A new functional assay for the diagnosis of X-linked inhibitor of apoptosis (XIAP) deficiency

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 nucleotide-binding and oligomerization domains (NOD)1/2 signalling, we evaluated the use of a simple flow cytometric assay assessing tumour necrosis factor (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, six 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 fewer 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.

Keywords: Crohn's disease; MDP; NOD2; XIAP; XLP.

Figure 1

(a) 6-O-stearoyl-MDP muramyldipeptide (L18-MDP) (lipidated derivative of muramyl dipeptide) is recognized by nucleotide-binding and oligomerization domain (NOD2) and induces cytokine production via recruitment of X-linked inhibitor of apoptosis (XIAP) and receptor-interacting serine–threonine kinase 2 (RIPK2). XIAP induces RIPK2 ubiquitilation leading to recruitment of the linear ubiquitin chain assembly complex (LUBAC) complex followed by nuclear factor-kappa B (NF-kB) activation [8]. (b) Gating strategy for tumour necrosis factor (TNF)-producing macrophages as defined by forward-/side-scatter, CD14 and human leucocyte antigen D-related (HLA-DR) expression. (c) TNF-producing monocytes from a healthy donor, an asymptomatic XIAP carrier and a XIAP patient cultured in the presence of medium alone, 200 ng/ml L18-MDP or 200 ng/ml lipopolysaccharide (LPS). The percentages indicate the percentage of TNF-positive cells of all HLA-DR⁺CD14⁺ monocytes.

Figure 2

(a) Overlay of staining with anti-X-linked inhibitor of apoptosis (XIAP) and an isotype control antibody, gated on CD3⁺ T lymphocytes. XIAP patients (upper panel) are shown with their day control (lower panel). (b,c) Overnight rested monocytes from healthy donors, XIAP carriers (mothers of affected patients), XIAP patients and other disease controls, as indicated, were stimulated either with 6-O-stearoyl-MDP muramyldipeptide (L18-MDP) (b) or with lipopolysaccharide (LPS) (c). Intracellular tumour necrosis factor (TNF) production was measured via flow cytometry. The numbers indicate the increase in the fraction of monocytes producing TNF upon stimulation, calculated by subtracting the percentage of monocytes producing TNF after incubation in medium alone from that measured after providing the stimulus. P-values were calculated with a two-tailed t-test.