The AML Tumor Suppressor Gene on Chromosome 5, Band q31: Localization and Evaluation of Novel Genes
Interstitial deletions or complete loss of the long arm of chromosome 5, centering at 5q31, are seen in a variety of hematologic malignancies, including acute myeloid leukemia (AML) and myelodysplasia (MDS). The consistent nature of these deletions, and their association with poor prognosis, suggests the presence of a tumor suppressor gene (TSG) whose mutational loss or inactivation causes this malignancy. Identification of this gene is being attempted by positional cloning. Previous studies have narrowed the likely site for this TSG site to a 600 kb interval between markers D5S500 and D5S594; thus, all genes lying in this interval are potential candidates for the suggested TSG. The objective of this project was to evaluate genes and expressed sequence tags (ESTs) obtained by database searching, to ascertain whether they are located within the interval, to confirm that they truly originate from expressed sequences, and to gather additional cDNA sequence so their assessment as TSGs can proceed. The first set of 23 genes/ESTs had been tentatively localized by analysis of Radiation Hybrid Database; the second set of 19 genes/ESTs was obtained by analysis of genomic sequence (from Human Genome Centers). Initially, all sequences were localized by PCR-based typing on a radiation hybrid breakpoint map that had been prepared for the interval. Among the first set of genes/ESTs, 9 of 23 were localized to the interval, including four known genes: CDC25C (cell division cycle 25C), HSPA9 (a cellular immortalization protein known as Mortalin), CTNNA1 (aE-Catenin), and EGR1 (Early Growth Response Gene 1). The remaining five transcript units are unknown ESTs: D5S1856, SGC32445, WI-15469, stSG8723, and WI-17966. RT-PCR and Northern hybridization show that all nine sequences are expressed in myeloid tissues and, therefore, represent potential TSG candidates. Additional cDNA sequence was then obtained by searching GenBank, Unigene, and TIGR databases, using multiple iterations to identify overlaps and extend the sequence; so far, we have obtained 5.5 kb for D5S1856, 2 kb for SGC32445, 1.3 kb for WI-15469, 0.8 kb for stSG8723, and 0.7 kb for WI-17966. As to the second set of genes/ESTs, confirmation of their localization to the interval is still in progress. For all of the candidate genes, further sequence will be obtained using Rapid Amplification of cDNA Ends (RACE) and cDNA
library screening. When full length cDNA sequences are obtained, all known and novel genes will be analyzed for mutations in clinical AML or MDS samples, and their function will be studied to identify the AML/MDS tumor suppressor gene.