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.     

Mapping of Xp21 translocation breakpoints in and around the DMD gene by pulsed field gel electrophoresis

Balanced translocations with a breakpoint in the Xp21 region are likely to disrupt the giant Duchenne muscular dystrophy (DMD) locus and can be demonstrated in females suffering from the disease. Pulsed field gel electrophoresis allows the positioning of these breakpoints by detecting junction fragments on the derived chromosomes; DNA probes hybridizing to these fragments may be located as many as several hundred kilobases away from the breakpoints. By using this approach, 11 translocation breakpoints from the Xp21 region have been analyzed. The localization of three previously examined breakpoints was confirmed. Six other breakpoints, including a breakpoint flanking the DMD gene and not associated with the DMD phenotype, could be positioned relative to SfiI sites on a 3.5-Mb restriction map of the region.     

Mapping of four translocation breakpoints within the Duchenne muscular dystrophy gene

There are over 20 females with Duchenne or Becker muscular dystrophy (DMD or BMD) who have X-autosome translocations that break the X chromosome within band Xp21. Several of these translocations have been mapped with genomic probes to regions throughout the large (approximately 2000 kb) DMD gene. In this report, a cDNA clone from the 5' end of the gene was used to further map the breakpoints in four X-autosome translocations. A t(X;21) translocation in a patient with BMD and a t(X;1) translocation in a patient with DMD were found to break within a large 110-kb intron between exons 7 and 8. Two other DMD translocations, t(X;5) and t(X;11), were found to break between the first and the second exon of the gene within a presumably large intron (greater than 100 kb). These results demonstrate that all four translocations have disrupted the DMD gene and make it possible to clone and sequence the breakpoints. This will in turn determine whether these translocations occur by chance in these large introns or whether there are sequences that predispose to translocations.     

Sequence analysis of the breakpoint regions of an X;5 translocation in a female with Duchenne muscular dystrophy.

X;autosome translocations in females with Duchenne muscular dystrophy (DMD) provide an opportunity to study the mechanisms responsible for chromosomal rearrangements that occur in the germ line. We describe here a detailed molecular analysis of the translocation breakpoints of an X;autosome reciprocal translocation, t(X;5)(p21;q31.1), in a female with DMD. Cosmid clones that contained the X-chromosome breakpoint region were identified, and subclones that hybridized to the translocation junction fragment in restriction digests of the patient's DNA were isolated and sequenced. Primers designed from the X-chromosomal sequence were used to obtain the junction fragments on the der(X) and the der(5) by inverse PCR. The resultant clones were also cloned and sequenced, and this information used to isolate the chromosome 5 breakpoint region. Comparison of the DNA sequences of the junction fragments with those of the breakpoint regions on chromosomes X and 5 revealed that the translocation arose by nonhomologous recombination with an imprecise reciprocal exchange. Four and six base pairs of unknown origin are inserted at the exchange points of the der(X) and der(5), respectively, and three nucleotides are deleted from the X-chromosome sequence. Two features were found that may have played a role in the generation of the translocation. These were (1) a repeat motif with an internal homopyrimidine stretch 10 bp upstream from the X-chromosome breakpoint and (2) a 9-bp sequence of 78% homology located near the breakpoints on chromosomes 5 and X.     

Characterization of patients with glycerol kinase deficiency utilizing cDNA probes for the Duchenne muscular dystrophy locus

Summary Genomic DNA from five previously unreported patients with glycerol kinase deficiency (GKD), dystrophic myopathy, and adrenal insufficiency were studied with genomic probes and cDNA probes for the Duchenne muscular dystrophy (DMD) locus. These individuals, together with those reported by ourselves and others, show that patients with a contigous gene syndrome involving the DMD, GK, and adrenal hypoplasia congenita (AHC) loci have a broader distribution of microdeletion breakpoints than those observed among patients with classical DMD. This study demonstrates the use of the DMD cDNA probes to delineate the centromeric deletion breakpoints for patients with Xp21 microdeletions extending beyond the DMD locus. It also shows the practical diagnostic application of the DMD cDNA probes when the diagnosis of GKD is entertained in a patient with known DMD and only DNA is available for study.     

Common sequence motifs at the rearrangement sites of a constitutional X/autosome translocation and associated deletion.

Reciprocal chromosome translocations are common de novo rearrangements that occur randomly throughout the human genome. To learn about causative mechanisms, we have cloned and sequenced the breakpoints of a cytologically balanced constitutional reciprocal translocation, t(X;4)(p21.2;q31.22), present in a girl with Duchenne muscular dystrophy (DMD). Physical mapping of the derivative chromosomes, after their separation in somatic cell hybrids, reveals that the translocation disrupts the DMD gene in Xp21 within the 18-kb intron 16. Restriction mapping and sequencing of clones that span both translocation breakpoints as well as the corresponding normal regions indicate the loss of approximately 5 kb in the formation of the derivative X chromosome, with 4-6 bp deleted from chromosome 4. RFLP and Southern analyses indicate that the de novo translocation is a paternal origin and that the father's X chromosome contains the DNA that is deleted in the derivative X. Most likely, deletion and translation arose simultaneously from a complex rearrangement event that involves three chromosomal breakpoints. Short regions of sequence homology were present at the three sites. A 5-bp sequence, GGAAT, found exactly at the translocation breakpoints on both normal chromosomes X and 4, has been preserved only on the der(4) chromosome. It is likely that the X-derived sequence GGAATCA has been lost in the formation of the der(X) chromosome, as it matches an inverted GAATCA sequence present on the opposite strand exactly at the other end of the deleted 5-kb fragment. These findings suggest a possible mechanism which may have juxtaposed the three sites and mediated sequence-specific breakage and recombination between nonhomologous chromosomes in male meiosis.     

Human X chromosome markers and Duchenne muscular dystrophy.

Two DNA markers, a random DNA fragment 754 and the cDNA sequence encoding the gene for ornithine transcarbamylase (OTC) have been studied in kindreds segregating for Duchenne muscular dystrophy. 754 and OTC are located close physically to the mutation in the region Xp21 below the breakpoints in two Duchenne females. The genetic distance was found to be approximately 10cM between 754 and DMD (two crossovers in 26 meioses) and to be approximately 10cM between OTC and DMD (two crossovers in 26 meioses). Physical data suggest the order DMD-754-OTC. The frequency of recombination compared to physical distance between these markers and DMD suggests that there may be a hot spot of recombination. The relevance of these observations for the isolation of the DMD mutation and clinical use of these probes is discussed.     

A physical map of 4 million bp around the Duchenne muscular dystrophy gene on the human X-chromosome

Employing pulsed field gradient electrophoresis, we constructed a 4.5 million bp (Mb) Sfil restriction map of the human X-chromosomal region p21, harboring genes for Duchenne (DMD) and Becker Muscular Dystrophy. In a DMD patient with additional chronic granulomatosis and retinitis pigmentosa, the proximal 3.5 Mb is deleted. Another DMD patient, with additional glycerol kinase deficiency and adrenal hypoplasia, lacks at least 3.3 Mb in the middle region, including marker C7 but not B24, placing C7 closer to DMD. Another DMD patient has a partial pERT-87 deletion of minimally 140 kb. Truncated Sfil fragments in a female X:21 translocation patient place the junction probe XJ1.1 115 kb from the distal end of the normal fragment. Probe pERT-84 maps to the same fragment, within 750 kb of XJ1.1.     

Isolation of a conserved sequence deleted in Duchenne muscular dystrophy patients.

We have isolated a DNA sequence (HIP25) by subtraction- hybridisation which is deleted in a number of Duchenne muscular dystrophy (DMD) patients. HIP25 is conserved in evolution and hybridises to human fetal and adult muscle mRNA. HIP25 is absent in human fetal fibroblast mRNA. Physical mapping data localise this sequence within Xp21 between the breakpoints of X;autosome translocations found in two females suffering from the disease. HIP25 is a candidate exon sequence for the basic defect in DMD boys deleted at this locus.     

Molecular analysis of the Duchenne muscular dystrophy region using pulsed field gel electrophoresis

Genetic and molecular studies show that the Duchenne muscular dystrophy (DMD) locus at Xp21 is large and complex. We have analyzed this region using pulsed field gel electrophoresis (PFGE) and have determined physical distances between Xp21 probes. The sum of the sizes of the Sfil restriction fragments detected by these probes is greater than 4000 kb. The deletion endpoints in two DMD patients were detected by observing changes in these restriction fragments. In addition, the Xp21 breakpoint for the X;1 translocation in an affected female was mapped. These results demonstrate the applicability of PFGE for analysis of Xp21, and should facilitate the mapping of other translocations and deletions in this region, some of which lead to glycerol kinase deficiency and adrenal hypoplasia as well as DMD.     

Germinal mosaicism from grand-paternal origin in a family with Duchenne muscular dystrophy

Summary We have identified a Duchenne muscular dystrophy (DMD) pedigree with an unexpected pattern of inheritance. Using marker restriction fragment length polymorphisms detected by probes that lie within and outside the DMD gene, we could demonstrate that the maternal grandfather has transmitted two distinct types of X chromosomes to his offspring. This original observation may be explained by postulating that the DMD mutation must have occurred during mitosis in early germline proliferation, leading to a germline mosaicism within this male ancestor.     

Localization of DNA sequences to a region within Xp11.21 between incontinentia pigmenti (IP1) X-chromosomal translocation breakpoints.

Incontinentia pigmenti (IP) is an X-linked dominant disorder characterized by developmental anomalies of the tissues and organs derived from embryonic ectoderm and neuroectoderm. An IP locus, designated IP1, probably resides in Xp11.21, since five unrelated patients with nonfamilial IP have been identified who possess constitutional de novo reciprocal X;autosome translocations involving Xp11.21. We have used a series of somatic cell hybrids containing the rearranged chromosomes derived from three of the five IP1 patients, along with other hybrid cell lines, to map probes in the vicinity of the IP1 locus. Five anonymous DNA loci--DXS422, DXS14, DXS343, DXS429, and DXS370--have been mapped to a region within Xp11.21, between two IP1 X-chromosomal translocation breakpoints; the IP1 t(X;17) breakpoint is proximal (centromeric) to this region, and the IP1 t(X;13) and t(X;9) X-chromosomal breakpoints lie distal to it. While no IP1 translocation breakpoint has yet been identified by pulsed-field gel electrophoretic (PFGE) analysis, an overlap between three probes--p58-1, 7PSH3.5, and cpX210--has been detected, placing these probes within 125 kb. Four probes--p58-1, 7PSH3.5, cpX210, and 30CE2.8--have been helpful in constructing a 1,250-kb PFGE map of the region between the breakpoints; these results suggest that the IP1 X-chromosomal translocation breakpoints are separated by at least this distance. The combined somatic cell hybrid and PFGE analyses we report here favor the probe order DXS323-(IP1 t(X;13), IP1, t(X;9]-(DXS422, DXS14, DXS343, DXS429, DXS370)-(IP1 t(X;17), DXZ1). These sequences provide a starting point for identifying overlapping genomic sequences that span the IP1 translocation breakpoints; the availability of IP1 translocation breakpoints should now assist the cloning of this locus.     

High resolution deletion breakpoint mapping in the DMD gene by whole cosmid hybridization.

The locus DXS269 (P20) defines a deletion hotspot in the distal part of the Duchenne Muscular Dystrophy gene. We have cloned over 90 kilobase-pairs of genomic DNA from this region in overlapping cosmids. The use of whole cosmids as probes in a competitive DNA hybridization analysis proves a fast and convenient method for identifying rearrangements in this region. A rapid survey of P20-deletion patients is carried out to elucidate the nature of the propensity to deletions in this region. Using this technique, deletion breakpoints are pinpointed to individual restriction fragments in patient DNAs without the need for tedious isolation of single copy sequences. Simultaneously, the deletion data yield a consistent restriction map of the region and permit detection of several RFLPs. A 176 bp exon was identified within the cloned DNA, located 3' of an intron exceeding 150 Kb in length. Its deletion causes a frameshift in the dystrophin reading frame and produces the DMD phenotype. This exon is one of the most frequently deleted exons in BMD/DMD patients and its sequence is applied in a pilot study for diagnostic deletion screening using Polymerase Chain Reaction amplification.     

Insights into extensive deletions around the XK locus associated with McLeod phenotype and characterization of two novel cases

The McLeod phenotype is derived from various forms of XK gene defects that result in the absence of XK protein, and is defined hematologically by the absence of Kx antigen, weakening of Kell system antigens, and red cell acanthocytosis. Individuals with the McLeod phenotype usually develop late-onset neuromuscular abnormalities known as the McLeod syndrome (MLS). MLS is an X-linked multi-system disorder caused by absence of XK alone, or when the disorder is caused by large deletions, it may be accompanied with Duchenne muscular dystrophy (DMD), chronic granulomatous disease (CYBB), retinitis pigmentosa (RPGR), and ornithine transcarbamylase deficiency (OTC). XK defects derived from a large deletion at the XK locus (Xp21.1) have not been characterized at the molecular level. In this study, the deletion breakpoints of two novel cases of McLeod phenotype with extensive deletions are reported. Case 1 has greater than 1.12 million base-pairs (mb) deletion around the XK locus with 7 genes affected. Case 2 has greater than 5.65 mb deletion from TCTE1L to DMD encompassing 20 genes. Phylogenetic analyses demonstrated that DMD, XK and CYBB have close paralogs, some of which may partially substitute for the functions of their counterparts. The loci around XK are highly conserved from fish to human; however, the disorders are probably specific to mammals, and may coincide with the translocation of the loci to the X chromosome after the speciation in birds. The non-synonymous to synonymous nucleotide substitution rate ratio (omega=dN/dS) in these genes was examined. CYBB and RPGR show evidence of positive selection, whereas DMD, XK and OTC are subject to selective constraint.     

Mapping of X chromosome translocation breakpoints in females with Duchenne muscular dystrophy with respect to exons of the dystrophin gene

There are rare female patients who suffer from Duchenne or Becker muscular dystrophy because they carry an X;autosome translocation with a breakpoint in the dystrophin gene. We have defined the positions of seven of these breakpoints with respect to exon-containing HindIII fragments detected by dystrophin cDNA. One breakpoint lies between exon-containing Hindlll fragments 7 and 8, five breakpoints between exon-containing HindIII fragments 31 to 41, and one lies close to exon-containing-HindIII fragment 50. The distribution of these and of a further seven translocation breakpoints whose positions are known is compared with that reported for deletions and duplications in affected males.     

Familial inheritance of a DXS164 deletion mutation from a heterozygous female.

Restriction-fragment-length-polymorphism analysis was used to examine a female who is segregating for Duchenne muscular dystrophy (DMD) and a deletion of the DXS164 region of the X chromosome. The segregating female has no prior family history of DMD, and she has two copies of the DXS164 region in her peripheral blood lymphocytes. The following two hypotheses are proposed to explain the coincidence of the DMD phenotype and deletion of the DXS164 region in her offspring: (1) she may be a gonadal mosaic for cells with two normal X chromosomes and cells with one normal X chromosome and an X chromosome with a deletion of the DXS164 region; and (2) she may carry a familial X;autosome translocation in which the DXS164 region is deleted from one X chromosome and translocated to an autosome. The segregation of DMD and the DXS164 deletion in this family illustrates the importance of extended pedigree analysis when DXS164 deletions are used to identify female carriers of the DMD gene.     

A new type of nonhomologous synapsis in T(X;4)1R1 translocation male mice

The synaptonemal complexes of T(X;4)1R1 (abbreviated R1) translocation heterozygotes have been examined by electron microscopy and compared with those of two X-7 translocations: R5 and R6. The X chromosome breakpoint of R1 is estimated to lie between 78 and 82% from the proximal end of the X, in the same general region as the R5 and R6 breakpoints. The position of the autosomal breakpoint of R1, like that of R6, is about 30% from the proximal end of the respective autosome. R1 is also similar to R6 in that there is extensive nonhomologous synapsis both in quadrivalents and heteromorphic bivalents. We have recently found that the location of breakpoints with respect to the position of the G-bands appears to be related to the synaptic behavior seen in translocation heterozygotes. If both breaks of a reciprocal translocation lie in G-light bands, as was the case with R5, synapsis is confined to homology. However, if one break lies in or immediately adjacent to a G-dark band, there is nonhomologous synapsis, as occurs with R1 and R6. Comparison of the synaptic behavior of R1 with R5 and R6 leads to the conclusion that this G-band-related nonhomologous synapsis is of a different type than the "synaptic adjustment" phenomenon that has been described by Moses (1977a). This G-band-related nonhomologous synapsis is not substage-specific, but competes with homologous synapsis during zygotene-early pachytene.     

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.     

Direct deletion analysis in two Duchenne muscular dystrophy symptomatic females using polymorphic dinucleotide (CA)n loci within the dystrophin gene

Duchenne muscular dystrophy (DMD) is the most common hereditary neuromuscular disease. It is inherited as an X-linked recessive trait in which males show clinical manifestations. In some rare cases, the disease can also be manifested in females. The aim of the present study was to determine the molecular alteration in two cases of nonrelated DMD symptomatic carriers with no previous history of DMD. Multiplex PCR is commonly used to search for deletion in the DMD gene of affected males. This method could not be used in females because the normal X chromosome masks the deletion of the mutated one. Therefore, we used a set of seven highly polymorphic dinucleotide (CA)(n) repeat markers that lie within the human dystrophin gene. The deletions were evidenced by hemizygosity of the loci under study. We localized a deletion in the locus 7A (intron 7) on the maternal X chromosome in one case, and a deletion in the region of introns 49 and 50 on the paternal X chromosome in the other. The use of microsatellite genotyping within the DMD gene enables the detection of the mutant allele in female carriers. It is also a useful method to provide DMD families with more accurate genetic counseling.     

Variant (6;15) translocations in murine plasmacytomas involve a chromosome 15 locus at least 72 kb from the c-myc oncogene.

The variant (6;15) translocations in murine plasmacytomas join the myc oncogene-bearing band of chromosome 15 and the immunoglobulin kappa band of chromosome 6. We recently cloned a region from chromosome 15 linked to C kappa and have now used probes from that region to define the major locus of plasmacytoma variant translocations, which we denote pvt-1. In five of nine plasmacytomas we analysed, the 6;15 translocation resulted from reciprocal recombination between the C kappa locus and a 4.5-kb region of pvt-1. Moreover, nearby we located the region shown by others to have undergone a complex (15;12;6) translocation in plasmacytoma PC7183. All the chromosome 6 breakpoints fell between 1 and 3 kb 5' to C kappa but only two were near J kappa genes. Thus the J kappa -C kappa region appears to be a recombination 'hot spot' in lymphocytes, but the breaks are unlikely to be mediated via V/J recombination enzymes. Comparison of a cloned 108-kb region across pvt-1 and another of 52 kb across c-myc established that the pvt-1 breakpoints lie at least 72 kb from the c-myc promoters. Since c-myc is expressed at a substantial level, the 6;15 translocation apparently activates c-myc. Activation may occur directly, at a remarkable distance along the chromosome, or indirectly, via a putative pvt-1 gene product.