The role of the gut microbiome in the healthy adult status

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The evolving role of genetic tests in reproductive medicine

Infertility is considered a major public health issue, and approximately 1 out of 6 people worldwide suffer from infertility during their reproductive lifespans. Thanks to technological advances, genetic tests are becoming increasingly relevant in reproductive medicine. More genetic tests are required to identify the cause of male and/or female infertility, identify carriers of inherited diseases and plan antenatal testing. Furthermore, genetic tests provide direction toward the most appropriate assisted reproductive techniques. Nevertheless, the use of molecular analysis in this field is still fragmented and cumbersome. The aim of this review is to highlight the conditions in which a genetic evaluation (counselling and testing) plays a role in improving the reproductive outcomes of infertile couples. We conducted a review of the literature, and starting from the observation of specific signs and symptoms, we describe the available molecular tests. To conceive a child, both partners' reproductive systems need to function in a precisely choreographed manner. Hence to treat infertility, it is key to assess both partners. Our results highlight the increasing importance of molecular testing in reproductive medicine

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

Cracking the Code of Human Diseases Using Next-Generation Sequencing: Applications, Challenges, and Perspectives

Next-generation sequencing (NGS) technologies have greatly impacted on every field of molecular research mainly because they reduce costs and increase throughput of DNA sequencing. These features, together with the technology's flexibility, have opened the way to a variety of applications including the study of the molecular basis of human diseases. Several analytical approaches have been developed to selectively enrich regions of interest from the whole genome in order to identify germinal and/or somatic sequence variants and to study DNA methylation. These approaches are now widely used in research, and they are already being used in routine molecular diagnostics. However, some issues are still controversial, namely, standardization of methods, data analysis and storage, and ethical aspects. Besides providing an overview of the NGS-based approaches most frequently used to study the molecular basis of human diseases at DNA level, we discuss the principal challenges and applications of NGS in the field of human genomics

A common microbial signature is present in the lower airways of interstitial lung diseases including sarcoidosis

Background: The etiology of pulmonary sarcoidosis is not well established. Although the mechanism triggering pulmonary sarcoidosis remains to be established, inflammatory reactions seem to play an important role in this process. Objectives: The aim of this study was to define the composition of the lower airway microbiota in the bronchoalveolar lavage (BAL) of patients affected by interstitial lung diseases, including sarcoidosis, to determine whether the bacterial signature differs among these diseases. Methods: Ten patients affected by pulmonary sarcoidosis and 9 patients affected by other interstitial lung diseases were enrolled. 16S rRNA next-generation sequencing was used to study BAL microbial composition of these patients, and were also compared with already published microbial content in higher airways of such diseases. Results: Four phyla dominated the lower airway microbiota, Bacteroidetes being the most abundant phylum in both groups (56.9%). Diversity analysis showed no significant differences between the various diseases, particularly between pulmonary sarcoidosis and other interstitial lung diseases affecting lower airways. Conclusions: Our data indicate that the bacterial lower airways microbiota share the same signature and, therefore, cannot be used as a diagnostic tool to discriminate among different interstitial lung diseases, including sarcoidosis, while microbial diversity is present when considering lower or higher respiratory airways

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

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