ASBMR 29th Annual Meeting

Abstract

The RUNX2 transcription factor is essential for osteoblast differentiation and chondrocyte maturation. Rare mutations within RUNXZ cause the dominantly inherited skeletal disorder cleidocranial dysplasia which is charaterized by gross dysgenesis of the skeleton. A unique feature of RUNX2 is the polyglutamine and polyalanine (poly Q/A)domain encoded by a trinucleotide repeat sequence. Such repeat sequences are prone to strand slippage resulting in high mutation rates. We hypothesized that mutations within the RUNX2 poly Q/A domain would exist and these variants would be associated with altered bone density and protein function. 4361 DNA samples were obtained from multiple epidemiological studies of bone density. Bone density was measured by DEXA and ultrasound and expressed as Z-scores around the appropriate age-adjusted mean. A total of 21 subjects were identified as being heterozygous for a wild type 23Q117A allele and a p l y Q/A repeat variant allele. Deletions (ISQ and 164) and insertions (304 and 23A) were identified. Collectively Q/A repeat variants presented with significantly lower Femoral Neck BMD (DEXA) displaying a 0.65 SD decrease (n = 21, p = 0.0004) and lower bone density as measured by quantitative ultrasound of similar magnitude (-0.79 SD, n = 8, p = 0.031). To understand the mechanism via which the rare mutations were decreasing bone density, functional analyses were carried out on 23Q (WT), 16Q and 304 RUNX2. The ability of the proteins to bind to the RUNX2 DNA binding site was assessed by EMSA. The analysis did not reveal any obvious changes in the DNA binding capacities of the mutant proteins. However, reporter gene analysis using the mouse osteocalcin promoter revealed significant decreases in the transactivation function of 169 and 30Q. Finally, the ability of the proteins to localise to the nucleus was assessed. Subcellular analysis revealed the 164 and 30Q proteins displayed defective nuclear localisation compared to WT. We have identified a new class of functionally relevant RUNX2 variants that occur at collective frequency of -0.5%. These mutations are associated with significantly lower bone density and altered protein function.