Neurocognitive Disorders Essay

HHS Public Access Author manuscript Author Manuscript Mol Psychiatry. Author manuscript; available in PMC 2018 March 23. Published in final edited form as: Mol Psychiatry. 2018 April ; 23(4): 963–972. doi:10.1038/mp.2017.81. Genetic Risk for Schizophrenia and Psychosis in Alzheimer Disease Author Manuscript Mary Ann A. DeMichele-Sweet, Ph.D.a, Elise A. Weamer, M.P.H.b, Lambertus Klei, Ph.D.a, Dylan T. Vranag, Deborah J. Hollingshead, M.S.d, Howard J. Seltman, M.D. Ph.D.h, Rebecca Sims, Ph.D.k, Tatiana Foroud, Ph.D.l, Isabel Hernandez, M.D., Ph.D.i, Sonia Moreno-Graui, Lluís Tárragai, Mercè Boada, M.D., Ph.D.i, Agustin Ruiz, Ph.D.i, Julie Williams, Ph.D.k, Richard Mayeux, MDe, Oscar L. Lopez, M.D.a,b, Etienne L. Sibille, Ph.D.a,j, M. Ilyas Kamboh, Ph.D.c, Bernie Devlin, Ph.D.a, and Robert A. Sweet, M.D.a,b,f aDepartment of Psychiatry, University of Pittsburgh, Pittsburgh, PA bDepartment of Neurology, University of Pittsburgh, Pittsburgh, PA cDepartment of Human Genetics, University of Pittsburgh, Pittsburgh, PA dGenomics Research Core of the Health Sciences Core Research Facilities, University of Pittsburgh, Pittsburgh, PA eDepartments of Neurology, Psychiatry and Epidemiology, Columbia University, New York, NY fVISN Author Manuscript 4 Mental Illness Research, Education and Clinical Center (MIRECC) VA Pittsburgh Healthcare System, Pittsburgh, PA gDepartment of Computational Biology, Carnegie Mellon University, Pittsburgh, PA hDepartment of Statistics, Carnegie Mellon University, Pittsburgh, PA iResearch Center and Memory Clinic of Fundació ACE, Institut Català de Neurociències Aplicades, Barcelona, Spain JDepartments of Psychiatry and of Pharmacology and Toxicology, University of Toronto, Toronto, ON, Canada; Campbell Family Mental Health Research Institute of CAMH, Toronto, ON, Canada kDivision of Psychological Medicine and Clinical Neuroscience, School of Medicine, Cardiff University, Cardiff, UK lMedical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN, USA Author Manuscript Abstract Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms For questions and correspondence please contact: Robert A. Sweet, M.D., Mail: Biomedical Science Tower, Rm W-1645, 3811 O’Hara Street, Pittsburgh, PA 15213-2593. Express Mail: Biomedical Science Tower, Rm W-1645, Lothrop and Terrace Streets, Pittsburgh, PA 15213-2593. Phone: 412-624-0064, Fax: 412-624-9910, sweetra@upmc.edu, Web: http://www.wpic.pitt.edu/research/ sweetlab/. Neurocognitive Disorders Essay

Conflict of Interest: MAAD-S, EAW, LK, DJH, DTV, DJH, HJS, RS, TF, IH, SM-G, LT, MB, AR, JW, RM, OLL, ELS, MIK, BD, and RAS have no conflicts to report. DeMichele-Sweet et al. Page 2 Author Manuscript Author Manuscript Psychotic symptoms, defined as the occurrence of delusions or hallucinations, are frequent in Alzheimer Disease, affecting ~ 40% to 60% of individuals with AD (AD with psychosis, AD+P). In comparison to AD subjects without psychosis, AD+P subjects have more rapid cognitive decline and poor outcomes. Prior studies have estimated the heritability of psychosis in AD at 61%, but the underlying genetic sources of this risk are not known. We evaluated a Discovery Cohort of 2876 AD subjects with (N=1761) or without psychosis (N=1115). All subjects were genotyped using a custom genotyping array designed to evaluate SNPs with evidence of genetic association with AD+P and include SNPs affecting or putatively affecting risk for schizophrenia and Alzheimer disease. Results were replicated in an independent cohort of 2194 AD subjects with (N=734) or without psychosis (N=1460). We found that AD+P is associated with polygenic risk for a set of novel loci and inversely associated with polygenic risk for schizophrenia. Among the biologic pathways identified by the associations of schizophrenia SNPs with AD+P are endosomal trafficking, autophagy, and calcium channel signaling. These findings provide the first clear demonstration that AD+P is associated with common genetic variation. In addition, they provide an unbiased link between polygenic risk for schizophrenia and a lower risk of psychosis in AD. This provides an opportunity to leverage progress made in identifying the biologic effects of schizophrenia alleles to identify novel mechanisms protecting against more rapid cognitive decline and psychosis risk in AD. Introduction Author Manuscript Psychotic symptoms, defined as the occurrence of delusions or hallucinations, are frequent in Alzheimer Disease (AD+Psychosis, AD+P), affecting ~ 40% to 60% of individuals with AD. In comparison to AD subjects without psychosis, AD+P subjects have more rapid cognitive decline and poor outcomes. Ropacki and Jeste1 comprehensively reviewed the literature on psychosis in AD from 1990 to 2003, identifying 55 studies comprised of 9,749 subjects. More rapid cognitive decline was the most consistent correlate of AD+P compared to AD without psychosis (AD-P). More recent studies have continued to support the relationship between greater cognitive impairment, more rapid cognitive decline, and AD+P. 2–8 AD+P is further associated with additional psychiatric and behavioral disturbances, the most frequent and troublesome of which are agitation9 and aggression10;11. AD+P leads to greater distress for family and caregivers12, greater functional impairment,13 higher institutionalization rates,14–17 worse health18 and increased mortality19 compared to AD-P patients. Neurocognitive Disorders Essay

Author Manuscript Treatment of psychosis in AD patients has been suboptimal due to the limited efficacy of available drugs and their high toxicity in this age group. First line treatments are atypical antipsychotics, which have efficacy similar to conventional antipsychotics for AD+P, with lower rates of motor side effects.20 However, atypical and conventional antipsychotics have been associated with an increased risk of all-cause mortality after even short-term treatment. 20;21 Other treatments, such as selective serotonin reuptake inhibitors, may have some efficacy22;23 and improved tolerability.24 Nevertheless, none of these treatments was derived to prevent or reverse an identified biology of AD+P, and there is no current data to suggest that any of these treatments effectively mitigate against the greater cognitive and functional decline associated with AD+P. It is thus imperative to develop an approach to promote Mol Psychiatry. Author manuscript; available in PMC 2018 March 23. DeMichele-Sweet et al. Page 3 Author Manuscript discovery regarding the biology of AD+P and identify opportunities to intervene to prevent its adverse trajectory. We initially observed familial aggregation of AD+P,25 since replicated in two independent cohorts.4;26 These studies show a remarkable consistency in the estimated 3–4 fold increased odds of psychosis in a family member with AD, given the presence of psychosis in a proband with AD. Similarly, we used two of these cohorts to estimate the heritability of psychosis in AD as 61%.27;28 Thus, AD+P is likely to be strongly influenced by genetic variation. In keeping with these observations, we recently reported the first Genome-Wide Association Study (GWAS) of AD+P, evaluating 1,299 cases with AD+P and 735 individuals characterized as AD-P. Although no single SNP demonstrated genome-wide significance, likely due to modest sample size, there was suggestive evidence for association with novel loci. Author Manuscript We further found a trend towards association with a group of 11 SNPs that had been identified in initial GWAS studies of schizophrenia and bipolar disorder.29 That latter finding also provided the biologically intriguing observation that the direction of 7/11 allelic effects on risk for AD+P were opposite that reported in the studies of psychiatric disorder subjects. Since the time of our prior report, genomic studies of schizophrenia risk have identified 128 SNPs in 108 loci that exceed genome-wide significance.30 Author Manuscript Recently, the use of polygenic risk scores has emerged as an important approach for summarizing genetic effects of a set of SNPs. A polygenic score is a simple, subject-specific summary of the additive effects of alleles on a trait. When computed to predict subjects’ risk for a disorder, it is called a polygenic risk score. The score can be obtained from a limited set of SNPs, such as those reaching genome wide significance in association studies, or a larger set based on some other threshold 313233, or the entire genome 34;35. For example, when alleles at the 108 schizophrenia-associated loci were combined in a polygenic risk score they explained 3.4% of the liability to schizophrenia. Neurocognitive Disorders Essay

30 For traits in which few or no individual SNPs reach genome wide significance, polygenic risk scores can provide initial evidence for true genetic association of the trait with the SNPs either included within the score or in close linkage disequilibrium 33, providing critical evidence in support of larger scale studies needed to identify the individual affected loci. Author Manuscript Here we follow up on our prior research in an expanded Discovery Cohort of 2876 AD subjects with and without psychosis. All subjects were genotyped using a custom chip designed to evaluate SNPs with evidence of genetic association, most prominently with AD +P, although SNPs affecting or putatively affecting risk for schizophrenia and Alzheimer disease were also assessed. Results were replicated in an independent cohort of 2194 AD subjects with and without psychosis. We found that AD+P is associated with polygenic risk for a set of novel loci and inversely associated with polygenic risk for schizophrenia. These findings provide the first clear demonstration that AD+P is associated with common genetic variation. In addition, they provide an unbiased link between polygenic risk for schizophrenia and a lower risk of psychosis in AD. As efforts to identify the biologic effects of schizophrenia alleles progress, it may be possible to leverage these results to identify novel mechanisms protecting against more rapid cognitive decline and psychosis risk in AD. Mol Psychiatry. Author manuscript; available in PMC 2018 March 23. DeMichele-Sweet et al. Page 4 Author Manuscript Materials and Methods An overview of the study design and workflow is shown in Figure 1. Subjects Author Manuscript This study analyzed samples obtained from subjects in two cohorts, an initial Discovery Cohort and an independent Replication Cohort (Table 1). All subjects were diagnosed with possible, probable,36 or definite37 AD. Importantly, subjects with a primary diagnosis of Dementia with Lewy bodies38 were excluded. The above diagnoses resulted from diagnostic evaluations, cognitive testing, and in some cases neuropathologic assessment, conducted during subjects’ participation in the following programs as previously described: the University of Pittsburgh Alzheimer Disease Research Center (ADRC),39;40 the Genetic and Environmental Risk in AD Consortium 1 (UK),29;41;42 the National Institute on Aging’s Late Onset Alzheimer’s Disease Family Study (NIA-LOAD),4;28 the National Institute of Mental Health Genetics Initiative AD Cohort (NIMH),25 the Fundació ACE Barcelona Alzheimer Treatment and Research Center (ACE),41;43, the Cardiovascular Health Study (CHS),3;41 and a consortium of National Institute on Aging Alzheimer Disease Centers (ADC).44 Collection of clinical data and genetic samples were approved by each sites local Institutional Review Board or Medical Ethics Committee, as appropriate. Additional detail of the individual cohorts and assessment methodology is available in Supplementary Methods and Tables S1–S13. Neurocognitive Disorders Essay

Characterization of Psychosis Author Manuscript Subjects were characterized for the presence or absence of delusions and hallucinations within the individual studies using the CERAD behavioral rating scale45 (ADRC and NIALOAD), Neuropsychiatric Inventory Questionnaire (NPI-Q,46 NIA-LOAD, ADC), NPI-Q Spanish Version47 (ACE), NPI48 (UK, CHS), and Brief Psychiatric Rating Scale49 (NIMH). Each of these instruments has established reliability in AD,4;50 and we have previously used all successfully in analyses of psychosis in AD subjects.3;4;6;27;39 Details of the application of these assessments for each cohort are provided in the Supplementary Methods. AD+P was defined by the presence of persistent hallucinations or delusions occurring during the course of the dementia, AD-P was defined by the absence of all symptoms at all assessments. Because psychotic symptoms typically emerge in the mild to moderate stages of AD4 individuals without psychosis but who were still in the early stages of disease at their last assessment (Clinical Dementia Rating51 score 20) were considered to be at substantial risk of developing AD+P later in their course. Thus, these individuals were excluded from the analysis. We have previously used these approaches to characterizing and defining AD+P and AD-P to demonstrate familial aggregation,4;25 heritability,27;28 genetic linkage,28;53 and suggestive genome-wide association29 with the AD+P phenotype. Author Manuscript Genotyping DNA Preparation—Samples from outside sources were shipped on dry ice, stored, and processed by the Genomic Core Lab at the University of Pittsburgh. ACE samples were supplied as whole blood and genomic DNA was extracted using the Qiamp Blood Mini kit Mol Psychiatry. Author manuscript; available in PMC 2018 March 23. DeMichele-Sweet et al. Page 5 Author Manuscript (Qiagen, Valencia, CA). All other centers provided genomic DNA (ADRC, NIA-LOAD, NIMH, UK, ADC) or whole genome amplified DNA (CHS). Custom Chip for Discovery Cohort—The Genomic Core Lab quantitated all samples by Pico Green (Thermo Fisher, Pittsburgh, PA) and diluted the DNA to 23ng/ul and shipped the plates on dry ice to Affymetrix (Los Angeles, CA) for genotyping. Plates also contained randomized duplicates. Affymetrix confirmed all DNA concentrations by Pico Green assay prior to genotyping. Genotyping used a custom designed Axiom® chip (see SNP selection below), and was performed using the Affymetrix GeneTitan® system as described in the axiom user manual54 with resultant genotype calls provided for QC and analysis. iPlex Assay for Genotyping SCZ risk score SNPs and Replication Cohort Testing Author Manuscript Author Manuscript iPlex Chemistry: Assays were designed with Assay Designer 4.0 (Agena) and analysis performed using iPlex Gold Genotyping Reagent Set (Agena, San Diego, CA) according to manufacturer’s instructions. Target loci were amplified within the samples by multiplex PCR in 1X PCR buffer containing 3.5 mM MgCl2, 25 mM dNTPs, 500 nM each of forward and reverse amplification primer within the multiplex pool and 2.5 U HotStar Taq. dNTPs and primers were removed by incubation with 0.5 U shrimp alkaline phosphotase (SAP) at 37 °C for 40 minutes. SAP was inactivated by incubation at 87 °C for 5 minutes. Single base extension was carried out in 0.2X iPLEX buffer plus, 1X termination mix (containing mass modified termination nucleotides), 1X iPLEX enzyme and primers at 0.84 μM, 1.04 μM and 1.25 μM as appropriate to the relative mass of each primer. Following thermocycling, clean resin and water was added to the MassExtend reaction products. Neurocognitive Disorders Essay

Samples were incubated in clean resin at room temperature with mixing for 5 minutes and centrifuged at 3200 × g for 5 minutes. Samples were then dispensed to a SpectraChip using the MassArray Nanodispenser according to manufacturer’s instructions. Spectra chips were loaded into the MassArray analyzer and spectra acquired for each sample. Genotype calls were made using Typer 4.0 (Agena) by mass identification of extended primer peaks. SNP Selection Author Manuscript Development of Custom Array for Discovery Cohort—The process of selecting SNPs for the genotyping array involved two principal stages. First SNPs were amalgamated based on genetic signal for association to a small set of phenotypes (Table S14). The bulk of the SNPs were included on the basis of association results from four contrasts reported in three genome-wide studies: a contrast of AD+P versus AD-P,29 AD+P versus controls,29 AD versus controls 55;56, and SCZ versus controls 32;57. An additional unpublished data set (described in58;59) of cis-eQTLs affecting gene expression and cis-eQTLs associated with age-related changes in gene expression was also used. For the first four genome-wide association studies (GWAS), SNPs with p-value less than a threshold of 0.01 were selected; for the eQTLs, the threshold was 0.001 and for the ‘aging’ eQTLs it was 0.05. Note that when a SNP was represented in more than one study, the minimum p-value in any of the 6 datasets was taken as representative for the SNP. To interrogate copy number regions shown Mol Psychiatry. Author manuscript; available in PMC 2018 March 23. DeMichele-Sweet et al. Page 6 Author Manuscript to be associated with schizophrenia, 1574 SNPs were included (1q21.1, 3q29, 15q11.2– 15q13.3, 16p13.1, 16p11.2 and 22q11.2, recently reviewed in 60; and 7q11.2361). Finally a small fraction of SNPs were chosen to cover four genes of interest regarding psychotic disorders (SCZ target genes: NRXN1,60 ERBB4,62 PAK2,63 CHRNA764) or were nominated from unpublished AD studies (UK SNPs). Author Manuscript Second, SNPs were retained for genotyping by a winnowing process. This process involved removing redundant SNPs, those that could not be genotyped on the Axiom platform, or SNPs not present in 1000 genomes. Of the SNPs passing this step, all SNPs with a minimum p-value < 0.0001 for any study were retained. For the remainder, by using a LD clumping process, we removed SNPs in LD with the retained SNPs (r2 > 0.9) and retained additional SNPs with the smallest p-value in “independent” clumps (r2 < 0.9) by pruning SNPs with Plink (maximum distance for pruning was 5 kb, window width was 25 SNPs, sliding step was 5 SNPs). SNP Selection for SCZ Risk Score Testing and Follow-up Genotyping in Replication Cohort—For SCZ risk score testing in each cohort we targeted the 128 GWA significant SNPs reported in 30, although not all could be genotyped. Follow-up genotyping in the Replication Cohort also selected SNPs from our custom array that passed Quality Control and with P < 0.0001 for the contrast of AD+P versus AD-P. For the replication cohort we selected Ancestry Informative Markers (AIMs) for European Ancestry based on the results in 65 Specifically, based on results found in with Supplementary Table 1 of Kosoy et al. 65, we selected their “Top 96” European AIMs, of which 82 could be genotyped on the Sequenom platform and 79 passed Quality Control. Author Manuscript Quality Control Author Manuscript QC was performed at the individual level first, then at the SNP level conditional on individual-level data passing QC and individuals of European ancestry. Details of QC are given in Supplementary Material. In brief, genetic data for samples were retained if their nominal sex agreed with genetically determined sex; heterozygosity rate, per subject, revealed no evidence of contamination by other samples; genetic data for subjects expected to be unrelated suggested this were true; and call rate of SNPs > 96.5% per sample. Next ancestry of subjects in the Discovery Cohort was determined using dacGem in GemTools based on 5712 autosomal markers with non-call rate ≤ 0.001, minor allele frequency (MAF) ≥ 0.05, and r2 ≤ 0.20. The samples were separated into 5 clusters based on 3 significant ancestry dimensions, four of which likely represent European ancestry and two of the…