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MUTATION SCREENING OF DOPAMINE AND SEROTONIN CANDIDATE GENES IN TOURETTE'S SYNDROME ANI) ALCOHOL
DEPENDENT PATIENTS
Miles Thompson
A Thesis submitted in conformity with the requirements for the Degree of Master of Science
Graduate Department of Pharmacology University of Toronto
O Copyright by Miles Thompson
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Mutation screening of dopamine and serotonin candidate genes in Tourette's syndrome and alcohol dependent patients
Miles Thompson, M.Sc. 1 997
Department of Pharmacology
University of Toronoto
PhamacoIogical evidence suggests that both dopamine and serotonin system genes may be
involved in the inheritance of Tourette's syndrome (TS) and alcohol dependence. Dopamine
transporter antagoniçts used to treat attention deficit-hyperactivity disorder (AD-HD) worsen
comorbid with TS, emphasizing possible TS dopamine dysregulation. Serotonergic
antidepressants treating obsessive compulsive disorder (OCD) comorbid with TS and dcohol
dependence withdrawal aiso implicating serotonin dysregulation in these disorders. Therefore,
we conducted a candidate gene study of the dopamine D l (DRDI), dopamine transporter
(DAT I ) and serotonin transporter (SHTT) genes in these disorders. The DRD 1 gene was found
to be non-polymorphic in patient and control groups. Comrnon conserved sequence variants of
the DATl gene were identified but were not associated with TS or alcohol dependence. The
short promoter 5HTT gene was significantly more cornrnon in the TS group compared with
controls (X2~=0.04), suggesting that the SHTT gene may be a susceptibility locus in TS.
1 would like to thank Drs. Bnan O'Dowd and Susan George for the oppomuiity to be part of their
research group. 1 thank our collaborators, Drs. George ühl and David Vandenbergh of the
Laboratory of Molecular Neurobiology, NIDA, for their contributions tn my work on the
dopamine transporter gene; Dr. Jeanette Holden, Department of Psychiatry, Queens University
for help interpreting her candidate gene studies; Dr. James Kennedy and Dr. Glen Sunohara,
Neurogenetics Section, Clarke Institute, for invaluable insight into the correct tools required to
smdy the inheritance of complex neuropsychiatrie disorders. 1 thank Mr. Tuan Nguyen, Ms. Mai
Nguyen. Mr. Yang Shen and Mr. Adriano Marchese for their excellent technical advice. Lastly, 1
thank my farnily and friends for encouraging me to pursue graduate studies.
Abstract
Acknowledgments
Table of Contents
List of Tables
List of Figures
Abbreviations
PAGE
i
ii
iii
vii
viii
ix
1. INTRC)DUCTIOIY
1.1 Neurotransmitter G protein coupled-receptors and transporters in disease 1
1.2 Clinical definition of TS, associated disorders and alcohol dependence 3
(a) TS and comorbid neuropsychiatrie disorden 3
(b) Alcohol dependent disorder and associated withdrawal symptoms 4
1.3 The pharmacology of TS, associated disorders and alcohol dependence 5
(a) Typical and atypical neuroleptics implicate dopamine pathways in TS 5
(b) SFüs implicate serotonin pathways in TS-OCD and TS-anxiety 6
(c) Methylphenidate implicates dopamine pathways in TS-AD-HD 7
(d) SRIs implicate serotonergic pathways in alcohol dependence withdrawal 8
(e) Dopamine is implicated in animai models of aicohol consumption 10
1.4 Mode of inheritance of TS, comorbid disorders and alcohol dependence 10
(a) Autosomal dominant mode1 of TS 1 I
i i i
(b) Polygenic models ofTS, AD-HD, OCD and alcohol dependence
Candidate gene hypothesis of TS, comorbidities and alcohol dependence
The dopamine hypotheses of TS, AD-HD and alcohol dependence
(a) The DATI and DRD 1 genes are candidates for TS
(b) The DATl and DRD 1 genes are candidates for AD-HD
(c) The DATl and DRDl genes are candidates for alcohol dependence
The serotonin hypothesis
(a) The 5HTT gene is a candidate for TS
(b) The SHTT gene is a candidate gene for aicohol dependence
Screening candidate genes for sequence variants
(a) Mutation screening by SSCP analysis
Research objective: to screen candidate genes for sequence variants
(a) Isolate DATl gene sequence variants in TS and alcohol dependence
(b) lsolate DRDI gene sequence variants in TS and alcohol dependence
(c) Genotype 5HTT gene sequence variants in TS and alcohol dependence
Research straterrv for candidate gene screenine and association studies
2- * 2.0 Materials
2.1 DNA extraction
2.2 Clinical sarnples
(a) Tourette's syndrome samples
(b) Alcohol dependence samples
(c) Control samples
2.3 PCR amplification of gene fragments subjected to SSCP mutation screening
(a) Amplification of GPR19 gene fragments used to refme SSCP method
(b) Amplification of DRD 1 fragments to be analyzed by SSCP
(c) Amplification of DATl exons to be analyzed by SSCP
(d) Genotyping the DATl 3'VNTR by PCR amplification
(e) Genotyping the SHTT polymorphisms by PCR amplification
2.7 SSCP mutation screening of GPR19, DRD1 and the DATl gene
(a) Overview of the SSCP methodology
(b) SSCP protocol adapted for use with Xcell II apparatus (Novex)
2.8 Identification of sequence variants by chah-termination DNA sequencing
2.9 S tatistical Methods
39 l3IEmLE
3.1 SSCP mutation screening revealed polymorphic variants of the GPRl9 gene
3.2 SSCP mutation screening of the DRD 1 gene
3 -3 SSCP mutation screening of the DAT 1 gene
3.4 Association study of DATl polymorphisms in TS and alcohol dependence
3.5 Association study of SHTT polymorphisms in TS and alcohol dependence
4. DISCUSSION
4.0 Interpreting candidate gene sequence variants in patient and control groups
(a) Conservation of the DRD 1 gene in TS and alcohol dependence
(b) DATl gene sequence variants in TS and alcohol dependence
( c ) Association studies of SHTT variants in TS and alcohol dependence
4.1 Conclusions
TABLE
1. Oligonucleotides Used in PCR-SSCP Analysis of Human GPR19 gene
2. Oligonucleotides Used in PCR-SSCP Analysis of Human DRD 1 gene
-i 3. Oligonucleotides Used in PCR-SSCP Analysis of Human DATl
gene
4. DAT l allele and genotype fiequencies: %'P values compare population fiequencies
5 . SHTT allele and genotype fiequencies: X Z ~ values compare patient and control fiequencies
PAGE
37
38
40
FIGURE
1 .
2.
The principal of PCR-SSCP analysis
PAGE
26
Optimization of SSCP temperature, running time and voltage to resolve 48 variants
The effect of SSCP ninning temperature on the resolution of sequence variants
Resolution of sequence variants in TM3 of GPRl9 50
Resolution of TM3 variant on 10% gels was inferior to that on 4-20% gels 52
Analysis of a fragment (350 bp) of the Dl receptor gene 54
SSCP anaiysis of DATl exon 2 in three control subjects 56
SSCP anaiysis of DAT1 exon 8 in three control subjects 57
SSCP analysis of DATl exon 9 in three TS subjects 58
SSCP analysis of DAT 1 exon 15 in three control subjects 59
PCR genotyping of the DATl 3'VNTR 6 1
Deletion/insertion SHTT promoter polymorphisrn genotyped on 4% agarose 65
Intron 2 VNTR polymorphism genotyped on 4% agarose 66
- 1.1 Neurotransmitter G protein coupled-receptors and transporters in disease
A number of diseases known to be caused by sequence variants in G protek coupled
receptors (GPCRs) have been described (Zastawny et al, 1997). Mutations causing both
loss and gain of receptor fûnction have been reported. Retinitis pigmentosa, one of the
earliest characterized GPCR pathologies. was found to be due to a gain of function
mutation in the rhodopsin gene (Dryja et al, 1990; Dryja et al, 1995). A loss of fünction
mutation in the rhodopsin receptor gene was recently found to cause a form of stationary
night blindness (Yamamoto et al, 1997). Other GPCR mutations have been identified.
These include a mutation in the V2 vasopressin receptor gene which causes nephrogenic
diabetes insipidus type 1 1 (van den Ouweland et al. 1992) and a mutation in the thyroid
stimulating hormone (TSH) receptor gene which causes hyperthyroidism (Kopp et al,
1995). Most recently, a mutation in the CCRS gene. a coreceptor for the hurnan
immunodeficiency virus (HIV) that is expressed on T helper cells, has been found to
confer resistance to HIV infection (Michael et al. 1997). The identification of simian
imrnunodeficiency virus (SIV) G protein-coupled coreceptors (Farzan et al, 1997), GPRl
and GPRI 5 (Heiber et al, 1996; Marchese et al, 1994). suggests that future studies of
GPCR genes in disease States may be relevant and fmitfùl.
Despite the rapidity with which the GPCR genes expressed in the central nervous
system (CNS) have been cloned (Marchese et al, 1997; O'Dowd, 1993; Seeman and Van
Tol, 1993), no mutations in these genes have yet been discovered to contribute to the
inheritance of neuropsychiatrie disorden (Petronis and Kennedy, 1995).
Pharmacogenetic studies have met with some success in identieing dopamine D 4
(DRD4) and serotonin 2A (5HT2A) receptor variants that coder different responses to
atypical neuroleptic drug treatment (Seeman et al, 1997; Rao et al, 1997). However, the
numerous attempts to utilize genetic markers in order to conduct linkage and association
studies of these disorders have not succeeded in convincingly associating GPCRs
expressed in the CNS with a neuropsychiatric disorder (Petronis and Kennedy, 1995).
In the work presented here, therefore, we set out to screen candidate genes implicated
in Tourette's syndrome (TS) and alcohol dependence for sequence variants that might
provide new insight into the inheritance of these disorders. The ernphasis of the work
was to screen DNA from patients with TS and alcohol dependence for mutations in genes
that had not been subjected to mutation screening previously. Therefore, we screened the
GPR19 gene which, while having no known ligand, shows high sequence homology and
similar tissue distribution to dopamine system genes including the dopamine DRD2 gene
(O'Dowd et al, 1996). From among the GPCRs, we also screened the DRûl gene for
sequence variants in two disorders that may have doparninergic etiologies: TS and
alcohol dependence.
We next elected to screen the dopamine transporter (DAT1) gene for sequence
variants in Tourette's syndrome (TS) and alcohol dependence. The final cornponent of
the work presented was based on the results of a similar mutation screening project which
identified a sequence variant in the 5' regulatory region of the serotonin transporter
(SHTT) gene that has been reported to alter 5HTT gene expression in vitro (Heils et al,
1996)
1.2 Clinical definition of TS, associated disorders and alcohol dependence
(a) TS and comorbid neuropsychiatrie disorders
TS is a relatively rare psychiatrie disorder affecting between 0.1 and 0.5 per thousand
people throughout the world. The cluster of symptoms that underlie TS have their onset
between the ages of 2 and 15. The diagnostic critena include recurrent, involuntary, rapid
motor movements with no purpose (tics) that affect multiple muscle groups. Symptoms
may Vary over weeks or months or may be suppressed in the short term but m u t be
otherwise unremittinz for at least a year in order to diagnose TS. The disorder is usually
IifeIong.
The tics are the essential feature of TS. Motor tics comrnonly involve the head, tono
and upper limbs. Vocal tics include sounds such as clicks, g m t s , yelps, barks, sniffs,
coughs and words. An irresistible urge to utter obscenities, coprolalia, is present in 60%
of cases. Symptoms are exacerbated by stress but disappear during sleep and absorbing
tas ks.
Associated features of TS include echokinesis, irnitating the movements of someone
being observed, and palildia, repetition of one's ovm phrases. Obsessive-compulsive or
ritual behaviors rnay also be present. These include obsessive thoughts of doubting,
compulsive rituals including complicated rnovements, impulses to touch objects, retrace
steps, squat and twirl when walking (Diagnostic and Statistical Manual of Mental
Disorders, Fourth Edition, 1 990).
TS is often diagnosed cornorbid with obsessive compulsive disorder (OCD)
(Rapoport and Inoff-Germain, 1997; Swedo and Leonard, 1996) or attention deficit-
hyperactivity disorder (AH-HD) (Castellanos et al. 1997; Schuerholz et al, 1996). Up to
50% of TS patients have been reported to be diagnosed comorbid with OCD. Comorbid
OCD or AD-HD in TS patients presents a challenge to treatment protocols because of the
need to simultaneously treat TS tics and the comorbid OCD or AD-HD. Treatment
modalities for OCD and AD-HD are distinctly different fiom each other and TS
treatments (Peterson, 1996). Major depression (Comings and Comings, 1993; Comings,
1994) and substance abuse disorders, such as alcohol dependence (Muller-Vahi et al,
1997; Comings, 1994), have also been diagnosed comorbid with TS. The contrasting
pharmacological interventions used to treat TS and associated comorbidities may provide
insight into the neuropathies that may underlie TS (Singer et al. 1993) and aicohol
dependence (Tiihonen et al, 1995).
(b) Alcohol dependent disorder and associated withdrawal symptoms
Alcohol dependence involves a pattern of pathologicd alcohol use or impainnents in
social or occupational functions due to alcohol use. The disorder is characterized by the
need for daily alcohol consumption and an inability to stop dnnking that is associated
with the need for markedly increased arnounts of alcohol in order to satisS, alcohol
craving (tolerance) (Diagnostic and Statistical Manual of Mental Disorders, Fourth
Edition, 1990). The consequence of a cessation of alcohol consumption for an alcohol
dependent patient is alcohol withdrawal, which involves severe anxiety, depression,
ataxia and muscle tremors (Lejoyeax, 1996; Anton, 19%; Diagnostic and statistical
manual of mental disorders, Fourth Edition, 1990).
1.3 The pbarmacology of TS, associated disorders and alcohol dependence
The neurotransmitters dopamine and serotonin have both been implicated in the
etiology of TS, associated comorbid disorders and alcohol dependence based on
pharmacological evidence (Lejoyeux et al, 1996; Anton et al, 1996; George et al, 1995,
Samson et al, 1993; Dyr et al, 1993). Treatment of these disorders is complex but only
those aspects salient to understanding the dopaminergic and serotonergic hypotheses of
TS and associated disorders will be reviewed here. However, the range of therapeutic
interventions used to treat these disorden provides strong evidence for their dysregulated
neurotransmitter etiology (Wolf et al, 1996; CastelIanos et al, 1997; Uhlenhuth et al,
1995; Narango and Bremner, 1 994).
(a) Typical and atypical neuroleptics implicate dopamine pathways in TS
Dopamine pathways are considered to be involved in TS pathology due to the
eficacy of typical neuroleptics, such as haloperidol and primozide, in the treatment of the
motor and vocal tics that are central to the disease (Peterson, 1996). Typical neuroleptics
are dopamine D2 receptor antagonists, although halopendol is reported to have some a,-
adrenergic effects, while primozole is reported to have some calcium channel blocking
properties. While side-effects often limit the use of haloperidol (Silva et al, 1996), the
fact that it remains a treatment of choice for managing TS motor and vocal tics (Silva et
al, 1996; Bruun and Budman, 1996; lancu et al, 1995) provides the best clinical rationale
for the hyperdopaminergic hypothesis of TS (Wolf et al, 1996).
The involvement of serotonin pathways in TS has less pharmacologicai evidence in
its support (Peterson, 1996). There is evidence that the atypical neuroleptic risperidone
has some eficacy in treating the tic symptoms of TS (Bruun and Budman, 1996).
Atypical neuroleptics, including rispendone and clozapine, antagonize both dopamine
and serotonin receptors. Risperidone, a 5HT2 receptor antagonist and a weak dopamine
D2 receptor antagonist (Peterson, 1996; Bruun and Budman, 1 W6), relieves the severity
of tics for some patients (Bruun and Budman, 1996), however, in 20% of TS patients
treated with risperidone, treatment is curtailed due to weight gain and sedating side
effects (Peterson, 1996).
Clozapine, the atypical neurolepic which antagonizes both 5HT2A and dopamine D4
receptors (Seeman et al, 1997), has not been shown to effectively treat tics (Caine et al,
1979) even though it is ofien effective in treating schizophrenia (Seeman et al, 1997; Rao
et al, 1996; Meltzer, 1995; Van TOI et al, 1992). In fact, exacerbation of tic symptoms
have been reported in some TS patients undergoing clozapine treatment (Caine et al,
1979). It is unclear whether the side effects of clozapine in TS result From serotonin or
dopamine receptor antagonisrn (Stein et al, 1997; Peterson, 1996). Taken together, there
is clinical and pharmacological evidence of a role for hyperserotonergic dysregulation in
the motor and vocal tics integral to TS.
(b) SRIs implicate serotonin pathways in TS-OCD and TS-anxiety
OCD comorbid with TS is often treated successfully with the combination of a
neuroleptic to control the tics and a serotonin antidepressant to relieve OCD symptoms
(Swedo and Leonard, 1994). Serotonin antidepressants al1 act, at least in part, to correct a
hyposerotonergic state that is hypothesized to underlie depression (Ogilvie et al, 1996;
Heils et al, 1996). By extension, the eficacy of serotonergic antidepressants in treating
OCD suggests that a hyposerotonergic state may also underlie OCD (Uhlenhuth et al,
1 995).
Both tricyclic antidepressants (TCAs) and serotonin reuptake inhibitors (SRIs) are
used to treat OCD comorbid with TS (Stein et al, 1997). The tricyclic antidepressant
(TCA) clornipramine (Stein et al, 1997; Peterson, 1996; Iancu et al, 1995), which
sensitizes serotonin receptors and releases norepinephrine in adrenergic pathways, has
been reported to be effective in treating OCD (Iancu et a[, 1995). The autonomic side
effects of TCA dnigs, however, limit the use of clomipramine treatrnent. Recently, SRI
inhibiton such as fluoxetine and fluoxamine (Stein et al, 1997; Peterson, 1996), which
antagonize the serotonin transporter, have been reported effective in treating OCD. In
order to treat TS-OCD, patients generally require an SRI or TCA for the relief of OCD
symptorns and rispendone augmentation for the relief of the motor and vocal tic
symptoms of TS.
(c) Methylphenidate implicates dopamine pathways in TS-AD-HD
Treatment of AD-HD commonly involves the use of dopamine psychotropic agents,
such as the dopamine transporter antagonist methylphenidate (MP) (Volkow et al, 1996;
Volkow et al, 1995; Geogiou et ai, 1995). AD-HD treatment, however, often complicates
the treatrnent of comorbid TS symptoms (Peterson, 1996). TS patients with comorbid
AD-HD require two contrasting dopaminergic interventions. Often the sedating
properties of typical neuroleptics are used to treat the tics associated with TS and a
dopamine psychotropic agent, such as MP, is used to treat AD-HD (Castellanos et al,
1997; Hetchman et al, 1993).
The tic symptoms in patients with TS-AD-HD are cornmonly reported to worsen
imrnediately after MP treatment commences (Daniels et al, 1996). However, once the
MP becomes effective in treating the AD-HD symptorns, tic exacerbation tends to
disappear. This makes MP the h g of choice for TS-AD-HD. Other dopamine
transporter antagonists, such as dextroarnphetamine, result in unabated tic exacerbation in
TS-AD-HD patients (Castellanos et al, 1997). Cocaine, a dopamine transporter
antagonist with a very high af3nity for the dopamine transporter by cornparison with MP,
results in extreme tic exacerbation (Castellanos et al, 1997). The complex pharmacology
of TS-AD-HD treatment suggests that the doparninergic disregulation in TS-AD-HD may
involve many aspects of doparninergic neurotransrnission .
(d) SRIs implicate serotonergic pathways in alcohol dependence withdrawai
SRIs are currently indicated not only for the treatment of obsessive compulsive
disorder (Peterson, 1996) but also depression (Ogilvie et al, 1996) and anxiety (Lesch et
al, 1996). Detoxification following clinical alcohol dependence is ofien associated with
alcohol related anxiety and depression (Lejoyeux, 1996; Anton, 1 996). The cornmon
alcohol dependence comorbidities, including affective disorders, often obscure whether
anxiety and depression cause or are caused by the withdrawal associated with alcohol
dependence (Anton, 1996). Regardless of their etiology. the symptoms of depression and
anxiety associated with detoxification have been reported to be improved when patients
are treated with SRIs (Lojoyewc, 1996; Anton, 1996; Oliver et al, 1993). This suggests
that a hyposerotonergic state underlies alcohol dependence.
Hurnan dxug trials and animal experiments also suggest that SRis may be effective in
reducing aicohol consurnption and implicate serotonin dysregulation in the pathways
involved with alcohol craving. A recent clinical trial of SRIs in non-depressed and
moderately depressed aicoholics found that alcohol consumption was decreased 1520%
in the SRI treatment groups compared to placebo control groups. SRI treatment of
alcohol preferring rats has also been s h o w to result in reduced alcohol consurnption.
However, evidence for long term reduction of alcohol consumption by SRIs is currently
lacking (Narango and Bremner, 1994).
The fhct that not al1 serotonergic antidepressants are reported to treat alcohol
dependence with the effectiveness of SRIs, however, suggests that much is still to be
learned about the significance of serotonergic dysregulation in alcohol dependence
(Lejoyeux, 1 996). Clinicai trials of other serotonergic drugs. including the 5HT 1 A
receptor agonist buspirone, the 5HT3 antagonist ondansetron and the 5HT2 antagonist
ritanserin, have met with less success than the SRIs in neating alcohol withdrawal or
ongoing alcoholism (Narango and Bremner, 1996). Therefore, although S R I drugs may
reverse a hyposerotonergic state that is common in alcoholics, the serotonergic pathology
that rnay underlie alcohol dependence remains unknown (Lejoyeux, 1996; Oliver et ai,
1 993).
(e) Dopamine is implicated in animal models of alcohol consumption
Alcohol dependence may also involve aberrant dopamine pathways (Ng et al, 1997;
Noble, 1996; Thiihonen et al, 1995; Rassnick et al, 1992). Treatment of the hi& ethanol
preferring CS7 mouse with the dopamine Dl receptor antagonist SKF 82958 or the
dopamine D2 antagonist quinpirole results in markedly decreased ethanol consurnption
for 24 hours (Ng et al, 1994). The resumption of ethanol consumption by mice treated
with these antagonists has been attributed to receptor desensitization (Ng el al, 1994;
George a2 et, 1995). While this animal model has been used to show the involvement of
dopaminergic pathways in alcohol consurnption, no neuroleptic treatment is currently
used to treat the primary symptoms of alcohol consurnption or withdrawal in human
alcohol dependent patients. A hypodopaminergic hypothesis of alcohol dependence has
been inferred frorn decreased ethanol consumption in the mouse model following
dopamine D 1 and D2 antagonist treatment (George et ai, 1995; Ng et al, 1994).
1.4 Mode of inheritance of TS, comorbid disorders and alcohol dependence
Debate about whether TS and a range of disorders including OCD, AD-HD and
alcohol dependence are part of a heritable TS spectnun disorder (Comings and Comings,
1993) remains controversial (Pauls et al. 199 1 ; Pauls et al, 1 988; Pauls et ai, 1 986). The
spectrum disorder hypothesis suggests that expression of TS genes results in not only the
tics charactenstic of TS but a broad range of associated behaviors including OCD, AD-
HD and aicohol dependence. Support for this idea cornes fiom a number of sources.
First, there is an unusually high incidence with which these disorders are diagnosed
comorbidly (Castellanos et al, 1 997; Stanley. 1 997; Cornings and Comings, 1993).
Second, pharmacological evidence suggests that this group of disorders share at least
some aspects of dopaminergic and serotonergic dysregulation (Petenon 1996). Third,
evidence exists that relatives of an affected individual are likely to be af5ected by the
same or one of the associated disorders at a rate exceeding that found in pedigrees of
nonpsychiatric individuals (Comings and Comings, 1993; Pauls et al. 1991 : Pauls et al,
1988). To date, however, studies that find evidence for a wide spectnim of related
disorders that share genetic liabilities (Comings et al, 1996) have not been replicated.
(a) Autosomal dominant model of TS
Studies of TS in affected pedigrees have resulted in the hypothesis that TS is inherited
as a single autosomal dominant gene that is subject to incomplete penetrance (Wolf ei al.
1996; Pauls et al, 1986; Comings et al. 1986). The single major locus hypothesis
suggests that the intermediate forms of TS. such as mild tic disorders. may result fiom the
inheritance of a single copy of the susceptibility gent (Comings et al. 1986). Thus. one
copy of the TS gene may confer some nsk while two copies may confer greater risk of
developing the severe tic disorder that characterizes TS (Comings et al. 1986). However.
the range of tic seventy found in TS patients (Pauls et al. 1996). combined with the fact
that tic severity worsens when one or both parents have a tic disorder (Comings ei al.
1986), suggests that TS may better fit a more complex model of inheritance (Walkup et
al, 1996). Models descnbing TS inhentance as semidominant, sernirecessive or as being
an example of incomplete dominance have also been proposed (Patel, 1996)
(b) Polygenic models of TS, AD-HD, OCD and alcohol dependence
Linkage studies on Tourette's syndrome have recently introduced evidence that tends
to exclude the possibility that a single major locus is responsible for the inheritance of TS
(Heutink et al, 1995). Complex segregation analysis suggests that susceptibility for TS is
conveyed by a major locus in concert with a multifactonal background, including
polygenetic and environmental contributions (Walkup et al, 1996). A revised mode1
proposes that the major locus is responsible for half of the observed phenotypic variation
described in the clinical literature of TS (Patel, 1996; Walkup et al, 1996). Models of TS
inheritance must be interpreted with caution, however, because none have yet met with
wide acceptance. The greatest complication to exarnining disorders such as TS is that its
heterogeneity may confound studies of its inheritance (Patel, 1996; Alsobrook and Pauls,
1996; Santengelo et al. 1996; Pauls et al. 199 1).
Similar problems must be accounted for when studying the genetic component of
alcohol dependence (George et al, 1993; Cloninger et al, 1985; Devor and Cloninger,
1989). While Type II alcoholics have been proposed to have a greater genetic liability,
which may include a hyposerotonergic phenotype (Cloninger et al, 1989; Cloninger,
1987), studies of this phenotype have not yet resulted in the identification of novel
candidate genes for alcohol dependence (Gordis, 1997). Studies associating alcohol
dependence with the dopamine Dl (DRDI), dopamine D2 (DRDL), dopamine D4
(DRD4) and DATl genes (to name but a few) al1 remain controversial (Comings et al,
1997; Muramatsu et al, 1995; Carlos et al, 1993; Uhl et al, 1992). The inheritance of
alcohol dependence, like that of TS (Patel, 1996), is likely to involve complex interaction
between many susceptibility loci and environmentai factors (Cloninger, 1987; Persico et
al, 1 993 ; Muramatsu et al, 1 995).
1.5 Candidate gene hypothesis of TS, comorbidities and alcohol dependence
Evidence fiom monozygotic twin studies suggests that a complex genetic liability for
TS, TS associated with AD-HD or OCD and alcohol dependence probably exists (Gordis,
1997; Patel, 1996). Because attempts to isolate candidate genes for TS, AD-HD, OCD
and alcohol dependence by linkage analysis have not been successful (Gordis, 1997;
Heutink et al, 1995; Gelemter et ai, I995), Mendelian inheritance of these disorders is
unlikely. However. failure to detect disease loci by linkage analysis does not exclude
polygenic models of the inheritance of TS or alcohol dependence.
In polygenic models, no single gene that contributes to the inheritance of a disorder is
sufficient in itself to cause the disorder phenotype. Each contributing gene, or
susceptibility loci (Nothen et al, 1992), may contribute additively (Comings et al, 1996)
to the inheritance of a suficient genetic liability to cause a recognizable phenotype.
Therefore, much of the recent work on the inheritance of TS, AD-HD, OCD and alcohol
dependence has abandoned the method of linkage analysis. the standard means of
identiQing candidate genes for a genetic disorder (Nothen et al, 1992; Hodge, 1994), in
favor of association studies (Owens, 1997; Berrettini, 1997; Hodge, 1994; Nothen et al,
I 992).
Association studies involve examining primary candidate genes: genes encoding
proteins that are known to be components of a pathway implicated in the pathology of a
disease. In their simplest fom, association studies involve comparing the frequency of a
genetic marker in patient and control groups (Owens, 1997; Hodge, 1994; Nothen et al,
1992). As a result, association studies are based on parametric models. The use of
parametic models to test the difference between patient ailele and genotype frequencies,
however, is correct only if certain assumption are true (Ott, 1 99 1 ).
Parametic models assume that while parameters cannot be estimated, standard
randorn variables (statistics), such as the X' statistic, can be calculated in order to test
whether significant differences exist between patient and control allele and genotype
frequency data. The X' statistic, the normaiized sum of squares of the differences
between patient and control allele or genotype frequencies, is assumed to fit a x2
distribution (Ott, 1991). The mode1 also assumes that allele and genotype frequencies in
patient and control groups are independent of other variables including the ethnic ongin
and sex of the members of each group (Hodge, 1994; Nothen et al, 1992). The fact that
association studies have often been found to be confounded by variables other than
disease (Kennedy et al, 1995; Barr and Kidd, 1993) suggests that the pararnetric models
used in association studies may not always be appropriate.
Linkage analysis, on the other hand, is a non-pararneuic approach that does not
require data to have a close fit to the normal distribution. Non-parametric models
accomodate complex systems for which only a few of many variables can be measured.
For these reasons, non-parametric linkage analysis is the best approach to isolate
candidate genes for Mendelian traits (Ott, 199 1). For cornplex nomMendelia traits,
however, the non-pararnetric approach has rarely suseeded in identifying susceptability
loci, probably due to the heterogeneity of these disorders (Heutink et al, 1995). The
occasions when linkage analysis has successfully identified susceptibility loci, such as for
severe bipolar disorder (Freimer ef al, 1996), the affected families studied have been
selected for the presence of a founder effect documented to be present by a history of
inter-rnarriage.
Given that linkage analysis of complex traits is only viable under some
circumstances, association studies remain a usefül if limited tool for the analysis of
complex traits. However, care must be taken in sample collection in order to be able to
accommodate the assumptions of the parametric mode1 employed in association studies.
In addition it should be remembered that d i k e linkage studies, association of a rnarker
with a disease, is not evidence that the gene under investigation, or an adjacent gene on
the same chromosome. contributes to the inheritance of a disorder (Berrettini, 1997;
Hodge, 1 994; Nothen et al, 1 992).
In psychiatnc genetic assocaition studies, candidate genes studied ofien encode an
important site of action for either an endogenous ligand or a drug used to relieve the
symptoms of a disorder (Nothen et al, 1992). This is the approach we have used to select
the DRDI, DATl and 5HTT genes for study in TS, AD-HD, OCD and alcohol
dependence. The rationale for selecting each candidate gene will be explained below.
1.6 The dopamine bypotheses of TS, AD-HD and alcohol dependence
Several lines of evidence suggest that TS, AD-HD and alcohol dependence are among
the neuropsychiatric disordee for which a dopamine hypothesis implicates the DRDl
gene and the DATl gene. The critical role of the DRDl and DATl gene products in
sustaining dopaminergic neurotransmission make these genes primary candidates for
neuropsychiatric disorders including TS (Gelemter et al. 1993), AD-HD (Cook et al,
1995; Cook et al, 1997) and alcohol dependence (George et al, 1995).
The DRD l gene, which encodes the dopamine D 1 receptor, is a member of the family
of genes that encode GPCRs. This receptor, while having 10 fold lower affinity for
dopamine than the dopamine D5 receptor (Sunahara et al, 1991), is important to
dopamine neurotransmission because of its greater expression in the striatal, cortical and
subcortical regions of the human brain (Sunahara et al, 1991). These regions are
implicated in ernotion, neuropsychiatric disorders and addictions (Weiner et al, 199 1).
The DATl gene, which encodes the dopamine transporter, is involved in terminating
dopaminergic neurotransmission at the synapse. The human dopamine transporter gene
(DATI) belongs to a family of twelve transmembrane domain ~a and Cl- dependent
neurotransmitter transporters (Uhl and Johnson, 1994). The dopamine transporter, like
the other members of this family. is a presynaptic re-uptake mechanism (Ding et al, 1994;
Nirenbergh et al, 1996) that is integral to the termination of dopaminergic synaptic
transmissions by ~ a + dependent reuptake (Kitayama et ai, 1992, Kilty et al, 199 1 ; Hom,
1990).
The DATI gene is implicated in the inheritance of TS (Comings et al, 1996;
Vandenbergh et al, 1992; Singer et al, 1991; Leckrnan et al, 1988) and alcohol
dependence (Muramatsu and Higushi, 1995; Thiihonen et al, 1995; Devor and Cloninger,
1989), in addition to attention deficit-hyperactivity disorder (AD-HD) (Gill et al, 1997;
Cook et al, 1997; Comings et al, 1996; Cook et al, 1995), Parkinson's disease (Uhl,
1990) and schizophrenia (Bordea-Pean et al, 1995; Daniels et al, 1995; Li et al, 1994;
Penico et al, 1993; Losonczy et al. 1987). Abnormalities of dopamine transporter
fhction have been proposed to contribute to both the hyperdopaminergic (Hom, 1990)
and hypodopaminergic hypotheses of the dopamine pathogenesis of these disorders.
(a) The DATl and DRDl genes are candidates for TS
The effîcacy of D2-iike receptor antagonists, such as haloperidol, in alleviating the
motor and vocal tic symptoms of Tourette's syndrome (Bruun and Budman, 1996; Wong
ef al, 1996; Wolf et al, 1996) suggests that, as in schizophrenia (Van Tol rf al, 1992). a
hyperdopaminergic pathology underlies TS (Barr e t al, 1 996). The possible
hyperdoparninergic state that underlies TS may be due to an increased number of
dopamine receptors, increased receptor affinity for dopamine (Wolf et al, 1996; Cohen er
al, 1978; Butler et al, 1978) or diminished termination of synaptic neurotransmission due
to dopamine transporter pathology (Malison et al, 1995; Vandenbergh er al, 1992; Kilty
et al, 199 1).
The DATl gene is a candidate gene in TS because its sequence variants may be
responsible for the dopamine transporter dysregulation that is proposed to account for the
hyperdopaminergic state that characterizes TS . The potential ro le of the dopamine
transporter in TS is emphasized by the fact that a variety of psychostimulant drugs, such
as cocaine (Kuhar et al, 1990; Wilson et al. 1996) and MP (Volkow et al, 1995) that bind
to the dopamine transporter have been reported to worsen the motor and vocal tics of TS
patients.
The DRDl gene is another candidate gene for TS because the hyperdoparninergic
state that is proposed to underlie TS may reflect the fact that TS patients have altered
dopamine D 1 receptor characteristics. Recent evidence from neuroimaging studies has
revealed that severely affected TS patients may have increased levels of DRD2
expression or aitered dopamine D2 receptor dupamine binding properties (Wong et al,
1997; Robertson, 1996: Wolf et al, 1 996). This suggests that dopamine receptors remain
valid candidates for TS. However, unlike the dopamine D2 receptor, no evidence of
altered DRDl gene expression in TS has yet been documented. The issue of whether
DRDl function is altered in TS can be investigated by attempting to isolate sequence
variants of the DRDl gene in TS patients as has been done previously in schizophrenic
(Ohara et al, 1993) and bipolar patients (Shah et al, 1995).
Dopamine receptor genes have been studied as candidate genes in numerous
association studies of sequence variants with TS. As with al1 association studies,
moderate associations of dopamine receptor sequence variants (markers) have been taken
as evidence that a hnctional polymorphic variant. perhaps in the 5' regulatory region.
was in linkage disequilibrium with the marker. However, studies finding moderate
association of the dopamine D2 (DRDZ), D3 (DRD3) and D4 (DRD4) genes with TS
(Comings et al, 1996; Grice et al, 1996; Comings et al, 1993; Cornings er al, 1991),
remain controversial (Nothen et al, 1994; Barr et al, 1996). No evidence for linkage of
the dopamine D 1 (DRD I ) and D5 (DRDS) genes with TS has yet been found (Gelemter
et al, 199 1 ; Barr et al, 1997).
Association studies. have not been conducted with DRDl in TS. However, snidies of
the 5' flanking region of DRD 1 gene, including the Taq 1A prornoter polymorphism, have
failed to show that the DRD1 gene is associated with schizophrenia (Cichon ei al, 1996)
or bipolar disorder (Coon et al, 1993; Nothen er al, 1992). Point mutation analysis has
recently confirmed that the DRDl gene coding region is non-polymorphic in
schizophrenia (Lui Q et al. 1995) and bipolar disorder (Shah et al, 1995). However,
studies searching for sequence variants of the DRDl gene coding region in TS patients
have not been reported.
(b) The DATl and DRDl genes are candidates for AD-HD
The DRDl and DATl genes have also been implicated in the dopamine pathology
that rnay underlie AD-HD. The hypodoparninergic hypothesis of AD-HD dopamine
pathology (Gill et al, 1997; Cook et al, 1997; Cornings et al, 1996; Cook et al. 1995) has
been inferred from the pharmacological evidence that psychotropic drugs, such as the
dopamine transporter antagonist rnethylphenidate (MP) (Kuhar et al, 1990; Volkow et al,
1995; Wilson et al, 1996), effectively treat AD-HD (Ding et al, 1994; Hetchman et al,
1994). The hypodoparninergic hypothesis suggests that, by antagonizing the dopamine
transporter, MP corrects the low synaptic dopamine levels that characterize AD-HD
(Ding et al, 1994), thereby prolonging dopamine receptor stimulation by dopamine. The
DRDl gene is therefore implicated in AD-HD because it encodes the dopamine Dl
receptor which has high &nity for dopamine (Sunahara et al, 1990) and is highly
expressed in the striatal and cortical brain region implicated in AD-HD (Volkow et al,
1995).
(c) The DATl and DRDl genes are candidates for alcohol dependence
Traits such as dcohol dependence may also involve aberrant dopamine pathways (Ng
er al, 1997; Noble, 1996; Tiihonen et al, 1995; Weiss et al. 1990). Dopaminergic
neurotransmission in the limbic and mesolirnbic pathways of reward located in the
midbrain, nucleus accurnbens and frontal cortex (Adamson et al, 1995) is implicated in
the pathways of dmg reinforcement and reward that characterize alcohol dependence
(Strange. 1993; Sibley et al. 1992; Wise and Rompre. 1989; Koob and Bloom. 1988;
Wise, 1980). A hypodoparninergic hypothesis of alcohol dependence has been advanced
(Ng et al, 1997), that suggests that ethanol releases dopamine in some brain regions.
thereby contributing to the rewarding and addictive properties of ethanol (Imperato and
Di Chiara, 1986; Ng et al, 1997). The identities of the dopamine system genes that may
contribute to alcohol dependence, however, remain obscured by complex environmental
factors (Cloninger, 1987; Persico et al, 1993; Muramatsu et al, 1995).
The DATl and DRDI genes are candidates for alcohol dependence because of their
role in sustaining dopaminergic neurotransmission in brain regions implicated in the
pathways of reward. Recent evidence that striatal dopamine transporter densities rnay be
altered in alcohol dependent subjects (Tiihonen et al, 1995) raises the question of whether
DATl sequence variations contribute to altered dopamine transporter f i c t i on in alcohol
dependence. It is tempting to suggest that DATl or DRDl sequence variants may
contribute to the hypothesized (Ng er al, 1997) effects of dopamine release into the
synapse by ethanol. However, no information on the frequency of DATl coding region
sequence variants is currently availabie. Information on the frequency of DRD 1 sequence
variants in alcohol dependent patients is limited to a mutation screening of eight patients
which, while revealing no mutations (Lui et al, 1995), cannot be considered to be
conclusive given the small sample size.
1.7 The serotonin bypothesis
A number of neuropsychiatric disorders are characterized by serotonergic
abnorrnalities. Severely affected TS patients have been found to excrete 5-
hydroxyindoleacetic acid (SHTAA), the major serotonin metabolite, at higher leveis than
found in controls (Bomstein and Baker, 1991). Post-mortern brain tissue from TS
(Anderson. 199 1) and depressed patients (Mann et al, 1996; Boyer and Feighner, 199 1)
have been found to contain altered concentrations of serotonin compared to control post-
mortem brain tissue. Evidence for serotonin metabolite dysregulation has aiso been
identified in Type II alcohol dependent patients (Goidman, 1999, those with early onset
and high genetic liability (Cloninger et al, 1989; Brown et al. 1985; Cloninger et al,
1985; Linnoila et al, 1983). In addition, serotonergic abnormalities have been noted in
obsessive compulsive disorder (OCD) (Heils et al, 1996; Swedo and Leonard, 1994).
The serotonin transporter (SHTT) gene is a candidate gene in the study of the etioiogy
of neuropsychiatric diseases considered to involve serotonergic abnorrnalities. Due to its
role in clearing serotonin from the synapse, the serotonin transporter is integral to
attenuating serotonergic neurotransmission (Heils et al, 1996). The efficacy of SRIs in
treating neuropsychiatrie disorders, including unipolar depression (Ogilivie et ai. 1996;
Anderson and Tomenson, 19941, OCD, anxiety and depression comorbid with TS (Riddle
et al, 1988), OCD (Swedo and Leonard, 1994) and alcohol dependence (Lejoyeux, 1996;
Narango and Bremer, 1994) suggests that the SHTT gene is a candidate gene in these
disorders.
(a) The SHTT gene is a candidate for TS
The frequency with which TS patients are treated with SRIs in order to control
comorbidities (Swedo and Leonard, 1994; Messiha. 1993), provides the pharmacological
rational for examining the SHTT gene as a candidate gene in TS-OCD patients. Mutation
screening, however, has shown that coding region variants of the 5HTT gene are very
rare (Di Bella et al, 1996) and probably do not contribute to psychiatrie disorders. In this
context the recently reported 44 base pair deletiodinsertion 5HTT promoter
polymorphism is of particular interest because it has been shown to reduce SHTT gene
expression in vitro (Heils et al, 1996; Lesch et al, 1996). Therefore, there is in vivo
evidence that the sequence variant of the 5HTT promoter rnay be a mutation of the
regulatory region of the 5HTT gene. This discovery raises the question of whether a
polymorphism that has Functional significance in vitro. is also functional in vivo
(Paterson, 1997; Heils et QI, 1996).
The hypothesis that the short promoter polymorphism may modulate serotonin
transporter expression. thereby influencing the rate of serotonin clearance from the
synapse in vivo (Heils et al, 1996), has not k e n tested. Evidence that 5HTT mRNA
expression may be increased in schizophrenics under neuroleptic treatment (Hemandez
and Sokolov, 1997) suggests that 5HTT regulatory pathology rnay underlie some
neuropsychiatric disease States. Similar evidence of altered 5HTT mRNA expression or
aitered binding sites for serotonin antidepressant dmgs (Owens and Nemeroff. 1994).
however, is not available for TS-OCD patients. However. nurnerous studies have used
the candidate gene approach to conduct association studies of the 5HTT promoter in
neuropsychiatnc disorder patient groups (Craddock and Owens. 1997).
Associations of the 5HlT gene promoter polymorphism with anxiety traits (Lesch et
al? 1996) and depression (Collier er al. 1996) remain controversial (Ebstein et al. 1997)
and unsupported. A snidy of the 5HTT promoter polymorphism in panic disorder found
no association (Deckert et al. 1997). The serotonin transporter remains a candidate gene
in anviety disorders and depression because of evidence for the efficacy of SRI treatment
(Ogilvie et al. 1996: Lejoyeux 1996) of the serotonin reuptake pathology (Collier et al.
1996; Iny et 02. 1994) that rnay underlie these disorders. The recent tentative association
of the 5HTT with autism (Cook et al, 1997). a disorder fkequently characterized by OCD
and anviety (Gordon et al' 1993; McDougle et al. 1996). suggests that other disorders
treated with SRIs, including TS and its comorbidities. rnay also be associated with
serotonegic dy sregulation (Messiha, 1 993) involving the 5 HlT gene.
(b) The SHTT gene is a candidate gene for alcohol dependence
Alcohol dependence withdrawal is charactenzed by severe anxiety and depression.
Both conditions can be relieved by SRIs. While anxiety has been associated with the
SHTT gene by studies of the 5HTT promoter alleles, depression has been associated with
the 5HTT gene (Ogilvie et al, 1996) not by genotyping the SHTT deletiodinsertion
polyrnorphism, but by genotyping the 5HTT intron 2 variable number tandem repeat
(VNTR). The fiequency of comorbid anxiety and depression in alcohol dependent
patients (Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, 1990)
makes the 5HW gene a candidate for alcohol dependence.
1.8 Screening candidate genes for sequence variants
(a) Mutation screening by SSCP analysis
Single stranded conformational polymorphism (SSCP) analysis is a cornrnonly used
method for the detection of gene sequence variants (Lee et al, 1992; Oto et al, 1993;
Hongyo et al, 1993). Cornrnon variants include single base pair changes, frameshift
mutations, microsatellite polymorphisms and nucleotide insertions. Sequence
information from muation screening is valuable because it provides evidence regarding
the extent of amino acid sequence conservation in disease and control populations in
addition to providing markers usehl in linkage and association studies (Weghorst, 1993;
Calvert et al, 1995). The method is based on the principle that when DNA is denatured,
the single strands containing sequence variants assume a conformation different from
those strands containing no sequence variants. The conformational differences in the
single stranded DNA cm be resolved on polyacrylamide gel (Giavac and Dean. 1995;
Weghorst, 1993) as depicted in Figure 1. When an SSCP band shift is noted, sequence
analysis is then performed in order to identifi the nuture of a sequence variant.
The availability of pre-cast gradient polyacrylamide gels (Novex, San Diego, CA,
USA) that are nin in the Novex Xcell II minice11 system has resulted in improved efficacy
of SSCP analysis (Hongyo et al. 1993). Many current protocols for SSCP, however, have
not been modified to accommodate these gels (Bardeesy and Pelletier. 1995: Lam et ni,
1996). The initial phase of experimental work described here involved optimizing SSCP
conditions for pre-cast gels.
1.9 Research objective: to screen candidate genes for sequence variants
(a) Isolate DATl gene sequence variants in TS and alcohol dependence
We set out to conduct the fint systematic mutation screening of the DATl gene in
controls, TS and alcohol dependent patients. The DATl screening project is the logical
sequel to the recent association of the DATl gene gene with TS (Comings et al. 1996),
AD-HD (Cook et al, 1997; Gill et al, 1997; Cook et al, 1995) and alcohol dependence
(Murarnatsu and Higushi, 1995). This study genotyped a variable number tandem repeat
located 3' (3'VNTR): the only DATl polymorphism reported prior to our effort to screen
the DATl gene (Vandenbergh el al, 1992). Therefore, the sequence information
obtained from screening the DATl gene may be a valuable addition to what is known
(3- Genornic DNA PCR - = - ,
Mutant
Denaturation
Single-stranded Conformations
Figure 1 The principle of PCR-SSCP analysis. Double stranded PCR products are generated for both wild-type (WT) and polymorphic/mutant (M) alleles. Follow-ing denaturation, the single strands refold to form specific secondary structures. Polyrnorphic/mutant single stranded DNA molecules have a different structure than the wild-type molecules. Polyacrylamide gel electrophoresis can resolve the two different wild-type strands and the two different polymorphic/mutant strands (Gdvac and Dean. 1995).
about the DATl gene conservation in human populations (Donovan et al, 1995;
Vandenbergh et al, 1992; Gros et al, 1992). This sequence information will help to
evaluate the contribution of sequence variability in dopamine system genes to the
etiology of neuropsychiatric disorders.
(b) lsolate DRDl gene sequence variants in TS and alcohol dependence
We continued our study of the role of GPCR sequence variants in the etiology of TS
and alcohol dependence (George et al, 1993; Lam et al. 1996) by screening the coding
region of the DRD 1 gene for sequence vaxiants. While linkage analysis has excluded the
DRDl gene From having a major gene effect in TS (Heutink et al. 1995; Gelemter et al,
1993), by genotyping the 5'Taq 1 A polymorphism no mutation screening of the DRDl
coding region in TS has been reported in the literature. Evidence of a role for the DRDl
gene in the inhentance of addictive disorders has recently been reported (Comings et al,
1997) but has not yet been replicated. No study has screened a Iage sarnple of alcohol
dependent patients for sequence variants in the coding region of the DRDl gene (Lui et
al, 1995).
(c) Genotype SHTT gene sequence variants in TS and alcohol dependence
Mutation screening of the SHTT gene in control and neuropsychiatric patient groups,
sirnilar to that which we conducted for the DATl gene, has shown that its amino acid
sequences are conserved in patient and control groups. Di Bella et al, 1996, conducted a
mutation screen in a number of neuropsychiatric patient groups. TS and alcohol
dependent patients, however, were not screened.
The evidence for conservation of SHTT coding region sequences in a number of
neuropsychiatric disorders suggested to us that rather than conducting a mutation
screenilg of the SHTT gene in our patient groups that it would be more f%utfuli to ca ry
out a candidate gene study of the 5HTT gene in TS and alcohol dependence. For this
purpose we genotyped the SHTT deletion.insertion polymorphism 5HTT and the intron 2
VNTR polymorphisms recently identified (Hiels et al. 1996; Lesch et al. 1994). The
5HTT deletionhsertion promoter polymorphism was of special interest because of
evidence of its possible functional relevance. The intron 2 VNTR was genotyped in order
to enable us to conduct haplotype analysis of the 5HTT gene in patient and control
groups.
1.10 Research strategy for candidate gene screening and association studies
The candidate gene studies presented here were conducted in order to identiQ gene
sequence variants unique to or significantly more common in patients compared to
controls. Mutation screening projects inevitably identiQ a plethora of sequence variants
that have no apparent functional significance. We therefore particularly sought to
identify sequence variants of the DATl and DRD 1 genes that would result in alterations
to the structure of their respective gene products. The discovery of novel sequence
changes of undetermined significance requires pharmacological characterization in vitro
by expression in ce11 lines (discussed by Hiels et al, 1996 and Lam el al, 1996) in order to
establish whether altered protien sequence alters its function. Association studies of TS
and alcohol dependence with the novel sequence variants were conducted while we
continued to seek sequence variants leading to altered protien structure for
characterization in vitro systems.
ERIALS AND METHODS
2.0 Materials
Alkaline Phosphatase (Calf intestinal) Pharmacia Biotech (Baie d'Urfe, PQ)
Agarose Bio-Rad Laboratones
Ampicillin Sigma (St. Louis, MO)
Bacto Agar DIFCO Laboratones (Detroit, MI)
Programmable Power Supply Biorad, Mode1 3000Xi
Control DNA (not psychiatric/addicted) Addiction Research Foundation (Toronto,ON)
Control DNA (not psychiatndaddicted) Psychopharmacology and Dependence Unit.
Women-s College Hospital (Toronto, ON) Control DNA (addicted, not psychiatric) Psychopharmacology and Dependence Unit,
Women's College Hospital (Toronto, ON) 7-deaza-deoxy-guanosine tri-phosphate Pharmacia Biotech (Baie d'Urfé, PQ)
Deionized Formamide Fluka (Ronkonkoma, NY)
Dimethyl sulfoxide (DMSO) Fluka (Ronkonkoma, NY)
DIOB Cornpetant Celts
DNA Ligase
Mr. Tuan Nguyen
Pharmacia Biotech (Baie d'Urfe, PQ)
Patient DNA (Tourette's syndrome) City of Hope Medical Centre (Durate, CA)
Patient DNA (Alcohol dependent) Addiction Research Foundation (Toronto, ON)
pBluescnpt Strategene (la Jolla, CA)
PCR Genearnp Kit Perkin-Elmer Cetus (Norwaik, CT)
T7 Sequencing Kit Pharmacia-Biotech (Uppsala, S W)
Polaroid Type 667 Film
Polyacrylamide Gel (6%)
Polynucleotide Kinase, FPLCpureB
Pre-cast 4-20% Acrylamide Gels
Pre-cast 10% Acrylarnide Gels
Restriction Endonucleases
SDS
Taq Polymerase
Temperature Controlled Water Bath
ThennoFlow SSCP System
Eastman Kodak Co. and Polariod
Helixx (Toronot, ON)
Pharmacia-Biotech (Baie d'Urne. PQ)
Novex (La Jolla, CA)
Novex (La Jolla, CA)
Pharrnacia/GIBCO B Ri,
ICN (Dorval. PQ)
GIBCO BRL (Gaithersburg, MD)
Haake (Karlsruhe, DR)
Novex (La Jolla CA)
Sequinase PCR Product Sequencing Kit USB (Cleveland, Ohio)
Ultrapure-dNTP Set Pharmacia-Biotech (Baie d'Urfie, PQ)
Xcell II mini-ceil system Novex (La Jolla CA)
2.1 DNA extraction
Following informed consent, blood samples were obtained from non-psychiatrie
control individuals, patients with TS, TS-AD-HD. TS-OCD and alcohol dependence with
no psychiatric comorbidity. The cnteria whereby these diagnoses were made are
described below. Genomic DNA was extracted using phenol extraction and ethanol
precipitation of DNA fiom patient blood as previously described (Bardeesy and Pelletier.
1995). Briefly. 5-10 ml of blood was added to an equal volume of lysis buffer (50 mbl
HEPES, pH 8. 50 mA4 NaCI. 5 mA4 MgC12. 10% sucrose. 05% Triton X-100) and the
mixture was kept on ice for 10 minutes. The mixture was then centrifùged at 3000 g for
10 minutes. The supernatant was discarded and the pellet was resuspended in a mixture
of TAE buffer and sodium dodecyl sulfate (SDS) before being incubated at 65 O C for 10
minutes. Fi@ ul of 10 mg/ml proteinase K and 50 lil of RNase A were then added before
incubating the mixture at 65 OC for 90 minutes.
Two extractions with equal volumes of phenol were performed by mixing the
phenoVaqueous mixture for 30 minutes on a platform shaker. The aqueous phase fiom
each extraction was saved and extracted rince more with equal volumes of phenol and
chloroform before final extraction with an equal volume of chloroform. A one-tenth
volume of 3 M sodium acetate in ethanol was then added to the aqueous phase. The DNA
was then removed by spooling with a g l a s rod and nnsing in 70% ethanol before
resuspending in TE buffer.
2.2 Clinical sam ples
(a) Tourette's syndrome sarnples
The TS group screened consisted of 109 unrelated patients recruited from the
Tourette's Syndrome Clinic of the City of Hope National Medical Center (COH). Durate.
California (Comings and Comings. 1993: Comings. 1994: Comings et al. 1996). The
patients were selected to be Caucasians of Xorthem and Western European descent. All
patients. and available relatives. completed a detailed behavioral questionnaire panemed
after the Diagnostic Interview Schedule (DIS) (Robbins et al. 1981). The entire DIS
questionnaire. and its use in other midies. is described elsewhere (Comings and Cornings.
1993: Comings et al. 1 996).
For al1 patients screened. the final diagnosis of TS. chronic motor and chronic vocal
tic disorder. was made by assessing the variables required according to the DSM-III-R
(Diagnostic and Statinical 1 Manual of .Mental Disorders. Third Edition-Revised. 1987)
and DS-M-IV (Diagnostic and Statistical .Manual of -Mental Disorders. Fourth Edition.
1990). In addition to the primary diagnosis of TS. 29 of the patients screened were
diagnosed comorbidly uith OCD (TS-OCD) and 33 of the patients were diagnosed
comorbidly with AD-HD (TS-AD-HD) according to the DSM-III-R (Diagnostic and
Statistical Manual of Mental Disorden. Thrd Edirion-Revised. 1987) and DSM-IV
(Diagnostic and Statistical Manual of .Mental Disorders. Fourth Editioa 1990) cnteria
Forty-seven of the TS patients had no comorbid diagnosis. The TS patients screened had
not been included in previous studies (David Comings. personal communication).
The TS sample
34
sizes varied for each gene that was screened or genotyped for two
reasons. First, DNA sarnples were still being obtained during the course of this study:
allowing sample sizes to increase &er the GPR19 and DRDl studies. Second, the PCR
reactions required to complete the SHTT gene study were less robust that those involved
in conducting the DATl study. As a result fewer patients were studied at the 5HTT locus
by cornparison with the DATl locus. The DATl gene was screened in al1 patients. The
DRDl gene was screened in 47 patients with a primary diagnosis of TS in addition to
three additional TS-OCD patients selected to be the next consecutive patients interviewed
(denoted by the COH patient identification nurnber).
(b) Alcohol dependence samples
DNA samples from 72 patients diagnosed with alcohol dependence were obtained
fiom the Addiction Research Foundation (AM) of Ontario. The patients were diagnosed
with alcohol dependence when admitted to the Toronto Hospital between 1989 and 1992
for alcohol-related chronic hedth problems. The assessrnent of alcohol dependence was
made according to DSM-III-R criteria (Diagnostic and Statistical Manual of Mental
Disorders, Third Edition, Revised. 1987). The alcohol dependent group consisted of
unrelated Caucasians of northem and western European descent who resided in the
Metropolitan Toronto Area (George et al. 1993).
A11 72 sarnples were screened for mutations in the GPR19 and DRDl genes. The
DATl gene was screened in 64 patients. The 5HTT gene was genotyped for the same 64
patients. The Bemoulie multiple testing corrections (discussed below) accounted for the
fact that previous studies by our group have genotyped the DRD4 exon 3 polymorphism
(George et al, 1993) and the DRD2 Taq 1A polymorphism in the alcohol dependent
group.
(c) Control samples
The GPR19 and DRDl mutation screening projects involved screening 50 non-
psychiatric control sarnples fiom the Addiction Research Foundation (ARF) of Ontario,
Toronto, Ontario. The subjects were described as being healthy volunteen From the
Metropolitan Toronto area with no history of psychiatnc treatment at the tirne blood was
drawn.
For the study of the DATl and 5HTT genes in TS, groups of 67 and 91 non-
psychiatric controls respectively, were selected from the data-base of the
Psychopharmacology and Dependence Research Unit, Women's College Hospital,
Toronto, Ontario. Each control was ethnically matched to the TS group. The controls
were genotyped for the 3'VNTR and the novel polymorphisms in exons 9 and exon 15 of
the DATl gene and the deletionlinsertion and intron 2 VNTR polymorphisms of the
5HTT genes.
For the study of the DATl and SHTT genes in alcohol dependent patients, 64 controls
were genotyped for the 3'VNTR and the novel polymorphisms of exons 9 and 15 of the
DATl gene. Of the total control group, the 15 exons of the DATl gene were screened for
42 controls obtained from the Addiction Research Foundation (ARF) of Ontario, Toronto.
Ontario. These controls were healthy volunteers from the Metropolitan Toronto area with
no history of psychiatnc treatment at the time blood sarnples were taken. The rernaining
22 controls, selected using the same criteria from the data-base of the
Psychopharmacology and Dependence Research Unit, Women's College Hospital,
Toronto, Ontario, were genotyped for the YVNTR and the comrnon novel
polymorphisms in exons 9 and 1 5 of the DAT 1 gene.
2.3 PCR amplification of gene fragments subjected to SSCP muation screening
(a) Amplification of GPR19 gene fragments used to refine SSCP method
SSCP analysis of the GPR19 gene was conducted by amplifying the 1245 base pair
open reading frame (O'Dowd et al. 1996) into five overlapping Fragments using primer
pairs designed designed de novo for this purpose (Table 1). The PCR reactions were
conducted using 1 ug of each oligonucleotide primer (Table l), 2 umol dNTPI 1.5 m o l
MgCI2 and 0.5 U Taq DNA polymerase (Life Technologies, Inc. Gaithersburg, MD) in a
total reaction volume of 100 ul. 200 ng of template was used per reaction. The PCR was
conducted in a Perkin Elmer Therocycler using 40 cycles, at 95 OC for 45 seconds. 55-6 1
OC for 45 seconds. and 72 *C for 1 minute 30 seconds. The arnplified DNA was analyzed
by agarose gel electrophoresis prior to mutation screening.
(b) Amplification of DRDl fragments to be analysed by SSCP
The pnmers used to amplifi the overlapping DRDl gene Fragments were designed de
novo for use in this screening project. SSCP analysis of the DRDl gene encoding the 446
amino acid dopamine Dl receptor (Sunahara et al, 1990) was conducted by arnplifying
the 1338 base pair DRDl gene coding region in five overlapping fragments using the
TABLE. 1 . Oligonucleotides Used in PCR-SSCP Analysis of Hurnan GPR19 ge
Fragrne Anneal S Primer N Primer Sequence (5'-3') (base pa Temp. O
1 1 -UP TTCTTTTCCCCAACCAGAAT 32 1 6 1 2 - D O W GGAATTGCCGAAGATAGAA
2 3-UP ACAGCCAGCATCTTCTTTGG 32 1 58 4-DO WN CCGGTCTATGCAGATGGA
3 5-UP ACTCCAGGTGTCCAGATCTA 32 1 60 6-DO WN TTGGTAAAATAAAATTATGAG
4 7-UP ACTGCCTACACTGTCATCCA 33 1 55 8-DO WN TTTCATCCCTCTCCGAAA
5 9-UP CCTACTCTGTATTCAATTTA 3 1 8 60 1 O-DO W AACAATTGAAAGAATGAG
TABLE. 2. Oligonucleotides Used in PCR-SSCP Analysis of Human DRD 1 ge -
Fragme Anneal S Primer N Primer Sequence (5'-3') (base pa Temp. O
1 Pl-UP CTGCTTAGGAACTTGAGGG 321 60 P2-DOW GAAGCCAGCAATCTCAGCCAC
2 P3-UP CTGGTCATGCCCTGGAAGG 309 55 P4-DO W GGTCTCAGCCAGGGAAGTGGC
3 P5-UP CCAGTGCAGCTCAGCTGGC 414 60 P6-DO W CAAAATGCAGTTCAAGATGAA
4 P7-UP GTGTGCTGTTGGCTACTTTT 352 55 P8-DOW CAGGTCCTCAGAGGAGCCCAC
5 P9-UP AAGGAGTGCAATCTGGTTT 26 1 58 P 1 O-DO GCAAACCCCAGAGCAATCTCC
primer pairs listed in Table 2. The PCR reactions were conducted using 200 ng human
genomic DNA, 1 ug of each oligonucleotide primer (Table 2), 2 m o l dNTP, 1.5 mm01
MgCl2 and 0.5 U Taq DNA polymerase (Life Technologies, Inc. Gaithersburg, MD).
PCR was conducted using 40 cycles, at 95 OC for 30 seconds. 55-60 OC for 40 seconds.
and 72 OC for 40 seconds in a Perkin Elmer Gene Arnp PCR Systern 2400 machine. The
arnplified DNA was analyzed by agarose gel electrophoresis prior to the mutation
screening.
(c) Amplification of DATl exons analyzed by SSCP
The complete set of PCR primers used to ampliS. each DATl exon and the 3 ' and 5'
flanking regions is listed in Table 3. The primers were designed from unpublished
sequence information made available to us through our collaboration with the laboratory
of George Uhl, National fnstitute on Dmg Abuse, Johns Hopkins University School of
Medicine (David Vandenbergh, personal communication). The PCR reactions were
conducted using 200 ng human genomic DNA. 0.5 ug of each oligonucIeotide primer
(Table l), 2 umol dNTP, 1.5 rnrnol MgCl,, 10% DMSO and 0.5 U Taq DNA polymerase
(Life Technologies, Inc. Gaithersburg, MD) (Lam et al, 1996; Heiber et al, 1996;
Marchese et al, 1994). The 15 pairs of PCR primers used to ampli@ each exon are listed
in Table 3. PCR was conducted using 40 cycles, at 95 OC for 30 seconds, 60-68 O C for 40
seconds, and 72 OC for 40 seconds in a Perkin Elmer System 2400 Gene Arnp PCR
machine. The total reaction volume was 50 til.
2C 1 DAT DAT 122s EX3FOR EX3REV EX4FOR EX4REV EXSFOR EXSREV EX6FOR EX6REV EX7FOR EX7REV EX8FOR EXSREV EX9FOR EX9REV Ex 1 ORO EXlORE EX1 lFO EXIlRE EX12FO EX12RE EX13FO EXl3RE EX14For EX 14Rev EXISFO HDAT2 1
CAAAGCCA ATGACGGACAG CGTGGGACTCATGTCTTCCGTGG CCCCGGCTGCACCTACGAC ACGAGGAGAGATGGGCCCITCC GTCACCACCATGATCCGCGC GTTGCTGATGGTGGCTCTGTGCT GTCGCGCCATCTCTCCCG TTCCAGGTGGGTTGACAGCCTC TTGGTGGCCCCATGTCTACAGG CCCACCAAGGGCCCTGCC TGGGAATGCCAGAGCCCCTG CTCAGGTCC'ITTGCCTGTGGC GACCTCTCCCTAGTATTGATGAGGCC ATTGCAGCTGCTGCAGCTCAGC CGGCGCTGGTGCTACACGG GGCAAGCAGGGCGGGTTCTG CGGGCGGTGCGGGTTACTC GGCCCAGGCTGCGGTCAG CCTGACAGTCCCAGCCAGGGC CCCCAGGCTGGGTTTACCTCTGG GAAGGGGAGTGGCACAGCCACC TGTCCAGCATCGGGGGAATG GTGCCAGAGTGGGGGCAGTG CCTGCTTTGTCCTGGCACCG GACACCCACGGAGCCTTTCTGG GGTCCTGACAGTGTGAGTCAGTGGTG GGGCTAAGAACACTGAGCTTGGGATC TGCTCTTAGCCACCTTCAGCTGCTC GGAGTCTTCTGCTTTGTTGTTTGTGTTTTCAG
TABLE. 3. Oligonucleotide Pnmers Used in PCR-SSCP Anaiysis of Human DATl g Frag Anneal
Name Primer Sequence (5'-3') Locati size ( temp " EX2ForB TGCCCTCCGCACCAGGTATG Exon 359 60
(d) Genotyping the DATl3'VNTR by PCR amplification
Amplification of the DATl 3'WTR was conducted using primers consisting of : T3-
SLong, 5'-TGTGGTGTAGGGAACGGCCTGAG-3' and T7-3aLong, 5'-
CTTCCTGGAGGTCACACGGCTCAAGG-3. Concentrations of PCR reagents in the
final reaction mixture were as described for amplification of DATI exons above. The
total PCR reaction volume was 50 irl. PCR was conducted using 40 cycles, at 94 OC for
30 seconds, 6 8 ' ~ for 40 seconds, and 72 OC for 40 seconds in a Perkin Elmer Gene Amp
PCR System 2400 machine. DNA was analyzed by 4% agarose gel electrophoresis in
order to separate VNTR alleles of different sizes. Amplifieci DNA was visualized by
ethidium bromide staining.
(e) Genotyping the SHTT polymorphisms by PCR amplification
The 5HTT promoter variant was arnplified using two sets of oligonucleotide primers.
Eighty-five DNA samples from TS patients and 9 1 TS controls. in addition to 61 of the
64 DNA samples fiom alcohol dependent patients and 32 controls were genotyped at the
5HTT promoter by ampliQing a 4841528-bp fragment using the prïmers stprj. 5'-
GGCGTTGCCGCTCTGPLATGC-3' and S t r P 3 5'-
GAGGGACTGAGCTGGACAACCCAC-3'. The alcohol dependent DNA samples were
genotyped at the SHTT prornoter by amplieing a 4061450-bp fragment using the primers
HTTpZA, 5'-TGAATGCCAGCACCTAACCC-3', and HTTpSB, j * -
TTCTGGTGCCACCTAGACGC-3 ' .
The amplification of the 5HTT promoter was conducted using as little as 100 ng
human genomic DNA. One ug of each oligonucleotide primer, 2.0 mm01 MgClz 0.6 U
Taq DNA polymerase and 1 X PCR buffer (Life Technologies, Inc. Gaithersburg, MD)
were used in each PCR reaction. Each reaction mixture contained 200 rmol of each
dNTP, with the exception of dGTP which was included at a concentration of 100 mole,
plus 100 zmole of 7'-deaza-dGTP. The total PCR reaction volume was 25 ul. PCR was
conducted using 40 cycles, at 94 OC for 30 seconds, 60 OC for 40 seconds. and 72 OC for
40 seconds in a Perkin Elmer Gene Amp PCR System 2400 machine. The amplified
DNA was analyzed by 4% agarose gel electrophoresis and the DNA amplified was
visualized by ethidiurn bromide staining pnor to photography over a UV light source.
The SHTT exon 2 VNTR was amplified for 75 controls matched to TS patients and
32 controls matched to alcohol dependent patients using the primers HTTZX, 5' -
TGGATTTCCTTCTCTCAGTGATTGG-3', and HTT2Y, y-
TCATGTTCCTAGTCTTACGCCAGTG-3'. to ampli@ the 345-bp (9 copy), 360-bp (10
copy) and 390-bp (12 copy) fragments of DNA encompassing the three alleles of the
VNTR. The VNTR of the 85 TS, 64 alcohol dependent, and 16 controls matched to TS
patients was amplified using the pnmers, S-CAATGTCTGGCGCTTCCCCTACATAT-
3' and 5'-GACATAATCTGTCTTCTGGCCTCTCAA-3'. The PCR products were
resolved in 4% acrylarnide gel and visualized by silver staining.
Amplification of the VNTR was conducted using 150 ng human genomic DNA, 1 ug
o f each oligonucleotide primer, 300 umol dNTP, 2.0 mm01 MgCl2, 0.6 U Taq DNA
polymerase and 10% PCR buffer (Life Technologies, Inc. Gaithersburg, MD) per PCR
reaction. The total PCR reaction volume was 25 ul. PCR was conducted using 40 cycles,
at 94 OC for 30 seconds, 57 OC for 40 seconds. and 72 OC for 40 seconds in a Perkin
Elmer Gene Arnp PCR System 2400 machine. DNA was analyzed by 4% agarose or 4%
acrylamide gel electrophoresis and then visualized by ethidium bromide staining or silver
staining respectively.
2.7 SSCP mutation screening of GPR19, DRDl and the DATl gene
(a) Overview of the SSCP methodology
Non-denaturing polyacrylamide gel electrophoresis was conducted on pre-cast 4-20%
gradient gels (Novex) in the Xcell II mini-ce11 system (Novex). In some cases the
ThermoFlow SSCP Electrophoresis System, consisting of an Xcell 11 mini-ce11 system
adapted to allow temperature regulated electrophoresis. was used to optimize SSCP
conditions. The T'hermoFlow re-circulates 1 XTBE buf3er fiom the mini-ceIl through a
condenser coi1 regulated by a temperature controlled water bath (Haake. Karlsruhe,
Gemany). Mutation screening efficiency was optimized (Oto et al. 1993) by screening
each PCR product under at least two temperature conditions by adjusting the TBE buffer
temperature. SSCP was routinely conducted at 4 OC in the cold room and at 24 "C (room
temperature). Intermediate or higher temperature SSCP (see results section) was
conducted in the ThemoFlow.
Initidly, electrophoresis was conducted at 200 volts for 2 hours at 24 O C in the
ThermoFlow as indicated by recent protocols (Calvert et al, 1995). However, the
majority of the SSCP analysis reported here was conducted at between 50-90 volts.
Electrophoresis at 4 OC was conducted at 85 volts for 15-18 hours. At 24 OC the
electrophoresis voltage was dropped to 55 volts in order to compensate for the faster
running time of SSCP conducted at 24 OC.
b) SSCP protocol adapted for use with Xcell II apparatus (Novex)
Between 3 and 6 u1 of the 50 ul PCR reaction was diluted 1 :4 with a formamide based
SSCP loading buffer in individual 0.2 uL micro-amp PCR tubes (O'Dowd et al, 1996).
The denaturing buffer was prepared before SSCP analysis by combining, in a 2 3 ratio, a
stock denaturing solution with a stock stop solution. Both stocks were stored at room
temperature. The denaturing solution was prepared with O. 1% SDS and 10 mM EDTA.
The stop solution was prepared with 95% formamide. 20 m . EDTA, 0.05%
bromophenol blue and 0.05% xylene cyanole FF. Samples were denatured at 95 O C for 5
minutes before being soaked at 4 OC in the PCR machine for 5 minutes prior to loading
on acrylamide geI (O'Dowd et al. 1996).
The entire 20 ul mixture of denaturing buffer and PCR product was loaded into the
separate wells of Novex 4-20 % TBE gels (Novex, La Jolla, California). Most SSCP
was run in the cold room or at room temperature, as described. DC Power was supplied
by a Biorad, Mode1 3000Xi Programmable Power Supply (Richmond, CA). A proportion
of the total PCR products amplified frorn patient and control DNA sarnples were selected
for repeated analysis in the ThemoFlow SSCP Electrophoresis System at other
temperatures. The Novex SilverXpress Staining kit was used to visualize SSCP band
shifts. Sequencing of the PCR products corresponding to SSCP band shifts was done as
descnbed below (O'Dowd et al. 1996).
2.8 Identification of sequence variants by chain-termination DNA sequencing
PCR products identified by SSCP to be potentially polymorphic were subcloned into
pBluescnpt. transformed into E. Coli competant cells (strain DIOB), grown ovemight in
LI3 broth with ampicillin before miniprepping and sequencing the PCR insert as
previously described (Marchese et al. 1994).
2.9 Statisticai Methods
The differences between the genotype and allele fiequencies of the TS and alcohol
dependent groups and theii respective control groups were analyzed by two-tailed a' tests
using the Statistical Package for the Social Sciences (SPSS). version 7. Hardy-
Weinbergh equilibrium for the genotypes at each locus was tested using x2 analysis as
done previously (Kennedy el al, 1995). The Bonferroni correction for multiple testing
was used in order to account for testing associations at three loci of the DATl gene and
two loci of the 5HTT gene (Aickin and Gensler, 1996). Analysis of sample power was
conducted using the Epi Infio, Version 5.01 a (Public Domain Sofrware for Epiderniolo~
and Disease Surveillance, March 199 1). Linkage disequilibriurn between haplotypes
consisting of the cornmon polymorphisms and the 3'VNTR was analyzed using the
maximum likelihood Equilibrium Haplotype (EH) (Terwilliger and On, 1994; Ott, 199 1 )
program.
RES_ULTS
3.1 SSCP mutation screening of the GPRl9 gene
The SSCP rnethodology used to screen candidate genes in this project was refined by
screening GPR19 in the 50 caucasian controls and 72 caucasian alcohol dependent
patients described in the context of the DRDl gene mutation screening project. The
purpose of this preliminary screening work was two-fold. First, we were interested in
establishing the optimal SSCP conditions for screening candidate genes for sequence
variants. Second, we were interested in determining whether polymorphic sequence
variants of GPR19 existed
GPR19 is an intronless gene that encodes a 414 amino acid gene producr. Our
interest in GPR19 stemmed fiom the fact that the GPR19 gene has many of the
chxacteristics of neurotransmitter G protein-coupled receptors (O'Dowd ei al, 1 993).
Hydrophobie analysis suggests that the deduced arnino acid sequence of GPR19 has the
seven transmembrane (TM) regions characteristic of GPCRs. The expression of GPRl9
in the olfactory tubercle, the islands of Calleja, the caudate-putarnen, and the pituitary
suggests that GPR19 encodes a neurotransmitter receptor with a pattern of expression
similar to the dopamine D2 receptor. The fact that 10 1 amino acids of the GPR19 gene
product were identical to those of the dopamine D2 receptor is further evidence that
GPR19 is likely to encode a neurotransmitter receptor. However, no ligand for the
GPR19 receptor has yet been identified.
Screening GRP19 revealed a number of single base pair nucleotide changes. The
combination of extended SSCP gel running time and reduced voltage was found to result
in more consistent resolution of DNA sequence variants by SSCP conducted on 4-20%
pre-cast polyacnlamide gels (Novex). SSCP detected a silent MG polymorphism at
nucleotide 1062 of the GPR19 gene of a control subject when samples were nin at 4 OC at
85 volts for 15 hours. This polymorphism was detected by the presence of an additional
band (Fig. 28, lane 2) in the portion of the SSCP gel resolving both single stranded (SS)
DNA and double stranded (DS) DNA. The polymorphism was poorly resolved, however,
when the samples were electrophoresed at 24 OC at 55 volts for 9 hours (Fig.2 C). The
rapid SSCP protocol (300 volts, 2 hours) did not resolve this polymorphism (Fig. 2A,
lane 2). niese results demcnstrate the importance of controlling temperature and voltage
conditions in order to ensure sensitive resolution.
The effect of SSCP running temperature on the resolution of sequence variants is also
illustrated in Figure 3. SSCP analysis run at 4 OC for 15 hours at 85 Volts resolved band
shifts that were revealed by sequencing to be homozygous (Fig 3A, lane 2) and
heterozygous (Fig 3B, lane 2) forms of a T/C nucleotide change at base pair 250 located
in the portion of GPR19 encoding the putative second transmembrane (TM2) domain.
The nucleotide change resuiting in the band shifis in Fig 3 was detected by SSCP in less
than 2% of the 114 DNA samples screened. The nucleotide change resulted in a
conservative Val l l6Ile amino acid change. The sarne heterozygous polymorphism
resolved in Fig 3B, lane 2 was also resolved by SSCP analysis run at 24 OC. however, the
pattern is very different.
Figure 4 shows the SSCP analysis of the portion of GPR19 encoding the putative
third transmembrane (TM3) domain of the receptor. The SSCP band shifis shown here
Figure 2 Optimization of SSCP temperature, ninning tirne and voltage to resolve variants. SSCP analysis of GPR19, run at 4 OC at 85 volts for 15 hours, detected a polymorphism (B, lane 2). Note the presence of an additional band in the portion of the SSCP gel resolving both single stranded (SS) and double stranded (DS) DNA. The polymorphism was poorly resolved at 24 OC at 55 volts for 9 hours (C, lane 2). Rapid SSCP (300 volts, 2 hours) did not resolve this polymorphism (A, lane 2).
Figure 3 The effect of SSCP ninning temperature on the resolution of sequence variants. The SSCP analysis run at 4 OC resolved band shifts revealed to be homozygous (A, lane 2) and heterozygous (B, lane 2) forms of a T/C nucleotide change in GPR19. The heterozygous polymorphism (B. lane 2) resolved into a different pattern when SSCP was conducted at 24 OC (C, lane 2).
Figure 4 Resolution of sequence variants in TM3 of GPR19. Variants were detected by SSCP nui at 24 OC but not at 4 O C . The SSCP gel was run at 50 volts for 15 hours using the SSCP conditions discussed.
were detected at 24 OC but not at 4 OC. The band shifis were present in nearly 7% of the
DNA sarnples anaiyzed. Sequencing showed that this nucleotide change of CiT at base
pair 300 resulted in a conservative arnino acid change fiom Ile289Val.
In the course of refining the SSCP protocol. various polyacrylamide gels were
compared for their ability to detect a single nucleotide change. Resolution of the
cornmon GPR19 polymorphism on pre-cast polyacrylamide non-gradient 10 % gels was
iderior to resolution on 4-20% gradient gels (Figure 5). The 10 % gels were
electrophoresed with 60% of the voltage used to run SSCP 4-20% graciient gels. however,
high SSCP band shift resolution was not maintained. Therefore, fùrther use of SSCP to
screen for sequence variants in candidate genes was undertaken using 440%
polyacrylarnide gels.
The above results represent our first experience with conducting SSCP on pre-cast
polyacrylamide gels. The resolution appeared superior compared with that used in Our
lab by Lam et al, 1996 to screen the SHTIA receptor for sequence variants in TS patients.
The GPRl9 gene was found to be highly conserved in patients and controls.
3.2 SSCP mutation screening of the DRDl gene
The SSCP analysis involved screening the entire coding region of the DRD 1 gene for
sequence variants using SSCP conditions that were found to be effective in screening the
GPR19 gene for sequence variants. No SSCP band shifts were noted in the TS (n=50),
the TS-AD-HD (n=35), the TS-OCD (n=30) and the alcohol dependent ( ~ 7 2 ) patient
groups when SSCP was conducted at 55 volts for 18 hours at 24 OC or at 100
Figure 5 Resolution of TM3 variant on 10% gels was inferior to that on 4-20% gels. When 10% gels were run at 60% of the voltage used to run gradient gels, SSCP resolution was not maintained.
volts for 18 hours at 4 O C . One band shift was observed in the SSCP analysis of the
portion of the gene coding for the amino terminus of the DRDl gene in one of the 50
controls matched to the Tourette's syndrome population (Figure 6. lane 2). The SSCP
analysis was conducted at 100 volts for 18 hours at 4 OC. Sequence analysis of the DRD 1
gene in 3 representative patients fiom each patient and control group confirmed that the
SSCP band shifi observed for the control subject analysed in figure 6, lane 2 was caused
by a base pair substitution of C for T which resulted in the conservation of 49Ile. This
sequence was not found among the patients screened.
3.3 SSCP mutation screening of the DATl gene
The DATl gene mutation screening project was ambitious because of the size and
complexity of the gene. The DATl cDNA encodes a large 620 amino acid protein.
encoded by 15 exons and introns of which al1 exons but the first are translated. The size
of the DAT 1 introns remains unknown. However, published (Donovan et al, 1995) and
unpublished (Vandenbergh, persona1 communication) intron-exon junction sequence
information enabled us to screen the DATl gene by subjecting the PCR products
amplified using the pnmers listed in Table 3 to SSCP analysis. This procedure presented
difficulties for those primer pairs that amplified more than one DNA product. For this
reason, the pnmers for exons 4, 10 and 12 were designed more than once before the set
presented in Table 3 successfûlly amplified the correct gene fragment.
The structure of the DATl gene was analyzed by SSCP in 225 subjects. Band shifts
were noted in al1 groups when exon 2 (using primer pair EX2ForB and 2C 1, Table
Figure 6 Analysis of a fragment (350 bp) of the D 1 receptor gene. This silent mutation was only detected by electrophoresis conducted at 85 volts for 24 hours at 1 OC. The arrow indicates the pattern of a polymorphic varient.
3) was analyzed by SSCP conducted at 55 volts for 18 hours at 24 OC (Fig. 7 A, lane 1).
Sequence analysis showed that the band shift was due to a sequence variant 171 C/T that
did not result in an amino acid change. The individual analyzed in lane 1 was revealed,
by sequencing, to be heterozygous.
In addition to the silent 171C/T polymorphism, a rare nucleotide change was also
discovered in exon 2 (Fig 7. lane 3) in a single control subject. Sequence analysis
showed that this band shifi was the result of a 164T/C nucleotide substitution that results
in a relatively conserved arnino acid change Va1 164AIa in this control subject.
A second conservative amino acid change was also discovered in another control
subject. Sequencing confirmed that the SSCP band shifi in exon 8 (Fig 8, lane 1).
resolved by 50 volt electrophoresis for 12 hours at 30 OC. resulted from a sequence
variant 1445TK. A Val 1 144Ala amino acid change resulted. The individual analyzed in
Fig 8' lane 1 was heterozygous for this sequence variant.
SSCP band shifts were also noted in al1 groups when exon 9 (Fig 9) and exon 15 (Fig
10) were analyzed by SSCP conducted at 100 volts for 18 hours at 4 OC on 440%
gradient gels. Sequencing revealed that the exon 9 SSCP band shifi (Fig 9, lane 2)
resulted from a hornozygous sequence variant 1215NG that did not result in an amino
acid change. Sequencing identified the individuals in lanes 1 and 3 to be homozygous
non-polymorphic and heterozygous for the polymorphism respectively. The exon 15
SSCP band shift (Fig. 10, lane 1) was confirmed to result fiom a homozygous sequence
variant 1898T/C downstream of the translated portion of exon 15. Sequencing confirmed
Figure 7 SSCP analysis of DATl exon 2 in three control subjects. Sequence analysis showed that the SSCP band shift in lane 1 was due to a silent sequence variant 171 C R . This individual was revealed, by sequencing, to be heterozygous. A rare nucleotide change was also discovered in the patient analysed in lane 3. Sequencing showed that this SSCP band shift reflects a 164TK nucleotide change in exon 2, resulting in a conservative arnino acid change Va1 1 64Ala in this control subject.
Figure 8 SSCP analysis of DATl exon 8 in three control subjects. The band shift in lane 1 was found to reflect a 445T/C sequence variant that results in a Va11 144Ala amino acid change. The individuai analyzed in lane 1 was heterozygous for this sequence variant. Individuals anaiyzed in lanes 2 and 3 were shown by sequencing to be non-polymorphic.
Figure 9 SSCP analysis of DATl exon 9 in d i r e TS subjects. Sequencing revealed that the exon 9 SSCP band shifi in lane 2 resulted from a homoqgous 1215NG sequence variant that did not result in an amino acid change. Sequencing identified the individuals in Ianes 1 and 3 to be hornozygous non-polymorphic and heterozygous for the polymorphism respectively.
Figure 10 SSCP analysis of DATl exon 15 in three control subjects. The band shift in lane 1 was confirmed, by sequencing. to result fiom a homozygous sequence variant 1898TIC Iocated downstream of the translated portion of exon 15. Sequencing confirmed that the individuals analyzed in lanes 2 and 3 were non-polymorphic and heterozygous for the exon 15 poiymorphism respectively.
that the individuals analyzed in lanes 2 and 3 were non-polymorphic and heterozygous
for the exon 1 5 polymorphisrn respectively (Giros er ai, 1992; Vandenbergh et a[, 1992).
In order to complete the project, patients and controls were genotyped for a
polymorphic 3'VNTR marker at the DATl locus (Vandenbergh et al, 1992). Three
alleles of the 3'VNTR were identified (Figure 1 1). Lane I shows the 480 base pair band
identified to be homozygous for the 10 repeat allele (1 0/10) of the 3'VNTR. Lane 2
shows the 480 base pair band and the 442 base pair band below it. This individual was
heterozygous for the 10 repeat and the 9 repeat aileles (1019) of the 3'VNTR. Lane 3
shows a 150 base pair band which identifies an individual homozygous for the 3 repeat
ailele (3/3) of the 3'VNTR.
3.4 Association study of DATl polymorphisms in TS and alcohol dependence
Allele frequencies of the novel polymorphisrns are shown in Table 4. The frequency
of the exon 2 polymorphism was between 3.7% and 6% in the TS (n=47), alcohol
dependent (n=64) and control groups (n=42) (Table 4). Because of its low frequency. we
did not genotype our remaining patient and control samples for the exon 2 polymorphism.
The frequencies of the cornmon polymorphisms in exons 9 and 15 were determined in al1
patient and control groups (Table 4).
The exon 9 A.G fiequencies were 19% and 23% in TS and control groups
respectively. The frequency of the exon 9 T/C polymorphism was 33% in the alcohol
dependent group and 24% in the matched control group. X h a l y s i s (Table 4), however,
Figure 11 PCR genotyping of the DATl 3'VNTR. Lane 1 was found to be a 480 base pair band homozygous for the 10 repeat allele ( 1 0110). Lane 2 shows the sepmation of the 480 base pair band and the 442 base pair band below it. This individual was found to be heterozygous for the 10 repeat and the 9 repeat alleles (1019). Lane 3 shows the 150 base pair band identifying an individual homozygous for the 3 repeat allele (313).
?'-LE 4. DATl allele and genotype frequencies: X'P values compare population frequencies Population Exon 2 N Frequency Genotype Frequencies x 2 p values
C/T 1-1 1 -2 2-2 3 x 2 3x3 Tourette's 1 09 0.037 0.926 0.074 O
Control 42 0.06 0.88 O. 12 O -
Alcoholics 64 0.047 0.906 0.094 O Control O - -
Exon 9 Allele 1 Genotype Frequencies x L p values Population MG N Frequency 1 - 1 1-2 - 3-2 3 x 2 2x2 Tourette's 1 09 0.77 0.59 0.37 O .O3 0.494 0.494
Control 67 0.8 1 0.64 0.3 0.06
Alcoholics 64 0.67 0.42 0.5 0.08 Control 64 0.76 0.53 0.36 0.0 1
Exon 15 Allele 1 Genotype Frequencies x2p values PopuIation T/C N Frequency 1-1 1-2 - 7-1 3 x 2 2 x 2 Tourette's 1 09 0.79 0.63 0.32 0.05 0.677 0.677
Control 67 0.75 0.57 0.37 0.06
Alcoholics 64 0.73 0.5 0.47 0.03 Control 64 0.76 0.56 0.46 0.0 I
S'VNTR 3'VNTR Genotype Frequencies X ~ P Population (48 bp), N 10/10 9/ 10 9/9 2/10 3 9 *Other 3 x 2 3x3 Tourette's 1 09 0.66 0.29 0.05 O O 0.05 0.767 0.767
Control 67 0.6 1 0.3 1 0.06 0.02 O 0.08
Alco holics 64 0.53 0.42 0.0; O 0.02 0.05 0.934 0.933 Control 64 0.56 0.3 8 0.03 0.03 O 0.06
3'VNTR compared by using 10/10, 9/10 and *Other genotpyes to generate X' P value.
indicates that the difference between exon 9 A/G allele frequencies and exon 9 A/G
genotype frequencies was not significant between patient and control groups.
The exon 15 T/C frequencies were between 21% and 27% for both patient (TS and
alcohol dependent) and control groups. X2 analysis (Table 4) indicated that the exon 15
TIC fiequencies and genotype frequencies were not significantly different between
patient and control groups. Genotype and allele fiequencies were also found not to be
significantly different between the TS group with no comorbidity, the TS-OCD group and
the TS-AD-HD ( X 2 ~ values ranging from 0.283 through 0.872). Al1 patient and control
groups were found to fit closely to Hardy-Weinbergh equilibnum at each of the three
polymorphisms genotyped, including the YVNTR. This concurs with the fact that
although the DNA was obtained fkom 4 sources, the patient and control subjects were
ethnicaily matched. The X 2 ~ values ranged fiorn 0.1 (exon 9 A/G in alcohol dependent
patients), through 0.97 (exon 15 T/C in TS).
Linkage disequilibrium between DAT 1 haplotypes was also analyzed. The lO/9
repeat polyrnorphism of the 3'VNTR (VNTR 10/9), found at similar allele and genotype
frequencies in each group (Table 4), was in linkage disequilibriurn with the exon 15
polymorphism in the TS group ( x2=6~ .9 1. 5df, pK1 the alcohol dependent group
(X1=19, jdf, p < l ~ 4 ) and in their respective control groups (X2=27.74. 5df. p<104:
X2=1 8.42, 5df, p<.104). For the exon 15 T/C-VNTR 10/9 haplotype, linkage
disequilibriurn coefficients 6 were O. 1 1 for TS patient and control groups and 0.10 for
alcohol dependent patient and control groups. The modest disequilibrium coefficient 6
values, on an absolute value scale fiom 0-0.25, suggest that, while significant
disequilibrium exists for the exon 15 A/G-VNTR 1019 haplotype, not al1 individuais with
the exon 15 TIC allele have the VNTR IO19 allele on the same chromosome.
In the TS group, linkage disequilibrium was aiso found between the exon 9
polymorphic allele and the 3'VNTR (X2=18.73. 5 df, p<4* 104) and between the exon 9
and exon 1 5 polymorphic alleles (X2= 12.72, 3 df, p<O.O4). The disequilibrium constants
6 were 0.7 and 0.6 for the exon 9 NG-VNTR 1019 and exon 9 NG-exon 15 T/C
haplotypes respectively. The more modest 6 values for these TS haplotypes compared to
those of the exon 15 T/C-VNTR 1019 haplotype suggests that, while statistically
significant, the exon 9 A/G-VNTR 1019 and exon 9 A/G-exon 15 TIC haplotypes are iess
fiequent. No evidence for linkage disequilibriurn was found between these alleles in
either the control groups or the alcohol dependent group.
3.5 Association study of SHTT polymorphisms in TS and alcohol dependeuce
The deletiordinsertion SHTT promoter polymorphism and the 5 H ï T intron 2 VNTR
polymorphism were genotyped in al1 patient and control groups (Figures 12 and 13). The
allele and genotype fiequencies are surnmarized in Table 5 . X 2 analysis showed that
genotype frequencies at both loci fit closely to Hardy-Weinbergh equilibrium for patient
and control groups. For the bi-allelic promoter locus, X Z ~ values ranged fiom 0.26 (for
the alcohol dependent group) to 0.92 (for the TS group). For the tri-allelic intron 2
VNTR locus, X 2 ~ values ranged corn 0.1 1 (for the TS group) to 0.80 (for the alcohol
dependent group).
Figure 12 Deletion/insertion SHTT promoter polymorphism genotyped on 4% agarose. Lane 1 shows the PCR product (-484 bp) of an individual homozygous for the short promoter. Lane 3 shows the PCR product (-528 bp) of an individual homozygous for the long promoter. Lane 2 shows PCR products for the long and short promoter arnplified from an individuai heterozygous for the long and short promoters.
Figure 13 Intron 2 VNTR polymorphism genotyped on 4% agarose. Lane 1 shows a PCR product (-390 bp) homozygous for the 12 copy (390 bp) . Lanes 2 and 3 show two band shifts for a patient heterozygous for the 12 copy and 9 copy (bp) alleles. Lane 4 shows the two band shifts of a patient heterozygous for the 12 copy and 10 copy (bp) alleles.
The SHTT promoter deletion (the short promoter) was significantly more common in
the TS group (51%) compared with the control group (36%) (X2=8.77, 2 df. p=0.04. two
tailed, Bonferroni corrected). Genotype fiequencies were not significantly different
between the TS and control groups (x'=8.45, 2 df. p=0.18, two tailed) afier Bonferroni
correction even though the homozygous shon genotype was twice as cornmon in the TS
group (26%) compared to the control group (13%). Odds Ratio for short promoter allele
association with TS was 1.88 [95% CI 1.2 1-2.931).
The short promoter allele was not significantly more fiequent (X'=1.52. 2 df, p=0.22,
two tailed) in the alcohol dependent group (45%) compared to its matched control group
(37.5%). Although the homozygous shon promoter alleie was more fiequent arnong
alcohol dependent patients (28%) compared with the matched control group (16%), the
genotype Frequencies were also not significantly different benveen the patient and control
groups (X2=1 83.2 df. p=0.40, two tailed).
The allele and genotype frequencies of the intron 2 VNTR were not significantly
different between the TS group and matched control group (X'=4.09. 2 df. ~ 0 . 1 2 9 . two
tailed; X2=6.37, 3 df, p=0.1, two tailed), as reported in Table 5 . Between alcohol
dependent and control groups, however, the allele frequencies (X'=20.56, 1 df, p=7.2* 10-
5 . two tailed, Bonferroni corrected) and genotype frequencies (X'=25.08, 3df, p=0.0006.
two tailed, Bonferroni corrected) were significantly different, as reported in Table 5. The
Odds Ratio for an alcohol dependent patient having the 10 copy allele was 5.80 [95% CI
2.42-14.371.
TABLE 5 . 5HTT allele and genotype frequencies: X'P values compare patient and control frequencies deletion/ N Frequency Genotype Frequencies X 2 p values
Group insertion 1 - 1 1-2 7-3 3 ? 2 2,V Tourette's 91 0.47 O .24 0.50 0.26 0.04 0.18
Control 91 0.64 O .42 0.45 O. 13
Intron 2 VNTR Genotype Frequencies X2p values Group VNTR N 12/12 10/12 10/10 9/ 12 9/10 919 9/AlI 4x2 2 .V Tou rette's 109 0.44 0.37 0.05 O O 0.05 0.05 0.10. O. 13.
Control 67 0.44 0.28 0.08 0.05 O 0.09 O. 14
Alcoholics 64 0.25 0.53 0.18 0.023 0.025 0 0.05 6'10'' 7.2'[0 -5
Control 32 0.62 0.20 0.03 O. 16 O O O- 16
SHTT innon VNTR genotype frequencies (4,V) compared 124'1 2, !O/ 12. i O / I O and 9iAll other alleles to jenerate X- value; SHTT allele frequencies (3x2) compared the fiequency of the 10 copy allele to the combined frequencies of al1 other alleles.
Linkage disequilibrium between the deletiodinsertion polymorphism and the intron 2
VNTR polymorphism was analyzed in each patient and control group. No evidence of
linkage disequilibrium between the polyrnorphisrns was found in the TS group or its
control group (%'=2.5 1. 5 df, p=0.5; X'=0.20, 5 df. p=O.j). Linkage disequilibrium was
significant between the polyrnorphisrns in the alcohol dependent group (X'= 1 5.67, 5 df,
p=0.004) but not in its control group (%'=O. 19, 5 df, p-0.5). In the alcohol dependent
group, the linkage disequilibriurn coefficient S was 0.1 for the haplotype consisting of the
(insertion) long promoter- 10 copy VNTR allele. This modest disequilibrium coefficient
6 value suggests that while statistically significant only partial linkage disequilibrium is
present for the short promoter-10 copy haplotype.
lDlSCUSSION
4.0 Interpreting candidate gene sequence vanants in patient and control groups
(a) Consenration of the DRDI gene in TS and in alcohol dependence
No systematic atternpt to associate strucniral variations in the coding region of the
DRDl gene with TS, aicohol dependence or related disorders has appeared in the
literature. The present midy has largely ruled out the possibility that mutations in the
coding region of the DRDl gene contribute to the etiology of TS or alcohol dependence
in the patients studied. The DRDl sequence conservation among the 72 alcohol
dependent patients reported here confirms the results of a previous study on the DRDI
gene in eight aicohol dependent patients (Lui et al, 1995). The integrity of the DRDl
gene coding region among TS patients confirms the results of a previous study on the role
of the DRDI gene in TS (Gelernter et al, 1993).
The previous work on the DRDl gene in TS was limited. however. by being confined
to analyzing RFLP sites f o n d in the regions flanking the DRDl gene (Gelemter et al.
1993). The present study. by contrast, effectively excludes the possibility that DRDl
coding region mutations contribute to the etiology of TS or aicohol dependence by
systematicdly screening the DRD I gene.
(b) DATl gene sequence variants in TS and in alcohol dependence
The DATl gene mutation screening examined whether sequence variants in the
coding region of this gene underlie the polygenic inheritance of TS (Gelemter et al, 1995:
Comings et al, 1996), AD-HD (Gill et ai, 1997; Cook e i al, 1995) and alcohol
dependence (Persico et al. 1993: Muramitsu et al. 1995). This study has succeeded in
identifying structurai variants in the coding region of the DATl gene that map help to
clari- association studies of the YWTR of the DATl gene with TS (Comings et al.
1996). .4D-HD (Cook et al. 1997: Gill et al. 1997: Cook et al. 1995) and alcohol
dependence (Muramatsu and H i p h i . 1995).
The DATl gene variants. however. are not likely to result in altered dopamine
transporter function. The two lare arnino acid changes. Val54Ala and Val164Ala are
consewative and were found in control subjects. Three common single nucleotide
polymorphisms were identified: exon 2 Cm. exon 9 A/G and exon 15 TIC. These
poly~oorphisms were not found at significantly different allele or genotype frequencies in
the TS or alcohol dependent groups bp %' analpis (Table 4). Therefore. the novel DATl
polymorphisms do not appear to be associated with TS or alcohol dependence. Power
analysis (specifuinp a type II error rate of ad.05 and power of 1 $=O. 80), indicates that
patient and control groups of the size used in this study are large enough to detect
hypothetical odds ratios of 2.3 and 3.35 for the DATI eson 9 A/G and exon 15 TIC
alleles to be risk alleles for TS (n=109) and alcohol dependence (n=64) respecrively.
Future studies should genotype larger patient and control sample groups in order to \-en@
that these polymorphisms are not associated with TS or alcohol dependence.
Previous studies (Comings et al. 1996: Gelemter el al. 1995) suppon the evidence
presented here that the DATl gene is not associated with the vocal and motor tics
considered to be the essential features of TS (Diagnostic and Statistical Manuai of Mental
Disorden. Fourth Edition. 1990). However. the earlier work by Comings et al. 1996. did
associate the DATl VNTR l0/10 genotype with 1-2% of AD-HD diagnosed comorbidly
with TS. In the present study. this association was not detected by analysis of the
frequency of the VNTR 1011 0 genotype in TS patients diagnosed comorbid with AD-KD
(n=33) compared with TS groups with no AD-HD cornorbidity (n=86). Future studies
should examine large samples of TS-AH-HD patients in order to confirm the lack of
association between DATl and TS-AD-HD found here.
Statistically significant, partial linkage disequilibriurn was found for the exon 15 TIC-
VNTR 1019 haplotype in al1 patient and control groups. This suggests that the exon 15
T/C marker may be of use in friture haplotype based Haplotvpe relative nsk (HH-RR)
studies of TS and aicohol dependent trios. Differences in linkage disequilibrium between
patient and control groups were noted. Partial linkage disequilibrium for the exon 9 NG-
VNTR 1019 and exon 9 NG-exon 15 T/C haplotypes was statistically significant in the
TS group but not in either control group or the alcohol dependent group. However.
linkage disequilibrium must be compared with caution between groups of unrelated
individuals because a control group unrelated to the patient group cannot control for
recombination events that result in different linkage disequilibria in different populations
(Tenvilliger and Ott. 1994). The linkage disequilibriurn between the novel DATl
polymorphisms, therefore, should be used to examine the family-based association of TS
with the DATl gene as done previously in studies of AD-HD (Gill et al, 1997; Cook et
al, 1997).
The results of the DATl candidate gene mutation screening represent a necessary step
(Gelman and Gelemter, 1993) toward understanding whether a candidate gene. such as
the DATl gene, contributes to the inheritance of a disease state. Our SSCP anaiysis of
the DATl gene, like the recent SSCP andysis of the norepinephrine transporter (NET)
(Stober et al, 1996), discovered sequence variants comrnon to control and disease States.
These polymorphisms rnay clarie studies of the phmacology of MP, cocaine (Volkow
et al, 1999, antidepressants and endogenous catecholamines (Buck and Amara, 1995)
because there is now evidence that amino acid variants, while comrnon in the NET gene,
are rare in the DATl gene. Research into the selective phmacology of the dopamine
and norepinephrine transporters using transporter chimeras (Buck and Amara, 1 995;
Buck and Amara, 1994) may be particularly usehl in determining the significance of
DATl sequence conservation.
The DATl sequence information, therefore, facilitates M e r studies of dopamine
transporter pharmacology (Volkow et al, 1995), the pharmacogenetics of cocaine
addiction (Gelemter et al, 1994; Persico et al, 1993) and future Iinkage studies of the
DATl gene in TS, alcohol dependence and other neuropsychiatrie disorders including
AD-HD. Should evidence of linkage or Iinkage disequilibrium between disorders and
the DATl gene be confi~rmed, a good case could be made for searching for
polymorphisms in the 5' regulatory sequences that may contribute to the dopamine
transporter pathology (Singer et al, 1991 ; Tiihonen ef al, 1995) that may underlie these
disorders.
(c) Association studies of SHTT variants in TS and in alcohol dependence
We have presented work that provides preliminary evidence that the 5HTT gene may
be a susceptibility loci for TS. X' analysis (Table 5) detected a significant difference
between allele fiequencies ( ~ ~ = 8 . 7 5 , 1 df. p=0.04, two tailed, Bonferroni corrected) but
not genotype fiequencies ($=8.45, 2 df, p=O. 18, two tailed, Bonferroni corrected) of the
5HTT promoter between TS patient and control groups. The Odds-Ratio of 1.88 for TS
patients to have at least one short 5 H ï T promoter allele, suggests that the 5HTT short
prornoter may be a risk ailele for TS. Ninety-five percent confidence intervals of 1.21-
2.73, indicate that the Odds-Ratio is above unity. Power analysis (speciSing a type II
error rate of a=0.05 and power of 1-B=0.80), indicates that patient and control groups of
the size used in this study are large enough to detect an odds ration of 2.4: a value toward
the upper Iimit of the 95% confidence interval of 1.21-2.73 of the Odds-Ratio for a TS
patient to have at least one short 5HTT promoter risk allele.
The discussion of the results of this study of the SHTT prornoter in TS would not be
complete without comparing the frequency of the SHTT short promoter allele in our
control group with that in control groups genotyped for other studies. In the control
group used for this study (n=9 l), the short promoter allele frequency was 36%. However,
other studies have found the short promoter at higher fiequencies. A study of the short
dlele in 90 German controls and 79 Itdian controls found this allele at frequencies of
41% and 42% respectively (Deckert et al, 1997). A study of the short allele in 104
English, 301 Italian and 95 German controls found its frequency to be 45%, 43% and
40% respectively (Collier et al, 1996). Therefore, replication of this work is necessary in
order to confimi the control frequency of the 5HTT short promoter allele in the caucasian
nonpsychiatrk control population. This issue must be considered because a common
weakness of association studies in psychiatric genetics has been the fact that statistically
significant associations can result from genotyping confounded control groups. even
when an attempt to match patients and controls for ethnic origin has been made (Pato et
al, 1993; Barr and Kidd, 1993).
The intron 2 VNTR, previously associated with unipolar depression (Evans et al,
1997; Ogilive et al, 1996) but not associated with manic depressive illness (Bellivier et
al, 1997), was also genotyped for both of our patient and control groups. VNTR allele
and genotype fiequencies were not significantly different in the TS group compared to
the control group, however, both the alleles and genotypes of this locus were significantly
different in the alcohol dependent group (n=64) compared to its control group (n=32)
(~~=22 .79 , 2 df, p=l .2* 1 O-', two tailed, Bonferroni corrected; X2=25.08, 4 df, p=7* 1 O-',
two taileci, Bonferroni corrected).
The control group assigned to the alcohol dependence group was small. Therefore,
we attempted to verify our analysis by comparing the alcohol dependent VNTR allele and
genotype fiequencies to those of the control group (n-91) that was originally assigned to
the TS patient group. This cornparison also found a significant difference between allele
and genotype frequencies (X'=16.64, 2 df, p=0.003, two tailed, Bonferroni corrected;
X2=20.83, 3 3df, p=0.0012, two tailed, Bonferroni corrected), although the X 2 ~ values are
larger. This analysis is not strictly valid because the large control group was not
originally assigned to the alcohol dependence group. However, it serves to codirm that
the VNTR allele and genotype frequencies are different between the alcohol dependent
patient group compared with both of our control groups.
Our VNTR findings for alcohol dependence are different From those reported (Ogilvie
et al, 1996) in the study associating the SHTT VNTR with depression. The depression
study found that the 9 copy VNTR allele was associated with risk for unipolar depression.
Our study, on the other hand, found that an excess of the 10 copy VNTR allele was
significantly more common than the combined 12 and 9 copy VNTR allele group (Table
5) in alcohol dependent patients (n=64) compared to controls (n=32). This analysis was
repeated using the larger control group (n=91) that was not originally assigned to the
alcohol dependent group. Again, the 10 copy VNTR allele was significantly more
cornmon that the combined 12 and 9 copy allele (%'=17-95, Idf, p=0.003, two tailed.
Bonferroni corrected), although the association was less significant ( X ' ~ values were
larger). The Odds Ratios of 5.80 [95% CI 2.42-14.371 and 2.85 [95% CI 1.69-4.8 11 were
calculated for the risk of an alcohol dependent patient having the 10 copy allele,
compared with controls from the small and large groups respectively. Therefore. our
study suggests that the 10 copy allele may confer risk for alcohol dependence by an
unknown mechanism that may invoive adjacent gene stuctures.
The VNTR association should be interpreted with caution, however, because the
intron 2 VNTR has no known effect on the 5HTT gene at the transcription level. While
alleles of the YVNTR of the insulin gene have been shown to correlate with its
transcription (Pugliese et al, 1997), there is no evidence as yet that the SHTT VNTR
alleles have any regulatory fünction (Ogilvie et al, 1996). The role of Activator Protein-l
(AP-1) motifs adjacent to the 5HTT WTR remains speculative (Ogilvie et al, 1996;
Lesch et al. 1994).
In addition, the SHTT intron 2 VNTR has not been genotyped in large enough
numbers (Evans et al, 1997; Ogilvie et al, 1996) to know the VNTR allele fiequency in
the caucasian population. Because depression is frequently comorbid with alcohol
dependence (Anton, 1996), our result showing an excess of the 10 copy VNTR allele in
alcohol dependence may conflict with the previous study showing that an excess of the 9
copy VNTR alleie is associated with depression (Ogilvie et al, 1996). Power analysis
(speciQing a type II error rate of a=0.05 and power of 1-P=0.80), however, suggests that
our alcohol dependent and control sarnples are large enough to be able to detect the Odds
Ratios for the 10 copy VNTR allele calculated. This work needs to be independently
repeated in order confirm these findings because the phenomenon of population
stratification (Barr and Kidd, 1993) has been known to confound studies that have
attempted to use unrelated controls matched for ethnic otigin (Barr and Kidd, 1993;
Kennedy et al, 1995).
It has previously been suggested (Ogilvie et al, 1996) that evidence of 5HTT VNTR
association with disease might indicate linkage disequilibrium of the VNTR with a
functional mutation (Ogilvie et al, 1996). This study found evidence that linkage
disequilibriurn for the short prornoter-VNTR IO copy haplotype is present in the alcohol
dependent group (6=0.1), but not in TS or control groups. However, the association of
the short promoter with alcohol dependence does not reach statistical significance. Power
analysis suggests that alcohol dependent and control groups are large enough to detect an
odds ratio of three if it were present.
While this sîudy does present evidence of linkage disequilibrium in the alcohol
dependent group that may be disease associated, it ought to be remembered that linkage
disequilibrium should be compared with caution between unrelated individuals
(Twenvlliger and Ott, 1994). A fùture study should genotype mother-father-proband
trios in order to assess whether the short promoter-VNTR 10 copy haplotype is associated
with alcohol dependence. Transmission of the short promoter allele in TS trios rnay also
be informative about the role of the 5HTT gene in TS.
4.1 Conclusions
We have succeeded in confirming the practicality of using SSCP to identifi sequence
variants of as little as a single base substitution under the conditions developed to screen
the GPR19 gene. n e fact that SSCP has previously been demonstrated to identim 85-
95% of single base pair substitutions (Jordanova et al, 1997; Schefield et al. 1993;
Weghorst et al, 1993) allows us to conclude that we have identified the majority of the
sequence variants of the candidate genes we screened in patient and control groups. We
have used SSCP to effectively exclude the role of sequence variants in the DATI and
DRDl gene coding regions fiom having a role in the etiology of TS or alcohol
dependence. The deletion/insertion polymorphism and the 10 copy VNTR allele of the
5HTT gene, discovered previously (Hiels et al 1996; Lesch et al, 1994), were found to be
significantly more cornmon in the TS and alcohol dependent groups, respectively,
compared with the control groups genotyped. The greater frequency of the 5HTT
deletiodinsertion polymorphism in the TS group is intriguing because there is in vitro
evidence that it may alter gene expression.
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