Skin Pigmentation represents the major natural protection against the deleterious effects of Ultraviolet light and involves a crosstalk between keratinocytes and melanocytes. Pigment synthesis or melanogenesis is initiated by the binding of \(\alpha\)-Melanocyte Stimulating Hormone \((\alpha-MSH)\) to the Melanocortin 1 Receptor (MC1R), expressed by the melanocytes. α-MSH is generated by cleavage of pro-opiomelanocortin hormone (POMC), produced by both melanocytes and keratinocytes. Activation of MC1R leads to an increase in cAMP levels, causing the expression of the transcription factor MITF. MITF regulates the expression of the enzymes involved in melanogenesis as well as genes important for the survival and proliferation of melanocytes. Pigment synthesis, which occurs in specialized organelles called melanosomes, involves the regulation of different proteins as well as fine homeostatic tuning such as melanosomal pH regulation. The POMC derivative, \(\alpha-MSH\), begins the pigmentation pathway by activating the MC1R signaling pathway, and OCA2 regulates the end of this pathway by controlling tyrosinase activity. The OCA2 gene has been shown to be important in the control of intra-melanosomal pH to allow optimal conditions for the activity of Tyrosinase, the limiting enzyme of pigment (melanin) production. OCA2 polymorphisms have been linked to oculocutaneous albinism type 2 and to blue eye color, demonstrating the importance of this gene in fine pH regulation on pigment production. Polymorphisms in POMC have also been linked to red-haired/fair-skin color in humans. Despite the effort to dissect the mechanisms involved in the control of pigmentation, the transcriptional regulation of POMC and OCA2 are still not fully understood. In this study, we investigate the relevance of the cAMP/CREB pathway in the transcriptional regulation of these two proteins. Our data shows that both POMC and OCA2 expression increases after stimulation of the cAMP/CREB pathway. We demonstrate that MITF transcriptionally regulates OCA2: the cAMP/CREB pathway therefore induces OCA2 in a MITF-dependent manner. On the other hand, our data reveals that POMC may be regulated by cAMP in a MITF-independent fashion but consistent with the hypothesis of a positive feedback loop within the MC1R signaling pathway.

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.