PMC9149281_01
Family pedigree and CARD11 domains in health and diseases. (A) Family pedigree. (B) Multiple species alignment of CARD11 protein (AA 52-101) showing high conservation of Arg75 across species. Red: full conservation. (C) Schematic representation of CARD11, with functional domains and reported variants, including monoallelic gain-of-function (GOF) variants in BENTA, biallelic LOF variants in SCID, monoallelic DN LOF variants in CARD11-associated atopy with dominant interference of NF-kappaB signaling (CADINS), additionally monoallelic DN LOF variants in CADINS and carcinoma. DN, dominant negative; LOF, loss of function; SCID, severe combined immunodeficiency, * truncating variant. (D) Sanger DNA sequencing electropherograms showing the novel heterozygote variant of the CARD11 gene in III.4, his mother II.2 and his cousin III.2.
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The variant was also confirmed by Sanger sequencing in the mother, aunt and cousins of the index patient ( Figure 1D ). Arg75 is a highly conserved amino acid residue located in the CARD domain, a region that is depleted of missense variation ( Figure 1C  and  Supplementary Figure 2 ).
Cutaneous findings. (A, B) Hand and foot eczema in patient III.4. (C, D) Extensive eczema in patient III.2. (E, F) Skin findings prior (E) and following (F) treatment with dupilumab in patient III.1. SCORAD: SCORing Atopic Dermatitis; Clinical scoring system to evaluate severity of atopic eczema. Interpretation: < 25 mild, 25-60 moderate, 61-103 severe atopic eczema.
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Severe eczema developed on his hands and feet ( Figures 2A, B ), as well as recurrent nail mycoses. Her son (III.1) and daughter (III.2) - the index patient's cousins - report various food allergies, bronchial asthma and severe AD ( Figures 2C, D ). Photodocumentation pre- and post-treatment is presented in  Figures 2E, F . Arg75 is a highly conserved amino acid residue located in the CARD domain, a region that is depleted of missense variation ( Figure 1C  and  Supplementary Figure 2 ).
Histopathology and immunohistochemistry of a squamous cell carcinoma of the anal canal in patient II.2. (A) Colorectal biopsy in H&E staining: left side colorectal mucosa, right side squamous cell carcinoma. (B) Anal squamous cell carcinoma in H&E staining with higher magnification with cellular atypia and dyskeratoses. (C) Anal squamous cell carcinoma showing positive p16 immunohistochemical staining of the tumor cells as a surrogate marker for HPV-infection in 7x magnification and (D) in 10.6x magnification. HPV, human papilloma virus.
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At age 45, she was diagnosed with anal squamous cell carcinoma induced by a local HPV18 infection ( Figure 3 ).
Functional testing of R75W CARD11. JPM50.6 cells were transfected with EV, WT or R75W CARD11-FLAG expression constructs, without (A) or with (B) a WT CARD11-V5 construct. After 24 hrs, transfectants were stimulated with anti-CD3/CD28 Abs or PMA/ionomycin for 24 hrs, and GFP expression was measured by flow cytometry. Histograms from one representative experiment (left panels) denote %GFP+ cells -/+ stimulation. Mean fluorescence intensity (MFI) of GFP+ cells was graphed for 3 independent experiments (middle panels); asterisks denote statistically significant decreases in GFP MFI for R75W relative to WT -/+ WT CARD11-V5 (paired t-test, p <0.04). CARD11 protein expression was measured in cell lysates by immunoblotting; beta-actin serves as a loading control.
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R75W CARD11 and measured NF-kappaB driven GFP expression following T-cell activation. Relative to WT, no appreciable NF-kappaB induction was noted with R75W CARD11 ( Figure 4A ). Moreover, R75W demonstrated mild DN activity in suppressing NF-kappaB activation when co-expressed with WT CARD11 ( Figure 4B ), with comparable expression of both proteins noted.