Figure 4. Identification of KDM5A mutations in patients with ASD.

(A) Pedigrees of seven families with KDM5A mutations in nine probands with ASD. Double lines: first cousin status; circles: females; squares: males; shaded symbols: affected individuals. (B) A schematic of KDM5A domains and location of the identified mutations. De novo and recessive mutations are indicated in red and blue, respectively. The Selbst mutation is a cysteine to premature stop codon substitution at position 322 of the protein. ARID, A-T rich interaction domain; JmjC, Jumonji C; JmjN, Jumonji N; PHD, plant homeodomain; PLU1, putative DNA/chromatin binding motif; Zn, zinc finger. (C) Western blot analysis of lymphoblastoid cell line lysates from affected individuals (KD-1–3, KD-2–4, KD-5–3, and KD-6–3) and unaffected family members (KD-1–1, KD-2–1, KD-2–2, KD-5–1, KD-5–2, KD-5–4, KD-6–1, and KD-6–2) showed reduced KDM5A protein in affected individuals KD-1–3, KD-5–3, and KD-6–3, and a truncated KDM5A protein in affected individual KD-2–4. Western blot analysis of HEK293T cells with knock-in of the splice site mutation present in affected individuals KD-4–3 and KD-4–4 (Var) and HEK293T cells which underwent transfection but kept the reference sequence (Ctrl 1 and Ctrl 2), showed a decrease in KDM5A protein level in the targeted cells compared to control cells. β-actin and vinculin were used as loading controls. The black arrowhead points to the KDM5A band (196 kDa) and the white arrowhead points to the truncated KDM5A band (174 kDa).

Figure 4—figure supplement 1. Related to Figure 4.

(A) Deletion map in family KD-2. Patients KD-2–3 and KD-2–4 have exons 6 through 9 of KDM5A deleted (~10 kb), which leads to the formation of a truncated protein. (B) Sanger sequencing in family KD-1. Patient KD-1–3 has a de novo missense mutation (A to T) in the first methionine of KDM5A, which gets substituted by a leucine. Data shown for reverse sequence. (C) Sanger sequencing of HEK293T cells with knock-in of the splice site mutation present in KD-4. The successful knock-in clone has a homozygous G to T substitution.

Figure 4—figure supplement 2. Related to Figure 4.

The Met1 (A), Arg1428 (B), and Phe477 (C) residues of KDM5A (denoted by the asterisks) are highly conserved across species. Met1, Arg1428, and Phe477 are mutated in KD-1–3, KD-3–3, and KD-5–3, respectively.

Figure 4—video 1. Related to Figure 4.

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Video recording of patient KD-4–3.

Figure 4—video 2. Related to Figure 4.

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Video recording of patient KD-4–4.