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Germline hypomorphic CARD11 mutations in severe atopic disease

Nature Genetics volume 49, pages 1192–1201 (2017)Cite this article

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A Corrigendum to this article was published on 27 October 2017

This article has been updated

Abstract

Few monogenic causes for severe manifestations of common allergic diseases have been identified. Through next-generation sequencing on a cohort of patients with severe atopic dermatitis with and without comorbid infections, we found eight individuals, from four families, with novel heterozygous mutations in CARD11, which encodes a scaffolding protein involved in lymphocyte receptor signaling. Disease improved over time in most patients. Transfection of mutant CARD11 expression constructs into T cell lines demonstrated both loss-of-function and dominant-interfering activity upon antigen receptor–induced activation of nuclear factor-κB and mammalian target of rapamycin complex 1 (mTORC1). Patient T cells had similar defects, as well as low production of the cytokine interferon-γ (IFN-γ). The mTORC1 and IFN-γ production defects were partially rescued by supplementation with glutamine, which requires CARD11 for import into T cells. Our findings indicate that a single hypomorphic mutation in CARD11 can cause potentially correctable cellular defects that lead to atopic dermatitis.

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Figure 1: Novel heterozygous CARD11 mutations in families with a history of severe atopic dermatitis.
Figure 2: Atopy-associated CARD11 mutations are hypomorphic and dominantly interfere with wild-type (WT) CARD11 signaling to NF-κB and mTORC1.
Figure 3: Impaired CBM complex formation leads to defective signaling in CARD11-mutant patient T cells.
Figure 4: Decreased surface expression of activation markers and defective proliferation after TCR stimulation in patients with CARD11 mutations.
Figure 5: Impaired IFN-γ, augmented TH2 cytokine production and Treg phenotype in patients with CARD11 mutations.
Figure 6: Effect of glutamine supplementation and cytokines on TCR-induced proliferation and IFN-γ defects in a patient with CARD11 mutation.

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Change history

  • 14 July 2017

    In the version of this article initially published online, the name of author Neil Romberg appeared incorrectly as Neil D Romberg, and the affiliation of author Nina Jones was incorrect and should have appeared as Clinical Research Directorate/Clinical Monitoring Research Program, Leidos Biomedical Research, Inc., NCI Campus at Frederick, Frederick, Maryland, USA. In addition, the following sentences were omitted from the Acknowledgments: "This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products or organizations imply endorsement by the US government." These errors have been corrected in the print, PDF and HTML versions of this article.

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Acknowledgements

We thank W. Tsai, M. Gadina and C. Malinverni for technical assistance. We thank the patients and their families for participating in this research. The patients were enrolled on an IRB-approved protocol and provided informed consent. CARD11-deficient Jurkat cells (JPM50.6) were originally provided by X. Lin (MD Anderson Cancer Center). This work was supported by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institutes of Health, the NIAID Clinical Genomics Program and grants from the National Institutes of Health (1R21AI109187 to A.L.S. and AI061093 to E.M.), the Henry M. Jackson Foundation (Val Hemming Fellowship to J.R.S.), Telethon (GGP13254 to E.R.), and the Joanne Siegel Fund (to E.W.G.). This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products or organizations imply endorsement by the US government.

Author information

Author notes

  1. Chi A Ma, Jeffrey R Stinson and Yuan Zhang: These authors contributed equally to this work.

  2. Erwin W Gelfand, Andrew L Snow and Joshua D Milner: These authors jointly directed this work.

Authors and Affiliations

  1. Laboratory of Allergic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA

    Chi A Ma, Yuan Zhang, Michael A Weinreich, Jonathan J Lyons, Celeste G Nelson, Thomas DiMaggio, Kelly D Stone & Joshua D Milner

  2. Department of Pharmacology and Molecular Therapeutics, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA

    Jeffrey R Stinson, Elisa Ruffo, Batsukh Dorjbal, Swadhinya Arjunaraja, Kelsey Voss & Andrew L Snow

  3. Department of Pediatrics, Immunodeficiency Diagnosis and Treatment Program, National Jewish Health, Denver, Colorado, USA

    Jordan K Abbott, Pia J Hauk, Paul R Reynolds & Erwin W Gelfand

  4. Department of Translational Medicine, University of Piemonte Orientale, Novara, Italy

    Elisa Ruffo

  5. Department of Immunobiology, Yale University School of Medicine, New Haven, Connecticut, USA

    Salomé Glauzy, Natsuko Yamakawa & Eric Meffre

  6. Department of Laboratory Medicine, Immunology Service, NIH Clinical Center, National Institutes of Health, Bethesda, Maryland, USA

    Jennifer Stoddard, Julie Niemela & Sergio D Rosenzweig

  7. Human Immunological Disease Section, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA

    Yu Zhang & Helen F Matthews

  8. Merck Research Laboratories, Merck and Co., Inc., Boston, Massachusetts, USA

    Joshua J McElwee

  9. Clinical Research Directorate/Clinical Monitoring Research Program, Leidos Biomedical Research, Inc., NCI Campus at Frederick, Frederick, Maryland, USA

    Nina Jones

  10. Servicio de Immunología y Reumatología, Hospital Nacional de Pediatría Prof. Dr. Juan P. Garrahan, Buenos Aires, Argentina

    Alejandro Palma, Matías Oleastro, Emma Prieto, Andrea R Bernasconi, Geronimo Dubra, Silvia Danielian, Jonathan Zaiat & Marcelo A Marti

  11. Division of Dermatology, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA

    Brian Kim

  12. Department of Pediatrics, Division of Rheumatology and Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, USA

    Megan A Cooper

  13. Department of Pediatrics, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania, USA

    Neil Romberg

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  1. Chi A Ma
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  18. Yu Zhang
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Contributions

C.A.M., Yuan Zhang, M.A.W. and S.G. performed experiments with primary patient cells. J.R.S. and B.D. produced all CARD11 mutant constructs. J.R.S., E.R., S.A., K.V. and B.D. conducted cell transfection experiments. J.J.L., C.G.N., T.D., K.D.S., H.F.M. and J.D.M. were involved in clinical workup of patient A.-I. J.S., J.N. and S.D.R. performed sequence analysis on patient A.-I. J.K.A., P.J.H., P.R.R. and E.W.G. were involved in clinical care and sequence analysis of family B. Yu Zhang, B.K., M.A.C., N.R., S.G. and E.M. were involved in clinical workup of family C. A.P., M.O., E.P., A.R.B., G.D. and S.D. were involved in clinical care and workup of family D. J.Z. and M.A.M. performed sequence analysis of family D. N.Y. performed regulatory T cell experiments. J.J.M. provided sequencing resources and data. N.J. provided patient care and information. C.A.M., M.A.W., J.R.S., A.L.S. and J.D.M. co-wrote the manuscript. E.W.G., A.L.S. and J.D.M. supervised the project. All authors discussed the results and contributed to the manuscript.

Corresponding author

Correspondence to Joshua D Milner.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Atopy-associated CARD11 mutants reduce the percentage of transfected cells signaling to NF-κB and mTORC1, and the Q945X mutant is functionally “null” and does not interfere with wild-type CARD11 signaling.

(a,b) Quantification of %κB-GFP+ JPM50.6 cells transfected in Figure 2a-d. Data are mean ± s.d. for 8 (a) and 5 (b) separate experiments. Asterisks denote statistically significant differences (Student’s t-test) for each LOF mutant versus wild-type (WT) with (black) or without (gray) stimulation, P < 0.05. (c) Flow cytometric histograms measuring NF-κB-driven GFP reporter expression in JPM50.6 cells transfected with empty vector (EV), WT, or Q945X CARD11 constructs, ± 24 h anti-CD3/CD28 stimulation. (d) Quantification of NF-κB-driven GFP reporter expression (MFI) in transfected JPM50.6 cells. Data are mean ± s.e.m. for three separate experiments. (e) Cropped immunoblot for CARD11-FLAG expression in transfected JPM50.6 lysates (c). Data representative of three independent experiments. (f) Flow cytometric histograms for JPM50.6 cells transfected with WT CARD11 plus EV, WT or mutant constructs and stimulated as in c. (g) GFP MFI quantification for JPM50.6 cells transfected in g. Data are mean ± s.e.m. (right) for five separate experiments; asterisks denote significance versus stimulated WT+WT (E57D P = 1.4 x 10−3; L194P P = 2.4 x 10−3; Q945X P = 0.113). (h) Cropped immunoblot for CARD11 expression in transfected JPM50.6 lysates described in h. Data representative of three independent experiments. (i) NF-κB-driven luciferase activity in WT Jurkat cells transfected with CARD11 (EV, WT, R975W, Q945X) plus luciferase reporter plasmids. Data represent mean ± s.d. fold change in κB-driven luciferase activity ± 24 h stimulation, normalized to Renilla luciferase activity for three separate experiments. (J) Quantification of % phospho-S6+ Jurkat cells transfected in Figure 2i,j. Data are mean ± s.d. for four separate experiments. Asterisks denote significance for each LOF mutant versus WT with stimulation (E57D P = 4.9 x 10−3; L194P P = 5.2 x 10−3; R975W P = 2.9 x 10−3; dup183_196 P = 0.013). (k) Percent inhibition of pS6 signal calculated for each mutant versus EV (gray) or WT (black) transfected cells in b, based on the change in % pS6+ cells ± stimulation. (l) Flow cytometric histograms measuring phospho-S6 in Jurkat cells transfected with EV, WT, L194P or Q945X CARD11 constructs, ± anti-CD3/CD28 stimulation for 20 min. Data are representative of three independent experiments.

Supplementary Figure 2 Increased incubation time of PMA diminishes the p-S6 activation defect in patient A-I compared to the healthy control.

PBMCs were rested and treated with 1 ng/mL PMA and stained with phospho-S6 antibody and gated on CD4+ cells as described in the Online Methods. Data are representative of two independent experiments (HC, healthy control; NS, no stimulation).

Supplementary Figure 3 CARD11 mutated patients show mildly impaired B cell signaling and efficient B cell development.

(a) PBMCs were stimulated with PMA and the signaling constituents were analyzed by intracellular flow cytometry (NS, no stimulation). (b) Representative analysis of CD19+CD27+ conventional memory B cells in a related healthy control (HC) subject and CARD11 mutated patients (left); summary of frequencies is depicted on the right. (c) Representative dot plots of CD21 and CD10 staining on CD19+CD27− naïve B cells in a related healthy control and CARD11 mutated patients (upper panels). Lower panels indicate frequencies of CD19+CD27−CD21loCD10hi transitional type 1 B cells, CD19+CD27−CD21+CD10+ transitional type 2 B cells, CD19+CD27−CD21+CD10− mature naïve B cells and CD19+CD27−CD21−/loCD10− B cells of CARD11 mutant patients compared with healthy control subjects. Each symbol represents a subject; solid lines display means, dashed lines show HC mean.

Supplementary Figure 4 Defective naive CD4 T cell proliferation in patient A-I, ELISA analysis of A-I and family B patients, and glutamine/cytokines rescue of IFN-γ in the family D patients.

(a) Blastogenesis and proliferation of isolated naïve CD4+ cells from representative healthy control (HC) vs. CARD11 patients after anti-CD3/CD28 activation for 5 days. CellTrace Violet (Violet) staining was used for tracking the number of the cell divisions. (b) Culture media from the PMA/ionomycin-treated patients’ PBMCs for 6 h were collected, and the IFNγ, IL-4, IL-13 and IL-5 secretions were measured by ProcartaPlex ELISA (eBioscience) (mean ± s.e.m.). (c) Intracellular flow cytometry identifying IFNγ/IL-4-producing cell ratio from CD3+CD8− CD45RO+ T cells within PBMC from CARD11 patients of family D and travel control (TC). PBMCs were cultured with anti-CD3/CD28 activation for 5 days, with or without cytokines (Th0) and plus 3 mM glutamine.

Supplementary Figure 5 CARD11 mutated patients show normal Treg cell frequencies, and CARD11 mutated patient Treg cells display normal suppressive function.

(a) Representative analysis of gated CD25+CD127lo (upper panels) and CD25+FOXP3+ (lower panels) on CD3+CD4+ T cells of a healthy (HC) control subject and CARD11 mutated patients (A-I, Family C) vs. control (C-II.2). (b) Quantitation of FOXP3+CD25+ Tregs among CD45RO+ CD127low CD4 T cells for family B members vs. healthy controls (HC). (c) Representative histograms of Treg-mediated suppression of autologous and heterologous CFSE labeled responder T cells (Tresp) from two CARD11 mutated patients compared to a healthy donor. Dashed line display non-stimulated Tresp cells. (d,e) Autologous and heterologous suppressive capacity of Treg cells of HC and CARD11 mutated patients. (f) Suppression of healthy control and CARD11 mutated patient Tresp cells by Tregs from healthy control. Full lines display the means and the dashed lines show the mean of the HC.

Supplementary Figure 6 Glutamine rescues of cell proliferation and IFN-γ production of the family D patients.

Blastogenesis and IFNγ+ cell ratio of PBMC from travel control vs. Family D patients after anti-CD3/CD28 activation for 5 days. Cytokines and glutamine were added as indicated.

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Full-length immunoblots (PDF 16601 kb)

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Ma, C., Stinson, J., Zhang, Y. et al. Germline hypomorphic CARD11 mutations in severe atopic disease. Nat Genet 49, 1192–1201 (2017). https://doi.org/10.1038/ng.3898

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