Characterization of Novel Pathogenic Variants Leading to Caspase-8 Cleavage-Resistant RIPK1-Induced Autoinflammatory Syndrome

Abstract

Pathogenic RIPK1 variants have been described as the cause of two different inborn errors of immunity. Biallelic loss-of-function variants cause the recessively inherited RIPK1 deficiency, while monoallelic variants impairing the caspase-8-mediated RIPK1 cleavage provoke a novel autoinflammatory disease (AID) called cleavage-resistant RIPK1-induced autoinflammatory (CRIA) syndrome. The aim of this study was to characterize the pathogenicity of two novel RIPK1 variants located at the cleavage site of caspase-8 detected in patients with dominantly-inherited, early-onset undefined AID. RIPK1 genotyping was performed by Sanger and next-generation sequencing. Clinical and analytical data were collected from medical charts, and in silico and in vitro assays were performed to evaluate the functional consequences. Genetic analyses identified two novel heterozygous RIPK1 variants at the caspase-8 cleavage site (p.Leu321Arg and p.Asp324Gly), which displayed a perfect intrafamilial phenotype-genotype segregation following a dominant inheritance pattern. Structural analyses suggested that these variants disrupt the normal RIPK1 structure, probably making it less accessible to and/or less cleavable by caspase-8. In vitro experiments confirmed that the p.Leu321Arg and p.Asp324Gly RIPK1 variants were resistant to caspase-8-mediated cleavage and induced a constitutive activation of necroptotic pathway in a similar manner that previously characterized RIPK1 variants causing CRIA syndrome. All these results strongly supported the pathogenicity of the two novel RIPK1 variants and the diagnosis of CRIA syndrome in all enrolled patients. Moreover, the evidences here collected expand the phenotypic and genetic diversity of this recently described AID, and provide interesting data about effectiveness of treatments that may benefit future patients.

Keywords: RIPK1; Receptor-interacting kinases; autoinflammatory diseases; cleavage-resistant RIPK1-induced autoinflammatory syndrome; necroptosis.

Fig. 1

Novel variants in the RIPK1 gene. a Pedigrees of enrolled families. Red arrows indicate the index patient of each family. Black symbols represent affected individuals, open symbols unaffected, squares males, and circles females. RIPK1 genotypes are shown below each analyzed individual. wt, wild-type; n.a., not available. b Sense Sanger chromatograms from a healthy individual (control), and from patients P1–P3 and P5 carrying heterozygous genotypes for the p.Leu321Arg and p.Asp324Gly RIPK1 variants, respectively. The gray arrows indicate the detected nucleotide exchanges. c Domains of human RIPK1. The sequence ³²¹LQLD³²⁴ represents a consensus binding and cleavage site of human caspase-8. Reported pathogenic variants in residue Asp324 causing CRIA syndrome are displayed in red, and novel RIPK1 variants here described are shown in blue. RHIM, receptor-interacting protein (RIP) homotypic interaction motif

Fig. 2

Functional analyses of novel RIPK1 variants. a Caspase-8 cleavage of RIPK1. Western blot for wild-type or variant RIPK1 expressed in HEK293T cells and incubated or not with recombinant caspase-8 as indicated. β-actin was used as a loading control. FL: full length; CL: cleaved. This Western blot is representative of three independent experiments. b Western blot showing the expression of RIPK3 and the different variants of RIPK1 in HeLa cells. β-actin was used as a loading control. Western blot is representative of three independent assays. Asterisks denote an unspecific band. c–d p.Leu321Arg and p.Asp324Gly RIPK1 variants induce cell death and cell permeabilization in response to TNF stimulation. Extracellular LDH release from HeLa cells (c) and Yo-Pro-1 uptake to HeLa cells (d) transfected with RIPK3 and empty pcDNA or the different RIPK1 variants after treatment with TNF and inhibitors of apoptosis proteins (IAP) antagonist, in the presence or absence of caspase-8 inhibitor (z-IETD-FMK) and/or necrosulfonamide (NSA); n: 4-8 different experiments. Each column represents the mean of all values, with bars in each column representing standard error; *denotes p < 0.05; **denotes p < 0.01; ***denotes p < 0.001; ****denotes p < 0.0001; Kruskal-Wallis test with Dunn’s multiple comparisons post-test

Fig. 3

Serum levels of circulating cytokines in CRIA patients during inactive intervals (blue columns for patients P3 and P4) and during active disease (red column for patient P5) compared with healthy controls (HC; n: 10). Each dot represents the value of duplicate experiments of a collected serum. Each column represents the mean of all values, with bars in each column representing standard deviation (SD). n.s. denotes not significant; *denotes p<0.05; **denotes p<0.01