Molecular characterization of an intronic RNASEH2B variant in a patient with Aicardi-Goutières syndrome.

Aicardi-Goutières syndrome (AGS) is a progressive multisystem disorder including encephalopathy with significant impacts on intellectual and physical abilities. An early diagnosis is becoming ever more crucial, as targeted therapies are emerging. A deep understanding of the molecular heterogeneity of AGS can help guide the early diagnosis and clinical management of patients, and inform recurrence risks. Here, we detail the diagnostic odyssey of a patient with an early presentation of AGS. Exome and genome sequencing detected an intronic RNASEH2B variant missed in a conventional leukodystrophy NGS gene panel. RNA studies demonstrated that a c.322-17 A > G variant affected splicing and caused 16-nucleotide intronic retention in the RNASEH2B transcript, introducing an out-of-frame early termination codon. RNASEH2B expression in the patient's blood was reduced when compared to controls. Our study highlights the pathogenicity of this intronic variant and the importance of its inclusion in variant assessment.

Mutations in RNU7-1 Weaken Secondary RNA Structure, Induce MCP-1 and CXCL10 in CSF, and Result in Aicardi-Goutières Syndrome with Severe End-Organ Involvement.

BACKGROUND: Aicardi-Goutières syndrome (AGS) is a type I interferonopathy usually characterized by early-onset neurologic regression. Biallelic mutations in LSM11 and RNU7-1, components of the U7 small nuclear ribonucleoprotein (snRNP) complex, have been identified in a limited number of genetically unexplained AGS cases. Impairment of U7 snRNP function results in misprocessing of replication-dependent histone (RDH) pre-mRNA and disturbance of histone occupancy of nuclear DNA, ultimately driving cGAS-dependent type I interferon (IFN-I) release. OBJECTIVE: We performed a clinical, genetic, and immunological workup of 3 unrelated patients with uncharacterized AGS. METHODS: Whole exome sequencing (WES) and targeted Sanger sequencing of RNU7-1 were performed. Primary fibroblasts were used for mechanistic studies. IFN-I signature and STAT1/2 phosphorylation were assessed in peripheral blood. Cytokines were profiled on serum and cerebrospinal fluid (CSF). Histopathology was examined on brain and kidney tissue. RESULTS: Sequencing revealed compound heterozygous RNU7-1 mutations, resulting in impaired RDH pre-mRNA processing. The 3' stem-loop mutations reduced stability of the secondary U7 snRNA structure. A discrete IFN-I signature in peripheral blood was paralleled by MCP-1 (CCL2) and CXCL10 upregulation in CSF. Histopathological analysis of the kidney showed thrombotic microangiopathy. We observed dysregulated STAT phosphorylation upon cytokine stimulation. Clinical overview of all reported patients with RNU7-1-related disease revealed high mortality and high incidence of organ involvement compared to other AGS genotypes. CONCLUSIONS: Targeted RNU7-1 sequencing is recommended in genetically unexplained AGS cases. CSF cytokine profiling represents an additional diagnostic tool to identify aberrant IFN-I signaling. Clinical follow-up of RNU7-1-mutated patients should include screening for severe end-organ involvement including liver disease and nephropathy.

A sensitive and affordable multiplex RT-qPCR assay for SARS-CoV-2 detection.

With the ongoing COVID-19 (Coronavirus Disease 2019) pandemic, caused by the novel coronavirus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2), there is a need for sensitive, specific, and affordable diagnostic tests to identify infected individuals, not all of whom are symptomatic. The most sensitive test involves the detection of viral RNA using RT-qPCR (quantitative reverse transcription PCR), with many commercial kits now available for this purpose. However, these are expensive, and supply of such kits in sufficient numbers cannot always be guaranteed. We therefore developed a multiplex assay using well-established SARS-CoV-2 targets alongside a human cellular control (RPP30) and a viral spike-in control (Phocine Herpes Virus 1 [PhHV-1]), which monitor sample quality and nucleic acid extraction efficiency, respectively. Here, we establish that this test performs as well as widely used commercial assays, but at substantially reduced cost. Furthermore, we demonstrate >1,000-fold variability in material routinely collected by combined nose and throat swabbing and establish a statistically significant correlation between the detected level of human and SARS-CoV-2 nucleic acids. The inclusion of the human control probe in our assay therefore provides a quantitative measure of sample quality that could help reduce false-negative rates. We demonstrate the feasibility of establishing a robust RT-qPCR assay at approximately 10% of the cost of equivalent commercial assays, which could benefit low-resource environments and make high-volume testing affordable.

cGAS-mediated induction of type I interferon due to inborn errors of histone pre-mRNA processing.

Inappropriate stimulation or defective negative regulation of the type I interferon response can lead to autoinflammation. In genetically uncharacterized cases of the type I interferonopathy Aicardi-Goutières syndrome, we identified biallelic mutations in LSM11 and RNU7-1, which encode components of the replication-dependent histone pre-mRNA-processing complex. Mutations were associated with the misprocessing of canonical histone transcripts and a disturbance of linker histone stoichiometry. Additionally, we observed an altered distribution of nuclear cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS) and enhanced interferon signaling mediated by the cGAS-stimulator of interferon genes (STING) pathway in patient-derived fibroblasts. Finally, we established that chromatin without linker histone stimulates cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) production in vitro more efficiently. We conclude that nuclear histones, as key constituents of chromatin, are essential in suppressing the immunogenicity of self-DNA.

PRIM1 deficiency causes a distinctive primordial dwarfism syndrome.

DNA replication is fundamental for cell proliferation in all organisms. Nonetheless, components of the replisome have been implicated in human disease, and here we report PRIM1 encoding the catalytic subunit of DNA primase as a novel disease gene. Using a variant classification agnostic approach, biallelic mutations in PRIM1 were identified in five individuals. PRIM1 protein levels were markedly reduced in patient cells, accompanied by replication fork asymmetry, increased interorigin distances, replication stress, and prolonged S-phase duration. Consequently, cell proliferation was markedly impaired, explaining the patients' extreme growth failure. Notably, phenotypic features distinct from those previously reported with DNA polymerase genes were evident, highlighting differing developmental requirements for this core replisome component that warrant future investigation.

Mutations in COPA lead to abnormal trafficking of STING to the Golgi and interferon signaling.

Heterozygous missense mutations in coatomer protein subunit α, COPA, cause a syndrome overlapping clinically with type I IFN-mediated disease due to gain-of-function in STING, a key adaptor of IFN signaling. Recently, increased levels of IFN-stimulated genes (ISGs) were described in COPA syndrome. However, the link between COPA mutations and IFN signaling is unknown. We observed elevated levels of ISGs and IFN-α in blood of symptomatic COPA patients. In vitro, both overexpression of mutant COPA and silencing of COPA induced STING-dependent IFN signaling. We detected an interaction between COPA and STING, and mutant COPA was associated with an accumulation of ER-resident STING at the Golgi. Given the known role of the coatomer protein complex I, we speculate that loss of COPA function leads to enhanced type I IFN signaling due to a failure of Golgi-to-ER STING retrieval. These data highlight the importance of the ER-Golgi axis in the control of autoinflammation and inform therapeutic strategies in COPA syndrome.

Analysis of U8 snoRNA Variants in Zebrafish Reveals How Bi-allelic Variants Cause Leukoencephalopathy with Calcifications and Cysts.

How mutations in the non-coding U8 snoRNA cause the neurological disorder leukoencephalopathy with calcifications and cysts (LCC) is poorly understood. Here, we report the generation of a mutant U8 animal model for interrogating LCC-associated pathology. Mutant U8 zebrafish exhibit defective central nervous system development, a disturbance of ribosomal RNA (rRNA) biogenesis and tp53 activation, which monitors ribosome biogenesis. Further, we demonstrate that fibroblasts from individuals with LCC are defective in rRNA processing. Human precursor-U8 (pre-U8) containing a 3' extension rescued mutant U8 zebrafish, and this result indicates conserved biological function. Analysis of LCC-associated U8 mutations in zebrafish revealed that one null and one functional allele contribute to LCC. We show that mutations in three nucleotides at the 5' end of pre-U8 alter the processing of the 3' extension, and we identify a previously unknown base-pairing interaction between the 5' end and the 3' extension of human pre-U8. Indeed, LCC-associated mutations in any one of seven nucleotides in the 5' end and 3' extension alter the processing of pre-U8, and these mutations are present on a single allele in almost all individuals with LCC identified to date. Given genetic data indicating that bi-allelic null U8 alleles are likely incompatible with human development, and that LCC is not caused by haploinsufficiency, the identification of hypomorphic misprocessing mutations that mediate viable embryogenesis furthers our understanding of LCC molecular pathology and cerebral vascular homeostasis.

Anti-MDA5 juvenile idiopathic inflammatory myopathy: a specific subgroup defined by differentially enhanced interferon-α signalling.

OBJECTIVES: JDM and juvenile overlap myositis represent heterogeneous subtypes of juvenile idiopathic inflammatory myopathy (JIIM). Chronic evolution can occur in up to 60% of cases, and morbidity/mortality is substantial. We aimed to describe the clinical, biological, histological and type I IFN status in JIIM associated with anti-melanoma differentiation-associated protein 5 (anti-MDA5) autoantibodies at presentation (group 1) in comparison with other JIIM (group 2). METHODS: This was a retrospective and prospective study of patients with JIIM ascertained from three French paediatric rheumatology reference centres between 2013 and 2019. Muscle biopsies were reviewed. Type I interferon pathway activity was assessed by dosage of IFNα serum protein and the expression of IFN-stimulated genes. RESULTS: Sixty-four patients were included, 13 in group 1 (54% JDM and 46% juvenile overlap myositis) and 51 in group 2 (76% JDM and 24% juvenile overlap myositis). Group 1 patients demonstrated more arthritis, skin ulcerations, lupus features and interstitial lung disease, and a milder muscular involvement. Serum IFNα levels were higher in group 1 than 2, and decreased after treatment or improvement in both groups. Outcome was similar in both groups. Unconventional treatment (more than two lines) was required in order to achieve remission, especially when skin ulceration was reported. CONCLUSION: This study indicates a higher frequency of arthritis, skin ulcerations and interstitial lung disease, but milder muscular involvement, in JIIM with positive anti-MDA5 autoantibodies compared with other JIIM. Our data support an important role of systemic IFNα in disease pathology, particularly in the anti-MDA5 auto-antibody-positive subgroup. In severe and refractory forms of JIIM, IFNα may represent a therapeutic target.

A Quantitative Chloride Channel Conductance Assay for Efficacy Testing of AAV.BEST1.

Mutations in the human BEST1 gene are responsible for a number of distinct retinal disorders known as bestrophinopathies, for which there are no current treatments. The protein product, bestrophin-1, is expressed in the retinal pigment epithelium (RPE) where it localizes to the basolateral membrane and acts as a Ca2+-activated chloride channel. Recent studies have shown successful BEST1-mediated gene transfer to the RPE, indicating human clinical trials of BEST1 gene therapy may be on the horizon. A critical aspect of such trials is the ability to assess the efficacy of vector prior to patient administration. Here, an assay is presented that enables the quantitative assessment of AAV-mediated BEST1 chloride conductance as a measure of vector efficacy. Expression of BEST1 following transduction of HEK293 cells with AAV.BEST1 vectors was confirmed by liquid chromatography, Western blot, and immunocytochemistry. Whole-cell patch-clamp showed increased chloride conductance in BEST1-transduced cells compared to sham-transduced and untransduced controls. Exogenous chloride current correlated to BEST1 expression level, with an enhanced AAV.BEST1.WPRE vector providing higher expression levels of BEST1 and increases in chloride conductance. This study presents in vitro electrophysical quantification of bestrophin-1 following AAV-mediated gene transfer, providing vital functional data on an AAV gene therapy product that will support a future application for regulatory approval.

Self-Awareness: Nucleic Acid-Driven Inflammation and the Type I Interferonopathies.

Recognition of foreign nucleic acids is the primary mechanism by which a type I interferon-mediated antiviral response is triggered. Given that human cells are replete with DNA and RNA, this evolutionary strategy poses an inherent biological challenge, i.e., the fundamental requirement to reliably differentiate self-nucleic acids from nonself nucleic acids. We suggest that the group of Mendelian inborn errors of immunity referred to as the type I interferonopathies relate to a breakdown of self/nonself discrimination, with the associated mutant genotypes involving molecules playing direct or indirect roles in nucleic acid signaling. This perspective begs the question as to the sources of self-derived nucleic acids that drive an inappropriate immune response. Resolving this question will provide fundamental insights into immune tolerance, antiviral signaling, and complex autoinflammatory disease states. Here we develop these ideas, discussing type I interferonopathies within the broader framework of nucleic acid-driven inflammation.

Autosomal-dominant early-onset spastic paraparesis with brain calcification due to IFIH1 gain-of-function.

We describe progressive spastic paraparesis in two male siblings and the daughter of one of these individuals. Onset of disease occurred within the first decade, with stiffness and gait difficulties. Brisk deep tendon reflexes and extensor plantar responses were present, in the absence of intellectual disability or dermatological manifestations. Cerebral imaging identified intracranial calcification in all symptomatic family members. A marked upregulation of interferon-stimulated gene transcripts was recorded in all three affected individuals and in two clinically unaffected relatives. A heterozygous IFIH1 c.2544T>G missense variant (p.Asp848Glu) segregated with interferon status. Although not highly conserved (CADD score 10.08 vs. MSC-CADD score of 19.33) and predicted as benign by in silico algorithms, this variant is not present on publically available databases of control alleles, and expression of the D848E construct in HEK293T cells indicated that it confers a gain-of-function. This report illustrates, for the first time, the occurrence of autosomal-dominant spastic paraplegia with intracranial calcifications due to an IFIH1-related type 1 interferonopathy.

Sort Your Self Out!

Discrimination between viral and self-derived nucleic acid species is crucial in maintaining effective antiviral immunity whilst avoiding autoinflammation. Ahmad et al. and Chung et al. delineate the consequences of MDA5 gain of function and loss of ADAR1 activity, highlighting the blurring of the concept of self and non-self when considering endogenous retroelements.

Type I interferon-mediated autoinflammation due to DNase II deficiency.

Microbial nucleic acid recognition serves as the major stimulus to an antiviral response, implying a requirement to limit the misrepresentation of self nucleic acids as non-self and the induction of autoinflammation. By systematic screening using a panel of interferon-stimulated genes we identify two siblings and a singleton variably demonstrating severe neonatal anemia, membranoproliferative glomerulonephritis, liver fibrosis, deforming arthropathy and increased anti-DNA antibodies. In both families we identify biallelic mutations in DNASE2, associated with a loss of DNase II endonuclease activity. We record increased interferon alpha protein levels using digital ELISA, enhanced interferon signaling by RNA-Seq analysis and constitutive upregulation of phosphorylated STAT1 and STAT3 in patient lymphocytes and monocytes. A hematological disease transcriptomic signature and increased numbers of erythroblasts are recorded in patient peripheral blood, suggesting that interferon might have a particular effect on hematopoiesis. These data define a type I interferonopathy due to DNase II deficiency in humans.

Brief Report: Blockade of TANK-Binding Kinase 1/IKKɛ Inhibits Mutant Stimulator of Interferon Genes (STING)-Mediated Inflammatory Responses in Human Peripheral Blood Mononuclear Cells.

OBJECTIVE: Gain-of-function mutations in TMEM173, encoding the stimulator of interferon (IFN) genes (STING) protein, underlie a novel type I interferonopathy that is minimally responsive to conventional immunosuppressive therapies and associated with high frequency of childhood morbidity and mortality. STING gain-of-function causes constitutive oversecretion of IFN. This study was undertaken to determine the effects of a TANK-binding kinase 1 (TBK-1)/IKKɛ inhibitor (BX795) on secretion and signaling of IFN in primary peripheral blood mononuclear cells (PBMCs) from patients with mutations in STING. METHODS: PBMCs from 4 patients with STING-associated disease were treated with BX795. The effect of BX795 on IFN pathways was assessed by Western blotting and an IFNß reporter assay, as well as by quantification of IFNα in cell lysates, staining for STAT-1 phosphorylation, and measurement of IFN-stimulated gene (ISG) messenger RNA (mRNA) expression. RESULTS: Treatment of PBMCs with BX795 inhibited the phosphorylation of IFN regulatory factor 3 and IFNß promoter activity induced in HEK 293T cells by cyclic GMP-AMP or by genetic activation of STING. In vitro exposure to BX795 inhibited IFNα production in PBMCs of patients with STING-associated disease without affecting cell survival. In addition, BX795 decreased STAT-1 phosphorylation and ISG mRNA expression independent of IFNα blockade. CONCLUSION: These findings demonstrate the effect of BX795 on reducing type I IFN production and IFN signaling in cells from patients with gain-of-function mutations in STING. A combined inhibition of TBK-1 and IKKɛ therefore holds potential for the treatment of patients carrying STING mutations, and may also be relevant in other type I interferonopathies.

Disease-associated mutations identify a novel region in human STING necessary for the control of type I interferon signaling.

BACKGROUND: Gain-of-function mutations in transmembrane protein 173 (TMEM173) encoding stimulator of interferon genes (STING) underlie a recently described type I interferonopathy called STING-associated vasculopathy with onset in infancy (SAVI). OBJECTIVES: We sought to define the molecular and cellular pathology relating to 3 individuals variably exhibiting the core features of the SAVI phenotype including systemic inflammation, destructive skin lesions, and interstitial lung disease. METHODS: Genetic analysis, conformational studies, in vitro assays and ex vivo flow-cytometry were performed. RESULTS: Molecular and in vitro data demonstrate that the pathology in these patients is due to amino acid substitutions at positions 206, 281, and 284 of the human STING protein. These mutations confer cGAMP-independent constitutive activation of type I interferon signaling through TBK1 (TANK-binding kinase), independent from the alternative STING pathway triggered by membrane fusion of enveloped RNA viruses. This constitutive activation was abrogated by ex vivo treatment with the janus kinase 1/2 inhibitor ruxolitinib. CONCLUSIONS: Structural analysis indicates that the 3 disease-associated mutations at positions 206, 281, and 284 of the STING protein define a novel cluster of amino acids with functional importance in the regulation of type I interferon signaling.

Restoration of mutant bestrophin-1 expression, localisation and function in a polarised epithelial cell model.

Autosomal recessive bestrophinopathy (ARB) is a retinopathy caused by mutations in the bestrophin-1 protein, which is thought to function as a Ca2+-gated Cl- channel in the basolateral surface of the retinal pigment epithelium (RPE). Using a stably transfected polarised epithelial cell model, we show that four ARB mutant bestrophin-1 proteins were mislocalised and subjected to proteasomal degradation. In contrast to the wild-type bestrophin-1, each of the four mutant proteins also failed to conduct Cl- ions in transiently transfected cells as determined by whole-cell patch clamp. We demonstrate that a combination of two clinically approved drugs, bortezomib and 4-phenylbutyrate (4PBA), successfully restored the expression and localisation of all four ARB mutant bestrophin-1 proteins. Importantly, the Cl- conductance function of each of the mutant bestrophin-1 proteins was fully restored to that of wild-type bestrophin-1 by treatment of cells with 4PBA alone. The functional rescue achieved with 4PBA is significant because it suggests that this drug, which is already approved for long-term use in infants and adults, might represent a promising therapy for the treatment of ARB and other bestrophinopathies resulting from missense mutations in BEST1.