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1.
Genet Med ; 26(5): 101076, 2024 05.
Artigo em Inglês | MEDLINE | ID: mdl-38258669

RESUMO

PURPOSE: Genome sequencing (GS)-specific diagnostic rates in prospective tightly ascertained exome sequencing (ES)-negative intellectual disability (ID) cohorts have not been reported extensively. METHODS: ES, GS, epigenetic signatures, and long-read sequencing diagnoses were assessed in 74 trios with at least moderate ID. RESULTS: The ES diagnostic yield was 42 of 74 (57%). GS diagnoses were made in 9 of 32 (28%) ES-unresolved families. Repeated ES with a contemporary pipeline on the GS-diagnosed families identified 8 of 9 single-nucleotide variations/copy-number variations undetected in older ES, confirming a GS-unique diagnostic rate of 1 in 32 (3%). Episignatures contributed diagnostic information in 9% with GS corroboration in 1 of 32 (3%) and diagnostic clues in 2 of 32 (6%). A genetic etiology for ID was detected in 51 of 74 (69%) families. Twelve candidate disease genes were identified. Contemporary ES followed by GS cost US$4976 (95% CI: $3704; $6969) per diagnosis and first-line GS at a cost of $7062 (95% CI: $6210; $8475) per diagnosis. CONCLUSION: Performing GS only in ID trios would be cost equivalent to ES if GS were available at $2435, about a 60% reduction from current prices. This study demonstrates that first-line GS achieves higher diagnostic rate than contemporary ES but at a higher cost.


Assuntos
Sequenciamento do Exoma , Exoma , Deficiência Intelectual , Humanos , Deficiência Intelectual/genética , Deficiência Intelectual/diagnóstico , Masculino , Feminino , Exoma/genética , Sequenciamento do Exoma/economia , Estudos de Coortes , Testes Genéticos/economia , Testes Genéticos/métodos , Sequenciamento Completo do Genoma/economia , Criança , Genoma Humano/genética , Variações do Número de Cópias de DNA/genética , Polimorfismo de Nucleotídeo Único/genética , Pré-Escolar
2.
Genome Res ; 28(5): 625-638, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29650553

RESUMO

The growth and progression of solid tumors involves dynamic cross-talk between cancer epithelium and the surrounding microenvironment. To date, molecular profiling has largely been restricted to the epithelial component of tumors; therefore, features underpinning the persistent protumorigenic phenotype of the tumor microenvironment are unknown. Using whole-genome bisulfite sequencing, we show for the first time that cancer-associated fibroblasts (CAFs) from localized prostate cancer display remarkably distinct and enduring genome-wide changes in DNA methylation, significantly at enhancers and promoters, compared to nonmalignant prostate fibroblasts (NPFs). Differentially methylated regions associated with changes in gene expression have cancer-related functions and accurately distinguish CAFs from NPFs. Remarkably, a subset of changes is shared with prostate cancer epithelial cells, revealing the new concept of tumor-specific epigenome modifications in the tumor and its microenvironment. The distinct methylome of CAFs provides a novel epigenetic hallmark of the cancer microenvironment and promises new biomarkers to improve interpretation of diagnostic samples.


Assuntos
Metilação de DNA , Epigenômica/métodos , Neoplasias da Próstata/genética , Microambiente Tumoral/genética , Fibroblastos Associados a Câncer/metabolismo , Células Cultivadas , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genoma Humano/genética , Humanos , Masculino , Regiões Promotoras Genéticas/genética , Neoplasias da Próstata/patologia , Sequenciamento Completo do Genoma/métodos
3.
Bioinformatics ; 35(4): 560-570, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30084929

RESUMO

MOTIVATION: A synoptic view of the human genome benefits chiefly from the application of nucleic acid sequencing and microarray technologies. These platforms allow interrogation of patterns such as gene expression and DNA methylation at the vast majority of canonical loci, allowing granular insights and opportunities for validation of original findings. However, problems arise when validating against a "gold standard" measurement, since this immediately biases all subsequent measurements towards that particular technology or protocol. Since all genomic measurements are estimates, in the absence of a "gold standard" we instead empirically assess the measurement precision and sensitivity of a large suite of genomic technologies via a consensus modelling method called the row-linear model. This method is an application of the American Society for Testing and Materials Standard E691 for assessing interlaboratory precision and sources of variability across multiple testing sites. Both cross-platform and cross-locus comparisons can be made across all common loci, allowing identification of technology- and locus-specific tendencies. RESULTS: We assess technologies including the Infinium MethylationEPIC BeadChip, whole genome bisulfite sequencing (WGBS), two different RNA-Seq protocols (PolyA+ and Ribo-Zero) and five different gene expression array platforms. Each technology thus is characterised herein, relative to the consensus. We showcase a number of applications of the row-linear model, including correlation with known interfering traits. We demonstrate a clear effect of cross-hybridisation on the sensitivity of Infinium methylation arrays. Additionally, we perform a true interlaboratory test on a set of samples interrogated on the same platform across twenty-one separate testing laboratories. AVAILABILITY AND IMPLEMENTATION: A full implementation of the row-linear model, plus extra functions for visualisation, are found in the R package consensus at https://github.com/timpeters82/consensus. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Assuntos
Biologia Computacional , Metilação de DNA , Genômica , Genoma Humano , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Software
4.
Bioinformatics ; 30(10): 1471-2, 2014 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-24470576

RESUMO

SUMMARY: The initial steps in the analysis of next-generation sequencing data can be automated by way of software 'pipelines'. However, individual components depreciate rapidly because of the evolving technology and analysis methods, often rendering entire versions of production informatics pipelines obsolete. Constructing pipelines from Linux bash commands enables the use of hot swappable modular components as opposed to the more rigid program call wrapping by higher level languages, as implemented in comparable published pipelining systems. Here we present Next Generation Sequencing ANalysis for Enterprises (NGSANE), a Linux-based, high-performance-computing-enabled framework that minimizes overhead for set up and processing of new projects, yet maintains full flexibility of custom scripting when processing raw sequence data. AVAILABILITY AND IMPLEMENTATION: Ngsane is implemented in bash and publicly available under BSD (3-Clause) licence via GitHub at https://github.com/BauerLab/ngsane. CONTACT: Denis.Bauer@csiro.au SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Automação Laboratorial , Humanos , Software
5.
Nat Commun ; 11(1): 54, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31911579

RESUMO

The architectural protein CTCF is a mediator of chromatin conformation, but how CTCF binding to DNA is orchestrated to maintain long-range gene expression is poorly understood. Here we perform RNAi knockdown to reduce CTCF levels and reveal a shared subset of CTCF-bound sites are robustly resistant to protein depletion. The 'persistent' CTCF sites are enriched at domain boundaries and chromatin loops constitutive to all cell types. CRISPR-Cas9 deletion of 2 persistent CTCF sites at the boundary between a long-range epigenetically active (LREA) and silenced (LRES) region, within the Kallikrein (KLK) locus, results in concordant activation of all 8 KLK genes within the LRES region. CTCF genome-wide depletion results in alteration in Topologically Associating Domain (TAD) structure, including the merging of TADs, whereas TAD boundaries are not altered where persistent sites are maintained. We propose that the subset of essential CTCF sites are involved in cell-type constitutive, higher order chromatin architecture.


Assuntos
Fator de Ligação a CCCTC/metabolismo , Cromatina/metabolismo , Epigênese Genética , Sítios de Ligação , Fator de Ligação a CCCTC/genética , Cromatina/química , Cromatina/genética , DNA/genética , DNA/metabolismo , Humanos , Regiões Promotoras Genéticas , Ligação Proteica , Domínios Proteicos
6.
Mamm Genome ; 20(9-10): 604-11, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19763687

RESUMO

Our knowledge of regulatory mechanisms of gene expression and other chromosomal processes related to DNA methylation and chromatin state is continuing to grow at a rapid pace. Understanding how these epigenomic phenomena vary between individuals will have an impact on understanding their broader role in determining variation in gene expression and biochemical, physiological, and behavioural phenotypes. In this review we survey recent progress in this area, focusing on data available from humans. We highlight the role of obligatory (sequence-dependent) epigenomic variation as an important mechanism for generating interindividual variation that could impact our understanding of the mechanistic basis of complex trait architecture.


Assuntos
Epigênese Genética , Variação Genética , Metilação de DNA , Genoma Humano , Humanos
7.
J Steroid Biochem Mol Biol ; 188: 48-58, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30529760

RESUMO

Breast cancer is a complex disease, and approximately 30% of cases are considered to be hereditary or familial, with a large fraction of this being polygenic. However, it is difficult to demonstrate the functional importance of genes of small effect in population studies, and these genes are not always easily targeted for prevention. The SuprMam (suppressor of mammary tumour) breast cancer susceptibility alleles were previously identified as contributors to spontaneous mammary tumour development in Trp53+/- mice. In this study, we have generated and characterised congenic mice that contain the BALB/c SuprMam1 (susceptibility) locus on a C57BL/6 (resistant) background and discovered a subtle impairment in the vitamin D/ calcium/ parathyroid hormone (PTH) pathway. This was evident as altered gene expression in the mammary glands of key players in this pathway. Further functional analysis of the mice revealed elevated PTH levels, reduced Cyp27b1 expression in kidneys, and reduced trabecular bone volume/ tissue volume percentage. Plasma 25(OH)D and serum calcium were unchanged. This impairment was a result of genetic differences and occurred only in females, but the elevated PTH levels could be overcome with either calcium or vitamin D dietary supplementation. Either low levels of active vitamin D (1,25(OH)2D) or chronically elevated PTH levels may contribute to increased breast cancer susceptibility. These indicators are not easily measured in human population studies, but either mechanism may be preventable with dietary calcium or vitamin D supplements. Therefore, SuprMam congenic mice could serve as a valuable model for studying the role of gene-hormone-environment interactions of the vitamin D/ calcium/ PTH pathway in cancer and other diseases and for testing preventive interventions.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Cálcio/metabolismo , Hormônio Paratireóideo/metabolismo , Transdução de Sinais , Vitamina D/metabolismo , Animais , Neoplasias da Mama/sangue , Neoplasias da Mama/patologia , Cálcio/sangue , Feminino , Loci Gênicos , Predisposição Genética para Doença , Masculino , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Hormônio Paratireóideo/sangue , Vitamina D/sangue
8.
Sci Rep ; 9(1): 9511, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266983

RESUMO

Adipocytes support key metabolic and endocrine functions of adipose tissue. Lipid is stored in two major classes of depots, namely visceral adipose (VA) and subcutaneous adipose (SA) depots. Increased visceral adiposity is associated with adverse health outcomes, whereas the impact of SA tissue is relatively metabolically benign. The precise molecular features associated with the functional differences between the adipose depots are still not well understood. Here, we characterised transcriptomes and methylomes of isolated adipocytes from matched SA and VA tissues of individuals with normal BMI to identify epigenetic differences and their contribution to cell type and depot-specific function. We found that DNA methylomes were notably distinct between different adipocyte depots and were associated with differential gene expression within pathways fundamental to adipocyte function. Most striking differential methylation was found at transcription factor and developmental genes. Our findings highlight the importance of developmental origins in the function of different fat depots.


Assuntos
Metilação de DNA , Epigênese Genética , Gordura Intra-Abdominal/metabolismo , Gordura Subcutânea/metabolismo , Transcriptoma , Adipócitos/citologia , Adipócitos/metabolismo , Adulto , Sítios de Ligação , Índice de Massa Corporal , Regulação para Baixo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Gordura Intra-Abdominal/citologia , Pessoa de Meia-Idade , Elementos Reguladores de Transcrição , Gordura Subcutânea/citologia , Fatores de Transcrição/metabolismo , Regulação para Cima
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