Ann Lab Med.  2013 May;33(3):159-166. 10.3343/alm.2013.33.3.159.

N-ras Mutation Detection by Pyrosequencing in Adult Patients with Acute Myeloid Leukemia at a Single Institution

• Jeong JH
• Park SH
• Park MJ
• Kim MJ
• Kim KH
• Park PW
• Seo YH
• Lee JH
• Park J
• Hong J
• Ahn JY

• KMID: 1781320
• DOI: http://doi.org/10.3343/alm.2013.33.3.159

Abstract

BACKGROUND
N-ras mutations are one of the most commonly detected abnormalities of myeloid origin. N-ras mutations result in a constitutively active N-ras protein that induces uncontrolled cell proliferation and inhibits apoptosis. We analyzed N-ras mutations in adult patients with AML at a particular institution and compared pyrosequencing analysis with a direct sequencing method for the detection of N-ras mutations.
METHODS
We analyzed 90 bone marrow samples from 83 AML patients. We detected N-ras mutations in codons 12, 13, and 61 using the pyrosequencing method and subsequently confirmed all data by direct sequencing. Using these methods, we screened the N-ras mutation quantitatively and determined the incidence and characteristic of N-ras mutation.
RESULTS
The incidence of N-ras mutation was 7.2% in adult AML patients. The patients with N-ras mutations showed significant higher hemoglobin levels (P=0.022) and an increased incidence of FLT3 mutations (P=0.003). We observed 3 cases with N-ras mutations in codon 12 (3.6%), 2 cases in codon 13 (2.4%), and 1 case in codon 61 (1.2%). All the mutations disappeared during chemotherapy.
CONCLUSIONS
There is a low incidence (7.2%) of N-ras mutations in AML patients compared with other populations. Similar data is obtained by both pyrosequencing and direct sequencing. This study showed the correlation between the N-ras mutation and the therapeutic response. However, pyrosequencing provides quantitative data and is useful for monitoring therapeutic responses.

Keyword

N-ras; AML; Pyrosequencing; Bone marrow

MeSH Terms

Adult
Aged
Aged, 80 and over
Antineoplastic Agents/therapeutic use
Bone Marrow/metabolism
Codon
Cytogenetic Analysis
Female
Hemoglobins/metabolism
Humans
Incidence
Leukemia, Myeloid, Acute/drug therapy/epidemiology/*genetics
Male
Middle Aged
Mutation
Sequence Analysis, DNA
fms-Like Tyrosine Kinase 3/genetics
ras Proteins/*genetics
Antineoplastic Agents
Codon
Hemoglobins
fms-Like Tyrosine Kinase 3
ras Proteins

Figure

• 

  Fig. 1 Schematic representation of the location of the direct sequencing primers on the N-ras codons 12, 13, and 61. Codons 12 and 13 are located in exon 1, and codon 61 is in exon 2. For the detection of mutations in codon 12 and 13, PCR products of 607 nucleotides were analyzed. Mutations in codon 61 were detected by sequencing the PCR product of exon 2 (248 nucleotides).

• 

  Fig. 2 N-ras mutations established by pyrosequencing and confirmed by direct sequencing. (A) Pyrogram showing the presence of mutation and quantitative data of mutated clones. The horizontal axis refers to the base, and the vertical axis represents the intensity of the fluorescent signal. The shaded regions represent the analyzed codon. The first box shows the analytic results of substitutions in the first base of the N-ras codons 12 and 13. The second box presents mutations in the second base of the N-ras codons 12 and 13. The third box shows a positive result for a mutation in the second base of codon 61. (B) All patient samples were confirmed by direct sequencing, and the plots showed the same results as pyrosequencing.

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