Rapid mutational analysis of N-ras proto-oncogene in hematologic malignancies: study of 77 Greek patient.
OBJECTIVE
N-ras mutations are the most commonly detected molecular abnormalities in hematologic malignancies, especially in those of myeloid origin. Different techniques have been used to detect N-ras mutations; however, most of them are either labor intensive or provide sequence data for only a limited number of codons. Consequently, study of the N-ras oncogene has not been convenient in every day clinical practice being restricted, as a rule, to retrospective analysis of patients.


METHODS
In this study we used a recently developed method that enables rapid and reliable detection of mutations at the cDNA level, namely, the non-isotopic RNase cleavage assay (NIRCA). Using this method we were able to screen the N-ras oncogene rapidly and determine the incidence and prognostic significance of N-ras mutations in 77 Greek patients with acute leukemia, myelodysplastic syndromes and chronic myeloproliferative disorders, both at the presentation and during relapse or progression of the disease.


RESULTS
Activating N-ras mutations were detected in 7 patients and our results were confirmed by direct sequencing. Interestingly, two novel alterations were identified, a mutation at codon 8 (characterized by a substitution of valine by leucine) in a patient with chronic myeloid leukemia during hematologic relapse of the disease and a polymorphism at codon 92 (1002T-->C, without amino acid substitution) in a patient with chronic myelomonocytic leukemia.


CONCLUSIONS
A rapid and easy protocol that allows the analyses of N-ras sequences has been developed. This reverse transcription-polymerase chain reaction (RT-PCR)/NIRCA protocol can allow the study of this proto-oncogene in every day clinical practice, rapidly facilitating the validation of the diagnostic and prognostic value of N-ras mutational analyses in patients with hematologic malignancies.
Speletas
M
M
First Department of Internal medicine, Democritus University of Thrace, Alexandroupolis, Greece. speletas@otenet.gr
Arvanitidi
K
K
Tzoanopoulos
D
D
Tsironidou
V
V
Pardali
E
E
Aggeli
C
C
Tsapogas
P
P
Kartalis
G
G
Sideras
P
P
Ritis
K
K
D000293:Adolescent; D000328:Adult; D000368:Aged; D002648:Child; D004252:DNA Mutational Analysis / Q000379:methods; D004722:Endoribonucleases / Q000378:metabolism; D011905:Genes, ras / Q000235:genetics*; D006115:Greece; D019337:Hematologic Neoplasms / Q000235:genetics*; D006801:Humans; D008875:Middle Aged; D009154:Mutation; D011110:Polymorphism, Genetic; D000090063:Proto-Oncogene Mas; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000592:standards*; D013997:Time Factors
C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D004722:Endoribonucleases