a highly sensitive quantitative real-time pcr assay for determination of mutant jak2 exon 12 allele burden一个高度敏感的实时定量pcr试验测定突变jak2外显子12个等位基因的负担.pdfVIP

A Highly Sensitive Quantitative Real-Time PCR Assay for Determination of Mutant JAK2 Exon 12 Allele Burden 1 2 1 3 4 Lasse Kjær *, Maj Westman , Caroline Hasselbalch Riley , Estrid Høgdall , Ole Weis Bjerrum , Hans Hasselbalch5 1 Department of Hematology, Herlev Hospital, Herlev, Denmark, 2 Department of Clinical Genetics, Rigshospitalet, Copenhagen, Denmark, 3 Department of Pathology, Herlev Hospital, Herlev, Denmark, 4 Department of Hematology, Rigshospitalet, Copenhagen, Denmark, 5 Department of Hematology, Roskilde Hospital, Copenhagen, Denmark Abstract Mutations in the Janus kinase 2 (JAK2) gene have become an important identifier for the Philadelphia-chromosome negative chronic myeloproliferative neoplasms. In contrast to the JAK2V617F mutation, the large number of JAK2 exon 12 mutations has challenged the development of quantitative assays. We present a highly sensitive real-time quantitative PCR assay for determination of the mutant allele burden of JAK2 exon 12 mutations. In combination with high resolution melting analysis and sequencing the assay identified six patients carrying previously described JAK2 exon 12 mutations and one novel mutation. Two patients were homozygous with a high mutant allele burden, whereas one of the heterozygous patients had a very low mutant allele burden. The allele burden in the peripheral blood resembled that of the bone marrow, except for the patient with low allele burden. Myeloid and lymphoid cell populations were isolated by cell sorting and quantitative PCR revealed similar mutant allele burdens in CD16+ granulocytes and peripheral blood. The mutations were also detected in B- lymphocytes in half of the patients at a low allele burden. In conclusion, our highly sensitive assay p