Abstract
During tumor development, loss of heterozygosity (LOH) often occurs. When LOH is preceded by an oncogene activating mutation, the mutant allele may be further potentiated if the wild-type allele is lost or inactivated. In myeloproliferative neoplasms (MPN) somatic acquisition of JAK2V617F may be followed by LOH resulting in loss of the wild type allele. The occurrence of LOH in MPN and other proliferative diseases may lead to a further potentiating the mutant allele and thereby increasing morbidity. A real time PCR based SNP profiling assay was developed and validated for LOH detection of the JAK2 region (JAK2LOH). Blood of a cohort of 12 JAK2V617F-positive patients (n = 6 25–50% and n = 6>50% JAK2V617F) and a cohort of 81 patients suspected of MPN was stored with EDTA and subsequently used for validation. To generate germ-line profiles, non-neoplastic formalin-fixed paraffin-embedded tissue from each patient was analyzed. Results of the SNP assay were compared to those of an established Short Tandem Repeat (STR) assay. Both assays revealed JAK2LOH in 1/6 patients with 25–50% JAK2V617F. In patients with >50% JAK2V617F, JAK2LOH was detected in 6/6 by the SNP assay and 5/6 patients by the STR assay. Of the 81 patients suspected of MPN, 18 patients carried JAK2V617F. Both the SNP and STR assay demonstrated the occurrence of JAK2LOH in 5 of them. In the 63 JAK2V617F-negative patients, no JAK2LOH was observed by SNP and STR analyses. The presented SNP assay reliably detects JAK2LOH and is a fast and easy to perform alternative for STR analyses. We therefore anticipate the SNP approach as a proof of principle for the development of LOH SNP-assays for other clinically relevant LOH loci.
Highlights
Neoplastic disorders may have a variety of underlying molecular and cytogenetic defects
JAK2V617F is caused by a G-toT transversion in the Janus Kinase 2 (JAK2) gene resulting in a valine-to-phenylalanine (V-F) amino acid substitution at codon 617 [1,2,3,4]
JAK2V617F-positive Patient Cohort To test our Single Nucleotide Polymorphism (SNP) assay for detection of JAK2LOH, 12 patients were selected based on JAK2V617F-positive mutational status
Summary
Neoplastic disorders may have a variety of underlying molecular and cytogenetic defects. In 2005, the involvement of the JAK2V617F somatic point mutation in myeloproliferative neoplasms (MPN) became apparent. JAK2V617F is caused by a G-toT transversion in the Janus Kinase 2 (JAK2) gene resulting in a valine-to-phenylalanine (V-F) amino acid substitution at codon 617 [1,2,3,4]. JAK2 is a non-receptor tyrosine kinase involved in the JAK-STAT signalling pathway [5]. The JAK2V617F mutation apparently alters the activity of the autoinhibitory pseudokinase JH2 domain resulting in a constitutive activation of the JAKSTAT signalling pathway, leading to a growth factor independent cell proliferation [6]. The diagnostic value of JAK2 mutational analysis in MPN is well established and has been endorsed in the classification of haematological malignancies by the World Health Organisation [7]
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