Large CTG Expansions Research Articles

P.269 - Association between mutation size and cardiac involvement in myotonic dystrophy type 1: an analysis of the DM1-heart registry

In myotonic dystrophy type 1 (DM1), the association between mutation size (CTG expansion) and the severity of cardiac involvement is controversial. We selected 855 patients with DM1 (women, 51%; median age, 37 years) with genetic testing performed at the moment of their initial cardiac evaluation, out of 1014 patients included in the DM1-Heart Registry between January 2000 and December 2015. We studied the association between CTG expansion size and other baseline characteristics and (i) cardiac involvement at baseline and (ii) the incidence of death, sudden death, and other cardiac adverse events. At initial presentation, the median CTG expansion size was 530 (interquartile range, 300–830). In multivariate analysis, larger expansions were associated with the presence at baseline of conduction defects on the electrocardiogram and left ventricular systolic dysfunction. In a median 11.5 years follow-up period, 210 patients died (25%), including 32 suddenly (4%). Supraventricular arrhythmias developed over lifetime in 166 patients (19%), sustained ventricular tachyarrhythmias in 17 (2%) and permanent pacemakers were implanted in 181 (21%). In Cox-regression analyses, larger CTG expansions were significantly associated with (i) total death, sudden death, and pacemaker implantation in a model including CTG expansion size, age, gender, diabetes and (ii) all endpoints except sudden death in a model including all baseline characteristics. The size of the CTG expansion in the blood of DM1 patients is associated with total and sudden deaths, conduction defects, left ventricular dysfunction and supraventricular arrhythmias.

Association Between Mutation Size and Cardiac Involvement in Myotonic Dystrophy Type 1: An Analysis of the DM1-Heart Registry.

In myotonic dystrophy type 1, the association between mutation size (CTG expansion) and the severity of cardiac involvement is controversial. We selected 855 patients with myotonic dystrophy type 1 (women, 51%; median age, 37 years), with genetic testing performed at the moment of their initial cardiac evaluation, out of 1014 patients included in the Myotonic Dystrophy Type 1-Heart Registry between January 2000 and December 2015. We studied the association between CTG expansion size and other baseline characteristics and (1) cardiac involvement at baseline and (2) the incidence of death, sudden death, and other cardiac adverse events. At initial presentation, the median CTG expansion size was 530 (interquartile range, 300-830). In multivariate analysis, larger expansions were associated with the presence at baseline of conduction defects on the ECG and left ventricular systolic dysfunction. In a median 11.5 years of follow-up period, 210 patients died (25%), including 32 suddenly (4%). Supraventricular arrhythmias developed over lifetime in 166 patients (19%), sustained ventricular tachyarrhythmias in 17 (2%), and permanent pacemakers were implanted in 181 (21%). In Cox regression analyses, larger CTG expansions were significantly associated with (1) total death, sudden death, and pacemaker implantation in a model, including CTG expansion size, age, sex, diabetes mellitus, and (2) all end points except sudden death in a model including all baseline characteristics. The size of the CTG expansion in the blood of myotonic dystrophy type 1 patients is associated with total and sudden deaths, conduction defects, left ventricular dysfunction, and supraventricular arrhythmias. URL: https://www.clinicaltrials.gov. Unique Identifier: NCT01136330.

Actinomycin D Specifically Reduces Expanded CUG Repeat RNA in Myotonic Dystrophy Models.

Myotonic dystrophy type 1 (DM1) is an inherited disease characterized by the inability to relax contracted muscles. Affected individuals carry large CTG expansions that are toxic when transcribed. One possible treatment approach is to reduce or eliminate transcription of CTG repeats. Actinomycin D (ActD) is a potent transcription inhibitor and FDA-approved chemotherapeutic that binds GC-rich DNA with high affinity. Here, we report that ActD decreased CUG transcript levels in a dose-dependent manner in DM1 cell and mouse models at significantly lower concentrations (nanomolar) compared to its use as a general transcription inhibitor or chemotherapeutic. ActD also significantly reversed DM1-associated splicing defects in a DM1 mouse model, and did so within the currently approved human treatment range. RNA-seq analyses showed that low concentrations of ActD did not globally inhibit transcription in a DM1 mouse model. These results indicate that transcription inhibition of CTG expansions is a promising treatment approach for DM1.

Trinucleotide repeat expansions in human breast cancer-susceptibility genes: relevant targets for aspirin chemoprevention?

Defining novel molecular mechanisms pertinent to aspirin chemoprevention of breast cancer (BC) and to explain controversial epidemiological results in this regard. Literature search in relevant databases with the following key words; aspirin, nucleotide repeat expansions, breast cancer. Human genome contains nucleotide repeat expansions and exon-1 of the androgen receptor gene AR contains a CAG string with an average of 20 repeats. Longer AR CAG repeats associate with lower AR protein functioning leading relatively higher estrogen receptor signals and higher risk of hormone receptor-positive BC. Nucleotide repeat expansions also exist in E2F4 and POLG genes in BC. In cell culture models, aspirin reduces CAG.CTG expansions in kidney cells and restores myogenic differentiation in cells obtained from tissues with myotonic dystrophy, a disorder caused by large CTG expansions. We hypothesize that aspirin reduction of trinucleotide repeat expansions in breast cancer-susceptibility genes may be one of the relevant mechanisms of its chemopreventive effects.

DM1 Patients with Small CTG Expansions are also at Risk of Severe Conduction Abnormalities

A high risk of cardiac arrhythmias was reported in myotonic dystrophy type 1 (DM1). The purpose of the study was to evaluate the risk of severe electrocardiographic abnormalities in DM1 patients with small CTG expansions. We assessed the ECG done at DM1 diagnosis for 127 patients with ≤200 CTG repeats and for 82 of them who had ≥1 ECG over a period of follow-up of 11.7 ± 7.6 years (mean ± SD). Criteria of severe ECG abnormality are at least one of the following features: PR interval ≥240 msec, QRS duration ≥120 msec, second-degree or third-degree atrioventricular block, atrial fibrillation or flutter, insertion of a pacemaker or cardioverter-defibrillator. At baseline, ECG was normal for 109 patients out of 127 (85.8%) and only 4 patients (3.1%) presented severe ECG abnormalities. At follow-up, 46 patients out of 82 (56.1%) had a normal ECG and 25 (30.5%) developed severe ECG abnormalities (p < 0.0001) including 6 of them who needed permanent pacemaker insertion. There were also 3 sudden deaths during the follow-up period. Using multivariate Cox regression analysis, age at entry (relative risk RR, 1.05; (95% CI 1.01-1.08; p = 0.012) and muscular weakness (MIRS) at the entry (RR, 2.03; 95% CI 1.28-3.22; p = 0.003) were significant risk factors for the development of severe ECG abnormality. An increased risk of severe ECG abnormalitiesy and cardiac events is observed even in DM1 patients with small CTG expansions and warrants close cardiac follow-up similar to DM1 patients with larger CTG expansions.

Perfil cardiovascular na distrofia muscular miotónica tipo 1: estudo de uma série de casos seguida num centro especializado

IntroductionMyotonic dystrophy is a multisystem disease associated with cardiac abnormalities that are responsible for high morbidity and mortality. It commonly affects conduction tissue, resulting in changes in heart rate that tend to progress with age. ObjectiveThe aim of the study was to assess overall cardiovascular risk and the risk of arrhythmias in patients with myotonic dystrophy type 1 (DM‐1) and to correlate them with genetic study (CTG expansion size). MethodsThis retrospective study included 31 DM‐1 patients referred to the cardiology department of Centro Hospitalar Entre Douro e Vouga by the neurology department for screening for heart disease. Patients’ medical records were consulted for the diagnostic tests performed in the diagnostic cardiology consultation: electrocardiogram (ECG), high‐resolution ECG, heart rate variability (HRV), Holter 24‐hour ambulatory ECG and transthoracic echocardiogram (TTE); results of genetic testing were also consulted. ResultsOf 31 patients studied, 38% had first‐degree atrioventricular block (AVB) and 51% had intraventricular conduction disturbances (62% had late potentials). TTE revealed no structural heart disease. Rare supraventricular and ventricular ectopic beats were the most common arrhythmias on 24‐hour Holter monitoring. The sample showed lower HRV, reflecting vagal dysfunction. Patients with larger CTG expansions had more cardiac abnormalities. ConclusionsPatients with DM‐1 had arrhythmic events, with AVB and more significantly intraventricular block, although none had malignant arrhythmias or structural heart disease. No patient died. Patients with larger CTG expansions had greater involvement of cardiac conduction tissue.

Cardiovascular profile in myotonic dystrophy type 1: Analysis of a case series in a specialized center

IntroductionMyotonic dystrophy is a multisystem disease associated with cardiac abnormalities that are responsible for high morbidity and mortality. It commonly affects conduction tissue, resulting in changes in heart rate that tend to progress with age. ObjectiveThe aim of the study was to assess overall cardiovascular risk and the risk of arrhythmias in patients with myotonic dystrophy type 1 (DM-1) and to correlate them with genetic study (CTG expansion size). MethodsThis retrospective study included 31 DM-1 patients referred to the cardiology department of Centro Hospitalar Entre Douro e Vouga by the neurology department for screening for heart disease. Patients’ medical records were consulted for the diagnostic tests performed in the diagnostic cardiology consultation: electrocardiogram (ECG), high-resolution ECG, heart rate variability (HRV), Holter 24-hour ambulatory ECG and transthoracic echocardiogram (TTE); results of genetic testing were also consulted. ResultsOf 31 patients studied, 38% had first-degree atrioventricular block (AVB) and 51% had intraventricular conduction disturbances (62% had late potentials). TTE revealed no structural heart disease. Rare supraventricular and ventricular ectopic beats were the most common arrhythmias on 24-hour Holter monitoring. The sample showed lower HRV, reflecting vagal dysfunction. Patients with larger CTG expansions had more cardiac abnormalities. ConclusionsPatients with DM-1 had arrhythmic events, with AVB and more significantly intraventricular block, although none had malignant arrhythmias or structural heart disease. No patient died. Patients with larger CTG expansions had greater involvement of cardiac conduction tissue.

Aberrant splicing and expression of the non muscle myosin heavy-chain gene MYH14 in DM1 muscle tissues

Myotonic dystrophy type 1 (DM1) is a complex multisystemic disorder caused by an expansion of a CTG repeat located at the 3′ untranslated region (UTR) of DMPK on chromosome 19q13.3. Aberrant messenger RNA (mRNA) splicing of several genes has been reported to explain some of the symptoms of DM1 including insulin resistance, muscle wasting and myotonia. In this paper we analyzed the expression of the MYH14 mRNA and protein in the muscle of DM1 patients (n=12) with different expansion lengths and normal subjects (n=7). The MYH14 gene is located on chromosome 19q13.3 and encodes for one of the heavy chains of the so called class II “nonmuscle” myosins (NMHCII). MYH14 has two alternative spliced isoforms: the inserted isoform (NMHCII-C1) which includes 8 amino acids located in the globular head of the protein, not encoded by the non inserted isoform (NMHCII-C0). Results showed a splicing unbalance of the MYH14 gene in DM1 muscle, with a prevalent expression of the NMHCII-C0 isoform more marked in DM1 patients harboring large CTG expansions. Minigene assay indicated that levels of the MBNL1 protein positively regulates the inclusion of the MYH14 exon 6. Quantitative analysis of the MYH14 expression revealed a significant reduction in the DM1 muscle samples, both at mRNA and protein level. No differences were found between DM1 and controls in the skeletal muscle localization of MYH14, obtained through immunofluorescence analysis. In line with the thesis of an “RNA gain of function” hypothesis described for the CTG mutation, we conclude that the alterations of the MYH14 gene may contribute to the DM1 molecular pathogenesis.

Abnormal prostaglandin E2 production blocks myogenic differentiation in myotonic dystrophy

The congenital form of myotonic dystrophy type 1 (DM1) is the most severe type of the disease associated with CTG expansions over 1500 repeats and delayed muscle maturation. The mechanistic basis of the congenital form of DM1 is mostly unknown. Here, we show that muscle satellite cells bearing large CTG expansions (>3000) secrete a soluble factor that inhibits the fusion of normal myoblasts in culture. We identified this factor as prostaglandin E2 (PGE2). In these DM1 cells, PGE2 production is increased through up-regulation of cyclooxygenase 2 (Cox-2), mPGES-1 and prostaglandin EP2/EP4 receptors. Elevated levels of PGE2 inhibit myogenic differentiation by decreasing the intracellular levels of calcium. Exogenous addition of acetylsalicylic acid, an inhibitor of Cox enzymes, abolishes PGE2 abnormal secretion and restores the differentiation of DM1 muscle cells. These data indicate that the delay in muscle maturation observed in congenital DM1 may result, at least in part, from an altered autocrine mechanism. Inhibitors of prostaglandin synthesis may thus offer a powerful method to restore the differentiation of DM1 muscle cells.

Large CTG Repeats Trigger p16-Dependent Premature Senescence in Myotonic Dystrophy Type 1 Muscle Precursor Cells

A CTG repeat amplification is responsible for the dominantly inherited neuromuscular disorder, myotonic dystrophy type 1 (DM1), which is characterized by progressive muscle wasting and weakness. The expanded (CTG)n tract not only alters the myogenic differentiation of the DM1 muscle precursor cells but also reduces their proliferative capacity. In this report, we show that these muscle precursor cells containing large CTG expansion sequences have not exhausted their proliferative capacity, but have entered into premature senescence. We demonstrate that an abnormal accumulation of p16 is responsible for this defect because the abolition of p16 activity overcomes early growth arrest and restores an extended proliferative capacity. Our results suggest that the accelerated telomere shortening measured in DM1 cells does not contribute to the aberrant induction of p16. We propose that a cellular stress related to the amplified CTG repeat promotes premature senescence mediated by a p16-dependent pathway in DM1 muscle precursor cells. This mechanism is responsible for the reduced proliferative capacity of the DM1 muscle precursor cells and could participate in both the impaired regeneration and atrophy observed in the DM1 muscles containing large CTG expansions.

Changes in Myotonic Dystrophy Protein Kinase Levels and Muscle Development in Congenital Myotonic Dystrophy

Myotonic dystrophy (DM1) is caused by the expansion of a CTG repeat in the noncoding region of a protein kinase, DMPK, expressed in skeletal and cardiac muscles. The aim of the present study was to determine the effects of very large CTG expansions on DMPK expression and skeletal muscle development. In fetuses suffering from the severe congenital form of DM1 with large CTG expansions (1800 to 3700 repeats), the skeletal muscle level of DMPK was reduced to 57% of control levels and a similar reduction was observed in cultured DM1 muscle cells relative to control cultures. These results are consistent with greatly reduced DMPK expression from the mutant allele and normal expression from the unaffected allele in this autosomal dominant disorder. In normal fetuses, DMPK protein levels increased dramatically between 9 and 16 weeks and remained high throughout the remaining gestation period. DM1 fetuses showed impaired skeletal muscle development, characterized by a persistence of embryonic and fetal myosin heavy chains and almost total absence of slow myosin heavy chains at the end of gestation. DMPK expression, however, was similar in both fast and slow fibers from normal adult muscle. The reduced DMPK and the delayed slow fiber maturation in congenital DM1 may be two separate consequences of nuclear retention of DMPK RNA transcripts with expanded CUG repeats.

Chromosomal localization of long trinucleotide repeats in the human genome by fluorescence in situ hybridization.

Trinucleotide microsatellites are widespread in the human and other mammalian genomes. Expansions of unstable trinucleotide repeats have been associated so far with a number of different genetic diseases including fragile X, myotonic dystrophy (DM) and Huntington disease. While ten possible trinucleotides can occur at the DNA level, only CTG and CCG repeats are involved in the disorders described so far. However, the repeat expansion detection (RED) technique has identified additional large repeats of ATG, CCT, CTT, and TGG of potentially pathological significance in the human genome. We now show that conclusive information about the chromosomal localization of long trinucleotide repeats can be achieved in a relatively short time using fluorescence in situ hybridization (FISH) with biotin-labelled trinucleotide polymers. Large CTG expansions (> 1 kb) in DM and an unstable (CTG)306 repeat in a patient with schizophrenia were detected by eye through the microscope without electronic enhancement. Digital imaging was used to analyse the chromosomal distribution of long CCA and AGG repeats. Our results suggest that long trinucleotide repeats occur in the normal human genome and that the size of individual repeat loci may be polymorphic.

Myotonic dystrophy patients have larger CTG expansions in skeletal muscle than in leukocytes

The genetic basis of myotonic dystrophy is an unstable expansion of CTG repeats located in a gene on chromosome 19 that encodes a putative serine/threonine protein kinase. We studied the somatic mosaicism of the (CTG)n expansion in myotonic dystrophy patients. (CTG)n expansions were 2- to 13-fold greater in DNA isolated from skeletal muscle than in DNA from leukocytes in 10 of 11 patients with myotonic dystrophy. Different muscles of the same individual showed similar (CTG)n expansions. In postmortem tissues from an adult patient, (CTG)n expansions in brain, skeletal muscle, cardiac muscle, testes, and liver were all greater than in leukocytes. Normal myotonic dystrophy gene alleles from 7 healthy subjects had the same number of CTG repeats in leukocytes and muscle. The myotonic dystrophy mutation displays pronounced heterogeneity in somatic cells. The (CTG)n expansion observed in peripheral blood leukocytes is not necessarily representative of the repeat expansion in affected tissues, such as skeletal muscle and myocardium. In some patients with myotonic dystrophy, the predictive value of genetic analysis based on leukocyte DNA may be limited.