Partial Loss of NEMO Function in a Female Carrier with No Incontinentia Pigmenti

Abstract

Background/Objectives: The nuclear factor (NF)-kB essential modulator (NEMO) has a crucial role in the NFκB pathway. Hypomorphic IKBKG pathogenic variants cause ectodermal dysplasia with immunodeficiency (EDA-ID) in affected males. However, heterozygous amorphic IKBKG variants could be responsible for Incontinentia Pigmenti (IP) in female carriers. Typically, IP patients do not exhibit immunodeficiency, although hypomorphic variants might lead to immunodeficiency in female IP patients. Here, we report the case of an IKBKG female carrier, with no IP but an unexpected picture of immunodeficiency. She had a positive family history for the same genetic condition. Methods: We performed immunological, molecular, and functional analysis to evaluate NEMO contribution. Results: The patient was healthy until the age of 25 when severe asthma and Hashimoto thyroiditis occurred. She had HLAB27-positive ankylosing spondylitis, non-tubercular mycobacteriosis, and pulmonary aspergillosis infections. We found CD19+ B cell lymphopenia and T cell subset alterations. Sanger sequencing revealed a heterozygous IKBKG variant at position +1 of the 5' UTR of the gene which disrupted the normal pre-mRNA splicing. We observed a decreased NEMO protein expression, a reduced level of mRNA, and a defective NF-κB pathway. Conclusions: These findings suggest a possible correlation between the partial loss of NEMO function and the immunodeficiency observed in this patient. This case could expand our understanding of NEMO deficiency in female carriers.

Keywords: NEMO deficiency; female carrier; immune dysregulation; incontinentia pigmenti.

Figure 1

Timeline showing patient’s clinical and treatment history.

Figure 2

Characterization of NEMO-III.2. (A) Family pedigree showing proband III.2 (black arrow), her carrier mother II.2, her brother III.1, and cousins III.3 and III.4 affected by NEMO deficiency. (B) Sanger sequencing performed on III.2, II.1, II.2, and HD genomic DNA showing the NG_009896:g.10503G>T variant indicated by “*”. (C) cDNA analysis of IKBKG fragments obtained from III.2 and II.2. Black arrows indicate the wt and wt + mt sequences of II.2 and III.2, respectively, on the agarose gel. Chromatograms showing the NG_009896:g.10503G>T variant causing the retention of the intron between exon 1 and 2 in III.2 compared to HD. (D) Schematic of the normal (upper panel) and alternative splicing of IKBKG exon 1 and 2 detected in III.2. (E) Real-time PCR analysis showing reduced NEMO mRNA levels, normalized to β-Actin, in LCLs from II.2, III.2, III.3, and HD. The graph represents the mean of two experiments.

Figure 3

Functional analysis of NEMO. (A) NEMO protein expression by WB performed on PBMCs derived from III.2, II.2, and one HD showing reduced protein expression in III.2 and a slight reduction in II.2. (B) NEMO protein expression by FACS on LCLs from one HD, II.2, III.2, and the NEMO-pt III.3. The gray histograms represent FMO, the orange histograms represent HD, the blue histogram represents the mother’s cells, the pink histogram shows reduced expression in III.2, and the green histogram represents nearly absent NEMO expression in III.3; the right graph shows the MFI. (C) Representative plots of IkBα degradation in CD4⁺ and CD8⁺ T cells from III.2, II.2, and HD. The numbers inside the plots represent the MFI. The gray histograms show IkBα expression at time 0 without stimulation. The colored histograms show IkBα degradation after stimulation with PMA/ionomycin.

Figure 4

Functional characterization of III.2. (A) Evaluation of NADPH oxidase activity on neutrophils showing normal respiratory burst. Filled gray histograms represent unstimulated III:2 neutrophils, empty gray histograms represent unstimulated HD neutrophils, filled pink histograms represent stimulated III.2 neutrophils, and empty black histograms represent stimulated HD neutrophils. The right graph shows the MFI (mean ± SEM of n = 2 experiments). (B) Monocytes’ cytokines. The left panel shows decreased monocytes’ TNFα production p value (*) 0.0244 (mean ± SEM of n = 2 experiments), the middle panel shows normal monocytes’ IL-8 production (mean ± SEM of n = 2 experiments), and the right panel displays decreased IFNγ production from CD3⁺CD45RA⁻ memory T cells (mean ± SEM of n = 2 experiments) after indicated stimulations.