Genomic DNA Methylation-Derived Algorithm Enables Accurate Detection of Malignant Prostate Tissues.

Introduction: The current methodology involving diagnosis of prostate cancer (PCa) relies on the pathology examination of prostate needle biopsies, a method with high false negative rates partly due to temporospatial, molecular, and morphological heterogeneity of prostate adenocarcinoma. It is postulated that molecular markers have a potential to assign diagnosis to a considerable portion of undetected prostate tumors. This study examines the genome-wide DNA methylation changes in PCa in search of genomic markers for the development of a diagnostic algorithm for PCa screening. Methods: Archival PCa and normal tissues were assessed using genomic DNA methylation arrays. Differentially methylated sites and regions (DMRs) were used for functional assessment, gene-set enrichment and protein interaction analyses, and examination of transcription factor-binding patterns. Raw signal intensity data were used for identification of recurrent copy number variations (CNVs). Non-redundant fully differentiating cytosine-phosphate-guanine sites (CpGs), which did not overlap CNV segments, were used in an L1 regularized logistic regression model (LASSO) to train a classification algorithm. Validation of this algorithm was performed using a large external cohort of benign and tumor prostate arrays. Results: Approximately 6,000 probes and 600 genomic regions showed significant DNA methylation changes, primarily involving hypermethylation. Gene-set enrichment and protein interaction analyses found an overrepresentation of genes related to cell communications, neurogenesis, and proliferation. Motif enrichment analysis demonstrated enrichment of tumor suppressor-binding sites nearby DMRs. Several of these regions were also found to contain copy number amplifications. Using four non-redundant fully differentiating CpGs, we trained a classification model with 100% accuracy in discriminating tumors from benign samples. Validation of this algorithm using an external cohort of 234 tumors and 92 benign samples yielded 96% sensitivity and 98% specificity. The model was found to be highly sensitive to detect metastatic lesions in bone, lymph node, and soft tissue, while being specific enough to differentiate the benign hyperplasia of prostate from tumor. Conclusion: A considerable component of PCa DNA methylation profile represent driver events potentially established/maintained by disruption of tumor suppressor activity. As few as four CpGs from this profile can be used for screening of PCa

Peripheral Blood Epi-Signature of Claes-Jensen Syndrome Enables Sensitive and Specific Identification of Patients and Healthy Carriers with Pathogenic Mutations in KDM5C.

Background: Claes-Jensen syndrome is an X-linked inherited intellectual disability caused by mutations in the KDM5C gene. Kdm5c is a histone lysine demethylase involved in histone modifications and chromatin remodeling. Males with hemizygous mutations in KDM5C present with intellectual disability and facial dysmorphism, while most heterozygous female carriers are asymptomatic. We hypothesized that loss of KDM5C function may influence other components of the epigenomic machinery including DNA methylation in affected patients. Results: Genome-wide DNA methylation analysis of 7 male patients affected with Claes-Jensen syndrome and 56 age- and sex-matched controls identified a specific DNA methylation defect (epi-signature) in the peripheral blood of these patients, including 1769 individual CpGs and 9 genomic regions. Six healthy female carriers showed less pronounced but distinctive changes in the same regions enabling their differentiation from both patients and controls. Highly specific computational model using the most significant methylation changes demonstrated 100% accuracy in differentiating patients, carriers, and controls in the training cohort, which was confirmed on a separate cohort of patients and carriers. The 100% specificity of this unique epi-signature was further confirmed on additional 500 unaffected controls and 600 patients with intellectual disability and developmental delay, including other patient cohorts with previously described epi-signatures. Conclusion: Peripheral blood epi-signature in Claes-Jensen syndrome can be used for molecular diagnosis and carrier identification and assist with interpretation of genetic variants of unknown clinical significance in the KDM5Cgene

Genetic and epigenetic profiling of BRCA1/2 in ovarian tumors reveals additive diagnostic yield and evidence of a genomic BRCA1/2 DNA methylation signature

Poly-ADP-ribose-polymerase inhibitor (PARPi) treatment is indicated for advanced-stage ovarian tumors with BRCA1/2 deficiency. The “BRCAness” status is thought to be attributed to a tumor phenotype associated with a specific epigenomic DNA methylation profile. Here, we examined the diagnostic impact of combined BRCA1/2 sequence, copy number, and promoter DNA methylation analysis, and evaluated whether genomic DNA methylation patterns can predict the BRCAness in ovarian tumors. DNA sequencing of 172 human tissue samples of advanced-stage ovarian adenocarcinoma identified 36 samples with a clinically significant tier 1/2 sequence variants (point mutations and in/dels) and 9 samples with a CNV causing a loss of function in BRCA1/2. DNA methylation analysis of the promoter of BRCA1/2 identified promoter hypermethylation of BRCA1 in two mutation-negative samples. Computational modeling of genome-wide methylation markers, measured using Infinium EPIC arrays, resulted in a total accuracy of 0.75, sensitivity: 0.83, specificity: 0.64, positive predictive value: 0.76, negative predictive value: 0.74, and area under the receiver’s operating curve (AUC): 0.77, in classifying tumors harboring a BRCA1/2 defect from the rest. These findings indicate that the assessment of CNV and promoter DNA methylation in BRCA1/2 increases the cumulative diagnostic yield by 10%, compared with the 20% yield achieved by sequence variant analysis alone. Genomic DNA methylation data can partially predict BRCAness in ovarian tumors; however, further investigation in expanded BRCA1/2 cohorts is needed, and the effect of other double strand DNA repair gene defects in these tumors warrants further investigations

Incidental findings from cancer next generation sequencing panels

Next-generation sequencing (NGS) technologies have facilitated multi-gene panel (MGP) testing to detect germline DNA variants in hereditary cancer patients. This sensitive technique can uncover unexpected, non-germline incidental findings indicative of mosaicism, clonal hematopoiesis (CH), or hematologic malignancies. A retrospective chart review was conducted to identify cases of incidental findings from NGS-MGP testing. Inclusion criteria included: 1) multiple pathogenic variants in the same patient; 2) pathogenic variants at a low allele fraction; and/or 3) the presence of pathogenic variants not consistent with family history. Secondary tissue analysis, complete blood count (CBC) and medical record review were conducted to further delineate the etiology of the pathogenic variants. Of 6060 NGS-MGP tests, 24 cases fulfilling our inclusion criteria were identified. Pathogenic variants were detected in TP53, ATM, CHEK2, BRCA1 and APC. 18/24 (75.0%) patients were classified as CH, 3/24 (12.5%) as mosaic, 2/24 (8.3%) related to a hematologic malignancy, and 1/24 (4.2%) as true germline. We describe a case-specific workflow to identify and interpret the nature of incidental findings on NGS-MGP. This workflow will provide oncology and genetic clinics a practical guide for the management and counselling of patients with unexpected NGS-MGP findings

The Different Effects of Skeletal Muscle and Fat Mass on Height Increment in Children and Adolescents Aged 6–11 Years: A Cohort Study From China

ObjectiveThis study aimed to investigate the contribution of body composition including skeletal muscle mass (SMM) and body fat mass (BFM) to longitudinal growth among children and adolescents aged 6–11 years old.MethodsThis cohort study was conducted from the annual health examination between 2019 and 2020. Annual height gain and weight gain and changes in SMM and BFM were calculated and compared between sexes, different nutritional status, and growth curve shifting mode. Spearman analyses and multiple linear regression analysis were performed to identify the impact of SMM, BFM, or body mass index (BMI) on height gain.ResultsOf the 584 subjects, the annual height gains of boys (4.76 cm in the 6–9-year group and 4.63 cm in the 10–11-year group) were significantly lower than those of girls (5.48 and 5.74 cm, respectively). Spearman analysis showed that SMM gain and height gain were positively and significantly correlated in each examination of all children (r = 0.535 for boys and 0.734 for girls, p < 0.001). Conversely, BFM and height gains were negatively (r = -0.5240 for boys and -0.232 for girls, p < 0.001) correlated. Multiple linear regression analysis identified SMM gain as an independent predictor (95% CI: 1.20,1.44) of height gain after adjusting for age, gender, BMI, BFM gain, and percentage of body fat (PBF).ConclusionSMM gains, rather than BFM gains, were associated with height gains in children and adolescents aged 6–11 years. Monitoring SMM changes in routine healthcare might motivate children and adolescents to achieve dietary and exercise recommendations, thereby growing taller without gaining excessive weight

Interface-engineered ferroelectricity of epitaxial Hf\u3csub\u3e0.5\u3c/sub\u3eZr\u3csub\u3e0.5\u3c/sub\u3eO\u3csub\u3e2\u3c/sub\u3e thin films

Ferroelectric hafnia-based thin films have attracted intense attention due to their compatibility with complementary metal-oxide-semiconductor technology. However, the ferroelectric orthorhombic phase is thermodynamically metastable. Various efforts have been made to stabilize the ferroelectric orthorhombic phase of hafnia-based films such as controlling the growth kinetics and mechanical confinement. Here, we demonstrate a key interface engineering strategy to stabilize and enhance the ferroelectric orthorhombic phase of the Hf0.5Zr0.5O2 thin film by deliberately controlling the termination of the bottom La0.67Sr0.33MnO3 layer. We find that the Hf0.5Zr0.5O2 films on the MnO2-terminated La0.67Sr0.33MnO3 have more ferroelectric orthorhombic phase than those on the LaSrO-terminated La0.67Sr0.33MnO3, while with no wake-up effect. Even though the Hf0.5Zr0.5O2 thickness is as thin as 1.5nm, the clear ferroelectric orthorhombic (111) orientation is observed on the MnO2 termination. Our transmission electron microscopy characterization and theoretical modelling reveal that reconstruction at the Hf0.5Zr0.5O2/ La0.67Sr0.33MnO3 interface and hole doping of the Hf0.5Zr0.5O2 layer resulting from theMnO2 interface termination are responsible for the stabilization of the metastable ferroelectric phase of Hf0.5Zr0.5O2. We anticipate that these results will inspire further studies of interface-engineered hafnia-based systems

Diagnostic Utility of Genome-wide DNA Methylation Testing in Genetically Unsolved Individuals with Suspected Hereditary Conditions.

Conventional genetic testing of individuals with neurodevelopmental presentations and congenital anomalies (ND/CAs), i.e., the analysis of sequence and copy number variants, leaves a substantial proportion of them unexplained. Some of these cases have been shown to result from DNA methylation defects at a single locus (epi-variants), while others can exhibit syndrome-specific DNA methylation changes across multiple loci (epi-signatures). Here, we investigate the clinical diagnostic utility of genome-wide DNA methylation analysis of peripheral blood in unresolved ND/CAs. We generate a computational model enabling concurrent detection of 14 syndromes using DNA methylation data with full accuracy. We demonstrate the ability of this model in resolving 67 individuals with uncertain clinical diagnoses, some of whom had variants of unknown clinical significance (VUS) in the related genes. We show that the provisional diagnoses can be ruled out in many of the case subjects, some of whom are shown by our model to have other diseases initially not considered. By applying this model to a cohort of 965 ND/CA-affected subjects without a previous diagnostic assumption and a separate assessment of rare epi-variants in this cohort, we identify 15 case subjects with syndromic Mendelian disorders, 12 case subjects with imprinting and trinucleotide repeat expansion disorders, as well as 106 case subjects with rare epi-variants, a portion of which involved genes clinically or functionally linked to the subjects\u27 phenotypes. This study demonstrates that genomic DNA methylation analysis can facilitate the molecular diagnosis of unresolved clinical cases and highlights the potential value of epigenomic testing in the routine clinical assessment of ND/CAs

BAFopathies\u27 DNA methylation epi-signatures demonstrate diagnostic utility and functional continuum of Coffin-Siris and Nicolaides-Baraitser syndromes.

Coffin-Siris and Nicolaides-Baraitser syndromes (CSS and NCBRS) are Mendelian disorders caused by mutations in subunits of the BAF chromatin remodeling complex. We report overlapping peripheral blood DNA methylation epi-signatures in individuals with various subtypes of CSS (ARID1B, SMARCB1, and SMARCA4) and NCBRS (SMARCA2). We demonstrate that the degree of similarity in the epi-signatures of some CSS subtypes and NCBRS can be greater than that within CSS, indicating a link in the functional basis of the two syndromes. We show that chromosome 6q25 microdeletion syndrome, harboring ARID1B deletions, exhibits a similar CSS/NCBRS methylation profile. Specificity of this epi-signature was confirmed across a wide range of neurodevelopmental conditions including other chromatin remodeling and epigenetic machinery disorders. We demonstrate that a machine-learning model trained on this DNA methylation profile can resolve ambiguous clinical cases, reclassify those with variants of unknown significance, and identify previously undiagnosed subjects through targeted population screening

Evaluation of DNA Methylation Episignatures for Diagnosis and Phenotype Correlations in 42 Mendelian Neurodevelopmental Disorders.

Genetic syndromes frequently present with overlapping clinical features and inconclusive or ambiguous genetic findings which can confound accurate diagnosis and clinical management. An expanding number of genetic syndromes have been shown to have unique genomic DNA methylation patterns (called episignatures ). Peripheral blood episignatures can be used for diagnostic testing as well as for the interpretation of ambiguous genetic test results. We present here an approach to episignature mapping in 42 genetic syndromes, which has allowed the identification of 34 robust disease-specific episignatures. We examine emerging patterns of overlap, as well as similarities and hierarchical relationships across these episignatures, to highlight their key features as they are related to genetic heterogeneity, dosage effect, unaffected carrier status, and incomplete penetrance. We demonstrate the necessity of multiclass modeling for accurate genetic variant classification and show how disease classification using a single episignature at a time can sometimes lead to classification errors in closely related episignatures. We demonstrate the utility of this tool in resolving ambiguous clinical cases and identification of previously undiagnosed cases through mass screening of a large cohort of subjects with developmental delays and congenital anomalies. This study more than doubles the number of published syndromes with DNA methylation episignatures and, most significantly, opens new avenues for accurate diagnosis and clinical assessment in individuals affected by these disorders