Fig. 2

Segregation analysis, schematic representation of CCDC88C protein, and functional validation of the variant NM_001080414.4:c.1993G > A (p.E665K). I. The family pedigree and chromatogram showing the mutant variant NM_001080414.4:c.1993G > A (p.E665K) (filled arrowhead) heterozygous in the patient F83–581 (filled circle) and the wild-type variant at the same position (empty arrowheads) homozygous in the healthy controls (coded circles and square). M: mutant allele; WT: wild type allele. II. Schematic representation of CCDC88C protein based on the Uniprot entry Q9P219 showing the locations of the reported pathogenic heterozygous variants in the protein. CC: coiled-coil region; CH: calponin-homology domain. III. Expression of CCDC88C^E665K mutant protein activated c-Jun N-terminal kinase (JNK) / caspase-3 apoptotic signaling pathway. a The wild-type and mutant pcDNA3.1(+)-myc-CCDC88C expression constructs were used to transfect Human Embryonic Kidney 293 (HEK293) cells to assess the effect of CCDC88C^E665K mutant protein on the activity of JNK / caspase-3 signaling pathway. Similar to the other two CCDC88C mutant proteins (CCDC88C^D43N and CCDC88C^R464H) that were previously reported to activate JNK / caspase-3 signaling [4, 5], overexpression of CCDC88C^E665K mutant protein caused a significant increase of JNK phosphorylation and caspase-3 cleavage. n = 3 biological replicates on 3 independent preparation of protein samples. Only representative blots were shown. b-d Quantification of the relative expression of p-JNK (b), cleaved caspase-3 (c), and CCDC88C (d) proteins. Error bars represent S.E.M. Statistical analysis was performed using one-way ANOVA followed by post hoc Tukey’s test. ns denotes no significant difference, * denotes p < 0.05, ** denotes p < 0.01 and *** denotes p < 0.001