An insertion/deletion polymorphism in the SERT linked promoter region (SERTLPR), previously reported to regulate mRNA expression in vitro, has been associated with mental disorders and response to psychotropic drugs. Contradictory evidence, however, has raised questions about the role of SERTLPR in regulating mRNA expression in vivo. We have used analysis of allelic expression imbalance (AEI) of SERT mRNA to assess quantitatively the contribution of SERTLPR to mRNA expression in human post-mortem pons tissue sections containing serotonergic neurons of the dorsal and median raphe nuclei. Any difference in the expression of one allele over the other indicates the presence of cis-acting elements that differentially affect transcription and/or mRNA processing and turnover. Using a marker SNP in the 3′ untranslated region of SERT mRNA, statistically significant differences in allelic mRNA levels were detected in nine out of 29 samples heterozygous for the marker SNP. While the allelic expression differences were relatively small (15–25%), they could nevertheless be physiologically relevant. Although previous results had suggested that the long form of SERTLPR yields higher mRNA levels than the short form, we did not observe a correlation between SERTLPR and allelic expression ratios. Also in contrast to previous results, we found no correlation between SERTLPR and allelic expression ratios or SERT mRNA levels in B-lymphocytes. This study demonstrates that regulation of SERT mRNA is independent of SERTLPR, but could be associated with polymorphisms in partial linkage disequilibrium with SERTLPR.
The neurotransmitter serotonin is an important modulator of physiology, behavior and psychological states.1, 2, 3, 4 Disturbances in serotonergic systems in brain have been implicated in mental illness, including schizophrenia,5, 6 anxiety disorders,7 obsessive-compulsive disorders,8 addiction,9, 10, 11, 12 depression13, 14, 15, 16 and suicide.15, 17
After release from axonal terminals of serotonergic neurons, synaptic effects of serotonin are terminated by reuptake into the nerve endings.18 The serotonin transporter mediating this reuptake, SERT, belongs to the Na+ and C1−-dependent family of neurotransporters.19 SERT mRNA is expressed mainly in serotonergic neurons in the raphe nuclei of the pons and upper brainstem, which project widely to various regions of the brain.20 Tricyclic antidepressants (TCAs) and selective serotonin-reuptake inhibitors (SSRIs) used in the treatment of anxiety disorders and depression directly inhibit SERT.21
The human gene encoding SERT, SLC6A4, is located on chromosome 17q11.2.22 The gene is organized in 15 exons spanning ∼39 kb, which encode a protein containing 630 amino acids.22, 23 Transcriptional activity is regulated by several positive and negative regulatory elements within the SERT promoter region23, 24, 25, 26 and by a 17 bp variable number tandem repeat (VNTR) element in intron 2.27 Differential splicing28, 29 and use of two 3′ polyadenylation sites30 may also contribute the regulation of SERT expression.31
Heils et al.25, 32 first described functional variants of a repetitive sequence in the SERT linked polymorphic region (SERTLPR) located 1.2 kb upstream of the transcription start site. The long (l) variant contains 16 copies of a 20–23 bp GC-rich sequence and the short (s) variant contains 14 copies of this sequence. Transfection studies with expression vectors containing the l or s variants linked to a reporter gene indicated that the l variant directs higher levels of transcription compared to the s variant when expressed in lymphoblast cell lines,24 a human placental choriocarcinoma cell line (JAR)25 and in the raphe nucleus-derived cell line RN46.33 In agreement with the reporter gene assays, higher SERT mRNA levels and higher rates of serotonin uptake were measured in lymphoblasts of l/l homozygotes compared to those containing at least one copy of the s allele.24 The l variant was also associated with higher rates (Vmax) of serotonin uptake34, 35 and higher levels of serotonin binding36 in platelets and higher serotonin levels in blood.37 In post-mortem human midbrain tissue sections containing the dorsal and median raphe nuclei, SERT mRNA levels were reported to be higher in subjects carrying two copies of the l allele compared to individuals with at least one s allele.38 Moreover, SERT ligand binding levels in the raphe region were higher in l/l individuals than in s allele carriers, with the exception of l/l alcoholics who had lower SERT ligand binding levels than l/s or s/s alcoholics.39
Not all studies, however, have reported a correlation between SERTLPR alleles and SERT expression. Analysis of SERT mRNA levels in 53 permanent lymphoblast cell lines with real-time PCR (using β-actin mRNA as a reference) failed to find a statistically significant correlation with SERTLPR, but did find evidence for a combined effect of SERTLPR and the intron 2 VNTR.40 Also, no correlation between SERTLPR alleles and promoter activity was observed with expression vectors assayed in COS-726, 33 or PC12 cells.26 There were no statistically significant correlations between SERTLPR alleles and binding of the SERT ligand [123I]CIT in the thalamus–hypothalamus or mesencephalon-pons measured in SPECT scans of healthy volunteers.41 In addition, no correlations were found between SERTLPR and binding of the SERT ligand [11C]McN5652 in healthy male subjects examined by PET.42
Surprisingly, most studies on SERTLPR-mediated regulation have focused on lymphocytes or heterologous cell systems, instead of the physiologically relevant cell populations, such as the serotonergic neurons in the raphe nuclei of the pons and upper brainstem. The functional effects of SERTLPR on mRNA expression in the brain therefore remain uncertain. In this study, we tested whether the SERTLPR influences mRNA expression in human pons tissue sections containing serotonergic neurons of the dorsal and median raphe nuclei, which are primary sites of SERT transcription in the CNS.43
Genetic variants that differentially affect mRNA expression from a given allele cause allelic expression imbalance (AEI), which can be quantitatively measured in individuals who are heterozygous for a marker SNP within the mRNA.44 The existence of AEI indicates the presence of cis-acting factors that influence intracellular levels of mRNA by modifying transcription, mRNA processing, or epigenetic effects.44, 45, 46, 47, 48 Genetic analyses suggest that cis-acting genetic variants are one of the main sources of variability in human phenotype, including susceptibility to disease.45, 47, 49, 50 Although a powerful approach, analysis of AEI has not been widely used, because of difficulties in obtaining reproducible measurements. We have developed a robust procedure for allele-specific measurement of mRNA expression using a fluorescence-based primer extension assay to quantify real-time PCR amplification.51, 52 We have successfully applied this approach to identifying functional cis-acting polymorphisms in MDR153 and OPRM.52 In the present study, we used this method to measure AEI for SERT mRNA in 48 post-mortem pons tissues sections from individuals heterozygous for a marker SNP (rs1042173) in the 3′UTR of SERT mRNA. Although we have detected a limited degree of allelic variations in mRNA expression, this did not correlate with the SERTLPR promoter polymorphism. Moreover, SERTLPR also did not account for AEI of SERT mRNA in immortalized B-lymphocytes.
Materials and methods
In total, 48 human pons sections were obtained from the Brain and Tissue Bank for Developmental Disorders (University of Maryland, Baltimore). The demographics of these samples are described in Table S1 in Supplementary Materials. PCR reagents were from Promega (Madison, WI, USA). Oligonucleotide primers were designed using OLIGO 4.0 (National Biosciences Inc., Plymouth, MN, USA) and synthesized by Integrated DNA Technologies (Coralville, IA, USA). SNaPshot reagents were from Applied Biosystems (Foster City, CA, USA). Other reagents for cell culture were from Sigma (St Louis, MO, USA) or Fisher Scientific (Pittsburgh, PA, USA).
Isolation of DNA and RNA from human pons
Individual sections of human pons were soaked overnight at −80°C in 10 volumes of RNA later-ICE Frozen Tissue Transition Solution (Ambion, Austin, TX, USA). Small pieces of tissue (∼500 mg) were removed for isolation of DNA and homogenized in 365 μl of nuclei lysis buffer containing 10 mM TRIS base, 400 mM NaCl, 2 mM Na2EDTA and 0.7% SDS. To digest protein contaminants, 35 μl proteinase K (10 mg/ml, Invitrogen, Carlsbad, CA, USA) were added and the mix incubated overnight at 55°C. The next day, digested proteins were precipitated with saturated NaCl (6 M), the supernatant fractions were transferred to fresh tubes and the DNA precipitated with ethanol. The remaining tissue was homogenized in Trizol reagent (1 ml Trizol reagent per 100 mg) for isolation of RNA. RNA samples were treated with 10 μl of RNase-Free DNase (2500 Kunitz units/mg, Qiagen, Valencia, CA, USA) for 15 min at room temperature, and total cellular RNA was purified using QIAGEN RNeasy columns. Complementary DNA (cDNA) was generated from 1 μg total RNA in a total volume of 20 with 1 μl (200 U) Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA), 1 μl of 1 μ M oligo(dT)20 primers (Invitrogen, Carlsbad, CA, USA), 1 μl of 10 mM dNTP mix (Invitrogen, Carlsbad, CA, USA), 0.5 μl of 1 μ M SERT gene-specific primer (5′-IndexTermTGGACACACTATTTTTCATTTTAG-3′) to facilitate reverse transcription of the target mRNA, 4 μl of 5 × first-strand buffer, 1 μl of RNaseOUT (40 U/μl), and RNase-free water to yield 20 μl. Typically, three independent cDNA preparations were made from each RNA sample. One of the 48 pons tissue samples failed to yield PCR products possibly due to degradation of genomic DNA and mRNA during the post-mortem interval or during processing of the sample.
Epstein–Barr virus transformed lymphoblastoid cell lines were obtained from the Coriell Cell Repositories (Coriell Institute for Medical Research, Camden, NJ, USA). Cells were cultured at 37°C in a humidified incubator at 5% CO2 in RPMI medium containing 2 mM L-glutamine plus 15% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin. When the growth medium turned yellow, the cells were supplied with fresh medium, doubling the volume. In total, 50 ml of cell culture typically yielded 50–100 million cells. Medium containing cells (20 ml) was centrifuged at 1000 × g, lysed in Sucrose-Triton solution prior to treatment with proteinase K (10 mg/ml), and incubated for 4–5 h at 55°C. NaCl (6 M) was added (125 μl) to precipitate the digested proteins. The supernatant fractions were transferred to new tubes and the DNA precipitated by addition of 1 ml of ethanol. RNA was isolated in Trizol reagent, followed by RNA purification with QIAGEN RNeasy mini prep kits. DNA in RNA samples was eliminated by treating with an RNase-Free DNase Set (2500 K U/mg, Qiagen, Valencia, CA, USA).
A marker SNP (G2651T; rs1042173) located in the 3′-untranslated region (UTR) of human SERT (Figure 1A) was genotyped for 48 pons and 12 lymphoblast genomic DNA samples using the SNaPshot assay. The l/s SERTLPR promoter polymorphism was genotyped by PCR amplification of the region using flanking oligonucleotide primers: 5′-IndexTermATGCCAGCACCTAACCCCTAATGT-3′ (forward primer) and 5′-IndexTermGGACCGCAAGGTGGGCGGGA-3′ (reverse primer). PCR amplification reactions were carried out using the GC-rich PCR System (Roche Applied Science, Indianapolis, IN, USA) with the following cycles: (1 × (30 s at 95°C); 35 × (30 s at 62°C, 45 s at 72°C)). For genotyping of the VNTR, we PCR-amplified the genomic DNA segment containing the VNTR using flanking oligonucleotide DNA primers. A modified forward primer containing fluorescent dye (6-FAM) covalently attached to its 5′-end was used in this reaction. The fluorescent PCR products were resolved by electrophoresis on an ABI3730 DNA sequence analyzer to determine the number of repeats in the VNTR. Amplification reaction for the 17-bp VNTR was performed in a total volume of 25 μl containing 25 ng DNA, 200 μ M dNTP mix, 200 nM of each of the primers, 10 × buffer and Taq DNA polymerase. Applied primer sequences were 5′-IndexTermGTCAGTATCACAGGCTGCGA-3′ (forward) and 5′-IndexTermTGTTCCTAGTCTTACGCCAGTG-3′ (reverse). The PCR program consisted of an initial denaturation at 94°C for 2 min, 35 cycles of 20 s at 95°C, 30 s at 62°C, 1 min at 72°C, and a final extension for 10 min at 72°C. For genotyping of the 3′-UTR SNP A2358C (rs 3813034), we performed allele-specific PCR amplification using an ABI 7000 DNA sequence detection system (Applied Biosystems, Foster City, CA, USA). Briefly, two reverse primers were designed, with the 3′ base of each primer matching only one of the biallelic SNP bases to be evaluated. To increase the specificity of the amplification from each allele, different mismatches were introduced into the third nucleotide from the 3′ end of each reverse primer.54 A common forward primer was designed to anneal upstream of the polymorphic site. Independent PCR amplification reactions were carried out with each allele-specific primer. The sequences of the primers were: 5′-IndexTermCAAATATATGAATT-CCCCAAATTTTTC-3′ (forward primer), 5′-IndexTermCACAATTGAGTTGGTAGAATTTGTGAA-3′ (allele-specific reverse primer 1) and 5′-IndexTermACACAATTGAGTTGGTAGAATTTGTAAC-3′ (allele-specific reverse primer 2). Amplification conditions consisted of a 10-min preincubation at 95°C to activate the Taq DNA polymerase, followed by 40 cycles of denaturation at 95°C for 15 sec and primer annealing and extension for 1 min at 60°C.
Allelic expression imbalance assays
A detailed protocol for this method has been previously described.55 Briefly, a segment of genomic DNA or cDNA (205 bp) spanning the marker SNP was amplified by PCR (1 × (30 s at 95°C); 35 × (30 s at 56°C, 45 s at 72°C)) using the following oligonucleotide primers: 5′-IndexTermTATCTGTTTGCTTCTAAAGGTTTC-3′ (forward primer); 5′-IndexTermTGGACACACTATTTTTCATTTTAG-3′ (reverse primer). Unincorporated dNTPs and excess primers were inactivated with 2 units of exonuclease I (New England Biolabs, Beverly, MA, USA) and 5 U of antarctic alkaline phosphatase (New England Biolabs, Beverly, MA, USA). The PCR products were then used as templates in SNaPshot (Applied Biosystems, Foster city, CA, USA) primer extension assays. Primer extension reactions were carried out independently in both directions (for verification) using primers that terminate immediately adjacent to the marker polymorphism (‘forward’ primer: 5′-IndexTermGCCATATATTTTCTGAGTAGCATATA-3′; ‘reverse’ primer: 5′-IndexTermGGTTCTAGT-AGATTCCAGCAATAAAATT-3′). Following incorporation of a single fluorophore-labeled dideoxyribonucleoside triphosphate (ddNTP) complementary to the nucleoside at the polymorphic site, the resulting primer extension products were resolved by capillary electrophoresis (ABI 3730 DNA Sequencer, Applied Biosystems) and analyzed using Gene Mapper 3.0 software (Applied Biosystems). To measure the expression ratios of the two alleles, we used DNA and cDNA from individuals who are heterozygous for the marker SNP. Incorporation of different fluorescently labeled dideoxynucleotides into the primers produced oligonucleotides with similar electrophoretic mobilities but distinct fluorescence spectra. Standard curves using peak areas and different ratios of allelic DNA were linear. Because different fluorophores differentially affect the efficiency of nucleotide incorporation and have different fluorescence yields, peak area ratios of genomic DNA diverge from the theoretical ratio of 1.0. The measured ratios for genomic DNA were therefore normalized to 1.0 using a correction factor based on the mean of the genomic DNA ratios, and the cDNA ratios of heterozygous samples adjusted accordingly. SNaPshot assays were performed 3 × with genomic DNA and 3 × with three independent cDNA preparations per sample.
Data are expressed as mean±standard deviation (s.d.). Statistical analyses were performed using Excel (Micosoft, Inc.) for ANOVA, t-tests (two-tailed) and linear regressions. Prism (GraphPad, San Diego, CA, USA) was used for evaluating differences in relative SERT mRNA expression levels for l/l, l/s and s/s genotypes. We assessed AEI in individual samples by comparing the mean allelic ratio for RNA with the mean allelic ratio for DNA. The mean allelic ratio for RNA was considered to significantly differ from that of DNA if (1) the means differed by more than 10% and (2) there was no overlap between two times the standard error for each mean. Calculations of pairwise linkage disequilibrium (D′) for three SERT polymorphisms and the contributions of these polymorphisms to SERT mRNA AEI were carried out using programs Linkage Disequilibrium View and Two Genetic Loci Plot programs in HelixTree® (GoldenHelix Inc., Bozeman, MT, USA).
The locations of the four SERT polymorphisms examined in this study are shown in Figure 1a. We genotyped the marker SNP in the 3′ UTR (G2651T) in 47 human pons samples using the SNaPshot primer extension assay. Fourteen individuals were T/T homozygotes, four were G/G homozygotes, and 29 were G/T heterozygotes. The l/s SERTLPR polymorphism was genotyped in the 29 G/T heterozygous individuals using agarose gel electrophoresis to distinguish the PCR products derived from the l (419 bp) and s variants (375 bp) (Figure 1b). Twelve individuals were l/l, 15 were l/s and 2 were s/s. We genotyped the intron 2 VNTR in the 29 G/T heterozygotes using capillary gel electrophoresis to distinguish the PCR products derived from the 10-repeat (265 bp) and 12-repeat variants (299 bp) (Figure 1c). Three individuals were homozygous for 10 repeats (10/10), nine were homozygous for 12 repeats (12/12), and 17 were heterozygous for 10 and 12 repeats (10/12). We also genotyped the 3′ UTR SNP (A2858C; rs3813034) located 207 bp downstream from the marker SNP within the second SERT mRNA polyadenylation site using the SNaPshot primer extension assay. Twenty-five individuals heterozygous for the marker SNP were also heterozygous (A/C) for rs3813034, three were homozygous C/C and one was homozygous A/A.
Analysis of mRNA expression ratios in human pons
To determine if there were sufficient numbers of SERT mRNA molecules to accurately measure allelic expression ratios, we used quantitative real-time PCR to determine the number of SERT mRNA molecules in 29 independent preparations of pons RNA. As shown in Figure S2–1, Supplementary Materials), genomic DNA was used to standardize our quantitative real-time PCR measurements. Based on this standard curve, we determined that our human pons RNA samples contained approximately 1000–64 000 molecules of SERT mRNA (Figure S2–2, Supplementary Materials), numbers sufficiently large for reliable measurements of allelic expression ratios. We also used quantitative real-time PCR to show that SERT mRNA levels are significantly higher in pons compared to cerebellum or cortex (regions that contain fewer serotonergic neurons), suggesting that the SERT mRNA in the pons derives from serotonergic neurons in the raphe nuclei (Figure S2–3, Supplemental Materials).
We next carried out a small-scale experiment to determine if allelic expression ratios could be accurately measured using human pons RNA. The four fluorescent (dR6G-, dTAMRA-, dR110- and dROX-) dideoxynucleotide derivatives used in the primer extension assays have different fluorescence yields, incorporation efficiencies, and electrophoretic mobilities. To determine if different combinations of fluorophores differentially affect the measured expression ratios, we carried out independent allelic expression ratio determinations with RNA or DNA isolated from six individuals heterozygous for the mRNA marker SNP using extension primers that anneal to the antisense or sense DNA strand, respectively. The ‘forward’ extension primer reaction produced G/T ratios derived from the fluorescence peak areas of dR110 and dROX (Figure 2a, top; 2b, left). The ‘reverse’ primer extension reaction produced C/A ratios derived from dTAMRA and dR6G fluorescence peaks (Figure 2b, bottom; 2b, right). Allelic ratios of PCR products derived from genomic DNA using the forward and reverse extension primers yielded an average ratio of 1.15±0.08 s.d.; reverse primer yielded an average ratio of 0.432±0.021 s.d.. No sample yielded genomic allele ratios that significantly deviated from the mean, suggesting an absence of chromosomal instability-related gene dosage effects. The small standard deviations (s.d.'s) of these measurements indicate that allelic ratios can be determined with good precision. Since each allele is present in equal amounts in genomic DNA, deviations from the expected allelic ratio of 1.0 likely arise from the measurement process itself, for example, from differences in the efficiency of incorporation between the fluorescently tagged dideoxynucleotide triphosphates used in the primer extension assay. To normalize the DNA-based allelic ratios, a correction factor was calculated as the inverse of the mean of all DNA allelic ratio measurements (n=18; F=1/(mean allelic ratio for DNA)). Correction factors were determined independently for the forward and backward primers. Each measured DNA allelic ratio was multiplied by this factor, and the mean of these values (±s.d.) tabulated. (Table 1A and B, left columns)
SERT mRNA expression ratios were also measured using RNA samples isolated from the pons of six individuals heterozygous for the 3′UTR marker SNP using both forward and reverse extension primers. The measured ratios were multiplied by the correction factors determined from the genomic DNA, and the average of these values (±s.d.) tabulated (Table 1A and B, right columns). As indicated, allelic mRNA ratios determined using the forward and reverse primers were not significantly different. Moreover, none of the measured allelic mRNA ratios deviated significantly from unity, indicating an absence of AEI in these samples, even though the samples included l/s heterozygotes where one would have expected AEI.
The above experiment demonstrates that we can reproducibly measure allelic ratios for SERT mRNA using total RNA isolated from sections of human pons. To look for evidence of AEI of SERT mRNA in a larger set of samples, we used RNA isolated from 29 individuals heterozygous for the 3′UTR marker SNP, including the six individuals analyzed above (Figure 3 and Table 2). After correction of the ratios as explained above, we found small differences in allelic mRNA expression in nine of the 29 samples (#1054 (l/l), 1103 (l/l), 1112 (l/l), 1209 (l/l), 1279 (l/l), 914 (l/s), 1027 (l/s), 1429 (l/s), 1500 (l/s); range of AEI=1.12–1.27). The samples that showed AEI for SERT mRNA, however, were not all heterozygous at the SERTLPR locus (i.e. l/s genotype), as would be expected if the l and s forms of the promoter were the dominant regulatory elements for this gene. Also, there were no significant differences in mRNA expression between G and T alleles in 11 out of 15 samples of individuals who are also heterozygous for the l/s promoter polymorphism. Finally, ANOVA revealed no significant differences in SERT AEI among the l/l, l/s and s/s genotypes (Table 2; P=0.381). Taken together, these results indicate that the SERTLPR genotype by itself does not predict measured SERT mRNA levels in these pons tissue sections.
AEI in 4 samples heterozygous for l/s suggests that there may be functional cis-acting polymorphisms in partial linkage disequilibrium with SERTLPR. Similarly, 5 out of 12 samples showed AEI for individuals homozygous for the l form, suggesting that the l allele associates with a functional polymorphism with a frequency of 20–25%. Since only two homozygous s carriers were among the study population, with no imbalance observed, we cannot ascertain whether this polymorphism is also associated with some s alleles.
Allele-specific mRNA expression in lymphoblasts
Our data using brain RNA differs from the results of a previous study of SERT expression in Epstein–Barr virus-transformed B-lymphoblasts, where the l promoter variant was found to produce 1.7-fold higher levels of endogenous SERT mRNA compared to the s promoter variant.24 To determine whether there are differences between SERT expression in pons and lymphocytes, we measured allelic expression ratios for SERT mRNA using RNA isolated from Epstein–Barr virus-transformed B-lymphoblasts prepared from nine individuals heterozygous for the 3′-UTR marker SNP. Genomic DNA from these cells was used to genotype SERTLPR. As for pons RNA samples, quantitative real-time PCR analysis demonstrated that there were sufficient numbers of SERT mRNA in the lymphoblast RNA samples to accurately determine allelic expression ratios (Figure S2–4, Supplemental Materials). Consistent with results from brain samples, we found small, statistically significant differences in allelic mRNA expression in five out of nine samples (Figure 4). Unlike the study of Lesch et al,24 however, these differences did not strictly correlate with l/l, l/s or s/s genotypes. Interestingly, one s/s homozygous sample (6991) showed the largest AEI observed in this study, indicating that a cis-acting polymorphism that regulates SERT expression could be in partial linkage disequilibrium with the s allele.
Estimated linkage between SERT polymorphisms and contributions of these polymorphisms to SERT AEI
Because the SERT polymorphisms are distributed within a 39 kb segment of the chromosome, it is experimentally difficult to determine which alleles are physically linked in any given individual. To determine the probability of physical linkage between alleles, we calculated estimated linkage disequilibrium for pairs of polymorphisms examined in this study. These results are summarized in Table 3A. Consistent with previously estimates,56 the data show that there is relatively low linkage disequilibrium between the SERTLPR and the three other markers (the VNTR, the marker SNP and polyA SNP). By contrast, the VNTR and 3′ UTR SNPs show a high degree of linkage disequilibrium. These results suggest that the SERTLPR and the three other polymorphisms are located on different haplotype blocks, a conclusion consistent with previous results56 and with the haplotype structure predicted for SERT from analysis of HapMAP57 SNPs (http://www.hapmap.org/index.html.en).
As mentioned above, a recent study by Hranilovic et al.40 reported a correlation between SERT mRNA expression in lymphocytes with the combined SERTLPR and VNTR markers. To determine if the VNTR contributes to the small AEI observed in pons sections, we examined the association of SERT mRNA AEI with VNTR alone and in combination with SERTLPR or the marker SNP. As shown in Table 3B, no individual marker showed a statistically significant association with SERT AEI. By contrast, SERTLPR and the VNTR in combination showed a weak, but statistically significant (in the absence of corrections for multiple testing) association with SERT AEI. Even in this case, however, the extent of SERT AEI was at most 1.27-fold (range 1.12–1.27), considerably less than the 1.7-fold difference in SERT mRNA expression previously reported for the l and s alleles in lymphocytes.
Previous studies have investigated the influence of l and s SERTLPR alleles on SERT promoter activity in heterologous expression systems,24, 25, 26, 33 SERT mRNA levels in lymphoblast cell lines24, 40 and brain,38 SERT ligand binding36 and transport activity in platelets,34 and SERT ligand binding levels in post-mortem brain sections38 and living brain.39, 41, 42, 58 Clinical association studies have further examined the relationship between SERTLPR alleles and psychiatric disorders and anxiety-related personality traits,59, 60, 61, 62, 63, 64, 65, 66 suicide,67, 68 alcoholism,69, 70 response to antidepressants71, 72, 73 and brain function as revealed by MRI scans.74, 75, 76, 77 Although many studies have reported correlations between SERTLPR alleles and SERT expression levels or clinical phenotype, almost an equal number of studies have not. Where correlations have been replicated or survived meta-analysis, the estimated contributions of the SERTLPR polymorphism are small.
Most studies on SERTLPR to date (more than 350 currently cited in PubMed) were motivated by early work showing that the l form of the SERT promoter is more active than the s form. Curiously, only a few studies have examined the relationship between SERTLPR genotype and SERT mRNA or protein expression in the relevant population of cells: serotonergic neurons in the pons and brainstem. The goal of the present study was to examine the relationship between SERTLPR genotype and SERT mRNA expression in tissue sections from the rostral pons that contain dorsal and median raphe nuclei.
Because it is difficult to make quantitative comparisons of mRNA levels between autopsy tissue sections, we decided to compare allele-specific expression of SERT mRNA using total cellular RNA isolated from the same tissue section. In this experimental design, the mRNA transcribed from one allele acts as a control for the other, since both are presumably exposed to the same trans-acting gene regulators, time to tissue harvest (post-mortem interval), and medication and medical histories. Allele-specific differences in mRNA expression in these samples are taken to reflect differences in cis-acting factors, that is, the presence of genetic variants located on the same chromosome, likely in the vicinity of the gene, that affect mRNA levels through modulation of mRNA transcription, splicing and/or degradation.
Recent studies have demonstrated that the expression of a large percentage of human genes are regulated by cis-acting genetic elements.44, 55 Given the paucity of functional polymorphisms in the coding region of SERT affecting protein structure and function,78, 79, 80 we suspect that functional differences in SERT expression among individuals likely arise from differences in regulation of the gene by cis-acting genetic elements located outside of the coding region.
To study allele-specific regulation of the SERT mRNA expression, we have developed an assay in which a common SNP (G2651T, rs1042173) in the 3′-UTR is used to quantify relative levels of mRNA transcribed from each allele (Figure 1A). This SNP represents the entire SERT gene region for each of the two alleles in a given individual, regardless of linkage disequilibrium, which is determined from a population average. As shown in Figure 2 and Table 1, this assay is highly reproducible, allowing us to reliably detect differences of 15–20% in expression between alleles. Using this assay, we demonstrated a statistically significant AEI in nine out of 29 pons samples heterozygous for the marker SNP, indicating the presence of a cis-acting factor in ∼30% of the population. While the extent of the imbalance was relatively small (15–25%), this could be sufficient for biological effects. For example, an AEI of maximally 30% was previously suggested to be sufficient to account for an association between catecholamine O-methyl transferase (COMT) and schizophrenia.81
On the basis of previous findings that the l promoter variant generates higher levels of mRNA expression compared to the s variant,24, 25 we had predicted that l/s heterozygous samples would show differences in allelic mRNA expression. Contrary to this prediction, however, we observed no consistent differences in levels of expression of individual SERT alleles in the pons of l/s individuals. It is possible that a cis-acting functional polymorphism is associated with the l form in a portion of alleles; however, we were unable to ascertain the phasing between the marker SNP and the l/s forms due to the low linkage disequilibrium between these markers (Table 3). This prevented unambiguous assignment of the l form to the more highly expressed allele. Our observation that 12/16 l/s heterozygous samples showed no allelic imbalance appears to rule out SERTLPR as the determining factor for AEI of SERT mRNA expression in the rostral pons.
Because the SNaPshot analysis of SERT mRNA in the pons produced unexpected results, we performed a series of control experiments to validate the assays. A critical variable is the quality of the mRNA extracted from post-mortem brain tissues. This problem is in part alleviated by the use of a ratio method, which as mentioned above, bypasses issues such as differences in mRNA decay between tissue samples. (We cannot completely rule out, however, the possibility of differential rates of decay between alleles during the postmortem interval or tissue processing.) As an extra precaution, we used gene-specific primers for the synthesis of cDNA (see Materials and methods), with the oligonucleotide targeting a sequence close to the marker SNP. This ensures a high yield of the region of interest, but possibly masks differences in splicing at other sites of the transcribed gene. The most likely sources of variability in the assay are in mRNA extraction, cDNA synthesis and/or PCR amplification. To account for this variability, we carried out three independent mRNA/cDNA preparations, and performed PCR in triplicate for each assay. Duplicate analysis of the PCR products using forward and backward primers in the SNaPshot reaction revealed that the detection step is precise and does not significantly contribute to overall measurement errors. Furthermore, we ascertained that the amount of mRNA available from pons tissue sections is in a range where we can expect reproducible results after PCR amplification. Allelic DNA ratios can be determined with an average s.d. of 4% (range: 0–11% for individual samples) across all samples, while the average s.d. for mRNA/cDNA measurements was 5% (range: 0–10% for individual samples).
To provide a direct comparison with previously published studies, we tested allelic mRNA expression of SERT in Epstein–Barr virus-transformed B-lymphocytes. Although we observed significant AEI in five out of nine samples (15–40%; Figure 4), this did not correlate with l/s heterozygosity in SERTLPR, consistent with our result with pons tissue sections. Absolute levels of SERT mRNA were also measured in B-lymphoblast cell lines (14 l/l, 12 l/s and 8 s/s) by real-time PCR, a more advanced technique than the competitive PCR method used previously.24 Large variations in SERT mRNA levels were observed, but again there was no significant correlation with the l or s promoter alleles (Figure S2–4).
Using quantitative real-time PCR to quantify levels of SERT mRNA, Hranilovic et al.40 reported a trend towards higher expression in EB virus-transformed lymphoblasts from l/l individuals compared to lymphoblasts from l/s and s/s individuals, but these differences were not statistically significant. These authors did observe a statistically significant correlation, however, between the combined SERTLPR and VNTR genotypes and SERT expression. By contrast, we observed no effect of VNTR by itself on AEI of SERT mRNA, and only a weak association for VNTR in combination with SERTPR (statistical significance was not observed following corrections for multiple testing). Taken together, these studies suggest that cis-acting polymorphisms regulate small allele-specific differences in SERT mRNA expression, but argue against SERTLPR or the VNTR per se as playing a significant role in regulating the SERT gene.
To our knowledge, only three previous studies have examined the relationship between SERTPR genotype and SERT mRNA or protein expression in serotonergic neurons of the dosal and median raphe nuclei. Little et al.38 observed higher levels of binding of the SERT ligand [123I]CIT in the dorsal and median raphe nuclei of l/l individuals (n=16) compared to l/s individuals (n=10). [123I]CIT binding levels in s/s individuals (n=4), however, were close to those of l/l individuals, an effect attributed to the small size of the s/s sample. Moreover, [123I]CIT binding levels were lower in l/l chronic alcohol users compared to l/s or s/s chronic alcohol users, which was taken to suggest that SERT expression is influenced by environmental factors. Analysis of SERT mRNA expression by in situ hybridization showed higher levels of SERT expression in l/l individuals (n=13) compared to l/s (n=8) and s/s individuals (n=3). Although these results are consistent with higher SERT expression in l/l compared to l/s and s/s individuals, the small number of individuals examined warrants caution in building upon these results.
Heinz et al.39 used [123I]CIT binding measured in vivo by SPECT to examine the relationship between SERTLPR genotype and SERT protein expression in the raphe area of the dorsal brainstem of alcohol users and abstinent volunteers. These studies revealed that l/l control subjects (n=3) had two-fold higher levels of [123I]CIT binding in the brainstem compared to l/s and s/s subjects (n=5). Consistent with the in vitro binding studies of Little et al.,38 [123I]CIT binding levels in l/l alcoholic individuals were lower than those in l/s and s/s alcoholic individuals. These results again emphasize that SERT expression may be modulated by environmental factors. Heinz and co-workers suggested that a possible explanation for the low [123I]CIT binding levels in l/l alcoholic individuals is that serotonergic neurons in l/l individuals are more susceptible to the neurotoxic effects of chronic alcohol use. In contrast to the results of Heinz and colleagues, a similar study by Willeit et al.41 failed to detect a correlation between SERTLPR genotype and [123I]CIT binding levels in the thalamus–hypothalamus or mesencephlon-pons measured by SPECT scans of healthy subjects (l/l (n=3); l/s (n=9); s/s (n=4)). Clearly, more work will be required to resolve these conflicting reports and to sort out the relative importance of SERTLPR genotype and environmental factors in determining SERT expression in the raphe nuclei.
While a direct association between SERTLPR and psychiatric traits remains elusive, other experiments suggest a gene–environmental interaction as a determining factor. In mice with disrupted SERT, homozygous and heterozygous strains displayed more fearful behavior and greater increases in the stress hormone adrenocorticotropin in response to stress compared to homozygous (SERT +/+) controls, but in the absence of stress no differences related to genotype were observed.82 In rhesus macaques, with an SERTLPR analogous to that of humans, the short allele is associated with decreased serotonergic function among monkeys reared in stressful conditions, but not among normally reared monkeys.83 In humans, individuals with one or two copies of short allele of the SERT promoter polymorphism exhibited more depressive symptoms, diagnosable depression, and suicidality in relation to stressful life events than individuals homozygous for the long allele; however, no direct association between the SERT gene and depression was observed.84 Taken together, these studies show that there may not be a direct relationship between SERTLPR and a variety of biochemical and clinical phenotypes; yet, intriguing results do imply a role for SERTLPR in behavioral disorders when environmental factors are taken into account.
While our results appear to contradict previous assertions that the SERTLPR regulates gene expression, the following possibilities need to be considered: (1) The l and s forms harbor functional polymorphisms of lower frequency (∼20%) that affect mRNA levels. Our study was not designed to identify these putative functional polymorphisms, which will require a larger sample size. (2) The pons tissue sections used in this study were from anonymous donors. It is possible that in afflicted individuals (e.g. with depression disorder), the frequency of another functional polymorphisms in linkage disequilibrium with SERTLPR is significantly higher, giving rise to clinical associations indirectly between SERTLPR and phenotypic trait. (3) AEI of SERT occurs only following exposure to stress, when different transcriptional regulation could involve the SERTLPR. (4) Differential regulation of SERT by SERTLPR occurs predominantly during the development of the nervous system, resulting in functional differences in neuronal circuits in the adult.
This is the first study to measure mRNA expression of SERT using an allele-specific assay to evaluate the potential role of cis-acting polymorphisms in human pons. Although we observed statistically significant differences in mRNA expression between alleles, our results argue against a role for SERTLPR in determining SERT mRNA expression. Our approach lends itself to finding functional polymorphisms in SERT and assessing their relative role in inter-individual variability.
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This work was supported by NIH research grants DA018744 (National Institute on Drug Abuse) and GM61390 (Pharmacogenetics Research Network, General Medical Sciences; UCSF Membrane Transporter Group) and by a grant from the Psychiatric Research Foundation, Columbus, Ohio. The data will be deposited into the Pharmacogenetics Research Network Knowledgebase at www.pharmgkb.org.
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Lim, J., Papp, A., Pinsonneault, J. et al. Allelic expression of serotonin transporter (SERT) mRNA in human pons: lack of correlation with the polymorphism SERTLPR. Mol Psychiatry 11, 649–662 (2006). https://doi.org/10.1038/sj.mp.4001797
- serotonin transporter
- allelic expression imbalance
- raphe nuclei
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