It's becoming increasingly challenging to efficiently visualize and extract useful insight from complex and big data sets. JavaScript stands out as a suitable programming choice that offers mature ...libraries, easy implementation, and extensive customization, all of which stay in the shadow of new and rapid developments in the language. To illustrate the use of JavaScript in a scientific context, this article elaborates on Lexicon, a collection of JavaScript libraries for generating interactive visualizations in bioinformatics and other custom libraries.
Predict whether a mutation is deleterious based on the custom 3D model of a protein.
We have developed MODICT, a mutation prediction tool which is based on per residue RMSD (root mean square ...deviation) values of superimposed 3D protein models. Our mathematical algorithm was tested for 42 described mutations in multiple genes including renin (REN), beta-tubulin (TUBB2B), biotinidase (BTD), sphingomyelin phosphodiesterase-1 (SMPD1), phenylalanine hydroxylase (PAH) and medium chain Acyl-Coa dehydrogenase (ACADM). Moreover, MODICT scores corresponded to experimentally verified residual enzyme activities in mutated biotinidase, phenylalanine hydroxylase and medium chain Acyl-CoA dehydrogenase. Several commercially available prediction algorithms were tested and results were compared. The MODICT PERL package and the manual can be downloaded from https://github.com/IbrahimTanyalcin/MODICT .
We show here that MODICT is capable tool for mutation effect prediction at the protein level, using superimposed 3D protein models instead of sequence based algorithms used by POLYPHEN and SIFT.
The JAK2 V617F mutation, the thrombopoietin receptor MPL W515K/L mutation and calreticulin (CALR) mutations are mutually exclusive in essential thrombocythemia and support a novel molecular ...categorization of essential thrombocythemia. CALR mutations account for approximately 30% of cases of essential thrombocythemia. In a retrospective study, we examined the frequency of MPL and CALR mutations in JAK2 V617F-negative cases of essential thrombocythemia (n=103). In addition, we compared the clinical phenotype and outcome of CALR mutant cases of essential thrombocythemia with a cohort of JAK2 V617F-positive essential thrombocythemia (n=57). CALR-positive cases represented 63.7% of double-negative cases of essential thrombocythemia, and most carried CALR type 1 or type 2 indels. However, we also identified one patient who was positive for both the JAK2 V617F and the CALR mutations. This study revealed that CALR mutant essential thrombocythemia is associated with younger age, higher platelet counts, lower erythrocyte counts, leukocyte counts, hemoglobin, and hematocrit, and increased risk of progression to myelofibrosis in comparison with JAK2 V617F-positive essential thrombocythemia. Analysis of the CALR mutant group according to indel type showed that CALR type 1 deletion is strongly associated with male gender. CALR mutant patients had a better overall survival than JAK2 V617F-positive patients, in particular patients of age 60 years or younger. In conclusion, this study in a Belgian cohort of patients supports and extends the growing body of evidence that CALR mutant cases of essential thrombocythemia are phenotypically distinct from JAK2 V617F-positive cases, with regards to clinical and hematologic presentation as well as overall survival.
Today's genome browsers and protein databanks supply vast amounts of information about proteins. The challenge is to concisely bring together this information in an interactive and easy to generate ...format.
We have developed an interactive CIRCOS module called i-PV to visualize user supplied protein sequence, conservation and SNV data in a live presentable format. I-PV can be downloaded from http://www.i-pv.org.
ibrahim.tanyalcin@i-pv.org, itanyalc@vub.ac.be or support@i-pv.org
Supplementary data are available at Bioinformatics online.
Bleeding and thrombotic events are major clinical complications of ET, together with progression to myelofibrosis and leukemic transformation (AML). The JAK2 V617F mutation, the thrombopoietin ...receptor mutation MPL W515K/L and the most recently discovered calreticulin (CALR) mutations are mutually exclusive in ET, account for up to 80-90 % of ET cases and support a novel molecular categorization of ET. However, the link between these driver mutations and ET pathogenesis and complications remains elusive.
We have studied megakaryocyte (MK) differentiation of peripheral blood progenitor cells in vitro as well as the platelet aggregation capacity in ET patients. Progenitors cells were grown under standard conditions in serum-free media supplemented with stem cell factor (SCF), FLT3 ligand, erythropoietin (Epo) and thrombopoietin (TPO), and gradually were switched to TPO only media after almost a week of expansion of the hematopoietic progenitors. ET MKs are larger in size, they are more granular and they display higher ploidy levels when compared to healthy MKs. They also co-express the megakaryocytic and erythroid markers, i.e. CD41 and CD71 at higher levels suggesting aberrant lineage specification from multipotent progenitors that can give rise to both the megakaryocytic and erythroid lineage. Remarkably, ET peripheral blood progenitors have a prolonged survival in vitro under the tested culture conditions when compared to healthy MKs. Megakaryocytic cultures from healthy donors last for 12-14 days in vitro while the ET MK cultures can be maintained for up to two extra weeks. Electron microscopy analysis of cultured MKs is consistent with the morphological observations mentioned above regarding size and granularity, which is higher in ET MKs, and hence support aberrant differentiation pathways. However, no differences were found between the molecular subtypes of ET. Therefore, we performed transcriptional analysis by RNA sequencing of megakaryocytic and erythroid cultures of progenitors from JAK2 V617F ET, CALR mutant ET or triple negative ET in order to identify the molecular cues that characterize abnormal megakaryopoiesis in ET subgroups. We have analyzed three biological replicates per genotype and we are currently confirming the results by RT-PCR and functional assays in primary cells and cell lines.
In parallel, we analyzed the aggregation capacity of JAK2 V617F, CALR mutant and triple negative ET platelets by a novel flow cytometry-based aggregation assay (De Cuyper IM et al. Blood 2013) in order to dissect potential signaling defects due to single receptor malfunction. We were able to pinpoint a significant defect in the response to collagen mediated by integrin a2b1 in ET platelets, regardless of the driver mutation, in comparison with platelets from healthy donors. However, convulxin-stimulated aggregation mediated by GPVI was normal. We also explored the degranulation capacity of ET platelets upon stimulation with thrombin peptide PAR-1. Platelets from CALR mutant or triple negative ET exhibited a reduced response as measured by the change of surface expression of the alpha granule protein CD62P compared to WT (healthy donors) and ET JAK2 V617F platelets. This was correlated with increased basal CD62P expression, suggesting that platelets in CALRmutant or triple negative ET degranulate spontaneously.
In summary, we have developed in vitro assays to explore peripheral blood progenitor cell differentiation and platelet function in ET. Our findings indicate functional differences between JAK2 V617F ET as compared with CALR mutant or triple negative ET. The correlation between these functional differences and the clinical behavior of the molecular subtypes of ET needs to be further investigated.
References
Cuyper IM, Meinders M, van de Vijver E, de Korte D, Porcelijn L, de Haas M, Eble JA, Seeger K, Rutella S, Pagliara D, Kuijpers TW, Verhoeven AJ, van den Berg TK, Gutiérrez L. A novel flow cytometry-based platelet aggregation assay. Blood 2013 Mar 7;121(10):e70-80
No relevant conflicts of interest to declare.
JAK2 V617F is the most common mutation in essential thrombocythemia (ET), occurring in approximately 50 % of cases. Second to JAK2 V617F is MPL W515K/L, accounting for about 10 % of cases. The ...molecular cause of the remaining ET cases is still largely unknown.
We sought to investigate JAK2 V617F-negative and MPL W515K/L-negative ET for regions of copy number variations (CNV) and loss of heterozygosity (LOH).
We studied blood or bone marrow samples from a series of 64 JAK2 V617F-negative and MPL W515K/L-negative ET cases. They were subjected to 2.7M SNP array by Affymetrix, which has 2,761,979 copy number markers including 400,103 SNP markers. The array data were analyzed for recurrent CNVs with Array Studio (OmicSoft), and for individual CNVs or recurrent LOHs (≥3 Mbs) with the Chromosome analysis suite (ChAS, Affymetrix).
Only 8 recurrent gains were identified, in 5/64 patients. Interestingly, the most common gain, occurring in 5 cases was a gain of chr7 q22.3, including the gene encoding Nicotinamide phosphoribosyltransferase (NAMPT). NAMPT is known to be overexpressed in several cancers such as multiple myeloma. It catalyzes the rate-limiting step of the nicotinamide adenine dinucleotide (NAD+) biosynthesis pathway. It is also required for cell growth and survival. We checked in the 5 patients for NAMPT amplification by quantitative PCR (qPCR) on genomic DNA in comparison to controls and by normalizing to ALB and RPPH1. We were able to validate the gain in 2/5 patients. The gain in these 2 patients was demonstrated to be acquired by qPCR of NAMPT in buccal swab DNA. Other recurrent gains involved regions of chromosomes 2, 5, 7, 12, 13, and 22. These gains included, amongst others, LCP1 on chr13 q14.3 and CYTIP on chr2 q24.1, occurring in 2/64 and in 3/64 respectively. We also checked for non-recurrent gains and losses in our cohort. This analysis generated a total of 8 CNVs in 6 different patients, comprising 5 regional gains in chromosomes 2, 8, 12, and 15 and 3 regional losses in chromosomes 5, 8 and 11. The array data were also analyzed for recurrent LOHs on ChAS, yielding 17 recurrent copy neutral LOHs (CN-LOH) in 35 patients (circos plot). The most common CN-LOH region was on chromosome 3 appearing in 8 patients. Other CN-LOH regions involved chromosomes 1, 2, 3, 4, 5, 6, 7, 10, 12, 15, and 17 and they occurred in 2-5/64 patients. However, as small regions of CN-LOH can be constitutional, we suspect that most of these CN-LOH regions are not acquired. The largest region of CN-LOH observed was 12 Mbs in size.
Previous studies in unselected series of BCR-ABL1-negative myeloproliferative neoplasms have shown that copy number alterations are rare in ET as well as in polycythemia vera. In this series of 64 JAK2 V617F-negative and MPL W515K/L-negative ET patients we found recurrent gains not reported previously in the database of genomic variants in only 8% of patients, and small areas of CN-LOH in ∼55% of cases. However, most of the latter probably are constitutional. Our SNP array study provides further evidence that gains, losses or CN-LOH of small genomic regions do not play an essential role in the pathogenesis of the majority of JAK2 V617F-negative and MPL W515K/L-negative ET. However, the low frequency of megakaryocytes and unknown level of clonal involvement of the myeloid compartment in JAK2 V617F-negative and MPL W515K/L-negative ET bone marrow remain a caveat. Next generation sequencing technology is expected to bring new insights on the molecular pathogenesis of this elusive ET subset.
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Left half represents a total of 35 patients carrying recurrent CN-LOHs and the right half represents the chromosomes and their associated properties. Right outermost layer depicts 1+log-gene density (min, 1; max 42) where cancer, OMIM and other genes are colored in red, blue and green respectively. Right middle and innermost layers designates SNP density (blue, values < 0,02; gray values, <0,06; red values >0,06; max scale 0,013) and absolute SNP numbers (min, 1; max, 12074) per windows of 50kb. Each 5Mb distance is marked with a tick underneath the innermost layer. Links from each chromosome are colored differently. Regions that are more confined on the same chromosome are least transparent and regions that are shared by more patients are drawn on top of lesser links.
No relevant conflicts of interest to declare.
Introduction: The JAK2 p.V617F, MPL p.W515K/L and CALR indels occur in a mutually exclusive pattern in 80-90% of cases with Essential Thrombocythemia (ET), but the driver mutations are unknown in the ...remaining 10-20%. In this study we aimed to identify driver mutations in the latter group of triple negative (TN) ET by exome sequencing of 10 such cases.
Results: We found 27 somatic variants, including indels, in 6 out of 10 TN ET patients (range: 1-10 mutations/case; mean: 2,7 mutations/case), none of which were recurrent. In one case, we found a MPL c.610T>C (p.S204P) mutation, which is located in the extracellular domain of the MPLreceptor. By Sanger sequencing of MPL exon 4 in 20 additional TN ET cases, an additional patient with the MPL S204P mutation was identified. Moreover, this mutation was previously reported in one case with idiopathic myelofibrosis1.
In order to study the effect of this mutation on the function of MPL, we produced stable Ba/F3 cell lines expressing MPL S204P, MPL W515K or MPL WT, and assessed the dependence of their growth on exogenous thrombopoietin (TPO). Only MPL W515K transduced Ba/F3 cells proliferated in the absence of TPO, but growth of MPL S204P Ba/F3 and of MPL WT Ba/F3 could be rescued by exogenous TPO, indicating the proper surface expression and the functionality of the transduced receptors. The levels of phospho-JAK2 and phospho-STAT5 were low in cytokine-deprived MPL S204P cells but increased upon TPO stimulation. In contrast, phospho-JAK2 and phospho-STAT5 were detectable in MPL W515K transduced Ba/F3 in the absence of cytokines as assessed by Western blotting. Culture of MPL S204P transduced Ba/F3 in the presence of TPO over a range of concentrations (0,01-10 ng/ml) yielded growth curves comparable with MPL WT transduced Ba/F3.
Using flow cytometry, we also explored cell surface marker expression on peripheral blood platelets from the two MPL S204P ET patients. Data were compared with healthy donors or ET patients with JAK2 or CALR mutations. MPL S204P ET platelets displayed higher expression of CD61 than platelets from healthy donors or from JAK2 or CALR mutated ET (p<0,01). In addition, there was a trend for higher expression of KIT, CD36 and CD42b on platelets from the MPL S204P ET cases. Moreover, following platelet activation through the protease activated receptor 1, the degranulation response of platelets from MPL S204P ET was decreased in comparison with JAK2 or CALR mutated ET.
Conclusion: The MPL S204P mutation is a recurrent mutation in TN ET, with a frequency of 7% (2/30) in this series, but this mutation does not induce TPO-independent growth nor increased TPO-sensitivity in Ba/F3 cells. However, preliminary phenotypic and functional evidence supports the notion that MPL S204P platelets display specific characteristics as compared with JAK2 or CALR mutated ET. The mechanisms by which the MPL S204P mutation influences megakaryopoiesis and platelet function remain to be elucidated.
1. Williams DM, et al. Phenotypic variations and new mutations in JAK2 V617F-negative polycythemia vera, erythrocytosis, and idiopathic myelofibrosis. Exp Hematol 2007; 35: 1641.
Graux:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees.
Bleeding and thrombotic events are major clinical complications of ET, together with progression to myelofibrosis and leukemic transformation (AML). The JAK2 V617F mutation, the thrombopoietin ...receptor mutation MPL W515K/L and the most recently discovered calreticulin (CALR) mutations are mutually exclusive in ET, account for up to 80-90 % of ET cases and support a novel molecular categorization of ET. In a retrospective study, we have examined the clinical phenotype and outcome of a Belgian cohort of 165 ET patients in relation to their mutational status. 38% of the patients were CALR mutated, 22% and 12% of whom carried Type 1 (p.L367fs*46) and Type 2 (p.K385fs*47) indels respectively. 35% were JAK2 V617F positive, 7% were MPL W515K/L positive, one patient was positive for both CALR and JAK2 V617F mutations, and 20% were triple negative (Fig. 1). We compared the hematological and clinical features between CALR mutant patients and JAK2 V617F positive patients. This revealed that CALR mutant ET is associated with younger age than JAK2 V617F positive ET (median age 56 y (range 23-84) versus 65 y (range 36-94), p<0.001), male gender (58% versus 39%, p=0.03), higher platelet count (988 ± 367*109/L versus 870 ± 291*109/L, p=0.04 (mean ± SD)), lower leukocyte count (9.1 ± 3.2*109/L versus 11.6 ± 5.8*109/L, p<0.001), lower erythrocyte count (4.36 ± 0.9*1012/L versus 4.98 ± 0.65*1012/L, p<0.001), hemoglobin (13.2 ± 1.8 g/dL versus 14.3 ±1.6 g/dL, p=0.001) and hematocrit (40 ± 6.2 % versus 44 ± 4.6 %, p<0.001). Analysis of the CALR mutant group according to the indel type showed that CALR Type 1 deletion is strongly associated with male gender (62%). Contrary to previously published findings, we did not find significant differences between CALR type I and II mutations with regard to age and platelet count.CALR mutant patients had a better overall survival than JAK2 V617F positive patients (mean survival 28 y versus 16 y, p=0.01). However, the better overall survival for CALR mutant ET was restricted to patients less than 60 years old (mean survival 29.2 y versus 18.5 y, p=0.02) while in the age group above 60 years, the overall survival was not significantly different (mean survival 18.3 y versus 13 y, p=0.32) (Fig. 2). In our cohort, no difference in myelofibrosis-free survival or leukemia-free survival was found between the molecular subtypes, although the risk of developing myelofibrosis was unexpectedly higher in the CALR mutant group (18% versus 4%, p=0.01). In contrast to other studies, we found no difference in the frequency of arteriovenous complications. In conclusion, this study on a Belgian cohort supports the notion that CALR mutant ET is phenotypically distinct from JAK2 V617F positive ET, with regard to the clinical and hematological presentation as well as the overall survival. In this cohort, the better overall survival was most marked in ET patients less than 60 y of age. Our study adds to the growing body of evidence that CALR mutant ET is a disease entity distinct from JAK2 V617F positive ET.
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No relevant conflicts of interest to declare.
Essential thrombocythemia (ET) is a myeloproliferative neoplasm featured by a sustained elevation of platelet count and a tendency for thrombosis and hemorrhage. Cytogenetic abnormalities are rare in ...ET. The most common molecular abnormality in ET is JAK2 V617F, found in approximately 50% of ET cases followed by MPL W515K/L, found in about 10% of cases. The molecular cause of the remaining ET cases is still largely unknown. As such, in a substantial fraction of ET cases, the underlying molecular cause is yet to be discovered. In a recent study by Hou et al., single cells derived from an ET JAK2 V617F-negative ET patient were sequenced using a method based on exome sequencing. Eight genes were identified as possible candidate drivers. However, their recurrence rate in ET was not established.
To establish the recurrence rate in JAK2 V617F-negative and MPL W515K/L-Negative ET of potential candidate driver mutations, as identified by Hou et al.
We studied unfractionated blood or bone marrow samples from a series of 64 cases of JAK2 V617F-negative and MPL W515K/L-negative ET. In this series, we used PCR and Sanger sequencing to detect the following mutations: SESN2 P87S, TOP1MT S479L, ST13 Q349*, and DNAJC17 A292P, as they exhibited the highest scores in the study of Hou et al. In addition, we included NTRK1 N323S, a mutant tyrosine kinase. None of the mutations reported by Hou et al. was detected in our patients. However, we identified a novel acquired heterozygous mutation in TOP1MT (c.1400 A>G, p.N467S) which is predicted to be damaging by polyphen 2. This mutation was not detected in the germline DNA from the buccal swab of the patient. TOP1MT is a mitochondrial topoisomerase encoded by the genomic DNA. It is a type IB enzyme, which sustains the appropriate conformation of DNA during replication, transcription, recombination, and repair. This mutation might affect the interaction of TOP1MT with the DNA molecule as suggested by the results of in silico analysis from I-Tasser. p.N467S mutation causes the gain of a helix and the loss of a β strand which are in close proximity to the bound DNA molecule. We screened exon 11 of TOP1MT gene in 38 additional JAK2 V617F-negative MPL W515K/L-negative ET cases, but did not find any additional cases.
In this series of 102 cases of JAK2 V617F-negative and MPL W515K/L-negative ET, only one case was identified with a mutation of TOP1MT. Mutations of SESN2, ST13, DNAJC17, or NTRK1, four other candidate driver genes as identified by Hou et al., could not be identified in a series of 64 cases. The functional role of TOP1MT in the pathogenesis of ET remains to be established. The absence of the mutations, as proposed by Hou et al., in our cohort raises questions about their role as potential driver mutations in JAK2 V617F-negative and MPL W515K/L-negative ET. The quest for the full complement of driver mutations in ET therefore remains open.
Hou, Y., et al., Single-cell exome sequencing and monoclonal evolution of a JAK2-negative myeloproliferative neoplasm. Cell, 2012. 148(5): p. 873-85.
No relevant conflicts of interest to declare.
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