Long non-coding RNAs: definitions, functions, challenges and recommendations.

Genes specifying long non-coding RNAs (lncRNAs) occupy a large fraction of the genomes of complex organisms. The term 'lncRNAs' encompasses RNA polymerase I (Pol I), Pol II and Pol III transcribed RNAs, and RNAs from processed introns. The various functions of lncRNAs and their many isoforms and interleaved relationships with other genes make lncRNA classification and annotation difficult. Most lncRNAs evolve more rapidly than protein-coding sequences, are cell type specific and regulate many aspects of cell differentiation and development and other physiological processes. Many lncRNAs associate with chromatin-modifying complexes, are transcribed from enhancers and nucleate phase separation of nuclear condensates and domains, indicating an intimate link between lncRNA expression and the spatial control of gene expression during development. lncRNAs also have important roles in the cytoplasm and beyond, including in the regulation of translation, metabolism and signalling. lncRNAs often have a modular structure and are rich in repeats, which are increasingly being shown to be relevant to their function. In this Consensus Statement, we address the definition and nomenclature of lncRNAs and their conservation, expression, phenotypic visibility, structure and functions. We also discuss research challenges and provide recommendations to advance the understanding of the roles of lncRNAs in development, cell biology and disease.

Long Noncoding RNAs CUPID1 and CUPID2 Mediate Breast Cancer Risk at 11q13 by Modulating the Response to DNA Damage.

Breast cancer risk is strongly associated with an intergenic region on 11q13. We have previously shown that the strongest risk-associated SNPs fall within a distal enhancer that regulates CCND1. Here, we report that, in addition to regulating CCND1, this enhancer regulates two estrogen-regulated long noncoding RNAs, CUPID1 and CUPID2. We provide evidence that the risk-associated SNPs are associated with reduced chromatin looping between the enhancer and the CUPID1 and CUPID2 bidirectional promoter. We further show that CUPID1 and CUPID2 are predominantly expressed in hormone-receptor-positive breast tumors and play a role in modulating pathway choice for the repair of double-strand breaks. These data reveal a mechanism for the involvement of this region in breast cancer.

Leveraging peer-based support to facilitate HIV care in Kenya.

Rakhi Karwa and colleagues discuss a program in which peer navigators support care for people with HIV at a Kenyan hospital.

Parallel laboratory evolution and rational debugging reveal genomic plasticity to S. cerevisiae synthetic chromosome XIV defects.

Synthetic chromosome engineering is a complex process due to the need to identify and repair growth defects and deal with combinatorial gene essentiality when rearranging chromosomes. To alleviate these issues, we have demonstrated novel approaches for repairing and rearranging synthetic Saccharomyces cerevisiae genomes. We have designed, constructed, and restored wild-type fitness to a synthetic 753,096-bp version of S. cerevisiae chromosome XIV as part of the Synthetic Yeast Genome project. In parallel to the use of rational engineering approaches to restore wild-type fitness, we used adaptive laboratory evolution to generate a general growth-defect-suppressor rearrangement in the form of increased TAR1 copy number. We also extended the utility of the synthetic chromosome recombination and modification by loxPsym-mediated evolution (SCRaMbLE) system by engineering synthetic-wild-type tetraploid hybrid strains that buffer against essential gene loss, highlighting the plasticity of the S. cerevisiae genome in the presence of rational and non-rational modifications.

Towards accurate and reliable resolution of structural variants for clinical diagnosis.

Structural variants (SVs) are a major source of human genetic diversity and have been associated with different diseases and phenotypes. The detection of SVs is difficult, and a diverse range of detection methods and data analysis protocols has been developed. This difficulty and diversity make the detection of SVs for clinical applications challenging and requires a framework to ensure accuracy and reproducibility. Here, we discuss current developments in the diagnosis of SVs and propose a roadmap for the accurate and reproducible detection of SVs that includes case studies provided from the FDA-led SEquencing Quality Control Phase II (SEQC-II) and other consortium efforts.

Ultra-deep sequencing data from a liquid biopsy proficiency study demonstrating analytic validity.

Recently we reported the accuracy and reproducibility of circulating tumor DNA (ctDNA) assays using a unique set of reference materials, associated analytical framework, and suggested best practices. With the rapid adoption of ctDNA sequencing in precision oncology, it is critical to understand the analytical validity and technical limitations of this cutting-edge and medical-practice-changing technology. The SEQC2 Oncopanel Sequencing Working Group has developed a multi-site, cross-platform study design for evaluating the analytical performance of five industry-leading ctDNA assays. The study used tailor-made reference samples at various levels of input material to assess ctDNA sequencing across 12 participating clinical and research facilities. The generated dataset encompasses multiple key variables, including a broad range of mutation frequencies, sequencing coverage depth, DNA input quantity, etc. It is the most comprehensive public-facing dataset of its kind and provides valuable insights into ultra-deep ctDNA sequencing technology. Eventually the clinical utility of ctDNA assays is required and our proficiency study and corresponding dataset are needed steps towards this goal.

Understanding the primary health care experiences of individuals who are homeless in non-traditional clinic settings.

BACKGROUND: Despite the widespread implementation of Health Care for the Homeless programs that focus on comprehensive, integrated delivery systems of health care for people experiencing homelessness, engaging and retaining people experiencing homelessness in primary care remains a challenge. Few studies have looked at the primary care delivery model in non-traditional health care settings to understand the facilitators and barriers to engagement in care. The objective of our study was to explore the clinic encounters of individuals experiencing homelessness receiving care at two different sites served under a single Health Care for the Homeless program. METHODS: Semi-structured interviews were conducted with people experiencing homelessness for an explorative qualitative study. We used convenience sampling to recruit participants who were engaged in primary care at one of two sites: a shelter clinic, n = 16, and a mobile clinic located in a church, n = 15. We then used an iterative, thematic approach to identify emergent themes and further mapped these onto the Capability-Opportunity-Motivation model. RESULTS: Care accessibility, quality and integration were themes that were often identified by participants as being important facilitators to care. Psychological capability and capacity became important barriers to care in instances when patients had issues with memory or difficulty with perceiving psychological safety in healthcare settings. Motivation for engaging and continuing in care often came from a team of health care providers using shared decision-making with the patient to facilitate change. CONCLUSION: To optimize health care for people experiencing homelessness, clinical interventions should: (1) utilize shared-decision making during the visit, (2) foster a sense of trust, compassion, and acceptance, (3) emphasize continuity of care, including consistent providers and staff, and (4) integrate social services into Health Care for the Homeless sites.

ADRAM is an experience-dependent long noncoding RNA that drives fear extinction through a direct interaction with the chaperone protein 14-3-3.

Here, we used RNA capture-seq to identify a large population of lncRNAs that are expressed in the infralimbic prefrontal cortex of adult male mice in response to fear-related learning. Combining these data with cell-type-specific ATAC-seq on neurons that had been selectively activated by fear extinction learning, we find inducible 434 lncRNAs that are derived from enhancer regions in the vicinity of protein-coding genes. In particular, we discover an experience-induced lncRNA we call ADRAM (activity-dependent lncRNA associated with memory) that acts as both a scaffold and a combinatorial guide to recruit the brain-enriched chaperone protein 14-3-3 to the promoter of the memory-associated immediate-early gene Nr4a2 and is required fear extinction memory. This study expands the lexicon of experience-dependent lncRNA activity in the brain and highlights enhancer-derived RNAs (eRNAs) as key players in the epigenomic regulation of gene expression associated with the formation of fear extinction memory.

A clinical laboratory-developed LSC17 stemness score assay for rapid risk assessment of patients with acute myeloid leukemia.

Leukemia stem cells (LSCs) are linked to relapse in acute myeloid leukemia (AML). The LSC17 gene expression score robustly captures LSC stemness properties in AML and can be used to predict survival outcomes and response to therapy, enabling risk-adapted, upfront treatment approaches. The LSC17 score was developed and validated in a research setting. To enable widespread use of the LSC17 score in clinical decision making, we established a laboratory-developed test (LDT) for the LSC17 score that can be deployed broadly in clinical molecular diagnostic laboratories. We extensively validated the LSC17 LDT in a College of American Pathologists/Clinical Laboratory Improvements Act (CAP/CLIA)-certified laboratory, determining specimen requirements, a synthetic control, and performance parameters for the assay. Importantly, we correlated values from the LSC17 LDT to clinical outcome in a reference cohort of patients with AML, establishing a median assay value that can be used for clinical risk stratification of individual patients with newly diagnosed AML. The assay was established in a second independent CAP/CLIA-certified laboratory, and its technical performance was validated using an independent cohort of patient samples, demonstrating that the LSC17 LDT can be readily implemented in other settings. This study enables the clinical use of the LSC17 score for upfront risk-adapted management of patients with AML.

Non-coding RNAs: regulators of disease.

For 50 years the term 'gene' has been synonymous with regions of the genome encoding mRNAs that are translated into protein. However, recent genome-wide studies have shown that the human genome is pervasively transcribed and produces many thousands of regulatory non-protein-coding RNAs (ncRNAs), including microRNAs, small interfering RNAs, PIWI-interacting RNAs and various classes of long ncRNAs. It is now clear that these RNAs fulfil critical roles as transcriptional and post-transcriptional regulators and as guides of chromatin-modifying complexes. Here we review the biology of ncRNAs, focusing on the fundamental mechanisms by which ncRNAs facilitate normal development and physiology and, when dysfunctional, underpin disease. We also discuss evidence that intergenic regions associated with complex diseases express ncRNAs, as well as the potential use of ncRNAs as diagnostic markers and therapeutic targets. Taken together, these observations emphasize the need to move beyond the confines of protein-coding genes and highlight the fact that continued investigation of ncRNA biogenesis and function will be necessary for a comprehensive understanding of human disease.

Integrating Prevention of Mother-to-Child Transmission of HIV Care into General Maternal Child Health Care in Western Kenya.

BACKGROUND: Health systems integration is becoming increasingly important as the global health community transitions from acute, disease-specific health programming to models of care built for chronic diseases, primarily designed to strengthen public-sector health systems. In many countries across sub-Saharan Africa, including Kenya, prevention of mother-to-child transmission of HIV (pMTCT) services are being integrated into the general maternal child health (MCH) clinics. The objective of this study was to evaluate the benefits and challenges for integration of care within a developing health system, through the lens of an evaluative framework. METHODS: A framework adapted from the World Health Organization's six critical health systems functions was used to evaluate the integration of pMTCT services with general MCH clinics in western Kenya. Perspectives were collected from key stakeholders, including pMTCT and MCH program leadership and local health providers. The benefits and challenges of integration across each of the health system functions were evaluated to better understand this approach. RESULTS: Key informants in leadership positions and MCH staff shared similar perspectives regarding benefits and challenges of integration. Benefits of integration included convenience for families through streamlining of services and reduced HIV stigma. Concerns and challenges included confidentiality issues related to HIV status, particularly in the context of high-volume, crowded clinical spaces. CONCLUSION AND GLOBAL HEALTH IMPLICATIONS: The results from this study highlight areas that need to be addressed to maximize the effectiveness and clinical flow of the pMTCT-MCH integration model. The lessons learned from this integration may be applied to other settings in sub-Saharan Africa attempting to integrate HIV care into the broader public-sector health system.

Projecting COVID-19 isolation bed requirements for people experiencing homelessness.

As COVID-19 spreads across the United States, people experiencing homelessness (PEH) are among the most vulnerable to the virus. To mitigate transmission, municipal governments are procuring isolation facilities for PEH to utilize following possible exposure to the virus. Here we describe the framework for anticipating isolation bed demand in PEH communities that we developed to support public health planning in Austin, Texas during March 2020. Using a mathematical model of COVID-19 transmission, we projected that, under no social distancing orders, a maximum of 299 (95% Confidence Interval: 223, 321) PEH may require isolation rooms in the same week. Based on these analyses, Austin Public Health finalized a lease agreement for 205 isolation rooms on March 27th 2020. As of October 7th 2020, a maximum of 130 rooms have been used on a single day, and a total of 602 PEH have used the facility. As a general rule of thumb, we expect the peak proportion of the PEH population that will require isolation to be roughly triple the projected peak daily incidence in the city. This framework can guide the provisioning of COVID-19 isolation and post-acute care facilities for high risk communities throughout the United States.

Advancing NGS quality control to enable measurement of actionable mutations in circulating tumor DNA.

The primary objective of the FDA-led Sequencing and Quality Control Phase 2 (SEQC2) project is to develop standard analysis protocols and quality control metrics for use in DNA testing to enhance scientific research and precision medicine. This study reports a targeted next-generation sequencing (NGS) method that will enable more accurate detection of actionable mutations in circulating tumor DNA (ctDNA) clinical specimens. To accomplish this, a synthetic internal standard spike-in was designed for each actionable mutation target, suitable for use in NGS following hybrid capture enrichment and unique molecular index (UMI) or non-UMI library preparation. When mixed with contrived ctDNA reference samples, internal standards enabled calculation of technical error rate, limit of blank, and limit of detection for each variant at each nucleotide position in each sample. True-positive mutations with variant allele fraction too low for detection by current practice were detected with this method, thereby increasing sensitivity.

Toward best practice in cancer mutation detection with whole-genome and whole-exome sequencing.

Clinical applications of precision oncology require accurate tests that can distinguish true cancer-specific mutations from errors introduced at each step of next-generation sequencing (NGS). To date, no bulk sequencing study has addressed the effects of cross-site reproducibility, nor the biological, technical and computational factors that influence variant identification. Here we report a systematic interrogation of somatic mutations in paired tumor-normal cell lines to identify factors affecting detection reproducibility and accuracy at six different centers. Using whole-genome sequencing (WGS) and whole-exome sequencing (WES), we evaluated the reproducibility of different sample types with varying input amount and tumor purity, and multiple library construction protocols, followed by processing with nine bioinformatics pipelines. We found that read coverage and callers affected both WGS and WES reproducibility, but WES performance was influenced by insert fragment size, genomic copy content and the global imbalance score (GIV; G > T/C > A). Finally, taking into account library preparation protocol, tumor content, read coverage and bioinformatics processes concomitantly, we recommend actionable practices to improve the reproducibility and accuracy of NGS experiments for cancer mutation detection.

Pervasive transcription of the eukaryotic genome: functional indices and conceptual implications.

Genome-wide analyses of the eukaryotic transcriptome have revealed that the majority of the genome is transcribed, producing large numbers of non-protein-coding RNAs (ncRNAs). This surprising observation challenges many assumptions about the genetic programming of higher organisms and how information is stored and organized within the genome. Moreover, the rapid advances in genomics have given little opportunity for biologists to integrate these emerging findings into their intellectual and experimental frameworks. This problem has been compounded by the perception that genome-wide studies often generate more questions than answers, which in turn has led to confusion and controversy. In this article, we address common questions associated with the phenomenon of pervasive transcription and consider the indices that can be used to evaluate the function (or lack thereof) of the resulting ncRNAs. We suggest that many lines of evidence, including expression profiles, conservation signatures, chromatin modification patterns and examination of increasing numbers of individual cases, argue in favour of the widespread functionality of non-coding transcription. We also discuss how informatic and experimental approaches used to analyse protein-coding genes may not be applicable to ncRNAs and how the general perception that protein-coding genes form the main informational output of the genome has resulted in much of the misunderstanding surrounding pervasive transcription and its potential significance. Finally, we present the conceptual implications of the majority of the eukaryotic genome being functional and describe how appreciating this perspective will provide considerable opportunity to further understand the molecular basis of development and complex diseases.

Ensuring Patient-Centered Access to Cardiovascular Disease Medicines in Low-Income and Middle-Income Countries Through Health-System Strengthening.

Cardiovascular disease (CVD) is the leading cause of global mortality and is expected to reach 23 million deaths by 2030. Eighty percent of CVD deaths occur in low-income and middle-income countries (LMICs). Although CVD prevention and treatment guidelines are available, translating these into practice is hampered in LMICs by inadequate health care systems that limit access to lifesaving medications. In this review article, we describe the deficiencies in the current LMIC supply chains that limit access to effective CVD medicines, and discuss existing solutions that are translatable to similar settings so as to address these deficiencies.

Opposing roles for p21(waf1/cip1) and p27(kip1) in enterocyte differentiation, proliferation, and migration.

BACKGROUND: Originating from proliferating stem cells of the intestinal crypt, enterocytes differentiate as they migrate up the crypt-villus axis. A regulatory role of the cyclin-dependent kinase inhibitors p21(waf1/cip1) and p27(kip1) in these processes has been suggested by in vitro models. We sought to determine the effect of p21(waf1/cip1) and p27(kip1) deficiency on enterocyte differentiation, proliferation and migration. METHODS: Three strains of mice including control (C57Bl/6), p27(kip1)-null, and p21(waf1/cip1)-null were studied. Enterocyte differentiation was evaluated by immunostaining for intestinal alkaline phosphatase, by colorimetric assaying for intestinal alkaline phosphatase and sucrase enzyme activity, and by polymerase chain reaction for intestinal fatty acid-binding protein and villin-messenger RNA in enterocytes extracted by laser capture microdissection. Rates of enterocyte proliferation and migration were determined by 5-bromo 2-deoxyuridine immunostaining after a 50% small-bowel resection (SBR). RESULTS: Compared with controls, p27(kip1)-null mice demonstrated minimal differentiation but maintained a normal proliferative response to SBR. Contrarily, p21(waf1/cip1)-null mice demonstrated greater enterocyte differentiation without significant increases in enterocyte proliferation after SBR. CONCLUSIONS: These findings suggest that p21(waf1/cip1) and p27(kip1) have distinctive and opposing roles in the pathogenesis of enterocyte differentiation, proliferation, and migration.

Selenium compounds regulate p53 by common and distinctive mechanisms.

Selenium compounds show much promise in the prevention of prostate and other human cancers. Various selenium chemical forms have been shown to differ widely in their anticancer properties. The main dietary form is selenomethionine, which we showed modulated p53 activity by causing redox regulation of key p53 cysteine residues. In the current study we included other selenium chemical forms, sodium selenite and methyl-seleninic acid. All three forms are relevant selenium sources in human populations. All three forms can affect p53 activity defined as trans-activation of a p53-dependent reporter gene. In addition to the reduction of cysteine sulfhydryl groups, p53 phosphorylation was also affected in cells treated with selenium compounds. Methyl-seleninic acid caused phosphorylation of one or more p53 threonine residues, but did not affect any known serine phosphorylation sites. By contrast sodium selenite caused phosphorylation of p53 serines 20, 37 and 46 known to mediate apoptosis. Selenomethionine did not cause detectable phosphorylation of p53 serines or threonines. Our data show that, although p53 modulation may be a common denominator of selenium compounds, specific mechanisms of p53 activation differ among selenium chemical forms. Post-translational modifications of p53 are determinants of p53 activity and probably affect the threshold for p53-mediated functions. Different selenium chemical forms may differentially modify p53 for DNA repair or apoptosis in conjunction with a given level of endogenous or exogenous DNA damage.

Mapping of mitochondrial RNA-protein interactions by digital RNase footprinting.

Human mitochondrial DNA is transcribed as long polycistronic transcripts that encompass each strand of the genome and are processed subsequently into mature mRNAs, tRNAs, and rRNAs, necessitating widespread posttranscriptional regulation. Here, we establish methods for massively parallel sequencing and analyses of RNase-accessible regions of human mitochondrial RNA and thereby identify specific regions within mitochondrial transcripts that are bound by proteins. This approach provides a range of insights into the contribution of RNA-binding proteins to the regulation of mitochondrial gene expression.