Myotonic Dystrophy: Sum and Substance

Ashok Kumar and Sarita Agarwal*

Department of Medical Genetics, SGPGIMS, India

*Corresponding author: Sarita Agarwal, Department of Medical Genetics, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India

Received: March 23, 2015; Accepted: May 04, 2015; Published: May 06, 2015

Abstract

Myotonic dystrophy is an autosomal dominant, multisystem disorder that is characterized by myotonic myopathy. The unstable repeat expansions of (CTG)n repeat in the 3’-UTR of the DMPK gene and a (CCTG)n repeat in intron 1 of the CNBP (formerly ZNF9) gene cause the two known subtypes of myotonic dystrophy: (i) Myotonic Dystrophy type 1 (DM1) and (ii) Myotonic Dystrophy type 2 (DM2) respectively. This review will focus on the molecular pathophysiology, genetics, diagnosis, management and therapeutics aspect of myotonic dystrophy. The length of the (CTG)n repeat expansion in DM1 correlates with disease severity and age of onset. The symptoms and severity of Myotonic Dystrophy Type 1 (DM1) ranges from severe and congenital forms. In adult patients, cardiac conduction abnormalities may occur and cause a shorter life span. In subsequent generations, the symptoms in DM1 may present at an earlier age and have a more severe course (anticipation). In Myotonic Dystrophy Type 2 (DM2), no anticipation is described, but cardiac conduction abnormalities as in DM1 are observed and patients with DM2 additionally have muscle pain and stiffness. Because of the disease characteristics in DM1 and DM2, appropriate molecular testing and reporting are very important for the optimal counselling in myotonic dystrophy. There is currently no cure but supportive management helps equally to reduce the morbidity and mortality and patients need close follow up to pay attention to their clinical problems.

Keywords: Myotonic dystrophy; Triplet repeat; TP-PCR; Gene therapy

Introduction

Myotonic Dystrophy (DM) is a chronic, slowly progressing, highly variable, inherited multisystem autosomal-dominant disease characterized by marked intrafamilial and interfamilial clinical variability. There are two types of DM, namely myotonic dystrophy types 1 and 2 (DM1 and DM2).

Myotonic dystrophy type 1 (DM1, MIM 160900) is the most frequent adult-onset muscular dystrophy. It was first clinically recognized by Steinert [1], Batten and Gibb [2]. The main characteristics of DM1 are myotonia, progressive muscle weakness and wasting and a broad spectrum of systemic symptoms [3]. Its clinical expression is unusual, characterized by a marked variability between and within pedigrees [3,4] and a striking genetic anticipation [5] where the age-at-onset typically decreases by 25 to 35 years per generation [6]. Based on clinical ascertainment, worldwide prevalence is estimated to be 12.5/100000 [3] but it can be higher as many patients in older generation remain undiagnosed. On the basis of clinical severity the disorder is divided into three groups mid, classical and congenital. Out of these three, the congenital form is the most severe [7].

DM2 (proximal myotonic myopathy) has similar disease manifestations, although they are generally less severe and usually of later onset [8].

In the present review we will emphasize on the molecular pathophysiology, genetics, diagnosis, management and therapeutics aspect of myotonic dystrophy.

Genetic Insight of Disease

DM1 is an autosomal dominant disorder caused by an expansion of an unstable CTG trinucleotide repeat in the 3’ Untranslated Region (UTR) of the gene DMPK (Myotonic Dystrophy Protein Kinase) located on chromosome 19q13.3, which codes for a myosin kinase expressed in skeletal muscled ‘myotonin protein kinase’ [9,10]. The DMPK gene is ~14 kb and encodes 2.3 kb of mRNA with 15 exons and the protein (cAMP-dependent serine-threonine kinase) of 624 amino acids [11,12].

Normal individuals have between 5 and 37 CTG repeats. CTG repeat lengths exceeding 37 are abnormal. Patients with between 38 and 49 CTG repeats are asymptomatic but are at risk of having children with larger, pathologically expanded repeats [13]. This is called a ‘pre-mutation’ allele. Full penetrance alleles occur with repeats greater than 50 CTGs and are nearly always associated with symptomatic disease although there are patients who have up to 60 repeats who are asymptomatic into old age and similarly patients with repeat sizes up to 500 who are asymptomatic into middle age. CTG repeat sizes in patients range from 50 to 4000. Molecular genetic testing detects mutations in 100% of affected individuals. Allele sizes were established by the Second International Myotonic Dystrophy Consortium (IDMC) in 1999 [14]. The disorder shows a phenomenon of genetic anticipation in which affected individuals in succeeding generations have an earlier age of onset and a more severe clinical course [15] due to the expansion of the repeat number during gametogenesis.

DM2 is an autosomal dominant disorder caused by a mutationin the ZNF9 (zinc finger protein 9) gene on chromosome 3q21. The first intron in ZNF9 contains a complex repeat motif (TG)n(TCTG) n(CCTG)n. Expansion of the CCTG repeat causes DM2 [16,17]. The repeat expansion for DM2 is much larger than for DM1, ranging from 75 to over 11000 repeats. Unlike DM1, the size of the repeated DNA expansion does not correlate with age of onset or disease severity in DM2. Anticipation is less evident clinically in DM2. A congenital form of DM2 has not been reported.

Molecular pathogenesis of disease

In DM1, the expanded CTG repeat is transcribed into DMPK mRNA but it does not affect DMPK protein structure. Rather, this expansion leads to production of a mutant DMPK mRNA which forms aggregates in affected nuclei [18] and the relative haploinsufficiency of the DMPK protein is not responsible for disease phenotype [19]. How does an accumulation of aberrant RNA lead to disease? It is proposed that many clinical manifestations of the disease are caused by alterations in the levels and activity of specific RNA-binding proteins involved in RNA splicing and those changes in levels and stability of these proteins then lead to aberrant splicing of many downstream RNA targets [20,21]. In general, the resultant spliceopathy leads to a splicing pattern more consistent with fetal expression than normal adult splicing patterns. Specifically, Muscleblind-like protein 1 (MBNL1) protein has been found to colocalize with mutant DMPK mRNA foci and this cause sequestration of MBNL1 by the RNA foci resulting in a loss of function that ultimately affects downstream targets [22,23]. Recent analysis has suggested that at least 200 targets are mispliced in a mouse model of DM1 and that >80% of these misplicing events are likely due to functional MBNL1 loss [24]. In support of this, genetic deletion of MBNL1 leads to a subset of the phenotypes seen in DM1, like cataracts, splicing alterations, myotonia and changes in muscle histology [25]. Interestingly, MBNL1 is sequestered with RNA foci in DM2 as well, and many aspects of the spliceopathy seen in DM1 are also found in DM2 [26]. In addition to MBNL1 sequestration, the levels of another RNA binding protein, CUG-Binding Protein 1 (CUGBP1) are increased in affected tissues. This is thought to be mediated by Protein Kinase C (PKC) phosphorylation and resultant stabilization of CUGBP1, although the details of this mechanism are poorly understood [27]. CUGBP1, like MBNL1, has important roles in RNA splicing and is thought to be antagonistic to MBNL1 for many of the splicing defects observed in DM1. Thus, the increase in CUGBP1 could synergize with the functional loss of MBNL1. CUGBP1 also has additional roles in RNA stability and translation [28-31]. Missplicing of certain downstream targets is then considered directly responsible for at least some of the observed phenotypes of DM1, and new targets are continually being discovered. For example, it has been elegantly shown that mis-splicing of the muscle-specific chloride channel ClC1 is responsible for the myotonia observed in DM1 models and patients. Aberrant splicing of the ClC-1 pre-mRNA leads to inclusion of exon 7a into the mature mRNA (a pattern more consistent with embryonic ClC-1 expression). Exon 7a inclusion ultimately results in a premature stop codon, resulting in rapid decay of the mis-spliced transcript [32,33]. Other splicing abnormalities may be responsible for insulin resistance (aberrant splicing of insulin receptor), some of the cardiac phenotypes (cTNT), and likely some of the central nervous system cognitive effects [34-37].

Diagnosis of DM1

System, muscular skeletal, cardiovascular, GI, Endocrinology, CNS etc. features and diagnosis: After contraction of the muscle relaxation is greatly delayed. This is the clinical manifestation of the disease. It may be troublesome in some while other patients may not be aware of it. Muscle weakness is variable. Weakness of facial muscles gives facial change, pain in abdomen and constipation may be symptoms in some of them. The severity and age of onset of symptoms correlates well with number of triplet repeats. The CTG repeat size is positively correlated with severity of the disease and inversely correlated with age of onset of symptoms [38-41]. The cases with 50 to 150 repeats have mild manifestations in the sixth decade or later while the classical cases manifesting at a young age have 150 to 1000 repeats. The third variety is congenital and is associated with prenatal hydramnios, joint contracture and severe hypotonia at birth. These cases have more than 2000 repeats and the diseased allele is transmitted through the mother. Some of the neonatal DM1 succumb to respiratory failures while some survive but have developmental disability. Other symptoms are also involved in myotonic dystrophy and features include cardiac rhythm abnormalities, cataract, diabetes mellitus, testicular atrophy and prenatal balding [3,42,43]. In cases without clinically obvious myotonia electromyogram is useful to demonstrate myotonic discharges. Creatinine phosphokinase may be mildly elevated in DM1 and muscle biopsy is only rarely required but required in cases with neuromuscular complaints and with negative genetic analysis [42,44]. However, clinical diagnosis is possible in most of the cases but molecular diagnosis is needed to differentiate between DM1 and DM2. In some cases it is difficult to differentiate from myotonia congenital and myopathies.

Molecular diagnosis: The detection of expansion of triplet repeat traditionally is done by Southern blot analysis [45,46]. However, Triplet Primed PCR (TP-PCR) based testing is found to be a reliable non-radioactive method as a replacement of the Southern blot [47- 50]. The number of triplet repeats correlates with the phenotype and hence, it is important for prediction of severity of the disease [38-41]. In the clinical scenario of DM, if DM1 mutation is not detected, the mutation in DM2 should be tested. The mutation detection helps in the confirmation of the diagnosis, predicting severity and providing genetic counselling. After molecular confirmation of the diagnosis in the proband, the family can be counselled for prenatal diagnosis and diagnosis can be offered to presymptomatic carriers in the family. Mutation detection is necessary to differentiate between DM1 and DM2 as in this era linkage analysis for prenatal or presymptomatic diagnosis has a limited role. The application of TP-PCR [49] and multiplex PCR over Southern blotting [45] for screening of triplet repeat expansion in DM1 is now recommended.

Molecular Pathogenesis of Disease

A summary of the steps in the pathogenesis of DM1 is shown in Figure 1, and the instances in which molecular or symptomatic therapy have been attempted are noted. General concepts in therapeutic design strategy are detailed in Figure 2. Targeting the earliest stage of aberrant pathophysiology is often the most difficult and technically challenging approach (in this case targeting DNA repeat expansion), although it has the potential for the greatest therapeutic benefit since all downstream effects are then modified.As one targets further down the cascade, potential therapies may be easier to design and implement, however, only a subset of pathology would be treated. For example, targeting splicing abnormalities in the chloride channel mRNAs in DM1 patients may be more feasible than targeting DNA repeat expansions. The resultant effect, however, would address only the myotonia in skeletal muscle, and other systemic complications would still be present. Thus, an ideal therapy would need to strike a pragmatic balance to allow for substantial benefit, relative ease of technical approach, and minimal effect on unrelated cellular mechanisms. Theoretically, and in practice, each of the abnormal steps in the pathogenesis of DM1 could be targeted for interventional modification. There is very little specific treatment that is distinct for DM2 and the multisystem pathologies of DM2 are similarly treated and monitored. Below we will discuss the varied therapeutic approaches as well as their potential applications and limitations to human disease.

Figure 1: Steps in pathogenesis and potential therapeutic targets in DM1. Myotonic Dystrophy results from the expansion of CUG repeats in the mutant RNA, rather than from reduced or dysfunctional protein levels. These repeats form a hairpin structure that can bind and sequester MBNL1 and potentially other RNA-binding proteins. Through mechanisms that are still poorly understood, this toxic RNA also causes hyperphosphorylation and stabilization of CUGBP1. The functional loss of MBNL1 and increase in available CUGBP1 are thought to result in misplicing of downstream RNA targets (largely to fetal isoforms) causing many, if not all, of the observed phenotypes. Attempts have been made to target each of the steps in pathogenesis, although the current effective therapy is limited solely to symptom management.

    
    
    Figure 1:  Steps in pathogenesis and potential therapeutic targets in DM1.
Myotonic Dystrophy results from the expansion of CUG repeats in the mutant RNA, rather than from reduced or dysfunctional protein levels. These repeats form a
hairpin structure that can bind and sequester MBNL1 and potentially other RNA-binding proteins. Through mechanisms that are still poorly understood, this toxic
RNA also causes hyperphosphorylation and stabilization of CUGBP1. The functional loss of MBNL1 and increase in available CUGBP1 are thought to result in
misplicing of downstream RNA targets (largely to fetal isoforms) causing many, if not all, of the observed phenotypes. Attempts have been made to target each of
the steps in pathogenesis, although the current effective therapy is limited solely to symptom management.

Figure 2: Therapeutic Design approach. DM1 is caused by a repeat expansion in the DNA which is then incorporated into a mutant RNA. This toxic RNA then leads to a cascade of mispliced RNA transcripts and multisystem manifestations of disease. The mechanism of pathogenesis is simplest at the stage of the expanded DNA repeat and becomes increasingly more complicated as more cellular processes and pathways are involved in the downstream effects. Thus, intervention at the level of the DNA provides the most therapeutic benefit but is least technically feasible. Conversely, intervention at the level of one mis-spliced target may be most technically approachable, but therapeutic impact is then limited to only one portion of the disease phenotype.

    
    
    Figure 2:  Therapeutic Design approach.
DM1 is caused by a repeat expansion in the DNA which is then incorporated into a mutant RNA. This toxic RNA then leads to a cascade of mispliced RNA
transcripts and multisystem manifestations of disease. The mechanism of pathogenesis is simplest at the stage of the expanded DNA repeat and becomes
increasingly more complicated as more cellular processes and pathways are involved in the downstream effects. Thus, intervention at the level of the DNA provides
the most therapeutic benefit but is least technically feasible. Conversely, intervention at the level of one mis-spliced target may be most technically approachable,
but therapeutic impact is then limited to only one portion of the disease phenotype.

DM1 and DM2 have 3 different problems: (1) myotonia, (2) muscle weakness, (3) systemic manifestations like cataracts, diabetes mellitus, cardiac arrhythmia, and others (Table 1). Both DM1 and DM2 progress through defect in chloride (Cl) channel. To date we do not have any medication to open Cl channels of the skeletal muscles; therefore we have been using Na channel blocker for myotonia. Mexiletine, Procainamide, Phenytoin, Carbamazepine are all Na channel blockers which were originally developed for cardiac arrhythmia, or seizure disorders. Since myotonic dystrophy has progressive muscle weakness due to the dystrophic nature of this disorder, even if myotonia is reduced, muscle weakness is a bigger problem. Na channel blockers tend to reduce muscle power by decreasing muscle action potentials and these unwanted effects of Na channel blockers are the problem. Therefore, it is important to develop medications for myotonic dystrophy which can reduce myotonia and yet maintain the muscle strength as it is.