RANK/RANKL/OPG pathway: Genetic associations with stress fracture period prevalence in elite athletes

Highlights

• Genetic associations with stress fracture injury were shown in elite athletes.

• SNPs located near the RANK/RANKL/OPG signalling pathway are associated with stress fracture injury.

• These data suggest a potential role for these SNPs in the regulation of bone strength and adaptation to mechanical loading.

Abstract

Context

The RANK/RANKL/OPG signalling pathway is important in the regulation of bone turnover, with single nucleotide polymorphisms (SNPs) in genes within this pathway associated with bone phenotypic adaptations.

Objective

To determine whether four SNPs associated with genes in the RANK/RANKL/OPG signalling pathway were associated with stress fracture injury in elite athletes.

Design, participants, and methods

Radiologically confirmed stress fracture history was reported in 518 elite athletes, forming the Stress Fracture Elite Athlete (SFEA) cohort. Data were analysed for the whole group and were sub-stratified into male and cases of multiple stress fracture groups. Genotypes were determined using proprietary fluorescence-based competitive allele-specific PCR assays.

Results

SNPs rs3018362 (RANK) and rs1021188 (RANKL) were associated with stress fracture injury (P < 0.05). 8.1% of the stress fracture group and 2.8% of the non-stress fracture group were homozygote for the rare allele of rs1021188. Allele frequency, heterozygotes and homozygotes for the rare allele of rs3018362 were associated with stress fracture period prevalence (P < 0.05). Analysis of the male only group showed 8.2% of rs1021188 rare allele homozygotes had suffered a stress fracture whilst 2.5% of the non-stress fracture group were homozygous. In cases of multiple stress fractures, homozygotes for the rare allele of rs1021188 and individuals possessing at least one copy of the rare allele of rs4355801 (OPG) were shown to be associated with stress fracture injury (P < 0.05).

Conclusions

The data support an association between SNPs in the RANK/RANKL/OPG signalling pathway and the development of stress fracture injury. The association of rs3018362 (RANK) and rs1021188 (RANKL) with stress fracture injury susceptibility supports their role in the maintenance of bone health and offers potential targets for therapeutic interventions.

Introduction

Stress fractures arise from the inability of bone to tolerate repeated mechanical loading and are characterised by damage to the bone's micro-architecture [1]. Repeated mechanical loading can cause an uncoupling of osteoblast bone formation and osteoclast bone resorption [1]. This can lead to bone loss and subsequent micro-damage that can result in localised bone weakening, prompting stress fracture development [2]. Stress fracture period prevalence in elite athletes and military recruits ranges from 14 to 21% [3], [4], and most commonly manifests in the lower limbs [5].

In athletes, stress fracture injury is likely to have a complex aetiology involving numerous factors, with, for example, prior training [6] and biomechanical variables (e.g., running kinematics) [7] being implicated in stress fracture risk (for a wider review of this issue please see Bennell et al. [4]). Susceptibility may also have genetic origins, supported by reports of monozygotic twins developing similar stress fracture injuries [8], multiple stress fractures occurring in the same individual [9], stress fractures occurring in some individuals but not in others undertaking identical training protocols [3], [4] and a family history of stress fracture injury acting as a risk factor [10].

Genetic associations with stress fracture period prevalence in military personnel have been investigated using a variety of single nucleotide polymorphisms (SNPs) previously associated with receptors known to influence bone mineralisation, remodelling [11] and endocrine abnormalities [12]. Associations were shown for SNPs and haplotype blocks within the vitamin D receptor [13], [16] and an androgen receptor repeat sequence [14]. However, other studies have shown no association for the same SNPs in similar military populations [15]. The reason for the disparity may be the range of SNPs analysed and small numbers of stress fracture cases in some studies (e.g., n = 64, [16]). There is a need to examine this in a large cohort of elite athletes given that the pathogenesis of stress fracture might be different due to the phenotypic differences, training variables and fitness.

Given that disturbances in bone remodelling and the inability of bone to withstand repeated bouts of mechanical loading are implicated in the development of stress fracture injury [1], SNPs repeatedly shown to be associated with these bone phenotypes in large-scale studies are worthy of focused study. As all previous studies have used military personnel, studies involving alternative groups, such as athletes with a similarly high period prevalence of stress fracture injury, are required to provide further insights into both the aetiology and genetic predisposition of stress fracture injury. Furthermore, no published literature exists in relation to genetic associations with stress fracture injury risk in elite athletes.

The receptor activator of nuclear factor-_KB (RANK) and its ligand (RANKL), a member of the tumour necrosis factor (TNF) superfamily, are integral to osteoclastogenesis as they stimulate osteoclast activation, formation and differentiation [17]. Osteoprotegerin (OPG) acts as a decoy receptor for RANKL leading to the prevention of osteoclast precursor development into mature osteoclasts, resulting in the subsequent attenuation of bone resorption [17]. These factors in combination make up the RANK/RANKL/OPG signalling pathway, an important system in the regulation of bone turnover, and in the potential mediation of stress fracture injury development in individuals with a high frequency and/or amplitude of mechanical loading.

The specific mechanisms of how SNPs within the RANK/RANKL/OPG signalling pathway influence bone health remain unknown. Several of these SNPs have been associated with bone phenotypic alterations, including changes in bone mineral density (BMD) [18], [19], [20], [21], bone cross sectional area [22], osteoporotic fracture risk [20] and bone resorption and formation [23], although none of these have been established in stress fracture injury.

This study examined whether SNPs within the RANK/RANKL/OPG signalling pathway were associated with stress fracture injury in elite athletes.