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. 2025 Jul 23:2025:3468799.
doi: 10.1155/humu/3468799. eCollection 2025.

Simultaneous Genotyping of Three Nonsynonymous SNVs, rs1042602, rs1426654, and rs16891982 Involved in Skin Pigmentation by Fluorescent Probe-Based Melting Curve Analysis

Mikiko Soejima  1 , Yoshiro Koda  1
Affiliations

Simultaneous Genotyping of Three Nonsynonymous SNVs, rs1042602, rs1426654, and rs16891982 Involved in Skin Pigmentation by Fluorescent Probe-Based Melting Curve Analysis

Mikiko Soejima et al. Hum Mutat. .

Abstract

Three nonsynonymous single nucleotide variations (SNVs), rs1042602 in TYR (p.S192Y), rs1426654 in SLC24A5 (p.A111T), and rs16891982 in SLC45A2 (p.L374F), were associated with human skin pigmentation variation and may have recently undergone positive natural selection. Furthermore, these three SNVs have been reported to correlate with the risk and prognosis of melanoma. To simultaneously determine these three SNVs, a triplex fluorescent probe-based melting curve assay (FMCA) was developed. The method was validated by analyzing genomic DNA from subjects with known genotypes. For rs16891982, triplex FMCA did not allow good separation of genotypes with the initial polymerase enzyme mix used, but by changing the enzyme mix used, the three genotypes could be clearly distinguished. Using this method, we definitively genotyped these three SNVs in 93 European, 58 Tamil, 54 Sinhalese, and 52 Bangladeshi subjects. This method allows genotyping of rs1042602, rs1426654, and rs16891982 in a relatively large number of samples to perform association studies on skin pigmentation variation or melanoma risk.

Keywords: melting curve genotyping; rs1042602; rs1426654; rs16891982; skin pigmentation.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Melting curve genotyping of selected subjects by triplex FMCA. Results of (a) rs1426654, (b) rs16891982, and (c) rs1042602 using Premix Ex Taq (Probe qPCR). Results of (d) rs1426654, (e) rs16891982, and (f) rs1042602 using Probe qPCR Mix MultiPlus. Negative controls are indicated by light blue.
Figure 2
Figure 2
Improving results of FMCA of rs16891982 by changing the enzyme mix used. (a) Melting peak and (d) melting curve by singleplex FMCA using Premix Ex Taq (Probe qPCR). (b) Melting peak and (e) melting curve by triplex FMCA using Premix Ex Taq (Probe qPCR). (c) Melting peak and (f) melting curve for genotyping by triplex FMCA using Probe qPCR Mix MultiPlus. Negative controls are indicated by light blue.
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