Genotype-phenotype relations in SF3B1 mutated myelodysplastic syndromes with ring sideroblasts

Myelodysplastic syndrome with ring sideroblasts (MDS-RS) is a clonal hematopoietic stem cell disorder characterized by hyperplastic and ineffective erythropoiesis, ring sideroblasts in the bone marrow, and anemia. Heterozygous mutations in the spliceosome gene SF3B1 are found in a majority of MDS-RS cases and are associated with the ring sideroblast phenotype and a favorable prognosis. MDS-RS is a usually slowly progressing disease of the elderly distinguished by an initial phase of erythroid hyperplasia and macrocytic anemia that can remain stable for several years, but which usually evolve into progressive erythroid failure and a need for regular red blood cell transfusions. The focus of this thesis was to study the genotype-phenotype relations in MDS-RS patients in order to understand the abnormal iron accumulation and erythroid failure, and in particular the role of SF3B1 in MDS pathogenesis. In study I, we tested whether ABCB7 was a key mediator of the aberrant iron accumulation, by modulating its expression in normal bone marrow. ABCB7 down-regulation reduced erythroid differentiation, growth and colony formation, and resulted in a gene expression pattern similar to that observed in MDS-RS erythroblasts, and in the accumulation of mitochondrial ferritin. Conversely, forced ABCB7 expression restored erythroid colony growth and decreased mitochondrial ferritin in MDS-RS CD34+ progenitor cells. Also, we showed altered exon usage of ABCB7 as possible explanation of the reduced expression in MDS-RS and a potential link of this gene with SF3B1. In study II we sought to identify potential downstream targets of SF3B1 mutations and understand how these affect RNA splicing and gene expression profile of MDS-RS patients. In particular, we detected a significant up-regulation of genes involved in hemoglobin synthesis and in the oxidative phosphorylation process, and down-regulation of mitochondrial ABC transporters compared to normal bone marrow. These findings together with mis-splicing of hemoglobin genes indicated a compromised hemoglobinization during MDS-RS erythropoiesis. Importantly, we demonstrated that anemia in MDS-RS patients develops during terminal differentiation into reticulocytes. Ultimately in study III, we investigated the mechanistic effects of SF3B1 mutation in MDS-RS pathogenesis and explored if the most frequent SF3B1 mutation K700E confers a loss-offunction with regard to gene expression and splicing of key genes in MDS-RS. Loss or reduction of SF3B1 normal protein in human myeloid cells resulted unexpectedly in an MDSRS-like phenotype with reduced ABCB7 expression and altered exon usage, increased levels of ALAS2 and mis-splicing of TMEM14C. Identical effects were observed when we expressed SF3B1 K700E mutation at physiological levels. Additionally, loss of SF3B1 compromised cell growth but did not increase apoptosis. Overall, our findings support an essential role of ABCB7 in the MDS-RS phenotype and offer insights into the mechanistic role of SF3B1 mutations. Via altered gene expression or mis-splicing of key genes in the heme and hemoglobin synthesis, these mutations disturb mitochondrial iron handling in a way that lead to mitochondrial iron accumulation in MDS-RS