Development of a Diagnostic Test for
Mutations in NPM1 Exon 12 in
Cytogenetically Normal Acute Myeloid
Leukaemia (AML) Patients
Alison Skinner
Wessex Regional Genetics Laboratory
NPM1 function
• Implicated in leukaemia as a translocation
partner for various oncogenes
• Nucleolar phosphoprotein present
predominantly in the nucleolus
• Regulates translational activity of p53 after
stress
• Involved in centrosome duplication in the
cell cycle via cyclin E/CDK2
phosphorylation
Effect of NPM1 mutations
• Gives prognostic information to the AML
patients with a normal karyotype
(phenotypically variable)
• Favourable prognosis in the absence of
the FLT3 ITD
• Better response to induction therapy and
have a better overall survival / longer
event free survival
Acquired Mutations in NPM1
Wildtype:
GCTATTCAAGATCTCTGGCAGTGGAGGAAGTCTCTTTAAgaaaatag
-A--I--Q--D--L--W--Q--W--R--K--S--L--*--------Mutation A:
GCTATTCAAGATCTCTGTCTGGCAGTGGAGGAAGTCTCTTTAAgaaaatag
-A--I--Q--D--L--C--L--A--V--E--E--V--S--L--R--K--*Mutation B:
GCTATTCAAGATCTCTGCATGGCAGTGGAGGAAGTCTCTTTAAgaaaatag
-A--I--Q--D--L--C--M--A--V--E--E--V--S--L--R--K--*Mutation D:
GCTATTCAAGATCTCTGCCTGGCAGTGGAGGAAGTCTCTTTAAgaaaatag
-A--I--Q--D--L--C--L--A--V--E--E--V--S--L--R--K--*-
Results of Direct Sequencing
From the original 66 samples
• 41 had no visible mutation
• 1 had an intronic mutation of unknown
significance
• 19 had a frameshift mutation:
–
–
–
–
13 had mutation ‘A’
1 had mutation ‘B’
3 had mutation ‘D’
2 had novel mutations which still produced the same
NES
Principles of Pyrosequencing
Image from www.pyrosequencing.com
Designing the Pyrosequencing
Assay
Wildtype
Mutation A
Mutation B
Mutation D
Testing the Pyrosequencing
Assay
Normal
Mutation A
Mutation B
Mutation D
Further Analysis of the
Pyrosequencing Results
Examples of Results Using
Analysis Spreadsheet
Validation – 1
Retesting the Original Cohort
• All 66 samples that were tested by direct
sequencing were re-tested using the
pyrosequencing assay
• 6 / 66 failed
– 1 sample failed for pyrosequencing but was normal
on direct sequencing
– 1 sample failed for direct sequencing but had
mutation ‘A’ on pyrosequencing,
– All other failures had failed for both techniques
• Results of all other samples matched the results
for direct sequencing
Validation – 2
Normal controls
• 3 / 96 failed
• There was no evidence of mutations in the
normal test plate
• A quantification below 10% should be
treated as normal / with caution
(depending on the quality of the data)
Validation – 3
Titration
• A titration of mutational load was set up
• Reliably sensitive to around 20% mutation
60
50
40
A
30
B
%
D
Linear (A)
Linear (D)
20
Linear (B)
10
0
Neat
9:1
8:2
7:3
6:4
5:5
-10
Dilution
4:6
3:7
2:8
1:9
Summary
• The test is sensitive and relatively highthroughput
• Identifies and quantifies the common
NPM1 exon 12 mutations
• Is able to identify other ‘novel’ mutations in
the region
• Helpful in identifying patients who have a
favourable prognosis
Acknowledgements
• Dr Helen White – NGRL (Wessex)
• Prof. Nick Cross – WRGL
• Christine Waterman - WRGL