Identification of homozygous missense mutations in ADD3 and KAT2B and effect of ADD3 and KAT2B mutations on protein levels in fibroblasts.

(A) Pedigree and segregation status of mutations found in ADD3 and KAT2B. Discovery of ADD3 mutations in family B and C was facilitated by GeneMatcher [44]. Half red coloured circles or squares denote patients with neurological defects and half blue coloured symbols denote patients with SRNS and cardiomyopathy. + symbols indicate non-mutated alleles. Mutations and segregation were confirmed by Sanger sequencing. (B) Exon structure of human ADD3 cDNA (long isoform NP_058432) and domains of adducin-γ protein. The relative position of ADD3 mutations to protein domains and exons are indicated (arrows). All mutations also affect the short isoform of ADD3 (NP_001112). Below each mutation, the phylogenetic conservation of the altered amino acid residues is shown. (C) Exon structure of human KAT2B cDNA and domains of KAT2B protein. PCAF-HD, p300/CBP-associated factor homology domain; AT, acetyl transferase domain; B, Bromo domain. The relative position of KAT2B variation to protein domains and exons is indicated (arrow). The phylogenetic conservation of the altered amino acid residue is shown. (D, E) Adducin-γ (D) and KAT2B (E) protein levels in control and patient fibroblasts. Lysates of patient II-3 and II-6 (family A) fibroblasts and age-matched control fibroblasts (Ctrl 1 and 2) were analyzed by western blotting. Results were normalized to the loading control α-tubulin. Each quantification is shown in the lower panel (n = 3 independent experiments, student’s t-test).