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.     

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.     

Carrier detection and prenatal molecular diagnosis in a Duchenne muscular dystrophy family without any affected relative available.

In this paper we report a family where the affected DMD patients were not available for study and a molecular strategy was used for female carriers detection and for prenatal diagnosis. Linkage analysis was performed with two markers within the DMD gene, in all family members screened. DMD markers used (pERT87.8/Taq1 and pERT87.15/Xmn1) seemed not to be informative because the propositas mother (II-2) was homozygous for the minor allele at each marker (T2 and X2), however, the proposita and one sister carried only the major allele, which was inherited from the father. These results suggested that a deletion involving both markers could be present, and was inherited from the mother to both daughters. Quantitative multiplex PCR confirmed the deletion in female carriers, involving at least exons 12 to 17. DNA studies of cultured amniotic fluid cells at 14 weeks gestation, by amplification of specific Y-chromosome sequences, followed by multiplex PCR, lead to the diagnosis of a male fetus affected by DMD.     

A very small frame-shifting deletion within exon 19 of the Duchenne muscular dystrophy gene

We report the molecular characterization of a Japanese Duchenne muscular dystrophy (DMD) patient. The analysis of genomic gene by polymerase chain reaction indicates that the individuals have a limited deletion within an amplified region, which encompasses exon 19 of DMD gene. The amplified region was sequenced. Comparison of the deletion joint sequence with the normal amplified region sequence indicates that both 5' and 3' deletion end points are present within exon 19 and the deletion removes total 52 bp out of 88 bp of exon 19. Both his mother and sister are carriers of the deletion-containing allele. The mutation introduces a termination codon at residue 791 in exon 20, and is predicted to result in the production of a severely truncated protein. This sort of deletion (designated as DMD-Kobe) might be classified as a new type of DMD gene abnormality.     

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.     

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.     

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.     

Fluorescent multiplex linkage analysis and carrier detection for Duchenne/Becker muscular dystrophy.

We have developed a fast and accurate PCR-based linkage and carrier detection protocol for families of Duchenne muscular dystrophy (DMD)/Becker muscular dystrophy (BMD) patients with or without detectable deletions of the dystrophin gene, using fluorescent PCR products analyzed on an automated sequencer. When a deletion is found in the affected male DMD/BMD patient by standard multiplex PCR, fluorescently labeled primers specific for the deleted and nondeleted exon(s) are used to amplify the DNA of at-risk female relatives by using multiplex PCR at low cycle number (20 cycles). The products are then quantitatively analyzed on an automatic sequencer to determine whether they are heterozygous for the deletion and thus are carriers. As a confirmation of the deletion data, and in cases in which a deletion is not found in the proband, fluorescent multiplex PCR linkage is done by using four previously described polymorphic dinucleotide sequences. The four (CA)n repeats are located throughout the dystrophin gene, making the analysis highly informative and accurate. We present the successful application of this protocol in families who proved refractory to more traditional analyses.     

Multiplex real-time PCR for detection of deletions and duplications in dystrophin gene

Genetic testing of Duchenne and Becker muscular dystrophies (DMD/BMD) is a difficult task due to the occurrence of deletions or duplications within dystrophin (DMD) gene that requires dose sensitive tests. We developed three multiplex quantitative real-time PCR assays for dystrophin exon 5, 45, and 51 within two major hotspots of deletion/duplication. Each exon was co-amplified with a reference X-linked gene and the copy number of the target fragment was calculated by comparative threshold cycle method (delta deltaC(t)). We compared the performance of this method with previously described end-point PCR fluorescent analysis (EPFA) by studying 24 subjects carrying DMD deletions or duplications. We showed that Q-PCR is an accurate and sensitive technique for the identification of deletions and duplications in DMD/BMD. Q-PCR is a valuable tool for independent confirmation of EPFA screening, particularly when deletions/duplications of single exons occur or for rapid identification of known mutations in at risk carriers.     

Physical mapping at a potential X-linked retinitis pigmentosa locus (RP3) by pulsed-field gel electrophoresis.

A genetic locus (RP3) for X-linked retinitis pigmentosa (XLRP) has been assigned to Xp21 by genetic linkage studies and has been supported by two Xp21 male deletion patients with XLRP. RP3 appears to be the most centromeric of several positioned loci, including chronic granulomatous disease (CGD), McLeod phenotype (XK), and Duchenne muscular dystrophy (DMD). In one patient, BB, the X-chromosome deletion includes RP3 and extends to within the DMD locus. Using a DMD cDNA, the centromeric endpoint of this patient was cloned and used as a starting point for chromosome walking along a normal X chromosome. A single-copy probe, XH1.4, positioned near the centromeric junction but deleted in BB, was used along with a CGD cDNA probe to establish a refined long-range physical map. Both probes recognized a common SfiI fragment of 205 kb. As the CGD gene covers approximately 30-60 kb, the RP3 locus has been restricted to approximately 150-170 kb. A CpG island, potentially marking a new gene, was identified within the SfiI fragment at a position approximately 35 kb from the deletion endpoint in BB.     

Common deletion of SMAD4 in juvenile polyposis is a mutational hotspot

Juvenile polyposis (JP) is an autosomal dominant syndrome in which affected patients develop upper- and/or lower-gastrointestinal (GI) polyps. A subset of families with JP have germline mutations in the SMAD4 (MADH4) gene and are at increased risk of GI cancers. To date, six families with JP have been described as having the same SMAD4 deletion (1244-1247delAGAC). The objective of the present study is to determine whether this deletion is a common ancestral mutation or a mutational hotspot. DNA from members of four families with JP, from Iowa, Mississippi, Texas, and Finland, that had this 4-bp deletion was used to genotype 15 simple tandem repeat polymorphism (STRP) markers flanking the SMAD4 gene, including 2 new STRPs within 6.3 and 70.9 kb of the deletion. Haplotypes cosegregating with JP in each family were constructed, and the distances of the closest markers were determined from the draft sequence of the human genome. No common haplotype was observed in these four families with JP. A 14-bp region containing the deletion had four direct repeats and one inverted repeat. Because no common ancestor was suggested by haplotype analysis and the sequence flanking the deletion contains repeats frequently associated with microdeletions, this common SMAD4 deletion in JP most likely represents a mutational hotspot.     

Analysis of sequence upstream of the endogenous H19 gene reveals elements both essential and dispensable for imprinting

Imprinting of the linked and oppositely expressed mouse H19 and Igf2 genes requires a 2-kb differentially methylated domain (DMD) that is located 2 kb upstream of H19. This element is postulated to function as a methylation-sensitive insulator. Here we test whether an additional sequence 5' of H19 is required for H19 and Igf2 imprinting. Because repetitive elements have been suggested to be important for genomic imprinting, the requirement of a G-rich repetitive element that is located immediately 3' to the DMD was first tested in two targeted deletions: a 2.9-kb deletion (Delta D MD Delta G) that removes the DMD and G-rich repeat and a 1.3-kb deletion (Delta G) removing only the latter. There are also four 21-bp GC-rich repetitive elements within the DMD that bind the insulator-associated CTCF (CCCTC-binding factor) protein and are implicated in mediating methylation-sensitive insulator activity. As three of the four repeats of the 2-kb DMD were deleted in the initial 1.6-kb Delta DMD allele, we analyzed a 3.8-kb targeted allele (Delta 3.8kb-5'H19), which deletes the entire DMD, to test the function of the fourth repeat. Comparative analysis of the 5' deletion alleles reveals that (i) the G-rich repeat element is dispensable for imprinting, (ii) the Delta DMD and Delta DMD Delta G alleles exhibit slightly more methylation upon paternal transmission, (iii) removal of the 5' CTCF site does not further perturb H19 and Igf2 imprinting, suggesting that one CTCF-binding site is insufficient to generate insulator activity in vivo, (iv) the DMD sequence is required for full activation of H19 and Igf2, and (v) deletion of the DMD disrupts H19 and Igf2 expression in a tissue-specific manner.     

The use of field-inversion gel electrophoresis for deletion detection in Duchenne muscular dystrophy.

Deletion is a common cause of Duchenne muscular dystrophy (DMD). Field-inversion gel electrophoresis, in conjunction with Southern blot hybridization, was used to detect large SfiI DNA fragments in the DMD locus. Two unrelated boys with DMD were found to have abnormal sized DNA fragments resulting from deletions. Some of the female relatives of these patients were also shown by this method to have deletions in the DMD locus.     

DNA studies in a family with Duchenne muscular dystrophy and a deletion at Xp21.

We have performed Southern blot analysis on a large, four-generation kindred with Duchenne muscular dystrophy (DMD). Probes 754 (DXS 84), pERT87-1, pERT87-8, pERT87-15 (DXS164), and pXJ-1.1 did not hybridize to digested genomic DNA of affected males. Obligate-carrier mothers and unaffected brothers showed signals of a single X-chromosome copy intensity, and suspected noncarrier sisters demonstrated either a single band of two-copy intensity or informative polymorphisms. Uniform hybridization was seen with probes C7 (DXS28) and D2 (DXS43), which map distal to the DMD locus, and with OTC, which maps proximally. This deletion was present in six affected individuals and has been transmitted through 3 generations to date. On high-resolution chromosome analysis, a deletion within band Xp21 was consistently observed in one affected male studied and in one of the two X chromosomes in obligate carriers. This large molecular and cytogenetically visible deletion in affected DMD individuals without glycerol kinase deficiency, chronic granulomatous disease, retinitis pigmentosa (RP), or ornithine transcarbamylase deficiency is a very rare finding and should prove useful in specifically cloning additional probes within and flanking the DMD locus.     

Mapping of 12 translocation breakpoints in the Xp21 region with respect to the locus for Duchenne muscular dystrophy

Over 20 females have been reported to carry reciprocal X; autosome translocations with breakpoints in Xp21 and to suffer from Duchenne muscular dystrophy (DMD). We have positioned nine of these breakpoints with respect to the Duchenne gene by mapping probes from the DMD region against a panel of somatic cell hybrids, each containing one of the translocation chromosomes from a different female patient; further information has also been obtained by in situ hybridization, including the breakpoint location in a tenth DMD patient. We have also characterized two translocation breakpoints that lie in the same chromosomal region but which are not associated with the expression of DMD. All the DMD-associated translocation breakpoints examined lie at several sites within the DMD locus and between the two non-DMD breakpoints.     

Uniparental disomy of the entire X chromosome in a female with Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a severe, progressive, X-linked muscle-wasting disorder with an incidence of approximately 1/3,500 male births. Females are also affected, in rare instances. The manifestation of mild to severe symptoms in female carriers of dystrophin mutations is often the result of the preferential inactivation of the X chromosome carrying the normal dystrophin gene. The severity of the symptoms is dependent on the proportion of cells that have inactivated the normal X chromosome. A skewed pattern of X inactivation is also responsible for the clinical manifestation of DMD in females carrying X;autosome translocations, which disrupt the dystrophin gene. DMD may also be observed in females with Turner syndrome (45,X), if the remaining X chromosome carries a DMD mutation. We report here the case of a karyotypically normal female affected with DMD as a result of homozygosity for a deletion of exon 50 of the dystrophin gene. PCR analysis of microsatellite markers spanning the length of the X chromosome demonstrated that homozygosity for the dystrophin gene mutation was caused by maternal isodisomy for the entire X chromosome. This finding demonstrates that uniparental isodisomy of the X chromosome is an additional mechanism for the expression of X-linked recessive disorders. The proband's clinical presentation is consistent with the absence of imprinted genes (i.e., genes that are selectively expressed based on the parent of origin) on the X chromosome.     

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.     

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.     

Why mice have lost genes for COL21A1, STK17A, GPR145 and AHRI: evidence for gene deletion at evolutionary breakpoints in the rodent lineage

The mouse genome has undergone extensive chromosome rearrangement relative to the human genome since these species last shared a common ancestor. One possible consequence of these rearrangements is the deletion of genes that are located within evolutionary breakpoint regions. In this article, we present evidence of four human genes (COL21A1, STK17A, GPR145 and ARHI) that are located in regions corresponding to evolutionary breakpoints in rodents and lack mouse and rat orthologues. We propose that "evolutionary breakpoint-associated gene deletion" is an unexpected consequence of evolutionary chromosome rearrangement, and we describe a novel mechanism through which genes can be lost during evolution.     

Direct carrier detection by in situ suppression hybridization with cosmid clones of the Duchenne/Becker muscular dystrophy locus

Summary A basic problem in genetic counseling of families with Duchenne/Becker muscular dystrophy (DMD/BMD) concerns the carrier status of female relatives of an affected male. In about 60% of these patients, deletions of one or more exons of the dystrophin gene can be identified. These deletions preferentially include exon 45, which can be detected by multiplex polymerase chain reaction (PCR) and Southern blot analysis of genomic cosmid clones that map to this critical region. As a new approach for definitive carrier detection, we have performed chromosomal in situ suppression (CISS) hybridization with these cosmid clones in female relatives of four unrelated patients. In normal females, most metaphases showed signals on both×chromosomes, whereas only one×chromosome was labeled in carriers. Our results demonstrate that CISS hybridization can define the carrier status in female relatives of DMD patients exhibiting a deletion in the dystrophin gene.