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Calreticulin gene exon 9 frameshift mutations in patients with thrombocytosis.

Chi J, Nicolaou KA, Nicolaidou V, Koumas L, Mitsidou A, Pierides C, Manoloukos M, Barbouti K, Melanthiou F, Prokopiou C, Vassiliou GS, Costeas P - Leukemia (2013)

View Article: PubMed Central - PubMed

Affiliation: The Center for the Study of Haematological Malignancies, Nicosia, Cyprus.

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Somatic frameshift mutations in exon 9 of the calreticulin (CALR) gene were recently identified in patients with BCR-ABL-negative myeloproliferative neoplasms (MPNs), particularly essential thrombocythemia and myelofibrosis... Calreticulin is a highly conserved endoplasmic reticulum (ER) luminal Ca-binding chaperone protein with a critical role in the process of glycoprotein folding and a number of other cellular functions both inside and outside the ER, and has three domains with different structural and functional properties; a globular N domain, a proline-rich P domain and an acidic C domain... However, until now the diagnosis of MPNs in the absence of JAK2 mutations has relied on specific clinicopathological criteria, and the frequency of CALR mutations in patients presenting with persistent thrombocytosis is unknown... Of 289 samples tested, 189 (65%) carried a JAK2 V617F mutation and 8 (3%) an MPL codon 515 mutation (7 W515L and 1 W515K)... Of the remaining 92 samples, 25 were found to carry a CALR exon 9 indel mutation (Figure 1a)... Also, patients with either JAK2 V617F or CALR exon 9 mutations had significantly higher platelet counts than patients with thrombocytosis and no mutations (Figure 1b)... Each of the 25 CALR mutant samples was found to harbor one of seven different indels; all leading to a +1 frameshift of the open reading frame, including two that have not been previously described... The most common mutation, found in 13 out of the 25 cases, was a 52-bp deletion of nt1172 to nt1223 of the CALR complementary DNA (cDNA; NM_004343.3)... The second most common mutation was a 5-bp insertion (TTGTC) after position nt1127 of the cDNA found in seven cases... In all five samples we were consistently able to detect the mutation after a 1:10 dilution, giving this assay a sensitivity to a mutant allele burden of 5% or less (e.g. Figure 2d)... Altogether, JAK2 V617F, CALR exon 9 indel and MPL codon W515 mutations were found in 77% of the patients referred to our laboratory for the investigation of persistent thrombocytosis.

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Characterization of CALR exon 9 mutations in patients with thrombocytosis. (a) Comparative sequences of the seven different types of CALR mutations found in this study against the wild-type CALR exon 9 cDNA. Repeat elements are highlighted by gray solid lined (simple) or dashed lined (tandem) boxes and inverted sequences are depicted by reverse arrows. Presumed genomic regions from which these inversions may have derived are depicted by matching solid/dashed forward arrows. A polymorphism (G) is depicted in red and this was also found in normal DNA from the same individual. (b) Predicted C-terminal amino-acid sequences resulting from the frameshift indel mutations compared with the wild-type calreticulin protein sequence. The common novel peptide sequence shared by all mutations described to date is underlined. The amino-acid variant resulting from the polymorphism above is shown in red (e). (c) Results of PCR fragment analysis of CALR exon 9 in three patients with different indel mutations. (d) Example of fragment analysis from a patient with a 5-bp insertion diluted into normal control DNA shows that the mutation is readily detectable after a 1:10 dilution. †Novel mutation variant, ††Novel mutant amino-acid sequence.
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fig2: Characterization of CALR exon 9 mutations in patients with thrombocytosis. (a) Comparative sequences of the seven different types of CALR mutations found in this study against the wild-type CALR exon 9 cDNA. Repeat elements are highlighted by gray solid lined (simple) or dashed lined (tandem) boxes and inverted sequences are depicted by reverse arrows. Presumed genomic regions from which these inversions may have derived are depicted by matching solid/dashed forward arrows. A polymorphism (G) is depicted in red and this was also found in normal DNA from the same individual. (b) Predicted C-terminal amino-acid sequences resulting from the frameshift indel mutations compared with the wild-type calreticulin protein sequence. The common novel peptide sequence shared by all mutations described to date is underlined. The amino-acid variant resulting from the polymorphism above is shown in red (e). (c) Results of PCR fragment analysis of CALR exon 9 in three patients with different indel mutations. (d) Example of fragment analysis from a patient with a 5-bp insertion diluted into normal control DNA shows that the mutation is readily detectable after a 1:10 dilution. †Novel mutation variant, ††Novel mutant amino-acid sequence.

Mentions: Each of the 25 CALR mutant samples was found to harbor one of seven different indels; all leading to a +1 frameshift of the open reading frame, including two that have not been previously described. The most common mutation, found in 13 out of the 25 cases, was a 52-bp deletion of nt1172 to nt1223 of the CALR complementary DNA (cDNA; NM_004343.3). The second most common mutation was a 5-bp insertion (TTGTC) after position nt1127 of the cDNA found in seven cases. Interestingly, these and all other mutations identified reside within a repetitive region containing two simple and three tandem repeats whose location and sequence context suggests that they have a role in the generation of CALR mutations by illegitimate local recombination associated with deletions, insertions and inversions (Figure 2a). In particular, the TTGTC, which starts at the end of the second simple repeat, whereas the 52-bp deletion occurs between two GCAGAGG heptanucleotide stretches (nt1092_1098 and nt1143_1150) (Figure 2a). One novel variant involved a complex 13 bp deletion and an inversion insertion of an AGACAA sequence, complementary to part of the deleted 13 bp. Another patient with a 31-bp deletion appeared to also carry a constitutional change of nt1145C>G such that the mutant protein sequence differed from any of those previously described (Figure 2b). Interestingly, the mutation hotspot contains a stretch of 38 nucleotides without a C or a T (such a stretch is expected to occur every 2.7 × 1011 nucleotides assuming random distribution of A, C, G, T). As observed by others, all CALR exon 9 mutations identified were associated with loss of the C-terminal KDEL moiety and led to the generation of a novel peptide sequence terminating with the same 36 amino acids. Crucially, this alteration transforms the negatively charged glutamic-acid-rich C terminus of calreticulin to a positively charged arginine-rich region (Figure 2b), and this may have a crucial role in mediating the effects of these mutants.


Calreticulin gene exon 9 frameshift mutations in patients with thrombocytosis.

Chi J, Nicolaou KA, Nicolaidou V, Koumas L, Mitsidou A, Pierides C, Manoloukos M, Barbouti K, Melanthiou F, Prokopiou C, Vassiliou GS, Costeas P - Leukemia (2013)

Characterization of CALR exon 9 mutations in patients with thrombocytosis. (a) Comparative sequences of the seven different types of CALR mutations found in this study against the wild-type CALR exon 9 cDNA. Repeat elements are highlighted by gray solid lined (simple) or dashed lined (tandem) boxes and inverted sequences are depicted by reverse arrows. Presumed genomic regions from which these inversions may have derived are depicted by matching solid/dashed forward arrows. A polymorphism (G) is depicted in red and this was also found in normal DNA from the same individual. (b) Predicted C-terminal amino-acid sequences resulting from the frameshift indel mutations compared with the wild-type calreticulin protein sequence. The common novel peptide sequence shared by all mutations described to date is underlined. The amino-acid variant resulting from the polymorphism above is shown in red (e). (c) Results of PCR fragment analysis of CALR exon 9 in three patients with different indel mutations. (d) Example of fragment analysis from a patient with a 5-bp insertion diluted into normal control DNA shows that the mutation is readily detectable after a 1:10 dilution. †Novel mutation variant, ††Novel mutant amino-acid sequence.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4017256&req=5

fig2: Characterization of CALR exon 9 mutations in patients with thrombocytosis. (a) Comparative sequences of the seven different types of CALR mutations found in this study against the wild-type CALR exon 9 cDNA. Repeat elements are highlighted by gray solid lined (simple) or dashed lined (tandem) boxes and inverted sequences are depicted by reverse arrows. Presumed genomic regions from which these inversions may have derived are depicted by matching solid/dashed forward arrows. A polymorphism (G) is depicted in red and this was also found in normal DNA from the same individual. (b) Predicted C-terminal amino-acid sequences resulting from the frameshift indel mutations compared with the wild-type calreticulin protein sequence. The common novel peptide sequence shared by all mutations described to date is underlined. The amino-acid variant resulting from the polymorphism above is shown in red (e). (c) Results of PCR fragment analysis of CALR exon 9 in three patients with different indel mutations. (d) Example of fragment analysis from a patient with a 5-bp insertion diluted into normal control DNA shows that the mutation is readily detectable after a 1:10 dilution. †Novel mutation variant, ††Novel mutant amino-acid sequence.
Mentions: Each of the 25 CALR mutant samples was found to harbor one of seven different indels; all leading to a +1 frameshift of the open reading frame, including two that have not been previously described. The most common mutation, found in 13 out of the 25 cases, was a 52-bp deletion of nt1172 to nt1223 of the CALR complementary DNA (cDNA; NM_004343.3). The second most common mutation was a 5-bp insertion (TTGTC) after position nt1127 of the cDNA found in seven cases. Interestingly, these and all other mutations identified reside within a repetitive region containing two simple and three tandem repeats whose location and sequence context suggests that they have a role in the generation of CALR mutations by illegitimate local recombination associated with deletions, insertions and inversions (Figure 2a). In particular, the TTGTC, which starts at the end of the second simple repeat, whereas the 52-bp deletion occurs between two GCAGAGG heptanucleotide stretches (nt1092_1098 and nt1143_1150) (Figure 2a). One novel variant involved a complex 13 bp deletion and an inversion insertion of an AGACAA sequence, complementary to part of the deleted 13 bp. Another patient with a 31-bp deletion appeared to also carry a constitutional change of nt1145C>G such that the mutant protein sequence differed from any of those previously described (Figure 2b). Interestingly, the mutation hotspot contains a stretch of 38 nucleotides without a C or a T (such a stretch is expected to occur every 2.7 × 1011 nucleotides assuming random distribution of A, C, G, T). As observed by others, all CALR exon 9 mutations identified were associated with loss of the C-terminal KDEL moiety and led to the generation of a novel peptide sequence terminating with the same 36 amino acids. Crucially, this alteration transforms the negatively charged glutamic-acid-rich C terminus of calreticulin to a positively charged arginine-rich region (Figure 2b), and this may have a crucial role in mediating the effects of these mutants.

View Article: PubMed Central - PubMed

Affiliation: The Center for the Study of Haematological Malignancies, Nicosia, Cyprus.

AUTOMATICALLY GENERATED EXCERPT
Please rate it.

Somatic frameshift mutations in exon 9 of the calreticulin (CALR) gene were recently identified in patients with BCR-ABL-negative myeloproliferative neoplasms (MPNs), particularly essential thrombocythemia and myelofibrosis... Calreticulin is a highly conserved endoplasmic reticulum (ER) luminal Ca-binding chaperone protein with a critical role in the process of glycoprotein folding and a number of other cellular functions both inside and outside the ER, and has three domains with different structural and functional properties; a globular N domain, a proline-rich P domain and an acidic C domain... However, until now the diagnosis of MPNs in the absence of JAK2 mutations has relied on specific clinicopathological criteria, and the frequency of CALR mutations in patients presenting with persistent thrombocytosis is unknown... Of 289 samples tested, 189 (65%) carried a JAK2 V617F mutation and 8 (3%) an MPL codon 515 mutation (7 W515L and 1 W515K)... Of the remaining 92 samples, 25 were found to carry a CALR exon 9 indel mutation (Figure 1a)... Also, patients with either JAK2 V617F or CALR exon 9 mutations had significantly higher platelet counts than patients with thrombocytosis and no mutations (Figure 1b)... Each of the 25 CALR mutant samples was found to harbor one of seven different indels; all leading to a +1 frameshift of the open reading frame, including two that have not been previously described... The most common mutation, found in 13 out of the 25 cases, was a 52-bp deletion of nt1172 to nt1223 of the CALR complementary DNA (cDNA; NM_004343.3)... The second most common mutation was a 5-bp insertion (TTGTC) after position nt1127 of the cDNA found in seven cases... In all five samples we were consistently able to detect the mutation after a 1:10 dilution, giving this assay a sensitivity to a mutant allele burden of 5% or less (e.g. Figure 2d)... Altogether, JAK2 V617F, CALR exon 9 indel and MPL codon W515 mutations were found in 77% of the patients referred to our laboratory for the investigation of persistent thrombocytosis.

Show MeSH
Related in: MedlinePlus