Mutations in the dystrophin gene (DMD) cause Duchenne and Becker muscular dystrophies and the majority of cases are due to DMD gene rearrangements. Despite the high incidence of these aberrations, ...little is known about their causative molecular mechanism(s). We examined 792 DMD/BMD clinical samples by oligonucleotide array-CGH and report on the junction sequence analysis of 15 unique deletion cases and three complex intragenic rearrangements to elucidate potential underlying mechanism(s). Furthermore, we present three cases with intergenic rearrangements involving DMD and neighboring loci. The cases with intragenic rearrangements include an inversion with flanking deleted sequences; a duplicated segment inserted in direct orientation into a deleted region; and a splicing mutation adjacent to a deletion. Bioinformatic analysis demonstrated that 7 of 12 breakpoints combined among 3 complex cases aligned with repetitive sequences, as compared to 4 of 30 breakpoints for the 15 deletion cases. Moreover, the inversion/deletion case may involve a stem-loop structure that has contributed to the initiation of this rearrangement. For the duplication/deletion and splicing mutation/deletion cases, the presence of the first mutation, either a duplication or point mutation, may have elicited the deletion events in an attempt to correct preexisting mutations. While NHEJ is one potential mechanism for these complex rearrangements, the highly complex junction sequence of the inversion/deletion case suggests the involvement of a replication-based mechanism. Our results support the notion that regional genomic instability, aided by the presence of repetitive elements, a stem-loop structure, and possibly preexisting mutations, may elicit complex rearrangements of the DMD gene.
Duplications of the Xq28 chromosome region resulting in functional disomy are associated with a distinct clinical phenotype characterized by infantile hypotonia, severe developmental delay, ...progressive neurological impairment, absent speech, and proneness to infections. Increased expression of the dosage-sensitive MECP2 gene is considered responsible for the severe neurological impairments observed in affected individuals. Although cytogenetically visible duplications of Xq28 are well documented in the published literature, recent advances using array comparative genomic hybridization (CGH) led to the detection of an increasing number of microduplications spanning MECP2. In rare cases, duplication results from intrachromosomal rearrangement between the X and Y chromosomes. We report six cases with sex chromosome rearrangements involving duplication of MECP2. Cases 1-4 are unbalanced rearrangements between X and Y, resulting in MECP2 duplication. The additional Xq material was translocated to Yp in three cases (cases 1-3), and to the heterochromatic region of Yq12 in one case (case 4). Cases 5 and 6 were identified by array CGH to have a loss in copy number at Xp and a gain in copy number at Xq28 involving the MECP2 gene. In both cases, fluorescent in situ hybridization (FISH) analysis revealed a recombinant X chromosome containing the duplicated material from Xq28 on Xp, resulting from a maternal pericentric inversion. These cases add to a growing number of MECP2 duplications that have been detected by array CGH, while demonstrating the value of confirmatory chromosome and FISH studies for the localization of the duplicated material and the identification of complex rearrangements.
Human artificial chromosomes (ACs) are non-integrating vectors that may be useful for gene therapy. They assemble in cultured cells following transfection of human centromeric α -satellite DNA and ...segregate efficiently alongside the host genome. In the present study, a 33 kilobase (kb) Factor IX (FIX) gene was incorporated into mitotically stable ACs in human HT1080 lung derived cells using co-transfection of a bacterial artificial chromosome (BAC) harboring synthetic α -satellite DNA and a P1 artificial chromosome(PAC) that spans the FIX locus. ACs were detected in ≥90% of chromosome spreads in 8 of 19 lines expanded from drug resistant colonies. FIX transgene copy number on ACs was determined by input DNA transfection ratios. Furthermore, a low level of FIX transcription was detected from ACs with multiple transgenes but not from those incorporating a single transgene, suggesting that reducing transgene number may limit misexpression. Their potential to segregate cross species was measured by transferring ACs into mouse and hamster cell lines using microcell-mediated chromosome transfer. Lines were obtained where ACs segregated efficiently. The stable segregation of ACs in rodent cells suggests that it should be possible to develop animal models to test the capacity of ACs to rescue FIX deficiency.
Human adipose stromal cells (ASCs) reside within the stromal-vascular fraction (SVF) in fat tissue, can be readily isolated, and include stem-like cells that may be useful for therapy. An important ...consideration for clinical application and functional studies of stem/progenitor cells is their capacity to maintain chromosome stability in culture. In this study, cultured ASC populations and ASC clones were evaluated at intervals for maintenance of chromosome stability. Uncultured SVF (uSVF) cells were included for comparison. G-banded chromosome analysis demonstrated that ASCs are diploid and have a normal karyotype. However since only approximately 20 cells are examined, low levels of chromosome instability would not be detected. To increase detection sensitivity, fluorescence in situ hybridization was employed, to permit chromosome enumeration in larger numbers of interphase cells. Seven cultured ASC populations, two ASC clones and four uSVF samples were examined. Chromosome X and 17 probes identified diploid, tetraploid, and aneuploid interphase cells. Both cultured ASC populations up to approximately 35 Population Doublings (PDs) and uSVF cells exhibited a similar level of diploidy (97.8% n = 6,355 and 98.83% n = 1,197, respectively) and numerical abnormalities, suggesting that cultured ASCs are genomically stable and supporting their suitability for transplantation applications. In comparison, cultured primary human chorionic villus cells exhibited marked genomic instability resulting in an 11.6% tetraploidy rate after 8-10 PD. Thus effects of culture on genomic stability may be cell type dependent and should be tested by appropriately scaled interphase fluorescence in situ hybridization analysis in any ex vivo expanded cell population destined for transplantation.
Mucopolysaccharidosis type II (MPS II) is caused by mutations in the IDS gene, which encodes the lysosomal enzyme iduronate-2-sulfatase. In ∼20% of MPS II patients the disorder is caused by gross IDS ...structural rearrangements. We identified two male cases harboring complex rearrangements involving the IDS gene and the nearby pseudogene, IDSP1, which has been annotated as a low-copy repeat (LCR). In both cases the rearrangement included a partial deletion of IDS and an inverted insertion of the neighboring region. In silico analyses revealed the presence of repetitive elements as well as LCRs at the junctions of rearrangements. Our models illustrate two alternative consequences of rearrangements initiated by non-allelic homologous recombination of LCRs: resolution by a second recombination event (that is, Alu-mediated recombination), or resolution by non-homologous end joining repair. These complex rearrangements have the potential to be recurrent and may be present among those MSP II cases with previously uncharacterized aberrations involving IDS.
Array-based comparative genomic hybridization (array CGH), a method used to detect gains or losses of genetic material, has recently been applied to prenatal diagnosis of genomic imbalance in the ...clinical laboratory setting. This new and exciting diagnostic tool represents a major technological step forward in cytogenetic testing and addresses many of the limitations of current cytogenetic methods. Conventional chromosome analysis, the current gold standard in prenatal diagnosis, focuses primarily on the detection of common aneuploidies and is limited by its capacity to detect only those copy number changes that are large enough to be microscopically visible (typically 5-6 Mb in size at the 500 band level). In contrast, array CGH analysis simultaneously evaluates regions across the entire genome and allows for detection of unbalanced structural and numerical chromosome abnormalities of less than one hundred kb. Array CGH analysis also overcomes some of the limitations of chromosome analysis, such as the requirement for cell culture and longer reporting time, by using direct uncultured fetal specimens. With many diagnostic laboratories now embracing this technology, the past year has seen tremendous growth in the use of array CGH analysis for prenatal diagnosis. This review aims to summarize array CGH methodology and its current applications in prenatal diagnosis.
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