Lecture 7: Mitochondrial genetic in Health L2

Question 1

Mitochondria are composed of proteins encoded by two genomes: 2

Answer 1

1. Nuclear DNA
2. Mitochondrial DNA

Question 2

Mitochondrial DNA features:

Answer 2

1. (mDNA) is found in cell mitochondria
2. contains genetic material ONLY FROM THE MOTHER

Question 3

Nuclear DNA features

Answer 3

1. (nuDNA) is found in the CELL NUCLEUS
2. Contains genetic material from BOTH PARENTS

Question 4

Which genome causes mitochondrial diseases..

Answer 4

1. Mutations in mtDNA or nuclear DNA genes that make up mitochondria can cause mitochondrial disease

Question 5

Mitochondrial dysfunction leads to diverse pathologies and diseases….7

Answer 5

1 * Neuromuscular diseases

2 * Dementia and neurological disorders

3 * Diabetes and obesity

4 * Liver disease

5 * Heart disease

6 * Cancer

7 * Ageing

Question 6

most common genetically inherited metabolic diseases:

Answer 6

Mitochondrial diseases

Question 7

ORGAN AND MITOCHONDRIAL DNA causes disease = 11

Answer 7

1. EYE:
   - optic neuropathy
   - ophthalmoplegia
   - retinopathy
2. LIVER:
   - Hepatopathy
3. KIDNEY:
   - Fanconi’s syndrome
   - glomerulpathy
4. PANCREAS
   - Diabetes mellitus
5. BLOOD
   - Pearson’s syndrome
6. INNER EAR
   - Sensorineural hearing loss
7. COLON
   - Pseudo-obstruction
8. BRAIN
   - seizures
   - myoclonus
   - ataxia
   - stoke
   - dementia
   - migraine
9. SKELETAL MUSCLE
   - weakness
   - fatigue
   - myopathy
   - neuropathy
10. HEART
    - Conduction disorder
    - wolf-parkinson-white syndrome
    - cardiomyopathy
11. NUCLEAR DNA —-SUBUNITS —-OXIDATIVE PHOSPHORYLATION —-NUCLEAR DNA

Question 8

Mitochondrial dysfunction leads to diverse pathologies and diseases - STATISTICS

Answer 8

1 in 8,000 at risk

1 in 15,000 to be affected

Question 9

Mitochondria are composed of proteins encoded by two genomes

Answer 9

UNDERSTAND SLIDE 5 DIAGRAM AND PROCESS

Question 10

Mitochondrial DNA mutations and heteroplasmy: 4

Answer 10

1. MtDNA mutations have maternal inheritance

2 *Stochastic distribution of mtDNA

3 *Severity of symptoms often can be linked to the
load of mutant mtDNA

4 *Transfer of mutant mtDNA is not well
understood

Question 11

*Diagnosis of mtDNA mutations in the clinic
relies on: 3

Answer 11

1. Sequencing

2 * Biopsies

3 * Enzymatic tests

Question 12

SLIDE 6 MITOCHONDRIAL INHERITANCE

Answer 12

STUDY FLOW CHART

Question 13

SLIDE 7 MITOCHONDRIAL…OOCYTE MATURATION AND mtDNA amplification + fertilisation

Answer 13

STUDY FLOW CHART

Question 14

SLIDE 8 Mitochondrial DNA mutations and heteroplasmy

Answer 14

STUDY FLOW CHART

Question 15

Mutations in mitochondrial genes cause disease…

Protein-encoding genes = 3

Answer 15

1 * Neurogenic weakness, ataxia and retinitis pigmentosa (NARP) – T8993G/C

2 * Maternally inherited Leigh syndrome (MILS) – T8993G/C

3 * Leber’s hereditary optic neuropathy (LHON) – G3460A, G11778A, T14484C and A14495G

Question 16

Mutations in mitochondrial genes cause disease

tRNA genes = 3

Answer 16

1 * Mitochondrial encephalopaty with lactic acidosis and stroke-like episodes (MELAS) – A3243G

2 * Myoclonic epilepsy with ragged red fibres (MERRF) – A8344G

3 * Non-syndromic sensorineural deafness – A7445G

Question 17

Mutations in mitochondrial genes cause disease

rRNA genes = 1

Answer 17

Aminoglycoside-induced non-syndromic deafness – A1555G

Question 18

Mutations in mitochondrial genes cause disease:

Rearrangements = 3

Answer 18

1 * Chronic progressive external ophthalmoplegia

2 * Kearns-Sayre syndrome

3 * Diabetes and deafness

Question 19

Mutations in mitochondrial genes cause mitochondrial diseases = 12

Answer 19

1. Mutations in mitochondrial genes:
2. Frequency 1:4000

3 *Destabilize the secondary or tertiary structure of RNAs
…4 * A3243G MELAS
…5 * A8344G MERRF

6 *Impair the processing of RNAs:
….7 * A3302G cardiomyopathy
…..8 * A5559G

9 *Impair the recognition by tRNA synthetases
…..10 * 5- taurinomethyl-2-thiouridine (τm5 s2 U)

11.*Impair protein synthesis
….12 * 5- taurinomethyl-2-thiouridine (τm5 s2 U)

Question 20

Mitochondrial Donation

• Eliminating mutant mitochondria = 4

Answer 20

1 *Precludes from transmitting inherited mitochondrial disease to future generations

2 *Already in practice in the United Kingdom

3 *Requires in vitro fertilization

4 *The risks are minimal and research into any
potential risks is carried out

Question 21

Mitochondrial Donation
Eliminating mutant mitochondria…DIAGRAM

Answer 21

UNDERSTAND FLOW CHART IN SLIDE 11

Question 22

Gene therapies for
mitochondrial diseases

Modulating the mutant heteroplasmy load: 4

Answer 22

1 *Using protein based therapeutic designed to target only the mutant mtDNA or mtRNA.

2 *Import into mitochondria using existing protein import pathways

3 *CRISPR technologies not possible in mammalian mitochondria as they don’t import RNA

4 *Current gene therapies are under research and development

Question 23

Gene therapies for
mitochondrial diseases
Modulating the mutant heteroplasmy load…2 FLOW CHARTS ARE IMPORTANT

Answer 23

UNDERSTAND SLIDE 12 DIAGRAMS

Question 24

Inheritance of mitochondrial diseases caused by mutations in nuclear encoded proteins….

Nuclear mutations follow autosomal dominant or recessive inheritance:

Answer 24

1 *Diagnosis of mitochondrial diseases caused by nuclear DNA mutations in the clinic is
challenging because the diseases are multisystemic and involve multiple different specialists.

2 *There are no treatments or cures to date only
palliative care for patients is available.

Question 25

Diagnosis of nuclear DNA mutations relies on a combination of: 3

Answer 25

1. Sequencing 2. Biopsies 3. Enzymatic tests

Question 26

Inheritance of mitochondrial diseases caused by mutations in nuclear encoded proteins = SLIDE 12

Answer 26

SLIDE 12 - 1. Autosomal recessive - 2. Consanguineous autosomal recessive - 3. autosomal dominant

Question 27

INTEGRATED APPROACH FOR MITOCHONDRIAL DISEASE DIAGNOSIS ...3

Answer 27

1. METABOLIC TESTING: -- Blood -- urine - CSF 2. Muscle testing - liver tissue respiromtry - muscle pathology - OXPHOSEnzymology - Protein chemistry - C0Q10 3. GENETIC TESTING: - mt DNA - Nuclear DNA

Question 28

Nuclear mutations in genes encoding mitochondrial proteins...8

Answer 28

1. Gene expression defects * Leigh syndrome – LRPPRC, TACO1 * Alpers, CMT – POLg * PEO – Twinkle 2. Assembly defects * Leigh syndrome – SURF1 * Cardioencephalomyopathy – SCO2 * Neonatal-onset hepatic failure and encephalopathy – SCO1 * Leigh and De Toni-FanconiDebre syndrome – COX10 3. Homeostasis and import * Friedreich’s ataxia –frataxin * Hereditary spastic paraplegia – paraplegin * Deafness-dystonia syndrome – DDP * Dominant optic atrophy – OPA1 4. Complex I deficiency * Leigh and Leigh-like syndrome NDUFS4, NDUFS7 and NDUFS8 * Hypertrophic cardiomyopathy and encephalomyopathy – NDUFS2 * Macrocephaly, leucodystrophy and myoclonic epilepsy – NDUFV1 5. Complex II deficiency * Leigh and Leigh-like syndrome 6. Complex III deficiency * Encephalopathies, GRACILE syndrome – BCS1L, UQCRB and UQCRQ 7. Complex IV deficiency * Leigh syndrome – COX15, COX8A 8. Complex V deficiency * Cardiomyopathy – ATPAF2

Question 29

Nuclear mutations in genes encoding mitochondrial proteins...DIAGRAM ..understand the process

Answer 29

SLIDE 14

Question 30

Mitochondrial Diseases affect different organs and have varying severity = 6

Answer 30

1. It is important to identify the genetic cause of mitochondrial diseases to understand how it leads to disease and identify potential treatments. 2 * Some diseases affect many organs and some few 3 * Some diseases are more severe than others 4 * Some diseases occur early in life and some later 5 * A same mutation can result in diverse phenotypes 6 * Different mutations can have a same phenotype

Question 31

Molecular and functional diagnosis of Mitochondrial Disease = IMPORTANT

Answer 31

KNOW FLOW CHART AND DIAGRAM ...SLIDE 16 TO 19

Question 32

Isolated Complex IV deficiency in a patient with a cytochrome oxidase subunit 1 (CO1) mutation = 5

Answer 32

1. Patient - a 10 year old girl 2 *Increased lactic acidosis 3 *Early onset retinitis pigmentosa 4 *Ataxia and peripheral neuropathy 5 *Mutation in CO1 gene coding for a scaffold protein required for Complex IV activity

Question 33

Limitations of cell models for mitochondrial disease research = 3

Question 34

Limitations of cell models for mitochondrial disease research = 3 **Animal models of disease are the best in vivo tool for clinical research

Answer 34

Animal models of disease are the best in vivo tool for clinical research 1 * C. elegans (worms), Drosophila (fruit flies), Mus musculus (mice) 2 * Availability of material, genetic manipulation and physiological relevance 3 * Investigating the disease in the affected tissues

Question 35

Impaired protein synthesis can generate diverse phenotypes = 7

Answer 35

1. Mrps34....Overall impaired translation...ORAGNS ..MITOCHONDRIAL DISEASE 2. Taco1...Impaired COXI translation...ORGANS...Mitochondrial Disease 3. Ptcd1 Het...Decreased translation... ORGANS ...HYPERTROPHIC, CARDIOMYOPATHY, METABOLIC DISEASE 4. Ptcd1, Elac2 and Mrpp3 KO...Loss of translation...DILATED CARDIOMYOPATHY 5. Mrps12...error-prone translation..ORGANS...Liver Regeneration 6. Mrps12....Hyper-accurate translation...ORGANS...DIALTED CARDIOMYOPATHY 7. Translation factor...uncoordinated translation...ORGANS....Myopathy and cardiomyopathy

Question 36

Mitochondrial RNA processing is required for energy production... 4

Answer 36

1.RNase P cleaves the 5’ end of mitochondrial tRNAs and is composed of 3 subunits: MRPP1, MRPP2 and MRPP3 *All three proteins are required for cleavage *No requirement for a catalytic RNA *Mutations in all three proteins cause disease 2. *Loss of MRPP3 is embryonic lethal and heart and skeletal muscle KO causes cardiomyopathy and lethality by 11 weeks 3. *5′ tRNA processing precedes 3′ tRNA processing 4 *RNA processing links transcription to translation via mitoribosome assembly and this essential for respiration

Question 37

RNase P cleaves the 5’ end of mitochondrial tRNAs and is composed of 3 subunits: MRPP1, MRPP2 and MRPP3 = 3

Answer 37

1 *All three proteins are required for cleavage 2 *No requirement for a catalytic RNA 3 *Mutations in all three proteins cause disease

Question 38

Mitochondrial RNA processing is required for energy production DIAGRAMS

Answer 38

IMPORTANT ..LOOK AT SLIDE 24 MRPP3 ..KNOW PROCESS AND DIAGRAM

Question 39

5′ tRNA processing in mitochondria ....RNA sequencing: 3

Answer 39

1 * RNA-Seq enabled capture of tRNAs contained within longer precursor transcripts 2 * PARE enabled capture of 5’ RNA ends 3 * Combined they identified impaired mitochondrial RNA processing

Question 40

Mutations in mitochondrial RNA processing enzymes cause disease.... ...Identified a mutation in MRPP1: 4

Answer 40

1. Early onset mitochondrial disease 2. * Combined OXPHOS defect, multi-systemic disorder, both children died at ~5 months 3 * Decreased stability of the MRPP1 protein 4 * Impaired mitochondrial RNA processing

Question 41

LRPPRC is a general RNA chaperone required for mRNA stability... ....The leucine-rich pentatricopeptide repeat cassette (LRPPRC) protein:

Answer 41

1 * A A354V mutation in LRPPRC causes the autosomal recessive French-Canadian variant Leigh Syndrome 2 * Loss of LRPPRC is embryonic lethal and heart and skeletal muscle-specific loss of LRPPRC causes dilated cardiomyopathy by 12 weeks of age in mice 3 * LRPPRC is in a stable complex with SLIRP that protects it from degradation 4 * The LRPPRC/SLIRP complex is localized in the mitochondrial matrix where it coordinates mRNA translation by relaxing their secondary structure

Question 42

LRPPRC is a general RNA chaperone required for mRNA stability... DIAGRAM

Answer 42

UNDERSTAND AND DRAW SLIDE 27

Question 43

Summary: 4

Answer 43

1. Mitochondrial diseases are the most common genetically inherited metabolic disorders. - They can affect young infants as well as adults and the severity of the diseases varies and most often is lethal. 2 * Mitochondrial diseases have several different inheritance modes. 3 * There are no cures for mitochondrial disease so there is an urgent need to find therapeutics and treatments. - Diagnosis for mitochondrial diseases has improved with the advances of next generation technologies, however there are still gaps in functional validation of putative pathogenic genes. 4 * There are many different models that can be used to understand the pathology of mitochondrial diseases and reveal therapeutic targets for drug development