An intronic VNTR affects splicing of ABCA7 and increases risk of Alzheimer's disease

Abstract

Mutations leading to premature termination codons in ATP-Binding Cassette Subfamily A Member 7 (ABCA7) are high penetrant risk factors of Alzheimer's disease (AD). The influence of other genetic variants in ABCA7 and downstream functional mechanisms, however, is poorly understood. To address this knowledge gap, we investigated tandem repetitive regions in ABCA7 in a Belgian cohort of 1529 AD patients and control individuals and identified an intronic variable number tandem repeat (VNTR). We observed strong association between VNTR length and a genome-wide associated signal for AD in the ABCA7 locus. Expanded VNTR alleles were highly enriched in AD patients [odds ratio = 4.5 (1.3-24.2)], and VNTR length inversely correlated with amyloid β_1-42 in cerebrospinal fluid and ABCA7 expression. In addition, we identified three novel ABCA7 alternative splicing events. One isoform in particular-which is formed through exon 19 skipping-lacks the first nucleotide binding domain of ABCA7 and is abundant in brain tissue. We observed a tight correlation between exon 19 skipping and VNTR length. Our findings underline the importance of studying repetitive DNA in complex disorders and expand the contribution of genetic and transcript variation in ABCA7 to AD.

Keywords: ATP-Binding Cassette, Sub-Family A, Member 7 (ABCA7); Alternative splicing; Alzheimer’s disease; Cerebrospinal fluid (CSF) biomarkers; Variable number tandem repeat (VNTR).

Fig. 1

Genomic location of the ABCA7 VNTR and its effect on ABCA7 splicing. a The ABCA7 genomic conformation (NM_019112.3) and positions of rs3764650, the VNTR, and rs78117248 are shown. Pairwise D′ and r² between rs3764650 and rs78117248 is noted on top. Black arrows superimposed on exons 45 and 46 indicate the primers used for qPCR. Below, ABCA7 exons 17–20 (blue rectangles), introns (black line), VNTR units (black rectangles), and primers in exons 17 and 20 for isoform quantification (black arrows) are represented. Twelve VNTR units are depicted, corresponding to the smallest observed VNTR length. The 25 bp VNTR unit sequence is shown in more detail, with the first two bases corresponding to a splice donor site. Caret-like exon connecting lines depict canonical (green), exon 18 cryptic splice acceptor (orange), retention of a single VNTR unit (purple) and exon 19 skipping (red) splicing. b Canonical spicing of the entire ABCA7 gene leads to a full length ABCA7 protein, containing two nucleotide binding domains (NBD). Usage of a cryptic splice acceptor in exon 18 and retention of a single VNTR unit lead to a frameshift, which causes a premature termination codon (PTC) that leads to either a truncated protein or degradation of the mRNA via nonsense mediated mRNA decay (NMD). Complete skipping of exon 19 causes a deletion of 44 amino acids embedded in the first ABCA7 NBD

Fig. 2

Association of ABCA7 VNTR lengths with AD and AD-associated SNPs. a The distribution of largest and smallest Southern blotted VNTR alleles is shown in relation to the genotype of rs3764650 and rs78117248 in three categories: homozygous reference allele (blue), heterozygous (green), and homozygous risk allele (red) carriers. These distributions are shown per phenotype in Fig. S5. b The main panel depicts patients (red) and healthy elderly control individuals (blue) according to their largest (x-axis) and smallest (y-axis) VNTR allele, as determined by Southern blotting. The upper and right panel show the Q–Q distance, respectively corresponding to the largest and smallest VNTR allele. A positive distance reflects enrichment in AD patients, while a negative distance corresponds to enrichment in controls

Fig. 3

Effect of the ABCA7 VNTR on CSF biomarkers of AD. Logarithmic levels (y-axis) of CSF Aβ_1–42 (a), P-tau_181P (b), and T-tau (c) in AD patients corresponding to the sum of VNTR allele lengths (x-axis) within those patients. A trendline (blue) is shown with standard error (shaded area)

Fig. 4

Quantification of ABCA7 VNTR effects on ABCA7 expression and splicing. a–c Quantification of ABCA7 expression in LCL according to the sum of the two VNTR alleles within an individual (x-axis). a Relative RT-PCR determined ABCA7 expression targeting all known isoforms of ABCA7 for patients (dots) and controls (triangles). A trendline (blue) is shown with standard error (shaded area). b The ratio of canonical (green), alternative exon 18 splicing (orange), VNTR unit retention (purple), and exon 19 skipping (red) shown for individuals (dots) and with a trendline per isoform. c Exon 19 skipping shown in more detail for patients (dots), and controls (triangles) with a trendline and standard error (shaded area). d–f Distribution of the four isoforms (x-axis) across three different tissues: LCL (d), hippocampus (e), and Brodmann area (BA) 10 (f)