SP3
Genome wide DNA methylation patterns in ANCA Associated Vasculitis
Brown N1, Williams S2, O’Sullivan J2, Venning M1, Brenchley P1
1. Renal Unit , Manchester Royal Infirmary, CMFT 2. Genetics department, St. Mary’s
Hospital, CMFT
Background: ANCA associated vasculitis (AAV) is an autoimmune disease with a
predilection for the renal tract and the potential to cause kidney injury and failure.
Autoantibodies directed against neutrophil proteins (PR3 and MPO) are known to have an
important role in disease pathogenesis but there are many additional mechanisms involved in
disease development that are not fully understood. There is now strong evidence for
underlying genetic predisposition (with genetic differences between MPO and PR3 positive
disease) but additional factors need to be present for disease development in the individual.
These may be epigenetic, referring to the process of altering genetic expression without
altering genetic sequence, often as a result of environmental exposure. The most frequently
observed of these regulatory processes is DNA methylation. We hypothesise that DNA
methylation patterns will differ between MPO and PR3 positive disease and that these
differences will help reveal more about the disease pathogenesis.
Patients and methods: Peripheral blood samples were taken from 20 patients with AAV, 10
with MPO positive disease and 10 with PR3 positive disease. Whole gene DNA methylation
sequencing was performed on all peripheral nucleated cells. The MDB based methylMiner
system was used for enrichment of methylated segments, with the subsequent library
generated using the TruSeq Nano library kit and sequenced on the Illumina HiSeq 2500. Data
was analysed using the MEDIPS Bioconductor package in R to calculate genome wide
differential coverage. Multiple testing was corrected for using Bonferroni correction.
Results: Comparison of the MPO positive versus the PR3 positive patients revealed 10
Differentially Methylated Regions (DMRs) reaching statistical significance (p<0.01) after
multiple correction testing. These included regions in the vicinity of the gene CLEC6A,
important in the regulation of Th17 differentiation and essential for immune regulation (see
Table 1). In addition there were DMRs involving non coding RNAs (microRNAs and Long
non-coding RNAs) another category of epigenetic control mechanisms with emerging
importance in immune regulation.
Window
Gene
Gene
co-ordinate
8608591
Gene function
P value
Th17 differentiation
3.48E-05
Within gene
body
109599284
RNA
protein
lncRNA
0.002752386
MicroRNA
0.006781731
Deubiquitinating
enzyme
0.007564336
chr12
8576001-8578000
CLEC6A
chr3
30016001-30018000
RBMS3
chr7
109538001-109540000
EIF3Ip1
chr8
30074001-30076000
mir548o-2
chr4
9162001-9164000
USP17L10
within
gene body
9212383
chr12
8586001-8588000
CLEC6A
8608591
chr8
9912001-9914000
MSRA
Within gene
body
binding
0.004535146
0.00802332
repair of oxidative
damage
0.009964491
Table 1. DMRs between MPO and PR3 positive disease.
Discussion: Study of DNA methylation status in leucocytes from patients with AAV
demonstrates significant differences between those with MPO and PR3 antibody positive
disease. Some of these genes are integral to immune control, in particular relevant to Th17
activity, a fairly new T helper subgroup that may be important in AAV. Further investigation
will include examination of the effects of disease activity on methylation status as well as
validation of these results in a larger cohort. We have demonstrated that the study of DNA
methylation in AAV has the potential to reveal more about underlying disease pathogenesis
which may, in the future, assist in identification of therapeutic targets.