Introduction
The etiology of major depressive disorder (MDD) remains unknown, despite many different etiologic hypotheses have been produced. A number of studies have suggested that neurodegenerative mechanisms are involved in MDD. Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophic superfamily and is responsible for promoting and modifying growth, development and survival of neurons Reference Duman and Monteggia(1). Lower BDNF mRNA levels have been reported in the hippocampus and anterior cingulate cortex from MDD patients Reference Dwivedi, Rizavi and Conley(2), and reduced serum (plasma) BDNF levels were also reported in some MDD patients Reference Shimizu, Hashimoto and Okamura(3,Reference Karege, Bondolfi and Gervasoni4). Some other studies further suggested that serum (plasma) BDNF levels were elevated in the MDD patients after treated by antidepressant drugs Reference Karege, Perret and Bondolfi(5). Thus, the BDNF gene located on chromosome 11p13 is a putative candidate gene for MDD taking aspects of the neurodevelopmental, neurodegenerative hypotheses. The single nucleotide polymorphism (SNP) rs6265, which causes a valine-to-methionine substitution at codon 66 (Val66Met) in the BDNF gene, has been shown to affect intracellular trafficking and activity-dependent secretion of BDNF Reference Egan, Kojima and Callicott(6).
Previous studies testing associations between BDNF Val66Met gene variants and MDD have produced contradictory results. Ribeiro et al. Reference Ribeiro, Busnello and Cantor(7) reported individuals homozygous for major allele (GG) had an increased chance of being depressed in Mexican-Americans (OR = 1.795% CI = 1.17 − 2.47). Two other genetic association studies, both in Chinese populations studies by Tsai et al. Reference Tsai, Cheng and Yu(8) and Hong et al. Reference Hong, Huo and Yen(9) failed to establish BDNF Val66Met gene as a risk factor for MDD. Schumacher et al. Reference Schumacher, Jamra and Becker(10) performed one large genetic case-control study for BDNF, including 1097 controls and 465 patients with major depressive disorder from a German population. They also did not find the positive association between BDNF Val66Met gene and MDD, but haplotypes G-174bp-A, G-170bp-A and C-174bp-G emerged as important discriminators between the patients and controls (p = 0.00065). In this study, we examined BDNF Val66Met gene in health controls and late-onset depressed Chinese Han populations to find whether specified gene type increase in late-onset depression (LOD) and to detect the relationship between the gene and BDNF plasma level.
Materials and methods
Subjects
A total of 99 patients (29 men and 70 women, mean age 69.69 ± 5.38) were recruited from the Affiliated Brain Hospital of Nanjing Medical University, China, between December 2005 and June 2007. All the patients met criteria for a current major depressive episode according to the Diagnostic and Statistical Manual of Mental Disorders, Fourth edition (DSM-IV) (11). The age of first episode for all the patients was 60 years or older, and the patients had scores of ≥ 17 on the 17-item Hamilton Depression Scale (HAM-D) Reference Hamilton(12). Exclusion criteria included medical illnesses, epilepsy, substance abuse disorders, including alcohol, active agents within the last year, schizophrenia, delusional disorder, psychotic disorders not elsewhere classified, bipolar disorder or antisocial personality disorder diagnosed by DSM-IV.
The 110 normal controls (33 men and 77 women, mean age 69.45 ± 5.14) according to the exclusion criteria were recruited from community population in Nanjing. Inclusion criteria for the control subjects were physical health and no history of mental disorder, neurological disease or drug abuse, and no undergoing psychotropic treatment. All controls had a score of ≤ 7 in the HAM-D. The sex and age between cases and controls are well matched which show no significant differences (p > 0.05).
The study was approved by the Ethics Committee of the Nanjing Medical University. All subjects participated after giving informed consent.
Measure of plasma BDNF level
Fasting venous blood samples (5 ml) from the patients and normal controls were collected in anticoagulant tubes between 7:30 a.m. and 8:00 a.m. After centrifugation (centrifugation at 2500 × g for 8 min), the samples were stored at −80°C until assayed.
BDNF concentrations were measured using the BDNF Emax Immunoassay System kit (Promega; Madison, WI, USA) according to the manufacturer's instructions. To minimise the assay variance, plasma BDNF was measured in all subjects on the same day. Briefly, anti-BDNF monoclonal antibody was coated into 96-well plates being used to capture the neurotrophins in the plasma. The captured BDNF bound specifically BDNF polyclonal antibody (pAb), which were washed and incubated with anti-IgY antibody conjugated to horseradish peroxidase to detect the amount of specifically bound pAb. The plates were incubated with a chromogenic substrate to produce a colour reaction. The amount of BDNF in the test solution is proportional to the colour generated in the redox reaction. The standard curve and the samples were analysed in triplicate and showed a direct relation between optical density and BDNF concentration. The intra-assay coefficient of variation was less than 6%.
Genotyping of BDNF Val66Met
Genomic DNA was extracted from 300 μl ethylenediamine tetraacetic acid (EDTA)- anticoagulated venous blood using Puregene DNA Purification Kit (Gentra, Minnesota, USA) according to the manufacturer's recommendations. Polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP) assay was performed to genotyping the DNA sequence variants (196 G/A) of the BDNF gene. The primer sequences used for analysis of 196 G/A were the sense primer 5′-ACTCTGGAGAGCGTGAAT-3′ and the antisense primer 5′-ATACTGTCACACACGCTC-3′. The amplification conditions were initiated at 94°C for 5 min, followed by 35 cycles consisting of denaturation at 94°C for 30 s, annealing at 60°C for 30 s and extension at 72°C for 30 s, with a final extension step of 5 min at 72°C. The PCR product was digested with NlaIII (Fermentas, Lithuania) at 37°C for 10 h. The products were electrophoresed on 12% polyacrylamide gel at 200 V for 2 h. The presence of 168 and 75 bp bands indicated the existence of A allele, the presence of 243 bp band indicated the existence of G allele, while the presence of 75, 168, and 243 bp indicated AG heterozygote.
Statistical analysis
The data were analysed using the SPSS for Window11.0 software. The categorical data were analysed using the chi-squared test or the Fisher's exact test, if necessary. Continuous variables were analysed using the Student's t-test for the comparison among the groups. The criterion for significance was set at p < 0.05 for all the tests.
Results
The data of BDNF genotypes and alleles were given in LOD patients and controls (Table 1). The Val66Met-genotype distributions for these two groups were in Hardy–Weinberg equilibrium. The genotype and allele distributions in the LOD patients did not show significant differences from those in the control subjects (p = 0.744 and 0.845, respectively). We also examined the association between BDNF genotypes and quantitative measures of the LOD, including BDNF plasma level, duration of illness, age-of-onset, HAM-D and Hamilton Anxiety Scale (HAM-A) Reference Hamilton(13) at baseline. Table 2 showed that no significant difference was observed among the three BDNF genotype groups of the LOD. Additionally, no significant effect was shown for BDNF genotype in terms of suicidal history and psychotic symptoms for the LOD patients (p = 0.688 and 0.199, respectively).
BDNF Val66Met genotype distributions and allele frequency in 99 late-onset depression patients and 110 normal controls
Demographic data and clinical characteristics of the three BDNF-genotype groups within the late-onset depression patients
SD, standard deviation.
Discussion
The main finding of the present study was that there was no association of BDNF Val66Met polymorphism with LOD. To our knowledge, it is firstly reported that Val66Met genotype has no association with BDNF plasma level in LOD. Increasing evidence suggested that BDNF serum or plasma level decreased at baseline and increased after treated by antidepressant or electro-convulsive treatment (ECT) in depressive patients Reference Huang, Lee and Liu(14,Reference Piccinni, Marazziti and Catena15). Egan et al. Reference Egan, Kojima and Callicott(6) showed that depolarisation-induced secretion was reduced in 66Met BDNF-transfected neurons compared with 66Val BDNF analogues. Thus, subjects carrying Met66Met may have lower BDNF activity, which leads to higher risk for the development of MDD Reference Gervasoni, Aubry and Bondolfi(16). Nevertheless, BDNF is secreted through both constitutive and regulated pathways; the latter is a key step to control of synaptic plasticity. However, the BDNF Val66Met variant only affects activity-dependent secretion of BDNF but not the constitutive pathway. Consequently, this provides plausible explanations for BDNF concentrations in blood being unaffected by the genotype Reference Yu, Zhang and Shi(17).
It should also be noted that the relation between plasma and brain BDNF levels is not very clear. One study reported that BDNF could cross the blood-brain barrier freely from the brain to the blood Reference Pan, Banks and Fasold(18). Piccinni et al. Reference Piccinni, Marazziti and Catena(15) reported that the plasma BDNF levels increased to the values found in control subjects in parallel with the clinical improvement, while antidepressant treatments did not induce any change in serum BDNF levels, which remained lower than those found in the control group at all assessment times. Thereafter, they suggest, while plasma BDNF would behave as a state-dependent marker, serum BDNF might represent a trait of illness. Similarly, considering the interactions among plasma, platelets and the blood-brain barrier, plasma BDNF levels may reflect brain BDNF levels Reference Lee and Kim(19).
Several genetic association studies have been carried out in MDD, bipolar affective disorder, Alzheimer's disease and schizophrenia. The majority of these studies have produced negative results (Reference Tsai, Cheng and Yu8,Reference Hong, Huo and Yen9,Reference Schumacher, Jamra and Becker10,Reference Yu, Zhang and Shi17). As all etiopathogenisis of these diseases are unclear, the heritability of diseases may derive from a number of genes influenced by environmental factor, each with a small effect. So the effect of only one gene SNP may be tinier. The previously ambiguous findings of association studies between the BDNF gene Val66Met polymorphisms and MDD or some other psychiatric diseases are not unusual for genetic analyses of complex genetic diseases. Given the fact that the positive association between BDNF gene Val66Met polymorphism and bipolar disorder has only been shown for a Caucasian population but not for a Japanese or our Chinese sample, it appears likely that this association is ethnicity dependent Reference Kaija, Sami and Martti(20). Shimizu et al. Reference Shimizu, Hashimoto and Iyo(21) had similar findings and pointed that the frequency of healthy individuals who carried Val/Val gene type was significantly decreased in Japanese (33.8%) than in Italians (48.7%) or in Americans (68.4%). So they presumed that ethnic difference of the polymorphism frequencies might contribute to inconsistent results in genetic association study. In our study, to reduce the risk of ethnic biases, all the cases and control subjects were of Chinese Han population. Hwang et al. Reference Hwang, Tsai and Hong(22) pointed that the positive associations between BDNF Val66Met genetic polymorphism and depression might have been due to the chance or a stratification effect in the sample collection. Although our study did not find positive association in LOD, the possibility could not be ruled out that other polymorphisms of the BDNF gene are involved in the pathogenesis of MDD. On the basis of the findings of animal, pharmacological and clinical studies, which mainly focused on depression, BDNF has been proposed as a susceptibility locus for MDD. Szeszko et al. Reference Szeszko, Lipsky and Mentschel(23) reported subjects with BDNF Val/Val homozygotes had a larger hippocampal formation than Val/Met heterozygotes. Decreased hippocampal volume had more chance in the development of depression especially geriatric depression Reference Hickie, Naismith and Ward(24).
BDNF gene Val66Met polymorphism only reflected the role of a single locus. Haplotype-based studies might be considered advantageous as they use combined genetic information from several markers. Schumacher et al. Reference Schumacher, Jamra and Becker(10) investigated a possible relationship among three genetic variants at the BDNF locus [SNPs rs988748 and rs6265 (Val66Met), and a dinucleotide repeat (GT) n] in large samples. Although there were no single-marker associations, haplotypes were differentially distributed between case and control subjects at a global significance of p = 0.0092. Thus, to determine whether or not the BDNF gene is associated with MDD, further studies on more polymorphic sites within and close to the gene in larger, independent samples are required.
Our current study found no association between BDNF genotype, age-of-onset of depression, duration of illness, psychotic symptoms and suicidal attempt history in LOD. Depression and anxiety severity, as evaluated by HAM-D and HAM-A, also did not differ significantly among the three BDNF genotype groups. These findings implied that the BDNF Val66Met genetic polymorphism did not play a major role in these clinical manifestations of LOD. The results were consistent with the study conducted in Taiwan patients with major depression Reference Hong, Huo and Yen(9). Shimizu et al. Reference Shimizu, Hashimoto and Koizumi(25) also reported that their results which did not support BDNF Val66Met genetic polymorphism may be implicated in the putative common pathophisiology of depression in panic disorder. Further work is continuing to explore more BDNF gene locus and elucidates the relationship in LOD.
Conclusion
This study indicated that allele and genotype association with BDNF Val66Met polymorphisms did not show significant differences between the patients and controls. The BDNF plasma level had no association with genotype in Chinese Han patients, suggesting that the SNP was not a robust genetic risk factor for LOD.
Acknowledgements
This study was supported by grants from Key Program of Medical Development of Nanjing (No: ZKX07018 Yonggui Yuan).
Declaration of Interests
The authors declare they have no biomedical financial or potential conflicts of interest.
