PMC6101191_01
Identification of a mutation in IKBKG in a family with female Behcet's disease and male immunodeficiency and intestinal inflammation. (A) Pedigree of the family with IKBKG gene mutation. Diagnosis of familial Behcet's disease was made in patients 1, 2 and 3. Patients 1 and 3 were HLA-B51 positive. Patient (Pt.) 5 (index patient) underwent HSCT from his sister (patient 3). (B) Western blot analysis of PBMC using a NEMO specific antibody. The 48kDa band representing full length NEMO protein is absent in the patient (black arrow). Instead, a truncated protein of approximately 40 kD is more prominent than in the control. (C) Gel electrophoresis of RT-PCR products upon amplification with primers flanking exon 3 to 7 of IKBKG. The approximately 600 bp product corresponds to the wildtype sequence of IKBKG, while in the patient a shorter, truncated product lacking exon 5 dominates (black arrow). (D) NK cell degranulation as assessed by CD107a expression of fresh NK cells before and after stimulation with K562 cells. DeltaCD107 represents the increase in the percentage of CD107 positive cells after stimulation; the normal range (grey area) starts above 10%. The assay was performed before (left plot) and after HSCT (right plot). (E) X-chromosomal inactivation pattern in the index patient (pt. 5) before and after HSCT, his mother (pt. 1) and sister (pt. 3, HSCT donor). The AR allele carrying the IKBKG mutation is depicted in red, wt alleles are depicted in black. Active (unmethylated) alleles are reduced after HhaI digestion (lower row). Please note: while the 276 bp fragment in pt. 3 corresponds to the paternally-inherited wt X-chromosome, the 284 bp fragment has the same fragment length as the maternal X-chromosome harboring the wild-type IKBKG-allele. However, sequencing the IKBKG-gene revealed that the sister was heterozygous for the same mutation present in pt. 1 and pt. 5. Further analysis of microsatellite markers revealed a X-chromosomal genetic recombination event as a cause of this unexpected finding in the sister (pt. 3) (supplementary figure 1).
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Two displayed incontinentia pigmenti (Fig. 1A). A western blot showed the virtual absence of the 48kDa band expected for full length NEMO in patient's cells, but instead displayed a truncated protein of approximately 40kDa (Fig. 1B). RT-PCR followed by sequencing indicated that this shorter isoform is generated from an alternatively spliced mRNA lacking exon 5 (Fig. 1C). Consistent with previous reports of NEMO deficiency, NK cell cytotoxic function was markedly impaired underscoring the functional relevance of the mutation identified (Fig. 1D). Mother and sister were heterozygous for the mutation and, strikingly, the sister showed skewed X-inactivation favoring the mutant NEMO allele (Fig. 1E). In our patient, HSCT resulted in full donor chimerism and functional immune reconstitution (Fig. 1D) without signs of graft-versus-host-disease. Skewed X-inactivation was even more pronounced in the recipient showing only 10% of cells with an active wild-type allele (Fig. 1E).
Severe inflammatory bowel disease after HSCT was successfully treated with Anti-TNF therapy.. (A) Small bowel (upper panel) and colonic (lower panel) pathology in NEMO deficient patient after HSCT (prior to anti-TNF treatment) as indicated by H&E staining. Upper panel: small bowel biopsy displaying irregular epithelial lining and epithelial cell tufting (arrow and inset) indicating apoptosis. Lower panel: epithelial ulcerations (black arrow) in the colon are characterized by severe epithelial cell damage with underlying mononuclear and lymphoid inflammatory infiltration of the lamina propria. The intact mucus layer above the ulcerations excludes a sampling artifact of the biopsy (dashed arrow). (B) Immunohistochemistry of colon tissue from the NEMO deficient patient (lower panel) and a pediatric control patient with Crohn's disease (upper panel) using anti-p65 antibodies. In the NEMO deficient patient intestinal epithelial cells show largely absent nuclear p65 staining (white arrow), while infiltrating donor-derived immune cells stain strongly for nuclear p65 (black arrows). In the control sample, strong p65 staining is shown in the intestinal epithelial cells and the infiltrating immune cells (black arrows). Sizing bar indicates 100 microm. (C) Photograph illustrating the extended perianal, scrotal and inguinal skin inflammatory lesions, which resolved completely upon escalated anti-TNF treatment.
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Several months after transplantation our patient developed erythema nodosum and severe inflammatory bowel disease (Fig. 2 A&C). Histological features included small intestinal epithelial cell tufting indicating apoptosis and multiple large intestinal ulcerations underneath an intact mucus layer (Fig. 2 A). In contrast, his intestinal epithelial cells displayed only weak staining for nuclear p65, whereas epithelial cells from 3 control patients with inflammatory bowel disease stained strongly (Fig. 2 B). In our patient complete remission of intestinal inflammation was only achieved upon escalated infliximab therapy (10 mg/kg every 3-4 weeks, trough levels 17 microg/ml (normal target range: 3.0-7.0 microg/ml), no anti-infliximab antibodies detectable) (Fig. 2 C).