Case report: Novel compound heterozygous IL1RN mutations as the likely cause of a lethal form of deficiency of interleukin-1 receptor antagonist

Abstract

Undiagnosed monogenic diseases represent a challenging group of human conditions highly suspicious to have a genetic origin, but without conclusive evidences about it. We identified two brothers born prematurely from a non-consanguineous healthy couple, with a neonatal-onset, chronic disease characterized by severe skin and bone inflammatory manifestations and a fatal outcome in infancy. We conducted DNA and mRNA analyses in the patients' healthy relatives to identify the genetic cause of the patients' disease. DNA analyses were performed by both Sanger and next-generation sequencing, which identified two novel heterozygous IL1RN variants: the intronic c.318 + 2T>G variant in the father and a ≈2,600-bp intragenic deletion in the mother. IL1RN mRNA production was markedly decreased in both progenitors when compared with healthy subjects. The mRNA sequencing performed in each parent identified two novel, truncated IL1RN transcripts. Additional experiments revealed a perfect intrafamilial phenotype-genotype segregation following an autosomal recessive inheritance pattern. The evidences shown here supported for the presence of two novel loss-of-function (LoF) IL1RN pathogenic variants in the analyzed family. Biallelic LoF variants at the IL1RN gene cause the deficiency of interleukin-1 receptor antagonist (DIRA), a monogenic autoinflammatory disease with marked similarities with the patients described here. Despite the non-availability of the patients' samples representing the main limitation of this study, the collected evidences strongly suggest that the patients described here suffered from a lethal form of DIRA likely due to a compound heterozygous genotype at IL1RN, thus providing a reliable genetic diagnosis based on the integration of old medical information with currently obtained genetic data.

Keywords: autoinflammatory diseases (AID); case report; interleukin-1; interleukin-1 receptor antagonist (IL-1 ra); whole-genome sequencing (WGS).

Figure 1

IL1RN variants detected in enrolled individuals. (A) Pedigree of the family. Black-filled symbols, affected subjects; open symbols, unaffected subjects; squares, male subjects; circles, female subjects; slash, deceased subjects. IL1RN genotypes are shown below each analyzed subject. n.a., not analyzed; wt, wild-type. (B) Sense Sanger chromatograms from subject I-2 carrying the heterozygous genotype for the c.318 + 2T>G IL1RN variant (left box) and from a healthy subject (right box). The arrows indicate the position where the nucleotide variant is located. (C) Genomic organization of isoform 1 of IL1RN gene (NM_173842.3). Green arrows represent the forward and reverse primers designed to generate a PCR amplicon specific of the genomic deletion. (D) Sense Sanger chromatogram showing the breakpoint and boundaries of the genomic deletion at IL1RN locus identified in subject I-1. The arrows indicate the nucleotides located at each side of the breakpoint site, and shown below are the respective genomic coordinates according to GRCh38. (E) Agarose gel electrophoresis of PCR products generated with the use of primers designed for genomic deletion. N, negative control; H1 and H2, healthy subjects. Black arrows indicate the specific bands of PCR amplicons of the IL1RN allele containing the deletion (top) and a positive control of PCR reaction (bottom).

Figure 2

| mRNA IL1RN analysis. (A) Scheme of IL1RN gene (isoform 1; RefSeq: NM_173842.3) and location of probes employed in the quantitative PCR assay. The red bar indicates the probe mapping in exon 1, whereas the green bars indicate the probe Hs00893626, which maps in the junction of exons 3 and 4. (B) Relative mRNA levels of IL1RN in the peripheral blood of healthy subjects (n = 8) and patients’ parents determined by quantitative PCR. The bars indicate the ratio of exon 3 versus exon 1, while T bars indicate the standard deviations, where applicable. (C) Relative quantification of exons of IL1RN determined by mRNA sequencing. The bars indicate the mean of the fold change among parents and healthy controls. (D) Schemes of novel IL1RN mRNA transcripts showing the exon junctions (blue) identified by mRNAseq in subject I-1 (upper panel) and subject I-2 (middle panel) compared with the normal mRNA transcripts identified in a healthy subject (bottom panel).