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Detecting copy number variations (CNVs) and alterations (CNAs) in the BRCA1 and BRCA2 genes is essential for testing patients for targeted therapy applicability. However, the available bioinformatics tools were initially designed for identifying CNVs/CNAs in whole-genome or -exome (WES) NGS data or targeted NGS data without adaptation to the BRCA1/2 genes. Most of these tools were tested on sample cohorts of limited size, with their use restricted to specific library preparation kits or sequencing platforms. We developed BRACNAC, a new tool for detecting CNVs and CNAs in the BRCA1 and BRCA2 genes in NGS data of different origin. The underlying mechanism of this tool involves various coverage normalization steps complemented by CNV probability evaluation. We estimated the sensitivity and specificity of our tool to be 100% and 94%, respectively, with an area under the curve (AUC) of 94%. The estimation was performed using the NGS data obtained from 213 ovarian and prostate cancer samples tested with in-house and commercially available library preparation kits and additionally using multiplex ligation-dependent probe amplification (MLPA) (12 CNV-positive samples). Using freely available WES and targeted NGS data from other research groups, we demonstrated that BRACNAC could also be used for these two types of data, with an AUC of up to 99.9%. In addition, we determined the limitations of the tool in terms of the minimum number of samples per NGS run (≥20 samples) and the minimum expected percentage of CNV-negative samples (≥80%). We expect that our findings will improve the efficacy of BRCA1/2 diagnostics. BRACNAC is freely available at the GitHub server. [ABSTRACT FROM AUTHOR]

In the last decade, new tumor entities have been described, including EWSR1/FUS::NFATC2-rearranged neoplasms of different biologic behavior. To gain further insights into the behavior of these tumors, we analyzed a spectrum of EWSR1/FUS::NFATC2-rearranged neoplasms and discuss their key diagnostic and molecular features in relation to their prognosis. We report five patients with EWSR1/FUS::NFATC2-rearranged neoplasms, including one simple bone cyst (SBC), two complex cystic bone lesions lacking morphological characteristics of SBC, and two sarcomas. In three cases, fluorescence in situ hybridization (FISH) and in all cases copy number variation (CNV) profiling and fusion analyses were performed. All patients were male, three cystic lesions occurred in children (aged 10, 14, and 17 years), and two sarcomas in adults (69 and 39 years). Fusion analysis revealed two FUS::NFATC2 rearrangements in two cystic lesions and three EWSR1::NFATC2 rearrangements in one complex cystic lesion and two sarcomas. EWSR1 FISH revealed tumor cells with break-apart signal without amplification in one complex cystic lesion and EWSR1 amplification in both sarcomas was documented. CNV analysis showed simple karyotypes in all cystic lesions, while more complex karyotypes were found in NFATC2-rearranged sarcomas. Our study supports and expands previously reported molecular findings of EWSR1/FUS::NFATC2-rearranged neoplasms. The study highlights the importance of combining radiology and morphologic features with molecular aberrations. The use of additional molecular methods, such as CNV and FISH in the routine diagnostic workup, can be crucial in providing a correct diagnosis and avoiding overtreatment. [ABSTRACT FROM AUTHOR]

The risk of chromosomal abnormalities in the child increases with increasing maternal age. Although non-invasive prenatal testing (NIPT) is a safe and effective prenatal screening method, the accuracy of the test results needs to be improved owing to various testing conditions. We attempted to achieve a more accurate and robust prediction of chromosomal abnormalities by combining multiple methods. Here, three different methods, namely standard Z-score, normalized chromosome value, and within-sample reference bin, were used for 1698 reference and 109 test samples of whole-genome sequencing. The logistic regression model combining the three methods achieved a higher accuracy than any single method. In conclusion, the proposed method offers a promising approach for increasing the reliability of NIPT. [ABSTRACT FROM AUTHOR]

Squamous esophageal carcinoma is a common pathological type of esophageal carcinoma around the world. The prognosis of esophageal carcinoma is usually poor and diagnosed at late stages. Recently, research suggested that genomic instability occurred in esophageal cells during the development of esophageal squamous cell carcinoma (ESCC). Identifying prognostic and specific genomic characteristics, especially at the early hyperplasia stage, is critical. Mice were given 4-nitroquinoline 1-oxide (4NQO) with drinking water to induce esophageal cancer. The immortalized human esophageal epithelial cell line (NE2) was also treated with 4NQO. We performed histologic analyses, immunofluorescence, and immunohistochemical staining to detect DNA damage at different time points. Whole-exome sequencing was accomplished on the esophagus tissues at different pathological stages to detect single-nucleotide variants and copy number variation (CNV) in the genome. Our findings indicate that all mice were tumor-forming, and a series of changes from simple hyperplasia (ESSH) to intraepithelial neoplasia (IEN) to esophageal squamous cell carcinoma (ESCC) was seen at different times. The expression of γ-H2AX increased from ESSH to ESCC. In addition, mutations of the Muc4 gene were detected throughout the pathological stages. Furthermore, CNV burden appeared in the esophageal tissues from the beginning of ESSH and accumulated more in cancer with the deepening of the lesions. This study demonstrates that mutations caused by the early appearance of DNA damage may appear in the early stage of malignant tissue before the emergence of atypia. The detection of CNV and mutations of the Muc4 gene may be used as an ultra-early screening indicator for esophageal cancer. [ABSTRACT FROM AUTHOR]

Rare copy number variations (CNVs) are part of the genetics of schizophrenia; they are highly heterogeneous and personalized. The CNV Analysis Group of the Psychiatric Genomic Consortium (PGC) conducted a large-scale analysis and discovered that recurrent CNVs at eight genetic loci were pathogenic to schizophrenia, including 1q21.1, 2p16.3 (NRXN1), 3q29, 7q11.23, 15q13.3, distal 16p11.2, proximal 16p11.2, and 22q11.2. We adopted a two-stage strategy to translate this knowledge into clinical psychiatric practice. As a screening test, we first developed a real-time quantitative PCR (RT-qPCR) panel that simultaneously detected these pathogenic CNVs. Then, we tested the utility of this screening panel by investigating a sample of 557 patients with schizophrenia. Chromosomal microarray analysis (CMA) was used to confirm positive cases from the screening test. We detected and confirmed thirteen patients who carried CNVs at these hot loci, including two patients at 1q21.1, one patient at 7q11.2, three patients at 15q13.3, two patients at 16p11.2, and five patients at 22q11.2. The detection rate in this sample was 2.3%, and the concordance rate between the RT-qPCR test panel and CMA was 100%. Our results suggest that a two-stage approach is cost-effective and reliable in achieving etiological diagnosis for some patients with schizophrenia and improving the understanding of schizophrenia genetics. [ABSTRACT FROM AUTHOR]

Mechanisms for somatic chromosomal mosaicism (SCM) and chromosomal instability (CIN) are not completely understood. During molecular karyotyping and bioinformatic analyses of children with neurodevelopmental disorders and congenital malformations (n = 612), we observed colocalization of regular chromosomal imbalances or copy number variations (CNV) with mosaic ones (n = 47 or 7.7%). Analyzing molecular karyotyping data and pathways affected by CNV burdens, we proposed a mechanism for SCM/CIN, which had been designated as "chromohelkosis" (from the Greek words chromosome ulceration/open wound). Briefly, structural chromosomal imbalances are likely to cause local instability ("wreckage") at the breakpoints, which results either in partial/whole chromosome loss (e.g., aneuploidy) or elongation of duplicated regions. Accordingly, a function for classical/alpha satellite DNA (protection from the wreckage towards the centromere) has been hypothesized. Since SCM and CIN are ubiquitously involved in development, homeostasis and disease (e.g., prenatal development, cancer, brain diseases, aging), we have metaphorically (ironically) designate the system explaining chromohelkosis contribution to SCM/CIN as the cytogenomic "theory of everything", similar to the homonymous theory in physics inasmuch as it might explain numerous phenomena in chromosome biology. Recognizing possible empirical and theoretical weaknesses of this "theory", we nevertheless believe that studies of chromohelkosis-like processes are required to understand structural variability and flexibility of the genome. [ABSTRACT FROM AUTHOR]

Tumors often show intra-tumor heterogeneity because of genotypic differences between all the cells that compose it and that derive from it. Recent studies have shown significant aspects of neuroblastoma heterogeneity that may affect the diagnostic-therapeutic strategy. Therefore, we developed a laboratory protocol, based on the combination of the advanced dielectrophoresis-based array technology and next-generation sequencing to identify and sort single cells individually and carry out their copy number variants analysis. The aim was to evaluate the cellular heterogeneity, avoiding overestimation or underestimation errors, due to a bulk analysis of the sample. We tested the above-mentioned protocol on two neuroblastoma cell lines, SK-N-BE(2)-C and IMR-32. The presence of several gain or loss chromosomal regions, in both cell lines, shows a high heterogeneity of the copy number variants status of the single tumor cells, even if they belong to an immortalized cell line. This finding confirms that each cell can potentially accumulate different alterations that can modulate its behavior. The laboratory protocol proposed herein provides a tool able to identify prevalent behaviors, and at the same time highlights the presence of particular clusters that deviate from them. Finally, it could be applicable to many other types of cancer. [ABSTRACT FROM AUTHOR]