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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

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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


    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

Katharine Evans1, Robert Goldin2 and Josu de la Fuente1,3

1Division of Haematology, Imperial College London London, 2Division of Histopathology, Imperial College London and 3Division of Paediatrics, St. Mary’s Hospital, London, United Kingdom

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).


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).


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) and could be due to a time lag between iron accumulation and fibrosis formation, particularly as the DBA patients were younger and had had shorter follow up (Table 2). Biochemical iron, ferritin level and MRI T2* had low, significant correlations with fibrosis in TM patients (0.547, p=0.001; 0.357, p=0.033; -0.430, p=0.011, respectively) and FerriScan in DBA patients (0.75, p=0.05) (Table 3).


In conclusion, DBA patients have a higher rate of iron accumulation with a trend to higher hepatic fibrosis.

Male cell in bone marrow adherent culture

Male cell in bone marrow before culture

CD45+ male cell

Vimentin+ male cell


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.



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 .

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.

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.

Iron Load Can Be Severe and Presents Early in DBA Patients Even When Receiving Adequate Chelation Treatment

Josu de la Fuente1,2, Yvonne Harrington1, Sonia Bonanomi1

1Division of Paediatrics, St. Mary’s Hospital, London and 2Centre for Haematology, Imperial College London,

United Kingdom

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 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.


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] .

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).


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 age of 6 years. Seven patients required intensification of chelation with continuous intravenous desferrioxamine, which was successful in all but one despite of the use of 50 mg/kg/day.


In summary, iron overload is a significant clinical problem in patients with DBA, even when receiving adequate chelation treatment with current guidelines and at a young age. It cannot be recognised by measurement of ferritin only and it requires an algorithm that uses all available techniques in an age appropriate manner from two years of age for its detection and management.

Male cell in bone marrow adherent culture

Male cell in bone marrow before culture

CD45+ male cell

Vimentin+ male cell




Figure 2.Relationship between ferritin and LIC in patients with severe hepatic iron load

Ferritin (mg/L) in X axis and FerriScan or liver biopsy LIC (mg/g DW) in Y axis

Table 2.Assessment of iron load in transfusion dependent patients

Transfusion dependent patients monitored with ferritin, MRI T2* and FerriScan

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.

Patients with Diamond Blackfan Anaemia have abnormalities of cellular and humoral immunity


  • Centre for Haematology, Imperial College, London, UK.

  • Department of Paediatrics, Imperial College Healthcare NHS Trust , London, UK.

V. Immune defects may be subclinical

Figure 2 and table 2: Lymphocyte abnormalities in patients with DBA

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.

Immunological parameters were available for 37 of the patients. At the time of inclusion in the study, all patients were alive and the median age was 7.8 years (range 18 months to 40.4 years).


Table 1. Characteristics of patients with DBA at Imperial College Healthcare Trust.

The male to female ratio was 1.1:1 and patients were from a broad range of ethnic backgrounds. A 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).

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 post-vaccination, but there was no correlation between age and response.

Figure 3: Immunity post-vaccination, indicated by unequivocally positive IgG against MMR and by Hib antibody titres.

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.


Patient characteristics

Figure 1: History of infections in 37 patients with DBA.



IV. Patients with DBA have suboptimal responses to vaccination



II. B lymphocyte deficiency is the most commonly detected immune abnormality


I. Infections are common in patients with DBA


  • 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 genotype and immunological phenotype.

III. Reduced immunoglobulins occur in patients with DBA

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.

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.

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).

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.

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.

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 neutropaenia and another 2 patients were receiving corticosteroids, but at low doses (0.15 and 0.12 mg/kg od).





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 Karadimitris11Centre 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. * Work funded by AHSC/IHBRC


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). Covarissonication 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 IlluminaMiSeq 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.


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 high-throughput 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).


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.


Target enrichment (Agilent SureSelect) and High-Throughput Sequencing (Illumina MiSeq) workflow for the screening of all 80 ribosomal protein genes in patients with DBA


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.

The authors confirm that there are no relevant conflicts of interest to disclose

TABLE 1. Gene variations flagged as loss of function and validated by Sanger sequencing.

Data now in press…

10 Identified DBA associated RP Genes

Mutations are mostly SNVs and indels, but large deletions & insertion are also seen

= 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

Extract DNA

Peripheral Blood







PCR target gene exons


Sanger Sequence

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

Target Enrichment

Hybridise and capture Ribosomal Protein Gene DNA

including exons, introns, & regulatory regions

Genomic DNA

10 patient-parent pairs

Fragment DNA

High-throughput Sequencing

Library quant, pool, clean up and cluster generation

Data analysis

Sanger seq validation

Results Summary

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


Ion Torrent High Throughput Seq

+ MLPA Assay for validation of large deletions

Current Understanding?

  • 70% of cases now have a known genetic basis

  • Cellular models have shown deficiency of RPS19 leads to a defect in erythroid differentiation

  • Thought to be between the CFU-e and proerythroblast stage

Why Study Bone Marrow Arrest?

  • Difficult to undertake DBA studies due to the rarity of the disease and thus difficulties in obtaining sufficient samples


  • 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

Materials and Methods

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

Megakaryocyte Morphology

Non lobulated

Normal lobulation

Megakaryocyte Lineage


  • 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

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