Genetic studies of hereditary myeloproliferative disorders

More than 300 billion blood cells are being replaced daily in a process called hematopoiesis. Hematopoiesis is orchestrated by hematopoietic stem cells (HSCs) residing in the bone marrow. HSCs produce multipotent and lineage-restricted progenitors, that are responsible for the supply of mature blood cells. Production of blood cells is governed by hematopoietic growth factors that are required for the survival and proliferation of blood cells at all stages of development. Mutations in genes responsible for the regulation of this fine-tuned system cause aberrant proliferation of different blood compartments. Myeloproliferative neoplasms (MPN) are characterized by the abnormal expansion of erythroid, megakaryocytic, and myeloid lineages, that is caused either by somatic mutations or by germline mutations transmitted through Mendelian inheritance within the family. The main topic of my doctoral research was the investigation of two distinct pedigrees diagnosed with erythrocytosis and thrombocytosis, respectively. Erythrocytosis occurred in ten individuals of Norwegian family that presented elevated hemoglobin and erythropoietin (EPO) serum levels. We performed genome-wide linkage analysis using SNP arrays coupled with targeted sequencing and identified a heterozygous single base deletion (ΔG) in exon 2 of the EPO gene as the sole candidate gene mutation in affected family members. EPO stimulates the proliferation of erythrocyte progenitors and prevents their apoptosis in order to produce mature erythrocytes. Surprisingly, ΔG introduces a frame-shift that generates a novel, 51-residue long polypeptide, which would predict a loss of erythropoietin function, and is at odds with the erythrocytosis phenotype. To elucidate the mechanism by which the loss-of-function mutation causes gain-off function phenotype, we utilized the CRISPR/Cas9 genome editing to introduce the ΔG mutation into Hep3B cells, a human hepatoma cell line that expresses EPO. We found that cells with ΔG mutation produce excessive amounts of biologically active EPO and reproduces the observation form the affected family members. On the molecular level, in addition to the known transcript originating from the physiologic promoter (P1), we identified novel transcripts that initiate in intron 1 of EPO from a putative alternative promoter (P2). Further functional analysis of P2 mRNAs revealed an alternative translational start site in exon 2 that P2 transcripts use to produce a biologically active EPO protein, by fusing a novel N-terminus to the EPO coding sequence through the ΔG single base deletion. Our data demonstrate for the first time, that a mutation in EPO cause familial erythrocytosis and explain how the ΔG mutation results in a gain-function phenotype. I also investigated a pedigree with autosomal-dominant. Targeted sequencing identified a novel activating mutation in exon 3 of the thrombopoietin (THPO) gene, a single nucleotide G->T substitution. Thrombopoietin stimulates the production of platelets from megakaryocytes. THPO expression is regulated on the translational level by seven upstream open reading frames (uORF1-7) in the exons 1-3 of THPO mRNA, that are interfering with the translation of TPO. G>T mutation maps to the Kozak sequence of the uORF7, the most critical negative regulator of TPO translation. We performed TPO overexpression and in vitro translation experiments
to demonstrate that the G>T mutation disrupts the negative regulation governed by uORF7 and allows for increased translation of THPO protein coding sequence, ultimately causing thrombocytosis. Collectively, in both studies we identified novel gain-of-function mutations in hematopoietic growth factors, that act at different steps of gene expression and result in the dysregulated production of EPO and TPO, causing erythrocytosis and thrombocytosis in respective pedigrees.