Myotonic disorders are diseases in which symptomatic individuals have difficulty in relaxing a muscle group after contraction. One hallmark feature of these diseases is the difficulty of affected individuals of releasing grip on objects like tools, cups and door knobs for example (Harper, 2001). In 1876, came one of the first documented accounts of a myotonic disorder from Dr Julius Thomsen by studying his most famous patient, himself. The disease he described, known today as Thomsen’s myotonia congenita , was of hereditary nature, caused myotonia and muscle hypertrophy but was not progressive in nature. Since no muscle weakness is observed in this disease, myotonia congenita is not considered to be a muscular dystrophy. Some thirty years later, in the early 20th century, another myotonic disorder was described with much more pronounced and degenerative symptoms. Myotonic dystrophy or dystrophia myotonica was first described by a German physician by the name of Hans Steinert in 1904 (Harper, 2001). He was the first physician to publish a detailed account of a new myotonic disorder in which he noted symptoms such as muscle weakness and wasting, facial weakness, ptosis, selective atrophy of the sternomastoids in the neck and testicular atrophy which underlined an endocrine disturbance. Steinert also conducted pathological studies on an autopsied case where he noted extensive fibrosis and degeneration of skeletal muscle. While Steinert died in 1910 shortly after publication of his paper, others by the names of Batten and Gibb pursued the work and clearly established myotonic dystrophy as a distinct degenerative neurological disorder. They documented that the disease affected several family members and all had the characteristic symptoms of progressive difficulty in walking with weakness of the legs and grip and also a striking lack of expression due to weakness of the facial muscles. Over the past century, through progress in medicine and technology, several clinical and physio-pathological discoveries helped paint a more defined picture of the characteristic symptoms of myotonic dystrophy. It was not however until 1992, that the genetic defect was coined by several teams showing that an unstable CTG repeat expansion in the 3’untranslated region of a gene could be the molecular basis for the DM pathology (Aslanidis et al., 1992; Brook et al., 1992; Buxton et al., 1992b; Fu et al., 1992; Harley et al., 1992a; Mahadevan et al., 1992).
There are three typical forms of myotonic dystrophy established according to the symptoms and the age of onset. Adult onset myotonic dystrophy is the most common and the less severe form of the disease with symptoms appearing in the early twenties to the late forties. Congenital myotonic dystrophy is the most severe form of the disease with symptoms present at birth. Childhood myotonic dystrophy is when the disease first appear early in life. Although less severe than the congenital cases, this form can be associated with mental retardation and retardation in muscle development. Childhood myotonic dystrophy will not be addressed here.
What is striking about myotonic dystrophy is the wide variability in both the symptoms and the age of onset. This neuromuscular disease not only alters normal skeletal muscle functions but also disrupts smooth muscle function, the cardio respiratory system, the endocrine system, the eyes and also the brain and personality (Harper, 2001). Myotonic dystrophy must thus be seen as a generalized disorder and not just one of the skeletal muscles. Table 2 resumes the clinical symptoms that are most frequently encountered within various organs and systems in patients with the adult onset form of myotonic dystrophy. Although many symptoms can be present simultaneously in any given affected individual, the severity of the disease and the extent of the symptoms is often dictated by the age of onset (Harper, 2001; Moxley, 1992). Other than the apparent clinical symptoms are the histological abnormalities in the tissues of myotonic dystrophy patients, especially in the eyes and muscle:
• Increased central nuclei (centronucleation)
• Corneal lesions and crystalline lens opacity
The severe form of the disease that is present at birth is called congenital myotonic dystrophy. This form is almost exclusively seen when the mother is the carrier of the disease (Harper, 2001). This devastating form of myotonic dystrophy is often regarded as a distinct neuromuscular disease altogether because of the likely involvement of developmental disturbances, the extreme severity of the symptoms and the heavy neurological component that is not present in adult onset cases. This form is associated with hypotonia and also cardiac and respiratory complications that very often lead to the death of the neonate (Harper, 2001). Neurological features include mental retardation, delayed motor development and difficulties in feeding, swallowing and speech. The condition of the few neonates that survive into childhood seems to improve at first, but rapidly regresses once the adult and degenerative features of the disease appear. Individuals with the congenital form are heavily disabled throughout their life and do not live beyond 30 years of age (Harper, 2001).
Table 2: Myotonic dystrophy clinical symptoms in adults
The first attempts of positioning the DM mutation were carried out in the early 70’s when linkage was first established with the Lutheran blood group utilizing family studies in the USA and Britain (Harper et al., 1972). The presence of the Lutheran antigen a+ Lu(a+) was consistently passed down with the DM mutation in the studied families (Harper et al., 1972). In the following years, several other loci (ABH secretor locus, peptidase D, Lewis blood group, complement C3, ApoE) were all linked with transmission of the DM mutation (Harper et al., 1972; O'Brien et al., 1983; Schrott and Omenn, 1975; Sistonen, 1984; Whitehead et al., 1982). Assignment of the complement C3 loci to chromosome 19 in 1982, made it clear that DM mutation was also located on that chromosome (Whitehead et al., 1982). The DM mutation was then further narrowed down to the 19p13-19q13 region when linkage to its closest markers, the genes for apolipoprotein C2 (ApoC2) and the muscle-specific creatine kinase (CKMM) were established by studying French Canadian, British French and Dutch families (Brunner et al., 1989; Shaw et al., 1985). The final positioning of the DM gene was done in 1991 by Harley and co-workers by establishing that the DM gene lies in region 19q13.2-q13.3, telomeric to the ApoC2 and CKMM genes (Fig. 1) (Harley et al., 1991).
2) Severity of the disease is variable but generally correlates with increased expansion length
Table 3: Trinucleotide repeat diseases
*General approximation. Each SCA disease has its own defined clinical threshold (Martin, 1999)
Anticipation in myotonic dystrophy and also in other triplet repeat diseases is a hallmark clinical feature that has been documented for more than 90 years (Harper, 2001; Moxley, 1992). It was not until the early 90’s, once the mutation had been identified, that extensive studies were conducted to monitor several key components of anticipation in myotonic dystrophy. These studies were done on several families and looked at parameters such as the age at onset of the affected parent, the age at onset of offspring, the number of repeats in affected parents and offspring and the influence of the sex of the affected parent on the outcome of the disease in the offspring (Ashizawa et al., 1994; Ashizawa et al., 1992a; Ashizawa et al., 1992b; Harley et al., 1993). The results revealed that although contraction of the mutation was observed in 10% of cases when the father carried the mutation and in 3% of cases when the mother had the mutation, the general tendency was that offspring had an increase in the length of the repeat tract and severity of the disease (Ashizawa et al., 1992b). The increment in CTG repeats in children with myotonic dystrophy is closely related to the length of the repeats in the parents, especially if the mother is a carrier of the mutation (Barcelo et al., 1993; Harper, 2001). Observations on families with myotonic dystrophy allowed thresholds linking CTG repeat number amplification and disease severity to be established (table 4).
Table 4: Critical CTG expansion thresholds for myotonic dystrophy onset and symptoms
Number of CTG repeats |
Phenotype |
Comments |
3 to 49 |
Normal individuals |
Over 90% of the general population has less than 35 CTG repeats in the DM1 locus. |
50 to 80 |
Very mild or no apparent symptoms except cataracts |
Expansions in this range are incremental from generation to generation. |
80 -1000 |
Full mutation, adult onset |
80 CTG repeats is the threshold for saltatory amplification. |
600 and more |
Full mutation, adult onset |
High risk of transmitting the congenital form if the mother is the carrier. |
1000 to 3000 |
Generally congenital |
Such expansions are most frequently seen in congenital cases. |
1) The expanded allele appears as a smear on Southern blots
2) Identical twins affected by myotonic dystrophy have different expansion patterns
3) Immortalized lymphocytes from patients show increased expansions in culture
4) Repeat lengths in patients blood and tissue increase with time
5) Repeat lengths in DNA vary from tissue to tissue in a same patient
Although the clinical and physiological perturbations of myotonic dystrophy have long been described, the more subtle molecular and cellular defects are much more elusive. It is still not clear today, twelve years after the initial identification of the mutation responsible for myotonic dystrophy, which are the molecular mechanisms involved in causing the vast range of symptoms seen in the disease.
Since the very beginning, it was shown that mutant DMPK RNA levels were altered in the disease. However, conflicting reports brought only confusion to whether mutant DMPK mRNA was reduced, elevated or unchanged in myotonic dystrophy (Carango et al., 1993; Fu et al., 1993; Sabouri et al., 1993; Wang et al., 1995). One group even reported complete absence of mutant DMPK mRNA from congenitally affected fetuses and infants (Hofmann-Radvanyi et al., 1993). Although the reasons for these variations were not understood at the time, it seemed to be clear that the lack or abundance of mutant DMPK mRNA was not the cause but a consequence of the disease.
Clues to understanding such variability in results came shortly afterwards with elegant work done by Taneja and co-workers showing that mutant DMPK mRNA with large CUG expansions were retained in the nucleus of myotonic dystrophy cells and form discrete foci when revealed by in situ hybridization (Fig. 3) (Taneja et al., 1995). Work performed by Davis and co-workers determined that mutant RNAs are linked to the nuclear matrix and were not exported to the cytoplasm (Davis et al., 1997). They showed by Northern analysis that this retention was not due to disruption of splicing or polyadenylation of the mutant transcripts (Davis et al., 1997). They also proposed that conflicting reports concerning mutant DMPK expression levels were most likely caused by improper RNA extraction and unreliable diagnosis methods.
Retention of the mutant DMPK mRNA causes a haplo-insufficiency of DMPK protein since only the normal transcripts are translated. Accordingly, it was demonstrated using specific anti-DMPK antibodies that DMPK protein levels were reduced to 50-57% of the levels in normal myoblasts (Furling et al., 2001a; Furling et al., 2001b). DMPK is a cyclic AMP-dependent serine/threonine protein kinase belonging to the Rho family. Fifteen exons predicts a 70.6 kDa protein with over 7 different tissue-specific splicing isoforms (Fig. 4a) (Groenen et al., 2000; Tiscornia and Mahadevan, 2000; Wansink et al., 2003). However, through strong post-translational modifications, certain isoforms of the protein can achieve an apparent molecular mass of up to ~ 80-86 kDa in skeletal muscle (Bush et al., 2000; Lam et al., 2000; Mahadevan et al., 1993; Wansink et al., 2003). These numerous DMPK isoforms exhibit cell-type and location-dependent substrate specificities possibly conferring to DMPK different physiological roles (Wansink et al., 2003). Figure 4b depicts the DMPK protein with its various structural domains. The function of these domains still needs to be defined, but one study has suggested that DMPK must be cleaved from it’s membrane associated domain in order to adopt an active conformation in the cytoplasm (Bush et al., 2000). Another study from the same group, suggests that phospholemman, a membrane-bound substrate for protein kinase A and C involved in ion transport, is also a substrate for DMPK phosporylation (Mounsey et al., 2000a). Modification of muscle Ca2+ and Na+ ion channels homeostasis could lead to an alteration of muscle excitability, as seen in myotonic dystrophy (Benders et al., 1997; Mounsey et al., 2000b). At this time, there are but speculations of the true biochemical role of DMPK in healthy individuals or in myotonic dystrophy (Wansink et al., 2003).
The development of specialized tools like mouse models and cell lines were necessary in order to examine the effects of the myotonic dystrophy mutation in live cells and tissues. In 1996, appeared the first mouse models to help understand myotonic dystrophy (Jansen et al., 1996). In these models, Jansen and colleagues disrupted the endogenous murine DMPK gene and/or overexpressed a normal human DMPK transgene. Their goal was to determine the influence of DMPK misregulation in the disease. Interestingly, the nullizygous mice they generated showed only mild atrophy in head and neck muscles, while the mice overexpressing human DMPK showed copy number-dependent cardiomyopathy (Jansen et al., 1996). In another study, the authors showed that these knockout mice showed cardiac conduction defects (Berul et al., 1999), which is in accordance with the previously published results and decreased muscular strength implicating a role for DMPK in maintenance of muscle structure (Reddy et al., 1996). However, none of these mouse models have exhibited the most prominent features of myotonic dystrophy such as the myotonia, cataracts and severe muscle wasting. These results clearly point out that the DMPK protein itself has but a subtle role in myotonic dystrophy pathogenesis in mice. Since murine and human DMPK share much similarity, it is not expected that DMPK plays any critical role in humans. Also, patients homozygous for the DM mutation do not exhibit symptoms more severe than heterozygous patients with a similar expansion length (Harper, 2001). This implies that the complete lack of DMPK protein in humans does not account for any visible phenotype at first glance.
• A high level of instability increasing with age in tissues and sperm
• Formation of foci revealed by a DMPK riboprobe
The initial idea of the gain-of-function effect of the RNA in myotonic dystrophy came in 1996 from Timchenko and co-workers when they identified CUG-BP, a heterogeneous nuclear ribonucleoprotein (hnRNP), that could bind to single-stranded CUG repeats and also accumulates in the nucleus of cells from myotonic dystrophy patients (Roberts et al., 1997; Timchenko et al., 1996a; Timchenko et al., 1996b; Timchenko et al., 2001a). CUG-BP is a phosphorylation substrate for the DMPK protein and exists as two phosphorylation isoforms that are ubiquitously expressed in both the cytoplasm and the nucleus (Roberts et al., 1997; Timchenko et al., 1996b). It is the hypophosphorylated isoform that accumulates in the nucleus of cells form myotonic dystrophy patients. The role hnRNPs, such as CUG-BP, is to perform multiple posttranscriptional regulatory functions such as splicing. CUG-BP is a member of the CELF family of RNA-processing factors that regulate alternative splicing (Ladd et al., 2001). Aberrant accumulation of the hypophosphorylated form of CUG-BP in the nucleus has been associated with abnormal splicing of several of its substrate mRNAs, ultimately lending support for a trans-dominant effect of expanded CUG repeats on RNA processing in myotonic dystrophy type 1 (Savkur et al., 2001). However, this accumulation is unlikely to be a titration effect from binding to the CUG repeat tract itself since accumulation of CUG-BP in the nucleus was not shown to be proportional to it’s length (Michalowski et al., 1999).
Since the discovery that mutant DMPK mRNA is fully transcribed and forms hairpin structures that are retained in the nucleus, much effort has been deployed to identify possible CUG-binding proteins (Michalowski et al., 1999; Taneja et al., 1995; Timchenko et al., 1996b). Identification of such proteins will give insight in the composition of nuclear foci that are now believed to be the cornerstone of the molecular defects involved in myotonic dystrophy. Four CUG binding protein candidates were identified from i n vitro binding studies: CUG-BP, PKR, ETR-3 and MBNL (Lu et al., 1999; Miller et al., 2000; Tian et al., 2000; Timchenko et al., 1996b). Only MBNL and certain of its family members have been shown to bind long double-stranded CUG repeats and co-localize with mutant DMPK mRNA-induced foci in vivo (Fardaei et al., 2002; Miller et al., 2000; Squillace et al., 2002). MBNL is a mammalian homologue of the Drosophila muscleblind (Mbl) protein essential for the terminal stages of muscle and photoreceptor differentiation (Begemann et al., 1997). MBNL is induced by myoblast differentiation in mice and is expressed in blood, eye, cardiac and skeletal muscle (Miller et al., 2000). Because of its involvement in muscle and eye development, cellular depletion of MBNL through binding to mutant DMPK mRNAs in the nucleus in myotonic dystrophy cells may provide some explanation to the myopathy and cataracts seen in the disease.
Myoblast differentiation is regulated by a sequential cascade of gene activation following the initial mitogen withdrawal (Fig. 7). Myoblasts are held in proliferation by presence of growth factors and of proteins Notch, Msx-1 and the bone morphogenic protein 4 (BMP-4), that block MyoD and Myf5 expression (Bailey et al., 2001). Transcription factors MyoD and Myf5 are the first genes to be activated since they are responsible for all downstream activation events in myogenesis. They bind the promoters of muscle-specific genes and activate their transcription (Rudnicki et al., 1993). Myogenin expression is the next key factor to be expressed and coincides with commitment of myoblasts to terminal differentiation (Sabourin and Rudnicki, 2000). Myogenin expression is crucial for fusion of myoblasts into multinucleated myotubes (Wright et al., 1989). Shortly following myogenin, the inhibitor p21 is expressed and marks the irreversible withdrawal from the cell cycle followed by expression terminal differentiation markers like myosin heavy chain (MyHC) and muscle creatine kinase (CK) (Sabourin and Rudnicki, 2000).
Trinucleotide CTG repeats in DNA form stable hairpin structure in vivo (Freudenreich et al., 1997; Mariappan et al., 1996). The chromatin structure in proximity of the repeat tract is highly perturbed and anomalously condensed from tight nucleosome assembly (Wang et al., 1994). This highly condensed region of the chromatin could result in transcription repression (Wang et al., 1994). In fact, the DMPK locus is flanked by two genes: upstream by DMWD and downstream by SIX5 (formerly known as DMAHP) (Fig. 8) (Alwazzan et al., 1999; Klesert et al., 1997). Both these genes have been shown to be downregulated in presence of large CTG expansions (Alwazzan et al., 1999; Klesert et al., 1997; Thornton et al., 1997). While the role of the DMWD gene product is suspected to be involved in brain-related symptoms in myotonic dystrophy, deficiency of SIX5 causes cataracts in knockout mouse models (Klesert et al., 2000; Sarkar et al., 2000; Westerlaken et al., 2003). While these results support a cis effect for the DM mutation, these mouse models did not present any muscle-related abnormalities (Klesert et al., 2000; Sarkar et al., 2000).
Since the discovery of the DM mutation, genetic testing has been used to validate clinical diagnosis for myotonic dystrophy. These tests revealed that 98% of patients who displayed clinical symptoms for myotonic dystrophy had in fact the DM mutation (Mahadevan et al., 1992). However, between 1-2 % of patients that displayed most of the classical symptoms of myotonic dystrophy did not have the characteristic CTG expansion in 3’UTR of the DMPK gene (Mahadevan et al., 1992).
1. The predominant distributions of muscle weakness is proximal in PROMM
2. PROMM is associated with muscle pain
3. PROMM patients display type II fiber atrophy as opposed to type I
4. Facial weakness is absent in PROMM
5. No CNS involvement has been documented for PROMM
The great heterogeneity of symptoms and defects at the molecular, cellular and metabolical level make myotonic dystrophy an extremely complicated disease to understand. Identification of the mutation in 1992 only confirmed that understanding the basis of the disease was still a long way down the road. There is now a general consensus that multiple factors contribute to the disease. Three theories have been put forward over the past 5 years to give some explanation on how triplet expansions in the untranslated region of a gene can cause myotonic dystrophy (Tapscott, 2000; Tapscott and Thornton, 2001):
2) Modification of the regional chromatin alters adjacent gene expression
Much of this three-level theory is based upon results generated with the three major mouse models:
1) DMPK knock-in/out mice (Jansen et al., 1996)
3) SIX5 knock-out mice (Klesert et al., 2000; Sarkar et al., 2000)
Since it is now clear that myotonic dystrophy type I is caused by a toxic mRNA, what are the therapeutic approaches available? While certain features of the disease phenotype such as the myotonia, arrhythmias and daytime drowsiness are treated today with drugs, surgery for other features such as cataracts and ptosis has been successful (Harper, 2001). Nonetheless, treatment of symptoms is only provisional since the disease is of neurodegenerative nature and worsens with time. A successful RNA-based gene therapy may be the one of the most effective ways at this time to attenuate symptoms of the disease.