Role of Wnt pathway genes in complex and monogenic phenotypes of low and high bone mass

Abstract

Osteoporosis is a complex disease characterized by low bone mass, microarchitectural deterioration, and increased fracture risk. It is the most common bone disease that affects more than 200 million people worldwide and is responsible for more than 8.9 million fractures every year, especially affecting the elderly. This makes it one of the major causes of morbidity and mortality and generates a high social-health cost, which is expected to grow as life expectancy has been increasing considerably over the last decades. Although nowadays there are different treatments available that reduce bone loss, few of them target bone formation enhancement. Therefore, it is essential to discover new anabolic therapeutic targets that restore bone mass and skeletal architecture. In this doctoral thesis, I have used two tools to identify new therapeutic targets by elucidating the genetic bases of the disease.

On the one hand, I have performed a search for variants in three Wnt pathway genes, that might explain the GWAS significant association with different bone parameters, and subsequently I characterized them functionally. Specifically, I have studied DKK1, SOST, and WNT16, identifying common and rare variants that may explain the susceptibility to the complex disease of osteoporosis or the extreme high bone mass (HBM) phenotype. Regarding DKK1, I have shown the loss of inhibitory function associated with missense mutations found in patients with HBM and/or in the general population. I have also detected for the first time a physical contact between the region enriched in BMD-GWAS signals and the DKK1 promoter and also with, LNCAROD, a lncRNA known to regulate DKK1 expression. For WNT16, I have identified a promoter region in intron 2 that interacts with different regulatory elements located in introns of the neighboring gene CPED1. Finally, in SOST, I have shown a decrease in gene expression associated with the minor allele of a variant that modifies an extended TATA box motif, a reduction in protein expression of a missense variant in the signal peptide found in an HBM women, and, for the first time, physical interactions between the SOST proximal promoter and ECR5 and several enhancer elements upstream of it. These functional studies reinforce the importance of the Wnt pathway in bone homeostasis and demonstrate the need to study in-depth the GWAS signals that often do not correspond to a functional variant.

On the other hand, using whole-exome sequencing (WES), I have also investigated a family case of HBM in the search for a causal gene. Manual filtering of the low- frequency variants found in this case suggests that mutated VAV3 - along with other variants in SIK3- could determine the HBM phenotype in the family due to an inhibitory effect on osteoclastogenesis. Importantly, the results have pointed at novel genes and regions which may constitute a new therapeutic target for osteoporosis.