At 8 years old, when anti-tuberculosis medications had already been stopped, and 4 months after stopping subcutaneous rIFN-gamma there was recurrence of infections including Pseudomonas aeruginosa pansinusitis and notable complications of bronchiectasis diagnosed on high resolution CT. Subcutaneous rIFN-gamma was recommenced at 50 mcg/m2 three times per week, however one month after she developed Mycobacterium abscessus left knee septic arthritis and tibial osteomyelitis for which she was treated with intravenous amikacin, cefoxitin and oral clarithromycin based on sensitivities. She has had no further infections on anti-tuberculosis therapy, rIFN-gamma, anti-microbial prophylaxis with Bactrim (after desensitization for challenge-proven allergy) and intravenous immunoglobulin replacement (due to poor pneumococcal antibody responses) and will continue until stem cell transplantation. Physical examination revealed an intellectually normal child with failure to thrive (height and weight less than 3rd percentile) and scarring over the face (a consequence of facial lupus vulgaris which she had at age 3). Respiratory examination reveals bilateral crepitations and wheezes in keeping with bilateral bronchiectasis. There were no features of anhidrotic ectodermal dysplasia (EDA) such as hypohidrosis, sparse hair, dental abnormalities, coarse skin or osteopetrosis.
The immunological results (Supplementary tables 1 and 2) show that the patient does not display an overt functional T cell defect and a specific defect in the IL-12/IFN-gamma loop was not identified. WES was thus performed on gDNA from the patient. We identified a heterozygous nucleotide substitution (c.107C>A) in exon 1 of NFKBIA (encoding IkappaBalpha), leading at the protein level to a missense mutation, replacing a Serine at position 36 by a Tyrosine (S36Y). The mutation was confirmed by Sanger sequencing, on PCR fragment amplified from gDNA extracted from granulocytes and dermal fibroblasts (Figure 1). The mutant and WT allele in the patient were of same amplitude, suggesting equal amount of both alleles. The mutation was not found in any public database (1,000G, ExAc, dbSNP). Familial segregation analysis (Figure 1) showed that both parents are wild-type for the substitution; the mutation thus appeared de novo in the patient (Figure 1). This mutation leads to a gain-of-function of IkappaBalpha and was reported in a patient with mild AD-EDA-ID and non-infectious systemic inflammation. We analyzed the IkappaBalpha phosphorylation and subsequent degradation in primary fibroblasts from the patient, a healthy control and a NEMO-deficient cell line after TNF-alpha (20 ng/ml) and IL-1beta (10 ng/ml) stimulation. We found that IkappaBalpha was phosphorylated on Serine 32 after TNF-alpha stimulation in control and in patient's cells, as compared with a NEMO-deficient patient. However, we observe an impaired, but not abolished IkappaBalpha degradation in the patient's cells compared to the healthy control (positive control) and the NEMO-deficient cells (negative control). The same results were obtained after stimulation with IL-1beta. Altogether, we have identified a de novo heterozygous gain-of-function mutation in NFKBIA, affecting the serine at position 36. The mutation does not impair the phosphorylation of IkappaBalpha on Serine 32, but affect partially its degradation, leading to impaired NF-kappaB signaling (Figure 1).
This is the first reported case of a female child with absolutely no features of EDA who was found to have a heterozygous missense variation mutation in IkappaBalpha (S36Y) resulting in significant immunodeficiency only. Immune cells as well as dermal fibroblasts from the patient carry the heterozygous missense mutation, apparently at the same level. Out of the eight patients previously reported (Supplementary Table 3), all, except a patient with mosaicism and our own, had at least mild features of EDA. The patient we report does not display any clinical signs of EDA. For reasons still unknown, there is a considerable variability in the extent of the severity of the EDA clinical signs in IkappaBalpha-mutated patient (Supplementary Table 3). In addition, variability in terms of EDA clinical signs is also observed in patients with mutation in another component of the NF-kappaB signaling pathway, IKBKB, as well as in NEMO. Recurrent mycobacterial diseases (tuberculous and non-tuberculous mycobacteria) is observed in our patient. Surprisingly, the other patient described with the exact same mutation (S36Y) by Yoshioka et al, display only mild EDA and had an episode of mycobacterial disease. All the other GOF-IkappaBalpha patients displayed EDA and were free of mycobacterial disease (Supplementary table 3). It is also interesting to note that our patient was free of all infections during three years (not only mycobacterial) when she was under IFN-gamma treatment. Altogether, IkappaBalpha mutations should be suspected in children with bacterial and mycobacterial infections even without EDA clinical presentation. In addition, treatment with rIFN-gamma injection may be proposed to prevent all infections.