BackgroundSomatic insertions/deletions in exon 9 of the Calreticulin (CALR) gene
have recently been identified in patients with Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) who are negative for both JAK2 and
MPL mutations. To date, over forty mutations in exon 9 have been discovered, over 80% of which consist of either type-1 52-bp deletion
(p.L367fs*46) or type-2 5-bp insertion (p.K385fs*47), with a further three mutations
(types 3 – 5) in conjunction with types 1 and 2 accounting for approximately
88% of identified CALR exon 9 mutations(1, 2). This
study aimed to develop a rapid, user friendly assay using LightCycler™ Hybridization Probes (HybProbes) and melt curve analysis for
the detection of type1-type 5 CALR mutations.
MaterialsA real-time PCR assay using HybProbes and melt curve analysis was
developed for the detection of type 1-type 5 mutations in CALR for use on the
LightCycler® 480. Limit of detection and specificity of the assay was
established using synthetic constructs. DNA was extracted from PBMCs from
twenty five patients with various myeloid malignancies including two patients
with ET and tested with the assay.
ResultsThe limit of detection of the assay was determined to be 10 copies of
target, while the ability of the assay to detect CALR mutations of types 1-5 was
confirmed using synthetic constructs. Twenty five samples from patients with
various haematological disorders were analysed.One sample was found to be
positive for type 2 5-bp TTGTC
insertion. This patient had been diagnosed with ET
and was JAK2 and MPL negative. The result
was confirmed by Sanger sequencing.
ConclusionsMelt curve analysis provides a sensitive, specific and a rapid method for
the detection of CALR mutations. CALR gene mutation detection can aid in the
specific diagnosis of a myeloproliferative neoplasm, and help distinguish this
clonal disease from a benign reactive process. CALR Diagnosis has been proposed
to become part of the world health organization diagnostic criteria for ET and
PMF in the same manner as Jak 2 did in 2008(3) and the assay developed as part of this study could provide a rapid,
reliable, sensitive, high throughput screening method which could be easily be
incorporated in a clinical setting.References 1. Klampfl
T, Gisslinger H, Harutyunyan AS, Nivarthi H, Rumi E, Milosevic JD, et al.
Somatic mutations of calreticulin in myeloproliferative neoplasms. N Engl J Med
Dec 19;369(25):2379-90.2. Nangalia
J, Massie CE, Baxter EJ, Nice FL, Gundem G, Wedge DC, et
al. Somatic CALR mutations in myeloproliferative neoplasms with nonmutated
JAK2. N Engl J Med Dec 19;369(25):2391-405.