Mutations in CECR1 associated with a neutrophil signature in peripheral blood

Abstract

Background: A reduction of ADA2 activity due to autosomal recessive loss of function mutations in CECR1 results in a newly described vasculopathic phenotype reminiscent of polyarteritis nodosa, with manifestations ranging from fatal systemic vasculitis with multiple strokes in children to limited cutaneous disease in middle-aged individuals. Evidence indicates that ADA2 is essential for the endothelial integrity of small vessels. However, CECR1 is not expressed, nor is the ADA2 protein detectable, in cultured human endothelial cells, thus implicating additional cell types or circulating factors in disease pathogenesis.

Methods: Considering the phenotypic overlap of ADA2 deficiency with the type I interferonopathy Aicardi-Goutières syndrome due to mutations in SAMHD1, we looked for the presence of an interferon signature in the peripheral blood of two newly ascertained ADA2-deficient patients.

Results: We identified biallelic CECR1 mutations in two patients consistent with ADA2 deficiency. Both patients demonstrated an upregulation of interferon stimulated gene transcripts in peripheral blood. More strikingly however, genome-wide analysis revealed a marked overexpression of neutrophil-derived genes, suggesting that the vasculitis seen in ADA2 deficiency may be an indirect effect resulting from chronic and marked activity of neutrophils.

Conclusions: We hypothesise that ADA2 may act as a regulator of neutrophil activation, and that a reduction of ADA2 activity results in significant endothelial damage via a neutrophil-driven process.

Keywords: ADA2; Adenosine deaminase; Aicardi-Goutières syndrome; CECR1; Neutrophil signature; SAMHD1; Type I interferon.

Figure 1

Clinical and radiological features. A-C. Generalized livedo of the back (A), leg (B) and foot (C). D. Right frontal insular haemorrhage. E. Left middle cerebral ischemic stroke. F. Left internal carotid artery stenosis.

Figure 2

Gene expression studies. Quantitative reverse transcription PCR (qPCR) of a panel of six interferon stimulated genes (ISGs) and six neutrophil-expressed genes in whole blood measured in CECR1 mutation-positive probands, SAMHD1 mutation-positive probands and controls. Bar graph showing relative quantification (RQ) values for a panel of six interferon stimulated genes (ISGs) (IFI27, IFI44L, IFIT1, ISG15, RSAD2, SIGLEC1) and six neutrophil-expressed genes (CEACAM6, CRISP3, DEFA4, LCN2, LTF, MMP8) measured in whole blood in two ADA2 mutation positive patients, and two SAMHD1 mutation positive patients, compared to a healthy control. RQ is equal to 2-∆∆Ct, with -∆∆Ct ± standard deviations (i.e. the normalized fold change relative to a calibrator). Each value is derived from three technical replicates. Numbers in brackets refer to decimalized age at sampling, followed by interferon score calculated from the median fold change in relative quantification value for the panel of six ISGs. Colors denote individuals, with repeat samples (biological replicates) denoted by different bars of the same color.

Figure 3

Genome-wide gene expression. Heatmap showing genome-wide expression array in patients with mutations in CECR1 and RNA-seq analysis in patients with mutations in SAMHD1. Genes shown had a fold change greater than 5 (mean patients versus mean controls in the microarrays). Normalised expression levels for the microarray were expressed as fold change versus control samples in log scale. Similarly, for RNA-seq, normalised counts were expressed as fold change versus control samples in log scale. To generate the heatmap these log ratios (and control values of zero) were normalised between genes by setting the standard deviation of each gene to 1. Blue low expression, grey no change, red high expression. Samples shown: C = control; 1 = F785 patient 1 (ADA2); 2 = F750 patient 2 (ADA2); 3 = F104 (SAMHD1); 4 = F116 (SAMHD1).