Mitochondrial disorders are clinically heterogeneous with causative mutations identified in over 250 genes. Whole exome sequencing has transformed diagnosis of these disorders but up to 50% of cases remain unsolved. This can be caused by failure to detect or interpret mutations in genes of interest and because some genomic regions remain refractory to analysis or interpretation. We aimed to identify novel mitochondrial disease genes and to exploit quantitative proteomic analyses to understand the underlying pathogenic mechanisms.
In one example, I will discuss how we identified large deletions in the ATAD3 locus encoding 3 highly homologous tandemly arrayed genes (ATAD3C, ATAD3B and ATAD3A), their homology severely complicating genetic analyses. Subjects carrying the deletions suffer congenital pontocerebellar hypoplasia and carried similar deletions of ~38 kbp that resulted in an ATAD3B/ATAD3A fusion that produced a protein 99% identical to ATAD3A, but under the control of the ATAD3B promoter. Quantitative proteomics confirmed this decrease and showed that peptides unique to ATAD3B were found only in the N-terminal region.
In the second example, I will describe a novel mitoribosomal protein defect. The synthesis of all 13 mitochondrial DNA (mtDNA)-encoded protein subunits of the human respiratory chain is carried out by mitochondrial ribosomes (mitoribosomes). We discovered pathogenic mutations in the gene encoding the small mitoribosomal subunit protein, MRPS34. Subjects carrying the mutations suffered Leigh syndrome, and exhibited a combined mitochondrial respiratory chain defect due to impaired translation of mtDNA encoded subunits. Examination of the mitoribosome profile and quantitative proteomics showed the translation defect to be caused by destabilization of the small mitoribosomal subunit and impaired monosome assembly.
In conclusion, quantitative proteomic analyses of patient cell lines have much potential for identifying and proving causation in mitochondrial and other inherited metabolic diseases.