1. Diamond-Blackfan Anemia Gene Discovery
Hanna T. Gazda, M.D., Ph.D.
Boston Children’s Hospital
Harvard Medical School
Boston, MA

2. Boston Children’s Hospital Boston, MA

3. Genetic DBA projects
DBA gene discovery
Modifier genes

4. DBA gene discovery project
Boston Children’s Hospital (Genetics)– Hanna Gazda, Daniel Yuan, Shideh Kazerounian, Lindsay Swanson
Broad Institute, Cambridge, MA – Vijay Sankaran, Eric Lander

5. Objectives of the presentation
Ribosomal protein genes mutated in DBA
GATA1 mutated in DBA
What does it mean for DBA families?

6. Ribosomal components 47 RPL 33 RPS 5S 5.8S rRNA 28S 18S rRNA 60S 40S

7. Hypothesis – Other ribosomal protein (RP) gene mutations may also cause DBA
Aim of the Study – To screen remaining 78 ribosomal protein genes for mutations in DBA patients without known RPS19 and RPS24 mutations

8. Methods – Screened DNA samples from DBA patients by direct sequencing of exons and intron/exon boundaries using DNA from 96 DBA patients
Sequence change identified
Sequencing of DNA from an additional 96 patients
Search the NCBI and HapMap SNP databases
Sequencing of DNA from control samples
Sequencing of DNA from family members

10. Analysis of sequencing data

11. Analysis of sequencing data

12. Summary of ribosomal protein genes mutated in DBA
Draptchinskaia et al 1999
Gazda et al 2006
Cmejla et al 2007
Farrar et al 2008
Gazda et al 2008
Doherty et al 2010
Gazda et al 2012

13. Large RP gene deletions in DBA
Dr. Bodine’s group (NIH); 9/51
RPS19, RPS17, RPS26 and RPL35A
Dr. Hamaguchi’s group (Japan); 7/27
RPS19, RPS17, RPL5 and RPL35A
Dr. Dianzani-Ramenghi’s group (Italy); 14/72
RPS19, RPS17, RPS26, RPL5, RPL11 and RPL35A
Our own data (BCH); 6/87
RPS19, RPS17, RPS24, RPS26 and RPL15

14. Ribosomal protein genes and DBA
Ribosomal protein gene mutations and large deletions are known in about 60-65% of DBA patients
~35-40% of patients do not have known pathogenic mutation(s)

15. Importance of genetic screening in DBA
To confirm the clinical diagnosis of DBA
For stem cell transplantation
For reproductive choices (pre-implantation genetic diagnosis)
For future gene therapy

16. RPL5 mutation in family with Aase syndromewt wt
Anemia
Triphalangeal thumb
VSD
Anemia
Triphalangeal thumb
Cleft lip
Aase JM & Smith DW, 1969 wt wt
II-1, II-3, III-3
Exon5 indel wt wt

17. Malformations in patients with RPL5, RPL11 and RPS19 mutations
*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 17

18. Mutations of ribosomal protein genes in DBA
Mutations of RPS19, RPL5, RPL11, RPS10, RPS26 and RPL35A are common causes of Diamond-Blackfan anemia, while RPS24, RPS7, RPS17, and RPL26 are sporadically mutated in DBA. All mutations are heterozygous and present in ~55% of patients.
Mutations in RPL5 are associated with multiple physical abnormalities including triphalangeal thumbs and cleft lip/cleft palate, while RPL11 mutations are predominantly associated with isolated abnormal thumbs
Large deletions are present in ~ 5-10% of patients. RPL15 is a novel gene associated with DBA. 18

19. Mutations of ribosomal protein genes in DBA
Majority are nonsense, splice site or frameshift (insertions, deletions)
Heterozygous (present on one copy of the gene) and indicate autosomal dominant inheritance

20. Karyogram of a human female

21. Autosomal dominant inheritance

22. Recurrence risk of DBA
Recurrence risk = 50%

23. Reduced penetrance and variable expressivity in DBAM
II-1
II-2
II-3
III-1
III-2
eADA
Normal eADA
eADA
MCV
Normal MCV
MCV

24. Variable expressivity in DBA
 eADA
 MCV
n HbF
II-1
II-2
II-3
II-4
II-5
II-6
II-7
n eADA
n eADA
n eADA
n eADA
n eADA
 eADA
n MCV
n MCV
n MCV
n MCV
n MCV
 MCV
n HbF
n HbF
n HbF
n HbF
n HbF
n HbF
III-1
III-2
III-3
n eADA
eADA
eADA
n MCV
 MCV
 MCV
n HbF
 HbF
 HbF

25. Germline mutations in DBA
N eADA
N eADA
N MCV
N MCV M M
 eADA
 eADA
 MCV
 MCV

26. Recurrence risk of DBA M M ? ? ? ? I-1 I-2 I-1 I-2 II-1 II-2 II-3 II-1

27. Recurrence risk of DBA
Recurrence risk is slightly higher than in general population

28. Ribosomal protein genes and DBA
Ribosomal protein gene mutations and large deletions are known in about 60-65% of DBA patients
~35-40% of patients do not have known pathogenic mutation(s)

29. Next step in DBA gene discoveries
Entire exome sequencing- (all exons) all coding regions of ~25,000 genes

30. New patients enrolled into our study
11 ribosomal protein gene screening
GATA1 gene screening
Screening of the new genes by exome sequencing

31. Study participation in DBA gene discovery and modifier genes
Consent form and Questionnaire –
Lindsay Swanson, genetic counselor;
ph ;
Blood draw at local doctor’s office
Blood sample sent to Boston Children’s Hospital
No charge to participate

32. ] ] ] ] ] ] Acknowledgements DBA Foundation DMA Foundation
Alan H. Beggs
Mee Rie Sheen
Natasha Darras
Leana Doherty
Mike Landowski
Chris Buros
Roxy Ghazvinian
Adrianna Vlachos
Jeffrey M. Lipton
Eva Atsidaftos
Colin A. Sieff
Sarah E. Ball
Edyta Niewiadomska
Michal Matysiak
Peter E. Newburger ] ]
Broad Institute,
Cambridge, MA
Genetics/Genomics
Children’s Hospital
Harvard Medical School
Boston, MA, USA
Vijay Sankaran
Eric Lander ]
Stanford University School of Medicine Stanford, CA
Bertil Glader ]
DBA Registry
Feinstein Institute for Medical Research,
Manhasset, NY ]
University of Freiburg, Freiburg, Germany
Charlotte Niemeyer
Joerg Meerpohl
Children’s Hospital
Boston, MA, USA
St.George's University of London, London, UK ]
DBA Foundation
DMA Foundation
University Medical School of Warsaw,
Warsaw, Poland
University of Massachusetts Medical School, Worcester, MA, USA
We thank the physicians, DBA patients and their family members for participating in the study! 32