DNA methylation is thought to be an important determinant of human phenotypic variation, but its inherent cell type specificity has impeded progress on this question. At exceptional genomic regions, ...interindividual variation in DNA methylation occurs systemically. Like genetic variants, systemic interindividual epigenetic variants are stable, can influence phenotype, and can be assessed in any easily biopsiable DNA sample. We describe an unbiased screen for human genomic regions at which interindividual variation in DNA methylation is not tissue-specific.
For each of 10 donors from the NIH Genotype-Tissue Expression (GTEx) program, CpG methylation is measured by deep whole-genome bisulfite sequencing of genomic DNA from tissues representing the three germ layer lineages: thyroid (endoderm), heart (mesoderm), and brain (ectoderm). We develop a computational algorithm to identify genomic regions at which interindividual variation in DNA methylation is consistent across all three lineages. This approach identifies 9926 correlated regions of systemic interindividual variation (CoRSIVs). These regions, comprising just 0.1% of the human genome, are inter-correlated over long genomic distances, associated with transposable elements and subtelomeric regions, conserved across diverse human ethnic groups, sensitive to periconceptional environment, and associated with genes implicated in a broad range of human disorders and phenotypes. CoRSIV methylation in one tissue can predict expression of associated genes in other tissues.
In addition to charting a previously unexplored molecular level of human individuality, this atlas of human CoRSIVs provides a resource for future population-based investigations into how interindividual epigenetic variation modulates risk of disease.
Abstract
Background
The traditional approach to studying the epigenetic mechanism CpG methylation in tissue samples is to identify regions of concordant differential methylation spanning multiple CpG ...sites (differentially methylated regions). Variation limited to single or small numbers of CpGs has been assumed to reflect stochastic processes. To test this, we developed software, Cluster-Based analysis of CpG methylation (CluBCpG), and explored variation in read-level CpG methylation patterns in whole genome bisulfite sequencing data.
Results
Analysis of both human and mouse whole genome bisulfite sequencing datasets reveals read-level signatures associated with cell type and cell type-specific biological processes. These signatures, which are mostly orthogonal to classical differentially methylated regions, are enriched at cell type-specific enhancers and allow estimation of proportional cell composition in synthetic mixtures and improved prediction of gene expression. In tandem, we developed a machine learning algorithm, Precise Read-Level Imputation of Methylation (PReLIM), to increase coverage of existing whole genome bisulfite sequencing datasets by imputing CpG methylation states on individual sequencing reads. PReLIM both improves CluBCpG coverage and performance and enables identification of novel differentially methylated regions, which we independently validate.
Conclusions
Our data indicate that, rather than stochastic variation, read-level CpG methylation patterns in tissue whole genome bisulfite sequencing libraries reflect cell type. Accordingly, these new computational tools should lead to an improved understanding of epigenetic regulation by DNA methylation.
Monozygotic twins have long been studied to estimate heritability and explore epigenetic influences on phenotypic variation. The phenotypic and epigenetic similarities of monozygotic twins have been ...assumed to be largely due to their genetic identity.
Here, by analyzing data from a genome-scale study of DNA methylation in monozygotic and dizygotic twins, we identified genomic regions at which the epigenetic similarity of monozygotic twins is substantially greater than can be explained by their genetic identity. This "epigenetic supersimilarity" apparently results from locus-specific establishment of epigenotype prior to embryo cleavage during twinning. Epigenetically supersimilar loci exhibit systemic interindividual epigenetic variation and plasticity to periconceptional environment and are enriched in sub-telomeric regions. In case-control studies nested in a prospective cohort, blood DNA methylation at these loci years before diagnosis is associated with risk of developing several types of cancer.
These results establish a link between early embryonic epigenetic development and adult disease. More broadly, epigenetic supersimilarity is a previously unrecognized phenomenon that may contribute to the phenotypic similarity of monozygotic twins.
DNA methylation regulates cell type-specific gene expression. Here, in a transgenic mouse model, we show that deletion of the gene encoding DNA methyltransferase Dnmt3a in hypothalamic AgRP neurons ...causes a sedentary phenotype characterized by reduced voluntary exercise and increased adiposity. Whole-genome bisulfite sequencing (WGBS) and transcriptional profiling in neuronal nuclei from the arcuate nucleus of the hypothalamus (ARH) reveal differentially methylated genomic regions and reduced expression of AgRP neuron-associated genes in knockout mice. We use read-level analysis of WGBS data to infer putative ARH neural cell types affected by the knockout, and to localize promoter hypomethylation and increased expression of the growth factor Bmp7 to AgRP neurons, suggesting a role for aberrant TGF-β signaling in the development of this phenotype. Together, these data demonstrate that DNA methylation in AgRP neurons is required for their normal epigenetic development and neuron-specific gene expression profiles, and regulates voluntary exercise behavior.
Our purpose was to analyze the effect on gastrointestinal (GI) toxicity models when their dose-volume metrics predictors are derived from segmentations of the peritoneal cavity after different ...contouring approaches.
A random forest machine learning approach was used to predict acute grade ≥3 GI toxicity from dose-volume metrics and clinicopathologic factors for 246 patients (toxicity incidence = 9.5%) treated with definitive chemoradiation for squamous cell carcinoma of the anus. Three types of random forest models were constructed based on different bowel bag segmentation approaches: (1) physician-delineated after Radiation Therapy Oncology Group (RTOG) guidelines, (2) autosegmented by a deep learning model (nnU-Net) following RTOG guidelines, and (3) autosegmented but spanning the entire bowel space. Each model type was evaluated using repeated cross-validation (100 iterations; 50%/50% training/test split). The performance of the models was assessed using area under the precision-recall curve (AUPRC) and the receiver operating characteristic curve (AUROCC), as well as optimal F1 score.
When following RTOG guidelines, the models based on the nnU-Net auto segmentations (mean values: AUROCC, 0.71 ± 0.07; AUPRC, 0.42 ± 0.09; F1 score, 0.46 ± 0.08) significantly outperformed (P < .001) those based on the physician-delineated contours (mean values: AUROCC, 0.67 ± 0.07; AUPRC, 0.34 ± 0.08; F1 score, 0.36 ± 0.07). When spanning the entire bowel space, the performance of the autosegmentation models improved considerably (mean values: AUROCC, 0.87 ± 0.05; AUPRC, 0.70 ± 0.09; F1 score, 0.68 ± 0.09).
Random forest models were superior at predicting acute grade ≥3 GI toxicity when based on RTOG-defined bowel bag autosegmentations rather than physician-delineated contours. Models based on autosegmentations spanning the entire bowel space show further considerable improvement in model performance. The results of this study should be further validated using an external data set.
Summary Objective To identify risk factors for radiographic signs of post-traumatic osteoarthritis (OA) 2–3 years after anterior cruciate ligament (ACL) reconstruction through multivariable analysis ...of minimum joint space width (mJSW) differences in a specially designed nested cohort. Methods A nested cohort within the Multicenter Orthopaedic Outcomes Network (MOON) cohort included 262 patients (148 females, average age 20) injured in sport who underwent ACL reconstruction in a previously uninjured knee, were 35 or younger, and did not have ACL revision or contralateral knee surgery. mJSW on semi-flexed radiographs was measured in the medial compartment using a validated computerized method. A multivariable generalized linear model was constructed to assess mJSW difference between the ACL reconstructed and contralateral control knees while adjusting for potential confounding factors. Results Unexpectedly, we found the mean mJSW was 0.35 mm wider in ACL reconstructed than in control knees (5.06 mm (95% CI 4.96–5.15 mm) vs 4.71 mm (95% CI 4.62–4.80 mm), P < 0.001). However, ACL reconstructed knees with meniscectomy had narrower mJSW compared to contralateral normal knees by 0.64 mm (95% C.I. 0.38–0.90 mm) ( P < 0.001). Age ( P < 0.001) and meniscus repair ( P = 0.001) were also significantly associated with mJSW difference. Conclusion Semi-flexed radiographs can detect differences in mJSW between ACL reconstructed and contralateral normal knees 2–3 years following ACL reconstruction, and the unexpected wider mJSW in ACL reconstructed knees may represent the earliest manifestation of post-traumatic osteoarthritis and warrants further study.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Background:
Anterior cruciate ligament (ACL) reconstruction can effectively return athletes to the playing field, but they are still at risk of developing posttraumatic osteoarthritis (PTOA). No ...studies have used multivariable analysis to evaluate the predictors of radiographic PTOA in the lateral compartment of the knee at short-term follow-up after ACL reconstruction.
Purpose:
To determine the predictors of radiographic joint space narrowing in the lateral compartment 2 to 3 years after ACL reconstruction in a young, active cohort.
Study Design:
Cohort study; Level of evidence, 2.
Methods:
A nested cohort of 358 patients from the Multicenter Orthopaedic Outcomes Network (MOON) prospective cohort who were aged ≤33 years, were injured playing a sport, and had never undergone surgery on the contralateral knee were followed up 2 years after ACL reconstruction with questionnaires and with weightbearing knee radiographs using the metatarsophalangeal (MTP) joint technique. The joint space width in the lateral compartment was measured using a semiautomatic computerized method, and multivariable predictive modeling was used to evaluate the relationship between meniscus treatment, cartilage injury, graft type, and joint space while adjusting for age, sex, body mass index, and Marx activity score.
Results:
The mean lateral joint space width was 0.11 mm narrower on the ACL-reconstructed knee compared with the contralateral healthy knee (7.69 mm vs 7.80 mm, respectively; P < .01). Statistically significant predictors of a narrower joint space width on the ACL-reconstructed knee included lateral meniscectomy (P < .001) and a Marx activity score less than 16 points (P < .001).
Conclusion:
This study identifies lateral meniscectomy and a lower baseline Marx activity score to be predictors of radiographic joint space narrowing in the lateral compartment 2 to 3 years after ACL reconstruction in young, active patients without a prior knee injury.
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FSPLJ, NUK, OILJ, SAZU, UKNU, UL, UM, UPUK
Automatic review of breast plan quality for clinical trials is time-consuming and has some unique challenges due to the lack of target contours for some planning techniques. We propose using an ...auto-contouring model and statistical process control to independently assess planning consistency in retrospective data from a breast radiotherapy clinical trial.
A deep learning auto-contouring model was created and tested quantitatively and qualitatively on 104 post-lumpectomy patients’ computed tomography images (nnUNet; train/test: 80/20). The auto-contouring model was then applied to 127 patients enrolled in a clinical trial. Statistical process control was used to assess the consistency of the mean dose to auto-contours between plans and treatment modalities by setting control limits within three standard deviations of the data’s mean. Two physicians reviewed plans outside the limits for possible planning inconsistencies.
Mean Dice similarity coefficients comparing manual and auto-contours was above 0.7 for breast clinical target volume, supraclavicular and internal mammary nodes. Two radiation oncologists scored 95% of contours as clinically acceptable. The mean dose in the clinical trial plans was more variable for lymph node auto-contours than for breast, with a narrower distribution for volumetric modulated arc therapy than for 3D conformal treatment, requiring distinct control limits. Five plans (5%) were flagged and reviewed by physicians: one required editing, two had clinically acceptable variations in planning, and two had poor auto-contouring.
An automated contouring model in a statistical process control framework was appropriate for assessing planning consistency in a breast radiotherapy clinical trial.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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