Mitochondrial donation Flashcards
Mitchondria - structure and function
Mitochondria are membrane bound cell organelles, consisting of an inner and outer membrane, and intermembrane space, the folds of the inner membrane are called cristae. Contain their own DNA which encodes transfer RNAs, ribosomal RNAs and some mitochondrial proteins (codes 37 genes only passed down maternally).
Functions:
Generation of metabolic energy
* Production of ATP, cellular respiration
* Replication, maintenance and transcriptions of mitochondrial DNA
* Calcium signalling, homeostasis
* Regulation of membrane potential
* Mitochondrial protein synthesis
* ROS production
Mitochondrial DNA disease
describes a diverse group of neuro-metabolic disorders characterised by impaired oxidative phosphorylation.
Causes debilitating and incurable disease. Can cause physical disability, metabolic prolems, abnormal organ function and early death for some patients.
1 in 5000 to 10000 people are affected.
Average lifespan of children with disease is 3-12years.
Mitchondrial disease is different from mitochondrial DNA disease in that it can be caused by nuclear DNA defects or within the mitchondrial DNA
Differences between nuclear and mitchondrial DNA
Nuclear DNA from both parents, mtDNA from mother only
nDNA is assembled in chromosomes contained within the nucleus of a cell, 46 chromsomes.
Amount of mDNA can vary greatly from mitochondrion to mitochondrion as well as from cell to cell. Some cells have <20 copies of mtDNA whereas the oocyte has >500 000
MtDNA held within mitchondria
Mitochondrial role in embryo development
Mt ATP needed for oocyte function
See this when take the function away - morula wont get to blast stage
Fertilisation stimulates mt ATP production
ROS - part of mt activity too
Oocytes have the most mitchondria
mitochondrial donation
two validated techniques available are Maternal spindle transfer (MST) and Pronuclear transfer (PNT)
Difference between heteroplasmy and homoplasmy
In patients who carry a pathogenic mtDNA mutation -
Homoplasmy - occurs when only on mtDNA type is present (wild type of mutant type)
Heteroplasmy - occurs when more than one mtDNA type are present in the same cell or tissue (mutant (pathogenic) and wildtype (“normal”) present together).
Threshold effect
When heteroplasmy exists - ratio of wild-type to mutant mtDNA that is important and clinical features of mt disease will only manifest once a critical threshold level has been exceeded.
Bottleneck effect with mt disease transmission
‘genetic bottleneck’ during development of the female germline can occur.
The primordial germ cell when it develops into a primary oocyte will transfer different percentages of mutant versus wildtype mt. Which means that women who carry a pathogenic mtDNA mutation can transmit different levels of mutant mtDNA to their children. This makes genetic counselling difficult as it is not always possible to predict the risk of disease in the child. Moreover, an asymptomatic woman carrying low levels of mutant mtDNA could have a child who is severely affected by mitochondrial disease.
Issues with mt donation
Carryover can occur - small amount of mutant mt can be transferred with the nuclear DNA. This can then develop preferential amplification in some tissues and if reaches threshold level can cause mt disease in offspring still. Also called reversion. Current investigations ongoing about how to reduce this risk.
Nuclear-cytoplasmic incompatibility - some evidence in animal models that the if nuclear and mt genome function is compromised if these two differ. Currently - haplogroup matching occuring where mt donor has a similar mtDNA sequence to the patient.
New territory -largely untested with short and long term safety data and consequence not well established.
Maeve’s law
2022 (Mitochondrial Donation Law Reform Bill)- allowed mitochondrial donation for the purpose of preventing mitochondrial disease transmission.
Required legislative change to allow this technology in Australia
mitoHOPE - $15million to conduct a pilot project - clinical trial to build evidence, safety, efficacy and feasibility of implementing mt donatoin in clinical practice settings.
Changes:
No longer an offence to create, for the purposes of reproduction, a human embryo that - contains the genetic material of more than two people, and contains heritable changes to the genome.
Two stage implementatio
Prenatal diagnosis
Women with homoplasmy - not indicated as 100% chance offspring has
Women with heteroplasmy - assessment of variant levels in at least two different tissues (blood, urine, buccal) and testing other maternal relatives and affected offspring.
Often testing not appropriate with high maternal heteroplasmy.
For low levels of maternal heteroplasmy - invasive testing can be performed. Shouldn’t culture as this can affect levels of mutant mtDNA.
Can be difficult to predict risk from mutant load particularly when correlation between variant level and disease severity is complex.
PGT
Involves quantification of the level of a pathogenic mtDNA variant to allow for the selection of the most appropriate embryo based on mutant load and quality.
Seem to do on cleavage stage embryo with one blastomere biopsied.
Fetal variant levels would be required to be below the critical threshold for severe disease expression and the prediction of the percentage threshold below which clinical symptoms are unlikely to occur later in adult life is challenging. A threshold of ≤18% has been proposed. However, the exact threshold would need to be considered on a case-by-case basis as it can be variable depending on the familial mtDNA variant involved, previous familial transmission, clinical presentations between family members, and local policies and procedures of the centre offering the PGT service. Therefore, provision of PGT should only be offered within specialised centres where comprehensive investigations of the threshold in each family can be undertaken. Moreover, as PGT is a risk reduction procedure it may only benefit women who are likely to produce oocytes with low mutant load, further highlighting that specialist reproductive counselling is required. PGT is not indicated for women that harbour variants at high mutant load.
AUGMENT
Autologous germline mitochondrial energy transfer (AUGMENT)
Ovarian cortex biopsy taken and mitochondria extracted.
Injected into egg at same time as ICSI insemination.
Thought it may improve outcome in patients with previously failed in vitro fertilization (IVF)
RCT published in Fert Stert 2019 showed lower D5 blast rate 23% and 41% between Augment and control ICSI. No difference in fert rate, CLBR observed.
Discontinued early as no benefit found.
How does mt DNA differ from nuclear DNA with respect to reproduction
Mutation rate higher- proximity to respiratory chain and absence of histones
Point mutations increase with aging
Oocyte mitochondria increase up until ovulation , mitochondrial DNA divided among cells therefore copy
number per cell decreases
Replication of mitochondrial DNA resumes at blastocyst stage
Replication only takes place after implantation
