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

摘要 面肩肱骨肌营养不良症(FSHD)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。先前的研究表明，转录去激活 D4Z4区表观遗传改变导致双同源盒蛋白4(DUX4)的抑制 FSHD。表观遗传变化是由D4Z4序列从11-150个重复序列收缩引起的 在大约95%的患者中，未受影响的个体的单位减少到1-10个重复单位(FSHD1)或突变 该区域的表观遗传调控因子(FSHD2)。目前的FSHD基因检测是劳动密集型的，而且 不评估DNA甲基化状态，这已被认为是 FSHD1中的疾病严重性。在这项应用中，我们建议使用一种新的方法，纳米孔长- 阅读测序，在一次测试中确定D4Z4区域的拷贝数和甲基化水平。 我们的初步数据表明，光学作图可以准确地提供D4Z4重复区域的大小。 在该项目的目标1中，我们将首先开发一种纳米孔长读测序分析来评估 D4Z4排列在4号染色体和10号染色体上，并为 数据。在目标2中，我们将使用该方法来确定D4Z4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人的DNA样本 早发性FSHD；然后确定甲基化状态与疾病发病之间的相关性 和严肃性。开发一种可同时评估遗传和表观遗传原因的单一检测方法 FSHD将显著改变FSHD的分子诊断，并为 研究人员调查4q35亚端粒区域的遗传和表观遗传变异如何影响 FSHD的发病和严重程度。

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.