Identification of novel pathogenic tandem repeat expansions using long read sequencing

使用长读长测序鉴定新型致病性串联重复扩增

• 批准号: 10225433
• 负责人: Andrew James Sharp
• 金额: $ 62.41万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-30 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10225433
• 关键词: Adult; Algorithms; Alleles; Ataxia; Baseline Surveys; Biological Assay; Biological Sciences; CAG repeat; Clinical Research; Code; Collaborations; Collection; Custom; DNA; DNA Sequence; DNA Sequence Alteration; Data; Diploidy; Disease; Etiology; Event; Family; Fragile X Syndrome; Generations; Genetic; Genetic Anticipation; Genetic Polymorphism; Genetic study; Genome; Genotype; Haplotypes; High-Throughput Nucleotide Sequencing; Human; Human Genome; Huntington Disease; Hybrids; Hypermethylation; Individual; Inherited; Length; Methods; Mutation; Myotonic Dystrophy; Neurodegenerative Disorders; Neuromuscular Diseases; Pathogenicity; Patients; Phase; Phenocopy; Population; Population Control; Repetitive Sequence; Research Personnel; Resources; Sampling; Shotguns; Structure; Surveys; Syndrome; Tandem Repeat Sequences; Technology; Trinucleotide Repeats; Variant; base; cohort; cost; exome sequencing; falls; genetic linkage; genetic pedigree; genome sequencing; genome-wide; human disease; nervous system disorder; novel; polyglutamine; single molecule real time sequencing; whole genome

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.

已知串联重复的（TR）DNA序列（例如多cag）的大量膨胀是> 30的基础 不同的人类神经系统疾病，包括亨廷顿氏病，脆弱的X和肌发育症。 在成年发作性退化性神经肌肉疾病中观察到了绝大多数已知TR扩张，并且 共济失调综合征。尽管已经取得了重大进展，使TR可以基因分型 从高通量测序中，当前用于对人类基因组进行测序的方法无法 确定TR扩展，因为它们仅查看DNA的很短的片段。但是，最近的进步和 太平洋生物科学等测序技术的下降成本SMRT测序产生了很多 更长的读数有望检测先前未发现的重复扩展。在这里，我们将表现完整 使用太平洋生物科学（PACBIO）的基因组测序在选定的患者队列上 未解决的共济失调和亨廷顿的疾病，其中所有已知的TR膨胀和其他突变均具有 被排除在外。这些样本中有许多来自具有主要继承的多代谱系 显示遗传预期和连锁信息，将致病突变定位到一个子集 基因组，因此代表了一个优化的队列，其中搜索未知的致病性TR膨胀。 为了能够识别人类疾病潜在的TR扩张，首先有必要 表征正常种群中串联重复变化的频谱。使用26的基因组 使用PACBIO测序的个体，我们将使用我们开发的名为MSPAC和的新型算法 PACMONST，以对正常人基因组中所有TR的大小分布进行调查。这将是 由HIPSTR从1,500个Illumina基因组产生的TR基因型补充。这些信息将 提供TR变化的基线调查，使我们确定具有 共济失调和神经退行性疾病，正如我们所表明的，还使我们能够识别候选TRS 可能会扩大人类疾病。使用这种方法，我们将首先执行四个的靶向基因分型 在250个SCA/HD-苯甲部的250个样品中显示出强烈不稳定性的聚谷氨酰胺TR。 接下来，我们将对来自未解决的40个谱系的100个个体进行PACBIO基因组测序 共济失调/高清样疾病，使用选定的样品队列，其中所有已知的遗传和环境 原因已经被排除在外。我们假设其中一些血统中的突变将是 新颖的扩展TR是以前的短阅读方法仍然看不见的。我们将寻找小说 在我们的对照人群中未观察到TR扩展。 使用这种优化的队列和新型混合式长阅读测序方法，该建议将导致 人类神经系统疾病构成的新型致病性TR扩张的鉴定，产生 我们对共济失调和神经退行性疾病的病因的理解取得了重大进展。