Contributions
Type: Publication Only
Background
An increase of allele burden and development of homozygosity with the JAK2 gene V617F mutation is well-known in polycythemia vera (PV) patients (Vannucchi AM et al, Leukemia 2008). However, the homozygosity development of specific for PV mutations in JAK2 exon 12 has been much less studied.
Aims
The aim of this study was detection and quantification of JAK2 exon 12 allele burden in JAK2 V617F- negative PV patients using a pyrosequencing method.
Methods
The 10 DNA samples from JAK2 V617F- negative patients with confirmed clinical diagnosis and bone marrow morphological features have been selected. The informed consents from these patients were obtained. The nucleotide sequencing of the JAK2 exon 12 fragment was performed by pyrosequencing method as described in (Mironov K et al, Spravochnik zav clin lab, 2011). The routine sample preparation and PCR kits («AmpliSens», Russia) we used. Pyrosequencing was carried out with «PyroMark Q24» using «PyroMark Gold» kit («Qiagen», Germany). To verify the presence of mutations, the DNA sequences extracted from the clinical samples were cloned into pGem-T vector using standard protocol («Promega», USA), and obtained clones were sequenced using reagents and equipment of the «Applied Biosystems» (USA).
Results
Among 10 of JAK2 V617F-negative PV patients, a N542-E543del mutation of JAK2 in exon 12 has been detected in one 62-year old male patient. On the onset of the disease in July 2013 he had a pronounced plethoric syndrome, Hb 204 g/l, HCT 49.9%, RBC 6.43?1012/l, WBC 8.0?109/l, PLT 250?109/l. The level of JAK2 exon 12 N542-E543del allele burden was 19%. The patient was treated phlebotomy only and did not received any cytoreductive treatment during 18 months. After these 18 months, the patient characterized with presence of moderate splenomegaly, level of Hb 171 g/l, HCT 61.3%, RBC 9.03?1012/l, WBC 7.47?109/l, PLT 500?109/l. The level of JAK2 exon 12 N542-E543del allele burden was increased up to 68%.
Summary
A pyrosequencing with «PyroMark Q24» was able to identified mutations in JAK2 exon 12 and it demonstrated a possibility that N542-E543del mutation is evolving into homozygous form in the same manner as described for the JAK2 V617F mutation.
Keyword(s): Mutation analysis, Myeloproliferative disorder, Polycythemia vera
Session topic: Publication Only
Type: Publication Only
Background
An increase of allele burden and development of homozygosity with the JAK2 gene V617F mutation is well-known in polycythemia vera (PV) patients (Vannucchi AM et al, Leukemia 2008). However, the homozygosity development of specific for PV mutations in JAK2 exon 12 has been much less studied.
Aims
The aim of this study was detection and quantification of JAK2 exon 12 allele burden in JAK2 V617F- negative PV patients using a pyrosequencing method.
Methods
The 10 DNA samples from JAK2 V617F- negative patients with confirmed clinical diagnosis and bone marrow morphological features have been selected. The informed consents from these patients were obtained. The nucleotide sequencing of the JAK2 exon 12 fragment was performed by pyrosequencing method as described in (Mironov K et al, Spravochnik zav clin lab, 2011). The routine sample preparation and PCR kits («AmpliSens», Russia) we used. Pyrosequencing was carried out with «PyroMark Q24» using «PyroMark Gold» kit («Qiagen», Germany). To verify the presence of mutations, the DNA sequences extracted from the clinical samples were cloned into pGem-T vector using standard protocol («Promega», USA), and obtained clones were sequenced using reagents and equipment of the «Applied Biosystems» (USA).
Results
Among 10 of JAK2 V617F-negative PV patients, a N542-E543del mutation of JAK2 in exon 12 has been detected in one 62-year old male patient. On the onset of the disease in July 2013 he had a pronounced plethoric syndrome, Hb 204 g/l, HCT 49.9%, RBC 6.43?1012/l, WBC 8.0?109/l, PLT 250?109/l. The level of JAK2 exon 12 N542-E543del allele burden was 19%. The patient was treated phlebotomy only and did not received any cytoreductive treatment during 18 months. After these 18 months, the patient characterized with presence of moderate splenomegaly, level of Hb 171 g/l, HCT 61.3%, RBC 9.03?1012/l, WBC 7.47?109/l, PLT 500?109/l. The level of JAK2 exon 12 N542-E543del allele burden was increased up to 68%.
Summary
A pyrosequencing with «PyroMark Q24» was able to identified mutations in JAK2 exon 12 and it demonstrated a possibility that N542-E543del mutation is evolving into homozygous form in the same manner as described for the JAK2 V617F mutation.
Keyword(s): Mutation analysis, Myeloproliferative disorder, Polycythemia vera
Session topic: Publication Only