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

Abstract

<jats:p>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.</jats:p>

Senescence plays a role in myotonic dystrophy type 1

“…See Table 4 for summary of donor subject and patient characteristics. To measure the CTG expansion size in fibroblasts, we performed small pool-PCR, described elsewhere (61). Some modifications were applied: 1) fragments were amplified with Long Amp® Taq (New England Biolabs; Ipswich, MA, USA) supplemented with 2% DMSO and 0,1% Tween20; and 2) DNA fragments were resolved by electrophoresis on a 1% agarose gel, followed by southern blot.…”

An Overview of Alternative Splicing Defects Implicated in Myotonic Dystrophy Type I

“…In recent years, interruptions at the 5′ and 3′ ends of the CUG tract of pathological DMPK transcripts have been described in 3–5% of DM1 patients [ 11 , 12 ]. Although these sequences are mainly composed by unstable CCG interruptions, CGG, CTC and CAG interruptions have also been reported, suggesting a mechanism of phenotypical variability, although further characterization studies are needed [ 11 , 12 , 13 , 14 ]. It is important to highlight that inherited mutation length increases during an affected individual lifespan, particularly in differentiated cells.…”

An Integrative Analysis of DNA Methylation Pattern in Myotonic Dystrophy Type 1 Samples Reveals a Distinct DNA Methylation Profile between Tissues and a Novel Muscle-Associated Epigenetic Dysregulation

“…They included three extra patients and two controls. All samples were obtained at the same time and processed as described previously by Ballester-López and collaborators [ 32 ]. Cell isolation and subsequent culturing was performed as previously described by Koehorst and collaborators [ 33 ].…”

“…Genomic DNA was isolated from peripheral blood by the use of the QIAamp DNA mini kit (Qiagen, Hilden, Germany), the PureLink genomic DNA mini kit (Thermo Fisher Scientific, Waltham, MA, USA) or a simple salting procedure, as previously described by Miller and collaborators [ 34 ]. Genomic DNA from muscle and skin tissue was extracted as previously described by Ballester-López and collaborators [ 32 ].…”