The unusual features and severity of the disease in this infant, who was born to consanguineous parents, prompted us to perform genomewide analysis of single-nucleotide polymorphisms. We found a 175-kb deletion at chromosome 2q13 that encompasses six interleukin-1 family members: IL1RN, IL1F5, IL1F6, IL1F8, IL1F9, and IL1F10 (). The parents were heterozygous for this deletion and were healthy ().
Genetic and Gene-Expression Characteristics of Our Patient with a Deletion at the IL1RN Locus and a Patient with Neonatal-Onset Multisystem Inflammatory Disease (NOMID)
To differentiate our patient's disease from NOMID, we compared global gene-expression profiles of mononuclear cells isolated from him, a control subject, and a patient with NOMID due to a mutation in NLRP3 (Phe525Leu). Since baseline gene expression varied considerably among these three subjects, we normalized the data to the overall mean log2 signal intensity for all conditions. Hierarchical clustering was conducted with the use of any probe set that exhibited a minimum difference by a factor of 16 among the three subjects. We found that the transcriptomes from unstimulated mononuclear cells from our patient with the deletion and the patient with NOMID were similar, but both differed from the control subject's transcriptome (). After lipopolysaccharide stimulation of mononuclear cells, the transcriptome of mononuclear cells from the our patient was more similar to that of the control than that of the patient with NOMID. The transcription of genes encoding interleukin-1β, interleukin-1α, interleukin-8, and interleukin-6 increased in cells from our patient with the deletion and the control but not in those from the patient with NOMID.
To confirm the results of the microarray experiments, we measured the amounts of interleukin-1β, interleukin-6, interleukin-8, interleukin-10, and TNF-α in the supernatants from unstimulated and lipopolysaccharide-stimulated mononuclear cells. Elevated amounts of interleukin-1β, interleukin-6, and interleukin-8 and TNF-α were found in unstimulated cells from the patient with NOMID and our patient (). After lipopolysaccharide stimulation, these cytokines increased markedly in the supernatants from cultured cells from the control and our patient but not in cells from the patient with NOMID.
Previous investigations showed that monocytes from patients with NOMID undergo apoptosis in response to lipopolysaccharide.11
We tested whether lipopolysaccharide stimulation also caused the death of monocytes from our patient with the deletion. shows dramatic loss of monocytes after lipopolysaccharide stimulation of mononuclear cells from the patient with NOMID, whereas no such loss was found in cultures of cells from controls (data not shown) and our patient.
Since both parents of our patient carried the deletion, we tested whether haploinsufficiency of the gene cluster on chromosome 2q13 increased the production of interleukin-1β. Interleukin-1β levels were measured in supernatants from lipopolysaccharide-stimulated mononuclear cells from the patient, both parents, a control, and a patient with NOMID. Cultures of unstimulated mono-nuclear cells from both parents and the control produced no interleukin-1β, whereas cultures of mononuclear cells from the patient with NOMID and our patient with the deletion showed elevated levels of interleukin-1β (). After lipopolysaccharide stimulation, mononuclear cells from the control, mother, father, and our patient markedly up-regulated interleukin-1β, with the highest levels after 24 hours, whereas the cells from the patient with NOMID failed to up-regulate interleukin-1β beyond the levels before stimulation.