Chromosome‐specific segmentation revealed by structural analysis of individually isolated chromosomes

Abstract

Analysis of structural rearrangements at the individual chromosomal level is still technologically challenging. Here we optimized a chromosome isolation method using fluorescent marker-assisted laser-capture and laser-beam microdissection and applied it to structural analysis of two aberrant chromosomes found in a lung cancer cell line. A high-density array-comparative genomic hybridization (array-CGH) analysis of DNA samples prepared from each of the chromosomes revealed that these two chromosomes contained 296 and 263 segments, respectively, ranging from 1.5 kb to 784.3 kb in size, derived from different portions of chromosome 8. Among these segments, 242 were common in both aberrant chromosomes, but 75 were found to be chromosome-specific. Sequences of 263 junction sites connecting the ends of segments were determined using a PCR/Sanger-sequencing procedure. Overlapping microhomologies were found at 169 junction sites. Junction partners came from various portions of chromosome 8 and no biased pattern in the positional distribution of junction partners was detected. These structural characteristics suggested the occurrence of random fragmentation of the entire chromosome 8 followed by random rejoining of these fragments. Based on that, we proposed a model to explain how these aberrant chromosomes are formed. Through these structural analyses, it was demonstrated that the optimized chromosome isolation method described here can provide high-quality chromosomal DNA for high resolution array-CGH analysis and probably for massively parallel sequencing analysis.

Whole-genome haplotyping by dilution, amplification, and sequencing

“…Several experimental phasing methods have been described. Examples include somatic cell hybrid construction to convert a diploid cell into haploid cell lines (2); large-insert cloning followed by screening of clone pools by either PCRbased genotyping (3) or sequencing (4)(5)(6) and isolation of individual chromosomes through chromosome microdissection of lysed metaphase cells on a glass slide (7)(8)(9); FACS-mediated single chromosome sorting (10); or the use of a custom microfluidic device (11) followed by amplification of the individual chromosomes and genotyping. The requirement of high-level expertise is a common denominator in these methods, which are not amenable to high-throughput studies or easily transferable to a clinical or diagnostic setting.…”

Coamplification of Multiple Regions of Chromosome 2, Including MYCN, in a Single Patchwork Amplicon in Cancer Cell Lines

“…This structure resembles a patchwork structure of multiple chromosomal segments which has been proposed to be generated by a single catastrophic event model. In this model, the entire chromosome or a part of a chromosome is massively fragmented, shuffled, and randomly rejoined [Kitada et al, 2011;Stephens et al, 2011]. In the lung cancer cell line SCLC-21H, two patchwork chromosomes (called ZZchr8s), composed of 1 300 different segments derived from chromosome 8, have been characterized [Kitada et al, 2011].…”

“…In this model, the entire chromosome or a part of a chromosome is massively fragmented, shuffled, and randomly rejoined [Kitada et al, 2011;Stephens et al, 2011]. In the lung cancer cell line SCLC-21H, two patchwork chromosomes (called ZZchr8s), composed of 1 300 different segments derived from chromosome 8, have been characterized [Kitada et al, 2011]. These two ZZchr8s, with similar but not identical structures, have no centromere of chromosome 8, but are stably maintained by being tethered to chromosomes 10 and 11, respectively.…”

“…Chromosome fragmentation in only 1 or 2 chromosomes in response to cellular stress was demonstrated in mitotic figures, and the relation of such simultaneous fragmentation to the generation of heterogeneous amplicons has been suggested [Stevens et al, 2007;Gibaud et al, 2010]. Recently, based on detailed structural analyses of severely rearranged chromosomes, a catastrophic event model was proposed to explain massive genomic rearrangement [Kitada et al, 2011;Stephens et al, 2011]. In this model, the entire chromosome or a part of a chromosome is massively fragmented and the resulting fragments are randomly rejoined as a single event.…”

Alu-Alu Fusion Sequences Identified at Junction Sites of Copy Number Amplified Regions in Cancer Cell Lines

“…In A431 (7p11.2), 2 signals with different intensities appeared in different-shaped chromosomes, suggesting that these 2 regional amplifications occurred independently. Further study, especially individual analysis of individually isolated chromosomes, as described before [Kitada et al, 2011], is needed for detailed characterization.…”