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01.12.2014 | Research | Ausgabe 1/2014 Open Access

Journal of Hematology & Oncology 1/2014

Splicing factor 3b subunit 1 (Sf3b1) haploinsufficient mice display features of low risk Myelodysplastic syndromes with ring sideroblasts

Zeitschrift:
Journal of Hematology & Oncology > Ausgabe 1/2014
Autoren:
Valeria Visconte, Ali Tabarroki, Li Zhang, Yvonne Parker, Edy Hasrouni, Reda Mahfouz, Kyoichi Isono, Haruhiko Koseki, Mikkael A Sekeres, Yogen Saunthararajah, John Barnard, Daniel Lindner, Heesun J Rogers, Ramon V Tiu
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​s13045-014-0089-x) contains supplementary material, which is available to authorized users.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

VV designed the study, performed the experiments, analyzed the data and wrote the manuscript; AT performed experiments and edited the manuscript; LZ performed statistical analysis; YP performed experiments and provided technical expertise; EH and RM performed experiments and edited the manuscript; KI and HK provided embryos of Sf3b1 mice; MAS, and YS edited the manuscript; JB analyzed RNA-sequencing data; DL provided expertise on mouse model and edited the manuscript; HJR interpreted the data and edited the manuscript; RVT designed the study, analyzed the data, interpreted the results and wrote the manuscript. All authors read and approved the final manuscript.

Abstract

Background

The presence of somatic mutations in splicing factor 3b subunit 1 (SF3B1) in patients with Myelodysplastic syndromes with ring sideroblasts (MDS-RS) highlights the importance of the RNA-splicing machinery in MDS. We previously reported the presence of bone marrow (BM) RS in Sf3b1 heterozygous (Sf3b1+/−) mice which are rarely found in mouse models of MDS. Sf3b1+/− mice were originally engineered to study the interaction between polycomb genes and other proteins.

Methods

We used routine blood tests and histopathologic analysis of BM, spleen, and liver to evaluate the hematologic and morphologic characteristics of Sf3b1+/− mice in the context of MDS by comparing the long term follow-up (15 months) of Sf3b1+/− and Sf3b1+/+ mice. We then performed a comprehensive RNA-sequencing analysis to evaluate the transcriptome of BM cells from Sf3b1+/− and Sf3b1+/+ mice.

Results

Sf3b1+/− exhibited macrocytic anemia (MCV: 49.5 ± 1.6 vs 47.2 ± 1.4; Hgb: 5.5 ± 1.7 vs 7.2 ± 1.0) and thrombocytosis (PLTs: 911.4 ± 212.1 vs 878.4 ± 240.9) compared to Sf3b1+/+ mice. BM analysis showed dyserythropoiesis and occasional RS in Sf3b1+/− mice. The splenic architecture showed increased megakaryocytes with hyperchromatic nuclei, and evidence of extramedullary hematopoiesis. RNA-sequencing showed higher expression of a gene set containing Jak2 in Sf3b1+/− compared to Sf3b1+/+.

Conclusions

Our study indicates that Sf3b1+/− mice manifest features of low risk MDS-RS and may be relevant for preclinical therapeutic studies.

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Zusatzmaterial
Additional file 1: Figure S1.: Genotyping of wild type (Sf3b1+/+) and Sf3b1 haploinsufficient (Sf3b1+/−) mice. Genomic DNA was extracted from tails and toes of Sf3b1 pups after rederivation and subjected to PCR amplification by using specific primers as described in Methods. Examples of Sf3b1+/− mice (#1, 2, 4, and 7) identified by the presence of amplicons corresponding to wild type (WT: 1.5 Kb) and knock-out (KO: 0.9 Kb) alleles on a 1.2% agarose gel. Lane marked with MM indicates 1Kb Plus DNA ladder. (JPEG 18 KB)
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Additional file 2: Figure S2.: Sf3b1+/− have no difference in the mast cell compartment compared to Sf3b1+/+ mice. Immunohistochemistry (IHC) was used to evaluate the presence of mast cells in Sf3b1+/− (n = 2) and Sf3b1+/+ (n = 2). Bone marrow cells (5 x 105) were spotted on cytospin slides and IHC for CD117 (c-Kit) was performed. (JPEG 78 KB)
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Additional file 3: Figure S3.: Sf3b1+/− mice have no difference in bone marrow cellularity compared to Sf3b1+/+ mice. (a) Hematoxylin/eosin (H&E) was performed on bone marrow (BM) cells (3–5 × 105) from Sf3b1+/− (n = 3) and Sf3b1+/+ (n = 3). An H&E representative image showed normal trilineage hematopoiesis and no changes in BM cellularity in both groups of mice. (b) A bar graft shows mean ± standard deviations of the number of BM cells at the end of the follow-up between Sf3b1+/− (n = 4) and Sf3b1+/+ (n = 3) mice. (JPEG 48 KB)
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Additional file 4: Figure S4.: Sf3b1+/− mice have dyserythropoietic features in the bone marrow. Bone marrow (BM) cells (3–5 × 105) were spotted on cytospin slides and iron stain (Prussian blue) was performed according to common pathology stain’s protocols. A representative image taken by light microscopy shows that BM cells from Sf3b1+/− showed specific dyserythropoietic features such as nuclear budding or nuclear irregularity (black arrows) that were not seen in Sf3b1+/− mice. (JPEG 35 KB)
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Additional file 5: Figure S5.: Sf3b1+/− mice have ring sideroblasts in the bone marrow. Iron stain (Prussian blue) was performed on bone marrow (BM) cells (3–5 x 105) derived from Sf3b1+/− and Sf3b1+/+ mice. Images taken by light microscopy show presence of ring sideroblasts (black arrows) in the BM of Sf3b1+/− (n = 3) and absence in Sf3b1+/+ (n = 2) mice. (JPEG 63 KB)
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Additional file 6: Figure S6.: Sf3b1+/− mice do not have any liver abnormalities. Representative images from Hematoxylin & Eosin stain of liver sections from 2 Sf3b1+/− and 2 Sf3b1+/+ mice show absence of hepatomegaly or abnormalities in the liver. (JPEG 91 KB)
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Additional file 7: Table S1.: Differential gene level between Sf3b1+/− and Sf3b1+/+ mice. The expression level of all genes is presented as mean fold change between Sf3b1+/− and Sf3b1+/+ mice. In total 10330 genes were found in the comparison Sf3b1+/− versus Sf3b1+/+ mice. (XLSX 1 MB)
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Additional file 8: Table S2.: Comparison of hematopoietic-related gene sets between Sf3b1+/− and Sf3b1+/+ mice. Gene set analysis shows hematopoietic –related genes between both groups of mice. (XLSX 423 KB)
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Additional file 9: Table S3.: Gene clustered in hematopoietic-related gene sets between Sf3b1+/− and Sf3b1+/+ mice. The genes related to the specific gene set are summarized. (XLSX 35 KB)
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Additional file 10: Figure S7.: Sf3b1+/− mice have features of low risk Myelodysplastic syndrome with ring sideroblasts. A table illustrating the criteria established by the hematopathology subcommittee of the Mouse Models of Human Cancers Consortium establishing the myeloid dysplasia in mice. The table has been adopted by Beachy, SM et al. (Hematol Oncol Clin North Am 2010) to summarize all the criteria and it shows the specific features harbored by the Sf3b1+/− mice (red color). (JPEG 82 KB)
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Authors’ original file for figure 1
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Authors’ original file for figure 2
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Authors’ original file for figure 3
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Authors’ original file for figure 4
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