Mechanism of ERBB2 gene overexpression by the formation of super-enhancer with genomic structural abnormalities in lung adenocarcinoma without clinically actionable genetic alterations.

BACKGROUND: In an extensive genomic analysis of lung adenocarcinomas (LUADs), driver mutations have been recognized as potential targets for molecular therapy. However, there remain cases where target genes are not identified. Super-enhancers and structural variants are frequently identified in several hundred loci per case. Despite this, most cancer research has approached the analysis of these data sets separately, without merging and comparing the data, and there are no examples of integrated analysis in LUAD. METHODS: We performed an integrated analysis of super-enhancers and structural variants in a cohort of 174 LUAD cases that lacked clinically actionable genetic alterations. To achieve this, we conducted both WGS and H3K27Ac ChIP-seq analyses using samples with driver gene mutations and those without, allowing for a comprehensive investigation of the potential roles of super-enhancer in LUAD cases. RESULTS: We demonstrate that most genes situated in these overlapped regions were associated with known and previously unknown driver genes and aberrant expression resulting from the formation of super-enhancers accompanied by genomic structural abnormalities. Hi-C and long-read sequencing data further corroborated this insight. When we employed CRISPR-Cas9 to induce structural abnormalities that mimicked cases with outlier ERBB2 gene expression, we observed an elevation in ERBB2 expression. These abnormalities are associated with a higher risk of recurrence after surgery, irrespective of the presence or absence of driver mutations. CONCLUSIONS: Our findings suggest that aberrant gene expression linked to structural polymorphisms can significantly impact personalized cancer treatment by facilitating the identification of driver mutations and prognostic factors, contributing to a more comprehensive understanding of LUAD pathogenesis.

Advances in cancer DNA methylation analysis with methPLIER: use of non-negative matrix factorization and knowledge-based constraints to enhance biological interpretability.

DNA methylation is an epigenetic modification that results in dynamic changes during ontogenesis and cell differentiation. DNA methylation patterns regulate gene expression and have been widely researched. While tools for DNA methylation analysis have been developed, most of them have focused on intergroup comparative analysis within a dataset; therefore, it is difficult to conduct cross-dataset studies, such as rare disease studies or cross-institutional studies. This study describes a novel method for DNA methylation analysis, namely, methPLIER, which enables interdataset comparative analyses. methPLIER combines Pathway Level Information Extractor (PLIER), which is a non-negative matrix factorization (NMF) method, with regularization by a knowledge matrix and transfer learning. methPLIER can be used to perform intersample and interdataset comparative analysis based on latent feature matrices, which are obtained via matrix factorization of large-scale data, and factor-loading matrices, which are obtained through matrix factorization of the data to be analyzed. We used methPLIER to analyze a lung cancer dataset and confirmed that the data decomposition reflected sample characteristics for recurrence-free survival. Moreover, methPLIER can analyze data obtained via different preprocessing methods, thereby reducing distributional bias among datasets due to preprocessing. Furthermore, methPLIER can be employed for comparative analyses of methylation data obtained from different platforms, thereby reducing bias in data distribution due to platform differences. methPLIER is expected to facilitate cross-sectional DNA methylation data analysis and enhance DNA methylation data resources.

A discrimination model by machine learning to avoid gastrectomy for early gastric cancer.

Aim: Gastrectomy is recommended for patients with early gastric cancer (EGC) because the possibility of lymph node metastasis (LNM) cannot be completely denied. The aim of this study was to develop a discrimination model to select patients who do not require surgery using machine learning. Methods: Data from 382 patients who received gastrectomy for gastric cancer and who were diagnosed with pT1b were extracted for developing a discrimination model. For the validation of this discrimination model, data from 140 consecutive patients who underwent endoscopic resection followed by gastrectomy, with a diagnosis of pT1b EGC, were extracted. We applied XGBoost to develop a discrimination model for clinical and pathological variables. The performance of the discrimination model was evaluated based on the number of cases classified as true negatives for LNM, with no false negatives for LNM allowed. Results: Lymph node metastasis was observed in 95 patients (25%) in the development cohort and 11 patients (8%) in the validation cohort. The discrimination model was developed to identify 27 (7%) patients with no indications for additional surgery due to the prediction of an LNM-negative status with no false negatives. In the validation cohort, 13 (9%) patients were identified as having no indications for additional surgery and no patients with LNM were classified into this group. Conclusion: The discrimination model using XGBoost algorithms could select patients with no risk of LNM from patients with pT1b EGC. This discrimination model was considered promising for clinical decision-making in relation to patients with EGC.

Analysis of super-enhancer using machine learning and its application to medical biology.

The analysis of super-enhancers (SEs) has recently attracted attention in elucidating the molecular mechanisms of cancer and other diseases. SEs are genomic structures that strongly induce gene expression and have been reported to contribute to the overexpression of oncogenes. Because the analysis of SEs and integrated analysis with other data are performed using large amounts of genome-wide data, artificial intelligence technology, with machine learning at its core, has recently begun to be utilized. In promoting precision medicine, it is important to consider information from SEs in addition to genomic data; therefore, machine learning technology is expected to be introduced appropriately in terms of building a robust analysis platform with a high generalization performance. In this review, we explain the history and principles of SE, and the results of SE analysis using state-of-the-art machine learning and integrated analysis with other data are presented to provide a comprehensive understanding of the current status of SE analysis in the field of medical biology. Additionally, we compared the accuracy between existing machine learning methods on the benchmark dataset and attempted to explore the kind of data preprocessing and integration work needed to make the existing algorithms work on the benchmark dataset. Furthermore, we discuss the issues and future directions of current SE analysis.

Variation of DNA methylation on the IRX1/2 genes is responsible for the neural differentiation propensity in human induced pluripotent stem cells.

Introduction: Human induced pluripotent stem cells (hiPSCs) are useful tools for reproducing neural development in vitro. However, each hiPSC line has a different ability to differentiate into specific lineages, known as differentiation propensity, resulting in reduced reproducibility and increased time and funding requirements for research. To overcome this issue, we searched for predictive signatures of neural differentiation propensity of hiPSCs focusing on DNA methylation, which is the main modulator of cellular properties. Methods: We obtained 32 hiPSC lines and their comprehensive DNA methylation data using the Infinium MethylationEPIC BeadChip. To assess the neural differentiation efficiency of these hiPSCs, we measured the percentage of neural stem cells on day 7 of induction. Using the DNA methylation data of undifferentiated hiPSCs and their measured differentiation efficiency into neural stem cells as the set of data, and HSIC Lasso, a machine learning-based nonlinear feature selection method, we attempted to identify neural differentiation-associated differentially methylated sites. Results: Epigenome-wide unsupervised clustering cannot distinguish hiPSCs with varying differentiation efficiencies. In contrast, HSIC Lasso identified 62 CpG sites that could explain the neural differentiation efficiency of hiPSCs. Features selected by HSIC Lasso were particularly enriched within 3 Mbp of chromosome 5, harboring IRX1, IRX2, and C5orf38 genes. Within this region, DNA methylation rates were correlated with neural differentiation efficiency and were negatively correlated with gene expression of the IRX1/2 genes, particularly in female hiPSCs. In addition, forced expression of the IRX1/2 impaired the neural differentiation ability of hiPSCs in both sexes. Conclusion: We for the first time showed that the DNA methylation state of the IRX1/2 genes of hiPSCs is a predictive biomarker of their potential for neural differentiation. The predictive markers for neural differentiation efficiency identified in this study may be useful for the selection of suitable undifferentiated hiPSCs prior to differentiation induction.

Repression of the PRELP gene is relieved by histone deacetylase inhibitors through acetylation of histone H2B lysine 5 in bladder cancer.

BACKGROUND: Proline/arginine-rich end leucine-rich repeat protein (PRELP) is a member of the small leucine-rich proteoglycan family of extracellular matrix proteins, which is markedly suppressed in the majority of early-stage epithelial cancers and plays a role in regulating the epithelial-mesenchymal transition by altering cell-cell adhesion. Although PRELP is an important factor in the development and progression of bladder cancer, the mechanism of PRELP gene repression remains unclear. RESULTS: Here, we show that repression of PRELP mRNA expression in bladder cancer cells is alleviated by HDAC inhibitors (HDACi) through histone acetylation. Using ChIP-qPCR analysis, we found that acetylation of lysine residue 5 of histone H2B in the PRELP gene promoter region is a marker for the de-repression of PRELP expression. CONCLUSIONS: These results suggest a mechanism through which HDACi may partially regulate the function of PRELP to suppress the development and progression of bladder cancer. Some HDACi are already in clinical use, and the findings of this study provide a mechanistic basis for further investigation of HDACi-based therapeutic strategies.

Introducing AI to the molecular tumor board: one direction toward the establishment of precision medicine using large-scale cancer clinical and biological information.

Since U.S. President Barack Obama announced the Precision Medicine Initiative in his New Year's State of the Union address in 2015, the establishment of a precision medicine system has been emphasized worldwide, particularly in the field of oncology. With the advent of next-generation sequencers specifically, genome analysis technology has made remarkable progress, and there are active efforts to apply genome information to diagnosis and treatment. Generally, in the process of feeding back the results of next-generation sequencing analysis to patients, a molecular tumor board (MTB), consisting of experts in clinical oncology, genetic medicine, etc., is established to discuss the results. On the other hand, an MTB currently involves a large amount of work, with humans searching through vast databases and literature, selecting the best drug candidates, and manually confirming the status of available clinical trials. In addition, as personalized medicine advances, the burden on MTB members is expected to increase in the future. Under these circumstances, introducing cutting-edge artificial intelligence (AI) technology and information and communication technology to MTBs while reducing the burden on MTB members and building a platform that enables more accurate and personalized medical care would be of great benefit to patients. In this review, we introduced the latest status of elemental technologies that have potential for AI utilization in MTB, and discussed issues that may arise in the future as we progress with AI implementation.

Application of non-negative matrix factorization in oncology: one approach for establishing precision medicine.

The increase in the expectations of artificial intelligence (AI) technology has led to machine learning technology being actively used in the medical field. Non-negative matrix factorization (NMF) is a machine learning technique used for image analysis, speech recognition, and language processing; recently, it is being applied to medical research. Precision medicine, wherein important information is extracted from large-scale medical data to provide optimal medical care for every individual, is considered important in medical policies globally, and the application of machine learning techniques to this end is being handled in several ways. NMF is also introduced differently because of the characteristics of its algorithms. In this review, the importance of NMF in the field of medicine, with a focus on the field of oncology, is described by explaining the mathematical science of NMF and the characteristics of the algorithm, providing examples of how NMF can be used to establish precision medicine, and presenting the challenges of NMF. Finally, the direction regarding the effective use of NMF in the field of oncology is also discussed.

Single-Cell Analysis Using Machine Learning Techniques and Its Application to Medical Research.

In recent years, the diversity of cancer cells in tumor tissues as a result of intratumor heterogeneity has attracted attention. In particular, the development of single-cell analysis technology has made a significant contribution to the field; technologies that are centered on single-cell RNA sequencing (scRNA-seq) have been reported to analyze cancer constituent cells, identify cell groups responsible for therapeutic resistance, and analyze gene signatures of resistant cell groups. However, although single-cell analysis is a powerful tool, various issues have been reported, including batch effects and transcriptional noise due to gene expression variation and mRNA degradation. To overcome these issues, machine learning techniques are currently being introduced for single-cell analysis, and promising results are being reported. In addition, machine learning has also been used in various ways for single-cell analysis, such as single-cell assay of transposase accessible chromatin sequencing (ATAC-seq), chromatin immunoprecipitation sequencing (ChIP-seq) analysis, and multi-omics analysis; thus, it contributes to a deeper understanding of the characteristics of human diseases, especially cancer, and supports clinical applications. In this review, we present a comprehensive introduction to the implementation of machine learning techniques in medical research for single-cell analysis, and discuss their usefulness and future potential.

Application of Artificial Intelligence in COVID-19 Diagnosis and Therapeutics.

The coronavirus disease 2019 (COVID-19) pandemic began at the end of December 2019, giving rise to a high rate of infections and causing COVID-19-associated deaths worldwide. It was first reported in Wuhan, China, and since then, not only global leaders, organizations, and pharmaceutical/biotech companies, but also researchers, have directed their efforts toward overcoming this threat. The use of artificial intelligence (AI) has recently surged internationally and has been applied to diverse aspects of many problems. The benefits of using AI are now widely accepted, and many studies have shown great success in medical research on tasks, such as the classification, detection, and prediction of disease, or even patient outcome. In fact, AI technology has been actively employed in various ways in COVID-19 research, and several clinical applications of AI-equipped medical devices for the diagnosis of COVID-19 have already been reported. Hence, in this review, we summarize the latest studies that focus on medical imaging analysis, drug discovery, and therapeutics such as vaccine development and public health decision-making using AI. This survey clarifies the advantages of using AI in the fight against COVID-19 and provides future directions for tackling the COVID-19 pandemic using AI techniques.

Epigenetic Mechanisms Underlying COVID-19 Pathogenesis.

In 2019, a novel severe acute respiratory syndrome called coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was reported and was declared a pandemic by the World Health Organization (WHO) in March 2020. With the advancing development of COVID-19 vaccines and their administration globally, it is expected that COVID-19 will converge in the future; however, the situation remains unpredictable because of a series of reports regarding SARS-CoV-2 variants. Currently, there are still few specific effective treatments for COVID-19, as many unanswered questions remain regarding the pathogenic mechanism of COVID-19. Continued elucidation of COVID-19 pathogenic mechanisms is a matter of global importance. In this regard, recent reports have suggested that epigenetics plays an important role; for instance, the expression of angiotensin I converting enzyme 2 (ACE2) receptor, an important factor in human infection with SARS-CoV-2, is epigenetically regulated; further, DNA methylation status is reported to be unique to patients with COVID-19. In this review, we focus on epigenetic mechanisms to provide a new molecular framework for elucidating the pathogenesis of SARS-CoV-2 infection in humans and of COVID-19, along with the possibility of new diagnostic and therapeutic strategies.

Integrated Analysis of Whole Genome and Epigenome Data Using Machine Learning Technology: Toward the Establishment of Precision Oncology.

With the completion of the International Human Genome Project, we have entered what is known as the post-genome era, and efforts to apply genomic information to medicine have become more active. In particular, with the announcement of the Precision Medicine Initiative by U.S. President Barack Obama in his State of the Union address at the beginning of 2015, "precision medicine," which aims to divide patients and potential patients into subgroups with respect to disease susceptibility, has become the focus of worldwide attention. The field of oncology is also actively adopting the precision oncology approach, which is based on molecular profiling, such as genomic information, to select the appropriate treatment. However, the current precision oncology is dominated by a method called targeted-gene panel (TGP), which uses next-generation sequencing (NGS) to analyze a limited number of specific cancer-related genes and suggest optimal treatments, but this method causes the problem that the number of patients who benefit from it is limited. In order to steadily develop precision oncology, it is necessary to integrate and analyze more detailed omics data, such as whole genome data and epigenome data. On the other hand, with the advancement of analysis technologies such as NGS, the amount of data obtained by omics analysis has become enormous, and artificial intelligence (AI) technologies, mainly machine learning (ML) technologies, are being actively used to make more efficient and accurate predictions. In this review, we will focus on whole genome sequencing (WGS) analysis and epigenome analysis, introduce the latest results of omics analysis using ML technologies for the development of precision oncology, and discuss the future prospects.

Identification of an epigenetic signature in human induced pluripotent stem cells using a linear machine learning model.

The use of human induced pluripotent stem cells (iPSCs), used as an alternative to human embryonic stem cells (ESCs), is a potential solution to challenges, such as immune rejection, and does not involve the ethical issues concerning the use of ESCs in regenerative medicine, thereby enabling developments in biological research. However, comparative analyses from previous studies have not indicated any specific feature that distinguishes iPSCs from ESCs. Therefore, in this study, we established a linear classification-based learning model to distinguish among ESCs, iPSCs, embryonal carcinoma cells (ECCs), and somatic cells on the basis of their DNA methylation profiles. The highest accuracy achieved by the learned models in identifying the cell type was 94.23%. In addition, the epigenetic signature of iPSCs, which is distinct from that of ESCs, was identified by component analysis of the learned models. The iPSC-specific regions with methylation fluctuations were abundant on chromosomes 7, 8, 12, and 22. The method developed in this study can be utilized with comprehensive data and widely applied to many aspects of molecular biology research.

Application of Artificial Intelligence Technology in Oncology: Towards the Establishment of Precision Medicine.

In recent years, advances in artificial intelligence (AI) technology have led to the rapid clinical implementation of devices with AI technology in the medical field. More than 60 AI-equipped medical devices have already been approved by the Food and Drug Administration (FDA) in the United States, and the active introduction of AI technology is considered to be an inevitable trend in the future of medicine. In the field of oncology, clinical applications of medical devices using AI technology are already underway, mainly in radiology, and AI technology is expected to be positioned as an important core technology. In particular, "precision medicine," a medical treatment that selects the most appropriate treatment for each patient based on a vast amount of medical data such as genome information, has become a worldwide trend; AI technology is expected to be utilized in the process of extracting truly useful information from a large amount of medical data and applying it to diagnosis and treatment. In this review, we would like to introduce the history of AI technology and the current state of medical AI, especially in the oncology field, as well as discuss the possibilities and challenges of AI technology in the medical field.

Predicting Deep Learning Based Multi-Omics Parallel Integration Survival Subtypes in Lung Cancer Using Reverse Phase Protein Array Data.

Mortality attributed to lung cancer accounts for a large fraction of cancer deaths worldwide. With increasing mortality figures, the accurate prediction of prognosis has become essential. In recent years, multi-omics analysis has emerged as a useful survival prediction tool. However, the methodology relevant to multi-omics analysis has not yet been fully established and further improvements are required for clinical applications. In this study, we developed a novel method to accurately predict the survival of patients with lung cancer using multi-omics data. With unsupervised learning techniques, survival-associated subtypes in non-small cell lung cancer were first detected using the multi-omics datasets from six categories in The Cancer Genome Atlas (TCGA). The new subtypes, referred to as integration survival subtypes, clearly divided patients into longer and shorter-surviving groups (log-rank test: p = 0.003) and we confirmed that this is independent of histopathological classification (Chi-square test of independence: p = 0.94). Next, an attempt was made to detect the integration survival subtypes using only one categorical dataset. Our machine learning model that was only trained on the reverse phase protein array (RPPA) could accurately predict the integration survival subtypes (AUC = 0.99). The predicted subtypes could also distinguish between high and low risk patients (log-rank test: p = 0.012). Overall, this study explores novel potentials of multi-omics analysis to accurately predict the prognosis of patients with lung cancer.

Critical Roles of N6-Methyladenosine (m6A) in Cancer and Virus Infection.

Studies have shown that epigenetic abnormalities are involved in various diseases, including cancer. In particular, in order to realize precision medicine, the integrated analysis of genetics and epigenetics is considered to be important; detailed epigenetic analysis in the medical field has been becoming increasingly important. In the epigenetics analysis, DNA methylation and histone modification analyses have been actively studied for a long time, and many important findings were accumulated. On the other hand, recently, attention has also been focused on RNA modification in the field of epigenetics; now it is known that RNA modification is associated with various biological functions, such as regulation of gene expression. Among RNA modifications, functional analysis of N6-methyladenosine (m6A), the most abundant RNA modification found from humans to plants is actively progressing, and it has also been known that m6A abnormality is involved in cancer and other diseases. Importantly, recent studies have shown that m6A is related to viral infections. Considering the current world situation under threat of viral infections, it is important to deepen knowledge of RNA modification from the viewpoint of viral diseases. Hence, in this review, we have summarized the recent findings regarding the roles of RNA modifications in biological functions, cancer biology, and virus infection, particularly focusing on m6A in mRNA.

Dog Steroidogenic Factor-1: Molecular cloning and analysis of epigenetic regulation.

Steroidogenic factor 1 (SF-1) is a nuclear receptor that is important in steroid hormone production, and adrenal and gonad development. The SF-1 gene is highly conserved among most vertebrates. However, dog SF-1 registered in public databases, such as CanFam3.1, lacks the 5' end compared to other mammals including mouse, human, bovine, and cat. Whether this defect is due to species differences or database error is unclear. Here, we determined the full-length dog SF-1 cDNA sequence and identified the missing 5' end sequence in the databases. The coding region of the dog SF-1 gene has 1,386 base pairs, and the protein has 461 amino acid residues. Sequence alignment analysis among vertebrates revealed that the 5' end sequence of dog SF-1 cDNA is highly conserved compared to other vertebrates. The genomic position of the first exon was determined, and its promoter region sequence was analyzed. The DNA methylation state at the basal promoter and the expression of dog SF-1 in steroidogenic tissues and non-steroidogenic cells were examined. CpG sites at the basal promoter displayed methylation kinetics inversely correlated with gene expression. The promoter was hypomethylated and hypermethylated in SF-1 expressing and non-SF-1 expressing tissues, respectively. In conclusion, we identified the true full sequence of dog SF-1 cDNA and determined the genome sequence around the first exon. The gene is under the control of epigenetic regulation, such as DNA methylation, at the promoter.

Uncovering Prognosis-Related Genes and Pathways by Multi-Omics Analysis in Lung Cancer.

Lung cancer is one of the leading causes of death worldwide. Therefore, understanding the factors linked to patient survival is essential. Recently, multi-omics analysis has emerged, allowing for patient groups to be classified according to prognosis and at a more individual level, to support the use of precision medicine. Here, we combined RNA expression and miRNA expression with clinical information, to conduct a multi-omics analysis, using publicly available datasets (the cancer genome atlas (TCGA) focusing on lung adenocarcinoma (LUAD)). We were able to successfully subclass patients according to survival. The classifiers we developed, using inferred labels obtained from patient subtypes showed that a support vector machine (SVM), gave the best classification results, with an accuracy of 0.82 with the test dataset. Using these subtypes, we ranked genes based on RNA expression levels. The top 25 genes were investigated, to elucidate the mechanisms that underlie patient prognosis. Bioinformatics analyses showed that the expression levels of six out of 25 genes (ERO1B, DPY19L1, NCAM1, RET, MARCH1, and SLC7A8) were associated with LUAD patient survival (p < 0.05), and pathway analyses indicated that major cancer signaling was altered in the subtypes.

Epigenetics Analysis and Integrated Analysis of Multiomics Data, Including Epigenetic Data, Using Artificial Intelligence in the Era of Precision Medicine.

To clarify the mechanisms of diseases, such as cancer, studies analyzing genetic mutations have been actively conducted for a long time, and a large number of achievements have already been reported. Indeed, genomic medicine is considered the core discipline of precision medicine, and currently, the clinical application of cutting-edge genomic medicine aimed at improving the prevention, diagnosis and treatment of a wide range of diseases is promoted. However, although the Human Genome Project was completed in 2003 and large-scale genetic analyses have since been accomplished worldwide with the development of next-generation sequencing (NGS), explaining the mechanism of disease onset only using genetic variation has been recognized as difficult. Meanwhile, the importance of epigenetics, which describes inheritance by mechanisms other than the genomic DNA sequence, has recently attracted attention, and, in particular, many studies have reported the involvement of epigenetic deregulation in human cancer. So far, given that genetic and epigenetic studies tend to be accomplished independently, physiological relationships between genetics and epigenetics in diseases remain almost unknown. Since this situation may be a disadvantage to developing precision medicine, the integrated understanding of genetic variation and epigenetic deregulation appears to be now critical. Importantly, the current progress of artificial intelligence (AI) technologies, such as machine learning and deep learning, is remarkable and enables multimodal analyses of big omics data. In this regard, it is important to develop a platform that can conduct multimodal analysis of medical big data using AI as this may accelerate the realization of precision medicine. In this review, we discuss the importance of genome-wide epigenetic and multiomics analyses using AI in the era of precision medicine.

Epigenetic-scale comparison of human iPSCs generated by retrovirus, Sendai virus or episomal vectors.

Human induced pluripotent stem cells (iPSCs) are established by introducing several reprogramming factors, such as OCT3/4, SOX2, KLF4, c-MYC. Because of their pluripotency and immortality, iPSCs are considered to be a powerful tool for regenerative medicine. To date, iPSCs have been established all over the world by various gene delivery methods. All methods induced high-quality iPSCs, but epigenetic analysis of abnormalities derived from differences in the gene delivery methods has not yet been performed. Here, we generated genetically matched human iPSCs from menstrual blood cells by using three kinds of vectors, i.e., retrovirus, Sendai virus, and episomal vectors, and compared genome-wide DNA methylation profiles among them. Although comparison of aberrant methylation revealed that iPSCs generated by Sendai virus vector have lowest number of aberrant methylation sites among the three vectors, the iPSCs generated by non-integrating methods did not show vector-specific aberrant methylation. However, the differences between the iPSC lines were determined to be the number of random aberrant hypermethylated regions compared with embryonic stem cells. These random aberrant hypermethylations might be a cause of the differences in the properties of each of the iPSC lines.