Etiology of Musculoskeletal Defects in a Sheep Model of Hypophosphatasia

Abstract

Hypophosphatasia (HPP) is a rare inherited disorder that is due to inactivation of the tissue-nonspecific alkaline phosphatase (ALP) gene ALPL. There are more than 400 different mutations in the human ALPL gene that contribute to the variable deficiencies in mineralization of bones and teeth. In addition to mineralization issues, children with HPP display a waddling gait and muscle weakness, as measured by reduced distances walked in the 6-Minute-Walk-Test and decreased distances in the standing long jump test compared with age-matched unaffected children. Using CRISPR-Cas 9, our lab developed a sheep model of HPP using the Rambouillet sheep by inducing a known human ALPL missense-mutation (c.1077C>G) into the Exon 10 of the ALPL gene of chromosome 2. Creating this mutation in sheep provides an ideal model in which to study the pathophysiology and etiology of HPP. Previous studies have shown that HPP sheep gluteal muscle have aberrant mitochondrial cristae and variable muscle fiber size. In addition, HPP sheep display the predicted bone phenotype with decreased mineralization and lower modulus and bone stiffness. Based on this data, we formulated and tested two hypotheses, namely that HPP sheep have smaller muscle fibers and delayed myoblast differentiation due to mitochondrial respiration deficiencies, and that HPP sheep have undermineralized bone resulting from the decreased differentiation of bone marrow stromal cells (BMSCs) towards osteoblasts as well as reduced oxidative phosphorylation and glycolysis capacities. Data collected during these dissertation studies have supported these hypotheses, demonstrating that HPP sheep have altered mitochondrial respiration and purinergic metabolism, which alters both myoblast morphogenesis and influences BMSC differentiation towards adipogenesis while forming osteoblasts with reduced function. The cellular deficiencies in myoblasts lead to delays in myogenesis and smaller, weaker muscle, whereas the cellular differences in BMSCs support a propensity toward adipogenesis and reduced osteoblast activity leading to undermineralized bone. These studies have uncovered some of the cellular mechanism(s) underlying the muscle phenotype of HPP, that has been difficult to study in human patients or other animal models. These compelling data provide added support for the utility of the HPP sheep model in dissecting the etiology of HPP.