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The discovery of highly recurrent somatic mutations in the calreticulin (CALR) gene in ~70-84% of subjects with Janus kinase 2 (JAK2)-negative myeloproliferative neoplasms MPN filled a significant gap in the mutational profile of theses heterogeneous disorders. 1 2 All CALR mutations reported in association with sporadic MPN are acquired, somatic mutations at the level of the haematopoietic stem cell and, thus, present with a highly variable allelic frequency (10-63%). 3 Such mutations involve indels in exon 9 that cause a frameshift to an alternative coding frame, resulting in a novel C-terminal sequence. 4 This endows mutant CALR with a gain-of-function property, ultimately triggering thrombopoietin receptor activation and downstream cellular signalling via the JAK-signal transducer and activator of transcription (JAK-STAT) pathway, as elegantly demonstrated in recent studies by Marty et al , 4 Chachoua et al 5 and Araki et al . 6
The two most common mutations accounting for ~85% of CALR-positive MPN are the type I, 52 bp deletion (p.L367fs*46), and the type II, 5 bp TTGTC insertion (p.K385fs*47), designated by Klampfl et al . 1 Here, we report for the first time the identification and confirmation of germline mutations in CALR (CALR-g) distinct from those somatic mutations (CALR-s), which define classical MPN.
We reviewed 2000 peripheral blood samples submitted to our centre for CALR mutational analysis between 2014 and 2016. The reasons for referral for testing were varied and included not only thrombocytosis, but also erythrocytosis, leukocytosis and miscellaneous causes, in certain cases, unrelated to MPN ( table 1 ). Assessment of the mutational status of exon 9 of the CALR gene was performed using fragment length analysis following a nucleic acid amplification test with fluorescent primers in accordance with the methods described by Klampfl et al . 1 A genetic analyser (ABI 3130, Life Technologies) was then used to determine the size of detected insertions and deletions. The non-standard 12 and 9 bp deletions in CALR were confirmed by Sanger sequencing. Mutation burden was assessed using a quantitative assay, which has been analytically validated to be highly reproducible and sensitive to a level of 1%. Analysis of wild-type and mutant C-terminal sequences and their corresponding isoelectric points was performed in selected cases using the SWISS-PROT protein sequence database.
Table 1
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