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ISBN 978-952-62-2397-1 (Paperback)ISBN 978-952-62-2398-8 (PDF)ISSN 0355-3221 (Print)ISSN 1796-2234 (Online)

U N I V E R S I TAT I S O U L U E N S I S

MEDICA

ACTAD

D 1538

AC

TASusanna Ylönen

OULU 2019

D 1538

Susanna Ylönen

GENETIC RISK FACTORSFOR MOVEMENT DISORDERS IN FINLAND

UNIVERSITY OF OULU GRADUATE SCHOOL;UNIVERSITY OF OULU,FACULTY OF MEDICINE;MEDICAL RESEARCH CENTER OULU;OULU UNIVERSITY HOSPITAL

ACTA UNIVERS ITAT I S OULUENS I SD M e d i c a 1 5 3 8

SUSANNA YLÖNEN

GENETIC RISK FACTORS FOR MOVEMENT DISORDERS IN FINLAND

Academic dissertation to be presented with the assentof the Doctoral Training Committee of Health andBiosciences of the University of Oulu for public defencein Auditorium 8 of Oulu University Hospital (Kajaanintie50), on 15 November 2019, at 12 noon

UNIVERSITY OF OULU, OULU 2019

Copyright © 2019Acta Univ. Oul. D 1538, 2019

Supervised byProfessor Kari Majamaa

Reviewed byAssociate Professor Andrea Carmine BelinProfessor Arto Mannermaa

ISBN 978-952-62-2397-1 (Paperback)ISBN 978-952-62-2398-8 (PDF)

ISSN 0355-3221 (Printed)ISSN 1796-2234 (Online)

Cover DesignRaimo Ahonen

JUVENES PRINTTAMPERE 2019

OpponentDocent Annakaisa Haapasalo

Ylönen, Susanna, Genetic risk factors for movement disorders in Finland. University of Oulu Graduate School; University of Oulu, Faculty of Medicine; MedicalResearch Center Oulu; Oulu University HospitalActa Univ. Oul. D 1538, 2019University of Oulu, P.O. Box 8000, FI-90014 University of Oulu, Finland

Abstract

Parkinson’s disease and Huntington’s disease are progressive neurodegenerative movementdisorders that typically manifest in adulthood. In this study, genetic risk factors contributing tothese two movement disorders were investigated in Finnish patients. Patients with early-onset orlate-onset Parkinson’s disease as well as population controls were examined. The p.L444Pmutation in GBA was found to contribute to the risk of Parkinson’s disease. POLG1 compoundheterozygous mutations were detected in two patients with Parkinson’s disease and rare lengthvariants in POLG1 were associated with Parkinson’s disease. Variants in SMPD1, LRRK2 orCHCHD10, previously detected in other populations, were not detected, suggesting that they arerare or even absent in the Finnish population. Patients with Huntington’s disease were investigatedfor HTT gene haplotypes as well as whether these haplotypes alter the stability of the elongatedCAG repeat. Haplogroup A was less common in Finns than in other European populations,whereas it was significantly more common in patients with Huntington’s disease than in thegeneral population. Certain HTT haplotypes as well as the parental gender were found to affect therepeat instability. We found that compound heterozygous mutations in POLG1 were causative ofParkinson’s disease, rare length variants in POLG1 were associated with Parkinson’s disease andGBA p.L444P was significantly more frequent in patients than in the controls, which suggests thatthese mutations are associated with the development of Parkinson’s disease. The low prevalenceof Huntington’s disease in Finland correlates with the low frequency of the disease-associatedHTT haplogroup A. Paternal inheritance combined with haplotype A1 increased the risk of repeatexpansion. Movement disorders in Finland were found to share some of the same genetic riskfactors found in other European populations, but some other recognized genetic variants could notbe detected.

Keywords: haplotype, Huntington’s disease, molecular epidemiology,neurodegenerative, Parkinson’s disease

Ylönen, Susanna, Liikehäiriösairauksien geneettisiä riskitekijöitä Suomessa. Oulun yliopiston tutkijakoulu; Oulun yliopisto, Lääketieteellinen tiedekunta; Medical ResearchCenter Oulu; Oulun yliopistollinen sairaalaActa Univ. Oul. D 1538, 2019Oulun yliopisto, PL 8000, 90014 Oulun yliopisto

Tiivistelmä

Parkinsonin tauti ja Huntingtonin tauti ovat hermostoa rappeuttavia eteneviä liikehäiriösairauk-sia, jotka tyypillisesti ilmenevät aikuisiällä. Tässä tutkimuksessa selvitettiin näiden kahden liike-häiriösairauden geneettisiä riskitekijöitä suomalaisilla potilailla. Tutkimme potilaita, joilla olivarhain alkava Parkinsonin tauti tai myöhään alkava Parkinsonin tauti sekä väestökontrolleja.GBA-geenin p.L444P mutaation havaittiin lisäävän Parkinsonin taudin riskiä. Kaksi Parkinsonintautia sairastavaa potilasta oli yhdistelmäheterotsygootteja haitallisten POLG1-geenin variant-tien suhteen ja harvinaiset POLG1 CAG toistojaksovariantit assosioituivat Parkinsonin tautiin.Tutkittuja variantteja SMPD1-, LRRK2- ja CHCHD10-geeneissä ei löydetty tästä aineistostalainkaan, mikä viittaa siihen, että ne puuttuvat suomalaisesta väestöstä tai ovat harvinaisia. Hun-tingtonin tautia sairastavilta potilailta tutkittiin HTT-geenin haploryhmiä ja niiden vaikutustaHuntingtonin tautia aiheuttavan pidentyneen toistojakson epästabiiliuteen. Haploryhmä A olisuomalaisessa väestössä harvinainen verrattuna eurooppalaiseen väestöön ja se oli huomattavas-ti yleisempi Huntingtonin tautipotilailla kuin väestössä. Toistojakson epästabiiliuteen vaikutti-vat tietyt HTT-geenin haplotyypit samoin kuin sen vanhemman sukupuoli, jolta pidentynyt tois-tojakso periytyy. POLG1 yhdistelmäheterotsygoottien katsottiin aiheuttavat Parkinsonin tautia jaharvinaisten POLG1 CAG toistojaksovarianttien todettiin assosioituvan Parkinsonin tautiin Suo-messa. GBA p.L444P mutaatio merkittävästi yleisempi Parkinsonin tautipotilailla kuin kontrol-leilla, mikä viittaa siihen, että se on Parkinsonin taudin riskitekijä. Huntingtonin tautiin assosioi-tuvan haploryhmä A:n matala frekvenssi selittää taudin vähäistä esiintyvyyttä Suomessa. Pater-naalinen periytyminen ja haplotyyppi A1 lisäsivät HTT-geenin toistojakson pidentymisen riskiä.Liikehäiriösairauksilla todettiin Suomessa osittain samanlaisia riskitekijöitä kuin muualla Euroo-passa, mutta kaikkia tutkittuja variantteja emme havainneet.

Asiasanat: haplotyyppi, Huntingtonin tauti, molekyyliepidemiologia, neuro-degeneratiivinen, Parkinsonin tauti

To my family

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9

Acknowledgements

My deepest gratitude goes to my supervisor Professor Kari Majamaa M.D., Ph.D.,

who has guided me through the whole process. He gave me the opportunity to take

part in this interesting scientific project. He has supported me throughout the

writing process. I appreciate his impressive scientific knowledge combined with

kindness and extraordinary patience.

I thank Associate Professor Andrea Carmine Belin and Professor Arto

Mannermaa Ph.D. for reviewing my thesis. I appreciate their valuable comments

on my work. I also want to thank Ewen MacDonald for the language review.

I want to thank all my co-authors in the original publications, especially Pauli

Ylikotila M.D., Ari Siitonen M.D. and Jussi Sipilä M.D., Ph.D.

I truly appreciate the technical assistance provided by Anja Heikkinen and Pirjo

Keränen and for their helping me find my way around the lab. I could always count

on their expertise and helpfulness.

I thank my colleagues from the Northern mitochondria project Laura

Kytövuori Ph.D., Antti Väisänen M.Sc., Tuomas Komulainen M.D., Ph.D., Paula

Widgren M.D., Johanna Krüger M.D., Ph.D., Maija Bolszac M.D., Jukka Kiiskilä

M.D., Milla-Riikka Hautakangas M.D., Naemeh Nayebzadeh M.Sc., Teija

Paakkola Ph.D., Anri Hurme-Niiranen M.Sc., Heidi Soini Ph.D., Tekla Järviaho

Ph.D., Joonas Lipponen M.D., Laura Luukkainen M.D., Eemeli Saastamoinen M.D.

(If I forgot to mention someone, insert your name here) as well as my roommates

from the dermatology research group Nina Kokkonen Ph.D., Antti Nätynki M.Sc.

and Jussi Tuusa Ph.D., for scientific and non-scientific conversations, peer support

and helpful tips.

I also want to thank Teekkaritorvet. My journey with them has been as long as

my academic one. With them, I have gained experience in performing in front of

an audience and several times been made aware that it is not so serious if you make

some mistakes.

I thank my parents who have done their best in bringing me up. My father

shares my interest in science and has taken me on bird watching trips as well as

showing me Halley’s Comet and the moons of Jupiter through the telescope when

I was a child. My mother who I appreciate for her practical sense - she taught me

how to read and to drive a car, as well as many other things.

I also thank my sisters Marianne, Annika and Annmari. Growing up with them

involved lots of playing together, but sometimes also bared teeth and cold war. They

10

are some of the most important people of my life and although they live far away,

they are always close to my heart.

I thank my in-laws Kari and Arja Ylönen for all the help they have given,

especially in childcare.

Finally, I want to thank my beloved husband Pekka for the love he has given

me and the life that we have created together, as well as my children, Viivi, Pauli

and Enna. They are my miracles who make me stop and wonder every once in a

while.

This work was funded by the Academy of Finland, Sigrid Juselius Foundation,

the Finnish Parkinson Foundation, the Finnish Brain Foundation. The research was

carried out in the Department of Neurology, Oulu University Hospital and Clinical

Research Center, University of Oulu.

Oulu, September 2019 Susanna Ylönen

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Abbreviations

ADP adenosine diphosphate

AHS Alpers-Huttenlocher syndrome

ATP adenosine triphosphate

CAG cytosine-adenine-guanine

CBD corticobasal degeneration

CHCHD10 coiled-coil-helix-coiled-coil-helix domain-containing protein

10 (gene)

CMT2 Charcot-Marie-Tooth disease type 2

COR C-terminal of ROC

DJ-1 parkinsonism associated deglycase (gene)

DNA deoxyribonucleic acid

EOPD early-onset Parkinson’s disease

ES embryonic stem

ExAC Exome Aggregation Consortium

FAD+ flavin adenine dinucleotide

FADH reduced flavin adenine dinucleotide

FTD-ALS frontotemporal dementia-amyotrophic lateral sclerosis

GBA glucosylceramidase beta

GCase glucocerebrosidase

HD Huntington’s disease

HDL Huntington’s disease-like

HMG high-mobility group

IBR in between RING domain

JPH3 junctophilin 3 (gene)

LHON Leber's hereditary optic neuropathy

LNA locked nucleic acid

LOPD late-onset Parkinson’s disease

LRR leucine-rich repeats

LRRK2 leucine-rich repeat kinase 2 (gene)

MAPT microtubule-associated protein tau (gene)

MIRAS mitochondrial recessive ataxia syndrome

MNGIE mitochondrial neurogastrointestinal encephalopathy syndrome

MPP+ 1-methyl-4-phenylpyridinium

MPTP 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine

MRI magnetic resonance imaging

12

MT-DN1 mitochondrially encoded NADH:ubiquinone oxidoreductase

core subunit 1(gene)

MT-DN4 mitochondrially encoded NADH:ubiquinone oxidoreductase

core subunit 4 (gene)

mtDNA mitochondrial DNA

MTHFR methylenetetrahydrofolate reductase (gene)

NAC non-amyloid β component domain

NAD+ nicotinamide adenine dinucleotide

NADH reduced nicotinamide adenine dinucleotide

NGS next generation sequencing

OMIM online Mendelian inheritance in man

OXPHOS oxidative phosphorylation

PD Parkinson’s disease

PEO progressive external ophthalmoplegia

PINK1 PTEN-induced putative kinase 1 (gene)

POLG1 polymerase γ (gene)

polyQ polyglutamine

PRNP prion protein gene

PSP progressive supranuclear palsy

REP repressor domain

RFLP restriction fragment length polymorphism

RING really interesting new gene

ROC Ras of complex protein

ROS reactive oxygen species

SANDO sensory ataxic neuropathy dysarthria and ophthalmoparesis

SAP sphingolipid activator protein

SMAJ late-onset spinal motor neuropathy

SMPD1 Sphingomyelin phosphodiesterase 1 (gene)

SNCA α-synuclein (gene)

SNP single nucleotide polymorphism

TFAM mitochondrial transcription factor A (gene)

WD40 Trp-Asp-40

WES whole exome sequencing

wt wild type

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Original publications

This thesis is based on the following publications, which are referred throughout

the text by their Roman numerals:

I Ylönen, S., Ylikotila, P., Siitonen, A., Finnilä, S., Autere, J., & Majamaa, K. (2013). Variations of mitochondrial DNA polymerase γ in patients with Parkinson's disease. Journal of Neurology, 260(12), 3144-3149.

II Ylönen, S., Siitonen, A., Nalls, M.A., Ylikotila, P., Autere, J., Eerola-Rautio, J., Gibbs, R., Hiltunen, M., Tienari, P.J., Soininen, H., Singleton, A.B., & Majamaa, K. (2017). Genetic risk factors in Finnish patients with Parkinson's disease. Parkinsonism and Related Disorders, 45, 39-43.

III Ylönen, S., Sipilä, J., Hietala, M. & Majamaa K. (2019) HTT haplogroups in Finnish patients with Huntington disease. Neurology Genetics, 5(3), e334.

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15

Table of contents

Abstract

Tiivistelmä

Acknowledgements 9 

Abbreviations 11 

Original publications 13 

Table of contents 15 

1  Introduction 17 

2  Review of the literature 19 

2.1  Parkinson’s disease ................................................................................. 19 

2.1.1  Clinical features ............................................................................ 19 

2.1.2  Pathogenesis ................................................................................. 20 

2.1.3  Genetic epidemiology of Parkinson’s disease .............................. 21 

2.1.4  Parkinson’s disease genes: Causative mutations .......................... 22 

2.1.5  Risk genes..................................................................................... 25 

2.1.6  Mitochondrial aspects of Parkinson’s disease .............................. 28 

2.2  Huntington’s disease ............................................................................... 33 

2.2.1  Clinical features ............................................................................ 34 

2.2.2  Pathogenesis ................................................................................. 34 

2.2.3  HTT gene ...................................................................................... 34 

3  Aims of the study 37 

4  Subjects and methods 39 

4.1  Patients and controls ............................................................................... 39 

4.2  Molecular methods .................................................................................. 39 

4.3  HTT haplotypes ....................................................................................... 40 

4.4  Sequencing and whole exome sequencing .............................................. 41 

4.5  Bioinformatics ......................................................................................... 42 

4.6  Statistics .................................................................................................. 42 

4.7  Ethics ....................................................................................................... 42 

5  Results 45 

5.1  POLG1 and Parkinson’s disease (I, II) ................................................... 45 

5.2  Genetic risk factors for Parkinson’s disease (II) ..................................... 45 

5.3  HTT haplotypes (III) ............................................................................... 46 

6  Discussion 49 

6.1  Mitochondria in Parkinson’s disease (I, II) ............................................. 49 

6.2  Genetic risk factors for Parkinson’s disease (II) ..................................... 52 

16

6.3  Huntington’s disease (III)........................................................................ 55 

6.4  Evaluation of the research and future prospects ...................................... 56 

7  Conclusions 59 

References 61 

Original publications 75 

17

1 Introduction

Parkinson’s disease (PD) and Huntington’s disease (HD) are classic movement

disorders. Parkinson’s disease is a heterogeneous disorder, where genetics and

environment act together to promote the development of the disease. PD was

previously thought to result mainly from environmental factors, but research is now

revealing an increasing number of genetic factors. A knowledge of genetic

backgrounds of diseases can help to clarify disease mechanisms and help in

developing novel treatments. Parkinson’s is a multifactorial disease in its origin,

and different cases may require different treatments. When it is known which

biochemical pathways are defective, it should be possible to develop personalized

therapy. Genetic research in PD has revealed several causative mutations and

genetic risk factors. The risk factors do not cause the disease by themselves, but

additional genetic factors or environmental triggers are needed to allow the disease

to develop. In addition, genetics has helped in identifying putative mechanisms of

dopaminergic cell degeneration in substantia nigra. The mechanisms underpinning

the neurodegeneration in PD include mitochondrial dysfunction, lysosomal

dysfunction, protein aggregation and oxidative stress. Huntington’s disease is

caused by a defect in a single gene. Despite the causative mutation being known,

there are still issues to be clarified in the genetics of Huntington’s disease. The

linkage disequilibrium detected in chromosome 4 markers in the early 1990s

suggested the presence of disease modifying factors and factors that affect the risk

of new mutations, but such factors have only recently become a focus of research.

The genetic factors in both of these disorders have been found to vary according to

the patient’s ethnic background. Our objective was to evaluate the contribution of

different genetic variants to these movement disorders in the Finnish population.

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19

2 Review of the literature

2.1 Parkinson’s disease

Parkinson’s disease (PD) is a neurodegenerative progressive movement disorder

that causes a deterioration of dopaminergic cells and a dopamine deficiency in basal

ganglia. It was first described by James Parkinson in 1817 (Parkinson, 1817). PD

is the second most common neurodegenerative disease, Alzheimer’s disease being

the first. PD is multifactorial, in other words, there are several genetic and

environmental factors which affect the development of the disease. Previously it

has been thought to be caused mainly by environmental factors, but research is

revealing more and more genetic factors that influence the development of PD.

(Kalia & Lang, 2015) The prevalence of PD in general is estimated at 300/100,000.

In Finland, the mean annual incidence of PD is 25.2/100,000. PD is typically a

disease of old age, affecting 1% of people older than 60 years, and about 4% of

people older than 80 years. The prevalence of PD is expected to rise, as the numbers

of old people increase in the population. In about 9% of the cases, the symptoms

begin before the age of 50 years; this is considered as early-onset Parkinson’s

disease (EOPD), which has a mean annual incidence of 3.3/100,000 (Ylikotila et

al., 2015). PD is slightly more common in men than in women with a ratio of three

affected men for every two affected women. The geographical distribution of the

birth places of the patients is uneven across Finland with cases clustering in

northern Ostrobothnia, northern Savo, north Carelia and parts of southern

Ostrobothnia, whereas EOPD patients with affected first degree relatives seem to

be clustered in western Finland and the province of Savo (Havulinna et al., 2008;

Ylikotila et al., 2015). Most often PD appears to be sporadic, but in around 10% of

the cases, it seems to be familial. Currently, no cure or treatment is available that

can slow down the progression of the disease. Only treatments that alleviate the

symptoms are in medical use.

2.1.1 Clinical features

PD is characterized by its motor symptoms, which include resting tremor, rigidity,

bradykinesia and postural instability. The clinical diagnosis is based on the

symptoms; the criteria have been described by the UK Parkinson’s Disease Society

Brain Bank Diagnostic Criteria (Gibb & Lees, 1988). First, the diagnosis of a

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parkinsonian syndrome is made according to the typical motor symptoms. Second,

exclusion criteria are applied to rule out possible other diagnoses. Third, supportive

criteria for Parkinson’s disease are sought. The sensitivity of PD diagnosis

according to these criteria is about 90% but the specificity is only 34% (Gibb &

Lees, 1988; Hughes, Daniel, & Lees, 2001). A diagnosis made by an expert

clinician is about 81.3% sensitive and 83.5% specific (Rizzo et al., 2016). There

are no reliable tests available that would be sufficient to confirm the PD diagnosis.

However, some ancillary examinations can be used to support the symptomatic

evidence of the disease. Imaging methods are widely used, and F-dopa-PET is a

highly sensitive and specific, but not a totally accurate method (Ibrahim et al.,

2016). Magnetic resonance imaging (MRI) can be used to distinguish between PD

and atypical parkinsonism (Obeso et al., 2017). The gold standard is considered to

be the post-mortem examination at autopsy. An accurate diagnosis is important not

only for prognosis and treatment but also for research purposes. PD is most

commonly confused with other tremor disorders, atypical parkinsonian conditions,

secondary parkinsonisms, and other dementias (Rizzo et al., 2016). The diagnostic

criteria are being updated as the understanding of PD develops (Postuma et al.,

2015). The mean age of onset is 64.8 years in the Finnish PD population (A. M.

Kuopio, Marttila, Helenius, & Rinne, 1999). In addition, there are numerous non-

motor symptoms, such as cognitive impairments, psychiatric symptoms, sleep

disturbances, constipation, olfactory dysfunction, autonomous dysfunction,

compulsive gambling, micrographia, pain and fatigue. These non-motor symptoms

can appear years before the onset of any motor symptoms. Sleep disturbances, for

example, can emerge 12 to 14 years prior to the motor symptoms. (Kalia & Lang,

2015) Parkinsonism is a broader concept; it refers to a manifestation of the

symptoms of PD but includes also conditions where all the criteria of PD are not

met.

2.1.2 Pathogenesis

The degeneration of dopaminergic cells in the substantia nigra and formation of

Lewy bodies are the hallmarks of PD pathogenesis. Neuronal loss occurs, however,

in many other regions as well. These include locus coeruleus, nucleus basalis of

Meynert, pedunculopontine nucleus, raphe nucleus, dorsal motor nucleus of the

vagus, amygdala and hypothalamus. Lewy bodies are protein inclusions that consist

mainly of α-synuclein, but they contain also at least 90 other proteins. Lewy bodies

are round structures with a diameter of 5-25 µm. They have a dense inner core and

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a less dense outer zone. They are located in the cytoplasm of the cells. Lewy bodies

are not restricted to brain but can be detected also in spine and the peripheral

nervous system. (Kalia & Lang, 2015) Protein aggregation, defect of mitochondrial

complex I, lysosomal impairment and oxidative stress are thought to be the

principal mechanisms leading to PD (Figure 1). Neuroinflammation occurs in PD,

but its role in the pathology of the disease is still controversial. The dopaminergic

cell loss can be as high as 70% before the motor symptoms emerge (Fearnley &

Lees, 1991). Thus, any possible disease-modifying treatment should be initiated at

the premotor phase of the disease.

Fig. 1. Different pathogenetic mechanisms leading to neurodegeneration. ROS, reactive

oxygen species (adapted from Federico et al., 2012).

2.1.3 Genetic epidemiology of Parkinson’s disease

Several studies have been conducted investigating the genetic epidemiology of PD

in different populations (Table 1). The studies have employed different methods in

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case ascertainment, ranging from case series to population-based studies. The

median of the proportion of familial cases is 11% in these studies.

Table 1. PD epidemiology in different populations.

Author Population Patients (N) Frequency of family

hx (%)

Risk measure

Semchuk et al. 1993 CAN 130 11 OR; 2.2

Payami et al. 1994 USA 114 16 OR; 3.5

Bonifati et al. 1995 IT 100 24 OR; 5.0

Marder et al. 1996 USA 233 N.D. RR; 2.3

Elbaz et al. 1999 FR, IT, NL 219 10 OR; 3.2

Autere et al. 2000 FI 268 10 RR; 2.9

Kuopio et al. 2001 FI 119 13 OR; 2.7

Chen et al. 2001 Taiwan 37 N.D. N.D.

Blanckenberg et al. 2013 SSA 311 1 N.D.

Blin et al. 2015 FR 2609 N.D. N.D.

Duncan et al. 2014 UK 155 N.D. N.D.

Gordon et al. 2015 Navajo 524 N.D. N.D.

SSA, sub-Saharan Africa; OR, odds ratio; RR, risk ratio; N.D. not defined.

2.1.4 Parkinson’s disease genes: Causative mutations

Causative mutations have been discovered in genes including SNCA, parkin,

PINK1 (PTEN-induced putative kinase 1), DJ-1 (Table 2). Variation in many other

genes contributes to the risk of PD. Many of the genes contributing to the

pathogenesis of PD are expressed in mitochondria or affect the function of this

organelle.

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SNCA (α-synuclein) is a gene with five exons located on chromosome 4. It

encodes a 140-amino acid protein, which consists of a N-terminal A2 lipid binding

alpha helix domain, a non-amyloid β component domain (NAC) and a C-terminal

acidic domain. SNCA is expressed predominantly in the brain, but also in the heart,

skeletal muscle and the pancreas; it is thought to be involved in the regulation of

dopamine release and transport, in the induction of MAPT fibrillization and in

neuroprotection. It has been reported that mutations reverse the neuroprotective

effect of SNCA. (Siddiqui, Pervaiz, & Abbasi, 2016) SNCA mutations are a rare

cause of PD. The first mutation discovered in this gene was p.A53T

(Polymeropoulos et al., 1997). It has been encountered mainly in families of Greek

origin (Tambasco et al., 2016). In the Finnish population, the p.A53E variant has

been detected in three families with PD (Martikainen, Päivärinta, Hietala, &

Kaasinen, 2015; Pasanen et al., 2014; Pasanen et al., 2017). These families seem to

share a common ancestor. (Pasanen et al., 2017)

Parkin gene consists of 12 exons located on chromosome 6 and it is one of the

largest genes in the human genome. It encodes the E3 ubiquitin protein ligase,

which is a 465 amino-acid protein has several domains including a ubiquitin-like

domain, really interesting new gene (RING0, RING1 and RING2), in between

RING domain (IBR) and repressor domain (REP) (Zheng & Shabek, 2017). It is

expressed especially in the brain, heart and skeletal muscle. It is a cytosolic protein,

but it has been shown to translocate to the outer mitochondrial membrane of

damaged mitochondria, where it has a function in mitophagy. Parkin protein is also

involved in the regulation of mitochondrial fission and fusion. (Gao et al., 2017)

Mutations cause early-onset Parkinson’s disease by a loss-of-function mechanism,

which causes the accumulation of defective mitochondria in the cell. More than

120 mutations in the Parkin gene have been discovered so far.

PINK1 on chromosome 1 harbors eight exons and encodes a 581-amino acid

protein. The PINK1 protein has an N-terminal mitochondrial targeting motif, a

transmembrane domain and a highly conserved kinase domain. The PINK1 protein

tags damaged mitochondria for mitophagy by accumulating on depolarized

mitochondria and recruiting Parkin. Like parkin, it participates in the regulation of

mitochondrial fission and fusion. (Gao et al., 2017) Furthermore, it inhibits the

fusion of damaged mitochondria, which is thought to prevent contamination of

healthy mitochondria. Mutations in PINK1 are associated with EOPD.

DJ-1 is also located on chromosome 1 and has eight exons. The protein is a

mitochondrial peroxiredoxin-like peroxidase which participates in control of

mitochondrial quality and reactions to oxidative stress. PD associated mutations are

24

postulated to lead to reduced lysosomal activity and the accumulation of damaged

mitochondria. (Dolgacheva, Berezhnov, Fedotova, Zinchenko, & Abramov, 2019)

Table 2. PD causative mutations.

Gene Chromosome Hereditary mode Mutation type Degree of

confidence Reference

SNCA 4 dominant missense,

multiplication causative

Polymeropoulos

et al. 1997

parkin 6 recessive

missense,

nonsense

splice-site, indel,

exon

rearrangements

causative Kitada et al.

1998

PINK1 1 recessive

missense,

nonsense, exon

rearrangements

causative Valente et al.

2004

DJ-1 1 recessive

missense,

splice-site, exon

rearrangements

causative Bonifati et al.

2003

ATP13A2 1 recessive causative Ramirez et al.

2006

UCH-L1 Leroy et al.

1998

VPS35 16 dominant missense causative

Vilariño-Güell et

al. 2011,

Zimprich et

al.2011

EIF4G1 3 dominant missense possibly

causative

Chartier-Harlin

et al. 2011

DNAJC13 3 dominant missense Vilariño-Güell et

al. 2014

CHCHD2 7 dominant missense,

splice-site

Funayama et al.

2015

25

2.1.5 Risk genes

LRRK2

The leucine-rich repeat kinase 2 (LRRK2) gene is located on chromosome 12 and

comprises 51 exons. LRRK2 encodes a large protein with 2527 amino acids. It has

several domains including leucine-rich repeats (LRR), a Ras of complex protein

(ROC) and C-terminal of ROC (COR) bidomain, a kinase domain, and a WD40

domain. LRRK2 has both kinase and GTPase activities and it is believed to be

involved in synaptic transmission and vesicular dynamics, especially autophagy.

(Cookson, 2015) LRRK2 is expressed in many tissues, but particularly in the brain,

the kidney, and the immune system. (Bae & Lee, 2015) The mutation p.G2019S is

considered to be the most common mutation and it is rather common in North

African Arab and Ashkenazi Jewish populations. It has been reported to be present

in 30-41% of North African Arab PD patients (Benamer & de Silva, 2010; Lesage

et al., 2006). It becomes less common in populations further from the

Mediterranean. In northern Europe, the mutation is found in about one per cent of

PD patients. In Asian populations, it is very rare, being found in less than 0.1% of

PD patients. The estimates of the penetrance of p.G2019S vary between 24 and 100%

and the penetrance may be affected by the genetic background in different

populations (Marder et al., 2015). Six other mutations have been proven to confer

a considerable risk of PD, p.N1437H, p.R1441C, p.R1441G, p.R1441H, p.Y1699C

and p.I2020T. The p.R1441G mutation is relatively common in the Basque

population, but otherwise it is rather rare. The kinase activity of the protein is

elevated in association with the p.G2019S mutation, while mutations in the ROC

and COR domains exhibit reduced GTPase activity. (Cookson, 2015) Impaired

protein synthesis and degradation of the autophagy-lysosomal pathway are the

pathological mechanisms behind the PD symptoms in patients with LRRK2

mutations. (Bae & Lee, 2015)

GBA

Glucosylceramidase beta (GBA) is located on chromosome 1 and it has 11 exons.

A pseudogene is located 16 kb downstream, and it occasionally recombines with

GBA, creating recombinant alleles. GBA encodes a lysosomal enzyme, which

metabolizes glucocerebroside into glucose and ceramide. (Schapira, 2015)

Homozygous mutations in GBA cause Gaucher’s disease, which is a lysosomal

26

storage disorder. In Gaucher’s disease, the deficiency of glucocerebrosidase

(GCase) leads to an accumulation of glucocerebrosides in the tissues which causes

various symptoms including hepatosplenomegaly, anemia, thrombocytopenia,

skeletal derangements and neurological disturbances. The estimated frequency of

Gaucher’s disease is 1:50,000 live births in general, but in the Ashkenazi Jewish

population, it is more common, being 1:850 live births. Mutations in GBA have

been found to be associated with an increased risk of PD (Aharon-Peretz,

Rosenbaum, & Gershoni-Baruch, 2004). PD patients with a GBA mutation have a

slightly earlier onset of motor symptoms when compared to patients without GBA

mutations. (Brockmann & Berg, 2014) The penetrance of PD with GBA mutations

is about 20% at 70 years and 30% at 80 years (Beavan & Schapira, 2013). About

5-10% of the PD patients are estimated to carry a GBA mutation (Schapira, 2015).

The most common mutations are p.L444P and p.N370S with the latter mutation

being the most common one in the Ashkenazi Jewish population. The heterozygous

p.L444P mutation has been found to disrupt mitophagy in a mouse model (H. Li et

al., 2018). The mutant GBA protein has a toxic gain of function, which inhibits

autophagy induction and mitochondrial priming. On the other hand, reduced GBA

activity hampers lysosomal clearance of autophagosomes and this means that

dysfunctional mitochondria will accumulate in the cell which in turn, increases

oxidative stress.

MAPT

The gene encoding microtubule-associated protein tau (MAPT) is located on

chromosome 17 and it has 16 exons. The microtubule-associated protein tau is

involved in the assembly and stabilization of microtubules. In Alzheimer’s disease

and frontotemporal dementia, tau forms neurofibrillary tangles. An inversion in the

chromosomal region where the MAPT gene is located has allowed two distinct

haplotypes, H1 and H2, to develop. The H1 haplotype has been reported to occur

more frequently in PD patients than in the general population (Zabetian et al., 2007).

A few mutations, such as p.N279K, have been found that may cause a parkinsonian

phenotype (van Swieten & Spillantini, 2007).

CHCHD10

The gene encoding coiled-coil-helix-coiled-coil-helix domain-containing protein

10 (CHCHD10) is located on chromosome 22 and has four exons. CHCHD10

27

belongs to a family of mitochondrial proteins characterized by conserved Cys-

(Xaa)n-Cys motifs, where two cysteine residues are spaced with a number of other

amino acids. The protein is located in the intermembrane space in mitochondria

and is enriched in the cristae junctions. It is involved in the maintenance of cristae

integrity. Mutations in the gene lead to deficiencies in the respiratory chain. This

can lead to different disorders including mitochondrial DNA instability disorder,

frontotemporal dementia-amyotrophic lateral sclerosis (FTD-ALS) (Bannwarth et

al., 2014) clinical spectrum, late-onset spinal motor neuropathy (SMAJ) (Penttilä

et al., 2015), and Charcot-Marie-Tooth disease type 2 (CMT2)(Auranen et al.,

2015). The p.S59L mutation in CHCHD10 has been associated with parkinsonism

(Bannwarth et al., 2014) whereas p.G66V has been found in patients with SMAJ

(Penttilä et al., 2015). The p.P34S variant has been detected in a few patients with

PD, but it is not considered to be pathogenic (Zhang et al., 2015). All the PD

associated mutations have been located in exon 2.

SMPD1

Sphingomyelin phosphodiesterase 1 (SMPD1) gene is located on chromosome 11

and consists of six exons. It encodes acid sphingomyelinase, a 631 amino-acid

protein with four functional domains including a sphingolipid activator protein

(SAP) domain, a proline-rich domain, a phosphoesterase domain and a C-terminal

domain. (Zampieri et al., 2016) Acid sphingomyelinase is a lysosomal enzyme

involved in ceramide metabolism. Mutations in SMPD1 evoke a decreased activity

of acid sphingomyelinase and are responsible for Niemann-Pick disease, a

lysosomal storage disorder. It is known that it is the accumulation of sphingolipids

that causes neuro- and/or dysmyelinative degeneration (Dagan et al., 2015).

Niemann-Pick disease has been divided into different types. Type A is a fatal

infantile neurodegenerative disease with hepatosplenomegaly and psychomotor

symptoms, which leads to death usually by the age of three years. Type B is a milder

disorder, manifesting with hepatosplenomegaly and respiratory problems and

usually without neurodegeneration. The onset is later, and most patients survive

into adulthood. (Schuchman, 2007) The first SMPD1 variant to be associated with

PD was p.L302P (Gan-Or et al., 2013). The association was considered strong

(odds ratio 9.4, 95% confidence interval 3.9–22.8, p < 0.0001). Also the variants

c.996delC (p.fsP330), p.P533L and p.R591C have been reported to have an

association with PD (Gan-Or et al., 2015).

28

2.1.6 Mitochondrial aspects of Parkinson’s disease

Mitochondria are the primary energy producing organelles of the cell. They are the

site of energy metabolism, where the citric acid cycle and beta-oxidation take place.

Mitochondria have other functions in the cell i.e. intracellular signalling, apoptosis

and the maintenance of Ca2+ homeostasis. (Venditti, Di Stefano, & Di Meo, 2013)

Mitochondria consist of an outer membrane, an inner membrane, an intermembrane

space and a matrix (Figure 2). The inner membrane is folded into cristae, which

increase the organelle’s surface area. The respiratory chain is located in the inner

membrane; it converts energy stored in glucose and fatty acids into the form of

adenosine triphosphate (ATP) which can be used to drive cellular functions. The

energy conversion takes place in a series of redox reactions (Giachin, Bouverot,

Acajjaoui, Pantalone, & Soler-López, 2016). The nervous system, including the

dopaminergic cells of substantia nigra, requires high amounts of energy and

therefore it is essential that the mitochondria in dopaminergic neurons are

functioning properly.

Mitochondria have their own DNA (mtDNA), which is circular and double-

stranded. It contains 37 genes, 13 of which encode subunits of the respiratory chain,

22 transfer RNA genes and two ribosomal RNA genes. (Craven, Alston, Taylor, &

Turnbull, 2017) There are multiple copies of mtDNA in each mitochondrion. The

number of mitochondria in a cell varies according to cell type. Cells that have high

energy demands have a greater number of mitochondria. Mitochondria form a

dynamic network via fusions and fissions. The citric acid cycle produces reduced

equivalents that are used by the respiratory chain. The respiratory chain is located

in the inner mitochondrial membrane and consists of five protein complexes

forming the oxidative phosphorylation system (OXPHOS). Complex I

(NADH:ubiquinone oxidoreductase) is the largest of the complexes. It comprises

45 subunits, seven of which are encoded by mtDNA with the remaining subunits

being encoded by the nuclear DNA. It starts the electron transport chain by

oxidizing NADH to NAD+ after which ubiquinone is reduced to ubiquinol.

Complex II (succinate:ubiquinone oxidoreductase) is the smallest unit consisting

of four subunits which are all coded by nuclear genes. It oxidizes FADH to FAD+.

Complex III (ubiquinol:cytochrome-c oxidoreductase) oxidizes ubiquinol and

reduces cytochrome c molecules, Complex IV (cytochrome-c oxidase) oxidizes

oxygen to water. In the final step, Complex V (FOF1-ATP-synthase) phosphorylates

ADP to ATP. Protons are pumped into the intermembrane space by Complexes I,

III and IV, and the resulting proton gradient provides the potential energy that

29

Complex V uses in the phosphorylation process. High energy production often

leads to the formation of reactive oxygen species (ROS), including superoxide

anion radical (O22−), hydroxyl radical (•OH) and hydrogen peroxide (H2O2), which

have functions in cell signaling and homeostasis. However, the excess production

of ROS creates a condition called oxidative stress, which can result in

mitochondrial damage. (Durcan & Fon, 2015)

Fig. 2. Schematic representation of a mitochondrion and the oxidative phosphorylation

system. OMM, the outer mitochondrial membrane; IMS, the intermembrane space; IMM,

the inner mitochondrial membrane; Asterisk, mitochondrial cristae; TCA, citric acid

cycle; ROS, reactive oxygen species; C, cytochrome c; Q, coenzyme Q (adapted from

Perier & Vila, 2012).

Complex I defects

Reduced Complex I activity was first reported in the substantia nigra of PD patients

(Schapira et al., 1989). In addition, Complex I activity is reduced in the brain,

platelets (Parker, Boyson, & Parks, 1989) and muscle of these patients (Banerjee,

Starkov, Beal, & Thomas, 2009; Bindoff, Birch-Machin, Cartlidge, Parker, &

Turnbull, 1991). However, a recent study has shown that the reduction in activity

applies to all brain regions and is not correlated with mtDNA damage (Flønes et al.,

2017). MPTP (1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine) is a synthetic

compound, the use of which has resulted in symptoms of parkinsonism as a

Complex I Complex II Complex III Complex IV Complex V

TCA

*

mtDNA

IMM

OMM

Matrix

IMS

ROS

ROS

ROS

IMM

IMS

Matrix

H+H+

H+

H+NADH NAD+

Succinate Fumarate

O₂ H₂O

ADP ATP

CQ

e‐ e‐e‐

e‐

e‐ e‐ e‐

30

consequence of neuronal loss in the substantia nigra (Langston, Ballard, Tetrud, &

Irwin, 1983). In dopaminergic cells, MPTP is metabolized into MPP+, which acts

as a Complex I inhibitor. In rats, the pesticide, rotenone, causes the degeneration of

dopaminergic cells with a selective effect on Complex I as well as producing

cytoplasmic inclusions that resemble Lewy bodies. Rotenone treated rats also

develop PD-like motor symptoms (Betarbet et al., 2000). Rotenone inhibits

electron transfer from the iron-sulphur centers in Complex I to ubiquinone,

blocking oxidative phosphorylation and reducing ATP synthesis (Heinz et al., 2017).

Rotenone also seems to increase the production of ROS in mitochondria, which can

lead to apoptosis (N. Li et al., 2003). Paraquat (1,1′-dimethyl-4,4′-

bipyridinium), a herbicide that has a chemical resemblance to MPTP, has been

epidemiologically associated with PD (Tangamornsuksan et al., 2018). It has also

been discovered to cause a selective loss of dopaminergic cells in mice. Paraquat

also oxidizes DJ-1, increasing the expression of the protein. (Thiruchelvam et al.,

2003)

mtDNA and PD

Some mutations in mtDNA genes coding subunits of Complex I have been

associated with parkinsonism. These include the Leber's hereditary optic

neuropathy (LHON) causing mutations m.11778G>A in MT-ND4 and m.3460G>A

in MT-ND1. A patient with m.3460G>A and with early-onset parkinsonism was

identified among 46 LHON patients (Nikoskelainen et al., 1995). In another study,

a family with a maternally inherited multisystem degeneration with parkinsonism

was investigated. The patients were found to harbor the m.11778G>A mutation.

(Simon et al., 1999) These studies suggest that mtDNA mutations can contribute to

the development of parkinsonism.

mtDNA is a maternally inherited haploid genome and there is no recombination

involved. The maternal inheritance pattern has enabled the formation of distinct

haplogroups characterized by a defined set of single nucleotide polymorphisms

(SNPs). Mitochondrial haplogroups have been observed to be associated with the

risk of PD. Haplogroups J, K and T and the superhaplogroup JT are associated with

a lower risk of PD and the superhaplogroup HV with a higher risk. This has been

confirmed in an association study of 3074 PD cases and 5659 controls from the

United Kingdom and a subsequent meta-analysis of 6140 PD cases and 13280

controls gathered from several published reports. (Hudson et al., 2013) Different

studies have reported variable results, but in several instances, the haplogroups J

31

and K have emerged as protective (Gaweda-Walerych et al., 2008; Ghezzi et al.,

2005; van der Walt, Joelle M et al., 2003). Different subhaplogroups may also have

different effects on the risk of PD. Thus, different subhaplogroup distributions in

different populations can account for differences between studies. (Gaweda-

Walerych et al., 2008) In Han Chinese, haplogroup B has been associated with a

lower risk of PD and haplogroup D with higher risk of PD in people younger than

50 years (Y. Chen et al., 2015).

Somatic mutations accumulate in mtDNA with age. This is thought to result

predominantly from replication errors by POLG (Szczepanowska & Trifunovic,

2017). In PD patients, the accumulation of these mutations is more extensive than

that encountered in normal aging, and the brains of PD patients are also more

vulnerable to mtDNA mutations. (Coxhead et al., 2016)

POLG1 and mtDNA maintenance

Several proteins are responsible for the maintenance of mtDNA. POLG1 is a

mitochondrial DNA polymerase. It is responsible for the replication of mtDNA.

Twinkle helicase unwinds the double stranded DNA in order to enable replication.

TFAM is the mitochondrial transcription factor A, and it also participates in mtDNA

replication and repair. Mitochondrial DNA polymerase will be described in more

detail.

The POLG1 gene is located in chromosome 15q25 and it has 23 exons. It

encodes the catalytic subunit of the enzyme polymerase γ, which is the only

mitochondrial DNA polymerase. The enzyme consists of an exonuclease, a linker

and polymerase domains. Defects in the enzyme affect replication and impair the

maintenance of mtDNA, leading to reduced copy number or multiple deletions. The

most common mutations include p.A467T and p.W748S. POLG1 mutations; these

cause a range of disorders that can manifest at any age. POLG1 mutations are often

associated with symptoms like epilepsy, ataxia, neuropathy, hepatic manifestations,

ophthalmoplegia, premature ovarian failure, or parkinsonism. (Rahman &

Copeland, 2018) Progressive external ophthalmoplegia (PEO) is a common

symptom in carriers of POLG1 mutations (Van Goethem, Dermaut, Löfgren,

Martin, & Van Broeckhoven, 2001). POLG1 mutations have also been associated

with sensory ataxic neuropathy, dysarthria and ophthalmoparesis (SANDO) (Van

Goethem et al., 2003a), Alpers-Huttenlocher syndrome (AHS) (Naviaux & Nguyen,

2004), and mitochondrial neurogastrointestinal encephalopathy syndrome

(MNGIE)(Van Goethem et al., 2003b).

32

Exon 2 of POLG1 harbors a CAG repeat that codes for a polyglutamine (polyQ)

tract consisting most commonly of ten glutamine moieties. Trinucleotide repeats

occur commonly in genomes and occasionally they can undergo expansions. There

are many neurological diseases caused by trinucleotide repeat expansions e.g.

Huntington’s disease, fragile X syndrome, spinocerebellar ataxias and

frontotemporal degeneration-amyotrophic lateral sclerosis (FTD-ALS). In the

POLG1 gene, variations in the CAG repeat ranging from 5 to 16 have been detected

but no large expansions have been identified (Anvret et al., 2010; Balafkan et al.,

2012). Male infertility has been reported in association with a CAG repeat number

different from ten (Rovio et al., 2001).

TFAM is located on chromosome 10 and it harbors eight exons. It contains two

high-mobility group (HMG) domains separated by a linker region and a C-terminal

tail. It is thought to act in the initiation of transcription and the packaging of the

mitochondrial genome (Hillen, Temiakov, & Cramer, 2018). It is expressed in

several tissues including the brain. Certain TFAM variants have been associated

with Alzheimer’s disease, PD, Parkinson's disease dementia, and age of onset in

HD (Kang, Chu, & Kaufman, 2018).

Mouse models of mtDNA maintenance

Trifunovic and colleagues have generated a mouse model with a defective mtDNA

polymerase (mtDNA-mutator mice) by performing a genetic knock-in procedure

(Trifunovic et al., 2004). They replaced a conserved aspartate residue in the second

exonuclease domain with an alanine in position p.257 in the PolgA subunit of the

mtDNA polymerase, the mouse equivalent of POLG1. They introduced the

p.D257A mutation into embryonic stem (ES) cells using plasmid vectors. The ES

cells were then injected into blastocysts in order to develop chimeric mice. They

selected mice that presented the mutation in the germline and then bred the mice to

obtain homozygous mice. The mice expressed symptoms of premature ageing

including kyphosis (curvature of the spine), progressive hair loss, reduced

subcutaneous fat, reduced fertility in both sexes, and a shorter lifespan. They had a

reduced body size, an enlarged spleen and heart, a lower hemoglobin concentration,

as well as the accumulation of enlarged mitochondria in the heart. Exonuclease

activity, but not that of the polymerase, was reduced. The mtDNA-mutator mice

expressed three to five times more somatic mtDNA point mutations than their wild

type counterparts.(Trifunovic et al., 2004)

33

Ekstrand and colleagues generated a conditional knockout mouse strain with a

disrupted mitochondrial transcription factor A (Tfam) in dopaminergic neurons

(MitoPark mice); these animals displayed signs of parkinsonism (Ekstrand et al.,

2007). The MitoPark mice were generated by directing cre-recombinase expression

in DA neurons. The resulting phenotype displayed an adult-onset, slowly

progressive deterioration of motor function, intraneuronal inclusions and

dopaminergic cell death. mtDNA expression was reduced and the respiratory chain

was deficient in midbrain dopaminergic neurons. Embryonic and neonatal lethality

was not found to be increased in MitoPark mice. Cytoplasmic inclusions were

detected at the age of six weeks. The inclusions formed independently of the

presence of α-synuclein and were found to contain mitochondrial protein and

membrane components. A loss of dopaminergic neurons was observable at 12

weeks of age and the motor symptoms started to appear at around 14-15 weeks of

age. Treatment with L-DOPA alleviated the symptoms, although the response was

less impressive in older mice. (Ekstrand et al., 2007)

2.2 Huntington’s disease

Huntington’s disease (HD) is another progressive neurodegenerative disease. It was

described in 1872 by George Huntington, who called it hereditary chorea. He

distinguished it from other types of chorea in that it was hereditary, it manifested

with a tendency to insanity and suicide and it was adult-onset. (Huntington, 1872)

In Western populations, the prevalence is 10.6–13.7/100 000. It is estimated to

affect 2.12/100 000 in the Finnish population, where its prevalence is lower than in

Europe in general. (Sipilä, Hietala, Siitonen, Päivärinta, & Majamaa, 2015) In East

Asian populations, HD is rare with a prevalence of 0.1–0.7/100 000. In South

Africa, the prevalence of HD has been estimated to be 5.1/100 000 in the white

population, 2.1/100 000 in the mixed ancestry population and 0.25/100 000 in the

black population (Baine, Krause, & Greenberg, 2016). There are also rare disorders

that resemble the symptoms of HD. These are called Huntington’s Disease-like

(HDL) syndromes. HDL-1 is a prion disease, caused by additional octapeptide

repeats in the prion protein gene (PRNP), HDL-2 is an autosomal dominant

disorder caused by a CTG-CAG triplet repeat expansion in the junctophilin 3

(JPH3) gene on chromosome 16 and HDL-3 is autosomal recessive and resembles

juvenile-onset HD. The gene causing HDL-3 has been mapped to chromosome 4.

(Gövert & Schneider, 2013)

34

2.2.1 Clinical features

HD manifests with motor disturbances, cognitive deterioration and behavioural

changes. The motor disturbance presents with involuntary movements such as

chorea and a deterioration of voluntary movements including rigidity, difficulties

in coordinating movements and bradykinesia. The cognitive deterioration begins

years before HD is diagnosed and progresses gradually, it includes learning

problems, cognitive slowing, impaired mental flexibility, planning, visuospatial

functions and emotion recognition and decreased attention. Behaviour becomes

more impulsive because of diminished inhibition, depression occurs commonly,

apathy and irritability also occur. (Ross et al., 2014) HD is usually adult-onset with

a mean onset at approximately 45 years. In a fraction of cases, the disease is

juvenile-onset. The disease progression in juvenile-onset HD is faster than in the

adult-onset form of the disease and the deterioration of voluntary movements is

more prominent. Chorea is less pronounced than in adult-onset HD. HD is

ultimately fatal. The median survival from the onset of motor symptoms is around

18 years.

2.2.2 Pathogenesis

Huntington’s disease is caused by an elongated CAG repeat in the first exon of the

HTT gene (MacDonald et al., 1993). In huntingtin, the protein encoded by HTT, the

elongated CAG repeat translates into an abnormally long polyQ stretch. This leads

to a misfolding of the protein and to post-translational alterations. In the brain of

HD patients, there is clear evidence of neuronal dysfunction and eventually

neuronal death. The medium spiny neurons of the striatum are especially affected,

but also globus pallidus, thalamus and hippocampus experience atrophy. Striatal

atrophy can be detected 15 years before diagnosis. There is atrophy of white matter

and later this also extends to the cortical grey matter. (Ross et al., 2014) In addition,

altered brain iron levels may be detected (Rosas et al., 2012).

2.2.3 HTT gene

HTT is located on chromosome 4, where it was mapped in 1983 with linkage

analysis using RFLP markers (Gusella et al., 1983). The causative change in HTT

was discovered ten years later (MacDonald et al., 1993). HTT is a large gene with

67 exons. Huntingtin, with the polyQ repeat length of 23 glutamines (23Q),

35

contains 3,144 amino acids. It is expressed ubiquitously at varying levels according

to the tissue. It is essential for early embryonic development and normal brain

development. Huntingtin is involved in vesicle transport, the regulation of gene

transcription and possibly in the regulation of protein synthesis and RNA

trafficking (Ross, Kronenbuerger, Duan, & Margolis, 2017).

Animal model of Huntington’s disease

Recently, HD knock-in pigs have been generated by introducing the exon 1 of

human HTT gene with 150 CAG repeats, using CRISPR/Cas9 and somatic cell

nuclear transfer (Yan et al., 2018). The age of onset of symptoms in these animals

was as early as four months, corresponding to juvenile HD in human patients. The

pigs displayed abnormal movements, altered gait, difficulties in running, irregular

breathing patterns and breathing difficulties. They gained less weight than their

wild type (wt) counterparts and their skin became wrinkled and sagging with

advancing age. They also had elevated mortality compared to wt pigs. The brain

was smaller, the cortex and striatum were reduced with a greater reduction in

caudate nucleus than in putamen. Further investigations revealed that the numbers

of medium spiny neurons were markedly reduced, whereas the interneurons were

mainly spared. The mutant huntingtin formed aggregates in the brain. Axon

degeneration was also evident with signs of demyelination. The pattern of

neurodegeneration was overall similar to that present in human HD patients. The

CAG repeat was found to be unstable, as the repeat number varied in different

individuals and generations and also in different tissues. (Yan et al., 2018)

CAG repeat

The normal length of the CAG repeat is 6-35 repeats. The disease emerges when

there are more than 35 repeats, with 36-39 repeats exhibiting reduced penetrance

and from upwards of 40 repeats, there is a full penetrance of the disease. The CAG

repeats longer than 17 are progressively unstable meaning that the repeat number

may experience alterations during meiosis. Expansions are more common in

paternal transitions and reductions and stable transitions are more common when

the inheritance is maternal. Age of onset correlates with the repeat length, the longer

the repeat, the earlier the onset. About 50–70% of the variation in age of onset of

motor symptoms can be explained with the CAG repeat length. There is a tendency

36

for the disease to start earlier in life in successive generations, a phenomenon called

anticipation. This happens especially when paternal transmission is involved.

Chromosome 4 haplogroups

Strong linkage disequilibrium was detected in early linkage analyses. Different

haplotypes have been associated with HD suggesting multiple mutational origins

(MacDonald et al., 1992). Expanded CAG repeats have been associated with

different haplotypes of chromosome 4 (Squitieri et al., 1994). There are three major

haplogroups A-C, defined by specific sets of SNPs. Haplogroup A is further divided

into variants A1-A5 using SNPs that are not used to define haplogroup A. (Warby

et al., 2009) The different haplogroups are thought to associate with CAG repeat

instability. Haplogroup A is especially common in Caucasian HD patients, whereas

haplogroup C is the most common in other populations. Haplogroup A is rare in

Asian populations, and haplogroups A1 and A2, which are considered to confer a

higher risk for an expanded CAG repeat, seem to be absent. Instead, Asian HD

cases are associated with haplogroup C. This is thought to explain the lower

prevalence of HD. Intermediate and high normal alleles have a similar haplotype

distribution to the disease alleles. (Warby et al., 2009)

37

3 Aims of the study

Parkinson’s disease is one of the movement disorders which affects individuals late

in life. Genetic studies have revealed several pathways in the pathogenesis of the

disease and hence in order to find suitable treatments, it is important to determine

the pathological mechanisms in different cases. The Finns differ genetically from

other European populations because of their population history. This means that the

genetic variants contributing to diseases can also be different from other

populations. Huntington’s disease is another movement disorder. This

neurodegenerative disease is monogenic, being caused by an expansion of a CAG

repeat in the HTT gene. Distinct haplotypes of HTT gene have been described in

association with HD.

In detail, the three aims of this thesis were:

1. To investigate the role of POLG1 variants in the risk of developing PD

2. To analyze the contribution of previously known risk alleles for PD in the

Finnish population

3. To examine the role of HTT haplotypes for CAG repeat instability in Finnish

HD patients

38

39

4 Subjects and methods

4.1 Patients and controls

We have previously collected a national cohort of 441 patients with EOPD, defined

by the fact that their PD diagnosis was made before they were 55 years of age.

(Ylikotila et al., 2015). In addition, we had a case series collected in the Kuopio

University Hospital during one year comprising 209 patients with late-onset

Parkinson's disease (LOPD) and 55 patients with EOPD (J. Autere et al., 2004),

and a case series collected in the Helsinki University Hospital comprising 116

patients with LOPD and 31 patients with EOPD (Luoma et al., 2007). Thus, a total

of 527 Finnish patients with EOPD and 325 patients with LOPD were included in

the study (Table 3). We had samples from 225 patients with Huntington’s disease

(Sipilä et al., 2015). As controls, we had samples from 403 healthy blood donors

from different regions of Finland.

Table 3. PD Patients.

Patient group EOPD LOPD

mitoPARK 441 -

Kuopio 55 209

Helsinki 31 116

Total 527 325

4.2 Molecular methods

DNA was extracted from blood by standard methods. Samples were screened for

previously known variants in POLG1, LRRK2, GBA, MAPT, CHCHD10 and

SMPD1 to examine whether they contributed to the PD risk in the Finnish

population. Mutation detection was performed using a PCR-restriction protocol or

allele specific amplification using locked nucleic acid (LNA) primers. The length

variation was detected with fragment length analysis. A FAM-labeled forward

primer CTCCGAGGATAGCACTTGC (CHLC.GCT14A01.P16693.1) and a

reverse primer CTGGGTCTCCAGCTCCGT CHLC.GCT14A01.P16693.2 were

used to amplify a fragment containing the CAG repeat. The fragment length was

124 bp, when it contained the allele with ten repeats. For the national EOPD cohort,

the fluorescent-labeled PCR products were separated on an ABI PRISM® 3100

Genetic Analyzer (Perkin Elmer, Foster City, CA, U.S.A.), using GeneScan™ −500

40

LIZ® Size Standard (Applied Biosystems, Foster City, CA, U.S.A.) as an internal

standard. Peak Scanner™ Software Version 1.0 (Applied Biosystems) was used to

identify the different alleles. For the case series from Kuopio and the controls, ABI

PRISM™ 377 DNA Sequencer (Perkin Elmer) was used with Genescan version

2.1 fragment analysis software (Perkin Elmer). As the internal standard,

GENESCAN-500™ TAMRA (Applied Biosystems) was used and the alleles were

identified using the Genotyper 2.0 program (Perkin Elmer). The mutations detected

with these methods were then confirmed by sequencing. The variants and methods

used to detect them are presented in table 4.

Table 4. Methods used for detecting variants in PD patients.

gene variant method enzyme reference

LRRK2 p.R1441C/G/H R Bsh12361 II

p.G2019S R BfmI II

GBA p.N370S R XhoI II

p.L444P R NciI II

MAPT rs1800547 R SduI II

CHCHD10 p.S59L R SduI II

p.G66V R ApeKI II

SMPD1 p.L302P R CaiI II

POLG1 p.T251I R BseNI I

p.A467T; p.N468D R MlsI I

p.G517V R BstXI I

p.P587L R SmaI I

p.R722H R MlsI I

p.W748S A - I

p.Y955C A - I

p.A1105T R TaaI I

p.E1143G A - I

CAG repeat F - II

R, restriction analysis; A, allele specific amplification; F, fragment length analysis.

4.3 HTT haplotypes

DNA samples from patients with HD were divided into haplogroups A-C and

further into haplogroup A variants 1-5, by genotyping selected SNPs (Kay et al.,

2015; Warby et al., 2009). SNPs and their detection methods are presented in table

5.

41

Table 5. Methods used in determining the HTT gene haplotypes.

variant rs number method enzyme

1 rs2857936 A -

11 rs762855 R PvuII

33 rs2798235 R XmiI

- rs1143646 S -

89 rs4690073 R AseI

94 rs363144 S -

95 rs3025838 R Alw26I

112 rs363096 A,S -

176 rs2776881 A,S -

182 rs362307 S -

R, restriction analysis; A, allele specific amplification; S, sequencing. Variant numbers are from Warby et

al. 2009.

4.4 Sequencing and whole exome sequencing

Sequencing was used to confirm the presence of variants which had been

discovered by other methods. Sequencing of all exons in POLG1 was performed

for samples with detected mutations to identify possible other mutations.

Appropriate DNA segments were first amplified, giving product sizes between 208

to 731bp. The PCR products were purified with shrimp alkaline phosphatase (SAP)

and ExoI. Sequencing was then performed by capillary electrophoresis using

ABI3500xL Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) In the

sequencing reaction, DNA was denatured, hybridized with a primer and lengthened

using a polymerase. Fluorescently labeled 2',3'-dideoxynucleotides, with a different

color label for each of the four bases, were added to the reaction, where they

randomly replaced the usual 2'-deoxynucleotides, thereby terminating the

extension of the DNA fragment. A range of DNA fragments with different lengths

was formed. The capillary electrophoresis separated the fragments according to

their molecular weight. The labels were excited with a laser beam and then read

with an optical detection device.

Samples of 225 randomly selected patients with EOPD were subjected to

whole exome sequencing (WES). The samples were prepared by using either

Nextera Rapid Capture Expanded Exome Kit or TruSeq Exome Library Prep Kit,

using 563 STAMPEED population controls from the North Finland Birth Cohort

1966 as controls. (Siitonen et al., 2017) The 10 x depth of the contigs was at least

90 % and the 30 x depth of the contigs was approximately 70 % on average. The

42

variants were filtered by using a 99.9% tranche and the variants that passed the

filter were then selected for subsequent analysis. No further filtering was made in

order to improve sensitivity. Functional annotations were made by Annovar,

SNPEff and SNPSift using default settings. Sequencing was used to verify

nonsynonymous variants in LRRK2 and SMPD1 that were more frequent in patients

than in controls.

4.5 Bioinformatics

Data of Finnish controls from the Exome Aggregation Consortium (ExAC) were

compared with the data we obtained from WES. The variants discovered by WES

were analysed with PredictSNP in order to estimate their pathogenicity. PredictSNP

is a consensus prediction tool that uses multiple prediction tools to estimate the

effect of amino acid changes. The PredictSNP score has values between −1 and +1.

The mutations are considered to be neutral, if the score is < 0, and deleterious, if

the score is ≥ 0. The absolute distance of the PredictSNP score from zero expresses

the confidence of the consensus classifier about its prediction. (Bendl et al., 2014)

4.6 Statistics

Fisher’s exact test was used to compare allele frequencies in patients and controls.

Exact test of population differentiation was used to analyse the distribution of CAG

length variants in POLG1.

4.7 Ethics

All samples were coded to prevent direct identification of individual patients. The

use of DNA samples from the controls was approved by the Ethics committee of

the Finnish Red Cross. The PD study was approved by the Ethics Committee of

Hospital District of Southwest Finland, the Ethics Committee of the Medical

Faculty of the University of Kuopio and the Ethics Committee of Helsinki

University Hospital. Written informed consent was obtained from the patients

participating in the study. The HD study has been approved by the Ethics

Committee of Hospital District of Southwest Finland (Dnro ETMK 19/180/2010)

and received the national study permits from the National Institute for Health and

Welfare (Dnro THL/1456/5.05.00/2010) and the National Supervisory Authority

43

for Welfare and Health, Valvira (Dnro 1195/06.01.03.01/2012). The study involved

no contact with patients and therefore, no informed consent was required.

44

45

5 Results

5.1 POLG1 and Parkinson’s disease (I, II)

Nine POLG1 mutations were screened in 441 patients with EOPD and in 263

patients belonging to a case series collected in the Kuopio University Hospital

(Table 3). Heterozygous mutations were detected in 32 patients (EOPD, N=20;

LOPD, N=12). The most common mutations were p.W748S in 12 patients,

p.G517V in nine patients and p.R722H in eight patients. The p.E1143G allele was

screened in patients with p.W748S and it was present in all these patients. The allele

frequencies among the patients were not significantly different from those in the

controls. One patient was compound heterozygous for the p.[(T251I; P587L)] allele

and the p.[(W748S; E1143G)] allele, which was confirmed with a segregation

analysis. Sequencing of the coding region in the 32 patients revealed three more

patients with more than one POLG1 mutant allele. One of these could be confirmed

as a compound heterozygote, but for the two others, a segregation analysis could

not be performed, and it remains unknown if the mutations were in trans. Clinically,

there was no difference between patients with or without POLG1 mutations.

However, the EOPD patients with POLG1 mutations had more often affected

siblings than those without these mutations.

POLG1 CAG repeat was analyzed in 496 patients with EOPD and 209 patients

with LOPD. The CAG repeat length was found to vary between eight and 13 repeats

in our samples with ten repeats being the most common repeat length. The non-

10Q alleles were significantly more common in EOPD patients than in controls (p=

0.0056 for difference). The non-10Q/non-10Q genotypes were found in seven

EOPD patients but in none of the controls.

5.2 Genetic risk factors for Parkinson’s disease (II)

Patients with EOPD were then screened for variants that have been reported to

increase the risk of PD. None of the variants (p.L302P in SMPD1, p.R1441C/G/H

or p.G2019S in LRRK2, p.S59L or p.G66V in CHCHD10) were detected. The

frequency of the MAPT haplotype H1 was 92.6% in EOPD patients and 95.0% in

292 controls (p = 0.068 for difference). The p.N370S variant in GBA was found in

0.4% of EOPD patients and 0.3% of controls and p.L444P was found in 2.0% of

EOPD patients and 0.5% of controls (p = 0.032, Fisher's exact test). Three of the

46

patients with p.L444P had an additional nonsynonymous p.A456P variant and the

synonymous rs1135675 variant at codon 460. These variants form together the

RecNciI allele. One patient harboring the p.N370S also had the POLG1 variants

p.N468D and p.A1105T mentioned earlier.

In a similar fashion, the variants in SMPD1, LRRK2 or CHCHD10 were not

found in 323 LOPD patients and the frequency of MAPT haplotype H1 was 94.4%

(p = 0.73 for difference). The p.N370S variant in GBA was found in 0.6% of LOPD

patients and p.L444P in 1.2%.

WES revealed five nonsynonymous variants that were not present in Finnish

ExAC controls. These variants were p.C1152F, p.S1627L and p.R1628P in LRRK2

and p.E358K and p.R542Q in SMPD1. Two of the variants, p.R1628P in LRRK2

and p.R542Q in SMPD1 were predicted by PredictSNP to be deleterious, while the

remaining variants were predicted to be neutral.

Table 6. PD patients with detected missense variants.

gene variant EOPD n (%) LOPD n (%) Controls %

GBA p.N370S 2 (0.4) 2 (0.6) 0.3

p.L444P 13 (2.5) 4 (1.2) 0.5

POLG1 p.T251I 2 (0.5) 0 (<0.4) 0.7

p.N468D 1 (0.2) 1 (0.4) <0.1

p.G517V 6 (1.4) 3 (1.1) 0.2

p.P587L 2 (0.5) 0 (<0.4) 0.7

p.R722H 5 (1.1) 3 (1.1) 0.7

p.W748S 8 (1.8) 4 (1.5) 0.8

p.A1105T 1 (0.2) 0 (<0.4) <0.1

SMPD1 p.E358K 1 (0.4) n.a. <0.02

p.R542Q 1 (0.4) n.a. <0.02

LRRK2 p.C1152F 1 (0.4) n.a. <0.2

p.S1627L 1 (0.4) n.a. <0.2

p.R1628P 2 (0.9) n.a. <0.02

EOPD, patients with early onset Parkinson’s disease; LOPD, patients with late onset Parkinson’s disease;

n.a., not analyzed.

5.3 HTT haplotypes (III)

The allele frequencies of the HTT haplotypes were significantly different in patients

and controls (p < 0.00001, exact test of population differentiation). The haplogroup

A was more common in patients, while haplogroups B and C were more common

in controls (See III table 1 for details). There were also significant differences in

47

the distribution of the haplotypes A1-A5 between patients and controls (p <

0.00001, exact test of population differentiation). Haplotypes A1 and A2 were more

common in patients whereas haplotypes A4 and A5 more often present in controls

(p = 0.003, exact test of population differentiation). Haplogroup A was less

common in our population samples as compared to other European populations

(Warby et al., 2011) (p = 0.0003, X2 test). The proportion of haplotypes A1 and A2

within the haplogroup A was similar to that in European populations.

The change in the CAG repeat length could be determined in 65 transmissions,

41 maternal and 24 paternal. In 50 of these transmissions, the CAG repeat could be

phased with the haplotype. There were 38 transmissions with haplogroup A, 10

with haplogroup C and two with other haplogroups. The mean change in CAG

repeat length was associated with haplotypes (table 7).

Table 7. The mean change (N) in CAG repeat lengths in intergenerational transmissions.

haplotype paternal maternal

A1 9.4 -1.5

A2 1.3 1

A3 6.2 0.5

A 7 0.3

C -3.1 -1.8

total 1.4 -0.2

48

49

6 Discussion

We investigated genetic risk factors in Finnish patients with well-known movement

disorders, i.e. Parkinson’s disease or Huntington’s disease. Two patients with

confirmed compound heterozygous mutations in POLG1 were detected among the

EOPD patients. We also found co-occurring POLG1 variants in two additional

patients, but we could not confirm if these variants were compound heterozygotes

or if they were located in the same allele. A significant difference in the POLG1

CAG repeat distribution could be detected between EOPD and controls, GBA

p.L444P was significantly more frequent in PD patients than in controls.

Interestingly, we did not detect the p.G2019S variant in LRRK2, although it has

been found in many European populations. We observed highly significant

differences in haplotype distribution between HD patients and controls.

Haplogroup A was less common in the Finnish population than in Caucasian

populations in general. Intergenerational CAG repeat instability was associated

with haplotypes and parental gender.

6.1 Mitochondria in Parkinson’s disease (I, II)

Two lines of evidence supporting the role of mitochondria in PD emerged from this

study. (1) We identified EOPD patients with compound heterozygous mutations in

POLG1, and (2) affected siblings were more frequent in EOPD patients with

heterozygous POLG1 mutations than in patients without these mutations.

Compound heterozygous mutations have been reported in patients with

parkinsonism previously (Davidzon et al., 2006; Luoma et al., 2004; Rempe et al.,

2016). Pathogenic mutations in POLG1 make the polymerase error prone, which

subsequently leads to mutations in mtDNA. This results in variable phenotypes

through mitochondrial failure. POLG1-related parkinsonism is often accompanied

with other symptoms or syndromes such as PEO or SANDO. It is responsive to

levodopa.

We searched PubMed for POLG or POLG1 and Parkinson or parkinsonism and

the subsequent review revealed 24 articles that had described POLG1 mutations in

patients with parkinsonism or PD. Fifteen of these articles were case reports, four

were small case series and five were epidemiological studies. In these articles, 41

different POLG1 variants were described in association with parkinsonism,

whereas in patients with idiopathic Parkinson’s disease, 17 different POLG1

variants were found at similar frequencies as in controls. Patients with

50

parkinsonism were usually also showing signs of PEO and occasionally there were

additional symptoms such as neuropathy and premature menopause. Each variant

had been described in one to five articles. The variants were distributed in all three

domains. The variants were usually found in either a heterozygous or compound

heterozygous state and only four studies reported homozygous variants. One of

them described a homozygous p.W748S mutation in a patient with ataxia,

parkinsonism, ophthalmoplegia, peripheral neuropathy, and sensorineural hearing

loss (Remes et al., 2008). The most commonly described POLG1 variants were

p.Y831C and p.Y955C, p.A467T, p.W748S. This list of variants is slightly different

from our list of screened variants, which were considered to be common variants

at the time of the study. However, p.Y831C has been found in controls at similar

frequencies as in patients and it is currently considered as a neutral polymorphism

(Gui et al., 2012; Hudson et al., 2009; Luoma et al., 2007; Tiangyou et al., 2006).

All of the described variants are not necessarily pathogenic. Twenty-two of the 41

variants have been described in association with PEO in Human DNA Polymerase

Gamma Mutation Database. Some of the variants have been reported together with

other variants. In the compound heterozygous state, different variants could act

together to form the pathogenic state or one of the variants could partly compensate

for the malfunction cause by some other variant. This review of the literature

suggests that there are no typical POLG1 variants causing parkinsonism, instead

there are several different variants scattered along the gene.

We found two patients with compound heterozygous mutations in the POLG1

gene among 450 patients with EOPD, suggesting it has a frequency of 4.4/1000.

One patient harbored p.T251I and p.P587L in one allele and p.W748S and

p.E1143G in the other allele, and the other patient harbored p.N468D and A1105T

in the two alleles. Two further patients with more than one heterozygous variant

were detected, but we could not determine if these were in cis or in trans. We

considered the compound heterozygotes to be causal for PD. The frequencies of

single heterozygous variants did not differ significantly from the controls

suggesting that they are not a risk factor of PD. Previous cohort studies on PD (Gui

et al., 2012; Hudson et al., 2009; Luoma et al., 2007; Tiangyou et al., 2006) or

atypical parkinsonism (Synofzik et al., 2012) have reported some cases in which

POLG1 variants seemed to be responsible for PD, but generally POLG1 variants

have been found at similar frequencies in PD patients as in controls. In case reports

and small case series, the phenotype was parkinsonism usually with PEO.

The three most common heterozygous variants among PD patients were

p.G517V (1.3%), p.R722H (1.1%) and p.W748S (1.7%). The frequencies of these

51

variants did not differ significantly from population frequencies. The p.G517V

variant has been reported to associate with mitochondrial disorders, but a

biochemical analysis has revealed that p.G517V decreases only slightly polymerase

activity and the affinity of p55 accessory subunit (Kasiviswanathan & Copeland,

2011). However, possible effects on fidelity were not investigated. p.R722H is

considered to be pathogenic, as it has been found to be homozygous in two Finnish

families with a mitochondrial disease phenotype. It is located in an evolutionarily

conserved domain in the linker region, although p.R722 itself is not extensively

conserved. The homologous region in E. coli is thought to be involved in DNA-

binding. Therefore, it has been speculated that the loss of arginine might affect the

DNA-binding properties and hence the fidelity of binding. The carrier frequency is

estimated to be 1:135 in the Finnish population. (Komulainen et al., 2010)

p.W748S and p.A467T are definitely pathogenic. They both are located in the

linker region of POLG1, and they have been identified previously in patients with

PD or parkinsonism (Gui et al., 2012; Hudson et al., 2009; Remes et al., 2008;

Synofzik et al., 2012; Tiangyou et al., 2006). The p.W748S variant was found with

p.E1143G in patients. This allele has a carrier frequency of 1:125 in the Finnish

population, and it is associated with mitochondrial recessive ataxia syndrome

(MIRAS) (Hakonen et al., 2005) as well as some other neurological phenotypes

when present in the homozygous state (Palin et al., 2010). It seems to be rare in PD

but has been found in parkinsonism (Remes et al., 2008). It is an ancient allele of

European origin possibly originating from Scandinavia, and it has been distributed

extensively during Viking times, later even spreading to Australia and New Zealand

via immigration from Europe (Hakonen et al., 2007). In Norway, its estimated

carrier frequency is 1:100 (Winterthun et al., 2005). p.A467T also seems to be of

ancient European origin. (Hakonen et al., 2007) It has been found to severely impair

the polymerase function (Luoma et al., 2005). The carrier frequency has been

estimated to be <1:500 in the Finnish population (Luoma et al., 2005), and 1:100

in Norway (Winterthun et al., 2005).

Several neurodegenerative diseases result from expanded trinucleotide repeats.

In the CAG repeat of POLG1, large expansions have not been detected, but instead

variations between 5 and 16 trinucleotides, with 10Q being the most frequent allele

(Anvret et al., 2010; Balafkan et al., 2012). We found an association between non-

10Q alleles and PD. Some previous studies have found an association either with

the non-10/11Q (Anvret et al., 2010; Balafkan et al., 2012; Gui et al., 2012; Luoma

et al., 2007) or non-10Q (Eerola et al., 2010), while others have not detected any

significant associations (Hudson et al., 2009; Tiangyou et al., 2006) In silico

52

analysis predicted that the length variation influences the folding energy of the

protein (Anvret et al., 2010). The non-10Q allele may predispose to PD or it could

act as a marker for a nearby predisposing allele. No studies investigating the

biochemical consequences of the CAG length variation in vivo could be found. The

deletion of the entire CAG repeat has no effect on the enzymatic properties of

POLG1, but induces a slight up-regulation of its expression (Spelbrink et al., 2000).

One study reported association with increased risk of PD when 9Q was with present

a distinct POLG1 haplotype rather than 9Q or the haplotype alone (Luoma et al.,

2007). In our patients, p.G517V was always accompanied with the POLG1 length

variant 11Q, and 8Q with p.G268A. It is not known, however, if these variants

belonged to the same allele or not.

Mitochondrial dysfunction is considered as one of the mechanisms leading to

PD. Neurons require a constant supply of energy and thus are sensitive to

mitochondrial failure. Some Complex I inhibiting chemicals, including MPTP,

have been observed to evoke parkinsonian symptoms (Langston et al., 1983),

respiratory chain defects have been detected in platelets, muscle and brain of PD

patients. Genes considered to cause PD, including Parkin, PINK1 and DJ-1, have

been shown to regulate mitochondrial functions. Parkin and PINK1 function in a

pathway that removes defective mitochondria from cells (Narendra et al., 2010).

DJ-1 protects cells from oxidative stress (Taira et al., 2004), acts as a transcriptional

activator of the PINK1 gene (Requejo-Aguilar et al., 2015), and interacts with

Parkin (Moore et al., 2005; Tang et al., 2006). Mouse models with a Tfam knockout

developed cytoplasmic inclusions and dopaminergic cell loss, and their symptoms

could be alleviated by levodopa treatment (Ekstrand et al., 2007). POLG1 variants

seem to make only a modest contribution to PD. The mitochondrial pathogenesis

of PD appears to consist of several factors, each adding its own small contribution

to the development of the disease. No single mitochondrial factor that would

explain a substantial portion of PD cases has so far been discovered.

6.2 Genetic risk factors for Parkinson’s disease (II)

Parkinson’s disease is a multifactorial disorder, where both genes and environment

influence the development of the disease. Environmental risk factors include rural

living, drinking well water and pesticide use. The p.G2019S variant in LRRK2 is

common in PD patients in northern African Arab and Ashkenazi Jewish populations

and in the Mediterranean countries. There is a gradient from southern to northern

Europe, where p.G2019S becomes rarer towards the north. In Swedish PD patients

53

with a mean age of 68 years and a mean age of onset of 60 years, the frequency

p.G2019S has been estimated to be 1.4% (Belin et al., 2006). In our study, however,

the p.G2019S was not detected, which indicates that its frequency in Finland is less

than 0.1%.

Putative pathogenic or risk variants in genes causing lysosomal storage

disorders have been found to be overrepresented in PD (Robak et al., 2017). GBA

is the best-known example of these genes. The two most common mutations in

GBA are p.N370S and p.L444P that are found at a frequency of 4%; these account

for 70% of GBA mutation alleles in European PD patients (Lesage et al., 2011). In

Ashkenazi Jewish PD patients, the frequency of the two mutations has been

reported to be as high as 15.3%, with the p.N370S mutation being more common.

In non-Ashkenazi Jewish patients, the frequency was 3.2% (Sidransky et al., 2009).

In a group of predominantly French PD patients, the frequencies were 2.9% for

p.N370S and 1.0% for p.L444P (Lesage et al., 2011). In Russian PD patients, the

frequencies have been estimated at 0.5% for p.N370S and 1.1% for p.L444P

(Emelyanov et al., 2018). In East Asian populations, the p.L444P is common, but

p.N370S seems to be either absent or at least rare (Pulkes et al., 2014). We found

that the frequency of the p.L444P mutation was 2.8% among Finnish PD patients,

being significantly different between patients and controls. In Sweden, the mutation

is clustered in the northern part of the country, where 4.1% of PD patients carry the

mutation, while in the southern Sweden, the mutation is found in 1% of PD patients.

The frequency of Gaucher’s disease is also elevated in northern Sweden (Ran et al.,

2016). The origin of the mutation cluster has been tracked back to the 16th century

to the county of Västerbotten, from where it has spread to Norrbotten (Dahl,

Hillborg, & Olofsson, 1993). PD patients with GBA mutations seem to have an

earlier onset of the disease in comparison to patients without GBA mutations

(Sidransky et al., 2009). The mean age of onset was 44.6 years in the Finnish EOPD

patients with GBA mutations, whereas the mean age of onset was 46.8 years in the

entire cohort.

The SMPD1 p.L302P is one of the mutations that causes Niemann-Pick type A

disease when present in a homozygous state. A heterozygous mutation has been

associated with PD. In an Ashkenazi Jewish population, the mutation was found in

0.1 % of the general population and in 1.0 % of PD patients (Gan-Or et al., 2013)

This mutation has not been found in Chinese or Taiwanese Populations (K. Li et

al., 2015; Mao et al., 2017; Wu, Lin, & Lin, 2014). We did not find the SMPD1

p.L302P among our samples, indicating that it is not a common risk factor for PD

54

in Finland (Kaasinen, Hietala, & Kuoppamäki, 2015). However, two other variants

that were not found in controls were detected by WES.

MAPT haplotype H1 was more common in Finnish population than in other

European populations. Unlike previous studies where the frequency of haplotype

H1 was 81.8% in PD patients and 77.4% in controls (Zabetian et al., 2007), we

could not detect an association between the MAPT haplotype and PD. We detected

haplotype H1 frequencies of 92.6% in EOPD patients and 95.0% in controls. MAPT

haplotype H1 has been associated with progressive supranuclear palsy (PSP) and

corticobasal degeneration (CBD), which are Parkinson-plus syndromes and present

with evidence of tau inclusions (Webb et al., 2008). It is possible that only part of

the variants of haplotype H1 are associated with PD, or alternatively that the genetic

background affects the association pattern. The methylenetetrahydrofolate

reductase gene (MTHFR) encodes for an enzyme involved in regulating

homocysteine levels. The variant C677T may cause elevated plasma homocysteine

levels, and it has been associated with an increased risk of PD in European, but not

in Asian, populations (Zhu, Zhu, He, Liu, & Liu, 2015). However, the analysis of

MTHFR was not included in this study.

The OMIM database lists 23 genes or loci that are associated with PD. Some

of these genes lead to the disease in a Mendelian fashion, while others are risk

factors. The currently known genetic factors, however, do not explain the total

estimated heritability of PD. The estimated heritability of PD is 27%, while GWAS

explains only about 5% (Keller et al., 2012). Rare variants could explain part of the

missing heritability. There is evidence that known PD genes could harbor rare

variants that contribute to PD pathogenicity in addition to the known pathogenic

and risk variants (Jansen et al., 2017). Indeed, we found three variants in LRRK2

and two variants in SMPD1 that were not found in controls. Two of these, one in

LRRK2 and another in SMPD1, were predicted to be deleterious by predictSNP.

Further studies, including functional studies, will be needed in order to evaluate

whether these variants are possible risk factors for PD or if the variants are specific

in the Finnish population. The Finnish PD population appears to differ from other

European populations in that some genetic variants that cause PD in Europe are

absent or rare in Finland. The SNCA mutation p.A53E has been found in three

Finnish families and it likely shares a common founder (Pasanen et al., 2017).

Parkin c.101_102delAG was described in two unrelated Finnish patients (Kaasinen

et al., 2015). This variant has been previously detected in European PD patients.

GBA mutations in Finnish population have similar frequencies as in non-Ashkenazi

55

Jewish populations in Europe. So far, no typically Finnish genetic variant has

emerged that would explain a substantial proportion of PD cases in Finland.

6.3 Huntington’s disease (III)

The prevalence of HD varies greatly in different populations, being high in most

European populations and low in East Asian populations. In Venezuela, around

Lake Maracaibo, there is a population with an exceptionally high prevalence of HD,

being 700/100 000; in contrast, in Iceland, the prevalence is very low, only 0.96/100

000 (Sveinsson, Halldórsson, & Olafsson, 2012). The prevalence in Finland is

lower than in most European populations. Finnish and Icelandic populations are

isolated and founder effects are thought to explain the lower prevalence.

We found that the frequency of haplogroup A was significantly higher in

Finnish HD patients than that in population controls and haplogroup C was more

common in controls. In particular, haplotypes A1 and A2 were common among HD

patients. The pattern resembles that seen in other European populations, where

haplotypes A1-3 have been associated with HD, whereas haplotypes A4 and A5 are

considered to be protective. The frequencies of haplotypes A1 and A2 have been

found to correlate with the prevalence of HD (Warby et al., 2009). In European

populations, HD occurs predominantly with haplogroup A. In East Asian

populations, haplogroup A is rare and HD-associated haplotypes A1 and A2 are

absent. Instead, most HD cases are associated with haplogroup C. In the Finnish

population, haplogroup A is less common than in other European populations and

this partly explains the lower prevalence of HD.

Paternal transmission has been shown to drive intergenerational HTT CAG

expansions, while maternal transmissions are more likely to be stable or lead to

contractions (Aziz, van Belzen, Coops, Belfroid, & Roos, 2011). Indeed, we

observed a tendency for expansions in paternal transmissions and for contractions

in maternal transmissions. The haplotype was also found to affect the changes in

the CAG length. In haplotype A1, the size of the CAG repeat tended to increase in

paternal transmissions and decrease in maternal transmission. Haplotypes A2 and

A3 were also associated with repeat expansion. In contrast, in haplogroup C, there

was a tendency towards contraction in both paternal and maternal transmission.

Stable transmissions or contractions have been observed similarly in some Cretan

families with late onset HD (Tzagournissakis, Fesdjian, Shashidharan, & Plaitakis,

1995). There is, however, no haplotype data of these families, so we cannot directly

compare them with our results. Differences in the intergenerational instability in

56

different haplotypes is probably caused by variable DNA sequence elements that

either stabilize or destabilize the CAG repeat. It is likely that differences in the

instability of paternal and maternal transmission reflect the gender differences in

germ cell generation.

6.4 Evaluation of the research and future prospects

Samples from EOPD patients were collected nationwide from those that possessed

the right to reimbursement of medication. About 40.9% of the patients who

received the invitation letter volunteered to participate in the study (Ylikotila et al.,

2015). It is possible that there may have been a slight bias towards patients that

were in better condition and lived closer to medical centers. The controls consisted

of anonymous blood donors (Meinilä, Finnilä, & Majamaa, 2001). The mean age

of the controls was 40 years, being a few years younger than the mean age at onset

of PD among the patients (46.8 yrs). At the time of the blood withdrawal, the

controls were neurologically healthy, but it is possible that some of them could

develop PD later in their life. Nonetheless, they do represent the general population

in terms of the frequencies of different variants. The genetics of the Finnish

population is regionally variable. We have taken this into account by choosing

population controls from multiple geographical areas that represent the areas in

which the patients were living. The POLG1 gene was screened only for selected

variants and sequenced in 32 patients. Therefore, we may have missed some of the

variants present in our subjects. We compared the frequencies of most of the

POLG1 variants in PD patients against population frequencies readily available in

the literature. Only some of the SNPs were screened in the 403 population controls,

so the information about combinations of the different variants in controls was

limited. We used in silico methods to evaluate the pathogenicity of those variants

that were not previously reported. These are, however, only predictions and cannot

be considered as definite proof of pathogenicity. Samples from HD patients were

collected retrospectively and consisted of samples that had remained in the

diagnostic laboratories after mutation analysis. The length of the HTT CAG repeat

was not determined in controls, so we could not compare the CAG length

distribution in different haplogroups.

We found heterozygous GBA mutations both among PD patients and controls.

Our controls were anonymous blood donors and, hence, it was not possible to

evaluate them clinically. If we had had access to national biobank databases, it

would have been possible to track subjects with heterozygous GBA mutations who

57

do not present with PD. Then, clinical examinations could be performed, or their

clinical records could be viewed to see if their phenotypes differed from that of

individuals without GBA mutations. The mutations found so far in Finnish PD

patients explain only a small fraction of familial PD or EOPD. The Finnish

population differs genetically from other European populations, and therefore

population specific research data is needed. Clarifying the genetic background in

Finnish PD patients would help when planning genetic diagnostic testing to

determine on which areas to concentrate. Many of the mutations found in other

populations have not been found in Finnish patients but typical Finnish PD

mutations are yet to be revealed. WES could be a suitable tool to search for potential

PD mutations in the Finnish population. Furthermore, the distribution of HTT CAG

repeat lengths in the Finnish population is not known. Determining the proportion

of intermediate alleles or alleles with reduced penetration would, however, be

essential in order to predict the rate of new mutations in the population. Our HD

research was retrospective; a prospective study would provide better information

about the genotype-phenotype correlation.

This study concentrated on genotyping risk variants of Parkinson’s disease and

Huntington’s disease. Attempts were made to find novel risk variants of PD using

WES. However, we only managed to scratch the surface in this project. Next

generation sequencing (NGS) techniques like WES could be applied to find more

possible risk variants in both known and novel risk genes. A biochemical

characterization of different variants could be made using cell cultures or animal

models. Understanding the biochemical pathways that are disrupted or changed

could be crucial in discovering novel treatments for these devastating diseases.

With prospective studies of HD, it could be possible to investigate if differences in

the clinical haplotype, e.g. age of onset or motor symptoms, can be detected in the

various haplogroups.

58

59

7 Conclusions

We investigated the genetic factors affecting the risk of PD and HD in Finnish

patients. We found both similarities and differences compared to other European

populations.

1. POLG1 variants are not a common cause of PD. The role of POLG1 variants

in PD is, however, supported by the identification of compound heterozygous

variants found in patients, and the elevated frequency of PD among siblings.

The POLG1 CAG repeat variants other than the common 10Q were

significantly more frequent in EOPD patients than in controls. This could mean

that the non-10Q variants increase the risk of PD or act as a marker for a nearby

PD risk allele.

2. The p.L444P variant in GBA increases the risk of PD. Other variants were

investigated including p.L302P in SMPD1, p.R1441C/G/H in LRRK2,

p.G2019S in LRRK2, p.S59L in CHCHD10 or p.G66V in CHCHD10 were not

found in our patients or controls suggesting that they are rare or even absent in

Finnish population. Five rare variants in LRRK2 and SMPD1 were detected

that might contribute to PD. No association of the MAPT haplotype H1 with

PD could be detected in our study.

3. The HTT haplogroup A, which has been associated with Huntington’s disease,

was less frequent in the Finnish population than in European populations in

general, explaining the lower incidence of HD in Finland. Paternal

transmissions were more prone to expansions, whereas contraction or stable

transmissions were more common in maternal cases. The HTT haplogroups

affected significantly the stability of the transmissions.

60

61

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Original publications

I Ylönen, S., Ylikotila, P., Siitonen, A., Finnilä, S., Autere, J., & Majamaa, K. (2013). Variations of mitochondrial DNA polymerase γ in patients with Parkinson's disease. Journal of Neurology, 260(12), 3144-3149.

II Ylönen, S., Siitonen, A., Nalls, M.A., Ylikotila, P., Autere, J., Eerola-Rautio, J., Gibbs, R., Hiltunen, M., Tienari, P.J., Soininen, H., Singleton, A.B., & Majamaa, K. (2017). Genetic risk factors in Finnish patients with Parkinson's disease. Parkinsonism and Related Disorders, 45, 39-43.

III Ylönen, S., Sipilä, J., Hietala, M. & Majamaa K. (2019) HTT haplogroups in Finnish patients with Huntington disease. Neurology Genetics, 5(3), e334.

Reprinted with permission from Springer Nature (I), Elsevier (II) and Wolters

Kluwer Health, Inc. (III).

Original publications are not included in the electronic version of the dissertation.

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1528. Honkanen, Tiia (2019) More efficient use of HER targeting agents in cancertherapy

1529. Arima, Reetta (2019) Metformin, statins and the risk and prognosis ofendometrial cancer in women with type 2 diabetes

1530. Sirniö, Kai (2019) Distal radius fractures : Epidemiology, seasonal variation andresults of palmar plate fixation

1531. Näpänkangas, Juha (2019) Pathogenesis of calcific aortic valve disease

1532. Luukkonen, Jani (2019) Osteopontin and osteoclasts in rheumatoid arthritis andosteoarthritis

1533. Niemelä, Maisa (2019) Temporal patterns of physical activity and sedentary timeand their association with health at mid-life : The Northern Finland Birth Cohort1966 study

1534. Hakala, Mervi (2019) Ihokontaktin, ensi-imetyksen, vierihoidon ja täysimetyksentoteutuminen synnytyssairaaloissa

UNIVERSITY OF OULU P .O. Box 8000 F I -90014 UNIVERSITY OF OULU FINLAND

A C T A U N I V E R S I T A T I S O U L U E N S I S

University Lecturer Tuomo Glumoff

University Lecturer Santeri Palviainen

Senior research fellow Jari Juuti

Professor Olli Vuolteenaho

University Lecturer Veli-Matti Ulvinen

Planning Director Pertti Tikkanen

Professor Jari Juga

University Lecturer Anu Soikkeli

Professor Olli Vuolteenaho

Publications Editor Kirsti Nurkkala

ISBN 978-952-62-2397-1 (Paperback)ISBN 978-952-62-2398-8 (PDF)ISSN 0355-3221 (Print)ISSN 1796-2234 (Online)

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TASusanna Ylönen

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Susanna Ylönen

GENETIC RISK FACTORSFOR MOVEMENT DISORDERS IN FINLAND

UNIVERSITY OF OULU GRADUATE SCHOOL;UNIVERSITY OF OULU,FACULTY OF MEDICINE;MEDICAL RESEARCH CENTER OULU;OULU UNIVERSITY HOSPITAL