Julie Williams and colleagues report a genome-wide association study for Alzheimer's disease. They identify variants at the CLU and PICALM loci associated with susceptibility to late-onset Alzheimer's disease. We undertook a two-stage genome-wide association study (GWAS) of Alzheimer's disease (AD) involving over 16,000 individuals, the most powerful AD GWAS to date. In stage 1 (3,941 cases and 7,848 controls), we replicated the established association with the apolipoprotein E (APOE) locus (most significant SNP, rs2075650, P = 1.8 × 10−157) and observed genome-wide significant association with SNPs at two loci not previously associated with the disease: at the CLU (also known as APOJ) gene (rs11136000, P = 1.4 × 10−9) and 5′ to the PICALM gene (rs3851179, P = 1.9 × 10−8). These associations were replicated in stage 2 (2,023 cases and 2,340 controls), producing compelling evidence for association with Alzheimer's disease in the combined dataset (rs11136000, P = 8.5 × 10−10, odds ratio = 0.86; rs3851179, P = 1.3 × 10−9, odds ratio = 0.86).

Julie Williams, Michael Owen and colleagues report staged follow-up and meta-analyses of genome-wide association studies for Alzheimer's disease from the GERAD+ consortium. They identify common variants at ABCA7 and MS4A6A/MS4A4E associated with Alzheimer's disease and support for several additional susceptibility loci. We sought to identify new susceptibility loci for Alzheimer's disease through a staged association study (GERAD+) and by testing suggestive loci reported by the Alzheimer's Disease Genetic Consortium (ADGC) in a companion paper. We undertook a combined analysis of four genome-wide association datasets (stage 1) and identified ten newly associated variants with P ≤ 1 × 10−5. We tested these variants for association in an independent sample (stage 2). Three SNPs at two loci replicated and showed evidence for association in a further sample (stage 3). Meta-analyses of all data provided compelling evidence that ABCA7 (rs3764650, meta P = 4.5 × 10−17; including ADGC data, meta P = 5.0 × 10−21) and the MS4A gene cluster (rs610932, meta P = 1.8 × 10−14; including ADGC data, meta P = 1.2 × 10−16) are new Alzheimer's disease susceptibility loci. We also found independent evidence for association for three loci reported by the ADGC, which, when combined, showed genome-wide significance: CD2AP (GERAD+, P = 8.0 × 10−4; including ADGC data, meta P = 8.6 × 10−9), CD33 (GERAD+, P = 2.2 × 10−4; including ADGC data, meta P = 1.6 × 10−9) and EPHA1 (GERAD+, P = 3.4 × 10−4; including ADGC data, meta P = 6.0 × 10−10).

Alzheimer's disease is a common debilitating dementia with known heritability, for which 20 late onset susceptibility loci have been identified, but more remain to be discovered. This study sought to identify new susceptibility genes, using an alternative gene-wide analytical approach which tests for patterns of association within genes, in the powerful genome-wide association dataset of the International Genomics of Alzheimer's Project Consortium, comprising over 7 m genotypes from 25,580 Alzheimer's cases and 48,466 controls. In addition to earlier reported genes, we detected genome-wide significant loci on chromosomes 8 (TP53INP1, p = 1.4×10⁻⁶) and 14 (IGHV1-67 p = 7.9×10⁻⁸) which indexed novel susceptibility loci. The additional genes identified in this study, have an array of functions previously implicated in Alzheimer's disease, including aspects of energy metabolism, protein degradation and the immune system and add further weight to these pathways as potential therapeutic targets in Alzheimer's disease.

Retinoblastoma is the most common eye malignancy in childhood caused by mutations in the RB1 gene. Both alleles of the RB1 gene must be mutated for tumour development. The initial RB1 mutation may be constitutional germline or somatic (originating in one retinal cell only). Distinguishing between these alternative mechanisms is crucial, with wider implications for management of the patient and family members. Bilateral retinoblastoma is nearly always due to a constitutional mutation; however, approximately 15% of unilateral cases also carry a germline mutation, and identifying these cases is important. This can be achieved by identifying both mutation types in tumour tissue and excluding their presence in blood. Modern eye-saving chemotherapy treatment (systemic, intra-arterial and intravitreal) has resulted in fewer enucleations. As a result, tumour tissue required to identify sporadic RB1 mutation(s) is not always available. Modern intravitreal chemotherapeutic techniques for retinoblastoma involve aspiration of aqueous humour (AH), providing a novel sample source for analysis. By analysing cell-free DNA present in the AH fluid of eyes affected with retinoblastoma, we have developed a screening test capable of detecting somatic RB1 mutations that is comparable to current tests on enucleated tumour tissue. The results obtained with fluid from enucleated eyes were concordant with tumour tissue in all 10 cases analysed. In addition, AH analysis from two patients undergoing intravitreal chemotherapy successfully identified somatic variants in both cases. Our findings suggest that AH fluid is a promising source of tumour-derived DNA in retinoblastoma for analysis.

Polymorphisms in the gene for phosphatidylinositol binding clathrin assembly protein (PICALM), an endocytic-related protein, are associated with a small, increased risk of developing Alzheimer’s disease (AD), strongly suggesting that changes in endocytosis are involved in the aetiology of the disease. We have investigated the involvement of PICALM in the processing of amyloid precursor protein (APP) to understand how PICALM could be linked to the development of AD. We used siRNA to deplete levels of PICALM, its isoforms and clathrin heavy chain in the human brain-derived H4 neuroglioma cell line that expresses endogenous levels of APP. We then used Western blotting, ELISA and immunohistochemistry to detect intra- and extracellular protein levels of endocytic-related proteins, APP and APP metabolites including β-amyloid (Aβ). Levels of functional endocytosis were quantified using ALEXA 488-conjugated transferrin and flow cytometry as a marker of clathrin-mediated endocytosis (CME). Following depletion of all the isoforms of PICALM by siRNA in H4 cells, levels of intracellular APP, intracellular β-C-terminal fragment (β-CTF) and secreted sAPPβ (APP fragments produced by β-secretase cleavage) were significantly reduced but Aβ40 was not affected. Functional endocytosis was significantly reduced after both PICALM and clathrin depletion, highlighting the importance of PICALM in this process. However, depletion of clathrin did not affect APP but did reduce β-CTF levels. PICALM depletion altered the intracellular distribution of clathrin while clathrin reduction affected the subcellular pattern of PICALM labelling. Both PICALM and clathrin depletion reduced the expression of BACE1 mRNA and PICALM siRNA reduced protein levels. Individual depletion of PICALM isoforms 1 and 2 did not affect APP levels while clathrin depletion had a differential effect on the isoforms, increasing isoform 1 while decreasing isoform 2 expression. The depletion of PICALM in brain-derived cells has significant effects on the processing of APP, probably by reducing CME. In particular, it affects the production of β-CTF which is increasingly considered to be an important mediator in AD independent of Aβ. Thus a decrease in PICALM expression in the brain could be beneficial to slow or prevent the development of AD.