Rare coding variants in the phospholipase D3 gene confer risk for Alzheimer's disease

Abstract

Genome-wide association studies (GWAS) have identified several risk variants for late-onset Alzheimer's disease (LOAD). These common variants have replicable but small effects on LOAD risk and generally do not have obvious functional effects. Low-frequency coding variants, not detected by GWAS, are predicted to include functional variants with larger effects on risk. To identify low-frequency coding variants with large effects on LOAD risk, we carried out whole-exome sequencing (WES) in 14 large LOAD families and follow-up analyses of the candidate variants in several large LOAD case-control data sets. A rare variant in PLD3 (phospholipase D3; Val232Met) segregated with disease status in two independent families and doubled risk for Alzheimer's disease in seven independent case-control series with a total of more than 11,000 cases and controls of European descent. Gene-based burden analyses in 4,387 cases and controls of European descent and 302 African American cases and controls, with complete sequence data for PLD3, reveal that several variants in this gene increase risk for Alzheimer's disease in both populations. PLD3 is highly expressed in brain regions that are vulnerable to Alzheimer's disease pathology, including hippocampus and cortex, and is expressed at significantly lower levels in neurons from Alzheimer's disease brains compared to control brains. Overexpression of PLD3 leads to a significant decrease in intracellular amyloid-β precursor protein (APP) and extracellular Aβ42 and Aβ40 (the 42- and 40-residue isoforms of the amyloid-β peptide), and knockdown of PLD3 leads to a significant increase in extracellular Aβ42 and Aβ40. Together, our genetic and functional data indicate that carriers of PLD3 coding variants have a twofold increased risk for LOAD and that PLD3 influences APP processing. This study provides an example of how densely affected families may help to identify rare variants with large effects on risk for disease or other complex traits.

Figure 1. Summary of the main genetic findings

The diagram represents the different steps to filter the variants identified by exome-sequencing, which lead to the identification of the PLD3-V232M variant. The diagram also shows the subsequent genetic analyses in large case-control datasets that validated the association of the V232M variant and PLD3 with risk for AD.

Figure 2

A) Schematic representation of PLD3 and the relative position of the PLD3 variants. PLD3 has two PLD phosphodiesterase domains, which contain an HKD signature motif (H-x-K-x(4)-D-x(6)-G-T-x-N, where x represents any amino acid residue). The scheme also shows the exon composition of the longest PLD3 mRNA and the position of the variants found in this study. Variants highlighted in red and noted with an “*” are significantly associated with AD risk. Variants noted with a “†” were found only in AD cases. Variants noted with a “ɣ” are more frequent in AD cases compared to controls. B) PLD3 neuronal gene expression is significantly lower in AD cases compared to controls. We used the GEO dataset GSE5281, in which neurons were laser-captured to analyze whether PLD3 mRNA expression levels are different between AD cases and cognitively normal elderly individuals.. C-D) The PLD3 A442A variant is associated with lower total PLD3 mRNA expression and lower levels of exon11 containing transcripts. C) Primers specific to exons 7, to 11 (two pairs of primers) were designed with PrimerExpress. cDNA from eight PLD3 A442A carriers and ten age, gender, APOE, CDR and PMI-matched individuals were obtained from parietal lobe. Relative expression of exon 11 compared to the other exons was calculated by the ΔCt method. Exon 11 containing transcripts in relation to exon 7-10 containing transcripts were 20% lower in A442A carriers (P<0.05). Graphs represent the mean±SEM. D). Real-time PCR was used to quantify total PLD3 mRNA and standardized using GADPH mRNA as a reference. P-value is for the gene-expression levels of major allele carriers vs. minor allele carriers after correcting for dementia severity.

Figure 3. PLD3 affects APP processing

A-B. Overexpression and knockdown of PLD3 produce opposing effects on extracellular Aβ levels. N2A cells stably expressing hAPP695-WT were transiently transfected with vectors containing no insert (pcDNA3), human PLD3-WT, scrambled shRNA (Origene), or mouse PLD3 shRNA (Origene) for 48 hours. Cell media were analyzed with Aβ40 and Aβ42 ELISAs and corrected for total intracellular protein. Aβ levels were then expressed relative to pcDNA3. Graphs represent the mean±SEM. A) Overexpression of human PLD3 produces significantly less extracellular Aβ42 and Aβ40. “*”, p<0.0001. B). Knockdown of endogenous PLD3 cells produces significantly more extracellular Aβ42 and Aβ40. “*”, p<0.002. C) Members of the PLD protein family have different effects on APP processing. HEK293T cells were transiently transfected with vectors containing hAPP-WT and an empty vector (pcDNA3), PLD1, PLD2, or PLD3-WT or PLD1, PLD2, PLD3 carrying a dominant negative mutation.. Left panel, PLD3 affects full-length APP levels. Cell lysates were extracted in non-ionic detergent, analyzed by SDS-PAGE and immunoblotting with antibodies to the myc-tag on APP (9E10) or β-tubulin. Middle (Aβ42) and right (Aβ40) panel, cell media were analyzed with Aβ40 and Aβ42 ELISAs and corrected for total intracellular protein. Graphs represent the mean±SEM. “*”, p<0.01, different from pcDNA3; “ο”, p=0.002, different from PLD1-WT; “•”, p<0.0001, different from PLD2-WT. Images are representative of at least three replicate experiments.

Extended Data Figure 1. PLD3 V232M is associated with age at onset for AD

. Age at onset was analyzed for association with the PLD3 V232M variant in 2,220 cases and 1,841 controls from the Knight-ADRC and NIA-LOAD, by the Kaplan-Meier method and tested for significant differences using the Log-rank test. A) Case only analysis. The carriers of the minor allele (AG) have an AAO 3 years lower than the non-carriers (69 vs 73; p=3×10^-3). B) Controls were included as censored data. The carriers of the minor allele (AG) have an AAO 8 years lower than the non-carriers (70 vs 78; p=3×10^-3).

Extended Data Figure 2

Forest plot for each case-control series for the V232M variant.

Extended Data Figure 3. PLD3 and APP mRNA expression are inversely correlated

PLD3 (probe 201050_at) and APP (probe 211277_x_at) expression levels were extracted from the GSE5281 dataset. PLD3 mRNA levels are significantly lower in AD cases compared to controls (p=8.10×10^-10), but APP is higher in AD cases (p=7.88×10^-8). PLD3 mRNA levels are inversely correlated with APP mRNA expression levels (p=1.00×10^-16). The correlation is stronger in AD cases (Person correlation coefficient = -0.55), than in controls (Person correlation coefficient = -0.44), but in both scenarios the correlation is highly significant.

Extended Data Figure 4. PLD3 interacts with APP

HEK293T cells were transiently transfected with vectors containing APP WT and an empty vector (pcDNA3) or PLD3 WT for 48 hours. Cell lysates were extracted in non-ionic detergent, pre-cleared with Protein A beads, and immunoprecipitated with an antibody to the myc-tag on APP (9E10). Immunoblots were probed with an antibody specific to human PLD3. PLD1 and PLD2 reportedly do not inmunoprecipitate with APP^,.