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Molecular Psychiatry (2005) 10, 69–78
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FEATURE REVIEW
Expanding the ‘central dogma’: the regulatory role of
nonprotein coding genes and implications for the genetic
liability to schizophrenia
DO Perkins1, C Jeffries2 and P Sullivan3
1
Department of Psychiatry, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; 2Genetic Control Research,
Research Triangle Park, NC, USA; 3Departments of Genetics, Psychiatry, and Epidemiology, Chapel Hill, NC, USA
It is now evident that nonprotein coding RNA (ncRNA) plays a critical role in regulating the
timing and rate of protein translation. The potential importance of ncRNAs is suggested by the
observation that the complexity of an organism is poorly correlated with its number of protein
coding genes, yet highly correlated with its number of ncRNA genes, and that in the human
genome only a small fraction (2–3%) of genetic transcripts are actually translated into proteins.
In this review, we discuss several examples of known RNA mechanisms for the regulation of
protein synthesis. We then discuss the possibility that ncRNA regulation of schizophrenia risk
genes may underlie the diverse findings of genetic linkage studies including that proteinaltering gene polymorphisms are not generally found in schizophrenia. Thus, inadequate or
mistimed expression of a functional protein may occur either due to mutation or other
dysfunction of the DNA coding base pair sequence, leading to a dysfunctional protein, or due
to post-transcriptional events such as abnormal ncRNA regulation of a normal gene. One or
more ‘schizophrenia disease genes’ may turn out to include abnormal transcriptional units
that code for RNA regulators of protein coding gene expression or to be proximal to such
units, rather than to be abnormalities in the protein coding gene itself. Understanding the
genetics of schizophrenia and other complex neuropsychiatric disorders might very well
include consideration of RNA and epigenetic regulation of protein expression in addition to
polymorphisms of the protein coding gene.
Molecular Psychiatry (2005) 10, 69–78. doi:10.1038/sj.mp.4001577
Published online 21 September 2004
Keywords: miRNA; ncRNA; schizophrenia; epigenetics; BDNF; DISC1; DRD2; RELN
Noncoding RNA, mRNA stability, and protein
coding gene regulation
The ‘central dogma’ of genetics is that DNA is
transcribed into messenger RNA (mRNA), and mRNA
in turn is translated into proteins. The central dogma is
certainly valid in that it defines what proteins are
possible to make, but in recent years it has become
evident that much more genetic information exists in the
genome, namely the information used in controlling the
timing and rates of the protein manufacturing processes.
In higher organisms only a minority of genetic transcripts
(2–3%) code for proteins.1,2 Nonprotein coding RNA
(ncRNA) refers to mRNA that is transcribed from DNA
but not translated into protein. Rather than being ‘junk’
DNA (ie an evolutionary relic) some nonprotein coding
transcripts may in fact play a critical role in regulating
gene expression and so organizing the development and
maintenance of complex life. For example, a study
Correspondence: DO Perkins, MD, MPH, Department of Psychiatry, CB 7160, University of North Carolina at Chapel Hill, Chapel
Hill, NC 27599-7961, USA. E-mail: [email protected]
Received 12 April 2004; revised 18 June 2004; accepted 26 July
2004
identified the highly conserved regions across 12
vertebrate species in a 12 mb orthologous genetic region
and found most (80%) of the highly conserved regions to
be located in introns or in intergentic regions, suggesting
that these nonprotein coding regions play an essential,
but as yet largely unknown regulatory role.4 In addition,
the potential importance of ncRNAs is suggested by the
observation that the complexity of an organism is poorly
correlated with its number of protein coding genes, but
highly correlated with its number of ncRNA genes.3
The rate and amount of protein synthesized is
related to the quantity of mRNA available, which in
turn is a function of the rate of DNA transcription, the
rate of mRNA degradation, and other factors. Both
transcription and mRNA degradation appear to be
tightly regulated, with ncRNAs potentially involved
in the developmental timing and quantity of protein
synthesis.4 For example, ncRNA may regulate protein
synthesis by decelerating or accelerating mRNA
degradation.5–8 Control mechanisms may target the
mRNA degradation enzymes or some other aspect of
the propensity of the mRNA to be degraded, thus
impacting the steady concentration (ie ‘half-life’) of
the mRNA.9 These newly discovered control mechan-
Expanding the ‘central dogma’
DO Perkins et al
70
isms for protein expression are often referred to as the
‘tip of the iceberg’, anticipating that yet undiscovered
regulatory functions of ncRNAs are likely to be
critical to the development and function of complex
organisms.2
In classical Mendelian disorders, mutations generally lead to striking alterations in protein structure
that impair function, resulting in disease. This
theoretical perspective has led investigators to emphasize investigation of nonsynonymous base pair
(bp) changes as candidate disease genes.10 However,
recent genome scans for complex diseases have found
that for the genomic location where statistical
evidence is strongest, the relevant markers are not
generally located in exons and so cannot directly
cause pathological change in the protein product. An
example includes the association of noninsulindependent diabetes mellitus with intronic genetic
variation in calpain 10 gene (CAPN10).11 Relevant to
this paper are the replicated findings of association of
schizophrenia with a specific noncoding singlenucleotide polymorphism (SNP) haplotype in the
gene for dysbindin (DTNBP1)12 and neuregulin 1
(NRG1).13 Furthermore, there is evidence that
nonsynonymous polymorphisms may alter the rate
of gene expression in humans through as yet
unknown mechanisms.14 It is thus becoming apparent
that altered regulatory control of the transcription or
the translation of a gene may contribute to disease
risk.
Below are described several noteworthy examples
of mRNA regulatory mechanisms that may play a role
in complex diseases. Several speculative examples
are then provided to illustrate how impairment of
mRNA regulation could potentially play a role in
schizophrenia.
Table 1
Micro-RNA (miRNA)
Small RNA segments form a critical cellular regulatory function, directly affecting the timing and rate of
protein expression (see Table 1). Science Magazine
highlighted the potential importance of these small
RNAs to understanding the evolution and operation
of the genome, naming this discovery the ‘Breakthrough of the Year’.7,15
A newly described ‘family’ of small ncRNA segments is miRNA. There are an estimated 200–255
human miRNA genes, constituting about 1% of
predicted human genes.16,17 Demonstrated function
of animal miRNAs includes the regulation of posttranscriptional processing of mRNA in neuronal and
general development, apoptosis, cell differentiation,
and cell proliferation.18,19
The precursor of a miRNA is initially transcribed
(from DNA) to a single strand of RNA, which then
folds over forming a double-stranded (ds) ‘hairpin’
precursor. The precursor miRNA contains a short
(B17–24) nucleotide ‘mature’ sequence imbedded in
the longer (B50–120) nucleotide sequence.18,16 The
mature sequence itself is highly conserved across
species, in contrast to the rest of the precursor miRNA
sequence, which shows evolutionary divergence.16
The precursor miRNA is exported from the nucleus to
the cytoplasm of a cell, where it is then processed by
the enzyme DICER into a dsRNA (from the stem of the
hairpin) that includes the mature sequence and its
partially homologous complement. This dsRNA is
further processed so that the mature sequence
becomes part of the RNA-induced silencing complex
(RISC). In animals, the nucleotides of the mature
miRNA sequence are imperfectly complementary to a
bp sequence of the target mRNA 30 -untranslated
region (UTR). Other types of small, ncRNA, such as
Definition of some recently described ncRNAs
Acronym
Name
Definition
ncRNA
Noncoding RNA
dsRNA
Double-stranded
RNA
miRNA
Micro-RNA
siRNA
Small interfering
RNA
RNAi
RNA interference
snRNA
Small nuclear
RNA
Small nucleolar
RNA
A group of RNAs that are transcribed but are not translated into a protein, some fraction
with regulatory functions.
Single-stranded RNA is unstable and quickly degrades unless associated with a protein
complex. Single-stranded transcribed RNA may fold over forming a double-stranded
‘hairpin’ that is protected from degrading enzymes.
Single strand of B17–25 nucleotides with regulatory functions when associated with a
protein complex. In animals, miRNAs inhibit translation by binding with imperfect
homology to the untranslated region of mRNA, inhibiting translation of mRNA into
protein.
Single strand of B21–23 nucleotides with regulatory functions when associated with a
protein complex. siRNAs bind to mRNA with perfect bp homology causing mRNA
degradation. Synthesized double-stranded RNA introduced into the cell may be processed
in a manner similar to endogenous siRNA and used to ‘silence’ a gene of interest.
The mechanism where double-stranded RNA is processed to a single strand that targets
mRNA resulting in mRNA degradation or translational inhibition.
Reside within the nucleus of eukaryotic cells and involved in RNA splicing or histone
mRNA termination.
Participate in methylation or ribosomal RNA modification or processing.
snoRNA
Molecular Psychiatry
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‘short interfering RNA’ (siRNA), as well as plant
miRNA are perfectly complementary to the target
mRNA. While ncRNA–target interactions with perfect
complementarity tend to result in mRNA cleavage
and degradation, ncRNA–target interactions with
imperfect complementarity tend to result in mRNA
translational inhibition. Since in animals, miRNA–
target interactions typically involve imperfect bp
complementarity, miRNAs act to regulate gene expression by binding to target mRNAs, thereby block-
ing ribosome processing and inhibiting mRNA
translation (see Figure 1).
The first description of miRNA:target interaction
and of miRNA function was in the nematode,
Caenorhabditis elegans.16 Two proteins important in
C. elegans development are lin-4, which is expressed
in the early larval stage, and lin-14, expressed in the
later larval stage. In a series of insightful studies, it
was discovered that a region of the lin-4 gene codes
for a miRNA (cel-lin-4) whose mature region binds to
71
Figure 1 ncRNA and mRNA interactions. mRNA transcribed from a protein coding gene (1) may be translated into a protein
(2). siRNA and miRNA interact with RISC (3). RISC ncRNA complex may then interact with the transcribed mRNA (4).
Perfectly complementary ncRNA and mRNA binding will then subject the mRNA to cleavage and degradation (5).
Imperfectly complementary ncRNA:mRNA may allow the mRNA to bind with ribosomes, but interferes with translation (6).
Either mechanism precludes protein expression (7).
Molecular Psychiatry
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the 30 -UTR of the mRNA from the lin-14 gene. Since
the 30 -UTR is needed for correct translation of mRNA
to protein, the binding of the mature region of the
miRNA to the target 30 -UTR prevented translation of
the lin-14 mRNA.20 The investigators demonstrated
that loss of the cel-lin-4 miRNA results in the
overexpression of lin-14 at a developmentally inappropriate time. Thus, an apparent function of cel-lin-4
miRNA is to regulate the lin-14 gene translation at
specific developmental time points.
Since this initial discovery, there have been
numerous descriptions of miRNA mature sequences
that are partially complementary to the 30 -UTR of a
protein coding gene.21,22 A regulatory role of miRNA
affecting mRNA degradation and stability (in plants)
and translation (in animals) is now known.16,18 In
addition to interfering with translation of mRNA,
miRNA may conversely increase gene expression by
binding with some other regulatory RNA, in effect
inhibiting inhibition.23
had very low levels of Makorin1 protein, Makorin1
mRNA and Makorin1p mRNA, resulting in the lethal
phenotype. Another experiment demonstrated that
Makorin1 mRNA has a much greater half-life in the
presence of Makorin1p as compared to the half-life in
the absence of Makorin1p. Thus, the pseudogene
somehow reduces the degradation rate of the gene
mRNA. The presence of the intact pseudogene is
needed for correct expression rate of the Makorin1
gene.
Potential explanations for Makorin1p regulation of
Makorin1 expression include that the Makorin1p
mRNA could interfere with the degradation of the
Makorin1 mRNA by acting as a ‘decoy’ for enzymes
degrading Makorin1 mRNA, thus increasing the
amount of mRNA available for translation into
protein.27 A second possible explanation is that the
pseudogene may act as a decoy for transcriptional
suppressor proteins, an instance of inhibiting inhibition of Makorin1 gene transcription to mRNA.28
Functional pseudogenes
A pseudogene has a similar bp sequence to a protein
coding gene, but is missing critical bp sequences
required for translation. A pseudogene may be
transcribed, but the resultant mRNA is not translated
into a protein. Numerous (B20 000) pseudogenes
have been identified in the human genome, and many
genes have multiple pseudogenes, often located on
different chromosomes than the gene itself.24,25 Until
very recently, pseudogenes were considered nonfunctioning genetic ‘mistakes’ or ‘fossilized’ remnants of
discarded genes. Evidence for a possible biological
role for at least some pseudogenes comes from the
observation that many pseudogenes appear to be
conserved across species and thus do not show the
pattern of gradual, evolutionary sequence degradation
as would be expected for a nonfunctioning genetic
region.25 Pseudogenes that reside near their gene are
thought to contribute to gene mutation through the
process of gene conversion. Gene conversion occurs
during meiotic crossover events, where there is
unequal exchange of genetic material between a gene
and near-homologous pseudogene that is in close
proximity to the gene. Gene conversion is a proposed
mechanism of PRODH gene mutation, implicated in
the etiology of schizophrenia.26
Direct evidence of pseudogene function has come
from the discovery of a functional pseudogene in
mice. The pseudogene was discovered fortuitously
while conducting a study which involved inserting a
Drosophila gene Sex-Lethal randomly into the mouse
genome.27 This gene insertion was fatal for one line of
mice. Surprisingly, the Sex-Lethal gene had been
inserted in the middle of a pseudogene, Makorin1p,
located on murine chromosome 5. This pseudogene is
located on a different chromosome than that of the
functional gene, Makorin1, located on chromosome 6.
Further study demonstrated that the Makorin1p was
normally transcribed during fetal and postnatal
development. Mice with the disrupted pseudogene
mRNA stability
The rate of mRNA degradation by various enzyme
systems impacts the rate of protein synthesis, since an
individual mRNA may be used in translation multiple
times, depending on how long the mRNA remains
intact in the cytosol. The steady-state concentration of
mRNA is thought to be tightly regulated, although the
mechanisms controlling mRNA concentration stability are not yet understood.9,29
The tertiary structure of mRNA may impact its rate
of degradation and translation. Some amino acids can
be specified in the translation process by multiple
RNA codons (eg CCU, CCC, CCA, and CCG all code
for proline). Thus, variations of DNA base sequences
among gene polymorphisms often do not impact the
translated amino-acid sequence and thus the synthesized protein. Such sets of codons coding for one
amino acid are termed ‘synonymous’ genetic polymorphisms. However, while the alternative codons do
not influence primary or secondary protein structure,
they may not be silent. Rather, alternative codons may
affect the tertiary structure of mRNA, thereby affecting mRNA degradation rate, mRNA half-life, and
hence the rate of protein expression.30
Molecular Psychiatry
Epigenetic factors
Epigenetic factors include a layer of genetic information and control embodied in the chromatin—the
proteins and other chemicals that surround a chromosome. The chromatin serves to isolate and protect
DNA from degradation, transcription, and interactions with other proteins including ncRNAs. Epigenetic information is remodeled in response to other
genetic events and environmental signals. The chromatin partitions active and silent chromosomal
regions, and the partition boundaries change in
development and also in response to environmental
factors.31 If genetic expression is altered through
epigenetic mechanisms by an environmental insult,
it may remain altered for the life of the individual,
Expanding the ‘central dogma’
DO Perkins et al
potentially causing disease by misexpression of a
protein.32 Chromatin boundary mechanics may be
either mutually reinforcing or mutually inhibitory.
The result may be polarization of chromatin domains,
so that they are fixed in a state of transcriptional
activity or silence.33 One prominent epigenetic mechanism is methylation, meaning the inactivation of a
gene by attachment of methyl radicals to instances of
C (cytosine) with following G (guanine).
Epigenetic proteins may control genetic expression
in a single locus, a chromosomal region, or an entire
chromosome.31 The epigenetic effect of ‘imprinting’
allows the activation of only one allele from the
copies in maternal and paternal chromosomes. Thus,
inheriting a chromatin structure that consistently
chooses for suppression of the same parental locus
of a trait may result in a parent-of-origins effect where
a trait can be inherited only from one parent.
Inheriting this chromatin structure means propagation of gene activity partitions from one cell generation to another (mitosis) or from one individual to
progeny (including meiosis). Environmental factors
early in life can modify chromatin structure and
thereby stably alter the expression of genes into
adulthood, that is, the genome reacts to its experiences.33 Also, identical twins exposed to different
environmental factors can differentially express disease due to the presence or absence of correct
imprinting.34 Hence, epigenetic effects may eventually help explain the complex roles of environment
and inheritance in the etiology of schizophrenia.
RNA mechanisms may be involved in epigenetic
gene regulation. Small (short), interfering RNAs,
siRNAs, might be involved in chromatin silencing or
DNA methylation, and they might also have roles in
the regulation of both conventional genes (that code
for proteins) and RNA-only transcriptional units.31
The genetics of schizophrenia
Schizophrenia is clearly a heritable disorder with
overwhelming evidence that genetic factors play an
important role.35,36 However, the specific genetic
factors that contribute to schizophrenia are not
known, despite decades of research. The basic
strategies used to investigate the genetic basis of
schizophrenia include family linkage studies and
candidate gene association studies. A recent metaanalysis of linkage studies defined 19 areas on 15
chromosomes where there is evidence across studies
for involvement in the etiology of schizophrenia (only
one area, 2p12-q22, met statistical significance when
adjusting for multiple comparisons).37 The areas
defined by these 19 linkage regions consists of about
565 million bp’s, representing approximately 18% of
the 3 billion bp’s that make up the human genome.
Additional chromosomal regions have also been
associated with schizophrenia risk in other linkage
studies.37,38
Converging findings from neurobiological studies
implicate abnormal function of prefrontal cortical and
limbic area circuits that involve dopamine, glutamate,
and GABA in the pathology of schizophrenia.39–42 In
addition, there is strong evidence that altered brain
development is involved in schizophrenia risk.42
Recent linkage studies using closely spaced SNPs
have further narrowed possible linkage areas and
then, based on SNP association, identified candidate
schizophrenia risk genes. The risk genes identified
notably include genes that are involved in the
metabolic pathways of these neurotransmitter or
implicated receptors, or in neurodevelopment. This
strategy has identified several schizophrenia risk
genes, with varying levels of genetic, molecular
biological, and theoretical evidence.43,44 In particular,
two leading schizophrenia risk genes, NRG1 and
DTNBP1, have disease-associated polymorphisms
that do not result in the expression of an abnormal
protein in the manner generally found for classical
Mendelian disorders.45,12
73
A third explanation
One hypothesized explanation for the diverse genetic
associations found for schizophrenia is a ‘polygene
model,’ where each gene locus has a small effect so
that schizophrenia arises as the aggregate consequence some mixture of multiple risk genes or
environmental factors. A second hypothesized explanation is a ‘locus heterogeneity model’, where the risk
is large, but only a small proportion of individuals are
vulnerable.
A third explanation for the diverse findings of
genetic linkage studies and the conflicting evidence
supporting a particular gene polymorphism is that
regulation of schizophrenia risk gene(s) expression is
disrupted. Dysregulation by ncRNA of the rate of
transcription or of translation of a normal gene may
lead to abnormal gene expression that is phenotypically similar to disruption of the gene itself. Thus,
inadequate or mistimed expression of a functional
protein may occur either due to disruption of the
DNA coding bp sequence, leading to a dysfunctional
protein, or due to abnormal ncRNA regulation of a
normal gene. ncRNA regulation of gene transcription
and translation may be particularly important during
brain development,19 and so is consistent with a
neurodevelopmental diathesis for schizophrenia.
ncRNA may also regulate gene expression in adulthood, and so may explain abnormal gene expression
in the presence of an apparently normal gene. One or
more schizophrenia disease ‘genes’ may turn out to
include abnormal transcriptional units that code for
RNA regulators of protein coding gene expression or
to be proximal to such units, rather than to be
abnormalities in the protein coding gene itself.
Regulating pseudogenes, miRNA, or other ncRNA
may exist at very different chromosomal locations
than the regulated gene itself. Thus, it may be that one
or more areas identified through genetic linkage
studies as associated with schizophrenia may in fact
be the location of a regulatory ncRNA.
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Could noncoding RNA regulate putative schizophrenia
genes?
The understanding of the importance of ncRNA
regulation of gene expression is an emerging area of
active research with many methodological challenges.5 The results of animal and plant research to
date suggest that ncRNAs are likely to be a major
regulatory mechanism of gene expression regulation
in humans. Pertinent examples are discussed above,
although numerous other ncRNA regulatory mechanisms exist.31,46,5,8 The following examples, while
speculative in nature, are meant to illustrate how
altered regulation of mRNA expression could potentially play a role in schizophrenia risk. Similar
mechanisms could also contribute to the risk of other
complex brain disorders, especially those with a
neurodevelopmental etiology.
ment and later in life. hsa-mir-1 maps to chromosome
20q13.33, which placed #21 in the Lewis metaanalysis, and is just distal to an area that was 11th.
hsa-mir-206 maps to chromosome 6p12.2, which
placed #36 in the Lewis meta-analysis, and is in
between two areas with significant linkage (#2 and
#5). Further study is needed to confirm whether this
hypothesized relationship between miRNAs, hsa-mir1 and hsa-mir-206, is etiologically important in
schizophrenia. For example, molecular genetic studies would be needed to demonstrate empirically that
either hsa-mir-1 or hsa-mir-206 (or both) is simultaneously present in a cell with mRNA for BDNF, and
that such miRNAs affect BDNF expression. If confirmed, further studies would also be needed to
determine if miRNA regulation of BDNF is altered
in schizophrenia.
Micro-RNA
GABA and glutamate neuronal circuits have been
implicated in schizophrenia. Brain-derived neurotrophic factor (BDNF) regulates the development,
survival, and synaptic maintenance of a variety of
neurons in the CNS, including GABA and glutamatergic neurons47,48 BDNF has been shown to increase
the survival of glutamate neurons and promote
glutamate pyramidal cell dendritic growth and spine
density. BDNF, through control of synaptic density,
may regulate long-term potentiation of glutamate
neurons. Cortical neuron dendrite growth and synapse formation is associated with an increase in
cortical BDNF mRNA. In addition, BDNF may
regulate type 3 dopamine receptor (DRD3) expression
during brain development and promote DRD3 receptor expression in adults.49,50
Recent studies have examined BDNF expression in
post-mortem brains. One study finds decreased BDNF
gene expression in the dorsolateral prefrontal cortex
of subjects with schizophrenia compared with
healthy subjects,51 although other studies have contradictory findings.52,53 Animal models suggest that
antipsychotics may impact BDNF expression,54 complicating the interpretation of these post-mortem
studies.
Genetic association studies between polymorphisms of the BDNF gene and schizophrenia are
inconclusive, with negative55–58 and positive59,60
studies. BDNF polymorphisms have been associated
with treatment responsivity61 and negative symptom
severity.62
Although findings are mixed, BDNF remains a
plausible candidate gene for schizophrenia. A theoretical mechanism that could explain altered mRNA
expression and protein production in the presence of
a normal gene is that ncRNA regulation of BDNF gene
expression is disrupted. Bioinformatic investigations
indicate that two newly described miRNAs, hsa-mir-1
and hsa-mir-206, may target the 30 -UTR of BDNF,22
consistent with the theory that miRNA could regulate
BDNF protein synthesis by interfering with BDNF
mRNA translation, potentially during brain develop-
Other ncRNA
The observation of an association of the genes
‘disrupted in schizophrenia 1’ (DISC1) and ‘disrupted
in schizophrenia 2’ (DISC2) with schizophrenia first
occurred in a family study where a balanced translocation between chromosomes 1 and 11 segregated
with schizophrenia.63 This dislocation was found to
disrupt the function of these two genes (hence the
name). DISC1 is expressed in neurons and glia, and
there is evidence that DISC1 may be involved in
neuronal migration during brain development,64–66
and may modulate signal transduction and glutamate
transmission.67 While several schizophrenia linkage
studies found significant signal in the 1q42 region,68–
70
overall the linkage evidence for involvement of this
area with schizophrenia is weak.37
DISC2 is known to be transcribed into ncRNA, but
the ncRNA transcript does not code a protein
product.71 The ncRNA product is partially complementary to DISC1, consistent with a regulatory role
for DISC1 gene expression. Further study would be
needed to determine if an actual regulatory role
exists, and if DISC2 regulation of DISC1 expression
is involved in schizophrenia.
Molecular Psychiatry
mRNA stability
The type 2 dopamine receptor gene (DRD2) is a
candidate gene for schizophrenia risk or disease
severity due to the overwhelming evidence that
schizophrenia pathology involves dysregulation of
the dopamine system, especially involving DRD2
receptors.72 However, genetic studies find no or only
a weak association between DRD2 gene polymorphisms and schizophrenia.73
Synonymous polymorphisms, in other words base
substitutions that do not have a predicted effect on
the synthesized protein, are often not examined in
association studies. In particular, known DRD2
synonymous polymorphisms have not been examined
in association studies because of the presumed lack of
potential to impact disease.74
Recent studies suggest, however, that these
synonymous polymorphisms may, in fact, not be
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DO Perkins et al
functionally neutral. Synonymous DNA polymorphisms may result in different three-dimensional folding patterns of the transcribed mRNA. The altered
tertiary structure of the mRNA may have consequences for mRNA degradation and translation, thus
impacting rate of protein synthesis.9 In a recent study,
the mRNA from one of the presumed synonymous
DRD2 DNA polymorphisms SNP C957T was found to
have altered tertiary structure that decreased the
stability of the mRNA and consequently the rate of
DRD2 protein synthesis.74 Furthermore, the SNP
C957T variant DRD2 receptor was less responsive to
dopamine induced upregulation. Interestingly, another synonymous polymorphism C1101A impacted
mRNA translation rate, but the combination of SNPs
C1101A and C957T did not. It remains to be shown
whether the synonymous SNPs actually impact
dopaminergic function in vivo or otherwise contributes to schizophrenia risk or disease severity.
However, there are other examples where alterations
in mRNA stability may be associated with brain
disease, notably Alzheimer’s disease.9,4
Epigenetic evidence
Factors operating independently of gene bp sequences are likely important in schizophrenia, as
monozygotic twins will have an estimated concordance for schizophrenia of only 41–65%.75 Environmental factors are presumed to be important,
including environmental events that occurred during
brain development (eg maternal infection, starvation,
obstetrical complications, maternal exposure to a
disaster) and environmental exposures that occurred
later in life (eg stressful life events, marijuana or
stimulant drug use).76,77 Several investigators have
proposed that environmental factors influence schizophrenia risk by epigenetic modification of protein
coding gene expression.78–80
Converging evidence suggests that schizophrenia
may be related to altered connectivity between brain
regions, especially at the level of the synapse.81 There
are numerous proteins involved in regulating and
maintaining synaptic integrity. One such protein is
Reelin (RELN), synthesized by GABA interneurons,
and thought to be involved in maintaining dendritic
spine density and function in neighboring pyramidal
cells.78 However, the location of the RELN gene on
chromosome 7q22.1 is not in a schizophrenia risk area
as defined by genetic linkage studies.37
There is convincing evidence from post-mortem
studies that RELN mRNA and protein expression are
significantly lower in the brains of individuals with
schizophrenia compared to healthy individuals.
Therefore, if altered RELN expression is involved in
schizophrenia, it may be due to abnormal regulation
of RELN expression. A recent study suggests that the
epigenetic regulation of RELN expression may be
altered.78 DNA cytosine-5 methyltransferase (DNMT)
is a protein critical to methylation and thus epigenetic
silencing of a gene. In the same brains where RELN
was underexpressed, a protein important in promot-
ing epigenetic gene silencing, DNA cytosine-5
methyltransferase 1 (DNMT1), was expressed at high
levels. The promoter region of the gene that codes for
RELN contains the binding sites for DNMT1 (CpG
islands), suggesting that epigenetic regulation is
possible for the RELN gene.
75
Conclusions
The recent sequencing of the human genome, advances in bioinformatics and molecular genetics, and
the pervasive availability of high-speed Internet
communication provide an unprecedented and readily accessible set of discovery tools. The tools have
initiated and accelerated much investigation of
regulation of gene expression, undoubtedly a phenomenon critical to the development and maintenance of complex life. The central dogma is now
known to describe only a part of the genetic information machinery, namely what proteins might possibly
be synthesized; additional genetic information controls the rates of syntheses.
Recent experimental evidence suggests a role for
ncRNA in other complex diseases, including cancers
and fragile X syndrome. Several studies now implicate miRNAs, in particular, with cancer etiology.82 For
example, in one study the levels of miRNAs hsa-mir143 and has-mir-145 were reduced in colorectal
cancer cells.83 Another investigation found that the
genes coding for hsa-mir-15 and hsa-mir-16, located
on chromosome 13q14, were deleted or expressed at
reduced levels in the tumor cells from 68% of patients
with B-cell chronic lymphocytic leukemia.84 Finally,
the BIC gene, containing a region that codes for hasmir-155, was overexpressed in patients with Hodgkin’s lymphoma,85 with a second study finding that
both hsa-mir-155 and the BIC gene were excessively
expressed in the cells from another cancer, Burkett’s
lymphoma.86 The possible role of miRNA in the
etiology of fragile X syndrome is suggested in an
animal model that found the etiologic fragile X mental
retardation protein to regulate synaptic plasticity via
a mechanism that involves miRNAs.87
To date there is no evidence linking ncRNAs and
schizophrenia, and so the examples provided above
are highly speculative. However, elucidation of the
genetic information that controls (directly or indirectly) the rate of protein synthesis might be fundamental to understanding disease expression,
especially with neurodevelopmental diseases. The
functions of disease risk genes might be disrupted
directly through mutation of protein coding regions
(that make protein production possible); indirectly
through mutation of ncRNA transcription sites (that
determine the rates of protein production); or through
environmental impact on epigenetic regulation of
gene expression. This perspective may provide the
basis of new interpretations of genetic association
studies, where markers may point to large genomic
regions (hundreds to tens of thousands of bp’s) that
are often in introns or to associations with exonic
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regions that do not result in protein-altering mutations.
Every gene implicated in the etiology of schizophrenia gives rise to multiple combinations of
potential regulatory mechanisms and their associated
sites of transcription. In order to focus investigative
efforts, the most likely potential regulatory mechanisms must be selected. As illustrated above, investigative strategies may now include bioinformatics and
may use mathematical modeling and genomic search
techniques to generate testable hypotheses (eg predict
new ncRNAs and their control functions, tertiary
structures of mRNA, etc). While progress can be
expected from existing molecular, clinical, and
epidemiological methodologies, the general elucidation of the foundations of genetic regulation may be
required before the genetic basis of schizophrenia and
other complex disorders is fully understood.
Acknowledgements
This work was supported in part USPHS Grants K23MH01905-01, P50-MH064065, and UO1 MH066069
(DOP)
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