Contributions
Abstract: PB2259
Type: Publication Only
Background
The principal phenotypic driver mutations of MPNs, occurring on JAK2, MPL and Calreticulin (CALR) genes, cause a direct or indirect deregulation of JAK/STAT signalling. Recurrent exon 9 CARL mutants, associated almost exclusively with ETs and PMFs, activate JAK/STAT by interacting with TPO-receptor. CALR mutations generally arise from a +1 frameshift that generates a common novel peptide lacking of KDEL ER-retrieval signal, converting the first 31 bases of CALR 3’UTR into coding sequence. However, the hematopoietic and regulative functions of this 3’UTR region are still unknown.
Aims
We aimed to analyse in 2 siblings the clinical and biological consequences of an unusual deletion of 3’UTR, designated c.1254+10_+33del24, occurring 10 bp downstream the stop codon, thus not altering the coding sequence. Interestingly, both these patients were diagnosed with JAK2-negative PV.
Methods
CALR mutations and mutant allele burden were detected by PCR screening, identified by Sanger sequencing and confirmed by fragment analysis on DNA from granulocytes, CD34+-hematopoietic progenitor cells (HPC) isolated from peripheral blood (PB) and saliva epithelium. CALR mRNA levels was measured by quantitative RT-PCR (ABI-PRISM-7000). CALR, STAT-3/-5 and phopho-STAT-3/-5 protein levels were assessed by immunoblotting both in PB cells and in myeloid cell lines. The colony assays were performed using PB mononuclear cells isolated by Ficoll.
Results
CALR 3’UTR deletion c.1254+10_+33del24 was found in two sibling PV patients. This deletion is located within an evolutionarily conserved region at nucleotides 10-33 of 3’UTR, thus not altering the KDEL domain. Such mutation was detected in granulocytes, CD34+-HPCs and oral epithelial cells from saliva samples, suggesting that the mutation is germline. In all samples, the mutant allele burden was >50%. Both siblings showed an enhanced erythropoiesis, also assessed by BFU-E growth at low erythropoietin conditions. Moreover, these patients and other MPNs cases affected by canonical CALR mutations showed increased CALR mRNA/protein levels and an aberrant activation of the JAK/STAT pathway, respect to healthy donors.
Conclusion
CALR mutations in MPNs have hitherto been observed only in ETs and PMFs, with a pathogenesis relying on TPO-receptor activation by the mutated novel peptide. Here we report that the mutation c.1254+10_+33del24, deleting 24 bp of CALR 3’UTR, is associated with PV. JAK/STAT signalling activation correlates with increased CALR expression and 3'UTR alteration. Our results suggest that CALR 3’UTR functional impairment, not altering KDEL sequence and operating for instance on RNA intracellular localization, miRNA binding, translational efficiency and mRNA editing, may affect erythropoiesis.
Session topic: 15. Myeloproliferative neoplasms – Biology & Translational Research
Keyword(s): Erythropoieisis, Myeloproliferative disorder
Abstract: PB2259
Type: Publication Only
Background
The principal phenotypic driver mutations of MPNs, occurring on JAK2, MPL and Calreticulin (CALR) genes, cause a direct or indirect deregulation of JAK/STAT signalling. Recurrent exon 9 CARL mutants, associated almost exclusively with ETs and PMFs, activate JAK/STAT by interacting with TPO-receptor. CALR mutations generally arise from a +1 frameshift that generates a common novel peptide lacking of KDEL ER-retrieval signal, converting the first 31 bases of CALR 3’UTR into coding sequence. However, the hematopoietic and regulative functions of this 3’UTR region are still unknown.
Aims
We aimed to analyse in 2 siblings the clinical and biological consequences of an unusual deletion of 3’UTR, designated c.1254+10_+33del24, occurring 10 bp downstream the stop codon, thus not altering the coding sequence. Interestingly, both these patients were diagnosed with JAK2-negative PV.
Methods
CALR mutations and mutant allele burden were detected by PCR screening, identified by Sanger sequencing and confirmed by fragment analysis on DNA from granulocytes, CD34+-hematopoietic progenitor cells (HPC) isolated from peripheral blood (PB) and saliva epithelium. CALR mRNA levels was measured by quantitative RT-PCR (ABI-PRISM-7000). CALR, STAT-3/-5 and phopho-STAT-3/-5 protein levels were assessed by immunoblotting both in PB cells and in myeloid cell lines. The colony assays were performed using PB mononuclear cells isolated by Ficoll.
Results
CALR 3’UTR deletion c.1254+10_+33del24 was found in two sibling PV patients. This deletion is located within an evolutionarily conserved region at nucleotides 10-33 of 3’UTR, thus not altering the KDEL domain. Such mutation was detected in granulocytes, CD34+-HPCs and oral epithelial cells from saliva samples, suggesting that the mutation is germline. In all samples, the mutant allele burden was >50%. Both siblings showed an enhanced erythropoiesis, also assessed by BFU-E growth at low erythropoietin conditions. Moreover, these patients and other MPNs cases affected by canonical CALR mutations showed increased CALR mRNA/protein levels and an aberrant activation of the JAK/STAT pathway, respect to healthy donors.
Conclusion
CALR mutations in MPNs have hitherto been observed only in ETs and PMFs, with a pathogenesis relying on TPO-receptor activation by the mutated novel peptide. Here we report that the mutation c.1254+10_+33del24, deleting 24 bp of CALR 3’UTR, is associated with PV. JAK/STAT signalling activation correlates with increased CALR expression and 3'UTR alteration. Our results suggest that CALR 3’UTR functional impairment, not altering KDEL sequence and operating for instance on RNA intracellular localization, miRNA binding, translational efficiency and mRNA editing, may affect erythropoiesis.
Session topic: 15. Myeloproliferative neoplasms – Biology & Translational Research
Keyword(s): Erythropoieisis, Myeloproliferative disorder