Identification of new genes in adult-onset mitochondrial diseases MRes Project 2012 Alexia Chrysostomou (083707160)
Identification of new genes in adult-onset
mitochondrial diseasesMRes Project 2012
Alexia Chrysostomou
(083707160)
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
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)
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
• 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
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
Exome sequencing
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
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) √
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
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
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:
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
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)
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)
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
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
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
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.
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