Premature termination codon (PTC) mutations in the ATP-Binding Cassette, Sub-Family A, Member 7 gene (ABCA7) have recently been identified as intermediate-to-high penetrant risk factor for late-onset Alzheimer's disease (LOAD). High variability, however, is observed in downstream ABCA7 mRNA and protein expression, disease penetrance, and onset age, indicative of unknown modifying factors. Here, we investigated the prevalence and disease penetrance of ABCA7 PTC mutations in a large early onset AD (EOAD)-control cohort, and examined the effect on transcript level with comprehensive third-generation long-read sequencing. We characterized the ABCA7 coding sequence with next-generation sequencing in 928 EOAD patients and 980 matched control individuals. With MetaSKAT rare variant association analysis, we observed a fivefold enrichment (p = 0.0004) of PTC mutations in EOAD patients (3%) versus controls (0.6%). Ten novel PTC mutations were only observed in patients, and PTC mutation carriers in general had an increased familial AD load. In addition, we observed nominal risk reducing trends for three common coding variants. Seven PTC mutations were further analyzed using targeted long-read cDNA sequencing on an Oxford Nanopore MinION platform. PTC-containing transcripts for each investigated PTC mutation were observed at varying proportion (5-41% of the total read count), implying incomplete nonsense-mediated mRNA decay (NMD). Furthermore, we distinguished and phased several previously unknown alternative splicing events (up to 30% of transcripts). In conjunction with PTC mutations, several of these novel ABCA7 isoforms have the potential to rescue deleterious PTC effects. In conclusion, ABCA7 PTC mutations play a substantial role in EOAD, warranting genetic screening of ABCA7 in genetically unexplained patients. Long-read cDNA sequencing revealed both varying degrees of NMD and transcript-modifying events, which may influence ABCA7 dosage, disease severity, and may create opportunities for therapeutic interventions in AD.

3rd Department of Neurology Medical School Aristotle University of Thessaloniki Thessaloniki Greece

Alzheimer's Disease and Other Cognitive Disorders Unit Neurology Department Hospital Clínic Institut d'Investigacions Biomediques August Pi i Sunyer Barcelona Spain

Center for Neuroscience and Cell Biology University of Coimbra Coimbra Portugal

Centro de Investigación Biomédica en Red de Enfermedades Neurodegenerativas Instituto de Salud Carlos 3 Madrid Spain

Department of Neurobiology Care Sciences and Society Division of Neurogeriatrics Center for Alzheimer Research Karolinska Institutet Huddinge Stockholm Sweden

Department of Neurology and Memory Clinic Hospital Network Antwerp Middelheim and Hoge Beuken Antwerp Belgium

Department of Neurology Antwerp University Hospital Edegem Belgium

Department of Neurology Clínica Universidad de Navarra University of Navarra School of Medicine Pamplona Spain

Department of Neurology Fundación Jiménez Díaz Madrid Spain

Department of Neurology IIB Sant Pau Hospital de la Santa Creu i Sant Pau Universitat Autònoma de Barcelona Barcelona Spain

Department of Neuroscience Psychology Drug Research and Child Health University of Florence Florence Italy

Department of Pathology 1st Medical Faculty Charles University Prague Czech Republic

Department of Pathology and Molecular Medicine Thomayer Hospital Prague Czech Republic

Department of Psychiatry and Psychotherapy Technische Universität München Munich Germany

Faculty of Medicine University of Lisbon Lisbon Portugal

Genetics Unit Department of Geriatric Medicine Karolinska University Hospital Stockholm Sweden

Institute Born Bunge University of Antwerp Antwerp Belgium

Institute of Pathology 3rd Medical Faculty Charles University Prague Czech Republic

IRCCS Don Gnocchi Florence Italy

Laboratory of Biochemistry Department of Chemistry Aristotle University of Thessaloniki Thessaloniki Greece

MAC Memory Center Istituto di Ricovero e Cura a Carattere Scientifico Istituto Centro San Giovanni di Dio Fatebenefratelli Brescia Italy

Memory Unit Department of Neurology University Hospital Mútua de Terrassa University of Barcelona School of Medicine Terrassa Barcelona Spain

Molecular Markers Laboratory Istituto di Ricovero e Cura a Carattere Scientifico Istituto Centro San Giovanni di Dio Fatebenefratelli Brescia Italy

Neurodegenerative Brain Diseases Group VIB Center for Molecular Neurology VIB Antwerp Belgium

Neuroimaging Laboratory Division of Neurosciences Center for Applied Medical Research University of Navarra Pamplona Spain

Neurological Tissue Bank of the Biobanc Hospital Clinic Institut d'Investigacions Biomediques August Pi i Sunyer Barcelona Spain

Neurology Unit Department of Clinical and Experimental Sciences Centre for Neurodegenerative Disorders University of Brescia Brescia Italy

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