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. 2021 Feb 6;23(1):52.
doi: 10.1186/s13075-021-02434-w.

Functional analysis of novel A20 variants in patients with atypical inflammatory diseases

Saori Kadowaki  1 , Kunio Hashimoto  2 , Toyoki Nishimura  3 , Kenichi Kashimada  4 , Tomonori Kadowaki  1   5 , Norio Kawamoto  1 , Kohsuke Imai  6 , Satoshi Okada  7 , Hirokazu Kanegane  8 , Hidenori Ohnishi  9   10
Affiliations

Functional analysis of novel A20 variants in patients with atypical inflammatory diseases

Saori Kadowaki et al. Arthritis Res Ther. .

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.

Keywords: A20 haploinsufficiency; In vitro assay; Missense variant; TNFAIP3 gene; Variant classification.

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Conflict of interest statement

No authors have any conflicts of interest to declare.

Figures

Fig. 1
Fig. 1
a Pedigree of the families with heterozygous TNFAIP3 variants. b The locations of the missense variants on the domain structure of the A20 protein. The panel includes the previously reported missense mutations (C243Y, M476I, and T647P) and the three variants evaluated in this study
Fig. 2
Fig. 2
NF-κB reporter gene activity of TNFAIP3 variants and the expression of A20. a–c NF-κB reporter gene analysis measuring activity of TNFAIP3 variants using A20-deficient HEK293 cells. NF-κB reporter gene activity was induced by a stimulation with 20 ng/mL TNF-α, b co-transfection with MYD88 mutant (L265P), and c co-transfection with CARD11 mutant (F130V). The variant E192K could not suppress the NF-κB reporter gene activity elevated by TNF-α stimulation or co-transfection of GoF mutants of MYD88 or CARD11. d Immunoblot analysis of A20 variants. The expression levels of the variant I310T and known mutants, C243Y and T647P, were reduced
Fig. 3
Fig. 3
NF-κB reporter gene activity of TNFAIP3 variants using A20-deficient HEK293 cells and the Tet-on system. The expression levels of A20 were controlled by the Tet-on system. The variant E192K could not suppress the increase of activity regardless of the A20 expression level compared with the WT
Fig. 4
Fig. 4
IKKβ-mediated A20 phosphorylation of the TNFAIP3 variants. Immunoblot of lysates from A20-deficient HEK293 cells transfected with the vectors encoding A20 WT or variants, with or without co-transfected IKKβ, and probed with antibodies to p-S381 A20, myc (detecting A20), FLAG (detecting IKKβ), and β-actin. a The loss of IKKβ-mediated S381 phosphorylation was observed with the known mutant C243Y. b None of the three variants (E192K, I310T, and Q709R) caused reductions in IKKβ-mediated phosphorylation of A20 S381
Fig. 5
Fig. 5
Immunoblot analysis to evaluate the deubiquitinase function of TNFAIP3 variants. A20 WT and variants’ constructs were co-transfected into A20-deficient HEK293 cells with plasmids expressing K63-linked ubiquitin and FLAG-TRAF6, which is the A20 ubiquitination target. Deubiquitinase functions of the variants E192K, I310T, and Q709R did not differ from that of the WT
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