Genotyping the Skin Color Gene

Skin pigmentation is one of the most phenotypically variant traits in humans, and its genetic basis is still not fully understood. While many genes have been linked to skin color and pigmentation-production pathways, this pigmentation remains a prime example of a trait influenced by polygenes, or genes which do not solely or even noticeably determine a phenotype, rather contributing to a larger polygenetic effort to influence phenotype. Recent research has found that variation in one of these genes, SLC24A5, is strongly correlated with skin color differences between people of European and African ancestry. The single nucleotide polymorphism rs1426654 on exon 3 of this gene contributes 25-38% of the variation in skin color, measured using a melanin index. The ancestral allele for SLC24A5, which has a guanine (G) at rs1426654, was found abundantly in Africa and East Asia, while the derived allele has an adenine (A) at this SNP and predominates in populations in Europe. The polymorphism from G to A causes a nonsynonymous amino acid substitution from Alanine to Threonine.

The gene SLC24A5 encodes for NCKX5, a potassium-dependent sodium-calcium exchanger. This protein is thought to regulate melanogenesis, which is the process where melanocyte cells in the epidermis produce melanin, the pigment responsible for skin and hair color. NCKX5 is located in an intracellular membrane, co-localized with the trans-Golgi network, in normal human epidermal melanocytes (NHM). Ginger et al show that NCKX5 cation-exchange activity in the TGN affects melanin synthesis, potentially by regulating enzyme activity via pH or Ca2+ gradients. The amino acid residue in NCKX5 altered by rs1426654 sits in a transmembrane domain and is likely critical for the protein's exchange function. Ginger et al also demonstrated that the threonine variant of NCKX5 (encoded by A allele) greatly reduced exchange activity, compared to that of the alanine variant (G allele). Further, knockdown of functional SLC24A5 in NHM reduced the melanin content of these cells by more than 20%. Thus, this SNP in SLC24A5 directly influences human skin color by regulating melanogenesis through NCKX5 exchanger activity.

Our hypothesis that Sneha and Piper will have the A allele while Sydnee will have the G allele is supported by both the alleles' respective associations with geographical location and the melanogenesis pathway controlled by SLC24A5. Since the G allele results in functional melanogenesis while the A allele reduces NCKX5 activity and thus melanin production, our hypothesized genotypes correspond with our skin color phenotypes.

Our first undigested gel showed DNA bands for all our samples, most clearly in Sneha's and Piper's, and no DNA bands other than primer dimers in the negative control, as expected. When compared to the MiniOne ladder, these bands measured approximately 500 bp long, which is close to expected length of the target PCR sequence, 443 bp. Thus, this gel suggests we successfully targeted and amplified the SLC24A5 gene in our DNA the first time we ran PCR.

However, in later gels such as the digested gel picture above, we did not observe bands for any of our samples. After re-extracting our DNA and rerunning PCR with both the mitochondrial DNA primers and SLC24A5 primers, we saw the expected bands for the mtDNA samples but no bands, other than primer dimers, for the SLC24A5 product. These results suggest an issue developed with the SLC24A5-targeting primers, such that they could not bind to our DNA in order to replicate the target sequence, since the mtDNA gel demonstrates that DNA extraction was successful and the other components of PCR and gel electrophoresis were functional. This conclusion about why there were no hits in the later SLC24A5 gels is also supported by how bright the primer material (<100 bp long) is in the digested gel above. The high concentration of primer dimers in our samples as well as the negative control group suggests that the primers did not bind to our DNA at all, so the sequence wasn't amplified. Thus, there wasn't enough PCR product for the Hin6I enzyme to cut in the digestion experiment and DNA bands did not form in the digested gel.

With functional SLC24A5 primers, we would expect to see two shorter bands, of lengths around 325 bp and 118 bp, in Sydnee's lane and the same 443 bp band in Sneha's and Piper's lanes in the digested gel. This is because, as shown in our benchling alignment, the A to G SNP in the SLC24A5 gene that influences skin pigmentation overlaps with the target cut site of Hin6I, the restriction enzyme we used to perform the digest. As per our hypothesis, we expect Sydnee to have the G allele, which contains the Hin6I restriction site, so in the digest the enzyme would be able to locate the sequence GCGC and cut the PCR product into two shorter segments. However, Sneha and Piper likely have the A allele; since Hin6I would not find its target site on that allele, no digestion would occur and the bands in lanes 2 and 3 should resemble the undigested gel -- one longer band. These gel electrophoresis results would have allowed us to determine our genotypes, as the length and number of bands that form in a sample would indicate whether the A or G allele of SLC24A5 is present in our DNA samples.

These expected results would suggest a correlation between SLC24A5 genotype and skin pigmentation phenotype & geographic ancestry. This would support the major hypothesis that skin color variation corresponds to surface-level UV radiation strata around the globe, as shown in the sepia rainbow below, because amount of melanin production evolved in response to ancient humans' migration across these strata. Although the sun's UV rays can cause harm like cell damage, they also are essential to vitamin D production in the skin, and vitamin D improves bone strength and immune function among other benefits. In tropical climates, geographically close to the equator, there is enough UV radiation to penetrate even the higher concentration of protective melanin in dark skin. Thus, the body receives the right amount of sunlight to prevent harmful effects of UV while still producing enough vitamin D. However, there is less UV radiation at higher latitudes -- not enough to penetrate dark skin and produce sufficient amounts of vitamin D. This change in environment may explain why the SLC24A5 A-variant evolved from the ancestral G allele and became predominant in places like Europe, since individuals with the A allele produce less melanin. When there is less UV radiation, less melanin is needed as protection from harmful effects and also allows enough UV rays to enter the body to trigger vitamin D production. This theory of the evolution of human skin color-- as a response to migration, local UV exposure, and vitamin D needs-- is based in the genetics of melanin production, including genes like SLC24A5.