Candidate gene expression and coding sequence variants in Warmblood horses with myofibrillar myopathy

Abstract

Background Myofibrillar myopathy (MFM) of unknown aetiology has recently been identified in Warmblood (WB) horses. In humans, 16 genes have been implicated in various MFM‐like disorders. Objectives To identify variants in 16 MFM candidate genes and compare allele frequencies of all variants between MFM WB and non‐MFM WB and coding variants with moderate or severe predicted effects in MFM WB with publicly available data of other breeds. To compare differential gene expression and muscle fibre contractile force between MFM and non‐MFM WB. Study design Case‐control. Animals 8 MFM WB, 8 non‐MFM WB, 33 other WB, 32 Thoroughbreds, 80 Quarter Horses and 77 horses of other breeds in public databases. Methods Variants were called within transcripts of 16 candidate genes using gluteal muscle mRNA sequences aligned to EquCab3.0 and allele frequencies compared by Fisher's exact test among MFM WB, non‐MFM WB and public sequences across breeds. Candidate gene differential expression was determined between MFM and non‐MFM WB by fitting a negative binomial generalised log‐linear model per gene (false discovery rate <0.05). The maximal isometric force/cross‐sectional area generated by isolated membrane‐permeabilised muscle fibres was determined. Results None of the 426 variants identified in 16 candidate genes were associated with MFM including 26 missense variants. Breed‐specific differences existed in allele frequencies. Candidate gene differential expression and muscle fibre‐specific force did not differ between MFM WB (143.1 ± 34.7 kPa) and non‐MFM WB (140.2 ± 43.7 kPa) (P = .8). Main limitations RNA‐seq–only assays transcripts expressed in skeletal muscle. Other possible candidate genes were not evaluated. Conclusions Evidence for association of variants with a disease is essential because coding sequence variants are common in the equine genome. Variants identified in MFM candidate genes, including two coding variants offered as commercial MFM equine genetic tests, did not associate with the WB MFM phenotype.

Myofibre Hyper-Contractility in Horses Expressing the Myosin Heavy Chain Myopathy Mutation, MYH1E321G

“…The number of horses evaluated in the present study was limited and should be expanded in the future. It is of note, however that specific force was previously assessed by our laboratory in a study of Warmblood horses (eight horses, 27 fibres) with values (140.2 ± 43.7 kPa) that were very similar to control horses in the present study [ 29 ]. Determining the proportion of mutated type IIx skeletal myosin heavy chains may be crucial in the development (or not) of MYHM and this should be addressed in a future study.…”

Commercial genetic testing for type 2 polysaccharide storage myopathy and myofibrillar myopathy does not correspond to a histopathological diagnosis

“…30 Furthermore, RNA-seq analysis of muscle from eight WB MFM and eight control WB horses found 26 missense variants, including P2, P3a and P3b, in 16 genes known to cause MFM or MFM-like diseases in humans; none of the variants, however, were associated with the MFM phenotype in the eight WB horses. 31 The relative ease with which genomes can be sequenced using high-throughput next-generation sequencing and the resultant identification of TA B L E 3 The number of horses homozygous for the reference allele, heterozygous or homozygous for each P variant by phenotype and breed as well as the percentage of horses in each classification possessing each P variant and the variant allele frequencies numerous variants in the genome underscore the importance of validating associations between a discovered variant and the gold standard for diagnosis of the associated disease.…”

Enriched Pathways of Calcium Regulation, Cellular/Oxidative Stress, Inflammation, and Cell Proliferation Characterize Gluteal Muscle of Standardbred Horses between Episodes of Recurrent Exertional Rhabdomyolysis

“…Total muscle RNA was isolated from snap frozen gluteus medius muscle from all horses as previously described [ 23 ]. Sequencing of mRNA (integrity score >7) was performed at the Michigan State University Research and Technology Support Facility Genomics Core (East Lansing MI, USA).…”