Abstract
Shwachman-Diamond syndrome (SDS), an autosomal recessive disorder, is characterized by bone marrow dysfunction, exocrine pancreatic insufficiency, congenital abnormalities, and leukemia predisposition (Myers et al., 2012). Most patients with SDS harbor biallelic mutations in the Shwachman-Bodian-Diamond syndrome (SBDS) gene. SBDS is known to play a role in ribosome biogenesis by enabling eviction of the ribosome anti-association factor eIF6 from the 60S ribosomal subunit, to allow formation of the 80S ribosome (Wong et al., 2011). SBDS-depleted cells are, therefore, defective in ribosome assembly. In addition, absence of SBDS sensitizes cells to ultraviolet irradiation, translation inhibitors, and endoplasmic reticulum (ER) stressors, such as tunicamycin (Ball et al., 2009). A recent report indicated that lymphoblastoid cell lines (LCLs) derived from two SDS patients accumulated more DNA damage after being exposed to ionizing radiation (IR) (Morini et al., 2015). A deficiency in DNA repair was alluded to as a possible cause, however, the mechanism underlying this previously unreported phenotype was not determined. In this study, we investigated LCLs derived from five SDS patients with biallelic SBDS mutations and found all to be hypersensitive to IR in a colony survival assay. In this assay, increasing doses of IR resulted in a significantly lower survival fraction in SDS-compared to control-LCLs. We found SBDS expression to increase in control-cells when stressed with IR, suggesting that SBDS is a stress response protein and its absence in SDS-LCLs induces hypersensitivity to IR. Because knockdown of SBDS in HEK293 cells induces an ER stress response (Ball et al., 2009), we examined the expression of the ER stress response factor phospho-eIF2α in untreated and IR exposed SDS-LCLs and found phospho-eIF2α expression to be markedly increased compared to controls. This result indicated that SDS-LCLs may have an activated ER stress response, as was further confirmed by exposing these cells to additional ER stressors, tunicamycin and H2O2, and observing a similar upregulation of phospho-eIF2α. Because ER stress is known to suppress DNA double strand break (DSBR) (Yamamori et al., 2013), we examined the expression of Rad51 and Ku70, which are required for the homology-directed and nonhomologous end-joining pathways of DSBR, respectively. Surprisingly, we found Rad51 and Ku70 protein levels to be repressed in SDS-LCLs compared to controls, both with and without exposure to IR. Collectively, these data support the hypothesis that, in addition to its role in ribosome biogenesis, SBDS is a stress response protein that plays an important role in regulating the ER stress response. In SDS-cells, where SBDS is lacking, activated ER stress represses DNA repair proteins rendering cells hypersensitive to IR and other stresses. This novel pathway to ER stress induction may contribute to the bone marrow failure and cancer predisposition seen in SDS patients.
Disclosures
No relevant conflicts of interest to declare.
Shwachman-Diamond Syndrome with Exocrine Pancreatic Dysfunction and Bone Marrow Failure Maps to the Centromeric Region of Chromosome 7