Immune-Mediated Myositis in Horses: From phenotype to genotype

Abstract

Background: Equine immune-mediated myositis (IMM) is a painful and debilitating condition of predominantly Quarter Horse (QH) and related breeds. The epaxial and gluteal muscles are most severely affected and muscle atrophy can be dramatic, with 50% of the affected muscle mass being lost in <72 hours. Diagnosis is based on a muscle biopsy of affected muscles and the identification of lymphocytes invading myofibers and in some cases surrounding blood vessels. The pathophysiology is presumed to be immune-mediated, but further evidence is needed to confirm this. Abnormal expression of major histocompatibility Complex (MHC) has been identified on muscle fibers from most human IMMs and provides the most consistent indication of an immune-mediated mechanism. The restriction of IMM to primarily QH and related breeds, particularly in certain bloodlines suggests that there is a genetic susceptibility underlying IMM. Hypothesis: Quarter Horses are genetically susceptible to an immune mediated myositis that is characterized by abnormal expression of MHC class I and/or class II on the sarcolemma of myofibers. Specific Aim 1: To determine if abnormal MHC class I and II expression is present on the sarcolemma of myofibers of horses with active IMM in the presence or absence of myofiber lymphocytic infiltrates. Specific Aim 2: To characterize the subtypes of lymphocytes in the myofibers of horses with active IMM and correlate this with MHC expression. Methods: Immunohistochemical staining for MHC I, II, CD4+, CD8+, CD20+ lymphocytes was performed on archived muscle samples of IMM (21 horses) and controls (3 healthy and 6 disease controls). Scores were given for MHC I and II and for lymphocytic subtypes. Results: A degree of sarcolemmal MHC I and II expression was present in 81% and 71% of IMM horses, respectively. CD4+, CD8+, and CD20+ cells were present in 20/21 IMM horses with a CD4+ predominance in 48% of cases. MHC I score was positively correlated with MHC II (r = 0.89, p = <0.001) and CD8+ (r= 0.64, p = 0.002) and CD20+ (r = 0.66, p = 0.001) lymphocyte and macrophage scores (r = 0.70, p = <0.001). MHC II scores were positively correlated to CD8+ (r = 0.59, p = 0.005), CD20+ (r = 0.61, p = 0.004) lymphocyte and macrophage (r = 0.70, p = <0.001) scores. Specific Aim 3: To determine if equine IMM is significantly associated with a region of the equine genome using a genome-wide association (GWA) study. Methods: DNA was extracted from blood and muscle of 36 IMM horses and 54 healthy controls of QH-related breeds that were housed in similar environments. Sequencing was performed on equine 50K and 70K single nucleotide polymorphism (SNP) arrays. A GWA was performed across 40,811 SNPs that passed quality control. To account for elevated genomic inflation, statistical analysis was performed using GEMMA and GRAMMAR-GC software. Results: A significant association was identified between IMM and a 2 MB region on equine chromosome 11. Five SNPs in 3 haplotype blocks reached genome-wide significance using the 2 different statistical methods to account for population stratification. The significant region contains 6 myosin heavy chain genes expressed in skeletal muscle, including MYH2, which has been associated with a human IMM. Conclusions: Equine IMM is characterized by MHC I and II expression on the sarcolemma of myofibers during an acute CD4+ and CD8+ lymphocytic inflammatory episode. There is an approximately 2MB region on equine chromosome 11 that is associated with the development of the disease. Sequencing of the MYH genes in IMM cases and unaffected controls is warranted to identify variants that cause IMM in Quarter Horses.