2. The Genetic Basis of Eye Color: OCA2 and HERC2 Polymorphisms
The eye color shows variable pigmentation depending on differences in the amount, type, and distribution of melanin obtained by the melanogenesis process in melanocytes
[7]. To understand the range of phenotypic variation in pigmentary traits, the regulation of melanogenesis must be considered, as its multi-step process is under genetic control involving multiple genes, which overall affect the outcome of eye color in humans. In the past,
OCA2 was considered the main gene involved in eye color
[8]. However, it was shown that the
HERC2 gene, positioned very close to
OCA2 (
Figure 1), plays a pivotal role in iris color determination, influencing the expression of
OCA2 [9][10].
Figure 1. Genomic organization of the
OCA2 and
HERC2 genes.
OCA2 and
HERC2 are very close to each other on human chromosome 15, with the 3′-end of
HERC2 adjacent to the promoter region of the
OCA2 gene. The red box in the chromosome ideogram indicates the position of the
OCA2-
HERC2 locus. Image from the UCSC Genome Browser (
http://genome.ucsc.edu, accessed on 2 June 2023). At the bottom, the positions of the SNPs related to eye color (with details described in the table 2 of the paper
10.3390/genes14081604) are indicated by red arrows.
Gene interactions are often involved in the determination of complex traits; thus, the relevance of a single gene on complex phenotypic features cannot be determined in a simple way due to epistatic effects among different genes
[11], even if the interaction between
HERC2 and
OCA2 genes cannot be compared to a classic epistatic effect. Indeed,
HERC2 influences the
OCA2 gene at the DNA level by modulating its transcriptional activity
[12], differently from other genes found to interplay in the determination of eye color in a different way
[10]. Indeed, in addition to the major eye color determinants
HERC2/
OCA2, with more than 50 SNPs related to eye color, other genes were shown to be involved in eye color. Together with
HERC2 and
OCA2, these genes are the best predictors for blue, brown, and intermediate eye color, bearing in mind that eye color phenotyping has followed several proposals, including light vs. dark and blue vs. brown
[8][13][14]. Only recently was another categorization proposed: blue (comprising light to dark blue), intermediate (comprising green and hazel), and brown (comprising light to dark brown).
The
OCA2 gene (NM_000275.3) is localized at the chromosomal band 15q13.1 and is composed of 24 exons spanning a region of 345 kb. It codes a protein of 838-aa (aa: aminoacids) (NP_000266.2), namely the human P protein, which is a homologue to the mouse pink-eyed dilution gene (p), an integral melanosomal membrane protein that contains 12 transmembrane-spanning regions and may stabilize the traffic of melanosomal proteins such as tyrosinase, regulate melanosomal pH, or serve as a melanosomal tyrosinase transporter. Altered P protein may therefore affect pigmentation characteristics via altered tyrosinase or tyrosinase bioavailability and function
[15].
This function appears to be conserved in an evolutionary manner, as mutations of
OCA2 result in oculocutaneous albinism, an autosomal recessive disorder that is characterized by reduced or absent biosynthesis of melanin pigment in melanocytes of the skin, hair follicles, and eye
[16]. The genetic variability of
OCA2 appears to be largely associated with eye color, with a contribution of 74% to the total phenotypic variance of eye color. Thus, the
OCA2 locus is considered the major genetic determinant for blue-brown eye color, and currently many polymorphisms of this gene have been largely studied and identified as directly responsible for variation in iris pigmentation
[17]. 44 apparently nonpathogenic variant alleles of the
OCA2 gene have been identified with distinct frequencies in varied populations, which explains differences in pigmentation phenotypes among ethnic groups
[18]. The large number of these SNPs (37 out of 44) are in the intronic regions of
OCA2, and only six are in the coding regions, with one of these being a synonymous substitution.
2.2. HERC2 Gene
The
HERC2 (NM_004667) gene is located on chromosomal band 15q13.1, adjacent to
OCA2, and contains 98 exons spanning a region of 211 kb. It is a member of the
HERC protein family, endowed by the presence of at least one regulator of chromosome condensation 1 (RCC1)-like domain (RLD) and a homologous E6AP carboxy terminus (HECT) domain characteristic of a group of ubiquitin ligases. Mutations on the
HERC2 gene have been described as being involved in severe neurological conditions, including syndromes of intellectual disability, autism, and a variable neurological deficit named Angelman syndrome
[23].
HERC2 is overexpressed in many tissues, and genetic variations in this gene are associated with pigmentation variability, but cellular activity and its regulation remain poorly understood.
In 2008, three studies highlighted the role of
HERC2 and described the SNP located in intron 86, rs12913832:A/G, as a major eye color predictor strongly associated with
OCA2 expression levels
[9][10][24][25]. More recently, it was experimentally demonstrated that this SNP acts as an enhancer, regulating
OCA2 transcription by distantly modulating chromatin folding
[12].
2.3. Model for OCA2 Gene Regulation
Different reports recognized that the SNP rs12913832:A/G accounted for blue-brown eye color (
Figure 2).
Figure 2. Phenotypic effect of the SNP rs12913832:A/G in eye color. Representative eye colors related to the three genotypes of SNP rs12913832. A/A homozygous genotype is related to the darker brown color (upper images). On the contrary, the G/G homozygous genotype is related to the lighter blue eye color (bottom images). The heterozygous A/G genotype is related to intermediate color of the eyes, namely by the iris with a lesser amount of melanin (respecting the A/A genotype) determining a lesser intensity of the brown or green-hazel color. These are unpublished images by the authors.
Additional evidence that supports the pivotal role of intron variants related to the pigmentary trait is the high level of evolutionary conservation of rs12913832 across many species of vertebrates. In fact, the rs12913832 polymorphism is contained in a region with 77% sequence identity between humans and mice, and specifically, it is located in a conserved 11-base sequence GACA(T/C)TTAAT, suggesting that this region may represent a consensus binding site for the helicase-like transcription factor (HLTF)
[10]. HLTF is a member of the SWI/SNF family and is implicated in many processes involving chromatin remodeling to permit the correct access of the transcriptional machinery. Thus, it was proposed a molecular basis for eye color determination involving the rs12913832:A allele via chromatin remodeling that leads to OCA2 expression and acts in the maturation pathway of the melanosome producing brown iris
[10]. The rs12913832:G allele, on the other hand, is related to a closed chromatin structure and is then unavailable for transcription of the OCA2 locus. This OCA2 transcriptional repression in human melanocyte cells results in the appearance of a light blue iris.
Experimental evidence to support the regulatory role of
HERC2 has been provided by demonstrating that rs12913832 functions as a human melanocyte-specific enhancer element that regulates
OCA2 transcription using various molecular approaches
[12]. In particular, rs12913832 acts as an enhancer, communicating with the
OCA2 promoter via a long-range chromatin loop, and this activity is mediated by the transcription factors HLTF, LEF1, and MITF.