SAT-283 A Mutation In Ccdc53 Affects Pth1r Trafficking And Causes Disproportionate Short Stature With Noonan-like Facies

In many children with short stature, the genetic etiology remains unknown. Short stature due to defects in trafficking of membrane-associated proteins, such as receptors, has not been reported. A mother and two daughters presented with disproportionate short stature and Noonan-like facies. Exome sequencing, confirmed by Sanger sequencing, identified a de novo dominant mutation (L69F) in CCDC53. CCDC53 normally binds to WASH1 within a protein complex termed the WASH complex. To study the effect of the mutation on this interaction, CCDC53 was first ablated in HEK293 cells (CCDC53KO-HEK) by CRISPER-Cas9 and then wild-type (WT) or mutant (mut) CCDC53 or empty vector (EV) was expressed in these cells. The identified mutation impaired CCDC53 interaction with WASH1 as assessed by co-immunoprecipitation. CCDC53 and the WASH complex are involved in trafficking of cell-surface proteins that have undergone endocytosis, including G-coupled protein receptors that contain a PDZ motif, such as PTH1R. We therefore hypothesized that the mutation would affect PTH1R endocytic trafficking. CCDC53KO-HEK cells were transfected with vectors to express PTH1R and either EV, WT or mut CCDC53 and treated with PTH, after which endosomes were extracted and PTH1R was detected by western blot. Compared to WT, EV and mut CCDC53 showed decreased PTH1R localization in endosomes. In this system, the mutation also decreased generation of cAMP by extracellular PTH (P=0.001 vs WT), indicating that the mutation interferes with PTH1R signal transduction. PTH1R acts as the receptor for PTHrP and prevents premature hypertrophy of growth plate chondrocytes. We therefore hypothesized that the CCDC53 mutation would allow premature hypertrophy of growth plate chondrocytes. Primary rat epiphyseal chondrocytes were transfected with siRNA against Ccdc53 and with vectors to express either WT or mut Ccdc53. Compared to WT, mut Ccdc53 led to increased expression of hypertrophy-associated genes: Col10a1 (P=0.001), Ihh (P=0.031), and Mmp13 (P=0.003), consistent with the hypothesized effect on hypertrophic differentiation. In conclusion, a mutation in CCDC53 was identified in a family with disproportionate short stature and Noonan-like facies. The mutation interfered with the ability of CCDC53 to participate in the WASH complex, affecting endosomal localization of PTH1R, PTH1R signal transduction, and chondrocyte hypertrophy. Because the timing of growth plate chondrocyte hypertrophy is critical for linear growth, the findings provide a potential explanation for the short stature in this disorder. More generally, the findings also indicate that endocytic trafficking of PTH1R by the WASH complex plays an important role in the regulation of human linear growth.