The inherited bone marrow failure syndromes (IBMFS) are rare genetic disorders caused by mutations in critical components of fundamental cellular processes such as ribosome biogenesis, DNA repair, and telomere maintenance. The IBMFS Shwachman-Diamond syndrome(SDS) and Diamond-Blackfan anemia (DBA) are classified as ribosomopathies due to etiologic mutations in genes encoding factors involved in ribosome biogenesis (SBDSin the majority of patients with SDS) or ribosomal proteins (RPS19most commonly in patients with DBA). Although these disorders can be distinguished clinically and from the other IBMFS, they share with each other and with other IBMFS increased predisposition to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Whereas genomic instability due to defective DNA repair or telomere maintenance is thought to underlie cancer predisposition in the IBMFS Fanconi anemia and dyskeratosis congenita, respectively, the molecular mechanisms driving cancer in SDS and DBA are not fully understood.

Our research has focused on DNA repair in SDS and DBA. A prior report suggested lymphoblastoid cell lines (LCLs) derived from patients with SDS arehypersensitive to ionizing radiation (IR). Consistent with this, we found SDS-LCLs had decreased survival following IR compared to control-LCLsin colony survival assays. To determine if this cellular phenotype was unique to SDS or present in the other IBMFS ribosomopathy, DBA, we examined LCLs derived from patients with DBA, including those with mutations in RPS19, RPS26, RPL5and RPL11. We found that the DBA-LCLs were similarly hypersensitive to IR as compared to control-LCLs. Further examination of γ-H2AX, a DNA damage response (DDR) factor and marker of DNA double strand breaks (DSBs), revealed that SDS- and DBA-LCLs had delayed resolution of γ-H2AX foci and increased protein levels at 24 hrs after IR as compared to control LCLs. p53, phospho-ATM, and DNA-PKcs protein levels were also higher in SDS-LCL compared to controls. The decreased survival and increased and sustained DDR following IR led us to hypothesize that SDS and DBA cells have a defect in DSB repair.

There are two major pathways of DSB repair in mammals, nonhomologous end-joining (NHEJ) and homology-directed repair (HDR), and loss of either results in hypersensitivity to IR. To examine each pathway, we employed U2OS (human osteosarcoma) and HCT116 (human colon cancer) cells containing an integrated green fluorescent protein HDR or NHEJ reporter transgene. Interestingly, we found that knockdown of either SBDS or RPS19 proteins resulted in an approximately 50% reduction in HDR efficiency but no change in NHEJefficiency compared to the scrambled control in both cell lines.

We next sought to determine the mechanism underlying the effect of SBDS and RPS19 deficiency on HDR. A survey of proteins required for HDR revealed a reduction in the recombinase RAD51 in SDS-LCLs and in SBDS-depleted HCT116 and U2OS cells, whereas, an initial survey in SDS-LCLs[e1] of factors involved in NHEJ did not reveal a specific NHEJ factor deficiency. Knockdown of eiF6 is known to rescue the defect in 40S and 60S ribosome subunit joining that manifests in SDS patient cells. However, we found eIF6 depletion failed to rescue the level of RAD51 protein and had no impact on HDR in SBDS-deficient cells. We conclude that decreased RAD51 levels in SBDS-deficient cells might contribute to impaired HDR, however, this decrease is independent of the ribosome subunit joining defect. Similarly, RPS19 knock down resulted in a reduction in RAD51 protein level, suggesting a potentially common pathway.

We also asked whether SBDS or RPS19 might be more directly involved in the DDR or repair of DSBs. Consistent with this, we found SBDS and RPS19 recruited to chromatin surrounding an I-Sce1 site following DSB induction in chromatin immunoprecipitation assays. Collectively, these findings provide evidence that SBDS and RPS19 may be directly involved in the DDR or DSB repair and raise the possibility that loss of this function may contribute to MDS/AML predisposition in SDS and DBA patients.


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.