Manfred Fliegauf, Vanessa L

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Haploinsufficiency of the NF-κB1 Subunit p50 in Common Variable Immunodeficiency Manfred Fliegauf, Vanessa L. Bryant, Natalie Frede, Charlotte Slade, See-Tarn Woon, Klaus Lehnert, Sandra Winzer, Alla Bulashevska, Thomas Scerri, Euphemia Leung, Anthony Jordan, Baerbel Keller, Esther de Vries, Hongzhi Cao, Fang Yang, Alejandro A. Schäffer, Klaus Warnatz, Peter Browett, Jo Douglass, Rohan V. Ameratunga, Jos W.M. van der Meer, Bodo Grimbacher The American Journal of Human Genetics Volume 97, Issue 3, Pages 389-403 (September 2015) DOI: 10.1016/j.ajhg.2015.07.008 Copyright © 2015 The American Society of Human Genetics Terms and Conditions

Figure 1 Autosomal-Dominant Inheritance of a Heterozygous NFKB1 Mutation in a Large CVID-Affected Family (A) The pedigree of FamNL152 was completed and revised after re-evaluation of clinical records and follow-up examinations. Circles, female; squares, male; filled symbols, affected individual with CVID; hatched symbols, affected individual with hypogammaglobulinemia; open symbol, healthy member with wild-type NFKB1; slash, deceased individual. (B) The mutation (c.730+4A>G) affects the intron 8 splice donor site, which perfectly aligns to the consensus splice site. The American Journal of Human Genetics 2015 97, 389-403DOI: (10.1016/j.ajhg.2015.07.008) Copyright © 2015 The American Society of Human Genetics Terms and Conditions

Figure 2 The Heterozygous Splice-Donor-Site Mutation in Intron 8 of NFKB1 Causes In-Frame Skipping of Exon 8 and Haploinsufficiency of the p50 Subunit (A) EBV-transformed B cell lines derived from a healthy control individual and affected individuals (n = 3) were analyzed by RT-PCR for correct NFKB1 mRNA splicing with primers located in exons 6 and 11. In addition to the expected band (649 bp) a shorter product is observed in affected individuals, which indicates the loss of either exon 7 (164 bp), 8 (159 bp), or 9 (105 bp). Representative results are shown. (B) Sequencing of the RT-PCR products shows normal splicing in the control individual (upper panels). The heterozygous mutation (c.730+4A>G) causes skipping of exon 8 and in-frame fusion of exon 7 to exon 9 (lower panels: left, forward; right, reverse), indicated by the double peaks at the exon borders. The mutant mRNA predicts the translation of a shortened NF-κB1 p105 (p105ΔEx8) with a 53-amino-acid internal deletion (p.Asp191_Lys244delinsGlu). (C) EBV B cell lines from affected persons (n = 3) harboring the heterozygous NFKB1 mutation (het), from unaffected (wt) family members (n = 4) and healthy control cells (n = 2) were stimulated with PMA plus ionomycin as indicated (−/+) and analyzed by western blot for NF-κB1 protein amounts with antibodies raised against the N terminus of both p150 and the processed form, p50. Representative results are shown. As depicted in the left panel, the altered protein p105ΔEx8 is only marginally preserved and processing to p50ΔEx8 is not detectable (expected size is indicated by the arrowhead). Overall amounts of both p105 and p50 are reduced to ∼50% in EBV B cells from affected individuals (het). As depicted in the right panel, activation and nuclear translocation of residual p50 (derived only from the non-mutant allele) in affected individuals and normal nuclear translocation of p50 in controls after PMA plus ionomycin stimulation for 45 min. Ponceau S staining was used to demonstrate equal loading. (D) PBMCs from affected family members (n = 2) and healthy control individuals (n = 4) were stimulated with PMA plus ionomycin as indicated, and cell lysates were analyzed for p105 and p50 amounts and for phosphorylation of p105 (Ser933, P-p105) by western blotting (representative results). In samples derived from affected individuals, both p105 and the altered p105ΔEx8 are detectable. Only the non-altered p105 is phosphorylated. Phosphorylation of p105ΔEx8 and processing to p50ΔEx8 is undetectable (arrowheads). Whether stimulated or not, p50 is sustained at reduced amounts in comparison to that in controls. β-actin was used as a loading control. The American Journal of Human Genetics 2015 97, 389-403DOI: (10.1016/j.ajhg.2015.07.008) Copyright © 2015 The American Society of Human Genetics Terms and Conditions

Figure 3 Rapid Elimination of Altered p50ΔEx8 and p105ΔEx8 in Transfected Cells (A) HEK293T cells were transiently transfected with the indicated constructs, and the amounts of the ectopic proteins were analyzed in crude lysates by western blot. The altered proteins gain only weak signals and processing of p105ΔEx8 to p50ΔEx8 is almost undetectable. The faint band at 50 kDa in all lanes is the endogeneous p50. Molecular weight markers are indicated on the right side. Ponceau S staining of the blotting membranes confirm equal loading. (B) Fluorescence imaging shows nuclear localization of GFP-p50 and cytoplasmic localization of GFP-p105. Only aberrant signals are detected with the mutant proteins. Green, GFP-fusion proteins as indicated; blue, nuclei. Scale bar represents 10 μm. The American Journal of Human Genetics 2015 97, 389-403DOI: (10.1016/j.ajhg.2015.07.008) Copyright © 2015 The American Society of Human Genetics Terms and Conditions

Figure 4 Haploinsufficiency of p50 Due to a Heterozygous Splice-Donor-Site Mutation in Intron 9 of NFKB1 (A) Autosomal-dominant inheritance of a heterozygous NFKB1 mutation in family Fam089. Shaded symbols, affected members; half-shaded symbol, healthy members harboring the mutation. (B) The mutation (c.835+2T>G) affects the splice-donor-site mutation in intron 9. (C) RT-PCR shows an additional shorter band in EBV-transformed B cells from the proband, indicating exon skipping. (D) cDNA sequencing demonstrates skipping of exon 9. In-frame fusion of exon 8 to exon 10 predicts a shortened NF-κB1 p105 (p105ΔEx9) with a 35-amino-acid internal deletion (p.Lys244_Asp279delinsAsn). (E) Western blotting shows reduced amounts of p105 and p50 in EBV B cell lines from affected individuals (n = 2) in comparison to those in unaffected family members (n = 2) and healthy control cells (n = 2). The predicted altered proteins p105ΔEx9 and p50ΔEx9 are undetectable (arrowheads), indicating rapid elimination or mRNA decay. Representative results are shown. The American Journal of Human Genetics 2015 97, 389-403DOI: (10.1016/j.ajhg.2015.07.008) Copyright © 2015 The American Society of Human Genetics Terms and Conditions

Figure 5 A Frameshift Mutation in NFKB1 Causes p50 Haploinsufficiency in a CVID-Affected Family from New Zealand (A) A heterozygous one-base-pair duplication (c.465dupA) in exon 7 was identified in the indicated family members. The three children in the third generation were not tested. Shaded symbols, affected members; half-shaded symbols, healthy members harboring the mutation. (B) The single-base-pair insertion predicts a shift of the reading frame and the premature termination of translation (p.Ala156Serfs∗12). (C) PBMCs from affected family members (n = 3) and healthy control individuals (n = 3) were stimulated with PMA plus ionomycin as indicated and analyzed by western blotting. Representative results are shown. In affected individuals, normal p105 can only be derived from the healthy allele. Thus, p105 amounts, phosphorylation of p105, and processing to p50 are severely reduced in comparison to controls. The mutation predicts the translation of a non-functional severely truncated protein (∼18 kDa in size), which is not detectable (arrowhead). The β-actin control confirms equal loading. The American Journal of Human Genetics 2015 97, 389-403DOI: (10.1016/j.ajhg.2015.07.008) Copyright © 2015 The American Society of Human Genetics Terms and Conditions