Rare catastrophes and evolutionary legacies: human germline gene variants in MLKL and the necroptosis signalling pathway

Abstract

Programmed cell death has long been characterised as a key player in the development of human disease. Necroptosis is a lytic form of programmed cell death that is universally mediated by the effector protein mixed lineage kinase domain-like (MLKL), a pseudokinase. MLKL's activating kinase, receptor interacting protein kinase 3 (RIPK3), is itself activated within context specific scaffolds of receptor interacting protein kinase 1 (RIPK1), Z-DNA Binding Protein-1 (ZBP1) or TIR domain-containing adaptor inducing interferon-β (TRIF). These core necroptosis modulating proteins have been comprehensively revealed as potent drivers and suppressors of disease in inbred mouse strains. However, their roles in human disease within the 'real world' of diverse genetic backgrounds, natural infection and environmental challenges remains less well understood. Over 20 unique disease-associated human germline gene variants in this core necroptotic machinery have been reported in the literature and human clinico-genetics databases like ClinVar to date. In this review, we provide an overview of these human gene variants, with an emphasis on those encoding MLKL. These experiments of nature have the potential to not only enrich our understanding of the basic biology of necroptosis, but offer important population level insights into which clinical indications stand to benefit most from necroptosis-targeted drugs.

Keywords: MLKL; loss of function gene variant; missense gene variant; necroptosis; pathogenic mutation.

Figure 1.. TNF-induced necroptosis occurs when upstream pro-survival and pro-apoptotic pathways are inhibited.

(A) The binding of TNF to TNFR1 stimulates downstream nuclear factor-κB activation and other pro-survival, proinflammatory signals. (B) When cIAP1/2 activity is low, signalling is diverted to the formation of a death-induced signalling complex termed Complex II (a/b). This physically distinct complex is variably composed of TRADD, FADD, RIPK1, RIPK3 and the apoptosis initiator Caspase-8. (C) Necroptosis is activated when cellular conditions curb Caspase-8 activity and favour the assembly of RIPK1 and RIPK3, via their RIP Homotypic Interaction Motif (RHIM) domains, into a high molecular mass complex termed the necrosome. Here, RIPK3 is activated by autophosphorylation and MLKL is recruited and phosphorylated by RIPK3. MLKL dissociates from RIPK3 and oligomerised MLKL is trafficked to biological membrane [84,85]. The precise molecular events that lead to lytic permeabilization of the cell are still a matter of contention [86]. Sites of some selected published human germline mutations shown to be associated with human disease due to gain (red) or loss/reduction in function (green) are shown.

Figure 2.. Frequency of gene variation and pathogenic gene variants in MLKL and upstream signalling modulators.

(A) Total unique germline and de novo variants reported in ClinVar [52] as ‘pathogenic’, ‘likely pathogenic’, ‘associated’ or ‘risk factor. Somatic gene variants, contiguous copy-number variants, and gene variants not accompanied by a human disease condition were excluded from these counts. It is important to note that some patient mutations reported in the scientific literature may not have been submitted or updated by authors to the ClinVar database at the time of writing and thus are not included here. Disease causing variants in more upstream MLKL signalling modulators including death receptors, pattern recognition receptors, interferons and the NF-κB pathway are not presented here but are described in recent reviews [87–90]. (B) Summed global Minor Allele Frequency (MAF) of Missense and Loss of Function (LOF) variants as annotated by gnomAD at time of writing, (n = >280 000 alleles sequenced) [54]. Missense and LOF alleles flagged as ‘low confidence’ or ‘variant quality/annotation dubious’, alleles unique to non-canonical transcripts (with exception of CFLAR, where both long and short forms were included) or alleles with MAFs > 0.5 were excluded. (C) The top 10 missense variants used in calculating summed missense allele frequency in (B) and their global allele frequency plotted according to position in protein. *TICAM1 pPro367dup MAF = 0.3.