Esteban J. Parra, Ph.D., University of Toronto

E-mail: eparra@utm.utoronto.ca

MYO7A

Eumelanosome

Pheomelanosome

UVR

- MSH

EDN3 - MSH

MC1R

MYO 5A

cAMP

PAX3MITFSOX10

ASIP

ATRN

OA1

OCA2TRP1 TYR

TYR

DCT

SILV POMCACTH

- MSH

EDN3

FGFR2

MGF

EDNRB

KIT

TT

DAG

CHS1

HPSADTB3A

SLC24A5

The genetic basis of normal The genetic basis of normal

pigmentation variation in humanspigmentation variation in humans

OverviewOverview

Distribution of skin pigmentation in humansDistribution of skin pigmentation in humans

Brief review of melanin synthesis Brief review of melanin synthesis

Main hypotheses explaining the distribution of skin Main hypotheses explaining the distribution of skin pigmentationpigmentation

The genetic basis and evolution of pigmentation in humansThe genetic basis and evolution of pigmentation in humans

Pigmentation, vitamin D and public health implicationsPigmentation, vitamin D and public health implications

Distribution of skin pigmentation in humansDistribution of skin pigmentation in humans

Skin pigmentation is one of the most variable Skin pigmentation is one of the most variable phenotypes in humansphenotypes in humans

88% of the total variation in skin color is explained by 88% of the total variation in skin color is explained by differences between geographic groupsdifferences between geographic groups

In contrast, for an “average” marker, the percentage of In contrast, for an “average” marker, the percentage of the total variation explained by differences between the total variation explained by differences between continents is just 10-15% continents is just 10-15%

The variation in skin pigmentation is highly The variation in skin pigmentation is highly correlated with latitudecorrelated with latitude

Global map of skin pigmentation levels based on the work of BiasuttiGlobal map of skin pigmentation levels based on the work of Biasutti

Source: D. O’Neil (http://anthro.palomar.edu) Source: D. O’Neil (http://anthro.palomar.edu)

Skin pigmentation and melanin Skin pigmentation and melanin Skin color is mainly due to the pigment Skin color is mainly due to the pigment melanin,melanin, which is produced by which is produced by specialized epidermal cells known as specialized epidermal cells known as melanocytes melanocytes

Within the melanocytes, melanin is synthesized in organelles known as Within the melanocytes, melanin is synthesized in organelles known as melanosomesmelanosomes. . TyrosinaseTyrosinase is the key enzyme involved in melanin is the key enzyme involved in melanin synthesis synthesis

MelanosomesMelanosomes are are transferredtransferred, through dendritic structures, from the , through dendritic structures, from the melanocytes to surrounding cells in the epidermis, known as melanocytes to surrounding cells in the epidermis, known as keratinocyteskeratinocytes

KeratinocytesMelanocyte

Melanosomes

Nucleus

Golgi Aparatus

Skin pigmentation variation Skin pigmentation variation

The number of melanocytes is similar The number of melanocytes is similar

in all human populations. in all human populations. Differences Differences

in skin pigmentationin skin pigmentation are mainly due to: are mainly due to:

Type of melanin synthesized by the melanocytesType of melanin synthesized by the melanocytes Eumelanin: brown/blackEumelanin: brown/black Pheomelanin: red/yellowPheomelanin: red/yellow

Number, size and distribution of melanosomesNumber, size and distribution of melanosomes In persons with In persons with light skinlight skin, , melanosomes are small and melanosomes are small and

packaged in groupspackaged in groups In In dark-skinned personsdark-skinned persons, there are , there are moremore melanosomes, melanosomes,

which are larger and packaged as a single entitywhich are larger and packaged as a single entity

The evolution of skin pigmentationThe evolution of skin pigmentationMany Many hypotheseshypotheses have been put forward to explain the have been put forward to explain the distribution of skin pigmentation in humans, and in particular, distribution of skin pigmentation in humans, and in particular, the strong correlation of pigmentation with latitude the strong correlation of pigmentation with latitude

Ultraviolet radiationUltraviolet radiation (UVR) seems to be the key environmental (UVR) seems to be the key environmental variable shaping pigmentation variationvariable shaping pigmentation variation

In equatorial and tropical environments with high UV radiation, In equatorial and tropical environments with high UV radiation, dark skin was probably selected because:dark skin was probably selected because:

Dark skin protects against Dark skin protects against skin cancerskin cancer

Dark skin protects against Dark skin protects against sunburnsunburn (severe sunburn can lead to infection (severe sunburn can lead to infection and interfere with an efficient sweating mechanism)and interfere with an efficient sweating mechanism)

Dark skin protects against Dark skin protects against folate destructionfolate destruction by UV radiation. by UV radiation.Folate is required for Folate is required for adequate fetus developmentadequate fetus development, and also , and also important in important in reproduction (lack of folatereproduction (lack of folate can result in can result in fetal defectsfetal defects and also and also male infertility)male infertility)

Out of Africa and the evolution of light skinOut of Africa and the evolution of light skin

When modern humans migrated out of Africa, they began to When modern humans migrated out of Africa, they began to occupy regions with lower UV radiation occupy regions with lower UV radiation

The protection provided by high melanin levels was The protection provided by high melanin levels was no longer no longer necessarynecessary

Less risk to skin cancer, sunburn and folate destructionLess risk to skin cancer, sunburn and folate destruction

Additionally, a new selective factor may also have played an Additionally, a new selective factor may also have played an important role, important role, vitamin D synthesisvitamin D synthesis

Vitamin DVitamin D is necessary for proper is necessary for proper absorption of calciumabsorption of calcium, and is , and is involved in many other physiological processes involved in many other physiological processes

In our body, In our body, vitamin D is synthesizedvitamin D is synthesized through the action of through the action of UV radiationUV radiation In environments with low UV radiation, dark skin would be harmful, In environments with low UV radiation, dark skin would be harmful,

because it would interfere with proper production of vitamin Dbecause it would interfere with proper production of vitamin D

The genetic basis of normal variation in The genetic basis of normal variation in skin pigmentationskin pigmentation

Studies in animal models (e.g. Studies in animal models (e.g. mouse) and pigmentation disorders mouse) and pigmentation disorders (e.g. albinism) have provided (e.g. albinism) have provided important insights on the genes important insights on the genes involved in the pigmentation involved in the pigmentation pathway pathway

127 loci have been linked to 127 loci have been linked to coat color variation in mice coat color variation in mice

However, the genetic basis of However, the genetic basis of normal pigmentation variation in normal pigmentation variation in humans remains largely unknownhumans remains largely unknown

Research carried out in the last five Research carried out in the last five years is shedding new light on this years is shedding new light on this topic. Below, I review the main topic. Below, I review the main genes involved in pigmentation genes involved in pigmentation variation in humansvariation in humans

MYO7A

Eumelanosome

Pheomelanosome

UVR

- MSH

EDN3 - MSH

MC1R

MYO 5A

cAMP

PAX3MITFSOX10

ASIP

ATRN

OA1

OCA2TRP1 TYR

TYR

DCT

SILV POMCACTH

- MSH

EDN3

FGFR2

MGF

EDNRB

KIT

TT

DAG

CHS1

HPSADTB3A

SLC24A5

MC1R and pigmentationMC1R and pigmentationBy far, the most extensively studied gene By far, the most extensively studied gene is the is the melanocortin 1 receptormelanocortin 1 receptor (MC1R). (MC1R). This gene regulates the This gene regulates the switch between switch between the production of eumelanin and the production of eumelanin and pheomelaninpheomelanin

Rana et al. (1999) and Harding et al. (2000) described that in dark-skinned Rana et al. (1999) and Harding et al. (2000) described that in dark-skinned populations living in tropical regions (West Africans, South Asians and Papuans), populations living in tropical regions (West Africans, South Asians and Papuans), the level of polymorphism in this gene is very low the level of polymorphism in this gene is very low

This has been interpreted as the result of This has been interpreted as the result of strong functional constraintstrong functional constraint on this gene, probably on this gene, probably to minimize the harmful effects of UVRto minimize the harmful effects of UVR

In contrast, in European, East Asian and Southeast Asian In contrast, in European, East Asian and Southeast Asian populations MC1R shows populations MC1R shows very high levels of polymorphismvery high levels of polymorphism, , with many non-synonymous variantswith many non-synonymous variants

Source: Makova and Norton (Peptides 26:1901,2005)Source: Makova and Norton (Peptides 26:1901,2005)

MC1R variants, red hair and sun sensitivityMC1R variants, red hair and sun sensitivityIn European populations, some non-synonymous MC1R variants (e.g. Arg151Cys, Arg160Trp and In European populations, some non-synonymous MC1R variants (e.g. Arg151Cys, Arg160Trp and Asp294His) show Asp294His) show decreased activitydecreased activity to stimulate adenylate cyclase to stimulate adenylate cyclase

These variants are These variants are strongly associatedstrongly associated with with red hair and fair skinred hair and fair skin, , tendency to burntendency to burn and increased and increased risk for risk for melanoma and non-melanoma skin cancersmelanoma and non-melanoma skin cancers

Interestingly, a recent study has described three novel variants of MC1R showing impaired activities Interestingly, a recent study has described three novel variants of MC1R showing impaired activities in Asian populations from relatively high latitudes (Nakayama et al. 2006) in Asian populations from relatively high latitudes (Nakayama et al. 2006)

Arg151CysArg151CysArg160TrpArg160TrpPhe147ΔPhe147ΔThr157IleThr157IlePro159ThrPro159Thr

IL2IL2 TM7TM7

Asp294HisAsp294His

SLC24A5 and pigmentation: finding SLC24A5 and pigmentation: finding answers in unexpected places…..answers in unexpected places…..

A) Wild type zebrafish B) Golden zebrafishC) Wild type melanosomes D) Golden melanosomes

The Zebrafish is widely used as a model organism

One of the best known zebrafish mutants, the golden zebrafish has much lighter pigmentation, with fewer, smaller, and less electron-dense melanosomes than the wild type

Recent research in zebrafish has brought new insights on pigmentation variation in humans

Source: Lamason et al. (Science 310: 1782, 2005) Source: Lamason et al. (Science 310: 1782, 2005)

Finding the “golden” gene…..Finding the “golden” gene…..

Using gene mapping techniques, Using gene mapping techniques, investigators from Penn State led by Keith investigators from Penn State led by Keith Chen recently found that the “golden” gene is Chen recently found that the “golden” gene is slc24a5slc24a5, a member of the potassium-, a member of the potassium-dependent, calcium exchanger family. The dependent, calcium exchanger family. The golden mutants have a premature stop codon golden mutants have a premature stop codon in this gene in this gene

This protein is highly conserved in This protein is highly conserved in vertebrates and injecting the human RNA vertebrates and injecting the human RNA rescues melanin pigmentation in rescues melanin pigmentation in goldengolden embryos!!embryos!!

Wild zebrafish embryo

Golden zebrafish embryo

Golden zebrafish embryoinjected with zebrafish slc24a5 RNA

Golden zebrafish embryoinjected with human SLC24A5 RNA

Source: Lamason et al. (Science 310: 1782, 2005) Source: Lamason et al. (Science 310: 1782, 2005)

Studying SLC24A5 in human populationsStudying SLC24A5 in human populations

Given the interesting results observed, the Given the interesting results observed, the research focus shifted to research focus shifted to human populationshuman populations. I . I was fortunate to collaborate in this stage of the was fortunate to collaborate in this stage of the project project

Exploring the distribution of single nucleotide Exploring the distribution of single nucleotide polymorphisms (SNPs) of SLC24A5 in human polymorphisms (SNPs) of SLC24A5 in human populations (HapMap database) revealed a populations (HapMap database) revealed a very very unusual pattern of variationunusual pattern of variation……..……..

Unusual distribution of SLC24A5 Unusual distribution of SLC24A5 variation in Europevariation in Europe

1. There is a non-synonymous SNP with an allele fixed in Europe (Thr) and the alternative allele (Ala) fixed in other populations

2. There is a dramatic reduction of heterozygosity in EuropeansSource: Lamason et al. (Science 310: 1782, 2005) Source: Lamason et al. (Science 310: 1782, 2005)

Is SLC24A5 involved in normal pigmentation Is SLC24A5 involved in normal pigmentation variation in humans? variation in humans?

p African Americans = 0.0000031; p African Caribbeans = 0.00023

We tackled this issue characterizing the Thr/Ala polymorphism We tackled this issue characterizing the Thr/Ala polymorphism in two admixed populations: African Americans and African in two admixed populations: African Americans and African Caribbeans Caribbeans

Source: Lamason et al. (Science 310: 1782, 2005) Source: Lamason et al. (Science 310: 1782, 2005)

In summaryIn summary

There is strong evidence that SLC24A5 has been subject to There is strong evidence that SLC24A5 has been subject to natural selectionnatural selection in European populations in European populations

The The Thr allele isThr allele is associated with lighter skin pigmentationassociated with lighter skin pigmentation. . We estimated that SLC24A5 explains around 30% of the We estimated that SLC24A5 explains around 30% of the pigmentation differences observed between European and pigmentation differences observed between European and West African populations West African populations

Importantly, the results of the SLC24A5 analysis indicate Importantly, the results of the SLC24A5 analysis indicate that lightening of skin pigmentation took place, at least that lightening of skin pigmentation took place, at least in in part, independently in European and East Asian populationspart, independently in European and East Asian populations

Other genes involved in normal Other genes involved in normal pigmentation variationpigmentation variation

In addition to MC1R and SLC24A5, several recent studies In addition to MC1R and SLC24A5, several recent studies have demonstrated the effect of other genes in normal have demonstrated the effect of other genes in normal variation in hair, eye and skin pigmentation. variation in hair, eye and skin pigmentation.

The list includes the genes The list includes the genes MATPMATP, skin, hair and iris pigmentation, skin, hair and iris pigmentation TYRTYR, skin and iris pigmentation, skin and iris pigmentation P (OCA2)P (OCA2), skin and iris pigmentation, skin and iris pigmentation ASIPASIP, skin and hair pigmentation. , skin and hair pigmentation.

Interestingly, three of these genes also play a role in Interestingly, three of these genes also play a role in albinismalbinism (TYR-OCA1, P-OCA2 and MATP-OCA4). The (TYR-OCA1, P-OCA2 and MATP-OCA4). The Agouti-signaling protein (ASIP) gene is an antagonist of the Agouti-signaling protein (ASIP) gene is an antagonist of the MC1R receptor. MC1R receptor.

New approaches to identify genes involved New approaches to identify genes involved in pigmentation variationin pigmentation variation

The availability of genome-wide SNP data resources from The availability of genome-wide SNP data resources from Perlegen Perlegen SciencesSciences (more than 1.5 million SNPs) and the (more than 1.5 million SNPs) and the HapMap projectHapMap project (more (more than 3 million SNPs) has made it possible to apply novel methods to than 3 million SNPs) has made it possible to apply novel methods to detect signatures of detect signatures of positive selectionpositive selection in pigmentation candidate genes in pigmentation candidate genes

A variety of methods have been proposed including: A variety of methods have been proposed including: Methods based on the allele frequency spectrum: Methods based on the allele frequency spectrum: Tajima’s D (TD), Fu and Tajima’s D (TD), Fu and

Li’s D and F, Fay and Wu’s HLi’s D and F, Fay and Wu’s H Methods based on measures of population differentiation: Methods based on measures of population differentiation: FFSTST, Locus-, Locus-

Specific Branch Length (LSBL)Specific Branch Length (LSBL) Methods based on the decay of Linkage Disequilibrium (LD): Methods based on the decay of Linkage Disequilibrium (LD): Long Range Long Range

Haplotype test (LRH), iHS, LD decay (LDD)Haplotype test (LRH), iHS, LD decay (LDD) Methods based on a reduction in heterozygosityMethods based on a reduction in heterozygosity: LnRH: LnRH

I will review briefly the results of three recent studies.I will review briefly the results of three recent studies. Voight et al. (Plos Biology 4:e72, 2006)Voight et al. (Plos Biology 4:e72, 2006) Izagirre et al. (Mol. Biol. Evol. In press, 2006)Izagirre et al. (Mol. Biol. Evol. In press, 2006) Shriver et al. (Hum. Mol. Genet. In press, 2006) Shriver et al. (Hum. Mol. Genet. In press, 2006)

Genes showing signatures of positive selection Genes showing signatures of positive selection in more than one studyin more than one study

In redIn red, genes for which genotype/phenotype association has been previously demonstrated., genes for which genotype/phenotype association has been previously demonstrated.

GENEGENE TESTTEST POPULATIONPOPULATION

SLC24A5SLC24A5 (Solute Carrier Family 24, Member 5)(Solute Carrier Family 24, Member 5) LSBL, TD, lnRH, FST, LSBL, TD, lnRH, FST, LRH, iHSLRH, iHS

EUREUR

MATPMATP (Solute carrier Family 45, Member 2)(Solute carrier Family 45, Member 2) LSBL, TD, lnRH, FST, LSBL, TD, lnRH, FST, LRHLRH

EUREUR

OCA2OCA2 (Oculocutaneous Albinism II)(Oculocutaneous Albinism II) LSBL, FST, LRHLSBL, FST, LRH EUR, EASEUR, EAS

DCT DCT (Dopachrome Tautomerase)(Dopachrome Tautomerase) LSBL, FST, LRHLSBL, FST, LRH EASEAS

TYRP1 TYRP1 (Tyrosinase-related Protein 1)(Tyrosinase-related Protein 1) LSBL, FST, EHH, iHSLSBL, FST, EHH, iHS EUREUR

LYST LYST (Lysosomal Trafficking Regulator)(Lysosomal Trafficking Regulator) LSBL, TD, lnRH, FST, LSBL, TD, lnRH, FST, LRHLRH

EASEAS

MITF MITF (Microphthalmia-Associated Transcription (Microphthalmia-Associated Transcription factor)factor)

LSBL, TD, lnRH, LRHLSBL, TD, lnRH, LRH EUR, EAS, AFREUR, EAS, AFR

KITLG KITLG (KIT Ligand)(KIT Ligand) TD, LRHTD, LRH EUREUR

ADAM17 ADAM17 (ADAM Metallopeptidase domain 17)(ADAM Metallopeptidase domain 17) LSBL, TD, lnRH, FST, LSBL, TD, lnRH, FST, LRHLRH

EASEAS

ADAMTS20 ADAMTS20 (ADAM Metallopeptidase with (ADAM Metallopeptidase with Thrombospondin Type 1 Motif)Thrombospondin Type 1 Motif)

LSBL, TD, lnRH, FST, LSBL, TD, lnRH, FST, LRHLRH

EASEAS

A cautionary note about tests of selectionA cautionary note about tests of selection

The evidence of positive selection seems to be strong for the The evidence of positive selection seems to be strong for the genes mentioned earlier: they are outliers compared to the rest genes mentioned earlier: they are outliers compared to the rest of the genome and the results are consistent using of the genome and the results are consistent using multiple multiple teststests

However, many of these genes have been implicated in a However, many of these genes have been implicated in a variety of biological processes other than pigmentationvariety of biological processes other than pigmentation

For example, ADAM17 has been implicated in many processes involved For example, ADAM17 has been implicated in many processes involved in cell-cell and cell-matrix interactions, including fertilization, muscle in cell-cell and cell-matrix interactions, including fertilization, muscle development and neurogenesisdevelopment and neurogenesis

The signatures of selection observed may be the result of The signatures of selection observed may be the result of selection related to other gene functions. In order to confirm selection related to other gene functions. In order to confirm their role in normal pigmentation variation, genotype/phenotype their role in normal pigmentation variation, genotype/phenotype association studies and/or direct functional studies will be association studies and/or direct functional studies will be requiredrequired

Pigmentation, vitamin D and public heathPigmentation, vitamin D and public heath

Pigmentation research is important, not only from the Pigmentation research is important, not only from the anthropological and evolutionary points of view, but also from anthropological and evolutionary points of view, but also from the the public healthpublic health perspective perspective

Because of the important role of pigmentation in Because of the important role of pigmentation in photoprotection and vitamin D synthesis, pigmentation levels photoprotection and vitamin D synthesis, pigmentation levels can have an influence in can have an influence in disease riskdisease risk. .

Light-skinned individuals have Light-skinned individuals have higher risks of melanoma and higher risks of melanoma and non-melanoma skin cancersnon-melanoma skin cancers in regions with high UVR levels in regions with high UVR levels (eg. Individuals of European ancestry in Australia).(eg. Individuals of European ancestry in Australia).

Dark-skinned individuals have Dark-skinned individuals have higher risk of vitamin D higher risk of vitamin D deficiency and insufficiencydeficiency and insufficiency in regions with low UVR levels in regions with low UVR levels (eg. Individuals of African or South Asian ancestry living in (eg. Individuals of African or South Asian ancestry living in Canada). Canada).

Pigmentation, vitamin D and healthPigmentation, vitamin D and healthIn recent years, it has become clear that the effects of vitamin In recent years, it has become clear that the effects of vitamin D go well beyond bone metabolism. Vitamin D D go well beyond bone metabolism. Vitamin D deficiency/insufficiency has been associated with:deficiency/insufficiency has been associated with:

Skeletal diseasesSkeletal diseases CancerCancer Autoimmune diseasesAutoimmune diseases Cardiovascular diseaseCardiovascular disease Microbial infectionsMicrobial infections

In Canada, there is In Canada, there is not enough UVnot enough UV to synthesize Vitamin D for to synthesize Vitamin D for several months of the year. several months of the year.

above 40°N (Toronto) from November to early Marchabove 40°N (Toronto) from November to early March above 52°N (Edmonton), mid-October to mid-March above 52°N (Edmonton), mid-October to mid-March

Two recent studies indicate that vitamin D insufficiency ranges Two recent studies indicate that vitamin D insufficiency ranges from 18-39% in Canada. Unfortunately, these studies have from 18-39% in Canada. Unfortunately, these studies have focused primarily on individuals of European ancestry. focused primarily on individuals of European ancestry.

More research is needed to evaluate vitamin D levels in More research is needed to evaluate vitamin D levels in individuals of diverse ancestry living in Canada. individuals of diverse ancestry living in Canada.

AcknowledgementsAcknowledgements.

Mark D. Shriver, Penn State UniversityMark D. Shriver, Penn State University

Keith Chen, Penn State UniversityKeith Chen, Penn State University

Heather Norton, University of ArizonaHeather Norton, University of Arizona

Funding agencies:Funding agencies:

NSERCNSERC