Modern Genomic Era Research Articles

Genomic aspects in reproductive medicine.

Infertility is a complex disease characterized by extreme genetic heterogeneity, compounded by various environmental factors. While there are exceptions, individual genetic and genomic variations related to infertility are typically rare, often family-specific, and may serve as susceptibility factors rather than direct causes of the disease. Consequently, identifying the cause of infertility and developing prevention and treatment strategies based on these factors remain challenging tasks, even in the modern genomic era. In this review, we first examine the genetic and genomic variations associated with infertility, and subsequently summarize the concepts and methods of preimplantation genetic testing in light of advances in genome analysis technology.

Management of Myeloproliferative Neoplasms in the Molecular Era: From Research to Practice

The 1960 discovery of the Philadelphia chromosome in chronic myeloid leukemia (CML) marked the beginning of the modern genomic era of oncology. In the following years, the molecular underpinnings of CML were unraveled, culminating in the development of the first molecularly targeted therapy: imatinib. Imatinib revolutionized CML management, inducing deep molecular responses for most patients and aligning survival curves with those of age-matched control participants. Five additional tyrosine kinase inhibitors are now approved for CML: dasatinib, nilotinib, bosutinib, ponatinib, and asciminib (approved October 2021). The 2005 discovery of JAK2 mutations in myelofibrosis (MF) sparked enthusiasm that molecularly targeted therapies could have a similar impact in that disease. Three JAK inhibitors are now available for MF: ruxolitinib, fedratinib, and pacritinib (approved February 2022). JAK inhibitors are helpful for improving symptoms and splenomegaly but still only scratch the surface of MF pathophysiology. Clinical research testing novel agents, next-generation JAK inhibitors, and combinations of JAK inhibitors plus novel agents is moving at a tremendous pace in the hope that outcomes for patients with MF may mirror those with CML one day. This review provides an update on the status of clinical care and research for MF and addresses ongoing issues related to CML management.

Personal Perspectives on Plant Ribosomal RNA Genes Research: From Precursor-rRNA to Molecular Evolution.

The history of rDNA research started almost 90 years ago when the geneticist, Barbara McClintock observed that in interphase nuclei of maize the nucleolus was formed in association with a specific region normally located near the end of a chromosome, which she called the nucleolar organizer region (NOR). Cytologists in the twentieth century recognized the nucleolus as a common structure in all eukaryotic cells, using both light and electron microscopy and biochemical and genetic studies identified ribosomes as the subcellular sites of protein synthesis. In the mid- to late 1960s, the synthesis of nuclear-encoded rRNA was the only system in multicellular organisms where transcripts of known function could be isolated, and their synthesis and processing could be studied. Cytogenetic observations of NOR regions with altered structure in plant interspecific hybrids and detailed knowledge of structure and function of rDNA were prerequisites for studies of nucleolar dominance, epistatic interactions of rDNA loci, and epigenetic silencing. In this article, we focus on the early rDNA research in plants, performed mainly at the dawn of molecular biology in the 60 to 80-ties of the last century which presented a prequel to the modern genomic era. We discuss – from a personal view – the topics such as synthesis of rRNA precursor (35S pre-rRNA in plants), processing, and the organization of 35S and 5S rDNA. Cloning and sequencing led to the observation that the transcribed and processed regions of the rRNA genes vary enormously, even between populations and species, in comparison with the more conserved regions coding for the mature rRNAs. Epigenetic phenomena and the impact of hybridization and allopolyploidy on rDNA expression and homogenization are discussed. This historical view of scientific progress and achievements sets the scene for the other articles highlighting the immense progress in rDNA research published in this special issue of Frontiers in Plant Science on “Molecular organization, evolution, and function of ribosomal DNA.”

OTC intron 4 variations mediate pathogenic splicing patterns caused by the c.386G>A mutation in humans and spfash mice, and govern susceptibility to RNA-based therapies

BackgroundAberrant splicing is a common outcome in the presence of exonic or intronic variants that might hamper the intricate network of interactions defining an exon in a specific gene context. Therefore, the evaluation of the functional, and potentially pathological, role of nucleotide changes remains one of the major challenges in the modern genomic era. This aspect has also to be taken into account during the pre-clinical evaluation of innovative therapeutic approaches in animal models of human diseases. This is of particular relevance when developing therapeutics acting on splicing, an intriguing and expanding research area for several disorders. Here, we addressed species-specific splicing mechanisms triggered by the OTC c.386G>A mutation, relatively frequent in humans, leading to Ornithine TransCarbamylase Deficiency (OTCD) in patients and spfash mice, and its differential susceptibility to RNA therapeutics based on engineered U1snRNA.MethodsCreation and co-expression of engineered U1snRNAs with human and mouse minigenes, either wild-type or harbouring different nucleotide changes, in human (HepG2) and mouse (Hepa1-6) hepatoma cells followed by analysis of splicing pattern. RNA pulldown studies to evaluate binding of specific splicing factors.ResultsComparative nucleotide analysis suggested a role for the intronic +10-11 nucleotides, and pull-down assays showed that they confer preferential binding to the TIA1 splicing factor in the mouse context, where TIA1 overexpression further increases correct splicing. Consistently, the splicing profile of the human minigene with mouse +10-11 nucleotides overlapped that of mouse minigene, and restored responsiveness to TIA1 overexpression and to compensatory U1snRNA. Swapping the human +10-11 nucleotides into the mouse context had opposite effects.Moreover, the interplay between the authentic and the adjacent cryptic 5′ss in the human OTC dictates pathogenic mechanisms of several OTCD-causing 5′ss mutations, and only the c.386+5G>A change, abrogating the cryptic 5′ss, was rescuable by engineered U1snRNA.ConclusionsSubtle intronic variations explain species-specific OTC splicing patterns driven by the c.386G>A mutation, and the responsiveness to engineered U1snRNAs, which suggests careful elucidation of molecular mechanisms before proposing translation of tailored therapeutics from animal models to humans.

Genome sequence of an uncharted halophilic bacterium Robertkochia marina with deciphering its phosphate-solubilizing ability.

The wide use of whole-genome sequencing approach in the modern genomic era has opened a great opportunity to reveal the prospective applications of halophilic bacteria. Robertkochia marina CC-AMO-30DT is one of the halophilic bacteria that was previously taxonomically identified without any inspection on its biotechnological potential from a genomic aspect. In this study, we present the whole-genome sequence of R. marina and demonstrated the ability of this bacterium in solubilizing phosphate by producing phosphatase. The genome of R. marina has 3.57Mbp and contains 3107 predicted genes, from which 3044 are protein coding, 52 are non-coding RNAs, and 11 are pseudogenes. Several phosphatases such as alkaline phosphatases and pyrophosphatases were mined from the genome. Further genomic study (phylogenetics, sequence analysis, and functional mechanism) and experimental data suggested that the alkaline phosphatase produced by R. marina could potentially be utilized in promoting plant growth, particularly for plants on saline-based agricultural land.

Galaxy and Apollo as a biologist-friendly interface for high-quality cooperative phage genome annotation.

In the modern genomic era, scientists without extensive bioinformatic training need to apply high-power computational analyses to critical tasks like phage genome annotation. At the Center for Phage Technology (CPT), we developed a suite of phage-oriented tools housed in open, user-friendly web-based interfaces. A Galaxy platform conducts computationally intensive analyses and Apollo, a collaborative genome annotation editor, visualizes the results of these analyses. The collection includes open source applications such as the BLAST+ suite, InterProScan, and several gene callers, as well as unique tools developed at the CPT that allow maximum user flexibility. We describe in detail programs for finding Shine-Dalgarno sequences, resources used for confident identification of lysis genes such as spanins, and methods used for identifying interrupted genes that contain frameshifts or introns. At the CPT, genome annotation is separated into two robust segments that are facilitated through the automated execution of many tools chained together in an operation called a workflow. First, the structural annotation workflow results in gene and other feature calls. This is followed by a functional annotation workflow that combines sequence comparisons and conserved domain searching, which is contextualized to allow integrated evidence assessment in functional prediction. Finally, we describe a workflow used for comparative genomics. Using this multi-purpose platform enables researchers to easily and accurately annotate an entire phage genome. The portal can be accessed at https://cpt.tamu.edu/galaxy-pub with accompanying user training material.

Ancient Plant DNA as a Window Into the Cultural Heritage and Biodiversity of Our Food System

Since the beginning of the ancient DNA revolution in the 1980s, archaeological plant remains and herbarium specimens have been analysed with molecular techniques to probe the evolutionary interface of plants and humans. In tandem with archaeobotany, ethnobiology, and other methods, ancient DNA offers tremendous insights into the co-evolution of people and plants, and the modern genomic era offers increasingly nuanced perspectives on plant use through time. Meanwhile, our global food system faces threats linked with declining biodiversity, an uncertain climate future, and vulnerable crop¬–wild relatives. Ancient plant DNA does not yield easy answers to these complex challenges, but we discuss how it can play an important role in ongoing conversations about resilience, sustainability, and sovereignty in our food system.

Knowledge-guided analysis of "omics" data using the KnowEnG cloud platform.

We present Knowledge Engine for Genomics (KnowEnG), a free-to-use computational system for analysis of genomics data sets, designed to accelerate biomedical discovery. It includes tools for popular bioinformatics tasks such as gene prioritization, sample clustering, gene set analysis, and expression signature analysis. The system specializes in "knowledge-guided" data mining and machine learning algorithms, in which user-provided data are analyzed in light of prior information about genes, aggregated from numerous knowledge bases and encoded in a massive "Knowledge Network." KnowEnG adheres to "FAIR" principles (findable, accessible, interoperable, and reuseable): its tools are easily portable to diverse computing environments, run on the cloud for scalable and cost-effective execution, and are interoperable with other computing platforms. The analysis tools are made available through multiple access modes, including a web portal with specialized visualization modules. We demonstrate the KnowEnG system's potential value in democratization of advanced tools for the modern genomics era through several case studies that use its tools to recreate and expand upon the published analysis of cancer data sets.

Genome Editing and Hematologic Malignancy.

The modern genomic era has seen remarkable advancement in our understanding of the molecular basis for disease, yet translation of basic discoveries into new disease treatments has arguably lagged behind. Recently, breakthroughs in genome editing technologies have created hope for their potential to directly treat the genetic causes of disease. Like any therapeutic intervention, genome editing should be considered in light of its potential risks and benefits. In this review, we highlight the promise of genome editing therapies, as well as the conceptual and technical barriers to their clinical application, with a special emphasis on hematologic malignancies.

NtEdit: scalable genome sequence polishing.

MotivationIn the modern genomics era, genome sequence assemblies are routine practice. However, depending on the methodology, resulting drafts may contain considerable base errors. Although utilities exist for genome base polishing, they work best with high read coverage and do not scale well. We developed ntEdit, a Bloom filter-based genome sequence editing utility that scales to large mammalian and conifer genomes.ResultsWe first tested ntEdit and the state-of-the-art assembly improvement tools GATK, Pilon and Racon on controlled Escherichia coli and Caenorhabditis elegans sequence data. Generally, ntEdit performs well at low sequence depths (<20×), fixing the majority (>97%) of base substitutions and indels, and its performance is largely constant with increased coverage. In all experiments conducted using a single CPU, the ntEdit pipeline executed in <14 s and <3 m, on average, on E.coli and C.elegans, respectively. We performed similar benchmarks on a sub-20× coverage human genome sequence dataset, inspecting accuracy and resource usage in editing chromosomes 1 and 21, and whole genome. ntEdit scaled linearly, executing in 30–40 m on those sequences. We show how ntEdit ran in <2 h 20 m to improve upon long and linked read human genome assemblies of NA12878, using high-coverage (54×) Illumina sequence data from the same individual, fixing frame shifts in coding sequences. We also generated 17-fold coverage spruce sequence data from haploid sequence sources (seed megagametophyte), and used it to edit our pseudo haploid assemblies of the 20 Gb interior and white spruce genomes in <4 and <5 h, respectively, making roughly 50M edits at a (substitution+indel) rate of 0.0024.Availability and implementation https://github.com/bcgsc/ntedit Supplementary information Supplementary data are available at Bioinformatics online.

Unsupervised clustering and epigenetic classification of single cells

Characterizing epigenetic heterogeneity at the cellular level is a critical problem in the modern genomics era. Assays such as single cell ATAC-seq (scATAC-seq) offer an opportunity to interrogate cellular level epigenetic heterogeneity through patterns of variability in open chromatin. However, these assays exhibit technical variability that complicates clear classification and cell type identification in heterogeneous populations. We present scABC, an R package for the unsupervised clustering of single-cell epigenetic data, to classify scATAC-seq data and discover regions of open chromatin specific to cell identity.

Brain metastasis from squamous cell carcinoma of the head and neck: a review of the literature in the genomic era.

Squamous cell carcinoma of the head and neck (HNSCC) affects nearly 500,000 individuals globally each year. With the rise of human papillomavirus (HPV) in the general population, clinicians are seeing a concomitant rise in HPV-related HNSCC. Notably, a hallmark of HPV-related HNSCC is a predilection for unique biological and clinical features, which portend a tendency for hematogenous metastasis to distant locations, such as the brain. Despite the classic belief that HNSCC is restricted to local spread via passive lymphatic drainage, brain metastases (BMs) are a rare complication that occurs in less than 1% of all HNSCC cases. Time between initial diagnosis of HNSCC and BM development can vary considerably. Some patients experience more than a decade of disease-free survival, whereas others present with definitive neurological symptoms that precede primary tumor detection. The authors systematically review the current literature on HNSCC BMs and discuss the current understanding of the effect of HPV status on the risk of developing BMs in the modern genomic era.

Treatment of brain metastases in the modern genomic era.

Development of brain metastasis (BM) portends a dismal prognosis for patients with cancer. Melanomas and carcinomas of the lung, breast, and kidney are the most common malignancies to metastasize to the brain. Recent advances in molecular genetics have enabled the identification of actionable, clinically relevant genetic alterations within primary tumors and their corresponding metastases. Adoption of genotype-guided treatment strategies for the management of systemic malignancy has resulted in dramatic and durable responses. Unfortunately, despite these therapeutic advances, central nervous system (CNS) relapses are not uncommon. Although these relapses have historically been attributed to limited blood brain barrier penetration of anti-neoplastic agents, recent work has demonstrated genetic heterogeneity such that metastatic sites, including BM, harbor relevant genetic alterations that are not present in primary tumor biopsies. This improved insight into molecular mechanisms underlying site specific recurrences can inform strategies for targeting these oncogenic drivers. Thus, development of rational, genomically guided CNS-penetrant therapies is crucial for ongoing therapeutic success.

Evolution, Medicine, and the Darwin Family

The common scientific roots of evolution and medicine are deep, as these fields of science developed in parallel from the Enlightenment in the late 1700s to the modern genomics era. The influence of the medical sciences on the discovery of evolution in the 1700s and 1800s is typified by how the medical family of Charles Darwin, including his grandfather Dr. Erasmus Darwin and father Dr. Robert Waring Darwin, directly and indirectly guided Charles’ scientific development and eventual discovery of natural selection. In particular, in the 1700s, Erasmus Darwin was a prolific writer, legendary doctor, and published extensive descriptions of both the process of adaptation and common descent among all of life (including humans). The influence began with Charles’ years in medical school at Edinburgh and is recorded in Charles Darwin’s own letters and notebooks. Despite scientific overlap, evolution and medicine have remained distant from each other, in part because of the same religious and political reasons that many oppose the view of a world changing via evolution. But evolution also has been limited in its influence on the biomedical sciences because of abuses and misunderstanding. The three issues discussed here are (1) typological application of medical “constitutions,” (2) teleological thinking in how adaptations evolve, and (3) the misapplication of evolution during the eugenics period up to the 1940s. The modern-day surge of interest and synthesis between evolutionary biology and the biomedical sciences, medical practice, and public health can build on a long legacy that spans more than two centuries. The large role played by the Darwin family of doctors can bring this history to life, can be used to illustrate potential pitfalls as the synthesis moves forward, and may be of interest to students both as undergraduates and in medical schools.

Comparative Linkage Meta-Analysis Reveals Regionally-Distinct, Disparate Genetic Architectures: Application to Bipolar Disorder and Schizophrenia

New high-throughput, population-based methods and next-generation sequencing capabilities hold great promise in the quest for common and rare variant discovery and in the search for ”missing heritability.” However, the optimal analytic strategies for approaching such data are still actively debated, representing the latest rate-limiting step in genetic progress. Since it is likely a majority of common variants of modest effect have been identified through the application of tagSNP-based microarray platforms (i.e., GWAS), alternative approaches robust to detection of low-frequency (1–5% MAF) and rare (<1%) variants are of great importance. Of direct relevance, we have available an accumulated wealth of linkage data collected through traditional genetic methods over several decades, the full value of which has not been exhausted. To that end, we compare results from two different linkage meta-analysis methods—GSMA and MSP—applied to the same set of 13 bipolar disorder and 16 schizophrenia GWLS datasets. Interestingly, we find that the two methods implicate distinct, largely non-overlapping, genomic regions. Furthermore, based on the statistical methods themselves and our contextualization of these results within the larger genetic literatures, our findings suggest, for each disorder, distinct genetic architectures may reside within disparate genomic regions. Thus, comparative linkage meta-analysis (CLMA) may be used to optimize low-frequency and rare variant discovery in the modern genomic era.

Roles of Mutation and Selection in Speciation: From Hugo de Vries to the Modern Genomic Era

One of the most important problems in evolutionary biology is to understand how new species are generated in nature. In the past, it was difficult to study this problem because our lifetime is too short to observe the entire process of speciation. In recent years, however, molecular and genomic techniques have been developed for identifying and studying the genes involved in speciation. Using these techniques, many investigators have already obtained new findings. At present, however, the results obtained are complex and quite confusing. We have therefore attempted to understand these findings coherently with a historical perspective and clarify the roles of mutation and natural selection in speciation. We have first indicated that the root of the currently burgeoning field of plant genomics goes back to Hugo de Vries, who proposed the mutation theory of evolution more than a century ago and that he unknowingly found the importance of polyploidy and chromosomal rearrangements in plant speciation. We have then shown that the currently popular Dobzhansky–Muller model of evolution of reproductive isolation is only one of many possible mechanisms. Some of them are Oka’s model of duplicate gene mutations, multiallelic speciation, mutation-rescue model, segregation-distorter gene model, heterochromatin-associated speciation, single-locus model, etc. The occurrence of speciation also depends on the reproductive system, population size, bottleneck effects, and environmental factors, such as temperature and day length. Some authors emphasized the importance of natural selection to speed up speciation, but mutation is crucial in speciation because reproductive barriers cannot be generated without mutations.

Updating the Model Definition of the Gene in the Modern Genomic Era with Implications for Instruction

Gericke and Hagberg (G & H, Sci Educ 16:849–881, 2007) recently published in this journal a thoughtful analysis of the historical progression of our understanding of the nature of the gene for use in instruction. This analysis, however, did not include the findings of the Human Genome Project (HGP), which must be included in any introductory genetics in the modern genomic era today. Many of these findings, especially the limited number of genes and the similarity of this number to that of primitive animals such as roundworms, were surprising and led to questions about the definition of the gene, many of which are addressed in this manuscript. The G & H models are also amended to include crucial concepts, including the history of determining that DNA and not protein is the molecule of inheritance, the work of Barbara McClintock and the discovery of transposons, polygenic/multi-factorial inheritance, and reverse transcription. The following discussion further extends the G & H work to include the more recent work of the ENCyclopedia Of DNA Elements (ENCODE) Project. The results of this work have resulted in even more fundamental questions about the gene. For example, large sections of the genome that were previously identified as non-protein-coding ‘junk’ have been shown to be transcribed into RNA that is likely involved in regulation of genome function that might be more crucial than the coding DNA itself in distinguishing simpler from more complex species. Should these transcribed but not translated sequences be recognized as genes? This level of questioning of our basic definition has not occurred since the modern synthesis of genetics based on the work of Watson and Crick and makes this one of the most exciting times for genetics and medicine.

One Medicine: An Introduction

All of nature has inherent value for biology. Consider how much we have already learned from the comparative biology of yeast,worms, flies, zebra fish and many other diverse organisms about the molecular machinery governing normal cell division and normal growth and development; knowledge that applies also to mammals. Plant systems have advanced our understanding of epigenetics that might also apply to cancer biology. Aberrations in these basic processes such as the cell cycle check points and chromosome alignment on the mitotic spindle contribute, of course, to cancer. Naturally occurring oncogenic retroviruses associated with tumors in animals – mainly domesticated chickens, mice and cats, gave us our first glimpse, about 30 years ago, of oncogenes, cellular genes highly conserved throughout mammalian evolution. I experienced the excitement of this era by helping to discover two oncogenes, both cytoplasmic tyrosine kinases (see below) in spontaneous sarcomas of domestic cats. In studying retroviruses in wild mice as a potential model for human cancer, I also learned the importance of natural history knowledge in contrast to the information gained from laboratory mice. In the 1980s, DNA tumor viruses in animals pointed the way to the key role of evolutionarily conserved tumor suppressor genes with which the viruses interacted and inactivated. In the modern genomic era, we now appreciate that all life forms, and mammals in particular, have evolutionarily related genomes and are governed by similar fundamental molecular processes. We know that oncogenes and tumor suppressor genes, for example, are normal cell genes that function in an intricate network together with many other genes, making up more than 30 signal transduction pathways and protein interaction domains that govern normal cell growth, differentiation and death (apoptosis). Hundreds of oncogenes and tumor suppressor genes and their protein products have already been identified as part of these pathways and domains. Point mutations or altered expression of these genes caused by chromosomal structural abnormalities may contribute to cancer in general and can be detected by modern techniques of cytogenetics, functional genomics and proteomics which allow the near simultaneous analyses of thousands of genes at the RNA and protein levels. Addition to or removal of phosphorous from proteins via protein kinases and phosphatases, respectively, is a key regulator of normal protein interactions in the signal transduction pathways and key driver of many forms of cancer. Immunoassays now permit the detection and quantitation of such intracellular factors and their extent of phosphorylation. Over 500 separate protein kinase genes are encoded in the mammalian genome, but relatively few of their targets are known. However, a good number of these kinases have already been linked to the development of cancer in general and as specific targets of therapeutic intervention. Other genes and proteins involved in signal transduction pathways, such as those controlling chromatin structure, telomere length, RNA transcription, RNA interference, protein folding and stability, cytoskelatal regulation, cell motility and angiogenesis are also highly conserved in evolution. Single nucleotide polymorphisms or larger structural variation in such genes could help explain the familial predisposition to certain cancers. Many other

Discovery of induced point mutations in maize genes by TILLING

BackgroundGoing from a gene sequence to its function in the context of a whole organism requires a strategy for targeting mutations, referred to as reverse genetics. Reverse genetics is highly desirable in the modern genomics era; however, the most powerful methods are generally restricted to a few model organisms. Previously, we introduced a reverse-genetic strategy with the potential for general applicability to organisms that lack well-developed genetic tools. Our TILLING (Targeting Induced Local Lesions IN Genomes) method uses chemical mutagenesis followed by screening for single-base changes to discover induced mutations that alter protein function. TILLING was shown to be an effective reverse genetic strategy by the establishment of a high-throughput TILLING facility and the delivery of thousands of point mutations in hundreds of Arabidopsis genes to members of the plant biology community.ResultsWe demonstrate that high-throughput TILLING is applicable to maize, an important crop plant with a large genome but with limited reverse-genetic resources currently available. We screened pools of DNA samples for mutations in 1-kb segments from 11 different genes, obtaining 17 independent induced mutations from a population of 750 pollen-mutagenized maize plants. One of the genes targeted was the DMT102 chromomethylase gene, for which we obtained an allelic series of three missense mutations that are predicted to be strongly deleterious.ConclusionsOur findings indicate that TILLING is a broadly applicable and efficient reverse-genetic strategy. We are establishing a public TILLING service for maize modeled on the existing Arabidopsis TILLING Project.