title: Functional and genetic analysis of haplotypic sequence variation at the nicastrin genomic locus.
abstract: Nicastrin (NCSTN) is a component of the γ-secretase complex and therefore potentially a candidate risk gene for Alzheimer's disease. Here, we have developed a novel functional genomics methodology to express common locus haplotypes to assess functional differences. DNA recombination was used to engineer 5 bacterial artificial chromosomes (BACs) to each express a different haplotype of the NCSTN locus. Each NCSTN-BAC was delivered to knockout nicastrin (Ncstn(-/-)) cells and clonal NCSTN-BAC(+)/Ncstn(-/-) cell lines were created for functional analyses. We showed that all NCSTN-BAC haplotypes expressed nicastrin protein and rescued γ-secretase activity and amyloid beta (Aβ) production in NCSTN-BAC(+)/Ncstn(-/-) lines. We then showed that genetic variation at the NCSTN locus affected alternative splicing in human postmortem brain tissue. However, there was no robust functional difference between clonal cell lines rescued by each of the 5 different haplotypes. Finally, there was no statistically significant association of NCSTN with disease risk in the 4 cohorts. We therefore conclude that it is unlikely that common variation at the NCSTN locus is a risk factor for Alzheimer's disease.
authors.affiliation: Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK. g.hamilton@ed.ac.uk
authors.forename: Gillian
authors.initials: G
authors.lastname: Hamilton
authors.forename: Richard
authors.initials: R
authors.lastname: Killick
authors.forename: Jean-Charles
authors.initials: JC
authors.lastname: Lambert
authors.forename: Philippe
authors.initials: P
authors.lastname: Amouyel
authors.forename: Minerva M
authors.initials: MM
authors.lastname: Carrasquillo
authors.forename: V Shane
authors.initials: VS
authors.lastname: Pankratz
authors.forename: Neill R
authors.initials: NR
authors.lastname: Graff-Radford
authors.forename: Dennis W
authors.initials: DW
authors.lastname: Dickson
authors.forename: Ronald C
authors.initials: RC
authors.lastname: Petersen
authors.forename: Steven G
authors.initials: SG
authors.lastname: Younkin
authors.forename: John F
authors.initials: JF
authors.lastname: Powell
authors.forename: Richard
authors.initials: R
authors.lastname: Wade-Martins
mesh_terms: D000544:Alzheimer Disease / Q000235:genetics*; D053829:Amyloid Precursor Protein Secretases / Q000235:genetics*; D001483:Base Sequence; D002874:Chromosome Mapping / Q000379:methods*; D056426:Genetic Loci / Q000235:genetics*; D006239:Haplotypes / Q000235:genetics*; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics*; D008969:Molecular Sequence Data
chemical_list: D008562:Membrane Glycoproteins; C413604:nicastrin protein; D053829:Amyloid Precursor Protein Secretases