Mitochondrion. 2016 Sep;30:236-47.
Mitochondrial Haplogroups and Expression Studies of commonly used Human Cell Lines
Christine Sabine Siegismund 1, Ingo Schäfer 2, Peter Seibel 2, Uwe Kühl 1,3, Heinz-Peter Schultheiss 1, and Dirk Lassner 1
1 Institute for Cardiac Diagnostics and Therapy (IKDT), Moltkestr. 31, D-12203 Berlin, Germany,
2 University of Leipzig, Centre for Biotechnology and Biomedicine (BBZ), Molecular Cell Therapy, Deutscher Platz 5, D-04103 Leipzig, Germany
3 Department of Cardiology, Campus Virchow, Charité – University Hospital Berlin, Augustenburger Platz 1, D-13353 Germany
Maternally inherited mitochondrial haplogroups are specific pattern of multiple single nucleotide polymorphisms on mitochondrial DNA (mtDNA). These evolutionary determined genetic markers allow the classification of human beings to distinct groups with defined properties and could be associated with certain diseases. The distribution of mitochondrial haplogroups among humans depends on the migration and the geographic area (Lott et al., 2013). We developed a multiplex fragment length analysis (MFLA) for clearly assigning mitochondrial haplogroups mostly endemic in Europe for future differentiation of patients with acquired cardiomyopathies. The most common 10 haplogroups in Europe defined by 27 variation sites are H, V, HV, J, T, U, and K of lineage R and I, W, and X of the lineage N. 23 commonly used human cell lines, as they carry the haplogroup of their donors, were haplotyped for the first time by MFLA and their functional characterisation were compared to peripheral blood mononuclear cells (PBMC) resp. endomyocardial biopsies (EMB) of cardiac patients. Determinations of mtDNA copies per cell revealed no correlation to haplogroups but a relatively high rate of mitochondria per cell and at the same time a very low expression of all mitochondrial and some nuclear encoded mitochondrial related genes. A low mitochondrial gene activity seems to be a side or essential effect of the immortalisation process to create long living cell lines. Established MFLA as an easy to handle method is suitable for determining European mitochondrial haplogroup markers for future studies which could be used to elucidate the diagnostic potential of mtDNA haplogroup analysis of cardiac patients (Schultheiss et al., 2011) and to verify if a haplogroup is predisposing a distinct disease form or dysfunction of mitochondria.
PMID: 27562426 DOI: 10.1016/j.mito.2016.08.012
Keywords: cell lines, multiplex fragment length analysis, mitochondrial haplogroup, mitochondria, mtDNA, gene expression analysis
1. Design of a multiplex system to define 27 positions in the mitochondrial genome
This system consists of two assays, a 13-plex and a 14-plex PCR. Each primer analyses a distinct position in mtDNA indicated by three specific fluorescence colours.
Figure 1: Example detection of position 7028T in the mitochondrial genome
2. SNP analysis of 23 commonly used cell lines by MFLA
After applying the analysis scheme, we found 6 cell lines with haplogroup H, 4 with haplogroup U, 3 with haplogroup J, 2 cell lines with haplogroup K and one with haplogroup X. Haplogroup H is the most common haplogroup in Europe (40-50 %) resp. Germany (44.8 %) (Roostalu et al., 2007) which represents our findings. With our analysis, 7 cell lines were correctly assigned to the superordinate haplogroup lineage LMN as they have origins in Asia or Africa.
3. Analysis scheme for haplotyping of mtDNA
This scheme uses only the 27 necessary positions in the mtDNA in respect to possible back mutations and relationship to close evolutionary groups to determine the correct mitochondrial haplogroup.
4. Comparison of mtDNA copies per cell
The number of mitochondria in a cell can vary widely by organism, and cell type. For instance, red blood cells have no mitochondria, whereas liver cells can have more than 2000 (Alzoubi et al., 2014; Ebrahimkhani et al., 2014). mtDNA copies per nuclear genome among all tested cell lines are ranging between 100 and 7000 (median±SD, 886 ± 1744). For comparison, five patient’s samples of PBMCs (median±SD, 94 ± 107) are showing the lowest ratios of mtDNA to gDNA, followed by fibroblasts (median±SD, 102 ± 46) and cell lines. The highest rates are present in EMB (median±SD, 4364±3737).
Figure 2: comparison of mtDNA copies per nuclear genome of the cell lines to example tissues such as EMB, fibroplasts and PBMCs of cardiac patients
5. Gene expression analysis of mitochondria related genes
Comparative analysis of haplogroups and transcriptional activity for human cells or tissues are limited. As shown for cancer cells, mitochondria can communicate with whole genome and regulate cellular gene expression (Kaipparettu et al., 2010; Thaker et al., 2016). Influence of different haplogroups on functional activity in cell lines were shown by cybrid models (Malik et al., 2014; Mueller et al., 2012; Thaker et al., 2016). It demonstrated impressively the sole influence of mtDNA carrying the haplogroup marker transferred to new target cells on cellular metabolism and physiology. Gene expression analyses of cell lines compared to PBMCs as cells in suspension and EMBs as cells of a tissue compartment of cardiac patients revealed a significant diminished expression of all mitochondrial genes in all cell lines.
To our knowledge, 19 cell lines were mtDNA haplotyped for the first time in this article by introduction of MFLA. In a comprehensive analysis we also measured the mtDNA copy numbers and the gene expression of mitochondria related proteins revealing influences of mtDNA on cellular metabolism and functions.
The consequence of this study should be the evaluation of mitochondrial haplogroup for all future cell culture experiments. The influence of SNPs to the mitochondrion as separate replication unit is not understood exactly and could not be overlooked for biomedical or pharmaceutical studies, especially if results from different cell lines were compared. The regulation of mtDNA copy number and cellular gene expression by mitochondrial haplogroup should be included in the interpretation of cell culture experiments. Furthermore, besides possible virus infection, cell type, ethnic origin and underlying disease of cell lines, the mitochondrial haplogroup should be provided as essential marker and main feature for every cell line.
Alzoubi, K., Calabrò, S., Egler, J., Faggio, C., Lang, F., 2014. Triggering of programmed erythrocyte death by alantolactone. Toxins (Basel). 6, 3596–612. doi:10.3390/toxins6123596
Brandon, M.C., Ruiz-Pesini, E., Mishmar, D., Procaccio, V., Lott, M.T., Nguyen, K.C., Spolim, S., Patil, U., Baldi, P., Wallace, D.C., 2009. MITOMASTER: a bioinformatics tool for the analysis of mitochondrial DNA sequences. Hum. Mutat. 30, 1–6. doi:10.1002/humu.20801
Ebrahimkhani, M.R., Neiman, J.A.S., Raredon, M.S.B., Hughes, D.J., Griffith, L.G., 2014. Bioreactor technologies to support liver function in vitro. Adv. Drug Deliv. Rev. 69-70, 132–57. doi:10.1016/j.addr.2014.02.011
Kaipparettu, B.A., Ma, Y., Wong, L.J.C., 2010. Functional effects of cancer mitochondria on energy metabolism and tumorigenesis: Utility of transmitochondrial cybrids, in: Annals of the New York Academy of Sciences. pp. 137–146.
Lott, M.T., Leipzig, J.N., Derbeneva, O., Xie, H.M., Chalkia, D., Sarmady, M., Procaccio, V., Wallace, D.C., 2013. mtDNA Variation and Analysis Using MITOMAP and MITOMASTER. Curr. Protoc. Bioinformatics 1, 1.23.1–1.23.26. doi:10.1002/0471250953.bi0123s44
Malik, D., Hsu, T., Falatoonzadeh, P., Cáceres-del-Carpio, J., Tarek, M., Chwa, M., Atilano, S.R., Ramirez, C., Nesburn, A.B., Boyer, D.S., Kuppermann, B.D., Jazwinski, S.M., Miceli, M. V., Wallace, D.C., Udar, N., Kenney, M.C., 2014. Human retinal transmitochondrial cybrids with J or H mtDNA haplogroups respond differently to ultraviolet radiation: Implications for retinal diseases. PLoS One 9.
Mueller, E.E., Brunner, S.M., Mayr, J.A., Stanger, O., Sperl, W., Kofler, B., 2012. Functional Differences between Mitochondrial Haplogroup T and Haplogroup H in HEK293 Cybrid Cells. PLoS One 7.
Roostalu, U., Kutuev, I., Loogväli, E.-L., Metspalu, E., Tambets, K., Reidla, M., Khusnutdinova, E.K., Usanga, E., Kivisild, T., Villems, R., 2007. Origin and expansion of haplogroup H, the dominant human mitochondrial DNA lineage in West Eurasia: the Near Eastern and Caucasian perspective. Mol. Biol. Evol. 24, 436–48. doi:10.1093/molbev/msl173
Schultheiss, H.-P., Kühl, U., Cooper, L.T., 2011. The management of myocarditis. Eur. Heart J. 32, 2616–25. doi:10.1093/eurheartj/ehr165
Thaker, K., Chwa, M., Atilano, S., Coskun, P., Cáceres-Del-Carpio, J Udar, N., Boyer, D., Jazwinski, S., Miceli, M., Nesburn, A., Kuppermann, B., Kenney, M., 2016. Increased expression of ApoE and protection from amyloid-beta toxicity in transmitochondrial cybrids with haplogroup K mtDNA. Neurobiol Dis Sept, 64–77.
Christine Siegismund, PhD
Institute Cardiac Diagnostics and Therapy