(E-F) Neither full-length nor truncated mutant IKK(R286X) protein is detectable in patients (PT), siblings, and normal peripheral blood mononuclear cells (E) and EBV-transformed B cells (F) by immunoblotting analysis with anti-N- and anti-C-terminal IKK antibodies

(E-F) Neither full-length nor truncated mutant IKK(R286X) protein is detectable in patients (PT), siblings, and normal peripheral blood mononuclear cells (E) and EBV-transformed B cells (F) by immunoblotting analysis with anti-N- and anti-C-terminal IKK antibodies. NK-cell numbers. Moreover, patients T cells were mostly CD45RA+-naive cells and were defective in activation after T-cell receptor stimulation. All patients contained the same S1RA homozygous nonsense mutation in (R286X), revealed by whole-exome sequencing with undetectable IKK and severely decreased NEMO proteins. Mutant IKK(R286X) was unable to complex with IKK/NEMO. Immortalized patient B cells displayed impaired IB phosphorylation and NFB nuclear translocation. These data indicate that S1RA mutated is the likely cause of immunodeficiency in these 4 patients. Introduction Mutations in genes important in T-cell or in both T- and B-cell development and function cause severe combined immunodeficiency (CID), with a majority of cases caused by mutations in in 4 patients with CID from 2 unrelated families and compare and contrast our findings with those in 2 other recent reports of mutations in this gene.15,16 Patient, materials, and methods Abbreviated information is presented here. Details are provided in the supplemental Data, available on the Web site. Patients All studies were performed with the approval of the Duke University Medical Center Institutional Review Board, and written informed consent of the patients parents was collected in accordance with the Declaration of Helsinki. The patients were members of 2 unrelated consanguineous families. Select clinical features are presented in Table 1, with further details available in the supplemental Data. Table 1 Clinical features, immunoglobulins, and lymphocyte subsets test was performed (*< .05; **< .01; ***< .001; Figure 1). Open in a separate window Figure 1 Contribution of IKK(R286X) mutation to CID. (A-C) Patients blood lymphocytes responded normally when stimulated with phytohemagglutinin (PHA) but had variably low responses to Rabbit Polyclonal to ELOVL5 concanavalin A (Con A) and pokeweed mitogen (A). However, they failed to respond when stimulated with candida and tetanus antigens (B) or soluble or immobilized anti-CD3 (C). (D) Impaired upregulation of T-cell activation markers in patients CD4 T cells after overnight stimulation with plate-bound anti-CD3 or phytohemagglutinin. (E-F) Neither full-length nor truncated mutant IKK(R286X) protein is detectable in patients (PT), siblings, and normal peripheral blood mononuclear cells (E) and EBV-transformed B cells (F) by immunoblotting analysis with anti-N- and anti-C-terminal IKK antibodies. (G) mRNA levels of IKK, IKK, and NEMO in PMCs detected by real-time quantitative polymerase chain reaction. (H) IKK(R286X) is not able to form a complex with IKK/NEMO. Cell lysates from Phoenix-Eco cells transfected with Flag-tagged-FL-IKK, Flag-tagged-IKK(R286X), or vector control were subjected to immunoblotting analysis directly (left) or after immunoprecipitation with anti-Flag antibody conjugated agarose beads (right). (I) Defective IB/NFB signaling in patient-derived B cells can be reverted by full-length (long) but not mutant IKK(R286X). EBV-transformed B cells of a sibling and patients stably infected with retrovirus expressing WT IKK or with control vector were rested in phosphate-buffered saline at 37C for 30 minutes, followed by PMA stimulation for 10 and 20 minutes. Immunoblotting analysis of cytosolic fractions (top) or nuclear extracts (bottom) with indicated antibodies. (J) Decreased expansion of patient-derived B cells can be corrected by full-length but not mutant IKK. *< .05; **< .01; ***< .001 determined by Student test. Results and discussion All 4 infants were hypogammaglobulinemic (Table 1). Three demonstrated normal or elevated numbers of T cells and normal numbers of B cells but low numbers of switched memory B cells and NK cells. Most T cells were CD45RA+. There were abnormally low numbers of CD45RO+ T cells and CD4+CD25bright or S1RA CD4+FOXP3+ T-regulatory cells (Treg) (supplemental Figure 1). All patient T cells displayed normal responses to phytohemagglutinin, but low responses to pokeweed mitogen or concanavalin A were seen in 3 of them (Figure 1A). Strikingly, all patients T cells failed to respond to candida or tetanus toxoid antigens or anti-CD3 stimulation (Figure 1B-C). Moreover, patients CD4 T cells were impaired for T-cell receptor-induced CD25 and CD69 upregulation (Figure 1D). NK-cell function, tested in 1 patient from each family, was impaired in both patients (data not shown). Whole exome sequencing on patients 1 and 2 representing the 2 2 unrelated families identified a single candidate nonsense homozygous variant in (R286X). The other 2 patients and the parents carried the same homozygous and heterozygous variant, respectively (supplemental Figure 1C-D), which was absent in the 998 sequenced controls and in the exome variant server public database (Exome Variant Server, National Heart, Lung and Blood Institute [NHLBI] Grand Oppportunity [GO] Exome Sequencing Project, Seattle, WA [URL: http://evs.gs.washington.edu/EVS/]). Contrary to in healthy controls and a heterozygous sibling, anti-N- or -C-terminal IKK antibodies failed to detect any full-length or truncated IKK in patients peripheral blood mononuclear cells and EBV-transformed B-cell lines (Figure 1E-F) that was not caused by possible low quality of the antibody (supplemental Figure 1E). Interestingly, NEMO.