No / P-cadherin serves as a major topobiological cue in mammalian epithelium. In human hair follicles (HFs), it is prominently expressed in the inner hair matrix that harbors the HF pigmentary unit. However, the role of P-cadherin in normal human pigmentation remains unknown. As patients with mutations in the gene that encodes P-cadherin show hypotrichosis and fair hair, we explored the hypothesis that P-cadherin may control HF pigmentation. When P-cadherin was silenced in melanogenically active organ-cultured human scalp HFs, this significantly reduced HF melanogenesis and tyrosinase activity as well as gene and/or protein expression of gp100, stem cell factor, c-Kit, and microphthalmia-associated transcription factor (MITF), both in situ and in isolated human HF melanocytes. Instead, epidermal pigmentation was unaffected by P-cadherin knockdown in organ-cultured human skin. In hair matrix keratinocytes, P-cadherin silencing reduced plasma membrane β-catenin, whereas glycogen synthase kinase 3 beta (GSK3β) and phospho-β-catenin expression were significantly upregulated. This suggests that P-cadherin-GSK3β/Wnt signaling is required for maintaining the expression of MITF to sustain intrafollicular melanogenesis. Thus, P-cadherin-mediated signaling is a melanocyte subtype-specific topobiological regulator of normal human pigmentation, possibly via GSK3β-mediated canonical Wnt signaling.

A cor da pele, olhos e cabelos são alguns dos traços fenotípicos mais aparentes quando nos referimos à identificação de características individuais. Essas características frequentemente são utilizadas na descrição de indivíduos em retratos falados usados em investigações policiais. Porém, em muitos casos as vítimas ou testemunhas não reconhecem o agressor, tornando inviável a produção desses retratos. Contudo, os vestígios biológicos deixados pelo criminoso poderiam ser utilizados na predição de suas características físicas, suprindo a falta ou complementando o retrato falado. Para que isso seja possível, é preciso conhecer as variáveis responsáveis pela formação desses fenótipos. No caso dos fenótipos de pigmentação há tanto um fator genético, quanto ambiental. Diversos genes participam da formação desses fenótipos, dentre eles está o gene MITF (Melanogenesis-associated transcription factor), um dos principais regulador da biossíntese de melanina nos melanócitos. Esse gene está fortemente associado às síndromes de Waadenburg e Tietz, as quais causam pigmentação anormal, principalmente na pele, e ao melanoma. No entanto, apesar do claro envolvimento do gene MITF na melanogênese, ainda não são conhecidas associações significativas de polimorfismos nesse gene com fenótipos de pigmentação. À vista disso, esse trabalho avaliou a relação da variabilidade do gene MITF com os fenótipos de pigmentação encontrados em uma amostra populacional do estado de São Paulo, por meio de sequenciamento de nova geração. Foram identificados 133 pontos de variação em toda a extensão do gene e sua região promotora, dos quais 21 estão associadas a pelo menos um fenótipo de pigmentação de pele, olhos ou cabelo. Adicionalmente foram encontradas associações com ao menos um fenótipo de pigmentação para 3 dos 17 haplótipos da região promotora, 7 dos 50 haplótipos da extensão que engloba a região 5UTR e codificante, e um dos 18 haplótipos encontrados na região 3UTR. Considerando os haplótipos encontrados para a extensão total do gene MITF e sua promotora, 20 dos 132 haplótipos encontrados estão associados a algum fenótipo de pigmentação. A maior parte das associações encontradas, tanto para alelos e genótipos quanto para haplótipos, são referentes a fenótipos mais escuros como cabelos castanhos escuros e pretos e pele escura. Associações com fenótipos mais claros, tais como olhos azuis e verdes e cabelos loiros e ruivos, também foram encontradas, porém envolvendo variantes e haplótipos de frequência baixa na população amostrada; tais associações, entretanto, representam achados falsos positivos. Os resultados confirmam a hipótese de que a variabilidade do gene MITF pode contribuir para a formação dos fenótipos de pigmentação de pele, olhos e cabelos dos indivíduos da população brasileira / Skin, eye and hair colors are some of the most noticeable phenotypes when referring to the identification of individual characteristics. These characteristics are often used to describe individuals in police sketches used in investigations. However, in many cases the victims or witnesses are unable to recognize the assaulter, making this sketches unfeasible. Nonetheless, biological traces left by the assaulter could be used to predict their physical characteristics, compensating or complementing these sketches. To make this possible, its necessary to know the variables responsible for the development of these traits. Pigmentation phenotype development relies on genetic and environmental aspects. A variety of genes contribute to the development of these phenotypes, among them MITF (Melanogenesis-associated transcription factor), one of the main regulators of melanin synthesis in melanocytes. This gene is strongly associated with Waardenburg and Tietz syndrome, which cause abnormal pigmentation, mostly in skin, and melanoma. Although MITFs clear involvement in melanogenesis, significant associations between this genes polymorphisms and pigmentation phenotypes are still unknown. Thus, this study evaluated the relation between MITF genetic variability and pigmentation phenotypes found in a population sample from the state of São Paulo, through next generation sequencing. There were identified 133 variation points through the whole gene and its promoter, from which 21 were associated with at least one skin, eye or hair pigmentation phenotype. Additionally, 3 of the 17 promoter haplotypes, 7 of the 50 haplotypes comprising the 5UTR and coding regions and one of the 18 3UTR haplotypes were associated with at least one pigmentation phenotype. Considering the haplotypes found for the whole gene and its promoter, 20 of the 132 haplotypes found were associated with at least one phenotype. The majority of the associations found for alleles, genotypes and haplotypes were related to darker phenotypes, like dark brown and black hair and dark skin. Associations with lighter phenotypes, like blue and green eyes and blonde and red hair, were also found, although involving variants and haplotypes with low frequencies in the studied population; these associations, however, represent false positives. The results corroborate the hypothesis that the MITF variability can contribute to the formation of pigmentation phenotypes in skin, eye and hair in the Brazilian population

A cor da pele, olhos e cabelos são alguns dos traços fenotípicos mais aparentes quando nos referimos à identificação de características individuais. Essas características frequentemente são utilizadas na descrição de indivíduos em retratos falados usados em investigações policiais. Porém, em muitos casos as vítimas ou testemunhas não reconhecem o agressor, tornando inviável a produção desses retratos. Contudo, os vestígios biológicos deixados pelo criminoso poderiam ser utilizados na predição de suas características físicas, suprindo a falta ou complementando o retrato falado. Para que isso seja possível, é preciso conhecer as variáveis responsáveis pela formação desses fenótipos. No caso dos fenótipos de pigmentação há tanto um fator genético, quanto ambiental. Diversos genes participam da formação desses fenótipos, dentre eles está o gene MITF (Melanogenesis-associated transcription factor), um dos principais regulador da biossíntese de melanina nos melanócitos. Esse gene está fortemente associado às síndromes de Waadenburg e Tietz, as quais causam pigmentação anormal, principalmente na pele, e ao melanoma. No entanto, apesar do claro envolvimento do gene MITF na melanogênese, ainda não são conhecidas associações significativas de polimorfismos nesse gene com fenótipos de pigmentação. À vista disso, esse trabalho avaliou a relação da variabilidade do gene MITF com os fenótipos de pigmentação encontrados em uma amostra populacional do estado de São Paulo, por meio de sequenciamento de nova geração. Foram identificados 133 pontos de variação em toda a extensão do gene e sua região promotora, dos quais 21 estão associadas a pelo menos um fenótipo de pigmentação de pele, olhos ou cabelo. Adicionalmente foram encontradas associações com ao menos um fenótipo de pigmentação para 3 dos 17 haplótipos da região promotora, 7 dos 50 haplótipos da extensão que engloba a região 5UTR e codificante, e um dos 18 haplótipos encontrados na região 3UTR. Considerando os haplótipos encontrados para a extensão total do gene MITF e sua promotora, 20 dos 132 haplótipos encontrados estão associados a algum fenótipo de pigmentação. A maior parte das associações encontradas, tanto para alelos e genótipos quanto para haplótipos, são referentes a fenótipos mais escuros como cabelos castanhos escuros e pretos e pele escura. Associações com fenótipos mais claros, tais como olhos azuis e verdes e cabelos loiros e ruivos, também foram encontradas, porém envolvendo variantes e haplótipos de frequência baixa na população amostrada; tais associações, entretanto, representam achados falsos positivos. Os resultados confirmam a hipótese de que a variabilidade do gene MITF pode contribuir para a formação dos fenótipos de pigmentação de pele, olhos e cabelos dos indivíduos da população brasileira / Skin, eye and hair colors are some of the most noticeable phenotypes when referring to the identification of individual characteristics. These characteristics are often used to describe individuals in police sketches used in investigations. However, in many cases the victims or witnesses are unable to recognize the assaulter, making this sketches unfeasible. Nonetheless, biological traces left by the assaulter could be used to predict their physical characteristics, compensating or complementing these sketches. To make this possible, its necessary to know the variables responsible for the development of these traits. Pigmentation phenotype development relies on genetic and environmental aspects. A variety of genes contribute to the development of these phenotypes, among them MITF (Melanogenesis-associated transcription factor), one of the main regulators of melanin synthesis in melanocytes. This gene is strongly associated with Waardenburg and Tietz syndrome, which cause abnormal pigmentation, mostly in skin, and melanoma. Although MITFs clear involvement in melanogenesis, significant associations between this genes polymorphisms and pigmentation phenotypes are still unknown. Thus, this study evaluated the relation between MITF genetic variability and pigmentation phenotypes found in a population sample from the state of São Paulo, through next generation sequencing. There were identified 133 variation points through the whole gene and its promoter, from which 21 were associated with at least one skin, eye or hair pigmentation phenotype. Additionally, 3 of the 17 promoter haplotypes, 7 of the 50 haplotypes comprising the 5UTR and coding regions and one of the 18 3UTR haplotypes were associated with at least one pigmentation phenotype. Considering the haplotypes found for the whole gene and its promoter, 20 of the 132 haplotypes found were associated with at least one phenotype. The majority of the associations found for alleles, genotypes and haplotypes were related to darker phenotypes, like dark brown and black hair and dark skin. Associations with lighter phenotypes, like blue and green eyes and blonde and red hair, were also found, although involving variants and haplotypes with low frequencies in the studied population; these associations, however, represent false positives. The results corroborate the hypothesis that the MITF variability can contribute to the formation of pigmentation phenotypes in skin, eye and hair in the Brazilian population

A pigmentação dos olhos, cabelos e pele, bem como presença ou ausência de sardas, está entre os exemplos mais visíveis da variação fenotípica humana. O estudo da diversidade genética em genes de pigmentação tem beneficiado diferentes áreas do conhecimento, como a área da genética e antropologia forense, bem como a área relacionada a saúde e bemestar. Adicionalmente, a presença de estruturas secundárias na íris tem sido reportada como importante fator na percepção de cor de olho observada que um indivíduo pode ter referente a íris e também a fatores de risco para algumas doenças oculares, ainda que as bases genéticas envolvidas nestas características sejam pouco conhecidas. Os genes OCA2 e HERC2 representam dois genes associados à variação normal da pigmentação. Este trabalho avaliou a relação de polimorfismos nas regiões regulatórias e codificantes destes dois genes com os fenótipos de pigmentação e estruturas secundárias presentes na íris encontrados em uma amostra populacional de 340 indivíduos do estado de São Paulo, por meio de sequenciamento de nova geração. Análises de regressão logística e linear para as variáveis qualitativas e quantitativas da cor dos olhos e estruturas secundárias presentes na íris foram realizadas. 170 pontos de variação ao longo das regiões estudadas foram identificados, dos quais 18 estão associadas a pelo menos um fenótipo de pigmentação e estruturas secundárias presentes na íris. Destaca-se a existência de muitos polimorfismos que não se mostrara-se associados quando avaliados independentemente, porém foram associados quando analisados sob a ótica de interações epistáticas, considerada uma possível explicação para a variabilidade encontrada nestes fenótipos, principalmente aqueles intermediários, como a cor dos olhos verdes e mel. O uso de variáveis quantitativas para os olhos revelou pela primeira vez a associação do polimorfismo não sinônimo rs201872292 no gene HERC2 com olhos claros, independente do efeito do polimorfismo rs12913832. Ainda, a associação do polimorfismo rs58358300 localizado em um íntron do gene HERC2 com pigmentação da esclera, o que representa a primeira vez que um polimorfismo é associado a esta característica. Este foi o primeiro estudo no Brasil que se propôs a analisar polimorfismos genéticos em genes candidatos à variação normal da pigmentação humana com estruturas secundárias presentes na íris. Os resultados confirmam a hipótese de que polimorfismos dos genes OCA2 e HERC2 podem contribuir para a formação dos fenótipos clássicos de pigmentação de olhos, pele, cabelos e estruturas secundárias presentes na íris humana dos indivíduos da população brasileira. / The pigmentation of the eyes, hair and skin, as well as the presence or absence of freckles, are amongst the most visible examples of human phenotypic variation. The study of genetic diversity in pigmentation genes has contributed greatly to the fields of forensics genetics, anthropological genetics and public health. In addition, the presence of iris features has been reported to influence the perception of overall iris color and also consists in risk factors for ocular diseases, although very little is known about the genetic basis of these traits. The OCA2 and HERC2 genes have been associated with normal variation of pigmentation in diverse populations. The present study evaluated the relationship of polymorphisms in the regulatory and coding regions of these two genes with the pigmentation phenotypes and iris features found in a population sample of 340 individuals from the state of São Paulo, Brazil, through next-generation sequencing. Logistic and linear regression analyzes for the qualitative and quantitative variables were performed. A total of 170 points of variation throughout the studied regions were identified, of which 18 were associated with at least one pigmentation phenotype when analyzed as qualitative and/or quantitative variables and iris features. It is worth mentioning that many associations that were not observed when evaluated independently, were indeed associated when analyzed from the perspective of epistatic effects, which is considered a possible explanation for the variability found in these phenotypes, especially those presented as intermediate, such as green and hazel eye colors. The use of quantitative variables to evaluate the eye color, acquired from photographs, revealed for the first time the association of the nonsynonymous mutation rs201872292 in the HERC2 gene with light eyes, independently of the effect of the rs12913832 polymorphism. We highlight the association of the polymorphism rs58358300 located in an intron of the HERC2 gene with sclera pigmentation, which was the first time that a polymorphism is associated with this feature. This was the first study in Brazil to analyze genetic polymorphisms in candidate genes related to normal variation of human pigmentation and iris features by next-generation sequencing. The results confirm the hypothesis that OCA2 and HERC2 genes may contribute to classic pigmentation phenotypes of eyes, skin, hair, freckles and iris features in the Brazilian population.

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.–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 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.