Oral Presentation HUPO 2019 - 18th Human Proteome Organization World Congress

From disease to biology: how quantitative proteomics can resolve the molecular diagnosis of mitochondrial disease patients and provide insights into mitochondrial biology (#162)

Daniella H. Hock 1 , Sumudu S. C. Amarasekera 2 3 , Alison G. Compton 2 3 , Ann E. Frazier 2 3 , Boris Reljić 1 , David R. Thorburn 2 3 4 , David A. Stroud 1
  1. Department of Biochemistry and Molecular Biology, University of Melbourne, Bio21 Molecular Science and Biotechnology Institute, Parkville, VIC, Australia
  2. Murdoch Children’s Research Institute, Royal Children’s Hospital, Melbourne, VIC, Australia
  3. Department of Paediatrics, University of Melbourne, Parkville, VIC, Australia
  4. Victorian Clinical Genetic Services, Royal Children’s Hospital, Parkville, VIC, Australia

Mitochondrial diseases are the most common type of inherited metabolic disorders, affecting approximately 1 in 5,000 live births. Most routine diagnoses for mitochondrial disease rely on targeted exome sequencing combined with enzyme activity and brain scans. Due to the complex genetics and phenotypic heterogenicity over 40% of patients remain undiagnosed, often harbouring variants of unknown significance (VUS). Quantitative proteomics is a powerful untargeted approach that quantifies the abundance of thousands of proteins, offering functional data in variant prioritisation. We have applied quantitative proteomics in primary cells from patients with unsolved molecular diagnoses. In the first example, we identified an intronic mutation in a novel mitoribosome disease gene (MRPL39), which resulted in destabilisation of the large mitoribosomal subunits. Our cDNA studies showed that the variant produced a stable mRNA missing exon 8, leading to a premature stop codon. In another example, the patient harboured novel variants in both MT-ATP6 and nuclear encoded ATAD3A. Our results clearly showed destabilisation of Complex V but no defect in ATAD3A transcript stability, protein abundance or pathways associated with known ATAD3 protein function. The MT-ATP6 variant has been reclassified from VUS to likely pathogenic, such reclassification allows this variant to be included in prenatal tests. In the final example, the patient had a variant in the mitochondrial alanyl tRNA synthetase (AARS2), which resulted in a Complex IV defect. Further analysis of the proteomics results led us to investigate HIGD2A function. Using CRISPR/Cas9 we generated HIGD2A knockout HEK293T cells, which presented with a clear Complex IV defect. Characterisation of HIGD2A function revealed that it is specifically involved in assembling the MT-CO3 module of Complex IV. Hence, our studies demonstrate the powerful contribution of quantitative proteomics for the molecular diagnosis of mitochondrial disease patients and potential discovery of novel protein function.