Chromosomal imbalances and rearrangements are common in most tumors. Likewise chromosome rearrangements are a hallmark of genomic differences between species (Noor et al., 2001; Navarro& Barton, 2003; Sankoff, 2003). It has often been speculated that there is a correlation between chromosome rearrangements in disease and evolution (Marinez et al., 2001). Even more intriguing is the possibility that the evolutionary history of chromosome rearrangements provides causal link behind at least some rearrangements in cancer (Sankoff et al, 2002). This link seems probable since genome rearrangements do not represent random events, but instead, relect higher order genomic features. An increasing number of diseases is now associated with unstable genomic regions. The clinical phenotype is a consequence of abnormal dosage of genes located within the rearranged fragments. These diseases are know as genomic disorders and they have wide-ranging effects on human health as detailed in recent reviews. Chromosome-specific low-copy repeats or duplicons occur in multiple regions of the human genome and that these sequences facilitate chromosome rearrangements. Sequence homology between duplicons provides a chance for misalignment during meiosis, leading to unequal exchange and chromosome rearrangements such as deletions, duplications, inversions, and inverted duplications, depending on the orientation of the recombining duplicons. This process and the subsequent divergence of duplicated segments are essential to the generation of diversity and new genes over evolutionary time; although the more typical, short-term effect is genetic disease.The ability to flow sort chromosomes present in the Core combined with microarray technology would expedite the identification and cloning of genes at the site of genomic rearrangements in both disease and evolution. Our research is designed to test the hypothesis that chromosome rearrangements in evolution and disease are intimately related. Initially, breakpoints of evolutionary rearrangements (translocations, inversions, and conserved fragile sites) of chromosome 1 (and to a lesser extent chromosome 3 and 7) are being investigated This research will further our understanding of the structure, function and fluidity of the genome and the relationship between evolution and disease. A future application of the research results would foresee the use of genome changes seen in both disease and evolution as biomarkers of cancer risk, diagnostics or prognostic assessments.Chromosome Flow sorting and """"""""array painting""""""""Rearranged chromosomes fixed in evolution or aberrant chromosomes from cancer cell lines can be sorted in high numbers. The DNA can then be extracted and hybridized to DNA microarrays to determine breakpoints with a degree of accuracy and ease previously unknown. Our collaborators at Cold Springs Laboratory now print human BAC clone arrays. These BAC arrays provide a hybridization platform that will allow assessment of regional genome copy number changes (gains and losses) at a 1-2 Mb resolution. The use of such arrays would allow a high definition of genome changes in human cancers, precancers, and analogously in murine cancer models.Flow sorting of evolutionarily rearranged chromosomes and their hybridization to human BAC arrays is a high-resolution equivalent of reciprocal chromosome painting in which chromosome paints are made from flow sorted chromosomes and then hybridized to human metaphases. A recent article (Fiegler et al., 2003) describes this method applied to aberrant tumor chromosomes as """"""""array painting"""""""". The resolution is governed only by the size and density of clones in the array. Our own work and that of others has shown that the clones spanning the breakpoints are easily identified and that the position of the breakpoint within the spanning clone can be estimated. Our recent results using multi-directional chromosome painting and gene mapping shows that human chromosome 1 represents an ancestral chromosomal synteny that became fragmented in the majority of species (Murphy et al, 2003). Human chromosome 1 is found intact in some higher primate species (humans, apes, some monkeys), but in most species it is found as two segments. One segment corresponds roughly to 1q21.3-pter, while the second segment is comprised of the remainder of the long arm (1q21.3-qter). We found that the number of segments of human chromosome 1 in a specific lineage reflects its general rate of genomic evolution with up to six segments found in some primates. Across mammals, chromosome fissioning appears to have been disproportionately clustered at two hotspots of the long arm (1q21-23 and 1q41-42). These bands are among the most commonly reported in the database of recurrent chromosome aberrations in caner. The part of the proposed research will characterize the evolutionary translocation breakpoints of chromosome 1. Cloning inversion breakpoint found by BAC FISH, screening libraries, sequencing. FISH can characterize inversion breakpoints with human subchromosomal DNA clones such as BACs and YACs. We recently defined some of the inversion breakpoints, which occurred during human phylogeny in the chromosomes 3 and 7 by FISH with human BACs in macaques. These human BACs can now be used to screen primate DNA libraries that will lead to a comparison of the breakpoints at the sequence level. Our recent research has identified human BACs, which span inversion breakpoints in the macaque homolog to human chromosome 3. Fragile SitesUntil recently the link between fragile sites (FSs) and cancer was highly speculative. Recently, ample evidence now shows that chromosome breaks associated with both cancer and evolution occur at fragile sites throughout the genome. Fragile sites are also found in primate chromosomes and a commonly repeated hypothesis is that they are evolutionarily conserved and implicated in chromosome rearrangements in phylogeny. We recently submitted a paper that tested the hyothesis of evolutionary conservation of FSs using FISH of BAC and YAC clones. The hybridizations showed that 85% of the fragile sites studied are located in homologous chromosomal bands in humans, macaques and baboons. Notably, two YAC clones known to span human chromosome regions containing fragile sites (FRA3B and FRAXB) were shown to also span fragile sites in macaques and mandrills. FRA3B (at 3p14.2) the most highly expressed and studied FS in humans is clearly associated with a variety of histologically distinct cancers. Up to now such comparative work to understand the evolution and function of this region has focused human/mouse comparisons. A comparative study of the evolution of this region in primates would be an important development in cancer research. It also may provide a key insight into the mechanisms of FS instability and eventually the impact of such instability on chromosome rearrangements, and gene function leading to new targets for cancer therapy.
