| Abstrakt: |
A single G-to-T missense mutation in the gene for the JAK2 tyrosine kinase, leading to a V617F amino acid substitution, is commonly found in several myeloproliferative neoplasms. Reliable quantification of this mutant allele is of increasing clinical and therapeutic interest in predicting and diagnosing this group of neoplasms. Because JAK2V617Fis somatically acquired and may be followed by loss of heterozygosity, the percentage of mutant versus wild-type DNA in blood can vary between 0% and almost 100%. Therefore, we developed a real-time PCR assay for detection and quantification of the low-to-high range of the JAK2V617Fallele burden. To allow the assay to meet these criteria, amplification of the wild-type JAK2was blocked with a peptide nucleic acid oligonucleotide. JAK2V617Fpatient DNA diluted in JAK2wild-type DNA could be amplified linearly from 0.05% to 100%, with acceptable reproducibility of quantification. The sensitivity of the assay was 0.05% (n= 3 of 3). In 9 of 100 healthy blood donors, a weak positive/background signal was observed in DNA isolated from blood, corresponding to approximately 0.01% JAK2V617Fallele. In one healthy individual, we observed this signal in duplicate. The clinical relevance of this finding is not clear. By inhibiting amplification of the wild-type allele, we developed a sensitive and linear real-time PCR assay to detect and quantify JAK2V617F. |
