Homozygous variant p. Arg90His in NCF1 is associated with early-onset Interferonopathy: a case report

Abstract

Background: Biallelic loss-of-function variants in NCF1 lead to reactive oxygen species deficiency and chronic granulomatous disease (CGD). Heterozygosity for the p.Arg90His variant in NCF1 has been associated with susceptibility to systemic lupus erythematosus, rheumatoid arthritis, and Sjögren's syndrome in adult patients. This study demonstrates the association of the homozygous p.Arg90His variant with interferonopathy with features of autoinflammation and autoimmunity in a pediatric patient.

Case presentation: A 5-year old female of Indian ancestry with early-onset recurrent fever and headache, and persistently elevated antinuclear, anti-Ro, and anti-La antibodies was found to carry the homozygous p.Arg90His variant in NCF1 through exome sequencing. Her unaffected parents and three other siblings were carriers for the mutant allele. Because the presence of two NCF1 pseudogenes, this variant was confirmed by independent genotyping methods. Her intracellular neutrophil oxidative burst and NCF1 expression levels were normal, and no clinical features of CGD were apparent. Gene expression analysis in peripheral blood detected an interferon gene expression signature, which was further supported by cytokine analyses of supernatants of cultured patient's cells. These findings suggested that her inflammatory disease is at least in part mediated by type I interferons. While her fever episodes responded well to systemic steroids, treatment with the JAK inhibitor tofacitinib resulted in decreased serum ferritin levels and reduced frequency of fevers.

Conclusion: Homozygosity for p.Arg90His in NCF1 should be considered contributory in young patients with an atypical systemic inflammatory antecedent phenotype that may evolve into autoimmunity later in life. The complex genomic organization of NCF1 poses a difficulty for high-throughput genotyping techniques and variants in this gene should be carefully evaluated when using the next generation and Sanger sequencing technologies. The p.Arg90His variant is found at a variable allele frequency in different populations, and is higher in people of South East Asian ancestry. In complex genetic diseases such as SLE, other rare and common susceptibility alleles might be necessary for the full disease expressivity.

Keywords: Autoimmunity; Autoinflammation; Interferons; NCF1; Systemic lupus erythematosus.

Fig. 1

Patient presentation and NCF1 p.Arg90His genotyping in the proband and her family. a: Atrophic skin lesion on lower abdomen of patient homozygous for NCF1, p.Arg90His. b: Residual oxidase activity of neutrophils and NCF1 expression in patient and healthy control. Left: Residual oxidase activity of neutrophils was determined using dihydrorhodamine (DHR) oxidation by flow cytometry. Right: NCF1 expression in patient and healthy control. NCF1 expression in neutrophils is presented as mean fluorescence intensity. Both panels: Red histograms represent neutrophils treated with buffer under basal conditions; blue histograms represent neutrophils in response to PMA (400 ng/mL). c: Pedigree of the family with the recessively inherited homozygous pathogenic variant p.Arg90His in the NCF1 gene. d: Sanger sequencing validation in proband and family. Sanger sequencing confirmed the homozygous variant NCF1, c.269G > A, p.Arg90His in the patient. Her parents and two brothers are heterozygous for the same variant

Fig. 2

Interferon signature analysis, inflammatory cytokine profile and immune phenotyping in patient and family. a: Nanostring analysis for IFN signature genes in the patient (Patient-F = sample taken during flare; Patient-NF, SLE patient with the complement C1R deficiency (SLE) and healthy controls (Control 1–4). Red, elevated expression compared to control; blue, reduced expression compared to control. b: Quantitative reverse transcription (qRT-PCR) analysis of 10 IFN-related genes shows strong upregulation in the proband compared to control. Shown is fold change to control normalized to GAPDH expression. c: Quantitative reverse transcription (qRT-PCR) analysis of IL15 in proband and healthy controls. Shown are the relative expression values of IL15/GAPDH. d: IFN-α2, IL-3, TNF-β and nerve growth factor β (NGFβ) cytokine measurement on whole blood cells from the proband, her parents and 2 brothers. Cells were untreated or stimulated with different stimuli (heat-killed Listeria monocytogenes [HKLM] at 107 bacteria/ml, Poly(I:C) at 10 μg/ml, lipopolysaccharide [LPS] at 1 μg/ml, flagellin [FLA] at 50 ng/ml, imiquimod [Imiqu] at 5 μg/ml, ODN2395 [2395] at 5 μM and Staphylococcal Enterotoxin B [SEB] at 1 μg/ml). e: Quantification of pDCs and intermediate monocytes in the proband, her parents and two brothers. PBMCs were extracted from whole blood and incubated with the monoclonal antibodies anti-CD11c and anti-CD123 for pDC analysis and anti-CD14 and anti-CD16 for the analysis of intermediate monocytes. The data shown here are the only comparisons that achieved nominal statistical significance by the Mann-Whitney U test, which would not withstand correction for multiple comparisons