Molecular characterization of common fragile sites as a strategy to discover cancer susceptibility genes

Abstract

The cytogenetic hypothesis that common fragile sites (cFSs) are hotspots of cancer breakpoints is increasingly supported by recent data from whole-genome profiles of different cancers. cFSs are components of the normal chromosome structure that are particularly prone to breakage under conditions of replication stress. In recent years, cFSs have become of increasing interest in cancer research, as they not only appear to be frequent targets of genomic alterations in progressive tumors, but also already in precancerous lesions. Despite growing evidence of their importance in disease development, most cFSs have not been investigated at the molecular level and most cFS genes have not been identified. In this review, we summarize the current data on molecularly characterized cFSs, their genetic and epigenetic characteristics, and put emphasis on less-studied cFS genes as potential contributors to cancer development.

Fig. 1

Strategies of cFS identification. Cloning cFS breakpoints using in vitro-induced rearrangements (blue segment): following induction of cFS breakage by low concentrations of aphidicolin in somatic cell hybrids containing a single human chromosome (red), cell clones are analyzed for the presence of genetic markers along the human chromosome, enabling the localization of breakpoints within the generated rearrangements. Induced cFS breaks can be targeted using insertional mutagenesis with different DNA vectors, allowing direct cloning of human DNA sequences flanking the vector integration sites. cFS-associated features in cancer cells (red segment): definition of the regions exhibiting recurrent loss of expression (LOE) and loss of heterozygosity (LOH) within chromosomal bands containing cFSs can be useful to narrow down cFS regions. Cloning viral integration sites, mainly of HPV in cervical tumors, has been implemented for pinpointing several cFS sequences. Mapping breakpoints of recurrent genomic alterations, including gross chromosomal deletions, translocations, and amplifications, which can be visualized by FISH painting methods, is also suitable to locate cFSs. Genetic and epigenetic landmarks of normal cells (green segment): two features in normal cells, large genes and a V-shaped replication timing pattern graphically representing Repli-Seq data have been used to search for cFSs. FISH-based mapping as a final step of cFS delineation (center): image demonstrating the principle of cFS mapping using six-color FISH

Fig. 2

Transcriptional map of fine-mapped cFS regions. Chromosome ideograms on the left indicate the cytogenetic position of each cFS. The genomic coordinates and corresponding chromosome bands are shown on the x-axis; dotted frames indicate the boundaries and size (kb) of each cFS. Displayed genes represent RefSeq genes according to the UCSC genome browser (GRCh37/hg19)

Fig. 3

The main types of recurrent cFS rearrangements in cancer. a Hemizygous deletions at cFSs. Loss of genetic material at FRA2H in HDC-133 nm colorectal cancer cells revealed by array CGH analysis (left) and validated by FISH (right), showing that genomic loss at FRA2H results from a hemizygous deletion (del) in one of the chromosome 2 homologues, as demonstrated by the absence of the red hybridization signal. b Duplications at cFSs. Copy number gain at FRA16D detected by array CGH in the NGP neuroblastoma cell line (left) originating from an intrachromosomal duplication in one of the chromosome 16 homologues displaying a double pink signal (dup) in metaphase spreads (right). c Amplifications at cFSs. FRA2C sequences partially overlap with MYCN amplicons in neuroblastoma cell lines on array CGH plots (left), corresponding to the high-level amplifications detected by FISH (left): homogeneously staining region (HSR) on a derivative chromosome in LA–N-2 cells, and extrachromosomal double minutes (DMs) in LA–N-5 metaphase spreads. d Translocations at cFSs. Multicolor FISH (mFISH) analysis revealing a derivative chromosome identified as a t(7;8;11) translocation in Caco-2 colorectal cancer cells (left). One of the translocation breakpoints in chromosome 8 maps within FRA8I, as confirmed by the absence of the red hybridization signal on a derivative chromosome using two FISH probes (right). Dotted frames indicate cFS boundaries; genomic coordinates are shown on the x-axis, the log₂ ratio of test to reference DNA on the y-axis. Arrowheads on array plots indicate the genomic position of the BAC probes used for FISH validation. Arrows highlight derivative chromosomes with cFS aberrations, n marks apparently normal copies of chromosome homologues