Roberta Bordoni

Whole-Genome Pyrosequencing of an Epidemic Multidrug-Resistant Acinetobacter baumannii Strain Belonging to the European Clone II Group

ABSTRACT The whole-genome sequence of an epidemic, multidrug-resistant Acinetobacter baumannii strain (strain ACICU) belonging to the European clone II group and carrying the plasmid-mediated blaOXA-58 carbapenem resistance gene was determined. The A. baumannii ACICU genome was compared with the genomes of A. baumannii ATCC 17978 and Acinetobacter baylyi ADP1, with the aim of identifying novel genes related to virulence and drug resistance. A. baumannii ACICU has a single chromosome of 3,904,116 bp (which is predicted to contain 3,758 genes) and two plasmids, pACICU1 and pACICU2, of 28,279 and 64,366 bp, respectively. Genome comparison showed 86.4% synteny with A. baumannii ATCC 17978 and 14.8% synteny with A. baylyi ADP1. A conspicuous number of transporters belonging to different superfamilies was predicted for A. baumannii ACICU. The relative number of transporters was much higher in ACICU than in ATCC 17978 and ADP1 (76.2, 57.2, and 62.5 transporters per Mb of genome, respectively). An antibiotic resistance island, AbaR2, was identified in ACICU and had plausibly evolved by reductive evolution from the AbaR1 island previously described in multiresistant strain A. baumannii AYE. Moreover, 36 putative alien islands (pAs) were detected in the ACICU genome; 24 of these had previously been described in the ATCC 17978 genome, 4 are proposed here for the first time and are present in both ATCC 17978 and ACICU, and 8 are unique to the ACICU genome. Fifteen of the pAs in the ACICU genome encode genes related to drug resistance, including membrane transporters and ex novo acquired resistance genes. These findings provide novel insight into the genetic basis of A. baumannii resistance.

Methods for detection of GMOs in food and feed

This paper reviews aspects relevant to detection and quantification of genetically modified (GM) material within the feed/food chain. The GM crop regulatory framework at the international level is evaluated with reference to traceability and labelling. Current analytical methods for the detection, identification, and quantification of transgenic DNA in food and feed are reviewed. These methods include quantitative real-time PCR, multiplex PCR, and multiplex real-time PCR. Particular attention is paid to methods able to identify multiple GM events in a single reaction and to the development of microdevices and microsensors, though they have not been fully validated for application.

Development of a Universal Microarray Based on the Ligation Detection Reaction and 16S rRNA Gene Polymorphism To Target Diversity of Cyanobacteria

ABSTRACT The cyanobacteria are photosynthetic prokaryotes of significant ecological and biotechnological interest, since they strongly contribute to primary production and are a rich source of bioactive compounds. In eutrophic fresh and brackish waters, their mass occurrences (water blooms) are often toxic and constitute a high potential risk for human health. Therefore, rapid and reliable identification of cyanobacterial species in complex environmental samples is important. Here we describe the development and validation of a microarray for the identification of cyanobacteria in aquatic environments. Our approach is based on the use of a ligation detection reaction coupled to a universal array. Probes were designed for detecting 19 cyanobacterial groups including Anabaena/Aphanizomenon, Calothrix, Cylindrospermopsis, Cylindrospermum, Gloeothece, halotolerants, Leptolyngbya, Palau Lyngbya, Microcystis, Nodularia, Nostoc, Planktothrix, Antarctic Phormidium, Prochlorococcus, Spirulina, Synechococcus, Synechocystis, Trichodesmium, and Woronichinia. These groups were identified based on an alignment of over 300 cyanobacterial 16S rRNA sequences. For validation of the microarrays, 95 samples (24 axenic strains from culture collections, 27 isolated strains, and 44 cloned fragments recovered from environmental samples) were tested. The results demonstrated a high discriminative power and sensitivity to 1 fmol of the PCR-amplified 16S rRNA gene. Accurate identification of target strains was also achieved with unbalanced mixes of PCR amplicons from different cyanobacteria and an environmental sample. Our universal array method shows great potential for rapid and reliable identification of cyanobacteria. It can be easily adapted to future development and could thus be applied both in research and environmental monitoring.

Rapid interactome profiling by massive sequencing

We have developed a high-throughput protein expression and interaction analysis platform that combines cDNA phage display library selection and massive gene sequencing using the 454 platform. A phage display library of open reading frame (ORF) fragments was created from mRNA derived from different tissues. This was used to study the interaction network of the enzyme transglutaminase 2 (TG2), a multifunctional enzyme involved in the regulation of cell growth, differentiation and apoptosis, associated with many different pathologies. After two rounds of panning with TG2 we assayed the frequency of ORFs within the selected phage population using 454 sequencing. Ranking and analysis of more than 120 000 sequences allowed us to identify several potential interactors, which were subsequently confirmed in functional assays. Within the identified clones, three had been previously described as interacting proteins (fibronectin, SMOC1 and GSTO2), while all the others were new. When compared with standard systems, such as microtiter enzyme-linked immunosorbant assay, the method described here is dramatically faster and yields far more information about the interaction under study, allowing better characterization of complex systems. For example, in the case of fibronectin, it was possible to identify the specific domains involved in the interaction.

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.

Whole Genome SNP Genotyping and Exome Sequencing Reveal Novel Genetic Variants and Putative Causative Genes in Congenital Hyperinsulinism

Congenital hyperinsulinism of infancy (CHI) is a rare disorder characterized by severe hypoglycemia due to inappropriate insulin secretion. The genetic causes of CHI have been found in genes regulating insulin secretion from pancreatic β-cells; recessive inactivating mutations in the ABCC8 and KCNJ11 genes represent the most common events. Despite the advances in understanding the molecular pathogenesis of CHI, specific genetic determinants in about 50 % of the CHI patients remain unknown, suggesting additional locus heterogeneity. In order to search for novel loci contributing to the pathogenesis of CHI, we combined a family-based association study, using the transmission disequilibrium test on 17 CHI patients lacking mutations in ABCC8/KCNJ11, with a whole-exome sequencing analysis performed on 10 probands. This strategy allowed the identification of the potential causative mutations in genes implicated in the regulation of insulin secretion such as transmembrane proteins (CACNA1A, KCNH6, KCNJ10, NOTCH2, RYR3, SCN8A, TRPV3, TRPC5), cytosolic (ACACB, CAMK2D, CDKAL1, GNAS, NOS2, PDE4C, PIK3R3) and mitochondrial enzymes (PC, SLC24A6), and in four genes (CSMD1, SLC37A3, SULF1, TLL1) suggested by TDT family-based association study. Moreover, the exome-sequencing approach resulted to be an efficient diagnostic tool for CHI, allowing the identification of mutations in three causative CHI genes (ABCC8, GLUD1, and HNF1A) in four out of 10 patients. Overall, the present study should be considered as a starting point to design further investigations: our results might indeed contribute to meta-analysis studies, aimed at the identification/confirmation of novel causative or modifier genes.

Two efficient polymeric chemical platforms for oligonucleotide microarray preparation.

ABSTRACT In this report we describe two robust procedures for oligonucleotide microarray preparation based on polymeric coatings. The proposed chemical approaches include: 1) a glass functionalisation step with appropriate silanes (γ-aminopropyltriethoxysilane-APTES or 3-glycid-oxypropyltrimethoxysilane-GOPS), 2) a coating step using polymers (poly-L-Lysine or poly(acrylic acid-co-acrylamide) copolymer) covalently bound to the modified glass and 3) a surface activation step to allow for the attachment of amino-modified oligonucleotides. Results obtained using these chemistries in oligo microarray preparation show: 1) an overall high loading capacity and availability to hybridisation against targets, 2) a good uniformity, 3) resistance to consecutive probing/stripping cycles, 4) stability to thermal cycles, 5) effectiveness in hybridisation-mediated mutation detection procedures and 6) the possibility to perform enzymatic reactions, such as ligation.

Detection of HLA polymorphisms by ligase detection reaction and a universal array format: a pilot study for low resolution genotyping.

We present our results in the identification of polymorphic sites within the second exon of the human leukocyte antigen A (HLA-A) region using the DNA microarray technology. Allele specific detection was performed by polymerase chain reaction followed by ligase detection reaction (LDR) in combination with a universal array, a powerful method for high throughput DNA sequence analysis. By this approach we confirmed 32 human samples previously characterized by direct DNA sequencing, thus demonstrating the interest of this approach.

Search for mutations in pancreatic sufficient cystic fibrosis Italian patients: detection of 90% of molecular defects and identification of three novel mutations

A cohort of 31 cystic fibrosis patients showing pancreatic sufficiency and bearing an unidentified mutation on at least one chromosome was analyzed through denaturing gradient gel electrophoresis of the whole coding region of the cystic fibrosis transmembrane conductance regulator gene, including intron-exon boundaries. Three new and 19 previously described mutations were detected. The combination of these with known mutations detected by other methods, allowed the characterization of mutations on 56/62 (90.3%) chromosomes. Among those identified, 17 can be considered responsible for pancreatic sufficiency, since they were found in patients carrying a severe mutation on the other chromosome. Among these presumed mild mutations, eight were detected more than once, R352Q being the most frequent in this sample (4.83%). Intragenic microsatellite analysis revealed that the six chromosomes still bearing unidentified mutations are associated with five different haplotypes. This may indicate that these chromosomes bear different mutations, rarely occurring among cystic fibrosis patients, further underlying the molecular heterogeneity of the genetic defects present in patients having pancreatic sufficiency.