RESUMEN
There are an estimated > 400 million people living with a rare disease globally, with genetic variants the cause of approximately 80% of cases. Next Generation Sequencing (NGS) rapidly identifies genetic variants however they are often of unknown significance. Low throughput functional validation in specialist laboratories is the current ad hoc approach for functional validation of genetic variants, which creating major bottlenecks in patient diagnosis. This study investigates the application of CRISPR gene editing followed by genome wide transcriptomic profiling to facilitate patient diagnosis. As proof-of-concept, we introduced a variant in the Euchromatin histone methyl transferase (EHMT1) gene into HEK293T cells. We identified changes in the regulation of the cell cycle, neural gene expression and suppression of gene expression changes on chromosome 19 and chromosome X, that are in keeping with Kleefstra syndrome clinical phenotype and/or provide insight into disease mechanism. This study demonstrates the utility of genome editing followed by functional readouts to rapidly and systematically validating the function of variants of unknown significance in patients suffering from rare diseases.
Asunto(s)
Anomalías Craneofaciales/diagnóstico , Edición Génica/métodos , Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes , Cardiopatías Congénitas/diagnóstico , N-Metiltransferasa de Histona-Lisina/genética , Discapacidad Intelectual/diagnóstico , Sistemas CRISPR-Cas , Deleción Cromosómica , Cromosomas Humanos Par 19/genética , Cromosomas Humanos Par 9/genética , Cromosomas Humanos X/genética , Anomalías Craneofaciales/genética , Diagnóstico Precoz , Regulación de la Expresión Génica , Variación Genética , Células HEK293 , Cardiopatías Congénitas/genética , Humanos , Discapacidad Intelectual/genética , Prueba de Estudio Conceptual , Análisis de Secuencia de ARNRESUMEN
BACKGROUND: With the move towards systems biology, we need sensitive and reliable ways to determine the relationships between transcription factors and their target genes. In this paper we analyze the regulatory relationships between 78 myeloid transcription factors and their coding genes by using the matrix RNAi system in which a set of transcription factor genes are individually knocked down and the resultant expression perturbation is quantified. RESULTS: Using small interfering RNAs we knocked down the 78 transcription factor genes in monocytic THP-1 cells and monitored the perturbation of the expression of the same 78 transcription factors and 13 other transcription factor genes as well as 5 non-transcription factor genes by quantitative real-time RT-PCR, thereby building a 78 x 96 matrix of perturbation and measurement. This approach identified 876 cases where knockdown of one transcription factor significantly affected the expression of another (from a potential 7,488 combinations). Our study also revealed cell-type-specific transcriptional regulatory networks in two different cell types. CONCLUSIONS: By considering whether the targets of a given transcription factor are naturally up- or downregulated during phorbol 12-myristate 13-acetate-induced differentiation, we could classify these edges as pro-differentiative (229), anti-differentiative (76) or neither (571) using expression profiling data obtained in the FANTOM4 study. This classification analysis suggested that several factors could be involved in monocytic differentiation, while others such as MYB and the leukemogenic fusion MLL-MLLT3 could help to maintain the initial undifferentiated state by repressing the expression of pro-differentiative factors or maintaining expression of anti-differentiative factors.
Asunto(s)
Modelos Genéticos , Monocitos/metabolismo , Interferencia de ARN , Factores de Transcripción/genética , Diferenciación Celular , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Redes Reguladoras de Genes/genética , Humanos , Modelos Biológicos , ARN Interferente Pequeño/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética , TransfecciónRESUMEN
EdgeExpressDB is a novel database and set of interfaces for interpreting biological networks and comparing large high-throughput expression datasets that requires minimal development for new data types and search patterns. The FANTOM4 EdgeExpress database http://fantom.gsc.riken.jp/4/edgeexpress summarizes gene expression patterns in the context of alternative promoter structures and regulatory transcription factors and microRNAs using intuitive gene-centric and sub-network views. This is an important resource for gene regulation in acute myeloid leukemia, monocyte/macrophage differentiation and human transcriptional networks.