Bone Marrow Cells from Patients with Shwachman-Diamond Syndrome Abnormally Express Ribosomal Protein and Ribosomal Biogenesis-Related Genes.

Abstract Background and rational: Shwachman-Diamond syndrome (SDS) is an autosomal recessive disorder characterized by varying degrees of cytopenia and high propensity for myelodysplastic syndrome and acute leukemia. SBDS, the gene associated with SDS, has recently been identified and is postulated to play a role in ribosomal biogenesis and RNA processing, but its functions are still unknown. Defects in ribosomal biogenesis can be characterized by abnormal synthesis of rRNA synthesis or ribosomal proteins or both. Determining the mRNA expression pattern of the various RP genes in SBDS deficient cells will help deciphering the role of SBDS in ribosomal biogenesis. Objectives: To determine whether the primary SDS marrow cells which carry homozygous SBDS mutations abnormally express genes which code for ribosomal proteins (RP) or for proteins that are involved in its transcription. Methods: Total RNA from marrow cells from 9 SDS patients who had hypocellular marrow with normal differential and no malignant transformation and 7 healthy age-matched donors of bone marrows for transplantation was extracted. RNA was labeled and hybridized to Affymetrix HG_U133_Plus2.0 GeneChip. Data were pre-processed using robust multichip analysis (RMA) and differentially expressed genes were identified with permutation-based methods. False discovery rate (FDR)-adjusted p-values were used to rank genes and cluster analysis grouped genes and samples. T-statistic values were used to screen for differentially expressed RP-related genes. Real-time PCR was performed to confirm differential expression of genes found by oligonucleotide microarray. Results: Of the 38,500 genes on the HG_133_Plus2.0 we analyzed 375 known ribosomal protein and RNA processing-related genes. Interestingly, there were differences in the expression pattern of the RP genes, suggesting differential regulation of these genes in Sbds-deficient cells. Interestingly, despite uniform decrease in RP gene expression in reduced cell growth conditions, only 27 of the 85 RP genes were downregulated. Downregulation of representative 2 genes was confirmed by real-time PCR. Further, one of the RP genes, RPL27L was upregulated. This gene, which is a target of p53, has a non-ribosomal function and lead to accelerated apoptosis. It is noteworthy that several genes involved in mRNA transcription such as GABPA and YY1were downregulated without dysregulation of genes involved in mRNA degradation, suggesting that the downregulation of the RP gene expression is at the transcription level. In addition to dysregulation of the RP mRNA we also found dysregulation of genes involved in rRNA transcription (e.g. MKI67IP) and pre rRNA processing (e.g. FBL). Conclusions: SBDS-deficiency results in dysregulation of selective group of RP genes as well as genes related to rRNA processing and rRNA transcription. Future studies should focus on the mechanism of the abnormal expression as well as its biological consequences.