Luciano Xumerle

Characterization of Transcriptional Complexity during Berry Development in Vitis vinifera Using RNA-Seq

The development of massively parallel sequencing technologies enables the sequencing of total cDNA (RNA-Seq) to derive accurate measure of individual gene expression, differential splicing activity, and to discover novel regions of transcription, dramatically changing the way that the functional complexity of transcriptomes can be studied. Here we report on the first use of RNA-Seq to gain insight into the wide range of transcriptional responses that are associated with berry development in Vitis vinifera ‘Corvina’. More than 59 million sequence reads, 36 to 44 bp in length, were generated from three developmental stages: post setting, véraison, and ripening. The sequence reads were aligned onto the 8.4-fold draft sequence of the Pinot Noir 40024 genome and then analyzed to measure gene expression levels, to detect alternative splicing events, and expressed single nucleotide polymorphisms. We detected 17,324 genes expressed during berry development, 6,695 of which were expressed in a stage-specific manner, suggesting differences in expression for genes in numerous functional categories and a significant transcriptional complexity. This exhaustive overview of gene expression dynamics demonstrates the utility of RNA-Seq for identifying single nucleotide polymorphisms and splice variants and for describing how plant transcriptomes change during development.

The Solanum commersonii Genome Sequence Provides Insights into Adaptation to Stress Conditions and Genome Evolution of Wild Potato Relatives

The draft genome and transcriptome sequences of the wild potato species S. commersonii demonstrate the usefulness of genome sequences from wild relatives for elucidating evolutionary mechanisms contributing to Solanum species diversity and understanding changes in response to cold. Here, we report the draft genome sequence of Solanum commersonii, which consists of ∼830 megabases with an N50 of 44,303 bp anchored to 12 chromosomes, using the potato (Solanum tuberosum) genome sequence as a reference. Compared with potato, S. commersonii shows a striking reduction in heterozygosity (1.5% versus 53 to 59%), and differences in genome sizes were mainly due to variations in intergenic sequence length. Gene annotation by ab initio prediction supported by RNA-seq data produced a catalog of 1703 predicted microRNAs, 18,882 long noncoding RNAs of which 20% are shown to target cold-responsive genes, and 39,290 protein-coding genes with a significant repertoire of nonredundant nucleotide binding site-encoding genes and 126 cold-related genes that are lacking in S. tuberosum. Phylogenetic analyses indicate that domesticated potato and S. commersonii lineages diverged ∼2.3 million years ago. Three duplication periods corresponding to genome enrichment for particular gene families related to response to salt stress, water transport, growth, and defense response were discovered. The draft genome sequence of S. commersonii substantially increases our understanding of the domesticated germplasm, facilitating translation of acquired knowledge into advances in crop stability in light of global climate and environmental changes.

Chromosome 7p linkage and GPR154 gene association in Italian families with allergic asthma.

Background Several genome scans have reported linkage of markers on chromosome 7p with asthma and related phenotypes in different populations. A fine mapping in Finnish and French‐Canadian populations has associated the GPR154 gene (also known as G‐protein‐coupled receptor for asthma susceptibility, GPRA) with elevated IgE or asthma.

Next generation sequencing: new tools in immunology and hematology

One of the hallmarks of the adaptive immune system is the specificity of B and T cell receptors. Thanks to somatic recombination, a large repertoire of receptors can be generated within an individual that guarantee the recognition of a vast number of antigens. Monoclonal antibodies have limited applicability, given the high degree of diversity among these receptors, in BCR and TCR monitoring. Furthermore, with regard to cancer, better characterization of complex genomes and the ability to monitor tumor-specific cryptic mutations or translocations are needed to develop better tailored therapies. Novel technologies, by enhancing the ability of BCR and TCR monitoring, can help in the search for minimal residual disease during hematological malignancy diagnosis and follow-up, and can aid in improving bone marrow transplantation techniques. Recently, a novel technology known as next generation sequencing has been developed; this allows the recognition of unique sequences and provides depth of coverage, heterogeneity, and accuracy of sequencing. This provides a powerful tool that, along with microarray analysis for gene expression, may become integral in resolving the remaining key problems in hematology. This review describes the state of the art of this novel technology, its application in the immunological and hematological fields, and the possible benefits it will provide for the hematology and immunology community.

The association of rs4307059 and rs35678 markers with autism spectrum disorders is replicated in Italian families

Objective The objective of this study was to replicate an association study on a newly collected Italian autism spectrum disorder (ASD) cohort by studying the genetic markers associated with ASDs from recent genome-wide and candidate gene association studies. Methods We have genotyped 746 individuals from 227 families of the Italian Autism Network using allelic discrimination TaqMan assays for seven common single-nucleotide polymorphisms: rs2292813 (SLC25A12 gene), rs35678 (ATP2B2 gene), rs4307059 (between CDH9 and CDH10 genes), rs10513025 (between SEMA5A and TAS2R1 genes), rs6872664 (PITX1 gene), rs1861972 (EN2 gene), and rs4141463 (MACROD2 gene). A family-based association study was conducted. Results A significant association was found for two of seven markers: rs4307059 T allele (odds ratio: 1.758, SE=0.236; P-value=0.017) and rs35678 TC genotype (odds ratio: 0.528, SE=0.199; P-value=0.0013). Conclusion A preferential allele transmission of two markers located at loci previously associated with social and verbal communication skill has been confirmed in patients of a new ASD family sample.

Wheat IgE profiling and wheat IgE levels in bakers with allergic occupational phenotypes

Objectives To characterise occupational wheat allergic phenotypes (rhino-conjunctivitis, asthma and dermatitis) and immunoglobulin (IgE) sensitisation to particular wheat allergens in bakers. Methods We conducted clinical and immunological evaluations of 81 consecutive bakers reporting occupational symptoms using commercial tests (skin prick test (SPT), specific IgE, ISAC microarray) and six additional dot-blotted wheat allergens (Tri a 39, Tri a Trx, Tri a GST, Tri a 32, Tri a 12, Tri a DH). Results Wheat SPT resulted positive in 29 bakers and was associated with work-related asthma (p<0.01). Wheat IgE was detected in 51 workers and was associated with work-related asthma (p<0.01) and rhino-conjunctivitis (p<0.05). ISAC Tri a 30 was positive in three workers and was associated with work-related dermatitis (p<0.05). Wheat dot-blotted allergens were positive in 22 bakers. Tri a 32 and Tri a GST were positive in 13 and three bakers, respectively, and both were associated with work-related dermatitis (p<0.05). This association increased (p<0.01) when Tri a 32, Tri a GST and Tri a 30 were analysed together (p<0.01). Wheat IgE levels were associated with work-related dermatitis (p<0.01). Conclusions Wheat IgE levels and wheat microarrayed allergens may be associated with some occupational allergic phenotypes. The extension of the panel of wheat allergens may be promising for discriminating the clinical manifestations of bakers allergy.

Association of childhood allergic asthma with markers flanking the IL33 gene in Italian families

To the Editor: Two recent, independent, large-scale genome-wide association studies described the association of asthma with single nucleotide polymorphisms (SNPs) mapping on chromosome 9 in a region flanking the IL33 gene. No family-based association study has yet been reported to confirm the association that was found in the case-control studies. We have previously reported the preliminary results of a genome-wide linkage study conducted on a sample of Italian asthmatic families that indicated linkage to the microsatellite marker D9S286, which is located in an extended region of chromosome 9p24 that includes the IL33 gene. We have now investigated whether the SNPs rs1342326 and rs928413,which showed association in the recent study byMoffatt et al, are associated with childhood asthma or related phenotypes in the abovementioned Italian families. The SNP rs928413 is in absolute linkage disequilibrium (LD; http://www.hapmap.org, rel#24) with rs3939286, which was associated to atopic asthma in the study by Gudbjartsson et al. Therefore this last marker was not genotyped because it is tagged by the marker rs928413. A family-based association study was performed in 137 family trios (father, mother, and asthmatic child) from northeastern Italy ascertained through an allergic asthmatic child. Details of the enrollment criteria were given elsewhere. Association analysis was conducted by using the transmission disequilibrium test (TDT) with the computer program PLINK. The following phenotypes were investigated: doctor-diagnosed asthma according to the Global Initiative for Asthma criteria, increased total serum IgE levels (defined as a qualitative trait with serum concentrations >200 kU/L for adults and >90th percentile in an age-adjusted standard curve value for subjects aged <10 years), bronchial hyperresponsiveness (BHR) to methacholine, positive skin prick test (SPT) responses to common aeroallergens, positive SPT responses to house dust mites, and positive SPT responses to graminae. Table I reports the phenotype distribution in asthmatic/atopic children and in their parents. Genotype data were available for all subjects (parents and offspring), and therefore missing parental genotypes did not represent an issue in this study. The SNPs rs1342326 and rs928413 were in Hardy-Weinberg equilibrium and LD with each other (D9 5 0.95, R 5 0.59). The observed minor allele frequency was 23% (minor allele, G; major allele, T) and 31% (minor allele, G; major allele, A) for rs1342326 and 928413, respectively. TDT analysis showed a preferential transmission of the rs928413 G allele to subjects with positive SPT responses (transmitted, 65; not transmitted, 43; P 5 .034) and in particular with positive SPT responses to graminae (transmitted, 54; not transmitted, 33; P 5 .024) but not with positive SPT responses to house dust mite (transmitted, 41; not transmitted, 33; P 5 .353). No significant association was observed between rs928413 and the other phenotypes or between rs1342326 and any of the phenotypes investigated. Even if the study had a limited dimension that might affect statistical power, we emphasize that the families were enriched for the genetic risk factor because they were in linkage with the 9p24 marker. A 2-loci haplotype TDT was performed. Table II reports the haplotype frequencies observed. Haplotype rs1342326rs928413/T-A was the most frequent haplotype observed (Table II). Table III shows the result of the haplotype association test. Haplotype rs1342326-rs928413/T-G was preferentially transmitted to children affected by asthma (P5 .018), BHR to methacholine (P 5 .006), increased IgE levels (P 5 .022), or positive SPT responses (P 5 .015), particularly positive SPT responses to graminae (P 5 .006). The TDT performed on 2 loci confirms the association found in the rs928413 analysis and adds the information that the rs1342326-rs928413/T-G haplotype (frequency, 8.8%) is associated to allergic phenotypes. This haplotype can therefore be considered the at-risk haplotype, and it might contain the susceptibility allele for the phenotypes investigated. It is worth noting that haplotype analysis showed a significant association for 5 of the 6 phenotypes, and this is far superior to what would be expected by chance (P5 .00002 when considering 5 associated phenotypes of the 6 phenotypes or P5 .001 for 5 significant results of 18 tests). It is therefore reasonable to conclude that the excess of associations might indicate the presence of a true association. These results are therefore in agreement with the previous studies because Moffatt et al indicated association with the rs938413 allele G and Gudbjartsson et al with the rs3939286 allele A, which is tagged by the rs928413 allele G. It is noteworthy that we observe a significant association of rs928413with positive SPT responses but not with asthma, and Gudbjartsson et al reported the association of rs3939286 with atopic asthma but not with nonatopic asthma. This could suggest that rs928413 (and rs3939286) could be mainly associated with the atopic component of the disease. The association to the most common aeroallergens was in particular because of positive SPT response to graminae, suggesting that rs928413 might be associated with the atopic component of seasonal epidemic asthma. We could not exclude that association with positive SPT responses is independent from asthmatic status because all the children were asthmatic. The 2 studied SNPsmap in an intergenic region, and the closest gene is IL33; the 59 side is located in an LD block containing the SNP rs928413 (http://www.hapmap.org, rel#24). No significant association was observed between genetic markers and total serum IgE levels by using the family-based association test statistic. We can speculate that SNP rs928413 might be in LD with variants affecting gene expression. For its characteristics and functions, IL33 represents an attractive candidate gene for the association with allergic asthma–related traits. The IL33 gene, a member of the IL-1 family, is an inducer of TH2 cytokine immunity and systemic inflammation through a complex including the membrane-bound ST2 protein. Binding of IL-33 to its receptor, which is present on mast cells, basophils, eosinophils, natural killer cells, and natural killer T cells, triggers the release of several proinflammatory mediators, induces systemic TH2-type inflammation in vivo, and contributes to allergen-induced airway inflammation and hyperresponsiveness. In conclusion, the results of this family-based association study in an Italian population extends the previously described asthma

Application of the whole-transcriptome shotgun sequencing approach to the study of Philadelphia-positive acute lymphoblastic leukemia

Although the pathogenesis of BCR–ABL1-positive acute lymphoblastic leukemia (ALL) is mainly related to the expression of the BCR–ABL1 fusion transcript, additional cooperating genetic lesions are supposed to be involved in its development and progression. Therefore, in an attempt to investigate the complex landscape of mutations, changes in expression profiles and alternative splicing (AS) events that can be observed in such disease, the leukemia transcriptome of a BCR–ABL1-positive ALL patient at diagnosis and at relapse was sequenced using a whole-transcriptome shotgun sequencing (RNA-Seq) approach. A total of 13.9 and 15.8 million sequence reads was generated from de novo and relapsed samples, respectively, and aligned to the human genome reference sequence. This led to the identification of five validated missense mutations in genes involved in metabolic processes (DPEP1, TMEM46), transport (MVP), cell cycle regulation (ABL1) and catalytic activity (CTSZ), two of which resulted in acquired relapse variants. In all, 6390 and 4671 putative AS events were also detected, as well as expression levels for 18 315 and 18 795 genes, 28% of which were differentially expressed in the two disease phases. These data demonstrate that RNA-Seq is a suitable approach for identifying a wide spectrum of genetic alterations potentially involved in ALL.

Identification of novel mutations in hemochromatosis genes by targeted next generation sequencing in Italian patients with unexplained iron overload

Hereditary hemochromatosis, one of the commonest genetic disorder in Caucasians, is mainly associated to homozygosity for the C282Y mutation in the HFE gene, which is highly prevalent (allele frequency up to near 10% in Northern Europe) and easily detectable through a widely available “first level” molecular test. However, in certain geographical regions like the Mediterranean area, up to 30% of patients with a HH phenotype has a negative or non‐diagnostic (i.e. simple heterozygosity) test, because of a known heterogeneity involving at least four other genes (HAMP, HJV, TFR2, and SLC40A1). Mutations in such genes are generally rare/private, making the diagnosis of atypical HH essentially a matter of exclusion in clinical practice (from here the term of “non‐HFE” HH), unless cumbersome traditional sequencing is applied. We developed a Next Generation Sequencing (NGS)‐based test targeting the five HH genes, and applied it to patients with clinically relevant iron overload (IO) and a non‐diagnostic first level genetic test. We identified several mutations, some of which were novel (i.e. HFE W163X, HAMP R59X, and TFR2 D555N) and allowed molecular reclassification of “non‐HFE” HH clinical diagnosis, particularly in some highly selected IO patients without concurring acquired risk factors. This NGS‐based “second level” genetic test may represent a useful tool for molecular diagnosis of HH in patients in whom HH phenotype remains unexplained after the search of common HFE mutations. Am. J. Hematol. 91:420–425, 2016.

On site DNA barcoding by nanopore sequencing

Biodiversity research is becoming increasingly dependent on genomics, which allows the unprecedented digitization and understanding of the planet’s biological heritage. The use of genetic markers i.e. DNA barcoding, has proved to be a powerful tool in species identification. However, full exploitation of this approach is hampered by the high sequencing costs and the absence of equipped facilities in biodiversity-rich countries. In the present work, we developed a portable sequencing laboratory based on the portable DNA sequencer from Oxford Nanopore Technologies, the MinION. Complementary laboratory equipment and reagents were selected to be used in remote and tough environmental conditions. The performance of the MinION sequencer and the portable laboratory was tested for DNA barcoding in a mimicking tropical environment, as well as in a remote rainforest of Tanzania lacking electricity. Despite the relatively high sequencing error-rate of the MinION, the development of a suitable pipeline for data analysis allowed the accurate identification of different species of vertebrates including amphibians, reptiles and mammals. In situ sequencing of a wild frog allowed us to rapidly identify the species captured, thus confirming that effective DNA barcoding in the field is possible. These results open new perspectives for real-time-on-site DNA sequencing thus potentially increasing opportunities for the understanding of biodiversity in areas lacking conventional laboratory facilities.