D 1220

OULU 2013

UNIV ER S IT Y OF OULU P. O. B R[ 00 FI-90014 UNIVERSITY OF OULU FINLAND

U N I V E R S I TAT I S

S E R I E S

SCIENTIAE RERUM NATURALIUM Professor Esa Hohtola

HUMANIORA University Lecturer Santeri Palviainen

TECHNICA Postdoctoral research fellow Sanna Taskila

MEDICA Professor Olli Vuolteenaho

SCIENTIAE RERUM SOCIALIUM University Lecturer Hannu Heikkinen

ACTA

MITOCHONDRIAL DNA SEQUENCE VARIATION IN FINNISH PATIENTS WITH MATERNALLY INHERITED TYPE 2 DIABETES, EPILEPSY AND MITOCHONDRIAL DISEASE: RISK AND NOVEL MUTATIONS

SCRIPTA ACADEMICA Director Sinikka Eskelinen

OECONOMICA Professor Jari Juga

EDITOR IN CHIEF Professor Olli Vuolteenaho PUBLICATIONS EDITOR Publications Editor Kirsti Nurkkala ISBN 978-952-62-0293-8 (Paperback) ISBN 978-952-62-0294-5 (PDF) ISSN 0355-3221 (Print) ISSN 1796-2234 (Online)

U N I V E R S I T AT I S O U L U E N S I S

Heidi Soini

E D I T O R S

Heidi Soini

A B C D E F G

O U L U E N S I S

ACTA

A C TA

D 1220

UNIVERSITY OF OULU GRADUATE SCHOOL; UNIVERSITY OF OULU, FACULTY OF MEDICINE, INSTITUTE OF CLINICAL MEDICINE, DEPARTMENT OF NEUROLOGY; OULU UNIVERSITY HOSPITAL, MEDICAL RESEARCH CENTER OULU

D

MEDICA

ACTA UNIVERSITATIS OULUENSIS

D Medica 1220

HEIDI SOINI

MITOCHONDRIAL DNA SEQUENCE VARIATION IN FINNISH PATIENTS WITH MATERNALLY INHERITED TYPE 2 DIABETES, EPILEPSY AND MITOCHONDRIAL DISEASE: RISK AND NOVEL MUTATIONS

Academic Dissertation to be presented with the assent of the Doctoral Training Committee of Health and Biosciences of the University of Oulu for public defence in Auditorium 1 of Oulu University Hospital, on 5 December 2013, at 12 noon

U N I VE R S I T Y O F O U L U , O U L U 2 0 1 3

Copyright © 2013 Acta Univ. Oul. D 1220, 2013

Supervised by Professor Kari Majamaa

Reviewed by Professor Antonio Salas Professor Wolfram Kunz

ISBN 978-952-62-0293-8 (Paperback) ISBN 978-952-62-0294-5 (PDF) ISSN 0355-3221 (Printed) ISSN 1796-2234 (Online)

Cover Design Raimo Ahonen

JUVENES PRINT TAMPERE 2013

Soini, Heidi, Mitochondrial DNA sequence variation in Finnish patients with maternally inherited type 2 diabetes, epilepsy and mitochondrial disease: risk and novel mutations. University of Oulu Graduate School; University of Oulu, Faculty of Medicine, Institute of Clinical Medicine, Department of Neurology; Oulu University Hospital, Medical Research Center Oulu Acta Univ. Oul. D 1220, 2013

Abstract Cellular energy is produced by the mitochondria via oxidative phosphorylation. In addition to nuclear DNA; the mitochondrion contains circular mitochondrial DNA (mtDNA) molecules. MtDNA is maternally inherited and encodes 37 genes that are crucial for the energy production of the cell. Mutations in the mtDNA cause mitochondrial diseases that manifest as maternally inherited energy metabolism disorders. Common symptoms include diabetes mellitus, myopathy, sensorineural hearing impairment, eye and vision problems, epilepsy and brain manifestations (encephalopathy). Mitochondrial mutations are often heteroplasmic; cells and tissues contain a mix of healthy and mutated mtDNA. The percentage of mutated mtDNA contributes to the severity of symptoms. Mitochondrial DNA also contains numerous polymorphisms; some of which have been reported to be non-neutral, thus contributing to the occurrence of common diseases. Whole mtDNA sequences were obtained from patients with diabetes mellitus (64), epilepsy (79) and unknown mitochondrial disease (66) using conformation-sensitive gel electrophoresis and direct sequencing. Whole mtDNA sequences of a Finnish family with ataxia were also obtained. Restriction fragment length analysis and cloning were used for heteroplasmy quantification. Whole mitochondrial genomes were organized into phylogenetic trees. All nonsynonymous mutations were analyzed with pathogenicity predicting algorithms (SNAP, PolyPhen-2, PMut, SIFT Blink). Non-neutral risk mutations were identified in diabetes mellitus and epilepsy patients. These patients had maternal relatives with diabetes, epilepsy and/or sensorineural hearing impairment. M. 3010A>G and m.16189T>C were found in increased frequency in diabetics and the haplogroup U5b variant m.15218A>G was detected more often among patients with epilepsy. These mutations were predicted to be deleterious in effect. Mitochondrial haplogroup V was found in increased frequency in matrilineal diabetes mellitus patients. We identified an m.8993T>C mutation in a Finnish family with ataxia. This mutation caused an adult-onset ataxic phenotype; previous studies have reported only juvenile onset phenotypes. Novel and rare mtDNA mutations were discovered in patients with an unspecified mitochondrial disease phenotype; these included an insertion m.7585insT and a novel MTTT mutation m. 15933G>A. This thesis emphasizes the importance of full mtDNA sequencing in patients with a suspected mitochondrial disease; novel mutations remain undetected if only the most common mutations are screened. In addition, the increasing importance of non-neutral mtDNA risk variants is supported by the findings of this thesis. In the future, individualized genetics and information on personal risk alleles will become even more important for maintaining health on a personal level. Keywords: diabetes mellitus, epilepsy, haplogroup, mitochondria, mitochondrial disease, mitochondrial DNA, mtDNA, nonsynonymous mutation, pathogenicity prediction, phylogeny, risk mutation

Soini, Heidi, Mitokondriaalisen DNA:n muutokset maternaalisesti periytyvää diabetesta, epilepsiaa ja mitokondriotautia sairastavilla potilailla. Oulun yliopiston tutkijakoulu; Oulun yliopisto, Lääketieteellinen tiedekunta, Kliinisen lääketieteen laitos, Neurologia; Oulun yliopistollinen sairaala, Medical Research Center Oulu Acta Univ. Oul. D 1220, 2013

Tiivistelmä Mitokondriot ovat energiaa tuottavia soluelimiä. Mitokondrioissa on oma rengasmainen mitokondriaalinen DNA (mtDNA), joka esiintyy solussa useana kopiona. MtDNA periytyy vain äidin kautta, joten kaikille lapsille periytyy sama mitokondriaalinen DNA. MtDNA koodaa 37:ää geeniä, jotka ovat tärkeitä solun energiantuotannolle. Geenimuutos mtDNA:ssa voi aiheuttaa äidiltä periytyvän mitokondriotaudin. Mitokondriotaudit ovat energia-aineenvaihdunnan sairauksia, joissa tavallisia oireita ovat diabetes mellitus, lihasoireet (esimerkiksi lihasten ennenaikainen väsymys, myopatia), sydänlihasoireet, maksaoireet, silmä- ja näköoireet, aistimistyyppinen kuulovika sekä aivo-oireet, kuten epilepsia. Oireet vaihtelevat huomattavasti, ja sama mutaatio voi aiheuttaa hyvin erilaisia taudinkuvia. Vakavimmillaan mitokondriotauti voi johtaa kuolemaan jo varhaislapsuudessa. Mutaation prosenttiosuus eli heteroplasmia-aste on usein oireiden vakavuutta määrittelevä tekijä. Mitokondriaalinen DNA muuntuu nopeasti evoluution aikana, joten siinä esiintyy paljon normaalia vaihtelua (polymorfioita). Osa näistä polymorfioista on kuitenkin todettu lievästi haitallisiksi, ja ne lisäävät riskiä sairastua kansanterveydellisesti yleisiin sairauksiin, kuten diabetekseen. Kartoitimme koko mitokondriogenomin muutokset eri potilasryhmiltä, joihin kuului diabetesta, epilepsiaa ja ataksiaa sairastavia potilaita. Lisäksi tutkittiin potilaita, joilla epäiltiin mitokondriotautia. Keskeiset käytetyt menetelmät olivat DNA:n rakenteellisia muutoksia havaitseva geelielektroforeesi ja sekvensointi. Määritimme heteroplasmian käyttäen restriktioentsyymianalyysia sekä kloonausta bakteerisoluihin. Järjestimme potilaiden mtDNA-sekvenssit fylogeneettisiksi puiksi ja kaikki proteiinin koodausta muuttavat geenimuutokset analysoimme haitallisuutta ennustavilla tietokoneohjelmilla (SNAP, PolyPhen-2, PMut, SIFT BLink). Diabetesta sairastavilla potilailla, joilla myös äidinpuoleisessa suvussa esiintyy diabetesta, havaitsimme useammin m.3010A>G- ja m.16189T>C-geenimuutoksia kuin väestöllä keskimäärin. Tutkimustulos tukee aikaisemmin julkaistuja tutkimustuloksia m.16189T>C-geenimuutoksen haitallisuudesta. Epilepsiapotilailta löytyi m.15218A>G-geenimuutos kahdessa U5a1-haploryhmän alatyypissä. Patogeenisyysanalyysien mukaan nämä geenimuutokset olivat haitallisia. Mitokondriaalinen haploryhmä V havaittiin useammin diabetesta sairastavilla kuin terveillä henkilöillä. Tutkimukseen valittiin potilaita, joiden äidinpuoleisilla lähisukulaisilla esiintyi yhtä tai useampaa seuraavista: diabetes, epilepsia tai aistimistyyppinen kuulovika. Väitöstutkimuksessa todetaan lisäksi, että m.8993T>C-mutaatio aiheuttaa aikuisiällä alkavaa ataksia-oireistoa. Kyseinen mutaatio on aiemmin yhdistetty vain lapsuusiän taudinkuviin. Kuvasimme uuden insertiomutaation (m.7585insT) kardiomyopatiasuvussa sekä uuden MTTT-geenin mutaation (m.15933G>A) tuntematonta mitokondriotautia sairastavalla potilaalla. Väitöskirjatutkimuksen tulokset osoittavat, että on tärkeää tutkia koko mitokondriogenomi, kun kyseessä on tuntemattomaksi jäänyt mitokondriaalinen taudinkuva. Uudet, tautia aiheuttavat, mtDNA:n geenimuutokset voivat jäädä tunnistamatta, jos tutkitaan ainoastaan raportoidut, tunnetut, mutaatiot. Lisäksi voi todeta, että mitokondriogenomissa esiintyy lievästi haitallisia geenimuutoksia tai niiden yhdistelmiä, jotka saattavat lisätä riskiä sairastua kansanterveydellisesti merkittäviin sairauksiin, kuten diabetekseen. Asiasanat: aistimistyyppinen kuulovika, ataksia, diabetes, epilepsia, fylogenia, haitallinen polymorfia, mitokondriaalinen DNA, mitokondrio, mitokondriotauti, mtDNA

To my Mother

8

Acknowledgements My sincere gratitude goes to my supervisor Kari Majamaa, whose inspiring guidance has made this thesis possible. Thank you for taking me inside the world of mitochondrial genetics and sharing your expertise, you are a wonderful teacher. Thank you for your vision, patience and understanding throughout this project. It has been a priviledge to complete this thesis under your guidance. My fellow research group members, I could not have done this without you! We have such a special athmosphere amongst us; it’s been a joy working with all of you! Huge thanks for my dear friends Anna-Leena, Johanna A-R, Reetta, Johanna K, Sanna, Johanna U, Anna-Kaisa, Anri, Laura and everyone else! Thank you for your loving support. You’ve picked me up on dark days and shared my joy on the good days! Pirjo and Anja, you’ve helped me so much, thank you for being the heart and spirit of our lab. It’s been a pleasure working with all the people of CRC; the lovely athmosphere in and outside the coffee room was always very supporting. I am grateful for all my collaborators for their expert contribution to my work. Thank you Billi for welcoming me to your lab in New York! You are the nicest man in New York for sure! Sara, my sincere thanks for taking me under your wing. Thank you Yin-Mei and Jorida for all our lovely lunch talks. Sindu and Orhan, I am grateful for all your help and support. Thank you professors Antonio Salas and Wolfran Kunz for the careful pre-examination of my thesis. Thank you Ryan Warner and Hermanni Honkimäki for the skilled English and Finnish language proofreading of my thesis. I’d like to express my deepest gratitude towards my family. You carried me through this. I cannot thank you enough! You all know how much I love you. I wish to thank my friends near and far, you are so special to me. Dearest Janne, thank you for all our adventures. On to the next, my love! Eemeli, Aatu and Emma, you are the light of my life! This thesis was supported by grants from the Graduate School of Population Genetics, the Sigrid Juselius Foundation, the University of Oulu and Oulu University Hospital. This study was carried out between 2002 and 2013 at the Department of Neurology, University of Oulu and the Clinical Research Center, Oulu University Hospital.

9

“Time is a keyhole, he thought as he looked up at the stars. Yes, I think so. We sometimes bend and peer through it. And the wind we feel on our cheeks when we do – the wind that blows through the keyhole – is the breath of all the living universe.” – Stephen King Love, always Heidi

10

Abbreviations ADP ANT1 ATP bp CNS CPEO CSGE del D-loop DM DUI indel ins KSS LHON LS MCRA MELAS MERFF mRNA mtDNA NADH NARP OXPHOS PCR PEO POLG1 RFLP RITOLS ROS SNHI TP tRNA

adenosine diphosphate gene encoding ATP/ADP translocator adenosine triphosphate base pair central nervous system Chronic external ophtalmoplegia Conformation sensitive gel electrophoresis deletion of nucleotide(s) displacement loop, mtDNA non-coding control region Diabetes mellitus doubly uniparental inheritance of mitochondrial DNA insertion or deletion mutation insertion mutation, insertion of extra nucleotide Kearns-Sayre syndrome Leber’s hereditary optic neuropathy Leigh syndrome most common recent ancestor mitochondrial encephalopathy, lactic acidosis and stroke-like episodes myoclonus epilepsy with ragged-red fibers messenger RNA mitochondrial DNA NADH dehydrogenase Neurogenic ataxia, retinitis pigmentosa and polyneuropathy oxidative phosphorylation polymerase chain reaction progressive external ophtalmoplegia polymerase gamma subunit 1 encoding gene Restriction fragment length polymorphism ribonucleotide incorporation throughout the lagging strand reactive oxygen species sensorineural hearing impairment Gene encoding thymidine phosphorylase transfer RNA 11

12

List of original papers This thesis is based on the following articles, which are referred to in the text by their Roman numerals: I

Rantamäki M, Soini HK, Finnilä SM, Majamaa K & Udd B (2005) Adult-onset ataxia caused by mitochondrial 8993T>C mutation. Ann Neurol 58: 337–340. II Soini HK, Moilanen JS, Finnilä S & Majamaa K (2012) Mitochondrial DNA sequence variation in Finnish patients with matrilineal diabetes mellitus. BMC Res Notes 5: 350. III Soini HK, Moilanen JS, Vilmi-Kerälä T, Finnilä S & Majamaa K (2013) Mitochondrial DNA variant m.15218A>G in Finnish epilepsy patients who have maternal relatives with epilepsy, sensorineural hearing impairment or diabetes. BMC Med Genetics 14: 73. IV Soini HK, Väisänen A, Kärppä M, Hinttala R, Kytövuori L, Uusimaa J & Majamaa K (2013) Analysis of mitochondrial DNA sequence variation in 66 patients with suspected mitochondrial disease reveals a novel pathogenic m.7585insT mutation in a family with cardiomyopathy. Manuscript.

13

14

Contents Abstract Tiivistelmä Acknowledgements 9 Abbreviations 11 List of original papers 13 Introduction 19 1 Review of the literature 21 1.1 Mitochondria ........................................................................................... 21 1.1.1 Endosymbiotic theory and evolution ............................................ 21 1.1.2 Structure ....................................................................................... 22 1.1.3 Oxidative phosphorylation and the many functions of the mitochondria................................................................................. 24 1.1.4 Other functions of the mitochondria ............................................. 27 1.2 Mitochondrial genome ............................................................................ 28 1.2.1 Mitochondrial sequence organization and conservation among species .............................................................................. 30 1.2.2 Maternal inheritance, heteroplasmy and the bottleneck effect ............................................................................................. 31 1.2.3 Replication of mtDNA ................................................................. 34 1.2.4 High mutation rate and the neutral theory .................................... 35 1.2.5 Molecular clock and phylogenetic applications of mtDNA ......... 37 1.3 Mitochondrial DNA variation in human populations .............................. 38 1.3.1 Benign polymorphisms and haplotypes ........................................ 38 1.3.2 The origin of modern humans ...................................................... 38 1.3.3 Haplogroups around the world ..................................................... 40 1.3.4 Finnish haplotypes and regional haplotype variation inside Finland.......................................................................................... 41 1.4 Mitochondrial diseases; syndromes, signs and symptoms ...................... 42 1.4.1 Point mutations ............................................................................. 44 1.4.2 Neuropathy, ataxia, retinitis pigmentosa (NARP) ........................ 44 1.4.3 MELAS ........................................................................................ 45 1.4.4 CPEO, LHON and eye symptoms of mitochondrial diseases ......................................................................................... 46 1.4.5 Frameshift mutations in mtDNA .................................................. 47 1.4.6 Multiple mtDNA deletions ........................................................... 47 15

1.4.7 Mitochondrial diseases caused by nuclear defects ....................... 48 1.4.8 Epilepsy in mitochondrial syndromes .......................................... 49 1.5 Mitochondrial DNA variation as a risk factor for disease ....................... 51 1.5.1 Risk haplogroups .......................................................................... 51 1.5.2 Predisposing mitochondrial mutations in diabetes mellitus.......... 52 1.6 Detection and analysis of novel mtDNA mutations in undetermined mitochondrial diseases ..................................................... 54 1.6.1 Functional assays for proving pathogenicity of novel mtDNA mutations......................................................................... 55 1.6.2 Prediction of the functional effect of nonsynonymous mtDNA mutations......................................................................... 56 2 Aims of the present study 59 3 Patients and methods 61 3.1 Patients (I-IV).......................................................................................... 61 3.2 Population controls (II-IV) ...................................................................... 62 3.3 Molecular methods .................................................................................. 62 3.3.1 DNA extraction (I-IV) .................................................................. 62 3.3.2 Haplogroup analysis (I – IV) ........................................................ 63 3.3.3 PCR and conformation sensitive gel electrophoresis (CSGE) (I-IV)............................................................................... 63 3.3.4 Sequencing (I-IV) ......................................................................... 64 3.3.5 Mutation confirmation and heteroplasmy analysis (I-IV) ............ 64 3.3.6 Multiple deletion detection (IV) ................................................... 65 3.4 Phylogenetic networks and statistical analyses (II-IV) ........................... 66 3.5 In silico analysis of nonsynonymous mtDNA mutations (III-IV) ........... 66 3.5.1 PolyPhen-2 ................................................................................... 66 3.5.2 SIFT BLink ................................................................................... 67 3.5.3 PMut ............................................................................................. 67 3.5.4 SNAP ............................................................................................ 67 4 Results 69 4.1 Haplogroup frequencies (II-IV) .............................................................. 69 4.2 Sequence variation and phylogenetic structure of mtDNA (II, III, IV) ........................................................................................................... 69 4.2.1 Heteroplasmic polymorphisms (I - IV)......................................... 71 4.2.2 Rare mtDNA variants (III) ............................................................ 71 4.3 Pathogenic and novel mutations (I – IV)................................................. 71 4.3.1 m.8993T>C (I) .............................................................................. 71 16

4.3.2 m.7585insT (IV) ........................................................................... 72 4.3.3 Novel tRNA and nonsynonymous mtDNA mutations (II, IV) ................................................................................................ 72 4.3.4 Novel synonymous mtDNA variants (II, III, IV) ......................... 73 4.4 Previously reported disease-associated mtDNA variants (II, III, IV) ........................................................................................................... 73 4.5 Prediction of the pathogenic effect of mtDNA mutations (II-IV) ........... 73 4.6 Mildly deleterious mutations (II, III) ...................................................... 75 4.6.1 m.15218A>G (III) ........................................................................ 75 4.6.2 m.16189T>C and m.3010G>A (II) ............................................... 75 4.7 Multiple deletions (IV)............................................................................ 76 4.7.1 POLG1 mutations (IV) ................................................................. 76 5 Discussion 77 5.1 Haplogroup V is more common in patients with maternally inherited diabetes mellitus in northern Finland ....................................... 77 5.1.1 Several disease-associated mutations detected in patients with matrilineal diabetes mellitus or matrilineal epilepsy ............ 77 5.2 Adult-onset ataxia in a family harbouring the heteroplasmic m.8993T>C mutation .............................................................................. 79 5.3 Novel heteroplasmic m.7585insT causes cardiomyopathy in a family with untimely deaths due to cardiac disease ................................ 80 5.4 Novel homoplasmic m.15933G>A in MTTT in a patient with ptosis ....................................................................................................... 81 5.5 Mildly deleterious mtDNA variants occur in patients with matrilineal diabetes or epilepsy as well as in population controls .......... 82 5.5.1 The mildly deleterious variant m.15218A>G in haplotype U5a1 occurs more often in patients with maternally inherited epilepsy ......................................................................... 83 5.5.2 The combination of m.16189T>C and m.3010G>A in haplotype H1b is more frequent among maternally inherited diabetes mellitus patients .............................................. 84 5.6 Multiple mtDNA deletions and mutations in POLG1 patients with suspected mitochondrial disease ..................................................... 85 6 Conclusions 87 References 89 Original papers 111 17

18

Introduction The mitochondrion is a crucial cellular organelle which transforms energy acquired from nutrients into adenosine triphosphate (ATP) molecules that can be used for the energy needs of the cell. The mitochondrion also participates in multiple tasks and metabolic pathways inside the cell that range from calcium homeostasis to programmed cell death. The mitochondrion has its own independent circular genome that is ~16 600 bp long. Mitochondrial DNA (mtDNA) encodes 37 genes that produce aerobic energy through an oxidative phosphorylation (OXPHOS) system (Kurland & Andersson 2000). Mutations in mtDNA can lead to mitochondrial diseases (a group of energy metabolism diseases with varying severity). Mitochondrial diseases often manifest in neurological symptoms, highlighting the extensive energy needs of the nervous system. Multi-organ systemic symptoms are common, such as diabetes mellitus, myopathy, cardiomyopathy, epilepsy, encephalomyopathy, lactic acidosis and eye symptoms such as ptosis. The most prevalent mitochondrial diseases include MELAS syndrome (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes), Leigh syndrome, NARP syndrome (neuropathy, ataxia and retinitis pigmentosa) and MERFF (myoclonic epilepsy with ragged-red-fibers) (Chinnery et al. 2012, DiMauro & Schon 2003, Schaefer et al. 2008, Schon et al. 2012). MtDNA variants have been observed to influence the function of the respiratory chain, both beneficially and adversely. These variants have been reported to raise the risk of certain diseases and modulate the phenotype of mitochondrial, viral and metabolic diseases (Achilli et al. 2011, Autere et al. 2004, Cho et al. 2011, Hendrickson et al. 2008, Khusnutdinova et al. 2008, Mueller et al. 2011, Palmieri et al. 2011, Park et al. 2008). Common multifactorial diseases, such as diabetes mellitus, have both a genetic and an environmental aspect to their etiology. It has been suggested that mildly deleterious mutations in mtDNA can raise the risk of degenerative diseases by compromising the function of the mitochondria, thus making it less effective and more prone to oxidation by the mitochondrial by-product reactive oxygen species (ROS) (Achilli et al. 2011, Federico et al. 2012, Kofler et al. 2009, Pichaud et al. 2012, Santoro et al. 2010). Detecting such deleterious variants is often difficult as they are also present in the population. Also, complex diseases are affected by several factors, such as nuclear gene background, nutrition and lifestyle (Liou et al. 2007, Prentice 2006). 19

These deleterious variants most likely vary from population to population (Breen & Kondrashov 2010) and this has to be taken into account.

20

1

Review of the literature

1.1

Mitochondria

Mitochondria are the batteries of a cell, producing most of the aerobic energy through oxidative phosphorylation (OXPHOS). These cytoplastic organelles are most abundant in tissues with high energy demands such as the brain, skeletal and cardiac muscle. According to the endosymbiotic theory, the mitochondrion originates from aerobic alpha-proteobacteria that were merged into a primordial eukaryotic cell about two billion years ago (Kurland & Andersson 2000). Recent studies have determined that this mitochondrial ancestor shares features of the Rickettsia order of intracellular parasitic bacteria (Thrash et al. 2011). The mitochondrion forms networks of varying shapes and sizes. Every cell has several mitochondria and the number of mitochondria depends on the energy requirements of the cell type in question. The mitochondrion has a doublestranded circular DNA molecule that is about 16 000 bp in size and together with nuclear mitochondria-targeted genes, it codes for the genes that are needed in oxidative phosphorylation. MtDNA is maternally inherited and independently replicated and translated. The mitochondrial genome is very compact; no introns are situated between genes. A non-coding control region, the D-loop or displacement loop, controls the replication and translation of the mitochondrial genome. Several copies of the mtDNA molecule exist in a single cell. The mitochondrial genome has a higher mutation rate than nuclear DNA because the mitochondrial genetic code is more flexible for the third base of the codon (Fonseca et al. 2012) and because spontaneous replication mistakes create point mutations. This steady mutation rate can be used as a phylogenetic tool that assesses divergence times between species and mtDNA haplogroup lineages. Mitochondrion has an active role not only in energy production but in other cellular functions, like calcium homeostasis, cell differentiation, cell death and cell cycle. 1.1.1 Endosymbiotic theory and evolution Two billion years ago in the Earth’s history, the level of atmospheric oxygen started to increase. This sudden rise in oxygen concentration forced early eukaryotic cells to adapt to aerobic energy production. This took place when an 21

oceanic α-proteobacterium entered the primordial eukaryotic cell to become a symbiotic cell organelle that produced energy from oxygen. The divergence of mitochondria from bacteria took place 1.5 to 2 billion years ago (Kurland & Andersson 2000). This endosymbiotic event in mitochondrial evolution is supposed to be very similar to how chloroplast evolved from cyanobacteria in plant cells (Nakayama & Archibald 2012). Most of the ancient α-proteobacterium genes have long since disappeared from the mitochondrial genome. Genes that were not essential to eukaryotic cytosolic life have been transferred to the nuclear genome, thus reducing the number of genes in mtDNA. Redundant genes were also deleted by purifying selection and random mutation during the course of evolution. A portion of the ancient bacterial genes have been replaced by non-orthologous genes with a similar function (Huynen et al. 2012). It has been proposed that the transferred genes that were embedded in nDNA form spliceosomal introns. Spliceosomal introns are segments of non-coding DNA that are spliced from pre-mRNA. These spliceosomal introns are only found in eukaryotes. Non-coding intron segments were added to the transferred mitochondrial genes after their introduction to nDNA (Ahmadinejad et al. 2010). Moreover, nuclear originated subunits were added to mitochondrial oxidative phosphorylation complexes, thus making them more complex than their ancestral analogues. It has been suggested that these additional subunits were responsible for balancing the accumulation of mutations in the bacterial ancestral genome (Gabaldon & Huynen 2007). The endosymbiotic addition of mitochondria to eukaryotic cells played a crucial role in eukaryotic evolution. Through multiple evolutionary steps, the mitochondrion has evolved into effective energy-producing machinery; nuclear and mitochondrial genomes function fluently together (Gabaldon & Huynen 2007, Huynen et al. 2012). 1.1.2 Structure Mitochondria are double-membrane cell organelles that form networks inside the cell. Depending on the energy needs of the cell, mitochondria are either in fusion or fission; this process happens continuously. This mitochondrial reticulum interacts with the cytoskeleton and the endoplasmic reticulum. The mitochondrion is composed of an outer membrane, inner boundary membrane, intermembrane space, cristal membranes, intracristal space and matrix (Fig. 1). 22

The cristal membrane forms cristae, which are folds inside the interboundary membrane. They are tubular in shape and are connected to the inner boundary membrane via crista junctions. ATP synthase particles and ribosomes are situated on the cristal membrane. The folded cristal membranes provide a larger surface area for oxidative phosphorylation (Frey & Mannella 2000, Logan 2006). The matrix space between the inner boundary membrane and the cristal membranes holds the multiple copies of mitochondrial DNA molecules and stores NAD, NADH, ATP and ADP molecules (Logan 2006). The number of crista junctions and the form of the intercristal space changes according to the metabolic state of the mitochondria (Mannella et al. 1997). Large intercristal spaces have been linked to a reduction in ATP production and a condensation of cristae that creates high levels of ATP energy production (Mannella 2006). Contact sites are special regions where the outer and inner membranes are in close proximity. Sometimes a bridge-like structure is formed to keep the membranes apart while maintaining a required close distance. Solutes and small molecules pass between these contact sites from the cytosol into the matrix. It is also suggested that pre-cursor proteins are bound to these sites (Schwaiger et al. 1987).

23

Fig. 1. Structure of the mitochondria.

1.1.3 Oxidative phosphorylation and the many functions of the mitochondria The main function of the mitochondria is to provide a constant level of ATP molecules to be used in cell functions. This aerobic energy formation is also called oxidative phosphorylation (OXPHOS). Other functions of the mitochondria include membrane potential maintenance, calcium homeostasis, heat production, cell death (apoptosis), intercellular signalling, cell differentiation and cell growth. The mitochondrion also participates in heme and steroid synthesis (McBride et al. 2006). Oxidative phosphorylation takes place on the inner membrane of the mitochondria. It is a very efficient way of producing energy in oxygen rich environments and is present in every eukaryotic organism from the simplest life forms to humans. Five enzymatic complexes (complexes I-V) carry out the redox reactions needed for converting energy from food to ATP. Enzyme complexes are a collaboration of nuclear and mitochondrial encoded subunits that come together 24

to form supercomplexes. Eukaryotes have five enzyme complexes. Subsequently, some prokaryotes have different enzymes and varying electron donors and acceptors. In oxidative phosphorylation (Fig. 2), electrons are transferred from electron donors to electron acceptors that release energy. The energy is then used to pump hydrogen protons into the intermembrane space via enzyme compslexes (I-IV). The citric acid cycle produces NADH and succinate substrates for the redox reactions. A large rotating ATP synthase protein (complex V) uses protons pumped into the intermembrane space for energy and pumps them back into the matrix. Complex V performs the final step in oxidative phosphorylation. It converts ADP molecules into ATP molecules that are ready to be used for the cells energy needs. As a byproduct of phosphorylation, reactive oxygen species (ROS) is formed. This comes in the form of peroxides and oxygen ions (Chandra & Singh 2011, Ylikallio & Suomalainen 2012). These molecules are very reaction sensitive and they easily convert to free radicals. ROS are harmful to the cells because they damage DNA, proteins and lipids. High amounts of ROS can be a disease causing mechanism for mitochondrial diseases and can also contribute to normal aging. However, it has been proposed that moderate amounts are important as a promoter of mitochondrial biogenesis and as a signaling molecule in cell differentiation (Valero et al. 2012). Cells also contain ROS counteracting agents (antioxidant vitamin C) and antioxidant enzymes (peroxidases). These minimize the damaging effect of ROS. Increasing age changes the efficiency of mitochondrial function. In cellular deterioration, mitochondria are especially vulnerable because they produce ROS as a side product and because they require constant biogenesis. Once the phosphorylation process starts to decline, increasing amounts of ROS are produced. Increased amounts of DNA damage and multiple deletions in mtDNA are commonly seen in aging tissues. Also, mitochondrial mass starts to decline and mitochondrial biogenesis becomes slower. An increase in mtDNA mutations is observed in aging cells (Peterson et al. 2012).

25

Fig. 2. Mitochondrial DNA and oxidative phosphorylation. a) MtDNA organization b) Oxidative phosphorylation complexes. Reprinted with permission from Macmillan Publishers Ltd: Nature Reviews Genetics (Schon et al. 2012).

26

1.1.4 Other functions of the mitochondria The mitochondrion also participates in programmed cell death (apoptosis). Cells that are no longer needed are programmed to be disposed. Two pathways lead to apoptosis: extrinsic, where the death stimulus becomes from outside the cell and intrinsic, where the signal for apoptosis comes from within the cell. The mitochondrion controls apoptosis in an intrinsic way. DNA damage, intracellular stress signals, oncogenes, hypoxia and so called death receptor stimuli starts the apoptosis process. The mitochondria will tell the cell to commence programmed cell death through many signaling caspases. Apoptosis removes the cell cleanly and recycles particles, whereas in necrosis, an inflammatory process takes place (Galluzzi et al. 2012). Heat production in brown fat tissue is a mitochondrial process because proton flow to the matrix is used for heat production, but no ATP molecules are produced. This is called non-shivering thermogenesis and it is most active in newborn babies and hibernating animals (Kitao & Hashimoto 2012). In humans, the amount of brown fat decreases with age. In small, cold climate adapted mammals, a drop in ambient temperature switches on non-shivering thermogenesis to increase body temperature (Zhang et al. 2012). The mitochondrion regulates calcium homeostasis (the mitochondrion functions as a deposit for Ca2+ ions). It can rapidly uptake calcium ions from the cytosol to stimulate calcium-sensitive dehydrogenases to regulate ATP synthesis to fit the needs of the cell. Calcium is also used for intracellular signaling (Raffaello et al. 2012, Rizzuto et al. 2012). Cell differentiation is controlled by the mitochondria. Several apoptosis factors that regulate programmed cell death also control cell differentiation. The mitochondrion regulates the self-renewal capacity of neural stem cells and mitochondrial involvement is an essential part of neuron differentiation, axon and dendrite growth and the organization of synapses (Sola et al. 2012). Heme and certain types of steroid synthesis are also linked to mitochondria. It has been proposed that oxygen carrying globins, such as hemoglobin (that contains a heme-group) in vertebrates and globins in plants, have evolved through mitochondrial and chloroplast endosymbiosis. Globins have highly conserved regions that are similar across a range of organisms. For example, from simple bacteria to mammals (Vazquez-Limon et al. 2012). A heme precursor is found in the mitochondria and this is where heme synthesis starts. Intermediate synthesis stages take place in the cytoplasm whilst the final stage, the formation of heme, 27

takes place in the mitochondria. Iron-sulphur cluster (Fe-S) biosynthesis is also a mitochondrial process (Lill et al. 2012). Like in heme synthesis, the precursor for steroids is manufactured in the mitochondria. Cytosolic cholesterol is transported to the mitochondria inner membrane where it is processed to form pregnenolone, a steroid precursor. The rest of the steroid biosynthesis takes place in the endoplasmic reticulum. Mitochondrial fusion is an important phenomenon in steroid biogenesis. An SHP2 phosphatase that is required for full steroid synthesis simultaneously modulates mitochondrial fusion (Duarte et al. 2012). 1.2

Mitochondrial genome

The mitochondria has an independent genome. This genome is a circular doublestranded DNA molecule that is 16 569 bp long in humans, but the length varies from 15 000 to 17 000 bp in other mammals. It is a compact molecule that lacks introns (non-coding areas in between genes that are seen in nuclear DNA). There are multiple copies of the genome inside the cell and there are anywhere from 100 to 10 000 copies per cell. It is most abundant in cells with high energy needs like neurons and muscle cells. Heteroplasmy occurs when a cell contains several types of mtDNA; a portion of mtDNA molecules harbouring a mutation/polymorphism and a portion of mtDNA molecules lacking it. Heteroplasmy is an important phenomenon. This is especially true for mitochondrial diseases where a certain threshold (percentage of mutated mtDNA) is often observed in association with disease symptom manifestation. MtDNA molecules can exist as monomeric circular molecules or as supercoiled circles. Two or more circular molecules can be connected to form catenanes. Catenanes are circles crossing each other at certain points. Also, two mtDNA molecules can be attached to form a head-to-tail dimer where two mtDNA molecules form a larger circle. Human heart mitochondria are most often organized in multiple mtDNA molecular catenanes, although in other tissues open-circular and supercoiled circular catenanes are the most common molecule form (Pohjoismäki & Goffart 2011). The two strands of the mtDNA molecule are called the heavy strand and the light strand. The heavy strand sequence is rich in guanine and the light strand contains large amounts of cytosine. A noncoding area, the D-loop (displacement loop), contains regulatory elements for replication and spans the region m.16024– 576. Mitochondrial DNA encodes a total of 37 genes, 13 of which encode the respiratory chain protein subunits that are needed in oxidative phosphorylation. 28

Two ribosomal RNA genes (MTRNR1 and MTRNR2) and 22 transfer RNA genes are encoded by genes in the mitochondrial DNA (Fig. 2) (DiMauro & Schon 2003, Ruiz-Pesini et al. 2007). The genetic code is mostly universal compared to nuclear DNA, but there are differences in the stop-codons and the codon for methionine. In nuclear DNA, the stop codons are UAA, UAG and UGA. In mitochondrial DNA, two additional stop codons exist (AGG and AGA). Emerging evidence has shown that human mitoribosomes invoke a -1 frameshift when alternate termination codons AGG/AGA in the mRNA template are encountered. The codon is re-positioned so that a UAG/UAA/UGA termination codon is created, after which the mtRF1a termination factor can recognize the stop codon UAG/UAA/UGA. MtRF1a continues to terminate the translation and detaches the newly synthesized polypeptide from the ribosome (Lightowlers & Chrzanowska-Lightowlers 2010, Soleimanpour-Lichaei et al. 2007, Temperley et al. 2010). Also, the nuclear stop codon UGA codes for tryptophan in mtDNA. The codon AUA codes for methionine in mitochondrial DNA and isoleucine in nuclear DNA. In the animal world, the mitochondrial genetic code also varies. For example, AAA codes for asparagine in Echinoderms and Platyhelminthes (sea stars, sea urchins, sea snails) and tRNA’s are imported from the cytoplasm in some species of the Cnidaria (jellyfish) phylym (Watanabe & Yokobori 2011). The medusozoan cnidarias, for example the winged box jellyfish (Alatina moseri), have linear ~ 16 000 bp long mtDNA molecules which are sometimes fragmented into several 4 000 to 8 000 bp long ‘chromosomes’. These ‘mitochondrial chromosomes’ have telomeres on both ends and inverted repeat sequences that sometimes contain pseudogenes and duplicate genes (Smith et al. 2012). Additionally, some yeast species (for example Candida parapilosis) have linear mtDNA (Rycovska et al. 2004). The biosynthesis of mitochondria requires collaboration between the nuclear and mitochondrial genes. For example, the polymerase gamma enzyme that is needed for mtDNA replication is encoded by the POLG-gene in the nuclear genome. During evolution, some mitochondrial genes have been transported to the nuclear genome and this phenomenon is still observed in nuclearmitochondrial pseudogenes. These are non-functional mtDNA sequence blocks that are integrated into nuclear DNA (Yao et al. 2008). Several interesting features can be seen in mitochondrial DNA. In particular, it is maternally inherited and male mitochondria are disintegrated after fertilization (Giles et al. 1980). Maternal inheritance allows for maternal lineage 29

tracking through many generations, therefore making mtDNA a powerful tool not only in population genetics and evolutionary studies, but in forensic science and genetic genealogy. Additionally, because of its steady faster mutation rate, divergence times can be calculated for the evolutionary differentiation events of speciation (molecular clock). 1.2.1 Mitochondrial sequence organization and conservation among species Large size differences in mitochondrial genomes can be seen between fungal, animal and plant mitochondria. In general, animal mtDNA is the most compact, approximately 16 kb in length throughout the animal world, while plant mtDNA is the longest, ranging from 200 up to 2500 kb in length. Fungal mtDNA length is in between animal and plant mtDNA size; fungal mtDNA ranges from 19 to 176 kb in length. The Atlantic comb jelly fish (Mnemiopsis leidyi) carries the smallest animal mtDNA. Its mtDNA is only about 10 kb in length (Pett et al. 2011). While the genes encoded in the mitochondrial DNA are similar, their organization and transcription varies from species to species. The size of the noncoding regions also displays a lot of variation. It has been suggested that endothermic animals like mammals, which are capable of producing their own body heat internally, have smaller mitochondrial genomes compared to ectothermic species. Ectothermic animals, e.g. reptiles, rely on environmental heat to maintain their body temperature and harbour larger mtDNA. The length of the mitochondrial genome also varies among mammalian species. In humans it is 16 567 bp long, while the bilby (Macrotis lagotis), a small Australian marsupial, has one of the smallest mammalian mitochondrial genomes (only 15 289 bp long). Larger mammalian mtDNAs are found in for example the European hare (Lepus europeansis); it has an mtDNA length of 17 734 bp (Rand 1993). Sequence conservation among mammals is an important tool in assessing the possible pathogenic effects of mitochondrial mutations in human disease. Many amino acids are very conserved with little or no variation among species. These amino acids are key parts of the protein in question and any changes in them can cause drastic damage to protein function. On the other hand, many amino acids are not conserved and show variation at the species level. These are considered to be functionally unimportant polymorphisms (Putz et al. 2007). The rate of evolution in mammalian mitochondrial DNA (nonsynonymous changes) is four to five times faster than in fish for example. Among mammalian species, primate 30

mtDNA evolves faster than any other order; baboons and orangutans being the fastest species with regards to nonsynonymous mtDNA evolution (Gissi et al. 2000). Most of the insect species encode the same 22 tRNA molecules, the same 13 protein encoding genes and the same rRNA molecules (small and large) that are common to the animal world. Melinopona bicolor (a stingless bee species) only encodes 19 tRNAs, Drosophila melanogaster encodes an extra protein coding gene in their mtDNA (Garesse 1988) and Caenorhabditis elegans (a parasitic nematode worm) lacks the MTATP8 gene altogether (Rea et al. 2010). 1.2.2 Maternal inheritance, heteroplasmy and the bottleneck effect Mitochondrial DNA is inherited almost exclusively from the mother. As a sperm cell enters the egg, its mitochondria-rich tail is left outside the outer membrane of the egg and the other paternal mitochondria are disposed of through mitophagy during early embryogenesis (Ding & Yin 2012). This way, most of the cytoplasm is inherited from the mother and along with it, the mitochondria. Mitochondrial DNA is therefore identical in a grandmother, mother and her children (Fig. 3).

Fig. 3. Inheritance of heteroplasmy.

In mitochondrial diseases, a mother with a pathogenic mutation passes on the mutated mtDNA to all her offspring. Since all paternal mitochondria from the 31

oocyte are discarded, a father with an mtDNA mutation does not pass on the mutation to his offspring. Pathogenic mtDNA mutations are often heteroplasmic and the mutation occurs in different quantities in different cells and tissues (Chalkia 2009). A threshold effect can be detected in many mitochondrial diseases. This means that a certain percentage of mutated mtDNA is needed for the symptoms to manifest. During egg development, each egg gets a random mixture of mtDNA molecules. Some eggs might get a high amount of mutated mtDNA and some eggs only get a small percentage of the mutation. This phenomenon is based on random genetic drift. During embryogenesis, when the primordial egg cells are developed, the amount of mtDNA molecules is temporarily reduced radically from tens of thousands to about 200 mtDNA molecules. This so called mitochondrial bottleneck determines the mitochondrial DNA mixture (population) that is then proliferated in a mature oocyte and eventually passed on to the child (Chalkia 2009, Payne et al. 2012). Predicting the disease severity in the offspring of an mtDNA mutation carrier mother is very difficult. The child can either inherit a high percentage of the mitochondrial mutation and develop a very serious mitochondrial disease, or the child can be symptom free if only a low percentage of the mutation has been passed on to the child (Fig. 4) (Chinnery et al. 2012). There is debate about whether the mtDNA mixture is divided randomly during oocyte development, or if certain mtDNA molecules are favoured through selection (Raap et al. 2012). Also, the strictly maternal inheritance of mitochondrial DNA has led to speculation about an increased male mutation load of age-related diseases (Camus et al. 2012). This could also partly explain why males in many species have a shorter lifespan (Wolff & Gemmell 2012). According to a recent ultra-deep sequencing study, very low heteroplasmy (0.2 to 2 %) is found in every healthy person; this was more common than originally suspected. These benign heteroplasmic variants were both somatic and inherited (Payne et al. 2012).

32

Fig. 4. Inheritance patterns of human hereditary diseases.

Non-maternal inheritance of mtDNA A different type of mitochondrial inheritance is observed in species like the saltwater mussel genus Mytiloida, saltwater clams (Veneroida) and freshwater 33

Unioneida mussels. In these species, both maternal and paternal mitochondria are inherited by the offspring. This phenomenon is called doubly uniparental inheritance (DUI) of mitochondria. Females are homoplasmic for their mtDNA, whereas males are heteroplasmic for their maternally inherited (F-mtDNA) and paternally inherited (M-mtDNA) mtDNA. These mtDNA molecules are a part of the sex determination process of an embryo (Breton et al. 2007). Despite the strictly maternal inheritance of mtDNA in mammals, a few reports of paternal mtDNA inheritance exist. A severe myopathy and exercise intolerance disease phenotype was reported to be caused by a paternally inherited 2 bp mtDNA deletion (Schwartz & Vissing 2003). Paternal inheritance is thought to be extremely rare among mammals, but it has been reported in sheep (Ovis aries) (Zhao et al. 2001) and mice (Gyllensten et al. 1991). In birds, paternal mtDNA inheritance was first detected in the great tit (Parus major) (Kvist et al. 2003) and is observed in plant mtDNA inheritance, for example in California redwood trees (Sequoia sempervirens) (Neale et al. 1989). In insects, paternal inheritance patterns of mtDNA have been reported in Drosophila simulans (Wolff et al. 2012), the honeybee (Apis mellifera L.) (Meusel & Moritz 1993) and is especially common in hybrid crosses between insect species (Arunkumar et al. 2006, Fontaine et al. 2007). 1.2.3 Replication of mtDNA Human mitochondrial DNA has two origins of replication, one for each strand of the mtDNA. The origin for heavy strand replication is situated in the D-loop (position m.110-441) and the origin for light strand replication is located in between tRNA asparagine and tRNA cysteine at position m.5721–5798. The transcription of mtDNA can proceed in both directions of the genome and open reading frames exist on both the heavy strand and the light strand; open reading frames are usually on the heavy strand. The non-coding region contains promoters for both strands (HSP and LSP), conserved regions for replication regulation and replication termination associated sequence (TAS). A third mtDNA strand, the so called 7S DNA (D-loop) sometimes occurs hybridized in the light strand. It is thought to be a replicated strand that starts from the origin of heavy strand replication and ends in the termination associated sequence. Individual mtDNA molecules are replicated randomly; some may replicate more than others and some do not replicate at all. Usually the number of mtDNA molecules inside the cell remains constant. Thus after cell division, an equal 34

number of mtDNA molecules exist in both daughter cells. The total number of mtDNA molecules in the cell is regulated according to the energy requirements of the cell (Kasamatsu et al. 1974). Three mitochondrial DNA replication mechanisms are explained below (the existence of all three is often debated). The first DNA replication mechanism is the strand-displacement model and it was first introduced in 1972 (Robberson et al. 1972). In this model, replication is initiated at the origin of heavy strand replication and an RNA primer is transcribed in the L-strand promoter. The primer is elongated by DNA polymerase γ which forms a larger loop structure. The lagging strand is meanwhile laid down as RNA. The lagging strand synthesis starts when the leading strand reaches the origin of light strand replication (OL). It is then continued until it is terminated at the termination associated region (Shadel & Clayton 1997). The second model of mtDNA replication is bi-directional; both strands are synthesized simultaneously (Bowmaker et al. 2003, Holt & Reyes 2012). No single-stranded RNA intermediates are produced like in the previous mode of replication. When the first replication fork of the replication loop structure reaches the termination associated region, the replication continues only in one direction until a whole double stranded mtDNA molecule is replicated. The replication of the lagging strand occurs simultaneously as short DNA fragments (Okazagi fragments) are joined to form a continuous DNA strand. The third model of mtDNA replication is the RITOLS model (Yasukawa et al. 2006). RNA molecules 200–600 nucleotides in length (RITOLS) are incorporated into the lagging strand and are later replaced or converted into DNA when the replication proceeds to OL (Holt & Reyes 2012, Yasukawa et al. 2006). Very little is still known about the regulation of mtDNA replication. All of the proteins that are needed for the replication of mtDNA are encoded in the nuclear genome. For example, POLG1, polymerase γ, the Twinkle helicase and the single-stranded DNA-binding protein (mtSSB) (Brown et al. 2005, Pohjoismäki & Goffart 2011). 1.2.4 High mutation rate and the neutral theory Mitochondrial DNA changes very rapidly compared to nuclear DNA even though mtDNA does not recombine like nuclear DNA. Benign polymorphisms sculpt sequence variation on the individual and species genome level. The mitochondrial DNA substitution rate has been calculated to be 0.02 substitutions per base for every million years. This is approximately ten times higher than in nuclear DNA 35

(Brown et al. 1979, Vawter & Brown 1986). Extremely fast evolving mtDNA has been reported in the Mnemiopsis leidyi (Atlantic comb jelly fish or sea walnut). This Atlantic comb jelly fish also holds the record for the smallest mtDNA in an animal (Pett et al. 2011). The higher mutation rate is due to oxidative lesions caused by ROS and intrinsic replication errors (Kujoth et al. 2007). The turnover rate of mtDNA is much faster compared to nuclear DNA and thus allows more chances for mutations to occur during replication (Miwa et al. 2008). Also, the third position of mitochondrial tRNA molecules has more tolerance for variation. This is called the wobble position of tRNA (Fonseca et al. 2012). Oxidative lesions due to reactive oxygen species in mtDNA also cause mutations (Kujoth et al. 2007). In mitochondrial DNA, all mutations are expected to be neutral so that no selection advantages or disadvantages exist between mutations (Kimura 1987). The neutral theory of evolution (Kimura 1979) was first suggested in the 1970’s. It states that random genetic drift is the driving force of molecular evolution and argues that all DNA variants are neutral and do not affect the fitness of the species. Adaptive mutations would therefore constitute only a small fraction of molecular changes and as a result, most changes would be silent. Random genetic drift is affected by the effective size of the population. The effects of random genetic drift are magnified in smaller populations. On a cellular level, the effective size of the mtDNA ‘population’ inside a cell can have a big impact on which mutations and mtDNA molecules become fixed and which ones disappear from the mtDNA during cell divisions. The somatic expansion of mtDNA mutations that occur in early life can play an important role in mitochondrial diseases (Mishmar & Zhidkov 2010). An increase in mutations due to the damaging effect of ROS on mtDNA is a common process in normal aging (Kujoth et al. 2007). Non-neutral mtDNA variation Mildly deleterious or advantageous mutations have been reported to exist in mtDNA. The m.150C>T polymorphism has been associated with living to an old age and has been found in abundance in Finnish and Japanese centenarians (Niemi et al. 2003, Niemi et al. 2005). The haplotypes in which this polymorphism occurs have been recently associated with a lower ROS production rate (Chen et al. 2012). Mildly deleterious mtDNA mutations and mitochondrial genome background have been reported to increase the risk for type 2 diabetes (Achilli et al. 2011, Park et al. 2008, Poulton et al. 2002a), Parkinson’s disease 36

(Autere et al. 2004, Coskun et al. 2012), sensorineural hearing loss (Finnilä et al. 2001, Lu et al. 2010a) and age-dependent hearing loss (Mutai et al. 2011). Since mitochondrial and nuclear proteins co-exist in molecular ‘symbiosis’, it has been suggested that they would also evolve as joint units. It has been proposed that most of the non-neutral variation is in between populations, as purifying selection inside the population would quickly eradicate any harmful variants. It has also been suggested that nuclear-mitochondrial interactions maintain variants that behave in a non-neutral way (Dowling et al. 2007, Dowling et al. 2008, Rand et al. 2001). 1.2.5 Molecular clock and phylogenetic applications of mtDNA Since mtDNA mutates at a predicted rate, it can be used for calculating divergence times between species and populations. The molecular clock theory states that changes in proteins occur at a similar rate in time (Zuckerkandl & Pauling 1965, Zuckerkandl 1987). This theory has opened up a multitude of applications for determining the divergence times for species, the phylogeographic differences between populations and the origins of species; even modern humans. Traditionally in animal research, the control region and MTCYTB sequences are often used to calculate phylogenetic distances. The cytochrome B gene evolves at quite a fast rate and it harbours many synonymous changes. This makes it ideal for depicting differences between populations and establishing haplogroups. It has very conserved regions too, so sometimes only a part of the MTCYB sequence is used. But it has its pitfalls too; phylogenetic trees with low resolution have been observed in certain species like the Cetacea family of dolphins (Duchene et al. 2011). In human population genetics, the use of control region mtDNA sequences was more common in the past, whereas today whole mtDNA sequences are increasingly easy to attain for both humans and animal species. By attaining whole mtDNA sequences, drawing more detailed and accurate phylogenetic trees has become possible. Mitochondrial DNA sequences are an important tool for paleobiologists (McMenamin & Hadly 2012) and forensic scientists. It is more likely to be preserved in fossils and decomposing remains where it is found in larger amounts compared to nuclear DNA. It is also more stable and resistant to degradation and provides evidence of maternal lineage (Butler & Levin 1998). Therefore, it is also used for maternity testing (e.g. in cases where children are separated from their 37

birthmothers due to adoption or other circumstances) and genetic genealogy purposes (Moreau et al. 2011). 1.3

Mitochondrial DNA variation in human populations

1.3.1 Benign polymorphisms and haplotypes The fast evolution rate of mitochondrial DNA means that mtDNA harbours a high amount of variation. Most of the variants are benign synonymous changes that do not affect the protein structure. The non-coding region harbours particularly frequent mtDNA variation compared to the coding region. MtDNA variants are most often transitions (A>G, G>A, T>C, C>T), while transversions are less common (A>C, G>T, C>G etc.). Simple deletions and repeat sequence length variation is observed in the D-loop area. A 9 bp deletion spanning nucleotides m.8272 – m.8281 is common in the non-coding nucleotides in between MTCO2 and MTTK. Mini deletions of single base pairs are also common in the D-loop. Insertions are seen as repeat length variants in the non-coding region and ribosomal RNA genes; especially at positions m.303, m.310, m.518, m.595 and m.16183. These benign changes form patterns that share common ancestral mtDNA variants that are called haplogroups. Haplogroups consist of similar haplotypes; haplotypes are groups which harbour a similar pattern of polymorphisms. Variation in both humans and animals can be classified into haplotypes according to the mtDNA variants they harbour. This is done by comparing them to commonly used reference sequences. These phylogenetic branch points can be used to study migration, evolution, and the origins of populations (human or animal). 1.3.2 The origin of modern humans Based on mtDNA data, the earliest modern human populations inhabited the southeast or east African regions around 160 000 to 200 000 years ago (Watson et al. 1997). ‘Mitochondrial Eve’, the most common matrilineal recent ancestor of modern humans, was a female to whom all living modern humans can be traced back to on their mother’s side. The earliest human mtDNA haplogroup has been reported to be L0. It is most common in Sub-Saharan Africa today and it is the 38

closest mtDNA haplotype in regards to the most common recent ancestor (MCRA). The oldest of the L0 subhaplotypes is L0d. L0 is extremely abundant (73 %) in the Khoisan people (the Khoi and San tribes). This population is considered to be genetically closest to the most common recent ancestor of all modern-day humans. This finding is supported by evidence from both mitochondrial and Y-chromosome data (Batini et al. 2011, Behar et al. 2008). The L1-haplotype diverged from L0 and is the founding haplotype for all other haplotypes observed today (Fig. 6). L1 is separated from L0 by 18 mtDNA polymorphisms. All other present-day haplotypes are descendants of the L1 haplotype (Behar et al. 2012). Since ancient DNA sequencing techniques have become better, more Homo neanderthalensis sequences are being used as an outgroup in the phylogenetic trees of human mtDNA evolution. Previously, Bonobo chimpanzee (Pan paniscus) mtDNA was used because it is the closest genetic match to modern humans. The use of Neanderthal human mtDNA as an outgroup has fine-tuned the human mtDNA tree because Neanderthals are more closely related to modern humans (Homo sapiens) than any primate species present today. The split between the Neanderthal human and Homo sapiens is becoming clearer as more ancient human sequences become available. Early interbreeding events between early modern humans and Neanderthals have been reported; both subspecies inhabited the same region for 130 000 years (Neves & Serva 2012).

39

Fig. 5. Phylogenetic tree of the origin of human mtDNA haplogroups. HN denotes Homo neanderthalensis, MRCA denotes the most recent common ancestor of modern humans.

1.3.3 Haplogroups around the world While L-haplotype and its subhaplotypes prevail in the African continent, each continent has its typical mtDNA haplotypes. Through the migration of ancient human populations, mtDNA diverged into various haplotypes with their special set of defining polymorphisms. The European haplotypes are H, V, U, K, T, J, W, I and X. Haplotypes N, M and R are prominent in the Far East and D, F, Z, G and Y are prominent in Asia. Native Americans harbour the A, B, C, D and X haplotypes, as do South American Indians (Baeta et al. 2012). But X2a and X2g are North American specific (Reidla et al. 2003). Haplotype B is also common in Pacific Island populations and in New Zealand’s Maori people (Knapp et al. 2012). Australian aboriginals harbour the N and M haplotypes (van Holst Pellekaan et al. 2006). Haplotype Z is observed in people of Russian descent and in the Saami people of Scandinavia; it is also found in central Asia, Korea and northern China (van Oven & Kayser 2009). 40

1.3.4 Finnish haplotypes and regional haplotype variation inside Finland There has been much debate about how Finns settled Finland in the thousands of years after the last glacial era. Linguistically, Finns belong to the Fenno-Ugric group of people that originate from the Volga-Ural region (Kittles et al. 1998). In genetic studies however, the genes of Finns have Y-chromosomal Scandinavian influence; this is especially true in the southern and western parts of Finland (Palo et al. 2009). Most studies agree that Finland was populated first in the coastal and river valley regions of southern and western Finland. Northern and eastern Finland were settled later (Norio 2003b). Several small waves of European settlers came from the west (‘Battle-axe people’) and southeastern Europe (‘Comb-ware people’) about 5000 years ago. In addition, small groups of Baltic, Scandinavian and German people immigrated to Finland (Norio 2003b). Geographic, linguistic and cultural isolation have sculpted the genetic landscape of the Finnish people. It is suggested that a genetic bottleneck happened approximately around the time when early agriculture arrived in Finland (Kere 2001). The founder effect, due to small settler groups with little intermixing, has reduced genetic variation of Finns and made around 40 diseases more common in Finland (Finnish disease heritage) (Norio 2003a, Norio 2003b). The frequency of these rare alleles increased as the population grew rapidly during the 1800s (Kere 2001). The European haplotypes H, V, U, K, J, T, W, I and X are the most common in the Finnish population. A subtle haplotype gradient can be seen inside Finland. The frequency of haplotypes H, J and I increase towards central and southern Finland, whereas haplotypes U, V, T and M are more prevalent in the north of Finland. Haplotypes V (37 %), U5b1b1 (41 %), H and D5 are reported in the Saami people of Sweden, Norway and Finland. The U5b1b1 haplotype contains the so called ‘Saami motif’. The ‘Saami motif’ is a combination of the mtDNA mutations m.16144, m.16189, and m.16270 (found in the HVR-1 region); it is a special feature of the Saami U5b1b1-subhaplotype. The frequency of the U5 haplotype is twice as high in Finland when compared to rest of Europe (Ingman & Gyllensten 2007, Meinilä et al. 2001). Similarities in the mtDNA of Finns, Saami people and southern European Basques have been reported (Torroni et al. 1998, Torroni et al. 2001). According to one theory, haplotype V could be a remnant of the paleolithic Cro-Magnon

41

hunters who might have been pushed to the remote parts of Europe by the rapidly spreading Indo-European speaking people (Barbujani et al. 1998). Haplotype Z exists in both Finns and the indigenous Saami people of the north (Meinilä et al. 2001a). Haplotype Z represents the Volga-Uralic contribution to the mtDNA gene pool of Finland. D5 is an East Asian haplotype and it is also present in the Saami population. These Asian mtDNA lineages are observed in low frequencies among the Saami. Both Z and D5 are absent in the rest of Europe (Ingman & Gyllensten 2007). It has been suggested that the occurrence of haplotype H among the Saami is a more recent admixture with the European population; H is the most common haplotype in Europe. For example as you move further north in Sweden, a gradual decrease in the frequency of haplotype H has been reported in Swedish Saami population. This points towards more admixture with European population in the south compared to the north (Ingman & Gyllensten 2007). Admixture between the Finnish and Saami population has also been observed (especially in the north of Finland). However, Saami haplotypes gradually fade as you move south to areas where European influence increases (Ingman & Gyllensten 2007, Meinilä et al. 2001). 1.4

Mitochondrial diseases; syndromes, signs and symptoms

Mitochondrial diseases are a clinically variable group of progressive diseases that affect the energy production and function of the mitochondria. Mitochondrial diseases are more common than first presumed: the prevalence has been estimated to be about 11.5/100 000 persons (adults and children) (Chinnery 1993, Schaefer et al. 2008). About 1/200 adults carries a pathogenic mtDNA mutation (Chinnery et al. 2012, Elliott et al. 2008). The prevalence of MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) has been calculated to be 5.7/100 000 in northern Finland (Majamaa et al. 1998). Mutations in mitochondrial and nuclear genes affecting mitochondrial function cause mitochondrial disorders. Multiple organs are usually affected. Common features include myopathy, diabetes mellitus, encephalopathy, cardiomyopathy, neuropathy, ptosis, external ophthalmoplegia, epilepsy, ataxia, sensorineural hearing impairment (SNHI), dementia, stroke-like episodes and exercise intolerance. The liver and kidneys can be also affected. In Leber’s hereditary optic neuropathy (LHON), the eyes are the main affected organ, but 42

additional neurological manifestations can occur in ‘Leber’s plus’ syndrome (Nikoskelainen et al. 1995). Mitochondrial diseases can be transmitted maternally (mtDNA mutations) or the autosomal recessive or autosomal dominant way (mitochondrial defects due to nuclear mutations). Diagnosing mitochondrial diseases is often challenging, unless the symptoms are well defined as part of a specific mitochondrial mutation. A good example of this is Leigh syndrome (LS). The most common mtDNA mutations can be easily screened for, but if the disease is caused by a rare or a novel mutation it is not as straight-forward. Whole mtDNA sequencing and nuclear exome sequencing is needed to diagnose and pinpoint the exact mutation when a novel mutation is presumed to be the cause of the disease. Functional biochemical testing is often needed to verify the pathogenic nature of the mutation before a definite association with the symptoms can be made. A detailed family history and the genetic testing of family members often takes place along with a large panel of medical tests. A respiratory chain enzyme activity test from a muscle biopsy is an important test for assessing the function of the respiratory chain (Chinnery et al. 2012, Pfeffer & Chinnery 2011). Symptom severity varies from mild (e.g. diabetes mellitus) to severe encephalopathy and intellectual disability. The severity and age of onset is highly correlated with mtDNA mutation heteroplasmy. Lower mtDNA mutation heteroplasmy is usually seen in the mother and a variable amount of heteroplasmy is observed in the children. A certain heteroplasmy threshold often must be reached for the symptoms of a mitochondrial disease to manifest. Genetic counselling is often difficult for mitochondrial diseases caused by mtDNA mutations because the mutation is inherited in random heteroplasmy levels. This makes it impossible to give a definite prognosis for children with a mitochondrial mutation. The m.8993T>C and m.8993T>G mutations are an exception because they are evenly spread in tissues and the heteroplasmy stays approximately the same over time. A prenatal test will show the mutation heteroplasmy level in the child for these mutations (White et al. 1999a, White et al. 1999b). Genetic testing is easier with nuclear defects because they are inherited in a predictable pattern. Also, mtDNA deletions can occur de novo where only a single family member is affected (Chinnery et al. 2012, DiMauro & Schon 2003). There is no cure for mitochondrial diseases, but the symptoms are treated with support and care (for example diabetes mellitus). Mitochondrial epilepsy is often difficult to treat and is less responsive to medication. Epileptic seizures cause cortical damage because of energy failure. The neuronal damage triggers 43

status epilepticus, a continuing seizure, which is common in mitochondrial diseases and is very difficult to control (Bindoff 2011). Coenzyme q10, creatine monohydrate, antioxidants and lipoic acid are some of the treatments being trialled for mitochondrial diseases (Pfeffer & Chinnery 2011). Gene therapy and embryo manipulation designed to prevent mtDNA mutation transmission may be possible in the future (Tachibana et al. 2012, Yabuuchi et al. 2012). 1.4.1 Point mutations The most common type of pathogenic mutation is a point mutation. Point mutations are usually transitions (and sometimes transversions). A large part of pathogenic mtDNA mutations are located in tRNA genes that are vital for the translation of the mitochondrial genome. More than 150 tRNA mutations have been reported so far (Ruiz-Pesini et al. 2007). The first one, the m.3243A>G MELAS mutation in the MTTL1 gene, was reported 22 years ago (Goto et al. 1990). Point mutations in rRNA genes are associated with SNHI; the m.1555A>G mutation in 12SrRNA is especially common (Prezant et al. 1993). Aminoglycoside use can trigger hearing loss in subjects harbouring m.1555A>G. This mutation is usually homoplasmic, but heteroplasmic forms have also been reported. The penetrance and clinical phenotype can vary and it has been suggested that additional modifier factors are involved in the pathogenesis of SNHI due to m.1555A>C mutation (Guan et al. 2001). All of the mitochondrial protein-encoding genes are reported to harbour pathogenic mutations. Clinical phenotypes and the mutations tend to vary quite a bit. A mutation in an evolutionary conserved base will cause a premature stop codon (or the rest of the gene will be coded wrong) and will eventually result in a non-functioning protein. 1.4.2 Neuropathy, ataxia, retinitis pigmentosa (NARP) Neurogenic muscle weakness, ataxia and retinitis pigmentosa (NARP) and Leigh syndrome are caused by mutations in the MTATP6 gene that encodes subunit 6 of ATPase (de Vries et al. 1993, Holt et al. 1990). The most common mutations are m.8993T>C (p.L156P) and m.8993T>G (p.L156R). Other Leigh syndrome causing pathogenic mutations in MTATP6 include m.9176T>C, m.9176T>G, 44

m.9185T>C and m.9191T>C (Carrozzo et al. 2000, Moslemi et al. 2005, Thyagarajan et al. 1995). Common symptoms of NARP usually start in early childhood. Neurological signs and symptoms include sensory neuropathy that causes numbness and pain in the extremities, muscle weakness, poor balance and ataxia and retinitis pigmentosa related vision deterioration. Night blindness is also common. Learning disabilities in children and dementia in adults are associated with NARP/Leigh mutations. Other features include hearing loss, epilepsy and cardiac symptoms. As with mitochondrial diseases in general, signs and symptoms vary tremendously among patients with the same mutation. Both the m.8993T>C and m.8993T>G mutations are usually heteroplasmic with the mother harbouring a smaller mutation load than the child. Mutation load correlates with disease severity. Compared to m.8993T>C mutation, the m.8993T>G mutation produces a more severe phenotype with an earlier onset. A particularly severe subtype of NARP syndrome is MILS (maternally inherited Leigh syndrome). It has been specifically identified in children with over 90% mutation load and it has an onset age of 3 to 12 months. 1.4.3 MELAS One of the most common mitochondrial diseases is MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes) (Goto et al. 1990, Pavlakis et al. 1984). Its prevalence is calculated to be 16.3/100 000 for adults (Majamaa et al. 1998) and 18.4/100 000 for children in northern Finland (Uusimaa et al. 2007). MELAS is usually caused by an m.3243A>G mutation in the MTTL1 gene. Some of the additional MELAS causing mutations include m.3243A>T, m.3244G>A, m.3252A>G, m.3256C>T, m.3258T>C, m.3271T>C and m.3291T>C; all of these mutations are in the same gene (Ruiz-Pesini et al. 2007). MELAS manifests in a number of different signs and symptoms that range from diabetes mellitus and hearing loss to severe encephalopathy. MELAS usually appears in childhood after a period of normal development. Muscle weakness, muscle pain, headaches, loss of appetite, vomiting and seizures are common symptoms. Diabetes mellitus and sensorineural hearing loss are often observed together. Lactic acid and creatine kinase also builds up in the blood. Stroke-like seizures are often the first sign of MELAS and the first episode is usually observed in childhood/young adulthood. Ataxia, short stature, migraines, 45

depression, intellectual disability, ophthalmoplegia and cardiomyopathy also belong to the wide spectrum of symptoms caused by MELAS. The heteroplasmy of the mutation strongly correlates with the severity of the symptoms. Mothers with less than ~ 50% heteroplasmy tend to have children with higher heteroplasmy, whereas children of high heteroplasmy mothers present with lower heteroplasmy (Uusimaa et al. 2007). 1.4.4 CPEO, LHON and eye symptoms of mitochondrial diseases Chronic progressive external ophthalmoplegia is a common eye condition caused by mitochondrial diseases. Progressive deterioration of the extraocular eye muscles causes bilateral ptosis and finally ophthalmoparesis (paralysis of eye muscles). Other common eye symptoms of mitochondrial diseases are diplopia, nystagmus, optic atrophy and pigmentary retinopathy. CPEO is the prevailing symptom in Kearns-Sayre syndrome (KSS), where the age of CPEO onset is before the age of 20. KSS is caused by large-scale mitochondrial deletions and duplications. The most common KSS deletion is a 4.9 kb deletion spanning nucleotides m.8469 – m.13147. Heteroplasmy levels correlate with disease severity. Additional symptoms of KSS are pigmentary retinopathy, cardiac conduction defects, elevated protein levels in cerebrospinal fluid and cerebellar defects. The adult-onset type of CPEO (CPEO plus) is often accompanied by multi-organ symptoms like myopathy and endocrine dysfunction (Pfeffer et al. 2011). Another mitochondrial disease that manifests in the eye is Leber’s hereditary optic neuropathy (LHON) (Wallace et al. 1988). This syndrome causes early onset (in the second or third decade of life) visual loss and is more often seen in males than in females. Retinal ganglion cells deteriorate and an acute loss of central vision or blindness is observed. It starts with one eye and progresses to both eyes over time. Mutations in MTND4 are particularly associated with LHON, but mutations in MTND1 and MTND6 have also been observed. All of the LHON mutations that have been reported cause changes in complex I amino acids. The penetrance of LHON mutations varies considerably. LHON mutations are usually homoplasmic (Kirches 2011). The penetrance of LHON is affected by certain secondary mutations. These mtDNA variants can increase the penetrance of LHON (Shu et al. 2012, Zhou et al. 2012). Haplotype J occurs with a high frequency among LHON patients. In particular, m.14484T>C and m.11778G>A in subhaplotypes J2b and Jc1 have 46

been linked to LHON (Cai et al. 2008, Huoponen et al. 1993, Kirches 2011). The sex-biased presentation could be linked to an X-chromosomal gene; a microsatellite marker has been associated with LHON in Finnish patients (Vilkki et al. 1991). 1.4.5 Frameshift mutations in mtDNA Insertion or deletion (indel) of a single or several nucleotides in a gene will disrupt the reading frame of the gene. The three base pair codons that are read in incorrect triplets and incorrect amino acids will be translated. A premature stop codon leads to a shorter protein or if a stop codon is skipped, a larger nonfunctional protein is translated. The functionality of the altered protein depends on the location of the indel in the gene. If the indel is at the beginning of the gene, a totally defective protein is being produced. Conversely, an indel at the end of the gene leads to a protein that may retain some activity. Frame shifting in bacterial and viral genomes can be used to translate overlapping open reading frames (van der Velden et al. 2013). Deletions and insertions in tRNA genes can disrupt the proper function of the tRNA, interfere with codon recognition etc. Insertions associated with deafness have been reported to occur in the MTRNR1 gene position m.960–961 (Ruiz-Pesini et al. 2007). Also, an m.3271delT deletion in MTTL1 has been reported to cause progressive encephalopathy (Shoffner et al. 1995), an m.5537insT insertion in MTTW was reported to cause Leigh syndrome (Santorelli et al. 1997) and a 5-base-pair deletion in MTCO1 m.6020_6025del has been reported to cause motor neuron disease (Comi et al. 1998). As a mutation type, pathogenic insertions in mtDNA are reported less often than mtDNA deletions (Ruiz-Pesini et al. 2007). Insertions frequently happen in the non-coding intergenic regions and in the D-loop (especially in the repeat sequences). An inversion mutation of 7 nucleotides has been reported in MTND1 by Musumeci et al. in 2000 (Musumeci et al. 2000). A duplication mutation of 4kb has also been previously reported (Tengan & Moraes 1998). 1.4.6 Multiple mtDNA deletions Large scale deletions occur when a substantial part of the mtDNA is deleted. Deletions present with variable heteroplasmy. Common deletion start points are repeat sequences or indirect repeats with a few mismatched nucleotides. It has 47

been suggested that replication stalling at these positions is the cause of mtDNA deletions (Wanrooij et al. 2004). Deletion junctions can occur at repeat boundaries or at a small distance from the repeat (Tanhauser & Laipis 1995). Mutations in the nuclear genes controlling mtDNA maintenance and synthesis, such as POLG (Komulainen et al. 2010), ANT1 (Kaukonen et al. 2000), C10orf2 (Twinkle-helicase) (Bohlega et al. 2009) and TP (Hirano et al. 2006) are often associated with the accumulation of multiple deletions (Naimi et al. 2006). As these disorders are not caused by mitochondrial genes, their inheritance is either autosomal recessive or autosomal dominant (Di Fonzo et al. 2003). Some syndromes associated with multiple deletions are KSS (Zeviani et al. 1998), PEO (Martikainen et al. 2012) and Pearson’s syndrome (Ayed et al. 2011). It has been suggested that mutations in POLG or C10orf2 can actually encourage the natural occurrence of multiple mtDNA deletions (Wanrooij et al. 2004). Multiple mtDNA deletions accumulate in postmitotic tissues like the brain during aging, but the proportion of deleted mtDNA is much smaller than in pathogenic mtDNA deletion syndromes like for example in KSS (Holt et al. 1989). The most common deletion is a 4977 bp deletion (‘common deletion’), but over 200 different age-related mtDNA deletions have been reported. The ‘common deletion’ m.8482_13460del removes parts of MTND5 and MTATP8 and completely eliminates MTND4, MTND4L, MTCO3, and MTND3 along with five tRNA genes (Meissner et al. 2008, Ruiz-Pesini et al. 2007b). It has been suggested that two 13 bp direct repeats at positions m.8470 and m.13447 would be the main contributors to human multiple mtDNA deletions (Samuels et al. 2004). Centenarians frequently lack the common deletion, thus providing further evidence of the role that multiple deletions play in the aging process (Guo et al. 2010). Multiple deletions can lead to permanent age-related mitochondrial dysfunction and can play a role in the degenerative diseases that affect the elderly (Bua et al. 2006, Gu et al. 2002, Yamasoba et al. 2007). 1.4.7 Mitochondrial diseases caused by nuclear defects Mitochondrial diseases with a Mendelian inheritance are caused by mutations in the nuclear mtDNA of the maintenance, structural, metabolic or import/transport genes for mitochondrial function (Angelini et al. 2009). These genes are responsible for the replication and maintenance of mtDNA, they form subunits of the oxidative phosphorylation complexes and they help with the assembly of the complexes. Defects in nuclear genes and the encoding factors of nuclear genes 48

that involve protein import and iron homeostasis also cause mitochondrial diseases. This happens along with the gene encoding factors that are related to mitochondrial integrity (e.g. mitochondrial fusion factors and mitochondrial RNA processing) (Table 1.) (Ruiz-Pesini et al. 2007). Table 1. Nuclear genes involved in mitochondrial disease. Gene

Function

Clinical phenotype

NDUFS1

Complex I structural

Leigh syndrome

NDUFS2

Complex I structural

Encephalopathy, cardiomyopathy

NDUFS3

Complex I structural

Leigh syndrome

NDUFS4

Complex I structural

Leigh syndrome

NDUFS7

Complex I structural

Leigh syndrome

NDUFS8

Complex I structural

Leigh syndrome

NDUFV1

Complex I structural

Leigh syndrome

NDUFV2

Complex I structural

Cardiomyopathy, encephalopathy, hypotonia

SDH-A

Complex II structural

Leigh syndrome

SDH-B

Complex II structural

Pheochromocytoma, paraganglioma

SDH-C

Complex II structural

Paraganglioma, autosomal dominant type 3

SDH-D

Complex II structural

Paraganglioma, autosomal dominant type 1

POLG

mtDNA replication

Alpers syndrome, progressive external opthalmoplegia

ANT1

Adenine nucleotide translocator Progressive external opthalmoplegia, multiple deletions

C10ORF2

Twinkle helicase

Progressive external opthalmoplegia, Sando syndrome

DGUOK

dNTP pool maintenance

Hepatocerebral mtDNA depletion syndrome

TK2

Thymidine kinase

Myopathic mtDNA depletion

FRDA

Iron-suphur cluster assembly

Friedreich ataxia, neuropathy, cardiomyopathy, diabetes

OPA1

Mitochondrial network

Optic atrophy

regulation MFN2

Mitochondrial network

Charcot-Marie-Tooth disease

regulation G4.5

Acyltransferase in lipid metab.

Barth syndrome

RMRP

Mitochondrial RNA processing

Metaphyseal chondrodysplasia, Cartilage-hair hypoplasia

MPV17

MtDNA copy number

Hepatocerebral mtDNA depletion syndrome

maintenance SUCLG1

Succinate-CoA ligase, α subunit Encephalomyopathy with methylmalonic aciduria

1.4.8 Epilepsy in mitochondrial syndromes Central nervous system (CNS) cells are highly differentiated cells that demand high amounts of energy and, therefore, this cell type depends heavily on properly functioning mitochondria. Respiratory chain dysfunction leads to lowered 49

amounts of ATP and this in turn will increase neuron excitability and decrease membrane potential. Mitochondrial dysfunction can also impair calcium homeostasis since intracellular calcium is stored in the mitochondria. All these factors make the neurons more susceptible to epileptic activity. Mitochondrial epilepsy is often progressive; seizures cause damage in the brain that can trigger future seizures more easily and eventually lead to longer status epilepticus. Stroke-like episodes are a common manifestation of mitochondrial diseases. This is most often seen in MELAS and POLG-mutation patients, whereas myoclonic epilepsy is most common the result of MERFF syndrome (Bindoff & Engelsen 2012). Typical mitochondrial epilepsy is a combination of different types of focal seizures: motor seizures, epilepsia partialis continua, bilateral convulsive seizures and myoclonic seizures (Bindoff & Engelsen 2012, Finsterer & Zarrouk Mahjoub 2012, Rahman 2012). Stroke-like episodes can cause stroke-like lesions (SLL) that can often form in the occipital and temporal lobes of the brain. These lesions can further aggravate the stroke-like episodes. Also, the seizures push the neuron’s energy demands even higher, which in turn can increase the risk of additional damage to the brain (Tzoulis et al. 2010). MERFF (myoclonic epilepsy with ragged red fibers) syndrome is a mitochondrial disorder that causes epilepsy. It is most commonly caused by m.8344A>G in the MTTK gene (Fukuhara et al. 1980, Rosing et al. 1985, Shoffner et al. 1990). The most prominent feature of MERFF is progressive myoclonic epilepsy. Other features include exercise intolerance, hearing loss, lactic acidosis and short stature. Epileptic seizures are generalized tonic-clonic seizures that are sometimes preceded by visual symptoms. Like in other mitochondrial disease, the manifestation of the diseases varies according to heteroplasmy of the mutation. Additional mutations in the MTTK gene are reported to cause MERFF (e.g. m.8356C>T and m.8361G>A) (Ruiz-Pesini et al. 2007). Also, MELAS/MERFF overlap syndromes have been reported (Nakamura et al. 2010). Mutations in the POLG gene can cause Alpers-Huttenlocher syndrome. This syndrome is characterized by psychomotor disability, liver failure and seizures (Uusimaa et al. 2008). Alpers-Huttenlocher syndrome usually starts in early infanthood, but cases with juvenile or early-adulthood onset have been reported (Uusimaa et al. 2003, Uusimaa et al. 2008, Wiltshire et al. 2008). Severe status epilepticus is often observed; the patient may not recover from this condition (Sofou et al. 2012). 50

Treatment of mitochondrial epilepsy can be difficult. Treatment is administered based on the symptoms, but the underlying pathology should be determined. Some epilepsy medicines act as mitochondrial toxins (e.g. sodium valproate) and they can induce fatal acute liver failure if certain POLG-mutations are present (Saneto et al. 2010, Stewart et al. 2010, Uusimaa et al. 2008). 1.5

Mitochondrial DNA variation as a risk factor for disease

Non-neutral polymorphisms are thought to occur more frequently in rapidly evolving mtDNA than in nuclear DNA (Dowling et al. 2008, Nachman et al. 1995). An increasing number of research papers deal with mtDNA haplogroups and polymorphisms as risk factors for complex and common diseases. It is suggested that mitochondrial background modifies the age of onset or the severity of symptoms; it is also thought to increase the overall risk of developing a common disease such as type 2 diabetes (Poulton et al. 2002a). Whether or not the pathogenic effect of deleterious mtDNA variants might be population-specific has also been discussed. Mixed results have been obtained for the same risk variant in different population backgrounds (Breen & Kondrashov 2010). 1.5.1 Risk haplogroups Mitochondrial DNA haplogroups have been associated with various diseases (Table 2.). Haplogroups contain a mixture of both neutral and mildly deleterious mtDNA variants that can have an additive effect on the function of the mitochondrial respiratory chain. These variants also affect the ability to cope with oxidative stress (Mueller et al. 2012, Pichaud et al. 2012). Also, mtDNA background may affect the outcome (and the associated complications) of viral and bacterial infections (De Luca et al. 2012, Hendrickson et al. 2008, Lorente et al. 2012). Mitochondrial haplotypes can modulate the phenotype and penetrance of certain mitochondrial diseases like deafness caused by m.1555A>G (Lu et al. 2010b). Because mitochondrial function depends on both mtDNA and nuclear DNA, possible epigenetic interactions between nuclear and mtDNA variants may also exist (Chinnery et al. 2012, Dowling et al. 2007).

51

Table 2. mtDNA haplogroups associated with disease. Haplogroup

Disease

T

Vascular diseases, coronary artery disease,

References Kofler et al. 2009, Mueller et al. 2012,

diabetic retinopathy, glaucoma, age-related

Achilli et al. 2011

macular degeneration

Santoro et al. 2010, Abu-Amero et al. 2011, SanGiovanni et al. 2009

Alzheimer’s disease, Parkinson’s disease,

Coskun et al. 2012, Fernandez-

osteoarthritis, multiple sclerosis, ischemic

Moreno et al. 2012, Ghabaee et al.

cardiomyopathy

2009, Fernandez-Caggiano et al. 2012

J

Age-related macular degeneration

Mueller et al. 2012, Kenney et al. 2013

M

Hepatocellular carcinoma

Guo et al. 2012

U

Maternally inherited diabetes, Schizophrenia,

Martikainen et al. 2012, Rollins et al.

bipolar disorder, major depressive disorder,

2009, Klemba et al. 2010()()()

H

vulvar carcinoma B K N

Human papilloma virus induced cervical

Guardado-Estrada et al. 2012, Luo et

cancer, high-altitude pulmonary edema

al. 2012

HIV induced lipodystrophy, ischaemic stroke,

De Luca et al. 2012, Chinnery et al.

Parkinson’s disease

2010, Gaweda-Walerych et al. 2008

Breast and esophageal cancer

Darvishi et al. 2007

A

Multiple sclerosis

Ghabaee et al. 2009

Y

Leigh syndrome

Hao et al. 2013

D

Lung cancer susceptibility

Zheng et al. 2012

1.5.2 Predisposing mitochondrial mutations in diabetes mellitus Diabetes mellitus is a chronic hyperglycemic disorder that affects the lives of millions worldwide. Type 2 diabetes constitutes 90% of diabetes cases. Diabetes mellitus is a common symptom of mitochondrial diseases and is frequently observed in for example the MELAS syndrome. Diabetes mellitus constitutes 0.2 to 2% of diabetic patients (Maassen et al. 2007, van den Ouweland et al. 1994). Type 2 diabetes is regarded as a complex multifactorial disease with both genetic and environmental factors (e.g. obesity) affecting the onset of the disease. Several studies have been done on the predisposing mitochondrial factors (Maasen 2008, Reiling et al. 2009, Reiling et al. 2010). The mechanisms by which mtDNA mutations impair glucose metabolism can be applied to studying the role of mtDNA in common type 2 diabetes. A weakened storage of fat and leakage of fatty acids in peripheral adiposytes is observed in diabetic individuals harbouring the m.3243A>G MELAS mutation 52

(Maassen et al. 2007). Fatty acid leakage is an inducing factor in the process of insulin resistance, whilst the mitochondrion protects against free fatty acids in the cytocol (Vamecq et al. 2012). Reduced ATP production, increased lactate production, reduced glucose oxidation and impaired NADH responses have been observed in m.3243A>G cells (de Andrade et al. 2006). Nuclear mitochondrial associated genes have also been associated with type 2 diabetes (e.g. LARS2 gene encoding mitochondrial leucyl tRNA synthetase) (T' Hart et al. 2005, Reiling et al. 2009). D-loop variant m.16189T>C and type 2 diabetes The common mtDNA D-loop variant m.16189T>C has been reported to be more frequent in type 2 diabetes in the United Kingdom (Poulton et al. 2002b). It is also more frequent in Asian type 2 diabetics (Liou et al. 2010, Park et al. 2008) and in Indian type 2 diabetics (Bhat et al. 2007). Furthermore, m.16189T>C and additional coding region mutations may have combined effects that raise the risk of type 2 diabetes (Liou et al. 2012). The same variant has also been associated with metabolic syndrome (Palmieri et al. 2011), thinness at birth (Casteels et al. 1999, Parker et al. 2005), endometriosis (Cho et al. 2011), coronary artery disease, myocardial infarction (Mueller et al. 2011) and alteration of mtDNA copy number (Liou et al. 2010). In contrast, a few studies have not found a connection between m.16189T>C and type 2 diabetes or metabolic syndrome (Aral et al. 2011, Das et al. 2007, Mohlke et al. 2005, Segre et al. 2010). This has led to the hypothesis that the pathogenicity of m.16189T>C may be population and mtDNA background dependent (Breen & Kondrashov 2010, Wang et al. 2009). Another study of a large cohort of diabetics concluded that the role of mtDNA mutations in the pathogenesis of type 2 diabetes is small (Segre et al. 2010). The m.16189T>C mutation is situated within a long cytosine stretch in the non-coding D-loop region of mtDNA spanning nucleotides m.16180 – m16195. The poly-C stretch is disrupted with a thymine at position m.16189T. It is suggested that this thymine acts as an mtDNA replication break to prevent replication slippage. This is important because the poly-C stretch is situated near the termination associated sequence of D-loop (Poulton et al. 1998). Individuals harbouring an uninterrupted poly-C have been reported to have a lower peripheral blood mtDNA copy number compared to the wild type interrupted poly-C subjects. It was also observed that certain variants of the m.16180–16195 53

sequence caused an increased mtDNA copy number (Liou et al. 2010). The slower metabolic rate due to a lower copy number of mtDNA in individuals harbouring m.16189T>C could increase the risk of developing type 2 diabetes when additional environmental factors such as oversupply of food are present (Liou et al. 2010). 1.6

Detection and analysis of novel mtDNA mutations in undetermined mitochondrial diseases

Diagnosing mitochondrial diseases is not always straightforward. This is especially true if a novel mtDNA mutation is causing the disease. Determining novel deleterious mtDNA mutations requires extensive whole mtDNA sequencing because no commonly used assays are available for unknown mtDNA mutations (Wang et al. 2012, Zaragoza et al. 2010). Novel nuclear mutation detection can be even more challenging due to the massive size of the human nuclear genome and the fact that not all genes associated with mitochondrial function are known. Sophisticated whole genome sequencing or exome sequencing techniques might be necessary (Ronchi et al. 2013). Distinguishing between either nuclear or mtDNA origin of the mutation is done by investigating inheritance patterns inside the family. Also, sporadic mutations in mtDNA do occur, so sometimes family information is not useful (Cardaioli et al. 2012). Since mtDNA is highly variable, discerning between a novel pathogenic mutation and a novel polymorphism can be difficult (Chinnery et al. 1999, Wang et al. 2012). Pathogenic mutations are more often heteroplasmic than polymorphisms, but pathogenic homoplasmic mutations also do occur (Giordano et al. 2013). Heteroplasmy level should correlate with symptom severity inside the family, hence higher heteroplasmy should present with a more severe disease phenotype (Sunami et al. 2011). Pathogenic mutations often occur in the conserved regions of the mitochondrial sequence and polymorphisms affect nonconserved regions, where a change in sequence does not compromise the whole protein or tRNA (Vilmi et al. 2005). Mutations in tRNA genes can be considered pathogenic if the mutation occurs in important parts of the tRNA molecule like the anticodon domain (Roos et al. 2012, Yarham et al. 2011). Special care should be taken when choosing animal species with which to compare sequence conservation (Yarham et al. 2012). In addition, each time a novel mutation is detected, at least 100 healthy population controls from the same region should be 54

tested to see if the mutation occurs naturally in the population; possible haplotype associations should still be considered (Benton et al. 2012). Human mtDNA variation databases Several online data collections of human mtDNA variants are available. They hold both benign and pathogenic mtDNA variants, phylogenetic haplogroup trees, sequences conservation tools and other useful applications. The most commonly cited database is Mitomap (http://www.mitomap.org/MITOMAP) (Ruiz-Pesini et al. 2007). Mitomap includes a list of reported/proven pathogenic mutations in combination with clinical phenotype and reference lists. Another database is the Uppsala University site mtDB (http://www.mtdb.igp.uu.se/). Searching for mtDNA variants is easy because there is a feature that allows you to search for sequences containing multiple mtDNA variants. However, this database has not been updated since 2007 (Ingman & Gyllensten 2006). The Giib Human Mitochondrial Genome Polymorphism database (http://mtsnp.tmig.or.jp/mtsnp/index_e.shtml) can be used for finding mtDNA polymorphisms, mtDNA sequence conservation analysis and mtDNA length polymorphism studies (Tanaka et al. 2004). The Human Mitochondrial DNA Database (http://www.hmtdb.uniba.it) on the other hand, is a robust tool for finding mtDNA variants and haplotypes according to country, disease phenotype and mtDNA mutation type (e.g. insertions and deletions) (Rubino et al. 2012). MtDNA haplogroup trees and haplotype defining mutations can be easily accessed using PhyloTree (http://www.phylotree.org/) (van Oven & Kayser 2009). Mamit tRNA, a compilation of mammalian mitochondrial tRNA genes (http://mamit-trna.u-strasbg.fr/), contains a selection of tools for the analysis of mitochondrial tRNA mutations (Putz et al. 2007). 1.6.1 Functional assays for proving pathogenicity of novel mtDNA mutations Ascertaining a biochemical defect to the novel mtDNA variant is very important when providing evidence for the pathogenic potential of new mutations. Tissue biopsies may reveal abnormal patterns in for example muscle cells, such as ragged-red-fibers, mitochondria clusters and COX-negative fibers (Lindal et al. 1992). MtDNA mutation heteroplasmy can also be analysed from individual single-fibers of a muscle sample (Kärppä et al. 2005). Assays of activities of the 55

mitochondrial respiratory chain complexes provide direct evidence of the complex in which the mutation affects, showing up as lowered enzyme activities of a certain respiratory chain complex. This can also be done in cell cultures of the patient’s fibroblasts (Hinttala et al. 2006, Ugalde et al. 2007). Increased blood lactate level is also a tell-tale sign of a mitochondrial disorder (Magner et al. 2011). Model organisms, such as Escherichia coli, have proven to be a valid tool for assessing the biochemical properties of novel mtDNA mutations (Kervinen et al. 2006, Pätsi et al. 2012). 1.6.2 Prediction of the functional effect of nonsynonymous mtDNA mutations Upon discovering a novel nonsynonymous mtDNA mutation, its functional effect can also be estimated using in silico protein function prediction algorithms (Table 3.). These programs calculate the proposed effect of the mutation (benign or pathogenic) on the protein using sequence conservation data and compare the properties of the changed amino acids such as secondary structure, solvent accessibility and polarity (Adzhubei et al. 2010, Bromberg et al. 2008, FerrerCosta et al. 2005, Ng & Henikoff 2001). The problems associated with prediction algorithms are that the reliability of a prediction is dependent of the number of protein sequences and the amount of data available for protein structure, thus making predictions for well-known proteins is more reliable than it is for more poorly described proteins. Also, the predictions of commonly used algorithms (Table 3.) can produce contradicting results for the same mutation depending on which algorithm is used (Hao da et al. 2011, Schaefer et al. 2012). Consequently, the in silico pathogenicity predictions should be used carefully; not relying solely on predictions, but instead using combined biochemical methods to provide plausible evidence of a damaging effect.

56

Table 3. Protein function prediction algorithms of nonsynonymous mutations. Program

Methodology basis

PolyPhen-2 Protein structure, interaction, evolution, multiple alignments of

Reference

Link

Adzhubei et al.

http://genetics.bwh.harvard.edu/pp

2010

h2/

homologous sequences PMut

Neural networks of disease-

Ferrer-Costa et http://mmb2.pcb.ub.es:8080/PMut/

associated and neutral mutations,

al. 2005

protein structure SIFT BLink Sequence homology, amino acid

Ng et al. 2009

http://sift.jcvi.org/

physical properties SNAP

Neural networks, SIFT results,

Bromberg et al. http://rostlab.org/services/snap/

conservation information from

2008

position-specific counts (PSIC)

57

58

2

Aims of the present study

The aim of this thesis was to detect mildly deleterious mtDNA variants and haplogroups that are associated with maternally inherited diabetes mellitus and epilepsy. We also screened the entire mtDNA in patients with suspected mitochondrial disease in order to detect novel pathogenic mtDNA mutations. The nonsynonymous mtDNA variants were consequently assessed for their pathogenic potential. Based on the mtDNA variation, phylogenetic trees were built to further investigate the occurrence of deleterious variants and haplotypes. Where previously reported or novel pathogenic mtDNA mutations were found, family studies on the inheritance of heteroplasmy within families were conducted. The specific aims were: 1. 2. 3.

4.

to identify the molecular etiology of slowly progressive adult-onset polyneuropathy and non-dominant ataxia in a family. to detect mildly deleterious mtDNA variants, combinations of variants or haplogroups that could increase the risk for maternally inherited diabetes. to detect mildly deleterious mtDNA variants in epilepsy patients that have maternal relatives with epilepsy, diabetes mellitus or sensorineural hearing impairment. to search for novel and pathogenic mtDNA mutations in patients with suspected mitochondrial disease, but who lack all the common pathogenic mtDNA mutations.

59

60

3

Patients and methods

3.1

Patients (I-IV)

A total of 217 patients had their entire mtDNA screened in this thesis (Table 4). Table 4. Patients (I-IV). Patient group

Symptoms in maternal relatives

Origin

Diabetes mellitus

Diabetes, hearing loss, epilepsy

Northern Finland 64

Epilepsy

Epilepsy, hearing loss, diabetes

Northern Finland 79

Ataxia, polyneuropathy

Ataxia, polyneuropathy, seizures

Suspected mitochondrial disease Mitochondrial disease symptoms

N

Western Finland 8 Finland

66

Total

217

Patients for studies II and III were obtained from the local-authority health care units in Northern Finland. The diabetes register (and the discharge diagnosis) of two hospitals were reviewed for the second original paper (II). Epilepsy patients (study III) were obtained from the outpatient register at the Department of Neurology, Oulu University Hospital. Diabetes (II) and epilepsy patients (III) filled out a family questionnaire that asked questions about the diabetes mellitus, epilepsy and hearing loss symptoms in their maternal first or second degree family members. No distinction between types of epilepsy was made in the third original paper. Diabetes patients were required to have started insulin treatment between the ages of 20 and 45. A family history score was calculated for patients participating in diabetes and epilepsy studies (II and III). The crude proportion of affected maternal relatives for each patient was calculated using the Naffected/Ntotal formula (van Esch et al. 1994). The diabetes and epilepsy patients that were ranked highest in family history scores (≥ 0.077 in diabetes patients, ≥ 0.1 in epilepsy patients) were selected for the study. Also, the epilepsy patients that had two or more maternal relatives with epilepsy, diabetes or sensorineural hearing impairment were selected. Both diabetes (II) and epilepsy (III) patients were screened to make sure that they did not harbour the m.3243A>G or m.8344A>G mutations. Blood samples of the family suffering from adult-onset ataxia and polyneuropathy (I) were obtained from Seinäjoki Central Hospital. The samples of the proband were investigated first, followed by samples provided from the rest

61

of the family. More detailed descriptions of the family’s clinical signs are presented in original paper I. Muscle and blood samples of suspected mitochondrial disease patients (IV) were obtained from various hospitals and centres in Finland. Common symptoms in the patients (IV) included exercise intolerance, myopathy, encephalomyopathy, cardiomyopathy, diabetes mellitus, sensorineural hearing impairment and progressive external ophtalmoplegia. These patients did not harbour any of the most common pathogenic mtDNA mutations and were therefore selected for whole mtDNA and mtDNA multiple deletion screening. Blood samples of the family of patient 16 (IV) harbouring m.7585insT were acquired after the discovery of the novel insertion. Their blood samples were obtained from the local health care units. More detailed clinical descriptions of the family members are presented in original paper IV. All participating patients signed informed consent forms (I-IV). The ethics committee of the University of Oulu approved the study protocols (I-IV). 3.2

Population controls (II-IV)

Population controls (II-IV) were anonymous blood donors at the Red Cross Office. It was required that they did not report diabetes, hearing loss or any neurological ailments in themselves or in their family. The controls and their mothers were born in the same province. We used 192 fully sequenced (Finnilä et al. 2001) and 403 haplotyped mtDNA population control samples (Meinilä et al. 2001) from Northern Finland. All participants signed an informed consent form before participating in the studies. The ethics committees of the Finnish Red Cross and the University of Oulu approved the study protocols (II-IV). 3.3

Molecular methods

3.3.1 DNA extraction (I-IV) Total DNA from blood samples was extracted using the QiAmp Blood Kit (Qiagen, Valencia, CA, U.S.A.). The Wizard® Genomic DNA purification kit (Promega, Madison, WI, U.S.A.) was used for muscle sample DNA extraction. The standard phenol and chloroform tissue DNA extraction method was used in parallel with the other methods. 62

3.3.2 Haplogroup analysis (I – IV) Haplogroups were determined using restriction fragment length analysis (Torroni et al. 1996). The polymorphisms and restriction enzymes used for detecting mtDNA haplotypes are presented in Table 5. The haplotype frequencies of patients (I-IV) were compared to those in population controls (Finnilä et al. 2001, Meinilä et al. 2001a). Differences in haplotype distribution were assessed using an exact test of population differentiation (as implemented in Arlequin 3.0) (Excoffier et al. 2007). Table 5. MtDNA haplogroup defining polymorphisms and restriction enzymes used for haplotyping patient samples. Haplotype H

Defining polymorphism

Restriction enzyme

m.7028C>T

Alu I

UK

m.12308A>G

Dde I

T

m.13368G>A

BamH I

J

m.13708A>T

BstN I

V

m.4580G>A

Nla III

WIX

m.8251G>A

Ava II

3.3.3 PCR and conformation sensitive gel electrophoresis (CSGE) (IIV) The standard PCR method and touch-down PCR were used to amplify 63 overlapping fragments of the mtDNA coding region (I-IV). PCR fragments were amplified in 30 cycles through denaturation at 94°C for 1 min, annealed at a primer-specific temperature and extended at 72°C for 1 min and finally extended one more time for 10 min. Total reaction volume was 30 µl and the size of the PCR fragment was approximately 350 bp. CSGE was performed for the mtDNA coding region spanning nucleotides m.577 – m.16023 (as previously described) (Finnilä et al. 2000, Körkkö et al. 1998). A more detailed description of the method is reported in original paper II. The primer sequences that were used for CSGE are reported in the supplemental data of original paper III. PCR fragments that differed in mobility on CSGE were selected for sequencing and further study (I-IV).

63

3.3.4 Sequencing (I-IV) Fragments that differed in mobility on CSGE were selected for sequencing. PCR fragments were first purified using exonuclease I and shrimp alkaline phosphatase (Werle et al. 1994). An ABI PRISM™ DYEnamic ET termination Cycle Sequencing kit (Amersham Pharmacia Biotech Inc., Buckinghamshire, U.K.) was used for sequencing (I-IV). The sequencing primers were the same 63 pairs that were used for the amplification of PCR fragments. MtDNA D-loop spanning nucleotides m.15975 – m.725 was sequenced directly from a separate unique PCR fragment (I-IV). Sequences were analyzed using Sequencher 5.0 (Gene Codes Corporation, Ann Arbor, MI, U.S.A.) and mtDNA variants were identified according to the revised Cambridge Reference Sequence (Andrews et al. 1999). 3.3.5 Mutation confirmation and heteroplasmy analysis (I-IV) Important and novel MtDNA mutations and variants were confirmed using RFLP (I-IV) and sequencing in both L and H directions by using a separate PCR product. CSGE was also used for heteroplasmy analysis. A heteroduplex mix containing only patient mtDNA was loaded on CSGE. This can separate heteroplasmic and homoplasmic variants because a PCR fragment containing a heteroplasmic variant migrates to form a double heteroduplex band on the CSGE gel. This provided a simple yes or no answer to whether a variant was heteroplasmic or not (as was reported in original paper IV, figure 2). RFLP was then used to further quantify the heteroplasmy levels (I, IV). A PCR reaction was performed in the presence of radioactively labelled 35S-dATP (Perkin-Elmer, Wellesley, MA, U.S.A.). This was followed by digestion with a suitable restriction enzyme (Table 6) and electrophoresis on 6% polyacrylamide gel. Finally, the digested DNA fragments were exposed on film. The intensities of the fragments containing the label were scanned using a Quantity One scanner (BioRad, Hercules, CA, U.S.A.). After the scanning process, the heteroplasmy levels were calculated (I, IV).

64

Table 6. Restriction enzymes used for heteroplasmy detection of pathogenic mtDNA mutations (I, IV). Mutation

Restriction enzyme

Restriction site

m.8993T>C

Eco52I

+8993 Eco52I

Original paper I

m.7585insT

FastDigest®MseI

-7585 MseI

IV

+ gain of restriction site, - loss of restriction site

Cloning (IV) The heteroplasmy of m.7585insT was quantified by cloning patient mtDNA into E. coli cells (IV). A PCR fragment containing m.7585insT spanning nucleotides m.7252 – m.7634 was ligated into a plasmid vector. A CloneJET™ PCR cloning kit (Fermentas, Thermo Fisher Scientific Inc., Waltham, MA, U.S.A.) was used for ligation. Plasmid containing the ligated mtDNA fragment was transformed into a Subcloning efficiency™ DH5α™ chemically competent E. coli strain (Invitrogen™, Life Technologies, Paisley, U.K.) according to the manufacturer instructions. The competent cells were grown for 16 h at 37 °C on LB-agarampicillin (20 mg/ml) plates. Bacterial clones were then used for a PCR template and the PCR fragments were sequenced. The primers used for the amplification of the PCR-fragment were used for sequencing. A total of 100 clones were collected and sequenced; these clones were used to calculate the heteroplasmy level of the mtDNA insertion in the patient (IV). 3.3.6 Multiple deletion detection (IV) Multiple deletions in muscle mtDNA (IV) were analysed using long PCR application, Phusion expand XL-PCR and an Expand Long Template PCR system kit (Boehringer Mannheim, Mannheim, Germany) as previously described (Remes et al. 2005). The primers spanned nucleotides m.10 – m.40 and m.16465 – m.16496. Total volume of the PCR reaction was 50 µl. It contained 1 – 2 ng of template muscle DNA, an expand PCR kit buffer III, 0.5 mmol/L of each nucleotide, primers (2 µ mol/L) and an expand PCR enzyme. The amplification was carried out using a program of initial denaturation of 2 min at 92 °C, denaturation cycles at 92 °C for 10 s and annealing/extension at 68 °C for 12 min in the first 10 cycles. This was followed by 5 cycles of 13 min and 15 cycles of 14 min. A final extension phase at 68 °C for 20 min was done and the amplified PCR fragments were then electrophoresed on 0.7% agarose gel. 65

POLG1 sequencing (IV) Two muscle DNA samples that harboured multiple mtDNA deletions were selected for POLG1 sequencing (IV). POLG1 sequencing was carried out using the same method that was used for mtDNA sequencing; an ABI PRISM™ DYEnamic ET termination Cycle Sequencing kit (Amersham Pharmacia Biotech Inc., Buckinghamshire, U.K.) was used. POLG1 variants were reported as differences compared to the NCBI reference sequence for POLG1 (NM_002693). 3.4

Phylogenetic networks and statistical analyses (II-IV)

Phylogenetic networks of mtDNA coding and D-loop regions were constructed based on the median algorithm (I-IV) (Bandelt et al. 1995, Bandelt et al. 1999). Variants were reported as deviations from the revised Cambridge reference sequence (GenBank: NC_012920) (Andrews et al. 1999). The variants were weighed equally. Fast evolving sites such as m.303, m.311 and m.16519 were not included in the D-loop networks. An African mtDNA sequence was used as an out group (GenBank: AF346980). The statistical differences for mtDNA variants in patient and population control data were calculated using Fisher’s exact two tailed test. 3.5

In silico analysis of nonsynonymous mtDNA mutations (III-IV)

Four methods of nonsynonymous mutation analysis for functional effect were used in this thesis (III-IV). These algorithms predict whether a nonsynonymous mutation will have either a pathogenic or a neutral effect in a protein. These algorithms are based on information about the conservation of the protein sequence, sequence divergence and the physical properties of amino acids. 3.5.1 PolyPhen-2 In original papers II, III and IV, PolyPhen-2 (version 2.2.2) (Hao da et al. 2011) was used to predict the pathogenicity of nonsynonymous mutations. PolyPhen-2 uses protein sequences available in the UniProtKB/UniRef100 database (release 2011_2012), protein structures reported in PDB/DSSP Snapshot 03-Jan-2012 and UCSC Multiz multiple alignments of 45 vertebrate genomes with the hg19/GRCh37 human genome. PolyPhen-2 assesses the function of the changed 66

protein structure (e.g. is the mutation situated in an active, binding etc. position) and identifies protein sequence homologues in a BLAST search of UniRef database. PolyPhen-2 utilizes machine-based learning methods. Protein 3D structures are assessed to detect changes in for example the hydrophobic properties of amino acids (Adzhubei et al. 2010). PolyPhen-2 is reported to be very specific when detecting benign polymorphisms (Hao da et al. 2011). 3.5.2 SIFT BLink SIFT BLink is based on the degree of conservation in sequence alignments that have been collected from related protein families using PSI BLAST. Amino acid substitutions are categorized as tolerant or intolerant. SIFT BLink assumes that functionally important sites are conserved and that a change in such amino acids would be regarded as intolerant. A multistep procedure is applied to first search for similar protein sequences, after which closely related sequences are chosen and normalized probabilities for all possible substitutions are finally calculated. SIFT BLink scores of < 0.05 are predicted to be deleterious and probabilities with a score of ≥ 0.05 are regarded as neutral (Ng & Henikoff 2001). 3.5.3 PMut PMut is a neural network application for predicting deleterious or neutral amino acid substitutions. Similar to PolyPhen-2 and SIFT Blink, it also searches its database for information on physical properties, secondary structure information and multiple sequence alignments of amino acids that are under comparison. A simple output from the prediction is produced (it has a pathogenicity index ranging from 0 to 1). Mutations with pathogenicity scores of > 0.5 are regarded as damaging. A confidence level of 0 (low) to 9 (high) is also provided (Ferrer-Costa et al. 2005). 3.5.4 SNAP The SNAP algorithm predicts whether an amino acid substitution is deleterious or neutral in effect (Bromberg et al. 2008). SNAP uses information in the neural networks of sequence alignment, evolutionary conservation and amino acid physical properties such as solvent accessibility. SNAP predicts whether the mutation will be neutral or non-neutral. This prediction is followed by a reliability 67

index with an expected accuracy ranging from 50 to 100 %. SNAP has been reported to be more specific than PolyPhen-2 when predicting pathogenic mutations (Bromberg et al. 2008, Hao da et al. 2011).

68

4

Results

4.1

Haplogroup frequencies (II-IV)

The frequencies of mtDNA haplogroups were determined in three patient groups (Table 7). Table 7. Haplogroup frequencies of patients with maternally inherited diabetes (II), maternally inherited epilepsy (III) and suspected mitochondrial disease (IV). Haplogroup

Diabetes mellitus (N)

Epilepsy (N)

Mitochondrial disease (N)

Total (N)

H

23

32

22

77

V

10

7

5

22

U

15

21

23

59

K

3

3

1

7

J

2

4

4

10

T

5

3

6

14

W

2

5

2

9

I

3

2

2

7

X

0

2

1

3

R

1

0

0

1

Total

64

79

66

209

We detected a trend towards increased frequency of haplogroup V when examining patients with maternally inherited diabetes (p = 0.0066, Fisher’s exact two-tailed test) (II: Table 1). Other patient groups (III, IV) revealed no differences in haplogroup distributions. A single patient with matrilineal diabetes (II) was found to belong to haplogroup R. This haplogroup is uncommon in the Finnish population. 4.2

Sequence variation and phylogenetic structure of mtDNA (II, III, IV)

MtDNA sequence variation in patient groups was different compared to population controls. Novel, disease-associated and rare variants occurred more frequently in patients with matrilineal diabetes, epilepsy or suspected mitochondrial disease. Several nonsynonymous variants were predicted to be deleterious and two clearly pathogenic mutations were discovered, one of which was a novel insertion. Branching of the phylogenetic tree (Figure 6) created 69

special patterns; specific branches that contained more patients than control samples.

Fig. 6. Phylogenetic network of mtDNA coding region sequence variation in diabetes mellitus, epilepsy and suspected mitochondrial disease patients. CRS = revised Cambridge reference sequence (GenBank: NC_012920), outgroup = African sequence (GenBank: AF346980), * = heteroplasmy, @ = back mutation, i = insertion, D9bp = common deletion of 9 bp spanning nucleotides m.8281 – m.8289. Underlined variants = pathogenic mutations and variants predicted to be deleterious. Fast evolving sites m.303, m.311 and m.514 – m.523 CA repeat variants were not included in the network.

70

4.2.1 Heteroplasmic polymorphisms (I - IV) Several heteroplasmic mtDNA variants (Table 8) and two pathogenic mutations were discovered. The remaining variants were not predicted to be deleterious. Table 8. Heteroplasmic mtDNA variants (I - IV). Heteroplasmic variant

Gene

Patient number

m.93A>G

noncoding

21

epilepsy

m.474T>C

noncoding

52

epilepsy

m.593T>C

MTTF

49

diabetes

m.4742T>C

MTND2

9

suspected mitochondrial disease

m.6968C>T

MTCO1

42

diabetes

m.7585insT1

MTTD - MTCO2

16

suspected mitochondrial disease

m.7604G>A

MTCO2

14

epilepsy

m.8993T>C1

MTATP6

1

adult-onset ataxia

m.9591G>A

MTCO3

31

suspected mitochondrial disease

m.9667A>G

MTCO3

40

epilepsy

m.9698T>C

MTCO3

54, 55, 56

epilepsy

m.11800A>G

MTND4

10

diabetes

m.14550T>C

MTND6

60

epilepsy

m.14998C>T

MTCYB

71

epilepsy

1

Disease phenotype

Pathogenic mutation

4.2.2 Rare mtDNA variants (III) Four rare mtDNA variants were discovered among matrilineal epileptic patients (III: Table 4). We considered variants to be rare if three or less identical sequences were found in public mtDNA databases. Three of the rare variants were previously reported among Finns. 4.3

Pathogenic and novel mutations (I – IV)

4.3.1 m.8993T>C (I) A previously reported pathogenic m.8993T>C (p.L156P, MTATP6) mutation for NARP syndrome was discovered in a family with adult-onset ataxia and polyneuropathy. This mutation occurred in the family at varying heteroplasmy levels and was maternally inherited (I: Figure 1). The proband of the family harboured the mutation at a level of 89% in their blood whilst her mother 71

harboured a blood heteroplasmy level of 59%. The heteroplasmy levels of her siblings and maternal cousin ranged from 28% to 79%. The mother (59% heteroplasmy) and her maternal cousin (28% heteroplasmy) reported no neuromuscular symptoms. One of the siblings had died in the 1970s; Friedreich’s ataxia was found during autopsy. All other siblings suffered from ataxia and polyneuropathy. 4.3.2 m.7585insT (IV) A novel heteroplasmic insertion (m.7585insT), situated after the last nucleotide of MTTD and before the first nucleotide of MTCO2, was discovered in a patient with a severe manifestation of mitochondrial disease that affected several organs. He suffered from intellectual disability, epilepsy, autoimmune haemolytic anemia, renal insufficiency, diabetes mellitus, dilating cardiomyopathy and hemosiderosis of the lungs. He also had an unsteady gait. Mitochondrial inclusion bodies and increased mitochondrial mass was detected in his muscle sample. The proband harboured m.7585insT at over 97% heteroplasmy in his muscle (IV: Figure 2). This was confirmed by subcloning 100 bacterial clones. He died at 49 years old of pneumonia-induced cardiac insufficiency. The proband belonged to a large family with nine siblings (IV: Figure 3). His mother had suffered from diabetes mellitus, cardiovascular disease and pulmonary embolisms. Five of his siblings had died of cardiovascular diseases at a fairly young age (~ 60y). Two siblings had died of undetermined lung disease. Three sisters remained and two of them suffered from rheumatoid arthritis. The sisters harboured m.7585insT at a 69% heteroplasmy level in blood, whilst the nephew of the proband harboured m.7585insT at a 67% heteroplasmy level in blood. He had a history of acute pericarditis. 4.3.3 Novel tRNA and nonsynonymous mtDNA mutations (II, IV) A novel homoplasmic m.15933G>A mutation in MTTT was discovered in a patient with ptosis, muscle weakness, myalgia, dysphagia and psoriasis. Raggedred fibers and COX-negative fibers were detected in her muscle sample. The m.15933G>A mutation is situated in a conserved position in the T-stem of MTTT, # 49. Another novel homoplasmic mutation, m.11322T>C (p.N188S, MTND4), was detected in two siblings (patients 21 and 22) (IV: Figure 2). Patient 21 suffered from sick sinus syndrome, hypertrophic cardiomyopathy and chronic 72

atrial fibrillation. Patient 22 had restricted eye movement, ptosis and muscle weakness. She also harboured multiple mtDNA deletions, but no POLG1 mutations. Nonsynonymous homoplasmic m.13762T>G (p.S476A, MTND5) was discovered in a patient with diabetes mellitus; this patient also had maternal relatives with diabetes, hearing loss or epilepsy. 4.3.4 Novel synonymous mtDNA variants (II, III, IV) Three novel synonymous mtDNA variants in the coding region were discovered. These variants had not been reported in any online databases at the time of investigation. The novel synonymous changes included MTND4, m.11266C>T (II: Figure 2), m.11392A>G (III: Figure 1) and m.13392A>G (IV: Figure 1). 4.4

Previously reported disease-associated mtDNA variants (II, III, IV)

Several mtDNA variants that had previously been reported in association with a disease were discovered in original papers II, III and IV (Table 9). Table 9. Previously reported disease associated mtDNA variants. Variant

Reported disease

Reference

Haplogroup

Original paper II, IV

association m.4659G>A

Parkinson’s disease

Khusnutdinova et al. 2008

H

m.5823A>G

Motor neuron disease

Kirches et al. 1999

R

II

m.6253T>C

Prostate cancer

Petros et al. 2005

H

III

m.6480G>A

Prostate cancer

Petros et al. 2005

H

II

m.6489C>A

Therapy resistant epilepsy

Varlamov et al. 2002

T

II, III

m.7444G>A

Hearing loss

II, III, IV

Yuan et al. 2005

V

m.12811T>C LHON, phenotype affecting

Cai et al. 2008

H

II, III

m.13637A>G LHON, phenotype affecting

Huoponen et al. 1993

U

III, IV

4.5

Prediction of the pathogenic effect of mtDNA mutations (II-IV)

SNAP, PMut, PolyPhen-2 and SIFT BLink were used in parallel to predict the deleterious effect of nonsynonymous mtDNA variants (II–IV). A total of 13 mtDNA variants predicted to have a deleterious effect were discovered among patients suffering from matrilineal diabetes mellitus, epilepsy or suspected 73

mitochondrial disease (Table 10). The nonsynonymous variants of 192 Finnish population controls (Finnilä et al. 2001) were also analysed with PolyPhen-2 and SIFT BLink. A total of 25 mtDNA variants in the population controls were predicted to have an adverse effect (Table 11). Table 10. Patient mtDNA variants predicted to be deleterious by SNAP, PolyPhen-2, PMut and SIFT BLink. Variant

Gene

Amino acid change

Patient group

Prediction algorithm

m.5095T>C

MTND2

p.I209T

M

PP2, SI, S, PM

m.8616G>T

MTATP6

p.L30F

M

PP2, SI, PM

m.9055G>A

MTATP6

p.A177T

EPI

PP2, SI, PM

m.9903T>C

MTATP6

p.F233L

DM, EPI

PP2. SI, PM

m.10192C>T

MTND3

p.S45F

M

SI, S, PM

m.12135C>A

MTND4

p.S459Y

M

PP2, SI, S, PM

m.12613G>A

MTND5

p.A93T

DM, EPI

PP2, SI, PM

m.14180T>C

MTND6

p.Y165C

DM, M

PP2, SI, S, PM

m.14198G>A

MTND6

p.T159M

DM, EPI

PP2, SI, PM

m.15218A>G

MTATP6

p.T158A

EPI

PP2, SI, PM

Abbreviations: DM = diabetes mellitus, EPI = epilepsy, M = suspected mitochondrial disease, S = SNAP, PP2 = PolyPhen-2, PM = PMut, SI = SIFT BLink

Table 11. Population control (N = 192) mtDNA variants predicted to be deleterious by both PolyPhen-2 and SIFT BLink. Variant

Gene

Amino acid change

m.3644T>G

MTND1

p.V113G

Haplogroup U

m.7706G>A

MTCO2

p.A41T

W

m.8567T>C

MTATP6

p.I14T

Z

m.8588T>C

MTATP6

p.V21E

H

m.8616G>T

MTATP6

p.L30F

I

m.8843T>C

MTATP6

p.I106T

H

m.8921G>A

MTATP6

p.G132D

J

m.8923A>G

MTATP6

p.T133A

J

m.8998G>A

MTATP6

p.V158M

T

m.9055G>A

MTATP6

p.A177T

K

m.9145G>A

MTATP6

p.A207T

V

m.9429G>A

MTCO3

p.V75I

W

m.9531A>G

MTCO3

p.T109A

I

m.9612G>A

MTCO3

p.V136M

W

m.9828G>A

MTCO3

p.V208I

J

m.10086A>G

MTND3

p.N10D

W

m.10237T>C

MTND3

p.I60T

J

74

Variant

Gene

Amino acid change

m.12126G>A

MTND4

p.G456E

Haplogroup H

m.12135C>A

MTND4

p.S459Y

U

m.12613G>A

MTND5

p.A93T

X

m.12842T>C

MTND5

p.I169T

V

m.14096A>G

MTND5

p.Y587C

K

m.14198G>A

MTND6

p.T159M

U

m.15218A>G

MTCYB

p.T158A

U

m.15780A>T

MTCYB

p.Y345F

Z

Controls (N=192) from the population of northern Finland (Finnilä et al. 2001).

4.6

Mildly deleterious mutations (II, III)

4.6.1 m.15218A>G (III) A common U5a1 polymorphism, m.15218A>G (p.T158A, MTCYB), was observed significantly more often in patients with epilepsy who had maternal relatives with epilepsy, diabetes mellitus or hearing impairment (p = 0.0077 for the difference between cases and controls, Fisher’s exact two-tailed test). The m.15218A>G variant was detected in five patients and in four out of 403 population controls (Meinilä et al. 2001). The m.15218A>G variant was present in two haplotypes within haplogroup U5a1 (III: Figure 4). It was also predicted to have a damaging effect by using three algorithms, PolyPhen-2, PMut and SIFT BLink (III: Table 2). The clinical features of the five patients harbouring m.15218A>G varied (III: Table 3). 4.6.2 m.16189T>C and m.3010G>A (II) A subhaplogroup H1b consisting of four matrilineal diabetes patients harbouring a the D-loop cytosine tract variant m.16189T>C and an MTRNR2 variant m.3010G>A was observed (II: Figure 4). No population controls were detected in this subhaplotype (p = 0.0038 for difference between patients and controls, Fisher’s exact two-tailed test). Only three similar subhaplogroup H1b sequences were discovered in the mtDB database; the database contains 1865 complete mtDNA sequences (Ingman & Gyllensten 2006). The length and composition of the cytocine tract around m.16189T>C was the same in both patients and population controls (II: Table 3). 75

4.7

Multiple deletions (IV)

Patients with a suspected mitochondrial disease were screened for multiple mtDNA deletions (IV). Two out of 30 patients were found to harbour multiple deletions in their muscle tissue (IV: Figure 1). Patient 59 (IV) suffered eye symptoms, such as bilateral progressive external ophthalmoplegia, diplopia, ptosis and also had mild exercise intolerance. Several ragged-red fibers and COXnegative fibers were discovered in his muscle histology. He belonged to haplotype U5a and harboured a private synonymous m.10101T>C mtDNA variant. Patient 22 (IV) also harboured multiple deletions. She suffered from both ocular and skeletal muscle weakness as well as ptosis. Patient 22 belonged to haplogroup U4 and harboured a novel m.11322C>T (p.N188S, MTND4). Her brother, patient 21 (IV) did not harbour multiple deletions, but had identical mtDNA (IV: Figure 1). He suffered from sick sinus syndrome, hypertrophic cardiomyopathy and chronic atrial fibrillation. 4.7.1 POLG1 mutations (IV) Patients 22 and 59 (IV), with suspected mitochondrial disease and multiple deletions, were screened for POLG1 mutations. Patient 59 harboured a heterozygous p.R722H allele in POLG1, whilst no POLG1 mutations were detected in patient 22.

76

5

Discussion

5.1

Haplogroup V is more common in patients with maternally inherited diabetes mellitus in northern Finland

A significant three-fold excessive increase of haplogroup V was found in patients with maternally inherited diabetes mellitus (in comparison to population-specific controls). Specifically, subhaplotype V8 was more frequent in patients than in controls. Haplogroup cluster HV has previously been reported to be more common in patients with type 2 diabetes, and in patients with diabetic complications such as retinopathy and renal failure (Achilli et al. 2011). Haplogroup V is one of the most common mitochondrial haplogroups among the Saami of northern Scandinavia. Approximately 40% of the Saami population in Finland belongs to haplogroup V (Meinilä et al. 2001, Tambets et al. 2004). Genetic admixture between the Saami and the Finns remains more pronounced in northern Finland than in other parts of the country (Meinilä et al. 2001). We therefore selected our population controls carefully from the same geographic area as the patients with maternally inherited diabetes mellitus. A significant excessive amount of haplogroup V was found in patients with maternally inherited diabetes mellitus. This suggests that haplogroup V is mildly deleterious. The high frequency of subhaplotype V8 suggests that populationspecific rare haplogroups could act as genetic risk factors for matrilineal diabetes mellitus. 5.1.1 Several disease-associated mutations detected in patients with matrilineal diabetes mellitus or matrilineal epilepsy Eight previously reported disease-associated mutations were discovered in patients with maternally inherited diabetes mellitus or epilepsy. Two of them were reported to be variants that affect the LHON phenotype: m.13637A>G (p.Q434R, MTND5) (Huoponen et al. 1993) and m.12811T>C (p.Y159H, MTND5) (Cai et al. 2008). Neither of these mutations was predicted to be deleterious in our analysis. The m.13637A>G variant was discovered in haplogroup U5b and is a homoplasic variant that occurs in haplotypes U5b2, U6b2, N1a3 and M1a3 (van Oven & Kayser 2009), while m.12811T>C was discovered in a haplogroup H patient; this variant also occurs in haplotypes M7b and A2 (van Oven & Kayser 2009). Our 77

data did not suggest a deleterious role for m.13637A>G or m.12811T>C in our patient groups. The m.7444G>A variant (p.*514K, MTCO1) has been associated with hearing loss caused by the MTRNR1 m.1555A>G mutation (Yuan et al. 2005, Yuan et al. 2007), but it is also reported to occur in haplotypes V7 and H40b (van Oven & Kayser 2009). This variant could not be analysed with pathogenicity prediction algorithms because it involves a stop-codon. It is interesting though that it is in haplogroup V; a haplotype that was found to be more common among maternally inherited diabetes mellitus patients. A patient with matrilineal diabetes belonged to haplogroup R1a (this is rare in Finland). This patient was also found to harbour the MTTC mutation m.5823A>G. Haplogroup R originates from India. In Europe it occurs in Kurdish, Russian and Polish populations (Palanichamy et al. 2004). The m.5823A>G mutation has been reported to be associated with motor neuron disease and temporal lobe epilepsy (Kirches et al. 1999), but unfortunately no information regarding the haplogroup is available on this patient. The mutation is situated in the amino acid acceptor stem of tRNACys. It alters a nonconserved base and three polymorphisms have been reported in nearby bases (Putz et al. 2007). Since this variant was homoplasmic in our patient and does not alter a conserved nucleotide, we consider it very likely that it is a rare mtDNA variant of haplogroup R. Two variants associated with prostate cancer (Petros et al. 2005) were discovered among patients with matrilineal diabetes mellitus or matrilineal epilepsy. It has been suggested that these variants increase the tumorigenicity of prostate cancer. The two MTCOI variants, m. 6480G>A (p.V193I, MTCO1) and m.6253T>C (p.M117T, MTCOI), were not predicted to be deleterious in effect. The m.6480G>A variant is homoplasic to haplotypes R31a, I2d, T2b, HV, L1, L2 and L3 (Ingman & Gyllensten 2006, van Oven & Kayser 2009). The other prostate cancer variant, m.6253T>C, was found in a matrilineal epilepsy patient that belonged to haplogroup H. The m.6253T>C variant is indeed present in haplotype H15. It is also present in haplotype D5b1, M13’ and L1c2 (van Oven & Kayser 2009). We did not find evidence supporting a pathogenic role for these homoplasmic MTCO1 variants. A patient with matrilineal diabetes and two patients with suspected mitochondrial disease harboured m.4659G>A (p.A64T, MTND2); a variant that has been associated with Parkinson’s disease (Khusnutdinova et al. 2008). This variant was classified as benign by our analyses and is present in haplotypes J, D, X2 and L0d (Ingman & Gyllensten 2006, van Oven & Kayser 2009). Both of our 78

patients belonged to haplogroup H, which has not been reported to harbour m.4659G>A. This variant was considered to be a homoplasic polymorphism. Three patients with matrilineal diabetes and one patient with matrilineal epilepsy harboured a homoplasmic m.6489C>A (p.L196I, MTCO1) transversion. They all belonged to haplogroup T2. This mutation has been previously reported in a patient with therapy-resistant epilepsy (Varlamov et al. 2002). It is present in several haplogroup T2 sequences in online databases (Ingman & Gyllensten 2006, Rubino et al. 2012); therefore we consider it to be a haplogroup T2 specific polymorphism, even though we also discovered it in an epilepsy patient. 5.2

Adult-onset ataxia in a family harbouring the heteroplasmic m.8993T>C mutation

A heteroplasmic m.8993T>C (p.L156P, MTATP6) was discovered in a patient with adult-onset slowly progressive ataxia and polyneuropathy. The mutation is known to cause childhood or juvenile-onset syndromic phenotype such as Leigh’s disease, but it is not associated with adult-onset ataxia (de Vries et al. 1993, Lopez-Gallardo et al. 2009, Santorelli et al. 1994, White et al. 1999b). Two mutations at position m.8993 cause either NARP or Leigh’s syndrome; m.8993T>G being associated with an earlier onset and a more serious phenotype than m.8993T>C (Vazquez-Memije et al. 1998). The proband and her two siblings were clinically examined; the fourth sibling had passed away earlier. The remaining three siblings suffered from peripheral axonal polyneuropathy and two presented with adult-onset slowly progressing gait ataxia and dysarthria. The ataxia was considered to be cerebellar and sensory in origin. Heteroplasmy levels of m.8993T>C correlated with clinical symptoms within the family. The deceased sibling had presented with an earlier-onset of disease. This was much like what was previously reported in association with m.8993T>C. She was diagnosed with Friedreich’s ataxia based on a post-mortem investigation, but she was later found not harbour the GAA repeat expansion (FRDA expansion) that causes Friedreich’s ataxia. Therefore, other causes for ataxia symptoms in the family had to be considered. Adult-onset ataxia and polyneuropathy have not been described in patients with the m.8993T>C mutation; therefore the correct diagnosis can be easily missed in such cases. Adult-onset non-dominant ataxias are less known and only a few autosomal recessive ataxias have been reported (Jayadev & Bird 2013). The 79

family described here suggests that the m.8993T>C mutation should be included in the differential diagnosis of adult-onset ataxia and axonal neuropathy. 5.3

Novel heteroplasmic m.7585insT causes cardiomyopathy in a family with untimely deaths due to cardiac disease

Pathogenic insertions are not very common in mtDNA; only five have been reported (Elstner et al. 2008, Santorelli et al. 1997, Simon et al. 2001, Tiranti et al. 1995, Valente et al. 2009). MtDNA insertions often arise somatically in cancer (Chatterjee et al. 2006). The novel heteroplasmic insertion m.7585insT was discovered in a patient with suspected mitochondrial disease. The proband, patient 16 (IV), was severely affected with multi-organ symptoms. He died from cardiac insufficiency after pneumonia at the age of 49. He harboured the insertion at a heteroplasmy level of ~ 97% in his muscle tissue. The proband had nine siblings. Their mother had been diagnosed with diabetes mellitus, cardiovascular disease and had suffered several pulmonary embolisms. Five out of the ten siblings had died of cardiovascular disease at around 60 years of age. Two siblings had an unidentified lung disease. Two sisters were diagnosed with rheumatoid arthritis and they harboured the insertion at a 69% heteroplasmy level in blood. One of the sister’s sons had a history of acute pericarditis and harboured the insertion at a 67% heteroplasmy level in blood. The inserted thymine is situated between the last nucleotide of MTTD and the first nucleotide of MTCO2. These genes are normally contiguous with no noncoding bases in between them (Ruiz-Pesini et al. 2007). The main symptoms in the family with m.7585insT were cardiac-related and untimely death. Cardiomyopathy is a common symptom of mitochondrial disease; it is particularly associated with tRNA mutations (Arredondo et al. 2012, Giordano et al. 2013). Only three pathogenic MTTD mutations have been reported: m.7520G>A (Bosley et al. 2008), m.7526A>G (Seneca et al. 2005) and m.7543A>G (Shtilbans et al. 1999). Several pathogenic mutations in MTCO2 have been published so far (RuizPesini et al. 2007). An encephalomyopathy causing m.7587T>C (p.M1T) affects the initiating methionine of MTCO2 (Clark et al. 1999). The m.7587T>C mutation resulted in a defective start of translation and a significant decrease in COX2 mRNA levels. Clark et al. highlighted the importance of mitochondrial COX subunits; they are first subunits to be needed for complete assembly of complex IV. 80

Three different mechanisms by which the insertion affects translation are possible. In the first scenario, MTTD is one nucleotide longer which could impair its structure and function. Since the insertion affects the last consensus nucleotide (position 73 of MTTD), potential problems could arise with amino acid attachment into the terminal adenine of the tRNA’s 3’ CCA sequence. The second mechanism would impair MTCO2 and create a shift in the reading frame. The initiating methionine is very conserved and its mutation to tyrosine would lead to a truncated non-functional protein after seven amino acids, or no protein product at all. The third possibility is that the RNAase cleavage site situated in between MTTD and MTCO2 would be unrecognizable for tRNAse Z and as such, both gene transcripts would be compromised. Polycistronic transcripts are processed by tRNAse Z by cleaving them into tRNA, rRNA and mRNA species after translation (Ojala et al. 1981). An mRNA fragment containing both the MTTD and MTCO2 transcripts would be created. No mtDNA insertions near polycistronic cleavage sites have been previously reported. Point mutations that involve nucleotides after a 3’-discriminator base of tRNA molecules have been reported to cause cleavage site shifting and lowered cleavage efficiency. As a result, a longer tRNA molecule is produced (Yan et al. 2006). The previously reported deafness-associated mutation m.7445T>C occurs right after the tRNA 3’-discriminator base of MTTS1 and results in reduced tRNA precursor rates; tRNA level is also lowered by 70%. The m.7445T>C mutation also affects MTND6 translation upstream because MTND6 is processed together with MTTS1 (Guan et al. 1998). An extra nucleotide in tRNA 3’-end could disrupt CCA-trailer addition, aminoacylation, conformational instability and eventually protein synthesis (Levinger et al. 2004). Further work is needed to clarify the mechanism that determines how m.7585insT affects translation. More work is also needed on how m.7585insT determines which gene (or genes) is impaired. 5.4

Novel homoplasmic m.15933G>A in MTTT in a patient with ptosis

A patient suffering from ptosis, muscle weakness, myalgia, dysphagia and psoriasis was found to harbour a novel homoplasmic m.15933G>A mutation in MTTT. This mutation changes a conserved nucleotide (nucleotide 49 in tRNA consensus structure) in the T-stem of tRNAThr. Evolutionary conservation of this nucleotide was very high; all species that were compared harboured an A in this 81

position (Putz et al. 2007, Vilmi et al. 2005). Only six pathogenic and four phenotype modulating mutations have been described in MTTT according to MITOMAP (Ruiz-Pesini et al. 2007) and Mamit tRNA (Putz et al. 2007). The mutation we discovered was homoplasmic (as polymorphisms often are). This suggests that the mutation has a polymorphic nature. There has been much discussion about the pathogenic nature of homoplasmic tRNA mutations in mtDNA (Giordano et al. 2013, Tuppen et al. 2008). To affirm a pathogenic status, homoplasmic mutations must meet stricter criteria than heteroplasmic mutations. They must be absent from the healthy population, be evolutionary conserved and show evidence of a functional defect. Homoplasmic pathogenic tRNA mutations often result in lowered steady-state levels of the tRNA molecule (Giordano et al. 2013, McFarland et al. 2004). The novelty and the conserved nature of the m.15933G>A mutation and the patient’s mitochondrial disease symptoms (with appropriate muscle histology findings) fulfill the first two criteria of a pathogenic homoplasmic tRNA mutation. Additional functional studies are needed to be able to undoubtedly define m.15933G>A as pathogenic. 5.5

Mildly deleterious mtDNA variants occur in patients with matrilineal diabetes or epilepsy as well as in population controls

Pathogenicity predictions were done with four different algorithms. This enabled us to describe several mildly deleterious mtDNA variants in both patients and controls. These variants can have an adverse effect on the function of the mitochondrial respiratory chain and make the cells more vulnerable to free radicals. Exhausted energy metabolism could increase the risk of developing several diseases; such as diabetes mellitus. This kind of an effect would be visible in maternal lineages as for example haplotype associated mildly deleterious variants that would be maternally inherited. Multiple prediction algorithms were used to screen for deleterious nonsynonymous mtDNA variation in patient groups. PolyPhen-2 predicted the highest number of benign polymorphisms, whereas SIFT BLink predicted the highest number of damaging variants. PolyPhen-2 has been reported to have a false negative rate of 5% for neutral variation and is considered to be very precise in predicting the benign effect of a variant. SIFT BLink on the other hand, has been reported to be very accurate in predicting damaging effects (Wang et al. 2012). We argue that the nonsynonymous changes that multiple prediction 82

algorithms identified as damaging are the best candidates for mildly deleterious variants. 5.5.1 The mildly deleterious variant m.15218A>G in haplotype U5a1 occurs more often in patients with maternally inherited epilepsy The m.15218A>G variant (p.T158A, MTCYB) was detected in five patients and four population controls (p = 0.0077). This variant was associated with epilepsy in patients with a maternal family history with of epilepsy and diabetes mellitus or SNHI. This variant is homoplasic and occurs in haplogroups M7, M10, HV and U5a1 (van Oven & Kayser 2009). The patients harbouring m.15218A>G all belonged to haplogroup U5a1 and the variant occurred in two branches of the phylogenetic tree. The major haplotype with m.15218A>G was identical to the haplotype of the controls and the minor m.15218A>G haplotype was identical to a patient with maternally inherited diabetes mellitus. The clinical features of the epilepsy patients with m.15218A>G were variable. The haplogroup U5a has been previously associated with occipital and migraneous stroke (Finnilä et al. 2001). Mitochondrial diseases often present with only epileptic seizures (Rahman 2012). However, stroke-like episodes can also resemble occipital epileptic seizures (Cesaroni et al. 2009). HIV-positive patients that harbour m.15218A>G and belong to haplogroup U5a have been associated with a rapid progression to AIDS and death (Hendrickson et al. 2008). Haplogroup U5 occurs more often in Finland than in the rest of Europe. The frequency of haplogroup U5 in Finland is estimated to be about 30%, whilst it is only 18 to 20% for the rest of Europe (Ruiz-Pesini et al. 2007). More so, haplogroup U5 is especially common in northern Finland where it occurs with less variation. This lack of variation has been suggested to have occurred because only a small settler group colonized northern Finland before the 16th century (Finnilä et al. 2000). Also, the indigenous Saami people of northern Scandinavia harbour a higher frequency of haplogroup U ranging from 32 to 52%. Admixture between the Saami and the Finns is more pronounced in the north (Meinilä et al. 2001, Sajantila et al. 1995). Bias due to geographical haplogroup differences was avoided because healthy controls were selected from the same area as the patients. Three algorithms predicted m.15218A>G to be deleterious in effect. This variant substitutes a conserved hydrophilic threonine with a hydrophobic alanine at position 158 of the cytochrome b subunit of complex III. Among 60 different 83

species, 53 harboured threonine in this position and only a macaque species, the Barbary macaque (Macaca sylvanus), harboured alanine at position 158 according to Giib mtSNP database (Tanaka et al. 2004). Cytochrome b forms the catalytic core of complex III. Changes in this subunit have been known to cause changes in the catalytic function of complex III, thus leading to complex III defects (Legros et al. 2001). Evolutionary conservation, the predicted damaging effect and the homoplasic nature of m.15218A>G suggest that it is a mildly deleterious variant rather than a neutral polymorphism. 5.5.2 The combination of m.16189T>C and m.3010G>A in haplotype H1b is more frequent among maternally inherited diabetes mellitus patients Several studies have suggested that there is an association between the D-loop variant m.16189T>C and type II diabetes (Liou et al. 2007, Palmieri et al. 2011, Park et al. 2008, Poulton et al. 2002a, Wang et al. 2009). This polymorphism creates a long uninterrupted cytocine tract in the control region of mtDNA. The length and composition of the polycytosine stretch has also been associated with a lower mtDNA copy number and it has been suggested that this region controls mtDNA replication (Liou et al. 2010). We did not find an association between m.16189T>C or polycytosine tract length variants and maternally inherited diabetes mellitus in northern Finland. However, we found a significant increase in the frequency of the combination of m.16189T>C and m.3010G>A in haplotype H1b. The 3010G>A variant is a defining polymorphism for haplotype H1b (van Oven & Kayser 2009). Four maternally inherited diabetes patients belonged to haplotype H1b and harboured m.16189T>C and m.3010G>A, whereas none of the controls belonged to this haplotype. The m.16189T>C and m.3010G>A combination is also present in the Asian haplotype D4b (Tanaka et al. 2004). Interestingly, this haplotype has been reported to increase the risk for type II diabetes among Korean men (Nishigaki et al. 2010). Subhaplotype H1f also harbours m.16189T>C and m.3010G>A, but also has additional variants such as m.4452T>C, m.7309T>C, m.9066A>G and m.16093T>C. All of our population controls in haplogroup H1 belonged to subhaplotype H1f. We suggest that the combination of m.16189T>C and m.3010G>A in subhaplotype H1b is damaging in effect and can increase the risk of maternally inherited diabetes mellitus, while the same effect cannot be seen in 84

subhaplotype H1f where additional variants are present. These results have to be regarded as population specific to northern Finland. 5.6

Multiple mtDNA deletions and mutations in POLG1 patients with suspected mitochondrial disease

Two patients with suspected mitochondrial disease were found to harbour multiple mtDNA deletions and one of these patients harboured a heterozygous p.R722H allele in the POLG1 gene. Multiple mtDNA deletions are a common phenomenon in elderly healthy individuals and in patients with POLG1 mutations. Around 48% of young patients with POLG1 mutations harbour multiple deletions (Ferreira et al. 2011). The carrier frequency for the p.R722H allele is 1:135 in the Finnish population (Komulainen et al. 2010). The homozygous p.R722H allele not only causes multiple mtDNA deletions but also myoclonic epilepsy, Alper’s syndrome and PEO (Bolszak et al. 2009, Isohanni et al. 2011, Tang et al. 2012). The clinical phenotype of our patient with the heterozygous p.R722H allele and multiple deletions consisted of mild exercise intolerance and eye manifestations including PEO, ptosis and diplopia. His muscle biopsy revealed a high number of RRFs and COX negative fibers. His symptoms are very similar to previously reported homozygous carriers of the POLG1 p.R722H allele, but a heterozygous p.R722H allele is not enough to specifically account for his symptoms. No additional POLG1 mutations were detected. The second patient with multiple deletions did not carry any POLG1 mutations. She suffered from ptosis, restricted eye movements and exercise intolerance. She was only 49 years old at the time of the investigation so agerelated multiple deletions are not very likely, although not at all impossible. Interestingly, she harboured a novel nonsynonymous mtDNA mutation, m.11322C>T (p.N188S, MTND4), which she shared with her brother; her brother did not have multiple deletions. This novel mutation on the other hand was predicted to be neutral by several damaging effect prediction algorithms, so no definite cause for either the multiple deletions or disease phenotype could be established.

85

86

6

Conclusions

We used CSGE and sequencing to screen the entire mtDNA of 217 patients with maternally inherited diabetes mellitus, maternally inherited epilepsy, adult-onset ataxia and suspected mitochondrial disease. We discovered pathogenic heteroplasmic mtDNA mutations, such as m.8993T>C, that cause adult-onset ataxia and polyneuropathy. We also discovered multiple mtDNA mutations with disease associations and several novel unreported mtDNA mutations. The novel mutation m.7585insT was detected in a family with cardiomyopathy and suspected mitochondrial disease. We determined that haplogroup V occurs more frequently among patients with maternally inherited diabetes. Specific subhaplotypes such as H1b and U5a1 occur at a higher frequency among patients with maternally inherited diabetes mellitus and maternally inherited epilepsy (when compared to population controls). These subhaplotypes were found to harbour mildly deleterious variants such as m.15218T>C, m.16189T>C and m.3010G>A. Four different algorithms were used to predict that several other polymorphisms were damaging in effect. These risk variants are most likely population-specific, which makes careful selection of population matched controls crucial. This thesis emphasizes the fact that deleterious variation exists in mtDNA and shows that this variation can increase the risk of developing complex diseases. This can be most clearly detected in families with maternally inherited symptoms of mitochondrial dysfunction such as diabetes mellitus or epilepsy. We also determined that only a small fraction of patients with suspected mitochondrial disease (excluding the most common mtDNA mutations) actually harbour a clearly pathogenic mtDNA mutation. Multiple deletions occurred in ~ 1% of patients with suspected mitochondrial disease, half of which were associated with a POLG1 variant.

87

88

References Achilli A, Olivieri A, Pala M, Hooshiar Kashani B, Carossa V, Perego UA, Gandini F, Santoro A, Battaglia V, Grugni V, Lancioni H, Sirolla C, Bonfigli AR, Cormio A, Boemi M, Testa I, Semino O, Ceriello A, Spazzafumo L, Gadaleta MN, Marra M, Testa R, Franceschi C & Torroni A (2011) Mitochondrial DNA backgrounds might modulate diabetes complications rather than T2DM as a whole. PLoS One 6(6): e21029. Adzhubei IA, Schmidt S, Peshkin L, Ramensky VE, Gerasimova A, Bork P, Kondrashov AS & Sunyaev SR (2010) A method and server for predicting damaging missense mutations. Nat Methods 7(4): 248–249. Ahmadinejad N, Dagan T, Gruenheit N, Martin W & Gabaldon T (2010) Evolution of spliceosomal introns following endosymbiotic gene transfer. BMC Evol Biol 10: 57– 2148–10–57. Andrews RM, Kubacka I, Chinnery PF, Lightowlers RN, Turnbull DM & Howell N (1999) Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA. Nat Genet 23(2): 147. Angelini C, Bello L, Spinazzi M & Ferrati C (2009) Mitochondrial disorders of the nuclear genome. Acta Myol 28(1): 16–23. Aral C, Akkiprik M, Caglayan S, Atabey Z, Ozisik G, Bekiroglu N & Ozer A (2011) Investigation of relationship of the mitochondrial DNA 16189 T>C polymorphism with metabolic syndrome and its associated clinical parameters in Turkish patients. Hormones (Athens) 10(4): 298–303. Arredondo JJ, Gallardo ME, Garcia-Pavia P, Domingo V, Breton B, Garcia-Silva MT, Sedano MJ, Martin MA, Arenas J, Cervera M, Garesse R & Bornstein B (2012) Mitochondrial tRNA valine as a recurrent target for mutations involved in mitochondrial cardiomyopathies. Mitochondrion 12(2): 357–362. Arunkumar KP, Metta M & Nagaraju J (2006) Molecular phylogeny of silkmoths reveals the origin of domesticated silkmoth, Bombyx mori from Chinese Bombyx mandarina and paternal inheritance of Antheraea proylei mitochondrial DNA. Mol Phylogenet Evol 40(2): 419–427. Autere J, Moilanen JS, Finnilä S, Soininen H, Mannermaa A, Hartikainen P, Hallikainen M & Majamaa K (2004) Mitochondrial DNA polymorphisms as risk factors for Parkinson's disease and Parkinson's disease dementia. Hum Genet 115(1): 29–35. Ayed IB, Chamkha I, Mkaouar-Rebai E, Kammoun T, Mezghani N, Chabchoub I, Aloulou H, Hachicha M & Fakhfakh F (2011) A Tunisian patient with Pearson syndrome harboring the 4.977kb common deletion associated to two novel large-scale mitochondrial deletions. Biochem Biophys Res Commun 411(2): 381–386. Baeta M, Nunez C, Sosa C, Bolea M, Casalod Y, Gonzalez-Andrade F, Roewer L & Martinez-Jarreta B (2012) Mitochondrial diversity in Amerindian Kichwa and Mestizo populations from Ecuador. Int J Legal Med 126(2): 299–302. Bandelt HJ, Forster P & Rohl A (1999) Median-joining networks for inferring intraspecific phylogenies. Mol Biol Evol 16(1): 37–48. 89

Bandelt HJ, Forster P, Sykes BC & Richards MB (1995) Mitochondrial portraits of human populations using median networks. Genetics 141(2): 743–753. Barbujani G, Bertorelle G & Chikhi L (1998) Evidence for Paleolithic and Neolithic gene flow in Europe. Am J Hum Genet 62(2): 488–492. Batini C, Ferri G, Destro-Bisol G, Brisighelli F, Luiselli D, Sanchez-Diz P, Rocha J, Simonson T, Brehm A, Montano V, Elwali NE, Spedini G, D'Amato ME, Myres N, Ebbesen P, Comas D & Capelli C (2011) Signatures of the preagricultural peopling processes in sub-Saharan Africa as revealed by the phylogeography of early Y chromosome lineages. Mol Biol Evol 28(9): 2603–2613. Behar DM, van Oven M, Rosset S, Metspalu M, Loogvali EL, Silva NM, Kivisild T, Torroni A & Villems R (2012) A "Copernican" reassessment of the human mitochondrial DNA tree from its root. Am J Hum Genet 90(4): 675–684. Behar DM, Villems R, Soodyall H, Blue-Smith J, Pereira L, Metspalu E, Scozzari R, Makkan H, Tzur S, Comas D, Bertranpetit J, Quintana-Murci L, Tyler-Smith C, Wells RS, Rosset S & Genographic Consortium (2008) The dawn of human matrilineal diversity. Am J Hum Genet 82(5): 1130–1140. Benton M, Macartney-Coxson D, Eccles D, Griffiths L, Chambers G & Lea R (2012) Complete mitochondrial genome sequencing reveals novel haplotypes in a Polynesian population. PLoS One 7(4): e35026. Bhat A, Koul A, Sharma S, Rai E, Bukhari SI, Dhar MK & Bamezai RN (2007) The possible role of 10398A and 16189C mtDNA variants in providing susceptibility to T2DM in two North Indian populations: a replicative study. Hum Genet 120(6): 821– 826. Bindoff LA (2011) Mitochondrial function and pathology in status epilepticus. Epilepsia 52 Suppl 8: 6–7. Bindoff LA & Engelsen BA (2012) Mitochondrial diseases and epilepsy. Epilepsia 53 Suppl 4: 92–97. Bohlega S, Van Goethem G, Al Semari A, Lofgren A, Al Hamed M, Van Broeckhoven C & Kambouris M (2009) Novel Twinkle gene mutation in autosomal dominant progressive external ophthalmoplegia and multisystem failure. Neuromuscul Disord 19(12): 845–848. Bolszak M, Anttonen AK, Komulainen T, Hinttala R, Pakanen S, Sormunen R, Herva R, Lehesjoki AE, Majamaa K, Rantala H & Uusimaa J (2009) Digenic mutations in severe myoclonic epilepsy of infancy. Epilepsy Res 85(2–3): 300–304. Bosley TM, Brodsky MC, Glasier CM & Abu-Amero KK (2008) Sporadic bilateral optic neuropathy in children: the role of mitochondrial abnormalities. Invest Ophthalmol Vis Sci 49(12): 5250–5256. Bowmaker M, Yang MY, Yasukawa T, Reyes A, Jacobs HT, Huberman JA & Holt IJ (2003) Mammalian mitochondrial DNA replicates bidirectionally from an initiation zone. J Biol Chem 278(51): 50961–50969. Breen MS & Kondrashov FA (2010) Mitochondrial pathogenic mutations are populationspecific. Biol Direct 5: 68.

90

Breton S, Beaupre HD, Stewart DT, Hoeh WR & Blier PU (2007) The unusual system of doubly uniparental inheritance of mtDNA: isn't one enough? Trends Genet 23(9): 465–474. Bromberg Y, Yachdav G & Rost B (2008) SNAP predicts effect of mutations on protein function. Bioinformatics 24(20): 2397–2398. Brown TA, Cecconi C, Tkachuk AN, Bustamante C & Clayton DA (2005) Replication of mitochondrial DNA occurs by strand displacement with alternative light-strand origins, not via a strand-coupled mechanism. Genes Dev 19(20): 2466–2476. Brown W, George M & Wilson AC (1979) Rapid evolution of animal mitochondrial DNA. Proc Natl Acad Sci U S A 76: 1967. Bua E, Johnson J, Herbst A, Delong B, McKenzie D, Salamat S & Aiken JM (2006) Mitochondrial DNA-deletion mutations accumulate intracellularly to detrimental levels in aged human skeletal muscle fibers. Am J Hum Genet 79(3): 469–480. Butler JM & Levin BC (1998) Forensic applications of mitochondrial DNA. Trends Biotechnol 16(4): 158–162. Cai W, Fu Q, Zhou X, Qu J, Tong Y & Guan MX (2008) Mitochondrial variants may influence the phenotypic manifestation of Leber's hereditary optic neuropathyassociated ND4 G11778A mutation. J Genet Genomics 35(11): 649–655. Camus MF, Clancy DJ & Dowling DK (2012) Mitochondria, maternal inheritance, and male aging. Curr Biol 22(18): 1717–1721. Cardaioli E, Malfatti E, Battisti C, Da Pozzo P, Rubegni A, Gallus GN, Malandrini A & Federico A (2012) Sporadic myopathy, myoclonus, leukoencephalopathy, neurosensory deafness, hypertrophic cardiomyopathy and insulin resistance associated with the mitochondrial 8306 T>C MTTK mutation. J Neurol Sci 321(1–2): 92–95. Carrozzo R, Murray J, Capuano O, Tessa A, Chichierchia G, Neglia MR, Capaldi RA & Santorelli FM (2000) A novel mtDNA mutation in the ATPase6 gene studied by E. coli modeling. Neurol Sci 21(5 Suppl): S983–4. Casteels K, Ong K, Phillips D, Bendall H & Pembrey M (1999) Mitochondrial 16189 variant, thinness at birth, and type-2 diabetes. ALSPAC study team. Avon Longitudinal Study of Pregnancy and Childhood. Lancet 353(9163): 1499–1500. Cesaroni E, Scarpelli M, Zamponi N, Polonara G & Zeviani M (2009) Mitochondrial encephalomyopathy lactic acidosis and strokelike episodes mimicking occipital idiopathic epilepsy. Pediatr Neurol 41(2): 131–134. Chalkia D (2009) Inheritance of mitochondrial DNA heteroplasmy: Does Kimura's solution work? Mitochondrial DNA : 1. Chandra D & Singh KK (2011) Genetic insights into OXPHOS defect and its role in cancer. Biochim Biophys Acta 1807(6): 620–625. Chatterjee A, Mambo E & Sidransky D (2006) Mitochondrial DNA mutations in human cancer. Oncogene 25(34): 4663–4674. Chen A, Raule N, Chomyn A & Attardi G (2012) Decreased reactive oxygen species production in cells with mitochondrial haplogroups associated with longevity. PLoS One 7(10): e46473.

91

Chinnery PF (1993) Mitochondrial Disorders Overview. In: Pagon RA, Bird TD, Dolan CR, Stephens K & Adam MP (eds) GeneReviews. Seattle (WA), University of Washington, Seattle. Chinnery PF, Elliott HR, Hudson G, Samuels DC & Relton CL (2012) Epigenetics, epidemiology and mitochondrial DNA diseases. Int J Epidemiol 41(1): 177–187. Chinnery PF, Howell N, Andrews RM & Turnbull DM (1999) Mitochondrial DNA analysis: polymorphisms and pathogenicity. J Med Genet 36(7): 505–510. Cho S, Lee YM, Choi YS, Yang HI, Jeon YE, Lee KE, Lim K, Kim HY, Seo SK & Lee BS (2011) Mitochondria DNA Polymorphisms Are Associated with Susceptibility to Endometriosis. DNA Cell Biol . Clark KM, Taylor RW, Johnson MA, Chinnery PF, Chrzanowska-Lightowlers ZM, Andrews RM, Nelson IP, Wood NW, Lamont PJ, Hanna MG, Lightowlers RN & Turnbull DM (1999) An mtDNA mutation in the initiation codon of the cytochrome C oxidase subunit II gene results in lower levels of the protein and a mitochondrial encephalomyopathy. Am J Hum Genet 64(5): 1330–1339. Comi GP, Bordoni A, Salani S, Franceschina L, Sciacco M, Prelle A, Fortunato F, Zeviani M, Napoli L, Bresolin N, Moggio M, Ausenda CD, Taanman JW & Scarlato G (1998) Cytochrome c oxidase subunit I microdeletion in a patient with motor neuron disease. Ann Neurol 43(1): 110–116. Coskun P, Wyrembak J, Schriner SE, Chen HW, Marciniack C, Laferla F & Wallace DC (2012) A mitochondrial etiology of Alzheimer and Parkinson disease. Biochim Biophys Acta 1820(5): 553–564. Das S, Bennett AJ, Sovio U, Ruokonen A, Martikainen H, Pouta A, Hartikainen AL, Franks S, Elliott P, Poulton J, Jarvelin MR & McCarthy MI (2007) Detailed analysis of variation at and around mitochondrial position 16189 in a large Finnish cohort reveals no significant associations with early growth or metabolic phenotypes at age 31 years. J Clin Endocrinol Metab 92(8): 3219–3223. de Andrade PB, Rubi B, Frigerio F, van den Ouweland JM, Maassen JA & Maechler P (2006) Diabetes-associated mitochondrial DNA mutation A3243G impairs cellular metabolic pathways necessary for beta cell function. Diabetologia 49(8): 1816–1826. De Luca A, Nasi M, Di Giambenedetto S, Cozzi-Lepri A, Pinti M, Marzocchetti A, Mussini C, Fabbiani M, Bracciale L, Cauda R & Cossarizza A (2012) Mitochondrial DNA haplogroups and incidence of lipodystrophy in HIV-infected patients on longterm antiretroviral therapy. J Acquir Immune Defic Syndr 59(2): 113–120. de Vries DD, van Engelen BG, Gabreels FJ, Ruitenbeek W & van Oost BA (1993) A second missense mutation in the mitochondrial ATPase 6 gene in Leigh's syndrome. Ann Neurol 34(3): 410–412. Di Fonzo A, Bordoni A, Crimi M, Sara G, Del Bo R, Bresolin N & Comi GP (2003) POLG mutations in sporadic mitochondrial disorders with multiple mtDNA deletions. Hum Mutat 22(6): 498–499. DiMauro S & Schon EA (2003) Mitochondrial respiratory-chain diseases. N Engl J Med 348(26): 2656–2668.

92

Ding WX & Yin XM (2012) Mitophagy: mechanisms, pathophysiological roles, and analysis. Biol Chem 393(7): 547–564. Dowling DK, Friberg U, Hailer F & Arnqvist G (2007) Intergenomic epistasis for fitness: within-population interactions between cytoplasmic and nuclear genes in Drosophila melanogaster. Genetics 175(1): 235–244. Dowling DK, Friberg U & Lindell J (2008) Evolutionary implications of non-neutral mitochondrial genetic variation. Trends Ecol Evol 23(10): 546–554. Duarte A, Poderoso C, Cooke M, Soria G, Cornejo Maciel F, Gottifredi V & Podesta EJ (2012) Mitochondrial fusion is essential for steroid biosynthesis. PLoS One 7(9): e45829. Duchene S, Archer FI, Vilstrup J, Caballero S & Morin PA (2011) Mitogenome phylogenetics: the impact of using single regions and partitioning schemes on topology, substitution rate and divergence time estimation. PLoS One 6(11): e27138. Elliott HR, Samuels DC, Eden JA, Relton CL & Chinnery PF (2008) Pathogenic mitochondrial DNA mutations are common in the general population. Am J Hum Genet 83(2): 254–260. Elstner M, Schmidt C, Zingler VC, Prokisch H, Bettecken T, Elson JL, Rudolph G, Bender A, Halmagyi GM, Brandt T, Strupp M & Klopstock T (2008) Mitochondrial 12S rRNA susceptibility mutations in aminoglycoside-associated and idiopathic bilateral vestibulopathy. Biochem Biophys Res Commun 377(2): 379–383. Excoffier L, Laval G & Schneider S (2007) Arlequin (version 3.0): an integrated software package for population genetics data analysis. Evol Bioinform Online 1: 47–50. Federico A, Cardaioli E, Da Pozzo P, Formichi P, Gallus GN & Radi E (2012) Mitochondria, oxidative stress and neurodegeneration. J Neurol Sci . Ferreira M, Evangelista T, Almeida LS, Martins J, Macario MC, Martins E, Moleirinho A, Azevedo L, Vilarinho L & Santorelli FM (2011) Relative frequency of known causes of multiple mtDNA deletions: two novel POLG mutations. Neuromuscul Disord 21(7): 483–488. Ferrer-Costa C, Gelpi JL, Zamakola L, Parraga I, de la Cruz X & Orozco M (2005) PMUT: a web-based tool for the annotation of pathological mutations on proteins. Bioinformatics 21(14): 3176–3178. Finnilä S, Autere J, Lehtovirta M, Hartikainen P, Mannermaa A, Soininen H & Majamaa K (2001) Increased risk of sensorineural hearing loss and migraine in patients with a rare mitochondrial DNA variant 4336A>G in tRNAGln. J Med Genet 38(6): 400–405. Finnilä S, Hassinen IE, Ala-Kokko L & Majamaa K (2000) Phylogenetic network of the mtDNA haplogroup U in Northern Finland based on sequence analysis of the complete coding region by conformation-sensitive gel electrophoresis. Am J Hum Genet 66(3): 1017–1026. Finnilä S, Hassinen IE & Majamaa K (2001) Phylogenetic analysis of mitochondrial DNA in patients with an occipital stroke. Evaluation of mutations by using sequence data on the entire coding region. Mutat Res 458(1–2): 31–39. Finnilä S, Lehtonen MS & Majamaa K (2001) Phylogenetic network for European mtDNA. Am J Hum Genet 68(6): 1475–1484. 93

Finsterer J & Zarrouk Mahjoub S (2012) Epilepsy in mitochondrial disorders. Seizure 21(5): 316–321. Fonseca MM, Rocha S & Posada D (2012) Base-pairing versatility determines wobble sites in tRNA anticodons of vertebrate mitogenomes. PLoS One 7(5): e36605. Fontaine KM, Cooley JR & Simon C (2007) Evidence for paternal leakage in hybrid periodical cicadas (Hemiptera: Magicicada spp.). PLoS One 2(9): e892. Frey TG & Mannella CA (2000) The internal structure of mitochondria. Trends Biochem Sci 25(7): 319–324. Fukuhara N, Tokiguchi S, Shirakawa K & Tsubaki T (1980) Myoclonus epilepsy associated with ragged-red fibres (mitochondrial abnormalities ): disease entity or a syndrome? Light-and electron-microscopic studies of two cases and review of literature. J Neurol Sci 47(1): 117–133. Gabaldon T & Huynen MA (2007) From endosymbiont to host-controlled organelle: the hijacking of mitochondrial protein synthesis and metabolism. PLoS Comput Biol 3(11): e219. Galluzzi L, Kepp O, Trojel-Hansen C & Kroemer G (2012) Mitochondrial control of cellular life, stress, and death. Circ Res 111(9): 1198–1207. Garesse R (1988) Drosophila melanogaster mitochondrial DNA: gene organization and evolutionary considerations. Genetics 118(4): 649–663. Giles RE, Blanc H, Cann HM & Wallace DC (1980) Maternal inheritance of human mitochondrial DNA. Proc Natl Acad Sci U S A 77(11): 6715–6719. Giordano C, Perli E, Orlandi M, Pisano A, Tuppen HA, He L, Ierino R, Petruzziello L, Terzi A, Autore C, Petrozza V, Gallo P, Taylor RW & d'Amati G (2013) Cardiomyopathies due to homoplasmic mitochondrial tRNA mutations: morphologic and molecular features. Hum Pathol . Gissi C, Reyes A, Pesole G & Saccone C (2000) Lineage-specific evolutionary rate in mammalian mtDNA. Mol Biol Evol 17(7): 1022–1031. Goto Y, Nonaka I & Horai S (1990) A mutation in the tRNA(Leu)(UUR) gene associated with the MELAS subgroup of mitochondrial encephalomyopathies. Nature 348(6302): 651–653. Gu G, Reyes PE, Golden GT, Woltjer RL, Hulette C, Montine TJ & Zhang J (2002) Mitochondrial DNA deletions/rearrangements in parkinson disease and related neurodegenerative disorders. J Neuropathol Exp Neurol 61(7): 634–639. Guan MX, Enriquez JA, Fischel-Ghodsian N, Puranam RS, Lin CP, Maw MA & Attardi G (1998) The deafness-associated mitochondrial DNA mutation at position 7445, which affects tRNASer(UCN) precursor processing, has long-range effects on NADH dehydrogenase subunit ND6 gene expression. Mol Cell Biol 18(10): 5868–5879. Guan MX, Fischel-Ghodsian N & Attardi G (2001) Nuclear background determines biochemical phenotype in the deafness-associated mitochondrial 12S rRNA mutation. Hum Mol Genet 10(6): 573–580. Guo X, Popadin KY, Markuzon N, Orlov YL, Kraytsberg Y, Krishnan KJ, Zsurka G, Turnbull DM, Kunz WS & Khrapko K (2010) Repeats, longevity and the sources of mtDNA deletions: evidence from 'deletional spectra'. Trends Genet 26(8): 340–343. 94

Gyllensten U, Wharton D, Josefsson A & Wilson AC (1991) Paternal inheritance of mitochondrial DNA in mice. Nature 352(6332): 255–257. Hao da C, Feng Y, Xiao R & Xiao PG (2011) Non-neutral nonsynonymous single nucleotide polymorphisms in human ABC transporters: the first comparison of six prediction methods. Pharmacol Rep 63(4): 924–934. Hendrickson SL, Hutcheson HB, Ruiz-Pesini E, Poole JC, Lautenberger J, Sezgin E, Kingsley L, Goedert JJ, Vlahov D, Donfield S, Wallace DC & O'Brien SJ (2008) Mitochondrial DNA haplogroups influence AIDS progression. AIDS 22(18): 2429– 2439. Hinttala R, Smeets R, Moilanen JS, Ugalde C, Uusimaa J, Smeitink JA & Majamaa K (2006) Analysis of mitochondrial DNA sequences in patients with isolated or combined oxidative phosphorylation system deficiency. J Med Genet 43(11): 881–886. Hirano M, Nishino I, Nishigaki Y & Marti R (2006) Thymidine phosphorylase gene mutations cause mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). Intern Med 45(19): 1103. Holt IJ, Harding AE, Cooper JM, Schapira AH, Toscano A, Clark JB & Morgan-Hughes JA (1989) Mitochondrial myopathies: clinical and biochemical features of 30 patients with major deletions of muscle mitochondrial DNA. Ann Neurol 26(6): 699–708. Holt IJ, Harding AE, Petty RK & Morgan-Hughes JA (1990) A new mitochondrial disease associated with mitochondrial DNA heteroplasmy. Am J Hum Genet 46(3): 428–433. Holt IJ & Reyes A (2012) Human mitochondrial DNA replication. Cold Spring Harb Perspect Biol 4(12): 10.1101/cshperspect.a012971. Huoponen K, Lamminen T, Juvonen V, Aula P, Nikoskelainen E & Savontaus ML (1993) The spectrum of mitochondrial DNA mutations in families with Leber hereditary optic neuroretinopathy. Hum Genet 92(4): 379–384. Huynen MA, Duarte I & Szklarczyk R (2012) Loss, replacement and gain of proteins at the origin of the mitochondria. Biochim Biophys Acta . Ingman M & Gyllensten U (2006) mtDB: Human Mitochondrial Genome Database, a resource for population genetics and medical sciences. Nucleic Acids Res 34(Database issue): D749–51. Ingman M & Gyllensten U (2007) A recent genetic link between Sami and the Volga-Ural region of Russia. Eur J Hum Genet 15(1): 115–120. Isohanni P, Hakonen AH, Euro L, Paetau I, Linnankivi T, Liukkonen E, Wallden T, Luostarinen L, Valanne L, Paetau A, Uusimaa J, Lonnqvist T, Suomalainen A & Pihko H (2011) POLG1 manifestations in childhood. Neurology 76(9): 811–815. Jayadev S & Bird TD (2013) Hereditary ataxias: overview. Genet Med . Kärppä M, Herva R, Moslemi AR, Oldfors A, Kakko S & Majamaa K (2005) Spectrum of myopathic findings in 50 patients with the 3243A>G mutation in mitochondrial DNA. Brain 128(Pt 8): 1861–1869. Kasamatsu H, Grossman LI, Robberson DL, Watson R & Vinograd J (1974) The replication and structure of mitochondrial DNA in animal cells. Cold Spring Harb Symp Quant Biol 38: 281–288.

95

Kaukonen J, Juselius JK, Tiranti V, Kyttala A, Zeviani M, Comi GP, Keranen S, Peltonen L & Suomalainen A (2000) Role of adenine nucleotide translocator 1 in mtDNA maintenance. Science 289(5480): 782–785. Kere J (2001) Human population genetics: lessons from Finland. Annu Rev Genomics Hum Genet 2: 103–128. Kervinen M, Hinttala R, Helander HM, Kurki S, Uusimaa J, Finel M, Majamaa K & Hassinen IE (2006) The MELAS mutations 3946 and 3949 perturb the critical structure in a conserved loop of the ND1 subunit of mitochondrial complex I. Hum Mol Genet 15(17): 2543–2552. Khusnutdinova E, Gilyazova I, Ruiz-Pesini E, Derbeneva O, Khusainova R, Khidiyatova I, Magzhanov R & Wallace DC (2008) A mitochondrial etiology of neurodegenerative diseases: evidence from Parkinson's disease. Ann N Y Acad Sci 1147: 1–20. Kimura M (1979) The neutral theory of molecular evolution. Sci Am 241(5): 98–100, 102, 108 passim. Kimura M (1987) Molecular evolutionary clock and the neutral theory. J Mol Evol 26(1–2): 24–33. Kirches E (2011) LHON: Mitochondrial Mutations and More. Curr Genomics 12(1): 44– 54. Kirches E, Winkler K, Vielhaber S, Michael M, Warich-Kirches M, von Bossanyi P, Plate I, Kunz WS, Szibor R, Feistner H & Dietzmann K (1999) Mitochondrial tRNA(Cys) mutation A5823G in a patient with motor neuron disease and temporal lobe epilepsy. Pathobiology 67(4): 214–218. Kitao N & Hashimoto M (2012) Increased thermogenic capacity of brown adipose tissue under low temperature and its contribution to arousal from hibernation in Syrian hamsters. Am J Physiol Regul Integr Comp Physiol 302(1): R118–25. Kittles RA, Perola M, Peltonen L, Bergen AW, Aragon RA, Virkkunen M, Linnoila M, Goldman D & Long JC (1998) Dual origins of Finns revealed by Y chromosome haplotype variation. Am J Hum Genet 62(5): 1171–1179. Knapp M, Horsburgh KA, Prost S, Stanton JA, Buckley HR, Walter RK & Matisoo-Smith EA (2012) Complete mitochondrial DNA genome sequences from the first New Zealanders. Proc Natl Acad Sci U S A 109(45): 18350–18354. Kofler B, Mueller EE, Eder W, Stanger O, Maier R, Weger M, Haas A, Winker R, Schmut O, Paulweber B, Iglseder B, Renner W, Wiesbauer M, Aigner I, Santic D, Zimmermann FA, Mayr JA & Sperl W (2009) Mitochondrial DNA haplogroup T is associated with coronary artery disease and diabetic retinopathy: a case control study. BMC Med Genet 10: 35–2350–10–35. Komulainen T, Hinttala R, Kärppä M, Pajunen L, Finnilä S, Tuominen H, Rantala H, Hassinen I, Majamaa K & Uusimaa J (2010) POLG1 p.R722H mutation associated with multiple mtDNA deletions and a neurological phenotype. BMC Neurol 10: 29. Körkkö J, Annunen S, Pihlajamaa T, Prockop DJ & Ala-Kokko L (1998) Conformation sensitive gel electrophoresis for simple and accurate detection of mutations: comparison with denaturing gradient gel electrophoresis and nucleotide sequencing. Proc Natl Acad Sci U S A 95(4): 1681–1685. 96

Kujoth GC, Bradshaw PC, Haroon S & Prolla TA (2007) The role of mitochondrial DNA mutations in mammalian aging. PLoS Genet 3(2): e24. Kurland CG & Andersson SG (2000) Origin and evolution of the mitochondrial proteome. Microbiol Mol Biol Rev 64(4): 786–820. Kvist L, Martens J, Nazarenko AA & Orell M (2003) Paternal leakage of mitochondrial DNA in the great tit (Parus major). Mol Biol Evol 20(2): 243–247. Legros F, Chatzoglou E, Frachon P, Ogier De Baulny H, Laforet P, Jardel C, Godinot C & Lombes A (2001) Functional characterization of novel mutations in the human cytochrome b gene. Eur J Hum Genet 9(7): 510–518. Levinger L, Morl M & Florentz C (2004) Mitochondrial tRNA 3' end metabolism and human disease. Nucleic Acids Res 32(18): 5430–5441. Lightowlers RN & Chrzanowska-Lightowlers ZM (2010) Terminating human mitochondrial protein synthesis: a shift in our thinking. RNA Biol 7(3): 282–286. Lill R, Hoffmann B, Molik S, Pierik AJ, Rietzschel N, Stehling O, Uzarska MA, Webert H, Wilbrecht C & Mühlenhoff U (2012) The role of mitochondria in cellular iron–sulfur protein biogenesis and iron metabolism. Biochimica et Biophysica Acta (BBA) Molecular Cell Research 1823(9): 1491–1508. Lindal S, Lund I, Torbergsen T, Aasly J, Mellgren SI, Borud O & Monstad P (1992) Mitochondrial diseases and myopathies: a series of muscle biopsy specimens with ultrastructural changes in the mitochondria. Ultrastruct Pathol 16(3): 263–275. Liou CW, Chen JB, Tiao MM, Weng SW, Huang TL, Chuang JH, Chen SD, Chuang YC, Lee WC, Lin TK & Wang PW (2012) Mitochondrial DNA coding and control region variants as genetic risk factors for type 2 diabetes. Diabetes 61(10): 2642–2651. Liou CW, Lin TK, Chen JB, Tiao MM, Weng SW, Chen SD, Chuang YC, Chuang JH & Wang PW (2010) Association between a common mitochondrial DNA D-loop polycytosine variant and alteration of mitochondrial copy number in human peripheral blood cells. J Med Genet 47(11): 723–728. Liou CW, Lin TK, Huei Weng H, Lee CF, Chen TL, Wei YH, Chen SD, Chuang YC, Weng SW & Wang PW (2007) A common mitochondrial DNA variant and increased body mass index as associated factors for development of type 2 diabetes: Additive effects of genetic and environmental factors. J Clin Endocrinol Metab 92(1): 235–239. Logan DC (2006) The mitochondrial compartment. J Exp Bot 57(6): 1225–1243. Lopez-Gallardo E, Solano A, Herrero-Martin MD, Martinez-Romero I, Castano-Perez MD, Andreu AL, Herrera A, Lopez-Perez MJ, Ruiz-Pesini E & Montoya J (2009) NARP syndrome in a patient harbouring an insertion in the MT-ATP6 gene that results in a truncated protein. J Med Genet 46(1): 64–67. Lorente L, Iceta R, Martin MM, Lopez-Gallardo E, Sole-Violan J, Blanquer J, Labarta L, Diaz C, Jimenez A, Montoya J & Ruiz-Pesini E (2012) Survival and mitochondrial function in septic patients according to mitochondrial DNA haplogroup. Crit Care 16(1): R10.

97

Lu J, Li Z, Zhu Y, Yang A, Li R, Zheng J, Cai Q, Peng G, Zheng W, Tang X, Chen B, Chen J, Liao Z, Yang L, Li Y, You J, Ding Y, Yu H, Wang J, Sun D, Zhao J, Xue L, Wang J & Guan MX (2010a) Mitochondrial 12S rRNA variants in 1642 Han Chinese pediatric subjects with aminoglycoside-induced and nonsyndromic hearing loss. Mitochondrion 10(4): 380–390. Lu J, Qian Y, Li Z, Yang A, Zhu Y, Li R, Yang L, Tang X, Chen B, Ding Y, Li Y, You J, Zheng J, Tao Z, Zhao F, Wang J, Sun D, Zhao J, Meng Y & Guan MX (2010b) Mitochondrial haplotypes may modulate the phenotypic manifestation of the deafnessassociated 12S rRNA 1555A>G mutation. Mitochondrion 10(1): 69–81. Maasen JA (2008) Mitochondria, body fat and type 2 diabetes: what is the connection? Minerva Med 99(3): 241–251. Maassen JA, 't Hart LM & Ouwens DM (2007) Lessons that can be learned from patients with diabetogenic mutations in mitochondrial DNA: implications for common type 2 diabetes. Curr Opin Clin Nutr Metab Care 10(6): 693–697. Magner M, Szentivanyi K, Svandova I, Jesina P, Tesarova M, Honzik T & Zeman J (2011) Elevated CSF-lactate is a reliable marker of mitochondrial disorders in children even after brief seizures. Eur J Paediatr Neurol 15(2): 101–108. Majamaa K, Moilanen JS, Uimonen S, Remes AM, Salmela PI, Kärppä M, MajamaaVoltti KA, Rusanen H, Sorri M, Peuhkurinen KJ & Hassinen IE (1998) Epidemiology of A3243G, the mutation for mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes: prevalence of the mutation in an adult population. Am J Hum Genet 63(2): 447–454. Mannella CA (2006) Structure and dynamics of the mitochondrial inner membrane cristae. Biochim Biophys Acta 1763(5–6): 542–548. Mannella CA, Marko M & Buttle K (1997) Reconsidering mitochondrial structure: new views of an old organelle. Trends Biochem Sci 22(2): 37–38. Martikainen MH, Hinttala R, Röyttä M, Jääskeläinen S, Wendelin-Saarenhovi M, Parkkola R & Majamaa K (2012) Progressive external ophthalmoplegia in southwestern Finland: a clinical and genetic study. Neuroepidemiology 38(2): 114–119. McBride HM, Neuspiel M & Wasiak S (2006) Mitochondria: more than just a powerhouse. Curr Biol 16(14): R551–60. McFarland R, Elson JL, Taylor RW, Howell N & Turnbull DM (2004) Assigning pathogenicity to mitochondrial tRNA mutations: when "definitely maybe" is not good enough. Trends Genet 20(12): 591–596. McMenamin SK & Hadly EA (2012) Ancient DNA assessment of tiger salamander population in Yellowstone National Park. PLoS One 7(3): e32763. Meinilä M, Finnilä S & Majamaa K (2001) Evidence for mtDNA admixture between the Finns and the Saami. Hum Hered 52(3): 160–170. Meissner C, Bruse P, Mohamed SA, Schulz A, Warnk H, Storm T & Oehmichen M (2008) The 4977 bp deletion of mitochondrial DNA in human skeletal muscle, heart and different areas of the brain: a useful biomarker or more? Exp Gerontol 43(7): 645–652. Meusel MS & Moritz RF (1993) Transfer of paternal mitochondrial DNA during fertilization of honeybee (Apis mellifera L.) eggs. Curr Genet 24(6): 539–543. 98

Mishmar D & Zhidkov I (2010) Evolution and disease converge in the mitochondrion. Biochim Biophys Acta 1797(6–7): 1099–1104. Miwa S, Lawless C & von Zglinicki T (2008) Mitochondrial turnover in liver is fast in vivo and is accelerated by dietary restriction: application of a simple dynamic model. Aging Cell 7(6): 920–923. Mohlke KL, Jackson AU, Scott LJ, Peck EC, Suh YD, Chines PS, Watanabe RM, Buchanan TA, Conneely KN, Erdos MR, Narisu N, Enloe S, Valle TT, Tuomilehto J, Bergman RN, Boehnke M & Collins FS (2005) Mitochondrial polymorphisms and susceptibility to type 2 diabetes-related traits in Finns. Hum Genet 118(2): 245–254. Moreau C, Vezina H, Jomphe M, Lavoie EM, Roy-Gagnon MH & Labuda D (2011) When genetics and genealogies tell different stories-maternal lineages in Gaspesia. Ann Hum Genet 75(2): 247–254. Moslemi AR, Darin N, Tulinius M, Oldfors A & Holme E (2005) Two new mutations in the MTATP6 gene associated with Leigh syndrome. Neuropediatrics 36(5): 314–318. Mueller EE, Brunner SM, Mayr JA, Stanger O, Sperl W & Kofler B (2012) Functional Differences between Mitochondrial Haplogroup T and Haplogroup H in HEK293 Cybrid Cells. PLoS One 7(12): e52367. Mueller EE, Eder W, Ebner S, Schwaiger E, Santic D, Kreindl T, Stanger O, Paulweber B, Iglseder B, Oberkofler H, Maier R, Mayr JA, Krempler F, Weitgasser R, Patsch W, Sperl W & Kofler B (2011) The mitochondrial T16189C polymorphism is associated with coronary artery disease in Middle European populations. PLoS One 6(1): e16455. Musumeci O, Andreu AL, Shanske S, Bresolin N, Comi GP, Rothstein R, Schon EA & DiMauro S (2000) Intragenic inversion of mtDNA: a new type of pathogenic mutation in a patient with mitochondrial myopathy. Am J Hum Genet 66(6): 1900–1904. Mutai H, Kouike H, Teruya E, Takahashi-Kodomari I, Kakishima H, Taiji H, Usami S, Okuyama T & Matsunaga T (2011) Systematic analysis of mitochondrial genes associated with hearing loss in the Japanese population: dHPLC reveals a new candidate mutation. BMC Med Genet 12: 135–2350–12–135. Nachman MW, Brown WM, Stoneking M & Aquadro CF (1995) Nonneutral mitochondrial DNA variation in humans and chimpanzees. Genetics 142: 953. Naimi M, Bannwarth S, Procaccio V, Pouget J, Desnuelle C, Pellissier JF, Rotig A, Munnich A, Calvas P, Richelme C, Jonveaux P, Castelnovo G, Simon M, Clanet M, Wallace D & Paquis-Flucklinger V (2006) Molecular analysis of ANT1, TWINKLE and POLG in patients with multiple deletions or depletion of mitochondrial DNA by a dHPLC-based assay. Eur J Hum Genet 14(8): 917–922. Nakamura M, Yabe I, Sudo A, Hosoki K, Yaguchi H, Saitoh S & Sasaki H (2010) MERRF/MELAS overlap syndrome: a double pathogenic mutation in mitochondrial tRNA genes. J Med Genet 47(10): 659–664. Nakayama T & Archibald JM (2012) Evolving a photosynthetic organelle. BMC Biol 10: 35. Neale DB, Marshall KA & Sederoff RR (1989) Chloroplast and mitochondrial DNA are paternally inherited in Sequoia sempervirens D. Don Endl. Proc Natl Acad Sci U S A 86(23): 9347–9349. 99

Neves AG & Serva M (2012) Extremely rare interbreeding events can explain neanderthal DNA in living humans. PLoS One 7(10): e47076. Ng PC & Henikoff S (2001) Predicting deleterious amino acid substitutions. Genome Res 11(5): 863–874. Niemi AK, Hervonen A, Hurme M, Karhunen PJ, Jylhä M & Majamaa K (2003) Mitochondrial DNA polymorphisms associated with longevity in a Finnish population. Hum Genet 112(1): 29–33. Niemi AK, Moilanen JS, Tanaka M, Hervonen A, Hurme M, Lehtimäki T, Arai Y, Hirose N & Majamaa K (2005) A combination of three common inherited mitochondrial DNA polymorphisms promotes longevity in Finnish and Japanese subjects. Eur J Hum Genet 13(2): 166–170. Nikoskelainen EK, Marttila RJ, Huoponen K, Juvonen V, Lamminen T, Sonninen P & Savontaus ML (1995) Leber's "plus": neurological abnormalities in patients with Leber's hereditary optic neuropathy. J Neurol Neurosurg Psychiatry 59(2): 160–164. Nishigaki Y, Fuku N & Tanaka M (2010) Mitochondrial haplogroups associated with lifestyle-related diseases and longevity in the Japanese population. Geriatr Gerontol Int 10 Suppl 1: S221–35. Norio R (2003a) Finnish Disease Heritage I: characteristics, causes, background. Hum Genet 112(5–6): 441–456. Norio R (2003b) Finnish Disease Heritage II: population prehistory and genetic roots of Finns. Hum Genet 112(5–6): 457–469. Ojala D, Crews S, Montoya J, Gelfand R & Attardi G (1981) A small polyadenylated RNA (7 S RNA), containing a putative ribosome attachment site, maps near the origin of human mitochondrial DNA replication. J Mol Biol 150(2): 303–314. Palanichamy MG, Sun C, Agrawal S, Bandelt HJ, Kong QP, Khan F, Wang CY, Chaudhuri TK, Palla V & Zhang YP (2004) Phylogeny of mitochondrial DNA macrohaplogroup N in India, based on complete sequencing: implications for the peopling of South Asia. Am J Hum Genet 75(6): 966–978. Palmieri VO, De Rasmo D, Signorile A, Sardanelli AM, Grattagliano I, Minerva F, Cardinale G, Portincasa P, Papa S & Palasciano G (2011) T16189C mitochondrial DNA variant is associated with metabolic syndrome in Caucasian subjects. Nutrition 27(7–8): 773–777. Palo JU, Ulmanen I, Lukka M, Ellonen P & Sajantila A (2009) Genetic markers and population history: Finland revisited. Eur J Hum Genet 17(10): 1336–1346. Park KS, Chan JC, Chuang LM, Suzuki S, Araki E, Nanjo K, Ji L, Ng M, Nishi M, Furuta H, Shirotani T, Ahn BY, Chung SS, Min HK, Lee SW, Kim JH, Cho YM, Lee HK & Study Group of Molecular Diabetology in Asia (2008) A mitochondrial DNA variant at position 16189 is associated with type 2 diabetes mellitus in Asians. Diabetologia 51(4): 602–608. Parker E, Phillips DI, Cockington RA, Cull C & Poulton J (2005) A common mitchondrial DNA variant is associated with thinness in mothers and their 20-yr-old offspring. Am J Physiol Endocrinol Metab 289(6): E1110-4.

100

Pätsi J, Maliniemi P, Pakanen S, Hinttala R, Uusimaa J, Majamaa K, Nyström T, Kervinen M & Hassinen IE (2012) LHON/MELAS overlap mutation in ND1 subunit of mitochondrial complex I affects ubiquinone binding as revealed by modeling in Escherichia coli NDH-1. Biochim Biophys Acta 1817(2): 312–318. Pavlakis SG, Phillips PC, DiMauro S, De Vivo DC & Rowland LP (1984) Mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes: a distinctive clinical syndrome. Ann Neurol 16(4): 481–488. Payne BA, Wilson IJ, Yu-Wai-Man P, Coxhead J, Deehan D, Horvath R, Taylor RW, Samuels DC, Santibanez-Koref M & Chinnery PF (2012) Universal heteroplasmy of human mitochondrial DNA. Hum Mol Genet . Peterson CM, Johannsen DL & Ravussin E (2012) Skeletal muscle mitochondria and aging: a review. J Aging Res 2012: 194821. Petros JA, Baumann AK, Ruiz-Pesini E, Amin MB, Sun CQ, Hall J, Lim S, Issa MM, Flanders WD, Hosseini SH, Marshall FF & Wallace DC (2005) mtDNA mutations increase tumorigenicity in prostate cancer. Proc Natl Acad Sci U S A 102(3): 719–724. Pett W, Ryan JF, Pang K, Mullikin JC, Martindale MQ, Baxevanis AD & Lavrov DV (2011) Extreme mitochondrial evolution in the ctenophore Mnemiopsis leidyi: Insight from mtDNA and the nuclear genome. Mitochondrial DNA 22(4): 130–142. Pfeffer G & Chinnery PF (2011) Diagnosis and treatment of mitochondrial myopathies. Ann Med . Pfeffer G, Sirrs S, Wade NK & Mezei MM (2011) Multisystem disorder in late-onset chronic progressive external ophthalmoplegia. Can J Neurol Sci 38(1): 119–123. Pichaud N, Ballard JW, Tanguay RM & Blier PU (2012) Naturally occurring mitochondrial DNA haplotypes exhibit metabolic differences: insight into functional properties of mitochondria. Evolution 66(10): 3189–3197. Pohjoismäki JL & Goffart S (2011) Of circles, forks and humanity: Topological organisation and replication of mammalian mitochondrial DNA. Bioessays 33(4): 290–299. Poulton J, Luan J, Macaulay V, Hennings S, Mitchell J & Wareham NJ (2002a) Type 2 diabetes is associated with a common mitochondrial variant: evidence from a population-based case-control study. Hum Mol Genet 11(13): 1581–1583. Poulton J, Marchington DR, Scott-Brown M, Phillips DI & Hagelberg E (1998) Does a common mitochondrial DNA polymorphism underlie susceptibility to diabetes and the thrifty genotype? Trends Genet 14(10): 387–389. Prentice AM (2006) The emerging epidemic of obesity in developing countries. Int J Epidemiol 35(1): 93–99. Prezant TR, Agapian JV, Bohlman MC, Bu X, Oztas S, Qiu WQ, Arnos KS, Cortopassi GA, Jaber L & Rotter JI (1993) Mitochondrial ribosomal RNA mutation associated with both antibiotic-induced and non-syndromic deafness. Nat Genet 4(3): 289–294. Putz J, Dupuis B, Sissler M & Florentz C (2007) Mamit-tRNA, a database of mammalian mitochondrial tRNA primary and secondary structures. RNA 13(8): 1184–1190.

101

Raap AK, Jahangir Tafrechi RS, van de Rijke FM, Pyle A, Wahlby C, Szuhai K, Ravelli RB, de Coo RF, Rajasimha HK, Nilsson M, Chinnery PF, Samuels DC & Janssen GM (2012) Non-Random mtDNA Segregation Patterns Indicate a Metastable Heteroplasmic Segregation Unit in m.3243A>G Cybrid Cells. PLoS One 7(12): e52080. Raffaello A, De Stefani D & Rizzuto R (2012) The mitochondrial Ca(2+) uniporter. Cell Calcium 52(1): 16–21. Rahman S (2012) Mitochondrial disease and epilepsy. Dev Med Child Neurol 54(5): 397– 406. Rand DM (1993) Endotherms, ectotherms, and mitochondrial genome-size variation. J Mol Evol 37(3): 281–295. Rand DM, Clark AG & Kann LM (2001) Sexually antagonistic cytonuclear fitness interactions in Drosophila melanogaster. Genetics 159(1): 173–187. Rea SL, Graham BH, Nakamaru-Ogiso E, Kar A & Falk MJ (2010) Bacteria, yeast, worms, and flies: exploiting simple model organisms to investigate human mitochondrial diseases. Dev Disabil Res Rev 16(2): 200–218. Reidla M, Kivisild T, Metspalu E, Kaldma K, Tambets K, Tolk HV, Parik J, Loogvali EL, Derenko M, Malyarchuk B, Bermisheva M, Zhadanov S, Pennarun E, Gubina M, Golubenko M, Damba L, Fedorova S, Gusar V, Grechanina E, Mikerezi I, Moisan JP, Chaventre A, Khusnutdinova E, Osipova L, Stepanov V, Voevoda M, Achilli A, Rengo C, Rickards O, De Stefano GF, Papiha S, Beckman L, Janicijevic B, Rudan P, Anagnou N, Michalodimitrakis E, Koziel S, Usanga E, Geberhiwot T, Herrnstadt C, Howell N, Torroni A & Villems R (2003) Origin and diffusion of mtDNA haplogroup X. Am J Hum Genet 73(5): 1178–1190. Reiling E, Ling C, Uitterlinden AG, Van't Riet E, Welschen LM, Ladenvall C, Almgren P, Lyssenko V, Nijpels G, van Hove EC, Maassen JA, de Geus EJ, Boomsma DI, Dekker JM, Groop L, Willemsen G & 't Hart LM (2010) The association of mitochondrial content with prevalent and incident type 2 diabetes. J Clin Endocrinol Metab 95(4): 1909–1915. Reiling E, van Vliet-Ostaptchouk JV, van 't Riet E, van Haeften TW, Arp PA, Hansen T, Kremer D, Groenewoud MJ, van Hove EC, Romijn JA, Smit JW, Nijpels G, Heine RJ, Uitterlinden AG, Pedersen O, Slagboom PE, Maassen JA, Hofker MH, 't Hart LM & Dekker JM (2009) Genetic association analysis of 13 nuclear-encoded mitochondrial candidate genes with type II diabetes mellitus: the DAMAGE study. Eur J Hum Genet 17(8): 1056–1062. Remes AM, Majamaa-Voltti K, Kärppä M, Moilanen JS, Uimonen S, Helander H, Rusanen H, Salmela PI, Sorri M, Hassinen IE & Majamaa K (2005) Prevalence of large-scale mitochondrial DNA deletions in an adult Finnish population. Neurology 64(6): 976–981. Rizzuto R, De Stefani D, Raffaello A & Mammucari C (2012) Mitochondria as sensors and regulators of calcium signalling. Nat Rev Mol Cell Biol 13(9): 566–578.

102

Robberson DL, Kasamatsu H & Vinograd J (1972) Replication of mitochondrial DNA. Circular replicative intermediates in mouse L cells. Proc Natl Acad Sci U S A 69(3): 737–741. Ronchi D, Di Fonzo A, Lin W, Bordoni A, Liu C, Fassone E, Pagliarani S, Rizzuti M, Zheng L, Filosto M, Ferro MT, Ranieri M, Magri F, Peverelli L, Li H, Yuan YC, Corti S, Sciacco M, Moggio M, Bresolin N, Shen B & Comi GP (2013) Mutations in DNA2 Link Progressive Myopathy to Mitochondrial DNA Instability. Am J Hum Genet . Roos S, Darin N, Kollberg G, Andersson Gronlund M, Tulinius M, Holme E, Moslemi AR & Oldfors A (2012) A novel mitochondrial tRNA Arg mutation resulting in an anticodon swap in a patient with mitochondrial encephalomyopathy. Eur J Hum Genet . Rosing HS, Hopkins LC, Wallace DC, Epstein CM & Weidenheim K (1985) Maternally inherited mitochondrial myopathy and myoclonic epilepsy. Ann Neurol 17(3): 228– 237. Rubino F, Piredda R, Calabrese FM, Simone D, Lang M, Calabrese C, Petruzzella V, Tommaseo-Ponzetta M, Gasparre G & Attimonelli M (2012) HmtDB, a genomic resource for mitochondrion-based human variability studies. Nucleic Acids Res 40(Database issue): D1150–9. Ruiz-Pesini E, Lott MT, Procaccio V, Poole JC, Brandon MC, Mishmar D, Yi C, Kreuziger J, Baldi P & Wallace DC (2007) An enhanced MITOMAP with a global mtDNA mutational phylogeny. Nucleic Acids Res 35(Database issue): D823–8. Rycovska A, Valach M, Tomaska L, Bolotin-Fukuhara M & Nosek J (2004) Linear versus circular mitochondrial genomes: intraspecies variability of mitochondrial genome architecture in Candida parapsilosis. Microbiology 150(Pt 5): 1571–1580. Sajantila A, Lahermo P, Anttinen T, Lukka M, Sistonen P, Savontaus ML, Aula P, Beckman L, Tranebjaerg L, Gedde-Dahl T, Issel-Tarver L, DiRienzo A & Pääbo S (1995) Genes and languages in Europe: an analysis of mitochondrial lineages. Genome Res 5(1): 42–52. Samuels DC, Schon EA & Chinnery PF (2004) Two direct repeats cause most human mtDNA deletions. Trends Genet 20(9): 393–398. Saneto RP, Lee IC, Koenig MK, Bao X, Weng SW, Naviaux RK & Wong LJ (2010) POLG DNA testing as an emerging standard of care before instituting valproic acid therapy for pediatric seizure disorders. Seizure 19(3): 140–146. Santorelli FM, Shanske S, Jain KD, Tick D, Schon EA & DiMauro S (1994) A T-->C mutation at nt 8993 of mitochondrial DNA in a child with Leigh syndrome. Neurology 44(5): 972–974. Santorelli FM, Tanji K, Sano M, Shanske S, El-Shahawi M, Kranz-Eble P, DiMauro S & De Vivo DC (1997) Maternally inherited encephalopathy associated with a singlebase insertion in the mitochondrial tRNATrp gene. Ann Neurol 42(2): 256–260.

103

Santoro A, Balbi V, Balducci E, Pirazzini C, Rosini F, Tavano F, Achilli A, Siviero P, Minicuci N, Bellavista E, Mishto M, Salvioli S, Marchegiani F, Cardelli M, Olivieri F, Nacmias B, Chiamenti AM, Benussi L, Ghidoni R, Rose G, Gabelli C, Binetti G, Sorbi S, Crepaldi G, Passarino G, Torroni A & Franceschi C (2010) Evidence for subhaplogroup h5 of mitochondrial DNA as a risk factor for late onset Alzheimer's disease. PLoS One 5(8): e12037. Schaefer AM, McFarland R, Blakely EL, He L, Whittaker RG, Taylor RW, Chinnery PF & Turnbull DM (2008) Prevalence of mitochondrial DNA disease in adults. Ann Neurol 63(1): 35–39. Schaefer C, Bromberg Y, Achten D & Rost B (2012) Disease-related mutations predicted to impact protein function. BMC Genomics 13 Suppl 4: S11–2164–13–S4–S11. Schon EA, DiMauro S & Hirano M (2012) Human mitochondrial DNA: roles of inherited and somatic mutations. Nat Rev Genet 13(12): 878–890. Schwaiger M, Herzog V & Neupert W (1987) Characterization of translocation contact sites involved in the import of mitochondrial proteins. J Cell Biol 105(1): 235–246. Schwartz M & Vissing J (2003) Paternal inheritance of mitochondrial DNA. Ugeskr Laeger 165(38): 3627–3630. Segre AV, DIAGRAM Consortium, MAGIC investigators, Groop L, Mootha VK, Daly MJ & Altshuler D (2010) Common inherited variation in mitochondrial genes is not enriched for associations with type 2 diabetes or related glycemic traits. PLoS Genet 6(8): 10.1371/journal.pgen.1001058. Seneca S, Goemans N, Van Coster R, Givron P, Reybrouck T, Sciot R, Meulemans A, Smet J & Van Hove JL (2005) A mitochondrial tRNA aspartate mutation causing isolated mitochondrial myopathy. Am J Med Genet A 137(2): 170–175. Shadel GS & Clayton DA (1997) Mitochondrial DNA maintenance in vertebrates. Annu Rev Biochem 66: 409–435. Shoffner JM, Bialer MG, Pavlakis SG, Lott M, Kaufman A, Dixon J, Teichberg S & Wallace DC (1995) Mitochondrial encephalomyopathy associated with a single nucleotide pair deletion in the mitochondrial tRNALeu(UUR) gene. Neurology 45(2): 286–292. Shoffner JM, Lott MT, Lezza AM, Seibel P, Ballinger SW & Wallace DC (1990) Myoclonic epilepsy and ragged-red fiber disease (MERRF) is associated with a mitochondrial DNA tRNA(Lys) mutation. Cell 61(6): 931–937. Shtilbans A, El-Schahawi M, Malkin E, Shanske S, Musumeci O & DiMauro S (1999) A novel mutation in the mitochondrial DNA transfer ribonucleic acidAsp gene in a child with myoclonic epilepsy and psychomotor regression. J Child Neurol 14(9): 610–613. Shu L, Zhang YM, Huang XX, Chen CY & Zhang XN (2012) Complete mitochondrial DNA sequence analysis in two southern Chinese pedigrees with Leber hereditary optic neuropathy revealed secondary mutations along with the primary mutation. Int J Ophthalmol 5(1): 28–31. Simon DK, Tarnopolsky MA, Greenamyre JT & Johns DR (2001) A frameshift mitochondrial complex I gene mutation in a patient with dystonia and cataracts: is the mutation pathogenic? J Med Genet 38(1): 58–61. 104

Smith DR, Kayal E, Yanagihara AA, Collins AG, Pirro S & Keeling PJ (2012) First complete mitochondrial genome sequence from a box jellyfish reveals a highly fragmented linear architecture and insights into telomere evolution. Genome Biol Evol 4(1): 52–58. Sofou K, Moslemi AR, Kollberg G, Bjarnadottir I, Oldfors A, Nennesmo I, Holme E, Tulinius M & Darin N (2012) Phenotypic and genotypic variability in Alpers syndrome. Eur J Paediatr Neurol 16(4): 379–389. Sola S, Morgado AL & Rodrigues CM (2012) Death receptors and mitochondria: Two prime triggers of neural apoptosis and differentiation. Biochim Biophys Acta . Soleimanpour-Lichaei HR, Kuhl I, Gaisne M, Passos JF, Wydro M, Rorbach J, Temperley R, Bonnefoy N, Tate W, Lightowlers R & Chrzanowska-Lightowlers Z (2007) mtRF1a is a human mitochondrial translation release factor decoding the major termination codons UAA and UAG. Mol Cell 27(5): 745–757. Stewart JD, Horvath R, Baruffini E, Ferrero I, Bulst S, Watkins PB, Fontana RJ, Day CP & Chinnery PF (2010) Polymerase gamma gene POLG determines the risk of sodium valproate-induced liver toxicity. Hepatology 52(5): 1791–1796. Sunami Y, Sugaya K, Chihara N, Goto Y & Matsubara S (2011) Variable phenotypes in a family with mitochondrial encephalomyopathy harboring a 3291T > C mutation in mitochondrial DNA. Neurol Sci 32(5): 861–864. T' Hart LM, Hansen T, Rietveld I, Dekker JM, Nijpels G, Janssen GM, Arp PA, Uitterlinden AG, Jorgensen T, Borch-Johnsen K, Pols HA, Pedersen O, van Duijn CM, Heine RJ & Maassen JA (2005) Evidence that the mitochondrial leucyl tRNA synthetase (LARS2) gene represents a novel type 2 diabetes susceptibility gene. Diabetes 54(6): 1892–1895. Tachibana M, Amato P, Sparman M, Woodward J, Sanchis DM, Ma H, Gutierrez NM, Tippner-Hedges R, Kang E, Lee HS, Ramsey C, Masterson K, Battaglia D, Lee D, Wu D, Jensen J, Patton P, Gokhale S, Stouffer R & Mitalipov S (2012) Towards germline gene therapy of inherited mitochondrial diseases. Nature . Tambets K, Rootsi S, Kivisild T, Help H, Serk P, Loogväli EL, Tolk HV, Reidla M, Metspalu E, Pliss L, Balanovsky O, Pshenichnov A, Balanovska E, Gubina M, Zhadanov S, Osipova L, Damba L, Voevoda M, Kutuev I, Bermisheva M, Khusnutdinova E, Gusar V, Grechanina E, Parik J, Pennarun E, Richard C, Chaventre A, Moisan JP, Barac L, Pericic M, Rudan P, Terzic R, Mikerezi I, Krumina A, Baumanis V, Koziel S, Rickards O, De Stefano GF, Anagnou N, Pappa KI, Michalodimitrakis E, Ferak V, Furedi S, Komel R, Beckman L & Villems R (2004) The western and eastern roots of the Saami--the story of genetic "outliers" told by mitochondrial DNA and Y chromosomes. Am J Hum Genet 74(4): 661–682. Tanaka M, Cabrera VM, Gonzalez AM, Larruga JM, Takeyasu T, Fuku N, Guo LJ, Hirose R, Fujita Y, Kurata M, Shinoda K, Umetsu K, Yamada Y, Oshida Y, Sato Y, Hattori N, Mizuno Y, Arai Y, Hirose N, Ohta S, Ogawa O, Tanaka Y, Kawamori R, ShamotoNagai M, Maruyama W, Shimokata H, Suzuki R & Shimodaira H (2004) Mitochondrial genome variation in eastern Asia and the peopling of Japan. Genome Res 14(10A): 1832–1850. 105

Tang S, Dimberg EL, Milone M & Wong LJ (2012) Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE)-like phenotype: an expanded clinical spectrum of POLG1 mutations. J Neurol 259(5): 862–868. Tanhauser SM & Laipis PJ (1995) Multiple deletions are detectable in mitochondrial DNA of aging mice. J Biol Chem 270(42): 24769–24775. Temperley R, Richter R, Dennerlein S, Lightowlers RN & Chrzanowska-Lightowlers ZM (2010) Hungry codons promote frameshifting in human mitochondrial ribosomes. Science 327(5963): 301. Tengan CH & Moraes CT (1998) Duplication and triplication with staggered breakpoints in human mitochondrial DNA. Biochim Biophys Acta 1406(1): 73–80. Thrash JC, Boyd A, Huggett MJ, Grote J, Carini P, Yoder RJ, Robbertse B, Spatafora JW, Rappe MS & Giovannoni SJ (2011) Phylogenomic evidence for a common ancestor of mitochondria and the SAR11 clade. Sci Rep 1: 13. Thyagarajan D, Shanske S, Vazquez-Memije M, De Vivo D & DiMauro S (1995) A novel mitochondrial ATPase 6 point mutation in familial bilateral striatal necrosis. Ann Neurol 38(3): 468–472. Tiranti V, Chariot P, Carella F, Toscano A, Soliveri P, Girlanda P, Carrara F, Fratta GM, Reid FM, Mariotti C & Zeviani M (1995) Maternally inherited hearing loss, ataxia and myoclonus associated with a novel point mutation in mitochondrial tRNASer(UCN) gene. Hum Mol Genet 4(8): 1421–1427. Torroni A, Bandelt HJ, D'Urbano L, Lahermo P, Moral P, Sellitto D, Rengo C, Forster P, Savontaus ML, Bonne-Tamir B & Scozzari R (1998) mtDNA analysis reveals a major late Paleolithic population expansion from southwestern to northeastern Europe. Am J Hum Genet 62(5): 1137–1152. Torroni A, Bandelt HJ, Macaulay V, Richards M, Cruciani F, Rengo C, Martinez-Cabrera V, Villems R, Kivisild T, Metspalu E, Parik J, Tolk HV, Tambets K, Forster P, Karger B, Francalacci P, Rudan P, Janicijevic B, Rickards O, Savontaus ML, Huoponen K, Laitinen V, Koivumaki S, Sykes B, Hickey E, Novelletto A, Moral P, Sellitto D, Coppa A, Al-Zaheri N, Santachiara-Benerecetti AS, Semino O & Scozzari R (2001) A signal, from human mtDNA, of postglacial recolonization in Europe. Am J Hum Genet 69(4): 844–852. Torroni A, Huoponen K, Francalacci P, Petrozzi M, Morelli L, Scozzari R, Obinu D, Savontaus ML & Wallace DC (1996) Classification of European mtDNAs from an analysis of three European populations. Genetics 144(4): 1835–1850. Tuppen HA, Fattori F, Carrozzo R, Zeviani M, DiMauro S, Seneca S, Martindale JE, Olpin SE, Treacy EP, McFarland R, Santorelli FM & Taylor RW (2008) Further pitfalls in the diagnosis of mtDNA mutations: homoplasmic mt-tRNA mutations. J Med Genet 45(1): 55–61. Tzoulis C, Neckelmann G, Mork SJ, Engelsen BE, Viscomi C, Moen G, Ersland L, Zeviani M & Bindoff LA (2010) Localized cerebral energy failure in DNA polymerase gamma-associated encephalopathy syndromes. Brain 133(Pt 5): 1428– 1437.

106

Ugalde C, Hinttala R, Timal S, Smeets R, Rodenburg RJ, Uusimaa J, van Heuvel LP, Nijtmans LG, Majamaa K & Smeitink JA (2007) Mutated ND2 impairs mitochondrial complex I assembly and leads to Leigh syndrome. Mol Genet Metab 90(1): 10–14. Uusimaa J, Finnilä S, Vainionpää L, Kärppä M, Herva R, Rantala H, Hassinen IE & Majamaa K (2003) A mutation in mitochondrial DNA-encoded cytochrome c oxidase II gene in a child with Alpers-Huttenlocher-like disease. Pediatrics 111(3): e262–8. Uusimaa J, Hinttala R, Rantala H, Päivärinta M, Herva R, Röytta M, Soini H, Moilanen JS, Remes AM, Hassinen IE & Majamaa K (2008) Homozygous W748S mutation in the POLG1 gene in patients with juvenile-onset Alpers syndrome and status epilepticus. Epilepsia 49(6): 1038–1045. Uusimaa J, Moilanen JS, Vainionpää L, Tapanainen P, Lindholm P, Nuutinen M, Lopponen T, Mäki-Torkko E, Rantala H & Majamaa K (2007) Prevalence, segregation, and phenotype of the mitochondrial DNA 3243A>G mutation in children. Ann Neurol 62(3): 278–287. Valente L, Piga D, Lamantea E, Carrara F, Uziel G, Cudia P, Zani A, Farina L, Morandi L, Mora M, Spinazzola A, Zeviani M & Tiranti V (2009) Identification of novel mutations in five patients with mitochondrial encephalomyopathy. Biochim Biophys Acta 1787(5): 491–501. Valero T, Moschopoulou G, Mayor-Lopez L & Kintzios S (2012) Moderate superoxide production is an early promoter of mitochondrial biogenesis in differentiating N2a neuroblastoma cells. Neurochem Int . Vamecq J, Dessein AF, Fontaine M, Briand G, Porchet N, Latruffe N, Andreolotti P & Cherkaoui-Malki M (2012) Mitochondrial dysfunction and lipid homeostasis. Curr Drug Metab 13(10): 1388–1400. van den Ouweland JM, Lemkes HH, Trembath RC, Ross R, Velho G, Cohen D, Froguel P & Maassen JA (1994) Maternally inherited diabetes and deafness is a distinct subtype of diabetes and associates with a single point mutation in the mitochondrial tRNA(Leu(UUR)) gene. Diabetes 43(6): 746–751. van der Velden GJ, Vink MA, Klaver B, Das AT & Berkhout B (2013) An AUG codon upstream of rev and env open reading frames ensures optimal translation of the simian immunodeficiency virus Env protein. Virology 436(1): 191–200. van Esch A, Steyerberg EW, Berger MY, Offringa M, Derksen-Lubsen G & Habbema JD (1994) Family history and recurrence of febrile seizures. Arch Dis Child 70(5): 395– 399. van Holst Pellekaan SM, Ingman M, Roberts-Thomson J & Harding RM (2006) Mitochondrial genomics identifies major haplogroups in Aboriginal Australians. Am J Phys Anthropol 131(2): 282–294. van Oven M & Kayser M (2009) Updated comprehensive phylogenetic tree of global human mitochondrial DNA variation. Hum Mutat 30(2): E386–94. Varlamov DA, Kudin AP, Vielhaber S, Schroder R, Sassen R, Becker A, Kunz D, Haug K, Rebstock J, Heils A, Elger CE & Kunz WS (2002) Metabolic consequences of a novel missense mutation of the mtDNA CO I gene. Hum Mol Genet 11(16): 1797–1805.

107

Vawter L & Brown WM (1986) Nuclear and mitochondrial DNA comparisons reveal extreme rate variation in the molecular clock. Science 234(4773): 194–196. Vazquez-Limon C, Hoogewijs D, Vinogradov SN & Arredondo-Peter R (2012) The evolution of land plant hemoglobins. Plant Sci 191–192: 71–81. Vazquez-Memije ME, Shanske S, Santorelli FM, Kranz-Eble P, DeVivo DC & DiMauro S (1998) Comparative biochemical studies of ATPases in cells from patients with the T8993G or T8993C mitochondrial DNA mutations. J Inherit Metab Dis 21(8): 829– 836. Vilkki J, Ott J, Savontaus ML, Aula P & Nikoskelainen EK (1991) Optic atrophy in Leber hereditary optic neuroretinopathy is probably determined by an X-chromosomal gene closely linked to DXS7. Am J Hum Genet 48(3): 486–491. Vilmi T, Moilanen JS, Finnilä S & Majamaa K (2005) Sequence variation in the tRNA genes of human mitochondrial DNA. J Mol Evol 60(5): 587–597. Wallace DC, Singh G, Lott MT, Hodge JA, Schurr TG, Lezza AM, Elsas LJ,2nd & Nikoskelainen EK (1988) Mitochondrial DNA mutation associated with Leber's hereditary optic neuropathy. Science 242(4884): 1427–1430. Wang J, Schmitt ES, Landsverk ML, Zhang VW, Li FY, Graham BH, Craigen WJ & Wong LJ (2012) An integrated approach for classifying mitochondrial DNA variants: one clinical diagnostic laboratory's experience. Genet Med 14(6): 620–626. Wang PW, Lin TK, Weng SW & Liou CW (2009) Mitochondrial DNA variants in the pathogenesis of type 2 diabetes - relevance of asian population studies. Rev Diabet Stud 6(4): 237–246. Wanrooij S, Luoma P, van Goethem G, van Broeckhoven C, Suomalainen A & Spelbrink JN (2004) Twinkle and POLG defects enhance age-dependent accumulation of mutations in the control region of mtDNA. Nucleic Acids Res 32(10): 3053–3064. Watanabe K & Yokobori S (2011) tRNA Modification and Genetic Code Variations in Animal Mitochondria. J Nucleic Acids 2011: 623095. Watson E, Forster P, Richards M & Bandelt HJ (1997) Mitochondrial footprints of human expansions in Africa. Am J Hum Genet 61(3): 691–704. Werle E, Schneider C, Renner M, Volker M & Fiehn W (1994) Convenient single-step, one tube purification of PCR products for direct sequencing. Nucleic Acids Res 22(20): 4354–4355. White SL, Collins VR, Wolfe R, Cleary MA, Shanske S, DiMauro S, Dahl HH & Thorburn DR (1999a) Genetic counseling and prenatal diagnosis for the mitochondrial DNA mutations at nucleotide 8993. Am J Hum Genet 65(2): 474–482. White SL, Shanske S, McGill JJ, Mountain H, Geraghty MT, DiMauro S, Dahl HH & Thorburn DR (1999b) Mitochondrial DNA mutations at nucleotide 8993 show a lack of tissue- or age-related variation. J Inherit Metab Dis 22(8): 899–914. Wiltshire E, Davidzon G, DiMauro S, Akman HO, Sadleir L, Haas L, Zuccollo J, McEwen A & Thorburn DR (2008) Juvenile Alpers disease. Arch Neurol 65(1): 121–124. Wolff JN & Gemmell NJ (2012) Mitochondria, maternal inheritance, and asymmetric fitness: Why males die younger. Bioessays .

108

Wolff JN, Nafisinia M, Sutovsky P & Ballard JW (2012) Paternal transmission of mitochondrial DNA as an integral part of mitochondrial inheritance in metapopulations of Drosophila simulans. Heredity (Edinb) . Yabuuchi A, Beyhan Z, Kagawa N, Mori C, Ezoe K, Kato K, Aono F, Takehara Y & Kato O (2012) Prevention of mitochondrial disease inheritance by assisted reproductive technologies: prospects and challenges. Biochim Biophys Acta 1820(5): 637–642. Yamasoba T, Someya S, Yamada C, Weindruch R, Prolla TA & Tanokura M (2007) Role of mitochondrial dysfunction and mitochondrial DNA mutations in age-related hearing loss. Hear Res 226(1–2): 185–193. Yan H, Zareen N & Levinger L (2006) Naturally occurring mutations in human mitochondrial pre-tRNASer(UCN) can affect the transfer ribonuclease Z cleavage site, processing kinetics, and substrate secondary structure. J Biol Chem 281(7): 3926– 3935. Yao YG, Kong QP, Salas A & Bandelt HJ (2008) Pseudomitochondrial genome haunts disease studies. J Med Genet 45(12): 769–772. Yarham JW, Al-Dosary M, Blakely EL, Alston CL, Taylor RW, Elson JL & McFarland R (2011) A comparative analysis approach to determining the pathogenicity of mitochondrial tRNA mutations. Hum Mutat 32(11): 1319–1325. Yarham JW, McFarland R, Taylor RW & Elson JL (2012) A proposed consensus panel of organisms for determining evolutionary conservation of mt-tRNA point mutations. Mitochondrion 12(5): 533–538. Yasukawa T, Reyes A, Cluett TJ, Yang MY, Bowmaker M, Jacobs HT & Holt IJ (2006) Replication of vertebrate mitochondrial DNA entails transient ribonucleotide incorporation throughout the lagging strand. EMBO J 25(22): 5358–5371. Ylikallio E & Suomalainen A (2012) Mechanisms of mitochondrial diseases. Ann Med 44(1): 41–59. Yuan H, Chen J, Liu X, Cheng J, Wang X, Yang L, Yang S, Cao J, Kang D, Dai P, Zhai S, Han D, Young WY & Guan MX (2007) Coexistence of mitochondrial 12S rRNA C1494T and CO1/tRNA(Ser(UCN)) G7444A mutations in two Han Chinese pedigrees with aminoglycoside-induced and non-syndromic hearing loss. Biochem Biophys Res Commun 362(1): 94–100. Yuan H, Qian Y, Xu Y, Cao J, Bai L, Shen W, Ji F, Zhang X, Kang D, Mo JQ, Greinwald JH, Han D, Zhai S, Young WY & Guan MX (2005) Cosegregation of the G7444A mutation in the mitochondrial COI/tRNA(Ser(UCN)) genes with the 12S rRNA A1555G mutation in a Chinese family with aminoglycoside-induced and nonsyndromic hearing loss. Am J Med Genet A 138A(2): 133–140. Zaragoza MV, Fass J, Diegoli M, Lin D & Arbustini E (2010) Mitochondrial DNA variant discovery and evaluation in human Cardiomyopathies through next-generation sequencing. PLoS One 5(8): e12295. Zeviani M, Moraes CT, DiMauro S, Nakase H, Bonilla E, Schon EA & Rowland LP (1998) Deletions of mitochondrial DNA in Kearns-Sayre syndrome. 1988. Neurology 51(6): 1525 and 8 pages following.

109

Zhang L, Zhang H, Zhu W, Li X & Wang Z (2012) Energy metabolism, thermogenesis and body mass regulation in tree shrew (Tupaia belangeri) during subsequent cold and warm acclimation. Comp Biochem Physiol A Mol Integr Physiol 162(4): 437–442. Zhao X, Chu M, Li N & Wu C (2001) Paternal inheritance of mitochondrial DNA in the sheep (Ovine aries). Sci China C Life Sci 44(3): 321–326. Zhou X, Qian Y, Zhang J, Tong Y, Jiang P, Liang M, Dai X, Zhou H, Zhao F, Ji Y, Mo JQ, Qu J & Guan MX (2012) Leber's Hereditary Optic Neuropathy Is Associated with the T3866C Mutation in Mitochondrial ND1 Gene in Three Han Chinese Families. Invest Ophthalmol Vis Sci 53(8): 4586–4594. Zuckerkandl E (1987) On the molecular evolutionary clock. J Mol Evol 26(1–2): 34–46. Zuckerkandl E & Pauling L (1965) Molecules as documents of evolutionary history. J Theor Biol 8(2): 357–366.

110

Original papers I

Rantamäki M, Soini HK, Finnilä SM, Majamaa K & Udd B (2005) Adult-onset ataxia caused by mitochondrial 8993T>C mutation. Ann Neurol 58: 337–340. II Soini HK, Moilanen JS, Finnilä S & Majamaa K (2012) Mitochondrial DNA sequence variation in Finnish patients with matrilineal diabetes mellitus. BMC Res Notes 5: 350. III Soini HK, Moilanen JS, Vilmi-Kerälä T, Finnilä S & Majamaa K (2013) Mitochondrial DNA variant m.15218A>G in Finnish epilepsy patients who have maternal relatives with epilepsy, sensorineural hearing impairment or diabetes. BMC Med Genet 14: 73. IV Soini HK, Väisänen A, Kärppä M, Hinttala R, Kytövuori L, Uusimaa J & Majamaa K (2013) Analysis of mitochondrial DNA sequence variation in 66 patients with suspected mitochondrial disease reveals a novel pathogenic m.7585insT mutation in a family with cardiomyopathy. Manuscript.

Reprinted with permissions from John Wiley & Sons Ltd. (I) and BioMed Central Ltd. (II, III). The original publications are not included in the electronic version of the dissertation.

111

112

ACTA UNIVERSITATIS OULUENSIS SERIES D MEDICA

1205. Ahonkallio, Sari (2013) Endometrial thermal ablation : a choice for treatment of heavy menstrual bleeding 1206. Penttilä, Matti (2013) Duration of untreated psychosis : association with clinical and social outcomes and brain morphology in schizophrenia 1207. Wikström, Erika (2013) Epidemiology of Chlamydia trachomatis infection in Finland during 1983–2009 1208. Jokela, Tiina (2013) Analyses of kidney organogenesis through in vitro and in vivo approaches : generation of conditional Wnt4 mouse models and a method for applying inducible Cre-recombination for kidney organ culture 1209. Hietikko, Elina (2013) Genetic and clinical features of familial Meniere’s disease in Northern Ostrobothnia and Kainuu 1210. Abou Elseoud, Ahmed (2013) Exploring functional brain networks using independent component analysis : functional brain networks connectivity 1211. Aro, Aapo (2013) Electrocardiographic risk markers of sudden cardiac death in middle-aged subjects 1212. Tikkanen, Jani (2013) Early repolarization in the inferolateral leads of the electrocardiogram : prevalence, prognosis and characteristics 1213. Merikallio, Heta (2013) Claudins and epitheliomesenchymal transition in lung carcinomas and chronic obstructive pulmonary disease 1214. Kaakinen, Pirjo (2013) Pitkäaikaissairaiden aikuisten ohjauksen laatu sairaalassa 1215. Pasanen, Anna Kaisa (2013) A translational study on the roles of redox molecules, cell cycle regulators and chemokine receptors as prognostic factors in diffuse large B-cell lymphoma 1216. Malo, Elina (2013) The role of low birth weight and resistin in metabolic syndrome 1217. Karjalainen, Jaana (2013) Cardiovascular autonomic function in coronary artery disease patients with and without type 2 diabetes : significance of physical activity and exercise capacity 1218. Peurala, Emmi (2013) Regulators of hypoxia response and the cell cycle in breast cancer 1219. Koskela, Sanna (2013) Granulosa cell anti-Müllerian hormone secretion in ovarian development and disease

Book orders: Granum: Virtual book store http://granum.uta.fi/granum/

D 1220

OULU 2013

UNIV ER S IT Y OF OULU P. O. B R[ 00 FI-90014 UNIVERSITY OF OULU FINLAND

U N I V E R S I TAT I S

S E R I E S

SCIENTIAE RERUM NATURALIUM Professor Esa Hohtola

HUMANIORA University Lecturer Santeri Palviainen

TECHNICA Postdoctoral research fellow Sanna Taskila

MEDICA Professor Olli Vuolteenaho

SCIENTIAE RERUM SOCIALIUM University Lecturer Hannu Heikkinen

ACTA

MITOCHONDRIAL DNA SEQUENCE VARIATION IN FINNISH PATIENTS WITH MATERNALLY INHERITED TYPE 2 DIABETES, EPILEPSY AND MITOCHONDRIAL DISEASE: RISK AND NOVEL MUTATIONS

SCRIPTA ACADEMICA Director Sinikka Eskelinen

OECONOMICA Professor Jari Juga

EDITOR IN CHIEF Professor Olli Vuolteenaho PUBLICATIONS EDITOR Publications Editor Kirsti Nurkkala ISBN 978-952-62-0293-8 (Paperback) ISBN 978-952-62-0294-5 (PDF) ISSN 0355-3221 (Print) ISSN 1796-2234 (Online)

U N I V E R S I T AT I S O U L U E N S I S

Heidi Soini

E D I T O R S

Heidi Soini

A B C D E F G

O U L U E N S I S

ACTA

A C TA

D 1220

UNIVERSITY OF OULU GRADUATE SCHOOL; UNIVERSITY OF OULU, FACULTY OF MEDICINE, INSTITUTE OF CLINICAL MEDICINE, DEPARTMENT OF NEUROLOGY; OULU UNIVERSITY HOSPITAL, MEDICAL RESEARCH CENTER OULU

D

MEDICA