The research proposed here will test the hypothesis that a critical gene encoded within the 300 kbp Yeast Artificial Chromosome (YAC) spanning the anonymous polymorphic locus D5S89 on human chromosome 5q3l, is inactivated in a subset of pre leukemic myelodysplasias (MDS) and acute myelogenous leukemias (AMLs). Acquired partial deletions of the long arm of chromosome 5 (5q-) are seen frequently in AMLs and MDS. Considerable evidence suggests that the 5q anomaly occurs in a myeloid precursor cell. Patients who undergo chemotherapy for primary malignancies of the ovaries or breast are at nine to twelve-fold increased risk for secondary MDS/AML. Fifty-percent of these cases present with a 5q- chromosome. Presence of the 5q deletion is a marker for poor prognosis in both de novo and secondary AML and MDS. Identification of a common deleted region at 5q31 in all of these cases, suggests that loss of a critical gene coupled mutation of the remaining allele contributes to leukemogenesis. The following lines of evidence suggest that the 300 kbp YAC harbors the critical gene: (i) the polymorphic D5S89 locus is consistently deleted in every single case of informative MDS and AML carrying the 5q- chromosome, although the cytogenetic limits of these deletions vary; (ii) the D5S89 is interrupted by Long Interspersed Nuclear Element (LINE) sequences on a marker chromosome of the AML cell line HL60; and (iii) Fluorescent in situ hybridization of metaphases from a myelodysplasia patient, with sequences representing 300 kbp of this locus, revealed deletion from the 5q-chromosome and interruption by inversion/duplication of the """"""""normal chromosome 5"""""""". This proposal describes experiments to isolate and characterize the myeloid tumor suppressor gene.
The specific aims of this project are: I. Narrow down the critical region of loss further; II. Isolate cDNAs encoded within the critical region; and III. Identify the MDS/AML candidate gene/s from the cDNAs and search for somatic mutation on the remaining alleles in cases of 5q- MDS and AML.
