scholarly journals High throughput screening for expanded CTG repeats in myotonic dystrophy type 1 using melt curve analysis

2021 ◽  
Author(s):  
Russell J. Butterfield ◽  
Carina Imburgia ◽  
Katie Mayne ◽  
Tara Newcomb ◽  
Diane M. Dunn ◽  
...  
Keyword(s):  
Myotonic Dystrophy ◽  
High Throughput ◽  
High Throughput Screening ◽  
Curve Analysis ◽  
Myotonic Dystrophy Type 1 ◽  
Myotonic Dystrophy Type ◽  
Melt Curve Analysis ◽  
Ctg Repeats

scholarly journals High Throughput Screening for Expanded CTG repeats in Myotonic Dystrophy Type 1 Using Melt Curve Analysis

2021 ◽  
Author(s):  
Russell J Butterfield ◽  
Carina Imburgia ◽  
Katie Mayne ◽  
Tara Newcomb ◽  
Diane M Dunn ◽  
...  
Keyword(s):  
Myotonic Dystrophy ◽  
High Throughput ◽  
Curve Analysis ◽  
Myotonic Dystrophy Type 1 ◽  
Myotonic Dystrophy Type ◽  
Melt Curve Analysis ◽  
Ctg Repeats ◽  
Repeat Expansions ◽  
Ctg Repeat

ABSTRACTBackgroundMyotonic dystrophy type 1 (DM1) is caused by CTG repeat expansions in the DMPK gene and is the most common form of muscular dystrophy. Patients can have long delays from onset to diagnosis, since clinical signs and symptoms are often non-specific and overlapping with other disorders. Clinical genetic testing by Southern blot or triplet-primed PCR (TP-PCR) is technically challenging and cost prohibitive for population surveys.MethodsHere, we present a high throughput, low-cost screening tool for CTG repeat expansions using TP-PCR followed by high resolution melt curve analysis with saturating concentrations of SYBR GreenER dye.ResultsWe determined that multimodal melt profiles from the TP-PCR assay are a proxy for amplicon length stoichiometry. In a screen of 10,097 newborn blood spots, melt profile analysis accurately reflected the tri-modal distribution of common alleles from 5 to 35 CTG repeats, and identified the premutation and full expansion alleles.ConclusionWe demonstrate that robust detection of expanded CTG repeats in a single tube can be achieved from samples derived from specimens with minimal template DNA such as dried blood spots (DBS). This technique is readily adaptable to large-scale testing programs such as population studies and newborn screening programs.

scholarly journals Two high-throughput screening assays for aberrant RNA–protein interactions in myotonic dystrophy type 1

2012 ◽  
Vol 402 (5) ◽  
pp. 1889-1898 ◽  
Author(s):  
Catherine Z. Chen ◽  
Krzysztof Sobczak ◽  
Jason Hoskins ◽  
Noel Southall ◽  
Juan J. Marugan ◽  
...  
Keyword(s):  
Myotonic Dystrophy ◽  
High Throughput ◽  
Protein Interactions ◽  
High Throughput Screening ◽  
Myotonic Dystrophy Type 1 ◽  
Myotonic Dystrophy Type ◽  
Screening Assays ◽  
Aberrant Rna

scholarly journals Neuropathology does not Correlate with Regional Differences in the Extent of Expansion of CTG Repeats in the Brain with Myotonic Dystrophy Type 1

2010 ◽  
Vol 43 (6) ◽  
pp. 149-156 ◽  
Author(s):  
Kyoko Itoh ◽  
Maki Mitani ◽  
Kunihiko Kawamoto ◽  
Naonobu Futamura ◽  
Itaru Funakawa ◽  
...  
Keyword(s):  
Myotonic Dystrophy ◽  
Regional Differences ◽  
Myotonic Dystrophy Type 1 ◽  
Myotonic Dystrophy Type ◽  
Ctg Repeats ◽  
The Brain

scholarly journals Inhibition of Postn Rescues Myogenesis Defects in Myotonic Dystrophy Type 1 Myoblast Model

2021 ◽  
Vol 9 ◽  
Author(s):  
Xiaopeng Shen ◽  
Zhongxian Liu ◽  
Chunguang Wang ◽  
Feng Xu ◽  
Jingyi Zhang ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Myotonic Dystrophy ◽  
Cell Model ◽  
Neutralizing Antibody ◽  
Underlying Mechanism ◽  
Myotonic Dystrophy Type 1 ◽  
Myotonic Dystrophy Type ◽  
Ctg Repeats ◽  
Myoblast Cells

Myotonic dystrophy type 1 (DM1) is an inherited neuromuscular disease caused by expanded CTG repeats in the 3′ untranslated region (3′UTR) of the DMPK gene. The myogenesis process is defective in DM1, which is closely associated with progressive muscle weakness and wasting. Despite many proposed explanations for the myogenesis defects in DM1, the underlying mechanism and the involvement of the extracellular microenvironment remained unknown. Here, we constructed a DM1 myoblast cell model and reproduced the myogenesis defects. By RNA sequencing (RNA-seq), we discovered that periostin (Postn) was the most significantly upregulated gene in DM1 myogenesis compared with normal controls. This difference in Postn was confirmed by real-time quantitative PCR (RT-qPCR) and western blotting. Moreover, Postn was found to be significantly upregulated in skeletal muscle and myoblasts of DM1 patients. Next, we knocked down Postn using a short hairpin RNA (shRNA) in DM1 myoblast cells and found that the myogenesis defects in the DM1 group were successfully rescued, as evidenced by increases in the myotube area, the fusion index, and the expression of myogenesis regulatory genes. Similarly, Postn knockdown in normal myoblast cells enhanced myogenesis. As POSTN is a secreted protein, we treated the DM1 myoblast cells with a POSTN-neutralizing antibody and found that DM1 myogenesis defects were successfully rescued by POSTN neutralization. We also tested the myogenic ability of myoblasts in the skeletal muscle injury mouse model and found that Postn knockdown improved the myogenic ability of DM1 myoblasts. The activity of the TGF-β/Smad3 pathway was upregulated during DM1 myogenesis but repressed when inhibiting Postn with a Postn shRNA or a POSTN-neutralizing antibody, which suggested that the TGF-β/Smad3 pathway might mediate the function of Postn in DM1 myogenesis. These results suggest that Postn is a potential therapeutical target for the treatment of myogenesis defects in DM1.

scholarly journals Molecular Genetics and Genetic Testing in Myotonic Dystrophy Type 1

2013 ◽  
Vol 2013 ◽  
pp. 1-13 ◽  
Author(s):  
Dušanka Savić Pavićević ◽  
Jelena Miladinović ◽  
Miloš Brkušanin ◽  
Saša Šviković ◽  
Svetlana Djurica ◽  
...  
Keyword(s):  
Genetic Testing ◽  
Molecular Genetics ◽  
Myotonic Dystrophy ◽  
Age At Onset ◽  
Gender Effect ◽  
Myotonic Dystrophy Type 1 ◽  
Myotonic Dystrophy Type ◽  
Ctg Repeats ◽  
Parental Gender

Myotonic dystrophy type 1 (DM1) is the most common adult onset muscular dystrophy, presenting as a multisystemic disorder with extremely variable clinical manifestation, from asymptomatic adults to severely affected neonates. A striking anticipation and parental-gender effect upon transmission are distinguishing genetic features in DM1 pedigrees. It is an autosomal dominant hereditary disease associated with an unstable expansion of CTG repeats in the 3′-UTR of theDMPKgene, with the number of repeats ranging from 50 to several thousand. The number of CTG repeats broadly correlates with both the age-at-onset and overall severity of the disease. Expanded DM1 alleles are characterized by a remarkable expansion-biased and gender-specific germline instability, and tissue-specific, expansion-biased, age-dependent, and individual-specific somatic instability. Mutational dynamics in male and female germline account for observed anticipation and parental-gender effect in DM1 pedigrees, while mutational dynamics in somatic tissues contribute toward the tissue-specificity and progressive nature of the disease. Genetic test is routinely used in diagnostic procedure for DM1 for symptomatic, asymptomatic, and prenatal testing, accompanied with appropriate genetic counseling and, as recommended, without predictive information about the disease course. We review molecular genetics of DM1 with focus on those issues important for genetic testing and counseling.

Somatic instability of CTG repeats in the cerebellum of myotonic dystrophy type 1

2013 ◽  
Vol 48 (1) ◽  
pp. 105-108 ◽  
Author(s):  
Kenji Jinnai ◽  
Maki Mitani ◽  
Naonobu Futamura ◽  
Kunihiko Kawamoto ◽  
Itaru Funakawa ◽  
...  
Keyword(s):  
Myotonic Dystrophy ◽  
Myotonic Dystrophy Type 1 ◽  
Myotonic Dystrophy Type ◽  
Ctg Repeats ◽  
Somatic Instability

Expanded CTG repeats trigger miRNA alterations in Drosophila that are conserved in myotonic dystrophy type 1 patients

2012 ◽  
Vol 22 (4) ◽  
pp. 704-716 ◽  
Author(s):  
Juan M. Fernandez-Costa ◽  
Amparo Garcia-Lopez ◽  
Sheila Zuñiga ◽  
Victoria Fernandez-Pedrosa ◽  
Amelia Felipo-Benavent ◽  
...  
Keyword(s):  
Myotonic Dystrophy ◽  
Myotonic Dystrophy Type 1 ◽  
Myotonic Dystrophy Type ◽  
Ctg Repeats

scholarly journals The Need for Establishing a Universal CTG Sizing Method in Myotonic Dystrophy Type 1

Genes ◽  
2020 ◽  
Vol 11 (7) ◽  
pp. 757 ◽  
Author(s):  
Alfonsina Ballester-Lopez ◽  
Ian Linares-Pardo ◽  
Emma Koehorst ◽  
Judit Núñez-Manchón ◽  
Guillem Pintos-Morell ◽  
...  
Keyword(s):  
Myotonic Dystrophy ◽  
Accurate Determination ◽  
Disease Onset ◽  
Clinical Severity ◽  
Myotonic Dystrophy Type 1 ◽  
Myotonic Dystrophy Type ◽  
Ctg Repeats ◽  
Long Pcr ◽  
Ctg Expansion

The number of cytosine-thymine-guanine (CTG) repeats (‘CTG expansion size’) in the 3′untranslated region (UTR) region of the dystrophia myotonica-protein kinase (DMPK) gene is a hallmark of myotonic dystrophy type 1 (DM1), which has been related to age of disease onset and clinical severity. However, accurate determination of CTG expansion size is challenging due to its characteristic instability. We compared five different approaches (heat pulse extension polymerase chain reaction [PCR], long PCR-Southern blot [with three different primers sets—1, 2 and 3] and small pool [SP]-PCR) to estimate CTG expansion size in the progenitor allele as well as the most abundant CTG expansion size, in 15 patients with DM1. Our results indicated variability between the methods (although we found no overall differences between long PCR 1 and 2 and SP-PCR, respectively). While keeping in mind the limited sample size of our patient cohort, SP-PCR appeared as the most suitable technique, with an inverse significant correlation found between CTG expansion size of the progenitor allele, as determined by this method, and age of disease onset (r = −0.734, p = 0.016). Yet, in light of the variability of the results obtained with the different methods, we propose that an international agreement is needed to determine which is the most suitable method for assessing CTG expansion size in DM1.

scholarly journals Correction of RNA-Binding Protein CUGBP1 and GSK3β Signaling as Therapeutic Approach for Congenital and Adult Myotonic Dystrophy Type 1

2019 ◽  
Vol 21 (1) ◽  
pp. 94 ◽  
Author(s):  
Lubov Timchenko
Keyword(s):  
Myotonic Dystrophy ◽  
Complex Disease ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Myotonic Dystrophy Type 1 ◽  
Myotonic Dystrophy Type ◽  
Ctg Repeats ◽  
Toxic Rna

Myotonic dystrophy type 1 (DM1) is a complex genetic disease affecting many tissues. DM1 is caused by an expansion of CTG repeats in the 3′-UTR of the DMPK gene. The mechanistic studies of DM1 suggested that DMPK mRNA, containing expanded CUG repeats, is a major therapeutic target in DM1. Therefore, the removal of the toxic RNA became a primary focus of the therapeutic development in DM1 during the last decade. However, a cure for this devastating disease has not been found. Whereas the degradation of toxic RNA remains a preferential approach for the reduction of DM1 pathology, other approaches targeting early toxic events downstream of the mutant RNA could be also considered. In this review, we discuss the beneficial role of the restoring of the RNA-binding protein, CUGBP1/CELF1, in the correction of DM1 pathology. It has been recently found that the normalization of CUGBP1 activity with the inhibitors of GSK3 has a positive effect on the reduction of skeletal muscle and CNS pathologies in DM1 mouse models. Surprisingly, the inhibitor of GSK3, tideglusib also reduced the toxic CUG-containing RNA. Thus, the development of the therapeutics, based on the correction of the GSK3β-CUGBP1 pathway, is a promising option for this complex disease.

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