Evaluating Bone and Mineral Metabolism in Patients with Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD), an X-linked recessive disorder due to a mutation of the dystrophin gene, is the most frequent muscular disease in childhood. The severe reduction or absence of dystrophin, depending on the type of mutation, leads to the progressive degeneration of striated muscle, which is slowly substituted by fat and connective tissue. Proximal muscle weakness becomes evident in early childhood, and after a few years the affected child becomes progressively unable to walk. Generally, ambulation is completely lost at around 15 years of age.¹

There is no cure, but glucocorticosteroids (GCs) improve muscle strength, maintain pulmonary function, prolong ambulation, reduce the severity of scoliosis and significantly extend life. GCs are currently started at a much earlier age than in the past, since the sooner they are given, the greater their effectiveness in slowing disease progression.² In recent years there has been greater awareness of bone problems (low bone mass and fragility fractures) in DMD. GCs may certainly play a role in this area, given their well-known negative effects on bone metabolism, but it is not clear whether the disease itself or other factors contribute to the weakening of bone.

While several specialists consider bone evaluation an essential aspect of DMD care, there are as yet no official guidelines or consensus statements. This is due, at least in part, to the lack of data: the relatively few studies addressing this problem evaluated only small numbers of patients, with a wide age range and at different pubertal and disease stage, and very often considered patients treated or not treated with steroids together, without clearly separating them in the analysis of the results. In addition, it is important to note that the study of bone and mineral metabolism and the evaluation of bone turnover, bone mass and bone density are always very complex in the growing skeleton of children and adolescents. Moreover, in patients affected by DMD, the bone effects of GCs must be considered: early childhood is thus the only time available to study the direct impact of DMD on bone and mineral metabolism, before the interference of these drugs.

Reasons for Evaluating Bone and Mineral Metabolism
In DMD, at least three factors can potentially affect bone, which is why a clear picture of a patient’s bone status should be obtained before starting GC treatment.

Reduced Mechanical Work
Muscle impairment significantly hinders weight-bearing activities during the crucial period of growth, and this can profoundly influence bone development and lead to disuse osteopenia, as in other pathological, traumatic or experimental conditions. Muscular strength is one of the major determinants of bone mass accrual during childhood and adolescence, ^3,4 and animal studies have demonstrated that lean mass (muscle) is a significant determinant of BMD and bone strength, even when accompanied by a dystrophic phenotype.⁵ In a study on boys with DMD, we found a significant reduction in the bone density of lower limbs and spine versus controls, and interpreted this as the consequence of reduced weight-bearing and muscular activity on bone. ⁶

Growth Hormone
Growth hormone (GH) acts on a variety of target cells, including those of the skeletal growth plates, through the release of insulin-like growth factor 1 (IGF-1). The GH/IGF-1 system and the androgens are the main stimulants of cell proliferation in the growth plate cartilages and of the linear growth of long bones. Moreover, GH increases bone mass acquisition, and during puberty the higher production of GH and IGF-1, stimulated by sex hormones, has a positive influence on peak bone mass (PBM).

Our understanding of the role of GH in DMD is still limited and partially contradictory. Short stature is common in boys with DMD,⁷ and in some studies the GH–IGF-1 axis was evaluated in an attempt to explain this phenomenon. The results were inconclusive, since GH deficiency was both not found⁸ and found.⁹ So, for the moment we do not know whether GH deficiency is present in DMD before the use of steroids.

Cytokines
Recently, several studies have investigated the effects of different cytokines on the production of osteoclasts and osteoblasts. Interleukin (IL)-1α, IL-1β, IL-6, IL-7 and tumour necrosis factor alpha (TNF-α) have all been found to promote osteoclastogenesis, whereas interferonbeta (INF-β), IL-3, IL-4, IL-10, IL-13, IL-12 and IL-18 inhibit it. Transforming growth factor-beta (TGF-β) seems to be able to either induce or suppress osteoclastogenesis. ¹⁰ Connective tissue growth factor (CTGF) promotes the endochondral ossification and the proliferation of osteoblasts. ¹¹ There are no published studies on the impact of these factors on bone in DMD. However, they seem to be involved in muscle degeneration and may have also an impact on bone. Increased levels of TNF-α, produced and expressed by muscle fibres, have been found in DMD. The upregulation of the TNF-α system has been associated with haemostatic activation and left ventricular dysfunction in DMD. ¹²


There are some preliminary studies on the involvement of cytokines in the replacement of the dystrophic muscle with fibrotic tissue. The
TGF-β pathway was strongly induced in symptomatic DMD patients, with the expression of TGF-β type II receptor and apoptotic signals in
mature DMD myofibres. A stage-specific remodelling of human dystrophin-deficient muscle was observed: in the early (presymptomatic) phases, inflammatory pathways were predominant, while in the overt phases of the disease, an acute activation of TGF-β and a failure of metabolic pathways were observed. ¹³ CTGF messenger RNA (mRNA) has been found in the muscle of DMD patients. Moreover, in the activated fibroblasts, TGF-β1 and CTGF were co-localised, suggesting that CTGF expression is regulated by TGF-β1 through a paracrine/ autocrine mechanism, and indicating that the TGF–TGF-β1–CTGF pathway is involved in muscle fibrosis.¹⁴