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 pigmentary anomalies and intestinal aganglionosis in humans, rodents and horses. We carried out a cytogenetic investigation in 2 ewes heterozygous for the deletion of the EDNRB gene and in 2 more females as control. The RBA-banding showed that all 4 ewes were karyologically normal. EDNRB gene-specific probes were produced by PCR and cloning. The application of the R-banding and propidium iodide-staining fluorescent in situ hybridization allowed mapping the gene to OAR 10q22 and confirmed the heterozygous status of the ewes investigated for the EDNRB gene deletion. For the fine estimation of the gene length in sheep and for the correct sizing of the chromosomal gap, a dual-color FISH was applied to high-resolution DNA fibers in combination with digital imaging microscopy. The comparison of the DNA fiber barcodes indicated a chromosomal deletion larger than the EDNRB gene itself. The length of the gene, not known for sheep until now, was estimated to be ∼21 kb, whereas the microchromosomal deletion was ∼100 kb. EDNRB is located in a chromosomal region previously shown to be a fragile site. The applied method allowed locating the potential breakpoints, thus permitting further interesting prospective investigations also in the field of the fragile sites in sheep.