Biochemical and molecular basis for inherited muscle disease in the horse

The first objective of this research was to determine if alterations in either the sarcoplasmic reticulum ryanodine receptor (RYR1) or the calcium ATPase, similar to alterations in human patients with disorders of muscle calcium regulation, could be identified in membrane vesicles isolated from muscles of Thoroughbred horses with recurrent exertional rhabdomyolysis (RER). Neither the time course of Ca2+-induced Ca2+ release, nor parameters of [3H]ryanodine binding to the RYR1, were found to differ between control and RER horse muscle membranes. Maximal rate of SR Ca2+-ATPase activity and its affinity for Ca 2+ also did not differ between control and RER horse membranes. We conclude that RER in Thoroughbred horses may represent a novel defect in muscle excitation-contraction coupling, calcium regulation, or contractility.; The second objective of this research was to characterize the biochemical basis for a newly discovered fatal neonatal and fetal glycogen storage disorder (GSD) in American Quarter Horses similar to GSD IV in humans. In ten suspect foals tested, glycogen branching enzyme (GBE) activity was absent. GBE activity in available parents of the foals was 50% of controls and GBE protein determined by Western immunoblot was markedly reduced to absent in affected foals. Fluorescent in situ hybridization assigned the equine GBE1 gene to ECA26q12--q13. An allele of a microsatellite (GBEms1) within this gene was found to be statistically associated with GSD IV. This data provided strong support for the candidacy of the GBE1 locus in equine GSD IV.; The third objective was to derive control and affected foal GBE1 cDNA sequence and identified a C to A substitution at position 102 in exon 1 that results in a tyrosine to a premature stop mutation in codon 34 (Y34X). All ten affected foals were homozygous for the X34 allele, their ten available dams and sires were heterozygous, and all 16 control horses were homozygous for the Y34 allele, supporting an autosomal recessive mode of inheritance. Defining the molecular basis of GSD IV as a Y34X GBE1 mutation will allow for accurate DNA testing and the ability to prevent occurrence of this devastating disease affecting American Quarter Horses and related breeds.