The Regulator of G protein signaling 4 (RGS4) gene is a candidate susceptibility gene for schizophrenia

(SCZ). Previous studies showed that the mRNA level of the longest splice variant RGS4-1 was decreased
in the dorsolateral prefrontal cortex (DLPFC) of SCZ patients compared with healthy controls. In this pilot
study, we examined the possible mechanisms of RGS4-1 mRNA reduction in SCZ. We genotyped SNP1
(rs10917670), rs2661347, SNP4 (rs951436), SNP7 (rs951439), SNP18 (rs2661319), and rs10799897
(SNP9897) and tested the methylation status of CpG islands of the RGS4 gene in the postmortem DLPFC
samples obtained from subjects with SCZ and bipolar disorder as well as healthy controls. RGS4-1 mRNA
level was associated with five SNPs (SNP1, rs2661347, SNP4, SNP7, and SNP18) and their haplotypes but
not with SNP9897. In addition, this study revealed that RGS4-1 mRNA was low in subjects with specific
genotypes of SNP1, rs2661347, SNP4, SNP7, and SNP18. Lower RGS4-1 mRNA expression in the DLPFC of
SCZ is associated with SNPs in the 5′ regulatory region of the RGS4 gene but not with the methylation
status of its CpG islands.

Introduction

The Regulator of G protein signaling 4 (RGS4) gene is associated with susceptibility to schizophrenia
(SCZ). A previous microarray study showed that RGS4 mRNA levels were decreased in prefrontal cortex
(PFC) of SCZ patients as compared to healthy subjects (1). The four common single-nucleotide
polymorphisms (SNP1, 4, 7, and 18) of the RGS4 gene, localized between the 5′ upstream sequence and
the second intron, as well as two haplotypes derived from these SNPs may confer SCZ risk (2). Some
other SNPs in the 20 kb genomic region of RGS4 are associated with SCZ (2), and the G-allele of SNP1 is
associated with non-deficit SCZ (3). Other linkage and association studies also indicated a connection
between RGS4 polymorphisms and SCZ risk (4–6). ---RGS4 polymorphisms (SNP1, 4, 7, and 18) were
associated with differences in the dorsolateral prefrontal cortex (DLPFC) volume in first episode SCZ
patients (7). Several RGS4 SNPs are associated with clinical symptoms of SCZ. For example, Positive and
Negative Symptoms Scale (PANSS) total and global psychopathology scores were associated with SNP4
(8). SNP18 (rs2661319) and rs2842030 were associated with more severe baseline PANSS total score (9).
At the baseline status, the A/A genotype at SNP7 of RGS4 was associated with a poorer social function
compared with the G/G genotype (10). SNP1 (rs10917670) was associated with the depression factor in
SCZ (11).

We previously cloned five splice variants of RGS4 mRNA expressed in the DLPFC of the human brain and
three splice variants of Rgs4 from the mouse brain and found that the transcriptional regulation of
human RGS4 differs from that of the mouse Rgs4. Our data showed that mouse Rgs4 encodes one
protein while human RGS4 encodes four different proteins, three of which are unique to humans (12).
We also found that the mRNA level of the longest isoform RGS4-3 was specifically deceased in DLPFC of
SCZ patients but not in healthy controls (NC) or subjects with bipolar disorder (BPD) (13). Since then,
RGS4-3 has been renamed RGS4-1 (NM_001102445.2), and we use the new nomenclature here.
How is RGS4-1 decreased in DLPFC of SCZ? One possibility is through epigenetic regulatory mechanisms
such as DNA hypermethylation. Bioinfomatics analysis found CpG islands (GC% >50%, >200 bp and the
ratio of observed/expected CpG >0.6) (14) spanning 246 bp located in the first intron and second exon
of the RGS4 gene. Hypermethylation of CpG islands leads to decrease in gene expression (15, 16), which
might explain the reduction in RGS4-1 mRNA level. To test whether the decrease in RGS4-1 mRNA
expression in the DLPFC of postmortem SCZ brains is due to epigenetic mechanisms, such as DNA
hypermethylation, we investigated the methylation status of CpG islands of RGS4.

RGS4-1 might also be decreased through mechanisms in which the regulatory region of the RGS4 gene
plays a role. Such a possibility might be revealed by testing whether the SNPs in the regulatory region of
the RGS4 gene are associated with RGS4 mRNA expression. Therefore, we genotyped a few SNPs in the
regulatory region of the RGS4 gene including SNP1 (rs10917670), rs2661347, SNP4 (rs951436), SNP7
(rs951439), SNP18 (rs2661319), and rs10799897 (SNP9897) to test for possible associations between the
SNPs and RGS4 mRNA expression. We present results showing that expression of RGS4-1 is associated
with five SNPs and that RGS4-1 expression is not correlated with the methylation status of CpG islands in
the RGS4 gene.

(https://www.frontiersin.org/articles/10.3389/fpsyt.2016.00026/full)

The significant role of serotonin (5-hydroxytryptamine [5-HT]) in the pathogenesis and early
development of schizophrenia has been established by contemporary research through the assessment
of structural and pharmacological neuroimaging, blood metabolites, cerebrospinal fluid, genome
polymorphisms, and other valid indicators of abnormal serotonergic activity in prodromal, ultra-high-
risk, and schizophrenic patient groups. A modern approach toward understanding the complex
psychophysiology behind schizophrenia will be outlined through the demonstration of 5-HT1A and 5-
HT2A receptors as key modulators within the spectrum of negative symptoms associated with
schizoaffective disorders, including a variety of disturbances in cognition, behavior, mood, social
function, perception of reality, and hormonal response to stressors. This paper will review the evidence
for attributing the risk of schizophrenia onset to early defects in serotonergic neurotransmission and
explore the perspective of selective serotonin receptor inhibitor (SSRI) pharmacotherapy as a method of
treatment and intervention for prodromal and ultra-high-risk patients by increasing 5-HT1A receptor
sensitivity levels and modifying the transcription of 5-HT1A receptor-associated gene expression in these
groups.

Keywords: prodrome, schizophrenia and other psychotic disorders, selective serotonin reuptake
inhibitor, receptor desensitization and downregulation, 5-hydroxytryptamine

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Editorial

Introduction
For several decades, dopamine (DA) has been considered the most important neurotransmitter in the
physiology and etiology of schizophrenia due to the blocking effect demonstrated by all traditional or
“typical” antipsychotics upon the dopamine D2 receptor. Although the dopamine hypothesis of
schizophrenia still remains the primary explanation behind the physiological basis and mechanism of
action in schizophrenia today, it has been increasingly demonstrated over time that a number of
additional neurotransmitter systems, including serotonin (5-HT), glutamate, gamma-aminobutyric acid
(GABA), opioids, and trace amines, are involved in the development of the disorder. 5-HT1A and 5-HT2A,
which are among the seven families of 5-HT receptors, are known to play a significant role in the
treatment of patients with various mood and/or psychotic disorders. Recent evidence for the naturally
occurring upregulation of 5-HT1A and downregulation of 5-HT2A receptors throughout the lifetime of
schizophrenic patients supports the role of serotonin as a primary factor in the developmental delay and
discourse of schizophrenia. New intervention strategies involving the functional relationship between 5-
HT1A and 5-HT2A receptors may be employed in the future to prevent, mitigate, or delay the onset of
positive and negative symptoms associated with full-blown schizophrenia before and after the first
episode of psychosis.

Pathogenesis and neurodevelopment

Evidence supporting the early predisposition for schizophrenia consists of a variety of structural
changes, including ventricular distension, increase in gliosis, reduced cortical folding, and loss of general
brain tissue. Furthermore, magnetic resonance imaging (MRI) studies have found that compared with
controls, patients with schizophrenia show significantly reduced folding in the anterior cingulate cortex
(ACC) that differs from conventional left ACC sulcal symmetry. Given that sulcal/gyral folding is almost
complete by the third trimester of gestation and remains relatively stable shortly after birth, the
structural anomalies found in ACC folding are likely to reflect early prenatal neurodevelopmental factors
in the etiology of schizophrenia [1].

Structural neuroimaging studies have revealed the limited progression of changes in the brain during the
prodromal, transitional, and onset stages of schizophrenia, thereby supporting the notion that
schizophrenia is a neurodevelopment disorder that may be characterized by early structural and
physiological abnormalities within the brain. Researchers have investigated brain structure in large
numbers of young people at-risk (AR) for the development of psychosis using MRI. Stone et al. (2011)
revealed that subjects at-risk for psychosis show significantly reduced activation in the left
parahippocampal gyrus during an episodic encoding task through fMRI and proton magnetic resonance
spectroscopy (1H-MRS) [2]. A separate comparison study between baseline and 12-month follow-up
scans for patients at-risk for psychosis indicates a reduction of gray matter in the left cerebellum. It is
unsurprising that the high-risk population had smaller volumes of the left hippocampus in comparison to
healthy controls; however, retrograde survival analyses have yielded unexpected results: the lack of
change in left hippocampal volume was associated with a future transition to psychosis, and a
significantly reduced left hippocampal volume was only seen in patients who not develop acute
psychotic symptoms [2]. These results imply that abnormalities in hippocampal volume and
transmission, among other structural defects, precede the clinical expression of psychosis and may serve
as predictive indicators for conversion to schizophrenia.
The ability to characterize premorbid neurostructural changes with the neurodevelopment of
schizophrenia or lack thereof supports the notion that prodromal, at-risk (AR), or ultra-high-risk (UHR)
groups may greatly benefit from early assessment and intervention strategies prior to the first episode
of psychosis.

Conclusion

The model of 5-HT as a significant guide for assessing prodromal predisposition to schizophrenia
suggests that pharmacotherapy using drugs that correct or restore disrupted 5-HT pathways and
abnormal receptor densities may be useful for the treatment of positive and negative symptoms in
prodromal and schizophrenic patients. Recently, early intervention approaches using atypical
antipsychotics and/or cognitive behavioral therapy have shown promising potential for decreasing the
duration of psychosis and improving short-term clinical outcomes. Although a pharmacogenetic test
response to clozapine has been proposed to predict response to treatment with antipsychotic drugs
[16], other methods of pharmacogenetic testing involving the 5-HT pathway should be developed to
predict response to treatment and likelihood for developing schizophrenia in prodromal and ultra-high-
risk patients. Such testing may help guide early intervention strategies towards the restoration or
correction of the most vulnerable brain regions involved during different stages of the illness.
Realistically, long-term studies that use a large sample of prodromal and ultra-high-risk patients with a
serotonergic component of vulnerability for developing schizophrenia are necessary to accurately
characterize the benefits and potential risks associated with this phase-specific psychopharmacological
intervention.

(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8294605/)

Candidate genes for schizophrenia (genes that encode products with neurobiological function such as
neurotransmitter receptors or enzymes) are commonly studied by way of association studies.
Pharmacological profiles have divided dopamine receptors into DRD1, DRD2, DRD3, DRD4 and DRD5
subtypes. DRD2 has been extensively studied as it shows high affinity for neuroleptics binding.2 DRD3 is
similar to DRD2 in that it shares common intronic sequences, cellular signaling pathways and has a
similar affinity for typical neuroleptics.14, 15 However, unlike DRD2, it shows a higher affinity for
atypical neuroleptics.16 DRD3 has a more restricted area of expression in the brain, which is known to
be associated with cognitive and emotional function.17

Association studies between DRD218, 19 and DRD320 receptor gene polymorphisms and schizophrenic
susceptibility have been carried out with conflicting results. The positive contribution of the A-206G
transition within exon 1 of the DRD3 receptor gene was first proposed by Lannnfelt21 and Crocq.20
More recent European multicenter reports also indicate the existence of a significant correlation
between increased homozygosity of either DRD3 allele and the disease; however, they present no
evidence of an allelic association.7, 22, 23 Although, controversial conclusions have been reported by a
significant number of other groups, which failed to replicate these findings of association with
homozygosity of the Ser9Gly variant.24, 25, 8, 9 In addition, thorough studies on the characterization,
mutation detection and association analysis of alternative promoters and 5′UTRs of the human DRD3
gene in schizophrenia imply that neither the coding nor the regulatory region of DRD3 plays a major role
in predisposition to schizophrenia.10 On the contrary, the contribution of the A-206G transition to the
development of Tardive Dyskinesia (TD) in schizophrenic patients treated with antipsychotics has been
maintained unanimously

With regard to DRD3 allele frequencies, no significant differences were obtained between controls and
patients. Nevertheless, our overall findings suggest that in our sample the genetic contribution of the A-
206 allele, presented in two copies in the genotype, might play a meaningful role in the appearance of
schizophrenia, as was proposed by other groups. However, it is worth to explore whether these
statistical differences result from true association or from nonsignificant departure from HW
distributions in controls or in cases.

Concerning the sample size, we recognize that was relative small; however, the main advantage of the
present study, in contrast to the other multicentre studies, was that our population was more
homogeneous, well-balanced and dispersed over a small geographical area and the results are likely to
provide a more accurate estimation of the frequency of these mutations in the 5-HTR2a and DRD3
genes.

(https://www.nature.com/articles/5201180)