Simplified PCR analysis of a mutation in the NHEJ1 gene causing collie eye anomaly in some dog breeds

Abstract

ABSTRACT:Collie eye anomaly (CEA) is an inherited eye disease affecting development of the choroids and sclera segregating in several, mostly herding breeds of dog. Phenotypic development of the disease varies greatly in the affected animals. Genetic control of its clinical variation is unknown so far. Affected dogs share a 7.8 kb deletion in intron 4 of the NHEJ1 gene. We report here population studies of 379 dogs (Australian Shepherd, Border Collie, Rough Collie, Smooth Collie, Shetland Sheep Dog, and Nova Scotia Duck Tolling Retriever) from breeders in the Czech Republic. A simple PCR method using a Piko TM Thermal Cycler and unclotted blood samples was employed for the analysis of the NHEJ1 gene. No isolation of DNA from blood samples before PCR was needed. The method is time-saving and gives excellent results. Frequencies of the disease allele in each breed were calculated (0. 045, 0.194, 0.797, 0.367, 0.429 and 0.244, respectively). An improvement of genetic health of the breeds on the basis of allele frequencies is discussed.

A novel multiplex polymerase chain reaction assay for the genotypic survey of the non-homologous end-joining factor 1 gene associated with Collie eye anomaly in Thailand

“…Almost all Shetland Sheepdogs tested in this survey had an intact NHEJ1 gene, and both NHEJ1 deletion patterns were detected in some individuals, with a frequency of 2.8%. This finding was extremely low compared to that described for the Czech Republic [13] and Denmark [19], where the mutation frequencies were 42.9% and 58.9%, respectively. Among the mutated cases examined in these investigations, the CEA/cea genotype was more frequent, except in Denmark, where the cea/cea genotype predominated [19].…”

“…Although NHEJ1 genotyping assay was available for CEA diagnosis, the geographic distribution of the NHEJ1 genotypic status across different countries remains undiscovered. In the Rough Collies, a remarkably high mutation frequency of 83.3% was observed in this study; this value is slightly higher compared to that reported in the Czech Republic, where the mutation frequency was 79.7% [13]. In Denmark, however, a considerably higher mutation frequency of 98.89% was reported [19].…”

“…The diagnosis of CEA by conventional PCRbased canine NHEJ1 genotyping was first introduced by Parker et al [6]. To date, several molecular genotyping assays for detecting NHEJ1 genotypes have been developed, including a rapid direct PCR amplification method without DNA extraction [13] and real-time PCR based on SYBR Green combined with Flinders Technology Associates cards [14]. In this study, we successfully established a novel multiplex PCR assay for identifying NHEJ1 genotypes in five purebred dog breeds in Thailand.…”

Progressive rod-cone degeneration (PRCD) in selected dog breeds and variability in its phenotypic expression

“…The frequency of the CEA mutant allele is 0.244 (Dostal et al, 2010). To select against both mutations in a population of the breed requires a thoroughly defined breeding policy and consideration of what the population needs, instead of simply taking into account the desires of breeders.…”

Collie eye anomaly: a review

“…Based on the previous work mentioned, Dostal et al (2010) performed simplified analysis of the deletion without DNA isolation and Chang at al. (2010) utilised a rapid genotyping technique based on SYBR Green Real Time PCR which was applied to blood and saliva specimens on Flinders Technology Associated Filter Papers.…”