Dissecting Intra-Tumor Heterogeneity by the Analysis of Copy Number Variations in Single Cells: The Neuroblastoma Case Study

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

Diagnosi molecolare di sindrome di Brugada in un giovane atleta mediante il sequenziamento di un pannello multigenico con tecniche di nuova generazione

Mutations in genes driving the molecular pathways that regulate myocardial functions can predispose to many independent cardiopathies and also to sudden cardiac death (SCD) even in asymptomatic subjects. The overlapping clinical signs or symptoms or even silent phenotypes make it difficult to diagnose these diseases, therefore the risk of undiagnosed disease could be high especially in young adults and athletes, which may then incur in SCD. We describe the case of a clinical asymptomatic eight-year-old child, practicing soccer game, who underwent a screening medical examination to undertake the path of an increasing physical activity to become a competitive athlete, where abnormal signs at ECG indicated a suspicion of an arrhythmogenic heart disease. Molecular screening analysis, to discriminate among the various predisposing gene alterations, was performed using a 75 gene-panel for arrhythmias customized in our laboratory. The child resulted carrier of a loss-of-function mutation in the SCN5A gene (c.1126C>T). About 25% of Brugada patients carry mutations in this gene coding for the cardiac sodium channel. The loss-of-function mutations in SCN5A gene induce alterations of sodium ion conduction in cardiomyocytes, compatible with the Brugada Syndrome. This case report highlights the importance of the implementation of a rapid, sensitive and wide molecular screening to shed light on possible genetic alterations present also in asymptomatic athletes with negative family history, which may often remain undiagnosed, thus exposed to high risk of sudden death

A novel pathogenic BRCA1 splicing variant produces partial intron retention in the mature messenger RNA

About 10% of all breast cancers arise from hereditary mutations that increase the risk of breast and ovarian cancers; and about 25% of these are associated with the BRCA1 or BRCA2 genes. The identification of BRCA1/BRCA2 mutations can enable physicians to better tailor the clinical management of patients; and to initiate preventive measures in healthy carriers. The pathophysiological significance of newly identified variants poses challenges for genetic counseling. We characterized a new BRCA1 variant discovered in a breast cancer patient during BRCA1/2 screening by next-generation sequencing. Bioinformatic predictions; indicating that the variant is probably pathogenetic; were verified using retro-transcription of the patient's RNA followed by PCR amplifications performed on the resulting cDNA. The variant causes the loss of a canonic donor splice site at position +2 in BRCA1 intron 21; and consequently the partial retention of 156 bp of intron 21 in the patient's transcript; which demonstrates that this novel BRCA1 mutation plays a pathogenetic role in breast cancer. These findings enabled us to initiate appropriate counseling and to tailor the clinical management of this family. Lastly; these data reinforce the importance of studying the effects of sequence variants at the RNA level to verify their potential role in disease onset

Celiac disease-associated Neisseria flavescens decreases mitochondrial respiration in CaCo-2 epithelial cells: Impact of Lactobacillus paracasei CBA L74 on bacterial-induced cellular imbalance

: We previously identified a Neisseria flavescens strain in the duodenum of celiac disease (CD) patients that induced immune inflammation in ex vivo duodenal mucosal explants and in CaCo-2 cells. We also found that vesicular trafficking was delayed after the CD-immunogenic P31-43 gliadin peptide-entered CaCo-2 cells and that Lactobacillus paracasei CBA L74 (L. paracasei-CBA) supernatant reduced peptide entry. In this study, we evaluated if metabolism and trafficking was altered in CD-N. flavescens-infected CaCo-2 cells and if any alteration could be mitigated by pretreating cells with L. paracasei-CBA supernatant, despite the presence of P31-43. We measured CaCo-2 bioenergetics by an extracellular flux analyser, N. flavescens and P31-43 intracellular trafficking by immunofluorescence, cellular stress by TBARS assay, and ATP by bioluminescence. We found that CD-N. flavescens colocalised more than control N. flavescens with early endocytic vesicles and more escaped autophagy thereby surviving longer in infected cells. P31-43 increased colocalisation of N. flavescens with early vesicles. Mitochondrial respiration was lower (P < .05) in CD-N. flavescens-infected cells versus not-treated CaCo-2 cells, whereas pretreatment with L. paracasei-CBA reduced CD-N. flavescens viability and improved cell bioenergetics and trafficking. In conclusion, CD-N. flavescens induces metabolic imbalance in CaCo-2 cells, and the L. paracasei-CBA probiotic could be used to correct CD-associated dysbiosis

The molecular analysis of BRCA1 and BRCA2: Next-generation sequencing supersedes conventional approaches

Abstract Background Accurate and sensitive detection of BRCA 1/2 germ-line mutations is crucial for the clinical management of women affected by breast cancer, for prevention and, notably, also for the identification of at-risk healthy relatives. The most widely used methods for BRCA1 / 2 molecular analysis are Sanger sequencing, and denaturing high performance liquid chromatography (dHPLC) followed by the Sanger method. However, recent findings suggest that next-generation sequencing (NGS)-based approaches may be an efficient tool for diagnostic purposes. In this context, we evaluated the effectiveness of NGS for BRCA gene analysis compared with dHPLC/Sanger sequencing. Methods Seventy women were screened for BRCA1/2 mutations by both dHPLC/Sanger sequencing and NGS, and the data were analyzed using a bioinformatic pipeline. Results Sequence data analysis showed that NGS is more sensitive in detecting BRCA 1/2 variants than the conventional procedure, namely, dHPLC/Sanger. Conclusion Next-generation sequencing is more sensitive, faster, easier to use and less expensive than the conventional Sanger method. Consequently, it is a reliable procedure for the routine molecular screening of the BRCA 1/2 genes

D-aspartate oxidase gene duplication induces social recognition memory deficit in mice and intellectual disabilities in humans

The D-aspartate oxidase (DDO) gene encodes the enzyme responsible for the catabolism of D-aspartate, an atypical amino acid enriched in the mammalian brain and acting as an endogenous NMDA receptor agonist. Considering the key role of NMDA receptors in neurodevelopmental disorders, recent findings suggest a link between D-aspartate dysmetabolism and schizophrenia. To clarify the role of D-aspartate on brain development and functioning, we used a mouse model with constitutive Ddo overexpression and D-aspartate depletion. In these mice, we found reduced number of BrdU-positive dorsal pallium neurons during corticogenesis, and decreased cortical and striatal gray matter volume at adulthood. Brain abnormalities were associated with social recognition memory deficit at juvenile phase, suggesting that early D-aspartate occurrence influences neurodevelopmental related phenotypes. We corroborated this hypothesis by reporting the first clinical case of a young patient with severe intellectual disability, thought disorders and autism spectrum disorder symptomatology, harboring a duplication of a chromosome 6 region, including the entire DDO gene

Genome sequencing strategies: the example of testicular cancer in a familial trio

Testicular cancer is the most common malignancy in men under the age of 50. In 1972 for the first time, the carcinoma in situ was mentioned and in particular the tumor of mixed germ cells in situ; this type of tumor, in fact, originates in the testicles but can also be found in other parts of the body. The diagnosis of these tumors involves the use of instrumental tests, usually carried out together with some laboratory tests including the determination of tumor markers; these, when increased, can be of help in indicating the presence of the disease. In recent years, there has been a sharp increase in the number of testicular cancer cases diagnosed especially in young males all over the world. Although the understanding of the molecular causes underlying this neoplasm is very limited, Next Generation Sequencing (NGS) strategies can help to shed light on complex and multifactorial pathologies such as in the case of testicular cancer. In addition, a new nucleic acid sequencing strategy is gaining ground: the third generation Oxford Nanopore Technology (ONT), which, allows to obtain a “picture” of the DNA in each individual as complete as possible, by providing very long genome sequences. The aim of this paper is to illustrate how the use of combined nucleic acid sequencing strategies has allowed to analyze the presence of mutations in a family trio where the boy (proband) was affected by testicular neoplasia. The combined sequencing of the genome and the methyloma can be of help in understanding the possible causative or predisposing mutations

The performance of multi-gene panels for breast/ovarian cancer predisposition

: BRCA1 and BRCA2 are the most mutated genes in breast cancer. We analyzed 48 breast cancer subjects using two methods that differ in terms of number of genes investigated and strategy used (primers: Panel A - 12 genes - vs probes: Panel B - 48 genes). Both the panels and procedures identified "pathogenic" or "likely pathogenic" variants in TP53, ATM, CHEK2 and BARD1 besides BRCA1 and BRCA2. Panel B identified two other putatively pathogenic variants in RNASEL and in RAD50. Identification of variants other than the BRCA genes can be useful in patient management. A total of 121 variants were distributed within the 12 genes and were correctly detected by both panels. However, the number of calls without divergence, namely ± 0.10 difference of allelic frequency, was 78.3%, while calls with a divergence below 0.10 was 16.7%, thus indicating that only 5% (n = 275) of 5,412 calls had a divergence above 0.10. Although these panels differ from each other, both are useful in different situations, particularly when patients should be tested for genes other than BRCA1/2 (as occurs in patients affected by a so called hereditary syndrome) or for therapeutic purposes

A Familial Novel Putative-Pathogenic Mutation Identified in Plaque-Psoriasis by a Multigene Panel Analysis

: Psoriasis is a chronic multifactorial skin disorder with an immune basis. It is characterized by patches of skin that are usually red, flaky and crusty, and that often release silvery scales. The patches appear predominantly on the elbows, knees, scalp and lower back, although they may also appear on other body areas and severity may be variable. The majority of patients (about 90%) present small patches known as "plaque psoriasis". The roles of environmental triggers such as stress, mechanical trauma and streptococcal infections are well described in psoriasis onset, but much effort is still needed to unravel the genetic component. The principal aim of this study was to use a next-generation sequencing technologies-based approach together with a 96 customized multigene panel in the attempt to determine if there are germline alterations that can explain the onset of the disease, and thus to find associations between genotypes and phenotypes. To this aim, we analyzed a family in which the mother showed mild psoriasis, and her 31-year-old daughter had suffered from psoriasis for several years, whereas an unaffected sister served as a negative control. We found variants already associated directly to psoriasis in the TRAF3IP2 gene, and interestingly we found a missense variant in the NAT9 gene. The use of multigene panels in such a complex pathology such as psoriasis can be of great help in identifying new susceptibility genes, and in being able to make early diagnoses especially in families with affected subjects