Gene dosage variations occur in many diseases. In cancer, deletions and copy number increases contribute to alterations in the expression of tumour-suppressor genes and oncogenes, respectively. Developmental abnormalities, such as Down, Prader Willi, Angelman and Cri du Chat syndromes, result from gain or loss of one copy of a chromosome or chromosomal region. Thus, detection and mapping of copy number abnormalities provide an approach for associating aberrations with disease phenotype and for localizing critical genes. Comparative genomic hybridization (CGH) was developed for genome-wide analysis of DNA sequence copy number in a single experiment. In CGH, differentially labelled total genomic DNA from a 'test' and a 'reference' cell population are cohybridized to normal metaphase chromosomes, using blocking DNA to suppress signals from repetitive sequences. The resulting ratio of the fluorescence intensities at a location on the 'cytogenetic map', provided by the chromosomes, is approximately proportional to the ratio of the copy numbers of the corresponding DNA sequences in the test and reference genomes. CGH has been broadly applied to human and mouse malignancies. The use of metaphase chromosomes, however, limits detection of events involving small regions (of less than 20 Mb) of the genome, resolution of closely spaced aberrations and linking ratio changes to genomic/genetic markers. Therefore, more laborious locus-by-locus techniques have been required for higher resolution studies. Hybridization to an array of mapped sequences instead of metaphase chromosomes could overcome the limitations of conventional CGH (ref. 6) if adequate performance could be achieved. Copy number would be related to the test/reference fluorescence ratio on the array targets, and genomic resolution could be determined by the map distance between the targets, or by the length of the cloned DNA segments. We describe here our implementation of array CGH. We demonstrate its ability to measure copy number with high precision in the human genome, and to analyse clinical specimens by obtaining new information on chromosome 20 aberrations in breast cancer.

We show here that quantitative measurement of DNA copy number across amplified regions using array comparative genomic hybridization (CGH) may facilitate oncogene identification by providing precise information on the locations of both amplicon boundaries and amplification maxima. Using this analytical capability, we resolved two regions of amplification within an approximately 2-Mb region of recurrent aberration at 20q13.2 in breast cancer. The putative oncogene ZNF217 (ref. 5) mapped to one peak, and CYP24 (encoding vitamin D 24 hydroxylase), whose overexpression is likely to lead to abrogation of growth control mediated by vitamin D, mapped to the other.

Elucidating the connection between genotype, phenotype, and adaptation in wild populations is fundamental to the study of evolutionary biology, yet it remains an elusive goal, particularly for microscopic taxa, which comprise the majority of life. Even for microbes that can be reliably found in the wild, defining the boundaries of their populations and discovering ecologically relevant phenotypes has proved extremely difficult. Here, we have circumvented these issues in the microbial eukaryote Neurospora crassa by using a "reverse-ecology" population genomic approach that is free of a priori assumptions about candidate adaptive alleles. We performed Illumina whole-transcriptome sequencing of 48 individuals to identify single nucleotide polymorphisms. From these data, we discovered two cryptic and recently diverged populations, one in the tropical Caribbean basin and the other endemic to subtropical Louisiana. We conducted high-resolution scans for chromosomal regions of extreme divergence between these populations and found two such genomic "islands." Through growthrate assays, we found that the subtropical Louisiana population has a higher fitness at low temperature (10°C) and that several of the genes within these distinct regions have functions related to the response to cold temperature. These results suggest the divergence islands may be the result of local adaptation to the 9°C difference in average yearly minimum temperature between these two populations. Remarkably, another of the genes identified using this unbiased, whole-genome approach is the well-known circadian oscillator frequency, suggesting that the 2.4°-10.6°difference in latitude between the populations may be another important environmental parameter.ecological genomics | genome scan | fungi | circadian clock D iscovering the genetic basis behind adaptive phenotypes has long been considered the holy grail of evolutionary genetics. Although there are now several studies that have succeeded in identifying genes responsible for such phenotypes, the majority of them use a "forward-ecology" approach whereby candidate genes are identified on the basis of their having a function related to conspicuous traits, such as pigmentation (1-4). A paucity of obvious phenotypic traits has been a major impediment for studying adaptation in microbes because these organisms are, by nature, inconspicuous. However, next-generation sequencing technology has made it possible for individual laboratories to acquire whole-genome sequence information across populations. This innovation has enabled an unbiased "reverse-ecology" approach whereby genes with functions related to ecologically relevant traits can be identified by examining patterns of genetic diversity within and between populations and identifying candidate genes as those showing the signature of recent positive selection and/or divergent adaptation between populations (5).

We report here the molecular cloning of an Ϸ1-Mb region of recurrent amplification at 20q13.2 in breast cancer and other tumors and the delineation of a 260-kb common region of amplification. Analysis of the 1-Mb region produced evidence for five genes, ZNF217, ZNF218, and NABC1, PIC1L (PIC1-like), CYP24, and a pseudogene CRP (Cyclophillin Related Pseudogene). ZNF217 and NABC1 emerged as strong candidate oncogenes and were characterized in detail. NABC1 is predicted to encode a 585-aa protein of unknown function and is overexpressed in most but not all breast cancer cell lines in which it was amplified. ZNF217 is centrally located in the 260-kb common region of amplification, transcribed in multiple normal tissues, and overexpressed in all cell lines and tumors in which it is amplified and in two in which it is not. ZNF217 is predicted to encode alternately spliced, Kruppel-like transcription factors of 1,062 and 1,108 aa, each having a DNA-binding domain (eight C2H2 zinc fingers) and a proline-rich transcription activation domain.Studies in which comparative genomic hybridization was used have revealed Ϸ20 regions of recurrent increased DNA sequence copy number in breast tumors (1-3). These regions are predicted to encode dominantly acting genes that may play a role in tumor progression or response to therapy. To date, three of these regions have been associated with established oncogenes: ERBB2 at 17q12, MYC at 8q24, and CCND1 and EMS1 at 11q13. In breast cancer, ERBB2 and CCND1͞EMS1 amplification and overexpression are associated with decreased life expectancy (4, 5), whereas MYC amplification has been associated with lymph node involvement, advanced stage, and an increased rate of relapse (6, 7). Efforts are now underway in several laboratories to identify oncogenes in the other regions of amplification.Amplification at 20q13 is particularly interesting because this aberration occurs in a variety of tumor types and is associated with aggressive tumor behavior. The initial comparative genomic hybridization study showed increased copy number involving 20q13 in 40% of breast cancer cell lines and 18% of primary breast tumors (2). Since then, other comparative genomic hybridization studies have revealed copynumber gains at 20q13 in greater than 25% of cancers of the ovary (8), colon (9), head and neck (10