overall responsibility for the publication;

3.there are no other authors of the publication according to these criteria;

4.potential conflicts of interest have been disclosed to (a) granting bodies, (b) the

editor or publisher of journals or other publications, and (c) the head of the

responsible academic unit, and

5.they agree to the use of the publication in the student’s thesis and its publication on

the Australasian Digital Thesis database consistent with any limitations set by

publisher requirements.

Contributor Statement of contribution*

Justin Graf

(candidate)

Involved in the conception and design of the study, collection of

samples, DNA extraction, genotyping all samples, RLM-RACE,

dHPLC screening, reporter assays, statistical analysis,

interpretation and analysis of data and composed the manuscript.

Ian Hughes* Involved in the design of the project, collection of samples,

interpretation and analysis of data, assisted in writing the

manuscript and critically reviewed the manuscript.

Joanne Voisey* Involved in the design of the project, interpretation and analysis of

data and critically reviewed the manuscript.

Angela van Daal* Involved in the conception and design of the project, collection

and phenotyping of samples, reviewed the manuscript.

99

Principal Supervisor Confirmation

I have sighted email or other correspondence providing certification from all co-authors.

_____________________ ____________________ ____________________

Name Signature Date

100

109

– Chapter 5 –

Functional characterisation of polymorphic variation

in the SLC45A2 (MATP) gene promoter

Justin Graf, Joanne Voisey, and Ian Hughes

Prepared for submission to Pigment Cell and Melanoma Research

110

Statement of joint authorship

In the case of this chapter:

Functional characterisation of polymorphic variation in the SLC45A2 (MATP)

gene promoter.

Prepared for submission to Pigment Cell and Melanoma Research.

The authors listed below have certified* that:

1.they meet the criteria for authorship in that they have participated in the conception,

execution, or interpretation, of at least that part of the publication in their field of

expertise;

2.they take public responsibility for their part of the publication, except for the

responsible author who accepts overall responsibility for the publication;

3.there are no other authors of the publication according to these criteria;

4.potential conflicts of interest have been disclosed to (a) granting bodies, (b) the

editor or publisher of journals or other publications, and (c) the head of the

responsible academic unit, and

5. they agree to the use of the publication in the student’s thesis and its publication on

the Australasian Digital Thesis database consistent with any limitations set by

publisher requirements.

Contributor Statement of contribution*

Justin Graf

(candidate)

Involved in the conception and design of the study, collection of

samples, DNA and RNA extraction, cell culture, genotyping all

samples, bioinformatic analysis, EMSA, reporter assays, gene

expression quantitation, statistical analysis, interpretation and

analysis of all data, and composed the manuscript.

Joanne Voisey* Involved in the design of the project, interpretation and analysis of

data and critically reviewed the manuscript.

Ian Hughes* Involved in the design of the project, collection of samples,

interpretation and statistical analysis of data, assisted in writing the

manuscript and critically reviewed the manuscript.

111

Principal Supervisor Confirmation

I have sighted email or other correspondence providing certification from all co-authors.

_____________________ ____________________ ____________________

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112

Summary

Non-synonymous polymorphisms (p.Glu272Lys and p.Phe374Leu) in the solute carrier

family 45 member 2 gene (SLC45A2, or previously named MATP) are strongly

associated with normal human pigmentation variation. Recently, we identified a novel

promoter duplication polymorphism (c.–1176_–1174dupAAT) and showed that it and

two previously-described SLC45A2 promoter polymorphisms (c.–1721C>G and c.–

1169G>A) are significantly associated with olive skin colour in Caucasians. We showed

through luciferase assays that these polymorphisms affect transcriptional activity and

postulated that transcription is affected through binding of MITF to the SLC45A2

promoter in the region surrounding the novel duplication polymorphism. Here, we show

that c.–1176_–1174dupAAT and the tightly linked c.–1169G>A polymorphisms

significantly affect DNA-protein binding. However, preliminary evidence suggests that

MITF was not the bound protein. Further promoter characterisation has shown that the

region –206 to –4 is sufficient to permit constitutive expression of SLC45A2.

Quantitative mRNA gene expression analysis of primary melanocytes and melanoblasts

suggests that SLC45A2 promoter polymorphisms may play a role in regulating the

expression of TYRP1. This report further strengthens the argument that SLC45A2

promoter polymorphisms play a functional role in determining normal pigmentation

variation in humans.

Keywords: MATP, SLC45A2, SNP, pigmentation, promoter, TYRP1

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Introduction

The membrane associated transporter protein gene (MATP) or solute carrier family 45

member 2 (SLC45A2) has been extensively studied for its role in the fourth and most

recently identified form of oculocutaneous albinism (OCA4) (Newton et al., 2001;

Baxter and Pavan, 2002; Costin et al., 2003; Inagaki et al., 2004; Rundshagen et al.,

2004; Inagaki et al., 2005; Suzuki et al., 2005; Inagaki et al., 2006). Due to the key role

of SLC45A2 in human pigmentation, polymorphisms in SLC45A2 have been the focus

of investigations to explain some of the normal human inter- and intra-population

pigmentation variation (Nakayama et al., 2002; Yuasa et al., 2004; Soejima and Koda,

2006; Yuasa et al., 2006).

SLC45A2 is located on chromosome 5p and has seven exons spanning 40 kb. The gene

expresses a 530 amino acid human SLC45A2 protein and is reportedly expressed in

numerous melanoma and melanocyte cell lines, a fibroblast cell line but not at

significant levels in a panel of 15 different normal tissues (Harada et al., 2001). The

human protein shares 82% identity with the mouse Slc45a2 protein and is predicted to

have 12 transmembrane domains (Newton et al., 2001). A 1.7 kb and 2.8 kb transcript

have been identified, with the larger transcript most likely resulting from an alternate

splicing event (Harada et al., 2001).

The initial focus of polymorphic variation in SLC45A2 has centred on two non-

synonymous polymorphisms, c.814G>A (p.Glu272Lys; NCBI dbSNP rs26722) and

c.1122C>G (p.Phe374Leu; NCBI dbSNP rs16891982, dbSNP -

http://www.ncbi.nlm.nih.gov/SNP). These two polymorphisms have been investigated in

white South African, Ghanaian, Japanese, and New Guinea Islander populations. It was

shown that the Phe374 allele was only present in the white South African population

and not in the three other, more darkly pigmented populations (Nakayama et al., 2002).

The 272Lys allele was only identified in the Japanese and New Guinea Islander

populations. A subsequent study showed that the Phe374 allele was not present in a

Japanese population and that a German population showed similar allele frequencies to

that seen in the white South African population studied by Nakayama et al. (2002)

(Yuasa et al., 2004). We also examined these polymorphisms and were the first to show

a significant association with normal pigmentation variation in a Caucasian population

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(Graf et al., 2005). Specifically, we reported highly significant associations for the

374Leu and 272Lys alleles with dark hair, skin, and eye pigmentation within a

Caucasian population. A distinctive distribution of allele frequencies of these

polymorphisms amongst four different population groups was also observed.

Subsequently, the allele frequency of the p.Phe374Leu polymorphism was examined in

14 different population groups (Yuasa et al., 2006) and the p.Glu272Lys in 5 population

groups (Soejima et al., 2006). Results confirmed the findings of previous studies and

suggest that the Phe374 allele is important in hypopigmentation in Caucasian

populations. In addition, admixture mapping studies have suggested that SLC45A2 has

played a predominant role in the evolution of light skin in Europeans (Norton et al.,

2007).

More recently, we undertook the first polymorphism screen of the human SLC45A2

promoter region and identified a novel three base pair duplication, c.–1176_–

1174dupAAT (Graf et al., 2007). This polymorphism, along with c.–1721C>G

(rs13289) and c.–1169G>A (rs6867641) were all found to be associated with normal

skin colour variation within a Caucasian population.

To date, little is understood about the function of SLC45A2 in human pigmentation,

although studies investigating the effects of mutations in murine Slc45a2have provided

a valuable tool for understanding the role SLC45A2 plays. A study of the mouse model

of OCA4 has shown that Slc45a2 plays a crucial role in the processing and intracellular

trafficking of tyrosinase and other melanosomal proteins (Costin et al., 2003). The basic

helix-loop-helix leucine zipper protein, microphthalmia-associated transcription factor

(MITF), has been shown to be important in the transcriptional regulation of SLC45A2

and a number of other pigmentation genes including Tyrosinase-related protein 1

(TYRP1), Dopachrome tautomerase (DCT or TYRP2) and Tyrosinase (TYR) ( Bentley et

al., 1994; Yasumoto et al., 1997; Bertolotto et al., 1998; Baxter and Pavan, 2002; Du

and Fisher, 2002).Direct transcriptional regulation through MITF usually involves

binding to consensus E-box motifs (CATGTG) within an 11 bp M-box (Lowings et al.,

1992; Hemesath et al., 1994; Aksan and Goding, 1998). However, in the SLC45A2

proximal promoter, it was shown that MITF does not bind recognised E-box binding

sites but was suggested to act via an indirect mechanism or at an alternate location (Du

and Fisher, 2002). Recently, we have shown that polymorphisms in the SLC45A2

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proximal promoter significantly affect transcriptional activity (Graf et al., 2007).

Bioinformatic analysis suggested that the –dup and –1169G alleles disrupt a Hox-A5

(Hox-1.3) binding site but create a MITF E-box binding site. We postulated that the

differential transcriptional activity seen in our luciferase assays could be due to altered

MITF binding at or near sequence position –1176 to –1169 of the SLC45A2 promoter.

In the present study, we further characterised the proximal promoter region of SLC45A2

by specifically investigating the functional consequences of the c.–1176_–1174dupAAT

and c.–1169G>A polymorphisms. Results of electromobility shift assays have provided

preliminary indications that a transcription factor other than MITF binds to the region

including the c.–1176_–1174dupAAT and c.–1169G>A polymorphisms. Binding of this

currently unidentified factor is decreased when DNA containing the –dup/G haplotype

was assayed. Further, through quantitative gene expression analysis of SLC45A2, MITF

and TYRP1 in primary melanocytes and melanoblasts, we show that SLC45A2 promoter

polymorphisms are associated with TYRP1 mRNA expression. In addition, luciferase

reporter assays demonstrated that increasing promoter length (construct size) was

associated with increasing transcriptional activity up to the largest construct

investigated which extended to –1935.

Results

Bioinformatic promoter analysis

Common promoter elements such as the TATA box, GC box, and CCAAT box are

normally located within 200 bp upstream of the transcription start site (TSS). However,

only GC boxes and CCAAT boxes were identified within this region of the proximal

SLC45A2 promoter. None were 100% identical to their respective consensus sequences.

Several TATA box sequences were identified but only upstream of –900 bp, effectively

suggesting that SLC45A2 has a TATA-less promoter. A CpG island scan revealed no

CpG islands in the promoter region investigated. Our tandem repeat search confirmed

the results of Du and Fisher (2001) identifying a large block of repetitive DNA

sequence. Specifically, the region between –299 and –1528 contained numerous repeat

regions of varying sizes. A long interspersed nuclear element (LINE2) was also

identified, encompassing the region –1914 to –1729. Further short interspersed nuclear

element (SINEs) and LINEs were identified upstream of –1935.

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Luciferase reporter assays

Promoter activity of five constructs (Figure 1), referred to as pGL3-Luc1 (smallest)

through to pGL3-Luc5 (largest), was evaluated by transfection into MM96 melanoma

cells. One-way analysis of variance (ANOVA) showed significant differences (P =

1.63×10-14) in luciferase activity existed between the five constructs and the promoter-

less pGL3-basic vector. Post-hoc t-tests (Least Significance for Difference) were

performed to test the differences between individual constructs. All constructs were

significantly different from each other, except for pGL3-Luc3 (–995 to –4) and pGL3-

Luc4 (–1284 to –4). The pGL3-Luc1 (–206 to –4) construct had significantly higher

luciferase activity than the pGL3-basic vector alone (P = 6×10-6) suggesting that the

region immediately upstream of the SLC45A2 5′ untranslated region (UTR) exhibits

constitutive promoter activity. Interestingly, the pGL3-Luc2 (–404 to –4) showed

approximately 3.5-fold higher activity than the pGL3-Luc1 construct (P = 1.78×10-4).

Promoter activity increased as the construct size increased, with the pGL3-Luc5

construct (–1935 to –4) showing the maximum activity, approximately 6-fold higher

than that of the pGL3-luc1 construct (P = 6.33×10-9). The pGL3-Luc4 construct, which

contained the –dup/G haplotype within a highly polymorphic region (Figure 1), did not

show significantly different luciferase activity to the pGL3-Luc3 construct (P = 0.069),

which did not include the polymorphic region.

Allele-specific gene expression analysis

To ascertain whether SLC45A2 promoter polymorphisms affect the expression of

SLC45A2 itself and other pigmentation genes, the levels of SLC45A2, MITF, Agouti

signalling protein (ASIP) and TYRP1 mRNA were determined in melanocyte and

melanoblast cell lines. Linear regression analysis revealed that TYRP1 expression was

positively correlated with presence of the –dup allele (r2 = 0.1938, P = 0.04) (Figure

2A). SLC45A2 and MITF showed a similar, but non-significant trend, with expression

increasing with an increasing number of –dup alleles present (Figure 2B). ASIP was not

expressed at detectable levels.

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Electrophoretic mobility shift assays (EMSA)

To investigate the possible effects of the –dup/G haplotype and the +dup/A haplotype of

the c.–1176_–1174dupAAT and c.–1169G>A (rs6867641) promoter polymorphisms on

DNA binding activity, electrophoretic mobility shift assays were performed. Two

labelled, double-stranded probes, one of each haplotype, were incubated with MM96

nuclear cell extracts in order to determine if the binding of transcription factors (TFs)

differed. Three separate biotin 3′ end-labelling reactions were performed to allow for

inconsistencies in biotin labelling efficiency, which could potentially result in false

variability between the two haplotype probes. Across three separate experiments, each

using independently labelled probes; the +dup/A haplotype consistently showed a

marked increase in DNA-nuclear protein binding affinity compared with the –dup/G

haplotype (Figure 3). A 100-fold molar excess of cold competitor (unlabelled probe) for

each probe was able to out compete binding of the labelled probe. To determine if the

DNA-protein shift was due to the binding of MITF, supershift experiments were

performed using monoclonal anti-MITF antibody (C5+D5). No detectable supershifts

were observed (data not shown) suggesting that the protein binding to the +dup/A

haplotype was not MITF. Two positive controls probes were used in this experiment,

that had previously been shown to exhibit a DNA-protein shift and specifically an MITF

supershift in a murine melanoma and human melanocyte cell line (Vetrini et al., 2004;

Schwahn et al., 2005). However, when using our MM96 melanoma cell extracts, neither

control probe showed any detectable DNA-protein complexes despite the presence of

MITF in our melanoma cell line being confirmed by Western-blot analysis (Figure 4).

Discussion

Polymorphic variation in the solute carrier family 45 member 2 (SLC45A2) gene has

been associated with both abnormal human pigmentation in the form of oculocutaneous

albinism type 4 (OCA4) as well as normal human pigmentation variation within

Caucasian populations (Newton et al., 2001; Graf et al., 2005). However, these studies

have yielded only a limited degree of information regarding the actual function of

SLC45A2 in normal pigmentation variation. Currently, it has been proposed that alleles

associated with a darker pigmentation phenotype, contribute to a melanosomal

environment that favours optimal tyrosinase activity possibly by altering

intramelanosomal pH (Newton et al., 2001; Graf et al., 2005). In addition, Costin et al.

118

(2003) showed that SLC45A2 plays a crucial role in the processing and intracellular

trafficking of tyrosinase, as well as other melanosomal proteins. As yet, no functional

effects have been shown for the p.Glu272Lys and p.Phe374Leu polymorphisms or other

coding region SLC45A2 polymorphisms.

Recently, we investigated the proximal promoter region of SLC45A2 by screening a

1300 bp region for novel polymorphisms and found that transcriptional activity is

altered by the three polymorphisms, c.–1721C>G (rs13289), the novel c.–1176_–

1174dupAAT, and c.–1169G>A (rs6867641). Bioinformatic analysis led to the

postulation that this altered transcriptional activity may be caused specifically by

disrupting the binding of MITF, since the –dup/G haplotype disrupts a Hox-A5 (Hox-

1.3) element creating a MITF binding site (E-box). Here, we tested the hypothesis that

differential transcriptional activity was due to the disruption of MITF binding at an

MITF consensus binding site which encompassed the c.–1176_–1174dupAAT and c.–

1169G>A polymorphisms. We found that the +dup/A haplotype (Hox-A5 element)

consistently showed a marked increase in DNA-nuclear protein binding affinity

compared with the –dup/G haplotype (MITF E-box), as demonstrated by an observed

gel mobility shift. When a monoclonal MITF antibody was used in supershift

experiments no additional gel retardation (supershift) was detected for either allele. The

MM96 cell line has previously been shown to express MITF (Carreira et al., 2000;

Cook et al., 2005) and we confirmed the presence of MITF by Western-blotting. It has

been shown that the ability of MITF to bind to an E-box relies on the presence of a 5′

flanking T residue immediately preceding the core E-box sequence (Aksan and Goding,

1998). The E-box sequence of the –dup/G haplotype investigated in this report does

contain a 5' flanking T residue. The absence of a T residue can therefore be ruled out as

a contributing factor for a lack of MITF binding. It is known that MITF binds with other

proteins such as p300 and LEF-1 (Yasumoto et al., 2002; Schwahn et al., 2005),

therefore it is possible that a protein-protein interaction in the MM96 cell line is

occurring that specifically prevents direct binding of MITF to the identified E-box, or to

the positive control E-boxes. Similarly, MITF is known to bind to DNA as both a

homodimer and heterodimer (Hemesath et al., 1994). Therefore, it is possible that the

process of heterodimerising with another b-HLH-Zip protein of the MiT family, could

block the antibody detection of MITF by obscuring the N-terminal epitope. It is

interesting to note that in the murine Oa1 gene, MITF specifically binds to a proximal

119

E-box of the Oa1 promoter but not to a more distal E-box (Vetrini et al., 2004).

Nonetheless, our preliminary results mirror those of Du and Fisher (2002) and provided

some evidence that MITF does not transcriptionally regulate SLC45A2 through the

direct binding of E-box motifs located within the SLC45A2 proximal promoter region.

Rather, MITF may transcriptionally regulate SLC45A2 expression indirectly or via a

currently uncharacterised alternate regulatory region, as initially proposed (Du and

Fisher, 2002).

Since our preliminary investigations suggest that MITF does not directly bind to the

region tested, another as yet unknown protein clearly binds to this region and its binding

is affected by the c.–1176_–1174dupAAT and c.–1169G>A polymorphisms. We cannot

rule out the possibility that the unknown protein is Hox-A5, however, this is unlikely

because Hox-A5 has only been implicated with the induction of apoptosis in breast

cancer cells, and no associations with pigmentation have been published (Chen et al.,

2004). In order to identity the unknown protein, a transcription factor pull-down assay

using a melanoma/melanocyte cell line could be combined with an array-based analysis

of DNA-binding proteins (Jiang et al., 2004). This approach would allow many

potential transcription factors to be simultaneously tested. Alternatively, investigating

the specific interactions of other known transcriptional regulators of pigmentation

genes, such as BRN2, SOX9 and 10, USF and PAX3, and SLC45A2 E-box motifs,

could identify novel mechanisms for transcriptional regulation of SLC45A2(Yasumoto

et al., 1994; Galibert et al., 2001; Cook et al., 2003; Cook et al., 2005; Corre and

Galibert, 2005; Corry and Underhill, 2005; Murisier et al., 2007).

To investigate the functional importance of SLC45A2 promoter polymorphisms, we

quantified the expression of SLC45A2, MITF, ASIP and TYRP1 in order to identify

allele-specific expression. While a trend of increased gene expression with presence of

the –dup allele was observed for SLC45A2, MITF and TYRP1, statistical significance

was only obtained for TYRP1. A large degree of variation between samples of the same

genotype was noted for all gene expression experiments. Since ancestry information and

coding region genotypes were not available for donors of foreskin samples, these two

factors may contribute to the large variation observed between samples. Based on our

previous results (Graf et al., 2007), where the highest luciferase expression was seen in

constructs containing the –dup allele, we expected to observe the highest SLC45A2

120

mRNA expression in melanocyte cell lines that were homozygous for the –dup allele.

This was indeed observed for SLC45A2, as well as for MITF and TYRP1. The allele-

specific correlation of expression levels between TYRP1 and SLC45A2 is interesting

because it suggests a coordinated expression of pigmentation genes and that SLC45A2

polymorphisms can alter the expression of TYRP1, most likely through altering the

levels of SLC45A2. The exact mechanism by which this can occur has not been

elucidated, but may be related to the role that SLC45A2 plays in trafficking TYRP1

from the trans-Golgi network to Stage II melanosomes (Costin et al., 2003). Subtle

changes in SLC45A2 expression may affect the trafficking and routing efficiency of

sorting vesicles that contain TYRP1. Thus, availability of TYRP1 for melanin

production would be affected, which could contribute to a currently unidentified

feedback loop that regulates TYRP1 expression. Our results suggest that the –dup allele,

which is more common in people with fair skin, increases SLC45A2 promoter activity

(Graf et al., 2007), which also leads to an increase in TYRP1 expression. An increased

SLC45A2 promoter activity and hence SLC45A2 expression, as well as increased TYRP1

expression would logically lead to an increase in melanin production, resulting in darker

rather than lighter skin. However, increased expression of SLC45A2 may imbalance the

levels of melanosomal proteins required for optimal melanin production, thereby

activating a mechanism that decreases the effectiveness of other melanosomal proteins

to contribute to melanin production.

To further characterise the SLC45A2 promoter, five deletion constructs were used to

investigate transcriptional activity in a melanoma cell line. The pGL3-Luc1 (–206 to –4)

and pGL3-Luc2 (–404 to –4) constructs were designed to incorporate a region

immediately upstream of the putative transcription start site. We recently identified two

transcription start sites for SLC45A2 located at –78 and –111 (Graf et al., 2007) while

another at –92 has also been reported (Genbank entry No. NM_016180.3). Indeed, the

pGL3-Luc1 fragment showed an activity almost 15-fold higher than the promoter-less

pGL3-basic vector, indicating the region –206 to –4 is sufficient to permit constitutive

expression of SLC45A2. Numerous putative SP1 sites were identified in the pGL3-Luc1

fragment, which are known to be crucial for basal transcription in TATA-less promoters

(Botella et al., 2001; Ross et al., 2002). Interestingly, the pGL3-Luc2 construct showed

an almost 3.5-fold higher activity than the pGL3-Luc1 construct indicating the region

121

between –404 and –206 may contain enhancer elements which further drive the

expression of SLC45A2.

The pGL3-Luc3 and pGL3-Luc4 constructs were designed to investigate the importance

of a polymorphic region of the SLC45A2 promoter located between –1203 and –1016.

This region contained a putative MITF binding site, which we have previously

suggested may be important in the transcriptional regulation of SLC45A2. Based on our

EMSA experiment results, which provided preliminary evidence for the differential

binding of nuclear proteins due to allelic variation in this region, and that the –dup allele

had been associated with increased transcriptional activity, we expected to see a

significant difference in luciferase activity between the pGL3-Luc4 which contained the

–dup allele and the shorter pGL3-Luc3 construct. However, although pGL3-Luc4 did

exhibit an increased luciferase activity over pGL3-Luc3, this increase only approached

significance (P = 0.069).

In this study we have extended the characterisation of the SLC45A2 promoter by using

in silico and in vitro methods. Our bioinformatic analysis revealed that the SLC45A2

promoter is TATA-less and contains many tandem repeats, confirming observations

previously made by Du and Fisher (2002). Luciferase assays with promoter constructs

of increasing size showed significant differences in transcriptional activity, suggesting

the existence of functionally important promoter elements. Smaller and more targeted

constructs will help to identify the exact location of these elements. The functional

impact of two promoter polymorphisms (c.–1176_–1174dupAAT and c.–1169G>A)

was investigated and it was shown that the +dup/A haplotype consistently exhibited a

higher DNA-nuclear protein binding affinity than the –dup/G haplotype. Consistent

with previous work (Du and Fisher, 2002), initial investigations concluded that the

bound protein was most likely not MITF. However, the absence of a supershift in MITF

control EMSA experiments creates some doubt as to the validity of this preliminary

conclusion. We have also shown that SLC45A2 promoter polymorphisms are

significantly correlated with altered TYRP1 mRNA expression, and possibly also with

MITF expression, further supporting the functional role of SLC45A2 promoter

polymorphisms in the coordinated expression of pigmentation genes. Taken together,

these results suggest that the SLC45A2 promoter and the polymorphisms within it, are

key contributors to normal human pigmentation variation.

122

Methods

Cell culture, RNA extraction and cDNA synthesis

MM96 cells were cultured in RPMI 1640 (Invitrogen, Mount Waverly, Victoria,

Australia) medium supplemented with 10% Fetal Bovine Serum (Invitrogen) and 1×

Antibiotic-Antimycotic (Invitrogen) and grown under standard conditions in a

humidified incubator at 37oC and 5% CO2. Human melanoblasts were established from

neonatal foreskin tissue and cultured in 5% O2 and 5% CO2 as described (Cook et al.,

2003), except that 50nM endothelin 3 was used. All cultures were propagated as

melanoblasts, and then subsequently differentiated to melanocytes through cultivation

in melanocyte growth medium for 7 days as described (Cook et al., 2008). RNA

extractions were carried out on differentiated cells (melanocytes) using the TRIZOL®

reagent (Invitrogen Corporation, Waverly, Victoria, Australia) as per manufacturer’s

instructions except for an extended precipitation of 1 hr at –20oC. Total RNA was

subject to rigorous DNase treatment with DNA-free™ (Ambion, Austin, TX), quantified

using UV spectrophotometry and 0.3 μg used for cDNA synthesis by SuperScript™ III

reverse transcriptase with random primers (Invitrogen).

Luciferase reporter assays

A 1932 bp (–1935 to –4) fragment of the SLC45A2 promoter was directionally cloned

into a pGL3-Basic vector (Promega, Annandale, NSW, Australia) as previously

described (Graf et al., 2005). This fragment contained the C, –dup, G haplotype for the

polymorphisms, c.–1721C>G (rs13289), c.–1176_–1174dupAAT, and c.–1169G>A

(rs6867641) and is referred to as pGL3-Luc5. SLC45A2 cDNA reference sequence with

Genbank accession No. NM_016180.3 was used, with the A of the ATG translation

initiation start site as nucleotide +1. Mutation nomenclature follows the HGVS

guidelines (www.hgvs.org/mutnomen) with all promoter polymorphisms numbering

using the cDNA as a reference. Four smaller fragments of decreasing size were

generated by Platinum® Pfx DNA Polymerase (Invitrogen) PCR amplification using the

pGL3-Luc5 as a template. Forward primers (Table 1), with an MluI restriction enzyme

site incorporated, were used with a common reverse primer with a BglII restriction

enzyme site incorporated (5'-TTCTAGATCTCACTGGGAGAGGAACCTTC-3'; BglII

site -TTCTAGATCT). The location of existing polymorphisms in the 1932 bp

fragment was a factor in designing the size of the five promoter constructs investigated

123

(Figure 1). The five promoter constructs will be referred to as pGL3-Luc1 (smallest)

through to pGL3-Luc5 (largest). Bi-directional sequencing confirmed all constructs

contained no introduced polymorphisms. Promoter activity of the five promoter

constructs was evaluated by transient transfection of melanoma cells (MM96) followed

by luciferase reporter assays as previously described (Graf et al., 2007). Data were

normalised for transfection efficiency and expressed as a fold change compared to cells

transfected with only the pGL3-basic vector. All experiments were performed four

times in triplicate. Data were analysed by one way analysis of variance (ANOVA)

followed by post-hoc Least Difference for Significance t-tests.

Bioinformatic promoter analysis

An extensive TF-binding search was undertaken using MatInspector

(http://www.genomatix.de/matinspector), TFBIND (http://tfbind.hgc.jp/) (Tsunoda and

Takagi, 1999), and Gene Promoter Miner (http://gpminer.mbc.nctu.edu.tw/) to identify

classical cis-acting sequence elements as well as other positive or negative regulatory

element motifs. The Repeat Masker program (http://www.repeatmasker.org/) and

Tandem Repeat Finder program (http://tandem.bu.edu/trf/trf.html) (Benson, 1999) were

used to identify repetitive DNA elements. The presence of CpG islands was assessed

using the Sequence Manipulation Suite CpG Islands program

(http://bioinformatics.org/sms2/cpg_islands.html). An additional region spanning from –

1935 to the 3' end of the alpha-methylacyl-CoA racemase (AMACR) gene

(approximately –3265) was also analysed using Repeat Masker.

Electrophoretic mobility shift assays (EMSA)

EMSAs were performed using the LightShift® Chemiluminescent EMSA kit (Pierce,

Rockford, USA) as per the manufacturer’s instructions. Nuclear protein extracts were

obtained from melanoma cells (MM96) using the NE-PER® Nuclear and Cytoplasmic

Extraction Kit (Pierce) and were quantified using the bicinchoninic acid (BCA) protein

assay kit (Pierce). Two double-stranded probes were designed that contained the –

dup/G haplotype or the alternate +dup/A haplotype of the c.–1176_–1174dupAAT and

c.–1169G>A polymorphisms. These probes included the entire MITF consensus binding

site. The –dup/G haplotype probe sequence was 5′-

CCATGTGTGTGGTCATGTGTAATGTGTGTGAGAGGGG-3′ whilst the +dup/A

alternate haplotype probe was 5′-

124

CCATGTGTGTGGTCATGTGTAATAATGTGTATGAGAGGGG-3′ (Polymorphic

sites are in bold). The inclusion of GGG at the 3′ end of each oligonucleotide ensured

end labelling did not interfere with complementary oligonucleotide annealing, as

instructed by the Biotin 3′ End DNA Labelling Kit (Pierce). Double-stranded probes

were prepared by mixing equal amounts of biotin-labelled single-stranded probes and

cooling from 95oC to 15oC over 1 hr. Binding reactions were prepared on ice and

contained 4 µg of nuclear extract, with or without a 100-fold molar excess of unlabelled

probe, 10 mM Tris (pH7.5), 50 mM KCl, 1 mM DTT, 1 µg Poly dI.dC and 50 fmol of

labelled probe. For supershift experiments nuclear extracts were pre-treated with 2 µg of

a monoclonal anti-MITF antibody (C5+D5) (NeoMarkers, Fremont, USA) in binding

buffer at 4oC overnight. Labelled probe was added to the reaction and further incubated

for 30 min at room temperature. DNA-protein complexes were resolved by

electrophoresis in a 6% polyacrylamide (29:1 acrylamide/bisacrylamide) gel in 0.5

TBE buffer for 2 hr at 100 V. The DNA-protein complexes were electrophoretically

transferred in chilled 0.5× TBE buffer to a positively charged nylon membrane

(Amersham) for 1 hr at 90 V, UV cross-linked for 2 min at 254 nm, and detected as per

the manufacturer’s instructions.

SNP Genotyping of c.–1721C>G (rs13289), c.–1176_–1174dupAAT, c.–1169G>A

(rs6867641) in primary melanocytes.

A total of 128 primary melanoblast and melanocyte cell lines were genotyped for the c.–

1721C>G (rs13289), c.–1176_–1174dupAAT and c.–1169G>A polymorphisms as

previously described (Graf et al., 2007).

Gene expression quantitation and analysis

Gene expression levels in twenty-two melanocyte or melanoblast cell lines were

quantitated using TaqMan® gene expression assays (Applied Biosystems, Foster City,

CA, USA) for SLC45A2 (Hs01125484_m1), MITF (Hs00165156_m1),ASIP

(Hs00181770_m1) and TYRP1 (Hs00167051_m1), together with TaqMan® PCR master

mix. Transcript levels, normalised against 18S ribosomal RNA that was quantified

using a Pre-Developed TaqMan® Assay for 18S (Applied Biosystems), were calculated

using the delta Ct method (delta Ct = Cttest gene– Ct18S). Assays were performed in 96-

well plates on an Applied Biosystems 7500 Real-Time PCR System using a two-step

thermal cycling protocol of 40 cycles of 95oC for 15 s and 60oC for 1 min. For analysis,

125

log-transformed expression level of the 22 samples was regressed on c.–1176_–

1174dupAAT genotype, coded as 0 for +dup/+dup (7 samples), 1 for –dup/+dup (11

samples), and 2 for –dup/–dup (4 samples).

Western-blot analysis

MM96 nuclear cell extracts were diluted 1:1 with SDS loading buffer and

electrophoretically separated on a 12% SDS-polyacrylamide gel at 50 mA for 1 hr.

Separated proteins were transferred at 350 mA for 1 hr to Hybond C nitrocellulose

membrane (Amersham Biosciences, North Ryde, NSW, Australia) and left to dry

overnight. The membrane was blocked at room temperature for 2 hr in 5% skimmed

milk in TBS (10 mM TRIS pH 7.5; 150 mM NaCl). MITF proteins were detected by

addition of a monoclonal anti-MITF antibody (C5+D5) (NeoMarkers, Fremont, USA) at

a concentration of 1 µg/ml of blocking buffer (1:670 dilution) for 1 hr at room

temperature. The membrane was washed for 20 min with TBST (20 mM TRIS pH 7.5;

500 mM NaCl; 0.05% Tween-20) replacing the buffer five times followed by washing

for 10 min with TBS replacing the buffer twice. The membrane was then incubated with

a polyclonal rabbit anti-mouse IgG-conjugated horseradish peroxidase (diluted 1:1000;

Dako, Botany, NSW, Australia) for 1 hr at room temperature in 5% skimmed milk +

TBS. The membrane was washed as described above and chemiluminescence was

detected by the ECL Plus Western-blotting Detection System (Amersham) and X-ray

film.

126

AcknowledgementsThis work was supported by a Queensland University of Technology Postgraduate

Research Award and Cooperative Research Centre for Diagnostics Postgraduate

Research Award to JG. We thank Associate Professor Rickard Sturm and Dr Anthony

Cook for providing melanoblast/melanocyte cell lines, assistance with laboratory

experiments and thoughtful discussion of this work. We also thank Dr John Lai for

technical assistance with EMSA, Dr Chris Barker for Western-blotting and also people

who kindly participated in the study.

127

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Tables

Table 1. Forward primers for SLC45A2 promoter fragments cloned into pGL3-basic

1 MluI site: GGCTACGCGT

FRAGMENT FORWARD PRIMER SEQUENCE1

pGL3-Luc1 5'-GGCTACGCGTATCTCTGTCATGCTTTCCAG-3'pGL3-Luc2 5'-GGCTACGCGTACTGTCTGAGAGAGCCCATG-3'pGL3-Luc3 5'-GGCTACGCGTTCATGTGTAATAACGTGAG-3'pGL3-Luc4 5'-GGCTACGCGTTGAGAGAAGCCATGAGTCC-3'pGL3-Luc5 5'-GGCTACGCGTGTATGGTTTATTCACTCAG-3'

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Figure legends

Figure 1. Luciferase reporter assays of SLC45A2 promoter constructs and

schematic of SLC45A2 promoter region.

Five deletion constructs, pGL3-Luc1 to pGL3-Luc5, were transiently transfected

individually into a MM96 melanoma cell line and data were normalised for transfection

efficiency to Renilla Luciferase activity and expressed as a fold change in relation to the

promoter-less pGL3-basic vector. Diagrammatic representations of the SLC45A2

promoter fragments fused to the luciferase reporter gene are shown with their

corresponding relative luciferase activity shown with open bars. The results are

expressed as means ± standard error for four experiments, in triplicate for each

construct. The schematic of the SLC45A2 promoter is in scale with the construct

schematic and highlights polymorphisms as white lines (alphabetised) on the SLC45A2

promoter region (solid black bar). 5' untranslated region (UTR) and exon one are also

shown but are not to scale. A table below the schematic summarises information

regarding the promoter polymorphisms including NCBI dbSNP rs number, type of

polymorphism and validation status.

Figure 2. Allele-specific gene expression

A) Correlation between the –dup allele of the c.–1176_–1174dupAAT polymorphism

and TYRP1 expression in melanocytes and melanoblasts, B) The highest SLC45A2 and

TYRP1 expression was observed in the samples which were homozygous for the –dup

allele. Columns represent the mean SEM.

132

Figure 3. Analysis of SLC45A2 promoter polymorphisms by EMSA.

A representative EMSA using double-stranded probes containing the –dup/G haplotype

or the alternate +dup/A haplotype of the c.–1176_–1174dupAAT and c.–1169G>A

polymorphisms. Probes were used in binding reactions with MM96 nuclear protein

extracts. Lane 1 represents the addition of –dup/G haplotype free probe without the

addition of nuclear extract. Lane 2 contains –dup/G haplotype probe plus MM96

nuclear extract and Lane 3 includes the addition of a 100-fold molar excess of

unlabelled –dup/G haplotype probe. Lanes 4–6 are identical to Lanes 1–3 except the

+dup/A haplotype probe was used instead. This experiment was duplicated two more

times using probes that were biotinylated in separate biotin 3′ end-labelling reactions to

account for differences in biotinylation efficiency. In all experiments, the +dup/A

haplotype probe showed consistently higher protein-binding affinity compared with the

–dup/G haplotype probe.

Figure 4. Western-blot analysis for MITF in MM96 melanoma cell line

MM96 melanoma cell nuclear extracts were tested to confirm the presence of MITF

using the monoclonal anti-MITF antibody (C5+D5). The two shorter forms of MITF

were detected (52 kDA and 56 kDa) as expected. Only one lane shown.

133

Figures

Figure 1. Luciferase reporter assays of SLC45A2 promoter constructs and schematic of SLC45A2 promoter region

134

Figure 2. Allele-specific gene expression

y = 0.3358x - 1.4036R2 = 0.1938

-2.5

-2

-1.5

-1

-0.5

0

TYRP1 mRNA level relative to 18s

+dup/+dup +dup/–dup –dup/–dup

B

A

SLC45A2 TYRP1

MITF

0.0

0.1

0.2

0.3

0.4

0.5

0.6

SLC45A2 promoter polymorphism

+dup/+dup

+dup/-dup

-dup/-dup

0

0.0005

0.001

0.0015

0.002

0.0025

SLC45A2 promoter polymorphism

MITF mRNA level relative to 18S .

+dup/+dup

+dup/-dup

-dup/-dup

0

0.0005

0.001

0.0015

0.002

0.0025

0.003

SLC45A2 promoter polymorphism

MATP mRNA level relative to 18S. +dup/+dup

+dup/-dup

-dup/-dup

135

Figure 3. Analysis of SLC45A2 promoter polymorphisms by EMSA.

136

Figure 4. Western-blot analysis for MITF in MM96 melanoma cell line

56 kDa

52 kDa

137

– Chapter 6 –

GENERAL DISCUSSION6.1 Introduction

Human pigmentation is one of the most distinguishable human physical characteristics,

with variation existing between and within populations. Human skin colour variation

exhibits geographical variation and has been used to define races as well as explain

evolutionary adaptations to environmental stimuli (Jablonski and Chaplin, 2000;

Millington and Levell, 2007; O'neill, 2007). Endeavouring to understand the genetic

variation that underlies this geographical variation will undoubtedly shed some light on

the evolution of human pigmentation, and more specifically how certain phenotypes

were preferentially selected (Sabeti et al., 2006). Since increased pigmentation offers

increased photoprotection, understanding the mechanisms controlling pigmentation is of

distinct interest to cancer researchers (Sturm et al., 2003). Being able to predict

pigmentation phenotype from DNA polymorphisms could enhance cancer susceptibility

estimates and provide a useful tool in forensic science (Jobling and Gill, 2004). Normal

pigmentation variation cannot be explained by simple Mendelian genetics. Human

pigmentation is a complex physical trait (also known as a quantitative trait loci) and

likely involves over 120 genes, of which several important genes contribute to the bulk

of variation observed (Oetting and Bennett, 2003). Therefore, unravelling the genetic

basis of normal human pigmentation variation will be decidedly difficult. Here, I

investigate the role of SLC45A2, a gene suggested to play a pivotal role in that variation.

Most of the knowledge regarding the role that SLC45A2 plays in pigmentaion has only

been generated recently. However, mutations in the orthologous mouse gene, Slc45a2,

originally known as underwhite, were first described in the early 1960s (Dickie, 1964).

Mutant underwhite alleles (Figure 9, Chapter 2) cause generalised hypopigmentation of

the eyes and fur by significantly reducing or abolishing tyrosinase activity and melanin

production (Sweet et al., 1998; Lehman et al., 2000). In humans, Newton et al. (2001)

were the first to identify SLC45A2 and show that mutations in it underlie a fourth type

of oculocutaneous albinism (OCA4). Newton and colleagues (2001) also identified the

non-synonymous polymorphism, p.Phe374Leu, and presumed it to be non-pathogenic

138

(neutral) because it was also detected in non-albino or normally-pigmented controls.

Evidence that the p.Phe374Leu polymorphism was involved in normal pigmentation

variation was first published by Nakayama et al. (2002), who also investigated another

non-synonymous polymorphism (p.Glu272Lys). Nakayama and colleagues (2002)

showed distinctive frequency distributions of these two non-synonymous SNPs between

White South African, Ghanaian, Japanese and New Guinea Islander populations,

suggesting that the observed differences were due to pigmentation differences between

the populations. However, they did not show intra-population allele frequency

differences associated with pigmentation variation. Thus, a significant knowledge gap

existed. Do the non-synonymous polymorphisms affect intra-population pigmentation

variation? Are there inter-population allele frequency differences between other

populations and do these differences correlate with pigmentation differences? Do other

SLC45A2 polymorphisms, such as those in the promoter, affect pigmentation variation?

How do they do it?

This thesis has attempted to answer these questions by carrying out two genetic

association studies and performing functional promoter experiments. Here, the key

findings of my research and what they mean to our understanding of human

pigmentation are discussed, as well as including discussion about some of the relevant

experimental considerations and limitations, future experiments, and the possible

applications of this work.

6.2 Experimental considerations and limitations

There are several key considerations when undertaking a candidate gene association

study (Chapter 3 and 4). These include polymorphism selection, sample size, collection

and phenotyping, and genotyping methodology. The ultimate aim of any association

study is to detect a significant difference in variant allele frequencies between different

groups. In order to maximise the chance of significance, the selection of polymorphisms

is a key consideration. Associations will occur for four reasons: (1) the polymorphism

has a causal role; (2) the polymorphism is not causal but is in linkage disequilibrium

with a nearby causal variant; (3) the association is a false positive due to random

chance; or (4) the association is due to underlying population stratification or admixture

(Cordell and Clayton, 2005). The association studies reported here have concentrated on

polymorphisms that are putatively causal. The most obvious targets are non-

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synonymous coding polymorphisms because they alter the amino acid sequence. At the

time of the study reported in Chapter 3, p.Phe374Leu and p.Glu272Lys were the only

non-synonymous polymorphisms identified in SLC45A2. The p.Cys318Arg

(rs35990319) polymorphism was only reported in September 2005. Causal

polymorphisms may also occur in the regulatory region of a gene, such as in the

promoter (usually immediately 5' to the transcription start site). Variations in regulatory

regions may cause differences in mRNA levels, timing and tissue-specificity of gene

expression, which ultimately lead to quantitative differences in phenotype (Mackay,

2001). The putative SLC45A2 promoter was screened and a novel duplication

polymorphism was identified. This, and other known polymorphisms in the vicinity,

were genotyped as described in Chapter 4.

Sample size is an important consideration in an association study because it determines

the magnitude of any frequency differences that can be thought of as being real, or

significant. Large sample sizes can detect small genetic differences at a statistically

significant level, while smaller sample sizes can only detect common genes with large

effects (Ioannidis et al., 2003). In the two association studies described in this thesis

(Chapters 3 and 4), 608 and 700 samples were used, respectively, enabling a power of

greater than 80%. To maximise the probability of detecting an association between

SLC45A2 genotype and pigmentation phenotype (i.e. increase the power of the study), a

focus on maximising sample size and volunteer recruitment was required in the early

stages of this project. In the interest of DNA quality and quantity, DNA samples were

obtained by collecting blood through venipuncture. Recruitment of volunteers was time-

consuming and difficult, which may have been due to the invasive method of blood

collection. The use of buccal swabs and other non-invasive DNA collection methods

were considered in order to increase recruitment, but at the time, methods would not

have provided DNA of suitable quality or quantity. Recently, DNA collection through

buccal cells and white blood cells in saliva has become a viable alternative. For

example, Oragene DNA self-collection kits (DNA Genotek Inc) can now collect

sufficient DNA of high quality from 2ml of saliva. Using such a method should increase

donor numbers, be easier to purify DNA, be safer to handle, and long-term storage can

be at room temperature. Therefore, future sample collection should involve these

recently developed DNA collection methods rather than invasive techniques to

maximise recruitment and ease of sample processing.

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Another important consideration in association studies is sample phenotyping. In this

study, pigmentation phenotype data were collected by way of questionnaire with all data

being subjectively determined by one person (as described in the materials and methods

section of Chapter 3). Whilst this method has some advantages over self-report data,

some subjectivity was inevitable with this phenotyping method. Therefore, having only

one person doing the phenotyping reduced the degree of subjectivity inherent with using

a method that is not objective. Ideally, there are several, more objective measures of

pigmentation that could have been used.

For skin colour, Fitzpatrick’s Skin Phototype scale can be used to classify skin colour,

not only by level of pigmentation but also by the effects of UVR exposure (Fitzpatrick,

1988; Astner and Anderson, 2004). Skin melanin content can also be non-invasively

determined using tristimulus reflectometry, narrow-band spectrometry and diffuse

reflectance spectrometry (Shriver and Parra, 2000; Makova and Norton, 2005). For

constitutive skin pigmentation analyses, measures are taken on non-exposed areas, such

as under the arm or on the buttocks. Skin reflectance measurements produce a

numerical, scalar measurement of skin colour, which can be analysed using more

powerful parametric statistics such as regression analysis and analysis of variance,

rather than being constrained by ordinal scale data. This technique has been used to

good effect in several analyses of skin pigmentation candidate genes (Akey et al., 2001;

Bonilla et al., 2005; Lamason et al., 2005). Spectrophotometric methods can also be

used for hair colour determination, providing the volunteer has not dyed their hair.

Alternatively, hair colour can be classified by comparison to a range of standard hair

colours, such as the Haafarbentafel Fischer-Saller hair colour swatches (Suter, 1979;

Duffy et al., 2004). If hair samples are collected, the level of eumelanin and

pheomelanin content can be determined by chemically degrading the pigment polymer

and using high performance liquid chromatography (HPLC) (Ito and Wakamatsu,

2003). Until recently, it was common for eye colour phenotyping to be carried out by a

single person, and classifications stratified into three or more categories (Rebbeck et al.,

2002; Duffy et al., 2007), which were later independently validated by a second person.

A new method for eye colour phenotyping has been developed using digital

photography and in silico spectrophotometry that quantifies the eumelanin content of

each iris. This method generates an Iris Colour Score (C) irrespective of iridial

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patterning whereby low C values are darker eyes and high C values are lighter eyes

(Frudakis et al., 2007).

More objective measures of phenotyping will inevitably decrease the likelihood of false

associations and increase the chance of independent replication of any identified

associations (Terwilliger and Goring, 2000). In light of the strong associations observed

in the two association studies in this research, the phenotyping method used here

probably did not lead to spurious associations. Admittedly, more object measures could

have been used, however, this would have added to the cost and complexity of

phenotyping volunteers, which may ultimately decrease the level of participation.

Nonetheless, it is recommended that future studies involving the genetic contribution of

SLC45A2 or any other pigmentation gene to pigmentation variation should ideally

utilise more objective measures than those used in the present study.

During the course of this research, the development of low-cost, high throughput

genotyping technology became available. Outsourced genotyping services now offer a

quicker and cheaper alternative to genotyping large numbers of samples and are

recommended over conventional genotyping methods. For example, the Australian

Genome Research Facility (AGRF) offers a custom SNP genotyping service by way of

homogenous MassExtend and iPlex assays on the Sequenom Autoflex Mass

Spectrometer (http://www.agrf.org.au/index.php?id=41).

6.3 Principal outcomes, significance and applications of this

research

The major outcomes of this thesis are as follows:

1.The p.Phe374Leu and p.Glu272Lys non-synonymous coding polymorphisms are

associated with normal human pigmentation variation. Specifically, the 374Leu and

272Lys alleles are more commonly seen in some darkly pigmented populations and

in Caucasians with darker skin, hair and eyes.

2.A novel three base pair duplication polymorphism (c.–1176_–1174dupAAT) was

identified in the SLC45A2 proximal promoter. The +dup allele, and other alleles (–

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1721G and –1169A) of previously reported promoter polymorphisms (c.–1721C>G

and c.–1169G>A), are strongly associated with olive skin colour in Caucasians.

3.The SLC45A2 gene has at least four transcription start sites, two of which were

identified in this thesis.

4.The c.–1176_–1174dupAAT polymorphism and the tightly linked c.–1169G>A

polymorphism were demonstrated to influence DNA-protein binding and SLC45A2

transcriptional activity.

5.Although bioinformatic analysis suggested that a MITF binding site may exist at the

c.–1176_–1174dupAAT polymorphism region, MITF was not observed to directly

bind to this site.

6.SLC45A2 promoter polymorphisms may affect the expression of TYRP1 and

potentially other melanosomal-protein coding genes.

The association study results in Chapters 3 and 4 showed for the first time that

SLC45A2 coding and regulatory region polymorphisms were associated with normal

intra-population pigmentation variation in Caucasians. Previously, SLC45A2 coding

region polymorphisms had only been shown to have different allele frequencies in

different population groups (Nakayama et al., 2002). The significance of these results

are relevant to several different facets of pigmentation research, including the evolution

of human pigmentation, genetic susceptibility to skin cancer, the development of

pigmentation altering therapeutics, and the genetic inference of human pigmentation

phenotype. As such, SLC45A2 is mentioned in numerous international patents, such as

those for eye and hair colour inference (WO/2005/079331; WO2002/097047), ancestry

inference (WO/2004/016768), ancestry inference by multiplex assay

(WO/2006/089238), cancer treatment response (WO/2003/045227) and cosmetic

enhancement (WO/2008/005533).

One of the major outcomes of finding that the Phe374 allele is present at very high

frequency in Caucasians and is involved in normal pigmentation variation within

Caucasians (Chapter 3), is that it has led to other researchers investigating the role of

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this polymorphism in the evolution of human pigmentation. The high frequency of the

Phe374 allele in Caucasians and low frequency in non-Caucasian populations raised the

possibility of directional selection that favours the Phe374 allele in some environmental

conditions, such as those of low sunlight in northern European latitudes (Soejima et al.,

2006). Indeed, several statistical tests of directional selection suggested that a 7.55 kb

region surrounding the F374L polymorphism in a European population was under

positive selection (Soejima et al., 2006). Studies such as this can provide genetic

evidence for the adaptive nature of human skin colour thereby supporting possible

explanations for variation due to photoprotection against sun-induced cancer, sexual

selection and vitamin D synthesis.

Given that people with lighter skin colour have an increased susceptibility to skin

cancer (Bliss et al., 1995), it follows that any polymorphisms associated with a lighter

phenotype will be of interest to cancer researchers. To date, no studies have been

published that investigate the relationship of SLC45A2 mutations with skin cancer risk.

Much of the work related to pigmentation phenotype and skin cancer risk has involved

MC1R and the red hair colour (RHC) phenotype characterised by red hair, fair skin, low

tanning ability and propensity to freckle (Sturm, 2002). This thesis has shown that

coding alleles (Phe374 and Glu272) and regulatory alleles (–1721C, –dup and –1169G)

of SLC45A2 polymorphisms are associated with lighter skin colour. Hence, it would be

logical that any future studies investigating genotype and skin cancer susceptibility

should include a screen for these SLC45A2 polymorphisms.

The significance of the work in Chapter 3 is evidenced by the strong interest shown

towards it by the multinational company, Unilever. Unilever’s interest in pigmentation

research lies in the demand that some dark-skinned populations have for lightening their

natural skin colour (Dixson et al., 2007). Lighter skin colour is perceived to be more

beautiful and hence is a much-desired physical attribute in some countries. Skin

lightening products are also used to achieve uniform pigmentation in people with

hyperpigmentation conditions. Therefore, investigating the mechanisms of skin

pigmentation may lead to skin lightening agents that could potentially be marketable. At

the 19th International Pigment Cell Conference in September 2005, Unilever researcher,

Dr Rebecca Ginger, presented data showing that Phe374 allele of the p.Phe374Leu

polymorphism is strongly associated with lighter skin in South Asian populations

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(India, Bangladesh, Sri Lanka and Pakistan) (Ginger et al., 2005). Recently, Unilever

have combined with Perlegen to use a genomewide association study approach to

confirm their earlier findings, and to identify novel SNPs associated with pigmentation

variation in South Asian populations (Stokowski et al., 2007). The finding that the

374Leu and 272Lys alleles are more commonly seen in Caucasians with darker skin,

hair and eyes in this thesis, highlights the significance of targeting SLC45A2 for

potential skin-lightening therapeutics as well as supporting the findings of Unilever and

Perlegen. Similarly, the finding that the –1721G, +dup and –1169A alleles are

associated with darker skin colour in Caucasians (Chapter 4) should be of interest to

Unilever. Given that the Phe374 allele is associated with lighter skin colour in both

Caucasians and South Asian populations, it could be expected that the promoter

associations observed in Caucasians (Chapter 4) would also be seen in South Asian

populations. Further, since the promoter polymorphisms are relatively common in the

other populations (African American, Australian Aboriginal and Spanish Basque), they

should also be expected to contribute to pigmentation variation within these

populations.

The genetic ancestry (DNAWitness™ 2.5 and Euro-DNA™ 2.0) and eye colour inference

(RETINOME™) tests available from DNAprint Genomics are the best examples of how

SLC45A2 polymorphism data have contributed to a commercialised product. For

example, the p.Phe374Leu polymorphism, and other SLC45A2 polymorphisms, are

included in a DNAprint patent (WO/2005/079331), which are used in a classification

tree solution for determining eye colour. After an extensive validation process,

DNAprint claim that the RETINOME™ test has an accuracy of 92%, thereby leaving

some room for improvement. With association study data becoming available regularly,

the test should have the scope to include new genetic association data, not only from

SLC45A2 but also from other pigmentation and non-pigmentation genes that provide

suitable discrimination. Therefore, further updates or additions to DNAprint’s ancestry

or pigmentation phenotype inference products, should test the feasibility of including

SLC45A2 promoter polymorphisms. Given that the SLC45A2 promoter polymorphisms

investigated in Chapters 4 and 5 have been shown to affect the function of the gene,

they may provide better candidates for phenotype inference than non-regulatory,

functionally unconfirmed polymorphisms.

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While companies like DNAprint are only concerned with statistical associations to infer

ancestry and pigmentation phenotype, and not the functional relevance of

polymorphisms, this project attempted to explain the underlying mechanisms by which

SLC45A2 promoter polymorphisms can cause pigmentation variation. Functional

experiments are intended to provide biological corroboration to statistical associations.

Therefore, in Chapters 4 and 5, the functional effects of three promoter polymorphisms

(c.–1721C>G, c.–1176_–1174dupAAT and c.–1169G>A) were investigated by

performing luciferase assays and electrophoretic mobility shift assays (EMSA).

Luciferase assays showed for the first time that the C, –dup, G haplotype significantly

increased transcriptional activity compared to the G, +dup, A haplotype (Chapter 4).

This result provided a possible mechanism to explain the statistical association of the G,

+dup, A alleles with darker Caucasian pigmentation, suggesting that decreased

SLC45A2 transcriptional activity, and hence decreased SLC45A2 expression, is

associated with darker Caucasian skin colour. This is somewhat counterintuitive and

raises interesting questions about the role that SLC45A2 plays in determining skin

colour variation. Of course, this result was only observed in one cell line and

confirming this finding in other cell lines would be worthwhile. Nonetheless, in

explaning this interesting finding, it is possible that if certain polymorphisms in

SLC45A2 result in diminished function or abundance of the protein, another

melanosomal protein compensates for this lack of function or abundance, thereby

increasing melanin production. An obvious candidate for such a molecular collaboration

is the OCA2 protein, which has been postulated to work inconjunction with SLC45A2

to regulate intramelanosomal pH (Newton et al., 2001). Further expression analyses

(mRNA and protein levels) that utilise primary melanocytes from lighter and more

darkly coloured Caucasian skin may shed some light on the regulation of key

melanosomal proteins in determining the variation seen in Caucasians and possibly

between different population groups.

EMSAs were used to investigate if the novel polymorphism, c.–1176_–1174dupAAT,

and the tightly linked polymorphism, c.–1169G>A, affected transcription factor protein

binding. These EMSA experiments revealed that the +dup/A haplotype DNA probe

consistently showed a marked increase in DNA-nuclear protein binding affinity

compared to the –dup/G haplotype probe (Chapter 5). Bioinformatic analysis suggested

that the +dup and –1169A alleles disrupt (or –dup and –1169G create) an MITF binding

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site, which is a known regulator of SLC45A2 and other pigmentation genes (Du and

Fisher, 2002). However, supershift EMSAs using an MITF antibody (recommended for

EMSA) could not confirm that the bound protein was in fact MITF, despite MITF

expression being confirmed in the MM96 cell line by Western-blot. This could simply

be explained by MITF not binding to the putative binding site because the sequence

surrounding the core-binding site was not conducive to MITF binding. Considering that

there are over 55 consensus MITF binding sites (E-boxes) located in the proximal

promoter, a mechanism to select certain sites must exist. One possible mechanism,

involving the presence or absence of a 5′ flanking T residue immediately preceding the

core E-box sequence, could not explain the lack of MITF binding since the probes used

in the supershift EMSA contained a T residue in the required location for binding

(Aksan and Goding, 1998). However, if MITF is in fact binding, there are several

explanations that can account for a lack of a supershift. MITF is known to bind to DNA

as both a homodimer and heterodimer (Hemesath et al., 1994). Therefore, it is possible

that the process of heterodimerising with another b-HLH-Zip protein of the MiT family,

could block the antibody detection of MITF by obscuring the N-terminal epitope.

Similarly, it is known that MITF binds with other proteins such as p300 and LEF-1

(Yasumoto et al., 2002; Schwahn et al., 2005), therefore it is possible that a protein-

protein interaction in the MM96 cell line is occurring that specifically prevents direct

binding of MITF to the identified E-box, or to the positive control E-boxes.

Attempts to replicate the conditions used for probes that have been shown to bind MITF

(Vetrini et al., 2004; Schwahn et al., 2005), failed to observe a shift or supershift. The

inability to validate known positive control probes that bind MITF, may be due to a

number of different methodological factors, including incorrect antibody concentration,

accidental exclusion of a vital reagent, lack of probe annealing, or the incompatibility of

buffers used in nuclear protein extraction with reagents used in the positive control

method. It is unfortunate that this could not be further investigated due to the time and

financial constraints of this PhD research. Given additional time, and the purchase of

different MITF antibodies, the reason for not observing MITF supershifts in positive

control probes may have been elucidated. Nonetheless, until the positive control

supershift EMSA can be optimised, it cannot be definitively stated that MITF does not

bind to the SLC45A2 promoter.

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A recent study has provided further evidence for the transcriptional regulation of

SLC45A2 by MITF (Cook and Sturm, manuscript under review). Using HEK293 cells,

this study showed increased luciferase activity of SLC45A2 promoter constructs that

were co-transfected with MITF-M; something that Du and Fisher (2002) did not find

when performing similar experiments. These results suggest that MITF is acting on the

SLC45A2 promoter, but how it is doing so was not investigated. While this thesis was

unable to show that MITF was directly binding to the SLC45A2 promoter E-box in the

vicinity of the c.–1176_–1174dupAAT and c.–1169G>A polymorphisms, this recent

evidence suggests that it is still possible that MITF is acting upon one or more of the

many E-boxes located in the SLC45A2 promoter region. Unless all possible E-box

sequences are tested for their ability to bind MITF, the regulation of SLC45A2 through

the direct binding of MITF cannot be ruled out entirely. The recent findings of Cook

and Sturm warrant further investigation for the direct regulation of SLC45A2 through

MITF binding.

Measuring the expression of SLC45A2, MITF, and TYRP1, in melanocyte/melanoblast

cell lines that were genotyped for SLC45A2 promoter polymorphisms, raised some

interesting questions about the regulation of SLC45A2 and melanogenesis. Firstly, the

absence of the duplication polymorphism (c.–1176_–1174dupAAT) showed an additive

trend towards increased SLC45A2 expression (Figure 2B, Chapter 5). While not

statistically significant, this preliminary result suggests that promoter polymorphisms

could be altering the expression of SLC45A2 and therefore, could be used as a

mechanism for altering human pigmentation phenotypes. More convincing evidence,

such as obtaining statistical significance when using more samples, would be required

before these initial results could be explored further. Secondly, an allele-specific

expression correlation was observed between SLC45A2 promoter polymorphisms and

TYRP1 expression (Figure 2A, Chapter 5). This is interesting because it suggests that a

coordinated expression of pigmentation genes is occurring and that SLC45A2 promoter

polymorphisms could play an important role in altering melanogenesis through the

regulation of other pigmentation genes such as TYRP1. This is not entirely surprising,

considering the crucial role that SLC45A2 plays in sorting TYRP1 from the trans-Golgi

network to stage II melanosomes (Costin et al., 2003). Therefore, promoter

polymorphisms may be influencing the abundance and timing of SLC45A2 expression,

which in turn affects the ability of TYRP1 to play its role in melanogenesis. These

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results have contributed to a greater understanding of the regulation of SLC45A2

expression, and other pigmentation genes. In doing this, a small piece of the puzzle has

been found which should, with further investigation, ultimately benefit the development

of future therapeutic interventions for OCA4 and other pigmentary disorders.

6.4 Future research

This thesis has provided some interesting results that should stimulate further research.

Outlined below are some possible avenues of investigation to further elucidate the role

of SLC45A2 and its polymorphisms.

Initial findings from genetic association studies are often not replicated in a larger

sample size or in different populations (Lohmueller et al., 2003). For this reason,

replication of any genetic association is an important step in validating initial

findings. By showing that the frequency of two non-synonymous polymorphisms

(p.Phe374Leu and p.Glu272Lys) differed between Caucasian, African American,

Australian Aborigine and Asian populations, the results of Chapter 3 have

substantiated initial findings that SLC45A2 coding polymorphisms exhibit

distinctive allele frequency distributions among different population groups

(Nakayama et al., 2002). Similarly, a recent study (Stokowski et al., 2007) has

confirmed initial data (Ginger et al., 2005), reporting that the 374Leu allele of the

p.Phe374Leu polymorphism is associated with darker skin colour in South Asian

populations. Given that the p.Phe374Leu polymorphism is not polymorphic

(monomorphic) in some East Asian populations it would seem unlikely that it

contributes to pigmentation variation in these populations as well as other

populations in which pPhe374Leu is monomorphic (e.g. Sub-Saharan African

populations). Further association studies, which explore the extent to which the

p.Phe374Leu polymorphism, and other SLC45A2 polymorphisms contribute to

normal intra-population pigmentation variation are suggested in other population

groups.

To date, there is no evidence that the p.Phe374Leu and p.Glu272Lys

polymorphisms are in fact causal. These two non-synonymous polymorphisms were

not investigated in this thesis because a collaborating laboratory was carrying out

experiments to determine their functional consequence concurrently with my

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research. It is hoped that any further functional work, possibly utilising primary

melanocyte cell lines that have been genotyped for p.Phe374Leu and p.Glu272Lys,

will help to elucidate the functional nature of these important polymorphisms. Since

SLC45A2 has been postulated to cotransport a sugar molecule with a proton, it has

been linked to regulation of pH within the melanosome (Newton et al., 2001). It

would therefore be logical to investigate if p.Phe374Leu and p.Glu272Lys

genotypes influence intra-melanosomal pH. This could be achieved using a suitable

fluorescent stain such as acridine orange, or another more sensitive and selective

method of melanosomal pH (Fuller et al., 2001). In this way, fluorescent staining

could be used to determine if variations in the distribution patterns and numbers of

acidic organelles (melanosomes) exist, due to SLC45A2 genotype and/or treatment

of cell lines with agents that affect pH (Ancans et al., 2001). In combination with

the results of acridine orange staining, tyrosinase activity and melanin content

assays could be carried out, in order to correlate any pH changes with

melanogenesis measures. Lastly, transmission electron microscopy could also be

used to observe changes in melanosome ultrastructure due to SLC45A2

polymorphisms affecting the maturation of the melanosome.

There is scope for a genetic association study, both within and between populations,

which includes polymorphisms from all major pigmentation candidate genes. This

would allow for the identification of not only individually associated alleles, but

also epistatic interactions and possibly, genotype by environment effects. Murray

Brilliant and colleagues have attempted such an analysis by genotyping 48

polymorphisms in 17 different genes, including SLC45A2, P, MC1R, ASIP, TYRP1

and TYRP2(Brilliant et al., 2005). In terms of pigmentation phenotype inference,

DNAPrint Genomics are also currently working towards a panel of pigmentation

gene polymorphisms for hair and skin colour prediction. Based on their previous

successes with eye colour prediction, it is assumed that it will not be long until a

companion to RETINOME™ is released.

In the work described here, the SLC45A2 promoter was partially characterised.

Further experiments should be carried out to fully understand the role that promoter

variation plays in the function of SLC45A2. Firstly, the SLC45A2 promoter was only

screened to –1287 bp. Therefore, another ~2 kb of upstream promoter sequence

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(before the 3' end of the AMACR gene) could be screened for novel polymorphisms.

In Chapter 4, the C, –dup, G promoter haplotype significantly increased

transcriptional activity compared to the G, +dup, A haplotype. It could not however,

be determined which of the polymorphisms was causal. Making constructs with

different combinations of promoter polymorphisms in addition to the two naturally

occurring hapltoypes, through site-directed mutagenesis, may reveal the causal

polymorphism/s. Other known promoter polymorphisms were identified through

dbSNP searches, and could also, potentially be causal. These polymorphisms were

not detected in our dHPLC screen because they were not polymorphic in the

samples used. Given this, they were assumed not to be causal for the pigment

variation observed here.

In Chapter 5, deletion constructs of the SLC45A2 promoter were created, spanning

the region –1935 bp to –4 bp. A significantly increased luciferase activity was

observed between pGL3-Luc 1 (–206 to –4) and pGL3-Luc2 (–404 to –4),

suggesting that the region between –206 to –404 contains transcription enhancer

elements. Using smaller deletion constructs encompassing this region could help to

narrow the search for important promoter elements that influence SLC45A2

expression. A significant difference in activity was expected to occur between

pGL3-Luc 3 (–995 to –4) and pGL3-Luc 4 (–1284 to –4), since the shorter construct

did not contain a polymorphic region (Figure 1, Chapter 5). An alternative approach

to investigating the signficance of this region may be to create a construct that has

had this polymorphic region deleted and comparing it to the non-deleted construct.

It was also shown that luciferase activity increased as the construct size increased.

To define the entire SLC45A2 promoter, it would be worthwhile designing longer

constructs to observe at which point transcriptional activity was decreased or

abrogated. Smaller constructs may also help to identify stimulatory and inhibitory

elements (in conjunction with bioinformatic analyses), thereby providing a further

source of investigation.

One of the major results in Chapter 5, detailed the differential binding of an

unknown protein to the region surrounding the novel polymorphism, c.–1176_–

1174dupAAT. Based on in silico analysis, it was postulated that the unknown

protein was MITF, however, supershift assays could not confirm this. While it is

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still possible that MITF is a direct transcriptional regulator of SLC45A2 and recent

evidence has provided additional support for this postulation (Cook and Sturm,

manuscript under review), it would be worthwhile exploring other potential

transcription factors that could bind to the SLC45A2 promoter. It is also possible

that Hox-A5 binding (despite the lack of evidence for the role of this protein in

pigmentation regulation) is disrupted and using an antibody to this protein would be

a worthwhile course of action to identify the unknown protein. Another approach

would be to investigate transcription factors that have been implicated with the

regulation of other pigmentation genes (such as MITF, TYR and DCT) or

pigmentation processes, and test these candidates individually with their

corresponding antibodies in supershift assays. Some possible candidates include

BRN2, SOX9 and 10, USF and PAX3 (Yasumoto et al., 1994; Galibert et al., 2001;

Cook et al., 2003; Cook et al., 2005; Corre and Galibert, 2005; Corry and Underhill,

2005; Murisier et al., 2007). Alternatively, a transcription factor protein array (e.g.

Protein/DNA arrays by Panomics) could be used to simultaneously screen a large

number of transcription factors.

6.5 Final conclusions

This thesis has significantly contributed to the field of human pigmentation by

investigating the role of polymorphisms in the SLC45A2 gene. This work has shown, for

the first time, that polymorphisms within the coding and promoter regions of SLC45A2

are associated with intra-population pigmentation variation. This information has been

used to good effect in attempting to explain the evolution of human pigmentation,

particularly the evolution of light skin colour in Europeans. From a commercialisation

perspective, the association data generated in this thesis could potentially be used to

enhance current and future products for the genetic inference of human skin, hair and

eye colour. In addition to providing statistical associations between pigmentation

phenotypes and SLC45A2 polymorphisms, this thesis has shown that SLC45A2

promoter polymorphisms affect the transcriptional activity of SLC45A2, and possibly

the expression of TYRP1, thereby suggesting an important relationship for the control of

melanogenesis. Unravelling the role that this key protein plays in delivering

melanosomal enzymes for melanogenesis will be vital for inventing potential treatments

for oculocutaneous albinism type 4, other pigmentary disorders, and skin colour altering

therapeutics.

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6.6 References

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