ABSTRACT: Spectrin deficiency is the most common deficiency found in HS. It is heterogeneous in terms of clinical expression, inheritance (dominant or recessive) and underlying genetic defects (related to alpha- or beta-spectrin gene defects or secondary to ankyrin gene defects). We studied a sampling of French dominant HS families, selected after linkage analyses, and found the presence of mutations resulting in the silencing of the mutant beta-spectrin allele. In three HS families, one haploid set of beta-spectrin mRNA was undectectable. In two families, a deletion of 8 bases (leading to a frameshift and a premature stop codon) and a nonsense mutation were identified, respectively. In the third HS family, we were unable to characterize a relevant mutation but the loss of heterozygosity at the cDNA level suggested the presence of a null mutation of the beta-spectrin allele. Sequencing of the beta-spectrin gene has also uncovered several new polymorphisms in the coding region of the beta- spectrin gene which will be very useful for detecting loss of heterozygosity at the cDNA level and designating the beta- spectrin gene as the culprit one.

Keywords: Dominant, erythrocytes, membrane, frameshift, nonsense.

Reprint requests to: Didier Dhermy, INSERM U409, Faculté X. Bichat, B.P. 416 75870 Paris Cedex 18, France, phone: (33)1 44856344, fax: (33)1 42264624, e-mail: dhermy@bichat.inserm.fr.

ABSTRACT: The primary role of protooncogene c-kit in mast cell differentiation is supported by the development of mast cells from CD34+/CD117+(c-kit) myeloid precursors. Growth factor independence, neoplastic transformation and differentiation of mast cells were found in association with c-kit activating mutations in both murine and human mastocytoma and mast cell diseases. We have identified a novel c-kit mutation (D816Y) in peripheral blood mononuclear cells from a patient with AML (M2), massive presence of mast cells in bone marrow and rapid progression of the disease. The mutation, a G T transversion at nt 2467 of the c-kit gene resulting in Asp816 Tyr substitution, corresponds to the D814Y and D817Y mutations identified and characterized in the murine P815 mastocytoma and the rat RBL-2H3 mast cell leukemia cell lines. The absence of SCF transcripts that we found by RTPCR in the patient's blasts indicates that, also in humans, this activating mutation leads to SCF independent growth. The expression of the mutant allele on Kit signaling may be further enhanced by trisomy of chromosome 4 (carrying the c- kit gene) in the patient's blasts. From these findings it is concluded that mast cells could be generated from a leukemic CD34/CD117-positive clone, that combines the antigenic expression of mast cell precursor to the growth and differentiation factor- independence which was derived by the c-kit D816Y mutation.

Keywords: C-kit, activating mutation, ligand independence, mast- cells, differentiation.

Reprint requests to: Lidia Larizza, M.D., Department of Biology and Genetics, Via Viotti 3/5 20133 Milano, Italy, phone: 39-2-23693226, fax: 39-2-70602472, e-mail: larizza@imiucca.csi.unimi.it.