Role of the RUNX2 rs59983488 variant in bone mass determination and fracture risk in Malta

Abstract

Osteoporosis is a multifactorial skeletal disease which is characterised by a reduced bone density (BMD) and compromised bone microarchitecture, leading to an increased fragility fracture risk. The Runt-related Transcription Factor 2 (RUNX2) protein encoded by the RUNX2 gene is involved in the maturation and differentiation of osteoblasts and chondrocytes that are, crucial for endochondral bone formation. The RUNX2 rs59983488 (G>T) variant, located -330bp from the transcriptional start site of the P1 Promoter of the RUNX2 gene, is predicted to alter vitamin D receptor (VDR) binding and several enhancer elements resulting in reduced gene expression and hence BMD. The aim of this study was to determine the genotype and allele frequencies of the RUNX2 rs59983488 variant in the Maltese population, and to investigate the effect of the variant on reduced BMD and fracture risk at specific anatomical sites. Genotyping was performed using Competitive Allele-Specific PCR (KASPTM) in the Malta Osteoporotic Fracture Study (MOFS), a case-control collection of 1,045 Maltese postmenopausal women aged 41-79 years. The Kruskal-Wallis and Mann-Whitney U tests were used to analyse genotype-phenotype associations with bone-specific measurements, whereas odds ratios (ORs) were computed using logistic regression analysis with a 95% confidence interval (CI) adjusted for confounders to determine exposure odds ratios. Genotyping was successfully conducted on 1,042 samples from MOFS, with the alternative T allele detected at a frequency of 12.9%. Allele frequencies were in HWE and in line with those demonstrated in the European non-Finnish populations from gnomAD. Heterozygosity for the RUNX2 rs59983488 variant was associated with a reduced BMD at the total hip (TH sBMD p = 0.023, TH T-Score p = 0.030) and the femoral neck (FN sBMD p = 0.042, FN T-Score p = 0.020) and serum ALP levels (p = 0.007) compared to homozygosity for the reference G allele. Women carrying one copy of the alternative T allele had a 1.45-fold increased risk of osteopenia at the total hip (Adjusted OR: 1.45 [95% CI: 1.02 – 2.04], p = 0.036). Homozygosity for the RUNX2 rs59983488 variant was however associated with a 3.07-fold increased risk for all-type of low-trauma fracture risk which was independent of BMD (Adjusted OR: 3.07 [95% CI: 1.06 – 8.85], p = 0.038). Furthermore, homozygosity for the T allele demonstrated a 6.70-fold increased risk for hip fractures (Adjusted OR: 6.70 [95% CI: 1.62 – 27.66], p =0.009) regardless of age and BMD. In conclusion, results indicate that the RUNX2 rs59983488 T allele negatively alters RUNX2 protein function, making it a potential genetic determinant contributing to the complex aetiology of osteoporosis and fracture risk in Malta.