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PubMed2005

Opposite roles of the dmd gene in the control of RNase E and RNase LS activities.

When the dmd gene of bacteriophage T4 is defective, expression of middle genes starts normally but drops abruptly. However, the residual expression of middle genes at late stages continues at a higher rate in cells infected with a dmd mutant than with the wild type. In order to understand the complex effects of the dmd gene, we followed changes in the quantity of mRNA from a middle gene, uvsY. The uvsY mRNA was degraded rapidly by RNase LS at middle stages but stabilized at late stages, suggesting that RNase LS targets middle-gene mRNAs only at middle stages. Furthermore, another RNase targeting middle mRNAs at late stages is also suggested to be inactivated when dmd is mutated. We found that RNase E was involved in the degradation of uvsY mRNA. Judging from the processing of gene-32 mRNA, RNase E activity declines after the beginning of the middle stage when dmd is defective.

Bacteriophage T4↗

PubMed2004

[Detection of micro mutation in dystrophin gene of DMD female carrier].

We attempted to identify a mutation in dystrophin gene in a female patient who was suspected a Duchenne muscular dystrophy (DMD) carrier with muscle weakness of upper limbs and congestive heart failure. We examined the mutation hot spots in DMD gene, exon 3, 6, 8, 13, 17, 19, 43, 44, 45, 47, 48, 49, 50, 52, 60 by multiplex PCR which had been a diagnostic screening strategy, and detected an extra band in exon 43 product. We also detected an extra band in exon 43 products by SSCP analysis for detection of small mutations which could not be detected by multiplex PCR. As a result of sequencing a PCR product of an exon 43, we confirmed an allele having the insertion of a 2 base of AT in Intron42, which is described for the first time. Although we can not conclude that this insertion is responsible for DMD, but it may cause abnormal splicing. In carrier detection of DMD without genetic information of proband, it is difficult to detect mutations by multiplex PCR solely. Therefore, SSCP of PCR products are recommended to detect mutation in DMD carrier.

Aged↗

PubMed2006

A different spectrum of DMD gene mutations in local Chinese patients with Duchenne/Becker muscular dystrophy.

BACKGROUND: Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked recessive, allelic disorders. This study was conducted to look into the spectrum of DMD gene mutations in Hong Kong Chinese patients with Duchenne or Becker muscular dystrophy (DMD/BMD), and to study genotype-phenotype correlation. METHODS: A retrospective review of 67 patients. RESULTS: Twenty-three (34.3%) patients had exon deletions; whereas 5 (7.5%) patients had exon duplications. Twenty-three (34.3%) patients had small mutations, including 17 point mutations and 6 small insertions or deletions. No correlation was found between the type of mutation and the muscle phenotype or mental retardation. Significantly fewer maternal carriers were found in patients with exon deletions, and a positive family history was more common in those with small mutations. DMD phenotype was significantly less common in patients with exon deletions/duplications at the 5' hotspot, whereas all 4 small mutations associated with mental retardation were located in the 3' end of the gene. CONCLUSIONS: The percentage of DMD exon deletions in local Chinese patients was significantly lower than the commonly quoted 60%. This indicated an ethnic or regional difference in predisposition to DMD exon deletions.

Asian People↗

PubMed1989

[Clinical implications of enhanced caffeine contracture in malignant hyperthermia (MH) and Duchenne muscular dystrophy (DMD)].

The caffeine contracture response was studied on the skinned muscle fiber in patients with MH episodes or with DMD. An abnormally enhanced caffeine contracture was observed in 12 out of 14 subjects with MH episodes and 11 out of 13 patients with DMD. The extent of abnormality was almost the same in both group of patients. Ca-induced Ca release (CICR) of the sarcoplasmic reticulum was also analyzed on the same specimens. CICR was increased in 3 out of 4 cases with MH episodes, but it was decreased or unchanged in 4 patients with DMD. It was suggested that the mechanism of abnormal caffeine contracture might not be the same between MH and DMD. Thus, adverse anesthetic reactions, which are occasionally reported in DMD, could be different from genuine MH in its pathophysiology. However, before concluding this issue, further studies seem necessary concerning the molecular mechanism of abnormal Ca release. In the meantime, studies of caffeine contracture on the skinned fiber are helpful as one of predictive tests of MH and related disorders.

Adolescent↗

PubMed1998

Successful transplantation of genetically corrected DMD myoblasts following ex vivo transduction with the dystrophin minigene.

Myoblast transplantation and gene therapy are two promising therapeutical approaches for the treatment of Duchenne Muscular Dystrophy (DMD). So far, both strategies have met many hurdles, mainly because of immune reactions. In this study, we investigated a third and novel strategy based on the combination of these two basic ones, i.e., transplantation of genetically modified myoblasts. We first derived a primary culture from a muscle biopsy of a young DMD patient (3 years old). Adenoviral-mediated dystrophin gene transfer into these DMD cultures and expression of the dystrophin transgene were achieved in vitro. The transduced cultures were then transplanted the same day in immunodeficient SCID mouse muscles. Three weeks following the graft, many human dystrophin-positive fibers were observed throughout sections of the injected muscles. However, many fibers expressed human MHC antigens without expressing human dystrophin due to the low percentage of infected primary muscle cells in vitro (even when a high MOI [400] was used) and to a reduction and even to a complete loss of transgene copy number during myoblast replication. From our results, we conclude that, although not at a high proportion, (1) DMD primary myoblast cultures are infectable by adenoviruses; (2) they can be efficiently transplanted back in a muscle, leading to normal fusion of infected myoblasts with the host fibers; and (3) they can correct the dystrophin deficiency in the host fibers by the expression of a mini-dystrophin transgene.

Adenoviridae↗

PubMed2006

Simultaneous determination of MHD and DMD in dog plasma by high-performance liquid chromatography with fluorescence detection and its application to pharmacokinetic studies.

A rapid, reproducible high-performance liquid chromatographic (HPLC) method with fluorescence detection for the simultaneous determination of 3(or 8)-(1-methoxyethyl)-8(or 3)-(1-hydroxyethyl)-deuteroporphyrin IX (MHD) and 3,8-di-(1-methoxyethyl)-deuteroporphyrin IX (DMD) in dog plasma was described. Fluorescein was used as an internal standard. A simple extraction step with ethyl acetate was performed before chromatography on a Diamonsil C18 column (5 microm, 4.6 mm x 150 mm). The chromatography used 0.02 mol L(-1) sodium acetate/tetrahydrofuran (66:34 v/v). The analytical curve was linear over the concentration range 0.025-2.5 microg mL(-1). For a 100 microL dog plasma sample, the limit of determination for both MHD and DMD was 0.025 microg mL(-1). The recoveries of MHD and DMD were more than 76% and 89%, respectively. The intra-assay (within-run) and interassay (between-run) coefficients of variation (precisions) for MHD and DMD were less than 15%. This method was found to be suitable for the analysis of biosamples and was successfully applied to pharmacokinetic studies of Deuxemether in dogs.

Animals↗

PubMed1998

Mutation analysis of the dystrophin gene in Southern French DMD or BMD families: from Southern blot to protein truncation test.

Data from 6 years of experience in molecular diagnosis of Duchenne (DMD) and Becker (BMD) muscular dystrophy in Southern France are reported. DMD and BMD patients have been extensively analyzed for deletions and for point mutations in the dystrophin gene. By scanning the whole coding sequence as reverse-transcribed from lymphocytes or muscular RNA by the protein truncation test, we have reached a minimum of an 86% detection rate for point mutations responsible for DMD; these mutations consist of nonsense, frameshifting, and splicing mutations. Four of 12 small alterations identified in our sample are novel and described in this study. We also present an improved protocol for the automated detection of fluorescently labeled duplex polymerase chain reactions of six known intragenic microsatellites (Dys II, TG 15, STRs 44, 45, 49, and 50). Accurate sizing of the alleles at each locus was performed, and we elucidated the sequence of several repeat units. Allele frequencies at each of the six microsatellite loci and at one restriction fragment length polymorphism site (intron 16/TaqI) were defined in a sample of normal, DMD, and BMD X chromosomes from Southern France. The determination of the grandparental origin of either deletions or point mutations revealed differences depending on the type of the mutation, with most of the deletions occurring in oogenesis and most of the point mutations occurring in spermatogenesis.

Blotting, Southern↗

PubMed1995

Sarcolemmal expression of dystrophin C-terminus but reduced expression of 6q-dystrophin-related protein in two DMD patients with large deletions of the dystrophin gene.

Partial deletions of the dystrophin gene are the predominant genetic lesions in Duchenne (DMD) and Becker (BMD) muscular dystrophies. According to the reading frame hypothesis [1], any deletion disrupting the translational reading frame of the mRNA cannot result in expression of the dystrophin molecule and should lead to severe phenotypes of DMD. In contrast, deletions which maintain the reading frame across the deleted exons may give rise to truncated, semifunctional proteins and milder courses of the disease (i.e. BMD). Among the notable exceptions of this hypothesis are very large "in-frame" deletions by which functionally indispensable domains of the dystrophin molecule have been removed. Here, we report on two DMD patients with large intragenic in-frame deletions. Grossly truncated, but stable dystrophin molecules with preserved C-terminal domains were detected at the sarcolemma on cryosections in both patients. However, dystrophin organization on single-teased muscle fibers revealed disarrangement of the costameric pattern, if compared to normal skeletal muscle fibers. Compared to dystrophin-deficient DMD muscle, expression of chromosome-6-encoded dystrophin-related protein (DRP) was greatly diminished in skeletal muscle of both patients. We show, that loss of more than 50% of dystrophin seems to be deleterious for the protein's function and therefore, the extent of the deletions may have an impact on construction of dystrophin mini genes. Moreover, these findings shed new light on the functional significance of the C-terminal domain of dystrophin. They also suggest a negative correlation between sarcolemmal expression of the dystrophin C-terminus and DRP expression at the sarcolemma.

Blotting, Western↗

PubMed2006

Detecting exon deletions and duplications of the DMD gene using Multiplex Ligation-dependent Probe Amplification (MLPA).

OBJECTIVES: To evaluate the efficacy of Multiplex Ligation-dependent Probe Amplification (MLPA) technique in comparison with the traditional multiplex PCR assay in detection of exon deletions and duplications of the DMD gene. DESIGN AND METHODS: The sensitivity and accuracy of MLPA were assessed and compared with the multiplex PCR in a total of 63 subjects including 43 subjects with Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD) and 20 female carriers. RESULTS: MLPA was able to detect all the known deletions and duplications; it detected four additional mutations that had been missed by multiplex PCR. In addition, the extent of the deletions and duplications could be more accurately defined which in turn facilitated a genotype-phenotype correlation. CONCLUSIONS: MLPA is superior to multiplex PCR. It should be the method of choice for the detection of exon deletions and duplications of the DMD gene in patients with DMD or BMD, as well as in female carriers.

Dystrophin↗

PubMed2006

Antisense oligonucleotide-induced exon skipping restores dystrophin expression in vitro in a canine model of DMD.

Manipulation of pre-mRNA splicing by antisense oligonucleotides (AOs) offers considerable potential for a number of genetic disorders. One of these is Duchenne muscular dystrophy (DMD), where mutations in the dystrophin gene typically result in premature termination of translation that causes a loss of functional protein. AOs can induce exon skipping such that the mutation is by-passed and the reading frame restored, producing an internally deleted protein similar to that found in the milder Becker muscular dystrophy. To date, this approach has been applied to the mdx mouse model in vitro and in vivo and in human myoblast cultures. Here, we report the application of AO-directed exon skipping to induce dystrophin expression in vitro in a canine model of DMD, golden retriever muscular dystrophy (GRMD). The efficacy of 2'-O-methyl phosphorothioate (2OMe), phosphorodiamidate morpholino oligomers (PMOs) and peptide-linked PMOs (PMO-Pep) to induce dystrophin expression was assessed. The 2OMe chemistry was only effective for short-term induction of corrected transcript and could not induce detectable dystrophin protein. The PMO chemistry generally induced limited exon skipping at only high concentrations; however, a low level of dystrophin protein was produced in treated cells. Use of the PMO-Pep, applied here for the first time to a DMD model, was able to induce high and sustained levels of exon skipping and induced the highest level of dystrophin expression with no apparent adverse effects upon the cells. The induction of dystrophin in the GRMD model offers the potential for further testing of AO delivery regimens in a larger animal model of DMD, in preparation for application in human clinical trials.

Alternative Splicing↗

PubMed2002

Gene expression comparison of biopsies from Duchenne muscular dystrophy (DMD) and normal skeletal muscle.

The primary cause of Duchenne muscular dystrophy (DMD) is a mutation in the dystrophin gene leading to the absence of the corresponding RNA transcript and protein. Absence of dystrophin leads to disruption of the dystrophin-associated protein complex and substantial changes in skeletal muscle pathology. Although the histological pathology of dystrophic tissue has been well documented, the underlying molecular pathways remain poorly understood. To examine the pathogenic pathways and identify new or modifying factors involved in muscular dystrophy, expression microarrays were used to compare individual gene expression profiles of skeletal muscle biopsies from 12 DMD patients and 12 unaffected control patients. Two separate statistical analysis methods were used to interpret the resulting data: t test analysis to determine the statistical significance of differential expression and geometric fold change analysis to determine the extent of differential expression. These analyses identified 105 genes that differ significantly in expression level between unaffected and DMD muscle. Many of the differentially expressed genes reflect changes in histological pathology. For instance, immune response signals and extracellular matrix genes are overexpressed in DMD muscle, an indication of the infiltration of inflammatory cells and connective tissue. Significantly more genes are overexpressed than are underexpressed in dystrophic muscle, with dystrophin underexpressed, whereas other genes encoding muscle structure and regeneration processes are overexpressed, reflecting the regenerative nature of the disease.

Adult↗

PubMed2002

Comprehensive detection of genomic duplications and deletions in the DMD gene, by use of multiplex amplifiable probe hybridization.

Duplications and deletions are known to cause a number of genetic disorders, yet technical difficulties and financial considerations mean that screening for these mutations, especially duplications, is often not performed. We have adapted multiplex amplifiable probe hybridization (MAPH) for the screening of the DMD gene, mutations in which cause Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy. MAPH involves the quantitative recovery of specifically designed probes following hybridization to immobilized genomic DNA. We have engineered probes for each of the 79 exons of the DMD gene, and we analyzed them by using a 96-capillary sequencer. We screened 24 control individuals, 102 patients, and 23 potential carriers and detected a large number of novel rearrangements, especially small, one- and two-exon duplications. A duplication of exon 2 alone was the most frequently occurring mutation identified. Our analysis indicates that duplications occur in 6% of patients with DMD. The MAPH technique as modified here is simple, quick, and accurate; furthermore, it is based on existing technology (i.e., hybridization, PCR, and electrophoresis) and should not require new equipment. Together, these features should allow easy implementation in routine diagnostic laboratories. Furthermore, the methodology should be applicable to any genetic disease, it should be easily expandable to cover >200 probes, and its characteristics should facilitate high-throughput screening.

Dystrophin↗

PubMed1993

Point mutations at the carboxy terminus of the human dystrophin gene: implications for an association with mental retardation in DMD patients.

Duchenne and Becker muscular dystrophies (DMD/BMD) are caused by mutations in the human dystrophin gene. About two-thirds of DMD/BMD patients exhibit gross rearrangements in the gene whereas the mutations in the remaining one third are thought to be point mutations or minor structural lesions. By means of various progressive PCR-based techniques hitherto a number of point mutations has been described that in most cases should cause premature translational termination. These data indicate a particular functional importance for the C-terminal region of dystrophin and consequently for its gene products Dp 71 and Dp 116. To screen for microheterogeneities in this gene region we applied PCR-SSCP analysis to exons 60-79 of twenty-six DMD/BMD patients without detectable deletions. The study identified seven point mutations and one intron polymorphism. Six point mutations, found in DMD patients, should cause premature translational termination. One point mutation, identified in a BMD patient, results in an amino acid exchange. Five of the DMD patients bearing a point mutation are mentally retarded suggesting that a disruption of the translational reading frame in the C-terminal region is associated with this clinical finding in DMD cases. Therefore our data raise the possibility, that Dp 71 and/or Dp 116, the C-terminal translational products of dystrophin, may be causally involved in cases of mental retardation that are associated with DMD.

Amino Acid Sequence↗

PubMed1998

Insertional mutation by transposable element, L1, in the DMD gene results in X-linked dilated cardiomyopathy.

X-linked dilated cardiomyopathy (XLDCM) is a clinical phenotype of dystrophinopathy which is characterized by preferential myocardial involvement without any overt clinical signs of skeletal myopathy. To date, several mutations in the Duchenne muscular dystrophy gene, DMD , have been identified in patients with XLDCM, but a pathogenic correlation of these cardiospecific mutations in DMD with the XLDCM phenotype has remained to be elucidated. We report here the identification of a unique de novo L1 insertion in the muscle exon 1 in DMD in three XLDCM patients from two unrelated Japanese families. The insertion was a 5'-truncated form of human L1 inversely integrated in the 5'-untranslated region in the muscle exon 1, which affected the transcription or the stability of the muscle form of dystrophin transcripts but not that of the brain or Purkinje cell form, probably due to its unique site of integration. We speculate that this insertion of an L1 sequence in DMD is responsible for some of the population of Japanese patients with XLDCM.

5' Untranslated Regions↗

PubMed1991

The value of deletion analysis for carrier detection in Duchenne muscular dystrophy (DMD).

We performed genetic analysis for carrier detection for several at-risk females in a four-generation Duchenne muscular dystrophy (DMD) pedigree using deletion analysis. We demonstrated that dosage analysis is a suitable alternative method to determine the carrier status of female relatives of DMD patients shown to have a deletion within the DMD gene. Subsequently, we diagnosed an affected male fetus for an at-risk female shown to be a DMD carrier by deletion analysis. The usefulness of deletion and linkage analysis are compared. In this family, linkage analysis was complicated by the unavailability of key family members, two recombination events and by previously undisclosed nonpaternity. We found that dosage analysis was more efficient than linkage for carrier evaluation in this family.

Adult↗

PubMed1981

Translocation (X;6) in a female with Duchenne muscular dystrophy: implications for the localisation of the DMD locus.

A female with Duchenne muscular dystrophy who was a carrier of a balanced translocation t(X;6)(p21;q21) is reported. Four other previously described (X;A) translocations associated with DMD share with the present case a breakpoint at Xp21. The extremely low probability of five independent (X;A) translocations having a breakpoint at Xp21 points to a non-rand association of this site with the DMD phenotype. A DMD locus at Xp21 could be damaged by the translocation, giving rise to Duchenne muscular dystrophy. Alternatively, a pre-existing DMD gene could weaken the chromosome, favouring breaks at Xp21.

Child↗

PubMed1995

Expression of the human Dp 71 (apo-dystrophin-1) gene from a 760-kb DMD-YAC transferred to mouse cells.

A 760-kb YAC was constructed by homologous recombination in yeast, containing the genes located in the distal portion of the DMD gene. The YAC was introduced in mouse LA-9 cells by PEG-mediated cell fusion. One transformant accommodated an intact DMD-YAC, i.e. a full copy of the DMD internal Dp 116, Dp 71 and Dp 40 genes (apo-dystrophin-2, -1 and -3, respectively). We have studied the expression of the various gene products derived from the introduced DMD-YAC. RT-PCR revealed expression of human Dp 71 but not of Dp 116 or Dp 40. Remarkably, differences were observed in processing of the 3' region of the endogenous mouse and the human transcripts, due to different splicing of exons 71 (absent in human and present in mouse transcript) and 78 (present in human and absent in mouse transcript). The splicing pattern of the human transcript is the same as that of the major Dp 71 (apo-dystrophin-1) product in human blood. The observed splicing differences may be caused by either species-specific exon use and/or by cis-acting factors, e.g. the upstream transcript composition, because we have no evidence for endogenous Dp 71 expression.

Animals↗

PubMed2002

Role of Escherichia coli Hfq in late-gene silencing of bacteriophage T4 dmd mutant.

When the dmd gene of bacteriophage T4 is mutated, many T4 late genes are post-transcriptionally silenced because of rapid mRNA degradation. Here we show that the host hfq gene is involved in the rapid mRNA degradation in a dmd mutant. A disruption of the hfq gene caused weak but significant effects on the stability of late-gene mRNA, the late-gene expression and the growth of a dmd mutant. By probing with the soc gene, we found that disruption of the hfq gene impaired the translation-independent mRNA degradation, one of two mechanisms promoting rapid mRNA degradation. We also showed that purified Hfq protein bound stoichiometrically to soc RNA. These results strongly suggest that the hfq gene has a stimulatory role in dmd mutant-specific mRNA degradation.

Bacteriophage T4↗