Identifying genetic susceptibility to fungal infectious diseases in Colombian patients

Abstract

ABSTRACT: Introduction: The landscape of human genes involved in the immune response is increasingly clear. Most mutations are associated with conventional Primary Immunodeficiency Diseases (PIDs), which confers a Mendelian predisposition to multiple infectious diseases; including fungal infections where each type of infection is highly suggestive of a specific type PID. However, the genetic susceptibility to fungal infections such as Paracoccidioides brasiliensis, Histoplasma capsulatum and Corynespora cassiicola is not fully undertood. Objective: To identify the molecular defects in a cohort of five unrelated Colombian patients with invasive fungal infections caused by Paracoccidiodes brasiliensis, Histoplasma capsulatum and Corynespora cassiicola. Methods: This is a descriptive study that presents a molecular characterization of five Colombian patients with confirmed invasive fungal infections (IFIs). Patient 1 (P1) presented with Corynespora cassiicola infection, P2 had disseminated histoplasmosis, and three more patients (P3, P4 and P5) presented juvenile Paracoccidioidomycosis (PMC). All patients were children with less than 15 years of age and all were HIV-negative. We reviewed medical records and performed PCR, tissues staining, western blotting, flow cytometry, immunological evaluation and whole exome sequencing (WES) in samples from all patients. Sanger sequencing was also used to confirm the genetic variants in patients and relatives. Results: Genetic analysis of CARD9 in patient P1 showed compound heterozygosity for a frameshift mutation with premature stop codon in exon 2 (c.23_29del; p.Asp8Alafs10X) and a nonsense mutation in exon 6 (c.C865T; p.Q289X). The p. Asp8Alafs10X has not been previously reported in the literature. CARD9 protein expression was absent in peripheral blood mononuclear cells (PBMCs) from the patient. Genetic analysis of patient P2, identified in NCF4 a homozygous genetic variation in exon 3 (c.C172T; p.R58C). Immunoblot analysis demonstrated a significant reduction of p40phox and p67phox proteins in Epstein Barr Viruses (EBV) transformed B cells line and neutrophils from the patient. His neutrophils and EBV-B cells showed a significant defect in superoxide production during phagocytosis and fMLF stimulation, whereas extracellular release of superoxide elicited by phorbol ester was unaffected. Finally, in patients P3, P4 and P5 affected with juvenile PMC, WES revealed candidate variants in IL17RA, IL18R1, NLRP2 and PIK3CA were in silico studies predicted to be deleterious or pathogenic. We are currently investigating the impact of these variations at the protein expression and functional levels. Conclusion: We studied five unrelated Colombian patients with severe IFIs: A patient with severe phaeohyphomycosis (P1) by C. cassiicola and compound heterozygous mutations, including the novel variant (p.Asp8Alafs10X) in the CARD9 gene. In a patient with disseminated histoplasmosis (P2), we identified a novel homozygous mutation in the NCF4 gene conferring susceptibility to microorganism, suggesting that p40phox protein plays an essential role during oxidative responses especially in fungal clearance. In the three patients with Juvenile PMC (P3, P4 and P5) although several candidate gene variants were selected to be good candidate genes according our analysis criteria, further re-analysis and functional assays are necessary. This information represents the first molecular characterization of Colombian patients with severe IFIs. This information can be useful to employ the specific and proper treatment and to promote in near future new therapeutic strategies in order to improve the life quality of these patients.