Role of MC1R gene in Skin Pigmentation in West Maharashtra, India. MC1R diversity in West Maharashtra

Objectives: The melanocortin-1 receptor gene (MC1R) is an important candidate pigmentation gene that controls the switch between the synthesis of black/brown eumelanin pigment and red/yellow pheomelanin pigment. In this study, we have looked at the whole MC1R gene sequence diversity in 102 individuals representing 6 (viz. 2 caste and 4 tribal) populations from the region of West Maharashtra, India to see if this gene contributes to skin pigmentation variation among populations who show broad distributions in skin pigmentary profiles. We analysed MC1R sequence (2648bp) among caste and tribal groups of this region to understand if variation in the MC1R gene has been constrained by purifying selection in these populations. This study also looked for associations between observed polymorphisms in the MC1R gene and quantitatively measured skin pigmentation phenotype in the populations under study.Methods: 102 samples of Genomic DNA corresponding to the MC1R gene were amplified, purified and sequenced using Illumina’s Nextera® XT DNA Library Preparation Kit (average depth 150x) and processed using published bioinformatic pipelines to yield individual FASTA files which were further analysed using DnaSP Ver. 6.12.03. Allele frequencies were computed and compared between populations using χ2 tests. Deviations from Hardy Weinberg Equilibrium were computed using Arlequin v. 3.5.2.2. Nucleotide diversity indices: π and ϴ and Tajima’s D were computed using DnaSP Ver. 6.12.03 and compared with populations of the 1000 genomes project. Haplotypes frequencies were computed and median-joining networks were constructed using Network 10.2.0.0. Pairwise FST statistics among West Maharashtra populations were computed using Arlequin v. 3.5.2.2. Association of MC1R variants and MI measures were tested using the additive (co-dominant model), dominant and recessive models using the program R. v. 4.0.0. Results: Non-synonymous variants rs2228479_A, rs1805007_T and rs885479_A show low polymorphisms in study populations. 38 haplotypes are observed within the study samples of which 3 haplotypes have a more common occurrence and are shared between the study populations. A number of mutations are seen to occur recurrently, creating a large number of unique haplotypes. Bhils present the lowest diversity (π = 0.00054, θ = 0.00071), whereas Warli’s show the highest diversity (π = 0.00141, θ = 0.00117) in West Maharashtra samples. Tajima’s D shows positive values are not significant at the 0.05% threshold for any of the individual study populations. 3 SNPs rs3212359, rs3212361 and rs76337330 were significantly associated with MI measures under the different models tested in this study.Conclusions: The MC1R gene shows high number of variable positions (22) and haplotypes (38) as compared to other populations who show high melanin levels. Allele frequencies vary significantly at 4 loci; rs3212359, rs76337330, rs2228478, and rs3212371 (p < 0.05) among the populations of West Maharashtra, however, low variation is observed in nonsynonymous variant positions. Tajima’s D indicate lack of departure from neutrality in these populations at these loci; indicating the possible roles of other pigmentation genes and demography influencing pigmentation variation in the absence of strong natural selection forces. Significant genotype – phenotype association of 3 loci with MI measures need to be validated by in-vitro functional studies to understand the true extent of these associations.