Michael J. Ciancanelli

Herpes simplex encephalitis in children with autosomal recessive and dominant TRIF deficiency

Herpes simplex encephalitis (HSE) is the most common sporadic viral encephalitis of childhood. Autosomal recessive (AR) UNC-93B and TLR3 deficiencies and autosomal dominant (AD) TLR3 and TRAF3 deficiencies underlie HSE in some children. We report here unrelated HSE children with AR or AD TRIF deficiency. The AR form of the disease was found to be due to a homozygous nonsense mutation that resulted in a complete absence of the TRIF protein. Both the TLR3- and the TRIF-dependent TLR4 signaling pathways were abolished. The AD form of disease was found to be due to a heterozygous missense mutation, resulting in a dysfunctional protein. In this form of the disease, the TLR3 signaling pathway was impaired, whereas the TRIF-dependent TLR4 pathway was unaffected. Both patients, however, showed reduced capacity to respond to stimulation of the DExD/H-box helicases pathway. To date, the TRIF-deficient patients with HSE described herein have suffered from no other infections. Moreover, as observed in patients with other genetic etiologies of HSE, clinical penetrance was found to be incomplete, as some HSV-1-infected TRIF-deficient relatives have not developed HSE. Our results provide what we believe to be the first description of human TRIF deficiency and a new genetic etiology for HSE. They suggest that the TRIF-dependent TLR4 and DExD/H-box helicase pathways are largely redundant in host defense. They further demonstrate the importance of TRIF for the TLR3-dependent production of antiviral IFNs in the CNS during primary infection with HSV-1 in childhood.

Herpes simplex virus encephalitis in a patient with complete TLR3 deficiency: TLR3 is otherwise redundant in protective immunity

A new autosomal recessive form of complete TLR3 deficiency reveals that human TLR3 is nonredundant in immunity against herpes simplex virus 1 in the central nervous system (CNS) but redundant in host defense against viruses outside the CNS.

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-β (IFN-β) 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-β 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-β ( IFN-α/β) but not IFN-λ1. Thus, impaired TLR3- and UNC-93B-dependent IFN-α/β 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.

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

A genetic cause for severe influenza Although chicken soup and plenty of rest get most kids through an influenza virus infection, some require hospitalization. Ciancanelli et al. report on one child who suffered severely from influenza because of null mutations in the gene for transcription factor IRF7. Cells isolated from this patient could not make enough secreted antiviral proteins, called interferons, to halt viral replication. The requirement for IRF7 seems quite specific, because this patient recovers normally from other common childhood viral infections. Science, this issue p. 448 A mutation that reduces antiviral interferons underlies certain cases of severe influenza in children. 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.

Mutation of YMYL in the Nipah Virus Matrix Protein Abrogates Budding and Alters Subcellular Localization

ABSTRACT Matrix (M) proteins reportedly direct the budding of paramyxoviruses from infected cells. In order to begin to characterize the assembly process for the highly lethal, emerging paramyxovirus Nipah virus (NiV), we have examined the budding of NiV M. We demonstrated that expression of the NiV M protein is sufficient to produce budding virus-like particles (VLPs) that are physically and morphologically similar to NiV. We identified in NiV M a sequence, YMYL, with similarity to the YPDL late domain found in the equine infectious anemia virus Gag protein. When the YMYL within NiV M was mutated, VLP release was abolished and M was relocalized to the nucleus, but the mutant M proteins retained oligomerization activity. When YMYL was fused to a late-domain mutant of the Ebola virus VP40 matrix protein, VP40 budding was restored. These results suggest that the YMYL sequence may act as a trafficking signal and a late domain for NiV M.

Heterozygous TBK1 mutations impair TLR3 immunity and underlie herpes simplex encephalitis of childhood

Two unrelated children with HSE carry distinct heterozygous mutations in the gene encoding TANK-binding kinase 1.

Human TYK2 deficiency: Mycobacterial and viral infections without hyper-IgE syndrome

Kreins et al. report the identification and immunological characterization of a group of TYK2-deficient patients.

TLR3 immunity to infection in mice and humans.

TLR3 is a receptor for dsRNA, which is generated during most viral infections. However, other cellular processes may also produce dsRNA and there are other receptors for dsRNA. The role of TLR3 in protective immunity to viruses has been investigated in mice and humans with genetically impaired TLR3 responses. TLR3-deficient mice responded to experimental challenge with 16 different viruses in various ways. They were susceptible to eight viruses, normally resistant to three other viruses, and their survival rates were higher than those of wild-type mice following infection with four other viruses. Conflicting results were obtained for the other virus tested. These data are difficult to understand in terms of a simple pattern based on virus structure or tissue tropism. Surprisingly, the known human patients with inborn errors of the TLR3 pathway have remained healthy or developed encephalitis in the course of natural primary infection with HSV-1. These patients display no clear susceptibility to other infections, including viral infections, such as other forms of viral encephalitis and other HSV-1 diseases in particular. This restricted susceptibility to viruses seems to result from impaired TLR3-dependent IFN-α/β production by central nervous system (CNS)-resident non-hematopoietic cells infected with HSV-1. These studies neatly illustrate the value of combining genetic studies of experimental infections in mice and natural infections in humans, to elucidate the biological function of host molecules in protective immunity.

The mutation significance cutoff: gene-level thresholds for variant predictions

Next-generation sequencing (NGS) has made it possible to identify about 20,000 variants in the protein-coding exome of each individual, of which only a few are likely to underlie a genetic disease. Variant-level methods such as PolyPhen-2, SIFT and CADD are useful for obtaining a prediction as to whether a given variant is benign/damaging1–3 or tolerant/intolerant1–3 (we hereafter use the terms benign/deleterious). These methods are commonly interpreted in a binary manner for filtering out benign variants from NGS data, with a single significance cutoff value across all protein-coding genes. PolyPhen-2 and SIFT integrate the fixed cutoff in the software. CADD proposed (but did not recommend for categorical usage) the fixed value of 15 (or another value between 10 and 20). Gene-level methods, such as RVIS, de novo excess and GDI are also useful4–6. Combining fixed gene-level and variant-level cutoffs is also applied in the RVIS hot zone approach4. However, owing to the diversity of medical and population genetic features between human genes and across populations, a uniform cutoff is unlikely to be accurate genome-wide.

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

Significance The protein-coding exome of a patient with a monogenic disease contains about 20,000 variations, of which only one or two are disease causing. When attempting to select disease-causing candidate mutation(s), a challenge is to filter out as many false-positive (FP) variants as possible. In this study, we describe the gene damage index (GDI), a metric for the nonsynonymous mutational load in each protein-coding gene in the general population. We show that the GDI is an efficient gene-level method for filtering out FP variants in genes that are highly damaged in the general population. 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.