Identification of somatic mutations in non-small cell lung carcinomas using whole-exome sequencing.

Lung cancer is the leading cause of cancer-related death, with non-small cell lung cancer (NSCLC) being the predominant form of the disease. Most lung cancer is caused by the accumulation of genomic alterations due to tobacco exposure. To uncover its mutational landscape, we performed whole-exome sequencing in 31 NSCLCs and their matched normal tissue samples. We identified both common and unique mutation spectra and pathway activation in lung adenocarcinomas and squamous cell carcinomas, two major histologies in NSCLC. In addition to identifying previously known lung cancer genes (TP53, KRAS, EGFR, CDKN2A and RB1), the analysis revealed many genes not previously implicated in this malignancy. Notably, a novel gene CSMD3 was identified as the second most frequently mutated gene (next to TP53) in lung cancer. We further demonstrated that loss of CSMD3 results in increased proliferation of airway epithelial cells. The study provides unprecedented insights into mutational processes, cellular pathways and gene networks associated with lung cancer. Of potential immediate clinical relevance, several highly mutated genes identified in our study are promising druggable targets in cancer therapy including ALK, CTNNA3, DCC, MLL3, PCDHIIX, PIK3C2B, PIK3CG and ROCK2.

Acquired copy number alterations in adult acute myeloid leukemia genomes.

Cytogenetic analysis of acute myeloid leukemia (AML) cells has accelerated the identification of genes important for AML pathogenesis. To complement cytogenetic studies and to identify genes altered in AML genomes, we performed genome-wide copy number analysis with paired normal and tumor DNA obtained from 86 adult patients with de novo AML using 1.85 million feature SNP arrays. Acquired copy number alterations (CNAs) were confirmed using an ultra-dense array comparative genomic hybridization platform. A total of 201 somatic CNAs were found in the 86 AML genomes (mean, 2.34 CNAs per genome), with French-American-British system M6 and M7 genomes containing the most changes (10-29 CNAs per genome). Twenty-four percent of AML patients with normal cytogenetics had CNA, whereas 40% of patients with an abnormal karyotype had additional CNA detected by SNP array, and several CNA regions were recurrent. The mRNA expression levels of 57 genes were significantly altered in 27 of 50 recurrent CNA regions <5 megabases in size. A total of 8 uniparental disomy (UPD) segments were identified in the 86 genomes; 6 of 8 UPD calls occurred in samples with a normal karyotype. Collectively, 34 of 86 AML genomes (40%) contained alterations not found with cytogenetics, and 98% of these regions contained genes. Of 86 genomes, 43 (50%) had no CNA or UPD at this level of resolution. In this study of 86 adult AML genomes, the use of an unbiased high-resolution genomic screen identified many genes not previously implicated in AML that may be relevant for pathogenesis, along with many known oncogenes and tumor suppressor genes.

Dynamic histone H3 epigenome marking during the intraerythrocytic cycle of Plasmodium falciparum.

Epigenome profiling has led to the paradigm that promoters of active genes are decorated with H3K4me3 and H3K9ac marks. To explore the epigenome of Plasmodium falciparum asexual stages, we performed MS analysis of histone modifications and found a general preponderance of H3/H4 acetylation and H3K4me3. ChIP-on-chip profiling of H3, H3K4me3, H3K9me3, and H3K9ac from asynchronous parasites revealed an extensively euchromatic epigenome with heterochromatin restricted to variant surface antigen gene families (VSA) and a number of genes hitherto unlinked to VSA. Remarkably, the vast majority of the genome shows an unexpected pattern of enrichment of H3K4me3 and H3K9ac. Analysis of synchronized parasites revealed significant developmental stage specificity of the epigenome. In rings, H3K4me3 and H3K9ac are homogenous across the genes marking active and inactive genes equally, whereas in schizonts, they are enriched at the 5' end of active genes. This study reveals an unforeseen and unique plasticity in the use of the epigenetic marks and implies the presence of distinct epigenetic pathways in gene silencing/activation throughout the erythrocytic cycle.

Perioperative genomic profiles using structure-specific oligonucleotide probes.

OBJECTIVES: Many complications in the perioperative interval are associated with genetic susceptibilities that may be unknown in advance of surgery and anesthesia, including drug toxicity and inefficacy, thrombosis, prolonged neuromuscular blockade, organ failure and sepsis. The aims of this study were to design and validate the first genetic testing platform and panel designed for use in perioperative care, to establish allele frequencies in a target population, and to determine the number of mutant alleles per patient undergoing surgery. DESIGN/SETTING/PARTICIPANTS AND METHODS: One hundred fifty patients at Marshfield Clinic, Marshfield, Wisconsin, 100 patients at the Medical College of Wisconsin Zablocki Veteran's Administration Medical Center, Milwaukee, Wisconsin, and 200 patients at the University of Wisconsin Hospitals and Clinics, Madison, Wisconsin undergoing surgery and anesthesia were tested for 48 polymorphisms in 22 genes including ABC, BChE, ACE, CYP2C9, CYP2C19, CYP2D6, CYP3A4, CYP3A5, beta2AR, TPMT, F2, F5, F7, MTHFR, TNFalpha, TNFbeta, CCR5, ApoE, HBB, MYH7, ABO and Gender (PRKY, PFKFB1). Using structure-specific cleavage of oligonucleotide probes (Invader, Third Wave Technologies, Inc., Madison, WI), 96-well plates were configured so that each well contained reagents for detection of both the wild type and mutant alleles at each locus. RESULTS: There were 21,600 genotypes confirmed in duplicate. After withdrawal of polymorphisms in non-pathogenic genes (i.e., the ABO blood group and gender-specific alleles), 376 of 450 patients were found to be homozygous for mutant alleles at one or more loci. Modes of two mutant homozygous loci and 10 mutant alleles in aggregate (i.e., the sum of homozygous and heterozygous mutant polymorphisms) were observed per patient. CONCLUSIONS: Significant genetic heterogeneity that may not be accounted for by taking a family medical history, or by obtaining routine laboratory test results, is present in most patients presenting for surgery and may be detected using a newly developed genotyping platform.

High-resolution genome-wide cytosine methylation profiling with simultaneous copy number analysis and optimization for limited cell numbers.

Many genome-wide assays involve the generation of a subset (or representation) of the genome following restriction enzyme digestion. The use of enzymes sensitive to cytosine methylation allows high-throughput analysis of this epigenetic regulatory process. We show that the use of a dual-adapter approach allows us to generate genomic representations that includes fragments of <200 bp in size, previously not possible when using the standard approach of using a single adapter. By expanding the representation to smaller fragments using HpaII or MspI, we increase the representation by these isoschizomers to more than 1.32 million loci in the human genome, representing 98.5% of CpG islands and 91.1% of refSeq promoters. This advance allows the development of a new, high-resolution version of our HpaII-tiny fragment Enrichment by Ligation-mediated PCR (HELP) assay to study cytosine methylation. We also show that the MspI representation generates information about copy-number variation, that the assay can be used on as little as 10 ng of DNA and that massively parallel sequencing can be used as an alternative to microarrays to read the output of the assay, making this a powerful discovery platform for studies of genomic and epigenomic abnormalities.

Chromosome 12p deletions in TEL-AML1 childhood acute lymphoblastic leukemia are associated with retrotransposon elements and occur postnatally.

TEL-AML1 (ETV6-RUNX1) is the most common translocation in the childhood leukemias, and is a prenatal mutation in most children. This translocation has been detected at a high rate among newborns ( approximately 1%); therefore, the rate-limiting event for leukemia seems to be secondary mutations. One such frequent mutation in this subtype is partial deletion of chromosome 12p, trans from the translocation. Nine del(12p) breakpoints within six leukemia cases were sequenced to explore the etiology of this genetic event, and most involved cryptic sterile translocations. Twelve of 18 del(12p) parent sequences involved in these breakpoints were located in repeat regions (8 of these in long interspersed nuclear elements). This stands in contrast with TEL-AML1, in which only 21 of 110 previously assessed breakpoints (19%) occur in DNA repeats (P=0.0001). An exploratory assessment of archived neonatal blood cards revealed significantly more long interspersed nuclear element CpG methylations in individuals at birth who were later diagnosed with TEL-AML1 leukemia, compared with individuals who did not contract leukemia (P=0.01). Nontemplate nucleotides were also more frequent in del(12p) than in TEL-AML1 junctions (P=0.004), suggesting formation by terminal deoxynucleotidyl transferase. Assessment of six archived neonatal blood cards indicated that no del(12p) rearrangements backtracked to birth, although two of these patients were previously positive for TEL-AML1 using the same assay with comparable sensitivity. These data are compatible with a two-stage natural history: TEL-AML1 occurs prenatally, and del(12p) occurs postnatally in more mature cells with a structure that suggests the involvement of retrotransposon instability.

An integrative genomic and epigenomic approach for the study of transcriptional regulation.

The molecular heterogeneity of acute leukemias and other tumors constitutes a major obstacle towards understanding disease pathogenesis and developing new targeted-therapies. Aberrant gene regulation is a hallmark of cancer and plays a central role in determining tumor phenotype. We predicted that integration of different genome-wide epigenetic regulatory marks along with gene expression levels would provide greater power in capturing biological differences between leukemia subtypes. Gene expression, cytosine methylation and histone H3 lysine 9 (H3K9) acetylation were measured using high-density oligonucleotide microarrays in primary human acute myeloid leukemia (AML) and acute lymphocytic leukemia (ALL) specimens. We found that DNA methylation and H3K9 acetylation distinguished these leukemias of distinct cell lineage, as expected, but that an integrative analysis combining the information from each platform revealed hundreds of additional differentially expressed genes that were missed by gene expression arrays alone. This integrated analysis also enhanced the detection and statistical significance of biological pathways dysregulated in AML and ALL. Integrative epigenomic studies are thus feasible using clinical samples and provide superior detection of aberrant transcriptional programming than single-platform microarray studies.

wuHMM: a robust algorithm to detect DNA copy number variation using long oligonucleotide microarray data.

Copy number variants (CNVs) are currently defined as genomic sequences that are polymorphic in copy number and range in length from 1000 to several million base pairs. Among current array-based CNV detection platforms, long-oligonucleotide arrays promise the highest resolution. However, the performance of currently available analytical tools suffers when applied to these data because of the lower signal:noise ratio inherent in oligonucleotide-based hybridization assays. We have developed wuHMM, an algorithm for mapping CNVs from array comparative genomic hybridization (aCGH) platforms comprised of 385 000 to more than 3 million probes. wuHMM is unique in that it can utilize sequence divergence information to reduce the false positive rate (FPR). We apply wuHMM to 385K-aCGH, 2.1M-aCGH and 3.1M-aCGH experiments comparing the 129X1/SvJ and C57BL/6J inbred mouse genomes. We assess wuHMM's performance on the 385K platform by comparison to the higher resolution platforms and we independently validate 10 CNVs. The method requires no training data and is robust with respect to changes in algorithm parameters. At a FPR of <10%, the algorithm can detect CNVs with five probes on the 385K platform and three on the 2.1M and 3.1M platforms, resulting in effective resolutions of 24 kb, 2-5 kb and 1 kb, respectively.

Methods in high-resolution, array-based comparative genomic hybridization.

A method of high resolution, array-based comparative genomic hybridization is described for the mapping of copy-number changes associated with chromosomal amplifications, deletions, and translocations. The method involves the design of whole-genome or targeted, fine-tiling arrays for synthesis on a high-density digital microarray-synthesis platform. The arrays can span entire eukaryotic genomes or be targeted to specific chromosomal regions for high-resolution identification of copy-number changes and the corresponding breakpoint locations. The methods described include the bioinformatics required for array design, and the protocols for DNA fragmentation, dual-color labeling, microarray hybridization, and array scanning. The processes for data extraction, normalization, and segmentation analysis are also described.

Discovery of a previously unrecognized microdeletion syndrome of 16p11.2-p12.2.

We have identified a recurrent de novo pericentromeric deletion in 16p11.2-p12.2 in four individuals with developmental disabilities by microarray-based comparative genomic hybridization analysis. The identification of common clinical features in these four individuals along with the characterization of complex segmental duplications flanking the deletion regions suggests that nonallelic homologous recombination mediated these rearrangements and that deletions in 16p11.2-p12.2 constitute a previously undescribed syndrome.

Characterization of a recurrent 15q24 microdeletion syndrome.

We describe multiple individuals with mental retardation and overlapping de novo submicroscopic deletions of 15q24 (1.7-3.9 Mb in size). High-resolution analysis showed that in three patients both proximal and distal breakpoints co-localized to highly identical segmental duplications (>51 kb in length, > 94% identity), suggesting non-allelic homologous recombination as the likely mechanism of origin. Sequencing studies in a fourth individual provided base pair resolution and showed that both breakpoints in this case were located in unique sequence. Despite the differences in the size and location of the deletions, all four individuals share several major features (growth retardation, microcephaly, digital abnormalities, hypospadias and loose connective tissue) and resemble one another facially (high anterior hair line, broad medial eyebrows, hypertelorism, downslanted palpebral fissures, broad nasal base, long smooth philtrum and full lower lip), indicating that this represents a novel syndrome caused by haploinsufficiency of one or more dosage-sensitive genes in the minimal deletion region. Our results define microdeletion of 15q24 as a novel recurrent genomic disorder.

Efficient high-resolution deletion discovery in Caenorhabditis elegans by array comparative genomic hybridization.

We have developed array Comparative Genomic Hybridization for Caenorhabditis elegans as a means of screening for novel induced deletions in this organism. We designed three microarrays consisting of overlapping 50-mer probes to annotated exons and micro-RNAs, the first with probes to chromosomes X and II, the second with probes to chromosome II alone, and a third to the entire genome. These arrays were used to reliably detect both a large (50 kb) multigene deletion and a small (1 kb) single-gene deletion in homozygous and heterozygous samples. In one case, a deletion breakpoint was resolved to fewer than 50 bp. In an experiment designed to identify new mutations we used the X:II and II arrays to detect deletions associated with lethal mutants on chromosome II. One is an 8-kb deletion targeting the ast-1 gene on chromosome II and another is a 141-bp deletion in the gene C06A8.1. Others span large sections of the chromosome, up to >750 kb. As a further application of array Comparative Genomic Hybridization in C. elegans we used the whole-genome array to detect the extensive natural gene content variation (almost 2%) between the N2 Bristol strain and the strain CB4856, a strain isolated in Hawaii and JU258, a strain isolated in Madeira.

A high-resolution map of segmental DNA copy number variation in the mouse genome.

Submicroscopic (less than 2 Mb) segmental DNA copy number changes are a recently recognized source of genetic variability between individuals. The biological consequences of copy number variants (CNVs) are largely undefined. In some cases, CNVs that cause gene dosage effects have been implicated in phenotypic variation. CNVs have been detected in diverse species, including mice and humans. Published studies in mice have been limited by resolution and strain selection. We chose to study 21 well-characterized inbred mouse strains that are the focus of an international effort to measure, catalog, and disseminate phenotype data. We performed comparative genomic hybridization using long oligomer arrays to characterize CNVs in these strains. This technique increased the resolution of CNV detection by more than an order of magnitude over previous methodologies. The CNVs range in size from 21 to 2,002 kb. Clustering strains by CNV profile recapitulates aspects of the known ancestry of these strains. Most of the CNVs (77.5%) contain annotated genes, and many (47.5%) colocalize with previously mapped segmental duplications in the mouse genome. We demonstrate that this technique can identify copy number differences associated with known polymorphic traits. The phenotype of previously uncharacterized strains can be predicted based on their copy number at these loci. Annotation of CNVs in the mouse genome combined with sequence-based analysis provides an important resource that will help define the genetic basis of complex traits.

Chromosome Conformation Capture Carbon Copy (5C): a massively parallel solution for mapping interactions between genomic elements.

Physical interactions between genetic elements located throughout the genome play important roles in gene regulation and can be identified with the Chromosome Conformation Capture (3C) methodology. 3C converts physical chromatin interactions into specific ligation products, which are quantified individually by PCR. Here we present a high-throughput 3C approach, 3C-Carbon Copy (5C), that employs microarrays or quantitative DNA sequencing using 454-technology as detection methods. We applied 5C to analyze a 400-kb region containing the human beta-globin locus and a 100-kb conserved gene desert region. We validated 5C by detection of several previously identified looping interactions in the beta-globin locus. We also identified a new looping interaction in K562 cells between the beta-globin Locus Control Region and the gamma-beta-globin intergenic region. Interestingly, this region has been implicated in the control of developmental globin gene switching. 5C should be widely applicable for large-scale mapping of cis- and trans- interaction networks of genomic elements and for the study of higher-order chromosome structure.

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.

Comparative isoschizomer profiling of cytosine methylation: the HELP assay.

The distribution of cytosine methylation in 6.2 Mb of the mouse genome was tested using cohybridization of genomic representations from a methylation-sensitive restriction enzyme and its methylation-insensitive isoschizomer. This assay, termed HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR), allows both intragenomic profiling and intergenomic comparisons of cytosine methylation. The intragenomic profile shows most of the genome to be contiguous methylated sequence with occasional clusters of hypomethylated loci, usually but not exclusively at promoters and CpG islands. Intergenomic comparison found marked differences in cytosine methylation between spermatogenic and brain cells, identifying 223 new candidate tissue-specific differentially methylated regions (T-DMRs). Bisulfite pyrosequencing confirmed the four candidates tested to be T-DMRs, while quantitative RT-PCR for two genes with T-DMRs located at their promoters showed the HELP data to be correlated with gene activity at these loci. The HELP assay is robust, quantitative, and accurate and is providing new insights into the distribution and dynamic nature of cytosine methylation in the genome.

Complex genomic alterations and gene expression in acute lymphoblastic leukemia with intrachromosomal amplification of chromosome 21.

We have previously identified a unique subtype of acute lymphoblastic leukemia (ALL) associated with a poor outcome and characterized by intrachromosomal amplification of chromosome 21 including the RUNX1 gene (iAMP21). In this study, array-based comparative genomic hybridization (aCGH) (n = 10) detected a common region of amplification (CRA) between 33.192 and 39.796 Mb and a common region of deletion (CRD) between 43.7 and 47 Mb in 100% and 70% of iAMP21 patients, respectively. High-resolution genotypic analysis (n = 3) identified allelic imbalances in the CRA. Supervised gene expression analysis showed a distinct signature for eight patients with iAMP21, with 10% of overexpressed genes located within the CRA. The mean expression of these genes was significantly higher in iAMP21 when compared to other ALL samples (n = 45). Although genomic copy number correlated with overall gene expression levels within areas of loss or gain, there was considerable individual variation. A unique subset of differentially expressed genes, outside the CRA and CRD, were identified when gene expression signatures of iAMP21 were compared to ALL samples with ETV6-RUNX1 fusion (n = 21) or high hyperdiploidy with additional chromosomes 21 (n = 23). From this analysis, LGMN was shown to be overexpressed in patients with iAMP21 (P = 0.0012). Genomic and expression data has further characterized this ALL subtype, demonstrating high levels of 21q instability in these patients leading to proposals for mechanisms underlying this clinical phenotype and plausible alternative treatments.

High-resolution mapping of DNA copy alterations in human chromosome 22 using high-density tiling oligonucleotide arrays.

Deletions and amplifications of the human genomic sequence (copy number polymorphisms) are the cause of numerous diseases and a potential cause of phenotypic variation in the normal population. Comparative genomic hybridization (CGH) has been developed as a useful tool for detecting alterations in DNA copy number that involve blocks of DNA several kilobases or larger in size. We have developed high-resolution CGH (HR-CGH) to detect accurately and with relatively little bias the presence and extent of chromosomal aberrations in human DNA. Maskless array synthesis was used to construct arrays containing 385,000 oligonucleotides with isothermal probes of 45-85 bp in length; arrays tiling the beta-globin locus and chromosome 22q were prepared. Arrays with a 9-bp tiling path were used to map a 622-bp heterozygous deletion in the beta-globin locus. Arrays with an 85-bp tiling path were used to analyze DNA from patients with copy number changes in the pericentromeric region of chromosome 22q. Heterozygous deletions and duplications as well as partial triploidies and partial tetraploidies of portions of chromosome 22q were mapped with high resolution (typically up to 200 bp) in each patient, and the precise breakpoints of two deletions were confirmed by DNA sequencing. Additional peaks potentially corresponding to known and novel additional CNPs were also observed. Our results demonstrate that HR-CGH allows the detection of copy number changes in the human genome at an unprecedented level of resolution.

Analysis of chromosome breakpoints in neuroblastoma at sub-kilobase resolution using fine-tiling oligonucleotide array CGH.

Understanding the genes and genetic pathways targeted by recurrent chromosomal imbalances in malignancy, along with the molecular mechanisms that generate the imbalances, are important problems in cancer biology. In this report, we demonstrate that oligonucleotide array CGH (oaCGH) analysis can routinely map chromosomal imbalance breakpoints at exon-level resolution, including imbalances that are single copy number genomic alterations. Different tiling-path array designs were used in this study: a whole-genome array with a 6-kb median probe spacing and fine-tiling arrays for selected genomic regions with either 50- or 140-bp median probe spacing. In both array formats, oligonucleotide probes were of isothermal design and were tiled through genic and inter-genic regions. Whole-genome oaCGH analysis of two neuroblastoma cell lines and three primary tumors led to the identification of 58 chromosomal breakpoints that generated 45 large-scale partial chromosomal imbalances (> 2 Mb). An unexpectedly high proportion (34%) of these breakpoint intervals mapped to regions containing segmental duplications. In addition, 88 smaller-sized regions (< 2 Mb) of imbalance were detected, the majority of which mapped to segmentally duplicated regions and may reflect constitutional copy number polymorphisms. The chromosomal breakpoints for 12 recurrent abnormalities exhibited in neuroblastoma tumors and cell lines, including MYCN amplicon boundaries, loss of 3p, loss of 11q, and gain of 17q, could be mapped to intervals ranging from 50 bp to 10 kb in size using high-density fine-tiling oligonucleotide microarrays. Fine-tiling oaCGH analysis provides an unprecedented level of resolution, allowing detailed mapping of recurrent unbalanced chromosomal abnormalities. Supplementary material for this article can be found on the Genes, Chromosomes, and Cancer website at http://www.interscience.wiley.com/jpages/1045-2257/suppmat/index.html.