Research Article: Mitochondrial DNA Variants Mediate Energy Production and Expression Levels for CFH, C3 and EFEMP1 Genes: Implications for Age-Related Macular Degeneration

Date Published: January 24, 2013

Publisher: Public Library of Science

Author(s): M. Cristina Kenney, Marilyn Chwa, Shari R. Atilano, Janelle M. Pavlis, Payam Falatoonzadeh, Claudio Ramirez, Deepika Malik, Tiffany Hsu, Grace Woo, Kyaw Soe, Anthony B. Nesburn, David S. Boyer, Baruch D. Kuppermann, S. Michal Jazwinski, Michael V. Miceli, Douglas C. Wallace, Nitin Udar, Walter Lukiw.


Mitochondrial dysfunction is associated with the development and progression of age-related macular degeneration (AMD). Recent studies using populations from the United States and Australia have demonstrated that AMD is associated with mitochondrial (mt) DNA haplogroups (as defined by combinations of mtDNA polymorphisms) that represent Northern European Caucasians. The aim of this study was to use the cytoplasmic hybrid (cybrid) model to investigate the molecular and biological functional consequences that occur when comparing the mtDNA H haplogroup (protective for AMD) versus J haplogroup (high risk for AMD).

Cybrids were created by introducing mitochondria from individuals with either H or J haplogroups into a human retinal epithelial cell line (ARPE-19) that was devoid of mitochondrial DNA (Rho0). In cybrid lines, all of the cells carry the same nuclear genes but vary in mtDNA content. The J cybrids had significantly lower levels of ATP and reactive oxygen/nitrogen species production, but increased lactate levels and rates of growth. Q-PCR analyses showed J cybrids had decreased expressions for CFH, C3, and EFEMP1 genes, high risk genes for AMD, and higher expression for MYO7A, a gene associated with retinal degeneration in Usher type IB syndrome. The H and J cybrids also have comparatively altered expression of nuclear genes involved in pathways for cell signaling, inflammation, and metabolism.

Our findings demonstrate that mtDNA haplogroup variants mediate not only energy production and cell growth, but also cell signaling for major molecular pathways. These data support the hypothesis that mtDNA variants play important roles in numerous cellular functions and disease processes, including AMD.

Partial Text

Mitochondria provide critical cellular energy using the tricarboxylic acid (TCA) cycle, oxidative phosphorylation (OXPHOS), and beta-oxidation of fatty acids for metabolism, cell division, production of reactive oxygen species (ROS), and apoptosis. Human mitochondrial (mt) DNA forms a circle of double stranded DNA with 16,569 nucleotide pairs. The non-coding mtDNA Dloop contains 1121 nucleotides and is important for replication and transcription. The coding region of mtDNA encodes for 37 genes including 13 protein subunits essential for OXPHOS, 2 ribosomal RNAs, and 22 transfer RNAs [1]–[3]. The mtDNA can be categorized into haplogroups that are defined by a set of specific SNP variants that have accumulated over tens of thousands of years and correspond to different geographic populations of the world. The H haplogroup is the most common European haplogroup, while the J haplogroup originates from the Northern European region and is defined by SNP variants that are associated with heat production as an adaptation to colder climates [4].

Studies were designed to first confirm the identity of each of the cybrids created from the common Rho0 cell line. Figure 1a shows that after PCR amplification and digestion with AluI enzyme, the H cybrids have mtDNA with the C allele in the SNP 7028 representing the H haplogroup (156 bp+152 bp bands; lanes 3 and 4). The non-H haplogroup mtDNA, represented by the T allele, shows bands at 152 bp+126 bp (lanes 1 and 5). Figure 1b shows the restriction digest with BstNI enzyme and reveals a single band at 1210 bp representing the A allele of J haplogroup mtDNA (lanes 4 and 5). The non-J haplogroup is represented by the G allele (874 bp+336 bp, lanes 1 and 3). The mtDNA is absent in the Rho0 cells (lane 2). Each donor mitochondrial haplogroup matched up with the corresponding cybrid, confirming their identity.

This study was designed to provide insights into the different molecular and functional outcomes of having the J haplogroup mtDNA variants versus the H mtDNA variants within cells that had identical nuclei. We found that the H and J cybrids have different modes of energy production. The H cybrids have higher levels of ATP production regardless of the plating density, indicating they utilize OXPHOS more effectively than the J cybrids. Elevated production of ATP levels has been reported in Huntington’s patients that have the H haplogroup compared to non-H individuals [23]. Our analyses also showed that the H cybrids produced significantly higher levels of ROS/RNS compared to the J cybrids. Endogenous production of ROS occurs as electrons leak from the electron transport chain (ETC) within the mitochondria [24]. Our findings suggest that the J cybrids may have lower efficiency of the ETC leading to lower ATP and ROS levels which is similar to that described in osteosarcoma cybrids with J haplogroups [25]. Similar relationships between the H and J haplogroups have been reported in human subjects. Marcuello studied 114 healthy males and showed that those with the J haplogroup had lower maximum oxygen consumption (VO2max) rates than the non-J haplogroups [26], but that a steady state of exercise could eliminate this disparity [27]. Further analyses showed that this disparity was because the H haplogroup had significantly higher VO2max and oxidative damage than the J haplogroup individuals [28]. The inefficiency of the OXPHOS mitochondrial energy production found in J haplogroups may lead to lower ROS production and less oxidative damage, which in part may explain high correlations between centenarians and the J haplogroup population [29]–[31].