A Study on the Association between Patients with Post-Traumatic Stress Disorder in Li and Han Ethnic Groups in Hainan Province and DNA Methylation of Brain-Derived Neurotrophic Factor Genes

Objective: To explore the pathogenesis of PTSD in the brain-derived neurotrophic factor (BDNF) gene methylation of patients with posttraumatic stress disorder (Posttraumatic Stress Disorder, PTSD) in Hainan Province, the relationship between the influence of BDNF gene methylation and the influence of PTSD. Methods: A case-control study method was adopted, strictly in accordance with DSM-IV and PTSD diagnosis, and 150 Li PTSD patients matched with gender and age of 300 Han PTSD patients were selected as the research objects. The peripheral venous whole blood of the subjects was drawn, genomic DNA was extracted, modified with bisulfite, and directly sequenced to quantitatively detect the methylation status of the CpG island in the promoter region of brain-derived neurotrophic factor (BDNF). Results: The results showed that the methylation levels of CPGl, CPG2, CPG3, CPG4, CPG5, CPG6, CPG7, CPG9, CPGl2, CPGl3, CPGl4, CPGl5, CPGl6, CPGl7, and CPGl8 in THE BDNF promoter were significantly different between the HAN PTSD group and the Li PTSD group (P Conclusion: It is suggested that CPG methylation in the promoter region of BDNF gene is closely related to patients with PTSD. There is a statistical difference in the level of CpG methylation in the promoter region of BDNF gene in PTSD between Li and Han ethnic groups in Hainan Province. CpG methylation in the promoter region of BDNF gene may be used as a biomarker for the diagnosis of PTSD.


Introduction
Posttraumatic Stress Disorder (PTSD) is the most typical type of stress mental illness [1]. It is mainly manifested as traumatic experience repeatedly intruding into consciousness or dreams, continuous increased alertness and avoidance of anything that can cause this trauma. In the scene of sex memory, the patient's psychological and social functions are seriously damaged. Epidemiological studies have shown that more than 1/2 of PTSD patients are often accompanied by comorbidities such as depression, other anxiety disorders, and drug abuse [2].
The suicide rate of PTSD patients is 6 times that of the general population, and it is one of the mental diseases that seriously impairs the ability to work. Among the many candidate genes for PTSD genetic susceptibility, BDNF is the second neurotrophic factor discovered after nerve growth factor (NGF) [3]. The precursor has 247 amino acid residues, and 119 are produced by post-translational processing [4]. A mature basic protein composed of amino acid residues has 3 pairs of disulfide bonds in the chain, which exist as dimers in the body. BDNF is the most abundant neurotrophic factor in the body. In the central nervous system, BDNF is mainly synthesized in neurons, transported from anterograde axoplasm to axon terminals, and after release; it mainly acts on target cells through the high-affinity receptor tyrosine protein kinase B (TrkB) [5]. In addition, BDNF can also be secreted by target cells acted on by neurons, and inversely nourish neurons. Immunohistochemistry confirmed that in the central nervous system, BDNF immunopositive neurons are widely distributed in the rat brain, especially in the hippocampus, thalamic striatum, and cortex. In the hippocampus, BDNF mRNA content is 20 -30 times higher than NGF mRNA content [6]. In the peripheral nervous system, more BDNF was found in the distal part of the nerve stump after nerve injury. BDNF is also expressed in small amounts in ovaries, heart, lungs, and skeletal muscles other than nerve tissues.
The content of BDNF is high in human serum, and a large amount of BDNF in whole blood is located in platelets, but the source of BDNF in platelets is still unclear. Due to the high content of BDNF in the blood, research on the repair and regeneration effects of BDNF on the peripheral nervous system has been focused on. Based on the weak areas of the blood-brain barrier, such as the hypothalamus, BDNF may pass through the blood-brain barrier. The mutual inductance between peripheral blood and BDNF in the nerve center cannot be ruled out [7]. It is necessary to explore whether changes in peripheral blood

Specimen Collection and Preservation
After completing the epidemiological investigation and test scale evaluation, all the research subjects will collect 5 -10 ml blood from the anterior cubital vein, EDTA anticoagulation, and send it to the Central Laboratory of Hainan Provincial People's Hospital at −20˚C Frozen.

DNA Extraction
Use the whole blood genomic DNA extraction kit (OMEGA, USA) to extract genomic DNA with a spin column method. The concentration and purity detection meet the PCR amplification requirements, and the DNA methylation research is carried out.

Experimental Method
The first IV BDNF gene launched the determination of DNA methylation: 1) CpGIsl and prediction: CpGplot is applied online.
2) Genomic DNA was treated with bisulfite and BDNF IV promoter fragment times, 720C final extension for 5 min, and termination at 40C. 15 L PCR product was taken and sent to sequencing company for sequencing (Shanghai Genomic).

Data Processing
Epidata3.1 was used for data entry, and SPSS19.0 was used for statistical analysis. The measurement data is expressed by and the counting data is expressed by frequency and percentage. For comparison of inter-group differences, if the measurement data of small samples meet the requirements of normality and homogeneity of variance, t-test is used for comparison of inter-group differences, and analysis of variance is used for comparison of multi-group differences. If normality or homogeneity of variance is not satisfied, use Rank sum test; t/z/F test for large sample measurement data. The chi-square test was used to compare the differences between the enumeration data groups. The test level a is 0.05, P ≤ 0.05, the difference is considered statistically significant.

1) Demographic Characteristics Analysis
The average age of PTSD group was 39.20 ± 6.30 years, 182 males, 118 females, 257 married and 43 unmarried. In the Li people group, there were 150 participants, aged 26 to 64, with an average age of 38.75 + 9.14 years, 79 males, 71 females, 132 married and 18 unmarried. There were no significant differences showed that the methylation levels of CPGl, CPG2, CPG3, CPG4, CPG5, CPG6, CPG7, CPG9, CPGl2, CPGl3, CPGl5, CPGl6, CPGl7, CPGl8 in the BDNF promoter were significantly different between the HAN PTSD group and the Li PTSD group (P < 0.001). It suggested that CPG methylation in BDNF gene promoter region was closely related to PTSD in Li and Han nationality patients in Hainan province. CpG methylation in the BDNF gene promoter region may be a biomarker for the diagnosis of PTSD (Table 1).

Discussion
With PTSD often suffer from depression, brain derived neurotrophic factor (BDNF) BDNF regulate pain and fear, a lack of will lead to posttraumatic stress disorder, its survival, differentiation, growth and development of neurons play an important role, and to prevent neuronal damage death, and improve the pathological state of neurons, and promote the regeneration of damaged neurons and the differentiation of biological effects [9]. As previously described, a gene-gene interaction between the DRD2 TaqlA locus (RSL800497) and the BDNF Val66 allele Val66Met (RS6265) predicts PTSD severity [10]. Although C270T, Val66Met) and PTSD [11].
DNA methyltransferase blockers can improve depression-like behaviors while causing hypomethylation [12]. These findings suggest that abnormal methyla- Many studies have shown that BDNF can be linked to depression through the serotonin nervous system. The levels of BDNF in the peripheral serum and plasma of patients with depression are reduced. For example, Karege et al. [17] found that the levels of BDNF in the serum and plasma of depression patients the severity of depression [18]. MDD patients with suicidal ideation and suicidal behavior also found elevated levels of methylation in the BDNF promoter region in the brain, similarly to MDD patients with suicidal ideation who were ineffective in antidepressant treatment [19]. DNA methyltransferase blockers can improve depression-like behaviors while causing hypomethylation [20]. These   Hainan Province, and the incidence of this disease There will be a deeper understanding of the mechanism, which will provide scientific basis for the clinical diagnosis of PTSD patients, provide basic data for the study of the pathogenesis of PTSD and its prevention and treatment, and provide genetic data for anthropology, forensic identification and PTSD association studies.

Conclusion
This study is more meaningful for larger samples. In addition, population differences, especially the degree of linkage disequilibrium among other potential variants in the tested genes, may have contributed to this inconsistent discovery.