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Loss-of-function mutations in DDRGK1 have been shown to cause Shohat type spondyloepimetaphyseal dysplasia. In zebrafish, loss-of-function of ddrgk1 lead to defects in early cartilage development. Ddrgk1−/− mice show delayed mesenchymal condensation in the limb buds and early embryonic lethality. Mechanistically, Ddrgk1 interacts with Sox9 and reduces ubiquitin mediated proteasomal degradation of Sox9 protein. To investigate the cartilage-specific role of DDRGK1, conditional knock-out mice were generated by intercrossing Prx1-Cre transgenic mice with Ddrgkfl/fl mice to delete its expression in limb mesenchymal cells. Mutant mice showed progressive severe shortening of the limbs and joint abnormalities. The growth plate showed disorganization with shortened proliferative zone and enlarged hypertrophic zone. In correlation with these findings Sox9 and Col2a1 protein levels were decreased while Col10a1 expression was expanded. These data demonstrate the importance of Ddrgk1 during growth plate development. In contrast, deletion of Ddrgk1 with the osteoblast-specific Osteocalcin-Cre and Leptin receptor-Cre lines did not show bone phenotypes suggesting the effect on limb development is cartilage-specific. To evaluate the role of DDRGK1 in cartilage postnatal homeostasis, inducible Agc1-CreERT2; Ddrgklfl/fl mice were generated. Mice in which Ddrgk1 was deleted at 3 months of age showed disorganized growth plate, with significant reduction in proteoglycan deposition. These data demonstrate a postnatal requirement for Ddrgk1 in maintaining normal growth plate morphology. Together, these findings highlight the physiological role of Ddrgk1 in development and maintenance of the growth plate cartilage. Furthermore, these genetic mouse models recapitulate the clinical phenotype of short stature and joint abnormalities observed in patients with Shohat type SEMD.

Haploinsufficiency of TGF-Beta Activated Kinase 1 (MAP3K7) Binding Protein 2 (TAB2) has been associated with congenital heart disease and more recently multiorgan structural abnormalities. Missense variant represents a major proportion of non-synonymous TAB2 variants reported in gnomAD (295/576) and Clinvar (16/73), most of which are Variants of uncertain significance (VUSs). However, interpretation of TAB2 missense variants remains challenging due to lack of functional assay. To address this issue, we established a cell-based luciferase assay that enables high-throughput screening of TAB2 variants to assess the functional consequence for predicting variant pathogenicity. Using this platform, we screened 47 TAB2 variants including 5 pathogenic controls and 1 benign control, and the results showed that the transcriptional activity of AP-1 but not NF-κB predicts the TAB2 variant pathogenicity. This assay provides accurate functional readout for both loss-of-function (LOF) and gain-of-function (GOF) variants, which are associated with distinct phenotypes. 22 out of 32 tested VUSs were reclassified. Genotype–phenotype association showed that most patients with partial LOF variants do not exhibit congenital heart disease but high frequency of developmental delay, hypotonia, and dysmorphic features, which suggests genetic testing for TAB2 is needed for a broader spectrum of patients with more diverse phenotypes. Molecular modeling with Npl4 zinc finger (NZF) domain variants revealed that the stability of the NZF domain in TAB2 protein is crucial for AP-1 activation. In conclusion, we developed a highly effective functional assay for TAB2 variant prediction and interpretation.