Multiplexed Functional Assessment of Genetic Variants in CARD11

Abstract

Genetic testing has increased the number of variants identified in disease genes, but the diagnostic utility is limited by lack of understanding variant function. CARD11 encodes an adaptor protein that expresses dominant-negative and gain-of-function variants associated with distinct immunodeficiencies. Here, we used a "cloning-free" saturation genome editing approach in a diploid cell line to simultaneously score 2,542 variants for decreased or increased function in the region of CARD11 associated with immunodeficiency. We also described an exon-skipping mechanism for CARD11 dominant-negative activity. The classification of reported clinical variants was sensitive (94.6%) and specific (88.9%), which rendered the data immediately useful for interpretation of seven coding and splicing variants implicated in immunodeficiency found in our clinic. This approach is generalizable for variant interpretation in many other clinically actionable genes, in any relevant cell type.

Keywords: B cells; CARD11; gene editing; immune dysregulation; lymphoma; primary immune deficiency; saturation genome editing; variant interpretation.

Figure 1

A Saturation Genome Editing Screen for Functional Assessment of CARD11 (A) A schematic of CARD11 containing domains and sites of clinically documented mutations (red, severe combined immunodeficiency [SCID]; orange, CARD11-associated atopy with dominant interference of NF-κB signaling [CADINS]; green, B cell expansion with NF-κB and T cell anergy [BENTA]; blue, diffuse large B cell lymphoma [DLBCL]; and purple, Sezary). (B) A schematic of B cell receptor signaling in TMD8 cells. Loss of CARD11 or BTK inhibition with ibrutinib leads to TMD8 cell death. Gain-of-function mutations in CARD11 lead to constitutive activation of NF-κB, allowing TMD8 cells to survive in the presence of ibrutinib. (C) HDR was used to introduce all possible amino acid variants into exons 3–5, including the CARD domain, where many variants associated with disease are located have been identified. gDNA from TMD8 lymphoma cells were sequenced 2 days after editing. A graph representing HDR rates for all replicates across each editing region plotted as a boxplot centered on the median with the whiskers representing the 5th-95th percentiles. (D) Table summarizing the number of variants introduced, scored, and classified in the SGE experiment. (E) TMD8 cells were edited at the CCR5 locus with increasing amounts of single-stranded oligonucleotide repair template to compare low, mid, and high editing rates. Monoclonal lines were isolated, and the identity of the other allele was quantified for cells containing at least one HDR allele. (F) Quantification of the percentage of HDR events that solely introduce silent Cas9-blocking variants, which is plotted as mean ± standard error.

Figure 2

Pathogenic Gain-of-Function Variants in CARD11 Grow Selectively in the Presence of Ibrutinib (A–D) Functional scores for all variants in the SGE experiment of CARD11 using the cell growth and ibrutinib assays were plotted as distributions. The thresholds used to score loss-of-function (dotted line, A and B) and gain-of-function (dotted line, C and D) were indicated. The distribution of scores was plotted for all missense (A and C) and synonymous and nonsense variants (C and D). The location of activating variants in CARD11 previously published to lead to BENTA were highlighted, or BENTA and DLBCL (light blue color) (C). (E) An X-Y scatterplot of functional scores from the growth (x axis) and ibrutinib (y axis) SGE. The functional classification of variants as gain-of-function or likely gain-of-function were annotated using the indicated colors. (F) Gain-of-function variants were primarily clustered at cysteine 49, and in the N-terminal coiled-coil and the latch domains. Individual substitutions that promote gain-of-function activity are color coded as in (E). (G) JPM50.6 cells were transfected with empty vector (EV), WT, or mutant CARD11 plasmids. NF-κB-driven GFP reporter expression ± 24 h anti-CD3/CD28 stimulation was quantified by flow cytometry and plotted as mean fluorescence intensity (MFI) ± standard error. Data are shown from three independent experiments; statistical significance versus WT (or WT+WT) was assessed using one-way ANOVA with the SIDAK correction for multiple comparisons ^∗∗∗∗p < 0.0001, ^∗∗p < 0.01. (H) Immunoblot showing CARD11-FLAG expression for each variant; ACTB serves as a loading control.

Figure 3

Functional Scoring from SGE Accurately Identify Variants that Decrease CARD11 Function (A) An X-Y scatterplot showing SGE functional scores from RNA (y axis) versus the growth assay (x axis). Correlation scores between the variant effect on RNA abundance and growth were quantified (pearson) for nonsense/splice (orange) and all missense (green) variants. (B) Violin plots showing the distribution of CADD scores for single nucleotide variants in each functional classification. (C and D) JPM50.6 cells were transfected with empty vector (EV), WT or mutant CARD11 plasmids alone (C) or in 50:50 ratio with WT (D). After stimulation with anti-CD3/CD28, NF-κB-driven GFP reporter expression was quantified by flow cytometry and plotted as mean ± standard error. NS, non-stimulated. Statistical significance versus WT (or WT+WT) was assessed using one-way ANOVA with the SIDAK correction for multiple comparisons ^∗∗∗∗p < 0.0001, ^∗∗p < 0.01. (E) Immunoblot showing CARD11-FLAG expression for each variant; actin serves as a loading control. (F) SGE functional scores were plotted for likely pathogenic/pathogenic variants and likely non-damaging variants present in gnomAD at a frequency greater than 1 in 300,000. (G) A ROC curve reveals the sensitivity and specificity of SGE functional scoring for identification of these disease variants.

Figure 4

Splice Junction Variants Can Lead to Exon Skipping and Dominant-Negative Activity CARD11 (A) Functional scores for all single-nucleotide variants in the growth assay were plotted by position in each of the CARD11 exons. Nonsense (orange) and intronic (red) variants were colored and exonic sequences spanning the intron-exon boundaries were labeled. Finally, we annotated the locations of two heterozygous variants identified in subjects with clinical suspicion for CARD11 dominant-negative disease. (B) A pedigree chart shows inheritance of the c.358+1G>A variant in family A. (C) PBMCs were isolated from healthy control subjects and case subjects (A-II.a and A-II.b) and stimulated with PMA for 0, 5, and 10 min. Ikb-alpha and phospho-ERK was quantified by flow cytometry and plotted as mean ± stardard deviation. (D–F) cDNA was isolated from PBMCs of affected individuals with CARD11 splice variants (A-II.a and F-I.a) and amplified with the primers indicated on the exon-intron diagram in (F). These amplicons were analyzed via DNA PAGE gel (D and E) and colony sequencing (F). (G) CARD11-deficient Jurkat cells were co-transfected with a plasmid expressing an NF-κB luciferase reporter and the indicated constructs. After stimulation with PMA/ionomycin, NF-κB reporter signal was quantified, normalized to cell viability, and plotted as mean ± standard deviation. (H) Immunoblot showing CARD11-FLAG expression for each variant; actin serves as a loading control. (I) Cas9 RNPs and HDR templates were used to introduce the indicated variants in place of c.358+1G in CARD11 into Ly7 lymphoma cells that express an NF-κB reporter. Following activation of CARD11-dependent signaling with PMA/ionomycin, reporter activity was normalized to cell viability, quantified, and plotted as mean ± starndard deveiation. For (G) and (I), we used one-way ANOVA with the SIDAK correction for multiple comparisons; ^∗∗∗∗p < 0.0001, ^∗∗p < 0.01.

Figure 5

Modeling Conservative Changes to Amino Acid R35, K41, K69, and R72 of CARD11 (A and B) Structure of CARD11 protein CARD domain modeled based upon the cryoEM structures of BCL10 filaments in an (A) unbound state and (B) together with BCL10 illustrating the type III interaction. Blue indicates positive charge and red indicates negative charges. (C and D) CARD11-deficient Jurkat cells were co-transfected with a plasmid expressing an NF-κB luciferase reporter and the indicated constructs. After stimulation with PMA/ionomycin, NF-κB reporter signal was quantified, normalized to cell viability, and plotted as mean ± standard deveiation. Variants highlighted in red have been previously identified in affected individuals. We used one-way ANOVA with the SIDAK correction for multiple comparisons; ^∗∗∗∗p < 0.0001, ^∗∗p < 0.01. (E) Immunoblots showing CARD11 expression for each construct; actin serves as a loading control. Images are representative of two repeat experiments.