Balkan Journal of Medical Genetics

ASSOCIATIONS BETWEEN VARIATIONS IN TPH1, TPH2 AND SLC6A4 GENES AND POSTPARTUM DEPRESSION: A STUDY IN THE JORDANIAN POPULATION
Khabour OF1, Amarneh BH2, Bani Hani EA3, Lataifeh IM4
*Corresponding Author: Dr. Omar F. Khabour, Associate Professor of Molecular Genetics, Department of Medical Laboratory, Sciences, Jordan University of Science and Technology, PO Box 3030, Irbid 22110, Jordan; Tel.: +962-2-720-1000 ext. 23784; Fax: +962-2-720-1087; E-mail: khabour@just.edu.jo
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MATERIALS AND METHODS

Subjects. This prospective cross-sectional study involved 370 randomly selected postpartum women (240 controls and 130 depressed cases) from five maternity centers in Irbid, Jordan. Random selection was carried out based on locations of the clinics in the city and type of clinic (public vs. private). Women with pre existing psychiatric disorders were excluded from the study. Women were recruited 4 to 6 weeks after child delivery. Informed written consent was obtained from all subjects in accordance with the requirements of the Institutional Review Boards of Jordan University of Science and Technology, Irbid, Jordan. Edinburgh Postnatal Depression Scale Questionnaire. An Arabic version of the Edinburgh Postnatal Depression Scale (EPDS) was used to screen postpartum women [21]. An EPDS score >13 was used as a criterion to indicate the presence of depression illness(es) [21]. This cut-off value has been reported to have high sensitivity and specificity. Several demographic variables were also included in the questionnaire to assess the possible correlation between PPD and some environmental factors such as maternal age, educational level, income satisfaction, number of children, delivery mode, depression history and pregnancy problems. DNA Extraction. Venous blood samples were collected in EDTA tubes and obtained from all subjects. DNA was extracted using Promega wizard genomic DNA purification kit (Promega, Madison, WI, USA) according to the protocol provided by the manufacturer. DNA concentrations were measured using Smart-SpectTM3000 (Bio-Rad Laboratories, Hemel Hempstead, Hertfordshire, UK). Extracted DNA was stored at –20°C until used. Genotyping of the TPH1 (218A>C) Polymorphism. TPH1 was analyzed using polymerase chain reaction (PCR) followed by the restriction fragment length polymorphism (RFLP) method. The TPH1 gene fragment was amplified using primers described previously [22]. The reaction mixture of 25 mL contained 5 ng of genomic DNA, 1 mM of each primer, green master mix (Promega), 1 mM of spermidine and H2O. The reaction mixture was initially denatured at 95°C for 5 min., followed by 30 cycles at 95°C for 30 seconds, 55°C for 30 seconds and 72°C for 30 seconds. The PCR was completed by a final extension cycle at 72°C for 5 min. Successful amplification of the fragment was confirmed by detection of a 847 bp band on a 2.0% agarose gel. The restriction enzyme digestion was carried out overnight at 37°C in a 20 mL volume reaction mixture containing 10 mL of the amplified PCR product and 1 unit of Bfa1. The digested fragments were then separated using 2.0% agarose gel electrophoresis and detected by ethidium bromide staining. The PCR product with the A allele remained uncut (847 bp), whereas the digested PCR product with the C allele gave fragments of 599 and 248 bp. Genotyping of the TPH2 (1463G>A) Polymorphism. Allele-specific PCR using amplification refractory mutation system (ARMS) method was used for analyzing the TPH2 G1463A polymorphism as described by Zhang et al. [12]. Only the G allele PCRspecific product was detected in the DNA obtained from participating women, while the A allele product was not detected. Validity of the ARMS results was achieved by sequencing of more than 10.0% of the PCR products using ABI PRISM™ 3.1 automated sequencer (Life Technologies, Applied Biosystems, Foster City, CA, USA) as previously described [23]. Genotyping of the SLC6A4 (L/S) Polymorphism. The SLC6A4 (L/S) polymorphism was analyzed using PCR. Desired DNA target sequences, 469 bp for the S allele and 513 bp for the L allele, were amplified as described by Middledorp et al. [24]. The reaction mixture of 20 mL contained 5 ng of genomic DNA, 1 mM of each primer, Promega green master mix, 2.0% DMSO and H2O. The reaction mixture was initially denatured at 94°C for 3 min., followed by 35 cycles at 94°C for 30 seconds, 59°C for 30 seconds and 72°C for 90 seconds. The PCR procedure was terminated by a final extension at 72°C for 6 min. Amplified 469 or 513 bp fragments were electrophoresed through 2.0% agarose and visualized by ultraviolet illumination upon ethidium bromide staining. Statistical Analyses. Data analysis was carried out using the statistical package for social studies (SPSS) version 17 (SPSS Inc., Chicago, IL, USA) to compute all de-scriptive statistics. The chi-squate (c2) test was used to identify the correlation between the studied demographic variables and PPD. If n <5, exact Fisher statistic test was used. In addition, the same tests were used to evaluate the genotype distribution and allele frequencies of the three studied polymorphisms. The test power was calculated for allele and genotype frequencies using Power and Sample Size Calculation Program (PS version 3.0.1; Vanderbilt University Medical Center, Nashville, TN, USA). For all analyses, the power was more than 75.0%. A p value <0.05 was considered significant. The Hardy-Weinberg equilibrium was assessed using the c2 test.

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