Utilising Uracil DNA Glycosylase to detect the presence of 5-Methylcytosine

DNA is regularly subjected to endogenous and exogenous reagents that cause mutations that can be detrimental to a cell if they are not repaired. One class of enzymes responsible for DNA repair is the family of DNA glycosylases and their role is to remove damaged bases. Uracil DNA Glycosylase (UDG) is a member of this family and is highly specific, removing only uracil, an RNA base, from DNA. Uracil arises in DNA through misincorporation of deoxyuridine monophosphate (dUMP) creating an A.U base pair, or through deamination of cytosine resulting in a G.U base pair. Though UDG acts on A.U pairs, this is not it’s primarily role as A.U pairings are not mutagenic. However the G.U mispair is highly mutagenic and leads to a G.C to A.T transition on subsequent rounds of replication. UDG only reacts with uracil and has no activity at thymine since the 5-methyl group on the base is excluded from the active site. This thesis examines mutants of UDG that can cleave cytosine but not 5-methylcytosine. Methylation of cytosine at CpG sites leads to gene silencing and is an important epigenetic signal. Knowing the methylation state of cytosines will therefore be important for understanding gene control and may be beneficial for treating many diseases. The most common method for detecting cytosine methylation uses a bisulphite reaction followed by normal DNA sequencing methods. This process has several drawbacks and the aim of this work is to create an enzyme that is capable of distinguishing between5-methylcytosine and cytosine. It has been reported that mutation of a critical asparagine in UDG to an aspartate allows the enzyme to accommodate cytosine into its active site; generating a cytosine DNA glycosylase (CDG). Using the natural ability of UDG to distinguish between uracil and thymine due to the presence of the 5-methyl group, we hypothesised that the mutant enzyme should be able to discriminate between5-methylcytosine and cytosine, which differ by the presence or absence of a methyl group in the same position. E. coli and human CDGs were prepared and their ability to remove cytosine or 5-methylcytosine examined when placed in different sequence contexts. hCDG was generated through complete gene synthesis of hUDG followed by the N204D mutation. The corresponding mutation in E.coli (N123D) generates a highly cytotoxic enzyme that cannot even be cloned in pUC19. As L191 aids base flipping, mutation to alanine (L191A) renders the enzyme inactive; activity can then be rescued using a bulky synthetic nucleoside that occupies the base pair and forces the target base into an extrahelical conformation. The L191A mutation was followed by N123D to generate an expressible and functional eCDG, denoted eCYDG. We demonstrate that these mutants have cytosine glycosylase activity when the cytosine is mispaired or unpaired, but not when paired with guanine, and show no activity against5-methylcytosine in any context. The activity of these CDGs varies with the stability of the base pair, with the fastest cleavage rates being obtained with the least stable base pairs, and also varies with the local sequence context. As CDGs are able to discriminate between cytosine and 5-methylcytosine we began development of a real-time PCR assay for detection of 5-methylcytosine. This employed a hexaethylene glycol (HEG) linker opposite the target cytosine, as this produces one of the fastest cleavage rates and cannot be read by a DNA polymerase

Association between TCF7L2 gene polymorphisms and susceptibility to Type 2 Diabetes Mellitus: a large Human Genome Epidemiology (HuGE) review and meta-analysis

<p>Abstract</p> <p>Background</p> <p>Transcription factor 7-like 2 (<it>TCF7L2</it>) has been shown to be associated with type 2 diabetes mellitus (T2MD) in multiple ethnic groups in the past two years, but, contradictory results were reported for Chinese and Pima Indian populations. The authors then performed a large meta-analysis of 36 studies examining the association of type 2 diabetes mellitus (T2DM) with polymorphisms in the <it>TCF7L2 </it>gene in various ethnicities, containing rs7903146 C-to-T (IVS3C>T), rs7901695 T-to-C (IVS3T>C), a rs12255372 G-to-T (IVS4G>T), and rs11196205 G-to-C (IVS4G>C) polymorphisms and to evaluate the size of gene effect and the possible genetic mode of action.</p> <p>Methods</p> <p>Literature-based searching was conducted to collect data and three methods, that is, fixed-effects, random-effects and Bayesian multivariate mete-analysis, were performed to pool the odds ratio (<it>OR</it>). Publication bias and study-between heterogeneity were also examined.</p> <p>Results</p> <p>The studies included 35,843 cases of T2DM and 39,123 controls, using mainly primary data. For T2DM and IVS3C>T polymorphism, the Bayesian <it>OR </it>for TT homozygotes and TC heterozygotes versus CC homozygote was 1.968 (95% credible interval (<it>CrI</it>): 1.790, 2.157), 1.406 (95% <it>CrI</it>: 1.341, 1.476), respectively, and the population attributable risk (PAR) for the TT/TC genotypes of this variant is 16.9% for overall. For T2DM and IVS4G>T polymorphism, TT homozygotes and TG heterozygotes versus GG homozygote was 1.885 (95%<it>CrI</it>: 1.698, 2.088), 1.360 (95% <it>CrI</it>: 1.291, 1.433), respectively. Four <it>OR</it>s among these two polymorphisms all yielded significant between-study heterogeneity (P < 0.05) and the main source of heterogeneity was ethnic differences. Data also showed significant associations between T2DM and the other two polymorphisms, but with low heterogeneity (<it>P </it>> 0.10). Pooled <it>OR</it>s fit a codominant, multiplicative genetic model for all the four polymorphisms of <it>TCF7L2 </it>gene, and this model was also confirmed in different ethnic populations when stratification of IVS3C>T and IVS4G>T polymorphisms except for Africans, where a dominant, additive genetic mode is suggested for IVS3C>T polymorphism.</p> <p>Conclusion</p> <p>This meta-analysis demonstrates that four variants of <it>TCF7L2 </it>gene are all associated with T2DM, and indicates a multiplicative genetic model for all the four polymorphisms, as well as suggests the <it>TCF7L2 </it>gene involved in near 1/5 of all T2MD. Potential gene-gene and gene-environmental interactions by which common variants in the <it>TCF7L2 </it>gene influence the risk of T2MD need further exploration.</p

Impact of primary kidney disease on the effects of empagliflozin in patients with chronic kidney disease: secondary analyses of the EMPA-KIDNEY trial

Background: The EMPA KIDNEY trial showed that empagliflozin reduced the risk of the primary composite outcome of kidney disease progression or cardiovascular death in patients with chronic kidney disease mainly through slowing progression. We aimed to assess how effects of empagliflozin might differ by primary kidney disease across its broad population. Methods: EMPA-KIDNEY, a randomised, controlled, phase 3 trial, was conducted at 241 centres in eight countries (Canada, China, Germany, Italy, Japan, Malaysia, the UK, and the USA). Patients were eligible if their estimated glomerular filtration rate (eGFR) was 20 to less than 45 mL/min per 1·73 m2, or 45 to less than 90 mL/min per 1·73 m2 with a urinary albumin-to-creatinine ratio (uACR) of 200 mg/g or higher at screening. They were randomly assigned (1:1) to 10 mg oral empagliflozin once daily or matching placebo. Effects on kidney disease progression (defined as a sustained ≥40% eGFR decline from randomisation, end-stage kidney disease, a sustained eGFR below 10 mL/min per 1·73 m2, or death from kidney failure) were assessed using prespecified Cox models, and eGFR slope analyses used shared parameter models. Subgroup comparisons were performed by including relevant interaction terms in models. EMPA-KIDNEY is registered with ClinicalTrials.gov, NCT03594110. Findings: Between May 15, 2019, and April 16, 2021, 6609 participants were randomly assigned and followed up for a median of 2·0 years (IQR 1·5–2·4). Prespecified subgroupings by primary kidney disease included 2057 (31·1%) participants with diabetic kidney disease, 1669 (25·3%) with glomerular disease, 1445 (21·9%) with hypertensive or renovascular disease, and 1438 (21·8%) with other or unknown causes. Kidney disease progression occurred in 384 (11·6%) of 3304 patients in the empagliflozin group and 504 (15·2%) of 3305 patients in the placebo group (hazard ratio 0·71 [95% CI 0·62–0·81]), with no evidence that the relative effect size varied significantly by primary kidney disease (pheterogeneity=0·62). The between-group difference in chronic eGFR slopes (ie, from 2 months to final follow-up) was 1·37 mL/min per 1·73 m2 per year (95% CI 1·16–1·59), representing a 50% (42–58) reduction in the rate of chronic eGFR decline. This relative effect of empagliflozin on chronic eGFR slope was similar in analyses by different primary kidney diseases, including in explorations by type of glomerular disease and diabetes (p values for heterogeneity all &gt;0·1). Interpretation: In a broad range of patients with chronic kidney disease at risk of progression, including a wide range of non-diabetic causes of chronic kidney disease, empagliflozin reduced risk of kidney disease progression. Relative effect sizes were broadly similar irrespective of the cause of primary kidney disease, suggesting that SGLT2 inhibitors should be part of a standard of care to minimise risk of kidney failure in chronic kidney disease. Funding: Boehringer Ingelheim, Eli Lilly, and UK Medical Research Council

Genetic mechanisms of critical illness in COVID-19.

Host-mediated lung inflammation is present1, and drives mortality2, in the critical illness caused by coronavirus disease 2019 (COVID-19). Host genetic variants associated with critical illness may identify mechanistic targets for therapeutic development3. Here we report the results of the GenOMICC (Genetics Of Mortality In Critical Care) genome-wide association study in 2,244 critically ill patients with COVID-19 from 208 UK intensive care units. We have identified and replicated the following new genome-wide significant associations: on chromosome 12q24.13 (rs10735079, P = 1.65 × 10-8) in a gene cluster that encodes antiviral restriction enzyme activators (OAS1, OAS2 and OAS3); on chromosome 19p13.2 (rs74956615, P = 2.3 × 10-8) near the gene that encodes tyrosine kinase 2 (TYK2); on chromosome 19p13.3 (rs2109069, P = 3.98 ×  10-12) within the gene that encodes dipeptidyl peptidase 9 (DPP9); and on chromosome 21q22.1 (rs2236757, P = 4.99 × 10-8) in the interferon receptor gene IFNAR2. We identified potential targets for repurposing of licensed medications: using Mendelian randomization, we found evidence that low expression of IFNAR2, or high expression of TYK2, are associated with life-threatening disease; and transcriptome-wide association in lung tissue revealed that high expression of the monocyte-macrophage chemotactic receptor CCR2 is associated with severe COVID-19. Our results identify robust genetic signals relating to key host antiviral defence mechanisms and mediators of inflammatory organ damage in COVID-19. Both mechanisms may be amenable to targeted treatment with existing drugs. However, large-scale randomized clinical trials will be essential before any change to clinical practice

Common, low-frequency, rare, and ultra-rare coding variants contribute to COVID-19 severity

The combined impact of common and rare exonic variants in COVID-19 host genetics is currently insufficiently understood. Here, common and rare variants from whole-exome sequencing data of about 4000 SARS-CoV-2-positive individuals were used to define an interpretable machine-learning model for predicting COVID-19 severity. First, variants were converted into separate sets of Boolean features, depending on the absence or the presence of variants in each gene. An ensemble of LASSO logistic regression models was used to identify the most informative Boolean features with respect to the genetic bases of severity. The Boolean features selected by these logistic models were combined into an Integrated PolyGenic Score that offers a synthetic and interpretable index for describing the contribution of host genetics in COVID-19 severity, as demonstrated through testing in several independent cohorts. Selected features belong to ultra-rare, rare, low-frequency, and common variants, including those in linkage disequilibrium with known GWAS loci. Noteworthily, around one quarter of the selected genes are sex-specific. Pathway analysis of the selected genes associated with COVID-19 severity reflected the multi-organ nature of the disease. The proposed model might provide useful information for developing diagnostics and therapeutics, while also being able to guide bedside disease management. © 2021, The Author(s)

Identification of multiple risk loci and regulatory mechanisms influencing susceptibility to multiple myeloma

Genome-wide association studies (GWAS) have transformed our understanding of susceptibility to multiple myeloma (MM), but much of the heritability remains unexplained. We report a new GWAS, a meta-analysis with previous GWAS and a replication series, totalling 9974 MM cases and 247,556 controls of European ancestry. Collectively, these data provide evidence for six new MM risk loci, bringing the total number to 23. Integration of information from gene expression, epigenetic profiling and in situ Hi-C data for the 23 risk loci implicate disruption of developmental transcriptional regulators as a basis of MM susceptibility, compatible with altered B-cell differentiation as a key mechanism. Dysregulation of autophagy/apoptosis and cell cycle signalling feature as recurrently perturbed pathways. Our findings provide further insight

Dimethyl fumarate in patients admitted to hospital with COVID-19 (RECOVERY): a randomised, controlled, open-label, platform trial

Dimethyl fumarate (DMF) inhibits inflammasome-mediated inflammation and has been proposed as a treatment for patients hospitalised with COVID-19. This randomised, controlled, open-label platform trial (Randomised Evaluation of COVID-19 Therapy [RECOVERY]), is assessing multiple treatments in patients hospitalised for COVID-19 (NCT04381936, ISRCTN50189673). In this assessment of DMF performed at 27 UK hospitals, adults were randomly allocated (1:1) to either usual standard of care alone or usual standard of care plus DMF. The primary outcome was clinical status on day 5 measured on a seven-point ordinal scale. Secondary outcomes were time to sustained improvement in clinical status, time to discharge, day 5 peripheral blood oxygenation, day 5 C-reactive protein, and improvement in day 10 clinical status. Between 2 March 2021 and 18 November 2021, 713 patients were enroled in the DMF evaluation, of whom 356 were randomly allocated to receive usual care plus DMF, and 357 to usual care alone. 95% of patients received corticosteroids as part of routine care. There was no evidence of a beneficial effect of DMF on clinical status at day 5 (common odds ratio of unfavourable outcome 1.12; 95% CI 0.86-1.47; p = 0.40). There was no significant effect of DMF on any secondary outcome

Dimethyl fumarate in patients admitted to hospital with COVID-19 (RECOVERY): a randomised, controlled, open-label, platform trial

Dimethyl fumarate (DMF) inhibits inflammasome-mediated inflammation and has been proposed as a treatment for patients hospitalised with COVID-19. This randomised, controlled, open-label platform trial (Randomised Evaluation of COVID-19 Therapy [RECOVERY]), is assessing multiple treatments in patients hospitalised for COVID-19 (NCT04381936, ISRCTN50189673). In this assessment of DMF performed at 27 UK hospitals, adults were randomly allocated (1:1) to either usual standard of care alone or usual standard of care plus DMF. The primary outcome was clinical status on day 5 measured on a seven-point ordinal scale. Secondary outcomes were time to sustained improvement in clinical status, time to discharge, day 5 peripheral blood oxygenation, day 5 C-reactive protein, and improvement in day 10 clinical status. Between 2 March 2021 and 18 November 2021, 713 patients were enroled in the DMF evaluation, of whom 356 were randomly allocated to receive usual care plus DMF, and 357 to usual care alone. 95% of patients received corticosteroids as part of routine care. There was no evidence of a beneficial effect of DMF on clinical status at day 5 (common odds ratio of unfavourable outcome 1.12; 95% CI 0.86-1.47; p = 0.40). There was no significant effect of DMF on any secondary outcome

A mutant of uracil DNA glycosylase that distinguishes between cytosine and 5-methylcytosine

We demonstrate that a mutant of uracil DNA glycosylase (N123D:L191A) distinguishes between cytosine and methylcytosine. Uracil DNA glycosylase (UDG) efficiently removes uracil from DNA in a reaction in which the base is flipped into the enzyme’s active site. Uracil is selected over cytosine by a pattern of specific hydrogen bonds, and thymine is excluded by steric clash of its 5-methyl group with Y66. The N123D mutation generates an enzyme that excises cytosine. This N123D:L191A mutant excises C when it is mispaired with A or opposite an abasic site, but not when it is paired with G. In contrast no cleavage is observed with any substrates that contain 5-methylcytosine. This enzyme may offer a new approach for discriminating between cytosine and 5-methylcytosine.<br/