Transcriptomics and epigenetic data integration learning module on Google Cloud.

Multi-omics (genomics, transcriptomics, epigenomics, proteomics, metabolomics, etc.) research approaches are vital for understanding the hierarchical complexity of human biology and have proven to be extremely valuable in cancer research and precision medicine. Emerging scientific advances in recent years have made high-throughput genome-wide sequencing a central focus in molecular research by allowing for the collective analysis of various kinds of molecular biological data from different types of specimens in a single tissue or even at the level of a single cell. Additionally, with the help of improved computational resources and data mining, researchers are able to integrate data from different multi-omics regimes to identify new prognostic, diagnostic, or predictive biomarkers, uncover novel therapeutic targets, and develop more personalized treatment protocols for patients. For the research community to parse the scientifically and clinically meaningful information out of all the biological data being generated each day more efficiently with less wasted resources, being familiar with and comfortable using advanced analytical tools, such as Google Cloud Platform becomes imperative. This project is an interdisciplinary, cross-organizational effort to provide a guided learning module for integrating transcriptomics and epigenetics data analysis protocols into a comprehensive analysis pipeline for users to implement in their own work, utilizing the cloud computing infrastructure on Google Cloud. The learning module consists of three submodules that guide the user through tutorial examples that illustrate the analysis of RNA-sequence and Reduced-Representation Bisulfite Sequencing data. The examples are in the form of breast cancer case studies, and the data sets were procured from the public repository Gene Expression Omnibus. The first submodule is devoted to transcriptomics analysis with the RNA sequencing data, the second submodule focuses on epigenetics analysis using the DNA methylation data, and the third submodule integrates the two methods for a deeper biological understanding. The modules begin with data collection and preprocessing, with further downstream analysis performed in a Vertex AI Jupyter notebook instance with an R kernel. Analysis results are returned to Google Cloud buckets for storage and visualization, removing the computational strain from local resources. The final product is a start-to-finish tutorial for the researchers with limited experience in multi-omics to integrate transcriptomics and epigenetics data analysis into a comprehensive pipeline to perform their own biological research.This manuscript describes the development of a resource module that is part of a learning platform named ``NIGMS Sandbox for Cloud-based Learning'' https://github.com/NIGMS/NIGMS-Sandbox. The overall genesis of the Sandbox is described in the editorial NIGMS Sandbox [16] at the beginning of this Supplement. This module delivers learning materials on the analysis of bulk and single-cell ATAC-seq data in an interactive format that uses appropriate cloud resources for data access and analyses.

CloudATAC: a cloud-based framework for ATAC-Seq data analysis.

Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) generates genome-wide chromatin accessibility profiles, providing valuable insights into epigenetic gene regulation at both pooled-cell and single-cell population levels. Comprehensive analysis of ATAC-seq data involves the use of various interdependent programs. Learning the correct sequence of steps needed to process the data can represent a major hurdle. Selecting appropriate parameters at each stage, including pre-analysis, core analysis, and advanced downstream analysis, is important to ensure accurate analysis and interpretation of ATAC-seq data. Additionally, obtaining and working within a limited computational environment presents a significant challenge to non-bioinformatic researchers. Therefore, we present Cloud ATAC, an open-source, cloud-based interactive framework with a scalable, flexible, and streamlined analysis framework based on the best practices approach for pooled-cell and single-cell ATAC-seq data. These frameworks use on-demand computational power and memory, scalability, and a secure and compliant environment provided by the Google Cloud. Additionally, we leverage Jupyter Notebook's interactive computing platform that combines live code, tutorials, narrative text, flashcards, quizzes, and custom visualizations to enhance learning and analysis. Further, leveraging GPU instances has significantly improved the run-time of the single-cell framework. The source codes and data are publicly available through NIH Cloud lab https://github.com/NIGMS/ATAC-Seq-and-Single-Cell-ATAC-Seq-Analysis. This manuscript describes the development of a resource module that is part of a learning platform named ``NIGMS Sandbox for Cloud-based Learning'' https://github.com/NIGMS/NIGMS-Sandbox. The overall genesis of the Sandbox is described in the editorial NIGMS Sandbox [1] at the beginning of this Supplement. This module delivers learning materials on the analysis of bulk and single-cell ATAC-seq data in an interactive format that uses appropriate cloud resources for data access and analyses.

Whole-genome bisulfite sequencing data analysis learning module on Google Cloud Platform.

This study describes the development of a resource module that is part of a learning platform named 'NIGMS Sandbox for Cloud-based Learning' https://github.com/NIGMS/NIGMS-Sandbox. The overall genesis of the Sandbox is described in the editorial NIGMS Sandbox at the beginning of this Supplement. This module is designed to facilitate interactive learning of whole-genome bisulfite sequencing (WGBS) data analysis utilizing cloud-based tools in Google Cloud Platform, such as Cloud Storage, Vertex AI notebooks and Google Batch. WGBS is a powerful technique that can provide comprehensive insights into DNA methylation patterns at single cytosine resolution, essential for understanding epigenetic regulation across the genome. The designed learning module first provides step-by-step tutorials that guide learners through two main stages of WGBS data analysis, preprocessing and the identification of differentially methylated regions. And then, it provides a streamlined workflow and demonstrates how to effectively use it for large datasets given the power of cloud infrastructure. The integration of these interconnected submodules progressively deepens the user's understanding of the WGBS analysis process along with the use of cloud resources. Through this module, we can enhance the accessibility and adoption of cloud computing in epigenomic research, speeding up the advancements in the related field and beyond. This manuscript describes the development of a resource module that is part of a learning platform named ``NIGMS Sandbox for Cloud-based Learning'' https://github.com/NIGMS/NIGMS-Sandbox. The overall genesis of the Sandbox is described in the editorial NIGMS Sandbox [1] at the beginning of this Supplement. This module delivers learning materials on the analysis of bulk and single-cell ATAC-seq data in an interactive format that uses appropriate cloud resources for data access and analyses.

NCI Cancer Research Data Commons: Lessons Learned and Future State.

More than ever, scientific progress in cancer research hinges on our ability to combine datasets and extract meaningful interpretations to better understand diseases and ultimately inform the development of better treatments and diagnostic tools. To enable the successful sharing and use of big data, the NCI developed the Cancer Research Data Commons (CRDC), providing access to a large, comprehensive, and expanding collection of cancer data. The CRDC is a cloud-based data science infrastructure that eliminates the need for researchers to download and store large-scale datasets by allowing them to perform analysis where data reside. Over the past 10 years, the CRDC has made significant progress in providing access to data and tools along with training and outreach to support the cancer research community. In this review, we provide an overview of the history and the impact of the CRDC to date, lessons learned, and future plans to further promote data sharing, accessibility, interoperability, and reuse. See related articles by Brady et al., p. 1384, Wang et al., p. 1388, and Pot et al., p. 1396.

Long term use of eltrombopag in children with chronic immune thrombocytopenia: extended real life retrospective multicenter experience of the Italian Association of Pediatric Hematology and Oncology.

Background: The present multicenter retrospective study on eltrombopag administration in Italian children with chronic ITP aims to extend follow-up of our previous study. Materials and methods: This retrospective multicenter study was conducted in 17 centers affiliated to the Italian Association of Pediatric Hematology and Oncology (AIEOP). Patients were classified into three subgroups: group 1 included patients who discontinued treatment due to a stable platelet count; group 2 included patients who discontinued treatment due to ineffectiveness; group 3 included patients who did not permanently discontinue treatment. Results: 56 patients were eligible for analysis. The median duration of eltrombopag treatment was 40 months (7-71 months). Twenty patients (36%) discontinued permanently eltrombopag. The reasons of permanent discontinuation were adverse effects (n = 1), inefficacy (n = 10), stable platelet count (n = 9). All patients of group 1 maintained a durable response without additional treatments after eltrombopag discontinuation. We found that patients of group 2 were on treatment for less time (median treatment time: 13.5 months, min: 6.0 - max: 56.0) than patients of group 1 (median treatment time: 34 months, min: 16.0 - max: 62.0) (p < 0.05). Patients of group 2 mostly did not achieve a stable platelet count in the first 6 months of treatment and underwent concomitant therapies during follow-up respect of group 1 and group 3 (p < 0.01). Conclusion: Our study found that the benefits of eltrombopag treatment, in terms of platelet count improvement and use of additional therapies, are identifiable from the first 6 months of treatment.

Autoimmune Lymphoproliferative Syndrome (ALPS) Disease and ALPS Phenotype: Are They Two Distinct Entities?

Autoimmune lymphoproliferative syndrome (ALPS) is an inherited disorder of lymphocyte homeostasis classically due to mutation of FAS, FASL, and CASP10 genes (ALPS-FAS/CASP10). Despite recent progress, about one-third of ALPS patients does not carry classical mutations and still remains gene orphan (ALPS-U, undetermined genetic defects). The aims of the present study were to compare the clinical and immunological features of ALPS-FAS/CASP10 versus those of ALPS-U affected subjects and to deepen the genetic characteristics of this latter group. Demographical, anamnestic, biochemical data were retrieved from medical record of 46 ALPS subjects. An enlarged panel of genes (next-generation sequencing) was applied to the ALPS-U group. ALPS-U subjects showed a more complex phenotype if compared to ALPS-FAS/CASP10 group, characterized by multiorgan involvement (P = 0.001) and positivity of autoimmune markers (P = 0.02). Multilineage cytopenia was present in both groups without differences with the exception of lymphocytopenia and autoimmune neutropenia that were more frequent in ALPS-U than in the ALPS-FAS/CASP10 group (P = 0.01 and P = 0.04). First- and second-line treatments were able to control the symptoms in 100% of the ALPS-FAS/CASP10 patients, while 63% of ALPS-U needed >2 lines of treatment and remission in some cases was obtained only after target therapy. In the ALPS-U group, we found in 14 of 28 (50%) patients 19 variants; of these, 4 of 19 (21%) were known as pathogenic and 8 of 19 (42%) as likely pathogenic. A characteristic flow cytometry panel including CD3CD4-CD8-+TCRαß+, CD3+CD25+/CD3HLADR+, TCR αß+ B220+, and CD19+CD27+ identified the ALPS-FAS/CASP10 group. ALPS-U seems to represent a distinct entity from ALPS-FAS/CASP10; this is relevant for management and tailored treatments whenever available.

"CHildren with Inherited Platelet disorders Surveillance" (CHIPS) retrospective and prospective observational cohort study by Italian Association of Pediatric Hematology and Oncology (AIEOP).

Background: Inherited thrombocytopenias (ITs) are rare congenital bleeding disorders characterized by different clinical expression and variable prognosis. ITs are poorly known by clinicians and often misdiagnosed with most common forms of thrombocytopenia. Material and methods: "CHildren with Inherited Platelet disorders Surveillance" study (CHIPS) is a retrospective - prospective observational cohort study conducted between January 2003 and January 2022 in 17 centers affiliated to the Italian Association of Pediatric Hematology and Oncology (AIEOP). The primary objective of this study was to collect clinical and laboratory data on Italian pediatric patients with inherited thrombocytopenias. Secondary objectives were to calculate prevalence of ITs in Italian pediatric population and to assess frequency and genotype-phenotype correlation of different types of mutations in our study cohort. Results: A total of 139 children, with ITs (82 male - 57 female) were enrolled. ITs prevalence in Italy ranged from 0.7 per 100,000 children during 2010 to 2 per 100,000 children during 2022. The median time between the onset of thrombocytopenia and the diagnosis of ITs was 1 years (range 0 - 18 years). A family history of thrombocytopenia has been reported in 90 patients (65%). Among 139 children with ITs, in 73 (53%) children almost one defective gene has been identified. In 61 patients a pathogenic mutation has been identified. Among them, 2 patients also carry a variant of uncertain significance (VUS), and 4 others harbour 2 VUS variants. VUS variants were identified in further 8 patients (6%), 4 of which carry more than one variant VUS. Three patients (2%) had a likely pathogenic variant while in 1 patient (1%) a variant was identified that was initially given an uncertain significance but was later classified as benign. In addition, in 17 patients the genetic diagnosis is not available, but their family history and clinical/laboratory features strongly suggest the presence of a specific genetic cause. In 49 children (35%) no genetic defect were identified. In ninetyseven patients (70%), thrombocytopenia was not associated with other clinically apparent disorders. However, 42 children (30%) had one or more additional clinical alterations. Conclusion: Our study provides a descriptive collection of ITs in the pediatric Italian population.

Elevated exposures to persistent endocrine disrupting compounds impact the sperm methylome in regions associated with autism spectrum disorder.

Environmental exposures to endocrine disrupting compounds (EDCs) such as the organochlorines have been linked with various diseases including neurodevelopmental disorders. Autism spectrum disorder (ASD) is a highly complex neurodevelopmental disorder that is considered strongly genetic in origin due to its high heritability. However, the rapidly rising prevalence of ASD suggests that environmental factors may also influence risk for ASD. In the present study, whole genome bisulfite sequencing was used to identify genome-wide differentially methylated regions (DMRs) in a total of 52 sperm samples from a cohort of men from the Faroe Islands (Denmark) who were equally divided into high and low exposure groups based on their serum levels of the long-lived organochlorine 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE), a primary breakdown product of the now banned insecticide dichlorodiphenyltrichloroethane (DDT). Aside from being considered a genetic isolate, inhabitants of the Faroe Islands have a native diet that potentially exposes them to a wide range of seafood neurotoxicants in the form of persistent organic pollutants (POPs). The DMRs were mapped to the human genome using Bismark, a 3-letter aligner used for methyl-seq analyses. Gene ontology, functional, and pathway analyses of the DMR-associated genes showed significant enrichment for genes involved in neurological functions and neurodevelopmental processes frequently impacted by ASD. Notably, these genes also significantly overlap with autism risk genes as well as those previously identified in sperm from fathers of children with ASD in comparison to that of fathers of neurotypical children. These results collectively suggest a possible mechanism involving altered methylation of a significant number of neurologically relevant ASD risk genes for introducing epigenetic changes associated with environmental exposures into the sperm methylome. Such changes may provide the potential for transgenerational inheritance of ASD as well as other disorders.

Association of Immune Thrombocytopenia and Celiac Disease in Children: A Retrospective Case Control Study

Objective: The association between celiac disease (CD) and immune thrombocytopenia (ITP) is still uncertain. The aim of this study was to characterize the coexistence of these two diseases in Italian children. Materials and Methods: This is a retrospective multicenter study investigating the occurrence of CD in 28 children with ITP diagnosed from January 1, 2000, to December 31, 2019. Results: The first diagnosis was ITP in 57.1% and CD in 32.1% of patients. In 3 patients (10.7%), the two diagnoses were simultaneous. All the potential and silent cases of CD in our cohort were diagnosed in the groups of "ITP first" and "simultaneous diagnosis". In all children ITP was mild, and in 2 out of 8 not recovered from ITP at the time of CD diagnosis a normalization of platelet counts (>100,000/µL) occurred 3 and 5 months after starting a gluten-free diet, respectively. Conclusion: We think that screening for CD should be considered in children with ITP regardless of the presence of gastrointestinal symptoms. Furthermore, some patients may recover from ITP after starting a gluten-free diet.

Electrocardiogram in Friedreich's ataxia: A short-term surrogate endpoint for treatment efficacy.

Friedreich's ataxia is a rare degenerative neuromuscular disorder, caused by a homozygous GAA triplet repeat expansion in the frataxin (FXN) gene, with a broad clinical phenotype characterized by progressive gait and limb ataxia, dysarthria, and loss of lower limb reflexes; cardiac involvement is represented by hypertrophic cardiomyopathy, ventricular arrhythmias, and sudden cardiac deaths. Currently, no definite therapy is available, while many drugs are under investigation; for this reasons, we need markers of short- and long-term treatment efficacy acting on different tissue for trial evaluation. We describe the case of a 21-year-old patient affected by Friedreich's ataxia on wheel-chair, with initial cardiac involvement and electrocardiographic features characterized by thiamine treatment-related negative T wave and QTc variations. We discuss plausible physiopathology and potential ECG role implications as an intermediate marker of treatment response in future clinical trials considering patients affected by Friedreich's ataxia.

Role of Subclinical Iatrogenic Hyperthyroidism in the Setting of Heart Disease and Arrhythmic Burden.

BACKGROUND: Subclinical hyperthyroidism is defined by a subnormal serum thyroidstimulating hormone (TSH) level with normal free thyroxine (FT4) and free triiodothyronine (FT3) levels. Its prevalence varies from 0.6% to 16% in the elderly and can increase to 20% in patients receiving thyroid hormone replacement therapy. Thyroid disease and/or replacement therapy are frequently associated with cardiovascular involvement. CASES PRESENTATION: We report three clinical cases of patients with initial subclinical hyperthyroidism and cardiological manifestations, including supraventricular and ventricular extrasystoles, prolapse of the mitral valve with severe regurgitation, higher mean heart rate and deterioration of the arrhythmias on arrhythmogenic dysplasia substrate. CONCLUSION: We discuss the role of appropriate and early correction of thyroid dysfunction in improving cardiological manifestations.

Electrical Remodeling of Ventricular Repolarization Abnormality after Treatment in Pheochromocytoma: U Wave Finding in a Retrospective Analysis.

Background: Pheochromocytoma is a rare neuroendocrine tumor, clinically characterized by high blood pressure, palpitations, and headache. It is often associated with abnormalities of the ventricular repolarization phase; the dispersion of ventricular repolarization is the basis for ventricular arrhythmias (torsion de point, ventricular tachycardia or ventricular fibrillation). Objectives: Analysis of abnormal ventricular repolarization focused on the presence and amount of U wave in patients affected by pheochromocytoma and its modification after surgery. Materials and Methods: We reviewed pathology records of 722 patients admitted for adrenal nodule or suspected chromaffin-cell tumor and identified 39 patients affected by pheochromocytoma. Metanephrine, normetanephrine, and 3-methoxytyramine have been assessed by determining concentrations in 24-hour urine collection. Standard 12-lead electrocardiogram records have been reviewed with analysis of heart rate, P wave, PR interval, QRS duration, QTc, and U wave. Then we selected and compared 22 patients of 39 affected by pheochromocytoma, with both clinical and electrocardiographic data before and after surgery. Results: In our cohort of 39 patients affected by pheochromocytoma, we found U wave in ECG, before treatment, in 82.8 percent of patients, while only 37.0 percent after treatment (p<0.001) and we observed a statistically significant correlation between this wave and the urinary metanephrine. After surgery, in the selected 22 patients, we observed a clear significant reduction in systemic blood pressure, fasting glucose, metanephrine, normetanephrine, and 3-methoxytyramine. We found a significant reduction of U wave presence and leads involved in these patients after surgery (90.9% versus 9%). We observed a linear correlation between the amount of U waves in 12-lead electrocardiogram and metanephrine (r2=0.333, p=0.015), 3-methoxytyramine levels (r2=0.458, p=0.006), and tumor size (r2=0.429, p=0.003). Conclusions: In our retrospective analysis, patients affected by pheochromocytoma presented U wave in electrocardiogram. The presence and amount of U wave were associated with the metanephrine levels and the tumor size with significant reduction after surgical removal.

A 3 Mb deletion in 14q12 causes severe mental retardation, mild facial dysmorphisms and Rett-like features.

The present report describes a 7-year-old girl with a de novo 3 Mb interstitial deletion of chromosome 14q12, identified by oligo array-CGH. The region is gene poor and contains only five genes two of them, FOXG1B and PRKD1 being deleted also in a previously reported case with a very similar phenotype. Both patients present prominent metopic suture, epicanthic folds, bulbous nasal tip, tented upper lip, everted lower lip and large ears and a clinical course like Rett syndrome, including normal perinatal period, postnatal microcephaly, seizures, and severe mental retardation. FOXG1B (forkhead box G1B) is a very intriguing candidate gene since it is known to promote neuronal progenitor proliferation and to suppress premature neurogenesis and its disruption is reported in a patient with postnatal microcephaly, corpus callosum agenesis, seizures, and severe mental retardation.