The association of anxiety and other clinical features with CACNA1C rs1006737 in patients with depression

2.1 Design

The patients were recruited from hospital departments where a high degree of psychopathology can be expected and the patients’ disease history will be well described from the treatment course.

The clinical data were collected in a standardized manner with a protocol including: age, sex, ethnicity, International Classification of Diseases version 10 (ICD-10) diagnoses, psychopathology including anxiety at admission, psychiatric illness in family, age for first psychiatric admission, age for first psychopharmacological treatment, number of admissions, suicide attempts, use of alcohol and drugs through life, treatment with lithium or electroconvulsive therapy (ECT) through life, and trauma in childhood. This information was obtained by a interview and/or extracted from patients’ file.

2.2 Patients and controls

Patients were selected from the Danish Psychiatric Biobank based on the diagnosis of unipolar depression according to the ICD-10 diagnostic criteria. The Danish Psychiatric Biobank is a collaborative effort between psychiatric departments in Copenhagen to collect blood samples from psychiatric patients from 2002 to 2015.

The majority (85%) of the patients were ethnical Danish, that is, the patients and both parents were born in Denmark, while in a minor fraction of the cases (15%), one parent was Caucasian and born outside Denmark in another North-western European country, primarily Sweden or Norway, secondarily in Germany, The Netherlands, England, or France [18].

Patients were selected from the Danish Psychiatric Biobank if they were given a clinical diagnosis of unipolar depression according to the ICD-10 when they were admitted to the psychiatric department. The ICD-10 diagnosis was later confirmed either 1) by a structured interview with “Scheduled Clinical Assessment Neuropsychiatry” by a trained research psychiatrist or 2) the diagnoses were approved from the file by a trained research psychiatrist or 3) the patient had been admitted at least 2 times with a clinical diagnosis of depression according to the ICD-10. A total of 754 patients participated (241 males and 513 females). The clinical information was obtained by interview with the patients or extracted from the patient’s file.

1. Ethical approval: The research related to human use has been complied with all the relevant national regulations, institutional policies and in accordance the tenets of the Helsinki Declaration, and has been approved by the Danish Scientific Committees (J.nr. 01-024/01) and the Danish Data Protection Agency.

2. Informed consent: Informed consent has been obtained from all individuals included in this study.

2.3 Controls

Controls were blood donors from the blood bank on Rigshospitalet in Copenhagen. To be a blood donor, the person had to be between 17 and 60 years of age, healthy, and with a weight of at least 50 kg. Blood donor samples were obtained according to the same protocol that was used to recruit patients [18]. The controls were anonymous and only the sex was known. Seven hundred and eight controls participated (272 males and 436 females).

2.4 Anxiety

The presence of anxiety was assessed in two ways.

It was recorded from the patients file whether there had been described anxiety or not in the first week after admission.

If the patient scored 2 or more on item 10 (feeling anxious) on Hamilton 17 Rating Scale [19]. The evaluation could be performed at any time under an admission or treatment in an outpatient clinic.

2.5 Alcohol abuse

It was recorded from the patient file if the patient had a diagnosis of alcohol abuse, had been in treatment with disulfiram, had been in treatment in an out-patients clinic for alcoholics, or if there was other information indicating abuse of alcohol.

2.6 Suicidality

It was extracted from the patient file on a 5-item scale ranging from no suicidal thoughts (0), suicidal thoughts (1), one nonserious suicidal attempt (2), more than one nonserious suicidal attempt (3), one serious suicidal attempt (4), and more than one serious suicidal attempt (5).

2.7 Treatment

It was recorded if the patients had been in treatment with ECT at any time in their life.

2.8 Start of treatment

Age of first medication and age at first admission were obtained by interview or extracted from the patient’s file.

2.9 Analysis

We used TaqMan^® SNP Genotyping Assay (Thermo Fisher Scientific, Waltham, MA, USA), a standard method for detecting single-nucleotide polymorphisms, to determine CACNA1C (A/G) genotype. Two labeled oligonucleotide probes are used to identify each allelic variant; in this study, the flurophoresVIC^® (4,7,2-trichloro-7-phenyl-6-carboxylfluorescein) and FAM™ (6-carboxy-fluorescein) reported the A/G SNP. The oligonucleotide probes are complementary to the sequence of interest, labeled at the 5′ end with a reporter dye and at the 3′ end with a quencher dye. The probe anneals to the DNA sequence of interest and during PCR amplification, the 5′–3′ exonuclease activity of the DNA TaqMan^® polymerase degrades the probe, separating the reporter and the quencher dye resulting in a fluorescent signal, increasing for each amplification round. The proximity between reporter and quencher in unbound probes will block the signal. The fluorescent intensity was measured by the Applied Biosystems^®7500 Fast Real-Time PCR System (Thermo Fisher Scientific) with fluorescent emission at 554–530 nm for VIC^® and FAM™. In the allelic discrimination analysis, the quantitative contribution of each dye for each sample was used to produce a scatterplot, where signal for FAM™ or VIC^® exclusively indicates homozygous samples and increased signal for both dyes indicates heterozygosity (Figure 1). Non-template controls were included to calibrate the scatterplot, and triplicates of each sample were used to ensure confident allelic determination. 5.4% of the sample failed the analysis. The Hardy–Weinberg equation was fulfilled.

Figure 1

Allelic discrimination plot. The X-axis is the VIC^® fluorescent intensity, and the Y-axis is the FAM™ fluorescent intensity. Samples showing high intensity on only one axis (relative to blank samples, seen in gray) are homozygous for either the A allele (VIC^®) or the G allele (FAM™), highlighted here in red or blue. Samples showing both VIC^® and FAM™ fluorescence intensities are heterozygous, highlighted here in green.

2.10 Statistics

Statistics was performed with SPSS 18. To compare frequencies, the chi square test was used. The analysis of variance (ANOVA) was used to compare means and the Bonferroni correction was used to compensate for multiple testing. Tests were performed for eight factors: depression, anxiety ×2, onset ×2, suicidality, alcohol, and ECT. A p-value of 0.006 was considered statistically significant.

3.1 Anxiety

3.1.1 At admission

No difference in frequency was found between genotypes in patients with or without anxiety (chi square, p = 0.061) (Table 2). A significant difference was found between genotypes of patients with anxiety and controls (chi square, p = 0.011) (Table 3).

Table 2

Distribution of genotypes in patients with and without anxiety at admission

	Anxiety	No anxiety
GG	51	69
AG	88	67
AA	18	16

Table 3

Distribution of genotypes in patients with anxiety at admission and controls

	Pt. with anxiety	Controls
AA	18	79
AG	88	309
GG	51	319

A significant difference was found for the A allele (AA + AG/GG) between patients with and without anxiety (chi square, p = 0.013) (Table 4) with the ODs ratio for the A allele of 1.45 (1.04–2.03).

Table 4

Distribution of genotypes in patients with and without depression at admission

	Anxiety	No anxiety
AA + AG	106	83
GG	51	69

A significant difference for the A allele was also found between patients with anxiety and controls (chi square, p = 0.0036).

When the patients were divided according to sex, the frequency of anxiety was significantly higher in females (121 anx/92 non-anx) than in males (36 anx/62 non-anx) (p = 0.001). A significant difference was found for the A allele between woman with and without anxiety (chi square, p = 0.014) (Figure 2). No significant difference was found for the A allele between males with and without anxiety (chi square, p = 0.59).

Figure 2

Age at first medication according to genotype.

3.2 Onset of illness

In this analysis, the age of first treatment and the age at first admission was taken as sign of start of illness. It is seen that, for both, the AA genotype had the highest age of incidence (Figures 2 and 3, Tables 5 and 6). By the ANOVA test, this reached significance for the age of first treatment (p = 0.010) but not for the age at first admission (p = 0.329) (Figure 4).

Figure 3

Age at first admission according to genotype.

Figure 4

ECT according to genotype.

3.3 Suicidal attempt

A total of 107 patients had performed a suicidal attempt and 357 had not. No significant difference in the frequency of suicidal attempts was found between the genotypes according to the chi square test (p = 0.356).

3.4 ECT

Fifty patients had received ECT and 87 had not. There was a significant difference in the frequency of ECT between the genotypes (p = 0.036) (Table 7). The OD ratio for the A allele was 1.51 (0.91–2.50).

3.5 Alcohol

A total of 104 patients had an abuse of alcohol and 360 did not have an abuse of alcohol. No difference in frequency of alcohol abuse was found between the different genotypes (p = 0.425).

4.1 Limitations

The only information about controls were their sex and that they were recruited from the bloodbank.

The clinical data were not complete for all patients. The patients were recruited from different hospitals and it was not possible to get access to all patient’s files.

The ICD-10 inclusion diagnosis was later confirmed in one of the three different ways: either by a structured interview Scheduled Clinical Assessment Neuropsychiatry by a trained research psychiatrist, the diagnoses were approved from the file by a trained research psychiatrist, or the patient had been admitted at least two times with a clinical diagnosis of depression according to the ICD-10.