A heterozygous N-terminal truncation mutation of NFKBIA results in an impaired NF-κB dependent inflammatory response.

Germline heterozygous gain-of-function (GOF) mutation of NFKBIA, encoding IκBα, would affect the activation of NF-κB pathway and cause an autosomal dominant (AD) form of anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID). Here we reported a Chinese patient with a heterozygous N-terminal truncation mutation of NFKBIA/IκBα. She presented recurrent fever, infectious pneumonia and chronic diarrhea with EDA-ID. Impaired NF-κB translocation and IL1R and TLR4 pathway activation were revealed in this patient. The findings suggested that the truncation mutation of IκBα caused medium impaired of activation of NF-κB but the early death. Furthermore, we reviewed all the reported patients with NFKBIA mutation to learn more about this disease.

Introduction

Nuclear factor kappa B (NF-κB) plays a vital role in innate and adaptive immunity, reflecting in the signal transduction in response to external stimuli as well as to internal stimuli within the cell., IκBα, encoded by NFKBIA, is one of the inhibitors of the NF-κB activation and binds to the NF-κB proteins p65 (RelA) and p50. Various stimuli could cause activation of the IkB kinase (IKK) complex, which phosphorylates IκBα on serines 32 and 36, leading to ubiquitination of lysines 21 and 22 and the subsequent degradation of IκBα. Mutation of NFKBIA causes impaired IκBα degradation and leads to an autosomal dominant (AD) form of anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID), characterized by sparse hair, conical teeth, reduced number of sweat glands, and susceptibility to severe infections. To date, 19 patients have been reported, including 15 point mutations at or adjacent to the S32 and S36 phosphorylation sites, 1 mosaicism point mutation at S32 site and 3 truncation mutations which introduce a premature stop codon and give rise to N-terminally truncated IκBα proteins through re-initiation of translation at downstream ATG sites.4, 5, 6, 7

In this study, we reported the clinical and immunological features of a Chinese patient with a truncation mutation of NFKBIA with EDA-ID. Since the disease with germline GOF NFKBIA mutation is a kind of rare primary immunodeficiency disease (PID), the clinical phenotypes are variable and the pathogenesis is not fully clear, we summarized the clinical, molecular and cellular phenotypes of the 20 reported patients since 2003 to recognize it better and help doctors to diagnose it as early as possible.

Materials and methods

Patient

The patient enrolled in this study was a 4-month-old girl born in a nonconsanguineous family. Clinical data and blood were collected when she first visited the Children's Hospital of Chongqing Medical University in January 2020. All research practices were approved by the Medical Ethics Committee of the Children's Hospital of Chongqing Medical University (approval number: 030/2013). Informed consent was obtained from guardians.

Genetic studies and conservative analysis of NFKBIA

Whole blood samples were sent to MyGenostics (Beijing, China) and subjected to medical whole-exome sequencing. Mutation in the NFKBIA gene was verified by Sanger sequencing. The conservation analysis of NFKBIA gene was analyzed on the weblogo.berkeley.edu.

Cell preparation and culture conditions

Peripheral blood mononuclear cells (PBMCs) were obtained from the patient and healthy adult volunteers by centrifugation of heparinized blood over Ficoll-Hypaque density gradient lymphocyte separation medium (GE Healthcare), with standard techniques. To measure IL-1β, IL-18, IL-6, IL-8(CXCL8), IL-12p70, MCP-1(CCL2), IFN-γ and TNF-α concentrations, 1 × 106 PBMCs were cultured in RPMI 1640 complete medium (1 ml) for 36 h. The following reagents were used: LPS from E. coli 01127:B8 (1 μg/ml; Sigma), human interferon (IFN)-γ (1000 U/ml; Peprotech), IL-1β (10 ng/ml; Peprotech). After 36 h, supernatants were collected. Cytokines concentrations were determined by Multi-Analyte Flow Assay Kit (Human Inflammation Panel(13-plex), Biolegend, 740118) according to the manufacturer's instructions. Data were collected with a FACS Canto II flow cytometer (BD Bioscience) and analyzed using LEGENDplexv8.0 software (Biolegend).

Immunofluorescence

PBMCs of the patient and healthy donor were stimulated with 100 ng/ml LPS at different time intervals (0, 15, and 30 min). Then incubated by primary antibody P-NF-κB p65 (Cell Signaling Technology), secondary antibody donkey-anti-rabbit AF488 (Abcam) and DAPI (Beyotime) at the room temperature. Then detected and analyzed by confocal microscope (Nikon C2 plus) and NIS-Elements BR software.

Statistical analysis

All statistical analyses were conducted in GraphPad Prism 8 software (GraphPad Software, Inc., San Diego, CA).

Results

Clinical manifestations of patient

A female Chinese infant of two healthy, non-consanguineous parents was born at term after an uncomplicated pregnancy. After birth, she was hospitalized for intrauterine infection and improved after treatment. She presented with sparse hair and no eyebrows with normal nail growth. She was fed initially with breast milk alone and then in combination with artificial milk. She received BCG and Hepatitis B vaccines at birth without complications. At 2 months of age, she began to have recurrent fever and severe infectious pneumonia (Acinetobacter baumannii, enterococcus faecalis, legionella, Klebsiella and fungi). By that time, she had evident growth retardation. During 2–4 months of age, the lab examination suggested that the platelet and hemoglobin were progressive reduction. Her hepatosplenomegaly became more obvious. At 4 months of age, she started suffering from gastrointestinal problem with feeding intolerance, chronic diarrhea and bloody stool. She was also noted to be heat intolerant and unable to sweat. The hemoglobin was persistent below the normal (Fig. 1A). The patient's serum IgG was normal, but the levels of IgA and IgM were below normal (Table 1). Cell counts of CD3+CD4+ T, NK, CD19+ B and the rate of CD4/CD8 were below normal, while cell count of CD3+CD8+ T was higher than normal (Table 2). After a combination of antibiotics, antifungal, anti-tuberculous and regular intravenous immunoglobulin (IVIG) treatment, the patient did not present significant infectious manifestations but still had gastrointestinal symptoms. At 6.5 months of age, she presented depressed spirit and less moving, about 15 days later, she unfortunately died of cardiac failure.

Figure 1

Clinical and genetic characterization of the patient. (A) The complete blood counts showed increased CRP (dark blue) and low hemoglobin levels (purple). The normal reference ranges are indicated as following: WBC 4–12×109/L; RBC 4.0–5.3×1012/L; PLT100–380×109/L; Hb 110–150 g/L; MCV 80–100 fL; MCH 26–32 pg; CRP <8 mg/L. (B) Family pedigrees of the patient with variant in NFKBIA. (C) Sequence analysis of the NFKBIA gene. The de novo mutation, c.40G>T, in the NFKBIA gene is indicated by the arrow. (D) Schematic domain structure of NFKBIA. All the reported mutations are in the upper part of the protein. The different domains are indicated in the lower part of the protein. (E) Evolutionary conservation of the site E14 in NFKBIA. Amino acid sequence of NFKBIA flanking E14 was aligned on the weblogo.berkeley.edu across various species.

Table 1

Immunoglobulin and complement.

	Result	Reference Range
lgG (g/L)	9.96	2.86–16.8
lgA (g/L)	0.097	0.1–1.29
lgM (g/L)	0.151	0.21–1.92
lgE (IU/ml)	<5.00	0–165
C3 (g/L)	1.54	0.74–1.86
C4 (g/L)	0.54	0.11–0.61
ESR (mm/h)	19	0–15

Table 2

Lymphocyte classification.

TBNK	Result	Reference Range
CD3+%	92.76	39–73
CD3+CD8+%	70.14	11–32
CD3+CD4+%	21.87	25–50
CD3+CD4+CD8+%	0.26
CD3+CD4–CD8–%	1.01
NK%	2.20	3–16
CD19%	5.03	7–41
CD4/CD8	0.31	0.98–1.94
CD3+# (cell/μl)	3694.10	1400–8000
CD3+CD8+# (cell/μl)	2793.31	400–2300
CD3+CD4+# (cell/μl)	870.86	900–5500
CD3+CD4+CD8+# (cell/μl)	10.36
NK# (cell/μl)	87.49	100–1400
CD19+# (cell/μl)	200.30	600–3100
CD45+# (cell/μl)	3982.47

Genetic analysis revealed NFKBIA mutation in patient

Based on the clinical symptoms and laboratory results, primary immunodeficiency was considered for the patient and whole-exome sequencing was performed. A heterozygous N-terminal truncation mutation in NFKBIA was found in this patient but not in her parents (Fig. 1B, C). The conservation analysis suggested that the site of 14E in NFKBIA is highly conserved across species (Fig. 1E).

Patient had impaired NF-κB dependent cytokine production

Because NFKBIA is an inhibitor of NF-κB, we evaluated whether the cells of the patient responded normally to the stimulants that active NF-κB. We examined cytokines produced by PBMCs after stimulation of TLR4 and IL-1 receptor as described by Lopes-Grandos et al and Yamamoto et al., The patient's PBMCs showed little response to LPS and IFN-γ in production of IL1β, IL-6, IL-8, IL-12p70, TNF-α, MCP-1 and IFN-γ, although we did detect a low response to IL1β in production of IL-18, IL-6, IL-8, TNF-α, MCP-1 (Fig. 2). These data indicated an impaired signal transduction downstream of the TLR receptor and IL-1 receptor families.

Figure 2

Impaired NF-κB dependent cytokine production in response to different stimuli by patient’s PBMCs. 1 × 106 PBMCs (control, grey bars; patient, black bars) were incubated with medium, LPS (1ug/ml), IFN-γ (1000 U/ml), IL-1β (10 ng/ml); IL-β (A), IL-18 (B), TNF-α (C), IFN-γ (D), IL-6 (E), IL-12 (F), IL-8 (CXCL8) (G) and MCP-1 (CCL2) (H) were quantitated 36 h after stimulation respectively. Data were representative of three independent experiments. ∗∗P < 0.01, ∗∗∗P < 0.001.

p.E14X leads to reduced nuclear translocation of NF-κB

Since the p.E14X mutation prevents IκB-α degradation, keeping the nuclear localization signals on the NF-κB subunits masked, we also detected the phosphorylation of RelA (p65) in response to LPS stimulated PBMCs at different time intervals. We found that the level of the patient's phosphorylation of p65 (p-P65) was below the healthy control (Fig. 3A, B). The result showed that the N-terminal truncation mutation in NFKBIA could lead to reduced nuclear translocation of NF-κB.

Figure 3

Impaired phosphorylation of RelA (p65) in response to LPS by patient’s PBMCs. PBMCs were stimulated with 100 ng/ml LPS at different time intervals (0, 15 and 30 min). (A) Average mean fluorescence intensity (MFI) of p-P65 was analyzed. (B) Shown are representative images and average MFI (±SD) from >30 cells for each time interval. ∗∗P < 0.01.

Discussion

The IκB (inhibitor of NF-κB) family of proteins includes IκBα, IκBβ, and IκBε. In resting cells, NF-κB proteins, including p65 (RelA), p105/p50, p100/p52, c-Rel, and RelB,, are retained in the cytoplasm by the IκB family of proteins, which could be activated by a wide variety of cell-surface receptors and finally result in NF-κB activation. Stimuli, including proinflammatory cytokines (TNF-α, IL-1) and Toll-like receptor (TLR) ligands, cause activation of the IκB kinase (IKK) complex, which phosphorylates IκBα on serines 32 and 36, leading to ubiquitination of lysines 21 and 22 and the subsequent degradation of IκBα. Serines 32 and 36, as well as lysines 21 and 22, are contained within an N-terminal 73-amino-acid sequence and designated the signal response domain because this region regulates the degradation of IκBα. The N-terminal sequence of IκBα is highly conserved across species, especially the six amino acid degron (DSGLDS) and the two serine residues located at positions 32 and 36 in humans. As a result, both of the point mutation and truncation mutation which relate to these sites could impair the degradation of IκBα, leading to the NF-κB translocation impairment.

In this work we described a patient with heterozygous N-terminal truncation mutation. Our patient had early onset age and presented with typical clinical features of EDA-ID, including sparse hair, susceptibility to severe infections and failure to thrive, however, she was not detected the development of sweat glands. Her high level of CD3+CD8+ T cell, low levels of CD19+ B cell, CD3+CD4+ T cell and CD4/CD8 suggested that the patient had evident immunodeficiency. She had low level of IgM and IgA with normal IgG, which was different with other patients.4, 5, 6, 7 Since truncation mutation of IκBα could give rise to re-initiation of translation at downstream ATG sites, severe disease and great impairment of NF-κB activation are more significant in IκBα point mutants versus truncation mutants. The patient showed absent or low response to TLR4 and IL-1R in production of inflammatory cytokines and chemokines, and presented with medium impaired phosphorylation of p65. These data suggested that the activation of NF-κB in this patient was not completely damaged, which was consistent with reported patient (P5: E14X, Table 3) and other truncation mutation patients (P4: W11X, P6: Q9X, Table 3).13, 14 However, our patient died at 7 months of age before hematopoietic stem cell transplantation (HSCT), much earlier than the other three truncation mutation patients. We supposed that the early death was caused by her chronic gastrointestinal syndrome and severe anemia without support treatment because of some special reasons.

Table 3

Genetic and clinical features of patients with heterozygous NFKBIA mutations.

Patient	Gender	Origin	Year of birth	Age of onset	Variant	Inheritance	Infection Manifestations
P1	M	Italian	nr	2 months	S32I	De novo	Recurrent LRTI: P. aeruginosa, Klebsiella, Serratia, S. aureusCMC
P2	M	Dutch	nr	2 years	S32Im	De novo	enteritis (Salmonella typhimurium) S. typhimurium infection persisted with recurrent manifestations in psoas muscle, pleural cavity, pericardial fluid, and ribs
P3	M	Dutch	nr	2 months	S32I	Inherited from his father	Meningitis: β-hemolytic group A Streptococcus: sepsis Respiratory infection pneumonia Pneumocystis jirovecii, mild CMC
P4	F	American	nr	Birth	W11X	Mother: WT; Father: nr	Recurrent pneumonia
P5	M	American	nr	1 month	E14X	De novo	Pneumonia (parainfluenza virus and Pneumocystis carinii). recurrent episodes of bacteremia oral candidiasis. Pyogenic bacteria sepsis, CMC
P6	M	Japanese	nr	1 month	Q9X	nr	Bacterial: pneumonia, respiratory syncytial virus. Bronchiolitis, acute otitis media, urinary tract infection. Cytomegalovirus: hepatitis, Rotavirus: enteritis. Bronchiolitis with respiratory syncytial virus
P7	M	Japanese	2007	4 months	S36Y	De novo	BCG skin infection
P8	M	German	nr	6 months	M37K	Mother: WT Father: nr	Haemophilus influenza: pneumonia, CMC
P9	F	Italian	2012	5 months	M37R	nr	Recurrent LRTISepsis: Klebsiella pneumonia, Candida parapsilosis, Stenotrophomonas maltophilia, osteomyelitis of skull and limb, CMC
P10	F	Chinese	2004	1 month	S36Y	De novo	LRTI: P. aeruginosa: bronchiectasis and sinusitisMycobacterium tuberculosis: abdominal lymphadenopathyM. abscessus: septic arthritis of the knee, osteomyelitisUrinary tract infection: K. pneumoniae
P11	F	Caucasian/Thai	2011	20 months	S32G	De novo	Salmonella enteritidis: osteomyelitis, hematocheziaCandida: esophagitisMycobacterium malmoense: blood and skinSapovirus and norovirus: stool
P12	M	Japanese	nr	2 months	S32R	De novo	S. aureus sepsis, CMC, Recurrent pneumonia
P13	nr	Japanese	nr	Birth	S32N	De novo	S. aureus and P. aeruginosa sepsis
P14	nr	nr	2007	nr	S32I	?	Recurrent infections
P15	M	nr	2007	nr	G33V	?	Recurrent infections
P16	M	Turkish	1982	early childhood	S36A	De novo	Recurrent gastrointestinal infections (Shigellosis, C. jejuni), recurrent upper respiratory tract infections (bronchitis, sinusitis, otitis media), recurrent pneumonias (S. pneumoniae, H. influenzae), bronchiectasis with chronic mucoid Pseudomonas aeruginosa infection meningitis (N. meningitidis), CNS tuberculosis with brain abscess, verruca vulgaris (HPV 9 and 57)
P17	F	Turkish	2010	early childhood	S36A	Inherited from her father	Recurrent upper respiratory tract infections (bronchitis, otitis media), recurrent pneumonias, bronchiectasis, Verruca vulgaris
P18	F	Turkish	2015	early childhood	S36A	Inherited from her father	Recurrent upper respiratory tract infections (bronchitis, otitis media), two pneumonias within two years
P19	M	Spain	2012	Birth	D31N	De novo	pustular deficiency of interleukin-1 receptor antagonist (DIRA)
P20∗	F	Chinese	2019	2 months	E14X	De novo	Recurrent bronchopneumonitis (Gram ± pyonenic)
Note: nr not report; P2 is father of P3; P16 is father of P17 and P18; P20∗ is our patient.

Note: nr not report; P2 is father of P3; P16 is father of P17 and P18; P20∗ is our patient.

Furthermore, we reviewed all the patients with mutation in NFKBIA and summarized their clinical features (Table 3), molecular and cellular phenotypes (Table 4). Including our patient, there have been 4 patients with heterozygous N-terminal truncation mutation of NFKBIA (P4, P5, P6 and P20). All of them had early onset age and similar infection manifestation with EDA and ID.,, P4 (W11X) was alive over 22 years old without HSCT. P6 (Q9X) was alive at 7 years old after HSCT. However, P5 died after HSCT because of pyogenic bacteria sepsis at 1 year old. These 4 patients had impaired IL-1R/TLR pathway activation, while P4 and P5 were confirmed to have impaired IκBα degradation and impaired NF-κB translocation. Pathogenicity of these three kinds of truncation mutation had been confirmed in cell lines. As for the point mutation patients, all of these patients had ID but P2 (S32Im), P16, P17, P18(S36A) had no EDA, indicating that there was no direct connection between NFKBIA mutation and EDA, which has not been studied before. However, these patients showed similar degree of activation of NF-κB. The pathogenicity of S32G, S32R, S32N and S36A was not confirmed in cell lines,,16, 17, 18, 19, 20, 21, 22, 23 and D31N was found in an infant (P19) who suffered from pustular and systemic inflammatory disease resembling the deficiency of interleukin-1 receptor antagonist (DIRA) through clinical exome sequencing screening. Unfortunately, in addition to symptomatic support treatment, HSCT is the only but not ideal therapy for these patients, which is a problem need to be improved.

Table 4

Molecular and cellular phenotypes of patients with heterozygous NFKBIA mutation.

Patient	IκBα degradation (agonist-cell type)	NF-κB translocation (dimer-agonist-cells)	IL-1R/TLR pathway activation (agonist-cell type)	TNFR pathway activation (agonist-cell type)	T-cell response in PBMCs (stimulus)	B-cell prolif (stimulus)
P6-Q9X	nr	nr	Impaired (LPS–monocyte)Impaired (LPS–fibroblast)	nr	Low prolif. (PHA; ConA)	nr
P4–W11X	Impaired (LPS-fibroblast)	Impaired (p50/p65-IL-1β-fibroblast)	Impaired (IL-1β, LPS-fibroblast)Impaired (poly(I:C), LPS, flagellin,CpG-PBMC)	nr	Normal prolif. (low α-CD3, α-CD3/α-CD28,PMA/iono, PHA, recall antigens)	nr
P5-E14X	Impaired (CD40L-EBV-B)	Impaired (p50; p65; c-Rel-CD40L-EBV-B)	Impaired (LPS, SAC OspA-PBMC)	Impaired (CD40L-EBV B cells)	Normal prolif. (PHA, ConA, and recall Ags)Impaired IFN-γ and TNF-α prod. (α-CD3)	nr
P20-E14X	nd	Impaired (p65-LPS-PBMC)	Impaired (LPS,IL-1β-PBMC)	nd	nd	nd
P19-D31N	nr	nr	nr	nr	nr	nr
P1–S32I	Impaired (TNF-α, LPS-fibroblast)	Impaired (p50/p65; p50/p50-TNF-α-fibroblast)	Impaired (LPS-PBMC)Impaired (LPS,IL-1β-fibroblast)	Impaired (TNF-α; LTα1β2-fibroblast)	Absent prolif. (low α-CD3, recall Ags) Normal prolif. (α-CD3/α-CD28, PMA, allogeneic cells) Normal IFN-γ prod. (α-CD3, α-CD3/α-CD28)	nr
P2–S32Im	nr	nr	Impaired (LPS, PAM3, zymosan-WB)Impaired (LPS-MdM)Impaired (LPS-fibroblast)	Impaired (TNF-α; LTα1β2-fibroblast)	Low prolif.a (low α-CD3, PHA)Normal prolif	Normal (CD40L + IL4)
P3–S32I	Impaired (LPS-fibroblast)	Impaired (p65-LPS-MdM)	Impaired (LPS, PAM3, zymosan-WB)Impaired (LPS-MdM)Impaired (LPS-fibroblast)	nr	Low prolif.a (low α-CD3, PHA)Absence prolif. (recall Ags)	Normal (CD40L + IL4)
P14–S32I	nr	nr	nr	nr	nr	nr
P11–S32G	Impaired (TNF-α-fibroblasts)	nr	Impaired (LPS–WB)	nr	Normal prolif. (PHA)	nr
P12–S32R	Impaired (TNF-α-fibroblast)	nr	nr	nr	nr	nr
P13–S32N	Impaired (CD40L-EBV-B)	nr	nr	nr	nr	nr
P15-G33V	Impaired (LPS-fibroblasts)	nr	Impaired (LPS–fibroblast)	nr	nr	nr
P7–S36Y	Impaired (TNF-α-T blast cells)	Impaired (p50/p65-TNF-α-fibroblast)	Impaired (LPS, IL-18–PBMC)	Impaired (TNF-α, LTα1β2-fibroblast)Impaired (CD40L-PBMC)	Low prolif. (low dose of α-CD3), Normal prolif. (high dose of α-CD3), Normal prolif. (PHA; PMA, recall Ags)	nr
P10–S36Y	Impaired (TNF-α, IL-1β-fibroblast)	nr	Impaired (IL-1β–fibroblast)	Impaired (TNF-α–fibroblast)	Impaired prolif. (high α-CD3) Normal prolif. α-CD3/α-CD28, PHA, ConA, PMA/iono Impaired (IFN-γ and IL-12 production; BCG, BCG/IL-12; BCG/IFN-γ)	nr
P16–S36A	Impaired (PMA/iono), anti-IgM and LPS-B cell)	nr	nr	nr	nr	nr
P17–S36A	nr	nr	nr	nr	nr	nr
P18–S36A	nr	nr	nr	nr	nr	nr
P8-M37K	Impaired (TNF-α, LPS, PAM3-fibroblast)	Impaired (p50/p65-TNF-α-HeLa cells)	Impaired (LPS, PAM3-fibroblasts)Normal (SAC-WB); impaired (IL-1β, SAC, LPS, PAM2, PMA/Iono-WB)	Impaired (TNF-α-fibroblast) Impaired (TNF-α-WB)	Low prolif. (OKT3, SAC), Normal prolif. (PHA, PWM, ConA, recall Ags, diphtheria, tetanus/streptolysin O/mumps)	nr
P9-M37R	nr	nr	nr	nr	Normal prolif. (PHA, PMA, α-CD3/α-CD28)	Decreased (CpG)

Note: nr not reported, nd not detected, WB whole blood, MdM macrophage-derived monocytes, Ags antigen, prolif. Proliferation.

Conflict of interests

The authors declare that they have no conflict of interest.