Research in DBA?
Josu de la Fuente
St Mary’s Hospital
Imperial College London
• A Genome-wide Approach to Investigate the Mechanism of
Glucocorticoid Effect on Erythroid Progenitors in Diamond-Blackfan
Anaemia.
Leukaemia & Lymphoma Research
November 2012
Value: £221,922
• Investigation of the cellular and molecular pathogenesis of
Diamond Blackfan Anaemia
DD McPhail Charitable Settlement
2011
Value: £35,000
• Development of a next generation sequencing-based test for
genetic diagnosis in Diamond-Blackfan Anaemia
NGS Award, Imperial College London
April 2011
Value: £30,000
Diamond Blackfan Anaemia Patients Have a Higher Rate of Hepatic Iron Accumulation than Thalassaemia
Major Patients Leading to Fibrosis
1Division
of Haematology, Imperial College London London,
Katharine Evans1, Robert Goldin2 and Josu de la Fuente1,3
of Histopathology, Imperial College London and 3Division of Paediatrics, St. Mary’s Hospital, London, United Kingdom
2Division
Background and Aims
Diamond Blackfan anaemia is an inherited bone marrow failure syndrome with haematological and systemic manifestations. The classical
presentation of the condition is the development of anaemia in infancy, which occurs in approximately 85-90% of the patients. Long-term 40%
require transfusions as they fail to maintain erythropoiesis at acceptable doses of steroids and only approximately 10% of the patients go into
remission. In the cohort of 64 patients who attend the specialist DBA clinic at St. Mary’s Hospital, London, we have identified that iron overload is
a significant clinical problem, even when receiving adequate chelation treatment with current guidelines and at a young age (Poster #1268).
Methods
To investigate the iron load caused by transfusions and its effect we studied the liver biopsies of 13 patients with DBA and compared them with 27
patients suffering from thalassaemia major (TM) (Table 1). The findings were correlated with the number of transfusions, chelation treatment,
ferritin level and MR techniques. Image analysis of the degree of fibrosis was performed using NIS-elements software after staining liver biopsy
slides with Sirius Red (Figure 1).
Figure 1. Fibrosis image analysis using NIS-elements software
Image analysis was performed using NIS-element software. Slides stained with Sirius Red
were photographed using the software at a magnification of 4x/0.10. The software was
programmed to differentiate background (white), hepatocytes (yellow) and collagen (red)
and the percentage of the are representing collagen was calculated.
Results
TM patients were significantly older [median age: TM 9 years (3-18), DBA 5 years (1-15); p=0.004], which was also reflected in the duration
(months) of transfusion [TM 105 (12-198), DBA 52 (12-130); p=0.004]. However, there was no difference in the frequency of transfusions (p = 0.51)
and in the length of time (months) between starting transfusions and chelation (TM 19, DBA 27; p=0.08). DBA patients received proportionately
more chelation per transfusion than TM patients at the time of biopsy (proportion of chelation time to transfusion time: TM 1.24, DBA 1.58;
p=0.015). Ferritin levels were higher in TM patients, though not significant [TM 2028 g/L (1292-4901), DBA 1324 g/L (535-2300); p=0.16].
Despite having significantly fewer transfusions, the grade of iron deposition was higher in the DBA group (TM 2, DBA 3; p=0.035). This was also
reflected in MRI T2* quantitation, which demonstrated a higher hepatic iron load in DBA patients [TM 3 ms (1-12), DBA 2 ms (1-3); p=0.59]. The
rate of biochemical iron accumulation (mg/g DW) for every month of transfusion was significantly higher in the DBA group (TM 0.05, DBA 0.11;
p=0.005). The rate of fibrosis accumulation was 60% higher in the DBA group, although this was not statistically significant (TM 0.1, DBA 0.16; p =
0.07) andMale
could
beindue
to marrow
a time lag
between ironMale
accumulation
and
fibrosisadherent
formation, particularly as the DBA patients were younger and had had
cell in bone
marrow
cell
bone
before
culture level and MRI T2* had low, significant correlations with fibrosis in TM patients (0.547,
shorter follow
culture up (Table 2). Biochemical iron, ferritin
p=0.001;CD45+
0.357, p=0.033;
and FerriScan
male cell-0.430, p=0.011, respectively)
Vimentin+
male in DBA patients (0.75, p=0.05) (Table 3).
cell
Conclusion
In conclusion, DBA patients have a higher rate of iron accumulation with a trend to higher hepatic fibrosis.
Beta Thalassemia major
Diamond Blackfan anaemia
Median (range)
Median (range)
P value
Number
Age (years)
Sex (% male)
Frequency of transfusions (weeks)
Duration of transfusion (months)
Duration of chelation (months)
36
15
-
9 (3-18)
5 (1-15)
0.004
58.3
53.3
0.40
4 (2-5)
4 (2-6)
0.51
105 (12-198)
52 (12-130)
0.004
71 (0-183)
16 (0-123)
0.016
Biochemical iron/
duration of
transfusion
Fibrosis/ duration of
transfusion
Beta
Thalassemia
major
Diamond
Blackfan
Anaemia
P
value
0.05
0.11
0.005
0.1
0.16
0.07
Table 2. Rate of iron and fibrosis accumulation in TM and DBA
The rate of iron accumulation for every month of transfusion was significantly higher in
DBA patients in comparison with patients with TM and there was a trend towards greater
and fibrosis formation.
Beta thalassemia Major
Diamond Blackfan Anaemia
Correlation
P-value
Correlation
Biochemical Iron (mg/g DW)
0.547
0.001
-0.105
P-value
0.75
Ferritin (μg/L)
0.357
0.033
0.446
0.095
T2* liver (ms)
-0.430
0.011
0.184
0.64
Ferriscan (mg/g DW)
0.029
0.96
0.750
0.05
Y
Table 3. Correlation between iron accumulation measured by different techniques and
fibrosis formation in TM and DBA
Biochemical iron, ferritin level and MRI T2* had low, significant correlations with fibrosis in TM
patients and FerriScan in DBA patients.
Y
XY-FISH
Table 1. Patient characteristics
TM patients were significantly older and had received blood transfusions for a longer period of time reflecting the
different referral pattern for both diseases to our institution. TM patients are referred for specialist advice of
chelation treatment and consideration of bone marrow transplantation. DBA patients attend the specialist DBA clinic
(n=64) from the moment of confirmation or strong suspicion of diagnosis. However, DBA patients received
proportionately more chelation per transfusion than TM patients at the time of biopsy .
Iron Load Can Be Severe and Presents Early in DBA Patients Even When Receiving
Adequate Chelation Treatment
1Division
Background and Aims
Josu de la Fuente1,2, Yvonne Harrington1, Sonia Bonanomi1
of Paediatrics, St. Mary’s Hospital, London and 2Centre for Haematology, Imperial College London,
United Kingdom
Diamond Blackfan anaemia is an inherited bone marrow failure syndrome with haematological and systemic manifestations. The classical
presentation of the condition is development of anaemia in infancy which occurs in approximately 85-90% of the patients. Long-term 40% require
transfusions as they fail to maintain erythropoiesis at acceptable doses of steroids and as approximately only 10% of the patients go into
remission . Historical data has shown that transfusion dependent patients are at risk of significant morbidity and mortality and there is evidence
that despite of adequate chelation treatment, they have significantly higher iron load than other transfusion dependent anaemias.
Patient
Age
years
Ferritin
g/L
1
2.8
1112
4.5
1090
T2* heart
ms
T2* liver
ms
FerriScan
mg/g DW
2
2.7
3
15.8
29
30.3
32.6
1.7
43
37.6
33.3
0.8
4
15.5
116
5
4.1
4649
6
3.7
979
5.0
13.17
10.21
28.8
4.86
1201
12.5
7
7.1
780
12.5
8
39.0
2692
5.5
Methods
30
1.6
826
-
3.6
9.7
15.1
1835
53.7
1.4
26.8
4.8
978
9
6.3
10
29.2
36
11
3.0
1600
12
5.5
13
14
15
16
Twenty-nine patients are transfusion dependent, 11 steroid responsive, 7 are in remission, 9 have undergone a bone marrow transplant achieving
normal haemopoiesis and 3 have never developed anaemia of sufficient severity to warrant treatment. Three have deceased (two transfusion
dependent patients due to overwhelming sepsis and one following unrelated bone marrow transplantation). Twenty two transfusion dependent
patients have initiated chelation treatment: 19 patients (82.5%) are currently taking deferasirox and 3 (13.6%) continuous intravenous
desferrioxamine as intensification treatment. Transfusion dependent patients have had their iron load assessed by a combination of techniques:
ferritin, MRI T2*, FerriScan and liver biopsy (Table 2).
Results
Seventeen patients had severe hepatic iron load (LIC > 10 mg/g DW, maximum 38.6 mg/g DW): four before initiation of chelation treatment, 8
following chelation with desferrioxamine and 5 following deferasirox treatment. Seven of the patients had severe hepatic iron load (maximum
29.17 mg/g DW) despite of maintaining the ferritin < 1500 g/L with adequate chelation treatment following guidelines for thalassaemia (Figure 1).
Severe hepatic iron load was seen as early as in the second year of life (2 years 6 months LIC 38.6 mg/g DW). In patients with severe hepatic
iron load, significant reductions achieved with chelation treatment as measured by liver biopsy or FerriScan were not reflected in an increase in
T2* measurement until the treatment was advanced. In addition, FerriScan showed higher LIC values than liver biopsy in keeping with its ability to
provide an overall measurement not affected by fibrosis. Three patients had cardiac iron load (T2* < 20 ms) in childhood, including 2 below the
Male cell inof
bone
marrowwith
adherent
age of 6Male
years.
required intensification
chelation
continuous intravenous desferrioxamine, which was successful in all but
cell Seven
in bone patients
marrow before
culture
culture
one despite
of the use of 50 mg/kg/day.
CD45+ male cell
Vimentin+ male
cell
Conclusion
16
1.7
6.3
789
21
1.8
7.1
888
41
3
7.2
1003
25.4
3.4
7.2
754
5.7
3592
5.0
3063
5.7
1324
6.1
2140
3.5
7154
6.1
3958
17
4.1
2597
18
20.2
87
4.2
131
19
20
n=59
in utero
2
0 -12 weeks
36
61%
>3 -12 months
12
20.3%
1-5 years
6
10.1%
2
3.3%
3.4%
10-18 years
0
0%
>18 years
1
1.6%
Steroids
11
18.6%
Remission
7
11.8%
No treatment
3
5%
BMT
8
9.01
22.2
34.4
2.2
8.65
2.8
766
1993
1503
22
49.8
1.4
23
24
24.3
24.9
714
25.5
543
32
2.85
3
9.1
1897
10.1
25
26
6.2
10.27
5.19
16
1.43
17
2.4
33
6
Chelation
desferrioxamine
3
13.6%
deferasirox
19
82.5%
Table 1. Patient characteristics of Imperial College Healthcare DBA Cohort.
Age of presentation, treatment and chelation of DBA patients attending specialist DBA Clinic at St. Mary’s
Hospital in London.
Figure 2. Relationship between ferritin and LIC in patients
with severe hepatic iron load
Ferritin (g/L) in X axis and FerriScan or liver biopsy LIC (mg/g
DW) in Y axis
11
2278
4.1
354
8.4
357
27
7.1
28
3.3
29
3.3
30
3.7
522
31
6.8
940
9.7
653
32
12.8
692
33
9.5
1600
22
20
7.58
5.54
42
60
40.3
2.8
23
6
21
1.8
29.7
3.5
3.29
9.8
10.1
10.2
34
35
4.84
378
6.6
7.0
1280
7.6
1071
7.8
983
9.1
433
9.3
8.6
13.3
8.49
16.3
36
20
2.27
8.7
10.3
10.8
16.1
2500
17.4
3279
17.9
1332
9.6
10.1
1410
10.8
800
15.8
1083
13
3.6
24
2.8
30
6.1
43
4.6
2.12
1.92
37
16.2
16.5
38
39
40
2606
3.6
2428
38.6
4.29
4.3
2096
4.8
1095
4.9
2.5
355
8.7
5.9
15.9
1128
700
11.3
12.5
42
28.1
2.8
10.3
1.3
13.9
2.3
15
2.9
10.4
350
5.7
550
89.5
3.8
125
49.5
19.1
5.8
2659
18.9
1200
22.4
5.2
1586
8.3
738
8.8
610
11.3
545
27.3
1.6
29.4
2.29
XY-FISH
29.5
8.8
1.3
24.5
25.6
44
45
Y
1634
6.6
10.3
41
43
5.9
6.1
2.6
6.2
Y
4.38
3.04
5.5
4.9
1794
47
8.5
2143
43.5
2
9.1
48
37.5
213
53.4
7.2
2.6
49
6.7
108
50
4.9
581
51
21.3
528
7.6
1180
52
5%
16.2
8
7.45
1.2
3.3
7.5
35
5
4.2
40.8
2.2
7.5
Deceased
19.3
10.3
3.35
2.8
3.01
1.2
22.8
1.8
7.87
10.09
30
23.9
25.8
29.17
8.4
21
46
13.5%
22.6
52
6.4
In summary, iron overload is a significant clinical problem in patients with DBA, even when receiving adequate chelation treatment with current
Treatment
guidelines and at a young age. It cannot be recognised by measurement of ferritin only and it requires an algorithm that uses all available
Transfusions
29
49.1%
Age
at
presentation
techniques in an age appropriate manner from two years of age for its detection and management.
5-10 years
4.6
1189
Fifty nine patients with clinical and laboratory features consistent with Diamond Blackfan anaemia attend the specialist DBA clinic at St. Mary’s
Hospital (Table 1).Thirty-five patients (59.3%) had systemic features [the heart was involved in 17 patients (27.1%)], 7 patients (11.8%) had short
stature only and 17 patients (28.8%) no systemic abnormalities. We have identified a ribosomal protein gene mutation in 18 patients with a novel
approach [Abstract #2369] .
Liver biopsy LIC
mg/g DW
2.18
7.67
7.9
53
1.6
2736
54
8.8
55
2.0
4483
56
27.7
3385
57
8.17
29.2
1.4
1300
58
4.0
1700
59
1.7
8.5
2.4
16.3
25
2.3
9.3
17.3
Table 2. Assessment of iron load in transfusion dependent patients
Transfusion dependent patients monitored with ferritin, MRI T2* and
FerriScan
Patients with Diamond Blackfan Anaemia have abnormalities of cellular
and humoral immunity
Deena ISKANDER1, Yvonne HARRINGTON2, Irene ROBERTS1,2 , Anastasios KARADIMITRIS1, Josu DE LA
FUENTE1,2.
1. Centre for Haematology, Imperial College, London, UK.
2. Department of Paediatrics, Imperial College Healthcare NHS Trust , London, UK.
Total…
43
5 420
1
0
20
Numb
Figure 2 and table 2: Lymphocyte abnormalities in patients with DBA
Introduction
Diamond Blackfan Anaemia (DBA) is an inherited bone marrow failure syndrome characterised by
anaemia, physical anomalies and an increased risk of malignancy. Although the hallmark of DBA is
anaemia due to pure red cell aplasia, some patients exhibit additional cytopaenias, suggestive of a
more widespread defect in haemopoiesis. In addition, aberrant immunity has been reported1, but the
scope and precise nature of these immunological defects is yet to be elucidated.
Median age (range)
Patient characteristics
Fifty-nine patients with clinical and laboratory
features consistent with DBA attend the
.
specialist
clinic at St. Mary’s Hospital,
London.
Their
characteristics
are
summarised in table 1.
Age at presentation
Systemic abnormalities
8.8 yrs (1.1-40.9)
3-12 mo
12/59 (20.3%)
1-5 yrs
6/59 (10.1%)
4 (10.8)
59.7 (38-94.4)
5-10 yrs
2/59 (3.4%)
CD56+ NK cells
8 (21.2)
75.5 (36.7-95.6)
>18 yrs
1/59 (1.6%)
CD19+ B lymphocytes
12 (32.4)
75 (20.5-96)
All
35/59 (59.3%)
Cardiac
17/59 (28.8%)
Clinical information was obtained retrospectively from patients’ medical records and from the Imperial
College Healthcare Trust electronic data system. Lymphocyte subsets were characterised by flow
cytometry and age-specific normal ranges were defined as previously described.2 Serology to identify
antibodies against specific pathogens was performed using Enzyme-Linked Immunosorbent Assays.
Results
I. Infections are common in patients with DBA
Sepsis was the cause of death in 2/3 patients with DBA who
died - immunological investigations had not been performed
prior to death in these cases.
No…
Figure 1: History of infections in 37 patients with
DBA.
84 (35.1-94.6)
36/59 (61%)
Methods
n=37
Median of deficiency % of lower limit of normal for age (range)
7 (18.9)
2/59 (3.3%)
0-12 wks
In 3 of the 37 patients investigated, a severe but non-fatal
septic episode was reported: Salmonella gastroenteritis,
Clostridium difficile colitis and neonatal pneumonia. Recurrent
infections occurred in an additional 13 patients: respiratory
7/16 (54%), multisystem 3/16 (23%), otitis media 2/6 (15%)
and urinary 1/37 (8%).
2 of the 16 patients with infections had longstanding
II. B lymphocyte deficiency is the
most commonly
detected
neutropaenia
and another
2 immune
patients abnormality
were receiving
corticosteroids, but at low doses (0.15 and 0.12 mg/kg od).
Consistent lymphopaenia was found in 7/37 (18.9%) patients, unaccounted for by concurrent medical
conditions or drug therapy. Abnormalities in one or more subsets were identified in the 7 patients with
low total lymphocyte counts and in a further 10 patients with normal total lymphocytes counts. A low
B lymphocyte fraction was the most frequent abnormality, present in 12/37 (32.4%) patients (figure 2
and table 2).
Lymphocyte subset
CD45+ total lymphocytes (low side scatter)
CD3+ T lymphocytes
n-
No. deficient patients (%)
[total patients n = 37]
In utero
Immunological parameters were available for
Short stature
7/59 (11.8%)
37 of the patients. At the time of inclusion in
Table 1. Characteristics of patients with DBA at Imperial College Healthcare
the study, all patients were alive and the
Trust.
median age was 7.8 years (range 18 months
The male to female ratio was 1.1:1 and patients were from a broad range of ethnic backgrounds. A
to 40.4 years).
ribosomal protein gene mutation was known in 18/37 (48.6%) patients. Of the 37 patients, 5 were in
remission, 20 transfusion-dependent, 9 steroid- responsive, 2 managed with both steroids and blood
transfusions and 1 treated with allogeneic stem cell transplantation (immunological investigations were
undertaken pre-transplant).
1…
3… 2…
40
At…
III. Reduced immunoglobulins occur in patients with DBA
Four of the 12 patients with reduced B lymphocytes also exhibited a defect in immunoglobulins (IgM
and IgG2 deficiency in 1, IgG3 deficiency in 3). In total, low levels of at least 1 Ig isotype were
detected in 4/34 (11.8%) patients. An additional 5/32 (15.6%) patients showed a selective deficiency
in 1 of the 4 IgG subclasses. Importantly, these abnormalities were masked by normal total IgG
levels.
IV. Patients with DBA have suboptimal responses to vaccination
Corticosteroid therapy in DBA is delayed beyond infancy to allow administration of routine immunisations
including measles, mumps and rubella (MMR) and H. Influenza type b (Hib) and minimise
musculoskeletal side effects. We investigated specific antibodies against these pathogens as a marker
for immunity3, as shown in figure 3. Measurements were performed at varying time points postvaccination, but there was no correlation between age and response.
M
MR
42112 13
All
Hi
b
514
11
Op…
n=23
n=30
Figure 3: Immunity post-vaccination, indicated by unequivocally positive IgG against MMR and by Hib antibody titres.
V. Immune defects may be subclinical
A defect in lymphocytes and/or Igs was detected in 10/16 (62.5%) patients with infections. Conversely, in
9/21 (42.9%) patients, immune defects were observed in the absence of a history of infections.
Conclusions
 Infections are a major cause of mortality and morbidity in DBA.
 This is the first report detailing immunological defects in DBA.
 Combined deficiencies in lymphocyte subsets and immunoglobulins, alongside clinical infections,
are present in 5/37 (13.5%) patients.
 Suboptimal responses to vaccination are observed in many patients.
 Further work should explore mechanisms underlying the observed defects and correlation between
References
genotype and immunological phenotype.
Acknowledgements
Target Enrichment and High-Throughput Sequencing of 80 Ribosomal Protein Genes to
Identify Mutations Associated with Diamond-Blackfan Anaemia
Gareth Gerrard1*, Mikel Valgañón1, Hui En Foong1, Dalia Kasperaviciute2, Deena Iskander1, Laurence Game3, Michael Müller2,
Irene Roberts1, Timothy J Aitman2, Letizia Foroni1, Josu de la Fuente1, Anastasios Karadimitris1
1Centre
for Haematology, Faculty of Medicine, Imperial College London, UK; 2Imperial NIHR Biomedical Research Centre, Imperial College London, UK; 3Genomics Laboratory,
Clinical Sciences Centre, Imperial College London, UK. *g.gerrard@imperial.ac.uk. Work funded by AHSC/IHBRC
Abstract
Methods
Diamond-Blackfan anaemia (DBA) is a rare congenital stem cell disorder associated with
inactivating mutations in ribosomal protein (RP) genes, causing defects in erythroid
progenitor and precursor cell development. Of the 80 or so RP genes, loss of function
mutations in 10 have been definitively associated with DBA. We used high-throughput
sequencing to screen all 80 RP genes in 20 DBA patient samples (3 of which were
controls) and found loss-of-function mutations in 15/17 (88.2%) of the test samples.
The coordinates for all 80 RP genes were used to generate custom SureSelect hybridisation
capture baits via the Agilent web portal. 3µg DNA was extracted from 20 PB samples (including 2
controls with known mutations; 6 members from 3 family groups: a mother-daughter pair, a sibling
pair and another sibling pair where one sibling was non-DBA and included as a control). Covaris
sonication was performed and the DNA fragments were hybridised with the capture baits for 48h.
After barcoded adapter ligation, the libraries were quantitated by qPCR and pooled into 2 runs of
10. The sequencing was performed on an Illumina MiSeq using 150bp paired-end chemistry. The
reads were aligned to the hg19 reference using BWA and the variant calls made using GATK;
ANNOVAR was used for functional annotations of the variants. Pipelines for both SNVs/indels
and large deletions/insertions were implemented, plus a separate analysis for RPS17 because of
its duplicate status. Mutation validation was by Sanger sequencing on an ABI 3500.
Introduction
Single nucleotide variations (SNV), small inversions/deletions (indels) and copy number
variations (CNV) have been found in 10 RP genes in 25-35% of DBA patients, meaning
that around 40% have no identifiable mutations (at least by conventional screening).
However, given that all mutations in DBA characterised so far (with the exception of 2
cases with GATA1 mutations) affect RP genes, it is likely that mutations in one of the 80
RP genes will be eventually identified in a significant proportion of patients. Current
screening methods are based on Sanger sequencing on a per-exon/per-gene basis, with
the associated time, labour and cost restrictions. We therefore aimed to evaluate highthroughput sequencing technology, including a bespoke target enrichment platform, to
screen all 80 known RP genes to facilitate rapid, cost-effective identification of DBA
associated mutations (Figure 1).
FIGURE 1
Results
Loss of function mutations were detected in RP genes in 17 of the 20 samples, including the 2
positive controls (Table 1). Of the 3 where no definitive mutation was seen, 1 was an unaffected
sibling. All mutations were in RP genes previous described as being involved in DBA, although 7
affected novel codons. No verifiable large deletions/insertions were seen. FastQC indicated good
quality sequencing metrics and all variations were subsequently validated by Sanger sequencing.
TABLE 1. Gene variations flagged as loss of function and validated by Sanger
sequencing.
Target enrichment (Agilent
SureSelect) and HighThroughput Sequencing
(Illumina MiSeq) workflow
for the screening of all 80
ribosomal protein genes in
patients with DBA
Summary
The authors confirm that there are no relevant conflicts of interest to disclose
Using custom designed target enrichment and high-throughput benchtop sequencing technology, mutations were
found in 17/20 samples and of the 17 test samples, 15 were found to have mutations in RP genes associated
with DBA (88.2%). Consequently, we are now optimising this approach for use as our primary screening
platform.
Data now in press…
10 Identified DBA associated RP Genes
Mutations are mostly SNVs and indels, but large deletions & insertion are also seen
RPS26
RPS24
RPS7
RPS17
RPL26
RPS35a
RPL11
RPS10
Unknown
RPL5
RPS19
= 7 genes in current molecular screen
Why Screen?
 Accurate diagnosis
 Reproductive counselling
 Exclude silent DBA from related BMT
donors
 Establish Genotype-Phenotype link
 Elucidate pathophysiology
Current
DBA
Screening
Pipeline
Measure & QC
Peripheral Blood
Extract DNA
RPS19
RPL5
RPL11
RPS24
RPS17
RPL35a
RPS7
Sanger Sequence
PCR target gene exons
Why Next Gen Seq (NGS)?
 Very high throughput
 Can look at all +80 targets at once
 Can multiplex many samples at once
 Potential to pick up large (allele-loss)
deletions & insertions
 Cost effective per-gene / per-sample
Next Generation
Sequencing Workflow
Genomic DNA
10 patient-parent pairs
Data analysis
Sanger seq validation
Target Enrichment
Fragment DNA
High-throughput
Sequencing
Hybridise and capture Ribosomal
Protein Gene DNA
including exons, introns, &
regulatory regions
Library quant, pool, clean up and
cluster generation
Results Summary
Gene
RPL5
RPS26
RPL11
RPS17
RPS7
RPS10
RPS24
RPS19
Tot Mut
NoMut
n= (17)
5(4)
3
2
2(1)
1
1
1
0
15(13)
2
%
29.4%
17.6%
11.8%
11.8%
5.9%
5.9%
5.9%
0.0%
88.2%
11.8%
Type
3(2) SG/2 FSD
SG/FSI/SL
FSD/FSI
2(1) SG
SSD
SG
SG
SG= Stop Gain SNV (Nonsense); FSD= Frame-shift Deletion; FSI= Frame-shift Insertion;
SL= Start Loss SNV (Missense); SSD= Splice Site Defect
Next Steps…..
Optimise NGS for upfront DBA screening
Agilent
Haloplex
Capture
+ MLPA Assay for validation of large deletions
Ion Torrent High
Throughput Seq
o70%
of cases now have a known
genetic basis
oCellular
models have shown
deficiency of RPS19 leads to a
defect in erythroid differentiation
oThought
to be between the CFUe and proerythroblast stage

Difficult to undertake DBA studies due to the
rarity of the disease and thus difficulties in
obtaining sufficient samples
Therefore….
 Stage of maturation is poorly characterised
 Struggle to make a diagnosis morphologically
 Cell morphology in the myeloid, lymphoid and
megakaryocytic cell lineages is poorly
documented
St Marys Hospital London has the largest cohort of
DBA patients worldwide
72 DBA patients currently registered at St
Mary’s Hospital
31 patients have had a bone marrow aspirate
29 samples suitable for analysis (n=29)
10 normal paediatric controls



500 cell differential count
500 cell myeloid:erythroid differential
conducted blind
50 megakaryocyte assessment also
conducted blind
Reduced erythrocyte % in DBA patients
The Erythroid
Lineage
Non lobulated
Normal lobulation



Significant reductions in the erythroid lineage
in concordance with DBA diagnosis
Demonstrated a stage of erythroid maturation
arrest at the level of the proerythroblast
RPL5/RPL11 positive patients have a greater
proportion of hypolobulated and non
lobulated megakaryocytes
Next steps………….
• Registry
• Genomewide approach
• Whole bone marrow study