PTPN11 mutations in JMML specimens. (A) DNA samples from 3 patients with JMML were amplified by using exon 3–specific primers, and the products were analyzed on DHPLC. (B) PTPN11 mutations corresponding to the abnormal DHPLC spectrographs shown in panel A. Abnormal nucleotides are marked with arrows. (Top) Mutation at nucleotide 181 (G>T); (middle) mutation at nucleotide 182 (A>T); (bottom) mutation at nucleotide 215 (C>T). (C) Absence of the normal PTPN11 allele in a JMML sample; (lane 1) 1-kb ladder, (lane 2) uncut (u) amplified DNA from healthy bone marrow, (lanes 3-5) DNA amplified from a healthy specimen (WT, lane 3), a patient with a heterozygous mutation (A, lane 4), and the patient with the homozygous mutation (B, lane 5). Note the presence of an abnormal (uncut) band (white asterisk) in both patients, with absence of the normal digested bands in patient B. (Lanes 6-8) The same 3 specimens were cut with MaeIII, which cleaves at a novel site within the mutant but not the normal allele (lane 6). (Lanes 7-8) An abnormal band (white asterisk) is visible in both patients A and B, and loss of the upper normal band is seen in patient B (lane 8).