title: Functional assessment of the mutational effects of human IRAK4 and MyD88 genes.
abstract: 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.
authors: Department of Pediatrics, Graduate School of Medicine, Gifu University, 1-1 Yanagido, Gifu 501-1194, Japan.
authors: Takahiro
authors: T
authors: Yamamoto
authors: Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan.
authors: Naotaka
authors: N
authors: Tsutsumi
authors: Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan. Electronic address: tochio@moleng.kyoto-u.ac.jp.
authors: Hidehito
authors: H
authors: Tochio
authors: Department of Pediatrics, Graduate School of Medicine, Gifu University, 1-1 Yanagido, Gifu 501-1194, Japan. Electronic address: ohnishih@gifu-u.ac.jp.
authors: Hidenori
authors: H
authors: Ohnishi
authors: Department of Pediatrics, Graduate School of Medicine, Gifu University, 1-1 Yanagido, Gifu 501-1194, Japan.
authors: Kazuo
authors: K
authors: Kubota
authors: Department of Pediatrics, Graduate School of Medicine, Gifu University, 1-1 Yanagido, Gifu 501-1194, Japan.
authors: Zenichiro
authors: Z
authors: Kato
authors: Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan; Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, 4-1-8 Hon-cho, Kawaguchi, Saitama 332-0012, Japan.
authors: Masahiro
authors: M
authors: Shirakawa
authors: Department of Pediatrics, Graduate School of Medicine, Gifu University, 1-1 Yanagido, Gifu 501-1194, Japan; Heisei College of Health Sciences, 180 Kurono, Gifu 501-1131, Japan.
authors: Naomi
authors: N
authors: Kondo
mesh_terms: D002460:Cell Line; D020022:Genetic Predisposition to Disease; D005838:Genotype; D057809:HEK293 Cells; D006801:Humans; D007153:Immunologic Deficiency Syndromes / Q000235:genetics*; D053592:Interleukin-1 Receptor-Associated Kinases / Q000235:genetics*; D009154:Mutation; D053594:Myeloid Differentiation Factor 88 / Q000235:genetics*; D016328:NF-kappa B / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide; D017434:Protein Structure, Tertiary; D015398:Signal Transduction / Q000235:genetics
chemical_list: C507779:MYD88 protein, human; D053594:Myeloid Differentiation Factor 88; D016328:NF-kappa B; C507773:IRAK4 protein, human; D053592:Interleukin-1 Receptor-Associated Kinases
keywords: DD; Death domain; ELISA; HEK; ID; IRAK; IRAK4; IRAK4-DD; IRAK4-DD+ID; Immune-deficiency; Interleukin-1 receptor-associated kinase; Mal; Mal-TIR; MyD88; MyD88 adaptor-like; MyD88-DD; MyD88-DD+ID; MyD88-TIR; Myddosome; NMR; SNP; TIR domain; TIR domain of Mal; TIR domain of MyD88; TNF receptor associated factor.; TRAF; Toll/interleukin-1 receptor homology domain; WT; death domain; death domain and internal domain of IRAK4; death domain and internal domain of MyD88; death domain of IRAK4; death domain of MyD88; enzyme-linked immunosorbent assay; human embryonic kidney; internal domain; myeloid differentiating factor 88; nuclear magnetic resonance; single nucleotide polymorphism; wild type