alterations in dopamine and benzodiazepine receptor binding precede overt neuronal pathology in mice...
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Brain Research 1039
Research report
Alterations in dopamine and benzodiazepine receptor binding
precede overt neuronal pathology in mice modelling early
Huntington disease pathogenesis
Laura Kennedya, Peggy F. Shelbournea, Deborah Dewarb,TaDivision of Molecular Genetics, Faculty of Biomedical and Life Sciences, University of Glasgow, Glasgow, G11 6NU, UK
bDivision of Clinical Neuroscience, Wellcome Surgical Institute, University of Glasgow, Garscube Estate, Glasgow, G61 1QH, UK
Accepted 10 January 2005
Abstract
Huntington disease (HD) is an inherited, late onset, progressive neurodegenerative disorder. Primary degeneration appears to selectively
occur in striatal medium spiny neurones but this is most likely preceded by a period of neuronal dysfunction. Altered levels of
neurotransmitter receptors may disrupt neuronal function and contribute to a toxic environment within the brain. In the present study, a
knock-in HD mouse modelling early stages of the disease was used to determine whether alterations in neurotransmitter receptor densities
occurred before overt neuronal loss. Receptor autoradiography demonstrated reduced dopamine D2 and increased benzodiazepine receptor
binding in the striatum of HD animals compared to wild-type littermates. The density of benzodiazepine receptor binding was also increased
in the cerebral cortex of the HD mice. Changes in opioid and dopamine D1 receptor densities were more subtle and influenced by the genetic
background of the mice. Our findings are consistent with the hypothesis that alterations in neurotransmitter receptor density precede cell loss
and may be an active cellular response to the initial stages of HD pathogenesis.
D 2005 Elsevier B.V. All rights reserved.
Theme: Disorders of the nervous system
Topic: Degenerative disease: other
Keywords: Huntington disease; Knock-in mouse; Neurotransmitter receptors
1. Introduction
The progressive neurodegenerative disorder, Huntington
disease (HD), is an autosomal dominant disease caused by
the expansion of a polymorphic CAG triplet repeat sequence
within exon 1 of the HD gene [17]. The expansion mutation
results in an extended polyglutamine stretch within the N-
terminus of the ubiquitously expressed protein called
huntingtin [17]. The initial pathological basis of HD is
selective degeneration of the medium spiny neurones within
the striatum [16], although progression of the disease is
0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.brainres.2005.01.029
T Corresponding author. Fax: +44 141 943 0215.
E-mail address: [email protected] (D. Dewar).
associated with degeneration of additional brain regions,
most prominently, the cerebral cortex [43]. Currently, it is
not understood how the presence of mutant huntingtin leads
to the cell-selective aspects of HD pathology.
The profound neuronal loss observed in end-stage
disease must clearly be a major causal factor in the clinical
picture. However, the disease course is so prolonged that
vulnerable neurones may be disposed to long periods of
dysfunction prior to cell death. The demonstration that a
small number of symptomatic HD patients show no overt
neuropathology (Grade 0) [42,43] argues that massive
striatal cell loss is not an absolute pre-requisite for onset
of disease. A number of functional imaging studies using
positron emission tomography (PET) have demonstrated
reduced striatal glucose metabolism in the early stages of
(2005) 14–21
L. Kennedy et al. / Brain Research 1039 (2005) 14–21 15
human HD [1], lending support to the idea that synaptic
activity and, therefore, neuronal function is compromised
early in HD. The precise nature of the cellular or
biochemical changes that mediate neuronal dysfunction is
not well understood. However, since neurotransmitters and
their receptors have a central role in maintaining the normal
operation of neostriatal circuitry, some effort has been
invested in determining whether neurotransmitter systems
are altered in HD brain tissue.
Studies of human postmortem brain tissue from HD
cases have indicated changes in a variety of neurotransmitter
receptors, including those associated with dopamine, ace-
tylcholine, GABA and glutamate [9,10,15,31]. Although
some early stage cases have been investigated [15], it is
possible that the deficits detected may reflect loss of specific
sub-populations of neurones rather than a primary role in the
disease process. The advent of functional imaging studies
has allowed investigation of HD patients throughout their
disease course. These studies have indicated that altered
brain neurochemistry is present early; PET studies have
revealed reduced levels of dopamine D2 receptors in the
caudate and putamen of asymptomatic mutation carriers,
coincident with reduced glucose metabolism [2,3]. Studies
using PET ligands have also showed altered opioid and
benzodiazepine receptor binding early in HD [19,46].
Whilst supporting the hypothesis that primary deficits in
neurotransmitter systems may contribute to neuronal dys-
function in early HD, subtle cell loss could still account for
the deficits.
In the past, one of the obstacles hampering studies in this
field of was the paucity of appropriate tissue for analysis.
However, the development of genetic models of HD
provides in vivo systems that can be used to examine
pathological cascades that culminate in clinical symptoms.
Accordingly, we have used a knock-in HD mouse model
[38] to investigate molecular and cellular changes involved
in early HD pathogenesis. The insertion of a perfect CAG
repeat tract into exon 1 of the mouse Hdh gene has provided
the opportunity to explore the consequences of the HD
mutation in its appropriate genomic and protein context.
Previous studies of these mice have demonstrated several
phenotypic changes including behavioural and motor
abnormalities [21,38], as well as abnormalities of long-term
potentiation in the hippocampus [41]. The phenotypic and
cellular changes appear to occur before frank neurodegen-
eration as histological and immunohistochemical analyses
have failed to reveal any evidence of overt neuronal loss in
the striatum of 18-month-old HD mice [38].
In the present study, we have determined the density of
dopamine, opioid and benzodiazepine receptor binding sites
in the brains of knock-in HD mice using quantitative ligand
binding autoradiography. The ligands selected for this study
were based on those used previously in human brain
imaging studies of pre-end stage disease patients and we
focused on the striatum as the region of primary pathology.
In order to investigate the presence of potential genetic
modifiers of the early disease process, a post hoc subgroup
analysis of animals with different genetic backgrounds was
performed.
2. Materials and methods
2.1. Animals
The generation of the knock-in HD mice used in the
current study has been described previously [38]. All
experiments were performed on female progeny of the
founder mouse line Hdh4/Q80 inbred onto either a C57BL/6
(N6–7 generation) or FVB/N (N4 generation) genetic
background. A total of 15 HD and 15 wild-type mice was
used. The genotype of the mice was determined by PCR
analysis of tail DNA biopsies using standard procedures
[38]. All experiments were carried out using 17- to 18-
month-old heterozygous mutant animals and wild-type
controls (littermates, where possible, or matched wild-type
mice from other litters). Mice were killed by rapid
dislocation of the neck, the brain removed, rapidly frozen
in dry-ice chilled iso-pentane and stored at �70 8C.
2.2. Receptor autoradiography
Quantitative receptor ligand binding autoradiography
was used so that multiple receptor binding sites could be
examined in adjacent sections from each animal. Receptor
binding assays and autoradiographic analyses were per-
formed by experimenters blinded to animal genotype.
Coronal sections (20 Am) were cut in a cryostat and
sections containing the striatum, motor and parietal cortices
corresponding to Bregma 0.98 mm [12], thaw-mounted onto
gelatin-coated microscope slides and stored at �70 8C. Priorto receptor autoradiography, sections were allowed to come
to room temperature for 15 min. For each ligand, triplicate
sections were used to determine levels of total binding and
duplicates to determine non-specific binding. Dopamine D1
receptors were labelled with 0.5 nM [3H]-SCH23390 (75.5
Ci/mmol, Amersham) by incubating sections in 10 mM
Tris–HCl, 1 mM EDTA, pH 7.4 for 150 min at room
temperature. Non-specific binding was determined in the
presence of cis-flupenthixol (1 AM). After incubation,
sections were washed twice for 10 min at 4 8C in buffer.
D2 receptors were labelled with 240 pM [3H]-YM09151-2
(85.5 Ci/mmol, Amersham) in 10 mM Tris–HCl, 1 mM
EDTA, pH 7.4 for 180 min at room temperature. Non-
specific binding was determined in the presence of
dopamine (50 AM). After incubation, sections were washed
twice for 10 min at 4 8C in buffer. For labelling
benzodiazepine receptors, slides were preincubated in 10
mM Tris–HCl, 1 mM EDTA, pH 7.4 at 4 8C for 10 min and
allowed to dry for 10 min. Sections were then incubated
with 1.5 nM [3H]-Ro15-1788 (70.8 Ci/mmol, Amersham) at
4 8C for 120 min. Non-specific binding was determined in
L. Kennedy et al. / Brain Research 1039 (2005) 14–2116
the presence of flunitrazepam (10 AM). After incubation,
sections were rinsed twice at 4 8C for 1 min in buffer. To
label opioid receptors, sections were preincubated for 15
min at room temperature in 0.17 M Tris–HCl, pH 7.4 and
allowed to dry for 10 min. Sections were then incubated
with 2 nM [3H]-diprenorphine (50 Ci/mmol, Amersham) at
room temperature for 60 min. Non-specific binding was
determined in the presence of naloxone hydrochloride (10
AM). [3H]-Diprenorphine binds to all three of the major
opioid receptor subtypes: mu, kappa and delta and has been
used previously in brain imaging studies of HD patients
[46]. After incubation, sections were washed twice for 5
min in buffer at 4 8C. For all ligands, sections received a
final rapid wash in distilled H2O before being dried in a
stream of air.
Dried slides were exposed to autoradiographic film
(Hyperfilmk-3H, Amersham or BioMax MR-1, Kodak
Scientific Imaging) along with a set of radioactive standards
(1.4–33.3 nCi/mg tissue equivalent, Amersham) for 2
(dopamine receptor binding) or 3 (opioid and benzodiaze-
pine receptor binding) weeks before being developed (D-19,
Kodak Scientific Imaging). Autoradiographic images were
quantified using an MCID M4 image analyser (Imaging
Research, Canada) with reference to the standards and the
specific activity of each ligand. Densitometric measure-
ments were made in the striatum for all four ligands and in
the cerebral cortex for opioid and benzodiazepine binding
only as dopamine receptor binding in the cortex was too
low for reliable analysis. Specific binding is expressed in
fmol/mg tissue equivalent.
2.3. Statistics
The effect of genotype and genetic background on
receptor binding was analysed by two-way ANOVA and
appropriate post hoc tests as indicated.
3. Results
Autoradiograms of total binding for the different ligands
are presented in Fig. 1 for illustrative purposes. On visual
inspection, differences in the levels of binding between HD
and wild-type mice were not marked. However, the
quantitative analysis data revealed there to be statistically
significant differences between the two groups.
3.1. Striatum
The level of dopamine D2 receptor binding in the
striatum of HD knock-in mice was significantly reduced
compared to wild-type controls (P b 0.05, Fig. 2). HD mice
also had significantly higher levels of benzodiazepine
receptor binding sites compared to wild-type mice (P b
0.05). The level of opioid receptor binding in the striatum
appeared to be slightly higher in HD compared to wild-type
mice although this trend did not reach statistical signifi-
cance, even when the data were subdivided according to
genetic background (Table 1) and compared across geno-
types using the unpaired, two-tailed, Student’s t test [HD
versus wild-type; P N 0.05 (FVB/N) and P N 0.05 (C57BL/
6)]. The effect of the HD mutation on D1 receptor binding
was less clear-cut. When data from the mice on different
genetic backgrounds were combined, little or no difference
in the level of dopamine D1 receptor binding was apparent
when HD and wild-type groups were compared (Fig. 2).
However, the mutation seemed to have a different effect on
D1 binding in the two genetic backgrounds used in this
study (Table 1). In the C57BL/6 lines of mice, D1 receptor
binding was significantly higher in the HD mice than their
wild-type littermates (P b 0.01, unpaired, two-tailed,
Student’s t test). By contrast, in the FVB/N lines, D1
receptor binding appeared to be lower in the HD mice than
their wild-type littermates, although this trend did not reach
statistical significance (P = 0.12, unpaired, two-tailed,
Student’s t test).
3.2. Cerebral cortex
In line with similar findings in the striatum, the level of
benzodiazepine receptor binding in the cerebral cortex was
significantly higher in the HD compared to wild-type mice
(P b 0.005, Fig. 3). There was no significant difference
between HD and wild-type mice in the level of opioid
receptor binding in the cerebral cortex. In comparison to the
striatum, very low levels of dopamine receptors are present
in the cerebral cortex and at the concentrations of the
ligands used in this study (chosen to assess striatal binding
levels), the levels of D1 and D2 binding were too low to be
accurately determined.
3.3. Influence of genetic background
The influence of genetic background on receptor
binding densities was analysed by two-way ANOVA
(Table 1). Although genetic background had no signifi-
cant influence on the density of dopamine D2 or
benzodiazepine receptors, it did have an influence on
opioid receptor density. Comparison of the wild-type
mouse cohorts indicated significantly higher levels of
opioid receptor binding in the striatum and cortex of the
FVB/N strain compared to the C57BL/6 strain [P b 0.05
(striatum), P b 0.05 (cortex), unpaired, two-tailed,
Student’s t test]. The presence of the HD mutation
appeared to have similar effects on opioid receptor
binding on both genetic backgrounds (slightly increased
although not statistically significant). By contrast, genetic
background appeared to differentially influence the effects
of the HD mutation on D1 receptor binding in the
striatum. A two-way ANOVA confirmed this interaction
(Table 1). Further work will be required to determine the
precise molecular nature of the influence and whether it
Fig. 1. Distribution of ligand binding sites in representative sections from wild-type mice. Images show levels of total binding in striatum and cerebral cortex at
a representative coronal plane used for densitometric analyses and are adjusted for maximum contrast. (A) 3H-YM09151-2 binding to D2 receptors; (B)3H-
Ro15-1788 binding to benzodiazepine receptors; (C) 3H-SCH23390 binding to D1 receptors; (D)3H-Diprenorphine binding to opioid receptors. Differences in
the densities of bindings sites between wild-type and HD mice could not be discerned on visual inspection of the autoradiograms although the quantitative
analysis showed that there were statistically significant differences.
L. Kennedy et al. / Brain Research 1039 (2005) 14–21 17
represents a bona fide modifier of mutant huntingtin
protein activity.
4. Discussion
The present study demonstrates that 18-month-old
knock-in HD mice expressing full-length mutant huntingtin
appropriately have reduced levels of D2 receptor binding
Fig. 2. Quantitative autoradiographic measurements in wild-type (WT) and
HD mice of 3H-YM09151-2 binding to dopamine D2 receptors (WT n = 14,
HD n = 10); 3H-SCH23390 binding to D1 receptors (WT n = 13, HD n =
11); 3H-diprenorphine binding to opioid receptors (WT n = 15, HD n = 14)
and 3H-Ro15-1788 binding to benzodiazepine receptors (BZ) (WT n = 15,
HD n = 15) in the striatum. Data are mean + SEM; *P b 0.05 WT compared
to HD by Student’s two-tailed t test.
sites in the striatum and increased levels of benzodiazepine
receptor binding sites in the striatum and cerebral cortex.
There is also a trend towards increased opioid binding
densities in the striatum and, to a lesser extent, in the cortex
of the HD mice, although these changes do not reach
statistical significance. All these changes appear to occur
independently of the genetic background (C57BL/6 and
FVB/N) of the mice tested. By contrast, dopamine D1
receptor binding densities in HD striatum are affected by the
genetic background, showing a significant increase in the
C57BL/6 strain and a slight decrease (statistically non-
significant) in the FVB/N strain. The reduced neurotrans-
mitter receptor densities observed may be the result of subtle
cell loss although a previous study has shown that the mice
used in this study do not exhibit significant levels of
neurodegeneration at 18 months of age [38]. However, HD
mice of this age do show significant motor deficits
compared to their wild-type littermates [21]. The increased
levels of benzodiazepine receptor binding observed in the
HD mice argues against cell loss as the GABAA/benzodia-
zepine receptor complex is highly expressed on virtually all
striatal neurones and axon terminals [29,30]. Thus, changes
in neurotransmitter receptor binding in the HD mice may
represent early cellular components of HD pathogenesis.
Medium spiny striatal neurones are selectively affected
early in the disease course. Those projecting to the external
segment of the globus pallidus preferentially express
dopamine D2 receptors, whilst neurones of the direct
pathway to the internal segment of the globus pallidus
preferentially express D1 receptors [13]. Comparison of
dopamine receptor binding levels has been previously
Table 1
Density of receptor binding sites in mice with different genetic backgrounds
Striatum Wild type HD ANOVA
FVB/N C57BL/6 FVB/N C57BL/6 Genotype Genetic background Factor interaction
Dopamine D2 138.9 F 7.0 (5) 124.7 F 5.3 (9) 116.1 F 7.4 (4) 113.0 F 6.0 (6) 0.02T 0.2 0.4
Benzodiazepine 86.1 F 6.7 (6) 88.1 F 3.7 (9) 97.7 F 3.8 (5) 97.7 F 3.35 (10) 0.03T 0.8 0.8
Dopamine D1 208.1 F 17.8 (6) 181.2 F 9.4 (7) 170.9 F 9.9 (5) 212.1 F 2.9 (6) 0.8 0.5 0.007TOpioid 140.5 F 6.4 (6) 112.6 F 8.2 (9) 167.0 F 15.6 (5) 123.9 F 9.8 (9) 0.07 0.002T 0.4
Cortex
Benzodiazepine 225.1 F 10.4 (6) 217.5 F 17.9 (9) 257.9 F 13.8 (5) 262.2 F 5.7 (10) 0.008T 0.9 0.7
Opioid 86.7 F 4.5 (6) 70.2 F 4.9 (9) 95.1 F 3.75 (5) 80.3 F 6.7 (10) 0.1 0.02T 0.9
Data are mean F SEM expressed as fmol/mg tissue; number of animals in parenthesis. FVB/N and C57BL/6 strains are shown for wild-type and HD groups.
ANOVA columns indicate P values.
T P b 0.05 by two-way ANOVA.
L. Kennedy et al. / Brain Research 1039 (2005) 14–2118
employed to investigate temporal patterns of selective
neurodegeneration within the HD brain. Some studies have
concluded that D2- and D1-expressing neurones are affected
in a similar time frame [14,23,40,45], whereas others
conclude that D1 neurones are affected before D2 neurones
[20,35]. Data from the present study (at least from mice on
an FVB/N strain background) are most consistent with
previous studies indicating the D2-expressing striatal effer-
ents are affected before the D1-expressing efferents [4,15],
an observation supported by studies of HD brain tissue
using additional cell-type selective immunohistochemical
markers [33,36,37]. Furthermore, one brain imaging study
showing a decline of both D1 and D2 receptor density in
early HD noted an earlier and greater annual loss of D2
receptors compared to that of D1 receptors in presympto-
matic HD gene carriers [2].
Discrepancies between the human studies mentioned
above may be due to differences in the stage of HD
pathogenesis at which receptor levels were assessed. How-
ever, it is interesting to note that data from the present study
suggest that one or more genetic modifiers may influence the
effect of the HD mutation on D1 receptor binding levels in
mice. A similar scenario in humans may contribute to
Fig. 3. Quantitative autoradiographic measurements of 3H-Ro15-1788
binding to benzodiazepine receptors (BZ) (WT n = 15, HD n = 15) and 3H-
diprenorphine binding to opioid receptors (WT n = 15, HD n = 15) in the
cerebral cortex. Data are mean + SEM; *P b 0.01 WT compared to HD by
Student’s two-tailed t test.
variability within the D1 data set and could help explain the
apparently inconsistent findings.
The results in this study are also consistent previous
reports of progressive dopamine D2 binding deficits in the
striatum of R6 mice [7,8], another mouse model of HD
generated by the random integration of a 5V fragment of the
mutant human HD gene [28]. However, in contrast to our
findings, Cha and colleagues also report a concomitant
reduction in D1 receptor binding in the R6 mice. Unlike the
knock-in HD mice used in this study, the R6 mice have
shortened lifespan and a dramatic symptom profile. The
discrepancy in D1 binding profiles may reflect the fact that
the knock-in mice provide a wider time window through
which to observe temporal patterns of pathological change.
Alternatively, D1 receptor binding profiles could be influ-
enced by other factors that differ between the HD mouse
lines, for example: genetic background, the CAG mutation
length and/or the size of the mutant protein expressed.
It has been suggested that reduced levels of D2 receptors
may play a role in the progression of HD pathology given
the proximity of dopaminergic and glutamatergic synapses
in striatal neurones and the ability of dopamine to modulate
medium spiny neurone excitability [18,26]. D2 receptors,
located either on corticostriatal inputs or postsynaptic sites,
attenuate neuronal responses to glutamate and therefore
reduced levels of D2 receptors could enhance excitotoxicity
in striatal neurones [5,11,24]. Interestingly, both increased
excitatory synaptic activity and enhanced release of
glutamate occurs in dopamine D2 receptor-deficient mice
[6]. Progressive loss of D2 receptors in HD may therefore
contribute to the toxic environment in the striatum that
eventually culminates in neuronal degeneration.
This study also showed that the HD mice had increased
levels of benzodiazepine receptor binding in the striatum
and cerebral cortex. Previous postmortem and imaging
studies of benzodiazepine receptor binding in human HD
cases suggest that, as with other receptor types, variations in
the extent and severity of neuronal loss are likely to exert a
major influence on the level of benzodiazepine receptor
binding observed. Accordingly, postmortem studies have
revealed reductions in GABAA/benzodiazepine receptor
L. Kennedy et al. / Brain Research 1039 (2005) 14–21 19
binding in the striatum of HD patients [10,31,34,44] whilst
brain imaging studies of early-stage HD patients have
reported increased GABAA/benzodiazepine receptor density
in the putamen, despite a reduced dopamine D2 receptor
density [22,32], and decreased benzodiazepine receptor
binding in the caudate nucleus [19,22,32].
Postmortem studies have also demonstrated a ~30–40%
increase in benzodiazepine receptor binding in frontal
cortex, globus pallidus and the substantia nigra pars
reticulata despite brain atrophy [15,31,34,39]. Our mouse
findings are therefore largely consistent with data from
early-stage human HD. As the GABAA/benzodiazepine
receptor complex is highly expressed on most cortical and
striatal neurones and axon terminals [29,30], one might
tentatively speculate that our observations reflect com-
pensatory up-regulation of these receptors by medium
spiny neurones to reduce the excessive firing brought
about by the loss of inhibitory D2 receptors. Similarly,
increased levels of benzodiazepine receptor binding in the
cerebral cortex might represent an attempt to counteract
increased firing of cortical neurones due to basal ganglia
dysfunction.
[3H]-Diprenorphine binding to opioid receptors in the
striatum was slightly, but not significantly higher in the
HD mice compared to wild-type. This contrasts with a
PET study in which [11C]-diprenorphine uptake was
reduced in the striatum of symptomatic HD patients [46].
The discrepancy between this human study and the present
study could most easily be accounted for by significant
degeneration of striatal neurones in the HD patients who
were exhibiting chorea at the time of their scan, compared
to the HD mice that exhibit minimal striatal degeneration
[38]. In addition, as [3H]-diprenorphine binds to all three
of the major opioid receptors (mu, kappa and delta), we
cannot exclude the possibility that the use of this ligand
may mask selective alterations of receptor subtypes in the
HD mice.
How does mutant huntingtin alter neurotransmitter
receptor levels? Previous studies in R6 mice have shown
that decreases in dopamine receptor mRNA levels occur
before detectable changes in the corresponding receptor
binding densities [7,8]. Similarly decreased levels of
dopamine receptor mRNA levels were also observed in
early stage human HD tissue [4]. Subsequent microarray
analyses of R6 HD mouse brains have revealed altered
expression levels of many genes prior to cell loss [27],
reinforcing the view that transcriptional dysregulation may
play a key role in the pathogenic mechanisms of HD [25].
Accordingly, future studies of D2, D1, GABAA/benzodia-
zepine and opioid receptor gene expression in knock-in
HD mice may be informative and would therefore be of
interest. As a final point, the HD mice used in the present
study had been backcrossed onto two different genetic
backgrounds. Our findings indicate that genetic back-
ground can influence certain receptor binding levels in the
presence or absence of the HD mutation. This illustrates
the importance of considering genetic background when
comparing any data obtained from different mouse models
of HD.
In summary, the present study has revealed altered
neurotransmitter receptor binding densities in the striatum
and cortex of HD mice. Differences in the spatial and
temporal profiles of each receptor type studied suggest a
sequence and pattern of neurodegenerative changes that
might inform discussions of early human HD patho-
genesis. Although the mechanistic basis of the observed
changes is not known, elucidating molecular aspects of the
pathogenic cascade that precedes onset of neuronal
degeneration may reveal potential targets for treatment
design in HD.
Acknowledgments
The authors are grateful to Colin Hughes, Dennis
Duggan, Margaret Ennis and staff at the Wellcome Surgical
Institute for technical assistance and The University of
Glasgow Dynamic Mutation Group and Professor J.
McCulloch for helpful discussion. This work was funded
by the Hereditary Disease Foundation. L.K. was supported
by a studentship from The Huntington’s Disease Associa-
tion of Great Britain.
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