Homozygous NLRP1 gain-of-function mutation in siblings with a syndromic form of recurrent respiratory papillomatosis

Abstract

Juvenile-onset recurrent respiratory papillomatosis (JRRP) is a rare and debilitating childhood disease that presents with recurrent growth of papillomas in the upper airway. Two common human papillomaviruses (HPVs), HPV-6 and -11, are implicated in most cases, but it is still not understood why only a small proportion of children develop JRRP following exposure to these common viruses. We report 2 siblings with a syndromic form of JRRP associated with mild dermatologic abnormalities. Whole-exome sequencing of the patients revealed a private homozygous mutation in NLRP1, encoding Nucleotide-Binding Domain Leucine-Rich Repeat Family Pyrin Domain-Containing 1. We find the NLRP1 mutant allele to be gain of function (GOF) for inflammasome activation, as demonstrated by the induction of inflammasome complex oligomerization and IL-1β secretion in an overexpression system. Moreover, patient-derived keratinocytes secrete elevated levels of IL-1β at baseline. Finally, both patients displayed elevated levels of inflammasome-induced cytokines in the serum. Six NLRP1 GOF mutations have previously been described to underlie 3 allelic Mendelian diseases with differing phenotypes and modes of inheritance. Our results demonstrate that an autosomal recessive, syndromic form of JRRP can be associated with an NLRP1 GOF mutation.

Keywords: NLRP1; genetics; human papillomavirus; inflammasome; recurrent respiratory papillomatosis.

Fig. 1.

A private homozygous missense mutation in siblings with JRRP and dermatologic abnormalities. (A) Pedigree showing NLRP1 genotype of individuals. (B) Clinical images of P1 showing (from left to right) larynx papillomas, atrophoderma vermiculata on cheeks, plantar warts, and keratosis pilaris on buttocks and thighs. (C) Micrographs of a larynx papilloma from P1 showing (from left to right) gross morphology of papillomas, areas of binucleated cells (Insets: enlarged), and focal areas of koilocytosis (arrows). (D) Schematic representation of NLRP1 protein showing functional domains, location of patients’ T755N mutations (red), and location of previously described NLRP1 mutations (blue) and their mode of inheritance (AD, AR, or codominant [CoD]). (E) Protein sequencing alignment of human NLRP1 to known orthologs, showing conservation of T755.

Fig. 2.

NLRP1 T755N is GOF for inflammasome activation. (A) Western blot showing similar expression of NLRP1 WT and T755N protein in HEK293T cells. A GAPDH Western blot is shown as a loading control. The image is representative of 3 independent experiments. (B) Western blot for HA-tagged NLRP1 after BN-PAGE or conventional SDS-PAGE and lysates from HEK293T cells overexpressing cDNA of NLRP1 WT (2 replicates), T755N, previously published GOF alleles (M77T and F878_R843del), or noncleavable NLRP1 T755N (T755N+F1212A), demonstrating oligomerization of T755N NLRP1 similar to other GOF mutations. GAPDH Western blot is shown as a loading control. (C) ELISA of IL-1β in supernatants of keratinocytes after transfection with NLRP1 alleles demonstrating that T755N is GOF for IL-1β production and that cleavage at the F1212 position is required for IL-1β production. NT, nontransfected cells; EV, empty vector. Data are an average of 4 replicates. ***P < 0.001, 1-way ANOVA.

Fig. 3.

Patient-derived keratinocytes show normal expression of NLRP1 protein, baseline inflammasome activation, and unresponsiveness to further NLRP1 activation. (A) Western blot (Top) and qPCR (Bottom) of NLRP1 expression in keratinocytes from P2, heterozygous father (I.1), and 3 controls. The image is representative of 3 independent experiments. (B) Relative expression of NLRP1 WT, T755N transcripts as assessed by TA cloning and Sanger sequencing of an NLPR1 cDNA from keratinocytes from control (CTL), heterozygous father (I.1), and P2. (Inset) Numbers correspond to the number of unique clones sequenced. (C) IL-1β ELISA of supernatants from keratinocytes that were untreated or treated with 3 μM of talabostat (Val-boroPro) for 16 h. (D) IL-18 ELISA of supernatants from keratinocytes that were untreated or treated with 3 μM of talabostat for 16 h. Bars represent mean ± 1 SD. The results are representative of 3 independent experiments.

Fig. 4.

Inflammasome activation in P1 and P2. (A) Luminex measurements of serum levels of IL-1β, IL-18, IL-1Ra, IL-6, and TNF-α from P1, P2, and 3 healthy controls. Results are representative of 2 independent experiments. (B) ELISA for IL-1β after stimulation of PBMCs from P1, P2, and 2 controls with TLR ligands that induce IL-1β in an NLRP1-independent manner demonstrating normal regulation of production. Bars represent mean ± 1 SD. The results are representative of 2 independent experiments.