No evidence for an association of the CTLA4 gene with bipolar I disorder
Dr Kyoung-Uk Lee, Department of Psychi-atry, College of Medicine, Catholic University of Korea, 62 Yoido-Dong, Youngdeungpo-Gu, Seoul 150-713, Korea. Email: firstname.lastname@example.org
Abstract The purpose of the present paper was to investigate the relationship between the first exon at position +49 (A/G) polymorphism of the cytotoxic T lymphocyte antigen 4 (CTLA4) gene and bipolar disorder. Among the Korean patients diagnosed with bipolar disorder according to Diagnostic and Statistical Manual of Mental Disorders (4th edn; DSM-IV), 90 patients without serious medical illness, neurologic illness, hormonal disorder, or concomitant mental illness were selected. The normal control group consisted of 149 age- and sex-matched subjects without current or past history of autoimmune diseases or mental disorder. DNA was extracted from peripheral blood using proteinase K; and the exon 1 region of the CTLA4 gene was amplified by polymerase chain reaction. Gene typing was performed using single strand conformation polymorphism. There were no significant differences in genotype frequencies of CTLA4*G/G, CTLA4*G/A, and CTLA4*A/A between the patients with bipolar disorder and the control group (48.9% vs 46.3%, 44.4% vs 39.6%, and 6.7% vs 14.1%, respectively). There were no significant differences in allelic frequencies of CTLA4*G and CTLA4*A between the patients with bipolar disorder and the control group (71.1% vs 66.1%; 28.9% vs 33.9%, respectively). In the present study an association was not found of exon 1 (+49) polymorphism of CTLA4 gene with bipolar disorder in the Korean population.
Bipolar disorder is one of the most common mental illnesses. The lifetime prevalence estimates are 1–6.5% of the general population, and mortality rates are increased as a result of high suicidal risks, accidents, comorbidity of substance abuse and increased fatality of stress-sensitive medical illnesses.1–4
Patients with bipolar disorder have been reported to have altered immune functions such as leukocytosis5 and higher prevalence of thyroid autoantibodies.6,7 Other immunological abnormalities such as increased plasma concentrations of soluble interleukin-2 receptor (sIL-2R)8,9 and sIL-6R,8 and lymphocyte proliferation to phytohemagglutinin,9 were reported in bipolar patients. These findings suggest that cell-mediated immunity activation is present in bipolar disorder.
Familial aggregation and the higher concordance rate of the monozygotic twin indicate the importance of genetic involvement in bipolar disorders.10 However, the mode of inheritance is complex and linkage findings have been difficult to replicate. Despite these limitations, advances in molecular genetics have provided the tools needed to identify genes involved in susceptibility to bipolar disorder.
Cytotoxic T lymphocyte antigen 4 (CTLA4) is a cell surface molecule that is expressed on the surface of activated T cells. It interacts with the B7 molecules on the surface of antigen-presenting cells.11 The binding of B7 by CTLA4 delivers negative signals to the T cell and thus, appears to be essential for the termination of an ongoing immune response and maintenance of T-cell homeostasis.12–14 Thus, disruption of the normal negative control by CTLA4 can contribute to the pathogenesis of bipolar disorder.
The CTLA4 gene is 7195 bp, located on chromosome 2q33 and has been known to have three polymorphic sites: a microsatellite (AT)n marker at position 642 of the 3′-untranslated region of exon 4;15 single nuceotide polymorphisms at position 49 of exon 1 (A/G);16 and in promoter region −318 (C/T).17 There has been much evidence that CTLA4 gene polymorphism resulted in immunologic abnormalities and was associated with certain autoimmune diseases.18–22 Although the A/G dimorphism at exon 1 position +49 results in an amino acid substitution (Thr/Ala) in the leader peptide of the expressed protein, the exact functional significance is not yet clearly understood.
With this in mind, we investigated if there was an association between the polymorphism of CTLA4 exon 1 (+49) and bipolar disorder, which is known to have immune alteration.
A total of 90 bipolar patients (43 men and 47 women) aged 16–65 years (mean age, 34.9 ± 11.0 years) were recruited on an outpatient and inpatient basis at St Mary's Hospital and Uijongbu St Mary's Hospital, College of Medicine, Catholic University of Korea. Diagnosis was carried out by consensus of two board-certified psychiatrists (WMB; CUP) according to Diagnostic and Statistical Manual of Mental Disorders (4th edn; DSM-IV),23 and based on individual interviews, clinical observation, medical records and family information. One hundred and forty-nine healthy native Koreans (68 male and 81 female) aged 18–63 years (mean age, 33.5 ± 11.0 ) from the Catholic Hemopoietic Stem Cell Information Bank were recruited as the controls. We assessed the controls with careful interviews to determine the presence of any psychiatric problems in the present or in the past history prior to sampling.
Both patients and controls who had serious medical illness, neurologic illness, hormonal disorder, autoimmune disorder, or concomitant mental illness were excluded from the study. As to screening for autoimmune or autoimmune-related disease, we excluded subjects through careful interview and referrals to experts if symptoms suggestive of autoimmune diseases or autoimmune-related diseases were found.
The two groups were also age- and sex-matched and were recruited from the same geographic area, the metropolitan area, which is located in the middle of Korea. The objectives and procedures of the study were explained to all subjects and written informed consent was obtained. The present study was also approved by the institutional review board.
Genotyping for the the exon 1 polymorphism of CTLA4 gene
DNA was extracted from peripheral blood by proteinase K using a method described elsewhere.24 The DNA samples were then stored at 4°C until used as template DNA in polymerase chain reaction (PCR).
Polymerase chain reaction
Amplification of the target DNA sequence in exon 1 of the CTLA4 gene was carried out by PCR using primers: 5′-GCT CTA CTT CCT GAA GAC CT-3′ and 5′-AGT CTC ACT CAC CTT TGC AG-3′. The PCR reagent mixture including 2 µL of 10X buffer (500 mmol/L KCl, 100 mmol/L Tris-HCl pH 8.3, 15 mmol/L MgCl2), 2 µL of 20 mmol/L oligonucleotide primers each, 2 µL of 2 mmol/L dNTP, 0.2 µL Taq DNA polymerase (5 U/µL; Boehringer Mannnheim, Mannheim, Germany) and 2 µL of genomic DNA was set up and the volume made up to 20 µL with distilled water. The PCR was carried out in a Perkin Elmer 9600 thermocycler (Foster City, CA, USA) under the following conditions: 4 min at 94°C and 30 cycles of 45 s at 94°C (denaturation), 45 s at 62°C (annealing), and 45 s at 72°C (elongation). The CTLA4 genotyping was performed by the PCR-single strand conformation polymorphism (SSCP) method.
Polymerase chain reaction–single strand conformation polymorphism
The PCR products were denatured for 5 min at 95°C to form single-stranded DNA, which was mixed with formamide containing stop buffer (95% formamide; 10 mmol/L NaOH; 0.25% bromophenol blue and 0.25% xylene cyanol) and then electrophoresed in MDETM gel (BioWhittaker Molecular Applications, Rockland, NY, USA). Gel electrophoreses were carried out at 4 W for 14 h at room temperature. Finally, conformational fragments were identified with subsequent silver staining and the results were assessed.
sas/PC version 6.0 (SAS Institute, Cary, NC, USA) was used for the statistical analysis. χ2 test was used to analyze the difference in allele and genotype frequencies between the patients with bipolar disorder and the control group. P < 0.05 was considered statistically significant.
The genotype distribution of the CTLA4 gene (in bipolar patients, χ2 = 0.59, P = 0.74; in controls, χ2 = 2.03, P = 0.36) was not significantly different from those expected based on the Hardy–Weinberg equilibrium.
There were no significant differences in genotype frequencies of CTLA4*G/G, CTLA4*G/A, and CTLA4*A/A between the patients with bipolar disorder and the control group (48.9% vs 46.3%, 44.4% vs 39.6%, 6.7% vs 14.1%, respectively). There were no significant differences in allelic frequencies of CTLA4*G and CTLA4*A between the patients with bipolar disorder and the control group (71.1% vs 66.1%, 28.9% vs 33.9%, respectively; Table 1).
Table 1. Frequencies of the genotypes and alleles of the CTLA4 gene in patients with bipolar disorder and controls
|Patients (n = 90)||6 (6.7)||40 (44.4)||44 (48.9)|| 52 (28.9)||128 (71.1)|
|Controls (n = 149)||21 (14.1)||59 (39.6)||69 (46.3)||101 (33.9)||197 (66.1)|
The present study was done to investigate the association of CTLA4 gene polymorphism with bipolar disorder, which is known to have immune alteration. To the best of our knowledge, this study is the first report on CTLA4 polymorphism in bipolar patients. It is disappointing that our study did not show an association between polymorphism of the CTLA4 exon 1 (+49) gene and bipolar disorder. This result suggests that the CTLA4 gene is not a major candidate gene for bipolar disorder in the Korean population.
Although our result showed a negative association, it does not mean absolute exclusion of the CTLA4 gene for the pathogenesis in bipolar disorder because of the possible association between other polymorphisms of CTLA4 with bipolar disorder. Previously, we studied the association of CTLA4 gene polymorphism with schizophrenia and major depression to investigate the possible roles of CTLA4 gene polymorphism in these disorders.25,26 and we reported a positive association of CTLA4 gene polymorphism with schizophrenia.25 Therefore, the present finding provides evidence that immune modulation may vary in each major psychiatric disorder.
Until recently, studies on the immune alteration in bipolar disorder have not yielded consistent results. Contrary to the evidence supporting immune activation, which is the theoretical basis for the present study, several reports showed no evidence of immune system activation in bipolar patients,27 such as reduced interferon production28 and significant reduction in mitogenic activity.29 These findings suggest a negative immunoregulatory status or impairment in cellular immune activity in bipolar patients. Thus, this could be one reason why the present study found no association of bipolar disorder with genetic polymorphism of CTLA4, which is a negative regulator of immune activation.
It is also important to suggest sample power when a negative result occurs. Our sample had a power of 0.80 to detect a small effect size (w = 0.13) between the allele variants under two-tailed P = 0.05 and to detect an odds ratio of 1.83.
In conclusion, our data showed that the polymorphism of the CTLA4 exon 1(+49) gene was not associated with susceptibility to bipolar disorder in the Korean population.
The present study was supported in part by a grant from the Korean Health 21 R&B Project (02-PJ1-PG3-20506–0001), Ministry of Health and Welfare, the Republic of Korea. The authors also thank Young-Mi Park and Hee-Ohk Kim for their support.