Cubilin gene mutation screening in Thai Border Collies using double-mismatch allele-specific and multiplex polymerase chain reaction: Implications for Imerslund–Gräsbeck syndrome

Background and Aim: Imerslund–Gräsbeck syndrome (IGS) is a rare autosomal recessive disorder characterized by selective intestinal cobalamin malabsorption in humans and several canine breeds. In Border Collies, IGS is caused by a single cytosine deletion in exon 53 of the CUBN gene (c.8392delC), resulting in a frameshift mutation and loss of Cubam receptor function. Although carrier frequencies have been reported in European and East Asian populations, region-specific genetic epidemiology data from Southeast Asia are lacking. This study aimed to determine the genotypic distribution and allele frequency of the CUBN c.8392delC mutation in Thai Border Collies and to validate a cost-effective molecular diagnostic approach combining double-mismatch allele-specific primers with multiplex polymerase chain reaction (PCR). Materials and Methods: A total of 107 clinically healthy Border Collies from private owners and breeding kennels in Thailand were enrolled. Genomic DNA extracted from peripheral blood was genotyped using a newly optimized double-mismatch allele-specific multiplex PCR assay designed to discriminate wild-type and mutant CUBN alleles, with an internal control amplicon included in each reaction. Synthetic mutant DNA fragments were used as positive controls for homozygous mutant detection. All PCR-based genotyping results were independently validated by Sanger sequencing. Genotype and allele frequencies were calculated, and 95% confidence interval (CI) values were estimated using the Wilson score method. Results: Of the 107 dogs examined, 106 were homozygous wild-type (WT; CUBN +/+) (99.1%; 95% CI: 94.8%–99.8%), one dog was a heterozygous carrier (CUBN +/−) (0.9%; 95% CI: 0.16–5.0%), and no homozygous mutant individuals (CUBN −/−) were identified (0%; 95% CI: 0%–4.3%). The estimated wild-type and mutant allele frequencies were 99.6% (95% CI: 98.2%–99.9%) and 0.4% (95% CI: 0.07%–1.8%), respectively. The double-mismatch allele-specific multiplex PCR assay demonstrated 100% concordance with Sanger sequencing, with no false-positive or false-negative results. Conclusion: This study provides the first population-level screening data for the CUBN c.8392delC mutation associated with IGS in Thai Border Collies and indicates a very low carrier and mutant allele frequency in this genetically diverse population. The validated double-mismatch allele-specific multiplex PCR assay offers a rapid, reliable, and cost-effective tool for routine genetic screening, supporting evidence-based breeding strategies and future large-scale genetic surveillance of hereditary disorders in dogs. Keywords: Border Collies, cubilin gene, double-mismatch allele-specific PCR, genetic screening, Imerslund–Gräsbeck syndrome, multiplex PCR, Thailand, veterinary genetics.