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Curs EpigeneticA

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Genomic Imprinting

Genomic imprintingis a genetic phenomenon by which certain genes are expressed in a parent-of-origin-specific

manner.

The imprinting processis explained by two main epigenetic processes:

1. the epigenetic mechanisms controlling gene expression and

2. the dynamic reprogramming o the epigenome through the ma!or embryogenesissteps.

Epigenome reprogramming is a physiological process that in"ol"es the dynamicerasure#reset maintenance o the epigenetic tags $chemical groups on %&A andhistones that do not a'ect %&A se(uence) by speci*c en+ymes.

The normal course o such reprogramming o the tags on the genome is controlled by the

cisand transacting actors that are *nally lin,ed both with endogenous genetic actors$IC-s and &/s)and external0 en"ironmental conditions $diet0 liestyle0 pollution etc).

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THE PROCESS REGULATINGGENOMIC IMPRINTING HAS FOURIMPORTANT PROPERTIES:

$1) The mar, must be able to in3uence transcription4

$2) it must be heritable in somatic lineages such that a memory o parentalorigin is aithully propagated into daughter cells during cell di"ision4

$5) the mar, is li,ely to be placed on the paternally and maternallyinherited chromosomes at a time when they are not in the same nucleus$i.e.0 during gametogenesis or perhaps immediately ater ertili+ation)4

$6) there must be a mechanism o erasure o the mar, in order thatpaternally inherited chromosomes in the emale germline can establish anew mar, indicati"e o their maternal origin and "ice "ersa.

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%&A 7ET89ATI;& is in"ersely correlated with gene expression4 itsignals heterochromatini+ation and gene reppression

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8istone modi*cation targets andthe epigenotypes speci*c or

silenced#acti"e state o a gene

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e y a e s gna s e reppress "es a e y a rac ng spec cproteins $7nmethylated %&Aattract other e'ector en+ymes that attach on

histones di'erent0 acti"ating tags: e.g. 8AT attaches acetyl groups

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%&A methylation is a relati"elystable e"ent in"ol"ed in long=termde"elopmental processes such astissue=speci*c gene expression0

gene imprinting and ? chromosomeinacti"ation

8istone proteins aid in pac,agingthe %&A into the nucleus and helpregulate interactions o transcriptionactors with the %&A .

7odi*cations to histones includeacetylation0 phosphorylation0ubi(uitination and methylation0which lead to a relaxed$transcriptionally acti"e) or

condensed $transcriptionallyrepressed) chromatin state.

DNA methylation and histone modifications. Two major epigenetic mechanisms, DNA methylation and histone

modifications, act in concert to regulate gene transcription. The DNA double helix backbone is shown in blue.after !alker "#$$%

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The imprinting process occurs in primordial germinalcells during embryogenesis $etal de"elopment) and

continues in neonatal period. It is crucial or the

de"elopment o the etusand in3uences the healthstatus in the o'spring.

%e'ectsin this process may arrise during the puberty oparentsor during the pregnancy. These de'ects re'ers

to erroneousestablishment o the epigenetic tags on thegenomeand thereore to the unproper parent speci*c

alleles@ expressionon critical chromosomal regions suchas 1(11(15.

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Epigenetic modi*cations are re"ersiblethereore theymay beattached and remo"edby the same en+ymes in

special conditions through a reprogrammingprocess.The normal inheritance and control of the imprinted state in&ol&es the correcterasure and reset of the imprinting marks on the DNA sequence epigenomic

reprogramming of the chemical groups on the DNA and histones% that is inherited

intact from the parental gametes. 'mprinting process occurs during se&eral specific

steps( primary imprinting takes place in parental gametes, secondary process during

offspring embryogenesis and third step during the de&elopment of )*+s primordial

germ cells%.

The de&elopment of the )*+ starts during the fetal stage and continues during the

postanal period, where an important contribution is the so-called social imprinting.

ach step comprises epigenetic reprogramming that acti&ate specific enymeacti&ities( one major class if enymes are DNTs DNT/a and /0 are essential for

the establishment of the imprints and DNT$ is essential for the maintenance of the

imprinting marks through mitoses and meiosis cycles%.

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-eprogramming o the %&A methylation tags: Critical stages othe %&A methylation process during the de"elopmental stages

Imprinting errors are explained through the transgenerational approach: imprinting process occurs duringse"eral de"elopmental steps and comprises more generations. /assing through a di'erent germline determines

an epigenomic reprogramming o the imprints. These processes re(uire erasure and reset o the imprint mar,son %&A and histones. Errors in en+yme acting or these processes determine the imprinting deects and theestablishment o the diseased state

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Cycle oImprinting

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7odel explaining the imprinting errors in critical region or/B#A due to de'ects in erasure#reset o epigenetic tags

cycles

IG. D. 7odel pentru erorile de imprinting in regiunea /B#A. In celulele somatice $dreptunghiuri"er+i)0 regiunea /B=-; a locusului IC- este metilata $cercul negru). In ciclul normal deimprinting $A)0metilarea este stearsa in celulele germinale primordiale $dreptunghiuri galbene). >ncomplex proteic ce contine cel putin una din proteinele speci*ce liniilor de o"ocite $stea) seasocia+a la /B=-; in timpul o"ogene+ei. Imediat dupa ertili+are $dreptunghiul albastru)0 acestcomplex conduce la metilarea CpG a regiunii materne /B=-;. Erorile de imprinting pot apare dinincapacitatea de a se sterge marca!ul prin metilare in linia germinala paterna $

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Introducere. indromul /rader=Billi $/B) este o boal rar$J1:2F0FFF)0 cu transmitere non=mendelian.

Este determinat de actori genetici Ki epigenetici care aectea+contribuLia monoalelic0 patern a genelor amprentate din regiunea1(11=(15.

Exist 5 tipuri principale de mecanisme moleculare ce conduc la /B:deleLii $6=M7b)0 disomia uniparental matern $>/%) Ki deect deimprinting $%I).

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Figura 1.1beitatea la pacien2i 3)! a% o feti2a

de ",4 ani 5i b% un b6rbat de "$ ani. De asemenea se

obser&6 tr6s6turile faciale tipice, 5i leiuni datorate

ciupirii tegumentului, cicatricea l6sat6 de tubul folosit

pentru a se hr6nii corespunator, pseudoginecomastie

5i genus &algus genunchi apropia2i% la b6rbat

*assidy5i Driscol, "##7%.

Figura 2. 8enotip facial clasic pentru 3)! la

adolescent6 5i maturitate. a% 9n b6iat de $4 ani. b% 1

femeie de :$ ani. 3e obser&6 diametru ;ngust bifrontal,

ochii ;n form6 de prun6 u5or ridica2i la coad6, punte

naal6 sub2ire 5i bua superioar6 sub2ire *assidy 5i

Driscol, "##7%.

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Figure 4. Harta regiunii 15q11q1. +enele imprintate paterne - patratele galbene, genele

imprintate de origine materna < patrate rosii. Alelele silentiate sunt repreentate prin patrate negre iar

cele cu expresie bialelica prin patrate &eri. 3tatusul de amprentare al ATP10A&ariaa intre indi&ii,

si este repreentat prin patrate moaicate. 3agetile punctate sunt situate deasupra clusterelor de

A=Nsno ce au expresie specifica in creier. 9n patrat galben s-a folosit pentru marcarea IPW, desiacesta face parte din transcriptul necodificator SNURF-SNRPN, nefiind o gena independenta. *entru

de imprinting '*% la )!3 si A3 sunt marcate prin bleu si rosu. *erculetele negre deasupra alelelor

silentiate ale SNURF-SNRPN, MKRN3siNDN indica insule *p+ metilate, iar cerculetele albe indica

alelele exprimate, nemetilate. >)$, >)", si >)/ repeinta onele principale ce delimitea majoritatea

rupturilor in caurile de deletie si sunt repreentate de linii in igag. =egiunea critica )!3 este

incadrata intr-un dreptunghi dup6 *hamberlain 5i 0alande "#$#%.

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Figur !" #e$ecte mo%ecu%re % SP&" rec"enNa cu care apare*ecare deect On sindromul /B Pi riscul de recurenN. 7etilarea genelorcu expresie patern este repre+entat printr=un cerc roPu. 7 = matern4 /= patern $dup

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Abordarea diagnosticului /B siA

A3area statusului de metilare doar la locusul &-/&

prin olosirea tehnicii /C- speci*c pentru metilare $7=/C-) sau anali+a outhern blot. Aceasta abordare "acon*rma diagnosticul dar nu "a oeri inormatiisuplimentare pri"ind mecanismul de aparitie0 acestlucru *ind re+ol"at de olosirea altor tehnici simultan$I8 si#sau anali+a microsatelitilor) .

Anali+a simultana a statusului de metilare si numarulde copii0 pentru numeroase situri din regiunea 1(11=(150 prin olosirea tehnicii multiplex ligation=dependent probe ampli*cation $7=7/A). Aceasta

abordare "a con*rma diagnosticul si "a identi*ca si oe"entuala deletie. Totusi daca nu este con*rmatapre+enta unei deletii trebuie eectuat un test deanali+a a microsatelitilor0 pentru a identi*ca daca esteimplicata o microdeletie in IC sau un >/%.

i 1 t t ii d t t t li l l l /B i A $i) li d til l l l

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igure 1. trategii de testare pentru anali+ele molecular /B si A $i) anali+a de metilare la locusul&-/& reali+ata initial si $ii) 7=7/A reali+ata initial $imon et al.0 2F1F)

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MS-PCR (Methylation specifc PCR)

/rincipiu metodei 7=/C- $ Toeng 2FFD)

/rincipiul acestei metode se ba+ea+ pe sensibilitatea dierit asitusurilor de cito+in metilat#nemetilat0 Pi pe eectulmutagen al bisul*tului. %oar situsurile nemetilate se transorm

On uracil0 care la urmatoarea rund de replicare /C- sunt

substituite cu timina0 On timp ce situsurile metilate pstrea+identitatea ba+ei a+otate.

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Figura 4!.)atternul electoforeei ;n gel de agaro6

"? ob2inut ;n urma analiei moleculare metilare%

pentru confirmarea caurilor suspecte clinic 3)!

prin amplificarea )*= ;n condi2iile cre5terii

cantit62ii ADN ;n mediul de reac2ie /,4 @l%,

temperatura de ata5are a primerilor fiind deasemenea ridicat6 la :B*. 0-0adder marker de

greutate molecular6%, godeurile $-: < pacien2i

suspec2i pentru sindromul )rader-!illi, godeurile

4- control persoan6 normal6, ,C-control poiti&

3)! 7-E"1.

Figura 41. )attern al electroforeei ;n gel de

agaroa "?, dup6 con&ersia cu bisulfit si

amplificare control poiti& 3)! - c% control

persoan6 normal6 - N c% normal N% " beni - cea

de /$/ pb repreint6 regiunea din exonul $ al alelei

3N)=N de origine matern6 ce ;n mod normal estemetilat6 % iar cea de ""$pb repreint6 o regiune din

exonul $ al alelei 3N)=N de origine patern6 ce ;n

mod normal este nemetilat6% pacien2ii confirma2i

molecular pentru 3)! - )!% -$ banda de /$/pb,

0-ladder.

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MethPrime diagram generated for CpG dinucleotide repeats density variation in

SNRPN (promotor/exon1) gene region comprising 2037 bp. It is grouped in three CpG

dinuceotide rich regions !ccording to their densit" (d!shed m!r#ed dom!ins)$ is!nd 1 o%

21& bp' is!nd 2 o% 71 bp !nd is!nd 3 o% 101 bp. !tern!/*!tern! !nd Common primer

se+uences th!t ,ere chosen !ccording to -eschnig# et !. protoco 1,ere !id!ted b" theeth *rime so%,!re !ccording to the st!nd!rd *C rues$ t,o primers' corresponding

to m!tern! !nd p!tern! !ees ,ere proided %rom the highest densit" region' ,hie the

common primer' %rom the ero densit" region. 4his di!gr!m ,!s gener!ted b" the

eth*rime so%t,!re.

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A"anta!ele si de+a"anta!ele metodei7=/C-

7etoda pre+entata aici pre+inta urmatoarele a"anta!e:5 $1) 7/C- se poate eectua in 2 +ile $Gillessen=Qaesbach et al.0

1RR)45 $2) Testarea se poate eectua chiar si cu Fng A%& genomic. %e

aceea se pot olosi alte surse de A%& genomic pentru anali+ecum ar * petele de sange uscat si celule din mucoasa bucala4

5 $5) 7/C- nu necesita olosirea radioacti"itatii. %e aceea0 oricelaborator ce oloseste tehnica /C-0 poate eectua si aceastaanali+a.

5 $6) Identi*ca peste RRS din ca+urile cu /B si aprox. FS dinca+urile A

%e+a"anta!e:

&u oera inormatii despre mecanismul implicat in aparitiasindromului

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MrMatze 2007

igura6. /rincipiul acestei metode este hibridi+area $legarea) princomplementaritate a unei sonde de A%&0 cu o anumit regiune a unuicromo+om. onda de A%& este On prealabil marcat cu a!utorul unui3uorocrom0 ceea ce o ace "i+ibila la microscopul cu 3uorescenta. Astel0

se stabilePte pre+enNa sau absenNa sec"enNei de A%& de interes$corespun+atoare sondei)0 numarul de copii ale sec"enNei respecti"e Pi

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Sonde olosite n tehnica FS!" pentr# detectareadele$iilor n dia%nostic#l P&S'S

igura . Alturi de sonda I8 pentru &/-& s=au olosit alte

dou sonde control CE/ 1 Pi I /7 pentru a e"idenNiabraNele scurte $CE/ 1 pe 1p11.20 semnal "erde)0 Pi braNelelungi $I /7 pe 1(220 semnal rosu) ale cromosomului 10

a!utndu=ne s detectm e"entuale transocaNii.

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onde olosite I %11F spectrum orange#/7 spectrum orange#CE/1

spectrum green$Uysis0 Abbott inc.)

A" #e%etie in 'on P&S(AS )" Nu s* con+rmt de%eti in regiuneP&S(AS

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A"anta!ele si de+a"anta!ele metodei I8

A"anta!eTehnica I8 se oloseste pentru identi*carea

anomaliilor de structur ale cromo+omilor:microdeleNii sau microduplicaNii0 translocaNii

/rin aceasta metoda0 se con*rma aproximati"DFS din ca+urile /B si AEste oarte olositoare in diagnosticul /B

deoarece deconspira translocatiile si e"entualele

ca+uri mo+aic%e+a"anta!eeste o metoda realti" costisitoarenecesita un timp mai mare de lucru

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Introducere

5 indromul /rader=Billi $/B) $7I7 1DM2DF) se caracteri+ea+a prinhipotonie in perioada postnatala0 obe+itate in copilarie0 trasaturidismor*ce0 retard mental si un risc crescut pentru aparitia cri+elor

5 /rocentele in care se impart clasele de deecte genetice ale

acestui sindrom sunt: $JDFS) deletie pe cromosomul 1(11=(15 .$J2S) apar datorita disomiei uniparentale materne $ m>/%).Aproximati" 2S dintre pacientii /B pre+inta o mutatie in centrulde imprinting0 re+ultand o metilare A%& si o expresie genicaanormala

5 /rin metoda 7/A se pot identi*ca doar ca+urile de deletie0 insaprin 7=7/A se identi*ca si aberatiile in paternul de metilare

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Figure 1. Strategii de testare pentru analizele molecular PWS si AS i! analiza

de metilare la locusul S"#P" realizata initial si ii! MS$M%PA realizata initial#amsden et al.& '(1(!

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4ehnic! 6*

8ig.2 9t!pee princip!e !e re!ctie 6* ((dup! nders et ! 200:))

M)*+,)

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3-0)A

6 6* #it 902 po!te %i %oositpentru ! detect! !ri!tii !e num!ruui de copii'

si de !semene! pentru ! !n!i! st!tusu de

meti!re ! insueor CpG din regiune! 1:+11

+13 intro m!nier! semic!ntit!ti!. cest #it

contine 32 de sonde speci%ice pentru secentee

din interioru s!u !propiere! regiunii critice*;/ din cromosomu 1:+11.Cinci din !ceste

sonde sunt speci%ice pentru o secent!

imprint!t! si contin un sit de recunostere pentru

enim! sensibi! ! meti!re

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n!i! !utom!ti!t! ! %r!gmenteor ! %ost re!i!t! printrun progr!m (Co%%!iser) ce

!n!ie!! %iec!re pe!# in %untie de pe!#u contro ce se !%! in !propiere! ui.

=um!ru re!ti de copii ! %ost obtinut prin comp!r!re! pe!#urior de ! >=u supus

digestiei cu

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G4 promotor meth"!tion

7G7T methylation is a prognostic biomar,er in oncologyClinical signi*cance:methylated 7G7T is correlated with genome instability

due to inability to repair al,ylated orms o %&A that may occur ater"arious en"ironmental exposures. 8owe"er0 this 7G7Tmethylation andsilencing might be exploited in treatment schemes including al,ylatingagents: an acti"e 7G7T en+yme may not allow an eVcient acti"ity o anal,ylating drug. Thereore0 methylation positi"e test in a tumor has a goodprognosis or such drugs eVciency.