Feature: Mitochondrial mysteries
Monday, 20 September, 2010
By Branwen Morgan
This feature appeared in the July/August 2010 issue of Australian Life Scientist. To subscribe to the magazine, go here.
They are diseases most people haven’t heard of: Kearns-Sayre syndrome; Leber’s hereditary optic neuropathy; Leigh syndrome or mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS). Neither had Associate Professor David Thorburn until he ‘inherited’ the mitochondrial disease research group at the Murdoch Childrens Research Institute (MCRI) in Melbourne on his return to Australia from the US in 1990. He is now recognised internationally as a leader in the field, has published over 100 related journal articles and been involved in the difficult diagnosis of more than 400 patients who have malfunctioning mitochondria.
Mitochondria are best known for their role in the generation of energy. But they are also involved in a range of other processes such as cell signalling – including calcium signalling, which is needed for proper muscle functioning – and porphyrin biosynthesis, which are compounds that form the basis of many reactions, including oxygen transport by the iron-containing porphyrin haemoglobin. Thus, when mitochondrial function is impaired, the consequences for an individual can be severe.
It’s been almost 30 years since the human mitochondrial genome was sequenced and 22 since the first disease-causing mitochondrial DNA (mtDNA) mutation was discovered. “Of the 37 genes in the mitochondrial genome, disease-causing mutations have been found in 34 of them. But there are also many nuclear genes, approximately 80 to date, with mutations that cause mitochondrial disease,” says Thorburn.
Prognosis depends on the type of mutation (whether it affects gene expression or protein function), the gene affected and the proportion of defective mitochondria in tissue cells. ‘Primary’ mutations, which are those that occur in the mtDNA genes, are maternally inherited, as an oocyte contains all of an individual’s progenitor mtDNA molecules – about 200,000 of them. The sperm’s mitochondria are destroyed during the fertilisation process.
“What’s particularly interesting is that a couple may have several children with dramatically different numbers of mutant mitochondria. One child could have 90 per cent defective mitochondria and another child zero per cent,” says Thorburn. “You could also have a child with 30 per cent mutant mitochondria who won’t develop mitochondrial disease.”
This ‘load shift’ indicates that early in germ cell development there is a bottleneck in the number of mtDNA molecules, similar to the founder effect. It seems that the primordial oocytes receive a small sub-population of the mother’s mtDNA, which are amplified to create the thousands found in the mature oocyte. It also means that the diversity of symptoms, including the time it takes for them to manifest clinically, is partly due to the random dispersion and segregation of the affected mtDNA during embryonic development.
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The mitochondrial cocktail
Every year in Australia around 50 children are born with mitochondrial disease, some will die within a few days of birth. Others may not show symptoms until their late teens or adulthood when a threshold level of mutant mitochondria in a specific tissue is surpassed. And it’s our power-hungry cells, such as nerve and muscle cells that suffer the most, leading to symptoms that include ataxia, seizures and gradual blindness.
“A muscle cell may contain more than 90 per cent mutant mtDNA when symptoms first appear,” says Thorburn. “Yet other cells in the same individual, such as white blood cells or skin cells, may contain very few malfunctioning mitochondria. So in some tissues there seems to be a negative selection process that favours healthy mitochondria.”
Mitochondria are continuously replicating and their numbers can be up-regulated independently of the cell cycle in response to the cell’s energy requirements. For example, physical exercise can lead to an increase in the numbers of mitochondria in muscle cells. In some disease cases, exercise is an effective form of therapy. “Twice as many mitochondria working at 40 per cent efficiency may generate enough energy for the cell to carry out its normal functions,” says Thorburn.
Other methods of compensating for mitochondrial inefficiency include supplementation with a ‘mito cocktail’, which usually contains coenzyme Q10, B vitamins and the amino acid arginine. These dietary supplements are usually given in conjunction with other medications such as anti-epileptics.
Karen Crawley, whose daughter Kara has MELAS, says that they can also limit their daughter’s fits by ensuring she doesn’t get cold (thus minimising the body’s need for thermogenesis), and that she never goes more than a couple of hours without food; Kara has a feeding tube while she is sleeping, which eliminates night-time fasting.
After Kara’s first stroke-like episode at age eight, diagnosis of MELAS quickly followed. But many others aren’t as diagnostically fortunate. Another child, Tom, who has been in and out of hospital since birth, was biochemically (and histologically) diagnosed with mitochondrial disease three years ago, at age five, but researchers still haven’t pinpointed the problem.
“More often than not with these diseases the clinical phenotype isn’t particularly helpful, so when mitochondrial disease is finally considered to be a possibility, we start by testing the activity of one or more of the individual enzymes involved in oxidative phosphorylation (the metabolic pathway that results in ATP production),” says Thorburn.
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MitoCarta
The MCRI receives up to a 150 tissue samples per year from around Australia for diagnostic testing. There are a number of biochemical genetics diagnostic labs in other states as well, but when the biological samples are liver or muscle biopsies they usually get sent to Melbourne. The biochemical tests will sometimes indicate which group of genes may harbour a mutation.
“This triaging is important, as no one has the resources to sequence all the potential genetic culprits and there’s no funding for molecular diagnosis of the vast majority of patients,” says Thorburn.
These undiagnosed samples will often become part of a ‘gene discovery’ research project. One of the significant research developments aiding identification of new mitochondrial disease gene mutations is MitoCarta, a mitochondrial compendium that details the expression of 1098 proteins across 14 mouse tissues. “It is an important reference point for systematic investigations,” says Thorburn, who was involved in the construction of the blueprint.
The MitoCarta inventory also revealed that of the approximately 1500 hundred proteins that comprise a mitochondrion, roughly half are ubiquitous and the other half specific to those mitochondria that reside in particular tissues. “We call this the mitochondrial proteome,” says Thorburn.
Most recently, the research group have used MitoCarta to predict which uncharacterised proteins are likely to be part of a multi-subunit enzyme that has a key role in energy generation and whose genes may thus harbour mutations that cause mitochondrial disease.
They used a high throughput sequencing strategy followed by in vivo validation studies to resolve 13 cases of mitochondrial disease where previously no mutations had been identified. Yet, this scale of sequencing is still relatively expensive and only available on a research basis, despite the continual decrease in costs and concomitant increase in capabilities of next generation sequencing technologies.
And while a definitive molecular diagnosis is comforting, the fact remains that there are no real treatments for this diverse array of disorders. Those in use are based around optimising mitochondrial efficiency and controlling symptoms.
However, should diagnosis reveal a female patient and or female relatives to have a primary mitochondrial defect, there are a number of reproductive options. They include IVF with pre-implantation genetic diagnosis and the possibility of using donor eggs in IVF. And, if predictions are correct, in three years time there may be a third related-option which will enable a couple to conceive their own biological child but with the mitochondria from a different mother.
This new method, previously only attempted in primates, shows that the pronucleus of a fertilised oocyte can be successfully transferred to a donor enucleated oocyte with minimal carry-over of mitochondria. The zygote was allowed to develop for eight days before licensing restrictions required it to be destroyed. The English scientists who are pioneering the technique liken it to changing the embryo’s batteries.
Thorburn says that it is an exciting procedure but more science needs to be done first. “It has only been trialled on abnormal human fertilised embryos that were discounted from further IVF procedures, it now needs to be carried out on normal fertilised eggs. We also need to know more about the interactions between nuclear and mitochondrial DNA within an oocyte as they’ve been coexisting for a long time.”
Regulatory and ethical issues aside, implementation of the nuclear transfer procedure could be hampered by the same barrier that currently exists: that of a donor egg shortage. But should the procedure become accepted, it could ensure those mitochondrial disorders remain rare enough that most people never need to hear of them.
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‘Primary’ mitochondrial disease
Kearns-Sayre syndrome
Caused by several large deletions in mtDNA. Symptoms include: eyesight defects, an abnormal heartbeat and central nervous system degeneration.
Leber’s hereditary optic neuropathy (LHON)
Caused by mutations in the NADH CoQ reductase subunit IV gene. Results in degeneration of the optic nerve, accompanied by vision difficulties and progressive blindness. Symptoms usually start in adulthood.
Mitochondrial encephalopathy, lactic acidosis, stroke-like episodes (MELAS)
Caused by mutations in a mitochondrial tRNA gene. Symptoms include headaches, stroke-like episodes and other neurological symptoms, and vomiting.
Leigh syndrome
Caused by mutations in over 30 different genes encoded by mtDNA or nuclear genes. Results in a progressive neurodegenerative disease. Symptoms usually start in infancy and most affected children die before five years of age.
Cyclical vomiting syndrome (CVS)
Believed to be caused by mutations in the MTTL1 gene, which codes for the mitochondrial transfer RNA for the amino acid leucine. Symptoms: recurring bouts of nausea and vomiting, and occasionally abdominal pain, as well as dizziness, headaches and migraines. It is believed that Charles Darwin may have suffered from CVS.
Case study
Rose has a form of mitochondrial disease caused by a mutation in a nuclear gene called POLG, which codes for the mitochondrial DNA polymerase gamma A subunit. POLG is a major locus for mitochondrial disease, with approximately 150 disease mutations identified in this gene, which is essential for mitochondrial DNA replication and repair.
Rose first started experiencing serious symptoms in the form of muscle twitching, followed by fits, at the age of 20, but she’d had regular stomach aches and vomiting episodes for years before. It took eight months to diagnose Rose with a mitochondrial disorder. Although Rose had an older brother who died five years before she was born, no one knew that he probably also had defective mitochondria.
Rose is now 23, has severe ataxia and tires easily, but she can still walk to the end of the street. Her diagnosis was based on an enzyme assay that assesses efficiency of energy generation in a muscle biopsy followed by DNA sequencing of known genes that carry mitochondrial disease causing mutations. Both of Rose’s parents have since been found to have different point mutations in POLG, giving rise to her compound heterozygosity.
Know your mitochondria
- Every cell has hundreds of mitochondria; the exact numbers depend on the energy requirement of the cell
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- Each mitochondrion contains multiple copies (2-10) of their circular DNA molecules in a compartment called the matrix
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- Mitochondria replicate like bacteria, when they become too large they split into two – this is independent of the cell cycle
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- The mitochondrion hosts about 1500 proteins, most of which are encoded in nuclear DNA. About half of these are tissue-specific
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- As well as coding for structural mitochondrial proteins, a number of nuclear DNA genes influence mitochondrial function, including mtDNA gene expression
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- Each mitochondrial genome contains 37 genes; only a third are involved in energy production (electron transport chain), 22 are transfer RNAs and two are ribosomal RNAs
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- Defects in 80-odd nuclear DNA genes, together with the known defects in 34 of the 37 mtDNA genes constitute the mitochondrial disease ‘family’
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- The large number of subtypes of mitochondrial disease, together with its wide-range of symptoms due to uneven distribution of mutant mitochondria in different tissues and organs, makes it extremely difficult to diagnose
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- Approximately one individual in 8000 has clinically-manifested mitochondrial disease where the primary mutation is in the mtDNA (the total incidence is 1:5000) and one in 200-250 individuals harbours a potentially disease-causing mtDNA mutation
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- Treatment options include dietary supplementation (B complex vitamins, coenzyme Q10), physical therapy, and specific medicines for symptom control, e.g. anti-epileptics to control seizures
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- Current reproductive options include: chorionic villus sampling (CVS), amniocentesis, IVF and pre-implantation genetic diagnosis, or donor egg IVF
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- New IVF-related processes have the potential to prevent a proportion of mitochondrial disease by transferring the fertilised egg’s nucleus to a donor e-nucleated oocyte containing ‘undamaged’ mitochondria
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- Next generation sequencing technologies will make the diagnosis of mitochondrial diseases much faster and cheaper
This feature appeared in the July/August 2010 issue of Australian Life Scientist. To subscribe to the magazine, go here.
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