Kadowaki S(1), Hashimoto K(2), Nishimura T(3), Kashimada K(4), Kadowaki T(1)(5), Kawamoto N(1), Imai K(6), Okada S(7), Kanegane H(8), Ohnishi H(9)(10). Author information:
(1)Department of Pediatrics, Gifu University Graduate School of Medicine, 1-1
Yanagido, Gifu, Gifu, 501-1194, Japan.
(2)Department of Pediatrics, Nagasaki University Graduate School of Biomedical
Sciences, Nagasaki, Japan.
(3)Division of Pediatrics, Developmental and Urological-Reproductive Medicine
Faculty of Medicine, University of Miyazaki, Miyazaki, Japan.
(4)Department of Pediatrics and Developmental Biology, Tokyo Medical and Dental
University (TMDU), Tokyo, Japan.
(5)Department of Pediatrics, National Hospital Organization, Nagara Medical
Center, Gifu, Japan.
(6)Department of Community Pediatrics, Perinatal and Maternal Medicine, Graduate
School of Medical and Dental Sciences, Tokyo Medical and Dental University
(TMDU), Tokyo, Japan.
(7)Department of Pediatrics, Hiroshima University Graduate School of Biomedical
and Health Sciences, Hiroshima, Japan.
(8)Department of Child Health and Development, Graduate School of Medical and
Dental Sciences, Tokyo Medical and Dental University (TMDU), Tokyo, Japan.
(9)Department of Pediatrics, Gifu University Graduate School of Medicine, 1-1
Yanagido, Gifu, Gifu, 501-1194, Japan. [Email]
(10)Clinical Genetics Center, Gifu University Hospital, Gifu, Japan.
BACKGROUND: A20 haploinsufficiency (HA20) is an early-onset autoinflammatory disease caused by mutations in the TNFAIP3 gene, which encodes the protein A20. Numerous truncating mutations in the TNFAIP3 gene have been reported in HA20 patients, whereas fewer missense variants have had their pathogenicity confirmed. Here, we evaluated the pathogenic significance of three previously unreported missense variants of the TNFAIP3 gene in suspected cases of HA20. METHODS: We obtained the clinical features and immunological data of three patients with missense variants (Glu192Lys, Ile310Thr, and Gln709Arg) of unknown significance of TNFAIP3. We then performed in vitro functional assays including analysis of nuclear factor (NF)-κB reporter gene activity, detection of A20 expression and phosphorylation of A20 by IκB kinase β (IKKβ), and K63-deubiquitination assay using TNFAIP3-deficient HEK293 cells. Three known pathogenic missense mutations reported previously were also investigated. RESULTS: The inhibitory effect on NF-κB reporter gene activity was significantly disrupted by A20 Glu192Lys and the three known mutations. The variants Ile310Thr and Gln709Arg did not show a difference from the wild type in any of the assays performed in this study. CONCLUSIONS: Among the three variants in the TNFAIP3 gene, Glu192Lys was interpreted as being likely pathogenic, but Ile310Thr and Gln709Arg as being not pathogenic (uncertain significance and likely benign, respectively), based on the American College of Medical Genetics and Genomics standards and guidelines. Our study highlights the necessity of performing in vitro functional assays, notably, NF-κB reporter gene assay, to evaluate the pathogenicity of TNFAIP3 missense variants for the accurate diagnosis of HA20.
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