Gene-Specific DNA Methylation Changes Predict Remission in Patients with ANCA-Associated Vasculitis

Abstract

ANCA-associated vasculitis is an autoimmune condition characterized by vascular inflammation and organ damage. Pharmacologically induced remission of this condition is complicated by relapses. Potential triggers of relapse are immunologic challenges and environmental insults, both of which associate with changes in epigenetic silencing modifications. Altered histone modifications implicated in gene silencing associate with aberrant autoantigen expression. To establish a link between DNA methylation, a model epigenetic gene silencing modification, and autoantigen gene expression and disease status in ANCA-associated vasculitis, we measured gene-specific DNA methylation of the autoantigen genes myeloperoxidase (MPO) and proteinase 3 (PRTN3) in leukocytes of patients with ANCA-associated vasculitis observed longitudinally (n=82) and of healthy controls (n=32). Patients with active disease demonstrated hypomethylation of MPO and PRTN3 and increased expression of the autoantigens; in remission, DNA methylation generally increased. Longitudinal analysis revealed that patients with ANCA-associated vasculitis could be divided into two groups, on the basis of whether DNA methylation increased or decreased from active disease to remission. In patients with increased DNA methylation, MPO and PRTN3 expression correlated with DNA methylation. Kaplan-Meier estimate of relapse revealed patients with increased DNA methylation at the PRTN3 promoter had a significantly greater probability of a relapse-free period (P<0.001), independent of ANCA serotype. Patients with decreased DNA methylation at the PRTN3 promoter had a greater risk of relapse (hazard ratio, 4.55; 95% confidence interval, 2.09 to 9.91). Thus, changes in the DNA methylation status of the PRTN3 promoter may predict the likelihood of stable remission and explain autoantigen gene regulation.

Keywords: ANCA; DNA methylation; epigenetics; gene expression; vasculitis.

Figure 1.

Relative DNMT1 expression and DNA methylation at PRTN3 and MPO loci comparing active to remission states. (A) Two-fold decrease in mean DNMT1 expression in active patients (red) compared with healthy controls (green); mean expression in remitting patients (blue) was 1.5-fold higher than active patients. Bars shown are mean and SD; P<0.03 is considered significant after accounting for multiple testing. (B) Three PRTN3 amplicons covering: the promoter, a CGI and intron 2, a CGI and exon 5. (C) Two MPO amplicons covering: a CGI and exon 7; a CGI and exon 5–6. Genes are shown in black, amplicons in blue, CGIs in green. Amplicon-wide cross-sectional DNA methylation patterns at the (D) PRTN3 promoter and (E) MPO CGI/exon 5–6. Green circles are healthy controls; squares are PR3-ANCA patients; triangles are MPO-ANCA patients; active patients are red; patients in remission are blue. Bars shown are median with interquartile range; P<0.01 is considered significant after accounting for multiple testing. HC, healthy control.

Figure 2.

Correlation of DNA methylation and mRNA expression at PRTN3 and MPO. Total leukocytes from healthy controls (green circles), MPO-ANCA patients (blue triangles), and PR3-ANCA patients (purple squares). Log transformed correlation between DNA methylation at the (A) PRTN3 promoter and PRTN3 expression (n=187; r=−0.2828) and (B) DNA methylation at MPO CGI/exon 5–6 and MPO expression (n=186; r=−0.3155).

Figure 3.

Longitudinal change in DNA methylation from disease activity to remission. Mean and SD shown; P values are as different from zero, where P<0.03 is significant after accounting for multiple testing. PR3-ANCA patients are squares and MPO-ANCA patients are triangles. Mean DNA methylation change at the (A) PRTN3 promoter: PR3-ANCA patients 4.03%, MPO-ANCA patients 3.69% and the (B) MPO CGI/exon 5–6: PR3-ANCA patients 3.06%, MPO-ANCA patients 4.93%.

Figure 4.

AAV patients stratified by DNA methylation increase or decrease. Paired patients with (A) increased DNA methylation and (B) decreased DNA methylation in disease remission at the PRTN3 promoter. (C) DNA methylation at the PRTN3 promoter compared with expression of PRNT3 in patients with increased methylation in disease remission (A) (r=−0.3390). (D) DNA methylation at the PRTN3 promoter compared with expression of PRNT3 in the samples with decreased methylation in disease remission (B) (r=−0.08322). Paired patients with (E) increased methylation and (F) decreased methylation in disease remission at MPO CGI/exon 5–6. (G) DNA methylation at MPO CGI/exon 5–6 compared with expression of MPO in patients with increased methylation in disease remission (E) (r=−0.3735). (H) DNA methylation at MPO CGI/exon 5–6 compared with expression of MPO in patients with decreased methylation in disease remission (F) (r=−0.08508). P values <0.05 are considered significant.

Figure 5.

DNA methylation and probability of relapse. AAV patients stratified by DNA methylation change and followed until next relapse or last clinic follow-up at the (A) PRTN3 promoter and (B) MPO CGI/exon 5–6; decreased DNA methylation in red, increased DNA methylation in blue. AAV patients stratified by DNA methylation change and serotype and followed until next relapse or last clinic follow-up at the (C) PRTN3 promoter and (D) MPO CGI/exon 5–6; MPO-ANCA patients with decreased DNA methylation in orange, MPO-ANCA patients with increased DNA methylation in dashed purple, PR3-ANCA patients with decreased DNA methylation in green, PR3-ANCA patients with increased DNA methylation in dashed gray. (E) AAV patients stratified by serotype and followed until next relapse or last clinic follow-up at the PRTN3 promoter; PR3-ANCA patients in pink, MPO-ANCA patients in black. Numbers at bottom of graphs correspond to the number of patients in each group who have not relapsed and have been followed up in clinic. P values <0.05 are considered significant.