Alessandro Pietrelli

Acquired copy-neutral loss of heterozygosity of chromosome 1p as a molecular event associated with marrow fibrosis in MPL-mutated myeloproliferative neoplasms.

We studied mutations of MPL exon 10 in patients with essential thrombocythemia (ET) or primary myelofibrosis (PMF), first investigating a cohort of 892 consecutive patients. MPL mutation scanning was performed on granulocyte genomic DNA by using a high-resolution melt assay, and the mutant allele burden was evaluated by using deep sequencing. Somatic mutations of MPL, all but one involving codon W515, were detected in 26/661 (4%) patients with ET, 10/187 (5%) with PMF, and 7/44 (16%) patients with post-ET myelofibrosis. Comparison of JAK2 (V617F)-mutated and MPL-mutated patients showed only minor phenotypic differences. In an extended group of 62 MPL-mutated patients, the granulocyte mutant allele burden ranged from 1% to 95% and was significantly higher in patients with PMF or post-ET myelofibrosis compared with those with ET. Patients with higher mutation burdens had evidence of acquired copy-neutral loss of heterozygosity (CN-LOH) of chromosome 1p in granulocytes, consistent with a transition from heterozygosity to homozygosity for the MPL mutation in clonal cells. A significant association was found between MPL-mutant allele burden greater than 50% and marrow fibrosis. These observations suggest that acquired CN-LOH of chromosome 1p involving the MPL location may represent a molecular mechanism of fibrotic transformation in MPL-mutated myeloproliferative neoplasms.

Deep sequencing reveals double mutations in cis of MPL exon 10 in myeloproliferative neoplasms

Somatic mutations of MPL exon 10, mainly involving a W515 substitution, have been described in JAK2 (V617F)-negative patients with essential thrombocythemia and primary myelofibrosis. We used direct sequencing and high-resolution melt analysis to identify mutations of MPL exon 10 in 570 patients with myeloproliferative neoplasms, and allele specific PCR and deep sequencing to further characterize a subset of mutated patients. Somatic mutations were detected in 33 of 221 patients (15%) with JAK2 (V617F)-negative essential thrombocythemia or primary myelofibrosis. Only one patient with essential thrombocythemia carried both JAK2 (V617F) and MPL (W515L). High-resolution melt analysis identified abnormal patterns in all the MPL mutated cases, while direct sequencing did not detect the mutant MPL in one fifth of them. In 3 cases carrying double MPL mutations, deep sequencing analysis showed identical load and location in cis of the paired lesions, indicating their simultaneous occurrence on the same chromosome.

An efficient rRNA removal method for RNA sequencing in GC-rich bacteria

BackgroundNext generation sequencing (NGS) technologies have revolutionized gene expression studies and functional genomics analysis. However, further improvement of RNA sequencing protocols is still desirable, in order to reduce NGS costs and to increase its accuracy. In bacteria, a major problem in RNA sequencing is the abundance of ribosomal RNA (rRNA), which accounts for 95-98% of total RNA and can therefore hinder sufficient coverage of mRNA, the main focus of transcriptomic studies. Thus, efficient removal of rRNA is necessary to achieve optimal coverage, good detection sensitivity and reliable results. An additional challenge is presented by microorganisms with GC-rich genomes, in which rRNA removal is less efficient.ResultsIn this work, we tested two commercial kits for rRNA removal, either alone or in combination, on Burkholderia thailandensis. This bacterium, chosen as representative of the important Burkholderia genus, which includes both pathogenic and environmental bacteria, has a rather large (6.72 Mb) and GC-rich (67.7%) genome. Each enriched mRNA sample was sequenced through paired-end Illumina GAIIx run in duplicate, yielding between 10 and 40 million reads. We show that combined treatment with both kits allows an mRNA enrichment of more than 238-fold, enabling the sequencing of almost all (more than 90%) B. thailandensis transcripts from less than 10 million reads, without introducing any bias in mRNA relative abundance, thus preserving differential expression profile.ConclusionsThe mRNA enrichment protocol presented in this work leads to an increase in detection sensitivity up to 770% compared to total RNA; such increased sensitivity allows for a corresponding reduction in the number of sequencing reads necessary for the complete analysis of whole transcriptome expression profiling. Thus we can conclude that the MICROBExpress/Ovation combined rRNA removal method could be suitable for RNA sequencing of whole transcriptomes of microorganisms with high GC content and complex genomes enabling at the same time an important scaling down of sequencing costs.

Comprehensive Genomic Characterization of Cutaneous Malignant Melanoma Cell Lines Derived from Metastatic Lesions by Whole-Exome Sequencing and SNP Array Profiling

Cutaneous malignant melanoma is the most fatal skin cancer and although improved comprehension of its pathogenic pathways allowed to realize some effective molecular targeted therapies, novel targets and drugs are still needed. Aiming to add genetic information potentially useful for novel targets discovery, we performed an extensive genomic characterization by whole-exome sequencing and SNP array profiling of six cutaneous melanoma cell lines derived from metastatic patients. We obtained a total of 3,325 novel coding single nucleotide variants, including 2,172 non-synonymous variants. We catalogued the coding mutations according to Sanger COSMIC database and to a manually curated list including genes involved in melanoma pathways identified by mining recent literature. Besides confirming the presence of known melanoma driver mutations (BRAFV600E, NRASQ61R), we identified novel mutated genes involved in signalling pathways crucial for melanoma pathogenesis and already addressed by current targeted therapies (such as MAPK and glutamate pathways). We also identified mutations in four genes (MUC19, PAICS, RBMXL1, KIF23) never reported in melanoma, which might deserve further investigations. All data are available to the entire research community in our Melanoma Exome Database (at https://155.253.6.64/MExDB/). In summary, these cell lines are valuable biological tools to improve the genetic comprehension of this complex cancer disease and to study functional relevance of individual mutational events, and these findings could provide insights potentially useful for identification of novel therapeutic targets for cutaneous malignant melanoma.

Gene and protein expression in response to different growth temperatures and oxygen availability in Burkholderia thailandensis.

Burkholderia thailandensis, although normally avirulent for mammals, can infect macrophages in vitro and has occasionally been reported to cause pneumonia in humans. It is therefore used as a model organism for the human pathogen B. pseudomallei, to which it is closely related phylogenetically. We characterized the B. thailandensis clinical isolate CDC2721121 (BtCDC272) at the genome level and studied its response to environmental cues associated with human host colonization, namely, temperature and oxygen limitation. Effects of the different growth conditions on BtCDC272 were studied through whole genome transcription studies and analysis of proteins associated with the bacterial cell surface. We found that growth at 37°C, compared to 28°C, negatively affected cell motility and flagella production through a mechanism involving regulation of the flagellin-encoding fliC gene at the mRNA stability level. Growth in oxygen-limiting conditions, in contrast, stimulated various processes linked to virulence, such as lipopolysaccharide production and expression of genes encoding protein secretion systems. Consistent with these observations, BtCDC272 grown in oxygen limitation was more resistant to phagocytosis and strongly induced the production of inflammatory cytokines from murine macrophages. Our results suggest that, while temperature sensing is important for regulation of B. thailandensis cell motility, oxygen limitation has a deeper impact on its physiology and constitutes a crucial environmental signal for the production of virulence factors.

Comparative genomics revealed key molecular targets to rapidly convert a reference rifamycin-producing bacterial strain into an overproducer by genetic engineering.

Rifamycins are mainstay agents in treatment of many widespread diseases, but how an improved rifamycin producer can be created is still incompletely understood. Here, we describe a comparative genomic approach to investigate the mutational patterns introduced by the classical mutate-and-screen method in the genome of an improved rifamycin producer. Comparing the genome of the rifamycin B overproducer Amycolatopsis mediterranei HP-130 with those of the reference strains A. mediterranei S699 and U32, we identified 250 variations, affecting 227 coding sequences (CDS), 109 of which were HP-130-specific since they were absent in both S699 and U32. Mutational and transcriptional patterns indicated a series of genomic manipulations that not only proved the causative effect of mutB2 (coding for methylmalonyl-CoA mutase large subunit) and argS2 (coding for arginyl tRNA synthetase) mutations on the overproduction of rifamycin, but also constituted a rational strategy to genetically engineer a reference strain into an overproducer.

High-throughput genetic characterization of a cohort of Brugada syndrome patients

Brugada syndrome (BrS) is an inherited cardiac arrhythmic disorder that can lead to sudden death, with a prevalence of 1:5000 in Caucasian population and affecting mainly male patients in their third to fourth decade of life. BrS is inherited as an autosomal dominant trait; however, to date genetic bases have been only partially understood. Indeed most mutations are located in the SCN5A gene, encoding the alpha-subunit of the Na(+) cardiac channel, but >70% BrS patients still remain genetically undiagnosed. Although 21 other genes have been associated with BrS susceptibility, their pathogenic role is still unclear. A recent next-generation sequencing study investigated the contribution of 45 arrhythmia susceptibility genes in BrS pathogenesis, observing a significant enrichment only for SCN5A. In our study, we evaluated the distribution of putative functional variants in a wider panel of 158 genes previously associated with arrhythmic and cardiac defects in a cohort of 91 SCN5A-negative BrS patients. In addition, to identify genes significantly enriched in BrS, we performed a mutation burden test by using as control dataset European individuals selected from the 1000Genomes project. We confirmed BrS genetic heterogeneity and identified new potential BrS candidates such as DSG2 and MYH7, suggesting a possible genetic overlap between different cardiac disorders.

Glucose availability enhances lipopolysaccharide production and immunogenicity in the opportunistic pathogen Acinetobacter baumannii

AIM Acinetobacter baumannii can cause sepsis with high mortality rates. We investigated whether glucose sensing might play a role in A. baumannii pathogenesis. MATERIALS & METHODS We carried out transcriptome analysis and extracellular polysaccharide determination in an A. baumannii clinical isolate grown on complex medium with or without glucose supplementation, and assessed its ability to induce production of inflammatory cytokines in human macrophages. RESULTS Growth in glucose-supplemented medium strongly enhanced A. baumannii sugar anabolism, resulting in increasing lipopolysaccharide biosynthesis. In addition, glucose induced active shedding of lipopolysaccharide, in turn triggering a strong induction of inflammatory cytokines in human macrophages. Finally, hemolytic activity was strongly enhanced by growth in glucose-supplemented medium. CONCLUSION We propose that sensing of exogenous glucose might trigger A. baumannii pathogenesis during sepsis.

Positioning the Red Deer (Cervus elaphus) Hunted by the Tyrolean Iceman into a Mitochondrial DNA Phylogeny

In the last years several phylogeographic studies of both extant and extinct red deer populations have been conducted. Three distinct mitochondrial lineages (western, eastern and North-African/Sardinian) have been identified reflecting different glacial refugia and postglacial recolonisation processes. However, little is known about the genetics of the Alpine populations and no mitochondrial DNA sequences from Alpine archaeological specimens are available. Here we provide the first mitochondrial sequences of an Alpine Copper Age Cervus elaphus. DNA was extracted from hair shafts which were part of the remains of the clothes of the glacier mummy known as the Tyrolean Iceman or Ötzi (5,350–5,100 years before present). A 2,297 base pairs long fragment was sequenced using a mixed sequencing procedure based on PCR amplifications and 454 sequencing of pooled amplification products. We analyzed the phylogenetic relationships of the Alpine Copper Age red deers haplotype with haplotypes of modern and ancient European red deer. The phylogenetic analyses showed that the haplotype of the Alpine Copper Age red deer falls within the western European mitochondrial lineage in contrast with the current populations from the Italian Alps belonging to the eastern lineage. We also discussed the phylogenetic relationships of the Alpine Copper Age red deer with the populations from Mesola Wood (northern Italy) and Sardinia.

Enly: Improving Draft Genomes through Reads Recycling.

The reconstruction of the complete genome sequence of an organism is an important point for comparative, functional and evolutionary genomics. Nevertheless, overcoming the problems encountered while completing the sequence of an entire genome can still be demanding in terms of time and resources. We have developed Enly, a simple tool based on the iterative mapping of sequence reads at contig edges, capable to extend the genomic contigs deriving from high-throughput sequencing, especially those deriving by Newbler-like assemblies. Testing it on a set of de novo draft genomes led to the closure of up to 20% of the gaps originally present. Enly is cross-platform and most of the steps of its pipeline are parallelizable, making easy and fast to improve a draft genome resulting from a de novo assembly.