Abstract. Human spleen-conditioned medium can induce the formation in vitro of large granulocyte colonies from normal human bone marrow cells. The granulocyte colonies contained cells in various stages of differentiation, from myeloblasts to mature neutrophile granulocytes. Human spleen-conditioned medium also induced colony formation with rodent bone-marrow cells, whereas rodent spleen-conditioned medium induced colony formation with rodent bone marrow but not with human cells.This in vitro system has been used to determine the potentialities for cell differentiation in bone-marrow and peripheral blood cells from patients with a block in granulocyte differentiation in vivo. The cloning efficiency, colony size, and number of mature granulocytes in bone-marrow colonies from patients with congenital neutropenia, whose bone marrow contained only 1% mature granulocytes, were not less than in people whose bone marrow had the normal level of about 40% mature granulocytes. The cloning efficiency of peripheral blood cells from patients with acute myeloid leukemia was 350 times higher, with 10 times larger colonies, than the cloning efficiency of peripheral blood cells from normal people. The cytochemical properties and number of mature granulocytes in colonies from the leukemic patients were the same as in colonies from nonleukemic people.The results indicate that a block in cell differentiation in vivo, in these cases with neutropenia and acute myeloid leukemia, was overcome in vitro, in the presence of an inducer in the conditioned medium. In patients with chronic myeloid leukemia, colony formation was induced only in some of the cases. This indicates that there are blast cells with different potentialities for the development of colonies in different patients.

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Congenital dyserythropoietic anemias (CDA) are a rare group of red-blood-cell disorders of unknown etiology that are characterized by ineffective erythropoiesis, pathognomonic cytopathology of the nucleated red blood cells in the bone marrow, and secondary hemochromatosis. In CDA type I, bone-marrow electron microscopy reveals characteristic findings in erythroid precursors, including spongy heterochromatin and enlarged nuclear pores. Since the genetic basis of CDA type I is not evident, we used homozygosity and linkage mapping to localize the genetic defect responsible for CDA type I in 25 Bedouins from four large consanguineous families. We report the linkage of this disease to markers on chromosome 15 located at q15. 1-q15.3. Fourteen markers within a 12-cM interval were typed in the relevant family members. Nine of the markers yielded maximum LOD scores of 1.625-12.928 at a recombination fraction of .00. Linkage disequilibrium was found only with marker D15S779. Haplotype analysis revealed eight different carrier haplotypes and highlighted the existence of a founder haplotype. Identification of historical crossover events further narrowed the gene location to between D15S779 and D15S778. The data suggest localization of the CDA type I gene within a 0.5-cM interval. The founder mutation probably occurred >/= 400 years ago. Sequence analysis of the coding region of protein 4.2, the only known erythroid-specific gene in the locus, did not reveal any change in the CDA type I patients. Future analysis of this locus may lead to the identification of a gene essential to normal erythropoiesis.

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Congenital dyserythropoietic anemia (CDA) type I is a rare macrocytic anemia of unknown etiology. In the present study, we redefined the clinical and laboratory picture of CDA type I, some of its pathogenic aspects, and the association with thalassemia-like features in 20 patients, all of whom belong to one Bedouin tribal group and are probably descended from a common ancestor. In each case ultrastructural studies of bone marrow (BM) erythroblasts showed the classic morphological findings of CDA type I. Serological tests for CDA type II were negative. The clinical picture was variable, but mostly benign. Some patients displayed elevated hemoglobin A2 levels or high ratio of alpha- to non-alpha- globin. However, neither family studies nor complete sequence analysis of the beta-globin was compatible with beta- thalassemia. Increased erythropoiesis was manifested by a high number of BM erythroid burst-forming units. Serum erythropoietin was also elevated. BM flow cytometry studies demonstrated arrest of erythroid precursors in the S phase of the cell cycle. The ultrastructural morphological features of the erythroid precursors, showing peripheral chromatin condensation, suggest apoptosis. Additional studies are indicated to define the molecular basis of this disease.