Mutations in Ribosomal Protein Genes of Diamond-Blackfan Anemia Patients in Japan.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3204-3204
Author(s):  
Yuki Konno ◽  
Tsutomu Toki ◽  
Satoru Tandai ◽  
Gang Xu ◽  
Kiminori Terui ◽  
...  

Abstract Abstract 3204 Poster Board III-141 Diamond-Blackfan anemia (DBA) is an inherited congenital bone marrow failure syndrome, characterized by red blood cell aplasia, macrocytic anemia, and increased risk of malignancy. Although anemia is the most prominent feature of DBA, the disease is also characterized by growth retardation and congenital malformations, which occur in about 40% of patients. Approximately 90% of patients present during the first year of life or in early childhood. Recent studies have shown that the disease is associated with heterozygous mutations in the ribosomal protein (RP) genes RPS19, RPS24, and RPS17, encoding small ribosomal subunit proteins, and in RPL5, RPL11 and RPL35a, encoding large ribosomal subunit proteins, in about 50% of patients with DBA in Western countries. There have been no studies to determine the incidence of these mutations in Asian patients with DBA. In this study, 44 probands (46 patients) with DBA in Japan were screened for mutations of the 6 known DBA genes RPS19, RPS24, RPS17, RPL5, RPL11, and RPL35a, in addition to RPS14, which is implicated in the 5q- syndrome, a subtype of myelodysplastic syndrome characterized by a defect in erythroid differentiation. Mutations in RPS19, which have been found in 25% of patients in Western countries, were detected in 6 probands (13.6%). Missense mutations were noted in 5 of these probands, and a frameshift mutation caused by a single-nucleotide insertion was found in 1 case. Three of 7 patients had multiple malformations. Novel mutations in RPL5 were identified in 3 probands (6.8%). Insertion of 2 nucleotides was found in 1 case, affecting the reading frame. Two cases had point mutations, which resulted in a loss of the first initiation codon. All 3 patients with RPL5 mutations had multiple physical anomalies. Remarkably, 2 of 3 patients with RPL5 mutations had cleft palate, whereas no other DBA patients presented with cleft palate. Mutations in RPL11 were identified in 2 patients (4.5%). Deletion of 1 or 2 nucleotides was found in each case, leading to a shift in the reading frame. In contrast to previous reports on patients with RPL11 mutations, thumb anomalies were not seen. Deletion of 1 nucleotide in RPS17 was identified in 1 patient (2.3%), resulting in introduction of a premature stop codon. RPS17 mutations are rare and have been only reported in 2 patients with DBA. Anomalies were not seen in our patient. In summary, RP gene mutations were identified in 27.3% of DBA index cases in Japan. No mutations were detected in RPS14, RPS24 and RPL35a. In Japan, the frequency of mutations in the RP genes appears to be lower than in Western countries. Mutations in RPL5 are associated with multiple physical abnormalities, including cleft palate. Disclosures No relevant conflicts of interest to declare.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 177-177 ◽  
Author(s):  
Elspeth Payne ◽  
Hong Sun ◽  
Barry H. Paw ◽  
A. Thomas Look ◽  
Arati Khanna-Gupta

Abstract Abstract 177 Diamond Blackfan Anemia (DBA) is a congenital autosomal dominant bone marrow failure syndrome of childhood manifested as profound anemia. The disease is characterized by enhanced sensitivity of hematopoietic progenitors to apoptosis with evidence of stressed erythropoiesis. In addition to bone marrow defects, DBA patients often have craniofacial, genitourinary, cardiac and limb abnormalities and have an increased risk of developing hematopoietic malignancies and osteosarcoma. Twenty-five percent of patients with DBA have heterozygous mutations in the ribosomal protein S19 (RPS19) gene, which encodes a component of the 40S ribosomal subunit. Additionally, a growing percentage of DBA patients lacking a mutation in the RPS19 gene have been shown to have mutations in other ribosomal protein genes. These observations support the hypothesis that DBA is a disease of altered ribosome assembly and function. It is unclear how defects in ribosomal proteins have such a specific effect on erythroid maturation and cause increased apoptosis in the erythroid compartment. An attempt to model DBA by homozygous deletion of the Rps19 gene in mice proved to be embryonic lethal, and heterozygous mice appeared to fully compensate for the loss of one Rps19 allele, in contrast to the disease observed in humans. However, two groups have successfully modeled DBA in zebrafish using an antisense morpholino (MO) approach. These studies demonstrated that similar to the human disease, rps19 deficiency leads to defective erythropoiesis, increased apoptosis and to developmental abnormalities. A central role for the tumor suppressor p53 was suggested in one of these studies. It has previously been shown that any MO injection into zebrafish embryos can lead to the activation of the p53 pathway. Therefore, in order to clarify whether p53-independent effects also contributed to the DBA phenotype in zebrafish, we utilized the p53e7/e7 line that harbors a mutation within the p53 DNA-binding domain. Splice site and validated 5'UTR MOs targeting zebrafish rps19 were injected into one-cell stage embyros that were wildtype (WT) for p53 (AB) or mutated p53e7/e7. Staining for hemoglobin at 48 hours post fertilization showed a profound reduction in circulating blood in both p53 wild-type and p53 mutant embryos. Although p53 mutants injected with rps19 MO show a similar reduction in hemoglobin expression to WT morphants, they have a marked improvement in their developmental defects. A 20% decrease in expression of the transcription factor GATA-1 was observed in the rps19 morphants in the p53 mutant background compared to control MO injection. The implications of this finding are being further investigated and extended to include a panel of additional erythroid-specific factors. We have observed no increase in the levels of cell death, as measured by acridine orange (AO) staining or expression of the p53-regulated apoptosis associated gene PUMA, in the p53 mutant background. Taken together, our observations indicate that the phenotype observed in DBA has both a p53-dependent and a p53-independent component. We hypothesize that the p53-dependent component of DBA is likely responsible for the increased apoptosis associated with DBA while the erythroid maturation defect is associated, in large part, with a p53-independent component. Our studies are currently focused on identifying the players in the latter pathway. These investigations should shed light on thus far undefined pathways that will likely open new avenues for drug design and development for DBA. Disclosures: No relevant conflicts of interest to declare.


Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 39-39
Author(s):  
Hanna T. Gazda ◽  
Mee Rie Sheen ◽  
Adrianna Vlachos ◽  
Valerie Choesmel ◽  
Marie-Francoise O’Donohue ◽  
...  

Abstract Diamond-Blackfan anemia (DBA), a congenital bone marrow failure syndrome, is characterized by red blood cell aplasia, macrocytic anemia, clinical heterogeneity, and increased risk of malignancy. Although anemia is the most prominent feature of DBA, the disease is also characterized by growth retardation and congenital anomalies, present in ~30–47% of patients. The disease is associated with mutations in six ribosomal protein (RP) genes, S19, S24, S17, L35A, L5, and L11, in about 40–45% of patients. To continue our large scale screen of RP genes in a DBA population, we sequenced 12 RP genes, S15, L36, L31, L37A, S7, S27A, S14, S23, L3, L23, S17, and L27A in our DBA patient cohort of 200 families. We identified the second known mutation in RPS17 and possible pathogenic single mutations in four more RP genes, S7, L36, S15, and S27A. These are a donor splice site mutation (intron 2) in RPS7, a deletion of two nucleotides causing frameshift in RPS17 and RPL36, and two missense changes in RPS15 and RPS27A. Northern blot analysis demonstrated that lymphoblastoid cells from the patient with RPS7 mutation displayed higher levels of 45S and 30S pre-rRNAs compared to normal cells, similar to results in HeLa cells with siRNA-based knock-down of RPS7. There is a strong defect in 5′-ETS processing, resulting in accumulation of 45S and 30S pre-rRNAs, and a strong drop of levels of the 41S, 21S and 18S-E intermediates, whereas the amount of precursors to the large ribosomal subunit RNAs were unchanged. These results suggest that mutation of RPS7 in this DBA patient directly affects maturation of pre-rRNA. In addition, review of available medical data of 20 patients with mutations in RPL5 revealed that majority of them (14/20) have physical malformations including craniofacial, thumb and heart anomalies. Similarly, 12/18 patients with RPL11 mutations presented with physical malformations, while among 76 reported DBA patients with RPS19 mutations, only 35 (46%) had physical abnormalities. Remarkably, 9 of 14 patients with RPL5 mutations and physical abnormalities have cleft lip/ palate or cleft soft palate, isolated or in combination with other facial malformations and/or with other physical abnormalities such as heart or thumb anomalies. In contrast, none of 12 patients with RPL11 mutations and malformations have craniofacial abnormalities (p=0.007, Fisher’s exact test [FET]). Moreover, none of the 35 reported patients with RPS19 mutations and malformations presented with cleft lip and/or palate (p=9.745×10−7for RPL5 vs RPS19, FET). We conclude craniofacial clefting is associated with mutations in RPL5. In addition, 8/20 patients with mutated RPL5 and 8/18 patients with mutated RPL11 have thumb abnormalities, compared to only 9% of patients with RPS19 mutations. Moreover, congenital heart defects were found more often among patients with RPL5 mutations (5/20) compared with RPL11 (3/18) and RPS19 (4/76) (p=0.017 for RPL5 vs RPS19, FET). Strikingly, the majority (11/20) of patients with RPL5 mutations presented with multiple, severe abnormalities, including craniofacial, heart and/or thumb malformations. In contrast, patients with RPL11 and RPS19 mutations who presented with multiple physical abnormalities were uncommon, three patients out of 18, and 16 out of 76, respectively (p=0.02 for RPL5 vs RPL11 and p=0.0047 for RPL5 vs RPS19, FET). In summary, we identified single mutations in four genes as well as the second mutation in RPS17, suggesting that sequence changes in RPS7, RPS17, RPL36, RPS15, and RPS27A are rare events in DBA. Mutations in RPL5 are associated with multiple physical abnormalities including craniofacial, thumbs and heart anomalies, while thumb malformations are predominantly present in patients carrying mutations in RPL11.


Blood ◽  
2011 ◽  
Vol 118 (23) ◽  
pp. 6087-6096 ◽  
Author(s):  
Pekka Jaako ◽  
Johan Flygare ◽  
Karin Olsson ◽  
Ronan Quere ◽  
Mats Ehinger ◽  
...  

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.


Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 175-175 ◽  
Author(s):  
Hanna T. Gazda ◽  
Mee Rie Sheen ◽  
Leana Doherty ◽  
Adrianna Vlachos ◽  
Valerie Choesmel ◽  
...  

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.


Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3439-3439
Author(s):  
Nadia Danilova ◽  
Kathleen M Sakamoto ◽  
Shuo Lin

Abstract Abstract 3439 Deficiency of ribosomal protein (RPs) is associated with Diamond Blackfan Anemia, (DBA) a congenital syndrome with bone marrow failure and variable malformations. Recent studies of ours and other labs established p53 network activation as a major contributor to DBA. Therefore, modulation of p53-dependent pathways emerges as a most promising approach to DBA treatment. Direct suppression of 53 may, however, lead to increased risk of malignant transformation. Search for more physiological modulators of p53 pathways is therefore warranted. p53 has been shown both to respond to metabolic alterations and to change metabolism. Specifically, nutrients can influence p53 status through mTOR and other pathways. Recently, Ortega et al reported that treatment of Caco-2 cells with exogenous nucleosides modulated expression and activity of many transcription factors including p53 (J Nutr Biochem, 22:595, 2011). We applied treatment with exogenous nucleosides to RP-deficient zebrafish and found positive effects. As a DBA model, we use zebrafish mutant for ribosomal protein RPL11; we also employ morpholino nucleotides to create deficiency of various ribosomal proteins including RPS19. Treatment of both models resulted in inhibition of p53-dependent pathways. Specifically, expression of genes associated with cell cycle arrest such as p21 and p53 targets inducing apoptosis such as puma, was decreased by nucleoside treatment. We hypothesize that nucleoside treatment affects p53 by modulating the activity of mTOR and MEK/Erk pathways. Previously, Gu et al. showed that nucleotide depletion strongly inhibited mTOR and activated MEK/Erk. We also study how nucleoside treatment influences other stress-response pathways and affects interaction of p53 and mTOR pathways. Our data indicate that nucleosides may be effective in modulation of p53-dependent signal transduction pathways and suggest their usefulness for treatment of p53-associated disorders. Disclosures: Sakamoto: Abbott: Research Funding; Genentech: Research Funding.


Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2033-2033
Author(s):  
Sioban B. Keel ◽  
Janis L. Abkowitz

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.


Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3103-3103
Author(s):  
Dagmar Pospisilova ◽  
Radek Cmejla ◽  
Jana Cmejlova ◽  
Helena Handrkova ◽  
Jan Stary ◽  
...  

Abstract Introduction: Diamond-Blackfan anemia (DBA) is a congenital red cell aplasia that is usually diagnosed during early infancy. Apart from defects in red cell maturation, the disorder is also associated with various physical anomalies in 40% of patients. Mutations in the ribosomal protein (RP) S19 were found in 25% of patients, while mutations in other proteins of a small ribosomal subunit (RPS17 and RPS24) were published only in a small fraction of patients. Recently, mutations in RPL5, RPL11 and RPL35a of a large ribosomal subunit were also disclosed in several DBA patients. Results: The Czech DBA registry currently comprises 31 patients. Mutations in RPL5 were identified in 8/31 patients (26%), and mutations in RPL11 in 2/31 patients (6.5%), implying that mutations in RPL5 account for more Czech DBA cases than mutations in RPS19 (22.6%). As for the classification of mutations, all types were identified, including a nonsense mutation (in RPL11), point mutations (in RPL11 and RPL5), a supposed splicing defect and a small insertion and deletions (all in RPL5). Except for point mutations, all other changes were predicted to cause frameshift with premature stop codon. Since identified alterations were found neither in dbSNP nor in 52 healthy controls, and in two families mutations segregate with the disease, we conclude that they represent true DBA-causative mutations. Although the Czech DBA Registry is rather small, we performed a direct comparison of the group of patients with RPS19 mutations (n=7) with the group of patients with RPL5 mutations (n=8). No differences were found in sex ratio, steroid responsiveness, severity or course of the disease or the treatment outcome. However, patients with RPL5 mutations were generally born small for gestational age (SGA) compared with patients from the RPS19-mutated group. Only one patient (12.5%) with an RPL5 mutation was born with normal birth weight compared to four patients (57.1%) with RPS19 mutations. The second difference was even more striking: all patients with RPL5 mutations had flat thenar and some also an additional thumb anomaly, while no thumb anomalies were observed in patients with RPS19 mutations. It is questionable whether normal RPL5 function is in some way more important for proper thumb development than RPS19. Discussion: The identification of mutations in the genes in DBA patients is also interesting from another point of view. Both proteins RPL5 and RPL11 have been reported to be implicated in the activation of p53 through the interaction with the MDM2 protein, suppressing its E3 ubiquitin ligase function that otherwise directs p53 to a rapid degradation. It is noteworthy that yet another RP of a large ribosomal subunit was described, having exactly the same function – RPL23. Because no RPL23 mutations in our DBA patients were found, the primary function of RPL5 and RPL11 in ribosome biogenesis and/or translation underlies DBA phenotype rather than the conjoint role of RPL5, RPL11 and RPL23 in the p53 regulation. Conclusions: We identified 6 and 2 different mutations in the RPL5 and RPL11 genes, respectively, expanding the repertoire of known DBA-associated mutations. No mutations in the RPL23 were identified, suggesting that aberrant p53 activation due to mutations in RPL5 and RPL11 seems unlikely to be the primary cause of DBA. Patients with RPL5 mutations are more commonly born SGA and have higer frequency of thumb anomalies.


2019 ◽  
Vol 48 (2) ◽  
pp. 770-787 ◽  
Author(s):  
Marco Lezzerini ◽  
Marianna Penzo ◽  
Marie-Françoise O’Donohue ◽  
Carolina Marques dos Santos Vieira ◽  
Manon Saby ◽  
...  

Abstract Variants in ribosomal protein (RP) genes drive Diamond-Blackfan anemia (DBA), a bone marrow failure syndrome that can also predispose individuals to cancer. Inherited and sporadic RP gene variants are also linked to a variety of phenotypes, including malignancy, in individuals with no anemia. Here we report an individual diagnosed with DBA carrying a variant in the 5′UTR of RPL9 (uL6). Additionally, we report two individuals from a family with multiple cancer incidences carrying a RPL9 missense variant. Analysis of cells from these individuals reveals that despite the variants both driving pre-rRNA processing defects and 80S monosome reduction, the downstream effects are remarkably different. Cells carrying the 5′UTR variant stabilize TP53 and impair the growth and differentiation of erythroid cells. In contrast, ribosomes incorporating the missense variant erroneously read through UAG and UGA stop codons of mRNAs. Metabolic profiles of cells carrying the 5′UTR variant reveal an increased metabolism of amino acids and a switch from glycolysis to gluconeogenesis while those of cells carrying the missense variant reveal a depletion of nucleotide pools. These findings indicate that variants in the same RP gene can drive similar ribosome biogenesis defects yet still have markedly different downstream consequences and clinical impacts.


2008 ◽  
Vol 83 (6) ◽  
pp. 769-780 ◽  
Author(s):  
Hanna T. Gazda ◽  
Mee Rie Sheen ◽  
Adrianna Vlachos ◽  
Valerie Choesmel ◽  
Marie-Françoise O'Donohue ◽  
...  

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5018-5018
Author(s):  
Yuanyuan Zhu ◽  
Honghu Li ◽  
Weiyan Zheng ◽  
Shuyang Cai ◽  
Yibo Wu ◽  
...  

Diamond Blackfan anemia (DBA) is a rare bone marrow failure disorder that usually manifests as severe macrocytic anemia and erythroid hypoplasia ,often accompanied by various physical malformations and increased frequency of cancer. The disease are associated with known ribosomal protein gene and rarely GATA1 mutations in most of DBA cases, with the genetic etiology unclear in the remaining patients. Here, we present one patient with Diamond-Blackfan anaemia who lacked documented mutations involving known DBA genes by screening.Whole-exome sequencing (WES) was performed and we identified a single missense mutation in RPL31.The missense variant c.254G>T is predicted to give rise to a gene product with substitution p. Arg85Leu ,which is regarded as causative because the unaffected parents and sister of the patient do not possess the mutation,implying that this mutation was de novo. Previous studies showed that nearly all of the causative variants of RP genes observed in DBA are loss-of function mutations and RP haploinsufficiency accounts for most cases of DBA. Particularly, The coding region of RPL31 shares 89.6% amino acid identities with its zebrafish ortholog, which is highly conserved between these two species.All of above led us to explore the effects of RPL31 deficiency on morphology and erythropoietic status during embryonic development. Therefore, morpholino antisense oligonucleotides (MOs) targeting zebrafish rpl31, orthologs of human RPL31,was injected at a proper concentration into one-cell-stage zebrafish embryos to mimic RPL31 haploinsufficiency in DBA. The specificity of the rpl31-MO was valitated by western blot detection.Compared with wild-type embryos, rpl31 deficiency embryos showed developmental abnormalities such as pericardial edema, microcephaly ,delayed pigmentation and bent tail. We also performed haemoglobin staining at 48 hr post fertilization (48hpf) and found a significant decrease of erythrocyte production in the cardial vein of the morphants. Meanwhile,the number of lcr+red blood cells was reduced about 50% in lcr:GFP transgenic embryos at 48 hpf by fluorescence microscopy and 3dpf by flow cytometry when compared to control embryos.However,the myeloid and lymphoid lineages were preserved as analyzed by using lyz:dsRed transgenic line and by observing expression of rag1,respectively. Lots of evidence showed that many other ribosomal protein genes deficiency can up-regulate p53 network,resulting in consequent cell cycle arrest or apoptosis.so we want to investigate the connection between RPL31 and p53 signaling in erythropoiesis.However,p53 null can not completely rescue the erythroid defect phenotype when we injected rpl31-MO into p53-/- embryos,suggesting alternative pathways may be involved in this process. In summary, we reported a brand new point mutation in RPL31 gene and we phonecopied loss of function of RPL31 observed in the DBA patient in vivo by constructing a RPL31 deficiency zebrafish model,indicating preliminarily that the loss of function mutation in RPL31 is a candidate responsible for DBA.However,underlying molecular mechanisms and reasons of the specific erythroid impairments in RPL31 deficiency are still needed to be clarified.we will focused on these in the future. Disclosures No relevant conflicts of interest to declare.


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