Variation in the Kozak sequence of WNT16 results in an increased translation and is associated with osteoporosis related parameters.

The importance of WNT16 in the regulation of bone metabolism was recently confirmed by several genome-wide association studies and by a Wnt16 (Wnt16(-/-)) knockout mouse model. The aim of this study was thus to replicate and further elucidate the effect of common genetic variation in WNT16 on osteoporosis related parameters. Hereto, we performed a WNT16 candidate gene association study in a population of healthy Caucasian men from the Odense Androgen Study (OAS). Using HapMap, five tagSNPs and one multimarker test were selected for genotyping to cover most of the common genetic variation in and around WNT16 (MAF>5%). This study confirmed previously reported associations for rs3801387 and rs2707466 with bone mineral density (BMD) at several sites. Furthermore, we additionally demonstrated that rs2908007 is strongly associated with BMD at several sites in the young, elderly and complete OAS population. The observed effect of these three associated SNPs on the respective phenotypes is comparable and we can conclude that the presence of the minor allele results in an increase in BMD. Additionally, we performed re-sequencing of WNT16 on two cohorts selected from the young OAS cohort, based on their extreme BMD values. On this basis, rs55710688 was selected for an in vitro translation experiment since it is located in the Kozak sequence of WNT16a. We observed an increased translation efficiency and thus a higher amount of WNT16a for the Kozak sequence that was significantly more prevalent in the high BMD cohort. This observation is in line with the results of the Wnt16(-/-) mice. Finally, a WNT luciferase reporter assay was performed and showed no activation of the β-catenin dependent pathway by Wnt16. We did detect a dose-dependent inhibitory effect of Wnt16 on WNT1 activation of this canonical WNT pathway. Increased translation of WNT16 can thus lead to an increased inhibitory action of WNT16 on canonical WNT signaling. This statement is in contrast with the known activating effect of canonical WNT signaling on bone formation and suggests a stimulatory effect on bone metabolism via noncanonical WNT signaling. More research is required to not only confirm this hypothesis, but also to further elucidate the role of non-canonical WNT pathways in bone metabolism and the general mechanisms of interplay between the different WNT signaling pathways.