Assessment of biochemical bone markers of osteoporosis in children with thalassemia major

This study shows that serum osteoprotegerin concentrations can be used as a biochemical marker in screening patients with beta-thalassemia major for the development of osteoporosis.

Beta thalassemia major (β-TM) is a common cause of skeletal morbidity and increased bone fracture risk in thalassemic patients [1]. Its pathogenesis is multifactorial and mainly includes bone marrow expansion, endocrine dysfunction and iron overload [2, 3]. The mechanisms through which these factors lead to bone loss have not been completely clarified [4]. Previous reports using dual-energy X-ray (DEXA) suggest that up to 70% of adults with β-TM have low bone mass [5]. The skeletal changes in thalassemic patients may include osteoporosis, growth failure, bone age delay and spondylometaphyseal abnormalities [6]. Some of the aetiological factors for thalassemic osteoporosis are dysfunction of the hypothalamic-pituitary-gonadal axis, growth hormone effects on the parathyroid gland, diabetes, hypothyroidism, ineffective haematopoiesis and direct toxicity of iron to osteoblasts. Iron chelation therapies may also cause osteopenia and osteoporosis via their effects on cartilage tissue [7, 8].

Osteoprotegerin (OPG) and the receptor activator of nuclear factor-kappa B (RANK)/receptor activator of nuclear factor-kappa B ligand (RANKL) are the major cytokines related to the regulation of bone resorption [9]. The receptor activator of the nuclear factor-kappa B (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) pathway has been recently recognised as the final, dominant mediator of osteoclast proliferation and activation [8‐11].

The aim of this study was to search for some potential biochemical markers [deoxypyridinoline (DPG), beta-crosslaps (β-CTX), OPG and RANKL]) as possible early predictors of BMD variations in children with β-TM.

Comparisons of major cytokines related to the regulation of bone resorption between patients with thalassemia major and healthy controls are presented in Table 1. Serum OPG concentrations were significantly lower in thalassemic children than in controls. The mean ratio of RANKL/OPG was significantly increased in children with beta-thalassemia compared to the control group (Table 1).

In this study, the values of DPD, OPG, beta-CTX and RANKL, DEXA, FSH, LH, oestradiol, vitamin D3, TSH, T4, dehydroepiandrosterone (DHEA-SO4), testosterone, AST, ALT, BUN, creatinine, calcium, phosphorus, alkaline phosphatase, parathormone, total protein, albumin, ferritin, white blood cells, haemoglobin and platelets were compared between the groups. There were significant negative correlations between the OPG value and DEXA, PTH and phosphorus. PTH/phosphorus ratio. (r: 0.55, p: 0.003) (phosphorus: r: 0.38, p: 0.029). There was a significantly positive correlation between RANKL and AST, and ALT. There was a significantly negative correlation between the DPD value and TSH and T4 levels. There was a significantly positive correlation between the beta-CTX value and alkaline phosphates (Table 2). There were no significant correlations among the other parameters (p > 0.05).

Femur and spine Z scores of the thalassemic patients were evaluated with DEXA. The mean value of the lumbar spine Z score was − 1.81 ± 0.93 (median − 1.81), and the femur Z score was − 1.6 ± 0.99 (median-1.60) in the thalassemic patients. Lumbar site osteopenia was present in 36.4% of the patients (12/33), and 33.3% (11/33) of them had osteopenia at the femur site. Osteoporosis was detected in 10 (3 female and 7 male) of 38 patients (26.3%) according to the femur Z score and in 6 of them (4 male and 2 female) (15.8%) according to the spine Z score.

When the groups were compared according to sex, the mean spine DEXA Z score results were higher in females than males (− 1.71 ± 0.86 SD versus − 1.89 ± 0.89 SD), while the mean femur Z score was higher in males than females (− 1.59 ± 0.99 SD versus − 1.74 ± 0.85). The spine Z score was negatively correlated with age and ferritin levels (r: 0.484 p: 0.002 for age and r: 0.380 p: 0.02 for ferritin).

Regression analysis was performed to assess the factors affecting the spine Z score. Age and ferritin levels significantly affected the spine Z score. The spine Z score was determined with Dubin Watson regression analyses with the formula “spine Z score = (− 0.125x age) + (9.4x ferritin level)”. According to this formula, the Z score declined as age and ferritin levels increased.

Beta-thalassemia major (β-TM) is a common cause of skeletal morbidity and increased bone fracture risk in thalassemic patients¹. Its pathogenesis is multifactorial and mainly includes bone marrow expansion, endocrine dysfunction and iron overload [2, 3]. In this study, we investigated the DPD, beta-CTX, OPG and RANKL values of our patients diagnosed with β-TM. We compared these values with DEXA and hormonal changes to observe whether there was a correlation between them.

OPG levels were significantly decreased in thalassemic patients, although no significant difference was detected between the serum RANKL levels of the groups. The most important result of this finding is an increased RANKL/OPG ratio in thalassemic children. Morabito et al. [10] reported that thalassemic patients showed no differences in plasma levels of OPG compared with controls and significantly higher plasma levels of RANKL, with a consequent significantly lower OPG/RANKL ratio (which is equal to a higher RANKL/OPG ratio) in an adult study. These results indicate that low bone mass can be associated with decreased osteoblastic activity in children despite increased osteoclastic activity in adults [10]. Our findings are correlated with Morabito’s study. Ozdemir et al. [11] reported that vitamin menaquinone-7 and calcitriol have beneficial effects on thalassemic osteopathy.

Alfaqih et al. [12] reported that plasma RANKL level was the most significant marker for bone resorption and strongly correlated with the N-terminal propeptide of type 1 collagen; lower in individuals with thalassemia intermedia.

Osteoblasts and osteoclasts are responsible for bone remodelling at the cellular level. In adult thalassemic patients, RANK/RANKL-OPG system disorder increases in favour of osteoclasts. Many studies have noted that the RANKL/OPG ratio increases in patients with thalassemia major, and this situation shows the role of this system in the pathogenesis of osteoporosis [13‐15]. Toumba and Skordis [6] reported that in thalassemia patients, progressive “ageing” of the bone starts even in childhood by the gradual development of an imbalance between augmented osteoclastic resorption and insufficient osteoblastic bone formation. We suggest that osteoblastic dysfunction is the dominant mechanism of osteopenia in prepubertal children.

We did not find any association with sex steroids, OPG, RANKL and DEXA, which agrees with different studies from Italy and Greece [16, 17].

Several studies have shown that PTH acts by enhancing the production of RANKL and by inhibiting the synthesis of OPG [18, 19]. However, we detected a positive correlation between PTH and OPG levels in this study. These results may be attributed to the unique characteristics of the growing bones and the high rate of bone turnover in childhood. There were no other significant correlations between the markers of bone turnover and PTH in this study.

In a study of 42 patients diagnosed with thalassemia major, the rate of osteoporosis by DEXA was 81%. β-CTX and PTH levels were high, but vitamin D levels were low in the same group [16]. This study similarly shows high levels of PTH and alkaline phosphatase and an increased ratio of RANKL/OPG in the thalassemic group. Although the difference was not statistically significant, the mean β-CTX levels of the thalassemic patients were higher than those in our study’s control group. These biochemical markers serve as a signal in the development of osteoporosis in patients with beta-thalassemia major.

It is not clear whether sex differences influence bone diseases [20]. Some studies report that sex differences have a role in developing osteoporosis syndrome in thalassemia and affect its severity. Males are more frequently and severely affected by osteopenia and osteoporosis than females, and some others report that sex differences are not significant [20, 21]. In a study conducted on patients with sickle cell anaemia, the mean lumbar spine Z score was significantly lower in prepubertal girls than in boys. However, there was no significant difference in the pubertal group [22]. When the prepubertal thalassemic patients in this study were evaluated according to sex, the mean spine DEXA Z score results were higher in females than males. In contrast, the mean femur Z score was higher in males than females.

Iron accumulation has a negative effect on bone mineralisation. BMD was significantly influenced by noneffective chelation in children with beta-thalassemia major [23]. According to the results of this study, it was confirmed that DEXA Z scores similarly decreased with increasing levels of ferritin.

Osteoporosis is a multifactorial disease and may occur early, especially in chronic diseases such as thalassemia. Because of the difficulties in diagnosis and follow-up, screening with DEXA and measuring ferritin level and RANKL/OPG ratios on a regular basis is essential. It should be kept in mind that osteoporosis may develop with advancing age in both sexes.