BDNF val66met genotype is not associated with psychological distress: A cross-sectional study in Indonesian young adults

Background The number of mental disorders has been increasing but has yet to receive sucient attention. Healthcare students and professionals tend to have high stress burden. Finding the root cause of psychological distress is important to formulate a method for early detection. The association of BDNF val66met polymorphism to neuropsychiatric disorders has been widely studied. The aim of this study was to interplay between BDNF val66met polymorphism and sociodemographic factors in the pathogenesis of psychological distress among Indonesian students. Methods Level of psychological distress and sociodemographic proling was assessed using the Kessler Psychological Distress Scale (K10) and sociodemographic questionnaires, respectively. Genotyping was performed using polymerase chain reaction-amplied refractory mutation system. Pearson’s chi square and binomial logistic tests were used to evaluate the correlation. (10.81%). >


Abstract Background
The number of mental disorders has been increasing but has yet to receive su cient attention. Healthcare students and professionals tend to have high stress burden. Finding the root cause of psychological distress is important to formulate a method for early detection. The association of BDNF val66met polymorphism to neuropsychiatric disorders has been widely studied. The aim of this study was to interplay between BDNF val66met polymorphism and sociodemographic factors in the pathogenesis of psychological distress among Indonesian students.

Methods
Level of psychological distress and sociodemographic pro ling was assessed using the Kessler Psychological Distress Scale (K10) and sociodemographic questionnaires, respectively. Genotyping was performed using polymerase chain reaction-ampli ed refractory mutation system. Pearson's chi square and binomial logistic tests were used to evaluate the correlation.

Conclusion
Psychological distress is not affected by genotypic and environmental factors. Further con rmatory research with larger and broader populations is required. Background Steel et al. reported a lifetime prevalence (nearly 30%) of mental disorders (substance use, anxiety, and mood) among adults in 59 countries [1]. The severity of mental disorders has prompted public awareness regarding the importance of mental health; in fact, mental health is highlighted in Sustainable Development Goals. The distinct long-term manifestations of mental disorders are depression and anxiety. Therefore, nding an approach to measure mental health condition predictively is crucial [2]. Psychological distress is a common indicator of mental health in epidemiological and clinical studies [3]. Self-administered or clinician-administered standardized scales, such as the general health questionnaires (GHQ-12,-20,-28,-30), the Kessler scales (K-6,-10), and the symptom checklists (SCL-5,-25; BSI-18), are regularly used to assess psychological distress [4][5][6][7]. The various tools and scales of measurement render the prevalence of psychological distress hard to determine, with an approximate estimation of around 5-27%. Factors affecting psychological distress are categorized as inborn and external. Typical inborn psychological distress factors include age, gender, ethnicity, and other sociodemographic factors. External factors, such as experiences, social behavior, income, and occupations, are widely varied among individuals [3]. In particular, work-related burden or occupational stress is associated with mental and medical disorders [8].
Psychological distress affects the neuronal plasticity on brain regions, such as the prefrontal cortex, the hippocampus, and the amygdala, thus altering cognitive processes, such as mood, emotion, learning, and memory [9,10]. Brain-derived neurotrophic factor (BDNF) is an abundant growth factor in the central nervous system is. It is highly in uential in mental disorders because of its critical roles in neuronal development and plasticity [11]. Reduced mRNA and protein expression levels of hippocampal BDNF have been found in depressive animal and human postmortem studies [12]. Genetic polymorphisms of BDNF (G196A, Val66Met, dbSNP: rs6265) result in the substitution of valine (val) to methionine (met), which modi es the secretion of BDNF and consequently affects mental health [13,14]. Despite its importance, the interaction between BDNF val66met polymorphism and sociodemographic pro le in psychological distress has yet to be studied in developing countries, such as Indonesia. In the present study, we aim to (1) determine the allele and genotype distribution, (2) analyze the association between BDNF val66met polymorphism and psychological distress, and (3) analyze the correlation between sociodemographic factors and psychological distress to evaluate the psychological distress gene environment interaction in an Indonesian student population.

Study design and ethical considerations
This cross-sectional study was approved by the Board of Ethics, Universitas Padjadjaran (727/UN6.C2.1.2/KE PK/PN.2014) and conducted in accordance with the Declaration of Helsinki. All participants were informed about the study and signed informed consent prior to their participation into the study.

Participants
Recruitment was done by using public notice boards in Universitas Padjadjaran, Sumedang, Indonesia. Healthy young adults aged 18-35 years were eligible to participate in this study. Subjects with a history of mental disorders were excluded.

Phenotype
Subjects were asked to ll in the Kessler Psychological Distress Scale (K10) and sociodemographic questionnaires. The Genotype DNA was extracted from blood samples using the Purelink Genomic DNA mini kit (Invitrogen®). The quality of DNA was checked using WPA Lightwave II (Biochrom®). Polymorphism genotyping was performed through polymerase chain reaction-ampli ed refractory mutation system (PCR-ARMS) analysis as described by Sheikh et al. [15]. This genotyping method utilized tetra-primer (Sigma Aldrich, Singapore) consisting of two forward and two reverse primers (Table 1)  Genotyping of BDNF val66met and its correlation with the K10 questionnaire Tetra-primer ARMS genotyping generated three bands for the control amplicon (401 bp) and G and A allele-speci c bands (253 and 201 bp, respectively) ( Fig. 1). Homozygous samples showed only the control and the G or A allele-speci c bands, whereas heterozygous samples showed all three bands. Thirty-eight participants (25.67%) were homozygous AA, 75 participants (50.68%) were heterozygous AG, and 35 participants (23.65%) were homozygous GG. The genotype frequencies were consistent with HWE as shown by p > 0.05 (Table 3).

BDNF val66met genotype and environmental factor interaction in psychological distress
No statistically signi cant association was found between BDNF val66met genotype and psychological distress. The correlation between sociodemographic factors was also not signi cant, except for the source of tuition fee in the female participants with p = 0.049 (Table 4).

Discussions
The number of suicidal healthcare professionals, such as physicians, pharmacists, nurses, dentists, and veterinarians, is increasing [16]. Pharmacy ranked top three among other healthcare professional degree students in terms of psychological distress and depression as assessed by using GHQ-12 and Beck Depression Inventory-II [17]. The psychological distress level in our study supports pharmacy as one of the top stressful healthcare degrees and professions, with nearly 75% of the students found psychologically distressed. Current results also aligned with previous ndings showing no signi cant correlation between psychological distress and sociodemographic factors, such as accommodation, GPA, source of living allowance, and tuition fee [18].
Our genotyping substantiated the variation of BDNF val66met genotype in different ethnicities, such as Asians and Caucasians [19]. Several neurodegenerative diseases studies also showed similar genotypic distribution of BDNF val66met polymorphism with Asian populations having higher heterogenous Val/Met genotype than homogenous genotypes [19][20][21][22]. Our nding suggests that BDNF val66met polymorphism and sociodemographic factors do not in uence the pathogenesis of psychological distress.
This insigni cant correlation is possibly due to the limitations of our study. First, our study is limited by the number of participants (also imbalanced between gender) and the population of participants, which are primarily highly stressburdened pharmacy students [16,17]. Second, the psychological distress questionnaire only focused on the short-term period (the last 4 weeks). Thus, it does not measure the psychological distress over time. Last, the students are subject to response bias, particularly the reluctance to express their true level of psychological distress due to negative image and public judgement toward mental disorders. This nding is supported by a study about depression in medical students where nearly 10% of the participants admitted giving false responses in the questionnaire due to the abovementioned reason [23].
Despite the non-associative ndings, our study is the rst gene-environment interaction (GxE) study in Indonesia that focused on psychological distress. GxE studies are useful in the development of personalized medicine in preventive (diagnostic) and therapeutic approaches [24]. Up till now, Indonesia is one of the developing countries that has yet to create its own genomic database. The BDNF val66met genotype pro le we obtained contributes not only to the genomic database of the Indonesian population but also to that of the Asian ethnicity. According to Indonesia's National Baseline Health Research report, in merely 5 years, the prevalence of emotional mental disorders increased nearly 4% from 6.0-9.8% among Indonesian citizens [25,26]. Our study also highlighted the importance of increasing public awareness and early screening of mental issues, speci cally among healthcare professions. We laid a basis for further research in GxE and provided suggestions that need to be considered in conducting mental health research.

Conclusions
In conclusion, we found no association between BDNF val66met polymorphism, sociodemographic factors, and psychological distress among Indonesian students. Further research with a larger gender-balanced and broader population of participants must be conducted to ascertain these ndings. A tool to measure long-term psychological distress and the environmental factors affecting it must also be devised. The clinical signi cance of BDNF val66met polymorphism has been widely studied for more than a decade in neurodegenerative and neuropsychiatric disorders. To establish rs6265 as a marker for mental health and neuro-disorders, studies should consider BDNF protein level, brain MRI and fMRI on brain structures in association with the val66met polymorphism [27,28].