Publications by authors named "Vincent Plagnol"

180 Publications

MIR-NATs repress MAPT translation and aid proteostasis in neurodegeneration.

Nature 2021 06 19;594(7861):117-123. Epub 2021 May 19.

Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden.

The human genome expresses thousands of natural antisense transcripts (NAT) that can regulate epigenetic state, transcription, RNA stability or translation of their overlapping genes. Here we describe MAPT-AS1, a brain-enriched NAT that is conserved in primates and contains an embedded mammalian-wide interspersed repeat (MIR), which represses tau translation by competing for ribosomal RNA pairing with the MAPT mRNA internal ribosome entry site. MAPT encodes tau, a neuronal intrinsically disordered protein (IDP) that stabilizes axonal microtubules. Hyperphosphorylated, aggregation-prone tau forms the hallmark inclusions of tauopathies. Mutations in MAPT cause familial frontotemporal dementia, and common variations forming the MAPT H1 haplotype are a significant risk factor in many tauopathies and Parkinson's disease. Notably, expression of MAPT-AS1 or minimal essential sequences from MAPT-AS1 (including MIR) reduces-whereas silencing MAPT-AS1 expression increases-neuronal tau levels, and correlate with tau pathology in human brain. Moreover, we identified many additional NATs with embedded MIRs (MIR-NATs), which are overrepresented at coding genes linked to neurodegeneration and/or encoding IDPs, and confirmed MIR-NAT-mediated translational control of one such gene, PLCG1. These results demonstrate a key role for MAPT-AS1 in tauopathies and reveal a potentially broad contribution of MIR-NATs to the tightly controlled translation of IDPs, with particular relevance for proteostasis in neurodegeneration.
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http://dx.doi.org/10.1038/s41586-021-03556-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7610982PMC
June 2021

Validation of an Integrated Risk Tool, Including Polygenic Risk Score, for Atherosclerotic Cardiovascular Disease in Multiple Ethnicities and Ancestries.

Am J Cardiol 2021 06 3;148:157-164. Epub 2021 Mar 3.

Genomics plc, Oxford, UK.

The American College of Cardiology / American Heart Association pooled cohort equations tool (ASCVD-PCE) is currently recommended to assess 10-year risk for atherosclerotic cardiovascular disease (ASCVD). ASCVD-PCE does not currently include genetic risk factors. Polygenic risk scores (PRSs) have been shown to offer a powerful new approach to measuring genetic risk for common diseases, including ASCVD, and to enhance risk prediction when combined with ASCVD-PCE. Most work to date, including the assessment of tools, has focused on performance in individuals of European ancestries. Here we present evidence for the clinical validation of a new integrated risk tool (IRT), ASCVD-IRT, which combines ASCVD-PCE with PRS to predict 10-year risk of ASCVD across diverse ethnicity and ancestry groups. We demonstrate improved predictive performance of ASCVD-IRT over ASCVD-PCE, not only in individuals of self-reported White ethnicities (net reclassification improvement [NRI]; with 95% confidence interval = 2.7% [1.1 to 4.2]) but also Black / African American / Black Caribbean / Black African (NRI = 2.5% [0.6-4.3]) and South Asian (Indian, Bangladeshi or Pakistani) ethnicities (NRI = 8.7% [3.1 to 14.4]). NRI confidence intervals were wider and included zero for ethnicities with smaller sample sizes, including Hispanic (NRI = 7.5% [-1.4 to 16.5]), but PRS effect sizes in these ethnicities were significant and of comparable size to those seen in individuals of White ethnicities. Comparable results were obtained when individuals were analyzed by genetically inferred ancestry. Together, these results validate the performance of ASCVD-IRT in multiple ethnicities and ancestries, and favor their generalization to all ethnicities and ancestries.
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http://dx.doi.org/10.1016/j.amjcard.2021.02.032DOI Listing
June 2021

Integrated Polygenic Tool Substantially Enhances Coronary Artery Disease Prediction.

Circ Genom Precis Med 2021 04 2;14(2):e003304. Epub 2021 Mar 2.

Genomics plc, Oxford, United Kingdom (F.R.-M., M.E.W., R.M., S.S., E.K., R.M.S., W.A.T., P.S., A.S.L., J.A.G., A.S., C.C.A.S., V.P., P.D.).

Background: There is considerable interest in whether genetic data can be used to improve standard cardiovascular disease risk calculators, as the latter are routinely used in clinical practice to manage preventative treatment.

Methods: Using the UK Biobank resource, we developed our own polygenic risk score for coronary artery disease (CAD). We used an additional 60 000 UK Biobank individuals to develop an integrated risk tool (IRT) that combined our polygenic risk score with established risk tools (either the American Heart Association/American College of Cardiology pooled cohort equations [PCE] or UK QRISK3), and we tested our IRT in an additional, independent set of 186 451 UK Biobank individuals.

Results: The novel CAD polygenic risk score shows superior predictive power for CAD events, compared with other published polygenic risk scores, and is largely uncorrelated with PCE and QRISK3. When combined with PCE into an IRT, it has superior predictive accuracy. Overall, 10.4% of incident CAD cases were misclassified as low risk by PCE and correctly classified as high risk by the IRT, compared with 4.4% misclassified by the IRT and correctly classified by PCE. The overall net reclassification improvement for the IRT was 5.9% (95% CI, 4.7-7.0). When individuals were stratified into age-by-sex subgroups, the improvement was larger for all subgroups (range, 8.3%-15.4%), with the best performance in 40- to 54-year-old men (15.4% [95% CI, 11.6-19.3]). Comparable results were found using a different risk tool (QRISK3) and also a broader definition of cardiovascular disease. Use of the IRT is estimated to avoid up to 12 000 deaths in the United States over a 5-year period.

Conclusions: An IRT that includes polygenic risk outperforms current risk stratification tools and offers greater opportunity for early interventions. Given the plummeting costs of genetic tests, future iterations of CAD risk tools would be enhanced with the addition of a person's polygenic risk.
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http://dx.doi.org/10.1161/CIRCGEN.120.003304DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8284388PMC
April 2021

A Comparison of Low Read Depth QuantSeq 3' Sequencing to Total RNA-Seq in FUS Mutant Mice.

Front Genet 2020 19;11:562445. Epub 2020 Nov 19.

UCL Genetics Institute, London, United Kingdom.

Transcriptomics is a developing field with new methods of analysis being produced which may hold advantages in price, accuracy, or information output. QuantSeq is a form of 3' sequencing produced by Lexogen which aims to obtain similar gene-expression information to RNA-seq with significantly fewer reads, and therefore at a lower cost. QuantSeq is also able to provide information on differential polyadenylation. We applied both QuantSeq at low read depth and total RNA-seq to the same two sets of mouse spinal cord RNAs, each comprised by four controls and four mutants related to the neurodegenerative disease amyotrophic lateral sclerosis. We found substantial differences in which genes were found to be significantly differentially expressed by the two methods. Some of this difference likely due to the difference in number of reads between our QuantSeq and RNA-seq data. Other sources of difference can be explained by the differences in the way the two methods handle genes with different primary transcript lengths and how likely each method is to find a gene to be differentially expressed at different levels of overall gene expression. This work highlights how different methods aiming to assess expression difference can lead to different results.
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http://dx.doi.org/10.3389/fgene.2020.562445DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7717943PMC
November 2020

Clinical Relevance of an Amplicon-Based Liquid Biopsy for Detecting and Fusion and Resistance Mutations in Patients With Non-Small-Cell Lung Cancer.

JCO Precis Oncol 2020 2;4. Epub 2020 Apr 2.

Department of Medical Oncology, Centre Léon Bérard Lyon, France.

Purpose: Liquid biopsy specimen genomic profiling is integrated in non-small-cell lung cancer (NSCLC) guidelines; however, data on the clinical relevance for alterations are scarce. We evaluated the clinical utility of a targeted amplicon-based assay in a large prospective cohort of patients with -positive NSCLC and its impact on outcomes.

Patients And Methods: Patients with advanced -positive NSCLC were prospectively enrolled in the study by researchers at eight French institutions. Plasma samples were analyzed using InVisionFirst-Lung and correlated with clinical outcomes.

Results: Of the 128 patients included in the study, 101 were positive for and 27 for alterations. Blood samples (N = 405) were collected from 29 patients naïve for treatment with tyrosine kinase inhibitors (TKI) or from 375 patients under treatment, including 105 samples collected at disease progression (PD). Sensitivity was 67% (n = 18 of 27) for fusion detection. Higher detection was observed for fusions at TKI failure (n = 33 of 74; 46%) versus in patients with therapeutic response (n = 12 of 109; 11%). -resistance mutations were detected in 22% patients (n = 16 of 74) overall; 43% of the total -resistance mutations identified occurred after next-generation TKI therapy. was the most common mutation detected (n = 7 of 16). Heterogeneity of resistance was observed. resistance was detected in 30% (n = 3 of 10). The absence of circulating tumor DNA mutations at TKI failure was associated with prolonged median overall survival (105.7 months). Complex -resistance mutations correlated with poor overall survival (median, 26.9 months NR for single mutation; = .003) and progression-free survival to subsequent therapy (median 1.7 6.3 months; = .003).

Conclusion: Next-generation, targeted, amplicon-based sequencing for liquid biopsy specimen profiling provides clinically relevant detection of fusions in TKI-naïve patients and allows for the identification of resistance mutations in patients treated with TKIs. Liquid biopsy specimens from patients treated with TKIs may affect clinical outcomes and capture heterogeneity of TKI resistance, supporting their role in selecting sequential therapy.
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http://dx.doi.org/10.1200/PO.19.00281DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7448797PMC
April 2020

FUS ALS-causative mutations impair FUS autoregulation and splicing factor networks through intron retention.

Nucleic Acids Res 2020 07;48(12):6889-6905

Department of Neuromuscular Diseases, UCL Queen Square Institute of Neurology, University College London, London WC1N 3BG, UK.

Mutations in the RNA-binding protein FUS cause amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative disease. FUS plays a role in numerous aspects of RNA metabolism, including mRNA splicing. However, the impact of ALS-causative mutations on splicing has not been fully characterized, as most disease models have been based on overexpressing mutant FUS, which will alter RNA processing due to FUS autoregulation. We and others have recently created knockin models that overcome the overexpression problem, and have generated high depth RNA-sequencing on FUS mutants in parallel to FUS knockout, allowing us to compare mutation-induced changes to genuine loss of function. We find that FUS-ALS mutations induce a widespread loss of function on expression and splicing. Specifically, we find that mutant FUS directly alters intron retention levels in RNA-binding proteins. Moreover, we identify an intron retention event in FUS itself that is associated with its autoregulation. Altered FUS levels have been linked to disease, and we show here that this novel autoregulation mechanism is altered by FUS mutations. Crucially, we also observe this phenomenon in other genetic forms of ALS, including those caused by TDP-43, VCP and SOD1 mutations, supporting the concept that multiple ALS genes interact in a regulatory network.
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http://dx.doi.org/10.1093/nar/gkaa410DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7337901PMC
July 2020

The complex genetic landscape of familial MDS and AML reveals pathogenic germline variants.

Nat Commun 2020 02 25;11(1):1044. Epub 2020 Feb 25.

UMC Utrecht Cancer Center, Universitair Medisch Centrum Utrecht, Huispostnummer, Utrecht, Netherlands.

The inclusion of familial myeloid malignancies as a separate disease entity in the revised WHO classification has renewed efforts to improve the recognition and management of this group of at risk individuals. Here we report a cohort of 86 acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) families with 49 harboring germline variants in 16 previously defined loci (57%). Whole exome sequencing in a further 37 uncharacterized families (43%) allowed us to rationalize 65 new candidate loci, including genes mutated in rare hematological syndromes (ADA, GP6, IL17RA, PRF1 and SEC23B), reported in prior MDS/AML or inherited bone marrow failure series (DNAH9, NAPRT1 and SH2B3) or variants at novel loci (DHX34) that appear specific to inherited forms of myeloid malignancies. Altogether, our series of MDS/AML families offer novel insights into the etiology of myeloid malignancies and provide a framework to prioritize variants for inclusion into routine diagnostics and patient management.
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http://dx.doi.org/10.1038/s41467-020-14829-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7042299PMC
February 2020

Targeted sequencing of plasma cell-free DNA to predict response to PD1 inhibitors in advanced non-small cell lung cancer.

Lung Cancer 2019 11 6;137:1-6. Epub 2019 Sep 6.

Thoracic Oncology Department, Larrey Hospital, University Hospital of Toulouse, France; Inserm, Centre de Recherche en Cancérologie de Toulouse, CRCT UMR-1037, Toulouse, France; Toulouse Paul Sabatier University, Toulouse, France.

Objectives: Tumor mutational burden is an emerging biomarker of response to immune checkpoint inhibitors (ICI), whose clinical adoption is challenging. We hypothesized that targeting limited but relevant genetic alterations in plasma cell-free DNA along with early monitoring may non-invasively predict response to ICI in advanced non-small cell lung cancer (NSCLC).

Material And Methods: Plasma samples from patients with progressive NSCLC collected before ICI initiation and at 1 month were profiled from responders (R: PFS > 6 months) and non-responders (NR: progressive disease at first evaluation) using amplicon sequencing of hotspots and coding regions from 36 genes. The molecular profile of ctDNA, and its early kinetics were analyzed.

Results: 97 patients were analyzed, of which 86 (39 R, 47 NR) were evaluable. Alterations in ctDNA were detectable in 67/86 baseline samples (78%). The detection of a targetable oncogenic driver was associated with a 2 months PFS. The presence of a PTEN or STK11 mutation was correlated with early progression (HR 8.9, p = 0.09 for PTEN, HR 4.7, p = 0.003 for STK11), while transversion mutations (Tv) in KRAS and TP53 predicted better outcomes (HR 0.36, p = 0.011 for TP53 Tv; HR 0.46, p = 0.11 for KRAS Tv). Patients with a low "immune score" (driver and/or PTEN or STK11 mutation and/or without KRAS or TP53 Tv) derived poor outcomes (median PFS 2 months), compared with patients with a high immune score (no driver, no PTEN or STK11 and with KRAS or TP53 Tv (median PFS 14 months, p = 0.0001, HR 2.96). Early changes in the ctDNA allele fraction (AF) of 65 specimens were correlated with clinical outcomes (14 months PFS if AF decreases vs. 2 months if AF increases, p < 0.0001).

Conclusion: Targeted sequencing of plasma ctDNA and monitoring its early variations can predict response to ICI.
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http://dx.doi.org/10.1016/j.lungcan.2019.09.005DOI Listing
November 2019

Gain-of-function CEBPE mutation causes noncanonical autoinflammatory inflammasomopathy.

J Allergy Clin Immunol 2019 11 13;144(5):1364-1376. Epub 2019 Jun 13.

Institute of Biotechnology, HiLIFE, University of Helsinki, Helsinki, Finland.

Background: CCAAT enhancer-binding protein epsilon (C/EBPε) is a transcription factor involved in late myeloid lineage differentiation and cellular function. The only previously known disorder linked to C/EBPε is autosomal recessive neutrophil-specific granule deficiency leading to severely impaired neutrophil function and early mortality.

Objective: The aim of this study was to molecularly characterize the effects of C/EBPε transcription factor Arg219His mutation identified in a Finnish family with previously genetically uncharacterized autoinflammatory and immunodeficiency syndrome.

Methods: Genetic analysis, proteomics, genome-wide transcriptional profiling by means of RNA-sequencing, chromatin immunoprecipitation (ChIP) sequencing, and assessment of the inflammasome function of primary macrophages were performed.

Results: Studies revealed a novel mechanism of genome-wide gain-of-function that dysregulated transcription of 464 genes. Mechanisms involved dysregulated noncanonical inflammasome activation caused by decreased association with transcriptional repressors, leading to increased chromatin occupancy and considerable changes in transcriptional activity, including increased expression of NLR family, pyrin domain-containing 3 protein (NLRP3) and constitutively expressed caspase-5 in macrophages.

Conclusion: We describe a novel autoinflammatory disease with defective neutrophil function caused by a homozygous Arg219His mutation in the transcription factor C/EBPε. Mutated C/EBPε acts as a regulator of both the inflammasome and interferome, and the Arg219His mutation causes the first human monogenic neomorphic and noncanonical inflammasomopathy/immunodeficiency. The mechanism, including widely dysregulated transcription, is likely not unique for C/EBPε. Similar multiomics approaches should also be used in studying other transcription factor-associated diseases.
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http://dx.doi.org/10.1016/j.jaci.2019.06.003DOI Listing
November 2019

Downregulated Wnt/β-catenin signalling in the Down syndrome hippocampus.

Sci Rep 2019 05 13;9(1):7322. Epub 2019 May 13.

Department of Pharmacology, UCL School of Pharmacy, University College London, 29-39 Brunswick Square, London, WC1N 1AX, UK.

Pathological mechanisms underlying Down syndrome (DS)/Trisomy 21, including dysregulation of essential signalling processes remain poorly understood. Combining bioinformatics with RNA and protein analysis, we identified downregulation of the Wnt/β-catenin pathway in the hippocampus of adult DS individuals with Alzheimer's disease and the 'Tc1' DS mouse model. Providing a potential underlying molecular pathway, we demonstrate that the chromosome 21 kinase DYRK1A regulates Wnt signalling via a novel bimodal mechanism. Under basal conditions, DYRK1A is a negative regulator of Wnt/β-catenin. Following pathway activation, however, DYRK1A exerts the opposite effect, increasing signalling activity. In summary, we identified downregulation of hippocampal Wnt/β-catenin signalling in DS, possibly mediated by a dose dependent effect of the chromosome 21-encoded kinase DYRK1A. Overall, we propose that dosage imbalance of the Hsa21 gene DYRK1A affects downstream Wnt target genes. Therefore, modulation of Wnt signalling may open unexplored avenues for DS and Alzheimer's disease treatment.
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http://dx.doi.org/10.1038/s41598-019-43820-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6513850PMC
May 2019

Prospective Clinical Validation of the InVisionFirst-Lung Circulating Tumor DNA Assay for Molecular Profiling of Patients With Advanced Nonsquamous Non-Small-Cell Lung Cancer.

JCO Precis Oncol 2019 25;3. Epub 2019 Apr 25.

Washington University School of Medicine, St. Louis, MO.

Purpose: Guidelines advocate molecular profiling in the evaluation of advanced non-small-cell lung cancer (NSCLC) and support the use of plasma circulating tumor DNA (ctDNA)-based profiling for patients with insufficient tissue. Thorough prospective clinical validation studies of next-generation sequencing (NGS)-based ctDNA assays are lacking. We report the multicentered prospective clinical validation of the InVision ctDNA assay in patients with advanced untreated NSCLC.

Methods: A total of 264 patients with untreated advanced NSCLC were prospectively recruited, and their plasma was analyzed using a ctDNA NGS assay for detection of genomic alterations in 36 commonly mutated genes. Tumor tissue was available in 178 patients for molecular profiling for comparison with plasma profiling. The remaining 86 patients were included to compare ctDNA profiles in patients with and without tissue for profiling.

Results: Concordance of InVisionFirst with matched tissue profiling was 97.8%, with 82.9% positive predictive value, 98.5% negative predictive value, 70.6% sensitivity, and 99.2% specificity. Considering specific alterations in eight genes that most influence patient management, the positive predictive value was 97.8%, with 97.1% negative predictive value, 73.9% sensitivity, and 99.8% specificity. Across the entire study, 48 patients with actionable alterations were identified by ctDNA testing compared with only 38 by tissue testing. ctDNA NGS reported either an actionable alteration or an alteration generally considered mutually exclusive for such actionable changes in 53% of patients.

Conclusion: The liquid biopsy NGS assay demonstrated excellent concordance with tissue profiling in this multicenter, prospective, clinical validation study, with sensitivity and specificity equivalent to Food and Drug Administration-approved single-gene ctDNA assays. The use of plasma-based molecular profiling using NGS led to the detection of 26% more actionable alterations compared with standard-of-care tissue testing in this study.
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http://dx.doi.org/10.1200/PO.18.00299DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7450945PMC
April 2019

Real-World Utility of an Amplicon-Based Next-Generation Sequencing Liquid Biopsy for Broad Molecular Profiling in Patients With Advanced Non-Small-Cell Lung Cancer.

JCO Precis Oncol 2019 6;3. Epub 2019 Mar 6.

Gustave Roussy, Villejuif, France.

Purpose: To assess the feasibility and utility of circulating tumor DNA (ctDNA) by amplicon-based next-generation sequencing (NGS) analysis in the daily clinical setting in a cohort of patients with advanced non-small-cell lung cancer (NSCLC), as an alternative approach to tissue molecular profiling.

Patients And Methods: In this single-center prospective study, treatment-naïve and previously treated patients with advanced NSCLC were enrolled. Clinical validation of ctDNA using amplicon-based NGS analysis (with a 36-gene panel) was performed against standard-of-care tissue molecular analysis in treatment-naïve patients. The feasibility, utility, and prognostic value of ctDNA as a dynamic marker of treatment efficacy was evaluated. Results of tissue molecular profile were blinded during ctDNA analysis.

Results: Of 214 patients with advanced NSCLC who were recruited, 156 were treatment-naïve patients and 58 were pretreated patients with unknown tissue molecular profile. ctDNA screening was successfully performed for 91% (n = 194) of all patients, and mutations were detected in 77% of these patients. Tissue molecular analysis was available for 111 patients (52%), and tissue somatic mutations were found for 78% (n = 87) of patients. For clinically relevant variants, concordance agreement between ctDNA and tumor tissue analysis was 95% among 94 treatment-naïve patients who had concurrent liquid and tumor biopsy molecular profiles. Sensitivity and specificity were 81% and 97%, respectively. Of the 103 patients with no tissue available, ctDNA detected potential actionable mutations in 17% of patients; of these, 10% received personalized treatment. ctDNA kinetics correlated with response rate and progression-free survival in 31 patients treated with first-line platinum-based chemotherapy.

Conclusion: These real-world data from a prospective study endorse ctDNA molecular profile by amplicon-based NGS as an accurate and reliable tool to detect and monitor clinically relevant molecular alterations in patients with advanced NSCLC.
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http://dx.doi.org/10.1200/PO.18.00211DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7446523PMC
March 2019

Novel Mutation in a Patient With APLAID and Cutis Laxa.

Front Immunol 2018 14;9:2863. Epub 2018 Dec 14.

Department of Medicine, University of Cambridge, Cambridge, United Kingdom.

The auto-inflammation and phospholipase Cγ2 (PLCγ2)-associated antibody deficiency and immune dysregulation (APLAID) syndrome is a rare primary immunodeficiency caused by a gain-of-function mutation S707Y in the gene previously described in two patients from one family. The APLAID patients presented with early-onset blistering skin lesions, posterior uveitis, inflammatory bowel disease (IBD) and recurrent sinopulmonary infections caused by a humoral defect, but lacked circulating autoantibodies and had no cold-induced urticaria, contrary to the patients with the related PLAID syndrome. We describe a new APLAID patient who presented with vesiculopustular rash in the 1st weeks of life, followed by IBD, posterior uveitis, recurrent chest infections, interstitial pneumonitis, and also had sensorineural deafness and cutis laxa. Her disease has been refractory to most treatments, including IL1 blockers and a trial with ruxolitinib has been attempted. In this patient, we found a unique heterozygous missense L848P mutation in the gene, predicted to affect the PLCγ2 structure. Similarly to S707Y, the L848P mutation led to the increased basal and EGF-stimulated PLCγ2 activity . Whole blood assays showed reduced production of IFN-γ and IL-17 in response to polyclonal T-cell stimulation and reduced production of IL-10 and IL-1β after LPS stimulation. Reduced IL-1β levels and the lack of clinical response to treatment with IL-1 blockers argue against NLRP3 inflammasome hyperactivation being the main mechanism mediating the APLAID pathogenesis. Our findings indicate that L848P is novel a gain-of-function mutation that leads to PLCγ2 activation and suggest cutis laxa as a possible clinical manifestations of the APLAID syndrome.
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http://dx.doi.org/10.3389/fimmu.2018.02863DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6302768PMC
October 2019

GWAS and colocalization analyses implicate carotid intima-media thickness and carotid plaque loci in cardiovascular outcomes.

Nat Commun 2018 12 3;9(1):5141. Epub 2018 Dec 3.

Department of Medicine, University of Mississippi Medical Center, Jackson, MS, 39216, USA.

Carotid artery intima media thickness (cIMT) and carotid plaque are measures of subclinical atherosclerosis associated with ischemic stroke and coronary heart disease (CHD). Here, we undertake meta-analyses of genome-wide association studies (GWAS) in 71,128 individuals for cIMT, and 48,434 individuals for carotid plaque traits. We identify eight novel susceptibility loci for cIMT, one independent association at the previously-identified PINX1 locus, and one novel locus for carotid plaque. Colocalization analysis with nearby vascular expression quantitative loci (cis-eQTLs) derived from arterial wall and metabolic tissues obtained from patients with CHD identifies candidate genes at two potentially additional loci, ADAMTS9 and LOXL4. LD score regression reveals significant genetic correlations between cIMT and plaque traits, and both cIMT and plaque with CHD, any stroke subtype and ischemic stroke. Our study provides insights into genes and tissue-specific regulatory mechanisms linking atherosclerosis both to its functional genomic origins and its clinical consequences in humans.
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http://dx.doi.org/10.1038/s41467-018-07340-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6277418PMC
December 2018

Effects of Collection and Processing Procedures on Plasma Circulating Cell-Free DNA from Cancer Patients.

J Mol Diagn 2018 11 28;20(6):883-892. Epub 2018 Aug 28.

Cancer Research UK Cambridge Institute, Li Ka Shing Centre, Robinson Way, University of Cambridge, Cambridge, United Kingdom; Cancer Research UK Major Centre, Cambridge, United Kingdom. Electronic address:

Circulating tumor DNA (ctDNA) offers new opportunities for noninvasive cancer management. Detecting ctDNA in plasma is challenging because it constitutes only a minor fraction of the total cell-free DNA (cfDNA). Pre-analytical factors affect cfDNA levels contributed from leukocyte lysis, hence the ability to detect low-frequency mutant alleles. This study investigates the effects of the delay in processing, storage temperatures, different blood collection tubes, centrifugation protocols, and sample shipment on cfDNA levels. Peripheral blood (n = 231) from cancer patients (n = 62) were collected into KEDTA or Cell-free DNA BCT tubes and analyzed by digital PCR, targeted amplicon, or shallow whole-genome sequencing. To assess pre-analytic effects, plasma was processed under different conditions after 0, 6, 24, 48, 96 hours, and 1 week at room temperature or 4°C, or using different centrifugation protocols. Digital PCR showed that cfDNA levels increased gradually with time in KEDTA tubes, but were stable in BCT tubes. KEDTA samples stored at 4°C showed less variation than room temperature storage, but levels were elevated compared with BCT. A second centrifugation at 3000 × g gave similar cfDNA yields compared with higher-speed centrifugation. Next-generation sequencing showed negligible differences in background error or copy number changes between KEDTA and BCT, or following shipment in BCT. This study provides insights into the effects of sample processing on ctDNA analysis.
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http://dx.doi.org/10.1016/j.jmoldx.2018.07.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6197164PMC
November 2018

Biallelic mutations in humans cause severe immunodeficiency, arthritis, and intestinal inflammation.

Science 2018 08 19;361(6404):810-813. Epub 2018 Jul 19.

Department of Medicine, University of Cambridge, Cambridge CB2 0QQ, UK.

RIPK1 (receptor-interacting serine/threonine kinase 1) is a master regulator of signaling pathways leading to inflammation and cell death and is of medical interest as a drug target. We report four patients from three unrelated families with complete RIPK1 deficiency caused by rare homozygous mutations. The patients suffered from recurrent infections, early-onset inflammatory bowel disease, and progressive polyarthritis. They had immunodeficiency with lymphopenia and altered production of various cytokines revealed by whole-blood assays. In vitro, RIPK1-deficient cells showed impaired mitogen-activated protein kinase activation and cytokine secretion and were prone to necroptosis. Hematopoietic stem cell transplantation reversed cytokine production defects and resolved clinical symptoms in one patient. Thus, RIPK1 plays a critical role in the human immune system.
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http://dx.doi.org/10.1126/science.aar2641DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6529353PMC
August 2018

Insights into the genetic epidemiology of Crohn's and rare diseases in the Ashkenazi Jewish population.

PLoS Genet 2018 05 24;14(5):e1007329. Epub 2018 May 24.

Department of Internal Medicine, University Hospital Schleswig-Holstein, Kiel, Germany.

As part of a broader collaborative network of exome sequencing studies, we developed a jointly called data set of 5,685 Ashkenazi Jewish exomes. We make publicly available a resource of site and allele frequencies, which should serve as a reference for medical genetics in the Ashkenazim (hosted in part at https://ibd.broadinstitute.org, also available in gnomAD at http://gnomad.broadinstitute.org). We estimate that 34% of protein-coding alleles present in the Ashkenazi Jewish population at frequencies greater than 0.2% are significantly more frequent (mean 15-fold) than their maximum frequency observed in other reference populations. Arising via a well-described founder effect approximately 30 generations ago, this catalog of enriched alleles can contribute to differences in genetic risk and overall prevalence of diseases between populations. As validation we document 148 AJ enriched protein-altering alleles that overlap with "pathogenic" ClinVar alleles (table available at https://github.com/macarthur-lab/clinvar/blob/master/output/clinvar.tsv), including those that account for 10-100 fold differences in prevalence between AJ and non-AJ populations of some rare diseases, especially recessive conditions, including Gaucher disease (GBA, p.Asn409Ser, 8-fold enrichment); Canavan disease (ASPA, p.Glu285Ala, 12-fold enrichment); and Tay-Sachs disease (HEXA, c.1421+1G>C, 27-fold enrichment; p.Tyr427IlefsTer5, 12-fold enrichment). We next sought to use this catalog, of well-established relevance to Mendelian disease, to explore Crohn's disease, a common disease with an estimated two to four-fold excess prevalence in AJ. We specifically attempt to evaluate whether strong acting rare alleles, particularly protein-truncating or otherwise large effect-size alleles, enriched by the same founder-effect, contribute excess genetic risk to Crohn's disease in AJ, and find that ten rare genetic risk factors in NOD2 and LRRK2 are enriched in AJ (p < 0.005), including several novel contributing alleles, show evidence of association to CD. Independently, we find that genomewide common variant risk defined by GWAS shows a strong difference between AJ and non-AJ European control population samples (0.97 s.d. higher, p<10-16). Taken together, the results suggest coordinated selection in AJ population for higher CD risk alleles in general. The results and approach illustrate the value of exome sequencing data in case-control studies along with reference data sets like ExAC (sites VCF available via FTP at ftp.broadinstitute.org/pub/ExAC_release/release0.3/) to pinpoint genetic variation that contributes to variable disease predisposition across populations.
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http://dx.doi.org/10.1371/journal.pgen.1007329DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5967709PMC
May 2018

Mice with endogenous TDP-43 mutations exhibit gain of splicing function and characteristics of amyotrophic lateral sclerosis.

EMBO J 2018 06 15;37(11). Epub 2018 May 15.

UCL Institute of Neurology and Francis Crick Institute, London, UK.

TDP-43 (encoded by the gene ) is an RNA binding protein central to the pathogenesis of amyotrophic lateral sclerosis (ALS). However, how mutations trigger pathogenesis remains unknown. Here, we use novel mouse mutants carrying point mutations in endogenous to dissect TDP-43 function at physiological levels both and Interestingly, we find that mutations within the C-terminal domain of TDP-43 lead to a gain of splicing function. Using two different strains, we are able to separate TDP-43 loss- and gain-of-function effects. TDP-43 gain-of-function effects in these mice reveal a novel category of splicing events controlled by TDP-43, referred to as "skiptic" exons, in which skipping of constitutive exons causes changes in gene expression. , this gain-of-function mutation in endogenous causes an adult-onset neuromuscular phenotype accompanied by motor neuron loss and neurodegenerative changes. Furthermore, we have validated the splicing gain-of-function and skiptic exons in ALS patient-derived cells. Our findings provide a novel pathogenic mechanism and highlight how TDP-43 gain of function and loss of function affect RNA processing differently, suggesting they may act at different disease stages.
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http://dx.doi.org/10.15252/embj.201798684DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5983119PMC
June 2018

A Transcriptomic Signature of the Hypothalamic Response to Fasting and BDNF Deficiency in Prader-Willi Syndrome.

Cell Rep 2018 03;22(13):3401-3408

University of Cambridge Metabolic Research Laboratories and NIHR Cambridge Biomedical Research Centre, Wellcome Trust-MRC Institute of Metabolic Science, Addenbrooke's Hospital, Cambridge CB2 0QQ, UK. Electronic address:

Transcriptional analysis of brain tissue from people with molecularly defined causes of obesity may highlight disease mechanisms and therapeutic targets. We performed RNA sequencing of hypothalamus from individuals with Prader-Willi syndrome (PWS), a genetic obesity syndrome characterized by severe hyperphagia. We found that upregulated genes overlap with the transcriptome of mouse Agrp neurons that signal hunger, while downregulated genes overlap with the expression profile of Pomc neurons activated by feeding. Downregulated genes are expressed mainly in neuronal cells and contribute to neurogenesis, neurotransmitter release, and synaptic plasticity, while upregulated, predominantly microglial genes are involved in inflammatory responses. This transcriptional signature may be mediated by reduced brain-derived neurotrophic factor expression. Additionally, we implicate disruption of alternative splicing as a potential molecular mechanism underlying neuronal dysfunction in PWS. Transcriptomic analysis of the human hypothalamus may identify neural mechanisms involved in energy homeostasis and potential therapeutic targets for weight loss.
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http://dx.doi.org/10.1016/j.celrep.2018.03.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5896230PMC
March 2018

Development of a highly sensitive liquid biopsy platform to detect clinically-relevant cancer mutations at low allele fractions in cell-free DNA.

PLoS One 2018 16;13(3):e0194630. Epub 2018 Mar 16.

Inivata Ltd, Granta Park, Cambridge, United Kingdom.

Introduction: Detection and monitoring of circulating tumor DNA (ctDNA) is rapidly becoming a diagnostic, prognostic and predictive tool in cancer patient care. A growing number of gene targets have been identified as diagnostic or actionable, requiring the development of reliable technology that provides analysis of multiple genes in parallel. We have developed the InVision™ liquid biopsy platform which utilizes enhanced TAm-Seq™ (eTAm-Seq™) technology, an amplicon-based next generation sequencing method for the identification of clinically-relevant somatic alterations at low frequency in ctDNA across a panel of 35 cancer-related genes.

Materials And Methods: We present analytical validation of the eTAm-Seq technology across two laboratories to determine the reproducibility of mutation identification. We assess the quantitative performance of eTAm-Seq technology for analysis of single nucleotide variants in clinically-relevant genes as compared to digital PCR (dPCR), using both established DNA standards and novel full-process control material.

Results: The assay detected mutant alleles down to 0.02% AF, with high per-base specificity of 99.9997%. Across two laboratories, analysis of samples with optimal amount of DNA detected 94% mutations at 0.25%-0.33% allele fraction (AF), with 90% of mutations detected for samples with lower amounts of input DNA.

Conclusions: These studies demonstrate that eTAm-Seq technology is a robust and reproducible technology for the identification and quantification of somatic mutations in circulating tumor DNA, and support its use in clinical applications for precision medicine.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0194630PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5856404PMC
July 2018

Analytical validation of a next generation sequencing liquid biopsy assay for high sensitivity broad molecular profiling.

PLoS One 2018 15;13(3):e0193802. Epub 2018 Mar 15.

Research and Development, Inivata Ltd, Granta Park, Cambridge, United Kingdom.

Circulating tumor DNA (ctDNA) analysis is being incorporated into cancer care; notably in profiling patients to guide treatment decisions. Responses to targeted therapies have been observed in patients with actionable mutations detected in plasma DNA at variant allele fractions (VAFs) below 0.5%. Highly sensitive methods are therefore required for optimal clinical use. To enable objective assessment of assay performance, detailed analytical validation is required. We developed the InVisionFirst™ assay, an assay based on enhanced tagged amplicon sequencing (eTAm-Seq™) technology to profile 36 genes commonly mutated in non-small cell lung cancer (NSCLC) and other cancer types for actionable genomic alterations in cell-free DNA. The assay has been developed to detect point mutations, indels, amplifications and gene fusions that commonly occur in NSCLC. For analytical validation, two 10mL blood tubes were collected from NSCLC patients and healthy volunteer donors. In addition, contrived samples were used to represent a wide spectrum of genetic aberrations and VAFs. Samples were analyzed by multiple operators, at different times and using different reagent Lots. Results were compared with digital PCR (dPCR). The InVisionFirst assay demonstrated an excellent limit of detection, with 99.48% sensitivity for SNVs present at VAF range 0.25%-0.33%, 92.46% sensitivity for indels at 0.25% VAF and a high rate of detection at lower frequencies while retaining high specificity (99.9997% per base). The assay also detected ALK and ROS1 gene fusions, and DNA amplifications in ERBB2, FGFR1, MET and EGFR with high sensitivity and specificity. Comparison between the InVisionFirst assay and dPCR in a series of cancer patients showed high concordance. This analytical validation demonstrated that the InVisionFirst assay is highly sensitive, specific and robust, and meets analytical requirements for clinical applications.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0193802PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5854321PMC
June 2018

Peripheral tissues reprogram CD8+ T cells for pathogenicity during graft-versus-host disease.

JCI Insight 2018 03 8;3(5). Epub 2018 Mar 8.

Haematology, UCL Cancer Institute and Institute of Immunity & Transplantation, London, United Kingdom (UK).

Graft-versus-host disease (GVHD) is a life-threatening complication of allogeneic stem cell transplantation induced by the influx of donor-derived effector T cells (TE) into peripheral tissues. Current treatment strategies rely on targeting systemic T cells; however, the precise location and nature of instructions that program TE to become pathogenic and trigger injury are unknown. We therefore used weighted gene coexpression network analysis to construct an unbiased spatial map of TE differentiation during the evolution of GVHD and identified wide variation in effector programs in mice and humans according to location. Idiosyncrasy of effector programming in affected organs did not result from variation in T cell receptor repertoire or the selection of optimally activated TE. Instead, TE were reprogrammed by tissue-autonomous mechanisms in target organs for site-specific proinflammatory functions that were highly divergent from those primed in lymph nodes. In the skin, we combined the correlation-based network with a module-based differential expression analysis and showed that Langerhans cells provided in situ instructions for a Notch-dependent T cell gene cluster critical for triggering local injury. Thus, the principal determinant of TE pathogenicity in GVHD is the final destination, highlighting the need for target organ-specific approaches to block immunopathology while avoiding global immune suppression.
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http://dx.doi.org/10.1172/jci.insight.97011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5922296PMC
March 2018

Familial Limbal Stem Cell Deficiency: Clinical, Cytological and Genetic Characterization.

Stem Cell Rev Rep 2018 Feb;14(1):148-151

Research Unit for Rare Diseases; Department of Pediatrics and Adolescent Medicine, First Faculty of Medicine, Charles University and General University Hospital in Prague, Ke Karlovu 2, Praha 2, 128 08, Prague, Czech Republic.

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http://dx.doi.org/10.1007/s12015-017-9780-yDOI Listing
February 2018

Humanized mutant FUS drives progressive motor neuron degeneration without aggregation in 'FUSDelta14' knockin mice.

Brain 2017 Nov;140(11):2797-2805

Department of Neurodegenerative Disease, UCL Institute of Neurology, Queen Square, London WC1N 3BG, UK.

Mutations in FUS are causative for amyotrophic lateral sclerosis with a dominant mode of inheritance. In trying to model FUS-amyotrophic lateral sclerosis (ALS) in mouse it is clear that FUS is dosage-sensitive and effects arise from overexpression per se in transgenic strains. Novel models are required that maintain physiological levels of FUS expression and that recapitulate the human disease-with progressive loss of motor neurons in heterozygous animals. Here, we describe a new humanized FUS-ALS mouse with a frameshift mutation, which fulfils both criteria: the FUS Delta14 mouse. Heterozygous animals express mutant humanized FUS protein at physiological levels and have adult onset progressive motor neuron loss and denervation of neuromuscular junctions. Additionally, we generated a novel antibody to the unique human frameshift peptide epitope, allowing specific identification of mutant FUS only. Using our new FUSDelta14 ALS mouse-antibody system we show that neurodegeneration occurs in the absence of FUS protein aggregation. FUS mislocalization increases as disease progresses, and mutant FUS accumulates at the rough endoplasmic reticulum. Further, transcriptomic analyses show progressive changes in ribosomal protein levels and mitochondrial function as early disease stages are initiated. Thus, our new physiological mouse model has provided novel insight into the early pathogenesis of FUS-ALS.
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http://dx.doi.org/10.1093/brain/awx248DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5841203PMC
November 2017

A recurrent splice-site mutation in EPHA2 causing congenital posterior nuclear cataract.

Ophthalmic Genet 2018 04 17;39(2):236-241. Epub 2017 Oct 17.

a Genetics, UCL Institute of Ophthalmology , London , UK.

Intoduction: Inherited cataract, opacification of the lens, is the most common worldwide cause of blindness in children. We aimed to identify the genetic cause of autosomal dominant (AD) posterior nuclear cataract in a four generation British family.

Methods: Whole genome sequence (WGS) was performed on two affected and one unaffected individual of the family and further validated by direct sequencing. Haplotype analysis was performed via genotying.

Results: A splice-site mutation c.2826-9G>A in the gene EPHA2, encoding EPH receptor A2 was identified and found to co-segregate with disease.

Conclusions: We have identified a recurrent splice-site mutation c.2826-9G>A in EPHA2 causing isolated posterior nuclear cataract, providing evidence of further phenotypic heterogeneity associated with this variant.
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http://dx.doi.org/10.1080/13816810.2017.1381977DOI Listing
April 2018

Missense variants in the X-linked gene PRPS1 cause retinal degeneration in females.

Hum Mutat 2018 01 17;39(1):80-91. Epub 2017 Oct 17.

UCL Institute of Ophthalmology, London, United Kingdom.

Retinal dystrophies are a heterogeneous group of disorders of visual function leading to partial or complete blindness. We report the genetic basis of an unusual retinal dystrophy in five families with affected females and no affected males. Heterozygous missense variants were identified in the X-linked phosphoribosyl pyrophosphate synthetase 1 (PRPS1) gene: c.47C > T, p.(Ser16Phe); c.586C > T, p.(Arg196Trp); c.641G > C, p.(Arg214Pro); and c.640C > T, p.(Arg214Trp). Missense variants in PRPS1 are usually associated with disease in male patients, including Arts syndrome, Charcot-Marie-Tooth, and nonsyndromic sensorineural deafness. In our study families, affected females manifested a retinal dystrophy with interocular asymmetry. Three unrelated females from these families had hearing loss leading to a diagnosis of Usher syndrome. Other neurological manifestations were also observed in three individuals. Our data highlight the unexpected X-linked inheritance of retinal degeneration in females caused by variants in PRPS1 and suggest that tissue-specific skewed X-inactivation or variable levels of pyrophosphate synthetase-1 deficiency are the underlying mechanism(s). We speculate that the absence of affected males in the study families suggests that some variants may be male embryonic lethal when inherited in the hemizygous state. The unbiased nature of next-generation sequencing enables all possible modes of inheritance to be considered for association of gene variants with novel phenotypic presentation.
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http://dx.doi.org/10.1002/humu.23349DOI Listing
January 2018

Digital PCR analysis of circulating tumor DNA: a biomarker for chondrosarcoma diagnosis, prognostication, and residual disease detection.

Cancer Med 2017 Oct 23;6(10):2194-2202. Epub 2017 Aug 23.

University College London Cancer Institute, London, United Kingdom.

Conventional chondrosarcoma is the most common primary bone tumor in adults. Prognosis corresponds with tumor grade but remains variable, especially for individuals with grade (G) II disease. There are currently no biomarkers available for monitoring or prognostication of chondrosarcoma. Circulating tumor DNA (ctDNA) has recently emerged as a promising biomarker for a broad range of tumor types. To date, little has been done to study the presence of ctDNA and its potential utility in the management of sarcomas, including chondrosarcoma. In this study, we have assessed ctDNA levels in a cohort of 71 patients, 32 with sarcoma, including 29 individuals with central chondrosarcoma (CS) and 39 with locally aggressive and benign bone and soft tissue tumors, using digital PCR. In patients with CS, ctDNA was detected in pretreatment samples in 14/29 patients, which showed clear correlation with tumor grade as demonstrated by the detection of ctDNA in all patients with GIII and dedifferentiated disease (n = 6) and in 8/17 patients with GII disease, but never associated with GI CS. Notably detection of ctDNA preoperatively in GII disease was associated with a poor outcome. A total of 14 patients with CS had ctDNA levels assessed at multiple time points and in most patients there was a clear reduction following surgical removal. This research lays the foundation for larger studies to assess the utility of ctDNA for chondrosarcoma diagnosis, prognostication, early detection of residual disease and monitoring disease progression.
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http://dx.doi.org/10.1002/cam4.1146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5633548PMC
October 2017
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