development of a diagnostic test for mutations in npm1 exon 12 in cytogenetically normal acute...
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Development of a Diagnostic Test for Mutations in NPM1 Exon 12 in
Cytogenetically Normal Acute Myeloid Leukaemia (AML) Patients
Alison SkinnerWessex 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 NPM1Wildtype:
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
100E S G C T C T G C A T A G T C G C A G A G T C T C
A:100.0%T:0.0%
T:100.0%C:0.0%
5 10 15 20
Normal
100
120
E S G C T C T G C A T A G T C G C A G A G T C T C
A:53.8%T:46.2%
T:53.5%C:46.5%
5 10 15 20
Mutation D
100
120
E S G C T C T G C A T A G T C G C A G A G T C T C
A:59.1%T:40.9%
T:57.8%C:42.2%
5 10 15 20
Mutation A
100
110
120
E S G C T C T G C A T A G T C G C A G A G T C T C
A:71.7%T:28.3%
T:64.2%C:35.8%
5 10 15 20
Mutation B
Further Analysis of the Pyrosequencing Results
Examples of Results Using Analysis Spreadsheet
Validation – 1Retesting 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 – 2Normal 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 – 3Titration
• A titration of mutational load was set up• Reliably sensitive to around 20% mutation
-10
0
10
20
30
40
50
60
Neat 9:1 8:2 7:3 6:4 5:5 4:6 3:7 2:8 1:9
Dilution
%
A
B
D
Linear (A)
Linear (D)
Linear (B)
Summary
• The test is sensitive and relatively high-throughput
• 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
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