Development of a Validated Interferon Score Using NanoString Technology

Abstract

Chronic elevation of interferon (IFN)-response genes (IRG) in a subset of patients with systemic immune-dysregulatory diseases, including the Mendelian Type-I IFN-mediated autoinflammatory diseases and some autoimmune diseases suggest a causative role of excessive IFN signaling in the disease pathogenesis and as target for treatment. We developed a 28-IFN response gene scoring system to calculate either a standardized or geomean score by customizing a NanoString assay to quantify the expression of putative IRGs. The gene targets were selected in patients with the IFN-mediated disease chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature (CANDLE) and an adult patient with chronic hepatitis C who received the first dose of pegylated interferon alpha-2a. The putative target genes were validated in patients with STING-associated vasculopathy with onset in infancy (SAVI), a monogenic autoinflammatory disease caused by gain-of-function mutations in TMEM173 that encodes the viral sensor stimulator of IFN genes (STING), and had low expression in clinically active patients with the monogenic IL-1-mediated autoinflammatory disease, neonatal-onset multisystem inflammatory disease (NOMID) and in healthy controls. The score calculation on the NanoString assay is rapid and showed high reproducibility and low intra-, and interassay variability. The utility of this 28-gene IFN score may be explored in the diagnosis of patients with presumed interferonopathies and as a biomarker to assess disease activity, long-term outcome, and treatment responses.

Trial registration: ClinicalTrials.gov NCT02974595 NCT00718172.

Keywords: IFN score; IFN-mediated autoinflammatory diseases; NanoString assay; assay validation; blood IFN signature; interferonopathies.

FIG. 1.

Comparison of IFN response genes included in the IFN score in CANDLE, SAVI, NOMID, and HCs. The figure panels depict the expression patterns of the 28 genes that were kept in the final score. For each example, a representative graph is shown with a list of the other genes with similar patterns. Box and whisker plots of log2 normalized counts of each gene are shown, with individual dots for each patient stratified by diagnosis (CANDLE, SAVI, and NOMID) and for healthy control subjects (HC). The middle line indicates the median. Uncorrected t-tests were performed to compare between diagnosis and control groups. At the right of each panel, each circle represents a diagnosis group, with the center at the level of the mean of the respective group. See Supplementary Fig. S2 legend for details. (A) Thirteen genes are highly expressed, significantly higher in CANDLE and SAVI than in NOMID and HC, expression in NOMID is significantly lower than in HCs. (P value range for CANDLE and NOMID were between 3.01 × 10⁻³⁰ and 7.24 × 10⁻¹⁸, for SAVI and HC between 8.70 × 10⁻²⁷ and 1.33 × 10⁻¹⁴), and SAVI and NOMID (P value range, 1.01 × 10⁻²⁸ and 1.40 × 10⁻¹⁶). There is no statistically significant difference between CANDLE and SAVI, but expression in NOMID patients is significantly lower than in HC (P value range, 7.46 × 10⁻⁶ to 0.0496). (B) Nine genes are highly expressed in CANDLE and SAVI with similar low expression in NOMID and HC. P values are significant for CANDLE versus NOMID (P value range, 4.34 × 10⁻²⁵ to 1.27 × 10⁻⁸), SAVI versus HC (P value range, 1.94 × 10⁻²² to 1.17 × 10⁻⁴), and SAVI versus NOMID (P value range, 8.61 × 10⁻²³ to 7.47 × 10⁻⁶). There is no significant difference between CANDLE and SAVI or NOMID and HC. (C) Three genes are significantly highly expressed in CANDLE than SAVI (P value range, 1.16 × 10⁻⁵ to 0.042), further expression in CANDLE patients is significantly higher than in NOMID patients (P value range, 1.08 × 10⁻²⁵ to 4.19 × 10⁻¹⁶), SAVI expression is significantly higher than in NOMID (P value range, 1.43 × 10⁻¹⁹ to 4.97 × 10⁻⁸) and in HC (P value range, 3.67 × 10⁻¹⁶ to 3.25 × 10⁻⁵). Gene expression in NOMID was significantly higher than in HC (P value range, 1.67 × 10⁻⁴ to 8.86 × 10⁻⁴) for SPATS2L and LAMP3, but the P value was not significant for GBP1. (D) One gene is highly expressed in SAVI than CANDLE (P value of 0.01), although both high, and NOMID lower than HC (P value of 0.0002), although both low. P values are as follows: CANDLE versus NOMID (2.05 × 10⁻²⁶), SAVI versus HC (3.38 × 10⁻²⁵), and SAVI versus NOMID (6.57 × 10⁻²⁷). (E) Two genes have NOMID higher than HC (P values 0.013 and 0.025), although both are lower than CANDLE and SAVI. P value are as follows: CANDLE versus NOMID (2.25 × 10⁻¹⁷ to 4.32 × 10⁻¹⁴), SAVI versus HC (6.25 × 10⁻¹⁹ to 4.10 × 10⁻⁸), and SAVI versus NOMID (7.09 × 10⁻⁵ and 1.21 × 10⁻¹⁵). For SOCS1, the gene expression is significantly higher in NOMID than in HC (02.47 × 10⁻⁴), but expression of IFIT5 is not significantly different. (F) Standardized 28-IFN gene scores in CANDLE and SAVI patients were each significantly higher than in NOMID and HC (P values <2 × 10⁻⁹), scores in CANDLE and SAVI patients are not significantly different and scores in NOMID patients and HC are not significantly different. (G) The geomean scores in patients with CANDLE and SAVI are significantly higher than in patients with NOMID and in HC (P values <2 × 10⁻¹³). CANDLE, chronic atypical neutrophilic dermatosis with lipodystrophy and elevated temperature; IFN, interferon; NOMID, neonatal-onset multisystem inflammatory disease; SAVI, STING-associated vasculopathy with onset in infancy; STING, stimulator of IFN genes.

FIG. 2.

Assessment of interassay and interoperator variability. (A) 11 samples were assayed at 2 different time points by the same operator. The counts for each gene were plotted. (B) Interoperator variability was compared in 4 samples that were assayed by 2 operators each.