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Blood, Vol. 113, Issue 8, 1749-1755, February 19, 2009
High-throughput sequencing screen reveals novel, transforming RAS mutations in myeloid leukemia patients
Blood Tyner et al.
113: 1749
Supplemental materials for: Tyner et al
Files in this Data Supplement:
- Table S1. M13 tagged and PCR sequencing primers* (PDF, 40.2 KB)
- Table S2. Relative activity of each mutant as assessed by each respective assay (PDF, 117 KB)
- Figure S1. Identification of RAS mutations in myeloid leukemia patients (JPG, 98.3 KB)
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Three cohorts of individuals comprising 192 AML patients, 32 CMML patients, and 96 normal individuals were sequenced for all coding exons in NRAS, KRAS, and HRAS. Displayed are four non-canonical RAS mutations identified in the leukemic but not the normal cohorts. The KRASA146T was identified as a heterozygous mutation in three patients and a homozygous mutation in one patient. All other alleles were heterozygous. Frozen viable cells were obtained from patients with NRASG60E, KRAST74P, and homozygous KRASA146T. Cells were immunostained with antibodies specific for cell surface markers that distinguish neoplastic from non-neoplastic cells (CD34+ vs. CD34− or CD33+ vs CD3+) and sorted by flow cytometry. Genomic DNA was isolated from each population of cells and the respective RAS exons were sequenced. All mutations were identified in neoplastic but not in non-neoplastic cells indicating that these are somatic mutations.
- Figure S2. Focus formations reveal novel mutations confer transforming capacity to Ras (JPG, 52.3 KB)
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Retroviruses expressing WT NRAS or KRAS as well as each mutant were used to infect A31 cells at equivalent multiplicity of infection. After 12 days, cells were stained with crystal violet for visualization of foci. Crystal violet-stained plates were scanned and displayed.
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