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
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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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