Association of FKBP5 Polymorphisms With Suicidal Events in the Treatment of Resistant Depression in Adolescents (TORDIA) Study

The two outcome measures for this pharmacogenetic study were adequate clinical response and suicidal events. Adequate clinical response after 12 weeks of treatment was defined a priori as a rating of much or very much improved on the improvement subscale of the Clinical Global Impressions scale (CGI-I) (22), a decrease >50% in the Children's Depression Rating Scale–Revised score, and a score <40 on the Children's Depression Rating Scale–Revised. Suicidal events were classified according to the Columbia Classification Algorithm of Suicide Assessment using the Brief Suicide Severity Rating Scale (23), which rates both ideation and behavior on a scale of 0 to 5. For ideation, 0 corresponds to no ideation and 5 to ideation with intent and a plan; for behavior, 0 corresponds to no behavior and 5 to multiple attempts. A suicidal event was defined as new-onset or worsened suicidal ideation or suicidal behavior requiring at least a 1-point increase from baseline on the Brief Suicide Severity Rating Scale. A suicide attempt was defined as "self-injurious behavior with explicit or implied intent to die" (23). All adverse events were discussed during weekly conference calls and were classified by consensus. In addition, interrater reliability in the assessment of suicidal events was high for both ideation (intraclass correlation coefficient=0.9, p<0.001) and behavior (100% agreement). Not counted as suicidal events were instances of nonsuicidal self-injury, which was defined as engagement in self-injury with no intent to die, motivated by a desire to relieve negative affect or to obtain social reinforcement (23, 24). Suicidal events were assessed by spontaneous report during the first half of the study and systematically at every visit during the second half of the study. Spontaneously reported events appeared to be representative of more serious suicidal adverse events (19).

The sample was also characterized with respect to duration of depression and presence of DSM-IV comorbid disorders using the Schedule for Affective Disorders and Schizophrenia for School-Age Children–Present and Lifetime Version, self-reported depression using the Beck Depression Inventory, suicidal ideation using the Suicidal Ideation Questionnaire–JR, and adolescent- and parent-reported family conflict using the Conflict Behavior Questionnaire, Adolescent Report and Parent Report versions (25–28). Self-ratings and interview ratings of symptoms and symptom severity were obtained at baseline, at 6 weeks, and at 12 weeks. In this sample, 83.2% completed treatment, and 96.1% and 96.8% were available for their 6-week and 12-week assessments, respectively.

Participants were cross-tapered from their entry SSRI over the first 2 weeks and were seen by pharmacotherapists weekly for the first 4 weeks and every 2 weeks for the next 8 weeks. Those assigned to an SSRI (fluoxetine, paroxetine, or citalopram) received 10 mg/day for the first week and 20 mg/day for the next 5 weeks, and then, if the CGI-I score was 3 or greater, the dose was increased to 40 mg/day. Those assigned to venlafaxine had the following dosage schedule: 37.5 mg/day for 1 week and weekly increases of 37.5 mg/day until a dosage of 150 mg/day was reached at 4 weeks, with an option to increase to a dosage of 225 mg/day if the CGI-I score was 3 or greater. By 12 weeks, the mean dosage for SSRIs was 32.0 mg/day (SD=9.9), and for venlafaxine 201.6 mg/day (SD=35.0). Participants assigned to receive CBT had up to 12 sessions lasting 60–90 minutes, as described previously (18). Participants in the pharmacogenetics study received an average of 9.6 CBT sessions (SD=2.5); there was no difference between medication cells. Adjunctive treatment (e.g., with trazodone or a benzodiazepine) was allowed for sleep and anxiety. In both the overall sample and in this subsample, the use of trazodone was associated with poorer antidepressant response and with self-harm (either nonsuicidal self-injury or suicidal) events (18, 19).

We targeted genes related to central serotonergic and noradrenergic neurotransmission, namely, tryptophan hydroxylase (TPH1, TPH2), the serotonin transporter (HTT, SLC6A4), serotonin receptors (HTR1A, HTR2A), the norepinephrine transporter (NET1, SLC6A2), the noradrenergic receptor (ADRB1), and monoamine metabolism (MAOA). We also studied genes involved in glutamatergic neurotransmission (GRIK2, GRIA3) and in the HPA axis (FKBP5) and genes that code for neurotrophic proteins (BDNF). Table 1 lists the polymorphisms that we tested for each gene (rs) and the frequency of the minor allele.

DNA was obtained from either blood or cheek swab from all participants. We collected 20 ml of whole blood in EDTA tubes, which were shipped to the Human Genetics Laboratory at the University of Pittsburgh for DNA extraction using the Puregene system (Gentra Systems, Minneapolis). Buccal cell DNA was extracted using a modification of this method. DNA was diluted to a standard 50 μg/ml, aliquoted, and frozen at –80°C until used.

All genotyping was carried out using standard polymerase chain reaction-based techniques that are routinely used in the Human Genetics Laboratory. DNA segments containing the target variable site were amplified using unique sequence flanking primers. Length polymorphisms (MAOA, DAT1 5′VNTR, HTT 5′VNTR) were genotyped by separation of amplimers on 3%–4% agarose gels and visualization of the ethidium bromide stained gel under ultraviolet light. All other sites are SNPs, which were genotyped by the fluorescence polarization method of Chen et al. (29). Genotypes were assigned by direct comparison to sequence-verified control samples. Across genotypes, a median of 7% of samples could not be genotyped (range=0.0%–13.1%). A random 5% subsample of individuals was regenotyped with concordance observed for all duplicates. For none of the genotypes was genotyping success related to either of the two outcome variables for this study.

Reported analyses were conducted on the 88.1% of the overall sample who were Caucasian (N=155), although all were repeated with the entire sample after controlling for ethnicity, which was assessed by participant self-report, with no difference in the results. The characteristics associated with treatment response and with the occurrence of suicidal events were assessed using standard univariate statistics. Hardy-Weinberg equilibrium was examined using exact tests (30). For sex-linked polymorphisms, Hardy-Weinberg equilibrium was examined in females. SNPs with a minor allele frequency <0.05 were excluded (ADRB rs3730284T). All SNPs were in Hardy-Weinberg equilibrium. Single-SNP analyses were conducted using PLINK (http://pngu.mgh.harvard.edu/~purcell/plink). For 80% power, an alpha of 0.001 to correct for the number of markers and outcomes, and a frequency of minor allele ranging from 20% to 40%, the power to detect an association between genotype and response was sufficient for a relative risk of 4 or higher, and the power to detect an association between genotype and suicidal events, given the lower frequency of the latter, was sufficient for a relative risk of 5.5 or higher.

Logistic regression analyses were used to examine the relationship between our outcome measures and the additive effects of each polymorphism, controlling for medication, treatment with CBT, and medication-by-CBT interaction and covariates that were significantly different on treatment response or suicidality (i.e., family conflict, trazodone). Because family conflict was associated with both response and suicidal events, we also controlled for family conflict-by-genotype interactions. For sex-linked polymorphisms, sex was included as a covariate in the models. We used permutation procedures (max[T] with 10,000 permutations) to generate significance levels empirically, which have the desirable properties of relaxing assumptions about Hardy-Weinberg equilibrium, dealing with rare alleles, and small sample sizes as well as providing a framework for correction for multiple testing. Two p values are computed: a pointwise estimate of the significance of each individual polymorphism and a p value that controls for the number of polymorphisms tested while preserving the correlation structure among them. We corrected the p value for examining two outcomes. This is conservative, since suicidal events were more common in those who did not respond to treatment (14/64 [21.9%] compared with 4/91 [4.4%]; χ2=11.2, df=1, p=0.001).

Patients who participated in the genetic study were similar to those who did not with regard to nearly all demographic and clinical variables and treatment assignment but were younger on average (15.7 years [SD=1.6] compared with 16.2 years [SD=1.5]; t=2.28, df=290, p=0.02) and were more likely to have at least one parent who graduated from college (48.8% versus 36.7%; χ²=3.94, df=1, p=0.047). Similar to the total sample, they were mostly female (70.5%), with moderate to severe chronic depression (CGI-S score, mean=4.4 [SD=0.6] and Children's Depression Rating Scale–Revised score, mean=58.8 [SD=9.8]) with a median duration of 2 years. A high proportion of participants entered the study with clinically significant suicidal ideation (59.1% had a score ≥31 on the Suicidal Ideation Questionnaire–JR) or a history of a previous suicide attempt (23.9%) or of nonsuicidal self-injury (38.7%). Anxiety disorders (31.7%) and attention deficit hyperactivity disorder (16.5%) were the two most common comorbid conditions. The 176 participants in the pharmacogenetics study were evenly distributed with respect to both CBT assignment (48.9%) and medication (50% to each medication condition), and the treatment groups did not differ with respect to baseline demographic or clinical variables.

Characteristics of those who responded. Participants who responded to treatment had a lower score for parent-child conflict on the Conflict Behavior Questionnaire–Adolescent Report (mean=6.7 [SD=5.9] compared with mean=10.1 [SD=6.2]; t=3.40, df=150, p=0.001), had a lower rate of previous nonsuicidal self-injury (29.2% versus 54.0%; χ²=9.46, df=1, p=0.002), and were less likely to have received trazodone (4.4% versus 25.0%; χ²=14.19, df=1, p<0.001). In the overall sample, CBT was associated with a greater response rate, which fell short of statistical significance in this subsample (54.9% versus 45.1%; χ²=3.08, df=1, p=0.08).

Characteristics of those with a suicidal event. A total of 18 participants experienced at least one suicidal event: four made suicide attempts, two communicated intent to attempt suicide, four had new-onset suicidal ideation (three with passive death wishes and one with ideation with a plan), and eight had worsening of their suicidal ideation by at least 2 points on the Brief Suicide Severity Rating Scale (four from passive death wish to ideation with a concrete plan, four from ideation to ideation with plan and intent). Those who experienced suicidal events were less likely to have a parent with a college education (23.5% versus 53.4%; χ²=5.38, df=1, p=0.02), had higher self-reported depression (Beck Depression Inventory score, mean=24.2 [SD=11.1] compared with mean=19.1 [SD=11.1]; t=–2.06, df=152, p=0.04), had higher suicidal ideation (Suicidal Ideation Questionnaire–JR score, mean=51.5 [SD=22.1] compared with mean=40.3 [SD=20.5]; t=–2.42, df=152, p=0.02), were more likely to have a history of nonsuicidal self-injury (77.8% compared with 34.3%; χ²=12.54, df=1, p<0.001), and had more child-reported parent-child conflict (Conflict Behavior Questionnaire–Adolescent Report score, mean=12.9 [SD=5.5] compared with mean=7.4 [SD=6.1]; t=3.61, df=150, p<0.001); those who had a suicidal event were more likely to have received trazodone (38.9% compared with 9.5%; Fisher's exact test, p<0.001).

There were no univariate relationships between genotype and treatment response, although there was a nonsignificant trend for the FKBP5 rs1360780 and rs3800373 genotypes to be associated with a lower response rate. After controlling for CBT, medication, medication-by-CBT interaction, Conflict Behavior Questionnaire–Adolescent Report score, SNP-by-Conflict Behavior Questionnaire–Adolescent Report score interaction, and use of trazodone, there were no significant relationships between genotype and treatment response (Table 2). There were no statistically significant gene-by-medication interactions with respect to genetic markers that we hypothesized would predict a differential response to venlafaxine (SNP-by-medication interactions for NET 1287 and ADRB SNPs).

There was a significant association between the FKBP5 rs1360780 TT genotype and suicidal events (7/15 compared with 4/64 and 7/71, respectively, for the CT and CC genotypes; Fisher's exact test, p=0.001; TT versus CC/CT, Fisher's exact test, p<0.001), with similar findings for the FKBP5 rs3800373 GG genotype (7/14 compared with 4/61 and 7/78, respectively, for the GT and TT genotypes; Fisher's exact test, p<0.001; GG versus TT/GT, Fisher's exact test, p<0.001).These associations did not differ across treatment groups and were statistically significant even when restricted to participants who did not respond to treatment. The FKBP5 rs1360780 and rs3800373 genotypes showed similar relationships with suicidal events when these were assessed using spontaneous or systematic reporting. Genotype was unrelated to a previous history of attempts, nonsuicidal self-injury, and baseline suicidal ideation. With respect to the occurrence of suicidal events, there was an additive effect for both the FKBP5 rs1360780 (odds ratio=59.3, p=0.0007, corrected p=0.025, 95% confidence interval [CI]=4.7–743.6) and FKBP5 rs3800373 alleles (odds ratio=36.8, p=0.0007, corrected p=0.025, 95% CI=3.6–371.5), even after controlling for the effects of medication, CBT, CBT-by-medication interaction, use of trazodone, family conflict, and family conflict-by-genotype interaction (Table 3). Follow-up analyses of the FKBP5 markers were consistent with a recessive effect for both the FKBP5 rs1360780TT (odds ratio=131.9, p=0.002, corrected p=0.004, 95% CI=6.4–2736) and for the FKBP5 rs3800373GG (odds ratio=79.1, p=0.003, corrected p=0.006, 95% CI=4.5–1389) genotypes. The addition of history of nonsuicidal self-injury as a covariate did not change these results. These two SNPs were in strong linkage disequilibrium (r=0.91).

A higher hazard ratio and an earlier onset of a suicidal event was associated with the FKBP5 rs1360780TT and rs3800373GG genotypes (hazard ratio=9.30, 95% CI=2.7–31.8, and hazard ratio=9.70, 95% CI=2.8–33.24, respectively, p values <0.0001 [see Figure 1]), and this effect persisted even after controlling for the effects of CBT, medication, medication-by-CBT interaction, trazodone use, Conflict Behavior Questionnaire–Adolescent Report score, and a Conflict Behavior Questionnaire–Adolescent Report score-by-genotype interaction (p values, 0.006 and 0.016, respectively).