Characterisation of genetic regulatory effects for osteoporosis risk variants in human osteoclasts

Abstract

Background: Osteoporosis is a complex disease with a strong genetic contribution. A recently published genome-wide association study (GWAS) for estimated bone mineral density (eBMD) identified 1103 independent genome-wide significant association signals. Most of these variants are non-coding, suggesting that regulatory effects may drive many of the associations. To identify genes with a role in osteoporosis, we integrate the eBMD GWAS association results with those from our previous osteoclast expression quantitative trait locus (eQTL) dataset.

Results: We identify sixty-nine significant cis-eQTL effects for eBMD GWAS variants after correction for multiple testing. We detect co-localisation of eBMD GWAS and osteoclast eQTL association signals for 21 of the 69 loci, implicating a number of genes including CCR5, ZBTB38, CPE, GNA12, RIPK3, IQGAP1 and FLCN. Summary-data-based Mendelian Randomisation analysis of the eBMD GWAS and osteoclast eQTL datasets identifies significant associations for 53 genes, with TULP4 presenting as a strong candidate for pleiotropic effects on eBMD and gene expression in osteoclasts. By performing analysis using the GARFIELD software, we demonstrate significant enrichment of osteoporosis risk variants among high-confidence osteoclast eQTL across multiple GWAS P value thresholds. Mice lacking one of the genes of interest, the apoptosis/necroptosis gene RIPK3, show disturbed bone micro-architecture and increased osteoclast number, highlighting a new biological pathway relevant to osteoporosis.

Conclusion: We utilise a unique osteoclast eQTL dataset to identify a number of potential effector genes for osteoporosis risk variants, which will help focus functional studies in this area.

Keywords: BMD; FBN2; Fracture; GWAS; Osteoclast; Osteoporosis; RIP3; RIPK3; SNP; eQTL.

Fig. 1

CIRCOS plot [20] displaying (from outside to inside) chromosome numbers, chromosome ideograms, scatterplot complete with gene labels representing the osteoclast eQTL associations presented in Table 1 and Additional file 1: Table S2 (red), scatterplot representing the eBMD GWAS results (green) and gene ontology (GO) biological process groupings relevant to osteoclast biology. Osteoclast eQTL and eBMD GWAS associations are displayed as −log₁₀P values ranging from 0.01 > P > 1.0 × 10⁻¹⁵ (osteoclast eQTL) and 0.001 > P > 1.0 × 10⁻⁵⁰ (eBMD GWAS). GO biological process groupings displayed include membrane organisation (blue), regulation of cell migration (red), regulation of catalytic activity (purple), cation transport (orange) and I-kappaB kinase/NF-kappaB signalling (green)

Fig. 2

Analysis of the eBMD GWAS and osteoclast eQTL datasets using the GARFIELD software [24] demonstrated significant enrichment of osteoporosis risk variants among osteoclast eQTL across four GWAS P value thresholds (P < 1 × 10⁻⁵, 1 × 10⁻⁶, 1 × 10⁻⁷ and 1 × 10⁻⁸), with the enrichment results for the trait neuroticism [25] included for comparison. The upper panels present the −log10 enrichment P values for each threshold, while the lower panels present the natural logarithm of the odds ratios for each threshold with 95% confidence intervals

Fig. 3

Micro-CT assessment of the distal femur from 15-week-old male Ripk3^−/− and WT mice. Representative 3D images of the distal femur demonstrate the following: a reduced extension of the trabecular network into the diaphysis, b expansion of the medullary cavity and c increased size of the trabecular bone compartment with no change in trabecular bone density in the Ripk3^-/- mice. Quantitative analysis of micro-CT parameters (mean + standard deviation) displays the following: d cortical volume, e bone marrow volume, f endosteal perimeter, g periosteal perimeter, h trabecular bone volume fraction and i trabecular extension in the Ripk3^−/− mice relative to WT mice. N = 5 for each group; WT, wildtype. **P < 0.01

Fig. 4

Quantitative histomorphometric analysis of femora from 15-week-old male WT and Ripk3^−/− mice (mean + standard deviation). a Osteoclast number (N.Oc). b Osteoclast surface relative to bone surface (Oc.S/BS). c Number of osteoclasts relative to bone surface (N.Oc/BS(mm⁻¹)). d Osteoblast number (N.Ob). e Number of osteoblasts relative to bone surface (N.Ob/BS(mm⁻¹)). f Representative low-power images (× 40 magnification) of TRAP-stained sections of the femur just below the growth plate. N = 4 and 5 for the WT and Ripk3^−/− groups, respectively; WT, wildtype