Novel mutations of dystrophin gene in DMD patients detected by rapid scanning in biplex exons DHPLC analysis

Mutations in the dystrophin gene result in both Duchenne and Becher muscular dystrophies (DMD and BMD). Approximately 65% of all mutations causing DMD are deletions (60%) or duplications (5%) of large segments of this gene, spanning one exon or more. Due to the large size of the dystrophin gene (79 exons), finding point mutations has been prohibitively expensive and laborious. Recent studies confirm the utility of pre-screening methods, as denaturing high-performance liquid chromatography (DHPLC) analysis in the identification of point mutations in the dystrophin gene, with an increment of mutation detection rate from 65% to more than 92%. Here we suggest an alternative and convenient method of DHPLC analysis in order to find mutations in a more rapid and less expensive way by introducing the analysis of 16 couples of dystrophin amplicons, in biplex exons DHPLC runs. Using this new protocol of biplex exons DHPLC screening, new mutations were identified in four male patients affected by DMD who had tested negative for large DNA rearrangements.     

Use of in silico tools for classification of novel missense mutations identified in dystrophin gene in developing countries

DMD gene which is composed of 79 exons is the largest known gene located on X chromosome (Xp21). Point mutations in the dystrophin gene are responsible for 30–35% of cases with DMD/BMD. Mutation analysis of all the exons of the DMD gene is costly in developing countries, therefore, a few of the exons are selected to be analyzed routinely in clinical laboratories. In this study, direct sequencing was used for detection of point mutations in 10 exons of dystrophin gene in patients affected with DMD without detectable large rearrangements. Freely available programs were used to predict the damaging effects of the mutations. Point mutations were successfully detected in three patients. Three novel mutations, two missense mutations located on nonconservative domains and a single nucleotide deletion, were detected. Missense mutations were predicted to change splicing efficiency. Detection of point mutations by DNA analysis followed by prediction of the pathogenecity by using bioinformatic tool might be an asset to provide proper diagnosis or genetic counseling to patients and their family.     

Molecular diagnosis of Duchenne/Becker muscular dystrophy by polymerase chain reaction and microsatellite analysis

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked recessive genetic disorders resulting from mutations in the dystrophin gene. About two-thirds of the affected patients have large deletions or duplications, which occur in the 5' and central region of the gene. The remaining DMD/BMD cases show no deletions, so they cannot be easily identified by current strategies. In these DMD/BMD families, a linkage analysis that involves DNA markers of the flanking and intragenic dystrophin gene are necessary for carrier and prenatal diagnosis. We analyzed eighteen deletion-prone exons of the gene by a polymerase chain reaction (PCR) in order to characterize the molecular defects of the dystrophin gene in Korean DMD/BMD families. We also performed a linkage analysis to assess the usefulness and application of six short tandem repeat markers for molecular diagnosis in the families. We observed a deletion that eliminated the exon 50. Also, a linkage analysis in the families with six short tandem repeat (STR) markers showed heterozygosity at most of the STR markers. The haplotype analysis was useful for detecting the carrier status. This study will be helpful for a molecular diagnosis of DMD/BMD families in the Korean population.     

Deletions in the dystrophin gene: analysis of Duchenne and Becker muscular dystrophy patients in Quebec

Summary We have analyzed patient DNA samples in 77 unrelated Duchenne (DMD) and Becker (BMD) muscular dystrophy families, 73 of which were of French Canadian origin. We show that the frequency (68%) and distribution of deletions within the dystrophin gene was neither random nor unique in this population. We localized 33% of the deletions to the proximal portion of the dystrophin gene while 63% involved the exons spanning introns 43 through 55 with breakpoint clusters occurring within introns 44 and 50. Whether the dystrophin open reading frame (ORF) is maintained constrains the distribution of DMD/BMD deletions such that BMD deletions tend to be strikingly homogeneous. Finally, the conservation of the dystrophin ORF and the severity of the clinical phenotype were concordant in 95% of the DMD/BMD deletions documented by this work.     

Assessment of the structural and functional impact of in-frame mutations of the DMD gene, using the tools included in the eDystrophin online database

ABSTRACT: BACKGROUND: Dystrophin is a large essential protein of skeletal and heart muscle. It is a filamentous scaffolding protein with numerous binding domains. Mutations in the DMD gene, which encodes dystrophin, mostly result in the deletion of one or several exons and cause Duchenne (DMD) and Becker (BMD) muscular dystrophies. The most common DMD mutations are frameshift mutations resulting in an absence of dystrophin from tissues. In-frame DMD mutations are less frequent and result in a protein with partial wild-type dystrophin function. The aim of this study was to highlight structural and functional modifications of dystrophin caused by in-frame mutations. Methods and results We developed a dedicated database for dystrophin, the eDystrophin database. It contains 209 different non frame-shifting mutations found in 945 patients from a French cohort and previous studies. Bioinformatics tools provide models of the three-dimensional structure of the protein at deletion sites, making it possible to determine whether the mutated protein retains the typical filamentous structure of dystrophin. An analysis of the structure of mutated dystrophin molecules showed that hybrid repeats were reconstituted at the deletion site in some cases. These hybrid repeats harbored the typical triple coiled-coil structure of native repeats, which may be correlated with better function in muscle cells. CONCLUSION: This new database focuses on the dystrophin protein and its modification due to in-frame deletions in BMD patients. The observation of hybrid repeat reconstitution in some cases provides insight into phenotype-genotype correlations in dystrophin diseases and possible strategies for gene therapy. The eDystrophin database is freely available: http://edystrophin.genouest.org/.     

Detection of gene deletions in Chinese patients with Duchenne/Becker muscular dystrophy using CDNA probes and the polymerase chain reaction method.

One hundred thirty-eight patients with Duchenne/Becker muscular dystrophy (DMD/BMD) were screened with complete cDNA probes and the multiplex polymerase chain reaction (mPCR) amplification of 18 pairs of oligonucleotide primers. Eighty-six deletions and 4 duplications were detected, the deletion frequency being 62.3%. Eighty-two deletions were detected with the two sets of primers described by Chamberlain et al. and Beggs et al, which was 95.4% of deletions detected by complete cDNA probes. Consistent with the deletion locations described previously, the deletions of dystrophin gene in Chinese individuals are clustered mainly in two high-frequency deletion regions of exons 44-52 (68.6%) of 3' side of the gene central regions and exons 1-19 (26.7%) in the 5' side. The distribution of deletions in dystrophin gene is associated with the phenotype of DMD/BMD. In the 25 cases with in-frame deletions, 15 deletions located in the region of exons 2-47 were milder BMD and intermediate patients, as the location of deletions was not the important region of the dystrophin gene.     

Multiplex real-time PCR for detection of deletions and duplications in dystrophin gene

Genetic testing of Duchenne and Becker muscular dystrophies (DMD/BMD) is a difficult task due to the occurrence of deletions or duplications within dystrophin (DMD) gene that requires dose sensitive tests. We developed three multiplex quantitative real-time PCR assays for dystrophin exon 5, 45, and 51 within two major hotspots of deletion/duplication. Each exon was co-amplified with a reference X-linked gene and the copy number of the target fragment was calculated by comparative threshold cycle method (delta deltaC(t)). We compared the performance of this method with previously described end-point PCR fluorescent analysis (EPFA) by studying 24 subjects carrying DMD deletions or duplications. We showed that Q-PCR is an accurate and sensitive technique for the identification of deletions and duplications in DMD/BMD. Q-PCR is a valuable tool for independent confirmation of EPFA screening, particularly when deletions/duplications of single exons occur or for rapid identification of known mutations in at risk carriers.     

The molecular basis for Duchenne versus Becker muscular dystrophy: Correlation of severity with type of deletion

About 60% of both Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) is due to deletions of the dystrophin gene. For cases with a deletion mutation, the "reading frame" hypothesis predicts that BMD patients produce a semifunctional, internally deleted dystrophin protein, whereas DMD patients produce a severely truncated protein that would be unstable. To test the validity of this theory, we analyzed 258 independent deletions at the DMD/BMD locus. The correlation between phenotype and type of deletion mutation is in agreement with the "reading frame" theory in 92% of cases and is of diagnostic and prognostic significance. The distribution and frequency of deletions spanning the entire locus suggests that many "in-frame" deletions of the dystrophin gene are not detected because the individuals bearing them are either asymptomatic or exhibit non-DMD/non-BMD clinical features.     

Detection of 98% of DMD/BMD gene deletions by polymerase chain reaction

Summary We describe oligonucleotide primer sequences that can be used to amplify eight exons plus the muscle promoter of the dystrophin gene in a single multiplex polymerase chain reaction (PCR). When used in conjunction with an existing primer set, these two multiplex reactions detect about 98% of deletions in patients with Duchenne or Becker muscular dystrophy (DMD, BMD). Furthermore, these primers amplify most of the exons in the deletion prone hot spot region around exons 44 to 53, allowing determination of deletion endpoints and prediction of mutational effects on the translational reading frame. Thus, use of these PCR-based assays will allow deletion detection and prenatal diagnosis for most DMD/BMD patients in a fraction of the time required for Southern blot analysis.     

Evaluation of point mutations in dystrophin gene in Iranian Duchenne and Becker muscular dystrophy patients: introducing three novel variants

Duchenne and Becker muscular dystrophies (DMD and BMD) are X-linked neuromuscular diseases characterized by progressive muscular weakness and degeneration of skeletal muscles. Approximately two-thirds of the patients have large deletions or duplications in the dystrophin gene and the remaining one-third have point mutations. This study was performed to evaluate point mutations in Iranian DMD/BMD male patients. A total of 29 DNA samples from patients who did not show any large deletion/duplication mutations following multiplex polymerase chain reaction (PCR) and multiplex ligation-dependent probe amplification (MLPA) screening were sequenced for detection of point mutations in exons 50–79. Also exon 44 was sequenced in one sample in which a false positive deletion was detected by MLPA method. Cycle sequencing revealed four nonsense, one frameshift and two splice site mutations as well as two missense variants.     

Molecular and phenotypic analysis of patients with deletions within the deletion-rich region of the Duchenne muscular dystrophy (DMD) gene

Eighty unrelated individuals with Duchenne muscular dystrophy (DMD) or Becker muscular dystrophy (BMD) were found to have deletions in the major deletion-rich region of the DMD locus. This region includes the last five exons detected by cDNA5b-7, all exons detected by cDNA8, and the first two exons detected by cDNA9. These 80 individuals account for approximately 75% of 109 deletions of the gene, detected among 181 patients analyzed with the entire dystrophin cDNA. Endpoints for many of these deletions were further characterized using two genomic probes, p20 (DXS269; Wapenaar et al.) and GMGX11 (DXS239; present paper). Clinical findings are presented for all 80 patients allowing a correlation of phenotypic severity with the genotype. Thirty-eight independent patients were old enough to be classified as DMD, BMD, or intermediate phenotype and had deletions of exons with sequenced intron/exon boundaries. Of these, eight BMD patients and one intermediate patient had gene deletions predicted to leave the reading frame intact, while 21 DMD patients, 7 intermediate patients, and 1 BMD patient had gene deletions predicted to disrupt the reading frame. Thus, with two exceptions, frameshift deletions of the gene resulted in more severe phenotype than did in-frame deletions. This is in agreement with recent findings by Baumbach et al. and Koenig et al. but is in contrast to findings, by Malhotra et al., at the 5' end of the gene.     

Analysis of deletions in the dystrophin gene in patients with Duchenne muscular dystrophy in the Bashkir Republic]

The deletion spectrum and distribution of deletion breakpoints (DBs) in 36 patients with Duchenne muscular dystrophy (DMD) from 33 families and in three patients with Becker muscular dystrophy (BMD) from one family from Bashkortostan were studied by amplifying 20 exons of the dystrophin gene by multiplex polymerase chain reaction (mPCR). Eight out of 34 unrelated DMD (BMD) patients (23.2%) were shown to carry a deletion varying in size from one to seven exons. Most DBs (15 out of 16, 93.7%) were in the distal region of the gene, commonly between exons 44-45, 45-47, and 50-52. Thus, high-polymorphic intergenic markers located in introns 44 (STR 44), 45 (STR 45), 49 (STR 49), and 50 (STR 50) can be used for indirect or direct carrier detection among women closely related to DMD patients that carry a deletion with DB located between exons 44-45, 45-47, and 50-52. Prenatal diagnosis of DMD is also possible in these families.     

Genotype-phenotype correlation and germline mosaicism in DMD/BMD patients with deletions of the dystrophin gene

Summary The molecular analysis of 127 DMD/BMD patients showed that 73 of them (57%) had deletions in the dystrophin gene. Two different methods were used in this study: (a) hybridization of HindIII-digested genomic DNA with nine cDNA probes corresponding to the entire 14 kb cDNA of the DMD gene; and (b) simultaneous amplification of nine exons of the DMD gene (multiplex DNA amplification) by the polymerase chain reaction (PCR). When the deletion breakpoints of the intragenic deletions were analyzed with regard to their phenotypic consequences, nine patients were found to represent exceptions to the reading-frame hypothesis. Information regarding mental development was also available for 61 of the 73 deleted patients and for 34 of the 54 non-deleted ones. The proportion of mentally retarded patients was found to be similar in the two groups (deleted, 15%; non-deleted, 18%). Finally, in one family, a junction fragment present in the patient was not found in the peripheral blood DNA of the mother but was present in the sister, thus indicating germline mosaicism in the mother.     

Analysis of a dystrophin gene deletion by amplification of mRNA isolated from DMD myotubes cultured in vitro

The most frequent causes for the X-linked muscular dystrophy of the allelic Duchenne (DMD) or Becker (BMD) type are partial deletions of the dystrophin gene. These mutations are accompanied either by disrupted or by preserved translational reading frames in mRNAs derived from the deleted genes. As a rule, the reading frame is destroyed in the more severe DMD, whereas it is preserved in the less severe BMD (M. Koenig et al., 1989, Am. J. Hum. Genet. 45, 498-506). We have analyzed in detail a deletion that was detected in a fetus at risk of DMD. The analysis of this mutation included the delineation of the altered subregion in the dystrophin mRNA. mRNA was isolated from myotubes derived from embryonic DMD myoblasts propagated in vitro. This study was based on enzymatic amplification by the polymerase chain reaction (PCR) of dystrophin mRNA and direct sequencing of the amplified cDNA. Exons 47 to 50 were found to be missing in the mRNA. The splicing of exon 46 to exon 51 resulted in a reading frameshift, indicating that this mutation is likely to be responsible for a DMD type of dystrophy. The clinical diagnosis of DMD for a 10-year-old patient in this family was compatible with the "reading frame" assumption.     

Point mutations and polymorphisms in the human dystrophin gene identified in genomic DNA sequences amplified by multiplex PCR

Summary About one third of Duchenne muscular dystrophy (DMD) patients have no gross DNA rearrangements in the dystrophin gene detectable by Southern blot analysis or multiplex exon amplification. Presumably, in these cases, the deficiency is caused by minor structural lesions of the dystrophin gene. However, to date, only a single human DMD case has been described where a point mutation, producing a stop codon, accounts for the DMD phenotype. To screen for microheterogeneities in the dystrophin gene, we applied analysis by chemical mismatch cleavage to thirteen exons amplified in multiplex sets by the polymerase chain reaction. This analysis covers approximately 20% of the dystrophin-coding sequence. Sixty DMD patients without detectable deletions or duplications were investigated, leading to the identification of two point mutations and four polymorphisms with a frequency higher than 5%. Both point mutations are frameshift mutations in exons 12 and 48, respectively, and are closely followed by stop codons, thus explaining the functional deficiency of the dystrophin gene products in both patients.     

Duchenne and Becker muscular dystrophy: from gene diagnosis to molecular therapy

Duchenne and Becker muscular dystrophy (DMD/BMD) are X-linked muscular dystrophies. The isolation of the defective gene in DMD/BMD has led to a better understanding of the disease process and has promoted studies regarding the application of molecular therapy. The purpose of this review is to present the progress made in this area of research with particular reference to dystrophin Kobe. Based on the results from the molecular analysis of dystrophin Kobe, we propose a novel molecular therapeutic method for DMD in which antisense oligonucleotides transform DMD into a milder phenotype by inducing exon skipping. In addition, current proposals for the molecular therapy of DMD are discussed.     

Strategy for comprehensive molecular testing for Duchenne and Becker muscular dystrophies

Comprehensive molecular testing for mutations in the DMD gene causing Duchenne and Becker muscular dystrophy (DMD/BMD) is challenging because of the large size of the gene and the variety of mutation types. There is an increasing demand for comprehensive DMD gene molecular testing, including deletion/duplication testing of 79 exons and direct sequencing of the 14-kb coding region from genomic DNA, to provide confirmation of clinical diagnoses in affected patients and to determine carrier risk for family members. To determine an efficient strategy to prioritize patients for comprehensive molecular testing of the DMD gene, we tested a consecutive cohort of 165 males referred over a 4-year period because of a suspicion of DMD or BMD using: (1) a new quantitative multiplex polymerase chain reaction (PCR) assay designed to detect deletions or duplications in all exons of the gene and the brain promoter and (2) direct sequencing of the coding region and intron/exon boundaries. For the patients being tested because of a suspicion of DMD, deletion/duplication testing followed by direct sequencing detected pathogenic mutations in 98% (106/108 total patients). However, of the patients tested because of a suspicion of BMD, only 60% (34/57 total patients) had causative mutations identified, all of which were deletions or duplications. Our results suggest that direct genomic sequence analysis of the DMD gene is a useful addition to deletion/duplication testing for diagnosis of DMD, but does not provide an improved sensitivity compared to deletion/duplication analysis alone for the diagnosis of BMD. In addition, due to the relatively common finding of single exon deletions and duplications (22%, 27 of 125 total patients with deletions/duplications), methods to examine all exons of the gene for deletions/duplications should be used as the initial molecular quantitative test for DMD and BMD.     

Direct carrier detection by in situ suppression hybridization with cosmid clones of the Duchenne/Becker muscular dystrophy locus

Summary A basic problem in genetic counseling of families with Duchenne/Becker muscular dystrophy (DMD/BMD) concerns the carrier status of female relatives of an affected male. In about 60% of these patients, deletions of one or more exons of the dystrophin gene can be identified. These deletions preferentially include exon 45, which can be detected by multiplex polymerase chain reaction (PCR) and Southern blot analysis of genomic cosmid clones that map to this critical region. As a new approach for definitive carrier detection, we have performed chromosomal in situ suppression (CISS) hybridization with these cosmid clones in female relatives of four unrelated patients. In normal females, most metaphases showed signals on both×chromosomes, whereas only one×chromosome was labeled in carriers. Our results demonstrate that CISS hybridization can define the carrier status in female relatives of DMD patients exhibiting a deletion in the dystrophin gene.     

Intragenic deletion patterns of dystrophin gene in Duchenne and Becker muscular dystrophy patients from Algeria

Duchenne and Becker muscular dystrophies (DMD and BMD) represent the most frequent neuromuscular diseases in humans (1/3,500–6,000 live male births), characterized by an X-linked recessive pattern of inheritance and therefore affecting mainly male individuals. DMD and BMD are allelic disorders resulting from genetic defects, mostly intragenic deletions, in the dystrophin gene. Using multiplex polymerase chain reaction (PCR), we have analyzed 170 male patients from unrelated families originating from Algeria, showing that 68 % of them harbored deletion events affecting the known 5′ or 3′ hot spot regions. The distal portion was predominantly involved (85 %), whereas 37 distinctive patterns of deletion were identified in our panel. The extent of deletion varied from 1 to 32 exons, although the average number was about four exons. The lack of seven exons (45, 46, 47, 48, 50, 51 and 52), each alone or in combination, represented about 78 % of the alterations encountered, while exon 48 was most frequently involved (50 %). The effect of the deletions showed that the reading frame rule proved mostly true, correlating with the clinical diagnosis suggested. Moreover, the c.525delT mutation in the γ-sarcoglycan gene was present in non-deleted patients (7 %), suggesting that clinical features can still be misleading. Finally, multiplex PCR proved to be a simple, fast and low-cost approach for the molecular diagnosis of dystrophinopathies in Algeria, whereas our data could contribute to the creation of a national registry of DMD/BMD patients in our country, which would give them hope to an access to already available genotype-based therapies.