Williams-Beuren Syndrome : a Model of CNV Affecting Dosage and Phenotypes Professor Lucy R. Osborne

Williams-Beuren Syndrome Locus: a Model of CNV Affecting Gene Dosage and Phenotypes

Professor Lucy R. Osborne Departments of Medicine and Molecular Genetics University of Toronto 1

Overview

• Deletions – Williams-Beuren syndrome • Genomic organization of 7q11.23 • Inversions • Duplications • Genotype-Phenotype

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Williams-Beuren Syndrome (WBS)

• Williams 1961 » Children with 'unusual' facial features, growth retardation, supravalvular aortic stenosis (SVAS) and mild mental retardation • Beuren 1962 » Children with these features plus dental anomalies and friendly personalities

Williams JC, Circulation 24:1311-8, 1961 Beuren AJ, Circulation 26:1235-40, 1962 3

The screen versions of these slides have full details of copyright and acknowledgements 1 Williams-Beuren Syndrome Locus: a Model of CNV Affecting Gene Dosage and Phenotypes Professor Lucy R. Osborne

Clinical Symptoms of WBS SYSTEM SYMPTOM INCIDENCE Developmental delaydelay, ADHD, 97% Nervous IQ<70, specific learning disability Supravalvular aortic stenosis (SVAS) 64% Cardiovascular Supravalvular pulmonary stenosis 24% Hypertension in adulthood 47% Ocular Stellate patterning of the iris 72% Impaired visual acuity 60% Auditory Hypersensitivity to sound 85% Malocclusion 85% Dental Enamel hypoplasia, microdontia 50% Genitourinary Renal abnormalities 52% Vomiting, constipation and colic in infancy 43% Gastrointestinal Hypercalcemia 67% Musculoskeletal Joint limitation, curvature of the spine 50% Dysmorphic featuresfeatures, hoarse voice Integumentary 96% Prematurely aging skin and hair 60% Morris CA, J. Pediatr. 113:318-26, 1982 4

Characteristic Facial Features

• Small upturned nose • Long philtrum • Stellate iris pattern • Puffiness around eyes • Full cheeks • Small jaw • Wide mouth, full lips • Small, widely spaced teeth

Photograph kindly provided by Barbara Pober Morris CA & Mervis CB, Annu. Rev. Genomics Hum. Genet. 1:461-84, 2000 5

Unique Cognitive and Behavioural Profile

• IQ of 55-60 • Relative strength in expressive language • Weakness in visuospatial abilities • Attention deficit /hyperactivity disorder • Anxiety and simple phobias • Hypersensitivity to sound • Over friendliness and disinhibition • Affinity for music

Morris CA & Mervis CB, Annu. Rev. Genomics Hum. Genet. 1:461-84, 2000 6

The screen versions of these slides have full details of copyright and acknowledgements 2 Williams-Beuren Syndrome Locus: a Model of CNV Affecting Gene Dosage and Phenotypes Professor Lucy R. Osborne

Different Spatial Deficits in Drawing by WBS and Trisomy 21 Children

handlebars bike basket seat wheels

chain

wheel person pedals pedals T21 age 11 WBS age 11

Matched in age and IQ

Bellugi U, Am. J. Med. Genet. Suppl.6:115-25, 1990 7

What Letter Do You See?

Y Y Y Y Y Y Y Y Y Y Y Y D Y Y Y 8

Seeing Either the Forest or the Trees

T21 Y Y Y Y Y Y Y 11 yrs 13 yrs 18 yrs Y Y Y Y Y Y Y Model WBS

10 yrs 13 yrs 16 yrs

Bihrle AM, Brain Cogn. 11:37-49, 1989 9

The screen versions of these slides have full details of copyright and acknowledgements 3 Williams-Beuren Syndrome Locus: a Model of CNV Affecting Gene Dosage and Phenotypes Professor Lucy R. Osborne

Genetic Basis of WBS

• Multisystem developmental disorder • Dominant, frequency 1/7,500

7q11.23 • Microdeletion (26-28 ) • Common deletion size • Haploinsufficiency of >1 gene

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Haploinsufficiency of Causes SVAS

• Translocations and deletions in SVAS » Curran, ME, Cell 73:159-68, 1993 » Ewart, AK, J.Clin. Invest. 93:1071-7, 1994 » Olson, TM, Hum. Mol. Genet. 4:1677-9, 1996 • Hemizygously deleted in WBS » Ewart, AK, Nat. Genet. 5:11-6 1993 • Null allele point mutations in SVAS » Tassabehji, M, Hum. Mol. Genet. 6:1029-36, 1997 » Li, DY, Hum. Mol. Genet. 6:1021-8, 1997 • Mutant allele mutation in AD cutis laxa » Tassabehji, M, Hum. Mol. Genet. 7:1021-8, 1998 » Zhang, MC, J. Biol. Chem. 274:981-6, 1999 11

Genomic Organization of 7q11.23

LCR 1 LCR 2 LCR 3 400 kb 280 kb 450 kb

NSUN5 BAZ1B VPS37D WBSCR27 GTF2IRD1 WBSCR16 FKBP6 BCL7B WBSCR28 RFC2 GTF2I HIP1 CALN1 STX1A LIMK1 CEN TEL FZD9 ELN CLIP2 CCL24 SDCR2B TRIM50 TBL2DNAJC30 CLDN4 LAT2 WBSCR23 GTF2IRD2 CCL26 SBDSP MLXIPL WBSCR22 CLDN3 EIF4H NCF1 ABHD11

C ABC B A B A C

FKBP6 POM121TRIM50 GTF2I GTF2IRD2 WBSCR19L PMS2L WBSCR20 NCF1 STAG3L 12

The screen versions of these slides have full details of copyright and acknowledgements 4 Williams-Beuren Syndrome Locus: a Model of CNV Affecting Gene Dosage and Phenotypes Professor Lucy R. Osborne

The Common WBS Deletion

1.5 Mb Common Deletion (26-28 genes)

NSUN5 BAZ1B VPS37D WBSCR27 GTF2IRD1 WBSCR16 FKBP6 BCL7B WBSCR28 RFC2 GTF2I HIP1 CALN1 STX1A LIMK1 CEN TEL FZD9 ELN CLIP2 CCL24 SDCR2B TRIM50 TBL2 DNAJC30 CLDN4 LAT2 WBSCR23 GTF2IRD2 CCL26 SBDSP MLXIPL WBSCR22 CLDN3 EIF4H NCF1 ABHD11

C ABC B A B A C

5%95% 95% 5% 13

Primate-Specific Evolution of WBS Segmental Duplications

~80 My ago mouse Evolutionary Time Scale ~25 My ago macaque A C ~14 My ago orangutan

~6 My ago chimpanzee B Human

Antonell A, Genome Res. 15:1179-88, 2005 14

Deletion Mechanism

Non-Allelic Homologous Recombination

Intra-chromosomal WBS deletions 33%

Inter-chromosomal WBS deletions 67%

Dutly, F & Schinzel,A, Hum. Mol. Genet. 5:1893-8, 1996 Baumer, A, Hum. Mol. Genet. 7:887-94, 1998 15

The screen versions of these slides have full details of copyright and acknowledgements 5 Williams-Beuren Syndrome Locus: a Model of CNV Affecting Gene Dosage and Phenotypes Professor Lucy R. Osborne

Direct and Inverted LCRs at 7q11.23 Blocks aligned in a direct orientation Blocks aligned in an inverted orientation

NSUN5 BAZ1B VPS37D WBSCR27 GTF2IRD1 WBSCR16 FKBP6 BCL7B WBSCR28 RFC2 GTF2I HIP1 CALN1 STX1A LIMK1 CEN TEL FZD9 ELN CLIP2 CCL24 SDCR2B TRIM50 TBL2 DNAJC30 CLDN4 LAT2 WBSCR23 GTF2IRD2 CCL26 SBDSP MLXIPL WBSCR22 CLDN3 EIF4H NCF1 ABHD11

C ABC B A B A C

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Fishing for Inversions

Three-colour interphase FISH

FISH probes Common Deletion

NSUN5 BAZ1B VPS37D WBSCR27 GTF2IRD1 WBSCR16 FKBP6 BCL7B WBSCR28 RFC2 GTF2I HIP1 CALN1 STX1A LIMK1 CEN TEL FZD9 ELN CLIP2 CCL24 SDCR2B TRIM50 TBL2 DNAJC30 CLDN4 LAT2 WBSCR23 GTF2IRD2 CCL26 SBDSP MLXIPL WBSCR22 CLDN3 EIF4H NCF1 ABHD11

Normal Inverted Osborne, LR, Nat. Genet. 29:321-5, 2001 17

Inversions Present in 33% of Transmitting WBS Parents

Non-transmitting parent D7S3197 Transmitting parent 11106 M P F N

N N INV

11105 11107

2 normal 7s 1 normal 1 inversion chromosome 7 Osborne, LR, Nat. Genet. 29:321-5, 2001 18

The screen versions of these slides have full details of copyright and acknowledgements 6 Williams-Beuren Syndrome Locus: a Model of CNV Affecting Gene Dosage and Phenotypes Professor Lucy R. Osborne

Inversion Does Not Cause Clinical Symptoms

• Breakpoints are outside the common deletion • is not altered • Present in 5%-6% of general population • Homozygous individual identified • Individuals with inversion and clinical symptoms are unlike individuals with WBS

Tam , E, Am. J. Med. Genet. A 146A:1797-806, 2008 19

Inversion Polymorphism is a Genetic Risk Factor for WBS

• Inversion of the WBS region is found at 6 times the frequency in transmitting parental from WBS families • This genomic variant (WBSinv-1) leads to disturbances in meiosis predisposing to chromosome rearrangements in future generations (deletions, duplications, inversions, translocations)

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Genomic Polymorphism as a Genetic Risk Factor for Genomic Disease

• Genomic polymorphism is an important contributor in other disease- associated recurrent chromosomal rearrangements, previously thought to be stochastic in nature » Williams-Beuren syndrome » Angelman syndrome » Sotos syndrome » 17q21.31 deletion syndrome » 15q13.3 deletion syndome

Osborne, LR, Nat. Genet. 29:321-5, 2001; Gimelli, G, Hum. Mol. Genet. 12:849-58, 2003 Visser, R, Am. J. Hum. Genet. 76:52-67, 2005; Koolen, DA, Nat. Genet. 38:999-1001, 2006 Sharp, AJ, Nat. Genet. 40:322-8, 2008 21

The screen versions of these slides have full details of copyright and acknowledgements 7 Williams-Beuren Syndrome Locus: a Model of CNV Affecting Gene Dosage and Phenotypes Professor Lucy R. Osborne

Familial Recurrence of WBS

French family US family

WBS WBS WBS WBS

Intrachromosomal Interchromosomal Maternal Paternal No inversion Father has WBSinv-1

Germline mosaicism NAHR between WBSinv-1 and normal chromosomes

Scherer, SW, Hum. Genet. 117:383-8, 2005 22

Risk Assessment in WBSinv-1 Carriers

• Population risk 1 in 7,500 • Risk increased 6-fold in WBSinv-1 carriers » WBSinv-1 carrier risk 1 in 1,300 • Perceived risk associated with WBSinv-1 carrier status may be even higher in WBS families • Risk of fetal loss due to invasive prenatal diagnosis 1 in 200-300

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Duplication of the WBS Region (1)

• Mechanism predicts duplications • 100’s of individuals with WBS-like symptoms and controls screened by FISH » Why no duplications? ¾ No phenotype ¾ Incompatible with life ¾ Phenotype different from Williams-Beuren syndrome

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The screen versions of these slides have full details of copyright and acknowledgements 8 Williams-Beuren Syndrome Locus: a Model of CNV Affecting Gene Dosage and Phenotypes Professor Lucy R. Osborne

First Duplication of the WBS Region

Referred for testing for VCFS duplication

Real-time PCR revealed copy number change at WBS locus

Somerville, MJ, N. Engl. J. Med. 353:1694-701, 2005 25

FISH Detection of WBS Duplication

Three colour interphase FISH Normal Duplicated Chr 7 Chr 7 N Chr7

WBS CTA-208H19 commonly deleted region RP5-1186P10

CTB-139P11

Dup Chr7

Somerville, MJ, N. Engl. J. Med. 353:1694-701, 2005 26

Duplication of the WBS Region is Reciprocal of the Common WBS Deletion Normal chromosome Markers cen tel Cc Ac Bc Cm Bm Am Bt At Ct 100 kb Proband cen tel Cc Ac Bc Cm Bm-BcCm Bm Am Bt At Ct

Markers BBSTR1 BBSTR2 Genes GTF2I/P1/P2 NCF1/P1/P2 GTF2lRD2/P1/P2 13 35 1 11 16 1 Bc A delGT TAG T G del del 9 bp SSNs Bm G GT C G G A G Bt A delGT C A G C A 246101389 11

Bmedial Bcentromeric B block junction Somerville, MJ, N. Engl. J. Med. 353:1694-701, 2005 27

The screen versions of these slides have full details of copyright and acknowledgements 9 Williams-Beuren Syndrome Locus: a Model of CNV Affecting Gene Dosage and Phenotypes Professor Lucy R. Osborne

Clinical Features Associated with Duplication of the WBS Region

Attention problems Attention problems Did not complete high school Difficulty at school

11 yr 1 mth 8 yr 10 mth

ADD (10mg Dexedrine/day) ADD (10mg Dexedrine/day) Mild developmental delay Mild developmental delay Mild dysmorphism Severe language delay 28

Developmental Assessment Revealed a Deficit in Expressive Language

Table 1. Standard Scores on Intellectual and Vocabulary Assessments

Assessment P S Differential Ability Scales (DAS) Verbala 65 93 Nonverbal reasoning 65 70 Spatial 70 68 General conceptual ability 62 73 Special Nonverbal Composite 65 67 Vocabulary PPVT-III 82 106 EVTb 40 104

Somerville, MJ, N. Engl. J. Med. 353:1694-701, 2005 29

Severe Expressive Language Delay

• Able to correctly pronounce only a very small number of words • Verbal responses, manual signs, gestures, pantomime, and drawing were all considered acceptable responses on Differential Ability Scales • Able to write words and sentences • Able to draw appropriate responses to questions

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The screen versions of these slides have full details of copyright and acknowledgements 10 Williams-Beuren Syndrome Locus: a Model of CNV Affecting Gene Dosage and Phenotypes Professor Lucy R. Osborne

Duplication of the WBS Region (2)

First example of the reciprocal WBS duplication A further 14 cases have been published since October 2005, including 13 reciprocal duplications of the WBS region Of the 15 known duplication cases, 10 were de novo events and 5 were inherited

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Frequency of 7q11.23 Duplication

Low frequency due to variable phenotype Deletions more common than duplications Single sperm analysis of meiotic recombinants Deletion = 2 x Duplication Hot-spot specific Duplication highly under-diagnosed

Turner, DJ, Nat. Genet. 40:90-5, 2008 Bayés M, Am. J. Hum. Genet. 73:131-51, 2003 32

Common Features of Dup(7)(q11.23q11.23)

• Expressive language delay • Intellectual impairment » Mild to moderate, ADHD • Motor delay » Gross motor skills, hypotonia • Mild dysmorphism » High broad nose » Posteriorly rotated ears » High arched palate » Small mouth and/or thin lips 33

The screen versions of these slides have full details of copyright and acknowledgements 11 Williams-Beuren Syndrome Locus: a Model of CNV Affecting Gene Dosage and Phenotypes Professor Lucy R. Osborne

Sporadic Features of Dup(7)(q11.23q11.23)

• Craniosynostosis • Macrocephaly • Brachycephaly • Cortical dysplasia • Cleft lip/palate •Seizures • Hyperphagia •Autism • Neutropenia •Glaucoma 34

Deletion and Duplication of 7q11.23 Result in Contrasting Phenotypes

Mild intellectual disability Delayed motor skills 9Short philtrum 9Thin lips 9Long philtrum 9Larger teeth 9Full lips 9Arched palate 9Small teeth 9Narrow 9Normal palate forehead 9Broad forehead 9Normal 9Periorbital periorbital area fullness

Severe expressive Relative strength language delay in expressive language Relative strength Very weak spatial skills in spatial skills 35

Molecular Basis of WBS (1)

NCF1

Hypertension

SVAS ELN Facial features Hernia

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The screen versions of these slides have full details of copyright and acknowledgements 12 Williams-Beuren Syndrome Locus: a Model of CNV Affecting Gene Dosage and Phenotypes Professor Lucy R. Osborne

NCF1 Copy Number Influences Risk of Developing Hypertension in WBS

WBS Control

Elastin deletion

Vascular narrowing

NCF1 not deleted NADPH NCF1 deleted activation Higher oxidative stress Lower oxidative stress OO2 2- High risk of angiotensin II- Lower risk of angiotensin II- mediated hypertension mediated hypertension

Del Campo, M, Am. J. Hum. Genet. 78:533-42, 2006 37

Molecular Basis of WBS (2)

NCF1

Hypertension Visual spatial deficits SVAS Dental ELN abnormalities Facial features Developmental delay Anxiety Hernia OTHER GENES Hyperaccusis Hypercalcemia ADHD Growth Endcocrine retardation abnormalities 38

Small Deletions of the WBS Region FKBP6 GTF2IRD2 TBL2 WBSCR23 WBSCR16 BAZ1B STX1A CLIP2 LAT2 ELN LIMK1 CLDN3 EIF4H GTF2IRD1 GTF2I HIP1 RFC2 FZD9 BCL7B MLXIPL ABHD11 DNAJC30 CLDN4 WBSCR22 Disease severity 1 Tassabehji 1999 2 Tassabehji 1999 3 Tassabehji 1999 SVAS 4 Morris 2003 5 Morris 2003 6 Morris 2003 SVAS 7 Morris 2003 WSCP 8 Morris 2003 9 Hirota 2003 10 Hirota 2003 11 Doyle 2004 12 Gagliardi 2003 Atypical 13 Howald 2005 WBS 14 Howald 2005 15 Van Hagen 2007 16 Hirota 2003 17 Tassabehji 2005 18 Heller 2003 Typical 19 Botta 1999 WBS 20 Botta 1999 21 WBS minimal critical interval 39

The screen versions of these slides have full details of copyright and acknowledgements 13 Williams-Beuren Syndrome Locus: a Model of CNV Affecting Gene Dosage and Phenotypes Professor Lucy R. Osborne

Dissecting Genotype-Phenotype Relationships

Single gene disorders ELN Point mutations e.g., SVAS

Deletion mapping Minimal Smaller deletions critical region Functional candidates Expression, domains

Genomic rearrangements Deletion, inversion, duplication

Animal models ELN, LIMK1, CLIP2, KO, transgenic, deletions GTF2IRD1, STX1A

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Summary

• 7q11.23 undergoes multiple rearrangements • Highly homologous flanking sequence blocks • Deletion – complex neurodevelopmental disorder • Duplications – contrasting neurodevelopment • Inversions increase the frequency of deletion • Genes at 7q11.23 affect cognition, behaviour and language

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Acknowledgements

Osborne Laboratory University of Nevada University of Western Ontario University of Toronto Colleen Morris Janis Cardy Edwin Young University of Louisville University of Alberta Jen O’Leary Ella Peregrine Martin Somerville Wayne Loo Carolyn Mervis Margaret Lilley Ariane Mandel Steven Bamforth Elaine Tam Boston Children’s Hospital Barbara Pober University Pompeu Fabra Sick Kids, Toronto Barcelona Stephen Scherer Alfred I. duPont Hospital for Children Miguel del Campo TCAG Karen Gripp Luis Pérez-Jurado Jennifer Skaug

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The screen versions of these slides have full details of copyright and acknowledgements 14 Williams-Beuren Syndrome Locus: a Model of CNV Affecting Gene Dosage and Phenotypes Professor Lucy R. Osborne

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The screen versions of these slides have full details of copyright and acknowledgements 15