SARS-CoV-2 brainstem encephalitis in human inherited DBR1 deficiency

Abstract

Inherited deficiency of the RNA lariat-debranching enzyme 1 (DBR1) is a rare etiology of brainstem viral encephalitis. The cellular basis of disease and the range of viral predisposition are unclear. We report inherited DBR1 deficiency in a 14-year-old boy who suffered from isolated SARS-CoV-2 brainstem encephalitis. The patient is homozygous for a previously reported hypomorphic and pathogenic DBR1 variant (I120T). Consistently, DBR1 I120T/I120T fibroblasts from affected individuals from this and another unrelated kindred have similarly low levels of DBR1 protein and high levels of RNA lariats. DBR1 I120T/I120T human pluripotent stem cell (hPSC)-derived hindbrain neurons are highly susceptible to SARS-CoV-2 infection. Exogenous WT DBR1 expression in DBR1 I120T/I120T fibroblasts and hindbrain neurons rescued the RNA lariat accumulation phenotype. Moreover, expression of exogenous RNA lariats, mimicking DBR1 deficiency, increased the susceptibility of WT hindbrain neurons to SARS-CoV-2 infection. Inborn errors of DBR1 impair hindbrain neuron-intrinsic antiviral immunity, predisposing to viral infections of the brainstem, including that by SARS-CoV-2.

Figure 1.

A patient with SARS-CoV-2 brainstem encephalitis homozygous for a DBR1 mutation. (A) Family pedigree of index patient 1 (P1) homozygous for the DBR1 I120T/I120T mutation. The segregations of the DBR1 (I120T) and MEFV (M694V, E148Q) variants are indicated. Pathogenic mutations are shown in red for DBR1 and in blue for MEFV. Siblings are labeled from sibling 1 (S1) to S6. (B) Estimation of a potential common haplotype surrounding the DBR1 I120T mutation, as predicted by EstiAge analysis, for P1 and a previously reported patient with HSV-1 BVE. (C) MRI fluid–attenuated inversion recovery images taken on day 2 of encephalitis in P1.

Figure S1.

Homozygosity for the I120T DBR1 variant in a child with isolated SCV-2 BVE. (A) VirScan test for antibodies against a wide range of viruses in the serum of P1, his siblings, and their parents. Hierarchically clustered (Pearson) heatmap showing PhIP-Seq antibody enrichment (z-score relative to mock immunoprecipitation [IP]) for each of the 30 viruses detected in at least one member of the family. All values are the means of technical duplicates. (B) Electropherogram showing the DBR1 gDNA sequence surrounding the I120T mutation, in P1 and his older siblings (S1 [born 2000], S2 [born 2002], and S3 [born 2004]). (C) IFN-α, -β, and -γ levels in the plasma of various members of the family and 30 other healthy controls, as measured by SIMOA digital ELISA. Statistical analysis was conducted with Mann–Whitney U tests. ns: not significant.

Figure 2.

Intronic RNA lariat levels in patient-derived fibroblasts homozygous for a DBR1 mutation. (A and B) DBR1 mRNA levels (A) and DBR1 protein levels (B) in fibroblasts from two healthy controls, a DBR1 WT/WT sibling (S3), a DBR1 WT/I120T sibling (S1), and a DBR1 I120T/I120T sibling (S2) of P1, P1, and a previously reported DBR1 I120T/I120T patient with HSV-1 brainstem encephalitis. (C) ID1 and DKK1 mRNA and intronic RNA lariat levels, in fibroblasts, as in A and B, as measured by RT-qPCR. Statistical analysis was performed with two-tailed Mann–Whitney U test. ***P < 0.001. (D and E) DBR1 mRNA levels (D) and DBR1 protein levels (E) in fibroblasts from one healthy control and P1 transduced with empty vector, I120T DBR1, or WT DBR1. (F) ID1 and DKK1 intronic RNA lariat levels, in fibroblasts, as in D and E, as measured by RT-qPCR. Statistical analysis was performed with two-tailed Mann–Whitney U test. **P < 0.01. Data from A, C, D, and F are presented as the means ± SEM from three independent experiments, with two biological replicates for each experiment. Data shown in B and E are representative of three independent experiments. Source data are available for this figure: SourceData F2.

Figure 3.

Intronic RNA lariat levels in hPSC-derived hindbrain neurons homozygous for a DBR1 mutation. (A) Angiotensin-converting enzyme 2 (ACE2) mRNA levels were determined by RT-qPCR in SV-40 transformed fibroblasts (SV40-F) from healthy controls (C1, C2) and P1, A549 lung carcinoma cells with or without ACE2 transduction, and hPSC-derived hindbrain neurons (HB neurons) from a healthy control (H9) and a previously reported patient with the DBR1 mutation (DBR1 I120T/I120T). The data shown are the mean ± SEM from two independent experiments, with two technical replicates for each experiment. The limit of detection (LOD) is set as the median of ACE2 mRNA levels in SV40-F, which does not express ACE2. (B and C) DBR1 mRNA levels (B) and ID1 and DKK1 RNA lariat levels (C) in hindbrain neurons derived from healthy control (H9) and DBR1 I120T/I120T patient hPSCs, as measured by RT-qPCR. The data from B and C are presented as means ± SEM from two independent experiments, with two biological replicates for each experiment. (D) DBR1 mRNA levels and ID1 and DKK1 RNA lariat levels in hindbrain neurons derived from healthy control and DBR1 I120T/I120T patient hPSCs transduced with empty vector, I120T DBR1, or WT DBR1, as measured by RT-qPCR. The data from D are presented as means ± SEM from two independent experiments, with two biological replicates for each experiment. (E) DKK1 lariat RNA levels in hindbrain neurons derived from healthy control and DBR1 I120T/I120T patient hPSCs with or without SARS-CoV-2 infection (MOI 1, 24 hpi). The data shown are the mean ± SEM from three independent experiments, with two technical replicates for each experiment.

Figure S2.

Characterization of hPSC-derived hindbrain neurons. (A and B) Abundance of mRNA for the neuronal markers GABAR1, TUBB2B, and SLC17A7 (A), and for the hindbrain neuron–specific markers GBX2, HOXA2, HOXB2, and HOXB4 (B), as assessed by RNAseq, in hPSC-derived hindbrain neurons derived from a healthy control (H9), a DBR1 I120T/I120T patient, an IFNAR1^−/− patient, and a TLR3^−/− patient. SV40-fibroblasts from a healthy control (C1), a DBR1 I120T/I120T patient, an IFNAR1^−/− patient, and a TLR3^−/− patient were included as negative controls for the detection of these neuron-specific markers. Triplicates were studied for each sample in A and B.

Figure 4.

SARS-CoV-2 infection in hPSC-derived hindbrain neurons. (A) Representative immunofluorescence images of hPSC-derived hindbrain neurons infected with SARS-CoV-2 (MOI 0.1) at 72 h post-infection (hpi) for a healthy control (H9), a previously reported patient with the DBR1 mutation (DBR1 I120T/I120T), and patients with complete TLR3 (TLR3^−/−) or IFNAR1 (IFNAR1^−/−) deficiency. Cells were stained with antibodies against the SARS-CoV-2 nucleocapsid protein (N, red) and a neuron-specific microtubule-associated protein 2 (MAP2, green). A/T-rich chromosomal DNA was stained with DAPI (blue). Bar: 150 µm. The data shown are representative of three independent experiments. (B and C) Percentage of hindbrain neurons (MAP2⁺) positive for the SARS-CoV-2 N protein, at various time points (hpi), with and without IFN-β pretreatment, for cells infected with SARS-CoV-2 at an MOI of 0.1 (B) or 10 (C). The data points are the means ± SEM from three independent experiments with three technical replicates per experiment. Statistical analysis was conducted with Kruskal–Wallis tests, with Dunn’s test for multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.001. (D) Scatterplots of the mean log₂ fold-changes in RNAseq-quantified gene induction following stimulation with 100 IU/ml of IFN-β for 8 h, in hPSC-derived hindbrain neurons from a healthy control (H9), a previously reported patient with the DBR1 mutation (DBR1 I120T/I120T), and patients with complete TLR3 (TLR3^−/−) or IFNAR1 (IFNAR1^−/−) deficiency. Each point represents a single gene. Genes with an absolute fold-change in expression >2 in response to IFN-β treatment relative to NS samples in the control (Ctrl) group are plotted. (E and F) ID1 and DKK1 intronic RNA lariat levels (E) and SARS-CoV-2 nucleocapsid 2 (SCV-2 N2) and RNA-dependent RNA polymerase (SCV-2 RdRp) mRNA levels (F), in hPSC-derived hindbrain neurons from a healthy control (H9) transduced with DKK1 lariat-expressing lentivirus, as measured by RT-qPCR, after infection with SARS-CoV-2 (MOI 0.1), 2 hpi, 24 hpi and 36 hpi. The data shown are the mean ± SEM from two independent experiments, with two biological replicates for each experiment.

Figure S3.

SARS-CoV-2 infection in hPSC-derived hindbrain neurons with and without IFN-β pretreatment. (A) Representative immunofluorescence images of hPSC-derived hindbrain neurons infected with SARS-CoV-2 (MOI 10) at 72 hpi, for a healthy control (H9), a previously reported patient with the DBR1 mutation (DBR1 I120T/I120T), and patients with complete TLR3 (TLR3^−/−) or IFNAR1 (IFNAR1^−/−) deficiency. Cells were stained with antibodies against the SARS-CoV-2 nucleocapsid protein (N, red) and a neuron-specific microtubule-associated protein 2 (MAP2, green). A/T-rich chromosomal DNA was stained with DAPI (blue). Bar: 150 µm. Data shown are representative of three independent experiments. (B) Quantification of the SARS-CoV-2 nucleocapsid (N2) (upper panel) and the RNA-dependent RNA polymerase (RdRp) (lower panel) by TaqMan real-time qPCR, at 2, 24, 48, 72, and 96 h after SARS-CoV-2 infection (MOI 1). Data are presented as the mean ± SEM and are representative of two independent experiments with biological triplicates in each experiment. (C) Heatmaps of RNAseq-quantified gene expression (z-score-scaled DESeq2 vst-normalization) in hPSC-derived hindbrain neurons from a healthy control (H9), a previously reported patient with the DBR1 mutation (DBR1 I120T/I120T), an IFNAR1^−/− patient, and a TLR3^−/− H patient, not stimulated (NS) or stimulated with IFN-β for 8 h. Duplicates were studied for each set of conditions and mean gene expression levels were used for subsequent analyses. The heatmap includes genes with a relative fold-change in expression >2 in response to IFN-β treatment relative to NS samples in the control group.