Project Summary/Abstract Investigating pathogenic mechanisms for rare Mendelian disorders is important not only to identify therapeutic strategies for lifelong debilitating diseases but also to understand fundamental biological mechanisms. In this renewal application, we propose mechanistic and translational studies for craniometaphyseal dysplasia (CMD), an understudied craniotubular bone disorder characterized by lifelong progressing hyperostosis of craniofacial bones and abnormal shape of long bones. Continued bone accrual in CMD can lead to excruciating headaches, blindness, deafness, and facial palsy. Severe cases can be life-threatening. CMD patients are treated with repetitive, costly and risky surgeries when corrections of facial deformity are needed or severe neurological symptoms occur. Mutations in the progressive ankylosis protein (ANKH) and connexin 43 (Cx43) have been identified as causes for autosomal dominant and recessive CMD, respectively. To study CMD, we have generated state-of-the-art research tools, which include mouse models carrying CMD mutations, isogenic human induced pluripotent stem cells (hiPSCs) with or without CMD mutations, and bone resorbing cells (osteoclasts) derived from these hiPSCs. In the past funding period, we have discovered the rapid degradation of mutant ANKH(Human)/ANK(Mouse) protein and studied negative effects of mutant ANKH/ANK on the cytoskeleton, which determinates cell shape, size, and polarity. We also identified differentially expressed proteins in CMD osteoclasts and preferential binding partners for mutant ANK protein. However, CMD pathogenesis is not fully understood and potential therapeutics have not been explored. Our long-term goal is to utilize our research findings for identifying potential therapeutic targets to reduce or prevent the lifelong bone deposition in craniofacial bones. In the next 5 years, we will use animal models and molecular and cellular methodologies that we have developed to focus on mechanistic investigations and prepare for future clinical studies. Based on our preliminary data and previous publications we propose three specific aims. We will study the impact of CMD-mutant ANK on cellular acidification of osteoclasts (Aim 1) and on the bi-directional regulation between the cytoskeleton and an energy metabolism regulator in CMD (Aim 2). These are likely novel dominant functions of mutant ANK leading to CMD. In Aim 3 we will identify biomarkers that can be used to monitor the disease progression in patients and mouse models. We will also evaluate shifts in biomarker expression in response to experimental treatment regimen in our model systems. We expect that the proposed studies will give deeper insight into pathogenic mechanisms of CMD, knowledge needed to discover candidate targets for therapeutics. Biomarkers that correspond to disease progression or treatment efficacy will be the basis for future clinical studies.