Here we report the clinical features of two siblings diagnosed with SMED-SL and a novel DDR2
missense mutation identified in these patients. This mutation (p.E113K) is located in the extracellular discoidin domain and affects a surface-exposed residue that was previously identified as an important amino acid in the collagen-binding site of DDR2 (6
). Additionally, we identified another mutation (p.R752C) in an Egyptian family with SMED-SL. This DDR2 mutation, localized in exon 17 of the DDR2
gene, which encodes part of the intracellular tyrosine kinase domain, is a recurring mutation previously found in five Arab families from Jerusalem affected with SMED-SL (11
Here we demonstrate the effects of all known SMED-SL causing missense mutations on DDR2. Our cellular and biochemical analysis of these four DDR2 mutations showed that these fall into two classes: all previously identified missense mutations cause a trafficking defect with the affected proteins mis-localizing to the ER, while the novel p.E113K-DDR2 mutation is localized in the cell similar to wild-type DDR2 but is defective in transmembrane signaling due to a ligand-binding defect. A previous report identified mutations in the DDR2
gene as causative genetic defects for SMED-SL (11
), but did not illuminate the mechanism of disease. Bargal et al
) speculated on the possible biochemical consequences of the disease mutations p.T713I, p.I726R and p.R752C. Because the affected amino acids are located in the active site of the DDR2 kinase, it was proposed that the substitutions may influence the stability of the activation loop, ATP binding affinity or substrate specificity, respectively (11
). The possibility that protein folding may be affected was not considered. DDR2 is a member of the RTK family of plasma membrane receptors and is phylogenetically related to ROR2 (24
). Missense mutations in the ROR2
gene were recently shown to cause retention of the mutated proteins in the ER as a result of the protein folding quality control within this organelle named ER-Associated Protein Degradation (ERAD) (15
). ERAD ensures that only properly folded proteins and fully assembled complexes are allowed to exit the ER to their final destinations. Proteins that fail to attain the required functional native conformations due to mutations or failure to acquire the needed posttranslational modifications, such as proteolytic cleavage of signal peptides, glycosylation and GPI-anchoring, are retained in the ER for a short period of time but then get targeted for re-translocation to the cytoplasm and degradation by the ubiquitin/proteasome systems (25
In this report, we show that three out of four missense mutations (p.T713I, p.I726R and p.R752C) found in SMED-SL patients result in the retention of the affected proteins in the ER. ER retention of the three mutants is strongly supported by the confocal images presented in Figures and and the biochemical findings on the sensitivity to Endo H treatment presented in Figure . We noticed lower levels of expression of the ER-retained mutant proteins in HEK293 cells, compared with wild-type DDR2, as can be seen in Figure B. These low steady-state levels of the mutant proteins may be due to ERAD degradation. Additionally, our findings add another genetic condition to the 60 or so known to be caused by ERAD (15
Unlike the ER-retained mutant DDR2 receptors, E113K-DDR2 was found at the cell surface, similar to wild-type DDR2 (Fig. A–F), indicating a different disease-causing mechanism. Glu113 is located in the DDR2 extracellular discoidin domain. The DDR2 discoidin domain adopts a β-barrel structure consisting of eight β-strands (23
). At the top of the barrel, five protruding loops create a trench that forms the collagen-binding site (6
). Glu113, which forms a salt bridge with Arg105, is one of several key residues that contact collagen (6
). Figure , which shows a cartoon drawing of the DDR2 discoidin domain bound to a collagen peptide, illustrates how Glu113 is involved in the DDR2-collagen interaction. Data presented in Figure demonstrate that E113K-DDR2 had lost most of the collagen-binding activity. This result agrees well with the previous observation that mutation of E113Q reduced collagen-binding to ~10% of wild-type DDR2 (23
). Thus, Glu113 is one of the main DDR2 residues contributing to collagen-binding. It is also noted that Glu113 and Arg105 are conserved in DDR1, further underscoring the importance of the Glu113-Arg105 salt bridge in the DDR-collagen interaction. This is the first report on the clinical relevance of substitutions at this site.
Figure 8. Cartoon drawing of the DDR2 discoidin domain (selected side chains: W52, cyan; R105, blue; E113, red) bound to a collagen peptide (selected side chains: M21 and O24 of leading chain, F23 of middle chain). Hydrogen bonds involving E113 are shown as dashed (more ...)
We noted some variations in the phenotype between the two families we are reporting here. For example, the clinical phenotype and the degree of chondral calcification of the second family (mutation p.R752C) were very severe (17
). Both children had extensive calcifications which appeared by 1 years of age and were very progressive and widespread involving almost all cartilages. In addition, both children had repeated respiratory infections requiring hospital admissions and leading to death in the older child. The younger sib had in addition symptoms of cord compression, which led to his sudden death. The phenotype in the affected children from the first family (mutation p.E113K), on the other hand, was less severe with very few mild respiratory infections not requiring hospital admissions. In addition, the chondral calcification was limited and much less severe than in the p.R752C family. This variability might be due to retention of some residual activity in the p.E113K mutant compared with the p.R752C mutant. Alternatively, cells expressing the ER-retained mutated protein (p. R752C) may experience ER stress, which would not be experienced by cells expressing the correctly targeted p.E113K protein. However, the clinical significance of these presumed differences in ER stress responses are unknown at this stage.
How does DDR2 control bone growth? Very little is known about the role of DDR2 in bone growth. DDR2 is expressed on chondrocytes and its elimination in the mouse leads to suppression of chondrocyte cell proliferation (9
). It is assumed that the interaction of DDR2 with collagen II in the proliferative zone of the growth plate is responsible for controlling chondrocyte proliferation. DDR2 is also a receptor for another collagen type with relevance to bone growth, collagen X (4
). While collagen II is found in all types of cartilage, collagen X expression is restricted to the hypertrophic zone of the growth plate. We show here that E113K-DDR2 is unable to interact with collagen II (Fig. C). Further studies are required to establish whether the mutant protein is also defective in binding to collagen X.
In mice with targeted DDR2 deletion, the growth plates were observed to be shortened (9
). In the only available histological study of a chostrochondral junction from a SMED-SL patient, extremely abnormal cartilage was found (12
). The growth plate was found to lack proliferating chondrocyte columns. Chondrocytes were small and sparse in the resting zone, often surrounded by a ‘ring-like’ amorphous matrix, which in turn could be surrounded by a more densely packed structure of fibrillar collagen. Clustering of chondrocytes within lacunae was also observed. Taken together with our data, it can be speculated that in SMED-SL patients, lack of DDR2 interaction with collagen II results in impaired DDR2 signaling, leading to severe suppression of chondrocyte proliferation, which may be responsible for poor linear bone growth. Further morphological studies on patient samples with known DDR2 mutations would be required to confirm this hypothesis.
In summary, we report a novel mutation in the DDR2 gene in patients affected with SMED-SL and elucidate the cellular and biochemical mechanisms underlying all reported SMED-SL disease mutations. Two different mechanisms, failure to reach the plasma membrane and impaired ligand binding, were found to result in the growth disorder. This study supports the notion that DDR2 signaling is important in human development and that the interaction of DDR2 with collagen is essential for proper skeletal growth.