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 The
neurotransmitter dopamine is released at the presynaptic terminal, diffuses
across the synaptic cleft, and binds to specific dopamine receptors on
the plasma membrane of the postsynaptic terminal. Five distinct dopamine
receptors have now been cloned, each produced by a different gene (D1D5
receptors). These different genes presumably arose by gene duplication
millions of years ago, and significant differences in their signaling
properties have evolved over time. Two general classes have emerged based
on pharmacological studies: the D1 and D5 receptors generally transmit
stimulatory signals, whereas the D2D4 receptors are inhibitory.
In
addition to these five separate dopamine genes, individual receptors may
vary to some extent between different populations and individuals. These
differences ultimately are based on differences in the nucleotide sequences
that also emerged over millions of years of evolution. The differences
in nucleotide sequences can now be directly determined in the laboratory
by sequencing of the gene in question. Obviously, when the sequences in
a gene from two individuals are compared, differences in the sequences
will emerge. These changes in sequences are termed polymorphisms.
Polymorphisms may occur as single base substitutions, as deletions or
insertions, or as multiple repetitions of a particular sequence of base
pairs (bp). This last type of polymorphism was the first polymorphism
discovered within the DRD4 gene, and it turned out to be a 48-bp
repeated sequence.
The
interest in polymorphisms arose when it was realized that they could be
used as markers to help localize genes that cause or contribute to the
expression of a particular illness. For example, if a particular polymorphism
is found to be passed on to the next generation and all individuals who
received that polymorphism develop a specific disease, then the investigators
would have considerable evidence that either that particular gene or one
that lies very close to it is responsible for the symptoms. This is in
fact the basis for genetic linkage studies.
Polymorphisms
may, or may not, change the functional properties of the protein in which
they occur. Whether the enzymatic properties of a protein are affected
depends on where the nucleotide changes occur within the gene. Changes
to the coding region of a gene are more likely to have functional consequences,
as many of these nucleotide changes will alter the amino acid sequence
of the protein.
Changes
outside of the coding region of a gene are less likely to have an effect
on the enzymatic activity as they are less likely to change its amino
acid structure. There are times, however, when even a polymorphism outside
of the coding region has a dramatic effect on the proteinšs functioning.
This occurs, for example, when the polymorphism arises within the promoter
region and affects the amount of transcription, either increasing or decreasing
the amount of message produced. Another example is when the polymorphism
falls within an intron and alters the nucleotide sequence that regulates
the correct splicing of the RNA message.
Since
the cloning of the gene for the dopamine D4 receptor (DRD4) in
1991, it has received considerable attention in genetic studies. Much
of the original research focused on the possible role of the D4 receptor
gene in schizophrenia because of the high affinity of the D4 receptor
for the atypical antipsychotic clozapine. Since then, DRD4 has
been investigated in a number of other behavioral phenotypes including
personality traits, bipolar affective disorder, alcohol and drug abuse,
and most recently attention-deficit/hyperactivity disorder (ADHD), the
focus of this review.
A
number of polymorphisms have been identified in the exons and introns
of DRD4, including a 12-bp repeat in the first exon and a 48-bp
repeat in exon III (Fig.
1). Less common DNA variants have also been identified in the coding
region of the gene including a 21-bp deletion and a 13-bp deletion both
located in exon 1, a substitution of arginine for glycine at position
11, and a substitution of glycine for valine at position 194. Additional
polymorphisms have been identified in the region upstream of the coding
sequence including a 120-bp repeat located approximately 1.2 kb upstream
of the first codon, as well as several single nucleotide polymorphisms.
The majority of genetic studies have used the 48-bp repeat polymorphism
located in the third exon mainly because it was the first polymorphism
identified and encodes for relatively large changes in the protein sequence.
The number of repeats varies from 2 to 10 in the human population, and
there is sequence and amino acid variation within the repeats as well.
Substantial genetic diversity of the repeats across different ethnic groups
has been documented.
In
1996, two studies reported an association between the personality trait
of novelty-seeking and the allele of the DRD4 with 7 copies of
the 48-bp repeat. This finding has not been replicated by other groups
and is still considered controversial. The issues concerning these reports
have been previously covered in detail and will therefore not be reviewed
here.
Regardless
of whether or not a genetic relationship between DRD4 and novelty-seeking
exists, these initial reports led to further investigations of DRD4
as a susceptibility factor for ADHD. The first study used a case-control
design and found an increased frequency of the 7-repeat allele in the
ADHD sample compared with an ethnically matched control group. Those authors
subsequently collected proband/parent trios and the same allele was found
to occur at a higher frequency in probands than in the nontransmitted
alleles of the parents. This type of family-based association study markedly
reduces the risk for false-positive associations as the control group
are the parents of the probands and not another group of potentially ethnically
diverse individuals in which normal polymorphisms are routinely found.
Further
support for DRD4 in ADHD has come from a number of additional published
studies using different sample ascertainment strategies and statistics.
Several different statistical approaches were used by Rowe and colleagues
to investigate the genetic relationship between DRD4 and ADHD.
Using a case-control approach, they found that the 7-repeat allele occurred
more frequently in children with the ADHD-inattentive subtype than in
controls. Using family data, the authors also examined the transmission
of alleles with both a categorical and a quantitative transmission disequilibrium
test (TDT). No significant evidence was found for biased transmission
of the 7-repeat allele when a diagnosis of ADHD was made by either a categorical
or a quantitative approach. However, a positive correlation was reported
for inattentive symptoms and the 7-repeat allele when a quantitative TDT
was used. The authors also investigated genetically discordant sibling
pairs and observed that the sibling with a greater number of 7-repeat
alleles displayed more inattentive symptoms than the respective cosibling
with fewer 7-repeat alleles. A larger sample size would help to further
clarify the relationship of DRD4 to the inattentive versus the
hyperactive subtype.
Using
a sample of 27 ADHD children and their parents that was collected through
parents referred with adult ADHD, Faraone et al. reported the biased transmission
of the 7-repeat allele compared with the 4-repeat allele (c2
= 7.4, df = 1, p = .007). In contrast, Smalley et al., using
genotypes from 133 families consisting of 49 families with a single ADHD
proband and 84 with at least one additional affected sibling, found no
significant evidence for identity-by-descent sharing of DRD4 48-bp
repeat alleles. The investigators then stratified the informative meioses
into those with and without the 7-repeat allele. When the data were analyzed
in this way, a trend emerged (p = .07) with increased allele-sharing
among the sibling pairs from parental meioses in which the 7-repeat was
present. An additional study also reported little evidence for an association.
This study used a case-control design with 41 children with ADHD and 56
controls group-matched for ethnicity and sex.
In
addition to the published studies, two unpublished studies out of Toronto
support the association. The first study used two samples ascertained
through probands with adult ADHD. Results were significant for a case-control
sample of 66 cases with ethnically matched controls (c2
= 5.65, p = .01). There was a trend for biased transmission of
the 7-repeat allele in a sample of 44 nuclear families with an ADHD adult
proband, but this was not significant (c2
= 2.00, p = .15). The results were significant after the samples
were combined (N = 110, z = 2.68, p = .003).
Our
group in Toronto has accumulated further support for biased transmission
of the 7-repeat allele by using the TDT test in an independent sample
of children. This sample of 107 families was ascertained through child
ADHD probands collected at the Hospital for Sick Children. Using a one-sided
test we observed biased transmission of the 7-repeat allele (c2
= 2.882, p = .045).
While
a number of studies thus far support DRD4 as a genetic susceptibility
factor in ADHD, the findings are not robust. It is clear that the 7-repeat
allele is neither necessary nor sufficient to cause ADHD and that this
is not a straightforward relationship. Therefore, it is still imperative
to consider that ADHD is a complex trait and much more evidence needs
to be accumulated before we can be conclusive about DRD4 or any
gene in this disorder.
Working
under the assumption that there is some type of relationship between the
DRD4 and ADHD, we are left with trying to find a biological explanation
for these observations. Functional differences in the intracellular signaling
system have been reported for the 48-bp repeat alleles, and the 7-repeat
allele may be less sensitive to endogenous dopamine. One could speculate
that a blunted response of the 7-repeat allele to dopamine is consistent
with the ameliorative effects of methylphenidate treatment. In other words,
methylphenidate raises synaptic dopamine levels, thus compensating for
the blunted response of the receptor. However, the functional differences
between the repeats are small and it is not yet clear how these subtle
effects could be related to the phenotype of ADHD.
A
second possible explanation is that an additional as yet unidentified
DNA variant in linkage disequilibrium with the 7-repeat allele is contributing
to the phenotype. My laboratory has been investigating the second possibility,
and we have some interesting preliminary findings that suggest that the
susceptibility may be more complicated than the simple inheritance or
not of the 7-repeat allele as a risk factor.
To
understand more about the molecular basis of the finding, we examined
the inheritance of other polymorphisms in the gene and also the inheritance
of the groupings of alleles on the chromosome (the haplotypes). We assembled
the haplotypes of individuals with the 48-bp repeat polymorphism, the
mononucleotide repeat in the first intron, and the 12-bp repeat in the
first exon. We observed biased transmission of two haplotypes formed from
these three polymorphisms. One of these was the only common haplotype
containing the 7-repeat allele (c2
= 4.900, df = 1, p = .027).
The
most surprising finding from the study of the haplotypes was the observation
that one haplotype containing a 4-repeat allele was not transmitted
to the affected probands more often than expected by chance. If the risk
conferred by DRD4 is simply related to the inheritance of the 7-repeat
allele, then there should be no bias as to what allele is not transmitted.
That is, for a two-allele marker, if one allele is preferentially transmitted,
then the other allele will not be transmitted. For a system with more
than one allele (in this case, more than one haplotype), then the nontransmitted
allele should be distributed across all of the other alleles. The finding
that one haplotype is preferentially not transmitted suggests that this
haplotype may provide some type of protective role. While this is an interesting
finding, the observation must be extensively replicated before any conclusion
can be made.
We
also searched for other known DNA variants that result in changes in the
protein sequence including two previously reported deletions, a 13-bp
and a 21-bp deletion in the first exon (see
Fig. 1). We did not observe either of these two deletions in our sample,
indicating that these deletions are not responsible for the reported association.
We also tested for linkage to the 12-bp insertion/deletion polymorphism
in the first exon. We did not observe biased transmission of the alleles
of this polymorphism.
Where
do we take this research from here? A number of investigators are concentrating
on dissecting the phenotype in relation to this gene, in particular the
components of the phenotype with respect to the inheritance of the 7-repeat
allele. If the number of 48-bp repeats is not responsible for the phenotype,
then identification of the DNA variant responsible for this finding is
necessary and the analysis of the components of the phenotype in relationship
to this allele may be premature. One line of inquiry that has not thus
far been pursued is the sequence variation within the repeats. It is possible
that different sequence variations or arrangement of variants within the
repeats may be more closely associated with the phenotype. The identification
of the responsible variant may be difficult, however, as it is likely
that there are a number of common variants found in individuals with and
without ADHD and proving causality is difficult with a common complex
phenotype.
Much
more work is needed to understand the molecular underpinnings of the DRD4
and ADHD findings, including the relationship of the genotype to the phenotype.
Investigating the interaction with other genetic and environmental susceptibility
factors and the relationship to comorbid disorders should also be the
goal of future research.
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