Wen-min Tian, Ke-rang Zhang, Juan Zhang, Yan Shen, and Qi Xu

1National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100005, China

2Department of Psychiatry, the First Hospital of Shanxi Medical University, Taiyuan 030001, China

MAJOR depression is an incapacitating disorder characterized by depressed mood, anhedonia, and an inhibition of both mental and physical activities.1,2The chronic, festering nature of depression results in huge emotional and financial consequences. One of the pathophysiological hypotheses states that abnormal neurogenesis leads to depression.3,4Blocking hippocampal neurogenesis inhibits the therapeutic efficacies of most antidepressant treatments in rodent models.1Several growth factors in the hippocampus influence the neurogenesis, including brain-derived neurotrophic factor (BDNF) and neuropeptide VGF (nonacronymic).1

Major depression is associated with cognitive impairments.5,6Epidermal growth factor (EGF), located on chromosome 4q25,7can stimulate proliferation of granule cell precursors in the dentate gyrus of adult rats.8It can also influence the synaptic plasticity and long-term potentiation in the hippocampus.9-11

Given the evidence supporting EGF not only as a susceptibility gene for major depression but also as a gene contributing to intellectual functioning, we hypothesized that variations in the EGF gene may contribute to the risk of major depression by affecting cognitive abilities. To date, association between genetic variation in the EGF gene and intellectual functioning has rarely been studied. Therefore, we investigated that association, focusing on four single nucleotide polymorphisms (SNPs) in the EGF gene.

SUBJECTS AND METHODS

Subjects

We recruited a total of 120 unrelated patients with major depression (55 males and 65 females, aged 33.8±12.7 years) and 46 control subjects (21 males and 25 females, aged 30.5±9.3 years). All the subjects were of Han nationality from Northern China. Clinical diagnosis of major depression was made by at least two experienced psychiatrists according to the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) published by the American Psychiatric Association in 1994. All subjects were assessed with Wechsler Adult Intelligence Scale (WAIS). Their blood samples were collected for DNA isolation using standard phenol-chloroform extraction.

SNP selection and genotyping

In this study, four SNPs, including rs2237051, rs2250724, rs6533482, and rs2237043 were selected from the National Center for Biotechnology Information SNP database (http://www.ncbi.nlm.nih.gov/SNP) (Table 1). Using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) to genotype rs6533482 and rs2237043, PCR amplification was performed with the GeneAmp PCR 2700 system (Applied Biosystems, Carlsbad, CA, USA) in a 25 μL reaction volume containing 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 1.5 mmol/L MgCl2, 0.001% (w/v) gelatin, 200 μmol/L of each dNTP, 0.4 μmol/L of each primer, 1.0 unit of Taq DNA polymerase (BioChain, USA), and 50 ng of genomic DNA. The conditions used for PCR amplification included initial denaturation at 95°C for 10 minutes, 35 cycles at 95°C for 30 seconds, at 55-60°C for 30 seconds, and at 72°C for 30 seconds, followed by a final extension at 72°C for 10 minutes. The genotypes were then determined by RFLP. Primer sequences, detailed PCR conditions and RFLP are shown in Table 2. Genotyping of rs2237051 and rs2250724 were preformed by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI- TOF-MS) on Mass ARRAY Analyzer Compact (Sequenom, USA).

Statistical analysis

The Hardy-Weinberg equilibrium was tested by the chi- square goodness-of-fit test for the genotypic distributions of SNPs. Quantitative trait analysis was performed using the UNPHASED program.12To circumvent the multi-point analysis problem, the significant P value was corrected for multiple testing by running 10 000 permutations. The Mann-Whitney test was employed to analyze the difference in WAIS scores between major depression patients and controls. A P value under 0.05 was considered statistically significant.

RESULTS

There were significant differences between patients and controls in score lang and score task, two subtests in the WAIS. The mean score lang of major depression patients was significantly lower than that of controls (62.00±14.54 vs. 72.98±9.48, P＜0.0001). Similarly, the mean score task in major depression patients was significantly lower than that in controls (48.08±10.42 vs. 61.65±8.60, P＜0.0001).

None of the 4 SNPs deviated from Hardy-Weinberg equilibrium (data not shown). rs2250724 showed close association with score lang and score task in patients with major depression, which remained significant after permutation tests (Tables 3, 4).

Table 1. Information of four SNPs in the EGF gene selected for analysis

Table 2. Primers and restriction enzymes for genotyping SNPs by PCR-RFLP

Table 3. Association of score lang scale with EGF alleles and genotypes among total population and major depression patients

Table 4. Association of score task scale with EGF alleles and genotypes among total population and major depression patients

DISCUSSION

To investigate the effect of EGF variant on intelligence in major depression, we applied WAIS to estimate general intelligence, which is one of the most commonly used test batteries for this purpose.5In our study, we found the levels of score lang and score task significantly decreased in major depression patients compared with those in controls, indicating impairment in semantic comprehension ability (represented by the score lang) and task planning ability (represented by the score task).

Several studies have demonstrated that major depression patients at rest exhibit abnormal blood flow in many brain regions involved in language processing, including prefrontal and temporal cortex. Furthermore, semantic deficits in depression patients are particularly obvious when the patients are asked to make decisions on the basis of what they have read or been told.13To study deficits of language comprehension in patients with major depression and its relation to the underlying neuropathophysiology is of crucial importance to our understanding of the etiology, manifestation, and treatment of depression.

One study showed that blocking the tyrosine kinase activity of EGF receptor contributes to depressive symptoms.14Therefore, abnormal conditions of EGF, the major ligand for that receptor, may associate with depression. Because of the small number of controls in our study, we did not analyze the association of EGF gene with all symptoms of major depression. As intelligence is one of the most obvious features in major depression patients, we chose this single feature for quantitative trait analysis. Of the four SNPs tested, rs2250724, located at intron 1 of the EGF gene, was the only one significantly associated with score lang and score task. Its mutant genotype might influence the function and quality of mature EGF by participating in the transcriptional process； or it could act just as a marker. The functional mutation in linkage disequilibrium leading to this phenotype cannot be determined based on our data.

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