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

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.

•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

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

Steroids treatmentSteroids 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.

Study subjectsAll 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.

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.

COPD Assessment Test (CAT)

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.

ResultsSubjects charactersThe 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.

Bisulfate conversion

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

Genomic location of selected methylation site

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

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-DPA1 LOC339788 GSTM3GAK NMNAT3 PPP1R2P9 RHPN1MYADML DNAH2 MIR1914 MAD1L1

SCGN MGMT RASA3 TCEA2HLA-DPA1 PSMD8 GOLIM4 ADAMTS17FLRT2 FOLR3 FLJ41941 TMEM41A

BST1 BAI1 F3 NR3C1

MCC ALOX5AP CMTM1 TP53INP2

ZNF235 B3GALT1 LOC388428 SH2D6

SLC38A7 MMP17

CHID1

False discovery rate adjust TargetID Gene FDR p diff(abs) Regulation

cg26848724 ALOX5AP 2.34E-17 4.82E-23 0.650512 up

cg01966791 MIR1914;UCKL1 2.05E-16 8.46E-22 0.483542 down

cg09152047 GSTM3 3.61E-16 2.23E-21 0.565655 down

cg08198265 BST1 7.33E-16 6.04E-21 0.607294 up

cg02296904 GAK 1.17E-15 1.45E-20 0.664047 up

cg18872426 PSMD8 1.17E-15 1.34E-20 0.582313 up

cg04972775 F3 7.49E-15 1.08E-19 0.451633 down

cg17251433 TMEM41A 1.42E-13 2.34E-18 0.496039 down

cg05129295 Not in gene region 9.50E-12 1.76E-16 0.358707 up

cg23464510 FOLR3 7.66E-08 1.74E-12 0.308588 up

cg00903950 MSTO2P 6.99E-06 1.87E-10 0.533629 up

cg24867279 Not in gene region 2.40E-05 6.91E-10 0.486841 down

cg07192612 Not in gene region 2.20E-04 7.24E-09 0.348538 down

cg23477406 MMP17 3.80E-04 1.57E-08 0.408409 down

cg02604560 GOLIM4 0.004467 2.67E-07 0.460664 down

cg02588809 RASA3 0.006741 4.86E-07 0.440774 down

cg07865444 Not in gene region 0.006741 5.14E-07 0.447221 down

cg08271318 ZNF235 0.006741 5.55E-07 0.339917 up

cg23109606 SH2D6 0.006741 5.48E-07 0.341878 down

cg17342132 NR3C1 0.008152 8.03E-07 0.475791 down

cg27370028 TCEA2 0.016469 2.17E-06 0.305391 down

cg07091346 CHID1 0.031651 5.81E-06 0.391012 down

cg05853503 LOC388428 0.03525 7.04E-06 0.408724 down

Several genes methylation status is powerful to distinguish different prognosis

gene Probe ID △ AVG AVG of good prognosis AVG of poor prognosis

mean max min mean max min

GSTM3 cg09152047 -0.5657 0.0308 0.0409 0.0233 0.5964 0.7018 0.5334

TMEM41A cg17251433 -0.4960 0.0323 0.0411 0.0252 0.5284 0.6252 0.4022

MIR1914 cg01966791 -0.4835 0.4653 0.5445 0.4075 0.9489 0.9640 0.9137

NR3C1 cg17342132 -0.4758 0.3405 0.8670 0.1670 0.8163 0.8647 0.7641

GOLIM4 cg02604560 -0.4607 0.3283 0.8013 0.1551 0.7890 0.8427 0.7214

F3 cg04972775 -0.4516 0.0331 0.0440 0.0187 0.4847 0.5788 0.4081

MSTO2P cg00903950 0.5336 0.5706 0.6806 0.0519 0.0369 0.0626 0.0247

PSMD8 cg18872426 0.5823 0.8471 0.8765 0.8170 0.2648 0.3767 0.1984

BST1 cg08198265 0.6073 0.8749 0.9095 0.8577 0.2676 0.3972 0.2046

ALOX5AP cg26848724 0.6505 0.9665 0.9722 0.9588 0.3160 0.3975 0.2414

GAK cg02296904 0.6640 0.9722 0.9865 0.9623 0.3081 0.4488 0.2261

AVG: methylation level;△ AVG =AVG of good prognosis – AVG of poor prognosis; max: maximum value; min: minimal value

Genomic location…..gene Probe ID UCSC_CPG_ISLANDS_N

AMEUCSC_REFGENE_GROUP

RELATION_TO_UCSC_CPG_ISLAND

REGULATORY_FEATURE_GROUP

GSTM3 cg09152047 chr1:110282351-110283306 Body Island Promoter_Associated_Cell_type_specific

TMEM41A cg17251433 chr3:185216310-185217131 TSS200 Island Unclassified

MIR1914 cg01966791 chr20:62571738-62572556 Body S_Shore Gene_Associated

NR3C1 cg17342132 chr5:142782071-142785071 Body N_Shore Promoter_Associated_Cell_type_specific

GOLIM4 cg02604560 Chr3:167789884 Body

F3 cg04972775 chr1:95006837-95008051 TSS1500 Island Unclassified

MSTO2P cg00903950 chr1:155715297-155715908 TSS1500 Island Promoter_Associated

PSMD8 cg18872426 chr19:38876070-38876332 3'UTR N_Shelf

BST1 cg08198265 chr4:15704640-15705000 Body S_Shelf

ALOX5AP cg26848724 Chr13:31326405(rs4769874) Body Gene_Associated_Cell_type_specific

GAK cg02296904 chr4:878714-878917 Body N_Shore Gene_Associated

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?

COPD methylation profiling : transcription factor analysis

50k 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 101 probe with count 73 probes with count 35 probes with count 238 probes with count 1

cg14302130

chr6chr8

chr8,2,7

chrX,4,4,5,13

methylation probes (>0.1)

ArntXbp1Tcfap4Elf1MaxSrebf1MycPax5Klf12PostnRunx2Tcf12Pax8Akr1b3Akr1b7AregMafkNfe2Elk1Sfpi1Zbtb6Nfkb1Nr1h2Nr1h3Pou2f1Pax2JunGcgrNr3c1Pitx2CrxPax3Mtf1Cebpb

RestCdx1Tcfap2aTcf12Akr1b3Akr1b7AregNfe2l1Egr1Egr2GcgrNr3c1

Nkx3-1JunCdx1CebpgCux1Myod1Zeb1Tcf3Pax5Klf12Tcfap2aPou3f2Gata6Elk1Sfpi1Zbtb6Pax2GcgrNr3c1Akr1b3Akr1b7AregSrebf1Pgr

HLA-DPA1

chr6:33032346-33041454 HLA-DPA1

-4552-4673 -4098-4219 -424-545

Human TFBS

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?

COPD vs. control

COPD p vs. control

COPD g vs. control

Control subjects without lung diseases were selected from another study.

COPD vs. control Methylation level down

ALOX5AP

BST1GAKPSMD8CEND1FAM20CMGMTPRDM16LRRK1CDK2AP1PRKCAGJA3MCF2LPCCASCARB1MCF2LFAM69BRNASE4ABRSPRR2DRFTN1UPF1FRG1BGOLIM4LOC388428MAST2TEKT5PRKAG2

Methylation level upGSTM3MIR1914HBE1GALNT9CLDN4DDX11RCAN1SLC14A1PYROXD1HLA-DPB2MYO3BUGT2B15SEPT9CLDN4UGT2B15;UGT2B17HLA-DQB1GSTM3MIR1914HBE1GALNT9CLDN4DDX1GJA3RCAN1SLC14A1PYROXD1HLA-DPB2MYO3B

UGT2B15;UGT2B17SEPT9CLDN4UGT2B15;UGT2B17HLA-DQB1MEGF6CCDC85CSNCAIPCYP2U1MIR518C;MIR520CDNAJA3MAGEB3HMOX2TIAL1EXOC7RGMAMPPED1ASAH2HSD3B2WDR90KCTD2OSBPL5TAP2ZFYVE28TAP2NME6CCDC46MCCTP73

MSTO2PFHOD3FHITSFRS8NRGNRAB11BAP4E1LYPD6BTAP2POLE

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

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)

Non-COPD vs. All COPD Top five significant genes

Gene FDR p note

OSBPL5 1.38E-18DNA methylation differences at growth related genes correlate with birth weight: a molecular signature linked to developmental origins of adult disease?

WDR6 1.19E-17WDR6 participates in insulin/IGF-I signaling and the regulation of feeding behavior and longevity in the brain.

PRKAG2 2.36E-17hypertrophic cardiomyopathy

NLRC5 3.05E-06NLRC5: a key regulator of MHC class I-dependent immune responses.

GIMAP4 1.53E-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.

ConclusionThe 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.

Future worksTo link the prognosis of COPD and

DNA methylation.

To find the new candidate gene(s) or pathological mechanism of COPD by DNA methylation approach

AcknowledgementDr. Shih-Wei Lee (General

Taoyuan Hospital)

Dr. Paul Wei-Che Hsu (Bioinformatics service center, IMB, Academia Sinica)

Dr. Jiu-Yao Wang (NCKU)

Thank you for your attention