Phd in HUMAN BIOLOGY AND MEDICAL GENETICS

Thesis title: Whole Exome Sequencing in medical genomics: potentialities and limitations

Next Generation Sequencing (NGS) technologies have completely changed the way to study molecular bases underlying human genetic diseases. Currently, Whole Exome Sequencing (WES) approach, that captures the coding portion of the human genome (1%), is the most used NGS method to study molecular bases of human phenotypes. WES data can also provide further meaningful amount of information, usually discarded by standard workflows of analysis, deriving from unintentionally sequenced regions that can provide information on introns, 5’UTR and 3’UTR sequences, intergenic regions, and mitochondrial genome. We analyzed WES data to evaluate the occurrence of reads aligning to mitochondrial genome. We disclosed that the coverage of mtDNA by exome sequencing in some cases is comparable to that of targeted mtDNA sequencing. To date, no comprehensive tool is available to annotate mitochondrial genome variants. We therefore developed a dedicate workflow to support the functional interpretation of coding and non-coding mitochondrial variants, integrating biological information in a comprehensive pipeline. Mitochondrial disorders are characterized by incomplete penetrance, variable expressivity and the lack of correlation between genotype and phenotype. To characterize potential phenotype-modifying factors depending on mitochondrial genomic context, we focused on epistatic interactions of mtDNA coding variants. We identified some cases of known pathogenic variants whose variable functional effect could be related to epistatic mechanisms. Exome sequencing has also some key limitations, indeed a significant portion of non-coding sequences, even if they are flanking a coding region, are not enriched using this approach. We systematically studied non-coding and regulatory regions of ACE2 gene, the main receptor for SARS-CoV-2. Single nucleotide variants and indels in introns, 5’ and 3’UTR sequences and promoter regions were retrieved, annotated and prioritized. Biological annotation was performed with tools specifically developed to integrate functional data on non-coding variants, as allele frequency, pathogenicity predictors and evolutionary conservation. We identified two polymorphic sites that displayed significative allele frequency among human populations and are predicted to be regulatory variants. Those sites are located in the promoter region for a specific ACE2 transcript that we confirmed is expressed in lung and lung-derived cell lines. We demonstrated that this region is not captured by exome enrichment kits, therefore, to evaluate Italian samples and COVID-19 patients we used a traditional Sanger sequencing approach as all the already available cases that have been sequenced in the context of international consortia, have been analyzed based on WES approach.