High-throughput sequencing of immunoglobulin heavy chain for minimal residual disease detection in B-lymphoblastic leukemia.

INTRODUCTION: Minimal residual disease (MRD) is a cornerstone for stratification of upfront B-lymphoblastic leukemia (B-ALL) treatment protocols to decrease relapse risk. Although its detection by flow cytometry (FC) and real-time quantitative polymerase has clinical usefulness, evidence suggests that methods with increased sensitivity could lead to improved outcomes. The aim of this study was to develop an amplicon-based assay followed by high-throughput sequencing of the immunoglobulin heavy chain variable region for MRD detection in B-ALL. METHODS: We analyzed 84 samples, 27 from diagnosis, 5 from relapse, 40 from post-treatment samples, and 12 from healthy controls. RESULTS: Our assay was able to identify more neoplastic clones at diagnosis than Sanger sequencing including incomplete DJ rearrangements. From the 40 MRD samples evaluated 21 were positive by our new approach on high-throughput sequencing assay, but only 15 of these were positive by FC. The remaining 19 were negative by the two techniques. CONCLUSION: We have developed a novel approach on high-sensitive assay for MRD detection in B-ALL, which could add clinical value in the management of patients, especially in cases negative for MRD by FC.

Heterogeneous phenotype of Hereditary Xerocytosis in association with PIEZO1 variants.

Hereditary Xerocytosis (HX) is an autosomal dominantly inherited congenital hemolytic anemia associated with erythrocyte dehydration due to decreased intracellular potassium content resulting in increased mean corpuscular hemoglobin concentration. The affected members of HX families show compensated anemia with splenomegaly, hemosiderosis, and perinatal edema but are in large part transfusion independent. Functional studies show a link between mutations in mechanosensitive ion channel, encoded by PIEZO1 gene and the HX. We identified new PIEZO1 variants that are likely pathogenic in three phenotypically characterized multi-generational HX Brazilian families. Interestingly, one missense variant of the PIEZO1 gene identified, p.E2494V was associated in trans with the previously reported most frequent pathogenic duplication p.E2496ELE. The three-dimensional structure of the human protein modeled using structural coordinates of the mouse Piezo1 solved by cryo-electron microscopy (Cryo-ME) showed that the two identified variants, p.M2007L and p.T2014I, are localized to an important mechanosensitive transmembrane domain suggesting a conformational mechanism for altered channel's gating. The p.E2496ELE variant identified alters the extension of helix α1 bringing it much closer to the beam affecting the position of it structure at the end of the pore.

Resistance-associated variants in HCV subtypes 1a and 1b detected by Ion Torrent sequencing platform.

BACKGROUND: As a result of increased understanding of the HCV life cycle, a new generation of drugs known as direct-acting antivirals (DAAs) was developed and is constantly being improved. At baseline, HCV variants resistant to DAA therapy may pre-exist, increasing the likelihood of treatment failure. The aim of this study was to investigate the presence of resistance-associated variants (RAVs) in treatment-naive patients infected with HCV subtypes 1a and 1b. METHODS: Next-generation sequencing was used to assess the frequencies of NS3-4A, NS5A and NS5B RAVs in 100 HCV monoinfected DAA-naive patients (HCV-1a: n=51; HCV-1b: n=49). RESULTS: Complete HCV sequence information was obtained for most samples. RAVs were detected in the NS3-4A (T54S, V55A, Q80K and R155K), NS5A (Q30H/R, H58P and Y93C/H/N) and NS5B (A421V) regions in 10%, 22% and 8%, respectively, of patients infected with HCV subtype-1a. Among the patients infected with HCV subtype-1b, mutations in the NS3-4A (F43I, T54S, Q80H, D168E and M175L), NS5A (L28M, R30Q, L31M, Q54H, A92T and Y93H) and NS5B (L159F, C316N, A421V and S556G) regions were observed in 12%, 53% and 31% of patients, respectively. CONCLUSIONS: High-throughput DNA sequencing allows an easier and more complete analysis of DAA RAVs, including mutations that represent only a minor variant of the whole viral population. RAVs to the three different classes of DAAs were found in our population. The characterization of their profile in the circulating virus is relevant to determine the better treatment option for infected individuals or to guide the implementation of treatment policies.

Deletion of copies of the gene encoding old yellow enzyme (TcOYE), a NAD(P)H flavin oxidoreductase, associates with in vitro-induced benznidazole resistance in Trypanosoma cruzi.

Old yellow enzyme (OYE) is a NAD(P)H flavin oxidoreductase that in Trypanosoma cruzi (TcOYE) catalyzes prostaglandin PGF2alpha synthesis and reduction of some trypanocidal drugs. We performed DNA microarray analysis and it revealed that the levels of transcription of the TcOYE gene were six-fold lower in a T. cruzi population with in vitro-induced resistance to benznidazole (BZ) (17LER) than in the wild-type (17WTS). Further we investigated the TcOYE levels in 15 T. cruzi strains and clones that were either susceptible or naturally resistant to BZ and nifurtimox, or had in vivo-selected resistance to BZ. Northern blot and real-time RT-PCR analyses confirmed our finding that TcOYE transcription levels were lower in 17LER than in 17WTS. In contrast, we detected no differences in TcOYE transcription levels between other T. cruzi samples. All T. cruzi strains contained four copies of TcOYE gene, except 17LER that contained only one. A 42kDa TcOYE protein was detected in all T. cruzi strains tested. The expression of this protein was similar for all samples, with the exception of 17LER for which the protein was nearly seven-fold less expressed. The chromosomal location of the TcOYE gene and the polymorphisms detected in TcOYE nucleotide and amino acid sequences of the T. cruzi strains are associated with the zymodeme but not with drug-resistance phenotype. Our data show that one of the mechanisms conferring in vitro-induced BZ resistance to T. cruzi correlates with deletion of copies of the TcOYE gene. In contrast, the in vivo and natural resistance to BZ are mediated by different mechanisms.

Gene expression profile of the plant pathogen Xylella fastidiosa during biofilm formation in vitro.

A biofilm is a community of microorganisms attached to a solid surface. Cells within biofilms differ from planktonic cells, showing higher resistance to biocides, detergent, antibiotic treatments and host defense responses. Even though there are a number of gene expression studies in bacterial biofilm formation, limited information is available concerning plant pathogen. It was previously demonstrated that the plant pathogen Xylella fastidiosa could grow as a biofilm, a possibly important factor for its pathogenicity. In this study we utilized analysis of microarrays to specifically identify genes expressed in X. fastidiosa cells growing in a biofilm, when compared to planktonic cells. About half of the differentially expressed genes encode hypothetical proteins, reflecting the large number of ORFs with unknown functions in bacterial genomes. However, under the biofilm condition we observed an increase in the expression of some housekeeping genes responsible for metabolic functions. We also found a large number of genes from the pXF51 plasmid being differentially expressed. Some of the overexpressed genes in the biofilm condition encode proteins involved in attachment to surfaces. Other genes possibly confer advantages to the bacterium in the environment that it colonizes. This study demonstrates that the gene expression in the biofilm growth condition of the plant pathogen X. fastidiosa is quite similar to other characterized systems.

Analysis of gene expression in two growth states of Xylella fastidiosa and its relationship with pathogenicity.

Xylella fastidiosa is a plant pathogen responsible for diseases of economically important crops. Although there is considerable disagreement about its mechanism of pathogenicity, blockage of the vessels is one of the most accepted hypotheses. Loss of virulence by this bacterium was observed after serial passages in axenic culture. To confirm the loss of pathogenicity of X. fastidiosa, the causing agent of citrus variegated chlorosis (CVC), freshly-isolated bacteria (first passage [FP] condition) as well as bacteria obtained after 46 passages in axenic culture (several passage [SP] condition) were inoculated into sweet orange and periwinkle plants. Using real time quantitative polymerase chain reaction, we verified that the colonization of FP cells was more efficient for both hosts. The sequence of the complete X. fastidiosa genome allowed the construction of a DNA microarray that was used to investigate the total changes in gene expression associated with the FP condition. Most genes found to be induced in the FP condition were associated with adhesion and probably with adaptation to the host environment. This report represents the first study of the transcriptome of this pathogen, which has recently gained more importance, since the genome of several strains has been either partially or entirely sequenced.

Microarray analyses of Xylella fastidiosa provide evidence of coordinated transcription control of laterally transferred elements.

Genetically distinct strains of the plant bacterium Xylella fastidiosa (Xf) are responsible for a variety of plant diseases, accounting for severe economic damage throughout the world. Using as a reference the genome of Xf 9a5c strain, associated with citrus variegated chlorosis (CVC), we developed a microarray-based comparison involving 12 Xf isolates, providing a thorough assessment of the variation in genomic composition across the group. Our results demonstrate that Xf displays one of the largest flexible gene pools characterized to date, with several horizontally acquired elements, such as prophages, plasmids, and genomic islands (GIs), which contribute up to 18% of the final genome. Transcriptome analysis of bacteria grown under different conditions shows that most of these elements are transcriptionally active, and their expression can be influenced in a coordinated manner by environmental stimuli. Finally, evaluation of the genetic composition of these laterally transferred elements identified differences that may help to explain the adaptability of Xf strains to infect such a wide range of plant species.

Competitive hybridization on spotted microarrays as a tool to conduct comparative genomic analyses of Xylella fastidiosa strains.

Xylella fastidiosa strains are responsible for several plant diseases and since such isolates display a broad host range and complex biological behavior, genomic comparisons employing microarray hybridizations may provide an effective method to compare them. Thus, we performed a thorough validation of this type of approach using two recently sequenced strains of this phytopathogen. By matching microarray hybridization results to direct sequence comparisons, we were able to establish precise cutoff ratios for common and exclusive sequences, allowing the identification of exclusive genes involved in important biological traits. This validation will enable the use of microarray-based comparisons across a wide variety of microorganisms