THE TWO SINGLE NUCLEOTIDE POLYMORPHISM HAPLOTYPES ON CHROMOSOME 15 OF THE HERC2 AND OCA2 GENES OF THE COLOR VARIATION OF THE HUMAN EYE IN A SAMPLE OF IRAQI POPULATION

Researcher Prof. 1 Dept. of Medical Laboratory Techniques, Ashur University Coll. Baghdad, Iraq. 2 Dept. of Biotechnology, Coll. of Sci. University of Baghdad, Baghdad, Iraq. Tabark.mundher@au.edu.iq ABSTRACT This study was aimed to identify the role of the single nucleotide polymorphism genes on the contrast of three groups (Dark-Brown, hazel and blue) of human eye color among a sample of Iraqi people specifically single nucleotide polymorphism (SNPs); the rs12913832 of HERC2 gene and the rs1800407 of OCA2 gene, for 90 human blood samples of different people were collected from College of Science, University of Baghdad and also people from different areas of Baghdad, Iraq. These SNPs were analyzed using quantitative real-time PCR (qPCR). The results of the rs12913832 and rs1800407 genotype showed significant differences (p <0.05) in the blue eye color groups. the rs12913832 of HERC2; rs1800407 of the OCA2 genotypes and allele frequencies in contrast to the dark brown and blue iris group showed that there was significant variation in the heterozygous genotype frequencies, while these SNPs in contrast to the dark brown and hazel iris group showed a non-significant effect in a sample of Iraqi population. The haplotype statistical analysis shows strongly Linkage disequilibrium (LD) between the rs12913832 of HERC2 gene and the rs1800407 of OCA2 gene in dark-brown, hazel and blue eye color subjects.


INTRODUCTION
The pigmentation of the human iris is mainly determined by the pigment melanin, which varies from yellow to black depending on the type of melanin (14). In humans, it is the main determinant of pigmentation of the skin, hair, and iris. Eye color has a high degree of heritability, ensuring that the variation in the human genome explains the difference in this phenotype. Many polymorphs are thought to play a role in determining eye color (4). The vast majority of iris pigment heterogeneity undergoes a mutation in a phenotype (single nucleotide polymorphism) called rs12913832 (6,17). This marker is thought to be responsible for determining the distinction between blue and brown eyes found in the intron 86 of the HERC2 gene. The mononucleotide polymorphism (SNP) may carry an allele for guanine or adenine. The guanine allele is present in the European groups of very high frequencies and is associated with blue irises. The allele for adenine is present in clusters of non-European origin at very high frequencies and is associated with a brown iris. This SNP works in a dominant / recessive manner. People with a homozygous guanine allele have a blue iris, but homozygous or heterozygous people with a homozygous guanine allele have a brown iris. One study group found that individuals with the G / G genotype only had a 1% chance of developing a brown iris, but those with the A / A genotype had an 80% chance of developing a brown iris. Comparative research shows that 98.3% of people with the A / A genotype have an 80% chance of staining, and suffer from a blue iris is homozygous for the generated allele (9; 16). The OCA2 expression in individuals with rs12913832: G which has melanocytes that are light-pigmented was found to be decreased in comparison with those in individuals with rs12913832: A, which have melanocytes that are darkly pigmented. According to the predominant theory, individuals with brown eyes were the outcome of persons with the rs12913832: AA or the rs12913832: GA genotype. However, this phenomenon was often not the case for individuals with the rs12913832: GA genotype, which can result in intermediate or blue eyes (10). The OCA2 study is performed subsequently in conjunction with the eye color, but common variants contribute to eye color variations. Single-nucleotide polymorphisms (SNPs) are first engaged in the inheritance of the eye color variation in Europeans in the molecular area of OCA2 (18,19) The SNP rs12913832 of the HERC2 gene may play the primary role in regulating the difference between brown and blue irises, and the complete incompatibility between HERC2 and iris pigmentation suggests that many other markers may be involved. During 2009, iris color was predicted in the Netherlands using DNA markers. They found six polymorphs that act as major genetic markers of eye color in six different shapes. The HERC2 genes include rs12913832, OCA2 rs1800407, and other SNPs. Moreover, an OCA2 coding SNP (rs1800407) acts as a penetration modifier for HERC2 rs12913832 SNP for eye color (4; 11). The rs1800407 of the OCA2 gene has little frequency but has good effects on eye color in all human species. This SNP is considered the second best indicator of eye color diversity in Europeans (9; 19). A major genetic determinant of blue, medium, and brown eye color was developed in the OCA2 / HERC2 region.
Several studies describe polymorphism, which may be directly responsible for the differences in pigmentation of the iris (3,7,17) .The present study aims to find out the relationship between rs12913832 and rs1800407 SNPs and their direct relationship to eye color.

MATERIALS AND METHODS Specimens' collection
The Ethics Committee has accepted this study, with the reference number (CSEC/0121/0009) in Department of Biotechnology, College of Science, University of Baghdad; Baghdad, Iraq. The sample groups consist of adult healthy males and females over 18 years old, involved 90 unrelated individuals from Baghdad. Samples were divided to three groups a dark-brown eye color (X), hazel eye color (Y) and blue eye color (Z). Single nucleotide polymorphisms of (OCA2 and HERC2) genes Designed primers and probes for OCA2 gene (rs1800407 G>A); HERC2 gene (rs12913832 A>G), they provided in a lyophilized state by Alpha-DNA Company (Canada) stored at (-20°C). The sequences of each of the probes and primers used in the allelic discrimination experiments are show in Table 1. ReliaPrep™ Blood gDNA Mini-prep System (Promega, Canada) was used to extract genomic DNA from leukocytes of the blood samples according to the manufacturer instructions.
Genotypes were detected by Taq-Man allelic discrimination assay on a quantitative realtime thermo-cycler (Qiagen Rotor Gene, Germany).
The components of the amplification reaction and their final concentrations are show in Table 2, qPCR was programmed after the optimization.   Fig. 1 represent the gender ratio of the samples for each group; with age 18 years and above. In addition, some pictures were taken by using High-resolution shots at ultra/Micro-close (Nikon D750) shown in Fig.2 to represent the phenotypes of pigmentation in each group

Genotyping of HERC2 (rs12913832) and OCA2 (rs1800407) genes
The SNP of the HERC2 gene (A > G; rs12913832) was presented with three genotypes (AA, AG, GG) and two alleles (A and G). Analysis of Hardy-Weinberg equilibrium (HWE) in (X) and (Z) groups revealed that the genotypes were consistent with equilibrium, and non-significant differences (p <0.05) and Hardy-Weinberg equilibrium (HWE) in the (Y) group revealed that the genotypes were not consistent with equilibrium, a significant differences (p =0.003) were detected between the observed and expected genotype frequencies in both groups ( Table 3).The SNP of the OCA2 gene (G > A; rs1800407) was presented with three genotypes (GG, GA, AA) and two alleles (G and A). Analysis of Hardy-Weinberg equilibrium (HWE) in (X), (Y) and (Z) revealed that the genotypes did not consistent with equilibrium, and significant differences (p <0.05) were detected between the observed and expected genotype frequencies in both groups (Table 3).  Table 4). In AG Polymorphism, the odds ratio for the AG genotype was 4.93 with Pvalue = 0.008 indicating that significant of AG of (R) group and the odd ratio of G allele was 3.74; so the P-value = 0.002 indicating that significant of G allele of (R) group, (Table 4). Inspecting OCA2 gene, genotypes and allele frequencies in dark-brown vs. hazel (H) group revealed that there was non-significant variation between these frequencies, although decreased frequencies of the A allele (70.0 vs. 65.0%) and increased frequencies of G allele (30 vs. 35.0 %) were observed in hazel eye color (Y) compared to dark-brown eye color (X) ( Table 4). In AG Polymorphism, the odds ratio for the AG was 1.15 with P-value = 0.546 indicating that non-significant of AG and of (H) group (Table 4). Inspecting HERC2 gene, genotypes and allele frequencies in darkbrown vs. blue (R) group revealed that there was significant variation between these frequencies, although decreased frequencies of the A allele (70 vs. 58.3%) and increased frequencies of G allele (30 vs. 41.7 %) were observed in blue eye color (Z) compared to dark-brown eye color (X) ( Table 4) In AG Polymorphism, the odds ratio for the AG genotype was 3.33 with P-value = 0.045 indicating that significant of AG of (R) group, ( Table 4). Analysis of haplotypes between alleles of two investigated SNPs revealed strong linkage disequilibrium (LD) as defined by the estimated LD coefficient (D') illustrate in (Table 5) and correlation coefficient (r 2 ). The LD was shown in Fig.3 Linkage disequilibrium coefficient between rs12913832 and rs1800407, the D' of darkbrown eye (X) was 0.69, hazel eye (Y) was 0.98 and blue eye (Z) was 0.80.    (rs12913832 -rs1800407). The haplotype A-A manifested an increased frequency in the hazel eye vs. the dark-brown eye (0.349 -0.143), the difference was significant (p-v=0.009), the OR=3.22; the haplotype A-G manifested an increased frequency in the hazel eye vs. the dark-brown (0.001-0.157), the difference was significant (p-v=0.002). The OR=0.01;the haplotype G-A manifested a decreased frequency in the hazel eye vs. the dark-brown (0.501-0.657), the difference was nonsignificant (p-v=0.083), the OR=0.52; the haplotype G-G manifested a decreased frequency in the hazel eye vs. the dark-brown (0.149-0.043), the difference was significant (p-v=0.048). The OR=3.94; which suggest the susceptibility role of such haplotype in increasing the recessive hazel eye color, (Table 6).  (rs12913832 -rs1800407). The haplotype A-A manifested an increased frequency in the blue eye vs. the dark-brown eye (0.042 -0.143), the difference was significant (p-v=0.057), the OR=0.27; the haplotype A-G manifested a decreased frequency in the blue eye vs. the dark-brown (0.375-0.157), the difference was significant (p-v=0.007), the OR=3.21; the haplotype G-A manifested a decreased frequency in the hazel eye vs. the dark-brown (0.475-0.657), the difference was significant (p-v=0.043), the OR=0.47; the haplotype G-G manifested a decreased frequency in the hazel eye vs. the dark-brown (0.109-0.043), the difference was non-significant (p-v=0.170), the OR=2.75; suggest the susceptibility role of such haplotype in increasing the recessive blue eye color (Table 7). Table 7 In previous study, sturm et al., (15) observed the regulatory region regulating constitutive expression of OCA2 was the conserved region around rs12913832 and the G allele of rs12913832 contributes to the reduction of OCA2 expression, especially within the pigment cells of the iris, which we assume is the ultimate cause of blue eye color, and the rs12913832 polymorphic was directly associated with blue eye color in Iranian population (12), LD haplotype containing these SNPs were also significantly associated with the three categories eye colors darkbrown/hazel and blue iris pigmentation. These deviations provide an indication of the association with Shapturenko et al, (13). A study in Saudi population (1) observed the SNP rs1800407 was not polymorphic in the Saudi population with the presence of the allele G. In this study, concluded the rs1800407 indicating of eye color with the rs12913832 because of the linkage disequilibrium coefficient between alleles of these SNPs strong associated with eye color. Irises exhibit extensive variation across Europe and a small degree of pigmentation from blue to green and brown in North Africa, Middle East, and Asia (8).