Familiar Hypopigmentation Syndrome in Sheep Associated with Homozygous Deletion of the Entire Endothelin Type-B Receptor Gene

Erhardt G (2012) Familiar Hypopigmentation Syndrome in Sheep Associated with Homozygous Deletion of the Entire Endothelin Type-B Receptor Gene. PLoS ONE 7(12): e53020. doi:10.1371/journal.pone.0053020 Familiar Hypopigmentation Syndrome in Sheep Associated with Homozygous Deletion of the Entire Endothelin Type-B Receptor Gene Gesine Lu hken 0 Katharina Fleck 0 Alfredo Pauciullo 0 Maike Huisinga 0 Georg Erhardt 0 Reiner Albert Veitia, Institut Jacques Monod, France 0 1 Department of Animal Breeding and Genetics, Justus-Liebig University of Giessen , Giessen, Germany , 2 Institute of Veterinary Pathology, Justus-Liebig University of Giessen , Giessen , Germany In humans, rodents and horses, pigmentary anomalies in combination with other disorders, notably intestinal aganglionosis, are associated with variants of the endothelin type-B receptor gene (EDNRB). In an inbred Cameroon sheep flock, five white lambs with light blue eyes were sired from the same ram and died within a few hours up to a few days after birth, some of them with signs of intestinal obstruction. The aim of this study was to investigate if the observed hypopigmentation and a possible lethal condition were associated with a molecular change at the ovine EDNRB locus, and to check if such a genetic alteration also occurs in other Cameroon sheep flocks. Sequence analysis revealed a deletion of about 110 kb on sheep chromosome 10, comprising the entire EDNRB gene, on both chromosomes in the two available hypopigmented lambs and on a single chromosome in the two dams and three other unaffected relatives. This micro-chromosomal deletion was also confirmed by quantitative real-time PCR and by fluorescence in situ hybridization. Genotyping of a total of 127 Cameroon sheep in 7 other flocks by duplex PCR did not identify additional carriers of the deletion. Although both hypopigmented lambs available for post-mortem examination had a considerably dilated cecum and remaining meconium, histopathological examination of intestinal samples showed morphologically normal ganglion cells in appropriate number and distribution. This is to our knowledge the first description of an ENDRB gene deletion and associated clinical signs in a mammalian species different from humans and rodents. In humans and rats it is postulated that the variable presence and severity of intestinal aganglionosis and other features in individuals with EDNRB deletion is due to a variable genetic background and multiple gene interactions. Therefore the here analyzed sheep are a valuable animal model to test these hypotheses in another species. - Lethal white foal syndrome (LWFS, OMIA #000629-9796) is an autosomal-recessively inherited condition of newborn foals born to American Paint Horse parents of the overo coat-pattern linage [1,2]. The foals are totally or almost totally white and affected with intestinal aganglionosis [35], leading to a functional obstruction (megacolon) and death. A mutation in the endothelin type-B receptor gene (EDNRB) was found to be the cause for LWFS in American [6,7] and Australian [8] Paint horses. Clues to this molecular basis came from man and rodents, where disorders are also observed that associate abnormal skin coloration and pigmentation patterns or white coat spotting, respectively, and intestinal aganglionosis. The inheritable human Waardenburg syndrome (WS) is characterized by the association of pigmentation abnormalities, including depigmented patches of the skin and hair, vivid blue eyes or heterochromia irides, and sensoneural hearing loss. The association of these disorders results from an abnormal proliferation, survival, migration, or differentiation of neural crestderived melanocytes [9]. Four subtypes of WS were defined on the basis of the presence or absence of additional clinical signs [10]. Type I WS (WS1) and WS2 are characterized by great variability of clinical signs, however both cover heterogeneous collection of melanocyte defects and they can be distinguished by the further presence of dystopia canthorum in WS1. WS with musculoskeletal abnormalities of the upper limbs and dystopia canthorum has been called Klein-Waardenburg syndrome or WS3. WS4, also known as Hirschsprung disease (HD) type II, or Shah-Waardenburg syndrome (OMIM #277580), is defined by the association with HD [1114]. HD or aganglionic megacolon is a congenital defect characterized by an absence of neural crest-derived intramural ganglia along varying lengths of the colon [15]. Single nucleotide substitutions and deletions in the gene encoding the endothelin type-B receptor are associated with a prominent portion of WS4 cases and a small percentage of WS2 cases, respectively [9]. A white coat colour in combination with intestinal aganglionosis is also observed in mice with targeted disruption or natural (piebald-lethal) mutations of the EDNRB locus [16], and in the spotting lethal rat, carrying an interstitial deletion of the EDNRB gene [17]. In an inbred flock of Cameroon sheep, five totally or almost totally white-coated lambs with light blue eyes were born. All died within few hours up to few days after birth, and signs of intestinal obstruction were noticed in some cases. The aim of this study was to investigate if the observed lethal hypopigmentation syndrome was associated with genetic variation at the EDNRB locus, and to check if such a possible genetic variant would also be found in other Cameroon sheep flocks. Evidence for Obstruction but not for Aganglionosis in Hypopigmented Lambs Two of the hypopigmented lambs were available for postmortem examination. Instead of the common brown phenotype of Cameroon sheep (figure 1A), one of the lambs was totally white, whereas the other was also white but had pigmented distal limb ends including the claws and black marks in a small perianal area (figure 1B). In both lambs, the irides were coloured light blue (figure 1C), instead of dark brown as it is usual for Cameroon sheep. Both showed a considerably dilated cecum (figure 1D) and remaining meconium. Histopathological examination of intestinal samples of both lambs revealed a normal number and distribution of ganglion cells which showed a homogenous faint labelling for synaptophysin by immunohistological investigation. By bacteriological examination of samples from both lambs E. coli could be cultured from the intestine, mesenterial lymph nodes, liver, spleen, kidneys and lung. The body of one of the lambs was rather fresh at postmortem examination (necropsy 24 hours post mortem). Therefore, the evidence of E. coli in many organs was interpreted as a final sepsis. The organs of the other lamb were already autolytic when necropsy was performed. Hence the detection of E. coli was without informative value. Amplification of EDNRB Sequences Failed Exclusively in Hypopigmented Lambs The coding regions including flanking parts of ovine EDNRB were amplified in order to sequence and to characterize the gene in affected lambs as (...truncated)