European Review for Medical and Pharmacological Sciences 2016; 20: 1852-1859 Effects of polymorphisms of serotonin transporter promoter (5-HTTLPR) and brain derived neurotrophic factor gene (G196A rs6265) on the risk of major depressive disorder in the Chinese Han population N. SUN1,2, C.-X. YANG1, Z.-F. LIU1, X.-R. LI1, Y. XU1, K.-R. ZHANG1 1Department of Psychiatry, First Hospital of Shanxi Medical University, Taiyuan, P.R. China 2Nuring College of Shanxi Medical University, Taiyuan, P.R. China Abstract. – OBJECTIVE: The etiology of Major Introduction depressive disorder (MDD) is multifactorial but the genetic risk is an important factor. Previous stud- Major depressive disorder (MDD) is a prev- ies have shown a significant interaction between alent form of mental illness and a leading cause serotonin and brain-derived neurotrophic fac- tor (BDNF) in brain function. The serotonin trans- of disability worldwide. The etiology of MDD is porter protein promoter polymorphism (5-HTTL- multifactorial but the genetic risk is an import- PR) and BDNF (rs6265) are two of the most studied ant factor1-4. A most studied genetic risk in MDD candidate gene polymorphisms in relation to MDD. manifested that neuroticism is the repeat-length However, the effect of 5-HTTLPR-BDNF (rs6265) polymorphism within promoter region (5-HT- interaction on MDD-risk is not consistent. TLPR) of genes encoding serotonin transport- PATIENTS AND METHODS: This study recruit- 5,6 ed 459 patients with MDD and 412 healthy volun- er (5-HTT) . 5-HTTLPR is made up of 44-bp teers in a Chinese Han population. Polymerase repeats. Insertion/deletion variable-number tan- chain reaction (PCR)-based genotyping was dem repeats leads to 5-HTTLPR polymorphism, used to detect polymorphisms. Logistic regres- which is primarily either as a shorter allele (S) sion was applied to estimate the effect of poly- of a segment of 14 complex repeats or a longer morphisms of 5-HTTLPR, BDNF (rs6265), and allele (L) with 16 repeats, which are associat- their interaction. ed with low and higher 5-HTT transcription, RESULTS: We observed a significant correla- tion between the heterozygous genotype of respectively. 5-HTT plays an important role in 5-HTTLPR and MDD [odds ratio (OR) = 1.42, 95% serotonergic signaling. Thus, polymorphism of CI: 1.05~1.91; p = 0.02]. The BDNF (rs6265) poly- 5-HTTLPR is expected to be of clinical signif- morphism showed that there is no correlation icance for psychiatric illnesses including MDD. with MDD. When interaction with BDNF was mod- However, the correlation between 5-HTTLPR eled, for individuals with BDNF (rs6265), geno- polymorphism and MDD with and other behav- type GG, cases in the heterozygous group had ioral traits of neuroticism is still controversial. even higher odds of MDD than those in the com- 7 bined homozygous group of 5-HTTLPR polymor- A large scale meta-analysis by Clarke , involv- phism (OR = 2.92, 95% CI: 1.43-5.95; p = 0.003). ing approximately 7800 cases and 16000 con- CONCLUSIONS: Our results suggested that trols, revealed a significant, but modest effect of 5-HTTLPR, may be associated with the suscep- the S allele (odds ratio = 1.076) using recessive tibility of MDD in an overdominant mode, and model. Odgerel et al8 did not find any significant there may be a significant interaction between association between 5-HTTLPR and another se- 5-HTTLPR and BDNF (rs6265) polymorphisms in relation to MDD. rotonin transporter polymorphism (rs25531) and MDD, anxiety disorders, or neuroticism scores Key Words: in either European Americans or African Ameri- Major depressive disorder, Serotonin transporter cans. Moreover, some studies suggested that the protein promoter, Brain-derived neurotrophic fac- 5-HTTLPR polymorphism plays an overdomi- tor, Gene polymorphism, Overdominant model. nant role in MDD9 or neuroticism10. 1852 Corresponding Author: Kerang Zhang, MD; e-mail: [email protected] 5-HTTLPR and BDNF gene G196A rs6265 Besides 5-HTTLPR, another widely stud- in Zhang’s study. Among subjects, 459 patients ied risk factor for susceptibility of MDD is a (male, n = 229; female, n = 230; age range, 18- brain-derived neurotrophic factor (BDNF), as a 60 years old) were recruited from clinical settings neurotropic factor in the brain and spinal cord that of the Department of Psychiatry, First Hospital promotes the survival of neurons11. The Val66Met of Shanxi Medical University. The diagnosis was (G196A, rs6265) polymorphism is the most im- made by at least two consultant psychiatrists ac- portant genetic variant within an exon of BDNF cording to criteria for MDD in “Diagnostic and gene which changes an amino acid at codon 66 Statistical Manual of Mental Disorders Fourth from Val to Met. Some investigations12-14 have Edition (DSM-IV)” (American Psychiatric Asso- suggested that Val66Met is positively associat- ciation, 1994). All patients were also diagnosed ed with MDD risk, although these have not been with the Chinese Version of the Modified Struc- confirmed in meta-analysis15,16. By recruiting 447 tured Clinical Interview for DSM-IV TR Axis I patients with MDD and 432 controls, we found Disorders Patient Edition (SCID-I/P, 11/2002 re- a significant effect of three-locus BDNF/GSK3β vision). Among these patients, 81.3% were suffer- interaction (GSK3β rs6782799, BDNF rs6265 ing their first major depressive episode (n = 373), and BDNF rs7124442) with MDD were found in while the remaining 18.7% were experiencing Chinese population and individuals carrying the a relapse (n = 86). Patients with pregnancy, sig- combination of three risk alleles gave the largest nificant medical conditions, abnormal laborato- OR value of 4.4617. ry baseline values, unstable psychiatric features Previous researches showed a significant in- (e.g., suicidal), a history of alcoholism or drug teraction between BDNF and serotonin in brain abuse, seizures or epilepsy, head trauma with loss function. Serotonergic transmission exerts pow- of consciousness, neurological illness, or con- erful control over BDNF gene expression, and comitant additional Axis I psychiatric disorders BDNF promotes the survival, differentiation and were excluded. maintenance of serotonergic neurons18. However, The control was consisted of 412 healthy vol- the effect of 5-HTTLPR-BDNF (rs6265) interac- unteers (male, n = 172; female, n = 240; age range, tion on MDD-risk is not consistent. In this paper, 18-60 years old). They were without any history it is aimed to explore 5-HTTLPR polymorphism of neuropsychiatric disorders and recruited from and the BDNF (rs6265) polymorphism and to the community or regular health screening visit- their possible interaction in relation to MDD-risk ing as controls. by using case-control data collected in the Han Chinese population. We were specifically inter- Genotyping ested in whether the effect of 5-HTTLPR poly- Two types of polymorphisms were selected morphism is overdominant, and how the effect is from the NCBI database (http://www.ncbi.nlm. influenced by BDNF polymorphism. nih.gov) and the HapMap database (http://www. hapmap.org), including 5-HTTLPR polymor- phism in the 5-HTT gene and rs6265 polymor- Patients and Methods phism in the BDNF gene. Genomic DNA was extracted from peripheral blood leukocytes by the Patients phenol-chloroform extraction. Polymerase chain All subjects were from the same geographical reaction (PCR)-based genotyping was used to de- areas in Northern China and were of Chinese Han tect polymorphisms. origin. All participants provided written informed Primers were designed with Primer 5.0 soft- consent. This work was approved by the Ethical ware. The primer pair for detecting 5HTTLPR Committee for Medicine of the First Hospital of polymorphism are forward 5’-GGCGTTGC- Shanxi Medical University, China. CGCTCTGAATGC-3’ and reverse 5’-GAGG- Cases were collected from March 2004 and GACTGAGCTGGACAACCAC-3’. The primer February 2008 in Shanxi Province of China. Sam- pair for detecting BDNF (rs6265) polymorphism ple collection and diagnosis for subjects had been are forward 5’-GCTTTCTCCCTACAGTTC- previously described in detail17,19. The sample of CAC-3’ and reverse 5’-TCTGCTGCCGTTAC- Zhang’s study included 401 patients with MDD CCAC-3’. PCR was performed in 25 μl volume and 391 controls in 2009, 447 patients with MDD containing 60 ng of genomic DNA, 200 μM and 432 controls in 2010. In the present study, dNTPs, 0.2 μM each primer, 2.5 μl 10×PCR buf- our sample completed were overlapped those fer, and 1 U of Taq DNA polymerase (Tiangen, 1853 N. Sun, C.-X. Yang, Z.-F. Liu, X.-R. Li, Y. Xu, K.-R. Zhang Beijing, China). PCR included initial denatur- Results ation at 94 °C for 5 min, followed by 35 cycles of 94 °C for 30 s, 60 °C for 5HTTLPR or 56 °C Hardy-Weinberg Equilibrium Analysis for BDNF (rs6265) annealing for 30 s, 72 °C for Genotype frequencies of BDNF polymorphism 30 s, and a final elongation at 72 °C for 10 min. rs6265 did not differ significantly from those ex- Products of 5HTTLPR were electrophoresed on pected under HWE. However, as shown in Table 12% polyacrylamide gels and products of rs6265 I, we observed a significant deviation from HWE were sequenced with the ABI 3730 DNA analyzer for 5-HTTLPR in the control group, but not in (Perkin-Elmer, Applied Biosystems, Foster City, the case group. In the control group, there were CA, USA). only 28% individuals carrying the heterozygous genotype (LS), which is far less than what should Statistical Analysis be expected (36%) based the observed allele fre- Genotype frequencies of the 5-HTTLPR and quency of the S-allele (76%) under HWE, leading BDNF polymorphisms in both cases and controls to a significant deviation from HWE (p = 1.62E- and tested for departure from Hardy-Weinberg 05). In the case group, the expected heterozygous equilibrium (HWE)20. Specifically, for the 5-HT- genotype frequency was 39%, which is very close TLPR polymorphism, the previously observed to the observed value of 36%, and the deviation heterosis might imply a selective advantage or from HWE was not significant p( = 0.11).