Mechanisms of genomic instabilities underlying two common fragile-site-associated loci, PARK2 and DMD, in germ cell and cancer cell lines - PubMed (original) (raw)
Comparative Study
. 2010 Jul 9;87(1):75-89.
doi: 10.1016/j.ajhg.2010.06.006.
Yuji Takahashi, Jun Goto, Hiroyuki Tomiyama, Shunpei Ishikawa, Hiroyo Yoshino, Narihiro Minami, David I Smith, Suzanne Lesage, Hiroyuki Aburatani, Ichizo Nishino, Alexis Brice, Nobutaka Hattori, Shoji Tsuji
Affiliations
- PMID: 20598272
- PMCID: PMC2896763
- DOI: 10.1016/j.ajhg.2010.06.006
Comparative Study
Mechanisms of genomic instabilities underlying two common fragile-site-associated loci, PARK2 and DMD, in germ cell and cancer cell lines
Jun Mitsui et al. Am J Hum Genet. 2010.
Abstract
Common fragile sites (CFSs) are specific chromosome regions that exhibit an increased frequency of breaks when cells are exposed to a DNA-replication inhibitor such as aphidicolin. PARK2 and DMD, the causative genes for autosomal-recessive juvenile Parkinsonism and Duchenne and Becker muscular dystrophy, respectively, are two very large genes that are located within aphidicolin-induced CFSs. Gross rearrangements within these two genes are frequently observed as the causative mutations for these diseases, and similar alterations within the large fragile sites that surround these genes are frequently observed in cancer cells. To elucidate the molecular mechanisms underlying this fragility, we performed a custom-designed high-density comparative genomic hybridization analysis to determine the junction sequences of approximately 500 breakpoints in germ cell lines and cancer cell lines involving PARK2 or DMD. The sequence signatures where these breakpoints occur share some similar features both in germ cell lines and in cancer cell lines. Detailed analyses of these structures revealed that microhomologies are predominantly involved in rearrangement processes. Furthermore, breakpoint-clustering regions coincide with the latest-replicating region and with large nuclear-lamina-associated domains and are flanked by the highest-flexibility peaks and R/G band boundaries, suggesting that factors affecting replication timing collectively contribute to the vulnerability for rearrangement in both germ cell and somatic cell lines.
Copyright 2010 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.
Figures
Figure 1
Determination of Breakpoint-Junction Sequences in PARK2 by Custom-Designed High-Density Array CGH Analysis (A) Scan data of array CGH analysis of a patient with AR-JP with 82 kb homozygous deletions (exon 4 of PARK2). The horizontal axis represents the nucleotide position. The vertical axis represents log2 (ratio of case to reference signal intensities on array CGH). Dots of log2 (ratio of case to reference signal intensities) larger than 0 are shown in red, and those smaller than 0 are shown in green. The physical map of PARK2 is also shown above the scan data. (B) Agarose gel electrophoresis of PCR products derived from the patient's genomic DNA obtained by employing primer pairs flanking the deletion. Amplifications did not occur in normal alleles because the segment between primers was too large (82 kb), while the band corresponding to the PCR products of 520 bp derived from the deletion allele was clearly visualized. (C) Design of primer pairs for specific amplification of the deletion allele by PCR. A pair of oligonucleotide primers (denoted by red and blue arrows) was designed to amplify the segment across the breakpoint junction. (D) Electropherogram of amplified segment encompassing breakpoint junctions. The nucleotide sequence corresponding to the segment upstream of the deletion is shown in blue, and the sequence corresponding to the segment downstream of the deletion is shown in red. The underlined inserted sequence not identical to either the upstream or the downstream segment is shown in black. (E) Scan data of array CGH analysis of a patient with AR-JP with a homozygous duplication (exons 6 of PARK2) that turned out to be a tandem duplication. The horizontal axis represents the nucleotide position. The vertical axis represents log2 (ratio of case to reference signal intensities on array CGH). Dots of log2 (ratio of case to reference signal intensities) larger than 0 are shown in red, and those smaller than 0 are shown in green. (F) Design of primer pairs for specific amplification of the duplicated allele by PCR based on head-to-tail, head-to-head, and tail-to-tail models. Oligonucleotide primers are denoted by red and blue arrows. (G) Agarose gel electrophoresis of the PCR products derived from patient's genomic DNA obtained by employing primer pairs flanking duplicated segment. The PCR products are generated only when appropriate primers are used for amplification of rearranged genomic DNA segments.
Figure 2
Histograms and Cumulative-Frequency Distributions of Breakpoint Positions (A) Histograms of breakpoint positions in PARK2 in AR-JP patients or cancer cell lines, and in DMD in patients with DMD/BMD or cancer cell lines. The horizontal axis represents nucleotide positions, and the vertical axis represents the number of breakpoints. The numbers of the positions of the upstream (toward the transcriptional initiation site) breakpoints are shown in white, and those of the downstream breakpoints are shown in black. (B) Cumulative-frequency distributions of breakpoint positions in PARK2 in patients with AR-JP or cancer cell lines, and those in DMD in patients with DMD/BMD or cancer cell lines: The horizontal axis represents the nucleotide positions of breakpoints. The vertical axis represents cumulative frequencies of breakpoints. The upstream breakpoints are shown in white, and the downstream breakpoints are shown in black. (C) Cumulative frequency distributions of breakpoint positions (PARK2 and DMD) in control subjects obtained from the Database of Genomic Variants. Physical maps of PARK2 and DMD, along with schematic representations of the center of FRA6E and FRAXC, are shown below.
Figure 3
Association of Breakpoint-Clustering Regions in PARK2 and DMD with Replication Timing, Flexibility Peak, R/G Band, and AT Content (A) Histograms of positional distributions of breakpoints in PARK2 and DMD in germ cell lines. Breakpoint-clustering regions are the regions with high frequencies (70%–78%) shown by arrows. The physical positions of PARK2, DMD, and the center of FRA6E are shown below. (B) Late-replicating regions, defined as S to G1 DNA ratios of 1.1–1.2. (C) Distributions of AT content (calculated with an average span of 500 bp and an average step of 100 bp). (D) Distributions of flexibility peaks of more than 13.7° in twist angle and more than 100 bp in length. Red bars are the highest peaks whose twist angles exceed 15.5. (E) Physical positions of LADs in PARK2 and DMD. (F) Chromosomal R and G bands are indicated by open and shaded boxes, respectively. (G) Recombination rates based on the deCODE map.
Figure 4
Schematic Representations of Mechanisms Underlying CFSs (A) The microhomology-mediated mechanism is predominantly involved in rearrangement processes at CFSs. (a) Detailed analysis of the nucleotide-sequence content flanking the breakpoints demonstrated that junctions with microhomologies (pink) are predominantly observed, compared with junctions without any homology (sky blue). Junctions with extended homologies (green) underlying NAHR are infrequent. (b) Schematic representation of MMEJ. (c) Schematic representation of NAHR. (B) Multiple factors affecting DNA-replication kinetics collectively contribute to fragility as a common molecular basis. The breakpoint-clustering region at CFSs is flanked by the high-flexibility peaks and the R/G band boundaries. The breakpoint-clustering region coincides with the late-replicating region and is embedded in large LADs.
Figure 5
Complex Rearrangements in DMD Considered to be Generated by MMBIR/FoSTeS Observed in One Patient with DMD An example of complex rearrangements in DMD with microhomology junctions leading to the deletion of approximately 5.7 kb, including exon 12 of DMD, is shown. (A) A map of a part of DMD. The colored boxes represent blocks of sequences. (B) A hypothetical series of four template switches leading to rearrangements, indicated by gray curved arrows and numbers; a gray curved arrow indicates resumption of replication on the original template. Numbers corresponding to the sequences are shown in (C). (C) Rearranged chromosomal region, in which tandem multiplications connect the green sequence to the brown sequence (1), the pink sequence to the brown sequence (2), and the tan sequence to the pink sequence (3), followed by gross deletion between the sky blue sequence and the purple sequence (4). The nucleotide sequences of the colored segments correspond to the colored boxes in (A), (B), and (C). The red boxes indicate the sequences of microhomologies. The gray box represents the inserted sequence of a junction. (1) The junction between the green and the brown sequences shows a 1 bp microhomology. (2) The junction between the pink and the brown sequences shows a 6 bp microhomology. (3) The junction between the tan and the pink sequences shows a 5 bp inserted sequence without microhomology. (4) The junction between the sky blue and the purple sequences shows a 2 bp microhomology. The sizes of the brown, pink, tan, sky blue, and green fragments are 4, 10, 5, 41, and 2 bp, respectively, including the microhomology sequences at both ends.
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