Fig. 1.

Genetic and molecular characterization of the CARD9 c.143 + 1 G > C mutation. A Pedigree of a family with the CARD9 mutation (upper panel). Half-filled symbols indicate the heterozygous CARD9 mutation and black filled symbols denote the homozygous mutation. The proband is indicated with an arrow; clinical expressivity and disease penetrance are discussed in the text. Chromatogram of CARD9 Sanger sequencing in a normal control (NC), patients (II.2 and II.3) with a homozygous change, and parents (I.1 and I.2) with a heterozygous change (lower panel). The blue color indicates the site of the mutation. B The agarose gel electrophoresis showing PCR products of WT and mutant alleles of CARD9 using cDNA from PBMCs. PCR was performed using exon-8-F and exon-13-R primers. C The PCR product from II.2 (in B) was cloned into a TA vector and sequenced 24 single colonies. Two transcript variants are detected: exon 11 deletion (c.1358–1434; Δ ex.11) and 18 nt deletion (c.1417–1434; Δ 18 nt). D Scatter plot showing quantitative real time PCR of CARD9 mRNA expression from PBMCs. The CARD9 TaqMan probe specific to exon 11 was used to discriminate the WT and mutant allele, and the expression was normalized to GAPDH as a housekeeping gene. Data represent the mean ± SD of three normal controls and the indicated individuals. E CARD9 protein expression from PBMCs from three different normal controls and indicated CARD9 mutation carriers. Immunoblotting with the CARD9 C-terminal antibody (Immunogen 467–480 amino acid) and N-terminal antibody (Immunogen 98–187 amino acid) are shown. CARD9 expression was not detected in a known CARD9 deficiency patient (CARD9 null) with compound heterozygous mutations (Q289*/D8Afs*10). Data are representative of three replicates. F Schematic diagram of WT and CARD9 mutant proteins (CARD9 Δ ex.11 and CARD9 Δ 18 nt); CARD9 domains and exon 11-coded regions are color identified. The protein resulting from the out-of-frame full exon 11 deletion (Mutant Δ ex.11) generates a missense C-terminal region, depicted by diagonal lines. The protein resulting from the in-frame exon 11 18 nucleotide deletion (Mutant Δ 18 nt) is identified by a smaller ex.11. Binding sites for CARD9 N- and C-terminal-directed antibodies used in (E) are also shown