RESUMEN
This perspective focuses on advances in genome technology over the last 25 years and their impact on germline variant discovery within the field of human genetics. The field has witnessed tremendous technological advances from microarrays to short-read sequencing and now long-read sequencing. Each technology has provided genome-wide access to different classes of human genetic variation. We are now on the verge of comprehensive variant detection of all forms of variation for the first time with a single assay. We predict that this transition will further transform our understanding of human health and biology and, more importantly, provide novel insights into the dynamic mutational processes shaping our genomes.
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Variación Estructural del Genoma , Genómica , Humanos , Genómica/métodos , Mutación de Línea Germinal , Mutación , TecnologíaRESUMEN
Metastatic progression is the main cause of death in cancer patients, whereas the underlying genomic mechanisms driving metastasis remain largely unknown. Here, we assembled MSK-MET, a pan-cancer cohort of over 25,000 patients with metastatic diseases. By analyzing genomic and clinical data from this cohort, we identified associations between genomic alterations and patterns of metastatic dissemination across 50 tumor types. We found that chromosomal instability is strongly correlated with metastatic burden in some tumor types, including prostate adenocarcinoma, lung adenocarcinoma, and HR+/HER2+ breast ductal carcinoma, but not in others, including colorectal cancer and high-grade serous ovarian cancer, where copy-number alteration patterns may be established early in tumor development. We also identified somatic alterations associated with metastatic burden and specific target organs. Our data offer a valuable resource for the investigation of the biological basis for metastatic spread and highlight the complex role of chromosomal instability in cancer progression.
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Genómica , Secuenciación de Nucleótidos de Alto Rendimiento , Metástasis de la Neoplasia/genética , Metástasis de la Neoplasia/patología , Estudios de Cohortes , Femenino , Humanos , Masculino , Especificidad de Órganos/genética , Estudios ProspectivosRESUMEN
We present deterministic barcoding in tissue for spatial omics sequencing (DBiT-seq) for co-mapping of mRNAs and proteins in a formaldehyde-fixed tissue slide via next-generation sequencing (NGS). Parallel microfluidic channels were used to deliver DNA barcodes to the surface of a tissue slide, and crossflow of two sets of barcodes, A1-50 and B1-50, followed by ligation in situ, yielded a 2D mosaic of tissue pixels, each containing a unique full barcode AB. Application to mouse embryos revealed major tissue types in early organogenesis as well as fine features like microvasculature in a brain and pigmented epithelium in an eye field. Gene expression profiles in 10-µm pixels conformed into the clusters of single-cell transcriptomes, allowing for rapid identification of cell types and spatial distributions. DBiT-seq can be adopted by researchers with no experience in microfluidics and may find applications in a range of fields including developmental biology, cancer biology, neuroscience, and clinical pathology.
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Código de Barras del ADN Taxonómico , Genómica , Especificidad de Órganos/genética , Animales , Automatización , Encéfalo/embriología , Análisis por Conglomerados , ADN Complementario/genética , Embrión de Mamíferos/metabolismo , Ojo/embriología , Femenino , Regulación del Desarrollo de la Expresión Génica , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Ratones Endogámicos C57BL , Microfluídica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reproducibilidad de los Resultados , Análisis de la Célula Individual , Transcriptoma/genéticaRESUMEN
CRISPR-Cas nucleoproteins target foreign DNA via base pairing with a crRNA. However, a quantitative description of protein binding and nuclease activation at off-target DNA sequences remains elusive. Here, we describe a chip-hybridized association-mapping platform (CHAMP) that repurposes next-generation sequencing chips to simultaneously measure the interactions between proteins and â¼107 unique DNA sequences. Using CHAMP, we provide the first comprehensive survey of DNA recognition by a type I-E CRISPR-Cas (Cascade) complex and Cas3 nuclease. Analysis of mutated target sequences and human genomic DNA reveal that Cascade recognizes an extended protospacer adjacent motif (PAM). Cascade recognizes DNA with a surprising 3-nt periodicity. The identity of the PAM and the PAM-proximal nucleotides control Cas3 recruitment by releasing the Cse1 subunit. These findings are used to develop a model for the biophysical constraints governing off-target DNA binding. CHAMP provides a framework for high-throughput, quantitative analysis of protein-DNA interactions on synthetic and genomic DNA. PAPERCLIP.
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Proteínas de Unión al ADN/análisis , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Unión Proteica , Análisis de Secuencia de ADN/métodos , Sistemas CRISPR-Cas , Ensayo de Cambio de Movilidad Electroforética , Microscopía Fluorescente , Motivos de NucleótidosRESUMEN
Early successes in identifying and targeting individual oncogenic drivers, together with the increasing feasibility of sequencing tumor genomes, have brought forth the promise of genome-driven oncology care. As we expand the breadth and depth of genomic analyses, the biological and clinical complexity of its implementation will be unparalleled. Challenges include target credentialing and validation, implementing drug combinations, clinical trial designs, targeting tumor heterogeneity, and deploying technologies beyond DNA sequencing, among others. We review how contemporary approaches are tackling these challenges and will ultimately serve as an engine for biological discovery and increase our insight into cancer and its treatment.
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Genómica , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Medicina de Precisión , Animales , Resistencia a Antineoplásicos , Heterogeneidad Genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Terapia Molecular Dirigida , Mutación Missense , Análisis de Secuencia de ADNRESUMEN
CRISPR-Cas9 is a powerful gene-editing technology; however, off-target activity remains an important consideration for therapeutic applications. We have previously shown that force-stretching DNA induces off-target activity and hypothesized that distortions of the DNA topology in vivo, such as negative DNA supercoiling, could reduce Cas9 specificity. Using single-molecule optical-tweezers, we demonstrate that negative supercoiling λ-DNA induces sequence-specific Cas9 off-target binding at multiple sites, even at low forces. Using an adapted CIRCLE-seq approach, we detect over 10,000 negative-supercoiling-induced Cas9 off-target double-strand breaks genome-wide caused by increased mismatch tolerance. We further demonstrate in vivo that directed local DNA distortion increases off-target activity in cells and that induced off-target events can be detected during Cas9 genome editing. These data demonstrate that Cas9 off-target activity is regulated by DNA topology in vitro and in vivo, suggesting that cellular processes, such as transcription and replication, could induce off-target activity at previously overlooked sites.
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Sistemas CRISPR-Cas , Edición Génica , Genoma , ADN/genética , Pinzas ÓpticasRESUMEN
The past decade has revolutionized our understanding of regulatory noncoding RNAs (ncRNAs). Among the most recently identified ncRNAs are downstream-of-gene (DoG)-containing transcripts that are produced by widespread transcriptional readthrough. The discovery of DoGs has set the stage for future studies to address many unanswered questions regarding the mechanisms that promote readthrough transcription, RNA processing, and the cellular functions of the unique transcripts. In this review, we summarize current findings regarding the biogenesis, function, and mechanisms regulating this exciting new class of RNA molecules.
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ARN no Traducido , Transcripción Genética , Procesamiento Postranscripcional del ARN , ARN no Traducido/genéticaRESUMEN
Gene delivery vehicles based on adeno-associated viruses (AAVs) are enabling increasing success in human clinical trials, and they offer the promise of treating a broad spectrum of both genetic and non-genetic disorders. However, delivery efficiency and targeting must be improved to enable safe and effective therapies. In recent years, considerable effort has been invested in creating AAV variants with improved delivery, and computational approaches have been increasingly harnessed for AAV engineering. In this review, we discuss how computationally designed AAV libraries are enabling directed evolution. Specifically, we highlight approaches that harness sequences outputted by next-generation sequencing (NGS) coupled with machine learning (ML) to generate new functional AAV capsids and related regulatory elements, pushing the frontier of what vector engineering and gene therapy may achieve.
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Dependovirus , Técnicas de Transferencia de Gen , Dependovirus/genética , Humanos , Terapia Genética/métodos , Vectores Genéticos/metabolismo , Ingeniería Genética , Animales , Biología Computacional/métodosRESUMEN
Lineage-based vaccine design is an attractive approach for eliciting broadly neutralizing antibodies (bNAbs) against HIV-1. However, most bNAb lineages studied to date have features indicative of unusual recombination and/or development. From an individual in the prospective RV217 cohort, we identified three lineages of bNAbs targeting the membrane-proximal external region (MPER) of the HIV-1 envelope. Antibodies RV217-VRC42.01, -VRC43.01, and -VRC46.01 used distinct modes of recognition and neutralized 96%, 62%, and 30%, respectively, of a 208-strain virus panel. All three lineages had modest levels of somatic hypermutation and normal antibody-loop lengths and were initiated by the founder virus MPER. The broadest lineage, VRC42, was similar to the known bNAb 4E10. A multimeric immunogen based on the founder MPER activated B cells bearing the unmutated common ancestor of VRC42, with modest maturation of early VRC42 intermediates imparting neutralization breadth. These features suggest that VRC42 may be a promising template for lineage-based vaccine design.
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Anticuerpos Neutralizantes/inmunología , Anticuerpos Anti-VIH/inmunología , VIH-1/inmunología , Vacunas contra el SIDA/inmunología , Secuencia de Aminoácidos , Linfocitos B/inmunología , Línea Celular , Células HEK293 , Infecciones por VIH/inmunología , Humanos , Leucocitos Mononucleares , Estudios LongitudinalesRESUMEN
DNA single-strand breaks (SSBs) are among the most common lesions in the genome, arising spontaneously and as intermediates of many DNA transactions. Nevertheless, in contrast to double-strand breaks (DSBs), their distribution in the genome has hardly been addressed in a meaningful way. We now present a technique based on genome-wide ligation of 3'-OH ends followed by sequencing (GLOE-Seq) and an associated computational pipeline designed for capturing SSBs but versatile enough to be applied to any lesion convertible into a free 3'-OH terminus. We demonstrate its applicability to mapping of Okazaki fragments without prior size selection and provide insight into the relative contributions of DNA ligase 1 and ligase 3 to Okazaki fragment maturation in human cells. In addition, our analysis reveals biases and asymmetries in the distribution of spontaneous SSBs in yeast and human chromatin, distinct from the patterns of DSBs.
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Mapeo Cromosómico/métodos , Replicación del ADN/genética , Análisis de Secuencia de ADN/métodos , Cromatina , ADN/genética , Roturas del ADN de Cadena Simple , Daño del ADN/genética , ADN Ligasa (ATP)/genética , Reparación del ADN/genética , Genoma/genética , Humanos , Nucleótidos , Saccharomyces cerevisiae/genéticaRESUMEN
Next-generation sequencing has revolutionized the speed of rare disease (RD) diagnoses. While clinical exome and genome sequencing represent an effective tool for many RD diagnoses, there is room to further improve the diagnostic odyssey of many RD patients. One recognizable intervention lies in increasing equitable access to genomic testing. Rural communities represent a significant portion of underserved and underrepresented individuals facing additional barriers to diagnosis and treatment. Primary care providers (PCPs) at local clinics, though sometimes suspicious of a potential benefit of genetic testing for their patients, have significant constraints in pursuing it themselves and rely on referrals to specialists. Yet, these referrals are typically followed by long waitlists and significant delays in clinical assessment, insurance clearance, testing, and initiation of diagnosis-informed care management. Not only is this process time intensive, but it also often requires multiple visits to urban medical centers for which distance may be a significant barrier to rural families. Therefore, providing early, "direct-to-provider" (DTP) local access to unrestrictive genomic testing is likely to help speed up diagnostic times and access to care for RD patients in rural communities. In a pilot study with a PCP clinic in rural Kansas, we observed a minimum 5.5 months shortening of time to diagnosis through the DTP exome sequencing program as compared to rural patients receiving genetic testing through the "traditional" PCP-referral-to-specialist scheme. We share our experience to encourage future partnerships beyond our center. Our efforts represent just one step in fostering greater diversity and equity in genomic studies.
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Pruebas Genéticas , Genómica , Accesibilidad a los Servicios de Salud , Enfermedades Raras , Población Rural , Humanos , Pruebas Genéticas/métodos , Enfermedades Raras/genética , Enfermedades Raras/diagnóstico , Genómica/métodos , Niño , Masculino , Secuenciación de Nucleótidos de Alto Rendimiento , FemeninoRESUMEN
The human brain contains a vast number of cells and shows extraordinary cellular diversity to facilitate the many cognitive and automatic commands governing our bodily functions. This complexity arises partly from large-scale structural variations in the genome, evolutionary processes to increase brain size, function, and cognition. Not surprisingly given recent technical advances, low-grade gliomas (LGGs), which arise from the glia (the most abundant cell type in the brain), have undergone a recent revolution in their classification and therapy, especially in the pediatric setting. Next-generation sequencing has uncovered previously unappreciated diverse LGG entities, unraveling genetic subgroups and multiple molecular alterations and altered pathways, including many amenable to therapeutic targeting. In this article we review these novel entities, in which oncogenic processes show striking age-related neuroanatomical specificity (highlighting their close interplay with development); the opportunities they provide for targeted therapies, some of which are already practiced at the bedside; and the challenges of implementing molecular pathology in the clinic.
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Neoplasias Encefálicas/genética , Encéfalo/crecimiento & desarrollo , Glioma/genética , Adulto , Factores de Edad , Encéfalo/patología , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/patología , Niño , Glioma/diagnóstico , Glioma/patología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Isocitrato Deshidrogenasa/genética , Técnicas de Diagnóstico Molecular , Mutación , Trastornos del Neurodesarrollo/genética , Trastornos del Neurodesarrollo/patología , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/genética , Quinasas raf/genéticaRESUMEN
Inborn errors of immunity (IEI) are a diverse group of monogenic disorders of the immune system due to germline variants in genes important for the immune response. Over the past decade there has been increasing recognition that acquired somatic variants present in a subset of cells can also lead to immune disorders or 'phenocopies' of IEI. Discovery of somatic mosaicism causing IEI has largely arisen from investigation of seemingly sporadic cases of IEI with predominant symptoms of autoinflammation and/or autoimmunity in which germline disease-causing variants are not detected. Disease-causing somatic mosaicism has been identified in genes that also cause germline IEI, such as FAS, and in genes without significant corresponding germline disease, such as UBA1 and TLR8. There are challenges in detecting low-level somatic variants, and it is likely that the extent of the somatic mosaicism causing IEI is largely uncharted. Here we review the field of somatic mosaicism leading to IEI and discuss challenges and methods for somatic variant detection, including diagnostic approaches for molecular diagnoses of patients.
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Autoinmunidad , Mosaicismo , Humanos , FenotipoRESUMEN
Establishing modular binders as diagnostic detection agents represents a cost- and time-efficient alternative to the commonly used binders that are generated one molecule at a time. In contrast to these conventional approaches, a modular binder can be designed in silico from individual modules to, in principle, recognize any desired linear epitope without going through a selection and hit-validation process, given a set of preexisting, amino acid-specific modules. Designed armadillo repeat proteins (dArmRP) have been developed as modular binder scaffolds, and we report here the generation of highly specific dArmRP modules by yeast surface display selection, performed on a rationally designed dArmRP library. A selection strategy was developed to distinguish the binding difference resulting from a single amino acid mutation in the target peptide. Our reverse-competitor strategy introduced here employs the designated target as a competitor to increase the sensitivity when separating specific from cross-reactive binders that show similar affinities for the target peptide. With this switch in selection focus from affinity to specificity, we found that the enrichment during this specificity sort is indicative of the desired phenotype, regardless of the binder abundance. Hence, deep sequencing of the selection pools allows retrieval of phenotypic hits with only 0.1% abundance in the selectivity sort pool from the next-generation sequencing data alone. In a proof-of-principle study, a binder was created by replacing all corresponding wild-type modules with a newly selected module, yielding a binder with very high affinity for the designated target that has been successfully validated as a detection agent in western blot analysis.
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Proteínas del Dominio Armadillo , Saccharomyces cerevisiae , Proteínas del Dominio Armadillo/genética , Proteínas del Dominio Armadillo/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Unión Proteica , Péptidos/metabolismo , Péptidos/genética , Péptidos/química , Epítopos/genética , Biblioteca de PéptidosRESUMEN
Inherited retinal diseases (IRDs) are a group of rare genetic eye conditions that cause blindness. Despite progress in identifying genes associated with IRDs, improvements are necessary for classifying rare autosomal dominant (AD) disorders. AD diseases are highly heterogenous, with causal variants being restricted to specific amino acid changes within certain protein domains, making AD conditions difficult to classify. Here, we aim to determine the top-performing in-silico tools for predicting the pathogenicity of AD IRD variants. We annotated variants from ClinVar and benchmarked 39 variant classifier tools on IRD genes, split by inheritance pattern. Using area-under-the-curve (AUC) analysis, we determined the top-performing tools and defined thresholds for variant pathogenicity. Top-performing tools were assessed using genome sequencing on a cohort of participants with IRDs of unknown etiology. MutScore achieved the highest accuracy within AD genes, yielding an AUC of 0.969. When filtering for AD gain-of-function and dominant negative variants, BayesDel had the highest accuracy with an AUC of 0.997. Five participants with variants in NR2E3, RHO, GUCA1A, and GUCY2D were confirmed to have dominantly inherited disease based on pedigree, phenotype, and segregation analysis. We identified two uncharacterized variants in GUCA1A (c.428T>A, p.Ile143Thr) and RHO (c.631C>G, p.His211Asp) in three participants. Our findings support using a multi-classifier approach comprised of new missense classifier tools to identify pathogenic variants in participants with AD IRDs. Our results provide a foundation for improved genetic diagnosis for people with IRDs.
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Simulación por Computador , Linaje , Enfermedades de la Retina , Humanos , Enfermedades de la Retina/genética , Femenino , Masculino , Mutación , Genes Dominantes , Predisposición Genética a la Enfermedad , Biología Computacional/métodos , Fenotipo , AdultoRESUMEN
Long-read sequencing (LRS) technologies have provided extremely powerful tools to explore genomes. While in the early years these methods suffered technical limitations, they have recently made significant progress in terms of read length, throughput, and accuracy and bioinformatics tools have strongly improved. Here, we aim to review the current status of LRS technologies, the development of novel methods, and the impact on genomics research. We will explore the most impactful recent findings made possible by these technologies focusing on high-resolution sequencing of genomes and transcriptomes and the direct detection of DNA and RNA modifications. We will also discuss how LRS methods promise a more comprehensive understanding of human genetic variation, transcriptomics, and epigenetics for the coming years.
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Genómica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Genómica/métodos , Análisis de Secuencia de ADN/métodos , Biología Computacional , Perfilación de la Expresión Génica/métodosRESUMEN
DNA variants that arise after conception can show mosaicism, varying in presence and extent among tissues. Mosaic variants have been reported in Mendelian diseases, but further investigation is necessary to broadly understand their incidence, transmission, and clinical impact. A mosaic pathogenic variant in a disease-related gene may cause an atypical phenotype in terms of severity, clinical features, or timing of disease onset. Using high-depth sequencing, we studied results from one million unrelated individuals referred for genetic testing for almost 1,900 disease-related genes. We observed 5,939 mosaic sequence or intragenic copy number variants distributed across 509 genes in nearly 5,700 individuals, constituting approximately 2% of molecular diagnoses in the cohort. Cancer-related genes had the most mosaic variants and showed age-specific enrichment, in part reflecting clonal hematopoiesis in older individuals. We also observed many mosaic variants in genes related to early-onset conditions. Additional mosaic variants were observed in genes analyzed for reproductive carrier screening or associated with dominant disorders with low penetrance, posing challenges for interpreting their clinical significance. When we controlled for the potential involvement of clonal hematopoiesis, most mosaic variants were enriched in younger individuals and were present at higher levels than in older individuals. Furthermore, individuals with mosaicism showed later disease onset or milder phenotypes than individuals with non-mosaic variants in the same genes. Collectively, the large compendium of variants, disease correlations, and age-specific results identified in this study expand our understanding of the implications of mosaic DNA variation for diagnosis and genetic counseling.
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Variaciones en el Número de Copia de ADN , Mosaicismo , Variaciones en el Número de Copia de ADN/genética , Pruebas Genéticas , Fenotipo , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , MutaciónRESUMEN
The island of St Helena played a crucial role in the suppression of the transatlantic slave trade. Strategically located in the middle of the South Atlantic, it served as a staging post for the Royal Navy and reception point for enslaved Africans who had been "liberated" from slave ships intercepted by the British. In total, St Helena received approximately 27,000 liberated Africans between 1840 and 1867. Written sources suggest that the majority of these individuals came from West Central Africa, but their precise origins are unknown. Here, we report the results of ancient DNA analyses that we conducted as part of a wider effort to commemorate St Helena's liberated Africans and to restore knowledge of their lives and experiences. We generated partial genomes (0.1-0.5×) for 20 individuals whose remains had been recovered during archaeological excavations on the island. We compared their genomes with genotype data for over 3,000 present-day individuals from 90 populations across sub-Saharan Africa and conclude that the individuals most likely originated from different source populations within the general area between northern Angola and Gabon. We also find that the majority (17/20) of the individuals were male, supporting a well-documented sex bias in the latter phase of the transatlantic slave trade. The study expands our understanding of St Helena's liberated African community and illustrates how ancient DNA analyses can be used to investigate the origins and identities of individuals whose lives were bound up in the story of slavery and its abolition.
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Pueblo Africano , Personas Esclavizadas , Humanos , Femenino , Masculino , ADN Antiguo , Población Negra/genética , GenotipoRESUMEN
Uterine leiomyomas (ULs) are benign smooth muscle tumors that are common in premenopausal women. Somatic alterations in MED12, HMGA2, FH, genes encoding subunits of the SRCAP complex, and genes involved in Cullin 3-RING E3 ligase neddylation are mutually exclusive UL drivers. Established predisposition genes explain only partially the estimated heritability of leiomyomas. Here, we examined loss-of-function variants across 18,899 genes in a cohort of 233,614 White European women, revealing variants in four genes encoding SRCAP complex subunits (YEATS4, ZNHIT1, DMAP1, and ACTL6A) with a significant association to ULs, and YEATS4 and ZNHIT1 strikingly rank first and second, respectively. Positive mutation status was also associated with younger age at diagnosis and hysterectomy. Moderate-penetrance UL risk was largely attributed to rare non-synonymous mutations affecting the SRCAP complex. To examine this disease phenotype more closely, we set out to identify inherited mutations affecting the SRCAP complex in our in-house sample collection of Finnish individuals with ULs (n = 860). We detected one individual with an ACTL6A splice-site mutation, two individuals with a YEATS4 missense mutation, and four individuals with DMAP1 mutations: one splice-site, one nonsense, and two missense variants. These individuals had large and/or multiple ULs, were often diagnosed at an early age, and many had family history of ULs. When a somatic second hit was found, ACTL6A and DMAP1 were silenced in tumors by somatic mutation and YEATS4 by promoter hypermethylation. Decreased H2A.Z staining was observed in the tumors, providing further evidence for the pathogenic nature of the germline mutations. Our results establish inactivation of genes encoding SRCAP complex subunits as a central contributor to moderate-penetrance UL predisposition.
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Leiomioma , Neoplasias Uterinas , Humanos , Femenino , Neoplasias Uterinas/genética , Neoplasias Uterinas/patología , Mutación de Línea Germinal , Penetrancia , Análisis Mutacional de ADN , Leiomioma/genética , Leiomioma/patología , Mutación , Complejo Mediador/genética , Actinas/genética , Proteínas Cromosómicas no Histona/genética , Proteínas de Unión al ADN/genética , Adenosina Trifosfatasas/genéticaRESUMEN
This article provides an in-depth review of computational methods for predicting transcriptional regulators (TRs) with query gene sets. Identification of TRs is of utmost importance in many biological applications, including but not limited to elucidating biological development mechanisms, identifying key disease genes, and predicting therapeutic targets. Various computational methods based on next-generation sequencing (NGS) data have been developed in the past decade, yet no systematic evaluation of NGS-based methods has been offered. We classified these methods into two categories based on shared characteristics, namely library-based and region-based methods. We further conducted benchmark studies to evaluate the accuracy, sensitivity, coverage, and usability of NGS-based methods with molecular experimental datasets. Results show that BART, ChIP-Atlas, and Lisa have relatively better performance. Besides, we point out the limitations of NGS-based methods and explore potential directions for further improvement.