Functional assessment of coding and regulatory variants from the DKK1 locus

Abstract

The DKK1 gene encodes an extracellular inhibitor of the Wnt pathway with an important role in bone tissue development, bone homeostasis, and different critical aspects of bone biology. Several BMD genome-wide association studies (GWASs) have consistently found association with SNPs in the DKK1 genomic region. For these reasons, it is important to assess the functionality of coding and regulatory variants in the gene. Here, we have studied the functionality of putative regulatory variants, previously found associated with BMD in different studies by others and ourselves, and also six missense variants present in the general population. Using a Wnt-pathway-specific luciferase reporter assay, we have determined that the variants p.Ala41Thr, p.Tyr74Phe, p.Arg120Leu, and p.Ser157Ile display a reduced DKK1 inhibitory capacity as compared with WT. This result agrees with the high-bone-mass (HBM) phenotype of two women from our cohort who carried mutations p.Tyr74Phe or p.Arg120Leu. On the other hand, by means of a circularized chromosome conformation capture- (4C-) sequencing experiment, we have detected that the region containing 24 BMD-GWA variants, located 350-kb downstream of DKK1, interacts both with DKK1 and the LNCAROD (LncRNA-activating regulator of DKK1, AKA LINC0148) in osteoblastic cells. In conclusion, we have shown that some rare coding variants are partial loss-of-function mutations that may lead to a HBM phenotype, whereas the common SNPs associated with BMD in GWASs belong to a putative long-range regulatory region, through a yet unknown mechanism involving LNCAROD. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Funds for the study included grants SAF2014‐56562‐R and SAF2016‐75948‐R (Spanish MINECO), and CIBERER (U720). NMG is a recipient of a FI predoctoral fellowship from AGAUR (Generalitat de Catalunya); NRA is a recipient of a FPU predoctoral fellowship from the Spanish Ministerio de Educación Cultura y Deporte. EB is a recipient of a postdoctoral fellowship (12A3814N) from the Research Foundation‐Flanders. We thank O. Monclús and M. Cozar for relevant technical assistance. Authorsʼ roles: NMG, WVH, DG, SB were involved in the study conception and design. Material preparation and data collection were performed by NMG, NRA, NA, NGG, EB, DO, LM, XN, and ADP. The analyses were performed by NMG. The statistical analyses were performed by MPM. The first draft of the manuscript was written by NMG, DG, SB, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.