Back in the 19th century, as the US grappled with the aftermath of the Civil War, Jesse James, leader of a notorious gang of outlaws, rose to prominence. He robbed banks and trains for over a decade before a fellow gang-member shot and killed him. His murder became a national sensation, and rumors of him faking death to escape justice soon followed.
In the late 1990s, Mark Stoneking, a molecular anthropologist at the Pennsylvania State University, and his colleagues sought to finally put the century-long rumors to rest. They set out to analyze genetic material from the body buried in the grave marked as James’s. Specifically, they looked at DNA present in the bean-shaped powerhouses of the cell, the mitochondria.1
The unique properties of mitochondrial DNA (mtDNA) have made it a useful tool for forensic studies. In addition, population geneticists have relied on mtDNA analysis to understand people’s histories and origins. However, some of mtDNA’s characteristics that make it useful also make people susceptible to mitochondrial disease, and scientists are leveraging information from mtDNA analysis to prevent and treat the diseases.
Sequencing Mitochondrial DNA to Map Ancestry
Mark Stoneking, a molecular anthropologist at the Max Planck Institute for Evolutionary Anthropology has used mtDNA to solve forensic cases and for studying population genetics.
Mark Stoneking
Mitochondria are unique organelles that have their own DNA–small, closed circular molecules containing about 16,500 base pairs (for reference, almost three billion based pairs make up nuclear DNA).
mtDNA also differs from its nuclear counterpart in that it follows a non-Mendelian inheritance pattern: Most sexually reproducing organisms acquire mtDNA exclusively from their mother. As mtDNA comes from only one parent, mitochondrial inheritance does not involve recombination, making mtDNA analysis straightforward, explained Stoneking.
In James’s case, Stoneking and his team could not rely on mtDNA samples taken from his descendants to confirm his identity. Instead, they found living relatives of his sister, Susan, who would have shared the same mtDNA from their mother, Zerelda. When Susan’s descendants’ mtDNA matched that from the exhumed remains, the team got their confirmation.
“That's a pretty strong indication that it was indeed Jesse James who was buried in the grave,” said Stoneking, now at the Max Planck Institute for Evolutionary Anthropology.
There was another reason why Stoneking and his team turned to mtDNA instead of nuclear DNA to solve the case. While there are only two copies of nuclear DNA per cell, there can be anywhere from 100 to 10,000 copies of the mitochondrial genome depending on the cell type.2
“So, if you're working with a very degraded old sample, or one that's been subject to a lot of environmental deterioration, you're more likely to be successful in analyzing mtDNA than in the nuclear DNA, just simply because there's more of it,” explained Stoneking. This makes mtDNA a useful tool in forensics, he noted.
There’s another interesting property of mtDNA that makes it useful in ancestry studies. Because mitochondrial genome is exposed to reactive oxygen species and possesses less efficient repair mechanisms compared to nuclear DNA, people rapidly acquire mutations in mtDNA.3 This results in genetic changes in the mtDNA between people, making it easier to identify related populations. “So, if there's only very few mutations, then that means [they] have a very recent common ancestor, but [if] there are more mutations, that means that [their] common ancestor lived further back in the past,” explained Stoneking.
Mitochondrial DNA Mutations in Health and Disease
While fast mutation rates in mtDNA come in handy for population geneticists and forensic experts, some mutations in mtDNA are known to cause diseases. Mitochondrial diseases affect one in 4,300 adults, making them among the most common genetic disorders.4
Shoukhrat Mitalipov, a mitochondrial biologist at Oregon Health & Science University, developed a technique to replace the cytoplasm and mitochondria in an egg with those obtained from a healthy donor’s egg.
Oregon Health & Science University
“[mtDNA] mutations affect the basic function of the cell, which is energy production,” said Shoukhrat Mitalipov, a mitochondrial biologist at Oregon Health & Science University. “That's why any mutation in the mitochondria is usually very severe on the embryo and then on the offspring.”
Although maternally-inherited mtDNA mutations can cause fatal or debilitating syndromes in children, disease severity depends on a number of factors.5 The specific gene that is mutated influences whether a disease will be lethal or cause symptoms such as muscle weakness, heart problems, and intellectual disabilities.
Most patients with mtDNA mutations also exhibit heteroplasmy, meaning that some copies of their mtDNA carry mutations while others do not. The proportion of normal and mutated DNA in each tissue influences the severity and nature of disease symptoms.5 However, assessing the extent of heteroplasmy is not straightforward, making it difficult for scientists to predict the risk of inheriting mitochondrial diseases. Mitalipov explained that the mitochondrial genome does not replicate equally in the embryo after fertilization. So, running genetic screens on embryos generated using in vitro fertilization (IVF) can help identify embryos with the lowest mutant load for transfer. This reduces the chances of selecting an embryo that carries disease-causing mitochondrial mutations but cannot completely eliminate it.
“With mitochondrial genes, unfortunately, [detecting disease] is very complicated, because inheritance is not Mendelian,” said Mitalipov. “We know so little about mitochondrial genetics.”
To help women with mtDNA mutations have healthy biological children, Mitalipov and his team developed a method for replacing the cytoplasm and mitochondria in an egg with those obtained from a healthy donor’s egg.
“Mito” and “Tracker” (whose names are in reference to the procedure used for imaging mitochondria) were the world’s first animals derived by spindle transfer for mitochondrial replacement.
Oregon Health & Science University
When they tested this mitochondrial replacement therapy in rhesus monkeys in 2009, they found that the monkeys gave birth to healthy offspring that did not show any adverse health effects, even a decade later.6,7 A few years ago, the researchers tested mitochondrial replacement therapy in clinical trials to improve IVF success, and reported the birth of healthy children.8 Although the participants did not carry a mtDNA disease, the trial presented evidence that the therapy could be safely used in humans.
Currently, clinical applications of this therapy are legal in only some clinics in the UK, Greece, Ukraine, and Australia. This is partly because of the inconsistent success of the therapy. Some researchers found that even if one percent of defective mitochondria are left behind in egg cells, this is enough to still be passed down to future generations.9 Moreover, scientists do not fully understand all the long-term consequences of “three-parent” techniques. For example, researchers found that mice carrying nuclear and mitochondrial genetic material from two different sources age faster, highlighting that more work needs to be done.10
Maternal Lineages Influence mtDNA Disease Susceptibility
While studying mitochondrial diseases, Francois Van der Westhuizen, a researcher at North-West University, came across children that showed typical signs and symptoms of a mitochondrial muscle disorder. However, he was surprised to find that mtDNA sequencing did not reveal any commonly reported syndrome-associated mutations.11
Joanna Elson, a mitochondrial geneticist at Newcastle University, studies how mitochondrial lineage influences the susceptibility of mitochondrial disease in people.
Joanna Elson
Van der Westhuizen’s colleague Joanna Elson, a mitochondrial geneticist with an evolutionary biology background at Newcastle University, had a hunch as to why this was the case. Data collected from European populations largely forms the basis of all mtDNA research, although African and European populations display mtDNA variations. She wondered whether the lack of mutations could be explained by the fact that the population they were studying was of South African descent.
“I started to consider, well, maybe there is an effect of the mitochondrial lineage on the penetrance of mutations, but also maybe there are some lineage specific mutations,” said Elson.
Disease-causing mtDNA mutations differ between mitochondrial lineages, or haplogroups, highlighting the importance of sequence context in the expression of mutations. To investigate whether the genetic background in which a mutation arises contributes to the disease manifestation, Elson and her team obtained sequences of the mitochondrial genome of more than 30 animals, ranging from primates to eels.12 They observed that mtDNA mutations that cause disease in humans do not cause disease in other animals. Further, some variants carried by the non-human animals were predicted to compensate for the disease-causing mutations, highlighting that the broader genomic context in which a mutation occurs influences the signs of disease.
Now, researchers, including Elson, have found that among other factors, an individual’s mitochondrial lineage influences their susceptibility to a variety of conditions including some mitochondrial disorders, diabetes mellitus, and even psychiatric disorders.13-16
“This means that when you're looking at different lineages that you haven't studied so much, you might need to consider variants as being linked with disease [that] you haven't before,” explained Elson.
Surita Meldau, a medical scientist specializing in mitochondrial genetics at the University of Cape Town, who is also a collaborator of Elson’s, believes that these results highlight the importance of accounting for genetic diversity in studying mitochondrial diseases.
Surita Meldau (center) and her lab members, Shrinav Dawlat (left) and Kashief Khan (right), run mtDNA diagnostics and haplogroup research in Cape Town, South Africa.
Surita Meldau
“We know a lot already about European haplogroups [because] there’s a lot of genetic data out there,” said Meldau. “There’s a lot we can still learn from the African haplogroups and disease context in African healthcare simply because we haven’t got a lot of data.”
To bridge this gap, Meldau and her team set up a laboratory—the only one of its kind in the country—where clinicians from across South Africa send patient samples for genetic testing. They hope to understand how mitochondrial disorders in the South African population differ from those in the rest of the world.
There is still a lot of work to be done to make genetic screening more accessible to people in Africa, said Meldau. With the advancement of sequencing technologies, “We are only really starting to try and understand [African haplogroups]. We are slowly making progress.”
Reducing these barriers will not only help the people in Africa but also provide important insights worldwide, she noted. “What we still need to find about mitochondrial disease is all lying in the unstudied populations. And there's a wealth of information still [that can teach us] how these diseases work.”
Although advancements in sequencing methods have advanced the field of mtDNA far from the days when Stoneking and his team resolved the Jesse James mystery, a lot remains to be explored. “We didn't pay too much attention to the mtDNA, and that's why we don't even know how it's transmitted,” said Mitalipov. Stoneking agreed. “There's some basic fundamental aspects about mtDNA that we still don't understand [but] I think the new techniques especially are opening things up.”
- Stone AC, et al. Mitochondrial DNA analysis of the presumptive remains of Jesse James. J Forensic Sci. 2001;46(1):173-176.
- Castellani CA, et al. Thinking outside the nucleus: Mitochondrial DNA copy number in health and disease. Mitochondrion. 2020;53:214-223.
- Gureev AP, et al. Long-range PCR as a tool for evaluating mitochondrial DNA damage: Principles, benefits, and limitations of the technique. DNA Repair (Amst). 2025;146:103812.
- Gorman GS, et al. Prevalence of nuclear and mitochondrial DNA mutations related to adult mitochondrial disease. Ann Neurol. 2015;77(5):753-759.
- Schon EA, et al. Human mitochondrial DNA: Roles of inherited and somatic mutations. Nat Rev Genet. 2012;13(12):878-90.
- Tachibana M, et al. Mitochondrial gene replacement in primate offspring and embryonic stem cells. Nature. 2009;461(7262):367-372.
- Ma H, et al. Germline transmission of donor, maternal and paternal mtDNA in primates. Hum Reprod. 2021;36(2):493-505.
- Costa-Borges N, et al. First pilot study of maternal spindle transfer for the treatment of repeated in vitro fertilization failures in couples with idiopathic infertility. Fertil Steril. 2023;119(6):964-973.
- Yamada M, et al. Genetic drift can compromise mitochondrial replacement by nuclear transfer in human oocytes. Cell Stem Cell. 2016;18(6):749-754.
- Latorre-Pellicer A, et al. Mitochondrial and nuclear DNA matching shapes metabolism and healthy ageing. Nature. 2016;535(7613):561-565.
- van der Walt EM, et al. Characterization of mtDNA variation in a cohort of South African pediatric patients with mitochondrial disease. Eur J Hum Genet. 2012 Jun;20(6):650-6.
- Queen RA, et al. Mitochondrial DNA sequence context in the penetrance of mitochondrial t-RNA mutations: A study across multiple lineages with diagnostic implications. PLoS One. 2017;12(11):e0187862.
- O'Keefe H, et al. Haplogroup context is less important in the penetrance of mitochondrial DNA complex I mutations compared to mt-tRNA mutations. J Mol Evol. 2018;86(6):395-403.
- Ji Y, et al. Mitochondrial haplotypes may modulate the phenotypic manifestation of the LHON-associated ND1 G3460A mutation in Chinese families. J Hum Genet. 2014;59(3):134-140.
- Alwehaidah MS, et al. Mitochondrial haplogroup reveals the genetic basis of diabetes mellitus type 2 comorbidity in psoriasis. Med Princ Pract. 2021;30(1):62-68.
- Chang X, et al. Mitochondrial DNA haplogroups and risk of attention deficit and hyperactivity disorder in European Americans. Transl Psychiatry. 2020;10(1):370.
