A Robust Serum Proteomic Signature of the E2 Allele of Apolipoprotein E

Abstract

A signature of 16 serum proteins that were previously profiled using the aptamer-based Somascan technology highlighted the roles of the e2 allele of APOE in lipid regulation via apolipoprotein B (APOB) and apolipoprotein E (APOE) and in inflammation. Here, the serum protein signature of APOE is validated and expanded using a combination of mass-spectrometry, ELISA, Luminex, blood transcriptomics, and antibody-based Olink serum proteomics. Some of the findings were replicated in the UK Biobank using antibody-based Olink serum proteomics. This analysis replicated the association between APOB and the e2 allele of APOE, detected a new, robust pattern of association between APOE genotypes and the serum level of APOE, and discovered new associations between APOE genotypes and the complex of apolipoproteins APOC1, APOC2, APOC3, APOC4, APOE, APOF, and APOL1. In addition, 13 new proteins correlated with APOE genotypes. This extended signature includes granule proteins CAMP, CTSG, DEFA3, and MPO secreted from neutrophils and points to olfactomedin 4 (OLFM4) as a new target for the prevention of Alzheimer's disease.

Keywords: APOE; aptameter‐based proteomics; gene expression; genetic variants; mass‐spectrometry; omics‐signatures.

Figure 1

Top left: We detected 398 proteins in 50 serum samples with nLC‐MS/MS, compared to the 3,889 proteins detected with the Somascan platform. There were 266 proteins in common. Bottom Left: the Venn diagram displays the number of proteins significantly associated with APOE genotypes at 1%FDR in the nLC‐MS/MS analysis (purple), and in Somascan‐based analysis of the same 50 samples (green). Right: The log2‐fold change relative to the mean expression of APOB and APOE in carriers of the e3e3 genotype. Both analyses detected APOB and APOE associations with APOE genotypes (purple: nLC‐MS/MS data; green Somascan). Samples per genotype group: e2e2: N = 7; e2e3: N = 13; e3e3: N = 20; e3e4: N = 10.

Figure 2

The distribution of apolipoproteins and CETP by APOE genotypes. Bar plots of the estimated log2‐fold changes of serum proteins comparing e2e2, e2e3, and e3e4 to e3e3 carriers. The bars denote SE. The density plot displays the pairwise correlations between these serum proteins in the four APOE genotype groups. Carriers of the e2e2 and e2e3 genotype have stronger pairwise correlations. Samples per genotype group: e2e2: N = 7; e2e3: N = 13; e3e3: N = 20; e3e4: N = 10.

Figure 3

The distribution of inflammatory markers by APOE genotypes. Bar plots of the estimated log2‐fold change of the six inflammatory markers comparing e2e2, e2e3, and e3e4 to e3e3 carriers. The density plot displays the pairwise correlations between these serum proteins in the four APOE genotype groups. Samples per genotype group: e2e2: N = 7; e2e3: N = 13; e3e3: N = 20; e3e4: N = 10.

Figure 4

a) Correlation of estimated log2‐fold change of gene expression in 1,348 blood samples from the LLFS (x‐axis), and serum proteins measured with nLC‐MS in 50 NECS participants (y‐axis). Each dot represents one of the 196 genes that we could map successfully between the two datasets. Left: The fold changes in the LLFS were calculated comparing the seven e2e2 carriers vs 851 e3e3 carriers. Right: The fold changes in the LLFS were calculated comparing 203 e2e3 carriers vs 851 e3e3 carriers. b) Correlation of APOE effects with gene expression in 1348 blood samples from the LLFS (x‐axis), and the serum proteins measured with the Somascan technology in 220 NECS participants (y‐axis). Each dot represents one of the 2,023 genes that we could map successfully between the two datasets. Left: The fold changes in the LLFS were calculated comparing 7 e2e2 carriers vs 851 e3e3 carriers. Right: The fold changes in the LLFS were calculated comparing 203 e2e3 carriers vs 851 e3e3 carriers. c) Replication of some of the effects detected with the LC‐MS/MS analysis and Luminex. d) Estimated hazard ratio (HR) and 95% confidence intervals (CI) for hypertension (HTN), death, and cancer for a log2‐fold change of APOB, APOC2, APOC3, APOE, CTSG, and MPO. Carriers of the genotype groups in the Luminex data set: e2e2: N = 6; e2e3: N = 19; e3e3: N = 49; e3e4: N = 23.