Preliminary study on the molecular structure of 3'''' region of Duchenne muscular dystrophy (DMD) gene in Chinese

Number and order of HindⅢ exon-containing fragments (Hd) at 3' region of DMD gene were studied systematically using 16 partly-overlapping cDNA subprobes which were produced from dystrophin cDNA 9- 14 with each of 9 restriction endonudeases. There are 25 Hd fragments corresponding to cDNA 9 -14 in DMD gene. Since then, the exact length and the new order of Hd fragments are established. A new 2.1 kb fragment (Hd 55) is revealed, a 5.2 kb fragment (formely designated as Hd 59) is excluded and the existence of a controversial 3.2 kb fragment (Hd 64) is confirmed. Besides, three new exons were revealed by comparing the PvuⅡ and the XbaⅠ hybridization patterns with the Hindlll hybridization patterns for these cDNA subprobes. It is concluded that there are at least 66 Hd fragments, or 79 exons in DMD gene basing on the discovery of three additional exons. The corresponding relationship between the 66 Hd fragments and the SfiⅠ large scale physical map has been studied, and at least 17 Hd fragments or 19 exo     

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.     

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.     

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.     

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.     

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.     

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.     

Topography of the Duchenne muscular dystrophy (DMD) gene: FIGE and cDNA analysis of 194 cases reveals 115 deletions and 13 duplications.

We have studied 34 Becker and 160 Duchenne muscular dystrophy (DMD) patients with the dystrophin cDNA, using conventional blots and FIGE analysis. One hundred twenty-eight mutations (65%) were found, 115 deletions and 13 duplications, of which 106 deletions and 11 duplications could be precisely mapped in relation to both the mRNA and the major and minor mutation hot spots. Junction fragments, ideal markers for carrier detection, were found in 23 (17%) of the 128 cases. We identified eight new cDNA RFLPs within the DMD gene. With the use of cDNA probes we have completed the long-range map of the DMD gene, by the identification of a 680-kb SfiI fragment containing the gene's 3' end. The size of the DMD gene is now determined to be about 2.3 million basepairs. The combination of cDNA hybridizations with long-range analysis of deletion and duplication patients yields a global picture of the exon spacing within the dystrophin gene. The gene shows a large variability of intron size, ranging from only a few kilobases to 160-180 kb for the P20 intron.     

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。     

Gene deletions in X-linked muscular dystrophy

Of the approximately 170 families with X-linked muscular dystrophy of the Duchenne (DMD) and Becker (BMD) type in Finland, we have studied 90 unrelated patients for intragenic deletions by using the cDNA probes described by Koenig et al. Forty-five patients (50%) had molecular deletions of one or several of the 65 exon-containing HindIII fragments. In six deletion cases junction fragments of altered size were seen. Thirty-eight (84%) of the 45 deletions were detected using only two (1–2a and 8) of the six cDNA subclones. Using a wheelchair age of 12 years to distinguish between DMD and BMD, we found that the proportions of patients with deletions were similar. Deletions were equally common in familial and sporadic disease. BMD was more commonly caused by deletions in the 5' end of the gene than was DMD. In at least three instances deletions of similar type resulted in diseases of similar severity. Of 14 patients with mental retardation seven had deletions; six of these comprised exons contained in probe 8. We conclude that cDNA hybridization studies provide a powerful diagnostic tool in DMD and BMD and that they promise to produce better insights into molecular-clinical correlations.     

Localisation of the endpoints of deletions in the 5'' region of the Duchenne gene using a sequence isolated by chromosome jumping.

We have used chromosome jumping technology to move from within a large intron sequence in the Duchenne muscular dystrophy (DMD) gene to a region adjacent to exons of the gene. The single copy jump clone, HH1, was used to characterise deletions in patients previously shown to be deleted for DNA markers in the 5' end of the gene. 12 out of 15 such patients have breakpoints which lie between HH1 and the genomic locus J-47. Thus the vast majority of the deletions in these patients have proximal breakpoints in a similar region distal to the 5' end of the gene. HH1 was mapped with respect to the X;1 translocation in a DMD female and was shown to lie at least 80 kb from the starting point of the chromosome jump, HIP25.     

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.     

Duchenne and Becker muscular dystrophy mutations: analysis using 2.6 kb of muscle cDNA from the 5'' end of the gene.

We have isolated overlapping human fetal muscle cDNAs encompassing 2.6kb which are localised very close to the 5' end of the Duchenne muscular dystrophy (DMD) gene. Using DNA from patients with deletions of previously reported genomic probes, we have mapped the exons across the region. Investigation of deletions in both DMD and Becker muscular dystrophy (BMD) patients shows the deletions to be present in 10% of cases and heterogeneous.     

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.     

Deletions and a translocation interrupt a cloned gene at the neurofibromatosis type 1 locus.

Three new neurofibromatosis type 1 (NF1) mutations have been detected and characterized. Pulsed-field gel and Southern blot analyses reveal the mutations to be deletions of 190, 40, and 11 kb of DNA. The 11 kb deletion does not contain any of the previously characterized genes that lie between two NF1 translocation breakpoints, but it does include a portion of a rodent/human conserved DNA sequence previously shown to span one of the translocation breakpoints. By screening cDNA libraries with the conserved sequence, we identified a number of cDNA clones from the translocation breakpoint region (TBR), one of which hybridizes to an approximately 11 kb mRNA. The TBR gene crosses at least one of the chromosome 17 translocation breakpoints found in NF1 patients. Furthermore, the newly characterized NF1 deletions remove internal exons of the TBR gene. Although these mutations might act by compromising regulatory elements affecting some other gene, these findings strongly suggest that the TBR gene is the NF1 gene.