Immunogenetics associated with severe coccidioidomycosis

Abstract

Disseminated coccidioidomycosis (DCM) is caused by Coccidioides, pathogenic fungi endemic to the southwestern United States and Mexico. Illness occurs in approximately 30% of those infected, less than 1% of whom develop disseminated disease. To address why some individuals allow dissemination, we enrolled patients with DCM and performed whole-exome sequencing. In an exploratory set of 67 patients with DCM, 2 had haploinsufficient STAT3 mutations, and defects in β-glucan sensing and response were seen in 34 of 67 cases. Damaging CLEC7A and PLCG2 variants were associated with impaired production of β-glucan-stimulated TNF-α from PBMCs compared with healthy controls. Using ancestry-matched controls, damaging CLEC7A and PLCG2 variants were overrepresented in DCM, including CLEC7A Y238* and PLCG2 R268W. A validation cohort of 111 patients with DCM confirmed the PLCG2 R268W, CLEC7A I223S, and CLEC7A Y238* variants. Stimulation with a DECTIN-1 agonist induced DUOX1/DUOXA1-derived hydrogen peroxide [H2O2] in transfected cells. Heterozygous DUOX1 or DUOXA1 variants that impaired H2O2 production were overrepresented in discovery and validation cohorts. Patients with DCM have impaired β-glucan sensing or response affecting TNF-α and H2O2 production. Impaired Coccidioides recognition and decreased cellular response are associated with disseminated coccidioidomycosis.

Keywords: Fungal infections; Genetics; Infectious disease; Innate immunity; Population genetics.

Figure 1. Fungal recognition variants in patients with DCM.

(A) Fold-enrichment of CLEC7A, c.714T>G; p.Y238* variant in patients with DCM compared with the gnomAD. Normalized to frequency of homozygous WT or variant carriers in gnomAD. P = 0.0303, Fisher’s exact test. (B) Parallel signaling pathways after β-glucan recognition by DECTIN-1 leading to activation of NF-κB and NFAT transcription factors and production of TNF-α. The figure was created using BioRender. (C) Confocal microscopy of lung from C. posadasii–infected C57BL/6 mouse showing DECTIN-1 (red) localized near endospores (blue) (left), LAMP-1 (green) localization near endospores (middle), and colocalization of DECTIN-1 and LAMP-1 (tan) around endospores (right). (D) Frequency of patients of European ancestry with DCM with PLCG2, c.802C>T; p.R268W genotype normalized to the non–Finnish European population in gnomAD. P = 0.0077 Fisher’s exact test. (E) Particulate β-glucan–induced TNF production by PBMCs from patients (n = 16) or healthy control (HC) participants (n = 12). DECTIN-1 variants (n = 4) include homozygous p.Y238* (filled) and heterozygous p.Y238* (open). Patients withPLCG2 variants (n = 6) include p.R268W heterozygotes (yellow symbols), p.M28L heterozygotes (green symbol), and p.R268W and p.K775R compound heterozygotes (open yellow symbol). Patients in the “other” category (n = 6) lack identified causal variants. P values were calculated using Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test. (F) Frequency of Y238* among East Asian patients from DCM validation cohort compared with the 1000G and gnomAD.

Figure 2. DECTIN-1 signaling drives H₂O₂ production by DUOX1 and DUOXA1 in HEK cells.

(A) DECTIN-1–activated PLCγ2 releases intracellular Ca⁺⁺, which activates the EF-hand domains of DUOX1, leading to H₂O₂ production. DUOX1 and DUOXA1 patient variants are highlighted by yellow stars. (B) DUOX1 variants identified in patients with DCM were transfected into HEK Flp-In cells stably expressing WT DUOXA1. Cells were stimulated with ionomycin, and H₂O₂ production was measured for 60 minutes. Results are the average of triplicate wells presented as the ratio of H₂O₂ production by variant or WT; each dot represents the average of triplicate wells from a unique experiment. ****P < 0.0001 by ordinary 1-way ANOVA and Dunnett’s multiple comparisons test. Western blot showing decreased protein abundance of several DUOX1 variants after transfection. (C) DUOXA1 variants, identified in patients with DCM, transfected into HEK Flp-In cells stably expressing DUOX1. Measured as in B. P = 0.016 by ordinary 1-way ANOVA and Dunnett’s multiple comparisons test. (D) Hydrogen peroxide production in HEK cells transfected with WT or patient variant DUOX1, DUOXA1, or DECTIN1, or lacking PLCG2 constructs. Results are the average of triplicate wells presented as the ratio of H₂O₂ production by variant/WT; each dot represents a unique experiment. ****P < 0.0001 using ordinary 1-way ANOVA and Dunnett’s multiple comparisons test.

Figure 3. Exploratory cohort: 34 patients with DCM with identified fungal pattern recognition pathway or DUOX1 / DUOXA1 variants.