Inherited human IFN-γ deficiency underlies mycobacterial disease
Gaspard Kerner, … , Jean-Laurent Casanova, Jacinta Bustamante
Gaspard Kerner, … , Jean-Laurent Casanova, Jacinta Bustamante
Published March 12, 2020
Citation Information: J Clin Invest. 2020;130(6):3158-3171. https://doi.org/10.1172/JCI135460.
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Research Article Genetics Immunology

Inherited human IFN-γ deficiency underlies mycobacterial disease

Abstract

Mendelian susceptibility to mycobacterial disease (MSMD) is characterized by a selective predisposition to clinical disease caused by the Bacille Calmette-Guérin (BCG) vaccine and environmental mycobacteria. The known genetic etiologies of MSMD are inborn errors of IFN-γ immunity due to mutations of 15 genes controlling the production of or response to IFN-γ. Since the first MSMD-causing mutations were reported in 1996, biallelic mutations in the genes encoding IFN-γ receptor 1 (IFN-γR1) and IFN-γR2 have been reported in many patients of diverse ancestries. Surprisingly, mutations of the gene encoding the IFN-γ cytokine itself have not been reported, raising the remote possibility that there might be other agonists of the IFN-γ receptor. We describe 2 Lebanese cousins with MSMD, living in Kuwait, who are both homozygous for a small deletion within the IFNG gene (c.354_357del), causing a frameshift that generates a premature stop codon (p.T119Ifs4*). The mutant allele is loss of expression and loss of function. We also show that the patients’ herpesvirus Saimiri–immortalized T lymphocytes did not produce IFN-γ, a phenotype that can be rescued by retrotransduction with WT IFNG cDNA. The blood T and NK lymphocytes from these patients also failed to produce and secrete detectable amounts of IFN-γ. Finally, we show that human IFNG has evolved under stronger negative selection than IFNGR1 or IFNGR2, suggesting that it is less tolerant to heterozygous deleterious mutations than IFNGR1 or IFNGR2. This may account for the rarity of patients with autosomal-recessive, complete IFN-γ deficiency relative to patients with complete IFN-γR1 and IFN-γR2 deficiencies.

Authors

Gaspard Kerner, Jérémie Rosain, Antoine Guérin, Ahmad Al-Khabaz, Carmen Oleaga-Quintas, Franck Rapaport, Michel J. Massaad, Jing-Ya Ding, Taushif Khan, Fatima Al Ali, Mahbuba Rahman, Caroline Deswarte, Rubén Martinez-Barricarte, Raif S. Geha, Valentine Jeanne-Julien, Diane Garcia, Chih-Yu Chi, Rui Yang, Manon Roynard, Bernhard Fleckenstein, Flore Rozenberg, Stéphanie Boisson-Dupuis, Cheng-Lung Ku, Yoann Seeleuthner, Vivien Béziat, Nico Marr, Laurent Abel, Waleed Al-Herz, Jean-Laurent Casanova, Jacinta Bustamante

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Figure 3

Levels of RNA and protein produced from the IFNG allele in an overexpression system and in vitro characterization.

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Levels of RNA and protein produced from the IFNG allele in an overexpres...
(A) Schematic representation of the WT protein and predicted proteins for P1 and P2. (B) qPCR on cDNA from HEK293T cells nontransfected (NT) or transfected with an empty plasmid (EV), WT-IFNG, or mutated IFNG. GUSB was used for normalization. The results shown are representative of 2 independent experiments. (C) Western blot analysis of IFN-γ in HEK293T cells left NT or that were transfected with an EV, WT-IFNG, or mutated IFNG, all inserted into p.CMV6 with a C-terminal DDK tag, with (left) or without (middle) brefeldin treatment and the addition of supernatants from HEK293T-transfected cells (right). The anti–IFN-γ antibodies used were a monoclonal mouse anti-IFN-γ antibody recognizing an N-terminal epitope between amino acids 20 and 50, and an antibody directed against the C-terminal DDK tag. An antibody against GAPDH (α-GAPDH) was used as a protein-loading control. The results shown are representative of 2 independent experiments. Different exposure times were used for each Western blot. (D) Induction of HLA-DR on SV-40 fibroblasts from a healthy control and from a patient with AR complete IFN-γR1 deficiency. Cells were activated with commercial IFN-γ or supernatants obtained from HEK293T cells transfected with different constructs. The results shown are representative of 2 independent experiments. (E) qPCR on cDNA from the HVS-T cells from healthy travel controls (C1 and C2), a heterozygous individual, and P1. GUSB was used for normalization. The results shown are representative of 2 independent experiments. (F) RT-PCR of exons 1–4 of the IFNG cDNA in PHA blasts from a healthy control (C+), 2 patients (P1 and P2), their relatives (Het1 and Het2), and a negative control (C–). The ACTB gene was used as a cDNA loading control.