Grapevine berries undergo complex biochemical changes during fruit maturation, many of which are dependent upon the variety and its environment. In order to elucidate the varietal dependent ...developmental regulation of primary and specialized metabolism, berry skins of Cabernet Sauvignon and Shiraz were subjected to gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) based metabolite profiling from pre-veraison to harvest. The generated dataset was augmented with transcript profiling using RNAseq.
The analysis of the metabolite data revealed similar developmental patterns of change in primary metabolites between the two cultivars. Nevertheless, towards maturity the extent of change in the major organic acid and sugars (i.e. sucrose, trehalose, malate) and precursors of aromatic and phenolic compounds such as quinate and shikimate was greater in Shiraz compared to Cabernet Sauvignon. In contrast, distinct directional projections on the PCA plot of the two cultivars samples towards maturation when using the specialized metabolite profiles were apparent, suggesting a cultivar-dependent regulation of the specialized metabolism. Generally, Shiraz displayed greater upregulation of the entire polyphenol pathway and specifically higher accumulation of piceid and coumaroyl anthocyanin forms than Cabernet Sauvignon from veraison onwards. Transcript profiling revealed coordinated increased transcript abundance for genes encoding enzymes of committing steps in the phenylpropanoid pathway. The anthocyanin metabolite profile showed F3'5'H-mediated delphinidin-type anthocyanin enrichment in both varieties towards maturation, consistent with the transcript data, indicating that the F3'5'H-governed branching step dominates the anthocyanin profile at late berry development. Correlation analysis confirmed the tightly coordinated metabolic changes during development, and suggested a source-sink relation between the central and specialized metabolism, stronger in Shiraz than Cabernet Sauvignon. RNAseq analysis also revealed that the two cultivars exhibited distinct pattern of changes in genes related to abscisic acid (ABA) biosynthesis enzymes.
Compared with CS, Shiraz showed higher number of significant correlations between metabolites, which together with the relatively higher expression of flavonoid genes supports the evidence of increased accumulation of coumaroyl anthocyanins in that cultivar. Enhanced stress related metabolism, e.g. trehalose, stilbene and ABA in Shiraz berry-skin are consistent with its relatively higher susceptibility to environmental cues.
Plants such as grapevine (Vitis spp.) display significant inter-cultivar genetic and phenotypic variation. The genetic components underlying phenotypic diversity in grapevine must be understood in ...order to disentangle genetic and environmental factors.
We have shown that cDNA sequencing by RNA-seq is a robust approach for the characterization of varietal diversity between a local grapevine cultivar (Corvina) and the PN40024 reference genome. We detected 15,161 known genes including 9463 with novel splice isoforms, and identified 2321 potentially novel protein-coding genes in non-annotated or unassembled regions of the reference genome. We also discovered 180 apparent private genes in the Corvina genome which were missing from the reference genome.
The de novo assembly approach allowed a substantial amount of the Corvina transcriptome to be reconstructed, improving known gene annotations by robustly defining gene structures, annotating splice isoforms and detecting genes without annotations. The private genes we discovered are likely to be nonessential but could influence certain cultivar-specific characteristics. Therefore, the application of de novo transcriptome assembly should not be restricted to species lacking a reference genome because it can also improve existing reference genome annotations and identify novel, cultivar-specific genes.
Abstract
Gatekeeper mutations are identified in only 50% of the cases at resistance to
Anaplastic Lymphoma Kinase
(
ALK
)-tyrosine kinase inhibitors (TKIs). Circulating tumor cells (CTCs) are ...relevant tools to identify additional resistance mechanisms and can be sequenced at the single-cell level. Here, we provide in-depth investigation of copy number alteration (CNA) heterogeneity in phenotypically characterized CTCs at resistance to ALK-TKIs in
ALK
-positive non-small cell lung cancer. Single CTC isolation and phenotyping were performed by DEPArray or fluorescence-activated cell sorting following enrichment and immunofluorescence staining (ALK/cytokeratins/CD45/Hoechst). CNA heterogeneity was evaluated in six
ALK
-rearranged patients harboring ≥ 10 CTCs/20 mL blood at resistance to 1
st
and 3
rd
ALK-TKIs and one presented gatekeeper mutations. Out of 82 CTCs isolated by FACS, 30 (37%) were ALK
+
/cytokeratins
-
, 46 (56%) ALK
-
/cytokeratins
+
and 4 (5%) ALK
+
/cytokeratins
+
. Sequencing of 43 CTCs showed highly altered CNA profiles and high levels of chromosomal instability (CIN). Half of CTCs displayed a ploidy >2n and 32% experienced whole-genome doubling. Hierarchical clustering showed significant intra-patient and wide inter-patient CTC diversity. Classification of 121 oncogenic drivers revealed the predominant activation of cell cycle and DNA repair pathways and of RTK/RAS and PI3K to a lower frequency. CTCs showed wide CNA heterogeneity and elevated CIN at resistance to ALK-TKIs. The emergence of epithelial
ALK
-negative CTCs may drive resistance through activation of bypass signaling pathways, while
ALK
-rearranged CTCs showed epithelial-to-mesenchymal transition characteristics potentially contributing to ALK-TKI resistance. Comprehensive analysis of CTCs could be of great help to clinicians for precision medicine and resistance to ALK-targeted therapies.
INTRODUCTION: Translocations of the anaplastic lymphoma kinase (ALK) can be effectively targeted in advanced non-small cell lung cancer by ALK-TKI inhibitors including Crizotinib. However, the ...development of acquired resistance often limits the duration of these therapies. While several mechanisms of secondary resistance have been already identified, little is known about molecular determinants of primary resistance. In our brief report we investigated the tumor molecular profile of a patient who failed to respond to Crizotinib. METHODS: Fluorescence in situ hybridization (FISH) and next-generation sequencing (NGS) were run on tumor specimen as well as search and characterization of circulating tumor cells (CTCs) in the blood. Confirmation of clinical findings was achieved using a translational cell-line in vitro model. RESULTS: We identified the amplification of MYC as a potential new mechanism of primary resistance to ALK inhibition. Human EML4-ALK rearranged cells infected with a lentiviral vector carrying full-length human MYC cDNA were treated in vitro with crizotinib and alectinib. Overexpression of MYC overexpression was associated with a reduced sensitivity to both ALK-inhibitors. MYC-overexpressing clones displayed also increased levels of both cyclin D and E and their growth was reduced by using Cdk4/6 inhibitors such as Palbociclib. CONCLUSIONS: We postulate that the MYC gene may be implicated in the mechanism of primary resistance to ALK inhibitors. We also suggest potential MYC-directed inhibition strategies to overcome primary resistance in advanced ALK-rearranged NSCLC.
Chromosomal instability and associated chromosomal aberrations are hallmarks of cancer and play a critical role in disease progression and development of resistance to drugs. Single-cell genome ...analysis has gained interest in latest years as a source of biomarkers for targeted-therapy selection and drug resistance, and several methods have been developed to amplify the genomic DNA and to produce libraries suitable for Whole Genome Sequencing (WGS). However, most protocols require several enzymatic and cleanup steps, thus increasing the complexity and length of protocols, while robustness and speed are key factors for clinical applications. To tackle this issue, we developed a single-tube, single-step, streamlined protocol, exploiting ligation mediated PCR (LM-PCR) Whole Genome Amplification (WGA) method, for low-pass genome sequencing with the Ion Torrent™ platform and copy number alterations (CNAs) calling from single cells. The method was evaluated on single cells isolated from 6 aberrant cell lines of the NCI-H series. In addition, to demonstrate the feasibility of the workflow on clinical samples, we analyzed single circulating tumor cells (CTCs) and white blood cells (WBCs) isolated from the blood of patients affected by prostate cancer or lung adenocarcinoma. The results obtained show that the developed workflow generates data accurately representing whole genome absolute copy number profiles of single cell and allows alterations calling at resolutions down to 100 Kbp with as few as 200,000 reads. The presented data demonstrate the feasibility of the Ampli1™ WGA-based low-pass workflow for detection of CNAs in single tumor cells which would be of particular interest for genome-driven targeted therapy selection and for monitoring of disease progression.
Precision medicine in oncology requires an accurate characterization of a tumor molecular profile for patient stratification. Though targeted deep sequencing is an effective tool to detect the ...presence of somatic sequence variants, a significant number of patient specimens do not meet the requirements needed for routine clinical application. Analysis is hindered by contamination of normal cells and inherent tumor heterogeneity, compounded with challenges of dealing with minute amounts of tissue and DNA damages common in formalin-fixed paraffin-embedded (FFPE) specimens. Here we present an innovative workflow using DEPArray™ system, a microchip-based digital sorter to achieve 100%-pure, homogenous subpopulations of cells from FFPE samples. Cells are distinguished by fluorescently labeled antibodies and DNA content. The ability to address tumor heterogeneity enables unambiguous determination of true-positive sequence variants, loss-of-heterozygosity as well as copy number variants. The proposed strategy overcomes the inherent trade-offs made between sensitivity and specificity in detecting genetic variants from a mixed population, thus rescuing for analysis even the smaller clinical samples with low tumor cellularity.
To analyze the association between age-related macular degeneration (AMD) and polymorphisms in vascular endothelial growth factor (VEGF) and VEGF receptor KDR gene polymorphisms. A complex, ...multifactorial disease in which genetic and environmental factors interact, AMD is the leading cause of blindness in the elderly. Vascular endothelial growth factor (VEGF), a key regulator of angiogenesis, is considered an important factor for the pathogenetic processes of AMD. Previous studies investigated the possible association between VEGF-A gene polymorphisms and AMD, with contrasting data. No study examined the possible role of VEGF receptor KDR gene polymorphisms.
Case-control study.
We enrolled 226 AMD cases and 248 controls from an ophthalmology hospital center.
Genotypying for 16 polymorphic markers (single nucleotide polymorphisms SNPs) in VEGF-A and KDR genes.
Distribution of genotypes in AMD cases and controls.
Two polymorphisms (rs833069 in intron 2 of the VEGF-A gene, rs2071559 in the promoter of the KDR gene) were significantly associated with risk of AMD. In particular, for VEGF-A rs833069 the AMD risk was increased >5-fold for G homozygotes compared with homozygous carriage of the A allele. For KDR rs2071559 the AMD risk was increased >3-fold for T homozygotes compared with homozygous carriage of the C allele. Carriers of risk alleles for both markers have a >6-fold increased risk of AMD with respect to carriers of non-risk alleles.
We expand previous data on the association of AMD with VEGF-A gene variations and identify for the first time an association with variations in the KDR gene. Because the SNP-604T-bearing KDR promoter has higher transcription activity, our findings further support the role of the VEGF pathway in the pathophysiology of AMD. It is possible that applications of haplotype/genotype analysis in these genes will play a role in risk assessment and pharmacogenomic approaches to AMD diagnosis and management.
The grapevine (Vitis vinifera) cultivar Tannat is cultivated mainly in Uruguay for the production of high-quality red wines. Tannat berries have unusually high levels of polyphenolic compounds, ...producing wines with an intense purple color and remarkable antioxidant properties. We investigated the genetic basis of these important characteristics by sequencing the genome of the Uruguayan Tannat clone UY11 using Illumina technology, followed by a mixture of de novo assembly and iterative mapping onto the PN40024 reference genome. RNA sequencing data for genome reannotation were processed using a combination of reference-guided annotation and de novo transcript assembly, allowing 5901 previously unannotated or unassembled genes to be defined and resulting in the discovery of 1873 genes that were not shared with PN40024. Expression analysis showed that these cultivar-specific genes contributed substantially (up to 81.24%) to the overall expression of enzymes involved in the synthesis of phenolic and polyphenolic compounds that contribute to the unique characteristics of the Tannat berries. The characterization of the Tannat genome therefore indicated that the grapevine reference genome lacks many genes that appear to be relevant for the varietal phenotype.
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