Serotoninergic neurotransmitter systems have been implicated in the pathogenesis of major psychoses. A functional polymorphism (5-HTTLPR) in the upstream regulatory region of the gene (SLC6A4) has been associated with a number of psychiatric disturbances, but conflicting replication followed. The aim of this study was to investigate the possibility that the 5-HTTLPR might be associated with major psychoses. One thousand, eight hundred and twenty inpatients (789 bipolars, 667 major depressives, 66 delusionals, 261 schizophrenics, 37 psychotics not otherwise specified—NOS) and 457 control subjects were included in this study. A subsample of 1235 patients (523 bipolars, 359 major depressives, 259 schizophrenics, 66 delusionals, 28 psychotic NOS) were evaluated for lifetime psychotic symptomatology using the Operational Criteria for Psychotic illness (OPCRIT) checklist. The subjects were also typed for 5-HTTLPR variants using PCR techniques. 5-HTTLPR allele frequencies were not significantly different between controls and bipolars, major depressives, schizophrenics, delusionals and psychotic NOS; genotype analysis also did not show any association. The analysis of symptomatology did not show significant differences. Consideration of possible stratification factors such as sex and age of onset did not significantly influence results. 5-HTTLPR variants are not therefore a liability factor for major psychoses or for major psychoses symptomatology.
The serotonin transporter is the major determinant of serotonin inactivation following release at synapses and it is the site of action of most antidepressants. The gene coding for the serotonin transporter (SLC6A4) has been therefore proposed as a possible candidate for involvement in the pathogenesis of major psychoses. The primary structure of the serotonin transporter gene did not prove to be associated with the diagnosis of mood disorders,1 however, a polymorphic region containing a 17-bp variable number of tandem repeat (VNTR) in the second intron was associated with major depressive disorder2,3,4 and a functional polymorphism in the upstream regulatory region of the gene has been associated with both major depressive and bipolar disorders,5,6,7 although subsequent studies did not replicate these results.8,9,10,11,12 The polymorphism in the upstream is a 44-bp deletion/insertion (5-HTTLPR) located exactly at the 5′-flanking regulatory region of the serotonin transporter gene on chromosome 17q11.2. In vitro studies evidenced that the basal activity of the long (l) variant was more than twice that of the short (s) form of the 5-HTTLPR, suggesting that serotonin transporter gene transcription is modulated by variants of the 5-HTTLPR with the s allele corresponding to low serotonin uptake activity.13
The gene also proved to be associated or showed conflicting evidence with a number of other conditions, like anxiety-related traits in normals and in depressed patients, seasonal affective disorder, anxiety disorders, autism, severe alcoholism, suicidal behavior, psychotic symptomatology in neuroleptic-free schizophrenics and schizophrenia.14,15,16,17,18,19,20,21
A possible reason for conflicting results is the use of psychiatric diagnoses which do not guarantee biological homogeneity.22 Diagnostic categories contain a high degree of heterogeneity (eg presence of delusional features, anxiety, melancholic features, variable time course and drug response), therefore genetic studies based on diagnostic criteria may furnish biased results based on a different rate of subgroup populations. Moreover abnormalities in the serotonin system have been described in the pathogenesis of a number of behaviors related to psychiatric disorders.23 This led to the suggestion that the influence of serotonin would be better analyzed at the level of symptoms rather than syndromes. For these reasons, in the present article we complemented the traditional association study with a phenotype definition that is based on the lifetime excitement, delusional, depressive, disorganized and negative symptomatology24,25 as an alternative to the traditional use of psychiatric diagnoses to define the affected phenotype for genetic studies.26 Our preliminary analyses on mood disorders and schizophrenia did not evidence associations, but the small sample size may lead to false negative findings.27,28 In fact a complex and oligogenic mode of transmission has been hypothesized for schizophrenia and mood disorders, with a small number of ‘minor effect’ interacting genes, each contributing to relative risks of about 2.29 Non-parametric analyses are the favorite tool to reveal ‘minor effect’ genes, but their low power compared to parametric analyses requires very large samples. Therefore a possible reason for conflicting results may be linked to sample size; samples in the range of 100–200 affected subjects do not allow enough power.30 In fact, evidence for association with bipolar disorder was observed with a large sample of hundreds of subjects.31 Our aim was to investigate the 5-HTTLPR polymorphism in a large, geographically homogeneous sample.
5-HTTLPR allele frequencies were not significantly different between controls and bipolars, major depressives, schizophrenics, delusionals and psychotic NOS (Table 1). Genotype analysis also showed no association (Table 2). Stratification factors such as early age of onset (<25 years) and sex did not modify 5-HTTLPR variant frequencies significantly.
The association between 5-HTTLPR variants and excitement, delusional, depressive, disorganized, and negative symptomatology was estimated using one-way analysis of variance (ANOVA) (Tables 3 and 4). The ANOVA on factor scores did not show significant differences. The control sample and all diagnostic groups were in Hardy–Weinberg equilibrium.
5-HTTLPR has been proposed as a candidate gene in major psychoses, as it modulates serotonin transporter gene transcription, with the short allele corresponding to low serotonin uptake activity.13 But mood disorders proved not to be associated unequivocally with both polymorphisms at SLC6A4.2,6,11,31 A linkage study on bipolar families using the PstI RFLP at the 5HTT locus did not support the presence of a susceptibility locus for bipolar disorder near the serotonin transporter32 and it did not show any association with schizophrenia.33 Finally, our preliminary studies did not evidence any association with major psychoses symptomatology.27,28 The present study suggests that no strong association exists between 5-HTTLPR variants and major psychoses, both for diagnoses and lifetime psychotic symptomatology.
A significant association was also reported between the 5-HTTLPR polymorphism and the Neuroticism factor in normals,14 even if conflicting results followed. This finding is in accordance with our report that 5-HTTLPR*s may be a risk factor for anxiety symptomatology among affective subjects.15 Moreover, we reported a worse response for the 5-HTTLPR*s/s subjects to antidepressant treatments.34,35,36,37,38
We may therefore hypothesize that a given genetic predisposition (5-HTTLPR*s) may confer susceptibility to anxiety features and a worse antidepressant response in subjects affected by mood disorder. Abnormalities in the serotonin transporter function may in fact confer only a small susceptibility to state conditions, because an adaptatory mechanism may partially compensate, while the dramatic alterations of serotonin turnover observed during antidepressant treatment may evidence partial transporter abnormalities, that lead to a worse antidepressant response. 5-HTTLPR l and s allele frequencies in controls are similar to those of previously published Caucasian samples,6,10,14 and are different from Asians.39
In this study we used psychopathology independently of clinical diagnoses and the use of symptoms for phenotype definition may be considered a limitation as symptomatologic presentation may vary across illness episodes, but follow-up studies showed that episodes are substantially stable over time.40,41 Moreover the validity of our factors is evidenced by the coherence with previous studies in schizophrenia and mixed samples,42 the moderate but significant heritability for all factors,43,44 and the correlation with regional anatomical abnormalities.45 The use of drugs could bias our ability to detect naive symptomatology, but this should not constitute a major obstacle.24 Specifically, while most subjects were admitted assuming lithium, antipsychotic, antidepressant or benzodiazepine treatments, we applied a lifetime perspective to rate the OPCRIT checklist, also using complementary sources of data like previous records and family informants.46 This strategy allowed us to describe early drug-free illness phases.
Further variants in the 5-HTTLPR area could be analyzed. Recently a 381-bp insert between the polymorphic region and the transcription start site had been described; the sequence analysis of this novel region reveals a number of transcription factor binding sites (eg AP1, Elk1, NFkappaB) that may be important in controlling the response of the 5HTT gene to regulatory factors.47,48
Another limitation is linked to the ethnic origin that is frequently a cause of stratification bias, but our sample was composed of subjects mainly collected in the North of Italy with Italian antecedents for at least two generations and Italy is characterized by a substantial genetic homogeneity.49 Controls were screened for the absence of mental illnesses, though this may introduce a bias on allelic frequencies. Association studies comparing very common diseases like major depression should exclude these cases from the control sample as they may obscure positive associations.
In conclusion, despite some limitation, the analysis of such a large sample of mood disorder subjects strongly supports the view that 5-HTTLPR variants are not associated with conditions such as the diagnoses of major psychoses or lifetime psychotic symptomatology. On the other hand converging evidence suggests a role in dynamic processes such as antidepressant treatments.
One thousand, eight hundred and twenty inpatients (age 46 ± 14.39 years; onset 32 ± 12.43 years; males 42%) consecutively admitted to the Department of Psychiatry at the Institute H. San Raffaele were included in this study (789 bipolars, 667 major depressives, 66 delusionals, 261 schizophrenics, 37 psychotic not otherwise specified—NOS). The present sample has been partly analyzed: 192 subjects (75 bipolars, 22 major depressives and 95 controls) were included in a previous collaborative paper,31 230 mood disorder subjects27 and 161 schizophrenics28 were analyzed for lifetime symptomatology. Three hundred and seventy-six (247 affected subjects and 129 controls) were collected in the context of the European Collaborative Project on Affective Disorders.50
Lifetime diagnoses were assigned by two independent psychiatrists on the basis of clinical interviews and medical records, according to DSM-IV criteria following a best estimate method.46 A subsample of 1235 patients (523 bipolars, 359 major depressives, 259 schizophrenics, 66 delusionals, 28 psychotic NOS) was evaluated using the Operational Criteria for Psychotic illness (OPCRIT) checklist with a lifetime perspective.51 Criteria for inclusion in the study were diagnosis of major psychoses. The presence of concomitant diagnoses of mental retardation or drug dependence, together with somatic or neurological illnesses that impaired psychiatric evaluation represented exclusion criteria. Four hundred and fifty-seven control subjects were recruited among both healthy department personnel and people attending the general lab of our hospital (age 46 ± 15.54 years; males 49%). Before drawing blood samples, controls were closely investigated to determine whether they or their first-degree relatives had psychiatric disturbances or previous psychiatric treatment, through interviews with subjects and relatives when possible. Only unaffected subjects with negative family histories were included in the study. Informed consent was obtained from all subjects; these were unrelated and of Italian descent, with antecedents from all parts of the country.
DNA was extracted from leucocytes by NaCl precipitation.52 PCR forward primer 5′-GGCGTTGCCGCT CTGAATGC-3′ and reverse primer 5′-GAGGGAC TGAGCTGGACAACCAC-3′ were employed and PCR products were analyzed as decribed previously.15
The factoring process of the OPCRIT checklist has been previously explained.24,25 The factor composition is as follows: Excitement (items: Excessive activity, Reduced need for sleep, Pressured speech, Elevated mood, Thoughts racing, Increased sociability, Increased self esteem, Irritable mood, Distractibility, Agitated activity, Dysphoria, Grandiose delusions, Reckless activity); Depression (items: Loss of pleasure, Loss of energy/tiredness, Diminished libido, Excessive self reproach, Slowed activity, Poor appetite, Poor concentration, Suicidal ideation, Weight loss, Diurnal variation, Early morning waking, Delusions of guilt); Delusion (items: Persecutory delusions, Well organized delusions, Delusions of influence, Widespread delusions, Primary delusional perception, Delusions and hallucinations last for one week, Persecutory/jealous delusions and hallucinations, Thought insertion, Thought withdrawal, Thought broadcast, Third person auditory hallucinations); Disorganization (items: Speech difficult to understand, Incoherent, Positive formal thought disorder, Inappropriate affect, Bizarre behavior, Bizarre delusions); and Negative (items: Blunted affect, Negative formal thought disorder, Restricted affect, Catatonia). Factor scores were calculated as the sum of binarized items divided by the number of items in each factor. They were considered to be dependent variables when investigating their distribution across variants. Data were analyzed with a Chi-square test for the association with diagnoses. Differences of factor scores among variants were assessed using one-way Analysis of Variance (ANOVA). The power of our sample to detect differences amongst 5-HTTLPR variants was calculated considering an alpha value of 0.01 (Bonferroni correction: 0.05/5 factors). With these parameters in our sample we had a power of 0.80 to detect a small effect size of d = 0.14, that corresponded to a difference of approximately 0.042 points between the two 5-HTTLPR variants on factor scores.53
This work was partially supported by the BIOMED 2 grant BMH4-CT97-2307.
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Monoamine Metabolites Level in CSF is Related to the 5-HTT Gene Polymorphism in Treatment-Resistant Depression