Article

Expanded CTG repeat demarcates a boundary for abnormal CpG methylation in myotonic dystrophy patient tissues

Genetics and Genome Biology, Department of Pediatrics, The Hospital for Sick Children, and Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
Human Molecular Genetics (Impact Factor: 6.39). 11/2010; 20(1):1-15. DOI: 10.1093/hmg/ddq427
Source: PubMed

ABSTRACT

Myotonic dystrophy (DM1) affects multiple organs, shows age-dependent progression and is caused by CTG expansions at the DM1 locus. We determined the DM1 CpG methylation profile and CTG length in tissues from DM1 foetuses, DM1 adults, non-affected individuals and transgenic DM1 mice. Analysis included CTCF binding sites upstream and downstream of the CTG tract, as methylation-sensitive CTCF binding affects chromatinization and transcription of the DM1 locus. In humans, in a given foetus, expansions were largest in heart and smallest in liver, differing by 40-400 repeats; in adults, the largest expansions were in heart and cerebral cortex and smallest in cerebellum, differing by up to 5770 repeats in the same individual. Abnormal methylation was specific to the mutant allele. In DM1 adults, heart, liver and cortex showed high-to-moderate methylation levels, whereas cerebellum, kidney and skeletal muscle were devoid of methylation. Methylation decreased between foetuses and adults. Contrary to previous findings, methylation was not restricted to individuals with congenital DM1. The expanded repeat demarcates an abrupt boundary of methylation. Upstream sequences, including the CTCF site, were methylated, whereas the repeat itself and downstream sequences were not. In DM1 mice, expansion-, tissue- and age-specific methylation patterns were similar but not identical to those in DM1 individuals; notably in mice, methylation was present up- and downstream of the repeat, but greater upstream. Thus, in humans, the CpG-free expanded CTG repeat appears to maintain a highly polarized pattern of CpG methylation at the DM1 locus, which varies markedly with age and tissues.

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Available from: Arturo López Castel, Feb 19, 2015
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    • "As a result of disrupting the function of these proteins, several mis-splicing defects have been described and have been linked to specific symptoms of the disease (Mankodi et al., 2002; Savkur et al., 2001; Tang et al., 2012; Fugier et al., 2011). However, the physiological consequences of alternative splicing, gene expression and microRNA alterations in the heart are yet to be clarified (Phillips et al., 1999; Rau et al., 2011; Kalsotra et al., 2014; Zu et al., 2011; Lopez Castel et al., 2011; Moseley et al., 2006; Perbellini et al., 2011; Fernandez-Costa et al., 2013; Wang et al., 2015). In general, cardiac involvement, which often precedes the skeletal muscle one, occurs in 80% of individuals with DM1 and represents the second most common cause of death of such individuals, after respiratory failure (Vinereanu et al., 2004). "
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    ABSTRACT: Up to 80% of myotonic dystrophy type 1 (DM1) patients will develop cardiac abnormalities at some point during the progression of their disease. The most common of them are varying degrees of heart blockage characterized by conduction defects and supraventricular and ventricular tachycardia, resulting in a high risk of sudden cardiac death. Despite its importance, very few animal model studies have focused on the heart dysfunction in DM1. Here, we describe the characterization of the heart phenotype in a Drosophila model expressing pure expanded CUG repeats under the control of the cardiomyocyte-specific driver GMH5-Gal4. Morphologically, expression of 250 CUG repeats caused abnormalities in the parallel alignment of the spiral myofibrils in dissected fly hearts revealed by phalloidin staining. Moreover, combined immunofluorescence and in situ hybridization of Muscleblind and CUG repeats, respectively, confirmed detectable ribonuclear foci and Muscleblind sequestration, characteristic features of DM1, exclusively in flies expressing the expanded CTG repeats. Similarly to what has been reported in human DM1 patients, heart-specific expression of toxic RNA resulted in reduced survival, increased arrhythmia, altered diastolic and systolic function and reduced heart tube diameters and contractility in the model flies. As a proof of concept that the fly heart model can be used for in vivo testing of promising therapeutic compounds, we fed flies with pentamidine, a compound previously described to improve DM1 phenotypes. Pentamidine not only released Muscleblind and reduced ribonuclear formation in the Drosophila heart but rescued heart arrhythmicity and contractility, and improved fly survival in animals expressing 250 CUG repeats.
    Full-text · Article · Oct 2015 · Disease Models and Mechanisms
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    • "This study suggested that hypermethylation would be age dependent , being higher in fetal vs adult tissues, and also documented inter and intra-individual variability in methylation levels not obviously related to the size of n(CTG) present in tissues. On the other hand, expression of DMPK and of adjacent genes in correlation to methylation status was not assessed in this study [22]. The CTG repeat at DMPK locus is usually uninterrupted in both healthy and expanded alleles, however so called " variant " repeats containing unstable CCG, CTC and CGG sequence interruptions at the 3′-end of the CTG array can be detected in a low percentage (3–5% of cases) of DM1 patients [23] [24] [25] [26]. "
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    ABSTRACT: A differential CpGmethylation profile upstreamof the expanded CTG array at the DMPK locus has been reported in patientswithmyotonic dystrophy type 1 (DM1), suggesting that hypermethylationmightmodulate DM1 phenotype, possibly affecting expression levels of DMPK and/or flanking genes. To clarify this issue,we characterized by methylation sensitive high resolution melting (MS-HRM) the CpG methylation pattern of DNA sequences flanking the pathological CTG expansion in 13 childhood-onset, 37 juvenile/adult-onset, 7 congenital DM1 pa- tients carrying uninterrupted CTG expansions and in 9 DM1 patients carrying variant expansions vs 30 controls. Association of methylation status with disease features (nCTG, age, sex, MIRS, disease duration) was also assessed. Finally, DMPK and SIX5 expression levels were evaluated in leukocytes from controls, methylated and unmethylated DM1 patients. We found hypermethylation involving upstreamsequences of DM1 locus in patientswith uninterrupted CTG ex- pansions N1000 CTG and affected by a congenital or childhood onset form. Besides the n(CTG) and early disease onset, hypermethylation was also significantly associated with maternal transmission. On the other hand, hypermethylation involved the 3' of the CTG array in DM1 patients carrying variant expan-sions. DMPK and SIX5 expression did not significantly differ in methylated vs unmethylated DM1 patients. Our results suggest that either the inherited size of the expanded allele and the presence of interruptions at the 3' end are associated with a highly polarized pattern of CpGmethylation at the DM1 locus and that, at least in leukocytes, DM1 locus hypermethylation would not significantly affect DMPK or SIX5 expression.
    Full-text · Article · Sep 2015 · Biochimica et Biophysica Acta
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    • "Moreover, they are consistent with the negative cis effect of large CTG expansions on the induction of hypermethylation in transgenic mice (Brouwer et al., 2013; Ló pez Castel et al., 2011), pointing to a role for chromatin modification in DM1 pathogenesis. However, it should be noted that hypermethylation patterns in transgenic mice are similar, but not identical, to those observed in DM1 individuals (Ló pez Castel et al., 2011) and that, in contrast to our study, the reduced expression in SIX5 is uncorrelated with expansion size in transgenic mice (Brouwer et al., 2013). The discrepancies between both studies may stem from epigenetic variations that exist between species or may be attributed to transgene integration site effects. "

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