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Myotonic dystrophy type 1 (DM1): A triplet repeat expansion disorder
Gene 522 (2013) 226–230
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Gene journal homepage: www.elsevier.com/locate/gene
Letter to the Editor
Myotonic dystrophy type 1 (DM1): A triplet repeat expansion disorder Ashok Kumar, Sarita Agarwal ⁎, Divya Agarwal, Shubha R. Phadke Department of Genetics, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow 226014, India
a r t i c l e
i n f o
Article history: Accepted 16 March 2013 Available online 6 April 2013 Keywords: CTG repeat DMPK gene Genetics Myotonic dystrophy type 1 (DM1) Triplet repeat disorder
a b s t r a c t Myotonic dystrophy is a progressive multisystem genetic disorder affecting about 1 in 8000 people worldwide. The unstable repeat expansions of (CTG)n or (CCTG)n in the DMPK and ZNF9 genes cause the two known subtypes of myotonic dystrophy: (i) myotonic dystrophy type 1 (DM1) and (ii) myotonic dystrophy type 2 (DM2) respectively. 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. This review will focus on the clinical features, molecular view and genetics, diagnosis and management of DM1. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Trinucleotide repeat disorder is a disorder caused by an increase in the number of triplet repeats occurring in some of the genes. The disease manifests when the number of repeats increases and crosses the limit of normal repeats and affects the gene function. These disorders are classified into 2 major categories: (i) Coding expansion disorder (Huntington disease (HD) and spirocerebellar ataxias) and (ii) non-coding expansion disorder (Myotonic Dystrophy (DM) and Friedreich ataxia (FRDA)). 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) which are caused by two different genes DMPK (dystrophia myotonica protein kinase) ZNF9 i.e. zinc finger protein (Table 1). On the basis of clinical severity the disorder is divided into three groups mid, classical and congenital (Table 2). Out of these three, the congenital form is the most severe (Harper, 1989). DM affects 1 in 8000 people worldwide and the relative prevalence rates of DM1 and DM2 are 98% and 2% respectively. 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 (Howeler et al., 1989) due to the expansion of the repeat number during gametogenesis. Repeat contraction events are rare and occur 4.2 to
⁎ Corresponding author. Tel.: +91 522 2494349(O), +91 9415336601; fax: +91 522 2668017. E-mail address: [email protected] (S. Agarwal). 0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2013.03.059
6.4% of the transmission (Musova et al., 2009). The triplet repeat numbers are unstable and the number of repeats varies from the tissue to tissue. 2. Genetic insight of disease 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 (Mahadevan et al., 1993; Shaw et al., 1993). The repeats in DMPK are repeats of CTG and vary in the normal population from 3 to 37. The size more than 50 CTG is associated with DM1 and severity correlates with CTG repeat number (Brook et al., 1992; Mahadevan et al., 1992). The mutant DMPK gene results in the following changes: 1. Dominantly inherited loss of function DMPK phosphorylates muscle-specific voltage gated Na + channels and regulates the excitability of muscle cells (Chahine and George, 1997). DM1 patients are heterozygous for the expansion mutation. The loss of protein production from the mutant allele is because the expanded CTG repeats in the 3′-UTR interfere with mRNA and/or protein synthesis (Botta et al., 2008; Karen, 2008). 2. Disruption of neighboring genes Expanded CTG repeats interfere with the local chromatin configuration which affects expression of both DMPK and neighboring genes DMAHP (dystrophia myotonica associated home domain protein)/SIX5, dystrophia myotonica containing WD repeat motif (DMWD), and muscle blind protein (MBNL1) (Frisch et al., 2001). MBNL1 gene is located on chromosome 3q25. The expanded number of CTG repeats within the 3′-UTR of the DMPK gene interferes with the transcription of the neighboring DMAHP (SIX5) gene by masking its enhancer that lies close to the CTG island (Klesert et
Letter to the Editor Table 1 Comparison of DM1 and DM2. Feature
DM 1
DM 2
Epidemiology Onset age (years) Anticipation and severity Congenital form Facial weakness Proximal onset Distal onset Muscle pain Myotonia Chromosome Mutated gene Mutation type Repeat size Cataracts Balding Cardiac arrhythmias Gonadal failure Hypersomnia
Widespread 0 to adult Moderate-severe Yes More Late Early less Yes 19q13.3 DMPK CTG repeats 50–4000 More More More More More
European 8–60 Mild-moderate No Less Early Late More Yes 3q21 ZNF9 CCTG repeats ~5000 Less Less Less Less Less
al., 1997). Therefore, CTG-repeat expansions can suppress local gene expression and SIX5/DMAHP may play a role in manifestation of DM (Klesert et al., 1997). If there are no further changes in local gene expression above a certain threshold of repeats, it may not be affect the phenotype of DM. The deficiency or decreased Six5 transcription is important in the etiology of DM like cataract formation (Klesert et al., 2000). As DM is a multisystemic and multigenic disorder, so SIX5 knockout mice may not show DM. 3. Defects in RNA metabolism In the normal cell, RNA is transcribed, processed in the nucleus and transported to the cytoplasm where it is bound by specific RNA binding proteins i.e. CUG-BP or CELF1 and then translated into the DM protein kinase (DMPK). In affected DM1 patients (Fig. 1) transcripts from the mutant DMPK allele and normal allele are retained within the nucleus because the expanded number of CUG repeats bind and sequester mRNA transport proteins (CUGBP1). DMPK transcripts bearing a long (CUG)n tract form hairpin-like structures
Table 2 Correlation of phenotype and CTG repeat length in DM1. Phenotype
Clinical signs
CTG repeata,b Size
Age of onset
Average age of death
Asymptomatic (premutation) Mild
None
35 to 49
NAc
NAc
Cataracts Mild myotonia
50 to ~150
20 to 70 yrs
Classical
Weakness, myotonia Cataracts Balding Cardiac arrhythmia Others Infantile hypotonia Respiratory deficits Intellectual disability
~100 to ~1000
10 to 30 yrs
60 yrs to normal life span 48 to 55 yrs
~100 to ~1000
From birth
48 to 55 yrs
Congenital
a b c
CTG repeat sizes are known to overlap between phenotypes. Normal CTG repeat size is 5–34. NA (not applicable).
227
(Tishkoff et al., 1997) and aggregate of DMPK RNA are observed in the ribonucleic foci in the nucleus and some of the mRNA with expanded repeats is also transported to the cytoplasm. The mechanism of toxic effects of these RNAs is not definitely established. The nuclear CUGn repeat aggregates sequester MBNL1 protein which is a muscle blind protein (Mankodi et al., 2001; Miller et al., 2000). This is likely the mechanism of phenotypic effect as MBNL1 knock out homozygous mice show myotonia and other symptoms of DM1 (Kanadia et al., 2003). The other cellular event is increase in CUGBP1. So both factors, reduction of MBNL1 and increase in CUGBP1 in DM1 cells as a result of RNA aggregates are likely to cause DM1 phenotype. Both MBNL1 and CUGBP1 regulate splicing of the same mRNAs (chloride channel 1, insulin receptor, troponin T) by binding to different sites (Lamusi and Artero, 2008). Altered splicing of RNAs affecting other proteins can explain multisystem nature of DM1 manifestation. MBNL1 and CUGBP1 also have several important functions like splicing, protein translation and RNA and their deregulation alter splicing of other RNAs and contribute to the disease manifestations (Miller et al., 2000; Timchenko et al., 2001). This suggests that their mechanism may be independent of each other (Ho et al., 2005). An increase in CUGBP1 causes degeneration in the drosophila model (de Haro et al., 2006). This suggests that therapeutics strategies at level of MBNL1 and CUGBP1 can be effective in DM1 management (de Haro et al., 2006). Thus, the altered RNA metabolism of both the mutant and normal DMPK RNA transcripts leads to deficiency of the DMPK protein, phosphorylation of proteins and symptoms such as myotonia. Phosphorylation of protein is a post-translational modification of proteins in which a serine, threonine or a tyrosine residue is phosphorylated by a protein kinase by the addition of a covalently bound phosphate group. In almost all cases of phosphoregulation, the protein switches between a phosphorylated and an unphosphorylated form and one is an active form while the other one is inactive. 3. Diagnosis of DM1 3.1. Clinical 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 myopathic face, 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 (Harley et al., 1992, 1993; Hunter et al., 1992; Redman et al., 1993). 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 (Harper, 2001; Schara and Schoser, 2006). 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 (Schara and Schoser, 2006; Schoser et al., 2004). 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 congenita and myopathies.
228
Letter to the Editor
Normal DMPK
DM
-
DMAHP DMPK
(CTG)n<37
DMAHP
DMPK
DMAHP
(CTG)n>50
(CTG)n<37
CUG-BP
DMPK
DMAHP
(CTG)n<37
CUG-BP CUG-BP (CTG)n<37
DMPK DMAHP Protein kinase Phosphorylation of Na+ channels
DMPK
DMAHP Homeobox Gene Expressed in brain and muscle
Altered CUG-BP Localization ? Endocrine Testicular atrophy Diabetes Frontal Balding ? Floppineess in C-DM
DMAH P 20% DMPK ?Myotonia
50% DMPH ? Mental retardation ? Dysmorpholgy
Processing of DMPK gene and DMAHP genes within normal and DM cells
Fig. 1. Processing of DMPK and DMAHP (SIX5) genes within normal and DM cells. In the normal cell, both DMPK genes are transcribed, and the RNAs are processed in the nucleus and transported to the cytoplasm where they are bound by specific RNA binding proteins (CUG-BP) and then translated into the DM protein kinase (DMPK). In DM cells, the DMPK gene is transcribed from both normal and mutant alleles but the transcripts are largely retained within the nucleus. The expanded numbers of CTG repeats within the 3′ UTR of the DMPK gene also interfere with the transcription of the neighboring DMAHP gene and thus lead to decreased amount of DMAHP transcript and protein. Thus, functional DMPK protein does not form in DM patients.
3.2. Molecular diagnosis The detection of expansion of triplet repeat traditionally is done by Southern blot analysis (Goossens et al., 2008; Sermon et al., 2001). However, triplet primed PCR (TP-PCR) based testing is found to be a reliable non-radioactive method as a replacement of the Southern blot (Addis et al., 2012; Khajavi et al., 2001; Warner et al., 1996). The number of triplet repeats correlates with the phenotype and hence, it is important for prediction of severity of the disease (Harley et al., 1992, 1993; Hunter et al., 1992; Redman et al., 1993). 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 counseling. After molecular confirmation of the diagnosis in the proband, the family can be counseled 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 (Warner et al., 1996), and multiplex PCR over Southern blotting (Goossens et al., 2008) for screening of triplet repeat expansion in DM1 is now recommended. 3.3. Therapeutics and management for DM1 No definitive treatment exists for the progressive weakness in individuals with DM1, some of the factors, drugs etc. which improve the different symptoms of DM1 are listed below:
1. Muscle weakness therapeutics DM1 may be associated with low circulating dehydroepiandrosterone (DHEA) levels (Nakazora and Kurihara, 2005) and a specific dose of DHEA may improve myotonia and muscle strength in DM1 (Sugino et al., 1998). Phosphocreatine has the potential to buffer the intracellular stores of ATP within muscle fibers. 2. Myotonia treatment Myotonia in DM1 is typically mild to moderate and rarely requires treatment (Ricker et al., 1999). Drugs such as sodium channel blockers (phenytoin, procainmide) tricyclic antidepressive drugs, benzodiazepines, calcium antagonists, taurine, prednisolone, clomipramine etc. have a significant effect in DM1 (Trip et al., 2007). 3. Respiratory problem therapeutics If the patient has normal sleep though symptoms persist in spite of treatment of sleep disordered breathing, then CNS stimulant drugs such as modafinil (200–400 mg/day) and dexamphetamine or methylphenidate can be used (Wintzen et al., 2007). 4. Cataracts Cataract formation occurs early in DM1 and loss of SIX5 gene is one of the possible causes of cataract formation in DM1 patient (Personius et al., 2005). In such cases surgery is recommended (Garrott et al., 2004). 5. Cardiac arrhythmias therapeutics Tachyarrhythmia and conduction block may be responsible for up to 30% of fatalities in DM1 (Mathieu et al., 1999). In more advance condition of fatal arrhythmias invasive electrophysiological testing of the heart may be required (Sovari et al., 2007).
Letter to the Editor
6. Gene therapy The development of targeted molecular treatments (especially antisense therapy) has achieved great success in vitro and in animal models. Preferential reduction of mutant DMPK causes reversal of the abnormal phenotype in DM1 myoblasts (Furling et al., 2003). Alterations in splicing of the muscle specific chloride channel 1 (ClCN-1) cause the myotonic phenotype of DM1. Morpholino (a small molecule to alter gene expression) reverses the altered splicing of ClCN-1 mRNA (Wheeler et al., 2007). Though still far away from successful clinical use, gene therapy may become a valuable therapeutic approach in future. Antisense therapy is a form of treatment for genetic disorders or infections. When the genetic sequence of a particular gene is known to be causative of a particular disease, it is possible to synthesize a strand of nucleic acid (DNA, RNA or a chemical analog) that will bind to the messenger RNA (mRNA) produced by that gene and inactivate it or turn it off. This is because mRNA has to be single stranded for it to be translated. Alternatively, the strand might be targeted to bind a splicing site on pre-mRNA and modify the exon content of an mRNA. This synthesized nucleic acid is termed an anti-sense oligonucleotide (AONs) because its base sequence is complementary to the gene's messenger RNA (mRNA) which is called the sense sequence (so that a sense segment of mRNA 5′-AAGGUC-3′, would be blocked by the anti-sense mRNA segment 3′-UUCCAG-5′). Morpholino, an antisense molecule (nucleotide like structure) carries a complementary base sequence to the expanded RNA. Antisense molecules of DNA on injection into affected muscle cells bind to the expanded RNA, which is the cause of myotonic dystrophy. The interaction prevents RNA from trapping and interacting with another molecule and muscle blind protein (MBNL) which is central to the disease process in DM1. 4. Conclusion and future outlook DM1 is unique among all triplet repeat disorders in which a mutation in the 3′-UTR of the DMPK gene expands with CTG repeats leading to the disorder. About 20 years after the discovery of the first DM1 mutation, there is better understanding of the molecular pathology. A series of promising and effective antisense oligonucleotides (AONs) and small molecules are in the pipeline of development but much work still needs to be done. A second important challenge in treating DM1 is its multisystemic nature which is highly variable and makes it difficult to define target tissues and to develop reliable biomarkers for clinical trials. In summary, although much progress has been made, additional basic and translational studies will be required to understand the molecular pathogenesis of DM1 and to develop safe and effective treatment strategies. Conflict of interest The authors declare that they have no conflict of interest to disclose. Acknowledgments The authors are thankful to Sanjay Gandhi Post Graduate institute of Medical Sciences, Lucknow for providing infrastructure facility to carry out laboratory work. AK is thankful to DBT — New Delhi for providing his fellowship. References Addis, M., Serrenti, M., Meloni, C., Cau, M., Melis, M.A., 2012. Triplet-primed PCR is more sensitive than Southern blotting-long PCR for the diagnosis of myotonic dystrophy type1. Genet. Test. Mol. Biomarkers 16 (12), 1428–1431.
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Botta, A., et al., 2008. The CTG repeat expansion size correlates with the splicing defects observed in muscles from myotonic dystrophy type 1 patients. J. Med. Genet. 45 (10), 639–646. Brook, J.D., et al., 1992. Molecular basis of myotonic dystrophy: expansion of a trinucleotide (CTG) repeat at the 3′ end of a transcript encoding a protein kinase family member. Cell 68, 799–808. Chahine, M., George Jr., A.L., 1997. Myotonic dystrophy kinase modulates skeletal muscle but not cardiac voltage-gated sodium channels. FEBS Lett. 412 (3), 621–624. de Haro, M., et al., 2006. MBNL1 and CUGBP1 modify expanded CUG-induced toxicity in a drosophila model of myotonic dystrophy 1. Hum. Mol. Genet. 15, 2138–2145. Frisch, R., et al., 2001. Effect of triplet repeat expansion on chromatin structure and expression of DMPK and neighboring genes, SIX5 and DMWD, in myotonic dystrophy. Mol. Genet. Metab. 74 (1–2), 281–291. Furling, D., et al., 2003. Viral vector producing antisense RNA restores myotonic dystrophy myoblast functions. Gene Ther. 10, 795–802. Garrott, H.M., Walland, M.J., O'Day, J., 2004. Recurrent posterior capsular opacification and capsulorhexis contracture after cataract surgery in myotonic dystrophy. Clin. Experiment. Ophthalmol. 32, 653–655. Goossens, V., et al., 2008. Diagnostic efficiency, embryonic development and clinical outcome after the biopsy of one or two blastomeres for preimplantation genetic diagnosis. Hum. Reprod. 223, 481–492. Harley, H.G., et al., 1992. Expansion of an unstable DNA region and phenotypic variation in myotonic dystrophy. Nature 355, 545–546. Harley, H.G., et al., 1993. Size of the unstable CTG repeat sequence in relation to phenotype and parental transmission in myotonic dystrophy. Am. J. Hum. Genet. 52, 1164–1174. Harper, P.S., 1989. Myotonic Dystrophy, 2nd edn. W.B.Saunders Co., Philadelphia PA. Harper, P.S., 2001. Major problems in neurology, 3rd ed. Myotonic Dystrophy, 37. W.B. Saunders, Philadelphia. Ho, T.H., Bundman, D., Amstrong, D.L., Cooper, T.A., 2005. Transgenic mice expressing CUG-BP1 reproduce splicing mis-regulation observed in myotonic dystrophy. Hum. Mol. Genet. 14, 1539–1547. Howeler, C.J., Busch, H.F.M., Geraedts, J.P.M., Neirmeijer, M.F., Staal, A., 1989. Anticipation in myotonic dystrophy: fact or fiction? Brain 112, 779–797. Hunter, A., et al., 1992. The correlation of age of onset with CTG trinucleotide repeat amplification in myotonic dystrophy. J. Med. Genet. 29, 774–779. Kanadia, R.N., et al., 2003. A muscleblind knockout model for myotonic dystrophy. Science 302, 1978–1980. Karen, Usdin, 2008. The biological effects of simple tandem repeats: lessons from the repeat expansion diseases. Genome Res. 18, 1011–1019. Khajavi, M., et al., 2001. “Mitotic drive” of expanded CTG repeats in myotonic dystrophy type (DM1). Hum. Mol. Genet. 10, 855–863. Klesert, T.R., Otten, A.D., Tapscott, S.J., 1997. Trinucleotide repeat expansion at the myotonic dystrophy locus reduces expression of DMAHP. Nat. Genet. 16 (4), 402–406. Klesert, T.R., et al., 2000. Mice deficient in Six5 develop cataracts: implications for myotonic dystrophy. Nat. Genet. 25, 105–109. Lamusi, B., Artero, R., 2008. Molecular effects of the CTG repeats in mutant dystrophia myotonica protein kinase gene. Curr. Genomics 9, 509–516. Mahadevan, M., et al., 1992. Myotonic dystrophy mutation: an unstable CTG repeat in the 3′ untranslated region of the gene. Science 255, 1253–1255. Mahadevan, M.S., et al., 1993. Structure and genomic sequence of the myotonic dystrophy (DM kinase) gene. Hum. Mol. Genet. 2, 299–304. Mankodi, A., et al., 2001. Muscleblind localizes to nuclear foci of aberrant RNA in myotonic dystrophy types 1 and 2. Hum. Mol. Genet. 10, 2165–2170. Mathieu, J., et al., 1999. 10-year study of mortality in a cohort of patients with myotonic dystrophy. Neurology 52, 1658–1662. Miller, J.W., et al., 2000. Recruitment of human muscleblind proteins to (CUG)n expansions associated with myotonic dystrophy. EMBO J. 19, 4439–4448. Musova, Z., et al., 2009. Highly unstable sequence interruptions of the CTG repeat in the myotonic dystrophy gene. Am. J. Med. Genet. 149A, 1365–1374. Nakazora, H., Kurihara, T., 2005. The effect of dehydroepiandrosterone sulfate (DHEAS) on myotonia: intracellular studies. Intern. Med. 44, 1247–1251. Personius, K.E., Nautiyal, J., Reddy, S., 2005. Myotonia and muscle contractile properties in mice with SIX5 deficiency. Muscle Nerve 31 (4), 503–505. Redman, J.B., Fenwick Jr., R.G., Fu, Y.H., Pizzuti, A., Caskey, C.T., 1993. Relationship between parental trinucleotide GCT repeat length and severity of myotonic dystrophy in offspring. JAMA 269, 1960–1965. Ricker, K., et al., 1999. Linkage of proximal myotonic myopathy to chromosome 3q. Neurology 52, 170–171. Schara, U., Schoser, B.G.H., 2006. Myotonic Dystrophies type 1 and 2 — a summary of current aspects. Semin. Pediatr. Neurol. 13, 71–79. Schoser, B., et al., 2004. Muscle pathology in 57 patients with myotonic dystrophy type 2. Muscle Nerve 29, 275–281. Sermon, K., Seneca, S., De Rycke, M., 2001. PGD in the lab for triplet repeat diseasesmyotonic dystrophy. Huntington's disease and fragile-X syndrome. Mol. Cell. Endocrinol. 183, S77–S85. Shaw, D.J., et al., 1993. Genomic organization and transcriptional units at the myotonic dystrophy locus. Genomics 18, 673–679. Sovari, A.A., Bodine, C.K., Farokhi, F., 2007. Cardiovascular manifestations of myotonic dystrophy-1. Cardiology 15 (4), 191–194. Sugino, M., et al., 1998. A pilot study of dehydroepiandrosterone sulfate in myotonic dystrophy. Neurology 51, 586–589. Timchenko, N.A., Cai, Z.-J., Welm, A.L., Reddy, S., Ashizawa, T., Timchenko, L.T., 2001. RNA CUG repeats sequester and alter protein levels and activity of CUGBP1. J. Biol. Chem. 276, 7820–7826.
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Tishkoff, D., Filosi, N., Gaida, G., Kolodner, R., 1997. A novel mutation avoidance mechanism dependent on S. cerevisiae RAD27 is distinct from DNA mismatch repair. Cell 88, 253–263. Trip, J., et al., 2007. Drug treatment for myotonia. Cochrane Database Syst. Rev. 4, CD004762. Warner, J.P., et al., 1996. A general method for the detection of large CAG repeat expansions by fluorescent PCR. J. Med. Genet. 33, 1022–1026.
Wheeler, T.M., et al., 2007. Correction of ClC-1 splicing eliminates chloride channelopathy and myotonia in mouse models of myotonic dystrophy. J. Clin. Invest. 117, 3952–3957. Wintzen, A.R., Lammers, G.J., van Dijk, J.G., 2007. Does modafinil enhance activity of patients with myotonic dystrophy? A double-blind placebo-controlled crossover study. J. Neurol. 254, 26–28.
Contents lists available at SciVerse ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Letter to the Editor
Myotonic dystrophy type 1 (DM1): A triplet repeat expansion disorder Ashok Kumar, Sarita Agarwal ⁎, Divya Agarwal, Shubha R. Phadke Department of Genetics, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow 226014, India
a r t i c l e
i n f o
Article history: Accepted 16 March 2013 Available online 6 April 2013 Keywords: CTG repeat DMPK gene Genetics Myotonic dystrophy type 1 (DM1) Triplet repeat disorder
a b s t r a c t Myotonic dystrophy is a progressive multisystem genetic disorder affecting about 1 in 8000 people worldwide. The unstable repeat expansions of (CTG)n or (CCTG)n in the DMPK and ZNF9 genes cause the two known subtypes of myotonic dystrophy: (i) myotonic dystrophy type 1 (DM1) and (ii) myotonic dystrophy type 2 (DM2) respectively. 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. This review will focus on the clinical features, molecular view and genetics, diagnosis and management of DM1. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Trinucleotide repeat disorder is a disorder caused by an increase in the number of triplet repeats occurring in some of the genes. The disease manifests when the number of repeats increases and crosses the limit of normal repeats and affects the gene function. These disorders are classified into 2 major categories: (i) Coding expansion disorder (Huntington disease (HD) and spirocerebellar ataxias) and (ii) non-coding expansion disorder (Myotonic Dystrophy (DM) and Friedreich ataxia (FRDA)). 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) which are caused by two different genes DMPK (dystrophia myotonica protein kinase) ZNF9 i.e. zinc finger protein (Table 1). On the basis of clinical severity the disorder is divided into three groups mid, classical and congenital (Table 2). Out of these three, the congenital form is the most severe (Harper, 1989). DM affects 1 in 8000 people worldwide and the relative prevalence rates of DM1 and DM2 are 98% and 2% respectively. 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 (Howeler et al., 1989) due to the expansion of the repeat number during gametogenesis. Repeat contraction events are rare and occur 4.2 to
⁎ Corresponding author. Tel.: +91 522 2494349(O), +91 9415336601; fax: +91 522 2668017. E-mail address: [email protected] (S. Agarwal). 0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2013.03.059
6.4% of the transmission (Musova et al., 2009). The triplet repeat numbers are unstable and the number of repeats varies from the tissue to tissue. 2. Genetic insight of disease 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 (Mahadevan et al., 1993; Shaw et al., 1993). The repeats in DMPK are repeats of CTG and vary in the normal population from 3 to 37. The size more than 50 CTG is associated with DM1 and severity correlates with CTG repeat number (Brook et al., 1992; Mahadevan et al., 1992). The mutant DMPK gene results in the following changes: 1. Dominantly inherited loss of function DMPK phosphorylates muscle-specific voltage gated Na + channels and regulates the excitability of muscle cells (Chahine and George, 1997). DM1 patients are heterozygous for the expansion mutation. The loss of protein production from the mutant allele is because the expanded CTG repeats in the 3′-UTR interfere with mRNA and/or protein synthesis (Botta et al., 2008; Karen, 2008). 2. Disruption of neighboring genes Expanded CTG repeats interfere with the local chromatin configuration which affects expression of both DMPK and neighboring genes DMAHP (dystrophia myotonica associated home domain protein)/SIX5, dystrophia myotonica containing WD repeat motif (DMWD), and muscle blind protein (MBNL1) (Frisch et al., 2001). MBNL1 gene is located on chromosome 3q25. The expanded number of CTG repeats within the 3′-UTR of the DMPK gene interferes with the transcription of the neighboring DMAHP (SIX5) gene by masking its enhancer that lies close to the CTG island (Klesert et
Letter to the Editor Table 1 Comparison of DM1 and DM2. Feature
DM 1
DM 2
Epidemiology Onset age (years) Anticipation and severity Congenital form Facial weakness Proximal onset Distal onset Muscle pain Myotonia Chromosome Mutated gene Mutation type Repeat size Cataracts Balding Cardiac arrhythmias Gonadal failure Hypersomnia
Widespread 0 to adult Moderate-severe Yes More Late Early less Yes 19q13.3 DMPK CTG repeats 50–4000 More More More More More
European 8–60 Mild-moderate No Less Early Late More Yes 3q21 ZNF9 CCTG repeats ~5000 Less Less Less Less Less
al., 1997). Therefore, CTG-repeat expansions can suppress local gene expression and SIX5/DMAHP may play a role in manifestation of DM (Klesert et al., 1997). If there are no further changes in local gene expression above a certain threshold of repeats, it may not be affect the phenotype of DM. The deficiency or decreased Six5 transcription is important in the etiology of DM like cataract formation (Klesert et al., 2000). As DM is a multisystemic and multigenic disorder, so SIX5 knockout mice may not show DM. 3. Defects in RNA metabolism In the normal cell, RNA is transcribed, processed in the nucleus and transported to the cytoplasm where it is bound by specific RNA binding proteins i.e. CUG-BP or CELF1 and then translated into the DM protein kinase (DMPK). In affected DM1 patients (Fig. 1) transcripts from the mutant DMPK allele and normal allele are retained within the nucleus because the expanded number of CUG repeats bind and sequester mRNA transport proteins (CUGBP1). DMPK transcripts bearing a long (CUG)n tract form hairpin-like structures
Table 2 Correlation of phenotype and CTG repeat length in DM1. Phenotype
Clinical signs
CTG repeata,b Size
Age of onset
Average age of death
Asymptomatic (premutation) Mild
None
35 to 49
NAc
NAc
Cataracts Mild myotonia
50 to ~150
20 to 70 yrs
Classical
Weakness, myotonia Cataracts Balding Cardiac arrhythmia Others Infantile hypotonia Respiratory deficits Intellectual disability
~100 to ~1000
10 to 30 yrs
60 yrs to normal life span 48 to 55 yrs
~100 to ~1000
From birth
48 to 55 yrs
Congenital
a b c
CTG repeat sizes are known to overlap between phenotypes. Normal CTG repeat size is 5–34. NA (not applicable).
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(Tishkoff et al., 1997) and aggregate of DMPK RNA are observed in the ribonucleic foci in the nucleus and some of the mRNA with expanded repeats is also transported to the cytoplasm. The mechanism of toxic effects of these RNAs is not definitely established. The nuclear CUGn repeat aggregates sequester MBNL1 protein which is a muscle blind protein (Mankodi et al., 2001; Miller et al., 2000). This is likely the mechanism of phenotypic effect as MBNL1 knock out homozygous mice show myotonia and other symptoms of DM1 (Kanadia et al., 2003). The other cellular event is increase in CUGBP1. So both factors, reduction of MBNL1 and increase in CUGBP1 in DM1 cells as a result of RNA aggregates are likely to cause DM1 phenotype. Both MBNL1 and CUGBP1 regulate splicing of the same mRNAs (chloride channel 1, insulin receptor, troponin T) by binding to different sites (Lamusi and Artero, 2008). Altered splicing of RNAs affecting other proteins can explain multisystem nature of DM1 manifestation. MBNL1 and CUGBP1 also have several important functions like splicing, protein translation and RNA and their deregulation alter splicing of other RNAs and contribute to the disease manifestations (Miller et al., 2000; Timchenko et al., 2001). This suggests that their mechanism may be independent of each other (Ho et al., 2005). An increase in CUGBP1 causes degeneration in the drosophila model (de Haro et al., 2006). This suggests that therapeutics strategies at level of MBNL1 and CUGBP1 can be effective in DM1 management (de Haro et al., 2006). Thus, the altered RNA metabolism of both the mutant and normal DMPK RNA transcripts leads to deficiency of the DMPK protein, phosphorylation of proteins and symptoms such as myotonia. Phosphorylation of protein is a post-translational modification of proteins in which a serine, threonine or a tyrosine residue is phosphorylated by a protein kinase by the addition of a covalently bound phosphate group. In almost all cases of phosphoregulation, the protein switches between a phosphorylated and an unphosphorylated form and one is an active form while the other one is inactive. 3. Diagnosis of DM1 3.1. Clinical 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 myopathic face, 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 (Harley et al., 1992, 1993; Hunter et al., 1992; Redman et al., 1993). 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 (Harper, 2001; Schara and Schoser, 2006). 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 (Schara and Schoser, 2006; Schoser et al., 2004). 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 congenita and myopathies.
228
Letter to the Editor
Normal DMPK
DM
-
DMAHP DMPK
(CTG)n<37
DMAHP
DMPK
DMAHP
(CTG)n>50
(CTG)n<37
CUG-BP
DMPK
DMAHP
(CTG)n<37
CUG-BP CUG-BP (CTG)n<37
DMPK DMAHP Protein kinase Phosphorylation of Na+ channels
DMPK
DMAHP Homeobox Gene Expressed in brain and muscle
Altered CUG-BP Localization ? Endocrine Testicular atrophy Diabetes Frontal Balding ? Floppineess in C-DM
DMAH P 20% DMPK ?Myotonia
50% DMPH ? Mental retardation ? Dysmorpholgy
Processing of DMPK gene and DMAHP genes within normal and DM cells
Fig. 1. Processing of DMPK and DMAHP (SIX5) genes within normal and DM cells. In the normal cell, both DMPK genes are transcribed, and the RNAs are processed in the nucleus and transported to the cytoplasm where they are bound by specific RNA binding proteins (CUG-BP) and then translated into the DM protein kinase (DMPK). In DM cells, the DMPK gene is transcribed from both normal and mutant alleles but the transcripts are largely retained within the nucleus. The expanded numbers of CTG repeats within the 3′ UTR of the DMPK gene also interfere with the transcription of the neighboring DMAHP gene and thus lead to decreased amount of DMAHP transcript and protein. Thus, functional DMPK protein does not form in DM patients.
3.2. Molecular diagnosis The detection of expansion of triplet repeat traditionally is done by Southern blot analysis (Goossens et al., 2008; Sermon et al., 2001). However, triplet primed PCR (TP-PCR) based testing is found to be a reliable non-radioactive method as a replacement of the Southern blot (Addis et al., 2012; Khajavi et al., 2001; Warner et al., 1996). The number of triplet repeats correlates with the phenotype and hence, it is important for prediction of severity of the disease (Harley et al., 1992, 1993; Hunter et al., 1992; Redman et al., 1993). 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 counseling. After molecular confirmation of the diagnosis in the proband, the family can be counseled 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 (Warner et al., 1996), and multiplex PCR over Southern blotting (Goossens et al., 2008) for screening of triplet repeat expansion in DM1 is now recommended. 3.3. Therapeutics and management for DM1 No definitive treatment exists for the progressive weakness in individuals with DM1, some of the factors, drugs etc. which improve the different symptoms of DM1 are listed below:
1. Muscle weakness therapeutics DM1 may be associated with low circulating dehydroepiandrosterone (DHEA) levels (Nakazora and Kurihara, 2005) and a specific dose of DHEA may improve myotonia and muscle strength in DM1 (Sugino et al., 1998). Phosphocreatine has the potential to buffer the intracellular stores of ATP within muscle fibers. 2. Myotonia treatment Myotonia in DM1 is typically mild to moderate and rarely requires treatment (Ricker et al., 1999). Drugs such as sodium channel blockers (phenytoin, procainmide) tricyclic antidepressive drugs, benzodiazepines, calcium antagonists, taurine, prednisolone, clomipramine etc. have a significant effect in DM1 (Trip et al., 2007). 3. Respiratory problem therapeutics If the patient has normal sleep though symptoms persist in spite of treatment of sleep disordered breathing, then CNS stimulant drugs such as modafinil (200–400 mg/day) and dexamphetamine or methylphenidate can be used (Wintzen et al., 2007). 4. Cataracts Cataract formation occurs early in DM1 and loss of SIX5 gene is one of the possible causes of cataract formation in DM1 patient (Personius et al., 2005). In such cases surgery is recommended (Garrott et al., 2004). 5. Cardiac arrhythmias therapeutics Tachyarrhythmia and conduction block may be responsible for up to 30% of fatalities in DM1 (Mathieu et al., 1999). In more advance condition of fatal arrhythmias invasive electrophysiological testing of the heart may be required (Sovari et al., 2007).
Letter to the Editor
6. Gene therapy The development of targeted molecular treatments (especially antisense therapy) has achieved great success in vitro and in animal models. Preferential reduction of mutant DMPK causes reversal of the abnormal phenotype in DM1 myoblasts (Furling et al., 2003). Alterations in splicing of the muscle specific chloride channel 1 (ClCN-1) cause the myotonic phenotype of DM1. Morpholino (a small molecule to alter gene expression) reverses the altered splicing of ClCN-1 mRNA (Wheeler et al., 2007). Though still far away from successful clinical use, gene therapy may become a valuable therapeutic approach in future. Antisense therapy is a form of treatment for genetic disorders or infections. When the genetic sequence of a particular gene is known to be causative of a particular disease, it is possible to synthesize a strand of nucleic acid (DNA, RNA or a chemical analog) that will bind to the messenger RNA (mRNA) produced by that gene and inactivate it or turn it off. This is because mRNA has to be single stranded for it to be translated. Alternatively, the strand might be targeted to bind a splicing site on pre-mRNA and modify the exon content of an mRNA. This synthesized nucleic acid is termed an anti-sense oligonucleotide (AONs) because its base sequence is complementary to the gene's messenger RNA (mRNA) which is called the sense sequence (so that a sense segment of mRNA 5′-AAGGUC-3′, would be blocked by the anti-sense mRNA segment 3′-UUCCAG-5′). Morpholino, an antisense molecule (nucleotide like structure) carries a complementary base sequence to the expanded RNA. Antisense molecules of DNA on injection into affected muscle cells bind to the expanded RNA, which is the cause of myotonic dystrophy. The interaction prevents RNA from trapping and interacting with another molecule and muscle blind protein (MBNL) which is central to the disease process in DM1. 4. Conclusion and future outlook DM1 is unique among all triplet repeat disorders in which a mutation in the 3′-UTR of the DMPK gene expands with CTG repeats leading to the disorder. About 20 years after the discovery of the first DM1 mutation, there is better understanding of the molecular pathology. A series of promising and effective antisense oligonucleotides (AONs) and small molecules are in the pipeline of development but much work still needs to be done. A second important challenge in treating DM1 is its multisystemic nature which is highly variable and makes it difficult to define target tissues and to develop reliable biomarkers for clinical trials. In summary, although much progress has been made, additional basic and translational studies will be required to understand the molecular pathogenesis of DM1 and to develop safe and effective treatment strategies. Conflict of interest The authors declare that they have no conflict of interest to disclose. Acknowledgments The authors are thankful to Sanjay Gandhi Post Graduate institute of Medical Sciences, Lucknow for providing infrastructure facility to carry out laboratory work. AK is thankful to DBT — New Delhi for providing his fellowship. References Addis, M., Serrenti, M., Meloni, C., Cau, M., Melis, M.A., 2012. Triplet-primed PCR is more sensitive than Southern blotting-long PCR for the diagnosis of myotonic dystrophy type1. Genet. Test. Mol. Biomarkers 16 (12), 1428–1431.
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