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1.  Polymorphisms in GRIK4, HTR2A, and FKBP5 Show Interactive Effects in Predicting Remission to Antidepressant Treatment 
Neuropsychopharmacology  2009;35(3):727-740.
Single-nucleotide polymorphisms (SNPs) in the FKBP5, GRIK4, and HTR2A genes have been shown to be associated with response to citalopram treatment in the STAR*D sample, but only associations with FKBP5 have so far been tested in the Munich Antidepressant Response Signature (MARS) project. Response and remission of depressive symptoms after 5 weeks of antidepressant treatment were tested against 82 GRIK4 and 37 HTR2A SNPs. Association analysis was conducted in about 300 depressed patients from the MARS project, 10% of whom had bipolar disorder. The most predictive SNPs from these two genes and rs1360780 in FKBP5 were then genotyped in a total of 387 German depressed in-patients to analyze potential additive and interactive effects of these variants. We could not replicate previous findings of the Sequenced Treatment Alternatives to Relieve Depression (STAR*D) study in our sample. Although not statistically significant, the effect for the best GRIK4 SNP of STAR*D (rs1954787, p=0.076, pcorrected=0.98) seemed to be in the same direction. On the other hand, the nominally significant association with the top HTR2A SNPs of STAR*D (rs7997012, allelic, p=0.043, pcorrected=0.62) was with the opposite risk allele. The GRIK4 SNP (rs12800734, genotypic, p=0.0019, pcorrected=0.12) and the HTR2A SNP (rs17288723, genotypic, p=0.0011, pcorrected=0.02), which showed the strongest association with remission in our sample, had not been reported previously. Associations across all genetic markers within the GRIK4 (genotypic, p=0.022) or HTR2A (genotypic, p=0.012) locus using the Fisher's product method (FPM) were also significant. In all 374 patients, the best predictive model included a main effect for GRIK4 rs12800734 and two significant interactions between GRIK4 rs12800734 and FKBP5 rs1360780, and GRIK4 rs12800734 and HTR2A rs17288723. This three SNP model explained 13.1% of the variance for remission after 5 weeks (p=0.00051 for the model). Analyzing a sub-sample of 194 patients, plasma ACTH (p=0.002) and cortisol (p=0.021) responses of rs12800734 GG (GRIK4) carriers, who also showed favorable treatment response, were significantly lower in the second combined dexamethasone (dex)/corticotrophin-releasing hormone (CRH) test before discharge compared with the other two genotype groups. Despite large differences in ethnicity and design compared with the STAR*D study, our results from the MARS study further support both independent and interactive involvement of GRIK4, HTR2A and FKBP5 in antidepressant treatment response.
doi:10.1038/npp.2009.180
PMCID: PMC3055621  PMID: 19924111
GRIK4; HTR2A; FKBP5; interaction; antidepressant treatment response; affective disorder; Pharmacogenetics/Pharmacogenomics; Depression; Unipolar/Bipolar; Glutamate; Serotonin; GRIK4; HTR2A; FKBP5; pharmacogenetic; antidepressant treatment response
2.  Genetic Variation in HTR2A Influences Serotonin Transporter Binding Potential as Measured using PET and [11C]DASB 
In a previous study we showed that genetic variation in HTR2A, which encodes the serotonin 2A receptor, influenced outcome of citalopram treatment in patients with major depressive disorder (MDD). Since chronic administration of citalopram, which selectively and potently inhibits the serotonin transporter (5-HTT), putatively enhances serotonergic transmission, it is conceivable that genetic variation within HTR2A also influences pretreatment 5-HTT function or serotonergic transmission. The present study used positron emission tomography (PET) and the selective 5-HTT ligand, [11C]DASB, to investigate whether the HTR2A marker alleles that predict treatment outcome also predict differences in 5-HTT binding.
Brain levels of 5-HTT were assessed in vivo using PET measures of the nondisplaceable component of the [11C]DASB binding potential (BPND). DNA from 43 patients and healthy volunteers, all unmedicated, was genotyped with 14 single nucleotide polymorphisms (SNPs) located within or around HTR2A. Allelic association with BPND was assessed in 8 brain regions, with covariates to control for race and ethnicity.
We detected allelic association between [11C]DASB BPND in thalamus and 3 markers in a region spanning the 3′ untranslated region and second intron of HTR2A (rs7333412, p=0.000045; rs7997012, p= 0.000086; rs977003, p=0.000069). The association signal at rs7333412 remained significant (p<0.05) after applying corrections for multiple testing via permutation.
Genetic variation in HTR2A that previously was associated with citalopram treatment outcome also was associated with thalamic 5-HTT binding. While further work is needed to identify the actual functional genetic variants involved, these results suggest that a relationship exists between genetic variation in HTR2A and either 5-HTT expression or central serotonergic transmission that influences the therapeutic response to 5-HTT inhibition in major depression.
doi:10.1017/S1461145709991027
PMCID: PMC3810474  PMID: 20047709
Genetic Association; Positron Emission Tomography; Serotonin Transporter; [11C]DASB; HTR2A
3.  Multiple regulatory variants modulate expression of 5-hydroxytryptamine 2A receptors in human cortex 
Biological psychiatry  2012;73(6):546-554.
Background
The 5-hydroxytryptamine 2A receptor, encoded by HTR2A, is a major post-synaptic target for serotonin in the human brain and a therapeutic drug target. Despite hundreds of genetic associations investigating HTR2A polymorphisms in neuropsychiatric disorders and therapies, the role of genetic HTR2A variability in health and disease remains uncertain.
Methods
To discover and characterize regulatory HTR2A variants, we sequenced whole transcriptomes from ten human brain regions with massively-parallel RNA sequencing and measured allelic expression of multiple HTR2A mRNA transcript variants. Following discovery of functional variants, we further characterized their impact on genetic expression in vitro.
Results
Three polymorphisms modulate the use of novel alternative exons and untranslated regions (UTRs), changing expression of RNA and protein. The frequent promoter variant rs6311, widely implicated in human neuropsychiatric disorders, decreases usage of an upstream transcription start site encoding a longer 5′UTR with greater translation efficiency. rs76665058, located in an extended 3′UTR and unique to individuals of African descent, modulates allelic HTR2A mRNA expression. The third SNP, unannotated and present in only a single subject, directs alternative splicing of exon 2. Targeted analysis of HTR2A in the Sequenced Treatment Alternatives to Relieve Depression (STAR*D) study reveals associations between functional variants and depression severity or citalopram response.
Conclusions
Regulatory polymorphisms modulate HTR2A mRNA expression in an isoform-specific manner, directing the usage of novel untranslated regions and alternative exons. These results provide a foundation for delineating the role of HTR2A and serotonin signaling in CNS disorders.
doi:10.1016/j.biopsych.2012.09.028
PMCID: PMC3582836  PMID: 23158458
serotonin; 5-HT2A; HTR2A; schizophrenia; depression; mRNA expression
4.  Association Between 5HT1b Receptor Gene and Methamphetamine Dependence 
Current Neuropharmacology  2011;9(1):163-168.
Several lines of evidence implicate serotonergic dysfunction in diverse psychiatric disorders including anxiety, depression, and drug abuse. Mice with a knock-out of the 5HT1b receptor gene (HTR1B) displayed increased locomotor response to cocaine and elevated motivation to self-administer cocaine and alcohol. Previous genetic studies showed significant associations of HTR1B with alcohol dependence and substance abuse, but were followed by inconsistent results. We examined a case-control genetic association study of HTR1B with methamphetamine-dependence patients in a Japanese population. The subjects were 231 patients with methamphetamine dependence, 214 of whom had a co-morbidity of methamphetamine psychosis, and 248 age- and sex-matched healthy controls. The three single nucleotide polymorphisms (SNPs), rs130058 (A-165T), rs1228814 (A-700C) and rs1228814 (A+1180G) of HTR1B were genotyped. There was no significant difference in allelic and genotypic distributions of the SNPs between methamphetamine dependence and the control. Genetic associations of HTR1B were tested with several clinical phenotypes of methamphetamine dependence and/or psychosis, such as age at first abuse, duration of latency from the first abuse to onset of psychosis, prognosis of psychosis after therapy, and complication of spontaneous relapse of psychotic state. There was, however, no asscocation between any SNP and the clinical phenotypes. Haplotype analyses showed the three SNPs examined were within linkage disequilibrium, which implied that the three SNPs covered the whole HTR1B, and distribution of estimated haplotype frequency was not different between the groups. The present findings may indicate that HTR1B does not play a major role in individual susceptibility to methamphetamine dependence or development of methamphetamine-induced psychosis.
doi:10.2174/157015911795017137
PMCID: PMC3137174  PMID: 21886584
Methamphetamine dependence; association study; HTR1B; haplotype.
5.  Resequencing of serotonin-related genes and association of tagging SNPs to citalopram response 
Pharmacogenetics and genomics  2009;19(1):1-10.
Several reports have been published investigating the relationship between common variants in serotonin-related candidate genes and antidepressant response, and most of the results have been equivocal. We previously reported a significant association between variants in serotonin-related genes and response to the selective serotonin reuptake inhibitor fluoxetine. Here, we attempt to expand upon and replicate these results by (i) resequencing the exonic and putatively regulatory regions of five serotonin-related candidate genes (HTR1A, HTR2A, TPH1, TPH2, and MAOA) in our fluoxetine-treated sample to uncover novel variants; (ii) selecting tagging single nucleotide polymorphisms (SNPs) for these genes from the resequencing data; and (iii) evaluating these tagging SNPs for association with response to the selective serotonin reuptake inhibitor citalopram in an independent sample of participants who are enrolled in the Sequenced Treatment Alternatives to Relieve Depression (STAR*D) clinical study (N=1953). None of the variants associated previously with fluoxetine response were found to be associated with citalopram response in the STAR*D sample set. Nor were any of the additional tagging SNPs found to be associated with citalopram response. An additional SNP in HTR2A (rs7997012), previously reported to be associated with outcome of citalopram treatment in this sample, but not well tagged by any of the other SNPs we studied, was also genotyped, and was associated with citalopram response (P=0.0002), strongly supporting the previous observation in the same STAR*D sample. Our results suggest that resequencing the serotonin-related genes did not identify any additional common SNPs that have not been identified previously. It appears that genetic variation in these five genes has a marginal effect on response to citalopram, although a previously observed association was supported and awaits replication in an independent sample.
doi:10.1097/FPC.0b013e3283163ecd
PMCID: PMC2896826  PMID: 19077664
association; citalopram; fluoxetine; pharmacogenetics; single nucleotide polymorphism
6.  Association, interaction, and replication analysis of genes encoding serotonin transporter and 5-HT3 receptor subunits A and B in alcohol dependence 
Human genetics  2013;132(10):1165-1176.
On the basis of the converging evidence showing regulation of drinking behavior by 5-HT3AB receptors and the serotonin transporter, we hypothesized that the interactive effects of genetic variations in the genes HTR3A, HTR3B, and SLC6A4 confer greater susceptibility to alcohol dependence (AD) than do their effects individually. We examined the associations of AD with 22 SNPs across HTR3A, HTR3B, and two functional variants in SLC6A4 in 500 AD and 280 healthy control individuals of European descent. We found that the alleles of the low-frequency SNPs rs33940208:T in HTR3A and rs2276305:A in HTR3B were inversely and nominally significantly associated with AD with odds ratio (OR) and 95 % confidence interval of 0.212 and 0.073, 0.616 (P = 0.004) and 0.261 and 0.088, 0.777 (P = 0.016), respectively. Further, our gene-by-gene interaction analysis revealed that two four-variant models that differed by only one SNP carried a risk for AD (empirical P < 1 × 10−6 for prediction accuracy of the two models based on 106 permutations). Subsequent analysis of these two interaction models revealed an OR of 2.71 and 2.80, respectively, for AD (P < 0.001) in carriers of genotype combinations 5′-HTT LPR:LL/LS(SLC6A4)–rs1042173:TT/TG(SLC6A4)–rs117 6744:AC(HTR3B)–rs3782025:AG(HTR3B) and 5′-HTTL PR:LL/LS(SLC6A4)–rs10160548:GT/TT(HTR3A)–rs1176 744:AC(HTR3B)–rs3782025:AG(HTR3B). Combining all five genotypes resulted in an OR of 3.095 (P = 2.0 × 10−4) for AD. Inspired by these findings, we conducted the analysis in an independent sample, OZ-ALC-GWAS (N = 6699), obtained from the NIH dbGAP database, which confirmed the findings, not only for all three risk genotype combinations (Z = 4.384, P = 1.0 × 10−5; Z = 3.155, P = 1.6 × 10−3; and Z = 3.389, P = 7.0 × 10−4, respectively), but also protective effects for rs33940208:T (χ2 = 3.316, P = 0.0686) and rs2276305:A (χ2 = 7.224, P = 0.007). These findings reveal significant interactive effects among variants in SLC6A4–HTR3A– HTR3B affecting AD. Further studies are needed to confirm these findings and characterize the molecular mechanisms underlying these effects.
doi:10.1007/s00439-013-1319-y
PMCID: PMC3775919  PMID: 23757001
7.  Risk-Taking Behavior in a Gambling Task Associated with Variations in the Tryptophan Hydroxylase 2 Gene: Relevance to Psychiatric Disorders 
Neuropsychopharmacology  2009;35(5):1109-1119.
Decision making, choosing the best option from the possible outcomes, is impaired in many psychiatric conditions including affective disorders. We tested the hypothesis that variations in serotonergic genes (TPH2, TPH1, SLC6A4, HTR1A), which influence serotonin availability, affect choice behavior in a probabilistic gambling task. A population cohort (N=1035) completed a paper-and-pencil gambling task, filled out personality and symptom questionnaires and gave consent for the use of their DNA in a genetic association study. A subgroup of subjects (N=69) also completed a computer version of the task. The gambling task was designed to estimate an individual's tendency to take a risk when choosing between a smaller but more certain ‘win' and a larger, less probable one. We genotyped seven haplotype tagging SNPs in the TPH2 gene, and previously reported functional polymorphisms from the other genes (rs1800532, 5HTTLPR, and rs6295). Carriers of the more prevalent TPH2 haplotype, which was previously associated with less active enzyme variant, showed reduced risk taking on both tasks compared with subjects not carrying the common haplotype. The effect of TPH2 haplotypes on risk-taking was independent of current depression and anxiety symptoms, neuroticism and impulsiveness scores. We did not find an association between functional polymorphisms in the TPH1, SLC6A4, HTR1A genes and risk-taking behavior. In conclusion, our study demonstrates the role of the TPH2 gene and the serotonin system in risk taking and suggests that TPH2 gene may contribute to the expression of psychiatric phenotypes through altered decision making.
doi:10.1038/npp.2009.216
PMCID: PMC3055398  PMID: 20043001
risk taking; TPH2; haplotype analysis; depression; psychiatric disorders; Serotonin; Neurogenetics; Behavioral Science; Mood/Anxiety/Stress Disorders; risk-taking; TPH2; haplotype analysis
8.  Serotonin Transporter and Receptor Genes Significantly Impact Nicotine Dependence through Genetic Interactions in both European American and African American Smokers* 
Drug and alcohol dependence  2013;129(3):217-225.
Background
Pharmacologic studies implicate a significant role of genes encoding the serotonin transporter (SLC6A4) and the 5-HT3AB subunits HTR3A and HTR3B in nicotine dependence (ND). However, whether they are involved in ND remains largely unknown.
Methods
Here, we examined the impact of variations in the three genes on ND in 1366 individuals from 402 African American (AA) and 671 individuals from 200 European American (EA) families. The ND of each smoker was assessed with smoking quantity (SQ), Heaviness of Smoking Index (HSI), and Fagerström Test for Nicotine Dependence (FTND).
Results
Association analysis revealed marginal association of rs10160548 in HTR3A with SQ and HSI in AA, 5-HTTLPR in SLC6A4 with FTND in EA, and rs11606194 in HTR3B with SQ and FTND in the pooled sample. Haplotype-based association analysis revealed a few major haplotypes in HTR3A that were significantly associated with ND in the AA, EA, and pooled samples. However, none of these associations remained significant after correcting for multiple testing except for a haplotype GC-C-T-A-T formed by SNPs rs1150226, rs1062613, rs33940208, rs1985242, rs2276302, and rs10160548 in HTR3A for the AA sample. Considering biological functions of the three genes, we examined interactive effects of variants in the three genes, which revealed significant interactions among rs1062613 and rs10160548 in HTR3A, rs1176744 in HTR3B, and 5-HTTLPR and rs1042173 in SLC6A4 in affecting ND in the three samples.
Conclusions
We conclude that SLC6A4, HTR3A and HTR3B play a significant role in ND through genetic interactions.
doi:10.1016/j.drugalcdep.2012.12.007
PMCID: PMC3628090  PMID: 23290502
Serotonin transporter; serotonin receptor 3AB; nicotine dependence; African and European American; gene-by-gene interactions; epistatic
9.  Association Study Between the Serotonin 1A Receptor (HTR1A) Gene and Neuroticism, Major Depression, and Anxiety Disorders 
The serotonin neurotransmitter system in general, and the serotonin 1A receptor in particular, has been broadly implicated in the pathophysiology of mood and anxiety disorders, although the results of genetic association studies have been mixed. In this study, we examined the serotonin 1A receptor gene, HTR1A, for its association with shared genetic risk across a range of anxiety and depression-related phenotypes. Using multivariate structural equation modeling, we selected twin pairs from the population-based Virginia Adult Twin Study of Psychiatric and Substance Use Disorders scoring at the extremes of a latent genetic risk factor that underlies susceptibility to neuroticism, major depression, and several anxiety disorders. One member from each selected pair was entered into a 2-stage, case-control association study for the HTR1A gene. In the resulting sample of 589 cases and 539 controls, four SNPs spanning the HTR1A locus, including the C(−1019)G functional promoter polymorphism (rs6295), were screened in stage 1, the positive results of which were tested for replication in stage 2. While one marker met threshold significance criteria in stage 1, this association was not replicated in stage 2. Post-hoc analyses did not reveal association to any of the specific psychiatric phenotypes. Our data suggests that the HTR1A gene may not play a major role in the genetic susceptibility underlying depressive and anxiety-related phenotypes.
doi:10.1002/ajmg.b.30656
PMCID: PMC2859302  PMID: 18163385
serotonin; depression; anxiety; personality; association study; genetics
10.  Association of serotonin-1A and 2A receptor promoter polymorphisms with depressive symptoms and functional recovery in elderly persons after hip fracture 
Journal of affective disorders  2008;111(1):61-66.
Background
Depression is common after hip fracture and is associated with poorer functional recovery. Polymorphisms of the serotonin 1a (5HTR1A) and 2a receptors (5HTR2A) are associated with depression; therefore, we examined their association with depressive symptoms and functional recovery after hip fracture.
Methods
145 elderly women were followed for 12 months after hip fracture. Depressive symptoms were measured with the 15-item Geriatric Depression Scale (GDS). Functional status was measured by Lower Extremity Physical and Instrumental Activity of Daily Living scales (LPADLs and IADLs). Time-adjusted general linear regression models compared mean GDS between those with and without risk alleles for 5HTR1A and 5HTR2A.
Results
Women with 1–2 copies of the 5HTR1A (−1019) G allele had higher GDS scores (Adjusted Mean Difference=0.59; 95% CI, 0.12–1.06), and poorer IADL scores (Adjusted Mean Difference=0.24; 95%CI −0.002–0.49), compared to those without this allele, controlling for potential confounders and 5HTR2A. Depressive symptoms partly accounted for poorer IADL recovery. Women with 1–2 copies of the 5HTR2A (−1438) C allele did not have significantly higher GDS scores (Adjusted Mean Difference=0.34; 95%CI, −0.20–0.87) and had better IADL scores (Adjusted Mean Difference=−0.40; 95%CI −0.74--0.06) than those with A/A genotype.
Limitations
The findings are limited by small sample size and the use of a screening scale to measure depression.
Conclusions
The 5HTR1A (−1019) G allele is associated with increased depressive symptoms after hip fracture, which in turn accounts for poorer functional recovery. These results suggest a role for serotonergic genetic variation in elderly persons’ resilience and recovery from medical events.
doi:10.1016/j.jad.2008.02.005
PMCID: PMC2610462  PMID: 18334271
11.  HTR3B is associated with alcoholism with antisocial behavior and alpha EEG power—an intermediate phenotype for alcoholism and co-morbid behaviors 
Alcohol (Fayetteville, N.Y.)  2009;43(1):73-84.
Alcohol use disorders (AUD) with co-morbid antisocial personality disorder (ASPD) have been associated with serotonin (5-HT) dysfunction. 5-HT3 receptors are potentiated by ethanol and appear to modulate reward. 5-HT3 receptor antagonists may be useful in the treatment of early-onset alcoholics with co-morbid ASPD. Low-voltage alpha electroencephalogram (EEG) power, a highly heritable trait, has been associated with both AUD and ASPD. A recent whole genome linkage scan in one of our samples, Plains American Indians (PI), has shown a suggestive linkage peak for alpha power at the 5-HT3R locus. We tested whether genetic variation within the HTR3A and HTR3B genes influences vulnerability to AUD with comorbid ASPD (AUD + ASPD) and moderates alpha power. Our study included three samples: 284 criminal alcoholic Finnish Caucasians and 234 controls; two independent community-ascertained samples with resting EEG recordings: a predominantly Caucasian sample of 191 individuals (Bethesda) and 306 PI. In the Finns, an intronic HTR3B SNP rs3782025 was associated with AUD + ASPD (P = .004). In the Bethesda sample, the same allele predicted lower alpha power (P = 7.37e-5). Associations between alpha power and two other HTR3B SNPs were also observed among PI (P = .03). One haplotype in the haplotype block at the 3′ region of the gene that included rs3782025 was associated with AUD + ASPD in the Finns (P = .02) and with reduced alpha power in the Bethesda population (P = .00009). Another haplotype in this block was associated with alpha power among PI (P = .03). No associations were found for HTR3A. Genetic variation within HTR3B may influence vulnerability to develop AUD with comorbid ASPD. 5-HT3R might contribute to the imbalance between excitation and inhibition that characterize the brain of alcoholics.
doi:10.1016/j.alcohol.2008.09.005
PMCID: PMC2754808  PMID: 19185213
Antisocial Personality Disorder; Serotonin; HTR3; Alcoholism; EEG
12.  A Putatively Functional Polymorphism in the HTR2C Gene is Associated with Depressive Symptoms in White Females Reporting Significant Life Stress 
PLoS ONE  2014;9(12):e114451.
Psychosocial stress is well known to be positively associated with subsequent depressive symptoms. Cortisol response to stress may be one of a number of biological mechanisms that links psychological stress to depressive symptoms, although the precise causal pathway remains unclear. Activity of the x-linked serotonin 5-HTR2C receptor has also been shown to be associated with depression and with clinical response to antidepressant medications. We recently demonstrated that variation in a single nucleotide polymorphism on the HTR2C gene, rs6318 (Ser23Cys), is associated with different cortisol release and short-term changes in affect in response to a series of stress tasks in the laboratory. Based on this observation, we decided to examine whether rs6318 might moderate the association between psychosocial stress and subsequent depressive symptoms. In the present study we use cross-sectional data from a large population-based sample of young adult White men (N = 2,366) and White women (N = 2,712) in the United States to test this moderation hypothesis. Specifically, we hypothesized that the association between self-reported stressful life events and depressive symptoms would be stronger among homozygous Ser23 C females and hemizygous Ser23 C males than among Cys23 G carriers. In separate within-sex analyses a genotype-by-life stress interaction was observed for women (p = .022) but not for men (p = .471). Homozygous Ser23 C women who reported high levels of life stress had depressive symptom scores that were about 0.3 standard deviations higher than female Cys23 G carriers with similarly high stress levels. In contrast, no appreciable difference in depressive symptoms was observed between genotypes at lower levels of stress. Our findings support prior work that suggests a functional SNP on the HTR2C gene may confer an increased risk for depressive symptoms in White women with a history of significant life stress.
doi:10.1371/journal.pone.0114451
PMCID: PMC4267787  PMID: 25514629
13.  The functional −1019C/G HTR1A polymorphism and mechanisms of fear 
Translational Psychiatry  2014;4(12):e490-.
Serotonin receptor 1A gene (HTR1A) knockout mice show pronounced defensive behaviour and increased fear conditioning to ambiguous conditioned stimuli. Such behaviour is a hallmark of pathological human anxiety, as observed in panic disorder with agoraphobia (PD/AG). Thus, variations in HTR1A might contribute to neurophysiological differences within subgroups of PD/AG patients. Here, we tested this hypothesis by combining genetic with behavioural techniques and neuroimaging. In a clinical multicentre trial, patients with PD/AG received 12 sessions of manualized cognitive-behavioural therapy (CBT) and were genotyped for HTR1A rs6295. In four subsamples of this multicentre trial, exposure behaviour (n=185), defensive reactivity measured using a behavioural avoidance test (BAT; before CBT: n=245; after CBT: n=171) and functional magnetic resonance imaging (fMRI) data during fear conditioning were acquired before and after CBT (n=39). HTR1A risk genotype (GG) carriers more often escaped during the BAT before treatment. Exploratory fMRI results suggest increased activation of the amygdala in response to threat as well as safety cues before and after treatment in GG carriers. Furthermore, GG carriers demonstrated reduced effects of CBT on differential conditioning in regions including the bilateral insulae and the anterior cingulate cortex. Finally, risk genotype carriers demonstrated reduced self-initiated exposure behaviour to aversive situations. This study demonstrates the effect of HTR1A variation on defensive behaviour, amygdala activity, CBT-induced neural plasticity and normalization of defence behaviour in PD/AG. Our results, therefore, translate evidence from animal studies to humans and suggest a central role for HTR1A in differentiating subgroups of patients with anxiety disorders.
doi:10.1038/tp.2014.130
PMCID: PMC4270311  PMID: 25514753
14.  Effects of HTR1A C(-1019)G on Amygdala Reactivity & Trait Anxiety 
Archives of general psychiatry  2009;66(1):33-40.
Background
5-HT1A autoreceptors mediate negative feedback inhibition of serotonergic neurons and play a critical role in regulating 5-HT signaling involved in shaping the functional response of major forebrain targets, such as the amygdala, supporting complex behavioral processes. A common functional variation (C(-1019)G) in the human 5-HT1A gene (HTR1A) represents one potential source of such inter-individual variability. Both in vitro and in vivo the -1019G blocks transcriptional repression leading to increased autoreceptor expression. Thus, the -1019G may contribute to relatively decreased 5-HT signaling at postsynaptic forebrain target sites via increased negative feedback.
Objectives & Design
To use imaging genetics to evaluate the effects of HTR1A C(-1019)G on amygdala reactivity in 89 healthy adults and employ path analyses to explore the impact of HTR1A-mediated variability in amygdala reactivity on individual differences in trait anxiety. We hypothesized that the -1019G, which potentially results in decreased 5-HT signaling, would be associated with relatively decreased amygdala reactivity and related trait anxiety.
Results
Consistent with prior findings, the -1019G was associated with significantly decreased threat-related amygdala reactivity. Importantly, this effect was independent of that associated with another common functional polymorphism impacting 5-HT signaling, namely the 5-HTTLPR. While there were no direct genotype effects on trait anxiety, the HTR1A C(-1019)G indirectly predicted 9.2% of interindividual variability in trait anxiety through its effects on amygdala reactivity.
Conclusions
Our findings further implicate relatively increased 5-HT signaling, associated with genetic variation mediating increased 5-HT1A autoreceptors, in driving amygdala reactivity and trait anxiety. Moreover, they provide empirical documentation of the basic premise that genetic variation impacts emergent behavioral processes related to psychiatric disease risk indirectly by biasing the response of underlying neural circuitries.
doi:10.1001/archpsyc.66.1.33
PMCID: PMC2736132  PMID: 19124686
15.  Evidence for interaction between 5-hydroxytryptamine (serotonin) receptor 2A and MHC type II molecules in the development of rheumatoid arthritis 
It has repeatedly been suggested that the development of complex diseases can be elucidated by gene–gene interactions. Recently, we found that HTR2A, a member of the serotonin receptor family, is associated with rheumatoid arthritis (RA). This study was aimed to investigate the possibility of a gene–gene interaction between HTR2A and the major genetic risk factor for RA, HLA-DRB1 shared epitope (SE) alleles. We studied 4095 RA cases and 3223 controls from three different populations – from Sweden, the United States and the Netherlands – to test for interaction between the protective HTR2A haplotype and HLA-DRB1 SE alleles. Further, we analyzed mRNA and/or protein expression of HTR2A and HLA-DR in biopsy samples and in synovial fibroblasts from RA patients. The interaction was defined as departure from additivity of effects using attributable proportion due to interaction. First, we could demonstrate and further replicate an interaction between a protective haplotype in HTR2A and HLA-DRB1 SE alleles regarding risk of developing autoantibody-positive RA. Second, we could show that both genes are constitutively expressed in fibroblasts from synovial tissue of RA patients, and, by double immunofluorescence staining, we demonstrated that these two proteins are colocalized in these cells. In conclusion, our data demonstrate a statistical interaction between HTR2A and HLA-DRB1 SE alleles and colocalization of the product of these two genes in inflamed synovial tissue, which suggest a possible biological relationship between these two proteins. This finding may lead to the development of treatment based on enhancing the protective features of 5-HT2A in individuals with a certain HLA genotype.
doi:10.1038/ejhg.2010.12
PMCID: PMC2987366  PMID: 20179740
serotonin receptor; MHC class II; RA; gene–gene interaction
16.  Patterns of genetic variation and the role of selection in HTR1A and HTR1B in macaques (Macaca) 
BMC Genetics  2014;15(1):116.
Background
Research has increasingly highlighted the role of serotonin in behavior. However, few researchers have examined serotonin in an evolutionary context, although such research could provide insight into the evolution of important behaviors. The genus Macaca represents a useful model to address this, as this genus shows a wide range of behavioral variation. In addition, many genetic features of the macaque serotonin system are similar to those of humans, and as common models in biomedical research, knowledge of the genetic variation and evolution of serotonin functioning in macaques are particularly relevant for studies of human evolution. Here, we examine the role of selection in the macaque serotonin system by comparing patterns of genetic variation for two genes that code for two types of serotonin receptors – HTR1A and HTR1B – across five species of macaques.
Results
The pattern of variation is significantly different for HTR1A compared to HTR1B. Specifically, there is an increase in between-species variation compared to within-species variation for HTR1A. Phylogenetic analyses indicate that portions of HTR1A show an elevated level of nonsynonymous substitutions. Together these analyses are indicative of positive selection acting on HTR1A, but not HTR1B. Furthermore, the haplotype network for HTR1A is inconsistent with the species tree, potentially due to both deep coalescence and selection.
Conclusions
The results of this study indicate distinct evolutionary histories for HTR1A and HTR1B, with HTR1A showing evidence of selection and a high level of divergence among species, a factor which may have an impact on biomedical research that uses these species as models. The wide genetic variation of HTR1A may also explain some of the species differences in behavior, although further studies on the phenotypic effect of the sequenced polymorphisms are needed to confirm this.
Electronic supplementary material
The online version of this article (doi:10.1186/s12863-014-0116-5) contains supplementary material, which is available to authorized users.
doi:10.1186/s12863-014-0116-5
PMCID: PMC4228068  PMID: 25376878
Serotonin; Macaca; Molecular evolution; HTR1A; HTR1B; Genetic variation
17.  The serotonin 1A receptor C(-1019)G polymorphism in relation to suicide attempt 
Background
Serotonergic neurotransmission has been implicated in suicidal behavior. Association between suicidal completers and a regulatory C(-1019)G polymorphism (rs6295) in the serotonin 1A receptor (HTR1A) gene was previously reported, whereas a following study showed no association in a sample of suicide attempters.
Methods
The involvement of the implicated G-allele of the 5-HTR1A C(-1019)G polymorphism (rs6295) was analyzed with the transmission disequilibrium test (TDT) in a sample of 272 suicide attempter families.
Results
No overtransmission of the G-allele was found in the entire sample of suicide attempters (p = 0.1460; n = 272 trios). However, a strong trend for overtransmission of the G-allele was observed in a sub-sample selected for a high level of previous traumatic and/or stressful life events prior to the suicide attempt (p = 0.0630, two-tail; n = 94 trios).
Conclusion
The current results show that variation at the rs6295 polymorphism of the HTR1A gene is not associated with suicide attempts generally. However, the results indicate a possible role of the G-allele in suicidal behavior in connection with high exposure to traumatic and/or stressful life events, which is in need of future investigation.
doi:10.1186/1744-9081-2-14
PMCID: PMC1459178  PMID: 16626484
18.  Transcriptional dysregulation of 5-HT1A autoreceptors in mental illness 
Molecular Brain  2011;4:21.
The serotonin-1A (5-HT1A) receptor is among the most abundant and widely distributed 5-HT receptors in the brain, but is also expressed on serotonin neurons as an autoreceptor where it plays a critical role in regulating the activity of the entire serotonin system. Over-expression of the 5-HT1A autoreceptor has been implicated in reducing serotonergic neurotransmission, and is associated with major depression and suicide. Extensive characterization of the transcriptional regulation of the 5-HT1A gene (HTR1A) using cell culture systems has revealed a GC-rich "housekeeping" promoter that non-selectively drives its expression; this is flanked by a series of upstream repressor elements for REST, Freud-1/CC2D1A and Freud-2/CC2D1B factors that not only restrict its expression to neurons, but may also regulate the level of expression of 5-HT1A receptors in various subsets of neurons, including serotonergic neurons. A separate set of allele-specific factors, including Deaf1, Hes1 and Hes5 repress at the HTR1A C(-1019)G (rs6295) polymorphism in serotonergic neurons in culture, as well as in vivo. Pet1, an obligatory enhancer for serotonergic differentiation, has been identified as a potent activator of 5-HT1A autoreceptor expression. Taken together, these results highlight an integrated regulation of 5-HT1A autoreceptors that differs in several aspects from regulation of post-synaptic 5-HT1A receptors, and could be selectively targeted to enhance serotonergic neurotransmission.
doi:10.1186/1756-6606-4-21
PMCID: PMC3130656  PMID: 21619616
19.  Serotonin receptor 3A polymorphism c.-42C > T is associated with severe dyspepsia 
BMC Medical Genetics  2011;12:140.
Background
The association between anxiety and depression related traits and dyspepsia may reflect a common genetic predisposition. Furthermore, genetic factors may contribute to the risk of having increased visceral sensitivity, which has been implicated in dyspeptic symptom generation. Serotonin (5-HT) modulates visceral sensitivity by its action on 5-HT3 receptors. Interestingly, a functional polymorphism in HTR3A, encoding the 5-HT3 receptor A subunit, has been reported to be associated with depression and anxiety related traits. A functional polymorphism in the serotonin transporter (5-HTT), which terminates serotonergic signalling, was also found associated with these psychiatric comorbidities and increased visceral sensitivity in irritable bowel syndrome, which coexistence is associated with higher dyspeptic symptom severity. We investigated the association between these functional polymorphisms and dyspeptic symptom severity.
Methods
Data from 592 unrelated, Caucasian, primary care patients with dyspepsia participating in a randomised clinical trial comparing step-up and step-down antacid drug treatment (The DIAMOND trial) were analysed. Patients were genotyped for HTR3A c.-42C > T SNP and the 44 bp insertion/deletion polymorphism in the 5-HTT promoter (5-HTTLPR). Intensity of 8 dyspeptic symptoms at baseline was assessed using a validated questionnaire (0 = none; 6 = very severe). Sum score ≥20 was defined severe dyspepsia.
Results
HTR3A c.-42T allele carriers were more prevalent in patients with severe dyspepsia (OR 1.50, 95% CI 1.06-2.20). This association appeared to be stronger in females (OR 2.05, 95% CI 1.25-3.39) and patients homozygous for the long (L) variant of the 5-HTTLPR genotype (OR 2.00, 95% CI 1.01-3.94). Females with 5-HTTLPR LL genotype showed the strongest association (OR = 3.50, 95% CI = 1.37-8.90).
Conclusions
The HTR3A c.-42T allele is associated with severe dyspeptic symptoms. The stronger association among patients carrying the 5-HTTLPR L allele suggests an additive effect of the two polymorphisms. These results support the hypothesis that diminished 5-HT3 mediated antinociception predisposes to increased visceral sensitivity of the gastrointestinal tract. Moreover, the HTR3A c.-42C > T and 5-HTTLPR polymorphisms likely represent predisposing genetic variants in common to psychiatric morbidity and dyspepsia.
doi:10.1186/1471-2350-12-140
PMCID: PMC3213216  PMID: 22014438
20.  Polymorphisms of the HTR1a Allele are linked to Frontal Brain Electrical Asymmetry 
Biological psychology  2009;83(2):153-158.
Polymorphic variations in genes related to serotonin synthesis, transport, recognition, or degradation may convey subtle changes in serotonin system architecture that may place an individual at risk for psychopathology when faced with life stressors. The relationship between three key serotonin alleles and frontal brain electrical asymmetry, a putative endophenotype of depression, was examined. Risk alleles were hypothesized to predict relatively greater right frontal brain activity regardless of current clinical state. A sample of 313 college-age individuals, spanning a range of depressive severity from no symptomotology to clinically meaningful levels, participated. Resting encephalographic (EEG) activity was recorded from 64 scalp sites on four occasions separated by at least 24 hours (two 8-min recording sessions occurring at each occasion). Alpha power asymmetry scores between homologous sites were calculated for each session and then averaged to form a trait metric of asymmetry for each pair. PCR based genotyping was conducted for the HTR1A, HTR2A, and HTTLPR genes. Variations in the HTR1A gene were related to trait EEG asymmetry, regardless of any history of depression. Compared to subjects with at least one non-risk allele, subjects with homozygous HTR1A risk alleles had significantly greater relative right frontal activity at sites F7/F8, F5/F6, & F1/F2. In conclusion, variation in HTR1A can influence trait level brain activity, which may ultimately be indicative of risk for psychopathology.
doi:10.1016/j.biopsycho.2009.12.002
PMCID: PMC2845287  PMID: 20025927
Serotonin; EEG; Asymmetry; HTR1a; Endophenotype; Gene
21.  The Haemophilus influenzae HtrA Protein Is a Protective Antigen 
Infection and Immunity  1998;66(3):899-906.
The htrA gene from two strains of nontypeable Haemophilus influenzae has been cloned and sequenced, and the encoded approximately 46-kDa HtrA proteins were found to be highly conserved. H. influenzae HtrA has approximately 55% identity with the Escherichia coli and Salmonella typhimurium HtrA stress response proteins, and expression of the H. influenzae htrA gene was inducible by high temperature. Recombinant HtrA (rHtrA) was expressed from E. coli, and the purified protein was found to have serine protease activity. rHtrA was found to be very immunogenic and partially protective in both the passive infant rat model of bacteremia and the active chinchilla model of otitis media. Immunoblot analysis indicated that HtrA is antigenically conserved in encapsulated and nontypeable H. influenzae species. Site-directed mutagenesis was performed on the htrA gene to ablate the endogenous serine protease activity of wild-type HtrA, and it was found that eight of nine recombinant mutant proteins had no measurable residual proteolytic activity. Two mutant proteins were tested in the animal protection models, and one, H91A, was found to be partially protective in both models. H91A HtrA may be a good candidate antigen for a vaccine against invasive H. influenzae type b disease and otitis media and is currently in phase I clinical trials.
PMCID: PMC107993  PMID: 9488373
22.  Serotonin-1A Receptor Polymorphism (rs6295) Associated with Thermal Pain Perception 
PLoS ONE  2012;7(8):e43221.
Background
Serotonin (5-HT) is highly involved in pain regulation and serotonin-1A (5-HT1A) receptors are important in determining central 5-HT tone. Accordingly, variation in the 5-HT1A receptor gene (HTR1A) may contribute to inter-individual differences in human pain sensitivity. The minor G-allele of the HTR1A single nucleotide polymorphism (SNP) rs6295 attenuates firing of serotonergic neurons and reduces postsynaptic expression of the receptor. Experiments in rodents suggest that 5-HT1A-agonism modulates pain in opposite directions at mild compared to high noxious intensities. Based upon this and several other similar observations, we hypothesized that G-carriers would exhibit a relative hypoalgesia at mild thermal stimuli but tend towards hyperalgesia at higher noxious intensities.
Methods
Fourty-nine healthy individuals were selectively genotyped for rs6295. Heat- and cold-pain thresholds were assessed along with VAS-ratings of a range of suprathreshold noxious heat intensities (45°C–49°C). Nociceptive-flexion reflex (NFR) thresholds were also assessed.
Results
Volunteers did not deviate significantly from Hardy-Weinberg equilibrium. G-carriers were less sensitive to threshold-level thermal pain. This relative hypoalgesia was abolished at suprathreshold noxious intensities where G-carriers instead increased their ratings of heat-pain significantly more than C-homozygotes. No differences with regard to NFR-thresholds emerged.
Conclusion/Significance
To the best of our knowledge this is the first study of human pain perception on the basis of variation in HTR1A. The results illustrate the importance of including a range of stimulus intensities in assessments of pain sensitivity. In speculation, we propose that an attenuated serotonergic tone may be related to a ‘hypo- to hyperalgesic’ response-pattern. The involved mechanisms could be of clinical interest as variation in pain regulation is known to influence the risk of developing pain pathologies. Further investigations are therefore warranted.
doi:10.1371/journal.pone.0043221
PMCID: PMC3432037  PMID: 22952650
23.  Polymorphisms of Genes in Neurotransmitter Systems Were Associated with Alcohol Use Disorders in a Tibetan Population 
PLoS ONE  2013;8(11):e80206.
Studies of linkage and association in various ethnic populations have revealed many predisposing genes of multiple neurotransmitter systems for alcohol use disorders (AUD). However, evidence often is contradictory regarding the contribution of most candidate genes to the susceptibility of AUD. We, therefore, performed a case-control study to investigate the possible associations of genes selected from multiple neurotransmitter systems with AUD in a homogeneous Tibetan community population in China. AUD cases (N = 281) with an alcohol use disorder identification test (AUDIT) score ≥10, as well as healthy controls (N = 277) with an AUDIT score ≤5, were recruited. All participants were genotyped for 366 single nucleotide polymorphisms (SNPs) of 34 genes selected from those involved in neurotransmitter systems. Association analyses were performed using PLINK version 1.07 software. Allelic analyses before adjustment for multiple tests showed that 15 polymorphisms within seven genes were associated with AUD (p<0.05). After adjustment for the number of SNPs genotyped within each gene, only the association of a single marker (rs10044881) in HTR4 remained statistically significant. Haplotype analysis for two SNPs in HTR4 (rs17777298 and rs10044881) showed that the haplotype AG was significantly associated with the protective effect for AUD. In conclusion, the present study discovered that the HTR4 gene may play a marked role in the pathogenesis of AUD. In addition, this Tibetan population sample marginally replicated previous evidence regarding the associations of six genes in AUD.
doi:10.1371/journal.pone.0080206
PMCID: PMC3842251  PMID: 24312204
24.  HUMAN HtrA1 IN THE ARCHIVED EYES WITH AGE-RELATED MACULAR DEGENERATION 
Purpose
HtrA1 belongs to the high temperature requirement factor A family of serine proteases, which are involved in protein quality control and cell fate. A single-nucleotide polymorphism (SNP), rs11200638, in the promoter of HtrA1 at chromosome 10q26 is reported as a likely causal variant for age-related macular degeneration (AMD). The SNP is located in the regulatory region and increases production of HtrA1 protein. This study investigates HtrA1 expression and SNP genotypes in archived ocular slides with AMD.
Methods
Macular, nonretinal, and peripheral retinal cells were microdissected from archived slides from 57 eyes with AMD and 16 age-matched, non-AMD controls. HtrA1 rs11200638 SNP genotyping was performed using polymerase chain reaction (PCR) and restriction fragment length polymorphism analysis. HtrA1 transcripts were measured using real-time reverse transcriptase–PCR. HtrA1 protein expression was evaluated using avidin-biotin complex immunohistochemistry.
Results
HtrA1 (G/A) SNP was successfully genotyped in 52 AMD cases and 13 non-AMD subjects. The frequencies of the risk allele (A) were 55 of 104 (52.9%) and 8 of 26 (30.8%) in AMD and control groups, respectively. HtrA1 mRNA was detected in normal peripheral and macular retinas, higher in the periphery than maculae. HtrA1 mRNA was much higher in the macula and a lot lower in the periphery of the AMD eyes as compared to control eyes. HtrA1 protein was expressed in normal retinal vascular endothelia and retinal pigment epithelia. Intense immunoreaction against HtrA1 was found in AMD lesions, slightly more in wet than dry AMD lesions.
Conclusion
This study successfully analyzes HtrA1 SNP and transcript expression in microdissected cells from archived paraffin fixed slides. Up-regulation of HtrA1 is detected in the macular lesions of AMD eyes. The data further suggest that rs11200638 in HtrA1 promoter is associated with AMD development.
PMCID: PMC2258134  PMID: 18427598
25.  AP-1 Transcription Factors, Mucin-Type Molecules and MMPs Regulate the IL-11 Mediated Invasiveness of JEG-3 and HTR-8/SVneo Trophoblastic Cells 
PLoS ONE  2012;7(1):e29745.
This study examines the IL-11 mediated activation of downstream signaling and expression of effector molecules to resolve the controversies associated with the IL-11 mediated regulation of the invasiveness of two commonly used trophoblastic cell models viz. JEG-3 and HTR-8/SVneo cells. It has been reported that IL-11 increases the invasiveness of JEG-3 cells while, reduces the invasiveness of HTR-8/SVneo cells. Invasion assay performed simultaneously for both the cell lines confirmed the above findings. In addition, HTR-8/SVneo cells showed a higher basal invasiveness than JEG-3 cells. Western blot showed the IL-11 mediated activation of STAT3(tyr705) and STAT1(tyr701) in both the cell lines. However, IL-11 activated the ERK1/2 phosphorylation in JEG-3 cells but, inhibited it in HTR-8/SVneo cells. Within 10 min of IL-11 treatment, p-STAT3(tyr705) was localized inside the nucleus of both the cell lines but, there was enhanced co-localization of protein inhibitor of activated STAT1/3 (PIAS1/3) and p-STAT3(tyr705) in HTR-8/SVneo cells and not in JEG-3 cells. This could be reason for the poor responsiveness of STAT3 responsive genes like mucin 1 (MUC1) in HTR-8/SVneo cells and not in JEG-3 cells. Further, microarray analysis of the IL-11 treated cells revealed differential responsiveness of JEG-3 as compared to HTR-8/SVneo cells. Several family of genes like activator protein-1 (AP-1) transcription factors (Jun and Fos), mucin-type molecules, MMP23B etc showed enhanced expression in IL-11 treated JEG-3 cells while, there was no response or decrease in their expression in IL-11 treated HTR-8/SVneo cells. Expression of these molecules was confirmed by quantitative RT-PCR. In addition, HTR-8/SVneo cells also showed a significant decrease in the expression of MMP2, MMP3 and MMP9 upon IL-11 treatment. Hence, IL-11 mediated differential activation of signaling and expression of effector molecules is responsible for the differential invasive response of JEG-3 and HTR-8/SVneo cells.
doi:10.1371/journal.pone.0029745
PMCID: PMC3250480  PMID: 22235337

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