CFT8634

Epigenetics of neuromuscular disorders

Fabio Coppede` *,1
1 Department of Translational Research & of New Surgical & Medical Technologies, University of Pisa, Via Roma 55, 56126 Pisa, Italy
*Author for correspondence: Tel.: +39 050 221 8544; [email protected]

Neuromuscular disorders are a heterogeneous group of conditions affecting the neuromuscular system. The aim of this article is to review the major epigenetic findings in motor neuron diseases and major hereditary muscular dystrophies. DNA methylation changes are observed in both hereditary and sporadic forms, and combining DNA methylation analysis with mutational screening holds the potential for better diagnostic and prognostic accuracy. Novel, less toxic and more selective epigenetic drugs are designed and tested in animal and cell culture models of neuromuscular disorders, and non-coding RNAs are being investigated as either disease biomarkers or targets of therapeutic approaches to restore gene expression levels. Overall, neuromuscular disorder epigenetic biomarkers have a strong potential for clinical applica- tions in the near future.

Keywords: amyotrophic lateral sclerosis • DNA methylation • Duchenne and Becker muscular dystrophy • epigenet- ics • facioscapulohumeral muscular dystrophy • miRNAs • myotonic dystrophy type 1 • spinal muscular atrophy

Background

Neuromuscular disorders (NMDs) include several hereditary or acquired conditions that impair the neuromuscular system and the functioning of the muscles. These disorders can result from a direct impairment of the skeletal muscle or from conditions affecting motor neurons, peripheral nerves or neuromuscular junctions [1]. Examples include but are not limited to the motor neuron diseases amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), hereditary muscular dystrophies and myopathies, peripheral neuropathies and the neuromuscular junction disorder myasthenia gravis [2]. These conditions are typically characterized by a progressive muscular weakness as well as a complex scenario of other symptoms and, taken individually, are often rare. However, because of the several forms and subtypes of NMDs, they collectively affect a large number of individuals [1,2]. Increasing evidence reveals the existence of epigenetic modifications in NMDs, opening the way for their application in clinical settings as either diagnostic or prognostic biomarkers or as targets of pharmacological interventions [3–6].

The major epigenetic mechanisms investigated in NMDs include DNA methylation, histone tail modifications and nucleosome positioning as well as the regulation of gene expression mediated by non-coding RNA (ncRNA) molecules, particularly miRNAs [3–6]. DNA methylation consists of the addition of a methyl group at the 5- carbon of the cytosine ring, causing cytosine to become 5-methylcytosine (5-mC) [7]. The reaction is catalyzed by enzymes called DNA methyltransferases (DNMTs) and usually occurs at CpG sites (i.e., sites where a cytosine is followed by a guanine) [8]. The methylation of CpG-rich regions (CpG islands) located at or near the promoter of a gene results in gene silencing and is one of the most investigated epigenetic marks. By contrast, DNA methylation of the gene body prevents spurious transcription initiation and is found in actively transcribed genes. DNA methylation has several other roles, including genomic imprinting, silencing of transposable and repetitive elements and inactivation of the X chromosome [7,8]. Histones are the most abundant proteins associated with DNA and aggregate each other, forming the histone octamer (2 copies each of the core histones H2A, H2B, H3 and H4), around which DNA is wrapped, creating the nucleosome. Nucleosomes are then connected by stretches of linker DNA and are further folded to produce a chromatin fiber [9]. The chromatin state is another important modulator of gene expression levels, and post-translational modifications of the N-terminal of histone tails in nucleosomes are associated with either open or condensed chromatin structures. These modifications include acetylation, methylation, phosphorylation, ubiquitylation, sumoylation and others that, overall, regulate DNA repair, replication and recombination processes as well as nucleosome positioning and gene expression levels via directly influencing the chromatin structure or by generating a “histone code” that regulates the binding of chromatin remodelers [9]. Several enzymes, including histone acetyltransferases, histone deacetylases (HDACs), histone lysine methyltransferases, protein arginine methyltransferases, histone demethylases and protein kinases and phosphatases as well as additional histone-modifying enzymes, are responsible for the establishment or removal of these marks [10]. Studies of higher-order chromatin arrangements and their dynamic interactions with other nuclear components have revealed that chromatin folding secures the positioning of chromosomes into discrete territories in the nucleus [11], with transcriptionally active loci mainly oriented toward the nuclear interior and heterochromatic loci that tend to be near the periphery [11,12]. Chromosome conformation capture technologies have shown that the genome is organized in a non-random fashion into higher-order chromatin domains, such as topologically associating domains, loop domains or insulated neighborhoods [12]. Topologically associating domains are defined as chromatin domains that exhibit high levels of internal interactions and are separated from each other by regions of low interaction termed boundary elements. Therefore, these units are believed to act by delineating the regions of enhancer–promoter contacts, thereby playing a pivotal role in transcriptional control [12]. Expansions or contractions of repetitive DNA sequences, including expanded triplets and contractions of macrosatellite repeats, have been shown to cause topological modifications of the genome [13,14]. A further layer of epigenetic regulation of gene expression and chromatin state exists at the level of short (<200 nt) and long (>200 nt) ncRNAs [15]. Among them, miRNAs are short molecules of about 22 nucleotides in length and the most investigated ncRNAs; they regulate gene expression at the post-transcriptional level by binding to the 3´ untranslated region of target mRNA molecules, leading to their degradation or translational inhibition, depending on the degree of sequence complementarity [16].

This article aims to summarize epigenetic findings in motor neuron disorders (ALS and SMA) and in some of the most common skeletal muscle disorders–namely, Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), myotonic dystrophy type 1 (DM1) and facioscapulohumeral muscular dystrophy (FSHD)– highlighting their emerging clinical potential.

Epigenetics of motor neuron disorders

Motor neuron disorders are a heterogeneous group of rare neurological conditions that selectively destroy motor neurons. They include both sporadic and hereditary diseases affecting either the upper (originating from the primary motor cortex) or lower motor neurons (originating from the brainstem or the spinal cord), or both, leading to progressive weakness and atrophy of voluntary skeletal muscles [17,18]. ALS is the most common of these conditions in adults and affects both upper and lower motor neurons [17]. By contrast, SMA is characterized by motor neuron degeneration only in the spinal cord [18]. Epigenetic modifications in ALS and SMA are summarized in Table 1.

Epigenetics of ALS

ALS has a worldwide incidence of about 1–3 new cases per 100,000 individuals per year, and the available drugs (riluzole and edaravone) only slow the progression of symptoms. There is no cure for the disease, and most patients die from respiratory failure within 2–3 years of onset [57,58]. Almost 90% of ALS cases are sporadic (sALS), and only about 10% of cases result from inherited gene mutations (familial ALS [fALS]). FALS and sALS are phenotypically and clinically indistinguishable, and 4 major genes account for most cases of fALS and a small percentage of the ‘apparently’ sporadic cases that are likely due to de novo mutations. The 4 major ALS genes are SOD1, coding for the antioxidant enzyme copper–zinc superoxide dismutase and accounting for 12% of cases of fALS and about 1% of sALS cases; TARDBP and FUS, both coding for RNA-interacting proteins and each accounting for about 4–5% of cases of fALS and less than 1% of sALS cases; and C9orf72, coding for the C9orf72 protein, which is abundant in motor neurons and involved in RNA processing [58–60]. A hexanucleotide GGGGCC repeat expansion in the C9orf72 gene is the most frequent cause of ALS in Europe and North America, accounting for up to 40% of cases of fALS and about 7% of sALS cases [60]. Expansions of hundreds or thousands of repeats are frequently observed in patients, but the pathological threshold is not yet clearly defined, and the proposed pathological mechanisms include loss of function of the C9orf72 protein and a toxic gain of function of the expanded RNAs and resulting proline–arginine dipeptide repeat proteins [60]. Other causative fALS genes have been identified, each accounting for less than 1% of cases [58–60]. In addition to the highly penetrant fALS loci, genome-wide association studies have identified more than 100 low-penetrance sALS loci, suggesting a polygenic inheritance model and a strong contribution of environmental factors in sALS [58,59].

A significant increase in global DNA methylation (5-mC content) was observed post-mortem in the spinal cord DNA of 11 ALS patients compared with healthy matched controls [19], and similar findings were observed when assessing whole blood global DNA methylation levels in a cohort of 96 ALS patients and 87 matched controls [20]. A subsequent large-cohort study confirmed a significant increase in peripheral blood DNA methylation levels in both sALS and fALS [21], and a study performed in ALS families with SOD1 mutations not only revealed a significant increase in 5-mC content in blood DNA from ALS patients but also showed that DNA methylation levels increased with the duration of the disease [22]. The epigenetic clock is based on the methylation analysis of several CpG sites and represents a valuable tool for estimating the biological age of a tissue, which can be older than the chronological age in the case of accelerated aging [23]. Studies in cohorts of monozygotic twins discordant for ALS revealed that unaffected twins had a younger DNA methylation age than the affected co-twins [23,24]. An increased methylation age was also observed in older ALS patients from a large cohort of more than 1000 cases and controls [25], and a recent genome-wide CpG methylation analysis in a cohort of similar sample size revealed that the methylation score generated by combining the top differentially methylated sites in whole blood DNA significantly classified the case–control status in a replication cohort [61]. Overall, there is substantial evidence of global changes in DNA methylation in both motor neurons and peripheral ALS tissues [19–25,61]. In this regard, research involving cell cultures and transgenic ALS mice has shown that motor neurons engage epigenetic mechanisms to drive apoptosis, involving upregulation of certain DNMTs, particularly DNMT3A and DNMT1, and increased global DNA methylation [62]. Similarly, the authors observed increased levels of DNMT1, DNMT3A and 5-mC in the brain and spinal cord of ALS patients, suggesting that they could contribute to motor neuron degeneration [62]. Impaired mitochondrial DNA methylation and mitochondrial Dnmt3a protein levels were observed in spinal cord and skeletal muscles of transgenic SOD1 mice [26], and a recent investigation in ALS families revealed that carriers of ALS-causative SOD1 mutations have a significant reduction in the methylation levels of the mitochondrial DNA regulatory region (D-loop region), resulting in increased mitochondrial DNA copy number [27].

The search for gene-specific methylation changes in ALS started with case–control studies addressing the methylation levels of genes linked to the familial or sporadic forms, including SOD1, VEGF, GLT1 and MT-coding genes, but these genes resulted demethylated in the investigated blood or brain tissues [63–65]. Subsequent studies revealed that the major ALS genes SOD1, FUS and TARDBP are demethylated in ALS tissues [22,25,31]. By contrast, the C9orf72 gene is frequently methylated in carriers of a GGGGCC repeat expansion [28–30,66]. In particular, hypermethylation of the C9orf72 promoter region upstream of the pathogenic repeats was observed in 10–30% of patients and linked to increased repeat length and reduced transcription of C9orf72 [28–30]. Moreover, the expanded hexanucleotide repeat itself is methylated in almost all cases [66]. The clinical utility of C9orf72 promoter methylation has been investigated in an attempt to better define the cutoff repeat size required for the initiation of neurodegeneration, but despite some evidence that intermediate alleles can display increased C9orf72 methylation and are associated with a higher frequency of neuropsychiatric complications, the utility of this biomarker is still debatable [67].

A genome-wide DNA methylation study in brain regions of 10 sALS patients and 10 neurologically healthy controls suggested that the differentially methylated genes were particularly involved in calcium homeostasis, neurotransmission and oxidative stress [31]. More recent genome-wide methylation and expression studies in ALS blood and spinal cord DNA revealed that hundreds of genes involved in inflammation and/or immune response show aberrant expression in ALS tissues, likely representing potential targets of environmentally induced epigenetic modifications [19,25,32]. Several environmental factors have been proposed to contribute to impaired DNA methylation in ALS tissues, including early-life events, organophosphate exposure, metal exposure and the patients’ workplace and lifestyle, but evidence is still inconclusive [68].

Histone tail modifications in ALS have been investigated less extensively compared with other neurodegenerative diseases such as Alzheimer’s disease or Parkinson’s disease [68]. A study performed in 2003 revealed loss of the histone acetyltransferase enzyme CREB-binding protein and decreased histone acetylation in motor neurons of transgenic ALS mice at disease onset [33], and most of the subsequent studies investigated the effects of targeting histone acetylation with an HDAC inhibitor (HDACi) in animal or cell culture models of the disease [68]. There are 18 HDACs in mammals, divided into 4 classes. Class I, II and IV enzymes (HDAC1–11) are Zn2+ dependent, whereas class III enzymes are NAD+ dependent and called sirtuins (SIRT1–7) [68]. Several HDACs, including HDAC1, HDAC2, HDAC4, HDAC6, HDAC11, SIRT1 and SIRT2, have been implicated in ALS-related neuroprotection and neurodegeneration in animal models, neuronal cultures and human tissues [3,34], even though a recent study showed no evidence of class I or II HDAC alterations in human post-mortem ALS tissues [35]. Early pharmacological interventions revealed that treatment with a pan-HDACi, such as sodium butyrate, valproate and trichostatin A, slightly delayed disease progression and/or increased animal survival in transgenic SOD1 mice [36–38]. More recently, class I and II HDACi showed neuroprotection in FUS models of the disease [39,40]. However, despite the fact that pharmacological HDAC inhibition has often shown a protective effect on motor neurons and slightly decreased disease progression, with a moderate effect on whole animal survival, it is unable to prevent the disruption of neuromuscular junctions, suggesting that these drugs might negatively influence other cell types involved in ALS, and that future research is warranted to increase their selectivity in an attempt to avoid deleterious effects in the periphery [41]. Moreover, although the modulation of histone tail acetylation remains an interesting strategy for motor neuron protection, other histone tail modifications could represent future targets of pharmacological intervention in ALS. In this regard, it has recently been demonstrated that mice overexpressing proline–arginine dipeptide repeat proteins resulting from C9orf72 GGGGCC hexanucleotide repeat expansion show global changes in both histone methylation and heterochromatin structure [69].

The expression levels of several hundred miRNAs are dysregulated in muscular tissue, nervous tissue, cerebrospinal fluid, serum, plasma and blood samples from ALS patients [42–44]. Several of these miRNAs have been proposed as potential diagnostic or prognostic biomarkers of the disease [44], but systematic reviews of the literature revealed that there is minimal overlap among the available studies, likely due to a large heterogeneity in sample size, tissue extraction and miRNA detection methodologies [42,43]. However, 9 miRNAs–miR-133a-3p, let-7a-5p, miR-127- 3p, miR-155-5p, miR-206-3p, miR-26a-5p, miR-455-3p, miR-9-5p and miR-124-3p–are repeatedly dysregulated in ALS tissues [42], and 4 of them–namely, miR-124-3p, miR-127-3p, let-7a-5p and miR-9-5p-overlap between tissues, particularly between cerebrospinal fluid, spinal cord/nervous tissue and blood, serum or plasma, thus representing potential disease biomarkers [42]. However, the limited sample size of several studies and the different methodological approaches employed for miRNA extraction, collection and analysis have limited the discovery of circulating ALS biomarkers [42–44]. Moreover, several of the detected miRNAs, such as miR-9-5p and let-7a-5p, are not specific to ALS but are frequently dysregulated in other neurodegenerative or neuromuscular disorders [42–44], so protocol standardization and large-cohort confirmation studies are required prior to translating these findings into clinical practice [42].

Epigenetics of SMA

Spinal muscular atrophy is an autosomal recessive disorder with a reported incidence of 1 in 6000–10,000 live births [70]. The disease results from the degeneration of spinal cord motor neurons and is classified into 4 main clinical types, ranging in severity from progressive infantile paralysis and premature death (type 1) to limited motor neuron loss and normal life expectancy (type 4). A very severe SMA form that develops prenatally (type 0) is also distinguished and results in respiratory distress at birth [70]. SMA is caused by a biallelic deletion or mutation of the SMN1 gene, coding for a protein involved in ribonucleoprotein assembly, RNA metabolism, macromolecular trafficking, actin dynamics and signal transduction [70]. The paralogous SMN2 gene is almost identical to SMN1, except for a few single nucleotide exchanges, including one in exon 7 leading to aberrant pre-mRNA splicing that results in the skipping of exon 7 in almost 90% of the transcripts so that only a small amount of SMN2 transcripts produce a full-length SMN protein [5]. Up to 4 SMN2 gene copies are present in SMA patients, and the SMN2 copy number is the main modifier of disease severity and age of symptom onset [5]. Indeed, patients with SMA type 0 harbor 1 SMN2 copy, and about 80% of patients with type 1 SMA carry 1 or 2 SMN2 copies, whereas most patients with less severe SMA (type 3–4) carry 3 or 4 SMN2 copies [70]. However, those with higher SMN2 copy numbers do not always have a mild disease phenotype, so additional factors regulating SMN2 gene expression have been suggested as modifiers of the SMA phenotype [5,70].

In 2009, Hauke et al. provided the first evidence that SMN2 gene methylation correlates with SMA severity [45]. The study authors demonstrated that the SMN2 gene is subject to gene silencing by DNA methylation, and the analysis of SMN2 methylation in SMA patients with identical SMN2 copy numbers but different disease severity revealed a correlation of SMN2 methylation with SMA severity. The study also revealed that the transcriptional repressor MeCP2 binds to the SMN2 promoter region in a methylation-dependent manner. A subsequent analysis of 35 children with SMA confirmed that SMN2 methylation might regulate the disease phenotype by modulating its transcription [46]. Both of these studies investigated the genomic region 3000 nucleotides upstream and downstream of the translational SMN2 start site (+1) and identified 4 CpG islands in this region containing several CpG sites. Hauke et al. detected 7 CpG sites that were correlated with disease severity, located at positions -871, -695, -296, -290, +855, +988 and +1103 [45]. The most pronounced differences in DNA methylation between SMA1 and SMA3 patients were observed at nucleotides -296 and -290, whose methylation levels were significantly higher in SMA1 patients and inversely correlated with the activity of the first transcriptional start site of SMN2 at position -296. The study authors also demonstrated that the transcriptional repressor MeCP2 binds to this region. Cao et al. identified 13 CpG sites that were more methylated in type 1 or 2 compared with type 3 SMA patients at positions -871, -735, -290, -288, -285, +890, +894, +898, +900, +938, +988, +999 and +1036, with the most pronounced differences at nucleotides -871, -735 and +999 [46]. Moreover, the study authors observed
that the methylation levels of nucleotide -871 and the -290/-288/-285 unit were negatively correlated with the expression of SMN2 full-length transcripts, but they did not investigate the binding to specific transcription factors. A genome-wide methylation analysis in SMA was also performed in blood DNA samples from 12 patients and 11 healthy controls [47]. The study authors identified significant differences in the methylation levels of 40 CpG sites between SMA patients and controls, including CpG sites in the regulatory regions of ARHGAP22, CHML and SLC23A2 genes, implicated in axonogenesis, cytoskeleton dynamics and neuronal development and maintenance, as well as CpG sites in many other genes, potentially acting as modulators of disease severity. To further address this issue, the same group investigated the methylation levels of some of these genes in a cohort of 96 SMA patients and searched for correlation with disease severity [48]. The study revealed significant differences in the methylation levels of SLC23A2 and NCOR2 genes between SMA type 1–2 and SMA type 3–4 groups. Interestingly, SLC23A2 encodes a sodium/ascorbate co-transporter that plays a role in neuronal maturation, whereas NCOR2 encodes a subunit of a complex that includes HDACs and plays a role in chromatin remodeling. More recently, the analysis of blood DNA samples from 76 SMA patients revealed a significantly decreased methylation of CpG islands within exon 37 of the DYNC1H1 gene in patients with severe SMA (type 1) compared with mildly affected SMA patients (type 3–4) [49]. The DYNC1H1 gene encodes a heavy chain of cytoplasmic dynein, a protein required for axonal transport, and DYNC1H1 gene mutations cause an extremely rare form of autosomal dominant SMA that predominantly affects the proximal lower extremities [71,72]. Collectively, increasing evidence suggests that DNA methylation differences in SMN2 and other genes could act as modifiers of disease severity in SMA patients.

The aforementioned studies, particularly the one by Hauke et al. [45], reveal that chromatin remodeling mediates DNA methylation-induced SMN2 gene silencing, and several approaches have been proposed to treat SMA, includ- ing the use of HDACi to enhance SMN2 promoter activity [50]. Mohseni et al. reviewed the literature in the field, observing that at least 11 compounds have been investigated in cell cultures or animal models of SMA, including synthetic HDAC inhibitors such as valproic acid, phenylbutyrate, suberoylanilide hydroxamic acid, trichostatin A, benzamide M344, entinostat (MS-275), romidepsin, panobinostat (LBH589) and the naturally derived compounds epigallocatechin gallate, resveratrol and curcumin [50]. Valproic acid, benzamide M344, resveratrol, epigallocatechin gallate and curcumin increased overall SMN2 expression through inhibition of targeted HDACs and by increasing the incorporation of exon 7 into the SMN2 transcripts through the activation of splicing factors [50], whereas an increase in SMN2 expression resulting from demethylation of its promoter was suggested for suberoylanilide hydroxamic acid, entinostat and romidepsin [42], in addition to inhibiting targeted HDACs [50]. However, most of these compounds, particularly the naturally derived ones, induced only a minimal increase in the total SMN2 full-length transcript level [50], and clinical trials using valproic acid or phenylbutyrate revealed that they showed only marginal or no effects in patients [51,52]. The current research in the field has moved to the investigation of novel, more specific and less toxic compounds. For example, it was recently shown that dacinostat, a novel and less toxic hydroxamate-based HDACi, increased SMN2 transcript and protein levels and promoted demethylation of the SMN2 gene in type 1 and 2 SMA fibroblasts [73]. Furthermore, previous efforts to identify HDAC inhibitors to treat SMA mainly employed non-neuronal cells as the screening platform [50]. Recently, several novel cyclic tetrapeptide HDAC inhibitors were able to increase SMN2 gene expression in neuronal cells derived from induced pluripotent stem cells of SMA patients [74], thus representing a promising approach for small molecule development in SMA.

The SMN protein has a fundamental role in RNA metabolism and in the biogenesis of miRNAs, and several miRNAs have been found to be dysregulated in SMA spinal cord, serum and muscular tissues, including miR- 9, miR-132, miR-206, miR-183, miR-335-5p, miR-431, miR-375 and miR-2, most of which are involved in neuronal outgrowth, regeneration and function [53]. MiRNAs are not only investigated as disease biomarkers or contributors to motor neuron degeneration in SMA but also as markers of response to effective experimental therapies [54]. Therapeutic approaches for SMA attempt to enhance SMN2 gene expression and avoid skipping of exon 7 during pre-mRNA splicing to increase full-length SMN protein levels [55]. These approaches are based on gene replacement therapy as well as antisense oligonucleotide-directed therapy and small molecules designed to alter the pattern of SMN2 splicing and/or enhance SMN2 gene expression [55]. The antisense oligonucleotide nusinersen (SpinrazaⓍR ) was the first approved treatment for SMA and is based on the correction of exon 7 splicing of the endogenous SMN2 pre-mRNA [56]. A recent study revealed that circulating levels of muscle-specific miRNAs (myomiRs) decreased over disease course upon nusinersen treatment in pediatric SMA patients, and that the serum levels of miR-133a predicted patient response to therapy, suggesting that circulating myomiRs can be used as noninvasive biomarkers to monitor disease progression and therapeutic response [54]. Two antisense long ncRNAs-namely, SMN-AS1 and SMN-AS1*–are generated from SMN loci and associated with repression of SMN2 transcription, thus representing potential targets of antisense oligonucleotide-directed therapy [55]. Circular RNAs (circRNAs) are another emerging class of stable ncRNAs with several proposed functions, including, among others, sponging of miRNAs, regulation of transcription, sequestration of proteins and assembly of multimeric complexes [75]. Increasing evidence suggests that SMN1 and SMN2 genes produce a large number of circRNAs that are likely to be part of the feedback mechanism that regulates SMN function and could serve as disease biomarkers as well as novel targets for therapeutic approaches in SMA [75].

Epigenetics of skeletal muscle disorders

Human disorders resulting from direct impairment of skeletal muscle are a vast and heterogeneous group of conditions, including muscular dystrophies and myopathies, often resulting from inherited gene mutations [2]. This review article will focus on the epigenetics of 4 major disorders of skeletal muscle, namely DMD, BMD, DM1 and FSHD (Table 2). Despite the fact that these are hereditary conditions, increasing evidence points to the involvement of ncRNA dysregulation in both DMD and BMD, with potential therapeutic applications [76], whereas the analysis of DNA methylation changes is gathering increasing interest in the diagnosis of DM1 and FSHD [4].

Epigenetics of DMD and BMD

DMD is one of the most common and severe muscular dystrophies, resulting from out-of-frame mutations of the X-linked DMD gene, leading to a complete loss of the encoded dystrophin protein, a structural protein that is part of a complex (DAPC) that connects the cytoskeleton of muscular cells to the extracellular matrix [108]. DMD shows an X-linked recessive inheritance pattern, with a reported incidence of 1 in 3500–5000 males [108]. Absence of a functional dystrophin protein in patients results in muscle inflammation and necrosis, followed by failure of muscle regeneration and fibrofatty substitution over time, leading to a progressive functional decline of skeletal, cardiac and respiratory muscles, with death occurring within the third decade of life because of heart or respiratory failure [76,108]. By contrast, BMD is caused by in-frame DMD mutations, resulting in the production of a truncated, partially functional dystrophin protein, leading to milder clinical phenotypes [108].

The search for DNA methylation impairments in DMD and BMD is limited to some evidence of a skewed X-chromosome inactivation in female carriers of DMD mutations, manifesting DMD-related phenotypes [109,110], but most of the epigenetic studies in these conditions are focused on the diagnostic, prognostic and therapeutic value of miRNAs [76,111]. The levels of some myomiRs (i.e., miR-1, miR-133 and miR-206) are increased in the serum of DMD patients compared with controls and correlated with the worsening of muscle damage [77]. Other circulating miRNAs-namely, miR-26a, miR-222 and miR-378a-5p-have been proposed as biomarkers of myocardial scars in DMD patients [78]. A recent longitudinal study was performed to investigate the potential use of miR-181a-5p, miR-30c-5p and miR-206 for long-term follow-up of DMD and BMD patients [79]. DMD, BMD and healthy control individuals were followed over 4 years, and serum levels of the 3 miRNAs were measured at different time points. The study revealed that miR-30c and miR-206 represent sensitive biomarkers for DMD, and their levels remained significantly elevated in DMD patients relative to controls over the entire study period. Furthermore, miR-206 expression levels were significantly capable of distinguishing DMD from BMD patients, suggesting that this may be particularly relevant for assessing the effectiveness of therapeutic approaches aimed at converting DMD to a milder (BMD-like) phenotype [79]. MiRNAs have also been investigated as potential targets of therapeutic interventions in DMD. For example, inhibition of miR-31, a miRNA that regulates dystrophin expression, resulted in increased dystrophin levels in DMD cell cultures [80]. Collectively, miRNAs have shown promise as diagnostic disease biomarkers, as tools for monitoring therapy efficacy and as potential therapeutic targets [76,111]. In addition to miRNAs, circRNAs are increasingly implicated in muscle function and disease [81], and research in DMD cells have revealed that they have a unique signature in terms of circRNA expression levels [82], opening the way to novel disease biomarkers and/or therapeutic targets [81].

Epigenetics of DM1

DM1 is an autosomal dominant disorder with an estimated prevalence of 1 in 2500 individuals and characterized by myopathy, progressive muscle weakness and multisystem complications [112]. The disease results from a CTG repeat expansion in the 3’ untranslated region of the DMPK gene, coding for a protein kinase that is necessary for the maintenance of skeletal muscle structure and function [112]. The expanded CTG tract ranges from 50 to several thousand repeats in patients, resulting in extremely variable age of disease onset and progression rates. Indeed, different clinical subtypes are recognized, including congenital forms (CDM1), childhood-onset cases, juvenile and adult-onset forms and late-onset DM1 [113]. The pathogenic mechanism of the repeat expansion likely results from a toxic gain in function of the resulting mRNA that contains a CUG repeat expansion able to sequester RNA-interacting proteins, particularly the alternative splicing factors muscleblind-like proteins (MBNL 1 and 2), thus leading to a global splicing impairment in muscular cells [112]. However, the DMPK locus harbors a long CpG island, and DNA methylation of this region is believed to alter the chromatin structure and gene expression levels at the locus, thus contributing to the phenotypic variability of DM1 [113]. CDM1 is the most severe form, presents distinct clinical features and large expansions and is characterized by a parent-of-origin effect, with almost all cases occurring when the mother transmits the expanded repeat [83]. Early studies in cells of CDM1 patients showed hypermethylation of the region upstream of the CTG expansion [84], and more recent investigations have revealed that hypermethylation of this region is a stronger indicator of CDM1 than the CTG repeat size, suggesting that DMPK methylation may account for the maternal bias for CDM1 transmission and may serve as a more accurate diagnostic indicator of CDM1 in prenatal screening than the CTG repeat length [83]. Hypermethylation of this region has also been observed in myoblasts of CDM1-affected fetuses and in skeletal muscle tissues of CDM1 infants and is associated with muscle immaturity, evaluated as variation in muscle fiber size and quantification of undifferentiated type 2C fibers [85,86]. With regard to the contribution of DMPK methylation to the clinical phenotype of DM1, analysis of patients with different age of symptom onset revealed DMPK hypermethylation in all congenital forms, 6 out of 13 childhood-onset forms and only 6 out of 37 adult-onset forms, and hypermethylation was associated with early onset of symptoms, with a larger CTG expansion and maternal origin of the expanded allele [87]. A more recent investigation performed in blood DNA from 90 patients with the adult form revealed that DMPK methylation contributes significantly to and independently from the CTG repeat length the variability of muscular strength and respiratory profiles [88]. Collectively, these data suggest that the analysis of DMPK locus methylation has a strong diagnostic potential in the prenatal screening for CDM1 and a prognostic potential in adult-onset forms.

Several studies have shown that the DMPK locus undergoes heterochromatinization upon repeat expansion [114– 116], and it has recently been shown that several short tandem repeats associated with human diseases, including the DMPK locus, co-localize with boundaries of topologically associating domains that are highly enriched on CpG islands, suggesting they might be hotspots for epigenetic misregulation or topological disruption upon expansion [13].

Several miRNAs are dysregulated in DM1 tissues, including upregulation of the serum myomiRs miR-1, miR-206, miR-133a and miR-133b, all proposed as disease biomarkers [117]. These miRNAs were also recently investigated to monitor rehabilitation efficacy in DM1 patients and showed a significant downregulation that occurred in parallel with improvement in endurance and gait speed during a few weeks of aerobic exercise rehabilitation [89]. A functional depletion of the MBNL splicing factors, resulting from the toxic gain in function of the expanded mRNAs from the DMPK locus, is at the basis of DM1 pathogenesis, and several miRNA-based approaches, including blocking endogenous miRNAs or delivering exogenous ones, have been investigated to restore MBNL levels in DM1 cells [90]. For example, antagonists of miR-23b and miR-218 enhanced MBNL protein levels and rescued pathogenic missplicing events in DM1 myoblasts [91], whereas restoration of miR-7 levels with agomiR- 7 was sufficient to rescue DM1 myoblast fusion defects and myotube growth in a MBNL-independent manner [118]. Despite its potential, miRNA-based therapeutic strategies have only recently been investigated in DM1 cells, and their development still needs time and technical improvements, especially with regard to delivery and stability issues [90]. CircRNAs have been recently identified in muscle biopsies of DM1 patients and correlated with skeletal muscle strength and disease severity [92,93].

Epigenetics of FSHD

FSHD is the third most common myopathy, with a reported prevalence ranging from 1 in 8500 to 1 in 21,000 [119]. The disease is caused by different genetic defects, leading to the loss of epigenetic control over the subtelomeric D4Z4 macrosatellite repeat on chromosome 4q35, resulting in overexpression of the retrogene DUX4 contained within the repeat [119]. DUX4 encodes a transcription factor required for early human development, after which the gene is silenced in most somatic cells [120]. Overexpression of DUX4 in FSHD skeletal muscle activates genes not normally expressed in this tissue, impairs RNA and protein metabolism and triggers inflammation, oxidative stress and apoptosis [120]. FSHD is an autosomal dominant disorder characterized by a slowly progressive and asymmetric dysfunction of facial and upper and lower extremity muscles, and symptoms usually begin in adolescence. However, incomplete penetrance and variable expressivity between and within families are observed, leading to early- and late-onset cases and disease severity ranging from essentially asymptomatic to wheelchair-bound cases [119,120].

FSHD1 accounts for >95% of FSHD cases and results from a contraction of the D4Z4 macrosatellite repeat to a size ranging from 1 to 10 units, in cis with the 4qA allele of chromosome 4q, containing a polyadenylation signal for DUX4. The D4Z4 contraction in FSHD1 individuals leads to chromatin relaxation and hypomethylation of the region, resulting in DUX4 full-length transcript overexpression from the last repeat unit, which is stabilized by the polyadenylation signal coded by the 4qA permissive allele [119,120]. In normal individuals, the length of the repeat can reach up to 100 units, and the D4Z4 repeat is heavily methylated [120]. A minority of FSHD (<5%), termed FSHD2, results from mutations in chromatin modifiers, particularly in the SMCHD1 and DNMT3B genes, leading to chromatin relaxation and hypomethylation of the D4Z4 macrosatellite on chromosome 4q35 and overexpression of the DUX4 full-length transcript [119,120]. Therefore, FSHD is caused either by the contraction of the D4Z4 macrosatellite repeat or by mutations in D4Z4 chromatin modifiers, and the two genetic forms (FSHD1 and FSHD2) converge into one disease characterized by common epigenetic alterations resulting in the derepression of the DUX4 gene in skeletal muscle [119,120]. The diagnosis of FSHD1 is established in a proband with characteristic clinical features by identification of a heterozygous pathogenic contraction of the D4Z4 repeat array [121]. There are two highly homologous subtelomeric D4Z4 regions, the first located on 4q35 and the latter on 10q26, and DNA methylation analysis has been proposed to discriminate between FSHD1 and FSHD2 cases because in FSHD1 only the D4Z4 macrosatellite on 4q is hypomethylated, whereas FSHD2 cases show hypomethylation of both 4q and 10q regions [94–97]. Global changes in chromatin organization have been observed upon D4Z4 contraction in FSHD cells compared with healthy ones, including chromatin relaxation and changes in the 3D organization of this region [98–105]. In particular, it has been shown that there are functional interactions between D4Z4, the nuclear lamina and the telomere, indicating that D4Z4 is involved in the organization and regulation of long-distance interactions at this locus [98–100]. Moreover, chromatin conformation capture approaches have revealed that contractions of D4Z4 units influence the higher-order 3D organization of this region [101–103]. Indeed, a recent investigation of the 3D organization of the 4q35 locus in cells from FSHD patients and healthy donors showed that D4Z4 contractions and/or SMCHD1 mutations impact the spatial organization of the 4q35 region and trigger changes in the expression of different genes in fibroblasts and muscular cells, supporting a role for the D4Z4 macrosatellite repeat in the topological organization of chromatin [104]. Similarly, integrated multiomics approaches have revealed that the normal D4Z4 array contacts several regions in a peripheral nuclear domain, controlling their transcription, whereas the shortened and hypomethylated D4Z4 array in FSHD1 patients causes an impairment of the chromatin conformation, resulting in the loss of contacts and transcriptional upregulation of genes primarily involved in muscular atrophy [105]. Collectively, these studies point to an altered genome topology as a pathogenic mechanism in FSHD. Several therapeutic strategies have been proposed for correcting the underlying epigenetic defect in FSHD, including inhibition of D4Z4 activators and enhancement of D4Z4 repressors as well as CRISPR/Cas9-based approaches [122,123], but no treatment for the disease is yet available [89,117]. Several miRNAs have been investigated in FSHD tissues, but because of the scarce overlap among different studies, it is still unclear which miRNAs are closely involved in FSHD [106]. A recent comparison of control and FSHD biopsies revealed 8 miRNAs exclusively expressed in FSHD1 samples (miR-330, miR-331-5p, miR-34a, miR-380-3p, miR-516b, miR-582-5p, miR-517* and miR-625) [107], and another study suggested that the small ncRNA transcriptome changes in FSHD2 might be different from those in FSHD1 [106], but further studies are required to confirm the findings and to asses the diagnostic and therapeutic potential of these biomarkers. Conclusion The examples discussed in this article clearly demonstrate the involvement of epigenetic mechanisms in the onset and progression of NMDs, highlighting their potential clinical utility. Despite the fact that DNA methylation changes are often secondary events to gene expansions, contractions or point mutations in hereditary NMDs, combining genetic screening with C9orf72, SMN2, DMPK or D4Z4 methylation analysis holds the potential for better diagnostic and prognostic accuracy in these conditions. Novel epigenetic molecules are under investigation to overcome the toxicity and scarce selectivity of pan-HDACi molecules, leading to novel therapeutic opportunities in combination with other approaches aimed at correcting the genetic and epigenetic defects in NMD cells. Alterations in genome topology are an emerging and attractive area of research in NMDs resulting from copy number variants of repeated sequences. Furthermore, active research is ongoing on miRNAs and other ncRNAs, which show the potential to represent future biomarkers to monitor disease status and response to therapeutic interventions or act themselves as novel therapeutic targets. The same is true for other NMDs that cannot be further discussed because of journal word limits. For example, epigenetic changes are increasingly observed in tissues from patients with neuromuscular junction disorders [124,125] as well as peripheral neuropathies, either hereditary [126] or induced by drugs or diabetes [127–129], paving the way for their future clinical applications. Future perspective Following standardization of protocols, the combined search for DNA methylation and point mutations, expansions or contractions at disease-specific loci is foreseen to represent an invaluable tool in diagnostics and genetic counseling for NMDs in the near future. Moreover, taking advantage of recent next-generation sequencing approaches, novel genetic and epigenetic factors acting as disease modifiers could be easily discovered, enabling a more accurate genotype–phenotype correlation. Novel inhibitors of DNMTs, HDACs or histone lysine methyltransferases are being designed and tested in cell culture and animal models of NMDs and will probably be explored in human clinical trials in the near future. MiRNA dysregulation often overlaps among different NMDs that share similar neuronal or skeletal muscle impairment, so the author predicts that certain miRNAs will be more useful for monitoring disease progression and treatment efficacy than as diagnostic biomarkers. NcRNAs will be further explored as targets of small molecules designed to regulate the expression of their target genes in NMD cells. Despite the fact that further effort is required to improve delivery and stability of these molecules, their application in combination with gene-editing approaches will represent one of the most attractive fields of research in these disorders. Moreover, novel epigenetic biomarkers, including mitochondrial epigenetic changes and circRNAs, require a deeper investigation to clarify their pathogenic contribution and translational potential in NMDs. Financial & competing interests disclosure The authors have no relevant affiliations or financialinvolvement with any organization or entity with a financial interest in or finan- cial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript. 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