Genome-wide analysis of DNA copy-number changes using cDNA microarrays.

Gene amplifications and deletions frequently contribute to tumorigenesis. Characterization of these DNA copy-number changes is important for both the basic understanding of cancer and its diagnosis. Comparative genomic hybridization (CGH) was developed to survey DNA copy-number variations across a whole genome. With CGH, differentially labelled test and reference genomic DNAs are co-hybridized to normal metaphase chromosomes, and fluorescence ratios along the length of chromosomes provide a cytogenetic representation of DNA copy-number variation. CGH, however, has a limited ( approximately 20 Mb) mapping resolution, and higher-resolution techniques, such as fluorescence in situ hybridization (FISH), are prohibitively labour-intensive on a genomic scale. Array-based CGH, in which fluorescence ratios at arrayed DNA elements provide a locus-by-locus measure of DNA copy-number variation, represents another means of achieving increased mapping resolution. Published array CGH methods have relied on large genomic clone (for example BAC) array targets and have covered only a small fraction of the human genome. cDNAs representing over 30,000 radiation-hybrid (RH)-mapped human genes provide an alternative and readily available genomic resource for mapping DNA copy-number changes. Although cDNA microarrays have been used extensively to characterize variation in human gene expression, human genomic DNA is a far more complex mixture than the mRNA representation of human cells. Therefore, analysis of DNA copy-number variation using cDNA microarrays would require a sensitivity of detection an order of magnitude greater than has been routinely reported. We describe here a cDNA microarray-based CGH method, and its application to DNA copy-number variation analysis in breast cancer cell lines and tumours. Using this assay, we were able to identify gene amplifications and deletions genome-wide and with high resolution, and compare alterations in DNA copy number and gene expression.     

Integrated genomic and epigenomic analyses pinpoint biallelic gene inactivation in tumors

Aberrant methylation of CpG islands and genomic deletion are two predominant mechanisms of gene inactivation in tumorigenesis, but the extent to which they interact is largely unknown. The lack of an integrated approach to study these mechanisms has limited the understanding of tumor genomes and cancer genes. Restriction landmark genomic scanning (RLGS; ref. 1) is useful for global analysis of aberrant methylation of CpG islands, but has not been amenable to alignment with deletion maps because the identity of most RLGS fragments is unknown. Here, we determined the nucleotide sequence and exact chromosomal position of RLGS fragments throughout the genome using the whole chromosome of origin of the fragments and in silico restriction digestion of the human genome sequence. To study the interaction of these gene-inactivation mechanisms in primary brain tumors, we integrated RLGS-based methylation analysis with high-resolution deletion maps from microarray-based comparative genomic hybridization (array CGH; ref. 3). Certain subsets of gene-associated CpG islands were preferentially affected by convergent methylation and deletion, including genes that exhibit tumor-suppressor activity, such as CISH1 (encoding SOCS1; ref. 4), as well as genes such as COE3 that have been missed by traditional non-integrated approaches. Our results show that most aberrant methylation events are focal and independent of deletions, and the rare convergence of these mechanisms can pinpoint biallelic gene inactivation without the use of positional cloning.     

Microdeletion encompassing MAPT at chromosome 17q21.3 is associated with developmental delay and learning disability

Recently, the application of array-based comparative genomic hybridization (array CGH) has improved rates of detection of chromosomal imbalances in individuals with mental retardation and dysmorphic features. Here, we describe three individuals with learning disability and a heterozygous deletion at chromosome 17q21.3, detected in each case by array CGH. FISH analysis demonstrated that the deletions occurred as de novo events in each individual and were between 500 kb and 650 kb in size. A recently described 900-kb inversion that suppresses recombination between ancestral H1 and H2 haplotypes encompasses the deletion. We show that, in each trio, the parent of origin of the deleted chromosome 17 carries at least one H2 chromosome. This region of 17q21.3 shows complex genomic architecture with well-described low-copy repeats (LCRs). The orientation of LCRs flanking the deleted segment in inversion heterozygotes is likely to facilitate the generation of this microdeletion by means of non-allelic homologous recombination.     

Mining the human genome using microarrays of open reading frames

To test the hypothesis that the human genome project will uncover many genes not previously discovered by sequencing of expressed sequence tags (ESTs), we designed and produced a set of microarrays using probes based on open reading frames (ORFs) in 350 Mb of finished and draft human sequence. Our approach aims to identify all genes directly from genomic sequence by querying gene expression. We analysed genomic sequence with a suite of ORF prediction programs, selected approximately one ORF per gene, amplified the ORFs from genomic DNA and arrayed the amplicons onto treated glass slides. Of the first 10,000 arrayed ORFs, 31% are completely novel and 29% are similar, but not identical, to sequences in public databases. Approximately one-half of these are expressed in the tissues we queried by microarray. Subsequent verification by other techniques confirmed expression of several of the novel genes. Expressed sequence tags (ESTs) have yielded vast amounts of data, but our results indicate that many genes in the human genome will only be found by genomic sequencing.     

Radiation hybrid map of the mouse genome.

Radiation hybrid (RH) maps are a useful tool for genome analysis, providing a direct method for localizing genes and anchoring physical maps and genomic sequence along chromosomes. The construction of a comprehensive RH map for the human genome has resulted in gene maps reflecting the location of more than 30,000 human genes. Here we report the first comprehensive RH map of the mouse genome. The map contains 2,486 loci screened against an RH panel of 93 cell lines. Most loci (93%) are simple sequence length polymorphisms (SSLPs) taken from the mouse genetic map, thereby providing direct integration between these two key maps. We performed RH mapping by a new and efficient approach in which we replaced traditional gel- or hybridization-based assays by a homogeneous 5'-nuclease assays involving a single common probe for all genetic markers. The map provides essentially complete connectivity and coverage across the genome, and good resolution for ordering loci, with 1 centiRay (cR) corresponding to an average of approximately 100 kb. The RH map, together with an accompanying World-Wide Web server, makes it possible for any investigator to rapidly localize sequences in the mouse genome. Together with the previously constructed genetic map and a YAC-based physical map reported in a companion paper, the fundamental maps required for mouse genomics are now available.     

Dichotomy of single-nucleotide polymorphism haplotypes in olfactory receptor genes and pseudogenes

Substantial efforts are focused on identifying single-nucleotide polymorphisms (SNPs) throughout the human genome, particularly in coding regions (cSNPs), for both linkage disequilibrium and association studies. Less attention, however, has been directed to the clarification of evolutionary processes that are responsible for the variability in nucleotide diversity among different regions of the genome. We report here the population sequence diversity of genomic segments within a 450-kb cluster of olfactory receptor (OR) genes on human chromosome 17. We found a dichotomy in the pattern of nucleotide diversity between OR pseudogenes and introns on the one hand and the closely interspersed intact genes on the other. We suggest that weak positive selection is responsible for the observed patterns of genetic variation. This is inferred from a lower ratio of polymorphism to divergence in genes compared with pseudogenes or introns, high non-synonymous substitution rates in OR genes, and a small but significant overall reduction in variability in the entire OR gene cluster compared with other genomic regions. The dichotomy among functionally different segments within a short genomic distance requires high recombination rates within this OR cluster. Our work demonstrates the impact of weak positive selection on human nucleotide diversity, and has implications for the evolution of the olfactory repertoire.     

Using FlyAtlas to identify better Drosophila melanogaster models of human disease

FlyAtlas, a new online resource, provides the most comprehensive view yet of expression in multiple tissues of Drosophila melanogaster. Meta-analysis of the data shows that a significant fraction of the genome is expressed with great tissue specificity in the adult, demonstrating the need for the functional genomic community to embrace a wide range of functional phenotypes. Well-known developmental genes are often reused in surprising tissues in the adult, suggesting new functions. The homologs of many human genetic disease loci show selective expression in the Drosophila tissues analogous to the affected human tissues, providing a useful filter for potential candidate genes. Additionally, the contributions of each tissue to the whole-fly array signal can be calculated, demonstrating the limitations of whole-organism approaches to functional genomics and allowing modeling of a simple tissue fractionation procedure that should improve detection of weak or tissue-specific signals.     

A copy number variation morbidity map of developmental delay

To understand the genetic heterogeneity underlying developmental delay, we compared copy number variants (CNVs) in 15,767 children with intellectual disability and various congenital defects (cases) to CNVs in 8,329 unaffected adult controls. We estimate that ～14.2% of disease in these children is caused by CNVs >400 kb. We observed a greater enrichment of CNVs in individuals with craniofacial anomalies and cardiovascular defects compared to those with epilepsy or autism. We identified 59 pathogenic CNVs, including 14 new or previously weakly supported candidates, refined the critical interval for several genomic disorders, such as the 17q21.31 microdeletion syndrome, and identified 940 candidate dosage-sensitive genes. We also developed methods to opportunistically discover small, disruptive CNVs within the large and growing diagnostic array datasets. This evolving CNV morbidity map, combined with exome and genome sequencing, will be critical for deciphering the genetic basis of developmental delay, intellectual disability and autism spectrum disorders.     

Large-scale analysis of the regulatory architecture of the mouse genome with a transposon-associated sensor

We present here a Sleeping Beauty-based transposition system that offers a simple and efficient way to investigate the regulatory architecture of mammalian chromosomes in vivo. With this system, we generated several hundred mice and embryos, each with a regulatory sensor inserted at a random genomic position. This large sampling of the genome revealed the widespread presence of long-range regulatory activities along chromosomes, forming overlapping blocks with distinct tissue-specific expression potentials. The presence of tissue-restricted regulatory activities around genes with widespread expression patterns challenges the gene-centric view of genome regulation and suggests that most genes are modulated in a tissue-specific manner. The local hopping property of Sleeping Beauty provides a dynamic approach to map these regulatory domains at high resolution and, combined with Cre-mediated recombination, allows for the determination of their functions by engineering mice with specific chromosomal rearrangements.     

Integrated detection and population-genetic analysis of SNPs and copy number variation

Dissecting the genetic basis of disease risk requires measuring all forms of genetic variation, including SNPs and copy number variants (CNVs), and is enabled by accurate maps of their locations, frequencies and population-genetic properties. We designed a hybrid genotyping array (Affymetrix SNP 6.0) to simultaneously measure 906,600 SNPs and copy number at 1.8 million genomic locations. By characterizing 270 HapMap samples, we developed a map of human CNV (at 2-kb breakpoint resolution) informed by integer genotypes for 1,320 copy number polymorphisms (CNPs) that segregate at an allele frequency >1%. More than 80% of the sequence in previously reported CNV regions fell outside our estimated CNV boundaries, indicating that large (>100 kb) CNVs affect much less of the genome than initially reported. Approximately 80% of observed copy number differences between pairs of individuals were due to common CNPs with an allele frequency >5%, and more than 99% derived from inheritance rather than new mutation. Most common, diallelic CNPs were in strong linkage disequilibrium with SNPs, and most low-frequency CNVs segregated on specific SNP haplotypes.     

Quantitative mapping of amplicon structure by array CGH identifies CYP24 as a candidate oncogene

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.     

The multidrug-resistant human pathogen Clostridium difficile has a highly mobile, mosaic genome

We determined the complete genome sequence of Clostridium difficile strain 630, a virulent and multidrug-resistant strain. Our analysis indicates that a large proportion (11%) of the genome consists of mobile genetic elements, mainly in the form of conjugative transposons. These mobile elements are putatively responsible for the acquisition by C. difficile of an extensive array of genes involved in antimicrobial resistance, virulence, host interaction and the production of surface structures. The metabolic capabilities encoded in the genome show multiple adaptations for survival and growth within the gut environment. The extreme genome variability was confirmed by whole-genome microarray analysis; it may reflect the organism's niche in the gut and should provide information on the evolution of virulence in this organism.     

Discovery of previously unidentified genomic disorders from the duplication architecture of the human genome

Genomic disorders are characterized by the presence of flanking segmental duplications that predispose these regions to recurrent rearrangement. Based on the duplication architecture of the genome, we investigated 130 regions that we hypothesized as candidates for previously undescribed genomic disorders. We tested 290 individuals with mental retardation by BAC array comparative genomic hybridization and identified 16 pathogenic rearrangements, including de novo microdeletions of 17q21.31 found in four individuals. Using oligonucleotide arrays, we refined the breakpoints of this microdeletion, defining a 478-kb critical region containing six genes that were deleted in all four individuals. We mapped the breakpoints of this deletion and of four other pathogenic rearrangements in 1q21.1, 15q13, 15q24 and 17q12 to flanking segmental duplications, suggesting that these are also sites of recurrent rearrangement. In common with the 17q21.31 deletion, these breakpoint regions are sites of copy number polymorphism in controls, indicating that these may be inherently unstable genomic regions.     

Genomic surveys by methylation-sensitive SNP analysis identify sequence-dependent allele-specific DNA methylation

Allele-specific DNA methylation (ASM) is a hallmark of imprinted genes, but ASM in the larger nonimprinted fraction of the genome is less well characterized. Using methylation-sensitive SNP analysis (MSNP), we surveyed the human genome at 50K and 250K resolution, identifying ASM as recurrent genotype call conversions from heterozygosity to homozygosity when genomic DNAs were predigested with the methylation-sensitive restriction enzyme HpaII. Using independent assays, we confirmed ASM at 16 SNP-tagged loci distributed across various chromosomes. At 12 of these loci (75%), the ASM tracked strongly with the sequence of adjacent SNPs. Further analysis showed allele-specific mRNA expression at two loci from this methylation-based screen--the vanin and CYP2A6-CYP2A7 gene clusters--both implicated in traits of medical importance. This recurrent phenomenon of sequence-dependent ASM has practical implications for mapping and interpreting associations of noncoding SNPs and haplotypes with human phenotypes.