Novel heterozygous NFKB1 mutation—variable penetrance in a family cohort

All patient data and samples were obtained in accordance with the Declaration of Helsinki and the Research Ethics Board at The Hospital for Sick Children. Patient data was compiled prospectively and retrospectively from medical records and entered into the Primary Immunodeficiency Registry and Tissue Bank (REB protocol No. 1000005598). Informed consent was obtained for genetic testing, including WES, as well as extensive investigations to unravel the functional effect of immune abnormalities.

Genomic DNA was isolated and extracted from peripheral blood lymphocytes (PBL) using the Geneaid genomic DNA extraction kit (Geneaid Mini Kit, ON, Canada), in accordance with the manufacturer’s instructions.

DNA was quantified using the Qubit DNA HS Assay (Life Technologies, Carlsbad, CA, USA) and 100 ng of input DNA subsequently used for library preparation (Ion AmpliSeq Exome Kit; Life Technologies). The exome library was immobilized on Ion PI™ Ion Sphere particles (Ion PI Template OT2 200 Kit v3; Life Technologies). Sequencing was performed utilizing the Ion PI™ Sequencing 200 Kit v3 and Ion PI™ Chip v2 in the Ion Proton semiconductor sequencing system, according to the manufacturer’s recommendations (Life Technologies).

Alignment and variant calling were performed with Torrent Suite (v4.0) on the Ion Proton Server using the Ion Proton AmpliSeq germline low-stringency setting and the hg19 reference genome. The variants were annotated using an in-house annotation pipeline 35 (based on Annovar; November 2014 version), 36 and RefSeq gene models (downloaded from UCSC on 1 August 2015).

B cell phenotypes were determined by flow cytometric analysis using the 4-laser BD analyzer LSR II CFI, BGRV (BD Biosciences, San Jose, CA, USA). Antibodies used included the following: CD19-BV711, CD27-BV421, IgD-PE-Cy7, CD24-BB515, CD38-allophycocyanin, CD21-PE-CF594, CD45RO-PE, CD197-BV421, and CD196-PE-Cy7 (BD Biosciences, San Jose, CA, USA).

Lymphocyte proliferative responses to mitogens, including PHA and the combination of anti-CD3 and anti-CD28 antibodies, and to a panel of recall antigens, including Candida species, tetanus toxoid, herpes zoster, and cytomegalovirus, were determined by H-thymidine incorporation (Arpaia et al. 1994). All assays were performed in triplicate and compared with simultaneously stimulated cells from healthy control subjects.

PBL was collected from patients and healthy controls, and T lymphocytes selectively cultured (CD3/CD28 Human T activator Dynabeads; Gibco, Life Technologies, Carlsbad, CA, USA) in the presence of IL-2. Cells were lysed on ice using lysis buffer (1% Triton X-Vanadate) and loaded onto 12% polyacrylamide gel for size fractionation (NuPage 4%–12% Bis-Tris Gel, Invitrogen, Rockford, IL, USA). Proteins were transferred onto polyvinylidene fluoride membrane in accordance with standard Western blotting protocols. p105 and p50 proteins were detected using rabbit anti-NF-κB1 p105/p50 antibody (#3035 Cell Signaling, Danvers, MA, USA; 1:1000 dilution). Anti-rabbit secondary antibody coupled to horseradish peroxidase (7074S, Cell Signaling; Danvers, MA, USA; 1:5000 dilution) was used for signal detection. Immunoreactive bands were visualized with ECL Western Blotting Substrate (ThermoScientific, Life Technologies, Carlsbad, CA, USA).

Serum concentrations of immunoglobulins (IgG, IgA, and IgM) were measured using nephelometry, and IgE concentrations were measured by using radioimmunoassay with the IgE PRIST kit (Pharmacia Diagnostics, Dorval, QC, Canada). Levels of serum tetanus toxoid and pneumococcus (pneumococcal capsular polysaccharide) were measured by means of ELISA, according to the manufacturer’s instructions (Binding Site, Birmingham, UK). Serum antibodies to measles, mumps, and rubella were measured by ELISA with kits available from Euroimmune (Gross-Groenau, Germany). Serum isohemagglutinin titers were determined by means of 2-fold serial dilution with erythrocytes and are reported as the antiglobulin phase, the dilution at which macroscopic agglutination is observed.

Patient 1 was a product of non-consanguineous parents of Irish and French descent. She presented at young adulthood, age 23 years, with recurrent prolonged upper respiratory tract infections (10/year), chest X-ray-proven pneumonias (5/year), chronic sinusitis, and recurrent otitis with purulent otorrhea, fatigue, and weight loss. Her childhood clinical history was unremarkable, with uneventful chickenpox, measles, and mumps infections. She had 1 episode of arthritis at age 2 years and normal echocardiography on examination. A tonsillectomy was performed at 5 years of age.

During her initial evaluation with our Immunology team at age 26 years, she presented with cylindrical bronchiectasis on bronchoscopy of the left lower lobe and the lingula. She had normal tuberculin test results and sweat chloride levels (7 mEq/L). Immunological evaluation (Table 1) showed pan-hypogammaglobulinemia, with low IgG, IgM, and IgA levels, as well as low anti-tetanus toxoid titer (0.03 ng/mL). IgE levels were normal, as were protein and albumin levels. Her CBC and differential was unremarkable. Lymphocyte counts were normal, however, B cell counts were almost absent with only 46 cells/μL of CD19+ cells alongside decreased NK cell numbers of 53 cells/μL. Assay for spontaneous suppressive cell activity assay was positive, and E-rosette and AET rosette assessment of T lymphocytes were normal at 58% and 70%, respectively. Results of mixed lymphocyte reaction were also normal. Lymphocyte proliferation responses to all mitogens except STA were normal, however, antigen stimulation responses to Candida and Tuberculin PPD were decreased.

Due to her clinical presentation and diminished immune responses, she was started on fresh frozen plasma (FFP), the only IgG replacement option that was available at the time she was diagnosed. She was eventually transferred to intramuscular and then intravenous immunoglobulin therapy. Laboratory assessments showed adequate IgG replacement. Unfortunately, the patient contracted blood product-related Hepatitis C infection resulting in liver cirrhosis, portal hypertension, massive ascites and varicose veins requiring multiply ligation and sclerotherapy due to bleeding, and eventually liver failure. Other immunodeficiency related clinical manifestations include chronic cough, yellow sputum, Mycoplasma colonization, chronic Salmonella gastroenteritis which resolved after 6 months of antibiotic therapy, chronic non-infectious diarrhea, and hepatosplenomegaly.

At the age of 36 years, the patient developed ophthalmic melanoma resulting in loss of vision in the affected eye. She suffered severe bronchiectasis with pyocele 2 years later for which she underwent left lower lobe lobectomy. Concurrently, she slowly developed progressing cerebellar symptoms with abnormal Romberg and heel/toe gait, however, CT imaging of the head was unremarkable.

Following a prolonged course of weight loss, intermittent fevers, and 1 episode of septicaemia, worsening productive cough, failed empiric antibiotic therapy, and increased fatigue, the patient was subsequently evaluated for malignancy. She was diagnosed with lymphoma and later died at age 45 years.

Patient 2, the son of patient 1, was evaluated at 1 year of age and found to have low IgG, IgM, and IgA levels (Table 1). At the age of 10 years he developed chronic immune thrombocytopenia and has been on regular monthly intravenous IgG replacement therapy since age 11 years. He was diagnosed with persistent abdominal lymphadenomegaly, and percutaneous needle aspiration biopsy showed follicular hyperplasia. His CBC and differential were unremarkable, with normal numbers of circulating CD3+, CD4+, CD8+, CD19+ and NK cells. Stimulation of T cells with anti-CD3 and anti-CD28 was markedly reduced, implying a T cell defect. In vitro lymphocyte responses to a series of antigens, including Candida species, tetanus toxoid, zoster, and cytomegalovirus, were also extremely low indicating the antigen specific memory T cells were either lacking or present at low frequencies (Figure 2). Assessment of B cell phenotype by flow cytometry showed increased number of IgD+ memory cells, but diminished class switching (CD38+/CD24+) (Figure 3). No transitional B cells and elevated number in CD21^low B cells were detected.

Patient 3, daughter of patient 2, is a 10 year old with a history of 3 episodes of chickenpox at ages 3, 5, and 7 years. Given the clinical history of her grandmother and father, live viral vaccines were avoided. Her CBC and differential were unremarkable, with normal B and T cell lymphocyte function. She had normal IgG levels with adequate specific antibody titers, but decreased levels of IgA and IgM. Her varicella zoster virus specific IgG antibody titers were negative. Despite 3 episodes of chickenpox, she was unable to mount an appropriate immune response.

Patient 4, daughter of patient 2, is a 7 year old female. She too suffered 3 episodes of chickenpox. Her immunological evaluations to date have been unremarkable. Like her sister, her varicella zoster virus specific antibody titers were negative.

Patient 5, the youngest sibling of patients 3 and 4, is a 3 year old female with eczema and repeated upper respiratory tract infection. While her T cell response to phytohemagglutinin was normal, the patient’s response to mitogen stimulation with anti-CD3 were decreased. She also had decreased IgA and IgM but normal IgG levels with adequate antibody responses to vaccination.

WES was performed on patient 2. We identified a novel heterozygous variant in the gene NFKB1 (NM_03998.3), c.1584dupG, resulting in p.M528fs (Figures 4 and 5). This was confirmed by Sanger sequencing of patients 2–5 and segregated with disease. The M528fs mutation in NFKB1 has not been previously reported in dbSNP, ClinVar, or HGMD. Due to unavailability of genetic material, presence of the NFKB1 variant in patient 1 could not be confirmed.

Western blot evaluation of samples obtained from our patients carrying the novel heterozygous NFKB1 mutation showed a marked decrease in p50 and p105 protein levels, indicating that this variant is a loss-of-function mutation (Figure 6).