NOTCH4 gene polymorphism and susceptibility to schizophrenia and schizoaffective disorder

Neuroscience Letters 301 (2001) 41±44

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NOTCH4 gene polymorphism and susceptibility to schizophrenia and schizoaffective disorder Hiroshi Ujike a,*, Yasushi Takehisa a, Manabu Takaki a, Yuji Tanaka a,b, Kenji Nakata a,c, Toshihiko Takeda c, Masafumi Kodama d, Yutaka Fujiwara e, Ayako Yamamoto a, Shigetoshi Kuroda a a

Department of Neuropsychiatry, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700-8558, Japan b Department of Psychiatry, Johns Hopkins University School of Medicine, Baltimore, MD, USA c Zikei Hospital, Okayama, Japan d Takami Hospital, Okayama, Japan e Takaoka Hospital, Himeji, Japan Received 11 January 2001; received in revised form 1 February 2001; accepted 5 February 2001

Abstract The NOTCH4 gene is located at 6p21.3, a site which several studies have shown to have signi®cant linkage with schizophrenia. Recently, an exceptionally strong association was reported between NOTCH4 gene polymorphisms and schizophrenia in British patients. We re-examined their ®ndings using a Japanese population. We genotyped three kinds of polymorphisms, SNP1 in the 5 0 ¯anking region, SNP2 in the promoter region and CTG repeats in exon 1 of the NOTCH4 gene of schizophrenics (N ˆ 188), patients with schizoaffective disorder (N ˆ 39) and controls (N ˆ 143). Genotypic distributions and allelic frequencies of SNP1, SNP2 and CTG repeats of the NOTCH4 gene did not show signi®cant associations with schizophrenia or schizoaffective disorder. Neither they showed association with schizophrenia subcategories, hebephrenic and paranoid type schizophrenia, nor with subgroups of schizophrenia with and without positive family history of psychoses. The present study found that the NOTCH4 gene does not confer susceptibility to schizophrenia and schizoaffective disorders, at least in Japanese subjects, in contrast to the ®ndings in British subjects. q 2001 Published by Elsevier Science Ireland Ltd. Keywords: Association study; NOTCH4 gene; Schizophrenia; Schizoaffective disorder; Japanese

Several lines of evidence from family-based, twin and adoption studies have indicated that both genetic and environmental factors must be involved in the major causes of schizophrenia and other endogenous psychoses [1,6]. Although several genes or genetic loci, such as D2 and D3 dopamine receptors, have been reported as candidates, only weak or inconsistent associations with schizophrenia have been seen [2,10,13]. To date, no gene has been con®rmed to confer certain susceptibility to schizophrenia. Recently, Wei and Hemmings [12] analyzed 13 loci spanning about 1.8 Mb in the MHC region located at 6p21.3, where signi®cant linkage was repeatedly identi®ed in previous schizophrenia linkage studies [4,5,7,11], and found several loci showing a signi®cant association with schizophrenia in Brit* Corresponding author. Tel.: 181-86-223-7151/7243; fax: 18186-235-7246. E-mail address: [email protected] (H. Ujike).

ish patients. Among these, the smallest P value, P ˆ 0:000036, was obtained for a CTG repeat polymorphism of exon 1 of the NOTCH4 gene. Such a strong association is exceptional in association studies examining endogenous psychoses like schizophrenia. The NOTCH family proteins play a role in cell-fate decisions, proliferation and apoptosis in metazoans [9]. In neurons, NOTCH signaling regulates extension and elaboration of neurites in vitro [8]. In humans, a missense mutation of the NOTCH 3 gene segregates with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, a type of dementia and stroke [3]. To con®rm the involvement of the NOTCH4 gene as a risk factor for schizophrenia, the ®ndings of the UK study must be replicated in other racial and ethnic groups. Therefore, we re-examined an association study between three kinds of NOTCH 4 gene polymorphisms, an A-to-G substitution in the 5 0 ¯anking region, a T-to-C substitution in the promoter region

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H. Ujike et al. / Neuroscience Letters 301 (2001) 41±44

(named SNP1 and SNP2 by Wei and Hemmings [12], originally described as T-to-C and A-to-G of complementary sequences, respectively) and polymorphic CTG repeats in exon 1 of the NOTCH4 gene. In this study, the subects were Japanese. The subjects were 188 patients with schizophrenia (F20, 109 males and 79 females, age 44.7 ^ 14.0 years old) and 39 with schizoaffective disorder (F25.1, 26 males and 13 females, age 46.1 ^ 13.2 years old) meeting ICD-10-DCR criteria who were outpatients or inpatients of psychiatric hospitals, and 143 age-matched normal controls (63 males and 80 females, age 42.6 ^ 15.1 years old) who had no known history of psychiatric disease in their families. Diagnosis was made by trained psychiatrists by interview. Almost normal controls were medical staffs. As to the subcategory of schizophrenia, hebephrenic (F20.1), paranoid (F20.0), catatonic (F20.2), undifferentiated type (F20.3) and residual type (F20.5) accounted for 103, 76, 4, 3 and 2 patients, respectively. Schizophrenics were also divided into two groups depending on family histories. A subgroup of schizophrenics with positive family histories was de®ned by the presence of a third degree or closer relative suffering from schizophrenia or schizoaffective disorder, and that without positive family histories was de®ned as lacking a third degree or closer relative suffering from schizophrenia, delusional disorder, schizoaffective disorder or bipolar disorders. Information about family history of parents was obtained from interviews with them, and that of the other family members was obtained from inquiries of patients and their parents. All subjects were Japanese, born and living in the middle western area of Japan. This study was performed after approval by the

ethics committee of Okayama University Medical School, Zikei hospital and Takaoka hospital, and all subjects provided written informed consent for the use of their DNA samples for this research. Genomic DNA was extracted from peripheral leukocytes by the standard phenol/CHCl3 method. Each region containing polymorphisms was ampli®ed by polymerase chain reaction (PCR) using primer sets as follows, SNP1: 5 0 -ATGCTTAAAAATGCCAGTATCG-3 0 , 5 0 -CAATTGCCCCATAATGTCCTTC-3 0 , SNP2: 5 0 -TAGTGTTCCTCCACTCTTCCTC-3 0 , 5 0 -AGTGAAGGGGGCTGCATTCCAC3 0 , CTG repeats: 5 0 -AAGAGGGGCAGTGGGAGCAGAG-3 0 , 5 0 -GAATCTCCTCCATCCAGCATCC-3 0 . PCR was performed in a ®nal volume of 15 ml with 10% dimethyl sulfoxide and 1 unit of Extaq (TAKARA Co., Japan) in the reaction mixture. PCR conditions were as follows: 958C for 5 min; 35 cycles of 988C for 20 s, 658C for 30 s, 728C for 1 min, and 728C for 5 min. SNP1 and SNP2 were analyzed on 3% agarose gel after digestion with Msp 1. For analysis of polymorphic CTG repeats, the forward primer was 5 0 -endlabelled with Texas Red, and PCR products and the Texas red-labelled size standard were electrophoretically run on 6% polyacrylamide gel using an SQ5500 DNA sequencer (Hitachi Co., Japan) and each length was calculated using Fragyls 2 (Hitachi Co., Japan) computer software. Repeat numbers of CTG were con®rmed by direct sequencing using PCR samples from subjects homozygous for the repeat. All genotyping was carried out in a blinded fashion with control and patient samples mixed randomly. Statistical analysis was done using the chi-square test. Genotypic distributions and allelic frequencies of SNP1, SNP2 and CTG repeats in normal controls, schizophrenics

Table 1 Genotypic distributions and allelic frequencies of SNP1 in the 5' ¯anking region and SNP2 in the promoter region of the NOTCH4 gene a SNP1

N

Genotypes (%) GG

AG

AA

G

A

60 (49.6) 74 (40.9) 40 (40.4) 33 (45.2) 29 (43.3) 45 (39.5) 17 (53.1)

18 (14.9) 30 (16.6) 21 (21.2) 7 (9.6) 12 (17.9) 18 (15.8) 4 (12.5)

146 (60.3) 228 (63.0) 116 (58.6) 99 (67.8) 81 (60.4) 147 (64.5) 39 (60.9)

96 (39.7) 134 (37.0) 82 (41.4) 47 (32.2) 53 (39.6) 81 (35.5) 25 (39.1)

Control Schizophrenia Sc, hebephrenic Sc, paranoid With FH Without FH Schizoaffective

121 181 99 73 67 114 32

43 (35.5) 77 (42.5) 38 (38.4) 33 (45.2) 26 (38.8) 51 (44.7) 11 (34.4)

SNP2

N

Genotypes (%)

Control Schizophrenia Sc, hebephrenic Sc, paranoid With FH Without FH Schizoaffective a

124 170 92 69 62 108 31

Alleles (%)

Alleles (%)

TT

TC

CC

T

C

38 (31.4) 48 (28.2) 23 (25.0) 23 (33.3) 18 (29.0) 30 (27.8) 9 (29.0)

54 (43.5) 80 (47.1) 42 (45.7) 34 (49.3) 26 (41.9) 50 (50.0) 15 (48.4)

32 (25.8) 42 (24.7) 27 (29.3) 12 (17.4) 18 (29.0) 24 (22.2) 7 (22.6)

130 (52.4) 176 (51.8) 88 (47.8) 80 (58.0) 62 (50.0) 114 (52.8) 33 (53.2)

118 (47.6) 164 (48.2) 96 (52.2) 58 (42.0) 62 (50.0) 102 (47.2) 29 (46.8)

Values in parentheses are percentages. FH, family history.

H. Ujike et al. / Neuroscience Letters 301 (2001) 41±44 Table 2 Allelic frequencies of the polymorphic CTG repeat in exoni of the NTOCH4 gene (CTG) n

Control Schizophrenia Sc, hebephrenic Sc, paranoid With FH Without FH Schizoaffective

N

286 376 206 152 152 224 72

%Alleles (number of CTG repeats) 5

6

9

10

11

12

13

0.3 0 0 0 0 0 0

20.6 14.9 16.0 14.5 13.2 16.1 22.2

29.7 35.1 37.4 32.9 38.8 32.6 27.8

40.2 38.6 33.5 43.4 35.5 40.6 44.4

8.7 11.2 13.1 8.6 12.5 10.3 5.6

0 0.3 0 0.7 0 0.4 0

0.3 0 0 0 0 0 0

and schizoaffective disorders are shown in Tables 1 and 2. Distributions of all three alleles in all groups were within the values expected from Hardy±Weinberg equilibrium. In control subjects (N ˆ 143), heterozygosity rates calculated according to each allele frequency of SNP1, SNP2 and CTG repeats were 0.48, 0.50 and 0.70, respectively. As to SNP1, there were no signi®cant genotypic differences between controls and the major diagnostic groups (schizophrenia; x2 ˆ 2:26, d:f: ˆ 2, P ˆ 0:32, schizoaffective disorders; x2 ˆ 0:17, d:f: ˆ 2., P ˆ 0:92) or among controls and two subcategories, hebephrenic and paranoid subtype schizophrenia (x2 ˆ 5:82, d:f: ˆ 4, P ˆ 0:21) or among controls and two subgroups of schizophrenia with and without of positive family histories of psychoses (x2 ˆ 2:90, d:f: ˆ 4, P ˆ 0:58), or for allelic frequencies between controls and major diagnostic groups (schizophrenia; x2 ˆ 0:36, d:f: ˆ 1, P ˆ 0:55, schizoaffective disorders; x2 ˆ 0:0, d:f: ˆ 1., P ˆ 1:0) or among controls and two subcategories (x2 ˆ 3:30, d:f: ˆ 2, P ˆ 0:19) or among controls and two subgroups of schizophrenia (x2 ˆ 1:01, d:f: ˆ 2, P ˆ 0:60). As to SNP2, no signi®cant genotypic differences were found between controls and major diagnostic groups (schizophrenia; x2 ˆ 0:37, d:f: ˆ 2, P ˆ 0:83, schizoaffective disorders; x2 ˆ 0:25, d:f: ˆ 2, P ˆ 0:88) or among controls and two subcategories (x2 ˆ 3:68, d:f: ˆ 4, P ˆ 0:45) or among controls and two subgroups of schizophrenia (x2 ˆ 1:68, d:f: ˆ 4, P ˆ 0:79), or for allelic frequencies between controls and major diagnostic groups (schizophrenia; x2 ˆ 0:01, d:f: ˆ 1, P ˆ 0:93, schizoaffective disorders; x2 ˆ 0:0, d:f: ˆ 1., P ˆ 1:0) or among controls and two subcategories (x2 ˆ 3:26, d:f: ˆ 2, P ˆ 0:19) or among controls and two subgroups of schizophrenia (x2 ˆ 0:27, d:f: ˆ 2, P ˆ 0:87). As to CTG repeats, schizophrenia or schizoaffective disorders did not differ signi®cantly from controls in terms of allelic frequencies (schizophrenia; x2 ˆ 5:57, d:f: ˆ 3, P ˆ 0:13, schizoaffective disorders; x2 ˆ 1:11, d:f: ˆ 3, P ˆ 0:77). Neither two subcategories of schizophrenia (x2 ˆ 9:93, d:f: ˆ 6, P ˆ 0:13), nor two schizophrenia subgroups (x2 ˆ 8:11, d:f: ˆ 6, P ˆ 0:23) showed to differ from controls. The present study revealed the genotypic distribution and allelic frequencies of the three polymorphisms of the

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NOTCH 4 gene in the Japanese control population, as shown in Tables 1 and 2. As Wei and Hemmings [12] did not present such data, it is unknown whether the values in the Japanese population are similar to or different from those in the British population. They reported only the heterozygosity values of SNP1 and 2 and CTG repeat polymorphisms as 0.16, 0.26 and 0.79, respectively [12]. Those of Japanese were 0.48, 0.50 and 0.70, respectively. Therefore, these data indicate that the genotypic distribution of the NOTCH 4 gene polymorphisms is likely to differ between the two races. Overall, the genotypic distributions and allelic frequencies of SNP1, SNP2 and CTG repeats in the NOTCH4 gene were not signi®cantly associated with schizophrenia or schizoaffective disorder. Neither was there any association with schizophrenia subcategories such as paranoid and hebephrenic types, nor subgroups of schizophrenia with/without positive family history. This ®nding suggests that the NOTCH4 gene has different roles in the pathogenesis of schizophrenia in the two races, Japanese and Caucasians. This is likely since the heterozygosity of the three polymorphisms of NOTCH4 differed markedly between the two races. Alternatively, the different analysis methods employed in the two studies may also have contributed to the inconsistent results. The British study [12] employed by transmission disequilibrium and halotype-based tests using parent-offspring trios, whereas we used a simple casecontrol association study, which is less sensitive. In conclusion, the present study revealed that the NOTCH4 gene does not confer susceptibility in schizophrenia and schizoaffective disorders, at least in Japanese, in contrast to the observations made in British patients. The present study was supported in part by a grant from Zikei Psychiatric Hospital (President; Dr H. Fujita, Okayama, Japan). [1] Cannon, T.D., Kaprio, J., Lonnqvist, J., Huttunen, M. and Koskenvuo, M., The genetic epidemiology of schizophrenia in a Finnish twin cohort. A population-based modeling study, Arch. Gen. Psychiatry, 55 (1998) 67±74. [2] Dubertret, C., Gorwood, P., Ades, J., Feingold, J., Schwartz, J.C. and Sokoloff, P., Meta-analysis of DRD3 gene and schizophrenia: ethnic heterogeneity and signi®cant association in Caucasians, Am. J. Med. Genet., 81 (1998) 318±322. [3] Joutel, A., Corpechot, C., Ducros, A., Vahedi, K., Chabriat, H., Mouton, P., Alamowitch, S., Domenga, V., Cecillion, M., Marechal, E., Maciazek, J., Vayssiere, C., Cruaud, C., Cabanis, E.A., Ruchoux, M.M., Weissenbach, J., Bach, J.F., Bousser, M.G. and Tournier-Lasserve, E., Notch3 mutations in CADASIL, a hereditary adult-onset condition causing stroke and dementia, Nature, 383 (1996) 707±710. [4] Kendler, K.S., Myers, J.M., O'Neill, F.A., Martin, R., Murphy, B., MacLean, C.J., Walsh, D. and Straub, R.E., Clinical features of schizophrenia and linkage to chromosomes 5q, 6p, 8p, and 10p in the Irish Study of High-Density Schizophrenia Families, Am. J. Psychiatry, 157 (2000) 402±408. [5] Nurnberger Jr., J.I. and Foroud, T., Chromosome 6 workshop report, Am. J. Med. Genet., 88 (1999) 233±238.

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