Familiar hypopigmentation syndrome in sheep associated with homozygous deletion of the entire endothelin type-B receptor gene

Abstract

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.

Figure 1. Phenotypically normal and hypopigmented Cameroon sheep.

(A) Brown Cameroon sheep ewe and lamb. (B) Hypopigmented Cameroon sheep lamb, with light blue irides (C), and dilated cecum (D).

Figure 2. Relative EDNRB copy numbers resulting from real-time PCR.

Values of single sheep are shown in classes: 1, dams and (great-) granddam of hypopigmented lambs (n = 4); 2, unrelated sheep (n = 12); 3, hypopigmented lambs (n = 2).

Figure 3. Schematic representation of a micro-chromosomal deletion on OAR 10 including the EDNRB locus.

Indicated positions refer to the bovine genomic sequence NC007310.4 (BTA 12, Btau4.0).

Figure 4. Fluorescence in situ hybridization of an EDNRB-spanning bovine BAC clone on metaphase chromosomes.

Results from a sheep related to hypopigmented lambs (A), and from an unrelated sheep (B). Arrows indicate positions of (expected) hybridization signals.

Figure 5. Agarose gel electrophoresis of DNA fragments resulting from duplex PCR.

Lane 3: sample from sheep homozygous for the deletion (EDNRB −/−), lane 4: sample from sheep heterozygous for the deletion (EDNRB +/−), lane 5: sample from sheep without the deletion (EDNRB +/+); lane 1: DNA size marker (Gene Ruler 100 bp Plus DNA Ladder, Fermentas, St. Leon-Rot, Germany); lane 2: no template control.

Figure 6. Pedigree of hypopigmented lambs.

Square: male; circle: female; rhomb: sex unknown; empty symbol: phenotypically normal; filled symbol: affected; symbol with dashed margin: not sampled.