Dual-Color High-Resolution Fiber-FISH Analysis on Lethal White Syndrome Carriers in Sheep

Molecular defects occurring in the endothelin receptor type-B (EDNRB) gene are known to be associated with pigmentaryanomalies and intestinal aganglionosis in humans, rodentsand horses. We carried out a cytogenetic investigation in 2ewes heterozygous for the deletion of the EDNRB gene andin 2 more females as control. The RBA-banding showed thatall 4 ewes were karyologically normal. EDNRB gene-specificprobes were produced by PCR and cloning. The applicationof the R-banding and propidium iodide-staining fluorescentin situ hybridization allowed mapping the gene to OAR10q22 and confirmed the heterozygous status of the ewesinvestigated for the EDNRB gene deletion. For the fine estimationof the gene length in sheep and for the correct sizingof the chromosomal gap, a dual-color FISH was applied tohigh-resolution DNA fibers in combination with digital imagingmicroscopy. The comparison of the DNA fiber barcodesindicated a chromosomal deletion larger than theEDNRB gene itself. The length of the gene, not known forsheep until now, was estimated to be ~ 21 kb, whereas themicrochromosomal deletion was ~ 100 kb. EDNRB is locatedin a chromosomal region previously shown to be a fragilesite. The applied method allowed locating the potentialbreakpoints, thus permitting further interesting prospectiveinvestigations also in the field of the fragile sites in sheep.