Genetic Variations of Brain Derived Neurotropic Factor Gene With Premenstrual Syndrome Among Pakistani Menarcheal Girls: A Pilot Study

Received: September 16, 2020 Accepted: October 20, 2020 Abstract: The premenstrual syndrome (PMS) comprises various physical and emotional symptoms which are associated with the menstrual cycle. The emotional changes might be due to the variation in the levels of BDNF especially lower levels of plasma BDNF may be present at the luteal phase of menstrual cycle. To date, no data were present to associate the link between BDNF gene polymorphism and PMS among young Menarcheal females. So, the goal of this investigation is to find out the possible relationship between BDNF gene polymorphism and PMS among young Menarcheal females of Pakistan. A total of 92 menarcheal girls with age range of 11-14yrs met the inclusion criteria. All participants were screened for the depression and PMS using Zung’s depression scale/ Diagnostic and Statistical Manual of Mental Disorders-IV (DSM IV) criteria and world health organization (WHO) criteria for PMS respectively. Genomic DNA was extracted from oral samples by Salting out method and BDNF genotyping was done using PCR-RFLP analysis. Results showed no significant association (χ=1.685, p-value=0.431) was observed between BDNF genotyping and PMS among menarcheal girls. BDNF (rs6265) AA and GA genotypes did not show significant association with the risk of PMS. Hence, it was concluded that BDNF gene polymorphism may not be associated with the PMS among Pakistani Menarcheal girls.


Materials and Methods Study participants
This study is based on schoole aged girls consisting of 92 participants (40 PMS cases and 52 healthy controls) recruited from different schools of Karachi, Pakistan from May-June 2014 with a mean age of 14±1years and had signed informed written consent form. Sample size was calculated using online calculator OpenEpi (https: //www.openepi.com/SampleSize/SSCC.htm). All participants were screened for the depression using Zung's depression scale [17] and DSMIV (Diagnostic and Statistical Manual of Mental Disorders IV) criteria. PMS was identified according to the World Health Organization (WHO) criteria for diagnosing PMS. In this way, 40 PMS and 52 Non PMS subjects were recruited. This study was conducted in accordance with the Helsinki declaration and approved by the Board of Advance Studies and Research, University of Karachi (01199/2010). All girls who were less than 11yrs and more than 16yrs, prepubertal and post-menarcheal girls and diseased females are excluded from this study.

Demographics
Information such as age, socio-economic status, life style, Basal metabolic index (BMI), waist to hip ratio (WHR) and dietary profile was obtained using questionnaire. Information regarding menstrual history i.e. menarcheal age, the regularity of cycles, length of cycle, and physical manifestations during menstruation was also collected.

DNA isolation
Saliva samples of all participants were collected into 1.5ml of eppendorf tube and genomic DNA was extracted using standard Salting out method.
RELP was carried out by adding 0.5 µl (5 units) of Eco72I (Fermentas, USA) restriction enzyme, 1ul buffer, 5ul PCR products and 3.5ul nuclease free water. The digested fragments of BDNF were electophoretically (80 volts) run on 3% of agarose gel and visualized under the same GelDoc with the same condition. The digested fragments were identified using 50 bp ladder (Thermo scientific, USA). The undigested BDNF products were defined as homozygous dominant having A allele (171 bp) in which restriction site was absent whereas two digested products were heterozygous and homozygous recessive with 171, 92, 72 bp and 92, 72 bp (G allele) respectively in which restriction sites was present.

Analysis of data
Any deviation of allele frequencies from predicted was calculated using Hardy-Weinberg equilibrium (HWE). The association between BDNF polymorphism and PMS was evaluated using Pearson Chi (χ 2 ) square test among menarcheal girls. Odds ratios (ORs) along with 95% confidence intervals (CIs) via binary logistic regression analysis were used to find out the risk of PMS associated with BDNF genotype. All analyses were executed on SPSS 22 and all values were considered significant at p<0.05.

Discussion
Premenstrual syndrome (PMS) is a multidimensional disorder affects various physiological systems. Several biological mechanisms that cause PMS have been proposed. Majority of them emphasize on the gonadal steroids, their metabolites and their communications with neurotransmitters and neuro-hormone systems including serotonin, gamma-Aminobutyric acid (GABA), cholecystokinin (CCK), and the renin-angiotensin-aldosterone system (RAAS). Alterations in the responses of these systems to gonadal steroidal fluctuations during the menstrual cycle of women and increased sensitivity to variations in steroids may be the cause PMS in susceptible females. Homeostatic disruption and poor adaptation may be the basic underlying mechanisms.
Our findings suggested that BDNF genotyping deviate significantly in accordance with the Hardy Weinberg principle which is in contrary to other findings [18]. No relationship between BDNF genotyping and PMS was found due to the small sample size. This pilot study first time report the association between PMS and BDNF genotyping which has not been previously testified. However a recent study was done on the plasma variations of BDNF and different phases of menstrual cycle among menstruating women with PMS and suggested that BDNF was found to be significantly lower during luteal phase of the menstrual cycle [10]. Whereas earlier report has determined the increase incidences of depressive episodes and BDNF gene polymorphism [19]. A meta-analysis was conducted on geriatric depression to compare the frequency of BDNF polymorphism among cases and psychiatrically healthy controls which suggested that individual possess Met allele were at increased risk for geriatric depression than Val/Val homozygotes [20]. However, this study is limited to the small sample size. Further research is needed with large sample cohort to confirm the results.
The study concluded that BDNF genotype may not associated the PMS among Pakistani Menarcheal girls.