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Investigating splicing variants uncovered by next-generation sequencing the Alzheimer’s disease candidate genes, CLU, PICALM, CR1, ABCA7, BIN1, the MS4A locus, CD2AP, EPHA1 and CD33

Clement, Naomi; Braae, Anne; Turton, James; Lord, Jenny; Guetta-Baranes, Tamar; Medway, Christopher; Brookes, Keeley; Barber, Imelda S.; Patel, Tulsi; Milla, Lucy; Azzopardi, Maria; Lowe, James; Mann, David; Pickering-Brown, Stuart; Kalsheker, Noor; Passmore, Peter; Chappell, Sally; Morgan, Kevin


Naomi Clement

Lucy Milla

Maria Azzopardi

James Lowe

David Mann

Stuart Pickering-Brown

Noor Kalsheker

Peter Passmore

Sally Chappell

Kevin Morgan

Anne Braae

James Turton

Jenny Lord

Tamar Guetta-Baranes

Christopher Medway

Keeley Brookes

Imelda S. Barber

Tulsi Patel


Late onset Alzheimer’s disease (LOAD), the most common cause of late onset dementia, has a strong genetic component. To date, 21 disease-risk loci have been identified through genome wide association studies (GWAS). However, the causative functional variant(s) within these loci are yet to be discovered. This study aimed to identify potential functional splicing mutations in the nine original GWAS-risk genes: CLU, PICALM, CR1, ABCA7, BIN1, the MS4A locus, CD2AP, EPHA1 and CD33. Target enriched next generation sequencing (NGS) was used to resequence the entire genetic region for each of these GWAS-risk loci in 96 LOAD patients and in silico databases were used to annotate the variants for functionality. Predicted splicing variants were further functionally characterised using splicing prediction software and minigene splicing assays. Following in silico annotation, 21 variants were predicted to influence splicing and, upon further annotation, four of these were examined utilising the in vitro minigene assay. Two variants, rs881768 A>G in ABCA7 and a novel variant 11: 60179827 T>G in MS4A6A were shown, in these cell assays, to affect the splicing of these genes. The method employed in the paper successfully identified potential splicing variants in GWAS-risk genes. Further investigation will be needed to understand the full effect of these variants on LOAD risk. However, these results suggest a possible pipeline in order to identify putative functional variants as a result of NGS in disease-associated loci although improvements are needed within the current prediction programme in order to reduce the number of false positives.

Journal Article Type Article
Journal Journal of Alzheimers Disease & Parkinsonism
Electronic ISSN 2161-0460
Publisher OMICS International
Peer Reviewed Peer Reviewed
Volume 6
Issue 6
Keywords Late onset Alzheimer’s disease; Next generation
sequencing; Splicing; Annotation; Functional variations; Minigene assays
Publisher URL
Copyright Statement Copyright information regarding this work can be found at the following address:


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