Diamond-Blackfan Anemia Gene Discovery

Hanna T. Gazda, M.D., Ph.D.Hanna T. Gazda, M.D., Ph.D.

Boston Children’s Hospital Boston Children’s Hospital Harvard Medical SchoolHarvard Medical School

Boston, MABoston, MA

Boston Children’s Hospital Boston, MA

Genetic DBA projects Genetic DBA projects DBA gene discoveryDBA gene discovery Modifier genesModifier genes

DBA gene discovery projectDBA gene discovery project

Boston Children’s Hospital (Genetics)– Boston Children’s Hospital (Genetics)– Hanna Gazda, Daniel Yuan, Shideh Hanna Gazda, Daniel Yuan, Shideh Kazerounian, Lindsay SwansonKazerounian, Lindsay Swanson

Broad Institute, Cambridge, MA – Vijay Broad Institute, Cambridge, MA – Vijay Sankaran, Eric LanderSankaran, Eric Lander

Objectives of the presentationObjectives of the presentation

Ribosomal protein genes mutated in DBARibosomal protein genes mutated in DBA GATA1GATA1 mutated in DBA mutated in DBA What does it mean for DBA families?What does it mean for DBA families?

60S

40S

47 RPL

33 RPS

Ribosomal components

5S5.8S rRNA28S

18S rRNARPS19

RPS24

Aim of the StudyAim of the Study – To screen – To screen remaining remaining 78 ribosomal protein78 ribosomal protein genes for mutations genes for mutations in DBA in DBA patientpatients without known s without known RPS19RPS19 and and RPS24 RPS24 mutationsmutations

HypothesisHypothesis – Other ribosomal protein – Other ribosomal protein (RP) gene(RP) gene mutations mutations may also cause may also cause DBADBA

MethodsMethods – – ScreenedScreened DNA samples from DBA DNA samples from DBA ppatients by datients by direct sequencing of exons and irect sequencing of exons and intron/exon boundaries usingintron/exon boundaries using DNA from 96 DBA DNA from 96 DBA ppatientatientss

Sequence change identifiedSequence change identified

1)1) Sequencing of DNA from an additional 96 Sequencing of DNA from an additional 96 patientspatients

2)2) Search the Search the NNCBI andCBI and HapMap SNP databases HapMap SNP databases

3)3) Sequencing of DNA from 150-200 control samplesSequencing of DNA from 150-200 control samples

4)4) Sequencing of DNA from family membersSequencing of DNA from family members

Analysis of sequencing data

Analysis of sequencing data

Gene symbol

(%) of mutated patients

RPS19 25%

RPL5 ~6.6%

RPS26 ~6.4%RPL11 ~4.8%

RPS10 ~2.6%

RPL35A ~3.5%

RPS24 ~2%

RPS17 ~1%

RPS7 ~1%

RPL26 ~1%

~53.9%

Summary of ribosomal protein Summary of ribosomal protein genes mutated in DBAgenes mutated in DBA

Draptchinskaia et al 1999Gazda et al 2006Cmejla et al 2007Farrar et al 2008Gazda et al 2008Doherty et al 2010Gazda et al 2012

Large RP gene deletions in DBALarge RP gene deletions in DBA

Dr. Bodine’s group (NIH); 9/51 Dr. Bodine’s group (NIH); 9/51 RPS19, RPS17, RPS26 RPS19, RPS17, RPS26 and and RPL35ARPL35A

Dr. Hamaguchi’s group (Japan); 7/27Dr. Hamaguchi’s group (Japan); 7/27 RPS19, RPS17, RPL5RPS19, RPS17, RPL5 and and RPL35ARPL35A

Dr. Dianzani-Ramenghi’s group (Italy); 14/72Dr. Dianzani-Ramenghi’s group (Italy); 14/72 RPS19, RPS17, RPS26, RPL5, RPL11RPS19, RPS17, RPS26, RPL5, RPL11 and and

RPL35ARPL35A Our own data (BCH); 6/87Our own data (BCH); 6/87

RPS19, RPS17, RPS24, RPS26RPS19, RPS17, RPS24, RPS26 and and RPL15RPL15

Ribosomal protein genes and Ribosomal protein genes and DBADBA

Ribosomal protein gene mutations and Ribosomal protein gene mutations and large deletions are known in about 60-65% large deletions are known in about 60-65% of DBA patientsof DBA patients

~35-40% of patients do not have known ~35-40% of patients do not have known pathogenic mutation(s)pathogenic mutation(s)

Importance of genetic screening Importance of genetic screening in DBAin DBA

To confirm the clinical diagnosis of DBATo confirm the clinical diagnosis of DBA For stem cell transplantation For stem cell transplantation For reproductive choices (pre-implantation For reproductive choices (pre-implantation

genetic diagnosis)genetic diagnosis) For future gene therapyFor future gene therapy

I-2

II-1 II-2 II-3

I-1

Aase JM & Smith DW, 1969

RPL5 mutation in family with Aase syndrome

II-1, II-3, III-3Exon5 indel

II-1 II-2 II-3

III-1 III-2 III-3

wt wt

wt wt

wt wt

Anemia Triphalangeal thumbCleft lip

Anemia Triphalangeal thumbVSD

Malformations in patients with Malformations in patients with RPL5, RPL5, RPL11RPL11 and and RPS19 RPS19 mutationsmutations

Mutated gene

Patients with

mutations

Patients with malformations

Cleft lip/palate

Thumb abnormality

Heart abnormality

Multiple malformations

RPL5 20 14 (70%) 9 (45%) 8 (40%) 5 (25%) 11 (55%)

RPL11 18 12 (67%) 0 (0%) 8 (44%) 3 (16%) 3 (16%)

RPS19* 76 35 (46%) 0 (0%) 7 (9%) 4 (5%) 16 (21%)

*Willig T-N et al, 1999; Rumenghi et al, 2000; Cmejla et al, 2000; Orfali et al, 2004

•Cleft lip/cleft palate RPL5 vs RPL11 p=0.007; RPL5 vs RPS19 p=9.745x10-7

•Thumb abnormalities RPL5 vs RPS19 p=0.0024; RPL11 vs RPS19 p=0.0012 •Congenital heart defects RPL5 vs RPS19 p=0.017•Multiple abnormalities RPL5 vs RPL11 p=0.02; RPL5 vs RPS19 p=0.0047

Mutations of ribosomal protein Mutations of ribosomal protein genes in DBAgenes in DBA

Mutations ofMutations of RPS19RPS19, , RPL5, RPL11, RPS10,RPL5, RPL11, RPS10, RPS26RPS26 and and RPL35ARPL35A aare re ccommon ommon ccauseausess of Diamond-Blackfan of Diamond-Blackfan aanemianemia, , while while RPS24RPS24, , RPRPS7S7, , RPRPS17S17, , and and RPL26RPL26 are sporadically are sporadically mutated in DBAmutated in DBA. All mutations are heterozygous and . All mutations are heterozygous and present in ~55% of patients.present in ~55% of patients.

Mutations in Mutations in RPL5RPL5 are associated with multiple physical are associated with multiple physical abnormalities including triphalangeal thumbs and cleft abnormalities including triphalangeal thumbs and cleft lip/cleft palatelip/cleft palate,, while while RPL11RPL11 mutations are predominantly mutations are predominantly associated with isolated abnormal thumbsassociated with isolated abnormal thumbs

Large deletions are present in ~ 5-10% of patients. Large deletions are present in ~ 5-10% of patients. RPL15RPL15 is a novel gene associated with DBA.is a novel gene associated with DBA.

Mutations of ribosomal protein Mutations of ribosomal protein genes in DBAgenes in DBA

Majority are nonsense, splice site or frameshift Majority are nonsense, splice site or frameshift (insertions, deletions)(insertions, deletions)

Heterozygous (present on one copy of the gene) Heterozygous (present on one copy of the gene) and indicate autosomal dominant inheritanceand indicate autosomal dominant inheritance

Karyogram of a human female

http://www.uic.edu/nursing/genetics/Lecture/Types/SingleGene/AutosomalDominant/AD.htm

Autosomal dominant inheritance

Recurrence risk of DBArisk of DBA

Recurrence risk = 50%

Reduced penetrance and Reduced penetrance and variable expressivity in DBAvariable expressivity in DBA

eADA Normal eADA

MCV Normal MCV

I-1 I-2

II-1 II-2 II-3

III-1 III-2

M

eADA

MCV

I-1 I-2

II-1 II-2 II-4 II-5 II-6 II-7II-3

III-1 III-3

n eADAn MCV

n HbF

eADA MCV

HbF

eADA MCV

HbF

III-2

n MCV n HbF

n eADA

n eADAn MCV

n HbF

n eADAn MCV

n HbF

n eADAn MCV

n HbF

n eADAn MCV

n HbF

eADA MCV n HbF

eADA MCV n HbF

Variable expressivity in DBA

M

eADA

MCV

M eADA

MCV

N eADA

N MCV

N eADA

N MCV

Germline mutations in DBA

Recurrence risk of DBArisk of DBA

I-1 I-2

II-1 II-2 II-3

III-1 III-2

M

I-1 I-2

II-1 II-2 II-3

III-1 III-2

M

? ? ? ?

Recurrence risk of DBA risk of DBA

Recurrence risk is slightly higher than in general population

Ribosomal protein genes and Ribosomal protein genes and DBADBA

Ribosomal protein gene mutations and Ribosomal protein gene mutations and large deletions are known in about 60-65% large deletions are known in about 60-65% of DBA patientsof DBA patients

~35-40% of patients do not have known ~35-40% of patients do not have known pathogenic mutation(s)pathogenic mutation(s)

Next step in DBA gene Next step in DBA gene discoveriesdiscoveries

Entire exome sequencing- (all exons) all Entire exome sequencing- (all exons) all coding regions of ~25,000 genescoding regions of ~25,000 genes

New patients enrolled into our New patients enrolled into our studystudy

11 ribosomal protein gene screening11 ribosomal protein gene screening GATA1GATA1 gene screening gene screening Screening of the new genes by exome Screening of the new genes by exome

sequencingsequencing

Study participation inStudy participation inDBA gene discovery and DBA gene discovery and

modifier genesmodifier genes Consent form and Questionnaire – Consent form and Questionnaire –

Lindsay Swanson, genetic counselor; Lindsay Swanson, genetic counselor;

ph. 617-919-2169; ph. 617-919-2169; lindsay.swanson@childrens.harvard.edulindsay.swanson@childrens.harvard.edu

Blood draw at local doctor’s officeBlood draw at local doctor’s office Blood sample sent to Boston Children’s Blood sample sent to Boston Children’s

Hospital Hospital No charge to participateNo charge to participate

AcknowledgementsAcknowledgementsAlan H. BeggsMee Rie SheenNatasha Darras Leana DohertyMike LandowskiChris BurosRoxy Ghazvinian

Adrianna VlachosJeffrey M. LiptonEva Atsidaftos

Colin A. Sieff

Sarah E. Ball

Edyta NiewiadomskaMichal Matysiak

Peter E. Newburger

Genetics/Genomics Children’s HospitalHarvard Medical School Boston, MA, USA]

]

Children’s HospitalBoston, MA, USA

St.George's University of London, London, UK

University of Massachusetts Medical School, Worcester, MA, USA

University Medical School of Warsaw,Warsaw, Poland

Vijay SankaranEric Lander

Broad Institute,Cambridge, MA

We thank the physicians, DBA patients and their family members for participating in the study!

]DBA RegistryFeinstein Institute for Medical Research, Manhasset, NY

Bertil GladerStanford University School of Medicine Stanford, CA

]Charlotte NiemeyerJoerg Meerpohl ]University of

Freiburg, Freiburg, Germany

]

DBA Foundation

DMA Foundation