Children and adolescents with NF1 had statistically significant increases in Dpd reflecting increased collagen degradation in children with NF1. In addition, the Dpd/Pyd ratio was elevated in the NF1 individuals consistent with increased bone turnover. Interestingly, individuals with Ehlers-Danlos syndrome type VI, who have normal amounts of pyridinium crosslinks but have a significantly increased Dpd/Pyd ratio (24
), are in part differentiated from other types of Ehlers-Danlos syndrome based on the presence of skeletal abnormalities characterized by a progressive kyphoscoliosis usually present in the first year of life, osteoporosis, and clubfeet (27
). Pyd is found in many tissues including both bone and cartilage, but Dpd is most abundant in bone and dentine and reflects more the status of bone (24
). The observation that the Dpd/Pyd ratio is elevated in NF1 children and adolescents indicates a preferential increase in bone resorption rather than a generalized collagen breakdown.
Although decreased bone mineral density has been reported in individuals with NF1 (11
), we are not aware that they display a substantially increased number of fractures compared with the general population, although long-bone fractures with poor bone healing is observed in patients with localized long-bone dysplasia. Potentially, decreased bone mineral density could predispose individuals with NF1 to clinically undetected microfractures. Microfractures are resorbed by osteoclasts and can alter trabecular architecture (30
). The observed increase in the excretion of urinary pyridinium crosslinks in our cohort of children and adolescents with NF1 may be the result of microfractures leading to an abnormal microarchitecture.
There is evidence that somatic loss of the nonmutant NF1
allele in NF1
heterozygous individuals leads to localized skeletal abnormalities as double inactivation of NF1
was observed in pseudarthrosis tissue from patients with NF1 with tibial bowing and fracture with nonunion (33
). In this cohort, 29% of individuals with NF1 had scoliosis, long-bone dysplasia, and/or sphenoid wing dysplasia. Although active fractures were not present, the contribution of scoliosis, long-bone dysplasia, and/or sphenoid wing dysplasia on the excretion of pyridinium crosslinks is unknown. Therefore, in this study NF1 individuals were divided into those with and without scoliosis, long-bone dysplasia, and/or sphenoid wing dysplasia. The NF1 individuals without a skeletal dysplasia still had increased Dpd and Dpd/Pyd ratios compared with controls, suggesting that NF1
haploinsufficiency contributes to increased bone resorption and represents a generalized abnormality of bone remodeling in the background of NF1. However, the precise role neurofibromin plays in the growth and development of bone in individuals with NF1 is not well understood.
Given the paucity of studies on bone remodeling in the NF1 human model, insights into the role of neurofibromin in osteoclast and osteoblast functioning must be taken from the murine model. It is known that neurofibromin directly impacts the Ras-signaling pathway, which interacts with multiple signaling pathways, several of which are important in bone. For example, transforming growth factor-beta increases neurofibromin mRNA (34
), murine Nf1±
mast cells secrete elevated concentrations of transforming growth factor-beta (35
), fibroblast growth factors activate the Ras/mitogen-activated phosphorylation kinase pathway (36
), and inactivation of the SHP2-Ras-mitogen-activated phosphorylation kinase pathway in mice results in enhanced bone formation after an increase in osteoclast activity suggesting a dissociation of the intercellular communications between osteoclasts and osteoblasts (37
). Specific investigations using the Nf1
haploinsufficient transgenic mouse model has shown abnormalities in myeloid cells. Yang et al.
) showed that monocytes from Nf1±
mice have an increased potential to mature into multinucleated osteoclasts, and that the osteoclasts have increased adhesive and lytic properties, which was also observed in a small cohort of two individuals with NF1. This increase in osteoclast formation and lytic activity in the murine model and the two NF1 individuals (39
) is in concert with our findings of increase bone resorption in children and adolescents with NF1.
Because of the complexities of bone remodeling, the observed increase in bone resorption in our cohort and the previously described abnormalities in the myeloid lineages do not fully explain the pathophysiology of the skeletal defects of NF1. Additional animal studies have shown that deficiency of neurofibromin also impacts differentiation of mesenchymal progenitor cells. Yu et al.
) reported that Nf1±
committed osteoprogenitors exhibited premature apoptosis and higher proliferation, and Wu et al.
) showed impaired osteoblast differentiation in Nf1±
mesenchymal stem/progenitor cells. The effects of neurofibromin deficiency on skeletal morphogenesis and remodeling likely vary depending on timing of expression, potentially impacted by early somatic inactivation of NF1
in the normal allele in a subset of mesenchymal cell lineages. This is evidenced by the discordant phenotypes observed in various Nf1
transgenic mouse models. For example, mice lacking neurofibromin in osteoblasts (Nf1ob−/−
) show increased bone formation without long-bone bowing (42
), whereas mice lacking neurofibromin in undifferentiated mesenchymal cells of the developing limb (Nf1Prx1
) display tibial bowing with a high degree of porosity (36
). The Nf1ob−/−
mice also showed increased urinary excretion of pyridinium crosslinks (42
), which is consistent with our results in children.
Investigation of the effects of downstream targets of the neurofibromin-Ras signal transduction pathway on osteoprogenitor cells will be important in selecting targeted therapies for the localized skeletal defects of NF1. With the advancement of transgenic mouse models that partially recapitulates the human NF1 skeletal phenotype such as the long-bone bowing in the Nf1Prx1
mouse model described by Kolanczyk et al.
), human clinical trials with therapeutic agents identified through animal studies are likely to develop. Given that decreased bone mineral density has been reported previously in individuals with NF1, and the herein reported data show evidence of increased bone resoprtion, currently used therapeutic agents within the general population such as bisphosphonates are potential candidates. Schindeler et al.
) used bone morphogenetic protein and bisphosphonate combination therapy in the mouse model showing an increase of net bone production in vivo
mice. Therefore, single agent therapy may not be the most appropriate treatment strategy, and further studies will be necessary to characterize the human NF1 skeletal phenotype more accurately, understand the effects of increased Ras signaling on bone remodeling, and understand the safety efficacy of each candidate agent before proceeding with clinical trials.
Pyridinium crosslinks may prove to be a good surrogate marker for future clinical trials. In addition, the analysis of pyridinium crosslinks may prove useful to identify individuals with NF1 who are at risk for clinical osseous complications. However, prospective studies measuring urinary pyridinium crosslinks before the development of an osseous complication will be needed to assess their clinical utility in individuals with NF1.