A Small Nucleolar RNA Linked to Diamond-Blackfan Anemia

Abstract Diamond-Blackfan anemia (DBA) is a rare congenital erythroblastopenia associated with haploinsufficiency of more than 15 ribosomal protein genes. We identified in the French registry a DBA patient exhibiting a large heterozygous deletion in ribosomal protein gene RPSA, making it a new candidate as a DBA-associated gene. The deletion was confirmed by qPCR using specific intron/exon primers that could discriminate the RPSA gene from pseudo-genes. Consistent loss of function of this allele, RPSA mRNAs were underrepresented in erythroid cultures and in lymphoblastoid cells (LCLs) derived from the patient. Additionally, an important decrease in erythroid proliferation was observed in erythroid progenitor and precursor cells derived from the patient's bone marrow CD34+ cells, together with a delay in erythroid differentiation, the activation of the p53 pathway, and a G0/G1 cell cycle arrest, which are regular features of DBA patient cells. Similar to other RP genes linked to DBA, interruption of RPSA expression in zebrafish embryos strongly affected development of the head and erythroid cell differentiation, supporting its relevance as a candidate DBA gene. Northern blot analysis also revealed impaired pre-ribosomal RNA (pre-rRNA) processing in patient LCLs compared to controls. But unexpectedly, the observed pre-rRNA profile was clearly different from that observed in HeLa cells upon RPSA knockdown and suggested a dysfunction of pre-rRNA processing upstream of that produced by a shortage of RPSA. We noticed that the RPSA gene hosts the intron-encoded H/ACA snoRNAs snoRA6 and snoRA62, the latter being included in the deletion. Q-RT-PCR analysis of patient cells showed decreased levels of snoRA62 when compared to controls and to other RP-mutated DBA patients. Strikingly, knocking out the snoRNA62 coding sequence in human haploid HAP1 cells with Crispr/Cas9 resulted in a pre-rRNA maturation defect similar to that observed in the RPSA+/mut DBA patient, thereby revealing a role for snoRA62 in human pre-rRNA processing. These data strongly suggest that, in addition to RPSA haploinsufficiency, the primum movens of DBA in this particular patient includes the deletion of the intronic snoRA62 hosted in the RPSA gene. To our knowledge, this is the first time that a snoRNA is linked to DBA. We will discuss this ongoing work in light of our most recent results in cells and zebrafish models. Disclosures No relevant conflicts of interest to declare.

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Insights Into Diagnosis and Etiology of Diamond Blackfan Anemia by Analysis of Pre-rRNA Processing

Blood ◽

10.1182/blood.v120.21.3476.3476 ◽

2012 ◽

Vol 120 (21) ◽

pp. 3476-3476

Author(s):

Ross Fisher ◽

Adrianna Henson ◽

Paola Quarello ◽

Anna Aspesi ◽

Jason E Farrar ◽

...

Keyword(s):

Ribosomal Protein ◽

Ribosomal Subunit ◽

Gene Discovery ◽

Rrna Processing ◽

Ribosomal Protein Genes ◽

Loss Of Function ◽

Activated Lymphocytes ◽

Ribosome Synthesis ◽

Diamond Blackfan Anemia ◽

Genes Encoding

Abstract Abstract 3476 Most cases of Diamond Blackfan anemia are caused by haploinsufficiency for genes encoding proteins of the large or small ribosomal subunit. All of the ribosomal proteins affected in DBA are essential components of the ribosome required for the assembly of their respective subunits, including processing of the primary pre-rRNA transcript to mature 18S, 5.8S, and 28S rRNAs. Pre-rRNA processing signatures associated with ribosomal protein haploinsufficiency demonstrate a role for individual proteins in subunit assembly and can differ depending on which protein is affected. A facile pre-rRNA processing assay that can discriminate between loss of function alleles for different ribosomal protein genes would be an invaluable aide to DBA diagnosis and gene discovery efforts. Such an assay could also provide insight into different aspects of DBA pathophysiology. We have developed a robust procedure to assess pre-rRNA processing patterns in activated lymphocytes from the peripheral blood of patients with known or suspected Diamond Blackfan anemia. This assay typically involves the electrophoretic separation of total RNA from activated lymphocytes followed by Northern blotting with various hybridization probes to different rRNA precursors. Using this assay, we have found a common 32S pre-rRNA processing intermediate present in RNA from DBA patients with mutations in virtually all known large ribosomal subunit genes. This 32S pre-rRNA can be visualized in situ in gels stained with ethidium bromide (see accompanying figure) greatly simplifying the identification of large subunit ribosomal protein genes harboring loss of function mutations. As more and more ribosomal protein genes are identified within the DBA population, it has become increasingly important to distinguish between variants that affect ribosomal protein function and benign polymorphisms. Therefore, an analysis of pre-rRNA processing can be used to identify causative genes in patients with complex genotypes where sequence variants are found in more than one ribosomal protein gene. We analyzed a patient with variants in genes encoding RPS19 and RPL11, two known DBA genes, plus a deletion containing the RPL31 gene that has not been previously linked to DBA. Data analyses overwhelmingly support the deletion of RPL31 as the causative lesion in this patient and identify RPL31 as a new DBA gene. Analysis of pre-rRNA processing can also guide additional gene discovery efforts. We performed pre-rRNA processing studies on two patients lacking mutations in known DBA genes. In one case, we observed a clear defect in the18S rRNA pathway, implicating a gene involved in the biogenesis of the small ribosomal subunit, whereas in a second patient there is no evidence of a ribosome biogenesis defect suggesting that the underlying mutation may not affect ribosome synthesis. These results will help guide further efforts to identify causative genes in this patient cohort. Finally, we have used pre-rRNA processing patterns to begin to examine the mechanisms underlying remission in DBA patients. To date, we have examined two samples from DBA patients in remission and showed the ribosome synthesis defect is retained even while in remission for one of these patients, whereas the pre-rRNA processing defect has resolved in the other patient. Disclosures: No relevant conflicts of interest to declare.

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Ribosomal Protein Genes S10 and S26 Are Commonly Mutated in Diamond-Blackfan Anemia.

Blood ◽

10.1182/blood.v114.22.175.175 ◽

2009 ◽

Vol 114 (22) ◽

pp. 175-175 ◽

Cited By ~ 2

Author(s):

Hanna T. Gazda ◽

Mee Rie Sheen ◽

Leana Doherty ◽

Adrianna Vlachos ◽

Valerie Choesmel ◽

...

Keyword(s):

Ribosomal Protein ◽

Large Scale ◽

Conflicts Of Interest ◽

Bone Marrow Failure ◽

Ribosomal Protein Genes ◽

Variants Of Unknown Significance ◽

Diamond Blackfan Anemia ◽

Increased Risk ◽

Unknown Significance ◽

Risk Of Cancer

Abstract Abstract 175 Diamond-Blackfan anemia (DBA) is a congenital bone marrow failure syndrome characterized by anemia usually presenting during infancy or in early childhood, birth defects, and increased risk of cancer. Although anemia is the most prominent feature of DBA, the disease is also characterized by growth retardation and congenital malformations, in particular craniofacial, upper limb, heart and urinary system defects, that are present in ∼30–50% of patients. To date, DBA has been associated with mutations in seven ribosomal protein (RP) genes, S19, S24, S17, L35A, L5, L11, and S7 in about ∼43% of patients. To complete our large scale screen of RP genes in a DBA population, we sequenced 49 ribosomal protein genes in our DBA patient cohort of 117 probands. Together with our previous efforts of screening 29 RP genes, this completes the analysis of all known RP genes (excluding RPS4Y on the Y chromosome). Here we report probable mutations in four more RP genes, RPS10, RPS26, RPL19, and RPL26, and variants of unknown significance in six more, RPL9, RPL14, RPL23A, RPL7, RPL35, and RPL3. RPS10 and RPS26 are now confirmed DBA genes as we identified five mutations in RPS10 in five probands and nine mutations in RPS26 in 12 probands. We found single small frameshifting deletions in RPL19 and RPL26 in single DBA families. Pre-rRNA analysis in lymphoblastoid cells from patients bearing mutations in RPS10 and RPS26 showed elevated levels of 18S-E pre-rRNA. This accumulation is consistent with the phenotype observed in HeLa cells after knock-down of RPS10 or RPS26 expression with siRNAs, which indicate that mutations in the RPS10 and RPS26 genes in DBA patients affect the function of the proteins. This brings to 11, the total number of RP genes mutated in ∼54% of patients with DBA, with nine more genes harboring variants of unknown significance requiring further study. Disclosures: No relevant conflicts of interest to declare.

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Ribosomal protein gene deletions in Diamond-Blackfan anemia

Blood ◽

10.1182/blood-2011-08-375170 ◽

2011 ◽

Vol 118 (26) ◽

pp. 6943-6951 ◽

Cited By ~ 87

Author(s):

Jason E. Farrar ◽

Adrianna Vlachos ◽

Eva Atsidaftos ◽

Hannah Carlson-Donohoe ◽

Thomas C. Markello ◽

...

Keyword(s):

Ribosomal Protein ◽

Ribosomal Proteins ◽

Protein Gene ◽

Single Nucleotide Polymorphism Array ◽

Ribosomal Protein Gene ◽

Small Subunit ◽

Ribosomal Protein Genes ◽

Gene Deletions ◽

Diamond Blackfan Anemia ◽

Genome Wide

Abstract Diamond-Blackfan anemia (DBA) is a congenital BM failure syndrome characterized by hypoproliferative anemia, associated physical abnormalities, and a predisposition to cancer. Perturbations of the ribosome appear to be critically important in DBA; alterations in 9 different ribosomal protein genes have been identified in multiple unrelated families, along with rarer abnormalities of additional ribosomal proteins. However, at present, only 50% to 60% of patients have an identifiable genetic lesion by ribosomal protein gene sequencing. Using genome-wide single-nucleotide polymorphism array to evaluate for regions of recurrent copy variation, we identified deletions at known DBA-related ribosomal protein gene loci in 17% (9 of 51) of patients without an identifiable mutation, including RPS19, RPS17, RPS26, and RPL35A. No recurrent regions of copy variation at novel loci were identified. Because RPS17 is a duplicated gene with 4 copies in a diploid genome, we demonstrate haploinsufficient RPS17 expression and a small subunit ribosomal RNA processing abnormality in patients harboring RPS17 deletions. Finally, we report the novel identification of variable mosaic loss involving known DBA gene regions in 3 patients from 2 kindreds. These data suggest that ribosomal protein gene deletion is more common than previously suspected and should be considered a component of the initial genetic evaluation in cases of suspected DBA.

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Characterization of the Hematologic Phenotype of rpS6 Heterozygous Null Mice as a Model of the Erythroid Failure In Diamond-Blackfan Anemia

Blood ◽

10.1182/blood.v116.21.2033.2033 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2033-2033

Author(s):

Sioban B. Keel ◽

Janis L. Abkowitz

Keyword(s):

Ribosomal Protein ◽

Ribosome Biogenesis ◽

Conflicts Of Interest ◽

Macrocytic Anemia ◽

Ribosomal Subunit ◽

T Test ◽

Ribosomal Protein Genes ◽

Diamond Blackfan Anemia ◽

Student’S T ◽

Student’S T Test

Abstract Abstract 2033 Diamond-Blackfan Anemia (DBA) is a congenital form of pure red cell aplasia characterized by a hypoproliferative, macrocytic anemia, congenital anomalies, and a predisposition to cancer. DBA, along with a growing number of human diseases, is linked to defects in ribosome biogenesis. Mutations in at least 10 ribosomal protein genes of both the 40S and 60S ribosomal subunits have now been identified in over 50% of patients with DBA (Narla A, et al. Blood 2010; 115) resulting in ribosomal protein haploinsufficency and in turn a defect in ribosome biogenesis. It remains, however, unknown how these events culminate in erythroid marrow failure. The study of this pathophysiology has been hindered by a lack of animal models. We became aware of the Rps6-deleted mouse as a potential murine model of DBA (Volarevic S, et al. Science 2000; 288). RPS6 is another 40S ribosomal subunit protein required for ribosomal subunit assembly. Haploinsufficiency of RPS6 causes a phenotype reminiscent of DBA during embryogenesis (Panic L, et al. Mol Cell Biol 2006; 26), however, the erythropoietic phenotype of the conditionally-deleted Rps6 heterozygous mouse was unknown. The purpose of these studies is to fully characterize the erythroid phenotype of this mouse as a model of DBA. We demonstrate that deletion of one Rps6 allele in mice results in a macrocytic anemia and leukopenia (an absolute neutropenia and lymphocytopenia, Table 1). Though this finding is not typical, neutropenia has been described in DBA. Like DBA, the anemia is hypoproliferative (corrected reticulocyte counts were equivalent in rpS6 heterozygous and control mice: 3.3% ± 0.21, n= 3 vs. 3.6 ± 0.33, n=3; two-tailed Student's t-test, p= 0.08, which is an inappropriately low value given the deleted animals’ anemia). Flow cytometric analyses of bone marrow and spleen double-stained for Ter119 and transferrin receptor (CD71) demonstrate impaired early erythroid differentiation, evidenced by a relative expansion in the proerythroblast and basophilic erythroblast populations. Hematopoietic colony assays confirm this early defect. These data suggest that haploinsufficiency of rpS6 impacts both erythropoiesis and granulopoiesis, and since the mice are not thrombocytopenic, the effect appears lineage specific, rather than occurring in a common progenitor cell. Polysome profiles to confirm a defect in ribosome biogenesis are pending. Since heterozygous mice recapitulate the erythroid phenotype of DBA, we treated the mice with standard and potential DBA therapies. Specifically, mice received 2 mg/kg/day of prednisone for 12 weeks. There was no improvement in the hemoglobin or MCV in treated animals. As DBA and 5q- syndrome myelodysplastic syndrome (MDS) share an erythroid phenotype and both result from a haploinsufficiency of a ribosomal protein, we also tested whether the macrocytic anemia in rpS6 heterozygous mice responds to lenalidomide (Revlimid®, gift from Celgene Corporation, San Diego, CA). Mice received 3 mg/kg/day of lenalidomide by oral gavage for 12 weeks. The hemoglobin increased in control mice and markedly increased in rpS6 heterozygous mice after 12 weeks of therapy (13.5 ± 0.4 to 14.9 ± 0.2, p= 0.0 and 7.9 g/dL ± 0.9 to 10.3 ± 0.8, p= 0.01, respectively; mean ± SEM, Student's t-test, paired). Additionally, the MCV decreased with therapy in both groups (49.1 fL ± 1.4 to 41.1 ± 0.2, p=0.005 and 57.4 ± 1.1 to 53.77 ± 1.4, p=0.08). With the caveat that we did not monitor drug levels achieved in vivo, these data suggest that lenalidomide improves hemoglobinization and deserves further study in DBA. Disclosures: No relevant conflicts of interest to declare.

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Haploinsufficiency for ribosomal protein genes causes selective activation of p53 in human erythroid progenitor cells

Blood ◽

10.1182/blood-2010-07-295238 ◽

2011 ◽

Vol 117 (9) ◽

pp. 2567-2576 ◽

Cited By ~ 237

Author(s):

Shilpee Dutt ◽

Anupama Narla ◽

Katherine Lin ◽

Ann Mullally ◽

Nirmalee Abayasekara ◽

...

Keyword(s):

Bone Marrow ◽

Myelodysplastic Syndrome ◽

Ribosomal Protein ◽

Progenitor Cells ◽

Bone Marrow Failure ◽

Ribosomal Protein Gene ◽

Ribosomal Protein Genes ◽

Erythroid Progenitor ◽

Bone Marrow Biopsies ◽

Erythroid Progenitor Cells

Abstract Haploinsufficiency for ribosomal protein genes has been implicated in the pathophysiology of Diamond-Blackfan anemia (DBA) and the 5q− syndrome, a subtype of myelodysplastic syndrome. The p53 pathway is activated by ribosome dysfunction, but the molecular basis for selective impairment of the erythroid lineage in disorders of ribosome function has not been determined. We found that p53 accumulates selectively in the erythroid lineage in primary human hematopoietic progenitor cells after expression of shRNAs targeting RPS14, the ribosomal protein gene deleted in the 5q− syndrome, or RPS19, the most commonly mutated gene in DBA. Induction of p53 led to lineage-specific accumulation of p21 and consequent cell cycle arrest in erythroid progenitor cells. Pharmacologic inhibition of p53 rescued the erythroid defect, whereas nutlin-3, a compound that activates p53 through inhibition of HDM2, selectively impaired erythropoiesis. In bone marrow biopsies from patients with DBA or del(5q) myelodysplastic syndrome, we found an accumulation of nuclear p53 staining in erythroid progenitor cells that was not present in control samples. Our findings indicate that the erythroid lineage has a low threshold for the induction of p53, providing a basis for the failure of erythropoiesis in the 5q− syndrome, DBA, and perhaps other bone marrow failure syndromes.

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Reduced ribosomal protein gene dosage and p53 activation in low-risk myelodysplastic syndrome

Blood ◽

10.1182/blood-2010-11-318584 ◽

2011 ◽

Vol 118 (13) ◽

pp. 3622-3633 ◽

Cited By ~ 37

Author(s):

Kelly A. McGowan ◽

Wendy W. Pang ◽

Rashmi Bhardwaj ◽

Marcelina G. Perez ◽

John V. Pluvinage ◽

...

Keyword(s):

Myelodysplastic Syndrome ◽

Ribosomal Protein ◽

Protein Gene ◽

Gene Dosage ◽

Critical Role ◽

Macrocytic Anemia ◽

Ribosomal Protein Gene ◽

Ribosomal Protein Genes ◽

Diamond Blackfan Anemia ◽

Ribosomal Protein S6

Abstract Reduced gene dosage of ribosomal protein subunits has been implicated in 5q− myelodysplastic syndrome and Diamond Blackfan anemia, but the cellular and pathophysiologic defects associated with these conditions are enigmatic. Using conditional inactivation of the ribosomal protein S6 gene in laboratory mice, we found that reduced ribosomal protein gene dosage recapitulates cardinal features of the 5q− syndrome, including macrocytic anemia, erythroid hypoplasia, and megakaryocytic dysplasia with thrombocytosis, and that p53 plays a critical role in manifestation of these phenotypes. The blood cell abnormalities are accompanied by a reduction in the number of HSCs, a specific defect in late erythrocyte development, and suggest a disease-specific ontogenetic pathway for megakaryocyte development. Further studies of highly purified HSCs from healthy patients and from those with myelodysplastic syndrome link reduced expression of ribosomal protein genes to decreased RBC maturation and suggest an underlying and common pathophysiologic pathway for additional subtypes of myelodysplastic syndrome.

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Post-Transcriptional Defects and Erythroid Pathobiology

Blood ◽

10.1182/blood.v124.21.sci-35.sci-35 ◽

2014 ◽

Vol 124 (21) ◽

pp. SCI-35-SCI-35

Author(s):

Vijay G. Sankaran

Keyword(s):

Gene Regulation ◽

Blood Cell ◽

Ribosomal Protein ◽

Messenger Rna ◽

Conflicts Of Interest ◽

Transcriptional Level ◽

Ribosomal Protein Genes ◽

Cell Production ◽

Therapeutic Implications ◽

Diamond Blackfan Anemia

A great body of work has focused on understanding the role that gene regulation at the transcriptional level plays in blood cell production and diseases that disrupt this process. However, until recently there has been limited insight on the role that post-transcriptional gene regulation has in both normal and pathological disorders of human hematopoiesis. Specifically, the regulation of messenger RNA translation can have a significant impact upon gene expression, and how this process affects hematopoiesis has only been explored in limited studies. In this talk, the role of ribosomal protein gene mutations in the specific disorder of red blood cell production, Diamond-Blackfan anemia, will be discussed. Recent findings from our laboratory show that mutations in the key hematopoietic transcription factor gene, GATA1, can result in Diamond-Blackfan anemia in rare cases. We have gone on to show that more common mutations in ribosomal protein genes can disrupt translation of GATA1 and thereby suggest a common underlying mechanism for the impaired erythropoiesis observed in Diamond-Blackfan anemia. We discuss both the mechanistic underpinnings of our observations and how these findings have important therapeutic implications. Other recent examples of how disordered translation can impair human hematopoiesis will also be examined. This talk will provide a cohesive framework to understand the implications of these recent findings for both normal and disordered human hematopoiesis. Disclosures No relevant conflicts of interest to declare.

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High Frequency of Large Gene Deletions Detected by Multiplex Ligation-Dependent Probe Amplification in Diamond Blackfan Anemia,

Blood ◽

10.1182/blood.v118.21.3428.3428 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3428-3428

Author(s):

Paola Quarello ◽

Emanuela Garelli ◽

Adriana Carando ◽

Patrizia Pappi ◽

Alfredo Brusco ◽

...

Keyword(s):

Ribosomal Protein ◽

Conflicts Of Interest ◽

Gene Dosage ◽

Mutation Screening ◽

Macrocytic Anemia ◽

Clinical Environment ◽

Ribosomal Protein Genes ◽

Genomic Deletions ◽

Diamond Blackfan Anemia ◽

Dependent Probe

Abstract Abstract 3428 Background: Diamond-Blackfan anemia (DBA,#MIM105650) is a rare congenital pure red cell aplasia characterized by normochromic macrocytic anemia, reticulocytopenia, and normocellular bone marrow with a selective deficiency of erythroid precursors. Although mutations of eleven ribosomal protein (RP) genes have been detected in more than 50% of DBA patients the remaining patients appear to have intact ribosomal protein genes using stadard sequencing methods (Boria et al. Hum Mut 2010). We previously described the detection of three large RPS19 deletions using the MLPA (Multiplex Ligation-dependent Probe Amplification) technique (Quarello et al. Haematologica 2008). As MLPA is an efficient and rapid technique that detects gene dosage alterations, we thus decided to apply this approach also to other RP genes. Aim: To search for unidentified RP large deletions we applied the MLPA technique in Italian DBA patients who have been found mutation-negative by sequencing. Methods: Italian DBA patients without RP genes mutations (73/156, 47%) were included in this study. The analysis was performed using a homemade MLPA kit following the recommendations provided by MRC Holland (Amsterdam, The Netherlands, www.mlpa.com). The probes were designed to detect deletions of six RP genes (RPS17, RPS19, RPS26, RPL5, RPL11, RPL35A). Deletions of probe recognition sequences were apparent by a 35–50% reduced relative peak area of the amplification product of that probe. Results: The results of the MLPA assay revealed that 13 out of the 73 probands (18%) had a multi-exonic deletion in one of the six DBA genes analyzed. We identified four deletions of the RPS17 gene, three of the RPS26 gene, three of the RPL35A, two of the RPL11 gene and one of the RPL5 gene. No additional RPS19 deletions were found. DBA patients with deletions showed a severe phenotype with a very high percentage of transfusion dependence (85%). Somatic malformations were observed only in two patients. Conclusion: We detected a high percentage of deletions of known DBA genes in a cohort of patients in whom no mutations were found by RP sequencing. Mutation screening of the RP genes with a combination of sequencing and MLPA reached an overall detection rate of 61.5% (96/156). In our cohort, large genomic deletions represent up to 18% of all mutations detected. In conclusion, we stress the high percentage of identified RP genes deletions in DBA patients. We also highlight that a gene-dosage technique, such as MLPA, should complement sequencing in a clinical environment since only a combined approach of this kind permits the comprehensive detection of all mutations in the DBA RP genes. Disclosures: No relevant conflicts of interest to declare.

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Molecular Analysis of Diamond Blackfan Anemia and Genotype-Phenotype Correlation: Experience from the Canadian Inherited Marrow Failure Registry

Blood ◽

10.1182/blood.v126.23.3621.3621 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3621-3621

Author(s):

Omri Avraham Arbiv ◽

Bozana Zlateska ◽

Robert J. Klaassen ◽

Conrad Fernandez ◽

Rochelle Yanofsky ◽

...

Keyword(s):

Ribosomal Protein ◽

Research Funding ◽

Gene Panel ◽

Ribosomal Protein Genes ◽

Severe Phenotype ◽

Hematopoietic Stem ◽

Diamond Blackfan Anemia ◽

Genetic Groups ◽

Number Of Patients ◽

The Impact

Abstract Background/Objectives: Diamond Blackfan anemia (DBA) is an inherited disorder characterized by chronic hypoproductive anemia, physical malformations, and an increased risk of malignancies. At least 12 DBA genes have been identified, which include various ribosomal protein genes and the transcription factor GATA1. The aims of our study were (1) to identify the mutation spectrum of DBA patients, utilizing a cohort of patients enrolled on the Canadian Inherited Marrow Failure Registry (CIMFR) and (2) to determine whether specific hematological abnormalities, malformations, and outcomes are associated with specific mutations. Methods: Patients were enrolled on the CIMFR, which is a multicenter cohort study of inherited bone marrow failure syndromes (IBMFS). Genetic testing was performed using one or more of the following tests: Sanger sequencing, next generation sequencing (NGS) DBA gene panel, a comprehensive NGS IBMFS gene panel developed in our laboratory, or comparative genetic hybridization (CGH). Severity of the hematological disease was dichotomized according to a patient's requirement for chronic treatment: those who were maintained on corticosteroids, blood transfusions, or received a hematopoietic stem cell transplantation were considered to have a more severe phenotype than those who did not require hematological treatment. Chi-square tests with a Fisher's exact test correction were used to compare genetic groups with at least 5 patients on observed phenotypes. Results: 71 patients with DBA have been enrolled in our registry. A causal mutation has been identified in 36 of these patients, with the following rates: RPS19 (n=11), RPL11 (n=7), RPL5 (n=6), RPS26 (n=5), RPL35a (n=2), RPS24 (n=2), and one of each RPS7, RPS29, RPS17. Remarkably, a substantial number of patients in our population-based cohort (19.4%) had mild hematological phenotype requiring no therapy. Patients with RPL11 mutations tended to have a less severe DBA phenotype, while patients with RPS19 mutations tended to have a more severe phenotype (p=0.04). In terms of non-hematological malformations, we found no differences in cardiac, stature and craniofacial malformations across the groups compared (all p>0.1). However, patients with RPL5 mutations had significantly more hand malformations (p=0.02), and patients with RPS26 mutations had more genitourinary malformations (p=0.04). To control for the impact of mutation severity on the observed phenotype, we compared the prevalence of mutations that are predicted to result in truncated or lack of protein from the respective allele (large copy-number variation, nonsense, or indel frameshift) to mutations that are predicted to be hypomorphic or affect function (splicing, indel/inframe and, missense) between mutation categories. There were no differences among genetic groups in the severity of their mutations (p=0.58). Conclusions: Mutations in a wide spectrum of ribosomal protein genes underlie DBA cases in Canada, which approximate those observed by other registries in Western countries. Patients with DBA caused by RPL11 mutations tended to have a milder hematological phenotype, while patients with RPS19 mutation tended to have a more severe phenotype. Mutations in RPS26 and RPL5 are associated with genitourinary and hand malformations, respectively. Our findings may help improve counseling of DBA patients and their family. Future studies are needed to replicate our results and determine whether these findings can help personalize care. Disclosures Lipton: Ariad: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Novartis Pharmaceuticals: Consultancy, Research Funding.

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Association of RAP1 binding sites with stringent control of ribosomal protein gene transcription in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.11.5.2723-2735.1991 ◽

1991 ◽

Vol 11 (5) ◽

pp. 2723-2735 ◽

Cited By ~ 1

Author(s):

C M Moehle ◽

A G Hinnebusch

Keyword(s):

Gene Expression ◽

Saccharomyces Cerevisiae ◽

Amino Acid ◽

Ribosomal Protein ◽

Binding Sites ◽

Protein Gene ◽

Ribosomal Protein Gene ◽

Ribosomal Protein Genes ◽

Biosynthetic Genes ◽

Stringent Control

An amino acid limitation in bacteria elicits a global response, called stringent control, that leads to reduced synthesis of rRNA and ribosomal proteins and increased expression of amino acid biosynthetic operons. We have used the antimetabolite 3-amino-1,2,4-triazole to cause histidine limitation as a means to elicit the stringent response in the yeast Saccharomyces cerevisiae. Fusions of the yeast ribosomal protein genes RPL16A, CRY1, RPS16A, and RPL25 with the Escherichia coli lacZ gene were used to show that the expression of these genes is reduced by a factor of 2 to 5 during histidine-limited exponential growth and that this regulation occurs at the level of transcription. Stringent regulation of the four yeast ribosomal protein genes was shown to be associated with a nucleotide sequence, known as the UASrpg (upstream activating sequence for ribosomal protein genes), that binds the transcriptional regulatory protein RAP1. The RAP1 binding sites also appeared to mediate the greater ribosomal protein gene expression observed in cells growing exponentially than in cells in stationary phase. Although expression of the ribosomal protein genes was reduced in response to histidine limitation, the level of RAP1 DNA-binding activity in cell extracts was unaffected. Yeast strains bearing a mutation in any one of the genes GCN1 to GCN4 are defective in derepression of amino acid biosynthetic genes in 10 different pathways under conditions of histidine limitation. These Gcn- mutants showed wild-type regulation of ribosomal protein gene expression, which suggests that separate regulatory pathways exist in S. cerevisiae for the derepression of amino acid biosynthetic genes and the repression of ribosomal protein genes in response to amino acid starvation.

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