Discovery of copy number variants by multiplex amplifiable probe hybridization (MAPH) in candidate pigmentation genes

<div><p></p><p><i>Background</i>: Copy Number Variants (CNVs) contribute to a large fraction of genetic diversity and some of them have been reported to offer an evolutionary advantage.</p><p><i>Aim</i>: To identify CNVs in pigmentary loci that could contribute to human skin pigmentation diversity.</p><p><i>Subjects and methods</i>: This study assessed the existence of CNVs in every exon of candidate genes: <i>TYR, TYRP1, DCT, MC1R</i> and <i>SLC24A5</i>, using the Multiplex Amplifiable Probe Hybridization technique (MAPH). This study analysed a total of 99 DNA samples of unrelated individuals from different populations. Validation and further analysis in a larger Spanish sample were performed by RT-qPCR.</p><p><i>Results</i>: Five CNVs were identified by MAPH: <i>DCT</i> exons 4 and 8, <i>TYR</i> exon 1 and <i>SLC24A5</i> exons 1 and 4. Real-time quantitative PCR (RT-qPCR) confirmed the CNV in exon 1 of <i>SLC24A5</i>. This study further analysed the 5′ promoter region of <i>SLC24A5</i> and found another CNV in this region. However, no association was found between the CNV and the degree of pigmentation.</p><p><i>Conclusion</i>: Although the functional role of these structural variants in pigmentation should be the subject of future work, the results emphasize the need to consider all classes of variation (both SNPs and CNVs) when exploring the genetics of skin pigmentation.</p></div

Principal Component Analysis.

<p>Charts a) and b) show the same 2-dimesional representation of the data according to the first 2 principal components, but colored according to different variables. Thus, in a) the effects of time (Squares: Time = 0; Dots: Time = 6 hours; Triangles: Time = 12 hours; Diamonds: Time = 24 hours) and pigmentation (Yellow = Light melanocytes; Brown = Dark melanocytes) are highlighted, while in b), it is the time (Squares: Time = 0; Dots: Time = 6 hours; Triangles: Time = 12 hours; Diamonds: Time = 24 hours) and the type of KCM used which are highlighted (Green: KCM-; Red: KCM+).</p

Top 50 upregulated genes in LM vs DM at 6 hours after UVB irradiation (non-coding RNAs are indicated with *) (Bonferroni-adjusted p-value <0.0001).

<p>Top 50 upregulated genes in LM vs DM at 6 hours after UVB irradiation (non-coding RNAs are indicated with *) (Bonferroni-adjusted p-value <0.0001).</p

Graphical scheme of the experimental design.

<p>Graphical scheme of the experimental design.</p

Proposed mechanism for the involvement of ribosomal proteins, MDM2 and p53 signaling pathway in the response to UVB irradiation.

<p>Proposed mechanism for the involvement of ribosomal proteins, MDM2 and p53 signaling pathway in the response to UVB irradiation.</p

Gene expression of genes <i>FDX6</i>, <i>RPL6</i>, <i>MDM2</i>, <i>TP53I3</i>, <i>ATP6VOB</i> and <i>MIF</i> assessed by RT-qPCR.

<p>Unpaired t-test; *** p<0.0001; ** p<0.001; * p<0.01.</p

Top 50 upregulated genes in LM vs DM under basal conditions (non- coding RNAs are indicated with *) (bonferroni-adjusted p-value <0.0001).

<p>Top 50 upregulated genes in LM vs DM under basal conditions (non- coding RNAs are indicated with *) (bonferroni-adjusted p-value <0.0001).</p

Frequency of 374F in Spain according to the intensity of annual UV irradiation.

<p>A colored map showing the frequency of 374F per UV intensity range and the correlation equations between UV intensity and frequency of 374F. UV ranges (J/m²) include: 23500–25500; 25501–27500; 27501–29500; 29501–31500; 31501–34500; and 43300. The green line corresponds to the correlation equation obtained when the Canary Islands were included. The red line corresponds to the correlation equation after excluding the Canary Islands.</p

SNPs found in the resequencing of <i>SLC45A2</i>.

<p>We show the location of the 3 SNPs found in the coding region of the gene: rs16891982, rs2287949 and rs26722, plus the most frequent SNP in intron 5: rs35397.</p

Estimation of selection coefficient (s) of allele 314F using the haplotipic data of 82 unrelated CEU individuals from the 1KGP.

<p>The highest likelihood was obtained for s = 0.0127 (95% CI, 0.0106–0.0148).</p