Early-stage idiopathic Parkinson's disease is associated with reduced circular RNA expression.

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 idiopathic 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 PD did not show any clear improvement over linear RNA, minimising its potential clinical utility. 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.

Feasibility and impact of haplogroup matching for mitochondrial replacement treatment.

Mitochondrial replacement technology (MRT) aims to reduce the risk of serious disease in children born to women who carry pathogenic mitochondrial DNA (mtDNA) variants. By transplanting nuclear genomes from eggs of an affected woman to enucleated eggs from an unaffected donor, MRT creates new combinations of nuclear and mtDNA. Based on sets of shared sequence variants, mtDNA is classified into ~30 haplogroups. Haplogroup matching between egg donors and women undergoing MRT has been proposed as a means of reducing mtDNA sequence divergence between them. Here we investigate the potential effect of mtDNA haplogroup matching on clinical delivery of MRT and on mtDNA sequence divergence between donor/recipient pairs. Our findings indicate that haplogroup matching would limit the availability of egg donors such that women belonging to rare haplogroups may have to wait > 4 years for treatment. Moreover, we find that intra-haplogroup sequence variation is frequently within the range observed between randomly matched mtDNA pairs. We conclude that haplogroup matching would restrict the availability of MRT, without necessarily reducing mtDNA sequence divergence between donor/recipient pairs.

Large-scale benchmarking of circRNA detection tools reveals large differences in sensitivity but not in precision.

The detection of circular RNA molecules (circRNAs) is typically based on short-read RNA sequencing data processed using computational tools. Numerous such tools have been developed, but a systematic comparison with orthogonal validation is missing. Here, we set up a circRNA detection tool benchmarking study, in which 16 tools detected more than 315,000 unique circRNAs in three deeply sequenced human cell types. Next, 1,516 predicted circRNAs were validated using three orthogonal methods. Generally, tool-specific precision is high and similar (median of 98.8%, 96.3% and 95.5% for qPCR, RNase R and amplicon sequencing, respectively) whereas the sensitivity and number of predicted circRNAs (ranging from 1,372 to 58,032) are the most significant differentiators. Of note, precision values are lower when evaluating low-abundance circRNAs. We also show that the tools can be used complementarily to increase detection sensitivity. Finally, we offer recommendations for future circRNA detection and validation.

Mismatch repair deficiency testing in Lynch syndrome-associated urothelial tumors.

Introduction: Lynch syndrome-associated cancer develops due to germline pathogenic variants in one of the mismatch repair (MMR) genes, MLH1, MSH2, MSH6 or PMS2. Somatic second hits in tumors cause MMR deficiency, testing for which is used to screen for Lynch syndrome in colorectal cancer and to guide selection for immunotherapy. Both MMR protein immunohistochemistry and microsatellite instability (MSI) analysis can be used. However, concordance between methods can vary for different tumor types. Therefore, we aimed to compare methods of MMR deficiency testing in Lynch syndrome-associated urothelial cancers. Methods: Ninety-seven urothelial (61 upper tract and 28 bladder) tumors diagnosed from 1980 to 2017 in carriers of Lynch syndrome-associated pathogenic MMR variants and their first-degree relatives (FDR) were analyzed by MMR protein immunohistochemistry, the MSI Analysis System v1.2 (Promega), and an amplicon sequencing-based MSI assay. Two sets of MSI markers were used in sequencing-based MSI analysis: a panel of 24 and 54 markers developed for colorectal cancer and blood MSI analysis, respectively. Results: Among the 97 urothelial tumors, 86 (88.7%) showed immunohistochemical MMR loss and 68 were successfully analyzed by the Promega MSI assay, of which 48 (70.6%) were MSI-high and 20 (29.4%) were MSI-low/microsatellite stable. Seventy-two samples had sufficient DNA for the sequencing-based MSI assay, of which 55 (76.4%) and 61 (84.7%) scored as MSI-high using the 24-marker and 54-marker panels, respectively. The concordance between the MSI assays and immunohistochemistry was 70.6% (p = 0.003), 87.5% (p = 0.039), and 90.3% (p = 1.00) for the Promega assay, the 24-marker assay, and the 54-marker assay, respectively. Of the 11 tumors with retained MMR protein expression, four were MSI-low/MSI-high or MSI-high by the Promega assay or one of the sequencing-based assays. Conclusion: Our results show that Lynch syndrome-associated urothelial cancers frequently had loss of MMR protein expression. The Promega MSI assay was significantly less sensitive, but the 54-marker sequencing-based MSI analysis showed no significant difference compared to immunohistochemistry. Data from this study alongside previous studies, suggest that universal MMR deficiency testing of newly diagnosed urothelial cancers, using immunohistochemistry and/or sequencing-based MSI analysis of sensitive markers, offer a potentially useful approach to identification of Lynch syndrome cases.

Constitutional Microsatellite Instability, Genotype, and Phenotype Correlations in Constitutional Mismatch Repair Deficiency.

BACKGROUND & AIMS: Constitutional mismatch repair deficiency (CMMRD) is a rare recessive childhood cancer predisposition syndrome caused by germline mismatch repair variants. Constitutional microsatellite instability (cMSI) is a CMMRD diagnostic hallmark and may associate with cancer risk. We quantified cMSI in a large CMMRD patient cohort to explore genotype-phenotype correlations using novel MSI markers selected for instability in blood. METHODS: Three CMMRD, 1 Lynch syndrome, and 2 control blood samples were genome sequenced to >120× depth. A pilot cohort of 8 CMMRD and 38 control blood samples and a blinded cohort of 56 CMMRD, 8 suspected CMMRD, 40 Lynch syndrome, and 43 control blood samples were amplicon sequenced to 5000× depth. Sample cMSI score was calculated using a published method comparing microsatellite reference allele frequencies with 80 controls. RESULTS: Thirty-two mononucleotide repeats were selected from blood genome and pilot amplicon sequencing data. cMSI scoring using these MSI markers achieved 100% sensitivity (95% CI, 93.6%-100.0%) and specificity (95% CI 97.9%-100.0%), was reproducible, and was superior to an established tumor MSI marker panel. Lower cMSI scores were found in patients with CMMRD with MSH6 deficiency and patients with at least 1 mismatch repair missense variant, and patients with biallelic truncating/copy number variants had higher scores. cMSI score did not correlate with age at first tumor. CONCLUSIONS: We present an inexpensive and scalable cMSI assay that enhances CMMRD detection relative to existing methods. cMSI score is associated with mismatch repair genotype but not phenotype, suggesting it is not a useful predictor of cancer risk.

Is HLA type a possible cancer risk modifier in Lynch syndrome?

Lynch syndrome (LS) is the most common inherited cancer syndrome. It is inherited via a monoallelic germline variant in one of the DNA mismatch repair (MMR) genes. LS carriers have a broad 30% to 80% risk of developing various malignancies, and more precise, individual risk estimations would be of high clinical value, allowing tailored cancer prevention and surveillance. Due to MMR deficiency, LS cancers are characterized by the accumulation of frameshift mutations leading to highly immunogenic frameshift peptides (FSPs). Thus, immune surveillance is proposed to inhibit the outgrowth of MMR-deficient cell clones. Recent studies have shown that immunoediting during the evolution of MMR-deficient cancers leads to a counter-selection of highly immunogenic antigens. The immunogenicity of FSPs is dependent on the antigen presentation. One crucial factor determining antigen presentation is the HLA genotype. Hence, a LS carrier's HLA genotype plays an important role in the presentation of FSP antigens to the immune system, and may influence the likelihood of progression from precancerous lesions to cancer. To address the challenge of clarifying this possibility including diverse populations with different HLA types, we have established the INDICATE initiative (Individual cancer risk by HLA type, http://indicate-lynch.org/), an international network aiming at a systematic evaluation of the HLA genotype as a possible cancer risk modifier in LS. Here we summarize the current knowledge on the role of HLA type in cancer risk and outline future research directions to delineate possible association in the scenario of LS with genetically defined risk population and highly immunogenic tumors.

Detection of Microsatellite Instability in Colonoscopic Biopsies and Postal Urine Samples from Lynch Syndrome Cancer Patients Using a Multiplex PCR Assay.

Identification of mismatch repair (MMR)-deficient colorectal cancers (CRCs) is recommended for Lynch syndrome (LS) screening, and supports targeting of immune checkpoint inhibitors. Microsatellite instability (MSI) analysis is commonly used to test for MMR deficiency. Testing biopsies prior to tumour resection can inform surgical and therapeutic decisions, but can be limited by DNA quantity. MSI analysis of voided urine could also provide much needed surveillance for genitourinary tract cancers in LS. Here, we reconfigure an existing molecular inversion probe-based MSI and BRAF c.1799T > A assay to a multiplex PCR (mPCR) format, and demonstrate that it can sample >140 unique molecules per marker from <1 ng of DNA and classify CRCs with 96−100% sensitivity and specificity. We also show that it can detect increased MSI within individual and composite CRC biopsies from LS patients, and within preoperative urine cell free DNA (cfDNA) from two LS patients, one with an upper tract urothelial cancer, the other an undiagnosed endometrial cancer. Approximately 60−70% of the urine cfDNAs were tumour-derived. Our results suggest that mPCR sequence-based analysis of MSI and mutation hotspots in CRC biopsies could facilitate presurgery decision making, and could enable postal-based screening for urinary tract and endometrial tumours in LS patients.

Interactions between nuclear and mitochondrial SNPs and Parkinson's disease risk.

Interactions between the products of the nuclear and mitochondrial genomes are critical for the function of most eukaryotic cells. Recently the introduction of mitochondrial replacement therapy has raised the question of incompatibilities between mitochondrial and nuclear variants, and their potential influence on the genetic makeup of human populations. Such interactions could also contribute to the variability of the penetrance of pathogenic DNA variants. This led us to investigate the frequencies of combinations of nuclear and mitochondrial SNP alleles (mitonuclear combinations) in healthy individuals (n = 5375) and in a cohort of patients with Parkinson's disease (PD, n = 2210). In the unaffected population, we were not able to find associations between nuclear and mitochondrial variants with a false discovery rate below 0.05 after accounting for multiple testing (i.e., the number of combinations examined). However, in the PD cohort, five combinations surpassed this threshold. Next, after combining both cohorts, we investigated whether these associations were modulated by disease status. All five combinations were significant (p < 10-3 for all tests). These combinations also showed significant evidence for an effect of the interaction between the mitochondrial and nuclear variants on disease risk. Their nuclear components mapped to TBCA, NIBAN3, and GLT25D1 and an uncharacterised intergenic region. In summary, starting from a single cohort design we identified combinations of nuclear and mitochondrial variants affecting PD disease risk.

How Should We Test for Lynch Syndrome? A Review of Current Guidelines and Future Strategies.

International guidelines for the diagnosis of Lynch syndrome (LS) recommend molecular screening of colorectal cancers (CRCs) to identify patients for germline mismatch repair (MMR) gene testing. As our understanding of the LS phenotype and diagnostic technologies have advanced, there is a need to review these guidelines and new screening opportunities. We discuss the barriers to implementation of current guidelines, as well as guideline limitations, and highlight new technologies and knowledge that may address these. We also discuss alternative screening strategies to increase the rate of LS diagnoses. In particular, the focus of current guidance on CRCs means that approximately half of Lynch-spectrum tumours occurring in unknown male LS carriers, and only one-third in female LS carriers, will trigger testing for LS. There is increasing pressure to expand guidelines to include molecular screening of endometrial cancers, the most frequent cancer in female LS carriers. Furthermore, we collate the evidence to support MMR deficiency testing of other Lynch-spectrum tumours to screen for LS. However, a reliance on tumour tissue limits preoperative testing and, therefore, diagnosis prior to malignancy. The recent successes of functional assays to detect microsatellite instability or MMR deficiency in non-neoplastic tissues suggest that future diagnostic pipelines could become independent of tumour tissue.

Constitutional mismatch repair deficiency is the diagnosis in 0.41% of pathogenic NF1/SPRED1 variant negative children suspected of sporadic neurofibromatosis type 1.

PURPOSE: Biallelic germline mismatch repair (MMR) gene pathogenic variants (PVs) cause constitutional MMR deficiency (CMMRD), a highly penetrant childhood cancer syndrome phenotypically overlapping with neurofibromatosis type 1 (NF1). CMMRD testing in suspected NF1 children without NF1/SPRED1 PVs enables inclusion of CMMRD positives into monitoring programs prior to tumor onset. However, testing is associated with potential harms and the prevalence of CMMRD among these children is unknown. METHODS: Using a simple and scalable microsatellite instability (MSI) assay of non-neoplastic leukocyte DNA to detect CMMRD, we retrospectively screened >700 children suspected of sporadic NF1 but lacking NF1/SPRED1 PVs. RESULTS: For three of seven MSI-positive patients germline MMR gene PVs confirmed the diagnosis of CMMRD. Founder variants NM_000535.5(PMS2):c.736_741delinsTGTGTGTGAAG, prevalent in Europe and North America, and NM_000179.2(MSH6):c.10C>G, affecting 1:400 French Canadians, represented two of five PVs. The prevalence of CMMRD was 3/735 (0.41%, 95% confidence interval [CI]: 0.08-1.19%). CONCLUSION: Our empirical data provide reliable numbers for genetic counseling and confirm previous prevalence estimations, on which Care for CMMRD consortium guidelines are based. These advocate CMMRD testing of preselected patients rather than offering reflex testing to all suspected sporadic NF1 children lacking NF1/SPRED1 PVs. The possibility of founder effects should be considered alongside these testing guidelines.

Circulating cell-free mitochondrial DNA levels in Parkinson's disease are influenced by treatment.

Several studies have linked circulating cell-free mitochondrial DNA (ccf-mtDNA) to human disease. In particular, reduced ccf-mtDNA levels in the cerebrospinal fluid (CSF) of both Alzheimer's and Parkinson's disease (PD) patients have raised the hypothesis that ccf-mtDNA could be used as a biomarker for neurodegenerative disease onset and progression. However, how a reduction of CSF ccf-mtDNA levels relates to neurodegeneration remains unclear. Many factors are likely to influence ccf-mtDNA levels, such as concomitant therapeutic treatment and comorbidities. In this study we aimed to investigate these factors, quantifying CSF ccf-mtDNA from the Parkinson's Progression Markers Initiative in 372 PD patients and 159 matched controls at two time points. We found that ccf-mtDNA levels appear significantly reduced in PD cases when compared to matched controls and are associated with cognitive impairment. However, our data indicate that this reduction in ccf-mtDNA is also associated with the commencement, type and duration of treatment. Additionally, we found that ccf-mtDNA levels are associated with comorbidities such as depression and insomnia, however this was only significant if measured in the absence of treatment. We conclude that in PD, similar to reports in HIV and sepsis, comorbidities and treatment can both influence ccf-mtDNA homeostasis, raising the possibility that ccf-mtDNA may be useful as a biomarker for treatment response or the development of secondary phenotypes. Given that, clinically, PD manifests often decades after neurodegeneration begins, predicting who will develop disease is important. Also, identifying patients who will respond to existing treatments or develop secondary phenotypes will have increased clinical importance as PD incidence rises.

A slipped-CAG DNA-binding small molecule induces trinucleotide-repeat contractions in vivo.

In many repeat diseases, such as Huntington's disease (HD), ongoing repeat expansions in affected tissues contribute to disease onset, progression and severity. Inducing contractions of expanded repeats by exogenous agents is not yet possible. Traditional approaches would target proteins driving repeat mutations. Here we report a compound, naphthyridine-azaquinolone (NA), that specifically binds slipped-CAG DNA intermediates of expansion mutations, a previously unsuspected target. NA efficiently induces repeat contractions in HD patient cells as well as en masse contractions in medium spiny neurons of HD mouse striatum. Contractions are specific for the expanded allele, independently of DNA replication, require transcription across the coding CTG strand and arise by blocking repair of CAG slip-outs. NA-induced contractions depend on active expansions driven by MutSß. NA injections in HD mouse striatum reduce mutant HTT protein aggregates, a biomarker of HD pathogenesis and severity. Repeat-structure-specific DNA ligands are a novel avenue to contract expanded repeats.

Sequencing-based microsatellite instability testing using as few as six markers for high-throughput clinical diagnostics.

Microsatellite instability (MSI) testing of colorectal cancers (CRCs) is used to screen for Lynch syndrome (LS), a hereditary cancer-predisposition, and can be used to predict response to immunotherapy. Here, we present a single-molecule molecular inversion probe and sequencing-based MSI assay and demonstrate its clinical validity according to existing guidelines. We amplified 24 microsatellites in multiplex and trained a classifier using 98 CRCs, which accommodates marker specific sensitivities to MSI. Sample classification achieved 100% concordance with the MSI Analysis System v1.2 (Promega) in three independent cohorts, totaling 220 CRCs. Backward-forward stepwise selection was used to identify a 6-marker subset of equal accuracy to the 24-marker panel. Assessment of assay detection limits showed that the 24-marker panel is marginally more robust to sample variables than the 6-marker subset, detecting as little as 3% high levels of MSI DNA in sample mixtures, and requiring a minimum of 10 template molecules to be sequenced per marker for >95% accuracy. BRAF c.1799 mutation analysis was also included to streamline LS testing, with all c.1799T>A variants being correctly identified. The assay, therefore, provides a cheap, robust, automatable, and scalable MSI test with internal quality controls, suitable for clinical cancer diagnostics.

Association of stroke and bleed events in non-valvular atrial fibrillation patients with direct oral anticoagulant prescriptions in NHS England between 2013 and 2016.

Prescription of direct oral anticoagulants (DOAC) compared to warfarin for treating atrial fibrillation patients have increased substantially since their introduction in the England's National Health Service. Assessment of the risk of strokes and bleeds in relation to the large-scale uptake in DOACs compared to warfarin at the clinical commissioning group (CCG) level needs to be carried out. Publicly available- aggregated, CCG level, multi-source health and prescription records data were interrogated to investigate the association between prescription rate of DOACs and stroke/ bleed events during the period of 2013 to 2016. Variability of prescription rates and patient numbers across 208 CCGs were used to infer the effect of DOACs on stroke and bleed risk. Relative risk (RR) and 95% credible intervals (CI) were estimated using Markov chain Monte Carlo approach in JAGS. During the study period, the proportion of DOAC prescriptions increased at an average rate of 122% per annum. DOAC prescription was association with a 50% reduction in ischaemic (RR = 0.48, 95% CI = 0.39, 0.57) and haemorrhagic stroke (RR = 0.50, 95% CI = 0.26-0.77). In contrast, DOAC prescription reached significant association with reduction in gastrointestinal bleeds (RR = 0.86, 95% CI = 0.73-0.98) but not clinically relevant bleeds (RR = 0.95, 95% CI = 0.85-1.05). Sex stratified data showed significant association between DOAC prescription and reduction in haemorrhagic stroke risk (RR = 0.40, 95% CI = 0.28-0.52) and gastrointestinal bleeds (RR = 0.76, 95% CI = 0.63-0.93) in males only. Age stratified data suggested significant association with reduction in risk of both ischaemic and haemorrhagic strokes in patients aged 70 years and above, and reduction in risk of clinically relevant and gastrointestinal bleeds in patients aged 70-79 years only. Publicly available health and prescription data for the English population indicates reduction in stroke and bleed risk in specific age and sex sub-groups with the uptake of DOACs compared to warfarin between 2013 and 2016.

Diverse presentations of cutaneous mosaicism occur in CYLD cutaneous syndrome and may result in parent-to-child transmission.

BACKGROUND: Clusters of rare cylindroma or spiradenoma tumors are a recurrent clinical presentation, yet conventional genetic testing results in individuals with these tumors are frequently normal. OBJECTIVE: To determine if genetic mosaicism accounts for such cases. METHODS: A study of 6 cases from a series of 55 patients who met criteria for diagnostic gene testing for pathogenic CYLD variants over a 5-year period (2012-2017) was performed. A novel genetic assay was used to study DNA from peripheral blood leukocytes and, where possible, matched skin and tumor tissue. RESULTS: Two patients had mosaic pathogenic CYLD variants in both the blood and skin. One of these patients transmitted a pathogenic variant to her daughter, and we report the novel phenotype of a contiguous gene deletion syndrome involving CYLD. Two patients had recurrent pathogenic variants in skin tumors from a single cluster but none detectable in the blood. LIMITATIONS: The remaining 2 patients had clinical features of mosaicism, but these cases were not solved with the assays used because of a lack of access of fresh tumor tissue. CONCLUSION: Genetic mosaicism should be considered in patients presenting with clustered cylindromas, because this may inform genetic testing and counseling of these patients.

An essential role for the Zn2+ transporter ZIP7 in B cell development.

Despite the known importance of zinc for human immunity, molecular insights into its roles have remained limited. Here we report a novel autosomal recessive disease characterized by absent B cells, agammaglobulinemia and early onset infections in five unrelated families. The immunodeficiency results from hypomorphic mutations of SLC39A7, which encodes the endoplasmic reticulum-to-cytoplasm zinc transporter ZIP7. Using CRISPR-Cas9 mutagenesis we have precisely modeled ZIP7 deficiency in mice. Homozygosity for a null allele caused embryonic death, but hypomorphic alleles reproduced the block in B cell development seen in patients. B cells from mutant mice exhibited a diminished concentration of cytoplasmic free zinc, increased phosphatase activity and decreased phosphorylation of signaling molecules downstream of the pre-B cell and B cell receptors. Our findings highlight a specific role for cytosolic Zn2+ in modulating B cell receptor signal strength and positive selection.

A sensitive and scalable microsatellite instability assay to diagnose constitutional mismatch repair deficiency by sequencing of peripheral blood leukocytes.

Constitutional mismatch repair deficiency (CMMRD) is caused by germline pathogenic variants in both alleles of a mismatch repair gene. Patients have an exceptionally high risk of numerous pediatric malignancies and benefit from surveillance and adjusted treatment. The diversity of its manifestation, and ambiguous genotyping results, particularly from PMS2, can complicate diagnosis and preclude timely patient management. Assessment of low-level microsatellite instability in nonneoplastic tissues can detect CMMRD, but current techniques are laborious or of limited sensitivity. Here, we present a simple, scalable CMMRD diagnostic assay. It uses sequencing and molecular barcodes to detect low-frequency microsatellite variants in peripheral blood leukocytes and classifies samples using variant frequencies. We tested 30 samples from 26 genetically-confirmed CMMRD patients, and samples from 94 controls and 40 Lynch syndrome patients. All samples were correctly classified, except one from a CMMRD patient recovering from aplasia. However, additional samples from this same patient tested positive for CMMRD. The assay also confirmed CMMRD in six suspected patients. The assay is suitable for both rapid CMMRD diagnosis within clinical decision windows and scalable screening of at-risk populations. Its deployment will improve patient care, and better define the prevalence and phenotype of this likely underreported cancer syndrome.