scholarly journals Minimizing carry-over PCR contamination in expanded CAG/CTG repeat instability applications

2017 ◽  
Author(s):  
Loréne Aeschbach ◽  
Vincent Dion
Keyword(s):  
Tandem Repeats ◽  
Neuromuscular Disorders ◽  
Pcr Amplification ◽  
Repeat Instability ◽  
Gold Standard Method ◽  
Ctg Repeats ◽  
Somatic Tissues ◽  
Small Pool ◽  
Carry Over ◽  
Ctg Repeat

AbstractExpanded CAG/CTG repeats underlie the aetiology of 14 neurological and neuromuscular disorders. The size of the repeat tract determines in large part the severity of these disorders with longer tracts causing more severe phenotypes. Expanded CAG/CTG repeats are also unstable in somatic tissues, which is thought to modify disease progression. Routine molecular biology applications involving these repeats, including quantifying their instability, are plagued by low PCR yields. This leads to the need for setting up more PCRs of the same locus, thereby increasing the risk of carry-over contamination. Here we aimed to reduce this risk by pre-treating the samples with a Uracil N-Glycosylase (Ung) and using dUTP instead of dTTP in PCRs. We successfully applied this method to the PCR amplification of expanded CAG/CTG repeats, their sequencing, and their molecular cloning. In addition, we optimized the gold-standard method for measuring repeat instability, small-pool PCR, such that it can be used together with Ung and dUTP-containing PCRs, without compromising data quality. We expect that the protocols herein to be applicable for molecular diagnostics of expanded repeat disorders and to manipulate other tandem repeats.

scholarly journals Expanded CAG/CTG repeats resist gene silencing mediated by targeted Epigenome editing

2021 ◽  
Author(s):  
Bin Yang ◽  
Alicia C Borgeaud ◽  
Marcela Buřičová ◽  
Lorène Aeschbach ◽  
Oscar Rodríguez-Lima ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Dna Methyltransferase ◽  
Rational Design ◽  
Repeat Instability ◽  
Efficient Treatment ◽  
Ctg Repeats ◽  
Epigenome Editing ◽  
Molecule Inhibitor ◽  
Gfp Reporter ◽  
Ctg Repeat

Abstract Expanded CAG/CTG repeat disorders affect over 1 in 2500 individuals worldwide. Potential therapeutic avenues include gene silencing and modulation of repeat instability. However, there are major mechanistic gaps in our understanding of these processes, which prevent the rational design of an efficient treatment. To address this, we developed a novel system, ParB/ANCHOR-mediated Inducible Targeting (PInT), in which any protein can be recruited at will to a GFP reporter containing an expanded CAG/CTG repeat. Previous studies have implicated the histone deacetylase HDAC5 and the DNA methyltransferase DNMT1 as modulators of repeat instability via mechanisms that are not fully understood. Using PInT, we found no evidence that HDAC5 or DNMT1 modulate repeat instability upon targeting to the expanded repeat, suggesting that their effect is independent of local chromatin structure. Unexpectedly, we found that expanded CAG/CTG repeats reduce the effectiveness of gene silencing mediated by targeting HDAC5 and DNMT1. The repeat-length effect in gene silencing by HDAC5 was abolished by a small molecule inhibitor of HDAC3. Our results have important implications on the design of epigenome editing approaches for expanded CAG/CTG repeat disorders. PInT is a versatile synthetic system to study the effect of any sequence of interest on epigenome editing.

scholarly journals Transcriptionally Repressive Chromatin Remodelling and CpG Methylation in the Presence of Expanded CTG-Repeats at the DM1 Locus

2013 ◽  
Vol 2013 ◽  
pp. 1-16 ◽  
Author(s):  
Judith Rixt Brouwer ◽  
Aline Huguet ◽  
Annie Nicole ◽  
Arnold Munnich ◽  
Geneviève Gourdon
Keyword(s):  
Transgenic Mice ◽  
Trinucleotide Repeat ◽  
Cpg Methylation ◽  
Chromatin Remodelling ◽  
Ctcf Binding ◽  
Type I ◽  
Repeat Instability ◽  
Ctg Repeats ◽  
Repressive Chromatin ◽  
Ctg Repeat

An expanded CTG-repeat in the 3′ UTR of theDMPKgene is responsible for myotonic dystrophy type I (DM1). Somatic and intergenerational instability cause the disease to become more severe during life and in subsequent generations. Evidence is accumulating that trinucleotide repeat instability and disease progression involve aberrant chromatin dynamics. We explored the chromatin environment in relation to expanded CTG-repeat tracts in hearts from transgenic mice carrying the DM1 locus with different repeat lengths. Using bisulfite sequencing we detected abundant CpG methylation in the regions flanking the expanded CTG-repeat. CpG methylation was postulated to affect CTCF binding but we found that CTCF binding is not affected by CTG-repeat length in our transgenic mice. We detected significantly decreasedDMPKsense andSIX5transcript expression levels in mice with expanded CTG-repeats. Expression of the DM1 antisense transcript was barely affected by CTG-repeat expansion. In line with altered gene expression, ChIP studies revealed a locally less active chromatin conformation around the expanded CTG-repeat, namely, decreased enrichment of active histone mark H3K9/14Ac and increased H3K9Me3 enrichment (repressive chromatin mark). We also observed binding of PCNA around the repeats, a candidate that could launch chromatin remodelling cascades at expanded repeats, ultimately affecting gene transcription and repeat instability.

scholarly journals DMPK gene DNA methylation levels are associated with muscular and respiratory profiles in DM1

2019 ◽  
Vol 5 (3) ◽  
pp. e338 ◽  
Author(s):  
Cécilia Légaré ◽  
Gayle Overend ◽  
Simon-Pierre Guay ◽  
Darren G. Monckton ◽  
Jean Mathieu ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Phenotypic Variability ◽  
Repeat Length ◽  
Ctg Repeats ◽  
Standardized Measures ◽  
Prognostic Accuracy ◽  
Small Pool ◽  
Linear Regression Modeling ◽  
Dystrophia Myotonica Protein Kinase ◽  
Ctg Repeat

ObjectiveTo assess the effects of dystrophia myotonica protein kinase (DMPK) DNA methylation (DNAme) epivariation on muscular and respiratory profiles in patients with myotonic dystrophy type 1 (DM1).MethodsPhenotypes were assessed with standardized measures. Pyrosequencing of bisulfite-treated DNA was used to quantify DNAme levels in blood from 90 patients with DM1 (adult form). Modal CTG repeat length was assessed using small-pool PCR. The presence of Acil-sensitive variant repeats was also tested.ResultsDNAme levels upstream of the CTG expansion (exon and intron 11) were correlated with modal CTG repeat length (rs = −0.224, p = 0.040; rs = −0.317, p = 0.003; and rs = −0.241, p = 0.027), whereas correlations were observed with epivariations downstream of the CTG repeats (rs = 0.227; p = 0.037). The presence of a variant repeat was associated with higher DNAme levels at multiple CpG sites (up to 10% higher; p = 0.001). Stepwise multiple linear regression modeling showed that DNAme contributed significantly and independently to explain phenotypic variability in ankle dorsiflexor (3 CpGs: p = 0.001, 0.013, and 0.001), grip (p = 0.089), and pinch (p = 0.028) strengths and in forced vital capacity (2 CpGs: p = 0.002 and 0.021) and maximal inspiratory pressure (p = 0.012).ConclusionsIn addition to the CTG repeat length, DMPK epivariations independently explain phenotypic variability in DM1 and could thus improve prognostic accuracy for patients.

scholarly journals Three-dimensional chromatin interactions remain stable upon CAG/CTG repeat expansion

2020 ◽  
Vol 6 (27) ◽  
pp. eaaz4012 ◽  
Author(s):  
Gustavo A. Ruiz Buendía ◽  
Marion Leleu ◽  
Flavia Marzetta ◽  
Ludovica Vanzan ◽  
Jennifer Y. Tan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Three Dimensional ◽  
Cag Repeat ◽  
Ctcf Binding ◽  
Chromatin Interaction ◽  
Chromatin Conformation ◽  
Repeat Instability ◽  
Ctg Repeats ◽  
Chromatin Interactions ◽  
Ctg Repeat

Expanded CAG/CTG repeats underlie 13 neurological disorders, including myotonic dystrophy type 1 (DM1) and Huntington’s disease (HD). Upon expansion, disease loci acquire heterochromatic characteristics, which may provoke changes to chromatin conformation and thereby affect both gene expression and repeat instability. Here, we tested this hypothesis by performing 4C sequencing at the DMPK and HTT loci from DM1 and HD–derived cells. We find that allele sizes ranging from 15 to 1700 repeats displayed similar chromatin interaction profiles. This was true for both loci and for alleles with different DNA methylation levels and CTCF binding. Moreover, the ectopic insertion of an expanded CAG repeat tract did not change the conformation of the surrounding chromatin. We conclude that CAG/CTG repeat expansions are not enough to alter chromatin conformation in cis. Therefore, it is unlikely that changes in chromatin interactions drive repeat instability or changes in gene expression in these disorders.

scholarly journals Expanded CAG/CTG Repeats Resist Gene Silencing Mediated by Targeted Epigenome Editing

2018 ◽  
Author(s):  
Bin Yang ◽  
Alicia C. Borgeaud ◽  
Lorène Aeschbach ◽  
Oscar Rodríguez-Lima ◽  
Gustavo A. Ruiz Buendía ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Dna Methyltransferase ◽  
Rational Design ◽  
Repeat Instability ◽  
Efficient Treatment ◽  
Ctg Repeats ◽  
Epigenome Editing ◽  
Molecule Inhibitor ◽  
Gfp Reporter ◽  
Ctg Repeat

AbstractExpanded CAG/CTG repeat disorders affect over 1 in 2500 individuals worldwide. Potential therapeutic avenues include gene silencing and modulation of repeat instability. However, there are major mechanistic gaps in our understanding of these processes, which prevent the rational design of an efficient treatment. To address this, we developed a novel system, ParB/ANCHOR-mediated Inducible Targeting (PInT), in which any protein can be recruited at will to a GFP reporter containing an expanded CAG/CTG repeat. Using PInT, we found no evidence that the histone deacetylase HDAC5 or the DNA methyltransferase DNMT1 modulate repeat instability upon targeting to the expanded repeat, suggesting that their effect is independent of local chromatin structure. Unexpectedly, we found that expanded CAG/CTG repeats reduce the effectiveness of gene silencing mediated by HDAC5 or DNMT1 targeting. The repeat-length effect in gene silencing by HDAC5 was abolished by a small molecule inhibitor of HDAC3. Our results have important implications on the design of epigenome editing approaches for expanded CAG/CTG repeat disorders. PInT is a versatile synthetic system to study the effect of any sequence of interest on epigenome editing.

scholarly journals Oxidized dNTPs and the OGG1 and MUTYH DNA glycosylases combine to induce CAG/CTG repeat instability

2016 ◽  
Vol 44 (11) ◽  
pp. 5190-5203 ◽  
Author(s):  
Piera Cilli ◽  
Ilenia Ventura ◽  
Anna Minoprio ◽  
Ettore Meccia ◽  
Alberto Martire ◽  
...  
Keyword(s):  
Dna Glycosylases ◽  
Repeat Instability ◽  
Ctg Repeat

Genetic diversity of Mycoplasma hyopneumoniae isolates from conventional farrow-to-finish pig farms in Serbia

2010 ◽  
Vol 58 (3) ◽  
pp. 297-308 ◽  
Author(s):  
Bozidar Savic ◽  
Vojin Ivetic ◽  
Vesna Milicevic ◽  
Ivan Pavlovic ◽  
Milenko Zutic ◽  
...  
Keyword(s):  
Tandem Repeats ◽  
Genetic Relatedness ◽  
Pcr Amplification ◽  
Mycoplasma Hyopneumoniae ◽  
Variable Number ◽  
Repeat Motif ◽  
Typing Method ◽  
Pig Farms ◽  
Different Ages ◽  
Swine Population

Mycoplasma hyopneumoniaeis a primary agent associated with mycoplasma pneumonia and the porcine respiratory disease complex (PRDC). Various reports have indicated that different strains ofM. hyopneumoniaeare circulating in the swine population. Lysates from lung swabs from naturally infected pigs of different ages were tested according to a new variable number of tandem repeats (VNTR) genetic typing method based on the polyserine repeat motif of the P146 lipoproteoadhesin, which can be applied directly on clinical material without isolation ofM. hyopneumoniae. The aim was to determine the diversity ofM. hyopneumoniaeisolates from conventional farrow-to-finish pig farms located in different geographical areas of Serbia. PCR amplification was carried out usingM. hyopneumoniae-specific designed, conserved primers (p146MH — L and p146MH — R) flanking the region encoding the repeat motif, followed by sequencing and cluster analysis. Five groups ofM. hyopneumoniaewith thirteen to twenty-four serine repeats were observed. Analysis of three samples from each farm indicated that the specific isolate is ubiquitous in pigs of different ages. Furthermore, seven clusters were observed within 27 tested samples. The results indicated a considerable diversity amongM. hyopneumoniaefield isolates in the swine population from conventional farrow-to-finish farms in Serbia and suggest close genetic relatedness of the corresponding isolates.

Characterization of the Gyrodactylus salaris Malmberg, 1957 (Platyhelminthes: Monogenea) ribosomal intergenic spacer (IGS) DNA

Parasitology ◽  
2000 ◽  
Vol 121 (5) ◽  
pp. 555-563 ◽  
Author(s):  
C. M. COLLINS ◽  
C. O. CUNNINGHAM
Keyword(s):  
Ribosomal Rna ◽  
Tandem Repeats ◽  
Gene Array ◽  
Pcr Amplification ◽  
Intergenic Spacer ◽  
Sequence Motifs ◽  
Ribosomal Rna Gene ◽  
Intergenic Spacers ◽  
Transcription Start Sites ◽  
Pcr Products

The intergenic spacer of the ribosomal RNA gene array from the monogenean Gyrodactylus salaris was isolated using PCR amplification. PCR products were cloned and sequenced. Three different fragments of 0·63, 1·0 and 2·62 kb, were consistently obtained. These showed homology at the 5′ and 3′ termini but differed in their overall size and intervening sequence. The 5′ end showed homology to various 28S ribosomal RNA gene sequences, suggesting that this represented the 3′ terminus of the G. salaris 28S ribosomal RNA gene. A number of features common to other eukaryotic intergenic spacers were found in the longest sequence, including A + T rich sequences, palindromic sequences and tandemly repeated elements. Two regions of 23 bp sequences arranged in non-identical tandem repeats were identified. There were 9 repeats in both regions, separated by 81 bp of non-repetitive sequence. The repeat units from the two regions shared some similarity at their 3′ ends. The G. salaris intergenic spacer sequence was examined for sequence motifs involved in the transcription of the ribosomal RNA genes in other species. Several regions with homology to transcription start sites were identified.

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