FAMILY HISTORY AS AN IMPORTANT FACTOR
FOR STRATIFYING PARTICIPANTS IN GENETIC
STUDIES OF MAJOR DEPRESSION Zalar B, Blatnik A, Maver A, Klemenc-Ketiš Z, Peterlin B *Corresponding Author: Professor Borut Peterlin, Clinical Institute of Medical Genetics, Division of Obstetrics and Gynecology,
University Medical Center Ljubljana, Šlajmerjeva 3, 1000 Ljubljana, Slovenia. Tel: +386-1-5401-137. E-mail:
borut.peterlin@guest.arnes.si page: 5
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MATERIALS AND METHODS
Subjects. Of the 133 patients participating in this
study, most were recruited via the Outpatient Psychiatry
Centre of the University Psychiatric Hospital in Ljubljana,
Slovenia. All had been previously diagnosed with
either MDD, or in rare cases GAD, by an experienced
psychiatrist, in accordance with the DSM-IV (Diagnostic
and Statistical Manual of Mental Disorders, 4th edition,
1994; American Psychiatric Association) classification
system criteria. A small number of patients was recruited
by their general practitioners; they had all been diagnosed
as having MDD in the past by a psychiatrist. No patients
with a history of schizophrenic, schizoaffective or bipolar
disorder or a diagnosis of personality disorder were included
in our study. Patients with MDD showing psychotic
symptoms were likewise excluded from further analysis.
A total of 279 healthy controls were enrolled in our
study while undergoing screening examinations at various
occupational medicine departments across Slovenia. Only
healthy volunteers with no personal or family history of
depression were recruited.
All participants were required to fill in a structured
questionnaire enquiring about their general health and
habits and about the family history of neuropsychiatric
disorders in their first-, second- and third-degree relatives.
They also completed the Zung Self-Rating Depression
Scale (SDS). No controls that scored 45 or more on the
SDS were included in our study.
Blood samples and, in a small number of MDD patients,
buccal swabs were obtained from all participants. All patients and controls were of Caucasian origin. They
all signed written informed consent forms. The study was
conducted in accordance with the Declaration of Helsinki
(1964) and was approved by the Slovenian National Ethics
Committee.
Genotyping. Genomic DNA was extracted from
blood samples and buccal swabs using standard protocols.
Genotyping of the COMT rs4680 and PCLO rs2522833
was performed using a real-time polymerase chain reaction
(RT-PCR) method on a 7000 Sequenced Detection System
(Applied Biosystems, Foster City, CA, USA). KASPar
(LGC Ltd., Teddington, Middlesex, UK) SNP genotyping
chemistry was utilized according to the manufacturer’s
protocols. Polymerase chain reaction was carried out in a
10 μL final volume containing 5 μL of a DNA sample, 5
μL of Reaction Mix 2 × and 0.14 μL of Assay Mix. After
initial denaturation at 94 °C for 15 min., 20 denaturation
cycles (94 °C for 10 seconds, 57 °C for 5 seconds, 72 °C for
20 seconds) were performed, followed by the final extension
step of 40 seconds at 72 °C. The allele discrimination
analysis was carried out using SDS Software Version 1.2
(Applied Biosystems) [25].
Genotyping of the 5-HTTLPR variant was performed
in accordance with procedures previously described in the
literature [26]. The 5-HTT gene regulatory region was
amplified by PCR with the following primers: 5’-GGC
GTT GCC GCT CTG AAT GC-3’ and 5’-GAG GGA CTG
AGCT GGA CAA CCA C-3’. Polymerase chain reaction
was performed in a 10 μL reaction mixture containing
1 μL of genomic DNA, 0.3 μL of each primer, 0.2 μL
of PCR Nucleotide Mix, 2 μL of GoTaq® Flexi Buffer,
0.8 μL of 25 mM MgCl2 solution, 0.1 μL of GoTaq®
DNA Polymerase and 5.3 μL of H2O. Denaturation was
performed at 94 °C for 2 min. and was followed by 30
cycles of amplification (98 °C for 10 seconds, 63 °C for
30 seconds and 68 °C for 30 seconds). The PCR products
were separated using electrophoresis in a 3.0% agarose
gel and visualized by UV after SYBR®Safe staining. A
484 bp band was observed for the short (S) allele, and a
528 bp band for the long (L) allele; heterozygous samples
showed both alleles.
Statistical Analyses. Genotype distributions were
tested for adherence to the Hardy-Weinberg disequilibrium
using the χ2 distribution test. The χ2 test was also used to
analyze the significance of associations between genotype
and allele frequencies and MDD. Patients with MDD were
compared to a control group consisting of healthy volunteers.
In addition, a group of patients with a positive family
history of depressive disorders was compared to patients
with no family history of depression, and both groups of
patients were separately compared to healthy controls.
Analyses were performed using R statistical language (R
3.1.1 for Windows; https://cran.r-project.org/bin/windows/
base/old/3.3.1). A nominal level of significance p = 0.05
was regarded as significant and corrected according to
Benjamini-Hochberg when multiple tests were performed.
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