Postzygotic NRAS Variants and Subsequent Copy-Neutral Loss of Heterozygosity Underlie Speckled-Pattern Formation in...

Nevus spilus-type congenital melanocytic nevus (NS-CMN) is characterized by a background brown macule with superimposed black macules or plaques; however, the mechanism underlying speckled-pattern formation in human skin remains unclear. We performed clinicopathological and molecular analyses of three NS-CMN cases using multiple biopsies from background and superimposed components. The epidermis and dermis were separated before DNA extraction, followed by whole-exome sequencing, Sanger sequencing of NRAS codons 13 and 61, and single-nucleotide polymorphism array analysis. In one patient, melanocyte-lineage cells were cultured separately from the epidermal and dermal compartments and genetically analyzed. Background lesions contained scattered spindle-shaped S100/Melan-A-positive dermal melanocytes, and the superimposed lesions contained dermal nests of nevus cells. Postzygotic NRAS variants were detected by Sanger sequencing in 10/11 dermis-derived DNA samples from background brown macules and in 16/16 dermis-derived DNA samples from superimposed black macules/plaques. Copy-neutral loss of heterozygosity (cn-LOH) encompassing the NRAS locus was confirmed in 14/16 dermal samples from superimposed lesions but not in those from background lesions. These findings support a two-step mosaic model wherein an early postzygotic NRAS variant underlies the background lesion, and independent secondary cn-LOH events generate homozygous NRAS-variant clones. This serves as a molecular explanation for speckled-pattern formation in human skin.