Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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There is considerable confusion in the literature about the size of the myotonic dystrophy protein kinase (DMPK) and its localization within tissues. We have used a new panel of monoclonal antibodies (mAbs) to begin to resolve these issues, which are important for understanding the possible role of DMPK in myotonic dystrophy. Antisera raised against the catalytic and coil domains of DMPK recognized a major 55 kDa protein and a minor 72-80 kDa doublet on western blots of human skeletal muscle. Ten mAbs, five against the catalytic domain and five against the coil region, recognized only the 72-80 kDa doublet. The 72 kDa protein was present in all tissues tested, whereas the 80 kDa component was variably expressed, mainly in skeletal and cardiac muscles. The 72 kDa protein was absent in a DMPK knockout mouse and was greatly increased in a transgenic mouse overexpressing human DMPK, confirming its identity as authentic DMPK. Two mAbs against the catalytic domain recognized only the more abundant 55 kDa protein, which was found only in skeletal muscle. Nine out of 10 mAbs located DMPK to intercalated discs in human heart, an affected tissue in myotonic dystrophy. However, co-localization of DMPK with acetylcholine receptors at neuromuscular junctions was not observed with any of the mAbs. Subcellular fractionation and sedimentation analysis suggest that a major proportion of the DMPK in skeletal muscle and brain is cytosolic.
Hum Mol Genet 1998 Nov
PMID:Localization of myotonic dystrophy protein kinase in human and rabbit tissues using a new panel of monoclonal antibodies. 981 41

We analyzed the expression of mouse DMAHP / Six5 (the myotonic dystrophy-associated homeodomain protein gene) during embryogenesis and in various tissues by northern blotting. Expression was observed as early as embryonic day 7 (E7) and continued to E17. Abundant expression was observed in neonatal heart and skeletal muscle with potential links to the phenotype of myotonic dystrophy. The transcription initiation sites of the gene were analyzed in mouse E11 and E15 embryos and in adult skeletal and heart muscle. Three major transcription initiation sites were identified, the proximal site was specific to the early E11 embryo, while the other two were common among the heart and skeletal muscle and E11 and E15 embryos. All transcription initiation sites were downstream of the corresponding CTG repeat locus of the mouse gene (-1195), excluding a possible inclusion of the CUG repeat sequence in mRNA leading to abnormal splicing or to translation of aberrant protein. For analysis of the regulatory elements in the promoter region, we used P19 embryonal carcinoma cells which abundantly express mouse DMAHP / Six5. Multiple positive and negative elements were identified in the promoter region. All positive elements were Sp1/Sp3 binding sites and one of the negative elements was a novel factor binding site. The transcription initiation sites and regulatory elements are conserved between human and mouse DMAHP.
Hum Mol Genet 1998 Dec
PMID:Promoter of mDMAHP/Six5: differential utilization of multiple transcription initiation sites and positive/negative regulatory elements. 981 28

The myotonic dystrophy (DM) expansion varies from 50 to 4000 CTG repeats in the 3' untranslated region of the DMPK gene. Direct analysis by Southern blot, after restriction enzyme digestion of genomic DNA, is the method of choice for studying the DM mutation. A long polymerase chain reaction (PCR)-formatted protocol, which involved a single genomic in vitro amplification followed by high concentration agarose gel electrophoresis and oligo-specific hybridization, was used to amplify normal alleles and DM alleles in all examined ranges of expansion (up to 3,700 CTGs) starting from a small amount of genomic DNA (> or = 15 pg). This method is quick, sensitive, and reproducible and reduces the cost of diagnostic laboratory processing.
Diagn Mol Pathol 1998 Jun
PMID:A single polymerase chain reaction-based protocol for detecting normal and expanded alleles in myotonic dystrophy. 983 67

Myotonic dystrophy (DM) is a neuromuscular disorder associated with CTG triplet repeat expansion in the myotonin protein kinase gene ( DMPK ). We previously proposed a hypothesis suggesting that the expanded CUG repeats sequester specific RNA-binding proteins and that such a sequestration results in abnormal RNA processing of several RNAs containing CUG repeats in multiple tissues affected in patients with DM. One of the members of the CUG-binding proteins, CUG-BP, has been identified previously. Here we describe the second member of this family, elav -type ribonucleoprotein (ETR-3), which is highly expressed in heart and is able to interact with CUG repeats. Screening of a mouse liver cDNA library with a CUG-BP probe identified two mETR-3 cDNAs. Two additional cDNAs from mouse heart were amplified by RT-PCR. These cDNAs differ by several insertions/deletions and might be generated via alternative splicing. Mouse ETR-3 has a mol. wt of 50 kDa and displays a high level of homology to CUG-BP protein. The organization of the RNA-binding domains (RBDs) within the ETR-3 molecule is similar to one within CUG-BP. A study of mETR-3 RNA-binding activity showed that the mETR-3 binds to (CUG)8repeats. Sequence analysis of mETR-3 indicates the presence of several CUG repeats within the mETR-3 mRNA. Both CUG-BP and mETR-3 bind to mETR-3 mRNA via CUG repeats, suggesting the possible involvement of CUG-BP-like proteins in the regulation of mETR-3 processing. Analysis of the tissue distribution of ETR-3 showed that in human cells, ETR-3 mRNA is highly expressed in heart, but is undetectable in other tissues examined. Our results suggest the existence of a family of proteins that bind to CUG repeats and might be affected in DM by expansion of CUG repeats.
Hum Mol Genet 1999 Jan
PMID:Cardiac elav-type RNA-binding protein (ETR-3) binds to RNA CUG repeats expanded in myotonic dystrophy. 988 31

The pathogenic mechanisms underlying myotonic dystrophy (DM), which results from a (CTG) n repeat expansion mutation in the 3'-untranslated region (3'-UTR) of the myotonic dystrophy protein kinase gene ( DMPK ), remain obscure. The multisystemic nature and variable expressivity of the symptoms are unlikely to be explained by a defect in this gene alone. However, the location of the DM-associated (CTG) n repeat in the promoter region of SIX5, immediately downstream of DMPK, implicates it as a second candidate with a pathological role in DM. We hypothesize that dysfunction of SIX5, which is homologous to the Drosophila eye development gene sine oculis ( so ), is primarily responsible for the ophthalmic features of DM. We report an expression pattern for SIX5 in the normal adult eye that matches the sites of the ocular pathology in DM. SIX5 transcripts were detected in the adult corneal epithelium and endothelium, lens epithelium, ciliary body epithelia, cellular layers of the retina and the sclera. SIX5 expression was not detected in fetal eyes. We also report a restricted but partially overlapping expression pattern for DMPK transcripts and DMPK protein in normal fetal and adult eyes. DMPK transcripts were detected in fetal eyes and in adult conjunctival and corneal epithelia, uvea, cellular layers of the retina, optic nerve and in the sclera. DMPK protein was detected in the adult retina, conjunctival and ciliary body epithelia and in the smooth muscle of the ciliary body, pupillary sphincter and uveal blood vessels. We propose that the expression patterns of these two genes indicate their relative contribution to the ophthalmological dysfunction seen in DM. Furthermore, the expression of SIX5 and not DMPK in the adult lens implicates a role for SIX5 dysfunction in the development of adult onset cataracts, the most frequently occurring eye phenotype in DM.
Hum Mol Genet 1999 Mar
PMID:Characterization of the expression of DMPK and SIX5 in the human eye and implications for pathogenesis in myotonic dystrophy. 994 7

Myotonic dystrophy (DM), the most common inherited muscle disorder, is caused by a CTG expansion in the 3"-untranslated region of a protein kinase gene ( DMPK ). The complex and variable phenotype is most likely caused by a complex molecular pathogenesis, including deficiency of the DMPK protein, a trans -dominant misregulation of RNA homeostasis and haploinsufficiency of a neighboring homeobox gene [DM locus-associated homeodomain protein (DMAHP )]. Here, we study the allele-specific transcriptional activity of the DMAHP/SIX5 gene in DM patient tissues. Using a quantitative fluorescent RT-PCR assay, we tested allele-specific accumulation of DMAHP/SIX5 transcripts in both total and poly(A)+pools. In muscle biopsies, we found that transcript reductions of DMAHP/SIX5 alleles in cis with CTG expansions correlated with the extent of expansion. A patient with approximately 90 CTG repeats in muscle DNA (normal n < 37) showed a 20% reduction of allele-specific transcript levels, while four other DM patients with larger expansions showed 80% reductions. The effects of the CTG expansions on DMAHP transcription were tissue specific: autopsy tissues from a patient with 1500 repeats showed 80% reductions in muscle and liver; however, RNA from other tissues (lung, aorta, heart conduction tissue, cerebellum) showed 0-20% reductions. Our results suggest that the effect of the CTG repeat on the DMAHP/SIX5 promoter is variable and tissue-specific. Our data are consistent with abnormalities of DMAHP/SIX5 probably having a more prominent role in disease pathogenesis in muscle, liver and brain, but being less important in other tissues.
Hum Mol Genet 1999 Jun
PMID:Myotonic dystrophy: tissue-specific effect of somatic CTG expansions on allele-specific DMAHP/SIX5 expression. 1033 33

An increasing number of human genetic disorders are associated with the expansion of trinucleotide repeats. The majority of these diseases are associated with CAG/CTG expansions, including Huntington's disease, myotonic dystrophy and many of the spinocerebellar ataxias. Recently, two new expanded CAG/CTG repeats have been identified that are not associated with a phenotype. Expanded alleles at all of these loci are unstable, with frequent length changes during intergenerational transmission. However, variation in the relative levels of instability, and the size and direction of the length change mutations observed, between the CAG/CTG loci is apparent. We have quantified these differences, taking into account effects of progenitor allele length, by calculating the relative expandability of each repeat. Since the repeat motifs are the same, these differences must be a result of flanking sequence modifiers. We present data that indicate a strong correlation between the relative expandability of these repeats and the flanking GC content. Moreover, we demonstrate that the most expandable loci are all located within CpG islands. These data provide the first insights into the molecular bases of cis -acting flanking sequences modifying the relative mutability of dispersed expanded human triplet repeats.
Hum Mol Genet 1999 Jun
PMID:Cis-acting modifiers of expanded CAG/CTG triplet repeat expandability: associations with flanking GC content and proximity to CpG islands. 1033 38

The causative mutation in the majority of cases of myotonic dystrophy has been shown to be the expansion of a CTG trinucleotide repeat, but the mechanism(s) by which this repeat leads to the very complex symptomatology in this disorder remains controversial. We have developed a highly sensitive and quantifiable assay, based on competitive RT-PCR, to test the hypothesis that the expansion disrupts the expression of the genes in its immediate vicinity, DMPK, 59 and DMAHP. In order to avoid cell culture-induced artifacts we performed these experiments using adult skeletal muscle biopsy samples and analysed total cytoplasmic poly(A)+mRNA levels for each gene simultaneously, as this is more physiologically relevant than allele-specific levels. There was considerable overlap between the expression levels of the three genes in myotonic dystrophy patient samples and samples from control individuals. However, in the myotonic dystrophy samples we detected a strong inverse correlation between the repeat size and the levels of expression of DMPK and 59. This is the first report of a possible effect of the CTG expansion on gene 59. Our results indicate that whilst a simple dosage model of gene expression in the presence of the mutation is unlikely to be sufficient in itself to explain the complex molecular pathology in this disease, the repeat expansion may be a significant modifier of the expression of these two genes.
Hum Mol Genet 1999 Jun
PMID:Simultaneous analysis of expression of the three myotonic dystrophy locus genes in adult skeletal muscle samples: the CTG expansion correlates inversely with DMPK and 59 expression levels, but not DMAHP levels. 1033 37

Myotonic dystrophy is caused by the expansion of a CTG repeat sequence. The mechanism by which this expanded repeat produces the pathophysiology of myotonic dystrophy is not clear. It has been shown previously that expansion of the repeat produces allele-specific effects on transcripts from two genes, DMPK and SIX5. We have examined the effect of repeat expansion on the level of RNA from a third gene, DMWD. We have identified a polymorphism in this gene and developed a quantitative allele-specific assay for DMWD RNA levels, which we have applied to nuclear and cytoplasmic fractions of RNA from DM cell lines. We have found that the level of the DM-associated allele in the cytoplasm of DM cell lines is reduced by 20-50% compared with the wild-type allele, similar to the level of reduction found for SIX5 in allele-specific analysis. However, no such reduction is observed in RNA from the nuclear fraction of DM cell lines. This may reflect the complex nature of processing transcriptional units at the DM locus.
Hum Mol Genet 1999 Aug
PMID:Myotonic dystrophy is associated with a reduced level of RNA from the DMWD allele adjacent to the expanded repeat. 1040 Sep 97

The mutation causing myotonic dystrophy (DM) has been identified as a CTG expansion in the 3'-untranslated region (3'-UTR) of the DM protein kinase gene ( DMPK ), but the mechanism(s) of pathogenesis remain unknown. Studies using DM patient materials have often produced confusing results. Therefore, to study the effects of the DM mutation in a controlled environment, we have established a cell culture model system using C2C12 mouse myoblasts. By expressing chimeric reporter constructs containing a reporter gene fused to a human DMPK 3'-UTR, we identified both cis and trans effects that are mediated by the DM mutation. Our data show that a mutant DMPK 3'-UTR, with as few as 57 CTGs, had a negative cis effect on protein expression and resulted in the aggregation of reporter transcripts into discrete nuclear foci. We determined by deletion analysis that an expanded (CTG) (n) tract alone was sufficient to mediate these cis effects. Furthermore, in contrast to the normal DMPK 3'-UTR mRNA, a mutant DMPK 3'-UTR mRNA with (CUG)(200)selectively inhibited myogenic differentiation of C2C12 myoblasts. Genetic analysis and the Cre- loxP system were used to clearly demonstrate that the myoblast fusion defect could be rescued by eliminating the expression of the mutant DMPK 3'-UTR transcript. Characterization of spontaneous deletion events mapped the inhibitory effect to the (CTG) (n) expansion and/or the 3' end of the DMPK 3'-UTR. These results provide evidence that the DM mutation acts in cis to reduce protein production (consistent with DMPK haploinsufficiency) and in trans as a 'riboregulator' to inhibit myogenesis.
Hum Mol Genet 1999 Oct
PMID:Cis and trans effects of the myotonic dystrophy (DM) mutation in a cell culture model. 1048 65


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