Identification of novel pathogenic tandem repeat expansions using long read sequencing

串联重复（TR）DNA序列的大扩增（例如，polyCAG）已知是>30 不同的人类神经系统疾病，包括亨廷顿病、脆性X染色体和强直性肌营养不良。的 绝大多数已知的TR扩张在成人发病的退行性神经肌肉疾病中观察到， 共济失调综合征虽然最近取得了重大进展，使TR的基因分型 从高通量测序来看，目前用于人类基因组测序的方法无法 鉴定TR扩增，因为它们只观察非常短的DNA片段。然而，最近的进展和 太平洋生物科学公司SMRT测序等测序技术的成本下降， 较长的读段有希望检测先前未检测到的重复扩增。在这里，我们将执行整个 使用联合太平洋生物科学公司（PacBio）对选定的患者队列进行基因组测序， 未解决的共济失调和亨廷顿样疾病，其中所有已知的TR扩展和其他突变都具有 被排除在外。这些样本中的许多来自多代系谱，具有显性遗传， 显示遗传预测和连锁信息，其将致病性突变定位于 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性TR扩增。 为了能够鉴定人类疾病潜在的TR扩增，首先需要 表征正常人群中串联重复序列变异的谱。使用26个基因组 与PacBio测序的个体，我们将使用一种新的算法，我们已经开发了所谓的MsPac， PacMONSTR，以生成正常人类基因组中所有TR的大小分布的调查。这将是 由来自1,500个Illumina基因组的HipSTR产生的TR基因型补充。这些信息将 提供TR变异的基线调查，使我们能够识别样本中的致病性TR扩增， 共济失调和神经退行性疾病，正如我们所展示的，也使我们能够识别候选TR， 很可能在人类疾病中传播。使用这种方法，我们将首先对四个基因进行靶向基因分型。 多聚谷氨酰胺TR在250个SCA/HD表型样本中显示出强烈的不稳定性特征。 接下来，我们将对来自40个家系的100名个体进行PacBio基因组测序， 共济失调/HD样疾病，使用选定的样本队列，其中所有已知的遗传和环境因素 原因已经排除。我们假设这些家系中的一些突变将是 新的扩展TR，这些TR对以前的短读方法仍然不可见。我们将寻找新的 在我们的对照人群中未观察到TR扩增。 使用这种优化的队列和新的混合长读序测序方法，该提议将导致 鉴定了人类神经系统疾病的新的致病性TR扩增， 我们对共济失调和神经退行性疾病病因学的理解有了重大进展。

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.