Hematopoiesis following disruption of the Pitx2 homeodomain gene.

OBJECTIVE: Study the effect of loss of expression of Pitx2, a homeodomain gene preferentially expressed in murine hematopoietic stem/progenitor cells, on hematopoietic stem cells (HSCs). METHODS: We examined the fetal livers of mouse embryos with homozygous disruption of the Pitx2 gene, using flow cytometry immunophenotyping analysis, as well as immunohistochemistry techniques. We further investigated the role of Pitx2 in HSCs using a chimeric mouse model system. Pitx2 null embryonic stem (ES) cell clones were generated from embryonic day 3.5 blastocysts of Pitx2 null embryos. The Pitx2 null donor ES cell contribution to the adult hematopoietic system was confirmed by identifying donor-specific glucose-phosphate isomerase isotype in the erythrocytes using cellulose acetate eletrophoresis, and by demonstrating donor-specific major histocompatibility complex antigen allotype on the granulocytes/monocytes and T and B lymphocytes of the chimeric mice using flow cytometry analysis. RESULTS: Pitx2 homozygous null fetal livers are decreased in size and overall cellularity. The erythroid cell component of these livers is further reduced as compared to that of their wild-type and heterozygous littermates. Detailed quantitative analysis of the chimeric mice revealed contribution of Pitx2 null ES cells to erythroid, myeloid, lymphoid, and megakaryocytic lineages. The quantitative level of ES cell contribution to the peripheral hematopoietic cells was proportional to the level of general chimerism as determined by coat color. CONCLUSION: Although the fetal livers of Pitx2 null embryos displayed signs of impaired erythropoiesis, Pitx2 gene disrupted HSCs can contribute to hematopoiesis under physiological conditions.

A novel mutation in the erythropoietin receptor gene is associated with familial erythrocytosis.

Primary familial erythrocytosis (familial polycythemia) is a rare myeloproliferative disorder with an autosomal dominant mode of inheritance. We studied a new kindred with autosomal dominantly inherited familial erythrocytosis. The molecular basis for the observed phenotype of isolated erythrocytosis is heterozygosity for a novel nonsense mutation affecting codon 399 in exon 8 of the erythropoietin receptor (EPOR) gene, encoding an EpoR peptide that is truncated by 110 amino acids at its C-terminus. The new EPOR gene mutation 5881G>T was found to segregate with isolated erythrocytosis in the affected family and this mutation represents the most extensive EpoR truncation reported to date, associated with familial erythrocytosis. Erythroid progenitors from an affected individual displayed Epo hypersensitivity in in vitro methylcellulose cultures, as indicated by more numerous erythroid burst-forming unit-derived colonies in low Epo concentrations compared to normal controls. Expression of mutant EpoR in interleukin 3-dependent hematopoietic cells was associated with Epo hyperresponsiveness compared to cells expressing wild-type EpoR.