detect the common Myeloproliferative Neoplasm- associated mutations - - PowerPoint PPT Presentation

Development of a NGS approach to simultaneously detect the common Myeloproliferative Neoplasm- associated mutations in JAK2, CALR, and MPL

• T. Frawley, C. O’Brien, M. Percy, E. Conneally, E. Vandenberghe, S. Langabeer, & K. Haslam

Myeloproliferative Neoplasms

MPN classification Overlapping clinical features of MPN

MPN driver mutations

PV ET PMF

JAK2 MPL CALR

Profile of mutations in MPN Molecular Diagnostic Algorithm

Next Generation Sequencing

Ion Torrent PGM

Materials and Methods

Primer Design PCR Optimisation Multiplex Optimisation Multiplex PCR Product Equalisation NGS of 35 samples positive for MPN associated mutations

Results - optimisation

NGS

Primer Concentration

GEL

No of Reads

JAK2 Ex 12 CALR MPL JAK2 Ex 14

NGS results

No of Samples Gene Positive Mutation

1 CALR 52bp del 1 52bp del 1 52bp del 12bp del 3 5bp ins 1 34bp del 1 49bp del 1 2bp ins 1 30bp del 1 46bp del 1 19bp del 5 MPL W515L 3 W515K 14 JAK2 V617F 1 K539L

Mutation Detected HGVS.c

52 bp del c.1099_1150del 52 bp del L367T c.1099_1150del c.1132G>C 52bp del 13bp del c.1099_1150del c.1126_1143delinsTG 5bp ins c.1154_1155insTTCTC 34bp del c.1099_1132del 49bp del c.1097_1149del 2bp ins c.1123_1125delinsTGTTT 31bp del c.1111_1141del 46bp del c.1100_1145del 19bp del c.1199_1218del W515L c.1544G>T W515K c.1543_1544TG>AA V617F c.1849G>T K539L c.1615_1616delinsTT

Known mutations Mutations sequenced 100% concordance

CALR 2bp ins c.1123_1125delinsTGTTT

Increased Resolution

AAA del TGTTT ins

Conclusion

• Single assay vs stepwise algorithms
• Simultaneous screening for rare and common mutations
• 100% specificity
• Good Sensitivity
• Increased resolution
• Sample preparation is labour intensive for high throughput
• Automation