Identification of a mutation in RELA that results in RelA haploinsufficiency. (A) Pedigree with RELA genotypes. (B) Sanger sequencing of the RELA mutation (arrow). (C) Schematic of WT and mutant RELA cDNA splicing (dotted blue lines) with the patients’ mutation (asterisk), interval nucleotides and aa (arrowheads), introns (red line). Alternative splicing of a cryptic splice site within exon 6 to the canonical acceptor splice site before exon 7 deletes 73 nucleotides at the 3′ of exon 6 and introduces a premature stop codon at residue 174. (D, left) RT-PCR, performed with saturating mRNA concentrations, identifies the alternatively spliced transcript (Mut RELA) in patient 1 (P1), but not in the control (Right) WT RELA transcript detected with RT-PCR under nonsaturating mRNA concentrations. Similar results were obtained in P2. Data are from one representative experiment of three independently performed. (E) WT RELA mRNA in P1 and P2 and three controls determined by qPCR using a primer complementary to the nucleotides in exon 6, specific to the WT RELA transcript. Gene expression was normalized to GAPDH. (F) Immunoblot of fibroblast lysates from controls (C1–C3) and patients (P1 and P2) using an N-terminal specific antibody against RelA, with densitometric quantification of RelA relative to β-actin. Data from E and F are pooled from three independent experiments. Columns and bars represent experiment means ± SEM. ***, P < 0.001, Student’s t test.