Novel Autoinflammatory Diseases Caused By RIPK1 Mutations And Somatic NLRC4 Mutation

Autoinflammatory diseases are inborn disorders of the innate immune system characterized by episodes of systemic inflammation that are mediated largely by myeloid cells.Autoinflammatory diseases are largely caused by genetic mutations.There remain a great number of patients with diseases for which we have no genetic explanation although over 50 autoinflammatory disease-causing genes have been identified in the past 30 years.Some of these patients have inflammatory conditions that are imprecisely defined on a clinical basis,became symptomatically apparent in adulthood,or both.Since many patients with autoinflammatory disease are sporadic which is hard to identify the disease-causing genes through traditional linkage methods,we adopt a genotype-first approach to dig out the disease-causing gene by next-generation sequencing.Patients with relevant symptoms were recruited to our undiagnosed program to conduct next generation sequencing.For those undiagnosed patients,variants segregate with phenotype are raised as candidates and those occurred in multiple patients will be carefully investigated by multiple strategy to reveal the disease mechanism.Through the genotype-first approach,we identified a new autoinflammatory disease caused by non-cleavable RIPK1 variants due to mutations at D324.The heterozygous RIPK1 D324 variants(D324V,D324 H and D324N)were found in 7patients from 4 unrelated families.These variants were not reported in any public database of human exomes and were predicted to be deleterious(CADD score > 24)for protein function by computational in silico modeling.We performed single cell RNA sequencing in patient PBMCs.The patient had a higher percentage of monocytes compared to an age-and sex-matched healthy control.We observed strong signals in both NF-?B and Type-I IFN inflammatory pathways in the patient’s monocytes.Our study reveals that patients’ PBMCs with RIPK1 variant are more sensitive to TNF triggered cell death which may explain the inflammatory condition in patients.In addition,novel somatic NLRC4 H443 Q mutation was reported in a patient with late-onset autoinflammatory disease.The mosaicism was occurred in a subset of leukocytes and enhanced inflammatory signaling was observed specifically in myeloid cells.The somatic NLRC4 H443 Q resulted in constitutive NLRC4 activation and mechanistic studies indicated that H443 Q somatic mutation resulted in stronger NLRC4 activation and more IL-1β and IL-18 production than other germline pathogenic mutations.Moreover,we provided proof of concept for blocking cytokine secretion by targeting gasdermin D with disulfiram.Taken together,by genotype-first approach,we identified a new disease-causing gene RIPK1 and first reported somatic NLRC4 mosaicism in late-onset autoinflammatory disease.Newly identified disease-causing gene RIPK1 and somatic form of NLRC4 mutation are sure to provide new insights in genetic testing of autoinflammatory disease and provide new therapeutic target in autoinflammatory disease.