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Reporting period: 2016-05-01 to 2018-04-30

Mitochondrial disorders are a group of genetically and clinically heterogeneous conditions that have emerged as the most common cause of metabolic disease in children and adults with a minimum estimated prevalence of 1/5000 live birth. Defects in mitochondrial DNA (mtDNA) metabolism (replication and translation) cause a substantial fraction of mitochondrial disorders, characterized by combined defective activities of mtDNA-dependent respiratory chain complexes. Genetically, they are Mendelian inherited as autosomal dominant, recessive or X-linked trait. Clinically, defect in mtDNA metabolism present as a spectrum of disorders ranging from: a) severe infantile multi-systemic disease, rapidly progressing to exitus; b) to childhood myopathy slowly progressing to severe motor dysfunction in adult age; c) or tissue-specific disorders manifesting at any age of life. The clinical and molecular genetics variability challenges the diagnosis and 60% of patients still lack of molecular diagnosis. In MITOBIOPATH project I aimed to discover new mitochondrial disease genes and proteins, and to elucidate new disease-associated metabolic pathways, which are key steps toward the development of treatment strategies. I achieved these major goals by applying whole exome sequencing (Work package 1) to DNA samples from a cohort of patients with suspected mitochondrial disorders and multiple OXPHOS defect and by studying the pathomechanism in in vitro models (Work package 2).
Other than a primary role in genetically inherited mitochondrial disorders, proteins operating the control of the mtDNA metabolism are involved in the mechanism of genetic or aging-related neurodegenerative disorders. Therefore, in the work package 3, I have focused my research activity on Fbxl4 protein that, based on preliminary studies on patients’ fibroblasts, has been hypothesized playing role in mtDNA biogenesis and mitochondrial network organization.
The overall project led to the discovery of new disease-causing genes for complex mitochondrial disorders, to the identification of new pathways involving proteins whose main function is outside of mitochondria (cytoskeleton or cytoplasm) and to the definition of new phenotypes and/or additional function for known mitochondrial proteins. Advances on Fbxl4 protein function were also made who resulted to be essential for life in the KO mouse model.
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.
The Marie Sklodowska Curie with MITOBIOPATH project contributed to the discovery of new mitochondrial disease genes and proteins, and to elucidate new disease-associated metabolic pathways operating in mitochondria. These new observations are extremely important for achieve the genetic diagnosis and better define the clinical spectrum of mitochondrial disorders. They have expanded our knowledge on mitochondrial DNA metabolism and they will be fundamental for designing new treatments for mitochondrial disorders.
Results from MITOBIOPATH project led to the development of new hypotheses that I will exploit in my future research career. We are currently applying to research funding for further investigating these pathways.
During my fellowship, I have also concluded previous collaborations by contributing to manuscripts preparation and submission on new gene defects and diseases pathways (work packages 1 and 2). Topics and collaborators: Tk2 deficiency, Columbia University (New York); ATAD3A mutations in collaboration with Bologna University, Columbia University and Baylor College of Medicine, Texas University; C1QBP mutations in collaboration with Columbia University, New Castle University and Mitochondrial Genetics Lab at MBU; and MRM2 mutations, Mitochondrial Genetics Lab at MBU; SSBP1 mutation in collaboration with New Castle University and Mitochondrial Genetics Lab at MBU.
I have been appointed as Guest-Editor for “Essay in Biochemistry” on “Mitochondrial Diseases” that is currently in press.
I supervised one Clinical trainee in Child Neurology and one PhD in Biological Sciences.
I have organized and attended public engagement events such as “Cambridge Science Festival”, “Anglia Ruskin University Science Day”, “Queen Edith Primary School Cambridge Science week”. In addition, I have been responsible for organizing event during the “Mitochondrial diseases awareness week” at the MRC Mitochondrial Biology Unit.