Short communication New test for endothelin receptor type B (EDNRB) mutation genotyping in horses Miguel Angel Ayala-Valdovinos * , Jorge Galindo-García, David S  anchez-Chipr  es, Theodor Duifhuis-Rivera Departamento de Producci on Animal, Divisi on de Ciencias Veterinarias, Centro Universitario de Ciencias Biol ogicas y Agropecuarias, Universidad de Guadalajara, A.P. 218 Zapopan 1, C.P. 45101, Zapopan, Jalisco, Mexico article info Article history: Received 6 February 2016 Received in revised form 18 March 2016 Accepted 24 March 2016 Available online xxx Keywords: Horse LWFS EDNRB gene Mutagenically separated polymerase chain reaction abstract Lethal white foal syndrome (LWFS) is an autosomal recessive disease of neonatal foals characterized by a white hair coat and a functional intestinal obstruction. Traditional techniques for identifying the dinu- cleotide mutation (TC/AG) of the endothelin receptor B gene (EDNRB) associated with LWFS are time- consuming. We developed a new technique based on mutagenically separated polymerase chain reac- tion (MS-PCR) for simple detection of the EDNRB genotype in horses. © 2016 Published by Elsevier Ltd. 1. Introduction Intestinal aganglionosis is an inherited abnormality seen in a number of species, such as mice, rats, horses and humans [1e4]. In mice, the aganglionic megacolon is associated with coat color spotting (piebald-lethal) [5,6]. In rats, the spotting lethal rat is a naturally occurring rodent model of congenital intestinal agan- glionosis [7,8]. In humans, the disorder is known as Hirschsprung's disease and affects about 1 in 5000 live births [4]. In horses, the variant of this disease is known as ileocolonic aganglionosis (ICA) or lethal white foal syndrome (LWFS) [9e11]. In all these species, the cause of the disease is a congenital disorder altering neural crest cell migration to the intestine [10]. Two overo spotted horses may produce an LWFS affected foal, but the LWFS affected foal will be white and not “overo spotted” [11,12]. Foals with this disease typically have white coats and may have blue eyes (iris depigmentation). All present abdominal distension, with signs of colic in the first 12e14 h after birth (Fig. 1). The white coat is due to an absence of skin melanocytes, and in- testinal obstruction is due to the absence of enteric nerve cells. There is no known treatment for this condition, which is lethal in all cases and, thus, necessitates humane euthanasia [1]. The genetic defect responsible for LWFS is a mutation in one dinucleotide (TC / AG) that changes the amino acid isoleucine to lysine in codon 118 of the endothelin receptor B gene (ENDRB) located in equine chromosome 17 [13,14]. Polymerase chain reac- tion (PCR)-based genetic tests are needed to identify the LWFS mutation. A test would also be useful for determining carrier status in spotted-coat overo and tobiano horses, as well as crop-out (white-spotted) Quarter Horses [11]. Different methods have been described to detect the EDNRB:c353_354delinsAG mutation, including allele-specific PCR (AS-PCR) and PCR primer-introduced restriction analysis (PIRA- PCR) [14,15]. These tests, which have been carried out by poly- acrylamide gel electrophoresis (PAGE), are time-consuming tech- niques that present some problems in detection sensitivity. In our study, we developed a new method based on mutagenically sepa- rated PCR (MS-PCR). Our method provides a fast and simple anal- ysis of the EDNRB horse gene. In the MS-PCR method, both normal and mutant alleles can be amplified in the same reaction tube by using allele-specific primers of different lengths without using a restriction endonuclease pro- cess. This method was originally developed for the easy analysis of familial hypercholesterolemia in humans [16]. We based our test on * Corresponding author. E-mail address: manayala@cucba.udg.mx (M.A. Ayala-Valdovinos). Contents lists available at ScienceDirect Molecular and Cellular Probes journal homepage: www.elsevier.com/locate/ymcpr http://dx.doi.org/10.1016/j.mcp.2016.03.005 0890-8508/© 2016 Published by Elsevier Ltd. Molecular and Cellular Probes xxx (2016) 1e3 Please cite this article in press as: M.A. Ayala-Valdovinos, et al., New test for endothelin receptor type B (EDNRB) mutation genotyping in horses, Molecular and Cellular Probes (2016), http://dx.doi.org/10.1016/j.mcp.2016.03.005