Wendy Roworth FRCPath course 17/09/10

1. Describe the mitochondrial genome and itsmajor features and review techniques employed in the genetics laboratory for the analysis of mitochondrial disease. Wendy Roworth FRCPath course 17/09/10

2. Mitochondrial disorders • Mitochondrial diseases are a clinically and genetically heterogeneous group of disorders characterised by biochemical abnormalities of the respiratory chain. • Mitochondrial disorders may be caused by mutations in the mitochondrial genome (mtDNA) or certain genes in the nuclear genome. • Mitochondrial disorders tend to affect tissues with a high energy requirement • More than 40 different types of mitochondrial disease • Affect at least 1 in 8000 • NCG funding covers most mitochondrial disease testing (in England and Scotland)

3. Mitochondrial genome • Distinct from nuclear genome. • Constitutes ~ 1% of cellular DNA • Each mitochondrion has 2-10 copies of mtDNA, each cell has 100’s of mitochondria, therefore several thousand copies of mtDNA per cell. • Some cells e.g. brain and muscle cells have high requirement for oxidative phosphorylation and therefore more mitochondria. These tissues more susceptible to effects of mtDNA mutations. • The mutation rate in mtDNA is ten times higher than in nuclear DNA; the mtDNA lacks the DNA repair mechanisms found in the nucleus. • Mitochondrial genome encodes only a small proportion (13/70) of the proteins that are needed for its functions; the bulk of the proteins are encoded by nuclear DNA and imported into the mitochondria • Different code to nuclear DNA

4. mtDNA genetic code

5. Human mitochondrial genome • Transcription occurs from both strands (heavy and light) • Two promoters IH1 and IH2 are on the heavy strand, just one promoter, IL is on the light strand. • All promoters and elements involved in replication initiation are found in the displacement (D) loop, the only major non-coding region. • The genome encodes 22 mt-tRNA (black diamonds), 2 mt-rRNA genes (purple) and 13 protein-coding genes (green)

6. mtDNA • 16,569 bp circular DNA • Encodes 37 genes • 13 polypeptides, 22 tRNA & 2 rRNA • No introns • Maternally inherited • Heavy & Light strands • Polycistronic • No recombination • High mutation rate (no proof reading mechanism, no histones). • Heteroplasmy

7. Nuclear vs mitochondrial genomes

8. Homoplasmy vs Heteroplasmy • Homoplasmy refers to a cell that has a uniform collection of mtDNA: either completely normal mtDNA or completely mutant mtDNA. • A cell can have some mitochondria that have a mutation in the mtDNA and some that do not. This is termed heteroplasmy. • In cells where heteroplasmy is present, each daughter cell may receive different proportions of mitochondria carrying normal and mutant mtDNA. • The proportion of mutant mtDNA molecules determines both the penetrance and severity of expression of some diseases: threshold effect

9. Inheritance of mtDNA • Maternally inherited • There has however been a case reported of paternal mtDNA inheritance: Normal elimination of sperm mitochondria was defective and 90% of patient’s muscle mitochondrial were inherited from his father who carried a 2bp deletion in the MTND2 gene (see refs). • An egg contains 100,000 to 1,000,000 mtDNA molecules, whereas a sperm contains only 100 to 1000 (in the tail). • The sperm tail often does not enter the egg. • Any sperm mtDNA are actively degraded in the fertilized egg. • Sperm therefore contribute nuclear but not mitochondrial genome to offspring.

10. Inheritance of mtDNA

11. Technical aspects • Mutation analysis depends on mitochondrial disorder and types of mutation involved. • Range of different mutations: mtDNA point mutations, mtDNA large rearrangements (single or multiple deletions or duplications), mtDNA depletion syndrome, degree of heteroplasmy, nuclear DNA mutations. • Sample type varies depending on disorder. Blood is suitable for some analyses, but urine or muscle biopsy samples are required for other types. • Other testing available: muscle biopsy and staining, biochemical testing, CSF protein concentration studies, brain imaging, ECG, nerve conduction studies.

12. Techniques: mtDNA point mutations • Targeted analysis for a particular mutation eg: m.8344A>G mutation in MERFF • Panel of mutations eg: LHON m.3460G>A in MTND1, m.11778G>A in MTND4 and m.1448T>C in MTND6 • Screening of particular gene/ genes eg: MTRNR1 (12S rRNA) (including m.1555A>G mutation) and MTTS1 in mitochondrial deafness syndrome. • Sequencing of entire mitochondrial genome may be appropriate in some cases (rare/ novel mutations) • Awareness of heteroplasmy in some disorders – may make detection of certain mutations difficult eg: m.3243A>G mutation • Methods suitable for detecting and quantifying heteroplasmic mutations include real-time PCR, pyrosequencing, last PCR cycle labelling followed by quantification & RFLP analysis (levels of m.3243A>G lower than 5% may not be detected).

13. Techniques: Real-time PCR assay for mtDNA depletion disorder • Used to test for mtDNA depletion syndromes (assessment of mtDNA copy number to screen for possible mtDNA depletion syndromes) • Taqman real-time PCR assay which coamplifies nuclear (18S rRNA) and mtDNA (MTND1) genes. DNA from affected tissues (e.g. muscle, liver) essential

14. Techniques: mtDNA rearrangement disorders (CPEO, KSS, Pearsons) • Long-range PCR for mtDNA rearrangements, followed by Southern blotting to characterise rearrangements detected. • Southern blotting using PvuII or BamHI or SnaBI/ BglII enzyme digestions (depends on whether looking for deletions or duplications). • Single mtDNA deletions and secondary multiple mtDNA deletions. • Multiple mtDNA deletions may be due to mutations in nuclear genes. • mtDNA rearrangements can also be detected using real-time PCR. • Muscle DNA essential (CPEO and KSS) • DNA from blood may be accepted for children with Pearsons syndrome

15. Techniques: Nuclear gene analysis • Mutations in the nuclear genes POLG, DGUOK, MPV17, RRM2B and ANT1 can cause mitochondrial disease. • Full screening or targeted mutation analysis of these genes may be appropriate. • Pyrosequencing is used to test for the 5 common mutations in the POLG1 gene. • POLG1 mutation screening (entire gene). POLG1 mutations cause mitochondrial disorder associated with multiple mtDNA deletions and depletion. • Mutations in PEO1 can cause multiple mtDNA deletions. Sequencing of the first 3 exons appropriate.

16. 2. In families with mitochondrial disorders, different family members can present with different clinical manifestations. Discuss this statement with respect to the principles defining mitochondrial genetics.

17. Variation of clinical presentation amongst families • Can be considerable variability in clinical presentation between family members. May have very mildly and very severely affected members of the same family. • For all mtDNA mutations, clinical expression depends on three factors: • Heteroplasmy • Tissue distribution of mutant mtDNAs • Threshold effect (see next slide) • Tissues have variable energy requirements. May have more/less or different organs affected to each other. • Respiratory function deteriorates with age so only severe defects manifest early on, while minor defects may only become be clinically evident in old age. • Aberrant mtDNA genomes may be selected against; this is particularly true of large deletions, which tend not to be inherited.

18. Threshold effect • Proportion of mutant mtDNA must exceed a critical threshold level before a cell expresses a biochemical abnormality of the mitochondrial respiratory chain. • Threshold may be different between different tissues which may have different requirement for mitochondrial energy supply. • Tissue order: brain>muscle>heart>kidney>lung • Mutant load may change over time. • Predicting severity, type and age of onset is often difficult due to variability between mutant loads and onset of symptoms. • Due to variation of heteroplasmy between tissues, the mutation may not be detectable in blood, and testing other tissues may be necessary e.g. cultured skin fibroblasts or skeletal muscle. • Variability in clinical manifestations may also depend on the nature of the mutation (its intrinsic pathogenicity and the gene affected).

19. Mitochondrial Bottleneck

20. MELAS (Mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes): Heteroplasmic mtDNA disorder • Mitochondrial myopathy, as seen by lactic acidosis and/or ragged red fibers (RRF) on muscle biopsy • Seizures and/ or dementia • Typical onset in the first decade of life. • Stroke-like episodes, usually before the age of 40 • Approximately 1.6 per 10,000 individuals are affected with MELAS. • Heteroplasmic mtDNA point mutations • The m.3243A>G (MTTL1) mutation is present in ~80% of patients with typical MELAS presentation • m.3243A>G mutation also seen in patients with CPEO, cardiomyopathy and maternally inherited diabetes and deafness • Severity is not clearly related to mutant load. • Prenatal diagnosis is not appropriate for several mutations in MELAS. • Muscle is preferable to blood sample; m.3243A>G levels decline in blood with age and low levels may be missed in older patients.

21. MERRF (Myoclonic epilepsy and ragged red fibres): Heteroplasmic mtDNA disorder • Myoclonus, epilepsy, muscle weakness and wasting with RRFs, cerebellar ataxia, deafness and dementia. • Range of symptoms affecting many different organs, different family members may have different clinical signs of the disorder. • Heteroplasmic point mutations. • Common practice for MERRF referrals is analysis of the m.8344A>G mutation only (80-90% of cases). • There is a relationship between mutant mtDNA load in the mother and the risk of an affected offspring in cases of MERRF – m.8344A>G. • Severe disease is rare in offspring of mothers with a load of <40% mutant mtDNA in blood. CVS should be offered to mothers with levels of >40%. Oocyte donation or PGD should also be considered in mothers with high mutant load. • Blood or muscle samples are acceptable; m.8344A>G levels in blood are reliable. • Genotype-phenotype correlation between MERRF syndrome and the m.8344A>G mutation is tighter than that of other mutations, but this same mutation has been reported in phenotypes as different as Leigh’s syndrome, isolated myoclonus, familial lipomatosis and isolated myopathy.

22. Homoplasmic mtDNA disorders • Variability in presentation within families with homoplasmic mutations • Clinical expression compared to heteroplasmic mutations often stereotypical and mainly restricted to a particular tissue • Presence of a pathogenic mtDNA mutation is necessary but not sufficient to cause disease • Penetrance is incomplete; possibly controlled by environmental factors, additional mitochondrial polymorphisms or the effect of nuclear gene(s) • Example: LHON

23. Leber’s Hereditary Optic Neuropathy (LHON): homoplasmic mtDNA disorder • Loss of central vision, vision deteriorates rapidly and suddenly in late teens or early 20’s. • Males more often affected than females. • 3 common mutations are indicated for LHON referrals (90% of cases). • Mutations are usually homoplasmic, although heteroplasmy can occasionally occur in some families. • Reduced penetrance: • Role of environmental factors - tobacco smoke and alcohol. • Certain haplogroups may increase penetrance of disease • A nuclear modifier could be a major determinant for both disease expression and male prevalence (search for X-linked modifier unsuccessful to date). • The presence of the mtDNA mutation does not predict the occurrence, age of onset, severity or the rate of progression of this typically adult-onset disease. • Presymptomatic testing for LHON should be undertaken cautiously and homoplasmy/ heteroplasmy should be stated on the report. • Not covered by NCG as LHON is deemed too common.

24. Mitochondrial DNA deletion disorders • Three overlapping phenotypes that may be observed in different members of the same family or may evolve in an individual over time. • Chronic progressive external ophthalmoplegia (CPEO): deletions confined to muscle biopsy • Kearns-Sayre Syndrome (KSS): found in lesser amounts in lymphocyte DNA as well as muscle • Pearson’s syndrome: comparable amounts between blood and muscle samples • Therefore muscle is the preferred tissue to the detection of rearrangements (apart from Pearson’s) • The severity of clinical symptoms increases as the tissue distribution becomes more widespread (CPEO<KSS<Pearsons)

25. Mitochondrial DNA deletion disorders • Most rearrangements occur at direct repeats within mitochondrial DNA • Common deletion of 4977 bp • mtDNA deletions are often sporadic, although familial cases have been reported. • Duplications have been associated with familial cases, risk of recurrence. • Multiple mtDNA deletions can be AD, AR or sporadic • Multiple mtDNA deletions may be due to mutations in the nuclear genes POLG, SLC25A4 or PEO1.

26. Mitochondrial DNA deletion disorders • No correlation exists between size or location of the mtDNA deletion and phenotype. • For all mtDNA mutations, clinical expressivity depends on the 3 following factors: • Mutational load (heteroplasmy) • Tissue distribution of mutant mtDNAs • Threshold effect - certain tissues may be more susceptible to mitochondrial dysfunction than others

27. Autosomal disorders of mitochondrial DNA maintenance • POLG, PEO1, DGUOK, MPV17, RRM2B & ANT1 nuclear genes all encode mitochondrial proteins. • These proteins function, at least in part, to maintain mitochondrial DNA. • Mutations in these genes have been associated with a number of disorders characterised by multiple mtDNA deletions and/or mtDNA depletion. • Autosomal dominant or recessive

28. References • www.geneclinics.org • www.mitomap.org • London ideas: Mitochondrial disorders • Best practice guidelines for molecular diagnosis of mitochondrial diseases (2008) • Mitochondrial DNA Mutations in Human Disease: Taylor & Turnbull. Nature Reviews Genetics (6) :389-402 (2005) • Mitochondrial DNA depletion syndromes – Many genes, common mechanisms. Suomalainen et al. Neuromuscular Disorders (2010) • Paternal inheritance of mitochondrial DNA: Schwarz & Vissing NEJM 347(8):576-580 (2002) • Mitochondrial disorders: Zeviani & Donato. Brain 127: 2153-2172 (2004)