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Y. Liu et al.
cycle arrest in G0/G1 phases leading to larger cell size.9,37 We also observed a significantly increased MCV in the mock group after induction with doxycycline for more than 4 weeks (Figures 3E and 5E). In particular, during the initial phase after doxycycline induction, the majority of red blood cells in the circulation were produced before the DBA phenotype was induced. Since old red blood cells are smaller than the newly produced cells, the MCV is decreased compared to normal for a short period during the first 2 weeks after doxycycline induction (Figure 2E). In addition to the severe anemia phenotype after induc- tion of Rps19 deficiency, we observed decreased white blood cell and platelet counts in the mock group. Mild thrombocytopenia and neutropenia (low levels of neu- trophilic granulocytes) have also been observed in about 25% of DBA patients during the course of the disease.4,37,38 In addition, several patients with RPS19-deficient DBA have developed myelodysplastic syndrome with multilin- eage cytopenia, which suggests a multilineage defect.37 This observation correlates with a reduction in the
absolute numbers of hematopoietic stem and progenitor cells in BM due to Rps19 deficiency,15 which led to the lethal BM failure we observed in untreated animals short- ly after doxycycline administration. This is also supported by the limited reconstitution ability of progenitor com- partments in the mock group (Figure 4C-I) and impaired erythroid differentiation of human primary RPS19-defi- cient cord blood cells. RPS19-deficient patients who develop thrombocytopenia and neutropenia also experi- ence similar progressive phenotypes of hypocellularity in the BM.38,39 Moreover, DBA is a very heterogeneous dis- ease. It is unknown why family members with the same genetic mutation in RPS19 may have very different phe- notypes, ranging from no anemia to severe anemia with progression to multilineage BM failure.40 The current understanding of phenotype-genotype correlations is far from comprehensive and needs to be studied further.
Although the majority of mice in the mock group died, a few mice did survive until the planned endpoint at 16 weeks. One of the possible reasons for this may be the
AB
C
D
Figure 7. Gene-corrected bone marrow cells show a vector integration pattern that indicates low risk of mutagenesis and a highly polyclonal insertion site pattern.
(A) The top ten integration sites in each sample (*indicates that the integration was within a transcription unit, ~ indicates that the insertion was within 50 kb of a cancer-related gene). (B, C) Percent of all integrations inside transcriptional units (B) and percent of integrations within 100 kb of proto-oncogenes compared to matched random control sites (C). (D) Genomic heatmap analysis of the insertion site profile. mrc: matched randon control. ***P<0.001 by an unpaired t-test.
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haematologica | 2022; 107(2)