A highly sensitive quantitative real-time pcr assay for determination of mutant jak2 exon 12 allele burden

L. Kjær; C.H. Riley; M. Westman; E. Høgdall; O.W. Bjerrum; H. Hasselbalch

doi:10.1371/journal.pone.0033100

A highly sensitive quantitative real-time pcr assay for determination of mutant jak2 exon 12 allele burden

L. Kjær, C.H. Riley, M. Westman, E. Høgdall, O.W. Bjerrum, H. Hasselbalch

Department of Clinical Medicine

16 Citations (Scopus)

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 provides an important tool for quantitative monitoring of the mutant allele burden and accordingly also for determining the impact of treatment with interferon-α-2, shown to induce molecular remission in JAK2V617F-positive patients, which may be a future treatment option for JAK2 exon 12-positive patients as well.

Original language	English
Journal	PLOS ONE
Volume	7
Issue number	3
ISSN	1932-6203
DOIs	https://doi.org/10.1371/journal.pone.0033100
Publication status	Published - 5 Mar 2012

Access to Document

10.1371/journal.pone.0033100

Cite this

@article{ad42d072599a4f92bae8f94622061351,

title = "A highly sensitive quantitative real-time pcr assay for determination of mutant jak2 exon 12 allele burden",

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 provides an important tool for quantitative monitoring of the mutant allele burden and accordingly also for determining the impact of treatment with interferon-α-2, shown to induce molecular remission in JAK2V617F-positive patients, which may be a future treatment option for JAK2 exon 12-positive patients as well.",

author = "L. Kj{\ae}r and C.H. Riley and M. Westman and E. H{\o}gdall and O.W. Bjerrum and H. Hasselbalch",

year = "2012",

month = mar,

day = "5",

doi = "10.1371/journal.pone.0033100",

language = "English",

volume = "7",

journal = "PLOS ONE",

issn = "1932-6203",

publisher = "Public Library of Science",

number = "3",

}

TY - JOUR

T1 - A highly sensitive quantitative real-time pcr assay for determination of mutant jak2 exon 12 allele burden

AU - Kjær, L.

AU - Riley, C.H.

AU - Westman, M.

AU - Høgdall, E.

AU - Bjerrum, O.W.

AU - Hasselbalch, H.

PY - 2012/3/5

Y1 - 2012/3/5

N2 - 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 provides an important tool for quantitative monitoring of the mutant allele burden and accordingly also for determining the impact of treatment with interferon-α-2, shown to induce molecular remission in JAK2V617F-positive patients, which may be a future treatment option for JAK2 exon 12-positive patients as well.

AB - 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 provides an important tool for quantitative monitoring of the mutant allele burden and accordingly also for determining the impact of treatment with interferon-α-2, shown to induce molecular remission in JAK2V617F-positive patients, which may be a future treatment option for JAK2 exon 12-positive patients as well.

UR - http://www.scopus.com/inward/record.url?scp=84857766125&partnerID=8YFLogxK

U2 - 10.1371/journal.pone.0033100

DO - 10.1371/journal.pone.0033100

M3 - Journal article

AN - SCOPUS:84857766125

SN - 1932-6203

VL - 7

JO - PLOS ONE

JF - PLOS ONE

IS - 3

ER -

A highly sensitive quantitative real-time pcr assay for determination of mutant jak2 exon 12 allele burden

Abstract

Access to Document

Other files and links

Fingerprint

Cite this