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1.
Cell ; 162(5): 1039-50, 2015 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-26300124

RESUMO

Chromatin state variation at gene regulatory elements is abundant across individuals, yet we understand little about the genetic basis of this variability. Here, we profiled several histone modifications, the transcription factor (TF) PU.1, RNA polymerase II, and gene expression in lymphoblastoid cell lines from 47 whole-genome sequenced individuals. We observed that distinct cis-regulatory elements exhibit coordinated chromatin variation across individuals in the form of variable chromatin modules (VCMs) at sub-Mb scale. VCMs were associated with thousands of genes and preferentially cluster within chromosomal contact domains. We mapped strong proximal and weak, yet more ubiquitous, distal-acting chromatin quantitative trait loci (cQTL) that frequently explain this variation. cQTLs were associated with molecular activity at clusters of cis-regulatory elements and mapped preferentially within TF-bound regions. We propose that local, sequence-independent chromatin variation emerges as a result of genetic perturbations in cooperative interactions between cis-regulatory elements that are located within the same genomic domain.


Assuntos
Cromatina/química , Regulação da Expressão Gênica , Variação Genética , Genoma Humano , Cromatina/metabolismo , Cromossomos Humanos/química , Genética Populacional , Humanos , Locos de Características Quantitativas , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/metabolismo
2.
Nature ; 578(7793): 129-136, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32025019

RESUMO

Transcript alterations often result from somatic changes in cancer genomes1. Various forms of RNA alterations have been described in cancer, including overexpression2, altered splicing3 and gene fusions4; however, it is difficult to attribute these to underlying genomic changes owing to heterogeneity among patients and tumour types, and the relatively small cohorts of patients for whom samples have been analysed by both transcriptome and whole-genome sequencing. Here we present, to our knowledge, the most comprehensive catalogue of cancer-associated gene alterations to date, obtained by characterizing tumour transcriptomes from 1,188 donors of the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA)5. Using matched whole-genome sequencing data, we associated several categories of RNA alterations with germline and somatic DNA alterations, and identified probable genetic mechanisms. Somatic copy-number alterations were the major drivers of variations in total gene and allele-specific expression. We identified 649 associations of somatic single-nucleotide variants with gene expression in cis, of which 68.4% involved associations with flanking non-coding regions of the gene. We found 1,900 splicing alterations associated with somatic mutations, including the formation of exons within introns in proximity to Alu elements. In addition, 82% of gene fusions were associated with structural variants, including 75 of a new class, termed 'bridged' fusions, in which a third genomic location bridges two genes. We observed transcriptomic alteration signatures that differ between cancer types and have associations with variations in DNA mutational signatures. This compendium of RNA alterations in the genomic context provides a rich resource for identifying genes and mechanisms that are functionally implicated in cancer.


Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , RNA/genética , Variações do Número de Cópias de DNA , DNA de Neoplasias , Genoma Humano , Genômica , Humanos , Transcriptoma
3.
Genome Res ; 2022 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-35794008

RESUMO

Analysis of allele-specific gene expression (ASE) is a powerful approach for studying gene regulation, particularly when sample sizes are small, such as for rare diseases, or when studying the effects of rare genetic variation. However, detection of ASE events relies on accurate alignment of RNA sequencing reads, where challenges still remain, particularly for reads containing genetic variants or those that align to many different genomic locations. We have developed the Personalised ASE Caller (PAC), a tool that combines multiple steps to improve the quantification of allelic reads, including personalized (i.e., diploid) read alignment with improved allocation of multimapping reads. Using simulated RNA sequencing data, we show that PAC outperforms standard alignment approaches for ASE detection, reducing the number of sites with incorrect biases (>10%) by ∼80% and increasing the number of sites that can be reliably quantified by ∼3%. Applying PAC to real RNA sequencing data from 670 whole-blood samples, we show that genetic regulatory signatures inferred from ASE data more closely match those from population-based methods that are less prone to alignment biases. Finally, we use PAC to characterize cell type-specific ASE events that would be missed by standard alignment approaches, and in doing so identify disease relevant genes that may modulate their effects through the regulation of gene expression. PAC can be applied to the vast quantity of existing RNA sequencing data sets to better understand a wide array of fundamental biological and disease processes.

5.
Nature ; 546(7658): 370-375, 2017 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-28489815

RESUMO

Technology utilizing human induced pluripotent stem cells (iPS cells) has enormous potential to provide improved cellular models of human disease. However, variable genetic and phenotypic characterization of many existing iPS cell lines limits their potential use for research and therapy. Here we describe the systematic generation, genotyping and phenotyping of 711 iPS cell lines derived from 301 healthy individuals by the Human Induced Pluripotent Stem Cells Initiative. Our study outlines the major sources of genetic and phenotypic variation in iPS cells and establishes their suitability as models of complex human traits and cancer. Through genome-wide profiling we find that 5-46% of the variation in different iPS cell phenotypes, including differentiation capacity and cellular morphology, arises from differences between individuals. Additionally, we assess the phenotypic consequences of genomic copy-number alterations that are repeatedly observed in iPS cells. In addition, we present a comprehensive map of common regulatory variants affecting the transcriptome of human pluripotent cells.


Assuntos
Variação Genética/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Células Cultivadas , Reprogramação Celular/genética , Variações do Número de Cópias de DNA/genética , Regulação da Expressão Gênica/genética , Genótipo , Humanos , Especificidade de Órgãos , Fenótipo , Controle de Qualidade , Locos de Características Quantitativas/genética , Transcriptoma/genética
7.
Nature ; 515(7526): 209-15, 2014 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-25363760

RESUMO

The genetic architecture of autism spectrum disorder involves the interplay of common and rare variants and their impact on hundreds of genes. Using exome sequencing, here we show that analysis of rare coding variation in 3,871 autism cases and 9,937 ancestry-matched or parental controls implicates 22 autosomal genes at a false discovery rate (FDR) < 0.05, plus a set of 107 autosomal genes strongly enriched for those likely to affect risk (FDR < 0.30). These 107 genes, which show unusual evolutionary constraint against mutations, incur de novo loss-of-function mutations in over 5% of autistic subjects. Many of the genes implicated encode proteins for synaptic formation, transcriptional regulation and chromatin-remodelling pathways. These include voltage-gated ion channels regulating the propagation of action potentials, pacemaking and excitability-transcription coupling, as well as histone-modifying enzymes and chromatin remodellers-most prominently those that mediate post-translational lysine methylation/demethylation modifications of histones.


Assuntos
Transtornos Globais do Desenvolvimento Infantil/genética , Cromatina/genética , Predisposição Genética para Doença/genética , Mutação/genética , Sinapses/metabolismo , Transcrição Gênica/genética , Sequência de Aminoácidos , Transtornos Globais do Desenvolvimento Infantil/patologia , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Exoma/genética , Feminino , Mutação em Linhagem Germinativa/genética , Humanos , Masculino , Dados de Sequência Molecular , Mutação de Sentido Incorreto/genética , Rede Nervosa/metabolismo , Razão de Chances
8.
Nature ; 501(7468): 506-11, 2013 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-24037378

RESUMO

Genome sequencing projects are discovering millions of genetic variants in humans, and interpretation of their functional effects is essential for understanding the genetic basis of variation in human traits. Here we report sequencing and deep analysis of messenger RNA and microRNA from lymphoblastoid cell lines of 462 individuals from the 1000 Genomes Project--the first uniformly processed high-throughput RNA-sequencing data from multiple human populations with high-quality genome sequences. We discover extremely widespread genetic variation affecting the regulation of most genes, with transcript structure and expression level variation being equally common but genetically largely independent. Our characterization of causal regulatory variation sheds light on the cellular mechanisms of regulatory and loss-of-function variation, and allows us to infer putative causal variants for dozens of disease-associated loci. Altogether, this study provides a deep understanding of the cellular mechanisms of transcriptome variation and of the landscape of functional variants in the human genome.


Assuntos
Variação Genética/genética , Genoma Humano/genética , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Sequência de RNA , Transcriptoma/genética , Alelos , Linhagem Celular Transformada , Éxons/genética , Perfilação da Expressão Gênica , Humanos , Polimorfismo de Nucleotídeo Único/genética , Locos de Características Quantitativas/genética , RNA Mensageiro/análise , RNA Mensageiro/genética
9.
Trends Genet ; 29(1): 23-30, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23103023

RESUMO

Progress in understanding the genetics of human disease is closely tied to technological developments in DNA sequencing. Recently, next-generation technology has transformed the scale of sequencing; compared to the methods used in the Human Genome Project, modern sequencers are 50000-fold faster. Complex disease genetics presents an immediate opportunity to use this technology to move from approaches using only partial information (linkage and genome-wide association studies, GWAS) to complete analysis of the relationship between genomic variation and phenotype. We first describe sequence-based improvements to existing study designs, followed by prioritization of both samples and genomic regions to be sequenced, and then address the ultimate goal of analyzing thousands of whole-genome sequences. Finally, we discuss how the same technology will also fundamentally change the way we understand the biological mechanisms underlying disease associations discovered through sequencing.


Assuntos
Doenças Genéticas Inatas/genética , Sequenciamento de Nucleotídeos em Larga Escala , Bases de Dados Genéticas , Estudos de Associação Genética/métodos , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Genômica/métodos , Genômica/tendências , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Projeto Genoma Humano , Humanos , Desequilíbrio de Ligação
10.
Bioinformatics ; 30(2): 165-71, 2014 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-24255646

RESUMO

MOTIVATION: High-throughput sequencing technologies enable the genome-wide analysis of the impact of genetic variation on molecular phenotypes at unprecedented resolution. However, although powerful, these technologies can also introduce unexpected artifacts. RESULTS: We investigated the impact of library amplification bias on the identification of allele-specific (AS) molecular events from high-throughput sequencing data derived from chromatin immunoprecipitation assays (ChIP-seq). Putative AS DNA binding activity for RNA polymerase II was determined using ChIP-seq data derived from lymphoblastoid cell lines of two parent-daughter trios. We found that, at high-sequencing depth, many significant AS binding sites suffered from an amplification bias, as evidenced by a larger number of clonal reads representing one of the two alleles. To alleviate this bias, we devised an amplification bias detection strategy, which filters out sites with low read complexity and sites featuring a significant excess of clonal reads. This method will be useful for AS analyses involving ChIP-seq and other functional sequencing assays. AVAILABILITY: The R package abs filter for library clonality simulations and detection of amplification-biased sites is available from http://updepla1srv1.epfl.ch/waszaks/absfilter


Assuntos
Imunoprecipitação da Cromatina/métodos , Genoma Humano , Linfócitos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , RNA Polimerase II/genética , Alelos , Sítios de Ligação , Feminino , Biblioteca Gênica , Humanos , Linfócitos/citologia , Masculino , Polimorfismo de Nucleotídeo Único/genética
11.
Hum Mol Genet ; 21(R1): R24-8, 2012 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-22976472

RESUMO

Much of the recent advances in functional genomics owe to developments in next-generation sequencing technology, which has contributed to the exponential increase of genomic data available for different human disease and population samples. With functional sequencing assays available to query both the transcriptome and the epigenome, annotation of the non-coding, regulatory genome is steadily improving and providing means to interpret the functional consequences of genetic variants associated with human complex traits. This has highlighted the need to better understand the normal variation in various cellular phenotypes, such as epigenetic modifications, and their transgenerational inheritance. In this review, we discuss different aspects of epigenetic variation in the context of DNA sequence variation and its contribution to complex phenotypes.


Assuntos
Epigênese Genética , Variação Genética , Genoma Humano , Característica Quantitativa Herdável , Epigenômica , Humanos , Fenótipo , Análise de Sequência de DNA
12.
Front Cell Dev Biol ; 11: 1090046, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36923252

RESUMO

Neurodevelopmental disorders encompass a group of debilitating diseases presenting with motor and cognitive dysfunction, with variable age of onset and disease severity. Advances in genetic diagnostic tools have facilitated the identification of several monogenic chromatin remodeling diseases that cause Neurodevelopmental disorders. Chromatin remodelers play a key role in the neuro-epigenetic landscape and regulation of brain development; it is therefore not surprising that mutations, leading to loss of protein function, result in aberrant neurodevelopment. Heterozygous, usually de novo mutations in histone lysine methyltransferases have been described in patients leading to haploinsufficiency, dysregulated protein levels and impaired protein function. Studies in animal models and patient-derived cell lines, have highlighted the role of histone lysine methyltransferases in the regulation of cell self-renewal, cell fate specification and apoptosis. To date, in depth studies of histone lysine methyltransferases in oncology have provided strong evidence of histone lysine methyltransferase dysregulation as a determinant of cancer progression and drug resistance. As a result, histone lysine methyltransferases have become an important therapeutic target for the treatment of different cancer forms. Despite recent advances, we still lack knowledge about the role of histone lysine methyltransferases in neuronal development. This has hampered both the study and development of precision therapies for histone lysine methyltransferases-related Neurodevelopmental disorders. In this review, we will discuss the current knowledge of the role of histone lysine methyltransferases in neuronal development and disease progression. We will also discuss how RNA-based technologies using small-activating RNAs could potentially provide a novel therapeutic approach for the future treatment of histone lysine methyltransferase haploinsufficiency in these Neurodevelopmental disorders, and how they could be first tested in state-of-the-art patient-derived neuronal models.

13.
Cell Genom ; 3(4): 100280, 2023 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-37082143

RESUMO

The use of induced pluripotent stem cells (iPSC) as models for development and human disease has enabled the study of otherwise inaccessible tissues. A remaining challenge in developing reliable models is our limited understanding of the factors driving irregular differentiation of iPSCs, particularly the impact of acquired somatic mutations. We leveraged data from a pooled dopaminergic neuron differentiation experiment of 238 iPSC lines profiled with single-cell RNA and whole-exome sequencing to study how somatic mutations affect differentiation outcomes. We found that deleterious somatic mutations in key developmental genes, notably the BCOR gene, are strongly associated with failure in dopaminergic neuron differentiation and a larger proliferation rate in culture. We further identified broad differences in cell type composition between incorrectly and successfully differentiating lines, as well as significant changes in gene expression contributing to the inhibition of neurogenesis. Our work calls for caution in interpreting differentiation-related phenotypes in disease-modeling experiments.

14.
Stem Cell Reports ; 17(2): 413-426, 2022 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-35063129

RESUMO

Conventional reprogramming methods rely on the ectopic expression of transcription factors to reprogram somatic cells into induced pluripotent stem cells (iPSCs). The forced expression of transcription factors may lead to off-target gene activation and heterogeneous reprogramming, resulting in the emergence of alternative cell types and aberrant iPSCs. Activation of endogenous pluripotency factors by CRISPR activation (CRISPRa) can reduce this heterogeneity. Here, we describe a high-efficiency reprogramming of human somatic cells into iPSCs using optimized CRISPRa. Efficient reprogramming was dependent on the additional targeting of the embryo genome activation-enriched Alu-motif and the miR-302/367 locus. Single-cell transcriptome analysis revealed that the optimized CRISPRa reprogrammed cells more directly and specifically into the pluripotent state when compared to the conventional reprogramming method. These findings support the use of CRISPRa for high-quality pluripotent reprogramming of human cells.


Assuntos
Reprogramação Celular/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Edição de Genes/métodos , Elementos Alu/genética , Perfilação da Expressão Gênica , Loci Gênicos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , MicroRNAs/genética , Análise de Célula Única
15.
Front Cell Dev Biol ; 10: 1023340, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36684426

RESUMO

Efficient and effective methods for converting human induced pluripotent stem cells into differentiated derivatives are critical for performing robust, large-scale studies of development and disease modelling, and for providing a source of cells for regenerative medicine. Here, we describe a 14-day neural differentiation protocol which allows for the scalable, simultaneous differentiation of multiple iPSC lines into cortical neural stem cells We currently employ this protocol to differentiate and compare sets of engineered iPSC lines carrying loss of function alleles in developmental disorder associated genes, alongside isogenic wildtype controls. Using RNA sequencing (RNA-Seq), we can examine the changes in gene expression brought about by each disease gene knockout, to determine its impact on neural development and explore mechanisms of disease. The 10-day Neural Induction period uses the well established dual-SMAD inhibition approach combined with Wnt/ß-Catenin inhibition to selectively induce formation of cortical NSCs. This is followed by a 4-day Neural Maintenance period facilitating NSC expansion and rosette formation, and NSC cryopreservation. We also describe methods for thawing and passaging the cryopreserved NSCs, which are useful in confirming their viability for further culture. Routine implementation of immunocytochemistry Quality Control confirms the presence of PAX6-positive and/or FOXG1-positive NSCs and the absence of OCT4-positive iPSCs after differentiation. RNA-Seq, flow cytometry, immunocytochemistry (ICC) and RT-qPCR provide additional confirmation of robust presence of NSC markers in the differentiated cells. The broader utility and application of our protocol is demonstrated by the successful differentiation of wildtype iPSC lines from five additional independent donors. This paper thereby describes an efficient method for the production of large numbers of high purity cortical NSCs, which are widely applicable for downstream research into developmental mechanisms, further differentiation into postmitotic cortical neurons, or other applications such as large-scale drug screening experiments.

16.
Hum Mol Genet ; 18(15): 2912-21, 2009 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-19454485

RESUMO

Population isolates, such as Finland, have proved beneficial in mapping rare causative genetic variants due to a limited number of founders resulting in reduced genetic heterogeneity and extensive linkage disequilibrium (LD). We have here used this special opportunity to identify rare alleles in autism by genealogically tracing 20 autism families into one extended pedigree with verified genealogical links reaching back to the 17th century. In this unique pedigree, we performed a dense microsatellite marker genome-wide scan of linkage and LD and followed initial findings with extensive fine-mapping. We identified a putative autism susceptibility locus at 19p13.3 and obtained further evidence for previously identified loci at 1q23 and 15q11-q13. Most promising candidate genes were TLE2 and TLE6 clustered at 19p13 and ATP1A2 at 1q23.


Assuntos
Transtorno Autístico/genética , Estudo de Associação Genômica Ampla , Desequilíbrio de Ligação , Linhagem , Cromossomos Humanos Par 1/genética , Cromossomos Humanos Par 15/genética , Cromossomos Humanos Par 19/genética , Feminino , Finlândia , Humanos , Masculino , Repetições de Microssatélites , População Branca/genética
17.
Nat Genet ; 53(3): 313-321, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33664507

RESUMO

Induced pluripotent stem cells (iPSCs) are an established cellular system to study the impact of genetic variants in derived cell types and developmental contexts. However, in their pluripotent state, the disease impact of genetic variants is less well known. Here, we integrate data from 1,367 human iPSC lines to comprehensively map common and rare regulatory variants in human pluripotent cells. Using this population-scale resource, we report hundreds of new colocalization events for human traits specific to iPSCs, and find increased power to identify rare regulatory variants compared with somatic tissues. Finally, we demonstrate how iPSCs enable the identification of causal genes for rare diseases.


Assuntos
Variação Genética , Células-Tronco Pluripotentes Induzidas/fisiologia , Locos de Características Quantitativas , Síndrome de Bardet-Biedl/genética , Canais de Cálcio/genética , Linhagem Celular , Ataxia Cerebelar/genética , Metilação de DNA , Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Polimorfismo de Nucleotídeo Único , Proteínas/genética , Doenças Raras/genética , Sequências Reguladoras de Ácido Nucleico , Análise de Sequência de RNA , Sequenciamento Completo do Genoma
18.
Elife ; 92020 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-32773033

RESUMO

Human disease phenotypes are driven primarily by alterations in protein expression and/or function. To date, relatively little is known about the variability of the human proteome in populations and how this relates to variability in mRNA expression and to disease loci. Here, we present the first comprehensive proteomic analysis of human induced pluripotent stem cells (iPSC), a key cell type for disease modelling, analysing 202 iPSC lines derived from 151 donors, with integrated transcriptome and genomic sequence data from the same lines. We characterised the major genetic and non-genetic determinants of proteome variation across iPSC lines and assessed key regulatory mechanisms affecting variation in protein abundance. We identified 654 protein quantitative trait loci (pQTLs) in iPSCs, including disease-linked variants in protein-coding sequences and variants with trans regulatory effects. These include pQTL linked to GWAS variants that cannot be detected at the mRNA level, highlighting the utility of dissecting pQTL at peptide level resolution.


Assuntos
Doença/genética , Variação Genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteoma , Transcriptoma , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Feminino , Genética Populacional , Genótipo , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Fenótipo , Proteômica , Locos de Características Quantitativas , RNA Mensageiro/genética , Adulto Jovem
20.
Nat Genet ; 50(1): 54-61, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29229984

RESUMO

Induced pluripotent stem cells (iPSCs), and cells derived from them, have become key tools for modeling biological processes, particularly in cell types that are difficult to obtain from living donors. Here we present a map of regulatory variants in iPSC-derived neurons, based on 123 differentiations of iPSCs to a sensory neuronal fate. Gene expression was more variable across cultures than in primary dorsal root ganglion, particularly for genes related to nervous system development. Using single-cell RNA-sequencing, we found that the number of neuronal versus contaminating cells was influenced by iPSC culture conditions before differentiation. Despite high differentiation-induced variability, our allele-specific method detected thousands of quantitative trait loci (QTLs) that influenced gene expression, chromatin accessibility, and RNA splicing. On the basis of these detected QTLs, we estimate that recall-by-genotype studies that use iPSC-derived cells will require cells from at least 20-80 individuals to detect the effects of regulatory variants with moderately large effect sizes.


Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Células Receptoras Sensoriais/metabolismo , Diferenciação Celular/genética , Linhagem Celular , Cromatina/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Técnicas de Genotipagem , Humanos , Locos de Características Quantitativas , Splicing de RNA , Células Receptoras Sensoriais/citologia , Análise de Sequência de RNA , Análise de Célula Única
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