Abstract
Diagnosis of mitochondrial disorders is still hampered by their phenotypic and genotypic heterogeneity. In many cases, exome sequencing, the state-of-the-art method for genetically diagnosing mitochondrial disease patients, does not allow direct identification of the disease-associated gene but rather results in a list of variants in candidate genes. Here, we present a method to validate the disease-causing variant based on functional complementation assays. First, cell lines expressing a wild-type cDNA of the candidate genes are generated by lentiviral infection of patient-derived fibroblasts. Next, oxidative phosphorylation is measured by the Seahorse XF analyzer to assess rescue efficiency.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Munnich A, Rustin P (2001) Clinical spectrum and diagnosis of mitochondrial disorders. Am J Med Genet 106(1):4–17. doi:10.1002/ajmg.1391
Haack TB, Haberberger B, Frisch EM, Wieland T, Iuso A, Gorza M, Strecker V, Graf E, Mayr JA, Herberg U, Hennermann JB, Klopstock T, Kuhn KA, Ahting U, Sperl W, Wilichowski E, Hoffmann GF, Tesarova M, Hansikova H, Zeman J, Plecko B, Zeviani M, Wittig I, Strom TM, Schuelke M, Freisinger P, Meitinger T, Prokisch H (2012) Molecular diagnosis in mitochondrial complex I deficiency using exome sequencing. J Med Genet 49(4):277–283. doi:10.1136/jmedgenet-2012-100846
Kornblum C, Nicholls TJ, Haack TB, Scholer S, Peeva V, Danhauser K, Hallmann K, Zsurka G, Rorbach J, Iuso A, Wieland T, Sciacco M, Ronchi D, Comi GP, Moggio M, Quinzii CM, DiMauro S, Calvo SE, Mootha VK, Klopstock T, Strom TM, Meitinger T, Minczuk M, Kunz WS, Prokisch H (2013) Loss-of-function mutations in MGME1 impair mtDNA replication and cause multisystemic mitochondrial disease. Nat Genet 45(2):214–219
Hildick-Smith GJ, Cooney JD, Garone C, Kremer LS, Haack TB, Thon JN, Miyata N, Lieber DS, Calvo SE, Akman HO, Yien YY, Huston NC, Branco DS, Shah DI, Freedman ML, Koehler CM, Italiano JE Jr, Merkenschlager A, Beblo S, Strom TM, Meitinger T, Freisinger P, Donati MA, Prokisch H, Mootha VK, DiMauro S, Paw BH (2013) Macrocytic anemia and mitochondriopathy resulting from a defect in sideroflexin 4. Am J Hum Genet 93(5):906–914. doi:10.1016/j.ajhg.2013.09.011
Wu M, Neilson A, Swift AL, Moran R, Tamagnine J, Parslow D, Armistead S, Lemire K, Orrell J, Teich J, Chomicz S, Ferrick DA (2007) Multiparameter metabolic analysis reveals a close link between attenuated mitochondrial bioenergetic function and enhanced glycolysis dependency in human tumor cells. Am J Physiol Cell Physiol 292(1):C125–C136. doi:10.1152/ajpcell.00247.2006
Moran M, Rivera H, Sanchez-Arago M, Blazquez A, Merinero B, Ugalde C, Arenas J, Cuezva JM, Martin MA (2010) Mitochondrial bioenergetics and dynamics interplay in complex I-deficient fibroblasts. Biochim Biophys Acta 1802(5):443–453. doi:10.1016/j.bbadis.2010.02.001
Brand MD, Nicholls DG (2011) Assessing mitochondrial dysfunction in cells. Biochem J 435(2):297–312. doi:10.1042/BJ20110162
Invernizzi F, D’Amato I, Jensen PB, Ravaglia S, Zeviani M, Tiranti V (2012) Microscale oxygraphy reveals OXPHOS impairment in MRC mutant cells. Mitochondrion 12(2):328–335. doi:10.1016/j.mito.2012.01.001
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Kremer, L.S., Prokisch, H. (2017). Identification of Disease-Causing Mutations by Functional Complementation of Patient-Derived Fibroblast Cell Lines. In: Mokranjac, D., Perocchi, F. (eds) Mitochondria. Methods in Molecular Biology, vol 1567. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6824-4_24
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6824-4_24
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6822-0
Online ISBN: 978-1-4939-6824-4
eBook Packages: Springer Protocols