Huntington Disease: Molecular Diagnostics Approach

Baştepe, Murat; Xin, Winnie

doi:10.1002/0471142905.hg0926s87

Cited by 15 publications

(8 citation statements)

References 29 publications

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“…Much work has been done to develop assays to measure repeat expansions (Warner et al 1996;Adler et al 2011;Bastepe and Xin 2015;Hayward et al 2016). However, with respect to next generation sequencing (NGS) methods, these are some of the most challenging regions of the genome due to the potential for very long repeating sequence and, in some cases, very high GC content (Loomis et al 2013;Ardui et al 2017) which hinders amplification kinetics.…”

Section: Introductionmentioning

confidence: 99%

“…These two factors also provide a significant challenge for PCR-based assays to faithfully reproduce the native genomic sequence during amplification. Furthermore, polymerase chain reaction (PCR) in patients heterozygous for a normal and large expansion allele can be problematic as only the normal allele may be amplified (Chakraborty et al 2016) and polymorphisms surrounding the repeat region can lead to allele bias, dropout, or misinterpretation of results (Bastepe and Xin 2015). To address these limitations, we have developed a novel targeted sequencing approach that does not rely on amplification and employs Single Molecule, Real-Time (SMRT) sequencing that does not suffer from the read length and GC-bias shortcomings present in first and second-generation sequencing technologies (Loomis et al 2013;Roberts et al 2013;Ross et al 2013;Shin et al 2013;Chaisson et al 2015).…”

Section: Introductionmentioning

confidence: 99%

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Amplification-free, CRISPR-Cas9 Targeted Enrichment and SMRT Sequencing of Repeat-Expansion Disease Causative Genomic Regions

Tsai

Greenberg

Powell

et al. 2017

Preprint

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Targeted sequencing has proven to be an economical means of obtaining sequence information for one or more defined regions of a larger genome. However, most target enrichment methods require amplification. Some genomic regions, such as those with extreme GC content and repetitive sequences, are recalcitrant to faithful amplification. Yet, many human genetic disorders are caused by repeat expansions, including difficult to sequence tandem repeats.We have developed a novel, amplification-free enrichment technique that employs the CRISPR-Cas9 system for specific targeting multiple genomic loci. This method, in conjunction with long reads generated through Single Molecule, Real-Time (SMRT) sequencing and unbiased coverage, enables enrichment and sequencing of complex genomic regions that cannot be investigated with other technologies. Using human genomic DNA samples, we demonstrate successful targeting of causative loci for Huntington's disease (HTT; CAG repeat), Fragile X syndrome (FMR1; CGG repeat), amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (C9orf72; GGGGCC repeat), and spinocerebellar ataxia type 10 (SCA10) (ATXN10; variable ATTCT repeat). The method, amenable to multiplexing across multiple genomic loci, uses an amplification-free approach that facilitates the isolation of hundreds of individual on-target molecules in a single SMRT Cell and accurate sequencing through long repeat stretches, regardless of extreme GC percent or sequence complexity content. Our novel targeted sequencing method opens new doors to genomic analyses independent of PCR amplification that will facilitate the study of repeat expansion disorders.peer-reviewed)

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Amplification-free, CRISPR-Cas9 Targeted Enrichment and SMRT Sequencing of Repeat-Expansion Disease Causative Genomic Regions

Tsai

Greenberg

Powell

et al. 2017

Preprint

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“…This individual bore one allele with 18 CAGs and another with 60 repeats, according to our Sanger sequencing. Using primers flanking the repeat tract (and including the polymorphic CGG repeat abutting the CAG repeat 22 ), we expected the amplification of fragments measuring 455 and 581 bp, respectively. To test for the sensitivity of the assay, we sized the repeat tracts in amplicons after 20, 25, 35, and 45 cycles of amplification, for which the total DNA concentration was 3.5, 5.7, 72, and 104 ng µL −1 , respectively, as inferred from absorbance spectroscopy.…”

Section: Resultsmentioning

confidence: 99%

“…Current diagnostic methods include the amplification of the repeat region from genomic DNA using a labelled primer followed by capillary electrophoresis 21 . In some instances, when the repeat tract is particularly large, a triplet-primed PCR is required in which one of the primers used anneals within the repeat tract 22 . This method has the advantage of being simple and thus most molecular biology laboratories have the necessary equipment.…”

Section: Introductionmentioning

confidence: 99%

µLAS: Sizing of expanded trinucleotide repeats with femtomolar sensitivity in less than 5 minutes

Malbec

Chami

Aeschbach

et al. 2019

Sci Rep

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We present µLAS, a lab-on-chip system that concentrates, separates, and detects DNA fragments in a single module. µLAS speeds up DNA size analysis in minutes using femtomolar amounts of amplified DNA. Here we tested the relevance of µLAS for sizing expanded trinucleotide repeats, which cause over 20 different neurological and neuromuscular disorders. Because the length of trinucleotide repeats correlates with the severity of the diseases, it is crucial to be able to size repeat tract length accurately and efficiently. Expanded trinucleotide repeats are however genetically unstable and difficult to amplify. Thus, the amount of amplified material to work with is often limited, making its analysis labor-intensive. We report the detection of heterogeneous allele lengths in 8 samples from myotonic dystrophy type 1 and Huntington disease patients with up to 750 CAG/CTG repeats in five minutes or less. The high sensitivity of the method allowed us to minimize the number of amplification cycles and thus reduce amplification artefacts without compromising the detection of the expanded allele. These results suggest that µLAS can speed up routine molecular biology applications of repetitive sequences and may improve the molecular diagnostic of expanded repeat disorders.

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“…Conventional diagnostic testing to assess repeat length involves triplet repeat primed PCR (TP-PCR) or Southern blotting ( Spector et al, 2021 ). These methods are imprecise when dealing with long expansions, are severely limited in their ability to detect minor alleles, and lack single-nucleotide resolution ( Warner et al, 1996 ; Nolin et al, 2003 ; Saluto et al, 2005 ; Filipovic-Sadic et al, 2010 ; Adler et al, 2011 ; Bastepe and Xin, 2015 ; Hayward et al, 2016 ; Ardui et al, 2018 ). More recently, third-generation sequencing technologies, such as Oxford Nanopore Technologies (ONT) and PacBio SMRT sequencing, have shown consistent benefits for the characterization of short tandem repeats in FXS and related disorders ( McFarland et al, 2014 , 2015 ; Tsai et al, 2017 ; Giesselmann et al, 2019 ; Mantere et al, 2019 ).…”

Section: Introductionmentioning

confidence: 99%

Characterization of FMR1 Repeat Expansion and Intragenic Variants by Indirect Sequence Capture

et al. 2021

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Traditional methods for the analysis of repeat expansions, which underlie genetic disorders, such as fragile X syndrome (FXS), lack single-nucleotide resolution in repeat analysis and the ability to characterize causative variants outside the repeat array. These drawbacks can be overcome by long-read and short-read sequencing, respectively. However, the routine application of next-generation sequencing in the clinic requires target enrichment, and none of the available methods allows parallel analysis of long-DNA fragments using both sequencing technologies. In this study, we investigated the use of indirect sequence capture (Xdrop technology) coupled to Nanopore and Illumina sequencing to characterize FMR1, the gene responsible of FXS. We achieved the efficient enrichment (> 200×) of large target DNA fragments (~60–80 kbp) encompassing the entire FMR1 gene. The analysis of Xdrop-enriched samples by Nanopore long-read sequencing allowed the complete characterization of repeat lengths in samples with normal, pre-mutation, and full mutation status (> 1 kbp), and correctly identified repeat interruptions relevant for disease prognosis and transmission. Single-nucleotide variants (SNVs) and small insertions/deletions (indels) could be detected in the same samples by Illumina short-read sequencing, completing the mutational testing through the identification of pathogenic variants within the FMR1 gene, when no typical CGG repeat expansion is detected. The study successfully demonstrated the parallel analysis of repeat expansions and SNVs/indels in the FMR1 gene at single-nucleotide resolution by combining Xdrop enrichment with two next-generation sequencing approaches. With the appropriate optimization necessary for the clinical settings, the system could facilitate both the study of genotype–phenotype correlation in FXS and enable a more efficient diagnosis and genetic counseling for patients and their relatives.

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Huntington Disease: Molecular Diagnostics Approach

Cited by 15 publications

References 29 publications

Amplification-free, CRISPR-Cas9 Targeted Enrichment and SMRT Sequencing of Repeat-Expansion Disease Causative Genomic Regions

Amplification-free, CRISPR-Cas9 Targeted Enrichment and SMRT Sequencing of Repeat-Expansion Disease Causative Genomic Regions

µLAS: Sizing of expanded trinucleotide repeats with femtomolar sensitivity in less than 5 minutes

Characterization of FMR1 Repeat Expansion and Intragenic Variants by Indirect Sequence Capture

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