Direct sequencing and quantitative mutational analysis of NRAS in JMML patients.NRAS mutations are detected by direct sequencing and quantified by pyrosequencing. Direct sequencing identified oncogenic NRAS mutations: for Patient 1, G12D, 35G > A; for Patient 2, G12S, 34G > A) in BM-MNCs at diagnosis of JMML and in the nails and buccal smear cells. Quantification by pyrosequencing revealed that the fractions of mutated allele varied among different tissue types. For Patient 1: BM, 50%; nail, 24%; and buccal smear, 43%. For Patient 2: BM, 48%; left-hand nail, 26%; and buccal smear, 21%.