Brain Research Bulletin, Vol. 56, Nos. 3/4, pp. 389 –395, 2001 Copyright © 2001 Elsevier Science Inc. Printed in the USA. All rights reserved 0361-9230/01/$–see front matter
PII S0361-9230(01)00656-6
Myotonic dystrophy—A multigene disorder Ken Larkin and Majid Fardaei* Department of Genetics, Queens Medical Centre, University of Nottingham, Nottingham, UK ABSTRACT: Myotonic dystrophy (DM1) is the most common form of adult muscular dystrophy with an estimated incidence of 1/8000 births. The mutation responsible for this condition is an expanded CTG repeat within the 3ⴕ untranslated region of the protein kinase gene DMPK. Strong nucleosome positioning signals created by this expanded repeat cause a reduction in gene expression within the region. This “field effect” is further confounded by the retention of DMPK expansion containing transcripts, which acquire a toxic gain of function. Thus, the various manifestations exhibited by DM1 patients can be explained as a result of gene silencing, nuclear retention and sequestration of nuclear factors by the CUG containing transcript. © 2001 Elsevier Science Inc.
hypersomnia, ocular cataracts and insulin dependent diabetes are also common, together with testicular atrophy and premature frontal balding in men. In fact, any possible combination of these is typical in classical cases of DM1. Congenital cases display the most severe phenotype and face a neonatal mortality rate of 25%. Features include reduced muscle tone (“floppy babies”), neonatal hypotonia, severe mental retardation, facial dysplegia, feeding difficulties and respiratory distress [23]. Histological examination of affected skeletal muscle tissue generally reveals type 1 fibre atrophy and type 2 fibre hypertrophy, increased numbers of centrally located nuclei occurring as chains within muscle fibres and ringed fibres with sarcoplasmic masses. Other observations include vacuolar degeneration of myocardial cells and conduction defects, ventricular dilation in the brain and pigmentary retinal degeneration in the eye. DM1 is associated with variable levels of penetrance, which could explain the extreme diversity of clinical symptoms. Earlier age of onset, together with an increase in severity of the disease symptoms through subsequent generations correlates with the number of CTG repeats. This observation has been used to explain the phenomenon of “anticipation” exhibited in DM1.
KEY WORDS: Myotonic dystrophy, DMPK, SIX5, DMWD, Field effect, CUG triplet repeat, EXP.
INTRODUCTION The causative agent for myotonic dystrophy (DM1) is known to be a large scale CTG expansion located within the 3⬘ UTR of DMPK (DM1 associated protein kinase) [9,18,35]. DM1 is one of a number of human diseases known to be caused by microsatellite instability. Although the reasons for this instability are not certain, several theories have arisen concerning the resulting pathological mechanisms. In this review we will consider how the manifestations of DM1 are caused by the CTG repeat expansion at a number of levels; firstly in cis by affecting the expression of DMPK and its neighbouring genes including SIX5 (a homeodomain transcription factor) and DMWD. Secondly, we will discuss the effects of the expansion in trans, i.e., at the level of mRNA. Since much emphasis has been placed on the pathological role of the expanded CUG transcript, the possibility of mRNA interference involving other genes or proteins required for tissue development and maturation will be discussed. We shall see that DM1 provides an interesting and complicated situation involving numerous genes at different levels and as such DM1 should be classed as a multigene disorder.
GENETIC BASIS FOR DM1 DM1 was one of the first autosomal diseases for which a causative genetic element was identified. Linkage analysis of DM1 families, mapped the region responsible to the chromosomal location of 19q 13.2–13.3 [4,10,22]. In 1992, DMPK and the mutation responsible for DM1 were discovered [9,18,35]. Rather than an alteration within the ORF of DMPK the mutation was shown to be an expansion of an unstable trinucleotide (CTG) repeat sequence within the 3⬘ UTR of the gene. This CTG repeat expansion is believed to be responsible for 98% of reported DM cases. In the remainder of patients where no expansion is detectable (DM2) the disease locus can be mapped to a 10-cM region on chromosome 3q [48]. At least 14 human disorders are now known to be caused by instability within simple DNA repeats—the most frequent being (CGG)n and (CAG)n. Fragile X syndrome, the most common cause of inherited mental retardation was one of the first trinucleotide disorders to be identified [17]. This occurs as a result of a CGG expansion within the 5⬘UTR of the X-linked FMR1 gene (Fragile X mental retardation 1). In contrast to normal alleles containing 5–50 CGGs, expansions greater than 200 repeats result in the hypermethylation of CpG islands within the promoter region and a reduction in gene expression. In comparison, spinobulbar muscular atrophy and Huntington’s disease, together with several spinocerebellar ataxias occur as a result of CAG expansions within the affected ORFs.
CLINICAL PHENOTYPE DM1 is an autosomal dominant neuromuscular disorder with an incidence of 1 in 8000 births and is characterised by an extremely variable clinical phenotype [23]. Symptoms usually appear in early adulthood with the most common being the delayed ability of patients to relax the grip of a clenched fist. Termed myotonia, this is present in the majority of symptomatic cases and can be followed by progressive weakness and wasting of muscle. Cardiac conduction defects, smooth muscle involvement, mental changes,
* Address for correspondence: Majid Fardaei, Department of Genetics, Queens Medical Center, University of Nottingham, Nottingham NG7 2UH, UK. Fax: ⫹44-115-9709906; E-mail:
[email protected]
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390 Translation of these CAG repeats results in the production of pathologically dominant proteins containing runs of polyglutamines (polyGln). These aberrant proteins are known to aggregate in vivo and have been observed in the neurons of a number of the polyGln expansion diseases [12]. DM1 on the other hand differs in comparison to the previous examples in that the CTG repeat is present within the 3⬘ UTR of DMPK. In the general population 5–27 repeats are associated with the normal allele, whereas in affected individuals several thousand repeats can be present [9]. In contrast to the coding CAG/polyGln disorders involving a “gain of function”, expansions of non-coding repeats as seen in Fragile X and DM1 are typically much larger. Therefore, it is generally accepted that the large scale DM1 expansion affects gene expression within this locus at a transcriptional or post-transcriptional level. DMPK HAPLOINSUFFICIENCY AND ITS ROLE IN THE DM1 PHENOTYPE As discussed, the expanded CTG repeat associated with DM1 occurs within the 3⬘ UTR of DMPK on chromosome 19. Thus, direct effects on DMPK levels were the first obvious suggestion for the cause of DM1. DMPK is comprised of 15 exons, it covers 14 kb of genomic DNA and encodes a protein of 629 amino acids which shares regions of homology with cyclic adenosine monophosphate (AMP) dependent protein kinases [9,18,27,35,53]. Subsequent studies have characterised DMPK as a serine/threonine protein kinase with a catalytic domain of ⬃43 kDa, followed by a helical region of ⬃12 kDa and a region of hydrophobicity thought to act as a transmembrane domain at the C-terminal end [18,19, 35,36]. DMPK is known to undergo autophosphorylation and phosphorylate the general protein kinase substrate histone H1 in vitro [13]. But, in contrast to other serine threonine protein kinases such as PKA and PKC, recombinant DMPK is unable to phosphorylate membrane associated phosphoproteins and is resistant to several known inhibitors of serine threonine kinases. These findings led to the idea that DMPK belonged to a new subgroup of protein kinases related to, but distinct from cyclic AMP dependent serine threonine kinases. More recent studies have shown that the actin cytoskeleton-linked GTPase Rac-1 can physically bind DMPK and co-expression of Rac1 with DMPK can activate the transphosphorylation activity of DMPK in a GTP-sensitive manner [54]. DMPK was also shown to be phosphorylated and activated by Raf-1 in this same study. Raf-1 kinase is thought to be activated by the small GTPase Ras which is considered to be part of a chemically stimulated signalling pathway linked to the MAP kinase cascade [38]. These findings suggest that activation of DMPK by Rac1 and Raf-1 regulates the transphosphorylation of target proteins by DMPK and permits “cross-talk” between different signalling pathways [54]. One such target where these pathways could converge is myosin phosphatase (MYPT1) [41]. DMPK has been shown to phosphorylate MYPT1 and inhibit its activity. An important consequence of this inhibition is an increase in the levels of phosphorylated myosin. This in turn results in Ca2⫹ sensitisation of smooth muscle and cytoskeletal changes in non-muscle cells [26]. In summary, MYPT1 represents only one of many potential substrates for DMPK and as such illustrates the complexity of the issue at hand. Subsequent identification of further downstream substrates for DMPK should provide valuable insight into the specific role of this protein and its contribution to the DM1 phenotype. As DM1 affects a number of cellular systems, attempts have been made to characterise the tissue specific localisation of DMPK. Early experiments using anti-sera proved extremely difficult due to the shared homology of DMPK with other protein
LARKIN AND FARDAEI kinases. These studies identified proteins ranging from 42– 85 kDa in a variety of tissues including brain, skeletal muscle and heart (as discussed by Lam et al. [34]). DMPK expression was specifically detected within intercalated discs of cardiac muscle and Purkinjie fibres, and at sites of neuromuscular and myotendinous junctions, implying that DMPK performs a role in intercellular communication. Initial studies also observed expression in the perinuclear cytoplasm of lens epithelial cells and in superficial subcapsular cortical fibres of the eye and so provided a link between lens degeneration and the formation of cataracts in DM1 [14]. In 1998 Pham and co-workers followed an alternative approach [46]. They utilised a panel of monoclonal antibodies recognising different epitopes on DMPK. It was proposed that if any protein on a western blot contained the majority of epitopes it was likely to be bona fide DMPK. Using 10 monoclonal antibodies (mAbs) against the catalytic and coil domains of DMPK they observed high levels of two discrete species (72 kDa and 80 kDa). The 72 kDa protein was present in all tissues tested whereas the 80 kDa form was differentially expressed, but found predominantly in skeletal and cardiac muscle. Subsequent characterisation of the mAb panel revealed that only the 80 kDa form expressed exclusively in muscle and heart was genuine DMPK [34]. The previously described 72 kDa form was believed to be a result of cross-reactivity. Both biochemical and cellular studies provide evidence linking DMPK dysfunction to the development of DM1. The argument for haploinsufficiency suggests that one DMPK allele is inactivated by the expansion and results in a reduction of cellular DMPK. However, whether haploinsufficiency is sufficient to cause DM1 is questionable because neither heterozygous nor homozygous DMPK (dm15) knock out mice show a profound phenotype, nor have they reproduced the myotonia, or cataracts characteristic of DM1 [29,49]. Although both these studies failed to reproduce myotonia or cataracts in either the heterozygous or homozygous form, DMPK ⫺/⫺ mice did develop late-onset skeletal myopathy and altered calcium ion homeostasis [6]. DMPK has also been shown to modulate cardiac conduction in a dose dependent manner [7]. Homozygous knock out mice were shown to develop first, second and third degree atrioventricular block, whereas heterozygous mice developed first degree heart block—a situation similar to the DM1 cardiac phenotype. Although these results provide good evidence that DMPK haploinsufficiency plays a contributory role in DM1, the lack of a complete DM1 phenotype in these models indicates that the inactivation of DMPK alone cannot be responsible for the development of DM1. DM1 IS A MULTIGENE DISEASE A possible explanation for the absence of phenotype demonstrated by knock out mice is that the expanded repeat affects a number of genes in addition to DMPK. This theory known as the “Field Effect” is likely to occur as a result of chromatin restructuring because CTG repeats are known to favour enhanced nucleosome assembly [8,24,27,60 – 62]. Chromatin structure plays a vital role in the accessibility and formation of transcription complexes. Therefore the CTG expansion may disrupt this process and prevent the binding of necessary factors for the expression of nearby genes. Work by Otten and Tapscott [45] showed that a DNaseI hypersensitive site 3⬘ of the triplet repeat was resistant in the expanded allele, suggesting the CTG repeat expansion was promoting the formation of a more condensed chromatin structure. Thus, one possibility is that the expanded CTG repeat exerts a direct effect on the transcription of DMPK and its neighbouring genes. A transcript map around the DM1 associated repeat was constructed using exon trap analysis [1]. This mapped a further five genes (DMWD (formerly 59), SIX5 (formerly DMAHP), SYMPLEKIN, 20-D7 and
MYOTONIC DYSTROPHY GIPR) to within 200 kb of DMPK and its associated repeat. A sixth gene RSHL1 has recently been discovered and is located upstream of DMWD [15]. As we shall see all six additional genes could play a contributory role in the DM1 phenotype. In addition to data obtained from DMPK mouse knock out studies, the idea of a “field effect” arose from results of numerous studies aimed at quantifying expression of DMPK, DMWD and SIX5 in both affected and normal individuals. Initial attempts to quantify levels of DMPK mRNA in DM1 patients produced extremely variable data. However, this problem was clarified by Taneja and co-workers [57] who used in situ hybridisation to show that expansion derived DMPK transcripts were retained within the nucleus of cells in distinct foci. The fact that previous experiments used intact cells or biopsied tissue and failed to take into account the altered distribution of expanded repeats was shown by Hamshere and co-workers [21]. In this study both nuclear and cytoplasmic RNA fractions were analysed by reverse transcriptionpolymerase chain reaction (RT-PCR). Here, it was noted that patients heterozygous for a BpmI restriction site polymorphism in exon 10 expressed mRNA from both alleles in the nuclear fraction, but subsequent analysis of the cytoplasmic fraction revealed the loss of the expanded repeat containing transcript. Again, this suggested that the expanded transcript was being retained within the nuclei. Finally, it was concluded that there was a threshold between 80 and 400 CTGs at which nuclear retention occurred. This was proposed since cell lines with one allele length of 80 CTGs showed a similar amount of cytoplasmic RNA as compared to the other wild type allele, whereas in the cytoplasm of the cell line with 400 repeats the expanded allele appeared to be absent. A similar result was also reported by Davis and co-workers [11] who found that expanded transcripts were only present within nuclear RNA fractions when analysed by Northern blot. In summary, these experiments add an important dimension to the DM1 story. The DM1 associated expansion may affect DMPK expression in cis by altering levels of transcription, but the most likely cause of haploinsufficiency appears to be the retention of expanded transcripts. In a further development Amack and colleagues [3] produced a cell culture model in which mouse C2C12 mouse myoblasts were used to express reporter genes fused to the human DMPK 3⬘UTR. Using a construct containing 57 CTG repeats a threefold decrease in protein was observed and similar to previous findings nuclear mRNA foci were observed using RNA-FISH analysis. These foci were found to be resistant to DNaseI, sensitive to RNaseA and were not detected using a sense (CTG) probe, thus confirming that the observed foci were in fact aggregates of the CUG containing transcript. In the same study a reporter construct containing 26 CTG repeats did not show nuclear retention, suggesting that retention occurs between 26 and 57 repeats. DM1 is also associated with reduced expression from the neighbouring gene SIX5 [31,33,58]. Using an allele specific RTPCR assay, Alwazzan and co-workers [2] were able to show a reduction in both nuclear and cytoplasmic fractions of the affected SIX5 allele. The observed decrease in SIX5 expression is possibly due to an association between the CTG repeat at the 3⬘ end of DMPK and the large CpG island which covers the 5⬘ end of SIX5 [8]. SIX5 encodes a homeodomain transcription factor with homology to sine oculis—required for eye development in Drosophila melanogaster. Although previous studies have shown DMPK to be expressed in the eye [14] a more recent study has shown that only SIX5 expression is detectable in the adult lens [63]. It was found that SIX5 transcripts were detected in the adult retina, scelera, cornea, uvea and lens epithelium but were absent in fetal eyes. In contrast, DMPK transcripts or protein were detected in the fetal eye and in the adult uvea and retina but not in the adult lens. Since, SIX5 expression was only detected in the adult eye and there
391 have been no reported cases of fetal ocular cataracts it is likely that SIX5 dysfunction rather than DMPK is responsible for the formation of cataracts commonly found in DM1 patients [63]. To determine whether this was the case two groups disrupted mouse SIX5. One strategy undertaken by Klesert and co-workers [32] involved replacing the first exon with the -galactosidase reporter gene. The other by Sarkar and colleagues involved replacing from 398 bp upstream of the SIX5 start codon to 180 bp downstream of the termination codon with a PGK-neo cassette [51]. Both groups reported that loss of SIX5 led to the formation of cataracts. -gal expression was observed in multiple tissues as well as the developing lens although examination of skeletal muscle from 3-month old mice showed normal fibre size distribution with no degeneracy, no centrally located nuclei or ringed fibres typically found in myotonic tissues. Electromyography on 3-month and 10-month muscle tissue also failed to show any abnormalities characteristic of DM1 [32]. Both these studies provided sound evidence that deficiency in SIX5 led to cataract formation in DM1 patients although the actual mechanism was not certain. To date six members of the SIX family have been identified. One potential target gene for SIX5 is ATP1A1. This encodes the ␣-1 subunit of Na⫹-K⫹-ATPase and has been shown to be regulated in vitro by Six5 [44]. On one hand Klesert et al. [32] found no difference in steady state levels of ATP1A1 mRNA in skeletal muscle or lens tissue from six5 knock out mice, whereas Sarkar et al. [51] observed an increase in steady state mRNA levels as a function of decreasing SIX5 dosage. If SIX5 does regulate ATP1A1, SIX5 dysfunction may be responsible for changes in osmotic balance within the lens and cause its degeneration [32,51]. It has also been suggested that SIX5 regulates DMPK since both genes are expressed in similar tissues [25]. In both studies an observed decrease in Dm15 (mouse DMPK homologue) expression was noted in six5 homozygous mice although it was not clear whether this was due to the loss of SIX5 or a negative cis-effect caused by its targeted disruption. Whether or not SIX5 regulates DMPK it has been hypothesised that as SIX5 is expressed in a number of tissues affected in DM1 it must regulate genes involved in the development of the DM1 phenotype [25]. DMWD, located 500 bp upstream of DMPK (Fig. 1) contains five exons and covers approximately 11 kb of genomic sequence [53]. A polymorphism was subsequently found within this gene and a quantitative allele-specific assay for DMWD mRNA levels was developed [2]. It was found that the DM1 associated allele was present in the nucleus at levels equivalent to the wild type allele but in the cytoplasmic fraction a 20 –50% reduction was observed. This was a similar situation to that observed for the affected SIX5 allele in that an overall reduction in cytoplasmic levels were observed, but, in contrast to SIX5 no reduction in either DMWD allele was found in the nucleus. DMWD is a member of a large family of eukaryotic WD-repeat (Trp-Asp) containing proteins in which the repeat unit occurs four to eight times within the polypeptide. Other WD-repeat family members include the -subunit of GTP-binding proteins involved in signal transduction, as well as proteins involved in neurological development, transcription, mRNA processing, vesicle transport and cytoskeletal assembly [43]. Northern analysis has shown strong expression of DMR-N9 (mouse DMWD homologue) in all neural tissues of mouse brain. Expression has also been detected in secondary spermatocytes of stages VIII–XII of the spermatogenic proliferation cycle in mature testis [28]. These observations, together with DMWD belonging to a protein family involved in a diverse range of cellular processes, suggests that DMWD may be partly responsible for the male sterility, testicular atrophy and mental retardation associated with DM1 [28,53].
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FIG. 1. The myotonic dystrophy (DM1) phenotype is likely to be caused by deficiencies in a number of gene products. CTG expansion repeats favour the formation of heterochromatin, leading to a decrease in transcription of neighbouring genes. The roles and potentially numerous targets of these neighbouring genes have to be fully elucidated. The expanded CUG triplet repeat within the DMPK 3⬘UTR is retained within the nucleus and could acquire a toxic gain of function. Inappropriate RNA/DNA, RNA/RNA or RNA/protein interactions may lead to the sequestration of factors necessary for the development and maturation of various tissues.
SYMPLEKIN, RSHL1, 20-D7 and GIPR also map to within 200 kb of the DM1 associated triplet repeat (Fig. 1). Whether these genes play a role in DM1 has yet to be determined, although ascribed protein function together with cellular location suggest a linkage to the DM1 phenotype for all four. SYMPLEKIN, was originally identified in a screen for novel proteins associated with tight junction plaques [30]. The fact that Symplekin has now been isolated as part of a large complex containing cleavage stimulation factor and shares significant similarity to the yeast protein PTA1 (implicated in pre-mRNA polyadenylation) led to the suggestion
that Symplekin functions in the assembly of the polyadenylation machinery [55]. Obvious implications therefore arise with respect to this gene and the multisystemic nature of DM1. Radial spokehead-like gene RSHL1 lies immediately upstream of DMWD [15] (Fig. 1). RSHL1 shares homology with proteins involved in ciliary or flagellar action from protozoa and sea urchin spermatozoa. Ciliary dysfunction has been found in human autosomal recessive disorders where respiratory tract infections and decreases in sperm motility are common. Thus, RSHL1 has now been implicated as a candidate gene for familial primary ciliary
MYOTONIC DYSTROPHY dyskinesia [15]. Although no role has been attributed to RSHL1 in connection with DM1, it would be interesting to see if DM1 was also associated with impaired sperm motility as well as testicular atrophy [15]. 20-D7, located downstream of SIX5 is expressed in the testis, therefore altered expression of this gene may result in testicular atrophy in males. Finally, human gastric inhibitory polypeptide receptor gene (GIPR) is involved in insulin secretion and so could be responsible for the increased incidence of diabetes observed in DM1 patients [1]. THE EXPANDED CUG REPEAT AND ITS TOXIC GAIN OF FUNCTION Previously described studies have shown that both DMPK and SIX5 knock out mice develop a partial DM1 phenotype. The fact that myotonia (the most common symptom of DM1) is absent in both cases provides two possible conclusions; firstly, DMPK haploinsufficiency in conjunction with additional gene dysfunction is required to develop the full DM1 phenotype. Alternatively, the expanded CTG repeat acquires a toxic gain of function at the RNA level. This creates an overall scenario where the effects of decreased protein levels DMPK, SIX5 etc. (caused by chromatin changes and retention of transcripts) are further confounded by the additional pathological role of the mutant mRNA. One piece of evidence showing that the CUG repeat was exerting a toxic effect on the cell came from Amack and coworkers [3]. Here, expression of the DMPK 3⬘UTR containing an expanded (CTG)200 repeat was shown to inhibit myogenic differentiation of mouse myoblasts as compared to a line expressing the wild type construct. This led to the suggestion that the expanded DMPK 3⬘UTR mRNA acts as a “riboregulator” in trans and thus inhibits myoblast differentiation and maturation. A definitive study illustrating the toxic properties of the expanded mRNA was subsequently carried out by Mankodi and colleagues [37]. Here, the human skeletal actin (HSA) gene was used to express an untranslated CUG repeat in the skeletal muscle of transgenic mice. Constructs were designed to mimic those found in the situation of DMPK, i.e., the placement of CTG repeats between the termination codon and the polyadenylation site. Mice expressing these expanded constructs showed normal development of non-muscle tissue but had a high mortality rate of 41% by 44 weeks. Electromyography showed discharges typical of DM1 patients exhibiting myotonia. In contrast to Dmpk or Six5 knock out mice, transgenic mice expressing the expanded repeat were observed to develop myotonia after only 4 weeks. Subsequent studies showed normal muscle histology at this stage providing evidence that these mice did in fact have myotonia and not non-specific hyperexitability related to muscle necrosis. Histologically defined myopathy was also observed in lines expressing the expanded repeats with increased central nuclei, ringed fibres and variation in fibre size in the absence of fibre necrosis [37]. Similar to previous studies [11,21,57], these expanded repeats were also found to be retained within the nucleus in distinct foci. Since the only similar sequence to DMPK is the long CUG repeat it was also concluded that it was this sequence alone which was responsible for the observed nuclear retention. Therefore in summary, this study was consistent with the idea that the CUG repeats are exerting a dominant toxic effect on muscle tissue. So the next question to determine is; by what mechanism does the CUG repeat induce myotonia and muscle degeneration? The RNA dominance model is believed to involve the formation of extended hairpin loops within the CUG tract which impair nuclear cytoplasmic transport resulting in nuclear retention. These expanded repeats could then exert a toxic gain of function through
393 their inappropriate association with DNA, RNA or proteins required for tissue development and maturation. In the case of RNA-RNA interactions the CUG repeats located within the 3⬘UTR of DMPK mRNA could pair with a CAG repeat from another gene transcript [20]. This was subsequently shown in experiments by Sasagawa and co-workers [52] who were able to demonstrate that expanded CUG repeats within DMPK transcripts were able to interact with CAG repeats contained within the TFIID/TATA binding protein mRNA. A similar situation has been suggested to explain the male infertility associated with DM1. In this case involving the CAG repeat contained within the androgen receptor mRNA [20]. According to the protein sequestration hypothesis, DMPK repeat expansion results in the chelation of CUG binding proteins and removal of these factors from other transcripts, which require them for processing. The first class of proteins thought to be sequestered by the retained transcripts belongs to a family known as hnRNPs. These factors associate with pre-mRNAs during transcription and are believed to play a subsequent role in processing (CUG-BP) [59], nuclear retention (hnRNP C) and export (hnRNP A) of mRNAs [42]. CUG-BP was identified in band shift assays using a (CUG)8 repeat probe [59]. CUG-BP provided a convincing candidate since it had been implicated in DM1 and alternative pre-mRNA splicing of cardiac troponin T [47,50,59]. However, recent data questions the role of this protein in the pathology of DM1. As discussed, DMPK transcripts accumulate as nuclear foci in DM1 cell lines, whereas the localisation pattern of CUG-BP in the same lines was unaltered. Furthermore, this protein has been shown to localise at the base of the RNA hairpin structure and not along the CUG repeat [39]. In a recent study, a combination of indirect immunofluorescence and GFP tagged proteins showed that neither CUG-BP nor hnRNP C colocalises with the expanded CUG repeat in DM1 cell lines [16]. It has also shown that CUG-BP preferentially binds to UG dinucleotide repeats in a yeast threehybrid system [56]. Expansion (EXP) double-stranded (ds) RNA binding proteins on the other hand specifically interact with CUG expansions in a length dependent manner and so belong to a second class of CUG binding proteins [40]. A subsequent study [16] has also shown colocalisation of EXP with CUG expanded repeats in vivo. These proteins are homologous to the Drosophila muscleblind (mbl) proteins which have roles in visual development as well as embryonic muscle differentiation and cells of the central nervous system [5]. Analysis of the mbl locus revealed at least 9 exons within 150 kb of genomic DNA. Alternative mRNA splicing gives rise to at least four protein isoforms with identical 5⬘ regions but different carboxyl terminal ends with each containing one or two Cys3His-type zinc finger motifs [5]. In humans, three mbl homologues have been identified hEXP42, hEXP40 and hEXP35 [40]. These isoforms are also believed to be produced by alternative splicing with each protein containing four zinc knuckle motifs. EXP expression using a polyclonal antibody was detected in skeletal muscle, in the heart and eye but was absent in brain or testes. Future experiments to determine the full range of targets normally associated with these proteins whether at the RNA or protein level should provide valuable insights into the development of DM1. SUMMARY DM1 provides a complicated problem caused primarily by the expansion of a CTG repeat within the 3⬘UTR of DMPK (Fig. 1). As we have seen the multiple manifestations exhibited by DM1 patients can be attributed to events occurring at several levels. The first involves a decrease in protein production from genes sur-
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rounding the expanded repeat. DMPK transcripts are retained in the nucleus and decreased expression caused by chromatin restructuring has been reported for SIX5 and DMWD. Dysfunction in both DMPK and SIX5 has been shown to play a dominant role in the development of certain symptoms associated with DM1. In addition SYMPLEKIN, DMWD, RSHL1, 20D7 and GIPR are implicated in DM1 since they are expressed in one or more tissues in affected patients. As yet the functions of these genes and their targets have yet to be fully elucidated. With DMPK’s role in signal transduction, SIX5 as a transcription factor and DMWD belonging to the WD-repeat family of proteins, a host of downstream effects can be envisaged. This reasoning probably explains why mouse models involving DMPK or SIX5 alone show only a few of the DM1 characteristics. Further characterisation of SYMPLEKIN, RSHL1, 20-D7 and GIPR together with expression studies will show whether these genes are also affected by the expanded repeat. Decreased expression around the CTG repeat and nuclear retention plays an important role in some aspects of DM1, however a second factor to take into account is the pathological “gain of function” of the expanded CUG repeat. This nuclear retained transcript has been proposed to act as a “riboregulator” in trans and to prevent the differentiation of mouse muscle cells. Toxic effects are also observed when CUG repeats are expressed as a transgene in mouse skeletal muscle. Here they are believed to sequester nuclear factors such as CAG containing mRNAs and CUG binding proteins such as EXP which could be vital in tissue development and maturation. Future work to determine the normal mRNA targets of these proteins will obviously help in the elucidation of the complicated pleiotropic nature of the DM1 phenotype. Although the CUG repeat containing mRNA is now known to play a dominant role in the pathogenesis of DM1, certain features of the DM1 phenotype were absent from the CUG expressing mouse model. This indicates that a decrease in at least DMPK and SIX5 protein levels together with the toxic effect of the expanded mRNA are required for the full DM1 phenotype. Future more comparable mouse models incorporating both gene knockouts and CUG repeat expressing transgenes will hopefully lead to a greater understanding of this complicated and debilitating disease. ACKNOWLEDGEMENTS
This work was supported by the Muscular Dystrophy Campaign, Muscular Dystrophy Association USA, Human Frontier Science and the Program Organization.
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