Impaired brain serotonin neurotransmission is a potential component of the diathesis of major depression. Tryptophan hydroxylase-2 (TPH2), is the rate limiting biosynthetic isoenzyme for serotonin that is preferentially expressed in the brain and a cause of impaired serotonin transmission. Here, we identify a novel TPH2 short isoform with truncated catalytic domain expressed in human brainstem, prefrontal cortex, hippocampus and amygdala. An exploratory study of 166 Caucasian subjects revealed association with major depression or suicide of a novel single nucleotide polymorphism (SNP) g.22879A>G located in exon 6 of this short isoform. This SNP and additional SNPs were discovered through a systematic characterization of the genetic architecture of the TPH2 gene for further genetic and functional investigations of its relationship to major depression and other psychopathology.
In the brain, serotonin is synthesized in the cell bodies of serotonergic neurons in the brainstem raphe nuclei that project to most cortical and subcortical structures. These brain regions include the prefrontal cortex, anterior cingulate, amygdala, hippocampus and thalamus, structures involved in regulating mood, memory and behavior and manifesting altered activity in major depression.1 Alterations in serotonergic function are implicated in the pathogenesis of major depression and in the diathesis for suicidal behavior.2, 3 Since genetic factors can contribute causally to both major depression and the proclivity to commit suicide4, 5 and since the serotonergic system is partially regulated by genetic factors,6, 7 candidate genes for both major depression and suicidal behavior could involve the serotonergic system. Although levels of serotonin and/or its main metabolite 5-hydroxyindoleacetic acid are reported to be low in suicides,8 we find that neuronal tryptophan hydroxylase-2, the rate-limiting serotonergic biosynthetic isoenzyme, is elevated in the dorsal raphe nuclei of depressed suicides at both the transcript9 and protein levels.10, 11 To reconcile these observations, we had suggested the presence of a gene variant that may affect the catalytic site resulting in the production of a lower activity enzyme. This hypothesis is consistent with recent reports of such a tryptophan hydroxylase-2 (TPH2) gene variant expressing an isoenzyme with impaired catalytic activity in the Balb/c mouse, an inbred strain that naturally exhibits a depressive behavioral phenotype and has low brain serotonin. Balb/c is reported to have a novel coding variant in a homologous region of a TPH2 human ortholog with apparent impaired catalytic activity.12 However, the report that this type of variant is present in 10% of human subjects diagnosed with unipolar depression has not been replicated in other datasets.13, 14, 15, 16, 17
We investigated the role of TPH2 in major depression and suicidal behavior using a collection of case and control postmortem brain samples. Through psychological autopsies using validated structured clinical interviews with family members18 we obtained detailed clinical information, including axis I and II psychiatric diagnoses based on DSM-IV diagnostic criteria. Recent studies report associations of TPH2 with major depression and with suicide, involving single nucleotide polymorphisms (SNPs) in intron 5 and exon 11.12, 13, 19, 20 Thus, while we explored the coding portion of the TPH2 gene and sections of each intron, we focused our analyses on the SNPs that lie in these two regions in an effort to replicate the previously reported positive findings in our dataset. In addition, we report on the first systematic examination of the TPH2 gene variants covering both the coding and non-coding regions and transcript variants in human brain.
Materials and methods
Samples and subjects
Brain tissue was obtained from autopsies conducted for medical or forensic reasons unrelated to the study. Next of kin gave informed consent to the use of brain tissue for research and to undergo a psychological autopsy interview.18 Clinical axis I and axis II diagnoses, including a major depressive disorder were based on DSM-IV criteria established from structured clinical interviews, SCID-I and SCID-II and when available, from review of medical records with the modified Diagnostic Evaluation After Death.21 These psychiatric diagnoses (or the absence of any psychiatric diagnosis), based on all available data, were confirmed as meeting published criteria,22, 23, 24 at a consensus conference by the authors (JJM, AJD and GR), which also reviewed the medical examiner's determination of suicide or other manner of death.
The samples in the study included: 72 suicides with major depression, four suicides without major depression; 10 non-suicides with major depression, 12 non-suicides with other psychiatric diagnoses and 68 non-suicides with no psychiatric diagnosis. With such a sample we carried out the following comparisons: (1) 82 major depression; or (2) 86 depressed or suicide were contrasted with a common control group of 80 non-major depression and non-suicide. The major depression group was 49.8±18.7 years at time of death, 61% were male and 87.8% died by suicide. In the non-major depression group there were 12 subjects with a psychiatric disorder, one subject had schizophrenia spectrum disorder and the remaining 11 subjects had other psychiatric disorders (not related to MDD). The group with MDD had an average of 2.4±2.7 prior episodes of major depression and 48.8% (40 subjects) had other comorbid axis I or II disorders. Brain tissue was either rapidly frozen after removal at autopsy and stored at −80 °C or fixed and stored in phosphate-buffered 10% formalin. Brain pH was measured from cerebellar tissue and was found to be in the physiological range (ranging from 6.0 to 7.0). Postmortem interval for all samples was under 24 h. All subjects for the quantitative studies were drug-free at the time of death as verified by peripheral and brain toxicology.
PCR and sequencing procedures
PCR primers for the human TPH2 gene were selected based on the published genomic sequence and SNP information from NCBI; primer sequences are provided as supplementary data. The 15 μl PCRs contained 50 ng genomic DNA extracted from brain tissue, 1X PCR buffer, 0.25 U Taq polymerase (AccuTaq, SIGMA, Sigma-Aldrich, St Louis, MO, USA), 0.1 mM dNTPs and 5 pmol forward and reverse primers. After PCR, 3 μl of each PCR product was run on 2% agarose gel with 1X TAE buffer to visualize the PCR product quality. PCR products were purified using shrimp alkaline phosphatase and exonuclease 1 to degrade the excess dNTPs and primers. Bi-directional DNA sequencing reactions were conducted with the ABI PRISM Big Dye Terminator Ready Reaction kit (Applied Biosystems, Foster City, CA, USA) on 1 μl of purified PCR products with the same primers used for PCR. Sequences were analyzed on an ABI 3730 × l sequencer. Raw sequences of both DNA strands were assembled and SNPs were checked against the ENSEMBL reference sequences using Seqman (DNASTAR, Madison, WI, USA).
Quantitative real-time RT-PCR
Quantitative real time PCR was carried out on the MJ Research DNA Engine Opticon System. Each 20 μl reaction contained 3 mM MgCl2, 1X PCR Buffer, 1 pmol of each primer, 0.5 Units of Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA), 2 μls of cDNA (100 ng) and 0.3 × Syber Green (Molecular Probes, Eugene, OR, USA) diluted from a 10 000 × stock solution, according to the manufacturer. PCR parameters were an initial denaturation at 95 °C for 30 s followed by seven cycles with an annealing temperature ramping down to 53 °C. This was followed by 40 cycles of 95 °C for 15 s, 53 °C for 20 s and 72 °C for 20 s. Each sample was run in triplicate using the primers described above and primers specific for β-actin and GAPDH mRNAs. The TPH2 results were normalized to curves obtained for amplification of actin cDNA. The fluorescence of the accumulated PCR products was acquired after each cycle. The lowest number of cycles yielding above background was compared between the long and short forms. These measurements were combined to a mean value.
All SNPs examined were tested for deviation from Hardy–Weinberg proportions, where no deviation was observed. Single-locus analyses were performed using both allelic and genotypic tests for association, comparing allele and genotype frequencies between cases and controls. Statistical evaluations were performed based on maximum likelihood methods, which are analogous to contingency table χ2 approaches, but allow for more straightforward nesting and combination of hypotheses. Statistical significance was determined empirically by randomization, where we held the multi-locus genotypes for the set of loci fixed, and randomized the assignment of individuals to case–control groups 10 000 times and re-evaluated each of the test statistics.25 The P-value for each statistic was defined as the proportion of replicates in which a given statistic exceeded the value observed in the actual data. As multiple hypotheses were tested for multiple loci, leading to a multiple testing problem that is not easily dealt with by the conservative Bonferroni approach, the 10 000 replicates were used to evaluate the effect of such a multiple testing as well. For each test in each of the 10 000 replicates, the ‘P-value’ was computed, and for each replicate, the minimum P-value over the tests evaluated in that replicate. The proportion of replicates with minimum P-value lower than that found in the real data over all tests evaluated at a given locus was used as the corrected P-value. These P-values were minimized over all loci in the real data, and likewise in each replicate, to evaluate P-values corrected for multiple loci considered. The global P-values were computed in a similar fashion, modelling all SNPs and hypotheses tested in each replicate and comparing to the observed data. Multilocus haplotype analysis was done using PHASE case–control option,26 and genetic power analyses were performed using the Genetic Power Calculator web resource.27
We conducted an exploratory study using 166 individuals who were genotyped for SNP loci spanning both intron 5,19, 20 and exon 11.12, 13 Exon 11 is of interest because it encodes the catalytic site of the enzyme and may reveal hypofunctional variants. Therefore, we sequenced that region in all individuals. We observed no polymorphic sites in exon 11 after extensive sequencing of the 166 individuals, as also observed in other studies.14, 15, 16 Thus, we did not replicate the reported polymorphism in this region that is associated with altered levels of serotonin in Balb/c mice and reported by the same group to be present in 10% of subjects diagnosed with major depressive disorder.13 We did identify five previously reported SNPs in intron 5: rs11316791, rs1843809, rs11386496, rs1386494 and rs1386493 and a novel SNP, g.22879A>G. This is the region of the TPH2 gene predicted to undergo splicing into two transcript isoforms, a short and a long form. As predicted by Ensemble annotation, these alternative transcripts, ENST00000266669 and ENST00000333850, have 7 and 11 coding exons, respectively.28 The predicted truncated transcript was based on evidence derived from a study characterizing all human cDNAs in a brain cDNA library.29 Depending on the annotation considered, the variant, g.22879A>G, is in exon 6 of the short transcript, or in intron 5 of the long transcript. We sought to validate the existence of the predicted truncated TPH 2 transcript isoform experimentally by RT-PCR. Primers specific to the short and long transcripts were designed and used to amplify each isoform from cDNA prepared from human brainstem. Both the long and short forms are present in human brainstem (Figure 1). To determine whether the short form is present in serotonergic nerve terminals, RT-PCR was used to amplify the short and long forms from the prefrontal cortex, hippocampus and amygdala from one representative case. Expression of the short transcript was also seen in these regions (data not shown). Quantitative RT-PCR of the short and long forms in four non-psychiatric subjects revealed that compared with the long transcript there is relatively lower abundance of the short transcript (Figure 2) that seemed unrelated to PMI or brain pH.
Since the major depression case sample population was drawn from a postmortem brain collection, the majority of the subjects with major depressive disorder had committed suicide. Thus, we assessed the potential associations between these gene variants and major depression separately and together with suicide, with the caveat that the major depression and suicide case samples in the two comparisons have substantial overlap. It is noteworthy that, this is the only TPH2 mapping study, to our knowledge, where the control sample is carefully ascertained to select subjects where a diagnosis of major depression has been ruled out and who have died from causes other than suicide. While the case sample may represent a spectrum of mood disorder and/or suicidal behavior, this control sample is an ideal complement. In this exploratory aim we sought to elucidate the potential relationship of such genetic variants to mood disorder and/or suicide. We used one narrowly defined and one more broadly defined clinical model, where affected individuals: (1) were diagnosed with major depressive disorder; and (2) had major depression or died by suicide. The number of affected (case) and unaffected (control) individuals as defined by each of these models is given in Table 1. We analyzed the SNP data by conducting allelic tests for association and found two SNPs yielding statistically significant results (Table 1). The first is locus rs1386493 that is 2636 bp downstream from rs1386494, the locus reported previously to be associated with depression and suicide.19, 20 The second is the novel variant g.22879A>G discovered in our present study. The strongest evidence for association was observed with the major depression clinical model (rs1386493—P=0.001 and g.22879A>G—P=0.05), that remained statistically significant after adjustment for multiple models and markers tested (global empirical P=0.01). The broader clinical model including individuals with either major depression or suicide as affected also gave significant results (rs1386493—P=0.003 and g.22879A>G—P=0.05, Table 1) with global empirical P=0.04. This is not surprising, since the major depression model alone and the broader depression or suicide model have a high degree of overlap. Therefore, for the subsequent multilocus association analyses of the data, we focused on the depression alone clinical model. For the loci with observed and reported significant associations (that is, rs1386494, rs1386493, g.22879A>G) we compared overall estimated haplotype frequencies among cases and controls and found a statistically significant difference with a global empirical P=0.01, where the haplotype CGG consisting of the putative risk variants, was almost three times more frequent in the case sample, with estimated frequencies of 0.065/0.024 in case–control samples respectively (Table 2).
To aid the process of a systematic study of this gene, we have characterized the genetic architecture of the TPH2 gene through systematic sequencing of the coding region, including exons 1 and 7 through 11 of the long transcript, and the complete coding region of exon 6 of the short transcript. Additionally, sequencing was fully completed for the 5′ UTR, and was partially completed for the 3′ UTR and the intronic regions throughout the gene. This effort was further augmented by inclusion of existing SNPs in the public repository, including 57 known SNPs evenly distributed across the gene for maximal genomic coverage. Of these, 39 SNPs displayed a polymorphism in our sample. In addition, we discovered 16 novel SNPs by direct sequencing. In total, approximately 13 000 base pairs of the TPH2 gene were sequenced for 78 individuals consisting of 48 cases with depression/suicide and 28 healthy controls. Given this sample size, we can expect to detect 95% of the polymorphic sites with minor allele frequency of at least 1 and 99% of the sites with minor allele frequency of at least 5%.30 As such, we have conducted an in-depth survey in an effort to detect potentially rare polymorphisms that may alter gene function through coding changes or other mechanisms. It is expected that the polymorphisms that contribute to phenotypic variation are generally rare, for example most coding SNPs that lead to amino acid changes have minor allele frequency under 5%.31, 32 Such low frequency variations are underrepresented in the public repositories and thus these data are a substantial resource for future genetic studies of depression and suicide within the Caucasian population from which our samples were drawn. To this end, we have estimated haplotype block structure of the TPH2 gene and generated tag-SNPs using our population-based controls (Figure 3). Although numerous haplotype block definitions are possible, we adopted the more conservative four-gamete test,33 yielding a total of nine blocks ranging from 88 to 15 335 bp in length. The associations with major depression reported previously19, 20 and in the present study, fall entirely within the second block. Within these haplotype blocks, spanning 44 SNPs, we identified 15 tag-SNPs (Table 3). Despite the block definition used, the tag-SNPs are generally consistent over 80% of the time,34 and we observed this trend in our data as well when exploring other block definitions (data not shown). Thus within each block that contains more than one SNP, only a few tag-SNPs need to be genotyped to capture the entire block. Thus this study provides a haplotype map to guide exploratory investigations of other disorders with which TPH2 is potentially associated.
These results provide evidence for association of TPH2 with major depression and/or suicide, since the majority of our cases had major depression and had committed suicide, and therefore support previous reports of associations of a TPH2 variant (rs1386494) with both depression19 and suicide20 in two separate samples of depressives and suicides (where depression status of the suicide subjects were not reported). We also examined our data in a suicide alone clinical model and found no significant association after multiple testing correction (data not shown). This might suggest that TPH2 plays a role in major depression, but not suicide.
Our findings with a downstream novel variant (g.22879A>G) suggest a potentially more important association. This is because a more critical evaluation indicates that the association with rs1386493 might be spurious. Specifically, the direction of association for marker rs1386493 in this study as well as previously reported studies19, 20 indicates the potential presence of a common risk allele, as observed in the pattern of allelic and genotypic frequencies in cases versus controls, where the major allele is more common in cases than controls. This trend is observed for both major depression and suicide (for example, 0.93/0.79 corresponding to cases/controls within the depression clinical model). In a model for disease susceptibility one would not predict that the more common allele would be even more common in the disease group, we computed the power of observing such a disease model in our sample. For the depression clinical model the power is 0.1% for Type I error rate of 0.001, assuming a lifetime prevalence of 5% for major depression35 (a conservative lower bound estimate used for the remaining power analyses for this discussion), and two-fold increase in relative risk for depression with a dominant model. Considering a recessive model with the same assumptions, the power only increases to 2.6% (the other clinical models follow similar trends). For such a model the sample necessary for 80% power is ∼189 500 cases and the equivalent number of controls, assuming a dominant model (755 is the estimate for the recessive model). Clearly, both this study, and the previously published studies,19, 20 are underpowered in detecting such common risk variants contributing to disease susceptibility given the available sample sizes. The low power raises the possibility that the observed findings for these loci might be spurious. In contrast, the novel variant g.22879A>G yields a power of 41% assuming a dominant model for relative risk of disease (using the same model parameters above). To detect such a variant with 80% power, a total of 215 cases and matched controls would be required, a far more feasible sample size than the previously published SNPs. Therefore, our findings with g.22879A>G are very promising and warrant investigation in a larger sample. Additionally, the comprehensive genetic characterization of the TPH2 candidate gene reveals that future studies of major depression should focus on the second haplotype block containing such variants of interest (Figure 3).
Cross-species examination revealed that human, chimpanzee, dog, mouse and rat show conserved non-coding sequences spanning the human TPH2 intron 5 region (as seen by the conservation track from UCSC Genome Browser based on the May 2004 draft of the human genome). This confers evidence for the presence of conserved elements with regulatory potential, and of particular interest is our finding that this newly discovered variant resides in a conserved genomic region. This variant and others in this region may affect generation of short and long isoforms of the enzyme with functional consequences for catalytic activity. We have demonstrated experimentally that both the short and long TPH2 isoforms are present in all brain regions tested including the brainstem. The role of these TPH2 transcripts in serotonergic target regions is unknown. One possibility is that the short form acts in a regulatory manner and has limited or no enzymatic function since it has a truncated catalytic site. Such a truncated isoform might regulate TPH2 activity via post-transcriptional interactions with the long isoform or with other mediators with which it may compete, given that the first 5 exons of the short and long TPH2 isoforms are identical. There are precedents for such interactions of truncated products.36, 37 Together the short and long forms may regulate local levels of serotonin in serotonergic terminal field targets. Further investigations of polymorphisms at the splice junction flanking exon 6 or in the coding/3′UTR regions of the short transcript such as g.22879A>G, may reveal variant isoforms with potentially aberrant functional effect. Thus, the possibility that the short isoform might play a critical role in the rate of brain serotonin synthesis and homeostasis, should motivate exploration of gene expression differences within brains of case and control subjects, which will be investigated in future studies. Taken together these results provide evidence for TPH2 as a candidate gene for depression and/or suicide for future investigations. To this end, we have constructed a haplotype map of the TPH2 gene and identified tag-SNPs that will be valuable in numerous genetic and functional studies investigating the role of TPH2 in disease etiology. In addition, the collection of 16 novel variants discovered in this study will significantly contribute towards future studies of such disorders as major depression, bipolar disorder, schizophrenia, alcoholism, drug abuse, aggression and suicide, with which TPH2 is associated.
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This study was supported by PHS Grants MH40210, MH64168, MH63749, HG002915 and MH62185. We thank Drs A Dumas, B Mancevski and T Serafimova for contributing to collection of specimens and psychological autopsies.
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