Introduction

The nuclear factor of kappa light chain gene enhancer in B-cells 1 (NF-κB1) plays a pivotal role in the regulation of immune responses and inflammation.^1,2 The NF-κB pathway regulates numerous cellular processes including proliferation, apoptosis, immune response, and inflammation. In the inactive state, NF-κB dimers are sequestrated in the cytoplasm and bound by a set of inhibitory proteins of the IκB family. Upon stimulation, these proteins undergo phosphorylation, ubiquitination, and proteasomal degradation. Subsequently, activated NF-κB complexes translocate to the nucleus where they bind to DNA at κB sites and elicit their effector functions by promoting or repressing the target gene expression.^2–5 The activation of NF-κB dimers occurs through either the classical (canonical) or the alternative (noncanonical) pathways. The classical pathway is initiated by cytokines, such as interleukin (IL)-1 and tumor necrosis factor alpha (TNFα), as well as bacterial products, e.g., lipopolysaccharide (LPS). The alternative pathway is stimulated by binding B-cell activating factor (BAFF, aka tumor necrosis factor ligand superfamily member 13B, TNFSF13B), lymphotoxin, or CD40 costimulatory molecule on antigen-presenting cells.^4,6

As a key regulator of inducible target gene expression, NF-κB plays an essential role in the development and function of the immune system. Lymphoid organogenesis and homeostasis of primary (bone marrow and thymus), as well as secondary lymphoid tissues (lymph nodes, Peyer patches, mucosa-associated lymphoid tissue (MALT), and the spleen) are reulated by NF-kB.^7,8 In the case of bacterial and viral infections, the canonical NF-κB signaling pathway is stimulated through the recognition of microbial pathogen-associated molecular patterns (PAMPs) by receptors, such as toll-like receptors (TLRs), that elicit subsequent stages of B and T lymphocyte activation, thereby linking the innate and adaptive immune responses.^9,10 The canonical NF-κB pathway, mediated by NF-κB1, has been a focus of interest because of its role in the pathogenesis of several inborn errors of immunity (IEI), such as caspase recruitment domain-containing protein 9 and 11 (CARD9 and CARD11, respectively), or NF-κB essential modulator (NEMO) deficiencies.^11,12 Monoallelic variants in the NFKB1 gene have been considered as the most common cause of antibody deficiency in the European cohort.13,14). Patients with identifiable variants in NFKB1 resulting in functional haploinsufficiency may develop common variable immunodeficiency (CVID). Also, a marked clinical individual heterogeneity and intrafamilial variability in genotype–phenotype correlations have been reported.^13–16 The leading clinical manifestations of the NF-κB1 deficiency are recurrent infections of the airways and gastrointestinal tract, with occasionally observable Epstein-Barr virus (EBV)-driven lymphoproliferative disease.^9,17 Since the expression of NF-κB1 is not confined to the immune system, and is an integral part of the homeostasis in the cellular skin components, inborn errors of NF-κB1 signaling may also be characterized by mucosal and cutaneous involvement.¹⁸

Herein, we report the case of a pediatric patient with an early onset antibody deficiency in whom the CVID diagnosis was established, according to current diagnostic guidelines. In this patient, the CVID diagnosis was supported by molecular genetic analysis revealing a novel heterozygous variant in the NFKB1 gene.¹⁹ In this report, we expand the clinical characteristics by adding viral meningitis and sepsis-like illness to the spectrum of the NF-κB1 deficiency. This clinical symptomatology is an unusual infectious complication, previously unreported in NF-κB1-deficient individuals.

Case Report

The patient is a 6-year-old girl who is the only child of a nonconsanguinous Caucasian couple. The family history is positive toward chronic diseases: maternal uncle and his son suffer from asthma, and galactosemia was diagnosed in two maternal cousins; furthermore, an infant had died the mother’s family with a syndromic disorder. Both parents occasionallysuffer from respiratory infections, but they do not present with autoimmune, allergic, or malignant diseases. During the pregnancy, the mother was treated for a genital herpes simplex (HSV) infection with acyclovir for 4 weeks before childbirth. The child was born at the 38th week gestational age, with an Apgar score of 9, and a birth weight 2980 g.

The child was first referred to the infectious diseases unit of our tertiary care university pediatric hospital at the age of 5 weeks because of fever of 39 degrees Celsius, apathy, and loss of appetite with a preliminary diagnosis of the neonatal sepsis. On admission, she presented with weakness, dehydration, pharyngitis, and stomatitis. However, there were no signs of abdominal organomegaly, lymphadenopathy, or meningismus during physical examination, and the lungs and heart were clear on auscultation. The infectious workup including blood cultures, tracheal aspirate, and blood real time-polymerase chain reaction (RT-PCR) viral and bacterial panels did not show any pathogens. In the cerebrospinal fluid, the parechovirus (HPeV)-RNA was detected thereby supporting the diagnosis of the neonatal sepsis-like illness with central nervous system (CNS) infection. The summary of an infectious workup performed in the patient is displayed in Table S1. The magnetic resonance imaging (MRI) examination with a contrast medium of the brain revealed abnormally increased enhancement of the leptomeninges (pia mater and arachnoid mater). The results of cerebral MRI consistent with meningitis are shown in Supplemental Figure 1. Because of hypogammaglobulinemia regarding all isotypes, IgG 230 mg/dL (N:270-780), IgA 2 mg/dL (N:6-58), and IgM 11 mg/dL (N:12-87), and anemia, hemoglobin 8.8 g/dL and red blood cell count 2.7x10⁶/mcL, transfusions of intravenous immunoglobulins and packed red blood cells were indicated. She recovered completely and her further psychomotor development was age-appropriate. She was vaccinated according to the national vaccination program with the live Bacille Calmette-Guerin (BCG) and anti-hepatitis B vaccines after birth, and then she continued the vaccination schedule with anti-tetanus, anti-diphtheria, inactivated poliomyelitis vaccine (IPV), and as a 13-month-old girl, she was vaccinated with a trivalent measles–mumps–rubella live vaccine without adverse effects following immunization (AEFI). At the age of 8 months, the girl was hospitalized in the department of pediatric nephrology for Proteus mirabilis urosepsis. Thereafter, by the age of 3 years, she suffered from recurrent urinary tract infections. Since the end of the first year of life, the girl also presented with recurrent respiratory tract infections: pneumonia, bronchitis, otitis media, tonsillitis, and laryngitis, requiring antibiotic therapy 6–8 times every year.

Figure 1 Cerebral MRI examination with a contrast medium showing abnormal increased enhancement of leptomeninges, pia, and arachnoid maters (marked with arrows).

Figure 2 Timeline showcasing the symptomatology, immunodiagnostics, imaging examinations, therapy, and diagnosis in the reported CVID-affected patient with a NFKB1 variant.

Referring to the patient’s history of very early onset of infections and hypogammaglobulinemia, a suspicion of an inborn IEI was raised. According to the European Society for Immunodeficiencies (ESID) definition,²⁰ major immunoglobulin class deficiency persisting beyond the age of 4 years, low IgG subclass levels, specific anti-diphtheria and anti-tetanus toxoids antibody deficiency, accompanied by maturational disorders within the B cell compartment prompted us to diagnose CVID. Table 1 summarizes the immunodiagnostics performed in the patient at the CVID diagnosis. To identify the genetic background of the IEI, whole exome sequencing (WES) was performed in the patient and her parents. A heterozygous c.258+5G>T variant in the NFKB1 gene was found in our patient affected with CVID. Since monoallelic alterations in NFKB1 are a well-known genetic cause of an early onset CVID,^19,21,22 the NFKB1 variant was corresponding with the infectious symptomatology and the IEI diagnosis.

Regular intravenous immunoglobulin (IVIg) replacement therapy (Ig-RT) has been implemented in the patient, intermittently accompanied by antibiotic prophylaxis with azithromycin and inhaled therapy with corticosteroids and bronchodilators to better control the respiratory tract infections and symptoms of bronchial obstruction, respectively.

To follow the patient’s history, including signs and symptoms; immunodiagnostics; imaging examinations; therapeutic interventions; and diagnosis, see timeline displayed in Figure 1.

Because of the rarity of the reported case with the initial inflammatory manifestations and consequent clinical and immunogenetic IEI diagnosis, an informed consent for publication was obtained from the patient’s parent.

Methods

Flow cytometric peripheral blood lymph cell immunophenotyping

Cells were labeled with the following murine fluorochrome-stained monoclonal antibodies: anti-CD45 FITC (fluorescein isothiocyanate), anti-CD14 PE (phycoerythrin), anti-CD19 PE, anti-CD19 PerCP (peridinin chlorophyll protein), anti-IgM FITC, anti-IgD FITC, anti-CD38 APC (allophycocyanin), anti-CD27 PE, anti-CD21 FITC, as well as anti-CD3 FITC, anti-CD4 FITC, CD45RA FITC, CD127 FITC, CD185 FITC, anti-CD8 PE, anti-CD16+CD56 PE, CD25 PE, CD31 PE, CD45RO PE, anti-CD3 PerCP, CD197 PerCP, anti-CD4 APC, and anti-CD8 APC (all Beckton-Dickinson Biosciences, USA). The acquisition of cells and analysis was carried out with the use of the flow cytometer FACSCanto and FACSDiva software (Beckton-Dickinson, USA). With sequential gating on biparametric scattering CD45+CD14- lymphocytes, the following lymphocyte subpopulations were identified:

• CD19+ B cells, immature CD19+CD21lo, immature activated CD19+CD38loCD21lo, transitional CD19+CD38hisIgMhi, nonswitched memory CD19+CD27+sIgD+, switched memory CD19+CD27+IgD- B cells, and CD19+CD38hisIgM-plasmablasts

• CD3+ T cells, CD3+CD4+ T helper cells, CD3+CD4+CD31+CD45RA+ recent thymic emigrants, naïve CD3+CD4+CD27+CD45RA+, regulatory CD3+CD4+CD25++CD27-, central memory CD3+CD4+CD27+CD45RO+, effector memory CD3+CD4+CD27-CD45RO+, terminally differentiated CD3+CD4+CD27-CD45RA+, follicular CD3+CD4+CD185+CD45RO+, and regulatory CD3+CD4+CD45RO+CD127-CD25++ T helper cells. Among CD3+CD8+ cytotoxic T cells, the following subsets were distinguished: naïve CD3+CD8+CD197+CD27+CD45RA+, central memory CD3+CD8+CD197+CD27+CD45RO+, effector memory CD3+CD8+CD197-CD27-CD45RO+, and terminally differentiated CD3+CD8+CD197-CD27-CD45RA+ cells.

• CD3-CD16+CD56+ NK cells.

The relative values of PB lymphocytes, the B, T, and NK cells of the total lymphocyte population as well as B and T cell subsets were calculated. The absolute counts of all cell subsets were calculated from the PB leukocyte counts. A comparative analysis was done with the reference cutoff values of B and T cell subsets for pediatric populations of different age groups.

Molecular genetic testing

DNA from the proband and her parents was obtained from peripheral blood and extracted using standard protocols. Library preparation for the WES was performed on the proband’s DNA sample with Twist Human Core Exome spiked-in with: Twist mtDNA Panel, Twist RefSeq Panel, and Custom Panel covering variants located in noncoding regions that have been linked to clinical phenotypes according to the ClinVar database (Twist Bioscience, San Francisco, CA, USA). Enriched library was paired-end sequenced (2×100 bp) on NovaSeq 6000 (Illumina, San Diego, CA, USA) to obtain 117 476 696 reads, resulting in mean depth of 133.82× (99.3% of target bases were covered at a minimum of 20×, whereas 99.4% had coverage of min. 10×). Bioinformatic analysis of raw WES data and variants prioritization were performed as previously described (PMID: 34448338). Reads were aligned to the hg38 reference genome sequence and visualized by an Integrative Genomic Viewer.

Presence of the selected variant was subsequently studied by amplicon deep sequencing (ADS) in the proband and her parents. The heterozygous variant in the NFKB1 gene (LRG_1316_t1:c.258+5G>T) has been indicated for further verification. The NFKB1 c.258+5G>T variant causes a noncoding transcript, intron, splice donor 5th base change. The variant was absent in null control chromosomes in the GnomAD project and in an in-house database of >10 000 WES of Polish individuals. No clinical diagnostic laboratories have submitted clinically significant assessments for this variant to the ClinVar database. ADS confirmed the presence of the heterozygous c.258+5G>T NFKB1 variant within the DNA of the proband and that of her father. In silico analysis of the pathogenicity using the dbscSNV engine (PMID: 25416802) indicated that the c.258+5G>T NFKB1 variant is deleterious (ADA score =0,9989) via impact on pre-mRNA splicing. Selected variant in the NFKB1 gene has not been described to date in The Human Gene Mutation Database (HGMD Professional 2021.4), although other deleterious variants within the NFKB1 gene are related to immunodeficiency, common variable, 12 (MIM# 616576), with an autosomal dominant model of inheritance.

Discussion

Herein, authors provide a comprehensive report of a pediatric patient with an ususual initial symptomatology and an early onset antibody deficiency in whom the CVID diagnosis was established and supported by molecular genetic analysis revealing a novel heterozygous variant in the NFKB1 gene. In light of the ever-increasing progress of insights into molecular genetics underpinning the pediatric CVID, we aimed to show the links between complex genetics and neonatal parechovirus meningitis and sepsis-like illness as a warning clinical symptomatology of an IEI. We also expand the phenotype-genotype characteristics by adding viral meningitis and sepsis-like illness to the spectrum of the NF-κB1 deficiency.

Individuals bearing heterozygous NFKB1 variants show a marked heterogeneity of clinical phenotypic expression, ranging from severe immunodeficiency with multiorgan autoinflammatory and EBV-mediated lymphoproliferative disease, through antibody deficiency with predisposition to infections, to asymptomatic course in variant-proven relatives.^21,23,24 In a Finnish cohort of carriers of distinct heterozygous variants in NFKB1, the clinical penetrance was not complete and with advancing age of affected patients, hypogammaglobulinemia in CVID was less prominent, whereas autoinflammatory disorders were more frequent.²⁵ However, despite considerable individual and intrafamilial variability of clinical manifestations in patients with CVID and variants in NFKB1, severe infectious and noninfectious complications have been reported. These include recurrent sinopulmonary and skin infections, and severe immune dysregulation disorders, such as autoimmune hemolytic anemia (AIH), autoimmune neutropenia,²⁶ an immune dysregulation, polyendocrinopathy, and enteropathy (IPEX)-like disease,²⁷ as well as malignant and nonmalignant proliferation.²⁸ The symptomatology of NFKB1-related disease-causing variants remarkably expand the immunogenetic landscape of this IEI.^29–31 These observations correspond with the phenotypic expression of the monoallelic c.258+5G>T NFKB1 variant in our CVID-affected proband and her mildly affected father, reflecting the incomplete penetrance of this NFKB1 variant and the resulting autosomal-dominant disease. Importantly, like other gene defects with impact on the immune system control, NFKB1 variants are characterized by highly variable penetrance (PMID 27365489, 29477724), thereby explaining the subtle symptoms in the proband’s father and absence of serious infections, and autoimmune, autoinflammatory, and lymphoproliferative disorders.

Noteworthy, while the neonatal and early childhood onset of clinical symptoms has been reported in NFKB1-haploinsufficient patients, age-dependent severity of clinical phenotypes has also been observed.^20,23 Indeed, our patient manifested viral sepsis-like illness and meningitis along with hypogammaglobulinemia as a 5-week-old young infant, yet presently, aged 6 years, she fulfills clinical and immunological criteria of the pediatric CVID. The diagnosis has been made because of recurrent respiratory tract infections, low serum immunoglobulin levels, IgG subclass deficiency, poor response to vaccines, and defective B cell development with low switched-memory B cells, without evidence of profound deficiency in the T cell pool.^19,23 However, because of the variant in NFKB1, she is at risk of age-related progressive depletion of B cells and hence, impaired class switch recombination, somatic hypermutation, antibody affinity maturation, abrogation of T cell functions, and severe infectious episodes, as well as autoimmune, autoinflammatory, lymphoproliferative, and malignant complications.^{8,20,23,28,30,32–36}

In this report, we expand the clinical phenotypic features of the NFKB1 gene-related disease by adding a neonatal viral sepsis and meningitis because of an HPeV infection to the symptomatology of the NF-κB1 deficiency. HPeV, in particular HPeV3 types are the major cause of meningitis in neonates and young infants. The HPeV3 strains are the most pathogenic among HPeV types. They replicate rapidly in neuronal cells, suggesting their relationship with neuropathogenicity.^37,38 In neonates, HPeV elicits a specifically modulated innate immune response profile, with several inflammatory mediator elevated levels, such as interleukin (IL)-15, a proinflammatory cytokine important in eradication of viral pathogens, fractalkine CXCL1, interferon (IFN)-α2, and IFN-γ-induced protein 10 (IP) CXCL10. Simultaneously, no significant increase in IFN-γ levels or downstream type I interferon pathway effectors were noted.

This phenomenon of immune response to HPeV may be associated with its tissue tropism and limitation of the CNS infection to smooth muscle cells of leptomeningeal blood vessels.³⁹ This apparently deficient innate immune response may be assumed to be a contributory factor to prolonged HPeV infection or long-term viral persistence despite good short-term clinical neurological outcomes. In children in whom CNS parechoviral infection and sepsis-like symptoms do not require intensive care, the disease is often not notifiable. However, the study on a large cohort of HPeV-infected infants indicate the need for long-term clinical and imaging monitoring as short-term evaluation does not specifically predict the neurodevelopmental outcomes.⁴⁰ Whereas NF-κB1 canonical pathway activates the components of the innate immune response, such as TLRs, important questions are then raised about the role of the monoallelic NFKB1 alterations and susceptibility to HPeV infection in our patient, and whether NF-κB deficiency is underpinning the less robust TLR sensitization, impaired adaptive immune response, and proneness to HPeV infection.

Importantly, regulation of NF-κB signaling pathways and its activation and controlling antiviral host defense is a ubiquitination-related process.^41,42 It may therefore be hypothesized that the ever-advancing knowledge on the regulatory mechanisms of NF-κB activity in virus-triggering inflammation will contribute to a better understanding of the risk of viral evasion in CVID patients with NFKB1 variants.

The patient’s perspective refers to CVID-related infectious and noninfectious complications. Infections of the respiratory tract, e.g., pneumonia and bronchitis, and upper airway infections, such as otitis media, sinusitis, and tonsillitis, are the leading CVID sequelae in pediatric patients, followed by gastrointestinal infections. Affected individuals are also at risk of developing severe infectious episodes, such as sepsis, osteomyelitis, septic arthritis, or CNS infections.²³ Furthermore, the spectrum of immunophenotypes also include CVID-related immune dysregulation, and autoimmune, autoinflammatory, and lymphoproliferative disorders. Among them autoimmune cytopenias, such as AIH, autoimmune thrombocytopenia, and autoimmune neutropenia (AIN), are the most frequently observable complications associated with specific NFKB1 gene variants.^43–46 It was also shown that the type of variants shapes the clinical phenotype in CVID, as more pronounced autoimmune and inflammatory disorders, such as enteropathy or bronchiectasis, occurred in patients bearing frameshift or nonsense NFKB1 variants.⁴⁷ Regular Ig-RT supplying specific antibodies and providing both antimicrobial and immunomodulatory activities, reinforced by antibiotic prophylaxis on as needed basis, are the mainstay of the management strategy. Vaccination strategy should be elaborated individually. Live attenuated vaccines are not recommended in children with CVID and in patients receiving IgRT as they may become inactivated. Inactivated vaccines, including intramuscular influenza vaccine, can be safely administered in CVID but may not elicit a protective antigen-specific immune response.^48–50 The lifelong risk of CVID-related infectious and immune dysregulation disorders requires a multidisciplinary care of specialists in immunology, infectious diseases, neurology, pulmonology, oncology, and hematology.

Conclusion

By including viral meningitis and sepsis-like illness in a child with antibody deficiency, this comprehensive clinical-molecular study expands the spectrum of warning signs of IEI. The manuscript is therefore meant not only for specialists in clinical immunology and medical genetics but also has an educational aspect for clinicians facing diagnostic challenges. Hopefully, it will contribute to increased awareness and recognition of IEI in children with hypogammaglobulinemia affected with viral meningitis.

Along with the ever-increasing role of immunogenetics in disease definition and delineation, this case report shows that pediatric patients with infectious symptomatology and antibody deficiency should be offered genetic testing. It might be helpful in anticipating the prognosis and making the space for novel patient-tailored therapeutic approaches.