BMC Genomics 2009, 10:442 http://www.biomedcentral.com/1471-2164/10/442 Microarray analysis was performed on samples from patients carrying two missense (p.Arg62Trp and p.Arg101His), one frameshift and one splice site muta- tions (c.58delG and c.1-1G>A) in RPS19 (table 1). We did not identify any gene showing a statistically significant differential expression in patients with missense muta- tions compared to other mutation types, indicating that changes due to the specific nature of the mutational status were not found in our study. Microarray analysis did not show any statistically signifi- cant difference in the mean expression of RPS19 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. Inspec- tion of the microarray data relative to each patient showed that patient 4 with the frameshift mutation c.del58G had reduced RPS19 levels (fold change 0.68) as expected by activation of NMD [17,18]. A slightly reduced RPS19 expression was found also in patient 3 carrying mutation c.1-1G>A (fold change 0.84). This was confirmed by qRT- PCR with a fold change of 0.76 as compared to five con- trols (data not shown). Mutation c.1-1G>A affects intron 1 acceptor splice site and is expected to cause exon 2 skip- ping. PCR using primers complementary to exon 1 (for- ward) and exon 4 (reverse) revealed the presence of two amplicons of 628 and 557 bp in patient 3 (figure 1a). Sequencing of PCR products confirmed that the smaller abnormal transcript lacks exon 2 which contains the canonical ATG (figure 1b). The first available ATG at posi- tion 96-98 in exon 3 is out-of-frame (nucleotide number- ing uses the A of the ATG translation initiation start site as nucleotide 1): this transcript is expected to be degraded by NMD [17,18]. However, we cannot rule out that the use of the in-frame ATG at codon 75 in exon 4 would generate a short protein of 71 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. Table 1: Clinical characteristics of DBA patients Patient Gender Mutation (RPS19) p.Arg62Trp p.Arg101His c1-1G>A c.del58G 1 2 3 4 M F F M 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 Biological processes altered in DBA patients In order to systematically detect impaired biological proc- esses and molecular functions, we analysed the dataset of genes differentially expressed in patients relative to con- trols through the use of Ingenuity Pathways Analysis [19]. The analysis identified a statistically significant enrich- ment of genes that belong to the following pathways (table 2): aminoacyl-tRNA biosynthesis (LARS, WARS, GARS, SARS, QARS, EPRS), glycine, serine and threonine metabolism (PSAT1, PHGDH, GARS, CTH, SARS, SHMT2), death receptor signaling (CASP9, NFKBIA, NFK- BIE, APAF1, NFKB2, NFKB1, BIRC3), role of PKR in inter- feron induction and antiviral response (CASP9, NFKBIA, NFKBIE, APAF1, NFKB2, NFKB1) and RAR activation (SRC, PRMT2, RDH11, ADCY3, PRKACA, NFKB2, NFKB1, MAPKAPK2, RBP1, MMP1, PTEN). 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 (RPL22, RPL27A, RPL29, RPL31, RPL14, RPL18A, RPS3, RPL18, RPL13, RPL3, RPL34, RPS2, RPS12, RPL15, RPL28), 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 (table 3) and diseases and disorders, which comprises cancer related genes, as well as genes involved in haematological, immunological, renal and skeletal disorders (table 4). Finally, we observed enriched clusters of genes related to the development and function of embryonic, skeletal and connective tissues (table 5). 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 AMPD3, CCND2, SOD2, TNFAIP3 (up-regulated in DBA patients) and Steroid Response R/Ia Rb NRc, d R Follow-up BMT/Reme TRT/Remf BMT/Rem SDg Congenital Abnormalities yes no no no Page 3 of 12 (page number not for citation purposes)