Detailed analysis of <i>HTT</i>
 repeat elements in human blood using targeted amplification-free long-read sequencing

Höijer, Ida; Tsai, Yu‐Chih; Clark, Tyson A.; Kotturi, Paul; Dahl, Niklas; Stattin, Eva‐Lena; Bondeson, Marie-Louise; Feuk, Lars; Gyllensten, Ulf; Ameur, Adam

doi:10.1002/humu.23580

Cited by 66 publications

(84 citation statements)

References 51 publications

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“…Therefore, additional tissue-specific effects may be revealed by the detection of rare expansions. Next-generation sequencing approaches can also be used to capture somatic CAG length variation ( 68 , 69 ). Methods based upon sequencing of PCR amplicons ( 68 ) capture similar profiles of somatic expansion to fragment size-based analyses of PCR products, as performed in this study.…”

Section: Discussionmentioning

confidence: 99%

Patterns of CAG repeat instability in the central nervous system and periphery in Huntington’s disease and in spinocerebellar ataxia type 1

Pinto

Arning

Giordano

et al. 2020

Human Molecular Genetics

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Abstract The expanded HTT CAG repeat causing Huntington’s disease (HD) exhibits somatic expansion proposed to drive the rate of disease onset by eliciting a pathological process that ultimately claims vulnerable cells. To gain insight into somatic expansion in humans we performed comprehensive quantitative analyses of CAG expansion in ~ 50 central nervous system (CNS) and peripheral postmortem tissues from seven adult-onset and one juvenile-onset HD individual. We also assessed ATXN1 CAG repeat expansion in brain regions of an individual with a neurologically and pathologically distinct repeat expansion disorder, spinocerebellar ataxia type 1 (SCA1). Our findings reveal similar profiles of tissue instability in all HD individuals, which, notably, were also apparent in the SCA1 individual. CAG expansion was observed in all tissues, but to different degrees, with multiple cortical regions and neostriatum tending to have the greatest instability in the CNS, and liver in the periphery. These patterns indicate different propensities for CAG expansion contributed by disease locus-independent trans-factors and demonstrate that expansion per se is not sufficient to cause cell type or disease-specific pathology. Rather, pathology may reflect distinct toxic processes triggered by different repeat lengths across cell types and diseases. We also find that the HTT CAG length-dependent expansion propensity of an individual is reflected in all tissues and in cerebrospinal fluid. Our data indicate that peripheral cells may be a useful source to measure CAG expansion in biomarker assays for therapeutic efforts, prompting efforts to dissect underlying mechanisms of expansion that may differ between the brain and periphery.

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Section: Discussionmentioning

confidence: 99%

Patterns of CAG repeat instability in the central nervous system and periphery in Huntington’s disease and in spinocerebellar ataxia type 1

Pinto

Arning

Giordano

et al. 2020

Human Molecular Genetics

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“…Using Cas9-based no-amp capture the region could be enriched and sequenced to high depth, showing no indication of repeat motif interruptions [67]. Similarly, enrichment was used to investigate somatic repeat size mosaicism and repeat motif interruptions in HTT [68] and ATXN10 expansions, with the latter shown to modify the phenotype in a family with Parkinsonism with or without epilepsy [69]. Repeat interruptions in ATXN1 reduce pathogenicity, in ATXN2 they cause FTD plus ALS rather than SCA, and in ATXN10 they are linked with seizures [70][71][72].…”

Section: Examples Of Svs In Neurodegenerationmentioning

confidence: 99%

Newest Methods for Detecting Structural Variations

Coster

Broeckhoven

2019

Trends in Biotechnology

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A substantial amount of structural variation in the human genome remains uninvestigated due to the limitations of existing technologies, the presence of repetitive sequences, and the complexity of a diploid genome. New technologies have been developed, increasing resolution and appreciation of structural variation and how it affects human diversity and disease. The genetic etiology of most patients with complex disorders such as neurodegenerative brain diseases is not yet elucidated, complicating disease diagnosis, genetic counseling, and understanding of underlying pathological mechanisms needed to develop therapeutic interventions. Here, we focus on innovative progress and opportunities provided by the newest methods such as linked read sequencing, strand-specific sequencing, and long-read sequencing. Finally, we describe a strategy for generating a comprehensive catalog of structural variations across populations. Structural Variation Has Been Systematically Missed Multiple projects have comprehensively cataloged small genetic variants [single nucleotide variants (SNVs)]; however, structural variants (SVs) remain largely underrepresented [1,2]. SVs are defined as regions of DNA larger than 50 bp showing a change in copy number or genomic location including copy number variants (CNVs; deletions and duplications), insertions, inversions, translocations, mobile elements, repetitive sequence expansions, and complex combinations thereof (Figure 1) [3]. SVs contribute $3.4 times more nucleotides to human genetic variation than the far more numerous SNVs [4]. Multiple sequencing technologies (see Glossary), notably longread sequencing and short-read sequencing library preparation methods such as strand-specific sequencing (strand-seq) and linked-read sequencing, have been developed, providing an unprecedented potential and accuracy of genome-wide structural variation. Here, we describe new improvements to SV detection methods, each with different strengths and shortcomings. One landmark study combined several technologies to obtain a complete haplotype-specific characterization in healthy human trios, yielding >30 000 SVs per genome, including >150 inversions and large unbalanced chromosomal rearrangements [5]. This work suggested that previous technologies missed most of the SVs due to technical limitations. The majority of SVs are novel and rare variants, implicating that structural variation databases are not saturated yet [2-7]. Highlights The genetic etiology of complex human diseases is still poorly understood, hampering diagnostics and therapeutic approaches.

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“…The level of enrichment by Xdrop technology is comparable to other long-read targeted enrichment strategies (Gabrieli et al, 2017;Gilpatrick et al, 2019;Tsai et al, 2017) interest, and they require micrograms of input DNA (Gilpatrick et al, 2019;Hoijer et al, 2018;Stangl et al, 2019;Tsai et al, 2017).…”

Section: Discussionmentioning

confidence: 99%

Xdrop: Targeted sequencing of long DNA molecules from low input samples using droplet sorting

et al. 2020

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Long‐read sequencing can resolve regions of the genome that are inaccessible to short reads, and therefore are ideal for genome‐gap closure, solving structural rearrangements and sequencing through repetitive elements. Here we introduce the Xdrop technology: a novel microfluidic‐based system that allows for targeted enrichment of long DNA molecules starting from only a few nanograms of DNA. Xdrop is based on the isolation of long DNA fragments in millions of droplets, where the droplets containing a target sequence of interest are fluorescently labeled and sorted using flow cytometry. The final product from the Xdrop procedure is an enriched population of long DNA molecules that can be investigated by sequencing. To demonstrate the capability of Xdrop, we performed enrichment of the human papilloma virus 18 integrated into the genome of human HeLa cells. Analysis of the sequencing reads resolved three HPV18‐chr8 integrations at base‐pair resolution, and the captured fragments extended up to 30 kb into the human genome at the integration sites. Further, we enriched the complete TP53 locus in a leukemia cell line and could successfully phase coexisting mutations using PacBio sequencing. In summary, our results show that Xdrop is an efficient enrichment technology for studying complex genomic regions.

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Detailed analysis of HTT repeat elements in human blood using targeted amplification-free long-read sequencing

Cited by 66 publications

References 51 publications

Patterns of CAG repeat instability in the central nervous system and periphery in Huntington’s disease and in spinocerebellar ataxia type 1

Patterns of CAG repeat instability in the central nervous system and periphery in Huntington’s disease and in spinocerebellar ataxia type 1

Newest Methods for Detecting Structural Variations

Xdrop: Targeted sequencing of long DNA molecules from low input samples using droplet sorting

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