Clinical and Analytical Validation of a NOD2 Functional Test for XIAP/XLP2 Diagnosis

Most pathogenic variants in the BIRC4/XIAP gene lead to absent or very low XIAP protein expression distinguishable via protein staining, but missense variants may allow expression of reduced or normal levels of XIAP. Thus, normal protein levels alone cannot fully exclude possible XIAP deficiency. As such, an assay examining function is needed to complement XIAP protein expression. We present clinical and analytical validation data of a NOD2 stimulation assay for the diagnosis of XIAP functional deficiency. Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized whole blood. Cells were stimulated for 2 h with either LPS as a positive control (signaling is independent of XIAP) or L18-MDP, which signals through NOD2 and requires XIAP for normal signaling. Cells were stained for viability and lineage markers before being fixed and permeabilized, followed by staining for TNF-α and IL-8. Results were acquired on a BD FACSLyric flow cytometer. The increase in TNF-α and IL-8 levels (delta) was calculated for LPS and MDP stimulations by subtracting levels found in untreated cells (Figure 1A). Figure 1. Functional characterization of an XIAP deficiency cohort. (A) Histograms depict monocyte responses to either PBS for mock stimulation, LPS as a positive control, and MDP as the test stimulation. Shown for a healthy adult and an XIAP patient bearing c.1141C>T, p.Arg381Ter. Percent positive cells gated displayed above each marker for both TNF-α and IL-8. (B) Tukey box plots comparing control and patient NOD2 functional readouts. Dotted lines represent calculated cutoffs to obtain 100% accuracy by ROC analysis. A total of 19 male patients were collected, of which there were 4 sets of siblings. Age at sampling ranged between 1 month and 56 years old. For controls, we ran 50 samples between 9 and 69 years of age. XIAP expression was normal in 11% of this cohort. All patients had defective TNF-α and IL-8 upregulation upon MDP stimulation (Figure 1B). The results were reproducible upon patent resampling over several years. No trends in age or sex were noted among controls. Our data demonstrate the importance of a functional NOD2 assay for the functional confirmation of XIAP deficiency. LPS stimulation produces robust TNF-α and IL-8 response, while response to MDP in XIAP-deficient cases is close to null. The test returned excellent accuracy and typically requires only 4-6 mL whole blood.