1. Identification of new genes
in adult-onset
mitochondrial diseases
MRes Project 2012
Alexia Chrysostomou
(083707160)

2.  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

3. 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)

4. 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

5. • 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

6. 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;NOS
PEO;
Ataxia
PEO;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

7. Exome sequencing

8. 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

9. Methodology Lane
1
Lane
5
Lane
6
Lane
3
Lane
7
Lane
8
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) √

10. Results
Gene
symbol
Variant 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
PANK2
chr20_3870334_T_C
chr20_3869911_T_G
polymorphism
polymorphism
1 FALSE
RARS2 chr6_88239365_C_T disease_causing; p.R258H 8 TRUE
TOP3A
chr17_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
NDUFV2
chr18_9104204_G__C_ins
chr18_9122529_G_A
NMD;p.H4P
polymorphism; p.V110I
8 TRUE
SUOX chr12_56398455_G_A disease_causing 3 FALSE
SYNE1 chr6_152702311_G_C disease_causing 8 FALSE
DNAH14
chr1_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
RRM1
chr11_4154851_T_C
chr11_4144575_C_A
disease_causing; p.M6555T
disease_causing; p.N427K
7
8
TRUE

11. 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

12. 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:

13. Gene
symbol
Variant 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
PANK2
chr20_3870334_T_C
chr20_3869911_T_G
polymorphism
polymorphism
1 FALSE
RARS2 chr6_88239365_C_T disease_causing; p.R258H 8 TRUE
TOP3A
chr17_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
NDUFV2
chr18_9104204_G__C_ins
chr18_9122529_G_A
NMD;p.H4P
polymorphism; p.V110I
8 TRUE
SUOX chr12_56398455_G_A disease_causing 3 FALSE
SYNE1 chr6_152702311_G_C disease_causing 8 FALSE
DNAH14
chr1_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
RRM1
chr11_4154851_T_C
chr11_4144575_C_A
disease_causing; p.M6555T
disease_causing; p.N427K
7
8
TRUE

14. 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, Feng
et al. 2010)
• Able to localize both in the nucleus
and mitochondria (Wang 2002)

15. 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)

16. Reference ID Position in chromosome
Region 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

17. 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

18. 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

19. 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 Genet
6(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 Biol
3(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.

20. 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