Abstract

Osteoporosis represents an important cause of morbidity in adult patients with thalassemia. Its pathogenesis is multifactorial, and includes mainly bone marrow expansion, endocrine dysfunction and iron overload. Bone metabolism is altered in thalassemia. Osteoclast function is elevated, while osteoblast activity seems to be reduced and thus the balance of bone remodeling is in favor of bone loss. The exact mechanisms of osteoblast dysfunction have not been fully clarified to-date. Wingless-type (Wnt) signaling is an important pathway for osteoblast differentiation. Dickkopf-1 (Dkk-1) protein is an inhibitor of Wnt pathway and is implicated in the pathogenesis of several bone disorders. Collagen type-I is the main structural protein of the bone. The collagen type-I alpha (COLIA)-1 specific protein (Sp)-1 polymorphism has been related to osteoporosis in thalassemia. The aim of this study was to evaluate the serum levels of Dkk-1 in patients with thalassemia-induced osteoporosis who receive therapy with zoledronic acid (ZOL) and evaluate possible correlations with clinical and laboratory data, including the COLIA-1 Sp1 polymorphism. Sixty-six patients (21M/45F; median age 35.5 years) with thalassemia and osteoporosis were studied. Patients were blindly randomized to receive ZOL at a dose of 4 mg, iv, in 15 min infusion, every 6 months (group A, n=23) or every 3 months (group B, n=21), or to receive placebo every 3 months (group C, n=22) for a period of one year. All patients received oral calcium (500 mg) during the treatment period. Dkk-1 was measured at baseline and after 12 months of therapy using ELISA methodology (Biomedica Medizinprodukte, Wien, Austria) along with a series of serum bone remodeling indices:

  • bone resorption markers [C-telopeptide of type-I collagen (CTX), tartrate-resistant acid phosphatase isoform-5b (TRACP-5b)],

  • bone formation markers [bone-specific alkaline phosphatase (bALP), osteocalcin, and C-terminal propeptide of collagen type-I (CICP)], and

  • osteoclast regulators [receptor activator of nuclear factor-kappa B ligand (RANKL), osteoprotegerin (OPG), and osteopontin].

The above bone markers were also evaluated in 30, age- and gender-matched, healthy controls. The G-->T mutation at base 1 of intron 1 at the binding site of the Sp1 transcription factor of the COLIA-1 gene was detected by polymerase chain reaction using mutagenesis primers followed by restriction enzyme analysis in all patients. BMD of the lumbar spine (L1-L4), femoral neck (FN) and wrist (W) was determined using DEXA, before and 12 months after treatment. At baseline, all patients had increased serum levels of Dkk-1 (mean±SD: 39±17.1 pmol/L) compared to controls (27.4±9.7 pmol/L; p<0.0001). Furthermore, thalassemia patients had increased values of CTX (p<0.0001), bALP (p<0.001), CICP (p=0.003), sRANKL (p=0.02), and OPG (p=0.001) compared to controls. Results for the COLIA-1 Sp1 polymorphism were available for 53 patients. Seventeen patients (32%) were G/T heterozygotes at the polymorphic Sp1 site (Ss), while 3 (5.6%) were T/T homozygotes (ss). Dkk-1 serum levels correlated with L1-L4 BMD (r=−0.290, p=0.022) and W-BMD (r=−0.415. p=0.001), but also with TRACP-5b (r=0.310, p=0.011) and bALP levels (r=−0.289, p=0.018). Ss and ss patients tended to have lower L1-L4 BMD compared with SS patients (p=0.09). No significant correlations were observed between Ss and ss patients with the measured bone markers or the response to ZOL. As reported previously, patients of group B experienced an increase of L1-L4 BMD, while no other alterations in BMD were observed in the 3 studied groups after 12 months of ZOL administration. Interestingly, patients of groups A+B showed a strong reduction of Dkk-1 after 12 months of ZOL (from 39.6±16.6 to 28.9±16.3 pmol/L; p=0.004); indeed they almost normalized Dkk-1 levels (no difference from control values). In contrast, patients of group C showed a borderline increase of Dkk-1 (from 33.1±16.8 to 40.1±23.2 pmol/L, p=0.08). These results show for the first time in the literature that Dkk-1 is increased in the serum of patients with thalassemia and osteoporosis, correlates with their BMD and is reduced post-ZOL therapy. This Dkk-1 elevation may be at least partially responsible for osteoblast dysfunction in thalassemia and reveal a novel possible target for the development of new agents for the management of bone loss in thalassemia patients.

Disclosures: Off Label Use: In this cohort of patients zoledronic acid has been given for the treatment of thalassemia-associated osteoporosis. The study was conducted with the approval of the hospital ethical committee and the Greek National Drug Organization (Ref. No Aa-KDelta-79/01/03) in keeping with the guidelines of Helsinki..

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