Our two patients had impaired antibody responses to pneumococcal carbohydrates. In both patients, poor clinical and biological inflammatory responses during the infectious episodes42
and antipolysaccharide antibody deficiency6
suggested genetic defects in toll like receptor (TLR)–NF‐κB‐mediated immunity.5,6
The broader antibody deficiency in patient 2 suggested a defect further downstream in NF‐κB activation.39
After the activation of whole‐blood cells with TNFα, IL‐10 production was impaired in patient 2, but not in patient 1 (fig 1A), whereas IL‐6 production by whole‐blood cells in response to stimulation with IL‐1β and lipopolysaccharide was abolished in patient 1, and mildly but reproducibly impaired in patient 2 (fig 1B).
Figure 1Stimulation of blood cells. (A) Interleukin (IL)10 production by whole‐blood cells from patient 1 (P1), patient 2 (P2), and 10 unrelated healthy controls (C) after stimulation with tumour necrosis factor (TNF)α (20 ng/ml) (more ...)
We then tested the response to IL‐1β, TNFα and PMA/ionomycin of SV40‐transformed skin‐derived fibroblasts from our patients, a healthy control (positive control, C+), and a NEMO‐deficient fetus (negative control, C–). Fibroblasts from patient 1 showed no response to IL‐1β but normal responses to TNFα (fig 2A, B), whereas fibroblasts from patient 2 showed impaired responses to both IL‐1β and TNFα (fig 2A, B). Overall, the pattern of response in patient 1 suggested a defect in the TLR and IL‐1R signalling pathways.4,5,6
By contrast, patient 2 probably had a defect downstream from the Toll/IL‐1R signalling pathway, affecting the TNFR pathway to a greater extent, possibly at the level of NF‐κB. The only known specific defect of the Toll/IL‐1R pathway is autosomal recessive IRAK4 deficiency.4
Mutations in NEMO3,43,44
are associated with defects in NF‐κB activation.
Figure 2Stimulation of fibroblasts. (A) Nuclear protein extracts from SV40‐transformed fibroblast lines from a healthy positive control (C+), a NEMO‐deficient fetus negative control (C–, bearing a large NEMO deletion (more ...)
In patient 1, we detected two heterozygotic mutations in IRAK4
in the intron located between exons 10 and 11 (1189–1 G→T and 1188+520 A→G) (fig 3A, B). We found no other mutation in the remaining exons. The 1189–1 G→T mutation was carried by the father and 1188+520 A→G by the mother (fig 3B). We did not detect these two mutations in 60 healthy Europeans. Reverse transcription PCR (RT‐PCR) of mRNA extracted from fibroblasts of patient 1 showed only trace amounts of full‐length wild‐type mRNA, with no detectable alternative splicing products (fig 4A). The paternal mutation, 1189–1 G→T, located in intron 10, probably affects mRNA splicing, whereas the effect of the maternal mutation, 1188+520 A→G, also located in intron 10, is less predictable. However, patient 1 and his mother produced an alternative mRNA product, corresponding to the retention of a fragment of the intron, as detected by RT‐PCR amplification of the region between exons 10 and 12 (fig 4B). This alternative mRNA was not detected in the patient's father. Western blotting showed that the IRAK4 protein was absent from the patient's fibroblasts (fig 4C), as in two previously described patients with IRAK4 deficiency.4
These data strongly suggest that the two single‐nucleotide mutations carried by the patient affect the splicing of IRAK4 mRNA, and are therefore disease‐causing mutations. Patient 1 is the first IRAK4‐deficient patient with non‐coding mutations to be identified.
Figure 3IRAK4 and NEMO mutations. (A) Mutant (bottom) and wild‐type (top) sequences from patient 1 (P1) (IRAK4, 1188+520 A→G/1189–1 G→T) and patient 2 (P2) (NEMO, R173G). The mutated nucleotides are indicated (more ...)
Figure 4IRAK4 and NEMO expression. (A) Left: IRAK4 expression (full‐length cDNA) in fibroblasts from a healthy positive control (C+), IRAK4‐deficient fibroblasts (C–, mutation Q293X4) and patient 1 (P1). Right: (more ...)
The sequencing of NEMO cDNA in patient 2 led to identification of the R173G mutation in exon 4 (nucleotide 518, C →G), two nucleotides away from the end of the exon (fig 3A, B). The mother of patient 2 was heterozygotic for this mutation, which was not found in 120 European controls (174 X chromosomes; fig 3B). We could not test for X inactivation in the mother's blood cells as she was homozygotic, and therefore not informative for the HUMARA and FMR1 loci (data not shown). Residue R173 in NEMO is conserved in all mammalian species studied to date—rats, mice and cows (NCBI GI40018574, GI31321959 and GI59858109, respectively)—and may help to stabilise the first coiled‐coil domain. RT‐PCR was carried out to amplify the full‐length NEMO cDNA, together with two additional lower‐molecular‐weight splicing products, corresponding to skipping of exons 4–6 and exons 5–6. The mutation therefore had an unexpected effect on mRNA splicing, probably owing to its location near the 3′ boundary of exon 4 (fig 4A). Western blotting showed that NEMO was produced but present in only small amounts in patient 2 (fig 4C). Our data show that patient 1 has autosomal recessive, complete IRAK4 deficiency, whereas patient 2 has X‐linked recessive, partial NEMO deficiency. As a result, both children had recurrent IPD.