Biochemical characterization of Indian hedgehog mutations associated with brachydactyly type A1 and acrocapitofemoral dysplasia

Abstract

Indian hedgehog (IHH) is involved in regulating bone development, playing a particularly important role in endochondral ossification. Mutations in the IHH gene have been implicated in two human genetic diseases, brachydactyly type A1 and acrocapitofemoral dylplasia. This project involves characterizing these mutations and elucidating the resulting defect in IHH function. Nine mutant IHH constructs were created using site-directed mutagenesis to induce point mutations into full-length IHH cDNA, and HEK293 cell lines stably expressing each of the mutant constructs were established. Attempts to characterize the mutant IHH constructs using in vitro models of chondrogenesis were unsuccessful due to a lack of long-term expression and low transfection efficiency. The HEK293[IHH] stable lines were co-cultured with SHH-Light II cells (a hedgehog responsive cell line) to measure the activity of the mutant constructs and compare to IHHWT. Secretion and processing of the mutant IHH constructs were assessed by ELISA and western blotting, respectively. Overall, the mutations result in decreased intracellular levels of the IHH, with additional defects in processing and signaling for several of the mutants.