A missense mutation in MYH1 is associated with susceptibility to immune-mediated myositis in Quarter Horses

Abstract

Background: The cause of immune-mediated myositis (IMM), characterized by recurrent, rapid-onset muscle atrophy in Quarter Horses (QH), is unknown. The histopathologic hallmark of IMM is lymphocytic infiltration of myofibers. The purpose of this study was to identify putative functional variants associated with equine IMM.

Methods: A genome-wide association (GWA) study was performed on 36 IMM QHs and 54 breed matched unaffected QHs from the same environment using the Equine SNP50 and SNP70 genotyping arrays.

Results: A mixed model analysis identified nine SNPs within a ~ 2.87 Mb region on chr11 that were significantly (P_unadjusted < 1.4 × 10^{- 6}) associated with the IMM phenotype. Associated haplotypes within this region encompassed 38 annotated genes, including four myosin genes (MYH1, MYH2, MYH3, and MYH13). Whole genome sequencing of four IMM and four unaffected QHs identified a single segregating nonsynonymous E321G mutation in MYH1 encoding myosin heavy chain 2X. Genotyping of additional 35 IMM and 22 unaffected QHs confirmed an association (P = 2.9 × 10^{- 5}), and the putative mutation was absent in 175 horses from 21 non-QH breeds. Lymphocytic infiltrates occurred in type 2X myofibers and the proportion of 2X fibers was decreased in the presence of inflammation. Protein modeling and contact/stability analysis identified 14 residues affected by the mutation which significantly decreased stability.

Conclusions: We conclude that a mutation in MYH1 is highly associated with susceptibility to the IMM phenotype in QH-related breeds. This is the first report of a mutation in MYH1 and the first link between a skeletal muscle myosin mutation and autoimmune disease.

Keywords: Equine; Genome-wide association; Immunology; Myopathy; Myosin heavy chain 1.

Fig. 1

a Normal muscle mass in an MYH1 E321G homozygote prior to developing IMM. b The same horse 4 months after an episode of IMM. The spine is prominent due to loss of epaxial muscles (arrow). c Atrophy of middle and superficial gluteal muscles (arrow) is present

Fig. 2

a Manhattan plot and (top right insert) QQ-plot demonstrating a genome-wide significant association with the IMM phenotype using GEMMA analysis [17] on chr11. Minimal genomic inflation was present. b Genotypes for the MYH1 E321G variant across IMM-affected horses (n = 71 GWA and follow-up cohorts combined), at risk horses (n = 75, housed on the same farms as IMM horses), a cohort of random QH (n = 28), and 21 other breeds (n = 179)

Fig. 3

a–d Pedigrees from four subsets of the larger pedigree (Additional file 2: Figure S1) demonstrating a founder effect of four stallions for the IMM phenotype. Circles = females, squares = males. Open circles = phenotype unknown. Colored circles = IMM-affected with red = G/G genotype, blue = G/A genotype, and gray = A/A phenotype. Two of these founder stallions were traced back to the stallion in family A within one to three generations. For family D, the dam line traced back to the stallion in family A within four generations (Additional file 2: Figure S1)

Fig. 4

Schematic representation of MYH1 (MyHC 2X) with wild type (E317E, wt) and MYH1 mutation (E317G, mut). a Wild-type (wt) variant is shown as light green (red arrow) and E317G mutation (mut) is shown on the adjacent panel as teal (red arrow). In both panels, helices are represented as blue and additionally loop, SWITCH regions and actin binding sites are labeled. b Detailed alignments of the query protein (upper sequence line) and structure hits showing the protein sequence (PDB SEQRES record) and the resolved coordinates (PDB ATOM record). The input variant is indicated by green arrows (below and above the alignment) and highlighted amino acids represent residues that appear in dbSNP as well as common associated somatic mutation in cancer (COSMIC) variants. c Side chain and stability analysis of MYH1 E317G mutation showing contact surface areas between atoms and solvent accessible surfaces. Table includes contact surface area (Ų) and minimal atomic distance (Å) between two atoms of the two residues for the 15 amino acid residues that were determined to be impacted by the mutation. d A pictorial presentation of contact/residues affected by the MYH1 E317G mutation and its association to SWITCH1, helix I and helix J regions of MYH1. The residues affected are represented with an asterisk (*). Helix K is partially represented. The input variant is indicated by green arrows (below and above the alignment) and highlighted amino acids represent residues that appear in dbSNP as well as common associated somatic mutation in cancer (COSMIC) variants. e G23D model displaying a close up of combined MYH1 mutation (teal only) and wild type (green). Blue represent the helices and shows the proximity of the MYH1 mutation to the SWITCH 1 region and to the G677 and P710 residues found between Loop2 and SH2 helix domain of MYH1. f Stability analysis of the E317G mutation using I-mutant-2 at physiological pH, which predicts decreased stability of the mutant protein

Fig. 5

a Marked CD4+ lymphocytic infiltrates (red) within myofibers of horse 1, an E321G MYH1 homozygote. IHC × 20. b Serial section to c demonstrating inflammatory cell infiltrates (arrows) in horse 1. HE × 20. c Serial section to b demonstrating that fibers infiltrated by lymphocytes (vertical arrows) are 2X fibers (red) and that some fibers with cellular infiltrates (horizontal arrows) do not have any remaining myosin staining. IF × 20. d Serial section to e from horse 2 showing inflammatory cells (arrows). HE × 10. e Serial section to D demonstrating a complete absence of 2X fibers. Dark regions with no staining (arrows) correspond to regions of inflammation in D. IF × 10. f Normal fiber type distribution and fiber sizes in another homozygous wild-type horse for comparison. IF × 20. g Section of muscle from horse 3 that had a 4-month history of IMM showing a small amount of inflammatory cell infiltrates. HE × 10. h Another section of horse 3 demonstrating atrophic type 2X fibers (brown). IF × 10. i Preponderance of type 2A (yellow) or 2AX fibers (intermediate) over type 2X fibers (brown) in another region of the sample from horse 3. IF × 10. j Normal fiber type distribution of muscle from a wild-type horse (type 1 blue, type 2A yellow, type 2X brown) showing type 2X fibers that are larger than type 2A fibers. IF × 10. k Normal fiber type distribution and fiber sizes in the semimembranosus muscle that lacks inflammation from horse 4 that was homozygous for the E321G variant. IF × 20. Bars in all figures represents 100 μm