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1.  Anti-tumor agent calixarene 0118 targets human galectin-1 as an allosteric inhibitor of carbohydrate binding 
Journal of medicinal chemistry  2012;55(11):5121-5129.
Calix[4]arene compound 0118 is an angiostatic agent that inhibits tumor growth in mice. Although 0118 is a topomimetic of galectin-1-targeting angiostatic amphipathic peptide anginex, we had yet to prove that 0118 targets galectin-1. Galectin-1 is involved in pathological disorders like tumor endothelial cell adhesion and migration and therefore presents a relevant target for therapeutic intervention against cancer. Here, 15N-1H HSQC NMR spectroscopy demonstrates that 0118 indeed targets galectin-1 at a site away from the lectin’s carbohydrate binding site, and thereby attenuates lactose binding to the lectin. Flow cytometry and agglutination assays show that 0118 attenuates binding of galectin-1 to cell surface glycans, and the inhibition of cell proliferation by 0118 is found to be correlated with the cellular expression of the lectin. In general, our data indicate that 0118 targets galectin-1 as an allosteric inhibitor of glycan/carbohydrate binding. This work contributes to the clinical development of anti-tumor calixarene compound 0118.
doi:10.1021/jm300014q
PMCID: PMC4242090  PMID: 22575017
NMR; protein; lectin; glycan; galactose
2.  TOPOMIMETICS OF AMPHIPATHIC β-SHEET AND HELIX-FORMING BACTERICIDAL PEPTIDES NEUTRALIZE LIPOPOLYSACCHARIDE ENDOTOXINS 
Journal of medicinal chemistry  2006;49(26):7754-7765.
Release of lipopolysaccharide (LPS) endotoxin from Gram negative bacterial membranes triggers macrophages to produce large quantities of cytokines that can lead to septic shock and eventual death. Agents that bind to and neutralize LPS may provide a means to clinically prevent septic shock upon bacterial infection. Previously, we reported the design of antibacterial helix peptide SC4 and β-sheet-forming βpep peptides that neutralize LPS in vitro. We hypothesized that the ability of these and other such peptides to neutralize LPS rested in the common denominator of positively charged amphipathic structure. Here, we describe the design and synthesis of non-peptide, calixarene-based helix/sheet topomimetics that mimic the folded conformations of these peptides in their molecular dimensions, amphipathic surface topology, and compositional properties. From a small library of topomimetics, we identified several compounds that neutralize LPS in the 10−8 M range, making them as effective as bactericidal/permeability increasing (BPI) protein and polymyxin B. In an endotoxemia mouse model, three of the most in vitro effective topomimetics are shown to be at least partially protective against challenges of LPS from different bacterial species. NMR studies provide mechanistic insight by suggesting the site of molecular interaction between topomimetics and the lipid A component of LPS, with binding being mediated by electrostatic and hydrophobic interactions. This research contributes to the development of pharmaceutical agents against endotoxemia and septic shock.
doi:10.1021/jm0610447
PMCID: PMC4242098  PMID: 17181157
3.  Polycationic calixarene PTX013, a potent cytotoxic agent against tumors and drug resistant cancer 
Investigational new drugs  2013;31(5):1142-1150.
Summary
Previously, we reported on the anti-tumor activities of two designed calix[4]arene-based topomimetics (PTX008 and PTX009) of the amphipathic, angiostatic peptide Anginex. Here, we chemically modified the hydrophobic and hydrophilic faces of PTX008 and PTX009, and discovered new calixarene compounds that are more potent, cytotoxic anti-tumor agents. One of them, PTX013, is particularly effective at inhibiting the growth of several human cancer cell lines, as well as drug resistant cancer cells. Mechanistically, PTX013 induces cell cycle arrest in sub-G1 and G0/G1 phases of e.g. SQ20B cells, a radio-resistant human head and neck carcinoma model. In the syngeneic B16F10 melanoma tumor mouse model, PTX013 (0.5 mg/Kg) inhibits tumor growth by about 50-fold better than parent PTX008. A preliminary pharmacodynamics study strongly suggests that PTX013 exhibits good in vivo exposure and a relatively long half-life. Overall, this research contributes to the discovery of novel therapeutics as potentially useful agents against cancer in the clinic.
doi:10.1007/s10637-013-9932-0
PMCID: PMC4242102  PMID: 23392775
Calixarenes; Galectin-1; Structure-activity relationships; Therapeutics
4.  Enhancement of T-cell mediated anti-tumor response: angiostatic adjuvant to immunotherapy against cancer 
Purpose
Tumor-released pro-angiogenic factors suppress endothelial adhesion molecule (EAM) expression and prevent leukocyte extravasation into the tumor. This is one reason why immunotherapy has met with limited success in the clinic. We hypothesized that overcoming EAM suppression with angiogenesis inhibitors would increase leukocyte extravasation and subsequently enhance the effectiveness of cellular immunotherapy.
Experimental Design
Intravital microscopy, multiple-color flow cytometry, immunohistochemistry, and various tumor mouse (normal and T cell-deficient) models were used to investigate the temporal dynamics of cellular and molecular events that occur in the tumor microenvironment during tumor progression and angiostatic intervention.
Results
We report that while EAM levels and T cell infiltration are highly attenuated early on in tumor growth, angiostatic therapy modulates these effects. In tumor models with normal and T cell-deficient mice, we demonstrate the active involvement of the adaptive immune system in cancer, and differentiate anti-angiogenic effects from anti-angiogenic-mediated enhancement of immuno-extravasation. Our results indicate that a compromised immune response in tumors can be obviated by the use of anti-angiogenic agents. Lastly, with adoptive transfer studies in mice, we demonstrate that a phased combination of angiostatic therapy and T cell transfer significantly (P<0.0013) improves tumor growth inhibition.
Conclusions
This research contributes to understanding the cellular mechanism of action of angiostatic agents and the immune response within the tumor microenvironment, in particular as a consequence of the temporal dynamics of EAM levels. Moreover, our results suggest that adjuvant therapy with angiogenesis inhibitors holds promise for cellular immunotherapy in the clinic.
doi:10.1158/1078-0432.CCR-10-2443
PMCID: PMC4242153  PMID: 21252159
tumor microenvironment; adaptive immune system; angiogenesis; endothelial cell anergy; anginex
5.  The Role of Variation at AβPP, PSEN1, PSEN2, and MAPT in Late Onset Alzheimer’s Disease 
Gerrish, Amy | Russo, Giancarlo | Richards, Alexander | Moskvina, Valentina | Ivanov, Dobril | Harold, Denise | Sims, Rebecca | Abraham, Richard | Hollingworth, Paul | Chapman, Jade | Hamshere, Marian | Pahwa, Jaspreet Singh | Dowzell, Kimberley | Williams, Amy | Jones, Nicola | Thomas, Charlene | Stretton, Alexandra | Morgan, Angharad R. | Lovestone, Simon | Powell, John | Proitsi, Petroula | Lupton, Michelle K. | Brayne, Carol | Rubinsztein, David C. | Gill, Michael | Lawlor, Brian | Lynch, Aoibhinn | Morgan, Kevin | Brown, Kristelle S. | Passmore, Peter A. | Craig, David | McGuinness, Bernadette | Todd, Stephen | Johnston, Janet A. | Holmes, Clive | Mann, David | Smith, A. David | Love, Seth | Kehoe, Patrick G. | Hardy, John | Mead, Simon | Fox, Nick | Rossor, Martin | Collinge, John | Maier, Wolfgang | Jessen, Frank | Kölsch, Heike | Heun, Reinhard | Schürmann, Britta | van den Bussche, Hendrik | Heuser, Isabella | Kornhuber, Johannes | Wiltfang, Jens | Dichgans, Martin | Frölich, Lutz | Hampel, Harald | Hüll, Michael | Rujescu, Dan | Goate, Alison M. | Kauwe, John S. K. | Cruchaga, Carlos | Nowotny, Petra | Morris, John C. | Mayo, Kevin | Livingston, Gill | Bass, Nicholas J. | Gurling, Hugh | McQuillin, Andrew | Gwilliam, Rhian | Deloukas, Panagiotis | Davies, Gail | Harris, Sarah E. | Starr, John M. | Deary, Ian J. | Al-Chalabi, Ammar | Shaw, Christopher E. | Tsolaki, Magda | Singleton, Andrew B. | Guerreiro, Rita | Mühleisen, Thomas W. | Nöthen, Markus M. | Moebus, Susanne | Jöckel, Karl-Heinz | Klopp, Norman | Wichmann, H-Erich | Carrasquillo, Minerva M | Pankratz, V Shane | Younkin, Steven G. | Jones, Lesley | Holmans, Peter A. | O’Donovan, Michael C. | Owen, Michael J. | Williams, Julie
Rare mutations in AβPP, PSEN1, and PSEN2 cause uncommon early onset forms of Alzheimer’s disease (AD), and common variants in MAPT are associated with risk of other neurodegenerative disorders. We sought to establish whether common genetic variation in these genes confer risk to the common form of AD which occurs later in life (>65 years). We therefore tested single-nucleotide polymorphisms at these loci for association with late-onset AD (LOAD) in a large case-control sample consisting of 3,940 cases and 13,373 controls. Single-marker analysis did not identify any variants that reached genome-wide significance, a result which is supported by other recent genome-wide association studies. However, we did observe a significant association at the MAPT locus using a gene-wide approach (p = 0.009). We also observed suggestive association between AD and the marker rs9468, which defines the H1 haplotype, an extended haplotype that spans the MAPT gene and has previously been implicated in other neurodegenerative disorders including Parkinson’s disease, progressive supranuclear palsy, and corticobasal degeneration. In summary common variants at AβPP, PSEN1, and PSEN2 and MAPT are unlikely to make strong contributions to susceptibility for LOAD. However, the gene-wide effect observed at MAPT indicates a possible contribution to disease risk which requires further study.
doi:10.3233/JAD-2011-110824
PMCID: PMC4118466  PMID: 22027014
Alzheimer’s disease; amyloid-β protein precursor; genetics; human; MAPT protein; PSEN1 protein; PSEN2 protein
6.  Lactose binding to human galectin-7 (p53-induced gene 1) induces long-range effects through the protein resulting in increased dimer stability and evidence for positive cooperativity 
Glycobiology  2013;23(5):508-523.
The product of p53-induced gene 1 is a member of the galectin family, i.e., galectin-7 (Gal-7). To move beyond structural data by X-ray diffraction, we initiated the study of the lectin by nuclear magnetic resonance (NMR) and circular dichroism spectroscopies, and molecular dynamics (MD) simulations. In concert, our results indicate that lactose binding to human Gal-7 induces long-range effects (minor conformational shifts and changes in structural dynamics) throughout the protein that result in stabilization of the dimer state, with evidence for positive cooperativity. Monte Carlo fits of 15N-Gal-7 HSQC titrations with lactose using a two-site model yield K1 = 0.9 ± 0.6 × 103 M−1 and K2 = 3.4 ± 0.8 × 103 M−1. Ligand binding-induced stabilization of the Gal-7 dimer was supported by several lines of evidence: MD-based calculations of interaction energies between ligand-loaded and ligand-free states, gel filtration data and hetero-FRET spectroscopy that indicate a highly reduced tendency for dimer dissociation in the presence of lactose, CD-based thermal denaturation showing that the transition temperature of the lectin is significantly increased in the presence of lactose, and saturation transfer difference (STD) NMR using a molecular probe of the monomer state whose presence is diminished in the presence of lactose. MD simulations with the half-loaded ligand-bound state also provided insight into how allosteric signaling may occur. Overall, our results reveal long-range effects on Gal-7 structure and dynamics, which factor into entropic contributions to ligand binding and allow further comparisons with other members of the galectin family.
doi:10.1093/glycob/cwt005
PMCID: PMC3608354  PMID: 23376190
circular dichroism; FRET; galectin; molecular dynamics; NMR
7.  C9ORF72 hexanucleotide repeat expansions in clinical Alzheimer’s disease 
JAMA neurology  2013;70(6):736-741.
Objective
Hexanucleotide repeat expansions in C9ORF72 underlie a significant fraction of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). This study investigates the frequency of C9ORF72 repeat expansions in clinically diagnosed late-onset Alzheimer’s disease (AD).
Design, setting and patients
This case-control study genotyped the C9ORF72 repeat expansion in 872 unrelated familial AD cases and 888 controls recruited as part of the NIA-LOAD cohort, a multi-site collaboration studying 1000 families with two or more individuals clinically diagnosed with late-onset-AD.
Main Outcome Measure
We determined the presence or absence of the C9ORF72 repeat expansion by repeat-primed PCR, the length of the longest non-expanded allele, segregation of the genotype with disease, and clinical features of repeat expansion carriers.
Results
Three families showed large C9ORF72 hexanucleotide repeat expansions. Two additional families carried more than 30 repeats. Segregation with disease could be demonstrated in 3 families. One affected expansion carrier had neuropathology compatible with AD. In the NIA-LOAD series, the C9ORF72 repeat expansions constituted the second most common pathogenic mutation, just behind the PSEN1 A79V mutation, highlighting the heterogeneity of clinical presentations associated with repeat expansions.
Interpretation
C9ORF72 repeat expansions explain a small proportion of patients with a clinical presentation indistinguishable from AD, and highlight the necessity of screening “FTD genes” in clinical AD cases with strong family history.
doi:10.1001/2013.jamaneurol.537
PMCID: PMC3681841  PMID: 23588422
8.  Cerebrospinal fluid APOE levels: an endophenotype for genetic studies for Alzheimer's disease 
Human Molecular Genetics  2012;21(20):4558-4571.
The apolipoprotein E (APOE) genotype is the major genetic risk factor for Alzheimer's disease (AD). We have access to cerebrospinal fluid (CSF) and plasma APOE protein levels from 641 individuals and genome-wide genotyped data from 570 of these samples. The aim of this study was to test whether CSF or plasma APOE levels could be a useful endophenotype for AD and to identify genetic variants associated with APOE levels. We found that CSF (P = 8.15 × 10−4) but not plasma (P = 0.071) APOE protein levels are significantly associated with CSF Aβ42 levels. We used Mendelian randomization and genetic variants as instrumental variables to confirm that the association of CSF APOE with CSF Aβ42 levels and clinical dementia rating (CDR) is not because of a reverse causation or confounding effect. In addition the association of CSF APOE with Aβ42 levels was independent of the APOE ɛ4 genotype, suggesting that APOE levels in CSF may be a useful endophenotype for AD. We performed a genome-wide association study to identify genetic variants associated with CSF APOE levels: the APOE ɛ4 genotype was the strongest single-genetic factor associated with CSF APOE protein levels (P = 6.9 × 10−13). In aggregate, the Illumina chip single nucleotide polymorphisms explain 72% of the variability in CSF APOE protein levels, whereas the APOE ɛ4 genotype alone explains 8% of the variability. No other genetic variant reached the genome-wide significance threshold, but nine additional variants exhibited a P-value <10−6. Pathway mining analysis indicated that these nine additional loci are involved in lipid metabolism (P = 4.49 × 10−9).
doi:10.1093/hmg/dds296
PMCID: PMC3459471  PMID: 22821396
9.  Structural features for α-galactomannan binding to galectin-1 
Glycobiology  2011;22(4):543-551.
Galectins have a highly conserved carbohydrate-binding domain to which a variety of galactose-containing saccharides, both β- and α-galactosides, can interact with varying degrees of affinity. Recently, we demonstrated that the relatively large α(1 → 6)-d-galacto-β(1 → 4)-d-mannan (Davanat) binds galectin-1 (gal-1) primarily at an alternative carbohydrate-binding domain. Here, we used a series of α-galactomannans (GMs) that vary in their mannose-to-galactose ratios for insight into an optimal structural signature for GM binding to gal-1. Heteronuclear single-quantum coherence nuclear magnetic resonance spectroscopy with 15N-labeled gal-1 and statistical modeling suggest that the optimal signature consists of α-d-galactopyranosyl doublets surrounded by regions of about four or more “naked” mannose residues. These relatively large and complex GMs all appear to interact with varying degrees at essentially the same binding surface on gal-1 that includes the Davanat alternative binding site and elements of the canonical β-galactoside-binding region. The use of two small, well-defined GMs [61-α(1 → 6)-d-galactosyl-β-d-mannotriaose and 63,64-di-α(1 → 6)-d-galactosyl-β-d-mannopentaose] helped characterize how GMs, in general, interact in part with the canonical site. Overall, our findings contribute to better understanding interactions of gal-1 with larger, complex polysaccharides and to the development of GM-based therapeutics for clinical use.
doi:10.1093/glycob/cwr173
PMCID: PMC3287016  PMID: 22156919
galactose; glycan; lectin; NMR; protein
10.  Structural aspects of binding of α-linked digalactosides to human galectin-1 
Glycobiology  2011;21(12):1627-1641.
By definition, adhesion/growth-regulatory galectins are known for their ability to bind β-galactosides such as Galβ(1 → 4)Glc (lactose). Indications for affinity of human galectin-1 to α-linked digalactosides pose questions on the interaction profile with such bound ligands and selection of the galactose moiety for CH–π stacking. These issues are resolved by a combination of 15N–1H heteronuclear single quantum coherence (HSQC) chemical shift and saturation transfer difference nuclear magnetic resonance (STD NMR) epitope mappings with docking analysis, using the α(1 → 3/4)-linked digalactosides and also Galα(1 → 6)Glc (melibiose) as test compounds. The experimental part revealed interaction with the canonical lectin site, and this preferentially via the non-reducing-end galactose moiety. Low-energy conformers appear to be selected without notable distortion, as shown by molecular dynamics simulations. With the α(1 → 4) disaccharide, however, the typical CH–π interaction is significantly diminished, yet binding appears to be partially compensated for by hydrogen bonding. Overall, these findings reveal that the type of α-linkage in digalactosides has an impact on maintaining CH–π interactions and the pattern of hydrogen bonding, explaining preference for the α(1 → 3) linkage. Thus, this lectin is able to accommodate both α- and β-linked galactosides at the same site, with major contacts to the non-reducing-end sugar unit.
doi:10.1093/glycob/cwr083
PMCID: PMC3219418  PMID: 21712397
agglutinin; glycolipid; glycoprotein; lectin; sugar code
11.  No consistent evidence for association between mtDNA variants and Alzheimer disease 
Hudson, G. | Sims, R. | Harold, D. | Chapman, J. | Hollingworth, P. | Gerrish, A. | Russo, G. | Hamshere, M. | Moskvina, V. | Jones, N. | Thomas, C. | Stretton, A. | Holmans, P.A. | O'Donovan, M.C. | Owen, M.J. | Williams, J. | Chinnery, P.F. | Harold, Denise | Abraham, Richard | Hollingworth, Paul | Sims, Rebecca | Gerrish, Amy | Chapman, Jade | Russo, Giancarlo | Hamshere, Marian | Pahwa, Jaspreet Singh | Moskvina, Valentina | Dowzell, Kimberley | Williams, Amy | Jones, Nicola | Thomas, Charlene | Stretton, Alexandra | Morgan, Angharad | Lovestone, Simon | Powell, John | Proitsi, Petroula | Lupton, Michelle K | Brayne, Carol | Rubinsztein, David C. | Gill, Michael | Lawlor, Brian | Lynch, Aoibhinn | Morgan, Kevin | Brown, Kristelle | Passmore, Peter | Craig, David | McGuinness, Bernadette | Todd, Stephen | Johnston, Janet | Holmes, Clive | Mann, David | Smith, A. David | Love, Seth | Kehoe, Patrick G. | Hardy, John | Mead, Simon | Fox, Nick | Rossor, Martin | Collinge, John | Maier, Wolfgang | Jessen, Frank | Heun, Reiner | Kölsch, Heike | Schürmann, Britta | van den Bussche, Hendrik | Heuser, Isabella | Kornhuber, Johannes | Wiltfang, Jens | Dichgans, Martin | Frölich, Lutz | Hampel, Harald | Hüll, Michael | Rujescu, Dan | Goate, Alison | Kauwe, John S.K. | Cruchaga, Carlos | Nowotny, Petra | Morris, John C. | Mayo, Kevin | Livingston, Gill | Bass, Nicholas J. | Gurling, Hugh | McQuillin, Andrew | Gwilliam, Rhian | Deloukas, Panagiotis | Holmans, Peter | O'Donovan, Michael | Owen, Michael J. | Williams, Julie
Neurology  2012;78(14):1038-1042.
Objective:
Although several studies have described an association between Alzheimer disease (AD) and genetic variation of mitochondrial DNA (mtDNA), each has implicated different mtDNA variants, so the role of mtDNA in the etiology of AD remains uncertain.
Methods:
We tested 138 mtDNA variants for association with AD in a powerful sample of 4,133 AD case patients and 1,602 matched controls from 3 Caucasian populations. Of the total population, 3,250 case patients and 1,221 elderly controls met the quality control criteria and were included in the analysis.
Results:
In the largest study to date, we failed to replicate the published findings. Meta-analysis of the available data showed no evidence of an association with AD.
Conclusion:
The current evidence linking common mtDNA variations with AD is not compelling.
doi:10.1212/WNL.0b013e31824e8f1d
PMCID: PMC3317529  PMID: 22442439
12.  Association and Expression analyses with SNPs in TOMM40 in Alzheimer’s Disease 
Archives of neurology  2011;68(8):1013-1019.
Objectives
Apolipoprotein E (APOE) is the most statistically significant genetic risk factor for late-onset Alzheimer’s disease (LOAD). The linkage disequilibrium pattern around the APOE gene has made it difficult to determine whether all of the association signal is derived from APOE or if there is an independent signal from a nearby gene. In this study we attempted to replicate a recently reported association of APOE 3-TOMM40 haplotypes with risk and age at onset.
Design
We used standard techniques to genotype several polymorphisms in the APOE-TOMM40 region in a large case-control series, in a series with cerebrospinal fluid biomarker data and in brain tissue.
Results
We failed to replicate the previously reported association of the polyT polymorphism (rs10524523) with risk and age at onset. We found a significant association between rs10524523 and risk for LOAD among APOE 33 homozygotes but in the opposite direction to the previously reported association (the very-long allele was underrepresented in cases compared to controls in our study (allele frequency: 0.41 vs. 0.48 respectively; p=0.004)). We found no association between rs10524523 and CSF tau or Aβ42 levels or TOMM40 or APOE gene expression.
Conclusions
Although we were not able to replicate the earlier association between the APOE 3-TOMM40 haplotypes and age at onset, we did observe that the polyT polymorphism is associated with risk for LOAD among APOE 33 homozygotes in a large case-control series, but in the opposite direction to the previous report. Additional studies in very large samples will be needed to confirm this association.
doi:10.1001/archneurol.2011.155
PMCID: PMC3204798  PMID: 21825236
14.  TMEM106B gene polymorphism is associated with age at onset in granulin mutation carriers and plasma granulin protein levels 
Archives of neurology  2011;68(5):581-586.
Objective
A recent genome-wide association study for frontotemporal lobar degeneration with TAR DNA-binding protein inclusions (FTLD-TDP), identified rs1990622 (TMEM106B) as a risk factor for FTLD-TDP. In this study we tested whether rs1990622 is associated with age at onset (AAO) in granulin (GRN) mutation carriers and with plasma GRN levels in mutation carriers and healthy elderly individuals.
Design
Rs1990622 was genotyped in GRN mutation carriers and tested for association with AAO using the Kaplan-Meier and a Cox proportional hazards model.
Subjects
We analyzed 50 affected and unaffected GRN mutation carriers from four previously reported FTLD-TDP families (HDDD1, FD1, HDDD2 and the Karolinska family). GRN plasma levels were also measured in 73 healthy, elderly individuals.
Results
The risk allele of rs1990622 is associated with a mean decrease of the age at onset of thirteen years (p=9.9×10−7), with lower plasma granulin levels in both healthy older adults (p = 4×10−4) and GRN mutation carriers (p=0.0027). Analysis of the HAPMAP database identified a non-synonymous single nucleotide polymorphism, rs3173615 (T185S) in perfect linkage disequilibrium with rs1990622.
Conclusions
The association of rs1990622 with AAO explains, in part, the wide range in the age at onset of disease among GRN mutation carriers. We hypothesize that rs1990622 or another variant in linkage disequilibrium could act in a manner similar to APOE in Alzheimer’s disease, increasing risk for disease in the general population and modifying AAO in mutation carriers. Our results also suggest that genetic variation in TMEM106B may influence risk for FTLD-TDP by modulating secreted levels of GRN.
doi:10.1001/archneurol.2010.350
PMCID: PMC3090529  PMID: 21220649
15.  Rare Variants in APP, PSEN1 and PSEN2 Increase Risk for AD in Late-Onset Alzheimer's Disease Families 
PLoS ONE  2012;7(2):e31039.
Pathogenic mutations in APP, PSEN1, PSEN2, MAPT and GRN have previously been linked to familial early onset forms of dementia. Mutation screening in these genes has been performed in either very small series or in single families with late onset AD (LOAD). Similarly, studies in single families have reported mutations in MAPT and GRN associated with clinical AD but no systematic screen of a large dataset has been performed to determine how frequently this occurs. We report sequence data for 439 probands from late-onset AD families with a history of four or more affected individuals. Sixty sequenced individuals (13.7%) carried a novel or pathogenic mutation. Eight pathogenic variants, (one each in APP and MAPT, two in PSEN1 and four in GRN) three of which are novel, were found in 14 samples. Thirteen additional variants, present in 23 families, did not segregate with disease, but the frequency of these variants is higher in AD cases than controls, indicating that these variants may also modify risk for disease. The frequency of rare variants in these genes in this series is significantly higher than in the 1,000 genome project (p = 5.09×10−5; OR = 2.21; 95%CI = 1.49–3.28) or an unselected population of 12,481 samples (p = 6.82×10−5; OR = 2.19; 95%CI = 1.347–3.26). Rare coding variants in APP, PSEN1 and PSEN2, increase risk for or cause late onset AD. The presence of variants in these genes in LOAD and early-onset AD demonstrates that factors other than the mutation can impact the age at onset and penetrance of at least some variants associated with AD. MAPT and GRN mutations can be found in clinical series of AD most likely due to misdiagnosis. This study clearly demonstrates that rare variants in these genes could explain an important proportion of genetic heritability of AD, which is not detected by GWAS.
doi:10.1371/journal.pone.0031039
PMCID: PMC3270040  PMID: 22312439
16.  Exome-Sequencing Confirms DNAJC5 Mutations as Cause of Adult Neuronal Ceroid-Lipofuscinosis 
PLoS ONE  2011;6(11):e26741.
We performed whole-exome sequencing in two autopsy-confirmed cases and an elderly unaffected control from a multigenerational family with autosomal dominant neuronal ceroid lipofuscinosis (ANCL). A novel single-nucleotide variation (c.344T>G) in the DNAJC5 gene was identified. Mutational screening in an independent family with autosomal dominant ANCL found an in-frame single codon deletion (c.346_348 delCTC) resulting in a deletion of p.Leu116del. These variants fulfill all genetic criteria for disease-causing mutations: they are found in unrelated families with the same disease, exhibit complete segregation between the mutation and the disease, and are absent in healthy controls. In addition, the associated amino acid substitutions are located in evolutionarily highly conserved residues and are predicted to functionally affect the encoded protein (CSPα). The mutations are located in a cysteine-string domain, which is required for membrane targeting/binding, palmitoylation, and oligomerization of CSPα. We performed a comprehensive in silico analysis of the functional and structural impact of both mutations on CSPα. We found that these mutations dramatically decrease the affinity of CSPα for the membrane. We did not identify any significant effect on palmitoylation status of CSPα. However, a reduction of CSPα membrane affinity may change its palmitoylation and affect proper intracellular sorting. We confirm that CSPα has a strong intrinsic aggregation propensity; however, it is not modified by the mutations. A complementary disease-network analysis suggests a potential interaction with other NCLs genes/pathways. This is the first replication study of the identification of DNAJC5 as the disease-causing gene for autosomal dominant ANCL. The identification of the novel gene in ANCL will allow us to gain a better understanding of the pathological mechanism of ANCLs and constitutes a great advance toward the development of new molecular diagnostic tests and may lead to the development of potential therapies.
doi:10.1371/journal.pone.0026741
PMCID: PMC3208569  PMID: 22073189
17.  Common variants in ABCA7, MS4A6A/MS4A4E, EPHA1, CD33 and CD2AP are associated with Alzheimer’s disease 
Hollingworth, Paul | Harold, Denise | Sims, Rebecca | Gerrish, Amy | Lambert, Jean-Charles | Carrasquillo, Minerva M | Abraham, Richard | Hamshere, Marian L | Pahwa, Jaspreet Singh | Moskvina, Valentina | Dowzell, Kimberley | Jones, Nicola | Stretton, Alexandra | Thomas, Charlene | Richards, Alex | Ivanov, Dobril | Widdowson, Caroline | Chapman, Jade | Lovestone, Simon | Powell, John | Proitsi, Petroula | Lupton, Michelle K | Brayne, Carol | Rubinsztein, David C | Gill, Michael | Lawlor, Brian | Lynch, Aoibhinn | Brown, Kristelle S | Passmore, Peter A | Craig, David | McGuinness, Bernadette | Todd, Stephen | Holmes, Clive | Mann, David | Smith, A David | Beaumont, Helen | Warden, Donald | Wilcock, Gordon | Love, Seth | Kehoe, Patrick G | Hooper, Nigel M | Vardy, Emma R. L. C. | Hardy, John | Mead, Simon | Fox, Nick C | Rossor, Martin | Collinge, John | Maier, Wolfgang | Jessen, Frank | Schürmann, Britta | Rüther, Eckart | Heun, Reiner | Kölsch, Heike | van den Bussche, Hendrik | Heuser, Isabella | Kornhuber, Johannes | Wiltfang, Jens | Dichgans, Martin | Frölich, Lutz | Hampel, Harald | Hüll, Michael | Gallacher, John | Rujescu, Dan | Giegling, Ina | Goate, Alison M | Kauwe, John S K | Cruchaga, Carlos | Nowotny, Petra | Morris, John C | Mayo, Kevin | Sleegers, Kristel | Bettens, Karolien | Engelborghs, Sebastiaan | De Deyn, Peter P | Van Broeckhoven, Christine | Livingston, Gill | Bass, Nicholas J | Gurling, Hugh | McQuillin, Andrew | Gwilliam, Rhian | Deloukas, Panagiotis | Al-Chalabi, Ammar | Shaw, Christopher E | Tsolaki, Magda | Singleton, Andrew B | Guerreiro, Rita | Mühleisen, Thomas W | Nöthen, Markus M | Moebus, Susanne | Jöckel, Karl-Heinz | Klopp, Norman | Wichmann, H-Erich | Pankratz, V Shane | Sando, Sigrid B | Aasly, Jan O | Barcikowska, Maria | Wszolek, Zbigniew K | Dickson, Dennis W | Graff-Radford, Neill R | Petersen, Ronald C | van Duijn, Cornelia M | Breteler, Monique MB | Ikram, M Arfan | DeStefano, Anita L | Fitzpatrick, Annette L | Lopez, Oscar | Launer, Lenore J | Seshadri, Sudha | Berr, Claudine | Campion, Dominique | Epelbaum, Jacques | Dartigues, Jean-François | Tzourio, Christophe | Alpérovitch, Annick | Lathrop, Mark | Feulner, Thomas M | Friedrich, Patricia | Riehle, Caterina | Krawczak, Michael | Schreiber, Stefan | Mayhaus, Manuel | Nicolhaus, S | Wagenpfeil, Stefan | Steinberg, Stacy | Stefansson, Hreinn | Stefansson, Kari | Snædal, Jon | Björnsson, Sigurbjörn | Jonsson, Palmi V. | Chouraki, Vincent | Genier-Boley, Benjamin | Hiltunen, Mikko | Soininen, Hilkka | Combarros, Onofre | Zelenika, Diana | Delepine, Marc | Bullido, Maria J | Pasquier, Florence | Mateo, Ignacio | Frank-Garcia, Ana | Porcellini, Elisa | Hanon, Olivier | Coto, Eliecer | Alvarez, Victoria | Bosco, Paolo | Siciliano, Gabriele | Mancuso, Michelangelo | Panza, Francesco | Solfrizzi, Vincenzo | Nacmias, Benedetta | Sorbi, Sandro | Bossù, Paola | Piccardi, Paola | Arosio, Beatrice | Annoni, Giorgio | Seripa, Davide | Pilotto, Alberto | Scarpini, Elio | Galimberti, Daniela | Brice, Alexis | Hannequin, Didier | Licastro, Federico | Jones, Lesley | Holmans, Peter A | Jonsson, Thorlakur | Riemenschneider, Matthias | Morgan, Kevin | Younkin, Steven G | Owen, Michael J | O’Donovan, Michael | Amouyel, Philippe | Williams, Julie
Nature genetics  2011;43(5):429-435.
We sought to identify new susceptibility loci for Alzheimer’s disease (AD) through a staged association study (GERAD+) and by testing suggestive loci reported by the Alzheimer’s Disease Genetic Consortium (ADGC). First, we undertook a combined analysis of four genome-wide association datasets (Stage 1) and identified 10 novel variants with P≤1×10−5. These were tested 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 provide compelling evidence that ABCA7 (meta-P 4.5×10−17; including ADGC meta-P=5.0×10−21) and the MS4A gene cluster (rs610932, meta-P=1.8×10−14; including ADGC meta-P=1.2×10−16; rs670139, meta-P=1.4×10−9; including ADGC meta-P=1.1×10−10) are novel susceptibility loci for AD. Second, we observed independent evidence for association for three suggestive loci reported by the ADGC GWAS, which when combined shows genome-wide significance: CD2AP (GERAD+ P=8.0×10−4; including ADGC meta-P=8.6×10−9), CD33 (GERAD+ P=2.2×10−4; including ADGC meta-P=1.6×10−9) and EPHA1 (GERAD+ P=3.4×10−4; including ADGC meta-P=6.0×10−10). These findings support five novel susceptibility genes for AD.
doi:10.1038/ng.803
PMCID: PMC3084173  PMID: 21460840
18.  Repression of Multiple Myeloma Growth and Preservation of Bone with Combined Radiotherapy and Anti-angiogenic Agent 
Radiation research  2010;173(6):809-817.
The effects of ionizing radiation, with or without the antiangiogenic agent anginex (Ax), on multiple myeloma growth were tested in a SCID-rab mouse model. Mice carrying human multiple myeloma cell-containing pre-implanted bone grafts were treated weekly with various regimens for 8 weeks. Rapid multiple myeloma growth, assessed by bioluminescence intensity (IVIS), human lambda Ig light chain level in serum (ELISA), and the volume of bone grafts (caliper), was observed in untreated mice. Tumor burden in mice receiving combined therapy was reduced to 59% (by caliper), 43% (by ELISA), and 2% (by IVIS) of baseline values after 8 weeks of treatment. Ax or radiation alone slowed but did not stop tumor growth. Four weeks after the withdrawal of the treatments, tumor burden remained minimal in mice given Ax + radiation but increased noticeably in the other three groups. Multiple myeloma suppression by Ax + radiation was accompanied by a marked decrease in the number and activity of osteoclasts in bone grafts assessed by histology. Bone graft integrity was preserved by Ax + radiation but was lost in the other three groups, as assessed by microCT imaging and radiography. These results suggest that radiotherapy, when primed by anti-angiogenic agents, may be a potent therapy for focal multiple myeloma.
doi:10.1667/RR1734.1
PMCID: PMC3137893  PMID: 20518660
19.  Tumour thermotolerance, a physiological phenomenon involving vessel normalisation 
The purpose of this study was to delineate the mechanisms by which stromal components of cancer may induce tumour thermotolerance and exploit alterations in stromal and tumour physiology to enhance radiation therapy. The vascular thermoresponse was monitored by daily one-hour 41.5°C heatings in two murine solid tumour models, SCK murine mammary carcinoma and B16F10 melanoma. A transient increase was seen in overall tumour oxygenation for 2–3 days, followed by a progressive decline in tumour pO2 upon continued daily heatings. Vascular thermotolerance was further studied by treating tumours with different heating strategies, i.e. (1) a single 60 min 41.5°C treatment; (2) two consecutive daily treatments of 41.5°C for 60 min; (3) a single 60 min 43°C treatment or (4) two days of 41.5°C for 60 min followed by treatment with 43°C for 60 min on the third day. Pre-heating tumours with mild temperature hyperthermia induced vascular thermotolerance, which was accompanied by evidence of vessel normalisation, i.e. a decrease in microvessel density and an increase in pericyte coverage. Rational scheduling of fractionated radiation during heat-induced increases in tumour oxygen levels rendered a significantly greater, synergistic, tumour growth inhibition. In vitro clonogenic survival responses of the individual cell types associated (endothelial cells, fibroblasts, pericytes and tumour cells) indicated only a direct cellular thermotolerance in endothelial cells. Overall, this suggests that tumour thermotolerance is a physiological phenomenon mediated through improvement of functional vasculature.
doi:10.3109/02656736.2010.510495
PMCID: PMC3086848  PMID: 21204622
Hyperthermia; radiation; thermotolerance; tumour microenvironment; vessel normalisation
20.  Ovarian tumor growth regression using a combination of vascular targeting agents anginex or topomimetic 0118 and the chemotherapeutic irofulven 
Cancer letters  2008;265(2):270-280.
Combination of chemotherapeutic agents and angiogenesis inhibitors is now commonly employed in the clinic to treat cancer. Here, we used angiostatic agents anginex and 0118, in combination with the chemotherapeutic irofulven, to treat human ovarian tumor xenografts in mice. General linear mixed models were used to statistically analyze tumor growth curves. Overall, combination of a low, non-toxic dose of irofulven with either angiogenesis inhibitor was more effective at inhibiting tumor growth than any of the single-agent therapies. For example, the anginex/irofulven and 0118/irofulven combinations inhibited tumor growth relative to controls by 92% (p<0.0001) and 96% (p<0.0001), respectively, with the 0118/irofulven combinations yielding 100% complete responses. This study suggests that combination therapy of 0118 or anginex and irofulven may be highly effective in the clinical setting.
doi:10.1016/j.canlet.2008.02.048
PMCID: PMC3042303  PMID: 18378392
anginex; irofulven; galectin-1; angiogenesis; chemotherapy
21.  Correction: Genetic Evidence Implicates the Immune System and Cholesterol Metabolism in the Aetiology of Alzheimer's Disease 
Jones, Lesley | Holmans, Peter A. | Hamshere, Marian L. | Harold, Denise | Moskvina, Valentina | Ivanov, Dobril | Pocklington, Andrew | Abraham, Richard | Hollingworth, Paul | Sims, Rebecca | Gerrish, Amy | Pahwa, Jaspreet Singh | Jones, Nicola | Stretton, Alexandra | Morgan, Angharad R. | Lovestone, Simon | Powell, John | Proitsi, Petroula | Lupton, Michelle K. | Brayne, Carol | Rubinsztein, David C. | Gill, Michael | Lawlor, Brian | Lynch, Aoibhinn | Morgan, Kevin | Brown, Kristelle S. | Passmore, Peter A. | Craig, David | McGuinness, Bernadette | Todd, Stephen | Holmes, Clive | Mann, David | Smith, A. David | Love, Seth | Kehoe, Patrick G. | Mead, Simon | Fox, Nick | Rossor, Martin | Collinge, John | Maier, Wolfgang | Jessen, Frank | Schürmann, Britta | van den Bussche, Hendrik | Heuser, Isabella | Peters, Oliver | Kornhuber, Johannes | Wiltfang, Jens | Dichgans, Martin | Frölich, Lutz | Hampel, Harald | Hüll, Michael | Rujescu, Dan | Goate, Alison M. | Kauwe, John S. K. | Cruchaga, Carlos | Nowotny, Petra | Morris, John C. | Mayo, Kevin | Livingston, Gill | Bass, Nicholas J. | Gurling, Hugh | McQuillin, Andrew | Gwilliam, Rhian | Deloukas, Panos | Al-Chalabi, Ammar | Shaw, Christopher E. | Singleton, Andrew B. | Guerreiro, Rita | Mühleisen, Thomas W. | Nöthen, Markus M. | Moebus, Susanne | Jöckel, Karl-Heinz | Klopp, Norman | Wichmann, H.-Erich | Rüther, Eckhard | Carrasquillo, Minerva M. | Pankratz, V. Shane | Younkin, Steven G. | Hardy, John | O'Donovan, Michael C. | Owen, Michael J. | Williams, Julie
PLoS ONE  2011;6(2):10.1371/annotation/a0bb886d-d345-4a20-a82e-adce9b047798.
doi:10.1371/annotation/a0bb886d-d345-4a20-a82e-adce9b047798
PMCID: PMC3039022
22.  Fine Mapping of Genetic Variants in BIN1, CLU, CR1 and PICALM for Association with Cerebrospinal Fluid Biomarkers for Alzheimer's Disease 
PLoS ONE  2011;6(2):e15918.
Recent genome-wide association studies of Alzheimer's disease (AD) have identified variants in BIN1, CLU, CR1 and PICALM that show replicable association with risk for disease. We have thoroughly sampled common variation in these genes, genotyping 355 variants in over 600 individuals for whom measurements of two AD biomarkers, cerebrospinal fluid (CSF) 42 amino acid amyloid beta fragments (Aβ42) and tau phosphorylated at threonine 181 (ptau181), have been obtained. Association analyses were performed to determine whether variants in BIN1, CLU, CR1 or PICALM are associated with changes in the CSF levels of these biomarkers. Despite adequate power to detect effects as small as a 1.05 fold difference, we have failed to detect evidence for association between SNPs in these genes and CSF Aβ42 or ptau181 levels in our sample. Our results suggest that these variants do not affect risk via a mechanism that results in a strong additive effect on CSF levels of Aβ42 or ptau181.
doi:10.1371/journal.pone.0015918
PMCID: PMC3036586  PMID: 21347408
23.  Validating predicted biological effects of Alzheimer’s disease associated SNPs using CSF biomarker levels 
Recent large-scale genetic studies of late-onset Alzheimer’s disease (LOAD) have identified risk variants in CALHM1, GAB2 and SORL1. The mechanisms by which these genes might modulate risk are not definitively known. CALHM1 and SORL1 may alter amyloid-beta (Aβ) levels and GAB2 may influence phosphorylation of the tau protein. In this study we have analyzed disease associated genetic variants in each of these genes for association with cerebrospinal fluid (CSF) Aβ or tau levels in 602 samples from two independent CSF series. We failed to detect association between CSF Aβ42 levels and SNPs in SORL1 despite substantial statistical power to detect association. While we also failed to detect association between variants in GAB2 and CSF tau levels, power to detect this association was limited. Finally, our data suggest that the minor allele of rs2986017, in CALHM1, is marginally associated with CSF Aβ42 levels. This association is consistent with previous reports that this non-synonymous coding substitution results in increased Aβ levels in vitro and provides support for an Aβ-related mechanism for modulating risk for AD.
doi:10.3233/JAD-2010-091711
PMCID: PMC3032214  PMID: 20634593
Alzheimer’s disease; genetics; association; endophenotypes; amyloid; tau; CALHM1; SORL1; GAB2
24.  Genetic Evidence Implicates the Immune System and Cholesterol Metabolism in the Aetiology of Alzheimer's Disease 
Jones, Lesley | Holmans, Peter A. | Hamshere, Marian L. | Harold, Denise | Moskvina, Valentina | Ivanov, Dobril | Pocklington, Andrew | Abraham, Richard | Hollingworth, Paul | Sims, Rebecca | Gerrish, Amy | Pahwa, Jaspreet Singh | Jones, Nicola | Stretton, Alexandra | Morgan, Angharad R. | Lovestone, Simon | Powell, John | Proitsi, Petroula | Lupton, Michelle K. | Brayne, Carol | Rubinsztein, David C. | Gill, Michael | Lawlor, Brian | Lynch, Aoibhinn | Morgan, Kevin | Brown, Kristelle S. | Passmore, Peter A. | Craig, David | McGuinness, Bernadette | Todd, Stephen | Holmes, Clive | Mann, David | Smith, A. David | Love, Seth | Kehoe, Patrick G. | Mead, Simon | Fox, Nick | Rossor, Martin | Collinge, John | Maier, Wolfgang | Jessen, Frank | Schürmann, Britta | van den Bussche, Hendrik | Heuser, Isabella | Peters, Oliver | Kornhuber, Johannes | Wiltfang, Jens | Dichgans, Martin | Frölich, Lutz | Hampel, Harald | Hüll, Michael | Rujescu, Dan | Goate, Alison M. | Kauwe, John S. K. | Cruchaga, Carlos | Nowotny, Petra | Morris, John C. | Mayo, Kevin | Livingston, Gill | Bass, Nicholas J. | Gurling, Hugh | McQuillin, Andrew | Gwilliam, Rhian | Deloukas, Panos | Al-Chalabi, Ammar | Shaw, Christopher E. | Singleton, Andrew B. | Guerreiro, Rita | Mühleisen, Thomas W. | Nöthen, Markus M. | Moebus, Susanne | Jöckel, Karl-Heinz | Klopp, Norman | Wichmann, H.-Erich | Rüther, Eckhard | Carrasquillo, Minerva M. | Pankratz, V. Shane | Younkin, Steven G. | Hardy, John | O'Donovan, Michael C. | Owen, Michael J. | Williams, Julie
PLoS ONE  2010;5(11):e13950.
Background
Late Onset Alzheimer's disease (LOAD) is the leading cause of dementia. Recent large genome-wide association studies (GWAS) identified the first strongly supported LOAD susceptibility genes since the discovery of the involvement of APOE in the early 1990s. We have now exploited these GWAS datasets to uncover key LOAD pathophysiological processes.
Methodology
We applied a recently developed tool for mining GWAS data for biologically meaningful information to a LOAD GWAS dataset. The principal findings were then tested in an independent GWAS dataset.
Principal Findings
We found a significant overrepresentation of association signals in pathways related to cholesterol metabolism and the immune response in both of the two largest genome-wide association studies for LOAD.
Significance
Processes related to cholesterol metabolism and the innate immune response have previously been implicated by pathological and epidemiological studies of Alzheimer's disease, but it has been unclear whether those findings reflected primary aetiological events or consequences of the disease process. Our independent evidence from two large studies now demonstrates that these processes are aetiologically relevant, and suggests that they may be suitable targets for novel and existing therapeutic approaches.
doi:10.1371/journal.pone.0013950
PMCID: PMC2981526  PMID: 21085570
25.  SNPs Associated with Cerebrospinal Fluid Phospho-Tau Levels Influence Rate of Decline in Alzheimer's Disease 
PLoS Genetics  2010;6(9):e1001101.
Alzheimer's Disease (AD) is a complex and multifactorial disease. While large genome-wide association studies have had some success in identifying novel genetic risk factors for AD, case-control studies are less likely to uncover genetic factors that influence progression of disease. An alternative approach to identifying genetic risk for AD is the use of quantitative traits or endophenotypes. The use of endophenotypes has proven to be an effective strategy, implicating genetic risk factors in several diseases, including anemia, osteoporosis and heart disease. In this study we identify a genetic factor associated with the rate of decline in AD patients and present a methodology for identification of other such factors. We have used an established biomarker for AD, cerebrospinal fluid (CSF) tau phosphorylated at threonine 181 (ptau181) levels as an endophenotype for AD, identifying a SNP, rs1868402, in the gene encoding the regulatory sub-unit of protein phosphatase B, associated with CSF ptau181 levels in two independent CSF series . We show no association of rs1868402 with risk for AD or age at onset, but detected a very significant association with rate of progression of disease that is consistent in two independent series . Our analyses suggest that genetic variants associated with CSF ptau181 levels may have a greater impact on rate of progression, while genetic variants such as APOE4, that are associated with CSF Aβ42 levels influence risk and onset but not the rate of progression. Our results also suggest that drugs that inhibit or decrease tau phosphorylation may slow cognitive decline in individuals with very mild dementia or delay the appearance of memory problems in elderly individuals with low CSF Aβ42 levels. Finally, we believe genome-wide association studies of CSF tau/ptau181 levels should identify novel genetic variants which will likely influence rate of progression of AD.
Author Summary
Alzheimer's disease (AD) is the most common neurodegenerative disease affecting more than 4.5 million people in the US. Genetic studies of AD have previously identified pathogenic mutations in three genes (APP, PSEN1 and PSEN2) and polymorphisms in APOE as risk factors. These findings have led to a better understanding of the underlying disease mechanisms. However, half of all AD cases have no known genetic risk factors for disease. Most studies are designed to identify variants associated with risk or age at onset, but rarely cover other important facets of AD, such as disease progression or duration. In this study we have used an established AD biomarker (cerebrospinal fluid tau phosphorylated at threonine 181, ptau181) to find genetic variants that influence levels of ptau181 in the cerebrospinal fluid. This novel and powerful approach has allowed us to identify a genetic factor located in the regulatory subunit of the calcineurin that is also strongly associated with rate of progression of AD. This study is important because it defines a strategy to find novel genetic factors influencing different facets of AD pathobiology including risk, onset and progression.
doi:10.1371/journal.pgen.1001101
PMCID: PMC2940763  PMID: 20862329

Results 1-25 (39)