A Microelectronic DNA Chip Detects the V617F JAK-2 Allelic Ratio in Myeloproliferative Disorders.

A single activating mutation in Janus Kinase (JAK)-2 is detected in 65%–97% patients with polycythemia vera (PV), in 23%–57% with essential thrombocytemia and in 35%–57% with idiopathic myelofibrosis. Recent studies have demonstrated the heterozygote population contains clonal and polyclonal granulopoiesis; clonality correlates with the Jak2V617F allelic ratio. We developed a microchip diagnostic platform (NMW NanoChip Molecular Biology Workstation, manufactured by Nanogen Inc, San Diego, CA) to identify the JAK-2 mutation in granulocyte-derived DNA and determine the allelic ratio. The system enables electronic deposition of biotinylated amplicons to selected pads. Twenty-three DNA samples from patients with PV were analysed by direct sequencing, AS-PCR and microelectronic chip. Sequencing results: a fragment of 345 genomic DNA was amplified by PCR under standard condition. 21/23 samples showed mutated JAK-2, 18/21 presented wild-type and mutated alleles; 3/21 had only the mutated allele. 21/23 samples showed mutated JAK-2. Microelectronic chip results: 21/23 samples showed mutated JAK-2, with a frequency of 91%. All samples were genotyped: at the first analysis 3 were identified as homozygotes; 18 were identified as heterozygotes. The allelic ratio in the 3 homozygotes shows residual wild-type allele due, in our view, to residual polyclonal granulopoiesis which the microchip detects. The allelic ratio of the 18 designated heterozygotes showed different T/G percentages, suggesting different disease stages. Combining microchip results with clonality assays could distinguish true heterozygotes. Overall genotyping was performed in 100% of positive cases. Sensitivity assay (on serial dilution of mutated HEL vs unmutated K562 cell lines) showed the electronic microchip detects the JAK-2 mutation in at least 1% of a total cell mixture. Our system is a fast, reliable and sensitive method alternative to DNA sequencing, AS-PCR or dHPLC. It overcomes the relative low sensitivity of direct sequencing and at the same time provides genotyping data even when sequencing fails or results are hard to interpret. This electronic microchip system is as sensitive as dHPLC but does not require an extra sequencing step for genotyping. Specific advantages over AS-PCR include automated calculation of the allelic ratio and consequently the genotype without any reduction in sensitivity.