SAMHD1-deficient fibroblasts from Aicardi-Goutières Syndrome patients can escape senescence and accumulate mutations.

In mammalian cells, the catabolic activity of the dNTP triphosphohydrolase SAMHD1 sets the balance and concentration of the four dNTPs. Deficiency of SAMHD1 leads to unequally increased pools and marked dNTP imbalance. Imbalanced dNTP pools increase mutation frequency in cancer cells, but it is not known if the SAMHD1-induced dNTP imbalance favors accumulation of somatic mutations in non-transformed cells. Here, we have investigated how fibroblasts from Aicardi-Goutières Syndrome (AGS) patients with mutated SAMHD1 react to the constitutive pool imbalance characterized by a huge dGTP pool. We focused on the effects on dNTP pools, cell cycle progression, dynamics and fidelity of DNA replication, and efficiency of UV-induced DNA repair. AGS fibroblasts entered senescence prematurely or upregulated genes involved in G1/S transition and DNA replication. The normally growing AGS cells exhibited unchanged DNA replication dynamics and, when quiescent, faster rate of excision repair of UV-induced DNA damages. To investigate whether the lack of SAMHD1 affects DNA replication fidelity, we compared de novo mutations in AGS and WT cells by exome next-generation sequencing. Somatic variant analysis indicated a mutator phenotype suggesting that SAMHD1 is a caretaker gene whose deficiency is per se mutagenic, promoting genome instability in non-transformed cells.

QueryOR: a comprehensive web platform for genetic variant analysis and prioritization.

BACKGROUND: Whole genome and exome sequencing are contributing to the extraordinary progress in the study of human genetic variants. In this fast developing field, appropriate and easily accessible tools are required to facilitate data analysis. RESULTS: Here we describe QueryOR, a web platform suitable for searching among known candidate genes as well as for finding novel gene-disease associations. QueryOR combines several innovative features that make it comprehensive, flexible and easy to use. Instead of being designed on specific datasets, it works on a general XML schema specifying formats and criteria of each data source. Thanks to this flexibility, new criteria can be easily added for future expansion. Currently, up to 70 user-selectable criteria are available, including a wide range of gene and variant features. Moreover, rather than progressively discarding variants taking one criterion at a time, the prioritization is achieved by a global positive selection process that considers all transcript isoforms, thus producing reliable results. QueryOR is easy to use and its intuitive interface allows to handle different kinds of inheritance as well as features related to sharing variants in different patients. QueryOR is suitable for investigating single patients, families or cohorts. CONCLUSIONS: QueryOR is a comprehensive and flexible web platform eligible for an easy user-driven variant prioritization. It is freely available for academic institutions at http://queryor.cribi.unipd.it/ .

Comprehensive transcript profiling of two grapevine rootstock genotypes contrasting in drought susceptibility links the phenylpropanoid pathway to enhanced tolerance.

In light of ongoing climate changes in wine-growing regions, the selection of drought-tolerant rootstocks is becoming a crucial factor for developing a sustainable viticulture. In this study, M4, a new rootstock genotype that shows tolerance to drought, was compared from a genomic and transcriptomic point of view with the less drought-tolerant genotype 101.14. The root and leaf transcriptome of both 101.14 and the M4 rootstock genotype was analysed, following exposure to progressive drought conditions. Multifactorial analyses indicated that stress treatment represents the main factor driving differential gene expression in roots, whereas in leaves the genotype is the prominent factor. Upon stress, M4 roots and leaves showed a higher induction of resveratrol and flavonoid biosynthetic genes, respectively. The higher expression of VvSTS genes in M4, confirmed by the accumulation of higher levels of resveratrol in M4 roots compared with 101.14, was coupled to an up-regulation of several VvWRKY transcription factors. Interestingly, VvSTS promoter analyses performed on both the resequenced genomes highlighted a significantly higher number of W-BOX elements in the tolerant genotype. It is proposed that the elevated synthesis of resveratrol in M4 roots upon water stress could enhance the plant's ability to cope with the oxidative stress usually associated with water deficit.

Genetic Layout of Melanoma Lesions Is Associated with BRAF/MEK-Targeted Therapy Resistance and Transcriptional Profiles.

The genetic landscape of melanoma resistance to targeted therapy with small molecules inhibiting BRAF and MEK kinases is still largely undefined. In this study, we portrayed in detail the somatic alterations of resistant melanoma and explored the associated biological processes and their integration with transcriptional profiles. By targeted next-generation sequencing and whole-exome sequencing analyses, a list of 101 genes showing imbalance in metastatic tumors from patients with a complete/durable response or disease progression during therapy with vemurafenib or with dabrafenib and trametinib was defined. Classification of altered genes in functional categories indicated that the mutational pattern of both resistant tumors and melanoma cell lines was enriched in gene families involved in oncogenic signaling pathways and in DNA repair. Integration of genomic and transcriptomic features showed that the enrichment of mutations in gene sets associated with anabolic processes, chromatin alterations, and IFN-α response determined a significant positive modulation of the same gene signatures at the transcriptional level. In particular, MTORC1 signaling was enriched in tumors from poorly responsive patients and in resistant tumors excised from treated patients. Results indicate that genetic patterns are associated with melanoma resistance to targeted therapy and disclose the underlying key molecular pathways to define drug combinations for improved personalized therapies.

Genetic Variants and Somatic Alterations Associated with MITF-E318K Germline Mutation in Melanoma Patients.

The MITF-E318K variant has been implicated in genetic predisposition to cutaneous melanoma. We addressed the occurrence of MITF-E318K and its association with germline status of CDKN2A and MC1R genes in a hospital-based series of 248 melanoma patients including cohorts of multiple, familial, pediatric, sporadic and melanoma associated with other tumors. Seven MITF-E318K carriers were identified, spanning every group except the pediatric patients. Three carriers showed mutated CDKN2A, five displayed MC1R variants, while the sporadic carrier revealed no variants. Germline/tumor whole exome sequencing for this carrier revealed germline variants of unknown significance in ATM and FANCI genes and, in four BRAF-V600E metastases, somatic loss of the MITF wild-type allele, amplification of MITF-E318K and deletion of a 9p21.3 chromosomal region including CDKN2A and MTAP. In silico analysis of tumors from MITF-E318K melanoma carriers in the TCGA Pan-Cancer-Atlas dataset confirmed the association with BRAF mutation and 9p21.3 deletion revealing a common genetic pattern. MTAP was the gene deleted at homozygous level in the highest number of patients. These results support the utility of both germline and tumor genome analysis to define tumor groups providing enhanced information for clinical strategies and highlight the importance of melanoma prevention programs for MITF-E318K patients.

Setup and Validation of a Targeted Next-Generation Sequencing Approach for the Diagnosis of Lysosomal Storage Disorders.

Lysosomal storage disorders (LSDs) are monogenic diseases, due to accumulation of specific undegraded substrates into lysosomes. LSD diagnosis could take several years because of both poor knowledge of these diseases and shared clinical features. The diagnostic approach includes clinical evaluations, biochemical tests, and genetic analysis of the suspected gene. In this study, we evaluated an LSD targeted sequencing panel as a tool capable to potentially reverse this classic diagnostic route. The panel includes 50 LSD genes and 230 intronic sequences conserved among 33 placental mammals. For the validation phase, 56 positive controls, 13 biochemically diagnosed patients, and nine undiagnosed patients were analyzed. Disease-causing variants were identified in 66% of the positive control alleles and in 62% of the biochemically diagnosed patients. Three undiagnosed patients were diagnosed. Eight patients undiagnosed by the panel were analyzed by whole exome sequencing: for two of them, the disease-causing variants were identified. Five patients, undiagnosed by both panel and exome analyses, were investigated through array comparative genomic hybridization: one of them was diagnosed. Conserved intronic fragment analysis, performed in cases unresolved by the first-level analysis, evidenced no candidate intronic variants. Targeted sequencing has low sequencing costs and short sequencing time. However, a coverage >60× to 80× must be ensured and/or Sanger validation should be performed. Moreover, it must be supported by a thorough clinical phenotyping.

Combining ontologies and workflows to design formal protocols for biological laboratories.

BACKGROUND: Laboratory protocols in life sciences tend to be written in natural language, with negative consequences on repeatability, distribution and automation of scientific experiments. Formalization of knowledge is becoming popular in science. In the case of laboratory protocols two levels of formalization are needed: one for the entities and individuals operations involved in protocols and another one for the procedures, which can be manually or automatically executed. This study aims to combine ontologies and workflows for protocol formalization. RESULTS: A laboratory domain specific ontology and the COW (Combining Ontologies with Workflows) software tool were developed to formalize workflows built on ontologies. A method was specifically set up to support the design of structured protocols for biological laboratory experiments. The workflows were enhanced with ontological concepts taken from the developed domain specific ontology.The experimental protocols represented as workflows are saved in two linked files using two standard interchange languages (i.e. XPDL for workflows and OWL for ontologies). A distribution package of COW including installation procedure, ontology and workflow examples, is freely available from http://www.bmr-genomics.it/farm/cow. CONCLUSIONS: Using COW, a laboratory protocol may be directly defined by wet-lab scientists without writing code, which will keep the resulting protocol's specifications clear and easy to read and maintain.

Muscle Research and Gene Ontology: New standards for improved data integration.

BACKGROUND: The Gene Ontology Project provides structured controlled vocabularies for molecular biology that can be used for the functional annotation of genes and gene products. In a collaboration between the Gene Ontology (GO) Consortium and the muscle biology community, we have made large-scale additions to the GO biological process and cellular component ontologies. The main focus of this ontology development work concerns skeletal muscle, with specific consideration given to the processes of muscle contraction, plasticity, development, and regeneration, and to the sarcomere and membrane-delimited compartments. Our aims were to update the existing structure to reflect current knowledge, and to resolve, in an accommodating manner, the ambiguity in the language used by the community. RESULTS: The updated muscle terminologies have been incorporated into the GO. There are now 159 new terms covering critical research areas, and 57 existing terms have been improved and reorganized to follow their usage in muscle literature. CONCLUSION: The revised GO structure should improve the interpretation of data from high-throughput (e.g. microarray and proteomic) experiments in the area of muscle science and muscle disease. We actively encourage community feedback on, and gene product annotation with these new terms. Please visit the Muscle Community Annotation Wiki http://wiki.geneontology.org/index.php/Muscle_Biology.

Alfalfa Mob 1-like genes are expressed in reproductive organs during meiosis and gametogenesis.

Mps-one-binder (Mob) proteins play an important role in chromosome separation and cell plate formation in yeast. We cloned two Mob 1-like genes from alfalfa (Medicago sativa L.) and show that one gene is constitutively expressed while the other is expressed only in flower buds during sporogenesis and gametogenesis. For the analysis of gene expression during reproduction in alfalfa wild-types and apomeiotic mutants, a specific antisense riboprobe was designed for MsMob 1 transcripts and a polyclonal antibody was raised against MsMob 1 proteins. In situ mRNA localization as well as protein immunolocalization proved that MsMob1-like genes are specifically expressed in degenerating megaspores of normal ovules and in enlarged megaspore mother cells and embryo sacs of apomeiotic ovules. Gene products were also found in microspore tetrads at the beginning of pollen development as well as in tapetum cells of anthers undergoing programmed cell death to allow pollen dispersal at maturity. Overall results suggest that MsMob 1-like genes can play a key role during the reproductive pathway in plants.