- Email: [email protected]
CAG repeats of CTG18.1 and KCNN3 in Korean patients with bipolar affective disorder
Journal of Affective Disorders 66 (2001) 19–24 www.elsevier.com / locate / jad
Research report
CAG repeats of CTG18.1 and KCNN3 in Korean patients with bipolar affective disorder Dong Kyu Jin a , Hye Zin Hwang b , Myung Ryurl Oh b , Jung Shim Kim a , Munhyang Lee a , b b c c c , ,1 Seonwoo Kim , Shinn-Won Lim , Min Young Seo , Ji-Hae Kim , Doh Kwan Kim * a
Department of Pediatrics, Sungkyunkwan University School of Medicine, 50 Ilwon-Dong, Kangnam-Ku, Seoul, 135 -710 South Korea b Clinical Research Center, Samsung Biomedical Research Institute, Seoul, 135 -710 South Korea c Department of Neuropsychiatry, Sungkyunkwan University School of Medicine, 50 Ilwon-Dong, Kangnam-Ku, Seoul, 135 -710 South Korea Received 9 October 1999; received in revised form 4 July 2000; accepted 4 July 2000
Abstract Background: Trinucleotide repetition combined with variable penetrance of expression could be responsible for the complex transmission pattern observed in bipolar affective disorder (BPAD). The purpose of this study was to investigate the association of excess longer allele of KCNN3 and CTG18.1 in the patients with BPAD. Methods: CAG / CTG repeat distribution in KCNN3, CTG 18.1 and ERDA1 was examined and the copy number of ligation product in repeat expansion detection (RED) was measured in Korean bipolar patients in comparison to ethnically matched healthy controls. Results: No significant difference was found in the allele distribution of those repeats between bipolar patients and controls. Ligation product size in RED was not increased in bipolar patients. However, the copy number of ligation product in RED was highly correlated with CAG / CTG copies of ERDA1 (P 5 0.0001), partly with CTG 18.1 (P 5 0.04), but not with KCNN3. Conclusions: A longer CAG repeat alleles of KCNN3 or CTG 18.1 may not be a risk factor for BPAD in Korean population and the copy number of ligation product in RED in the patients with BPAD is influenced by the longer allele of CAG / CTG of ERDA1 or CTG 18.1. 2001 Elsevier Science B.V. All rights reserved. Keywords: Bipolar affective disorder; Genetics; Trinucleotide repeats
1. Introduction *Corresponding author. Tel.: 1 82-2-3410-3582; fax: 1 82-23410-0050. E-mail address: [email protected] (D.K. Kim). 1 D.K.J. and D.K.K. contributed equally to this work.
Since the first demonstration of association of longer alleles of CAG / CTG with major psychiatric disorders using the repeat expansion detection meth-
0165-0327 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0165-0327( 00 )00291-3
20
D.K. Jin et al. / Journal of Affective Disorders 66 (2001) 19 – 24
od (RED), much interest has been focused on the possibility of the unstable dynamic trinucleotide repeats (TNR) as an etiology of bipolar affective disorder (BPAD). Reports have been divided, with some papers showing the presence of expanded CAG / CTG (O’Donovan et al., 1995; Mendlewicz et al., 1997) and others not (Zander et al., 1998). Previously, several interesting CAG containing loci in human genome have been identified. The second polyglutamine repeat of hSKCa3 / KCNN3( KCNN3) was highly polymorphic, and the overall allele distribution showed different trend in bipolar patients compared to ethnically matched controls, with CAG repeats longer than the modal value being over-represented in patients (Chandy et al., 1998). The CTG repeat locus, termed CTG 18.1, was reported to confer an odds ratio of 2.6–2.8 as a vulnerability factor for BPAD (Lindblad et al., 1998). On the other hand, a novel, long and unstable CAG / CTG TNR sequence (ERDA1 ) was identified and regarded to account for most expansions detected by the RED technique (Nakamoto et al., 1997; Sidransky et al., 1998). In this study, we investigated the association of excess longer allele of KCNN3 and CTG 18.1 in patients with BPAD among the Korean population. Furthermore, we examined a correlation of CAG / CTG repeats of KCNN3, CTG 18.1 and ERDA1 with the anonymous CAG / CTG repeats of RED.
twenty healthy volunteers with no history of psychiatric illness and a normal profile on the Minnesota Multiphasic Personality Inventory (MMPI), whose clinical and content scale score ranging from 35 to 65, were studied. The male / female ratio was 51 / 69, and their mean age was 39.369.4 years.
2.2. Genotyping For the quantitation of CAG repeats in KCNN3, we used the primers F1: 59-AAGTGCCCCTGTCCATCCTCT-39 and R1: 59-GCCAAGCAAGTGGTCATTGAG-39 which were 59-labelled with g 32 P-ATP using T4 polynucleotide kinase. PCR conditions were same as previously described (Chandy et al., 1998). PCR products were analyzed onto 4.5% denaturing PAGE and autoradiography was taken. pUC 19 sequence was used for allele sizing. For the quantitation of CAG repeat numbers of CTG 18.1, we tried to measure it with the primers F1: 59-AATCCAAACCGCCTTCCAAGT-3 and R1: 59-ACTTCC-GAAAGCCATTTCT-3 (Breschel et al., 1997). And for the quantitation of CAG repeat numbers of ERDA1, we used the primers F1: 59-ATGGATTGTTCCAAGGA-G-39 and R1: 59-AGGTGGAAGGAAGGTCTT-39 (Nakamoto et al., 1997).
2.3. Southern blotting
2. Material and methods
2.1. Subjects All subjects including controls were unrelated and of Korean ancestry. One hundred forty nine patients with BPAD meeting the criteria of DSM-IV for a life-time diagnosis of bipolar I or II disorder were selected from the clinical services of Department of Neuropsychiatry, Samsung Medical Center in Seoul, Korea. This study was performed upon approval by the local Ethics Committee, and all subjects provided an informed consent permitting use of their DNA for research. The male / female ratio was 65 / 84, and their mean age was 43.5612.4 years. One hundred
Southern blotting was performed to detect the enlarged alleles that surpass the PCR amplification range. 5 mg of genomic DNAs were digested with EcoRI (BMS) enzyme at 378C overnight. After ethanol precipitation, the restricted genomic DNAs were electrophoresed onto 1% agarose gel and transferred to Nylon Membrane (Amersham). 1.3 Kb SEF2 -1 fragment generated by PCR cloning (249 bp-1546 bp, F1: 59-GCTTTCGGAAGTTTTGCCAGG-39, R1: 59-GCCTCTAAGATTAGCAA-GAGGC-39; Breschel et al., 1997) was labelled with a 32 P-dCTP and probed in Rapid Hyb buffer (Amersham) at 658C. First washing buffer composed of 5 3 SSC, 0.1% SDS was employed at room temperature and proceeded to 2nd wash with 0.1 3 SSC and 0.1% SDS at 658C.
D.K. Jin et al. / Journal of Affective Disorders 66 (2001) 19 – 24
2.4. RED reaction conditions All reactions were performed on a GeneAmp PCR System (Perkin Elmer Cetus). Each reaction mixture contained 1 mg of genomic DNA, 0.7 pM of labelled oligonucleotide, 10 3 ligase buffer, 5U of Ampligase (Epicentre Technologies, Madison, Wisconsin) with supplied Ampligase buffer and were taken through 497 cycles of 948C for 10 s and 768C for 30
21
s. We used (CTG) 17 as an oligonucleotide and it was labelled with g 32 P-ATP. This oligonucleotide was added to the RED reaction directly. This modification obviates the need for hybridization but gives a same resolution as the hybridized one. Samples were heat denatured for 5 min before loading on a 6% polyacrylamide gel containing 6M urea in a TBE buffer. The autoradiogram was taken from the gel and analyzed by phosphoimage analyzer.
Fig. 1. Distribution of allele frequency having different CAG repeat length in KCNN3 and CTG 18.1. Black bars represent the patients with bipolar disorder (n 5 298 chromosomes) and white bars normal control (n 5 240 chromosomes). (a) The number of occurrences at each CAG repeat length of KCNN3 is plotted as a percentage of the total within each group. (b) The number of occurrences at each CAG repeat length of CTG 18.1 is plotted as a percent of the total within each group.
22
D.K. Jin et al. / Journal of Affective Disorders 66 (2001) 19 – 24
Fig. 2. RED scores plotted against ERDA1 repeat size in the patients with bipolar affective disorder (A) and normal controls (B). The RED assay results was strongly affected by the CAG copy number of ERDA1 in BPAD patients (r 5 0.86, P 5 0.0001) as well as in normal controls (r 5 0.82, P , 0.001).
D.K. Jin et al. / Journal of Affective Disorders 66 (2001) 19 – 24
2.5. Statistical analysis The Wilcoxon’s Rank Sum Test was used to investigate if the copy numbers of ligation product in RED was significantly increased in BPAD patients, and if median length of KCNN3, CTG18.1, or ERDA1 in BPAD patients is significantly longer compared with normal controls. The Fisher’s Exact Test was used to see if there was a significant difference in the frequency of the individuals with CAG repeats longer than 100 repeats between BPAD patients and normal controls. The Spearman’s correlation analysis was applied to present how the copy number of ligation product in RED was correlated with CAG / CTG copies of ERDA1, CTG18.1, and KCNN3.
3. Results Neither the relative frequency of copy number of ligation product in RED nor the allele frequency of KCNN3 and CTG 18.1 was found significantly altered in patients with BPAD compared to normal control (P . 0.1, Fig. 1). And we could not find any significant difference in the frequency of the individuals with CAG repeats longer than 100 repeats, between bipolar patients and healthy controls (7 / 298, 5 / 240, respectively; P . 0.1). In our population, there was a striking CAG allele polymorphism in ERDA1. There was no significant difference in the median length of ERDA1 repeat numbers between BPAD patients and controls (P . 0.1). TNR expansions detected by RED in the patients with BPAD was strongly affected by the CAG copy number of ERDA1 (r 5 0.86, P 5 0.0001; Fig. 2A), and partly by CTG18.1 (r 5 0.18, P 5 0.04), but not by KCNN3 (r 5 0.01, P 5 0.90). The RED assay results in normal controls was also affected by CAG copy number of ERDA1 (r 5 0.82, P , 0.001; Fig. 2B).
4. Discussion Although our results failed to confirm the presence of expanded CAG / CTG repeats in the Korean patients with BPAD, several points must be empha-
23
sized. Our results show a similar trend of allele distribution of KCNN3 as shown in Caucasian populations in previous study (Chandy et al., 1998), while the present data are aggregated at the modal value and sparse at the other CAG alleles. As for the CTG 18.1, our data are not consistent with the recent report that expanded alleles at the CTG 18.1 locus may act as a vulnerability factor for affective disorder (Lindblad et al., 1998). It is not known whether a very enlarged expansion of CTG 18.1 up to CTG 800 – CTG 2100 cause a phenotype or not. In our study, there was no individual who had very enlarged expansion up to CTG 800 –CTG 2100 . The longest CAG / CTG allele of CTG18.1 in our population pertains to moderately enlarged one which was reported to be present in 3% in the bipolar population (Lindblad et al., 1998). We found this allele of CTG 18.1, which contains more than 200 repeats, in the control population who showed normal profile in MMPI. We could not demonstrate the presence of expanded CAG / CTG in BPAD. However, we found the RED assay results was affected by the CAG copy number of ERDA1 (P 5 0.0001), and partly by CTG 18.1 (P 5 0.04), but not by KCNN3 (P 5 0.98). This result supports that TNR expansions detected by RED could be influenced by PCR analysis of two specific loci, ERDA1 and CTG18.1. The combination of RED analysis with PCR of the ERDA1 and CTG18.1 loci may improve the ability to identify other neuropsychiatric illnesses associated with TNR expansions. Although a longer CAG repeat alleles of KCNN3 or CTG 18.1 seems not to be a risk factor for BPAD in Korean population, a further study might be needed to evaluate the importance of TNR in major psychiatric disorders as novel CAG / CTG containing genes are cloned every year.
Acknowledgements This work was supported by the grants from Genome-1G-3 of Ministry of Science and Technology, HMP-98-N-2-0014 of the Good Health R&D Project, Ministry of Health and Welfare, R.O.K. and 98-006 / 508 of Samsung Biomedical Research Institute.
24
D.K. Jin et al. / Journal of Affective Disorders 66 (2001) 19 – 24
References Breschel, T.S., Mclnnis, M.G., Margolis, R.L., Sirugo, G., Corneliussen, B., Simpson, S.G., McMahon, F.J., MacKinnon, D.F., Xu, J.F., Pleasant, N., Huo, Y., Ashworth, R.G., Grundstrom, T., Kidd, K.K., DePaulo, J.R., Ross, C.A., 1997. A novel, heritable, expanding CTG repeat in an intron of the SEF2-1 gene on chromosome 18q21.1. Hum. Mol. Genet. 6, 1855–1863. Chandy, K.G., Fantino, E., Wittekindt, O., Kalman, K., Tong, L.L., Ho, T.H., Gutman, G.A., Crocq, M.A., Ganguli, R., Nimgaonkar, V., Morris-Rosendahl, D.J., Gargus, J.J., 1998. Isolation of a novel potassium channel gene hSKCa3 containing a polymorphic CAG repeat: A candidate for schizophrenia and bipolar disorder? Mol. Psychiatry 3, 32–37. Lindblad, K., Nylander, P.O., Zander, C., Yuan, Q.P., Stahle, L., Engstrom, C., Balciuniene, J., Pettersson, U., Breschel, T., McInnis, M., Ross, C.A., Adolfsson, R., Schalling, M., 1998. Two commonly expanded CAG / CTG repeat loci: Involvement in affective disorders? Mol. Psychiatry 3, 405–410. Mendlewicz, J., Lindbald, K., Souery, D., Mahieu, B., Nylander, P.O., De Bruyn, A., Zander, C., Engstrom, C., Adolfsson, R.,
Van Broeckhoven, C., Schalling, M., Lipp, O., 1997. Expanded trinucleotide CAG repeats in families with bipolar affective disorder. Biol. Psychiatry 15, 1115–1122. Nakamoto, M., Takebayashi, H., Kawaguchi, Y., Narumiya, S., Taniwaki, M., Nakamura, Y., Ishikawa, Y., Akiguchi, I., Kimura, J., Kakizuka, A., 1997. A CAG / CTG expansion in the normal population. Nature Genet. 17, 385–386. O’Donovan, M.C., Guy, C., Craddock, N., Murphy, K.C., Cardno, A.G., Jones, L.A., Owen, M.J., McGuffin, P., 1995. Expanded CAG repeats in schizophrenia and bipolar disorder. Nature Genet. 10, 380–381. Sidransky, E., Burgess, C., Ikeuchi, T., Lindblad, K., Long, R.T., Philibert, R.A., Rapoport, J., Schalling, M., Tsuji, S., Ginns, E.I., 1998. A triplet repeat on 17q accounts for most expansions detected by the repeat–expansion–detection technique. Am. J. Hum. Genet. 62, 1548–1551, (Letter). Zander, C., Schurhoff, F., Laurent, C., Chavand, O., Bellivier, F., Samolyk, D., Leboyer, M., Allilaire, J.F., Cann, H., Neri, C., Mallet, J., 1998. CAG repeat sequences in bipolar affective disorder: No evidence for association in a French population. Am. J. Med. Genet. 81, 338–341.
Research report
CAG repeats of CTG18.1 and KCNN3 in Korean patients with bipolar affective disorder Dong Kyu Jin a , Hye Zin Hwang b , Myung Ryurl Oh b , Jung Shim Kim a , Munhyang Lee a , b b c c c , ,1 Seonwoo Kim , Shinn-Won Lim , Min Young Seo , Ji-Hae Kim , Doh Kwan Kim * a
Department of Pediatrics, Sungkyunkwan University School of Medicine, 50 Ilwon-Dong, Kangnam-Ku, Seoul, 135 -710 South Korea b Clinical Research Center, Samsung Biomedical Research Institute, Seoul, 135 -710 South Korea c Department of Neuropsychiatry, Sungkyunkwan University School of Medicine, 50 Ilwon-Dong, Kangnam-Ku, Seoul, 135 -710 South Korea Received 9 October 1999; received in revised form 4 July 2000; accepted 4 July 2000
Abstract Background: Trinucleotide repetition combined with variable penetrance of expression could be responsible for the complex transmission pattern observed in bipolar affective disorder (BPAD). The purpose of this study was to investigate the association of excess longer allele of KCNN3 and CTG18.1 in the patients with BPAD. Methods: CAG / CTG repeat distribution in KCNN3, CTG 18.1 and ERDA1 was examined and the copy number of ligation product in repeat expansion detection (RED) was measured in Korean bipolar patients in comparison to ethnically matched healthy controls. Results: No significant difference was found in the allele distribution of those repeats between bipolar patients and controls. Ligation product size in RED was not increased in bipolar patients. However, the copy number of ligation product in RED was highly correlated with CAG / CTG copies of ERDA1 (P 5 0.0001), partly with CTG 18.1 (P 5 0.04), but not with KCNN3. Conclusions: A longer CAG repeat alleles of KCNN3 or CTG 18.1 may not be a risk factor for BPAD in Korean population and the copy number of ligation product in RED in the patients with BPAD is influenced by the longer allele of CAG / CTG of ERDA1 or CTG 18.1. 2001 Elsevier Science B.V. All rights reserved. Keywords: Bipolar affective disorder; Genetics; Trinucleotide repeats
1. Introduction *Corresponding author. Tel.: 1 82-2-3410-3582; fax: 1 82-23410-0050. E-mail address: [email protected] (D.K. Kim). 1 D.K.J. and D.K.K. contributed equally to this work.
Since the first demonstration of association of longer alleles of CAG / CTG with major psychiatric disorders using the repeat expansion detection meth-
0165-0327 / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0165-0327( 00 )00291-3
20
D.K. Jin et al. / Journal of Affective Disorders 66 (2001) 19 – 24
od (RED), much interest has been focused on the possibility of the unstable dynamic trinucleotide repeats (TNR) as an etiology of bipolar affective disorder (BPAD). Reports have been divided, with some papers showing the presence of expanded CAG / CTG (O’Donovan et al., 1995; Mendlewicz et al., 1997) and others not (Zander et al., 1998). Previously, several interesting CAG containing loci in human genome have been identified. The second polyglutamine repeat of hSKCa3 / KCNN3( KCNN3) was highly polymorphic, and the overall allele distribution showed different trend in bipolar patients compared to ethnically matched controls, with CAG repeats longer than the modal value being over-represented in patients (Chandy et al., 1998). The CTG repeat locus, termed CTG 18.1, was reported to confer an odds ratio of 2.6–2.8 as a vulnerability factor for BPAD (Lindblad et al., 1998). On the other hand, a novel, long and unstable CAG / CTG TNR sequence (ERDA1 ) was identified and regarded to account for most expansions detected by the RED technique (Nakamoto et al., 1997; Sidransky et al., 1998). In this study, we investigated the association of excess longer allele of KCNN3 and CTG 18.1 in patients with BPAD among the Korean population. Furthermore, we examined a correlation of CAG / CTG repeats of KCNN3, CTG 18.1 and ERDA1 with the anonymous CAG / CTG repeats of RED.
twenty healthy volunteers with no history of psychiatric illness and a normal profile on the Minnesota Multiphasic Personality Inventory (MMPI), whose clinical and content scale score ranging from 35 to 65, were studied. The male / female ratio was 51 / 69, and their mean age was 39.369.4 years.
2.2. Genotyping For the quantitation of CAG repeats in KCNN3, we used the primers F1: 59-AAGTGCCCCTGTCCATCCTCT-39 and R1: 59-GCCAAGCAAGTGGTCATTGAG-39 which were 59-labelled with g 32 P-ATP using T4 polynucleotide kinase. PCR conditions were same as previously described (Chandy et al., 1998). PCR products were analyzed onto 4.5% denaturing PAGE and autoradiography was taken. pUC 19 sequence was used for allele sizing. For the quantitation of CAG repeat numbers of CTG 18.1, we tried to measure it with the primers F1: 59-AATCCAAACCGCCTTCCAAGT-3 and R1: 59-ACTTCC-GAAAGCCATTTCT-3 (Breschel et al., 1997). And for the quantitation of CAG repeat numbers of ERDA1, we used the primers F1: 59-ATGGATTGTTCCAAGGA-G-39 and R1: 59-AGGTGGAAGGAAGGTCTT-39 (Nakamoto et al., 1997).
2.3. Southern blotting
2. Material and methods
2.1. Subjects All subjects including controls were unrelated and of Korean ancestry. One hundred forty nine patients with BPAD meeting the criteria of DSM-IV for a life-time diagnosis of bipolar I or II disorder were selected from the clinical services of Department of Neuropsychiatry, Samsung Medical Center in Seoul, Korea. This study was performed upon approval by the local Ethics Committee, and all subjects provided an informed consent permitting use of their DNA for research. The male / female ratio was 65 / 84, and their mean age was 43.5612.4 years. One hundred
Southern blotting was performed to detect the enlarged alleles that surpass the PCR amplification range. 5 mg of genomic DNAs were digested with EcoRI (BMS) enzyme at 378C overnight. After ethanol precipitation, the restricted genomic DNAs were electrophoresed onto 1% agarose gel and transferred to Nylon Membrane (Amersham). 1.3 Kb SEF2 -1 fragment generated by PCR cloning (249 bp-1546 bp, F1: 59-GCTTTCGGAAGTTTTGCCAGG-39, R1: 59-GCCTCTAAGATTAGCAA-GAGGC-39; Breschel et al., 1997) was labelled with a 32 P-dCTP and probed in Rapid Hyb buffer (Amersham) at 658C. First washing buffer composed of 5 3 SSC, 0.1% SDS was employed at room temperature and proceeded to 2nd wash with 0.1 3 SSC and 0.1% SDS at 658C.
D.K. Jin et al. / Journal of Affective Disorders 66 (2001) 19 – 24
2.4. RED reaction conditions All reactions were performed on a GeneAmp PCR System (Perkin Elmer Cetus). Each reaction mixture contained 1 mg of genomic DNA, 0.7 pM of labelled oligonucleotide, 10 3 ligase buffer, 5U of Ampligase (Epicentre Technologies, Madison, Wisconsin) with supplied Ampligase buffer and were taken through 497 cycles of 948C for 10 s and 768C for 30
21
s. We used (CTG) 17 as an oligonucleotide and it was labelled with g 32 P-ATP. This oligonucleotide was added to the RED reaction directly. This modification obviates the need for hybridization but gives a same resolution as the hybridized one. Samples were heat denatured for 5 min before loading on a 6% polyacrylamide gel containing 6M urea in a TBE buffer. The autoradiogram was taken from the gel and analyzed by phosphoimage analyzer.
Fig. 1. Distribution of allele frequency having different CAG repeat length in KCNN3 and CTG 18.1. Black bars represent the patients with bipolar disorder (n 5 298 chromosomes) and white bars normal control (n 5 240 chromosomes). (a) The number of occurrences at each CAG repeat length of KCNN3 is plotted as a percentage of the total within each group. (b) The number of occurrences at each CAG repeat length of CTG 18.1 is plotted as a percent of the total within each group.
22
D.K. Jin et al. / Journal of Affective Disorders 66 (2001) 19 – 24
Fig. 2. RED scores plotted against ERDA1 repeat size in the patients with bipolar affective disorder (A) and normal controls (B). The RED assay results was strongly affected by the CAG copy number of ERDA1 in BPAD patients (r 5 0.86, P 5 0.0001) as well as in normal controls (r 5 0.82, P , 0.001).
D.K. Jin et al. / Journal of Affective Disorders 66 (2001) 19 – 24
2.5. Statistical analysis The Wilcoxon’s Rank Sum Test was used to investigate if the copy numbers of ligation product in RED was significantly increased in BPAD patients, and if median length of KCNN3, CTG18.1, or ERDA1 in BPAD patients is significantly longer compared with normal controls. The Fisher’s Exact Test was used to see if there was a significant difference in the frequency of the individuals with CAG repeats longer than 100 repeats between BPAD patients and normal controls. The Spearman’s correlation analysis was applied to present how the copy number of ligation product in RED was correlated with CAG / CTG copies of ERDA1, CTG18.1, and KCNN3.
3. Results Neither the relative frequency of copy number of ligation product in RED nor the allele frequency of KCNN3 and CTG 18.1 was found significantly altered in patients with BPAD compared to normal control (P . 0.1, Fig. 1). And we could not find any significant difference in the frequency of the individuals with CAG repeats longer than 100 repeats, between bipolar patients and healthy controls (7 / 298, 5 / 240, respectively; P . 0.1). In our population, there was a striking CAG allele polymorphism in ERDA1. There was no significant difference in the median length of ERDA1 repeat numbers between BPAD patients and controls (P . 0.1). TNR expansions detected by RED in the patients with BPAD was strongly affected by the CAG copy number of ERDA1 (r 5 0.86, P 5 0.0001; Fig. 2A), and partly by CTG18.1 (r 5 0.18, P 5 0.04), but not by KCNN3 (r 5 0.01, P 5 0.90). The RED assay results in normal controls was also affected by CAG copy number of ERDA1 (r 5 0.82, P , 0.001; Fig. 2B).
4. Discussion Although our results failed to confirm the presence of expanded CAG / CTG repeats in the Korean patients with BPAD, several points must be empha-
23
sized. Our results show a similar trend of allele distribution of KCNN3 as shown in Caucasian populations in previous study (Chandy et al., 1998), while the present data are aggregated at the modal value and sparse at the other CAG alleles. As for the CTG 18.1, our data are not consistent with the recent report that expanded alleles at the CTG 18.1 locus may act as a vulnerability factor for affective disorder (Lindblad et al., 1998). It is not known whether a very enlarged expansion of CTG 18.1 up to CTG 800 – CTG 2100 cause a phenotype or not. In our study, there was no individual who had very enlarged expansion up to CTG 800 –CTG 2100 . The longest CAG / CTG allele of CTG18.1 in our population pertains to moderately enlarged one which was reported to be present in 3% in the bipolar population (Lindblad et al., 1998). We found this allele of CTG 18.1, which contains more than 200 repeats, in the control population who showed normal profile in MMPI. We could not demonstrate the presence of expanded CAG / CTG in BPAD. However, we found the RED assay results was affected by the CAG copy number of ERDA1 (P 5 0.0001), and partly by CTG 18.1 (P 5 0.04), but not by KCNN3 (P 5 0.98). This result supports that TNR expansions detected by RED could be influenced by PCR analysis of two specific loci, ERDA1 and CTG18.1. The combination of RED analysis with PCR of the ERDA1 and CTG18.1 loci may improve the ability to identify other neuropsychiatric illnesses associated with TNR expansions. Although a longer CAG repeat alleles of KCNN3 or CTG 18.1 seems not to be a risk factor for BPAD in Korean population, a further study might be needed to evaluate the importance of TNR in major psychiatric disorders as novel CAG / CTG containing genes are cloned every year.
Acknowledgements This work was supported by the grants from Genome-1G-3 of Ministry of Science and Technology, HMP-98-N-2-0014 of the Good Health R&D Project, Ministry of Health and Welfare, R.O.K. and 98-006 / 508 of Samsung Biomedical Research Institute.
24
D.K. Jin et al. / Journal of Affective Disorders 66 (2001) 19 – 24
References Breschel, T.S., Mclnnis, M.G., Margolis, R.L., Sirugo, G., Corneliussen, B., Simpson, S.G., McMahon, F.J., MacKinnon, D.F., Xu, J.F., Pleasant, N., Huo, Y., Ashworth, R.G., Grundstrom, T., Kidd, K.K., DePaulo, J.R., Ross, C.A., 1997. A novel, heritable, expanding CTG repeat in an intron of the SEF2-1 gene on chromosome 18q21.1. Hum. Mol. Genet. 6, 1855–1863. Chandy, K.G., Fantino, E., Wittekindt, O., Kalman, K., Tong, L.L., Ho, T.H., Gutman, G.A., Crocq, M.A., Ganguli, R., Nimgaonkar, V., Morris-Rosendahl, D.J., Gargus, J.J., 1998. Isolation of a novel potassium channel gene hSKCa3 containing a polymorphic CAG repeat: A candidate for schizophrenia and bipolar disorder? Mol. Psychiatry 3, 32–37. Lindblad, K., Nylander, P.O., Zander, C., Yuan, Q.P., Stahle, L., Engstrom, C., Balciuniene, J., Pettersson, U., Breschel, T., McInnis, M., Ross, C.A., Adolfsson, R., Schalling, M., 1998. Two commonly expanded CAG / CTG repeat loci: Involvement in affective disorders? Mol. Psychiatry 3, 405–410. Mendlewicz, J., Lindbald, K., Souery, D., Mahieu, B., Nylander, P.O., De Bruyn, A., Zander, C., Engstrom, C., Adolfsson, R.,
Van Broeckhoven, C., Schalling, M., Lipp, O., 1997. Expanded trinucleotide CAG repeats in families with bipolar affective disorder. Biol. Psychiatry 15, 1115–1122. Nakamoto, M., Takebayashi, H., Kawaguchi, Y., Narumiya, S., Taniwaki, M., Nakamura, Y., Ishikawa, Y., Akiguchi, I., Kimura, J., Kakizuka, A., 1997. A CAG / CTG expansion in the normal population. Nature Genet. 17, 385–386. O’Donovan, M.C., Guy, C., Craddock, N., Murphy, K.C., Cardno, A.G., Jones, L.A., Owen, M.J., McGuffin, P., 1995. Expanded CAG repeats in schizophrenia and bipolar disorder. Nature Genet. 10, 380–381. Sidransky, E., Burgess, C., Ikeuchi, T., Lindblad, K., Long, R.T., Philibert, R.A., Rapoport, J., Schalling, M., Tsuji, S., Ginns, E.I., 1998. A triplet repeat on 17q accounts for most expansions detected by the repeat–expansion–detection technique. Am. J. Hum. Genet. 62, 1548–1551, (Letter). Zander, C., Schurhoff, F., Laurent, C., Chavand, O., Bellivier, F., Samolyk, D., Leboyer, M., Allilaire, J.F., Cann, H., Neri, C., Mallet, J., 1998. CAG repeat sequences in bipolar affective disorder: No evidence for association in a French population. Am. J. Med. Genet. 81, 338–341.