Variation and plasticity in life-history traits and fitness of wild Arabidopsis thaliana populations are not related to their genotypic and ecological diversity.

BACKGROUND: Despite its implications for population dynamics and evolution, the relationship between genetic and phenotypic variation in wild populations remains unclear. Here, we estimated variation and plasticity in life-history traits and fitness of the annual plant Arabidopsis thaliana in two common garden experiments that differed in environmental conditions. We used up to 306 maternal inbred lines from six Iberian populations characterized by low and high genotypic (based on whole-genome sequences) and ecological (vegetation type) diversity. RESULTS: Low and high genotypic and ecological diversity was found in edge and core Iberian environments, respectively. Given that selection is expected to be stronger in edge environments and that ecological diversity may enhance both phenotypic variation and plasticity, we expected genotypic diversity to be positively associated with phenotypic variation and plasticity. However, maternal lines, irrespective of the genotypic and ecological diversity of their population of origin, exhibited a substantial amount of phenotypic variation and plasticity for all traits. Furthermore, all populations harbored maternal lines with canalization (robustness) or sensitivity in response to harsher environmental conditions in one of the two experiments. CONCLUSIONS: Overall, we conclude that the environmental attributes of each population probably determine their genotypic diversity, but all populations maintain substantial phenotypic variation and plasticity for all traits, which represents an asset to endure in changing environments.

The trichome pattern diversity of Cardamine shares genetic mechanisms with Arabidopsis but differs in environmental drivers.

Natural variation in trichome pattern (amount and distribution) is prominent among populations of many angiosperms. However, the degree of parallelism in the genetic mechanisms underlying this diversity and its environmental drivers in different species remain unclear. To address these questions, we analyzed the genomic and environmental bases of leaf trichome pattern diversity in Cardamine hirsuta, a relative of Arabidopsis (Arabidopsis thaliana). We characterized 123 wild accessions for their genomic diversity, leaf trichome patterns at different temperatures, and environmental adjustments. Nucleotide diversities and biogeographical distribution models identified two major genetic lineages with distinct demographic and adaptive histories. Additionally, C. hirsuta showed substantial variation in trichome pattern and plasticity to temperature. Trichome amount in C. hirsuta correlated positively with spring precipitation but negatively with temperature, which is opposite to climatic patterns in A. thaliana. Contrastingly, genetic analysis of C. hirsuta glabrous accessions indicated that, like for A. thaliana, glabrousness is caused by null mutations in ChGLABRA1 (ChGL1). Phenotypic genome-wide association studies (GWAS) further identified a ChGL1 haplogroup associated with low trichome density and ChGL1 expression. Therefore, a ChGL1 series of null and partial loss-of-function alleles accounts for the parallel evolution of leaf trichome pattern in C. hirsuta and A. thaliana. Finally, GWAS also detected other candidate genes (e.g. ChETC3, ChCLE17) that might affect trichome pattern. Accordingly, the evolution of this trait in C. hirsuta and A. thaliana shows partially conserved genetic mechanisms but is likely involved in adaptation to different environments.

Genomic and immune landscape Of metastatic pheochromocytoma and paraganglioma.

The mechanisms triggering metastasis in pheochromocytoma/paraganglioma are unknown, hindering therapeutic options for patients with metastatic tumors (mPPGL). Herein we show by genomic profiling of a large cohort of mPPGLs that high mutational load, microsatellite instability and somatic copy-number alteration burden are associated with ATRX/TERT alterations and are suitable prognostic markers. Transcriptomic analysis defines the signaling networks involved in the acquisition of metastatic competence and establishes a gene signature related to mPPGLs, highlighting CDK1 as an additional mPPGL marker. Immunogenomics accompanied by immunohistochemistry identifies a heterogeneous ecosystem at the tumor microenvironment level, linked to the genomic subtype and tumor behavior. Specifically, we define a general immunosuppressive microenvironment in mPPGLs, the exception being PD-L1 expressing MAML3-related tumors. Our study reveals canonical markers for risk of metastasis, and suggests the usefulness of including immune parameters in clinical management for PPGL prognostication and identification of patients who might benefit from immunotherapy.

Comprehensive molecular analysis of immortalization hallmarks in thyroid cancer reveals new prognostic markers.

BACKGROUND: Comprehensive molecular studies on tumours are needed to delineate immortalization process steps and identify sensitive prognostic biomarkers in thyroid cancer. METHODS AND RESULTS: In this study, we extensively characterize telomere-related alterations in a series of 106 thyroid tumours with heterogeneous clinical outcomes. Using a custom-designed RNA-seq panel, we identified five telomerase holoenzyme-complex genes upregulated in clinically aggressive tumours compared to tumours from long-term disease-free patients, being TERT and TERC denoted as independent prognostic markers by multivariate regression model analysis. Characterization of alterations related to TERT re-expression revealed that promoter mutations, methylation and/or copy gains exclusively co-occurred in clinically aggressive tumours. Quantitative-FISH (fluorescence in situ hybridization) analysis of telomere lengths showed a significant shortening in these carcinomas, which matched with a high proliferative rate measured by Ki-67 immunohistochemistry. RNA-seq data analysis indicated that short-telomere tumours exhibit an increased transcriptional activity in the 5-Mb-subtelomeric regions, site of several telomerase-complex genes. Gene upregulation enrichment was significant for specific chromosome-ends such as the 5p, where TERT is located. Co-FISH analysis of 5p-end and TERT loci showed a more relaxed chromatin configuration in short telomere-length tumours compared to normal telomere-length tumours. CONCLUSIONS: Overall, our findings support that telomere shortening leads to a 5p subtelomeric region reorganization, facilitating the transcription and accumulation of alterations at TERT-locus.

Spatial control of potato tuberization by the TCP transcription factor BRANCHED1b.

The control of carbon allocation, storage and usage is critical for plant growth and development and is exploited for both crop food production and CO2 capture. Potato tubers are natural carbon reserves in the form of starch that have evolved to allow propagation and survival over winter. They form from stolons, below ground, where they are protected from adverse environmental conditions and animal foraging. We show that BRANCHED1b (BRC1b) acts as a tuberization repressor in aerial axillary buds, which prevents buds from competing in sink strength with stolons. BRC1b loss of function leads to ectopic production of aerial tubers and reduced underground tuberization. In aerial axillary buds, BRC1b promotes dormancy, abscisic acid responses and a reduced number of plasmodesmata. This limits sucrose accumulation and access of the tuberigen protein SP6A. BRC1b also directly interacts with SP6A and blocks its tuber-inducing activity in aerial nodes. Altogether, these actions help promote tuberization underground.

Chronological and biological aging of the human left ventricular myocardium: Analysis of microRNAs contribution.

Aging is the main risk factor for cardiovascular diseases. In humans, cardiac aging remains poorly characterized. Most studies are based on chronological age (CA) and disregard biological age (BA), the actual physiological age (result of the aging rate on the organ structure and function), thus yielding potentially imperfect outcomes. Deciphering the molecular basis of ventricular aging, especially by BA, could lead to major progresses in cardiac research. We aim to describe the transcriptome dynamics of the aging left ventricle (LV) in humans according to both CA and BA and characterize the contribution of microRNAs, key transcriptional regulators. BA is measured using two CA-associated transcriptional markers: CDKN2A expression, a cell senescence marker, and apparent age (AppAge), a highly complex transcriptional index. Bioinformatics analysis of 132 LV samples shows that CDKN2A expression and AppAge represent transcriptomic changes better than CA. Both BA markers are biologically validated in relation to an aging phenotype associated with heart dysfunction, the amount of cardiac fibrosis. BA-based analyses uncover depleted cardiac-specific processes, among other relevant functions, that are undetected by CA. Twenty BA-related microRNAs are identified, and two of them highly heart-enriched that are present in plasma. We describe a microRNA-gene regulatory network related to cardiac processes that are partially validated in vitro and in LV samples from living donors. We prove the higher sensitivity of BA over CA to explain transcriptomic changes in the aging myocardium and report novel molecular insights into human LV biological aging. Our results can find application in future therapeutic and biomarker research.

MYB transcription factors drive evolutionary innovations in Arabidopsis fruit trichome patterning.

Both inter- and intra-specific diversity has been described for trichome patterning in fruits, which is presumably involved in plant adaptation. However, the mechanisms underlying this developmental trait have been hardly addressed. Here we examined natural populations of Arabidopsis (Arabidopsis thaliana) that develop trichomes in fruits and pedicels, phenotypes previously not reported in the Arabidopsis genus. Genetic analyses identified five loci, MALAMBRUNO 1-5 (MAU1-5), with MAU2, MAU3, and MAU5 showing strong epistatic interactions that are necessary and sufficient to display these traits. Functional characterization of these three loci revealed cis-regulatory mutations in TRICHOMELESS1 and TRIPTYCHON, as well as a structural mutation in GLABRA1. Therefore, the multiple mechanisms controlled by three MYB transcription factors of the core regulatory network for trichome patterning have jointly been modulated to trigger trichome development in fruits. Furthermore, analyses of worldwide accessions showed that these traits and mutations only occur in a highly differentiated relict lineage from the Iberian Peninsula. In addition, these traits and alleles were associated with low spring precipitation, which suggests that trichome development in fruits and pedicels might be involved in climatic adaptation. Thus, we show that the combination of synergistic mutations in a gene regulatory circuit has driven evolutionary innovations in fruit trichome patterning in Arabidopsis.

Genetic variation and association analyses identify genes linked to fruit set-related traits in grapevine.

Grapevine is one of the most valuable fruit crops in the world. Adverse environmental conditions reduce fruit quality and crop yield, so understanding the genetic and molecular mechanisms determining crop yield components is essential to optimize grape production. The analysis of a diverse collection of grapevine cultivars allowed us to evaluate the relationship between fruit set-related components of yield, including the incidence of reproductive disorders such as coulure and millerandage. The collection displayed a great phenotypic variation that we surveyed in a genetics association study using 15,309 single nucleotide polymorphisms (SNPs) detected in the sequence of 289 candidate genes scattered across the 19 grapevine linkage groups. After correcting statistical models for population structure and linkage disequilibrium effects, 164 SNPs from 34 of these genes were found to associate with fruit set-related traits, supporting a complex polygenic determinism. Many of them were found in the sequence of different putative MADS-box transcription factors, a gene family related with plant reproductive development control. In addition, we observed an additive effect of some of the associated SNPs on the phenotype, suggesting that advantageous alleles from different loci could be pyramided to generate superior cultivars with optimized fruit production.

VviUCC1 Nucleotide Diversity, Linkage Disequilibrium and Association with Rachis Architecture Traits in Grapevine.

Cluster compactness is a trait with high agronomic relevance, affecting crop yield and grape composition. Rachis architecture is a major component of cluster compactness determinism, and is a target trait toward the breeding of grapevine varieties less susceptible to pests and diseases. Although its genetic basis is scarcely understood, a preliminary result indicated a possible involvement of the VviUCC1 gene. The aim of this study was to characterize the VviUCC1 gene in grapevine and to test the association between the natural variation observed for a series of rachis architecture traits and the polymorphisms detected in the VviUCC1 sequence. This gene encodes an uclacyanin plant-specific cell-wall protein involved in fiber formation and/or lignification processes. A high nucleotide diversity in the VviUCC1 gene promoter and coding regions was observed, but no critical effects were predicted in the protein domains, indicating a high level of conservation of its function in the cultivated grapevine. After correcting statistical models for genetic stratification and linkage disequilibrium effects, marker-trait association results revealed a series of single nucleotide polymorphisms (SNPs) significantly associated with cluster compactness and rachis traits variation. Two of them (Y-984 and K-88) affected two common cis-transcriptional regulatory elements, suggesting an effect on phenotype via gene expression regulation. This work reinforces the interest of further studies aiming to reveal the functional effect of the detected VviUCC1 variants on grapevine rachis architecture.

Hsa-miR-139-5p is a prognostic thyroid cancer marker involved in HNRNPF-mediated alternative splicing.

It is critical to identify biomarkers and functional networks associated with aggressive thyroid cancer to anticipate disease progression and facilitate personalized patient management. We performed miRNome sequencing of 46 thyroid tumors enriched with advanced disease patients with a median follow-up of 96 months. MiRNome profiles correlated with tumor-specific histopathological and molecular features, such as stromal cell infiltration and tumor driver mutation. Differential expression analysis revealed a consistent hsa-miR-139-5p downexpression in primary carcinomas from patients with recurrent/metastatic disease compared to disease-free patients, sustained in paired local metastases and validated in publicly available thyroid cancer series. Exogenous expression of hsa-miR-139-5p significantly reduced migration and proliferation of anaplastic thyroid cancer cells. Proteomic analysis indicated RICTOR, SMAD2/3 and HNRNPF as putative hsa-miR-139-5p targets in our cell system. Abundance of HNRNPF mRNA, encoding an alternative splicing factor involved in cryptic exon inclusion/exclusion, inversely correlated with hsa-miR-139-5p expression in human tumors. RNA sequencing analysis revealed 174 splicing events differentially regulated upon HNRNPF repression in our cell system, affecting genes involved in RTK/RAS/MAPK and PI3K/AKT/MTOR signaling cascades among others. These results point at the hsa-miR-139-5p/HNRNPF axis as a novel regulatory mechanism associated with the modulation of major thyroid cancer signaling pathways and tumor virulence.

Integrative multi-omics analysis identifies a prognostic miRNA signature and a targetable miR-21-3p/TSC2/mTOR axis in metastatic pheochromocytoma/paraganglioma.

Rationale: Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors that present variable outcomes. To date, no effective therapies or reliable prognostic markers are available for patients who develop metastatic PPGL (mPPGL). Our aim was to discover robust prognostic markers validated through in vitro models, and define specific therapeutic options according to tumor genomic features. Methods: We analyzed three PPGL miRNome datasets (n=443), validated candidate markers and assessed them in serum samples (n=36) to find a metastatic miRNA signature. An integrative study of miRNome, transcriptome and proteome was performed to find miRNA targets, which were further characterized in vitro. Results: A signature of six miRNAs (miR-21-3p, miR-183-5p, miR-182-5p, miR-96-5p, miR-551b-3p, and miR-202-5p) was associated with metastatic risk and time to progression. A higher expression of five of these miRNAs was also detected in PPGL patients' liquid biopsies compared with controls. The combined expression of miR-21-3p/miR-183-5p showed the best power to predict metastasis (AUC=0.804, P=4.67·10-18), and was found associated in vitro with pro-metastatic features, such as neuroendocrine-mesenchymal transition phenotype, and increased cell migration rate. A pan-cancer multi-omic integrative study correlated miR-21-3p levels with TSC2 expression, mTOR pathway activation, and a predictive signature for mTOR inhibitor-sensitivity in PPGLs and other cancers. Likewise, we demonstrated in vitro a TSC2 repression and an enhanced rapamycin sensitivity upon miR-21-3p expression. Conclusions: Our findings support the assessment of miR-21-3p/miR-183-5p, in tumors and liquid biopsies, as biomarkers for risk stratification to improve the PPGL patients' management. We propose miR-21-3p to select mPPGL patients who may benefit from mTOR inhibitors.

WeReview: CRISPR Tools-Live Repository of Computational Tools for Assisting CRISPR/Cas Experiments.

Computational tools are essential in the process of designing a CRISPR/Cas experiment for the targeted modification of an organism's genome. Among other functionalities, these tools facilitate the design of a guide-RNA (gRNA) for a given nuclease that maximizes its binding to the intended genomic site, while avoiding binding to undesired sites with similar sequences in the genome of the organism of interest (off-targets). Due to the popularity of this methodology and the rapid pace at which it evolves and changes, new computational tools show up constantly. This rapid turnover, together with the intrinsic high death-rate of bioinformatics tools, mean that many of the published tools become unavailable at some point. Consequently, the traditional ways to inform the community about the landscape of available tools, i.e., reviews in the scientific literature, are not adequate for this fast-moving field. To overcome these limitations, we have developed "WeReview: CRISPR Tools," a live, on-line, user-updatable repository of computational tools to assist researchers in designing CRISPR/Cas experiments. In its web site users can find an updated comprehensive list of tools and search for those fulfilling their specific needs, as well as proposing modifications to the data associated with the tools or the incorporation of new ones.

Recurrent Germline DLST Mutations in Individuals with Multiple Pheochromocytomas and Paragangliomas.

Pheochromocytomas and paragangliomas (PPGLs) provide some of the clearest genetic evidence for the critical role of metabolism in the tumorigenesis process. Approximately 40% of PPGLs are caused by driver germline mutations in 16 known susceptibility genes, and approximately half of these genes encode members of the tricarboxylic acid (TCA) cycle. Taking as a starting point the involvement of the TCA cycle in PPGL development, we aimed to identify unreported mutations that occurred in genes involved in this key metabolic pathway and that could explain the phenotypes of additional individuals who lack mutations in known susceptibility genes. To accomplish this, we applied a targeted sequencing of 37 TCA-cycle-related genes to DNA from 104 PPGL-affected individuals with no mutations in the major known predisposing genes. We also performed omics-based analyses, TCA-related metabolite determination, and 13C5-glutamate labeling assays. We identified five germline variants affecting DLST in eight unrelated individuals (∼7%); all except one were diagnosed with multiple PPGLs. A recurrent variant, c.1121G>A (p.Gly374Glu), found in four of the eight individuals triggered accumulation of 2-hydroxyglutarate, both in tumors and in a heterologous cell-based assay designed to functionally evaluate DLST variants. p.Gly374Glu-DLST tumors exhibited loss of heterozygosity, and their methylation and expression profiles are similar to those of EPAS1-mutated PPGLs; this similarity suggests a link between DLST disruption and pseudohypoxia. Moreover, we found positive DLST immunostaining exclusively in tumors carrying TCA-cycle or EPAS1 mutations. In summary, this study reveals DLST as a PPGL-susceptibility gene and further strengthens the relevance of the TCA cycle in PPGL development.

The Major Origin of Seedless Grapes Is Associated with a Missense Mutation in the MADS-Box Gene VviAGL11.

Seedlessness is greatly prized by consumers of fresh grapes. While stenospermocarpic seed abortion determined by the SEED DEVELOPMENT INHIBITOR (SDI) locus is the usual source of seedlessness in commercial grapevine (Vitis vinifera) cultivars, the underlying sdi mutation remains unknown. Here, we undertook an integrative approach to identify the causal mutation. Quantitative genetics and fine-mapping in two 'Crimson Seedless'-derived F1 mapping populations confirmed the major effect of the SDI locus and delimited the sdi mutation to a 323-kb region on chromosome 18. RNA-sequencing comparing seed traces of seedless and seeds of seeded F1 individuals identified processes triggered during sdi-determined seed abortion, including the activation of salicylic acid-dependent autoimmunity. The RNA-sequencing data set was investigated for candidate genes, and while no evidence for causal cis-acting regulatory mutations was detected, deleterious nucleotide changes in coding sequences of the seedless haplotype were predicted in two genes within the sdi fine-mapping interval. Targeted resequencing of the two genes in a collection of 124 grapevine cultivars showed that only the point variation causing the arginine-197-to-leucine substitution in the seed morphogenesis regulator gene AGAMOUS-LIKE11 (VviAGL11) was fully linked with stenospermocarpy. The concurrent postzygotic variation identified for this missense polymorphism and seedlessness phenotype in seeded somatic variants of the original stenospermocarpic cultivar supports a causal effect. We postulate that seed abortion caused by this amino acid substitution in VviAGL11 is the major cause of seedlessness in cultivated grapevine. This information can be exploited to boost seedless grape breeding.

Gain-of-function mutations in DNMT3A in patients with paraganglioma.

PURPOSE: The high percentage of patients carrying germline mutations makes pheochromocytomas/paragangliomas the most heritable of all tumors. However, there are still cases unexplained by mutations in the known genes. We aimed to identify the genetic cause of disease in patients strongly suspected of having hereditary tumors. METHODS: Whole-exome sequencing was applied to the germlines of a parent-proband trio. Genome-wide methylome analysis, RNA-seq, CRISPR/Cas9 gene editing, and targeted sequencing were also performed. RESULTS: We identified a novel de novo germline mutation in DNMT3A, affecting a highly conserved residue located close to the aromatic cage that binds to trimethylated histone H3. DNMT3A-mutated tumors exhibited significant hypermethylation of homeobox-containing genes, suggesting an activating role of the mutation. CRISPR/Cas9-mediated knock-in in HeLa cells led to global changes in methylation, providing evidence of the DNMT3A-altered function. Targeted sequencing revealed subclonal somatic mutations in six additional paragangliomas. Finally, a second germline DNMT3A mutation, also causing global tumor DNA hypermethylation, was found in a patient with a family history of pheochromocytoma. CONCLUSION: Our findings suggest that DNMT3A may be a susceptibility gene for paragangliomas and, if confirmed in future studies, would represent the first example of gain-of-function mutations affecting a DNA methyltransferase gene involved in cancer predisposition.

Catastrophic Unbalanced Genome Rearrangements Cause Somatic Loss of Berry Color in Grapevine.

Grape (Vitis vinifera) color somatic variants that can be used to develop new grapevine cultivars occasionally appear associated with deletion events of uncertain origin. To understand the mutational mechanisms generating somatic structural variation in grapevine, we compared the Tempranillo Blanco (TB) white berry somatic variant with its black berry ancestor, Tempranillo Tinto. Whole-genome sequencing uncovered a catastrophic genome rearrangement in TB that caused the hemizygous deletion of 313 genes, including the loss of the functional copy for the MYB transcription factors required for anthocyanin pigmentation in the berry skin. Loss of heterozygosity and decreased copy number delimited interspersed monosomic and disomic regions in the right arm of linkage groups 2 and 5. At least 11 validated clustered breakpoints involving intrachromosomal and interchromosomal translocations between three linkage groups flanked the deleted fragments, which, according to segregation analyses, are phased in a single copy of each of the affected chromosomes. These hallmarks, along with the lack of homology between breakpoint joins and the randomness of the order and orientation of the rearranged fragments, are all consistent with a chromothripsis-like pattern generated after chromosome breakage and illegitimate rejoining. This unbalanced genome reshuffling has additional consequences in reproductive development. In TB, lack of sexual transmission of rearranged chromosomes associates with low gamete viability, which compromises fruit set and decreases fruit production. Our findings show that catastrophic genome rearrangements arise spontaneously and stabilize during plant somatic growth. These dramatic rearrangements generate new interesting phenotypes that can be selected for the improvement of vegetatively propagated plant species.

Targeted Exome Sequencing of Krebs Cycle Genes Reveals Candidate Cancer-Predisposing Mutations in Pheochromocytomas and Paragangliomas.

Purpose: Mutations in Krebs cycle genes are frequently found in patients with pheochromocytomas/paragangliomas. Disruption of SDH, FH or MDH2 enzymatic activities lead to accumulation of specific metabolites, which give rise to epigenetic changes in the genome that cause a characteristic hypermethylated phenotype. Tumors showing this phenotype, but no alterations in the known predisposing genes, could harbor mutations in other Krebs cycle genes.Experimental Design: We used downregulation and methylation of RBP1, as a marker of a hypermethylation phenotype, to select eleven pheochromocytomas and paragangliomas for targeted exome sequencing of a panel of Krebs cycle-related genes. Methylation profiling, metabolite assessment and additional analyses were also performed in selected cases.Results: One of the 11 tumors was found to carry a known cancer-predisposing somatic mutation in IDH1 A variant in GOT2, c.357A>T, found in a patient with multiple tumors, was associated with higher tumor mRNA and protein expression levels, increased GOT2 enzymatic activity in lymphoblastic cells, and altered metabolite ratios both in tumors and in GOT2 knockdown HeLa cells transfected with the variant. Array methylation-based analysis uncovered a somatic epigenetic mutation in SDHC in a patient with multiple pheochromocytomas and a gastrointestinal stromal tumor. Finally, a truncating germline IDH3B mutation was found in a patient with a single paraganglioma showing an altered α-ketoglutarate/isocitrate ratio.Conclusions: This study further attests to the relevance of the Krebs cycle in the development of PCC and PGL, and points to a potential role of other metabolic enzymes involved in metabolite exchange between mitochondria and cytosol. Clin Cancer Res; 23(20); 6315-24. ©2017 AACR.

PheoSeq: A Targeted Next-Generation Sequencing Assay for Pheochromocytoma and Paraganglioma Diagnostics.

Genetic diagnosis is recommended for all pheochromocytoma and paraganglioma (PPGL) cases, as driver mutations are identified in approximately 80% of the cases. As the list of related genes expands, genetic diagnosis becomes more time-consuming, and targeted next-generation sequencing (NGS) has emerged as a cost-effective tool. This study aimed to optimize targeted NGS in PPGL genetic diagnostics. A workflow based on two customized targeted NGS assays was validated to study the 18 main PPGL genes in germline and frozen tumor DNA, with one of them specifically directed toward formalin-fixed paraffin-embedded tissue. The series involved 453 unrelated PPGL patients, of whom 30 had known mutations and were used as controls. Partial screening using Sanger had been performed in 275 patients. NGS results were complemented with the study of gross deletions. NGS assay showed a sensitivity ≥99.4%, regardless of DNA source. We identified 45 variants of unknown significance and 89 pathogenic mutations, the latter being germline in 29 (7.2%) and somatic in 58 (31.7%) of the 183 tumors studied. In 37 patients previously studied by Sanger sequencing, the causal mutation could be identified. We demonstrated that both assays are an efficient and accurate alternative to conventional sequencing. Their application facilitates the study of minor PPGL genes, and enables genetic diagnoses in patients with incongruent or missing clinical data, who would otherwise be missed.

Early transcriptional response to biotic stress in mixed starter fermentations involving Saccharomyces cerevisiae and Torulaspora delbrueckii.

Advances in microbial wine biotechnology have led to the recent commercialization of several non-Saccharomyces starter cultures. These are intended to be used in either simultaneous or sequential inoculation with Saccharomyces cerevisiae. The different types of microbial interactions that can be stablished during wine fermentation acquire an increased relevance in the context of these mixed-starter fermentations. We analysed the transcriptional response to co-cultivation of S. cerevisiae and Torulaspora delbrueckii. The study focused in the initial stages of wine fermentation, before S. cerevisiae completely dominates the mixed cultures. Both species showed a clear response to the presence of each other, even though the portion of the genome showing altered transcription levels was relatively small. Changes in the transcription pattern suggested a stimulation of metabolic activity and growth, as a consequence of the presence of competitors in the same medium. The response of S. cerevisiae seems to take place earlier, as compared to T. delbrueckii. Enhanced glycolytic activity of the mixed culture was confirmed by the CO2 production profile during these early stages of fermentation. Interestingly, HSP12 expression appeared induced by co-cultivation for both of S. cerevisiae and Torulaspora delbrueckii in the two time points studied. This might be related with a recently described role of Hsp12 in intercellular communication in yeast. Expression of S. cerevisiae PAU genes was also stimulated in mixed cultures.