The term 5q- syndrome has been applied to a subset of patients with myelodysplasia, particularly refractory anemia, and a deletion of 5q as the sole karyotypic abnormality detected in bone marrow cells. The majority of these patients are elderly women with macrocytic anemia resistant to therapy, normal to elevated platelet counts and characteristic megakaryocyte abnormalities. The clinical course is typically mild, and transformation to acute leukemia is rare. Assignments of breakpoints on the deleted chromosome 5 have varied, but those most commonly cited are 5q12-q14 proximally and 5q31-q33 distally. Based upon the work of LeBeau et al., the minimal commonly deleted segment on 5q was mapped to 5q31. Subsequent efforts to find a putative tumor suppressor gene in this region have not yet been successful. As it is known that dihydrofolate reductase is involved in the DNA synthetic pathway, that patients with folic acid deficiency develop megaloblastic and macrocytic anemias probably due to impairment of thymidilate synthesis, and that congenital dihydrofolate reductase deficiency is associated with megaloblastic anemia; we hypothesized that deletion of the DHFR gene, which has been mapped to proximal 5q, could play a role in pathogenesis of the 5q- syndrome in some patients. Our studies tested this hypothesis. The specific aims of the study were: to do high-resolution G-banding to fluorescence in-situ hybridization (FISH) mapping of a locus-specific dihydrofolate reductase (DHFR) gene probe; to determine the frequency of loss of the DHFR gene, using metaphase and interphase FISH techniques, in patients who have clonal deletions of the long arm of chromosome 5 by G-banded analysis; and to investigate the possible pathogenetic role of loss of DHFR in patients with the 5q- syndrome by correlating the cytogenetics results with clinical and morphologic features, RT-PCR analysis of the DHFR gene, hematopoietic progenitor cell assays, and response to treatment with Leucovorin. Using high-resolution G-banding to FISH techniques, we mapped the DHFR locus to subband 5q13.3. G-banded metaphase chromosome analysis of bone marrow cells from eight patients entered on-study revealed clonal deletions in the long arm of chromosome 5 with proximal breakpoints ranging from 5q13 to 5q31 and distal breakpoints from 5q33 to 5q35. FISH screening showed that the DHFR locus was present on both the normal and the deleted chromosomes 5 in all five patients analyzed. All patients had a normal sequence of the DHFR gene amplified from bone marrow RNA. Furthermore, there was no difference in the findings of G-banded chromosome analyses, in-vitro colony formation assays, marrow morphology, or transfusion requirements before and after Leucovorin treatment. These data suggest DHFR is not lost or structurally altered in bone marrow cells from patients with the 5q- syndrome. The study was therefore terminated on 4/1/02.