A novel CARD11 heterozygous missense variant in a CADINS patient

Abstract

Background: CARD11-associated atopy with dominant interference of NF-κB signaling (CADINS) is developed as a result of heterozygous loss-of-function variants in CARD11 that function as strong dominant-negative alleles. In lymphocytes, CARD11 encodes a scaffold protein that links activation of the antigen receptor with downstream signaling. Patients with CADINS generally experience severe atopic dermatitis, asthma, recurrent pneumonia and other upper respiratory tract infections, skin infections, and allergies to a variety of dietary and environmental antigens. Additionally, patients experience elevated levels of serum IgE, but low to normal levels of other immunoglobulin types.

Objective: We performed genetic diagnosis of a patient of nonconsanguineous descent presenting at 11 years of age with severe atopic dermatitis, asthma, food allergy, skin and recurrent infections, and an extremely elevated level of serum IgE.

Methods: We performed whole genome sequencing of samples obtained from the patient and his entire family.

Results: Clinical, laboratory, genetic, and functional findings suggested CADINS. Genetic evaluation revealed a novel heterozygous missense variant (c.2913C>G, p.Cys971Trp) in the CARD11 gene as the potential underlying defect. Expression of CARD11 variant-stimulated constitutive NF-κB activity in T-cell lines demonstrated both loss-of-function and dominant-negative activity.

Conclusion: A novel germline heterozygous missense variant (c.2913C>G) in CARD11 potentially leads to CADINS.

Keywords: CADINS; CARD11; CBM complex; asthma; atopic dermatitis; dominant negative; eczema; food allergies; heterozygous variant; loss-of-function variant.

Fig 1

Clinical phenotype and genotype of patient. (A) Family pedigree shows affected patient with black square.(B) CDC growth charts for patient’s height (ages 5-19) and body surface area (ages 2-20). (C) CARD11 protein diagram highlights novel C971W variant (red) compared to known LOF variants (black) and DN variants (blue/orange).(D) C971 residue in CARD11 is highly conserved across species. (E) MAF versus CADD scores for CARD11 variants using gnomAD data (gnomad.broadinstitute.org). CADD, Combined annotation-dependent depletion; CDC, Centers for Disease Control and Prevention; MAF, minor allele frequency.

Fig 2

C971W is LOF CARD11 variant with mild DN activity. (A) Flow cytometry shows NF-κB–driven GFP reporter expression in CARD11-deficient JPM506 cells, which were transfected with different CARD11 constructs and subjected to stimulation with CD3/CD28 or PMA/ionomycin. (B) LOF assay indicates lower GFP MFI in C971W-transfected cells compared to WT, indicating statistical significance (P < .05). (C) Immunoblotting confirms CARD11-Flag expression, with β-actin used as loading control. (D) Flow cytometry of cells transfected with WT or C971W constructs shows GFP expression after stimulation. (E) DN assay shows slight MFI reduction in cells transfected with C971W + WT versus WT + WT. (F) Immunoblot verifies CARD11-Flag expression. GFP, Green fluorescent protein; MFI, mean fluorescence intensity; PMA, phorbol 12-myristate 13-acetate; WT, wild type.

Fig 3

Comparison of CARD11 C971W mutation with 23 other CADINS mutations from published case reports. Left, Colored bars represent CARD11 protein domains. CARD11 mutations are clustered in colored bars that reflect their location in CARD11 domains. Reported functional defect for CADINS mutations were DN, LOF, GOF, and weak DN. Because asthma, AD, and infections are critical clinical phenotypes associated with CADINS, number of reported cases is listed under each phenotype; 0 indicates absence of clinical phenotype for specific mutation. IgE serum levels are indicated as follows: ↑, high; N, normal levels, ND, not determined. ∗Refers to this report.