Saori Kadowaki1, Kunio Hashimoto2, Toyoki Nishimura3, Kenichi Kashimada4, Tomonori Kadowaki1,5, Norio Kawamoto1, Kohsuke Imai6, Satoshi Okada7, Hirokazu Kanegane8, Hidenori Ohnishi9,10. 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. ohnishih@gifu-u.ac.jp. 10. Clinical Genetics Center, Gifu University Hospital, Gifu, Japan. ohnishih@gifu-u.ac.jp.
Abstract
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.
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.
Authors: David L Boone; Emre E Turer; Eric G Lee; Regina-Celeste Ahmad; Matthew T Wheeler; Colleen Tsui; Paula Hurley; Marcia Chien; Sophia Chai; Osamu Hitotsumatsu; Elizabeth McNally; Cecile Pickart; Averil Ma Journal: Nat Immunol Date: 2004-08-29 Impact factor: 25.606
Authors: Nathan W Zammit; Owen M Siggs; Christopher C Goodnow; Shane T Grey; Paul E Gray; Keisuke Horikawa; David B Langley; Stacey N Walters; Stephen R Daley; Claudia Loetsch; Joanna Warren; Jin Yan Yap; Daniele Cultrone; Amanda Russell; Elisabeth K Malle; Jeanette E Villanueva; Mark J Cowley; Velimir Gayevskiy; Marcel E Dinger; Robert Brink; David Zahra; Geeta Chaudhri; Gunasegaran Karupiah; Belinda Whittle; Carla Roots; Edward Bertram; Michiko Yamada; Yogesh Jeelall; Anselm Enders; Benjamin E Clifton; Peter D Mabbitt; Colin J Jackson; Susan R Watson; Craig N Jenne; Lewis L Lanier; Tim Wiltshire; Matthew H Spitzer; Garry P Nolan; Frank Schmitz; Alan Aderem; Benjamin T Porebski; Ashley M Buckle; Derek W Abbott; John B Ziegler; Maria E Craig; Paul Benitez-Aguirre; Juliana Teo; Stuart G Tangye; Cecile King; Melanie Wong; Murray P Cox; Wilson Phung; Jia Tang; Wendy Sandoval; Ingrid E Wertz; Daniel Christ Journal: Nat Immunol Date: 2019-09-18 Impact factor: 25.606
Authors: Sue Richards; Nazneen Aziz; Sherri Bale; David Bick; Soma Das; Julie Gastier-Foster; Wayne W Grody; Madhuri Hegde; Elaine Lyon; Elaine Spector; Karl Voelkerding; Heidi L Rehm Journal: Genet Med Date: 2015-03-05 Impact factor: 8.822