IMPI: An Interface for Low-Frequency Point Mutation Identification Exemplified on Resistance Mutations in Chronic Myeloid Leukemia

Background: In genomics, highly sensitive point mutation detection is particularly relevant for cancer diagnosis and early relapse detection. Next-generation sequencing combined with unique molecular identifiers (UMIs) is known to improve the mutation detection sensitivity. Methods: We present an open-source bioinformatics framework named Interface for Point Mutation Identification (IMPI) with a graphical user interface (GUI) for processing especially small-scale NGS data to identify variants. IMPI ensures detailed UMI analysis and clustering, as well as initial raw read processing, and consensus sequence building. Furthermore, the effects of custom algorithm and parameter settings for NGS data pre-processing and UMI collapsing (e.g., UMI clustered versus unclustered (raw) reads) can be investigated. Additionally, IMPI implements optimization and quality control methods; an evolution strategy is used for parameter optimization. Results: IMPI was designed, implemented, and tested using BCR::ABL1 fusion gene kinase domain sequencing data. In summary, IMPI enables a detailed analysis of the impact of UMI clustering and parameter setting changes on the measured allele frequencies. Conclusions: Regarding the BCR::ABL1 data, IMPI’s results underlined the need for caution while designing specialized single amplicon NGS approaches due to methodical limitations (e.g., high PCR-mediated recombination rate). This cannot be corrected using UMIs

Proportion of Respiratory Syncytial Virus, SARS-CoV-2, Influenza A/B, and Adenovirus Cases via Rapid Tests in the Community during Winter 2023—A Cross Sectional Study

Respiratory infections constitute a major reason for infants and children seeking medical advice and visiting health facilities, thus remaining a significant public threat with high morbidity and mortality. The predominant viruses causing viral respiratory infections are influenza A and B viruses (Flu-A, Flu-B), respiratory syncytial virus (RSV), adenovirus and coronaviruses. We aimed to record the proportion of RSV, SARS-CoV-2, influenza A/B and adenovirus cases with rapid antigen tests and validate the results with RT-PCR assays of upper respiratory specimens with a wide range of viral loads and (co)-infection patterns in children. Clinical samples were collected from early symptomatic children (presenting with fever and/or cough and/or headache within 5–7 days). The surveillance program was conducted in five private pediatric dispensaries and one pediatric care unit, from 10 January 2023 to 30 March 2023 in central Greece. The total sample of specimens collected was 784 young children and infants, of which 383 (48.8%) were female and 401 were male (51.2%). The mean age of participants was 7.3 + 5.5 years. The sensitivity of the FLU A & B test was 91.15% (95% CI: 84.33–95.67%), and the specificity was 98.96% (95% CI: 97.86–99.58%). The sensitivity and specificity of the adenovirus and RSV test was {92.45% (95% CI: 81.79–97.91%), 99.32% (95% CI: 98.41–99.78%)} and {92.59% (95% CI: 75.71–99.09%), 99.47% (95% CI: 98.65–99.86%)} respectively. Lastly, the sensitivity of the SARS-CoV-2 test was 100.00% (95% CI: 79.41–100.00%) and the specificity was 99.74% (95% CI: 99.06–99.97%). We recorded a proportion of 14.3% and 3.44% for influenza A and B, respectively, followed by a proportion of 6.9% for adenovirus, a proportion of 3.7% for RSV, and finally, a proportion of 2.3% for SARS-CoV-2. The combination of a new multiple rapid test with multiple antigens will probably be a useful tool with a financial impact for health systems targeting the early detection and appropriate treatment of respiratory infections in emergency departments in primary health care facilities

Overexpression of the proneural transcription factor ASCL1 in chronic lymphocytic leukemia with a t(12;14)(q23.2;q32.3)

Abstract Background Translocations of the IGH locus on 14q32.3 are present in about 8% of patients with chronic lymphocytic leukemia (CLL) and contribute to leukemogenesis by deregulating the expression of the IGH-partner genes. Identification of these genes and investigation of the downstream effects of their deregulation can reveal disease-causing mechanisms. Case presentation We report on the molecular characterization of a novel t(12;14)(q23.2;q32.3) in CLL. As a consequence of the rearrangement ASCL1 was brought into proximity of the IGHJ-Cμ enhancer and was highly overexpressed in the aberrant B-cells of the patient, as shown by qPCR and immunohistochemistry. ASCL1 encodes for a transcription factor acting as a master regulator of neurogenesis, is overexpressed in neuroendocrine tumors and a promising therapeutic target in small cell lung cancer (SCLC). Its overexpression has also been recently reported in acute adult T-cell leukemia/lymphoma. To examine possible downstream effects of the ASCL1 upregulation in CLL, we compared the gene expression of sorted CD5+ cells of the translocation patient to that of CD19+ B-cells from seven healthy donors and detected 176 significantly deregulated genes (Fold Change ≥2, FDR p ≤ 0.01). Deregulation of 55 genes in our gene set was concordant with at least two studies comparing gene expression of normal and CLL B-lymphocytes. INSM1, a well-established ASCL1 target in the nervous system and SCLC, was the gene with the strongest upregulation (Fold Change = 209.4, FDR p = 1.37E-4). INSM1 encodes for a transcriptional repressor with extranuclear functions, implicated in neuroendocrine differentiation and overexpressed in the majority of neuroendocrine tumors. It was previously shown to be induced in CLL cells but not in normal B-cells upon treatment with IL-4 and to be overexpressed in CLL cells with unmutated versus mutated IGHV genes. Its role in CLL is still unexplored. Conclusion We identified ASCL1 as a novel IGH-partner gene in CLL. The neural transcription factor was strongly overexpressed in the patient’s CLL cells. Microarray gene expression analysis revealed the strong upregulation of INSM1, a prominent ASCL1 target, which was previously shown to be induced in CLL cells upon IL-4 treatment. We propose further investigation of the expression and potential role of INSM1 in CLL

Additional file 3: Figure S1. of Overexpression of the proneural transcription factor ASCL1 in chronic lymphocytic leukemia with a t(12;14)(q23.2;q32.3)

Hierarchical Clustering. (DOCX 115 kb

Additional file 4: Table S2. of Overexpression of the proneural transcription factor ASCL1 in chronic lymphocytic leukemia with a t(12;14)(q23.2;q32.3)

List of deregulated genes (Fold Change ≥2, FDR p ≤ 0.01). (DOCX 474 kb

Systemic oxidative stress in patients with pulmonary sarcoidosis

Background: A local redox imbalance has been reported in pulmonary sarcoidosis. However so far no, study has described a systemic redox imbalance in this context. The aim of the present study was to evaluate the systemic oxidative stress in patients with sarcoidosis and determine its relationship to treatment and indices of disease severity. Methods: 35 patients with histologically proven pulmonary sarcoidosis and 13 healthy volunteers were included in the study. All patients were studied during a stable phase of their disease. Systemic oxidative stress was quantified in serum with the use of a commercially available spectrophotometric method (D-ROM test) which determines overall oxidative stress, by measuring total hydroperoxides. Oxidative stress was expressed in conventional units, i.e. Carratelli Units (UCarr), where 1 UCarr corresponds to 0 8 mg/L. H(2)O(2). Results: Serum oxidative stress levels were significantly higher in patients with sarcoidosis compared to those of normal subjects (390 +/- 25 vs 300 +/- 18 UCarr respectively, p = 0.04). Patients not receiving systemic corticosteroids had higher levels of oxidative stress compared to steroid-treated patients (461.5 +/- 38 vs 315 +/- 20, p < 0.01) and compared to controls (461.5 +/- 38 vs 300 +/- 18 UCarr, p < 0.01). Oxidative stress did not correlate with diffusion lung capacity (DLCO), partial arterial oxygen tension (PaO(2)), MRC dyspnoea scale or chest X-ray stage. Conclusions: Systemic oxidative stress is increased in patients with stable pulmonary sarcoidosis who do not receive systemic corticosteroids. This finding suggests a sustained oxidative burden even when clinical, functional and radiological criteria indicate disease stability. (c) 2009 Elsevier Ltd. All rights reserved