1. Whole genome methylation profiling difference in PBMC between responder and nonresponder of acute exacerbations of COPD patients treated with corticosteroid Lawrence Wu, Ph.D Associate Professor Institute of Medical Sciences Tzu Chi University

2. COPD Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and mortality throughout the world, and further increases in its prevalence and mortality can be predicted in the coming decades. The World Health Organization has predicted that it will be the third leading cause of death in the world by the year 2020. The clinical course of the disease is characterized by progressive, irreversible airflow obstruction associated with chronic inflammation of the respiratory tract. However, there are still no effective drug therapies for COPD that alter disease progression.

4. a cough that lasts a long time, or coughing up mucus feeling short of breath, especially when you are making an effort (climbing stairs, exercising) many lung infections that last a long time (the flu, acute bronchitis, pneumonia, etc.) wheezing (a whistling sound when you breathe) feeling tired (fatigue) losing weight without trying

6. AECOPD Acute exacerbations are triggered mainly by respiratory tract infections. According to evidence-based reviews and current guidelines, systemic glucocorticoid therapy is an integral part of the management of COPD exacerbations

7. Steroids treatment Steroids are often used in the treatment of AECOPD. Use of corticosteroids has been shown to shorten recovery time, hasten improvement in lung function, reduce the risk of early relapse and reduce length of hospital stay. The existing guidelines suggest that oral administration of corticosteroids in a dose of 30– 40 mg prednisolone per day for 10–14 days is preferable.

8. Study subjects All 60 enrolled patients with COPD exacerbation were received medicine including Predisolone 2 tablet (5mg/tablet) three times a day, Medicon 1 tablet three times a day, Ventolin 1 tablet three times a day, and Bisolvon 1 tablet three time a day. The treatment duration is two weeks (14 days). Subjects were improved all evaluation (CAT, spirometry test) after treatment and defined as responder of corticosteroid treatment. Other subjects without improved CAT and spirometry test after treatment were defined as non-responder of corticosteroid treatment.

9. CAT is usefulness in evaluating COPD exacerbation Mackay AJ et al. Am J Respir Crit Care Med Vol 185, Iss. 11, pp 1218–1224, Jun 1, 2012 The CAT provides a reliable score of exacerbation severity. Baseline CAT scores are elevated in frequent exacerbators. CAT scores increase at exacerbation and reflect severity as determined by lung function and exacerbation duration.

10. COPD Assessment Test (CAT)

11. COPD is progressive disease, the FEV1and FVC is no significant alteration in many COPD patients in acute exacerbation during the two-week medical treatment FEV1, FEV1% and FVC are objective measurements. CAT is subjective questionnaire. Patients with better FEV1, FVC and CAT score (more than 5 points decrease) after treatment were defined as response to corticosteroid. The poor response group was defined as that FEV1 and FVC after 2 week treatment did not better than before treatment and CAT score didn’t decrease (more than 5 points) or increased after treatment.

12. Results Subjects characters The total 24 COPD patients were enrolled and DNA samples were obtained from subjects’ PBMC. The good response group included 9 males and 3 females and the poor response group was all males. All subjects were diagnosis to COPD at first time and never treated with corticosteroid before. The subject counts with different lung function in good and poor response groups are mild 4/5, moderate 5/4 and severe obstruction 3/3, respectively. The average age of two groups was no significant difference.

16. Bisulfate conversion

18. Genome-wide methylation chip 24 selected patients’ PBMC DNA were subjected to genome-wide methylation analysis 500 ng of each sample underwent bisulfite conversion using the EZ DNA methylation kit. Bisulfite converted DNA samples were then subjected to methylation profiling on the Infinium® HumanMethylation450 BeadChips

20. Genomic location of selected methylation site

21. Figure 1. heatmap of methylation pattern, left side (12 subjects): good prognosis patients, right side (12 subjects) : poor prognosis patients

22. Gene with different methylation level between two study groups Genes with low methylation level in poor prognosis group Genes with higher methylation level in poor prognosis group MSTO2P HLA-DPA1LOC339788GSTM3 GAK NMNAT3PPP1R2P9RHPN1 MYADML DNAH2MIR1914 MAD1L1 SCGN MGMTRASA3TCEA2 HLA-DPA1 PSMD8GOLIM4ADAMTS17 FLRT2FOLR3 FLJ41941TMEM41A BST1BAI1 F3 NR3C1 MCCALOX5AP CMTM1 TP53INP2 ZNF235 B3GALT1LOC388428 SH2D6 SLC38A7 MMP17 CHID1

23. False discovery rate adjust TargetIDGeneFDRpdiff(abs)Regulation cg26848724ALOX5AP2.34E-174.82E-230.650512up cg01966791MIR1914;UCKL12.05E-168.46E-220.483542down cg09152047GSTM33.61E-162.23E-210.565655down cg08198265BST17.33E-166.04E-210.607294up cg02296904GAK1.17E-151.45E-200.664047up cg18872426PSMD81.17E-151.34E-200.582313up cg04972775F37.49E-151.08E-190.451633down cg17251433TMEM41A1.42E-132.34E-180.496039down cg05129295Not in gene region9.50E-121.76E-160.358707up cg23464510FOLR37.66E-081.74E-120.308588up cg00903950MSTO2P6.99E-061.87E-100.533629up cg24867279Not in gene region2.40E-056.91E-100.486841down cg07192612Not in gene region2.20E-047.24E-090.348538down cg23477406MMP173.80E-041.57E-080.408409down cg02604560GOLIM40.0044672.67E-070.460664down cg02588809RASA30.0067414.86E-070.440774down cg07865444Not in gene region0.0067415.14E-070.447221down cg08271318ZNF2350.0067415.55E-070.339917up cg23109606SH2D60.0067415.48E-070.341878down cg17342132NR3C10.0081528.03E-070.475791down cg27370028TCEA20.0164692.17E-060.305391down cg07091346CHID10.0316515.81E-060.391012down cg05853503LOC3884280.035257.04E-060.408724down

24. Several genes methylation status is powerful to distinguish different prognosis geneProbe ID △ AVG  AVG  of good prognosisAVG  of poor prognosis meanmaxminmeanmaxmin GSTM3cg09152047-0.56570.03080.04090.02330.59640.70180.5334 TMEM41Acg17251433-0.49600.03230.04110.02520.52840.62520.4022 MIR1914cg01966791-0.48350.46530.54450.40750.94890.96400.9137 NR3C1cg17342132-0.47580.34050.86700.16700.81630.86470.7641 GOLIM4cg02604560-0.46070.32830.80130.15510.78900.84270.7214 F3cg04972775-0.45160.03310.04400.01870.48470.57880.4081 MSTO2Pcg009039500.53360.57060.68060.05190.03690.06260.0247 PSMD8cg188724260.58230.84710.87650.81700.26480.37670.1984 BST1cg081982650.60730.87490.90950.85770.26760.39720.2046 ALOX5APcg268487240.65050.96650.97220.95880.31600.39750.2414 GAKcg022969040.66400.97220.98650.96230.30810.44880.2261 AVG: methylation level;  △ AVG =AVG of good prognosis – AVG of poor prognosis; max: maximum value; min: minimal value

25. Genomic location….. geneProbe IDUCSC_CPG_ISLANDS_N AME UCSC_REF GENE_GRO UP RELATION_ TO_UCSC_C PG_ISLAND REGULATORY_FEATURE _GROUP GSTM3cg09152047 chr1:110282351-110283306BodyIsland Promoter_Associated_Cell_type_specific TMEM41Acg17251433 chr3:185216310-185217131 TSS200Island Unclassified MIR1914cg01966791 chr20:62571738-62572556BodyS_Shore Gene_Associated NR3C1cg17342132 chr5:142782071-142785071BodyN_Shore Promoter_Associated_Cell_type_specific GOLIM4cg02604560 Chr3:167789884Body F3cg04972775 chr1:95006837-95008051TSS1500Island Unclassified MSTO2Pcg00903950 chr1:155715297-155715908TSS1500Island Promoter_Associated PSMD8cg18872426 chr19:38876070-388763323'UTRN_Shelf BST1cg08198265 chr4:15704640-15705000BodyS_Shelf ALOX5APcg26848724 Chr13:31326405(rs4769874)Body Gene_Associated_Cell_type_specific GAKcg02296904 chr4:878714-878917BodyN_Shore Gene_Associated

26. Some thought about candidate genes FLAP (ALOX5AP) inhibitors for the treatment of inflammatory diseases ( Sampson AP. Curr Opin Investig Drugs. 2009 Nov;10(11):1163-72. ). Does patients with high level methylation reduce the ALOX5AP expression in PBMC cell and obtain the result similar to FLAP inhibitors treatment? Gene expression profiling of lung from emphysema patients identified seven candidate genes associated with emphysema severity including GSTM3. ( Francis SM et al. Respir Res. 2009 Sep 2;10:81.) Glutathione S-transferases (GSTs) detoxify toxic compounds in tobacco smoke via glutathione-dependent mechanisms. Few studies have also found an increase in GSTM3 expression in mild/moderate COPD smokers; this strengthens their role as protective intracellular and extracellular lung mediators ( Bentley AR et al. Thorax 2008, 63(11):956-61. Harju T et al. Respiratory research 2008, 9:80.) Does low level methylation increase the GSTM3 expression in PBMC cell and protect the lung function decline? Many cases of glucocorticoid resistance may be due to mutations or polymorphisms present in the glucocorticoid receptor gene (GR/NR3C1). ( Bray PJ and Cotton RG. Hum Mutat. 2003;21:557-68. ) Does high level methylation decrease NR3C1 expression in PBMC cell and increase the risk of glucocorticoid resistance?

27. COPD methylation profiling : transcription factor analysis 50k 485566 probes 50 probes > 0.25 △ AVG Probe >0.25 Count probes (>0.1 △ AVG ) among upstream 50k and downstream 50k 3 probes with count 10 1 probe with count 7 3 probes with count 3 5 probes with count 2 38 probes with count 1 cg14302130 chr6 chr8 chr8,2,7 chrX,4,4,5,13

28. methylation probes (>0.1)

29. Arnt Xbp1 Tcfap4 Elf1 Max Srebf1 Myc Pax5 Klf12 Postn Runx2 Tcf12 Pax8 Akr1b3 Akr1b7 Areg Mafk Nfe2 Elk1 Sfpi1 Zbtb6 Nfkb1 Nr1h2 Nr1h3 Pou2f1 Pax2 Jun Gcgr Nr3c1 Pitx2 Crx Pax3 Mtf1 Cebpb Rest Cdx1 Tcfap2a Tcf12 Akr1b3 Akr1b7 Areg Nfe2l1 Egr1 Egr2 Gcgr Nr3c1 Nkx3-1 Jun Cdx1 Cebpg Cux1 Myod1 Zeb1 Tcf3 Pax5 Klf12 Tcfap2a Pou3f2 Gata6 Elk1 Sfpi1 Zbtb6 Pax2 Gcgr Nr3c1 Akr1b3 Akr1b7 Areg Srebf1 Pgr HLA-DPA1 chr6:33032346-33041454 HLA-DPA1 -4552-4673 -4098-4219 -424-545 Human TFBS

30. Further thinking……. Do the different response groups indicate two subtypes of COPD? Is the pharmacoepigenetics helpful to reveal heterogeneity of COPD? HLA-DPA1 vs. COPD: MHC class II antigen involving pathological mechanism of COPD?

31. COPD vs. control COPD p vs. control COPD g vs. control Control subjects without lung diseases were selected from another study.

32. COPD vs. control Methylation level down ALOX5AP BST1 GAK PSMD8 CEND1 FAM20C MGMT PRDM16 LRRK1 CDK2AP1 PRKCA GJA3 MCF2L PCCA SCARB1 MCF2L FAM69B RNASE4 ABR SPRR2D RFTN1 UPF1 FRG1B GOLIM4 LOC388428 MAST2 TEKT5 PRKAG2 Methylation level up GSTM3 MIR1914 HBE1 GALNT9 CLDN4 DDX11 RCAN1 SLC14A1 PYROXD1 HLA-DPB2 MYO3B UGT2B15 SEPT9 CLDN4 UGT2B15;UGT2B17 HLA-DQB1 GSTM3 MIR1914 HBE1 GALNT9 CLDN4 DDX1 GJA3 RCAN1 SLC14A1 PYROXD1 HLA-DPB2 MYO3B UGT2B15;UGT2B17 SEPT9 CLDN4 UGT2B15;UGT2B17 HLA-DQB1 MEGF6 CCDC85C SNCAIP CYP2U1 MIR518C;MIR520C DNAJA3 MAGEB3 HMOX2 TIAL1 EXOC7 RGMA MPPED1 ASAH2 HSD3B2 WDR90 KCTD2 OSBPL5 TAP2 ZFYVE28 TAP2 NME6 CCDC46 MCC TP73 MSTO2P FHOD3 FHIT SFRS8 NRGN RAB11B AP4E1 LYPD6B TAP2 POLE

33. Preliminary functional analysis by bioinformatics methods using DAVID Poor response COPD group: related genes located to membrane and associated to glycoprotein (p <0.02) Good response COPD group: related genes associated to Ubl conjugation pathway (p<0.003), nicotinamide nucleotide metabolism (p<0.008), alkaloid metabolic process (p<0.009) and regulation of glucose metabolic process (p<0.006) *DAVID: The Database for Annotation, Visualization and Integrated Discovery

34. 35 genes plasma membrane (p=0.03) cell junction(p=0.04) serine/threonine-protein kinase (p=0.04) 43 genes negative regulation of kinase activity (p=0.004) purine ribonucleotide binding (p=0.03) DNA metabolic process/Purine metabolism(p=0.02) 43 genes (overlap of 3 circles) positive regulation of apoptosis (p=0.09) steroid metabolic process (p=0.02)

35. Non-COPD vs. All COPD Top five significant genes GeneFDR pnote OSBPL51.38E-18 DNA methylation differences at growth related genes correlate with birth weight: a molecular signature linked to developmental origins of adult disease? WDR61.19E-17 WDR6 participates in insulin/IGF-I signaling and the regulation of feeding behavior and longevity in the brain. PRKAG22.36E-17 hypertrophic cardiomyopathy NLRC53.05E-06 NLRC5: a key regulator of MHC class I-dependent immune responses. GIMAP41.53E-05 1.Gimap4 accelerates T-cell death. 2.Knock-down of PHF11 also decreased cell viability and was accompanied by reduced expression of GIMAP4 and 5 genes required for T-cell differentiation, viability and homeostasis.

36. Conclusion The DNA methylation should be a good biomarker for investigating the pharmaco- epigenetics of COPD. Methylation status of COPD susceptibility gene(s), inflammatory gene(s) and glucocorticoid receptor gene associate to outcome of 2-week corticosteroid treatment in AECOPD patients Responsiveness of corticosteroids, should reflect COPD heterogeneity, especially in pathology involving DNA methylation.

37. Future works To link the prognosis of COPD and DNA methylation. To find the new candidate gene(s) or pathological mechanism of COPD by DNA methylation approach

38. Acknowledgement Dr. Shih-Wei Lee (General Taoyuan Hospital) Dr. Paul Wei-Che Hsu (Bioinformatics service center, IMB, Academia Sinica) Dr. Jiu-Yao Wang (NCKU)

39. Thank you for your attention