W4 - The Mitochondrial Genome Flashcards

Question

How is electrophoresis used?

Answer 1

Blue-native gel electrophoresis of OXPHOS complexes

Answer 2

Electron micrography of abnormal muscle mitochondria The shape of the mitochondria would change and become large and unusual swollen. Can also be seen with paracrystalline inclusions (PCI)

Answer 3

Next Gen sequencing is a game changer for mitochondrial disease diagnosis. There is a point mutation on gene m8969 converting G to A. Eg. Only 2 children suffer from the disease but no predecessors do. How?

Answer 4

A single cell can contain thousands of DNA copies. Homoplasmy - The presence of just one single mitochondrial variant. This could be healthy or pathogenic. Heteroplasmy - When there are two or more variants of mitochondrial DNA present. Someone could equally have two healthy variants as opposed to two problematic ones.

Answer 5

Heteroplasmy levels determine disease manifestation Relative quantity of pathogenic patients in mitochondria determines if the disease manifests or not (>80%) Referring back to the former example, it shows that the 2 children had high levels of the variant as opposed to low levels of the variant by other family members.

Answer 6

Heteroplasmic mutations = Inheritance of mutation load is random Homoplasmic mutations = Females carrying homoplasmic mutation transmit to all children. Mitochondrial diseases are maternally inherited. But can also be transmitted through autosomal recessive and autosomal dominant modes of inheritance and X-linked inheritance and de novo mutations. Most are caused by mutations in nuclear genes.

Answer 7

* m8993T>G * >90% = leigh syndrome * 60-75% = NARP * <60% asymptomatic * Variable penetrance also in homoplasmic mutations * LHON * 50% males affected * 10% females affected

Answer 8

This answers why in families the heteroplasmy variants differ so much. During early development, there were a group of cells that specialised to become primordial germ cells. These then differentiate to become primary oocytes - this means there is a reduction of mitochondria when it divides as well as asymetric division itself. This leads to different levels of inheritance.

Answer 9

* Blood, urine, fibroblasts, tissue * Next-generation sequencing of mtDNA * Increased reliability and sensitivity * More accurate detection of low level heteroplasmy * Muscle and liver may be necessary for tissue-specific mutations not present in blood (e.g. A3243G MELAS/MIDD mutation) * Detect SNVs, single or multiple deletions, duplications * Quantitative PCR for mtDNA depletion (caused by mutations in nuclear genes affecting mtDNA replication/maintenance)

Answer 10

Mutations in any mitochondrial DNA subunits of Oxidative Phosphorylation complexes can lead to disease. Requires mitochondrialDNA replication with transcription, translation. Mutations in proteins involved in these steps can lead to secondary mitochondrial disease.

Answer 11

* mtDNA deletions * mtDNA depletion * Occurs in post-mitotic tissues: * Brain * Muscle * Heart * Liver

Answer 12

Dominant mutations in TWINKLE (one of the replicating proteins) cause mtDNA deletions and late-onset mitochondrial myopathy. * Multiple mtDNA deletions in muscle, brain and heart * Progressive external ophthalmoplegia (PEO), muscle weakness, exercise intolerance * No cure and lack of treatments * Lack of biomarkers for diganosis and as outcome measures in clinical trials * Reasons for tissue-specificity unknown

Answer 13

Sanger sequencing can be performed on known genes known to cause clinically recognisable syndromes. This would be laborious and the diagnostic yield would be very low. Since Next Gen sequencing had been on the rise, there are nearly 400 known gene mutations that causes mitochondrial disease.

Answer 14

>300 mitochondrial disease genes Genes coloured blue are mtDNA genes All others are nuclear genes

Answer 15

* Sequencing panels of disease specific genes * Fewer genes but better sequence coverage. Good for clinical practice but new disease genes overlooked. * Whole exome sequencing * All coding genes but some important regions not well sequenced * Optimised Whole exome sequencing for capture of all known disease associated regions * Costs more * Whole genome sequencing * Sequencing everything but even more expensive and data analysis complex

Answer 16

* Homoplasmic or heteroplasmic? * Prognosis depends on mutation, heteroplasmy levels, variable penetrance of homoplasmic mutations and therefore difficult to predict * Recurrance risks * Strictly maternally inherited (new evidence suggests paternal inheritance possible) * Homoplasmic mutations passed to all children * Heteroplasmic mutations passed in variable amounts * Low for de novo mutations (risk for potential germline mosaicism)

Answer 17

* Oocyte donation * Prenatal diagnosis * Preimplantation genetic diagnosis (PGD) * Mitochondrial replacement therapy

Answer 18

MRI and MRS (Magnetic Resonance Spectroscopy) can be used for disease identification. Neural imaging is important. MRI - Can be used to see bilateral lesions of the basal ganglia. Atrophes of the cerebellum, brainstems and white matter disease. MRS - Can be used to investigate specific metabolites in the brain like lactate.

Answer 19

The aim is to remove the mitochondrial DNA of the mother that has a pathogenic variant to an oocyte of the donor. The children born will have the nuclear DNA of the mother and the father and the mitochondrial DNA from the donor. Aka - 3 parent babies.

W4 - The Mitochondrial Genome Flashcards

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