A Dinucleotide Mutation in the Endothelin-B Receptor Gene Is Associated with Lethal White Foal Syndrome (LWFS); A Horse Variant of Hirschsprung Disease (HSCR)

G. C. Yang 0 D. Croaker 0 A. L. Zhang 0 P. Manglick 0 T. Cartmill 0 D. Cass 0 0 Department of Surgical Research, Royal Alexandra Hospital for Children , Westmead, NSW 2145, Australia Lethal white foal syndrome (LWFS) is a congenital anomaly of horses characterized by a white coat colour and aganglionosis of the bowel, which is similar to Hirschsprung disease (HSCR). We decided to investigate possible mutations of the endothelin-B receptor gene (EDNRB) in LWFS as recent studies in mutant rodents and some patients have demonstrated EDNRB defects. First, we identified a full-length cDNA for horse EDNRB. This cDNA fragment contained a 1329 bp open reading frame which encoded 443 amino acid residues. The predicted amino acid sequence was 89, 91 and 85% identical to human, bovine and mouse as well as rat EDNRB respectively, but only 55% identical to the human, bovine and rat endothelin A receptor (EDNRA). Secondly, sequence analysis, together with allele-specific PCR and the amplification-created restriction site (ACRS) technique, revealed a dinucleotide TCAG mutation, which changed isoleucine to lysine in the predicted first transmembrane domain of the EDNRB protein. This was associated with LWFS when homozygous and with the overo phenotype when heterozygous. - Hirschsprung disease (HSCR) is a congenital human disorder characterized by the absence of enteric ganglion cells (aganglionosis) in the distal gastrointestinal tract (1). The disease is thought to be multifactorial, modified by sex, and there is a positive family history of HSCR in 15% of patients. Recent studies in HSCR patients have demonstrated gene mutations in two membrane receptorligand pairs: RETglial-derived neurotrophic factor (GDNF) (24), and endothelin-B receptor (EDNRB)endothelin 3 (EDN3) (5,6), as well as a transcription factor SOX10 (7). All these are considered to be loss-of-function mutations. Aganglionosis occurs in rodents (8,9), cats (10), pigs (11) and horses (1214). The rodent models also have a characteristic coat colour pattern suggesting that the genetic defect resides in a common mechanism involved in regulation of development of two neural crest cell-derived cell lineages, namely the enteric nervous system (ENS) and epidermal melanocytes. When the RET gene was functionally deleted in mice, the animals produced had extensive aganglionosis and a pigmented coat (15); whereas when EDNRB or EDN3 genes were knocked out the mice produced had colonic aganglionosis and changes in the coat colour resulting in a phenotype identical to piebald-lethal and lethal spotting mice. Subsequent studies identified an absence of EDNRB in piebald-lethal mice, and a point mutation of pro-EDN3 in lethal spotting mice (16). The spotting lethal rat has a 301 bp deletion in EDNRB exonintron 1 (17). It too has a white coat and aganglionosis, which often extends into the ileum. The combination of white coat with little or no patches of colour and extensive aganglionosis has also been described in the congenital abnormality, lethal white foal syndrome (LWFS), which presents as a fatal neonatal intestinal obstruction (12,13). LWFS most commonly results from mating overo overo paint horses. The similarity in clinical, histological and pigmentary phenotype to that observed in rodent models led us to suspect that a defect in the EDNRB gene may cause LWFS. To decide whether an EDNRB defect causes the LWFS, we first isolated and identified a full-length cDNA for EDNRB from standard bred horses. When we compared this sequence with that of LWFS horses, we found a TCAG dinucleotide mutation in the EDNRB gene of LWFS horses, changing the amino acid isoleucine to lysine in the predicted first transmembrane domain of the EDNRB protein. Association analysis confirmed the homozygous mutation was present in all LWFS. Complete cDNA sequence for the horse EDNRB As no sequence data or gene structure information were available for horse EDNRB, we applied a strategy of cross-species RT-PCR combined with a technique for isolation of full-length cDNA to determine the coding sequence of horse EDNRB. Two pairs of primers, ho1/pr4 and pr3/ho2, were selected and designed so as to have high homology with EDNRB cDNA in species including human, rodents, bovine and pig, but weak homology with corresponding sequences of EDNRA. The central part of a 1282 bp cDNA framed by oligonucleotide sequence ho1 and ho2 was isolated in two cDNA fragments (ho1/pr4 and pr3/ho2) from random-primed first-strand cDNA (Fig. 1). Sequences determined Figure 1. Schematic diagram showing the location on cDNA and direction of primers utilized in RT-PCR and inverse PCR used for isolation of full-length horse EDNRB cDNA. The translated region is shown by the box. The non-translated region is a single line. The sequences encoding the seven predicted transmembrane domains are displayed as black areas. Figure 2. A dendrogram of multiple sequence alignment showing homology of amino acid sequence of horse EDNRB with other species. Sequences were extracted from the database using the Entrez network server. The notations are as follows: ednr = endothelin receptors; the prefix A = EDNRA, prefix B = EDNRB, hum = human, hor = horse, bov = bovine, mou = mouse. in the ho1/pr4 cDNA fragment and the pr3/ho2 cDNA fragment overlapped. The isolated 5- and 3-terminal cDNA contained 130 bp of 5- and 228 bp of 3-untranslated regions respectively. Sequence analysis indicated that these sequences also overlapped with the initially isolated central part of the cDNA segment framed by oligonucleotides ho1 and ho2. In order to isolate the whole molecule of cDNA, a pair of primers, her1 and her2, were generated from the 5- and 3-untranslated region and used to amplify the cDNA sequence from the first strand cDNA pool. A 1667 bp cDNA fragment was isolated and cloned into plasmid vector. The complete cDNA sequence is composed of a 1329 bp open reading frame which encodes a 443 amino acid peptide. By using a probe which contained a 113 bp 5-untranslated region and a 237 bp 5-coding region, a 5 kb mRNA band was found on northern blot hybridization (data not shown). In the 3-noncoding region of this sequence, there were two potential polyadenylation signals AATAA and ATTAAA which were at 123 and 24 bp upstream from the end of this sequence where there is thought to be a polyadenylation site for EDNRB mRNA. Amino acid sequence analysis showed that this peptide consists of seven hydrophobic domains, a typical feature of the G protein-coupled receptor family (GPCR). The DNA and deduced amino acid sequence were aligned with EDNRB and EDNRA from several species. Sequence comparison revealed that the coding region sequence of the cDNA has most homology with EDNRB, with 85% identity in humans. The predicted amino acid sequence shares 91% identity with bovine, 89% with humans, and 85% with rat and mouse EDNRB; but has only 55% homology to human, bovine and rat EDNRA. A dendogram of receptor sequences sho (...truncated)