kloster et al. 2013_cnr1
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Experimental research
CNR1 variation is associated with the age at onset in Huntington
disease
Eugen Kloster a,b, Carsten Saft c, Jrg T. Epplen a,b, Larissa Arning a,*
a Department of Human Genetics, Ruhr-University Bochum, Germanyb International Graduate School of Neuroscience, Ruhr-University Bochum, Germanyc Department of Neurology, Ruhr-University Bochum, St. Josef-Hospital, Germany
a r t i c l e i n f o
Article history:
Received 27 February 2013
Accepted 27 May 2013
Available online 7 June 2013
Keywords:
Huntington disease
Modier genes
Age at onset
CNR1
miRNAs
a b s t r a c t
Huntington disease (HD) is caused by the expansion of a CAG repeat within exon 1 of the HTT gene.
Although the variation in age at onset (AO) is partly explained by the length of the expanded repeat
blocks, the unexplained variation in AO is highly heritable, emphasizing the role of modi er genes on
disease expression. Since down-regulation of type 1 cannabinoid (CB1) receptors is a key pathogenic
event in HD, it has been suggested that activation of these receptors in patients may attenuate disease
progression. In order to evaluate whether variations in the cannabinoid receptor 1 ( CNR1) gene encoding
the CB1 receptor protein have modifying effects on the AO of HD, we performed an association study
between CNR1 polymorphisms and AO in HD patients. A (AAT)n repeat and a total of nine single
nucleotide polymorphisms (SNPs) in the CNR1 gene were selected for genotyping in a cohort of 473
German HD patients recruited in the Huntington Center NRW in Bochum. The AO was signicantly
associated with the longest alleles (17 AAT) of the (AAT)n repeat polymorphism downstream of the
CNR1gene (p 0.007) as well as with one SNP in the 30UTR ofCNR1(rs4707436,p 0.05). Interestingly,
the allelic variation of rs4707436 affects different microRNA (miRNA) binding sites which could alter
gene regulation and consequently inuence protein expression. These ndings support the idea that
CNR1variation may have modifying effects on the AO in HD. 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
Huntington disease (HD) is an autosomal dominantly inherited,
neurodegenerative disorder characterized by adult-onset of motor
dysfunctions, psychiatric changes and cognitive decline. The caus-
ative mutation is an expansion of an unstable CAG repeat in therst
exon of the HTTgene that results in an elongated polyglutamine
tract in the huntingtin protein (htt [1],). The neuropathological
signs are most evident within the striatum, a structure of the basal
ganglia involved in motor behavior. The pathological hallmark ofthe disease is a predominant and selective loss of striatal projection
neurons [2] that leads to various motor symptoms, including
choreiform movements, rigidity and abnormal posture[3].
Age at onset (AO) in HD is determined mainly by the size of the
expanded CAG repeat allele,i.e.the longer the repeat is the earlier
the onset of symptoms. This correlation explains about 70% of the
variability in AO, the remaining unexplained variation is highly
heritable, emphasizing the role of modier genes on disease
expression[4]. Up to now, a wide range of HD modier genes have
been examined related to different biochemical pathways like
dysregulation in energy metabolism, altered neurotransmitter re-
ceptor function, htt protein interactions and regulation of gene
expression [5].
Since cannabinoid receptors are highly abundant in the basal
ganglia and play a pivotal role in the control of motorbehavior, theyrepresent candidates as HD modiers. This assumption is further
supported by a range of studies: signicant down-regulation of
type 1 cannabinoid receptor (CB1) binding and messenger RNA
levels has been documented in the basal ganglia of HD patients and
animal models [6]. Interestingly, this down-regulation seems to
occur in advance of other receptor changes at early stages of the
disease and prior to the appearance of overt clinical symptoms
following neurodegeneration [6e8]. The precise pathophysiolog-
ical impact of this loss of receptors in HD is as yet unknown;
however, recent results showed that the mutant htt-dependent
down-regulation of the CB1 receptors involves the control of the
* Corresponding author. Ruhr-University Bochum, Universittsstr, 150, MA5/39,
44801 Bochum, Germany. Tel.: 49 (0) 234 32 23831; fax: 49 (0) 234 32 14196.
E-mail address:[email protected](L. Arning).
Contents lists available at SciVerse ScienceDirect
European Journal of Medical Genetics
j o u r n a l h o m e p a g e : h t t p : / / w w w . e l s e vi e r . c o m / l o c a t e / e j m g
1769-7212/$e see front matter 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmg.2013.05.007
European Journal of Medical Genetics 56 (2013) 416e419
mailto:[email protected]://www.sciencedirect.com/science/journal/17697212http://www.elsevier.com/locate/ejmghttp://dx.doi.org/10.1016/j.ejmg.2013.05.007http://dx.doi.org/10.1016/j.ejmg.2013.05.007http://dx.doi.org/10.1016/j.ejmg.2013.05.007http://dx.doi.org/10.1016/j.ejmg.2013.05.007http://dx.doi.org/10.1016/j.ejmg.2013.05.007http://dx.doi.org/10.1016/j.ejmg.2013.05.007http://www.elsevier.com/locate/ejmghttp://www.sciencedirect.com/science/journal/17697212http://crossmark.dyndns.org/dialog/?doi=10.1016/j.ejmg.2013.05.007&domain=pdfmailto:[email protected] -
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CNR1 promoter by repressor element 1 silencing transcription
factor (REST), a transcriptional repressor of neuronal genes [9].
Under normal condition wild-type htt sequesters REST in the
cytoplasm, resulting in gene-silencing prevention, whereas the HTT
mutation is associated with a loss-of-function in its ability to
regulate the REST [9,10]. Here we sought to determine whether
variation inCNR1, encoding CB1, is associated with AO in HD.
2. Materials and methods
2.1. Study population
We analyzed HD patients with known AO of overt motor
symptoms. The major part of the study population has been
described before [11]. Since extreme CAG lengths could have a
disproportionate impact on the results[12], we restricted our main
analyses on individuals with repeats range of the 40 e 54 CAGs
(n 473). However, in order to provide an additional impression on
the inuence of the potential modier variations on extreme CAG
lengths, the calculations have also been performed on a larger
cohort including CAGs up to 73. The study was performed in a
manner that fully complies with the Code of Ethics of the World
Medical Association (Declaration of Helsinki) and was approved bythe ethics review board of the Ruhr-University Bochum (Germany).
2.2. Genotyping
Genotyping was performed by PCR-RFLP techniques. For frag-
ment analysis of the (AAT)ntriplet repeat polymorphism we used
uorescence 50FAM labeled, tailed oligonucleotide added to the 50-
part of the sequence specic primer as described before [13]. All
primers were designed with the Primer Express 2.0 Software
(Applied Biosystems, Foster City, USA). All other details of the
methodology and primer sequences are available upon request.
2.3. Statistical analysis
Variability in AO attributable to CAG repeat number was
controlled by linear regression using the logarithmically trans-
formed AO as the dependent variable and the genotypes as inde-
pendent variables. All analyses were performed assuming a
dominant or an additive effect for each polymorphism. In the
dominant model, both, the heterozygous and the rarely observed
homozygous variation were combined. In the additive model, both,
rare homozygous and heterozygous variation effects were esti-
mated using two dummy variables.
The (AAT)ntriplet repeat was tested as a continuous trait, based
upon the larger of the two alleles present in each patient. Addi-
tionally, tests were performed for presence of each individual allele
and at least one of the two longest alleles (17 or 18 AATs). SPSS
Ver.21.0 (SPSS Inc.) was used for all statistical analyses with nom-inal signicance assigned when p 0.05. HardyeWeinberg equi-
librium (HWE) was tested for each SNP. The D 0 values for each pair
ofCNR1 markers were calculated and visualized through the pro-
gram Haploview 3.0[14]. The Probability of Interaction by Target
Accessibility (PITA) algorithm and miRNASNP analyses were used
for miRNA target site prediction in the CNR130UTR[15,16].
3. Results
In the cohort of 473 unrelated German patients (233 men and
240 women) the expanded CAG blocks ranged from 40 e 54 and
accounted for nearly 68% of the variance in motor AO (R2 0.675,
p < 0.0001). Potential modier effects were tested by determining
whether adding the different CNR1 genotypes has a signi
cant
impact on a linear regression model relating the natural log-
transformed AO to the CAG repeat length. In our cohort, the CNR1
(AAT)nrepeat displayed 10 alleles, ranging from 9 to 18 AATs. Most
frequently the (AAT)12allele was observed (30.9%), followed by the
(AAT)16allele (27.6%), the (AAT)15 allele (16.9%) and the (AAT)14allele (16.4%). (AAT)17 repeats were relatively rarely (2.9%)
encountered as well as those of other lengths (0.0001
rs6454674 128/256/89 0.17
rs806380 221/206/46 0.55
rs1049353 238/184/51 0.59
rs4707436_012 248/188/37 0.677 0.002 0.6 0.05
rs4707436_011 248/ 225 0. 677 0.002 0.6 0.05
rs12720071 389/80/4 0.68
rs806368 238/216/19 0.44
rs806366 127/230/116 0.14
rs7766029 121/256/96 0.18
rs806365 140/238/95 0.38
CNR1(AAT)n 17 445/28 0.679 0.004 1.2 0.007
The variability in motoric AO attributable to the CAG repeat length was assessed by
linear regression using the logarithmically transformed onset age as the dependent
variableand genotypes as independent variables. Delta (D)R2 quanties the relative
improvement of the regression model when the genotypes are considered in
addition to the CAG repeats.
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hand, CB1 receptor deletion in HD mouse models aggravates the
symptoms of the disease[9,21].CNR1has therefore been chosen ascandidate gene in order to investigate the impact of naturally
occurring variation in this gene on the AO. Numerous association
studies were published, and variation in CNR1 has been associated
with a wide range of phenotypes like alcohol and substance use
disorders, schizophrenia, depression and Parkinson disease [22,23].
Since there is no evidence that the (AAT)n repeat itself has any
functional impact, this polymorphism may rather be in LD with
other functional variations in CNR1 or in not yet identied brain-
specic isoforms, like it is postulated for PPARGC1A[24].
Further analyzing the most frequent SNPs in CNR1 revealed a
weakerassociation with rs4707436 in the 30UTR. Since SNPs located
in this region may affect miRNA binding sites, we analyzed
rs4707436in silicofor its ability to modify miRNA binding sites and
thus gene regulation via miRNAs. Rs4707436 G allele appears as a
putative miRNA binding site for hsa-miR-652, thus suggesting a
functional role for this variation in mRNA expression ofCNR1. The
PITA algorithm, a thermodynamic model of miRNA binding, sup-
ports allelic differences. PITA models miRNA targeting as a
competition between the free energygained by miRNA binding and
the energetic cost of displacing existing RNA secondary structure at
the target site [15]. The predicted regulatory strength is thereby
expressed in the form of an energy-based score termed DDG, where
lower values indicate stronger miRNA binding. Analyzing the sur-
rounding sequence of the rs4707436 G/A alleles with PITA revealed
DDGvalues of13.48 and 8.72, thus suggesting reduced binding
of hsa-miR-652 to CNR1 for the minor A-allele. Conversely, the A
allele creates new target sites for hsa-miR-183 and hsa-miR-767-
5p, which are absent in the more common G allele [16]. MiRNAs
are potent regulators of gene expression and thus involved in abroad range of biological processes. SNPs within human miRNAs
sequences have been shown to have impact on various phenotypes
like the Parkinson disease, Friedreich ataxia, schizophrenia,
aggressive human behaviors and various types of cancers [25e30].
Thus, we submit that variation in CNR1 may be associated with
altered miRNA binding heralding AO modifying effects in HD.
However, the underlyingndings warrant independent replication
and further investigations since HD modier studies, like all asso-
ciation studies, are susceptible to various faults[12,31].
Conict of interests
The authors declare that they have no competing interests.
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Fig. 1. Graphical representation of the CNR1 polymorphisms in relation to the exoneintron structure (top) and the Haploview pairwise linkage disequilibrium (LD) structure
(bottom) of part of the gene. The Exon is indicated by solid black boxes, and the numbered vertical lines indicate positions of the variations analyzed inCNR1. The Haploview plot
shows the pairwise LD (D0 values) for all 9 SNPs and the (AAT)nrepeatsn17 based on genotypes of 473 HD patients of the study. Each square plots the level of LD between a pair of
SNPs, comparisons between neighboring SNPs are arranged along the rst line under the names of the SNPs. Dark red coloring indicates strong LD, medium red shading indicates
less strong LD, light red indicates intermediate LD and white indicates weak LD.
E. Kloster et al. / European Journal of Medical Genetics 56 (2013) 416e419418
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E. Kloster et al. / European Journal of Medical Genetics 56 (2013) 416e419 419
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