What are mitochondria?

1. Describe the mitochondrial genome and its major features and review techniques employed in the genetics laboratory for the analysis of mitochondrial disease.

2. What are mitochondria? • Semiautonomous cellular organelles • numbers range from 2 to 100’s depending cell type • Thought to possibly have started life as bacteria that invaded a nucleated cell • Power-houses of aerobic eukaryotic cells • They contain the electron transport chain which transfers electrons to oxygen by means of a process called oxidativephosphorylation. • releases energy for the production of ATP by forming a pH and electrical gradient across the inner mitochondrial membrane

3. Mitochondrial genome, Major features 1 • Mitochondrial genome is 16,560bp • 44% GC • Double stranded circular • Two strands very different in sequence • Heavy strand = Rich in guanines • Light strand rich in cytosines • Compact, encodes only 37 genes • 2 = Ribosomal RNAs (Heavy strand) • 22 = tRNAs (14 Heavy, 8 Light) • 13 = polypeptides, all components of the respiratory chain/oxidative phosphorylation system. (12 Heavy, 1 Light) • Maternal inheritance • Oocyte mtDNA from mother to offspring • No paternal contribution (generally although there are some suggestions that this may occur very very rarely)

4. Transcription and replication • Transcription of H and L strands is from promoters in the triple stranded, non-coding D-loop region of the genome • Transcription from Heavy strand is CLOCKWISE • Transcription from Light strand is ANTICLOCKWISE • No introns, genes separated by 1 or 2 non-coding bases • Multigenic transcripts generated then cleaved • H-strand origin of replication is in D-loop • Replication of H-strand involves using the L-strand as a template and displacing the old H strand. • L-strand replication starts after 2/3 of daughter H-strand has been replicated • L-strand replication proceeds in the opposite direction using the H strand as a template

5. Mitochondrial genetic code • Mitochondrial genome encodes all rRNA and tRNA molecules it needs for protein synthesis • Need nuclear encoded genes to provide all other components • Mitochondrial genetic code has drifted from the universal code due to small functional load • 60 sense codons, not 61 • 4 stop codons for instead of three • Two Trp codons instead of one • Four Arg codons instead of six • Two Met codons instead of one • Two Ile instead of three • Genetic wobble hypothesis means 22 tRNA species can code for 60 codons

6. Mitochondrial disease • Definition: Heterogeneous group of disorders that arise as a result of dysfunction of the mitochondrial respiratory chain • Mitochondrial disease can be caused by: • Deletions / duplications in the mitochondrial genome • Kearns Sayre syndrome • CPEO (Chronic progressive external opthalmoplegia) • Pearsons syndrome • Point mutations in the mitochondrial genome • MELAS • MERF • Missense mutations in nuclear DNA altering the function of proteins in the respiratory chain. • Leigh syndrome • Encephalopathy

7. Major features 2 • Mitochondrial genome is polyploid • Each cell has 100’s of mitochondria with several copies of mtDNA • In normal conditions individuals have one species of mtDNA • HOMOPLASMY • Diseased individuals can have a mixture of mutant and wild type mtDNA • HETERPLASMY • Varied clinical phenotype with individuals within the same family probably due to heteroplasmy • There is a critical threshold of mutant mtDNA before disease is seen • A heteroplasmic female may transmit variable amount of mutant mtDNA to offspring due ‘bottle neck’ hypothesis

8. The mitochondrial genetic bottle neck

9. Mutability • Mitochondrial genome has a high mutation rate (approximately 10 times higher than nuclear DNA) • They do not have an efficient repair system • They lack protective HISTONES • The genome is physically associated with the inner mitochondrial membrane, so is in close proximity to highly mutagenic oxygen radicals. • No introns so any mutation is likely to affect the codon region • Mutations can be sporadic or maternally inherited • Mutations can be detected in the mitochondrial genome to confirm diagnosis of mitochondrial disease • Sample type important (muscle biopsy rather than blood is preferred for many mutations) • Rearrangements not readily detectable in blood of adults • MELAS mutation 3243A>G may be missed in blood of older patients

10. Testing for point mutationsin mtDNA • Targeted mutation analysis or genome scanning • e.g. RFLP, SSCP, DGGE, sequencing, WAVE-dHLCP (Transgenomic kit) • Problems encountered can include: • Reporting levels of heteroplasmy • Low, medium, high? • ? Use quantitative methods such as real time PCR, pyrosequencing or known controls with restriction digests • Detecting low level heteroplasmic variants • Sequencing will not detect mutations at heteroplasmic levels of less than 30% • Transgenomic kit reports detection down to 0.5%!! • Variable phenotype with the same mutations causing different diseases. E.g. A3242G can cause MELAS, Cardiomyopathy and Diabetes and Deafness • Whole genome screen • Target tests according clinical information provided

11. Testing for large scale rearrangements in mtDNA 1 • Techniques: • Long range PCR • Provides rapid reliable exclusion of rearrangements • Artefacts eliminated by DNA dilution • Rearrangements confirmed by second technique (Southern blotting) • Can detect levels of rearrangements that are not clinically significant • Southern blotting • Digestion of DNA with SnaB1 to detect duplications • Digestion of DNA with PvuII or BamH1 to detect deletions • Can also be used to detect depletion disorders • Time consuming

12. Real time PCR Specialised equipment MLPA? MRC Holland kit exists, however, No control probes Changes of less than 50% decrease or increase in probe signal expected Deletions / duplications probably only detected when more that one probe is located within the area Testing for large scale rearrangements in mtDNA 2 Testing for point mutations in the nuclear genome • Targeted mutation analysis or genome scanning • SURF1, COX10, SCO2, COX15, POLG , ANT1, Twinkle etc

13. Risks to offspring • mtDNA deletions generally occur de novo, recurrence risk is low (1/24) • mtDNA point mutations and duplications may be transmitted through the maternal line • All offspring of females with a mtDNA mutation are at risk ofinheriting the mutation (variable amounts of mutant mtDNA) • Offspring of males with mutant mtDNA are not at risk • Offspring of patients with nuclear gene mutations are either: • Heterozygotes (autosomal recessive disease) • At 50% risk of inheriting the mutation (autosomal dominant)

14. Mitochondrial pedigree

15. Prenatal diagnosis 1 • mtDNA mutations difficult because of mtDNA heteroplasmy • % mtDNA in CVS may not reflect level in other fetal tissue • Mt DNA levels may change during development and life • Therefore for most mtDNA mutations prenatal diagnosis is not recommended • Mutations T8993G/C that cause NARP and MILS does not appear to change significantly over time • Successful prenatal diagnosis has been carried for these two mutations • Relationship between mutant load in mother and disease in offspring for MERF 8344 A>G • Mutant levels below 40% in mother rarely give rise to severe disease in offspring • CVS offered to mothers with mutant load of more than 40%

16. Prenatal diagnosis 2 • Prenatal diagnosis of MELAS (3243A>G) is not indicated • Severity is not clearly related to mutant load • Prenatal diagnosis recommended in patients with Kearns Sayre syndrome where rearrangement is detectable in blood at >5% • Prenatal diagnosis not usually recommended in other deletion / syndromes • Prenatal diagnosis of mutations in nuclear genes causing mitochondrial disease is possible providing the disease causing mutation has previously been identified