Recessive inborn errors of type I IFN immunity in children with COVID-19 pneumonia

Abstract

Recessive or dominant inborn errors of type I interferon (IFN) immunity can underlie critical COVID-19 pneumonia in unvaccinated adults. The risk of COVID-19 pneumonia in unvaccinated children, which is much lower than in unvaccinated adults, remains unexplained. In an international cohort of 112 children (<16 yr old) hospitalized for COVID-19 pneumonia, we report 12 children (10.7%) aged 1.5-13 yr with critical (7 children), severe (3), and moderate (2) pneumonia and 4 of the 15 known clinically recessive and biochemically complete inborn errors of type I IFN immunity: X-linked recessive TLR7 deficiency (7 children) and autosomal recessive IFNAR1 (1), STAT2 (1), or TYK2 (3) deficiencies. Fibroblasts deficient for IFNAR1, STAT2, or TYK2 are highly vulnerable to SARS-CoV-2. These 15 deficiencies were not found in 1,224 children and adults with benign SARS-CoV-2 infection without pneumonia (P = 1.2 × 10-11) and with overlapping age, sex, consanguinity, and ethnicity characteristics. Recessive complete deficiencies of type I IFN immunity may underlie ∼10% of hospitalizations for COVID-19 pneumonia in children.

Graphical abstract

Figure 1.

Recessive inborn errors of the type I IFN pathway underlie life-threatening viral infections. (A) Chest computed tomography scan on day 6 after disease onset in P1, showing ground-glass opacification and consolidation in both lungs. (B) PCA analysis of patients and controls. KGP, 1000 Genomes Project database. (C) Pedigrees and familial segregation of the variants identified. Black symbols, patients with moderate to critical COVID-19 pneumonia; symbols with vertical bars, individuals with asymptomatic SARS-CoV-2 infection; blue + and − symbols, seropositive and seronegative for SARS-CoV-2, respectively.

Figure 2.

Novel deleterious variants of STAT2 and TLR7 underlie life-threatening COVID-19 in children. (A) STAT2 protein levels in HEK293T cells, with and without transfection with WT or mutant STAT2 plasmids, as assessed by Western blotting. The known LOF variant R510X served as an LOF control. (B) ISG induction upon stimulation with IFN-α2b, in STAT2-deficient U2A fibrosarcoma cells with and without transfection with the WT or STAT2 variants. qPCR results were normalized against WT. The known LOF variant R510X served as an LOF control. Experiments were repeated twice. (C) Phosphorylated STAT1 (p-STAT1) and p-STAT2 levels following stimulation with IFN-α2a or IFN-γ, as assessed by Western blotting, in SV40 fibroblasts from P1 and previously published patients with AR complete IFNAR1, STAT1, or STAT2 deficiencies and cells from two healthy controls (C1 and C2). (D) HEK293T cells were transfected with TLR7 variants, the firefly luciferase gene regulated by NF-κB, and the constitutively expressed Renilla luciferase gene, and were then stimulated with the TLR7 agonist R848. Firefly luciferase activity levels were first normalized against Renilla luciferase activity, and then against WT activity. The known LOF variant F670fs served as an LOF control. Experiments were repeated four times. Error bars indicate the SD of repeats. (E) HEK293T cells were transfected with TLR7 variants, and protein levels were assessed by Western blotting.

Figure 3.

Functional tests of biallelic variants identified in the control cohort. (A) HEK293T cells were transfected with MDA5 variants, the firefly luciferase gene regulated by IFN-β, and the constitutively expressed Renilla luciferase gene for 24 h and were then stimulated with 2 μg/ml poly(I:C) with Lipofectamine. Firefly luciferase activities were first normalized against Renilla luciferase activity and then against WT values. The known LOF variant K365E served as an LOF control. EV, empty vector. Experiments were repeated three times. Error bars indicate the SD of repeats. (B) HEK293T cells were transfected with MDA5 variants, and protein levels were assessed by Western blotting. (C) HEK293T cells were transfected with IRF7 variants, the firefly luciferase gene regulated by IFN-β, and the constitutively expressed Renilla luciferase gene for 24 h and were then stimulated with Sendai viruses or left unstimulated. Firefly luciferase activity was first normalized against Renilla luciferase activity and then against unstimulated EV. Two known LOF variants, F410V and Q421X, served as LOF controls. Experiments were repeated three times. Error bars indicate the SD of repeats. (D) HEK293T cells were transfected with IRF7 variants, and protein levels were assessed by Western blotting.

Figure 4.

SARS-CoV-2 infection in patient cells. (A) Patient SV40 fibroblasts expressing ACE2 were pretreated with 1,000 IU/ml IFN-α2b or left untreated for 16 h and were then infected with SARS-CoV-2 for 72 h. N-protein and cell nuclei were stained with specific anti-N-protein antibody and Hoechst 33342, respectively. The percentage of cells positive for N-protein was determined automatically. We tested SV40 fibroblasts from P1 and a patient with the same genotype as P2–P4 (homozygous for P216fs*14). SV40 fibroblasts from a patient with complete STAT2 deficiency (G522R/R506*) and another patient with complete IFNAR1 deficiency (homozygous V225fs) served as positive controls, whereas SV40 fibroblasts from two healthy donors (C1 and C2) served as negative controls. Experiment was done once with four technical replicates. Error bars indicate the SD of four technical repeats. (B) The fold-induction of ISGs (IFIT1, MX1, and IFI27) was determined by qPCR, with normalization against the housekeeping gene GUSB, and then untreated mock infection, with experiments performed in parallel with A. Experiments were repeated twice. Error bars indicate the SD of the repeats.