Our primary research involves diagnostic studies of primary mitochondrial disorders. Mitochondrial disorders are oxidative phosphorylation disorders that affect the function of the respiratory chain and the main substrates through the Krebs cycle. The complex dual genomic origin (both mitochondrial DNA and nuclear DNA) results in extreme phenotypic and genetic heterogeneity with already nearly 300 genetic causes identified. The past decade has seen a shift to diagnosis using next-gen sequencing (whole exome or whole genome sequencing as well as mitochondrial DNA next-gen sequencing). This has greatly improved diagnostic identification including the identification of multiple new potential genetic causes. However, it frequently results in identification of variants of unknown significance (VOUS). To resolve these VOUS, our research laboratory develops and explores functional assays and uses them to identify and confirm new genetic causes of mitochondrial dysfunction.
Diagnostic Assay Studies
We have been developing several techniques that could assist in the diagnostic evaluation of genetic causes using minimally invasive tissue of fibroblasts. Fibroblasts were rarely used in the past due to its reported low sensitivity of only about 50% in known cases of mitochondrial disorders using respiratory chain enzyme assays, although a systematic study was never done. By expanding the functional assays with several new assays, we have been able to greatly increase the diagnostic yield in fibroblasts (to the high 80%).
In addition to respiratory chain enzyme assays, we have added in fibroblasts: blue native PAGE with in-gel activity staining, western blot of mitochondrial proteins, complex I assembly assay, complex V ATPase enzyme assay, mitochondrial translation assay, lipoate western blot assay, aconitase mitochondrial and cytosolic, and respirometry. We are in the process of adding additional assays including complex V dependent ATP synthesis (Complex V forward reaction, supported by a Mitochondrial Gateway grant. Testing for ARS2 defects is planned.
We validate our assays in > 40 control fibroblast cell lines providing a good grasp of the range of normal. We are in the process of analyzing > 100 fibroblast cell lines from patients with genetically proven primary mitochondrial disorders which will allow us to derive the data on sensitivity. We have also collected more than 30 cell lines of patients with either clinical similarities to mitochondrial diseases or of disorders with known, presumed or potential secondary mitochondrial dysfunction to analyze the specificity of the functional tests. This has already shown surprising results such as finding of abnormal complex I and complex V assembly in EIF2AK3 mutant cell line, a cause of Wolcott-Ralison syndrome, in which thus far mitochondrial dysfunction had not yet been suspected, let alone documented. Overall, this comprehensive study of the diagnostic utility will evaluate over 100 patient cell lines, over 30 normal controls and 40 disease controls evaluated with at least five groups of tests to derive sensitivity and specificity (involving a total of >2300 data sets), providing a first comprehensive and systematic study of clinical utility of mitochondrial functional testing.
This study is funded by the NAMDC U54 grant with added support from CHCO Foundation Summits for Samantha, CU Foundation Mitochondrial Fund, and Miracles for Mito.
New Genetic Causes of Mitochondrial Dysfunction
Through our diagnostic work and our clinic we have come into contact with patients with new tentative genetic causes of mitochondrial dysfunction. We have provided functional assays to support this finding and to begin to explore the biochemical mechanisms for the pathological dysfunction. We have thus far already evaluated 7 new nuclear genes that had not yet been identified or 7 genes for whom only a single or very few prior publications existed (for instance new genes: CARS2, TMEM126B, NDUFB10, ATP5FD1, PMPCB, GatCAB complex, SQOR, with a new complex V defect in writing. Other genes with functional characterization include: MRPL44, HSD10, GFM1, FARS2, NUBPL, and a few newly recognized mitochondrial DNA mutations.
We have been a diagnostic center for the mitochondrial hepatopathies (mitochondrial liver diseases) as part of the ChiLDREN consortium directed by Dr. Ron Sokol. We are currently evaluating the mitochondrial protein biomarkers FGF21 and GDF15 for their use in mitochondrial liver diseases, whereas past studies have exclusively focused on myopathies.
Other Aspects of Mitochondrial Diseases
We are also studying the one-carbon metabolism aspect of the universal mitochondrial stress response in muscle from patients with single mtDNA deletion syndromes (supported by a pilot grant of the NAMDC consortium awarded to Dr. Mike Swanson).