Molecular Psychiatry (2016) 21, 831–836 © 2016 Macmillan Publishers Limited All rights reserved 1359-4184/16 www.nature.com/mp ORIGINAL ARTICLE SORL1 rare variants: a major risk factor for familial early-onset Alzheimer’s disease G Nicolas1,2,3,16, C Charbonnier2,3,16, D Wallon2,3,4, O Quenez2,3, C Bellenguez5,6,7, B Grenier-Boley5,6,7, S Rousseau3, A-C Richard3, A Rovelet-Lecrux2, K Le Guennec2, D Bacq8, J-G Garnier8, R Olaso8, A Boland8, V Meyer8, J-F Deleuze8,9, P Amouyel5,6,7, HM Munter10, G Bourque10, M Lathrop10, T Frebourg1,2, R Redon11,12, L Letenneur13, J-F Dartigues13, E Génin14, J-C Lambert5,6,7, D Hannequin1,2,3,4, D Campion2,3,15 and The CNR-MAJ collaborators17 The SORL1 protein plays a protective role against the secretion of the amyloid β 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. Molecular Psychiatry (2016) 21, 831–836; doi:10.1038/mp.2015.121; published online 25 August 2015 INTRODUCTION set of families, we next aimed to determine whether predicted SORL1 (Sortilin-related receptor, L(DLR class A) repeats containing, damaging rare SORL1 variants may contribute to EOAD genetic risk. also known as SORLA or LR11) plays a key role in the trafficking of To address this issue we performed a case–control study on rare the amyloid β (Aβ) precursor protein (APP) as an APP neuronal variants using whole-exome sequencing (WES) data from 484 sorting receptor. By binding to the retromer complex, SORL1 unrelated French EOAD patients and 498 ethnically matched redirects APP from the cell membrane to the Golgi apparatus controls. through the recycling pathway.1 Sorting APP through this pathway results in reduced amyloidogenic processing of APP.1–3 Moreover, SORL1 was recently shown to direct nascent Aβ peptides to the MATERIALS AND METHODS lysosomal compartment, a mechanism that may also contribute to Patients the reduction of the amyloid load.4 SORL1 therefore plays a β Patients were recruited over a 20-year period by the French National CNR- protective role against A neuronal secretion, a central event in MAJ consortium, a memory clinical network including 24 expert centers Alzheimer’s disease (AD) pathogenesis. We previously reported dedicated to the EOAD patient study and care. Diagnoses were performed potentially pathogenic SORL1 rare variants in 5/14 early-onset AD according to the NINCDS-ADRDA (National Institute of Neurological and (EOAD, defined by an age of onset before 65 years) patients who Communicative Disorders and Stroke and the Alzheimer's Disease and were negatively screened for mutations in known autosomal Related Disorders Association) criteria.6 All patients underwent a compre- dominant EOAD genes, namely APP, PSEN1 or PSEN2, despite the hensive clinical examination including personal medical and family history fact that they belonged to pedigrees in which inheritance was assessment, neurological examination, neuropsychological assessment and fl consistent with autosomal dominant transmission.5 The identifica- neuroimaging. When available, cerebrospinal uid (CSF) AD biomarkers had to be consistent with an AD profile. The cutoffs used to define a tion of a nonsense variant among one of these patients supported − 1 − 1 biochemical AD signature were: Aβ42 o550 pg ml , Tau 4350 pg ml the hypothesis that these variants may exert a proamyloidogenic 4 − 1 β 5 and P-Tau 60 pg ml . We also calculated the Tau/A 42 ratio and a effect through a loss of SORL1 function. This hypothesis was value of 40.52 was considered abnormal.7 Two criteria differing by recently reinforced by the observation that one of the reported stringency were used to classify CSF samples as supportive of an AD missense variants led to a decrease in SORL1 ability to mediate Aβ diagnosis: either (1) all three biomarkers were abnormal or (2) two out of 4 lysosomal degradation. Beyond these results obtained in a small three biomarkers and the Tau/Aβ42 ratio were abnormal. When patients 1Department of Genetics, Rouen University Hospital, Rouen, France; 2Inserm U1079, Rouen University, IRIB, Normandy University, Rouen, France; 3CNR-MAJ, Rouen University Hospital, Rouen, France; 4Department of Neurology, Rouen University Hospital, Rouen, France; 5Inserm, U1167, Lille, France; 6Institut Pasteur de Lille, Lille, France; 7Université Lille- Nord de France, Lille, France; 8Centre National de Génotypage, Institut de Génomique, CEA, Evry, France; 9Fondation Jean Dausset, Centre d’études du Polymorphisme Humain, Paris, France; 10McGill University and Génome Québec Innovation Centre, Montréal, QC, Canada; 11Inserm UMR 1087, l'institut du Thorax, CHU Nantes, Nantes, France; 12CNRS, UMR 6291, Université de Nantes, Nantes, France; 13Inserm U897, Univ Bordeaux, Bordeaux, France; 14Inserm UMR1078, CHU Brest, Univ Bretagne Occidentale, Brest, France and 15Department of Research, Rouvray Psychiatric Hospital, Sotteville-lès-Rouen, France. Correspondence: Dr D Campion, Inserm U1079, Faculté de Médecine, 22 Boulevard Gambetta, Rouen 76183, France. E-mail: [email protected] 16These two authors contributed equally to this work. 17CNR-MAJ collaborators are listed before references. Received 16 April 2015; revised 13 July 2015; accepted 14 July 2015; published online 25 August 2015 SORL1 rare variants G Nicolas et al 832 had negative CSF results, the diagnosis of AD was not retained and they were not included. All patients gave informed, written consent for genetic analyses. This study was approved by our ethics committee. Blood samples were neuropath extracted using the Qiagen (Hilden, Germany) DNA blood kit. APOE Percentage genotyping was performed by sequencing. Only patients with no PSEN1, PSEN2 or APP mutation (detected either by Sanger sequencing or by WES), rmed AD diagnosis. fi no APP duplication (detected by QMPSF (quantitative multiplex PCR of CSF fl 8 short uorescent fragments) and no C9ORF72 expansion (detected Percentage following the protocol described by DeJesus-Hernandez et al.9) were included in this study. We finally included a total of 485 unrelated French patients with EOAD (age of onset ≤ 65 years). Of note, 52.9% had a disease 0.0213) 54.1% 1% ≤ 0.0162) 51.5% 2% onset at 55 years. All but one passed quality controls (see below): 196 0.0117) NA NA – – men (40.5%) and 288 women (59.5%). We verified that none of them – carried a pathogenic variant in a list of 20 other dementia-causing genes.10 Ten of them (2%) had a definite diagnosis of AD (neuropathological confirmation) and the other 474 (98%) were diagnosed with probable AD. MAF (95% CI) Among them, 249 had CSF biomarkers indicative of AD (51.4% from all 0.005 (0.0021 0.0073 (0.0025 patients). The remaining 225 (46.5%) had no CSF biomarker available and 0.0083 (0.0042 were carefully selected using neuropsychological assessments (evidence of a progressive hippocampal amnestic syndrome associated with another 24 31 10 = = cognitive dysfunction) and evidence of neuronal injury using imaging = (6.4%) (2.0%) N N N criteria (AD pattern of a cortical atrophy on magnetic resonance imaging (11.7%) and/or decreased 18flurodesoxyglucose uptake on positron emission tomography). 110 214 Family history was obtained by family interview. When available, medical 102 = = reports of the relatives were examined. A positive family history was defined = (53.7%) (44.2%) (20.5%) N N by the presence of at least one first-degree relative diagnosed with AD or with N a reported phenotype consistent with AD, whatever the age of onset in the affected relative. Among the 484 cases, 205 (42%) had a positive family 60 206 history, 230 (48%) had sporadic AD and the family history was unknown in 315 = = = fi N (29.3%) (42.6%) (63.3%) N 49 (10%). The 205 cases with positive family history included 2 de nite and N 203 probable AD cases, 111 of whom had CSF biomarkers indicative of AD. Among the 484 patients, the distribution of APOE genotypes was 6 as follows: ε2/ε2(n = 1, 0.2%), ε2/ε3(n = 21, 4.3%), ε3/ε3(n = 206, 42.6%), 4 11 = = ε2/ε4(n = 11, 2.3%), ε3/ε4(n = 214, 44.2%) and ε4/ε4(n = 31, 6.4%) = N N APOE distribution TREM2 p.R47H variant (1.2%) (1.9%) (2.3%) (Table 1). We included only 31 out of 188 APOE ε4ε4 EOAD patients initially N eligible for this study. These patients were either sporadic cases with an age of onset at ≤ 55 years (7 patients) or had a positive family history 6 63 that had to occur in at least two generations (24 patients). 21 = = = N (4.3%) (2.9%) N N s disease; MAF, minor allele frequency; NA, not available; Percentage CSF: percentage of patients with cerebrospinal ’ (12.6%) Controls A total of 500 controls were recruited in 5 different French cities and were 2 1 all of French ancestry (498 passed the quality control, see below). Of these, 1 = = ≥ = N N 301 had an age at inclusion of 55 years and had normal Mini Mental N (0.4%) (0.2%) (0.5%) State Examination score, according to age and education level.