Detection of JAK2 V617F Mutation in Myeloproliferative Neoplasms (MPNs) Assistant Prof. Dr. Nibras Saleam Al-Ammar PhD in Clinical Immunology Pathology & Forensic Medicine Department

Myeloproliferative Neoplasms (MPNs) A group of conditions that cause platelets, white blood, and red blood cells to grow abnormally in the bone marrow.

Polycythemia vera Normal range 4-6 million RBCs /µL Polycythemia 11 million RBCs /µL

Essential Thrombocythemia Platelet count > 450 x 103/µL for at least 3 months Normal count 150-400 x 103/µL

Myelofibrosis (Too many collagen or fibrous tissue in bone marrow).

Chronic myelogenous leukemia (CML) (Abnormal granulocytes, a type of WBCs).

2008 WHO Classification of Myeloproliferative Neoplasms (MPNs) & the Diagnostic criteria (Major criteria and Minor criteria) Reference: Link springer.com/article/10. 1007/s11899-013-0186-x

Traditionally, Diagnosis of MPNs The discovery of a disease-specific molecular marker results in both simplification of the process and increased diagnostic accuracy. clinical bone marrow histology cytogenetic criteria

JAK2 V617F mutation has been reported in: Chronic myelogenous leukemia (CML) (0%)!(3-5%)! Myelofibrosis (50%) Essential thrombocythemia (50-60%) Polycythemia vera (>95%)

Janus kinase (JAK) A family of intracellular, non receptor thyrosine kinases. That transduce cytokine-mediated signals via JAK-STAT pathway.

Four family members JAK1 JAK2 JAK3 TYK2

The JAK-STAT system consists of three main components: a receptor (2) Janus kinase (JAK) (3) Signal Transducer and Activator of Transcription (STAT), which carries the signal into the nucleus and DNA.

► After the cytokine binds to the receptor, ► JAK adds a phosphate to (phosphorylates) the receptor. ► This attracts the STAT proteins, which are also phosphorylated and bind to each other, ► forming a pair (dimer). ► The dimer moves into the nucleus, ► binds to the DNA, and ► causes transcription of genes. Enzymes that add phosphate groups are called protein kinases.

JAK2 V617F mutation has been identified in 2005 JAK2 V617F mutation has been identified in 2005. The mutation corresponds to a single-nucleotide change of JAK2 nucleotide 1849 in exon 14, resulting in a unique valine (V) to phenylalanine (F) substitution at position 617 of the protein (JH2 domain). It leads to constitutive activation of JAK2.

JAK2 V617F represents a key driver in the transformation of hematopoitic cells in MPNs.

Testing Method:

DNA based Real-time PCR JAK2 MutaScreen Kit, lpsogen Sample: 3 ml peripheral blood (EDTA). Amplified mutated JAK2 region Amplification Portion of JAK2 mutated region

Principle of the procedure

Multiplex assay by using Hydrolysis probes. TaqMan probe 1 match to wild-type allele match to mutated allele

Each probe is labeled with a fluorescent dye at its 5’ end (Reporter dye), and a non-fluorescent (quencher) at the 3’ end.

Fluorescence signal is collected at the end of the PCR indicating the presence of the targeted sequence in the sample (Wild-type allele, mutated allele or both). During extension phase of PCR, the matched probe is cleaved by the 5’→3’ exonuclease activity of TaqMan DNA polymerase separating reporter dye from the quencher & releasing detectable fluorescence. No reporter dye is released if there is no matching.

We can also use Hybridization probes

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