Specific Aim 1 (WP1): I have collected biological sample from 13 patients with clinical and metabolic characteristics of childhood mitochondrial disorders. Multiple OXPHOS defects were found in the muscle homogenate of 10 probands suggesting a defect in mtDNA metabolism (replication, translation, transcription). By applying whole exome sequencing, internal algorithm for variants filtering, manual inspection and Sanger sequencing analysis of the family, we have identified: a) new variants/phenotypes for known disease causing gene; b) large rearrangement including mitochondrial genes; c) three new potential disease causing genes. The analysis of 2 families has not identified a clear candidates and it is going to be re-evaluate with new bioinformatic tools.
Specific Aim 2 (WP2): in the specific Aim 2, we performed functional studies in in vitro models for confirming the pathogenicity of the new variant and explore the underlined disease pathway.
Additional discoveries will be further explored in future research program or as part of collaboration with other research institutes.
Specific Aim 3 (WP3): in order to identify FBXL4 protein function, we have generated different transgenic FBXL4 cell lines that have allowed to study the protein in different conditions. We have performed proteomics studies, cell fractionation experiments, shRNA of FBXL4 and other proteins involved in fission and fusion. Results from these studies led us to conclude that that Fbxl4 is a mitochondrial outer membrane protein, playing role in the ubiquitination pathways and potentially involved in mitophagy. However, additional studies are required to clearly define the pathway. We have also generated a fbxl4 knockout mouse model with cre-lox technology. The mice are embryonically letal with only 2% of birth rate. Fbxl4 KO mice that are born alive can survive up to 8 months life but they present growth failure, parkinsonism and very mild biochemical defect with increased mitochondrial mass. MEFs fbxl4 Ko cell lines have been generated from the embryos and they will be object of future research development.
In the last year, we have also set-up a collaboration with expert in protein ubiquitination at Dundee University and we are currently analyzing results from additional proteomic studies.
Data from MITOBIOPATH project were discussed with expert in the field at EUROMIT 2017 in Cologne. In addition, new gene defects in the mtDNA metabolism pathways were presented as poster at the EMBO workshop “Molecular biology of mitochondrial gene expression” (Sweden) and “Mitochondrial Medicine” meeting (UK).
During my fellowship, I have been invited speakers at several national and international meetings (Cambridge Mitochondrial meeting, UK, 2016; British Pediatric Neurology Association, UK, 2017; ENMC Workshop “Recommendations for treatment of Mitochondrial DNA maintenance disorders”, The Netherland, 2017; Mitocon, Italy, 2017). I have been also appointed as Faculty member at European School of Human Genetics with teaching lessons on Mitochondrial Diseases at “Clinical genomics and NGS” course.
