Six genetic variants that can disrupt muscle structure and/or function have been identified in horses with symptoms of Exertional Myopathy. Previously described as subtypes of PSSM2, these variants are properly classified as Muscle Integrity Myopathies.
The Myotilin gene (MYOT, equine mutation P2) encodes the structural protein Myotilin (MYOT). This protein plays an important role in the structural stability and organization of the smallest functional unit of striated muscle, the Z-disc, which is essential for muscle contractions. Myotilin binds F-actin and α-actinin-2 and allows actin cross-linking for Z-disc assembly.
Humans with mutations in the Myotilin gene develop Myofibrillar Myopathy 3 (MFM3), a rare, hereditary, adult-onset disease. Most affected human patients develop symptoms between 50-77 years of age; the most reported symptoms are progressive weakness of the distal (furthest from the body) muscles and peripheral neuropathy. In horses, a mutation (chr14:37,818,823 A/G) in the orthologous MYOT gene results in an amino acid change that interferes with the binding of myotilin to actins.
The Filamin C gene (FLNC, equine mutation P3) encodes an actin-binding protein that is involved in linking the Z-disc and actin filaments. The Z-disc is located between sarcomeres. In horses, the dominant mutation (chr4:83,840,299 G/A) leads to a loss of function. Humans with mutations in the FLNC gene develop Myofibrillar Myopathy 5 (MFM5), an adult-onset hereditary disease characterized by progressive skeletal muscle weakness that can sometimes extend to muscles in the heart and respiratory system. Appearance on biopsy is dependent on the muscle and the stage of the disease process.
Myozenin 3 is an intracellular binding protein which connects other proteins that are active in the Z-disc (eg. a-Alpha-Aktin, g-Filamin, TCAP/Telethonin, LDB3/ZASP). Myozenin also plays an important role in the actions of Calcineurin in the sarcomere. No disease-associated myozenin mutation has yet been described in humans; the mutation in horses is chr14:26,710,261 G/A.
The PYROXD1 gene (pyridine nucleotide-disulphide oxidoreductase domain 1) encodes a protein important in oxidative defence in the body. Normal cellular metabolism and signalling in the body results in the production of reactive oxygen species (ROS) which can cause damage to DNA. Antioxidant molecules are therefore produced by the body to scavenge ROS and alleviate oxidative stress. PYROXD1 is natural antioxidant and an important part of the oxidative defence system.
Experiments in yeast show that mutations in PYROXD1 reduce reductase activity, and zebrafish knockdown models show reduced swimming ability. In humans, mutations in PYROXD1 lead to myofibrillar myopathy 8 (MFM8), which can be child- or adult-onset. It is characterized by slowly progressive proximal muscle wasting and weakness. The equine mutation (chr6:48,924,749 G/C) causes an amino acid substitution at a highly conserved site in the PYROXD1 protein and reduces its functionality.
The CACNA2D3 gene (Calcium voltage-gated channel auxiliary subunit alpha2 delta 3 (equine mutation Px: chr16: 34635494 T/C) encodes a protein that is part of a regulatory subunit of the calcium channel DHPR (dihydropyridine receptor) that helps regulate signaling to trigger muscle contractions. It is important to note, however, that the Px mutation causes no change in the encoded protein. The mutation is a change in the codon but conserves the amino acid. It is hypothesized that the consequence is either a direct effect via the modulation of splicing mechanisms, or that the Px mutation is an indirect marker of a coupled pathogenic mutation. Px is considered a risk factor for recurrent exercise-related rhabdomyolysis (RER) in several Thoroughbred and Arabian horse families. In the context of Muscle Integrity Myopathy (“PSSM2”), the Px variant appears to exacerbate the effects of other variants.
The COL6A3 gene encodes the Collagen type 6 alpha 3 protein. This chain-like protein binds with two other collagen proteins (usually COL6A1 and COL6A2) to form a triple helical Collagen type 6 molecule. COL6 is an essential structural protein in the extracellular matrix of the entire protein, especially in areas of fibroblast synthesis. In muscle, COL6 is a key protein of the Endomysium. The clinical symptoms of COL6-associated myopathies are dependent on the type and location of the gene mutation.
In humans there are multiple genetic diseases caused by defects in type 6 collagens; these include Bethlem Myopathy and congenital Ullrich Muscular dystrophy, both of which are caused by mutations in the COL6A3 gene.
In horses, the K1 variant (equine Mutation K1: chr6:23.416.882 C/G) causes a substitution of an amino acid in the COL6A3 protein. This disrupts the binding of the collagen 6 triple helix, resulting in the production of an aberrant COL6 molecule with reduced function.