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Myotonic dystrophy (DM) is a complex, slowly progressing, highly variable, mutisystemic disorder which occurs in patients of any age. It is an autosomal dominant clinical syndrome affecting approximately 1 in 8000 individuals worldwide and is characterised by myotonia in skeletal muscles, progressive myopathy, cateracts, gonadal dysfunction, mental retardation, and cardiac conduction defects (Machuca-Tzili et al. 2005; Harper 2001). DM is caused by nucleotide expansion repeats above a critical size. There are two main types of the disease, myotonic dystrophy type-1 (DM1) and myotonic dystrophy type-2 (DM2), caused by tandem repeat expansions in different genes. DM1, the most common form of the disease, is caused by cytosine-thymine-guanine (CTG) trinucleotide repeats, whereas DM2 is caused by cytosine-cytosine-thymine-guanine (CCTG) tetranucleotide repeats.
The DM1 mutation was identified in 1992 to be expanded CTG repeats located within the 3'-untranslated region (3'-UTR) of the DMPK (dystrophia myotonica protein kinase) gene located on chromosome 19 at position 13.3 (J. David Brook et al. 1992). In healthy, unaffected individuals there are between 5-38 CTG repeats here. Symptoms can occur in DM1 patients with as few as 50 repeats, with larger expansions (up tp >4000) correlating with an increased severity of the disease and also a lower age of onset (Tsilfidis et al. 1992). DM1 also shows an increased severity in phenotype through inheritance in successive generations, due to the repeats in the DNA becoming larger through meiosis and replication. This is termed "anticipation", and can occur in many nucleotide repeat disorders when DNA polymerase "slips" on the repeat sequence during replication, forming a hairpin structure. This is incorporated into DNA and over each replication cycle the repeat sequence grows larger. DM1 is considered the more severe of the two disease types, exhibiting adult-onset, child-onset, and congenital (the most severe) forms of the disease. Only the adult-onset form has been reported in DM2.
Unlike DM1, the repeat size does not appear to affect the severity or age of onset for DM2. The CCTG expansions in DM2 are generally much larger than in DM1, ranging from 75 - 11,000 repeats in length, with a mean size of 5000 repeats (Liquori et al. 2001). The fact that increased repeat length does not affect disease severity in DM2 may be because of a "ceiling" effect, where any further increase to repeat size will have no effect on disease severity because the repeat is already so long it results in maximal effects. DM1 repeats are generally much shorter, and below this theoretical ceiling threshold, so that an increase in length will still have an effect on severity.
There have been several hypotheses proposed to explain DM pathogenesis. Initially, when only one disease type (DM1) was known, the DMPK gene was investigated. The idea being that the CTG repeats inhibited DMPK mRNA or protein production, which resulted in DMPK haploinsufficiency. Expression of DMPK mRNA and protein was shown to be reduced in DM1 muscle (Fu et al. 1993). DMPK knockout mice were also shown to exhibit cardiac conduction defects (Berul et al. 1999), a characteristic symptom of DM. (insert diagram from this cardiac conduction defects paper?) However, this study only focused on cardiac conduction problems, and no findings of other DM symptoms were reported. Mice only exhibited mild myopathy in another DMPK-knockout study by Jansen et al. (1996), and still lacked many of the multisystemic symptoms of the disease. Neither of these studies produced mice that exhibited myotonia, one of the characteristic features of the disease, and in fact to date, no DMPK loss-of-function mutation has been reported in DM1 individuals. The multisystemic features of the disease cannot therefore be explained by DMPK haploinsufficiency alone.
Another model behind the pathogenesis of DM1 involves the CTG repeats in the 3'UTR of the DMPK gene affecting the expression of adjacent genes. Otten & Tapscott (1995) demonstrated that a DNase I hypersensitive site is found 3' of the CTG repeat in wild-type alleles. DM1 individuals with large expansions exhibited alleles with DNase I resistance, as well as inaccessibility of nucleases to the adjacent hypersensitive site. This suggested the CTG repeat expansion alters the chromatin structure, causing a region of condensed chromatin, which could affect expression of adjacent genes. A homeodomain-encoding gene, SIX5, is located in this region and its mRNA level has been shown to be decreased in DM1 individuals (Charles A. Thornton et al. 1997). SIX5-knockout mice have also been shown develop cateracts, another symptom of DM, however no muscle pathology was observed in these mice (Klesert et al. 2000), which means decreased SIX5 expression cannot be the primary cause of DM1. A second gene, DMWD, is also located next to the DMPK gene and is has been shown to have decreased levels of RNA expression in some DM1 cell lines (Alwazzan et al. 1999). However, no specific link was determined between reduced DMWD expression and phenotype, or between repeat length and DMWD expression, probably due to the small sample population used and the lack of variation in repeat lengths within it. With regards to the effect on adjacent genes, more has been reported and is known about SIX5 than DMWD.
A RNA gain-of-function hypothesis was proposed in which the mutant RNA transcribed from the expanded repeat-containing allele is sufficient to induce disease symptoms. This hypothesis was based on several observations. The fact that the loss of function of DMPK or surrounding genes did not reproduce the major symptoms of the disease, which meant another main mechanism must be present. Also, it was shown that expression of only the DMPK 3'-UTR region with 200 CTG repeats was sufficient to inhibit myogenesis (Amack et al. 1999). It is also known that the expanded CTG repeats are transcribed into CUG repeats, which accumulate in discrete nuclear foci (Davis et al. 1997), a hallmark of dominant RNA-mediated diseases. The repeats are thought to form imperfect hairpins, becoming more stable as the repeat length increases. The association of RNA binding proteins leads to the accumulation of these repeat containing RNAs within nuclear foci. The result of this is ineffective metabolism of mutant mRNA, and the bound proteins can no longer perform their normal functions. A study by (A Mankodi et al. 2000) gave direct experimental support for a RNA gain-of-function mechanism behind DM. In this study they used a mouse model containing 250 CTG repeats in the 3'UTR regions of the human skeletal Î±-actin gene. This gene is not linked with DM, and yet the mice developed myotonia and showed muscle histology similar to DM1 individuals. This demonstrated that on their own, the repeats were enough to induce pathogenic features of DM1, irrelevant of the gene context.
A second type of DM, termed DM2, was identified in 1998 (Ranum et al. 1998) and in 2001 it was attributed to CCTG repeats within intron 1 of the zinc finger 9 (ZNF9) gene on chromosome 3 (Liquori et al. 2001). Patients with DM2 showed similar symptoms to those with DM1, although subtle differences existed and no congenital form has been reported in DM2. The fact these two repeat sequences are located in completely different genes and on different chromosomes, yet can still cause such similar symptoms supports the idea of RNA gain-of-function as a common pathogenic mechanism.
Repeat containing RNA can cause pathogenic disease features through interaction with RNA-binding proteins. Two important proteins have been identified by their ability to bind CUG repeats in RNA: CUGBP (CUG-binding protein) and MBNL1 (Muscleblind-like 1). CUG repeat expansions can fold into hairpin-like structures (Napierala & Krzyzosiak 1997) and particular proteins can be sequestered to these hairpins, altering the levels at which they are present in the nucleoplasm.
MBNL1, was identified in a study by Miller et al. (2000) when looking for proteins that bind CUG repeat expansions. In DM1 cells, MBNL1 is sequestered to the nuclear foci in DM1 cells containing CUG repeat RNA, thereby depleting it from the nucleoplasm (Jiang et al. 2004). Mice with a modified MBNL1 gene that had a deletion of exon 3 (containing a RNA-binding motif) developed some of the characteristic DM1 features, including cateracts and myotonia, as well as exhibiting alternative splicing defects (Kanadia et al. 2003). Another study by Kanadia et al. (2006) showed that overexpression of MBNL1 in a mouse model for DM was shown to restore normal splicing and reverse myotonia. Disrupted alternative splicing is one of the most important molecular features of DM and the mis-regulated splicing events are believed to be behind many of the symptoms of DM. Mis-regulation of splicing of the skeletal muscle-specific chloride channel 1 (ClC-1) leads to the myotonia seen in DM1, for example (Mankodi et al. 2002; Charlet-B et al. 2002). The two studies by Kanadia et al. suggested that depletion of the MBNL1 protein, a regulator of alternative splicing, resulted in many of the downstream splicing problems that gave rise to multiple DM phenotypes. Another study by (Ho et al. 2004) showed decreased expression of MBNL1 in cultured cells caused aberrant splicing in both the cardiac troponin T (cTNT) and insulin receptor (IR) genes, which is consistent with DM1 individuals.
While there is much evidence supporting MBNL1 sequestration as a cause of DM1 pathogenesis, it is not the only mechanism involved. It should be noted that in the study by Kanadia (2003), while mice did display symptoms such as cateracts and myotonia, no signs of muscle wastage appeared. This therefore suggests some features of the disease are due to other causes and not MBNL1 loss-of-function. Supporting this idea is a study by (Ho et al. 2005) that showed MBNL1 was sequestered to both CUG- and CAG-containing nuclear foci with similar affinity, however only the CUG repeats caused mis-regulation of splicing in the cTNT and IR genes. This therefore suggests sequestration of MBNL1 alone is not enough to cause all the splicing changes involved in DM, and other factors are in force.
Another protein involved in the modulation of splicing events is CUGBP. It was identified by (Timchenko et al. 1996) by its ability to bind short single-stranded CUG repeats. However, CUGBP does not in fact bind double-stranded repeats (such as those present in the hairpin structures), or co-localise with the foci of expanded repeat transcripts (Fardaei et al. 2001), and so is not sequestered like MBNL1. Instead, CUGBP levels are increased in DM1 muscle cells and heart tissue (Philips et al. 1998; Timchenko et al. 2001). A mouse model overexpressing CUGBP in muscle exhibited splicing defects and muscle problems similar to DM1 (Ho et al. 2005), which further supported the idea that CUGBP is involved in abnormal splicing and disease pathogenesis. Many other studies have reported similar findings, providing further evidence that increased CUGBP expression (and the subsequent downstream splicing problems) is one of the main effects of the DMPK repeat expansion (see Wang et al. 2007 and Orengo et al. 2008, for example).
A study by Timchenko et al. (2001) revealed that DM myoblasts become incapable of cell cycle withdrawal during differentiation. Their findings suggested that alterations in CUGBP levels disrupted a CUGBP translational target, p21, resulting in impaired withdrawal from the cell cycle in DM patients. Another mouse model study by Timchenko et al. (2004) revealed CUGBP to have a role in the regulation of translation. It interacts with the mRNA transcript of MEF2A (myocyte enhancer factor 2A), increasing its translation. MEF2A is a transcription factor involved in myogenesis, and its levels are found to be increased in DM1 skeletal muscle, which is believed to contribute towards delayed myogenesis.
The entire mechanism through which CUGBP levels are increased because of the repeat containing RNAs is not fully known, although it has been shown that PKC (protein kinase C) mediates phosphorlyation of CUGBP (Kuyumcu-Martinez et al. 2007). The expression of expanded CUG repeats in DMPK RNA induces hyperphosphorylation of CUGBP, which causes an increased protein half-life and steady-state levels. This study used a phorbol ester to activate PKC, and the direct phosphorylation of CUGBP by both PKCÎ± and PKCÎ²II was observed in vitro. PKCÎ± and PKCÎ²II were also found to be activated in cells expressing DMPK-CUG-repeat containing RNA in DM1 cell cultures, DM1 tissues, and also a DM1 mouse model. The mechanism through which the repeat containing RNA activates the PKC pathway remains unknown, although this will surely be an area for future work and it could lead to some potential treatment areas for the disease.
There is a wide range of effects the aberrant splicing, caused by altered levels of CUGBP and MBNL1, can have (figure #). For example, DM patients are predisposed to diabetes type II, due to an unusual form of insulin resistance caused by a defect in skeletal muscle. DM patients express inappropriate levels of the IR-A (insulin receptor-A) isoform, due to mis-regulation of splicing resulting in increased skipping of the IR exon 11. This abnormal splicing causes a switch from IR-B to IR-A (Savkur et al. 2001). The IR-A isoform has a twofold higher affinity for insulin, as well as a faster internalisation and recycling time than the B isoform. The mis-regulation of IR alternative splicing can be reproduced in normal cells by the expression of CUG-repeat RNA, and also the expression of CUGBP in normal cells induces the change to IR-A as well (Savkur et al. 2001).
Aberrant splicing events, such as that observed for IR, cause a variety of different symptoms. The exact mechanism behind some of the phenotypes still remain relatively unknown (figure #) and are areas of future work. The aberrant splicing events involved in the cTNT, ClC-1, and IR are among the better understood pathways behind DM phenotypes (see Ho et al. 2004, Mankodi et al. 2002, and Savkur et al. 2001, for example). For these three examples (cTNT, CLC-1, and IR), when CUGBP induces inclusion of an exon, MBNL1 increases exclusion of that exon, and vice-versa.
Interestingly, in these three examples (cTNT, IR and ClC-1), when CUGBP1 induces inclusion of an exon, MBNL1 increases exclusion of the same exon and vice versa. Kalsotra et al.  performed a large screen to identify alternative splicing events regulated during mouse heart development and identified CUGBP1 and MBNL1 as regulators of more than half of the splicing transitions tested. Many of these developmentally regulated splicing events are modulated exclusively by CUGBP1 or MBNL1, but all events regulated by both proteins exhibit antagonistic responses . In addition, while MBNL1 nuclear levels increase during development, CUGBP1 nuclear levels decrease [22,42], supporting the hypothesis that the level and localization of these two proteins control a fetal to adult splicing transition, which is reversed in DM1 tissues (Figure 2).
Potential causes of clinical distinctions: One possible mechanism for modulating the toxic RNA effect could be that CUG-BP and MBNL affinity for CUG differs from their affinity for transcripts containing CCUG.
Clinical differences could reflect variation in the RNA effects produced by dissimilarities in temporal and cell specific expression patterns of DMPK and ZNF9.
Alternatively, there could be a separate mechanism accounting for phenotypic differences differences between DM1 and DM2 such as alterations in the expression of locus specific genes including DMPK and SIX5 in DM1.
Mention the DM3 thing and criticise it.