Resequencing Efforts Research Articles

Ubtf- Tandem Duplications Are Recurrent Somatic Genetic Events in Adult AML and Associate with Additional Structural Variants and a Distinct Gene Expression Profile (Alliance)

Background The gene upstream binding transcription factor ( UBTF ) encodesa nucleolar protein that is a key regulator of the epigenetic status of ribosomal DNA and RNA transcription. Tandem duplications (TDs) of the UBTF gene have been initially identified in pediatric patients (pts) with acute myeloid leukemia (AML) and were recently also reported as relatively rare, but recurrent genetic events in adult AML based on targeted re-sequencing efforts (1.2%-3%). Their presence was associated with dysplastic bone marrow features, and co-occurrence of select genetic features, including FLT3-internal tandem duplications (ITDs) and WT1 mutations, and inferior outcome. However, associated biologic features and affected pathways have not yet been thoroughly investigated, and may provide further insights into leukemogenesis and identify potentially targetable vulnerabilities. Thus, we set out to assess the frequency of UBTF-TDs in a large cohort of adult AML pts (n=807) treated on frontline protocols of the Alliance for Clinical Trials in Oncology with complete RNA sequencing data, comprehensive targeted mutation profiling, centrally reviewed cytogenetic data and clinical information available, and to gain further insights into UBTF-TDs via comprehensive integrated genomic profiling. Methods UBTF-TD detection was performed via the RNAseq-based RNAmut and EnFusion algorithm. To gain insights into associated molecular features, all UBTF-TD + pts underwent paired tumor-normal whole-exome sequencing. To understand associated pathways and downstream effects, differential gene expression (DGE) and gene set enrichment analyses (GSEA) were performed. Results UBTF-TDs were detected in 6/807 pts (0.7%). In line with previous studies, these 6 UBTF-TD + pts were young, with a median age of 35y (range: 19-59y). Three pts had extramedullary disease, 2 with skin and 1 with gingival involvement. All but 1 of the 6 pts were cytogenetically normal. Clinically, pts did poorly after treatment with intensive chemotherapy induction, with a CR rate of only 33%. No pt was alive 2.5 y after diagnosis. In paired tumor/normal exome sequencing, UBTF-TD + pts had a median mutation burden of 21 non-synonymous variants in coding genes (range: 17-27), with a notable presence of larger insertions/deletions (indels), loss of heterozygosity (LOH) or TDs as co-occurring somatic genetic lesions (median: 4, range: 2-5). There was a 100% co-occurrence of UBTF-TDs with FLT3-ITDs. In addition, pts harbored larger indels in WT1 (n=2), BPTF, CEBPA, Corf53, KRTAP5-7, NFE2, PTPRN2, RNF144B, SOWAHC and ZCCHC14. Also, analysis of copy-number variations and LOH revealed LOH in 5 pts, in chromosomes 13 (n=3), 15 (n=1) and 18 (n=1), suggestive of a propensity to the acquisition of structural variants. In unsupervised clustering analysis of the RNAseq data, UBTF-TD + pts clustered with FLT3-ITD + pts but formed a relatively distinct subcluster in principal component and t-distributed stochastic neighbor embedding analyses. In supervised clustering comparing 6 UBTF-TD +/ FLT3-ITD + and 196 UBTF-TD -/ FLT3-ITD + pts, 2 UBTF-TD - pts clustered together with the UBTF-TD +/ FLT3-ITD + group, suggesting either presence of an undetected TD or genetic phenocopy. UBTF expression was higher in UBTF-TD pts. DGE analysis of UBTF-TD +/ FLT3-ITD + and UBTF-TD -/ FLT3-ITD + pts (Fig 1) showed distinct upregulation of the locus of the stem cell and chemoresistance marker ALDH1A3, including the associated long non-coding RNAs AC015712.7/AC015712.2 in UBTF-TD + pts, which was confirmed by a comparison of those pts to AML pts who had neither TD (n=605). GSEA indicated a downregulation of inflammation pathways and several metabolic pathways of UBTF-TD +/ FLT3-ITD + compared to FLT3-ITD + pts (Fig 2). Conclusions UBTF-TDs are rare, but recurrent genetic events in adult AML, associated with high chemotherapy resistance. Our results highlight a susceptibility of those pts to the acquisition of structural variants and suggest a distinct associated biology, with upregulation of ALDH1A3 as possible contributing resistance mechanism. Support: U10CA180821, U10CA180882, U24CA196171; Clinicaltrials.gov Identifiers: NCT00048958 (CALGB 8461), NCT00899223 (CALGB 9665), NCT00900224 (CALGB 20202); https://acknowledgments.alliancefound.org

Reference Genome of the California Sheephead, Semicossyphus pulcher (Labridae, Perciformes), A Keystone Fish Predator in Kelp Forest Ecosystems.

Keystone species are known to play a critical role in kelp forest health, including the well-known killer whales, sea otter, sea urchin, kelp trophic cascade in the Aleutian Islands, Alaska, USA. In California, a major player in the regulation of sea urchin abundance, and in turn, the health of kelp forests ecosystems, is a large wrasse, the California Sheephead, Semicossyphus pulcher. We present a reference genome for this ecologically important species that will serve as a key resource for future conservation research of California's inshore marine environment utilizing genomic tools to address changes in life-history traits, dispersal, range shifts, and ecological interactions among members of the kelp forest ecological assemblages. Our genome assembly of S. pulcher has a total length of 0.794 Gb, which is similar to many other marine fishes. The assembly is largely contiguous (N50 = 31.9 Mb) and nearly complete (BUSCO single-copy core gene content = 98.1%). Within the context of the California Conservation Genomics Project (CCGP), the genome of S. pulcher will be used as an important reference resource for ongoing whole genome resequencing efforts of the species.

Pear genetics: Recent advances, new prospects, and a roadmap for the future.

Pear, belonging to the genus Pyrus, is one of the most economically important temperate fruit crops. Pyrus is an important genus of the Rosaceae family, subfamily Maloideae, and has at least 22 different species with over 5000 accessions maintained or identified worldwide. With the release of draft whole-genome sequences for Pyrus, opportunities for pursuing studies on the evolution, domestication, and molecular breeding of pear, as well as for conducting comparative genomics analyses within the Rosaceae family, have been greatly expanded. In this review, we highlight key advances in pear genetics, genomics, and breeding driven by the availability of whole-genome sequences, including whole-genome resequencing efforts, pear domestication, and evolution. We cover updates on new resources for undertaking gene identification and molecular breeding, as well as for pursuing functional validation of genes associated with desirable economic traits. We also explore future directions for "pear-omics".

Phenotype to genotype in Neurospora crassa: Association of the scumbo phenotype with mutations in the gene encoding ceramide C9-methyltransferase

Using a legacy of genetic mutants of Neurospora crassa, paired with resequencing efforts through JGI, we have identified the gene responsible for the 'scumbo' mutant. This early morphological mutant was described as "Irregular flat, spreading growth with knobby protrusions and abnormal conidiation, but no free conidia. Mycelium usually appears yellowish rather than orange. Female fertile." (Perkins, Radford etal. 2000). Our further investigation has found new insights into the identity and associated functions of scumbo as a ceramide C9 methyltransferase, previously annotated as "similar to cyclopropane-fatty-acyl-phospholipidsynthase", encoded by the gene NCU07859. This enzyme performs a fungal-specific methyl modification of glycosyl-ceramides and has implications for membrane homeostasis and hyphal polarity in filamentous fungi.

Genome assembly of the popular Korean soybean cultivar Hwangkeum.

Massive resequencing efforts have been undertaken to catalog allelic variants in major crop species including soybean, but the scope of the information for genetic variation often depends on short sequence reads mapped to the extant reference genome. Additional de novo assembled genome sequences provide a unique opportunity to explore a dispensable genome fraction in the pan-genome of a species. Here, we report the de novo assembly and annotation of Hwangkeum, a popular soybean cultivar in Korea. The assembly was constructed using PromethION nanopore sequencing data and two genetic maps and was then error-corrected using Illumina short-reads and PacBio SMRT reads. The 933.12 Mb assembly was annotated as containing 79,870 transcripts for 58,550 genes using RNA-Seq data and the public soybean annotation set. Comparison of the Hwangkeum assembly with the Williams 82 soybean reference genome sequence (Wm82.a2.v1) revealed 1.8 million single-nucleotide polymorphisms, 0.5 million indels, and 25 thousand putative structural variants. However, there was no natural megabase-scale chromosomal rearrangement. Incidentally, by adding two novel subfamilies, we found that soybean contains four clearly separated subfamilies of centromeric satellite repeats. Analyses of satellite repeats and gene content suggested that the Hwangkeum assembly is a high-quality assembly. This was further supported by comparison of the marker arrangement of anthocyanin biosynthesis genes and of gene arrangement at the Rsv3 locus. Therefore, the results indicate that the de novo assembly of Hwangkeum is a valuable additional reference genome resource for characterizing traits for the improvement of this important crop species.

Seeing the Forest through the (Phylogenetic) Trees: Functional Characterisation of Grapevine Terpene Synthase (VviTPS) Paralogues and Orthologues.

Gene families involved in specialised metabolism play a key role in a myriad of ecophysiological and biochemical functions. The Vitis vinifera sesquiterpene synthases represent the largest subfamily of grapevine terpene synthase (VviTPS) genes and are important volatile metabolites for wine flavour and aroma, as well as ecophysiological interactions. The functional characterisation of VviTPS genes is complicated by a reliance on a single reference genome that greatly underrepresents this large gene family, exacerbated by extensive duplications and paralogy. The recent release of multiple phased diploid grapevine genomes, as well as extensive whole-genome resequencing efforts, provide a wealth of new sequence information that can be utilised to overcome the limitations of the reference genome. A large cluster of sesquiterpene synthases, localised to chromosome 18, was explored by means of comparative sequence analyses using the publicly available grapevine reference genome, three PacBio phased diploid genomes and whole-genome resequencing data from multiple genotypes. Two genes, VviTPS04 and -10, were identified as putative paralogues and/or allelic variants. Subsequent gene isolation from multiple grapevine genotypes and characterisation by means of a heterologous in planta expression and volatile analysis resulted in the identification of genotype-specific structural variations and polymorphisms that impact the gene function. These results present novel insight into how grapevine domestication likely shaped the VviTPS landscape to result in genotype-specific functions.

Bioinformatic Extraction of Functional Genetic Diversity from Heterogeneous Germplasm Collections for Crop Improvement

Efficient utilization of genetic variation in plant germplasm collections is impeded by large collection size, uneven characterization of traits, and unpredictable apportionment of allelic diversity among heterogeneous accessions. Distributing compact subsets of the complete collection that contain maximum allelic diversity at functional loci of interest could streamline conventional and precision breeding. Using heterogeneous population samples from Arabidopsis, Populus and sorghum, we show that genomewide single nucleotide polymorphism (SNP) data permits the capture of 3–78 fold more haplotypic diversity in subsets than geographic or environmental data, which are commonly used surrogate predictors of genetic diversity. Using a large genomewide SNP data set from landrace sorghum, we demonstrate three bioinformatic approaches to extract functional genetic diversity. First, in a “candidate gene” approach, we assembled subsets that maximized haplotypic diversity at 135 putative lignin biosynthetic loci, relevant to biomass breeding programs. Secondly, we applied a keyword search against the Gene Ontology to identify 1040 regulatory loci and assembled subsets capturing genomewide regulatory gene diversity, a general source of phenotypic variation. Third, we developed a machine-learning approach to rank semantic similarity between Gene Ontology term definitions and the textual content of scientific publications on crop adaptation to climate, a complex breeding objective. We identified 505 sorghum loci whose defined function is semantically-related to climate adaptation concepts. The assembled subsets could be used to address climatic pressures on sorghum production. To face impending agricultural challenges and foster rapid extraction and use of novel genetic diversity resident in heterogeneous germplasm collections, whole genome resequencing efforts should be prioritized.

Evolutionary superscaffolding and chromosome anchoring to improve Anopheles genome assemblies

BackgroundNew sequencing technologies have lowered financial barriers to whole genome sequencing, but resulting assemblies are often fragmented and far from ‘finished’. Updating multi-scaffold drafts to chromosome-level status can be achieved through experimental mapping or re-sequencing efforts. Avoiding the costs associated with such approaches, comparative genomic analysis of gene order conservation (synteny) to predict scaffold neighbours (adjacencies) offers a potentially useful complementary method for improving draft assemblies.ResultsWe evaluated and employed 3 gene synteny-based methods applied to 21 Anopheles mosquito assemblies to produce consensus sets of scaffold adjacencies. For subsets of the assemblies, we integrated these with additional supporting data to confirm and complement the synteny-based adjacencies: 6 with physical mapping data that anchor scaffolds to chromosome locations, 13 with paired-end RNA sequencing (RNAseq) data, and 3 with new assemblies based on re-scaffolding or long-read data. Our combined analyses produced 20 new superscaffolded assemblies with improved contiguities: 7 for which assignments of non-anchored scaffolds to chromosome arms span more than 75% of the assemblies, and a further 7 with chromosome anchoring including an 88% anchored Anopheles arabiensis assembly and, respectively, 73% and 84% anchored assemblies with comprehensively updated cytogenetic photomaps for Anopheles funestus and Anopheles stephensi.ConclusionsExperimental data from probe mapping, RNAseq, or long-read technologies, where available, all contribute to successful upgrading of draft assemblies. Our evaluations show that gene synteny-based computational methods represent a valuable alternative or complementary approach. Our improved Anopheles reference assemblies highlight the utility of applying comparative genomics approaches to improve community genomic resources.

Linking a role of lncRNAs (long non-coding RNAs) with insulin resistance, accelerated senescence, and inflammation in patients with type 2 diabetes

BackgroundStudying epigenetics is expected to provide precious information on how environmental factors contribute to type 2 diabetes mellitus (T2DM) at the genomic level. With the progress of the whole-genome resequencing efforts, it is now known that 75–90% of the human genome was transcribed to generate a series of long non-coding RNAs (lncRNAs). While lncRNAs are gaining widespread attention as potential and robust biomarkers in the genesis as well as progression of several disease states, their clinical relevance and regulatory mechanisms are yet to be explored in the field of metabolic disorders including diabetes. Despite the fact that Asian Indians are highly insulin resistant and more prone to develop T2DM and associated vascular complications, there is virtually lack of data on the role of lncRNAs in the clinical diabetes setting. Therefore, we sought to evaluate a panel of lncRNAs and senescence-inflammation signatures in peripheral blood mononuclear cells (PBMCs) from patients with type 2 diabetes (T2DM; n = 30) compared to individuals with normal glucose tolerance (NGT; n = 32).ResultsCompared to control subjects, expression levels of lncRNAs in PBMCs from type 2 diabetes patients showed significantly (p < 0.05) increased levels of HOTAIR, MEG3, LET, MALAT1, MIAT, CDKN2BAS1/ANRIL, XIST, PANDA, GAS5, Linc-p21, ENST00000550337.1, PLUTO, and NBR2. In contrast, lncRNA expression patterns of THRIL and SALRNA1 were significantly (p < 0.05) decreased in patients with T2DM compared to control subjects. At the transcriptional level, senescence markers (p53, p21, p16, and β-galactosidase), proinflammatory markers (TNF-α, IL6, MCP1, and IL1-β), and epigenetic signature of histone deacetylase-3 (HDAC3) were significantly (p < 0.05) elevated in patients with type 2 diabetes compared to control subjects. Interestingly, mRNA expression of Sirt1 and telomere length were significantly (p < 0.05) decreased in patients with type 2 diabetes compared to control subjects. Majority of the altered lncRNAs were positively correlated with poor glycemic control, insulin resistance, transcriptional markers of senescence, inflammation, and HDAC3 and negatively correlated with telomere length. Logistic regression analysis revealed a significant association of altered lncRNA signatures with T2DM, but this association was lost after adjusting for insulin resistance (HOMA-IR) and senescence markers.ConclusionOur study provides a clinically relevant evidence for the association of altered lncRNAs with poor glycemic control, insulin resistance, accelerated cellular senescence, and inflammation.

Assessing the between-background stability of metabolic effects arising from lignin-related transgenic modifications, in two Populus hybrids using non-targeted metabolomics

The advances in 'high-throughput' biology have significantly expanded our fundamental understanding of complex biological processes inherent to tree growth and development. Relative to the significant achievements attained with whole genome re-sequencing and transcriptomics efforts, the development and power of post-transcriptional tools such as proteomics and metabolomics continue to lag behind in tree biology. However, the inclusion of these powerful functional genomics platforms should substantially enable systems biology assessments of tree development, physiology and response(s) to biotic and abiotic stresses. Herein, we employ a non-targeted metabolomics platform to elucidate the metabolic plasticity of xylem lignification in distinct hybrid poplar genetic backgrounds, as well as in transgenic trees in these backgrounds expressing two common constructs: the first construct (C4H::F5H) augments monolignol content (syringyl:guaiacyl (S:G) ratio), while the second construct (C3'H-RNAi) reduces cell wall lignification. The results clearly show that genotype-specific metabolism exists, and provide an appropriate foundation for properly comparing the influence of background on the relationships between metabolic and specific phenotypic traits. Moreover, it was apparent that transgene-induced phenotypic gradients in cell wall chemical wood can be associated with global metabolism of secondary xylem biosynthesis, however in a genotype-specific manner. This result implies that the same may be true for phenotypic gradients arising through natural genetic variation, intensive breeding or environmental factors. It is also apparent that while distinct, at a global level the wood-forming metabolisms of different poplar hybrids can, to some extent, respond similarly to the influences of genetic manipulation of lignin-related genes. This further implies that with the correct approach, it may be possible to associate the emergence of specific wood traits from different genetic backgrounds-be they transgene-induced or otherwise-with stable metabolic signatures.

Short template switch events explain mutation clusters in the human genome.

Resequencing efforts are uncovering the extent of genetic variation in humans and provide data to study the evolutionary processes shaping our genome. One recurring puzzle in both intra- and inter-species studies is the high frequency of complex mutations comprising multiple nearby base substitutions or insertion-deletions. We devised a generalized mutation model of template switching during replication that extends existing models of genome rearrangement and used this to study the role of template switch events in the origin of short mutation clusters. Applied to the human genome, our model detects thousands of template switch events during the evolution of human and chimp from their common ancestor and hundreds of events between two independently sequenced human genomes. Although many of these are consistent with a template switch mechanism previously proposed for bacteria, our model also identifies new types of mutations that create short inversions, some flanked by paired inverted repeats. The local template switch process can create numerous complex mutation patterns, including hairpin loop structures, and explains multinucleotide mutations and compensatory substitutions without invoking positive selection, speculative mechanisms, or implausible coincidence. Clustered sequence differences are challenging for current mapping and variant calling methods, and we show that many erroneous variant annotations exist in human reference data. Local template switch events may have been neglected as an explanation for complex mutations because of biases in commonly used analyses. Incorporation of our model into reference-based analysis pipelines and comparisons of de novo assembled genomes will lead to improved understanding of genome variation and evolution.

Rs2802292 polymorphism in the FOXO3A gene and exceptional longevity in two ethnically distinct cohorts

ObjectivesPrevious studies have indicated that the rs2802292 polymorphism in the human forkhead box O3A (FOXO3A) gene might be associated with exceptional longevity (EL, i.e., living 100+ years), although the results are conflicting. Study design and main outcome measuresUsing a case-control design, we investigated the distribution of the rs2802292 polymorphism in two ethnically distinct cohorts of centenarians (cases) and younger adults (controls). The first cohort included Japanese individuals (733 centenarians and 820 controls) and the second was from Northern Italy (79 disease-free centenarians and 316 controls). ResultsNo statistically significant association was found between the rs2802292 polymorphism and EL in either cohort (either examined in their entirety or in a sex-based analysis). ConclusionsIn light of our negative findings, further research and resequencing efforts are needed to shed more light on the potential association between EL and FOXO3A polymorphisms.

The genetic landscape of Alzheimer disease: clinical implications and perspectives.

The search for the genetic factors contributing to Alzheimer disease (AD) has evolved tremendously throughout the years. It started from the discovery of fully penetrant mutations in Amyloid precursor protein, Presenilin 1, and Presenilin 2 as a cause of autosomal dominant AD, the identification of the ɛ4 allele of Apolipoprotein E as a strong genetic risk factor for both early-onset and late-onset AD, and evolved to the more recent detection of at least 21 additional genetic risk loci for the genetically complex form of AD emerging from genome-wide association studies and massive parallel resequencing efforts. These advances in AD genetics are positioned in light of the current endeavor directing toward translational research and personalized treatment of AD. We discuss the current state of the art of AD genetics and address the implications and relevance of AD genetics in clinical diagnosis and risk prediction, distinguishing between monogenic and multifactorial AD. Furthermore, the potential and current limitations of molecular reclassification of AD to streamline clinical trials in drug development and biomarker studies are addressed.Genet Med 18 5, 421–430.

Connecting SNPs in Diabetes: A Spatial Analysis of Meta-GWAS Loci.

Meta-analyses of genome-wide association studies (GWAS) have improved our understanding of the genetic foundations of a number of diseases, including diabetes. However, single nucleotide polymorphisms (SNPs) that are identified by GWAS, especially those that fall outside of gene regions, do not always clearly link to the underlying biology. Despite this, these SNPs have often been validated through re-sequencing efforts as not just tag SNPs, but as causative SNPs, and so must play a role in disease development or progression. In this study, we show how the 3D genome (spatial connections) and trans-expression Quantitative Trait Loci connect diabetes loci from different GWAS meta-analyses, informing the backbone of regulatory networks. Our findings include a three-way functional–spatial connection between the TM6SF2, CTRB1–BCAR1, and CELSR2–PSRC1 loci (rs201189528, rs7202844, and rs7202844, respectively) connected through the KCNIP3 and BCAR1/BCAR3 loci, respectively. These spatial hubs serve as an example of how loci in genes with little biological connection to disease come together to contribute to the diabetes phenotype.

Does reverse transport of dopamine play a role in autism?

genetic variants affecting the reverse transport of DA might influence the risk of common, sporadic forms of autism remains open. It is now widely accepted that rare genetic variants could play an important role in susceptibility to common diseases. In light of Cartier et al.'s results, major resequencing efforts of the SLC6A3 and STX1A genes should be undertaken in large cohorts of individuals with autism, with significant insights into disease biology likely from these results. Another interesting implication of the work by Cartier and colleagues lies in unraveling the therapeutic potential of DA reverse transport in autism. Starting from the observation that rare autism-causing variants are associated with inhibition of reverse transport of DA, it could be hypothesized that psychostimulants may serve as a potential therapeutic aid in autism. Notably, not only have psychostimulants been suggested to be effective for ADHD-like symptoms in autism spectrumdisorderindividuals,buttheprevalenceratesoftheiruseinautism

GeneFriends: a human RNA-seq-based gene and transcript co-expression database.

Co-expression networks have proven effective at assigning putative functions to genes based on the functional annotation of their co-expressed partners, in candidate gene prioritization studies and in improving our understanding of regulatory networks. The growing number of genome resequencing efforts and genome-wide association studies often identify loci containing novel genes and there is a need to infer their functions and interaction partners. To facilitate this we have expanded GeneFriends, an online database that allows users to identify co-expressed genes with one or more user-defined genes. This expansion entails an RNA-seq-based co-expression map that includes genes and transcripts that are not present in the microarray-based co-expression maps, including over 10 000 non-coding RNAs. The results users obtain from GeneFriends include a co-expression network as well as a summary of the functional enrichment among the co-expressed genes. Novel insights can be gathered from this database for different splice variants and ncRNAs, such as microRNAs and lincRNAs. Furthermore, our updated tool allows candidate transcripts to be linked to diseases and processes using a guilt-by-association approach. GeneFriends is freely available from http://www.GeneFriends.org and can be used to quickly identify and rank candidate targets relevant to the process or disease under study.

Targeted resequencing of the microRNAome and 3'UTRome reveals functional germline DNA variants with altered prevalence in epithelial ovarian cancer.

Ovarian cancer is a major cause of cancer deaths, yet there have been few known genetic risk factors identified, the best known of which are disruptions in protein coding sequences (BRCA1 and 2). Recent findings indicate that there are powerful genetic markers of cancer risk outside of these regions, in the noncoding mRNA control regions. To identify additional cancer-associated, functional non-protein-coding sequence germline variants associated with ovarian cancer risk, we captured DNA regions corresponding to all validated human microRNAs and the 3' untranslated regions (UTRs) of ~6000 cancer-associated genes from 31 ovarian cancer patients. Multiple single-nucleotide polymorphisms in the 3'UTR of the vascular endothelial growth factor receptor/FLT1, E2F2 and PCM1 oncogenes were highly enriched in ovarian cancer patients compared with the 1000 Genome Project. Sequenom validation in a case-control study (267 cases and 89 controls) confirmed a novel variant in the PCM1 3'UTR is significantly associated with ovarian cancer (P=0.0086). This work identifies a potential new ovarian cancer locus and further confirms that cancer resequencing efforts should not ignore the study of noncoding regions of cancer patients.

Regulatory variation: an emerging vantage point for cancer biology

Transcriptional regulation involves complex and interdependent interactions of noncoding and coding regions of the genome with proteins that interact and modify them. Genetic variation/mutation in coding and noncoding regions of the genome can drive aberrant transcription and disease. In spite of accounting for nearly 98% of the genome comparatively little is known about the contribution of noncoding DNA elements to disease. Genome-wide association studies of complex human diseases including cancer have revealed enrichment for variants in the noncoding genome. A striking finding of recent cancer genome re-sequencing efforts has been the previously underappreciated frequency of mutations in epigenetic modifiers across a wide range of cancer types. Taken together these results point to the importance of dysregulation in transcriptional regulatory control in genesis of cancer. Powered by recent technological advancements in functional genomic profiling, exploration of normal and transformed regulatory networks will provide novel insight into the initiation and progression of cancer and open new windows to future prognostic and diagnostic tools.

Abstract 1218: Development of comprehensive molecular portraits of lobular breast cancer within the RATHER (Rational Therapy for Breast Cancer) Consortium.

Abstract Background: The RATHER Consortium aims to identify novel kinase targets for therapy in poor-prognosis subtypes of breast cancer for which there are currently no targeted therapies available; namely invasive lobular carcinomas (ILC) which represent 10% of breast tumors. The RATHER approach differs in two fundamental ways from the ongoing cancer genome re-sequencing efforts. Firstly, the major cancer genome re-sequencing efforts focus on DNA sequence analysis of the whole genome. In RATHER, we identify and validate novel kinases as therapy targets in a more comprehensive way by analyzing the DNA sequence and by applying state-of-the-art proteomics, copy number analysis and gene expression profiling technologies to identify activated/altered kinases. Secondly, RATHER also differs by focusing on the lobular subtype of breast cancer. Methods: One hundred and fifty ILC samples (fresh frozen) with &amp;gt;5years follow-up were collected from two institutes (Cambridge, NKI). All samples were processed following one standard operating protocol to isolate RNA, DNA and protein of high quality. We used a five-pronged approach to identify and validate novel kinase targets for therapy in ILC breast cancer, namely i) direct re-sequencing of the kinome of 150 ILC breast tumors, ii) determination of abundance and activation status of kinases in these tumors by reverse phase protein lysate array (RPPA) technology, iii) determination of copy number aberration (CNA) in kinase genes by SNP arrays, iv) mRNA quantitation of both genome and kinome using DNA microarrays and v)RNAseq of a subset of ILC tumors. Results: Data from these independent genome-scale technologies were integrated, yielding a priority list of potential kinase targets for therapy in ILC breast cancer. Deep sequencing of the kinome has revealed somatic mutations characteristic of ILC, which are currently being validated via mass spectrometry-based genotyping technology and their possible effects confirmed with gene expression, protein expression and phosphorylation changes. In addition, a subset of the ILC samples was assessed via RNA sequencing approach to confirm expression of particular mutants. Known gene mutations in ILC such as loss of CDH1 were confirmed; furthermore, a high frequency of PI3K pathway alterations (50%) was observed. Gene expression analysis of such a large set of ILC can reveal subsets of these breast cancers that significantly regulate alternate biological processes. Such biological subsets are currently being validated with clinical and follow-up data. Conclusion: The RATHER project aims to deliver proof-of-concept for novel therapeutic interventions, together with companion molecular diagnostic assays for patient stratification, for up to 10% of breast cancer patients, where current treatment options are unsatisfactory. Citation Format: Justine K. Peeters, Ian Majewski, Leanne de Koning, Yue Fan, Finbarr Tarrant, Darran O'Connor, Jeroen Heijmans, Mireille Snel, Tesa Severson, Astrid Bosma, Magali Michaut, Lorenza Mittempergher, Suet-Feung Chin, Thierry Dubois, William Gallagher, Carlos Caldas, Rene Bernards, Iris Simon, RATHER Consortium. Development of comprehensive molecular portraits of lobular breast cancer within the RATHER (Rational Therapy for Breast Cancer) Consortium. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1218. doi:10.1158/1538-7445.AM2013-1218

Karyotype polymorphism and chromosomal rearrangement in populations of the phytopathogenic fungus, Ascochyta rabiei

The fungus Ascochyta rabiei is the causal agent of Ascochyta blight of chickpea and the most serious threat to chickpea production. Little is currently known about the genome size or organization of A. rabiei. Given recent genome sequencing efforts, characterization of the genome at a population scale will provide a framework for genome interpretation and direction of future resequencing efforts. Electrophoretic karyotype profiles of 112 isolates from 21 countries revealed 12–16 chromosomes between 0.9 Mb and 4.6 Mb with an estimated genome size of 23 Mb–34 Mb. Three general karyotype profiles A, B, and C were defined by the arrangement of the largest chromosomes. Approximately one-third of isolates (group A) possessed a chromosome larger than 4.0 Mb that was absent from group B and C isolates. The ribosomal RNA gene (rDNA) cluster was assigned to the largest chromosome in all except four isolates (group C) whose rDNA cluster was located on the second largest chromosome (3.2 Mb). Analysis of progeny from an in vitro sexual cross between two group B isolates revealed one of 16 progeny with an rDNA-encoding chromosome larger than 4.0 Mb similar to group A isolates, even though a chromosome of this size was not present in either parent. No expansion of the rDNA cluster was detected in the progeny, indicating the increase in chromosome size was not due to an expansion in number of rDNA repeats. The karyotype of A. rabiei is relatively conserved when compared with published examples of asexual ascomycetes, but labile with the potential for large scale chromosomal rearrangements during meiosis. The results of this study will allow for the targeted sequencing of specific isolates to determine the molecular mechanisms of karyotype variation within this species.