PMC4720548_01
Identification and functional characterization of a heterozygous de novo NFKBIA mutation. (A-B) Electropherogram and (C) familial segregation showing de novo heterozygous S36Y (c.107C>A) mutation (indicated in red) in the patient's gDNA from granulocytes (A) and dermal fibroblasts (B), and wild-type (WT) alleles in both parents. (D) Picture of the patient's face and teeth (E) Immunoblot analysis of phospho-Ser32 IkappaBalpha, IkappaBalpha degradation and NEMO from a healthy control and the patient s fibroblasts after stimulation with TNF-alpha (20 ng/ml) and IL-1beta (10 ng/ml). Numbers below the western blot indicate the ratio between GAPDH and IKBA in the patient compared with the control after stimulation, and analyzed by densitometry.
nihms746824f1b.jpg
PMC4720548_02_nihms746824f1b.jpg
F1
The mutation was confirmed by Sanger sequencing, on PCR fragment amplified from gDNA extracted from granulocytes and dermal fibroblasts (Figure 1). Familial segregation analysis (Figure 1) showed that both parents are wild-type for the substitution; the mutation thus appeared de novo in the patient (Figure 1). The mutation does not impair the phosphorylation of IkappaBalpha on Serine 32, but affect partially its degradation, leading to impaired NF-kappaB signaling (Figure 1).
PMC4720548_02
Identification and functional characterization of a heterozygous de novo NFKBIA mutation. (A-B) Electropherogram and (C) familial segregation showing de novo heterozygous S36Y (c.107C>A) mutation (indicated in red) in the patient's gDNA from granulocytes (A) and dermal fibroblasts (B), and wild-type (WT) alleles in both parents. (D) Picture of the patient's face and teeth (E) Immunoblot analysis of phospho-Ser32 IkappaBalpha, IkappaBalpha degradation and NEMO from a healthy control and the patient s fibroblasts after stimulation with TNF-alpha (20 ng/ml) and IL-1beta (10 ng/ml). Numbers below the western blot indicate the ratio between GAPDH and IKBA in the patient compared with the control after stimulation, and analyzed by densitometry.
nihms746824f1b.jpg
PMC4720548_02_nihms746824f1b.jpg
F1
The mutation was confirmed by Sanger sequencing, on PCR fragment amplified from gDNA extracted from granulocytes and dermal fibroblasts (Figure 1). Familial segregation analysis (Figure 1) showed that both parents are wild-type for the substitution; the mutation thus appeared de novo in the patient (Figure 1). The mutation does not impair the phosphorylation of IkappaBalpha on Serine 32, but affect partially its degradation, leading to impaired NF-kappaB signaling (Figure 1).