Molecular genetics of congenital myotonic dystrophy

Molecular genetics of congenital myotonic dystrophy

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Neurobiology of Disease xxx (xxxx) xxxx

Contents lists available at ScienceDirect

Neurobiology of Disease journal homepage: www.elsevier.com/locate/ynbdi

Review

Molecular genetics of congenital myotonic dystrophy Stella Lannia, Christopher E. Pearsona,b,



a Program of Genetics & Genome Biology, The Hospital for Sick Children, The Peter Gilgan Centre for Research and Learning, 686 Bay Street, Toronto M5G 0A4, Ontario, Canada b University of Toronto, Program of Molecular Genetics, Canada

A R T I C LE I N FO

A B S T R A C T

Keywords: Myotonic Dystrophy type 1 Congenital Myotonic Dystrophy Repeat instability Parent-of-origin effect DNA methylation Epigenetics

Myotonic Dystrophy type 1 (DM1) is a neuromuscular disease showing strong genetic anticipation, and is caused by the expansion of a CTG repeat tract in the 3′-UTR of the DMPK gene. Congenital Myotonic Dystrophy (CDM1) represents the most severe form of the disease, with prenatal onset, symptoms distinct from adult onset DM1, and a high rate of perinatal mortality. CDM1 is usually associated with very large CTG expansions, but this correlation is not absolute and cannot explain the distinct clinical features and the strong bias for maternal transmission. This review focuses upon the molecular and epigenetic factors that modulate disease severity and might be responsible for CDM1. Changes in the epigenetic status of the DM1 locus and in gene expression have recently been observed. Increasing evidence supports a role of a CTCF binding motif as a cis-element, upstream of the DMPK CTG tract, whereby CpG methylation of this site regulates the interaction of the insulator protein CTCF as a modulating trans-factor responsible for the inheritance and expression of CDM1.

1. Myotonic dystrophy type 1: repeat size and disease subtypes Myotonic Dystrophy type 1 (DM1; OMIM# 160900) is a multisystemic disorder due to the expansion of the trinucleotide CTG repeat in the 3′ untranslated region of the serine-threonine kinase gene DMPK on chromosome 19q13.3 (Aslanidis et al., 1992; Brook et al., 1992; Mahadevan et al., 1992). The expanded DNA is transcribed to form CUG expanded RNAs that sequester RNA processing factors, like MBNL proteins, resulting in splicing defects (Batra et al., 2014; Masuda et al., 2012; Wang et al., 2012). In the non-affected population the DMPK CTG repeat length is between 7 and 37 CTG repeats, DM1 families inherit an expanded tract of 50–6500 repeats, with disease age-of-onset and progression rates varying with the inherited CTG tract length (Lopez Castel et al., 2010). The expanded repeat is genetically unstable and tends to expand further during transmission leading to a decrease in the age of disease onset and an increase in disease severity from parents to the offspring (genetic anticipation)(Ashizawa et al., 1992; Tsilfidis et al., 1992). The CTG tract continues to expand during the lifetime of an individual, inducing worsening of the disease symptoms with age (Lopez Castel et al., 2010). The rate of somatic instability varies in different tissues of the same individual with differences of up to 6000 CTG repeats, and larger expansions in the most affected tissues, such as the heart, muscle and brain (Ashizawa et al., 1993; Jansen et al., 1994;

Lopez Castel et al., 2011b). Apart from the myopathy and the progressive muscle weakness, DM1 also presents with a variety of other symptoms, including heart failure, insulin resistance, sleep disorders and cognitive deficits. The clinical aspects of CDM1 have recently been reviewed (Berggren et al., 2018; Campbell et al., 2013; Johnson et al., 2016a; Johnson et al., 2016b; Prasad et al., 2016; Pucillo et al., 2017; Zapata-Aldana et al., 2018). Based on the onset of the main symptoms, five clinical subtypes of DM1 are recognized: congenital (CDM1), infantile, juvenile, adult, and late-onset DM1 (Aslanidis et al., 1992; De Antonio et al., 2016) (Fig. 1). The inheritance of a larger repeat expansion is thought to be the cause of the different clinical manifestations, nonetheless some overlap in the repeat size ranges has been observed in the different DM1 subtypes (Barbe et al., 2017; De Antonio et al., 2016; Lagrue et al., 2019) (Fig. 1). Adult-onset DM1 patients with very large expansions (> 1000 CTG) have been described, as well as, congenital patients with a shorter CTG tract (Barbe et al., 2017; Barcelo et al., 1994; Clark et al., 1998; Cobo et al., 1993; Lopez de Munain et al., 1995; Spranger et al., 1999). Ongoing somatic expansions throughout the lifetime of DM1 and CDM1 individuals may determine a bias for an over- and under-estimate of the CTG repeat length in late-onset/adult DM1 patients and infantile/ CMD1 individuals, especially since the former are often sampled at older ages while the latter are typically sampled at birth (Joseph et al.,

⁎ Corresponding author at: Program of Genetics & Genome Biology, The Hospital for Sick Children, The Peter Gilgan Centre for Research and Learning, 686 Bay Street, Toronto M5G 0A4, Ontario, Canada. E-mail address: [email protected] (C.E. Pearson).

https://doi.org/10.1016/j.nbd.2019.104533 Received 26 March 2019; Received in revised form 29 June 2019; Accepted 11 July 2019 0969-9961/ Crown Copyright © 2019 Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

Please cite this article as: Stella Lanni and Christopher E. Pearson, Neurobiology of Disease, https://doi.org/10.1016/j.nbd.2019.104533

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Fig. 1. DM1 clinical subtypes. The bar graphs represent the CTG expansion length in the 5 different subtypes of DM1. Overlapping in repeat size is evident (repeat numbers are estimates, as many individuals do not have absolute sizes). An increase in disease severity and decrease in the ageof-onset is observed through generations. CDM1 presents with the most severe phenotype and prenatal onset. A clear bias for maternal transmission of CDM1 is also observed.

1997; Di Costanzo et al., 2009; Nakagawa et al., 1994; Ohya et al., 1994; Tachi et al., 1997; Tanaka et al., 2000; Zeesman et al., 2002). It is possible that this is an underestimate, as many paternal transmissions may have not been reported in the literature. Two recent studies from the French DM-Scope registry looking at 314 DM1-affected children and 1409 adult DM1 patients, observed a paternal CDM1 transmission rate of 12.5% and 9% of all CDM1 cases, respectively (Dogan et al., 2016; Lagrue et al., 2019). The same studies reported a paternal transmission rate of 42-50% for infantile DM1, 68-72% for juvenile DM1 and around 70% for adult and late-onset DM1 (Dogan et al., 2016; Lagrue et al., 2019) (Fig. 1). CTG lengths between 50 and 500 repeats show a paternal expansion bias, while for CTG > 500 repeats it is maternal transmission that shows the stronger expansion bias to exceedingly large repeat expansions (> 1000–6500 repeats), often leading to CDM1 (Harley et al., 1993; Lavedan et al., 1993; Pearson, 2003). The majority of DM1 patients who inherit expansions from their father do not develop CDM1, even if the repeat length is greater than expansion lengths typically found in CDM1 (Campbell et al., 2013; Novelli et al., 1995; Passos-Bueno et al., 1995; Spranger et al., 1999; Tsilfidis et al., 1992). All these evidences indicate that some other factors, together with the CTG repeat expansion itself, contribute to the most severe symptoms and to the maternal transmission bias observed in CDM1 patients. Myotonic dystrophy type 2, caused by the expansion of a CCTG tract in the ZFN9 gene, produces a toxic CCUG, that like the DM1 toxic CUG RNA, avidly binds MBNL, and shows similar (but not identical) adult phenotypes. Curiously, there is effectively no congenital DM2 phenotype even with very large repeats (Day et al., 2003). This further supports the possibility that some factor other than the large expansions at either loci must be contributing to the CDM1 phenotype.

1997; Lopez Castel et al., 2011b; Martorell et al., 2004; Martorell et al., 1998; Morales et al., 2012; Ohya et al., 1995; Tachi et al., 1995; Thornton et al., 1994; Wong and Ashizawa, 1997; Zatz et al., 1995). A more direct comparison would be possible by measuring the estimated progenitor allele (ePAL), representing the repeat size the patient was born with, essentially accounting for the confounding effect of age-atsampling due to somatic instability (Morales et al., 2012, 2015). A recent study revealed that an accurate measure of ePAL, provides a strict correlation between repeat size and phenotype, that could be extremely useful for patients stratification in clinical trials (Overend et al., 2019). ePAL estimation is technically demanding and currently performed by one group (Morales et al., 2012, 2015). Importantly, most of the studies analyzing the effect of repeat size on the clinical phenotype assessed blood DNA, where somatic instability is considerably lower than other tissues like muscles or heart. 2. Congenital myotonic dystrophy: phenotype and transmission CDM1 is not merely an early onset and severe form of DM1, but it exhibits distinct clinical features not observed in DM1 patients (Johnson et al., 2016a; Zapata-Aldana et al., 2018). Prenatally CDM1 is characterized by polyhydramnios and reduced fetal movement, while newborn infants often have pre-term deliveries, present as floppy infants, show severe muscle fibers immaturity, hypotonia, respiratory insufficiency and cognitive impairment (De Antonio et al., 2016; Ho et al., 2015; Johnson et al., 2016a; Lagrue et al., 2019; Verrijn Stuart et al., 2000). CDM1 represents one of the most extreme examples of parent-of-origin effect, with the disease occurring almost exclusively when the expanded repeat is transmitted by affected mothers (Harper and Dyken, 1972). Only 9 cases of CDM1 transmitted from affected fathers were reported (Bergoffen et al., 1994; de Die-Smulders et al., 2

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3. Maternal bias for large expansions in CDM1

differential methylation was found in CDM1 patient samples in the DMPK gene body (Shaw et al., 1993). Later studies on a clinically characterized cohort of patients and with a different set of methylation sensitive restriction enzymes, identified hypermethylation in the region upstream the CTG expansion in cells of CDM1 tissues (Steinbach et al., 1998). More recently, CpG hypermethylation of the same region of the maternally inherited DM1 locus has been associated with CDM1 in a larger cohort of patients (Barbe et al., 2017). This region of hypermethylation contains a binding site for the insulator protein CTCF (CTCF1 site). CpG methylation flanking the CTG repeat was analysed in 79 blood DNAs from DM1 affected individuals, of which 20 had CDM1. Almost all (19/20) patients with CDM1 had DNA hypermethylation at the CTCF1 binding site, while only 4% of DM1 individuals and none of the unaffected controls had DNA methylation at the same site (Barbe et al., 2017). The presence of the aberrant methylation in the region upstream the repeat was assessed also in human DM1 embryonic stem cells (hESCs) and it was associated with larger expansions (> 300CTG) (Yanovsky-Dagan et al., 2015). A closer analysis to the CTCF1 region revealed hypermethylation of this specific site only in hESCs derived from DM1 affected mothers and not from affected fathers (Barbe et al., 2017). The same bias for methylation for the maternal inherited allele was observed also in chorionic villus samples (CVS), the tissue used for prenatal diagnosis, supporting a maternal oocyte-specific aberrant epigenetic mark for CDM1 (Barbe et al., 2017). If confirmed in a larger cohort of patients, the assessment of CTCF1 hypermethylation in CVS can be a precious diagnostic indicator for an accurate prenatal diagnosis of CDM1. A definite prenatal diagnosis based only upon repeat length is complicated by the fact that prenatal tissues from pregnancies that led to the birth of CDM1 children can have repeat lengths considerably shorter than 1000 repeats, even shorter than the length in the blood of the transmitting mother (Campbell et al., 2013; Cobo et al., 1993; Dalphin et al., 1992; DiRocco et al., 1996; Geifman-Holtzman and Fay, 1998; Hojo et al., 1995; Verrijn Stuart et al., 2000). Hypermethylation of the same CTCF1 site has been described in tissues from CDM1 affected infants and in myoblasts from CDM1 fetuses, and it has been associated with muscle immaturity and repeat instability (Lopez Castel et al., 2011b; Nakamori et al., 2017; Rizzo et al., 2018). Non-CpG methylation occurs primarily in human stem cells, mostly at CpNpG sites including CAG and CTG (Lister et al., 2009; Ramsahoye et al., 2000). The function of CpNpG methylation is poorly characterized (Pinney, 2014). The absence of non-CpG methylation within the repeat tract in DM1 was confirmed using a series of methylation sensitive restriction enzymes (Lopez Castel et al., 2011a). Further studies are necessary to exclude non-CpG methylation in DM1 hESCs, affected tissues, and to clarify the role of this epigenetic mark in the pathogenesis of DM1 and CDM1. The DMPK - CTG repeat region is in a gene-rich area and in nondiseased tissues displays strong enhancer chromatin features, enriched to varying degrees in H3K4me3, H3K27ac and H3K4me1 (Buckley et al., 2016). Two binding sites for the insulator protein, CTCF, surround the CTG repeat and regulate anti-sense transcription as well as replication fork progression (Cleary et al., 2010; Filippova et al., 2001; Nakamori et al., 2017; Otten and Tapscott, 1995). In all cells studied, the CTCF1 site (upstream of the repeat) was found by ChIP-seq to be bound by CTCF (Buckley et al., 2016; Filippova et al., 2001; YanovskyDagan et al., 2015). CpG methylation of CTCF1 prevents binding of CTCF, potentially affecting chromatin dynamics (Cho et al., 2005; Filippova et al., 2001). CTCF binding to the downstream site, CTCF2, seems controversial. Weaker CTCF binding to CTCF2 was reported in DM1 mice (Brouwer et al., 2013), but interaction of any kind could not be confirmed in hESCs (Yanovsky-Dagan et al., 2015). DNA methylation of the CTCF2 region also presents high variability amongst different tissues of the same patient and different DM1 subtype, and it is not correlated to either CTG length, DM type or parental transmission (Barbe et al., 2017; Lopez Castel et al., 2011b).

Prior to the discovery of the DM-causing gene mutation in 1992, clinicians and scientists had already identified the extreme bias for maternal transmission of CDM1 and had associated the risk of CDM1 with complications during gestation and delivery (Harper and Dyken, 1972; Jaffe et al., 1986). Cases of CDM1 affected children were more frequent in families with history of CDM1, leading to the hypothesis of a maternal environmental or intrauterine factor (Harper and Dyken, 1972; Koch et al., 1991; Webb et al., 1978). The chance of having a more severely affected child was shown to be increased with greater severity of the maternal disease, leading to the hypothesis of maternal metabolites acting on the development of the fetus (Koch et al., 1991). Subsequent studies could not identify any specific intrauterine factors and showed no correlation with the maternal state of the disease, even if the risk of giving birth to CDM1 child was considered higher when the maternal allele was greater than 300 repeats (Cobo et al., 1995; Morales et al., 2015). Most women were asymptomatic and diagnosed with DM1 only after giving birth to congenital affected babies (Erikson et al., 1995). These studies pointed out to the possibility of a secondary mutation or other factors specific of the mothers with CDM1 children (Erikson et al., 1995; Lavedan et al., 1993). While studies failed to identify any specific intrauterine factors driving the maternal bias of CDM1 (Cobo et al., 1995; Morales et al., 2015), the absence of evidence is not evidence of absence – some factor may yet be revealed. The possible contribution of mitochondrial DNA mutations to CDM1 was long disputed (Poulton, 1992) and subsequently disregarded by sequence analysis of the mitochondrial genome in CDM1 (Poulton et al., 1995; Thyagarajan et al., 1991, 1993). While a definitive exclusion of a contribution of a mitochondrial contribution seems likely, recent awareness of the biparental contribution of the mitochondrial complement, varying levels of heteroplasmy, and mitochondrial gene variants, coupled with increased depth of mitochondrial sequencing, may invite a reassessment of this (Luo et al., 2018; McCormick et al., 2018; McWilliams and Suomalainen, 2019). The high infertility rate in males with longer expansions together with the observed expansions transmitted only from fathers with repeats larger than 1.5 kb, led to the hypothesis of some mechanisms inhibiting paternal transmission of a repeat tract large enough to cause the congenital form (Cobo et al., 1993; Lavedan et al., 1993; Mulley et al., 1993). In particular, as spermatogenesis involves more rapid cell divisions than oogenesis does (Pearson, 2003), the role of mitotic repeat instability was proposed to explain the differential effects on CTG sizes in males and females (Lavedan et al., 1993; Mulley et al., 1993). A greater instability in female meiosis was also suggested by two different studies, looking at the size of the repeat in embryos as well as oocytes from DM1 affected donors (Dean et al., 2006; Rakocevic-Stojanovic et al., 2005). 4. Epigenetic characterization of CDM1 Apart from the first wave of repeat instability in the germ line, somatic instability has been suggested to occur between week 13 and 16 of gestation, leading to the heterogeneity between the tissues observed in a series of 13 congenitally affected fetuses and 4 neonates (Hecht et al., 1993; Jansen et al., 1994; Lavedan et al., 1993; Martorell et al., 1997; Wohrle et al., 1995). Interestingly, these two periods of repeat instability coincide with the two major cycles of epigenetic reprogramming that occur during development. Immediately after fertilization, DNA methylation is erased and then restored in a tissue-specific manner, and later during germline development, a second wave of epigenetic erasure occurs (Bergman and Cedar, 2013; Reik, 2007). This coincident timing of epigenetic changes and repeat expansion lead to the hypothesis that the two phenomena might be related. The possible role of imprinting in the maternal bias for CDM1 was hypothesized in 1992 (Harper et al., 1992), but the idea was later abandoned when no 3

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5. Epigenetics and parent-of-origin effect

observed in CDM1 (Laurent et al., 1997; Thomas et al., 2017). The major molecular mechanism underlying DM1 pathogenesis is the production of CUG expanded RNAs that sequester RNA processing factors, like MBNL proteins, resulting in mis-regulation of alternative splicing and in changes in RNA localization and turnover (Batra et al., 2014; Masuda et al., 2012; Wang et al., 2012). The overexpression of DMPK during fetal development may induce more MBNL to be sequestered into foci and less free MBNL to regulate proper splicing, leading to the more severe mis-splicing events in CDM1 muscles compared to adult DM1, and therefore to the more severe phenotype (Thomas et al., 2017). Together with the increase in DMPK sense transcript, a decrease of the antisense DM1-AS has been observed in hypermethylated CDM1 muscles (Nakamori et al., 2017). CAG containing transcripts can from hairpins that are recognized by the ribonuclease Dicer and cleaved to form short ~21-nt CAG-repeat RNAs (Krol et al., 2007). These short 21nt-RNAs seem to have a protective role against muscle pathology in CDM1, and correlate with muscle maturity. Furthermore, overexpression of the DM1 antisense transcript in a cell model with 800 repeats induces increased levels of 21nt RNAs and reduction in missplicing events as well as in CUG RNA foci (Nakamori et al., 2017). These data support a role for CTCF1 hypermethylation in the severe phenotype observed in CDM1. Peptides generated by non-AUG translation across the repeats (RAN translation) have been observed in DM1 myoblasts, skeletal muscle and blood, as well as in DM1 mouse models (Zu et al., 2011). RAN proteins have been observed in several repeat expanded diseases, including Huntington disease (HD), DM2, fragile X tremor ataxia syndrome (FXTAS), C9orf72 amyotrophic lateral sclerosis (ALS) (Banez-Coronel et al., 2015; Mori et al., 2013; Todd et al., 2013; Zu et al., 2013, 2017), and they have been reported to induce cell toxicity and apoptosis. The role of RAN proteins in DM1 pathogenesis is not well understood yet and there are currently no studies investigating a possible role in the pathogenesis of CDM1. Studies in HD showed that RAN peptide aggregates increase with the size of the repeat tract and that they occur at the longer repeat expansions typically associated with the more severe juvenile onset cases of HD (Banez-Coronel et al., 2015). Similarly, accumulation of RAN peptides could be involved in the severe phenotype observed in CDM1, where longer repeats are present. Further studies are required to evaluate this possibility.

The CTG repeat resides in a CpG island in the 3′-UTR of DMPK, within the overlapping antisense DM1-AS gene and in the promoter of SIX5. In non-affected individuals with non-expanded CTG tracts, the region surrounding the repeat was found to be unmethylated in 16 different tissues (Buckley et al., 2016). The methylation-free zone spans from 800 bp upstream and 2.2 kb downstream of the CTG tract in all tissues analysed apart from sperm, where it is further extended from 1.6 kb upstream to 3.3 kb downstream of the repeat (Buckley et al., 2016). Methylation/demethylation events are differentially regulated in oogonia and spermatogonia (Clarke and Vieux, 2015; Lucifero et al., 2002; Ly et al., 2015; Maatouk and Resnick, 2003) and, as observed in other diseases, like fragile X syndrome, this regulation can be perturbed by the presence of a repeat expansion (Malter et al., 1997; Salat et al., 2000; Sun and Baumer, 1999; Willemsen et al., 2002). A threshold FMR1 CGG expansion length that induces aberrant methylation was recently detected in fragile X cells (Brykczynska et al., 2016), and the same phenomenon could have a role in CDM1. A strong correlation between repeat length and methylation has not been identified in CDM1, but it could depend on the cohort analyzed and further studies are necessary to fully evaluate this link. Sperm of affected DM1 fathers may escape aberrant CpG methylation due to a differential methylation/demethylation program, that is responsible for the different methylation boundaries compared to all the other tissues analyzed (Barbe et al., 2017; Buckley et al., 2016). This could explain the bias for maternal transmission in CDM1 (Barbe et al., 2017). In hypermethylated hESCs as well as in CDM1 muscles it has also been reported a reduction in the expression of the neighbouring SIX5 gene (Thomas et al., 2017; Yanovsky-Dagan et al., 2015), responsible of the “christmas tree” cataracts typical of DM1 patients (Klesert et al., 2000; Sarkar et al., 2000). The Six5 protein is involved in spermatogenesis survival and spermiogenesis (Sarkar et al., 2004). If CTCF1 site gets hypermethylated in male germ line, this will induce a decrease in the levels of SIX5, leading to reduced survival of those spermatozoa and reduced chances of CDM1 progeny. Notably, while this may explain the azoospermia common to DM1 fathers (Vazquez et al., 1990), and the maternal bias of CDM1, this hypothesis remains to be tested. 6. Gene expression and transcript processing in CDM1

7. Role of epigenetics in CDM1 pathogenesis CTCF1 binding site overlaps with a DNaseI-hypersensitive site (DNAseI-HS) detected in all non-affected tissues studied (Buckley et al., 2016). DNaseI-hypersensitive sites are small regions of open chromatin that often overlap transcription regulatory elements, regions compacted by modified histones, and often reflect active promoters and/or enhancers. This chromatinization is consistent with the CTG repeat region being coincident with the promoter of both SIX5 and the antisense DM1-AS gene (Cho et al., 2005). Loss of these DNaseI-HS sites was observed in DM1 patients and associated with altered gene expression at the locus, but it was not assessed relative to DNA hypermethylation at CTCF1 site (Frisch et al., 2001; Otten and Tapscott, 1995). Tissues of non-affected individuals showed strand-specific RNAs predominantly derived from the sense CUG-containing DMPK strand. Only hESC had considerable poly(A)+ antisense (DM1-AS) RNA in this region, especially downstream of the 3′ end of DMPK extending into SIX5. Recent analysis of two CDM1 fetal terminations (12-33 weeks gestation) revealed abundant RNA foci due to sense and anti-sense DMPK transcripts in heart and brain (Michel et al., 2015). Expression of both strands was greater in heart than brain and the antisense expression was higher in CDM1 brain than in the same tissue from non-affected controls (Michel et al., 2015). DNA methylation analysis was not performed (Michel et al., 2015). DMPK transcripts levels were found to be particularly high in CDM1 fetal muscles as well as in muscle precursor cells from CDM1 affected patients and were associated with the most severe pathogenesis

The current model describing the role of epigenetics in the pathogenesis of CDM1, suggests that CpG methylation at CTCF1 site is inhibiting the proper binding of CTCF protein, which may alter the chromatin structure (DNAseI-HS and histone marks) and gene expression at the locus (Fig. 2). In particular, the increased DMPK mRNA and decreased DM1-AS during myogenesis may be responsible of the more severe mis-splicing events, and therefore of the more severe phenotype (Nakamori et al., 2017; Thomas et al., 2017). The reduction of SIX5 transcript, instead, may be involved in the maternal bias for transmission of CDM1 and possibly other symptoms (Barbe et al., 2017; Yanovsky-Dagan et al., 2015). A role for CTCF protein in repeat instability has been described in mouse models of spinocerebellar ataxia type 7 (SCA7), a disease caused by the expansion of an unstable CAG tract in the ataxin-7 gene (Libby et al., 2008). In particular, mutations of the CTCF binding site adjacent to the expanded SCA7 repeat enhanced repeat instability in the germ-line and in somatic tissues of SCA7 mice (Libby et al., 2008). In mice containing functional CTCF binding site, in some tissues, methylation of the site ablated CTCF binding and was coincident with enhanced CAG instability in that tissue (Libby et al., 2008). Furthermore, CTCF binding at the expanded locus was shown to be necessary for the transcription of the antisense noncoding RNA at the ataxin-7 locus (Sopher et al., 2011). Similarly, CTCF binding has been associated with the regulation of sense and antisense transcription at the CGG-expanded FMR1 locus in cell lines from fragile 4

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Fig. 2. Current model for the role of CTCF1 site methylation in the pathogenesis of CDM1. Binding of CTCF protein is disrupted in CDM1 patients due to hypermethylation of the CTCF1 site upstream of the CTG repeat. Expression levels are reflected by the thickness of the promoter arrows. Increased transcription of DMPK sense mRNA, and reduction in the expression levels of the antisense DM1-AS and the neighbour gene SIX5, are associated with the more severe mis-splicing events and pathogenesis of CDM1. Figure created with BioRender.

The Marigold Foundation (CEP). The authors declare no competing financial interests. Correspondence should be addressed to CEP ([email protected]).

X syndrome affected patients (Lanni et al., 2013). Site-specific mutagenesis experiments will be necessary to define the exact role of the CTCF1 binding site in regulating gene expression in DM1/CDM1, as well as in other repeat expanded disorders.

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Many questions still need to be answered: What is the cause of CTCF1 site methylation? Might CTG expansions cause CTCF1 methylation, and if so, how? Is CTCF1 methylation affecting CTG repeat instability? Is aberrant CTCF1 methylation regulated in a developmental and/or tissue-specific manner? Why is this mechanism specific of CDM1? Is there a threshold repeat length that signals for CTCF1 methylation? Are these epigenetic changes involved in the altered gene expression at the expanded DMPK locus? Might CTCF1 methylation be present in the rare cases of paternally-transmitted CDM1? Can an epigenetic model of CDM1 be produced to recapitulate the severe aspects of the disease? Clearly, further research into the etiology of CDM1 is centered upon epigenetics. Acknowledgements This work was supported by the Muscular Dystrophy Canada (CEP), Canadian Institutes of Health Research (CEP), Tribute Communities (CEP), The Petroff Family Fund (CEP), The Kazman Family Fund (CEP), 5

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