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Ashdin Publishing
Journal of Drug and Alcohol Research
Vol. 2 (2013), Article ID 235714, 11 pages
doi:10.4303/jdar/235714
ASHDIN
publishing
Review Article
Cannabinoid Receptor Gene Variations in Drug Addiction and
Neuropsychiatric Disorders
E. S. Onaivi,1,3 H. Ishiguro,2S. Sgro,1and C. M. Leonard1
1Department of Biology, College of Science and Health, William Paterson University, 300 Pompton Road, Wayne, NJ 07470, USA
2Department of Neuropsychiatry and Clinical Ethics, Graduate School of Medical Science, University of Yamanashi, Ch¯
u¯
o-shi,
Yamanashi 409-3898, Japan
3National Institute on Drug Abuse (NIDA), National Institutes of Health (NIH), 251 Bayview Blvd., Baltimore, MD 21224, USA
Address correspondence to E. S. Onaivi, onaivie@wpunj.edu
Received 5 March 2013; Revised 10 August 2013; Accepted 12 August 2013
Copyright © 2013 E. S. Onaivi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract Cannabinoid receptors (CBRs) are involved in neuropsy-
chiatric disturbances including drug addiction. Studies show that
single nucleotide polymorphisms (SNPs) of CNR1 and FAAH may
contribute to drug addiction and other neuropsychiatric disorders.
However, cannabinoid type-2 receptors (CB2Rs) in the CNS and their
role in drug addiction and neuropsychiatric disorders have been much
less well characterized. Features of CBR gene structures and their
variants in drug abuse and neuropsychiatric disorders and in rodent
models were studied. Association studies were performed between
polymorphisms in CNR2 gene and neuropsychiatric disorders in two
independent case-control populations. We identified novel human and
rodent CB2R isoforms with differential tissue expression patterns and
regulation by CBR ligands. There is association between polymor-
phisms of CB2R gene and neuropsychiatric disorders investigated with
increased risk of schizophrenia, depression, drug abuse, and eating
and autism spectrum disorders in low CB2R function. CBR variants
may provide a deeper insight and novel targets for the effects of
cannabinoids in drug addiction and other neuropsychiatric disorders.
Keywords cannabinoid; CNR2 gene; variants; drug addiction; neuro-
psychiatry
1. Introduction
The ubiquitous cannabinoid receptors (CBRs)—probably
the most abundant binding sites in the CNS—are known to
be involved in a number of neuropsychiatric disturbances
including drug addiction. CBRs are coded in human
chromosomes 1 and 6 and activated by endocannabinoids,
phytocannabinoids and marijuana use (medical/recreational
use). The components of the endocannabinoid system
(ECS) include CNR1 and CNR2 genes encoding these
CBRs (CB1Rs and CB2Rs), endocannabinoids (eCBs),
and their synthesizing and degradation enzymes (Table 1)
which are major targets of investigation for their impact in
neuropsychiatry. The discovery that specific genes, codes
for CBRs are activated by marijuana use and that the
human body makes its own marijuana-like substances—
endocannabinoids [67,74], that also activate CBRs have
provided surprising new knowledge about endocannabinoid
system. Our remarkable new understanding indicates that
the cellular, biochemical, and behavioral responses to
marijuana, which remains one of the most widely used
and abused drugs in the world, are coded in our genes
and chromosomes. With increasing new information from
the decoding of the human genome, many aspects of
genetic risk factors in marijuana use including age of
initiation, continuation, and problem use undoubtedly will
interact with environmental factors such as availability
of marijuana along with the individual’s genotype and
phenotype. These remarkable advances in understanding
the biological actions of marijuana, cannabinoids, and
endocannabinoids are unraveling the genetic basis of
marijuana use with implication in human health and disease.
The two well-characterized cannabinoid receptors, CB1Rs
and CB2Rs, are encoded by CNR1 and CNR2 genes
that have been mapped to human chromosomes 6 and 1,
respectively (Figures 1and 2). A number of polymorphisms
in cannabinoid receptor genes have been associated with
human disorders including ADHD and PTSD [56], drug
dependency [71], obesity [17,43], depression [71,85]
and other neuropsychiatric disorders (see Table 2). Thus,
because of the ubiquitous distribution and role of the
endocannabinoid system in the regulation of a variety
of normal human physiology, drugs that are targeted to
different aspects of this system are already benefiting cancer
subjects and those with AIDs and metabolic syndromes [43].
In the coming era of personalized medicine, genetic variants
and haplotypes in CNR1 and CNR2 genes associated with
obesity or addiction phenotypes may help identify specific
targets in conditions of endocannabinoid dysfunction. Our
previous investigations had defined a number of features of
the CNR1 gene’s structure, regulation, and variation [102],
2Journal of Drug and Alcohol Research
Table 1: Subtypes of cannabinoid receptors.
CB1-R CB2-R
Amino acids 472 AA 360 AA
Chromosome location 6q14-q15 1p34-p35
Gene name CNR1 CNR2
Endogenous ligand 2-AG 2-AG
CNS distribution Yes Yes
Peripheral distribution Yes Yes
Subtypes∗CB1, CB1A-CBin CB2A and CB2B
∗See text for isoforms and variants of CB1 and CB2 receptors.
CBin and n=A–E variants.
but many features of CNR2 gene structure, regulation,
and variation still remain poorly defined. However, we
and others have now demonstrated and reported that
variants of the CNR1 gene are associated with a number
of disorders and substance abuse vulnerability in diverse
ethnic groups including European-American, African-
American, and Japanese subjects [6,18,32,33,34,35,102].
Most strikingly, variants of CNR genes co-occur with
other genetic variations and share biological susceptibility
that underlies comorbidity in most neuropsychiatric
disturbances [8]. Thus, emerging evidence indicates that
the endocannabinoid system exerts a powerful modulatory
action on retrograde signaling associated with inhibition of
synaptic transmission [75]. Interestingly a role for variations
in CNR1 gene has been associated with striatal responses
to happy but not to disgust faces [11] with implication that
functional variation of CNR1 genotypes may be associated
with disturbances of the brain involving emotional and
social stimuli, such as autism [11] and depression [19,70].
Here we review and present additional data that focuses
on these recent advances in cannabinoid genomics and the
surprising new fundamental roles that the ECS plays in the
genetic basis of marijuana use and cannabinoid pharma-
cogenomics [67], and pharmacotherapeutics. The powerful
influence of cannabinoid-induced retrograde signaling
modulates GABAergic and glutamatergic systems, which
indicates that the main excitatory and inhibitory systems
are in part under the influence of the endocannabinoid
system. Thus, the genetic basis of compulsive marijuana use
may involve an interaction of CNR genes with other genes
and environmental factors. As with other dependences
with genetic risk factors, the risk for marijuana use is
likely to be the result of CNR genes and other genes and
environmental factors, each contributing a small fraction
of the overall risk [94]. Additional evidence is provided
for the complex CNR1 and CNR2 gene structures and their
associated regulatory elements. In our current ongoing
studies, many features of CNR gene structures, single
nucleotide polymorphisms (SNPs), copy number variations
(CNVs), CPG islands, microRNA regulation, and the impact
of CNR gene variants in neuropsychiatry and where possible
in rodent models are assessed. Although CNR1 gene has
(a)
(b)
Figure 1: Human CB2 (CNR2, 1p36.1) genomic structure
and alternative spliced transcripts: (a) The gene size is
marked in bp; vertical bars represent exons; triangles rep-
resent splicing patterns; arrows represent nonsynonymous
SNPs. (b) CB2R subtypes, hCB2A and hCB2B, alterna-
tively spliced variants are shown under the gene structure.
more CPG islands than CNR2 gene, both have CPG islands
less than 300 bases, and may also be regulated by DNA
methylation amongst regulatory mechanisms. MicroRNA
binding to the 3untranslated region of the CNR1 gene
with two polyadenylation sites may also potentially regulate
CB1R expression. CNR1 gene has 4 exons and there are 135
SNPs reported in more than 1% of the population with no
common SNP that changes amino acids of CB1R currently
known or reported. A copy number variant (CNV) which
is 19.5 kb found in 4 out of 2,026 people covers exons 3
and 4 and codes amino acid that could alter the expression
of CB1Rs. CNR2 has 4 exons with CB2A with 3 exons
and CB2B with 2 exons; and there are about 100 SNPs
found in more than 1% of the population, which include
common cSNPs that change amino acids of the CB2R,
including R63Q, Q66R, and H316Y. CNVs in Asian and
Yoruba population have been reported. Therefore, studying
the CBR genomic structure, its polymorphic nature, subtype
specificity, their variants, and associated regulatory elements
that confer vulnerabilities to a number of neuropsychiatric
disturbances may provide a deeper insight in unraveling
the underlining mechanisms, as discussed below. Thus,
understanding the ECS in the human body and brain will
contribute to elucidating this natural regulatory mechanism
in health and disease.
2. Variation in cannabinoid receptor genes in drug
addiction and other neuropsychiatric disorders
While the expression of CBRs in humans varies according
to ethnicity and gender [69], variations in other mam-
malian species are also notable. Therefore, a number of
Journal of the International Drug Abuse Research Society 3
Table 2: Genetic polymorphisms of cannabinoid receptor genes (CNR genes).
CNR genes polymorphism Linkage or association References
CB1, Two allele DNA polymorphism Associated with CNR1 gene [66]
CNR1 rs16880261 Associated with cannabis dependence [6]
CNR1 rs4707436 Associated with endocannabinoid effects [6]
CNR1 rs806377 Associated with endocannabinoid effects [6,33]
CNR1 rs1049353 Associated with addictive disorders [6,9,18]
CNR1 rs2023239 Associated with endocannabinoid effects [6,18,21]
CNR1 rs12720071 Associated with endocannabinoid effects [6,18]
CNR1 rs806375, rs806371, rs806368 Associated with drug addiction [16,103]
1359 G/A CNR1 variant Associated with alcohol dependence [22,84]
1359 G/A CNR1 variant Not associated with Tourette syndrome [23]
1359 G/A CNR1 variant Not associated with alcohol withdrawal tremens [80]
1359 G/A CNR1 variant Associated with weight loss [1,2]
3813 A/G and 4895 A/G variant Associated with obesity in men [83]
CNR1 SNPs Not associated with obesity in German children [59]
CNR1 SNPs Associated with obesity and BMI [10,24,42,77]
CNR1, FAAH, DRD2 gene Associated with comorbidity of alcoholism and antisocial [32]
(AAT)nrepeat of CNR1 gene Conflicting associations with drug dependence [28,43]
CNR1 variants, SNPs, “TAG” haplotype Associated with polysubstance abuse [102]
CNR1 SNPs Not associated with polysubstance abuse [30]
CNR1 SNPs Associated with cannabis dependence [3,4,5]
CBR haplotype Associated with fewer cannabis dependence symptoms [33,34,35]
CNR1 SNPs Associated with alcohol and nicotine dependence [13,37]
CNR1 SNPs No association with anorexia nervosa [59,60]
CNR1 (AAT)nrepeats Associated with restricting and binging/purging anorexia nervosa [86]
CNR1 (AAT)nrepeats Associated with depression in Parkinson’s disease [8]
CNR1 SNPs Associated to striatal responses to facial exp [11]
(AAT)nrepeats Association with ADHD in alcoholics [56,79]
CNR1 SNP haplotype Risk factor for ADHD and PTSD [56]
1359 G/A CNR1 variant Associated with schizophrenia [51]
(AAT)nrepeats Not associated with schizophrenia and mood disorders [52,92,93]
(AAT)nrepeats Associated with schizophrenia [57]
(AAT)nrepeats Associated with hebephrenic schizophrenia [12,95]
CNR1 variants Associated with depression and anxiety [19]
CNR1 variants and (AAT)nrepeats Associated with impulsivity [20]
1359 G/A CNR1 tag SNP Associated with antipsychotic response but not schizophrenia [27]
CNR1 SNPs No association with cognitive impairment in MS [99]
CB2, CNR2 SNPs and haplotypes Associated with human osteoporosis [47]
CNR2 SNPs Not associated with cardiovascular risk factors [82]
CNR2 SNPs Associated with bone mass [100]
CNR2 (Q63R) SNP Risk factor for autoimmune disorders [87]
CNR2 (Q63R) but not (H316Y) Associated with alcoholism and depression [40]
CNR2 (rs41311993) Associated with bipolar disorder [58]
confounding factors and disparities arise in different studies
due to the variations in human CBRs dependent on gender
and ethnicity. A number of variations have been found in
genes associated with the ECS including those encoding
the CBRs, and those involved in the synthesizing enzymes
of endocannabinoids including fatty acid amide hydrolase
(FAAH) and metabolizing enzymes like diacylglycerol
lipase alpha (DAGLA). There are a number of reported
mutations in the genes associated with the ECS that lead
to altered mRNA stability and transcription rate with
modification of the encoded proteins. These functional
variations have been associated in a number of studies and
meta-analysis with neuropsychiatric disturbances (Table 2).
We and others have reported that the human CB1R have
a number of splice variants, which may in part account
for the myriad behavioral effects of smoking marijuana.
Up to five isoforms including the canonical/long and
short isoforms are known to be produced by alternative
splicing of the CNR1 transcript [102]. Some effects of
marijuana and other cannabinoids may include actions
at CB2Rs that have received much less attention than
CB1Rs. However, we and others have now identified
4Journal of Drug and Alcohol Research
Figure 2: CNR1 gene structure showing 4 exons with some introns. A number of ESTs have been identified and some of the
SNPs discussed and in Table 1are shown. The CNR1 gene is in human chromosome 6q15. The currently identified structures
of CNR1 isoforms are indicated.
and characterized glial and neuronal CB2Rs in the brain.
Nonetheless, many features of the CNR2 gene structure,
regulation, and variation remain poorly characterized
compared to the CNR1. In humans, the CNR2 gene is
reported to consist of a single translated exon flanked by
5and 3untranslated regions and a single untranslated
exon [87] (see Figure 2). Most regions of the CNR2 gene
are highly conserved, but the human has glutamine at
position 63 instead of arginine [47,87] and another SNP
H316Y has been reported and linked to autoimmune
disorders [47,87]. There has been little or no data on the
role of CB2Rs in neuropsychiatric disorders. However,
in neurological disorders associated with inflammation,
the expression of CB2Rs has been reported in limited
populations of microglial including plaque-associated glia
in Alzheimer’s disease brains [64,76]. Indeed our studies
provide the first evidence for a role of CB2Rs in depression,
schizophrenia, and substance abuse [39,40,66,71,72]. We
and others have identified splice variants of the human
CB1Rs and CB2Rs but they have thus far been poorly
characterized for functional specificity apart from the
broad roles associated with CB1R and CB2R subtypes.
Alternative splicing of RNAs appears to be more common
than previously thought in people, and can generate a variety
of proteins, with most genes producing at least two variants.
The characterization of CBR variants will add validity
to the functional evidence for the existence of multiple
cannabinoid receptor subtypes. It has been demonstrated
in vitro that amino-terminal processing of the hCB1R may
involve a rapid N-terminal truncation in the cytoplasm prior
to translocation to the endoplasmic reticulum membrane.
It was suggested that such a truncation process might be a
way to create a novel type of CB1R isoforms but exactly
how the truncated CB1R may be formed and how the
processing is regulated remains to be determined [63]. In
comparison to the monoaminergic system, the application
of modern techniques to cannabinoid research is new. For
example, molecular cloning has revealed the presence of
serotonin (5-hydroxytryptamine; 5-HT) receptor subtypes,
which can be subdivided in seven subfamilies [25,94]
serotonin (5-HT) receptor subtypes and growing. New
knowledge on cannabinoid post-transcriptional and post-
translational modifications, such as alternate splicing and
perhaps RNA editing, may indicate formation of multiple
proteins that could unravel specific mechanisms associated
with numerous behavioral and physiological effects of
marijuana use. The cloning and sequencing of CNR1 gene
from 62 species has also been reported [61] and awaits
full characterization. As predicted here, the identification
and characterization of these putative CBR isozymes and
different elements of the ECS may reveal novel targets for
medication development. However, the limitless signaling
capabilities and the endless complexity of the cannabinoid
system require a continuous intensive investigation. Specific
genetic variants and polymorphisms in multiple genes
including variations in the ECS genes have been associated
with neuropsychiatric and other pathophysiology of human
diseases [97]. It is to be noted that depending on the nature
Journal of the International Drug Abuse Research Society 5
of classification, other CBRs exist. The vanilloid receptor 1
(VRI), the site at which capsaicin in hot chili peppers acts, is
a site at which anandamide is a full agonist. As anandamide
is a partial agonist at the CBRs, some have suggested that
VR1 be classified as a CBR subtype. .. may be CB3. In
fact, the endocannabinoid that is a full agonist at the CBRs
is 2-Arachidonyl glycerol (2-AG) [26,90,91]. Another
putative CBR, GPR55, has been suggested as a CBR that
increases intracellular calcium and inhibits mM current [49].
However, using a strategy for defining cannabinoid receptor
functional fingerprints from mutagenesis and molecular
recognition literature data, it was noted that hGPR55 does
not appear to share a similar fingerprint with the hCB1R and
hCB2R [78]. While this could not be considered as a proof
to exclude GPR55 from the CBR family, the data from other
studies strongly suggest that GPR55 is a specific functional
receptor for lysophosphatidylinositol receptor [29,65]. Thus
far, it appears that GPR55 is quite distinct from other GPCRs
and represents an intriguing and unique therapeutic target
whose functional receptor requires a further validation and
characterization [29]. The implication of variations in other
putative CBR genes will certainly contribute to unraveling
of the genetic basis of the ECS in neuropsychiatric
disorders. We are mainly concerned here with the variations
associated with CNR genes. However, a number of putative
endocannabinoids have been identified and anandamide and
2-AG are better characterized. These endocannabinoids are
known to act as retrograde messengers and are released
on demand and undergo enzymatic hydrolysis. While 2-
AG is metabolized by monoglyceride lipase (MGL) and
cyclooxygenase-2 (COX2), anandamide is metabolized by
FAAH and N-acylethanolamine acid amidase (NAAA). The
FAAH1 gene is located on human chromosome 1p35-34
and FAAH2 gene, recently identified, has been mapped to
chromosome Xp11.21 or Xp11.1, while MGL gene is on
3q21.3. The results of studies conducted thus far on the
polymorphisms and haplotype blocks in endocannabinoid
metabolizing enzymes and neuropsychiatric disorders
appear to vary due to disparities and confounding factors
associated with ethnicity, gender, and phenotypes of the
population studied [41,55]. Firm conclusions on the role(s)
of variations and polymorphisms in endocannabinoid
metabolizing enzymes in neuropsychiatry and their
diagnostic value and use in pharmacogenomics needs
more study.
3. CNR1 and CNR2 gene variations in drug addiction and
other neuropsychiatric disorders
CBRs and especially CB1Rs have been described as one
of the most abundant binding sites in the human brain and
many studies have focused on the CNR1 gene variants
in neuropsychiatric disturbances. Hence CNR1 gene is a
candidate for association and linkage studies not only in
the effects of substance abuse and addiction but also with
other neuropsychiatric disorders. However, polymorphisms
in CNR2 gene in neuropsychiatry gained less attention as
CB2Rs were previously thought to be mainly expressed
in immune cells and not expressed in neurons contrary to
new research [38,54,66,67,68,70,71,72,73]. To date many
CNR1 variants have been studied and implicated in different
populations for their impact on a number of neuropsy-
chiatric disorders including substance abuse and addic-
tion, depression, schizophrenia, anxiety, ADHD, PTSD,
impulsivity, neurological disorders including Alzheimer’s,
Parkinson’s Huntington’s, Multiple Sclerosis, Amyotrophic
lateral sclerosis and more (Table 2). Earlier studies on
CNR1 gene variations were on the triplet repeat polymor-
phism (the (AAT)nrepeats) and on the nonsynonymous
1359 A >G polymorphism (rs1049353). For the (AAT)n
triplet repeat polymorphism, and with other variants studied,
caution is required as neuropsychiatric disorders appear to
vary due to disparities and confounding factors associated
with ethnicity, gender, and phenotypes of the population
studied [41,55]. These initial studies found associations
of these variants with schizophrenia, P300 event-related
potentials and substance dependence [14,23,45,51,84].
In our previous mapping of the CNR1 gene locus [102],
we conducted association studies between polymorphisms
and haplotype-specific expression patterns in three human
populations. Common human CNR1 variants assessed
in this study reveal patterns of linkage disequilibrium in
European- and in African-American populations. It was also
shown that a 5CNR1 “TAG” haplotype displays significant
allelic frequency differences between substance abusers
and controls in European-American, African-American,
and Japanese samples [102]. In a review and meta-
analysis of study conducted on three of the most studied
CNR1 gene polymorphisms, rs1049353, rs806379, and the
(AAT)nin addictive disorders, it was reported that only the
(AAT)nrepeats (n>16) in the Caucasian population were
significantly associated with substance dependence [9].
However, specifically the rs1049353 SNP in the CNR1 gene
was found to be associated with heroin addiction only in
Caucasian population [81]. While some polymorphisms in
the CNR1 gene have been associated with some aspects of
drug abuse and addiction such as the (AAT)ntriplet repeat,
rs64546774, rs1049353, and rs806368 (Table 2); many other
polymorphisms were not replicable probably due to various
confounding and co-morbidity factors in the different
studies. As CB2Rs were previously thought to be expressed
in immune cells and referred to as peripheral CB2Rs, the
functional neuronal expression and its variants were less
investigated for roles in neuropsychiatric disorders. Indeed
our studies from mice to human subjects provided the
first evidence for a role of CB2Rs in depression, eating
disorders, autism substance abuse [40,66,71,72], and other
6Journal of Drug and Alcohol Research
neuropsychiatric disorders. CNR2 has 4 exons with CB2A
with 3 exons and CB2B with 2 exons; and there are about
100 SNPs found in more than 1% of the population, which
include common cSNPs that change amino acids of the
CB2R, including R63Q, Q66R, and H316Y. CNVs in Asian
and Yoruba population have been reported. Association
studies were also performed between polymorphisms in
CNR2 gene and schizophrenia [38], eating disorders [38,
39], depression [40,66,70,72], and alcoholics [40,41]in
two independent case-control populations. We also report
on the identification of novel human and rodent CB2R
isoforms, their differential tissue expression patterns and
regulation by CBR ligands. There are associations between
polymorphisms of CNR2 gene and the neuropsychiatric
disorders investigated. Our findings also indicate increased
risk of schizophrenia, depression, drug abuse, and eating
and autism spectrum disorders in low CB2R function and
polymorphisms in CNR2 gene associated with disease
type, ethnicity, and gender. In an Italian population using a
case control study, the association of bipolar disorder was
investigated with three missense SNPs of CNR2 gene [58].
Genetic association between bipolar disorder and 524 A >C
polymorphism was reported and the investigators suggested
that the CB2R may play a role in bipolar disorders. With the
significant association of marijuana use and cannabinoids in
modulating the physiological effects of the ECS, the CNR1
gene has been investigated not only in food intake and the
current obesity epidemic worldwide, but also in a number
of neuropsychiatric problems. Many studies have also
demonstrated CNR1 gene polymorphisms and haplotype
blocks to investigate a number of parameters associated
with eating disorders and obesity [55,97]. Human CNR1
gene polymorphisms associated with eating disorders are
presented in Table 2. Marijuana and cannabinoid induced
psychoactivity is well documented in animal and human
studies, and both CNR1 and CNR2 gene polymorphisms
have been associated with psychosis, multiple sclerosis,
depression, bipolar and ADHD disorders (Table 2). We and
others have studied haplotype blocks in both the CNR1 and
CNR2 genes in human population and disease and addiction
vulnerability [31,41,102].
4. CNVs of cannabinoid receptor gene
A copy number variation (CNV) is a structural variation
in the genome when the number of copies of a gene(s)
varies in the population and this is a source of diversity
and uniqueness between the genomes of individual
humans [101]. These structural variations accounting
for about 12% of human genomic DNA alterations result
in the cell with abnormal copies due to insertions, deletions
or duplications [96]. Normally in the human genome, we
inherit one copy from each parent, but the copy number
varies from two to several copies for some genes. Following
the completion of the human genome sequence, recent
evidence indicates that chunks of DNA and gene(s) can vary
in copy-number (with duplications and/or deletions) and
in some rare instances the gene(s) may not be expressed.
Such CNVs may have functional implications in gene
dosage imbalances by loss or gain in the level of gene
expression [48], and contribute to various complex human
diseases. When CNVs alter the dose of genes critical for
normal brain development and adult brain functioning, they
may cause severe disorders such autism and schizophre-
nia [88]. But the vast majorities of most CNVs are harmless
and impact human health when they alter gene expression
or change gene dosage [88]. Significant advances have been
made in mapping gene variations due to SNPs which were
previously thought to be the most prevalent form of genetic
variations. With advances in genomic technologies, analyses
of CNVs of individual human genomes have been identified
as a major cause of structural variations in those genomes
that are more than the changes caused by SNPs [48]. Indeed
the HapMap project shows that CNVs encompass more
nucleotide content per genome than SNPs, underscoring
CNV’s significance to genetic diversity [89]. It is important
to study CNVs that encompass genes involving duplication
and deletions of sequences and their role in human health,
disease, pharmacotherapeutic and pharmacogenomic
responses. It turned out that CNVs are an important form of
human genetic variation, contributing more than SNPs to the
number of bases differing between human genomes [44].
While CNR1 and CNR2 SNPs have been associated with
a number of neuropsychiatric disorders (Table 2), it is
still unclear to what extent CNR gene CNVs are involved in
neuropsychiatric disorders. Numerous CNVs have now been
identified with various genome analysis platforms [101].
In our studies, many features of CBR gene structures,
SNPs, CNVs, CPG islands, microRNA regulation, and the
impact of CNR gene variants in neuropsychiatry and where
possible in rodent models are assessed. A copy number
variant (CNV) which is 19.5 kb found in 4 out of 2,026
people covers exons 3 and 4 and codes amino acid that
could alter the expression of CB1Rs. For example CNVs
in Asian and Yoruba population have been reported. In our
preliminary CNR2 gene CNV studies, we analyzed one of
the CNV regions located in intron of the CNR2 gene in a
human population of Japanese alcoholics DNA samples in
comparison to non-alcoholic controls. The CNVs in CNR2
gene region were confirmed to be relatively common in
10 out of 420 Japanese people [data not published]. It was
difficult to make a conclusion from the high CNVs of the
CNR2 gene in alcoholics; and more alcoholic DNA samples
and samples from other neuropsychiatric disorders and in
other ethnic populations should be analyzed to understand
and determine the nature of elevated copy numbers of CNR2
gene in neuropsychiatric disease risk. Whether the larger
Journal of the International Drug Abuse Research Society 7
CNR2 gene CNVs in Japanese alcoholics compared to non-
alcoholics are associated with the disease is unknown and
the phenotypic effects are often unclear and unpredictable,
with larger CNVs [48,89]. However, the bigger the CNV,
the more likely it will cause a change in gene dosage [88].
Therefore, the underlying pathogenic mechanism for the
larger CNR2 gene CNV obtained in the sample analyzed in
the alcoholics is currently unknown.
5. Consequences of CNR1 and CNR2 variants
Many CNR gene SNPs and their role in predisposing to
disease have been well documented and studied (Table 1),
but studies on CNR gene CNVs have been less studied
and our understanding of the functional impact of CNVs
in neuropsychiatry is still limited [44]. Many studies have
focused on analysis of regions in the human genome that
vary in copy number in specific disorders, but others have
focused on analysis on regions of which the copy number
never seems to vary in the general population [44]. With
such a strategy, significant associations between some
copy number stable regions have been identified in some
patients with intellectual disability or autism, but not in
controls [44]. It was therefore proposed that copy number
stable regions can be used to complement maps of known
CNVs to facilitate interpretation of patient data [44].
Overall, some CNVs, which may be either inherited or
caused by de novo mutations, have been shown to explain
some of the genetic contribution to common diseases
and may also explain rare uncharacterized disorders [44,
98]. Other factors associated with consequences of CNVs
include whether the copy number variant changes the
sequence or relative location of specific segments of
genomic DNA that act as enhancers or suppressors of
gene expression [15,44]. The higher the number of CNR
gene CNVs and the length of the CNR1 trinucleotide, the
higher the AAT repeats may be associated with aberrant
CNR gene expression and probably modify cannabinoid
induced biological function. CNVs which are a highly
prevalent form of genomic variation can also depend on the
phenotypic and cellular context, and on the environmental
background [15,98]. For example, CNVs in chromosomes
6q14.1 and 5q13.2 have been reported to be associated with
alcohol dependence [53]. The endocannabinoid system is
involved in neuropsychiatric disorders and CB1Rs appear
to be the most abundant binding receptor protein in many
brain regions. A number of CNR1 gene SNPs (Table 2)
are involved in many neuropsychiatric conditions. CNR1
and CNR2 gene polymorphisms are also associated with
the effects of drugs of abuse and addiction and withdrawal
process. The clinical consequences of CNV in the coding
and non-coding CNR gene sequences associated with
human phenotypes and disorders are unknown, but with new
microarray and sequencing technologies, the (epi)genetic
contributions to CNR2 gene CNV can be determined. With
advances in genomic technologies and the analysis and
identification of CNR gene CNVs we may uncover the
relationship between CNR gene CNVs and phenotype and
disease. A significant progress in understanding the nature
of CNVs in the human genome has been achieved, but not
yet extended to CNR gene CNVs apart from our pilot study
described above. Yet accumulating evidence suggests the
importance of CNVs in the etiology of neuropsychiatric
disorders [36]. More studies are needed to determine the
role and contribution of CNR gene CNV to conditions
of endocannabinoid system disorders. We do not know
if CNR gene CNVs will affect the entire subtype CNR
genes and function and whether this may be a factor
with marijuana use as medicine or in the biological
effects after smoking marijuana and the propensity for
its addictive potential in humans. But precise and accurate
data from new genomic technologies will facilitate not
only CNR gene CNVs but also other structural variants
in individual genomes to disease susceptibilities and drug
responses [7,46]. Many CNVs have been reported to affect
complex diseases including autism, schizophrenia, bipolar
disorder, obesity, Crohn’s disease, neurological disorders,
cardiovascular disease, nicotine metabolism and tobacco-
related diseases and more [7]. Ultimately creating animal
models of neuropsychiatric disorders that reflect human
CNV will provide insight into human neuropsychiatric
disorders that will contribute to novel drug screening for
these disorders [62]. Great potential exists for CNVs along
with other genomic variants including SNPs to explain and
predict disorders and traits in the future, but great challenges
exist for understanding the relationship between genomic
changes and the phenotypes that might be predicted and
may be treated or prevented [50].
6. Summary, conclusions, and future perspectives
We now know that CNVs and other variants of the human
genome are more prevalent than SNPs that have been
well studied and analyzed and have been linked to human
disorders. With many thousands of SNPs in the human
genome, and some associated with CNRs, it appears
that their contributions to the genetic basis of complex
diseases are relatively small effects. This has created the
possibility of other genomic variants, epigenetic, and other
nongenetic contributions to complex human diseases. For
the endocannabinoid system many SNPs for both CB1 and
CB2 receptors have been identified and characterized in
a number of neuropsychiatric disorders. Our preliminary
data indicated high CNVs in the CNR2 gene in Japanese
alcoholic patients compared to controls. It was difficult
to make a conclusion from the high CNVs of the CNR2
gene in alcoholics; and more alcoholic DNA samples and
samples from other neuropsychiatric disorders and in other
8Journal of Drug and Alcohol Research
ethnic populations should be analyzed to understand and
determine the nature of elevated copy numbers of CNR2
gene in neuropsychiatric disease risk. Numerous CNVs have
now been identified with various genome analysis platforms.
Whether the larger CNR2 gene CNVs in Japanese alcoholics
compared to non-alcoholics is associated with the disease
is unknown and the phenotypic effects are often unclear
and unpredictable, with larger CNVs [48,89]. However,
the bigger the CNV, the more likely it will cause a change
in gene dosage [15]. Therefore, the underlying pathogenic
mechanism for the larger CNR2 gene CNV obtained in the
sample analyzed in the alcoholics is currently unknown.
While CNR1 and CNR2 SNPs have been associated with
a number of neuropsychiatric disorders (Table 2), it is still
unclear to what extent CNR gene CNVs are involved in neu-
ropsychiatric disorders. Thus, it is important to study CNVs
that encompasses genes involving duplication and deletions
of sequences and their role in human health, disease,
pharmacotherapeutic and pharmacogenomic responses.
Acknowledgments We acknowledge Dr. Qing-Rong Liu for the
pivotal work on cannabinoid receptor gene structures and revision of
CNR2 gene features and for helpful comments and suggestions. E. S.
Onaivi, S. Sgro, and C. M. Leonard are supported by WPUNJ and E.
S. Onaivi is also supported by NIH grant DA032890. We are forever
indebted to Norman Schanz and Dr. Robert Benno for laboratory
animal support and the Dean Dr. Ken Wolf for support of student
workers for the maintenance of research animals. The authors have no
conflict of interest and have received no payments in the preparation
of this manuscript. The findings and conclusions in this manuscript are
those of the authors.
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