1-like Mutations Research Articles

Effects of CALR-Mutant Type and Burden on the Phenotype of Myeloproliferative Neoplasms.

Somatic CALR mutations occur in approximately 70% of patients with JAK2 V617F-negative essential thrombocythemia (ET) and primary myelofibrosis (PMF). We evaluated the effects of the CALR mutant type and burden on the phenotype of CALR-mutated myeloproliferative neoplasms (MPN). Of the 510 patients with suspected or diagnosed MPN, all 49 patients detected with CALR mutations were diagnosed with ET (n = 32) or PMF (n = 17). The CALR mutant burden was significantly higher in PMF than in ET (45% vs. 34%), and type 1-like and type 2-like mutations were detected in 49% and 51% patients, respectively. Patients with MPN and type 2-like mutation showed a significantly higher median platelet count than those with type 1-like mutation. Particularly, patients with ET and type 2-like mutation had no thrombotic events, despite higher platelet counts. The effect of CALR mutant burden differed depending on the mutant type. A higher mutant burden tended to be associated with a cytopenic phenotype (i.e., lower hemoglobin levels and platelet counts) in patients with the type 1-like mutation and a proliferative hematological phenotype (i.e., higher platelet and neutrophil counts) in patients with the type 2-like mutation. This study suggests that the disease phenotype of MPN may be altered through CALR mutant burden and mutant type.

Study of CALR, MPL, and c-kit Gene Mutations in Thai Patients with JAK2 V617F Negative Myeloproliferative Neoplasms.

Objective:The aim of this study to determine the prevalence of CALR, MPL and c-kit gene mutations in JAK2 V617F negative-MPN patients. Methods:The retrospective study of CALR, MPL and c-kit mutations were analyzed in 113 samples collected from March 2010 to May 2017 and identified as JAK2 V617F–negative MPN Thai patients. The samples were analysis by gel electrophoresis and direct sequencing. Results:28.3% of JAK2 V617F–negative MPN patients showed CALR gene mutations. Within the MPN patients with CALR mutation, 46.9% were classified as essential thrombocythemia (ET) and 20.9% were classified as primary myelofibrosis (PMF). Previous studies classified CALR mutations into three types using negatively charged amino acid stretches at the C-terminal domain. Type 1-like mutations were observed in 12 of 49 (24.5%) ET patients and type 2-like mutations were observed in 10 of 49 (20.4%) patients. In addition, 8 of 43 (18.6%) PMF patients showed type 1-like mutations and 1 of 43 (2.3%) showed type 2-like CALR mutation. Interestingly, platelet counts were higher in patients with CALR gene mutation than in patients without CALR gene mutation. MPL mutations (W515K and W515L) were identified in 2 of 109 (1.8%) MPN patients; the MPL mutations were only found in ET patients, which was consistent with previous studies. We did not detect exon 17 c-kit mutation in JAK2-negative MPN patients but detected intronic single nucleotide polymorphisms at c.74,978 and c.75,255 in these samples. Approximately 66% of patients did not have mutations in CALR and MPL genes, in addition to lacking JAK2 gene mutation, and these cases are classified as triple-mutations. Conclusion:Our results showed that 66% of cases were triple-negative mutation MPN because they lacked mutations in JAK2, CALR and MPL genes. The frequencies of CALR and MPL mutation in this study are similar to other CALR and MPL patient data.

Rare Type 1-like and Type 2-like Calrecticulin Mutants Induce Similar Myeloproliferative Neoplasms Than Type 1 and Type 2 Mutants in Mice

IntroductionThe acquisition of somatic mutations in the hematopoietic stem cell (HSC) compartment leading to the constitutive activation of the JAK-STAT signaling pathway drives the development of myeloproliferative neoplasms (MPNs). After the gain-of-function mutation JAK2 V617F, the second most frequent driver mutations inducing 30% of essential thrombocythemia and mylofibrosis (MF) are located in the calreticulin (CALR) gene. The two most frequent CALR mutations are a deletion of 52 base pairs (del52) L367fs*46 also called type 1 and an insertion of 5 base pairs (ins5) K385fs*47 or type 2. They represent together more than 80% of all CALR mutations. In retroviral mouse models CALR mutants induce a thrombocytosis, but to a lesser extent in presence of ins5 compared to del52. Moreover, del52 mice evolved more frequently into MF than ins5 mice, mimicking what is observed in patients. Many other rare mutations of CALR exist and each represents less than 2% of CALR mutations in patients. They are structurally classified as type 1- and type 2-like CALR mutations according to the absence or presence of a residual wild-type calcium-binding motif and the modification of the alpha helix structure but it is not known whether they are equally functionally related to CALRdel52 and CALRins5, respectively.Material and MethodsWe modeled two type 1-like mutations, del34 (E369fs*50) and del46 (L367fs*48) and of one type 2-like mutation del19 (K374fs*50) in cell lines and in mice by bone marrow transplantation after retroviral transduction with CALR mutation constructs.ResultIn Ba/F3 cell lines, all CALR mutants specifically activated cytokine-independent cell growth in presence of the thrombopoietin (TPO) receptor (MPL) with close efficiency, and induced a constitutive activation of JAK2 and STAT5/3/1 in a similar way.Lethally irradiated mice that were engrafted with bone marrow transduced with the wild-type (CALR wt) or the different CALR mutations developed pure thrombocytosis but to a much lesser extent in presence of type 2 and type 2-like CALR mutations. The thrombocytosis was associated with megakaryocyte hyperplasia in the bone marrow and in the spleen seen by Von Willebrand factor immunostaining and flow cytometry. In contrast to type 2-like mice, type 1-like mice developed a MF after 10 months of engraftment associated with splenomegaly, a marked osteogenesis in bone marrow and a thickening of reticulin fiber network seen in histology.Like observed for type 1 mutant, type 1-like CALR mutations induced an expansion at early stage of hematopoiesis compare to type 2 and type 2-like mutations with a significant increased frequency in immature SLAM, LSK and MkP progenitors. Altogether, type 1-like CALR mutants harbor a stronger phenotype than the type 2-like recapitulating thrombocytosis and progression to MF.Thus, type 1-like and type 2-like CALR mutants structurally and functionally resemble type 1 and type 2, respectively. The mechanisms responsible for these differences remain to be determined and could be related to a MPL independent process. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract 16997: Reaffirmation of GPD1L-A280V as a Brugada Syndrome Disease-Causing Variant

Introduction: Brugada Syndrome (BrS) is an inherited arrhythmia syndrome characterized by ST-segment elevation in the right precordial leads (V1-V3) and sudden cardiac death; ~20% of cases are caused by mutations in SCN5A . Over the past two decades, over 20 genes that may cause BrS have been identified. We reported that a Glycerol-3-Phosphate Dehydrogenase 1-Like mutation (GPD1L;p.A280V, chromosome 3p22.3) was linked to BrS in a large, multigenerational family with a LOD score ~4. Recent efforts in the field have called into question the pathogenicity of non- SCN5A genetic variants in BrS. Hypothesis: We sought to confirm the role of GPD1L in this family using a high density SNP array to narrow the linkage region and whole exome sequencing (WES) of the proband to rule out other possible mutations. We also sought to test whether BrS susceptibility SNPs in SCN5A and SCN10A linked to GPD1L may play a role in this family. Methods: SNPs were identified on an Illumina Global Screening Array and used for fine mapping. The exome of the proband was captured with the Agilent V6+UTR kit. SNPs were called using GATK3. BrS susceptibility SNPs were confirmed by PCR amplification and Sanger Sequencing. Results: The linkage region in this family resolved to ~3.07 MB (hg19; 3:29,899,567-32,970,637; Figure). This region contained 14 genes including GPD1L , and lacking SCN5A , SCN10A , or any other BrS-associated gene. WES identified GPD1L;p.A280V as the only coding variant in the linkage region with an allele frequency ≤ 1%. Previously identified BrS risk alleles in SCN5A and SCN10A were linked to the GPD1L;p.A280V allele in most family members (Figure). Conclusions: Our data suggests that GPD1L;p.A280V causes BrS in this family. The pathogenicity of GPD1L;p.A280V may be increased by linkage with SCN5A and SCN10A risk SNPs. The role of GPD1L mutations in the pathogenesis of BrS should be reaffirmed.

Significance of calreticulin gene mutations classification in BCR-ABL1 negative myeloproliferative neoplasms

Objective To investigate calreticulin (CALR) gene mutations classification in BCR-ABL1 negative myeloproliterative neoplasms (MPN), and its relationship with clinical manifestations. Methods Genomic DNA polymerase chain reaction (PCR) amplification product Sanger sequencing method was used to detect the mutation of exon 9 of CALR gene in 236 patients with BCR-ABL1 negative MPN (excluding polycythemia vera and negative CALR mutations) in Ruijin Hospital of Shanghai Jiao Tong University School of Medicine from November 2015 to November 2018. The mutations were classified into 52 bp deletion (type 1) mutation, 5 bp insertion (type 2) mutation and other mutation types according to PCR sequencing analysis. The clinical characteristics of the carriers with two kinds of mutations in 198 patients with essential thrombocythemia (ET) and 38 primary myelofibrosis (PMF) were compared. For the types of mutations that could not be determined, they were classified according to the α-helix propensity score of the mutant protein peptide chain or the degree of retention of the negatively charged amino acid residues, and the differences between the two classification methods were also compared. Results Among 236 patients, the CALR gene type 1 or type 2 mutation was detected in 206 cases (87.3%), including 173 ET patients (99 cases of type 1 mutation and 74 cases of type 2 mutation) and 33 PMF patients (28 cases of type 1 mutation and 5 cases of type 2 mutation). The CALR non-type 1 or non-type 2 mutation was detected in 30 cases, including 25 ET patients and 5 PMF patients. Among 173 ET patients with CALR gene mutation, the white blood cell count (WBC) of patients with type 1 mutation was higher than that of patients with type 2 mutation [(8.6±2.7)×109/L vs. (7.6±2.4)×109/L, t = 2.45, P = 0.015]. Among 33 PMF patients with CALR gene mutation, the age of patients with type 1 mutation was older than that of patients with type 2 mutation [(58±13) years old vs. (41±16) years old, t = 2.51, P = 0.018]. According to the α-helix propensity score of mutant protein peptide chain and the degree of retention of the negatively charged amino acid residues, 27 kinds of non-type 1 or non-type 2 mutations were classified by using sequencing method, and there were differences between the two methods. According to the α-helix propensity score of the mutant protein peptide chain, the proportion of type 1/type 1-like mutation in PMF patients was higher than that in ET patients [78.9% (30/38) vs. 56.6% (112/198), P < 0.01]. According to the degree of retention of negatively charged amino acid residues in the mutant protein peptide chain, the isoelectric point (pI) value of the mutant protein peptide chain was higher than that of the wild type sequence. The pI value of the type 1-like mutant protein peptide chain was higher than that of the type 2-like mutation (11.79±0.15 vs. 10.02±0.42, t = 11.51, P < 0.01). Conclusions Type 1 mutated ET patients may be closely related to the high risk of myelofibrosis transformation. The results of the classification of CALR mutations are different according to the α-helix propensity score of the mutant protein peptide chain and the degree of retention of the negatively charged amino acid residues. Further study is necessary to identify the pathogenesis of MPN caused by CALR mutation, and to determine the relationship between mutation type and prognosis of disease. Key words: Myeloproliferative neoplasms; Calreticulin; Thrombocythemia, essential; Primary myelofibrosis; Mutation; DNA mutational analysis

Knock-Ins of Type-2 Calr Mutants Cause Myeloproliferative Neoplasm (MPN)-like Hematopoiesis in Mice

MPNs, including polycythemia vera, essential thrombocythemia (ET) and myelofibrosis (MF), are characterized by proliferation of mature myeloid cells. A somatic mutation in a hematopoietic stem cell (HSC) that activates JAK/STAT pathway drives MPN. Following the JAK2V617F, the CALR insertion/deletion mutations (indels) are the second most frequent driver and present in 20-30% ET and primary MF. Two major indels, a 52-bp deletion (type 1, p.L367fs*46) and a 5-bp insertion (type 2, p.K385fs*47), account for 80% of the CALR mutations. In addition, there have been more than 100 other indels, which can be classified as type 1- and type 2-like mutations based on the length of negatively-charged amino acid (AA) stretch at the C-terminal side of mutated CALR. Patients with type 1/type 1-like CALR mutations exhibit more incidence of MF while type 2/type 2-like mutations are associated with higher platelet counts in ET (Petra et al, Leukemia, 2016). In previous studies, mice carrying type 1/type 1-like mutations, including knock-in (KI) models, showed mild ET- or MF-like hematopoiesis. Although wild-type (WT) CALR AA sequences are highly conserved between human and mouse, there have been no KI models of type 2/type 2-like CALR mutations. Here, we generated 2 lines of KI mice carrying type 2-like Calr mutations, 2-bp insertion (CR2i, p.K378fs*53) and 10-bp deletion (CR10d, p.K375fs*52), using the CRISPR/Cas9 method. Both KIs removed KDEL, altered AA charges and increased values for isoelectric point, which are similar to type 2/type 2-like mutations in MPN patients. Compared with WT mice, peripheral platelets (1277 ± 228 vs 1560 ± 344 x 109/L, p = 0.004) and leukocytes (14.4 ± 3.7 vs 18.7 ± 4.9 x 109/L, p = 0.006) were increased in CR10d mice, whereas blood cell counts were not different between CR2i and WT mice. In FACS, both CR10d (p = 0.04) and CR2i (p = 0.04) mice exhibited an increased myeloid-cell ratio in bone marrow (BM). Splenomegaly was not present, but histopathological study showed a significant increase and accumulation of large megakaryocytes in BM and spleen of both KI mouse lines. BM fibrosis was not present in any sample. Therefore, CR10d and CR2i mice mimicked ET-like and unclassifiable MPN-like hematopoiesis, respectively. Next, we studied the basis of MPN-like hematopoiesis in CR10d and CR2i mice. Colony forming-cell assay in the presence of cytokines showed reduced growth of CFU-E, especially in CR2i mice (p = 0.01) compared with WT mice, while there was no difference in growth of CFU-Mk between CR10d or CR2i mice and WT mice. TPO-independent colony growth was not observed in both KI mice. Correspondingly, FACS showed comparable expression of phospho-STAT3 (pSTAT3) in BM cells between CR10d or CR2i mice and WT mice in the absence of TPO. However, pSTAT3 was significantly upregulated both in CR10d and CR2i mice compared with WT mice in the presence of TPO, suggesting that high sensitivity of HSCs or progenitor cells to TPO contributes to MPN phenotype in these mice. Thus, we investigated HSC function by a competitive repopulation assay, in which we transplanted a mixture of BM cells from KI mice (Ly5.2) and Ly5.1 mice at a 1:1 ratio into lethally irradiated Ly5.1 mice, showed reduced repopulating capacity, especially in CR2i mice. In the second transplant recipients, cells derived from either CR2i or CR10d mice were markedly diminished, suggesting the reduced self-renewal capacity of an HSC carrying a type 2/type 2-like Calr mutation. Finally, we performed RNAseq for FACS-sorted HSC-enriched lineage-Sca1+Kit+ (LSK) cells, which revealed that approximately 70% of genes among differentially expressed genes were commonly upregulated or downregulated in CR2i and CR10d mice, suggesting a similarity in gene expression profile of LSK cells of these KI mouse lines. As a result, there were several pathways commonly affected in both CR2i and CR10d mice in gene set enrichment analysis, including upregulation of JAK/STAT pathway (FDRq = 0.060 in CR2i and 0.111 in CR10d). On the other hand, targets of polycomb recessive complex 2, which are important for HSC functions in MPNs (Ueda et al, Blood Adv, 2017), were downregulated in both KI mouse lines (FDRq = 0 in both CR2i and CR10d), possibly explaining the reduced repopulating capacities of CR2i and CR10d HSCs. In conclusion, our data indicate that type 2/type 2-like Calr mutation can cause MPN-like hematopoiesis. For disease progression, further mechanism may be required. Disclosures Yokokawa: Actelion Pharmaceuticals Ltd: Other: Donated Fund Laboratory. Ikeda:Kyokuto Pharmaceutical: Research Funding; Hokuyo Denki: Research Funding; Novartis Pharma: Honoraria; Takeda Pharmaceutical: Honoraria, Research Funding.

Rare type 1-like and type 2-like calreticulin mutants induce similar myeloproliferative neoplasms as prevalent type 1 and 2 mutants in mice.

Frameshift mutations in the calreticulin (CALR) gene are present in 30% of essential thrombocythemia and myelofibrosis patients. The two most frequent mutations are CALR del52 (type 1, approximately 60%) and CALR ins5 (type 2, around 30%), but many other rarer mutations exist accounting each for less than 2% of all CALR mutations. Most of them are structurally classified as type 1-like and type 2-like CALR mutations according to the absence or presence of a residual wild-type calcium-binding motif and the modification of the alpha-helix structure. Yet, several key questions remain unanswered, especially the reason of such low frequencies of these other mutations. In an attempt to investigate specific pathogenic differences between type 1-like and type 2-like CALR mutations and del52 and ins5, we modeled two type 1-like (del34 and del46) and one type 2-like (del19) mutations in cell lines and in mice. All CALR mutants constitutively activate JAK2 and STAT5/3/1 in a similar way in the presence of the thrombopoietin receptor (MPL) and induced cytokine-independent cell growth but to a lesser extent with rare mutants over time. This correlates with reduced expression levels of rare CALR mutants compared to del52 and ins5. Lethally irradiated mice that were engrafted with bone marrow transduced with the different CALR mutations developed thrombocytosis, but to a much lesser extent with ins5 and the type 2-like CALR mutation. In contrast to type 2-like mice, type 1-like mice developed marked myelofibrosis and splenomegaly 10 months after engraftment. Similar to del52, type 1-like CALR mutations induced an expansion at an early stage of hematopoiesis compared to ins5 and type 2-like mutation. Thus, type 1-like and type 2-like CALR mutants structurally and functionally resemble del52 and ins5 mutants, respectively.

Mutational subtypes of JAK2 and CALR correlate with different clinical features in Japanese patients with myeloproliferative neoplasms.

The majority of patients with Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) harbor JAK2, CALR, or MPL mutations. We compared clinical manifestations of different subtypes of JAK2 and CALR mutations in Japanese patients with MPNs. Within our cohort, we diagnosed 166 patients as polycythemia vera (PV), 212 patients as essential thrombocythemia (ET), 23 patients as pre-primary myelofibrosis (PMF), 65 patients as overt PMF, and 27 patients as secondary myelofibrosis following the 2016 WHO criteria. Compared to patients with JAK2V617F-mutated PV, JAK2 exon 12-mutated PV patients were younger, showed lower white blood cell (WBC) counts, lower platelet counts, higher red blood cell counts, and higher frequency of thrombotic events. Compared to JAK2-mutated ET patients, CALR-mutated ET patients were younger, showed lower WBC counts, lower hemoglobin levels, higher platelet counts, and fewer thrombotic events. CALR type 1-like mutation was the dominant subtype in CALR-mutated overt PMF patients. Compared with JAK2V617F-mutated ET patients, JAK2V617F-mutated pre-PMF patients showed higher LDH levels, lower hemoglobin levels, higher JAK2V617F allele burden, and higher frequency of splenomegaly. In conclusion, Japanese patients with MPNs grouped by different mutation subtypes exhibit characteristics similar to those of their Western counterparts. In addition, ET and pre-PMF patients show different characteristics, even when restricted to JAK2V617F-mutated patients.

The clinical and prognostic relevance of driver mutations in 203 Taiwanese patients with primary myelofibrosis

AimsWe investigated the clinical and prognostic relevance of the mutational status of driver genes with allele burden and endogenous erythroid colony (EEC) growth in 203 Taiwanese patients with primary myelofibrosis...

Driver Mutations and Prognosis in 1118 Patients with Primary Myelofibrosis

▪Background: In primary myelofibrosis (PMF), ̴ 88% of patients harbor JAK2, CALR or MPL, with respective mutational frequencies of approximately 60%, 22% and 6%; ̴ 12% are wild type for all three driver mutations and are labelled as being "triple negative" (Blood. 2014;124:2507). It is now well-established that CALR-mutated patients with PMF display significantly better survival, compared to all the other mutational categories; in addition, preliminary data have suggested inferior survival for "triple-negative" cases (Leukemia. 2014;28:1472; Blood. 2014;124:1062) and the possibility that the favorable impact of CALR mutations might be restricted to CALR type 1 or type 1-like variants (Blood. 2014;124:2465). In the current study, we sought to clarify the prognostic impact of driver mutations, and CALR variants, on overall (OS) and leukemia-free (LFS) survival, in a two-center study involving 1118 patients.Methods: A total of 1118 patients with PMF from the Mayo Clinic (n =722) and University of Florence, Italy (n =396) were included in the current study. Study patients were selected based on availability of mutation information. PMF diagnosis was according to World Health Organization criteria (Blood. 2009;114:937). Previously published methods were used for CALR, JAK2 and MPL mutation analyses and determination of CALR variants (Blood. 2014;124:2465). Kaplan-Meier survival analysis was considered from the date of first referral for the Mayo cohort and date of diagnosis for the Florence cohort. Leukemic transformation events replaced death as the uncensored variable during LFS analysis and Cox proportional hazard regression model was used for multivariable analysis.Results:Analysis was first conducted on the Mayo cohort of 722 patients (median age 64 years; 64% males). DIPSS-plus risk distributions were 14% low, 17% intermediate-1, 37% intermediate-2 and 33% high. All patients were annotated for JAK2/CALR/MPL mutations as well as CALR variants; 477 harbored JAK2, 139 CALR and 41 MPL mutations; 65 patients were triple-negative. The 139 CALR -mutated patients included type 1/type 1-like (n =115) and type 2/type 2-like (n =24).At a median follow-up time of 3.1 years, 439 (61%) deaths and 63 (8.7%) blast transformations were documented. In univariate analysis, survival in patients with CALR type 1/type 1-like mutations was significantly longer than every other mutational category: HR (95% CI) were for triple-negative 2.7 (1.8-4.0), JAK2 2.7 (2.0-3.7), CALR type 2/type 2-like 2.3 (1.3-4.2) and MPL 1.9 (1.1-3.1) (Figure 1); these differences were sustained during multivariable analysis that included ASXL1 mutations (n =480) and DIPSS-plus, and were also apparent in the Florence cohort of 396 cases that included 251 JAK2, 53 CALR type 1/type 1-like, 21 CALR type 2/type 2-like, 50 triple-negative and 21 MPL mutated cases (Figure 2). There was no difference in survival among the Mayo cohort across mutational categories not including CALR type 1/type 1-like variants (p=0.33). Because specific driver mutations in PMF differentially cluster with age and sex, multivariable analysis including these two parameters was also performed and confirmed the significant survival advantage of CALR type 1/type 1-like over triple-negative, JAK2 and CALR type 2/type 2-like mutated cases.Based on the above information, we divided driver mutational categories into "type 1/type 1-like" (median survival 10.3 years) and "all other mutational categories" (median survival 3.9 years; p<0.01). The difference in survival between these two groups remained significant (HR 2.1, 95% CI 1.5-2.9) during multivariable analysis that included both DIPSS-plus (P<0.01) and ASXL1 mutation (HR 1.8, 95% CI 1.4-2.2). LFS was similar between these two groups (p=0.4) but it was inferior in triple-negative cases compared to CALR type 1/type 1-like variants (HR 2.8, 95% CI 1.3-6.2), MPL (HR 4.6; 95% CI 1.04-20.4) and JAK2 (HR 2.5, 95% CI 1.3-4.8) but not CALR type 2/type 2-like variants (p=0.56). There was no difference in LFS among mutational categories not including triple-negative cases (p=0.33).Conclusions: In PMF, driver mutations might be classified into two prognostically distinct categories: "favorable" (type 1/type 1-like) and "unfavorable" (all other mutational categories). In addition, triple-negative cases might be at a higher risk of leukemic transformation. [Display omitted] [Display omitted] DisclosuresPardanani:Stemline: Research Funding. Vannucchi:Novartis: Other: Research Funding paid to institution (University of Florence), Research Funding; Shire: Speakers Bureau; Baxalta: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Differential clinical effects of different mutation subtypes in CALR-mutant myeloproliferative neoplasms.

A quarter of patients with essential thrombocythemia or primary myelofibrosis carry a driver mutation of CALR, the calreticulin gene. A 52-bp deletion (type 1) and a 5-bp insertion (type 2 mutation) are the most frequent variants. These indels might differentially impair the calcium binding activity of mutant calreticulin. We studied the relationship between mutation subtype and biological/clinical features of the disease. Thirty-two different types of CALR variants were identified in 311 patients. Based on their predicted effect on calreticulin C-terminal, mutations were classified as: (i) type 1-like (65%); (ii) type 2-like (32%); and (iii) other types (3%). Corresponding CALR mutants had significantly different estimated isoelectric points. Patients with type 1 mutation, but not those with type 2, showed abnormal cytosolic calcium signals in cultured megakaryocytes. Type 1-like mutations were mainly associated with a myelofibrosis phenotype and a significantly higher risk of myelofibrotic transformation in essential thrombocythemia. Type 2-like CALR mutations were preferentially associated with an essential thrombocythemia phenotype, low risk of thrombosis despite very-high platelet counts and indolent clinical course. Thus, mutation subtype contributes to determining clinical phenotype and outcomes in CALR-mutant myeloproliferative neoplasms. CALR variants that markedly impair the calcium binding activity of mutant calreticulin are mainly associated with a myelofibrosis phenotype.

Mutation Type As a Major Determinant of Clinical Phenotype in Myeloproliferative Neoplasms Associated with Mutant Calreticulin

▪About 25% of patients with essential thrombocythemia (ET) or primary myelofibrosis (PMF) carry a somatic mutation of CALR, the calreticulin gene [N Engl J Med. 2013;369:2379-90]. So far, more than 50 different indels in CALR exon 9 have been found, but a 52-bp deletion (type 1 mutation) and a 5-bp insertion (type 2) are the most common lesions. All indels generate a novel C-terminus of the mutant protein, in which the endoplasmic reticulum retention signal KDEL is lost and the negatively charged amino acids are replaced by neutral and positively charged amino acids, disrupting the Ca-binding site. This suggests that both cellular dislocation and impaired Ca-binding activity may be involved in the abnormal proliferation of cells expressing a mutant calreticulin. It is still unclear, however, why the same mutant gene is associated with 2 different disease phenotypes (ET and PMF). In particular, little in known about the effect of the mutant protein on megakaryocyte biology and bone marrow collagen deposition.We studied the relationships between CALR mutation type, megakaryocyte biology, and clinical phenotype in patients with myeloproliferative neoplasms. According to the 2008 WHO criteria, 716 out of 892 patients had ET and 176 had PMF. Overall, 578 (65%) patients carried JAK2 (V617F), 230 (26%) had a CALR indel, and 84 (9%) had nonmutated JAK2 and CALR. Patients with MPL mutations were excluded.Twenty-six different types of CALR lesions were identified: 120 (52%) patients had type 1 mutation, 75 (33%) had type 2, and 35 (15%) carried other indels. The frequency of type 1 mutation was significantly higher in PMF than in ET (71% vs 46%, P=.004). All these variants involved 3 different stretches of negatively charged amino acids, with an increase in the isoelectric points (pI) of the mutant protein. As type 1 and type 2 mutations affected stretch I and III, respectively, the 26 indels were categorized into 3 groups on the basis of the stretch they affected: i) type 1-like (61%), affecting stretch I; ii) type 2-like (36%), stretch III; iii) and other types (3%), stretch II. The pI values were significantly different in the 3 groups (P<.001). The frequency of type-1 like mutations was significantly higher in PMF than in ET (82% vs 55%, P=.001).In vitro differentiated megakaryocytes from CALR-mutant patients displayed a significant increase in the extent of both intracellular Ca2+ release from the endoplasmic reticulum and extracellular Ca2+ entry inside the cytoplasm, as compared with healthy controls. Megakaryocytes carrying type 1-like CALR mutations exhibited the highest amplitude of Ca2+ flows regardless of the type of disease. In ET, impaired Ca2+ homeostasis was accompanied by atypical proplatelet architecture (ie, more branches and bifurcations).With respect to clinical phenotype at diagnosis, ET patients with type 2-like CALR mutation showed a trend towards higher PLT count (P=.063) and lower age (P=.053), and significantly lower LDH values (P=.021) than those with type 1-like mutation. In a hierarchical cluster analysis including demographic, clinical and molecular data, CALR mutation type (1 vs 2) identified the 2 clusters with the highest dissimilarity.Considering all patients, those with type 2-like CALR lesions had a better survival than those with JAK2 (V617F) (96.1% vs 84.4% at 10 years, P=.039), while no difference was found between the 2 CALR mutation types. ET patients with type 2-like CALR mutations showed a lower risk of thrombosis than those with JAK2 (V617F) (P=.010). By contrast, ET patients with type 1-like CALR mutations had a higher risk of myelofibrotic transformation that those with type 2-like CALR mutations (P=.029) and especially those with JAK2 (V617F) (P=.011). Finally, PMF patients with type 1-like CALR variants had a better survival than those with JAK2 (V617F) (80.1% vs 48% at 10 years, P=.008).In summary, abnormalities in megakaryocyte calcium metabolism and proplatelet architecture are found in patients with CALR-mutant myeloproliferative neoplasms, and their extent is related to mutation type. Type 2-like CALR mutations are more likely to be associated with isolated thrombocytosis without bone marrow fibrosis, ie, with an ET phenotype. By contrast, type 1-like CALR mutations are generally associated with bone marrow fibrosis, ie, with a PMF phenotype. Thus, in CALR-mutant myeloproliferative neoplasms, the mutation type is a major determinant of the clinical phenotype. DisclosuresNo relevant conflicts of interest to declare.

Mutations in the Yeast LCB1 and LCB2Genes, Including Those Corresponding to the Hereditary Sensory Neuropathy Type I Mutations, Dominantly Inactivate Serine Palmitoyltransferase

It was recently demonstrated that mutations in the human SPTLC1 gene, encoding the Lcb1p subunit of serine palmitoyltransferase (SPT), cause hereditary sensory neuropathy type I . As a member of the subfamily of pyridoxal 5'-phosphate enzymes known as the alpha-oxoamine synthases, serine palmitoyltransferase catalyzes the committed step of sphingolipid synthesis. The residues that are mutated to cause hereditary sensory neuropathy type I reside in a highly conserved region of Lcb1p that is predicted to be a catalytic domain of Lcb1p on the basis of alignments with other members of the alpha-oxoamine synthase family. We found that the corresponding mutations in the LCB1 gene of Saccharomyces cerevisiae reduce serine palmitoyltransferase activity. These mutations are dominant and decrease serine palmitoyltransferase activity by 50% when the wild-type and mutant LCB1 alleles are coexpressed. We also show that serine palmitoyltransferase is an Lcb1p small middle dotLcb2p heterodimer and that the mutated Lcb1p proteins retain their ability to interact with Lcb2p. Modeling studies suggest that serine palmitoyltransferase is likely to have a single active site that lies at the Lcb1p small middle dotLcb2p interface and that the mutations in Lcb1p reside near the lysine in Lcb2p that is expected to form the Schiff's base with the pyridoxal 5'-phosphate cofactor. Furthermore, mutations in this lysine and in a histidine residue that is also predicted to be important for pyridoxal 5'-phosphate binding to Lcb2p also dominantly inactivate SPT similar to the hereditary sensory neuropathy type 1-like mutations in Lcb1p.

The RET protooncogene in sporadic pheochromocytomas: frequent MEN 2-like mutations and new molecular defects.

To assess the pathophysiological role of the RET protooncogene in sporadic pheochromocytomas, we examined the 2 regions of the gene in which molecular defects are specifically associated with the multiple endocrine neoplasias (MEN) type 2A (the cysteine-rich domain encoded by exons 10 and 11), and type 2B (the tyrosine kinase domain encoded by exon 16). The sequences of both regions were amplified by reverse transcriptase-polymerase chain reaction (PCR) or PCR from tumor RNA and/or leukocyte DNA. The amplified fragments were analyzed by denaturing gradient gel electrophoresis using chemical clamps. In 28 patients with unilateral sporadic tumors, 6 RET mutations were found, 3 in the MEN 2A region, 3 in the MEN 2B region. Five patients had missense mutations: 2 in the MEN 2A region (C634W and D631Y), and 3 in the MEN 2B region (M918T). Analysis of leukocyte DNA in 3 of these patients confirmed that RET mutations were only present in tumor DNA. The sixth patient had lost exon 10 in the tumor complementary DNA as a result of the deletion of the dinucleotide -AG- at the 3'splice acceptor site of intron 9; this molecular defect was only found in the tumor DNA. Thus RET mutations of the MEN 2A and 2B regions are also found in about 20% of sporadic pheochromocytomas. We describe new types of molecular defects of the RET protooncogene in the MEN 2A region that involve noncysteine residues and loss of exon 10. Further studies should be extended to analyze the entire RET protooncogene. These findings have a profound clinical impact for the management of patients with supposedly sporadic pheochromocytomas.