Epigenetic regulation, including DNA methylation, histone modifications, non-coding RNAs, and higher-order chromatin remodeling, plays a central role in the biology of neuroendocrine neoplasms (NENs). Advances in high-throughput profiling have uncovered epigenetic alterations across pheochromocytomas/paragangliomas (PPGLs), gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs), lung neuroendocrine neoplasms (LNENs), medullary thyroid carcinoma (MTC), and pituitary NETs (PitNETs). These alterations converge on pathways governing cell cycle control, telomere maintenance, hypoxia signaling, epithelial-mesenchymal transition, and chromatin architecture. Importantly, epigenetic signatures not only mirror genetic backgrounds (eg, SDHx, MEN1, ATRX, RET) but also provide independent layers of prognostic and predictive information. Distinct methylation profiles, histone modification patterns, and deregulated microRNA/long non-coding RNA networks have been consistently linked to tumor aggressiveness, metastatic potential, and therapeutic resistance. The clinical translation of these insights is rapidly evolving. Methylation-based classifiers and circulating epigenetic markers are emerging as promising tools for early diagnosis, risk stratification, and longitudinal monitoring. Moreover, epigenetic pathways represent attractive therapeutic targets, with DNA methyltransferase inhibitors, histone deacetylase inhibitors, and RNA-modifying enzyme modulators under active investigation. However, significant challenges remain, including methodological heterogeneity, small or retrospective cohorts, and the limited functional validation of candidate biomarkers. Future priorities include prospective multi-institutional validation, integration of epigenomics with other molecular layers in multidimensional classifiers, and the application of spatial and single cell approaches to resolve intra-tumoral heterogeneity. Ultimately, epigenetic research has redefined our understanding of NEN pathogenesis and progression, positioning the epigenome as a promising frontier in precision oncology. Through robust validation, epigenetic biomarkers and therapies may transform the clinical management of NENs.

Eneboparatide, a long-acting modified form of parathyroid hormone, increases serum calcium levels without increasing urine calcium in mice with autosomal dominant hypocalcemia type 1.

BACKGROUND:Multiple endocrine neoplasia type 1 (MEN1) is a rare hereditary disorder characterised by the combined occurrence of parathyroid, pancreatic, and pituitary tumours. Current treatments are based on very low-quality evidence. Our aims were to determine treatment outcomes in patients with MEN1 for: subtotal parathyroidectomy versus less than subtotal parathyroidectomy for primary hyperparathyroidism (Q1); surgery versus active surveillance for non-functioning pancreatic neuroendocrine tumours sized 2 cm or less (Q2); and dopamine agonist responses of prolactinomas in patients with MEN1 versus patients without MEN1 (Q3). METHODS:We conducted three systematic reviews and one meta-analysis. Four electronic databases (MEDLINE Ovid, Embase Ovid, The Cochrane Library, and Web of Science) were searched from Dec 1, 2001, to Feb 13, 2023, with no language restrictions. Study designs included randomised controlled trials, prospective and retrospective cohort studies, and case-controlled and case series. Adults and children with MEN1-associated tumours were included in all three systematic reviews. For each clinical question, three pairs of authors independently screened abstracts and assessed the full text for final inclusion, discordant views were resolved by senior authors. Dichotomous outcomes were calculated using risk ratios or hazard ratios for time-to-event analyses, with 95% CIs. Continuous outcomes were ascertained using mean difference with 95% CIs. Where feasible, outcomes from individual studies were analysed through meta-analysis, using a random-effects model. The systematic reviews were prospectively registered (PROSPERO reference numbers CRD42023409912, CRD42023409936, and CRD42023409949). FINDINGS:For primary hyperparathyroidism (Q1), 990 non-duplicate records were screened for title and abstract, of which 23 studies with 1073 patients were eligible for meta-analysis. These studies showed that subtotal parathyroidectomy had a significantly lower risk of persistent primary hyperparathyroidism (RR 0·32, 95% CI 0·20-0·52; I2=0%) and recurrent primary hyperparathyroidism (RR 0·78, 0·62-0·97; I2=27%), when compared with less than subtotal parathyroidectomy, although the risk of post-operative hypoparathyroidism was higher (RR 2·64, 1·63-4·29; I2=0%). For non-functioning pancreatic neuroendocrine tumours sized 2 cm and less (Q2), 1583 non-duplicate records were screened for title and abstract, of which three cohort studies were eligible for analysis. These studies showed that combined metastatic disease and mortality rates were comparable between patients in the surgery group (two [7%] of 27 to three [20%] of 15) and patients in the active surveillance group (one [3%] of 33 to four [8%] of 50]). For prolactinomas (Q3), 475 non-duplicate records were screened for title and abstract, of which ten studies with 505 patients were eligible for analysis. These studies showed that dopamine agonist treatment failure rates to normalise serum prolactin were similar between patients with MEN1 (zero of one to one [33%] of three) and patients without MEN1 (four [6%] of 68 to nine (82%) of 11), n=23 studies). GRADE certainty scores for all were low or very low. INTERPRETATION:In patients with MEN1, subtotal parathyroidectomy achieved greater reductions in persistence and recurrence of primary hyperparathyroidism than less than subtotal parathyroidectomy; for non-functioning pancreatic neuroendocrine tumours sized 2 cm or less, the few available studies suggest that active surveillance might be comparable to surgical resection; and for prolactinomas, dopamine agonist therapy appears to have comparable efficacy as in patients without MEN1. FUNDING:None.

Multiple endocrine neoplasia type 1 (MEN1) is characterised by combined occurrence of parathyroid tumours, duodenopancreatic neuroendocrine tumours, and anterior pituitary adenomas. Some patients might also develop thymic and bronchopulmonary neuroendocrine tumours, and adrenal tumours. MEN1 is an autosomal dominant disorder caused by mutations in the tumour-suppressor gene MEN1, which encodes a scaffold protein, menin. Without treatment, patients with MEN1 have high morbidity and premature mortality, which can be mitigated by early tumour detection and intervention. Identification of individuals at high risk for MEN1 can be facilitated by genetic testing of patients and their first-degree relatives, and undertaking periodic clinical, biochemical, and radiological screening in patients and MEN1 mutation carriers. However, no consensus exists regarding the optimal assessment and management of MEN1. To provide such recommendations, a multidisciplinary group was convened to undertake systematic reviews and a meta-analysis of the literature, and to use a Delphi approach for the development of consensus statements. 55 clinical recommendations were developed to guide clinicians, patients, and stakeholders about approaches for MEN1 in adults and children.

Neuroendocrine tumors (NETs) occur sporadically or as part of rare endocrine tumor syndromes (RETSs) such as multiple endocrine neoplasia 1 and von Hippel–Lindau syndromes. Due to their relative rarity and lack of model systems, NETs and RETSs are difficult to study, hindering advancements in therapeutic development. Causal or mechanistic mathematical modeling is widely deployed in disease areas such as breast and prostate cancers, aiding the understanding of observations and streamlining in vitro and in vivo modeling efforts. Mathematical modeling, while not yet widely utilized in NET research, offers an opportunity to accelerate NET research and therapy development. To illustrate this, we highlight examples of how mathematical modeling associated with more common endocrine cancers has been successfully used in the preclinical, translational and clinical settings. We also provide a scope of the limited work that has been done in NETs and map how these techniques can be utilized in NET research to address specific outstanding challenges in the field. Finally, we include practical details such as hardware and data requirements, present advantages and disadvantages of various mathematical modeling approaches and discuss challenges of using mathematical modeling. Through a cross-disciplinary approach, we believe that many currently difficult problems can be made more tractable by applying mathematical modeling and that the field of rare diseases in endocrine oncology is well poised to take advantage of these techniques.

Gain-of-function mutations of the calcium-sensing receptor (CaSR) result in autosomal dominant hypocalcemia type 1 (ADH1), which may cause symptomatic hypocalcemia with low parathyroid hormone concentrations. Negative allosteric CaSR modulators, known as calcilytics, have potential as a targeted ADH1 therapy and comprise two main classes, which are the amino alcohols and the quinazolinones. Amino alcohol calcilytics have been assessed as ADH1 therapies but may not be effective for all ADH1-causing mutations. We therefore evaluated quinazolinone calcilytics (ATF936 and AXT914) as an alternate ADH1 treatment. Calcilytic docking studies were performed using reported cryo-EM CaSR structures. In vitro dose-response studies were performed using CaSR-expressing HEK293 cells and in vivo studies undertaken in mice with a gain-of-function CaSR mutation, Leu723Gln, known as Nuf. ATF936 and AXT914, as well as the amino alcohol calcilytics, NPS 2143 and NPSP795, were shown to bind at a common region within the CaSR transmembrane domain, which is also an ADH1 mutational hotspot. Treatment of cells expressing the Nuf mutant (Gln723) CaSR with 1 to 20 nM AXT914 caused dose-dependent decreases in CaSR-mediated intracellular calcium responses with 10 nM AXT914 normalizing the gain of function. Oral administration of 10 mg/kg AXT914 to Nuf mice increased parathyroid hormone to 104 ± 29 pmol/l compared with 23 ± 4 pmol/l for vehicle-treated mice, p < 0.05; and increased plasma albumin-adjusted calcium to 2.03 ± 0.02 mmol/l compared with 1.84 ± 0.02 mmol/l for vehicle-treated mice, p < 0.001. These studies indicate that quinazolinone calcilytics may have potential for treating ADH1.

Gastroenteropancreatic neuroendocrine tumours (GEP-NETs), which may be hormone secreting (e.g., gastrinomas and insulinomas) or non-secreting (also known as non-functioning NETs) are associated with severe morbidity and have a median overall survival of 75-124 months. Studies have highlighted the importance of epigenetic mechanisms in GEP-NETs pathogenesis, with the most frequently mutated genes being the epigenetic regulators, MEN1, DAXX, and ATRX. However, the consequences of these aberrant epigenetic mechanisms are poorly understood. The calcium sensing receptor (CASR), a G protein coupled-receptor, is epigenetically silenced in cancers, and therefore we examined its role in GEP-NET subtypes. Using RNA-Scope and quantitative PCR analyses in two independent tumour cohorts from Europe (n = 18 patients) and the USA (n = 46 patients) we showed that CASR mRNA is almost completely absent in gastrinomas, insulinomas and non-functioning pancreatic NETs. Furthermore, immunohistochemical staining confirmed a significant reduction in CaSR protein expression in all GEP-NET subtypes, compared to normal islets. DNA methylationEPIC and ATAC-seq analyses in the pancreatic NET cell line QGP-1 showed the CaSR promoter was both hypermethylated and in a region of closed chromatin. Furthermore, transfection of wild type CaSR into QGP-1 cells decreased cell viability, in keeping with the CaSR having a role in cellular proliferation. In summary, our study reveals that CaSR expression is decreased in GEP-NETs and that this reduced expression is likely due to DNA methylation and chromatin changes. Moreover, we demonstrate that transfection of the CaSR into a PNET cell line reduces cell viability, thereby indicating that the CaSR acts as a tumour suppressor in this tumour type.

Nuclear factor I/X (NFIX) mutations are associated with 2 skeletal dysplasias, Marshall-Smith (MSS) and Malan (MAL) syndromes. NFIX encodes a transcription factor that regulates expression of genes, including Bobby sox (BBX) and glial fibrillary acidic protein (GFAP) in neural progenitor cells and astrocytes, respectively. To elucidate the role of NFIX mutations in MSS, we studied their effects in fibroblast cell lines obtained from 5 MSS unrelated patients and 3 unaffected individuals. The 5 MSS NFIX frameshift mutations in exons 6-8 comprised 3 deletions (c.819-732_1079-948del, c.819-471_1079-687del, c.819-592_1079-808del), an insertion (c.1037_1038insT), and a duplication (c.1090dupG). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot analyses using MSS and unrelated control fibroblasts and in vitro expression studies in monkey kidney fibroblast (COS-7) cells showed that frameshift mutations in NFIX exons 6-8 generated mutant transcripts that were not cleared by nonsense-mediated-decay mechanisms and encoded truncated NFIX proteins. Moreover, BBX or GFAP expression was unaffected in the majority of MSS fibroblasts. To identify novel NFIX downstream target genes, RNA sequencing and proteomics analyses were performed on mouse embryonic fibroblast (MEF) cells derived from control Nfix+/+, Nfix+/Del2, Nfix+/Del24, NfixDel24/Del24, Nfix+/Del140, and NfixDel140/Del140 mice, compared with NfixDel2/Del2 mice which had developmental, skeletal, and neural abnormalities. This identified 191 transcripts and 815 proteins misregulated in NfixDel2/Del2 MEFs with ≥2-fold-change (P <0 .05). Validation studies using qRT-PCR and western blot analyses confirmed that 2 genes, cellular retinoic acid binding protein 2 (Crabp2) and vascular cell adhesion molecule 1 (Vcam1), were misregulated at the RNA and protein levels in NfixDel2/Del2 MEFs, and that CRABP2 and VCAM1 expressions were altered in 60%–100% of MSS fibroblast cells. Furthermore, in vitro luciferase reporter assays confirmed that NFIX directly regulates CRABP2 promoter activity. Thus, these altered genes and pathways may represent possible targets for drugs as potential treatments and therapies for MSS.

Adaptor-Related Protein Complex 2 Sigma-1 Subunit (AP2S1) encodes AP2σ2, which forms part of the heterotetrameric AP2 complex that is composed of α, β2, μ2, and σ2 subunits and has a pivotal role in clathrin-mediated endocytosis (CME). AP2S1 variants involving the Arg15 residue are associated with familial hypocalciuric hypercalcaemia type 3 (FHH3). Here, we report 5 different AP2S1 variants (AP2σ2: p.Arg10Trp, p.Arg10Gln, p.Lys18Glu, p.Lys18Asn and p.Arg61His) in 26 patients with neurodevelopmental delay, of whom >70% had epilepsy, 50% had brain abnormalities, and none had hypercalcaemia. All 5 variants decreased cell viability, 4 reduced CME transferrin uptake, and 4 disrupted interactions with other AP2 complex subunits, thereby affecting AP2 formation. Furthermore, AP2σ2 p.Arg10Trp had reduced interactions with 44 human proteins including intersectin 1, a component required for clathrin-coated pit formation and synaptic vesicle dynamics in neurones. Thus, our results show that AP2σ2 variants may disrupt CME and be associated with neurodevelopmental disorders. ### Competing Interest Statement AB, DAC, JJ and JJK are employees of GeneDx, LLC ### Funding Statement This work was supported by a Wellcome Trust Senior Clinical Investigator Award (RVT), and National Institute for Health Research (NIHR) Oxford Biomedical Research Centre Programme grant. RVT is a Wellcome Trust Investigator and NIHR Senior Investigator. For the purpose of Open Access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript (AAM) version arising from this submission. We are grateful to the patients and families that participated in this research. The authors are grateful to the participants of the MyCode Community Health Initiative for the use of their genomic and electronic health information. The patient enrolment and exome sequencing for the DiscovEHR study were funded by the Regeneron Genetics Center. We would like to acknowledge the Geisinger-Regeneron DiscovEHR Collaboration for making the genotype data and phenotype available for this project. This research was also made possible through access to data in the National Genomic Research Library, which is managed by Genomics England Limited (a wholly owned company of the Department of Health and Social Care). The National Genomic Research Library holds data provided by patients and collected by the NHS as part of their care and data collected as part of their participation in research. The National Genomic Research Library is funded by the National Institute for Health Research and NHS England. The Wellcome Trust, Cancer Research UK and the Medical Research Council have also funded research infrastructure. Mass spectrometry analysis was performed at the Discovery Proteomics Facility (headed by Roman Fischer) which is part of the TDI MS Laboratory (led by Benedikt Kessler). KGC is Chairholder of the Emil von Behring Chair for Neuromuscular and Neurodegenerative Disorders by CSL Behring. KGC is member of the European Reference Network for Rare Neuromuscular Diseases (ERN EURO-NMD) and of the European Reference Network for Rare Neurological Diseases (ERN-RND). KGC reports no disclosures relevant to the manuscript. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The London Multi-Centre Research Ethics Committee (MREC) gave ethical approval for this work (MREC/02/2/93). Informed consent was obtained from individuals or their family/guardians/parents, using protocols approved by the local and national ethics committees. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors

Nuclear factor I/X (NFIX) mutations are associated with 2 skeletal dysplasias, Marshall-Smith (MSS) and Malan (MAL) syndromes. NFIX encodes a transcription factor that regulates expression of genes, including Bobby sox (BBX) and glial fibrillary acidic protein (GFAP) in neural progenitor cells and astrocytes, respectively. To elucidate the role of NFIX mutations in MSS, we studied their effects in fibroblast cell lines obtained from 5 MSS unrelated patients and 3 unaffected individuals. The 5 MSS NFIX frameshift mutations in exons 6-8 comprised 3 deletions (c.819-732_1079-948del, c.819-471_1079-687del, c.819-592_1079-808del), an insertion (c.1037_1038insT), and a duplication (c.1090dupG). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot analyses using MSS and unrelated control fibroblasts and in vitro expression studies in monkey kidney fibroblast (COS-7) cells showed that frameshift mutations in NFIX exons 6-8 generated mutant transcripts that were not cleared by nonsense-mediated-decay mechanisms and encoded truncated NFIX proteins. Moreover, BBX or GFAP expression was unaffected in the majority of MSS fibroblasts. To identify novel NFIX downstream target genes, RNA sequencing and proteomics analyses were performed on mouse embryonic fibroblast (MEF) cells derived from control Nfix(+/+), Nfix(+/Del2), Nfix(+/Del24), Nfix(Del24/Del24), Nfix(+/Del140), and Nfix(Del140/Del140) mice, compared with Nfix(Del2/Del2) mice which had developmental, skeletal, and neural abnormalities. This identified 191 transcripts and 815 proteins misregulated in Nfix(Del2/Del2) MEFs with >= 2-fold-change (P <0 .05). Validation studies using qRT-PCR and western blot analyses confirmed that 2 genes, cellular retinoic acid binding protein 2 (Crabp2) and vascular cell adhesion molecule 1 (Vcam1), were misregulated at the RNA and protein levels in Nfix(Del2/Del2) MEFs, and that CRABP2 and VCAM1 expressions were altered in 60%-100% of MSS fibroblast cells. Furthermore, in vitro luciferase reporter assays confirmed that NFIX directly regulates CRABP2 promoter activity. Thus, these altered genes and pathways may represent possible targets for drugs as potential treatments and therapies for MSS.

Current models for the study of neuroendocrine tumours (NETs) are severely limited. While in vitro (e.g. cell lines), ex vivo (e.g. organoids) and in vivo (e.g. mice) models all exist, each has limitations. To address these limitations and collectively identify strategies to move the NET models field forward, we held an inaugural NET models meeting, hosted by our founding group: Dr Lines (Oxford), Prof. Quelle (Iowa), Dr Dayton (Barcelona), Dr Ear (Iowa), Dr Marinoni (Bern) and Dr Guenter (Alabama). This two-day meeting in Oxford (UK) was organised and supported by Bioscientifica Ltd and was solely dedicated to the discussion of NET models. The meeting was attended by ∼30 international researchers (from the UK, EU, Israel, USA and Canada). Plenary talks were given by Prof. Thakker, who summarised NET research over the past few decades, and Dr Schrader, who described the process and pitfalls of generating new cell lines. Eight researchers also presented their work on topics ranging from human cell 3D bioprinting to zebrafish models and included novel ideas and improvements on current concepts. This was followed by an interactive workshop, where discussion topics included a summary of currently available NET models, limitations of these models, barriers to developing new models, and how we can address these issues going forward. This white paper summarises the key points raised in these discussions and the future aspirations of the NET Models Consortium. The next meeting will take place in Oxford (UK) in 2025; contact contact@netcancerfoundation.com for more information.

Mosaic variants in genes GNAQ or GNA11 lead to a spectrum of vascular and pigmentary diseases including Sturge-Weber syndrome, in which progressive postnatal neurological deterioration led us to seek biologically targeted therapeutics. Using two cellular models, we find that disease-causing GNAQ/11 variants hyperactivate constitutive and G-protein coupled receptor ligand-induced intracellular calcium signaling in endothelial cells. We go on to show that the aberrant ligand-activated intracellular calcium signal is fueled by extracellular calcium influx through calcium-release-activated channels. Treatment with targeted small interfering RNAs designed to silence the variant allele preferentially corrects both the constitutive and ligand-activated calcium signaling, whereas treatment with a calcium-release-activated channel inhibitor rescues the ligand-activated signal. This work identifies hyperactivated calcium signaling as the primary biological abnormality in GNAQ/11 mosaicism and paves the way for clinical trials with genetic or small molecule therapies.

We have previously shown that expression of S100PBP, an S100P binding partner, gradually decreases during progression of pancreatic ductal adenocarcinomas (PDAC). Here, we show that loss of S100PBP leads to oncogenic transformation of pancreatic cells; after deregulation of S100PBP expression, both in silico and in vitro analyses highlighted alterations of genes known to modulate cytoskeleton, cell motility and survival. Overexpression of S100P reduced S100PBP expression, while co-immunoprecipitation indicated the interaction of S100P with S100PBP-p53-ubiquitin protein complex, likely causing S100PBP degradation. The doxycycline-induced Kras G12D activation resulted in decreased S100PBP levels, while low-dose treatment with HDAC inhibitor MS-275 rescued its expression in both human and mouse PDAC cell lines. This indicates Kras G12D as an upstream epigenetic regulator of S100PBP. Finally, analysis of TCGA PanCancer Atlas PDAC datasets demonstrated poor prognosis in patients with high S100P and low S100PBP expression, suggesting that S100PBP is a novel tumour suppressor gene with potential clinical utility.

Primary hyperparathyroidism (PHPT), a relatively common disorder characterized by hypercalcemia with raised or inappropriately normal serum parathyroid hormone (PTH) concentrations, may occur as part of a hereditary syndromic disorder or as a non-syndromic disease. The associated syndromic disorders include multiple endocrine neoplasia types 1–5 (MEN1-5) and hyperparathyroidism with jaw tumor (HPT-JT) syndromes, and the non-syndromic forms include familial hypocalciuric hypercalcemia types 1–3 (FHH1-3), familial isolated hyperparathyroidism (FIHP), and neonatal severe hyperparathyroidism (NS-HPT). Such hereditary forms may occur in > 10% of patients with PHPT, and their recognition is important for implementation of gene-specific screening protocols and investigations for other associated tumors. Syndromic PHPT tends to be multifocal and multiglandular with most patients requiring parathyroidectomy with the aim of limiting end-organ damage associated with hypercalcemia, particularly osteoporosis, nephrolithiasis, and renal failure. Some patients with non-syndromic PHPT may have mutations of the MEN1 gene or the calcium-sensing receptor ( CASR ), whose loss of function mutations usually cause FHH1, a disorder associated with mild hypercalcemia and may follow a benign clinical course. Measurement of the urinary calcium-to-creatinine ratio clearance (UCCR) may help to distinguish patients with FHH from those with PHPT, as the majority of FHH patients have low urinary calcium excretion (UCCR < 0.01). Once genetic testing confirms a hereditary cause of PHPT, further genetic testing can be offered to the patients’ relatives and subsequent screening can be carried out in these affected family members, which prevents inappropriate testing in normal individuals.

Pancreatic neuroendocrine tumours (PNETs) are the second most common pancreatic tumour. However, relatively little is known about their tumourigenic drivers, other than mutations involving the multiple endocrine neoplasia 1 (MEN1), ATRX chromatin remodeler, and death domain-associated protein genes, which are found in ~40% of sporadic PNETs. PNETs have a low mutational burden, thereby suggesting that other factors likely contribute to their development, including epigenetic regulators. One such epigenetic process, DNA methylation, silences gene transcription via 5'methylcytosine (5mC), and this is usually facilitated by DNA methyltransferase enzymes at CpG-rich areas around gene promoters. However, 5'hydroxymethylcytosine, which is the first epigenetic mark during cytosine demethylation, and opposes the function of 5mC, is associated with gene transcription, although the significance of this remains unknown, as it is indistinguishable from 5mC when conventional bisulfite conversion techniques are solely used. Advances in array-based technologies have facilitated the investigation of PNET methylomes and enabled PNETs to be clustered by methylome signatures, which has assisted in prognosis and discovery of new aberrantly regulated genes contributing to tumourigenesis. This review will discuss the biology of DNA methylation, its role in PNET development, and impact on prognostication and discovery of epigenome-targeted therapies.

Mosaic mutations in genes GNAQ or GNA11 lead to a spectrum of diseases including Sturge-Weber syndrome. Progressive post-natal neurological deterioration led us to seek biologically-targeted therapeutics, and neurovascular calcification to hypothesise that calcium metabolism was pathogenetically central. Calcium signaling was assessed in two cell models expressing GNAQ(c.548G>A,p.(R183Q)) and GNA11(c.547C>T,p.(R183C)). We designed mutant-allele-specific siRNAs and employed these and CRAC channel inhibitor CM4260 to test the biological pathways. We show here that GNAQ/11 mutations cause constitutive and hyperactive GPCR ligand-induced intracellular calcium signaling, which in turn drives extracellular calcium influx and abnormal angiogenesis. Treatment with CM4260 or mutant-specific siRNAs rescues the cellular abnormalities. We go on to demonstrate previously unsuspected disruption of systemic calcium metabolism in 42 patients, despite normally functioning parathyroid, skeletal and renal systems. Critically, levels of serum ionised calcium in the patients cohort as a whole were significantly associated with the presence of seizures (p=0.013) and occurrence of status epilepticus (p=0.017). These data demonstrate that GNAQ/11 mosaicism are fundamentally abnormalities of calcium signaling and handling, that it is related to serious neurological adverse outcomes, and that at cellular level it is druggable. These findings will lead directly to clinical trials.

Background Whole genome sequencing is increasingly being used for the diagnosis of patients with rare diseases. However, the diagnostic yields of many studies, particularly those conducted in a healthcare setting, are often disappointingly low, at 25–30%. This is in part because although entire genomes are sequenced, analysis is often confined to in silico gene panels or coding regions of the genome. Methods We undertook WGS on a cohort of 122 unrelated rare disease patients and their relatives (300 genomes) who had been pre-screened by gene panels or arrays. Patients were recruited from a broad spectrum of clinical specialties. We applied a bioinformatics pipeline that would allow comprehensive analysis of all variant types. We combined established bioinformatics tools for phenotypic and genomic analysis with our novel algorithms (SVRare, ALTSPLICE and GREEN-DB) to detect and annotate structural, splice site and non-coding variants. Results Our diagnostic yield was 43/122 cases (35%), although 47/122 cases (39%) were considered solved when considering novel candidate genes with supporting functional data into account. Structural, splice site and deep intronic variants contributed to 20/47 (43%) of our solved cases. Five genes that are novel, or were novel at the time of discovery, were identified, whilst a further three genes are putative novel disease genes with evidence of causality. We identified variants of uncertain significance in a further fourteen candidate genes. The phenotypic spectrum associated with RMND1 was expanded to include polymicrogyria. Two patients with secondary findings in FBN1 and KCNQ1 were confirmed to have previously unidentified Marfan and long QT syndromes, respectively, and were referred for further clinical interventions. Clinical diagnoses were changed in six patients and treatment adjustments made for eight individuals, which for five patients was considered life-saving. Conclusions Genome sequencing is increasingly being considered as a first-line genetic test in routine clinical settings and can make a substantial contribution to rapidly identifying a causal aetiology for many patients, shortening their diagnostic odyssey. We have demonstrated that structural, splice site and intronic variants make a significant contribution to diagnostic yield and that comprehensive analysis of the entire genome is essential to maximise the value of clinical genome sequencing.

Abstract Disclosure: K.A. English: None. A. Selberherr: None. O.A. Shariq: None. E. O'Neill: None. K.E. Lines: None. R.V. Thakker: None. Background: DNA methylation and hydroxymethylation (5hmC) are epigenetic alterations that can silence or maintain gene transcription, respectively. Pancreatic neuroendocrine tumours (PNETs), which can be hormone-secreting (e.g. insulinomas) or non-functioning (NF-PNETs), have a low mutational burden and are reported to be associated with DNA hypermethylation, but the role of 5hmC in PNETs is unknown. Aim: To determine the patterns of DNA 5hmC in NF-PNETs, insulinomas, and normal pancreatic islets. Methods: NF-PNETs (n=31, mean age 54 ± 13 years (y), 14 males (M), tumour grade (G) G1=15, G2=13, G3=3) and insulinomas (n=21, mean age 50 ± 21y, 9M, G1=17 and G2=4) were collected from patients who did not have a genetic syndrome associated with PNETs together with 14 normal pancreata (mean age 63 ± 12y, 11M). Samples were obtained following ethical approval from the Universities of Oxford and Vienna. DNA 5hmC was assessed by immunohistochemistry (IHC) and MethylationEPIC array (that interrogates 5hmC CpG sites). Multivariate analysis was used to determine variables associated with 5hmC. Transcriptome analysis was undertaken by RNA-Seq. Results: NF-PNETs had reduced 5hmC when compared to insulinomas (p<0.01) and to normal adjacent islets (p<0.001). Thus, 5hmC was ∼3 fold lower in NF-PNETs compared to normal adjacent islets (n=12; p<0.001), whilst 5hmC between insulinomas and normal adjacent islets (n=15) was not significantly different. Multivariate analysis confirmed this finding, with NF-PNETs being associated with lower 5hmC when controlled for patient age, sex and tumour grade (p<0.01). In normal pancreatic tissue 5hmC was ∼2 fold higher in normal islets when compared to exocrine tissue (51 islets assessed in total, range 1-9 islets/sample from 34 fields of 305,866um2 studied; p<0.001). MethylationEPIC array analysis, using DNA from NF-PNETs (n=5), insulinomas (n=4) and normal pancreatic tissue (n=7), revealed NF-PNETs and insulinomas to have a significant decrease in 5hmC compared to normal pancreatic tissue (p<0.001). Furthermore, subtype analysis revealed that NF-PNETs had lower 5hmC compared to insulinomas (0% vs 0.003%, adjusted p<0.08). To determine possible mechanisms underlying these differences in tumour subtype 5hmC, RNA-Seq analysis using NF-PNETs (n=7) and insulinomas (n=6) was performed. This revealed 79 genes to be differentially expressed (adjusted p<0.01), of which 33 were upregulated (including Calcium/Calmodulin Dependent Protein Kinase 2 Gamma (CAMK2G)); and 46 were down-regulated (including Glucagon Like peptide 1 receptor (GLP1R)) in NF-PNETs. Interestingly, CAMK2G and GLP1R are both involved in DNA 5hmC modulation via cAMP. Conclusions: DNA 5hmC, an epigenetic mechanism, is significantly lower in NF-PNETs when compared to normal islets and insulinomas and may involve dysregulation of cAMP-mediated signalling pathways. Presentation: Friday, June 16, 2023