Multifactorial causes of chronic mortality in juvenile sturgeon (Huso huso)

This investigation focused on an episode of chronic mortality observed in juvenile Huso huso sturgeons. The examined subjects underwent pathological, microbiological, molecular, and chemical investigations. Grossly severe body shape deformities, epaxial muscle softening, and multifocal ulcerative dermatitis were the main observed findings. The more constant histopathologic findings were moderate to severe rarefaction and disorganization of the lymphohematopoietic lymphoid tissues, myofiber degeneration, atrophy and interstitial edema of skeletal epaxial muscles, and degeneration and atrophy of the gangliar neurons close to the myofibers. Chemical investigations showed a lower selenium concentration in affected animals, suggesting nutritional myopathy. Other manifestations were nephrocalcinosis and splenic vessel wall hyalinosis. Septicemia due to bacteria such as Aeromonas veronii, Shewanella putrefaciens, Citrobacter freundii, Chryseobacterium sp., and pigmented hyphae were found. No major sturgeon viral pathogens were detected by classical methods. Next-generation sequencing (NGS) analysis confirmed the absence of viral pathogens, with the exception of herpesvirus, at the order level; also, the presence of Aeromonas veronii and Shewanella putrefaciens was confirmed at the family level by the metagenomic classification of NGS data. In the absence of a primary yet undetected biological cause, it is supposed that environmental stressors, including nutritional imbalances, may have led to immune system impairment, facilitating the entry of opportunistic bacteria and mycotic hyphae

NEXT GENERATION TARGETED DEEP SEQUENCING OF HEMOCHROMATOSIS GENES IN IRON OVERLOADED PATIENTS: A PILOT STUDY

INTRODUCTION: Hereditary Hemocromatosis (HH) is a common and genetically heterogeneous disorder with relevant geographical differences. While in people of North European descent nearly all cases (up to 95%) are associated with homozygosity for the C282Y mutation in the HFE gene, in the Mediterranean area up to one third of patients do not carry this genotype. Other genes associated with HH include HFE2, HAMP, TFR2 and SLC40A1, but mutations are generally \u201cprivate\u201dand require sequencing. The advent of high 12performance next generation sequencing (NGS) techniques, whose costs are rapidly declining, represents a possible approach for future molecular diagnosis in patients with iron overload. This pilot study was aimed to explore this approach in patients attending a tertiary care center for iron overload disorders. MATERIALS AND METHODS: We re 12sequenced targeted regions (340Kb) of 5 HH 12related genes (HFE, HFE2, HAMP, TFR2, SLC40A1) in 27 patients attending to our Regional Referral Centre for Iron Overload Disorders. Of them, some had known mutations in the HH genes (3 C282Y homozygotes, 1 C282Y/H63D compound heterozygote, 2 with mutations in TFR2, 3 with mutations in SLC40A1), previously determined by standard techniques or traditional sequencing. The remaining had relevant biochemical signs of iron overload that could be considered of \u201cunknown origin\u201d(including some with simple C282Y or H63D heterozygosity). A similar number (n=27) of subjects with normal serum iron parameters were included as controls. Exon capture was performed by a new technology, i.e. the HaloPlex Target Enrichment System (Agilent), which produces circularized fragments suitable for Illumina paired 12end sequencing (using the Illumina HiSeq 1000 platform). Raw reads were filtered by quality and then aligned against human reference HG19 version 37 using BWA with standard parameters. On average we had a 354x sequence coverage for captured regions. These alignments were used to detect variants in each sample using Genome Analysis ToolKit (GATK): on average, GATK found about 1500 variants in each sample. The putative effects of the called variants were evaluated using the Variant Effect Predictor by the EnsEMBL consortium. RESULTS: In the iron overloaded patients, a total of 49 potentially deleterious variants were found in the 5 selected genes (2 missense and 1 splicing variants for HFE, 10 missense variants for HFE2, 0 variants for HAMP, 28 missense and 5 splicing variants for TFR2, 2 missense and 1 splicing variants for SLC40A1). Comparison with control samples as well as the functional relevance of these variants and their possible pathogenic role in each individual patient is currently under further investigation. CONCLUSIONS: high 12coverage selective exon capture is a powerful methodology for rapid DNA analysis in patients with iron overload of uncertain origin. Due to the high number of variants found, proper bioinformatic analyses are needed to reveal variants that are interesting for further clinical investigation. Anyway, as costs for NGS analyses keep to decline, they are expected to become useful tools in the near future for clinicians

Pyrenochaeta lycopersici genome assembly showed interesting feature related to its lifestyle and interaction with the host

Pyrenochaeta lycopersici is a soil-borne pathogen that causes corky root rot disease in tomato (Solanum lycopersicum) and other Solanaceous crops, reducing fruit yields by up to 75%. De novo assembly of the P. lycopersici genome was based on Illumina sequencing and the functional characterization of the draft sequence by integrating RNA-Seq data was followed by an in-depth analysis of the virulence mechanisms and potential pathogenicity effectors encoded by this pathogen. We assembled a 54.9Mb P. lycopersici draft genome and annotated approximately 17,000 genes. The P. lycopersici genome is closely related to hemibiotrophs and necrotrophs, in agreement with the phenotypic characteristics of the fungus and its lifestyle. P. lycopersici genome reveals a significative expansion of specific genes families related both to pathogenesis and to reproduction mechanisms, including those responsible for plant cell wall degradation, nutrient absorption and fungicide detoxification. We also observed a significant expansion of the gene families associated with heterokaryon incompatibility (HI), which represents an important mechanism for this imperfect fungus for increasing genetic variability. The assembly constitutes an important resource to understand the molecular bases of corky root rot and more in general to enrich current knowledge of plant-pathogen interaction mechanisms

Tarrgeted Exome Sequencing of 5 Hemochromatosis Genes in Iron Overloaded Patients by Next Generation Sequencing (NGS) Platform.

Background and Aim. Defects in several proteins involved in iron metabolism can lead to pathological iron accumulation. Autosomal dominant HFE-related Hereditary Hemochromatosis (HH) is the most frequent form of genetically determined iron overloaded (60-90% of cases) disorders, whose penetrance is modulated by both genetic and environmental factors.\u201cClassic\u201d HFE-related HH is due to homozygosity for the C282Y mutation or to compound heterozygosity between the C282Y and the H63D mutation. The search for these two common mutations, easily and cheaply detectable with standard PCR-based techniques, represents the so-called \u201cfirst level\u201d genetic test for the diagnosis of HH. However, other rare HFE variants have been occasionally reported, and the HH phenotype can also be due to mutations in at least four other genes (HFE2, HAMP, TFR2 and SLC40A1). Of note, mutations in these genes are typically \u201cprivate\u201d and require sequencing, making the molecular diagnosis of \u201cnon-HFE\u201d HH quite difficult and expensive. This pilot study was designed to develop a high-performance next generation sequencing (NGS) technique for the molecular diagnosis of \u201cnon-classic\u201d HH, including those cases due to digenic inheritance or to the compound heterozygosity for mutations in any of the five HH genes and concurrent beta-thalassemia trait, a condition relatively frequent in Italy. Methods and Population. The exomic region of 340Kb comprises of 5 HH-related genes (HFE, HFE2, HAMP, TFR2, SLC40A1) was captured and re-sequenced in 61 patients attending our tertiary care center for iron overload disorders. Among them, some had known mutations in the HH genes determined by either standard PCR-based techniques (i.e. the C282Y and the H63D mutation on HFE) or traditional Sanger Sequencing (i.e. mutations in \u201cnon-HFE\u201d genes previously described). The remaining had relevant biochemical signs of iron overload that could be considered of \u201cunknown origin\u201d (including some with simple C282Y or H63D heterozygosity). A similar number (n=72) of subjects with normal serum iron parameters were included as controls. Halo-Plex\u2122 Technology, which produces circularized fragments suitable for Illumina paired-end sequencing (using the IlluminaHiSeq 1000 platform) was used to capture the targeted region. Raw reads were filtered by quality and then aligned against human reference HG19 version 37using BWA with standard parameters. On average we obtained 354x sequence coverage for captured regions. These alignments were used to detect variants in each sample using GoldenHelix\u2122 software . Results. In the iron overload patients the known and reported mutations were confirmed, and several new \u201cnon-synonymous variants\u201d (according to bioinformatics tools based on publicly available databases including the 1000-genomes project) were found. Of these, 3 were in the HFE gene, 15 in the HFE2 gene, 13 in the SCL40A1 gene, 42 in the TFR2 gene, and 1 in the HAMP gene. Many of these variants were relatively frequent and detected also in controls, thus being likely not pathogenetic. However, we also found some \u201cprivate\u201d variants potentially pathogenetic that are currently under further characterization. Significance and Future Perspectives. Halo-Plex\u2122 targeted exome capturing technique combined with high resolution NGS technology is a promising approach for increasing the knowledge of the molecular basis of non-HFE HH, with potential implementation as \u201csecond level\u201d diagnostic test in those cases that remain unexplained after application of standard first level molecular testing for HFE C282Y and H63D mutations

The high polyphenol content of grapevine cultivar tannat berries is conferred primarily by genes that are not shared with the reference genome.

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