Hypoxia-induced transcriptional stress is mediated by ROS-induced R-loops.

Hypoxia is a common feature of solid tumors and is associated with poor patient prognosis, therapy resistance and metastasis. Radiobiological hypoxia (<0.1% O2) is one of the few physiologically relevant stresses that activates both the replication stress/DNA damage response and the unfolded protein response. Recently, we found that hypoxia also leads to the robust accumulation of R-loops, which led us to question here both the mechanism and consequence of hypoxia-induced R-loops. Interestingly, we found that the mechanism of R-loop accumulation in hypoxia is dependent on non-DNA damaging levels of reactive oxygen species. We show that hypoxia-induced R-loops play a critical role in the transcriptional stress response, evidenced by the repression of ribosomal RNA synthesis and the translocation of nucleolin from the nucleolus into the nucleoplasm. Upon depletion of R-loops, we observed a rescue of both rRNA transcription and nucleolin translocation in hypoxia. Mechanistically, R-loops accumulate on the rDNA in hypoxia and promote the deposition of heterochromatic H3K9me2 which leads to the inhibition of Pol I-mediated transcription of rRNA. These data highlight a novel mechanistic insight into the hypoxia-induced transcriptional stress response through the ROS-R-loop-H3K9me2 axis. Overall, this study highlights the contribution of transcriptional stress to hypoxia-mediated tumorigenesis.

Induction of the alternative lengthening of telomeres pathway by trapping of proteins on DNA.

Telomere maintenance is a hallmark of malignant cells and allows cancers to divide indefinitely. In some cancers, this is achieved through the alternative lengthening of telomeres (ALT) pathway. Whilst loss of ATRX is a near universal feature of ALT-cancers, it is insufficient in isolation. As such, other cellular events must be necessary - but the exact nature of the secondary events has remained elusive. Here, we report that trapping of proteins (such as TOP1, TOP2A and PARP1) on DNA leads to ALT induction in cells lacking ATRX. We demonstrate that protein-trapping chemotherapeutic agents, such as etoposide, camptothecin and talazoparib, induce ALT markers specifically in ATRX-null cells. Further, we show that treatment with G4-stabilising drugs cause an increase in trapped TOP2A levels which leads to ALT induction in ATRX-null cells. This process is MUS81-endonuclease and break-induced replication dependent, suggesting that protein trapping leads to replication fork stalling, with these forks being aberrantly processed in the absence of ATRX. Finally, we show ALT-positive cells harbour a higher load of genome-wide trapped proteins, such as TOP1, and knockdown of TOP1 reduced ALT activity. Taken together, these findings suggest that protein trapping is a fundamental driving force behind ALT-biology in ATRX-deficient malignancies.

A key feature of all cancer cells is their ability to divide indefinitely, and this is dependent on circumvention of telomere shortening through induction of a telomere maintenance mechanism, such as the telomerase-independent, Alternative Lengthening of Telomeres (ALT) pathway. The ALT pathway is characterised by loss of the ATRX chromatin remodeler. The current study provides evidence that, in the absence of ATRX, increased trapping of proteins on DNA leads to replication fork stalling and collapse. At telomeres, this leads to ALT pathway activity. These results help to better understand ALT tumours and might, eventually, be instrumental in developing new therapeutic strategies.

Risk and Lag-time for Development of Lacrimal Gland Carcinoma Expleomorphic Adenoma: Observations and Conjectural Study.

AIMS: To estimate the proportion of de novo lacrimal gland pleomorphic adenomas (PAs) and carcinomas expleomorphic adenomas (CEPAs), together with age at presentation and first symptom. Conjectural models of tumor growth are considered. METHODS: Retrospective review of patients with orbital lobe PA or CEPA. The presenting age was examined for conformation to a Gaussian distribution and the cumulative distribution function derived for both tumor types. The risk of CEPA with age was estimated by logistic regression. RESULTS: About one-sixth (27/172; 16%) of these primary orbital lobe tumors were CEPAs, with 145 PAs (76/145 male; 52%) and 27 CEPAs (12/27 male; 44%). The mean presenting age for PAs was 48.3 years (median 47.7; range 11-84 years) and 57.7 years for CEPAs (median 61.2, range 27-91 years) ( p = 0.0062), and the standard deviations for each group are almost identical (16.3 for PAs, 15.9 for CEPAs; p = 0.92). Five (3.4%) PAs and 1 (3.7%) CEPA were asymptomatic: otherwise, the median symptom duration was 24 months for both PAs and CEPAs ( U test: p = 0.65). The odds of CEPA rises significantly with age, increasing 1.04-fold annually ( p = 0.0079). CONCLUSION: The almost identical measures of dispersion for the presenting ages of PA and CEPA suggests that, once malignant transformation occurs, there might be a relatively constant period before it is evident. CEPAs present about a decade after PAs, this unexpectedly later presentation for the malignancy possibly being explained by a gradual replacement of the PA by the newly arising carcinoma within the preceding benign tumor.

Detection of mutations in SF3B1, EIF1AX and GNAQ in primary orbital melanoma by candidate gene analysis.

BACKGROUND: Ocular melanoma is a rare but often deadly malignancy that arises in the uvea (commonest primary site), conjunctiva or the orbit. Primary orbital melanoma (POM) is exceedingly rare, with approximately 60 cases reported to date. Despite recent advances in our understanding of the genetics of primary uveal and conjunctival melanomas, this information is lacking for POM. METHODS: DNA was extracted from 12 POM tissues, with matched germline DNA (where available). MLPA was conducted to detect chromosomal alterations and Sanger sequencing used to identify point mutations in candidate melanoma driver genes (BRAF, NRAS, KRAS, GNA11, GNAQ), and other genes implicated in melanoma prognosis (EIF1AX, SF3B1). Immunohistochemistry was performed to analyse BAP1 nuclear expression. RESULTS: MLPA detected copy number alterations in chromosomes 1p, 3, 6 and 8. Sequencing of melanoma driver genes revealed GNAQ (p.Q209L) mutations in two samples; although it is possible that these samples represent extraocular spread of an occult uveal melanoma. A recurrent mutation in SF3B1 (p.R625H) was observed in indolent, but not aggressive, tumours; a mutation in EIF1AX (p.N4S) was detected in one patient with non-aggressive disease. CONCLUSIONS: EIF1AX and SF3B1 mutations appear have a role in determining the clinical course of POM and detection of these changes could have clinical significance. Further in depth analysis of this rare group using differing 'omic technologies will provide novel insights into tumour pathogenesis.

CNOT3 is a modifier of PRPF31 mutations in retinitis pigmentosa with incomplete penetrance.

Heterozygous mutations in the PRPF31 gene cause autosomal dominant retinitis pigmentosa (adRP), a hereditary disorder leading to progressive blindness. In some cases, such mutations display incomplete penetrance, implying that certain carriers develop retinal degeneration while others have no symptoms at all. Asymptomatic carriers are protected from the disease by a higher than average expression of the PRPF31 allele that is not mutated, mainly through the action of an unknown modifier gene mapping to chromosome 19q13.4. We investigated a large family with adRP segregating an 11-bp deletion in PRPF31. The analysis of cell lines derived from asymptomatic and affected individuals revealed that the expression of only one gene among a number of candidates within the 19q13.4 interval significantly correlated with that of PRPF31, both at the mRNA and protein levels, and according to an inverse relationship. This gene was CNOT3, encoding a subunit of the Ccr4-not transcription complex. In cultured cells, siRNA-mediated silencing of CNOT3 provoked an increase in PRPF31 expression, confirming a repressive nature of CNOT3 on PRPF31. Furthermore, chromatin immunoprecipitation revealed that CNOT3 directly binds to a specific PRPF31 promoter sequence, while next-generation sequencing of the CNOT3 genomic region indicated that its variable expression is associated with a common intronic SNP. In conclusion, we identify CNOT3 as the main modifier gene determining penetrance of PRPF31 mutations, via a mechanism of transcriptional repression. In asymptomatic carriers CNOT3 is expressed at low levels, allowing higher amounts of wild-type PRPF31 transcripts to be produced and preventing manifestation of retinal degeneration.

Late presentation to hospital services necessitates greater community-based care for malnourished children.

The records for all paediatric deaths (ages 0-14) in a large hospital in urban Southern Africa were examined for a 3 year period (January 2007 to February 2010), to explore the role of malnutrition in paediatric mortality in this region. A total of 516 records were obtained, demonstrating that malnutrition was the primary or secondary cause of death in 35% of cases. It was also found that children presented very late to hospital services, with an average length of final admission of only 0-3 days. The rate of human immunodeficiency virus (HIV) infection was found to be very high, although low testing rates limits the analysis of these figures. Malnutrition remains an important factor in paediatric mortality in southern Africa, contributing to approximately 35% of deaths. Furthermore, fatal cases presented very late to hospital services. In light of this, increased community-based therapy would be beneficial. Implementation of universal HIV testing would also be valuable.

Expression of PRPF31 and TFPT: regulation in health and retinal disease.

PRPF31, a gene located at chromosome 19q13.4, encodes the ubiquitous splicing factor PRPF31. The gene lies in a head-to-head arrangement with TFPT, a poorly characterized gene with a role in cellular apoptosis. Mutations in PRPF31 have been implicated in autosomal dominant retinitis pigmentosa (adRP), a frequent and important cause of blindness worldwide. Disease associated with PRPF31 mutations is unusual, in that there is often non-penetrance of the disease phenotype in affected families, caused by differential expression of PRPF31. This study aimed to characterize the basic promoter elements of PRPF31 and TFPT. Luciferase reporter constructs were made, using genomic DNA from an asymptomatic individual with a heterozygous deletion of the entire putative promoter region. Fragments were tested by the dual-luciferase reporter assay in HeLa and RPE-1 cell lines. A comparison was made between the promoter regions of symptomatic and asymptomatic mutation-carrying individuals. A patient (CAN493) with adRP was identified, harbouring a regulatory region mutation; both alleles were assayed by the dual-luciferase reporter assay. Luciferase assays led to the identification of core promoters for both PRPF31 and TFPT; despite their shared gene architecture, the two genes appear to be controlled by slightly different regulatory regions. One functional polymorphism was identified in the PRPF31 promoter that increased transcriptional activation. The change was not, however, consistent with the observed symptomatic-asymptomatic phenotypes in a family affected by PRPF31-adRP. Analysis of the mutant promoter fragment from CAN493 showed a >50% reduction in promoter activity, suggesting a disease mechanism of functional haploinsufficiency-the first report of this disease mechanism in adRP.

Dominant PRPF31 mutations are hypostatic to a recessive CNOT3 polymorphism in retinitis pigmentosa: a novel phenomenon of "linked trans-acting epistasis".

Mutations in PRPF31 are responsible for autosomal dominant retinitis pigmentosa (adRP, RP11 form) and affected families show nonpenetrance. Differential expression of the wildtype PRPF31 allele is responsible for this phenomenon: coinheritance of a mutation and a higher expressing wildtype allele provide protection against development of disease. It has been suggested that a major modulating factor lies in close proximity to the wildtype PRPF31 gene on Chromosome 19, implying that a cis-acting factor directly alters PRPF31 expression. Variable expression of CNOT3 is one determinant of PRPF31 expression. This study explored the relationship between CNOT3 (a trans-acting factor) and its paradoxical cis-acting nature in relation to RP11. Linkage analysis on Chromosome 19 was performed in mutation-carrying families, and the inheritance of the wildtype PRPF31 allele in symptomatic-asymptomatic sibships was assessed-confirming that differential inheritance of wildtype chromosome 19q13 determines the clinical phenotype (P < 2.6 × 10(-7) ). A theoretical model was constructed that explains the apparent conflict between the linkage data and the recent demonstration that a trans-acting factor (CNOT3) is a major nonpenetrance factor: we propose that this apparently cis-acting effect arises due to the intimate linkage of CNOT3 and PRPF31 on Chromosome 19q13-a novel mechanism that we have termed "linked trans-acting epistasis."

Overcoming resistance to belief revision and correction of misinformation beliefs: psychophysiological and behavioral effects of a counterfactual mindset.

In a series of experiments involving beliefs and misinformation beliefs, we find that individuals who are prompted with a counterfactual mindset are significantly more likely to change their existing beliefs when presented with evidence that contradicts their beliefs. While research finds that beliefs that are considered part of one's identity are highly resistant to change in the face of evidence that challenges these beliefs, four experiments provide evidence that counterfactual generation causes individuals to adjust beliefs and correct misinformation beliefs in response to contradicting evidence. Indeed, we find that a counterfactual mindset was effective in promoting incorporation of accurate facts and causing individuals to revise misinformation beliefs about COVID vaccination safety for a large sample of individuals who have rejected COVID vaccinations. Finally, the results of the psychophysiological experiment reveal that counterfactual generation alters decision makers' search strategies, increases their cognitive arousal in response to evidence that challenges their beliefs, and increases their desire to seek out disconfirming evidence. Overall, the four experiments indicate that counterfactual generation can effectively activate mindsets that increase individuals' willingness to evaluate evidence that contradicts their beliefs and adjust their beliefs in response to evidence.

A rare case of orbital haemangiopericytoma arising in childhood.

Haemangiopericytoma (HPC) is a rare soft tissue tumour of fibroblastic origin and is part of the solitary fibrous tumour spectrum. The tumour is generally considered to be benign, but can behave clinically as if sarcomatous -- with relentless infiltrative local growth. HPC generally presents in adulthood (median age 45 years for orbital disease) and is equally frequent in both sexes. HPC can arise in any site in the body and presents as a slowly growing, painless mass. We report a case of a 20 year old African male seen at Kikuyu Eye Unit, Kenya, with a 12 year history of a gradually enlarging, painless orbital mass. The patient underwent skin-sparing orbital exenteration with complete tumour excision; histology confirmed diagnosis of HPC.

The emergence of Fanconi anaemia type S: a phenotypic spectrum of biallelic BRCA1 mutations.

BRCA1 is involved in the Fanconi anaemia (FA) pathway, which coordinates repair of DNA interstrand cross-links. FA is a rare genetic disorder characterised by bone marrow failure, cancer predisposition and congenital abnormalities, caused by biallelic mutations affecting proteins in the FA pathway. Germline monoallelic pathogenic BRCA1 mutations are known to be associated with hereditary breast/ovarian cancer, however biallelic mutations of BRCA1 were long predicted to be incompatible with embryonic viability, hence BRCA1 was not considered to be a canonical FA gene. Despite this, several patients with biallelic pathogenic BRCA1 mutations and FA-like phenotypes have been identified - defining a new FA type (FA-S) and designating BRCA1 as an FA gene. This report presents a scoping review of the cases of biallelic BRCA1 mutations identified to date, discusses the functional effects of the mutations identified, and proposes a phenotypic spectrum of BRCA1 mutations based upon available clinical and genetic data. We report that this FA-S cohort phenotype includes short stature, microcephaly, facial dysmorphisms, hypo/hyperpigmented lesions, intellectual disability, chromosomal sensitivity to crosslinking agents and predisposition to breast/ovarian cancer and/or childhood cancers, with some patients exhibiting sensitivity to chemotherapy. Unlike most other types of FA, FA-S patients lack bone marrow failure.

Epistasis and immunity: the role of genetic interactions in autoimmune diseases.

Autoimmune disorders are a complex and varied group of diseases that are caused by breakdown of self-tolerance. The aetiology of autoimmunity is multi-factorial, with both environmental triggers and genetically determined risk factors. In recent years, it has been increasingly recognized that genetic risk factors do not act in isolation, but rather the combination of individual additive effects, gene-gene interactions and gene-environment interactions determine overall risk of autoimmunity. The importance of gene-gene interactions, or epistasis, has been recently brought into focus, with research demonstrating that many autoimmune diseases, including rheumatic arthritis, autoimmune glomerulonephritis, systemic lupus erythematosus and multiple sclerosis, are influenced by epistatic interactions. This review sets out to examine the basic mechanisms of epistasis, how epistasis influences the immune system and the role of epistasis in two major autoimmune conditions, systemic lupus erythematosus and multiple sclerosis.

Epistasis: the key to understanding immunological disease?

Epistasis is fast becoming central to the understanding of the complex relationship between genotype and phenotype observed in autoimmune disease. A study in this issue of the European Journal of Immunology uses in-depth analysis of genome-wide mapping by polymorphic microsatellite markers to shed light on the genomic control of autoimmunity and self-tolerance.

Gene of the month: PRPF31.

Pre-mRNA splicing is an essential process in eukaryotic cells where the transcribed intronic sequences are removed, prior to translation into protein. PRPF31 is a ubiquitously expressed splicing factor, which aids in the assembly of the macromolecular spliceosome. Mutations in PRPF31 cause autosomal dominant retinitis pigmentosa (adRP), a form of retinal degeneration that causes progressive visual impairment. Interestingly, mutations in PRPF31 are non-penetrant, with some mutation carriers being phenotypically unaffected. In this review, the gene organisation, protein structure and biological function of PRPF31 are discussed, and the mechanisms of non-penetrance in PRPF31-associated adRP are discussed.

Primary Orbital Melanoma: Presentation, Treatment, and Long-term Outcomes for 13 Patients.

BACKGROUND: Periocular melanoma is a rare but often deadly malignancy that arises in the uvea (commonest origin), conjunctiva or orbit (rarest primary site). Melanoma accounts for 5-10% of metastatic/secondary orbital malignancies, but only a tiny proportion of primary orbital neoplasia. Primary orbital melanoma (POM) is exceedingly rare, with approximately 50 cases reported to date. METHODS: All patients seen in the orbital unit at a tertiary referral hospital (1991-2016) with a biopsy-proven diagnosis of POM were identified from a diagnostic database and were studied. The case notes, imaging, surgical approach, and histology were reviewed. RESULTS: Thirteen patients (five male; 38%) presented with isolated malignant melanoma of the orbit, for which no other primary site was identified at presentation or during an average follow-up of 44 months (median 22; range 0-13 years). The patients presented between the ages of 40 and 84 years (mean 55.5; median 48 years) and typically gave a short history of rapidly increasing proptosis and eyelid swelling. On the basis of history, a malignant lesion was suspected in most patients and all underwent incisional biopsy, with debulking of the mass in 10 (77%) patients, and skin-sparing exenteration in 3/13 (23%). Ten patients underwent orbital radiotherapy and the survival to date ranged from 9 months to 14 years (mean 55 months; median 23 months); two patients received solely palliative care for widespread disease and one patient refused orbital radiotherapy. Five of the 13 (38%) patients died from the disease. DISCUSSION: POM is a very rare malignancy, but clinical analysis of this cohort gives insight into disease presentation and prognosis. The tumor typically presents with a rapidly progressive, well-defined mass that is, in some cases, amenable to macroscopically intact excision. Unusual for malignant melanoma, some of these patients can show an unusually long period of quiescent disease after surgical debulking and radiotherapy.

Presentation, Treatment, and Prognosis of Secondary Melanoma within the Orbit.

BACKGROUND: Ocular melanoma is a rare but often deadly malignancy that arises in the uvea, conjunctiva, or orbit. Uveal melanoma is the most common type, with conjunctival melanoma being the second most frequently observed. Melanoma accounts for 5-10% of metastatic or secondary orbital malignancies, but only a minute proportion of primary orbital neoplasia. The aim of this study was to characterize the clinical presentation, treatment, and prognosis in patients presenting with melanoma metastatic to, or secondary within, the orbit. METHODS: A retrospective cohort study of patients presenting to a tertiary referral orbital unit from 1982 to 2016 was performed. Eighty-nine patients with biopsy-proven diagnosis of melanoma within the orbit were included in the study. The clinical notes, radiological imaging, histology, surgical notes, and outcome data for the patients were reviewed. The main outcome measures of interest were the interval between primary malignant melanoma and orbital presentation, survival after orbital presentation, and clinical parameters (such as gender, age at presentation, and treatment approach). RESULTS: The commonest primary source of tumor was choroidal melanoma, with conjunctival and cutaneous melanomas being relatively common; eyelid and naso-sinus tumors occurred in a few cases. The mean age at presentation with orbital disease was 65 years (31-97 years). The interval between primary malignancy and orbital disease (either local spread/recurrence or true metastatic disease) showed wide variability, with almost one-third of patients having orbital disease at the time of primary diagnosis, but others presenting many years later; indeed, the longest orbital disease-free interval was over 34 years. Twenty-three patients were considered to have had late orbital metastases-that is, at more than 36 months after primary tumor. The median survival following presentation with orbital involvement was 24 months. Patients with tumors of cutaneous origin had worst survival, whereas those with conjunctival tumors had the best prognosis. CONCLUSION: A high index of suspicion for orbital recurrence should be maintained in any patient with prior history of melanoma, however distant the primary tumor is in site or time. Furthermore, giving a prognosis for orbital melanoma remains problematic due to highly variable survival, and further investigation will be necessary to understand the likely genetic basis of this phenomenon.

Transcriptional regulation of PRPF31 gene expression by MSR1 repeat elements causes incomplete penetrance in retinitis pigmentosa.

PRPF31-associated retinitis pigmentosa presents a fascinating enigma: some mutation carriers are blind, while others are asymptomatic. We identify the major molecular cause of this incomplete penetrance through three cardinal features: (1) there is population variation in the number (3 or 4) of a minisatellite repeat element (MSR1) adjacent to the PRPF31 core promoter; (2) in vitro, 3-copies of the MSR1 element can repress gene transcription by 50 to 115-fold; (3) the higher-expressing 4-copy allele is not observed among symptomatic PRPF31 mutation carriers and correlates with the rate of asymptomatic carriers in different populations. Thus, a linked transcriptional modifier decreases PRPF31 gene expression that leads to haploinsufficiency. This result, taken with other identified risk alleles, allows precise genetic counseling for the first time. We also demonstrate that across the human genome, the presence of MSR1 repeats in the promoters or first introns of genes is associated with greater population variability in gene expression indicating that copy number variation of MSR1s is a generic controller of gene expression and promises to provide new insights into our understanding of gene expression regulation.