Functional assessment of the mutational effects of human IRAK4 and MyD88 genes.

Human interleukin-1 receptor-associated kinase 4 (IRAK4) deficiency and myeloid differentiating factor 88 (MyD88) deficiency syndromes are two primary immune-deficiency disorders with innate immune defects. Although new genetic variations of IRAK4 and MyD88 have recently been deposited in the single nucleotide polymorphism (SNP) database, the clinical significance of these variants has not yet been established. Therefore, it is important to establish methods for assessing the association of each gene variation with human diseases. Because cell-based assays, western blotting and an NF-κB reporter gene assay, showed no difference in protein expression and NF-κB activity between R12C and wild-type IRAK4, we examined protein-protein interactions of purified recombinant IRAK4 and MyD88 proteins by analytical gel filtration and NMR titration. We found that the variant of IRAK4, R12C, as well as R20W, located in the death domain of IRAK4 and regarded as a SNP, caused a loss of interaction with MyD88. Our studies suggest that not only the loss of protein expression but also the defect of Myddosome formation could cause IRAK4 and MyD88 deficiency syndromes. Moreover a combination of in vitro functional assays is effective for confirming the pathogenicity of mutants found in IRAK4 and MyD88-deficiency patients.