RESUMO
Glutaric aciduria type II (GAII) is a heterogeneous genetic disorder affecting mitochondrial fatty acid, amino acid and choline oxidation. Clinical manifestations vary across the lifespan and onset may occur at any time from the early neonatal period to advanced adulthood. Historically, some patients, in particular those with late onset disease, have experienced significant benefit from riboflavin supplementation. GAII has been considered an autosomal recessive condition caused by pathogenic variants in the gene encoding electron-transfer flavoprotein ubiquinone-oxidoreductase (ETFDH) or in the genes encoding electron-transfer flavoprotein subunits A and B (ETFA and ETFB respectively). Variants in genes involved in riboflavin metabolism have also been reported. However, in some patients, molecular analysis has failed to reveal diagnostic molecular results. In this study, we report the outcome of molecular analysis in 28 Australian patients across the lifespan, 10 paediatric and 18 adult, who had a diagnosis of glutaric aciduria type II based on both clinical and biochemical parameters. Whole genome sequencing was performed on 26 of the patients and two neonatal onset patients had targeted sequencing of candidate genes. The two patients who had targeted sequencing had biallelic pathogenic variants (in ETFA and ETFDH). None of the 26 patients whose whole genome was sequenced had biallelic variants in any of the primary candidate genes. Interestingly, nine of these patients (34.6%) had a monoallelic pathogenic or likely pathogenic variant in a single primary candidate gene and one patient (3.9%) had a monoallelic pathogenic or likely pathogenic variant in two separate genes within the same pathway. The frequencies of the damaging variants within ETFDH and FAD transporter gene SLC25A32 were significantly higher than expected when compared to the corresponding allele frequencies in the general population. The remaining 16 patients (61.5%) had no pathogenic or likely pathogenic variants in the candidate genes. Ten (56%) of the 18 adult patients were taking the selective serotonin reuptake inhibitor antidepressant sertraline, which has been shown to produce a GAII phenotype, and another two adults (11%) were taking a serotonin-norepinephrine reuptake inhibitor antidepressant, venlafaxine or duloxetine, which have a mechanism of action overlapping that of sertraline. Riboflavin deficiency can also mimic both the clinical and biochemical phenotype of GAII. Several patients on these antidepressants showed an initial response to riboflavin but then that response waned. These results suggest that the GAII phenotype can result from a complex interaction between monoallelic variants and the cellular environment. Whole genome or targeted gene panel analysis may not provide a clear molecular diagnosis.
Assuntos
Flavoproteínas Transferidoras de Elétrons , Deficiência Múltipla de Acil Coenzima A Desidrogenase , Humanos , Feminino , Masculino , Criança , Adulto , Pré-Escolar , Flavoproteínas Transferidoras de Elétrons/genética , Adolescente , Deficiência Múltipla de Acil Coenzima A Desidrogenase/genética , Queensland , Riboflavina/uso terapêutico , Adulto Jovem , Lactente , Proteínas Ferro-Enxofre/genética , Estudos de Coortes , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Recém-Nascido , Mutação , Sequenciamento Completo do GenomaRESUMO
Retrotransposons are mobile DNA sequences duplicated via transcription and reverse transcription of an RNA intermediate. Cis-regulatory elements encoded by retrotransposons can also promote the transcription of adjacent genes. Somatic LINE-1 (L1) retrotransposon insertions have been detected in mammalian neurons. It is, however, unclear whether L1 sequences are mobile in only some neuronal lineages or therein promote neurodevelopmental gene expression. Here we report programmed L1 activation by SOX6, a transcription factor critical for parvalbumin (PV) interneuron development. Mouse PV interneurons permit L1 mobilization in vitro and in vivo, harbor unmethylated L1 promoters and express full-length L1 mRNAs and proteins. Using nanopore long-read sequencing, we identify unmethylated L1s proximal to PV interneuron genes, including a novel L1 promoter-driven Caps2 transcript isoform that enhances neuron morphological complexity in vitro. These data highlight the contribution made by L1 cis-regulatory elements to PV interneuron development and transcriptome diversity, uncovered due to L1 mobility in this milieu.
Assuntos
Interneurônios , Elementos Nucleotídeos Longos e Dispersos , Parvalbuminas , Animais , Interneurônios/metabolismo , Interneurônios/fisiologia , Camundongos , Elementos Nucleotídeos Longos e Dispersos/genética , Parvalbuminas/metabolismo , Retroelementos/genética , Masculino , Neurogênese/fisiologia , Neurogênese/genética , Camundongos Endogâmicos C57BL , Regulação da Expressão Gênica no Desenvolvimento/genéticaRESUMO
INTRODUCTION: Previous studies have reported that neonates less than the 25th BWC especially if they were male, were more likely to be associated with birth complications suggesting small neonates often identified as appropriately grown are at risk of adverse outcomes. We have questioned whether smaller neonates not typically categorized as "small for gestational age" may not reach their genetically determined growth due to placental insufficiency. METHODS: RNA-Seq was performed on the Illumina NovaSeq 600 using term placentae from neonates that were less than the 10th birthweight centile (BWC) (n = 39), between the 10th and the 30th BWC (n = 15) or greater than the 30th BWC (n = 23). Bioinformatic analyses were conducted and statistical significance was assessed at a level of P < 0.05 for single comparisons or FDR <0.05 unless otherwise noted. RESULTS: Gene set enrichment analysis revealed differences between BWC groups and in relation to the sex of the placenta. Genes associated with hypoxia, inflammatory responses, estrogen responsive genes, and androgen responsive genes were enriched (FDR <0.1) for in placentae of neonates <10th BWC regardless of sex and also in male placentae of neonates between the 10th-30th BWC. Female placenta of neonates between the 10th-30th BWC were comparable to placentae of neonates >30th BWC. DISCUSSION: These findings provide evidence that small male neonates may be at a greater risk of an adverse outcome than females due to changes in gene expression that are associated with placental dysfunction. The current data raises questions of whether placental pathology for smaller appropriately grown neonates should be scientifically and clinically examined in more depth.
Assuntos
Placenta , Insuficiência Placentária , Recém-Nascido , Gravidez , Feminino , Masculino , Humanos , Placenta/metabolismo , Insuficiência Placentária/patologia , Idade Gestacional , Recém-Nascido Pequeno para a Idade Gestacional , Retardo do Crescimento Fetal/metabolismo , Perfilação da Expressão GênicaRESUMO
BACKGROUND: Vitamin C (vitC) enhances the activity of 2-oxoglutarate-dependent dioxygenases, including TET enzymes, which catalyse DNA demethylation, and Jumonji-domain histone demethylases. The epigenetic remodelling promoted by vitC improves the efficiency of induced pluripotent stem cell derivation, and is required to attain a ground-state of pluripotency in embryonic stem cells (ESCs) that closely mimics the inner cell mass of the early blastocyst. However, genome-wide DNA and histone demethylation can lead to upregulation of transposable elements (TEs), and it is not known how vitC addition in culture media affects TE expression in pluripotent stem cells. RESULTS: Here we show that vitC increases the expression of several TE families, including evolutionarily young LINE-1 (L1) elements, in mouse ESCs. We find that TET activity is dispensable for L1 upregulation, and that instead it occurs largely as a result of H3K9me3 loss mediated by KDM4A/C histone demethylases. Despite increased L1 levels, we did not detect increased somatic insertion rates in vitC-treated cells. Notably, treatment of human ESCs with vitC also increases L1 protein levels, albeit through a distinct, post-transcriptional mechanism. CONCLUSION: VitC directly modulates the expression of mouse L1s and other TEs through epigenetic mechanisms, with potential for downstream effects related to the multiple emerging roles of L1s in cellular function.
Assuntos
Ácido Ascórbico , Células-Tronco Embrionárias Murinas , Humanos , Animais , Camundongos , Ácido Ascórbico/farmacologia , Células-Tronco Embrionárias Murinas/metabolismo , Elementos Nucleotídeos Longos e Dispersos , Metilação de DNA , Histona Desmetilases/metabolismo , DNA/metabolismo , Desmetilação , Histona Desmetilases com o Domínio Jumonji/genética , Histona Desmetilases com o Domínio Jumonji/metabolismoRESUMO
Mice harbor â¼2800 intact copies of the retrotransposon Long Interspersed Element 1 (L1). The in vivo retrotransposition capacity of an L1 copy is defined by both its sequence integrity and epigenetic status, including DNA methylation of the monomeric units constituting young mouse L1 promoters. Locus-specific L1 methylation dynamics during development may therefore elucidate and explain spatiotemporal niches of endogenous retrotransposition but remain unresolved. Here, we interrogate the retrotransposition efficiency and epigenetic fate of source (donor) L1s, identified as mobile in vivo. We show that promoter monomer loss consistently attenuates the relative retrotransposition potential of their offspring (daughter) L1 insertions. We also observe that most donor/daughter L1 pairs are efficiently methylated upon differentiation in vivo and in vitro. We use Oxford Nanopore Technologies (ONT) long-read sequencing to resolve L1 methylation genome-wide and at individual L1 loci, revealing a distinctive "smile" pattern in methylation levels across the L1 promoter region. Using Pacific Biosciences (PacBio) SMRT sequencing of L1 5' RACE products, we then examine DNA methylation dynamics at the mouse L1 promoter in parallel with transcription start site (TSS) distribution at locus-specific resolution. Together, our results offer a novel perspective on the interplay between epigenetic repression, L1 evolution, and genome stability.
Assuntos
Desenvolvimento Embrionário , Elementos Nucleotídeos Longos e Dispersos , Camundongos , Animais , Retroelementos/genética , Metilação de DNA , Regiões Promotoras GenéticasRESUMO
Hypoglycemia in type 1 diabetes associates with changes in the pancreatic islet α cells, where the receptor for advanced glycation end products (RAGE) is highly expressed. This study compared islet RAGE expression in donors without diabetes, those at risk of, and those with type 1 diabetes. Laser-dissected islets were subject to RNA bioinformatics and adjacent pancreatic tissue were assessed by confocal microscopy. We found that islets from type 1 diabetes donors had differential expression of the RAGE gene (AGER) and its correlated genes, based on glucagon expression. Random forest machine learning revealed that AGER was the most important predictor for islet glucagon levels. Conversely, a generalized linear model identified that glucagon expression could be predicted by expression of RAGE signaling molecules, its ligands and enzymes that create or clear RAGE ligands. Confocal imaging co-localized RAGE, its ligands and signaling molecules to the α cells. Half of the type 1 diabetes cohort comprised of adolescents and a patient with history of hypoglycemia-all showed an inverse relationship between glucagon and RAGE. These data confirm an association between glucagon and islet RAGE, its ligands and signaling pathways in type 1 diabetes, which warrants functional investigation into a role for RAGE in hypoglycemia.
Assuntos
Diabetes Mellitus Tipo 1 , Células Secretoras de Glucagon , Hipoglicemia , Receptor para Produtos Finais de Glicação Avançada , Adolescente , Humanos , Diabetes Mellitus Tipo 1/genética , Glucagon , Células Secretoras de Glucagon/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , Ligantes , Receptor para Produtos Finais de Glicação Avançada/metabolismoRESUMO
Here, we present a novel case of a patient with chronic lymphocytic leukemia (CLL) who received CTLA-4 and then PD-1 immune-checkpoint blockade (ICB) as treatment for concomitant metastatic melanoma. Whereas the metastatic melanoma was responsive to ICB, the CLL rapidly progressed (but responded to ICB cessation and ibrutinib). There were no new genetic mutational drivers to explain the altered clinical course. PD-1/PD-L1/PD-L2 and CTLA-4/CD80/CD86 expression was not increased in CLL B cells, CD8+ or CD4+ T-cell subsets, or monocytes. The patient's CLL B cells demonstrated strikingly prolonged in vitro survival during PD-1 blockade, which was not observed in samples taken before or after ICB, or with other patients. To our knowledge, a discordant clinical course to ICB coupled with these biological features has not been reported in a patient with dual malignancies.
Assuntos
Antineoplásicos , Inibidores de Checkpoint Imunológico , Leucemia Linfocítica Crônica de Células B , Melanoma , Receptor de Morte Celular Programada 1 , Neoplasias Cutâneas , Humanos , Antígeno CTLA-4/antagonistas & inibidores , Antígeno CTLA-4/imunologia , Progressão da Doença , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/patologia , Melanoma/tratamento farmacológico , Melanoma/etiologia , Melanoma/patologia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/imunologia , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/etiologia , Neoplasias Cutâneas/patologia , Antígeno B7-H1 , Inibidores de Checkpoint Imunológico/imunologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Antineoplásicos/imunologia , Antineoplásicos/uso terapêuticoRESUMO
Induced pluripotent stem cells (iPSCs) can in principle differentiate into any cell of the body, and have revolutionized biomedical research and regenerative medicine. Unlike their human counterparts, mouse iPSCs (miPSCs) are reported to silence transposable elements and prevent transposable element-mediated mutagenesis. Here we apply short-read or Oxford Nanopore Technologies long-read genome sequencing to 38 bulk miPSC lines reprogrammed from 10 parental cell types, and 18 single-cell miPSC clones. While single nucleotide variants and structural variants restricted to miPSCs are rare, we find 83 de novo transposable element insertions, including examples intronic to Brca1 and Dmd. LINE-1 retrotransposons are profoundly hypomethylated in miPSCs, beyond other transposable elements and the genome overall, and harbor alternative protein-coding gene promoters. We show that treatment with the LINE-1 inhibitor lamivudine does not hinder reprogramming and efficiently blocks endogenous retrotransposition, as detected by long-read genome sequencing. These experiments reveal the complete spectrum and potential significance of mutations acquired by miPSCs.
Assuntos
Células-Tronco Pluripotentes Induzidas , Humanos , Camundongos , Animais , Retroelementos/genética , Elementos de DNA Transponíveis/genética , Mutação , Elementos Nucleotídeos Longos e Dispersos/genéticaRESUMO
INTRODUCTION: The pathological decrease of fetal growth during gestation can lead to subsequent poor health outcomes for the fetus. This process is commonly controlled by the placenta, the interface between mother and baby during gestation. Sex-specific gene expression has been implicated in placental function, therefore, there is a need to determine if it is important during reduced fetal growth. We therefore aimed to characterise placental gene expression at term to evaluate sex-specific genetic changes that occur in small for gestational age (SGA) infants. METHODS: RNA-sequencing of twelve human placental tissue samples collected from pregnancies yielding either term appropriate for gestational age (AGA) or SGA infants identified at delivery. Candidate genes associated with fetal size and fetal sex were identified using differential gene expression and weighted gene co-expression network analyses. Single-cell sequencing data was used for candidate validation and to estimate candidate transcript expression in specific placental cell populations. RESULTS: Differential gene expression and weighted gene co-expression network analyses identified 403 candidate transcripts associated with SGA infants. One hundred and three of these transcripts showed sex-specific expression. . Published placental sequencing datasets were used to validate the key expression results from the twelve placental samples initially studied; the sex-independent transcript expression for genes involved in cell cycle processes in males (7 transcripts) and endoplasmic reticulum stress in females (17 transcripts). DISCUSSION: This study identified the activation of multiple molecular mechanisms involved in the placental response to an adverse environmental stressor. Mechanisms such as disrupted protein synthesis were shared between infant biological sex when comparing AGA to SGA, whilst other pathways such as cell cycle and endoplasmic reticulum stress appear as independent/specific to either males or females when investigating reduced fetal growth. This data suggests that sexual dimorphism is an important consideration when examining placental dysfunction and poor fetal growth.
Assuntos
Doenças Placentárias , Placenta , Feminino , Retardo do Crescimento Fetal/metabolismo , Humanos , Lactente , Recém-Nascido , Recém-Nascido Pequeno para a Idade Gestacional/metabolismo , Masculino , Placenta/metabolismo , Doenças Placentárias/patologia , Gravidez , RNA/metabolismoRESUMO
The retrotransposon LINE-1 (L1) is central to the recent evolutionary history of the human genome and continues to drive genetic diversity and germline pathogenesis. However, the spatiotemporal extent and biological significance of somatic L1 activity are poorly defined and are virtually unexplored in other primates. From a single L1 lineage active at the divergence of apes and Old World monkeys, successive L1 subfamilies have emerged in each descendant primate germline. As revealed by case studies, the presently active human L1 subfamily can also mobilize during embryonic and brain development in vivo. It is unknown whether nonhuman primate L1s can similarly generate somatic insertions in the brain. Here we applied approximately 40× single-cell whole-genome sequencing (scWGS), as well as retrotransposon capture sequencing (RC-seq), to 20 hippocampal neurons from two rhesus macaques (Macaca mulatta). In one animal, we detected and PCR-validated a somatic L1 insertion that generated target site duplications, carried a short 5' transduction, and was present in â¼7% of hippocampal neurons but absent from cerebellum and nonbrain tissues. The corresponding donor L1 allele was exceptionally mobile in vitro and was embedded in PRDM4, a gene expressed throughout development and in neural stem cells. Nanopore long-read methylome and RNA-seq transcriptome analyses indicated young retrotransposon subfamily activation in the early embryo, followed by repression in adult tissues. These data highlight endogenous macaque L1 retrotransposition potential, provide prototypical evidence of L1-mediated somatic mosaicism in a nonhuman primate, and allude to L1 mobility in the brain over the past 30 million years of human evolution.
Assuntos
Encéfalo , Elementos Nucleotídeos Longos e Dispersos , Retroelementos , Animais , Proteínas de Ligação a DNA/genética , Macaca mulatta/genética , Neurônios , Retroelementos/genética , Fatores de Transcrição/genéticaRESUMO
SUMMARY: Methylartist is a consolidated suite of tools for processing, visualizing and analysing nanopore-derived modified base calls. All detectable methylation types (e.g. 5mCpG, 5hmC, 6mA) are supported, enabling integrated study of base pairs when modified naturally or as part of an experimental protocol. AVAILABILITY AND IMPLEMENTATION: Methylartist is implemented in Python and is installable via PyPI and bioconda. Source code and test data are available at https://github.com/adamewing/methylartist. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Nanoporos , SoftwareRESUMO
Genome-wide association studies (GWAS) have been highly informative in discovering disease-associated loci but are not designed to capture all structural variations in the human genome. Using long-read sequencing data, we discovered widespread structural variation within SINE-VNTR-Alu (SVA) elements, a class of great ape-specific transposable elements with gene-regulatory roles, which represents a major source of structural variability in the human population. We highlight the presence of structurally variable SVAs (SV-SVAs) in neurological disease-associated loci, and we further associate SV-SVAs to disease-associated SNPs and differential gene expression using luciferase assays and expression quantitative trait loci data. Finally, we genetically deleted SV-SVAs in the BIN1 and CD2AP Alzheimer's disease-associated risk loci and in the BCKDK Parkinson's disease-associated risk locus and assessed multiple aspects of their gene-regulatory influence in a human neuronal context. Together, this study reveals a novel layer of genetic variation in transposable elements that may contribute to identification of the structural variants that are the actual drivers of disease associations of GWAS loci.
Assuntos
Elementos de DNA Transponíveis , Estudo de Associação Genômica Ampla , Elementos Alu , Elementos de DNA Transponíveis/genética , Predisposição Genética para Doença , Variação Genética , Genoma Humano , Humanos , Polimorfismo de Nucleotídeo Único , Locos de Características QuantitativasRESUMO
Age is one of the strongest risk factors for the development of neurodegenerative diseases, the majority of which involve misfolded protein aggregates in the brain. These protein aggregates are thought to drive pathology and are attractive targets for the development of new therapies. However, it is unclear how age influences the onset of pathology and the accompanying molecular response. To address this knowledge gap, we used a model of seeded tau pathology to profile the transcriptomic changes in 3 and 12 month old mice in response to developing tau hyperphosphorylation and aggregation. First, we found the burden of hyperphosphorylated tau pathology in mice injected at 12 months of age was moderately reduced compared to animals injected at 3 months. On a molecular level, we found an inflammation-related subset of genes, including C3 and the disease-associated microglia genes Ctsd, Cst7, and Clec7a, were more expressed early in disease in 12 but not 3 month old mice. These findings provide evidence of an early, age-specific response to tau pathology, which could serve as a marker for the severity of downstream pathology.
Assuntos
Doença de Alzheimer , Proteínas tau , Fatores Etários , Doença de Alzheimer/patologia , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Transgênicos , Microglia/patologia , Agregados Proteicos , Proteínas tau/genética , Proteínas tau/metabolismoRESUMO
A recent study proposed that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) hijacks the LINE-1 (L1) retrotransposition machinery to integrate into the DNA of infected cells. If confirmed, this finding could have significant clinical implications. Here, we apply deep (>50×) long-read Oxford Nanopore Technologies (ONT) sequencing to HEK293T cells infected with SARS-CoV-2 and do not find the virus integrated into the genome. By examining ONT data from separate HEK293T cultivars, we completely resolve 78 L1 insertions arising in vitro in the absence of L1 overexpression systems. ONT sequencing applied to hepatitis B virus (HBV)-positive liver cancer tissues located a single HBV insertion. These experiments demonstrate reliable resolution of retrotransposon and exogenous virus insertions by ONT sequencing. That we find no evidence of SARS-CoV-2 integration suggests that such events are, at most, extremely rare in vivo and therefore are unlikely to drive oncogenesis or explain post-recovery detection of the virus.
Assuntos
COVID-19/virologia , DNA Viral/genética , Genoma Humano , SARS-CoV-2/genética , Análise de Sequência de DNA , Integração Viral , Idoso , Animais , COVID-19/diagnóstico , Carcinoma Hepatocelular/virologia , Chlorocebus aethiops , Células HEK293 , Vírus da Hepatite B/genética , Interações Hospedeiro-Patógeno , Humanos , Neoplasias Hepáticas/virologia , Elementos Nucleotídeos Longos e Dispersos , Masculino , Sequenciamento por Nanoporos , Células VeroRESUMO
Basal cell nevus syndrome (also known as Gorlin Syndrome; MIM109400) is an autosomal dominant disorder characterized by recurrent pathological features such as basal cell carcinomas and odontogenic keratocysts as well as skeletal abnormalities. Most affected individuals have point mutations or small insertions or deletions within the PTCH1 gene on human chromosome 9, but there are some cases with more extensive deletion of the region, usually including the neighboring FANCC and/or ERCC6L2 genes. We report a 16-year-old patient with a deletion of approximately 400,000 bases which removes only PTCH1 and some non-coding RNA genes but leaves FANCC and ERCC6L2 intact. In spite of the small amount of DNA for which he is haploid, his phenotype is more extreme than many individuals with longer deletions in the region. This includes early presentation with a large number of basal cell nevi and other skin lesions, multiple jaw keratocysts, and macrosomia. We found that the deletion was in the paternal chromosome, in common with other macrosomia cases. Using public databases, we have examined possible interactions between sequences within and outside the deletion and speculate that a regulatory relationship exists with flanking genes, which is unbalanced by the deletion, resulting in abnormal activation or repression of the target genes and hence the severity of the phenotype.
Assuntos
Síndrome do Nevo Basocelular/genética , DNA Helicases/genética , Proteína do Grupo de Complementação C da Anemia de Fanconi/genética , Receptor Patched-1/genética , Adolescente , Síndrome do Nevo Basocelular/epidemiologia , Síndrome do Nevo Basocelular/patologia , Criança , Pré-Escolar , Transtornos Cromossômicos/genética , Transtornos Cromossômicos/patologia , Cromossomos Humanos Par 9/genética , Predisposição Genética para Doença , Humanos , Lactente , Recém-Nascido , Masculino , Recidiva Local de Neoplasia/epidemiologia , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Cistos Odontogênicos/genética , Cistos Odontogênicos/patologia , Fenótipo , Índice de Gravidade de DoençaRESUMO
Pseudogenes are gene copies presumed to mainly be functionless relics of evolution due to acquired deleterious mutations or transcriptional silencing. Using deep full-length PacBio cDNA sequencing of normal human tissues and cancer cell lines, we identify here hundreds of novel transcribed pseudogenes expressed in tissue-specific patterns. Some pseudogene transcripts have intact open reading frames and are translated in cultured cells, representing unannotated protein-coding genes. To assess the biological impact of noncoding pseudogenes, we CRISPR-Cas9 delete the nucleus-enriched pseudogene PDCL3P4 and observe hundreds of perturbed genes. This study highlights pseudogenes as a complex and dynamic component of the human transcriptional landscape.
Assuntos
DNA Complementar/genética , Pseudogenes , Análise de Sequência de DNA , Transcriptoma/genética , Linhagem Celular , Deleção de Genes , Haploidia , Humanos , Regiões Promotoras Genéticas/genéticaRESUMO
Transposable elements (TEs) drive genome evolution and are a notable source of pathogenesis, including cancer. While CpG methylation regulates TE activity, the locus-specific methylation landscape of mobile human TEs has to date proven largely inaccessible. Here, we apply new computational tools and long-read nanopore sequencing to directly infer CpG methylation of novel and extant TE insertions in hippocampus, heart, and liver, as well as paired tumor and non-tumor liver. As opposed to an indiscriminate stochastic process, we find pronounced demethylation of young long interspersed element 1 (LINE-1) retrotransposons in cancer, often distinct to the adjacent genome and other TEs. SINE-VNTR-Alu (SVA) retrotransposons, including their internal tandem repeat-associated CpG island, are near-universally methylated. We encounter allele-specific TE methylation and demethylation of aberrantly expressed young LINE-1s in normal tissues. Finally, we recover the complete sequences of tumor-specific LINE-1 insertions and their retrotransposition hallmarks, demonstrating how long-read sequencing can simultaneously survey the epigenome and detect somatic TE mobilization.
Assuntos
Metilação de DNA , Elementos de DNA Transponíveis , DNA de Neoplasias , Epigênese Genética , Epigenoma , Regulação Neoplásica da Expressão Gênica , Elementos Nucleotídeos Longos e Dispersos , Sequenciamento por Nanoporos , Neoplasias , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Neoplasias/genética , Neoplasias/metabolismo , Especificidade de ÓrgãosRESUMO
The mammary epithelium is indispensable for the continued survival of more than 5,000 mammalian species. For some, the volume of milk ejected in a single day exceeds their entire blood volume. Here, we unveil the spatiotemporal properties of physiological signals that orchestrate the ejection of milk from alveolar units and its passage along the mammary ductal network. Using quantitative, multidimensional imaging of mammary cell ensembles from GCaMP6 transgenic mice, we reveal how stimulus evoked Ca2+ oscillations couple to contractions in basal epithelial cells. Moreover, we show that Ca2+-dependent contractions generate the requisite force to physically deform the innermost layer of luminal cells, compelling them to discharge the fluid that they produced and housed. Through the collective action of thousands of these biological positive-displacement pumps, each linked to a contractile ductal network, milk begins its passage toward the dependent neonate, seconds after the command.
Assuntos
Sinalização do Cálcio , Glândulas Mamárias Animais/fisiologia , Ejeção Láctea , Animais , Células Epiteliais/fisiologia , Humanos , Microscopia Intravital , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/diagnóstico por imagem , Glândulas Mamárias Humanas/metabolismo , Camundongos , Camundongos Transgênicos , Cadeias Leves de Miosina/metabolismoRESUMO
We report an actionable secondary finding from whole-genome sequencing (WGS) of a 10-year-old boy with autism. WGS identified non-synonymous variants in several genes, including a nonsense mutation in the ANOS1 gene which is an X-linked cause of Kallmann syndrome. WGS can provide insights into complex genetic disorders such as autism, and actionable incidental findings can offer the potential for therapeutic interventions.
RESUMO
Tumor DNA sequencing data can be interpreted by computational methods that analyze genomic heterogeneity to infer evolutionary dynamics. A growing number of studies have used these approaches to link cancer evolution with clinical progression and response to therapy. Although the inference of tumor phylogenies is rapidly becoming standard practice in cancer genome analyses, standards for evaluating them are lacking. To address this need, we systematically assess methods for reconstructing tumor subclonality. First, we elucidate the main algorithmic problems in subclonal reconstruction and develop quantitative metrics for evaluating them. Then we simulate realistic tumor genomes that harbor all known clonal and subclonal mutation types and processes. Finally, we benchmark 580 tumor reconstructions, varying tumor read depth, tumor type and somatic variant detection. Our analysis provides a baseline for the establishment of gold-standard methods to analyze tumor heterogeneity.