Novel proteins associated with risk for coronary heart disease or stroke among postmenopausal women identified by in-depth plasma proteome profiling

Abstract

Background: Coronary heart disease (CHD) and stroke were key outcomes in the Women's Health Initiative (WHI) randomized trials of postmenopausal estrogen and estrogen plus progestin therapy. We recently reported a large number of changes in blood protein concentrations in the first year following randomization in these trials using an in-depth quantitative proteomics approach. However, even though many affected proteins are in pathways relevant to the observed clinical effects, the relationships of these proteins to CHD and stroke risk among postmenopausal women remains substantially unknown.

Methods: The same in-depth proteomics platform was applied to plasma samples, obtained at enrollment in the WHI Observational Study, from 800 women who developed CHD and 800 women who developed stroke during cohort follow-up, and from 1-1 matched controls. A plasma pooling strategy, followed by extensive fractionation prior to mass spectrometry, was used to identify proteins related to disease incidence, and the overlap of these proteins with those affected by hormone therapy was examined. Replication studies, using enzyme-linked-immunosorbent assay (ELISA), were carried out in the WHI hormone therapy trial cohorts.

Results: Case versus control concentration differences were suggested for 37 proteins (nominal P < 0.05) for CHD, with three proteins, beta-2 microglobulin (B2M), alpha-1-acid glycoprotein 1 (ORM1), and insulin-like growth factor binding protein acid labile subunit (IGFALS) having a false discovery rate < 0.05. Corresponding numbers for stroke were 47 proteins with nominal P < 0.05, three of which, apolipoprotein A-II precursor (APOA2), peptidyl-prolyl isomerase A (PPIA), and insulin-like growth factor binding protein 4 (IGFBP4), have a false discovery rate < 0.05. Other proteins involved in insulin-like growth factor signaling were also highly ranked. The associations of B2M with CHD (P < 0.001) and IGFBP4 with stroke (P = 0.005) were confirmed using ELISA in replication studies, and changes in these proteins following the initiation of hormone therapy use were shown to have potential to help explain hormone therapy effects on those diseases.

Conclusions: In-depth proteomic discovery analysis of prediagnostic plasma samples identified B2M and IGFBP4 as risk markers for CHD and stroke respectively, and provided a number of candidate markers of disease risk and candidate mediators of hormone therapy effects on CHD and stroke.

Clinical trials registration: ClinicalTrials.gov identifier: NCT00000611.

Figure 1

Identification and quantitative analysis of peptides in plasma. From CHD cases and controls in eight experiments for (a) beta-2 microglobulin (B2M) and (b) alpha-1-acid glycoprotein 1 (ORM1); and from stroke cases and controls in eight experiments for (c) peptidyl-prolyl isomerase A (PPIA) and (d) insulin-like growth factor binding protein 4 (IGFBP4). Tryptic peptides from the amino terminus (1) to the carboxyl terminus are shown at the top. S, C and G indicate signal peptide, cysteine-containing and glycosylated peptides, respectively. Peptides identified, but which lack cysteine for quantification, are shown in gray. The log2 case/control ratio is shown for cysteine-containing peptides with the number of MS events for that peptide shown in parentheses. The number of plasma fractions where each peptide was quantified is indicated.

Figure 2

Glycolysis/gluconeogenesis pathway. Enzymes identified in stroke experiments are indicated by shading. Red and yellow indicate increased and no change in cases compared to controls, respectively. Gray indicates proteins identified but not quantified.

Figure 3

Baseline plasma B2M concentrations for CHD cases and controls, and IGFBP4 concentrations for stroke cases and controls, from the Women's Health Initiative hormone therapy trials. Individual ELISA-based concentrations are shown along with boxplots showing the median (dark line) and the 25th and 75th percentiles (bottom and top of box). The notches indicate 95% confidence intervals for the median.