Long-read sequencing the HTT CAG repeat

亨廷顿病（HD）是由HTT基因中CAG重复扩增引起的常染色体显性遗传神经变性。在HD中，重复次数越多，疾病发作越早，然而，重复次数仅解释症状发作中约50%的方差。一个重要的顺式因子似乎是CAG重复序列：纯CAG片段与较早的疾病发作相关，而其他密码子中断CAG片段与较晚的疾病发作相关（Massey，麦卡利斯特，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，等位基因定相是重要的，然而，由于短的读取长度，我们不能在基于Illumina的外显子组测序中可靠地定相HTT等位基因。我们正在与格拉斯哥的Monckton教授合作，使用Illumina miSeq技术，该技术可以在HTT重复序列中读取300 bp的片段，但这并不适用于所有HTT等位基因，也不足以读取包含>120个串联重复序列的细胞和动物模型等位基因。因此，迫切需要建立能够可靠地读取HTT扩增等位基因和其他长重复等位基因的其他方法。我们建议在最先进的PacBio Sequel机器上使用长读测序来确定HD患者HTT等位基因中CAG重复序列的大小和分布以及疾病修饰CAG重复序列中断的阶段。此外，关于HTT位点的表观遗传变异在HD发病机制中的作用知之甚少，然而，由于其在调节基因表达中的关键作用，与重复长度相关的不同表观遗传变化可能与疾病有关。新出现的证据表明，这样一种类型的变异，DNA甲基化，与发病年龄的HD和HD的跨代效应具有重要意义。当与基于Cas9的靶捕获技术配对时，下一代测序平台可用于检测特定基因或基因组的甲基化状态，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：研究来自HD患者的样品中HTT CAG重复序列的扩增、相位和表观遗传状态

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whereas interruption of the CAG tract by other codons is associated with later disease onset (Massey, McAllister, Jones unpublished data).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