Genome-wide haplotype association study identifies the FRMD4A gene as a risk locus for Alzheimer's disease.

Recently, several genome-wide association studies (GWASs) have led to the discovery of nine new loci of genetic susceptibility in Alzheimer's disease (AD). However, the landscape of the AD genetic susceptibility is far away to be complete and in addition to single-SNP (single-nucleotide polymorphism) analyses as performed in conventional GWAS, complementary strategies need to be applied to overcome limitations inherent to this type of approaches. We performed a genome-wide haplotype association (GWHA) study in the EADI1 study (n=2025 AD cases and 5328 controls) by applying a sliding-windows approach. After exclusion of loci already known to be involved in AD (APOE, BIN1 and CR1), 91 regions with suggestive haplotype effects were identified. In a second step, we attempted to replicate the best suggestive haplotype associations in the GERAD1 consortium (2820 AD cases and 6356 controls) and observed that 9 of them showed nominal association. In a third step, we tested relevant haplotype associations in a combined analysis of five additional case-control studies (5093 AD cases and 4061 controls). We consistently replicated the association of a haplotype within FRMD4A on Chr.10p13 in all the data set analyzed (OR: 1.68; 95% CI: (1.43-1.96); P=1.1 × 10(-10)). We finally searched for association between SNPs within the FRMD4A locus and Aß plasma concentrations in three independent non-demented populations (n=2579). We reported that polymorphisms were associated with plasma Aß42/Aß40 ratio (best signal, P=5.4 × 10(-7)). In conclusion, combining both GWHA study and a conservative three-stage replication approach, we characterised FRMD4A as a new genetic risk factor of AD.

A polymorphism in the regulatory region of APOE associated with risk for Alzheimer's dementia.

The epsilon4 allele of the apolipoprotein E gene (APOE) has been associated with an increased risk of developing Alzheimer's disease (AD; refs 1,2). However, it is apparent that the APOEepsilon4 allele alone is neither necessary nor sufficient to cause the disease. We have recently found three new polymorphisms within the APOE transcriptional regulatory region (M.J.A. et al., manuscript submitted) and now establish an association between one of these polymorphisms (-491A/T) and dementia as observed in Alzheimer's disease, in two independent clinical populations. The results suggest that homozygosity of a common variant (-491A) is associated with increased risk for AD, and that this association is independent of APOEepsilon4 status. In vitro studies suggest that the -491A/T polymorphism may increase risk for AD by altering the level of ApoE protein expression.

APOE and Alzheimer disease: a major gene with semi-dominant inheritance.

Apolipoprotein E (APOE) dependent lifetime risks (LTRs) for Alzheimer Disease (AD) are currently not accurately known and odds ratios alone are insufficient to assess these risks. We calculated AD LTR in 7351 cases and 10 132 controls from Caucasian ancestry using Rochester (USA) incidence data. At the age of 85 the LTR of AD without reference to APOE genotype was 11% in males and 14% in females. At the same age, this risk ranged from 51% for APOE44 male carriers to 60% for APOE44 female carriers, and from 23% for APOE34 male carriers to 30% for APOE34 female carriers, consistent with semi-dominant inheritance of a moderately penetrant gene. Using PAQUID (France) incidence data, estimates were globally similar except that at age 85 the LTRs reached 68 and 35% for APOE 44 and APOE 34 female carriers, respectively. These risks are more similar to those of major genes in Mendelian diseases, such as BRCA1 in breast cancer, than those of low-risk common alleles identified by recent GWAS in complex diseases. In addition, stratification of our data by age groups clearly demonstrates that APOE4 is a risk factor not only for late-onset but for early-onset AD as well. Together, these results urge a reappraisal of the impact of APOE in Alzheimer disease.

Cholesterol dysregulation in peripheral blood mononuclear cells of Alzheimer's disease.

Cholesterol and the immune system are involved in Alzheimer's Disease (AD). To investigate the relations among them, we compared the cholesterol content in peripheral blood mononuclear cells (PBMC) of cognitively healthy controls and patients with mild cognitive impairment (MCI) and AD in two independent samples. Free cholesterol content of PBMC was lower in MCI and AD patients, and was modulated by APOE genotype. A decrease of CD8+ and an increase of CD16+ was also found in AD patients. These results suggest that cholesterol levels in PBMCs may represent an early signature of the disease and support the involvement of immune system in AD.

Alzheimer's amyloid precursor protein is expressed on the surface of hematopoietic cells upon activation.

A4-amyloid is the major component of senile plaques and neurofibrillary tangles found in the brain of patients suffering Alzheimer's disease. This 39-42 amino acid peptide is derived from a larger precursor protein (APP). Since APP gene encodes for a putative membrane protein, the study of APP expression at the cell surface may provide useful data for understanding its physiological function. In this report, we present data on APP expression, that was detected by APP specific mAbs in cells of the hematopoietic system. APP was weakly expressed on the cell surface of resting human lymphocytes and monocytes, but it could be induced to the surface of those cells upon stimulation. The cell activators capable of inducing APP membrane expression comprehended mitogenic lectins, calcium ionophores, phosphatase inhibitors, and anti mu-chain or anti-CD3 antibodies in B and T cells, respectively. Interestingly, phorbol esters were able to induce APP membrane expression in monocytic, but not in lymphoid cells. In contrast to lymphocytes and monocytes, granulocytes never expressed cell surface or cytoplasmic APP, even after the activation. The induction of membrane APP in response to lymphocyte activation signals, including antibodies to the antigen receptor of B and T cells, raises the possibility that APP might play the role of a cell surface receptor in the immune system.

Location of an epitope shared by Alzheimer's amyloid peptide and brain creatine kinase using a newly developed monoclonal antibody.

Amyloid plaques, composed mainly by a peptide termed A4-amyloid, derived by proteolytic processing from the amyloid precursor protein (APP), are a hallmark in the brain of Alzheimer's disease patients. We have prepared a collection of monoclonal antibodies as tools to study APP expression and proteolysis in different systems. One of these, 5AH10, raised against residues 9-22 of A4-peptide, was selected for its ability to recognize only A4 subpeptides having the intact APP-secretase target sequence, as well as whole recombinant APP. By using synthetic subpeptides, we have located 5AH10 epitope between amino acids 15 and 22 of A4. In addition, 5AH10 showed a strong immunoreactivity to a 47 kDa protein present in rat brain extracts, that was identified as the B (brain specific) subunit of creatine kinase by immunochemical data and direct N-terminal sequencing. The cross-reaction observed is most probably due to a high degree of sequence identity between amino acids 15 to 22 of A4 peptide and amino acids 9 to 16 of rat B creatine kinase. 5AH10 did not recognize the muscle specific isoform (M subunit) of rat creatine kinase, nor the B subunit of human and rabbit creatine kinase, suggesting that glutamine at first position of the epitope is essential for antigen recognition by 5AH10.

Proteolysis of Alzheimer's disease beta-amyloid precursor protein by factor Xa.

Amyloid beta-protein is a 4-kDa peptide which originates from proteolysis of a larger protein precursor (APP) and accumulates in senile plaques in brains of Alzheimer's disease (AD) patients. Since secreted APP inhibits factors IXa, Xa and XIa, and thrombin appears to play a role in APP secretion and proteolysis, a relationship between hemostasis system and APP metabolism seems to exist. In this work we investigate the susceptibility to proteolytic cleavage by factor Xa of a fusion construct containing full-length APP prepared in bacteria, and demonstrate that both APP695 and APP770 are substrates for this protease. Factor Xa was found to cleave APP after arginines 102, 268, 510, 573 and 601 (APP695 numeration); most of these sites appear to be common for different coagulation factors. In addition, APP incubation with factor Xa generates an array of six potentially amyloidogenic fragments. Comparative kinetic analysis of APP695 and APP770 cleavage by factor Xa suggests that Kunitz-type inhibitor-containing isoforms exert an inhibitory effect on the protease. However, this inhibition is far from complete even at a 5-fold molar excess of inhibitor. Our results raise the possibility that proteases from the coagulation cascade may contribute to APP proteolysis, and support the notion that these proteases play a role in AD pathogenesis.

Allelic polymorphisms in the transcriptional regulatory region of apolipoprotein E gene.

In this work, we explored the existence of genetic variants within the apolipoprotein E gene transcriptional regulatory region, using a denaturing gradient gel electrophoresis screening of a region comprising nucleotides -1017 to +406. Upon a population study, three new polymorphic sites (-491, -427 and -219) and two mutations were found. Functional effects of the polymorphisms, assayed by transient transfection and electrophoretic mobility shift assays in a human hepatoma cell line, showed that polymorphisms at sites -491 and -219 of the APOE promoter produce variations in the transcriptional activity of the gene, most probably through differential binding of nuclear proteins.

Contribution of APOE promoter polymorphisms to Alzheimer's disease risk.

OBJECTIVE: To determine whether the effects of APOE promoter polymorphisms on AD are independent of the APOE-epsilon4 allele. BACKGROUND: Recently, the -491 A-->T and -219 G-->T polymorphisms located in the APOE promoter have been suggested to be risk factors for AD. However, the effects of these polymorphisms have not always been reproduced in case-control studies, possibly because of the strong linkage disequilibrium existing at this locus or the characteristics of the populations studied. METHODS: Data collection was performed from six independent samples (1,732 patients with AD and 1,926 control subjects) genotyped for APOE exon 4 and the two APOE promoter polymorphisms. The risks associated with the APOE polymorphisms for developing AD were estimated using logistic regression procedures and calculation of odds ratios with 95% CI adjusted by age, sex, and collection center. Independence of the APOE promoter polymorphisms was tested by stratification for APOE-epsilon4 and tertile design was used for age stratification. RESULTS: The independence of the -491 AA genotype was observed in the whole sample whereas the independence of the -219 TT genotype was observed only in the oldest population. CONCLUSION: The -491 and -219 APOE promoter polymorphisms incur risk for AD in addition to risk associated with the APOE-epsilon4 allele, with age accentuating the effect of the -219 TT genotype. Because these polymorphisms appear to influence apoE levels, these results suggest that APOE expression is an important determinant of AD pathogenesis.

Antigenic structure of the E2 glycoprotein from transmissible gastroenteritis coronavirus.

The antigenic structure of transmissible gastroenteritis (TGE) virus E2 glycoprotein has been defined at three levels: antigenic sites, antigenic subsites and epitopes. Four antigenic sites (A, B, C and D) were defined by competitive radioimmunoassay (RIA) using monoclonal antibodies (MAbs) selected from 9 fusions. About 20% (197) of the hybridomas specific for TGE virus produced neutralizing MAbs specific for site A, which was one of the antigenically dominant determinants. Site A was differentiated in three antigenic subsites: a, b and c, by characterization of 11 MAb resistant (mar) mutants, that were defined by 8, 3, and 3 MAbs, respectively. These subsites were further subdivided in epitopes. A total of 11 epitopes were defined in E2 glycoprotein, eight of which were critical for virus neutralization. Neutralizing MAbs were obtained only when native virus was used to immunize mice, although to produce hybridomas mice immunizations were made with antigen in the native, denatured, or mixtures of native and denatured form. All neutralizing MAbs reacted to conformational epitopes. The antigenic structure of the E2-glycoprotein has been defined with murine MAbs, but the antigenic sites were relevant in the swine, the natural host of the virus, because porcine sera reacted against these sites. MAbs specific for TGE virus site C reacted to non-immune porcine sera. This reactivity was not directed against porcine immunoglobulins. These results indicated that TGE virus contains epitope(s) also present in some non-immunoglobulin component of porcine serum.

Cooperation between transmissible gastroenteritis coronavirus (TGEV) structural proteins in the in vitro induction of virus-specific antibodies.

Following infection of haplotype defined NIH-miniswine with virulent transmissible gastroenteritis coronavirus (TGEV), isolated mesenteric lymph node CD4+ T-cells mounted a specific proliferative response against infectious or inactivated purified virus in secondary in vitro stimulation. A specific, dose-dependent response to the three major recombinant viral proteins: spike (S), membrane (M), and nucleoprotein (N), purified by affinity chromatography, was characterized. Induction of in vitro antibody synthesis was analyzed. The purified recombinant viral proteins induced the in vitro synthesis of neutralizing TGEV-specific antibodies when porcine TGEV-immune cells were stimulated with each of the combinations made with two of the major structural proteins: S + N, S + M, and to a minor extent with M + N, but not by the individual proteins. S-protein was dissociated from purified virus using NP-40 detergent and then micellar S-protein oligomers (S-rosettes) were formed by removing the detergent. These occurred preferentially by the association of more than 10 S-protein trimmers. These S-rosettes in collaboration with either N or M-proteins elicited TGEV-specific antibodies with titers up to 84 and 60%, respectively, of those induced by the whole virus. N-protein could be partially substituted by a 15-mer peptide that represents a T helper epitope previously identified in N-protein (Antón et al. (1995)). These results indicate that the induction of high levels of TGEV-specific antibodies requires stimulation by at least two viral proteins, and that optimum responses are induced by a combination of S-rosettes and the nucleoprotein.

Apolipoprotein E gene promoter polymorphisms in Alzheimer's disease.

Alzheimer's disease, the most frequent form of senile dementia, presents in the vast majority of cases as a multifactorial trait, where a series of genetic and environmental risk factors converge. The increasing body of data, both epidemiological and functional, is strengthening the evidence that apolipoprotein E (APOE, gene; apoE, protein) is a true susceptibility factor for the onset of the common form of Alzheimer's disease. The E4 isoform of apoE remains to date as the main genetic risk factor for the disease, although the mechanisms responsible for this association are not well understood. It is also clear that apoE4 is not necessary or sufficient to cause the disease, indicating that other risk and protecting factors exist. ApoE is upregulated in response to nervous system injury, suggesting that it could have a neuroprotective role; on the other hand, there is evidence indicating that apoE is neurotoxic when present at high levels. Thus, apoE levels seem to be relevant for the functionality of the protein. The APOE proximal promoter hosts numerous regulatory elements, raising the possibility that polymorphisms in this region could produce variation in apoE levels by altering APOE transcriptional activity, which could finally result in AD susceptibility. We will review here the current evidence on the relationship between APOE proximal promoter polymorphisms, APOE gene transcriptional activity and apoE protein levels, and risk for AD.

Identification of a novel mutation (Leu282Arg) of the human presenilin 1 gene in Alzheimer's disease.

Many different mutations, causative of Alzheimer's disease, have been found in the presenilin-1 gene (PS-1). We have developed a screening method based on denaturing gradient gel electrophoresis (DGGE), which allows the mutational analysis of the whole exon 9 of PS-1. Upon the screening of a Spanish sample of early onset familial Alzheimer disease cases, we have found a novel mutation in the PS-1 gene. The mutation (a T to G transition) results in a change of the amino acid at position 282 of the presenilin protein from leucine to arginine. This mutation is located in the hydrophobic domain number 7 (exon 9) close to the site of physiological cleavage processing. The average of onset of the affected members of this family is 43+/-5 years, and the average age of exitus of affected members is 56+/-3 years. The possibility to determine the specific pathologic mechanisms of this mutation is now open.

A polymorphism in the tau gene associated with risk for Alzheimer's disease.

Searching for tau genetic variations which could be associated with risk for Alzheimer's disease (AD), we have performed a mutational analysis of a region containing the whole exon 11 of the tau gene, which encodes a microtubule binding region critical for tau self-assembly, and we have found a biallelic polymorphism at position +34 of intron 11 (IVS11 + 34G/A). We have analyzed the allelic frequencies of this polymorphism in a case-control sample (167 clinically diagnosed AD and 194 controls) and found that the presence of any G allele (genotypes AG + GG) is associated with a five-fold AD risk in individuals carrying the apolipoprotein E4 allele, strongly suggesting that the combined effect of tau and apoE is relevant in relation with AD pathogenesis.

Alzheimer's risk associated with human apolipoprotein E, alpha-2 macroglobulin and lipoprotein receptor related protein polymorphisms: absence of genetic interactions, and modulation by gender.

Apolipoprotein E (apoE), the lipoprotein receptor related protein (LRP) and alpha-2 macroglobulin (alpha2M) have been proposed as a functional complex involved in amyloid clearance, a crucial event for Alzheimer's disease development. In this work, we present an epidemiological approach aimed to study the interactions among these genes, age and gender. This approach did not reveal significant associations between the genes; however, the present study indicated that the risk associated with APOE promoter and LRP gene polymorphisms is modulated by gender.

The -491 A/T polymorphism in the regulatory region of the apolipoprotein E gene and early-onset Alzheimer's disease.

The -491 polymorphism in the promoter region of the apolipoprotein E gene (APOE) has been suggested to be associated with increased risk for Alzheimer's disease (AD) independent of APOE status. We studied the association between the -491 polymorphism and risk for early-onset Alzheimer's disease in 99 Dutch and 78 Spanish patients. In patients with early-onset AD, we found no consistent relationship with a single allele of the -491 polymorphism. Linkage disequilibrium between the polymorphism and the APOE gene was found which most likely might explain the inconsistent findings.

Apolipoprotein E promoter and alpha2-macroglobulin polymorphisms are not genetically associated with Chinese late onset Alzheimer's disease.

In this study, we investigated two newly reported polymorphisms in association with late onset Alzheimer's disease (AD) in Chinese. They were a -491 A/T polymorphism in the Apolipoprotein E (APOE) promoter region and a five base pair deletion at exon 18 of alpha2-Macroglobin (A2M). There were 196 AD and 180 normal controls (N), which were age- and sex-matched. APOE epsilon4 alleles were significantly increased in AD vs. N (chi2 = 33.3, P < 0.000001). However, neither the -491 A/T (chi2 = 1.13, P = 0.29) nor A2M (chi2 = 0.18, P = 0.67) polymorphism was associated with AD risk, suggesting that these polymorphisms do not represent risk factors for AD in the Chinese population.

APOE genotype in cerebrovascular disease and vascular dementia.

BACKGROUND: The fact that the allele epsilon 4 of the Apolipoprotein E (APOE) gene could act like a risk factor not only in late-onset familial and sporadic Alzheimer's disease (AD) but also in cerebrovascular disease (CVD) and vascular dementia (VaD) is still controversial. METHODS: In order to study if epsilon 4 allele is overrepresented not only in AD but also in CVD and VaD, APOE genotyping was undertaken in a series of 247 patients: 26 cases with VaD, 41 cases with CVD but without cognitive impairment (CVD-C), 83 cases with AD and 97 aged-matched "healthy controls" (HC). RESULTS: Percentages of subjects bearing one or two copies of the epsilon 4 allele was much higher in AD patients (54%) than in either CVD-C (29%) (p<0.05), VaD (15%) (p<0.001) or HC (13%) (p<0.001). CONCLUSIONS: These results strengthen the hypothesis that involves the APOE epsilon 4 allele as a predisposing factor for AD, but not for CVD or VaD.