Long-read sequencing the HTT CAG repeat

HTT CAG 重复序列的长读长测序

• 批准号: 1947177
• 金额: --
• 依托单位: CARDIFF UNIVERSITY
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2017
• 资助国家: 英国
• 起止时间: 2017 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1947177
• 关键词: Long; read; sequencing; HTT; CAG

Huntington's disease (HD) is an autosomal dominant neurodegeneration caused by a CAG repeat expansion in the HTT gene. In HD, the greater the repeat number the earlier the onset of disease, however, repeat number only explains approximately 50% of the variance in symptom onset. One important cis factor seems to be the sequence of the CAG repeat: pure CAG tracts are associated with earlier disease onset, whereas interruption of the CAG tract by other codons is associated with later disease onset (Massey, McAllister, Jones unpublished data). Furthermore, allelic phasing is important when trying to untangle the role of genetic variants in disease, however we are not able to phase HTT alleles reliably in our Illumina-based exome sequencing due to short read lengths. We are collaborating with Prof Monckton in Glasgow to use Illumina miSeq technology which gives reads of 300bp across the HTT repeat, but this will not work in all HTT alleles and is not long enough to read our cell and animal model alleles which contain >120 tandem repeats. Hence there is a pressing need to establish other methods that can reliably read through the HTT expanded alleles and other long repetitive alleles. We propose to use long-read sequencing on the state-of-the-art PacBio Sequel machine to establish the size and distribution of CAG repeats and the phase of disease-modifying CAG repeat interruptions in HTT alleles from HD patients.In addition, little is known about the role of epigenetic variation at the HTT locus in HD pathogenesis, however, due to its critical role in regulating gene expression, differential epigenetic changes correlated with repeat length may well be relevant to disease. Emerging evidence suggests that one such type of variation, DNA methylation, is associated with age of onset of HD and has important implications for transgenerational effects in HD. When paired with Cas9-based target capture technology, next-generation sequencing platforms can be used to detect the methylation status of a specific gene or gene panel without PCR. Furthermore, the throughput of this technique can be scaled up to hundreds of samples with the inclusion of barcoded adaptors and provide thousands of reads per sample.Hypothesis and aimsRepeat sequence, structure and epigenetic modification affect the somatic stability of the HTT CAG repeat and pathogenesis of HDAim: to characterise the expansion, phase and epigenetic status of the HTT CAG repeat in samples derived from HD patients

亨廷顿病(HD)是一种常染色体显性遗传性神经变性，由HTT基因CAG重复扩增引起。在HD中，重复次数越多，发病越早，然而，重复次数只能解释症状发作差异的大约50%。一个重要的顺式因素似乎是CAG重复序列：单纯的CAG区段与较早的发病有关，而其他密码子阻断CAG区段与较晚的发病有关(Massey，McAllister，Jones未发表的数据)。此外，当试图解开遗传变异在疾病中的作用时，等位基因分期是重要的，然而，由于阅读长度较短，我们无法在基于Illumina的外显子组测序中可靠地对HTT等位基因进行分期。我们正在与格拉斯哥的蒙克顿教授合作，使用Illumina MiSeq技术，该技术可以在HTT重复序列上提供300个基点的读数，但这并不适用于所有HTT等位基因，也不足以读取我们的细胞和动物模型中包含&gt；120串联重复序列的等位基因。因此，迫切需要建立其他方法来可靠地读取HTT扩展等位基因和其他长重复等位基因。我们建议使用最先进的PacBio续集机器上的长读测序来确定HD患者HTT等位基因中CAG重复的大小和分布以及疾病修饰性CAG重复中断的阶段。此外，关于HTT基因座的表观遗传变异在HD发病中的作用尚不清楚，然而，由于其在调节基因表达方面的关键作用，与重复长度相关的差异性表观遗传学变化很可能与疾病相关。新出现的证据表明，其中一种类型的变异，DNA甲基化，与HD的发病年龄有关，并对HD的跨代影响具有重要意义。当与基于Cas9的靶标捕获技术配合使用时，下一代测序平台可以用于检测特定基因或基因面板的甲基化状态，而无需PCR。假设和目的重复序列、结构和表观遗传修饰影响HTT CAG重复序列的体细胞稳定性和HDAIM的发病机制：表征HD患者样本中HTT CAG重复序列的扩展、时相和表观遗传学状态