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Review article
Ribosomopathies: human disorders of ribosome dysfunction
Anupama Narla1-3 and Benjamin L. Ebert1,2,4
1Dana-Farber
Cancer Institute, Harvard Medical School, Boston, MA; 2Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School,
Boston, MA; 3Department of Medicine, Children’s Hospital Boston, MA; and 4Harvard Stem Cell Institute, Cambridge, MA
Ribosomopathies compose a collection
of disorders in which genetic abnormalities cause impaired ribosome biogenesis
and function, resulting in specific clinical
phenotypes. Congenital mutations in
RPS19 and other genes encoding ribosomal proteins cause Diamond-Blackfan
anemia, a disorder characterized by hypoplastic, macrocytic anemia. Mutations in
other genes required for normal ribosome
biogenesis have been implicated in other
rare congenital syndromes, Schwachman-
Diamond syndrome, dyskeratosis congenita, cartilage hair hypoplasia, and
Treacher Collins syndrome. In addition, the
5qⴚ syndrome, a subtype of myelodysplastic syndrome, is caused by a somatically
acquired deletion of chromosome 5q, which
leads to haploinsufficiency of the ribosomal
protein RPS14 and an erythroid phenotype
highly similar to Diamond-Blackfan anemia.
Acquired abnormalities in ribosome function have been implicated more broadly in
human malignancies. The p53 pathway pro-
vides a surveillance mechanism for protein
translation as well as genome integrity and
is activated by defects in ribosome biogenesis; this pathway appears to be a critical
mediator of many of the clinical features of
ribosomopathies. Elucidation of the mechanisms whereby selective abnormalities in
ribosome biogenesis cause specific clinical
syndromes will hopefully lead to novel therapeutic strategies for these diseases. (Blood.
2010;115(16):3196-3205)
Identification of ribosomal abnormalities in human disease
In 1999, Draptchinskaia et al reported recurrent mutations in a ribosomal
protein gene, RPS19, in patients with Diamond-Blackfan anemia
(DBA), a rare congenital bone marrow failure syndrome with a striking
erythroid defect.1 Since that initial discovery, mutations in a number of
ribosomal proteins have been identified in up to 50% of patients with
DBA. Moreover, other congenital syndromes have been linked to
defective ribosome biogenesis, including Schwachman-Diamond syndrome (SDS), X-linked dyskeratosis congenita (DKC), cartilage hair
hypoplasia (CHH), and Treacher Collins syndrome (TCS).2 In the 5q⫺
syndrome, a subtype of adult myelodysplastic syndrome, acquired
haploinsufficiency for RPS14 resulting from an interstitial chromosomal
deletion causes a severe refractory anemia.3 Each of these disorders is
associated with specific defects in ribosome biogenesis, which cause
distinct clinical phenotypes, most often involving bone marrow failure
and/or craniofacial or other skeletal defects. The disorders of ribosome
dysfunction have become collectively known as ribosomopathies.
Overview of ribosome biogenesis
The eukaryotic ribosome is composed of 40S and 60S subunits,
which associate to form the translationally active 80S ribosome.
This process requires the coordinated synthesis of 4 ribosomal
RNAs (rRNAs), approximately 80 core ribosomal proteins, more
than 150 associated proteins, and approximately 70 small nucleolar
RNAs (snoRNAs).4,5 The 40S subunit contains 18S rRNA and the
60S subunit contains 28S, 5.8S, and 5S rRNAs. In eukaryotes,
assembly of rRNA and ribosomal proteins, along with associated
proteins and snoRNAs, occurs in the nucleolus, leading to the
production of pre-60S and pre-40S preribosomal particles. These
particles are exported to the cytoplasm where the final steps in
assembly and maturation of ribosomes occur.6 A simplified overview of this process is outlined in Figure 1.
Submitted October 26, 2009; accepted January 27, 2010. Prepublished online as
Blood First Edition paper, March 1, 2010; DOI 10.1182/blood-2009-10-178129.
3196
Mature rRNAs are produced from precursor rRNAs after a
series of chemical modifications and nucleolytic cleavages. Whereas
RNA polymerase III transcribes the 5S rRNA, RNA polymerase I
transcribes a 45S precursor transcript that is processed into the
5.8S, 18S, and 28S rRNAs. Ribosomal proteins assemble onto
precursor rRNA transcripts and facilitate a series of endonucleolytic and exonucleolytic cleavages required for generation of the
mature rRNAs. Haploinsufficiencies for distinct ribosomal proteins
have been linked to defects at distinct steps in pre-rRNA processing, which are outlined in Figure 1.2 Several ribosomal proteins
have extraribosomal functions, including replication and DNA
repair, so mutations in ribosomal proteins may have effects that are
independent of the protein translation machinery.7,8
Clinical syndromes
Although there is an abundance of genetic and experimental
evidence that mutations in ribosomal genes cause impaired erythropoiesis in DBA and the 5q⫺ syndrome, ribosome dysfunction may
also play a role in other congenital syndromes, which are summarized in Table 1. Clinical images of selected physical abnormalities
seen in these syndromes are shown in Figure 2.
Diamond-Blackfan anemia
DBA was originally described by Josephs in a review of anemia in
infancy and childhood in 193612 and later categorized by Diamond and
Blackfan as a congenital hypoplastic anemia.12,13 The disorder is
characterized by anemia, macrocytosis, reticulocytopenia, and a selective decrease or absence of erythroid precursors in an otherwise
normocellular bone marrow.9 The incidence is estimated to be 4 to 5
© 2010 by The American Society of Hematology
BLOOD, 22 APRIL 2010 䡠 VOLUME 115, NUMBER 16
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BLOOD, 22 APRIL 2010 䡠 VOLUME 115, NUMBER 16
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Figure 1. Simplified schematic of eukaryotic ribosome biogenesis. Adapted from Liu and Ellis2 with
permission.
cases per million live births, and there are 595 patients currently
registered in the Diamond-Blackfan Anemia Registry of North America
(A. Vlachos, Schneider Children’s Hospital, Division of Hematology/
Oncology, personal written communication, January 2010). The majority of patients are diagnosed in the first year of life, with pallor and
lethargy being the most common presenting symptoms. There is often a
family history of the disease, and approximately 45% of cases are
thought to be autosomal dominant.14 Other notable characteristics of the
disease include an elevated red blood cell adenosine deaminase level,
the presence of fetal membrane antigen “i,” and a range of physical
abnormalities seen in 40% to 62% of patients and ranging from short
stature to thumb abnormalities to cardiac defects.9 Current therapies
include steroids and chronic transfusions, with the only definitive
treatment being bone marrow transplantation.15
Since the initial description in 1999 by Draptchinskaia, mutations in
RPS19, RPS24, RPS17, and RPL35A have been identified in approximately one-third of patients with DBA.16-19 More recently, Gazda et al
identified mutations in RPL5 and RPL11 in an additional 11.4% of
patients and noted that mutations in RPL5 were associated with a higher
frequency of physical abnormalities, including cleft lip and/or palate,
whereas mutations in RPL11 had more isolated thumb abnormalities
compared with patients with mutations in RPS19.20 Subsequent work by
the Czech DBA registry revealed that, in the 10 patients with either a
RPL5 or RPL11 mutation, there was a thumb abnormality, whereas none
Table 1. Summary of known and suspected ribosomopathies with details about clinical characteristics, cancer
risk, and diagnostic work-up
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BLOOD, 22 APRIL 2010 䡠 VOLUME 115, NUMBER 16
A
B
C
Figure 2. Selected physical abnormalities seen in ribosomopathies. (A) Left panel: Patient with DBA, illustrating the characteristic craniofacial abnormalities (courtesy of J.
Lipton). Note the absent lower eyelashes, deformed external ears, and micrognathia. Second panel: An example of the triphalangeal thumb seen in DBA patients (courtesy of
E. Atsidaftos and E. Muir). First panel: Reprinted from Lipton and Ellis9 with permission. (B) These panels illustrate the diagnostic triad of DKC, including dystrophic fingernails,
lacy/reticular pigmentation on neck and trunk, and oral leukoplakia (courtesy of B. Alter). Reprinted from Savage and Alter10 with permission. (C) The craniofacial abnormalities
of a patient with TCS are illustrated (courtesy of M. R. Passos Bueno). Note the down slanting palpebral fissures, malar and maxillary hypoplasia, and malformation of the ears.
Reprinted from Passos-Bueno et al11 with permission.
of the 7 patients with RPS19 mutations in the registry exhibited such an
anomaly.21 This may indicate that mutations in different ribosomal
genes lead to distinct clinical phenotypes. A large-scale screen of RP
genes in the DBA population has also revealed candidate mutations in
RPS7, RPL36, RPS15, and RPS27A.20 Despite extensive investigation,
mutations have not been discovered in any genes other than those
encoding ribosomal proteins for patients with DBA.
Mutations in RPS19 and in RPS24, the first 2 mutations
identified in DBA, impair pre-rRNA processing of the 18S rRNA,
which leads to decreased production of the 40S ribosomal subunit.22-25 Moreover, there is evidence that depletion of any of the
RPS proteins causes a reduction in the amount of free 40S subunits
and a significant reduction in the amount of mature 80S ribosomes
(with the exception of RPS25, which has not been shown to be
mutated in DBA).4 In contrast, when RPL proteins, including
RPL35A, are depleted, the amount of the 60S subunit is reduced, as
is the level of mature 80S ribosomes.4 Overall, it is clear that
mutations in a range of ribosomal proteins ultimately lead to a
decrease in the number of mature ribosomes, which would be
expected to have a number of consequences for a cell.
5qⴚ syndrome
The 5q⫺ syndrome was first described in 1974 by van Den Berghe
in 3 patients with refractory anemia and an interstitial deletion of
the long arm of chromosome 5.26 Patients with the 5q⫺ syndrome,
an independent subtype of myelodysplastic syndrome (MDS) in the
World Health Organization classification system in which del(5q)
is the sole cytogenetic abnormality, characteristically have a severe
macrocytic anemia, normal/elevated platelets with hypolobulated
micromegakaryocytes, and a relatively low rate of progression to
acute myeloid leukemia (AML) compared with other types of
MDS.27 Lenalidomide, a derivative of the immunomodulatory
agent thalidomide, is highly effective for patients with the 5q⫺
syndrome. In a phase 2 trial in low-risk MDS patients with 5q
deletions, lenalidomide treatment decreased transfusion requirement in 76% of patients, and 61% of patients had a complete
cytogenetic response.28 Lenalidomide has many biologic activities,
including the promotion of erythropoiesis, modulation of cytokine
production, inhibition of 2 phosphatases on 5q (CDC25C, and
PP2A) and the activation of Rac1 and RhoA.28-32
RPS14 was identified as a 5q⫺ syndrome gene in an RNA
interference screen of each gene within the 5q⫺ syndrome
common deleted region.3,33 In patients with the 5q⫺ syndrome,
1 allele of RPS14 is deleted,3 and haploinsufficient expression of
RPS14 has been confirmed in patient samples.3,34 Decreased
expression of RPS14 causes impaired erythropoiesis, with relative
preservation of the other lineages. Conversely, overexpression of
RPS14 in samples from patients with the 5q⫺ syndrome rescues
erythropoiesis. It has also been shown that RPS14 deficiency
causes a block in the processing of the 18S rRNA and the formation
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BLOOD, 22 APRIL 2010 䡠 VOLUME 115, NUMBER 16
of the 40S ribosomal subunit.3 A murine model has now been
developed in which coordinate deletion of loci syntenic with the
common deleted region of the 5q⫺ syndrome, including haploinsufficiency for Rps14, recapitulates the macrocytic anemia characteristic of the disease.35 Acquired haploinsufficiency for RPS14 in the
5q⫺ syndrome is therefore analogous to the inactivating mutations
in RPS19 and other ribosomal genes in DBA.
Heterozygous deletions of chromosome 5q in MDS are large,
and haploinsufficiency for multiple genes probably contributes to
the phenotype of the 5q⫺ syndrome.33 For example, haploinsufficiency for miR-145 and miR-146a may contribute to thrombocytosis, and haploinsufficiency for EGR1 may contribute to the clonal
advantage of the del(5q) clone.36-39 Nevertheless, in vitro and in
vivo studies indicate that the erythroid defect, the aspect of the 5q⫺
syndrome phenotype most analogous to DBA, is caused by RPS14
haploinsufficiency.3,35
Schwachman-Diamond syndrome
SDS was first reported in 1964 by Schwachman et al in a group of 5
children being followed in a cystic fibrosis clinic at Harvard
University.40 SDS is an autosomal recessive disease, with an
incidence estimated at 1 in 50 000 births, characterized by exocrine
pancreatic insufficiency, ineffective hematopoiesis, and an increased risk of leukemia. Patients often present with steatorrhea
and failure to thrive from the pancreatic insufficiency. Patients may
also present with serious infections as a result of neutropenia, the
most common hematologic problem in SDS. Other signs may
include anemia, thrombocytopenia, short stature, and skeletal
abnormalities.41 Patients with SDS are also at an increased risk for
myelodysplasia and malignant transformation. Supportive measures for patients with SDS include pancreatic enzymes, antibiotics, transfusions, and granulocyte colony-stimulating factor. The
only definitive therapy is bone marrow transplantation.42
In 2003, Boocock et al reported causal mutations in the SBDS
gene, named after Schwachman-Bodian-Diamond.43 Approximately 90% of SDS patients have been found to have biallelic
mutations in SBDS.43 It was also shown that 75% of these
mutations are the result of a gene conversion with an adjacent
pseudogene, SBDSP, which shares 97% homology with SBDS.43
The 250-amino acid SBDS protein is highly conserved through
evolution, and the SBDS mRNA is ubiquitously expressed. The
structure and function of SBDS are not yet known, but mounting
evidence suggests that the gene plays a role in ribosome biogenesis
and RNA processing.44 In patient bone marrow samples, translationrelated gene expression is the most prominently regulated gene
cluster compared with a number of other pathways known to be
defective in various marrow failure conditions, including hematopoietic transcription factors and telomere maintenance.45 SDS cells
have also been noted to have abnormal expression of multiple
genes involved in ribosome biogenesis and rRNA and mRNA
processing and to have decreased expression of several ribosomal
protein genes involved in cell growth and survival, including RPS9,
RPS20, RPL6, RPL15, RPL22, RPL23, and RPL29.45 Furthermore,
SBDS has been shown to cosediment with the 60S ribosomal
precursor subunit in sucrose gradients and to associate with the 28S
rRNA, which is a component of the 60S subunit.44
Although these findings provide evidence for a role of SBDS in
ribosome biogenesis, SBDS is a multifunctional protein, and
nonribosomal activities may play a dominant role in the clinical
phenotype. In particular, SBDS has been shown to have a role in
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stabilizing the mitotic spindle,46,47 which might indicate a role for
SBDS in proliferation and/or chromosome segregation, thereby
contributing to chromosomal instability and some component of
bone marrow failure.
Dyskeratosis congenita
The first case report that linked the classic triad of abnormal skin
pigmentation, oral leukoplakia, and nail dystrophy was in 1910.48
Subsequently, bone marrow failure and a range of other abnormalities were identified in similar patients, and the disorder became
known as DKC.49 Patients usually present during the first decade of
life with skin hyperpigmentation and nail changes. Almost 90% of
patients will eventually develop a peripheral cytopenia of 1 or more
lineages, and bone marrow failure is the major cause of death. The
estimated prevalence of DKC is 1 case in 1 million people, and the
male-to-female ratio is approximately 3:1.50,51 Patients with DKC
have a high risk of developing leukemia, solid tumors, and
pulmonary fibrosis. Management includes steroids and supportive
measures; the only curative option is stem cell transplantation, but
the success rate of stem cell transplantation is limited because of a
high prevalence of fatal pulmonary complications.10
DKC is a heterogeneous disorder, but in all characterized cases,
the causative mutations are in components of the telomerase
complex. Telomerase is an enzyme complex composed of telomerase reverse transcriptase (TERT), telomerase RNA (TERC), and
dyskerin, which adds specific DNA sequence repeats to the ends of
chromosomes and counters some of the normal shortening that
occurs during DNA replication.52 X-linked DKC, which has a more
severe phenotype compared with the autosomal dominant form of
DKC, is caused by a mutation in DKC1, which encodes dyskerin.53
One of the functions of dyskerin is to act as a nucleolar protein
associated with the snoRNPs involved in rRNA modification.
Dyskerin associates with a specific group of snoRNPs known as
H/ACA, which function in the pseudo-uridylation of rRNAs.2
However, the functional consequences of this defect in pseudouridylation remain unclear.
Patients with the autosomal dominant form of DKC have been
found to have mutations in TERC, and 2 families with the
autosomal recessive form of DKC have been found to have
mutation in TERT, suggesting that disruption of the telomerase
complex alone could result in defective hematopoiesis.54 Also of
note, mutations in the TERT gene have been identified in some
patients with aplastic anemia and short telomeres.55 Further
confusing the picture is a hypomorphic Dkc1 mutant mouse, which
demonstrated impaired ribosomal RNA pseudo-uridylation before
the onset of clinical features of DKC, whereas reductions in
telomere length became evident only in later generations.56 Although it is clear that telomeres play a significant role in the
pathogenesis of DKC, there may be a distinct contribution from
ribosomal defects. Ongoing work examining genotype/phenotype
correlations and basic mechanisms will hopefully elucidate the true
contributions of telomerase activity and impaired ribosomal biogenesis to the pathophysiology of DKC.
Cartilage hair hypoplasia
CHH was first described in several Amish families by McKusick et
al in 1965 as a form of short-limbed dwarfism resulting from
skeletal dysplasia.57 The incidence in Old Order Amish population
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is 1 in 1300 newborns57 and in people of Finnish descent, 1 in
20 000.58 Outside of these populations, the condition is rare and the
specific incidence is not known.59 The autosomal recessive syndrome is also characterized by hypoplastic hair, immune dysfunction, and an increased predisposition to malignancies, especially
non-Hodgkin lymphoma and basal cell carcinoma.41 Hematologic
abnormalities can include a macrocytic anemia and lymphopenia.
Management is again supportive, with stem cell transplantation
being the only curative option. Transplantation is particularly
indicated in patients with severe T-cell immunodeficiency with or
without concomitant B-cell disease, although transplantation does
not correct the skeletal abnormalities.60
In 2001, Ridanpää et al identified mutations in the untranslated
RMRP gene as causative for this pleiotropic disease, with an
ancient founder mutation in Finland.61 RMRP encodes the RNA
component of the mitochondrial RNA processing complex (RNase
MRP), which is primarily located in the nucleolus and is classified
as a snoRNA. snoRNAs form small nucleolar ribonucleoprotein
complexes (snoRNPs) within the nucleolus and are involved in
various steps in the synthesis of ribosomal RNA.62 One of the
important functions of the RNase MRP complex is to cleave the
precursor rRNA, which contributes to the maturation of the 5⬘ end
of the 5.8S rRNA. RNase MRP also shares protein subunits and
contains a structurally related RNA subunit with another snoRNP,
which is involved in tRNA precursor maturation.
Mutations in RMRP have also been associated with anauxetic
dysplasia, an autosomal recessive growth disorder without marrow
involvement.63 Thiel et al showed that, in cells overexpressing the
RMRP mutants seen in anauxetic dysplasia, there were decreased
cyclin A2 levels.63 In contrast, cells with the CHH founder RMRP
mutation had significantly increased levels of cyclin B2 mRNA.63
Cyclin B2 is known to contribute to chromosomal instability
through alterations of the mitotic spindle checkpoint,64 which
suggests another possible explanation for the bone marrow dysfunction seen in CHH. Recent work by Maida et al has revealed a
connection between RMRP and TERT.65 The clinical heterogeneity
seen in CHH and in anauxetic dysplasia might be attributable to
differences in the magnitude and type of alteration in ribosomal
assembly, telomere dysfunction, and cyclin B2 levels caused by
various RMRP mutations.63
Treacher Collins syndrome
In 1900, the British ophthalmologist Edward Treacher Collins
described the essential features of a syndrome, which included
characteristic craniofacial changes66 that arise from symmetrically
and bilaterally diminished growth of the structures derived from
the first and second pharyngeal arch, groove, and pouch.67 TCS is
now known to be an autosomal dominant condition with an
estimated incidence of 1 in 50 000 live births.68 Patients often have
complications from the craniofacial dystosis, which can include
issues with airway, swallowing, brain development, and hearing.67
Management of these patients requires a multidisciplinary approach involving craniofacial surgeons, orthodontists, ophthalmologists, otolaryngologists, and speech pathologists.67
In 1996, TCOF1 was identified as the gene responsible for TCS;
TCOF1 encodes a protein known as treacle.69 Immunofluorescence
studies demonstrated that treacle colocalizes with upstream binding
factor and RNA polymerase I in the nucleolus and that treacle is a
constituent of one of the preribosomal ribonucleoprotein (preRNP) complexes. Further data have shown that treacle is essential
BLOOD, 22 APRIL 2010 䡠 VOLUME 115, NUMBER 16
for the transcription of ribosomal DNA and that it may also play a
role in the methylation of rRNA.70 Mice haploinsufficient for Tcof1
exhibit diminished production of ribosomes, and this deficiency
correlates with decreased proliferation of both neural ectoderm and
neural crest cells.71 Interestingly, a study by Jones et al showed that
chemical and genetic inhibition of p53 activity in these mice can
prevent the craniofacial abnormalities.72 This is of special note, and
the link between p53 and ribosomopathies is discussed in greater
detail later in this review.
Although the craniofacial defects in TCS are highly similar to
those seen in some cases of DBA and there is a suggestion of a
ribosomal basis for the pathophysiology, there are no hematologic
abnormalities associated with TCS. This adds yet another fascinating wrinkle to our understanding of the ribosomopathies.
Diagnosis of ribosomopathies: current and
future strategies
When a child is first suspected of having a bone marrow failure
syndrome, he or she undergoes a personal and family history,
physical examination, and a series of radiographic and laboratory
evaluations. The laboratory data includes a CBC with differential,
smear review, reticulocyte count, erythropoietin levels, and hemoglobin electrophoresis to assess the severity of anemia and the level
of ineffective erythropoiesis. Ultimately, a bone marrow aspiration
and biopsy is required. In addition, there are now specific
diagnostic tests for the ribosomopathies ranging from SBDS gene
testing for SDS and RMRP sequencing in CHH. These tests are
summarized in Table 1. In the future, given the shared ribosomal
pathogenesis of these disorders, other diagnostic tests might be
developed. These may include sequencing of the relevant ribosomal genes or analysis of aberrant preribosomal RNA precursor
accumulation by quantitative RT-PCR. After the diagnosis of an
inherited bone marrow failure syndrome, patients and families can
be referred to a number of resources. Useful links can be found at
www.marrowfailure.cancer.gov.
Cellular consequences of ribosomal
haploinsufficiency
It is clear that mutations in genes encoding ribosomal proteins or
other proteins involved in ribosome biogenesis cause specific
phenotypes, restricted to particular cell types, despite the universal
expression of ribosomal proteins and the ubiquitous requirement
for protein synthesis. Specifically, defects in ribosome biogenesis
or function appear to be capable of causing anemia and other
hematologic phenotypes, defects in growth and development, and
congenital anomalies, such as craniofacial defects and thumb. The
fundamental question of how a mutation in a ribosomal protein,
which would be expected to have widespread and diverse effects
throughout an organism, can lead to such selective defects is not
fully answered, but possible mechanisms are outlined in this
section.
A leading hypothesis, depicted in Figure 3A, posits that
ribosomal haploinsufficiency leads to disrupted ribosome biogenesis and an accumulation of free ribosomal proteins that bind
MDM2, a repressor of p53. The consequent activation of p53 leads
to apoptosis and cell-cycle arrest, which ultimately leads to
anemia.74,75 Another potential mechanism, depicted in Figure 3B, is
that defective maturation of ribosomal subunits could delay
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BLOOD, 22 APRIL 2010 䡠 VOLUME 115, NUMBER 16
A
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B
40S
60S
p53
-
MDM2
-
40S
60S
p53
-
MDM2
-
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}
}
Figure 3. Potential mechanisms for the cellular consequences of ribosomal haploinsufficiency. (A) Top panel: Normal cell in unstressed conditions, with unperturbed
ribosome biogenesis and steady levels of p53. Bottom panel: Ribosomal haploinsufficiency leads to up-regulation of rpL11, which binds to MDM2 causing p53 activation, which
results in apoptosis and cell-cycle arrest. (B) Top panel: Normal hemoglobin synthesis, with the coordinated production of heme and globin. Bottom panel: Relative excess of
free heme leads to oxidative stress and hemolysis through a variety of mechanisms.73
translation of globin genes, resulting in a relative excess of free
heme, which would also lead to erythroid-specific apoptosis and
anemia.76 Both of these potential mechanisms are discussed in
more detail in “Ribosomal haploinsufficiency in human disease.”
Alternate mechanisms include pathogenic functions for the aberrantly accumulated ribosomal precursors and aberrant translation
by defective ribosomes.77 Further understanding of the molecular
basis of ribosomopathies should lead to more insights into the
clinical phenotypes of these diseases and to the development of
novel therapeutic strategies.
haploinsufficiency of RPL35A is inferred based on genomic
deletions or a nonsense splicing defect in 1 allele.19 In the 5q⫺
syndrome, 1 allele of RPS14 is deleted, without mutation or
epigenetic silencing of the intact allele, resulting in haploinsufficient expression of RPS14.3,34 To date, there is no evidence of
biallelic inactivation of any ribosomal genes in human disease.
Ribosomal haploinsufficiency in human
disease
The central hematopoietic defects in DBA are thought to be the
hypoproliferation of erythroid cells and the enhanced sensitivity of
hematopoietic progenitors to apoptosis.80 Both in vitro and in vivo
models of ribosomal haploinsufficiency recapitulate the hematopoietic phenotype of DBA. These models have informed the biology
of ribosomopathies and will be critical resources for the development of and testing of novel therapies. A limitation to note,
particularly with the in vitro models, is that the degree of
knockdown of ribosomal proteins is often greater than 50%;
therefore, these are not perfect models for studying haploinsufficiency. It is very possible that the mechanism and phenotype of
these disorders are influenced by the precise level of ribosomal
activity, which is why ongoing work to generate optimal models
will be essential for further progress in the field.
Decreased expression of RPS19 in vitro is sufficient to phenocopy many aspects of the DBA phenotype. Knockdown of RPS19
RPS19 was the first ribosomal gene implicated in human disease
and is the most frequently mutated gene in DBA with a total of
77 mutations having been described.78 The majority are whole gene
deletions, translocations, or truncating mutations (nonsense or
frameshift) universally present in only a single allele.16,78 One
group of missense mutations in RPS19 alters the nucleolar localization signal with a resulting dramatic decrease in protein expression.16 This suggests that haploinsufficiency for a ribosomal gene
in the nucleolus is the basis for the pathology.78 Cmejlova et al
showed that the level of translation was on average 48% to 73% of
controls in both unstimulated and phytohemagglutinin-stimulated
DBA lymphocytes irrespective of mutations in RPS19.79 In the
DBA patients who have been found to have RPL35A mutations,
In vitro and in vivo models of ribosomal
haploinsufficiency
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using RNA interference in primary human hematopoietic progenitor cells from normal persons causes a severe defect in the
differentiation and proliferation of erythroid progenitor cells.81,82
Similarly, primary CD34⫹ cells from DBA patients have a higher
number of apoptotic cells compared with normal CD34⫹ cells,83
and mononuclear cells derived from patients with DBA fail to
proliferate in response to erythropoietin and form smaller erythroid
colonies in methylcellulose compared with normal patients.84 The
hematopoietic defect caused by RPS19 deficiency can be rescued
by forced overexpression of RPS19. Expression of a RPS19
transgene by retroviral vectors in CD34⫹ bone marrow cells from
DBA patients significantly improved erythropoiesis.85 In addition,
the erythroid defect can be partially rescued by treatment with
corticosteroids, the primary pharmacologic treatment for DBA.82
Both zebrafish and murine models have been generated with
mutations in or decreased expression of ribosomal genes. Two
groups have knocked down expression of RPS19 in zebrafish using
morpholinos with similar findings.86,87 The RPS19 morphants have
hematopoietic and developmental abnormalities that resemble
DBA, and this phenotype is partially rescued by treatment with
corticosteroids.86 As with RNA interference studies, morpholinos
may decrease ribosomal gene expression by greater than 50%,
potentially resulting in more severe hematopoietic defects.
Genetically engineered mice have the potential to model
ribosomal haploinsufficiency more precisely. An initial Rps19
knockout mouse model was lethal in the homozygous state and did
not have a DBA phenotype in the heterozygous state, although the
heterozygous mice did not have the expected decline in RPS19
levels.88 Murine models now exist with heterozygous missense
mutations in Rps19 or Rps20 or conditional deletion of Rps6. The
RPS19 and RPS20 mice have only a mild macrocytic anemia, although
this relatively benign phenotype may be explained by the fact that the
mutations cause hypomorphic alleles rather than true haploinsufficiency.74 Mice with conditional deletion of a set of genes on 5q,
including Rps14, develop a severe macrocytic anemia, consistent with
the phenotype of the 5q⫺ syndrome and DBA.35
Studies using the RPS6 mice provide further insight into the biology
of ribosome dysfunction. Conditional homozygous deletion of RPS6
using CD4-Cre abolished T-cell development. In contrast, haploinsufficiency did not have any effect on T-cell maturation in the thymus.
However, once T cells were activated and induced to proliferate, T cells
with RPS6 haploinsufficiency were unable to proliferate.89 Thus, cells
with ribosomal haploinsufficiency may synthesize intact ribosomes
sufficiently when the cells proliferate slowly but have more defective
ribosome biogenesis in states of rapid proliferation, including hematopoiesis, which might explain some of the lineage specificity seen in
ribosomopathies.
The role of p53
Evidence from a variety of model systems indicates that p53 is
activated by ribosome dysfunction. Among its myriad of roles, p53
is known to play a fundamental role in the surveillance of protein
translation.90 The murine double minute 2 protein (MDM2, or
HDM2 in humans) functions as a link between ribosome biogenesis and the p53 pathway. MDM2 is a central regulator of p53,
acting as a ubiquitin ligase that leads to the degradation of p53.91
MDM2 has also been shown to bind specifically to several free
ribosomal proteins, including RPL5, RPL23, RPL11, RPS7, and
RPL26.92-98 In an elegant series of experiments, nucleolar disruption, experimentally induced by treatment with actinomycin D, was
BLOOD, 22 APRIL 2010 䡠 VOLUME 115, NUMBER 16
shown to lead to the release of RPL11 and other ribosomal proteins
into the nucleoplasm, the binding of RPL11 to MDM2, the
inhibition of MDM2 activity, and the consequent accumulation of
p53.75,95 A schematic view of this pathway is shown in Figure 3A.
In the Rps19 mutant mouse model described previously, induction of p53 and p53 target genes were identified in the hyperpigmented foot pads of the mice. These mice also had a decreased
hematocrit and increased MCV. When the Rps19 mutant mouse line
had 1 allele of p53 genetically inactivated, there was an increase in
red blood cell count and decrease in MCV. Homozygous inactivation of p53 in Rps19 mutant mice fully corrected the hematologic
phenotype.74 Furthermore, when the mice with the conditional
deletion of a set of genes found in the common deleted region of the
5q⫺ syndrome were crossed with p53 null mice, there was a
complete rescue of the erythroid phenotype.35 These findings
indicate that p53 is critical for the macrocytic anemia caused by
ribosomal dysfunction.
Studies in mice with conditional inactivation of Rps6 have
further elucidated the mechanism of MDM2-mediated induction of
p53 by ribosomal haploinsufficiency.75 Conditional deletion of
Rps6 in murine liver inhibited 40S (but not 60S) ribosomal
biogenesis, and the liver in affected mice did not regenerate
normally after partial hepatectomy resulting from cell-cycle arrest.99 The increased free RPL11 that was generated by the
mechanism shown in Figure 3A was found to be the result of the
increased translation of rpL11 mRNAs through derepression of
5⬘-TOP mRNA translation.75 If specific agents that target the
signaling components involved in rpL11 up-regulation and/or that
regulate 5⬘-TOP mRNA translation could be developed, there is the
potential to alleviate the block in ribosomal biogenesis without
involving other pathways involved in p53 induction.
Balance of heme and globin
An alternative or potentially complementary hypothesis for the
erythroid specificity of DBA involves an imbalance between heme
and globin production, as represented in Figure 3B. Erythroid
progenitor cells are notable for their incredibly rapid rate of
proliferation, doubling every 12 to 24 hours,100 and for their need to
synthesize globin at a tremendous pace. This elevated rate of
proliferation requires a very high level of ribosome biogenesis and
ribosomal activity. Cells with impaired ribosome biogenesis may
not be able to maintain the level of globin synthesis required by
erythroid progenitor cells, which raises the possibility that the
erythroid phenotype seen in many ribosomopathies is the result of a
decrease in globin synthesis, which leads to a relative excess of free
heme resulting in apoptosis and anemia.
In a murine model with conditional inactivation of FLVCR, a heme
export protein, animals develop a severe macrocytic anemia, indicating
that erythroid heme toxicity can cause a phenotype similar to ribosomal
haploinsufficiency.101 FLVCR is the receptor for the feline leukemia
virus, subgroup C (FeLV-C), which causes a red cell aplasia in cats.101
Pro-erythroblasts may need FLVCR both to prevent heme toxicity and
to preserve adequate heme supply. Homozygous conditional inactivation of FLVCR in the murine bone marrow causes a macrocytic anemia,
reticulocytopenia, and maturation arrest at the pro-erythroblast stage,
phenocopying the hematologic phenotype in DBA patients. Of note, the
mutant mouse embryos were also smaller than controls and had digit
abnormalities, wide-spaced eyes, and flattened facies. The conditionally
deleted FLVCR⫺/⫺ mice were also noted to accumulate iron.76
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BLOOD, 22 APRIL 2010 䡠 VOLUME 115, NUMBER 16
RIBOSOMOPATHIES
These experiments indicate that mutations in ribosomal proteins
could cause defective ribosomal subunit maturation, leading to
delayed globin translation and a relative excess of free heme, with
FLVCR acting as a safety valve. This hypothesis implies that the
severity of the heme toxicity may modulate the clinical phenotype
of patients with ribosomal haploinsufficiency, although accumulation of heme in erythroid progenitor cells from patients with DBA
has not been examined. To date, no mutations have been identified
in FLVCR in any of the ribosomopathies.
Ribosomal dysfunction and cancer
All of the pediatric bone marrow failure syndromes linked to
ribosomal dysfunction appear to have an increased incidence of
cancer, although the type and frequency vary considerably, adding
yet another intriguing piece to the story.
For patients with SDS, a 2005 review of the French Severe
Chronic Neutropenia Registry estimated the risk of MDS or AML
at 19% at 20 years and 36% at 30 years.102 A recent report from the
National Cancer Institute dyskeratosis congenita cohort revealed a
ratio of observed to expected cancers of 11-fold compared with the
general population with the observed/expected ratios being significantly elevated for tongue cancer and AML.103 Of the 123 Finnish
patients with CHH who were followed for a mean 19.2 years,
14 cases of cancer were diagnosed. Non-Hodgkin lymphoma was
the most frequent tumor, with 9 cases reported, followed by
squamous cell carcinoma, leukemia, and Hodgkin lymphoma. Nine
of the 14 tumors were in patients younger than 45 years. In
addition, 10 patients in the cohort developed basal cell carcinoma
of the skin.104 The authors concluded that there was a 7-fold
increase overall cancer rate in patients with CHH compared with
the normal population, with the risk of non-Hodgkin lymphoma
and skin cancer being even higher. A predisposition for cancer has
not been reported in TCS, which is not associated with hematologic
manifestations.
For DBA, the predisposition to cancer is less clear. In the latest
update from the DBA Registry, of the 568 patients registered, there
were 15 cancers in 13 patients. These included 4 cases of
MDS/AML, 3 cases of osteosarcoma, 2 colon cancers, 1 soft tissue
sarcoma, 2 squamous cell carcinomas (1 oral and 1 vaginal),
2 breast cancers, and 1 uterine cancer (A. Vlachos, Schneider
Children’s Hospital, Division of Hematology/Oncology, personal
written communication, January 2010).
An animal model that ties together ribosomopathies and tumorogenesis was developed in 2004 by Amsterdam et al.105 They
generated several hundred lines of zebrafish, each of which was
heterozygous for an embryonic lethal recessive mutation. Eleven of
the 12 lines with elevated cancer incidence had a mutation in
different types of ribosomal protein genes.105 Of note, the distribution and penetrance of cancers in these zebrafish lines phenocopy
the malignancies in p53 null zebrafish. Further work indicates that
the tumors in zebrafish with ribosomal haploinsufficiency lose
expression of p53.106
3203
Studies in human cancer specimens have also yielded some
tantalizing clues. The gene encoding nucleophosphomin (NPM1 or
B23) has been show to bind to p53 and its associated proteins,
including MDM2.107 NPM1 has also been shown to be mutated in
up to 60% of normal karyotype adult AML107 and to be involved in
certain cases of anaplastic large cell lymphoma, MDS, and acute
promyelocytic leukemia.108 NPM1 is an abundant protein located
in the nucleolus and functions as an RNA-binding nucleolar
phosphoprotein involved in preribosomal assembly. It also serves
as a shuttle between the nucleoplasm and the cytoplasm for both
nucleic acids and proteins.107 NPM1 heterozygous mice develop a
hematologic syndrome similar to MDS.109
Although provocative links have been drawn between bone
marrow failure syndromes, ribosome dysfunction, p53 activation,
and tumor development, key mechanisms remain incompletely
understood.
In conclusion, disease-causing mutations in a collection of
congenital and acquired syndromes result from defective ribosome
biogenesis and function. In at least some of these cases, ribosomal
dysfunction appears to be central to the molecular pathology of the
disorder. Different defects in ribosome biogenesis have now been
associated with distinct clinical phenotypes. These phenotypes,
including impaired erythropoiesis and craniofacial abnormalities,
have been linked to p53 activation. The identification of additional
disease alleles and characterization of their functional effects will
further inform the biology of ribosomopathies and will hopefully
lead to novel therapeutic strategies for the treatment of
ribosomopathies.
Acknowledgments
The authors thank Eva Atsidaftos (Diamond-Blackfan Anemia
Registry), Ellen Muir (Diamond-Blackfan Anemia Surveillance
and Awareness Program), Blanche Alter, Jeffrey Lipton, Adrianna
Vlachos, and Maria Rita Passos Bueno for clinical images and
registry data; Gretchen Jones and Rachel Murphy for assistance
with figure generation; and Steven Ellis for critical review of this
manuscript.
This work was supported by the National Institutes of Health
(R01 HL82945).
The authors regret the omission of many important references
because of length constraints.
Authorship
Contribution: A.N. and B.L.E. wrote the manuscript.
Conflict-of-interest disclosure: B.L.E. received research funding from GlaxoSmithKline. A.N. declares no competing financial
interests.
Correspondence: Benjamin L. Ebert, Brigham and Women’s
Hospital, Karp Research Bldg, 1 Blackfan Cir, 5th Fl, CHRB
05.211, Boston, MA 02115; e-mail: [email protected].
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From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
2010 115: 3196-3205
doi:10.1182/blood-2009-10-178129 originally published
online March 1, 2010
Ribosomopathies: human disorders of ribosome dysfunction
Anupama Narla and Benjamin L. Ebert
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