5-HT polymorphisms and response to SSRIs in OCD Corregiari FM et al. treatment (clomipramine, fluoxetine, fluvoxamine, citalo- pram, sertraline or paroxetine) as either non-responders (NRs) or responders (RPs) according to the following criteria: – NR: no more than a 25% reduction in the Y-BOCS (6) score after at least: two different selective serotonin reuptake inhibitors (SSRIs) at a maximum or maximum tolerated dose for at least 8 weeks and (2) clomipramine at a maximum or maximum tolerated dose for at least 8 weeks (the NRs in this study have received an average of 13.27¡9.5 years of treatment). – RP: a Y-BOCS score of less than 9 and a Sheehan Disability Scale (7) score of less than 10 after treatment with an SRI and stable for at least 2 months. We evaluated 239 OCD patients from our own service or referred to us for specialized treatment. From these, 60 patients met inclusion criteria. OCD patients who did not meet the criteria for either NR or RP were excluded from this study. Patients with severe depression (HAMD-21.25), affective bipolar disorder, schizophrenia, schizoaffective disorder, active substance abuse or dependence were excluded. Patients currently taking fluoxetine were excluded because of the drug’s long half-life. We selected 30 age- and gender-matched subjects who were free of mental and medical disorders as a control group (CN). The control subjects had never used psycho- tropic medications and were free from any medication for at least one month. The clinical characteristics of patients and controls are presented elsewhere (4). All medications including SRIs (except for low-dose bedtime benzodiazepines, which were maintained at a consistent dose throughout the study) were gradually tapered off, and the patients were observed over a one-week washout period before the challenge test. Females were tested in the early follicular stage of their menstrual cycle (defined as the period between the third and tenth day of the cycle). Challenge test procedure Challenge test procedures began at 8:00 AM, after an overnight fast. After a peripheral intravenous insertion and 60 minutes of rest, blood sampling began at 9:00 AM. The samples were collected at 20-minutes intervals during 180 minutes. The citalopram infusion began 20 minutes after the first blood sample had been collected. Twenty milligrams of citalopram were diluted in 250 ml of saline and infused over 60 minutes. Subjects were tested in a reclining position and were kept awake. Plasma cortisol, prolactin and growth hormone (GH) levels were quantified by standard immunoradiometric assays at -20, 0 (onset of the citalopram infusion), 20, 40, 60, 80, 100, 120, 140, and 160 minutes. The serotonin concentration in platelet-rich plasma was measured through high-performance liquid chromatography with a Shimadzu 10Vp system in conjunction with a fluorescence detector (Shimadzu RF-10A XL). The platelets were counted on a cell counter Ac T diff Coulter (Beckman Coulter) to normalize the results. Investigated polymorphisms The investigated polymorphisms included the G861C polymorphism (rs6296) of the serotonin receptor subtype 336 CLINICS 2012;67(4):335-340 1Db (HTR1B) gene and T102C (rs6313) and C516T (rs6305) polymorphisms of the serotonin receptor subtype 2A (HTR2A) gene. Genotyping Genomic DNA was extracted using a salting out method (8). There were technical problems when genotyping three responders and three control subjects. Therefore, some of the gene variants were not analyzed for these samples. However, two resistant patients (excluded in the challenge test because of pairing issues) were included in the gene frequency analysis. HTR1B (G861C). The G861C polymorphism of the gene coding for the serotonin receptor subtype 1Db was genotyped according to the procedures described by Mundo et al. (9). The 548-bp PCR fragment was digested with Hinc II restriction enzyme. The alleles were detected after separation on an agarose gel, and the G allele was the undigested fragment. HTR2A (T102C). Genotyping of the T102C polymorphism of the gene coding for the serotonin receptor subtype 2A was performed using the primers and conditions described by Warren al. (10). After amplification, the PCR products were digested with Msp I. The fragment sizes were a single 342-bp band for the 102T allele and two bands (216 bp and 126 bp) for the 102C allele. HTR2A (C516T). The primers and PCR conditions used for the analysis of the C516T polymorphism of the gene coding for the serotonin receptor subtype 2A were described by Arranz et al. (11). The amplified DNA products from the patient and control samples were digested with Sau 96I restriction enzyme (New England Biolabs Inc., cat. R0165S). The C516 allele (digested PCR product) showed 109-bp and 87-bp fragments, whereas the T516 allele remained uncut. To confirm the genotyping results for all the investigated markers, we used a commercial 50–bp DNA ladder (GE Healthcare Life Sciences, product code: 27-4005-01). After the restriction enzyme cleavage reaction, the fragments of each marker were separated electrophoretically on an agarose gel and visualized under a UV light. All of the genotyping results were interpreted by two independently trained research technicians. The same person genotyped all samples. The genotyping process was repeated when a disagreement arose (e.g., the T516C genotyping of two heterozygous individuals was repeated during the analysis). Statistical analysis The prolactin and cortisol responses to citalopram were measured as maximal percentage variation (max%D). Because of the values observed at baseline, GH responses were measured as the maximal variation over baseline (maxD). The continuous variables were analyzed using an analysis of variance (ANOVA) or a paired t-test when appropriate. Friedman, Mann-Whitney or Wilcoxon matched tests were used when a non-parametric analysis was required. A general linear procedure was used to perform a two-way ANOVA and post-hoc analysis. Categorical data were analyzed using a chi-square test and a chi-square partition. The significance level was 5% and based on two-tailed tests. A standard deviation was calculated to indicate variability. A test for deviations from the Hardy-Weinberg equilibrium was performed using the