Epigenetic mechanisms underlying the imprinting

defects:
implications for the establishment of diagnostic
testing schemes for Prader Willi syndrome in
Romanian population.

Natalia Cucu (University of Bucharest), Cosmin Arsene (Univesrsty of Bucharest),

Gabriela Anton (Institute of Virology, Bucharest), Anca Botezatu (Institute of
Virology Bucharest), Maria Puiu (UMFTimisoara), Corin Badiu (Institute of
Endocrinology bucharest), Vasilica Plaiasu (IOMC), Radu Stefanescu
(Genexplore), Narcis Dobre (VitroBioChem)
PWS [MIM – Mendelian Inheritance of Man- no. 176270] is an inherited human
disorder characterized by a complex phenotype, including: decreased muscle tone
and failure to thrive at birth; also, later during specific developmental stages, the
syndrome symptoms appear more evidently: mental retardation, short stature,
hypogonadism, sleep apnoea, behavioural problems and hyperphagia or insatiable
appetite that can lead to severe obesity (Nicholls and Knepper, 2001).

Prader Willi syndrome (PWS) is a complex disease which therefore is nost

easily diagnosed only on clinical criteria; its mechanism involves both
genetic and epigenetic factors
The diagnosis is difficult due to individual variations of associated
phenotypes and because these phenotypes appear during the offspring
development
Desciphering the molecular mechanism involved in the establishment of the
imprinting defects in PWS is essential for a proper diagnosis and also for the
genetic councelling
Prader-Willi syndrome is a model to study the
epigenetic defects in imprinting process
 PWS is a syndrome classified together with its sister: Angelmann syndrome;
untill recently these syndromes were diagnosed based on genetic analyses
which envisaged the detection of the deletions in the critical region of
chromosome 15 (15q11-q13).These confirmed only 75% of cases.
 New insights into the molecular mechanisms of these syndromes highlighted
the common process named genetic imprinting. It implies that the different
parental alleles are marked by different moleculatr tags, however they carry
the same DNA sequence or genetic information. Consequently these tags
instruct the precise gene to express or to be silent. In the syndromes, the
normal contribution of the parental alleles is erroneously established during
the pregnancy as a consequence of the wrong imprinting patterns
established in parental gametes. The embryo development is affected by the
imprinting process.
 During the offspring development, the effects of the unproper parental gene
expression appear gradually, determining new phenotypes, characteristic to
the disscussed syndromes.
The genotype-phenotype relationship in PWS
is complex
The critical chromosomal region for PWS is 15q11q13, in humans;

It contains several exclusively paternally expressed genes:

-Mkrn3 (makorin ring-finger protein 3)
-Magel 2 (MAGE like protein 2)
-NdN (necdin)
-Snurf-Snrpn(Snrpn-small nuclear ribonucleoprotein N; Snurf-Snrpn
upstream reading frame)
and two exclusively maternally expressed genes:
-Atp1(a (ATPase, classV, type 10A)
-Ube3A (ubiquitin protein ligase E3A isoform 3).

This region is also characterized by many ncRNA genes, including numerous repeated
C/D RNA genes (for ex. HBII-85 and HBII 52 clusters) and Ube3A, as an antisense transript
to Ube3A gene.
Imprinting process occurs by enzymatic attachment of precise
chemical tags on chromatin that instruct it to express or to be silen
This process does not affect the DNA sequence, hence it is named
“epigenetic”

It is dynamic and requires several reprogrammings of the epigene

profiles in the germlines

It requires several steps that include different generations:

-parental germ cells
-offspring embryogenesis (development of the PCGs, primordial
germ cells),
-offspring neonatal development
What is Epigenetics and the epigenetic profiles? Key words:
chromatin conformation, gene expression
DNA sequence is not the only hereditary information
that is transmitted through generations of cells and
organisms: there are also transmitted molecular
signals such as methyl groups on DNA and histones
and these ones represent key factors for the
chromatin conformation. These tags form an
epigenetic pattern that instructs chromatin
conformation on a gene to adopt a specific
transcriptional state.

Transcriptional state of a gene is therefore not encoded in the DNA sequence; this codes
for the aminoacid sequence in proteins. When the epigenetic signals indicate an active
state, the gene expression is started only after the decondensation of the chromatin
conformation. This structure enables the transcription machinery (TF and ARN
polymerases) to act on DNA sequence. Condensation/decondensation of chromatin is
controlled by epigenetic (independent of DNA sequence) modification of the chromatin
components: DNA and histones. Special enzymes for these biochemical modifications
target specific chromatin regions and contribute to condesation/decondensation
processes which therefore determine reppression/activation of gene expression.
Chromatin compaction steps

Major processes that control the

chromatin conformation
compaction (chromatin
remodelling) are:

-DNA methylation
-histone modification
(acetylation, methylation,
phospholylation etc)
-noncoding RNA molecules
Covalent DNA modification: attachment of methyl
tags on DNA sequence, respectivelly on cytosine
rings (DNA methylation)

DNA methylation is a biologic process involving

enzymes: DNMTs- DNA methyltransferases
Methylated DNA signals the reppressive state by attracting specific proteins
(MBDPs) that further recruit remodelling complexes containing enzymes that
modify the histones (HMT-histone methylases, HDAC-histone deacetylases
etc).
Unmethylated DNA attract other effector enzymes that attach on histones
different, activating tags: e.g. HAT attaches acetyl groups
Histone modification targets and the
epigenotypes specific for silenced/active state
of a gene
DNA METHYLATION is inversely correlated with gene expression; it signals
heterochromatinization and gene reppression
The imprinting process occurs in primordial germinal cells
during embryogenesis (fetal development) and continues in
neonatal period. It is crucial for the development of the
fetus and influences the health status in the offspring.

Deffects in this process may arrise during the puberty of

parents or during the pregnancy. These deffects reffers to
erroneous establishment of the epigenetic tags on the
genome and therefore to the unproper parent specific
alleles’ expression on critical chromosomal regions such as
15q11q13.
Epigenetic modifications are reversible therefore they may be
attached and removed by the same enzymes in special conditio
through a reprogramming process.
The normal inheritance and control of the imprinted state involves the correct
erasure 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. Imprinting process occurs during several specific
steps: primary imprinting takes place in parental gametes, secondary process during
offspring embryogenesis and third step during the development of PCGs (primordial
germcells).

The development of the PCG starts during the fetal stage and continues during the
postanal period, where an important contribution is the so-called “social imprinting”.

Each step comprises epigenetic reprogramming that activate specific enzyme

activities: one major class if enzymes are DNMTs (DNMT3a and 3L are essential for
the establishment of the imprints and DNMT1 is essential for the maintenance of the
imprinting marks through mitoses and meiosis cycles).
 Reprogramming of the DNA methylation tags: Critical stages of the
DNA methylation process during the developmental stages

Imprinting errors are explained through the transgenerational approach: imprinting process
occurs during several developmental steps and comprises more generations. Passing through
a different germline determines an epigenomic reprogramming of the imprints. These
processes require erasure and reset of the imprint marks on DNA and histones. Errors in
 Model explaining the imprinting errors in critical region for PWS/AS
due to deffects in erasure/reset of epigenetic tags cycles

Methylated allele
Somatic cells/p
Is maternal one

PGcells

Germinal cells
Erasure

Zygote
Reset
Maintenance Somatic cells/o

FIG. 7. Model pentru erorile de imprinting in regiunea PWS/AS. In celulele somatice (dreptunghiuri verzi),
regiunea PWS-SRO a locusului ICR este metilata (cercul negru). In ciclul normal de imprinting (A), metilarea
este stearsa in celulele germinale primordiale (dreptunghiuri galbene). Un complex proteic ce contine cel putin
una din proteinele specifice liniilor de ovocite (stea) se asociaza la PWS-SRO in timpul ovogenezei. Imediat dupa
fertilizare (dreptunghiul albastru), acest complex conduce la metilarea CpG a regiunii materne PWS-SRO. Erorile
de imprinting pot apare din incapacitatea de a se sterge marcajul prin metilare in linia germinala paterna (B), din
incapacitatea de a se restabili metilarea dupa ovogeneza si fertilizare (C), sau din incapacitatea de a mentine
paternul de metilare dupa fertilizare (D) ceea ce duce la mozaicism somatic (Horsthemke B. si Wagstaff J.,
2008).
Cis factors influencing the imprinting process- ICR, DNA / histone
methylation ,nonhistone proteins activity and nc RNA
A. ICRs- imprinting controlling region required for bidirectional
activation
of the potentially expressed genes. It is located at the 5’end of Snurf-
Snrpn
EXPRESIA GENELOR IMPRINTATE SI MARKERi EPIGENETICI IN REGIUNEA 15q11-q13 UMANA

Expresia genelor imprintate si markeri epigenetici in regiunea 15q11-q13 umana. Coloratia patratelelor indica daca
genele au numai expresie paterna, expresie paterna > materna, materna > paterna, sau expresie egala a alelelor
paterne si materne. PWRN1 si C15Orf2 au expresie monoalelica in creierul fatului, dar originea parentala a expresiei
monoalelice inca nu s-a determinat; datorita faptului ca genele fac parte din clusterul numai cu expresie paterna,
aceste gene sunt notate cu expresie paterna > maternal. Modificarile epigenetice specific genelor materne si paterne
sunt expuse ca simboluri pe linii negre verticale. Figura nu este desenata la scara ((Horsthemke B. si Wagstaff J.,
2008).).
 Recent findings

Researchers’ problem: finding the minimal critical gene or region on the

critical 15 chromosome, as the PWS, contrary to its sister syndrome: AS,
is a complex disease, comprising the errors in multiple genes or, probably,
the error in a minimal, controlling region, that influence the expression of
a cluster of genes.

Although it is still a matter of debate whether PWS results from loss of function of
a single gene or several ones, the study of rare translocations identified in human
genome, as well as several mice models have identified a minimal critical region
believed to play a pivotal role in the aetiology of the disease. The only
characterized and conserved genes within this 121kb-long genomic interval are
the numerous HBII-85 gene copies. Loss of expression of these repeated small
C/D RNA genes might play a role in conferring some (or even all) phenotypes of
the human disease and PWS like phenotypes in mice (neonatal letality, growth
retardation and hypotonia).
 Imprinted ncRNA genes

Upper panel: an imprinted ncRNA gene (purple line) is transcribed from a DMR that acts as an IC. The non-coding gene (e.g. Air, Kcnq1ot1) is
expressed from the unmethylated chromosome, whereas the flanking protein-coding genes (rectangles) display a reciprocal imprinted status and
are only expressed from the methylated chromosome. Expression of full-length ncRNA gene (e.g. Air or Kcnq1ot1) is thought to act in cis to
silence the flanking protein-coding genes. Note that ncRNA can also share antisense homology with one of the protein-coding genes. Lower
panel: imprinted small RNAs (purple vertical bars) are processed from large poorly characterized non-coding transcription units (e.g. at the
Snurf–Snrpn and Dlk1–Gtl2 domains), the expression of which is controlled by the IC. These imprinted small RNAs are believed to regulate gene
expression in trans, mainly at the post-transcriptional level by site-specific 2-O-methylation (e.g. C/D small RNAs) or RNA-mediated gene
silencing (e.g. miRNAs). It is important to note that the above described genomic features do not apply to all imprinted gene clusters
 Small imprinted ncRNA genes are mainly
located at the Snurf–Snrpn

Representation of the human Snurf–Snrpn imprinted domain (at 15q11q13), also referred as the
PWS/AS locus. This locus contains several paternally and maternally expressed protein-coding
genes (blue and pink rectangles respectively), as well as numerous C/D RNA genes (vertical
blue bars) and Ube3A-as that are only expressed from the paternal chromosome. The Snurf–
Snrpn, C/D small RNAs and Ube3A-as are thought to be part of a single large transcription unit
(blue line). Four copies of miR-344 genes also map between the Ndn and Magel2 genes in
rodents, but apparently not in human (their imprinted status is unknown)
One controlling region that is critical for the proper imprinting process to occur and the
correct order of the parental expression of imprinted genes to be established, is named
imprinting control region or IC/ICR. This region takes care of the critical DNA
sequences on the 15 chromosome to be correctly inherited through germlines and
properly prepared for the expression only from the parental allele.

Although the mode of action of ICRs is still poorly understood and might differ from one
cluster to the other, several common imprinting, that is silencing mechanisms have
emerged: DNA methylation, histone modification (methylation being the major process)
and ncRNA (noncoding RNA) genes.
One such region was named: SNURF-SNRPN (SNRPN- small nuclear ribonucleoprotein
N, SNURF- SNRPN upstream reading frame) and it is part of the IC region. The
promoter and the first exon of SNURF-SNRPN was up to now the ubiquitous target for
the diagnosis of PWS.
The imprinting process is explained by two main epigenetic processes:
1. the epigenetic gene expression controlling mechanisms and
2. the dynamic reprogramming of the epigenome through the major embryogenesis steps.

Epigenome reprogramming is a physiological process that involves the dynamic erasure/reset

and maintenance of the epigenetic tags (chemical groups on DNA and histones that do not
affect DNA sequence) by specific enzymes.
The normal course of such reprogramming of the tags on the genome is controlled by the
cis and trans acting factors that are finally linked both with endogenous genetic factors (ICRs and
SNPs) and external, environmental conditions (diet, lifestyle, pollution etc).

Different types of programmes have been detected during perinatal, fetal and postnatal
developmental processes
IC activity for controlling the activation/inactivation of paternal/maternal gene expression on the
critical chromosomal region 15q11-q13 d

Model ce priveste controlul expresiei genelor in 15q11-q13 de catre PWS/AS-IC. Pe copia paterna a cromosomului 15, PWS-SRO
este nemetilat si activ. Acest PWS-SRO actioneaza prin mecanisme necunoscute pentru activarea transcriptiei lui MKRN3,
MAGEL2, si NDN, si realizeaza silentierea specifica creierului pentru UBE3A, printr-un mecanism in care este folosit ca promotor in
expresia unui transcript antisens UBE3A. Pe copia materna a cromosomului 15, PWS-SRO este metilat si inactiv. In absenta unui
PWS-SRO activ, MKRN, MAGEL2, NDN, si SNRPN sunt inactive, si UBE3A este activa in creier. La pacientii cu deletii PWS-SRO,
consecintele pentru expresia genica sunt aceleasi ca si pe cromosomul matern unde PWS-SRO este metilat. La pacientii cu deletii
AS-SRO, ce au PWS-SRO intact, PWS-SRO nu este metilat, iar dupa trasmiterea materna, consecintele pentru expresia genica
sunt aceleasi ca si pe cromosomul de origine paterna. La pacientii cu deletii in ambele zone, lipsa unui PWS-SRO activ conduce la
un patern de expresie matern (Horsthemke B. si Wagstaff J., 2008).
Regulation of the imprinting process and expression
of critical genes involves also trans-acting
mechanisms
DNMTs are proteins and therefore their corresponding gene
expression may be influenced by certain, so called-trans factors.
SNPs in DNMT promoter is one of the possible cause that may
influence the activity of the DNMT during embryogenesis and
PCG development.

Errors in the enzymatic activities for the erasure and reset of the
imprinting marks introduce the imprinting defects which nfluence
the organismal development of the offspring
The enzymatic activities are equally influenced not only by the
corresponding genes’ activities, but also by the environmental
factors, such as diet (nutrition, lifestyle, customs etc) and
endocrine disruptors (pollution). A major factor is the methyl
group containing foods, such as choline, folates, methionine and
the B vitamins that control the correspondin pathways of the
cellular methylome. Their metabolism include the pathway of
DNA and histone methylation.
Stil de viata indulgent Epigenetica
Dezechilibru energetic
Stress oxidativ
Imbatranire …
CH3
CH3
CH3

Genotip

Perturbarea ritmurilor Programarea Alterarea

oscilator, circadian, defectuoasa Disfunctii structurii
sezonal metabolica si mitocondriale cromozoma
neuronala le
(ADN)
Environmental exposure can threaten fetal health: the impact of genetic (SNPs) and
epigenetic factors (DNA methylation, histone modifications, ncRNA, nonhistone
proteins-CTCF, BORIS etc).

-Concerns not only for exposure to medications, illicit drugs, chemical infectious or
physical agents, but also for parental diet (lifestyle: nutrition, customs etc) and the so-
called new toxicants such as endocrine disruptors from dietary stuffs (detergents,
water, food etc).

Many of these anxieties and frequently real risks could be avoided through
preconception care. Reasons for genetic councelling before and during pregnancy.
Recently is apparently important also the so-called “social imprinting” during the
neonatal period
We can estimate the wrong and correct methylation pathways

The mthfr gene

Critical SNPs that are trans acting factors and influence the
imprinting process:
-Genes linked with the cell methylome- Mthfr gene, Mtrr gene, etc may present SNPs
-genes coding the DNMTs (especially DNMT3L and 3a) may present SNPs or
epigenetic factors controlling their promoters

SNPs and MVPs may

influence each other: SNPs
may derive from epigenetic
modifications (MVPs) and
epigenetic modifications as
MVPs may arise due to the
effect of certain SNPs in
critical genes

The overall effect is the

genome instability
One of the newly emerged mechanism of imprinting involves
ncRNA genes. Each imprinted gene cluster express one or
several large nc RNA genes, that display reciprocal imprinted
expression relative to the neighbouring protein coding genes. For
instance, if the ncRNA genes are expressed from paternally
inherited chromosome, then the flanking protein-coding genes
are expressed from the maternally inherited allele. Also, some of
these nc RNA are transcribed in an antisense orientation, thus
influencing their neighbouring protein coding genes expression/

C/D RNAs are 80-300nt long nuclear RNAs that concentrate

either in the nucleolus or in the Cajal bodies. They form specific
RNA duplexes with their cellular RNA targets to act either as
RNA chaperones to facilitate rRNA processing or as RNA
methylation guides to direct in a sequence-specific manner the
biosynthesis of 2’-O- methylation into rRNAs or spliceosomal U-
snRNAs (type U small nuclear RNAs).
 Impact on diagnosis approaches
The assays for PWS diagnosis are detecting the errors that caused the lack of normal
expression of the paternal genes

The lack of expression may be caused by different genome structures:

-lack of the DNA sequence encoding the critical genes due to deletion of an entir
chromosomal region comprising the critical paternal genes or only a minimal critica
chromosomal region that control the expression of a cluster of critical genes (named imprintin
control region: IC or ICR); this type of genome modification comprises the lack of the geneti
information encoded by the lacking DNA sequence; this type of error is due to a genetic deffect

Corresponding technique approached so far: FISH (fluorescent labeling the probes for th
critical deletion region and monitoring their presence on the chromosomal spread
microscopically)
Classes of alterations registered for PWS and AS

Paternal chromosome: blue color

Maternal chromosome- red color
Nicholls et al., 2001
 FISH(fluorescent in situ hybridization) for the
region 15q11-q13

A. Deletion in PWS/AS critical region B. Normal PWS/AS critical region

The probes used: LSI D15S10 spectrum orange/PML spectrum orange/CEP15

spectrum green(Vysis, Abbott inc.)
-lack of the paternal allele + the compensation of it by a maternal
contribution (same region provided by a second segment of the maternal
germline): this is called UPD (uniparental disomy); the DNA sequence
encoding the critical genetic information does not come from the paternal
germline, but from the maternal one; this type of error is called a genetic
defect

Corresponding technique approached so far: qPCR for the quantitation,

through DNA sequencing methods, the maternal sequences contribution (a
double quantity means UPD); it requires the comparison of the offspring
DNA with at least maternal DNA
-the presence of the paternal DNA sequence encoding the critical genes, however it
is repressed or its normal chromatin structure may be transformed so that it is repelling
the transcriptional machinery; hence the paternal gene is present in the genome and
is not expressed or silenced- this type of error is named epigenetic imprinting defect
that may be formed de novo or may be inherited from parents or grandparents or; the
chromatin structure is normal when it is permissive for the transcriptional machinery
and is repressive when it becomes repellant, and the erroneous modification of this
conformation is due to the epigenetic modifications (attachments to the unmodified
DNA sequence of the critical genes of the methyl groups on DNA or methyl, and other
chemical groups to the corresponding histones)

Corresponding technique approached so far: molecular methylation analysis

(methylation specific PCR or MSPCR) for the detection of the erroneously attached
methyl groups on the critical genes
 Clinical diagnostic validation of PWS using MS-PCR

After the bisulfit conversion and amplification it resulted the following electroforetic
pattern:
-normal (N) 2 bends - the 313 bp represents the region from exon 1 of the SNPRN alelle
wich is methilated (maternal origin), the 221 bp represents the region from exon 1 of
the SNPRN alelle wich is unmethilated (paternal origin)
-PWS (PW) 1 banda de 313pb – shows the lack of activity in the paternal alelle
Principiul metodei MS-PCR (folosita pentru a vedea
statusul de metilare ADN)
 Validarea cazurilor de PWS prin metoda MS-PCR

Dupa conversia cu bisulfit si amplificare, s-a obtinut urmatorul pattern de ampliconi:

-normal (N) 2 benzi - cea de 313 pb reprezinta regiunea din exonul 1 al alelei SNPRN de
origine materna (ce in mod normal este metilata ) iar cea de 221pb reprezinta o regiune
din exonul 1 al alelei SNPRN de origine paterna ( ce in mod normal este nemetilata)
-PWS (PW) 1 banda de 313pb – ce ne arata inactivarea prin metilare a alelei de origine
paterna.
-AS 1 banda de 221pb- ar fi fost prezenta in gel doar banda specifica patternului de
metilare al cromosomului de origine paterna
Testing strategies for the
molecular analysis of PWS and
AS based upon (i) an initial
methylation analysis at the
SNRPN locus
and (ii) an initial MLPA analysis
(Simon et al., 2010).
Approaches of the histone code and nonhistone
proteins activity are our future research goals, as
these ones may be informative for the subtle
mechanisms of erasure and reset of the imprinting
marks during the primordial germ cells development.

Appraches of ncRNAs in the chromosome 15 ICR are

presently reported for PWS diagnosis through RTPCR
methods.

These methods and the previously described ones

are envisaged to be introduced in a prental,
preventive diagnosis scheme.
Preliminary results after molecular analyses
Of 19 cases clinically diagnosed, 9 were confirmed by MS-PCR si
FISH . 8 cases were due to deletions (FISH positives). One case had an
imprinting deffect without deletion (FISH negative). The qPCR method excluded
the presence of UPD.
 Analysis of microdeletion causing PWS and expression
studies.

(a) A high-resolution oligonucleotide array-CGH plot is shown with loss of a segment in

15q11.2 from position B22,835,000 bp to B23,010,100 bp (red arrows). (b) A schematic
physical map of the 15q11– q13 genomic interval is shown, highlighting the deleted
segment with respect to SNRPN, UBE3A and snoRNAs within the interval(Trilochan
Sahoo, et al. 2008).
Epigenetic approaches and analyses were financed
by the National Research Programme, the project
42-113/2008, coordinated by UMFTimisoara,
Professor Dr Maria Puiu

And the partners:

University of Bucharest, Institute of Virology
Bucharest, Endocrinology Institute Bucharest

Collaborators: IOMC
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