A Long-read Sequencing Approach for Investigating Repeat Number and DNA Methylation of the D4Z4 Region

用于研究 D4Z4 区域重复数和 DNA 甲基化的长读长测序方法

• 批准号: 10093171
• 负责人: YI-WEN CHEN
• 金额: $ 8.93万
• 依托单位: CHILDREN'S RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-01 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10093171
• 关键词: 4q35; Address; Affect; Age; Age of Onset; Alleles; Appearance; Biological Assay; CRISPR/Cas technology; Chromosome 10; Chromosome 4; Chromosomes; Clinical; Clinical Data; Cohort Studies; Complex; Consumption; Contracts; D4Z4; DNA; DNA Methylation; DNA Sequence Alteration; Data; Data Analyses; Development; Diagnosis; Diagnostic; Disease; Epigenetic Process; Facioscapulohumeral Muscular Dystrophy; Gender; Genes; Genetic; Genetic Transcription; Genetic Variation; Genome; Genomic DNA; Genomics; Goals; Homeobox; Homeodomain Proteins; Individual; Lead; Manuals; Measurement; Methods; Methylation; Molecular Diagnosis; Muscle; Mutation; Myopathy; Neurology; Onset of illness; Optics; Patients; Prognosis; Protocols documentation; Pulsed-Field Gel Electrophoresis; Regulation; Repression; Research; Research Personnel; Role; Sampling; Severities; Severity of illness; Southern Blotting; Surveys; Test Result; Testing; Time; base; bioinformatics pipeline; cohort; early onset; epigenetic variation; genetic testing; improved; insight; nanopore; new therapeutic target; novel; novel strategies; therapeutic development; tool

ABSTRACT Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal dominant muscle disorder caused by complex genetic and epigenetic mechanisms. Previous studies showed that transcription de- repression of double homeobox protein 4 (DUX4) due to epigenetic changes in the D4Z4 region causes FSHD. The epigenetic changes are caused by either contraction of the D4Z4 array from 11-150 repeat units in unaffected individuals to 1-10 repeat units in roughly 95% of patients (FSHD1) or mutations in epigenetic regulators of the region (FSHD2). Current genetic testing for FSHD is labor intensive and does not assess DNA methylation status, which has been suggested to be the primary determinant of disease severity in FSHD1. In this application, we propose to use a novel approach, Nanopore long- read sequencing, to determine the copy number and methylation level of D4Z4 region in a single test. Our preliminary data showed that optical mapping can accurately provide sizing for D4Z4 repeat region. In aim 1 of the project, we will first develop a Nanopore long-read sequencing assay to evaluate the D4Z4 arrays on both chromosome 4 and chromosome 10 and built a data analysis workflow for the data. In aim 2, we will use the approach to determine D4Z4 repeat size and DNA methylation status of FSHD DNA samples and DNA samples from unaffected individuals collected from a recent study of early onset FSHD; then determine the correlation between the methylation status and disease onset and severities. Development of a single test that could assess both genetic and epigenetic causes of FSHD will significantly transform the molecular diagnosis of FSHD as well as provide a roadmap for researcher to investigate how genetic and epigenetic variations in subtelomeric region of 4q35 affect FSHD onset and severity.

摘要 面肩肱型肌营养不良症是一种常染色体显性遗传性肌肉疾病 由复杂的遗传和表观遗传机制引起。以前的研究表明，转录去- 由于D4 Z4区域的表观遗传变化导致双同源框蛋白4（DUX 4）的抑制， FSHD。表观遗传变化是由D4 Z4阵列从11-150个重复的收缩引起的， 未受影响个体中的1-10个重复单位到大约95%患者中的1-10个重复单位（FSHD 1）或 表观遗传调节因子区域（FSHD 2）。目前FSHD的基因检测是劳动密集型的， 不评估DNA甲基化状态，这被认为是主要的决定因素， FSHD 1中的疾病严重程度。在这个应用中，我们建议使用一种新的方法，纳米孔长- 读取测序，以在单次测试中确定D4 Z4区域的拷贝数和甲基化水平。 我们的初步数据表明，光学定位可以准确地提供D4 Z4重复区的大小。 在该项目的目标1中，我们将首先开发一种纳米孔长读序测序测定法，以评估 D4 Z4阵列在染色体4和染色体10上，并建立了数据分析工作流程， 数据在目标2中，我们将使用该方法来确定D4 Z4重复序列大小和DNA甲基化状态。 FSHD DNA样本和从最近的一项研究中收集的未受影响个体的DNA样本， 早发性FSHD;然后确定甲基化状态与疾病发作之间的相关性 和严重性。开发一种单一的测试，可以评估遗传和表观遗传的原因， FSHD将显著改变FSHD的分子诊断，并为FSHD的诊断提供路线图。 研究人员研究4 q35亚端粒区域的遗传和表观遗传变异如何影响 FSHD发作和严重程度。