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.

RIPK1 Biallelic Activating Variants lead to Autoinflammatory Disease Driven by T cell Death

“…The condition in our study is inherited in an autosomal recessive (AR) pattern, which is different from autosomal dominantly inherited CRIA syndrome even if they share similar manifestations. Mutation in RIPK1 D324 does not impair NF-κB pathway 3–5 ; in contrast, compound heterozygous mutations in RIPK1 K377/R390 suppressed NF-κB activation, indicating that the autoinflammation caused by these mutations is not directly induced by dysregulation of NF-κB. The evidence that PBMCs and MEFs carrying heterozygous mutations in D324 as well as T cells carrying compound heterozygous variants in RIPK1 K377/R390 instead of heterozygous variant are hypersensitive to TNF-induced cell death support the inheritance pattern of the two conditions 3,4 .…”

“…The list of genes whose mutations lead to SAIDs are enriched with key regulators of programmed cell death (PCD). Enhanced PCD is one of the characteristics in the SAID patients caused by the cleavage resistance mutations in RIPK1 3–5 or deficiencies of RIPK1 6,7 , TANK-binding kinase 1 ( TBK1 ) 8 , OTU deubiquitinase with linear linkage specificity ( OTULIN ), HOIL1-interacting protein ( HOIP ), Heme-oxidized IRP2 ubiquitin ligase-1 ( HOIL1 ) and Shank-associated RH domain-interacting protein ( SHARPIN) 9 . Pathogenic variants of these genes have been shown to activate apoptosis and necroptosis in cellular or murine models 10 .…”

Very-early-onset Inflammatory Bowel Disease in an Infant with a Partial RIPK1 Deletion

“…While impaired T- and B-cell differentiation, significant lymphopenia, and decreased production of proinflammatory cytokines such as IL-6, TNF, and IL-12 lead to immunodeficiency, active inflammasome formation and necroptosis might be related to the inflammatory component of the disease [ 9 – 12 , 15 ]. Interestingly, recent studies show that variants which impair the caspase-8-mediated RIPK1 cleavage, confer a gain-of-function effect, leading to the autosomal dominant cleavage-resistant RIPK1 -induced autoinflammatory (CRIA) syndrome [ 15 – 18 ].…”

Very-early-onset inflammatory bowel disease with a partial RIPK1/ BPHL deletion in an infant

“…While impaired T and B cell differentiation, signi cant lymphopenia, and decreased production of proin ammatory cytokines such as IL-6, TNF, and IL-12 lead to immunode ciency, active in ammasome formation and necroptosis might be related to the in ammatory component of the disease [9][10][11]14]. Interestingly, recent studies show that dominantly inherited variants which impair the caspase-8-mediated RIPK1 cleavage, confer a gain-of-function effect, has been associated to cleavage-resistant RIPK1-induced autoin ammatory (CRIA) syndrome [14][15][16][17].…”