PMC8738076_01
Monoallelic NFKB1 missense mutation in a family with late-onset antibody deficiency. (A) Segregation of NFKB1variant was analyzed by sequencing genomic PCR product and revealed an autosomal-dominant inheritance in families with reduced clinical penetrance. The analysis excluded the mother of patients because of material lack. (B) Sanger sequencing of genomic PCR products results in the chromatogram of missense variant and wild type (WT) (C) Structure of NFkappaB protein showing the position of the identified mutation. (D) Immunoblotting was performed in PBMCs of subjects (S1, S2, and S3) and healthy control (HC), and the expression of p105/50 was evaluated. The expression of p105 was reduced for all the subjects compared to the control. However, the expression of p50 was reduced in S2. (E) PBMCs from HC and S1, S2 and S3 were stimulated with PMA; 50 ng/ml and ionomycin; 1 mug/ml and the expression of p105/50 evaluated. There were no significant changes in the p105/50 expression after stimulation in the subjects; however, p105 phosphorylation at serine 933 was detected in only the HC but not in the subject. Beta-actin was used as a cytoplasmic loading control.
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Both the two brothers (S2/3) were identified to carry the missense variant 691 C>T, p.R230C in NFKB1 located on the N-terminal of the RHD ( Figure 1C ). A reduced mRNA level and a drastically reduced protein level (approximately 92%) were observed for p105/50 ( Figures 2B ;  Supplementary Figure 1 ) in transfected HEK293 cells. More importantly, the expression of p50 using the EGFP-fused p50-R230C mutant in HEK293 showed that p50 expression in the mutant was reduced compared to the wild type ( Figure 1C ). Representative results are shown ( Figures 1D, E ). p105 amounts, phosphorylation of p105, and processing to p50 are severely reduced compared to control.
NFkB1 expression in and nuclear translocation in transfected HEK293 cells. Total proteins were extracted from transfected HEK293 cells to determine the expression of p105/50. (A) For microscopic analysis of NFkB1-p50 translocation into the nucleus, HEK293 cells transiently expressing the eGFP-p50 wild type and mutant were used. Nuclei were stained with DAPI. The wild-type p50-construct localizes into the nucleus while the mutant p50-construct was retained in the cytoplasm. (B) Western blot to analyze the effect of the variant on the expression of p105/50 in the transfected HEK293 cells. The expression in the wild type was prominent in the wild type, while no noticeable expression in the variant was observed. (C) p50 expression in HEK293 showed an increase in wild-type and a decrease in the mutant (p. R230C) in the nuclear extract.
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A reduced mRNA level and a drastically reduced protein level (approximately 92%) were observed for p105/50 ( Figures 2B ;  Supplementary Figure 1 ) in transfected HEK293 cells. In transfected HEK293 cells, the EGFP-fused p50-R230C mutant showed an aberrant localization into the nucleus while the wild-type localization was normal ( Figure 2A ).