Whole-genome sequencing reveals activation-induced cytidine deaminase signatures during indolent chronic lymphocytic leukaemia evolution.

Patients with chromosome 13q deletion or normal cytogenetics represent the majority of chronic lymphocytic leukaemia (CLL) cases, yet have relatively few driver mutations. To better understand their genomic landscape, here we perform whole-genome sequencing on a cohort of patients enriched with these cytogenetic characteristics. Mutations in known CLL drivers are seen in only 33% of this cohort, and associated with normal cytogenetics and unmutated IGHV. The most commonly mutated gene in our cohort, IGLL5, shows a mutational pattern suggestive of activation-induced cytidine deaminase (AID) activity. Unsupervised analysis of mutational signatures demonstrates the activities of canonical AID (c-AID), leading to clustered mutations near active transcriptional start sites; non-canonical AID (nc-AID), leading to genome-wide non-clustered mutations, and an ageing signature responsible for most mutations. Using mutation clonality to infer time of onset, we find that while ageing and c-AID activities are ongoing, nc-AID-associated mutations likely occur earlier in tumour evolution.

Multiclass cancer diagnosis using tumor gene expression signatures.

The optimal treatment of patients with cancer depends on establishing accurate diagnoses by using a complex combination of clinical and histopathological data. In some instances, this task is difficult or impossible because of atypical clinical presentation or histopathology. To determine whether the diagnosis of multiple common adult malignancies could be achieved purely by molecular classification, we subjected 218 tumor samples, spanning 14 common tumor types, and 90 normal tissue samples to oligonucleotide microarray gene expression analysis. The expression levels of 16,063 genes and expressed sequence tags were used to evaluate the accuracy of a multiclass classifier based on a support vector machine algorithm. Overall classification accuracy was 78%, far exceeding the accuracy of random classification (9%). Poorly differentiated cancers resulted in low-confidence predictions and could not be accurately classified according to their tissue of origin, indicating that they are molecularly distinct entities with dramatically different gene expression patterns compared with their well differentiated counterparts. Taken together, these results demonstrate the feasibility of accurate, multiclass molecular cancer classification and suggest a strategy for future clinical implementation of molecular cancer diagnostics.

Classification of human lung carcinomas by mRNA expression profiling reveals distinct adenocarcinoma subclasses.

We have generated a molecular taxonomy of lung carcinoma, the leading cause of cancer death in the United States and worldwide. Using oligonucleotide microarrays, we analyzed mRNA expression levels corresponding to 12,600 transcript sequences in 186 lung tumor samples, including 139 adenocarcinomas resected from the lung. Hierarchical and probabilistic clustering of expression data defined distinct subclasses of lung adenocarcinoma. Among these were tumors with high relative expression of neuroendocrine genes and of type II pneumocyte genes, respectively. Retrospective analysis revealed a less favorable outcome for the adenocarcinomas with neuroendocrine gene expression. The diagnostic potential of expression profiling is emphasized by its ability to discriminate primary lung adenocarcinomas from metastases of extra-pulmonary origin. These results suggest that integration of expression profile data with clinical parameters could aid in diagnosis of lung cancer patients.

A radiation hybrid map of mouse genes.

A comprehensive gene-based map of a genome is a powerful tool for genetic studies and is especially useful for the positional cloning and positional candidate approaches. The availability of gene maps for multiple organisms provides the foundation for detailed conserved-orthology maps showing the correspondence between conserved genomic segments. These maps make it possible to use cross-species information in gene hunts and shed light on the evolutionary forces that shape the genome. Here we report a radiation hybrid map of mouse genes, a combined project of the Whitehead Institute/Massachusetts Institute of Technology Center for Genome Research, the Medical Research Council UK Mouse Genome Centre, and the National Center for Biotechnology Information. The map contains 11,109 genes, screened against the T31 RH panel and positioned relative to a reference map containing 2,280 mouse genetic markers. It includes 3,658 genes homologous to the human genome sequence and provides a framework for overlaying the human genome sequence to the mouse and for sequencing the mouse genome.

Genetic variation in the 5q31 cytokine gene cluster confers susceptibility to Crohn disease.

Linkage disequilibrium (LD) mapping provides a powerful method for fine-structure localization of rare disease genes, but has not yet been widely applied to common disease. We sought to design a systematic approach for LD mapping and apply it to the localization of a gene (IBD5) conferring susceptibility to Crohn disease. The key issues are: (i) to detect a significant LD signal (ii) to rigorously bound the critical region and (iii) to identify the causal genetic variant within this region. We previously mapped the IBD5 locus to a large region spanning 18 cM of chromosome 5q31 (P<10(-4)). Using dense genetic maps of microsatellite markers and single-nucleotide polymorphisms (SNPs) across the entire region, we found strong evidence of LD. We bound the region to a common haplotype spanning 250 kb that shows strong association with the disease (P< 2 x 10(-7)) and contains the cytokine gene cluster. This finding provides overwhelming evidence that a specific common haplotype of the cytokine region in 5q31 confers susceptibility to Crohn disease. However, genetic evidence alone is not sufficient to identify the causal mutation within this region, as strong LD across the region results in multiple SNPs having equivalent genetic evidence-each consistent with the expected properties of the IBD5 locus. These results have important implications for Crohn disease in particular and LD mapping in general.

Chemosensitivity prediction by transcriptional profiling.

In an effort to develop a genomics-based approach to the prediction of drug response, we have developed an algorithm for classification of cell line chemosensitivity based on gene expression profiles alone. Using oligonucleotide microarrays, the expression levels of 6,817 genes were measured in a panel of 60 human cancer cell lines (the NCI-60) for which the chemosensitivity profiles of thousands of chemical compounds have been determined. We sought to determine whether the gene expression signatures of untreated cells were sufficient for the prediction of chemosensitivity. Gene expression-based classifiers of sensitivity or resistance for 232 compounds were generated and then evaluated on independent sets of data. The classifiers were designed to be independent of the cells' tissue of origin. The accuracy of chemosensitivity prediction was considerably better than would be expected by chance. Eighty-eight of 232 expression-based classifiers performed accurately (with P < 0.05) on an independent test set, whereas only 12 of the 232 would be expected to do so by chance. These results suggest that at least for a subset of compounds genomic approaches to chemosensitivity prediction are feasible.

Association analysis of NOTCH4 loci in schizophrenia using family and population-based controls.

A genetic association between NOTCH4 and schizophrenia has previously been proposed. Unsing all markers previously shown to be associated, we found no evidence for such in three independent family-based samples (n=519 parent-offspring trios), and a case-control sample derived from the same ethnic background as the original observation. These data strongly suggest that NOTCH4 is not a significant susceptibility allele for schizophrenia.

Growth factor-specific signaling pathway stimulation and gene expression mediated by ErbB receptors.

The mechanisms by which receptor tyrosine kinases (RTKs) utilize intracellular signaling pathways to direct gene expression and cellular response remain unclear. A current question is whether different RTKs within a single cell target similar or different sets of genes. In this study we have used the ErbB receptor network to explore the relationship between RTK activation and gene expression. We profiled growth factor-stimulated signaling pathway usage and broad gene expression patterns in two human mammary tumor cell lines expressing different complements of ErbB receptors. Although the growth factors epidermal growth factor (EGF) and neuregulin (NRG) 1 similarly stimulated Erk1/2 in MDA-MB-361 cells, EGF acting through an EGF receptor/ErbB2 heterodimer preferentially stimulated protein kinase C, and NRG1beta acting through an ErbB2/ErbB3 heterodimer preferentially stimulated Akt. The two growth factors regulated partially overlapping yet distinct sets of genes in these cells. In MDA-MB-453 cells, NRG1beta acting through an ErbB2/ErbB3 heterodimer stimulated prolonged signaling of all pathways examined relative to NRG2beta acting through the same heterodimeric receptor species. Surprisingly, NRG1beta and NRG2beta also regulated partially overlapping but distinct sets of genes in these cells. These results demonstrate that the activation of different RTKs, or activation of the same RTKs with different ligands, can lead to distinct profiles of gene regulation within a single cell type. Our observations also suggest that the identity and kinetics of signaling pathway usage by RTKs may play a role in the selection of regulated genes.

Deletion of cytosolic phospholipase A(2) suppresses Apc(Min)-induced tumorigenesis.

Although nonsteroidal antiinflammatory drugs (NSAIDs) show great promise as therapies for colon cancer, a dispute remains regarding their mechanism of action. NSAIDs are known to inhibit cyclooxygenase (COX) enzymes, which convert arachidonic acid (AA) to prostaglandins (PGs). Therefore, NSAIDs may suppress tumorigenesis by inhibiting PG synthesis. However, various experimental studies have suggested the possibility of PG-independent mechanisms. Notably, disruption of the mouse group IIA secretory phospholipase A(2) locus (Pla2g2a), a potential source of AA for COX-2, increases tumor number despite the fact that the mutation has been predicted to decrease PG production. Some authors have attempted to reconcile the results by suggesting that the level of the precursor (AA), not the products (PGs), is the critical factor. To clarify the role of AA in tumorigenesis, we have examined the effect of deleting the group IV cytosolic phospholipase A(2) (cPLA(2)) locus (Pla2g4). We report that Apc(Min/+), cPLA(2)(-/-) mice show an 83% reduction in tumor number in the small intestine compared with littermates with genotypes Apc(Min/+), cPLA(2)(+/-) and Apc(Min/+), cPLA(2)(+/+). This tumor phenotype parallels that of COX-2 knockout mice, suggesting that cPLA(2) is the predominant source of AA for COX-2 in the intestine. The protective effect of cPLA(2) deletion is thus most likely attributed to a decrease in the AA supply to COX-2 and a resultant decrease in PG synthesis. The tumorigenic effect of sPLA(2) mutations is likely to be through a completely different pathway.

Axonemal beta heavy chain dynein DNAH9: cDNA sequence, genomic structure, and investigation of its role in primary ciliary dyskinesia.

Dyneins are multisubunit protein complexes that couple ATPase activity with conformational changes. They are involved in the cytoplasmatic movement of organelles (cytoplasmic dyneins) and the bending of cilia and flagella (axonemal dyneins). Here we present the first complete cDNA and genomic sequences of a human axonemal dynein beta heavy chain gene, DNAH9, which maps to 17p12. The 14-kb-long cDNA is divided into 69 exons spread over 390 kb. The cDNA sequence of DNAH9 was determined using a combination of methods including 5' rapid amplification of cDNA ends, RT-PCR, and cDNA library screening. RT-PCR using nasal epithelium and testis RNA revealed several alternatively spliced transcripts. The genomic structure was determined using three overlapping BACs sequenced by the Whitehead Institute/MIT Center for Genome Research. The predicted protein, of 4486 amino acids, is highly homologous to sea urchin axonemal beta heavy chain dyneins (67% identity). It consists of an N-terminal stem and a globular C-terminus containing the four P-loops that constitute the motor domain. Lack of proper ciliary and flagellar movement characterizes primary ciliary dyskinesia (PCD), a genetically heterogeneous autosomal recessive disorder with respiratory tract infections, bronchiectasis, male subfertility, and, in 50% of cases, situs inversus (Kartagener syndrome, KS). Dyneins are excellent candidate genes for PCD and KS because in over 50% of cases the ultrastructural defects of cilia are related to the dynein complex. Genotype analysis was performed in 31 PCD families with two or more affected siblings using a highly informative dinucleotide polymorphism located in intron 26 of DNAH9. Two families with concordant inheritance of DNAH9 alleles in affected individuals were observed. A mutation search was performed in these two "candidate families," but only polymorphic variants were found. In the absence of pathogenic mutations, the DNAH9 gene has been excluded as being responsible for autosomal recessive PCD in these families.

Loss-of-heterozygosity analysis of small-cell lung carcinomas using single-nucleotide polymorphism arrays.

Human cancers arise by a combination of discrete mutations and chromosomal alterations. Loss of heterozygosity (LOH) of chromosomal regions bearing mutated tumor suppressor genes is a key event in the evolution of epithelial and mesenchymal tumors. Global patterns of LOH can be understood through allelotyping of tumors with polymorphic genetic markers. Simple sequence length polymorphisms (SSLPs, or microsatellites) are reliable genetic markers for studying LOH, but only a modest number of SSLPs are used in LOH studies because the genotyping procedure is rather tedious. Here, we report the use of a highly parallel approach to genotype large numbers of single-nucleotide polymorphisms (SNPs) for LOH, in which samples are genotyped for nearly 1,500 loci by performing 24 polymerase chain reactions (PCR), pooling the resulting amplification products and hybridizing the mixture to a high-density oligonucleotide array. We characterize the results of LOH analyses on human small-cell lung cancer (SCLC) and control DNA samples by hybridization. We show that the patterns of LOH are consistent with those obtained by analysis with both SSLPs and comparative genomic hybridization (CGH), whereas amplifications rarely are detected by the SNP array. The results validate the use of SNP array hybridization for tumor studies.

Genomic analysis of metastasis reveals an essential role for RhoC.

The most damaging change during cancer progression is the switch from a locally growing tumour to a metastatic killer. This switch is believed to involve numerous alterations that allow tumour cells to complete the complex series of events needed for metastasis. Relatively few genes have been implicated in these events. Here we use an in vivo selection scheme to select highly metastatic melanoma cells. By analysing these cells on DNA arrays, we define a pattern of gene expression that correlates with progression to a metastatic phenotype. In particular, we show enhanced expression of several genes involved in extracellular matrix assembly and of a second set of genes that regulate, either directly or indirectly, the actin-based cytoskeleton. One of these, the small GTPase RhoC, enhances metastasis when overexpressed, whereas a dominant-negative Rho inhibits metastasis. Analysis of the phenotype of cells expressing dominant-negative Rho or RhoC indicates that RhoC is important in tumour cell invasion. The genomic approach allows us to identify families of genes involved in a process, not just single genes, and can indicate which molecular and cellular events might be important in complex biological processes such as metastasis.

The Mom1AKR intestinal tumor resistance region consists of Pla2g2a and a locus distal to D4Mit64.

The Mom1 (Modifier of Min-1) region of distal chromosome 4 was identified during a screen for polymorphic modifiers of intestinal tumorigenesis in ApcMin/+ mice. Here, we demonstrate that the Mom1AKR allele consists of two genetic components. These include the secretory phospholipase Pla2g2a, whose candidacy as a Mom1 resistance modifier has now been tested with several transgenic lines. A second region, distal to Pla2g2a, has also been identified using fine structure recombinants. Pla2g2aAKR transgenic mice demonstrate a modest resistance to tumorigenesis in the small intestine and a very robust resistance in the large intestine. Moreover, the tumor resistance in the colon of Pla2g2aAKR animals is dosage-dependent, a finding that is consistent with our observation that Pla2g2a is expressed in goblet cells. By contrast, mice carrying the distal Mom1 modifier demonstrate a modest tumor resistance that is confined to the small intestine. Thus, the phenotypes of these two modifier loci are complementary, both in their quantitative and regional effects. The additive effects and tight linkage of these modifiers may have been necessary for the initial identification of the Mom1 region.

Genomewide search in Canadian families with inflammatory bowel disease reveals two novel susceptibility loci.

The chronic inflammatory bowel diseases (IBDs)-Crohn disease (CD) and ulcerative colitis (UC)-are idiopathic, inflammatory disorders of the gastrointestinal tract. These conditions have a peak incidence in early adulthood and a combined prevalence of approximately 100-200/100,000. Although the etiology of IBD is multifactorial, a significant genetic contribution to disease susceptibility is implied by epidemiological data revealing a sibling risk of approximately 35-fold for CD and approximately 15-fold for UC. To elucidate the genetic basis for these disorders, we undertook a genomewide scan in 158 Canadian sib-pair families and identified three regions of suggestive linkage (3p, 5q31-33, and 6p) and one region of significant linkage to 19p13 (LOD score 4.6). Higher-density mapping in the 5q31-q33 region revealed a locus of genomewide significance (LOD score 3.9) that contributes to CD susceptibility in families with early-onset disease. Both of these genomic regions contain numerous genes that are important to the immune and inflammatory systems and that provide good targets for future candidate-gene studies.

Expression analysis with oligonucleotide microarrays reveals that MYC regulates genes involved in growth, cell cycle, signaling, and adhesion.

MYC affects normal and neoplastic cell proliferation by altering gene expression, but the precise pathways remain unclear. We used oligonucleotide microarray analysis of 6,416 genes and expressed sequence tags to determine changes in gene expression caused by activation of c-MYC in primary human fibroblasts. In these experiments, 27 genes were consistently induced, and 9 genes were repressed. The identity of the genes revealed that MYC may affect many aspects of cell physiology altered in transformed cells: cell growth, cell cycle, adhesion, and cytoskeletal organization. Identified targets possibly linked to MYC's effects on cell growth include the nucleolar proteins nucleolin and fibrillarin, as well as the eukaryotic initiation factor 5A. Among the cell cycle genes identified as targets, the G1 cyclin D2 and the cyclin-dependent kinase binding protein CksHs2 were induced whereas the cyclin-dependent kinase inhibitor p21(Cip1) was repressed. A role for MYC in regulating cell adhesion and structure is suggested by repression of genes encoding the extracellular matrix proteins fibronectin and collagen, and the cytoskeletal protein tropomyosin. A possible mechanism for MYC-mediated apoptosis was revealed by identification of the tumor necrosis factor receptor associated protein TRAP1 as a MYC target. Finally, two immunophilins, peptidyl-prolyl cis-trans isomerase F and FKBP52, the latter of which plays a role in cell division in Arabidopsis, were up-regulated by MYC. We also explored pattern-matching methods as an alternative approach for identifying MYC target genes. The genes that displayed an expression profile most similar to endogenous Myc in microarray-based expression profiling of myeloid differentiation models were highly enriched for MYC target genes.

Molecular classification of cancer: class discovery and class prediction by gene expression monitoring.

Although cancer classification has improved over the past 30 years, there has been no general approach for identifying new cancer classes (class discovery) or for assigning tumors to known classes (class prediction). Here, a generic approach to cancer classification based on gene expression monitoring by DNA microarrays is described and applied to human acute leukemias as a test case. A class discovery procedure automatically discovered the distinction between acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) without previous knowledge of these classes. An automatically derived class predictor was able to determine the class of new leukemia cases. The results demonstrate the feasibility of cancer classification based solely on gene expression monitoring and suggest a general strategy for discovering and predicting cancer classes for other types of cancer, independent of previous biological knowledge.

Identification of the Finnish founder mutation for diastrophic dysplasia (DTD).

Diastrophic dysplasia (DTD) is especially prevalent in Finland and the existence of a founder mutation has been previously inferred from the fact that 95% of Finnish DTD chromosomes have a rare ancestral haplotype found in only 4% of Finnish control chromosomes. Here we report the identification of the Finnish founder mutation as a GT-> GC transition (c.-26 + 2T > C) in the splice donor site of a previously undescribed 5'-untranslated exon of the diastrophic dysplasia sulfate transporter gene (DTDST); the mutation acts by severely reducing mRNA levels. Among 84 DTD families in Finland, patients carried two copies of the mutation in 69 families, one copy in 14 families, and no copies in one family. Roughly 90% of Finnish DTD chromosomes thus carry the splice-site mutation, which we have designated DTDST(Fin). Unexpectedly, we found that nine of the DTD chromosomes having the apparently ancestral haplotype did not carry DTDST(Fin), but rather two other mutations. Eight such chromosomes had an R279W mutation and one had a V340del deletion. We consider the possible implications of presence of multiple DTD mutations on this rare haplotype.

Transcript identification on the CLN5 region on chromosome 13q22.

Our efforts to clone the CLN5 gene, mutated in a severe children's brain disease, variant late infantile neuronal ceroid lipofuscinosis (vLINCL, MIM256731), resulted in large-scale sequencing of genomic clones flanking the critical chromosomal region on 13q22. Computational and traditional transcript identification analyses of the resulting sequence were used to identify the disease gene. In addition to the identification of the CLN5 gene, this effort produced a large amount of genomic sequence data. Here, we report a transcription map of the 107 kb sequence in the CLN5 region, based on traditional and computational gene identification strategies. Several transcripts were identified in this sequence. Queries against the database of expressed sequence tags proved to be the most powerful tool for gene identification from large-scale sequence.

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.