Abstract
Nβras gene activation occurs via single base substitutions in codons 12, 13, and 61. We have developed a rapid screening method, termed allele specific restriction analysis (ASRA), for detection of Nβras mutations at these three critical codons in acute myeloid leukemia (AML). Patient DNA samples are amplified by the polymerase chain reaction (PCR) by using primers that induce restriction sites in normal but not mutant Nβras alleles. We have used ASRA to identify 5 point mutations in four out of 19 patients at initial presentation of de novo AML. Three patients had one mutation at codon 12, 13, or 61 respectively, while a fourth patient had concurrent mutations at codons 12 and 13. Nβras mutations were more common in patients over 65 years of age (P < 0.04), but did not correlate with FAB classification, attainment of complete remission, disease free survival, or overall survival. ASRA can also be used as the first step in a more sensitive approach to the detection of ras mutations. When ASRA was combined with allele specific oligonucleotide (ASO) hybridization the sensitivity and specificity of these assays were increased. This allowed identification of additional low level mutations in two patients. The data presented here constitute the first complete analysis of Nβras mutations in leukemia by ASRA and include the first identification of three concurrent Nβras mutations in a single leukemic patient. By facilitating sensitive sequential studies, ASRA should contribute to our understanding of the role of Nβras mutations in leukemogenesis.