Myotonic dystrophy and myotonic dystrophy protein kinase

Myotonic dystrophy protein kinase (DMPK) was designated as a gene responsible for myotonic dystrophy (DM) on chromosome 19, because the gene product has extensive homology to protein kinase catalytic domains. DM is the most common disease with multisystem disorders among muscular dystrophies. The genetic basis of DM is now known to include mutational expansion of a repetitive trinucleotide sequence (CTG)n in the 3'-untranslated region (UTR) of DMPK. Full-length DMPK was detected and various isoforms of DMPK have been reported in skeletal and cardiac muscles, central nervous tissues, etc. DMPK is localized predominantly in type I muscle fibers, muscle spindles, neuromuscular junctions and myotendinous tissues in skeletal muscle. In cardiac muscle it is localized in intercalated dises and Purkinje fibers. Electron microscopically it is detected in the terminal cisternae of SR in skeletal muscle and the junctional and corbular SR in cardia muscle. In central nervous system, it is located in many neurons, especially in the cytoplasm of cerebellar Purkinje cells, hippocampal interneurons and spinal motoneurons. Electron microscopically it is detected in rough endoplasmic reticulum. The functional role of DMPK is not fully understood, however, it may play an important role in Ca2+ homeostasis and signal transduction system. Diseased amount of DMPK may play an important role in the degeneration of skeletal muscle in adult type DM. However, other molecular pathogenetical mechanisms such as dysfunction of surrounding genes by structural change of the chromosome by long trinucleotide repeats, and the trans-gain of function of CUG-binding proteins might be responsible to induce multisystemic disorders of DM such as myotonia, endocrine dysfunction, etc.     

Myogenic defects in myotonic dystrophy

Myogenesis is the developmental program that generates and regenerates skeletal muscle. This process is impaired in patients afflicted with myotonic dystrophy type 1 (DM1). Muscle development is disrupted in infants born with congenital DM1, and recent evidence suggests that defective regeneration may contribute to muscle weakness and wasting in affected adults. DM1 represents the first example of a human disease that is caused, at least in part, by pathogenic mRNA. Cell culture models have been used to demonstrate that mutant DM1 mRNA takes on a gain-of-function and inhibits myoblast differentiation. Although the molecular mechanism(s) by which this mutant mRNA disrupts myogenesis is not fully understood, recent findings suggest that anomalous RNA-protein interactions have downstream consequences that compromise key myogenic factors. In this review, we revisit morphological studies that revealed the nature of myogenic abnormalities seen in patients, describe cell culture systems that have been used to investigate this phenotype and discuss recent discoveries that for the first time have identified myogenic events that are disrupted in DM1.     

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. BioEssays 20: 901–912, 1998. © 1998 John Wiley & Sons, Inc.     

Towards cloning the gene responsible for myotonic dystrophy

For the purpose of cloning the gene of myotonic dystrophy (DM) using the technique of reverse genetics, we have introduced new methods such as microdissection, a YAC library and a Not I linking library and cloned many DNA fragments derived from the region of 19q13.2. Then we have assigned these to chromosome 19 by linkage map (CEPH families and linkage disequilibrium) and physical map (PFGE and in situ hybridization). Here we have described these methods.     

Radiation-reduced hybrids for the myotonic dystrophy locus.

The myotonic dystrophy (DM) gene maps to the long arm of human chromosome 19 and is flanked by markers ERCC1 and D19S51. Also mapping to this region is the polio virus receptor gene (PVS). To produce more markers for this interval, we have constructed radiation-reduced hybrids by selecting for the retention of ERCC1 and for the loss of PVS. One of the cell lines produced has been characterized extensively and contains about 2 Mb of human DNA derived exclusively from chromosome 19, and includes ERCC1 and D19S51. Phage libraries constructed from DNA of this cell line have been screened and several new markers identified, including two for which cDNAs have been isolated. These represent candidate genes for DM. The new markers have also been used to extend the long-range restriction map of this region.     

Dynamic docking of small molecules targeting RNA CUG repeats causing myotonic dystrophy type 1

Expansion of RNA CUG repeats causes myotonic dystrophy type 1 (DM1). Once transcribed, the expanded CUG repeats strongly attract muscleblind-like 1 (MBNL1) proteins and disturb their functions in cells. Because of its unique structural form, expanded RNA CUG repeats are prospective drug targets, where small molecules can be utilized to target RNA CUG repeats to inhibit MBNL1 binding and ameliorate DM1-associated defects. In this contribution, we developed two physics-based dynamic docking approaches (DynaD and DynaD/Auto) and applied them to nine small molecules known to specifically target RNA CUG repeats. While DynaD uses a distance-based reaction coordinate to study the binding phenomenon, DynaD/Auto combines results of umbrella sampling calculations performed on 1 × 1 UU internal loops and AutoDock calculations to efficiently sample the energy landscape of binding. Predictions are compared with experimental data, displaying a positive correlation with correlation coefficient (R) values of 0.70 and 0.81 for DynaD and DynaD/Auto, respectively. Furthermore, we found that the best correlation was achieved with MM/3D-RISM calculations, highlighting the importance of solvation in binding calculations. Moreover, we detected that DynaD/Auto performed better than DynaD because of the use of prior knowledge about the binding site arising from umbrella sampling calculations. Finally, we developed dendrograms to present how bound states are connected to each other in a binding process. Results are exciting, as DynaD and DynaD/Auto will allow researchers to utilize two novel physics-based and computer-aided drug-design methodologies to perform in silico calculations on drug-like molecules aiming to target complex RNA loops.     

Insulin resistance in myotonic dystrophy.

The aim of the present study was to obtain a comprehensive picture of the rate of insulin secretion and of tissue sensitivity to the endogenous hormone in myotonic dystrophy patients (MyD). The minimal model approach was utilized for the analysis of frequently sampled intravenous glucose tolerance test data (FSIGT). This method provided the characteristic parameters: SI, insulin sensitivity index; SG fractional glucose disappearance independent of dynamic insulin; n, fractional insulin clearance; phi 1 and phi 2 first and second phase insulin delivery sensitivities to glucose stimulation. In MyD patients SI was reduced (p less than 0.01) by 71% to 1.4 +/- 0.3 x 10(-4) min-1/(microU/ml), whereas in controls it was 4.85 +/- 0.77; SG was within the normal range: 0.044 +/- 0.012 min-1 in MyD patients and 0.036 +/- 0.017 min-1 in controls; phi 1 increased in MyD patients (7.4 +/- 1.3 min (microU/ml)/(mg/dl) versus 4.1 +/- 1.2 in controls); phi 2 increased in MyD patients (126 +/- 47 x 10(4) min-2/(microU/ml)/(mg/dl) versus 17 +/- 6 in controls; p less than 0.05). MyD patients showed a normal tolerance with the glucose disappearance constant, KG within the normal range: 2.75 versus 2.62% min-1 in controls. In MyD patients insulin resistance was associated with a higher than normal insulin delivery for both secretory phases, although the second phase was responsible for releasing a greater amount of hormone. In conclusion MyD patients try to compensate for overall insulin resistance by a more marked pancreatic response.     

Genetic risks for children of women with myotonic dystrophy

In genetic counseling, the recommended risk estimate that any heterozygous woman with myotonic dystrophy (DM) will have a congenitally affected child is 3%-9%. However, after already having had such an offspring, a DM mother's risk increases to 20%-37%. The risks of 10% and 41%, respectively, calculated in this study are similar to the estimates in the literature. However, our data on clinical status of the mothers demonstrate that only women with multisystem effects of the disorder at the time of pregnancy and delivery are likely to have congenitally affected offspring. No heterozygous woman with polychromatic lens changes but no other clinically detectable multisystem involvement had a congenitally affected child. In addition, our data suggest that the chance of having a more severely affected child increases with greater severity of maternal disease. The findings of this study are relevant for genetic counseling, as the risk of having a congenitally affected child for women with classical manifestations of the disease is shown to be higher than predicted by the overall risk estimate for any heterozygous woman. We consider it appropriate to give these classically affected women risk figures which approach the recurrence risk given to mothers with congenitally affected children. However, the risk of having a congenitally affected child for heterozygous women with no multisystem involvement appears to be minimal. Our findings support the earlier proposed hypothesis of maternal metabolites acting on a heterozygous offspring. Neither genomic imprinting nor mitochondrial inheritance is able to explain the correlation between the clinical status of heterozygous mothers and that of their children.     

Phospholemman is a substrate for myotonic dystrophy protein kinase

The genetic abnormality in myotonic muscular dystrophy, multiple CTG repeats lie upstream of a gene that encodes a novel protein kinase, myotonic dystrophy protein kinase (DMPK). Phospholemman (PLM), a major membrane substrate for phosphorylation by protein kinases A and C, induces Cl currents (I(Cl(PLM))) when expressed in Xenopus oocytes. To test the idea that PLM is a substrate for DMPK, we measured in vitro phosphorylation of purified PLM by DMPK. To assess the functional effects of PLM phosphorylation we compared I(Cl(PLM)) in Xenopus oocytes expressing PLM alone to currents in oocytes co-expressing DMPK, and examined the effect of DMPK on oocyte membrane PLM expression. We found that PLM is indeed a good substrate for DMPK in vitro. Co-expression of DMPK with PLM in oocytes resulted in a reduction in I(Cl(PLM)). This was most likely a specific effect of phosphorylation of PLM by DMPK, as the effect was not present in oocytes expressing a phos(-) PLM mutant in which all potential phosphorylation had been disabled by Ser --> Ala substitution. The biophysical characteristics of I(Cl(PLM)) were not changed by DMPK or by the phos(-) mutation. Co-expression of DMPK reduced the expression of PLM in oocyte membranes, suggesting a possible mechanism for the observed reduction in I(Cl(PLM)) amplitude. These data show that PLM is a substrate for phosphorylation by DMPK and provide functional evidence for modulation of PLM function by phosphorylation.     

Molecular basis for impaired muscle differentiation in myotonic dystrophy

Differentiation of skeletal muscle is affected in myotonic dystrophy (DM) patients. Analysis of cultured myoblasts from DM patients shows that DM myoblasts lose the capability to withdraw from the cell cycle during differentiation. Our data demonstrate that the expression and activity of the proteins responsible for cell cycle withdrawal are altered in DM muscle cells. Skeletal muscle cells from DM patients fail to induce cytoplasmic levels of a CUG RNA binding protein, CUGBP1, while normal differentiated cells accumulate CUGBP1 in the cytoplasm. In cells from normal patients, CUGBP1 up-regulates p21 protein during differentiation. Several lines of evidence show that CUGBP1 induces the translation of p21 via binding to a GC-rich sequence located within the 5' region of p21 mRNA. Failure of DM cells to accumulate CUGBP1 in the cytoplasm leads to a significant reduction of p21 and to alterations of other proteins responsible for the cell cycle withdrawal. The activity of cdk4 declines during differentiation of cells from control patients, while in DM cells cdk4 is highly active during all stages of differentiation. In addition, DM cells do not form Rb/E2F repressor complexes that are abundant in differentiated cells from normal patients. Our data provide evidence for an impaired cell cycle withdrawal in DM muscle cells and suggest that alterations in the activity of CUGBP1 causes disruption of p21-dependent control of cell cycle arrest.