Functional Studies of a Missense Mutation in the CRADD Gene in Mennonite Patients With Autosomal Recessive Non-Syndromic Mental Retardation
The Clinic for Special Children recently associated a novel variant (c.382G>C) in CRADD with autosomal recessive non-syndromic mental retardation in Mennonite patients. The CRADD gene, encodes a death-adaptor protein with a caspase recruitment domain (CARD) and a death domain (DD). CRADD interacts with caspase-2, a cysteine protease, and PIDD, a p53- induced protein with a DD, to form the PIDDosome. CRADD and PIDD interact within the PIDDosome to recruit and activate caspase-2, initiating apoptosis (Berufae et aL, 2005). Previous work has shown that a single amino acid residue change on an interface of CRADD DD and PIDD DD interaction inhibits formation of the PIDDosome (Jang et aL, 2010). The c.382G>C variant is predicted to change the highly conserved Gly128 to Arg128 in the CRADD DD. If the Glyl28Arg variant alters interaction between the DDs of CRADD and PIDD, then formation of the PIDDosome may be disrupted. Mouse (m) Cradd Glyl28Arg in mIMCD3 cells and human (h) CRADD Gly 128Arg in hARPE-19 cells formed clusters when co-overexpressed with mPidd or hPIDD, which were not observed in wild-type (wt) overexpression, suggesting a change in CRADD and PIDD interaction. Semi-quantitative western blotting demonstrated that overexpressed hCRADD Glyl28Arg was 82.9 ± 2.3% (n=3) less abundant than overexpressed wt hCRADD, suggesting that hCRADD Gly 128Arg may be unstable or that cells containing the Gly 128Arg variant undergo apoptosis more readily. Mouse Cradd DD GIyl28Arg did not coimmunoprecipitate mPidd DD, suggesting that the Gly 128Arg variant may alter one of the interaction interfaces of CRADD DD to increase CRADD DD homo-oligomerization and decrease CRADD DDnulls affinity for PIDD DD. Disruption of the PIDDosome and subsequent neurotrophin-mediated apoptosis in the developing central nervous system may underlie the cognitive impairment in CRADD c.382G>C patients.
Franklin and Marshall College Archives, Undergraduate Honors Thesis 2012
- F&M Theses Collection