Laboratory evaluation of the IFN-γ circuit for the molecular diagnosis of Mendelian susceptibility to mycobacterial disease

Abstract

The integrity of the interferon (IFN)-γ circuit is necessary to mount an effective immune response to intra-macrophagic pathogens, especially Mycobacteria. Inherited monogenic defects in this circuit that disrupt the production of, or response to, IFN-γ underlie a primary immunodeficiency known as Mendelian susceptibility to mycobacterial disease (MSMD). Otherwise healthy patients display a selective susceptibility to clinical disease caused by poorly virulent mycobacteria such as BCG (bacille Calmette-Guérin) vaccines and environmental mycobacteria, and more rarely by other intra-macrophagic pathogens, particularly Salmonella and M. tuberculosis. There is high genetic and allelic heterogeneity, with 19 genetic etiologies due to mutations in 10 genes that account for only about half of the patients reported. An efficient laboratory diagnostic approach to suspected MSMD patients is important, because it enables the establishment of specific therapeutic measures that will improve the patient's prognosis and quality of life. Moreover, it is essential to offer genetic counseling to affected families. Herein, we review the various genetic and immunological diagnostic approaches that can be used in concert to reach a molecular and cellular diagnosis in patients with MSMD.

Keywords: MSMD; Mycobacteria; diagnosis; interferon gamma; intracellular pathogens; primary immunodeficiency.

Figure 1. IFN-γ circuit

Summary of molecules implicated in the IFN-γ circuit. Molecules represented with bold characters are known to cause of MSMD. GAS: γ-interferon activated site; GAF: γ-activated factor.

Figure 2. Diagram of the laboratory analysis of MSMD defects with examples

IFN-γR and STAT1 phosphorylation detection is performed in whole blood assay. IL-12Rβ2 detection is performed in PBMCs after 72 h stimulation with PHA. STAT4 phosphorylation detection is performed in PBMCs after 72 h stimulation with PHA and at least 48 h of culture in the presence of IL-2 or PHA + IL-2. An example of a healthy control is shown for each technique. Cytokine production is detected after 18 h of culture (for IL-12p70) and 48 h of culture (for IFN-γ and IL-12p70) in the gold standard procedure, BCG with or without IFN-γ or IL-12p70 co-stimulation. Control cohort is shown.

Figure 3. IFN-α and IFN-γ signaling

GAS: γ-interferon activated site; ISRE: interferon-sensitive response element.