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Simple Summary: Tumours of the colon and endometrium (lining of the womb) often have defects in genes that repair our DNA. Identifying these tumours is important for patient treatment and identifying Lynch syndrome (LS), an inherited cancer predisposition. However, the two methods most commonly used to identify these defects often give inconsistent results for endometrial tumours. Here, we investigate these inconsistencies by re-analysing 361 tumour samples from clinical trials with a third assay and establish that they are largely due to false positives in one assay (immunohistochemistry) and false negatives in the other (Promega MSI assay). In addition, the DNA repair defects seem to have a reduced impact within endometrial tumours. However, we find that tumours with defects in a gene called MSH6, especially when associated with LS, are only detected efficiently with immunohistochemistry. This supports current guidelines specifically recommending the use of this method for endometrial tumours. Background/Objectives: Mismatch repair (MMR) deficiency can be indicative of Lynch syndrome (LS) and guide treatment with immune checkpoint inhibitors. Colorectal cancers (CRCs) and endometrial cancers (ECs) are routinely screened to identify LS, primarily using immunohistochemistry (IHC) or microsatellite instability (MSI) testing, but concordance between these methods is variable in ECs. Here, we investigate this variability in 361 ECs from the Ohio OCCPI/OPTEC (n = 196) and Manchester PETALS (n = 165) trials, where concordance between assays differed significantly. Methods: Samples were re-tested using the amplicon-sequencing-based Newcastle MSI assay (NCL_MSI), and analysed with respect to existing IHC, MSI and MLH1 promoter hypermethylation data. Results: NCL_MSI showed consistency with the Ohio results (94% and 97% concordance with IHC and original MSI assays, respectively) and increased concordance within the Manchester cohort from 78% to 86% (MSI) and 84% (IHC). Among discordant Manchester samples, NCL_MSI was significantly associated with MLH1 promoter methylation status (p = 0.0028) and had the highest concordance with methylation, (62/69 samples, 90%), indicating utility as a screening tool in this tumour type. However, tumours with germline MSH6 defects were only detected efficiently with IHC; seven out of eight LS tumours classified as MSS by either MSI assay had isolated MSH6 loss, compared to four out of twelve classified as MSI-H by both (p = 0.028). Furthermore, reduced MSI signal was observed in tumours with isolated MSH6 loss (p = 0.009 Ohio, p = 6.2 × 10−5 Manchester) and in both ECs and CRCs with germline defects, although this only reached significance in CRCs (p = 0.002). Conclusions: These results provide further evidence that ECs with MSH6 loss in particular and LS tumours in general have an attenuated MSI signal, providing support for current guidelines specifically recommending IHC for LS detection and immune checkpoint therapy assessment in EC. [ABSTRACT FROM AUTHOR]

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. Synopsis: Haplogroup‐defining mtDNA sequence variants do not preclude within‐haplogroup variation. Comparison of variant counts between haplogroup matched and random mtDNA pairings indicates that matching would not necessarily reduce sequence divergence between egg donors and women undergoing MRT. Women belonging to rare haplogroups may have to wait several years for a suitable haplogroup matched egg donor.High levels of sequence divergence within many rare haplogroups limits the potential of haplogroup matching to reduce sequence divergence between egg donors and women belonging to rare haplogroups. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A previous literature review found at least 4/9 (>=44.4%) CMMRD-associated ALL were T-ALL [[1]], and 8/88 (9.1%, 95% CI: 0.4-17.1%) childhood T-LBL patients were diagnosed with CMMRD in a consecutive series between 2007 and 2020 [[15]], suggesting an association of CMMRD with T cell lineage malignancies. For example, PCR-fragment length analysis has been shown to be insensitive to MSH6 deficiency in ALL relapse samples [[3]], and detected increased MSI in only 27% of CMMRD haematological malignancies and in 0% of non-neoplastic CMMRD tissues [[9]]. Here, 1/17 (5.9%, 95% CI: 0.2-28.7%) ALL patients with SMN had CMMRD, which is similar to the 14/189 (7.4%, 95% CI: 4.1-12.1%) pediatric non-Hodgkin lymphoma patients with SMN who had CMMRD in another recent study [[16]]. Constitutional mismatch repair deficiency (CMMRD, MIM #276300) is a rare, recessive pediatric and adolescent cancer syndrome, caused by pathogenic variants in a mismatch repair (MMR) gene: I MLH1 i , I MSH2 i , I MSH6 i , or I PMS2 i . [Extracted from the article]

Simple Summary: Lynch syndrome is caused by inherited defects in genes which repair DNA, and predisposes affected individuals to cancers of the bowel, endometrium (uterus lining), and urinary tract, among others. Most of these cancers exhibit a characteristic pattern of DNA instability in Lynch syndrome patients. Here, we adapt an existing DNA instability test for use with small clinical samples, and show that it works with tumour biopsies taken during colonoscopic investigation just as effectively as with larger samples taken during surgery. We also analyse preoperative postal urine samples where DNA recovery is limited, identifying instability in samples from a Lynch syndrome patient with a urinary tract cancer, and a patient with a suspected endometrial cancer. The assay could enable earlier detection of these tumours, benefiting these patients. 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. [ABSTRACT FROM AUTHOR]

Introduction and aims Lynch syndrome (LS) is a common heritable cancer predisposition syndrome. Tumours from patients with LS demonstrate microsatellite instability (MSI-high), a genetic signature detectable by low-cost DNA sequencing assays that is currently utilized in colorectal cancer screening. The utility of MSI assays for LS detection has not been extensively studied in unselected sebaceous skin tumours or duodenal tumours, where currently immunohistochemical (IHC) expression of mismatch repair (MMR) proteins is used. In addition, it has recently been suggested that cutaneous squamous cell carcinoma (cSCC) could be linked to LS; however MSI status has not been studied in patients with early-onset cSCC. Methods We investigated MSI status in archival formalin-fixed paraffin-embedded cases with ethical approval. We examined 110 sebaceous tumours (STs) (45 female patients, 65 male patients; median age 77 years), 50 duodenal tumours (DTs) (28 female patients, 22 male patients; median age 64 years), and 148 cSCCs selected from patients under 50 years of age (69 female patients, 79 male patients; median age 49 years). Immunohistochemistry (IHC) was performed on each MSI-high sample to determine the expression of MMR proteins MLH1, MSH2, MSH6 and PMS2. Samples that passed quality control were included in the analysis. Results A total of 42 of 106 STs (39.6%) were MSI-high. All STs had loss of at least one MMR protein. Overall, 10 of 41 (24%) DTs were MSI-high. All DTs had loss of at least one MMR protein. We found that 12 of 148 (8.1%) cSCCs were MSI-high and 5 of 12 SCCs exhibited loss of at least one MMR protein expression (41%). Conclusions MSI-high status correlated well with loss of MMR protein expression in STs and DTs samples. Future studies will help establish MSI status in tumours initially selected by MMR loss on IHC, informing its use as an adjunct to MMR IHC in LS detection in patients with STs and DTs. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Early diagnosis of Parkinson's disease and mild cognitive impairment is important to enable prompt treatment and improve patient welfare, yet no standard diagnostic test is available. Metabolomics is a powerful tool used to elucidate disease mechanisms and identify potential biomarkers.Objectives: The objective of this study was to use metabolic profiling to understand the pathoetiology of Parkinson's disease and to identify potential disease biomarkers.Methods: This study compared the serological metabolomic profiles of early-stage Parkinson's patients (diagnosed < 12 months) to asymptomatic matched controls using an established array based detection system (DiscoveryHD4™, Metabolon, UK), correlating metabolite levels to clinical measurements of cognitive impairment.Results: A total of 1434 serological metabolites were assessed in early-stage Parkinson's disease cases (n = 41) and asymptomatic matched controls (n = 40). Post-quality control, statistical analysis identified n = 20 metabolites, predominantly metabolites of the fatty acid oxidation pathway, associated with Parkinson's disease and mild cognitive impairment. Receiver operator curve assessment confirmed that the nine fatty acid oxidation metabolites had good predictive accuracy (area under curve = 0.857) for early-stage Parkinson's disease and mild cognitive impairment (area under curve = 0.759).Conclusions: Our study indicates that fatty acid oxidation may be an important component in the pathophysiology of Parkinson's disease and may have potential as a diagnostic biomarker for disease onset and mild cognitive impairment. © 2017 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Somatic mutations in mononucleotide repeats are commonly used to assess the mismatch repair status of tumours. Current tests focus on repeats with a length above 15bp, which tend to be somatically more unstable than shorter ones. These longer repeats also have a substantially higher PCR error rate, and tests that use capillary electrophoresis for fragment size analysis often require expert interpretation. In this communication, we present a panel of 17 short repeats (length 7–12bp) for sequence-based microsatellite instability (MSI) testing. Using a simple scoring procedure that incorporates the allelic distribution of the mutant repeats, and analysis of two cohort of tumours totalling 209 samples, we show that this panel is able to discriminate between MMR proficient and deficient tumours, even when constitutional DNA is not available. In the training cohort, the method achieved 100% concordance with fragment analysis, while in the testing cohort, 4 discordant samples were observed (corresponding to 97% concordance). Of these, 2 showed discrepancies between fragment analysis and immunohistochemistry and one was reclassified after re-testing using fragment analysis. These results indicate that our approach offers the option of a reliable, scalable routine test for MSI. [ABSTRACT FROM AUTHOR]

Background: Circular RNAs (circRNAs) are predominantly derived from protein coding genes, and some can act as microRNA sponges or transcriptional regulators. Changes in circRNA levels have been identified during human development which may be functionally important, but lineage-specific analyses are currently lacking. To address this, we performed RNAseq analysis of human embryonic stem (ES) cells differentiated for 90 days towards 3D laminated retina. Results: A transcriptome-wide increase in circRNA expression, size, and exon count was observed, with circRNA levels reaching a plateau by day 45. Parallel statistical analyses, controlling for sample and locus specific effects, identified 239 circRNAs with expression changes distinct from the transcriptome-wide pattern, but these all also increased in abundance over time. Surprisingly, circRNAs derived from long non-coding RNAs (lncRNAs) were found to account for a significantly larger proportion of transcripts from their loci of origin than circRNAs from coding genes. The most abundant, circRMST:E12-E6, showed a > 100X increase during differentiation accompanied by an isoform switch, and accounts for > 99% of RMST transcripts in many adult tissues. The second most abundant, circFIRRE:E10-E5, accounts for > 98% of FIRRE transcripts in differentiating human ES cells, and is one of 39 FIRRE circRNAs, many of which include multiple unannotated exons. Conclusions: Our results suggest that during human ES cell differentiation, changes in circRNA levels are primarily globally controlled. They also suggest that RMST and FIRRE, genes with established roles in neurogenesis and topological organisation of chromosomal domains respectively, are processed as circular lncRNAs with only minor linear species. [ABSTRACT FROM AUTHOR]

Regular aspirin use is associated with reduced risk of colorectal cancer (CRC). Variation in aspirin’s chemoprevention efficacy has been attributed to the presence of single nucleotide polymorphisms (SNPs). We conducted a meta-analysis using two large population-based case-control datasets, the UK-Leeds Colorectal Cancer Study Group and the NIH-Colon Cancer Family Registry, having a combined total of 3325 cases and 2262 controls. The aim was to assess 42 candidate SNPs in 15 genes whose association with colorectal cancer risk was putatively modified by aspirin use, in the literature. Log odds ratios (ORs) and standard errors were estimated for each dataset separately using logistic regression adjusting for age, sex and study site, and dataset-specific results were combined using random effects meta-analysis. Meta-analysis showed association between SNPs rs6983267, rs11694911 and rs2302615 with CRC risk reduction (All P<0.05). Association for SNP rs6983267 in the CCAT2 gene only was noteworthy after multiple test correction (P = 0.001). Site-specific analysis showed association between SNPs rs1799853 and rs2302615 with reduced colon cancer risk only (P = 0.01 and P = 0.004, respectively), however neither reached significance threshold following multiple test correction. Meta-analysis of SNPs rs2070959 and rs1105879 in UGT1A6 gene showed interaction between aspirin use and CRC risk (Pinteraction = 0.01 and 0.02, respectively); stratification by aspirin use showed an association for decreased CRC risk for aspirin users having a wild-type genotype (rs2070959 OR = 0.77, 95% CI = 0.68–0.86; rs1105879 OR = 0.77 95% CI = 0.69–0.86) compared to variant allele cariers. The direction of the interaction however is in contrast to that published in studies on colorectal adenomas. Both SNPs showed potential site-specific interaction with aspirin use and colon cancer risk only (Pinteraction = 0.006 and 0.008, respectively), with the direction of association similar to that observed for CRC. Additionally, they showed interaction between any non-steroidal anti-inflammatory drugs (including aspirin) use and CRC risk (Pinteraction = 0.01 for both). All gene x environment (GxE) interactions however were not significant after multiple test correction. Candidate gene investigation indicated no evidence of GxE interaction between genetic variants in genes involved in aspirin pathways, regular aspirin use and colorectal cancer risk. [ABSTRACT FROM AUTHOR]

Response to 'Cutaneous squamous cell carcinoma is associated with Lynch syndrome: widening the spectrum of Lynch syndrome-associated tumours' It should also be noted that tumour MMR deficiency alone does not prove a causative link between LS and cSCC as the presence of a heterozygous pathogenic variant in all LS patient cells makes a secondary breakdown in MMR more likely in any tumour. Dear Editor, We read with interest the recent paper by Ykema I et al i .1 suggesting that cutaneous squamous cell carcinoma (cSCC) may be part of the Lynch syndrome (LS) tumour spectrum. [Extracted from the article]

Purpose: We aimed to achieve a retrospective molecular diagnosis by applying state-of-the-art genomic sequencing methods to past patients with T-B+NK+ severe combined immunodeficiency (SCID). We included identification of copy number variations (CNVs) by whole exome sequencing (WES) using the CNV calling method ExomeDepth to detect gene alterations for which routine Sanger sequencing analysis is not suitable, such as large heterozygous deletions. Methods: Of a total of 12 undiagnosed patients with T-B+NK+ SCID, we analyzed eight probands by WES, using GATK to detect single nucleotide variants (SNVs) and small insertions and deletions (INDELs) and ExomeDepth to detect CNVs. Results: We found heterozygous single- or multi-exon deletions in IL7R, a known disease gene for autosomal recessive T-B+NK+ SCID, in four families (seven patients). In three families (five patients), these deletions coexisted with a heterozygous splice site or nonsense mutation elsewhere in the same gene, consistent with compound heterozygosity. In our cohort, about a quarter of T-B+NK+ SCID patients (26%) had such compound heterozygous IL7R deletions. Conclusions: We show that heterozygous IL7R exon deletions are common in T-B+NK+ SCID and are detectable by WES. They should be considered if Sanger sequencing fails to detect homozygous or compound heterozygous IL7R SNVs or INDELs. [ABSTRACT FROM AUTHOR]

Summary The brain is made up of trillions of synaptic connections that together form neural networks needed for normal brain function and behavior. SLM2 is a member of a conserved family of RNA binding proteins, including Sam68 and SLM1, that control splicing of Neurexin1-3 pre-mRNAs. Whether SLM2 affects neural network activity is unknown. Here, we find that SLM2 levels are maintained by a homeostatic feedback control pathway that predates the divergence of SLM2 and Sam68. SLM2 also controls the splicing of Tomosyn2, LysoPLD/ATX, Dgkb, Kif21a, and Cask , each of which are important for synapse function. Cortical neural network activity dependent on synaptic connections between SLM2-expressing-pyramidal neurons and interneurons is decreased in Slm2 -null mice. Additionally, these mice are anxious and have a decreased ability to recognize novel objects. Our data reveal a pathway of SLM2 homeostatic auto-regulation controlling brain network activity and behavior. [ABSTRACT FROM AUTHOR]

The article examines the role of human developmental biology resource (HDBR) expression for studying prenatal human brain development. Topics include obtaining of human embryonic and fetal tissues from the medical research council (MRC)/Wellcome-Trust funded HDBR, carrying out of single nucleotide polymorphisms (SNP) genotyping according to the Illumina Infinium LCG Quad Assay protocol; quantifying of DNA was quantified on the QuBit system; and extraction of RNA from human embryonic.

The single nucleotide polymorphism (SNP) rs835487 is associated with hip osteoarthritis (OA) at the genome-wide significance level and is located within CHST11, which codes for carbohydrate sulfotransferase 11. This enzyme post-translationally modifies proteoglycan prior to its deposition in the cartilage extracellular matrix. Using bioinformatics and experimental analyses, our aims were to characterise the rs835487 association signal and to identify the causal functional variant/s. Database searches revealed that rs835487 resides within a linkage disequilibrium (LD) block of only 2.7 kb and is in LD (r2 ≥ 0.8) with six other SNPs. These are all located within intron 2 of CHST11, in a region that has predicted enhancer activity and which shows a high degree of conservation in primates. Luciferase reporter assays revealed that of the seven SNPs, rs835487 and rs835488, which have a pairwise r2 of 0.962, are the top functional candidates; the haplotype composed of the OA-risk conferring G allele of rs835487 and the corresponding T allele of rs835488 (the G-T haplotype) demonstrated significantly different enhancer activity relative to the haplotype composed of the non-risk A allele of rs835487 and the corresponding C allele of rs835488 (the A-C haplotype) (p < 0.001). Electrophoretic mobility shift assays and supershifts identified several transcription factors that bind more strongly to the risk-conferring G and T alleles of the two SNPs, including SP1, SP3, YY1 and SUB1. CHST11 was found to be upregulated in OA versus non-OA cartilage (p < 0.001) and was expressed dynamically during chondrogenesis. Its expression in adult cartilage did not however correlate with rs835487 genotype. Our data demonstrate that the OA susceptibility is mediated by differential protein binding to the alleles of rs835487 and rs835488, which are located within an enhancer whose target may be CHST11 during chondrogenesis or an alternative gene. [ABSTRACT FROM AUTHOR]

Recent reports have questioned the accepted dogma that mammalian mitochondrial DNA (mtDNA) is strictly maternally inherited. In humans, the argument hinges on detecting a signature of inter-molecular recombination in mtDNA sequences sampled at the population level, inferring a paternal source for the mixed haplotypes. However, interpreting these data is fraught with difficulty, and direct experimental evidence is lacking. Using extreme-high depth mtDNA re-sequencing up to ~1.2 million-fold coverage, we find no evidence that paternal mtDNA haplotypes are transmitted to offspring in humans, thus excluding a simple dilution mechanism for uniparental transmission of mtDNA present in all healthy individuals. Our findings indicate that an active mechanism eliminates paternal mtDNA which likely acts at the molecular level. [ABSTRACT FROM AUTHOR]

Inherited ataxias are difficult to diagnose genetically. Pyle et al. use whole-exome sequencing to provide a likely molecular diagnosis in 14 of 22 families with ataxia. The approach reveals de novo mutations, broadens the phenotype of other disease genes, and is equally effective in young and older-onset patients.Inherited ataxias are clinically and genetically heterogeneous, and a molecular diagnosis is not possible in most patients. Having excluded common sporadic, inherited and metabolic causes, we used an unbiased whole exome sequencing approach in 35 affected individuals, from 22 randomly selected families of white European descent. We defined the likely molecular diagnosis in 14 of 22 families (64%). This revealed de novo dominant mutations, validated disease genes previously described in isolated families, and broadened the clinical phenotype of known disease genes. The diagnostic yield was the same in both young and older-onset patients, including sporadic cases. We have demonstrated the impact of exome sequencing in a group of patients notoriously difficult to diagnose genetically. This has important implications for genetic counselling and diagnostic service provision. [ABSTRACT FROM AUTHOR]

End-stage renal disease (ESRD) presenting in a familial autosomal dominant pattern points to an underlying monogenic cause. Nail-patella syndrome (NPS) is an autosomal dominant disorder that may lead to ESRD caused by mutations in the transcription factor LMX1B. Renal-limited forms of this disease, termed nail-patella-like renal disease (NPLRD), and LMX1B nephropathy have recently been described. We report a large family, from the North East of England, with seven affected members with varying phenotypes of renal disease, ranging from ESRD at 28 years of age to microscopic haematuria and proteinuria and relatively preserved renal function. In this family, there were no extra-renal manifestations to suggest NPS. Genome-wide linkage studies and inheritance by descent (IBD) suggested disease loci on Chromosome 1 and 9. Whole exome sequencing (WES) analysis identified a novel sequence variant (p.R249Q) in the LMX1B gene in each of the three samples submitted, which was confirmed using Sanger sequencing. The variant segregated with the disease in all affected individuals. In silico modelling revealed that R249 is putatively located in close proximity to the DNA phosphoskeleton, supporting a role for this residue in the interaction between the LMX1B homeodomain and its target DNA. WES and analysis of potential target genes, including CD2AP, NPHS2, COL4A3, COL4A4 and COL4A5, did not reveal any co-inherited pathogenic variants. In conclusion, we confirm a novel LMX1B mutation in a large family with an autosomal dominant pattern of nephropathy. This report confirms that LMX1B mutations may cause a glomerulopathy without extra-renal manifestations. A molecular genetic diagnosis of LMX1B nephropathy thus provides a definitive diagnosis, prevents the need for renal biopsies and allows at risk family members to be screened. [ABSTRACT FROM AUTHOR]

Motivation: During the past 4 years, whole-exome sequencing has become a standard tool for finding rare variants causing Mendelian disorders. In that time, there has also been a proliferation of both sequencing platforms and approaches to analyse their output. This requires approaches to assess the performance of different methods. Traditionally, criteria such as comparison with microarray data or a number of known polymorphic sites have been used. Here we expand such approaches, developing a maximum likelihood framework and using it to estimate the sensitivity and specificity of whole-exome sequencing data.Results: Using whole-exome sequencing data for a panel of 19 individuals, we show that estimated sensitivity and specificity are similar to those calculated using microarray data as a reference. We explore the effect of frequency misspecification arising from using an inappropriately selected population and find that, although the estimates are affected, the rankings across procedures remain the same.Availability and implementation: An implementation using Perl and R can be found at busso.ncl.ac.uk (Username: igm101; Password: Z1z1nts).Contact: Darren.Houniet@ogt.com; mauro.santibanez-koref@newcastle.ac.uk [ABSTRACT FROM PUBLISHER]

Identifying the genetic basis for mitochondrial diseases is technically challenging given the size of the mitochondrial proteome and the heterogeneity of disease presentations. Using next-generation exome sequencing, we identified in a patient with severe combined mitochondrial respiratory chain defects and corresponding perturbation in mitochondrial protein synthesis, a homozygous p.Arg323Gln mutation in TRIT1. This gene encodes human tRNA isopentenyltransferase, which is responsible for i6A37 modification of the anticodon loops of a small subset of cytosolic and mitochondrial tRNAs. Deficiency of i6A37 was previously shown in yeast to decrease translational efficiency and fidelity in a codon-specific manner. Modelling of the p.Arg323Gln mutation on the co-crystal structure of the homologous yeast isopentenyltransferase bound to a substrate tRNA, indicates that it is one of a series of adjacent basic side chains that interact with the tRNA backbone of the anticodon stem, somewhat removed from the catalytic center. We show that patient cells bearing the p.Arg323Gln TRIT1 mutation are severely deficient in i6A37 in both cytosolic and mitochondrial tRNAs. Complete complementation of the i6A37 deficiency of both cytosolic and mitochondrial tRNAs was achieved by transduction of patient fibroblasts with wild-type TRIT1. Moreover, we show that a previously-reported pathogenic m.7480A>G mt-tRNASer(UCN) mutation in the anticodon loop sequence A36A37A38 recognised by TRIT1 causes a loss of i6A37 modification. These data demonstrate that deficiencies of i6A37 tRNA modification should be considered a potential mechanism of human disease caused by both nuclear gene and mitochondrial DNA mutations while providing insight into the structure and function of TRIT1 in the modification of cytosolic and mitochondrial tRNAs. [ABSTRACT FROM AUTHOR]

A genetic deficit mediated by SNP rs143383 that leads to reduced expression of GDF5 is strongly associated with large-joint osteoarthritis. We speculated that this deficit could be attenuated by the application of exogenous GDF5 protein and as a first step we have assessed what effect such application has on primary osteoarthritis chondrocyte gene expression. Chondrocytes harvested from cartilage of osteoarthritic patients who had undergone joint replacement were cultured with wildtype recombinant mouse and human GDF5 protein. We also studied variants of GDF5, one that has a higher affinity for the receptor BMPR-IA and one that is insensitive to the GDF5 antagonist noggin. As a positive control, chondrocytes were treated with TGF-β1. Chondrocytes were cultured in monolayer and micromass and the expression of genes coding for catabolic and anabolic proteins of cartilage were measured by quantitative PCR. The expression of the GDF5 receptor genes and the presence of their protein products was confirmed and the ability of GDF5 signal to translocate to the nucleus was demonstrated by the activation of a luciferase reporter construct. The capacity of GDF5 to elicit an intracellular signal in chondrocytes was demonstrated by the phosphorylation of intracellular Smads. Chondrocytes cultured with TGF-β1 demonstrated a consistent down regulation of MMP1, MMP13 and a consistent upregulation of TIMP1 and COL2A1 with both culture techniques. In contrast, chondrocytes cultured with wildtype GDF5, or its variants, did not show any consistent response, irrespective of the culture technique used. Our results show that osteoarthritis chondrocytes do not respond in a predictable manner to culture with exogenous GDF5. This may be a cause or a consequence of the osteoarthritis disease process and will need to be surmounted if treatment with exogenous GDF5 is to be advanced as a potential means to overcome the genetic deficit conferring osteoarthritis susceptibility at this gene. [ABSTRACT FROM AUTHOR]

Abstract: The molecular diagnosis of adult-onset autosomal recessive cerebellar ataxias remains challenging because of genetic heterogeneity. However, recently developed molecular genetic techniques will potentially revolutionize the diagnostic approach. Here we set out to define the genetic basis of the ataxia in two brothers with no molecular diagnosis. Clinical evaluation was followed by whole-exome second-generation sequencing and comparative genomic hybridization to determine the diagnosis. Whole-exome sequencing identified a hemizygous novel spastic ataxia of Charlevoix-Saguenay ( SACS) stop-codon mutation in both brothers (c.13048G→T, p.E4350*) that was present in the mother, but not the father. Comparative genomic hybridization revealed a 0.7-Mb deletion on chromosome 13q12.12 in both brothers, which included SACS and was heterozygous in the asymptomatic father. The milder phenotype, caused by a whole-gene deletion and a stop-codon mutation in SACS, indicates a loss-of-function mechanism in late-onset autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS), and illustrates the importance of chromosomal rearrangements in the investigation of adult-onset ataxia. [ABSTRACT FROM AUTHOR]

In 2001, we described an autosomal dominant myopathy characterized by neuromuscular ventilatory failure in ambulant patients. Here we describe the underlying genetic basis for the disorder, and we define the neuromuscular, respiratory and radiological phenotype in a study of 31 mutation carriers followed for up to 31 years. A combination of genome-wide linkage and whole exome sequencing revealed the likely causal genetic variant in the titin (TTN) gene (g.274375T>C; p.Cys30071Arg) within a shared haplotype of 2.93 Mbp on chromosome 2. This segregated with the phenotype in 21 individuals from the original family, nine subjects in a second family with the same highly selective pattern of muscle involvement on magnetic resonance imaging and a third familial case with a similar phenotype. Comparing the mutation carriers revealed novel features not apparent in our original report. The clinical presentation included predominant distal, proximal or respiratory muscle weakness. The age of onset was highly variable, from early adulthood, and including a mild phenotype in advanced age. Muscle weakness was earlier onset and more severe in the lower extremities in nearly all patients. Seven patients also had axial muscle weakness. Respiratory function studies demonstrated a gradual deterioration over time, reflecting the progressive nature of this condition. Cardiomyopathy was not present in any of our patients despite up to 31 years of follow-up. Magnetic resonance muscle imaging was performed in 21 affected patients and revealed characteristic abnormalities with semitendinosus involvement in 20 of 21 patients studied, including 3 patients who were presymptomatic. Diagnostic muscle histopathology most frequently revealed eosinophilic inclusions (inclusion bodies) and rimmed vacuoles, but was non-specific in a minority of patients. These findings have important clinical implications. This disease should be considered in patients with adult-onset proximal or distal myopathy and early respiratory failure, even in the presence of non-specific muscle pathology. Muscle magnetic resonance imaging findings are characteristic and should be considered as an initial investigation, and if positive should prompt screening for mutations in TTN. With 363 exons, screening TTN presented a major challenge until recently. However, whole exome sequencing provides a reliable cost-effective approach, providing the gene of interest is adequately captured. [ABSTRACT FROM PUBLISHER]

There is emerging evidence that people with successfully treated HIV infection age prematurely, leading to progressive multi-organ disease, but the reasons for this are not known. Here we show that patients treated with commonly used nucleoside analog anti-retroviral drugs progressively accumulate somatic mitochondrial DNA (mtDNA) mutations, mirroring those seen much later in life caused by normal aging. Ultra-deep re-sequencing by synthesis, combined with single-cell analyses, suggests that the increase in somatic mutation is not caused by increased mutagenesis but might instead be caused by accelerated mtDNA turnover. This leads to the clonal expansion of preexisting age-related somatic mtDNA mutations and a biochemical defect that can affect up to 10% of cells. These observations add weight to the role of somatic mtDNA mutations in the aging process and raise the specter of progressive iatrogenic mitochondrial genetic disease emerging over the next decade. [ABSTRACT FROM AUTHOR]

We present a new statistical method to identify genes in which one or more variants influence quantitative traits. We use the Genetic Analysis Workshop 17 (GAW17) data set of unrelated individuals as a test of the method on the raw GAW17 phenotypes and on residuals after fitting linear models to individual-based covariates. By performing appropriate randomization tests, we found many significant results for a proportion of the genes that contain variants that directly contribute to disease but that have an increased type I error for analyses of raw phenotypes. Power calculations show that our methods have the ability to reliably identify a subset of the loci contributing to disease. When we applied our method to derived phenotypes, we removed many false positives, giving appropriate type I error rates at little cost to power. The correlation between genome-wide heterozygosity and the value of the trait Q1 appears to drive much of the type I error in this data set. [ABSTRACT FROM AUTHOR]

Germline defects in the MLH1 gene are associated with Lynch syndrome. A substantial proportion of these mutations leads to premature termination codons and can induce nonsense mediated decay (NMD) of the corresponding transcript. Resulting allelic expression differences represent a fast and inexpensive method to identify patients carrying MLH1 mutations. In patients and controls, we show that allelic expression imbalance (AEI) can be readily detected in RNA extracted from whole blood from patients carrying mutations expected to elicit NMD using mass spectrometry. Mutations closer to the 5′ end of the gene tend to show smaller imbalances. AEI can also be detected in normal controls. Analysis of allelic expression in controls and individuals with mutations not expected to exhibit NMD revealed that MLH1 expression is influenced by sequence variation acting in cis. A maximum likelihood framework was used to identify two SNPs, rs1799977 (c.655G>A; p.I219V) and rs1800734 (c.-93 G>A) that are independently associated with expression. These influences are, however, small compared to the differences associated with pathological variants. [ABSTRACT FROM AUTHOR]

Alkyltransferase-like proteins (ATLs) share functional motifs with the cancer chemotherapy target O6-alkylguanine-DNA alkyltransferase (AGT) and paradoxically protect cells from the biological effects of DNA alkylation damage, despite lacking the reactive cysteine and alkyltransferase activity of AGT. Here we determine Schizosaccharomyces pombe ATL structures without and with damaged DNA containing the endogenous lesion O6-methylguanine or cigarette-smoke-derived O6-4-(3-pyridyl)-4-oxobutylguanine. These results reveal non-enzymatic DNA nucleotide flipping plus increased DNA distortion and binding pocket size compared to AGT. Our analysis of lesion-binding site conservation identifies new ATLs in sea anemone and ancestral archaea, indicating that ATL interactions are ancestral to present-day repair pathways in all domains of life. Genetic connections to mammalian XPG (also known as ERCC5) and ERCC1 in S. pombe homologues Rad13 and Swi10 and biochemical interactions with Escherichia coli UvrA and UvrC combined with structural results reveal that ATLs sculpt alkylated DNA to create a genetic and structural intersection of base damage processing with nucleotide excision repair. [ABSTRACT FROM AUTHOR]

Background: Concurrent tumors can be synchronous, independently derived, non-metastatic tumors or metastatic tumors. The prognosis and clinical management of patients with these different concurrent tumor types are different. Methods: DNA from normal and tumor tissues of 62 patients with synchronous endometrial and ovarian, bilateral ovarian, or endometrial and bilateral ovarian tumors was analyzed for loss of heterozygosity and microsatellite instability using eight polymorphic microsatellite markers at loci frequently deleted in ovarian and/or endometrial cancers. A statistical algorithm was designed to assess the clonal relationship between the tumors. Results: The original histopathology reports classified 26 (42%) case patients with single primary tumors and related metastatic lesions and 21 (34%) with independent primary tumors; 15 (24%) were unclassified. Genetic data identified 35 (56%) case patients with single primary tumors and related metastatic lesions, 18 (29%) with independent primary tumors, and nine (15%) that could not be typed. Excluding case patients with histopathology reports for which a clonal relationship was uncertain or was not reported, there was 53% concordance between genetic and histopathology diagnoses. Increasing the stringency of the statistical analysis increased the number of uncertain diagnoses but did not affect the proportion of discordant genetic and histologic diagnoses. Conclusions: We have developed a rapid and robust combined genetic and statistical method to establish whether multiple tumors from the same patient represent distinct primary tumors or whether they are clonally related and therefore metastatic. For the majority of case patients, histopathology reports and genetic analyses were in agreement and diagnostic confidence was improved. Importantly, in approximately one-fourth of all case patients, genetic and histopathologic analyses suggested alternative diagnoses. The results suggest that genetic analysis has implications for clinical management and can be performed rapidly as a diagnostic test with paraffin-embedded tissues. [ABSTRACT FROM AUTHOR]

Simple Summary: Carriers of Lynch syndrome (LS) have an increased risk for several types of tumour, in particular bowel and uterine cancers. LS is caused by inheritance of a faulty mismatch repair (MMR) gene, and, in accordance with clinical guidelines, bowel cancers are tested for MMR deficiency to screen for LS. We review the significant barriers to following these guidelines in routine practice, and discuss guideline limitations. We also look at the advances in our knowledge and technology that may address some of these barriers and limitations. We further discuss additional LS screening strategies, in particular MMR deficiency testing of other tumour types and normal tissues to identify LS gene carriers. 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. [ABSTRACT FROM AUTHOR]