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 [22
]. An abnormal proliferation rate has been found also in fibroblasts from patients with mutations in RPS19
. 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 [11
To characterise the molecular abnormalities in DBA non-haematopoietic cells, we evaluated gene expression profiles of fibroblasts isolated from DBA patients carrying mutations in RPS19. Patients 1 and 2 carried mutations leading to amino acid substitutions (p.Arg62Trp and p.Arg101His), the mutation in patient 3 (c.1-1G>A) impairs the correct splicing of the first intron and abrogates the ATG start codon, whereas the mutation in patient 4 (c.del58G) 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 RPS19
and in remission from the disease (i.e. without any treatment for at least 10 years), compared with healthy controls [23
], and on CD4+
peripheral blood mononuclear cells from two DBA patients with unknown mutations compared with two acquired aplastic anaemia patients [24
]. Our study demonstrates for the first time a global alteration of several biological processes in non-haematopoietic DBA cells. In agreement with previous studies [23
], we identified a cluster of 22 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 (EIF3
), EIF3 and EIF4 interacting proteins (EIF3S6IP
) and the eukaryotic elongation factor 1 δ (EEF1D
). 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 (QARS
). Aminoacyl-tRNA synthetases (ARS) 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 p18, p38 and p43, and interacting core proteins are required to form a functional multisynthetase complex [25
]. The gene encoding p18 (CDKN2C
) 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 (WARS
) is up-regulated after IFN-gamma treatment, together with indoleamine 2,3-dioxygenase (IDO
), the enzyme responsible for tryptophan degradation, thus creating a pool of Trp-tRNA and providing a reservoir of Trp available for protein synthesis [26
]. Interestingly, haem stimulates Trp catabolism enhancing both IDO and WARS enzymatic activities [29
]. An abnormal haem catabolism has been suggested to occur in DBA [31
]. Finally, non canonical functions have been proposed for glutamyl-prolyl-tRNA synthetase (EPRS) in the translational regulation of specific genes containing a GAIT element in the 3'UTR [32
]. 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 (eADA) 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 [14
]. Interestingly, we observed an increased expression of adenosine monophosphate deaminase (AMPD3
), 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 [33
]. The maintenance of an appropriate intracellular purine nucleotide concentration range is necessary for cell survival. The increased expression of AMPD3
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 [5
]. 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 (CASP9, APAF1
) and oncosuppressor genes (PTEN
), coupled to the increased expression of some oncogenes (SRC, CYLD
) and pro-survival genes (CALR
) may suggest a predisposition for RPS19
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 [35
Finally, patient fibroblasts differentially express several genes involved in embryonic and tissue development, including ZIC1
, strongly down-regulated in patients. In mice, deletion of zic1
gene results in cerebellar malformations and axial skeletal abnormalities [36
]. It is worthwhile stressing that 30-48% of DBA patients display congenital malformations, including abnormalities affecting the skeletal axis, such as preaxial polydactily.