Motivation: The sequencing of tumors and their matched normals is frequently used to study the genetic composition of cancer. Despite this fact, there remains a dearth of available software tools designed to compare sequences in pairs of samples and identify sites that are likely to be unique to one sample.

Results: In this article, we describe the mathematical basis of our SomaticSniper software for comparing tumor and normal pairs. We estimate its sensitivity and precision, and present several common sources of error resulting in miscalls.

Availability and implementation: Binaries are freely available for download at http://gmt.genome.wustl.edu/somatic-sniper/current/, implemented in C and supported on Linux and Mac OS X.

Contact: delarson@wustl.edu; lding@wustl.edu

Supplementary information: Supplementary data are available at Bioinformatics online.

Background

Although the relationship between allergy and cancer has been investigated extensively, the role of allergy in head and neck cancer (HNC) appears less consistent. It is not clear whether allergies can independently influence the risk of HNC in the presence of known strong environmental risk factors, including consumption of alcohol, betel quid, and cigarette.

Methods

The current paper reports results from: 1) an original hospital-based case-control study, which included 252 incident cases of HNC and 236 controls frequency-matched to cases on sex and age; and 2) a meta-analysis combining the results of the current case-control study and 13 previously published studies (9 cohort studies with 727,569 subjects and 550 HNC outcomes and 5 case-control studies with 4,017 HNC cases and 10,928 controls).

Results

In the original case-control study, we observed a strong inverse association between allergies and HNC [odds ratio = 0.41, 95% confidence interval (CI): 0.27–0.62]. The meta-analysis also indicated a statistically significant inverse association between HNC and allergies [meta-relative risk (RR) = 0.76, 95% CI: 0.63–0.91], particularly strong for allergic rhinitis (meta-RR = 0.55, 95% CI: 0.40–0.76). In addition, the inverse association between allergies and HNC was observed only among men (meta-RR = 0.67, 95% CI: 0.54–0.84) but not among women (meta-RR = 0.98, 95% CI: 0.81–1.18).

Conclusions

These findings suggest that immunity plays an influential role in the risk of HNC. Future studies investigating immune biomarkers, including cytokine profiles and genetic polymorphisms, are warranted to further delineate the relationship between allergies and HNC. Understanding the relationship between allergies and HNC may help devise effective strategies to reduce and treat HNC.

Summary

To correlate the variable clinical features of estrogen receptor positive (ER+) breast cancer with somatic alterations, we studied pre-treatment tumour biopsies accrued from patients in a study of neoadjuvant aromatase inhibitor (AI) therapy by massively parallel sequencing and analysis. Eighteen significantly mutated genes were identified, including five genes (RUNX1, CBFB, MYH9, MLL3 and SF3B1) previously linked to hematopoietic disorders. Mutant MAP3K1 was associated with Luminal A status, low grade histology and low proliferation rates whereas mutant TP53 associated with the opposite pattern. Moreover, mutant GATA3 correlated with suppression of proliferation upon AI treatment. Pathway analysis demonstrated mutations in MAP2K4, a MAP3K1 substrate, produced similar perturbations as MAP3K1 loss. Distinct phenotypes in ER+ breast cancer are associated with specific patterns of somatic mutations that map into cellular pathways linked to tumor biology but most recurrent mutations are relatively infrequent. Prospective clinical trials based on these findings will require comprehensive genome sequencing.

We developed CREST (Clipping REveals STructure), an algorithm that uses next-generation sequencing reads with partial alignments to a reference genome to directly map structural variations at the nucleotide level of resolution. Application of CREST to whole-genome sequencing data from five pediatric T-lineage acute lymphoblastic leukemias (T-ALLs) and a human melanoma cell line, COLO-829, identified 160 somatic structural variations. Experimental validation exceeded 80% demonstrating that CREST had a high predictive accuracy.

BACKGROUND

The myelodysplastic syndromes are a group of hematologic disorders that often evolve into secondary acute myeloid leukemia (AML). The genetic changes that underlie progression from the myelodysplastic syndromes to secondary AML are not well understood.

METHODS

We performed whole-genome sequencing of seven paired samples of skin and bone marrow in seven subjects with secondary AML to identify somatic mutations specific to secondary AML. We then genotyped a bone marrow sample obtained during the antecedent myelodysplastic-syndrome stage from each subject to determine the presence or absence of the specific somatic mutations. We identified recurrent mutations in coding genes and defined the clonal architecture of each pair of samples from the myelodysplastic-syndrome stage and the secondary-AML stage, using the allele burden of hundreds of mutations.

RESULTS

Approximately 85% of bone marrow cells were clonal in the myelodysplastic-syndrome and secondary-AML samples, regardless of the myeloblast count. The secondary-AML samples contained mutations in 11 recurrently mutated genes, including 4 genes that have not been previously implicated in the myelodysplastic syndromes or AML. In every case, progression to acute leukemia was defined by the persistence of an antecedent founding clone containing 182 to 660 somatic mutations and the outgrowth or emergence of at least one subclone, harboring dozens to hundreds of new mutations. All founding clones and subclones contained at least one mutation in a coding gene.

CONCLUSIONS

Nearly all the bone marrow cells in patients with myelodysplastic syndromes and secondary AML are clonally derived. Genetic evolution of secondary AML is a dynamic process shaped by multiple cycles of mutation acquisition and clonal selection. Recurrent gene mutations are found in both founding clones and daughter subclones. (Funded by the National Institutes of Health and others.)

Background

Ultrasound (US) is a commonly-used intraoperative imaging modality for guiding percutaneous renal access (PRA). However, the anatomy identification and target localization abilities of the US imaging are limited. This paper evaluates the feasibility and efficiency of a proposed image-guided PRA by augmenting the intraoperative US with preoperative magnetic resonance (MR) planning models.

Methods

First, a preoperative surgical planning approach is presented to define an optimal needle trajectory using MR volume data. Then, a MR to US registration is proposed to transfer the preoperative planning into the intraoperative context. The proposed registration makes use of orthogonal US slices to avoid local minima while reduce processing time. During the registration, a respiratory gating method is used to minimize the impact of kidney deformation. By augmenting the intraoperative US with preoperative MR models and a virtual needle, a visual guidance is provided to guarantee the correct execution of the surgical planning. The accuracy, robustness and processing time of the proposed registration were evaluated by four urologists on human data from four volunteers. Furthermore, the PRA experiments were performed by the same four urologists on a kidney phantom. The puncture accuracy in terms of the needle-target distance was measured, while the perceptual quality in using the proposed image guidance was evaluated according to custom scoring method.

Results

The mean registration accuracy in terms of the root mean square (RMS) target registration error (TRE) is 3.53 mm. The RMS distance from the registered feature points to their average is 0.81 mm. The mean operating time of the registration is 6'4". In the phantom evaluation, the mean needle-target distance is 2.08 mm for the left lesion and 1.85 mm for the right one. The mean duration for all phantom PRA tests was 4'26". According to the custom scoring method, the mean scores of the Intervention Improvement, Workflow Impact, and Clinical Relevance were 4.0, 3.3 and 3.9 respectively.

Conclusions

The presented image guidance is feasible and promising for PRA procedure. With careful setup it can be efficient for overcoming the limitation of current US-guided PRA.

Intimate Partner Violence (IPV) is a major public health issue occurring in the United States and globally. While little is known in general about IPV, understanding about the prevalence of physical IPV among gay men is even more obscure. There is a clear disparity in violence research attention focused on this vulnerable segment of society. This cross-sectional survey study was conducted to examine the feasibility of enrolling 100 gay men from Atlanta into an IPV survey study. The survey was administered via Facebook. Ninety-nine usable surveys were collected. Chi-square tests reveal that minority ethnic status, illicit drug use, and non-disclosed orientation status were all significantly associated with positive IPV reports--in terms of both victimization as well as perpetration. Overall, the majority of the study sample indicated that they believe IPV is a health problem in the Atlanta gay community. These findings bear importance for the Atlanta gay community and public health professionals who must address this nearly invisible yet increasing public health issue.

Summary

Most patients with acute myeloid leukemia (AML) die from progressive disease after relapse, which is associated with clonal evolution at the cytogenetic level1,2. To determine the mutational spectrum associated with relapse, we sequenced the primary tumor and relapse genomes from 8 AML patients, and validated hundreds of somatic mutations using deep sequencing; this allowed us to precisely define clonality and clonal evolution patterns at relapse. Besides discovering novel, recurrently mutated genes (e.g. WAC, SMC3, DIS3, DDX41, and DAXX) in AML, we found two major clonal evolution patterns during AML relapse: 1) the founding clone in the primary tumor gained mutations and evolved into the relapse clone, or 2) a subclone of the founding clone survived initial therapy, gained additional mutations, and expanded at relapse. In all cases, chemotherapy failed to eradicate the founding clone. The comparison of relapse-specific vs. primary tumor mutations in all 8 cases revealed an increase in transversions, probably due to DNA damage caused by cytotoxic chemotherapy. These data demonstrate that AML relapse is associated with the addition of new mutations and clonal evolution, which is shaped in part by the chemotherapy that the patients receive to establish and maintain remissions.

Angelman syndrome (AS) is a human neurological disorder caused by lack of maternal UBE3A expression in the brain. UBE3A is known to function as both an ubiquitin-protein ligase (E3) and a coactivator for steroid receptors. Many ubiquitin targets, as well as interacting partners, of UBE3A have been identified. However, the pathogenesis of AS, and how deficiency of maternal UBE3A can upset cellular homeostasis, remains vague. In this study, we performed a genome-wide microarray analysis on the maternal Ube3a-deficient (Ube3am−/p+) AS mouse to search for genes affected in the absence of Ube3a. We observed 64 differentially expressed transcripts (7 upregulated and 57 downregulated) showing more than 1.5-fold differences in expression (P<0.05). Pathway analysis shows that these genes are implicated in three major networks associated with cell signaling, nervous system development and cell death. Using quantitative reverse-transcription PCR, we validated the differential expression of genes (Fgf7, Glra1, Mc1r, Nr4a2, Slc5a7 and Epha6) that show functional relevance to AS phenotype. We also show that the protein level of melanocortin 1 receptor (Mc1r) and nuclear receptor subfamily 4, group A, member 2 (Nr4a2) in the AS mice cerebellum is decreased relative to that of the wild-type mice. Consistent with this finding, expression of small-interfering RNA that targets Ube3a in P19 cells caused downregulation of Mc1r and Nr4a2, whereas overexpression of Ube3a results in the upregulation of Mc1r and Nr4a2. These observation help in providing insights into the genesis of neurodevelopmental phenotype of AS and highlight specific area for future research.

BACKGROUND

The full complement of DNA mutations that are responsible for the pathogenesis of acute myeloid leukemia (AML) is not yet known.

METHODS

We used massively parallel DNA sequencing to obtain a very high level of coverage (approximately 98%) of a primary, cytogenetically normal, de novo genome for AML with minimal maturation (AML-M1) and a matched normal skin genome.

RESULTS

We identified 12 acquired (somatic) mutations within the coding sequences of genes and 52 somatic point mutations in conserved or regulatory portions of the genome. All mutations appeared to be heterozygous and present in nearly all cells in the tumor sample. Four of the 64 mutations occurred in at least 1 additional AML sample in 188 samples that were tested. Mutations in NRAS and NPM1 had been identified previously in patients with AML, but two other mutations had not been identified. One of these mutations, in the IDH1 gene, was present in 15 of 187 additional AML genomes tested and was strongly associated with normal cytogenetic status; it was present in 13 of 80 cytogenetically normal samples (16%). The other was a nongenic mutation in a genomic region with regulatory potential and conservation in higher mammals; we detected it in one additional AML tumor. The AML genome that we sequenced contains approximately 750 point mutations, of which only a small fraction are likely to be relevant to pathogenesis.

CONCLUSIONS

By comparing the sequences of tumor and skin genomes of a patient with AML-M1, we have identified recurring mutations that may be relevant for pathogenesis.

Context

The identification of patients with inherited cancer susceptibility syndromes facilitates early diagnosis, prevention, and treatment. However, in many cases of suspected cancer susceptibility, the family history is unclear and genetic testing of common cancer susceptibility genes is unrevealing.

Objective

To apply whole-genome sequencing to a patient with suspected cancer susceptibility (and lacking a clear family history of cancer and no BRCA1 and BRCA2 mutations) to identify rare or novel germline variants in cancer susceptibility genes.

Design, Setting, and Participant

Skin (normal) and bone marrow (leukemia) DNA were obtained from a patient with early-onset breast and ovarian cancer and therapy-related acute myeloid leukemia (t-AML), and analyzed with: 1) whole genome sequencing using paired end reads; 2) SNP genotyping; 3) RNA expression profiling; and 4) spectral karyotyping.

Main Outcome Measures

Structural variants, copy number alterations, single nucleotide variants and small insertions and deletions (indels) were detected and validated using the above platforms.

Results

Whole genome sequencing revealed a novel, heterozygous 3 Kb deletion removing exons 7-9 of TP53 in the patient’s normal skin DNA, which was homozygous in the leukemia DNA as a result of uniparental disomy. In addition, a total of 28 validated somatic single nucleotide variations or indels in coding genes, 8 somatic structural variants, and 12 somatic copy number alterations were detected in the patient’s leukemia genome.

Conclusions

Whole genome sequencing can identify novel, cryptic variants in cancer susceptibility genes in addition to providing unbiased information on the spectrum of mutations in a cancer genome.

All living organisms exhibit autonomous daily physiological and behavioural rhythms to help them synchronize with the environment. Entrainment of circadian rhythm is achieved via activation of cyclic AMP (cAMP) and mitogen-activated protein kinase signaling pathways. NonO (p54nrb) is a multifunctional protein involved in transcriptional activation of the cAMP pathway and is involved in circadian rhythm control. Rasd1 is a monomeric G protein implicated to play a pivotal role in potentiating both photic and nonphotic responses of the circadian rhythm. In this study, we have identified and validated NonO as an interacting partner of Rasd1 via affinity pulldown, co-immunoprecipitation and indirect immunofluorescence studies. The GTP-hydrolysis activity of Rasd1 is required for the functional interaction. Functional interaction of Rasd1-NonO in the cAMP pathway was investigated via reporter gene assays, chromatin immunoprecipitation and gene knockdown. We showed that Rasd1 and NonO interact at the CRE-site of specific target genes. These findings reveal a novel mechanism by which the coregulator activity of NonO can be modulated.

Context

Whole genome sequencing (WGS) is becoming increasingly available for research purposes, but it has not yet been routinely used for clinical diagnosis.

Object

To determine whether whole genome sequencing can identify cryptic, actionable mutations in a clinically relevant time frame.

Design, Setting, and Patient

We were referred a difficult diagnostic case of acute promyelocytic leukemia with no pathogenic X-RARA fusion identified by routine metaphase cytogenetics or interphase FISH. The patient was enrolled in an IRB approved protocol, with consent specifically tailored to the implications of whole genome sequencing. The protocol employs a ‘movable firewall,’ which maintains patient anonymity within the entire research team, but allows the research team to communicate medically relevant information to the treating physician.

Main Outcome Measure

Clinical relevance of whole genome sequencing and time to communicate validated results to the treating physician.

Results

Massively parallel paired-end sequencing allowed us to identify a cytogenetically cryptic event: 77 kilobases from chromosome 15 was inserted en bloc into the second intron of the RARA gene on chromosome 17, resulting in a classic bcr3 PML-RARA fusion gene. RT-PCR subsequently validated the expression of the fusion transcript. Novel FISH probes identified two additional cases of t(15;17)-negative acute promyelocytic leukemia that had cytogenetically invisible insertions. Whole genome sequencing and validation were completed in seven weeks, and changed the treatment plan for the patient.

Conclusions

Whole genome sequencing can identify cytogenetically invisible oncogenes in a clinically relevant timeframe.

Summary

Genomic structural variants (SVs) are abundant in humans, differing from other variation classes in extent, origin, and functional impact. Despite progress in SV characterization, the nucleotide resolution architecture of most SVs remains unknown. We constructed a map of unbalanced SVs (i.e., copy number variants) based on whole genome DNA sequencing data from 185 human genomes, integrating evidence from complementary SV discovery approaches with extensive experimental validations. Our map encompassed 22,025 deletions and 6,000 additional SVs, including insertions and tandem duplications. Most SVs (53%) were mapped to nucleotide resolution, which facilitated analyzing their origin and functional impact. We examined numerous whole and partial gene deletions with a genotyping approach and observed a depletion of gene disruptions amongst high frequency deletions. Furthermore, we observed differences in the size spectra of SVs originating from distinct formation mechanisms, and constructed a map constructed a map of SV hotspots formed by common mechanisms. Our analytical framework and SV map serves as a resource for sequencing-based association studies.

Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia (AML). It is characterized by the t(15;17)(q22;q11.2) chromosomal translocation that creates the promyelocytic leukemia–retinoic acid receptor α (PML-RARA) fusion oncogene. Although this fusion oncogene is known to initiate APL in mice, other cooperating mutations, as yet ill defined, are important for disease pathogenesis. To identify these, we used a mouse model of APL, whereby PML-RARA expressed in myeloid cells leads to a myeloproliferative disease that ultimately evolves into APL. Sequencing of a mouse APL genome revealed 3 somatic, nonsynonymous mutations relevant to APL pathogenesis, of which 1 (Jak1 V657F) was found to be recurrent in other affected mice. This mutation was identical to the JAK1 V658F mutation previously found in human APL and acute lymphoblastic leukemia samples. Further analysis showed that JAK1 V658F cooperated in vivo with PML-RARA, causing a rapidly fatal leukemia in mice. We also discovered a somatic 150-kb deletion involving the lysine (K)-specific demethylase 6A (Kdm6a, also known as Utx) gene, in the mouse APL genome. Similar deletions were observed in 3 out of 14 additional mouse APL samples and 1 out of 150 human AML samples. In conclusion, whole genome sequencing of mouse cancer genomes can provide an unbiased and comprehensive approach for discovering functionally relevant mutations that are also present in human leukemias.

Motivation: DNA copy number aberration (CNA) is a hallmark of genomic abnormality in tumor cells. Recurrent CNA (RCNA) occurs in multiple cancer samples across the same chromosomal region and has greater implication in tumorigenesis. Current commonly used methods for RCNA identification require CNA calling for individual samples before cross-sample analysis. This two-step strategy may result in a heavy computational burden, as well as a loss of the overall statistical power due to segmentation and discretization of individual sample's data. We propose a population-based approach for RCNA detection with no need of single-sample analysis, which is statistically powerful, computationally efficient and particularly suitable for high-resolution and large-population studies.

Results: Our approach, correlation matrix diagonal segmentation (CMDS), identifies RCNAs based on a between-chromosomal-site correlation analysis. Directly using the raw intensity ratio data from all samples and adopting a diagonal transformation strategy, CMDS substantially reduces computational burden and can obtain results very quickly from large datasets. Our simulation indicates that the statistical power of CMDS is higher than that of single-sample CNA calling based two-step approaches. We applied CMDS to two real datasets of lung cancer and brain cancer from Affymetrix and Illumina array platforms, respectively, and successfully identified known regions of CNA associated with EGFR, KRAS and other important oncogenes. CMDS provides a fast, powerful and easily implemented tool for the RCNA analysis of large-scale data from cancer genomes.

Availability: The R and C programs implementing our method are available at https://dsgweb.wustl.edu/qunyuan/software/cmds.

Contact: qunyuan@wustl.edu

Supplementary information: Supplementary data are available at Bioinformatics online.

Background

The Rasd1 protein is a dexamethasone induced monomeric Ras-like G protein that oscillates in the suprachiasmatic nucleus (SCN). Previous studies have shown that Rasd1 modulates multiple signaling cascades. However, it is still unclear exactly how Rasd1 carries out its function. Studying protein-protein interactions involving Rasd1 may provide insights into its biological functions in different contexts.

Results

To further explore the molecular function of Rasd1, we performed a yeast two-hybrid screen and identified Ear2, a negative regulator of renin transcription, as an interaction partner of Rasd1. We validated the interaction in vitro and in transfected COS-7 cells. We further confirmed the interaction of endogenous Rasd1 and Ear2 from HEK293T cell and mouse brain extract. Rasd1 inhibited transcriptional repression by Ear2 on a renin promoter-luciferase reporter construct both in the presence and absence of all-trans-retinoic acid. Moreover, real-time RT-PCR showed upregulation of endogenous renin transcription in As4.1 cells over-expressing Rasd1. We demonstrated that the ligand binding domain of Ear2 is required for physical and functional interaction between the two proteins. In addition, we demonstrated that shRNA-mediated knockdown of Rasd1 results in further repression of Ear2-mediated renin transcription, whereas induction of Rasd1 by dexamethasone counteracts the effects of shRNA-mediated Rasd1 knockdown. Finally, our study showed that Rasd1 missense mutations not only attenuate their physical interaction with Ear2 but also abolish their ability to counteract repression of renin transcription mediated by Ear2.

Conclusions

Our study provides evidence for physical and functional interactions between Rasd1 and Ear2. The results suggest that their interactions are involved in renin transcriptional regulation. These findings not only reveal a novel role for Rasd1-medated signaling but also provide the basis for potential intervention of renin expression.

Due to the inherent immune evasion properties of the HIV envelope, broadly neutralizing HIV-specific antibodies capable of suppressing HIV infection are rarely produced by infected individuals. We examined the feasibility of utilizing genetic engineering to circumvent the restricted capacity of individuals to endogenously produce broadly neutralizing HIV-specific antibodies. We constructed a single lentiviral vector that encoded the heavy and light chains of 2G12, a broadly neutralizing anti-HIV human antibody, and that efficiently transduced and directed primary human B cells to secrete 2G12. To evaluate the capacity of this approach to provide protection from in vivo HIV infection, we used the humanized NOD/SCID/γcnull mouse model, which becomes populated with human B cells, T cells, and macrophages after transplantation with human hematopoietic stem cells (hu-HSC) and develops in vivo infection after inoculation with HIV. The plasma of the irradiated NOD/SCID/γcnull mice transplanted with hu-HSC transduced with the 2G12-encoding lentivirus contained 2G12 antibody, likely secreted by progeny human lymphoid and/or myeloid cells. After intraperitoneal inoculation with high-titer HIV-1JR-CSF, mice engrafted with 2G12-transduced hu-HSC displayed marked inhibition of in vivo HIV infection as manifested by a profound 70-fold reduction in plasma HIV RNA levels and an almost 200-fold reduction in HIV-infected human cell numbers in mouse spleens, compared to control hu-HSC-transplanted NOD/SCID/γcnull mice inoculated with equivalent high-titer HIV-1JR-CSF. These results support the potential efficacy of this new gene therapy approach of using lentiviral vectors encoding a mixture of broadly neutralizing HIV antibodies for the treatment of HIV infection, particularly infection with multiple-drug-resistant isolates.

Massively parallel DNA sequencing technologies provide an unprecedented ability to screen entire genomes for genetic changes associated with tumor progression. Here we describe the genomic analyses of four DNA samples from an African-American patient with basal-like breast cancer: peripheral blood, the primary tumor, a brain metastasis, and a xenograft derived from the primary tumor. The metastasis contained two de novo mutations and a large deletion not present in the primary tumor, and was significantly enriched for 20 shared mutations. The xenograft retained all primary tumor mutations, and displayed a mutation enrichment pattern that paralleled the metastasis (16 of 20 genes). Two overlapping large deletions, encompassing CTNNA1, were present in all three tumor samples. The differential mutation frequencies and structural variation patterns in metastasis and xenograft compared to the primary tumor suggest that secondary tumors may arise from a minority of cells within the primary.

Summary: Massively parallel sequencing technologies hold incredible promise for the study of DNA sequence variation, particularly the identification of variants affecting human disease. The unprecedented throughput and relatively short read lengths of Roche/454, Illumina/Solexa, and other platforms have spurred development of a new generation of sequence alignment algorithms. Yet detection of sequence variants based on short read alignments remains challenging, and most currently available tools are limited to a single platform or aligner type. We present VarScan, an open source tool for variant detection that is compatible with several short read aligners. We demonstrate VarScan's ability to detect SNPs and indels with high sensitivity and specificity, in both Roche/454 sequencing of individuals and deep Illumina/Solexa sequencing of pooled samples.

Availability and Implementation: Source code and documentation freely available at http://genome.wustl.edu/tools/cancer-genomics implemented as a Perl package and supported on Linux/UNIX, MS Windows and Mac OSX.

Contact: dkoboldt@genome.wustl.edu

Supplementary information: Supplementary data are available at Bioinformatics online.

Determining the genetic basis of cancer requires comprehensive analyses of large collections of histopathologically well-classified primary tumours. Here we report the results of a collaborative study to discover somatic mutations in 188 human lung adenocarcinomas. DNA sequencing of 623 genes with known or potential relationships to cancer revealed more than 1,000 somatic mutations across the samples. Our analysis identified 26 genes that are mutated at significantly high frequencies and thus are probably involved in carcinogenesis. The frequently mutated genes include tyrosine kinases, among them the EGFR homologue ERBB4; multiple ephrin receptor genes, notably EPHA3; vascular endothelial growth factor receptor KDR; and NTRK genes. These data provide evidence of somatic mutations in primary lung adenocarcinoma for several tumour suppressor genes involved in other cancers—including NF1, APC, RB1 and ATM—and for sequence changes in PTPRD as well as the frequently deleted gene LRP1B. The observed mutational profiles correlate with clinical features, smoking status and DNA repair defects. These results are reinforced by data integration including single nucleotide polymorphism array and gene expression array. Our findings shed further light on several important signalling pathways involved in lung adenocarcinoma, and suggest new molecular targets for treatment.

Lay Summary

Acute myeloid leukemia is a highly malignant hematopoietic tumor that affects about 13,000 adults yearly in the United States. The treatment of this disease has changed little in the past two decades, since most of the genetic events that initiate the disease remain undiscovered. Whole genome sequencing is now possible at a reasonable cost and timeframe to utilize this approach for unbiased discovery of tumor-specific somatic mutations that alter the protein-coding genes. Here we show the results obtained by sequencing a typical acute myeloid leukemia genome and its matched normal counterpart, obtained from the patient’s skin. We discovered 10 genes with acquired mutations; two were previously described mutations thought to contribute to tumor progression, and 8 were novel mutations present in virtually all tumor cells at presentation and relapse, whose function is not yet known. Our study establishes whole genome sequencing as an unbiased method for discovering initiating mutations in cancer genomes, and for identifying novel genes that may respond to targeted therapies.

We used massively parallel sequencing technology to sequence the genomic DNA of tumor and normal skin cells obtained from a patient with a typical presentation of FAB M1 Acute Myeloid Leukemia (AML) with normal cytogenetics. 32.7-fold ‘haploid’ coverage (98 billion bases) was obtained for the tumor genome, and 13.9-fold coverage (41.8 billion bases) was obtained for the normal sample. Of 2,647,695 well-supported Single Nucleotide Variants (SNVs) found in the tumor genome, 2,588,486 (97.7%) also were detected in the patient’s skin genome, limiting the number of variants that required further study. For the purposes of this initial study, we restricted our downstream analysis to the coding sequences of annotated genes: we found only eight heterozygous, non-synonymous somatic SNVs in the entire genome. All were novel, including mutations in protocadherin/cadherin family members (CDH24 and PCLKC), G-protein coupled receptors (GPR123 and EBI2), a protein phosphatase (PTPRT), a potential guanine nucleotide exchange factor (KNDC1), a peptide/drug transporter (SLC15A1), and a glutamate receptor gene (GRINL1B). We also detected previously described, recurrent somatic insertions in the FLT3 and NPM1 genes. Based on deep readcount data, we determined that all of these mutations (except FLT3) were present in nearly all tumor cells at presentation, and again at relapse 11 months later, suggesting that the patient had a single dominant clone containing all of the mutations. These results demonstrate the power of whole genome sequencing to discover novel cancer-associated mutations.

Posttranslational modifications of transcription factors provide alternate protein interaction platforms that lead to varied downstream effects. We have investigated how the acetylation of EKLF plays a role in its ability to alter the β-like globin locus chromatin structure and activate transcription of the adult β-globin gene. By establishing an EKLF-null erythroid line whose closed β-locus chromatin structure and silent β-globin gene status can be rescued by retroviral infection of EKLF, we demonstrate the importance of EKLF acetylation at lysine 288 in the recruitment of CBP to the locus, modification of histone H3, occupancy by EKLF, opening of the chromatin structure, and transcription of adult β-globin. We also find that EKLF helps to coordinate this process by the specific association of its zinc finger domain with the histone H3 amino terminus. Although EKLF interacts equally well with H3.1 and H3.3, we find that only H3.3 is enriched at the adult β-globin promoter. These data emphasize the critical nature of lysine acetylation in transcription factor activity and enable us to propose a model of how modified EKLF integrates coactivators, chromatin remodelers, and nucleosomal components to alter epigenetic chromatin structure and stimulate transcription.

Three research prototype speech recognition systems are described, all of which use recently developed methods from artificial intelligence (specifically support vector machines, dynamic Bayesian networks, and maximum entropy classification) in order to implement, in the form of an automatic speech recognizer, current theories of human speech perception and phonology (specifically landmark-based speech perception, nonlinear phonology, and articulatory phonology). All three systems begin with a high-dimensional multiframe acoustic-to-distinctive feature transformation, implemented using support vector machines trained to detect and classify acoustic phonetic landmarks. Distinctive feature probabilities estimated by the support vector machines are then integrated using one of three pronunciation models: a dynamic programming algorithm that assumes canonical pronunciation of each word, a dynamic Bayesian network implementation of articulatory phonology, or a discriminative pronunciation model trained using the methods of maximum entropy classification. Log probability scores computed by these models are then combined, using log-linear combination, with other word scores available in the lattice output of a first-pass recognizer, and the resulting combination score is used to compute a second-pass speech recognition output.

AIM: To investigate the effects of Tat-NEMO-binding domain (NBD) peptide on taurocholate-induced pancreatitis and lipopolysaccharide (LPS)-stimulated AR42J acinus cells inflammatory response in rats.

METHODS: Sodium taurocholate (5%) was used to induce the pancreatitis model. Forty-eight rats from the taurocholate group received an intravenous bolus of 13 mg/kg Tat-NBD (wild-type, WT) peptide, Tat-NBD (mutant-type, MT) peptide, NBD peptide or Tat peptide. The pancreatic histopathology was analyzed by hematoxylin staining. LPS was added to the culture medium to stimulate the AR42J cells. For pretreatment, cells were incubated with different peptides for 2 h before LPS stimulation. Expression of IL-1β and TNF-α mRNA was analyzed using a semi-quantitative reverse-transcript polymerase chain reaction (RT-PCR) method. IL-1β and TNF-α protein in culture medium were detected by enzyme linked immunosorbent assay (ELISA). NF-κB DNA-binding in pancreas was examined by electrophoretic mobility shift assays. P65 expression of AR42J was determined by Strept Actividin-Biotin Complex (SABC) method.

RESULTS: Pretreatment with Tat-NBD (WT) peptide at a concentration of 13 mg/kg body wt showed beneficial effect in pancreaitis model. LPS (10 mg/L) resulted in an increase of IL-1β mRNA, IL-1β protein, TNF-α mRNA and TNF-α protein, whereas significantly inhibitory effects were observed when cells were incubated with Tat-NBD (WT). Consisting with p65 expression decrease analyzed by SABC method, NF-κB DNA-binding activity significantly decreased in Tat-NBD (WT) pretreatment group, especially at the largest dose. No significant changes were found in the control peptide group.

CONCLUSION: Our result supports that active NF-κB participates in the pathogenesis of STC-induced acute pancreatitis in rats. Tat-NBD (WT) peptide has anti-inflammatory effects in this model and inhibits the inflammation of acinus simulated by LPS.