PMC6383679_01
Characterization of MYD88-CARD9 deficiency patient: part 1. (A) Family pedigree showing proband (II3), carrier parents (I1, I2), and sisters (II1, II2). Genotype + and - indicate in the figure wild type and mutated, respectively. (B) Percentage of CD3+CD4+IL17+ cells after PMA stimulation (mean +- SEM of n = 2). (C) Sanger sequencing confirmed a homozygous in-frame deletion (c.195_197delGGA) in MYD88 gene and a homozygous splice-donor mutation (c.1434+1G>C) in CARD9 gene. (D) Western Blot of CARD9 and MYD88 proteins performed on PBMC, EBVB, and PHA derived T cell lines. (E) TNFalpha production by monocytes after LPS stimulation (mean +- SEM of n = 2). (F) Phenotypic analysis of iDC and mDC differentiated in vitro. Results indicate the mean percentage +- SD of CD83 maturation/activation markers in gated CD14-CD1a+HLA-DR+ subsets of iDC and mDC. (G) Capacity of MDM to kill (right panel) BCG (*P < 0.05).
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Her sisters deceased, respectively, for meningitis and sepsis of unknown origin (Figure 1A). HD (Pt 0.36 +- 0.14 vs. HDs 1.23 +- 0.19%) (Figure 1B). HaloPlex targeted sequencing panel, including 630 causative and candidate PID genes (Cifaldi et al., manuscript submitted) identified two homozygous mutations in two different genes (Figures 1C,D and Supplementary Table 1). Patient's monocytes showed a marked reduction of TNFalpha-production (7-fold decreased) after LPS-stimulation (Figure 1E), although PB-monocytes (classical, intermediate, and non-classical), and major subtypes of dendritic cells (mDC1, mDC2, and pDC) were normally represented (data not shown). Moreover, immature monocytes-derived DC had a defective ability to become mature/activated-DC (mDC) (3.5-fold decrease) (Figure 1F), while maintaining their phagocytic capacity (data not shown). Additionally, patient's monocytes-derived macrophages (MDMs) failed to kill Bacillus Calmette-Guerin (BCG) in vitro (Figure 1G) after engulfment.
PMC6383679_02
Characterization of MYD88-CARD9 deficiency patient: part 2. (A,B) NADPH oxidase activity evaluated by dihydrorhodamine (DHR) assay in patient's neutrophils and monocytes stimulated with PMA or E. coli. The production of ROS is determined by the oxidation of DHR into the fluorescent rhodamine. (C) INFgamma production by patient's CD3 cells stimulated with PMA or LPS/monocytes. Left panel shows representative plots and right panel shows mean +- SEM of n = 2.
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CARD9 deficient patients. Conversely, we found an impaired capacity to produce ROS in patient's monocytes selectively stimulated with E. coli (7-fold decreased respect to HD) (Figure 2B). To confirm this inability, we tested the capacity of monocytes to stimulate T cells via LPS to produce INFgamma and found a defective LPS/monocytes-triggered INFgamma production by patient's CD3 cells (7-fold decreased) as compared to HD (Figure 2C).