Novel Insights into the Clinical Features, Genetic Spectrum and Clonal Evolution of Patients Carrying NLRP3 Mosaicism

Abstract

NLRP3 mosaicism is a well-established mechanism causing the monogenic autoinflammatory disease named cryopyrin-associated periodic syndromes (CAPS). The number of reported patients with NLRP3 mosaicism is small, and the knowledge about the long-term disease behavior is limited. Herein we assembled the largest cohort of individuals with NLRP3 mosaicism reported to date to obtain additional evidence that strengthens the understanding of this disease. The novel genetic data were obtained by using Sanger and next-generation sequencing methods, whereas in vitro analyses determined the functional consequences of detected variants. A total of seventeen individuals with NLRP3 mosaicism were enrolled, with 16/17 experiencing different CAPS phenotypes. An overrepresentation of late-onset forms was detected (37.5%). Overall, clinical manifestations, analytical results, and outcomes of treatments were markedly similar to those detected in patients with germline variants. A large mutational diversity was identified, with 16 different variants among 17 individuals. Two main patterns of mosaicism (extended vs. myeloid-restricted) were detected, with the last one overrepresented in the late-onset group. The evaluation of mosaicism over time identified three different patterns, being the group with stable mosaicism the largest one. Collected evidence supports the marked similarities among patients carrying somatic or germline NLRP3 variants. The overrepresentation of NLRP3 mosaicism in late-onset forms should be considered in patients with inflammatory manifestations starting in adulthood. Analysis of mosaicism at the biological level confirms the two known patterns of corporal distribution and reveals that mosaicism remains stable over time in most patients, but it may also vary during the course of the disease.

Keywords: NLRP3 gene; Autoinflammatory diseases; Inflammasome; Interleukin-1; Interleukin-1 inhibitors; Mosaicism.

Fig. 1

Panel A. Distribution of enrolled patients according to their age at disease onset. White bars indicate those patients belonging to the late-onset group. Panel B. Delay of diagnosis detected since disease onset until genetic confirmation. The decades on the X-axis indicate the patients’ year of birth. White bars indicate those patients belonging to the late-onset group. Panel C. Variant allele frequency (VAF) of detected NLRP3 variants at diagnosis. Green bar indicates the asymptomatic individual, whereas gray bars represent the symptomatic patients. Horizontal dotted red line represents the mean VAF

Fig. 2

Main Phenotypic Features in Patients with NLRP3 mosaicism. Black columns indicate the features detected in all enrolled patients (n: 16), red columns those features detected in the patients of the early-onset group (n: 6) and blue columns the features detected in the patients of the late-onset group (n: 6). Abbreviatures: EO, early-onset; LO, late-onset

Fig. 3

Domain organization and functionality of cryopyrin and localization of known post-zygotic NLRP3 pathogenic variants. Panel (A) Above the protein scheme are indicated those post-zygotic NLRP3 variants reported elsewhere, and below the protein scheme those post-zygotic variants detected in enrolled individuals. The novel NLRP3 variants are highlighted in red letters, whereas those post-zygotic variants detected in healthy individuals are highlighted in green letters. Boxes indicates the regions spanning the amino acid resides 300–310 (left) and 560–570 (right), where the post-zygotic NLRP3 variants concentrate. Red dots indicate the number of patients identified as carriers of the concrete post-zygotic NLRP3 variant. PYD: Pyrin domain; NBD: Nucleotide binding domain (Walker A and B marked in orange); HD: Helix domain; WHD: Wind helix domain; LRR: Leucine rich repeat domain. Panel (B) Chain A of the NLRP3 cryo-EM structure, corresponding to the active inflammasome disk (PDB: 8EJ4, left) and the inactive decamer bound to MCC950 (PDB: 7PZC, right), regions corresponding to the regions where the post-zygotic NLRP3 variants are concentrated are denoted in red and green. Panel (C) Percentage of ASC-specking HEK293T cells expressing different NLRP3 variants as indicated in the presence or absence of the NLRP3 inhibitor MCC950

Fig. 4

Clonal dynamics of NLRP3 mosaicism over time. Every panel represents the evolution of the frequency of the mutant NLRP3 allele in each analyzed individual over time. X-axes represent the time in months (M) from diagnosis to sample collection, with M0 being the results obtained at genetic diagnosis. Abbreviations: VAF, variant allele frequency

Fig. 5

Patterns of Mosaicism Distribution in Leukocyte Subpopulations. Each panel shows the results of amplicon-based deep sequencing using DNA extracted from isolated leukocyte subpopulations. Each bar represents the mean variant allele frequency (VAF) from three independent experiments in the indicated leukocyte subpopulation and the whiskers on top indicate the standard deviation (SD)