Spondylo-meta-epiphyseal dysplasia (SMED), short limb-hand type (SMED-SL, OMIM 271665) is a rare autosomal recessive disorder affecting human skeletal growth. The condition is characterized by disproportionately short stature, platispondyly, abnormal epiphyses and metaphyses, shortening of the lower and upper limbs, short broad fingers and punctate calcifications [1–4]. This bone dysplasia is progressive, with serious complications leading to death in some cases. Atlantoaxial instability resulting in cord damage has been the most reported cause of death [2, 5]. Homozygous mutations occurring in the Discoidin domain receptor 2 gene (DDR2, MIM 191311) have been identified as the cause for this severely dwarfing condition [6, 7]. The DDR2 gene encodes one of the two members of a unique receptor tyrosine kinase (RTK) subfamily known as the discoidin domain containing receptors (DDRs), which recognize collagen as their ligands [8, 9]. Upon collagen binding, the receptor displays delayed and sustained tyrosine phosphorylation which further elicits downstream signalling to cellular metabolic pathways that cross-talk at various points [10, 11]. In addition, dysregulation of DDR2 has been shown to be associated with various human diseases such as fibrosis, arthritis and cancer and like other RTKs, DDRs are emerging as potential therapeutic targets .
DDR2 is commonly expressed in cells of mesenchymal origin and is activated by fibrillar collagens [8, 9] and collagen X . DDR2 has been shown to play a critical role in cell invasion and collagen remodelling through the regulation of matrix metalloproteases and collagen fibrillogenesis [10, 14–16]. The involvement of DDR2 in skeletal growth was demonstrated by the DDR2 knockout mice, which display skeletal abnormalities that reflect the SMED-SL phenotype of humans . The abnormal skeletal development in DDR2 deficient mice was due to reduced chondrocyte proliferation in the growth plate. A spontaneous, autosomal recessive mutation in a mouse colony was characterised that resulted from deletion of most of the Ddr2 gene . These mutant mice were also found to be infertile due to gonadal dysfunction including impaired spermatogenesis and ovulation, which were attributed to defects in overall endocrine function [18–20].
DDRs are plasma membrane RTKs comprising an extracellular domain (ECD), a transmembrane domain (TM), a large cytosolic juxtamembrane domain and a C-terminal catalytic tyrosine kinase domain. The ECD is necessary and sufficient for ligand binding and consists of an N-terminal discoidin homology (DS) domain followed by a region unique to the DDRs that contains a DS-like domain [21–23]. The DDRs exist as preformed homodimers on the cell membrane even in the absence of collagen [24, 25]. The exact signaling pathways and binding partners through which DDR2 controls bone growth remain unknown despite significant progress made in recent years in understanding the structural basis of collagen recognition . In recent reports it was shown that DDR2 modulates the phosphorylation of Runx2, a master transcription factor involved in skeletal development [27, 28]. In addition, DDR2 has been shown to mediate the secretion of lysyl oxidase , an enzyme that catalyzes cross-linking of collagen fibers, an essential modification to strengthen bone.
A recent study has shown that the DDR2 ECD aids the formation of mineralized calcium deposits in vitro, and this activity is independent of the tyrosine kinase activity . The authors proposed that the presence of DDR2 ECD in the mutant proteins along with impaired signaling leading to increased calcification could be a potential mechanism in SMED-SL. Mutations prevalent in SMED-SL disorder have been mapped to the extracellular domain and tyrosine kinase domain of DDR2 [6, 7]. Recently we have shown that DDR2 missense mutations occurring at the kinase domain (p.T713I, p.I176R, p.R752C) result in retention of the mutant protein in the endoplasmic reticulum (ER), while the ectodomain mutant (p.E113K) is expressed on the cell surface, but failed to bind to collagen, thus elucidating two different cellular mechanisms resulting in loss of function of the protein leading to disease .
ER retention of misfolded proteins and subsequent degradation (ERAD) is a recurring theme in many skeletal disorders [30–33] and numerous other monogenic diseases [31, 34–36]. Inhibition of ERAD coupled with proteostasis modulation has been shown to enhance folding, trafficking and activity of unstable proteins and is emerging as a potential strategy to combat many protein misfolding disorders . Defining the potential contribution of specific mutations and intracellular stress to the pathophysiology of the disease will pave avenues for targeted therapeutic intervention and disease management in the future.
We report here the clinical and molecular findings in SMED-SL patients of consanguineous origin from Oman and the identification of a novel disease-causing truncating mutation in DDR2. The sub-cellular localization and functional status of the mutant and wild type proteins were compared in a mammalian expression system. Our results indicate that the novel mutation results in defective trafficking of the protein and loss of its activation by collagen.