Diamond-Blackfan Anemia
Gene Discovery
Hanna T. Gazda, M.D., Ph.D.
Boston Children’s Hospital
Harvard Medical School
Boston, MA
Boston Children’s Hospital
Boston, MA
Genetic DBA projects


DBA gene discovery
Modifier genes
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
Objectives of the presentation



Ribosomal protein genes mutated in DBA
GATA1 mutated in DBA
What does it mean for DBA families?
Ribosomal components
60S
5S
5.8S rRNA
28S
47 RPL
33 RPS
RPS19
18S rRNA
RPS24
40S
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
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
1) Sequencing of DNA from an additional 96 patients
2) Search the NCBI and HapMap SNP databases
3) Sequencing of DNA from 150-200 control samples
4) Sequencing of DNA from family members
Analysis of sequencing data
Analysis of sequencing data
Summary of ribosomal protein
genes mutated in DBA
Gene (%) of mutated
symbol
patients
RPS19
25%
RPL5
~6.6%
RPS26
RPL11
~6.4%
~4.8%
RPS10
~2.6%
RPL35A
~3.5%
RPS24
~2%
RPS17
RPS7
RPL26
~1%
~1%
~1%
~53.9%
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
Large RP gene deletions in DBA

Dr. Bodine’s group (NIH); 9/51


Dr. Hamaguchi’s group (Japan); 7/27


RPS19, RPS17, RPL5 and RPL35A
Dr. Dianzani-Ramenghi’s group (Italy); 14/72


RPS19, RPS17, RPS26 and RPL35A
RPS19, RPS17, RPS26, RPL5, RPL11 and
RPL35A
Our own data (BCH); 6/87

RPS19, RPS17, RPS24, RPS26 and RPL15
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)
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
RPL5 mutation in family with
Aase syndrome
I-1
I-2
II-1
wt
wt
II-1
II-2
II-3
II-2
Anemia
Triphalangeal thumb
VSD
II-3
Anemia
Triphalangeal thumb
Cleft lip
Aase JM & Smith DW, 1969
wt
wt
II-1, II-3, III-3
Exon5 indel
III-1
wt
III-2
wt
III-3
Malformations in patients with RPL5,
RPL11 and RPS19 mutations
M utated
gene
Patients
Patients w ith
Cle ft
Thum b
Heart
M ultiple
w ith
m alform ations lip/palate abnorm ality abnorm ality m alform ations
m utations
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
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.
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
Karyogram of a human female
Autosomal dominant inheritance
http://www.uic.edu/nursing/genetics/Lecture/Types/SingleGene/AutosomalDominant/AD.htm
Recurrence risk of DBA
Recurrence risk = 50%
Reduced penetrance and
variable expressivity in DBA
I-1
II-1
III-1
II-2
M
I-2
II-3
III-2
eADA
Normal eADA
eADA
MCV
Normal MCV
MCV
Variable expressivity in DBA
I-1
I-2
 eADA
 MCV
n HbF
II-1
II-2
n eADA
n MCV
n HbF
n eADA
n MCV
n HbF
III-1
n eADA
n MCV
n HbF
II-3
II-4
n eADA
n MCV
n HbF
III-2
eADA
 MCV
 HbF
III-3
eADA
 MCV
 HbF
II-5
II-6
II-7
n eADA
n MCV
n HbF
n eADA
n MCV
n HbF
 eADA
 MCV
n HbF
Germline mutations in DBA
N eADA
N eADA
N MCV
N MCV
M
M
 eADA
 eADA
 MCV
 MCV
Recurrence risk of DBA
I-1
II-1
II-2
M
III-1
?
I-1
II-3
III-2
?
I-2
II-1
III-1
?
II-2
M
?
II-3
III-2
I-2
Recurrence risk of DBA
Recurrence risk is
slightly higher than in
general population
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)
Next step in DBA gene
discoveries

Entire exome sequencing- (all exons) all
coding regions of ~25,000 genes
New patients enrolled into our
study



11 ribosomal protein gene screening
GATA1 gene screening
Screening of the new genes by exome
sequencing
Study participation in
DBA gene discovery and
modifier genes




Consent form and Questionnaire –
Lindsay Swanson, genetic counselor;
ph. 617-919-2169;
lindsay.swanson@childrens.harvard.edu
Blood draw at local doctor’s office
Blood sample sent to Boston Children’s
Hospital
No charge to participate
Acknowledgements
Alan H. Beggs
Mee Rie Sheen
Natasha Darras
Leana Doherty
Mike Landowski
Chris Buros
Roxy Ghazvinian
Adrianna Vlachos
Jeffrey M. Lipton
Eva Atsidaftos
]
Genetics/Genomics
Children’s Hospital
Harvard Medical School
Boston, MA, USA
]
Vijay Sankaran
Eric Lander
Bertil Glader
DBA Registry
Feinstein Institute for Medical Research,
Manhasset, NY
Colin A. Sieff Children’s Hospital
]
]
Stanford University
School of Medicine
Stanford, CA
Charlotte Niemeyer
Joerg Meerpohl
Boston, MA, USA
University of London,
Sarah E. Ball St.George's
London, UK
Edyta Niewiadomska
Michal Matysiak
]
Peter E. Newburger
University of Massachusetts Medical
School, Worcester, MA, USA
University Medical School of Warsaw,
Warsaw, Poland
Broad Institute,
Cambridge, MA
]
University of
Freiburg, Freiburg,
Germany
DBA Foundation
DMA Foundation
We thank the physicians, DBA patients and their family members for participating in the study!