Neurodegeneration in Parkinson’s disease (PD) precedes diagnosis by years. Early neurodegeneration may be reflected in RNA levels and measurable as a biomarker. Here, we present the largest quantification of whole blood linear and circular RNAs (circRNA) in early-stage PD, using RNA sequencing data from two cohorts (PPMI = 259 PD, 161 Controls; ICICLE-PD = 48 PD, 48 Controls). We identified a replicable increase in TMEM252 and LMNB1 gene expression in PD. We identified novel differences in the expression of circRNAs from ESYT2 , BMS1P1 and CCDC9 , and replicated trends of previously reported circRNAs. Overall, using circRNA as a diagnostic biomarker in Parkinson’s did not show any clear improvement over linear RNA. More interestingly, we observed a general reduction in circRNA expression in both PD cohorts, accompanied by an increase in RNASEL expression. This imbalance implicates the activation of an innate antiviral immune response and suggests a previously unknown aspect of circRNA regulation in PD.

Introduction Lynch Syndrome accounts for 2–3% of all colorectal cancers. 1 in 200 individuals are thought to be affected but fewer than 5% of these know they have the condition. NICE guidance in 2017 recommended that all people diagnosed with colorectal cancer are tested for Lynch Syndrome. This study explores the implementation and efficacy of a testing pathway, with a focus on identifying barriers to achieving high levels of test coverage and onward referral when indicated. Methods The initial part of the study is a retrospective analysis of all patients diagnosed with colorectal cancer at a large NHS Trust in a 5-year period that encompassed the implementation and refinement of a fast-track microsatellite instability (MSI) testing pathway. The second part of the study involves ascertaining the viability of using fresh endoscopic biopsies for molecular MSI testing with a multiplex 12-marker polymerase chain reaction (mPCR) assay. Results In the study period there were 1688 new diagnoses of colorectal cancer, of whom 906 (62.7%) underwent an MSI assay. 283 (31.2%) of MSI tests were performed on a fresh endoscopic biopsy. The remainder were performed on a paraffin curl of the endoscopic biopsy containing >30% of pathologically confirmed malignant tissue. The median turn-around time (TAT) from sample submission to result of MSI testing was 13 days (IQR 10, 19). 40 MSI high, BRAF negative cases were identified. 29 (72.5%) of these were referred for assessment by the regional genetic service. DNA analysis was performed in 19 (65.5%) of the referred cases with an MMR mutation identified in 7 cases. A greater proportion of new cases were tested following introduction of reflex MSI testing by the histopathology lab. Analysis of four LS composite biopsy blocks demonstrated a clear MSI signal regardless of tumour content of individual endoscopic biopsies. MSI testing was less likely to occur in frailer patients, advanced stage at presentation and those diagnosed radiologically. Conclusions This study demonstrates a model for fast-track MSI testing to facilitate screening for Lynch Syndrome and to inform surgical and oncological decision making early in the colorectal cancer pathway. Identifying factors that impact negatively on testing coverage and onward referral enables the pathway to be modified to optimise detection of Lynch Syndrome.

Abstract 3-5% of CRCs show microsatellite instability (MSI) caused by germline defects in mismatch repair genes (MMR) predisposing to Lynch syndrome. In addition, 12% of sporadic CRCs show MSI. Currently, MSI is tested using a fragment analysis based assay not suitable for high throughput testing with suboptimal sensitivity and specificity. Knowledge of microsatellite instability affects prognosis (MSI is a positive prognostic factor in stage II CRC), risk stratification (for the affected and at risk relatives in Lynch syndrome), prediction of lymph node involvement (lymph node metastasis is unlikely in stage I MSI positive CRC) and treatment of CRCs (MMR deficient tumours showed observable benefit from PD-1 blocking agent pembrolizumab). For all these important benefits, MSI testing is now recommended for all newly diagnosed CRCs. As a result, development of a high throughput approach is desirable. We have developed and validated a high throughput sequence based MSI assay. In this study, we tested 17 short (7-12bp) mononucleotide markers (previously identified by our team via an in silico analyses of whole genome sequencing data). These 17 markers were able to discriminate between MSI-high (MSI-H) and microsatellite stable (MSS) cases. To define the optimal parameters to discriminate between MSI-H and MSS samples, we tested these 17 markers across a panel of 141 CRC samples. This allowed us to define a scoring scheme for the 17 markers using allelic imbalance based on a linked SNP (called weighted scoring scheme), which achieved 96% sensitivity and 100% specificity. This scoring scheme was then validated using an independent cohort of 70 CRCs without knowing their MSI status. The assay achieved a 100% sensitivity and specificity. We provide here a high throughput tool to detect microsatellite instability that is less costly, uses short mononucleotide markers (eliminating the need to test matched normal tissue) and is validated on formalin fixed paraffin embedded (FFPE) samples (similar to routine samples). The ability to test the microsatellite instability status in all the newly diagnosed CRC cases would have a great clinical impact on prognosis, risk stratification and treatment of CRCs. Citation Format: Ghanim Alhilal, Lisa Redford, Angel Alonso, Sira Moreno, Mark Arends, Anca Oniscu, Ottilia O'Brien, Stephanie Needham, John Burn, Michael Jackson, Mauro Santibanez-Koref. A next generation sequencing based microsatellite instability assay suitable for routine risk stratification in colorectal cancer. [abstract]. In: Proceedings of the AACR Special Conference: Improving Cancer Risk Prediction for Prevention and Early Detection; Nov 16-19, 2016; Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(5 Suppl):Abstract nr A09.

The increase in identification of putative biomarkers and opportunities to develop tailored treatments are due to emergence of omics technologies. Application of pharmacogenetic knowledge with the help of quick and cheap companion diagnostics in the primary care setting is expected to deliver improved treatment and reduced heathcare costs. Warfarin and aspirin are the two most widely prescribed drugs for preventing cardiovascular diseases. Long term aspirin use has also been shown to reduce risk, recurrence and mortality from colorectal cancer. However, they both have narrow therapeutic windows and several genetic polymorphisms have been noted to influence their dose and efficacy. We therefore have launched two collaborative projects: first, to study the genetics of warfarin safety in the Gujarati Indian population and second, to identify further polymorphisms that modulates aspirin’s colorectal cancer chemopreventive efficacy. Understanding the impact of polymorphisms on dose and efficacy for these drugs would lead to development of a combined panel of markers that would predict accurate therapeutic dose with minimal risk for adverse reactions. These markers will be deployed at the point of care settings using a novel handheld genotyping device which will use disposable microfluidic cassettes and silicon nanowires currently developed by QuantuMDx. Results, future work, opportunities and barriers will be examined.

Human HNRNPGT, encoding the protein hnRNP G-T, is one of several autosomal retrogenes derived from RBMX. It has been suggested that HNRNPGT functionally replaces the sex-linked RBMX and RBMY genes during male meiosis. We show here that during normal mouse germ cell development, hnRNP G-T protein is strongly expressed during and after meiosis when proteins expressed from Rbmx or Rbmx-like genes are absent. Amongst these Rbmx-like genes, DNA sequence analyses indicate that two other mouse autosomal Rbmx-derived retrogenes have evolved recently in rodents and one already shows signs of degenerating into a non-expressed pseudogene. In contrast, orthologues of Hnrnpgt are present in all four major groups of placental mammals. The sequence of Hnrnpgt is under considerable positive selection suggesting it performs an important germ cell function in eutherians. To test this, we inactivated Hnrnpgt in ES cells and studied its function during spermatogenesis in chimaeric mice. Although germ cells heterozygous for this targeted allele could produce sperm, they did not contribute to the next generation. Chimaeric mice with a high level of mutant germ cells were infertile with low sperm counts and a high frequency of degenerate seminiferous tubules and abnormal sperm. Chimaeras made from a 1:1 mix of targeted and wild-type ES cell clones transmitted wild-type germ cells only. Our data show that haploinsufficiency of Hnrnpgt results in abnormal sperm production in the mouse. Genetic defects resulting in reduced levels of HNRNPGT could, therefore, be a cause of male infertility in humans.

Human centromeric regions are enriched for segmental duplications, which elsewhere in the genome precipitate both genetic disease and gene formation. Molecular cytogenetic analyses of primate chromosomes have established that centromeres frequently move without altering the surrounding gene order. Recently, the positions of two ancestral centromeres have been mapped to regions of the human genome that are both rich in segmental duplications and are associated with duplication-based clinical phenotypes. This suggests a model for the evolution of euchromatic segmental duplication families involving the localised elevation of recombination rates within the duplication-rich heterochromatin of recently inactivated centromeres, and raises the possibility that the distribution of duplication/deletion syndromes within our genome has been heavily influenced by such events. The relaxation of the heterochromatin environment that must accompany centromere inactivation would also increase the transcriptional activity within previously pericentromeric DNA, increasing the likelihood of chimaeric gene creation through pericentromeric-directed duplication events.