Pancreatic lipomatosis in Diamond-Blackfan anemia: The importance of genetic testing in bone marrow failure disorders

Abstract Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia caused by a functional haploinsufficiency of genes encoding for ribosomal proteins. Among these genes, ribosomal protein S19 (RPS19) is mutated most frequently. Generation of animal models for diseases like DBA is challenging because the phenotype is highly dependent on the level of RPS19 down-regulation. We report the generation of mouse models for RPS19-deficient DBA using transgenic RNA interference that allows an inducible and graded down-regulation of Rps19. Rps19-deficient mice develop a macrocytic anemia together with leukocytopenia and variable platelet count that with time leads to the exhaustion of hematopoietic stem cells and bone marrow failure. Both RPS19 gene transfer and the loss of p53 rescue the DBA phenotype implying the potential of the models for testing novel therapies. This study demonstrates the feasibility of transgenic RNA interference to generate mouse models for human diseases caused by haploinsufficient expression of a gene.

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Detection of Mutations in Inherited Bone Marrow Failure and Myelodysplastic Syndrome Genes Using Genomic Capture and Massively Parallel Sequencing in Clinical Diagnostics

Blood ◽

10.1182/blood.v128.22.1507.1507 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1507-1507

Author(s):

Siobán B. Keel ◽

Tom Walsh ◽

Colin Pritchard ◽

Akiko Shimamura ◽

Mary-Claire King ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Genetic Testing ◽

Hematopoietic Stem Cell ◽

Copy Number ◽

Bone Marrow Failure ◽

Copy Number Variants ◽

Donor Selection ◽

Hematopoietic Stem ◽

Pathogenic Mutations

Abstract Accurate and timely diagnosis of inherited bone marrow failure (BMF) and myelodysplastic syndromes (MDS) ensures appropriate clinical management. The correct diagnosis allows appropriate monitoring for both hematopoietic (i.e. clonal evolution and progressive marrow failure) and extra-hematopoietic complications, informs the timing of hematopoietic stem cell transplant, donor selection and transplant regimen planning, and ensures appropriate genetic counseling of family members. Substantial phenotypic overlap among these disorders and the variable expressivity within syndromes complicate their diagnosis based purely on physical exam and standard laboratory testing and provide the rationale for comprehensive genetic diagnostic testing. We report here our initial one-year experience utilizing a targeted capture assay of known inherited BMF/MDS genes for clinical diagnostic purposes at the University of Washington. The assay sequences all exons and 20 base pairs of intronic sequence flanking each exon, as well as several regulatory and intronic regions of specific genes containing known pathogenic variants of 85 known inherited BMF/MDS genes (Zhang M. et al. Haematologica 2016). Between June 2015 and July 2016, 81 individual patients were referred for clinical testing (median age: 15 years-old, range: 0.6-76 years-old). For all samples evaluated, median coverage across the 383kb targeted region was 1887X. This depth of coverage enabled identification of all classes of mutations, including point mutations, small indels, copy number variants, and genomic rearrangements. Pathologic mutations in known inherited BMF/MDS genes were identified in 12 of 82 (14.6%) individuals (median age 13 years-old, range: 1.25-43 years-old) across a broad number of genes and of multiple classes including copy number variants (Table). Among the twelve patients with pathogenic mutations in inherited BMF/MDS genes, genetic testing was consistent with the prior clinical diagnoses of eight patients, including two Fanconi anemia patients subtyped as complementation group A, one of whom demonstrated reversion to wild-type resulting in mosaicism in the peripheral blood. Importantly, four patients carried no specific inherited BMF/MDS diagnosis prior to testing and were found to have pathogenic mutations in RPS10, RTEL1 and RUNX1 (ID 005, 008, 009, 010), suggesting additional diagnostic value to a multiplexed genetic approach in the clinical setting. Detailed clinical information was available for nine of the patients diagnosed with pathogenic mutations, two of whom have or will undergo a sibling or haploidentical hematopoietic stem cell transplantation (009 and 012, respectively) and thus genetic testing informed donor selection. To improve diagnostic accuracy, we are now updating the capture design to include newly discovered inherited BMF/MDS genes and intronic regions to optimize copy number variant detection. We are additionally pursuing CLIA-certified RNA analyses to characterize whether several variants bioinformatically predicted to affect splicing are functionally deleterious. Next-generation sequencing for mutations involved in hereditary marrow failure and MDS may also become increasingly important in the context of precision-medicine in which germline mutations are unexpectedly identified in somatic testing. Disclosures No relevant conflicts of interest to declare.

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Enforced expression of RPS19 derived from human elongation factor 1A short (EFS) promoter corrects the anemia and lethal bone marrow failure in mouse model for RPS19-deficient diamond-blackfan anemia

Experimental Hematology ◽

10.1016/j.exphem.2015.06.106 ◽

2015 ◽

Vol 43 (9) ◽

pp. S59

Author(s):

Shubhranshu Debnath ◽

Pekka Jaako ◽

Kavitha Siva ◽

Ute Modlich ◽

Axel Schambach ◽

...

Keyword(s):

Bone Marrow ◽

Mouse Model ◽

Bone Marrow Failure ◽

Elongation Factor ◽

Diamond Blackfan Anemia ◽

Human Elongation Factor

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Case Report of an Adolescent Male With Unexplained Pancytopenia:GATA2-Associated Bone Marrow Failure and Genetic Testing

Global Pediatric Health ◽

10.1177/2333794x17744947 ◽

2017 ◽

Vol 4 ◽

pp. 2333794X1774494 ◽

Cited By ~ 1

Author(s):

Lauren Azevedo ◽

Allison Jay ◽

Adonis Lorenzana ◽

Sioban Keel ◽

Roshini S. Abraham ◽

...

Keyword(s):

Bone Marrow ◽

Case Report ◽

Genetic Testing ◽

Bone Marrow Failure ◽

Adolescent Male

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Gene Therapy Corrects the Lethal Bone Marrow Failure in a Mouse Model for RPS19-Deficient Diamond-Blackfan Anemia

Blood ◽

10.1182/blood.v120.21.513.513 ◽

2012 ◽

Vol 120 (21) ◽

pp. 513-513

Author(s):

Pekka Jaako ◽

Shubhranshu Debnath ◽

Karin Olsson ◽

Axel Schambach ◽

Christopher Baum ◽

...

Keyword(s):

Gene Therapy ◽

Bone Marrow ◽

Mouse Model ◽

Progenitor Cells ◽

Bone Marrow Cells ◽

Bone Marrow Failure ◽

Hematopoietic Stem ◽

Diamond Blackfan Anemia ◽

Stem And Progenitor Cells

Abstract Abstract 513 Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia associated with physical abnormalities and predisposition to cancer. Mutations in genes that encode ribosomal proteins have been identified in approximately 60–70 % of the patients. Among these genes, ribosomal protein S19 (RPS19) is the most common DBA gene (25 % of the cases). Current DBA therapies involve risks for serious side effects and a high proportion of deaths are treatment-related underscoring the need for novel therapies. We have previously demonstrated that enforced expression of RPS19 improves the proliferation, erythroid colony-forming potential and differentiation of patient derived RPS19-deficient hematopoietic progenitor cells in vitro (Hamaguchi, Blood 2002; Hamaguchi, Mol Ther 2003). Furthermore, RPS19 overexpression enhances the engraftment and erythroid differentiation of patient-derived hematopoietic stem and progenitor cells when transplanted into immunocompromised mice (Flygare, Exp Hematol 2008). Collectively these studies suggest the feasibility of gene therapy in the treatment of RPS19-deficient DBA. In the current project we have assessed the therapeutic efficacy of gene therapy using a mouse model for RPS19-deficient DBA (Jaako, Blood 2011; Jaako, Blood 2012). This model contains an Rps19-targeting shRNA (shRNA-D) that is expressed by a doxycycline-responsive promoter located downstream of Collagen A1 gene. Transgenic animals were bred either heterozygous or homozygous for the shRNA-D in order to generate two models with intermediate or severe Rps19 deficiency, respectively. Indeed, following transplantation, the administration of doxycycline to the recipients with homozygous shRNA-D bone marrow results in an acute and lethal bone marrow failure, while the heterozygous shRNA-D recipients develop a mild and chronic phenotype. We employed lentiviral vectors harboring a codon-optimized human RPS19 cDNA driven by the SFFV promoter, followed by IRES and GFP (SFFV-RPS19). A similar vector without the RPS19 cDNA was used as a control (SFFV-GFP). To assess the therapeutic potential of the SFFV-RPS19 vector in vivo, transduced c-Kit enriched bone marrow cells from control and homozygous shRNA-D mice were injected into lethally irradiated wild-type mice. Based on the percentage of GFP-positive cells, transduction efficiencies varied between 40 % and 60 %. Three months after transplantation, recipient mice were administered doxycycline in order to induce Rps19 deficiency. After two weeks of doxycycline administration, the recipients transplanted with SFFV-RPS19 or SFFV-GFP control cells showed no differences in blood cellularity. Remarkably, at the same time-point the recipients with SFFV-GFP homozygous shRNA-D bone marrow showed a dramatic decrease in blood cellularity that led to death, while the recipients with SFFV-RPS19 shRNA-D bone marrow showed nearly normal blood cellularity. These results demonstrate the potential of enforced expression of RPS19 to reverse the severe anemia and bone marrow failure in DBA. To assess the reconstitution advantage of transduced hematopoietic stem and progenitor cells with time, we performed similar experiments with heterozygous shRNA-D bone marrow cells. We monitored the percentage of GFP-positive myeloid cells in the peripheral blood, which provides a dynamic read-out for bone marrow activity. After four months of doxycycline administration, the mean percentage of GFP-positive cells in the recipients with SFFV-RPS19 heterozygous shRNA-D bone marrow increased to 97 %, while no similar advantage was observed in the recipients with SFFV-RPS19 or SFFV-GFP control bone marrow, or SFFV-GFP heterozygous shRNA-D bone marrow. Consistently, SFFV-RPS19 conferred a reconstitution advantage over the non-transduced cells in the bone marrow. Furthermore, SFFV-RPS19 reversed the hypocellular bone marrow observed in the SFFV-GFP heterozygous shRNA-D recipients. Taken together, using mouse models for RPS19-deficient DBA, we demonstrate that the enforced expression of RPS19 rescues the lethal bone marrow failure and confers a strong reconstitution advantage in vivo. These results provide a proof-of-principle for gene therapy in the treatment of RPS19-deficient DBA. Disclosures: No relevant conflicts of interest to declare.

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Pearson Marrow Pancreas Syndrome In a Cohort Of Diamond Blackfan Anemia Patients

Blood ◽

10.1182/blood.v122.21.1226.1226 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1226-1226

Author(s):

Katelyn E Gagne ◽

Roxanne Ghazvinian ◽

Daniel Yuan ◽

Rebecca L. Zon ◽

Kelsie Storm ◽

...

Keyword(s):

Bone Marrow ◽

Bone Marrow Failure ◽

Pancreatic Insufficiency ◽

Diagnostic Evaluation ◽

Clinical Samples ◽

Study Cohort ◽

Deletion Mapping ◽

Deletion Event ◽

Diamond Blackfan Anemia ◽

Mtdna Deletions

Abstract Pearson marrow pancreas syndrome (PS) is a congenital multisystem disorder characterized by sideroblastic anemia, pancreatic insufficiency, metabolic acidosis, and other defects, and is caused by mitochondrial DNA (mtDNA) deletions. Diamond Blackfan anemia (DBA) is a congenital hypoproliferative anemia with associated physical malformations, and in which mutations in ribosomal protein (RP) genes and GATA1 have been implicated. The clinical presentation of both of these bone marrow failure (BMF) syndromes shares several features including early onset of severe anemia, sporadic genetic inheritance, variable penetrance and manifestations, and episodes of spontaneous hematologic improvement. PS is less frequently occurring than DBA, with estimated incidences of < 1/1,000,000 versus 1/100,000 respectively, and therefore less often encountered by hematologists. We hypothesized that some patients in whom the leading clinical diagnosis is DBA actually have PS. To test this hypothesis, we retrospectively evaluated DNA samples from a cohort of patients that were submitted to a research study for DBA genetic testing. The study cohort consists of clinical samples and/or data from 362 patients, with a primary inclusion criterion of known or suspected congenital anemia. Prior genetic studies from this cohort have yielded the novel identification or confirmation of mutations and deletions in several genes implicated in DBA (e.g. RP genes, GATA1), which are to date identifiable in 175/362 samples (48%), a proportion consistent with that found in independent DBA registries. We screened peripheral blood DNA samples available from 173 genetically uncharacterized patients using a long PCR strategy, and found that 8 samples (4.6%) contained large mtDNA deletions. Deletion mapping and Southern blot analysis on DNA from these 8 patients confirmed the presence of a single deletion event within each patient, ranging in size from 2.3 - 7.0 kb of the 16.6 kb mitochondrial genome, existing as monomer or multimer mtDNA species, and in a proportion ranging from 55-80% of total mtDNA, all of which are consistent with the molecular diagnosis of PS. Follow-up with referring providers in the 1 month to 8 year time span since sample submission revealed that 2 of the 8 patients (25%) were subsequently diagnosed with PS. Of the remaining 6 undiagnosed patients, 2 had died from complications of bone marrow transplantation, performed for worsening cytopenias and concern for myelodysplasia; one patient died from bacterial sepsis; and 3 were alive with the provisional diagnosis of DBA. One of the 3 patients had become transfusion-independent. Review of bone marrow examinations revealed that the pathological hallmarks of ringed sideroblasts and/or vacuolization of precursors described in PS were inconsistently present or reported in the diagnostic evaluation. We conclude that PS is frequently overlooked in the diagnostic evaluation of children with congenital anemia. Establishing the diagnosis of PS, as distinct from DBA and other BMF disorders, holds important implications for patient management and family counseling. mtDNA deletion testing should be performed in the initial genetic evaluation of all patients with congenital anemia. Disclosures: Szczepanski: Octapharma AG: Investigator Other.

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Novel and Known Ribosomal Causes of Diamond-Blackfan Anemia Identified through Comprehensive Genomic Characterization

Blood ◽

10.1182/blood.v128.22.1495.1495 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1495-1495 ◽

Cited By ~ 1

Author(s):

Payal Khincha ◽

Lisa Mirabello ◽

Steven R Ellis ◽

Neelam Giri ◽

Seth Brodie ◽

...

Keyword(s):

Genetic Testing ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Pathogenic Variant ◽

Comparative Genomic ◽

Rrna Processing ◽

Nonsynonymous Variant ◽

Germline Mosaicism ◽

Diamond Blackfan Anemia ◽

Pathogenic Variants

Abstract Introduction: Diamond-Blackfan anemia (DBA) is a rare inherited bone marrow failure syndrome (IBMFS) characterized by erythroid hypoplasia. It is associated with a number of congenital anomalies and a high risk of developing specific cancers. DBA is caused by germline mutations or deletions in genes affecting ribosomal biogenesis and function, with autosomal dominant or X-linked recessive patterns of inheritance. The most commonly mutated gene is RPS19, seen in approximately 25% of patients. About 45% of DBA families have no known disease-causing pathogenic variant. Methods: Affected and unaffected individuals from families with DBA were ascertained through the IRB-approved NCI IBMFS retrospective/prospective cohort study (ClinicalTrials.gov Identifier: NCT00027274). Study participants completed detailed family and medical history questionnaires, medical records were reviewed, and a subset of families underwent clinical evaluations at the NIH Clinical Center. DBA patients enrolled prior to 2014 underwent routine clinical mutation testing for the established DBA genes; beginning in 2014, DBA patient samples (buccal and blood DNA) were evaluated by whole exome sequencing (WES) for mutation identification. We incorporated WES with deletion analyses and copy number variant (CNV) assessment to uncover the genetic changes causative of DBA. Deletion analyses performed included SNP genotyping and array comparative genomic hybridization. Functional effects of the genetic variants were proven by pre-rRNA processing defect analysis by Northern blot. Controls for functional studies were healthy mutation-negative individuals from the IBMFS study. Results: Genetic testing information was available in 61 of the 87 families with DBA enrolled in the IBMFS study. Thirty-five of the 61 families did not have a known genetic cause at enrollment. Our combined approach of WES, deletion and CNV analyses identified the causative pathogenic variant in 18 of the 35 (51%) uncharacterized DBA families. We discovered pathogenic variants in two previously undescribed genes in two DBA families. One family had a nonsynonymous variant (p.K77N) in RPL35; the second family had a nonsynonymous variant (p. L51S) in RPL18. Both of these variants result in characteristic pre-rRNA processing defects. Our analyses also uncovered germline mosaic deletions in known DBA genes in both buccal and blood cells of two patients from two different families. One was a 1.8 Mb mosaic deletion in chromosome 15 including RPS17; the other was a large 2.5 Mb mosaic deletion on chromosome 3 including RPL35A. In addition to these findings, we found variants in previously known DBA-associated ribosomal genes in 14 of the 35 families. We further evaluated the genomic characteristics of the entire DBA cohort. Pathogenic variants in ribosomal DBA genes were found in a total of 44 of the 61 families (72%) on whom genetic testing information and/or biospecimens were available. RPS19 was the most frequently mutated gene and accounted for 36% of families, followed by RPL35A and RPS26, accounting for 14% and 11% each, respectively. Notably, 30% of the variation in disease-causing genes in our cohort was due to a single copy or mosaic gene deletion. We had complete parental testing and inheritance information on 23 (52%) of the 44 families whose gene was identified. Ten of the 23 (43%) had an inherited mutation and 13 (57%) had a de novo change in the causative gene (both parents were negative for the affected child's disease-associated mutation). At this time, 17 of 61 families tested (28%) do not have a characterized disease-associated mutation. Conclusion: This efficient comprehensive genomic approach was the basis for our discovery of two novel causes of DBA, characterization of ribosomal gene deletions not previously described to be disease-associated, and of DBA-associated germline mosaicism. We identified the disease-associated mutations in 51% (18 of 35) of our families without a known genetic cause of DBA. A total of 74% (44 of 61) of our families are now genetically characterized. Our comprehensive approach appears to provide more genomic information than other methods since pathogenic variants of DBA genes have been reported previously in about 55% of DBA patients. Disclosures No relevant conflicts of interest to declare.

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Dexamethasone and lenalidomide have distinct functional effects on erythropoiesis

Blood ◽

10.1182/blood-2010-11-318543 ◽

2011 ◽

Vol 118 (8) ◽

pp. 2296-2304 ◽

Cited By ~ 52

Author(s):

Anupama Narla ◽

Shilpee Dutt ◽

J. Randall McAuley ◽

Fatima Al-Shahrour ◽

Slater Hurst ◽

...

Keyword(s):

Bone Marrow ◽

Expression Profiles ◽

Bone Marrow Failure ◽

Gene Expression Profiles ◽

Absolute Number ◽

Red Blood Cell Transfusion ◽

Erythroid Progenitor ◽

Diamond Blackfan Anemia ◽

Cd34 Cells

Abstract Corticosteroids and lenalidomide decrease red blood cell transfusion dependence in patients with Diamond-Blackfan anemia (DBA) and myelodysplastic syndrome (MDS), respectively. We explored the effects of dexamethasone and lenalidomide, individually and in combination, on the differentiation of primary human bone marrow progenitor cells in vitro. Both agents promote erythropoiesis, increasing the absolute number of erythroid cells produced from normal CD34+ cells and from CD34+ cells with the types of ribosome dysfunction found in DBA and del(5q) MDS. However, the drugs had distinct effects on the production of erythroid progenitor colonies; dexamethasone selectively increased the number of burst-forming units-erythroid (BFU-E), whereas lenalidomide specifically increased colony-forming unit-erythroid (CFU-E). Use of the drugs in combination demonstrated that their effects are not redundant. In addition, dexamethasone and lenalidomide induced distinct gene-expression profiles. In coculture experiments, we examined the role of the microenvironment in response to both drugs and found that the presence of macrophages, the central cells in erythroblastic islands, accentuated the effects of both agents. Our findings indicate that dexamethasone and lenalidomide promote different stages of erythropoiesis and support the potential clinical utility of combination therapy for patients with bone marrow failure.

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Acquired ribosomopathies in leukemia and solid tumors

Hematology ◽

10.1182/asheducation-2017.1.716 ◽

2017 ◽

Vol 2017 (1) ◽

pp. 716-719 ◽

Cited By ~ 6

Author(s):

Adrianna Vlachos

Keyword(s):

Bone Marrow ◽

Ribosomal Protein ◽

Solid Tumors ◽

Ribosomal Proteins ◽

Bone Marrow Failure ◽

Ribosomal Protein Gene ◽

Small Subunit ◽

Gene Encoding ◽

Bone Marrow Failure Syndrome ◽

Diamond Blackfan Anemia

AbstractA mutation in the gene encoding the small subunit-associated ribosomal protein RPS19, leading to RPS19 haploinsufficiency, is one of the ribosomal protein gene defects responsible for the rare inherited bone marrow failure syndrome Diamond Blackfan anemia (DBA). Additional inherited and acquired defects in ribosomal proteins (RPs) continue to be identified and are the basis for a new class of diseases called the ribosomopathies. Acquired RPS14 haploinsufficiency has been found to be causative of the bone marrow failure found in 5q– myelodysplastic syndromes. Both under- and overexpression of RPs have also been implicated in several malignancies. This review will describe the somatic ribosomopathies that have been found to be associated with a variety of solid tumors as well as leukemia and will review cancers in which over- or underexpression of these proteins seem to be associated with outcome.

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