BACKGROUND
protein synthesis is essential to the survival and growth of cells.
ribosomes, the sites where translation occurs, therefore play a fundamental role in cell biology.
in human cells, ribosome biogenesis occurs in the nucleolus: it requires the transcription of four ribosomal rna  species and their assembly with  <dig> ribosomal proteins  in order to produce the small  and large  ribosomal subunits.
these subunits are independently exported to the cytoplasm and joined to obtain mature ribosomes  <cit> .
several inherited or acquired bone marrow failure syndromes are due to mutations in genes encoding proteins involved in ribosome biogenesis.
they include diamond-blackfan anaemia , shwachman-diamond syndrome , dyskeratosis congenita  and 5q- syndrome   <cit> .
dba is an inherited erythroid hypoplasia which usually develops within the first year of life and is characterised by a severe normochromic macrocytic anaemia caused by a defect in the maturation of erythroid progenitors.
haematological signs include paucity of bone marrow progenitors, reticulocytopenia, elevated erythrocyte adenosine deaminase  activity and high levels of foetal haemoglobin.
patients are prone to develop malignancies and one third of them present congenital abnormalities  <cit> .
mutations causing dba have been found so far in six rp genes, encoding both small and large subunit components: rps <dig> , rps <dig> , rps <dig> , rpl35a , rpl <dig>  and rpl <dig> , overall accounting for about 50% of cases  <cit> .
all patients are heterozygous with respect to mutations and haplo-insufficiency is thought to be responsible for the pathogenetic mechanism of the disease.
the main hypothesis to explain dba implies that a defect in ribosome biogenesis and protein synthesis would trigger apoptotic processes in erythroid progenitors.
this is supported by several studies showing that mutations in rps impair rrna processing, both in cd34+ cells from dba patients and in erythroid cells with knock-down of the known dba genes  <cit> .
mutations in different genes impair different steps of rrna maturation, but they all lead to the accumulation of rrna precursors and to a reduction in mature ribosomes.
similar alterations have also been demonstrated in yeast and human cells deficient for other rps, not yet found mutated in dba  <cit> .
however, the link between the haplo-insufficiency of an rp and the erythroid defect occurring in dba has not been clarified yet.
dba has been considered a disease that affects only erythroid progenitors and thus the prototype of a pure erythroid aplasia.
however, several lines of evidence suggest that erythroid progenitors, though apparently more sensitive to rp haplo-insufficiency than other cell types, are not the only cell type affected in dba.
some patients evolve trilinear aplasia  <cit> , showing that haematopoiesis in general can be affected, while the presence of congenital malformations demonstrates that some organogenetic processes are affected by dba mutations.
moreover, patients show short statures well below their genetic potentials  <cit> .
to gain a better insight into the biological processes and functions involved in the pathogenesis of dba, we performed a global gene expression analysis of fibroblasts isolated from dba patients carrying mutations in rps <dig>  our data reveal for the first time the presence of abnormal gene expression in non-haematopoietic dba cells.
protein synthesis, nucleotide and amino acid metabolism and apoptosis are the most affected biological processes.
RESULTS
gene expression profiling of cells from dba patients
to identify genes associated with a defect of rps <dig>  a whole genome expression profiling study was performed using affymetrix genechip human genome u133a  <dig>  arrays which allow the screening of  <dig>  transcripts, including  <dig>  well-characterised genes.
we analysed the gene expression profiles of dermal fibroblasts isolated from four dba patients carrying mutations in rps <dig>  in comparison to those obtained from six healthy individuals.
of the  <dig>  probes present on the chip,  <dig>  had affymetrix "present" detection calls and intensity values more than  <dig> in all arrays and have been used for data analysis.
a list of  <dig> statistically significantly differentially expressed probes was generated,  <dig> of which are up-regulated and  <dig> down-regulated in dba patients .
these probes correspond to  <dig> differentially expressed genes  in patients compared to controls.
we also evaluated whether the observed differences in gene expression may be due to a gender-dependent bias.
the comparison between the array data of female to male individuals irrespective of the health status yielded a list of  <dig> differentially expressed probes , corresponding to genes mainly located on chromosomes x or y .
moreover, we identified only five differentially expressed genes in female dba patients relative to male dba patients .
these results rule out the presence of gender-dependent defects in dba patients.
microarray analysis was performed on samples from patients carrying two missense , one frameshift and one splice site mutations  in rps <dig> .
we did not identify any gene showing a statistically significant differential expression in patients with missense mutations compared to other mutation types, indicating that changes due to the specific nature of the mutational status were not found in our study.
a r/i responsive and subsequent interruption of therapy
b r responsive
c this patient was transfusion dependent
d nr non responsive
e bmt/rem remission after bone marrow transplant
f trt/rem remission after treatment
g sd steroid dependent
microarray analysis did not show any statistically significant difference in the mean expression of rps <dig> when the group of patients is compared to the group of controls.
this is due to the fact that two patients carried missense mutations that are expressed similarly to controls.
inspection of the microarray data relative to each patient showed that patient  <dig> with the frameshift mutation c.del58g had reduced rps <dig> levels  as expected by activation of nmd  <cit> .
a slightly reduced rps <dig> expression was found also in patient  <dig> carrying mutation c.1-1g>a .
this was confirmed by qrt-pcr with a fold change of  <dig>  as compared to five controls .
mutation c.1-1g>a affects intron  <dig> acceptor splice site and is expected to cause exon  <dig> skipping.
pcr using primers complementary to exon  <dig>  and exon  <dig>  revealed the presence of two amplicons of  <dig> and  <dig> bp in patient  <dig> .
sequencing of pcr products confirmed that the smaller abnormal transcript lacks exon  <dig> which contains the canonical atg .
the first available atg at position 96- <dig> in exon  <dig> is out-of-frame : this transcript is expected to be degraded by nmd  <cit> .
however, we cannot rule out that the use of the in-frame atg at codon  <dig> in exon  <dig> would generate a short protein of  <dig> aminoacids.
either way, a loss of function effect is expected to occur by this mutation in agreement with the assumed haploinsufficiency theory of dba pathogenesis.
biological processes altered in dba patients
in order to systematically detect impaired biological processes and molecular functions, we analysed the dataset of genes differentially expressed in patients relative to controls through the use of ingenuity pathways analysis  <cit> .
the analysis identified a statistically significant enrichment of genes that belong to the following pathways : aminoacyl-trna biosynthesis , glycine, serine and threonine metabolism , death receptor signaling , role of pkr in interferon induction and antiviral response  and rar activation .
in addition, analysis of the molecular function of the annotated genes revealed enrichment of gene clusters with roles in protein synthesis, including a large cluster of rp genes , cell death, cellular development, lipid metabolism and molecular transport.
additionally, we identified clusters of genes belonging to the "top bio functions" category of molecular and cellular functions  and diseases and disorders, which comprises cancer related genes, as well as genes involved in haematological, immunological, renal and skeletal disorders .
finally, we observed enriched clusters of genes related to the development and function of embryonic, skeletal and connective tissues .
*fischer's exact test was used to calculate a p-value determining the probability that each canonical pathway assigned to that data set is due to chance alone.
**number of genes from the dataset that map to the pathway divided by the total number of molecules that exist in the canonical pathway
*fischer's exact test was used to calculate a p-value determining the probability that each biological function assigned to that data set is due to chance alone.
*fischer's exact test was used to calculate a p-value determining the probability that each biological function and/or disease assigned to that data set is due to chance alone.
*fischer's exact test was used to calculate a p-value determining the probability that each biological function assigned to that data set is due to chance alone.
quantitative rt-pcr validation of microarray data
in order to corroborate the microarray gene expression results, we selected seven genes among those differentially expressed in dba patients relative to controls.
real-time rt-pcr was performed on the same rna samples used for microarray analysis.
the expression of ampd <dig>  ccnd <dig>  sod <dig>  tnfaip <dig>  and comp, wars and zic <dig>  was tested.
all genes were found to be differentially expressed in patients relative to controls and data are statistically significant .
the correlation between qrt-pcr and microarray data is  <dig>  , which corresponds to the value obtained in similar studies  <cit> .
figure 2c shows the comparison of fold changes from qrt-pcr and microarray analysis.
microarray data showed the same trend as qrt-pcr results for all the examined genes in each sample, as represented in figure  <dig>  these data overall indicate that the expression patterns detected by microarray analysis are in good agreement with those detected by qrt-pcr and validate our study.
discussion
the most prominent feature in dba is anaemia due to paucity of erythroid progenitors.
however, other cellular types also display molecular alterations.
lymphocytes from patients show reduced proliferation and impaired translational rates irrespective of the nature of the mutation  <cit> .
an abnormal proliferation rate has been found also in fibroblasts from patients with mutations in rps <dig>  additionally, both lymphocytes and fibroblasts are characterised by impaired pre-rrna processing with accumulation of 21s species.
fibroblasts show abnormal nucleoli, which have irregular shape and disorganised dense fibrillar centers, the compartments where early pre-ribosome maturation takes place  <cit> .
to characterise the molecular abnormalities in dba non-haematopoietic cells, we evaluated gene expression profiles of fibroblasts isolated from dba patients carrying mutations in rps <dig>  patients  <dig> and  <dig> carried mutations leading to amino acid substitutions , the mutation in patient  <dig>  impairs the correct splicing of the first intron and abrogates the atg start codon, whereas the mutation in patient  <dig>  causes a frameshift of the open reading frame with insertion of a premature stop codon.
regardless of the heterogeneity of the mutational types, we did not observe any significant difference when missense cases were compared to the other mutations, although this may be due to the limited number of patient samples of each mutational class in our study.
gene expression profiling has been previously performed on bone marrow cd34+ cells isolated from three dba patients with mutations in rps <dig> and in remission from the disease , compared with healthy controls  <cit> , and on cd4+ peripheral blood mononuclear cells from two dba patients with unknown mutations compared with two acquired aplastic anaemia patients  <cit> .
our study demonstrates for the first time a global alteration of several biological processes in non-haematopoietic dba cells.
in agreement with previous studies  <cit> , we identified a cluster of  <dig> ribosomal protein genes down-regulated in dba patients relative to controls.
patients exhibit significant down-regulation of genes encoding proteins important for translation, including several eukaryotic translation initiation factors , eif <dig> and eif <dig> interacting proteins  and the eukaryotic elongation factor  <dig> δ .
this may be caused either by the co-regulated transcription of rp genes or by their coordinated post-transcriptional regulation.
most interestingly, among down-regulated genes we identified a large cluster of aminoacyl-trna synthetases .
aminoacyl-trna synthetases  catalyse the aminoacylation of their cognate trnas and thus are key enzymes to maintain the fidelity of protein synthesis.
in mammals, additional cofactors, i.e.
proteins p <dig>  p <dig> and p <dig>  and interacting core proteins are required to form a functional multisynthetase complex  <cit> .
the gene encoding p <dig>  is also down-regulated in dba fibroblasts.
moreover, ars contribute to the regulation of amino acid metabolism, which is tightly regulated and essential for ribosome biogenesis and function.
human tryptophanyl-trna synthetase  is up-regulated after ifn-gamma treatment, together with indoleamine  <dig> -dioxygenase , the enzyme responsible for tryptophan degradation, thus creating a pool of trp-trna and providing a reservoir of trp available for protein synthesis  <cit> .
interestingly, haem stimulates trp catabolism enhancing both ido and wars enzymatic activities  <cit> .
an abnormal haem catabolism has been suggested to occur in dba  <cit> .
finally, non canonical functions have been proposed for glutamyl-prolyl-trna synthetase  in the translational regulation of specific genes containing a gait element in the 3'utr  <cit> .
we demonstrate here that these key regulators are defective in fibroblasts from dba patients.
this suggests that a differential regulation of specific mrnas may have a role in dba.
high levels of erythrocyte adenosine deaminase  activity are a common clinical feature in dba patients, suggesting that adenine catabolism is stimulated.
this most likely happens since dba cells show the impaired processing of rrna precursor species, which markedly accumulate in the nucleoli and need to be degraded  <cit> .
interestingly, we observed an increased expression of adenosine monophosphate deaminase , an enzyme of nucleotide break-down involved in the regulation of energetic metabolism in mammalian cells.
it catalyses the irreversible deamination of adenylic acid and represents a branchpoint of adenylate nucleotides catabolism, regulating the size of the purine nucleotide pool  <cit> .
the maintenance of an appropriate intracellular purine nucleotide concentration range is necessary for cell survival.
the increased expression of ampd <dig> may indicate the need to dispose of an excess purine pool in dba fibroblasts.
about 4% of dba patients develop cancer, most frequently acute myeloid leukemia, myelodysplastic syndrome and osteosarcoma  <cit> .
this prevalence is much higher than that in the general population.
thus dba patients seem to have an increased risk of developing malignancies.
gene expression analysis performed on fibroblasts from dba patients revealed dysregulation of genes involved in cell death and cancer.
the decrease in pro-apoptotic  and oncosuppressor genes , coupled to the increased expression of some oncogenes  and pro-survival genes  may suggest a predisposition for rps <dig> mutated fibroblasts to carcinogenesis.
it is interesting to note that several zebrafish lines carrying heterozygous mutations for rp genes are also prone to develop malignancies  <cit> .
finally, patient fibroblasts differentially express several genes involved in embryonic and tissue development, including zic <dig>  strongly down-regulated in patients.
in mice, deletion of zic <dig> gene results in cerebellar malformations and axial skeletal abnormalities  <cit> .
it is worthwhile stressing that 30-48% of dba patients display congenital malformations, including abnormalities affecting the skeletal axis, such as preaxial polydactily.
CONCLUSIONS
the global gene expression analysis we performed shed light for the first time on the impaired biological processes in a non-haematopoietic cell type in dba.
we revealed a dysregulation of genes involved in ribosome biogenesis and protein synthesis, as well as amino acid and nucleotide metabolism.
these data support the hypothesis that dba may be due to a defect in general or specific protein synthesis.

 protein synthesis is essential to the survival and growth of cells. ribosomes, the sites where translation occurs, therefore play a fundamental role in cell biology. in human cells, ribosome biogenesis occurs in the nucleolus: it requires the transcription of four ribosomal rna species and their assembly with ribosomal proteins in order to produce the small and large ribosomal subunits. these subunits are independently exported to the cytoplasm and joined to obtain mature ribosomes. several inherited or acquired bone marrow failure syndromes are due to mutations in genes encoding proteins involved in ribosome biogenesis. they include diamond-blackfan anaemia, shwachman-diamond syndrome, dyskeratosis congenita and 5q- syndrome. dba is an inherited erythroid hypoplasia which usually develops within the first year of life and is characterised by a severe normochromic macrocytic anaemia caused by a defect in the maturation of erythroid progenitors. haematological signs include paucity of bone marrow progenitors, reticulocytopenia, elevated erythrocyte adenosine deaminase activity and high levels of foetal haemoglobin. patients are prone to develop malignancies and one third of them present congenital abnormalities. mutations causing dba have been found so far in six rp genes, encoding both small and large subunit components: rps, rps, rps, rpl35a, rpl and rpl, overall accounting for about 50% of cases. all patients are heterozygous with respect to mutations and haplo-insufficiency is thought to be responsible for the pathogenetic mechanism of the disease. the main hypothesis to explain dba implies that a defect in ribosome biogenesis and protein synthesis would trigger apoptotic processes in erythroid progenitors. this is supported by several studies showing that mutations in rps impair rrna processing, both in cd34+ cells from dba patients and in erythroid cells with knock-down of the known dba genes. mutations in different genes impair different steps of rrna maturation, but they all lead to the accumulation of rrna precursors and to a reduction in mature ribosomes. similar alterations have also been demonstrated in yeast and human cells deficient for other rps, not yet found mutated in dba. however, the link between the haplo-insufficiency of an rp and the erythroid defect occurring in dba has not been clarified yet. dba has been considered a disease that affects only erythroid progenitors and thus the prototype of a pure erythroid aplasia. however, several lines of evidence suggest that erythroid progenitors, though apparently more sensitive to rp haplo-insufficiency than other cell types, are not the only cell type affected in dba. some patients evolve trilinear aplasia, showing that haematopoiesis in general can be affected, while the presence of congenital malformations demonstrates that some organogenetic processes are affected by dba mutations. moreover, patients show short statures well below their genetic potentials. to gain a better insight into the biological processes and functions involved in the pathogenesis of dba, we performed a global gene expression analysis of fibroblasts isolated from dba patients carrying mutations in rps our data reveal for the first time the presence of abnormal gene expression in non-haematopoietic dba cells. protein synthesis, nucleotide and amino acid metabolism and apoptosis are the most affected biological processes.  gene expression profiling of cells from dba patients to identify genes associated with a defect of rps a whole genome expression profiling study was performed using affymetrix genechip human genome u133a arrays which allow the screening of transcripts, including well-characterised genes. we analysed the gene expression profiles of dermal fibroblasts isolated from four dba patients carrying mutations in rps in comparison to those obtained from six healthy individuals. of the probes present on the chip, had affymetrix "present" detection calls and intensity values more than in all arrays and have been used for data analysis. a list of statistically significantly differentially expressed probes was generated, of which are up-regulated and down-regulated in dba patients. these probes correspond to differentially expressed genes in patients compared to controls. we also evaluated whether the observed differences in gene expression may be due to a gender-dependent bias. the comparison between the array data of female to male individuals irrespective of the health status yielded a list of differentially expressed probes, corresponding to genes mainly located on chromosomes x or y. moreover, we identified only five differentially expressed genes in female dba patients relative to male dba patients. these results rule out the presence of gender-dependent defects in dba patients. microarray analysis was performed on samples from patients carrying two missense, one frameshift and one splice site mutations in rps. we did not identify any gene showing a statistically significant differential expression in patients with missense mutations compared to other mutation types, indicating that changes due to the specific nature of the mutational status were not found in our study. a r/i responsive and subsequent interruption of therapy b r responsive c this patient was transfusion dependent d nr non responsive e bmt/rem remission after bone marrow transplant f trt/rem remission after treatment g sd steroid dependent microarray analysis did not show any statistically significant difference in the mean expression of rps when the group of patients is compared to the group of controls. this is due to the fact that two patients carried missense mutations that are expressed similarly to controls. inspection of the microarray data relative to each patient showed that patient with the frameshift mutation c.del58g had reduced rps levels as expected by activation of nmd. a slightly reduced rps expression was found also in patient carrying mutation c.1-1g>a. this was confirmed by qrt-pcr with a fold change of as compared to five controls. mutation c.1-1g>a affects intron acceptor splice site and is expected to cause exon skipping. pcr using primers complementary to exon and exon revealed the presence of two amplicons of and bp in patient. sequencing of pcr products confirmed that the smaller abnormal transcript lacks exon which contains the canonical atg. the first available atg at position 96- in exon is out-of-frame: this transcript is expected to be degraded by nmd. however, we cannot rule out that the use of the in-frame atg at codon in exon would generate a short protein of aminoacids. either way, a loss of function effect is expected to occur by this mutation in agreement with the assumed haploinsufficiency theory of dba pathogenesis. biological processes altered in dba patients in order to systematically detect impaired biological processes and molecular functions, we analysed the dataset of genes differentially expressed in patients relative to controls through the use of ingenuity pathways analysis. the analysis identified a statistically significant enrichment of genes that belong to the following pathways: aminoacyl-trna biosynthesis, glycine, serine and threonine metabolism, death receptor signaling, role of pkr in interferon induction and antiviral response and rar activation. in addition, analysis of the molecular function of the annotated genes revealed enrichment of gene clusters with roles in protein synthesis, including a large cluster of rp genes, cell death, cellular development, lipid metabolism and molecular transport. additionally, we identified clusters of genes belonging to the "top bio functions" category of molecular and cellular functions and diseases and disorders, which comprises cancer related genes, as well as genes involved in haematological, immunological, renal and skeletal disorders. finally, we observed enriched clusters of genes related to the development and function of embryonic, skeletal and connective tissues. *fischer's exact test was used to calculate a p-value determining the probability that each canonical pathway assigned to that data set is due to chance alone. **number of genes from the dataset that map to the pathway divided by the total number of molecules that exist in the canonical pathway *fischer's exact test was used to calculate a p-value determining the probability that each biological function assigned to that data set is due to chance alone. *fischer's exact test was used to calculate a p-value determining the probability that each biological function and/or disease assigned to that data set is due to chance alone. *fischer's exact test was used to calculate a p-value determining the probability that each biological function assigned to that data set is due to chance alone. quantitative rt-pcr validation of microarray data in order to corroborate the microarray gene expression results, we selected seven genes among those differentially expressed in dba patients relative to controls. real-time rt-pcr was performed on the same rna samples used for microarray analysis. the expression of ampd ccnd sod tnfaip and comp, wars and zic was tested. all genes were found to be differentially expressed in patients relative to controls and data are statistically significant. the correlation between qrt-pcr and microarray data is, which corresponds to the value obtained in similar studies. figure 2c shows the comparison of fold changes from qrt-pcr and microarray analysis. microarray data showed the same trend as qrt-pcr results for all the examined genes in each sample, as represented in figure these data overall indicate that the expression patterns detected by microarray analysis are in good agreement with those detected by qrt-pcr and validate our study. discussion the most prominent feature in dba is anaemia due to paucity of erythroid progenitors. however, other cellular types also display molecular alterations. lymphocytes from patients show reduced proliferation and impaired translational rates irrespective of the nature of the mutation. an abnormal proliferation rate has been found also in fibroblasts from patients with mutations in rps additionally, both lymphocytes and fibroblasts are characterised by impaired pre-rrna processing with accumulation of 21s species. fibroblasts show abnormal nucleoli, which have irregular shape and disorganised dense fibrillar centers, the compartments where early pre-ribosome maturation takes place. to characterise the molecular abnormalities in dba non-haematopoietic cells, we evaluated gene expression profiles of fibroblasts isolated from dba patients carrying mutations in rps patients and carried mutations leading to amino acid substitutions, the mutation in patient impairs the correct splicing of the first intron and abrogates the atg start codon, whereas the mutation in patient causes a frameshift of the open reading frame with insertion of a premature stop codon. regardless of the heterogeneity of the mutational types, we did not observe any significant difference when missense cases were compared to the other mutations, although this may be due to the limited number of patient samples of each mutational class in our study. gene expression profiling has been previously performed on bone marrow cd34+ cells isolated from three dba patients with mutations in rps and in remission from the disease, compared with healthy controls, and on cd4+ peripheral blood mononuclear cells from two dba patients with unknown mutations compared with two acquired aplastic anaemia patients. our study demonstrates for the first time a global alteration of several biological processes in non-haematopoietic dba cells. in agreement with previous studies, we identified a cluster of ribosomal protein genes down-regulated in dba patients relative to controls. patients exhibit significant down-regulation of genes encoding proteins important for translation, including several eukaryotic translation initiation factors, eif and eif interacting proteins and the eukaryotic elongation factor δ. this may be caused either by the co-regulated transcription of rp genes or by their coordinated post-transcriptional regulation. most interestingly, among down-regulated genes we identified a large cluster of aminoacyl-trna synthetases. aminoacyl-trna synthetases catalyse the aminoacylation of their cognate trnas and thus are key enzymes to maintain the fidelity of protein synthesis. in mammals, additional cofactors, i.e. proteins p p and p and interacting core proteins are required to form a functional multisynthetase complex. the gene encoding p is also down-regulated in dba fibroblasts. moreover, ars contribute to the regulation of amino acid metabolism, which is tightly regulated and essential for ribosome biogenesis and function. human tryptophanyl-trna synthetase is up-regulated after ifn-gamma treatment, together with indoleamine -dioxygenase, the enzyme responsible for tryptophan degradation, thus creating a pool of trp-trna and providing a reservoir of trp available for protein synthesis. interestingly, haem stimulates trp catabolism enhancing both ido and wars enzymatic activities. an abnormal haem catabolism has been suggested to occur in dba. finally, non canonical functions have been proposed for glutamyl-prolyl-trna synthetase in the translational regulation of specific genes containing a gait element in the 3'utr. we demonstrate here that these key regulators are defective in fibroblasts from dba patients. this suggests that a differential regulation of specific mrnas may have a role in dba. high levels of erythrocyte adenosine deaminase activity are a common clinical feature in dba patients, suggesting that adenine catabolism is stimulated. this most likely happens since dba cells show the impaired processing of rrna precursor species, which markedly accumulate in the nucleoli and need to be degraded. interestingly, we observed an increased expression of adenosine monophosphate deaminase, an enzyme of nucleotide break-down involved in the regulation of energetic metabolism in mammalian cells. it catalyses the irreversible deamination of adenylic acid and represents a branchpoint of adenylate nucleotides catabolism, regulating the size of the purine nucleotide pool. the maintenance of an appropriate intracellular purine nucleotide concentration range is necessary for cell survival. the increased expression of ampd may indicate the need to dispose of an excess purine pool in dba fibroblasts. about 4% of dba patients develop cancer, most frequently acute myeloid leukemia, myelodysplastic syndrome and osteosarcoma. this prevalence is much higher than that in the general population. thus dba patients seem to have an increased risk of developing malignancies. gene expression analysis performed on fibroblasts from dba patients revealed dysregulation of genes involved in cell death and cancer. the decrease in pro-apoptotic and oncosuppressor genes, coupled to the increased expression of some oncogenes and pro-survival genes may suggest a predisposition for rps mutated fibroblasts to carcinogenesis. it is interesting to note that several zebrafish lines carrying heterozygous mutations for rp genes are also prone to develop malignancies. finally, patient fibroblasts differentially express several genes involved in embryonic and tissue development, including zic strongly down-regulated in patients. in mice, deletion of zic gene results in cerebellar malformations and axial skeletal abnormalities. it is worthwhile stressing that 30-48% of dba patients display congenital malformations, including abnormalities affecting the skeletal axis, such as preaxial polydactily.  the global gene expression analysis we performed shed light for the first time on the impaired biological processes in a non-haematopoietic cell type in dba. we revealed a dysregulation of genes involved in ribosome biogenesis and protein synthesis, as well as amino acid and nucleotide metabolism. these data support the hypothesis that dba may be due to a defect in general or specific protein synthesis. 
this analysis reports for the first time an abnormal gene expression profile in a non-haematopoietic cell type in dba. dba lymphocytes show reduced protein synthesis and fibroblasts display abnormal rrna processing and impaired proliferation. these genes include a large cluster of ribosomal proteins and factors involved in protein synthesis and amino acid metabolism, as well as genes associated to cell death, cancer and tissue development. these data support the hypothesis that dba may be due to a defect in general or specific protein synthesis. to evaluate the involvement of non-haematopoietic tissues in dba, we have analysed global gene expression in fibroblasts from dba patients compared to healthy controls. diamond-blackfan anaemia is a rare inherited red cell hypoplasia characterised by a defect in the maturation of erythroid progenitors and in some cases associated with malformations. microarray expression profiling using affymetrix genechip human genome u133a arrays revealed that genes are differentially expressed in dba patient fibroblasts. studies in haematopoietic progenitors from patients show that haplo-insufficiency of an rp impairs rrna processing and ribosome biogenesis.
 diamond-blackfan anaemia is a rare inherited red cell hypoplasia characterised by a defect in the maturation of erythroid progenitors and in some cases associated with malformations. patients have an increased risk of solid tumors. mutations have been found in several ribosomal protein genes, i.e rps rps rps rpl rpl rpl35a. studies in haematopoietic progenitors from patients show that haplo-insufficiency of an rp impairs rrna processing and ribosome biogenesis. dba lymphocytes show reduced protein synthesis and fibroblasts display abnormal rrna processing and impaired proliferation.  to evaluate the involvement of non-haematopoietic tissues in dba, we have analysed global gene expression in fibroblasts from dba patients compared to healthy controls. microarray expression profiling using affymetrix genechip human genome u133a arrays revealed that genes are differentially expressed in dba patient fibroblasts. these genes include a large cluster of ribosomal proteins and factors involved in protein synthesis and amino acid metabolism, as well as genes associated to cell death, cancer and tissue development.  this analysis reports for the first time an abnormal gene expression profile in a non-haematopoietic cell type in dba. these data support the hypothesis that dba may be due to a defect in general or specific protein synthesis. 