# Evaluation of the Prevalence of Genetic Variants at the Nebulette Locus in Cavalier King Charles Spaniels

## TL;DR

This study examines the frequency of healthy genetic variants in Cavalier King Charles Spaniels to understand their potential role in reducing heart disease.

## Contribution

The study provides the first assessment of wild-type NEBL allele prevalence in asymptomatic breeding populations of Cavalier King Charles Spaniels.

## Key findings

- No dogs were homozygous for the wild-type NEBL allele variants.
- Only one Dutch and nine Australian dogs were heterozygous for the wild-type allele.
- Selective breeding for wild-type alleles would reduce breeding stock and worsen genetic bottlenecks.

## Abstract

The Cavalier King Charles Spaniel is a dog breed in which mitral valve disease is very common. A potential link to this disease for risk allele variants near the heart-specific nebulette gene has been identified, where wild-type (i.e., healthy) allele variants are associated with less severe heart disease. The frequency of these wild-type allele variants in the asymptomatic breeding population is, however, unknown. Therefore, the aim of this study was to investigate the frequency of wild-type allele variants through genetic testing in dogs that were intended for breeding in both the Netherlands and Australia. No dog was homozygous (carrying two identical copies) for the wild-type allele variants. Only one dog from the Netherlands was heterozygous (carrying one wild-type and one risk allele variant), while nine dogs from Australia were heterozygous. The prevalence of heterozygous dogs in the Australian breeding population was low (4.6%), but significantly higher compared to the prevalence in the Dutch breeding population (0.57%). In conclusion, selective breeding for the wild-type allele variants on its own would significantly reduce the number of breeding individuals and would add to the existing genetic bottleneck. The selective breeding of Cavalier King Charles Spaniels for wild-type allele variants should not be undertaken on its own due to the low prevalence in this breed and the polygenic character of the disease.

The Cavalier King Charles Spaniel (CKCS) exhibits an unusually high prevalence of myxomatous mitral valve disease (MMVD). A potential link to MMVD for risk allele variants near the heart-specific nebulette (NEBL) gene has been identified. Although these risk allele variants seemed fixed in the CKCS, wild-type (i.e., healthy) allele variants at NEBL1-3 have likewise been found in a larger cohort, in which it was associated with less severe heart enlargement. The frequency of the wild-type allele variants in the asymptomatic breeding population is unknown. Therefore, the aim of this study was to investigate the wild-type allele variants frequency through prospective genetic testing in a large sample of CKCS that were intended for breeding in both the Netherlands and Australia. Blood samples of 370 CKCS with an unknown genetic status were collected, of which 175 from the Netherlands, and 195 from Australia. Deoxyribonucleic acid (DNA) was extracted for the genotyping of NEBL allele variants. No dog was homozygous for the wild-type allele variants. Only one dog from the Netherlands was heterozygous, while nine dogs from Australia were heterozygous. The prevalence of heterozygous dogs in the Australian breeding population was low (4.6%), but significantly higher compared to the prevalence in the Dutch breeding population (0.57%). In conclusion, selective breeding for the wild-type allele variants on its own would significantly reduce the number of breeding individuals and would add to the existing genetic bottleneck. The selective breeding of CKCS for wild-type allele variants should not be undertaken on its own due to the low prevalence in this breed and the polygenic character of the disease.