Disruption of CTCF binding by germline non-coding variants in CDKN2B suppress CDKN2A expression and predispose to melanoma

Some melanoma-prone families linked to the 9p21 locus, harboring the established susceptibility gene CDKN2A, lack pathogenic protein-coding variants. Using whole-exome and targeted sequencing, we identified three rare single-nucleotide variants in two melanoma-prone families and one sporadic melanoma case. Variants map to a conserved CTCF-bound region within the first intron of CDKN2B that physically interacts with CDKN2A. Analysis of UK Biobank showed significant enrichment of variants in this region in melanoma cases. Variants result in diminished CTCF binding in vitro. CTCF ChIP-seq in fibroblasts from the carriers of the largest family demonstrated loss of CTCF binding, accompanied by weakened promoter interactions and allele-specific reduction of CDKN2A p16 transcript expression from the variant haplotype. CRISPR-based perturbation of this region and editing of the large family variant into melanocytes resulted in reduced expression of p14 and p16 CDKN2A transcripts. These findings suggest that non-coding regulatory variants function as high-penetrance susceptibility alleles in melanoma families by altering CDKN2A function.