Differential expression and parent-of-origin effect of the 5-HT2A receptor gene C102T polymorphism Analysis of suicidality in schizophrenia and bipolar disorder.код для вставкиСкачать
American Journal of Medical Genetics Part B (Neuropsychiatric Genetics) 144B:370 –374 (2007) Brief Research Communication Differential Expression and Parent-of-Origin Effect of the 5-HT2A Receptor Gene C102T Polymorphism: Analysis of Suicidality in Schizophrenia and Bipolar Disorder Vincenzo De Luca, Olga Likhodi, James L. Kennedy, and Albert H.C. Wong* Centre for Addiction and Mental Health, Department of Psychiatry, University of Toronto, Toronto, Ontario, Canada The serotonin 2A (5-HT2A) receptor gene has been implicated in the pathogenesis of suicidal behavior by a genetic association between the 5-HT2A C102T silent polymorphism and suicidality in patients with major depression. However, a recent metaanalysis failed to confirm this association. We developed an improved quantitative assay for the measurement of allele-specific expression of the 5-HT2A gene, and find that the ratio of C/T allele expression in the pre-frontal cortex of heterozygous suicide victims (n ¼ 10) was significantly decreased in comparison with the non-suicide group (n ¼ 10) (P ¼ 0.049). Because the 5-HT2A gene is subject to imprinting, the parent-of-origin may affect the inheritance of suicidal behavior. Thus we examined the parental origin of specific alleles for genetic association in a genetic family-based sample of major psychoses in which information on suicidal behavior was available. No association between the 5-HT2A C102T polymorphism and suicidal behavior in major psychoses was detected with the transmission/disequilibrium test (TDT). ß 2007 Wiley-Liss, Inc. KEY WORDS: suicide; 5-HT2A; psychoses; imprinting; allele ratio; postmortem brain Please cite this article as follows: De Luca V, Likhodi O, Kennedy JL, Wong AHC. 2007. Differential Expression and Parent-of-Origin Effect of the 5-HT2A Receptor Gene T102C Polymorphism: Analysis of Suicidality in Schizophrenia and Bipolar Disorder. Am J Med Genet Part B 144B:370–374. Suicide and suicidal behavior are responsible for considerable morbidity and mortality in schizophrenia (SCZ) and bipolar disorder (BD). The lifetime suicide risk is between 4% CIHR Clinician-Scientist Fellow and NARSAD Young Investigator. Grant sponsor: Canadian Institutes for Health Research (CIHR); Grant sponsor: National Alliance for Research in Schizophrenia and Depression (NARSAD). *Correspondence to: Dr. Albert H.C. Wong, Centre for Addiction and Mental Health, 250 College Street, Room 711, Toronto, Ont., Canada M5T 1R8. Received 9 May 2006; Accepted 20 September 2006 DOI 10.1002/ajmg.b.30458 ß 2007 Wiley-Liss, Inc. and 13% in SCZ [Meltzer, 2005] and 19% in BD [Goodwin and Jamison, 1990]. Between 25% and 60% of bipolar patients make at least one suicide attempt during the course of their illness [Chen and Dilsaver, 1996] and up to 20–50% of schizophrenic patients attempt suicide [Roy et al., 1984; Radomsky et al., 1999]. The contribution of genetics to suicidal behavior is demonstrated by a higher average concordance for suicidal behavior among monozygotic twins compared to dizygotic twins or other relatives of suicidal subjects [Baldessarini and Hennen, 2004]. The observation that 10/26 monozygotic co-twins of suicide completers also attempted suicide, in comparison to 0/9 dizygotic co-twins, reinforces that suicidal behavior is influenced by genetic factors [Roy et al., 1995]. These data also suggest that a complex inheritance pattern is present and that epigenetic effects may be relevant. The serotonin (5-HT) neurotransmitter system regulates anxiety, impulsivity, and aggression [Mann, 1995], and so disturbances in the functioning of the 5-HT system have been hypothesized to influence suicidal behavior. Several studies have reported altered serotonin system function in people who die by suicide. For example, low concentrations of the serotonin metabolite 5-hydroxyl-indole-acetic-acid (5-HIAA) in cerebrospinal fluid (CSF) have been associated with both suicide attempts [Asberg et al., 1976] and completed suicide [Nordstrom et al., 1994]. The 5-HT2A receptor (HRT2A gene) is one of many 5-HT receptor subtypes, and it has been of particular interest in studies of suicide and major psychoses. The 5-HT2A-receptor gene is located on chromosome 13q14-q21 in humans, and consists of three exons separated by two introns, spanning over 20 kb [Chen et al., 1992]. A series of post-mortem studies found greater 5-HT2A-binding in the prefrontal cortex of suicide victims in comparison to controls, with similar findings in platelets of individuals with major depression who attempted suicide [Mann, 1999]. There have been many genetic association studies of polymorphic variants in 5-HT system genes and suicidality. In particular, the HTR2A C102T gene variant has been analyzed extensively [Du et al., 2000], but a large metaanalysis (including 1,599 suicidal patients) did not find a significant association between suicide attempts or suicide completion and the C102T polymorphism [Anguelova et al., 2003]. Other family-based genetic association studies using the transmission disequilibrium test have examined suicidal behavior, but none of these studies focused on the HTR2A C102T polymorphism [Zalsman et al., 2001; Wasserman et al., 2005]. The biological function of the HTR2A C102T polymorphism is unclear because it is synonymous and therefore does not alter amino acid sequence, although it may affect mRNA secondary structure [Arranz et al., 1995]. Another polymorphism in the HTR2A promoter region (1438A/G), is in complete linkage disequilibrium with C102T and could mediate the differential expression of the HTR2A C102T alleles. However, Sokolov and Polesskaya  found that this promoter SNP does not affect transcription Imprinting of 5-HT2A in Suicidality in vitro. The presence of a cytosine (C) at position 102 and a guanosine (G) at the position –1,438 creates two additional CpG islands, which are present in the C102 chromosomes, but are absent in the 102T chromosomes and these CpG sites may be methylated in the brain. Furthermore, carriers of the HRT2A 102T allele had a higher density of 5-HT2A receptors than carriers of the HRT2A C102 allele, in a post-mortem and genetic study that replicated the finding of greater 5-HTR2A binding in subjects who committed suicide [Turecki et al., 1999]. Analysis of mRNA expression in human post-mortem brain tissue suggests that the expression of the HTR2A C102 allele in the cortex of heterozygous individuals is significantly lower than that of the T allele [Polesskaya and Sokolov, 2002]. This group found no evidence of polymorphic imprinting, suggesting that if imprinting does occur, it must be a rare event. In light of these findings, our aim was to investigate the possibility that genomic imprinting of the HTR2A gene may play a role in suicidal behavior by examining both HTR2A C102T DNA variants and allele-specific mRNA expression in post-mortem brain samples from patients with SCZ or BD. Genomic imprinting is an epigenetic mechanism in which the parental origin of the allele can influence the level of gene expression [Surani et al., 1986]. The existence of genomic imprinting can be suggested by the presence of parent-of-origin effect (POE) in transmissions from maternal versus paternal meiosis to an affected offspring, thus we also investigated the possibility of genomic imprinting in suicidality by analyzing the C102T polymorphism in nuclear families of psychotic patients with suicidal behavior. The goals of the current study are: (1) to investigate the presence of genomic imprinting in suicidal behavior through POE analysis; (2) to document frequencies of biallelic and monoallelic expression in post-mortem brains from victims of suicide and matched controls; (3) to investigate the ratio of expression of the C102T SNP alleles in completed suicide. mRNA levels were quantified in samples donated by the Stanley Medical Research Institute [Torrey et al., 2000]. The Stanley Array Collection consists of mRNA and genomic DNA samples extracted from dorsolateral prefrontal cortex (DLPFC) (Brodmann’s area 46) from 35 individuals in each of three diagnostic groups: SCZ, BD, and unaffected controls. These specimens were collected, with informed consent from next-of-kin, by participating medical examiners between January 1995 and June 2002. The specimens were all collected, processed, and stored in a standardized way. Exclusion criteria for all specimens included: (a) significant structural brain pathology on post-mortem examination by a qualified neuropathologist, or by pre-mortem imaging, (b) a history of significant focal neurological signs pre-mortem, (c) a history of central nervous system disease that could be expected to alter gene expression in a persistent way, (d) documented IQ < 70, and (e) poor RNA quality (vide infra). Additional exclusion criteria for unaffected controls included: (a) age less than 30 (thus, still in the period of maximum risk), and (b) substance abuse within 1 year of death or evidence of significant alcohol-related changes in the liver. Diagnoses were made by two senior psychiatrists, using DSM-IV criteria, based on medical records, and when necessary, telephone interviews with family members. Diagnoses of unaffected controls were based on structured interviews by a senior psychiatrist with family member(s) to rule out Axis I diagnoses. Information about lifetime alcohol and drug abuse was recorded using a non-parametric scale ranging from 0 ¼ absent to 5 ¼ heavy at time of death. Of the 105 samples in total, there were 10 individuals who died by suicide, who were also heterozygous at the HTR2A T102C locus. We matched these suicide cases for genotype, diagnosis, age of death and sex to 10 controls who died from other causes. 371 The control group consisted of seven bipolar and three schizophrenia subjects (four male and six female) with a mean age of 43.6 11.04 (Table I). We have no information about whether the suicide attempts were violent or not but we do have information about medication treatment (Table II). The number of BD subjects with psychotic features was four in both groups. Genomic DNA was analyzed from the same samples and the single nucleotide polymorphism of the 5-HT2A receptor gene at position 102 was genotyped with TaqMan1 probes (ABI: Applied Biosystems Inc., Foster City, CA) to identify heterozygous subjects. HTR2A mRNA levels were measured with quantitative PCR (Q-PCR; ABI PRISM1 7000 Sequence Detection System) using TaqMan1 Assays-by-Design (ABI). The forward primer sequence was 50 -GACACCAGGCTCTACAGTAATGAC-30 and the reverse primer was 50 -CGACTGT CCAGTTAAATGCATCAGA-30 . Differential allelic expression and analysis was carried out on the ABI PRISM1 7000 with the following allele-specific, fluorescent-labeled probes: VIC- 50 CTTCTCCGGAGTTAAA-30 and 6FAM-50 - CTTCTCCAGAG TTAAA-30 . All reactions were performed in quadruplicate, and the investigators performing the PCR (V.D.L. and O.L.) were blind to the diagnosis of the tissue donors until after the data were analyzed. Differential allele expression was calculated by subtracting real-time PCR threshold cycle (Ct) values for the ‘‘C’’ and ‘‘T’’ alleles (DCt). However, assays based on allelespecific probes may generate heterozygote ratios that deviate from 1 as a result of differential binding efficiencies of the probes rather than differences in expression [Hoogendoorn et al., 2000; Bray et al., 2003]. To correct for this potential bias, the average DCt of the genomic DNA sample was subtracted from the DCt of the respective cDNA sample (DDCt). Relative levels of ‘‘C’’ to ‘‘T’’ were calculated as 2DDCt . Total 5-HT2A mRNA levels were calculated by combining the measured values of both alleles: ‘‘C’’ and ‘‘T’’. Relative mRNA levels in tissue are commonly expressed as a ratio between the gene of interest and a control or housekeeping gene. This procedure controls for differences in the amount of tissue from which the RNA is extracted, the amount of RNA in each sample, and the efficiency of reverse transcription to cDNA, since any distortion of these variables should affect the target gene and the control gene equally. In our study, we were interested in quantitative differences in mRNA specific to each allele of the HTR2A C102T SNP, and thus these factors are controlled for because both alleles would be affected TABLE I. Demographic Characteristics of the Subjects From Stanley Sample Suicide subjects Control subjects Sex M/F Principal Axis I Diagnosis, No. (%) Schizophrenia Bipolar disorder Psychotic bipolar Mean SD Age of death Lifetime alcohol abuse* Lifetime drug abuse* Time in the hospital (years) Age of onset (years) PMI (hours) Brain pH 4:6 4:6 1 (10%) 9 (90%) 4 3 (30%) 7 (70%) 4 40.4 10.68 2.40 2.01 1.70 1.83 0.200 0.163 26.6 11.2 41.0 17.6 6.45 0.31 43.6 11.04 2.10 2.31 2.10 2.08 0.267 0.269 24.2 6.3 26.9 14.8 6.30 0.22 *Lifetime alcohol and drug abuse were assessed on a semi-quantitative scale, with the following order: 0¼ little or none; 1 ¼ social; 2 ¼ moderate in the past; 3 ¼ moderate at time of death; 4 ¼ heavy past in the past; 5 ¼ heavy at time of death. 372 De Luca et al. TABLE II. Medications of the Subjects From Stanley Sample Stanley ID 133* 192 199 280 354 356 381 424 465 39 42 113 189 204 212 217 290 357 391 Suicide No No No No No No No No No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Antipsychotics Mood stabilizers Antidepressants Clozapine Fluoxetine, Trazodone, Doxepin Haloperidol Thiothixene Quetiapine, Olanzapine Quetiapine Quetiapine Risperidone Perphenazine Olanzapine Risperidone Quetiapine Risperidone Lithium Valproate Valproate, Gabapentin Carbamazepine Topiramate Lithium Lithium, Valproate Lithium Valproate Valproate Carbamazepine, Gabapentin Lithium Venlafaxine Trazodone, Sertraline Amitriptyline, Venlafaxine Fluoxetine, Venlafaxine Paroxetine, Trazodone Paroxetine Amitriptyline Venlafaxine Fluoxetine *This subject was on benztropine. equally. The differential expression ratio for each diagnostic group was described as a mean standard deviation (Fig. 1). Group differences were evaluated using independent and paired t-tests. Agonal status is one of the major limitations of post-mortem brain studies [Iwamoto and Kato, 2006], and thus we examined the effect of brain pH and post-mortem interval (PMI) on the allelic ratio. The correlation between mRNA expression and confounding factors was calculated by the Pearson coefficient (PMI and pH) and where appropriate (alcohol and illicit drug abuse) by non-parametric Spearman correlations. All test P values were two-tailed. The family-based DNA sample consisted of 102 nuclear families in which probands had a DSM-IV diagnosis of major psychoses (52 Bipolar I; 21 Bipolar II; 9 Schizoaffactive; 20 Schizophrenia) and who had attempted suicide. The patients were recruited from several hospitals in Toronto and across central Canada. Genomic DNA was extracted using standard high salt methods from white blood cells. A sexspecific parent analysis of the transmission of the common alleles was conducted using the ETDT package [Sham and Curtis, 1995]. This analysis allows us to detect a POE. In heterozygous individuals, the ‘‘C’’ and ‘‘T’’ alleles are expressed in the same cell. The most commonly used approach for quantifying the C/T ratio is the use of PCR amplification Fig. 1. Mean and SD of the C/T allele ratio for suicide and control groups. followed by HpaII digestion that distinguishes the C102T site [Polesskaya and Sokolov, 2002], but this method is biased by the efficiency of restriction endonuclease digestion. Another approach is to measure the relative expression of each allele, comparing the peak height ratio of each allele-specific extension product derived from SnaPshot analysis, giving a measurement of relative allele quantity [Bray et al., 2004]. We used instead a Taqman genotyping method, which allows us to circumvent the problem of incomplete digestion. The method employs PCR-amplification of cDNA and separate measurements of the amount of each allele through the use of sequence-specific oligonucleotide probes labeled with different fluorescent markers. Examining the raw Ct value for the ‘‘C’’ and ‘‘T’’ alleles (not normalized by subtraction from the average genomic DNA sample), confirmed lower levels of the ‘‘C’’ allele cDNA in overall sample (n ¼ 20). The average Ct was 31.63 1.76 for the ‘‘C’’ allele and 30.98 1.83 for the ‘‘T’’ allele (t ¼ 13.188; 19 df; P < 0.001). The average Ct in the genomic DNA was 29.10 1.53 for the ‘‘C’’ allele and 28.09 1.27 for the ‘‘T’’ allele (t ¼ 7.415; 18 df; P < 0.001). However, comparisons of the raw measurements of Ct for the two alleles should be treated with caution since differences in PCR amplification efficiency could affect this parameter. On the other hand, the fact that the same trend was seen in another study using a different technique [Polesskaya and Sokolov, 2002] suggests that C < T allele expression may indeed reflect differences in the amount of the two versions of the cDNA. The clinical and demographic variables for both suicide and non-suicide groups are shown in Table I. The groups were quite similar for all clinical and tissue variables, with only PMI showing a trend towards longer PMI in the suicide group (t ¼ 1.937; 19 df; P ¼ 0.069). However, PMI did not have an effect on the C/T ratio (r ¼ 0.274; P ¼ 0.242). The DCt in the genomic DNA was 1.05 0.65. The C/T allele cDNA level ratio in the 10 heterozygous suicide victims was 1.25 0.15, after the correction for the calibrator (genomic DNA DCt). The DCt in suicide victims cDNA was 0.73 0.17, significantly lower than the expected DCt of 1.05 (t ¼ 5.734; 9 df; P < 0.001). The DCt was <1.05 in cDNA from all 10 heterozygous suicide victims. The relative expression of the ‘‘C’’ to ‘‘T’’ allele in heterozygous non-suicide Imprinting of 5-HT2A in Suicidality controls was 1.42 0.21 after the correction. Also this group DCt (0.55 0.23) was significantly lower than the expected 1.05 (t ¼ 6.748; 9 df; P < 0.001). The mean ratios for the suicide and non-suicide groups were significantly different (t ¼ 2.107; 18 df; P ¼ 0.049), with the ‘‘C’’ allele showing lower cDNA levels in the suicide group in comparison to the control subjects. The allele ratio is not associated with demographic variables such as gender (t ¼ 1.216; 18 df; P ¼ 0.240), age at death (r ¼ 0.20; P ¼ 0.984), PMI (r ¼ 0.272; P ¼ 0.242), or brain pH (r ¼ 0.259; P ¼ 0.270). Furthermore, the C/T ratio was not influenced by the clinical variables of diagnosis (schizophrenia vs. bipolar: t ¼ 1.222; 18 df; P ¼ 0.238), lifetime alcohol abuse (r ¼ 0.107; P ¼ 0.653), lifetime drug abuse (r ¼ 0.039; P ¼ 0.870), age of onset of illness (r ¼ 0.118; P ¼ 0.621) or time spent in hospital (r ¼ 0.071; P ¼ 0.774). The total levels of the 5-HT2A receptor mRNA were 2.14 0.08 in the non-suicide controls, and 2.06 0.06 in the suicide victims, and this difference is statistically significant (t ¼ 2.406; 18 df; P ¼ 0.027). Comparison of the total level of 5-HT2A receptor mRNA between heterozygous suicide victims and non-suicide controls confirmed lower levels of the ‘‘C’’ allele in suicide subjects. The analysis of the C102T SNP did not reveal preferential transmission for either allele overall (35 vs. 29; Chisquare ¼ 0.563; 1 df; P ¼ 0.452). The separate analysis of individual alleles from maternal meiosis showed a trend (Chi-square ¼ 2.164; 1 df; P ¼ 0.141); the allele C was maternally transmitted only eight times while not transmitted 15 times. The C allele was paternally transmitted 13 times while not transmitted 12 times (Chi-square ¼ 0.040; 1 df; P ¼ 0.841). This particular data sample of post-mortem brain was not previously analyzed using this differential allelic expression technique. Our main findings are that decreased levels of the HTR2A C102 allele mRNA may be associated with suicide in major psychoses, and that suicide victims have a lower C102/ 102T allele expression ratio than controls. Few studies have explored the HTR2A C102T SNP in combination with other HTR2A gene sequence variants in the context of suicide. Previous studies have shown lower levels of the C102 allele, but did not account for the possible confounder of diagnosis [Polesskaya and Sokolov, 2002]. The molecular mechanism causing the HTR2A C102 allele mRNA to be decreased versus the 102T allele is unclear. One possibility is that epigenetic mechanisms may be involved, specifically differential methylation of this gene in suicide victims in comparison to non-suicide control subjects. Sokolov and Polesskaya  found that only 50% of the C102 sites are methylated in brain tissue; thus, lower HTR2A C102 allele mRNA expression in suicide victims could be the result of greater methylation. Furthermore, the HTR2A promoter polymorphism (1438G) that is linked to the C102T SNP may not directly affect transcription [Sokolov and Polesskaya, 2002]. We did not find evidence of genomic imprinting (complete inactivation of one allele in some individuals) in our sample of 10 heterozygous suicide victims and 10 heterozygous nonsuicide controls. This is in contrast with findings of complete imprinting of one allele in the analysis of 18 human adult brains, with four individuals showing monoallelic expression while others had biallelic expression [Bunzel et al., 1998]. It is possible that complete polymorphic imprinting of the 5-HTR2A gene may be a rare event. Consistent with this lack of imprinting at RNA level in suicide victims, the ETDT did not show a POE in suicide attempters, even though the 102T allele was transmitted more frequently from the mother. We also found decreased total 5-HT2A receptor mRNA in suicide victims, in contrast with some previous findings, but our methods were different and we analyzed only heterozygous 373 5-HT2A T102C subjects. Another issue is the type of brain tissue studied: we examined only DLPFC, whereas Bray et al.  found no significant effect of C102T polymorphism on mRNA expression in RNA from different cortical regions (frontal, parietal, and temporal). One potential limitation is that the sample used to analyze the differential allelic expression is small, but the observed power for this analysis was 63.7% considering the not so large effect size observed in this study (http://calculators.stat.ucla.edu/powercalc/normal/ n-2-unequal/index.php). Our results suggest that differential allele-specific expression of the HTR2A gene, influenced by the C102T polymorphism may be involved in suicidal behavior in major psychiatric disorders. Further, work to directly test the epigenetic regulation of this gene is required to confirm this hypothesis. In summary, the three main results presented (lower expression of 5-HT2A mRNA, lower HTR2A 102C vs. T102 allele expression and the absence of imprinting), contradict some previous findings. Our finding of decreased HTR2A mRNA in suicide victims is inconsistent with Turecki et al. , and could be explained by the fact that we analyzed only subjects heterozygous for the C102T polymorphism. However, our observation that the C102T polymorphism affects mRNA levels is concordant with Turecki et al. . With regards to the issue of imprinting, our results are consistent with two previous reports [Polesskaya and Sokolov, 2002; Bray et al., 2004] that found no imprinting, with one published study reporting evidence for imprinting [Bunzel et al., 1998]. Our data show decreased expression of the HTR2A C102 allele, which is in the same direction as one paper [Polesskaya and Sokolov, 2002] but not another [Bray et al., 2004]. It is possible that differences in the brain region studied (Bray’s sample: temporal and frontal cortex) could be responsible for these inconsistencies. Other limitations of our study include the unavailability of information on suicide method, previous suicide attempts, and concomitant personality disorders. Of course these clinical factors could increase sample heterogeneity, but it is unlikely that these factors influence the allelic ratio. The suicide and non-suicide groups are matched overall but not individually case-matched (there are more bipolar patients in the suicide group). However, the number of BD subjects with psychotic features in both groups is four, so the groups are matched on this feature. Furthermore, major axis I co-morbidity was not present in either group. Although the male–female and bipolar-schizophrenia distribution is not the same in the suicide and non-suicide groups, the C102T allele ratio is not significantly different between the two diagnostic groups or between male and female subjects. Another limitation is the fact that different medication treatment may influence the HTR2A allelic ratio even though a specific effect of psychotropic drugs on differential allelic expression is not yet demonstrated. Differences in allele-specific PCR amplification efficiency could have affected the comparison, even though we have compared allelic ratios and not the raw measurement Ct values. 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