Functional screening of Alzheimer risk loci identifies PTK2B as an in vivo modulator and early marker of Tau pathology.

A recent genome-wide association meta-analysis for Alzheimer's disease (AD) identified 19 risk loci (in addition to APOE) in which the functional genes are unknown. Using Drosophila, we screened 296 constructs targeting orthologs of 54 candidate risk genes within these loci for their ability to modify Tau neurotoxicity by quantifying the size of >6000 eyes. Besides Drosophila Amph (ortholog of BIN1), which we previously implicated in Tau pathology, we identified p130CAS (CASS4), Eph (EPHA1), Fak (PTK2B) and Rab3-GEF (MADD) as Tau toxicity modulators. Of these, the focal adhesion kinase Fak behaved as a strong Tau toxicity suppressor in both the eye and an independent focal adhesion-related wing blister assay. Accordingly, the human Tau and PTK2B proteins biochemically interacted in vitro and PTK2B co-localized with hyperphosphorylated and oligomeric Tau in progressive pathological stages in the brains of AD patients and transgenic Tau mice. These data indicate that PTK2B acts as an early marker and in vivo modulator of Tau toxicity.

17q21.31 duplication causes prominent tau-related dementia with increased MAPT expression.

To assess the role of rare copy number variations in Alzheimer's disease (AD), we conducted a case-control study using whole-exome sequencing data from 522 early-onset cases and 584 controls. The most recurrent rearrangement was a 17q21.31 microduplication, overlapping the CRHR1, MAPT, STH and KANSL1 genes that was found in four cases, including one de novo rearrangement, and was absent in controls. The increased MAPT gene dosage led to a 1.6-1.9-fold expression of the MAPT messenger RNA. Clinical signs, neuroimaging and cerebrospinal fluid biomarker profiles were consistent with an AD diagnosis in MAPT duplication carriers. However, amyloid positon emission tomography (PET) imaging, performed in three patients, was negative. Analysis of an additional case with neuropathological examination confirmed that the MAPT duplication causes a complex tauopathy, including prominent neurofibrillary tangle pathology in the medial temporal lobe without amyloid-ß deposits. 17q21.31 duplication is the genetic basis of a novel entity marked by prominent tauopathy, leading to early-onset dementia with an AD clinical phenotype. This entity could account for a proportion of probable AD cases with negative amyloid PET imaging recently identified in large clinical series.

SORL1 rare variants: a major risk factor for familial early-onset Alzheimer's disease.

The SORL1 protein plays a protective role against the secretion of the amyloid ß peptide, a key event in the pathogeny of Alzheimer's disease. We assessed the impact of SORL1 rare variants in early-onset Alzheimer's disease (EOAD) in a case-control setting. We conducted a whole exome analysis among 484 French EOAD patients and 498 ethnically matched controls. After collapsing rare variants (minor allele frequency ≤1%), we detected an enrichment of disruptive and predicted damaging missense SORL1 variants in cases (odds radio (OR)=5.03, 95% confidence interval (CI)=(2.02-14.99), P=7.49.10(-5)). This enrichment was even stronger when restricting the analysis to the 205 cases with a positive family history (OR=8.86, 95% CI=(3.35-27.31), P=3.82.10(-7)). We conclude that predicted damaging rare SORL1 variants are a strong risk factor for EOAD and that the association signal is mainly driven by cases with positive family history.

A novel Alzheimer disease locus located near the gene encoding tau protein.

APOE ɛ4, the most significant genetic risk factor for Alzheimer disease (AD), may mask effects of other loci. We re-analyzed genome-wide association study (GWAS) data from the International Genomics of Alzheimer's Project (IGAP) Consortium in APOE ɛ4+ (10 352 cases and 9207 controls) and APOE ɛ4- (7184 cases and 26 968 controls) subgroups as well as in the total sample testing for interaction between a single-nucleotide polymorphism (SNP) and APOE ɛ4 status. Suggestive associations (P<1 × 10(-4)) in stage 1 were evaluated in an independent sample (stage 2) containing 4203 subjects (APOE ɛ4+: 1250 cases and 536 controls; APOE ɛ4-: 718 cases and 1699 controls). Among APOE ɛ4- subjects, novel genome-wide significant (GWS) association was observed with 17 SNPs (all between KANSL1 and LRRC37A on chromosome 17 near MAPT) in a meta-analysis of the stage 1 and stage 2 data sets (best SNP, rs2732703, P=5·8 × 10(-9)). Conditional analysis revealed that rs2732703 accounted for association signals in the entire 100-kilobase region that includes MAPT. Except for previously identified AD loci showing stronger association in APOE ɛ4+ subjects (CR1 and CLU) or APOE ɛ4- subjects (MS4A6A/MS4A4A/MS4A6E), no other SNPs were significantly associated with AD in a specific APOE genotype subgroup. In addition, the finding in the stage 1 sample that AD risk is significantly influenced by the interaction of APOE with rs1595014 in TMEM106B (P=1·6 × 10(-7)) is noteworthy, because TMEM106B variants have previously been associated with risk of frontotemporal dementia. Expression quantitative trait locus analysis revealed that rs113986870, one of the GWS SNPs near rs2732703, is significantly associated with four KANSL1 probes that target transcription of the first translated exon and an untranslated exon in hippocampus (P ⩽ 1.3 × 10(-8)), frontal cortex (P ⩽ 1.3 × 10(-9)) and temporal cortex (P⩽1.2 × 10(-11)). Rs113986870 is also strongly associated with a MAPT probe that targets transcription of alternatively spliced exon 3 in frontal cortex (P=9.2 × 10(-6)) and temporal cortex (P=2.6 × 10(-6)). Our APOE-stratified GWAS is the first to show GWS association for AD with SNPs in the chromosome 17q21.31 region. Replication of this finding in independent samples is needed to verify that SNPs in this region have significantly stronger effects on AD risk in persons lacking APOE ɛ4 compared with persons carrying this allele, and if this is found to hold, further examination of this region and studies aimed at deciphering the mechanism(s) are warranted.

A genome-wide association meta-analysis of plasma Aß peptides concentrations in the elderly.

Amyloid beta (Aß) peptides are the major components of senile plaques, one of the main pathological hallmarks of Alzheimer disease (AD). However, Aß peptides' functions are not fully understood and seem to be highly pleiotropic. We hypothesized that plasma Aß peptides concentrations could be a suitable endophenotype for a genome-wide association study (GWAS) designed to (i) identify novel genetic factors involved in amyloid precursor protein metabolism and (ii) highlight relevant Aß-related physiological and pathophysiological processes. Hence, we performed a genome-wide association meta-analysis of four studies totaling 3 528 healthy individuals of European descent and for whom plasma Aß1-40 and Aß1-42 peptides levels had been quantified. Although we did not observe any genome-wide significant locus, we identified 18 suggestive loci (P<1 × 10(-)(5)). Enrichment-pathway analyses revealed canonical pathways mainly involved in neuronal functions, for example, axonal guidance signaling. We also assessed the biological impact of the gene most strongly associated with plasma Aß1-42 levels (cortexin 3, CTXN3) on APP metabolism in vitro and found that the gene protein was able to modulate Aß1-42 secretion. In conclusion, our study results suggest that plasma Aß peptides levels are valid endophenotypes in GWASs and can be used to characterize the metabolism and functions of APP and its metabolites.

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.

Increased expression of BIN1 mediates Alzheimer genetic risk by modulating tau pathology.

Genome-wide association studies (GWAS) have identified a region upstream the BIN1 gene as the most important genetic susceptibility locus in Alzheimer's disease (AD) after APOE. We report that BIN1 transcript levels were increased in AD brains and identified a novel 3 bp insertion allele ∼28 kb upstream of BIN1, which increased (i) transcriptional activity in vitro, (ii) BIN1 expression levels in human brain and (iii) AD risk in three independent case-control cohorts (Meta-analysed Odds ratio of 1.20 (1.14-1.26) (P=3.8 × 10(-11))). Interestingly, decreased expression of the Drosophila BIN1 ortholog Amph suppressed Tau-mediated neurotoxicity in three different assays. Accordingly, Tau and BIN1 colocalized and interacted in human neuroblastoma cells and in mouse brain. Finally, the 3 bp insertion was associated with Tau but not Amyloid loads in AD brains. We propose that BIN1 mediates AD risk by modulating Tau pathology.

Alzheimer risk associated with a copy number variation in the complement receptor 1 increasing C3b/C4b binding sites.

Two multicentre genome-wide association (GWA) studies provided substantial evidence, implicating the complement receptor 1 gene (CR1) in Alzheimer disease (AD) genetic etiology. CR1 encodes a large transmembrane receptor with a crucial role in the immune complement cascade. We performed a genetic follow-up of the GWA CR1 association in a Flanders-Belgian cohort (n=1883), and investigated the effect of single-nucleotide polymorphisms (SNPs) located in the CR1 locus on AD risk and cerebrospinal fluid (CSF) biomarker levels. We obtained significant association (P(adj)<0.03; odds ratio (OR)=1.24 (95% confidence interval (CI): 1.02-1.51)) for one CR1 risk haplotype, and haplotype association was strongest in individuals carrying apolipoprotein E (APOE) ɛ4 alleles (P(adj)<0.006; OR=1.50 (95% CI: 1.08-2.09)). Also, four SNPs correlated with increased CSF amyloid Aß1₋42 levels, suggesting a role for the CR1 protein in Aß metabolism. Moreover, we quantified a low-copy repeat (LCR)-associated copy number variation (CNV) in CR1, producing different CR1 isoforms, CR1-F and CR1-S, and obtained significant association in carriers of CR1-S. We replicated the CR1 CNV association finding in a French cohort (n=2003) and calculated in the combined cohorts, an OR of 1.32; 95% CI: 1.10-1.59 (P=0.0025). Our data showed that the common AD risk association may well be explained by the presence of CR1-S increasing the number of C3b/C4b and cofactor activity sites and AD risk with 30% in CR1-S carriers. How precisely the different functional role of CR1-S in the immune complement cascade contributes to AD pathogenesis will need additional functional studies.

High frequency of potentially pathogenic SORL1 mutations in autosomal dominant early-onset Alzheimer disease.

Performing exome sequencing in 14 autosomal dominant early-onset Alzheimer disease (ADEOAD) index cases without mutation on known genes (amyloid precursor protein (APP), presenilin1 (PSEN1) and presenilin2 (PSEN2)), we found that in five patients, the SORL1 gene harbored unknown nonsense (n=1) or missense (n=4) mutations. These mutations were not retrieved in 1500 controls of same ethnic origin. In a replication sample, including 15 ADEOAD cases, 2 unknown non-synonymous mutations (1 missense, 1 nonsense) were retrieved, thus yielding to a total of 7/29 unknown mutations in the combined sample. Using in silico predictions, we conclude that these seven private mutations are likely to have a pathogenic effect. SORL1 encodes the Sortilin-related receptor LR11/SorLA, a protein involved in the control of amyloid beta peptide production. Our results suggest that besides the involvement of the APP and PSEN genes, further genetic heterogeneity, involving another gene of the same pathway is present in ADEOAD.

Association between 9p21-23 Locus and Frailty in a Community-Dwelling Greek Population: Results from the Hellenic Longitudinal Investigation of Ageing and Diet.

BACKGROUND: Frailty is a complex geriatric syndrome arising from a combination of genetic and environmental factors and is associated with adverse health outcomes and mortality. A recent study reported an association between variants of the 9p21-23 locus, associated with a number of age-related disorders, including Alzheimer's disease (AD), and frailty. Frailty has been associated with increased risk of developing AD and it has been proposed that frailty burden may modify AD clinical presentation. In view of the overlapping genetic architecture between the two disorders, it is noteworthy to conduct studies to uncover risk variants that contribute to both AD and frailty. The purpose of this study is to test the reproducibility of the association of 9p21-23 locus with frailty in a population that is ethnically different from previous work and in the context of multidimensional definitions of frailty that will allow us to examine the potential impact to domains pertaining to AD pathology. METHODS: We operationalized frailty according two definitions and the corresponding instruments, the Frailty Index (FI) and the Tilburg Frailty Indicator (TFI) and we determined genotypes of eight alleles previously identified as risk increasing for frailty in 1172 community-dwelling older participants (57% females) from the HELIAD study with a mean age of 74 years old. We cross-sectionally investigated the association between risk alleles and frailty, as well as with specific components of each definition using linear regression analyses adjusted for age, sex and years of education. RESULTS: Compared to non-carriers, carriers of rs7038172 C risk allele, were associated with a higher FI Score (ß=0.089, p=0.002). Similarly, we found a positive association between the presence of at least one rs7038172 C variant and TFI score (ß=0.053, p=0.04). Moreover, the rs7038172 variant was associated, irrespectively of dementia status, with the memory and psychological domain of FI and TFI, respectively. CONCLUSION: Our study confirms the association of the rs7038172 C allele with the frailty syndrome in a Greek population and in the context of multidimensional definitions of frailty. Furthermore, we report novel associations between this allele and the memory domain of FI and the psychological domain of TFI, that includes memory problems on its components. Given that frailty burden has been shown to modify the AD clinical presentation, it is likely that rs7038172 C allele may accelerate the transition of AD or frailty to dementia Overall, our study corroborates the role of the 9p21-23 region in frailty development and draw potential links with AD pathology.

Ultracold neutron properties of the Eljen-299-02D deuterated scintillator.

In this paper, we report studies of the Fermi potential and loss per bounce of ultracold neutrons (UCNs) on a deuterated scintillator (Eljen-299-02D). These UCN properties of the scintillator enable its use in a wide variety of applications in fundamental neutron research.

Evidence for induction of the ornithine transcarbamylase expression in Alzheimer's disease.

To more rapidly identify candidate genes located within chromosomal regions of interest defined by genome scan studies in Alzheimer's disease (AD), we have developed a customized microarray containing all the ORFs (n=2741) located within nine of these regions. Levels of gene expression were assessed in total RNA from brain tissue of 12 controls and 12 AD patients. Of all genes showing differential expression, we focused on the ornithine transcarbamylase (OTC) gene on Xp21.1., a key enzyme of the urea cycle which we found to be expressed in AD brains but not in controls, as confirmed by RT-PCR. We also detected mRNA expression of all the other urea cycle enzymes in AD brains. Immunochemistry experiments revealed that the OTC expression was strictly restricted to vascular endothelial cells in brain. Furthermore, OTC activity was 880% increased in the CSF of probable AD cases compared with controls. We analysed the association of the OTC -389 G/A and -241 A/G promoter polymorphisms with the risk of developing AD. We observed that rare haplotypes may be associated with the risk of AD through a possible modulation of the methylation of the OTC promoter. In conclusion, our results suggest the involvement of a new pathway in AD brains involving the urea cycle.

Transcriptomic and genetic studies identify IL-33 as a candidate gene for Alzheimer's disease.

The only recognized genetic determinant of the common forms of Alzheimer's disease (AD) is the epsilon 4 allele of the apolipoprotein E gene (APOE). To identify new candidate genes, we recently performed transcriptomic analysis of 2741 genes in chromosomal regions of interest using brain tissue of AD cases and controls. From 82 differentially expressed genes, 1156 polymorphisms were genotyped in two independent discovery subsamples (n=945). Seventeen genes exhibited at least one polymorphism associated with AD risk, and following correction for multiple testing, we retained the interleukin (IL)-33 gene. We first confirmed that the IL-33 expression was decreased in the brain of AD cases compared with that of controls. Further genetic analysis led us to select three polymorphisms within this gene, which we analyzed in three independent case-control studies. These polymorphisms and a resulting protective haplotype were systematically associated with AD risk in non-APOE epsilon 4 carriers. Using a large prospective study, these associations were also detected when analyzing the prevalent and incident AD cases together or the incident AD cases alone. These polymorphisms were also associated with less cerebral amyloid angiopathy (CAA) in the brain of non-APOE epsilon 4 AD cases. Immunohistochemistry experiments finally indicated that the IL-33 expression was consistently restricted to vascular capillaries in the brain. Moreover, IL-33 overexpression in cellular models led to a specific decrease in secretion of the A beta(40) peptides, the main CAA component. In conclusion, our data suggest that genetic variants in IL-33 gene may be associated with a decrease in AD risk potentially in modulating CAA formation.

Rapid identification of mitochondrial DNA (mtDNA) mutations in neuromuscular disorders by using surveyor strategy.

Mutations of mitochondrial genome are responsible for respiratory chain defects in numerous patients. We have used a strategy, based on the use of a mismatch-specific DNA endonuclease named " Surveyor Nuclease", for screening the entire mtDNA in a group of 50 patients with neuromuscular features, suggesting a respiratory chain dysfunction. We identified mtDNA mutations in 20% of patients (10/50). Among the identified mutations, four are not found in any mitochondrial database and have not been reported previously. We also confirm that mtDNA polymorphisms are frequently found in a heteroplasmic state (15 different polymorphisms were identified among which five were novel).

Kinetics of cell proliferation in benigh and premalignant tumors of the human epidermis.

A double labeling method with two levels of tritiated thymidine was used to study 6 patients with seborrheic keratosis, 1 with a fibroepithelial tumor of Pinkus, and 1 with basal cell nevus syndrome manifesting three pits on the palm of the hand. The two latter types of lesions, known to be able to evolve into basal cell carcinoma, had an increased S-phase duration (18 hr for the germ cells of the palmar pits) as compared with normal epidermis (10 hr). This situation was similar to that observed in basal cell carcinoma, However, the S phase was not lengthened in seborrheic keratosis (9.2 plus or minus 1.6), a benign tumor in which malignant transformation is extremely rare. S phase was also of normal duration in the benign eosinophilic septa of the tumor of Pinkus.

Establishment, characterization, chemosensitivity, and radiosensitivity of two different cell lines derived from a human breast cancer biopsy.

In vitro culture of a human breast cancer biopsy fragment gave rise to two permanent cell lines, CAL 18 A and CAL 18 B, which were differentiated by both morphological and ultrastructural analysis. The karyotypic and growth properties of these two cell lines also differed, providing further evidence of cell heterogeneity within a given tumor. Both cell lines lost their hormone receptors in vitro. CAL 18 A cells grew in agar and were tumorigenic after inoculation into nude mice; neither of these properties was observed in CAL 18 B cells. The chemosensitivity of 12 antineoplastic drugs was assessed by a short-term assay, using inhibition of tritiated thymidine incorporation by the cells after contact with the drugs as the end point. Only a few drugs were active at moderate concentrations. The overall responses of both cell lines were similar. The cell survival curves, established by the colony method following a single dose of radiation, were also very similar, despite the greater heterogeneity of CAL 18 B cells. The two cell lines appear to be interrelated, since CAL 18 B cells were occasionally observed to emerge from CAL 18 A clones, suggesting that malignant cell redifferentiation may occur spontaneously in vitro.

Ground-based assessment of the bias and long-term stability of fourteen limb and occultation ozone profile data records.

The ozone profile records of a large number of limb and occultation satellite instruments are widely used to address several key questions in ozone research. Further progress in some domains depends on a more detailed understanding of these data sets, especially of their long-term stability and their mutual consistency. To this end, we made a systematic assessment of fourteen limb and occultation sounders that, together, provide more than three decades of global ozone profile measurements. In particular, we considered the latest operational Level-2 records by SAGE II, SAGE III, HALOE, UARS MLS, Aura MLS, POAM II, POAM III, OSIRIS, SMR, GOMOS, MIPAS, SCIAMACHY, ACE-FTS and MAESTRO. Central to our work is a consistent and robust analysis of the comparisons against the ground-based ozonesonde and stratospheric ozone lidar networks. It allowed us to investigate, from the troposphere up to the stratopause, the following main aspects of satellite data quality: long-term stability, overall bias, and short-term variability, together with their dependence on geophysical parameters and profile representation. In addition, it permitted us to quantify the overall consistency between the ozone profilers. Generally, we found that between 20-40 km the satellite ozone measurement biases are smaller than ±5 %, the short-term variabilities are less than 5-12% and the drifts are at most ±5% decade-1 (or even ±3 % decade-1 for a few records). The agreement with ground-based data degrades somewhat towards the stratopause and especially towards the tropopause where natural variability and low ozone abundances impede a more precise analysis. In part of the stratosphere a few records deviate from the preceding general conclusions; we identified biases of 10% and more (POAM II and SCIAMACHY), markedly higher single-profile variability (SMR and SCIAMACHY), and significant long-term drifts (SCIAMACHY, OSIRIS, HALOE, and possibly GOMOS and SMR as well). Furthermore, we reflected on the repercussions of our findings for the construction, analysis and interpretation of merged data records. Most notably, the discrepancies between several recent ozone profile trend assessments can be mostly explained by instrumental drift. This clearly demonstrates the need for systematic comprehensive multi-instrument comparison analyses.

The allelic modulation of apolipoprotein E expression by oestrogen: potential relevance for Alzheimer's disease.

BACKGROUND: The epsilon4 allele of the apolipoprotein E (APOE) gene is a major genetic risk factor for Alzheimer's disease but appears to be associated with greater risk in women than in men. Some studies suggest that the level of APOE may of its own modulate the risk for Alzheimer's disease. Sex differences and an apparent benefit of oestrogen therapy suggest a role for oestrogen. APOE expression is influenced by oestrogen and oestrogen therapy may not benefit women bearing an APOE epsilon4 allele. These findings suggest an interaction between oestrogen and APOE in the Alzheimer's disease process. AIM: To explore the hypothesis that APOE expression is regulated by a genomic mechanism and is modified by the polymorphisms in APOE associated with risk for Alzheimer's disease. METHODS: In vitro binding studies were undertaken between oestrogen receptors and fragments of the human APOE gene. APOE gene expression was studied to investigate a possible functional interaction. RESULTS: APOE epsilon2/epsilon3/epsilon4 coding and -219 G/T promoter polymorphisms influenced binding to the oestrogen receptor and altered transcriptional activity in response to oestrogen. CONCLUSIONS: An allele dependent modulation of oestrogen induced regulation of APOE might be involved in the increased risk for Alzheimer's disease in women bearing an epsilon4 allele.