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

2017 ◽  
Author(s):  
Yu-Chih Tsai ◽  
David Greenberg ◽  
James Powell ◽  
Ida Höijer ◽  
Adam Ameur ◽  
...  
Keyword(s):  
Repetitive Sequences ◽  
Gc Content ◽  
Repeat Expansion ◽  
Cag Repeat ◽  
Targeted Sequencing ◽  
Spinocerebellar Ataxia Type ◽  
Sequence Information ◽  
Smrt Sequencing ◽  
Cgg Repeat ◽  
Genomic Regions

AbstractTargeted 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.

scholarly journals A clinical report of the massive CAG repeat expansion in spinocerebellar ataxia type 2: Severe onset in a Mexican child and review previous cases

2020 ◽  
Vol 43 (3) ◽  
Author(s):  
José Sánchez-Corona ◽  
Sergio Alberto Ramirez-Garcia ◽  
Gema Castañeda-Cisneros ◽  
Susan Andrea Gutiérrez-Rubio ◽  
Víctor Volpini ◽  
...  
Keyword(s):  
Spinocerebellar Ataxia ◽  
Repeat Expansion ◽  
Cag Repeat ◽  
Spinocerebellar Ataxia Type ◽  
Clinical Report ◽  
Spinocerebellar Ataxia Type 2 ◽  
Cag Repeat Expansion ◽  
Mexican Child

CAG Repeat Expansion in an Italian Family with Spinocerebellar Ataxia Type 2 (SCA2): A Clinical and Genetic Study

1998 ◽  
Vol 40 (3) ◽  
pp. 164-168 ◽  
Author(s):  
Alessandro Malandrini ◽  
Lucia Galli ◽  
Marcello Villanova ◽  
Silvia Palmeri ◽  
Emma Parrotta ◽  
...  
Keyword(s):  
Spinocerebellar Ataxia ◽  
Genetic Study ◽  
Repeat Expansion ◽  
Cag Repeat ◽  
Spinocerebellar Ataxia Type ◽  
Spinocerebellar Ataxia Type 2 ◽  
Cag Repeat Expansion ◽  
Italian Family

Spinocerebellar ataxia type 6

Neurology ◽  
1997 ◽  
Vol 49 (5) ◽  
pp. 1238-1243 ◽  
Author(s):  
R. Matsumura ◽  
N. Futamura ◽  
Y. Fujimoto ◽  
S. Yanagimoto ◽  
H. Horikawa ◽  
...  
Keyword(s):  
Family History ◽  
Cerebellar Ataxia ◽  
Age At Onset ◽  
Repeat Expansion ◽  
Repeat Length ◽  
Cag Repeat ◽  
Cag Repeats ◽  
Spinocerebellar Ataxia Type ◽  
Cag Repeat Expansion ◽  
Spinocerebellar Ataxia Type 6

Spinocerebellar ataxia type 6 (SCA6) is a newly classified autosomal-dominant cerebellar ataxia (ADCA) associated with CAG repeat expansion. We screened 111 patients with cerebellar ataxia for the SCA6 mutation. Of these, 35 patients were found to have expanded CAG repeats in the SCA6 gene, indicating that second to SCA3, SCA6 is the most common ADCA in Japan. Expanded alleles ranged from 21 to 29 repeats, whereas normal alleles had seven to 17 repeats. There was no change in the CAG repeat length during meiosis. The age at onset was inversely correlated with the repeat length. The main clinical feature of the 35 patients with SCA6 was slowly progressive cerebellar ataxia; multisystem involvement was not common. The 35 patients included nine cases without apparent family history of cerebellar ataxia. The sporadic cases had smaller CAG repeats (21 or 22 repeats) and a later age at onset (64.9 ± 4.9 years) than the other cases with established family history. We also identified one patient who was homozygous for the SCA6 repeat expansion. The homozygote showed an earlier age of onset and more severe clinical manifestations than her sister, a heterozygote carrying an expanded allele with the same repeat length as the homozygote. This finding suggests that the dosage of the CAG repeat expansion plays an important role in phenotypic expression in SCA6.

scholarly journals Polyglutamine-expanded ataxin-3: a target engagement marker for Spinocerebellar ataxia type 3 in peripheral blood

2021 ◽  
Author(s):  
Jeannette Huebener-Schmid ◽  
Kirsten Kuhlbrodt ◽  
Julien Peladan ◽  
Jennifer Faber ◽  
Magda M Santana ◽  
...  
Keyword(s):  
Spinocerebellar Ataxia ◽  
Repeat Expansion ◽  
Cag Repeat ◽  
Spinocerebellar Ataxia Type ◽  
Clinical Parameters ◽  
Target Engagement ◽  
Spinocerebellar Ataxia Type 3 ◽  
Curative Therapy ◽  
Cag Repeat Expansion ◽  
Type 3

Abstract Spinocerebellar ataxia type 3 is a rare neurodegenerative disease, caused by a CAG repeat expansion leading to polyglutamine elongation in the ataxin-3 protein. While no curative therapy is yet available, preclinical gene silencing approaches to reduce polyglutamine-toxicity demonstrate promising results. In view of upcoming clinical trials, quantitative and easily accessible molecular markers are of critical importance as pharmacodynamic and particularly as target engagement markers. We developed a novel ultrasensitive immunoassay to measure specifically polyQ-expanded ataxin-3 in plasma and cerebrospinal fluid. Statistical analyses revealed a correlation with clinical parameters and a stability of polyglutamine-expanded ataxin-3 during conversion from the pre-ataxic to the ataxic phase.

scholarly journals FAN1, a DNA Repair Nuclease, as a Modifier of Repeat Expansion Disorders

2021 ◽  
Vol 10 (1) ◽  
pp. 95-122
Author(s):  
Amit L. Deshmukh ◽  
Antonio Porro ◽  
Mohiuddin Mohiuddin ◽  
Stella Lanni ◽  
Gagan B. Panigrahi ◽  
...  
Keyword(s):  
Dna Repair ◽  
Age Of Onset ◽  
Disease Onset ◽  
Repeat Expansion ◽  
Cag Repeat ◽  
Common Mechanism ◽  
Clinical Effects ◽  
Single Nucleotide Variants ◽  
Cgg Repeat ◽  
Repeat Expansions

FAN1 encodes a DNA repair nuclease. Genetic deficiencies, copy number variants, and single nucleotide variants of FAN1 have been linked to karyomegalic interstitial nephritis, 15q13.3 microdeletion/microduplication syndrome (autism, schizophrenia, and epilepsy), cancer, and most recently repeat expansion diseases. For seven CAG repeat expansion diseases (Huntington’s disease (HD) and certain spinocerebellar ataxias), modification of age of onset is linked to variants of specific DNA repair proteins. FAN1 variants are the strongest modifiers. Non-coding disease-delaying FAN1 variants and coding disease-hastening variants (p.R507H and p.R377W) are known, where the former may lead to increased FAN1 levels and the latter have unknown effects upon FAN1 functions. Current thoughts are that ongoing repeat expansions in disease-vulnerable tissues, as individuals age, promote disease onset. Fan1 is required to suppress against high levels of ongoing somatic CAG and CGG repeat expansions in tissues of HD and FMR1 transgenic mice respectively, in addition to participating in DNA interstrand crosslink repair. FAN1 is also a modifier of autism, schizophrenia, and epilepsy. Coupled with the association of these diseases with repeat expansions, this suggests a common mechanism, by which FAN1 modifies repeat diseases. Yet how any of the FAN1 variants modify disease is unknown. Here, we review FAN1 variants, associated clinical effects, protein structure, and the enzyme’s attributed functional roles. We highlight how variants may alter its activities in DNA damage response and/or repeat instability. A thorough awareness of the FAN1 gene and FAN1 protein functions will reveal if and how it may be targeted for clinical benefit.

scholarly journals Massive CAG Repeat Expansion and Somatic Instability in Maternally Transmitted Infantile Spinocerebellar Ataxia Type 7

2015 ◽  
Vol 72 (2) ◽  
pp. 219 ◽  
Author(s):  
Heather Trang ◽  
Sabrina Y. Stanley ◽  
Paul Thorner ◽  
Hannaneh Faghfoury ◽  
Andreas Schulze ◽  
...  
Keyword(s):  
Spinocerebellar Ataxia ◽  
Repeat Expansion ◽  
Cag Repeat ◽  
Spinocerebellar Ataxia Type ◽  
Somatic Instability ◽  
Cag Repeat Expansion ◽  
Spinocerebellar Ataxia Type 7

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

2020 ◽  
Vol 29 (15) ◽  
pp. 2551-2567 ◽  
Author(s):  
Ricardo Mouro Pinto ◽  
Larissa Arning ◽  
James V Giordano ◽  
Pedram Razghandi ◽  
Marissa A Andrew ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Huntington's Disease ◽  
Spinocerebellar Ataxia ◽  
Repeat Expansion ◽  
Cag Repeat ◽  
Spinocerebellar Ataxia Type ◽  
Spinocerebellar Ataxia Type 1 ◽  
Cag Expansion

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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