Distribution of CTG repeats at the DMPK gene in myotonic distrophy patients and healthy individuals from the Mexican population

Myotonic dystrophy type 1 (DM1), the most common form of adult muscular dystrophy, is caused by anormal expansion of CTG trinucleotide repeats located in the 3′-untranslated region of the DMPK gene. The clinical features of DM1 are multisystemic and highly variable, and the unstable nature of CTG expansion causes wide genotypic and phenotypic presentations. In this study, we described to our knowledge for the first time the molecular diagnosis of myotonic dystrophy type 1 patients in the Mexican population, applying a fluorescent PCR method in combination with capillary electrophoresis analysis of the amplified products. We identified expanded alleles in 45 out of 50 patients (90%) with clinical features of myotonic disease. Furthermore, genotyping of 400 healthy subjects revealed the presence of 25 different alleles, ranging in size from 5 to 34 repeats. The most frequent allele was 13 CTG repeats (38.87%) and the frequency for alleles over 18 CTG repeats was 6.7%. Molecular test is essential for DM1 diagnosis and distribution of the CTG repeat alleles present in the Mexican population are significantly different from those of other populations.     

Cardiac conduction abnormalities and Stokes-Adams attacks in myotonic dystrophy.

Myotonic dystrophy is a well known cause of cardiomyopathy. While various cardiac conduction abnormalities have been described in patients with myotonic dystrophy, so far only sporadic cases of Stokes-Adams attacks have been reported. Of 27 patients with this disease various conduction disturbances were detected in 17 (63%), 5 of whom presented with Stokes-Adams attacks and were found to have intracardiac conduction defects. The prognosis in four of the five patients was greatly improved with permanent pacemaker implantation.     

Leptin in type 2 diabetic or myotonic dystrophic women.

BACKGROUND: Insulin resistance is an important determinant of circulating leptin concentrations in humans, but its independent contribution on plasma leptin levels are controversial. In the present study, we characterized plasma leptin levels and their regulation in women with 2 different insulin resistance states: type 2 diabetes and myotonic dystrophy disease, and in controls. MATERIAL AND METHODS: We studied 3 groups of women: 21 type 2 diabetic patients, 20 myotonic dystrophic patients and a control group of 20 normoglycemic subjects, matched in age and body mass index. Body composition, fasting glucose and insulin, IGF-I, IGF-binding protein-3 and leptin were studied. Body composition was measured using a bioelectrical impedance analyser. Insulin sensitivity (in percentage) was modeled according to a computer-based homeostasis model assessment model. Data are expressed in mean +/- SEM. RESULTS: In both groups of patients, glucose concentrations were higher in type 2 diabetic patients than in myotonic dystrophic patients, and insulin concentrations and insulin sensitivity were similar in the 2 groups of patients (82.4 +/- 18.6% in type 2 diabetic patients vs. 69.7 +/- 9.7% in myotonic dystrophic patients, p = 0.2) and lower than in controls. Serum leptin and leptin/fat mass ratio were higher in myotonic dystrophic patients than in type 2 diabetic patients (30 +/- 4.9 ng/ml vs. 17.7 +/- 2.6 ng/ml, p = 0.03 and 2.32 +/- 0.69 ng/ml/kg vs. 1.07 +/- 0.2 ng/ml/kg, p = 0.02, respectively) or those found in controls. In type 2 diabetic patients, leptin concentrations were correlated with body mass index and body fat, and in myotonic dystrophic patients leptin concentrations were correlated with age, body mass index, fasting insulin and lower insulin sensitivity, whereas leptin concentrations were not correlated with body fat. CONCLUSIONS: These findings suggest that leptin concentrations and regulation in myotonic dystrophic patients are different from type 2 diabetes.     

Size of the unstable CTG repeat sequence in relation to phenotype and parental transmission in myotonic dystrophy.

A clinical and molecular analysis of 439 individuals affected with myotonic dystrophy, from 101 kindreds, has shown that the size of the unstable CTG repeat detected in nearly all cases of myotonic dystrophy is related both to age at onset of the disorder and to the severity of the phenotype. The largest repeat sizes (1.5-6.0 kb) are seen in patients with congenital myotonic dystrophy, while the minimally affected patients have repeat sizes of < 0.5 kb. Comparison of parent-child pairs has shown that most offspring have an earlier age at onset and a larger repeat size than their parents, with only 4 of 182 showing a definite decrease in repeat size, accompanied by a later age at onset or less severe phenotype. Increase in repeat size from parent to child is similar for both paternal and maternal transmissions when the increase is expressed as a proportion of the parental repeat size. Analysis of congenitally affected cases shows not only that they have, on average, the largest repeat sizes but also that their mothers have larger mean repeat sizes, supporting previous suggestions that a maternal effect is involved in the pathogenesis of this form of the disorder.     

Electrocardiographic abnormalities in patients with myotonic dystrophy.

In examining the incidence and progression of electrocardiographic abnormalities in 45 patients with myotonic dystrophy, 26 (58%) of whom at entry had at least 1 electrocardiographic abnormality, we found conduction abnormalities in 17 (38%). In 21 patients (47%), new abnormalities developed during follow-up (mean, 4.6 years). The overall incidence of electrocardiographic abnormalities increased to 78%, and the incidence of conduction defects increased to 62%. Second-degree or complete atrioventricular block did not develop in any of the patients. Pseudoinfarction patterns were common at entry and during follow-up and were not correlated with evidence of clinical coronary artery disease. There was no correlation between the presence of electrocardiographic abnormalities and apparent disease severity.     

Patients with dystrophinopathy show evidence of increased oxidative stress

Duchenne muscular dystrophy (DMD) is associated with an increase in oxidative stress. We measured 24 h 8-hydroxy-2'-deoxyguanosine (8-OHdG) excretion in 24 patients with MD (DMD + Becker's MD), 23 with myotonic dystrophy, and 34 healthy controls. The 8-OHdG/creatinine ratio was higher in patients with dystrophinopathy ( upward arrow 48%, p <.01) but not myotonic dystrophy, as compared to healthy controls. These results indicate that 8-OHdG excretion can be used as a marker of oxidative stress in clinical trials with dystrophinopathy.     

Application of a closely linked polymorphism of restriction fragment length to counselling and prenatal testing in families with myotonic dystrophy.

The close genetic linkage between the loci for apolipoprotein CII (ApoC2) and myotonic dystrophy makes ApoC2 the closest fully validated marker for prediction of myotonic dystrophy. Application to genetic counselling and presymptomatic and prenatal prediction is reported in seven families with myotonic dystrophy, including one case in which the disorder was excluded prenatally. Only one of the families did not have members with ApoC2 genotypes that allowed prediction, but careful clinical study of older family members was found to be an important factor. ApoC2 typing of families with myotonic dystrophy should be of practical help both in prediction for asymptomatic relatives and for prenatal diagnosis in pregnancies of an affected parent.     

Coiled-coil interactions modulate multimerization, mitochondrial binding and kinase activity of myotonic dystrophy protein kinase splice isoforms

The myotonic dystrophy protein kinase polypeptide repertoire in mice and humans consists of six different splice isoforms that vary in the nature of their C-terminal tails and in the presence or absence of an internal Val-Ser-Gly-Gly-Gly motif. Here, we demonstrate that myotonic dystrophy protein kinase isoforms exist in high-molecular-weight complexes controlled by homo- and heteromultimerization. This multimerization is mediated by coiled-coil interactions in the tail-proximal domain and occurs independently of alternatively spliced protein segments or myotonic dystrophy protein kinase activity. Complex formation was impaired in myotonic dystrophy protein kinase mutants in which three leucines at positions a and d in the coiled-coil heptad repeats were mutated to glycines. These coiled-coil mutants were still capable of autophosphorylation and transphosphorylation of peptides, but the rates of their kinase activities were significantly lowered. Moreover, phosphorylation of the natural myotonic dystrophy protein kinase substrate, myosin phosphatase targeting subunit, was preserved, even though binding of the myotonic dystrophy protein kinase to the myosin phosphatase targeting subunit was strongly reduced. Furthermore, the association of myotonic dystrophy protein kinase isoform C to the mitochondrial outer membrane was weakened when the coiled-coil interaction was perturbed. Our findings indicate that the coiled-coil domain modulates myotonic dystrophy protein kinase multimerization, substrate binding, kinase activity and subcellular localization characteristics.     

Myotonic dystrophy is closely linked to the gene for muscle-type creatine kinase (CKMM)

Summary We have studied genetic linkage between the gene for creatine kinase muscle type (CKMM) and the gene for myotonic dystrophy (DM). In a panel of 65 myotonic dystrophy families from Canada and the Netherlands, a maximum lod score (Z_max) of 22.8 at a recombination frequency (Θ) of 0.03 was obtained. Tight linkage was also demonstrated for CKMM and the gene for apolipoprotein C2 (ApoC2). This establishes CKMM as a useful marker for myotonic dystrophy.     

Misregulated alternative splicing of BIN1 is associated with T tubule alterations and muscle weakness in myotonic dystrophy

Myotonic dystrophy is the most common muscular dystrophy in adults and the first recognized example of an RNA-mediated disease. Congenital myotonic dystrophy (CDM1) and myotonic dystrophy of type 1 (DM1) or of type 2 (DM2) are caused by the expression of mutant RNAs containing expanded CUG or CCUG repeats, respectively. These mutant RNAs sequester the splicing regulator Muscleblind-like-1 (MBNL1), resulting in specific misregulation of the alternative splicing of other pre-mRNAs. We found that alternative splicing of the bridging integrator-1 (BIN1) pre-mRNA is altered in skeletal muscle samples of people with CDM1, DM1 and DM2. BIN1 is involved in tubular invaginations of membranes and is required for the biogenesis of muscle T tubules, which are specialized skeletal muscle membrane structures essential for excitation-contraction coupling. Mutations in the BIN1 gene cause centronuclear myopathy, which shares some histopathological features with myotonic dystrophy. We found that MBNL1 binds the BIN1 pre-mRNA and regulates its alternative splicing. BIN1 missplicing results in expression of an inactive form of BIN1 lacking phosphatidylinositol 5-phosphate-binding and membrane-tubulating activities. Consistent with a defect of BIN1, muscle T tubules are altered in people with myotonic dystrophy, and membrane structures are restored upon expression of the normal splicing form of BIN1 in muscle cells of such individuals. Finally, reproducing BIN1 splicing alteration in mice is sufficient to promote T tubule alterations and muscle weakness, a predominant feature of myotonic dystrophy.     

Haploinsuffciency for Znf9 in Znf9+/- mice is associated with multiorgan abnormalities resembling myotonic dystrophy

Myotonic dystrophy type 2 is caused by a (CCTG)/(CCUG)n repeat expansion in the first intron of the ZNF9 gene. The pathomechanism for the myotonic dystrophies is not well understood and the role of ZNF9 in myotonic dystrophy type 2 pathogenesis has not been fully clarified. We characterized Znf9+/- mice, in which the expression of Znf9 was significantly decreased, and found that their phenotype reflects many of the features of myotonic dystrophy, including muscle histological morphology, and myotonic discharges and heart conduction abnormalities, shown by electromyography and electrocardiogram analysis, respectively. Znf9 is normally highly expressed in heart and skeletal muscle, where skeletal muscle chloride channel 1 (Clc1) plays an important role. Clc1 expression was dramatically decreased in Znf9+/- mice. Znf9 transgenic mice raised Znf9 and Clc1 expression and rescued the myotonic dystrophy phenotype in Znf9+/- mice. Our results suggest that the Znf9 haploinsufficiency contributes to the myotonic dystrophy phenotype in Znf9+/- mice.     

Identification of a novel protein, DMAP, which interacts with the myotonic dystrophy protein kinase and shows strong homology to D1 snRNP

The most common adult form of muscular dystrophy, myotonic dystrophy, is due to a triplet repeat (CTG) expansion in the 3 untranslated region of the myotonic dystrophy gene. Although this gene is known to encode a protein kinase, the mechanism by which a defect in this gene results in a disease state is not understood. To gain insight into this mechanism, the yeast two hybrid system was utilized to identify proteins which interact with myotonic dystrophy protein kinase. Eight positive clones were identified that interact specifically with the myotonic dystrophy protein kinase. One clone, which encodes a novel protein interacting with myotonic dystrophy protein kinase bothin vivo in yeast andin vitro, was characterized further. The gene encoding this protein may represent a member of a small gene family, and the protein (95 amino acids) exhibits a high degree of homology to an snRNP protein, D1. This novel protein may be a member of the signal transduction pathway which is responsible for the manifestation of this disease.     

Does quantitative EMG differ myotonic dystrophy type 2 and type 1?

Genetic testing is considered the only reliable diagnostic approach in myotonic dystrophy. However it has recently been reported that a considerable number of patients with genetically proven types of the disease have unusual phenotypic presentation. The aim of our study was to evaluate motor unit reorganization reflected by various electrophysiological abnormalities in myotonic dystrophies and to compare findings between type 1 (DM 1) and type 2 myotonic dystrophy (DM2). Quantitative electromyography (EMG) recordings in 63 patients (33 with DM1 and 30 with DM2) from the biceps brachii (BB), rectus femoris (RF), first dorsal interosseus (FDI), and tibialis anterior (TA) muscles were analyzed. Mean amplitude and size index (SI) of motor unit potentials recorded in TA and RF muscles, mean potential duration in TA, and mean SI and the number of outliers with amplitude above the normal range in BB were significantly increased in DM2 as compared to DM1. Myotonic discharges were recorded more frequently in DM1 than in DM2. EMG findings significantly differ between DM1 and DM2. The presence of high amplitude potentials in lower limb muscles in DM2 patients, atypical for myogenic muscle lesions, could be explained by muscle fiber hypertrophy observed in muscle biopsies.     

Polymorphism of CTG-repeats in the DMPK gene in populations of Yakutia and central Asia

Allele frequency distribution of CTG-repeat in the 3'-flanking region of DMPK gene was analyzed in populations of Yakutia (three ethnogeographical groups of Yakuts, Evenks, Evens, Yukaghirs, Dolgans) and Central Asia (Kazakhs, Uzbeks, Uighurs). Frequencies of CTG alleles were found to be significantly different in two regions. Allele frequency distribution in populations of Yakutia was similar to Asian populations, whereas Central Asian populations showed similarity to European populations. The features of allele spectrum in Yakut populations were discussed in terms of high prevalence of myotonic dystrophy in Yakuts. Our result supports the hypothesis of founder effect in spread of myotonic dystrophy in Yakuts. The phylogenetic relationships between the investigated populations based on polymorphism of CTG-locus of the DMPK gene have been analyzed as well.     

The apolipoprotein CII gene: Subchromosomal localisation and linkage to the myotonic dystrophy locus

Summary The human apolipoprotein CII gene probe detects a restriction fragment length polymorphism located on chromosome 19. We have investigated the linkage of this polymorphism to the myotonic dystrophy locus in families. The two lici are closely linked with a maximum Lod score of 7.877 at 4% recombination. The close linkage and informativeness of the APOC2 polymorphism suggest that this probe may be of use for presymptomatic diagnosis of the myotonic dystrophy gene. The APOC2 gene was localised to the region 19p13–19q13 using somatic cell hybrids, providing further evidence that the myotonic dystrophy locus is situated in the central region of chromosome 19.     

Myotonic dystrophy: RNA pathogenesis comes into focus

Myotonic dystrophy (DM)--the most common form of muscular dystrophy in adults, affecting 1/8000 individuals--is a dominantly inherited disorder with a peculiar and rare pattern of multisystemic clinical features affecting skeletal muscle, the heart, the eye, and the endocrine system. Two genetic loci have been associated with the DM phenotype: DM1, on chromosome 19, and DM2, on chromosome 3. In 1992, the mutation responsible for DM1 was identified as a CTG expansion located in the 3' untranslated region of the dystrophia myotonica-protein kinase gene (DMPK). How this untranslated CTG expansion causes myotonic dystrophy type 1(DM1) has been controversial. The recent discovery that myotonic dystrophy type 2 (DM2) is caused by an untranslated CCTG expansion, along with other discoveries on DM1 pathogenesis, indicate that the clinical features common to both diseases are caused by a gain-of-function RNA mechanism in which the CUG and CCUG repeats alter cellular function, including alternative splicing of various genes. We discuss the pathogenic mechanisms that have been proposed for the myotonic dystrophies, the clinical and molecular features of DM1 and DM2, and the characterization of murine and cell-culture models that have been generated to better understand these diseases.     

cDNA and genomic cloning of HRC, a human sarcoplasmic reticulum protein, and localization of the gene to human chromosome 19 and mouse chromosome 7

Histidine-rich calcium binding protein (HRC) is a luminal sarcoplasmic reticulum (SR) protein of 165 kDa identified by virtue of its ability to bind 125I-labeled low-density lipoprotein with high affinity after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Hofmann et al., J. Biol. Chem. 264: 8260-8270, 1989). Its role in SR function is unknown. In this report, the gene encoding human HRC was localized to human chromosome 19 and mouse chromosome 7 by hybridization of a human HRC cDNA fragment to a panel of somatic cell hybrids. Known synteny between a portion of human chromosome 19 and a portion of mouse chromosome 7 and in situ hybridization of a biotin-labeled HRC probe to human chromosomes suggest a localization to a region corresponding to 19q13.3. The locus for myotonic dystrophy resides in the region 19q13.2-13.3. Therefore, we considered HRC, a muscle-specific gene, to possibly represent a "candidate gene" for myotonic muscular dystrophy. As a first step toward localizing HRC in relation to the myotonic dystrophy locus, we report the cloning of the human HRC gene, its intron-exon organization, and characterization of several informative polymorphisms to be used in future linkage studies in families with myotonic dystrophy. Of particular interest is an Alu-associated poly-d(GA) sequence located in an intron in the middle of the gene, and two stretches of acidic amino acids in the coding region of exon 1 that vary in length among different individuals.     

Myotonic dystrophy type 1 (DM1): A triplet repeat expansion disorder

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.     

Analysis of expansion of the triplet repeat (CTG)n in myotonic dystrophy patients from Bashkir

A method was elaborated for simple and rapid diagnosis of myotonic dystrophy (MD). The method consists in estimating expansion of the CTG repeat in the myotonin protein kinase gene by means of PCR amplification of a gene fragment from genomic DNA and Southern hybridization of the amplified fragments with probe (CTG)9. Bashkir patients with Rossolimo-Steinert-Batten-Kurshmann MD were examined with this method.