1. Sanger sequencing results
Implementation and improvement of molecular diagnostics of polycythemia vera in Latvia
PAPER ID 23010
Z. Dobele1 (Presenter ), B. Janicka-Kupra2,3, K. Bernate2,3, L. Belajeva3, K. Mikuda3 , L. Zarina3, D. Rots1 , A. Tutane1, S. Lejniece2,3 , L. Gailīte1
1 Rīga Stradiņš University,Scientific Laboratory of Molecular Genetics
2 Rīga Stradiņš University
3 Riga East University Hospital, Clinic of Chemotherapy and Haematology
Introduction
Results
Molecular testing significantly improve diagnostic criteria and prognostic relevance of entities included in Word Health Organization classification of myeloproliferative neoplasms (MPN).
Pathogenic allelic variation presence in genes JAK2, CALR and MPL have been presented as major criteria for polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). JAK2-associated myeloproliferative neoplasms (MPNs) major criteria shown in Table 1.
Three patients initially diagnosed with PV were excluded from the PV group based on allelic variations that were found in CALR gene, which are typical only in ET and PMF groups.
In Figure 2 molecular findings present that from all other PV patients allele V617F was found for 69 (83%) patients.
In one patient among V617F-negative patients allele F537-F547dup11(33 bp duplication, see Figure 3) in exon 12 of JAK2 gene was found. In the group of PV 12% were negative for performed genetic testing.
Figure 2.
Table 1. Adapted from «The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia» Daniel A. Arber et al., 2016
Aim of the project
F537-F547dup11 in JAK2 exon 12
Sanger sequencing results
Figure 3.
In the last two years we have successfully introduced JAK2 V617F and CALR gene testing for patients with BCR-ABL1 negative myeloproliferative neoplasms (MPN), but currently our aim was to improve molecular diagnostics of JAK2 gene analysis for PV patients.
Materials and Methods
A total of 291 patient samples were sent to RSU Scientific laboratory of Molecular Genetics for the analysis of the most common MPN genetic causes. Only 175 of them were confirmed as MPNs and classified in four groups (see Figure 1): PV (n=86; 49%), ET (n=23; 13%), PMF (n=15; 9%) and unclassifiable MPN (n=51; 29%).
JAK2 gene allele V617F and indels in 9th exon of CALR gene was analyzed for all MPN patients by using qPCR and fluorescent fragment length analysis, respectively.
Sanger sequencing was used for JAK2 exon 12 screening that was performed only for patients who presented as JAK2 V617F-negative PV.
Conclusions
Results show high number of unclassifiable MPN and V617F-negative PV patients. Three patients were misdiagnosed having PV before molecular testing, therefore proving the usefulness of genetic testing also for differential diagnostics.
As still there are many patients who lack any molecular findings in analyzed genes, there is a need for improvement of sensitivity of used methods as well as introduction of MPL gene analysis in routine molecular testing of MPN.
The growing body of genetic data have not only altered the classification of myeloid neoplasms, but are also impacting patient management. Genetically-defined disease categories have characteristic prognoses and predicted clinical behavior.
Figure 1.