A homozygous CARD9 mutation in a family with susceptibility to fungal infections

Abstract

Background: Chronic mucocutaneous candidiasis may be manifested as a primary immunodeficiency characterized by persistent or recurrent infections of the mucosa or the skin with candida species. Most cases are sporadic, but both autosomal dominant inheritance and autosomal recessive inheritance have been described.

Methods: We performed genetic studies in 36 members of a large, consanguineous five-generation family, in which 4 members had recurrent fungal infections and an additional 3 members died during adolescence, 2 after invasive infection of the brain with candida species. All 36 family members were enrolled in the study, and 22 had blood samples taken for DNA analysis. Homozygosity mapping was used to locate the mutated gene. In the 4 affected family members (patients) and the 18 unaffected members we sequenced CARD9, the gene encoding the caspase recruitment domain-containing protein 9, carried out T-cell phenotyping, and performed functional studies, with the use of either leukocytes from the patients or a reconstituted murine model of the genetic defect.

Results: We found linkage (lod score, 3.6) to a genomic interval on chromosome 9q, including CARD9. All four patients had a homozygous point mutation in CARD9, resulting in a premature termination codon (Q295X). Healthy family members had wild-type expression of the CARD9 protein; the four patients lacked wild-type expression, which was associated with low numbers of Th17 cells (helper T cells producing interleukin-17). Functional studies based on genetic reconstitution of myeloid cells from Card9(-/-) mice showed that the Q295X mutation impairs innate signaling from the antifungal pattern-recognition receptor dectin-1.

Conclusions: An autosomal recessive form of susceptibility to chronic mucocutaneous candidiasis is associated with homozygous mutations in CARD9.

Figure 1. Pedigree of an Iranian Family with Chronic Mucocutaneous Candidiasis

In Panel A, circles denote female family members; squares male family members; solid circles and squares patients with chronic mucocutaneous candidiasis, who were homozygous for the Q295X mutation; half-solid circles and squares members who were heterozygous for the Q295X mutation; open circles and squares healthy members with wild-type CARD9, and double horizontal lines consanguinity in a married couple. A slash denotes a deceased family member. Asterisks indicate family members whose samples were sequenced. In Panel B, the sequence at the top is for a healthy family member with wild-type CARD9. The middle sequence, obtained from Patient 1M, is characteristic of a person with a homozygous CARD9 mutation, in which a single-nucleotide exchange (C→T) in exon 6 of CARD9 results in a premature stop codon (Q295X). The bottom sequence, from Patient 1G1, is characteristic of a healthy heterozygous person.

Figure 2. Western Blot Detection of CARD9 in Peripheral-Blood Mononuclear Cells

Macrophages from Card9 wild-type mice (Card9^+/+) and Card9 knockout mice (Card9^−/−) were used as positive and negative controls (lanes 1 and 2), respectively. The blot in lane 4 is from Relative 4F, that in lane 5 from Relative 2G2, and that in lane 6 from Patient 2M. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control.

Figure 3. Analysis of the Functional Defect and Genetic Reconstitution of the Card9 Q295X Mutation

Bone-marrow–derived macrophages from wild-type mice and Card9^−/− mice were stimulated for 16 hours with both the dectin-1 agonist curdlan (300 μg per milliliter) and the toll-like-receptor agonist lipopolysaccharide (LPS, 100 ng per milliliter) (Panel A). The concentration of secreted tumor necrosis factor (TNF-α) was determined in the cell supernatant with the use of an enzyme-linked immunosorbent assay. To examine the effect of mutated CARD9 Q295X on signal transduction (Panel B), we cloned human wild-type CARD9 complementary DNA (cDNA) and mutated CARD9 Q295X cDNA into a retroviral expression vector. Using these constructs, we retrovirally transduced primary bone marrow cells from Card9^−/− mice with either wild-type CARD9 or the mutated CARD9 Q295X. To establish a control, we transduced bone marrow cells from Card9^+/+ wild-type mice and Card9^−/− mice with a control vector only. The transduced bone marrow cells were then differentiated into macrophages in vitro. After stimulation of the macrophages with curdlan for 16 hours, the concentration of secreted TNF-α was determined. The macrophages of Card9^−/− mice transduced with human wild-type CARD9 secreted as much TNF-α upon stimulation with curdlan as did the control macrophages of wild-type Card9^+/+ mice with the control vector. In contrast with these cells, the macrophages of Card9^−/− mice transduced with mutated human CARD9 Q295X or with a control vector only did not respond with increased secretion of TNF-α upon stimulation with curdlan, a finding showing that CARD9 Q295X is a loss-of-function mutation that cannot correct the dectin-1/Card9 signaling pathway in the cells of Card9^−/− mice. T bars indicate standard deviations in three independent experiments.

Figure 4. Proportion of Interleukin-17–Producing CD4⁺CD45RO⁺ Cells That Were Negative for Interferon-γ Production in Four Patients with a Homozygous Mutation in CARD9 as Compared with Unrelated Healthy Controls

Cells were surface-stained for CD4 and CD45RO and then subjected to intracellular staining for interleukin-17 and interferon-γ. CMC denotes chronic mucocutaneous candidiasis.