Flow cytometry is a valuable tool for the detection and characterization of proteins expressed by individual cells. Flow cytometry can be used to measure cell expression of 2 intracellular proteins that are involved in the regulation of immune homeostasis, SLAM-associated protein (SAP) and X-linked inhibitor of apoptosis (XIAP). These proteins are defective in patients with the immune deficiency X-linked lymphoproliferative disease (XLP), due to mutations in the SH2D1A and XIAP/BIRC4 genes, respectively (Coffey et al. Nat Genet 20:129-135 1998; Nichols et al. Proc Natl Acad Sci U S A 95:13765-13770, 1998; Sayos et al. Nature 395:462-469, 1998; Rigaud et al. Nature 444:110-114, 2006). This procedure describes a technique that can be efficiently used to detect SAP and XIAP by flow cytometry.
[Show abstract][Hide abstract] ABSTRACT: X-linked inhibitor of apoptosis (XIAP) deficiency caused by mutations in BIRC4 was initially described in patients with X-linked lymphoproliferative syndrome (XLP) who had no mutations in SH2D1A. In the initial reports, EBV-associated hemophagocytic lymphohistiocytosis (HLH) was the predominant clinical phenotype. Among 25 symptomatic patients diagnosed with XIAP deficiency, we identified 17 patients who initially presented with manifestations other than HLH. These included Crohn-like bowel disease (n=6), severe infectious mononucleosis (n=4), isolated splenomegaly (n=3), uveitis (n=1), periodic fever (n=1), fistulating skin abscesses (n=1) and severe Giardia enteritis (n=1). Subsequent manifestations included celiac-like disease, antibody deficiency, splenomegaly and partial HLH. Screening by flow cytometry identified 14 of 17 patients in our cohort. However, neither genotype nor protein expression nor results from cell death studies were clearly associated with the clinical phenotype. Only mutation analysis can reliably identify affected patients. XIAP deficiency must be considered in a wide range of clinical presentations.
[Show abstract][Hide abstract] ABSTRACT: X-linked lymphoproliferative syndromes (XLP) are rare primary immunodeficiencies. Mutations within the XIAP/BIRC4 gene characterize XLP type 2 and cause XIAP deficiency. We present the case of a 5-year-old boy with a novel mutation of the XIAP/BIRC4 gene and describe the immunological phenotype for the first time. We characterized the distinct immunological phenotype and evaluated the family history accordingly.
[Show abstract][Hide abstract] ABSTRACT: X-linked Inhibitor of Apoptosis (XIAP) deficiency, caused by mutations in BIRC4, is an immunodeficiency associated with immune dysregulation and a highly variable clinical presentation. Current diagnostic screening tests such as flow cytometry for XIAP expression or lymphocyte apoptosis assays have significant limitations. Based on recent evidence that XIAP is essential for NOD1/2 signalling, we evaluated the use of a simple flow cytometric assay assessing TNF production of monocytes in response to NOD2 stimulation by muramyl dipeptides (L18-MDP) for the functional diagnosis of XIAP deficiency. We investigated 12 patients with XIAP deficiency, 6 female carriers and relevant disease controls. Irrespective of the diverse clinical phenotype, the extent of residual protein expression or the nature of the mutation, the TNF response was severely reduced in all patients. On average, L18-MDP induced TNF production in 25% of monocytes from healthy donors or female carriers, while less than 6% of monocytes responded in affected patients. Notably, the assay clearly discriminated affected patients from disease controls with other immunodeficiencies accompanied by lymphoproliferation, hypogammaglobulinaemia or inflammatory bowel disease. Functional testing of the NOD2 signalling pathway is an easy, fast and reliable assay in the diagnostic evaluation of patients with suspected XIAP deficiency.
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