Gillian I. Rice

The X-linked α-thalassaemia intellectual disability syndrome (ATRX) protein is a chromatin remodeller involved in transcriptional regulation and genome stability. While the importance of ATRX in development and malignancy is well recognised, its role in innate immunity is less well defined. Here we describe three unrelated patients with ATR-X syndrome carrying missense mutations in the ATPase domain of ATRX, each manifesting features of severe inflammation accompanied by persistent upregulation of interferon-stimulated gene expression in whole blood. Studies in patient-derived cells, CRISPR-engineered fibroblasts and neuronal models demonstrate that ATRX loss-of-function mutations drive enhanced type I interferon signalling through a cGAS-dependent mechanism uncoupled from the DNA sensing activity of cGAS. Mechanistically, ATRX deficiency disrupts the chromatin distribution of DAXX and H3.3, with cGAS essential for the changes in nucleosome composition and gene expression mediated by loss of ATRX. Thus, our study highlights a link between ATRX dysfunction and inflammation involving a non-canonical role of cGAS.

Objective: To characterize the demographic, clinical, serological, genetic background, course and value of the interferon (IFN) score in a cohort of patients with rheumatoid factor (RF)-positive polyarticular juvenile idiopathic arthritis (pJIA). Methods: Monocentric retrospective study of patients with RF-positive pJIA. Demographic, clinical and laboratory data were collected. The IFN score was calculated based on the expression levels of 24 IFN stimulated genes. Whole exome sequencing was performed in 22 patients. Results: Thirty-two patients were included. The IFN score was positive in 18 patients (56.2%) and negative in 14 patients (43.7%). An increased IFN score was associated with a significantly higher family history of autoimmunity (P = 0.0004), parental first-degree consanguinity (P = 0.05) and prevalence of antibodies to anti-cyclic citrullinated peptide (ACPA) (P = 0.01). Three patients with interstitial lung disease exhibited a positive IFN score. Conversely, demographic features and characteristics of polyarthritis did not differ between the two groups. Clinically inactive disease was achieved in 21/32 (66%) patients, with no significant difference according to the IFN score. Remission was never achieved with methotrexate alone. We identified one patient with a pathogenic de novo mutation in COPA. Additionally, ultra-rare variants in genes related to IFN and/or innate immunity were found in 21/22 (95.5%) patients. Conclusion: The IFN signature identified two distinct subgroups in RF-positive pJIA according to family history and immunological features. Our study suggests that patients with an elevated IFN score should be screened for pulmonary involvement. Future prospective studies are required to validate the use of the IFN score as a biomarker in RF-positive pJIA.

TBK1 kinase is a central regulator of type I IFN production. Upon activation of the IFN-β induction pathway, TBK1-adaptor proteins (NAP1, SINTBAD, TANK) form liquid condensates. We show that NAP1 condensates concentrate TBK1. Using NAP1 ^KO cell lines, we demonstrate that NAP1 exerts a dual effect on TBK1 activity. Initially, NAP1 binds TBK1 and increases its activity, promoting IFN pathway activation. Subsequently, TBK1-mediated phosphorylation of NAP1 induces the formation of condensates. These NAP1 condensates concentrate both TBK1 and the phosphatase PP2A, which dephosphorylates and consequently deactivates TBK1, thus limiting IFN induction. Additionally, in patients with lupus or interferonopathies, we identify NAP1 variants unable to form condensates upon danger signal exposure, which sustain TBK1 activation without limiting its activity. This study reveals a mode of regulating a signaling pathway through condensate formation and provides a potential molecular explanation for immune dysregulation associated with NAP1 variants in certain patients with interferonopathies.

Mutations that enhance type I interferon (IFN-I) activity cause monogenic autoinflammatory disorders termed type I interferonopathies. Along with the typical neurologic and rheumatologic manifestations, severe pulmonary disease is increasingly recognized yet poorly understood. We studied three siblings presenting with early-onset, life-threatening pulmonary alveolar proteinosis (PAP) and autoinflammatory stigmata. Genetic analysis uncovered a novel homozygous variant (R223Q) in STAT2, a key mediator of IFN-I signaling, which also facilitates feedback inhibition via USP18. R223Q STAT2 preserved signal transduction and viral control in vitro. However, cells homozygous for the R223Q variant failed to terminate IFN-I responses, owing to impaired localization of USP18. Unlike in classical forms of PAP, GM-CSF signaling remained intact. Instead, persistent IFN-I signaling antagonized monocyte migration toward chemokines essential for lung trafficking. Informed by these findings, the youngest sibling received JAK inhibitor and anti-IFN-I receptor therapy with marked clinical improvement. Collectively, type I interferonopathy by mutation of STAT2 (TIMS2) compromises monocyte chemotaxis and underlies a novel mechanism of PAP.

COPA syndrome is a rare monogenic autoinflammatory disease due to heterozygous mutations in COPA, encoding the coatomer subunit α. COPA syndrome demonstrates phenotypic overlap with STING-associated vasculopathy with onset in infancy (SAVI), the latter due to gain-of-function mutations in STING1. Indeed, STING activation is a key driver of the pathogenesis of COPA syndrome, and a recent report suggested that the presence of the common HAQ STING allele confers complete protection against the development of clinical disease in the context of pathogenic heterozygous mutations in COPA. Given the potential significance of this result for patient management, we investigated the STING HAQ haplotype status of a separate cohort of individuals segregating pathogenic mutations in COPA. In doing so, we ascertained five HAQ-negative, clinically asymptomatic individuals aged 30, 39, 39, 42, and 43 years at last evaluation, and an HAQ-positive male with kidney disease that we consider most likely attributable to the recurrent R233H mutation in COPA. Our findings challenge the suggestion that STING haplotype status is the sole determinant of clinical penetrance in COPA syndrome.

Small nuclear RNAs (snRNAs) combine with specific proteins to generate small nuclear ribonucleoproteins (snRNPs), the building blocks of the spliceosome. U4 snRNA forms a duplex with U6 and, together with U5, contributes to the tri-snRNP spliceosomal complex. Variants in RNU4-2, which encodes U4, have recently been implicated in neurodevelopmental disorders. Here we show that heterozygous inherited and de novo variants in RNU4-2 and in four RNU6 paralogs (RNU6-1, RNU6-2, RNU6-8 and RNU6-9), which encode U6, recur in individuals with nonsyndromic retinitis pigmentosa (RP), a genetic disorder causing progressive blindness. These variants cluster within the three-way junction of the U4/U6 duplex, a site that interacts with tri-snRNP splicing factors also known to cause RP (PRPF3, PRPF8, PRPF31), and seem to affect snRNP biogenesis. Based on our cohort, deleterious variants in RNU4-2 and RNU6 paralogs may explain up to ~1.4% of otherwise undiagnosed RP cases. This study highlights the contribution of noncoding RNA genes to Mendelian disease and reveals pleiotropy in RNU4-2, where distinct variants underlie neurodevelopmental disorder and retinal degeneration.

R-loops are DNA–RNA hybrid structures that may promote mutagenesis. However, their contribution to human Mendelian disorders is unexplored. Here we show excess de novo variants in genomic regions that form R-loops (henceforth, ‘R-loop regions’) and demonstrate enrichment of R-loop region variants (RRVs) in ribozyme, snoRNA and snRNA genes, specifically in rare disease cohorts. Using this insight, we report neurodevelopmental disorders (NDDs) caused by rare variants in two major spliceosomal RNA encoding genes, RNU2-2 and RNU5B-1. These, along with the recently described RNU4-2-related ReNU syndrome, provide a genetic explanation for a substantial proportion of individuals with NDDs.

Patients heterozygous for germline CBL loss-of-function (LOF) variants can develop myeloid malignancy, autoinflammation, or both, if some or all of their leukocytes become homozygous for these variants through somatic loss of heterozygosity (LOH) via uniparental isodisomy. We observed an upregulation of the inflammatory gene expression signature in whole blood from these patients, mimicking monogenic inborn errors underlying autoinflammation. Remarkably, these patients had constitutively activated monocytes that secreted 10 to 100 times more inflammatory cytokines than those of healthy individuals and CBL LOF heterozygotes without LOH. CBL-LOH hematopoietic stem and progenitor cells (HSPCs) outgrew the other cells, accounting for the persistence of peripheral monocytes homozygous for the CBL LOF variant. ERK pathway activation was required for the excessive production of cytokines by both resting and stimulated CBL-LOF monocytes, as shown in monocytic cell lines. Finally, we found that about 1 in 10,000 individuals in the UK Biobank were heterozygous for CBL LOF variants and that these carriers were at high risk of hematological and inflammatory conditions.

UNC93B1 is a transmembrane domain protein mediating the signaling of endosomal Toll-like receptors (TLRs). We report five families harboring rare missense substitutions (I317M, G325C, L330R, R466S, and R525P) in UNC93B1 causing systemic lupus erythematosus (SLE) or chilblain lupus (CBL) as either autosomal dominant or autosomal recessive traits. As for a D34A mutation causing murine lupus, we recorded a gain of TLR7 and, to a lesser extent, TLR8 activity with the I317M (in vitro) and G325C (in vitro and ex vivo) variants in the context of SLE. Contrastingly, in three families segregating CBL, the L330R, R466S, and R525P variants were isomorphic with respect to TLR7 activity in vitro and, for R525P, ex vivo. Rather, these variants demonstrated a gain of TLR8 activity. We observed enhanced interaction of the G325C, L330R, and R466S variants with TLR8, but not the R525P substitution, indicating different disease mechanisms. Overall, these observations suggest that UNC93B1 mutations cause monogenic SLE or CBL due to differentially enhanced TLR7 and TLR8 signaling.