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.

E. Kloster et al. / European Journal of Medical Genetics 56 (2013) 416e419 417


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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.

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[32] PITA: http://genie.weizmann.ac.il/pubs/mir07/mir07_prediction.html, miR-NASNP: http://www.bioguo.org/miRNASNP/geneTargets.php.

E. Kloster et al. / European Journal of Medical Genetics 56 (2013) 416e419 419
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