Mosaic genetic variants can have major clinical impact. We systematically analyse trio exome sequence data from 4,293 probands from the DDD Study with severe developmental disorders for pathogenic ...postzygotic mosaicism (PZM) in the child or a clinically-unaffected parent, and use ultrahigh-depth sequencing to validate candidate mosaic variants. We observe that levels of mosaicism for small genetic variants are usually equivalent in both saliva and blood and ~3% of causative de novo mutations exhibit PZM; this is an important observation, as the sibling recurrence risk is extremely low. We identify parental PZM in 21 trios (0.5% of trios), resulting in a substantially increased sibling recurrence risk in future pregnancies. Together, these forms of mosaicism account for 40 (1%) diagnoses in our cohort. Likely child-PZM mutations occur equally on both parental haplotypes, and the penetrance of detectable mosaic pathogenic variants overall is likely to be less than half that of constitutive variants.
Macroalgae is an under-utilised tool as a bioindicator of anthropogenic nitrogen loading to the coastal environment in the UK. This study compared two island systems-Jersey (Channel Islands) and St ...Mary's (Isles of Scilly) to assess how differing sewerage infrastructure affects nitrogen loading. A total of 831 macroalgae samples of Fucus vesiculosus and Ulva sp. were analysed for nitrogen isotopes (δ
N). Elevated δ
N values were recorded for Jersey (> 9‰) in St Aubin's Bay-caused by the outflow of the Bellozanne Sewerage Treatment Works (STW). δ
N isoplots maps indicate low diffusion of nitrogen out of St Aubin's Bay. St Mary's produced a varied δ
N isoplot map in comparison. δ
N was typically lower and is attributed to a smaller population and inefficient STW. Outflow of sewage/effluent at Morning Point, Hugh Town and Old Town produced elevated δ
N values in comparison to the island average. St Mary's inefficient sewerage treatment and reliance on septic tanks/soakaways complicates δ
N interpretation although it still indicates that nitrogen pollution is an island-wide issue. Future sewerage development and upgrades on islands are required to prevent similar effluent environmental issues as recorded in St Aubin's Bay. This study advocates the use of macroalgae as a bioindicator of nitrogen effluent in the marine environment.
Abstract Background Multidisciplinary cancer conferences (MCCs) are a forum for health care providers to discuss diagnostic and treatment aspects of a cancer patient’s care. In Ontario, we have found ...that very few hospitals have developed cancer conferences or forums for the prospective discussion of patient cancer care. In this paper, we describe the process of creating a province-wide standards document for MCCs. Methods A systematic review and environmental scan were conducted to evaluate the literature regarding the impact of MCCs on physician practice patterns and patient outcomes, using the methodology of the Practice Guidelines Development Cycle. An Expert Panel was created to develop draft MCC standards. Ontario administrators and practitioners were surveyed to elicit feedback regarding the standards document. The findings were collated, and practice standards were developed. Results Multidisciplinary care, predominantly in the form of multidisciplinary clinics, has been shown to improve patient outcomes. While only limited evidence suggested a benefit for MCCs, they have been documented as influential in changing patient management plans. MCCs were also found to be part of standard cancer care on an international level. Ontario practitioners surveyed generally supported MCC implementation. Discussion We have described the process of creating an Ontario MCC standards document, including a literature review and an examination of the attitudes of Ontario practitioners and hospital administrators regarding the development and implementation of a MCC Standards document.
The use of in silico pathogenicity predictions as evidence when interpreting genetic variants is widely accepted as part of standard variant classification guidelines. Although numerous algorithms ...have been developed and evaluated for classifying missense variants, in-frame insertions/deletions (indels) have been much less well studied.
We created a dataset of 3964 small (< 100 bp) indels predicted to result in in-frame amino acid insertions or deletions using data from gnomAD v3.1 (minor allele frequency of 1-5%), ClinVar and the Deciphering Developmental Disorders (DDD) study. We used this dataset to evaluate the performance of nine pathogenicity predictor tools: CADD, CAPICE, FATHMM-indel, MutPred-Indel, MutationTaster2021, PROVEAN, SIFT-indel, VEST-indel and VVP.
Our dataset consisted of 2224 benign/likely benign and 1740 pathogenic/likely pathogenic variants from gnomAD (n = 809), ClinVar (n = 2882) and, DDD (n = 273). We were able to generate scores across all tools for 91% of the variants, with areas under the ROC curve (AUC) of 0.81-0.96 based on the published recommended thresholds. To avoid biases caused by inclusion of our dataset in the tools' training data, we also evaluated just DDD variants not present in either gnomAD or ClinVar (70 pathogenic and 81 benign). Using this subset, the AUC of all tools decreased substantially to 0.64-0.87. Several of the tools performed similarly however, VEST-indel had the highest AUCs of 0.93 (full dataset) and 0.87 (DDD subset).
Algorithms designed for predicting the pathogenicity of in-frame indels perform well enough to aid clinical variant classification in a similar manner to missense prediction tools.
BACKGROUND Cell-free fetal nucleic acids (cffNA) can be detected in the maternal circulation during pregnancy, potentially offering an excellent method for early non-invasive prenatal diagnosis ...(NIPD) of the genetic status of a fetus. Using molecular techniques, fetal DNA and RNA can be detected from 5 weeks gestation and are rapidly cleared from the circulation following birth. METHODS We searched PubMed systematically using keywords free fetal DNA and NIPD. Reference lists from relevant papers were also searched to ensure comprehensive coverage of the area. RESULTS Cell-free fetal DNA comprises only 3–6% of the total circulating cell-free DNA, therefore diagnoses are primarily limited to those caused by paternally inherited sequences as well as conditions that can be inferred by the unique gene expression patterns in the fetus and placenta. Broadly, the potential applications of this technology fall into two categories: first, high genetic risk families with inheritable monogenic diseases, including sex determination in cases at risk of X-linked diseases and detection of specific paternally inherited single gene disorders; and second, routine antenatal care offered to all pregnant women, including prenatal screening/diagnosis for aneuploidy, particularly Down syndrome (DS), and diagnosis of Rhesus factor status in RhD negative women. Already sex determination and Rhesus factor diagnosis are nearing translation into clinical practice for high-risk individuals. CONCLUSIONS The analysis of cffNA may allow NIPD for a variety of genetic conditions and may in future form part of national antenatal screening programmes for DS and other common genetic disorders.
To determine whether the sensitivity and specificity of SNP chips are adequate for detecting rare pathogenic variants in a clinically unselected population.
Retrospective, population based diagnostic ...evaluation.
49 908 people recruited to the UK Biobank with SNP chip and next generation sequencing data, and an additional 21 people who purchased consumer genetic tests and shared their data online via the Personal Genome Project.
Genotyping (that is, identification of the correct DNA base at a specific genomic location) using SNP chips versus sequencing, with results split by frequency of that genotype in the population. Rare pathogenic variants in the
and
genes were selected as an exemplar for detailed analysis of clinically actionable variants in the UK Biobank, and BRCA related cancers (breast, ovarian, prostate, and pancreatic) were assessed in participants through use of cancer registry data.
Overall, genotyping using SNP chips performed well compared with sequencing; sensitivity, specificity, positive predictive value, and negative predictive value were all above 99% for 108 574 common variants directly genotyped on the SNP chips and sequenced in the UK Biobank. However, the likelihood of a true positive result decreased dramatically with decreasing variant frequency; for variants that are very rare in the population, with a frequency below 0.001% in UK Biobank, the positive predictive value was very low and only 16% of 4757 heterozygous genotypes from the SNP chips were confirmed with sequencing data. Results were similar for SNP chip data from the Personal Genome Project, and 20/21 individuals analysed had at least one false positive rare pathogenic variant that had been incorrectly genotyped. For pathogenic variants in the
and
genes, which are individually very rare, the overall performance metrics for the SNP chips versus sequencing in the UK Biobank were: sensitivity 34.6%, specificity 98.3%, positive predictive value 4.2%, and negative predictive value 99.9%. Rates of BRCA related cancers in UK Biobank participants with a positive SNP chip result were similar to those for age matched controls (odds ratio 1.31, 95% confidence interval 0.99 to 1.71) because the vast majority of variants were false positives, whereas sequence positive participants had a significantly increased risk (odds ratio 4.05, 2.72 to 6.03).
SNP chips are extremely unreliable for genotyping very rare pathogenic variants and should not be used to guide health decisions without validation.
Aims/hypothesis
The mechanisms for diet-induced intramyocellular lipid accumulation and its association with insulin resistance remain contentious. In a detailed time-course study in rats, we ...examined whether a high-fat diet increased intramyocellular lipid accumulation via alterations in fatty acid translocase (FAT/CD36)-mediated fatty acid transport, selected enzymes and/or fatty acid oxidation, and whether intramyocellular lipid accretion coincided with the onset of insulin resistance.
Methods
We measured, daily (on days 1–7) and/or weekly (for 6 weeks), the diet-induced changes in circulating substrates, insulin, sarcolemmal substrate transporters and transport, selected enzymes, intramyocellular lipids, mitochondrial fatty acid oxidation and basal and insulin-stimulated sarcolemmal GLUT4 and glucose transport. We also examined whether upregulating fatty acid oxidation improved glucose transport in insulin-resistant muscles. Finally, in
Cd36
-knockout mice, we examined the role of FAT/CD36 in intramyocellular lipid accumulation, insulin sensitivity and diet-induced glucose intolerance.
Results
Within 2–3 days, diet-induced increases occurred in insulin, sarcolemmal FAT/CD36 (but not fatty acid binding protein FABPpm or fatty acid transporter FATP1 or 4), fatty acid transport and intramyocellular triacylglycerol, diacylglycerol and ceramide, independent of enzymatic changes or muscle fatty acid oxidation. Diet-induced increases in mitochondria and mitochondrial fatty acid oxidation and impairments in insulin-stimulated glucose transport and GLUT4 translocation occurred much later (≥21 days). FAT/CD36 ablation impaired insulin-stimulated fatty acid transport and lipid accumulation, improved insulin sensitivity and prevented diet-induced glucose intolerance. Increasing fatty acid oxidation in insulin-resistant muscles improved glucose transport.
Conclusions/interpretations
High-fat feeding rapidly increases intramyocellular lipids (in 2–3 days) via insulin-mediated upregulation of sarcolemmal FAT/CD36 and fatty acid transport. The 16–19 day delay in the onset of insulin resistance suggests that additional mechanisms besides intramyocellular lipids contribute to this pathology.
A host of new technologies are under development to improve the quality and reproducibility of cryoelectron microscopy (cryoEM) grid preparation. Here we have systematically investigated the ...preparation of three macromolecular complexes using three different vitrification devices (Vitrobot, chameleon, and a time-resolved cryoEM device) on various timescales, including grids made within 6 ms (the fastest reported to date), to interrogate particle behavior at the air-water interface for different timepoints. Results demonstrate that different macromolecular complexes can respond to the thin-film environment formed during cryoEM sample preparation in highly variable ways, shedding light on why cryoEM sample preparation can be difficult to optimize. We demonstrate that reducing time between sample application and vitrification is just one tool to improve cryoEM grid quality, but that it is unlikely to be a generic “silver bullet” for improving the quality of every cryoEM sample preparation.
Display omitted
•Particles partition to the air-water-interface even during fast (ms) grid making•Higher grid-making speeds can result in greater angular spread of the specimen•Higher grid-making speeds may reduce particle damage and subunit dissociation•Different grid-making apparatus can affect sample concentration in the ice
Here, we take advantage of new grid-making procedures to investigate the effect of speed in cryoEM grid production on protein particle behavior. These results allow us to better understand how different grid-making approaches can affect the resultant grid with respect to particle damage, preferred orientation, and resultant concentration.
BACKGROUND AND PURPOSE The G protein‐coupled receptor 119 (GPR119) mediates insulin secretion from pancreatic β cells and glucagon‐like peptide 1 (GLP‐1) release from intestinal L cells. While ...GPR119‐mediated insulin secretion is glucose dependent, it is not clear whether or not GPR119‐mediated GLP‐1 secretion similarly requires glucose. This study was designed to address the glucose‐dependence of GPR119‐mediated GLP‐1 secretion, and to explore the cellular mechanisms of hormone secretion in L cells versus those in β cells.
EXPERIMENTAL APPROACH GLP‐1 secretion in response to GPR119 agonists and ion channel modulators, with and without glucose, was analysed in the intestinal L cell line GLUTag, in primary intestinal cell cultures and in vivo. Insulin secretion from Min6 cells, a pancreatic β cell line, was analysed for comparison.
KEY RESULTS In GLUTag cells, GPR119 agonists stimulated GLP‐1 secretion both in the presence and in the absence of glucose. In primary mouse colon cultures, GPR119 agonists stimulated GLP‐1 secretion under glucose‐free conditions. Moreover, a GPR119 agonist increased plasma GLP‐1 in mice without a glucose load. However, in Min6 cells, GPR119‐mediated insulin secretion was glucose‐dependent. Among the pharmacological agents tested in this study, nitrendipine, an L‐type voltage‐dependent calcium channel blocker, dose‐dependently reduced GLP‐1 secretion from GLUTag cells, but had no effect in Min6 cells in the absence of glucose.
CONCLUSIONS AND IMPLICATIONS Unlike that in pancreatic β cells, GPR119‐mediated GLP‐1 secretion from intestinal L cells was glucose‐independent in vitro and in vivo, probably because of a higher basal calcium tone in the L cells.
PDF
