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)

Epigenetic mechanisms underlying the imprinting defects:

implications for the establishment of diagnostic testing schemes for Prader Willi syndrome in

Romanian population.

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 factorsThe diagnosis is difficult due to individual variations of associated phenotypes and because these phenotypes appear during the offspring developmentDesciphering 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

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 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 silent. This process does not affect the DNA sequence, hence it is named“epigenetic”

It is dynamic and requires several reprogrammings of the epigenetic profiles in the germlines

It requires several steps that include different generations:-parental germ cells-offspring embryogenesis (development of the PCGs, primordialgerm cells), -offspring neonatal development

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.

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.

What is Epigenetics and the epigenetic profiles? Key words: chromatin conformation, gene expression

Major processes that control the chromatin conformation compaction (chromatin remodelling) are:

-DNA methylation-histone modification (acetylation, methylation, phospholylation etc)-noncoding RNA molecules

Chromatin compaction steps

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 conditionsthrough 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 enzyme acting for these processes determine the imprinting defects and the establishment of the diseased state

Model explaining the imprinting errors in critical region for PWS/AS due to deffects in erasure/reset of epigenetic

tags cycles

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).

Somatic cells/p

Germinal cells

Zygote

Somatic cells/o

Methylated alleleIs maternal one

Erasure

Reset

PGcells

Maintenance

Cis factors influencing the imprinting process- ICR, DNA / histone methylation ,nonhistone proteins activity and nc RNAA. 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).).

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).

Recent findings

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

Imprinted ncRNA genes

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

mechanismsDNMTs 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.

Disfunctii mitocondriale

Epigenetica

CH3CH3

CH3

Alterarea structurii

cromozomale

(ADN)

Programarea defectuoasa metabolica si

neuronala

Perturbarea ritmurilor oscilator, circadian,

sezonal

Stil de viata indulgentStil de viata indulgentDezechilibru energeticDezechilibru energetic

Stress oxidativStress oxidativImbatranire …Imbatranire …

GenotipGenotip

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

The mthfr gene

We can estimate the wrong and correct methylation pathways

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 approachesThe 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 entire chromosomal region comprising the critical paternal genes or only a minimal critical chromosomal region that control the expression of a cluster of critical genes (named imprinting control region: IC or ICR); this type of genome modification comprises the lack of the genetic 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 the critical deletion region and monitoring their presence on the chromosomal spreads microscopically)

Paternal chromosome: blue colorMaternal chromosome- red colorNicholls et al., 2001

Classes of alterations registered for PWS and AS

FISH(fluorescent in situ hybridization) for the region 15q11-q13

The probes used: LSI D15S10 spectrum orange/PML spectrum orange/CEP15 spectrum green(Vysis, Abbott inc.)

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

-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 locusand (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

Referinte

Horsthemke B, Wagstaff J. (2008) Mechanisms of imprinting of the Prader–Willi/Angelman region. Am J Med Genet Part A 146A:2041–2052.

Suzanne B Cassidy and Daniel J Driscoll (2009) Prader–Willi syndrome. European Journal of Human Genetics 17, 3–13

Griet Van Buggenhout and Jean-Pierre Fryns (2009) Angelman syndrome (AS, MIM 105830). European Journal of Human Genetics 17, 1367–1373

Williams CA, Beaudet AL, Clayton-Smith J et al (2006) Angelman syndrome 2005: updated consensus for diagnostic criteria. Am J Med Genet A 140: 413– 418.

Gunay-Aygun M, Schwartz S, O’Riordan MA, Cassidy SB (2001) The changing purpose of Prader-Willi syndrome clinical diagnostic criteria and proposed revised criteria. Pediatrics 108: E92.