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Prevalence and demographics of the MYBPC3-mutations in ragdolls and Maine coons in the British Isles D. Casamian-Sorrosal, S. K. Chong, S. Fonfara and C.Helps* Langford Cardio-Respiratory Unit, Langford Veterinary Services and School of Veterinary Science, University of Bristol, Langford, Bristol BS40 5DU *Molecular Diagnostic Unit, Langford Veterinary Services, University of Bristol, Langford, Bristol BS40 5DU

OBJECTIVES: To determine prevalence and demographics of two myosin-binding protein C (MYBPC3) mutations that affect ragdolls (R820W) and Maine coons (A31P) in the British Isles. METHODS: From the database of a genetic testing laboratory samples from 2018 ragdolls and 742 Maine coons were analysed with respect to mutation status, age, sex and county of origin. The actual prevalence was compared to the expected Hardy–Weinberg prevalence by chi-squared test. RESULTS: The prevalence of the R820W mutation in ragdolls was 27% (25·6% heterozygous, 1·4% homozygous), and that of the A31P mutation in Maine coons was 39·4% (36·4% homozygous, 3% heterozygous). There were more female cats (69·5% ragdoll, 70·3% Maine coon). The median age was 6·4 months (ragdolls) and 5·9 months (Maine coons). Cats from more than 60 counties were represented for each breed. The difference between the expected and observed allele frequency was significant in Maine coons (P=0·047) but not in ragdolls (P=0·092). CLINICAL SIGNIFICANCE: This is the first report of prevalence and demographics of the R820W and A31P mutations in ragdolls and Maine coons, respectively, in the British Isles. The prevalence is high, which is of relevance for breeding and screening programmes. The significant difference in genetic distribution may suggest early death of homozygous Maine coons. Journal of Small Animal Practice (2014) 55, 269–273 DOI: 10.1111/jsap.12201 Accepted: 26 January 2014; Published online: 7 March 2014

INTRODUCTION Hypertrophic cardiomyopathy (HCM) is characterised by a hypertrophied non-dilated left ventricle in the absence of other cardiac or systemic conditions (e.g. aortic valve stenosis, systemic hypertension) (Fox 1999, Maron 2012). It is a common disease in humans (Maron 2012) and cats (Ferasin et al. 2003) and has a genetic basis (Meurs et al. 2005, 2007, Maron 2012). In humans, mutations of the beta-myosin heavy chain and myosinbinding protein C (MYBPC3) proteins (Maron 2012) are most frequent. In cats, in Maine coons (Meurs et al. 2005) and ragdolls (Meurs et al. 2007) two different causative mutations affecting the MYBPC3 protein and resulting in A31P and R820W amino acid substitutions, respectively, have been identified. The prevalence of the A31P MYBPC3 mutation in Maine coons has been shown to vary between countries, ranging from Journal of Small Animal Practice



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May 2014



22% in Germany (Wess et al. 2010) to 46% in Australia-New Zealand (Fries et al. 2008). In the UK the prevalence has been previously reported as 34% (Fries et al. 2008) in 215 cats from a population with unknown demographic characteristics or subgeographical background. No information is available about the prevalence or demographics of the R820W MYBPC3 mutation in the ragdolls of the British Isles. Elsewhere, a recent study reported prevalences of 17 and 23% in small cohorts of ragdolls in Italy (n=171) and the United States (n=25), respectively (Longeri et al. 2013). As in human HCM, an autosomal dominant mode of inheritance with incomplete penetrance is suspected in Maine coons (Longeri et al. 2013). Maine coon cats heterozygous for A31P show a low penetrance by middle age, while most homozygous affected cats suffer from the disease by the same age (Godiksen et al. 2011, Longeri et al. 2013) and might have reduced survival

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times (Godiksen et al. 2011). In ragdolls, the nature of phenotypic penetrance is still not fully understood, but a recent study (Borgeat et al. 2013) showed that ragdolls homozygous for R820W died at a younger age compared to heterozygous or wild-type cats. The primary aim of this study was to describe the prevalence and demographics of the R820W MYBPC3 mutation in ragdolls and the A31P MYBPC3 mutation in Maine coons in a large and geographically diverse population of cats within the British Isles. Furthermore, the genetic distribution of the mutations within the respective populations were compared to the expected Hardy–Weinberg distribution to investigate whether early death of affected individuals occurred (Wigginton et al. 2005, Godiksen et al. 2011).

MATERIALS AND METHODS Samples submitted to a genetic testing laboratory (Molecular Diagnostic Unit, Langford Veterinary Services, University of Bristol) for identification of the R820W MYBPC3 mutation in ragdolls and the A31P MYBPC3 mutation in Maine coons between June 2009 and May 2013 were used for this study. In most cases the cats’ phenotypic status was unknown at the time of genetic testing. Samples had been submitted by breeders or owners, or had been collected randomly at breed shows as part of a previous study (Casamian-Sorrosal et al. 2010). Mutation analysis was performed by polymerase chain reaction (PCR) followed by pyrosequencing. Briefly, genomic (g)DNA was extracted from buccal swab or ethylenediaminetetraacetic acid (EDTA) blood samples using a commercial Kit (Nucleospin Blood; MachereyNagel). The region of the feline MYBPC3 gene containing the relevant mutation (A31P or R820W) was amplified by PCR as follows: 12·5 µL 2× GoTaq Master Mix (Promega), 0·5 µL of 10 µM each primer mix (Table 1), 5 µL gDNA and 7 µL water. Thermal cycling was undertaken in a PeqStar 2× thermal cycler (PeqLab) for 2 minutes at 95°C followed by 38 cycles of 20 seconds at 95°C and 40 seconds at 58°C. Pyrosequencing was undertaken using PyroMark Gold Q24 reagents (Qiagen), gene-specific sequencing primers (Table 1) and a PyroMark Q24 pyrosequencer (Qiagen) as described by the manufacturer. The sequence to analyse was C/TGGCTGAA (dispensation order GCTGCTGA) for R820W and C/GCTCGAAC (dispensation order TGCTCGAC) for A31P. Results were analysed using PyroMark Q24 software ver 2.0.6 (Qiagen). The database was also queried for information about age and sex of the cats at the time of submission, and county of origin within the British Isles (counties within the UK or the Republic of Ireland). Cats were excluded from analysis if no identification

(name or microchip number) was available, if samples were tested twice (repeated identification), were submitted from outside the British Isles or had erroneous data in any other way. Descriptive statistics were applied to the prevalence of homozygous, heterozygous and wild-type genotype in respect to the mutation for each breed, the percentage of either sex or the age distribution. Age was given as median, interquartile (IQ) range and 5th to 95th percentiles. For the age of homozygous affected cats the latter could not be applied because of the low numbers and the total range was given instead. Geographical distribution of the data is given in percentages according to the location within five geographical areas: South of England, North of England, Wales, Scotland and Ireland (Northern Ireland and Republic of Ireland). Any county in England north of Norfolk, Cambridgeshire, Northamptonshire, Warwickshire, West Midlands, Worcestershire or Shropshire was considered North of England. Cats from the Isle of Man were also included within the North of England population. A chi-squared test was applied to compare the observed mutation prevalence in ragdolls and Maine coons with the expected prevalence as determined using the Hardy– Weinberg equation. Significance was taken at P

Prevalence and demographics of the MYBPC3-mutations in ragdolls and Maine coons in the British Isles.

To determine prevalence and demographics of two myosin-binding protein C (MYBPC3) mutations that affect ragdolls (R820W) and Maine coons (A31P) in the...
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