Haematologica, Vol 93, Issue 11, 1601-1604 doi:10.3324/haematol.2008.000513
Copyright © 2008 by Ferrata Storti Foundation
This Article
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Dianzani, I.
Right arrow Articles by Loreni, F.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Dianzani, I.
Right arrow Articles by Loreni, F.
Related Collections
Right arrowRelated Articles

Editorials and Perspectives

Diamond-Blackfan anemia: a ribosomal puzzle

Irma Dianzani1, Fabrizio Loreni2

1 Dipartimento di Scienze Mediche, Università del Piemonte Orientale, Novara
2 Dipartimento di Biologia, Università di Roma "Tor Vergata", Rome, Italy. E-mail:irma.dianzani{at}med.unipmn.it;loreni{at}uniroma2.it

Diamond-Blackfan anemia (DBA; MIM 105650) has long been a puzzle for hematologists. Even the diagnosis has been difficult, because other diseases and syndromes may present a similar phenotype and because there is still no specific biochemical assay available.1 The identification of RPS19 (ribosomal protein S19) as the first DBA gene has done little to solve the mystery surrounding the pathophysiology of this syndrome, characterized by defective erythropoiesis, low stature and malformations.2 Classically, DBA has been included within the inherited bone marrow aplastic syndromes and shares many aspects with other syndromes that may present erythroblastopenia, such as Fanconi anemia, Shwachman-Diamond syndrome (SD), cartilage-hair hypoplasia syndrome and dyskeratosis congenita (DC). Interestingly, the pathogenesis of SD and DC has been connected in some ways to ribosomal failure.3

DBA is the first, and so far only known, inherited disease due to a defect in a structural ribosomal protein. The identification of the role of another RP, RPS14, in the pathogenesis of an acquired myelodysplastic disease, the 5q- syndrome has recently attracted great interest.4 The definition of ribosomapathy to describe DBA has been strengthened by the identification of detrimental mutations in several RP genes other than RPS19. The actual involvement of mutated ribosomal components includes 25% RPS19,2 3% RPL35A,5 2% RPS24,6 1% RPS17,7 although mutations have also been frequently found in RPL11 and RPL58. At present, these six genes seem to account for more than 50% of DBA cases. It is apparent that other genes are involved and a systematic survey of all the 80 RP genes has started in the DBA community in the hope of diagnosing all DBA patients.

The paper of Quarello et al. in this issue of the journal reports an increased incidence of mutations in RPS19 compared to that found previously.9 Since the disease is due to loss-of-function mutations, the Authors decided to evaluate the role of gene deletions in the pathogenesis of DBA. These types of mutations are not screened by methods usually employed for mutation analyses. Since the disease is autosomal dominant, the presence of the wild-type allele masks deletions when a PCR-based methodology, such as Sanger sequencing, is used.

Quarello et al. used a technique well-known among geneticists: Multiplex Ligation-dependent Probe Amplification (MLPA) that detects all types of deletions, both those affecting the whole gene and those that affect a single exon. Using MLPA they showed that whole gene deletions are more frequent than expected in RPS19. Taking all mutation types into consideration, RPS19 is mutated in 28% in their panel of 123 Italian patients. They recommend MLPA, or other methods able to detect deletions, such as FISH, as necessary tools for an accurate molecular diagnosis of DBA due to RPS19 deficiency. Such techniques should probably also be implemented for the other frequently mutated DBA genes.

Even though most mutations affecting DBA genes are loss-of-function mutations thought to cause NMD (i.e. deletion, nonsense and frameshift), missense mutations are also well represented. Several papers have addressed the effect of DBA mutations; most of them are focused on RPS19 (recently reviewed in Campagnoli et al. 2008;10 A database reporting mutations and their functional consequences for DBA genes is available at www.dbagenes.unito.it.11 Analysis of the properties of mutated RPS19 proteins expressed in cultured cells showed that protein stability is more or less decreased according to the different mutations.12 Moreover, nucleolar localization appears compromised in some cases and, importantly, none of the mutated RPS19 proteins are found assembled into functional ribosomes.12

The paper of Crètien et al. in this issue of the journal13 addresses the same aspect, studying the fate of 12 GFP-tagged mutated RPS19 proteins expressed in vitro in Cos-7 cells. According to their analysis, the mutated RPS19 proteins fall into two groups: 1) proteins with a slightly decreased stability and normal nucleolar localization; and 2) proteins which show a drastic reduction in stability and an alteration of nucleolar localization. The results match previous studies and are also consistent with the crystal structure of RPS19 from Archea.14 Mutations of the first class are located in positions which are not predicted to influence the folding of RPS19; mutations of the second class, on the other hand, are supposed to affect the general structure of the protein and therefore its stability. Crètien et al. then address the mechanism of degradation of the mutated RPS19 by using proteasome inhibitors. Proteasome is involved in the maturation of ribosome precursors and in the degradation of RPs which are normally produced in excess.15 By inhibiting proteasomal degradation the researchers obtained an increase in the levels of mutated RPS19 proteins and restored nucleolar localization. Extending the results of previous studies,12 their findings, therefore, indicate that the fate of the mutated RPS19 is to be degraded by the proteasome.

Although, as decribed above, several studies suggest that DBA is a ribosomapathy, the link between the gene dosage defect for an RP and the defective erythropoiesis remains unsolved. A defect in the processing of ribosomal RNA (rRNA) has been identified both in CD34+ cells from DBA patients and in erythroid cells down-regulated for RPS19.16 Similar alterations in the rRNA maturation pathway have also been shown in the case of deficiency (experimentally induced or due to mutations) of the other DBA-associated RPs: RPS24, RPS17, RPL35A, RPL5 and RPL11.5,17,18 The processing step affected is different among the various RPs (summarized in Figure 1) but results, in all cases, in a ribosome biogenesis defect. This observation supports the hypothesis of a defect in general protein synthesis to explain the pathogenesis of DBA: reduced capacity of protein production would affect proliferation mostly evident in tissues with a high turnover such as the erythron and the embryo. Indeed, decreased protein synthesis has been shown in lymphocytes from DBA patients.19 This hypothesis, though, is not completely convincing and alternative views have been proposed to explain the tissue specificity. The amount of RP expression could be tissue-specific, as such certain RPs would be a limiting factor in a definite tissue.20


Figure 1931601
View larger version (23K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 1. Simplified scheme of rRNA processing in human cells. Only the major pathway and cleavage sites are shown. The name of precursors is indicated on the side of each molecule. The mature rRNA forms (18S, 5.8S, 28S), and the external (5' ETS, 3' ETS) and internal (ITS1, ITS2) spacers are indicated on the first precursor (45S). The principal cleavage sites are indicated by the black triangles. The processing steps possibly affected in DBA cells with mutations in RPS17,18 RPS19,16 RPS24,17 RPL35A,5 RPL5,18 RPL1118 (Gleizes PE, Gazda HT, personal communication), are indicated by red lines.

Another hypothesis focuses on a non-ribosomal function for RPs, that would be decreased by haploin-sufficiency. The identification of the RPS19 interactome has increased speculation about other possible roles for this protein, but has not solved the DBA issue.21

The paper by Rey et al. in this issue of the journal22 presents another suggestive hypothesis based on the observation of increased abnormally spliced forms of FLVCR1 in DBA immature erythroid cells. This has been obtained in cells from patients that did not carry RPS19 mutations, but the same scenario was shown by K562 cells down-regulated for RPS19. The FLVCR1 protein is a heme exporter and the receptor for the subgroup C leukemia retrovirus. This virus causes anemia in infected cats because its Env protein blocks the heme exporter activity. A phenotype similar to DBA (i.e. anemia and craniofacial and limb deformities) is shown by Flvcr1 null mice.23 Although Flvcr1 is not mutated in DBA patients, the similarity among the two phenotypes is striking. The paper by Rey et al. now suggests a possible functional link between the two syndromes that could explain the similar phenotype. The abnormally spliced forms of FLVCR1, that could not produce a normal protein, could play a role in the defective erythropoiesis of DBA patients. The authors suggest that a disruption of heme transport leads to heme toxicity and apoptosis of early erythroid progenitors. Actually, a reduction of CFUe has been found in Flvcr1 null mice.23 Interestingly, human erythroid precursors down-regulated for FLVCR1 exhibit features of defective erythropoiesis, but normal myelopoiesis: an exact recapitulation of DBA pathophysiology.

The definite role of FLVCR1 in regulating erythropoiesis is still unknown. Neither is it known how exactly a defect in RPS19 or other DBA genes can influence FLVCR1 splicing. The identification of splicing factors among RPS19 interactors21 can suggest interesting speculation about a role for RPs in controlling the spliceosome. More experiments are needed to explain the abnormal FLVCR1 splicing in DBA, but the observation is intriguing.

A further interesting hypothesis to explain the pathophysiology of DBA proposes that the impairment of ribosome biogenesis induced by the decrease of an RP (nucleolar or ribosomal stress) might cause p53 activation with a consequent cell growth arrest and/or apoptosis, as schematized in Figure 2.3 Recently, vertebrate models for DBA have been produced that support this hypothesis. Two studies published in Human Molecular Genetics24 and Blood25 propose zebrafish as a model system for studying the molecular mechanism underlying DBA. In both cases the researchers induced RPS19 deficiency by injecting antisense oligonucleotide analogs (morpholinos) into one-cell-stage embryos. Uechi et al.24 report that the decrease of RPS19 causes a dramatic reduction in blood cells and deformities in the head and tail region of the injected embryos. Importantly, the phenotype can be rescued by the injection of rps19 mRNA, but not if it contains mutations found in DBA patients. The authors of the study then induced deficiency for other 20 RPs and grouped the mutants according to the severity of the phenotype. They found that the knock-down of three other RPs causes blood cell defects similar to rps19 mutants. Interestingly, among them is rpl35a which has been recently found mutated in DBA patients.5 In the second study, Danilova et al.,25 after finding that rps19 deficiency results in hematopoietic and developmental abnormalities similar to DBA, address the molecular mechanism of the defects. They found that the decrease of rps19 in the embryos injected with morpholinos induces changes in the transcription of p53 family members. Particularly evident is the increase of deltaNp63 in red blood cell progenitors purified from embryos. Similar alterations of expression of the p53 family were observed in zebrafish with mutations in rps8, rps11 and rps18. Therefore, the Authors propose that ribosomal stress caused by rps19 deficiency in zebrafish results in the upregulation of the p53 family with a consequent imbalance between the network components. This would lead to the observed alterations in development and differentiation. Consistent with their model, the authors could alleviate the phenotype of rps19 mutants by treatment with an inhibitor of p53 function, so suggesting the possibility of a novel drug treatment for DBA by targeting p53.


Figure 2931601
View larger version (36K):
[in this window]
[in a new window]
[Download PPT slide]
  		Figure 2. Ribosomal stress hypothesis. A decrease in the synthesis of an RP and/or rRNA triggers stabilization and/or activation of p53 and, possibly, unbalance with the other p53 family members. According to the susceptibility of the cell type to this alteration the induced response could be apoptosis or cell cycle arrest. This in turn can generate different phenotypical alterations: anemia, hyperpigmentation, growth retardation, malformations.

In addition to these zebrafish models, a new mouse recapitulating DBA has been produced.26 This mouse, obtained by random chemical mutagenesis, presents a R32L mutation in Rps19 and a different phenotype as compared to the RPS19 knock-out mouse produced a few years ago.27

Indeed both mice, Rps19 knock-out and the R32L mutant, are homozygous lethal in the embryo. By contrast, the heterozygous KO mouse is identical to the WT, whereas the heterozygous R32L mouse shows a slightly reduced erythrocyte level, increased apoptosis of bone marrow erythroid precursors, reduced body weight and, most strikingly, dark spots in the limb and tail (dark skin). This phenotype is reminiscent of DBA, that is characterized by defective erythropoiesis, apoptosis of erythroid precursors and reduced body weight. However, this mouse does not carry physical malformations. Moreover, discromy is not normally present in DBA, although sometimes it has been reported in DBA and in Fanconi patients. McGowan et al. have made great efforts to explain the mice discromy. Epitheliocytes from mice defective in rps6 show an increased expression of p53 targets and, especially, Mdm2 and Kitl. This scenario supports the hypothesis that stabilization of p53 is due to the increased availability of free RPs, not used to assemble ribosomal subunits. Some RPs, such as RPL5 and RPL11, were shown to interact with MDM2 and prevent MDM2-dependent p53 ubiquitination and degradation. Kitl, a p53 target, is known to regulate melanocyte migration from the neural crest and their function. Interestingly, the increased number and activity of melanocytes in the epidermis of dark skin mice is due to a p53 effect, because the mouse Trp53–/– Rps19 R32L–/+ shows a phenotype reversal and no dark spots. Authors state that an increased Kitl cannot be involved in the defective erythropoiesis, because mice defective in Kitl (the so-called Steel mice) are anemic and their anemia is not cured by bone marrow transplantation, unlike DBA patients. It is interesting to note that erythroid progenitors from DBA patients show a dramatic response in vitro by the addition of KITL to EPO and IL-3.28 Moreover, the heterozygous defect in the KIT gene is the cause of piebaldism (MIM 172800) in humans, a condition characterized by discromy, but not by anemia.

In conclusion, many strides have been made towards solving DBA mystery. The most striking successes have been obtained in genetics, diagnostics and clinical treatment. In this regard, for the sake of brevity, we refer to the consensus document generated by the DBA community.1

The pathophysiology of DBA has been addressed by a number of elegant and systematic approaches, including the production of cell and animal models. These strategies have shed light on several aspects of DBA and proposed several not mutually exclusive hypotheses, but have failed to obtain a comprehensive picture. Therefore, DBA still remains a ribosomal puzzle.

References

  1. Vlachos A, Ball S, Dahl N, Alter BP, Sheth S, Ramenghi U, et al. Diagnosing and treating Diamond Blackfan anaemia: results of an international clinical consensus conference. Br J Haematol 2008, Jul 30, [Epub ahead of print].
  2. Draptchinskaia N, Gustavsson P, Andersson B, Pettersson M, Willig TN, Dianzani I, et al. The gene encoding ribosomal protein S19 is mutated in Diamond-Blackfan anaemia. Nat Genet 1999;21:169-75.[CrossRef][Web of Science][Medline]
  3. Liu JM, Ellis SR. Ribosomes and marrow failure: coincidental association or molecular paradigm?. Blood 2006;107:4583-8.[Abstract/Free Full Text]
  4. Ebert BL, Pretz J, Bosco J, Chang CY, Tamayo P, Galili N, et al. Identification of RPS14 as a 5q- syndrome gene by RNA interference screen. Nature 2008;451:335-9.[CrossRef][Web of Science][Medline]
  5. Farrar JE, Nater M, Caywood E, McDevitt MA, Kowalski J, Takemoto CM, et al. Abnormalities of the large ribosomal subunit protein, Rpl35a, in Diamond-Blackfan anemia. Blood 2008;112:1582-92.[Abstract/Free Full Text]
  6. Gazda HT, Grabowska A, Merida-Long LB, Latawiec E, Schneider HE, Lipton JM, et al. Ribosomal protein S24 gene is mutated in Diamond-Blackfan anemia. Am J Hum Genet 2006;79:1110-8.[CrossRef][Web of Science][Medline]
  7. Cmejla R, Cmejlova J, Handrkova H, Petrak J, Pospisilova D. Ribosomal protein S17 gene (RPS17) is mutated in Diamond-Blackfan anemia. Hum Mutat 2007;28:1178-82.[CrossRef][Web of Science][Medline]
  8. Gazda HT, Sheen MR, Darras N, Shneider H, Sieff CA, Ball SE, et al. Mutations of the genes for Ribosomal Proteins L5 and L11 are common cause of Diamond-Blackfan anemia [ASH Annual Meeting Abstracts]. Blood 2007;110:421.
  9. Quarello P, Garelli E, Brusco A, Carando A, Pappi P, Barberis M, et al. Multiplex ligation-dependent probe amplification enhances molecular diagnosis of Diamond Blackfan anemia due to RPS19 deficiency. Haematologica 2008;93:1748-50.[CrossRef][Medline]
  10. Campagnoli MF, Ramenghi U, Armiraglio M, Quarello P, Garelli E, Carando A, et al. RPS19 mutations in patients with Diamond-Blackfan anemia. Hum Mutat 2008;29:911-20.[CrossRef][Web of Science][Medline]
  11. Boria I, Quarello P, Avondo F, Garelli E, Aspesi A, Carando A, et al. A new database for ribosomal protein genes which are mutated in Diamond-Blackfan anemia. Hum Mutat 2008, Sep 9, [Epub ahead of print].
  12. Angelini M, Cannata S, Mercaldo V, Gibello L, Santoro C, Dianzani I, et al. Missense mutations associated with Diamond-Blackfan anemia affect the assembly of ribosomal protein S19 into the ribosome. Hum Mol Genet 2007;16:1720-7.[Abstract/Free Full Text]
  13. Crétien A, Hurtaud C, Moniz H, Proust A, Marie I, Wagner-Ballon O, et al. Study of the effects of proteasome inhibitors on ribosomal protein S19 mutants, identified in patients with Diamond-Blackfan anemia. Haematologica 2008;93:1627-34.[CrossRef][Medline]
  14. Gregory LA, Aguissa-Toure AH, Pinaud N, Legrand P, Gleizes PE, Fribourg S. Molecular basis of Diamond-Blackfan anemia: structure and function analysis of RPS19. Nucleic Acids Res 2007;35:5913-21.[Abstract/Free Full Text]
  15. Lam YW, Lamond AI, Mann M, Andersen JS. Analysis of nucleolar protein dynamics reveals the nuclear degradation of ribosomal proteins. Curr Biol 2007;17:749-60.[CrossRef][Web of Science][Medline]
  16. Flygare J, Aspesi A, Bailey JC, Miyake K, Caffrey JM, Karlsson S, et al. Human RPS19, the gene mutated in Diamond-Blackfan anemia, encodes a ribosomal protein required for the maturation of 40S ribosomal subunits. Blood 2007;109:980-6.[Abstract/Free Full Text]
  17. Choesmel V, Fribourg S, Aguissa-Toure AH, Pinaud N, Legrand P, Gazda HT, et al. Mutation of ribosomal protein RPS24 in Diamond-Blackfan anemia results in a ribosome biogenesis disorder. Hum Mol Genet 2008;17:1253-63.[Abstract/Free Full Text]
  18. Robledo S, Idol RA, Crimmins DL, Ladenson JH, Mason PJ, Bessler M. The role of human ribosomal proteins in the maturation of rRNA and ribosome production. RNA 2008;14:1918-29.[Abstract/Free Full Text]
  19. Cmejlova J, Dolezalova L, Pospisilova D, Petrtylova K, Petrak J, Cmejla R. Translational efficiency in patients with Diamond-Blackfan anemia. Haematologica 2006;91:1456-64.[Abstract/Free Full Text]
  20. Ellis SR, Massey AT. Diamond Blackfan anemia: A paradigm for a ribosome-based disease. Med Hypotheses 2006;66:643-8.[CrossRef][Web of Science][Medline]
  21. Orrù S, Aspesi A, Armiraglio M, Caterino M, Loreni F, Ruoppolo M, et al. Analysis of the ribosomal protein S19 interactome. Mol Cell Proteomics 2007;6:382-93.[Abstract/Free Full Text]
  22. Rey MA, Duffy SP, Brown JK, Kennedy JA, Dick JE, Dror Y, et al. Enhanced alternative splicing of the FLVCR1 gene in Diamond Blackfan Anemia disrupts FLVCR1 expression and function that is critical for erythropoiesis. Haematologica 2008;93:1617-26.[CrossRef][Medline]
  23. Keel SB, Doty RT, Yang Z, Quigley JG, Chen J, Knoblaugh S, et al. A heme export protein is required for red blood cell differentiation and iron homeostasis. Science 2008;319:825-8.[Abstract/Free Full Text]
  24. Uechi T, Nakajima Y, Chakraborty A, Torihara H, Higa S, Kenmochi N. Deficiency of ribosomal protein S19 during early embryogenesis leads to reduction of erythrocytes in a zebrafish model of Diamond-Blackfan anemia. Hum Mol Genet 2008;[Epub ahead of print].
  25. Danilova N, Sakamoto KM, Lin S. Ribosomal protein S19 deficiency in zebrafish leads to developmental abnormalities and defective erythropoiesis through activation of p53 protein family. Blood 2008, May 30, [Epub ahead of print].
  26. McGowan KA, Li JZ, Park CY, Beaudry V, Tabor HK, Sabnis AJ, et al. Ribosomal mutations cause p53-mediated dark skin and pleiotropic effects. Nat Genet 2008;40:963-70.[CrossRef][Web of Science][Medline]
  27. Matsson H, Davey EJ, Frojmark AS, Miyake K, Utsugisawa T, Flygare J, et al. Erythropoiesis in the Rps19 disrupted mouse: analysis of erythropoietin response and biochemical markers for Diamond-Blackfan anemia. Blood Cells Mol Dis 2006;36:259-64.[CrossRef][Web of Science][Medline]
  28. Bagnara GP, Zauli G, Vitale L, Rosito P, Vecchi V, Paolucci G, et al. In vitro growth and regulation of bone marrow enriched CD34+ hematopoietic progenitors in Diamond-Blackfan anemia. Blood 1991;78:2203-10.[Abstract/Free Full Text]

Related Articles

Study of the effects of proteasome inhibitors on ribosomal protein S19 (RPS19) mutants, identified in patients with Diamond-Blackfan anemia
Aurore Crétien, Corinne Hurtaud, Hélène Moniz, Alexis Proust, Isabelle Marie, Orianne Wagner-Ballon, Valérie Choesmel, Pierre-Emmanuel Gleizes, Thierry Leblanc, Jean Delaunay, Gil Tchernia, Narla Mohandas, Lydie Da Costa
Haematologica 2008 93: 1627-1634. [Abstract] [Full Text] [PDF]

Enhanced alternative splicing of the FLVCR1 gene in Diamond Blackfan anemia disrupts FLVCR1 expression and function that are critical for erythropoiesis
Michelle A. Rey, Simon P. Duffy, Jennifer K. Brown, James A. Kennedy, John E. Dick, Yigal Dror, Chetankumar S. Tailor
Haematologica 2008 93: 1617-1626. [Abstract] [Full Text] [PDF]




This Article
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Dianzani, I.
Right arrow Articles by Loreni, F.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Dianzani, I.
Right arrow Articles by Loreni, F.
Related Collections
Right arrowRelated Articles