ALBERT

Description: Key Points Germline activating STAT3 mutations were detected in 3 patients with autoimmunity, hypogammaglobulinemia, and mycobacterial disease. T-cell lymphoproliferation, deficiency of regulatory and helper 17 T cells, natural killer cells, dendritic cells, and eosinophils were common.

Description: The NFkB signaling pathway is a master regulator of the immune response. Recently, dominant mutations in NFKB2 have shown to cause antibody deficiency with adrenal insufficiency. We investigated two families with antibody deficiency and autoinflammatory features, with onset in teenage years and early adulthood (Fig. 1 and Table 1). In family 1, all patients available to study presented with respiratory tract infections and B cell dysfunction marked by hypogammaglobulinemia, poor antibody response to vaccines, or low switched memory B cell counts. Several experienced recurrent episodes of aphthous mucositis in upper gastrointestinal and genital area, sometimes accompanied by abdominal pain, monoarthritis, or fever with elevated inflammatory markers (peripheral blood leukocytes 〉10x106 cells/ml, C reactive protein 〉100 mg/L). A lesional biopsy revealed lymphocytic small cell vasculitis reminiscent of Behcet's disease. In family 2, all patients presented with respiratory tract infections, hypogammaglobulinemia, and poor vaccine responses. Based on the pedigrees, we assumed autosomal dominant inheritance. For family 1, we performed genotyping and linkage analysis coupled with whole genome sequencing to pinpoint the causative variant. Linkage analysis showed a 15.4 Mb haplotype on chr4 segregating with the phenotype in all affected individuals. When focusing our search on novel, heterozygous variants negatively affecting conserved residues, we identified only one variant in this genomic location, localizing to NFKB1 gene (p.H67R). In family 2, we exome sequenced one affected individual and filtered the data for novel heterozygous variants negatively affecting conserved residues. This resulted in 17 variants, out of which the I553M NFKB1 variant was considered the best candidate. Several functional analyses showed that the mutations had a negative impact on NFKB1 function. The H67R variant increased NFKB1 affinity to NEMO and delayed protein entry to nucleus, when assessed by affinity purification mass spectrometry and immunofluorescence microscopy. The mutation also decreased NFKB1 transcriptional activity, when expressed together with an NFkB-responsive luciferase reporter, and showed a neomorphic DNA binding pattern on chromatin immunoprecipitation-sequencing. Furthermore, the cell growth was hampered when the mutant NFKB1 was overexpressed in cultured HEK293 cells. The I553M showed a similar negative effect on cultured cell proliferation. Furthermore, the I553M variant negatively affected serine S907 and S893 phosphorylation, leading to defective posttranslational processing of the full-length NFKB1 upon TNF stimulation. To conclude, we show that hypogammaglobulinemia and Behcet's disease-like autoinflammatory syndrome can be caused by mutations in NFKB1. This suggests that a subset of Behcet's disease cases may be of monogenic origin, and highlights the role of NFkB complex in B cell maturation and innate immune regulation. Table 1. Patient characteristics Patient 1 2 3 4 5 6 7 8 9 10 11 12 Age 55 43# 25 29 40 30 37 7 10 60# 61 32 Sex F M F F F F M F M M F M Mutation status H67R ND H67R H67R H67R H67R H67R H67R H67R ND I553M I553M Immunodeficiency Infections URTI RTI URTI - URTI URTI URTI URTI - RTI RTI RTI Antibody deficiency Hypogammaglobulinemia, SAD ND Hypogammaglobulinemia, SAD - IgG subclass deficiency, SAD Hypogammaglobulinemia, SAD Hypogammaglobulinemia, SAD Hypogammaglobulinemia Hypogammaglobulinemia Hypogammaglobulinemia Hypogammaglobulinemia, SAD SAD Immunoglobulin replacement + - + - - + + - - - + - Immune dysregulation Febrile attacks - + + + - - + + - - - - Complex aphtae Mouth, genitalia ND Mouth, genitalia Mouth, genitalia - Mouth Esophagus Mouth, esophagus - - - - Arthritis Monoarthritis - Monoarthritis Monoarthritis - - - - - - Oligoarthritis - Gut disease Periodic abdominal pain, chronic idiopathic diarrhea ND Periodic abdominal pain, microscopic colitis Periodic abdominal pain - - - Periodic abdominal pain - ND Chronic idiopathic diarrhea Coeliac disease Other - Hyperinflammatory response and death after gallbladder surgery - Iritis, Hyperinflammatory response to tooth excision Kidney tumor - Rudimentary left kidney - - - Astma, hypothyreosis - URTI, upper respiratory tract infections; RTI, respiratory tract infections including recurrent pneumonia; SAD, Specific antibody deficiency; ND, No data; #Death age Figure 1. Family pedigrees. Figure 1. Family pedigrees. Disclosures Mustjoki: Sigrid Juselius Foundation: Research Funding; Signe and Ane Gyllenberg Foundation: Research Funding; the Finnish Cancer Societies: Research Funding; Novartis: Honoraria, Research Funding; Academy of Finland: Research Funding; Finnish Cancer Institute: Research Funding; Pfizer: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding. Saarela:Roche: Honoraria.