Screening exons 16 and 17 of the amyloid precursor protein gene in sporadic early-onset Alzheimer's disease

Abstract

Early-onset Alzheimer's disease (EOAD) can be familial (FAD) or sporadic EOAD (sEOAD); both have a disease onset ≤65 years of age. A total of 451 sEOAD samples were screened for known causative mutations in exons 16 and 17 of the amyloid precursor protein (APP) gene. Four samples were shown to be heterozygous for 1 of 3 known causative mutations: p.A713T, p.V717I, and p.V717G; this highlights the importance of screening EOAD patients for causative mutations. Additionally, we document an intronic 6 base pair (bp) deletion located 83 bp downstream of exon 17 (rs367709245, IVS17 83-88delAAGTAT), which has a nonsignificantly increased minor allele frequency in our sEOAD cohort (0.006) compared to LOAD (0.002) and controls (0.002). To assess the effect of the 6-bp deletion on splicing, COS-7 and BE(2)-C cells were transfected with a minigene vector encompassing exon 17. There was no change in splicing of exon 17 from constructs containing either wild type or deletion inserts. Sequencing of cDNA generated from cerebellum and temporal cortex of a patient harboring the deletion found no evidence of transcripts with exon 17 removed.

Keywords: APP; Alzheimer's disease; Early-onset; Screening; Sporadic; rs367709245.

Figure 1. Sequence chromatograms from individuals harbouring known causative APP mutations

Each sample is presented one per column, with sample ID and mutation ID stated at the top and sequence chromatographs underneath. The chromatographs were generated using Sequence Scanner version 2 (Applied Biosystems) and show the heterozygous causative mutation at the centre with 8 surrounding bases. The top image in each column shows the sequence in the forward direction using primer E17F and the bottom image sequence in the reverse direction using primer E17R.

Figure 2. RT-PCR products from cells transfected with vector containing wild type or variant insert (rs367709245)

RT-PCR products from COS7 cells (A) and Be(2)-C cells (B) transfected with pET01 vector containing wild type or variant insert (rs367709245). Lane 1 contains the DNA ladder GeneRuler 1 kb Plus (Life Technologies) which has two reference bands; 1000 bp (top) and 500 bp (bottom). Lanes 2 contains the RT-PCR product from a colony harbouring vector with wild type insert, lane 3 being its respective RT-PCR negative control (no enzyme). Lanes 4 contains the RT-PCR product from a colony harbouring a variant insert (rs367709245) with lane 5 being its respective RT-PCR negative control (no enzyme). Lane 6 contains the RT-PCR negative control (no RNA).

Figure 3. RT-PCR products from individual O0483 who harbours rs367709245

Reverse transcriptase polymerase chain reaction (RT-PCR) products generated using Oligo(dT) or random primers and RNA from individual (O0483) cerebellum (C) and Temporal cortex (TC). Lanes 1 and 12 contain GeneRuler 100 bp Plus DNA ladder (ThemoFisher Scientific) containing one reference band marked at 500bp. Lanes 2 and 3 contain the RT-PCR products generated using Oligo(dT) primers. Lanes 4 and 5 contain the RT-PCR product generated using random primers. Lanes 6 to 9 are the same as lanes 2 to 5 but with no reverse transcriptase enzyme (cDNA –ve). Lane 10 contains an RNA negative control (-ve) and lastly lane 11 contains a PCR positive control (+ve), which was cDNA generated from Lymphoblastoid cell lines from two individuals obtained from the NHGRI sample repository.