Fragilome

Fragile sites appear as a gap or break in chromosome structure (Fig.1). This chromosome breakage tends to occur at the same loci on metaphase chromosomes from different cells.


Such non random chromosomal fragility represents a well documented phenomenon, which was first described in 1965. Only now its molecular basis is beginning to be understood. Up to date, approximately 120 different fragile sites have been identified in the human genome (listed in the Genome Database) and classified as rare or common, according to their frequency within the population.

Rare fragile sites segregate in families in a Mendelian codominant fashion and range from an incidence of one in several thousand to one in 20 individuals. The DNA at several rare fragile sites has been cloned and characterised. The molecular basis for their expression is an expansion of the repetitive DNA elements. Sequence data for the rare fragile sites revealed the expansion of CGG/CCG trinucleotide repeats or the AT-rich minisatellite repeats. Both CGG/CCG and AT-rich repeats have an ability to form unusual, non-B DNA structures, such as hairpins, which can perturb and delay the DNA replication. Therefore, fragility at these regions might be due to an inability of the incomplete replicated DNA to condence for mitosis. The significance of rare fragile sites in human genetic diseases is associated with FRAXA (fragile X syndrome), and with FRAXE (a mild form of mental retardation).

In contrast to the rare fragile sites, the common fragile sites are seen on chromosomes of all individuals and have been suggested to be ubiquitous in human populations. For several common fragile sites the unstable DNA involved has been determined and characterized. Sequence analysis of these regions reveals no expanded repeats or other specific sequences that could account for their fragility. The molecular basis for common fragile sites expression is far from being understood. Studies of replication timing, together with a recent evidence of an involvement of the checkpoint proteins of the ATR-dependent checkpoint pathway, suggest that common fragile sites represent single-stranded unreplicated chromosomal regions caused by stalled or collapsed replication forks.

Different strategies, such as cloning of translocation breakpoints coincident with fragile site in tumor cells, cloning of the integration sites of exogenous DNA, and FISH mapping of induced gaps and breaks, have been used to clone DNA at common fragile sites. In our lab we are using the six-colour fluorescence in situ hybridization (FISH) with YAC and BAC probes labelled with fluorescence-conjugated nucleotides. Such simultaneous use of six different DNA probes allowed us in a few steps to narrow down the region of fragility from several megabases to the fragile sequence. Finally, this approach allowed us to detect the individual BAC bridging a break in each metaphase (see an example in Fig.2).

Highly recombinogenic DNA sequence encompassing these sites may contribute to both germinal and somatic mutations. The genomic instability at these regions can be associated with a range of human diseases, depending on the individual gene function and tissue type in which the fragile site is expressed. The majority of cloned common fragile sites have been involved in somatic rearrangements found in the chromosomes of cancer cells. The best studied genes, FHIT and WWOX do not appear to be traditional mutation targets in cancer because point mutations are rarely found within them, although many tumors have larger heterozygous deletions within these genes.

There is growing evidence that several common fragile sites are localized within the genes involved in development and function of the central nervous system. Dysfunction of such genes is associated with severe neurological disorders. For instance, FRA13A common fragile site encompassing a 650 kb region within the neurobeachin (NBEA) gene (Fig.3).

NBEA encodes a neuron-specific multidomain protein implicated in membrane trafficking that is predominantly expressed in the brain and during development. It is intriguing that , NBEA has been considered a candidate gene for autism. However, it has yet to be determined if damage of the NBEA gene through activation of FRA13A may have a role in neurodevelopmental disorders.

Common fragile sites appear to be highly conserved during evolution. They have been found in a number of other mammalian species (Fig.4) as well as in lower eukaryotes like the yeast, Saccharomyces cerevisiae.

The best studied mouse fragile sites contain a number of characteristics of human fragile sites, including AT-rich sequence. Moreover, the large non-coding sequences within the mouse fragile sites show high similarity to their human ortholog, suggesting that they serve a conserved function in the cell. It has been hypothesised that they may be among the last sites to replicate and serve for signalling to the cell that replication is complete.

People from the FRAGILOME group (Fig. 5) are using and developing novel experimental approaches to identify DNA sequences of common fragile sites regions. We are searching for the causes and consequences of their fragility, and looking for the possible function of fragile sites in genome.