Frequency and Geographic Distribution of CARD9 Mutations in Patients With Severe Fungal Infections

Autosomal recessive deficiency in the caspase recruitment domain containing protein 9 (CARD9) results in susceptibility to fungal infections. In the last decade, infections associated with CARD9 deficiency are more reported due to the advent of genome sequencing. The aim of this study was to evaluate the frequency, geographic distribution and nature of mutations in patients with CARD9 deficiency. We identified 60 patients with 24 mutations and different fungal infections. The presence of the homozygous (HMZ) p.Q295X (c.883C > T) and HMZ p.Q289X (c.865C > T) mutations were associated with an elevated risk of candidiasis (OR: 1.6; 95% CI: 1.18–2.15; p = 0.004) and dermatophytosis (OR: 1.85; 95% CI: 1.47–2.37; p < 0.001), respectively. The geographical distribution differed, showing that the main mutations in African patients were different Asian patients; HMZ p.Q289X (c.865C > T) and HMZ p.Q295X (c.865C > T) accounted for 75% and 37.9% of the African and Asian cases, respectively. The spectrum of CARD9 mutations in Asian patients was higher than in African. Asia is the most populous continent in the world and may have a greater genetic burden resulting in more patients with severe fungal infections. The presence of a high diversity of mutations revealing 24 distinct variations among 60 patients emphasize that the unique genetic alteration in CARD9 gene may be associated with certain geographical areas.


INTRODUCTION
Susceptibility to fungal infections in otherwise healthy individuals with Mendelian disorders are increasingly being recognized (Vinh, 2011) than before the widespread use of genome sequencing. Primary immunodeficiencies consist of various genetic defects that affect the innate and adaptive immune systems. In addition, evaluation of previously healthy, fungus infected patients, suspected of having a primary genetic immunodeficiency may give valuable insights on the role of specific proteins in the immune system for protection from these infections (Wang et al., 2014;Corvilain et al., 2018). Caspase recruitment domain containing protein 9 (CARD9) is a central regulator of innate immunity that is highly expressed in neutrophils, macrophages, dendritic cells, and during cell apoptosis in low-serum conditions (Bertin et al., 2000;Liang et al., 2015). Mutations in several proteins involved in the CARD9 signaling protein have been demonstrated to cause primary immunodeficiencies in humans. These mutations cause a decreased production of cytokines from innate immune cells, leading to deficiencies of TH17 and accordingly predispose patients to severe disseminated infections (Conti and Gaffen, 2015). Severe fungal infections in healthy patients have recently been reported from a few countries, i.e., Algeria, Brazil, France, China, Iran, Morocco and Tunisia (Glocker et al., 2009;Drewniak et al., 2013;Lanternier et al., 2013;Wang et al., 2014;Grumach et al., 2015) and linked to autosomal recessive CARD9 deficiency. The species involved in these infections are Trichophyton violaceum, Trichophyton rubrum, Candida species, Exophiala species, Phialophora verrucosa, Aspergillus fumigatus, Prototheca zopfii, and Mucor irregularis. Some of those etiological agents are plant pathogens, which rarely have been associated with human infection. Highly diverse clinical manifestations from cutaneous to disseminated and progressive infections are observed (Boudghène-Stambouli and Mérad-Boudia, 1991;Boudghène-Stambouli et al., 1992;Pruszkowski et al., 1995). Our aim was to evaluate the global frequency, geographic distribution and nature of mutations in patients with CARD9 deficiency associated with fungal infections.

MATERIALS AND METHODS
The review process involved study of existing published literature of all reported cases with fungal infection due to CARD9 deficiency. To search the published literature, Medline database through PubMed, Embase through Scopus, ISI Web of Science, Science Direct and Google Scholar were used to explore the published literature of patients with severe fungal infection and CARD9 deficiency using the key words "caspase recruitment domain deficiency, " "CARD9 deficiency, " "autosomal recessive CARD9 deficiency, " "primary immunodeficiency, " "mutations, " "fungal infection" or "invasive fungal diseases, " "candidiasis, " "deep dermatophytosis, " "disseminated phaeohyphomycosis, " and "chronic mucocutaneous candidiasis" in different combinations.
A total of 21 relevant articles were found using these key words. The extracted data were analyzed using R software version 3.4.1. The chi-square test was utilized to evaluate associations between nominal variables and the p-value was estimated using the Monte Carlo method. To compare the differential prevalence of CARD9 mutations and determine differences in causative agents of fungal infections, odds ratios (ORs) were used. The significance of all ORs, using a 95% Bayesian credible interval (CI), was calculated using Bayesian logistic regression.

A Relationship Between CARD9 Gene Mutations and Specific Geographic Distribution
The pattern of distribution was differed by geographical region in reported cases with CARD9 mutations. The main mutations in African patients were different from those in Asians; HMZ p.Q289X (c.865C > T) and HMZ p.R101C (c.C301T), accounting for 75% and 10%, respectively, were the common mutations in Africa. The three most common mutations in Asia were HMZ p.Q295X (c.883C > T), HMZ p.D274fsX60 (c.819-820insG), and HMZ p.R70W (c.208C > T), which accounted for 34.5%, 17.2%, and 13.8% of the Asian cases, respectively. Notably, HMZ p.Q289X (c.865C > T) was the most common mutation observed in 75% of the Algerian patients (9 out of 12), while the HMZ p.Q295X (c.883C > T) mutation was reported in 8 out of 10 Iranian patients (80%). This finding is important as it provides a relationship between mutation and specific geographic occurrence in these patients.

DISCUSSION
CARD9 deficiency is inherited in an autosomal recessive manner. CARD9 plays an important role in the activation of antifungal mechanisms leading to expression of gene products that initiate the inflammatory cascade (Liang et al., 2015;Drummond and Lionakis, 2016). The importance of the CARD9 signaling protein in host defense has been demonstrated in a murine CARD9−/− model with targeted disruptions of innate signaling from the antifungal pattern-recognition receptor, dectin-1, that identifies the β-glucan component of the fungal cell (Taylor et al., 2007). Defective antifungal clearance and latently infected cells could be the result of impaired CARD9 function (Yamamoto et al., 2014;Drummond and Lionakis, 2016). We analyzed the characteristics, distribution, frequency, and relationship between the genotype of the CARD9 gene mutations and fungal infections among the reported cases. Since the first mutation described in 1989 from Algeria (Boudghène-Stambouli and Mérad-Boudia, 1989), several mutations have been reported from Africa. However, only few reports are from Europe and America. Glocker et al. (2009) United Kingdom, and Turkey, respectively, which suggests that mutations may be specific in particular populations or geographic regions. Another possible explanation is the high rate of consanguinity in many closed groups. Although this autosomal recessive disorder which is rare on a world-wide scale, it may not be rare in some countries. The variations in the gene, which are associated with a specific fungal infection, remain unknown. Asia is the most populous continent in the world and may have a greater genetic burden resulting in more patients with severe fungal infections. Although we cannot exclude other causative factors, our data support the notion that some CARD9 mutations, circulating in specific geographic regions, could be the contributing factor for fungal infections. However, because of the small sample size, future screening should be conducted to confirm these conclusions. Studying the impact of genetic variation on severe fungal infection will improve our understanding of pathogenesis and may ultimately aid future interventions. CARD9 deficiency should be considered in patients with unexplained progressive fungal infection, as it may allow early initiation of appropriate antifungal treatment. Regular medical follow-up and identification of patients with CARD9 deficiencies is recommended including family members.

CONCLUSION
In recent years, interest in primary immunodeficiency disorders and opportunistic infections has grown. The current study reviewed 60 reported cases with CARD9 mutations and severe fungal infections, which may provide more information about the relationship between these mutations, the specific geographic presence and the unique predisposition to a particular fungal disease.

AUTHOR CONTRIBUTIONS
AV, HB, and JM conceptualized the study, gathered resources, and wrote, reviewed, and edited the manuscript. AV, HF, ZA, MG, SK, and AA curated the data. AV, HB, and AA performed the formal analysis of the study. HB contributed to funding acquisition, project administration, and data validation. AV and HF investigated the data. AV, HF, ZA, MG, and SK provided methodology for this study. HB and JM supervised the study. AV, HF, ZA, MG, SK, AA, JM, and HB wrote the original draft of the manuscript.