Detrimental NFKB1 missense variants affecting the Rel-homology domain of p105/p50

Most of the currently known heterozygous pathogenic NFKB1 (Nuclear factor kappa B subunit 1) variants comprise deleterious defects such as severe truncations, internal deletions, and frameshift variants. Collectively, these represent the most frequent monogenic cause of common variable immunodeficiency (CVID) identified so far. NFKB1 encodes the transcription factor precursor p105 which undergoes limited proteasomal processing of its C-terminal half to generate the mature NF-κB subunit p50. Whereas p105/p50 haploinsufficiency due to devastating genetic damages and protein loss is a well-known disease mechanism, the pathogenic significance of numerous NFKB1 missense variants still remains uncertain and/or unexplored, due to the unavailability of accurate test procedures to confirm causality. In this study we functionally characterized 47 distinct missense variants residing within the N-terminal domains, thus affecting both proteins, the p105 precursor and the processed p50. Following transient overexpression of EGFP-fused mutant p105 and p50 in HEK293T cells, we used fluorescence microscopy, Western blotting, electrophoretic mobility shift assays (EMSA), and reporter assays to analyze their effects on subcellular localization, protein stability and precursor processing, DNA binding, and on the RelA-dependent target promoter activation, respectively. We found nine missense variants to cause harmful damage with intensified protein decay, while two variants left protein stability unaffected but caused a loss of the DNA-binding activity. Seven of the analyzed single amino acid changes caused ambiguous protein defects and four variants were associated with only minor adverse effects. For 25 variants, test results were indistinguishable from those of the wildtype controls, hence, their pathogenic impact remained elusive. In summary, we show that pathogenic missense variants affecting the Rel-homology domain may cause protein-decaying defects, thus resembling the disease-mechanisms of p105/p50 haploinsufficiency or may cause DNA-binding deficiency. However, rare variants (with a population frequency of less than 0.01%) with minor abnormalities or with neutral tests should still be considered as potentially pathogenic, until suitable tests have approved them being benign.

is depicted, with reporter activity (red) and p50 expression (green). RelA amounts were: 5ng (top rows each), 6ng (middle rows) or 7.5ng (bottom rows). Mean intensities were calculated as an x-fold of background values (with the non-transfected samples set as 1) and are indicated by bar graphs, separately for each experiment. Average fold values of reporter activities and p105 expression are given in Supplementary Table 2. Please note the ductile effect of some variants on the reporter activity with increasing RelA.  Figure 5B is depicted, with reporter activity (red) and p50 expression (green). RelA amount: 5ng each. Mean intensities were calculated as an x-fold of background values (with the non-transfected samples set as 1) and are indicated by bar graphs, separately for each experiment. Average fold values of reporter activities and p50 expression are given in Supplementary Table 2. R57C/1. This patient was born in 2000 and presented with recurrent bolus events at the age of 12 years, when he tried to swallow pieces of meat. An esophagogastroduodenoscopy revealed eosinophilic esophagitis with quite severe strictures. In the past history the mother reported her son was prone to infections of the upper and lower respiratory tract and suffered from severe food allergic reactions. He had been instructed on how to use an epinephrine-pen after a life-threatening systemic reaction related to seafood. Unfortunately, at that time no further immunological investigations had been done. His eosinophilic esophagitis was treated with topical budesonide and strictures expanded by repeated transesophageal bougienage.

Supplementary
Laboratory tests in the primary immunodeficiency clinic showed blood eosinophilia in the absence of elevated or specific reactive IgE in serum, severe hypogammaglobulinemia (IgG 3.7 g/l, IgA <0.5 g/l, IgM <0.25 g/l, and IgE 26 IU/l) expending over all IgG subclasses (IgG1 2.4 g/l, IgG2 0.4 g/l, IgG3 0.2 g/l, and IgG4 0.04 g/l) and complete absence of any specific immune responses to standard vaccinations, still six weeks after re-challenging. Lymphocyte phenotyping was compatible with CVID, EURO Class B+smB+TrnormCD21norm. No evidence of protein loss in urine or stool sampling was found. Genetic panel-testing of known PID genes revealed a new mutation in NFKB1.
Initiation of IgG replacement therapy was challenging, due to moderate systemic allergic reactions to different immunoglobulin products even when adequate premedication (glucocorticoids and antihistamines) was used. Fortunately, after the invention of subcutaneous IgG substitution all allergic reactions subsided completely and no infections occurred ever since. Problems with swallowing and histologic eosinophilic esophagitis recurred after termination of budesonide therapy and seemed not to be beneficially influenced by IgG replacement. Therefore, the patient is on long-term topical budesonide treatment and otherwise in good general condition.
c.191G>T; exon 5; p.Gly64Val G64V/2. This female patient was born to non-consanguineous parents in 1976, and had suffered since childhood from recurrent upper and lower respiratory tract infections often needing antibiotics, and had been diagnosed with allergic asthma. At 34 years, she was admitted with a severe pneumonia, and further diagnostic testing was done. Her blood chemistry showed low IgG (5.6 g/L), low IgA (0.6 g/L) and normal IgM (0.96 g/L) levels. The overall antibody response to vaccination with Pneumovax® was adequate, however antibody responses against separate pneumococcal serotypes were not assessed. After further immunological evaluation, she was diagnosed with CVID EuroClass SmB+CD21norm and immunoglobulin replacement therapy was started. Since then, the frequency and severity of her infections has markedly decreased, with only a limited need for antibiotics. Thus far, she has not developed bronchiectasis or chronic lung disease. However, she suffers from severe fatigue and neuro-psychiatric symptoms. At the age of 39, she had bilateral optic neuritis/papillitis causing loss of vision and eye pain. She was treated with corticosteroids with good response, although her vision was only partially restored. Extensive neurological workup remained negative; multiple sclerosis was excluded. Corticosteroids could be successfully weaned, and no other immunosuppressive drugs were started. In the next years, she presented with several episodes of weakness and paresthesia in the upper or lower limbs, each time with normal MRI findings and spontaneous recovery. These episodes were considered to be triggered by severe psychological stress.
At the age of 40, genetic testing revealed the heterozygous mutation in NFKB1, regarded as likely pathogenic by all in silico prediction tools. The same NFKB1 mutation was also detected in her mother as well as in one of her sons, confirming an autosomal dominant inheritance across three generations.
G64V/1. This female patient was born in 1947 and is the mother of patient G64V/2. She had breast cancer at the age of 64, which is currently in remission and she is in good general condition. The NFKB1 mutation was detected at age 68. She was found to have very low IgG levels (3.7 g/dL), normal IgA (2.2 g/L) and IgM (1.0 g/L) levels, and inadequate antibody response to vaccination with Pneumovax® (serotype analysis

R157P/3. This female patient was born in 2006
and is the first of two children of patient R157P/1. Both parents were unrelated. The pregnancy was uneventful, apart from Caesarean section due to HELLP-syndrome of the mother. Since 2 years of age, she has been suffering from relapsing abdominal pain and diarrhea (5-7 stools per day). Gastroenterologic diagnostic was not able to detect a cause for the symptoms and she had no failure to thrive. Treatment with loperamid was commenced with improvement of the symptoms. At 2 years of age, an ophthalmological examination showed reduced marginal sharpness of the optic nerve on both sides. MRI-scan of the head and lumbar puncture including measurement of the intracranial pressure revealed no abnormalities. Except for recurrent sinusitis no invasive infections or other signs of immune dysregulation appeared. Immunological evaluation found normal immunoglobulin levels for IgG (inclusive IgG-subclasses), IgA and IgM, normal T-and B-cellimmunity and measurable vaccination levels (diphtheria, tetanus, haemophilus, pneumococcus). Molecular genetic examination revealed the same variant in NFKB1 as in the girl's father, uncle and the mother of the father.

c.586C>G; exon 8; p.Leu196Val
L196V/1. This male patient was born in 1998. He was born to non-consanguineous parents. At birth, he received supplementary oxygen due to hypoglycemia, neonatal sepsis and hemolytic anemia (Coombs positive). During infancy he developed spasmodic cough, workup for cystic fibrosis was negative. At 1 year he presented failure to thrive. He was hospitalized at 3 years of age with severe neutropenia, suspected Evans syndrome, and cellulitis with Pseudomonas aeruginosa. Schwachman-Diamond syndrome investigations were negative. Laboratory tests showed neutropenia, hypogammaglobulinemia and low IgG1 levels. Anemia and thrombocytopenia were intermittent, antiplatelet-antibodies and positive Coombs were detected in each episode. He was treated with intravenous immunoglobulin, red cell concentrates transfusion, G-CSF plus antibiotics. CVID was diagnosed due to hypogammaglobulinemia (IgG 2.84g/l, IgM 0.38 g/l, IgA not detectable), T cell lymphopenia and no response to tetanus or pneumococcal vaccines. Immunoglobulin replacement therapy was started. Panel sequencing was negative for X-linked syndromes, type 2 and 3 Hyper IgM Syndromes. Bone marrow aspirate showed richly cellular marrow with a marked left shift. There were no morphologic abnormalities. Multilineage hematopoiesis was normal for age. He had a microcytic, hypochromic anemia with monocytosis, but no evidence of leukemia. He continued to have leukopenia and thrombocytopenia throughout his childhood. His bone age was determined to be 5 years when he was only 3 years and 7 months old (about 2 standard deviations above the chronological age). He received live viral vaccines, including VVZ. At 4 years, old he developed a vesicular lesion on the right small finger which progressively tracked up the right arm in a dermatomal fashion. He was hospitalized with a vaccine strain herpes zoster with no neurologic symptoms. Lumbar puncture showed pleocytosis but was negative for VZV. He had frequent infections including pneumonia, chronic sinusitis, epididymitis, but were later well controlled since switching to subcutaneous immunoglobulin. He was diagnosed with growth hormone deficiency and supplemented with it until 14 years of age. He developed retinopathy due to an unspecified cone dystrophy and is legally blind. At 19 years, he presented with a severe autoimmune hemolytic anemia, and was treated with prednisone and solumedrol. He later relapsed, and was treated with rituximab. WES revealed three variants of unknown significance: in NFKB1 (c.586C>G; L196V, heterozygous), in RIMS1 (c.3182C>A; W1061X, heterozygous) and in TNIK (c.1220A>T; E407V, heterozygous). At 20 years, he was hospitalized with fever, severe neutropenia, recurrent painful genital and oral ulcers. He received acyclovir. Blood CMV, EBV, HHV6, an HSV PCR were all negative. CRP was elevated. Bone marrow biopsy due to severe neutropenia was performed showing megakaryocytic hyperplasia with dysplastic changes noted among the maturing megakaryocytes, maturing myeloid lineage cells demonstrating relative maturation arrest with extremely rare maturing granulocytes. Neupogen was given as treatment. At age 21, he was re-evaluated due to worsening T cell lymphopenia and autoimmune hemolytic anemia. A PID panel showed a heterozygous pathogenic variant in SPINK5 (c.2671C>T; R891X), and a heterozygous VUS in CARD9 (c.923_925del; L308del) and another heterozygous VUS in CHD7 (c.1496_1497delinsGC; Q499R). He developed painful recurrent herpetic lesions of feet treated with antivirals. During the pandemic he had no COVID infection, and has received both Regeneron and Evusheld. He continues to be monitored closely for autoimmune hemolytic anemia, recurrent viral infections, and is maintained on subcutaneous immunoglobulin replacement and prophylactic antibiotics s/p rituximab with continued B and T cell lymphopenia.
c.592C>T; exon 8; p.Arg198Cys (protein defect unclear) R198C/1. This male patient was born in 1982 to non-consanguineous parents without a previous family history of immune system disorders. Vaccination history and childhood development were unremarkable. Disease onset was at age 7 with a recurrent and chronic cough. Low body temperature and feeling cold even in the warm seasons have been documented since the age of 10. The patient also had a history of recurrent upper respiratory tract infections. He had herpes zoster at age 25. At age 28, he was hospitalized because of kidney stones. At the time, the patient had submandibular and axillary lymphadenopathy, associated with enteropathy and weight loss but no hepatosplenomegaly. Endocrine, rheumatology, and hematology work-up were normal without any evidence of malignancy. Immunological analyses revealed decreased immunoglobulin levels (IgG 3.0 g/l, IgA 0.1 g/l, IgM 0.4 g/l), absence of specific antibody response (anti-tetanus <0.01 IU/ml and antidiphtheria <0.01 IU/ml), but normal B cell counts (CD3+ 66%, CD19+ 12%), indicating the clinical diagnosis of CVID. The B cell subset analysis of this patient was compatible with CVID Freiburg Class Ia, EURO B+ smB+ CD21 low Tr norm . After intravenous immunoglobulin replacement, lymphadenopathies improved and he had a good infection control, however, the gastrointestinal manifestation did not respond to immunoglobulin treatment or other immunomodulators including corticosteroid, azathioprine, sirolimus and mesalamine.
c.641G>A; exon 8; p.Arg214Gln (protein defect unclear) R214Q/1. This male patient was born in 1992 to a non-consanguineous couple. He has two healthy siblings. Shortly after birth he developed bronchitis and an immunodeficiency was suspected. However, he did not have any increased susceptibility to infection until he became 17. Since that age, he started having recurrent bronchitis 3 to 4 times per year, which required antibiotic therapy. He did not have other major infections nor lymphadenopathy or hepatosplenomegaly. When he was 20, he had an abscess of 1.5 cm in the right gluteus, which was successfully treated with ammonium bituminosulfonate. At age 21 he started having intermittent diarrhea. At age 22 he was immunologically evaluated. Immunoglobulin levels showed decreased immunoglobulin levels (IgG 3.9 g/l, IgA 0.22 g/l, IgM 0.23 g/l), and subclasses IgG1 and IgG2 were reduced. Response to tetanus and diphtheria vaccination was normal, whereas his pneumococcal polysaccharide response was non-protective. Lymphocyte phenotyping revealed decreased transitional and class-switched memory B cells, absent plasmablasts and increased CD21low B cells. The patient was therefore diagnosed with CVID, EURO Class B + smB + CD21 low Tr norm . Since then, he has received antibody replacement therapy. Currently he has a good exercise tolerance and good infection control, his only problems being low grade esophageal reflux, and intermittent diarrheal episodes without weight loss. The patient also reports skin folliculitis. M216V/2. This male patient was born in 1956 to a non-consanguineous couple and is the father of patient M216V/1. He suffered from bronchial asthma, coronary heart disease and compression of nerve roots due to intervertebral disc degeneration but lacked a history of susceptibility to infections or autoimmune phenomena. At age 57, he had an episode of fatigue and joint pain with elevated inflammatory markers. At age 60, the same NFKB1 variant as in his son was identified, which triggered an immunological evaluation. Immunoglobulin levels showed decreased IgG and IgM (IgG 6.17 g/l, IgA 1.70 g/l, IgM 0.24 g/l), with a selective IgG2 reduction (0.90 g/L). Tetanus antibodies were protective, but diphtheria vaccination response was nonprotective. Lymphocyte phenotyping revealed an expansion of CD4+ T cells and a light increase in CD21low B cells. Currently, he is in a good general condition without IgG replacement. were found to be decreased (IgG 2.6 g/l, IgA 0.28 g/l, IgM 1.16 g/l). After further immunological evaluation, he was diagnosed with CVID Freiburg Class Ia, EURO Class B+ smB-Tr high and immunoglobulin replacement therapy was started. At age 40 splenomegaly was observed and at age 43 he presented with lymphoproliferation of cervical lymphnodes from unknown significance. In the context of his immunodeficiency, the patient also developed autoimmune sprue-like enteropathy, initially treated with cyclosporin and later with tacrolimus after cyclosporine-induced leukopenia. When 53, bronchiectasis was observed on his thorax CT scan. A sinobronchial syndrome was suspected and at age 54 he underwent sinus surgery with septoplasty. At the same age osteoporosis was diagnosed and two years later he had a femur fracture. He had idiopathic immune thrombocytopenia and recurrent autoimmune hemolytic anemia, treated with several cycles of rituximab since age 56. The patient developed liver cirrhosis and portal hypertension. When 62-years old he required hospitalization because of progressive ascites, an umbilical hernia and grade III esophageal varices, treated several times with esophageal banding. Nodular regenerative hyperplasia was suspected but not confirmed by histology. In addition, he had 12 kg of weight loss, associated with 6-month chronic diarrhea, which improved after change of tacrolimus to mycophenolate. During the hospitalization he had another episode of autoimmune hemolytic anemia. Serology revealed a past hepatitis B infection. Seven months later he had an incarceration of his umbilical hernia with subsequent necrosis of the intestinal mucosa and a loop ileostomy and enteral nutrition were required. He underwent transjugular intrahepatic portosystemic shunt placement. The patient also suffered from chronic kidney disease and corticosteroid-induced diabetes at age 62. He died at age 63 following a catheter sepsis. c.1156G>A; exon 12; p.Gly386Arg (protein defect unclear) G386R/1. This female Caucasian patient was born in 2009, with no family history of consanguinity. Her father has IgA-deficiency, vitiligo, pernicious anemia, brachial neuritis, recurrent pneumonia, bronchiectasis and gastric metaplasia; her mother has Crohn's disease; and her 12 year-old brother had recurrent infectious bronchitis up to 5 years of age and hypogammaglobulinemia. From the age of 3 years she had recurrent supurative otitis with persistent purulent rhinorrhea and 2 episodes of tonsillitis. At 6 years, adenoidectomy was performed with amygdalar reduction and placement of bilateral transtympanic tubes. Six months later, she had lost the right ear tube, restarting recurrent otitis media. At age 7, during a work-up for tube replacement, thrombopenia was detected. She developed abdominal pain accompanied by vaginal bleeding, frequent epistaxis and hematomas as well as habitual asthenia, which was aggravated with sports. A hematological study was performed, with a diagnosis of autoimmune thrombocytopenia (platelets between 18,000 and 100,000/mm 3 ). A decrease in the levels of IgG and IgA was detected and she was referred for immunological studies. Isoagglutinins were negative. An initial response to tetanus and anti-pneumococcal 23 vaccination was lost in less than a year. She was subsequently diagnosed with CVID. At 8 years and 11 months immunoglobulin replacement therapy was started, with clinical improvement and increase in the platelet count to 98,000/mm 3 . She developed intermittent inguinal lymphadenopathy, usually increasing in size the week before intravenous immunoglobulin. Due to the persistence of the adenopathies, a lymph node biopsy was performed at 9 years of age with a final diagnosis of reactive lymphoid hyperplasia. At age 10, she started having episodes of dysarthria and paresthesia episodes. At 12 she was diagnosed with migraine with aura and began prophylactic amitriptyline. Immunological studies of B cell subpopulations between age 8 to 12 revealed a progressive decrease in the percentage of switched memory cells and an increase in transitional B cells and an expansion of CD21 low B cells. At 10 years, splenomegaly with an accessory spleen was detected. At 11 years, the frequency of respiratory infections and suppurative otitis increased and she had lymphadenopathies in other locations (axillary, retroperitoneal and paraaortic). Splenectomy was performed due to severe hypersplenism and doubt of an underlying lymphoma, leaving the accessory spleen. Histology revealed a reactive lymphoid follicular hyperplasia with expansion of CD4 + PD1 + T cells. Two months later she was admitted for febrile gastroenteritis with PCR in stool positive for Salmonella, Giardia and Escherichia coli, she was treated with good recovery effect. Following diagnosis of the NFKB1 variant c.1156G>A (p.Gly386Arg), rapamycin treatment was started. Five months later, MRI showed a decrease in the chronic sinupathy with a stability in the size of the lymphadenopathies. A family study confirmed both, father and brother, carrying the same mutation.