BMC Genomics 2009, 10:442 http://www.biomedcentral.com/1471-2164/10/442 FGiegnuerex2pression by qRT-PCR Gene expression by qRT-PCR. a) The mean relative expression in the group of DBA patients relative to the group of controls (set equal to 1) is reported for each ana- lysed gene. Beta actin was used to normalise data. P values less than 0.05 were considered as statistically significant. b) Correlation of fold changes in the expression of analysed genes by microarray and qRT-PCR. The R2 coefficient of cor- relation is reported. c) The mean relative expression in the group of DBA patients relative to the group of controls is reported for each analysed gene. Fold changes obtained from qRT-PCR and microarray analysis are shown for each ana- lysed gene. oncogenes (SRC, CYLD) and pro-survival genes (CALR) may suggest a predisposition for RPS19 mutated fibrob- lasts 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 malforma- tions and axial skeletal abnormalities [36,37]. It is worth- while stressing that 30-48% of DBA patients display congenital malformations, including abnormalities affecting the skeletal axis, such as preaxial polydactily. Conclusion 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 nucle- otide metabolism. These data support the hypothesis that DBA may be due to a defect in general or specific protein synthesis. Methods Patients and cell culture This study was performed on four DBA patients, carrying two missense (p.Arg62Trp and p.Arg101His), one frameshift and a splice site mutations (c.58delG and c.1- 1G>A), respectively, in RPS19. Table 1 reports the main clinical characteristics of these patients. Further informa- tion about these mutations and their functional character- isation may be found in [4] and in the DBA genes Database [38,39]. Research was carried out in compliance with the Helsinki Declaration. Dermal biopsies were obtained from the four DBA patients and from six healthy controls after informed consent during surgery for medical reasons that were not connected with this study. Fibroblasts were cultured in IMDM (Iscove's Modified Dulbecco's Medium) supple- mented with 4 mM L-glutamine, 10% foetal calf serum, 0.1 mg/ml streptomycin, 100 U/ml penicillin (Sigma- Aldrich) at 37°C and 5% CO2. Total RNA was extracted from 106 cells upon reaching 80% confluence, within 2-5 tissue culture passages. RNA isolation, microarray processing and data analysis Total RNA was isolated using the TRIzol reagent (Invitro- gen) according to manufacturer's instructions, and puri- fied with the RNeasy Mini kit (QIAGEN). The quality of total RNA was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies). RNA was quantified with a Nano- Drop 1000 spectrophotometer (Thermo Scientific). A 6 μg-amount of each total RNA sample was labelled accord- ing to the standard one-cycle amplification and labelling protocol developed by Affymetrix (Santa Clara, CA). Labelled cRNA was hybridised on Affymetrix GeneChip Human Genome U133A 2.0 Arrays containing over 18,000 transcripts. Hybridized arrays were stained and washed (GeneChip Fluidics Station 450) and then Page 8 of 12 (page number not for citation purposes)