doi: 10.1111/j.1469-1809.2007.00407.x Association of Polymorphic Sites in the OCA2 Gene with Eye Colour Using the Tree Scanning Method W. Branicki 1, * , U. Brudnik 2 , T. Kupiec 1 , P. Wola´ nska-Nowak 1 , A. Szczerbi ´ nska 1 and A. Wojas-Pelc 2 1 Institute of Forensic Research, Section of Forensic Genetics, Westerplatte 9, 31-033 Krakow, Poland 2 Collegium Medicum of the Jagiellonian University, Department of Dermatology, Kopernika 19, 31-501, Krakow, Poland Summary A number of genes are considered to affect normal variation in human pigmentation. Recent studies have indicated that OCA2 is the crucial gene involved in the high variation of iris colour present among populations of European descent. In this study, eleven polymorphisms of the OCA2 gene were examined in search of their association with different pigment traits. The evolutionary tree scanning method indicated that the strongest phenotypic eye colour variation is associated with the branch defined by nonsynonymous change rs1800407, which refers to amino acid causing change Arg419Gln located in exon 13. Single SNP analysis indicated that allele 419Gln is associated with green/hazel iris colour (p < 0.001). According to tree scanning analysis, the proportion of eye colour variation ex- plained by this nucleotide position is merely 4%. Thus, additional variation present in the OCA2 gene and perhaps some other pigment related genes must be taken into account in order to explain the high phenotypic variation in iris colour. Keywords: OCA2, association study, eye colour, genetic prediction, tree scanning Introduction Pigmentation is one of most striking human physical traits. Although it is assumed that pigment variation in man is controlled by more than a hundred genes, until now only the role of one locus has been explained fully (Bennett & Lamoreux, 2003). It has been shown that the melanocortin 1 receptor gene (MC1R, MIM# 155555) is responsible for most cases of red hair colour in humans. Population stud- ies have disclosed a few nonsynonymous polymorphisms which strongly affect receptor performance and, in conse- quence, cause a significant excess of red or yellow pheome- lanin relative to black or brown eumelanin pigment. This state is expressed in a characteristic phenotype of red hair colour and lighter skin often associated with freckling (e.g. Valverde et al. 1995; Box et al. 1997; Rana et al. 1999). The impact of the MC1R gene on pigmentation is very distinct, especially in populations of Northern European origin. Over the past several years, a number of important genes involved in pigmentation in man have been identi- ∗ Corresponding author: Wojciech Branicki, Fax: +4812 422 38 50. E-mail: wbranicki@ies.krakow.pl fied, often on the basis of DNA sequence homology with widely studied mouse pigmentary genes. They have been linked to different human Mendelian pigment disorders of which the best known example is oculocutaneous albinism (Rees, 2003). It is assumed that genes involved in such ex- treme phenotypes might also be responsible for normal variation in human pigmentation. Their role in determi- nation of high variation in skin, hair and eye colour among humans is currently the subject of extensive studies. Work in this area has led to significant progress in the under- standing of the genetic basis for eye colour determination (Sturm & Frudakis, 2004). Eye colour was formerly con- sidered as a simple Mendelian recessive trait (Duffy et al. 2007). Although currently it is accepted that multiple genes contribute to the variety of iris hues, most recent findings suggest that a single gene plays the predominant role. A whole genome scan indicated that more than 70% of eye colour variation is due to the quantitative trait locus located on chromosome 15q (Zhu et al. 2004). This conclusion corresponds with some previous data indicating that vari- ation within the OCA2 gene (MIM# 203200) located on chromosome 15q11.2-12 may be involved in differences in eye colour among humans (Rebbeck et al. 2002; Frudakis et al. 2003). We further addressed this issue by conducting 184 Annals of Human Genetics (2008) 72,184–192 C  2007 The Authors Journal compilation C  2007 University College London