A structural variant in the 5’-flanking region of the TWIST2 gene affects melanocyte development in belted cattle
(A) Haplotype analysis defined a critical interval of 37 kb indicated by blue shading for the belt causative variant (Chr3:118,578,893–118,616,348, UMD3.1 assembly, S1 Table). The belt locus mapped to a gene poor region containing TWIST2 as the only known gene (NCBI annotation release 105; protein coding genes are shown in black, predicted non-coding RNA genes in grey). (B) A 6 kb CNV is located within the critical interval and approximately 16 kb upstream of the transcription start site of TWIST2. The CNV is flanked by two highly homologous LINE sequences that share 94% sequence identitiy over 650 bp. (C) Experimental identification of the CNV. IGV screenshots of the illumina short read sequences illustrate a ~4-fold increased coverage in a homozygous belted (bt/bt) cattle with respect to a control animal (wt/wt) and several read-pairs with incorrect read-pair orientation at the boundaries of the CNV (indicated in green). (D) The CNV is largely composed of interspersed repeats. However, it has a short single copy region, which is highly conserved in mammals. (E) Inverse PCR strategy to confirm the presence of tandemly repeated copies. (F) Agarose gel showing the expected 4845 bp amplicon that is diagnostic for the amplified CNV in belted animals. (G) Allele-specific quantification of TWIST2 mRNA expression in adult skin. RNA-seq data were analyzed from non-belted (wt/wt) and heterozygous belted (bt/wt) animals. All animals were heterozygous for an A/G SNV in the 3’-UTR of TWIST2. In wt/wt animals the two TWIST2 alleles were expressed at equal amounts. In bt/wt animals, the G-allele transcribed from the bt haplotype was reduced by 35% compared to the A-allele (p = 0.046, two-sided t-test).