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

Abstract

Background and Aim: Collie eye anomaly (CEA) is a hereditary and congenital ocular disorder, which affects several dog breeds, including Collies, Collie-related breeds, and other purebreds. An intronic deletion of 7799-bp in the non-homologous end-joining factor 1 (NHEJ1) gene has been identified as the genetic defect causing CEA. This study aimed to investigate the prevalence of CEA based on NHEJ1 genotyping assay in Thailand. Materials and Methods: We clarified the prevalence of CEA in 224 dogs from five purebred dog breeds using a novel multiplex polymerase chain reaction (PCR)-based technique and confirmed the genotypic status with direct DNA sequencing. Results: The highest frequency of the mutated NHEJ1 allele was 83.3% for Rough Collies, followed by 7.8% for Border Collies, 5.1% for Australian Shepherds, and 2.8% for Shetland Sheepdogs. The heterozygous mutated NHEJ1 genotype detected for Rough Collies, Border Collies, Australian Shepherds, and Shetland Sheepdogs was 33.3%, 15.6%, 10.3%, and 3.3%, respectively. The homozygous mutated NHEJ1 genotype was detected only in Rough Collies and Shetland Sheepdogs, accounting for 66.7% and 1.1%, respectively. Thai Ridgeback Dogs were not affected by this mutation. Conclusion: This study describes, for the 1st time, the genotypic survey of the NHEJ1 gene associated with CEA in dogs in Thailand. In addition, we successfully developed a new multiplex PCR assay with high accuracy, reproducibility, and cost-efficiency and validated its usefulness for determining NHEJ1 genotypes.

Multiplex allele-specific polymerase chain reaction-based prevalence study of canine trapped neutrophil syndrome in Thailand

“…As expected, no mutations in the VPS13B gene were identified in any of the 30 dogs evaluated, indicating that this native breed has little or no incidence of TNS. Interestingly, a similar finding has been reported by previous genotypic surveys of the MDR1 and NHEJ1 genes in Thailand [14,15]. Although the population of Thai Ridgebacks in these studies was relatively small, our findings provide valuable evidence that this native breed has a relatively healthy genetic profile and is not susceptible to genetic disorders commonly associated with sheepdog lineages.…”

“…Based on the previous studies and the present survey, it is clear that VPS13B genotypic assays reveal that the prevalence of TNS in Border Collies is relatively low in many different countries. In addition to the results published here, we recently investigated the prevalence of two additional gene mutations, including multi-drug resistance 1 ( MDR1 ) and non-homologous end-joining factor 1 ( NHEJ1 ) genes in several dog breeds in Thailand [ 14 , 15 ]. In the sample of 45 Border Collies examined for these surveys, the mutated MDR1 allele was not observed in any of the dogs tested, indicating a remarkably low prevalence of the MDR1 gene mutation in this breed [ 14 ].…”

“…In the sample of 45 Border Collies examined for these surveys, the mutated MDR1 allele was not observed in any of the dogs tested, indicating a remarkably low prevalence of the MDR1 gene mutation in this breed [ 14 ]. Furthermore, the prevalence of the NHEJ1 gene mutation associated with Collie eye anomaly was relatively low in this population, as indicated by a carrier frequency of 15.6%, and a mutant allele frequency of 7.8% [ 15 ]. Taken together, our findings therefore provide compelling evidence that the risk of Thai Border Collies developing these common inherited genetic disorders is relatively low.…”

The incidence of genetic disease alleles in Australian Shepherd dog breed in European countries

“…According to OFA (Orthopaedic Foundation for Animals) statistics based on CEA test results of 128 AS from the North American population, the incidence is 1.98%, which is much lower than in Europe [ 37 ]. The frequency of the mutant allele, revealed by the CEA survey in Thailand, was 5.1% [ 38 ]. It could be an effect of genetic drift, but further large-scale studies are required to confirm such a hypothesis.…”

“…frequency of the mutant allele, revealed by the CEA survey in Thailand, was 5.1% [38]. It could be an effect of genetic drift, but further large-scale studies are required to confirm such a hypothesis.…”