Characterization of human disease phenotypes associated with mutations in TREX1, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, ADAR, and IFIH1.

Aicardi-Goutières syndrome is an inflammatory disease occurring due to mutations in any of TREX1, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, ADAR or IFIH1. We report on 374 patients from 299 families with mutations in these seven genes. Most patients conformed to one of two fairly stereotyped clinical profiles; either exhibiting an in utero disease-onset (74 patients; 22.8% of all patients where data were available), or a post-natal presentation, usually within the first year of life (223 patients; 68.6%), characterized by a sub-acute encephalopathy and a loss of previously acquired skills. Other clinically distinct phenotypes were also observed; particularly, bilateral striatal necrosis (13 patients; 3.6%) and non-syndromic spastic paraparesis (12 patients; 3.4%). We recorded 69 deaths (19.3% of patients with follow-up data). Of 285 patients for whom data were available, 210 (73.7%) were profoundly disabled, with no useful motor, speech and intellectual function. Chilblains, glaucoma, hypothyroidism, cardiomyopathy, intracerebral vasculitis, peripheral neuropathy, bowel inflammation and systemic lupus erythematosus were seen frequently enough to be confirmed as real associations with the Aicardi-Goutieres syndrome phenotype. We observed a robust relationship between mutations in all seven genes with increased type I interferon activity in cerebrospinal fluid and serum, and the increased expression of interferon-stimulated gene transcripts in peripheral blood. We recorded a positive correlation between the level of cerebrospinal fluid interferon activity assayed within one year of disease presentation and the degree of subsequent disability. Interferon-stimulated gene transcripts remained high in most patients, indicating an ongoing disease process. On the basis of substantial morbidity and mortality, our data highlight the urgent need to define coherent treatment strategies for the phenotypes associated with mutations in the Aicardi-Goutières syndrome-related genes. Our findings also make it clear that a window of therapeutic opportunity exists relevant to the majority of affected patients and indicate that the assessment of type I interferon activity might serve as a useful biomarker in future clinical trials.

A type I interferon signature identifies bilateral striatal necrosis due to mutations in ADAR1.

BACKGROUND: We recently observed mutations in ADAR1 to cause a phenotype of bilateral striatal necrosis (BSN) in a child with the type I interferonopathy Aicardi-Goutières syndrome (AGS). We therefore decided to screen patients with apparently non-syndromic BSN for ADAR1 mutations, and for an upregulation of interferon-stimulated genes (ISGs). METHODS: We performed Sanger sequencing of ADAR1 in a series of patients with BSN presenting to us during our routine clinical practice. We then undertook detailed clinical and neuroradiological phenotyping in nine mutation-positive children. We also measured the expression of ISGs in peripheral blood from these patients, and in children with BSN who did not have ADAR1 mutations. RESULTS: Nine ADAR1 mutation-positive patients from seven families demonstrated an acute (five cases) or subacute (four cases) onset of refractory, four-limb dystonia starting between 8 months and 5 years of age. Eight patients were developmentally normal at initial presentation. In seven cases, the disease was inherited as an autosomal recessive trait, while two related patients were found to have a heterozygous (dominant) ADAR1 mutation. All seven mutation-positive patients assayed showed an upregulation of ISGs (median: 12.50, IQR: 6.43-36.36) compared to controls (median: 0.93, IQR: 0.57-1.30), a so-called interferon signature, present many years after disease onset. No interferon signature was present in four children with BSN negative for mutations in ADAR1 (median: 0.63, IQR: 0.47-1.10). CONCLUSIONS: ADAR1-related disease should be considered in the differential diagnosis of apparently non-syndromic BSN with severe dystonia of varying evolution. The finding of an interferon signature provides a useful screening test for the presence of ADAR1 mutations in this context, and may suggest novel treatment approaches.

Protein kinase cδ deficiency causes mendelian systemic lupus erythematosus with B cell-defective apoptosis and hyperproliferation.

OBJECTIVE: Systemic lupus erythematosus (SLE) is a prototype autoimmune disease that is assumed to occur via a complex interplay of environmental and genetic factors. Rare causes of monogenic SLE have been described, providing unique insights into fundamental mechanisms of immune tolerance. The aim of this study was to identify the cause of an autosomal-recessive form of SLE. METHODS: We studied 3 siblings with juvenile-onset SLE from 1 consanguineous kindred and used next-generation sequencing to identify mutations in the disease-associated gene. We performed extensive biochemical, immunologic, and functional assays to assess the impact of the identified mutations on B cell biology. RESULTS: We identified a homozygous missense mutation in PRKCD, encoding protein kinase δ (PKCδ), in all 3 affected siblings. Mutation of PRKCD resulted in reduced expression and activity of the encoded protein PKCδ (involved in the deletion of autoreactive B cells), leading to resistance to B cell receptor- and calcium-dependent apoptosis and increased B cell proliferation. Thus, as for mice deficient in PKCδ, which exhibit an SLE phenotype and B cell expansion, we observed an increased number of immature B cells in the affected family members and a developmental shift toward naive B cells with an immature phenotype. CONCLUSION: Our findings indicate that PKCδ is crucial in regulating B cell tolerance and preventing self-reactivity in humans, and that PKCδ deficiency represents a novel genetic defect of apoptosis leading to SLE.

Synonymous mutations in RNASEH2A create cryptic splice sites impairing RNase H2 enzyme function in Aicardi-Goutières syndrome.

Aicardi-Goutières syndrome is an inflammatory disorder resulting from mutations in TREX1, RNASEH2A/2B/2C, SAMHD1, or ADAR1. Here, we provide molecular, biochemical, and cellular evidence for the pathogenicity of two synonymous variants in RNASEH2A. Firstly, the c.69G>A (p.Val23Val) mutation causes the formation of a splice donor site within exon 1, resulting in an out of frame deletion at the end of exon 1, leading to reduced RNase H2 protein levels. The second mutation, c.75C>T (p.Arg25Arg), also introduces a splice donor site within exon 1, and the internal deletion of 18 amino acids. The truncated protein still forms a heterotrimeric RNase H2 complex, but lacks catalytic activity. However, as a likely result of leaky splicing, a small amount of full-length active protein is apparently produced in an individual homozygous for this mutation. Recognition of the disease causing status of these variants allows for diagnostic testing in relevant families.

Differential regulation of oestrogen receptor ß isoforms by 5' untranslated regions in cancer.

Oestrogen receptors (ERs) are critical regulators of the behaviour of many cancers. Despite this, the roles and regulation of one of the two known ERs - ERß- are poorly understood. This is partly because analyses have been confused by discrepancies between ERß expression at mRNA and proteins levels, and because ERß is expressed as several functionally distinct isoforms. We investigated human ERß 5' untranslated regions (UTRs) and their influences on ERß expression and function. We demonstrate that two alternative ERß 5'UTRs have potent and differential influences on expression acting at the level of translation. We show that their influences are modulated by cellular context and in carcinogenesis, and demonstrate the contributions of both upstream open reading frames and RNA secondary structure. These regulatory mechanisms offer explanations for the non-concordance of ERß mRNA and protein. Importantly, we also demonstrate that 5'UTRs allow the first reported mechanisms for differential regulation of the expression of the ERß isoforms 1, 2 and 5, and thereby have critical influences on ERß function.

Intracerebral large artery disease in Aicardi-Goutières syndrome implicates SAMHD1 in vascular homeostasis.

AIM: To describe a spectrum of intracerebral large artery disease in Aicardi-Goutières syndrome (AGS) associated with mutations in the AGS5 gene SAMHD1. METHOD: We used clinical and radiological description and molecular analysis. RESULTS: Five individuals (three males, two females) were identified as having biallelic mutations in SAMHD1 and a cerebral arteriopathy in association with peripheral vessel involvement resulting in chilblains and ischaemic ulceration. The cerebral vasculopathy was primarily occlusive in three patients (with terminal carotid occlusion and basal collaterals reminiscent of moyamoya syndrome) and aneurysmal in two. Three of the five patients experienced intracerebral haemorrhage, which was fatal in two individuals. Post-mortem examination of one patient suggested that the arteriopathy was inflammatory in origin. INTERPRETATION: Mutations in SAMHD1 are associated with a cerebral vasculopathy which is likely to have an inflammatory aetiology. A similar disease has not been observed in patients with mutations in AGS1 to AGS4, suggesting a particular role for SAMHD1 in vascular homeostasis. Our report raises important questions about the management of patients with mutations in SAMHD1.

Assessment of interferon-related biomarkers in Aicardi-Goutières syndrome associated with mutations in TREX1, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, and ADAR: a case-control study.

BACKGROUND: Aicardi-Goutières syndrome (AGS) is an inflammatory disorder caused by mutations in any of six genes (TREX1, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, and ADAR). The disease is severe and effective treatments are urgently needed. We investigated the status of interferon-related biomarkers in patients with AGS with a view to future use in diagnosis and clinical trials. METHODS: In this case-control study, samples were collected prospectively from patients with mutation-proven AGS. The expression of six interferon-stimulated genes (ISGs) was measured by quantitative PCR, and the median fold change, when compared with the median of healthy controls, was used to create an interferon score for each patient. Scores higher than the mean of controls plus two SD (>2·466) were designated as positive. Additionally, we collated historical data for interferon activity, measured with a viral cytopathic assay, in CSF and serum from mutation-positive patients with AGS. We also undertook neutralisation assays of interferon activity in serum, and looked for the presence of autoantibodies against a panel of interferon proteins. FINDINGS: 74 (90%) of 82 patients had a positive interferon score (median 12·90, IQR 6·14-20·41) compared with two (7%) of 29 controls (median 0·93, IQR 0·57-1·30). Of the eight patients with a negative interferon score, seven had mutations in RNASEH2B (seven [27%] of all 26 patients with mutations in this gene). Repeat sampling in 16 patients was consistent for the presence or absence of an interferon signature on 39 of 41 occasions. Interferon activity (tested in 147 patients) was negatively correlated with age (CSF, r=-0·604; serum, r=-0·289), and was higher in CSF than in serum in 104 of 136 paired samples. Neutralisation assays suggested that measurable antiviral activity was related to interferon α production. We did not record significantly increased concentrations of autoantibodies to interferon subtypes in patients with AGS, or an association between the presence of autoantibodies and interferon score or serum interferon activity. INTERPRETATION: AGS is consistently associated with an interferon signature, which is apparently sustained over time and can thus be used to differentiate patients with AGS from controls. If future studies show that interferon status is a reactive biomarker, the measurement of an interferon score might prove useful in the assessment of treatment efficacy in clinical trials. FUNDING: European Union's Seventh Framework Programme; European Research Council.

Mutations in CTC1, encoding conserved telomere maintenance component 1, cause Coats plus.

Coats plus is a highly pleiotropic disorder particularly affecting the eye, brain, bone and gastrointestinal tract. Here, we show that Coats plus results from mutations in CTC1, encoding conserved telomere maintenance component 1, a member of the mammalian homolog of the yeast heterotrimeric CST telomeric capping complex. Consistent with the observation of shortened telomeres in an Arabidopsis CTC1 mutant and the phenotypic overlap of Coats plus with the telomeric maintenance disorders comprising dyskeratosis congenita, we observed shortened telomeres in three individuals with Coats plus and an increase in spontaneous γH2AX-positive cells in cell lines derived from two affected individuals. CTC1 is also a subunit of the α-accessory factor (AAF) complex, stimulating the activity of DNA polymerase-α primase, the only enzyme known to initiate DNA replication in eukaryotic cells. Thus, CTC1 may have a function in DNA metabolism that is necessary for but not specific to telomeric integrity.

Mutations in ADAR1 cause Aicardi-Goutières syndrome associated with a type I interferon signature.

Adenosine deaminases acting on RNA (ADARs) catalyze the hydrolytic deamination of adenosine to inosine in double-stranded RNA (dsRNA) and thereby potentially alter the information content and structure of cellular RNAs. Notably, although the overwhelming majority of such editing events occur in transcripts derived from Alu repeat elements, the biological function of non-coding RNA editing remains uncertain. Here, we show that mutations in ADAR1 (also known as ADAR) cause the autoimmune disorder Aicardi-Goutières syndrome (AGS). As in Adar1-null mice, the human disease state is associated with upregulation of interferon-stimulated genes, indicating a possible role for ADAR1 as a suppressor of type I interferon signaling. Considering recent insights derived from the study of other AGS-related proteins, we speculate that ADAR1 may limit the cytoplasmic accumulation of the dsRNA generated from genomic repetitive elements.

Tartrate-resistant acid phosphatase deficiency causes a bone dysplasia with autoimmunity and a type I interferon expression signature.

We studied ten individuals from eight families showing features consistent with the immuno-osseous dysplasia spondyloenchondrodysplasia. Of particular note was the diverse spectrum of autoimmune phenotypes observed in these individuals (cases), including systemic lupus erythematosus, Sjögren's syndrome, hemolytic anemia, thrombocytopenia, hypothyroidism, inflammatory myositis, Raynaud's disease and vitiligo. Haplotype data indicated the disease gene to be on chromosome 19p13, and linkage analysis yielded a combined multipoint log(10) odds (LOD) score of 3.6. Sequencing of ACP5, encoding tartrate-resistant acid phosphatase, identified biallelic mutations in each of the cases studied, and in vivo testing confirmed a loss of expressed protein. All eight cases assayed showed elevated serum interferon alpha activity, and gene expression profiling in whole blood defined a type I interferon signature. Our findings reveal a previously unrecognized link between tartrate-resistant acid phosphatase activity and interferon metabolism and highlight the importance of type I interferon in the genesis of autoimmunity.