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Alternating hemiplegia of childhood (AHC) is a rare and severe neurological disorder. ATP1A3 was recently identified as the causative gene. Here we report the first genetic study in Chinese AHC cohort. We performed whole-exome sequencing on three trios and three unrelated patients, and screened additional 41 typical cases and 100 controls by PCR-Sanger sequencing. ATP1A3 mutations were detected in 95.7% of typical AHC patients. At least 93.3% were de novo. Four late onset, atypical AHC patients were also mutation positive, suggesting the need for testing ATP1A3 mutations in atypical cases. Totally, 13 novel missense mutations (T370N, G706R, L770R, T771N, T771I, S772R, L802P, D805H, M806K, P808L, I810N, L839P and G893R) were identified in our study. By homology modeling of the mutant protein structures and calculation of an extensive list of molecular features, we identified two statistically significant molecular features, solvent accessibility and distance to metal ion, that distinguished disease-associated mutations from neutral variants. A logistic regression classifier achieved 92.9% accuracy by the average of 100 times of five-fold cross validations. Genotype-phenotype correlation analysis showed that patients with epilepsy were more likely to carry E815K mutation. In summary, ATP1A3 is the major pathogenic gene of AHC in Chinese patients; mutations have distinctive molecular features that discriminate them from neutral variants and are correlated with phenotypes.

Repair of cartilage-bone interface tissue remains challenging, because it combines different cell types and gradients of composition and properties. To enable simultaneous regeneration of bone, cartilage, and especially their interface, a conically graded scaffold of chitosan-gelatin hydrogel/poly(l-lactide-co-glycolide) (PLGA) was facilely prepared in the study. The chitosan-gelatin hydrogel containing transforming growth factor β1 (TGF-β1) was used for chondrogenesis, while the PLGA scaffold loading bone morphogenetic protein-2 (BMP-2) for osteogenesis. The conically graded transition from the hydrogel to PLGA scaffold and graded variation in amount of growth factors from TGF-β1 to BMP-2 benefited the cartilage-bone interface reconstruction. The graded scaffold exhibited spatio-temporal delivery of TGF-β1 and BMP-2. Preliminary results of in vitro cell culture demonstrated that the hydrogel and PLGA phases could promote bone marrow mesenchymal stem cells toward chondrogenic and osteogenic differentiation, respectively. From the result of the pilot in vivo experiment, it showed that the regenerated hyaline-like cartilage surface and subchondral bone excellently integrated with the native tissues were found by using the TGF-β1 and BMP-2 double-loaded hydrogel/PLGA graded scaffold via H&E and immunohistochemical stainings of collagen I, collagen II, and osteocalcin at 2 months. The obtained preliminary experiment results showed that the hydrogel/PLGA graded scaffold combining multiphasic composition and spatial dual growth-factor delivery would be useful for cartilage-bone interface tissue defect repair.