Molecular Psychiatry (2015) 20, 1046–1056 © 2015 Macmillan Publishers Limited All rights reserved 1359-4184/15 www.nature.com/mp IMMEDIATE COMMUNICATION De novo deleterious genetic variations target a biological network centered on Aβ peptide in early-onset Alzheimer disease A Rovelet-Lecrux1,11, C Charbonnier1,2,11, D Wallon1,2,3,11, G Nicolas1,2,4,11, MNJ Seaman5, C Pottier1, SY Breusegem5, PP Mathur6,7, P Jenardhanan6, K Le Guennec1, AS Mukadam5, O Quenez1,2, S Coutant1, S Rousseau1,2, A-C Richard1,2, A Boland8, J-F Deleuze8, T Frebourg1,4, D Hannequin1,2,3,4, D Campion1,2,9 and CNR-MAJ collaborators10 We hypothesized that de novo variants (DNV) might participate in the genetic determinism of sporadic early-onset Alzheimer disease (EOAD, onset before 65 years). We investigated 14 sporadic EOAD trios first by array-comparative genomic hybridization. Two patients carried a de novo copy number variation (CNV). We then performed whole-exome sequencing in the 12 remaining trios and identified 12 non-synonymous DNVs in six patients. The two de novo CNVs (an amyloid precursor protein (APP) duplication and a BACE2 intronic deletion) and 3/12 non-synonymous DNVs (in PSEN1, VPS35 and MARK4) targeted genes from a biological network centered on the Amyloid beta (Aβ) peptide. We showed that this a priori-defined genetic network was significantly enriched in amino acid-altering DNV, compared with the rest of the exome. The causality of the APP de novo duplication (which is the first reported one) was obvious. In addition, we provided evidence of the functional impact of the following three non- synonymous DNVs targeting this network: the novel PSEN1 variant resulted in exon 9 skipping in patient’s RNA, leading to a pathogenic missense at exons 8–10 junction; the VPS35 missense variant led to partial loss of retromer function, which may impact neuronal APP trafficking and Aβ secretion; and the MARK4 multiple nucleotide variant resulted into increased Tau phosphorylation, which may trigger enhanced Aβ-induced toxicity. Despite the difficulty to recruit Alzheimer disease (AD) trios owing to age structures of the pedigrees and the genetic heterogeneity of the disease, this strategy allowed us to highlight the role of de novo pathogenic events, the putative involvement of new genes in AD genetics and the key role of Aβ network alteration in AD. Molecular Psychiatry (2015) 20, 1046–1056; doi:10.1038/mp.2015.100; published online 21 July 2015 INTRODUCTION with large effect could account for a fraction of sporadic cases The genetic architecture of Alzheimer disease (AD) is still incompletely affected by these conditions. Consequently, a 'de novo'experimental characterized. Rare autosomal dominant forms of early-onset Alzheimer design has become an attractive paradigm for the discovery of 4 disease (EOAD) are caused by mutations within three genes (APP (amyloid relevant genes in complex diseases. Different types of de novo precursor protein), PSEN1 and PSEN2). In addition, genome-wide genomic events have been studied. Although earlier studies focused association studies have implicated common variants at ~ 20 genetic on copy number variations (CNVs), more recent whole-exome loci.1 Notably, with the exception of the ε4alleleoftheAPOE gene, these sequencing studies have allowed for the detection of de novo single- variants have a modest effect on AD risk (OR ranging from 0.77 to 1.22). or multiple-nucleotide variants (MNV) and indels. It is now estimated With the advent of whole-exome sequencing, which consists in the that CNVs larger than 100 kb occur de novo in ~ 1 of every 50 massive parallel sequencing of all protein-coding exons of a genome, it individuals, whereas each individual carries an average of one exonic has become possible to interrogate low-frequency variants in case– de novo single-nucleotide variants.5 These studies are based on the control studies, and rare missense variants in TREM2 and PLD3 have been simultaneous analysis of the whole-exome sequence of a patient reported to increase risk for AD.2,3 However, despite these findings, it is and of his/her unaffected parents in family trios to detect mutation acknowledged that a substantial part of the genetic component of the (s) present in the patient but not in his/her parents. Owing to the disease remains unidentified. age structure of AD pedigrees, such a design is difficult to apply Recently, the impact of de novo mutations in common neuro- when dealing with diseases affecting elderly subjects. We never- psychiatric diseases including mental retardation, autism or theless succeeded in sampling 14 EOAD patients (age of onset schizophrenia has been highlighted by several studies. Together, younger than 65) with no history of the disease in first- or second- these studies have provided arguments that germline mutations degree relatives, and their living parents (Table 1). 1Inserm U1079, Institute for Research and Innovation in Biomedicine, University of Rouen, Rouen, France; 2CNR-MAJ, Rouen University Hospital, Rouen, France; 3Department of Neurology, Rouen University Hospital, Rouen, France; 4Department of Genetics, Rouen University Hospital, Rouen, France; 5Cambridge Institute for Medical Research/Dept of Clinical Biochemistry, University of Cambridge, Addenbrookes Hospital, Cambridge, UK; 6Centre of Bioinformatics and Department of Biochemistry & Molecular Biology, School of Life Sciences, Pondicherry University, Pondicherry, India; 7KIIT University, Bhubaneshwar, Odisha, India; 8Centre National de Génotypage, Evry, France and 9Department of Research, Centre Hospitalier du Rouvray, Sotteville-Les-Rouen, France. Correspondence: D Campion, Inserm U1079, Faculté de Médecine, 22 boulevard Gambetta, Rouen 76183 France. E-mail: [email protected] 11These authors contributed equally to this work. 10CNR-MAJ collaborators are listed in Appendix A. Received 20 March 2015; revised 27 May 2015; accepted 16 June 2015; published online 21 July 2015 De novo mutations in early onset Alzheimer disease A Rovelet-Lecrux et al 1047 Table 1. Demographic and clinical data of the study participants Patient code Sex AOO APOE Neuropathology CSF Aβ42 CSF Tau CSF P-Tau Tau/Aβ Age of father (years) Age of mother (years) pg ml − 1 pg ml − 1 pg ml − 1 42 At At N4550 No350 No60 No0.52 Inclusion Childbirth Inclusion Childbirth EXT 804 M 50 3/4 678 1028 96 1.52 81 26 80 25 EXT 773 M 44 3/3 AD+ CAA 85 30 76 21 EXT 235 F 46 2/3 481 777 96 1.62 87 32 78 24 ROU 682 F 54 3/3 AD 92 27 85 20 ROU 1331 M 58 3/4 366 612 73 1.67 92 27 86 21 ROU 1322 F 50 3/3 573 624 100 1.09 94 35 80 21 EXT 395 F 50 3/4 248 525 124 2.12 81 27 81 27 EXT 488 F 52 3/3 588 627 80 1.07 86 26 85 25 EXT 687 M 49 3/3 396 628 123 1.59 79 28 76 25 EXT 306 F 49 3/4 430 744 101 1.73 76 23 76 23 EXT 186 F 58 3/3 463 1200 145 2.59 91 26 89 24 EXT 344 M 46 3/4 608 900 82 1.48 76 25 77 26 EXT 573 M 46 3/3 390 658 27 1.69 72 24 68 20 EXT 576 F 45 3/4 497 1013 143 2.04 80 30 73 23 Abbreviations: Aβ, Amyloid beta; AD, Alzheimer disease; AOO, age of onset; APOE, APOE genotype; CAA, cerebral amyloid angiopathy; CSF, cerebrospinal fluid. MATERIALS AND METHODS literature searches were subsequently used to maintain the data For detailed methods see Supplementary Information. set and identify new interactors. We only retained genes for which experimental evidence based on cell or animal models had been published. No further exclusion criteria were used except for the Subjects Aβ toxicity category for which genes involved in non-specific late Patients were recruited by a French memory clinical network. stages of cell death were not retained. Direct protein–protein Diagnoses (neuropathologically proven AD, n = 2; probable AD interaction was not a prerequisite but was noted, when with evidence of the AD pathophysiological process, n = 12) were 6 documented. For that purpose, data retrieved during this search performed according to the NINCDS-ADRDA criteria. Inclusion were compared with the extensive list of APP interactors criteria are described in Supplementary Methods and the previously compiled by Perreau et al.7 demographic characteristics and APOE genotypes of the sample are displayed in Table 1. Only patients with no affected relative Testing for enrichment in de novo mutations of the Aβ network among first- or second-degree relatives were included in β the study. The objective was to test whether the A network was enriched in de novo mutations among our 12 EOAD patients, compared with the expected relative mutation rate of this network in the general CNV analysis population, conditional on the total number of de novo mutations High-resolution array-comparative genomic hybridization analysis observed. We considered several candidates of the network was performed using the Human High-Resolution Discovery relative baseline mutation rate, either based on the proportion of Microarray Kit 1 × 1 M (Agilent Technologies, Santa Clara, CA, bases jointly covered at 20 × that fell within the Aβ network, or USA) using standard protocols. All CNVs encompassing genes and based on the proportion of variants (synonymous or missense, not previously reported in large array-comparative genomic rare or of any minor allele frequency) carried by the unaffected hybridization studies in the database of genomic variants parents that fell within the Aβ network. (http://dgv.tcag.ca) were confirmed in patients and their inheri- We tested for the enrichment of the network in de novo tance was assessed by quantitative multiplex PCR of short mutations among AD patients using a one-sided binomial test and fluorescent fragments (Supplementary Figure S1).