MOLECULAR ALTERATIONS IN HUMAN GENETIC DISEASES THROUGH NEXT GENERATION SEQUENCING TECHNOLOGIES

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D’Argenio, Valeria

(2015)

MOLECULAR ALTERATIONS IN HUMAN GENETIC DISEASES THROUGH NEXT GENERATION SEQUENCING TECHNOLOGIES.

[Tesi di dottorato]

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PhD Thesis D’Argenio V.pdf

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Abstract

Next Generation Sequencing (NGS) technologies have greatly impacted every field of molecular research, reducing costs and simultaneously increasing throughput of DNA sequencing. These features, together with technology’s flexibility, have open the way to a variety of applications, especially for the study of the molecular basis of human diseases. So far, several analytical approaches have been developed to selectively enrich regions of interest from the whole genome, both to identify germinal and/or somatic sequence variants. All of these have assessed their potential in research area and are now being improved also in routine molecular diagnostics. Thanks to the improvement due to NGS methods introduction, also the metagenomic field has achieved very exciting results, increasing our knowledge about the microbiome and its mutually beneficial relationships with the human host. If microbiome plays a role in the maintenance of a healthy status, it is conceivable to suppose that its quantitative and/or qualitative alterations could lead to pathological dysbiosis, as shown in an increasing number of intestinal and extra-intestinal diseases. The aim of this project was to use NGS-based strategies to study the molecular basis of human diseases. In particular, two analytic approaches were used: DNA sequence capture and metagenomics. A DNA sequence capture approach was used to analyze a large panel of genes possibly related to inherited cardiomyopathies. The obtained results indicate that this approach is useful to analyze, in a time and cost effective manner, heterogeneous diseases allowing the identification not only of the disease-causing mutation, but also of other variants involved in disease-phenotypic expression. Finally, methods reliability was higher than traditional, currently used techniques. All the above data indicate that this validated NGS-based approach can be used to improve the molecular analysis of inherited cardiomyopathies, such as of other inherited diseases, also in routine diagnostic settings. With regard to metagenomics, a 16S rRNA pyrotag analysis was carried out to deeply investigate the gut microbiome composition of Crohn and celiac diseases. Specific microbial signatures were identified in the patients. Moreover, the effects of Crohn nutritional therapy on gut microbial composition were also verified. These results suggest a role of gut microbiome in diseases pathogenesis and could, in turn, make possible to develop novel diagnostic, prognostic and, most important, therapeutic strategies. Taken together, all the above results indicate that the used NGS-based procedures can be easily applied to increase our understanding of the molecular basis of human diseases and that they can be useful also for routine diagnostic purposes.

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