Download Mitochondrial DNA mutations

Mitochondrial
DNA mutations
ASSOCIATE PROFESSOR DAN MISHMAR
Associate Professor Dan Mishmar explains the significance of
research into mitochondrial DNA and the direction of his work at
the Ben-Gurion University of the Negev in Israel
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Mitochondria have their own distinct
genome. What accounts for the sequence
variations among individuals around
the globe?
Unlike the nuclear genome, mitochondrial
DNA (mtDNA) resides in multiple copies
per cell, ranging from ~100,000 copies in
the ovum to ~100 copies in the sperm.
Mutations that occur in our body’s cells result
in increased intracellular sequence variation;
if such mutations occur in the ovum (but
not the sperm) they will be transferred to
the next generation, and will either be fixed
or lost during cell divisions depending on a
bottleneck that occurs during the maturation
of the ova. Therefore, cells that divide a lot,
such as those in the haematopoietic system,
may lose much of their intracellular variation
as compared to slow-dividing post-mitotic
cells, such as neurons and skeletal muscle
cells. It is estimated that mtDNA has an order
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ASSOCIATE PROFESSOR DAN MISHMAR
of magnitude higher mutation rate than the
nuclear genome on the evolutionary timescale.
To these pieces of evidence, one could add
evolutionary forces such as Darwinian selection
that shape variation both within the cell and
between individuals. We recently exemplified
all this through the study of intracellular
mtDNA variation in human identical twins.
Can you elucidate your hypothesis
surrounding the functionality of haplogroupdefining mutations within the mtDNA
transcription/replication regulatory region?
MtDNA is inherited solely through the maternal
lineage and therefore genetic variation in the
population stems from the accumulation of
mutations during the course of evolution. Since
mtDNA does not undergo recombination, one
cannot assess the functionality of individual
mtDNA mutations independent of their
linked genetic background. Moreover, there is
currently no available technology to mutate
the human mtDNA sequence at will in the cell.
Therefore, to assess the functionality of
individual mutations we sought an in vitro assay
to overcome the aforementioned obstacles.
Since many of the evolutionary variants lie
within or adjacent to mtDNA promoters, we
chose in vitro transcription as an assay to assess
the functionality of mtDNA variants. This led
us to discover that certain ancient variants
in the vicinity of mtDNA promoters affect
in vitro transcription, the binding capacity of
mitochondrial transcription factor A (TFAM)
and the replication efficiency of mtDNA
(mtDNA copy number in cells). With this in
mind, we asked ourselves whether the mtDNA
transcription machinery is confined to the
mtDNA promoter or if there are additional
undiscovered transcriptional regulatory
elements in other human mtDNA regions.
This question led us to screen for alternative
transcription regulatory elements throughout
the mtDNA sequence in a hypothesis-free
manner, which yielded the exciting preliminary
identification of novel transcription factors that
bind and regulate human mtDNA.
You and your collaborators screened
thousands of complete human mtDNA
sequences for natural variation in
experimentally established protein and
RNA-coding genes, and regulatory regions.
Have your investigations uncovered new
information on mutations?
We recently published a comprehensive
analysis of more than 9,800 whole human
mtDNA sequences representing all major
human populations. In that study, we aimed at
identifying the mutations that were retained
in the human phylogeny in certain ‘branches’
of the phylogenetic tree, ie. mutations that
define genetic backgrounds. We also identified
mutations that independently recurred in
distant mtDNA lineages.
Strikingly, we found mutations with a high
functional potential both among the large
repertoire of lineage-defining and recurrent
‘nodal’ mutations. This raises a question: if
those mutations are found in the general
population and they are similar to diseasecausing mutations, how are we still healthy?
The answer is still open, but our hypothesis is
that these mutations were likely compensated
by changes elsewhere in the genome, including
the nucleus. If this is true, certain combinations
of mutations involving these functional
variants will be beneficial and others will alter
susceptibility to diseases. Our preliminary
results indicate just that.
What do your team members contribute to
your investigations? Does being based at
Ben-Gurion University offer advantages in
terms of collaboration?
I am blessed by a group of outstanding PhD and
MSc students and a very capable technician
who not only perform the experiments but
contribute intellectually to the design and
experimental testing of our hypotheses.
For disease association studies, especially
in relation to Type 2 diabetes, we have a
wonderful ongoing collaboration with the
Israeli Diabetes Research Group (IDRG),
to which I am most grateful for medical
information and DNA samples of hundreds of
patients and controls. In the frame of other
projects we have excellent local, European and
US collaborators.
Ben-Gurion University is a dynamic and
relatively young research institute which is
eager to provide a vivid working environment
for researchers, lively discussions with
colleagues and cutting-edge research facilities.
This atmosphere is essential for my work.
Mechanisms of disease and evolution
A team of Israeli life scientists is intensively investigating mitochondrial DNA and its
relationship to two phenomena determined by the same principles – evolution and disease
MITOCHONDRIA ARE THE powerhouse of
mammalian life. Of the many different organelles
in a human cell, mitochondria are responsible for
providing us with most of our energy; being the
hosts of oxidative phosphorylation (OXPHOS),
a key process in aerobic respiration. They are
also assigned many other essential tasks, such
as playing a role in apoptosis, the programmed
death of cells. This process is vital to embryo
development and to our existence, and its
malfunction leads to uncontrolled cellular growth
found in cancer.
Beyond fulfilling their share of the cellular division
of labour, mitochondria stand out from other
organelles in one vital respect – they are the only
organelles in animals, apart from the nucleus,
to contain their own DNA. This mitochondrial
DNA (mtDNA) contains genes that are vital for
the functioning of the mitochondrion, such as
programming for enzymes involved in OXPHOS,
as well as being involved in the creation of transfer
RNA (tRNA) and ribosomal RNA (rRNA) involved
in uniting amino acids to form proteins.
mtDNA exists in a double-stranded ring and has
a very high mutation rate due to a poor repair
mechanism and high number of replication
cycles. It thus evolves rapidly, making it useful
in phylogeny – the study of evolutionary
relationships by comparing DNA sequences
between species. As it is inherited from the
mother only, mtDNA allows maternal lineage to
be traced, and is thus affected purely by mutation
accumulation rather than by recombination.
Because there are many mitochondria within a
cell, mutations in mtDNA permit variation to
occur both between and within cells. Associate
Professor Dan Mishmar, an expert in mtDNA from
the Ben-Gurion University of the Negev in Israel,
explains: “This is the beauty of mitochondrial
genetics: it exhibits inter-individual variation, but
unlike the nucleus it also displays intracellular
sequence variation that may accumulate over the
lifetime of the individual. It plays a role not only
in the generation of population genetic variants
but also alters mitochondrial function during the
ageing process and plays a role in age-related
disorders such as Parkinson’s disease”.
EVOLVING DISEASE
Genetic variation in mtDNA was, until recently,
considered a mere academic curiosity. However,
the work of Mishmar and colleagues is part
of a momentous shift which emphasises the
significance of mtDNA mutations on the
human phenotype. For the team, understanding
mtDNA is vital to an understanding of human
complex diseases, as well as major evolutionary
transitions and the emergence of new species.
Mutations in mtDNA, just as in nuclear DNA
(nDNA), have the potential to cause disease. The
researchers are driven by their unique perspective
that sees disease through the lens of evolution,
understanding disease-causing mutations and
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INTELLIGENCE
MtDNA
OBJECTIVES
To provide evidence for the hypothesis
that mitochondrial genetic variability and
evolutionary dynamics play a role in major
evolutionary transitions including the
emergence of new species and also in the
tendency of humans to develop complex
disease phenotypes.
KEY COLLABORATORS/PARTNERS
The Israeli Diabetes Research Group
(IDRG)
Dr Raz Zarivach; Professor Amir Aharoni;
Professor Amos Bouskila; Professor
Ofer Ovadia, Ben-Gurion University of the
Negev, Israel
Professor Eran Meshorer, Hebrew
University, Israel
Dr Leo Nijtmans, Radboud University,
The Netherlands
FUNDING
Israeli Science Foundation
Israeli Ministry of Health
Israeli Cancer Association
CONTACT
Associate Professor Dan Mishmar
Group leader
Ben-Gurion University of the Negev
Department of Life Sciences
Building 40, Room 005
Beer Sheva, 84105
Israel
T +972 8 646 1355
E [email protected]
www.plosgenetics.org/article/
info%3Adoi%2F10.1371%2Fjournal.
pgen.1000474
ASSOCIATE PROFESSOR DAN
MISHMAR obtained a BA in Archaeology
from Hebrew University in 1992. He was
then supervised by Professor Batsheva
Kerem whilst completing a PhD in Human
Genetics also at Hebrew University.
His postdoctoral training mentor was
Professor Douglas C Wallace. Since 2004,
he has been Principle Investigator (Senior
Lecturer) at the Ben-Gurion University
of the Negev, Israel, before becoming
Associate Professor in 2011.
Mishmar’s work has the potential
to shed light on the functioning of
the mitochondrion as an organelle
vital to life as well as to pick apart
the intricacies of its high degree of
interdependency with the nucleus
genetic variations as being governed by the same
evolutionary principles.
Much of their work concerns complex diseases,
which are determined by multiple genes in
multiple locations as well as by environmental
factors, as Mishmar underlines: “Complex diseases
could be caused by combinations of variants, each
of which contributes only little to the disease
phenotype”. This active gene-environment
interface draws the forces of natural selection into
play. A simple example of the tight relationship
between evolution and disease would be the
positive selection of efficient energy-producing
genotypes in an ancestral age when food was
scarce. This same adaptation, upon encountering
the abundance of food in the modern world, has
led to the rise of obesity and diabetes.
Whilst it could be assumed that evolutionary
processes would negatively select diseasecausing mutations in mtDNA, there is evidence
that in some contexts they could present an
evolutionary advantage. The group, hypothesising
that recurrent nodal mutations are retained due
to positive selection pressures, have investigated
the functions of these variants. By looking at
the recurrence of mtDNA ancient variants in a
different genetic context, they have shown that
the genetic landscape in which a mutation occurs
can have a strong influence on its effect on the
development of mitochondrial disorders such as
the legal blindness caused by Leber’s hereditary
optic neuropathy (LHON).
explain the roots of mtDNA. Over the billions
of years that have passed since this event, the
nucleus and mitochondria have become symbiotic
in their functioning.
This interdependence is highlighted in the
experimental generation of cytoplasmic hybrids –
cybrids – in which mitochondria from one species
are present within a nucleus from another. Such
hybrid cells often display reduced mitochondrial
functionality, providing evidence that mtDNA
and nDNA must thus remain compatible despite
the rapid mutation rate of mtDNA, ie. the
mitochondria and nucleus coevolve.
TWO SIDES OF THE SAME COIN
Mishmar’s team has focused on protein subunits
of complex I that are encoded by the mtDNA
and nDNA. The researchers have provided
evidence of how these subunits encoded by
nDNA and mtDNA directly interact. They have
also uncovered evidence that mtDNA is not only
regulated by specific mitochondrial regulatory
factors but is integrated into the general cellular
regulatory system; editing occurs in transcripts
of mtDNA just as it does in the nucleus. In
addition to these important results, Mishmar’s
group has explored beyond the known mtDNA
transcription factors, discovering that several
nDNA transcription factors are also used in
mitochondria. These are significant contributions
to our still hazy knowledge of mitochondrial
transcription and translation.
The tight relationship between nDNA and mtDNA
has also been shown to be pivotal to disease
susceptibility. Mishmar’s group was one of the
first to show that the significance of mtDNA in
determining susceptibility to Type 2 diabetes
depends on the combination of mtDNA variants
and as yet unknown variants in nDNA, rather
than being dependent on variants in mtDNA
or in nDNA alone, as Mishmar elaborates: “The
latter finding exemplified the importance of
functional cooperation between the nuclear and
mitochondrial genomes not only during evolution
but also for human health”.
TOGETHER FOREVER
THE MITOCHONDRIAL FUTURE
Mishmar and his team have also investigated
the interdependence of mitochondria and the
nucleus. It is generally believed that mitochondria
originated from a process of endosymbiosis;
they were originally independent bacteria which
became absorbed into the cell. This may partially
Since the functional impact of a genetic variant is
likely to be only slight, the team is now searching
for experimental methods sensitive enough to
determine this. They are also currently working
to unravel the mysteries of mtDNA transcription,
identifying factors and their binding sites involved
in the regulation of this process. In addition,
the researchers are pioneering methods of
identifying the signatures of natural selection
both for variants between individuals and for the
intracellular mtDNA variants.
Electron microscope image of mitochondria
in human cells.
Mishmar’s work has the potential to shed light
on the functioning of the mitochondrion as an
organelle vital to life, as well as to unpick the
intricacies of its high degree of interdependency
with the nucleus. Through this new knowledge, it
is hoped that further insights will be gleaned into
both evolutionary processes and susceptibility
to a host of diseases, paving the way towards
preventative medicines.
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