alexia chrysostomou (083707160)
TRANSCRIPT
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Identification of new genes in adult-onset
mitochondrial diseasesMRes Project 2012
Alexia Chrysostomou
(083707160)
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Introduction Mitochondria and diseases Progressive External Ophthalmoplegia (PEO) Patients cohort
Methodology Exome sequencing Variant filtering criteria Sanger sequencing
Results RRM1 TOP3A
Conclusions Discussion and Future Work
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Mitochondria and diseases
• Subcellular organelles required for maintenance and survival
• Production of the majority of energy demand through oxidative phosphorylation (Kim, Kim et al. 1989)
• Contain circular double-stranded DNA (mtDNA)
• Wide spectrum of disorders linked to them
• Primary mtDNA defects
• Secondary changes due to nuclear-encoded genes (Taylor and Turnbull 2005; Copeland 2008)
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Progressive External Ophthalmoplegia (PEO)
• Commonest mitochondrial myopathy in adults
• “Facial expression with eyes motionless and dropping lids giving the impression that the patient is half asleep” (Hutchinson 1879)
• Characterized by ptosis and ophthalmoparesis
• Symptoms include: proximal limb muscle weakness, ataxia, axonal neuropathy and cardiomyopathy
• Disease progression
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• Genetic causes: primary mtDNA defects or nuclear DNA mutations leading to multiple mtDNA deletions
• Muscle biopsy demonstrates cytochrome c oxidase (COX) inactivity
Progressive External Ophthalmoplegia (PEO)
1 2 3
- 9.9 kb
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Patients cohort
• Recruitment of an initial cohort of 8 patients
• Similar disease phenotype, mainly PEO
• Multiple mtDNA deletions and COX-negative fibers
• Exclusion of known genes (POLG, POLG2, ANT1, Twinkle, RRM2B)
• Exome sequenced
• We had a panel of 48 further patients for testing of any candidate genes
Patient 1 2 3 4 5 6 7 8
Phenotype PEO;NOSPEO;
AtaxiaPEO;NOS
PEO; Ataxia
PEO; Ataxia; Neuropathy;
Cardiomyopathy
PEO; OPMD-like
PEO; Ataxia
PEO; OPMD-like
Suspected mode of
inheritance
Autosomal Recessive
Autosomal Recessive
Autosomal Dominant
Autosomal Recessive
Autosomal Recessive
Autosomal Recessive
Autosomal Dominant
Autosomal Dominant
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Exome sequencing
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Methodology• Exome sequencing-Filtering criteria
1. Selection of genes predicted to be mitochondrial
2. Exclusion of known polymorphisms, mutations reported in the Thousand Genomes Projects and other non-coding changes
3. For sporadic cases, assumed with autosomal recessive inheritance: homozygote or compound heterozygote coding changes -> 106 candidates
4. For familial cases, inherited the disease in a dominant fashion: single heterozygous coding changes -> 533 genes
5. From (3) and (4), evaluated the genes according to function (biological plausibility-mtDNA replication and mitochondrial dynamics) -> final list of 13 genes
• Sanger sequencing
• Verification of mutations that came up from exome sequencing
• Whole (candidate) gene sequencing
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Methodology Lane1
Lane5
Lane6
Lane3
Lane7
Lane8
Genes Function
PANK2 May be the master regulator of the CoA biosynthesis √
TTN Assembly and functioning of vertebrate striated muscles √
CPT1B Enzyme of the long-chain fatty acid beta-oxidation √
DNAH14 Microtubule-dependent motor ATPase √ √ √
SUOX Oxidation of sulfite to sulfate √
TOP3A Control and alteration of the topologic states of DNA √ √
SACS regulator of the Hsp70 chaperone machinery √
RARS2 Arginyl-tRNA synthetase √
DMWD Could have a regulatory function in meiosis √
SYNE1 Maintenance of subcellular spatial organization √
RRM1 Provides the precursors necessary for DNA synthesis √ √
SPG11 Phosphorylated upon DNA damage-defects cause spastic paraplegia type 11 √
NDUFV2 Subunit of the mitochondrial membrane respiratory chain NADH dehydrogenase (Complex I) √
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ResultsGene
symbolVariant Prediction Patient
Sanger Sequencing
TTN
chr2_179428370_C_T chr2_179454530_C_T chr2_179455731_C_G chr2_179500777_C_T
disease_causing;p.G18622R disease_causing;p.R11766Q disease_causing;p.E11366Q
polymorphism;p.D4968N
5 TRUE
PANK2chr20_3870334_T_C chr20_3869911_T_G
polymorphism polymorphism
1 FALSE
RARS2 chr6_88239365_C_T disease_causing; p.R258H 8 TRUE
TOP3Achr17_18208522_G_A chr17_18211681_T_C chr17_18196087_G_A
NMD; p.R135* polymorphism; p.M100V
disease_causing;rs139068958; p.P385S
5 5 7
TRUE
NDUFV2chr18_9104204_G__C_ins
chr18_9122529_G_ANMD;p.H4P
polymorphism; p.V110I8 TRUE
SUOX chr12_56398455_G_A disease_causing 3 FALSE
SYNE1 chr6_152702311_G_C disease_causing 8 FALSE
DNAH14chr1_225393676_T_A chr1_225231636_G_T chr1_225270424_A_T
polymorphism;p.F1972Y disease_causing
polymorphism;p.N1104Y
3 7 8
TRUE
SACS chr13_23906739_G_A disease_causing; p.T3759M 8 TRUE
CPT1B chr22_51014487_C_T disease_causing; p.V252M 3 TRUE
DMWD chr19_46294291_T_G disease_causing 3 FALSE
RRM1chr11_4154851_T_C chr11_4144575_C_A
disease_causing; p.M6555T disease_causing; p.N427K
7 8
TRUE
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RRM1• Ribonucleotide Reductase
large subunit (RNR1)
• Normal partner of RRM2B, known to cause ad PEO, for supplying resting cells with deoxynucleotides for DNA repair
• Baruffinni and colleagues (2006) demonstrated that overexpression of RNR1 (or deletion of its inhibitor-SML1) is able to rescue yeast petite colonies
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Reference ID Position in chromosome Region in gene
rs111548639 g.412A>C; Chr11_4116335 Intron
rs725518 g.12922G>A;Chr11_4128845 Intron
rs56336381 g.17394C>A;Chr11_4133317 Intron
rs183484 c.850C>A;Chr11_4141132 CDS
rs9937 c.2223A>G;Chr11_4159457 CDS
rs1042858 c.2232G>A;Chr11_4159466 CDS
Screening the remaining 48 patients in the panel did not indicate further changes in any of the gene’s exons. Common polymorphisms were detected instead:
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Genesymbol
Variant Prediction PatientSanger
Sequencing
TTN
chr2_179428370_C_T chr2_179454530_C_T chr2_179455731_C_G chr2_179500777_C_T
disease_causing;p.G18622R disease_causing;p.R11766Q disease_causing;p.E11366Q
polymorphism;p.D4968N
5 TRUE
PANK2chr20_3870334_T_C chr20_3869911_T_G
polymorphism polymorphism
1 FALSE
RARS2 chr6_88239365_C_T disease_causing; p.R258H 8 TRUE
TOP3Achr17_18208522_G_A chr17_18211681_T_C chr17_18196087_G_A
NMD; p.R135* polymorphism; p.M100V
disease_causing;rs139068958; p.P385S
5 5 7
TRUE
NDUFV2chr18_9104204_G__C_ins
chr18_9122529_G_ANMD;p.H4P
polymorphism; p.V110I8 TRUE
SUOX chr12_56398455_G_A disease_causing 3 FALSESYNE1 chr6_152702311_G_C disease_causing 8 FALSE
DNAH14chr1_225393676_T_A chr1_225231636_G_T chr1_225270424_A_T
polymorphism;p.F1972Y disease_causing
polymorphism;p.N1104Y
3 7 8
TRUE
SACS chr13_23906739_G_A disease_causing; p.T3759M 8 TRUE
CPT1B chr22_51014487_C_T disease_causing; p.V252M 3 TRUE DMWD chr19_46294291_T_G disease_causing 3 FALSE
RRM1chr11_4154851_T_C chr11_4144575_C_A
disease_causing; p.M6555T disease_causing; p.N427K
7 8
TRUE
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TOPOISOMERASE 3A (TOP3A)• Maintaining genome integrity,
through the resolution of DNA replication and recombination intermediates (Holliday junctions)
• Shown to be crucial for Drosophila and Arabidopsis cell viability and normal development (Wu, Feng et al. 2010;Hartung, Suer et al. 2008), also involved in mtDNA depletion in Drosophila (Wu, Fenget al. 2010)
• Able to localize both in the nucleus and mitochondria (Wang 2002)
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Patient5 chr17_18211681_T_C_ENST00000412083
Patient45 chr17_18211681_T_C_ENST00000412083
TOP3A was the preferred candidate for sporadic cases (compound heterozygous changes in patient 5).
Screening for the presence of the 3 changes found from exome sequencing revealed the presence of one of them in patient 45 (p.M100V)
That same change was not found in any of the 102 regionally- and ethnically-matched controls (204 chromosomes)
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Reference ID Position in chromosomeRegion in
gene
rs17805992 g.386C>G;Chr17_18217903 intron
rs7212337 c.331G>A;Chr17_18217958 CDS
rs 6502645 g.23927G>A;Chr17_18194362 intron
rs7213789 g.29574G>A;Chr17_18188715 intron
rs7207123 g.9745C>T;Chr17_18208544 intron
rs2294913 g.15293G>A;Chr17_18202996 intron
rs2230154 c.1723C>T;Chr17_18193941 CDS
rs3817992 g.24278G>T;Chr17_18194011 intron
rs6502644 g.34278C>A;Chr17_18184011 intron
rs140837737 c.3016C>T;Chr17_18180996 CDS
Sequencing all of the gene’s exons in a panel of 19 clinically well-characterized patients did not indicate the existence of any further variants
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Conclusions• Exome sequencing identified novel sequence variants in RRM1
and TOP3A
• Conventional Sanger sequencing did not reveal the presence of any further variants, expect for the p.M100V mutation in TOP3A (patient 5,45)
• Patient 45 is a sporadic case, thus autosomal recessive inheritance is expected (compound heterozygote changes). No new variants were detected, apart from the p.M100V one
• The p.M100V change did not appear in any of the 102 regionally-and ethnically-matched controls
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Future work• Sequence the remaining patients in the panel for TOP3A
• Revise the gene list
Discussion• The control group size is still small, since the p.M100V change
could be a polymorphism with low frequency• Patient 7 was subsequently diagnosed with Spinocerebellar ataxia
type 28, hence the RRM1 variant is unlikely to be of significance• Possible reasons for missing out the disease gene(variants):• Lack of family data• Stringent filtering criteria• Low call rates • Coverage of each gene
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References• Baruffini, E., T. Lodi, et al. (2006). "Genetic and chemical rescue of the Saccharomyces cerevisiae phenotype
induced by mitochondrial DNA polymerase mutations associated with progressive external ophthalmoplegia in humans." Human Molecular Genetics 15(19): 2846-2855.
• Copeland, W. C. (2008). Inherited mitochondrial diseases of DNA replication. 59: 131-146.
• Gorman, G. S. and R. W. Taylor (2011). "Mitochondrial DNA abnormalities in ophthalmological disease." Saudi Journal of Ophthalmology 25(4): 395-404.
• Hartung, F., S. Suer, et al. (2008). "Topoisomerase 3α and RMI1 Suppress Somatic Crossovers and Are Essential for Resolution of Meiotic Recombination Intermediates in <italic>Arabidopsis thaliana</italic>." PLoS Genet 4(12): e1000285.
• Hutchinson, J. (1879). "On Ophthalmoplegia Externa, or Symmetrical Immobility (partial) of the Eyes, with Ptosis." Med Chir Trans 62: 307-329.
• Kim, J. S., C. J. Kim, et al. (1989). "Chronic progressive external ophthalmoplegia (CPEO) with 'ragged red fibers': a case report." J Korean Med Sci 4(2): 91-96.
• Singleton, A. B. (2011). "Exome sequencing: a transformative technology." The Lancet Neurology 10(10): 942-946.
• Taylor, R. W. and D. M. Turnbull (2005). "Mitochondrial DNA mutations in human disease." Nat Rev Genet6(5): 389-402.
• Thelander, L. (2007). "Ribonucleotide reductase and mitochondrial DNA synthesis." Nat Genet 39(6): 703-704.
• Wang, J. C. (2002). "Cellular roles of DNA topoisomerases: a molecular perspective." Nat Rev Mol Cell Biol3(6): 430-440.
• Wu, J., L. Feng, et al. (2010). "Drosophila topo IIIα is required for the maintenance of mitochondrial genome and male germ-line stem cells." Proceedings of the National Academy of Sciences 107(14): 6228-6233.
• Yang, J., C. Z. Bachrati, et al. (2010). "Human Topoisomerase IIIα Is a Single-stranded DNA Decatenase That Is Stimulated by BLM and RMI1." Journal of Biological Chemistry 285(28): 21426-21436.
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Acknowledgments
Professor Patrick Chinnery
Professor Robert Taylor
• Dr. Gerald Pfeffer
• Dr. Angela Pyle
• Dr. Gavin Hudson
• Dr. Helen Griffin
• Dr. Grainne Gorman
• Mrs. Tania Smertenko
• Everyone in PFC lab