Edited by: Stojan Z. Peric, University of Belgrade, Serbia
Reviewed by: Giovanni Meola, University of Milan, Italy; Ralf Krahe, University of Texas MD Anderson Cancer Center, United States
†These authors have contributed equally to this work and share first authorship
This article was submitted to Neurogenomics, a section of the journal Frontiers in Neuroscience
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Myotonic dystrophy type 1 (DM1) is an autosomal dominant multisystemic disorder caused by unstable CTG-repeat expansions in the
Myotonic dystrophy type 1 (DM1) is caused by CTG-repeat expansions in the 3′ UTR of the
Up to 35 CTG triplets in blood derived DNA are normal, a repeat length between 35 and 49 is considered to be a premutation (
Clinical testing for DM1 is challenging due to the nature of the mutation. The standard method to detect DM1 repeat expansions is still Southern blot of genomic DNA, which is usually performed on DNA isolated from leukocytes (
To date there is no therapy available for DM1. The most advanced approach, an antisense–oligonucleotide treatment used for post–transcriptional silencing of
Considering the predominant muscle involvement in DM1, the usage of primary human myoblasts has several potential advantages. As they can be obtained from several patients, a number of those cultures can be considered to stratify phenotypic variability observed in DM1 patients. Furthermore, they differentiate into myotubes, thus shifting the gene expression profile toward the mature muscle, and proliferating cells allow to investigate cell cycle effects. The latter is also a disadvantage, primary cells enter into replicative senescence after a define number of divisions which is inversely correlated with the age of the donor (
Here we describe the characterization of the extended CTG–repeat in primary human myoblasts cultures gained from adult onset DM1 patients, using an adaption of a small–pool PCR/Southern blot protocol (
Primary human myoblasts (
Primary myoblast cultures used in this study.
Phenotype | Age of onset | Age at biopsy | Sex | Source of the muscle biopsy | CTG–repeat length in |
||
Blood | Muscle | ||||||
DM1–1 | DM1 | 3rd decade | 42 | ♂ | Biceps brachii muscle | 50–70 | |
DM1–2 | DM1 | 2nd decade | 33 | ♀ | Unknown | 300–500 | |
DM1–3 | DM1 | 2nd decade | 34 | ♂ | Deltoid muscle | 240–430 | ∼600 |
Con–1 | Unaffected | 43 | ♂ | Biceps brachii muscle | – | ||
Con–2 | Unaffected | 36 | ♀ | Tibialis anterior muscle | – |
Primary human myoblast from DM1 patients were obtained from patients with adult onset (
Myoblasts were grown at 37°C with 5% CO2 in 10 cm tissue culture plates. For cell growth skeletal muscle cell growth medium (PeloBiotech, Munich, Germany), supplemented with 40 U/ml Penicillin and 0.04 mg/ml Streptomycin was used, cells were kept from reaching confluency by splitting at a density of about 80%. Myoblasts were harvested between passages 6 and 8.
To obtain DNA from the primary myoblast cultures cells were trypsinized until they detached from the plate. Cells were pelleted by centrifugation (260 rcf, 5 min) and the supernatant was discarded. The cell pellet was washed twice with PBS, each washing step followed by centrifugation (16,100 rcf for 1 min) and stored at −80°C till further processing. For DNA isolation the Quick—DNATM Miniprep Plus Kit (ZymoResearch) was used according to the manufacturer’s instructions. DNA concentration was measured by a Nanodrop (Thermo Fisher Scientific).
To generate a marker that allowed us to measure the number of CTG-repeats we first amplified a 171 bp DNA fragment, containing 20 CTG repeats, using the DM-C and DM-DR primer published by
Plasmids used to create the size marker by restriction digest with endonucleases and the resulting sizes of the CTG-repeat containing fragments.
Dig-probes were designed to directly target the repeat and ordered from Eurofins. The probes were labeled 5′ and 3′ with digoxigenin (DIG). Following sequences were used for the sense and antisense strand probes:
5′-[DIG]-GAATGCTGCTGCTGCTGCTGCTGCTGCTG CTG-[DIG]-3′
5′-[DIG]-CAGCAGCAGCAGCAGCAGCAGCAGCAGC ATTC-[DIG]-3′.
Amplification of the repeat containing DNA was modified after the original protocol by
Forward primer: 5′-CAGTTCACAACCGCTCCGAGC-3′
Reverse primer: 5′-CGTGGAGGATGGAACACGGAC-3′
Following settings were used for the PCR:
∗Time increment of 15 s per cycle.
The PCR was performed in a Biometra Tadvanced thermocycler (Analytik Jena).
The PCR products were size-separated by gel electrophoresis. For this a 1% agarose gel of 14 cm length was prepared using Tris-acetate-EDTA (TAE) buffer. The gel was loaded with 30 μl of PCR product mixed with 10 μl of loading buffer. Initial the electrophoresis was run for 10 min at 100 V (volt), followed by 60 min at 140 V.
After the electrophoresis the DNA was transferred via vacuum blot to a nylon membrane (Amersham HybondTM-XL). The gel was washed in ddH2O and placed in depurination buffer for 15 min. After the depurination and a washing step with ddH2O the gel was placed for 30 min in denaturation buffer. Before the gel was equilibrated in 20× SSC buffer for 10 min it was incubated two times in neutralizing buffer for 15 min. The equilibrated membrane (in 20× SSC) was placed on the blotting apparatus (vacuum blot, Analytik Jena) and on top the gel. Stepwise the low vacuum of 90–100 mBar was established. During the blotting phase of 1–1.5 h 20× SSC buffer was constantly added to the top of the gel. Following this, the membrane was equilibrated for 2 min in 2× SSC buffer and then dried for 5 min at 65°C (UVP Hybrilinker Oven, Analytik Jena). Following the drying process, the DNA was cross-linked via UV-light (UVP Hybrilinker Oven, Analytik Jena). Solutions were prepared the following:
Depurination buffer HCl 0.25 M
Denaturation buffer NaCl 1.5 M; NaOH 0.5 M
SSC buffer (20×) NaCl 3 M; Ma-Citrate 0.3 M (pH 7.5)
Neutralizing buffer NaCl 1.5 M; Tris/HCl 0.5 M; EDTA 1 mM (pH 7.2).
The membrane was transferred into a prewarmed hybridization tube with 10 ml PerfectHybTM Plus hybridization buffer (Sigma Aldrich) and equilibrated for 15–30 min. In the meantime, 20 μl of the two probes (9 ng/μl) where incubated with 80 μl ddH2O for 10 min at 100°C. After the denaturation step the probes where immediately placed on ice for 5 min and then transferred into the hybridization solution. After the overnight incubation the membrane was washed for 20 min at 65°C with prewarmed (65°C) washing solution (1× SSC + 0.2% SDS). After the second washing step the membrane was equilibrated for 5 min in wash-buffer (100 mM maleic acid + 150 mM NaCl + 0.3% Tween20 pH 7.5). Blocking solution (Roche DIG-detection Kit) was set up in maleic buffer (100 mM maleic acid + 150 mM NaCl pH 7.5) and then put on the membrane for 30 min. The DIG-antibody conjugate (Roche DIG-detection Kit) was diluted in blocking solution and the membrane was incubated for another 30 min. Following two 15 min washing steps the membrane was equilibrated in detection buffer (Roche DIG-detection Kit) for 5 min. The membrane was incubated for 15 min at 37°C in the detection solution (Roche DIG-detection Kit) before the signals were detected for 10 min using an Odyssey® Fc imaging system (Licor).
To calculate the actual repeat length the image studioTM software (Licor) was used to measure the size of the bands detected in samples based on the size of the marker bands. Based on these data the following formula was used:
x = number of repeats.
y = fragment length (measured using the image studioTM software, Licor).
z = flanking gene sequence (311 bp).
i = number of the repeats within the marker sequence (20 repeats).
Using two control myoblast cultures we could detect small repeats with the calculated sizes of about 20 CTG-repeats for both (
Southern blot for
To test if the repeat length in primary myoblast cultures is comparable to the muscle biopsy they were grown from, we tested material from the cryo-preserved biopsy of DM1-3 which was part of the biopsy the cell line was grown from and compared directly to the myoblast culture (
Southern blot comparing DNA derived from a muscle biopsy and a myoblast culture of the same DM1 patient (both samples taken at the same time). For each lane the amount of used DNA in pg (as input for the small pool PCR) is shown. M = size-marker.
With our modified method, we can successfully identify extended CTG-repeats in primary human myoblast cultures in comparison to DNA extracted from the original muscle specimen it was grown from. We show that there is a range of different repeat lengths in myoblast cultures. Thus, human myoblast cultures reflect rather well the repeat-length of mature muscle. Furthermore, it seems that the repeat length in myoblasts and muscle does, for the patient samples tested, not differ significantly from each other. Consequently, we show that primary human DM1 myoblast cultures are a well-suited model to investigate repeat-length related preclinical aspects of the DM1 pathology and are a useful platform to do first-line treatment interventions.
Presently, there are several distinct methods for the detection of CTG-repeats published (
The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.
The studies involving human participants were reviewed and approved by the Ethical Review Committee at the Ludwig-Maximilians-University, Munich, Germany. The patients/participants provided their written informed consent to participate in this study.
SH, BS, and PM contributed to the conception and design of the experiments. PM and SH wrote the manuscript. SH, RM, and LK performed the experiments. All authors contributed to the article and approved the submitted version.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.