Mean age of onset in familial Alzheimer's disease is determined by amyloid beta 42

More than 130 known mutations in the presenilin-1 (PS1) gene result in familial Alzheimer's disease (FAD) with a mutation specific age of disease onset. These mutations increase amyloid beta 42 (A beta42) levels, and this increase has been validated in recent years as one pathogenic factor in FAD. However, further malfunctions of mutant presenilin-1 are discussed as well. In order to assess the weight of A beta42 regarding the pathogenesis of FAD, we expressed mutant forms of PS1 (30-65 years onset age) in COS-7 cells and analyzed amyloid beta levels by a novel ELISA. We found a strong correlation (r = 0.98; p<0.001) between the A beta40/42-ratio and mean age of disease onset indicating a substantial extent of A beta42 contribution to FAD pathology. Our data strongly suggest that A beta42 is the decisive factor for age of onset in FAD.     

Knockout of plasminogen activator inhibitor 1 gene reduces amyloid beta peptide burden in a mouse model of Alzheimer's disease

Accumulation of amyloid beta peptide (Aβ) in the brain is a pathological hallmark of Alzheimer's disease (AD); the underlying mechanism, however, is not well understood. In this study, we show that expression of plasminogen activator inhibitor 1 (PAI-1), a physiological inhibitor of tissue type and urokinase type plasminogen activators (tPA and uPA), increases with age in the brain of wild type and Aβ precursor protein-presenilin 1 (APP/PS1) transgenic mice as well as in AD patients. Most importantly, we show that knocking out the PAI-1 gene dramatically reduces Aβ burden in the brain of APP/PS1 mice but has no effect on the levels of full-length APP, alpha or beta C-terminal fragments. Furthermore, we show that knocking out the PAI-1 gene leads to increases in the activities of tPA and plasmin, and the plasmin activity inversely correlates with the amounts of SDS insoluble Aβ40 and Aβ42. Together, these data suggest that increased PAI-1 expression/activity contributes importantly to Aβ accumulation during aging and in AD probably by inhibiting plasminogen activation and thus Aβ degradation.     

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.     

Single nucleotide polymorphisms in protein tyrosine phosphatase 1beta (PTPN1) are associated with essential hypertension and obesity

Protein tyrosine phosphatase 1beta (PTP-1beta) is involved in the regulation of several important physiological pathways. It regulates both insulin and leptin signaling, and interacts with the epidermal- and platelet-derived growth factor receptors. The gene is located on human chromosome 20q13, and several rare single nucleotide polymorphisms (SNPs) have been shown to be associated with insulin resistance and diabetes in different populations. As part of our ongoing investigations into the genetic basis of hypertension, we examined common sequence variants in the gene for association with hypertension, obesity and altered lipid profile in two populations of Japanese and Chinese descent. We re-sequenced all exons, selected intronic sequences and the promoter region in 24 individuals from our cohort. Fourteen SNPs were discovered, and six of these spanning 78 kb were genotyped in 1553 individuals from 672 families. All six SNPs were in linkage disequilibrium, and we found strong association of common risk haplotypes with hypertension in Chinese and Japanese (P<0.0001). In addition, individual SNPs showed association to total plasma cholesterol, LDL-cholesterol and VLDL-cholesterol levels, as well as obesity measures (body mass index). This analysis supports that PTP-1beta affects plasma lipid levels, and may lead to obesity and hypertension in Japanese and Chinese. Given similar associations found in other populations to insulin resistance and diabetes, this gene may play a crucial role in the development of the characteristic metabolic changes seen in patients with the metabolic syndrome.     

A functional polymorphism within plasminogen activator urokinase (PLAU) is associated with Alzheimer's disease

A number of susceptibility loci for Alzheimer's disease (AD) have been identified including a region on Chromosome 10q21-q22. Within this region the plasminogen activator urokinase gene (PLAU) was considered as a reasonable candidate from its functional implication in plasmin generation, a serine protease capable of degrading beta-Amyloid (Abeta) protein. We screened 56 single nucleotide polymorphisms (SNPs) around PLAU using 1751 individuals from four independent case-control samples (Munich, N=679; Bonn N=282; Brescia (Italy) N=219; Perth (Australia) N=557 and one discordant sib-pair sample (Munich N=622). In brain tissue samples of neuropathologically confirmed cases with AD (N=33) we analyzed plaque counts according to the risk allele. We identified that one functional exonic SNP (rs2227564) is associated with development of AD using the four independent case-control samples (Munich, P=0.02; Bonn, P=0.005; Brescia (Italy), P=0.001; Perth (Australia), P=0.03) and the discordant sib-pair sample (P=0.001). In brain tissue, from neuropathologically confirmed cases with AD, we identified significantly higher plaque counts in carriers of the risk allele (N=6; 60.3+/-16.9) compared with non-carriers (N=9; 26.3+/-8.8; P=0.007). This study provides compelling evidence of a genetic and functional involvement of a common PLAU variant into the pathogenesis of AD. Further functional investigations are warranted to elucidate the specific role of PLAU, respectively, PLAU variants in the metabolism of Abeta proteins.     

Dinucleotide repeat polymorphisms in the neprilysin gene are not associated with sporadic Alzheimer's disease

In the pathological process of Alzheimer's disease (AD), deposition of amyloid beta-peptide (A beta) in the brain parenchyma plays an important role. Neprilysin (NEP), a neutral endopeptidase, degrades A beta, and it is postulated that decreased NEP activity may contribute to the development of AD by promoting the accumulation of A beta. The human NEP gene possesses four dinucleotide repeat polymorphisms, and it is possible that these polymorphisms regulate the NEP expression levels and influence the pathological cascade of AD. Therefore, we investigated the association of these polymorphisms with AD. We performed genotyping of each polymorphism in 201 Japanese sporadic AD patients and 208 Japanese controls. There were no significant differences between the AD and control groups in allele frequencies of each polymorphism. We conclude that these polymorphisms in the NEP gene do not contribute to genetic risk factors for sporadic AD.     

Two exonic single nucleotide polymorphisms in the microsomal epoxide hydrolase gene are jointly associated with preeclampsia

This study determined whether genetic variability in exons 3 and 4 of the microsomal epoxide hydrolase gene jointly modifies individual preeclampsia risk. The study also determined whether genetic variability in the gene encoding for microsomal epoxide hydrolase (EPHX) contributes to individual differences in susceptibility to the development of preeclampsia. The study involved 133 preeclamptic and 115 healthy control pregnant women who were genotyped for two single nucleotide polymorphisms (SNPs), T-->C (Tyr113His) in exon 3 and A-->G (His139Arg) in exon 4, in the EPHX gene. Chi-square analysis was used to assess genotype and allele frequency differences between the preeclamptic and control groups. In addition, single-point analysis was expanded to pair of loci haplotype analysis to examine the estimated haplotype frequencies of the two SNPs, of unknown phase, among the preeclamptic and control groups. Estimated haplotype frequencies were assessed using the maximum-likelihood method, employing an expectation-maximization (EM) algorithm. Single-point allele and genotype distributions in exons 3 and 4 of the EPHX gene were not statistically different between the groups. However, according to the haplotype estimation analysis, we observed a significantly elevated frequency of haplotype T-A (Tyr113-His139) among the preeclampsia group vs the control group (P=0.01). The odds ratio for preeclampsia associated with the high-activity haplotype T-A (Tyr113-His139) was 1.61 (95% CI: 1.12-2.32). The use of two intragenic SNPs jointly in haplotype analysis of association demonstrated that the genetically determined high-activity haplotype T-A (Tyr113-His139) was significantly associated with preeclampsia.     

Single nucleotide polymorphisms in protein tyrosine phosphatase 1{beta} (PTPN1) are associated with essential hypertension and obesity

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Predominant deposition of amyloid-beta 42(43) in plaques in cases of Alzheimer's disease and hereditary cerebral hemorrhage associated with mutations in the amyloid precursor protein gene.

Amyloid (A beta) deposition was investigated in cases of Alzheimer's disease and hereditary cerebral hemorrhage with amyloidosis, Dutch type, due to mutations in the amyloid precursor protein (APP) gene using the end-specific monoclonal antibodies BA27 and BC05 that recognize A beta 40 or A beta 42(43), respectively. In cases of APP717 mutation the predominant A beta species within plaques terminate at A beta 42(43) with relatively little A beta 40 being present. The total amount of A beta deposited as A beta 42(43) is significantly greater than in sporadic Alzheimer's disease, consistent with the suggestion that this mutation might influence the processing of APP so as to produce more of the highly aggregatable form, A beta 1-42. In cases of APP670/671 mutation the major peptide in plaques is also A beta 42(43), although the proportion of plaques containing A beta 40, and the total A beta load is similar to that in sporadic Alzheimer's disease. As in sporadic Alzheimer's disease, the vascular amyloid in APP670/671 and APP717 and in cases of hereditary cerebral hemorrhage with amyloidosis, Dutch type is predominantly A beta 40 in this latter disorder, however, parenchymal deposits are exclusively A beta 42(43). Although the various APP mutations may influence the type, quantity, and location of A beta deposited, the predominant, and possibly the initial, species deposited in the brain parenchyma is A beta 42(43).     

PERB11 (MIC): a polymorphic MHC gene is expressed in skin and single nucleotide polymorphisms are associated with psoriasis

The susceptibility genes for psoriasis remain to be identified. At least one of these must be in the major histocompatibility complex (MHC) to explain associations with alleles at human leucocyte antigen (HLA)-A, -B, -C, -DR, -DQ and C4. In fact, most of these alleles are components of just two ancestral haplotypes (AHs) designated 13.1 and 57.1. Although relevant MHC gene(s) could be within a region of at least 4 Mb, most studies have favoured the area near HLA-B and -C. This region contains a large number of non-HLA genes, many of which are duplicated and polymorphic. Members of one such gene family, PERB11.1 and PERB11.2, are expressed in the skin and are encoded in the region between tumour necrosis factor and HLA-B. To investigate the relationship of PERB11.1 alleles to psoriasis, sequence based typing was performed on 97 patients classified according to age of onset and family history. The frequency of the PERB11.1*06 allele is 44% in type I psoriasis but only 7% in controls (Pc = 0.003 by Fisher's exact test, two-tailed). The major determinant of this association is a single nucleotide polymorphism (SNP) within intron 4. In normal and affected skin, expression of PERB11 is mainly in the basal layer of the epidermis including ducts and follicles. PERB11 is also present in the upper keratin layers but there is relative deficiency in the intermediate layers. These findings suggest a possible role for PERB11 and other MHC genes in the pathogenesis of psoriasis.     

Cholesteryl ester transfer protein gene polymorphisms are associated with coronary artery disease in Corsican population (France)

The aim of the present study was to investigate the association between coronary artery disease (CAD) and Cholesterol Ester Transfer Protein (CETP) (gaaa)n polymorphisms of the CETP gene in Central Corsica island (France). The study group was composed by 300 unrelated Corsican patients with angiographically documented CAD and 300 unrelated healthy blood donors. Significant differences were observed in the distribution of CETP (gaaa)n alleles between the groups under study (p=0.03; chi(2): 16.8, df: 8). The occurrence of a long allele (408 bp) was higher in cases (12%) than in control group (2%), showing a 6.75-fold increased risk for CAD in Corsica patients (p=0.0055; OR=6.750; 95% CIs=1.47-31.00). The correlation of this polymorphism with the lipid profile (cholesterol, low density lipoprotein-cholesterol, high density lipoprotein-cholesterol and triglycerides) in the patients group was determined. There was a significant association of the long alleles of CETP (gaaa)n with HDL-C levels. In the patient and in the control groups the LL genotypes had lower HDL-C compared with the SS and SL genotypes (p<0.0001). In summary our results suggest that the genetic variation at the CETP gene may play an important role in determining CAD in Corsican population.     

Evaluation of plasma Abeta(40) and Abeta(42) as predictors of conversion to Alzheimer's disease in patients with mild cognitive impairment

Numerous studies have shown a marked decrease of beta-amyloid(42) (Abeta(42)) in the cerebrospinal fluid (CSF) of patients with incipient Alzheimer's disease (AD). However, studies on Abeta in plasma are contradictory, and show very marginal differences between patients and controls. Here, we analyzed plasma samples using a new multiplex immunoassay for simultaneous analysis of Abeta(1-40), Abeta(n-40), Abeta(1-42), and Abeta(n-42). The plasma samples were obtained at baseline from two independent cohorts of patients with mild cognitive impairment (MCI) and age-matched controls. In the first cohort, 41% of the 117 MCI cases converted to AD during a clinical follow-up period of 4-7 years. In the second cohort, 14% of the 110 MCI subjects developed AD during a clinical follow-up period of 2-4 years. None of the plasma Abeta isoforms differed between MCI patients that subsequently developed AD and healthy controls or stable MCI patients. The Cox proportional hazards model did not reveal any differences in the probability of progression from MCI to AD related to plasma Abeta levels. In contrast, low levels of Abeta(1-42) in CSF were strongly associated with increased risk of future AD. The absence of a change in plasma Abeta in incipient AD, despite the marked change in CSF, may be explained by the lack of a correlation between the levels of Abeta(1-42) in CSF and plasma. In conclusion, the results show that CSF biomarkers are better predictors of progression to AD than plasma Abeta isoforms.     

Estrogen receptor beta gene variants are associated with increased risk of Alzheimer's disease in women

We investigated the association of five intronic single-nucleotide polymorphism (SNP) at the estrogen receptor beta (ESR2) gene locus and the susceptibility of developing Alzheimer's disease (AD) in 387 subjects with clinically diagnosed probable AD and 467 cognitively normal individuals derived from eastern Finland. According to our results, variation in the ESR2 gene is associated with an increased risk of AD in women, whereas it does not contribute to the disease susceptibility in men. More specifically, in women, the allele T and the genotype T/T of two of the studied ESR2 gene SNPs (SNP2 and SNP3) were more frequent in AD women than in cognitively normal control women (P=0.012 and P=0.016, respectively). The ESR2 SNP2 T/T genotype and the SNP3 T/T genotype were associated with a significant, nearly two-fold increase in the risk of AD in women (OR=1.87, 95% CI=1.21-2.90), and remained significant after adjustment with the APOE genotype and age (OR=1.63, 95% CI, 1.00-1.68). The combined effect of the ESR2 SNP2 T/T or SNP3 T/T genotype and female gender increases the risk of the disease (OR=3.2, 95% CI=1.3-7.7). Consistent with these results, also the frequency of the haplotype containing the two above ESR2 gene risk alleles was elevated in AD women (P=0.027, OR=1.3, 95% CI=1.02-1.65). Results show that variation in ESR2 gene may be linked with increased AD susceptibility and furthermore, this association is gender specific.     

Immunity-related gene single nucleotide polymorphisms associated with Rhodococcus equi infection in foals

In previous work, we found significant associations of horse polymorphic microsatellite and immunity-related (IR) gene markers with Rhodococcus equi infection of foals. Here, a statistically significant association between a single nucleotide polymorphism (SNP) within the interleukin 7 receptor-encoding gene (IL7R) with high R. equi burden in transtracheal aspirates was found (Fisher’s F = 0.043, odds ratio: 8.00, 95% confidence interval: 1.127–56.795). Further positional and/or functional candidate genes investigated TLR2, IL13, IL17A, IL28R, TACE/ADAM 17 and GBP1, were not associated with infection in this study. SNPs analysed were found by sequencing and appropriate restriction fragment length polymorphism markers were developed. Their associations with R. equi infection were tested by genotyping thoroughbred foals from the original study. The association was confirmed by analysing genotypes composed with genes previously reported to be associated with R. equi infection in the same group.     

Expression of Met protein and urokinase-type plasminogen activator receptor (uPA-R) in papillary carcinoma of the thyroid

Met protein encoded by MET oncogene is the high affinity receptor for hepatocyte growth factor (HGF)/scatter factor (SF). HGF/SF has to be cleaved in its heterodimeric form by the urokinase-type plasminogen activator (uPA) to become active as a ligand for Met receptor. The expression of Met protein and of the high affinity receptor for uPA (uPA-R) was investigated in 39 samples of papillary carcinoma using immunohistochemistry. Reactivity for Met protein was present in 33 of 34 tumours, mostly with a diffuse pattern of staining. Reactivity for uPA-R was present in 78 per cent of papillary tumours and exhibited a pattern of staining similar to that of Met protein. Staining for uPA-R was present in 23 of 25 cases (92 per cent) of papillary carcinoma with prominent sclerosis, and in only 1 of 7 cases (14 per cent) without sclerosis. Peritumoural normal thyroid, follicular adenomas, and follicular carcinomas were negative for Met protein and for uPA-R. Hyperfunctioning tall thyroid cells showed weak membrane reactivity for uPA-R and for Met protein. The findings of immunohistochemistry were confirmed at the mRNA level using in situ hybridization, since the signal for uPA-R and Met RNAs was detected in most tumour cells of five cases of papillary carcinoma.     

Extremes of L-ficolin concentration in children with recurrent infections are associated with single nucleotide polymorphisms in the FCN2 gene

L-ficolin (also called ficolin-2, P35 or hucolin) is a soluble pattern recognition molecule of suspected importance in anti-microbial immunity. It activates the lectin pathway of complement and acts as an opsonin. l-ficolin, encoded by the FCN2 gene, recognizes microbial polysaccharides and glycoconjugates rich in GlcNAc or GalNAc. We report here data concerning four single nucleotide polymorphisms (SNPs) of the FCN2 gene and their relationship to l-ficolin serum concentrations. There are two pairs of SNPs in linkage disequilibrium: ss32469536 (located in promoter) with rs7851696 (in exon 8) and ss32469537 (promoter) with ss32469544 (exon 8). We selected groups possessing low or high serum l-ficolin concentrations (or= 4.5 microg/ml, respectively) from Polish children suffering from recurrent respiratory infections (n = 146). Low l-ficolin levels were associated with variant alleles for ss32469536 and rs7851696 and normal alleles for ss32469537 and ss32469544. Conversely, high l-ficolin levels were associated with variant alleles of ss32469537 and ss32469544. FCN2 genotyping should be a valuable additional tool for disease association studies.     

Association of tagSNPs in the urokinase-plasminogen activator (PLAU) gene with Alzheimer's disease and associated quantitative traits.

The gene coding for urokinase-plasminogen activator (PLAU) is a strong biological and positional candidate gene for Alzheimer's disease (AD). Previously some studies have examined the role of common variation in the PLAU gene with AD risk but the results have been inconsistent and this inconsistency could have been due to the use of relatively small sample sizes. In this study we evaluated the distribution of four tagSNPs (rs2227562 in intron 5, rs2227564 in exon 6, rs2227571 in intron 9, and rs4065 in 3'UTR) in the PLAU gene in a large case-control study consisting of up to 1,000 AD patients and 697 white control subjects. We examined the role of these tagSNPs with AD risk and quantitative traits of AD, including age-at-onset (AAO), disease duration, and mini-mental state examination (MMSE) scores. The 3'UTR SNP revealed modest significant association with risk (OR = 0.71, 95% CI: 0.53-0.95; P = 0.02), AAO (P = 0.036) and disease duration (P = 0.04) of AD. In addition, the intron 9 SNP also revealed a significant association with AAO (P = 0.01) and disease duration (P = 0.006). Our data on a large number of AD cases and controls suggest that genetic variation in PLAU may affect the risk and AAO of AD.     

Regulatory region single nucleotide polymorphisms of the apolipoprotein E gene and the rate of cognitive decline in Alzheimer's disease

The aim of this study was to investigate whether single nucleotide polymorphisms (SNPs) in the regulatory regions of the apolipoprotein E (APOE) gene modify the well-established epsilon4-associated risk for Alzheimer's disease (AD). Sequencing of the APOE gene regulatory regions revealed four previously reported promoter SNPs and one novel SNP in the previously described macrophage enhancer (ME.1). In addition, we also studied the two classic allelic missense SNPs that define epsilon2/epsilon3/epsilon4 status in a case-control association study. Analysis of pair-wise linkage disequilibrium (LD) of the five regulatory region SNPs with classic APOE SNPs revealed a previously unreported 7 kb LD block covering the entire APOE gene, part of the promoter and 3' enhancer region. We report here that in a case-control association study (N=719) of the seven SNPs, the genotype at codon 112 captures all the information required to assess disease risk. To explore correlations with quantitative traits, 169 patients were studied in whom rates of cognitive decline were available. In addition to the epsilon4 allele, two regulatory region SNPs were associated with the rate of cognitive decline in AD patients. This study highlights the effect of APOE gene variation on risk of AD and rate of cognitive decline and demonstrates that a single SNP, which confers epsilon4 status, captures all of the risk of developing AD but two SNPs in the regulatory region may affect the rate of cognitive decline in AD patients.     

Phospholipase D1 is associated with amyloid precursor protein in Alzheimer's disease

Amyloid precursor protein (APP) is a widely expressed transmembrane protein of unknown function that is involved in the pathogenesis of Alzheimer's disease (AD). We investigated the involvement of phospholipase D (PLD) in the pathophysiology of AD. We showed dramatic upregulation of PLD1 immunoreactivity in reactive astroglial cells in brain tissue sections from authentic AD patients. Expression and activity of PLD1 were up-regulated in brain tissues from AD patients, especially caveolae membrane fraction, compared with those of control brains. Interestingly, PLD1 physically interacts and colocalizes with APP and caveolin-3. We found that APP was associated with the pleckstrin homology domain of PLD1, and the amyloid region of APP interacted with PLD. Elevated expression of APP stimulated PLD activity in human astroglioma cells. These results suggest that up-regulation of PLD might have a role in the neuronal pathology associated with AD.     

In vitro and in vivo oxidative stress associated with Alzheimer's amyloid beta-peptide (1-42)

The amyloid beta-peptide (A beta)-associated free radical oxidative stress model for neuronal death in Alzheimer's disease (AD) brain predicts that neuronal protein oxidation is a consequence of A beta-associated free radicals [8]. In this study we have used both in vitro and in vivo models of beta-amyloid (A beta) toxicity to detect free radical induced oxidative stress by the measure of protein carbonyl levels. These model systems employed cultured hippocampal neurons exposed to exogenous synthetic A beta(1-42) and transgenic Caenorhabditis elegans (C. elegans) animals expressing A beta(1-42). We also investigated the importance of the A beta(1-42) Met35 residue for free radical formation in peptide solution and for peptide-induced protein oxidation and neuronal toxicity in these model systems. A beta(1-42) in solution yielded an EPR spectrum, suggesting that free radicals are associated with this peptide; however, neither the reverse [A beta(42-1)] nor methionine-substituted peptide [A beta(1-42)Met35Nlc] gave significant EPR spectra, suggesting the importance of the methionine residue in free radical formation. A beta(1-42) addition to cultured hippocampal neurons led to both neurotoxicity (30.1% cell death, p < 0.001) and increased protein oxidation (158% of controls, p < 0.001). and both of those effects were not observed with reverse or Met35Nle substituted peptides. C. elegans transgenic animals expressing human A beta(1-42) also had significantly increased in vivo protein carbonyls (176% of control animals, p < 0.001), consistent with our model. In contrast, transgenic animals with a Met35cys substitution in A beta(1-42) showed no increased protein carbonyls in vivo, in support of the hypothesis that methionine is important in A beta-associated free radical oxidative stress. These results are discussed with reference to the A beta-associated free radical oxidative stress model of neurotoxicity in AD brain.