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Genetic Investigation of Tourette Syndrome

in the French Canadian Population

A thesis submitted t o McGill University in partial fulfillment of the

requirements of the degree of Doctor i n Philosophy, Biology Department

ADRIANA D ~ A Z

ANZALDUA

Department of Biology

McGill University, Montreal

August,

2004

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ACKNOWLEGEMENTS

First and foremost, I want to express my gratitude to Dr. Guy A. Rouleau, my supervisor, for his guidance, advice, and support in all the stages of my studies.

I am also very grateful to each and every one at Dr. Guy Rouleau's laboratory, for their support during the past five years. In particular, I thank Jean-Baptiste Rivikre and Sylvie Toupin for their valuable help, Janet Laganikre for her expertise and friendship, and my dear friends Liliane Karemera, Claudia Gaspar, Amelie Brunet, Annie Levert, Chantal Carlos, Christiane Messaed, and Karine Lachapelle for their encouragement. My thanks also go to Nicolas SatgC, Judith Saint-Onge, Daniel Rochefort, Andr6 Toulouse, Pascale Thibodeau, Claude Marineau, Lan Xiong, Sandra Laurent, Julie Gauthier, Aida Abu-Baker, Viji Shanmugam, Collete Hand, Inge Meijer, Patrick Dion, and Julie Roussel for their help in the laboratory and during the clinical-assessment days.

As well, I want to thank Drs. Ridha Joober, Paul Lasko, William Foulkes, Rima Slim, Marie-Pierre DubC, and Neil Malik for their comments and suggestions; the Biology Department, especially Ms. Susan Bocti, for her warn welcoming and help.

I extend my deep gratitude to all the families that participated in the study and to all the members of the Montreal Tourette Syndrome Study Group; Drs. Fran~ois Richer, Yves Dion, Sylvain Chouinard, Paul LespCrance, and Ms. Marie-Paule Desrochers for their feedback and vital contribution to the recruitment and assessment of the patients.

I am also grateful to CONACyT (Consejo Nacional de Ciencia y Tecnologia, Mexico) for their support through a 5-year scholarship.

I dedicate this thesis to my dear parents, Dr. Alejandro Diaz Martinez and Dr. Blanca A. Anzaldua Quintana, whose love and support are always essential.

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CONTRIBUTORS TO THE THESIS

Guy Rouleau. He guided and supervised all stages of this study. He participated in the Tourette syndrome clinic assessment days as head of the Montreal Tourette Syndrome Study Group.

Ridha Joober. He reviewed and contributed to the production of the manuscripts and participated in the Tourette syndrome clinical assessment.

Jean-Baptiste Riviere. He did some of the genotyping on chromosome 7, and helped to complete the genotyping on chromosomes X and 11. He translated into French the abstract of the thesis.

Yves Dion. He participated in the recruitment of patients and clinical assessment of patients and their families.

Paul Lesperance. He participated in the Tourette syndrome clinical assessment.

Francois Richer. He participated in the clinical assessment and reviewed the manuscripts.

Sylvain Chouinard. He participated in the recruitment of patients and clinical assessment of the patients and their families.

All other members of the Montreal Tourette Syndrome Study Group. They helped in the clinical assessment and database maintenance.

Sylvie Toupin, Nicolas Satge and Marie-Paule Desrochers. They participated in the coordination of the clinical assessment days, drawing of blood, preparation of pedigree information, and the database.

Judith Saint-Onge. She did part of the final genotyping on chromosome 11.

Marie-Pierre Dube. She helped on the statistical analysis for chromosome 11.

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ABSTRACT

Tourette syndrome (TS) is a childhood-onset neuropsychiatric disorder characterized by multiple motor and one or more phonic tics. TS affects all races and ethnic groups and its prevalence is estimated to be 1% of school children who are 6 to 17 years old. The etiology of this disorder is unknown, but there is strong evidence that supports the involvement of genetic factors. No specific gene unequivocally increasing the risk for TS has been identified. However, positive linkage and association studies, as well as chromosomal anomalies have been reported in the past.

The aim of this study was to search for genetic variants associated with TS as a step towards the elucidation of the causes of the disorder. This study represents the first population-based genetic analysis of TS in the French Canadian population, a genetically homogenous group with respect to typical outbred human populations.

We found associations between TS and variants in two dopaminergic genes, the dopamine D4 receptor and the Monoamine oxidase genes. Furthermore, we identified a new locus on chromosome 7, flanked by Dock 4 and FoxP2 genes, within a larger region previously suggested as candidate for TS by cytogenetic studies.

Our results illustrate the multifactorial etiology of TS in the French Canadian population. It is probable that there is locus heterogeneity for TS even in homogenous populations such as the French Canadians in Quebec, which adds to the complexity of the involvement of non-genetic factors.

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RESUME

Le syndrome Gilles de la Tourette (SGT) est une affection neuropsychiatrique

caracttride par de multiples tics moteurs ainsi qu'un ou plusieurs tics vocaux. Chez les Ccoliers de 6 B 17 ans, la frkquence de la maladie est estimke i environ 1%. Elle touche tous les groupes ethniques. La cause du syndrome n'est pas connue, mais des facteurs gCnCtiques jouent tres probablement un r6le important, m&me si aucun gene augmentant les risques d'exprimer le SGT n'a encore CtC identifiC formellement. Seules des dudes de

linkage positives ainsi que des anomalies chromosomiques ont pour l'instant CtC rapport6es.

La recherche de variants gknbtiques associCs au SGT Ctait le but de cette Ctude; et ce, dans I'optique d'une meilleure comprChension des causes de cette maladie. Cette ttude pksente les premieres analyses ginttiques du SGT basees sur une population precise : les Canadiens-fran~ais. La relative homogCnCit6 g6nktique de ce groupe est un facteur important pour ce type de recherche.

Plusieurs variants appartenant

B

deux genes dopaminergiques se sont avCr6s &we associCs

i3la maladie chez les Canadiens-franqais. Ces deux genes sont le rkcepteur de dopamine D4 ainsi que le gene de la monoamme oxydase. De plus, nous proposons un nouveau locus sur le chromosome 7 comprenant les gknes Dock4 et FoxP2, dans une region auparavant suggCr& par des Ctudes cytogCnktiques.

Les rksultats pdsentes ici illustrent bien les multiples causes du syndrome de Tourette dans la population canadienne-franpise. L'htttrog6nCitk des locus est trks probable, m&me dans une population homogene comme c'est le cas ici. Cette

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hktkrogknkitk doublke de I'influence de facteurs environnementaux rend les ktudes gin6tiques beaucoup plus complexes.

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CONTENTS

CHAPTER 1 : INTRODUCTION: TOURElTE SYNDROME

History 2 Classification 4

Prevalence of TS and frequent comorbidity with other psychiatric disorders 6 Description of tics 7

Evidence found through Imaging studies 8

The involvement of dopamine in the etiology of the disorder 9

Possible involvement of other neurotransmitters in the etiology of the disorder 1 0 The role of genetics in the etiology of TS 12 Rationale and objectives of the research 14

Objective 1 14

Objective 2a and figure 1 18

Objective 2b 21 Objective 3 22 Objective 4 23

CHAPTER 2: FORWARD GENETICS APPROACH Section 2.1

:

Tourette syndrome and dopaminergic genes: a family based association

study i n the French Canadian founder population

2.1.1 Abstract 25 2.1.2 Introduction 2 6

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2.1.3 Materials and methods 28 Subjects 2 8

Genotyping 28

SLC6A. DRD2, DRD3, DRD4, MAO-A 28

2.1.4 Results 30

DRD4, MAO-A, Other dopaminergic genes 30

2.1.5 Discussion 33

2.1.6 Acknowledgements 37 2.1.7 Tables 1-6 37

Section 2.2:

Further analysis of dopaminergic genes 2.2.1 Tyrosine hydroxylase 41

2.2.2 Dopamine beta hydroxylase 42

2.2.3 Acid phosphatase locus 43

2.2.4 Tables 7-12 44

CHAPTER 3: REVERSE GENETICS, CHROMOSOME 7

Section 3.1

:

Association between 7q31 markers and Tourette syndrome 3.1.1 Abstract 48

3.1.2 Introduction 49

3.1.3 Materials and Methods 50

3.1.4 Results 51

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3.1.6 Acknowledgements 55 3.1.7 Tables 13-17 55

Section 3.2:

Extended analysis of the 7q31 region

3.2.1 Tables 18-22; figures 2 and 3 62

CHAPTER 4: REVERSE GENETICS, CHROMOSOME 11

Chromosome 11 -q24 region in Tourette syndrome: Association and Linkage disequilibrium study in the French Canadian population

4.1. Abstract 66 4.2. Introduction 66

4.3. Materials and Methods 68 4.4. Results 69

4.5. Discussion 70

4.6. Acknowledgements 71 4.7. Tables 24-28; figure 4 72

CHAPTER 5: GENERAL DISCUSSION AND CONCLUSIONS Section 5.1

Forward Genetics

5.1.1 Dopamine D4 receptor (DRD4) gene 78

5.1.2 Monoamine oxidase A (MAO-A) gene, figure 5 79 5.1.3 Other dopaminergic genes 81

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Section 5.2

Reverse Genetics

5.2.1 Chromosome 7, q31 region, figure 6 82

5.2.2 Chromosome 1 1, q24 region 84

REFERENCES 88

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LIST OF

TABLES

CHAPTER 2: FORWARD GENETICS APPROACH

Table 1 Table 2 Table 3 Table 4 Table 5 Table 6 Table 7 Table 8 Table 9 Table 10 Table 11 Table 12

Previous associations between candidate gene polymorphisms and TS 31

TDT analysis for the DRD4 exon 3 VNTR 32 TDT analysis for the DRD4 exon 3 VNTR for

TS patients with no comorbidity with ADHD or OCD 32

TDT analysis for the DRD4 7L haplotype vs. all the other haplotypes 32 TDT analysis for MAO-A 33

TDT analysis for the two common haplotypes of the MAO-A gene 33 Marker information at the tyrosine hydroxylase (TH), dopamine

beta-hydroxylase (DBH), and acid phosphatase (ACP1) loci. 38 Multi-allelic TDT analysis for TH (1). ETDT program v.2.4. 39

Multi-allelic TDT analysis for TH (2), ETDT program v.2.4. 39 TDT analysis for DBH (I), ETDT program v.2.4. 39

TDT analysis for DBH (2), ETDT program v.2.4. 40 TDT analysis for ACP1, ETDT program v.2.4. 40

CHAPTER 3: REVERSE GENETICS, CHROMOSOME 7 Table 13 Marker information 49

Table 14 TDT analysis for marker D7S522 50

Table 15 TDT analysis for marker D7S522 with no comorbidity 50 Table 16 Multiallelic TDT analysis for marker D7S523 51

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Table 18 Marker information at the 7q31 region 56

Table 19 TDT analysis (FBAT v.1.4.2) for marker rs2040627, using the additive model, 110 FC nuclear families 56 Table 20 TDT analysis (FBAT v.1.4.2) for marker D7S2554,

using the bi-allelic additive model, 110 FC nuclear families. 56 Table 21 Haplotype analysis (HBAT v.1.4.2) for markers

rs2040627 and D7S523, additive model, EM algorithm for haplotype inference, 110 FC nuclear families. 5 7 Table 22 Haplotype analysis (HBAT v.1.4.2) for markers rs2040627,

D7S523, and D7S2554, additive model, EM algorithm for haplotype inference, 110 FC nuclear families

CHAPTER 4: REVERSE GENETICS, CHROMOSOME 11

Table 23 Table 24 Table 25 Table 26 Table 27 Table 28

Marker information at 1 lq24 region 66

TDT analysis (FBAT v.1.4.2) using the bi-allelic additive model for marker D l 1S1377, 199 FC tourette syndrome trios 66 TDT analysis (FBAT v.1.4.2) using the bi-allelic additive

model for marker D l lS933 on 199 FC tourette syndrome trios 67 TDT analysis (FBAT v.1.4.2) using the multi-allelic additive

model for all the markers on 199 FC tourette syndrome trios 68

Haplotype analysis (HBAT v.1.4.2) for markers D11S1377 and D11S933, with additive model and haplotype inference with the EM algorithm 69 Pairwise linkage disequilibrium estimates in parents of

199 FC Tourette syndrome trios 70

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LIST OF FIGURES

Figure 1 Diagram of dopamine metabolism and transmission 57

Figure 2 Multiallelic TDT analysis for 16 markers, region 7q31 57

Figure 3 Linkage disequilibrium pattern, region 7q31 58

Figure 4 Linkage disequilibrium pattern, region 1 lq24 71

Figure 5 Diagram of dopamine metabolism and transmission according to the genetic associations found in TS patients 74

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ABBREVIATIONS

a. a. ACPl ADHD APA BP BS A CAMP CAV 1 CAV2 cM CONACYT D A DARPP-32 dATP Dellins DBH DDC DNA DRD 1 DRD2 DRD3 DRD4 Amino acid

Low-molecular weight tyrosine phosphatase, acid phosphatase locus 1 Attention deficit1 hyperactivity disorder

American Psychiatric Association Base pair, base pairs

Bovine serum albumin

Cyclic adenosine monophosphate Caveolin 1 gene

Caveolin 2 gene

Centimorgan, centimorgans

National Council for Science and Technology, Mexico Dopamine

Dopamine and CAMP-regulated phosphoprotein Deoxyadenosine triphosphate

Deletion1 insertion

Dopamine-beta hydrox ylase DOPA decarboxylase Deoxy-ribonucleic acid Dopamine receptor D l Dopamine receptor D2 Dopamine receptor D3 Dopamine receptor D4

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DRDS Dopamine receptor D5 DTBZ ETDT FAH FBAT FC GAB A HVA IFRD 1 IMMP2L Kb Dihydrotetrabenazine

Extended transmission disequilibrium test (e-TDT) Fumarylacetoacetate hydrolase

Family based association test French Canadian

y-aminobutyric acid Homovanillic acid

Interferon-regulated developmental regulator 1 Inner mitochondria1 membrane peptidase 2-like gene Kilobase, kilobases

KIAA0716 N-ethylmaleimide sensitive factor attachment protein L LD L-DOPA LOD MAO-A MAO-B Mb mRNA NE OCD PAPBNl

Long allele, dopamine receptor D4 promoter polymorphism Linkage disequilibrium

L-dyhydroxyphenylalanine Log of the odds

Monoamine oxidase-A Monoamine oxidase-B Megabase, megabases Messenger ribonucleic acid Norepinephrine

Obsessive-compulsive disorder

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PBGD Porphobilinogen deaminase gene PCR PKA PP 1 RNA

s

SACS SD SGT SLC6A3 SNP Subs. TDT

Polymerase chain reaction CAMP-dependent protein kinase Protein phosphatase 1

Ribonucleic acid

Short allele, dopamine receptor D4 promoter polymorphism Sacsin

Standard deviation

Syndrome de Gilles de la Tourette Dopamine transporter 1

Single nucleotide polymorphism Substitution

Transmission disequilibrium test Tetranucl. Rep. Tetranucleotide repeat

TS Tourette syndrome

TSAIGC Tourette Syndrome Association International Genetics Consortium TSCSG Tourette Syndrome Classification Study Group

TH Tyrosine hydroxylase

VNTR Variable number of tandem repeats

ZNF277 Zinc finger protein 277

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CHAPTER

1

:

INTRODUCTION

Tourette syndrome

History 2 Classification 4

Prevalence of TS and frequer Tics 7

~t comorbidity with psychiatri

Evidence found through imaging studies 8

c disorde

The involvement of dopamine in the etiology of the disorder 9 The role of genetics 10

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CHAPTER 1: INTRODUCTION

1 .I History

Gilles de la Tourette syndrome, now commonly called Tourette syndrome (TS), was formally described at the end of the 19Ih century. Nevertheless, there were earlier reports of cases that matched its current diagnostic criteria indicating it may have been recognized in humanity at least two thousand years ago. In ancient Greece, the physician Aretaeus of Cappadocia recorded cases of barking, twitching, and cursing, according to Oliver Sacks (1996). In 1489, Sprenger and Kraemer described a priest who also showed motor and phonic tics, which are the main features of TS. A similar condition was recorded in the 17" century, concerning the Prince de Cond6, a member of Louis XIV court, who had phonic tics that forced him to stuff objects in his mouth in order to avoid the production of noises (Goetz et al., 2001).

Perhaps one of the most known and well-documented cases refers to the 18"- century English writer and lexicographer, Samuel Johnson, author of the English Language Dictionary. According to Johnson's friends, some of them physicians, he showed repetitive body twitches, facial grimaces, barks and grunts, among other tics (Murray, 2003). In one of his biographies, James Boswell wrote about Johnson's tics and compulsive behaviour, describing him holding his head towards his right shoulder and shaking it, moving his body backwards and forwards repetitively, rubbing his left knee in the same direction with his hand. As for the phonic tics, Boswell described Johnson making sounds similar to ruminating, whistling, clucking like a hen, protruding his tongue against the upper gums in front, as if pronouncing: "too, too, too", etc. Johnson

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CHAPTER 1: INTRODUCTION

was observed going in or out of a door using a certain number of steps, from a certain point (Murray, 1979).

Still controversial is the case of Wolfgang A. Mozart who, according to letters written by himself as well as other records, may have had tics, inclination to nonsense words, sudden impulses, and hyperactivity (Simkin, 1992; Aterman, 1994). The features

of Tourette syndrome, or at least the presence of chronic tics and comorbid conditions such as hyperactivity, obsessive-compulsive behaviour or rage attacks were also observed in other famous artists and world leaders including Napoleon Bonaparte, Peter the Great, Molikre, and more recently, the French writer Andre Malraux (Guidotti, 1985; Lees, 1985). Some authors have even proposed that the creative, determined, competitive, and

persistent nature of these people may be related to the presence of TS (Sacks, 1992). The

traits of Tourette syndrome may somehow be associated with elaboration, humorous mimicry, uninhibited inventiveness, and artistic creativity (Bradshaw and Sheppard, 2000). There are reports of patients for whom the disorder was a source of inspiration in languages, music, art, athletics, and games (Sacks, 1992). Furthermore, clinicians have

observed that some patients are particularly sensitive to the feelings and experiences of others, and more prone to outside stimuli. In this way, they are considered more empathic (Leckman, 1999).

Given the conspicuous and fascinating features of Tourette syndrome, some non- medical descriptions of the disorder are also found in the literature since the 1 9 ' ~ century. Charles Dickens described, for example, Mr. Pancks in Little Dorrit, a character who had phonic and motor tics, as well as obsessive-compulsive behaviour and trichotillomania.

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CHAPTER I : INTRODUCTION

Leo Tolstoy in Anna Karenina also described the TS features of the character Nikolai Levin (Lamer, 2003).

In 1825, Jean Marc Gaspard Itard made the first medical description of TS based on two cases, one of which was later followed by the French Neurologist Jean-Marie Charcot (Teive et al., 2001). When Charcot began studying tics, the disorder became widely recognised as a neurological disease. Finally, the most formal and detailed description of TS was written by Charcot's student, Georges Gilles de la Tourette in

1885, based primarily on a famous marquise described by Itard. Gilles de la Tourette put together information from previous fragmented reports and wrote a complete and formal description of TS, thus establishing a novel clinical entity (Lajonchere et al., 1996).

Sigmund Freud, who was also Charcot's student, described the case of Frau Emmy von N in 1893. This patient had a complex emotional disorder that included striking tic symptoms (Kushner, 1998). Later, during the first half of the 201h century, the neurological point of view of Gilles de la Tourette and the psychological hypothesis of Freud remained almost invariably separated. Nevertheless, during the last decades of the century, the disorder became known as a neuropsychiatric entity.

I

.Z

Classification

Georges Gilles de la Tourette defined TS as a hereditary childhood onset disorder characterized by motor and phonic tics (Lajonchere et al., 1996). Behavioural abnormalities such as obsessions, compulsions, inattentiveness and hyperactivity, usually observed in TS patients, were considered mental tics at the time. From the 9 cases

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CHAPTER I : INTRODUCTION

reported by Gilles de la Tourette, 6 of them fulfill the current diagnostic criteria for TS; and the diagnosis of the 3 other cases would now be considered chronic motor tics, given that there were no phonic tics reported (Dugas, 1986).

The current classification, largely based on Gilles de la Tourette's work, defines TS by the presence of two or more motor, and at least one phonic tic. The diagnostic criteria for TS in the 4th edition of the Diagnostic and Statistical Manual of Psychiatry (APA,

1994) include the following:

a) Both multiple motor and one or more phonic tics have been present at some time during the illness, although not necessarily concurrently.

b) The tics occur many times a day (usually in bouts) nearly every day or intermittently throughout a period of more than 1 year, and during this period there was never a tic-free period of more than 3 consecutive months.

c) The disturbance causes marked distress or significant impairment in social, occupational, or other important areas of functioning.

d) The onset is before the age of 18 years.

e) The disturbance is not due to the direct physiologic effects of a substance or a general medical condition.

A slightly different classification is based on the Tourette syndrome classification study group, and indicates that the onset of tics should be prior to 21 years (instead of IS), and there is no mention of the necessity of marked distress or significant impairment

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CHAPTER 1: INTRODUCTION

caused by tics that the DSM-IV classification defines. Finally, this classification differentiates definite Tourette syndrome (motor andlor phonic tics witnessed by a reliable examiner) from Tourette syndrome by history (in case tics were not witnessed by a reliable examiner, but by a relative or close friend, and the description is accepted by the examiner) (Leckman et al., 1999).

The anatomic location, number, frequency, complexity, type, and severity of tics change over time (TSCSG, 1993). The onset is observed during the first or second decade of life, and the mean age of onset is 6 to 7 years of age (Brunn, 1988). In some cases, there is a pre-pubertal exacerbation, post-pubertal attenuation and adult stabilization of the symptoms (Faridi and Suchowersky, 2003).

1.3 Prevalence of TS and frequent comorbidity with other psychiatric disorders

The prevalence of tic disorders ranges from 4 to 18% in school-aged children, and in most cases a prevalence of 10% has been reported for this age group (Robertson, 2003). For TS, the prevalence is estimated to be 1% of school children who are from 6 to 17 years old, and it is even reported higher (up to 7%) in children with special educational needs (Kurlan et al., 2001) and autistic spectrum disorders (Baron-Cohen et al., 1999). TS is estimated to be present in 0.1 to 0.5% of the general population (Tanner and Goldman, 1997), and about 80% of the cases of TS are males (Apter et al., 1992). TS is commonly associated with obsessive-compulsive disorder (OCD) and attention deficidhyperactivity disorder (ADHD) (Singer, 1997). Obsessive compulsive symptoms or OCD have been

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CHAPTER 1: INTRODUCTION

reported in 20-60% of TS patients; also, OCD patients have a 7% risk of having TS, and 20% risk of having tics (Cohen and Leckrnan 1994.33: 2-15 JAACAP). ADHD has been

observed in 50 to 90% of TS subjects (Leckman et al., 1999). Other comorbidities seen in TS cases are non-OCD anxiety disorders, phobias, major depression, oppositional defiant disorder, rage, restless leg syndrome, and bipolar disorder (Eapen et al., 1997; Kurlan et al., 2002). Freeeman and colleagues studied 3,500 TS individuals in 22 countries. They found that 15% had comorbid oppositional defiant disorder, 37% had a history of anger control problems, 20% had social skills problems, and 6% had sexually inappropriate behaviours (Freeman et al. 2000).

1.4 Description of tics

Tics are sudden, repetitive, non-rythmic, and stereotyped movements and sounds that affect discrete muscle groups. Motor tics usually begin between ages of 3 and 8 years with eye blinking andlor other simple movements of the upper part of the body, including elevation of the eyebrows, grimacing, or a brief shrug of a shoulder. With time motor tics may become more complex, and involve different muscle groups organized in sequence. Motor tics can incorporate any voluntary movement by any portion of the body. These coordinated movements may resemble normal motor gestures. Examples of complex motor tics are sustained looks, facial gestures, obscene gestures (copropraxia) or the imitation of the movements that someone else just did (echopraxia) (Lees et al., 1984).

Simple phonic tics, such as sniffing, throat clearing or coughing, can begin as early as 3 years of age, but typically follow the onset of motor tics. Phonic tics are

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CHAPTER 1: INTRODUCTION

produced at the respiratory apparatus by the contractions of laryngeal, oral, and nasal musculature. Complex phonic tics include repetitive syllables, words, phrases, changes in rhythm or volume of speech, repetitions of what other people say (echolalia), repetition of own words (palilalia), and intrusion of offensive words into normal speech (coprolalia).

Sometimes tics are preceded or accompanied by uncomfortable sensations or an urge to move, which are sometimes called 'sensory tics'. Berardelli and colleagues (2003) suggested that tics appear because patients cannot inhibit an irresistible urge to move in response to internal signals.

1.5 Evidence found through imaging studies

Magnetic resonance studies have provided evidence of subtle basal ganglia abnormalities in TS. It has been suggested, for example, that normal asymmetry of the basal ganglia, larger on the left, is lost in TS. The caudate or the lenticular nuclei in TS patients have been shown to be abnormal in volume (Peterson et al., 1993; Singer et al., 1993). In a study of 154 children and adults with TS and 130 healthy controls, caudate nucleus volumes were found to be significantly smaller (p=0.008) in TS children and adults; lenticular nucleus volumes were smaller in TS adults only (Peterson, Thomas et al., 2003). TS subjects also exhibited abnormalities of the corpus callosum (Baumgardner et al., 1996).

Positron emission tomography (PET) and single photon emission computed tomography (SPECT) studies also identified frontal lobe metabolic differences between TS and normal subjects (Braun et a]., 1993; Moriarty et al., 1995).

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CHAPTER 1 : INTRODUCnON

1.6 The involvement of dopamine in the etiology of the disorder

Dopamine (DA) has a critical role in controlling basal ganglia output, and it contributes significantly to the initiation of movement. Evidence of the involvement of this neurotransmitter in the pathogenesis of TS originally comes from observations of the effects of pharmacological agents on TS patients. The initial successful treatments of TS in the 1960s were obtained with the neuroleptic Haloperidol. Since then, it is known that drugs that block the action of DA, including typical and atypical neuroleptics (Lombroso et al., 1995; Sallee et al., 1997), tetrabenazine and a-methylpara-tyrosine (Sweet et al., 1974; Jankovic et al., 1984) tend to reduce tic symptoms in TS. On the contrary, agents and actions that increase the function of the dopaminergic system, such as L-DOPA, stimulant medication, or neuroleptic withdrawal, tend to exacerbate tics (Golden, 1974; Singer et al., 1981). These observations are consistent with the hypothesis of a hyper- functional dopaminergic system in TS.

No significant differences between TS cases and controls have been reported for Doparnine receptors D l and D2 binding in postmortem striatal tissue. However, a postmortem study reported increased striatal expression of the dopamine transporter by measures of [ 3 ~ ] mazindol binding (Singer et al., 1991). Similar results were obtained in vivo, by means of SPECT, using the dopamine transporter ligand [lZ31] PCIT (Malison et al., 1995; Muller-Vahl et al., 2000). Furthermore, one study reported enhanced putaminal dopamine release following amphetamine administration in subjects with TS (Singer et al., 2002). In a recent study, PET was used with the type 2 vesicular monoamine transporter ligand ["c] dihydrotetrabenazine (DTBZ) in 19 patients with TS and 27 controls. In this study an increase in right ventral striatal DTBZ binding in TS subjects

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CHAPTER 1: lNTRODUCTION

was found, suggesting that abnormal ventral striatal dopaminergic innervation may be an underlying factor in tics (Albin et al., 2003).

1.7 Possible involvement of other neurotransmitters in the etiology of the disorder

It has been observed that emotional stress affects the intensity and frequency of tics in TS patients; given that the central noradrenergic neurons have an important role in stress response, norepinephrine has also been considered in the investigation of TS etiology. It has been suggested that this neurotransmitter plays at least a modulating role in TS because some studies showed that the a2-noradrenergic agonist clonidine tends to reduce tic symptoms (Leckman et al., 1983; Chappell et al., 1997).

The serotonergic system may also be involved in TS. Serotonergic neurons of the raphe nuclei project particularly to limbic areas and the basal ganglia, which are implicated in the etiology of TS. Furthermore, selective serotonin reuptake inhibitors, and sometimes monoamine oxidase inhibitors, are an effective treatment for OCD, which is a common comorbidity observed in TS patients (Micallef and Blin, 2001); serotonin is also implicated in other common comorbidities of TS such as impulse control, and ADHD. In addition, low plasma levels of serotonin precursor tryptophan have been found in TS subjects (Comings, 1990); other researchers have reported reduced tryptophan in subcortical and cortical regions from TS patients (Anderson et al., 1992b); also, reduced levels of serotonin and its metabolite 5-hydroxyindoleacetic acid were observed in nearly

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CHAPTER I : INTRODUCTION

all the brain areas that were examined. This means that decreases in serotonin, its precursor and its metabolite may be involved in TS.

Other neurotransmitters with an important role in the basal ganglia have been considered as candidates in the study of TS; these neurotransmitters are glutamate, y-

aminobutyric acid (GABA), and acetylcholine. In a postmortem study, reduced levels of glutamate were found in the medial globus pallidus of 4 TS patients (Anderson et a]., 1992a). A transgenic mouse model was created with characteristics that may be similar to OCD and TS. This model was obtained by expressing a neuropotentiating cholera toxin transgene in a subset of dopamine receptor D l neurons that are thought to induce glutamate output. The effects of glutamate receptor binding drugs were examined in one transgenic mice line (DlCT-7). In this study, it was found that a glutamatergic drug (MK-

801) exacerbated the OCD and TS-like behaviors in the transgenic mouse; the authors concluded that these behaviors may be mediated by cortical-limbic glutamate (McGrath et a]., 2000).

In studies with GABA and acetylcholine, no significant differences have been found between cases and controls, but the number of studies have been limited (Anderson et al., 1992a).

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CHAPTER 1: INTRODUCTION

1.8 The role of genetics in the etiology of TS

Georges Gilles de la Tourette suggested that TS was an inherited disorder. Later, throughout the 20th century, numerous familial studies confirmed that genetics plays an important role in the etiology of this disease (Friel, 1973; Kidd et al., 1980; Walkup et al., 1996). Most of these studies have been done with families from the United States and Europe, with the exception of one that analyzed Japanese subjects. According to these investigations, the risk of developing TS for first-degree relatives of TS patients ranges from 9.8 to 15%. When tics are also considered, the risk ranges from 15 to 20%, a clearly higher risk than the one reported by estimates in the general population. In previous studies, it was shown that while 50 to 70% of monozygotic (identical) twins were concordant for TS, this concordance was only observed in 8 to 10% of dizygotic (fraternal) twins (Shapiro et a]., 1978; Price et al., 1985); this reflects a heritability for TS of 90%. When tics were also included in the concordance, the rates were 75 to 90% for monozygotics and 23% for dizygotics. This demonstrates that genes have an important role in the etiology of TS, given that the genetically identical twin of a TS subject has 5 times more risk to develop the disorder compare to a fraternal twin. Added to the important role of genetic factors in TS, there are other critical non-genetic factors involved, since not all the identical twins are fully concordant for the disorder. One adoption study also showed that hereditary factors are crucial for the development of TS. By means of the family history method, the adoptive relatives of 22 TS cases were found to have no history of tics at all. On the contrary, two of these TS patients had biological relatives available and the biological father of one and the biological grandfather of the other one had a tic disorder (Shapiro et al., 1988). In addition, the authors of this study

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CHAPTER 1 : INTRODUCTlON

examined the family history of tics of another 641 TS cases. They found that 35% of them had a first-degree relative with tics, which contrasted with the complete negative history of tics in the adoptive relatives of the first group. These results represented new proof of the importance of genetic factors in TS.

In family studies, it was also shown that OCD was frequent in first-degree relatives of TS patients. It has been suggested that OCD may be genetically related with TS, but it is still not clear whether the same relation with TS applies to ADHD (Pauls et al., 1991). Pauls and colleagues (1993) investigated the presence of ADHD in first degree relatives of TS patients with or without ADHD. They found cases were ADHD is independent of TS, but also cases were ADHD is secondary to TS.

The initial segregation analyses suggested that there was a major locus for TS, which was transmitted in an autosomal dominant way, with variable penetrances (Pauls and Leckman, 1986; Eapen et al., 1993). However, other studies failed to confirm this model (Kidd and Pauls, 1982; Comings et al., 1984). Recent studies have found that an intermediate model of inheritance, with one or few major loci and a multifactorial background, may be more adequate for TS. One study found evidence for a mixed model of inheritance in their complex segregation analysis of 53 TS probands and their first degree relatives, where a single major locus and an undefined non-genetic multifactorial background explained the transmission on TS (Walkup et al., 1996). About 40% of the phenotypic variance observed was accounted for by this multifactorial background. Another study on 108 German TS probands found that TS inheritance cannot be explained by any model of Mendelian inheritance (Seuchter et a]., 2000). In conclusion,

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CHAPTER 1: INTRODUCTION

most of the studies provide strong evidence for the role of major genes in the expression of TS.

1.9 Rationale and objectives of the research

The central aim of this investigation was to search for genetic variants associated with TS, as a step towards the elucidation of the etiology of the disorder.

Objective 1. To determine whether previously reported genetic associations with

TS could be replicated and extended in a sample from the French Canadian (FC)

population, using a suitable genetic methodology and a strict phenotyping scheme.

Theoretically, i n order to determine if an association between a genetic variant and a given trait is true, there must be some replication in independent samples (Anonymous, 1999). For this reason, the first objective of this research was to determine whether some of the previously reported associations could be confirmed and even extended with new markers in our sample. This research represents the first population-based genetic investigation of Tourette syndrome in French Canadians. Some of the advantages of studying this population are the following:

a) Decreased genetic heterogeneity.

This is caused by the small number of founders who are direct ancestors of most of the 6 million French Canadians in Quebec (Scriver, 2001). Thus, at the genetic level, there may be much more similarity (or less heterogeneity) among French Canadian

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CHAPTER 1: INTRODUCTION

patients than that observed among cases from outbred populations; this may facilitate the search for genetic variants that are associated with TS. Examples of loci for which a founder effectlor genetic dnft in the French Canadians was demonstrated are FAH (encodes for fumarylacetoacetate hydrolase), SACS (for sacsin), and PAPBNl (for polyadenilate-binding protein nuclear l)(Scriver, 2001).

b) Increased linkage disequilibrium (Boehnke, 2000)

It is possible that patients of this population share longer stretches of DNA around at least some of the genetic variants associated with TS, which would also facilitate the identification of relevant genes or regulatory regions.

c) Environmental exposure on this kind of population is more homogenous (Boehnke, 2000).

This may be important, given that non-genetic factors are known to be involved in the etiology of the disorder. If French Canadians have a relatively more homogenous environment, with respect to people from outbred populations (diet, health programs, etc.), then there may be less variables to consider and it may be easier to identify genetic factors. However, the possibility of a more homogenous environment affecting TS in French Canadians is only a hypothesis that would have to be proven; and it is still probable that other populations that are not isolated have similar environmental factors affecting TS.

TS is an etiologically complex disorder, influenced by both genetic and environmental factors. While variants that cause many Mendelian traits have been

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CHAPTER 1 : INTRODUCTION

successfully identified using the traditional model-based linkage approach, this technique has generally failed in the study of TS and other neuropsychiatric disorders. In contrast with Mendelian disorders, the mode of transmission of TS is still unclear, and above all, it is very probable that there is locus heterogeneity, along with the possibility of bilineal transmission of TS-related variants to an affected offspring. This may explain the many failed attempts to find linkage with parametric (model-based) methods in large pedigrees, with the premise that one single mutation relates to TS with a clear Mendelian mode of transmission.

The second method used for the study of TS has been non-parametric linkage analysis that is model-free, and a few suggestive results have been obtained for regions on chromosomes 8 and 4. In a genome scan study of 76 families with 110 total sib-pairs, maximum likelihood scores >2.0 were obtained in 4q and 8p regions (TSAIGC, 1999). A third approach is association analysis, which has been considered a good alternative for complex traits such as TS. Nevertheless, the traditional case-control approach has the disadvantage of being susceptible to population structure, resulting in an increase of false positive results due to ethnic differences between the case and the control group. It has been shown, for example, that allele frequencies at some loci vary considerably among sub-groups of the white population of the United States (Kang et al., 1999).

It has been suggested that the ideal situation in the study of complex traits is to try to replicate results in independent families, but also to perform new studies with different methodologies that might complement previous findings (Anonymous, 1999). Family- based association methods have rarely been used in the study of TS. These methods compare the alleles that the parents transmit to their TS-offspring (case alleles) to the

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CHAPTER 1: lNTRODUCTlON

alleles that these parents don't transmit to their TS-offspring (control alleles). This allows to test whether there is an excess of transmission of certain alleles to the affected offspring (transmission disequilibrium test), which would give, in this case, information of linkage andlor association with TS. Family-based association studies have a potentially higher power to map complex disease genes, such as TS, and are more robust in the presence of population stratification because the non-transmitted alleles of the parents belong to the same ethnic pool than the alleles of their TS offspring (Zhao, 2000).

The definition of the TS phenotype has been relatively inconsistent in previous studies. In some of them TS, chronic motor or phonic tics, OCD, transient tic disorder, and even tic disorder not otherwise specified (APA, 1994) were considered as affected (broad definition). Others used an intermediate phenotype with the inclusion of TS, chronic motor or phonic tics, and OCD in the same category. Finally, there were some researchers who made a distinction between TS cases with or without comorbid OCD. In this investigation, we had a complete phenotyping done by experienced specialists (Neurologists, Psychiatrists and Neuropsychologists) from the Montreal Tourette Syndrome Study Group who made a thorough clinical assessment for TS, OCD, ADHD and other psychiatric comorbidities. In this way, it was possible to include only definite TS cases in the study, from which we could make subgroups according to the presence or absence of OCD and ADHD.

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CHAPTER 1: INTRODUCTION

Objective 2a. To analyze specific genes belonging to the dopaminergic system,

with special emphasis on the genes already reported to be associated with TS.

As previously mentioned, several lines of research and clinical observations have suggested that the dopaminergic system is involved in the etiology of TS. Doparnine accounts for approximately half the catecholamines in the brain. It is widely distributed throughout the brain, particularly in the striatum, which receives major input from the substantia nigra and plays an essential role in the coordination of body movements. Doapamine is also believed to be involved in motivation, reward, and reinforcement (Keitz et a]., 2003).

In the dopaminergic pathway, as shown in figure 1 , the amino acid tyrosine is

metabolized by the tyrosine hydroxylase (TH), an enzyme that synthesizes doparnine. TH uses molecular oxygen and tyrosine as its substrates and biopterin as its cofactor (Shiman et a]., 1971). TH catalyzes the addition of a hydroxyl group to the meta- position of tyrosine, forming 3-4-dihydroxy-L-phenylalanine (L-DOPA). TH can also hydroxylate phenylalanine to form tyrosine, which is then converted to LDOPA. Once L-DOPA is formed, DOPA decarboxylase converts it to dopamine (Christenson et a]., 1970). This represents the final step in the pathway of dopaminergic neurons, but dopamine can also be transported by vesicles into adrenergic terminals where it is metabolized to norepinephrine by the Dopamine (3-hydroxylase (DBH); this enzyme is concentrated within the vesicles that store catecholamines, most of the time bound to the inner vesicular membrane, but sometimes free within the vesicles. Norepinephrine can be converted to epinephrine (also called adrenaline), which is fhe catecholamine present at lower levels in the brain (Olschowka et a]., 1981).

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CHAPTER I: RJTRODUCnON Tyrosine

Pre-synaptic

neuron

ACP 1 Doparnine Transporter

.

.

m.

. .

+ *. MAO-B

.

.

.

amine Receptors

Figure 1 Diagram of dopamine metabolism and transmission.

TH (tyrosine hydroxylase); DDC (DOPA decarboxylase); DA (dopamine); MAO-A (monoamine oxidase-A); HVA (homovanillic acid); ACPl (tyrosine phosphatase, from acid phosphatase locus 1); MAO-B (monoamine oxidase-B); DBH (dopamine-beta hydroxylase); NE (norepinephrine); DRDl (dopamine receptor Dl); DRD2 (dopamine receptor D2); DRD3 (dopamine receptor D3); DRD4 (dopamine receptor D4); CAMP (cyclic adenosine monophosphate); PKA (CAMP-dependent protein kinase); DARPP-32 (dopamine and CAMP-regulated phosphoprotein); PPI (protein phosphatase 1).

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CHAPTER I: INmODUCTION

Once DA is liberated from the pre-synaptic neuron, it binds to dopamine receptors. Dopamine Dl-class receptors are D l (DRD1) and D5 (DRDS); the D2-class receptors are D2 (DRD2), D3 (DRD3), and D4 (DRD4). D l receptors are present on dynorphinl substance P-containing medium spiny neurons. Both D l and D2 receptors are abundant on a subpopulation of neurons in the striatum. The D2 receptors are present on the terminals of corticostriatal projections, on enkephalin-containing medium spiny neurons and cholinergic interneurons and, as autoreceptors, on dopaminergic neurons on the substantia nigra pars compacta. Dl-class receptors are excitatory and they stimulate the adenylyl cyclase (CAMP) activity; they are coupled to adenylyl cyclase and the CAMP-dependent protein kinase (PKA) cascade. D2-like receptors, on the contrary, are inhibitory and they reduce adenylyl cyclase activity; their signaling pathways seem to be more complex, with a negative coupling to cAMP and inositol phospholipids metabolism (Sibley et al., 1993). The expression of dopamine receptors and their signal transduction machinery seem to be regulated by tyrosine kinases and phosphatases. Dopamine and cAMP regulated phosphoprotein, molecular weight 32 Kilodaltons (DARPP-32) was found to function as a regulatory element for the protein phosphatase 1 (PP1). When D l receptors are stimulated and PKA is activated, there is phophorylation that converts DARPP-32 into a potent inhibitor of PPI. As PP1 can antagonize many actions of PKA, DARPP-32 can remove the opposition to the actions of activated PKA, thus providing necessary signal amplification (Greengard et a]., 1999). Other known phosphatase is encoded by the acid phosphatase locus 1 (ACP 1); this phosphatase is involved in several intracellular signal transduction pathways and is mainly expressed in the brain (enriched at the synaptic terminals). Functional studies related to the basal ganglia demonstrated

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CHAPTZR 1: INTRODUCTION

that in order to have a motor behavior, there most be a synergistic action of both D l , and mainly D2 receptors (Gingrich et al., 1992: Palermo-Neto, 1997).

DA is deaminated by the monoamine oxidase and o-methylated by the catechol-o- methyl transferase. The final metabolite is homovanillic acid. The Monoamine oxidase (MAO) is a protein present in the outer mithochondrial membrane. M A 0 type A (MAO- A) is present in catecholaminergic neurons and their axons; M A 0 type B (MAO-B) is more abundant in serotonin-containing neurons and astrocytes. (Vitalis et al., 2002).

At first, we analyzed doparninergic genes that had been previously associated or linked to TS in the past (Chapter 2, Section 2.1, article published in Molecular

Psychiatry). Then, we analyzed variants from three extra dopaminergic genes (not previously reported to be associated with TS): Tyrosine hydroxylase (TH), dopamine-

beta-hydroxylase (DBH), and a low-molecular weight tyrosine phosphatase (ACP1).

Objective 2b. To analyze potentially functional variants in dopaminergic genes.

It has been suggested that among all the polymorphisms, those that appear to have a functional effect on the gene product should be preferentially analyzed in the study of complex traits. Genetic variants with significant functional effects most likely are related to an amino acid substitution i n the gene product, a deletion or insertion that results in a frameshift in the coding region, the complete or partial deletion/duplication of a gene, the polymorphism directly affecting gene transcription, RNA splicing, mRNA stability or mRNA translation. In particular, polymorphisms in promoter regions upstream of genes

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CHAPTER 1 : INTRODUCTION

may potentially affect the process of transcription and as such, the levels of expression of the gene product. A variation in the DNA sequence at the promoter region may alter the affinities of existing protein-DNA interactions or even create new binding sites for proteins. Thus, the specificity and kinetics of transcription may be altered. We selected a polymorphism in the Dopamine D4 receptor (DRD4) gene that affects the amino acid sequence and the length of the third transmembrane domain of the protein altering the conformation and possibly the function of the receptor. Furthermore, a second putative functional polymorphism in the promoter region of the DRD4 was chosen, and it contains the consensus binding sequences for several known transcription factors (Seaman et al., 1999). In addition, the analysis of the monoamine oxidase A (MAO-A) gene included a promoter polymorphism known to affect the transcription levels of the gene (Sabol et al., 1998), as well as two other markers within the gene, which are not associated with an amino acid substitution, but have indirectly been associated with differences in MAO-A activity (Hotamisligil and Breakefield, 1991).

Objective 3. To analyze markers contained within a TS candidate region on

chromosome 7.

Previously, some genomic regions had been associated with TS through cytogenetic reports of translocations and duplications, but rarely has a result of this nature been replicated or extended with molecular genetic approaches (Chapter 3, article published in American Journal of Medical Genetics).

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CHAPTER I : INTRODUCTION

Objective 4. To analyze markers on chromosome 11-q24 region.

According to three previous results obtained with independent samples of an Afrikaner population and a large French Canadian family respectively, there is association and linkage between genetic variants in a region flanked by markers DllS1377 andD11S933 (Simonic et al., 1998; Merette et a]., 2000; Simonic et a]., 2001).

No further studies for TS have been reported in this region, and no other French Canadian families have been reported to be studied by other groups (Chapter 4, article to be submitted).

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CHAPTER 2: FORWARD GENETICS APPROACH

In this chapter, I will describe the results obtained with a "forward genetics" approach, which initially takes into consideration previous knowledge of the TS phenotype (an imbalance in the dopaminergic system). Section 2.1 is an article that describes the results on loci for which there were previous reports of association andlor linkage with TS. Section 2.2 describes the results with other dopaminergic genes.

SECTION 2.1

:

Tourette syndrome and dopaminergic genes: A

family based association study i n the French

Canadian founder population.

Adriana Diaz-~nzaldtia', Ridha ~oober', Jean-Baptiste Rivikre', Yves Dion', Paul L'espirance3, Franqois ~ i c h e r ~ , Sylvain ~ h o u i n a r d ~ . ~ , Guy A. ~ o u l e a u " ~ and the Montreal Tourette Syndrome Study ~ r o u ~ ' . ~ ~ ~ ~ ~ .

' ~ c ~ i l l University Health Centre, 'Douglas Hospital Research Centre, 3 ~ e n t r e Hospitalier de I'Universitk de Montrkal, 4 ~ a i n t e Justine Hospital. Montreal, Quebec, Canada.

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CHAPTER 2: FORWARD GENETICS

2.1.1 ABSTRACT

Tourette syndrome (TS) is a genetically complex disorder for which no causative genes have been unequivocally identified. Nevertheless, a number of molecular genetic studies have investigated several candidate genes, particularly those implicated in dopamine modulation. Results of these studies were inconclusive, which may be due, at least in part, to the variable ethnicity of the patients included in different studies and the chosen research design. In this study we used a family-based association approach to investigate the implication of dopamine-related candidate genes, that had been previously reported as possibly associated with TS [genes that encode for the dopamine receptors DRD2, DRD3, and DRD4, the dopamine transporter 1 (SLC6A3) and the Monoamine oxidase-A (MAO- A)]. The studied group was composed of 110 TS patients. These patients were selected from the French Canadian population, which displays a founder effect. Excess of transmission of the 7-repeat allele of the DRD4 exon-3 VNTR polymorphism (x' TDT =4.93, 1 df, p=0.026), and the putative "high activity" alleles of the MAO-A promoter VNTR polymorphism (x* TDT =7.124, 1 df p=0.0076) were observed. These results were confirmed in a subgroup of patients with no attention deficithyperactivity or obsessive- compulsive comorbid disorders. Haplotype analysis using one or two supplemental polymorphisms in each of these genes confirmed these associations and allowed to identify risk haplotypes. No associations were found for DRD2, DRD3 or SLC6A3. These data support the notion that DRD4 and MOA-A genes may confer an increased risk for developing TS in the French Canadian population.

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CHAPTER 2: FORWARD GENETICS

2.1.2 INTRODUCTION

At the end of the XIX Century, the French Neurologist Georges Gilles de la Tourette described in detail the disorder that was later named after him. Since then, Tourette syndrome (also known as Tourette's or Gilles de la Tourette syndrome) is recognized mainly by the presence of chronic tics, which are both motor and phonic. Tourette syndrome is generally a childhood-onset disorder, although in some cases the first symptoms are observed during adolescence, and always before 21 years of age. It is three to four times more common in boys than in girls and its prevalence may be as high as 0.5% of the population (Faridi and Suchowersky, 2003).

Since its early description by Gilles de la Tourette, the hereditary nature of this disorder was suspected and was subsequently confirmed during the second half of the XX

century (Friel, 1973); (Walkup et al., 1996). Indeed, while identical twin pairs were shown to be highly concordant for TS (50-56%), only 8% of fraternal twins were concordant for this disorder (Price et al., 1985), suggesting that part of the risk to develop the disease is genetically determined.

To date, molecular genetic studies on TS have been based on different assumptions, and positive results have been obtained by linkage (parametric and non- parametric) and association analyses (Pauls, 2001). Overall, the results of these studies are difficult to interpret given that the replication of positive findings was relatively infrequent. If multiple genes are implicated in TS, the lack of replication may be due to the small effects produced by each one of these genes. In addition, other study-design features such as ethnic heterogeneity among the analyzed populations and the use of case-

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CHAPTER 2 FORWARD GENETICS

control studies (sensitive to population structure biases) may also contribute to this non- replication problem.

We used the transmission disequilibrium test (TDT), which has proven to be a robust family-based association analysis that compares the number of times an allele is transmitted (T) versus non-transmitted (NT) from a heterozygous parent to an affected offspring (Spielman et al., 1993). For the multi-allelic markers we used the extended TDT (Sham and Curtis, 1995). We only included patients from a French Canadian origin. This ethnically homogeneous population has proven to be a very powerful tool to identify genes implicated both in monogenic Mendelian disorders (Brais et al., 1998), as well as complex disorders such as schizophrenia (Chumakov et al., 2002). It has also been suggested that testing functional polymorphisms in candidate genes relevant to the disorder under investigation should be considered whenever possible. This approach may cast some biological validity on statistically significant results of association studies.

Genes involved in dopamine transmission are suitable candidates for TS. Indeed, neuroleptics, both typical and atypical, have been proven to improve the symptoms of TS in a dose dependent fashion (Lavenstein, 2003). In addition, neuroimaging studies have found abnormalities in brain structures that are functionally related to the dopamine neurotransmission system (Peterson, 2001; Peterson et al., 2003). By searching the literature, we obtained a list of candidate genes coding for proteins implicated in dopamine pathways that have been positively associated andlor linked with TS (table I). The genes are: the dopamine transporter gene (SLC6A3). the three D2-like dopamine receptor genes (DRD2, DRD3 and DRD4). and the Monoamine oxidase-A gene (MAO- A).

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CHAPTER 2: FORWARD GENETICS

2.1.3 MATERIALS AND METHODS

Subjects

9

Three hundred and thirty DNA samples, belonging to 110 unrelated TS patients and their parents, were analyzed. The patients and their families were ascertained at the TS Clinic of the Allan Memorial Institute, and the Montreal General Hospital. All the patients had definite TS defined by the TS Classification Study Group (TSCSG, 1993; Freeman et al., 1995). The members of each family were interviewed by an interdisciplinary group, which was formed by a neurologist, a psychiatrist and a neuropsychologist. Clinical information was obtained for TS, attention deficithyperactivity disorder (ADHD) and

obsessive-compulsive disorder (OCD). All the patients had full French Canadian ancestry, defined as having four French Canadian grandparents. Subjects were excluded from the study if there was any evidence of other neurological disorder that could mimic TS, if they had tics secondary to drug abuse or head injury, if they had a psychotic disorder, or neuroleptic induced tardive dyskinesia. All subjects gave a written informed consent after the study was explained to them. For children who accepted to participate in the study, the consent was obtained from their parents also.

Genotyping

DNA was extracted from blood using standard phenollchlorophorm methods. PCR reaction for the 40 bp VNTR was performed according to the method described by

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CHAPTER 2: FORWARD GENETICS

Vandenbergh and colleagues (Vandenbergh et al., 1992). The PCR products were electrophoresed on a 2.5% agarose gel and stained with ethidium bromide.

The amplification for the Taq I polymorphism was performed according to a previously described method (Grandy et al., 1993). Ten microliters of PCR product were digested with 5 U of Taq I enzyme (New England Biolabs) at 65OC for 4 hours and run on 2% agarose gels.

PCR amplification for the Msc I polymorphism was performed as previously described (Joober et al., 2000). PCR products were digested with MscI (New England Biolabs) for 4 hours at 37OC and run on 3% agarose gels.

DRD4

PCR amplification for the exon 3, 48 bp-VNTR was performed according to Lichter et al. (Lichter et al., 1993), modified by Grice et al. (Grice et al., 1996). PCR products were run on 2.2% Metaphor agarose gels. All the samples were amplified at least two times in order to confirm the genotypes. A 25 bp ladder was used as a molecular weight marker (Invitrogen Life Technologies). The 120 bp duplication polymorphism at position -1200 in the promoter region was analyzed as previously described (Seaman et

(46)

CHAPTER 2: FORWARD GENETICS

al., 1999), with a modified annealing temperature of 56-53°C. Electrophoresis of PCR products was done on 1.5% agarose gels.

MA 0 - A

Although the previously reported association for TS was not done with the promoter VNTR polymorphism, we chose to focus on this marker because of its suspected functional relevance. For this polymorphism, PCR amplification was performed as previously described (Sabol et al., 1998). adapted for a 20p1 reaction. PCR products were run on 2.5% metaphor agarose gels (FMC Bioproducts). Two other polymorphisms, the Fnu

4HI

and Eco RV SNPs, were analyzed; PCR amplifications were done according to previously described methods (Hotamisligil and Breakefield, 1991). PCR products were digested with 5 U of the corresponding restriction enzyme (Eco RV and Fnu4H1 respectively). PCR products were run on 2% agarose gels.

2.1.4 RESULTS

There were 88 male and 22 female patients (4:l male: female ratio). The mean age was 19 years. In addition to TS, 56% of the patients had OCD, and 40% had ADHD.

Genotyping of exon 3 VNTR in the DRD4 gene revealed 7 alleles corresponding 2

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CHAPTER 2: FORWARD GENETICS

0.033) and genotype-wise (TDT

x2

= 19.23, df = 10, p = 0.038) transmission disequilibrium was observed. Because previous studies had found association andlor linkage specifically between the 7 repeat allele (7) and TS, we focused mainly on its transmission (table 2) and found that it was significantly more transmitted to affected individuals (transmitted in 64 % and non-transmitted in 36% of the cases, XZ = 5.88, df = 1, p = 0.015). As shown in table 3, we analyzed a subset of 38 TS patients who did not

have comorbid ADHD or OCD and we also found an excess of transmission of the 7- repeat allele (X'TDT =4.57, df=l, p=0.032).

Regarding the second DRD4 polymorphism analyzed, the 120 bp duplication at the promoter region, the frequencies of the short (S) allele and long (L) allele were 0.23 and 0.77 respectively in the TS unrelated subjects. Although no association was found with this polymorphism, a haplotype analysis focusing on allele 7 (exon 3) showed that this allele was generally co-transmitted with allele L (promoter). As indicated in table 4, the haplotype "7L" was transmitted to TS patients more frequently than expected (xZ= 4.74, 1 df, p= 0.023) when compared to all the other haplotypes. Haplotype "7/Sn was only observed 5 times, which prevented a statistical analysis. In contrast, allele S was observed in combination with alleles 2 or 4 in 84% of the times. In total, 11 haplotypes were obtained (data not shown).

MAO-A

In the case of the MAO-A gene promoter VNTR polymorphism, genotyping revealed 5 different alleles distinguished by the number of their 30 bp repeat sequence: 2- , 3-, 3.5-, 4- and 5- repeat alleles. We compared the "high" activity vs. the "low activity"

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CHAPTER 2: FORWARD GENETICS

alleles reported by Sabol and colleagues (Sabol et al., 1998). We found that heterozygous parents preferentially transmitted the "high activity" alleles. From the total "high activity" alleles found in heterozygote parents, 68% were transmitted to the TS patients. In contrast, from the "low activity" alleles, only 33% were transmitted (table 5). We then analyzed the Fnu 4H1 and EcoRV SNPs to test whether we could find a commonly transmitted haplotype. We found a significant excess of transmission of a "high-activity" haplotype (3-1-1) formed by alleles of the promoter VNTR and both SNPs (table 6). Haplotype "1-2-2", on the contrary was significantly less transmitted to TS offspring.

OTHER DOPAMINERGIC GENES

For the dopamine transporter gene (SLC6A3) we identified alleles of 4, 6, 9, 10 and 11 copies. Alleles 4 and 6 were rarely observed, and they were never transmitted. As previously reported, allele 10 was frequently transmitted, but this was not statistically significant.

Similarly, we could not find any association with the DRD2 Taq I A polymorphism, although there was a slight trend towards a higher allele A1 transmission

(data not shown).

Concerning the DRD3 Msc I polymorphism, we also failed to find an association with TS using the TDT test.

Figure

Table  14  TDT analysis for marker D7S522  50
Figure  1  Diagram of dopamine metabolism and transmission.
Table 1. Previous associations between candidate gene polymorphisms and  TS
Table  2.  TDT analysis for the DRD4 exon  3  VNTR.
+7

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