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

Jan 23, 2018

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Page 1: Alexia Chrysostomou (083707160)

Identification of new genes in adult-onset

mitochondrial diseasesMRes Project 2012

Alexia Chrysostomou

(083707160)

Page 2: 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

Page 3: Alexia Chrysostomou (083707160)

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)

Page 4: Alexia Chrysostomou (083707160)

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

Page 5: Alexia Chrysostomou (083707160)

• 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

Page 6: Alexia Chrysostomou (083707160)

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

Page 7: Alexia Chrysostomou (083707160)

Exome sequencing

Page 8: Alexia Chrysostomou (083707160)

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

Page 9: Alexia Chrysostomou (083707160)

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) √

Page 10: Alexia Chrysostomou (083707160)

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

Page 11: Alexia Chrysostomou (083707160)

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

Page 12: Alexia Chrysostomou (083707160)

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:

Page 13: Alexia Chrysostomou (083707160)

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

Page 14: Alexia Chrysostomou (083707160)

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)

Page 15: Alexia Chrysostomou (083707160)

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)

Page 16: Alexia Chrysostomou (083707160)

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

Page 17: Alexia Chrysostomou (083707160)

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

Page 18: Alexia Chrysostomou (083707160)

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

Page 19: Alexia Chrysostomou (083707160)

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.

Page 20: Alexia Chrysostomou (083707160)

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