Differentially expressed alternatively spliced genes in Malignant Pleural Mesothelioma identified using massively parallel transcriptome sequencing

<p>Abstract</p> <p>Background</p> <p>Analyses of Expressed Sequence Tags (ESTs) databases suggest that most human genes have multiple alternative splice variants. The alternative splicing of pre-mRNA is tightly regulated during development and in different tissue types. Changes in splicing patterns have been described in disease states. Recently, we used whole-transcriptome shotgun pryrosequencing to characterize 4 malignant pleural mesothelioma (MPM) tumors, 1 lung adenocarcinoma and 1 normal lung. We hypothesized that alternative splicing profiles might be detected in the sequencing data for the expressed genes in these samples.</p> <p>Methods</p> <p>We developed a software pipeline to map the transcriptome read sequences of the 4 MPM samples and 1 normal lung sample onto known exon junction sequences in the comprehensive AceView database of expressed sequences and to count how many reads map to each junction. 13,274,187 transcriptome reads generated by the Roche/454 sequencing platform for 5 samples were compared with 151,486 exon junctions from the AceView database. The exon junction expression index (EJEI) was calculated for each exon junction in each sample to measure the differential expression of alternative splicing events. Top ten exon junctions with the largest EJEI difference between the 4 mesothelioma and the normal lung sample were then examined for differential expression using Quantitative Real Time PCR (qRT-PCR) in the 5 sequenced samples. Two of the differentially expressed exon junctions (ACTG2.aAug05 and CDK4.aAug05) were further examined with qRT-PCR in additional 18 MPM and 18 normal lung specimens.</p> <p>Results</p> <p>We found 70,953 exon junctions covered by at least one sequence read in at least one of the 5 samples. All 10 identified most differentially expressed exon junctions were validated as present by RT-PCR, and 8 were differentially expressed exactly as predicted by the sequence analysis. The differential expression of the AceView exon junctions for the ACTG2 and CDK4 genes were also observed to be statistically significant in an additional 18 MPM and 18 normal lung samples examined using qRT-PCR. The differential expression of these two junctions was shown to successfully classify these mesothelioma and normal lung specimens with high sensitivity (89% and 78%, respectively).</p> <p>Conclusion</p> <p>Whole-transcriptome shotgun sequencing, combined with a downstream bioinformatics pipeline, provides powerful tools for the identification of differentially expressed exon junctions resulting from alternative splice variants. The alternatively spliced genes discovered in the study could serve as useful diagnostic markers as well as potential therapeutic targets for MPM.</p

Effects of the flavanone combination hesperetin-naringenin, and orange and grapefruit juices, on airway inflammation and remodeling in a murine asthma model

We investigated whether flavanones, hesperetin-naringenin, orange, and grapefruit juices reduce airway inflammation and remodeling in murine chronic asthma model. To establish chronic asthma, mice received house dust mite (HDM) for 3days in 2weeks, followed by twice per week for 4weeks. Concurrently, during the last 4weeks, mice received hesperetin plus naringenin (HN), orange plus grapefruit juice (OGJ), orange juice (OJ), or grapefruit juice (GJ); whereas the asthmatic control (AC) group and non-asthmatic control (NC) group consumed water ad libitum. In histopathological examination, no goblet cells metaplasia was observed in the HN, OJ, and GJ groups; also, intra-alveolar macrophages decreased compared with those of the AC group. Hesperetin plus naringenin significantly decreased subepithelial fibrosis, smooth muscle hypertrophy in airways, and lung atelectasis compared with the AC group. Also, there was a reduction of subepithelial fibrosis in airways in OJ and GJ groups compared with AC group, but it was not noticed in OGJ group. In bronchoalveolar lavage fluid, macrophages numbers decreased in OJ and OGJ groups, whereas eosinophil numbers were increased in OJ group compared with NC group. Our finding revealed that hesperetin plus naringenin ameliorate airway structural remodeling more than orange juice and grapefruit juice in murine model of HDM-induced asthma. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd

Chromosomal aberrations in patients suspected with the risk of Fanconi anemia

Background and Objective: Fanconi anemia is the most prevalent inherited aplastic anemia. Diagnosis based solely on the recognition of clinical symptoms is not reliable. This study was done to determine chromosomal aberrations in patients suspected with the risk of Fanconi anemia in the Eastern Azarbaijan province- Iran. Materials and Methods: This descriptive study was conducted on 20 patients in the Eastern Azarbaijan province-Iran. The cytogenetic method was used to determine type and number of chromosomal disorders. Results: Nine eight and nine patients had co-morbid anemia, platelet deficiency and 9 patients had hand and finger deformities, respectively. Using cytogenetic method, Fanconi anemia was confirmed in 5 (25%) of the cases. The percentage of mitotic abnormalities in the chromosomes without administration of mitomycin C varied between 5-30% in the cultures of the 5 affected and between 0-4% in the 15 unaffected patients with the administration of mitomycin C, the percentages were increased up to 35-78% and 0-20% in affected and unaffected patients, respectively. Conclusion: Fanconi anemia is confirmed precisely in 25% of suspected patients using cytogenetic method

Comparison of gene‐expression profiles in parallel bone marrow and peripheral blood samples in acute myeloid leukaemia by real‐time polymerase chain reaction

BACKGROUND: Gene signatures (Indicator genes) in bone marrow that provide more precise prognostication in haematological malignancy have been identified by microarray expression studies. It would be beneficial to measure these diagnostic signatures in peripheral blood. AIMS: To determine the degree of correspondence of gene expression for a set of Indicator genes between bone marrow and peripheral blood in acute myeloid leukaemia (AML). METHODS: Parallel bone marrow aspirate and peripheral blood samples were obtained from 19 patients diagnosed with AML and mononuclear cells isolated from both sample types. mRNA was globally amplified by polyadenylated real‐time polymerase chain reaction (polyA RT‐PCR); the expression of 15 AML Indicator genes, identified from previous microarray studies, was measured by RT‐PCR. All values were normalised to the mean expression of three housekeeping genes (IF2‐β, GAP and RbS9) and were statistically compared using SPSS software. RESULTS: No significant difference in expression between bone marrow and peripheral blood was observed for 10 of the genes (leptin receptor, CD33, adipsin, proteoglycan 1, MB‐1, cyclin D3, hSNF2b, proteasome iota, HkrT‐1 and E2A), indicating its possible use in monitoring disease activity in peripheral blood samples, whereas c‐myb, HOXA9, LYN, cystatin c and LTC4s showed significantly different expression between bone marrow and peripheral blood samples. CONCLUSION: These results indicate a possible use for the method in monitoring AML in peripheral blood by RT‐PCR measurement of Indicator genes. In addition, the initial use of polyA PCR facilitates translation to very small clinical samples, including fractionated cell populations, of particular importance for monitoring haematological malignancy