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. 2009 Sep 18:10:442.
doi: 10.1186/1471-2164-10-442.

Fibroblasts from patients with Diamond-Blackfan anaemia show abnormal expression of genes involved in protein synthesis, amino acid metabolism and cancer

Federica Avondo  1 , Paola Roncaglia, Nicoletta Crescenzio, Helena Krmac, Emanuela Garelli, Marta Armiraglio, Carlotta Castagnoli, Maria Francesca Campagnoli, Ugo Ramenghi, Stefano Gustincich, Claudio Santoro, Irma Dianzani
Affiliations

Fibroblasts from patients with Diamond-Blackfan anaemia show abnormal expression of genes involved in protein synthesis, amino acid metabolism and cancer

Federica Avondo et al. BMC Genomics. .

Abstract

Background: Diamond-Blackfan anaemia (DBA) 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 (RP) genes, i.e RPS19, RPS24, RPS17, RPL5, RPL11, 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.

Results: 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 2.0 Arrays revealed that 421 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.

Conclusion: 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.

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Figures

Figure 1
Figure 1
RPS19 expression in patient 3. a) RPS19 expression in patient 3 and controls is shown. Primers complementary to exon 1 (forward) and exon 4 (reverse) were used. Two PCR products are indicated in lane 1 at 628 and 557 bp. b) Sequencing of RPS19 in patient 3. Exon 2 is 71 nt long and the first three nucleotides represent the translation initiation start site (bold). The chromatogram shows deletion of exon 2 in c.1-1G>A allele compared to the wild-type sequence in patient 3.
Figure 2
Figure 2
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 analysed 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 correlation 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 analysed gene.
Figure 3
Figure 3
Comparison between qRT-PCR and microarray fold changes. The relative expression of genes analysed by qRT-PCR is reported for each sample. The mean expression of controls was used as calibrator (set equal to 1) and beta actin was used to normalise data. Fold changes obtained from microarray analysis are shown for each analysed gene.
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