A critical region of A20 unveiled by missense TNFAIP3 variations that lead to autoinflammation
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eLife assessment
This manuscript is of big interest to physicians and geneticists who may struggle with interpreting the clinical significance of novel or rare missense variants in the TNFAIP3 gene in patients with systemic inflammatory diseases. There is also much debate about the potential mechanisms by which these missense mutations might be pathogenic. El Khour et al. addressed these questions by using a combination of in silico analysis and in vitro functional assays. However, their conclusions require additional experimental support and should be expanded to include other reported likely pathogenic missense variants.
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Abstract
A20 haploinsufficiency (HA20) is an autoinflammatory disease caused by heterozygous loss-of-function variations in TNFAIP3 , the gene encoding the A20 protein. Diagnosis of HA20 is challenging due to its heterogeneous clinical presentation and the lack of pathognomonic symptoms. While the pathogenic effect of TNFAIP3 truncating variations is clearly established, that of missense variations is difficult to determine. Herein, we identified a novel TNFAIP3 variation, p.(Leu236Pro), located in the A20 ovarian tumor (OTU) domain and demonstrated its pathogenicity. In the patients’ primary cells, we observed reduced A20 levels. Protein destabilization was predicted in silico for A20_Leu236Pro and enhanced proteasomal degradation was confirmed in vitro through a flow cytometry-based functional assay. By applying this approach to the study of another missense variant, A20_Leu275Pro, for which no functional characterization has been performed to date, we showed that this variant also undergoes enhanced proteasomal degradation. Moreover, we showed a disrupted ability of A20_Leu236Pro to inhibit the NF-κB pathway and to deubiquitinate its substrate TRAF6. Structural modeling revealed that two residues involved in OTU pathogenic missense variations (i.e. Glu192Lys and Cys243Tyr) establish common interactions with Leu236. Interpretation of newly identified missense variations is challenging, requiring, as illustrated here, functional demonstration of their pathogenicity. Together with functional studies, in silico structure analysis is a valuable approach that allowed us (i) to provide a mechanistic explanation for the haploinsufficiency resulting from missense variations and (ii) to unveil a region within the OTU domain critical for A20 function.
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Author Response
Reviewer #3 (Public Review):
The authors described the one family showing autoinflammatory phenotypes with L236P variant of TNFAIP3 gene. The variant has not been reported on and they evaluated the function of this variant using in vitro and in silico methods. I think this is well-written manuscript and I agree with their interpretation about the pathogenicity of this variant, but the new finding is poor. The variant information was only a new finding.
I recommend the revision of the following points.
In Table 2, T647P seemed to be pathogenic which was evaluated with in vitro assay by Kadowaki.
The Kadowaki study indeed showed reduced NFκB activity for the Thr647Pro variant. This information has now been added to Table 2. However, the variant is highly frequent in the control population. According the ACMG …
Author Response
Reviewer #3 (Public Review):
The authors described the one family showing autoinflammatory phenotypes with L236P variant of TNFAIP3 gene. The variant has not been reported on and they evaluated the function of this variant using in vitro and in silico methods. I think this is well-written manuscript and I agree with their interpretation about the pathogenicity of this variant, but the new finding is poor. The variant information was only a new finding.
I recommend the revision of the following points.
In Table 2, T647P seemed to be pathogenic which was evaluated with in vitro assay by Kadowaki.
The Kadowaki study indeed showed reduced NFκB activity for the Thr647Pro variant. This information has now been added to Table 2. However, the variant is highly frequent in the control population. According the ACMG guidelines, the variant does not fulfil all the conditions to be considered as pathogenic as its allele frequency is not compatible with the disease frequency. Therefore, as we cannot conclude on the pathogenic effect of the variation, we have described it as a variant of unknown significance.
Two other missense variants, V377I (Niwano, Rheumatology 2022) and T602S (Jiang W, Cellular Immunol 2022) were recently reported. These should be included in the discussion.
We have analyzed all additional missense variations, including the V377M (we found the report of a variation involving V377, but it was V377M and not V377I) and T602S variations and have added them to Table 2.
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eLife assessment
This manuscript is of big interest to physicians and geneticists who may struggle with interpreting the clinical significance of novel or rare missense variants in the TNFAIP3 gene in patients with systemic inflammatory diseases. There is also much debate about the potential mechanisms by which these missense mutations might be pathogenic. El Khour et al. addressed these questions by using a combination of in silico analysis and in vitro functional assays. However, their conclusions require additional experimental support and should be expanded to include other reported likely pathogenic missense variants.
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Reviewer #1 (Public Review):
In this manuscript, the authors describe a novel HA20-causing missense mutation, p.(Leu236Pro), in three patients from one family with periodic fevers, GI symptoms, urogenital ulcers, arthritis, and pustular rash. The patients had elevated levels of multiple proinflammatory cytokines, including IL-1b, IL-6, and TNFa. Patients had reduced A20 expression levels, and in silico analysis suggested that the p.Leu236Pro mutation destabilized the A20 protein. Using transfection assays, the authors determined that steady state protein expression of mutant A20 protein was lower, and that the half-life of the mutant protein was shorter. Treatment with MG132 increased the half life of mutant A20, suggesting that the mutant protein underwent degradation through the proteasome. Further experiments in the transfection …
Reviewer #1 (Public Review):
In this manuscript, the authors describe a novel HA20-causing missense mutation, p.(Leu236Pro), in three patients from one family with periodic fevers, GI symptoms, urogenital ulcers, arthritis, and pustular rash. The patients had elevated levels of multiple proinflammatory cytokines, including IL-1b, IL-6, and TNFa. Patients had reduced A20 expression levels, and in silico analysis suggested that the p.Leu236Pro mutation destabilized the A20 protein. Using transfection assays, the authors determined that steady state protein expression of mutant A20 protein was lower, and that the half-life of the mutant protein was shorter. Treatment with MG132 increased the half life of mutant A20, suggesting that the mutant protein underwent degradation through the proteasome. Further experiments in the transfection system revealed that mutant A20 failed to suppress TNFα-induced NF-κB activity.
This paper will be of great interest to the field. HA20 is a novel disease (first described in 2016), and although the effects of frameshift/truncating mutations are quite evident, there is quite a lot of debate about the potential effects of missense mutations. It is not really clear which missense mutations cause disease and why, and clinicians who treat this disease are frequently faced with the dilemma of evaluating a patient with a rare missense variant of unknown significance. Thus, a paper that can explain the potential mechanisms by which missense mutations cause disease is highly relevant -- and this is an area of active investigation by several groups.
The strengths of this study include the thorough functional assessment of this novel mutation: the authors have collected quite a lot of data to show the effects of their mutation on protein stability and function. Another strength is the comparison with other similar mutations in the OTU and other domains. However, the data are not currently sufficient to support the conclusions of the authors about the effects of their mutation on protein folding. Similarly, the data do not sufficiently support the generalizability of this mechanism to other mutations in the OTU domain.
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Reviewer #2 (Public Review):
The authors identified a novel TNFAIP3 variation Leu236Pro located in the A20 OUT domain and demonstrated its pathogenicity. Proinflammatory cytokines were substantially elevated in the patients. In vitro study showed decreased stability of the Leu236Pro A20 protein and Leu236Pro mutant failed to suppress TNF induced NF-κB activity. Review of previously reported TNFAIP3 missense variations revealed that only 3/7 are pathogenic. Truncating A20 mutations are easy to determine the pathogenicity, while missense TNFAIP3 variants require more functional studies to determine the pathogenicity. The results of this study can help interpretation of TNFAIP3 missense variations.
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Reviewer #3 (Public Review):
The authors described the one family showing autoinflammatory phenotypes with L236P variant of TNFAIP3 gene. The variant has not been reported on and they evaluated the function of this variant using in vitro and in silico methods. I think this is well-written manuscript and I agree with their interpretation about the pathogenicity of this variant, but the new finding is poor. The variant information was only a new finding.
I recommend the revision of the following points.
In Table 2, T647P seemed to be pathogenic which was evaluated with in vitro assay by Kadowaki.
Two other missense variants, V377I (Niwano, Rheumatology 2022) and T602S (Jiang W, Cellular Immunol 2022) were recently reported. These should be included in the discussion.
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