Inborn Errors of RNA Lariat Metabolism in Humans with Brainstem Viral Infection.

Viruses that are typically benign sometimes invade the brainstem in otherwise healthy children. We report bi-allelic DBR1 mutations in unrelated patients from different ethnicities, each of whom had brainstem infection due to herpes simplex virus 1 (HSV1), influenza virus, or norovirus. DBR1 encodes the only known RNA lariat debranching enzyme. We show that DBR1 expression is ubiquitous, but strongest in the spinal cord and brainstem. We also show that all DBR1 mutant alleles are severely hypomorphic, in terms of expression and function. The fibroblasts of DBR1-mutated patients contain higher RNA lariat levels than control cells, this difference becoming even more marked during HSV1 infection. Finally, we show that the patients' fibroblasts are highly susceptible to HSV1. RNA lariat accumulation and viral susceptibility are rescued by wild-type DBR1. Autosomal recessive, partial DBR1 deficiency underlies viral infection of the brainstem in humans through the disruption of tissue-specific and cell-intrinsic immunity to viruses.

Impaired intrinsic immunity to HSV-1 in human iPSC-derived TLR3-deficient CNS cells.

In the course of primary infection with herpes simplex virus 1 (HSV-1), children with inborn errors of toll-like receptor 3 (TLR3) immunity are prone to HSV-1 encephalitis (HSE). We tested the hypothesis that the pathogenesis of HSE involves non-haematopoietic CNS-resident cells. We derived induced pluripotent stem cells (iPSCs) from the dermal fibroblasts of TLR3- and UNC-93B-deficient patients and from controls. These iPSCs were differentiated into highly purified populations of neural stem cells (NSCs), neurons, astrocytes and oligodendrocytes. The induction of interferon-ß (IFN-ß) and/or IFN-λ1 in response to stimulation by the dsRNA analogue polyinosinic:polycytidylic acid (poly(I:C)) was dependent on TLR3 and UNC-93B in all cells tested. However, the induction of IFN-ß and IFN-λ1 in response to HSV-1 infection was impaired selectively in UNC-93B-deficient neurons and oligodendrocytes. These cells were also much more susceptible to HSV-1 infection than control cells, whereas UNC-93B-deficient NSCs and astrocytes were not. TLR3-deficient neurons were also found to be susceptible to HSV-1 infection. The rescue of UNC-93B- and TLR3-deficient cells with the corresponding wild-type allele showed that the genetic defect was the cause of the poly(I:C) and HSV-1 phenotypes. The viral infection phenotype was rescued further by treatment with exogenous IFN-α or IFN-ß ( IFN-α/ß) but not IFN-λ1. Thus, impaired TLR3- and UNC-93B-dependent IFN-α/ß intrinsic immunity to HSV-1 in the CNS, in neurons and oligodendrocytes in particular, may underlie the pathogenesis of HSE in children with TLR3-pathway deficiencies.

The human gene damage index as a gene-level approach to prioritizing exome variants.

The protein-coding exome of a patient with a monogenic disease contains about 20,000 variants, only one or two of which are disease causing. We found that 58% of rare variants in the protein-coding exome of the general population are located in only 2% of the genes. Prompted by this observation, we aimed to develop a gene-level approach for predicting whether a given human protein-coding gene is likely to harbor disease-causing mutations. To this end, we derived the gene damage index (GDI): a genome-wide, gene-level metric of the mutational damage that has accumulated in the general population. We found that the GDI was correlated with selective evolutionary pressure, protein complexity, coding sequence length, and the number of paralogs. We compared GDI with the leading gene-level approaches, genic intolerance, and de novo excess, and demonstrated that GDI performed best for the detection of false positives (i.e., removing exome variants in genes irrelevant to disease), whereas genic intolerance and de novo excess performed better for the detection of true positives (i.e., assessing de novo mutations in genes likely to be disease causing). The GDI server, data, and software are freely available to noncommercial users from lab.rockefeller.edu/casanova/GDI.

Severe influenza pneumonitis in children with inherited TLR3 deficiency.

Autosomal recessive IRF7 and IRF9 deficiencies impair type I and III IFN immunity and underlie severe influenza pneumonitis. We report three unrelated children with influenza A virus (IAV) infection manifesting as acute respiratory distress syndrome (IAV-ARDS), heterozygous for rare TLR3 variants (P554S in two patients and P680L in the third) causing autosomal dominant (AD) TLR3 deficiency. AD TLR3 deficiency can underlie herpes simplex virus-1 (HSV-1) encephalitis (HSE) by impairing cortical neuron-intrinsic type I IFN immunity to HSV-1. TLR3-mutated leukocytes produce normal levels of IFNs in response to IAV. In contrast, TLR3-mutated fibroblasts produce lower levels of IFN-ß and -λ, and display enhanced viral susceptibility, upon IAV infection. Moreover, the patients' iPSC-derived pulmonary epithelial cells (PECs) are susceptible to IAV. Treatment with IFN-α2b or IFN-λ1 rescues this phenotype. AD TLR3 deficiency may thus underlie IAV-ARDS by impairing TLR3-dependent, type I and/or III IFN-mediated, PEC-intrinsic immunity. Its clinical penetrance is incomplete for both IAV-ARDS and HSE, consistent with their typically sporadic nature.

Human SNORA31 variations impair cortical neuron-intrinsic immunity to HSV-1 and underlie herpes simplex encephalitis.

Herpes simplex virus-1 (HSV-1) encephalitis (HSE) is typically sporadic. Inborn errors of TLR3- and DBR1-mediated central nervous system cell-intrinsic immunity can account for forebrain and brainstem HSE, respectively. We report five unrelated patients with forebrain HSE, each heterozygous for one of four rare variants of SNORA31, encoding a small nucleolar RNA of the H/ACA class that are predicted to direct the isomerization of uridine residues to pseudouridine in small nuclear RNA and ribosomal RNA. We show that CRISPR/Cas9-introduced bi- and monoallelic SNORA31 deletions render human pluripotent stem cell (hPSC)-derived cortical neurons susceptible to HSV-1. Accordingly, SNORA31-mutated patient hPSC-derived cortical neurons are susceptible to HSV-1, like those from TLR3- or STAT1-deficient patients. Exogenous interferon (IFN)-ß renders SNORA31- and TLR3- but not STAT1-mutated neurons resistant to HSV-1. Finally, transcriptome analysis of SNORA31-mutated neurons revealed normal responses to TLR3 and IFN-α/ß stimulation but abnormal responses to HSV-1. Human SNORA31 thus controls central nervous system neuron-intrinsic immunity to HSV-1 by a distinctive mechanism.

Deciphering Human Cell-Autonomous Anti-HSV-1 Immunity in the Central Nervous System.

Herpes simplex virus 1 (HSV-1) is a common virus that can rarely invade the human central nervous system (CNS), causing devastating encephalitis. The permissiveness to HSV-1 of the various relevant cell types of the CNS, neurons, astrocytes, oligodendrocytes, and microglia cells, as well as their response to viral infection, has been extensively studied in humans and other animals. Nevertheless, human CNS cell-based models of anti-HSV-1 immunity are of particular importance, as responses to any given neurotropic virus may differ between humans and other animals. Human CNS neuron cell lines as well as primary human CNS neurons, astrocytes, and microglia cells cultured/isolated from embryos or cadavers, have enabled the study of cell-autonomous anti-HSV-1 immunity in vitro. However, the paucity of biological samples and their lack of purity have hindered progress in the field, which furthermore suffers from the absence of testable primary human oligodendrocytes. Recently, the authors have established a human induced pluripotent stem cells (hiPSCs)-based model of anti-HSV-1 immunity in neurons, oligodendrocyte precursor cells, astrocytes, and neural stem cells, which has widened the scope of possible in vitro studies while permitting in-depth explorations. This mini-review summarizes the available data on human primary and iPSC-derived CNS cells for anti-HSV-1 immunity. The hiPSC-mediated study of anti-viral immunity in both healthy individuals and patients with viral encephalitis will be a powerful tool in dissecting the disease pathogenesis of CNS infections with HSV-1 and other neurotropic viruses.

Infectious disease. Life-threatening influenza and impaired interferon amplification in human IRF7 deficiency.

Severe influenza disease strikes otherwise healthy children and remains unexplained. We report compound heterozygous null mutations in IRF7, which encodes the transcription factor interferon regulatory factor 7, in an otherwise healthy child who suffered life-threatening influenza during primary infection. In response to influenza virus, the patient's leukocytes and plasmacytoid dendritic cells produced very little type I and III interferons (IFNs). Moreover, the patient's dermal fibroblasts and induced pluripotent stem cell (iPSC)-derived pulmonary epithelial cells produced reduced amounts of type I IFN and displayed increased influenza virus replication. These findings suggest that IRF7-dependent amplification of type I and III IFNs is required for protection against primary infection by influenza virus in humans. They also show that severe influenza may result from single-gene inborn errors of immunity.

Feeder-free derivation of neural crest progenitor cells from human pluripotent stem cells.

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

TLR3 deficiency in herpes simplex encephalitis: high allelic heterogeneity and recurrence risk.

OBJECTIVE: To determine the proportion of children with herpes simplex encephalitis (HSE) displaying TLR3 deficiency, the extent of TLR3 allelic heterogeneity, and the specific clinical features of TLR3 deficiency. METHODS: We determined the sequence of all exons of TLR3 in 110 of the 120 patients with HSE enrolled in our study who do not carry any of the previously described HSE-predisposing mutations of TLR3 pathway genes (TLR3, UNC93B1, TRIF, TRAF3, and TBK1). All the new mutant TLR3 alleles detected were characterized experimentally in-depth to establish the causal relationship between the genotype and phenotype. RESULTS: In addition to the 3 previously reported TLR3-deficient patients from the same cohort, 6 other children or young adults with HSE carry 1 of 5 unique or extremely rare (minor allele frequency <0.001) missense TLR3 alleles. Two alleles (M374T, D592N) heterozygous in 3 patients are not deleterious in vitro. The other 3 are deleterious via different mechanisms: G743D+R811I and L360P heterozygous in 2 patients are loss-of-function due to low levels of expression and lack of cleavage, respectively, and R867Q homozygous in 1 patient is hypomorphic. The 3 patients' fibroblasts display impaired TLR3 responses and enhanced herpes simplex virus 1 susceptibility. Overall, TLR3 deficiency is therefore found in 6 (5%) of the 120 patients studied. There is high allelic heterogeneity, with 3 forms of autosomal dominant partial defect by negative dominance or haploinsufficiency, and 2 forms of autosomal recessive defect with complete or partial deficiency. Finally, 4 (66%) of the 6 TLR3-deficient patients had at least 1 late relapse of HSE, whereas relapse occurred in only 12 (10%) of the total cohort of 120 patients. CONCLUSIONS: Childhood-onset HSE is due to TLR3 deficiency in a traceable fraction of patients, in particular the ones with HSE recurrence. Mutations in TLR3 and TLR3 pathway genes should be searched and experimentally studied in children with HSE, and patients with proven TLR3 deficiency should be followed carefully.