Functional analysis of naturally occurring amino acid substitutions in human IFN-γR1

IFN-γ plays an essential role in the IL-12/IL-23/IFN-γ pathway that is required for the defense against intracellular pathogens. In the IFN-γR1 several amino acid substitutions have been reported that abrogate IFN-γ signaling. These substitutions can lead to a null phenotype and enhanced susceptibility to infection by poorly pathogenic mycobacteria, a disorder known as Mendelian Susceptibility to Mycobacterial Disease (MSMD). More common amino acid variations in the IFN-γR1 may also influence IFN-γR function, albeit more subtle. To determine the effect of various amino acid substitutions on IFN-γR1 expression and function we cloned two newly identified amino acid substitutions (S149L, I352M), four common variations (V14M, V61I, H335P, L467P), seven reported missense mutations (V61Q, V63G, Y66C, C77Y, C77F, C85Y, I87T) and the 818delTTAA mutation in a retroviral expression vector. IFN-γR1 expression was determined as well as responsiveness to IFN-γ stimulation. The two newly discovered variants, and the four common polymorphisms could be detected on the cell surface, however, the V14M, H335P and I352M variants were significantly lower expressed at the cell membrane, compared to the wild type receptor. Despite the variance in cell surface expression, these IFN-γR1 variants did not affect function. In contrast to literature, in our model the expression of the V63G variant was severely reduced and its function was severely impaired but not completely abrogated. In addition, we confirmed the severely reduced function of the I87T mutant receptor, the completely abrogated expression and function of the V61E, V61Q, C77F, C77Y and the C85Y mutations, as well as the overexpression pattern of the 818delTTAA mutant receptor. The Y66C mutation was expressed at the cell surface, it was however, not functional. We conclude that the V14M, V61I, S149L, H335P, I352M and L467P are functional polymorphisms. The other variants are deleterious mutations with V61E, V61Q, Y66C, C77F, C77Y and C85Y leading to complete IFN-γR1 deficiency, while V63G and I87T lead to partial IFN-γR1 deficiency.