ECTS Abstracts (2015) 1 P244

Search and Functional Evaluation of Rare Variants in RSPO3 Gene

Igor Fijalkowski1, Eveline Boudin1, Torben Leo Nielsen2, Marianne Andersen2, Kim Brixen2 & Wim Van Hul1

1University of Antwerp, Antwerp, Belgium; 2Odense University Hospital, Odense, Denmark.

R-spondin 3 is a member of the RSPO family of secreted Wnt-signalling agonists, an important pathway in the regulation of bone metabolism. Furthermore, genetic variation in RSPO3 is previously reported to be associated with BMD. Therefore, the aim of this study was to confirm the previously reported association and to identify a possible causative variant by screening the exons of RSPO3 using Sanger sequencing in two subpopulations of the Odense Androgen Study (OAS). The two subpopulations contain the 64 individuals with the lowest and highest BMD-values (t-score <-1.38 and t-score >1,54). We identified 4 known common intronic variants, however, genotype frequencies didn’t differ significantly between the high and the low BMD cohort. In addition, we identified one rare coding variant (rs140821794) located in exon 1. The variant was found in one individual (T-score=-1,515) and according to the prediction program SIFT, the variant has a deleterious effect on the protein function while Polyphen2 predicts that is benign. Consequently, we tested the effect of the variant on canonical Wnt-signalling with an in vitro reporter assay in HEK293 and Saos-2 cells. Although we were able to show cell-type dependent differences in the activation of the pathway by RSPO3, we didn’t find a significant difference in the effect of the WT or mutated RSPO3 on Wnt-signalling activity. In conclusion, we were not able to replicate the previously reported associations of common genetic variation in RSPO3 with BMD in our population. Although we did identify a possible interesting rare variant, we were not able to show an effect of the variant on the regulation of canonical Wnt signalling using functional studies. This can be due to the fact that the effect is too small to detect with our assay. Therefore, more studies are needed to elucidate the role of RSPO3 in maintaining bone mass.

Disclosure: The authors declared no competing interests.

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