Marichela Schembri ^1* Melissa M. Formosa ^1,2*

• ¹ Department of Applied Biomedical Science, Faculty of Health Sciences, University of Malta, Msida, Malta
• ² Centre for Molecular Medicine and Biobanking, University of Malta, Msida, Malta

Osteoporosis is a multifactorial bone disease characterised by reduced bone mass and increased fracture risk. Family studies have made significant contribution in unravelling the genetics of osteoporosis. Yet, most of the underlying molecular and biological mechanisms remain unknown prompting the need for further studies. This review outlines the proper phenotyping and advanced genetic techniques in the form of high-throughput DNA sequencing used to identify genetic factors underlying monogenic osteoporosis in a family-based setting. The steps related to variant filtering prioritisation and curation are all also described. From an evolutionary perspective, deleterious risk variants with higher penetrance tend to be rare as a result of negative selection. High-throughput sequencing (HTS) can identify rare variants with large effect sizes which are likely to be missed by candidate gene analysis or genome-wide association studies (GWAS) wherein common variants with small to moderate effect sizes are identified. We also describe the importance of replicating implicated genes, and possibly variants, identified following HTS to confirm their causality. Replication of the gene in other families, singletons or independent cohorts confirms that the shortlisted genes and/or variants are indeed causal. Furthermore, novel genes and/or variants implicated in monogenic osteoporosis require a thorough validation by means of in vitro and in vivo assessment. Therefore,