Association Between 50 bp Insertion / Deletion Polymorphism in Promoter of the Superoxide Dismutase-1 and Temperament

Introduction: Temperament (mizaj) plays an important role in society’s general health as well as in diagnosis and treatment of diseases. Despite its effectiveness, the mechanisms of temperament-dependent treatment in practical Iranian Traditional Medicine (ITM) has not been well understood yet. This study aimed to evaluate the association between the temperament and superoxide dismutase-1 (SOD1) polymorphism. Methods: The study was carried out on healthy blood donors. All participants were from Fars province, southern Iran. To determine the participants’ temperaments, a self-reported identification scale was used. Two hundred eighty males with any of 4 simple temperaments (Warm, Cold, Moist, and Dry) were included. The SOD1 genotypes were determined using a polymerase chain reaction (PCR)-based method. Multinomial logistic regression was used to calculate odds ratios (ORs) and 95% CI for the association of temperament with SOD1


Introduction
Temperament (mizaj), one of the basic concepts of Iranian Traditional Medicine (ITM), plays an important role in maintaining the healthy state of an individual as well as in diagnosis and treatment of diseases.Temperament is believed to be a dominant quality resulting from the interaction of elements with different qualities (hot, cold, wet, dry) in the human body.In ITM, based on warmness and wetness intensity, the temperament is classified into 9 major groups: equilibrium group with equilibrium for both warmness and wetness; 4 simple groups (Warm, Cold, Moist, and Dry), each of them is in equilibrium for one quality and out of that for another quality; and 4 combined groups (Warm/ Moist, Warm/Dry, Cold/Moist, Cold/Dry), each of them is out of equilibrium for both qualities.Healthy state is believed to be the result of the balanced temperament, and most of the diseases occur when this factor is disturbed. 1,2 riation in the temperament, which influences phenotypic traits such as morphological, physiological and psychological features, 2 can be partially attributed to genetic differences.Considering the fact that genetic polymorphisms are involved in biodiversity, it is therefore of interest to explore the distribution pattern of genetic polymorphisms in different temperament modes.Definitely, identification of the genes related to this factor could help us better understand the molecular mechanism of ITM based treatments.Superoxide dismutase-1 or SOD1 (EC 1.15.1.1;SOD1, OMIM: 147450) is an antioxidant enzyme localized in the cytosol, nucleus, peroxisomes, and mitochondria.It is a metaloenzyme which binds to copper and zinc and catalyzes the conversion of toxic superoxide radicals to hydrogen peroxide (H 2 O 2 ) and molecular oxygen (O 2 ). 3 The loss of SOD1 activity, thus, could lead to the accumulation of reactive oxygen species (ROS).ROS, which play an important role in many physiological functions, are regularly produced in cells. 4However in excess, ROS may react with fatty acids, proteins and DNA, thereby causing damage or death to the cells. 4][7][8][9][10] There is a 50 bp insertion/deletion (Ins/Del) polymorphism in the promoter region of this gene.][13] Previously, it was shown that the frequency of DNA damage (nuclear buds) and cell death biomarkers (condensed chromatin, karyorrhexis, pyknotic changes, and karyolitic cells) significantly differ between certain temperament groups. 14Moreover, the association of polymorphisms in antioxidant enzymes of glutathione S-transferase M1 and T1 and warm temperament has been reported. 15These findings support the idea that the antioxidant system may affect the temperament modes.Therefore, the present study was carried out to evaluate the association of temperament with SOD1 50 bp Ins/Del polymorphism as a potential genetic candidate.

Materials and Methods
The study was carried out on healthy individuals referred for blood donation to Shiraz Blood Transfusion Organization from November to December 2016.All participants were from Fars province, southern Iran, and aged between 20-40 years.To determine the participants' temperaments, a self-reported identification scale constructed for healthy individuals was used. 16Two hundred eighty males with equilibrium or any of 4 simple temperaments (warm, cold, moist, and dry) were included in the study.The primers and polymerase chain reaction (PCR) conditions for determining the SOD1 genotypes were the same as those reported previously. 10Multinomial logistic regression was used to calculate odds ratios (ORs) and 95% CI for the association of temperament with SOD1 polymorphism.

Results
As revealed by the data of age, no significant difference was observed between the means of age in different groups.The allele frequencies of SOD1 gene 50 bp variant in each of the temperament groups is shown in Table 1.Because of low prevalence of the Del allele, Del/Del and Ins/Del genotypes were combined with each other as Del/-genotype for statistical comparisons.To control the possible effect of age, the association between SOD1 and temperament was examined using multinomial logistic regression while controlling for the age, and the equilibrium group was selected as the reference group.Neither the Ins/Ins nor the Del/-genotypes were associated with any of the simple groups (Table 2).

Discussion
There are increasing evidence indicating the association of temperament with biological factors such as autonomic and immune systems, blood groups, enzymatic variation, and body composition. 17-202][23] The present results indicated that the temperament was not affected by the SOD1 Ins/Del polymorphism.This is the first study on the association of SOD1 polymorphism with temperament.
The sample size which might not be large enough to reach a convincing conclusion is the limitation of the present study.Another limitation of this study which should be taken into consideration is the unavailability of general health status of participants.However, despite the fact that physicians ignore some diseases during screening, it should be noticed that usually most of volunteers verified for blood donation are in well physical and mental health.

Conclusion
In conclusion, this study did not support the association between SOD1 Ins/Del polymorphism and any of the temperament modes.However, Ins/Del polymorphism of SOD1 needs to be further analyzed in future studies to verify our observation.Performing larger studies with greater samples and with other SOD1 polymorphisms as well as other polymorphisms in genes of antioxidant system is recommended.

Ethical Approval
This study was approved by Ethics Committee of Shiraz University (ECBDE-SU-9-6177616), and each subject was informed about the objectives of the study.

Competing interests
Not declared.

Table 1 .
Allele Frequencies of SOD1 Gene 50 bp Variant in Each Temperament Group