Resources and insights

ContentsIntroductionThe K1 genetic variant that has been part of EquiSeq’s Myopathy Panel since October 2019 is a missense allele of COL6A3, a gene encoding a collagen [1]. Collagens are a family of proteins that are the main structural components of the connective tissue. Collagens also guide bone formation and play an important role in many other tissues. They are the most abundant proteins in the body of mammals.There are thirty types of collagen, made up of collagen proteins encoded by 44 different genes. In humans, mutations in different collagen genes are responsible for a wide variety of inherited disorders. The phenotype produced by mutations in different collagens gives us an insight into the role of each of these collagens.The COL6A3 gene encodes one of the type VI collagens that are components of the endomysium, a layer of connective tissue that covers individual muscle fibers. Mutations in human COL6A1, COL6A2, and COL6A3 are associated with Bethlem myopathy and Ullrich congenital muscular dystrophy [1-11].The K1 allele of equine COL6A3 is defined by its coordinates in the current genome assembly for horse, chr6:23,416,882 C/G in EquCab3.0 [12]. For purposes of this blog post, we will use the protein model XP_023498413.1, which would make this allele G2178A, the substitution of an alanine (A) for…

This blog post is a discussion of a paper published by Williams et al. in a recent issue of Equine Veterinary Journal [1]. The paper aims to identify genetic variants that might be responsible for Myofibrillar Myopathy (MFM) in Warmbloods. The authors use a candidate gene approach, searching for variants of eight of the nine genes known to be associated with MFM in human patients and eight additional genes. Variants are identified by RNA-Seq of 16 warmbloods typed by muscle biopsy (8 affected and 8 control), and by analysis of additional whole genome sequence data available to the authors and in public databases. The citation is: Zoe J. Williams (will3084@msu.edu), Deborah Velez-Irizarry (no contact information), Jessica L. Petersen (jessica.petersen@unl.edu), Julien Ochala (julien.ochala@kcl.ac.uk), Carrie J. Finno (cjfinno@ucdavis.edu) and Stephanie J. Valberg (valbergs@cvm.msu.edu) (2020) Candidate gene expression and coding sequence variants in Warmblood horses with myofibrillar myopathy. Equine Vet J. 00: 1-10. PMID: 32453872. Contents Summary Ninety variants were identified in exons of thirteen of the sixteen genes (ACTA1, BAG3, DES, DNAJB6, FLNC, HSPB8, KY, LDB3, LMNA, MYOT, PLEC, PYROXD1, and SQSTM1). No coding variants were found for CRYAB, FHL1, or TIA1. Of the 90 variants, 62 were synonymous substitutions, 2 were splice region variants, and 26 were missense alleles. All 26 missense alleles (in 11 genes) were predicted to have moderate effects…

Paul Szauter, PhD, Chief Scientific Officer of EquiSeq, gave a presentation titled “Genetic Basis of Exercise Intolerance in Arabians” at the Al Khamsa Annual Meeting and Convention in Fayetteville, Arkansas on October 13, 2019. EquiSeq is a company based in Albuquerque, New Mexico, that develops and sells genetic tests for horses. Exercise intolerance is known in Arabians (1, 2) and limits the performance of some affected horses in endurance events. Symptoms of exercise intolerance (tying up) include sudden lameness, myoglobinuria, and greatly increased serum levels of creatine kinase (CK), a muscle enzyme released into the bloodstream when muscles are damaged. Researchers from EquiSeq began studying exercise intolerance in Arabians at an endurance event at the Biltmore estate in 2017. They were stationed at the vet trailer in order to study horses pulled from the event for symptoms of lameness. The weather was unusually cold and rainy, and it wasn’t long before the first horse showed up at the vet trailer with urine the color of black coffee and obvious symptoms of pain and lameness. A test for serum CK (normally in the range of 500 or less) showed levels in excess of 20,000. Eight hours and thirty liters of IV…

A Genome Wide Association Study (GWAS) of Recurrent Exertional Rhabdomyolysis (RER) identified a region on chromosome 16 as the only part of the genome associated with RER [1]. One of the populations in the study showed an association between RER and five Single Nucleotide Polymorphisms (SNPs) that define a 71.6 kb interval within the 822 kb CACNA2D3 gene. The CACNA2D3 gene encodes a regulatory subunit of the voltage-gated calcium channel called the dihydropyridine receptor (DHPR) that functions in excitation-contraction coupling, the process through which signals from motor neurons initiate muscle contraction [2]. The alpha(2)delta subunit encoded by CACNA2D3 sets the abundance of voltage-gated calcium channels by acting as a molecular chaperone. The alpha(2)delta subunit is bound to the plasma membrane through a glycosylphosphatidylinositol (GPI) anchor [3,4]. The level of expression of alpha(2)delta subunits sets the abundance of voltage-gated calcium channels in both neuronal and transfected nonneuronal cells [5,6]. The dihydropyridine receptor, of which the CACNA2D3 protein is a part, is known to physically interact with the ryanodine receptor encoded by the RYR1 gene. Malignant Hyperthermia (MH) in horses is caused by mutations in the RYR1 gene [7]. The RYR1-R2454G allele that causes MH in horses acts as an enhancer of the GYS1-R309H allele of glycogen synthase…

Several recent studies address the mechanism of muscle damage in filaminopathy. Filaminopathy is a term that describes muscle disorders resulting from mutations in the FLNC gene, which encodes filamen C. Two different clinical disease states are included in filaminopathy: Myofibrillar Myopathy 5 (MFM5) and Distal Myopathy 4 (DM4). Studies on the mechanism of muscle damage in filaminopathy suggest that there is an overlap of symptoms between these two disease states, and that the same mechanism of muscle damage takes place in both. Muscle tissue from patients with MFM5 shows cytoplasmic protein aggregates and rimmed vacuoles; electron microscopy shows Z disc fragmentation, Z disc streaming, nemaline rods, and autophagic vacuoles. Muscle tissue from patients with DM4 shows nonspecific changes, not including the Z disc fragmentation and protein aggregates seen in MFM5. Studies of Human Patients A study of muscle tissue from human filaminopathy patients with the W2710X or V930-T933del mutations shows that the large protein aggregates that are seen in Myofibrillar Myopathy contain not only Z disc proteins, but also proteins involved in clearing protein aggregates [1]. Double immunofluorescence with antibodies to either FLNC or MYOT and a series of other proteins shows that the aggregates contain heat shock proteins (HSP20, HSP27, HSP40,…

How can you figure out the chances of different genotypes when breeding horses? Monohybrid Cross It’s easy for one gene. Let’s say that there is a stallion that has one copy of one of the genetic variants associated with PSSM2, the P2 variant (MYOT-S232P). This stallion is heterozygous (n/P2), meaning that he has one normal copy of the MYOT gene and one copy with the P2 mutation. If the stallion is bred to a mare that is clear (n/n) for the P2 allele, the Punnett square below shows the odds. Gametes (sperm and eggs) contain a single copy of each gene in the horse. The stallion produces two kinds of sperm in equal frequency: sperm with P2, and sperm with the normal allele (n). The mare produces eggs with one of the two normal alleles (n). This means that there is a 50% chance of a foal that is n/P2, and a 50% chance of a foal that is n/n. Dihybrid Cross What if we are following two genes? In general, two gene pairs will show independent assortment. This means that the segregation of one pair of alleles into gametes will not influence the segregation of another pair of alleles….