Whole exome sequencing (WES) is currently the most practical approach for the investigation of coding variations in medical research. However, in several cases, it remains difficult to identify causative mutations and this could be due to different technical and biological reasons. Despite these limitations, exome experiments could provide other information that is generally filtered out, and that could at least partially explain negative results. The aim of this project is to analyse exome data focusing on biological information that is usually discarded and that could be helpful in the comprehension of molecular bases of genetic diseases: mitochondrial DNA and microRNAs variants. Mitochondria are eukaryotic semi-autonomous organelles of endosymbiotic origin and they constitute a dynamic network, which is intimately interconnected with other cell components: an intense crosstalk with the nucleus provides mitochondria with nuclear-encoded proteins as well as noncoding RNAs, which function as a guide molecules in RNA silencing required for mitochondrial homeostasis and function. Preliminary results suggest that at least a portion of miRNAs is efficiently captured by exome enrichment kits. Now we propose to evaluate the exome enrichment systems' capture efficiency of mitochondrial DNA sequences too. This analysis will be performed on a cohort of patients with different diseases and the results will be analyzed to improve our knowledge about the crosstalk between nuclear and mitochondrial genome and the role of these variants to explain the genetic variability. We will focus our attention on variants potentially altering nuclear-coded miRNAs' acting in the nucleus or in cytosol on genes encoding mitochondrial proteins. Finally, we will also analyze predicted nuclear-encoded miRNAs acting in mitochondria too. A list of selected mitochondrial and microRNA variants will be experimentally validated using functional assays to evaluate their biological effect on phenotype.
The development of high throughput sequencing technologies has revolutionized molecular diagnosis of human genetic diseases. The exome represents about 1%-1,5% of the human genome but accounts over 85% of all mutations that have been identified in Mendelian disorders (Gilissen et al., 2012). In medical genetics both whole genome sequencing (WGS) and whole exome sequencing (WES) are the most useful techniques to understand genetic causes in both extremely rare diseases and common heterogeneous disorders whose molecular basis are unknown but, because of the complexity and the greater cost of WGS, WES is currently the most practical approach for the investigation of coding variations in medical research. Currently, whole exome sequencing is successful in diagnosing only around 25-40% of cases (Sawyer et al., 2016). In undiagnosed cases the pathogenic variant may occur in unknown genes or the variant may be missed for a number of reasons, including: extra-exonic localization, non-coverage, oligogenic contributions, complex or large-scale insertions, deletions, rearrangements or mutations in non-protein coding genes. In this context, analyzing miRNAs could be useful in the comprehension of molecular bases of genetic diseases (Mencía et al., 2009; Hughes et al., 2011; Peng and Croce, 2016) and the knowledge of variants' position in miRNA will be helpful in investigation of their potential biological role, as they could lead to different effects. Furthermore, the ability to detect variants in mitochondrial DNA sequences from whole-exome sequence data, raise the possibility of a comprehensive single experiment to detect pathogenic point mutations in nuclear genome both coding regions and non-coding regions like microRNA sequences and mitochondrial DNA. In this perspective, we plan to analyze WES data through our integrated approach, focusing on variants potentially altering nuclear-coded miRNAs' acting in the nucleus or in cytosol on genes encoding mitochondrial proteins to retrieve the hidden information about miRNAs and mitochondrial DNA variants. Finally, the results will be analyzed at the same time to improve our knowledge about the crosstalk between nuclear and mitochondrial genome and the role of these variants to explain the variability in human genetic diseases.