The Role of RANKL/Osteoprotegerin and Wnt Signaling Pathways in the Development of Osteoporosis in Patients with Hemophilia
Authors: Anagnostis Panagiotis , Vakalopoulou Sofia , Christoulas Dimitrios , Karras Spyridon , Papatheodorou Athanasios , Katodritou Eirini , Kokkoris Panagiotis , Garipidou Vasilia , Terpos Evangelos 
Published: October 2019

Abstract
Hemophilia A and B is associated with reduced bone mineral density (BMD). However, the exact mechanisms of bone loss in hemophilia have not been fully elucidated. The Wnt signaling enhances osteoblast differentiation; its canonical pathway is inhibited by sclerostin and Dickkopf-1 (Dkk-1). The RANKL/osteoprotegerin (OPG) system is the major regulator of osteoclastogenesis. The primary aim of this prospective study was to assess, for the first time, circulating sclerostin, Dkk-1, RANKL and OPG in hemophilia patients and to examine possible correlations with BMD. Secondary endpoint was to evaluate the effect of ibandronate on BMD and bone turnover markers of hemophilia patients with osteoporosis.
Eighty-nine male patients with hemophilia (median age 44 years; 76 with hemophilia A) and 30 age-matched, healthy, male controls participated in this study. BMD of the lumbar spine (LS), total hip (TH) and femoral neck (FN) was assessed by Dual-energy X-ray absorptiometry (DXA). Low BMD was defined when T-score was <-1 SD in patients >50 years of age (between -1 and -2.5 SD as osteopenia and <-2.5 SD as osteoporosis) and for patients <50 years of age when Z-score was <-2 SD. Sclerostin, Dkk-1, RANKL and OPG were measured in the serum of patients and controls along with: i) bone resorption markers [C-terminal telopeptide of collagen type-1 (CTX) and tartrate resistant-acid phosphatase-5b] and ii) bone formation markers [bone-alkaline phosphatase (bALP) and osteocalcin (OC)]. Ten patients (median age 45 years, 7 with hemophilia A) with T-score <-2.5 SD or Z-score <-2 and/or increased risk of fracture (according to the FRAX model) received 150 mg oral ibandronate, once monthly. The above bone regulators and bone turnover markers were also measured in the serum of these patients on the day of ibandronate initiation and 12 months post-therapy.
Patients with hemophilia had lower circulating sclerostin (48.7±20.8 vs. 314.7±204.5 pg/ml, p<0.001), Dkk-1 (23.3±15.5 vs. 27.4±9.7 ng/ml, p=0.04), bALP (p<0.001) and OC (p=0.003) and higher levels of RANKL (0.31±0.26 vs. 0.15±0.21 pmol/l, p=0.001), RANKL/OPG ratio (0.09±0.10 vs. 0.03±0.05, p=0.001) and CTX (p<0.001) compared to healthy controls. Twenty five patients (28.1%) had low BMD in at least one of the measured sites. Patients with hemophilia A had lower incidence of low BMD compared to patients with hemophilia B (23.7% vs 53.8% respectively, p=0.042). Patients with at least one affected joint had lower TH-BMD versus all others (0.89±0.15 vs. 0.98±0.14 g/cm2, p=0.01). Patients with low BMD had higher OPG levels (5.87±3.16 vs. 4.48±2.85 pmol/L, p=0.045) compared to those with normal BMD. Sclerostin correlated positively with LS-BMD (r=0.302, p=0.004) and TH-BMD (r=0.235, p=0.028), while RANKL/OPG ratio correlated positively with BMD of the FN (r=0.314, p=0.003) and of the TH (r=0.252, p=0.018).
Patients with severe hemophilia had lower sclerostin levels compared with those with mild or moderate disease (38.9±19.6 vs. 50.9±20.6 pg/ml, respectively, p=0.021), and increased bone turnover, as reflected by the high circulating OPG, CTX and bALP levels (p<0.01). The degree of arthropathy (assessed by total Pettersson score) negatively correlated with sclerostin (r=-0.254, p=0.034) and Dkk-1 (r=-0.319, p=0.007).
The patients who received ibandronate, showed an increase in LS-BMD (from 0.886±0.169 to 0.927±0.176 g/cm2; 4.7%; p=0.004) after 12 months of therapy. Serum Dkk-1 and CTX significantly decreased: from 18.9±9.1 to 13.7±7.8 ng/ml (p=0.04) and from 0.520±0.243 to 0.347±0.230 ng/ml (p=0.042), respectively. No significant effect on TH-BMD, FN-BMD, sclerostin and RANKL/OPG was noticed after 12 months of ibandronate treatment.
We conclude that increased osteoclastic activity is present in patients with hemophilia, as indicated by the increased RANKL/OPG and CTX levels. Disease severity and hemophilic arthropathy are associated with increased bone turnover and reduced sclerostin and Dkk-1. Ibandronate did not affect RANKL/OPG ratio, but it decreased osteoclastic activity (by decreasing CTX) and it increased osteoblastic activity (by decreasing Dkk-1) and BMD. It seems that increased osteoclastic activity and not decreased osteoblastic activity is the main mechanism for low bone mass in hemophilia. These findings provide new insights in the pathogenesis of bone loss in hemophilia, but need further confirmation in future studies.

Disclosures
Terpos: Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel expenses; Celgene: Honoraria, Other: travel expenses; Novartis: Honoraria.