ORIGINAL ARTICLEDNA methylation changes between relapse and
remissionof minimal change nephrotic syndromeYasuko Kobayashi &
Akira Aizawa & Takumi Takizawa &Chikage Yoshizawa &
Hiromi Horiguchi & Yuka Ikeuchi &Satoko Kakegawa &
Toshio Watanabe &Kenichi Maruyama & Akihiro Morikawa
&Izuho Hatada & Hirokazu ArakawaReceived: 5 January 2012 /
Revised: 31 May 2012 / Accepted: 11 June 2012#The Author(s) 2012.
This article is published with open access at
Springerlink.comAbstractBackgroundDNA methylation of gene promoters
is associ-atedwithtranscriptional inactivation. Changes
inDNAmethylation can lead to differences in gene expression
levelsandtherebyinfluencediseasedevelopment. Wehypothe-sized that
epigenetics underlies the pathogenesis of minimalchange nephrotic
syndrome (MCNS).MethodsGenome-wide DNA methylation changes
betweenrelapseandremissioninmonocytes (n06) andnaiveThelper cells
(Th0s) (n04) isolated from patients with MCNSwere investigated
using the microarray-based integrated
anal-ysisofmethylationbyisochizomers(MIAMI)method. Weconfirmed the
MIAMI results using bisulfite-pyrosequencinganalysis. Expression
analysis was performed using quantita-tive real-time
PCR.ResultsThreegeneloci (GATA2, PBX4, andNYX)
weresignificantlylessmethylatedinTh0sduringrelapsethaninremission,
comparedtonone inmonocytes. Inaddi-tion,
thedistancedistributionfromtheregressionlineofall
probesinMIAMIwassignificantlydifferent betweenmonocytes and Th0s.
The mRNAlevels of the threegenes in Th0s were not significantly
different betweenrelapseandremission.ConclusionsOur results
demonstrate that thechangeinDNAmethylationpatterns
fromremissiontorelapseinMCNSoccurspredominantlyinTh0sratherthaninmono-cytes
and suggest that epigenetic regulation in Th0s underliesthe
pathogenesis of MCNS.KeywordsDNAmethylation.Nephrotic
syndrome.Monocytes .Naive T helper cells
.Microarray-basedintegratedanalysis of methylationbyisoschizomers
(MIAMI)method .Genome-wide
.ChildrenIntroductionEpigeneticsisthestudyofmitoticallyheritablechangesingeneexpressionthat
occur without direct DNAsequencealterations. DNA methylation, one
of the principal epigenet-ic mechanisms in mammals, involves the
covalent additionof a methyl group to a cytosine residue that is
followed by aElectronic supplementary material The online version
of this article(doi:10.1007/s00467-012-2248-z) contains
supplementary material,which is available to authorized users.Y.
Kobayashi (*): A. Aizawa:T. Takizawa:S. Kakegawa:T. Watanabe: H.
ArakawaDepartment of Pediatrics,Gunma University Graduate School of
Medicine,3-39-22 Showa-machi,Maebashi, Gunma 371-8511, Japane-mail:
[email protected]. Yoshizawa: H. Horiguchi: Y.
IkeuchiDepartment of Pediatrics, Gunma Chuo General
Hospital,Maebashi, Gunma, JapanK. MaruyamaGunma Childrens Medical
Center,Hokkitsumura, Seta-gun, Gunma, JapanA. MorikawaKitakanto
Allergy Institute,Midorishi, Gunma, JapanI. HatadaLaboratory of
Genome Science,Biosignal Genome Resource Center,Gunma University
Institute for Molecular and Cellular Regulation,Maebashi, Gunma,
JapanPediatr NephrolDOI 10.1007/s00467-012-2248-zguanine (CpG) [1].
DNA methylation regulates gene expres-sion and is essential for
differentiation, embryonic develop-ment [2], genomic imprinting[3],
andX-chromosomeinactivation[4].DNAmethylationwithinthepromoterre-gion
of a gene is commonly associated with transcriptionalinactivation,
whereas demethylationcontributes totran-scriptional activation.
Changes
intheDNAmethylationprofilecanalsoleadtodifferencesingeneexpressionpat-terns
andtherebyinfluencethedevelopment of diseases,such as cancer
[5].Minimal change nephrotic syndrome (MCNS) is themost commoncause
of nephrotic syndrome inchildrenandis
characterizedbymassiveproteinuriaandhypoal-buminemia ina
relapse/remissioncourse without histo-logical evidence of
immune-mediated inflammatorydamage. These manifestations are
typically reversiblewith the use of corticosteroid therapy.
Although thepathogenesisof MCNSremainstobeelucidated,
immu-nological disruptionhas beenimplicatedinthis disease[6] as
Tcell-derivedvascular permeabilityfactors
havebeenshowntoberesponsiblefor alterations inglomer-ular
permeability [79]. The incidence of MCNS inchildhoodis
twofoldhigher inboys, witha prevalencethat is inversely
proportional to age, and recurrent re-lapse
tendstolessenafteradolescence[10,11].Sincethecharacteristic
features of MCNSinclude (1) a recurrentrelapse/remissioncourse, (2)
gender preference, (3) agepreferenceofonset andrelapse,
and(4)steroidresponsein most patients, a genetic defect cannot
explain thepathogenesis of this disease; however, epigenetic
alter-ationsmayoccur without adirect changeinthegeneticsequence.
DNAmethylationchangeswithageandenvi-ronmental factors, even in the
same individual, and isinvolvedwithX-chromosomeinactivation.Audard
et al. reported that NFRKB (nuclear factor
relatedtokappaBbindingprotein) was
highlyexpressedinthenuclearcompartmentduringrelapseandthatNFRKBpro-moteshypomethylationofgenomicDNA,
suggestingepi-genetic involvement in the pathogenesis of MCNS [12].
Theepigenotype is influenced by the environment and alters
theregulation of gene expression, leading Elie et al. to suggest
aprobable impact of epigenetic modifications in infected cellssince
MCNS relapses are frequently triggered by external orinternal
environmental factors, includingviral infection[13]. Zhang et al.
reported significant differences in histoneH3 lysine 4
tri-methylation of peripheral blood mononuclearcells(PBMCs)
fromadult patientswithMCNScomparedwith those from healthy subjects.
Their results indicate thatalterations in epigenotype are
associated with the pathogenesisof MCNS [14].The aimof this study
is to elucidate whether theDNAmethylation profile changes between
relapse andremission in MCNS cases and whether this process
isimmune-competent cell-type-specific. Ultimately, wewished to
determine whether epigenetics underlies thepathogenesis of
MCNS.Patients and
methodsPatientsSamplesformicroarray-basedintegratedanalysisofmeth-ylationbyisochizomers(MIAMI)
analysiswereobtainedfrom six male patients with MCNS (Table 1),
while samplesfor quantitativereal-timePCR(qRT-PCR)
wereobtainedfromanadditional sevenpatientswithMCNS(3boys, 4girls)
(Table 2) at relapse andalsofollowingcompleteremission. All
patients werediagnosedaccordingtothecriteriaof theInternational
Studyof KidneyDiseaseinChildren [15] and had developed nephrotic
syndrome priorto 16 years of age. Informed consent was obtained
from theparents of each child and from older children/adolescents
asnecessary. This study was approved by the Ethics Commit-teeof
GunmaUniversityGraduateSchool of Medicine,Japan (Receipt Number
89).Cell separationWeusedmonocytes,
whichareprecursorsoftheantigen-presentingcellsderivedfromthemyeloidcell
series, andnaiveThelper cells (Th0s),
whicharederivedfromthelymphoid system, as material for the
analyses. PBMCs wereisolatedfrom20-mL samples of anti-coagulated
blood thathad been obtained by gradient separation using the
Lymph-prep Tube system (Axis-Shield PoC AS, Oslo, Norway).Monocytes
and Th0s were separated from PBMCs that hadbeen magnetically
labeled with CD14, CD4, and CD45ROmicroBeads (Miltenyi Biotec)
using an autoMACS Pro Sep-arator (Miltenyi Biotec, Bergisch
Gladbach, Germany) . Toobtain monocytes, the CD14-positive (CD14+)
fraction wascollected as monocytes, whereas the CD14-negative,
CD4-positive, andCD45RO-negative
(CD14-CD4+CD45RO-)fractionswerecollectedasTh0s.FlowcytometricanalysiswithaMACSQuantAnalyzer(MiltenyiBiotec)revealedthat
the precision of cell separation was 96.2 % for
CD14+cellsand94.46%forCD4-positiveandCD45RA-positivecells as a
CD45RO-negative fraction.CD14+monocytes were obtained from six
patients, whileCD14-CD4+CD45RO-Th0cells werealsoobtainedfromfour of
these patients (Table 1) for the MIAMI analysis bothat relapse and
following complete remission. Genomic DNA(gDNA)
wasextractedfromthesecellsaspreviouslyde-scribed [16]. Consistent
amounts of extracted gDNA(300 ng from monocytes and 250 ng from
Th0s) from eachselectedcell typewerepooledandusedfor
subsequentPediatr NephrolDNA methylation analysis using MIAMI in
order to excludethe influences of epigenetic differences amongthe
celltypes, toidentifyspecificchangesduetoclinical coursesof MCNS,
andtoreducethenoisecausedbyindividualdifferences. Total
RNAwasextractedfromTh0spurifiedfromsevenother patients
withMCNS(Table2) usingaToTALLY RNA kit (Ambion, Austin, TX).
Complemen-tary DNA (cDNA) was synthesized from total RNA of
eachTh0s sample using the High Capacity RNA-to-cDNA Mas-ter Mix
(Applied Biosystems, Foster City, CA) for the qRT-PCR.MIAMI
analysisThe MIAMI method, which provides high-throughput
globalanalysisofDNAmethylation,
wasperformedasdescribedpreviouslyusing1.8and1.0gofpooledgDNAisolatedfrom
the monocytes of six patients and from the Th0s of fourpatients,
respectively [17, 18]. Briefly, this technique
utilizesisoschizomers(HpaII andMspI) that
recognizethesamerecognitionsite(CCGG). PooledgDNA
wasdigestedwithHpaII, a methylation-sensitive restriction enzyme
that cleavesonly unmethylated DNA, and then adapter-ligated and
ampli-fiedbyPCRwithprimers designedagainst theadaptersequences. The
samples were then further digested with
MspI,amethylation-insensitiveenzymethat
digestsCCGGsitesirrespectiveoftheirmethylationstatus,andamplifiedagainwiththesameset
ofprimers(HpaIIMspI treatment). Thesecond treatment with MspI
yields amplicons from unmethy-latedDNAfragments only. Hence,
onlyHpaII cleavableunmethylatedDNAfragmentsareamplified,
andthesecanthenbequantifiedbasedontheir
respectivefluorescenceintensity by microarray analysis. The
amplified products
werethenlabeledwithCy3(remissionsamples)orCy5(relapsesamples)
andco-hybridizedtoamicroarrayspottedwith38,172sixty-mer
oligonucleotidescoveringthevicinityofthe transcription start sites
(TSSs) of 14,978 genes.Following hybridization, the microarray was
scanned, andthe obtained fluorescence intensities were quantified
and nor-malized. The same pooled gDNA samples were treated
firstwith MspI instead of HpaII (MspIMspI treatment) and ana-lyzed
on a duplicate array to correct for false-positives causedby single
nucleotide polymorphisms or incomplete
digestion.Bisulfite-pyrosequencing analysisIn the
bisulfite-pyrosequencing method, unmethylated cyto-sine residues
are converted into uracil, whereas methylatedcytosines
remainunchanged. Analysisof themethylationstatus inthis manner
exploits thequantitativenatureofpyrosequencing by reporting the
ratio of cytosine to
thymineateachanalyzedCpGsite,whichreflectstheproportionofmethylated
DNA. This analysis was performed using pooled
Table1CharacteristicsofpatientswithminimalchangenephroticsyndromeatsamplingwhoseDNAwasisolatedformethylationanalysisPatientno.SexAgeatonsetTotalfollow-upperioda(months)FrequentrelapserBiopsySamplingageatrelapseRelapsetimesatsamplingIntervalsb(weeks)PSLatrelapse(mg/day)PLSatremission(mg/day)ISatrelapse(mg/day)ISatremission(mg/day)CD14+(n06)RO(n04)1M10y4m78YesMC13y0m3rd5401000TakenTaken2M5y4m35YesMC6y0m3rd160350CyA90TakenTaken3M5y11m186YesMC18y6m9th23101500TakenTaken4M7y3m188YesMC19y10m13th2360500TakenTaken5M9y9m109YesND11y7m4th1200CyA180CyA160TakenNT6M11y11m61YesND12y5m4th71020MZV125MZV75TakenNTMean8y4m109.513y5m6th25PSL,prednisoloneIS,immunosuppressant;M,Male;y,years;m,months;MC,minimalchange;ND,notdone;CyA,cyclosporinA;MZB,mizoribine;NT,nottakenaFollow-upperiodtillJune2011bIntervalsofsamplingbetweenrelapseandremissionPediatr
Nephrolsamplesinaccordancewithestablishedprotocols.
Briefly,gDNAextracts frompatients weredigestedwithEcoRI(Takara Bio,
Otsu, Japan) and subjected to bisulfite
treatmentusingtheEZDNAMethylation-GoldKit(ZymoResearch,Orange, CA).
TheanalyzedCpGsiteswereintheclosestHpaII recognition sites at the
5- and 3- ends of the probesdesigned in the TSSs vicinity of GATA2
(A_17_P02574948),PBX4(A_17_P10909964),
andNYX(A_17_P11717994)usedintheMIAMIanalysis.Amplificationandsequencingprimersfor
pyrosequencingweredesignedwithPyroMarkAssayDesign software
ver.2.0(Qiagen, Venlo,the Nether-lands) (Electronic Supplementary
Material Table 1).ThetargetedDNAsegmentswereamplifiedusingahotstart
protocol with a touchdown PCR system (Veriti
Ther-malCycler;AppliedBiosystems),andthe strand servingasthe
pyrosequencing template was biotinylated. Following de-naturation,
thebiotinylatedsingle-strandedPCRampliconswereisolatedandallowedtohybridizewithasequencingprimer.
Pyrosequencing (PyroMark Gold Q96 Reagents;
Qia-gen)wasthenperformedusingthePyroMarkQ24system(Qiagen)accordingtothemanufacturersprotocol.
These-quencing assay was validated using an internal control (a
non-CpG cytosine within the target methylation sequence
region).Quantitative real-time
PCRTheqRT-PCRanalysiswasperformedusingtheTaqManPCRmethodwitha7900HTFastReal-TimePCRSystem(AppliedBiosystems)
withcDNAfromTh0s separatedfromtheother patientswithMCNSat
relapseandsubse-quent remission. The three genes previously
analyzed usingMIAMI, namely, GATA2 (Hs00231119_m1),
PBX4(Hs00257935_m1), andNYX(Hs00360869_m1), wereassayed. Each gene
was assayed four times for each sample.Aliquots of cDNA equivalent
to 300 ng of total RNA wereusedinthe RT-PCR reactions.
GAPDH(Hs99999905_m1)was used as an endogenous control for
normalizing the RNAconcentrations.
DifferencesintheCTvaluesbetweenthetested genes and endogenous
control (CT) were calculatedand used for subsequent statistical
analyses.Statistical analysisStatistical analyses of
thedistancedistributions
fromtheMIAMIregressionlineforeachoftheprobesusedintheassays was
performed using a non-parametric MannWhit-neyUtest (Fig. 1).
Comparisonsbetweentheexpressionlevels for
eachgroupwereanalyzedusingtheWilcoxonmatched-pairs signed-ranktest
(Fig. 4a, b). All statisticalanalyses were performed using GraphPad
PRISM5 software(GraphPadSoftware, LaJolla, CA)
withthesignificancelevel set at P