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Rothmund-Thomson Syndrome (RTS) is a rare autosomal recessive disorder characterized by skin rash, skeletal dysplasias, premature ageing, and caused by mutations in RECQL4. RTS is a familial cancer syndrome, with hematological cancers amongst the most common reported malignancies. We previously reported that mice with somatic deletion of Recql4, resulting in null alleles, developed a fully penetrant bone marrow failure syndrome. RTS patients, however, have truncating mutations that result in hypomorphic alleles, not null alleles. To understand the impact of Recql4 mutations on haematopoiesis, we established mice with three distinct point mutations: K525A, which results in an ATP-dependent helicase-inactive but full-length protein, and G522EfsX43 and R347X, which result in truncated protein products. We crossed the point mutant mice to the R26-CreER Recql4 fl/fl line, and at 8-10 weeks of age fed tamoxifen containing food for 30 days to delete the wild-type Recql4 floxed allele leaving only the mutation expressed. Analysis of peripheral blood and bone marrow by FACS showed that only mice carrying truncating mutations developed bone marrow failure. To validate these results in vitro, we immortalized R26-CreER Recql4 point mutant cells with Hoxb8 retrovirus and treated them with tamoxifen for 14 days. In this setting, we also found that truncating mutations caused a marked decrease in cell proliferation, while the helicase-inactive mutation did not. Finally, we performed in vitro complementation assays to determine the capacity of human RECQL4 mutations, similar to our murine alleles, to rescue Hoxb8 R26-CreER Recql4-depleted myeloid cells. Results showed that the helicase-inactive mutant successfully rescued the RECQL4-depleted cells, while the truncating human mutants did not. Collectively, these data demonstrate that the helicase function of RECQL4 is dispensable for hematopoiesis but that the deletion of both the helicase and the C-terminal domains in truncating mutations has deleterious effects on hematopoietic cells. |
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