A zebrafish functional genomics model to investigate the role of human A20 variants in vivo

Abstract

Germline loss-of-function variation in TNFAIP3, encoding A20, has been implicated in a wide variety of autoinflammatory and autoimmune conditions, with acquired somatic missense mutations linked to cancer progression. Furthermore, human sequence data reveals that the A20 locus contains ~ 400 non-synonymous coding variants, which are largely uncharacterised. The growing number of A20 coding variants with unknown function, but potential clinical impact, poses a challenge to traditional mouse-based approaches. Here we report the development of a novel functional genomics approach that utilizes a new A20-deficient zebrafish (Danio rerio) model to investigate the impact of TNFAIP3 genetic variants in vivo. A20-deficient zebrafish are hyper-responsive to microbial immune activation and exhibit spontaneous early lethality. Ectopic addition of human A20 rescued A20-null zebrafish from lethality, while missense mutations at two conserved A20 residues, S381A and C243Y, reversed this protective effect. Ser381 represents a phosphorylation site important for enhancing A20 activity that is abrogated by its mutation to alanine, or by a causal C243Y mutation that triggers human autoimmune disease. These data reveal an evolutionarily conserved role for TNFAIP3 in limiting inflammation in the vertebrate linage and show how this function is controlled by phosphorylation. They also demonstrate how a zebrafish functional genomics pipeline can be utilized to investigate the in vivo significance of medically relevant human TNFAIP3 gene variants.

Environmental and genetic disease modifiers of haploinsufficiency of A20

“…Analysis of human sequence data from people without early onset disease (gnomAD v2.1.1) revealed only 2 from 141,456 individuals with heterozygous protein-truncating TNFAIP3 mutations, compared to an expected rate of 32 individuals based on coding region size and composition (pLI=1, LOEUF=0.2) (28). Studies in vertebrate model organisms also reveal that HA20 does not always drive a disease phenotype, as mice and zebrafish heterozygous for Tnfaip3 deletion present as healthy when housed under specific pathogen free (SPF) conditions (1, 3). This suggested that additional, but hitherto unknown, disease modifiers cooperate with TNFAIP3 haploinsufficiency in these families to drive their disease phenotypes.…”

“…TNFAIP3 (tumor necrosis factor, alpha-induced protein 3) encodes A20, a highly conserved and evolutionarily ancient protein that serves as a key immune tolerance checkpoint by negatively regulating tissue inflammation and immune responses by inhibiting NF-κB signaling (1)(2)(3)(4). A20 inhibits NF-κB signalling though catalytic and non-catalytic mechanisms.…”

Selection of a novel AAV2/TNFAIP3 vector for local suppression of islet xenograft inflammation

“…Similar to rodent and human islets, 32,34‐36 A20 expression is induced in NPIs by inflammatory stress, and once expressed in NPIs A20 negatively regulates NF‐κB signaling and pro‐inflammatory gene expression. The preservation of A20's cytoprotective function in mouse, human and porcine islets reflects conservation in its protein structure across mammalian species, 33,68 but also the high degree of sequence homology across the TNFAIP3 gene locus in vertebrates 68,69 . Forced expression of A20 in mouse islet allografts led to the expression of TGFβ and IL‐10, rather than pro‐inflammatory TNF and IL‐6, in the graft microenvironment and engendered the local accumulation of Tregs 32 .…”

In Vitro, In Vivo, and In Silico Models of Lymphangiogenesis in Solid Malignancies

“…Its high fecundity and the low cost of maintenance make it an ideal actor for disease modeling. Indeed, thanks to its ability to grow rapidly and its transparency during the early stages of life, it is ideal for development study [109], in addition to being largely used in genomics [110,111]. More recently, the use of zebrafish in cancer research became the aim of several studies and reviews [112,113].…”