Association of Polymorphisms in the Angiotensin-Converting Enzyme Gene with Alzheimer Disease in an Israeli Arab Community
2006; Elsevier BV; Volume: 78; Issue: 5 Linguagem: Inglês
10.1086/503687
ISSN1537-6605
AutoresYan Meng, Clinton T. Baldwin, Abdalla Bowirrat, Kristin Waraska, Rivka Inzelberg, Robert P. Friedland, Lindsay A. Farrer,
Tópico(s)Genetic Associations and Epidemiology
ResumoSeveral lines of evidence support for a role of angiotensin converting enzyme (ACE) in Alzheimer disease (AD). Most genetic studies have focused on an Alu insertion/deletion (I/D) polymorphism in the ACE gene (DCP1) and have yielded conflicting results. We evaluated the association between 15 single-nucleotide polymorphisms (SNPs) in DCP1, including the I/D variant, and AD in a sample of 92 patients with AD and 166 nondemented controls from an inbred Israeli Arab community. Although there was no evidence for association between AD and I/D, we observed significant association with SNPs rs4343 (P=.00001) and rs4351 (P=.01). Haplotype analysis revealed remarkably significant evidence of association with the SNP combination rs4343 and rs4351 (global P=7.5×10−7). Individuals possessing the haplotype "GA" (frequency 0.21 in cases and 0.01 in controls) derived from these SNPs had a 45-fold increased risk of developing AD (95% CI 6.0–343.2) compared with those possessing any of the other three haplotypes. Longer range haplotypes including I/D were even more significant (lowest global P=1.1×10−12), but the only consistently associated alleles were in rs4343 and rs4351. These results suggest that a variant in close proximity to rs4343 and rs4351 modulates susceptibility to AD in this community. Several lines of evidence support for a role of angiotensin converting enzyme (ACE) in Alzheimer disease (AD). Most genetic studies have focused on an Alu insertion/deletion (I/D) polymorphism in the ACE gene (DCP1) and have yielded conflicting results. We evaluated the association between 15 single-nucleotide polymorphisms (SNPs) in DCP1, including the I/D variant, and AD in a sample of 92 patients with AD and 166 nondemented controls from an inbred Israeli Arab community. Although there was no evidence for association between AD and I/D, we observed significant association with SNPs rs4343 (P=.00001) and rs4351 (P=.01). Haplotype analysis revealed remarkably significant evidence of association with the SNP combination rs4343 and rs4351 (global P=7.5×10−7). Individuals possessing the haplotype "GA" (frequency 0.21 in cases and 0.01 in controls) derived from these SNPs had a 45-fold increased risk of developing AD (95% CI 6.0–343.2) compared with those possessing any of the other three haplotypes. Longer range haplotypes including I/D were even more significant (lowest global P=1.1×10−12), but the only consistently associated alleles were in rs4343 and rs4351. These results suggest that a variant in close proximity to rs4343 and rs4351 modulates susceptibility to AD in this community. Alzheimer disease (AD [MIM #104300]) is a progressive, neurodegenerative disease characterized clinically by gradual loss of memory and pathologically by neurofibrillary tangles and amyloid plaques in the brain. Currently, the apolipoprotein E (APOE [MIM +107741]) ɛ4 allele is the only broadly recognized genetic risk factor for late-onset AD (LOAD) in most populations.1Farrer LA Cupples LA Haines JL Hyman BT Kukull WA Mayeux R Pericak-Vance MA Risch N van Duijn CM APOE and Alzheimer Disease Meta Analysis Consortium Effects of age, gender and ethnicity on the association of apolipoprotein E genotype and Alzheimer disease.JAMA. 1997; 278: 1349-1356Crossref PubMed Google Scholar Much attention has been focused on the connection between angiotensin I converting enzyme (ACE [MIM +106180]) and AD. ACE is a dipeptidyl carboxypeptidase that plays an important role in regulation of blood pressure by converting angiotensin I to biologically active angiotensin II. Several studies show that an Alu insertion/deletion (I/D) polymorphism in the ACE gene (symbol DCP1) is associated with plasma level of ACE,2Rigat B Hubert C Alhenc-Gelas F Cambien F Corvol P Soubrier F An insertion/deletion polymorphism in the angiotensin I-converting enzyme gene accounting for half the variance of serum enzyme levels.J Clin Invest. 1990; 86: 1343-1346Crossref PubMed Scopus (3387) Google Scholar, 3Tiret L Rigat B Visvikis S Breda C Corvol P Cambien F Soubrier F Evidence, from combined segregation and linkage analysis, that a variant of the angiotensin I-converting enzyme (ACE) gene controls plasma ACE levels.Am J Hum Genet. 1992; 51: 197-205PubMed Google Scholar although the genetic regulation of ACE levels in the brain is poorly understood. Studies showing association between the I/D polymorphism and cardiovascular disease risk4Samani NJ Thompson JR O'Toole L Channer K Woods KL A meta-analysis of the association of the deletion allele of the angiotensinconverting enzyme gene with myocardial infarction.Circulation. 1996; 94: 708-712Crossref PubMed Scopus (424) Google Scholar, 5Staessen JA Wang JG Ginocchio G Petrov V Saavedra AP Soubrier F Vlietinck R Fagard R The deletion/insertion polymorphism of the angiotensin converting enzyme gene and cardiovascular-renal risk.J Hypertens. 1997; 15: 1579-1592Crossref PubMed Scopus (397) Google Scholar, 6Sharma P Meta-analysis of the ACE gene in ischaemic stroke.J Neurol Neurosurg Psychiatry. 1998; 64: 227-230Crossref PubMed Scopus (156) Google Scholar, 7Keavney B McKenzie C Parish S Palmer A Clark S Youngman L Delepine M Lathrop M Peto R Collins R Large-scale test of hypothesised associations between the angiotensinconverting-enzyme insertion/deletion polymorphism and myocardial infarction in about 5000 cases and 6000 controls.Lancet. 2000; 355: 434-442Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar, 8Sayed-Tabatabaei FA Houwing-Duistermaat JJ van Duijn CM Witteman JC Angiotensin-converting enzyme gene polymorphism and carotid artery wall thickness: a meta analysis.Stroke. 2003; 34: 1634-1639Crossref PubMed Scopus (85) Google Scholar and evidence suggesting cardiovascular risk factors promote AD9Skoog I Lernfelt B Landahl S Palmertz B Andreasson LA Nilsson L Persson G Oden A Svanborg A 15-year longitudinal study of blood pressure and dementia.Lancet. 1996; 347: 1141-1145Crossref PubMed Scopus (869) Google Scholar, 10Snowdon DA Greiner LH Mortimer JA Riley KP Greiner PA Markesbery WR Brain infarction and the clinical expression of Alzheimer disease: The Nun Study.JAMA. 1997; 277: 813-817Crossref PubMed Google Scholar, 11Kivipelto M Helkala EL Laakso MP Hanninen T Hallikainen M Alhainen K Soininen H Tuomilehto J Nissinen A Midlife vascular risk factors and Alzheimer's disease in later life: longitudinal, population based study.Brit Med J. 2001; 322: 1447-1451Crossref PubMed Scopus (1209) Google Scholar are consistent with the idea that ACE might play a role in AD via a cardiovascular mechanism. However, the observation that ACE degrades Aβ, the pathological hallmark of AD, in vitro12Hu J Igarashi A Kamata M Nakagawa H Angiotensin-converting enzyme degrades Alzheimer amyloid beta-peptide (A beta) retards A beta aggregation, deposition, fibril formation; and inhibits cytotoxicity.J Biol Chem. 2001; 276: 47863-47868Crossref PubMed Scopus (301) Google Scholar, 13Hemming ML Selkoe DJ Amyloid beta-protein is degraded by cellular angiotensin-converting enzyme (ACE) and elevated by an ACE inhibitor.J Biol Chem. 2005; 280: 37644-37650Crossref PubMed Scopus (252) Google Scholar, 14Oba R Igarashi A Kamata M Nagata K Takano S Nakagawa H The N-terminal active centre of human angiotensin-converting enzyme degrades Alzheimer amyloid beta-peptide.Eur J Neurosci. 2005; 21: 733-740Crossref PubMed Scopus (114) Google Scholar suggests that variation in DCP1 may directly modulate susceptibility to AD. Nearly all investigations of the association between DCP1 and AD have examined the I/D polymorphism. Of the published reports on 41 independent samples,15Kolsch H Jessen F Freymann N Kreis M Hentschel F Maier W Heun R ACE I/D polymorphism is a risk factor of Alzheimer's disease but not of vascular dementia.Neurosci Lett. 2005; 377: 37-39Crossref PubMed Scopus (55) Google Scholar, 16Sleegers K den Heijer T van Dijk EJ Hofman A Bertoli-Avella AM Koudstaal PJ Breteler MM van Duijn CM ACE gene is associated with Alzheimer's disease and atrophy of hippocampus and amygdala.Neurobiol Aging. 2005; 26: 1153-1159Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 17Lehmann DJ Cortina-Borja M Warden DR Smith AD Sleegers K Prince JA van Duijn CM Kehoe PG Large meta-analysis establishes the ACE insertion-deletion polymorphism as a marker of Alzheimer's disease.Am J Epidemiol. 2005; 162: 305-317Crossref PubMed Scopus (165) Google Scholar 11 showed significant association with the I allele, 1 showed significant association with the D allele, and 29 found no association with this marker. These conflicting results prompted four meta-analyses of ACE studies, which considered 39 samples published before September 2004.17Lehmann DJ Cortina-Borja M Warden DR Smith AD Sleegers K Prince JA van Duijn CM Kehoe PG Large meta-analysis establishes the ACE insertion-deletion polymorphism as a marker of Alzheimer's disease.Am J Epidemiol. 2005; 162: 305-317Crossref PubMed Scopus (165) Google Scholar, 18Kehoe PG Katzov H Feuk L Bennet AM Johansson B Wiman B de Faire U Cairns NJ Wilcock GK Brookes AJ Blennow K Prince JA Haplotypes extending across ACE are associated with Alzheimer's disease.Hum Mol Genet. 2003; 12: 859-867Crossref PubMed Scopus (109) Google Scholar, 19Narain Y Yip A Murphy T Brayne C Easton D Evans JG Xuereb J Cairns N Esiri MM Furlong RA Rubinsztein DC The ACE gene and Alzheimer's disease susceptibility.J Med Genet. 2000; 37: 695-697Crossref PubMed Scopus (105) Google Scholar, 20Elkins JS Douglas VC Johnston SC Alzheimer disease risk and genetic variation in ACE: a meta-analysis.Neurology. 2004; 62: 363-868Crossref PubMed Scopus (98) Google Scholar Two SNPs in the DCP1 promoter region (rs4291 and rs1800764) and one synonymous coding SNP (rs4343) proximate to I/D have been associated with AD in a combined sample of four case-control samples from Sweden and the United Kingdom.18Kehoe PG Katzov H Feuk L Bennet AM Johansson B Wiman B de Faire U Cairns NJ Wilcock GK Brookes AJ Blennow K Prince JA Haplotypes extending across ACE are associated with Alzheimer's disease.Hum Mol Genet. 2003; 12: 859-867Crossref PubMed Scopus (109) Google Scholar The rs4343 A allele was associated with both risk and age at onset of AD.21Kehoe PG Katzov H Andreasen N Gatz M Wilcock GK Cairns NJ Palmgren J de Faire U Brookes AJ Pedersen NL Blennow K Prince JA Common variants of ACE contribute to variable age-at-onset of Alzheimer's disease.Hum Genet. 2004; 114: 478-483Crossref PubMed Scopus (37) Google Scholar In this study, we evaluated the association between DCP1 and AD in Wadi Ara, an Israeli Arab community with a high prevalence of AD.22Bowirrat A Treves TA Friedland RP Korczyn AD Prevalence of Alzheimer's type dementia in an elderly Arab population.Eur Neurol. 2001; 8: 119-123Crossref Scopus (60) Google Scholar A total of 92 individuals meeting DSM-IV criteria for AD23McKhann G Drachman D Folstein M Katzman R Price D Stadlan EM Clinical diagnosis of Alzheimer's disease: report of the NINCDS-ADRDA Work Group under the auspices of Department of Health and Human Services Task Force on Alzheimer's Disease.Neurology. 1984; 34: 939-944Crossref PubMed Google Scholar and 166 nondemented controls aged 60 and older were included in this study. Although most members of this community of >81,500 people trace their ancestors to ∼14 founder families, investigation of family history by use of multiple informants (since genealogical records do not exist) revealed that most subjects belonged to distinct multigenerational pedigrees. Thus, for analytical purposes, this group was treated as a case-control sample. A detailed description of subject ascertainment and evaluation is provided elsewhere.24Farrer LA Bowirrat A Friedland RP Waraska K Korczyn AD Baldwin CT Identification of multiple loci for Alzheimer disease in a consanguineous Israeli-Arab Community.Hum Mol Genet. 2003; 12: 415-422Crossref PubMed Scopus (116) Google Scholar We profiled the sample for the I/D polymorphism and 21 SNPs spanning a 575.53-kb region including the 5′ and 3′ portions of DCP1 which were selected from public databases, primarily dbSNP. Four nonsynonymous coding SNPs (rs4317, rs4318, rs4364, and rs4976) were almost completely monomorphic. Assays for SNPs rs4342 and rs13030 were unsuccessful. SNPs available for the statistical analyses include 11 within the coding region, 2 within the promoter region, and 2 in the flanking regions (see table 1). Genotyping was perfomed either by the MassARRAY Homogenous MassEXTEND (hME) Assay (Sequenom) or by TaqMan SNP Genotyping Assays (Applied Biosystems).Table 1Characteristics of DCP1 Markers and Their Association with ADMinor Allele FrequencyMarker NameMap PositionLocation or TypeMinor AlleleCasesControlsPrs151877258,679,848Upstream of 5′ UTRT.45.40.36rs180076458,904,261Promoter SNPA.37.39.57rs429158,907,926Promoter SNPT.36.33.56rs429558,910,030IntronC.42.39.61rs431158,914,495IntronC.47.53.26rs432958,917,190IntronG.27.33.15rs433558,918,757IntronA.43.45.75Alu I/D58,919,636IntronI.27.31.44rs434358,919,763T776TG.42.18.00001rs435158,923,464IntronA.44.32.01rs435358,924,154IntronG.38.51NTaNT = not tested (see text).rs436258,927,493F1129FC.49.40.06rs457559558,930,947IntronA.37.30.18rs426738558,937,488IntronC.30.31.73rs89440759,255,373Downstream of 3′ UTRA.35.41.30a NT = not tested (see text). Open table in a new tab Analyses of individual SNPs were performed using SAS software, release 9.1. A χ2 test was used to determine whether the genotype distribution in control subjects conformed to Hardy-Weinberg equilibrium. Genotype and allele frequencies were compared between cases and controls by χ2 analysis. Fisher's exact test was used when the expected frequency of one or more cells was too small for the χ2 test. Differences were considered significant if the P value was ≤.05. A Bonferroni correction was applied to results of analyses with individual SNPs. The linkage disequilibrium (LD) structure among SNPs was examined with the program Haploview.25Barrett JC Fry B Maller J Daly MJ Haploview: analysis and visualization of LD and haplotype maps.Bioinformatics. 2005; 21: 263-265Crossref PubMed Scopus (11431) Google Scholar Haplotype blocks were defined using an algorithm which created 95% confidence boundaries on D′ to define SNP pairs in strong LD. Haplotype analysis was carried out using Haplo.stats v1.1.1.26Schaid DJ Rowland CM Tines DE Jacobson RM Poland GA Score tests for association between traits and haplotypes when linkage phase is ambiguous.Am J Hum Genet. 2002; 70: 425-434Abstract Full Text Full Text PDF PubMed Scopus (1538) Google Scholar Haplotypes were considered significant if the empirical global P value was below .05. Of the 15 polymorphic DCP1 markers, only the genotypes for rs4353 were not in Hardy-Weinberg equilibrium in controls and therefore excluded from further analysis. The remaining panel of 14 markers displayed variable and often weak intermarker linkage disequilibrium (LD) (fig. 1). SNPs rs1518772 and rs894407 are respectively located in the genes immediately proximal (TANC2) and distal (FTJS3) to DCP1, and are not in LD with any of the DCP1 intragenic markers. The LD block structure derived from this sample of AD cases and controls from Wadi Ara is nearly identical to the structure obtained for the Caucasian sample in the HapMap project27The International HapMap Consortium The International HapMap Project.Nature. 2003; 426: 789-796Crossref PubMed Scopus (4688) Google Scholar although not all SNPs in this study are included in HapMap. Analysis of individual markers revealed significant disease association with SNPs rs4343 and rs4351 (table 1). The allele frequency difference between cases and controls for rs4343 remained significant after correcting for multiple testing (adjusted P=.0002). To assess whether a particular marker profile including rs4343 accounts for or clarifies the association with AD, we first performed haplotype analysis of all marker pair combinations including rs4343. The most significant haplotypes included rs4343 and rs4351 (data not shown). Next, to assess whether adding more markers would significantly modify the effect, we evaluated 3–5 marker haplotypes, including these two SNPs using a sliding-window approach. Compared to the haplotype containing only rs4343 and rs4351 (global P=7.5×10−7), noticeable improvement was obtained by extending the haplotypes in the 5′ direction (most significant global P=1.1×10−12), however, the same alleles in these proximal SNPs (including the I/D polymorphism) were present in both the risk and protective haplotypes suggesting that the source of the effect on AD risk is more proximate to rs4343 and rs4351 than to the I/D site (table 2). Close examination of the haplotype containing rs4343 and rs4351 showed a significant enrichment of the haplotype G-A in AD cases (21%) compared to controls (1%). Subjects possessing this haplotype versus all other rs4343–rs4351 haplotypes had 45-fold increased risk of developing AD (table 3). Haplotype analyses of DCP1 using sliding windows excluding rs4343 showed unremarkable results.Table 2Association of AD with Haplotypes Containing rs4343 and rs4351Haplotypers4329rs4335Alu I/Drs4343rs4351rs4362rs4575595FrequencyPGlobalPaGlobal P value is based on comparison of frequency distribution of all haplotypes for the combination of SNPs indicated among cases and controls.GA.104.0×10−87.5×10−7DGA.101.8×10−91.6×10−11GAC.102.8×10−96.4×10−7GDGA.111.5×10−93.9×10−12DGAC.101.4×10−105.9×10−10GACA.083.5×10−84.0×10−5AGDGA.101.1×10−101.1×10−12GDGAC.101.4×10−102.5×10−10DGACA.081.0×10−91.9×10−10AG.435.3×10−47.5×10−7DAG.327.8×10−88.0×10−13GDAG.226.6×10−103.9×10−12Note.—Protective haplotypes are shaded, and risk haplotypes are not.a Global P value is based on comparison of frequency distribution of all haplotypes for the combination of SNPs indicated among cases and controls. Open table in a new tab Table 3Risk of AD Associated with rs4343 and rs4351 HaplotypesHaplotypeFrequencyrs4343rs4351CasesControlsOdds Ratio (95% CI)PAA.23.32GG.20.18AG.35.50GA.21.0145.2 (5.95–343.23)3.4×10−9Note.—The first three haplotypes shown are the reference group. Open table in a new tab Note.— Protective haplotypes are shaded, and risk haplotypes are not. Note.— The first three haplotypes shown are the reference group. The unusually high prevalence of AD in and the consanguineous nature of Wadi Ara make this population attractive for investigating AD susceptibility genes. Remarkably, AD is not associated with APOE because the frequency of the ɛ4 allele is very low in both nondemented (2.4%) and demented elders (3.6%).28Bowirrat A Friedland RP Chapman J Korczyn AD The very high prevalence of Alzheimer's disease in an Arab population is not explained by the APOE e4 allele frequency.Neurology. 2000; 55: 731Crossref PubMed Scopus (34) Google Scholar Previously, we carried out an unconventional yet efficient 10-cM genome scan, using a small sample of cases and controls from this community, and confirmed the existence and narrowed substantially the locations of previously reported AD loci on chromosomes 9, 10, and 12.24Farrer LA Bowirrat A Friedland RP Waraska K Korczyn AD Baldwin CT Identification of multiple loci for Alzheimer disease in a consanguineous Israeli-Arab Community.Hum Mol Genet. 2003; 12: 415-422Crossref PubMed Scopus (116) Google Scholar In this study, we observed in this population significant evidence of association with two adjacent polymorphisms (rs4343 and rs4351) in the DCP1 gene and AD. One of these markers (rs4343) is located 127 bp from an Alu I/D polymorphism which has been reported to be associated with AD in some but not all studies.17Lehmann DJ Cortina-Borja M Warden DR Smith AD Sleegers K Prince JA van Duijn CM Kehoe PG Large meta-analysis establishes the ACE insertion-deletion polymorphism as a marker of Alzheimer's disease.Am J Epidemiol. 2005; 162: 305-317Crossref PubMed Scopus (165) Google Scholar, 18Kehoe PG Katzov H Feuk L Bennet AM Johansson B Wiman B de Faire U Cairns NJ Wilcock GK Brookes AJ Blennow K Prince JA Haplotypes extending across ACE are associated with Alzheimer's disease.Hum Mol Genet. 2003; 12: 859-867Crossref PubMed Scopus (109) Google Scholar, 19Narain Y Yip A Murphy T Brayne C Easton D Evans JG Xuereb J Cairns N Esiri MM Furlong RA Rubinsztein DC The ACE gene and Alzheimer's disease susceptibility.J Med Genet. 2000; 37: 695-697Crossref PubMed Scopus (105) Google Scholar, 20Elkins JS Douglas VC Johnston SC Alzheimer disease risk and genetic variation in ACE: a meta-analysis.Neurology. 2004; 62: 363-868Crossref PubMed Scopus (98) Google Scholar Notably, I/D is not associated in this population,29Bowirrat A Cui J Waraska K Friedland RP Oscar-Berman M Farrer LA Korczyn A Baldwin CT Lack of association between angiotensin-converting enzyme (ACE) and dementia of the Alzheimer's type (DAT) in an elderly Arab population in Wadi Ara, Israel.Neuropsych Dis Treatment. 2005; 1: 73-76Crossref PubMed Google Scholar despite the fact that it is in strong LD (D′=1) with rs4343 (fig. 1) and that markers encompassing this region are in the same LD block in the African, Asian, and European ancestry samples included in the HapMap project.27The International HapMap Consortium The International HapMap Project.Nature. 2003; 426: 789-796Crossref PubMed Scopus (4688) Google Scholar However, the findings that the minor allele frequencies differ markedly (table 1) and the correlation of alleles at these two sites is low (r2=0.22; see fig. 1) are consistent with the observation that AD is associated with only one of two polymorphisms in very close proximity to each other. Explanations for this phenomenon include recent admixture, local variation in recombination rates, gene conversion, and small chromosomal inversions.30Pritchard JK Przeworski M Linkage disequilibrium in humans: models and data.Am J Hum Genet. 2001; 69: 1-14Abstract Full Text Full Text PDF PubMed Scopus (860) Google Scholar, 31Jeffreys AJ Neumann R Panayi M Myers S Donnelly P Human recombination hot spots hidden in regions of strong marker association.Nat Genet. 2005; 37: 601-606Crossref PubMed Scopus (143) Google Scholar The observed genetic association between DCP1 and AD is unlikely to be a consequence of population stratification, because the individuals in our sample descended from a small number of founders in a community which until recently was genetically isolated. It is also possible that the significance of our results is overestimated because we were unable to account for extended familial relationships. However, the impact of population structure on conclusions from genetic association analyses in a genetic isolate, which has been investigated in the context of a genomewide scan,32Newman DL Abney M McPeek MS Ober C Cox NJ The importance of genealogy in determining genetic associations with complex traits.Am J Hum Genet. 2001; 69: 1146-1148Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar is probably minor in this study because we tested a prior hypothesis and obtained an extraordinarily significant result. It is also unlikely that the association between AD and DCP1 is due to LD with pathogenic SNPs in a neighboring gene, because the SNPs associated with AD were located within the central portion of DCP1, whereas SNPs in the flanking genes TANC2 and FTSJ3, which are in haplotype blocks different from DCP1, showed no association. Although the association between AD and the I/D polymorphism has been intensively scrutinized, few studies have examined other variants in DCP1 in the context of AD susceptibility. Kehoe et al. evaluated eight DCP1 markers in three late-onset AD and one early-onset AD case-control data sets.18Kehoe PG Katzov H Feuk L Bennet AM Johansson B Wiman B de Faire U Cairns NJ Wilcock GK Brookes AJ Blennow K Prince JA Haplotypes extending across ACE are associated with Alzheimer's disease.Hum Mol Genet. 2003; 12: 859-867Crossref PubMed Scopus (109) Google Scholar Five of these markers (rs1800764, rs4291, I/D, rs4343, and rs4362) were common to our study, and all were contained within the region of our SNP panel. None of the markers was significantly associated with AD in any of the individual data sets; however, significant evidence was obtained for rs1800764, rs4291, and rs4343 in the combined data sets composed of samples from Sweden, England, and Scotland. Curiously, the most significant result from this previous study was obtained with the promoter SNP rs4291 (P=2×10−5), which was derived from a weighted odds ratio comparing AA+AT versus TT subjects. We did not detect association with this SNP or its immediate neighbors, rs1800764 and rs4295, which are located 3,665 bp upstream and 2,104 bp downstream, respectively. Moreover, in the Kehoe et al. study, rs4343 exhibited relatively weak association with AD, and the pattern of association was opposite to that observed in the Wadi Ara sample. These conflicting results might reflect that rs4343 is in tight LD with another variant with pathogenic alleles or, as has been suggested,33Cox R Bouzekri N Martin S Southam L Hugill A Golamaully M Cooper R Adeyemo A Soubrier F Ward R Lathrop GM Matsuda F Farrall M Angiotensin-1-converting enzyme (ACE) plasma concentration is influenced by multiple ACE-linked quantitative trait nucleotides.Hum Mol Genet. 2002; 11: 2969-2977Crossref PubMed Google Scholar that there exist pathogenic alleles at multiple locations within DCP1. Our haplotype analysis suggests that the functional variant responsible for the AD association in this population is located distal to I/D and proximate to rs4343 and rs4351. This region of the ACE gene is shared by all three transcript isoforms. Because Alu insertions can be associated with alternative splicing, many studies have suggested that the I/D polymorphism may explain differences in ACE plasma levels. However, recent in vitro minigene studies have shown that the I/D polymorphism and rs4343 do not affect alternative splicing of DCP1.34Lei H Day IN Vorechovsky I Exonization of AluYa5 in the human ACE gene requires mutations in both 3′ and 5′ splice sites and is facilitated by a conserved splicing enhancer.Nucleic Acids Res. 2005; 33: 3897-3906Crossref PubMed Scopus (24) Google Scholar Our in silico examination of the rs4351 using the program RESCUE-ESE35Fairbrother WG Yeh RF Sharp PA Burge CB Predictive identification of exonic splicing enhancers in human genes.Science. 2002; 297: 1007-1013Crossref PubMed Scopus (817) Google Scholar suggests that it could affect alternative splicing; however, this has not been demonstrated in vitro or in vivo. There are no common sequence variants between rs4343 and rs4351 that have obvious pathogenic alleles. It is possible that rs4343, rs4351, or other SNPs in LD with these SNPS affect alternative isoform production, tissue specificity, or ACE activity for substrates (such as Aβ) that have not been studied extensively. This may explain the controversial evidence of association between I/D and other SNPs in this region with disease. In summary, our results indicate that a genetic variant near rs4343 and rs4351 has a major influence on AD susceptibility in the Wadi Ara community. In view of (1) the preponderance of data supporting a genetic association between DCP1 and AD, (2) evidence showing association between CSF Aβ42 levels and both APOE genotype36Galasko D Chang L Motter R Clark CM Kaye J Knopman D Thomas R Kholodenko D Schenk D Lieberburg I Miller B Green R Basherad R Kertiles L Boss MA Seubert P High cerebrospinal fluid tau and low amyloid beta42 levels in the clinical diagnosis of Alzheimer disease and relation to apolipoprotein E genotype.Arch Neurol. 1998; 55: 937-945Crossref PubMed Scopus (398) Google Scholar and DCP1 haplotypes,18Kehoe PG Katzov H Feuk L Bennet AM Johansson B Wiman B de Faire U Cairns NJ Wilcock GK Brookes AJ Blennow K Prince JA Haplotypes extending across ACE are associated with Alzheimer's disease.Hum Mol Genet. 2003; 12: 859-867Crossref PubMed Scopus (109) Google Scholar (3) the observation of significantly smaller hippocampal and amygdalar volumes in women homozygous for the DCP1 I allele independent of vascular factors,16Sleegers K den Heijer T van Dijk EJ Hofman A Bertoli-Avella AM Koudstaal PJ Breteler MM van Duijn CM ACE gene is associated with Alzheimer's disease and atrophy of hippocampus and amygdala.Neurobiol Aging. 2005; 26: 1153-1159Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar and (4) the growing recognition that vascular factors including inflammation contribute to the development of AD,37Gorelick PB Risk factors for vascular dementia and Alzheimer disease.Stroke. 2004; 35: 2620-2622Crossref PubMed Scopus (264) Google Scholar, 38Scacchi R Ruggeri M Gambina G Martini MC Ferrari G Corbo RM Plasma alpha1-antichymotrypsin in Alzheimer's disease; relationships with APOE genotypes.Neurobiol Aging. 2001; 22: 413-416Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 39Manelli AM Stine WB Van Eldik LJ LaDu MJ ApoE and Abeta1-42 interactions: effects of isoform and conformation on structure and function.J Mol Neurosci. 2004; 23: 235-246Crossref PubMed Scopus (79) Google Scholar, 40Rozemuller AJ van Gool WA Eikelenboom P The neuroinflammatory response in plaques and amyloid angiopathy in Alzheimer's disease: therapeutic implications.Curr Drug Targets CNS Neurol Disord. 2005; 4: 223-233Crossref PubMed Scopus (77) Google Scholar, 41Das UN Is angiotensin-II an endogenous pro-inflammatory molecule?.Med Sci Monit. 2005; 11: RA155-RA162PubMed Google Scholar multiple pathways linking APOE and ACE to AD risk can be proposed (fig. 2). Increased plasma levels of particular ACE and APOE isoforms may lead to increased production of the toxic form of Aβ (Aβ42).12Hu J Igarashi A Kamata M Nakagawa H Angiotensin-converting enzyme degrades Alzheimer amyloid beta-peptide (A beta) retards A beta aggregation, deposition, fibril formation; and inhibits cytotoxicity.J Biol Chem. 2001; 276: 47863-47868Crossref PubMed Scopus (301) Google Scholar, 13Hemming ML Selkoe DJ Amyloid beta-protein is degraded by cellular angiotensin-converting enzyme (ACE) and elevated by an ACE inhibitor.J Biol Chem. 2005; 280: 37644-37650Crossref PubMed Scopus (252) Google Scholar, 14Oba R Igarashi A Kamata M Nagata K Takano S Nakagawa H The N-terminal active centre of human angiotensin-converting enzyme degrades Alzheimer amyloid beta-peptide.Eur J Neurosci. 2005; 21: 733-740Crossref PubMed Scopus (114) Google Scholar Alternatively, ACE and APOE might influence AD risk through action on vascular risk factors (including blood pressure, lipids, and the endothelium) that alter metabolism of Aβ or perhaps through another factor leading to inflammatory response associated with AD.41Das UN Is angiotensin-II an endogenous pro-inflammatory molecule?.Med Sci Monit. 2005; 11: RA155-RA162PubMed Google Scholar, 42Sjogren M Blennow K The link between cholesterol and Alzheimer's disease.World J Biol Psychiatry. 2005; 6: 85-97Crossref PubMed Scopus (38) Google Scholar Our hypothesis predicts that the DCP1/AD association may be more evident in populations like Wadi Ara showing a weak influence of APOE genotype. Studies of DCP1 and APOE in other populations will help to distinguish and elaborate these pathways. This work was supported by grants from Fonds de la Recherche en Santé, the National Institutes of Health (U01-AG17173, R01-AG09029, and P30-AG13846), the Institute for the Study of Aging, the GOJO Corp (Akron, OH), the Fullerton Family Charitable Trust, the Nickman family, and the Florence and Joseph Mandel Research Fund. Accession numbers and URLs for data presented herein are as follows: Daniel J. Schaid's Web site, http://mayoresearch.mayo.edu/mayo/research/biostat/schaid.cfm (for Haplo.stats v1.1.1) dbSNP, http://www.ncbi.nlm.nih.gov/projects/SNP/ (primary source for SNP information) International HapMap Project, http://www.hapmap.org/ Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/Omim/ (for AD, APOE, and ACE) RESCUE-ESE Web Server, http://genes.mit.edu/burgelab/rescue-ese/
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