Sequences of junction fragments in the deletion-prone region of the dystrophin gene

The Duchenne muscular dystrophy locus is remarkable in that it shows a high mutation rate and the majority of mutations found are deletions. These deletions are generated as meiotic as well as mitotic events and occur preferentially in the central region of the gene. Nothing is known so far about the mechanisms involved. This paper reports the first sequencing of deletion junctions in the dystrophin gene. The data from a study of two patients with deletions in the central region of dystrophin show the breakpoints to lie in regions of introns in which stretches of dA-dT are seen. The relationship between these observations and possible mechanisms for the mutations is discussed.     

A transposon-like element in the deletion-prone region of the dystrophin gene.

The central portion of the dystrophin gene locus is a preferential site for deletions causing progressive muscular dystrophy of the Duchenne type (DMD). The nucleotide sequence of a deletion junction fragment from a DMD patient was determined, revealing that the proximal breakpoint of the deletion in intron 43 fell within the sequence of a transposon-like element. This segment, belonging to the THE-1 family of human transposable elements, is normally present in a complete form in intron 43 of the dystrophin gene. The deletion mutation was maternally transmitted and eliminated two-thirds of the THE-1 element. Analysis of DNA from additional DMD patients revealed a second deletion with the proximal breakpoint mapping within the same THE-1 element.     

An unusual extended DNA loop attachment region is located in the human dystrophin gene

We have found and characterized an unusual extended area of DNA association with the nuclear matrix in the human dystrophin gene. This extended DNA loop anchorage region (LAR) has been mapped and characterized using a variety of biochemical and microscopy techniques. It spans approximately 200 kbp at chromosomal locations 950-1,150 Kb downstream to the beginning of the first exon of the dystrophin gene Dp427m and covers a part of the intron 43, exon 44, and most of intron 44. The extended LAR harbors the major recombination hot spot of the dystrophin gene and a replication origin. We propose a model where DNA topoisomerase II-mediated cleavage at the nuclear matrix may enhance recombination events within this extended LAR.     

Exon edited dystrophin rods in the hinge 3 region

We have studied the properties of a panel of proteins engineered to be end-products of envisioned exon skipping therapy by antisense oligonucleotides, AONs, directed at exon 51 applied to relevant dystrophin defects causing Duchenne muscular dystrophy, DMD. Exon skipping therapy is a leading therapeutic strategy being investigated for the treatment of this devastating genetic disease. AONs targeting exon 51 have progressed furthest in human clinical trials. Exon 51 skipping is applicable to a variety of dystrophin defects found in different patients. Due to the differences in original defect, the end result of the therapy will be different in each case. An open question is whether these differences will produce significant differences in the dystrophin protein so edited. In this study we have identified differences in the stability, structure and lipid binding properties of these end-product proteins produced by exon 51 skipping repair.     

Rapid direct sequence analysis of the dystrophin gene

Mutations in the dystrophin gene result in both Duchenne and Becker muscular dystrophy (DMD and BMD), as well as X-linked dilated cardiomyopathy. Mutational analysis is complicated by the large size of the gene, which consists of 79 exons and 8 promoters spread over 2.2 million base pairs of genomic DNA. Deletions of one or more exons account for 55%-65% of cases of DMD and BMD, and a multiplex polymerase chain reaction method-currently the most widely available method of mutational analysis-detects approximately 98% of deletions. Detection of point mutations and small subexonic rearrangements has remained challenging. We report the development of a method that allows direct sequence analysis of the dystrophin gene in a rapid, accurate, and economical fashion. This same method, termed "SCAIP" (single condition amplification/internal primer) sequencing, is applicable to other genes and should allow the development of widely available assays for any number of large, multiexon genes.     

Spectrum of small mutations in the dystrophin coding region.

Duchenne and Becker muscular dystrophies (DMD and BMD) are caused by defects in the dystrophin gene. About two-thirds of the affected patients have large deletions or duplications, which occur in the 5' and central portion of the gene. The nondeletion/duplication cases are most likely the result of smaller mutations that cannot be identified by current diagnostic screening strategies. We screened approximately 80% of the dystrophin coding sequence for small mutations in 158 patients without deletions or duplications and identified 29 mutations. The study indicates that many of the DMD and the majority of the BMD small mutations lie in noncoding regions of the gene. All of the mutations identified were unique to single patients, and most of the mutations resulted in protein truncation. We did not find a clustering of small mutations similar to the deletion distribution but found > 40% of the small mutations 3' of exon 55. The extent of protein truncation caused by the 3' mutations did not determine the phenotype, since even the exon 76 nonsense mutation resulted in the severe DMD phenotype. Our study confirms that the dystrophin gene is subject to a high rate of mutation in CpG sequences. As a consequence of not finding any hotspots or prevalent small mutations, we conclude that it is presently not possible to perform direct carrier and prenatal diagnostics for many families without deletions or duplications.     

Mapping dystrophin gene recombinants in Greek DMD/BMD families: low recombination frequencies in the STR region

A systematic study of 42 Greek DMD/BMD families using 14 polymorphic markers that span the dystrophin gene was performed in order to assess the position and frequency of recombinants in the Greek population and to test whether hot spots of recombination and deletions coincide when exclusively studying DMD/BMD families. We report a low percentage of recombination between markers STR44 and STR50; otherwise, the distribution of recombination events in other parts of the gene is largely in agreement with previously published data on Centre d'Etude du Polymorphisme Humaine families. We therefore propose that recombination frequencies and the correlation between recombination and deletion hot spots should be evaluated on DMD/BMD families exclusively.     

Identification of a mutation in the promoter region of the dystrophin gene in a patient with atypical Becker muscular dystrophy

Summary We have identified 7 patients with Becker muscular dystrophy (BMD) in whom analysis of dystrophin by immunoblotting shows a full-sized molecule produced at reduced abundance compared with controls. They have no detectable deletion in their dystrophin cDNA. One patient presented atypically with unusually severe cramps as his only symptom for 25 years. These patients were investigated using the polymerase chain reaction (PCR) with 3 sets of primers within the promoter region of the dystrophin gene, followed by dot blot and restriction analysis. In the patient with the atypical history, one of the expected fragments on PCR failed to amplify. A large deletion was excluded by the finding of normally sized fragments on amplification with the other primer sets. The mutation was localised to the 3 end of the forward primer binding site by dot blot and restriction analysis. This result supports the hypothesis that, in patients with a full-sized dystrophin molecule produced at reduced abundance, the phenotype may result from a mutation in the promoter region of the dystrophin gene. The atypical history of the patient in whom this was detected adds to the variety of phenotypes now known to exist as BMD.     

Rapid detection of CA polymorphisms in cloned DNA: application to the 5'' region of the dystrophin gene.

To identify CA repeats in genomic sequences which had been previously subcloned into plasmids, we performed PCR using a (CA)n primer and a flanking vector primer on the genomic inserts. By incorporation of a restriction enzyme site into the (CA)n primer, we have been able to subclone the genomic DNA so that the sequence flanking the CA repeat is readily determined. Primers can then be designed to amplify across the CA repeat in patient DNA samples. Application of this technique to genomic DNAs surrounding the upstream "brain" promoter of the dystrophin gene has led to the discovery of four new CA repeats. Three of these repeats are highly polymorphic, with PICs ranging from .586 to .768. The location of these markers at the extreme 5' terminus of the dystrophin gene, together with their high degree of polymorphism and ease of assay, makes them ideal for linkage analysis in families with Duchenne muscular dystrophy.     

Polymorphisms of three new microsatellite sites of the dystrophin gene

To look for novel microsatellites in the dystrophin gene for the diagnosis of Duchenne muscular dystrophy, candidate microsatellite sites in the dystrophin gene were analyzed with the SSRHunter software and were also genotyped. Among the 15 candidate microsatellite sites, three novel microsatellite sites in the 60th, 30th, and 2nd intron were found to have a high degree of polymorphism. We submitted these three new loci to the European Molecular Biology Laboratory, under accession Nos. FN547040, FN547041 and FN557526, which were called DXSDMD-in60, DXSDMD-in30 and DXSDMD-in2, respectively. In these three loci, we found 9, 6 and 11 alleles, respectively, in the 205 individuals. In addition, we also detected 20, 19 and 20 genotypes for the three loci in female samples, with a polymorphism information content of more than 0.600. In conclusion, the three microsatellite sites in the intron region of the dystrophin gene have a high degree of polymorphism, and they can be used in population genetics, as well as to provide a theoretical basis for genetic diagnosis and elucidation of molecular mechanisms in Duchenne muscular dystrophy.     

A PvuII polymorphism near exon 2 of the dystrophin gene

We report a PvuII polymorphism near exon 2 of the dystrophin gene with a heterozygosity frequency of 0.5.     

Comparative analysis of the human dystrophin and utrophin gene structures

We present analysis of intronic sequences in the human DMD and UTRN genes. In both genes accumulation of repeated elements could account for intron expansion. Out-of-frame rod-domain exons have stronger splice sites and are separated by significantly longer introns as compared to in-frame exons. These features are unique for the two homologs and not shared by other spectrin superfamily genes.     

Detection of an exon 53 polymorphism in the dystrophin gene

We utilized a heteroduplex method to screen for small mutations in Duchenne muscular dystrophy patients who did not have deletions or duplications. A dystrophin exon 53 heteroduplex band was identified in 14.4% of the affected patients. Direct sequencing of the amplified product from DNA producing the heteroduplex revealed the presence of a polymorphism in the coding region. The codon for asparagine was converted from AAT to AAC.     

A polymorphic STS in intron 44 of the dystrophin gene

A 300-bp EcoRV polymorphism, detected with P20 (DXS269) in intron 44 of the human dystrophin gene, is due to an insertion or deletion. To make this restriction fragment length polymorphism (RFLP) available for polymerase chain reaction (PCR) analysis, we sequenced both alleles of this polymorphism and synthesized primers flanking the mutation site. The origin of the mutation is a single Alu repeat insertion. The 300-bp polymorphism can now be successfully detected by PCR and provides an excellent tool to detect female carriers in this deletion prone region of the dystrophin gene.     

Comparative mapping of the DiGeorge syndrome region in mouse shows inconsistent gene order and differential degree of gene conservation

We have constructed a comparative map in mouse of the critical region of human 22q11 deleted in DiGeorge (DGS) and Velocardiofacial (VCFS) syndromes. The map includes 11 genes potentially haploinsufficient in these deletion syndromes. We have localized all the conserved genes to mouse Chromosome (Chr) 16, bands B1-B3. The determination of gene order shows the presence of two regions (distal and proximal), containing two groups of conserved genes. The gene order in the two regions is not completely conserved; only in the proximal group is the gene order identical to human. In the distal group the gene order is inverted. These two regions are separated by a DNA segment containing at least one gene which, in the human DGS region, is the most proximal of the known deleted genes. In addition, the gene order within the distal group of genes is inverted relative to the human gene order. Furthermore, a clathrin heavy chain-like gene was not found in the mouse genome by DNA hybridization, indicating that there is an inconsistent level of gene conservation in the region. These and other independent data obtained in our laboratory clearly show a complex evolutionary history of the DGS-VCFS region. Our data provide a framework for the development of a mouse model for the 22q11 deletion with chromosome engineering technologies. Received: 8 July 1997 / Accepted 11 August 1997