The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 62:350-351 (2007)
© 2007 The Gerontological Society of America
Genetic Association Between Notch4 Polymorphisms and Alzheimer's Disease in the Japanese Population
Nobuto Shibata,
Tohru Ohnuma,
Shinji Higashi,
Maiko Higashi,
Chie Usui,
Taku Ohkubo,
Tomoko Watanabe,
Ritsuko Kawashima,
Akiyoshi Kitajima,
Akira Ueki,
Masatsugu Nagao and
Heii Arai
1 Department of Psychiatry, Juntendo University School of Medicine, Tokyo, Japan.
2 Department of Neurology, Omiya Medical Center, Jichi Medical School, Saitama-shi Japan.
3 Department of Psychiatry, Nagao Hospital, Hiroshima, Japan.
Address correspondence to Nobuto Shibata, MD, Department of Psychiatry, Juntendo University School of Medicine. 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421 Japan. E-mail: nobuto.shibata{at}nifty.ne.jp
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Abstract
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It is shown that Notch 4 plays important roles in the pathogenesis of Alzheimer's disease (AD). To investigate whether three single nucleotide polymorphisms (SNPs) of the Notch4 gene are associated with AD, the three SNPs were genotyped by a polymerase chain reaction–restriction fragment length polymorphism method for 243 AD patients and 130 age-matched controls. We also confirmed the linkage disequilibrium among these three SNPs of the gene using the EH program. The three SNPs did not seem to alter risk for AD. Our study suggests that SNPs studied are not associated with AD. The linkage disequilibrium of this locus indicates that there is genetic heterogeneity in the Notch4 gene. We could not confirm the previous synergetic associations of the 5' untranslated region (rs367398) C/C genotype in apolipoprotein E 
4 bearers in AD patients. Potential markers nearby the 5' untranslated region polymorphism might affect risk for AD.
THE genes HLA-DR3, CREBLI, RAGE (the receptor for advanced glycosylation end products), and Notch4 are located within the major histocompatibility complex (MHC) 6p21.3 locus. This genetic region seems to provide good candidates for Alzheimer's disease (AD) (1–3). Missense mutations of the Notch3 gene cause CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy), a form of dementia with stroke (4). In addition, AD might have a pathogenesis similar to that of CADASIL (5). Symptoms of dementia observed in two diseases have suggested a relationship between the Notch family and AD. The presenilins are known to interact with several proteins including the Notch 1–4 proteins, and a dysfunction of these interactions may play a role in AD pathogenesis (6,7). Although Lambert and colleagues (8) showed negative results for two single nucleotide polymorphisms (SNPs) of the gene, genetic studies of the gene were not fully performed. In this study, we focused on three SNPs—rs367398, rs2071282, and rs422951 to confirm the genetic association. These three SNPs cover a 3.3-kb region from the 5' untranslated region to exon 4 of the Notch4 gene, and were studied in a Japanese cohort of AD cases with age-matched controls.
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MATERIALS AND METHODS
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Sporadic AD cases (n = 243, ratio of men to women = 106:137) were obtained from the in-/outpatients of the hospitals where the authors work. All the AD cases were diagnosed according to National Institute of Neurological and Communication Disorders and Stroke–Alzheimer's Disease and Related Disorders Association (NINCDS–ADRDA) criteria. The controls (n = 130, ratio of men to women = 56:74) were obtained from healthy volunteers with no history of neuropsychiatric diseases or dementia. The mean age of the AD group (69.4 years, standard deviation 9.9, range: 38–90) was not significantly different from that of the control group (70.3 years, standard deviation 9.0, range: 51–93). Japanese samples are genetically homogenous, and the collected case–control samples are geographically matched. The purpose and significance of this study was explained in detail to each patient and family, and all participants provided their written informed consent. The study protocol was approved by the Ethics committee of the Juntendo University School of Medicine.
Genomic DNA was extracted from white blood cells of peripheral bloods by using the Nucleon II kit (Scott Lab Bioscience, U.K.). Information on the SNPs of each gene was derived from the SNP database (dbSNP) established by the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/SNP/). The polymorphisms of the Notch4 gene were studied by a polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analysis method (Table 1). The distribution of genotypes between the AD cases and their controls were compared using chi-square tests (a p value of <.05 was considered statistically significant). For SNP rs2071282, Fisher's exact test was performed. To assess linkage disequilibrium (LD) between each SNP, the estimate haplotype frequencies (EH) program (http://linkage.rockefeller.edu/ott/eh.htm) was used.
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RESULTS
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The distribution of the three polymorphisms is shown in Table 2. Logistic regression tests have shown that there were no associations among age, sex, and each polymorphism. There was no significant difference in genotypic distribution for any polymorphism (rs367398: df = 2, 
2 = 0.24, p =.89; rs2071282: df = 2, 2 = 0.0006, p =.99; rs422951: df = 2, 2 = 0.14, p =.93) between the AD participants and their controls. Analysis of the distribution of the allele of three SNPs showed negative results (rs367398: df = 1, 2 = 0.10, p =.81; rs2071282: df = 1, 2 = 0.0001, p =.99; rs422951: df = 1, 2 = 0.03, p =.93). Strong LD was shown between rs367398 and rs422951 in our Japanese participants (p <.001). LD between rs367398 and rs2071282 (p <.001) was weaker than LD between rs367398 and rs422951. Two SNPs, rs2071282 and 422951, were also in weak LD in Japanese samples (p <.02).
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DISCUSSION
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To the best our knowledge, this is the first follow-up study evaluating the association between SNPs of the Notch4 gene and AD. Our results indicate that the three SNPs of the Notch4 gene did not alter risk for AD in our Japanese cohort. In a previous genetic study, Lambert and colleagues (8) analyzed two SNPs, rs387071 (in the promoter region) and rs367398. They reported an increased risk for AD associated with the C/C genotype of rs367398 in apolipoprotein E (Apo E) 4 bearers. In the present study, logistic regression analysis by Apo E 4 allele were performed to each SNP of the Notch4 gene, and suggested no associations between any of the Notch4 SNPs and the Apo E 4 allele (data not shown). The discrepancy between their results and ours might be attributed to the differences between ethnic groups or a low penetrance effect. Lambert and colleagues observed no relationship between rs367398 and amyloid ß loads. It is possible that a marker yet to be studied near this locus, and not rs367398 itself, might play a synergetic role with Apo E 4. We also confirmed that rs367398 was in strong LD with rs422951 in the AD and the control groups. Rs2071282 showed weaker LD with rs367398 and rs422951. Because it was suggested that there might be more variants between rs2071282 and rs367398, other SNPs around this genetic locus should be searched in other populations.
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Acknowledgments
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This study was funded in part by a High Technology Research Center Grant from the Japanese Ministry of Education, Culture, Sports, Science and Technology and by a research grant from the Japanese Ministry of Health, Labour and Welfare.
We are grateful for the technical assistance of K. Yamamoto and S. Itakura.
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Footnotes
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Decision Editor: LaDora V. Thompson, PhD
Received January 24, 2006
Accepted September 4, 2006
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References
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