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.     

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.     

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 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.     

两个DMD cDNA片段在进行性肌营养不良症基因诊断中的应用

本文使用了缺失热点区的两个DMD cDNA片段1b-2a及8为探针检测Duc-henne型及Becker型肌营养不良(DMD/BMD)患者的基因缺失。在34例不相关患者中分别检测到5例及8例基因片段缺失,缺失检测率分别为14.7％及23.5％,总检出率为38.2％。结果表明,中国肌营养不良患者的基因缺失也不是随机分布的,主要集中于基因中心附近,其次在基因5′侧。     

Intragenic deletions in 21 Duchenne muscular dystrophy (DMD)/Becker muscular dystrophy (BMD) families studied with the dystrophin cDNA: location of breakpoints on HindIII and BglII exon-containing fragment maps, meiotic and mitotic origin of the mutations.

Following the strategy outlined in an accompanying paper, we studied 32 X-linked muscular dystrophy families (29 Duchenne [DMD] and three Becker [BMD] type) for abnormalities of HindIII and BglII fragments detected by the entire dystrophin cDNA. Twenty-one different single-intragenic deletions, and no duplications, were identified. The deletion endpoints were precisely mapped on the published HindIII fragment map. Detailed analysis of overlapping deletions led to clarification of the fragment order for some previously unsettled regions of the HindIII map and to the construction of a partial map of exon-containing BglII fragments. For the regions involved in deletions, the corresponding HindIII and BglIII fragments could be identified. Noncontiguous comigrating fragments were detected in two regions by careful analysis of the patterns in deletion patients. Four of the 21 deletions generated novel restriction fragments that facilitated detection of female carriers in these families. Twelve of the deletions had a breakpoint in one of the two large introns known to be the sites of breakpoint clusters. By combining deletions and RFLP analyses, we unequivocally identified the gamete that first carried the mutation in 13 families: eight oocytes and five sperm. Germ-line mosaicism previously detected in one male was confirmed by cDNA studies. In two additional families gonadal mosaicism was found in females. As evidence is accumulating for frequent mitotic origin of these deletion mutations, this phenomenon has to be considered when postulating mutational mechanisms and in genetic counseling of DMD/BMD families.     

Detection of Duchenne muscular dystrophy carriers by dosage analysis using the DMD cDNA clone 8

Deletion screening in 11 unrelated DMD patients has been performed using DMD cDNA clones 1-8. Of these 11 patients, 6 exhibit deletions of the cDNA clone 8. The carriership of 18 female relatives from these six DMD families has been investigated by dosage analysis. It is shown that dosage analysis is an available method to determine the carrier status of the female relatives of DMD patients showing a deletion within a DMD cDNA clone.     

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.     

RFLPs for Duchenne muscular dystrophy cDNA clones 9 and 10.

The complete 14-kb cDNA for the gene causing the X-linked recessive muscular dystrophy (MD) type Duchenne (DMD) and Becker (BMD) has recently been cloned and made available for deletion/duplication screening in patients. It detects 65 exon-containing nonpolymorphic HindIII fragments spread over a gene locus of about 2,000 kb. When the entire DMD cDNA is used, deletions/duplications can be found in about 65%-70% of affected patients, permitting direct carrier detection by densitometric scanning. But in cases where no deletion/duplication is detectable, RFLP analysis, specially favored within the gene, will be the method for carrier-status determination. Clones 9 and 10-1.2-kb and 0.7-kb fragments, respectively, of the 14-kb DMD cDNA--have been hybridized with human genomic DNA digested by nine different restriction enzymes. Five RFLPs, involving Asp700, PvuII, XbaI, and EcoRV sites, were detected, and Mendelian inheritance could be demonstrated. Since clones 9 and 10 are localized telomeric to the mutation-hot-spot region, their polymorphisms are thought to be very helpful as flanking markers for indirect carrier detection in families with a family history of DMD/BMD. Moreover, these RFLPs can be used for direct carrier detection or exclusion in families with patients showing a deletion/duplication in the region of p9 or p10.     

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.     

Deletion mutations in Duchenne muscular dystrophy (DMD) in Western Saudi children

Duchenne and Becker muscular dystrophy (DMD and BMD) are caused, in the majority of cases, by deletions in the dystrophin gene (DMD). The disease is an X-linked neuromuscular diseases typically caused by disrupting (DMD) or non-disrupting (BMD) the reading frame in the dystrophin (DMD) gene. In the present study, amplifications of the genomic DNAs of unrelated 15 Saudi DMD males were carried out using multiplex polymerase chain reaction (PCR) for nine-hotspot regions of exons 4, 8, 12, 17, 19, 44, 45, 48 and 51. We detected six Saudi patients having deletions in a frequency of 40%. The frequency of deletions in exon 51 (20%) was the most common deletion frequently associated with our Saudi sample males. Exons 19, 45, and 48 were present in a frequency of 6.7% each. All deletions were recognized as an individual exonic deletions, while no gross deletion where detected. Finally, the molecular deletions in the Saudi males was expected to be characterized by a moderate frequency among different populations due to the geographical KSA region, which it is in the crossroad of intense migrations and admixture of people coming from continental Asia, Africa, and even Europe. In conclusion, attempts to include an extra DNA samples might reflect a valid vision of the deletions within the high frequency deletion regions (HFDR’s) in the DMD gene mutations in KSA.     

Molecular genetic analysis of 67 patients with duchenne/becker muscular dystrophy

Summary A total of 56 Duchenne muscular dystrophy (DMD) patients and 11 Becker muscular dystrophy (BMD) patients was analyzed by extended multiplex amplification of the DMD/BMD gene; deletions were found in 60% of these patients. The data obtained were used to test the frameshift hypothesis and to compare the distribution of familial versus isolated cases. A significant correlation was found between deletions and isolated cases. Additional experiments were performed in order to determine the deletion breakpoints more precisely. These data are a prerequisite for carrier analysis in the respective families by detection or exclusion of aberrant cDNA fragments derived from ectopic lymphocyte RNA. This diagnostic technique is illustrated by 5 examples.     

DNA deletions in mild and severe Becker muscular dystrophy

Summary The DNA of 33 patients diagnosed as suffering from Becker muscular dystrophy (BMD) has been probed with cloned DNA sequences from Xp21, known to reveal DNA deletions in patients suffering from the more severe Duchenne muscular dystrophy (DMD). Two BMD cases showed clear deletions. A third case gave aberrant band sizes, which further analysis showed to be caused by a small deletion. This suggests that deletions in DXS164 occur approximately as frequently in BMD as they do in DMD. Of the two cases showing large deletions, one is at the severe end of the Becker clinical spectrum, whilst the other is a classical Becker-type dystrophy. The fact that loci defined by probes commonly deleted in classical DMD patients are also deleted in BMD patients of varying severity is strong additional evidence that these disorders are allelic, and further justifies the use of probes with defined linkage relationships to DMD also being used for counselling in BMD families.     

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.     

Improved Detection of Deletions and Duplications in the DMD Gene Using the Multiplex Ligation-Dependent Probe Amplification (MLPA) Method

The multiplex ligation-dependent probe amplification (MLPA) assay is the most powerful tool in screening for deletions and duplications in the dystrophin gene in patients with Duchenne and Becker muscular dystrophy (DMD/BMD). The efficacy of the assay was validated by testing 20 unrelated male patients with DMD/BMD who had already been screened by multiplex PCR (mPCR). We detected two duplications that had been missed by mPCR. In one DMD patient showing an ambiguous MLPA result, a novel mutation (c.3808_3809insG) was identified. MLPA improved the mutation detection rate of mPCR by 15 %. The results of our study (1) confirmed MLPA to be the method of choice for detecting DMD gene rearrangements in DMD/BMD patients, (2) showed that ambiguous MLPA amplification products should be verified by other methods, and (3) indicated that the MLPA method could be used in screening even for small mutations located in the probe-binding regions.     

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.     

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.     

A deletion hot spot in the Duchenne muscular dystrophy gene

We have made a detailed study of a deletion hot spot in the distal half of the Duchenne muscular dystrophy (DMD) gene, using intragenic probe P20 (DXS269), isolated by a hybrid cell-mediated cloning procedure. P20 detects 16% deletions in patients suffering from either DMD or Becker muscular dystrophy (BMD), in sharp contrast to the adjacent intragenic markers JBir (7%) and J66 (less than 1%), mapping respectively 200-320 kb proximal and 380-500 kb distal to P20. Of the P20 deletions, 30% start within a region of 25-40 kb, the majority extending distally. P20 was confirmed to map internal to a distal intron of the DMD gene. This region was recently shown by both cDNA analysis (M. Koenig et al., 1987; Cell 50: 509-517), and field inversion electrophoresis studies (J.T. Den Dunnen et al., 1987, Nature (London) 329: 640-642) to be specifically prone to deletions. In addition, P20 detects MspI and EcoRV RFLPs, informative in 48% of the carrier females. Together, these properties make P20 useful for carrier detection, prenatal diagnosis, and the study of deletion induction in both DMD and BMD.     

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.     

DMD Mutations in 576 Dystrophinopathy Families: A Step Forward in Genotype-Phenotype Correlations

Recent advances in molecular therapies for Duchenne muscular dystrophy (DMD) require precise genetic diagnosis because most therapeutic strategies are mutation-specific. To understand more about the genotype-phenotype correlations of the DMD gene we performed a comprehensive analysis of the DMD mutational spectrum in a large series of families. Here we provide the clinical, pathological and genetic features of 576 dystrophinopathy patients. DMD gene analysis was performed using the MLPA technique and whole gene sequencing in blood DNA and muscle cDNA. The impact of the DNA variants on mRNA splicing and protein functionality was evaluated by in silico analysis using computational algorithms. DMD mutations were detected in 576 unrelated dystrophinopathy families by combining the analysis of exonic copies and the analysis of small mutations. We found that 471 of these mutations were large intragenic rearrangements. Of these, 406 (70.5%) were exonic deletions, 64 (11.1%) were exonic duplications, and one was a deletion/duplication complex rearrangement (0.2%). Small mutations were identified in 105 cases (18.2%), most being nonsense/frameshift types (75.2%). Mutations in splice sites, however, were relatively frequent (20%). In total, 276 mutations were identified, 85 of which have not been previously described. The diagnostic algorithm used proved to be accurate for the molecular diagnosis of dystrophinopathies. The reading frame rule was fulfilled in 90.4% of DMD patients and in 82.4% of Becker muscular dystrophy patients (BMD), with significant differences between the mutation types. We found that 58% of DMD patients would be included in single exon-exon skipping trials, 63% from strategies directed against multiexon-skipping exons 45 to 55, and 14% from PTC therapy. A detailed analysis of missense mutations provided valuable information about their impact on the protein structure.