Elevated levels of autoantibodies against DNAJC2 in sera of patients with atherosclerotic diseases

Abstract

Background: Serum antibody markers have been increasingly identified not only for cancer and autoimmune diseases but also for atherosclerosis-related diseases such as acute ischemic stroke (AIS), acute myocardial infarction (AMI), diabetes mellitus (DM), and chronic kidney disease (CKD). Biomarkers for transient ischemic attack (TIA) and non-ST segment elevation acute coronary syndrome (NSTEACS) are potentially useful for detection of early phase of atherosclerotic changes against AIS and AMI, respectively.

Methods: We utilized serological identification of antigens by recombinant cDNA expression cloning (SEREX) using a human aortic endothelial cell cDNA phage library and sera from patients with TIA or NSTEACS. Serum antibody levels were measured by amplified luminescent proximity homogeneous assay-linked immunosorbent assay (AlphaLISA) using purified recombinant antigens.

Results: Screening of sera from patients with TIA identified DnaJ heat shock protein family (Hsp40) member C2 (DNAJC2) as a candidate antigen, which was also isolated by SEREX screening using sera of patients with NSTEACS. The validation cohort revealed significantly higher DNAJC2 antibody (DNAJC2-Ab) levels in the sera of patients with TIA or AIS than those in healthy donors (HDs). Multivariate logistic regression analysis indicated that the predictive odds ratios (OR) of DNAJC2-Ab levels for TIA and AIS were 2.54 (95% confidence interval [CI]: 1.36-4.74, p = 0.0034) and 2.14 (95% CI: 1.39-3.30, p = 0.0005), respectively. Serum DNAJC2-Ab levels were also higher in patients with AMI, DM, and CKD than those in HDs.

Conclusion: Serum DNAJC2-Ab level may be useful for early detection of atherosclerotic lesions, which lead to AIS and AMI.

Keywords: Acute ischemic stroke; Acute myocardial infarction; Atherosclerosis; Autoantibody biomarker; Biomarkers; Cardiology; Cardiovascular system; Clinical research; DNAJC2; Diagnostics; Hematological system; Neurology; Neurosurgery.

Figure 1

Western blot analysis. Glutathione-S-transferase (GST) (lane 1) and affinity-purified GST-tagged DnaJ heat shock protein family (Hsp40) member C2 (DNAJC2) (lane 2) proteins were separated on sodium dodecyl sulfate-polyacrylamide gels and blotted using an anti-GST antibody (a); the sera of healthy donors (HDs) (b); or the sera of patients with transient ischemic attack (TIA) (c, d), acute ischemic stroke (AIS) (e), non-ST segment elevation acute coronary syndrome (NSTEACS). (f), and acute myocardial infarction (AMI) (g). Arrows at 58 kDa and 28 kDa indicate GST-DNAJC2 and GST proteins, respectively. Asterisks indicate partially degraded proteins. Molecular weights are shown on the left. The full, non-adjusted image of Figure 1 is shown in the supplementary figure S1.

Figure 2

Serum levels of DNAJC2 antibodies (DNAJC2-Abs) in stroke patients examined by amplified luminescent proximity homogeneous assay-linked immunosorbent assay (AlphaLISA) in the validation cohort. The DNAJC2-Ab levels measured as Alpha photon counts were compared between the HDs and the patients with TIA, AIS, or chronic-phase cerebral infarction (cCI) in box-whisker plots displaying the 10^th, 20^th, 50^th, 80^th, and 90^th percentiles (a). ∗∗∗p < 0.001 by the Mann–Whitney U test with type I error adjustment using the Bonferroni procedure, not significant (n.s.), p = 1 by the Kruskal–Wallis test with type I error adjustment using the Bonferroni procedure. Receiver operating characteristic curve (ROC) analysis was performed to assess the ability of DNAJC2-Abs to detect TIA (b), AIS (c), and chronic cerebral infarction (cCI) (d). Table 3 summarizes areas under the curves (AUCs), 95% confidence intervals (CIs), cutoff values, sensitivity, specificity, and p values calculated using ROC analysis.

Figure 3

Association between DNAJC2-Ab levels and other clinical parameters in stroke patients. Correlations between DNAJC2-Ab levels and age (a), sex (b), hypertension (c), diabetes mellitus (DM) (d), hyperlipidemia (e), cardiovascular disease (CVD) (f), obesity (body mass index ≥25) (g), and smoking (h) were examined using Spearman's correlation analysis (a) or the Mann–Whitney U test (b–h).

Figure 4

Association between DNAJC2-Ab levels and other atherosclerotic diseases including AMI and DM. (a) Serum DNAJC2-Ab levels of the HDs and the patients with AMI or DM determined by AlphaLISA are shown as box-whisker plots, as described in the legend of Figure 2. The mean ages (±standard deviation) of the HDs and the patients with AMI and DM were 58.29 ± 5.63, 58.28 ± 8.5, and 58.37 ± 9.11 years, respectively. Results of the ROC analysis of DNAJC2-Abs to detect AMI (b), and DM (c) are also shown. ∗∗∗p < 0.001 by the Mann–Whitney U test with type I error adjustment using the Bonferroni procedure.

Figure 5

Association between DNAJC2-Ab levels and chronic kidney disease (CKD). (a) Serum DNAJC2-Ab levels of the HDs and the patients with CKD are shown as box-whisker plots, as described in the legend of Figure 2. CKD patients were divided into three groups: type 1, diabetic kidney disease; type 2, nephrosclerosis; and type 3, glomerulonephritis. The mean age (±standard deviation) of the HDs and the patients with type 1, 2, and 3 CKD were 45.82 ± 11.66, 65.78 ± 10.28, 75.97 ± 9.94, and 66.05 ± 14.60 years, respectively. Results of the ROC analysis of DNAJC2-Abs to detect type 1, 2, and 3 CKD (b–d) are also shown. ∗∗∗p < 0.001 by the Mann–Whitney U test with type I error adjustment using the Bonferroni procedure.