Germline Mutations Occurring Outside the Negatively Charged Amino Acid Stretches of the Exon 9 of Calr : A Predisposition to Hematological Malignancies?

Introduction:

Germline predisposition to hematological malignancies is an increasingly recognized clinical entity, and several genes with germline mutations, such as GATA2, RUNX1 or CEBPA can lead to autosomal inheritance of hematological malignancies. Recurrent CALR somatic mutations are detected in a large majority of V617F JAK2 negative myeloproliferative neoplasms. Kampfl et al. have described 36 different types of insertions/deletions of the C-terminal domain of CALR in ET and PMF that create a novel protein with altered isoelectric point (Pi). Moreover, germline multiple of 3bp deletions have been reported in MPN and in patients devoid of any hematological malignancies (Szuber et al., 2016). In order to describe in an unbiased way the mutations occurring in then exon 9 of CALR, we analyzed using NGS a cohort of 214 patients with suspected MPN and a second cohort of 159 AML cases.

Methods:

The exon 9 of CALR was sequenced using the amplicon Illumina TruSight Myeloid Sequencing Panel©. Libraries were sequenced on Illumina Miseq apparatus (2X 250bp) with V2 chemistry. A mean coverage of 910X was obtained. All non-type 1 or type 2 mutations were checked using fragment length analysis and/or Sanger sequencing. Somatic status of variants, when the variant allele frequency was comprised between 45 and 55% were confirmed after informed consent.

Results:

A mutation was detected in 29 out of 214 (14%) of these patients. Mutation subtypes detected in the 3 stretches of negatively charged amino acids of the C-terminal domain were the following, according to Klampfl T et al. (NEJM 2013): type 1 (del52bp, n=14, 6 ET, 5 MF, 3 N/A), type 2 (ins5bp, n=10, 3 ET, 2 MF , 5 N/A), type3 (del45bp, n= 1, MF) type 12 (del34bp, n=1, ET), 1 type 22 (del4bp, n=1, ET), type 34 (delinsCTTGTC, n=1, ET) (Table 1). All these mutations induced a frameshift and modified the isoelectric point (Pi) of the C-terminal domain: 11.66 to 20.9 instead of 3.98 for the WT protein, as assessed by theScripps Institute's on-line Protein Calculator v.3.

Moreover we detected in a MPN-U case with normal karyotype an in-frame deletion of 3 bases (codon 395) in 50% of the sequences, not previously described. The Pi of the C-terminal domain was 3.99, very close to the WT protein. No additional mutation was observed in the gene panel and the 3bp deletion deletion was detected in a buccal cell sample at the same frequency than the bone marrow, confirming its germline status.

In order to look if such in frame deletions may occur in AML, we screened 159 AML cases that benefited from a NGS analysis at diagnosis using the same library preparation. In this cohort, we detected one type 1 and one type 2 mutation in 2 post-TE AML cases. We detected also in an undifferentiated AML with normal karyotype one in-frame deletion of 9 bases (p393_395del), leaving the acidic Pi of the protein unmodified (Pi=4.02). The mutation was detected in the buccal cell sample and the bone marrow sample at the same frequency (50%). This germline mutation was previously reported by Szuber et al. (J Clin Pathol 2016) in 2 patients devoid of hematological malignancy.

Discussion:

This study demonstrates that NGS, using specifically designed data analysis tools, can reliably detect mutations and indels up to 52 bases in the exon 9 of CALR gene. In the 373 AML and MPN cases studied, we detected a range of previously identified somatic mutations and a non-reported 3bp germline deletion that did not modified the Pi. We also detected another germline multiple of 3 bp deletion involving codon 395 in one de novo AML case. Multiple of 3 bp germline deletions reported in the literature so far (in patients with or without MPN) are localized in the vicinity of codon 395. In our study and in Szuber et al. paper, such deletions were reported at a frequency of 5.36 10-3 and 6 10-3 respectively. They are not reported in 1000 genomes nor in 6500 exomes NHLBI-ESP databases, but in-frame deletion of the exon 9 of CALR are reported at a frequency ranging from 10^-4 to 10^-5 in the Exome Aggregation Consortium browser. Germline predisposition of these deletion can only be suspected at this time, and further studies are necessary to evaluate the impact of such germline variants.