Myotonic dystrophy phenotype without expansion of (CTG)n repeat: an entity distinct from proximal myotonic myopathy (PROMM)?

Myotonic dystrophy (DM) is associated with an expansion of an unstable (CTG)n repeat in the 3' untranslated region of the DM protein kinase (DMPK) gene on chromosome 19q13.3. We studied six patients from two families who showed no expansions of the repeat, in spite of their clinical diagnosis of DM. These patients had multi-systemic manifestations that were distinguishable from those seen in other myotonic disorders, including proximal myotonic myopathy (PROMM). In one additional family, two symptomatic members showed no expanded (CTG)n repeats, while their affected relatives had the expanded repeats. DM haplotype analysis failed to exclude the DMPK locus as a possible site of mutation in each family; however, DMPK mRNA levels were normal. We conclude that a mutation(s) other than the expanded (CTG)n repeat can cause the DM phenotype. The mutation(s) in these families remain(s) to be mapped and characterized.     

Expression of myotonic dystrophy protein kinase gene during in vivo and in vitro mouse myogenesis

Myotonic dystrophy (DM) is an autosomal dominant neuromuscular disorder characterized by a great variability in its clinical manifestations. The mutational basis underlying DM consists of an unstable (CTG)n trinucleotide repeat in the 3' untranslated region of the myotonic dystrophy protein kinase gene (DMPK). Conflicting results on DMPK gene expression in congenitally affected infants (CDM) have been published. Moreover, the prominence of satellite cells seen in muscle of CDM infants supports the notion that the congenital form is associated with an arrest in muscle development and suggests a role for the DMPK gene during differentiation and maturation of muscle. In order to clarify these findings, a comparative study of DMPK and myogenic factor mRNA levels was performed in developing mouse muscle tissues and cultured muscle cells at different developmental stages. Results show that DMPK gene expression is upregulated at a late stage of muscular development. This upregulation does not seem to depend on a given muscle specific bHLH factor.     

Expanding complexity in myotonic dystrophy

Myotonic dystrophy (DM) is a highly variable multisystemic disease belonging to the rather special class of trinucleotide expansion disorders. DM results from dynamic expansion of a perfect (CTG)n repeat situated in a gene-dense region on chromosome 19q. Based on findings in patient materials or cellular and animal models, many mechanisms for the causes and consequences of repeat expansion have been proposed; however, none of them has enjoyed prolonged support. There is now circumstantial evidence that long (CTG)n repeats may affect the expression of any of at least three genes, myotonic dystrophy protein kinase (DMPK), DMR-N9 (gene 59), and a DM-associated homeodomain protein (DMAHP). Furthermore, the new findings suggest that DM is not a simple gene-dosage or gain-or-loss-of-function disorder but that entirely new pathological pathways at the DNA, RNA, or protein level may play a role in its manifestation.     

Urinary bladder wall repair: what suture to use?

We report the mapping of a second myotonic dystrophy locus, myotonic dystrophy type 2 (DM2). Myotonic dystrophy (DM) is a multi-system disease and the most common form of muscular dystrophy in adults. In 1992, DM was shown to be caused by an expanded CTG repeat in the 3' untranslated region of the dystrophia myotonica-protein kinase gene (DMPK) on chromosome 19 (refs 2-6). Although several theories have been put forth to explain how the CTG expansion causes the broad spectrum of clinical features associated with DM, it is not understood how this mutation, which does not alter the protein-coding region of a gene, causes an affect at the cellular level. We have identified a five-generation family (MN1) with a genetically distinct form of myotonic dystrophy. Affected members exhibit remarkable clinical similarity to DM (myotonia, proximal and distal limb weakness, frontal balding, cataracts and cardiac arrhythmias) but do not have the chromosome-19 D CTG expansion. We have mapped the disease locus (DM2) of the MN1 family to a 10-cM region of chromosome 3q. Understanding the common molecular features of two different forms of the disease should shed light on the mechanisms responsible for the broad constellation of seemingly unrelated clinical features present in both diseases.     

Altered phosphorylation and intracellular distribution of a (CUG)n triplet repeat RNA-binding protein in patients with myotonic dystrophy and in myotonin protein kinase knockout mice

Myotonic dystrophy (DM) is associated with expansion of CTG repeats in the 3'-untranslated region of the myotonin protein kinase (DMPK) gene. The molecular mechanism whereby expansion of the (CUG)n repeats in the 3'-untranslated region of DMPK gene induces DM is unknown. We previously isolated a protein with specific binding to CUG repeat sequences (CUG-BP/hNab50) that possibly plays a role in mRNA processing and/or transport. Here we present evidence that the phosphorylation status and intracellular distribution of the RNA CUG-binding protein, identical to hNab50 protein (CUG-BP/hNab50), are altered in homozygous DM patient and that CUG-BP/hNab50 is a substrate for DMPK both in vivo and in vitro. Data from two biological systems with reduced levels of DMPK, homozygous DM patient and DMPK knockout mice, show that DMPK regulates both phosphorylation and intracellular localization of the CUG-BP/hNab50 protein. Decreased levels of DMPK observed in DM patients and DMPK knockout mice led to the elevation of the hypophosphorylated form of CUG-BP/hNab50. Nuclear concentration of the hypophosphorylated CUG-BP/hNab50 isoform is increased in DMPK knockout mice and in homozygous DM patient. DMPK also interacts with and phosphorylates CUG-BP/hNab50 protein in vitro. DMPK-mediated phosphorylation of CUG-BP/hNab50 results in dramatic reduction of the CUG-BP2, hypophosphorylated isoform, accumulation of which was observed in the nuclei of DMPK knockout mice. These data suggest a feedback mechanism whereby decreased levels of DMPK could alter phosphorylation status of CUG-BP/hNab50, thus facilitating nuclear localization of CUG-BP/hNab50. Our results suggest that DM pathophysiology could be, in part, a result of sequestration of CUG-BP/hNab50 and, in part, of lowered DMPK levels, which, in turn, affect processing and transport of specific subclass of mRNAs.     

Myotonic dystrophy protein kinase is involved in the modulation of the Ca2+ homeostasis in skeletal muscle cells

Myotonic dystrophy (DM), the most prevalent muscular disorder in adults, is caused by (CTG)n-repeat expansion in a gene encoding a protein kinase (DM protein kinase; DMPK) and involves changes in cytoarchitecture and ion homeostasis. To obtain clues to the normal biological role of DMPK in cellular ion homeostasis, we have compared the resting [Ca2+]i, the amplitude and shape of depolarization-induced Ca2+ transients, and the content of ATP-driven ion pumps in cultured skeletal muscle cells of wild-type and DMPK[-/-] knockout mice. In vitro-differentiated DMPK[-/-] myotubes exhibit a higher resting [Ca2+]i than do wild-type myotubes because of an altered open probability of voltage-dependent l-type Ca2+ and Na+ channels. The mutant myotubes exhibit smaller and slower Ca2+ responses upon triggering by acetylcholine or high external K+. In addition, we observed that these Ca2+ transients partially result from an influx of extracellular Ca2+ through the l-type Ca2+ channel. Neither the content nor the activity of Na+/K+ ATPase and sarcoplasmic reticulum Ca2+-ATPase are affected by DMPK absence. In conclusion, our data suggest that DMPK is involved in modulating the initial events of excitation-contraction coupling in skeletal muscle.     

Somatic mosaicism of p(CTG)n expansion in a case of myotonic dystrophy with parotid tumor

Myotonic dystrophy (MD) is an autosomal dominant systemic disorder with an unstable expansion of the CTG triplet repeat in the 3'-untranslated region of the gene encoding myotonine protein kinase (DMPK) which maps to chromosome 19q13.3. Somatic mosaicism of CTG repeats in MD has been reported; and it has been observed that CTG repeats in tumor tissues associated with MD are more expanded than the other tissues. It is not rare that parotid tumors are found in patients with MD. We performed Southern blot analysis for tissues from the parotid tumor, the normal parotid gland, the skeletal muscles, and the leukocyte from a 60-year-old patient with MD. CTG repeat was most expanded in the parotid tumor, and the normal parotid gland had longer expansion of CTG repeat than the skeletal muscles. The leukocyte had the shortest expansion of CTG repeat. The expansion of CTG repeat in the parotid tumor may be related to active cell division and may underlie the occurrence of tumors in MD.     

Normal levels of DM RNA and myotonin protein kinase in skeletal muscle from adult myotonic dystrophy (DM) patients

A major question about the pathogenesis of myotonic dystrophy (DM) is how the (CTG)n repeat mutation alters expression of the DM gene and how that is related to disease causation. Most previous studies have found a decrease in DM RNA and protein in patient tissue. In contrast to these reports we find, unexpectedly, that independent of the size of the CTG repeat: (1) there are equal levels of RNA products of mutant and normal alleles, and (2) levels of Mt-PK in skeletal muscle from DM patients is unaltered from normal. These findings are consistent with the recent hypothesis that mutant DM DNA or RNA may cause disease by disrupting the function of other, yet unidentified, genes.     

Instability of normal (CTG)n alleles in the DM kinase gene

We report on a myotonic dystrophy (DM) family exhibiting instability of normal sized (CTG)n alleles in the DM kinase gene on the non-DM chromosome. At least two mutational events involving normal DM alleles must have occurred in this family; one was characterised as a 34-35 (CTG)n repeat mutation. These findings represent a dissociation between (CTG)n repeat instability and myotonic dystrophy. Furthermore, this family highlights genetic counselling issues relating to the pathogenicity of alleles at the upper end of the normal size range and the risk of further expansion into the disease range.     

Over expression of the murine myotonic dystrophy protein kinase in the mouse myogenic C2C12 cell line leads to inhibition of terminal differentiation

Myotonic dystrophy (DM) is an autosomal dominant human disorder, caused by the abnormal expansion of a CTG trinucleotide repeat in the 3' untranslated region of a protein kinase gene (DMPK). Muscle symptoms are a common feature of the disorder and in the adult onset cases there are increased patterns of muscle fibre degeneration and regeneration. In the congenitally affected infants there is a failure of muscle maturation, with the histological presence of numerous immature fibres. However, the pathological mechanism in both forms of the disease is unclear. We report that over-expression of the murine dmpk gene, in a murine myogenic cell line, leads to markedly reduced levels of fusion to the terminally differentiated state. These findings complement recently published data using a heterologous expression/cell system and may have implications for the understanding of the disease process in this disorder.     

Diameter of the inferior vena cava as an index of dry weight in patients undergoing CAPD

Trinucleotide microsatellites are widespread in the human and other mammalian genomes. Expansions of unstable trinucleotide repeats have been associated so far with a number of different genetic diseases including fragile X, myotonic dystrophy (DM) and Huntington disease. While ten possible trinucleotides can occur at the DNA level, only CTG and CCG repeats are involved in the disorders described so far. However, the repeat expansion detection (RED) technique has identified additional large repeats of ATG, CCT, CTT, and TGG of potentially pathological significance in the human genome. We now show that conclusive information about the chromosomal localization of long trinucleotide repeats can be achieved in a relatively short time using fluorescence in situ hybridization (FISH) with biotin-labelled trinucleotide polymers. Large CTG expansions (> 1 kb) in DM and an unstable (CTG)306 repeat in a patient with schizophrenia were detected by eye through the microscope without electronic enhancement. Digital imaging was used to analyse the chromosomal distribution of long CCA and AGG repeats. Our results suggest that long trinucleotide repeats occur in the normal human genome and that the size of individual repeat loci may be polymorphic.     

Relative stability of a minimal CTG repeat expansion in a large kindred with myotonic dystrophy

The genetic basis for myotonic dystrophy (DM) is a CTG trinucleotide repeat expansion. The number of CTG repeats commonly increases in affected individuals of successive generations, in association with anticipation. We identified a large DM family in which multiple members had minimal CTG repeat expansions, and in which the number of CTG repeats remained in the minimally expanded range through at least three, and possibly four, generations. This relative stability of minimal CTG repeat expansions may help to maintain the DM mutation in the population.     

Jozef B. Cohen: 1921-1995

The distribution of alleles with various CTG-repeat numbers was studied and the haplotypes for polymorphic sites HhaI and HinfI of mouse muscle protein kinase (DMPK) were analyzed in inhabitants of northwestern Russia and in patients with myotonic dystrophy (90 and 18 chromosomes, respectively). Twelve normal alleles with the triplet-repeat number from 5 to 24 were identified and the alleles with five (42.5%) and 11-13 (37%) repeats were found to be predominant. The bimodal distribution revealed is similar to those described earlier for other populations, however, the frequencies of individual alleles differed from those in populations of Europe and Central Russia. No significant differences in frequencies of CTG alleles were found in 32 normal chromosomes involved in compounds with the mutant chromosomes (i.e., in patients with myotonic dystrophy) as compared to their frequencies in the population. However, almost all mutant chromosomes (16 of 18) had the same haplotype for intragenic polymorphic sites: HhaI-; HinfI+. This haplotype was also inherent in 91% of all chromosomes with CTG5 and all chromosomes with a CTG number more than 15. Possible evolution of chromosomes with different numbers of triplet repeats mediating their expansion and impairing the function are discussed.     

Myotonic dystrophy: antisense oligonucleotide inhibition of DMPK gene expression in vitro

Antisense phosphorothioate oligonucleotides, targeted against the first codon starting region of DMPK mRNA, were successfully used in K562 and HepG2 cells to decrease DMPK expression. The most effective antisense oligo, MIO1, when added to K562 cells, shows a 75% reduction of the DMPK gene expression 6 hours after addition. The same molecule, when encapsulated in liposomes, delays myotonin mRNA decrease at 24 hours after cell treatment. This considerable success with such inhibition in vitro could be utilised to generate a cell model to study myotonic dystrophy (DM) chemio-physiological alterations.     

Molecular genetics of myotonic dystrophy--population genetics of the CTG repeat expansion of the MTPK gene

The CTG repeat number in the 3'-untranslated region of the myotonin protein kinase (MTPK) gene varies between 5 and 37 in normal individuals, whereas myotonic dystrophy (DM) patients have expansions from 50 to 3000 copies. However little is known about the molecular mechanisms or the genetic control of the expansion of triplet repeats. To explain the dynamic mutation mechanism and high prevalence in the population, slippage theory, multistep model and meiotic drive hypothesis have been proposed. Recent studies have shown that repeat expansion may affect neighboring genes (59 gene and DMAHP gene), or exert its effect at the RNA level by modulating the binding of (CUG)n-RNA binding proteins which are required for the maturation, stability and translation of specific mRNAs.     

Accommodation of a D-Phe residue into a right-handed 3(10)-helix: structure of Boc-D-Phe-(Aib)4-Gly-L-Leu-(Aib)2-OMe, an analogue of the amino terminal segment of antiamoebins and emerimicins

Recently an unstable trinucleotide CTG repeat, located within the 3' untranslated region of a gene on 19q13.3 was discovered in kindreds with myotonic dystrophy (DM). The age-of-onset/severity of DM shows a good correlation with CTG repeat size, and pedigrees and data reported to date have shown a striking trend toward amplification of the size of the CTG repeat during transmission from parent to child. The amplification has been accepted as the biological explanation for anticipation in the clinical severity observed in many families with DM. In this paper we report on 3 families where CTG amplification decreased during transmission from parent to child. In one case there was a gene conversion event, while in the remaining 2 there was a simpler reduction in the size of the repeat length. The changes appear to have been accompanied by a reduction in clinical severity in the child when compared to the parent. These observations are discussed in terms of their clinical implications and the biases that may exist in much of the reported data.     

MKBP, a novel member of the small heat shock protein family, binds and activates the myotonic dystrophy protein kinase

Muscle cells are frequently subjected to severe conditions caused by heat, oxidative, and mechanical stresses. The small heat shock proteins (sHSPs) such as alphaB-crystallin and HSP27, which are highly expressed in muscle cells, have been suggested to play roles in maintaining myofibrillar integrity against such stresses. Here, we identified a novel member of the sHSP family that associates specifically with myotonic dystrophy protein kinase (DMPK). This DMPK-binding protein, MKBP, shows a unique nature compared with other known sHSPs: (a) In muscle cytosol, MKBP exists as an oligomeric complex separate from the complex formed by alphaB-crystallin and HSP27. (b) The expression of MKBP is not induced by heat shock, although it shows the characteristic early response of redistribution to the insoluble fraction like other sHSPs. Immunohistochemical analysis of skeletal muscle cells shows that MKBP localizes to the cross sections of individual myofibrils at the Z-membrane as well as the neuromuscular junction, where DMPK has been suggested to be concentrated. In vitro, MKBP enhances the kinase activity of DMPK and protects it from heat-induced inactivation. These results suggest that MKBP constitutes a novel stress-responsive system independent of other known sHSPs in muscle cells and that DMPK may be involved in this system by being activated by MKBP. Importantly, since the amount of MKBP protein, but not that of other sHSP family member proteins, is selectively upregulated in skeletal muscle from DM patients, an interaction between DMPK and MKBP may be involved in the pathogenesis of DM.