Imprinting diseases in humans Beckwith Wiedemann and Silver Russell syndromes

Dr Sylvie Rossignol,MD, PhDExplorations Fonctionnelles Endocriniennes

Hôpital Armand-Trousseau, Paris Centre de Recherche St-Antoine

UMRS. 938, équipe 4sylvie.rossignol@trs.aphp.fr

Epigenetics and genomic imprinting

11p15 region

Beckwith Wiedemann Syndrome

Silver Russell Syndrome

EPIGENETICS

Epigenetics refers to chromatin modifications, which donot involve the DNA sequence itself, resulting in theregulation of gene expression.

development, cells differentiation

X-chromosome inactivation

genomic imprinting

pathological processes such as cancer and other diseases

DNA methylation

Histone tail

modifications

Acetylation

Methylation

Ubiquination

Sumoylation

Phosphorylation

nc RNA

EPIGENETIC MARKS

GENOMIC IMPRINTING

Fonctional hemizygosity

Epigenetic modifications leading to the expressionor the absence of expression of a gene dependingon its parental origin

pat mat pat matmatpat

Paternal imprint Maternal imprint

HDAC: histone deacetylase

HMT : histone methyltransferase

MBD : methyl CpG binding protein

HAT : histone acetyltransferase

CTCF : CCCTC binding factor

IMPRINT MARKS:Allele specific epigenetic modifications

DNA methylation

ICR

ICR

Clustering of imprinted genes

CpG rich domains : DMR

Imprinting center region ICR

Repeat elements

Non coding RNA

CHARACTERISTICS OF IMPRINTED REGIONS

IMPRINTED LOCI AND HUMAN DISEASES

11p15: Silver Russell,Beckwith Wiedemann

6q24: TNDM

7p11-13/7q32:Silver Russell

15q11: Angelman,Willi Prader

20q13 : PHPA, B

14q32 : maternal & paternal UPD14

The Beckwith-Wiedemann syndrome (BWS) isan overgrowth disorder involving developmentalabnormalities, tissue and organ hyperplasia,and an increased risk of childhood tumours(10% of patients)

The BWS results from various genetic orepigenetic defects within the imprinted 11p15region

Beckwith-Wiedemann Syndrome

Beckwith-Wiedemann syndrome

tumours --- 9%

macroglossiamacrosomia

94%88%

------

abdominal wall defects --- 63%organomegaly --- 44%

ear abnormalities --- 38%neonatal hypoglycaemia

facial naevushemihyperplasia

---------

44% 45%33%

Pathogenesis of the Beckwith-Wiedemann syndrome

Alterations of the 11p15 region in BWS are various: cytogenetic, genetic, epigenetic

They can involve the whole 11p15 region, one subdomains of the region or only one gene

They almost always display a mosaicismpattern

mat

pat

methylation

CDKN1C KCNQ1 H19

KCNQ1OT1 IGF2

ICR2 ICR1

enhancers

11p15 REGION

Centromeric domain Telomeric domain

pat

pat

11p15 paternal isodisomy : 20-25%mutation of the maternal

CDKN1C gene : 5%

loss of methylation of the

maternal ICR2 : 60%

gain of methylation of the

maternal ICR1 : 10%

CDKN1C ICR2 KCNQ1OT1

mat

pat

mat

pat

ICR1ICR2

CDKN1C KCNQ1 H19

KCNQ1OT1 IGF2

MOLECULAR DEFECT IN BWS

MECHANISM OF LOI IN BWS

ICR1 GOM

Microdeletions in the human H19 DMR result in loss of IGF2 imprinting and Beckwith-Wiedemann syndrome.

Sparago et al. Nat Genet. 2004;36:958-60

Analysis of the IGF2/H19 imprinting control region uncovers new genetic defect, including mutations of OCT-binding sequences, in patients with 11p15 fetal growth disorders.

Demars J, Shmela Me, Rossignol S, Okabe J, Netchine I, Azzi S, Cabrol S, Le Caignec C, David A, Le Bouc Y, El-Osta A, Gicquel C.Hum Mol Genet. 2010 Mar 1;19(5):803-14.

Frequency in BWS with ICR1 GOM ? 30% ?

MECHANISM OF LOI IN BWS

ICR2 LOM

Microdeletions of Lit1 in familial Beckwith-Wiedemann Syndrome.Niemitz et al. Am J Hum Genet. 2004;75:844-49

Impact of early development environment

- IVF and imprinting diseases- BWS and monozygotic twins- Multiloci defects

Mesure de l’IM par ASMM RTQ-PCR

Quantification Absolue avec une gamme standard

Efficacité de PCR est > à 90%

La quantité de fluorescence est proportionnelle à la quantité initiale de chaque allèle

tACCGGAACGtCtACGAAttG

tACCGGAACGtCtACGAAttG

CH3 CH3 CH3

tACCGGAACGtCtACGAAttG

CH3 CH3 CH3

tATCGGAACGtTtACGAAttG

tATTGGAATGtTtATGAAttG

Traitement

Bisulfite

M

UM

M

UM

M

UM

M

UM

controlLOM

Disruption of the probes

after primer extension and

release of the fluorochromes

NFQF

MGB

NFQ MGBV

V

FNFQ MGB

NFQ MGB

Disruption of the probes

after primer extension and

release of the fluorochromes

NFQF

MGB

NFQ MGBV

NFQF

MGB

NFQ MGBV NFQ MGBV

V

FNFQ MGB

NFQ MGB

V

FNFQ MGB

NFQ MGB

Azzi S et al. Hum Mutat. 2011 Feb;32(2):249-58

ETAPES DU DIAGNOSTIC MOLECULAIRE

Détermination de l’IM ICR1 et ICR2

ICR1 GOM ou

ICR2 LOM

Recherche d’une anomalie du nb de copies par MLPA (kit MRC-Holland)

ICR1 GOM et

ICR2 LOM=

UPD probable

Eliminer une rare anomalie cytogénétique (surtout si index proche de 2/3)

Normal +

Anomalie de paroi

Séquençage de CDKN1C

UPD~40%upd(11)pat.arr11p15.5p11.2(193,788-47,303,516)x2 htz~hmz

Puces SNP : confirmation, bornage de l’UPD, calcul du % de mosaïcisme, éliminer anomalie cytogénétique

Keren B et al. Apport des puces SNP dans la détection et la caractérisation des anomalies moléculaires et chromosomiques responsables du syndrome de Beckwith-Wiedemann. ACLF 2010

ICR2 LOM

257

99

PatientsPatients withtumoursTumours

Wilms tumour

Hépatoblastoma

Adrenocortical T

Neuroblastoma

Rhabdomyosarcoma

Others

ICR1 GOM

35

1010

10

UPD

81

1417

102

2

1

11

CDKN1C

Mutationn

411

3841

Centromeric Telomeric

411 patients

38patients/41 tumours

Total 9% 8,8% 3,5%*/2,3% 29% 17% 50%

cytogen

4

22

1

1

2

2

1

4*

34

3

3

1

2

Follow-up of BWS patients with 11p15 defect (n=411)*

ICR1 defectn=35

ICR2 defectn=257

CDKN1Cmutation

n=34

Low risk, no Wilms High tumour risk

11 Wilms,2 adrenocartical T2 hépatoblastomas

1 rhabdomyosarcoma1 neuroblastoma,1 ganglioneuroma

1 other

1 neuroblastoma1 ganglioneuroma

1 ALL

10 Wilms2 hepatoblastomas, 1 liver sarcoma

2 neuroblastomas1 rhabdomyosarcoma

3 others

-0 to 12 months: clinical examination/3 months and abdominal ultrasound/3 months -1 to 6 years: abdominal ultrasound/3 months and regular clinical examinations->6 years: clinical examination every year

-0 to 12 months: clinical examination each month-One abdominal ultrasound at 3 months-1 to 6 years:clinical examination every year

11p15 UDPn=81 and

cytogenetic defects

Low risk ?

?

C Gicquel, S Rossignol, G Audry, F Auber, L Brugières, C Patte, M Gauthier-Villar, Y Le Bouc

Picture from Hannula et al. J Med Genet 2001

(1)IUGR/SGA (birth weight or length ≤-2 SDS)(2) frequent asymmetry(3)poor postnatal growth ≤-2 SDS(4) preservation of occipitofrontal head

circumference (5) classic facial phenotype(6) severe feeding problems(7) clinodactyly of the fifthfingers/camptodactyly(8)“café au lait” spots(9) genital abnormalities

Many other signs….

Price et al. J med Genet 1999Wollmann et al. Eur J Pediatr 1995

Difficult to assessEasier to diagnose when the patienthas body asymmetry

SILVER RUSSELL SYNDROME Clinical spectrum

SILVER RUSSELL SYNDROME Molecular defects

• implication of imprinted genes

• rare duplications or partial UPD :

7p11.2-p13 (GRB10),

7q31-qter (PEG1/MEST1)

Rare cytogenetic anomalies

Chromosome 7 maternal UPD (mUPD7) ≈ 7-10%

Rare cytogenetic anomalies

mUPD7 ≈ 7-10%

11p15 ICR1 LOM ≈ 50-60%

SILVER RUSSELL SYNDROME Molecular defects

mat

patGicquel, Rossignol et al. Nat Genet 2005

Mandatory: SGA (BW and /or BL ≤-2SDS)

With at least 3 out of these 5 criteria: prominent forehead (before 3 years) relative macrocephaly at birth postnatal growth retardation body asymmetry

feeding difficulties and /or BMI ≤-2SDS

SILVER RUSSELL SYNDROME: A CLINICAL SCORE

Netchine, Rossignol et al. JCEM 2007

127 SGA

69 non SRS SGA

No mUPD7, No 11p15 epimutation

58 SRS

37 (63%) 11p15 ICR1 LOM

3 (5.2 %) mUPD7

Mandatory: SGA (BW and /or BL ≤-2SDS) With at least 3 out of these 5 criteria: prominent forehead (before 3 years) relative macrocephaly at birth postnatal growth retardation body asymmetry feeding difficulties and /or BMI ≤-2SDS

SILVER RUSSELL SYNDROME: A CLINICAL SCORE

Netchine, Rossignol et al. JCEM 2007* Binder 2006, Bartholdi 2008, Bruce 2008

• 11p15 ICR1 LOM is specific of SRS*

• 11p15 ICR1 LOM is the most frequent molecular anomalie in SRS*

• Clinical score usefull to propose SRS molecular investigation

EPIGENOTYPE/PHENOTYPE CORRELATIONS

11p15 ICR1 LOMn=44

mUPD7 n=20

Classic SRS features 61% 20%

Asymmetry 68% 30%

Global developmental 20% 65%delay

Congenital abnormalities 36% 10%

SGA (BW<2SDS) 82% 70%

Post-natal height 57% 65%<-2SDS

CONCLUSIONS

• Dysregulation of the expression of imprintedgenes plays a crucial role in BWS and SRS (>70%cases)IGF2 and H19 on 11p15 region, still unidentified

genes on chromosome 7

• Interaction between 11p15 region and chro 7 ?

• The molecular mechanism is still unknown in 15%of WBS cases and 30% of SRS cases

To the colleagues who referred patients, DNA and clinical data

Madeleine Harbison, NYC, USA

Christine Gicquel Baker Institute, Australia

Irène NetchineSalah Azzi Virginie SteunouThuy-Ai Vu-HongLaurence PerinFabienne Danton Nathalie ThibaudMaryline Le Jules Annick BlaiseEvelyne TagodoeSylvie CabrolYves Le BoucPediatric Endocrinology

Beatrice DubernNutrition

Acknowledgements

To the patients and their families