Identification of novel pathogenic tandem repeat expansions using long read sequencing

重复(Tr)DNA序列的大规模扩展(例如，PolyCAG)是已知的基础 不同的人类神经系统疾病，包括亨廷顿病、脆性X和强直性肌营养不良。这个 绝大多数已知的TR扩张见于成人起病的退行性神经肌肉疾病和 共济失调综合征。尽管最近取得了重大进展，使TRs能够进行基因分型 从高通量测序来看，目前用于对人类基因组进行测序的方法无法 识别tr扩增，因为它们只查看非常短的dna片段。然而，最近的进展和 测序技术的成本下降，如太平洋生物科学公司的SMRT测序 更长的读取有望检测到以前未检测到的重复扩展。在这里，我们将完整地表演 联合太平洋生物科学(PacBio)在选定的一组患者中的基因组测序 未解决的共济失调和类亨廷顿病，在这些疾病中，所有已知的TR扩张和其他突变都具有 被排除在外。这些样本中的许多来自具有显性遗传的多代家系 显示遗传预测和连锁信息，将致病突变定位到 基因组，因此代表了一个优化的队列，在其中搜索未知的致病TR扩展。 为了能够识别人类疾病背后的TR扩展，首先有必要 描述正常人群中串联重复序列变异的谱。使用26个基因组 使用PacBio对个体进行测序，我们将使用我们开发的名为MsPac的新算法和 PacMONSTR，以生成正常人类基因组中所有TRs的大小分布的调查。这将是 由HipSTR从1,500个灯盏花属基因组中产生的TR基因型补充。此信息将 提供tr变异的基线调查，使我们能够识别样本中的致病tr扩展。 共济失调和神经退行性疾病，正如我们所展示的，也使我们能够识别出 很可能会在人类疾病中扩大。使用这种方法，我们将首先进行四个目标基因分型 在250个有SCA/HD表型的样本中，多谷氨酰胺受体显示出强烈的不稳定特征。 接下来，我们将对来自40个未解决的家系的100个个体进行PacBio基因组测序 共济失调/类HD疾病，使用选定的样本队列，其中所有已知的遗传和环境 原因已经被排除了。我们推测，这些家系中的一些突变将是 新奇的扩展的RR，以前的短文方法仍然看不见。我们将寻找小说 在我们的对照人群中没有观察到TR值的扩张。 使用这种优化的队列和新的混合长读测序方法，这项建议将导致 人类神经系统疾病基础上新的致病树突起的鉴定，产生 我们对共济失调和神经退行性疾病病因的理解取得了重大进展。

Large expansions of tandemly repeated (TR) DNA sequences (eg. polyCAG) are known to underlie >30 different human neurological diseases, including Huntington’s disease, Fragile X, and Myotonic dystrophy. The vast majority of known TR expansions are observed in adult onset degenerative neuromuscular disorders and ataxia syndromes. Although significant recent advances have been made that enable TRs to be genotyped from high-throughput sequencing, the methods currently used to sequence human genomes are unable to identify TR expansions, as they only look at very short fragments of DNA. However, recent advances and falling costs of sequencing technologies like Pacific Biosciences SMRT sequencing that generates much longer reads hold the promise to detect previously undetected repeat expansions. Here we will perform whole genome sequencing using combined Pacific Biosciences (PacBio) on a selected cohort of patients with unsolved ataxias and Huntington’s-like disease, in which all known TR expansions and other mutations have been excluded. Many of these samples come from multi-generation pedigrees with dominant inheritance that show genetic anticipation and linkage information that localizes the pathogenic mutation to a subset of the genome, thus representing an optimized cohort in which to search for unknown pathogenic TR expansions. In order to be able to identify TR expansions underlying human disease, it is first necessary to characterize the spectrum of tandem repeat variation within the normal population. Using genomes of 26 individuals sequenced with PacBio, we will use a novel algorithms we have developed called MsPac and PacMONSTR, to generate a survey of the size distribution of all TRs in the normal human genome. This will be supplemented by TR genotypes generated by HipSTR from 1,500 Illumina genomes. This information will provide a baseline survey of TR variation that will allow us identify pathogenic TR expansions in samples with ataxia and neurodegenerative disease, and as we show, also enables us to identify candidate TRs that are likely to expand in human disease. Using this approach, we will first perform targeted genotyping of four polyglutamine TRs that show strong signatures of instability in 250 samples with SCA/HD-phenocopies. We will next perform PacBio genome sequencing of 100 individuals from 40 pedigrees with unsolved ataxia/HD-like disease, using a selected cohort of samples in which all known genetic and environmental causes have already been excluded. We hypothesize that the mutation in some of these pedigrees will be novel expanded TRs that have remained invisible to previous short-read approaches. We will search for novel TR expansions not observed in our control population. Using this optimized cohort and novel hybrid long-read sequencing approach, this proposal will lead to the identification of novel pathogenic TR expansions that underlie human neurological diseases, yielding significant advances in our understanding of the etiology of ataxia and neurodegenerative disease.