A structural variant in the 5’-flanking region of the TWIST2 gene affects melanocyte development in belted cattle

Belted cattle have a circular belt of unpigmented hair and skin around their midsection. The belt is inherited as a monogenic autosomal dominant trait. We mapped the causative variant to a 54 kb segment on bovine chromosome 3. Whole genome sequence data of 2 belted and 130 control cattle yielded only a single genetic variant in the critical interval, which was private to the two belted animals. The belt-associated variant was a copy number variant (CNV) involving the quadruplication of a 6 kb non-coding sequence located approximately 16 kb upstream of the TWIST2 gene. Increased copy numbers at this CNV were strongly associated with the belt phenotype in a cohort of 239 cases and 1303 controls (p = 1exp-265). We hypothesized that the CNV causes aberrant expression of TWIST2 during neural crest development, which might negatively affect melanoblasts . Functional studies showed that ectopic expression of bovine TWIST2 in developing melanoblasts in transgenic zebrafish led to a decrease in melanocyte numbers. Our results thus implicate an unsuspected involvement of TWIST2 in pigmentation and reveal a non-coding CNV underlying a captivating Mendelian character.