Download FRAGMENT LENGTH ANALYSIS SCREENING FOR CEBPa

FRAGMENT LENGTH ANALYSIS SCREENING FOR CEBPa MUTATIONS
DETECTION IN ACUTE MYELOID LEUKEMIA
Fuster O, Barragán E, Bolufer P, Cervera J, Duch E, Montesinos P, Sanz MA.
BACKGROUND
CEBPa encodes a protein member of the basic region lucine zipper (bZIP) transcription
family that is essential for myeloid differentiation. CEBPa gene consists of two N-terminal
transactivating domains (TAD1 and TAD2) and a C-terminal basic and leucine zipper
region (bZIP). Inactivating CEBPa mutations have been reported predominantly in normal
karyotype acute myeloid leukaemia (AML) and have been related with a favourable
outcome.
AIMS
Our objective was to set up a rapid fragment analysis method for the screening of CEBPa
mutations and to validate this method in a group of AML patients.
METHODS
The study included 70 AML patients with normal or intermediate-risk karyotype. Twentynine patients (41%) showed FLT3-ITD, NPM1 mutations or both.
CEBPa mutations study was performed on genomic DNA extracted from bone marrow
samples collected at diagnosis. Three PCR were performed using three primer pairs to
amplify TAD1, TAD2 (including region between the two domains) and bZIP domains with
forward primers fluorescently labelled with FAM.
Thirty nanograms of DNA were amplified in a 25 µL reaction containing 7% DMSO and
0.3U of Taq Expand High Fidelity for TAD1 and bZIP fragments and 10% DMSO, 1x Pfx
Amp Buffer, 1x PCR Enhancer Solution and 2U Platinum Taq DNA Polimerase for TAD2
fragment.
Initial denaturation at 95ºC for 3 min was followed by 35 cycles of denaturation at 95ºC 1
min, annealing at 55ºC for 40 sec and extension at 60ºC for 1 min. A final extension of
94ºC 30 sec and 60ºC 45 min was performed.
The mixtures were electrophoresed on an ABI PRISM 3130 Genetic Analyzer and results
were analysed using the GENEMAPPER software (Applied Biosystems).
Results were confirmed by sequence analysis using ABI PRISM terminator cycle
sequencing kit v1.1 (Applied Biosystems) on the ABI PRISM 3130 Genetic Analyzer.
RESULTS
Eleven out of 70 (16%) patients showed altered electropherograms. Sequence analysis
confirmed nucleotide sequence variation in all the cases. These variations were the
polymorphism P194_H195dup in two patients and CEBPa mutations in the remaining nine
patients (13%) (Table 1). Six out of nine patients had two mutations and three patients had
a single mutation. All patients were sequenced in parallel and no additional mutations were
detected. When we consider FLT3 and NPM1 status we observed that 41 patients without
FLT3 or NPM1 mutations showed 9 (22%) CEBPa mutations. By contrast no CEBPa
mutations were detected in any of the 29 patients with FLT3 and/or NPM1 mutations. The
subgroup of patients FLT3 and NPM1 negatives with CEBPa mutated showed a trend to a
better relapse free survival than wild-type patients (75% vs 23%, p=0,09) at 2 years.
CONCLUSIONS
Fragment analysis is a rapid, specific and sensitive method for CEBPa mutation screening.
The incidence of CEBPa mutations was 22% in the group of AML with normal or
intermediate karyotype and without FLT3 or NPM1 mutations. In this group without
molecular markers CEBPa mutations may establish a subgroup of patients with better
prognosis.
ACKNOWLEDGEMENTS
This work was partially supported by the grants No. 06/0657 and RD06/0020/0031 [Fondo
de Investigación Sanitaria (FIS)/Instituto de Salud Carlos III, Ministry of Health (Spain)].