Molecular deletion patterns in families from southern France with Duchenne/Becker muscular dystrophies

Summary We studied 38 unrelated patients from southern France with Duchenne (DMD) or Decker (BMD) muscular dystrophy for intragenic deletions of the DMD/ BMD gene. We used both multiplex amplification of selected exons and cDNA probes. Of the 26 (68%) unrelated individuals found to have deletions, 24 (92%) were detected by multiplex polymerase chain reaction. All these deletions have been delineated with regard to the exon-containing HindIII fragments revealed by cDNA probes, and in two cases, junction fragments of altered size were seen. The correlation between phenotype and type of deletion agreed with the reading frame theory, except for two BMD and two DMD cases.     

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.     

Molecular analysis of Duchenne and Becker muscular dystrophy

Duchenne muscular dystrophy (DMD) is a progressive and lethal neuromuscular disorder caused by a defective gene on the X chromosome. There is no effective treatment and the biochemical defect is yet unknown. Mapping of the DMD locus to band Xp21 in the short arm of the X chromosome has given rise to strategies to clone the gene from its known location in the chromosome. Two cloning strategies have led to the isolation of a gene that is the largest of any yet described. Portions of the gene are deleted in about 8% of affected males, and rare translocations that disrupt the gene cause the disease in females. The isolation of expressed sequences from the DMD locus will undoubtedly lead to isolation of the gene product and ultimately to an understanding of the basic defect. In the meantime, DNA probes from the DMD locus provide a new and accurate approach for carrier identification and prenatal diagnosis of this dreaded disease.     

Further studies of gene deletions that cause Duchenne and Becker muscular dystrophies

Fetal muscle cDNA clones covering at least 11.4 kb of the Duchenne muscular dystrophy (DMD) gene sequence were used to identify a deletion-prone region in DNA from DMD and Becker muscular dystrophy (BMD) patients. Of 36 BMD cases, 17 (47%) had deletions and all of the deletions began in the same intron of the gene. Of 107 DMD patients, 27 (25%) were deleted for this region, and 19 deletions originate in the same intron. Using a cDNA probe for an adjacent region of the gene, 32 new deletions were detected in DMD patients (total 44%). No new BMD deletions were detected. The DMD deletions were very heterogeneous. Thus two cDNA probes covering 2.4 kb could detect 53% of these deletions. Considering the whole locus, DMD and BMD are caused by a deletion of the gene sequence in at least 67% of cases.     

Molecular pathology of Duchenne and Becker muscular dystrophy]

Duchenne and Becker muscular dystrophies (DMD and BMD) are two allelic recessive X-linked disorders. Molecular deletions of various regions of the dystrophin gene are the main mutations detected in DMD and BMD patients. Molecular study of DMD and BMD DNA are instrumental to understand the pathological molecular mechanisms and the function of the protein. We describe here dystrophin and its interaction with a glycoprotein complex and we then focus on two particular patients with partial deletions of the dystrophin gene: 1) a typical Becker patient, who shows an intragenic deletion disrupting the reading frame. We describe in this case alternative splicings restoring the reading frame, which might explain the mild clinical phenotype of this patient, 2) a deletion of the distal part of the DMD gene coding for the carboxyterminal domain of the dystrophin in a young patient. The normal localization of dystrophin at the inner face of the plasma membrane in the muscle of this patient suggests that the last domain of this protein is not sufficient to anchor dystrophin at the membrane.     

Pharmacological treatments for Duchenne and Becker dystrophies

Duchenne muscular dystrophy (DMD) is a severe X-linked genetic disease affecting 1 boy out of 3500. DMD is due to the lack of a submembranous cytoskeletal protein named dystrophin, leading to the progressive degeneration of skeletal, cardiac and smooth muscle tissue. A milder form of the disease, Becker muscular dystrophy (BMD), is characterised by the presence of a semi-functional truncated dystrophin, or the full-length dystrophin at reduced level. Three different therapeutic approaches are currently under study, gene therapy, cellular therapy and pharmacological therapy. One of the chosen strategies consists of the overexpression of utrophin, a protein 80% homologous with dystrophin, and able to perform similar functions. In this review, we shall consider studies of pharmacological therapy, the aims of which can be classified in three categories: reversal of dystrophic phenotype, dystrophin expression, utrophin overexpression.     

Molecular deletion patterns in Duchenne and Becker type muscular dystrophy

Summary DNA from 80 Duchenne (DMD) and 15 Becker (BMD) index patients was analyzed with 12 genomic probes and the total cDNA. Deletions were detected in 24 DMD (30%) and 10 BMD patients (67%) by genomic probes alone, mostly p20, pXJ, and/or pERT87. All deletions were confirmed by cDNA probes, and an additional 29 DMD deletions were detected, resulting in a total of 63/95 deletions (66%). The majority of the deletions are localized between kb 6.7 and 9.7 of the cDNA; a smaller group, between kb 0.5 and 3.5. Of the deletions, 90% are detected by the three cDNA probes 1–2a, 7, and 8. This can be applied to strategies for carrier detection and prenatal diagnosis. The order of 13 exon-containing HindIII fragments in the region between probes 7 and 9–10, where most of the deletions are found, could be defined. The deletion patterns in DMD and BMD patients are different and well in accordance with the “reading frame theory” of Monaco and coworkers. Thus our findings indicate that a DMD or BMD phenotype may be predicted according to the breakpoint position and the number of deleted exons.     

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.     

Analysis of molecular deletions with cDNA probes in patients with Duchenne and Becker muscular dystrophies

In the course of a systematic survey of DMD and BMD patients with intronic probes and with cDNA probes covering three-fourths of the coding sequence, 45 molecular deletions within the DMD gene were investigated. Forty-two percent of the breakpoints were located in the intronic sequence containing probe P20, whereas the other deletions were widespread around the more proximal part of the gene. Most of the BMD deletions were in the P20 region. Pulsed field gel electrophoresis was used to determine the size of some deletions and allowed us to estimate the physical distance between the intronic probes JBir and P20. The reading frame was checked in 11 cases with proximal deletions and found to be disrupted in 6 of 7 DMD patients, in 1 intermediate case, and, unexpectedly, in 3 BMD 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.     

多重连接探针扩增方法在假肥大性肌营养不良产前基因诊断中的应用

假肥大性肌营养不良(Duchenne/Becker muscular dystrophy, DMD/BMD)是一种由于DMD基因突变导致的X连锁隐性致死性遗传病。目前没有有效的治疗方法。为建立一种既可以对携带者进行检测又可以进行产前基因诊断的方法, 文章联合应用多重连接探针扩增技术(Multiplex ligation-dependent probe amplification, MLPA)和短串联重复序列(Short tandem repeats , STR)为遗传标记连锁分析的方法对26例有高风险再生育患儿的假肥大性肌营养不良家系的孕妇通过羊水穿刺进行产前基因诊断。26例进行产前基因诊断的羊水标本中有7例诊断为男性患儿, 4例诊断为女性携带者。MLPA可以作为筛查DMD基因缺失和重复突变的首选方法。联合应用MLPA和STR连锁分析, 可以提高假肥大性肌营养不良的产前基因诊断率。     

Estimate of the intrafamilial correlation for serum creatine kinase and pyruvate kinase in females at risk for Duchenne and Becker muscular dystrophies.

In order to verify the possibility of nonrandom X-inactivation in females heterozygous for Duchenne (DMD) and Becker (BMD) muscular dystrophies, intrafamilial correlations and the heritabilities for serum creatine kinase (CK) and pyruvate kinase (PK) were estimated in a large sample of females belonging to families with affected patients. The results of the present investigation suggest that the apparent intrafamilial correlations for serum CK reported in previous studies in DMD families are not related with the presence of the DMD/BMD gene. Our data do not seem to support the hypothesis of a gene leading to a preferential inactivation of the X-chromosome in females at risk for the dystrophin gene.     

Patterns of exon deletions in Duchenne and Becker muscular dystrophy

Summary A panel of patients with Duchenne and Becker muscular dystrophy (DMD and BMD) has been screened with the cDNA probes Cf56a and Cf23a, which detect exons in the central part of the DMD gene. One or more exons were deleted in 60% of patients. The deletions were mapped and prove to be heterogeneous in size and extent, particularly in DMD. Deletions specific to DMD and to BMD are described. Half of all BMD patients have a deletion of one particular small group of exons; smaller deletions within this same group produce the more severe DMD.     

Carrier detection and prenatal diagnosis in Duchenne and Becker muscular dystrophy families, using dinucleotide repeat polymorphisms.

To improve carrier detection and prenatal diagnosis for Duchenne and Becker muscular dystrophy families, we determined allele frequencies and measures of variation for four (dC-dA)n.(dG-dT)n loci identified within a deletion-prone region of the human dystrophin gene. The loci are highly polymorphic, with predicted heterozygosities of 71.6%-93.3%. Direct DNA sequence analysis of the (dC-dA)n.(dG-dT)n locus in intron 49 revealed an additional length polymorphism which varies by single-basepair increments, is adjacent to the dinucleotide repeat block, and enhances the polymorphic content of this marker. The four (dC-dA)n.(dG-dT)n loci are each easily amplified by PCR in two diplex reactions. The variability of allele lengths at these loci makes them ideal for carrier detection and prenatal diagnosis, often providing diagnostic information when RFLP analysis is uninformative. These markers have aided in identification of deletion mutations, exclusion of maternal cell contamination of chorionic villus samples, confirmation of paternity, and mapping of gene recombinations. The allele identification of these loci can be performed either with a radiolabel or with an automated, nonradioactive, fluorescent gel detection system.     

DNA analysis in Turkish Duchenne/Becker muscular dystrophy families

Summary The molecular genetics of Duchenne/Becker muscular dystrophy was investigated in 81 affected Turkish families. Deletions were detected by multiplex polymerase chain reaction assays and cDNA Southern analyses. The distribution of the deletions along the gene and their correlation to clinical phenotype were different from the studies reported on other populations. Moreover, DNA polymorphisms in mothers were determined using 8 DNA probes and three CA repeat sequences, and a high degree of informativeness was observed.     

Frameshift deletion mechanisms in Egyptian Duchenne and Becker muscular dystrophy families

Partial gene deletion is the major type of mutation leading to Duchenne muscular dystrophy (DMD) and its mild allelic form, Becker muscular dystrophy (BMD). Amplification of the genomic DNAs of 152 unrelated dystrophin patients using multiple primers detected 78 (51.3%) probands with deletion mutations. We predicted the translational reading frame for all the deletions in Egyptian dystrophin males. The frameshift rule was confirmed positively ranging for 50 to 67% of the cases depending on the type of disease. We discuss ways of accounting for some exceptions from the frameshift hypothesis in the central and proximal regions. These explanations may help in developing procedures for reducing the severity of dystrophin phenotypes to restore the correct frame by disrupting the translational fidelity. Great efforts have been put into the development of effective 'gene correction' procedures via such intrinsic mechanisms. In addition, we mapped regional difference in deletion mutation frequencies within the DMD gene locus between the different Egyptian governorates. There were no double deletions in the Egyptian dystrophin males.     

Microlesions and polymorphisms in the Duchenne/Becker muscular dystrophy gene

One third of mutations responsible for Duchenne or Becker muscular dystrophy (DMD/BMD) represent point mutations or other small sequence alterations not readily detectable by Southern blot analysis or multiplex amplification. Here, we report results of a comprehensive point mutation search that yielded seven new sequence variations and one novel polymorphism. We also summarize known mutations, polymorphisms and other small nucleotide variations in the DMD gene. To date, 12 nonsense mutations, two missense mutations, six microdeletions and one microinsertion have been reported in the coding sequence and a further six mutations in splice sites all of which were made responsible for the disease. Twelve polymorphisms with frequencies suitable for diagnostic purposes have been detected. A further 28 differences from the published sequence of the coding sequence or the promotor region are described.