Deep sequencing reveals double mutations in cis of MPL exon 10 in myeloproliferative neoplasms.

PubMed

Pietra, Daniela; Brisci, Angela; Rumi, Elisa; Boggi, Sabrina; Elena, Chiara; Pietrelli, Alessandro; Bordoni, Roberta; Ferrari, Maurizio; Passamonti, Francesco; De Bellis, Gianluca; Cremonesi, Laura; Cazzola, Mario

2011-04-01

Somatic mutations of MPL exon 10, mainly involving a W515 substitution, have been described in JAK2 (V617F)-negative patients with essential thrombocythemia and primary myelofibrosis. We used direct sequencing and high-resolution melt analysis to identify mutations of MPL exon 10 in 570 patients with myeloproliferative neoplasms, and allele specific PCR and deep sequencing to further characterize a subset of mutated patients. Somatic mutations were detected in 33 of 221 patients (15%) with JAK2 (V617F)-negative essential thrombocythemia or primary myelofibrosis. Only one patient with essential thrombocythemia carried both JAK2 (V617F) and MPL (W515L). High-resolution melt analysis identified abnormal patterns in all the MPL mutated cases, while direct sequencing did not detect the mutant MPL in one fifth of them. In 3 cases carrying double MPL mutations, deep sequencing analysis showed identical load and location in cis of the paired lesions, indicating their simultaneous occurrence on the same chromosome.

[Expression of JAK2V617F and MPLW515L/K mutation in 30 suspected cases of early myeloproliferative disorders].

PubMed

Fan, Zheng; Zhang, Ri; Shen, Yi-Min; Fei, Hai-Rong; Zhu, Zi-Ling; Cen, Jian-Nong

2008-09-01

To investigate the prevalence of JAK2V617F and MPLW515L/K mutation in patients with slightly elevated platelets (BPC) or hemoglobin (Hb) not meeting the criteria of polycythemia vera (PV) or essential thrombocythemia (ET). Genomic DNA from bone marrow or blood mononuclear cells was screened with allele specific polymerase chain reaction (AS-PCR) for JAK2V617F and MPLW515L/K mutation. The history of thrombosis was assessed retrospectively by patients files. Of 30 patients, 14 (46.7%) were positive for the JAK2V617F mutation, none of them had the MPLW515L/ K. Five of these 14 patients had a history of thrombosis. Follow-up results were available in 22 patients. Among them, 12 patients with JAK2V617F mutation turned out to be MPD in 6-24 months; only 2 out of 10 patients without this mutation evolved to MPD. JAK2V617F mutation could be one of the diagnosis criteria of early MPD. No MPLW515L/K expression was found in early MPD.

Estimation of diagnosis and prognosis in ET by assessment of CALR and JAK2V617F mutations and laboratory findings: a meta-analysis.

PubMed

Saki, N; Shirzad, R; Rahim, F; Saki Malehi, A

2017-07-01

Essential thrombocythemia (ET) is a benign disease with slow progress in which thrombosis is a cause of mortality. JAK2 V617F and calreticulin (CALR) are the most frequent mutations in this disease. In this systematic review and meta-analysis, we compared the prevalence of JAK2 V617F and CALR mutations in ET and examined the incidence of thrombosis and other hematologic indices. After choosing MeSH keywords, including essential thrombocythemia, JAK2 V617F , calreticulin, prognosis, and diagnosis, as well as searching Medline/PubMed and Scopus, 12 papers were selected. Data were pooled, and summary prevalence and OR were estimated using either a random-effects model or a fixed-effects model. The frequency of JAK2 V617F and CALR shows heterogeneity in Caucasian population [JAK2 V617 I 2 % = 84.3, P < 0.001, 95% CI 0.56 (0.51-0.61)], [CALR I 2 % = 96.1, P < 0.001, 95% CI 0.23 (0.15-0.31)]. The prevalence of JAK2 V617F and CALR was 0.57 (95% CI 0.53-0.61), I 2 % = 79.3 and 0.22 (95% CI 0.16-0.27), I 2 % = 94, respectively. JAK2 V617F positive ET was associated with increasing odds of thrombosis [OR 2.35 (95% CI 1.83-3.02), P < 0.001]. The incidence of splenomegaly was not statistically different between these two mutations. Hemoglobin, platelet, and WBC count did not affect the risk of thrombosis. Detection of CALR mutation is helpful for molecular diagnosis of ET patients as well as JAK2 V617F . Due to reduction of thrombosis in CALR-positive patients, it can be stated that such patients have less thrombotic disorders and better prognosis relative to patients bearing JAK2 V617F mutation. Therefore, detection of mutation in CALR and JAK2 V617F may contribute to diagnosis and prognosis of ET patients.

Low frequency of c-MPL gene mutations in Iranian patients with Philadelphia-negative myeloproliferative disorders.

PubMed

Ghotaslou, A; Nadali, F; Chahardouli, B; Alizad Ghandforosh, N; Rostami, S H; Alimoghaddam, K; Ghavamzadeh, A

2015-01-01

Myeloproliferative disorders are a group of diseases characterized by increased proliferation of myeloid lineage. In addition to JAK2V617F mutation, several mutations in the c-MPL gene have been reported in patients with philadelphia-negative chronic myeloproliferative disorders that could be important in the pathogenesis of diseases. The aim of the present study was to investigate the frequency of c-MPL and JAK2V617F mutations in Iranian patients with Philadelphia-negativemyeloproliferative disorders. Peripheral blood samples were collected from 60 patients with Philadelphia-negative MPD) Subgroups ET and PMF) and 25 healthy subjects as control group. The mutation status of c-MPL and Jak2V617F were investigated by using Amplification-refractory mutation system (ARMS) and Allele-Specific PCR (AS-PCR), respectively. The results were confirmed by sequencing. Among 60 patients, 34 (56.6%) and 1(1.7%) had Jak2V617F and c-MPL mutation, respectively. Patients with Jak2V617F mutation had higher WBC counts and hemoglobin concentration than those without the mutation (p= 0.005, p=0.003). In addition, for all healthy subjects in control group, mutations were negative. The present study revealed that the c-MPL mutations unlike the Jak2V617F mutations are rare in Iranian patients with Ph-negative MPNs and the low mutation rate should be considered in the design of screening strategies of MPD patients.

Low frequency of c-MPL gene mutations in Iranian patients with Philadelphia-negative myeloproliferative disorders

PubMed Central

Ghotaslou, A; Nadali, F; Chahardouli, B; Alizad Ghandforosh, N; Rostami, SH; Alimoghaddam, K; Ghavamzadeh, A

2015-01-01

Background Myeloproliferative disorders are a group of diseases characterized by increased proliferation of myeloid lineage. In addition to JAK2V617F mutation, several mutations in the c-MPL gene have been reported in patients with philadelphia-negative chronic myeloproliferative disorders that could be important in the pathogenesis of diseases. The aim of the present study was to investigate the frequency of c-MPL and JAK2V617F mutations in Iranian patients with Philadelphia-negativemyeloproliferative disorders. Material and Methods Peripheral blood samples were collected from 60 patients with Philadelphia-negative MPD) Subgroups ET and PMF) and 25 healthy subjects as control group. The mutation status of c-MPL and Jak2V617F were investigated by using Amplification-refractory mutation system (ARMS) and Allele-Specific PCR (AS-PCR), respectively. The results were confirmed by sequencing. Results Among 60 patients, 34 (56.6%) and 1(1.7%) had Jak2V617F and c-MPL mutation, respectively. Patients with Jak2V617F mutation had higher WBC counts and hemoglobin concentration than those without the mutation (p= 0.005, p=0.003). In addition, for all healthy subjects in control group, mutations were negative. Conclusions The present study revealed that the c-MPL mutations unlike the Jak2V617F mutations are rare in Iranian patients with Ph-negative MPNs and the low mutation rate should be considered in the design of screening strategies of MPD patients. PMID:25914801

Structural effects of clinically observed mutations in JAK2 exons 13-15: comparison with V617F and exon 12 mutations

PubMed Central

Lee, Tai-Sung; Ma, Wanlong; Zhang, Xi; Kantarjian, Hagop; Albitar, Maher

2009-01-01

Background The functional relevance of many of the recently detected JAK2 mutations, except V617F and exon 12 mutants, in patients with chronic myeloproliferative neoplasia (MPN) has been significantly overlooked. To explore atomic-level explanations of the possible mutational effects from those overlooked mutants, we performed a set of molecular dynamics simulations on clinically observed mutants, including newly discovered mutations (K539L, R564L, L579F, H587N, S591L, H606Q, V617I, V617F, C618R, L624P, whole exon 14-deletion) and control mutants (V617C, V617Y, K603Q/N667K). Results Simulation results are consistent with all currently available clinical/experimental evidence. The simulation-derived putative interface, not possibly obtained from static models, between the kinase (JH1) and pseudokinase (JH2) domains of JAK2 provides a platform able to explain the mutational effect for all mutants, including presumably benign control mutants, at the atomic level. Conclusion The results and analysis provide structural bases for mutational mechanisms of JAK2, may advance the understanding of JAK2 auto-regulation, and have the potential to lead to therapeutic approaches. Together with recent mutation profiling results demonstrating the breadth of clinically observed JAK2 mutations, our findings suggest that molecular testing/diagnostics of JAK2 should extend beyond V617F and exon 12 mutations, and perhaps should encompass most of the pseudo-kinase domain-coding region. PMID:19744331

JAK2 46/1 haplotype is associated with JAK2 V617F--positive myeloproliferative neoplasms in Brazilian patients.

PubMed

Macedo, L C; Santos, B C; Pagliarini-e-Silva, S; Pagnano, K B B; Rodrigues, C; Quintero, F C; Ferreira, M E; Baraldi, E C; Ambrosio-Albuquerque, E P; Sell, A M; Visentainer, J E L

2015-10-01

This study aimed to verify the association between the JAK2 46/1 haplotype (V617F positive) and some hematological parameters in BCR-ABL-negative chronic myeloproliferative neoplasms (cMPNs) in our population. The blood samples obtained from the patients with cMPN were genotyped for the JAK2 V617F mutation and JAK2 rs10974944 SNP screening using a PCR-RFLP assay. The JAK2 V617F mutation was detected in 80.15% of patients. The G variant of rs10974944 was more frequent in all MPNs, especially those that were JAK2 V617F positive, than in the control population. We also compared the 46/1 haplotype status in each MPN disease entity, polycythemia vera (PV), essential thrombocythemia (ET), primary myelofibrosis (PMF), and MPNu with controls. The G allele frequency relative to controls was significantly enriched in patients with PV and ET, but not in those with PMF and MPNu. PV and ET patients especially, all of whom had the JAK2 V617F mutation, showed significant excess of the G allele. The frequency of JAK2 V617F mutation was associated with elevated hematological parameters, but when we analyze the occurrence of the mutation and the presence of the G allele, just the high hemoglobin was significantly. In agreement with previous reports, JAK2 46/1 haplotype for JAK2 V617F was associated with cMPN positive in Brazilian patients. © 2015 John Wiley & Sons Ltd.

Molecular analyses of 15,542 patients with suspected BCR-ABL1-negative myeloproliferative disorders allow to develop a stepwise diagnostic workflow

PubMed Central

Schnittger, Susanne; Bacher, Ulrike; Eder, Christiane; Dicker, Frank; Alpermann, Tamara; Grossmann, Vera; Kohlmann, Alexander; Kern, Wolfgang; Haferlach, Claudia; Haferlach, Torsten

2012-01-01

We investigated 15,542 patients with suspected BCR-ABL1- negative myeloproliferative or myelodysplastic/myeloproliferative neoplasm (including 359 chronic myelomonocytic leukemia) by a molecular marker set. JAK2V617F was detected in the suspected categories as follows: polycythemia vera 88.3%, primary myelofibrosis 53.8%, essential thrombocythemia 50.2%, and not further classifiable myeloproliferative neoplasms 38.0%. JAK2 exon 12 mutations were detected in 40.0% JAK2V617F-negative suspected polycythemia vera, MPLW515 mutations in 13.2%JAK2V617F-negative primary myelofibrosis and 7.1% JAK2V617F-negative essential thrombocythemia. TET2 mutations were distributed across all entities but were most frequent in suspected chronic myelomonocytic leukemia (77.8%). CBL mutations were identified in suspected chronic myelomonocytic leukemia (13.9%), primary myelofibrosis (8.0%), and not further classifiable myeloproliferative neoplasm (7.0%). This leads to a stepwise workflow for suspected myeloproliferative neoplasms starting with JAK2V617F and investigating JAK2V617F-negative patients for JAK2 exon 12 or MPL mutations, respectively. In cases in which a myeloproliferative neoplasm cannot be established, analysis for TET2, CBL and EZH2 mutations may be indicated. PMID:22511494

TG101209, a small molecule JAK2-selective kinase inhibitor potently inhibits myeloproliferative disorder-associated JAK2V617F and MPLW515L/K mutations.

PubMed

Pardanani, A; Hood, J; Lasho, T; Levine, R L; Martin, M B; Noronha, G; Finke, C; Mak, C C; Mesa, R; Zhu, H; Soll, R; Gilliland, D G; Tefferi, A

2007-08-01

JAK2V617F and MPLW515L/K represent recently identified mutations in myeloproliferative disorders (MPD) that cause dysregulated JAK-STAT signaling, which is implicated in MPD pathogenesis. We developed TG101209, an orally bioavailable small molecule that potently inhibits JAK2 (IC(50)=6 nM), FLT3 (IC(50)=25 nM) and RET (IC(50)=17 nM) kinases, with significantly less activity against other tyrosine kinases including JAK3 (IC(50)=169 nM). TG101209 inhibited growth of Ba/F3 cells expressing JAK2V617F or MPLW515L mutations with an IC(50) of approximately 200 nM. In a human JAK2V617F-expressing acute myeloid leukemia cell line, TG101209-induced cell cycle arrest and apoptosis, and inhibited phosphorylation of JAK2V617F, STAT5 and STAT3. Therapeutic efficacy of TG101209 was demonstrated in a nude mouse model. Furthermore, TG101209 suppressed growth of hematopoietic colonies from primary progenitor cells harboring JAK2V617F or MPL515 mutations.

Detection of CALR and MPL Mutations in Low Allelic Burden JAK2 V617F Essential Thrombocythemia.

PubMed

Usseglio, Fabrice; Beaufils, Nathalie; Calleja, Anne; Raynaud, Sophie; Gabert, Jean

2017-01-01

Myeloproliferative neoplasms are clonal hematopoietic stem cell disorders characterized by aberrant proliferation and an increased tendency toward leukemic transformation. The genes JAK2, MPL, and CALR are frequently altered in these syndromes, and their mutations are often a strong argument for diagnosis. We analyzed the mutational profiles of these three genes in a cohort of 164 suspected myeloproliferative neoplasms. JAK2 V617F mutation was detected by real-time PCR, whereas high-resolution melting analysis followed by Sanger sequencing were used for searching for mutations in JAK2 exon 12, CALR, and MPL. JAK2 V617F mutation was associated with CALR (n = 4) and MPL (n = 4) mutations in 8 of 103 essential thrombocytosis patients. These cases were harboring a JAK2 V617F allelic burden of <4% and a significantly higher platelet count compared with JAK2 V617F (P < 0.001) and CALR (P = 0.001) single-mutation patients. The findings from this study support the possibility of coexisting mutations of the JAK2, CALR, and MPL genes in myeloproliferative neoplasms and suggest that CALR and MPL should be analyzed not only in JAK2-negative patients but also in low V617F mutation patients. Follow-up of these double-mutation cases will be important for determining whether this group of patients presents particular evolution or complications. Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Significance of combined detection of JAK2V617F, MPL and CALR gene mutations in patients with essential thrombocythemia

PubMed Central

Ji, Liying; Qian, Mengyao; Wu, Nana; Wu, Jianmin

2017-01-01

The aim of this study was to analyze the mutation rate of JAK2V617F, MPLW515L/K and CALR genes in adult patients with essential thrombocythemia (ET) and the accuracy of the combined detection by the receiver operating curve. Three hundred and forty-two cases with high-platelets (≥300×109/l) were consecutively selected. The patients were analyzed for routine blood examination, bone marrow biopsy and genetic testing. One hundred and fifty-four cases (45.03%) were diagnosed with ET and 188 cases of secondary thrombocythemia according to the hematopoietic and lymphoid tissue tumor classification standards of 2008. It was found that the mutant type of three genes showed three bands, whereas only one band for wild-type. The JAK2V617F and MPL mutations did not cause a change in the open reading frame and the CALR mutation resulted in its change. The mutation rate of JAK2V617F and CALR in ET group was significantly higher than that in the secondary thrombocythemia group (p<0.05). The positive mutation rate of MPL was only 4.55%. JAK2V617F-positive mutation alone was used to diagnose with ET. The area under the curve (AUC) was 0.721. The sensitivity was 72.4%, the specificity was 79.5% and the cut-off value was 0.25. When CALR-positive mutation alone was used to diagnose ET, the AUC, sensitivity, specificity and cut-off value were 0.664, 68.4, 82.4 and 0.09%, respectively. JAK2V617F combined with CALR mutation were used for diagnosis of ET. The AUC was 0.862, the sensitivity was 85.9%, the specificity was 87.8%, and the cut-off values were 0.21 and 0.07. In conclusion, the positive mutation rate of JAK2V617F and CALR in ET was higher, and the sensitivity, specificity and accuracy of the diagnosis of ET were significantly improved using the detection of JAK2V617F and CALR. PMID:28450924

Significance of combined detection of JAK2V617F, MPL and CALR gene mutations in patients with essential thrombocythemia.

PubMed

Ji, Liying; Qian, Mengyao; Wu, Nana; Wu, Jianmin

2017-03-01

The aim of this study was to analyze the mutation rate of JAK2V617F, MPLW515L/K and CALR genes in adult patients with essential thrombocythemia (ET) and the accuracy of the combined detection by the receiver operating curve. Three hundred and forty-two cases with high-platelets (≥300×10 9 /l) were consecutively selected. The patients were analyzed for routine blood examination, bone marrow biopsy and genetic testing. One hundred and fifty-four cases (45.03%) were diagnosed with ET and 188 cases of secondary thrombocythemia according to the hematopoietic and lymphoid tissue tumor classification standards of 2008. It was found that the mutant type of three genes showed three bands, whereas only one band for wild-type. The JAK2V617F and MPL mutations did not cause a change in the open reading frame and the CALR mutation resulted in its change. The mutation rate of JAK2V617F and CALR in ET group was significantly higher than that in the secondary thrombocythemia group (p<0.05). The positive mutation rate of MPL was only 4.55%. JAK2V617F-positive mutation alone was used to diagnose with ET. The area under the curve (AUC) was 0.721. The sensitivity was 72.4%, the specificity was 79.5% and the cut-off value was 0.25. When CALR-positive mutation alone was used to diagnose ET, the AUC, sensitivity, specificity and cut-off value were 0.664, 68.4, 82.4 and 0.09%, respectively. JAK2V617F combined with CALR mutation were used for diagnosis of ET. The AUC was 0.862, the sensitivity was 85.9%, the specificity was 87.8%, and the cut-off values were 0.21 and 0.07. In conclusion, the positive mutation rate of JAK2V617F and CALR in ET was higher, and the sensitivity, specificity and accuracy of the diagnosis of ET were significantly improved using the detection of JAK2V617F and CALR.

JAK2 Exon 12 Mutations in Polycythemia Vera and Idiopathic Erythrocytosis

PubMed Central

Scott, Linda M.; Tong, Wei; Levine, Ross L.; Scott, Mike A.; Beer, Philip A.; Stratton, Michael R.; Futreal, P. Andrew; Erber, Wendy N.; McMullin, Mary Frances; Harrison, Claire N.; Warren, Alan J.; Gilliland, D. Gary; Lodish, Harvey F.; Green, Anthony R.

2010-01-01

BACKGROUND The V617F mutation, which causes the substitution of phenylalanine for valine at position 617 of the Janus kinase (JAK) 2 gene (JAK2), is often present in patients with polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis. However, the molecular basis of these myeloproliferative disorders in patients without the V617F mutation is unclear. METHODS We searched for new mutations in members of the JAK and signal transducer and activator of transcription (STAT) gene families in patients with V617F-negative polycythemia vera or idiopathic erythrocytosis. The mutations were characterized biochemically and in a murine model of bone marrow transplantation. RESULTS We identified four somatic gain-of-function mutations affecting JAK2 exon 12 in 10 V617F-negative patients. Those with a JAK2 exon 12 mutation presented with an isolated erythrocytosis and distinctive bone marrow morphology, and several also had reduced serum erythropoietin levels. Erythroid colonies could be grown from their blood samples in the absence of exogenous erythropoietin. All such erythroid colonies were heterozygous for the mutation, whereas colonies homozygous for the mutation occur in most patients with V617F-positive polycythemia vera. BaF3 cells expressing the murine erythropoietin receptor and also carrying exon 12 mutations could proliferate without added interleukin-3. They also exhibited increased phosphorylation of JAK2 and extracellular regulated kinase 1 and 2, as compared with cells transduced by wild-type JAK2 or V617F JAK2. Three of the exon 12 mutations included a substitution of leucine for lysine at position 539 of JAK2. This mutation resulted in a myeloproliferative phenotype, including erythrocytosis, in a murine model of retroviral bone marrow transplantation. CONCLUSIONS JAK2 exon 12 mutations define a distinctive myeloproliferative syndrome that affects patients who currently receive a diagnosis of polycythemia vera or idiopathic erythrocytosis. PMID:17267906

JAK2V617F influences epigenomic changes in myeloproliferative neoplasms.

PubMed

Chen, Chih-Cheng; Chiu, Chia-Chen; Lee, Kuan-Der; Hsu, Chia-Chen; Chen, Hong-Chi; Huang, Tim H-M; Hsiao, Shu-Huei; Leu, Yu-Wei

2017-12-16

Negative valine (V) to phenylalanine (F) switch at the Janus kinase (JAK2) 617 codon (V617F) is the dominant driver mutation in patients with myeloproliferative neoplasms (MPNs). JAK2V617F was proved to be sufficient for cell transformation; however, independent mutations might influence the following epigenomic modifications. To assess the JAK2V617F-induced downstream epigenomic changes without interferences, we profiled the epigenomic changes in ectopically expressed JAK2V617F in Ba/F3 cells. Antibodies against phosphorylated signal transducer and activator of transcription 3 (pSTAT3) and enhancer of zeste homolog 2 (EZH2) were used for chromatin-immunoprecipitation sequencing (ChIP-seq) to detect the downstream epigenomic targets in the JAK2-STAT3 signaling pathway. To confirm the JAK2V617F-induced epigenetic changes in vivo, DNA methylation changes in the target loci in patients with MPNs were detected through methylation-specific polymerase chain reaction and were clustered against the changes within controls. We found that ectopically expressed JAK2V617F in Ba/F3 cells reduced the binding specificity; it was associated with cis-regulatory elements and recognized DNA motifs in both pSTAT3-downstream and EZH2-associated targets. Overlapping target loci between the control and JAK2V617F were <3% and 0.4%, respectively, as identified through pSTAT3 and EZH2 ChIP-seq. Furthermore, the methylation changes in the direct target loci (FOXH1, HOXC9, and SRF) were clustered independently from the control locus (L1TD1) and other mutation genes (HMGA2 and Lin28A) in the analyzed MPN samples. Therefore, JAK2V617F influences target binding in both pSTAT3 and EZH2. Without mutations in epigenetic regulators, JAK2V617F can induce downstream epigenomic modifications. Thus, epigenetic changes in JAK2 downstream targets might be trackable in vivo. Copyright © 2017 Elsevier Inc. All rights reserved.

Impact of genotype on leukaemic transformation in polycythaemia vera and essential thrombocythaemia.

PubMed

Alvarez-Larrán, Alberto; Senín, Alicia; Fernández-Rodríguez, Concepción; Pereira, Arturo; Arellano-Rodrigo, Eduardo; Gómez, Montse; Ferrer-Marin, Francisca; Martínez-López, Joaquín; Camacho, Laura; Colomer, Dolors; Angona, Anna; Navarro, Blanca; Cervantes, Francisco; Besses, Carlos; Bellosillo, Beatriz; Hernández-Boluda, Juan Carlos

2017-09-01

The influence of driver mutations on leukaemic transformation was analysed in 1747 patients with polycythaemia vera or essential thrombocythaemia. With a median follow-up of 7·2 years, 349 patients died and 62 progressed to acute leukaemia or myelodysplastic syndrome. Taking death as a competing risk, CALR genotype was associated with a lower risk of transformation [subdistribution hazard ratio (SHR): 0·13, 95% confidence interval (CI): 0·2-0·9, P = 0·039], whereas JAK2 V617F showed borderline significance for higher risk (SHR: 2·05, 95% CI: 0·9-4·6, P = 0·09). Myelofibrotic transformation increased leukaemic risk, except in CALR-mutated patients. Next generation sequencing of 51 genes at the time of transformation showed additional mutations (median number: 3; range: 1-5) in 25 out of 29 (86%) assessable cases. Mutations (median: 1; range: 1-3) were detected in 67% of paired samples from the chronic phase. Leukaemia appeared in a JAK2 V617F negative clone in 17 (58%) cases, eleven of them being previously JAK2 V617F-positive. JAK2 V617F-mutated leukaemia was significantly associated with complex karyotype and acquisition of TP53 mutations, whereas EZH2 and RUNX1 mutations were more frequent in JAK2 V617F-negative leukaemia. Survival was longer in JAK2 V617F-unmutated leukaemia (343 days vs. 95 days, P = 0·003). In conclusion, CALR genotype is associated with a lower risk of leukaemic transformation. Leukaemia arising in a JAK2 V617F-negative clone is TP53 independent and shows better survival. © 2017 John Wiley & Sons Ltd.

Clinical features of Japanese polycythemia vera and essential thrombocythemia patients harboring CALR, JAK2V617F, JAK2Ex12del, and MPLW515L/K mutations.

PubMed

Okabe, Masahiro; Yamaguchi, Hiroki; Usuki, Kensuke; Kobayashi, Yutaka; Kawata, Eri; Kuroda, Junya; Kimura, Shinya; Tajika, Kenji; Gomi, Seiji; Arima, Nobuyoshi; Mori, Sinichiro; Ito, Shigeki; Koizumi, Masayuki; Ito, Yoshikazu; Wakita, Satoshi; Arai, Kunihito; Kitano, Tomoaki; Kosaka, Fumiko; Dan, Kazuo; Inokuchi, Koiti

2016-01-01

The risk of complication of polycythemia vera (PV) and essential thrombocythemia (ET) by thrombosis in Japanese patients is clearly lower than in western populations, suggesting that genetic background such as race may influence the clinical features. This study aimed to clarify the relationship between genetic mutations and haplotypes and clinical features in Japanese patients with PV and ET. Clinical features were assessed prospectively among 74 PV and 303 ET patients. There were no clinical differences, including JAK2V617F allele burden, between PV patients harboring the various genetic mutations. However, CALR mutation-positive ET patients had a significantly lower WBC count, Hb value, Ht value, and neutrophil alkaline phosphatase score (NAP), and significantly more platelets, relative to JAK2V617F-positive ET patients and ET patients with no mutations. Compared to normal controls, the frequency of the JAK246/1 haplotype was significantly higher among patients with JAK2V617F, JAK2Ex12del, or MPL mutations, whereas no significant difference was found among CALR mutation-positive patients. CALR mutation-positive patients had a lower incidence of thrombosis relative to JAK2V617F-positive patients. Our findings suggest that JAK2V617F-positive ET patients and CALR mutation-positive patients have different mechanisms of occurrence and clinical features of ET, suggesting the potential need for therapy stratification in the future. Copyright © 2015 Elsevier Ltd. All rights reserved.

Increased risk of recurrent thrombosis in patients with essential thrombocythemia carrying the homozygous JAK2 V617F mutation.

PubMed

De Stefano, Valerio; Za, Tommaso; Rossi, Elena; Vannucchi, Alessandro M; Ruggeri, Marco; Elli, Elena; Micò, Caterina; Tieghi, Alessia; Cacciola, Rossella R; Santoro, Cristina; Vianelli, Nicola; Guglielmelli, Paola; Pieri, Lisa; Scognamiglio, Francesca; Cacciola, Emma; Rodeghiero, Francesco; Pogliani, Enrico M; Finazzi, Guido; Gugliotta, Luigi; Leone, Giuseppe; Barbui, Tiziano

2010-02-01

Evidence suggests that the JAK2 V617F mutation is associated with an increased risk of first thrombosis in patients with essential thrombocythemia (ET). Whether this mutation is also a risk factor for recurrent thrombosis is currently unknown. To investigate the impact of the JAK2 V617F mutation on the risk of recurrent thrombosis in patients with ET, we carried out a multicentre retrospective cohort study. We recruited 143 patients with previous arterial (64.4%) or venous major thrombosis (34.8%) or both (0.8%); 98 of them (68.5%) carried the mutation. Thrombosis recurred in 43 of the patients (30%); overall, after adjustment for sex, age, presence of vascular risk factors, and treatment after the first thrombosis, the presence of the JAK2 mutation did not predict recurrence (multivariable hazard ratio, HR, 0.88, 95% CI 0.46-1.68). Indeed, the individuals homozygous for the JAK2 V617F (allele burden >50%) mutation had an increased risk of recurrence in comparison with wild-type patients (HR 6.15, 95% CI 1.51-24.92). In conclusion, a homozygous JAK2 V617F mutation is an independent risk factor for recurrent thrombosis in patients with ET.

An accurate, simple prognostic model consisting of age, JAK2, CALR, and MPL mutation status for patients with primary myelofibrosis.

PubMed

Rozovski, Uri; Verstovsek, Srdan; Manshouri, Taghi; Dembitz, Vilma; Bozinovic, Ksenija; Newberry, Kate; Zhang, Ying; Bove, Joseph E; Pierce, Sherry; Kantarjian, Hagop; Estrov, Zeev

2017-01-01

In most patients with primary myelofibrosis, one of three mutually exclusive somatic mutations is detected. In approximately 60% of patients, the Janus kinase 2 gene is mutated, in 20%, the calreticulin gene is mutated, and in 5%, the myeloproliferative leukemia virus gene is mutated. Although patients with mutated calreticulin or myeloproliferative leukemia genes have a favorable outcome, and those with none of these mutations have an unfavorable outcome, prognostication based on mutation status is challenging due to the heterogeneous survival of patients with mutated Janus kinase 2. To develop a prognostic model based on mutation status, we screened primary myelofibrosis patients seen at the MD Anderson Cancer Center, Houston, USA, between 2000 and 2013 for the presence of Janus kinase 2, calreticulin, and myeloproliferative leukemia mutations. Of 344 primary myelofibrosis patients, Janus kinase 2 V617F was detected in 226 (66%), calreticulin mutation in 43 (12%), and myeloproliferative leukemia mutation in 16 (5%); 59 patients (17%) were triple-negatives. A 50% cut-off dichotomized Janus kinase 2-mutated patients into those with high Janus kinase 2 V617F allele burden and favorable survival and those with low Janus kinase 2 V617F allele burden and unfavorable survival. Patients with a favorable mutation status (high Janus kinase 2 V617F allele burden/myeloproliferative leukemia/calreticulin mutation) and aged 65 years or under had a median survival of 126 months. Patients with one risk factor (low Janus kinase 2 V617F allele burden/triple-negative or age >65 years) had an intermediate survival duration, and patients aged over 65 years with an adverse mutation status (low Janus kinase 2 V617F allele burden or triple-negative) had a median survival of only 35 months. Our simple and easily applied age- and mutation status-based scoring system accurately predicted the survival of patients with primary myelofibrosis. Copyright© Ferrata Storti Foundation.

Extending Jak2V617F and MplW515 mutation analysis to single hematopoietic colonies and B and T lymphocytes.

PubMed

Pardanani, Animesh; Lasho, Terra L; Finke, Christy; Mesa, Ruben A; Hogan, William J; Ketterling, Rhett P; Gilliland, Dwight Gary; Tefferi, Ayalew

2007-09-01

JAK2V617F and MPLW515L/K are myeloproliferative disorder (MPD)-associated mutations. We genotyped 552 individual hematopoietic colonies obtained by CD34+ cell culture from 16 affected patients (13 JAK2V617F and 3 MPLW515L/K) to determine (a) the proportion of colonies harboring a particular mutation in the presence or absence of cytokines, (b) the lineage distribution of endogenous colonies for each mutation, and (c) the differences (if any) in the pattern of mutation among the various MPDs, as established by genotyping of individual colonies. Genotyping analysis revealed cohabitation of mutation-negative and mutation-positive endogenous colonies in polycythemia vera as well as other MPDs. Culture of progenitor cells harboring MPLW515L/K yielded virtually no endogenous erythroid colonies in contrast to JAK2V617F-harboring progenitor cells. The mutation pattern (i.e., relative distribution of homozygous, heterozygous, or wild-type colonies) was not a distinguishing feature among the MPDs, and MPLW515 mutations were detected in B and/or T lymphocytes in all three patients tested. These observations suggest that clonal myelopoiesis antedates acquisition of JAK2V617F or MPLW515L/K mutations and that the latter is acquired in a lympho-myeloid progenitor cell.

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

PubMed

Misawa, Kyohei; Yasuda, Hajime; Araki, Marito; Ochiai, Tomonori; Morishita, Soji; Shirane, Shuichi; Edahiro, Yoko; Gotoh, Akihiko; Ohsaka, Akimichi; Komatsu, Norio

2018-06-01

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.

MPL W515L/K Mutations in Chronic Myeloproliferative Neoplasms.

PubMed

Akpınar, Timur Selçuk; Hançer, Veysel Sabri; Nalçacı, Meliha; Diz-Küçükkaya, Reyhan

2013-03-01

The MPL gene encodes the thrombopoietin receptor. Recently MPL mutations (MPL W515L or MPL W515K) were described in patients with essential thrombocythemia (ET) and primary (idiopathic) myelofibrosis (PMF). The prevalence and the clinical importance of these mutations are not clear. In the present study, we aimed to investigate the frequency and clinical significance of MPL W515L/K mutations in our patients with ET and PMF. A total of 77 patients (66 were diagnosed with ET and 11 with PMF) and 42 healthy controls were included in the study. Using peripheral blood samples, the presence of MPL W515L/K mutations and JAK-2 V617F mutation were analyzed by real-time polymerase chain reaction. In our study, MPL W515L/K or JAK-2 V617F mutations were not observed in healthy controls. JAK-2 V617F mutation was present in 35 patients, of whom 29 had ET (43.9%, 29/66) and 6 had PMF (54.5%, 6/11). In the patient group, MPL W515L/K mutations were found in only 2 PMF cases, and these cases were negative for JAK-2 V617F mutation. The prevalence of MPL W515L/K mutations in the patient group was 2.6%, and the prevalence of MPL W515L/K mutations among the cases negative for the JAK-2 V617F mutation was found to be 4.8%. The 2 cases with MPL W515L/K mutations had long follow-up times (124 months and 71 months, respectively), had no thrombotic or hemorrhagic complications, and had no additional cytogenetic anomalies. MPL W515L/K mutations may be helpful for identifying clonal disease in MPN patients with no established Ph chromosome or JAK-2 V617F mutation. None declared.

JAK2 V617F, MPL, and CALR mutations in essential thrombocythaemia and major thrombotic complications: a single-institute retrospective analysis.

PubMed

Pósfai, Éva; Marton, Imelda; Király, Péter Attila; Kotosz, Balázs; Kiss-László, Zsuzsanna; Széll, Márta; Borbényi, Zita

2015-07-01

Thrombo-haemorrhagic events are the main cause of morbidity and mortality in essential thrombocythemia. The aim of this study was to estimate the incidence of thrombotic events and the impact of the JAK2V617F, MPL (W515L, W515K, W515R, W515A and S505N) and CALR (type-1, type-2) mutations on 101 essential thrombocythaemia patients (72 females and 29 males with a mean age of 61 years) diagnosed in a Southern Hungarian regional academic centre. The incidence of major thrombosis was 13.86 %. Sixty percent of the patients carried the JAK2V617F mutation. The MPL mutations were analysed by sequencing and the W515L was the only one we could identify with an incidence of 3.96 %. Type-2 CALR mutation could be identified in 3 cases among the patients who had JAK2/MPL-unmutated ET. Statistical analyses revealed that the JAK2V617F mutation was associated with significantly increased levels of platelet (p = 0.042), haemoglobin (p = 0.000), red blood cell (p = 0.000) and haematocrit (p = 0.000) and hepatomegaly (p = 0.045) at diagnosis compared to JAK2V617F negative counterparts, however there was no significant association between the JAK2V617F mutation status (relative risk: 1.297, 95 % CI 0.395-4.258; p = 0.668) and subsequent thrombotic complications. The impact of JAK2V617F, MPL W515L and CALR mutations on the clinical findings at the diagnosis of ET was obvious, but their statistically significant role in the prediction of thrombotic events could not be proven in this study. Our results indirectly support the concept that, besides the quantitative and qualitative changes in the platelets, the mechanisms leading to thrombosis are more complex and multifactorial.

Splanchnic Vein Thrombosis in the Myeloproliferative Neoplasms.

PubMed

Akpan, Imo J; Stein, Brady Lee

2018-06-01

To review the epidemiology, diagnostic challenges, pathogenesis, and treatment strategies for patients with myeloproliferative neoplasm-associated splanchnic vein thrombosis. The epidemiology of myeloproliferative neoplasm-associated splanchnic vein thrombosis (MPN-SVT) has been well characterized. While typical MPN-associated thrombosis affects older patients and involves the arterial circulation, MPN-SVT mostly impacts younger women. An association with JAK2 V617F is well-known; recent studies have demonstrated only a weak association with CALR mutations. JAK inhibition may represent a novel treatment strategy, complementing anticoagulation, and management of portal hypertension. While the epidemiology has been well characterized, more work is needed to identify novel contributors to disease pathogenesis, beyond the JAK2 V617F mutation itself, and endothelial compromise. Testing for MPN mutations in the setting of non-cirrhotic SVT is commonplace; JAK2 V617F is the most likely to be identified. Testing for CALR or MPL mutations requires clinical judgement, though not unreasonable. The mainstay of therapy is indefinite anticoagulation; the role of direct oral anticoagulants is unclear. JAK inhibition may play a role in addressing associated splenomegaly and portal hypertension.

Hmga2 promotes the development of myelofibrosis in Jak2V617F knockin mice by enhancing TGF-β1 and Cxcl12 pathways.

PubMed

Dutta, Avik; Hutchison, Robert E; Mohi, Golam

2017-08-17

Myelofibrosis (MF) is a devastating blood disorder. The JAK2V617F mutation has been detected in ∼50% cases of MF. Elevated expression of high-mobility group AT hook 2 (HMGA2) has also been frequently observed in patients with MF. Interestingly, upregulation of HMGA2 expression has been found in association with the JAK2V617F mutation in significant cases of MF. However, the contribution of HMGA2 in the pathogenesis of MF remains elusive. To determine the effects of concurrent expression of HMGA2 and JAK2V617F mutation in hematopoiesis, we transduced bone marrow cells from Jak2 V617F knockin mice with lentivirus expressing Hmga2 and performed bone marrow transplantation. Expression of Hmga2 enhanced megakaryopoiesis, increased extramedullary hematopoiesis, and accelerated the development of MF in mice expressing Jak2 V617F Mechanistically, the data show that expression of Hmga2 enhances the activation of transforming growth factor-β1 (TGF-β1) and Cxcl12 pathways in mice expressing Jak2 V617F In addition, expression of Hmga2 causes upregulation of Fzd2, Ifi27l2a, and TGF-β receptor 2. Forced expression of Cxcl12, Fzd2, or Ifi27l2a increases megakaryocytic differentiation and proliferation in the bone marrow of Jak2 V617F mice, whereas TGF-β1 or Cxcl12 stimulation induces collagen deposition in the bone marrow mesenchymal stromal cells. Together, these findings demonstrate that expression of Hmga2 cooperates with Jak2 V617F in the pathogenesis of MF. © 2017 by The American Society of Hematology.

Detection of MPL exon10 mutations in 103 Chinese patients with JAK2V617F-negative myeloproliferative neoplasms.

PubMed

Chen, Xiuhua; Qi, Xiling; Tan, Yanhong; Xu, Zhifang; Xu, Aining; Zhang, Linlin; Wang, Hongwei

2011-06-15

JAK2V617F mutation has been reported in 90% of patients with polycythemia vera (PV) and about 50% of patients with essential thromobocythemia (ET) and primary myelofibrosis (PMF). Recently, acquired mutations in the transmembrane-juxtamembrane region of MPL (MPLW515 mutations) have been reported in approximately 5% of JAK2V617F-negative PMF and about 1% of all cases of ET. MPL is the receptor for thrombopoietin that regulates the production of platelets by bone marrow. It is likely that some mutations more closely related to ET in MPL exon10 may have been missed by current assays. We inferred that there might be other mutations in MPL exon10 for MPN patients in addition to MPLW515 mutations. To investigate its mutation types and prevalence in Chinese patients with myeloproliferative neoplasms (MPN), we performed mutation detection on MPL exon10 in 103 JAK2V617F-negative MPN patients by single strand conformation polymorphism (SSCP) and allele-specific PCR (AS-PCR) combined with sequencing. As a result, one previously unrecognized MPL mutation (12-bp in-frame insertion) was identified in one patient with ET in addition to an MPLW515K mutation identified in one PMF patient. This confirms our hypothesis that BCR/ABL negative and JAK2V617F-negative MPN patients have other mutations besides W515 mutation in MPL exon10 and mutations other than single nucleotide exchange also exist. In addition, MPL mutation was associated with Chinese MPN patients. Copyright © 2011 Elsevier Inc. All rights reserved.

Oncogenic JAK2V617F requires an intact SH2-like domain for constitutive activation and induction of a myeloproliferative disease in mice.

PubMed

Gorantla, Sivahari P; Dechow, Tobias N; Grundler, Rebekka; Illert, Anna Lena; Zum Büschenfelde, Christian Meyer; Kremer, Marcus; Peschel, Christian; Duyster, Justus

2010-11-25

The oncogenic JAK2V617F mutation is found in myeloproliferative neoplasms (MPNs) and is believed to be critical for leukemogenesis. Here we show that JAK2V617F requires an intact SH2 domain for constitutive activation of downstream signaling pathways. In addition, there is a strict requirement of cytokine receptor expression for the activation of this oncogene. Further analysis showed that the SH2 domain mutation did not interfere with JAK2 membrane distribution. However, coimmunoprecipitated experiments revealed a role for the SH2 domain in the aggregation and cross-phosphorylation of JAK2V617F at the cell membrane. Forced overexpression of cytokine receptors could rescue the JAK2V617F SH2 mutant supporting a critical role of JAK2V617F abundance for constitutive activation. However, under physiologic cytokine receptor expression the SH2 domain is absolutely necessary for oncogenic JAK2V617F activation. This is demonstrated in a bone marrow transplantation model, in which an intact SH2 domain in JAK2V617F is required for the induction of an MPN-like disease. Thus, our results points to an indispensable role of the SH2 domain in JAK2V617F-induced MPNs.

MPL mutation profile in JAK2 mutation-negative patients with myeloproliferative disorders.

PubMed

Ma, Wanlong; Zhang, Xi; Wang, Xiuqiang; Zhang, Zhong; Yeh, Chen-Hsiung; Uyeji, Jennifer; Albitar, Maher

2011-03-01

Mutations in the thrombopoietin receptor gene (myeloproliferative leukemia, MPL) have been reported in patients with JAK2 V617F-negative chronic myeloproliferative disorders (MPDs). We evaluated the prevalence of MPL mutations relative to JAK2 mutations in patients with suspected MPDs. A total of 2790 patient samples submitted for JAK2 mutation analysis were tested using real-time polymerase chain reaction and bidirectional sequencing of plasma RNA. JAK2 V617F-negative samples were tested for JAK2 exons 12 to 14 mutations, and those with negative results were then tested for mutations in MPL exons 10 and 11. Of the 2790 patients, 529 (18.96%) had V617F, 12 (0.43%) had small insertions or deletions in exon 12, and 7 (0.25%) had other JAK2 mutations in exons 12 to 14. Of the 2242 JAK2 mutation-negative patients, 68 (3.03%) had MPL mutations. W515L was the predominant MPL mutation (n=46; 68%), and 10 (15%) patients had other W515 variants. The remaining MPL mutations (n=12, 17%) were detected at other locations in exons 10 and 11 and included 3 insertion/deletion mutations. The S505N mutation, associated with familial MPD, was detected in 3 patients. Overall, for every 100 V617F mutations in patients with suspected MPDs, there were 12.9 MPL mutations, 2.3 JAK2 exon 12 mutations, and 1.3 JAK2 exons 13 to 14 mutations. These findings suggest that MPL mutation screening should be performed before JAK2 exons 12 to 14 testing in JAK2 V617F-negative patients with suspected MPDs.

Concurrent MPL515 and JAK2V617F mutations in myelofibrosis: chronology of clonal emergence and changes in mutant allele burden over time.

PubMed

Lasho, Terra L; Pardanani, Animesh; McClure, Rebecca F; Mesa, Ruben A; Levine, Ross L; Gilliland, D Gary; Tefferi, Ayalew

2006-12-01

MPLW515L/K and JAK2V617F can co-exist in myelofibrosis with myeloid metaplasia (MMM). The chronology of clonal emergence was studied in three such cases using serially stored bone marrow. At diagnosis, a major MPL515 mutant clone was accompanied by a minor JAK2V617F clone in all three instances. At 25 time points over a period of 4-8 years, allele burden fluctuated but remained high for MPLW515L/K and low for JAK2V617F. We conclude that MPLW515L/K and JAK2V617F are both early events in MMM and allele burden, rather than the mere presence of these mutations, might be relevant to phenotypic variation in myeloproliferative disorders.

The JAK2 46/1 haplotype predisposes to MPL-mutated myeloproliferative neoplasms

PubMed Central

Jones, Amy V.; Campbell, Peter J.; Beer, Philip A.; Schnittger, Susanne; Vannucchi, Alessandro M.; Zoi, Katerina; Percy, Melanie J.; McMullin, Mary Frances; Scott, Linda M.; Tapper, William; Silver, Richard T.; Oscier, David; Harrison, Claire N.; Grallert, Harald; Kisialiou, Aliaksei; Strike, Paul; Chase, Andrew J.; Green, Anthony R.

2010-01-01

The 46/1 JAK2 haplotype predisposes to V617F-positive myeloproliferative neoplasms, but the underlying mechanism is obscure. We analyzed essential thrombocythemia patients entered into the PT-1 studies and, as expected, found that 46/1 was overrepresented in V617F-positive cases (n = 404) versus controls (n = 1492, P = 3.9 × 10−11). The 46/1 haplotype was also overrepresented in cases without V617F (n = 347, P = .009), with an excess seen for both MPL exon 10 mutated and V617F, MPL exon 10 nonmutated cases. Analysis of further MPL-positive, V617F-negative cases confirmed an excess of 46/1 (n = 176, P = .002), but no association between MPL mutations and MPL haplotype was seen. An excess of 46/1 was also seen in JAK2 exon 12 mutated cases (n = 69, P = .002), and these mutations preferentially arose on the 46/1 chromosome (P = .029). No association between 46/1 and clinical or laboratory features was seen in the PT-1 cohort either with or without V617F. The excess of 46/1 in JAK2 exon 12 cases is compatible with both the “hypermutability” and “fertile ground” hypotheses, but the excess in MPL-mutated cases argues against the former. No difference in sequence, splicing, or expression of JAK2 was found on 46/1 compared with other haplotypes, suggesting that any functional difference of JAK2 on 46/1, if it exists, must be relatively subtle. PMID:20304805

The role of serum erythropoietin level and JAK2 V617F allele burden in the diagnosis of polycythaemia vera.

PubMed

Ancochea, Agueda; Alvarez-Larrán, Alberto; Morales-Indiano, Cristian; García-Pallarols, Francesc; Martínez-Avilés, Luz; Angona, Anna; Senín, Alicia; Bellosillo, Beatriz; Besses, Carles

2014-11-01

Low serum erythropoietin (EPO) is a minor criterion of Polycythaemia Vera (PV) but its diagnostic usefulness relies on studies performed before the discovery of JAK2 V617F mutation. The objective of the present study was to evaluate the diagnostic accuracy of serum EPO and JAK2 V617F allele burden as markers of PV as well as the combination of different diagnostic criteria in 287 patients (99 with PV, 137 with Essential Thrombocythaemia and 51 with non-clonal erythrocytosis). Low EPO showed good diagnostic accuracy as a marker for PV, with the area under the curve (AUC) of the chemiluminescent-enhanced enzyme immunoassay (CEIA) being better than that of radioimmunoassay (RIA) (0·87 and 0·76 for CEIA and RIA, respectively). JAK2 V617F quantification displayed an excellent diagnostic accuracy, with an AUC of 0·95. A haematocrit >52% (males) or >48% (females) plus the presence of the JAK2 V617F mutation had a sensitivity and specificity of 79% and 97%, respectively. Adding low EPO or the JAK2 V617F allele burden did not improve the diagnostic accuracy for PV whereas the inclusion of both improved the sensitivity up to 83% and maintaining 96% specificity. Haematocrit and qualitative JAK2 V617F mutation allow a reliable diagnosis of PV. Incorporation of EPO and/or JAK2 V617F mutant load does not improve the diagnostic accuracy. © 2014 John Wiley & Sons Ltd.

Vascular events in Korean patients with myeloproliferative neoplasms and their relationship to JAK2 mutation.

PubMed

Bang, Soo-Mee; Lee, Jong-Seok; Ahn, Jeong Yeal; Lee, Jae Hoon; Hyun, Myung Soo; Kim, Bong Seog; Park, Moo Rim; Chi, Hyun-Sook; Kim, Ho Young; Kim, Hyo Jung; Lee, Moon Hee; Kim, Hwak; Won, Jong Ho; Yoon, Hwi Joong; Oh, Do-Yeun; Nam, Eun-Mi; Bae, Sung Hwa; Kim, Byoung-Kook

2009-03-01

Evaluation of the Janus kinase 2 (JAK2) V617F mutation has been widely used for the diagnosis of myeloproliferative neoplasms (MPN). However, its prognostic relevance to clinical outcome is not completely understood. We investigated the association of JAK2 V617F with vascular events in Korean patients with myeloproliferative neoplasms (MPN). We studied 283 patients from 15 centers, who were diagnosed with MPN. The JAK2 V617F status was evaluated by allele-specific polymerase chain reaction (PCR) and sequencing. The patients' diagnoses were essential thrombocythemia (ET n = 146), polycythemia vera (PV n = 120), primary myelofibrosis (n = 12), and unclassifiable MPN (MPNu n = 5). JAK2 V617F was detected in 89 (61%) patients with ET, 103 (86%) with PV, four (33%) with myelofibrosis, and four (80%) with MPNu. A higher number of leukocytes, haemoglobin levels and BM cellularity as well as an older age, lower platelet counts, and diagnosis of PV were significantly correlated with JAK2 V617F. Eighty-three and 43 episodes of thrombosis and bleeding occurred in 100 patients each before and after the diagnosis. Vascular events more frequently occurred in 37% of patients with JAK2 V617F than in 29% of those without the mutation (p = 0.045). Among 175 patients whose samples were available for sequencing, 28 patients with homozygous JAK2 V617F had vascular events more frequently (57%) than those who were heterozygotes (39%) or had the wild type (27%) (p = 0.03). The multivariate analysis showed that a JAK2 homozygous mutation, hypercholesterolemia and older age were independent risk factors for a vascular event. The results of this study showed that Korean patients with MPN had a similar JAK2 mutation rate and frequency of vascular events when compared to Western patients. The presence of V617F was significantly related to vascular events. Therefore, initial evaluation for the JAK2 mutation and careful monitoring for vascular events should be performed in MPN patients.

[MPLW515L point mutation in patients with myeloproliferative disease].

PubMed

Xia, Jun; Xu, Wei; Zhang, Su-Jiang; Fan, Lei; Qiao, Chun; Li, Jian-Yong

2008-12-01

In order to investigate the frequency of MPLW515L and JAK2V617F point mutations of the patients with myeloproliferative disease (MPD) in Nanjing area, MPLW515L and JAK2V617F point mutations were simultaneously detected by alleles specific polymerase chain reaction (AS-PCR) and sequencing in 190 MPD patients. The results showed that MPLW515L point mutation was detected in 1 out of 102 essential thrombocythemia (ET) patients (1.0%) and was not detected in 32 polycythemia vera (PV) patients, 13 idiopathic myelofibrosis (IMF) patients, 43 chronic myelogenous leukemia (CML) patients. JAK2V617F point mutation was detected in 20 out of 32 PV patients (62.5%), 43 out of 102 ET patients (42.2%), 5 out of 13 IMF patients (38.5%), and was not detected in 43 CML patients. It is concluded that MPLW515L point mutation exists in ET patient, but is not found in PV, IMF and CML. JAK2V617F point mutation exists in PV, ET and IMF, but not in CML.

Clinical Implications of Quantitative JAK2 V617F Analysis using Droplet Digital PCR in Myeloproliferative Neoplasms

PubMed Central

Lee, Eunyoung; Lee, Kyoung Joo; Park, Hyein; Chung, Jin Young; Lee, Mi-Na; Chang, Myung Hee; Yoo, Jongha; Lee, Hyewon

2018-01-01

Background JAK2 V617F is the most common mutation in myeloproliferative neoplasms (MPNs) and is a major diagnostic criterion. Mutation quantification is useful for classifying patients with MPN into subgroups and for prognostic prediction. Droplet digital PCR (ddPCR) can provide accurate and reproducible quantitative analysis of DNA. This study was designed to verify the correlation of ddPCR with pyrosequencing results in the diagnosis of MPN and to investigate clinical implications of the mutational burden. Methods Peripheral blood or bone marrow samples were obtained from 56 patients newly diagnosed with MPN or previously diagnosed with MPN but not yet indicated for JAK2 inhibitor treatment between 2012 and 2016. The JAK2 V617F mutation was detected by pyrosequencing as a diagnostic work-up. The same samples were used for ddPCR to determine the correlation between assays and establish a detection sensitivity cut-off. Clinical and hematologic aspects were reviewed. Results Forty-two (75%) and 46 (82.1%) patients were positive for JAK2 V617F by pyrosequencing and ddPCR, respectively. The mean mutated allele frequency at diagnosis was 37.5±30.1% and was 40.7±31.2% with ddPCR, representing a strong correlation (r=0.9712, P<0.001). Follow-up samples were available for 12 patients, including eight that were JAK2 V617F-positive. Of these, mutational burden reduction after treatment was observed in six patients (75%), consistent with trends of hematologic improvement. Conclusions Quantitative analysis of the JAK2 V617F mutation using ddPCR was highly correlated with pyrosequencing data and may reflect the clinical response to treatment. PMID:29214759

Different expression patterns of LGALS1 and LGALS3 in polycythemia vera, essential thrombocythemia and primary myelofibrosis.

PubMed

Moura, L G; Tognon, R; Nunes, N S; Rodrigues, L Cataldi; Ferreira, A F; Kashima, S; Covas, D T; Santana, M; Souto, E X; Perobelli, L; Simões, B P; Dias-Baruffi, M; Castro, F A

2016-10-01

Despite all the knowledge, the cellular and molecular mechanisms involved in myeloproliferative neoplasm (MPN) pathophysiology remain unclear. Authors have shown galectin-1 (Gal-1) and 3 playing roles in tumour angiogenesis and fibrosis, which were correlated with poor prognosis in patients with MPN. In the present study LGALS1 and LGALS3 were differently expressed between polycythemia vera, essential thrombocythemia (ET) and primary myelofibrosis (PMF) diseases. Increased LGALS3 expression was associated with a negative JAK2 V617F status mutation in leucocytes from PMF but not in patients with ET without this mutation. However, a positive Janus kinase 2 (JAK2) V617F cell line established from patients with ET (SET-2 cells) when treated with JAK inhibitor presented high levels of LGALS3. Additionally, high LGALS1 expression was found in CD34(+) cells but not in leucocytes from patients with PMF, in absence of JAK2 V617F mutation, and also in SET-2 cells treated with JAK inhibitor. Thus, our findings indicate that differential expression of LGALS1 and/or LGALS3 in patients with MPN is linked with JAK2 V617F status mutation in these diseases and state of cell differentiation. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

Driver mutations (JAK2V617F, MPLW515L/K or CALR), pentraxin-3 and C-reactive protein in essential thrombocythemia and polycythemia vera.

PubMed

Lussana, Federico; Carobbio, Alessandra; Salmoiraghi, Silvia; Guglielmelli, Paola; Vannucchi, Alessandro Maria; Bottazzi, Barbara; Leone, Roberto; Mantovani, Alberto; Barbui, Tiziano; Rambaldi, Alessandro

2017-02-22

The driver mutations JAK2V617F, MPLW515L/K and CALR influence disease phenotype of myeloproliferative neoplasms (MPNs) and might sustain a condition of chronic inflammation. Pentraxin 3 (PTX3) and high-sensitivity C-reactive protein (hs-CRP) are inflammatory biomarkers potentially useful for refining prognostic classification of MPNs. We evaluated 305 with essential thrombocythemia (ET) and 172 polycythemia vera (PV) patients diagnosed according to the 2016 WHO criteria and with full molecular characterization for driver mutations. PTX3 levels were significantly increased in carriers of homozygous JAK2V617F mutation compared to all the other genotypes and triple negative ET patients, while hs-CRP levels were independent of the mutational profile. The risk of haematological evolution and death from any cause was about 2- and 1.5-fold increased in individuals with high PTX-3 levels, while the thrombosis rate tended to be lower. High hs-CRP levels were associated with risk of haematological evolution, death and also major thrombosis. After sequential adjustment for potential confounders (age, gender, diagnosis and treatments) and the presence of JAK2V617F homozygous status, high hs-CRP levels remained significant for all outcomes, while JAK2V617F homozygous status as well as treatments were the factors independently accounting for adverse outcomes among patients with high PTX3 levels. These results provide evidence that JAK2V617F mutation influences MPN-associated inflammation with a strong correlation between allele burden and PTX3 levels. Plasma levels of hs-CRP and PTX3 might be of prognostic value for patients with ET and PV, but their validation in future prospective studies is needed.

Telomere length is severely and similarly reduced in JAK2V617F-positive and -negative myeloproliferative neoplasms

PubMed Central

Bernard, L; Belisle, C; Mollica, L; Provost, S; Roy, D-C; Gilliland, DG; Levine, RL; Busque, L

2015-01-01

Myeloproliferative neoplasms (MPNs) are clonal stem cell disorders characterized by chronic proliferation of hematopoietic progenitors. We studied the telomere length (TL) of 335 MPN patients and 93 gender- and age-matched controls using a quantitative PCR method (relative TL calculated as the ratio of the amount of telomere DNA vs single-copy DNA: T/S ratio). TL was markedly reduced in MPN patients compared with controls (T/S 0.561 vs 0.990, P<0.001). In JAK2V617F MPN patients, TL correlated inversely with allelic burden (P<0.001). Patients homozygous for the mutation (allelic burden 90–100%) had the shortest TL, even when compared with patients with lower allele burdens consistent with a dominant heterozygous population (allelic burden 55–65%) (T/S 0.367 vs 0.497, P = 0.037). This suggests that the high degree of proliferation of the MPN clone reduces TL and suggests the possibility that TL shortening may be indicative of progressive genomic instability during MPN progression. The TL of JAK2V617F-negative MPN patients was similar to JAK2V617F-positive counterparts (T/S 0.527 vs 0.507, P = 0.603), suggesting that the yet-to-be-discovered causative mutation(s) impact the mutated stem cell similarly to JAK2V617F, and that TL measurement may prove useful in the diagnostic workup of JAK2V617F-negative MPN. PMID:19005480

The development of gastric cancer in a patient with polycythemia Vera, 3P deletion, and JAK2 V617F mutation.

PubMed

Ayvaz, Ozlem; Yavasoglu, Irfan; Kadikoylu, Gurhan; Meydan, Nezih; Barutca, Sabri; Bolaman, Zahit

2010-12-01

3p deletion which is frequently associated with solitary tumors and hematological malignancies is a chromosomal abnormality. Recently, Janus kinase-2 (JAK2) V617F mutation has an important role in the diagnosis of myeloproliferative disorders, especially in polycythemia vera (PV). We reported the development of gastric cancer in a 75-year-old patient with PV, 3p 12-14 deletion and JAK2 V617F mutation. PV was diagnosed according to the classification of World Health Organization. JAK2 V617F mutation with polymerase chain reaction and 3p12-14 deletion with cytogenetic examination of the bone marrow were detected. We investigated solitary tumors in the patient using computed tomographies of thorax, neck, ear, nose, and throat. However, they were normal. After 2 years, gastric cancer appeared in the patient. In conclusion, cytogenetic examination may be important in both the development and the diagnosis of hematological malignancies and solitary tumors. So the patients should be followed closely.

[High resolution melting analysis for detecting of JAK2V617F mutation in patients with myeloproliferative neoplasms].

PubMed

Chen, Hai-Hua; Yang, Ji-Long; Lu, Hui-Fang; Zhou, Wei-Jun; Yao, Fei; Deng, Lan

2014-02-01

This study was purposed to investigate the feasibility of high resolution melting (HRM) in the detection of JAK2V617F mutation in patients with myeloproliferative neoplasm (MPN). The 29 marrow samples randomly selected from patients with clinically diagnosed MPN from January 2008 to January 2011 were detected by HRM method. The results of HRM analysis were compared with that detected by allele specific polymerase chain reaction (AS-PCR) and DNA direct sequencing. The results showed that the JAK2V617F mutations were detected in 11 (37.9%, 11/29) cases by HRM, and its comparability with the direct sequencing result was 100%. While the consistency of AS-PCR with the direct sequencing was moderate (Kappa = 0.179, P = 0.316). It is concluded that the HRM analysis may be an optimal method for clinical screening of JAK2V617F mutation due to its simplicity and promptness with a high specificity.

The JAK2V617 mutation induces constitutive activation and agonist hypersensitivity in basophils from patients with polycythemia vera

PubMed Central

Pieri, Lisa; Bogani, Costanza; Guglielmelli, Paola; Zingariello, Maria; Rana, Rosa Alba; Bartalucci, Niccolò; Bosi, Alberto; Vannucchi, Alessandro M.

2009-01-01

Background The JAK2V617F mutation has been associated with constitutive and enhanced activation of neutrophils, while no information is available concerning other leukocyte subtypes. Design and Methods We evaluated correlations between JAK2V617F mutation and the count of circulating basophils, the number of activated CD63+ basophils, their response in vitro to agonists as well as the effects of a JAK2 inhibitor. Results We found that basophil count was increased in patients with JAK2V617F -positive myeloproliferative neoplasms, particularly in those with polycythemia vera, and was correlated with the V617F burden. The burden of V617F allele was similar in neutrophils and basophils from patients with polycythemia vera, while total JAK2 mRNA content was remarkably greater in the basophils; however, the content of JAK2 protein in basophils was not increased. The number of CD63+ basophils was higher in patients with polycythemia vera than in healthy subjects or patients with essential thrombocythemia or primary myelofibrosis and was correlated with the V617F burden. Ultrastructurally, basophils from patients with polycythemia vera contained an increased number of granules, most of which were empty suggesting cell degranulation in vivo. Ex vivo experiments revealed that basophils from patients with polycythemia vera were hypersensitive to the priming effect of interleukin-3 and to f-MLP-induced activation; pre-treatment with a JAK2 inhibitor reduced polycythemia vera basophil activation. Finally, we found that the number of circulating CD63+ basophils was significantly greater in patients suffering from aquagenic pruritus, who also showed a higher V617F allele burden. Conclusions These data indicate that the number of constitutively activated and hypersensitive circulating basophils is increased in polycythemia vera, underscoring a role of JAK2V617F in these cells’ abnormal function and, putatively, in the pathogenesis of pruritus. PMID:19608683

Myeloproliferative neoplasms: Current molecular biology and genetics.

PubMed

Saeidi, Kolsoum

2016-02-01

Myeloproliferative neoplasms (MPNs) are clonal disorders characterized by increased production of mature blood cells. Philadelphia chromosome-negative MPNs (Ph-MPNs) consist of polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). A number of stem cell derived mutations have been identified in the past 10 years. These findings showed that JAK2V617F, as a diagnostic marker involving JAK2 exon 14 with a high frequency, is the best molecular characterization of Ph-MPNs. Somatic mutations in an endoplasmic reticulum chaperone, named calreticulin (CALR), is the second most common mutation in patients with ET and PMF after JAK2 V617F mutation. Discovery of CALR mutations led to the increased molecular diagnostic of ET and PMF up to 90%. It has been shown that JAK2V617F is not the unique event in disease pathogenesis. Some other genes' location such as TET oncogene family member 2 (TET2), additional sex combs-like 1 (ASXL1), casitas B-lineage lymphoma proto-oncogene (CBL), isocitrate dehydrogenase 1/2 (IDH1/IDH2), IKAROS family zinc finger 1 (IKZF1), DNA methyltransferase 3A (DNMT3A), suppressor of cytokine signaling (SOCS), enhancer of zeste homolog 2 (EZH2), tumor protein p53 (TP53), runt-related transcription factor 1 (RUNX1) and high mobility group AT-hook 2 (HMGA2) have also identified to be involved in MPNs phenotypes. Here, current molecular biology and genetic mechanisms involved in MNPs with a focus on the aforementioned factors is presented. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Cytogenetics, JAK2 and MPL mutations in polycythemia vera, primary myelofibrosis and essential thrombocythemia.

PubMed

Dos Santos, Leonardo Caires; Ribeiro, Juliana Corrêa da Costa; Silva, Neusa Pereira; Cerutti, Janete; da Silva, Maria Regina Regis; Chauffaille, Maria de Lourdes Lopes Ferrari

2011-01-01

The detection of molecular and cytogenetic alterations is important for the diagnosis, prognosis and classification of myeloproliferative neoplasms. THE AIM OF THIS STUDY WAS TO DETECT THE FOLLOWING MUTATIONS: JAK2 V617F, JAK2 exon 12 and MPL W515K/L, besides chromosomal abnormalities. Furthermore, molecular and cytogenetic alterations were correlated with the leukocyte and platelet counts, hemoglobin levels and age in all patients and with the degree of fibrosis in primary myelofibrosis cases. Twenty cases of polycythemia vera, 17 of essential thrombocythemia and 21 of primary myelofibrosis were selected in the Hematology Department of the Universidade Federal de São Paulo (UNIFESP) between February 2008 and December 2009. The JAK2 V617F, JAK2 exon 12 mutations, MPL W515K and MPL W515L mutations were investigated by real-time PCR and direct sequencing. G-band karyotyping and fluorescence in situ hybridization were used to detect chromosomal abnormalities. Chromosomal abnormalities were observed only in polycythemia vera (11.8%) and primary myelofibrosis cases (17.6%), without correlation to clinical data. Chromosomal abnormalities were not detected by fluorescence in situ hybridization. The JAK2 V617F mutation was observed in polycythemia vera (90%), primary myelofibrosis (42.8%) and essential thrombocythemia (47%). Patients with JAK2 V617F-negative polycythemia vera had lower platelet and leukocyte counts compared to V617F-positive polycythemia vera (p-value = 0.0001 and p-value = 0.023, respectively). JAK2 V617F-positive and MPL W515L-positive primary myelofibrosis cases had a higher degree of fibrosis than V617F-negative cases (p-value = 0.022). JAK2 exon 12 mutations were not detected in polycythemia vera patients. The MPL W515L mutation was observed in one case of primary myelofibrosis and in one of essential thrombocythemia. The MPL W515K mutation was not found in patients with essential thrombocythemia or primary myelofibrosis. The MPL W515L-positive patient with primary myelofibrosis had more severe anemia than other patients with primary myelofibrosis. This study demonstrates that karyotyping for JAK2 and MPL mutations is useful in the diagnosis of myeloproliferative neoplasms. The precise pathogenetic contribution of these alterations is still unclear. However, this study adds more information about the pathophysiology of polycythemia vera, essential thrombocythemia and primary myelofibrosis.

Development and inter-laboratory validation of unlabeled probe melting curve analysis for detection of JAK2 V617F mutation in polycythemia vera.

PubMed

Wu, Zhiyuan; Yuan, Hong; Zhang, Xinju; Liu, Weiwei; Xu, Jinhua; Zhang, Wei; Guan, Ming

2011-01-01

JAK2 V617F, a somatic point mutation that leads to constitutive JAK2 phosphorylation and kinase activation, has been incorporated into the WHO classification and diagnostic criteria of myeloid neoplasms. Although various approaches such as restriction fragment length polymorphism, amplification refractory mutation system and real-time PCR have been developed for its detection, a generic rapid closed-tube method, which can be utilized on routine genetic testing instruments with stability and cost-efficiency, has not been described. Asymmetric PCR for detection of JAK2 V617F with a 3'-blocked unlabeled probe, saturate dye and subsequent melting curve analysis was performed on a Rotor-Gene® Q real-time cycler to establish the methodology. We compared this method to the existing amplification refractory mutation systems and direct sequencing. Hereafter, the broad applicability of this unlabeled probe melting method was also validated on three diverse real-time systems (Roche LightCycler® 480, Applied Biosystems ABI® 7500 and Eppendorf Mastercycler® ep realplex) in two different laboratories. The unlabeled probe melting analysis could genotype JAK2 V617F mutation explicitly with a 3% mutation load detecting sensitivity. At level of 5% mutation load, the intra- and inter-assay CVs of probe-DNA heteroduplex (mutation/wild type) covered 3.14%/3.55% and 1.72%/1.29% respectively. The method could equally discriminate mutant from wild type samples on the other three real-time instruments. With a high detecting sensitivity, unlabeled probe melting curve analysis is more applicable to disclose JAK2 V617F mutation than conventional methodologies. Verified with the favorable inter- and intra-assay reproducibility, unlabeled probe melting analysis provided a generic mutation detecting alternative for real-time instruments.

Involvement of mast cells by the malignant process in patients with Philadelphia chromosome negative myeloproliferative neoplasms.

PubMed

Wang, J; Ishii, T; Zhang, W; Sozer, S; Dai, Y; Mascarenhas, J; Najfeld, V; Zhao, Z J; Hoffman, R; Wisch, N; Xu, M

2009-09-01

The Philadelphia chromosome negative myeloproliferative neoplasms (MPNs) are clonal hematologic malignancies frequently characterized by a mutation in JAK2 (JAK2V617F). Peripheral blood (PB) CD34(+) cells from patients with polycythemia vera (PV) and primary myelofibrosis (PMF) generated in vitro significantly fewer mast cells (MCs) than normal PB CD34(+) cells. The numbers of MC progenitors assayed from MPN CD34(+) cells were, however, similar to that assayed from normal CD34(+) cells. A higher percentage of the cultured MPN MCs expressed FcvarepsilonRIalpha, CD63 and CD69 than normal MCs, suggesting that cultured MPN MCs are associated with an increased state of MC activation. Further analysis showed that a higher proportion of cultured PV and PMF MCs underwent apoptosis in vitro. By using JAK2V617F, MplW515L and chromosomal abnormalities as clonality markers, we showed that the malignant process involved MPN MCs. JAK2V617F-positive MC colonies were assayable from the PB CD34(+) cells of each of the 17 JAK2V617F positive MPN patients studied. Furthermore, erlotinib, a JAK2 inhibitor, was able to inhibit JAK2V617F-positive PV MC progenitor cells, indicating that malignant MC progenitor cells are a potential cellular target for such JAK2 inhibitor-directed therapy.

The Prevalence of JAK2, MPL, and CALR Mutations in Chinese Patients With BCR-ABL1-Negative Myeloproliferative Neoplasms.

PubMed

Lin, Yani; Liu, Enbin; Sun, Qi; Ma, Jiao; Li, QingHua; Cao, Zeng; Wang, Jun; Jia, Yujiao; Zhang, Hongju; Song, Zhen; Ai, Xiaofei; Shi, Lihui; Feng, Xiaofang; Li, Chenwei; Wang, Jianxiang; Ru, Kun

2015-07-01

To evaluate the mutation frequency of JAK2 V617F, JAK2 exon 12, MPL exon 10, and CALR exon 9 and the value of the combined tests in the diagnosis of BCR-ABL1-negative myeloproliferative neoplasms (MPNs). In the current study, mutations of JAK2 V617F, JAK2 exon 12, MPL exon 10, and CALR exon 9 were analyzed in 929 Chinese patients with BCR-ABL1-negative MPN, including 234 cases of polycythemia vera (PV), 428 ETs, 187 PMFs, and 80 unclassifiable MPNs (MPN-Us). Our result showed that the positive rate of any of four mutations in patients with PV, ET, PMF, and MPN-U was 89.3%, 83.4%, 87.2%, and 77.5%, respectively, which significantly improved the diagnostic rate, especially in ET and PMF. Meanwhile, we also found that the patients without any of four mutations were younger than those with one or more mutations. Unexpectedly, the coexistence of JAK2 V617F and CALR exon 9 was identified in six (0.6%) patients, and JAK2 V617F and MPL exon 10 were present simultaneously in two (0.2%) patients. In addition, we also identified several novel mutation types in CALR exon 9. The combined genetic tests of JAK2 V617F, JAK2 exon 12, MPL exon 10, and CALR exon 9 help improve the diagnostic rate for BCR-ABL1-negative MPN. Copyright© by the American Society for Clinical Pathology.

Erythrocytes from patients with myeloproliferative neoplasms and splanchnic venous thrombosis show greater expression of Lu/BCAM.

PubMed

Novitzky-Basso, I; Spring, F; Anstee, D; Tripathi, D; Chen, F

2018-05-13

Lutheran/BCAM protein (Lu) on the surface of erythrocytes is key for their adhesion to the endothelium, and erythrocytes from individuals with JAK2V617F-mutated myeloproliferative neoplasms (MPN) have increased endothelial adhesion. Splanchnic vein thrombosis (SVT) is a devastating thrombotic complication of MPN, and frequently, the only diagnostic feature is the JAK2V617F mutation. We sought to examine whether erythrocytes from patients with JAK2V617F mutated SVT (MPN-SVT) exhibited increased Lu expression, thereby supporting a mechanistic contribution to the development of thrombosis. We report the validation of a novel flow cytometry assay for Lu expression on erythrocytes. We examined the expression of Lu on erythrocytes from a cohort of MPN patients with and without SVT, and healthy controls. Samples were obtained from 20 normal individuals, 22 with MPN (both JAK2V617F-mutated and wild-type) and 8 with JAK2V617F-mutated MPN-SVT. Lu expression by erythrocytes from patients with MPN and MPN-SVT is significantly increased compared to erythrocytes from healthy individuals (P < .05), but there was no significant difference between patients with MPN-SVT and MPN. Patients with MPN have increased expression of the red cell Lu/BCAM adhesion molecule. Further work is required to determine the role of the increased Lu/BCAM adhesion to the endothelium in the development of thrombosis in MPN of all genotypes. © 2018 John Wiley & Sons Ltd.

JAK2-V617F-induced MAPK activity is regulated by PI3K and acts synergistically with PI3K on the proliferation of JAK2-V617F-positive cells

PubMed Central

Wolf, Alexandra; Eulenfeld, René; Gäbler, Karoline; Rolvering, Catherine; Haan, Serge; Behrmann, Iris; Denecke, Bernd; Haan, Claude; Schaper, Fred

2013-01-01

The identification of a constitutively active JAK2 mutant, namely JAK2-V617F, was a milestone in the understanding of Philadelphia chromosome-negative myeloproliferative neoplasms. The JAK2-V617F mutation confers cytokine hypersensitivity, constitutive activation of the JAK-STAT pathway, and cytokine-independent growth. In this study we investigated the mechanism of JAK2-V617F-dependent signaling with a special focus on the activation of the MAPK pathway. We observed JAK2-V617F-dependent deregulated activation of the multi-site docking protein Gab1 as indicated by constitutive, PI3K-dependent membrane localization and tyrosine phosphorylation of Gab1. Furthermore, we demonstrate that PI3K signaling regulates MAPK activation in JAK2-V617F-positve cells. This cross-regulation of the MAPK pathway by PI3K affects JAK2-V617F-specific target gene induction, erythroid colony formation, and regulates proliferation of JAK2-V617F-positive patient cells in a synergistically manner. PMID:24069558

Renovascular hypertension associated with JAK2 V617F positive myeloproliferative neoplasms treated with angioplasty: 2 cases and literature review.

PubMed

Mishima, Eikan; Suzuki, Takehiro; Takeuchi, Yoichi; Seiji, Kazumasa; Fukuhara, Noriko; Takase, Kei; Harigae, Hideo; Abe, Takaaki; Ito, Sadayoshi

2018-04-01

Myeloproliferative neoplasms (MPNs) with Janus kinase 2 (JAK2) mutation are associated with a high risk for occlusive vascular diseases. We report 2 cases of renovascular hypertension associated with JAK2 V617F mutation-positive MPNs and provide a literature review. In Case 1, a 63-year-old woman had resistant hypertension, massive proteinuria, and erythrocytosis. Evaluations revealed right renal artery stenosis causing renovascular hypertension and polycythemia vera with JAK2 V617F mutation. Renin-angiotensin system inhibitors and subsequent angioplasty controlled the blood pressure and the proteinuria resolved. In Case 2, a 74-year-old woman had resistant hypertension and thrombocytosis. Evaluations confirmed left renal artery stenosis and essential thrombocythemia with JAK2 V617F. Angioplasty cured the hypertension. A literature review of 18 cases revealed the following as the most common characteristics of MPN-associated renovascular hypertension: manifests primarily in women; is associated with untreated polycythemia vera and essential thrombocythemia, concomitant leukocytosis, and JAK2 mutation positivity; and is responsive to angioplasty. This report demonstrates that JAK2 mutation-positive MPNs are a less common but important underlying cause of adult renovascular hypertension. ©2018 Wiley Periodicals, Inc.

Molecular genetic tests for JAK2V617F, Exon12_JAK2 and MPLW515K/L are highly informative in the evaluation of patients suspected to have BCR-ABL1-negative myeloproliferative neoplasms.

PubMed

dos Santos, Marcos Tadeu; Mitne-Neto, Miguel; Miyashiro, Kozue; Chauffaille, Maria de Lourdes L Ferrari; Rizzatti, Edgar Gil

2014-02-01

Polycythaemia vera (PV), essential thrombocythemia (ET) and idiopathic myelofibrosis (MF), are the most common myeloproliferative neoplasms (MPN) in patients without the BCR-ABL1 gene rearrangement. They are caused by clonal expansion of haematopoietic stem cells and share, as a diagnostic criterion, the identification of JAK2V617F mutation. Classically, when other clinical criteria are present, a JAK2V617F negative case requires the analysis of Exon12_JAK2 for the diagnosis of PV, and of MPL515K/L mutations for the diagnosis of ET and MF. Here, we evaluated 78 samples from Brazilian patients suspected to have MPN, without stratification for PV, ET or MF. We found that 28 (35.9%) are JAK2V617F carriers; from the 50 remaining samples, one (2%) showed an Exon12_JAK2 mutation, and another (2%) was positive for MPLW515L mutation. In summary, the investigation of JAK2V617F, Exon12_JAK2 and MPLW515K/L was relevant for the diagnosis of 38.4% of patients suspected to have BCR-ABL1-negative MPN, suggesting that molecular genetic tests are useful for a quick and unequivocal diagnosis of MPN.

Molecular genetic tests for JAK2V617F, Exon12_JAK2 and MPLW515K/L are highly informative in the evaluation of patients suspected to have BCR-ABL1-negative myeloproliferative neoplasms

PubMed Central

dos Santos, Marcos Tadeu; Mitne-Neto, Miguel; Miyashiro, Kozue; Chauffaille, Maria de Lourdes L Ferrari; Rizzatti, Edgar Gil

2014-01-01

Polycythaemia vera (PV), essential thrombocythemia (ET) and idiopathic myelofibrosis (MF), are the most common myeloproliferative neoplasms (MPN) in patients without the BCR-ABL1 gene rearrangement. They are caused by clonal expansion of haematopoietic stem cells and share, as a diagnostic criterion, the identification of JAK2V617F mutation. Classically, when other clinical criteria are present, a JAK2V617F negative case requires the analysis of Exon12_JAK2 for the diagnosis of PV, and of MPL515K/L mutations for the diagnosis of ET and MF. Here, we evaluated 78 samples from Brazilian patients suspected to have MPN, without stratification for PV, ET or MF. We found that 28 (35.9%) are JAK2V617F carriers; from the 50 remaining samples, one (2%) showed an Exon12_JAK2 mutation, and another (2%) was positive for MPLW515L mutation. In summary, the investigation of JAK2V617F, Exon12_JAK2 and MPLW515K/L was relevant for the diagnosis of 38.4% of patients suspected to have BCR-ABL1-negative MPN, suggesting that molecular genetic tests are useful for a quick and unequivocal diagnosis of MPN. PMID:23986553

CALR, JAK2 and MPL mutation status in Argentinean patients with BCR-ABL1- negative myeloproliferative neoplasms.

PubMed

Ojeda, Mara Jorgelina; Bragós, Irma Margarita; Calvo, Karina Lucrecia; Williams, Gladis Marcela; Carbonell, María Magdalena; Pratti, Arianna Flavia

2018-05-01

To establish the frequency of JAK2, MPL and CALR mutations in Argentinean patients with BCR-ABL1-negative  myeloproliferative neoplasms (MPN) and to compare their clinical and haematological features. Mutations of JAK2V617F, JAK2 exon 12, MPL W515L/K and CALR were analysed in 439 Argentinean patients with BCR-ABL1-negative MPN, including 176 polycythemia vera (PV), 214 essential thrombocythemia (ET) and 49 primary myelofibrosis (PMF). In 94.9% of PV, 85.5% ET and 85.2% PMF, we found mutations in JAK2, MPL or CALR. 74.9% carried JAK2V617F, 12.3% CALR mutations, 2.1% MPL mutations and 10.7% were triple negative. In ET, nine types of CALR mutations were identified, four of which were novel. PMF patients were limited to types 1 and 2, type 2 being more frequent. In ET, patients with CALR mutation were younger and had higher platelet counts than those with JAK2V617F and triple negative. In addition, JAK2V617F patients had high leucocyte and haemoglobin values compared with CALR-mutated and triple-negative patients. In PMF, patients with mutant CALR were associated with higher platelet counts. Our study underscores the importance of JAK2, MPL and CALR genotyping for accurate diagnosis of patients with BCR-ABL1-negative MPN.

Influence of the JAK2 V617F mutation and inherited thrombophilia on the thrombotic risk among patients with essential thrombocythemia

PubMed Central

De Stefano, Valerio; Za, Tommaso; Rossi, Elena; Fiorini, Alessia; Ciminello, Angela; Luzzi, Claudia; Chiusolo, Patrizia; Sica, Simona; Leone, Giuseppe

2009-01-01

It is uncertain whether the JAK2 V617F mutation increases the thrombotic risk in patients with essential thrombocythemia, and it is unknown whether inherited thrombophilia is an additive risk factor in mutated subjects. We studied 132 patients with essential thrombocythemia, 38 of them (29%) with a history of thrombosis. The JAK2 mutation was present in 83 (63%), and inherited thrombophilia in 7. The mutated patients <60 years had a relative risk (RR) for thrombosis at any time of 3.83 (95%CI 1.27–11.49) in comparison with wild-type patients; in those with both the mutation and thrombophilia the RR was 2.23 (95%CI 1.57–3.18) and 7.66 (95%CI 2.66–22.03) in comparison with mutated or wild-type patients without thrombophilia, respectively. During the follow-up, only the homozygotes for JAK2 V617F were more prone to thrombosis (RR 17.25, 95%CI 2.33–127.4). Among the patients >60 years, no increase in RR was associated with the JAK2 mutation. In conclusion, in the younger patients with ET the thrombotic risk is higher in the JAK2 V617F-mutated and is further increased by the presence of inherited thrombophilia. PMID:19336736

[Clinical Analysis of Driver Mutations in Patients with Ph Negative Myeloproliferative Neoplasms].

PubMed

He, Zhi-Peng; Tian, Hui-Yun; Tan, Ming; Wu, Yong

2018-06-01

To explore the relationship between driver mutations and clinical characteristics in patients with Philadelphia chromosome (Ph) negative myeloproliferative neoplasms (MPN), so as to provide evidence for diagno-sis and treatment of the disease. The clinical data of 410 patients with classic Ph negative MPN including 150 cases of polycythemia vera (PV), 188 cases of essential thrombocythemia (ET) and 72 cases of primary myelofibrosis (PMF) from January 2013 to December 2016 in Fujian Medical University Union Hospital were retrospectively analyzed. The PCR or DNA sequencing were used for JAK2 V617F, JAK2 exon12, CALR and MPL W515L/K mutation analyses, and follow-up information on patients was updated by direct phone call or follow-up in outpatient. Among the 410 patients with Ph negative MPN, 136 (33.2%) cases were asymptomatic at diagnosis. 389 cases were sequenced and JAK2 V617F was detected in 87.1% (122/140) of PV, 64.1% (118/184) of ET, 64.6% (42/65) of PMF; JAK2 exon 12 mutation in 1 case of PV; MPL W515L/K mutation in 1 case of ET and PMF, respectively; CALR mutation in 18(9.8%) cases of ET and 5 (7.7%) cases of PMF. JAK2 V617F mutated PV patients ocourred in older age: the white blood cell count, platelet count and incidence of splenomegaly were higher than JAK2-negative PV cases(P<0.05). Compared with JAK2 V617F mutated ET patients, CALR mutated ET cases displayed younger age, lower leukocyte count, higher platelet count and lower incidence of thrombosis; JAK2-negative ET cases had younger age, lower leukocyte count, lower hemoglobin level, higher platelet count and lower incidence of thrombosis(P<0.05). The incidence of splenomegaly in JAK2 V617F or CALR mutated PMF patients was both higher than that in JAK2-negative PMF cases, but the incidence of leukemia transformation in JAK2-negative PMF patients was higher than that in JAK2 V617F mutated cases (P<0.05). The types of driver mutations are closely related with the clinical features and prognosis in Ph - negative MPN patients.

Cytogenetics, JAK2 and MPL mutations in polycythemia vera, primary myelofibrosis and essential thrombocythemia

PubMed Central

dos Santos, Leonardo Caires; Ribeiro, Juliana Corrêa da Costa; Silva, Neusa Pereira; Cerutti, Janete; da Silva, Maria Regina Regis; Chauffaille, Maria de Lourdes Lopes Ferrari

2011-01-01

Background The detection of molecular and cytogenetic alterations is important for the diagnosis, prognosis and classification of myeloproliferative neoplasms. Objectives The aim of this study was to detect the following mutations: JAK2 V617F, JAK2 exon 12 and MPL W515K/L, besides chromosomal abnormalities. Furthermore, molecular and cytogenetic alterations were correlated with the leukocyte and platelet counts, hemoglobin levels and age in all patients and with the degree of fibrosis in primary myelofibrosis cases. Methods Twenty cases of polycythemia vera, 17 of essential thrombocythemia and 21 of primary myelofibrosis were selected in the Hematology Department of the Universidade Federal de São Paulo (UNIFESP) between February 2008 and December 2009. The JAK2 V617F, JAK2 exon 12 mutations, MPL W515K and MPL W515L mutations were investigated by real-time PCR and direct sequencing. G-band karyotyping and fluorescence in situ hybridization were used to detect chromosomal abnormalities. Results Chromosomal abnormalities were observed only in polycythemia vera (11.8%) and primary myelofibrosis cases (17.6%), without correlation to clinical data. Chromosomal abnormalities were not detected by fluorescence in situ hybridization. The JAK2 V617F mutation was observed in polycythemia vera (90%), primary myelofibrosis (42.8%) and essential thrombocythemia (47%). Patients with JAK2 V617F-negative polycythemia vera had lower platelet and leukocyte counts compared to V617F-positive polycythemia vera (p-value = 0.0001 and p-value = 0.023, respectively). JAK2 V617F-positive and MPL W515L-positive primary myelofibrosis cases had a higher degree of fibrosis than V617F-negative cases (p-value = 0.022). JAK2 exon 12 mutations were not detected in polycythemia vera patients. The MPL W515L mutation was observed in one case of primary myelofibrosis and in one of essential thrombocythemia. The MPL W515K mutation was not found in patients with essential thrombocythemia or primary myelofibrosis. The MPL W515L-positive patient with primary myelofibrosis had more severe anemia than other patients with primary myelofibrosis. Conclusions This study demonstrates that karyotyping for JAK2 and MPL mutations is useful in the diagnosis of myeloproliferative neoplasms. The precise pathogenetic contribution of these alterations is still unclear. However, this study adds more information about the pathophysiology of polycythemia vera, essential thrombocythemia and primary myelofibrosis. PMID:23049357

[Evidence on chronic myeloproliferative neoplasms from landmark clinical trials].

PubMed

Yamaguchi, Hiroki

2015-08-01

Mutations in the JAK2 gene are thought to underlie the development of chronic myeloproliferative neoplasms (cMPN). Indeed, ≥95% of polycythemia vera patients, and half or more of essential thrombocythemia and primary myelofibrosis (PMF) patients, harbor the JAK2V617F mutation. Besides the JAK2V617F mutation, the JAK2 exon 12 deletion, the MPLW515L/K, and CALR mutation have been discovered and shown to be involved in the pathogenesis of these diseases. Based on these advancements in the study of cMPN, the JAK2 inhibitor was developed as a new therapy for PMF. Moreover, recent advancements in our ability to diagnose cMPN have paralleled the development of large clinical trials for patients with cMPN. This article provides explanatory information from these large clinical trials that is useful for the actual clinical practice of caring for patients with cMPN in Japan.

JAK2 Exon 14 Deletion in Patients with Chronic Myeloproliferative Neoplasms

PubMed Central

Ma, Wanlong; Kantarjian, Hagop; Zhang, Xi; Wang, Xiuqiang; Zhang, Zhong; Yeh, Chen-Hsiung; O'Brien, Susan; Giles, Francis; Bruey, Jean Marie; Albitar, Maher

2010-01-01

Background The JAK2 V617F mutation in exon 14 is the most common mutation in chronic myeloproliferative neoplasms (MPNs); deletion of the entire exon 14 is rarely detected. In our previous study of >10,000 samples from patients with suspected MPNs tested for JAK2 mutations by reverse transcription-PCR (RT-PCR) with direct sequencing, complete deletion of exon 14 (Δexon14) constituted <1% of JAK2 mutations. This appears to be an alternative splicing mutation, not detectable with DNA-based testing. Methodology/Principal Findings We investigated the possibility that MPN patients may express the JAK2 Δexon14 at low levels (<15% of total transcript) not routinely detectable by RT-PCR with direct sequencing. Using a sensitive RT-PCR–based fluorescent fragment analysis method to quantify JAK2 Δexon14 mRNA expression relative to wild-type, we tested 61 patients with confirmed MPNs, 183 with suspected MPNs (93 V617F-positive, 90 V617F-negative), and 46 healthy control subjects. The Δexon14 variant was detected in 9 of the 61 (15%) confirmed MPN patients, accounting for 3.96% to 33.85% (mean  = 12.04%) of total JAK2 transcript. This variant was also detected in 51 of the 183 patients with suspected MPNs (27%), including 20 of the 93 (22%) with V617F (mean [range] expression  = 5.41% [2.13%–26.22%]) and 31 of the 90 (34%) without V617F (mean [range] expression  = 3.88% [2.08%–12.22%]). Immunoprecipitation studies demonstrated that patients expressing Δexon14 mRNA expressed a corresponding truncated JAK2 protein. The Δexon14 variant was not detected in the 46 control subjects. Conclusions/Significance These data suggest that expression of the JAK2 Δexon14 splice variant, leading to a truncated JAK2 protein, is common in patients with MPNs. This alternatively spliced transcript appears to be more frequent in MPN patients without V617F mutation, in whom it might contribute to leukemogenesis. This mutation is missed if DNA rather than RNA is used for testing. PMID:20730051

The thrombopoietin receptor, MPL, is critical for development of a JAK2V617F-induced myeloproliferative neoplasm

PubMed Central

Sangkhae, Veena; Etheridge, S. Leah; Kaushansky, Kenneth

2014-01-01

The most frequent contributing factor in Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) is the acquisition of a V617F mutation in Janus kinase 2 (JAK2) in hematopoietic stem cells (HSCs). Recent evidence has demonstrated that to drive MPN transformation, JAK2V617F needs to directly associate with a functional homodimeric type I cytokine receptor, suggesting that, although acquiring JAK2V617F may promote disease, there are additional cellular components necessary for MPN development. Here we show that loss of the thrombopoietin (TPO) receptor (MPL) significantly ameliorates MPN development in JAK2V617F+ transgenic mice, whereas loss of TPO only mildly affects the disease phenotype. Specifically, compared with JAK2V617F+ mice, JAK2V617F+Mpl−/− mice exhibited reduced thrombocythemia, neutrophilia, splenomegaly, and neoplastic stem cell pool. The importance of MPL is highlighted as JAK2V617FMpl+/− mice displayed a significantly reduced MPN phenotype, indicating that Mpl level may have a substantial effect on MPN development and severity. Splenomegaly and the increased neoplastic stem cell pool were retained in JAK2V617F+Tpo−/− mice, although thrombocytosis was reduced compared with JAK2V617F+ mice. These results demonstrate that Mpl expression, but not Tpo, is fundamental in the development of JAK2V617F+ MPNs, highlighting an entirely novel target for therapeutic intervention. PMID:25339357

The thrombopoietin receptor, MPL, is critical for development of a JAK2V617F-induced myeloproliferative neoplasm.

PubMed

Sangkhae, Veena; Etheridge, S Leah; Kaushansky, Kenneth; Hitchcock, Ian S

2014-12-18

The most frequent contributing factor in Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) is the acquisition of a V617F mutation in Janus kinase 2 (JAK2) in hematopoietic stem cells (HSCs). Recent evidence has demonstrated that to drive MPN transformation, JAK2V617F needs to directly associate with a functional homodimeric type I cytokine receptor, suggesting that, although acquiring JAK2V617F may promote disease, there are additional cellular components necessary for MPN development. Here we show that loss of the thrombopoietin (TPO) receptor (MPL) significantly ameliorates MPN development in JAK2V617F(+) transgenic mice, whereas loss of TPO only mildly affects the disease phenotype. Specifically, compared with JAK2V617F(+) mice, JAK2V617F(+)Mpl(-/-) mice exhibited reduced thrombocythemia, neutrophilia, splenomegaly, and neoplastic stem cell pool. The importance of MPL is highlighted as JAK2V617FMpl(+/-) mice displayed a significantly reduced MPN phenotype, indicating that Mpl level may have a substantial effect on MPN development and severity. Splenomegaly and the increased neoplastic stem cell pool were retained in JAK2V617F(+)Tpo(-/-) mice, although thrombocytosis was reduced compared with JAK2V617F(+) mice. These results demonstrate that Mpl expression, but not Tpo, is fundamental in the development of JAK2V617F(+) MPNs, highlighting an entirely novel target for therapeutic intervention. © 2014 by The American Society of Hematology.

Concomitant presence of JAK2V617F mutation and BCR‑ABL translocation in two patients: A new entity or a variant of myeloproliferative neoplasms (Case report).

PubMed

Mousinho, Filipa; Azevedo, Ana P; Mendes, Tatiana; Santos, Paula Sousa E; Cerqueira, Rita; Matos, Sónia; Santos, Sónia; Ramos, Sância; Viana, João Faro; Lima, Fernando

2018-05-17

Myeloproliferative neoplasms (MPNs) are classically divided into BCR RhoGEF and GTPase activating protein (BCR)-ABL proto‑oncogene 1 non‑receptor tyrosine kinase (ABL) positive chronic myeloid leukemia (CML) and BCR‑ABL negative MPNs, including essential thrombocythemia (ET). One of the major diagnostic criteria for ET is the absence of the philadelphia chromosome, thus when present it is almost indicative of CML. ET and CML are considered to be mutually exclusive; however, there are rare situations in which patients with ET present positive BCR‑ABL without the features of CML. Although from the literature review, the frequency of JAK2V617F mutation and BCR‑ABL translocation coexistence in MPNs is low, it may be higher than expected. The current study reported cases of two patients with an initial diagnosis of ET in the presence of JAK2V617F mutation and BCR‑ABL translocation by fluorescent in situ hybridization. Both patients presented with a heterozygous BCR‑ABL translocation, and absence of p190 and p210 transcripts, seemingly a der(9) in the background of an ET JAK2V617F mutation.

Clinical utility of routine MPL exon 10 analysis in the diagnosis of essential thrombocythaemia and primary myelofibrosis.

PubMed

Boyd, Elaine M; Bench, Anthony J; Goday-Fernández, Andrea; Anand, Shubha; Vaghela, Krishna J; Beer, Phillip; Scott, Mike A; Bareford, David; Green, Anthony R; Huntly, Brian; Erber, Wendy N

2010-04-01

Approximately 50% of essential thrombocythaemia and primary myelo-fibrosis patients do not have a JAK2 V617F mutation. Up to 5% of these are reported to have a MPL exon 10 mutation but testing for MPL is not routine as there are multiple mutation types. The ability to routinely assess both JAK2 and MPL mutations would be beneficial in the differential diagnosis of unexplained thrombocytosis or myelofibrosis. We developed and applied a high resolution melt (HRM) assay, capable of detecting all known MPL mutations in a single analysis, for the detection of MPL exon 10 mutations. We assessed 175 ET and PMF patients, including 67 that were JAK2 V617F-negative by real time polymerase chain reaction (PCR). Overall, 19/175 (11%) patients had a MPL exon 10 mutation, of whom 16 were JAK2 V617F-negative (16/67; 24%). MPL mutation types were W515L (11), W515K (4), W515R (2) and W515A (1). One patient had both W515L and S505N MPL mutations and these were present in the same haemopoietic colonies. Real time PCR for JAK2 V617F analysis and HRM for MPL exon 10 status identified one or more clonal marker in 71% of patients. This combined genetic approach increases the sensitivity of meeting the World Health Organization diagnostic criteria for these myeloproliferative neoplasms.

MPL515 mutations in myeloproliferative and other myeloid disorders: a study of 1182 patients.

PubMed

Pardanani, Animesh D; Levine, Ross L; Lasho, Terra; Pikman, Yana; Mesa, Ruben A; Wadleigh, Martha; Steensma, David P; Elliott, Michelle A; Wolanskyj, Alexandra P; Hogan, William J; McClure, Rebecca F; Litzow, Mark R; Gilliland, D Gary; Tefferi, Ayalew

2006-11-15

Recently, a gain-of-function MPL mutation, MPLW515L, was described in patients with JAK2V617F-negative myelofibrosis with myeloid metaplasia (MMM). To gain more information on mutational frequency, disease specificity, and clinical correlates, genomic DNA from 1182 patients with myeloproliferative and other myeloid disorders and 64 healthy controls was screened for MPL515 mutations, regardless of JAK2V617F mutational status: 290 with MMM, 242 with polycythemia vera, 318 with essential thrombocythemia (ET), 88 with myelodysplastic syndrome, 118 with chronic myelomonocytic leukemia, and 126 with acute myeloid leukemia (AML). MPL515 mutations, either MPLW515L (n = 17) or a previously undescribed MPLW515K (n = 5), were detected in 20 patients. The diagnosis of patients with mutant MPL alleles at the time of molecular testing was de novo MMM in 12 patients, ET in 4, post-ET MMM in 1, and MMM in blast crisis in 3. Six patients carried the MPLW515L and JAK2V617F alleles concurrently. We conclude that MPLW515L or MPLW515K mutations are present in patients with MMM or ET at a frequency of approximately 5% and 1%, respectively, but are not observed in patients with polycythemia vera (PV) or other myeloid disorders. Furthermore, MPL mutations may occur concurrently with the JAK2V617F mutation, suggesting that these alleles may have functional complementation in myeloproliferative disease.

[JAK2 V617F and exon 12 genetic variations in Korean patients with BCR/ABL1-negative myeloproliferative neoplasms].

PubMed

Kim, Jeong Tae; Cho, Yong Gon; Choi, Sam Im; Lee, Young Jin; Kim, Hye Ran; Jang, Sook Jin; Moon, Dae Soo; Park, Young Jin; Park, Geon

2010-12-01

JAK2 genetic variations have been described in a high proportion of patients with BCR/ABL1-negative myeloproliferative neoplasms (MPN). This study was designed to analyze the frequencies of JAK2 V617F and exon 12 variations, and their correlations with clinical characteristics of Korean patients with BCR/ABL1-negative MPN. We examined a total of 154 patients with BCR/ABL1-negative MPN that included 24, 26, 89, and 15 patients with polycythemia vera (PV), primary myelofibrosis (PMF), essential thrombocythemia (ET), and unclassified myeloproliferative neoplasms (MPNU), respectively. We performed allele-specific PCR to detect V617F in all BCR/ABL1-negative patients, and performed direct sequencing to detect exon 12 variations in 47 V617F-negative MPN patients. JAK2 c.1641+179_183del5 variation was detected by restriction fragment length polymorphism assay in 176 healthy subjects. JAK2 V617F was detected in 91 patients (59.1%): PV (91.6%), PMF (46.2%), ET (52.8%), and MPNU (66.7%). In V617F-negative MPN patients, no mutations were found in exon 12. The c.1641+179_183del5 was detected in 68.1% of V617F-negative MPN patients and 45.4% of healthy subjects (P=0.008). JAK2 V617F was closely correlated with age and leukocytosis in BCR/ABL1-negative MPN patients (P<0.05). However, c.1641+179_183del5 was not related to age, sex, or complete blood cell count parameters in V617F-negative MPN patients and healthy subjects. The c.1641+179_183del5 was associated with an increased odds ratio for MPN (odds ratio, 2.6; 95% confidences interval, 1.3-5.1; P=0.007). Frequencies of V617F are similar to reported results. JAK2 exon 12 mutations may be rare and c.1641+179_183del5 may influence the occurrence of MPN in Korean patients with V6 17F-negative MPN.

[Clinical significance of JAK2、CALR and MPL gene mutations in 1 648 Philadelphia chromosome negative myeloproliferative neoplasms patients from a single center].

PubMed

Li, M Y; Chao, H Y; Sun, A N; Qiu, H Y; Jin, Z M; Tang, X W; Han, Y; Fu, C C; Chen, S N; Wu, D P

2017-04-14

Objective: To explore the prevalences of JAK2, CALR and MPL gene mutations and the mutation types in patients with Philadelphia chromosome negative myeloproliferative neoplasms (MPNs) , and to compare their clinical characteristics of different mutation types with each other and mutation negative group. Methods: The mutations of JAK2 V617F, JAK2 gene at exon 12, CALR gene at exon 9 and MPL gene at exon 10 in 1 648 Ph negative MPNs patients were detected by direct sequencing. Results: ① The JAK2V617F mutation was found in 471 (92.7%) of 508 PV patients, 819 (78.1%) of 1 049 ET patients and 74 (81.3%) of 91 PMF patients respectively, with the total mutation rate as 82.8% (1 364/1 648) . The JAK2 exon12 mutation was found in 9 (1.7%) of 508 PV patients, none was found in ET or PMF patients, with the total mutation rate as 0.5% (9/1 648) . The CALR mutation was found in 132 (12.6%) of 1 049 ET patients and 11 (12.1%) of 91 PMF patients respectively, with the total mutation rate as 8.7% (143/1 648) ; the MPL mutation was found in 9 (0.9%) of 1 049 ET patients and 1 (1.1%) of 91 PMF patients respectively, with the total mutation rate as 0.6% (10/1 648) . The co-occurrence of any two types of driver gene mutations was not detected by direct sequencing. ②The median onset age of patients with JAK2V617F[61 (15-95) y] was significant higher than of with JAK2 exon12 mutation[49 (33-62) y] or without mutations[42 (3-78) y] ( P <0.001) , but not for patients with CALR[57 (17-89) y] or MPL mutation[59 (22-71) y] ( P >0.05) . Patients with JAK2V617F had higher white blood cell count and hemoglobin level ( P <0.05) when compared with patients with CALR mutation or without mutations, or only significantly higher white blood cell count when compared with patients with MPL mutation ( P =0.013) . The platelet count of patients with CALR mutation was significantly higher than of with JAK2V617F[966 (400-2 069) ×10(9)/L vs 800 (198-3 730) ×10(9)/L, P <0.001]. ③Karyotype analysis was conducted in 1 160 patients with MPNs, the rates of karyotype abnormality of patients with and without CALR mutation were 9.8% (8/82) and 7.4% (80/1 078) ( P =0.441) respectively; The rates of karyotype abnormality of patients with and without JAK2V617F mutation were 7.7% (75/971) and 6.9% (13/189) ( P =0.688) respectively. The incidence of karyotype abnormality of patients with CALR mutation was higher than of with JAK2V617F[9.8% (8/82) vs 7.7% (75/971) ] without statistically significant difference ( P =0.512) . The karyotype analysis of 7 cases of JAK2 exon12 mutation and 6 ones with MPL gene mutation revealed normal karyotype. Conclusions: Driver gene mutations detection could ensure the diagnosis and prognosis judgment of MPN more reliable, different subtypes of MPNs had different profiles of driver gene mutations, the latter lead to unique clinical phenotype.

After 10years of JAK2V617F: Disease biology and current management strategies in polycythaemia vera.

PubMed

Grinfeld, Jacob; Godfrey, Anna L

2017-05-01

The JAK2V617F mutation accounts for the vast majority of patients with polycythaemia vera (PV) and around half of those with other Philadelphia-negative myeloproliferative neoplasms. Since its discovery in 2005, numerous insights have been gained into the pathways by which JAK2V617F causes myeloproliferation, including activation of JAK-STAT signalling but also through other canonical and non-canonical pathways. A variety of mechanisms explain how this one mutation can be associated with distinct clinical disorders, demonstrating how constitutional and acquired factors may interact in the presence of a single mutation to determine disease phenotype. Important biological questions remain unanswered in PV, in particular how JAK2V617F affects stem cell function and what mechanisms drive myelofibrotic and leukaemic transformation. Whilst current management is largely centred on prevention of cardiovascular events, future therapies must aim to target the JAK2-mutant clone, to reverse the underlying marrow pathology and to address the risk of transformation events. Copyright © 2016 Elsevier Ltd. All rights reserved.

Mechanisms of mutations in myeloproliferative neoplasms.

PubMed

Levine, Ross L

2009-12-01

In recent years, a series of studies have provided genetic insight into the pathogenesis of myeloproliferative neoplasms (MPNs). It is now known that JAK2V617F mutations are present in 90% of patients with polycythaemia vera (PV), 60% of patients with essential thrombocytosis (ET) and 50% of patients with myelofibrosis (MF). Despite the high prevalence of JAK2V617F mutations in these three myeloid malignancies, several questions remain. For example, how does one mutation contribute to the pathogenesis of three clinically distinct diseases, and how do some patients develop these diseases in the absence of a JAK2V617F mutation? Single nucleotide polymorphisms at various loci and somatic mutations, such as those in MPLW515L/K, TET2 and in exon 12 of JAK2, may also contribute to the pathogenesis of these MPNs. There are likely additional germline and somatic genetic factors important to the MPN phenotype. Additional studies of large MPN and control cohorts with new techniques will help identify these factors.

Myeloproliferative neoplasms: JAK2 signaling pathway as a central target for therapy.

PubMed

Pasquier, Florence; Cabagnols, Xenia; Secardin, Lise; Plo, Isabelle; Vainchenker, William

2014-09-01

The discovery of the JAK2V617F mutation followed by the discovery of other genetic abnormalities allowed important progress in the understanding of the pathogenesis and management of myeloproliferative neoplasms (MPN)s. Classical Breakpoint cluster region-Abelson (BCR-ABL)-negative neoplasms include 3 main disorders: essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF). Genomic studies have shown that these disorders are more heterogeneous than previously thought with 3 main entities corresponding to different gene mutations: the JAK2 disorder, essentially due to JAK2V617F mutation, which includes nearly all PVs and a majority of ETs and PMFs with a continuum between these diseases and the myeloproliferative leukemia (MPL) and calreticulin (CALR) disorders, which include a fraction of ET and PMF. All of these mutations lead to a JAK2 constitutive activation. Murine models either with JAK2V617F or MPLW515L, but also with JAK2 or MPL germ line mutations found in hereditary thrombocytosis, have demonstrated that they are drivers of myeloproliferation. However, the myeloproliferative driver mutation is still unknown in approximately 15% of ET and PMF, but appears to also target the JAK/Signal Transducer and Activator of Transcription (STAT) pathway. However, other mutations in genes involved in epigenetics or splicing also can be present and can predate or follow mutations in signaling. They are involved either in clonal dominance or in phenotypic changes, more particularly in PMF. They can be associated with leukemic progression and might have an important prognostic value such as additional sex comb-like 1 mutations. Despite this heterogeneity, it is tempting to target JAK2 and its signaling for therapy. However in PMF, Adenosine Tri-Phosphate (ATP)-competitive JAK2 inhibitors have shown their interest, but also their important limitations. Thus, other approaches are required, which are discussed in this review. Copyright © 2014 Elsevier Inc. All rights reserved.

Primary myelofibrosis with or without mutant MPL: comparison of survival and clinical features involving 603 patients.

PubMed

Pardanani, A; Guglielmelli, P; Lasho, T L; Pancrazzi, A; Finke, C M; Vannucchi, A M; Tefferi, A

2011-12-01

MPL and JAK2V617F mutation analysis was performed in 603 patients with primary myelofibrosis (PMF) seen at the Mayo Clinic, USA (n=329) or University of Florence, Italy (n=274). Mutant MPL was detected in 49 (8.1%) patients and JAK2V617F in 350 (58%); 4 patients showed both mutations. MPLW515L/K was the commonest mutation; 2 patients showed novel mutations (L513ins and Q516-P518insAAAA). The US and Italy patient cohorts were separately analyzed for comparison of survival and clinical features between MPL-mutated, JAK2-mutated and JAK2/MPL-unmutated cases. JAK2/MPL-unmutated patients were significantly younger than their JAK2-mutated counterparts, in both patient cohorts (P<0.01). In the Florence only cohort, the presence of mutant MPL was associated with older age (P<0.01) and constitutional symptoms (P=0.04) and JAK2V617F with higher hemoglobin (P<0.01) and leukocyte (P=0.03) count; neither patient cohort showed significant associations with platelet count, hemoglobin <10 g/dl, abnormal/unfavorable karyotype, spleen size or prognostic score distribution. To date, 240 deaths and 79 leukemic transformations have been documented among all 603 study patients. Multivariable analysis disclosed no significant difference in overall or leukemia-free survival between the three molecular subgroups. We conclude that the presence of mutant MPL has narrow and inconsistent phenotypic effect in PMF and does not influence overall or leukemia-free survival.

IRS2 silencing increases apoptosis and potentiates the effects of ruxolitinib in JAK2V617F-positive myeloproliferative neoplasms

PubMed Central

de Melo Campos, Paula; Machado-Neto, João A.; Eide, Christopher A.; Savage, Samantha L.; Scopim-Ribeiro, Renata; da Silva Souza Duarte, Adriana; Favaro, Patricia; Lorand-Metze, Irene; Costa, Fernando F.; Tognon, Cristina E.; Druker, Brian J.; Saad, Sara T. Olalla; Traina, Fabiola

2016-01-01

The recurrent V617F mutation in JAK2 (JAK2V617F) has emerged as the primary contributor to the pathogenesis of myeloproliferative neoplasms (MPN). However, the lack of complete response in most patients treated with the JAK1/2 inhibitor, ruxolitinib, indicates the need for identifying pathways that cooperate with JAK2. Activated JAK2 was found to be associated with the insulin receptor substrate 2 (IRS2) in non-hematological cells. We identified JAK2/IRS2 binding in JAK2V617F HEL cells, but not in the JAK2WT U937 cell line. In HEL cells, IRS2 silencing decreased STAT5 phosphorylation, reduced cell viability and increased apoptosis; these effects were enhanced when IRS2 silencing was combined with ruxolitinib. In U937 cells, IRS2 silencing neither reduced cell viability nor induced apoptosis. IRS1/2 pharmacological inhibition in primary MPN samples reduced cell viability in JAK2V617F-positive but not JAK2WT specimens; combination with ruxolitinib had additive effects. IRS2 expression was significantly higher in CD34+ cells from essential thrombocythemia patients compared to healthy donors, and in JAK2V617F MPN patients when compared to JAK2WT. Our data indicate that IRS2 is a binding partner of JAK2V617F in MPN. IRS2 contributes to increased cell viability and reduced apoptosis in JAK2-mutated cells. Combined pharmacological inhibition of IRS2 and JAK2 may have a potential clinical application in MPN. PMID:26755644

Role of JAK-STAT signaling in the pathogenesis of myeloproliferative disorders.

PubMed

Levine, Ross L; Wernig, Gerlinde

2006-01-01

The identification of JAK2V617F mutations in polycythemia vera (PV), essential thrombocytosis (ET), and myelofibrosis (MF) represents an important advance in our understanding of these myeloproliferative disorders (MPD). Most, if not all, patients with PV and a significant number of patients with ET and MF are JAK2V617F positive, and the mutation likely arises in the hematopoietic stem cell compartment. JAK2V617F is a constitutively active tyrosine kinase that is able to activate JAK-STAT signaling most efficiently when co-expressed with the erythropoietin receptor (EPOR), the thrombopoietin receptor (MPL), or the granulocyte colony-stimulating factor receptor (GCSFR). Data from murine models supports the central role of JAK2V617F in the pathogenesis of MPD, as expression of JAK2V617F in a bone marrow transplantation assay results in polycythemia and myelofibrosis in recipient mice. Activation of JAK-STAT signaling by JAK2V617F in some, but not all MPD patients with ET and MF led to the identification of the constitutively active MPLW515L allele in ET and MF. Small molecule inhibitors of JAK-STAT signaling are currently being developed, which offer potential for molecularly targeted therapy for patients with PV, ET, and MF. Despite these advances, many questions remain regarding the role of a single disease allele in three phenotypically distinct MPD, the potential clinical efficacy of JAK2 inhibitors, and the identity of oncogenic alleles in JAK2V617F/MPLW515-negative MPD.

PO-19 - Platelet (PLT) adhesion under flow condition in essential thrombocythemia (ET) and polycythemia vera (PV) is variably influenced according to patient mutational status.

PubMed

Vignoli, A; Tessarolo, S; Marchetti, M; Gamba, S; Piras, F; Finazzi, G; van der Meijden, P E J; Swieringa, F; Ten Cate, H; Heemskerk, J W M; Rambaldi, A; Falanga, A

2016-04-01

The myeloproliferative neoplasms ET and PV are characterized by a high incidence of both arterial and venous thrombosis, and/or microcirculatory disturbances. Three somatic mutations, i.e. JAK2-V617F, Calreticulin (CalR) and MPL, commonly found in these diseases, correlate with different thrombotic risk levels. To analyze the influence of JAK2-V617F, CalR and MPL mutations on PLT adhesion, evaluated by a dynamic method under flow conditions in a group of patients with ET and PV. 86 patients, i.e. 51 ET (19 M/32 F; age range 32-86 years) and 35PV (22 M/13 F; 41-83 yrs.), and 24 healthy controls (13 M/11 F; 28-61 yrs.) were enrolled upon informed consent. For the adhesion assay, peripheral venous whole blood was perfused over collagen for 4' at a 1,000 s-1 shear rate. PLTs were then stained with an anti-P-selectin-FITC antibody to evaluate PLT activation, and annexin V-AlexaFluor647 to detect procoagulant phosphatidylserine expression. Then, images of adherent PLTs in random fields were taken using phase contrast and fluorescence imaging by EVOS® fluorescence microscope. Results are mean±SEM of the % area covered by PLTs, or as the % of adherent PLTs positive for P-selectin or phosphatidylserine. Main hematological parameters and mutational status were recorded. PLT adhesion was significantly (p<0.01) greater in ET (44.6±1.6%) and PV patients (49.0±1.9%) compared to controls (37.9±1.7%). In ET, PLT adhesion was highest in JAK2-V617F mutation carriers (n=23), followed by CalR-positive (n=16) and triple negative subjects (n=9), and lowest in the MPL-positive patients (n=3). In PV, no difference in PLT adhesion was observed between JAK2-V617F heterozygous and homozygous subjects. P-selectin expression by adherent PLTs was not statistically different between patients and controls. Differently, phosphatidylserine expression on adherent PLTs was significantly reduced (p<0.01) in both ET and PV compared to healthy subjects. In ET patients, a significant (p<0.05) correlation was found between PLT adhesion and PLT count in JAK2-V617F and CalR-positive mutation carriers. Multivariate regression analysis adjusted for age and sex, confirmed PLT count as a significant determinant of PLT adhesion in JAK2-V617F positive patients only. ET and PV platelets show an increased adhesion to collagen in vitro, particularly in those carrying the JAK2-V617F mutation. A prospective study is ongoing to evaluate the predictive value of our PLT thrombus formation dynamic model for the thrombotic risk in ET and PV patients. Project funded by "AIRC-IG2013" grant Nr. 14505 from the "Italian Association for Cancer Research" (A.I.R.C.). © 2016 Elsevier Ltd. All rights reserved.

The C allele of JAK2 rs4495487 is an additional candidate locus that contributes to myeloproliferative neoplasm predisposition in the Japanese population

PubMed Central

2012-01-01

Background Polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) are myeloproliferative neoplasms (MPNs) characterized in most cases by a unique somatic mutation, JAK2 V617F. Recent studies revealed that JAK2 V617F occurs more frequently in a specific JAK2 haplotype, named JAK2 46/1 or GGCC haplotype, which is tagged by rs10974944 (C/G) and/or rs12343867 (T/C). This study examined the impact of single nucleotide polymorphisms (SNPs) of the JAK2 locus on MPNs in a Japanese population. Methods We sequenced 24 JAK2 SNPs in Japanese patients with PV. We then genotyped 138 MPN patients (33 PV, 96 ET, and 9 PMF) with known JAK2 mutational status and 107 controls for a novel SNP, in addition to two SNPs known to be part of the 46/1 haplotype (rs10974944 and rs12343867). Associations with risk of MPN were estimated by odds ratios and their 95% confidence intervals using logistic regression. Results A novel locus, rs4495487 (T/C), with a mutated T allele was significantly associated with PV. Similar to rs10974944 and rs12343867, rs4495487 in the JAK2 locus is significantly associated with JAK2-positive MPN. Based on the results of SNP analysis of the three JAK2 locus, we defined the "GCC genotype" as having at least one minor allele in each SNP (G allele in rs10974944, C allele in rs4495487, and C allele in rs12343867). The GCC genotype was associated with increased risk of both JAK2 V617F-positive and JAK2 V617F-negative MPN. In ET patients, leukocyte count and hemoglobin were significantly associated with JAK2 V617F, rather than the GCC genotype. In contrast, none of the JAK2 V617F-negative ET patients without the GCC genotype had thrombosis, and splenomegaly was frequently seen in this subset of ET patients. PV patients without the GCC genotype were significantly associated with high platelet count. Conclusions Our results indicate that the C allele of JAK2 rs4495487, in addition to the 46/1 haplotype, contributes significantly to the occurrence of JAK2 V617F-positive and JAK2 V617F-negative MPNs in the Japanese population. Because lack of the GCC genotype represents a distinct clinical-hematological subset of MPN, analyzing JAK2 SNPs and quantifying JAK2 V617F mutations will provide further insights into the molecular pathogenesis of MPN. PMID:22251709

Association of TNF polymorphisms with JAK2 (V617F) myeloproliferative neoplasms in Brazilian patients.

PubMed

Macedo, Luciana Conci; de Cesare Quintero, Fernanda; Pagliari-E-Silva, Sara; Pagnano, Katia Borgia Barbosa; Rodrigues, Camila; de Alencar, Josiane Bazzo; Sell, Ana Maria; Visentainer, Jeane Eliete Laguila

2016-03-01

The classical chromosome Philadelphia-negative myeloproliferative neoplasms (MPNs) are a group of disorders that share clinical, hematological, and histological features. Proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) are elevated in patients with MPN. The aim of this study was to verify the association between the polymorphisms of TNF gene (-308G/A and -238 G/A) in BCR-ABL-negative MPN in our population. Blood samples obtained from MPN patients were genotyped for the JAK2V617F mutation and both TNF polymorphisms using PCR-RFLP. Thirty three (26.8%) patients with polycythemia vera (PV), 35 (28.7%) essential thrombocythemia (ET), 22 (17.7%) primary myelofibrosis (PMF), and 33 (26.8%) with unclassifiable MPN (MPNu) were included in the study. The JAK2 V617F mutation was detected in 94 (76.42%) patients. Were observed a significant increase on the frequency of the TNF-238 GA genotype in MPN patients compared to controls (OR=2.21, 95% CI=1.02-4.80, P<0.04). The distribution of the genotypes and allelic frequencies of TNF-308 was significantly different among the MPNs, JAK2V617F positive, PV and PMF, and controls. Our data has demonstrated that the polymorphisms on TNF-238 GA, TNF-308 GA were associated to MPN development in this population, triggered by JAK2 V617F mutation. Copyright © 2015. Published by Elsevier Inc.

Establishing optimal quantitative-polymerase chain reaction assays for routine diagnosis and tracking of minimal residual disease in JAK2-V617F-associated myeloproliferative neoplasms: a joint European LeukemiaNet/MPN&MPNr-EuroNet (COST action BM0902) study.

PubMed

Jovanovic, J V; Ivey, A; Vannucchi, A M; Lippert, E; Oppliger Leibundgut, E; Cassinat, B; Pallisgaard, N; Maroc, N; Hermouet, S; Nickless, G; Guglielmelli, P; van der Reijden, B A; Jansen, J H; Alpermann, T; Schnittger, S; Bench, A; Tobal, K; Wilkins, B; Cuthill, K; McLornan, D; Yeoman, K; Akiki, S; Bryon, J; Jeffries, S; Jones, A; Percy, M J; Schwemmers, S; Gruender, A; Kelley, T W; Reading, S; Pancrazzi, A; McMullin, M F; Pahl, H L; Cross, N C P; Harrison, C N; Prchal, J T; Chomienne, C; Kiladjian, J J; Barbui, T; Grimwade, D

2013-10-01

Reliable detection of JAK2-V617F is critical for accurate diagnosis of myeloproliferative neoplasms (MPNs); in addition, sensitive mutation-specific assays can be applied to monitor disease response. However, there has been no consistent approach to JAK2-V617F detection, with assays varying markedly in performance, affecting clinical utility. Therefore, we established a network of 12 laboratories from seven countries to systematically evaluate nine different DNA-based quantitative PCR (qPCR) assays, including those in widespread clinical use. Seven quality control rounds involving over 21,500 qPCR reactions were undertaken using centrally distributed cell line dilutions and plasmid controls. The two best-performing assays were tested on normal blood samples (n=100) to evaluate assay specificity, followed by analysis of serial samples from 28 patients transplanted for JAK2-V617F-positive disease. The most sensitive assay, which performed consistently across a range of qPCR platforms, predicted outcome following transplant, with the mutant allele detected a median of 22 weeks (range 6-85 weeks) before relapse. Four of seven patients achieved molecular remission following donor lymphocyte infusion, indicative of a graft vs MPN effect. This study has established a robust, reliable assay for sensitive JAK2-V617F detection, suitable for assessing response in clinical trials, predicting outcome and guiding management of patients undergoing allogeneic transplant.

Self-Renewal of Single Mouse Hematopoietic Stem Cells Is Reduced by JAK2V617F Without Compromising Progenitor Cell Expansion

PubMed Central

Kent, David G.; Li, Juan; Tanna, Hinal; Fink, Juergen; Kirschner, Kristina; Pask, Dean C.; Silber, Yvonne; Hamilton, Tina L.; Sneade, Rachel; Simons, Benjamin D.; Green, Anthony R.

2013-01-01

Recent descriptions of significant heterogeneity in normal stem cells and cancers have altered our understanding of tumorigenesis, emphasizing the need to understand how single stem cells are subverted to cause tumors. Human myeloproliferative neoplasms (MPNs) are thought to reflect transformation of a hematopoietic stem cell (HSC) and the majority harbor an acquired V617F mutation in the JAK2 tyrosine kinase, making them a paradigm for studying the early stages of tumor establishment and progression. The consequences of activating tyrosine kinase mutations for stem and progenitor cell behavior are unclear. In this article, we identify a distinct cellular mechanism operative in stem cells. By using conditional knock-in mice, we show that the HSC defect resulting from expression of heterozygous human JAK2V617F is both quantitative (reduced HSC numbers) and qualitative (lineage biases and reduced self-renewal per HSC). The defect is intrinsic to individual HSCs and their progeny are skewed toward proliferation and differentiation as evidenced by single cell and transplantation assays. Aged JAK2V617F show a more pronounced defect as assessed by transplantation, but mice that transform reacquire competitive self-renewal ability. Quantitative analysis of HSC-derived clones was used to model the fate choices of normal and JAK2-mutant HSCs and indicates that JAK2V617F reduces self-renewal of individual HSCs but leaves progenitor expansion intact. This conclusion is supported by paired daughter cell analyses, which indicate that JAK2-mutant HSCs more often give rise to two differentiated daughter cells. Together these data suggest that acquisition of JAK2V617F alone is insufficient for clonal expansion and disease progression and causes eventual HSC exhaustion. Moreover, our results show that clonal expansion of progenitor cells provides a window in which collaborating mutations can accumulate to drive disease progression. Characterizing the mechanism(s) of JAK2V617F subclinical clonal expansions and the transition to overt MPNs will illuminate the earliest stages of tumor establishment and subclone competition, fundamentally shifting the way we treat and manage cancers. PMID:23750118

Acute Myeloid Leukemia with MYC Rearrangement and JAK2 V617F Mutation

PubMed Central

Ohanian, Maro; Bueso-Ramos, Carlos; Ok, Chi Young; Lin, Pei; Patel, Keyur; Alattar, Mona Lisa; Khoury, Joseph D.; Rozovski, Uri; Estrov, Zeev; Huh, Yang O.; Cortes, Jorge; Abruzzo, Lynne V.

2016-01-01

Little is known about MYC dysregulation in myeloid malignancies, and we can find no published studies that have evaluated MYC protein expression in primary cases of myelodysplastic syndromes (MDS) or acute myeloid leukemias (AML). We describe the clinical, morphologic, immunophenotypic, cytogenetic, and molecular genetic findings in two MDS/AML cases that contained both MYC rearrangement and JAK2-V617F mutation. We demonstrate MYC protein expression by immunohistochemistry in both patients. PMID:26382622

Phenotypic variability within the JAK2 V617F-positive MPD: The roles of progenitor cell and neutrophil allele burdens

PubMed Central

Moliterno, Alison R.; Williams, Donna M.; Rogers, Ophelia; Isaacs, Mary Ann; Spivak, Jerry L.

2008-01-01

(1) Objective The myeloproliferative disorders (MPD), polycythemia vera (PV), essential thrombocytosis (ET) and primary myelofibrosis (PMF) differ phenotypically but share the same JAK2V617F mutation. We examined the relationship of the quantitative JAK2V617F allele burden to MPD disease phenotype among the three MPD classes and within PV. (2) Methods We measured the JAK2V617F allele percentage in genomic DNA from neutrophils, CD34+ cells, and cloned progenitors in 212 JAK2V617F –positive MPD patients and correlated the allele burdens to both disease class and disease features. (3) Results In ET and PV, the mean CD34+ cell JAK2V617F allele burdens were lower than the corresponding neutrophil allele burdens, but these were equivalent in PMF. JAK2WT progenitors were present in ET and PV when the CD34+ JAK2V617F allele burden was lower than the neutrophil allele burden, but not in PV and PMF subjects in whom the CD34+ cell and neutrophil allele burdens were similar. CD34+ cell JAK2V617F clonal dominance, defined as coherence between the CD34+ cell and neutrophil JAK2V617F allele burdens, was present in 24% of ET, 56% of PV and 93% of PMF patients, and was independent of the CD34+ cell JAK2V617F genotype. Clonally-dominant PV patients had significantly longer disease durations, higher white cell counts and larger spleens than nondominant PV patients. (4) Conclusions We conclude that the extent of JAK2V617F CD34+ cell clonal dominance is associated with disease phenotype within the MPD, and in PV, is associated with extramedullary disease, leukocytosis and disease duration. PMID:18723264

Effect of Mutation Order on Myeloproliferative Neoplasms

PubMed Central

Nangalia, Jyoti; Silber, Yvonne; Wedge, David C.; Grinfeld, Jacob; Baxter, E. Joanna; Massie, Charles E.; Papaemmanuil, Elli; Menon, Suraj; Godfrey, Anna L.; Dimitropoulou, Danai; Guglielmelli, Paola; Bellosillo, Beatriz; Besses, Carles; Döhner, Konstanze; Harrison, Claire N.; Vassiliou, George S.; Vannucchi, Alessandro; Campbell, Peter J.; Green, Anthony R.

2015-01-01

BACKGROUND Cancers result from the accumulation of somatic mutations, and their properties are thought to reflect the sum of these mutations. However, little is known about the effect of the order in which mutations are acquired. METHODS We determined mutation order in patients with myeloproliferative neoplasms by genotyping hematopoietic colonies or by means of next-generation sequencing. Stem cells and progenitor cells were isolated to study the effect of mutation order on mature and immature hematopoietic cells. RESULTS The age at which a patient presented with a myeloproliferative neoplasm, acquisition of JAK2 V617F homozygosity, and the balance of immature progenitors were all influenced by mutation order. As compared with patients in whom the TET2 mutation was acquired first (hereafter referred to as “TET2-first patients”), patients in whom the Janus kinase 2 (JAK2) mutation was acquired first (“JAK2-first patients”) had a greater likelihood of presenting with polycythemia vera than with essential thrombocythemia, an increased risk of thrombosis, and an increased sensitivity of JAK2-mutant progenitors to ruxolitinib in vitro. Mutation order influenced the proliferative response to JAK2 V617F and the capacity of double-mutant hematopoietic cells and progenitor cells to generate colony-forming cells. Moreover, the hematopoietic stem-and-progenitor-cell compartment was dominated by TET2 single-mutant cells in TET2-first patients but by JAK2–TET2 double-mutant cells in JAK2-first patients. Prior mutation of TET2 altered the transcriptional consequences of JAK2 V617F in a cell-intrinsic manner and prevented JAK2 V617F from up-regulating genes associated with proliferation. CONCLUSIONS The order in which JAK2 and TET2 mutations were acquired influenced clinical features, the response to targeted therapy, the biology of stem and progenitor cells, and clonal evolution in patients with myeloproliferative neoplasms. (Funded by Leukemia and Lymphoma Research and others.) PMID:25671252

Germ line variants predispose to both JAK2 V617F clonal hematopoiesis and myeloproliferative neoplasms

PubMed Central

Hinds, David A.; Barnholt, Kimberly E.; Mesa, Ruben A.; Kiefer, Amy K.; Do, Chuong B.; Eriksson, Nicholas; Mountain, Joanna L.; Francke, Uta; Tung, Joyce Y.; Nguyen, Huong (Marie); Zhang, Haiyu; Gojenola, Linda; Zehnder, James L.

2016-01-01

We conducted a genome-wide association study (GWAS) to identify novel predisposition alleles associated with Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) and JAK2 V617F clonal hematopoiesis in the general population. We recruited a web-based cohort of 726 individuals with polycythemia vera, essential thrombocythemia, and myelofibrosis and 252 637 population controls unselected for hematologic phenotypes. Using a single-nucleotide polymorphism (SNP) array platform with custom probes for the JAK2 V617F mutation (V617F), we identified 497 individuals (0.2%) among the population controls who were V617F carriers. We performed a combined GWAS of the MPN cases plus V617F carriers in the control population (n = 1223) vs the remaining controls who were noncarriers for V617F (n = 252 140). For these MPN cases plus V617F carriers, we replicated the germ line JAK2 46/1 haplotype (rs59384377: odds ratio [OR] = 2.4, P = 6.6 × 10−89), previously associated with V617F-positive MPN. We also identified genome-wide significant associations in the TERT gene (rs7705526: OR = 1.8, P = 1.1 × 10−32), in SH2B3 (rs7310615: OR = 1.4, P = 3.1 × 10−14), and upstream of TET2 (rs1548483: OR = 2.0, P = 2.0 × 10−9). These associations were confirmed in a separate replication cohort of 446 V617F carriers vs 169 021 noncarriers. In a joint analysis of the combined GWAS and replication results, we identified additional genome-wide significant predisposition alleles associated with CHEK2, ATM, PINT, and GFI1B. All SNP ORs were similar for MPN patients and controls who were V617F carriers. These data indicate that the same germ line variants endow individuals with a predisposition not only to MPN, but also to JAK2 V617F clonal hematopoiesis, a more common phenomenon that may foreshadow the development of an overt neoplasm. PMID:27365426

The mutation profile of JAK2, MPL and CALR in Mexican patients with Philadelphia chromosome-negative myeloproliferative neoplasms.

PubMed

Labastida-Mercado, Nancy; Galindo-Becerra, Samantha; Garcés-Eisele, Javier; Colunga-Pedraza, Perla; Guzman-Olvera, Valeria; Reyes-Nuñez, Virginia; Ruiz-Delgado, Guillermo J; Ruiz-Argüelles, Guillermo J

2015-03-01

By using molecular markers, it is possible to gain information on both the classification and etiopathogenesis of chronic myeloproliferative neoplasias (MPN). In a group of 27 Mexican mestizo patients with MPNs, we studied seven molecular markers: the BCR/ABL1 fusion gene, the JAK2 V617F mutation, the JAK2 exon 12 mutations, the MPL W515L mutation, the MPL W515K mutation, and the calreticulin (CALR) exon 9 deletion or insertion. Patients with the BCR/ABL1 fusion gene were excluded. We studied 14 patients with essential thrombocythemia (ET), eight with polycythemia vera (PV), four with primary myelofibrosis (MF), and one with undifferentiated MPN. We found twelve individuals with the JAK2 V617F mutation; five of them had been clinically classified as PV, five as ET, and one as MF. One patient with the MPL W515L was identified with a clinical picture of ET. Five patients with the CALR mutation were identified, four ET and one MF. No individuals with either the MPL W515K mutation or the JAK2 exon 12 mutations were identified. The most consistent relationship was that between PV and the JAK2 V617F mutation (p=.01). Despite its small size, the study shows much less prevalence of JAK2 mutation in PV, ET and MF, which does not match international data. Copyright © 2015 King Faisal Specialist Hospital & Research Centre. Published by Elsevier B.V. All rights reserved.

Risk factors for vascular complications and treatment patterns at diagnosis of 2389 PV and ET patients: Real-world data from the Swedish MPN Registry.

PubMed

Abdulkarim, Khadija; Samuelsson, Jan; Johansson, Peter; Andréasson, Björn

2017-06-01

The study mainly aimed at investigating possible correlations between peripheral blood counts, erythropoietin (EPO), JAK2 V617F mutation, and vascular complications prior to diagnosis of a population-based cohort of newly diagnosed patients with myeloproliferative neoplasms (MPN). The study comprises 1105 patients with polycythemia vera (PV) and 1284 patients with essential thrombocythemia (ET) registered in the Swedish MPN Registry. Vascular complications, prior to diagnosis, were registered in 37% of PV patients. In multivariate analysis, low hemoglobin was the only significant risk factor (P=.0120). Among ET patients, 35% had encountered a vascular complication. Risk factors for thromboembolic complications in ET were identified as age>65 years, white cell count>12×10 9 /L, and the presence of JAK2 V617F mutation (P=.0004, P=.0038, and P=.0016, respectively). A JAK2 V617F mutation was present in 71% of ET patients with vascular complications, compared to 60% in patients without. A majority of complications were thromboembolic, in both PV and ET. We conclude that vascular complications among newly diagnosed patients had affected more than one-third of our study population. Risk factors for vascular complications prior to diagnosis were lower hemoglobin in PV, and the presence of JAK2 V617F mutation, higher age, and leukocytosis in ET. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Screening and monitoring of MPL W515L mutation with real-time PCR in patients with myelofibrosis undergoing allogeneic-SCT.

PubMed

Alchalby, H; Badbaran, A; Bock, O; Fehse, B; Bacher, U; Zander, A R; Kröger, N

2010-09-01

Monitoring of minimal residual disease (MRD) after allogeneic (allo)-SCT for myelofibrosis (MF) allows recognizing the depth of remission and thus guides application of appropriate therapeutic interventions. MPL W515L/K mutations, which are detected in 5-10% of JAK2V617F-negative patients, may be useful for this purpose. Using a highly sensitive quantitative PCR method, we tested 90 patients with MF who underwent allo-SCT for the presence of MPL W515L/K mutations. Two patients with primary MF were found to harbor MPLW515L while no patient was positive for MPLW515K mutation. Both patients were JAK2V617F negative and cleared the mutation rapidly after allo-SCT and remained negative for a median follow-up of 19 months. The results of molecular monitoring correlated well with other remission parameters such as normalization of peripheral blood counts and morphology and complete donor chimerism. We conclude that MPLW515L can be cleared after allo-SCT and hence may be used as an MRD marker in a proportion of JAK2V617F-negative MF patients.

Molecular drug targets in myeloproliferative neoplasms: mutant ABL1, JAK2, MPL, KIT, PDGFRA, PDGFRB and FGFR1

PubMed Central

Tefferi, Ayalew

2009-01-01

Abstract Therapeutically validated oncoproteins in myeloproliferative neoplasms (MPN) include BCR-ABL1 and rearranged PDGFR proteins. The latter are products of intra- (e.g. FIP1L1-PDGFRA) or inter-chromosomal (e.g.ETV6-PDGFRB) gene fusions. BCR-ABL1 is associated with chronic myelogenous leukaemia (CML) and mutant PDGFR with an MPN phenotype characterized by eosinophilia and in addition, in case of FIP1L1-PDGFRA, bone marrow mastocytosis. These genotype-phenotype associations have been effectively exploited in the development of highly accurate diagnostic assays and molecular targeted therapy. It is hoped that the same will happen in other MPN with specific genetic alterations: polycythemia vera (JAK2V617F and other JAK2 mutations), essential thrombocythemia (JAK2V617F and MPL515 mutations), primary myelofibrosis (JAK2V617F and MPL515 mutations), systemic mastocytosis (KITD816V and other KIT mutations) and stem cell leukaemia/lymphoma (ZNF198-FGFR1 and other FGFR1 fusion genes). The current review discusses the above-listed mutant molecules in the context of their value as drug targets. PMID:19175693

JAK2 haplotype is a major risk factor for the development of myeloproliferative neoplasms

PubMed Central

Jones, Amy V; Chase, Andrew; Silver, Richard T; Oscier, David; Zoi, Katerina; Wang, Y Lynn; Cario, Holger; Pahl, Heike L; Collins, Andrew; Reiter, Andreas; Grand, Francis; Cross, Nicholas C P

2014-01-01

Chronic myeloproliferative neoplasms (MPNs) are a group of related conditions characterized by the overproduction of cells from one or more myeloid lineages. More than 95% of cases of polycythemia vera, and roughly half of essential thrombocythemia and primary myelofibrosis acquire a unique somatic 1849G>T JAK2 mutation (encoding V617F) that is believed to be a critical driver of excess proliferation1–4. We report here that JAK2V617F-associated disease is strongly associated with a specific constitutional JAK2 haplotype, designated 46/1, in all three disease entities compared to healthy controls (polycythemia vera, n = 192, P = 2.9 × 10−16; essential thrombocythemia, n = 78, P = 8.2 × 10−9 and myelofibrosis, n = 41, P = 8.0 × 10−5). Furthermore, JAK2V617F specifically arises on the 46/1 allele in most cases. The 46/1 JAK2 haplotype thus predisposes to the development of JAK2V617F-associated MPNs (OR = 3.7; 95% CI = 3.1–4.3) and provides a model whereby a constitutional genetic factor is associated with an increased risk of acquiring a specific somatic mutation. PMID:19287382

Targeting glutamine metabolism in myeloproliferative neoplasms

PubMed Central

Zhan, Huichun; Ciano, Kristen; Dong, Katherine; Zucker, Stanley

2016-01-01

JAK2V617F mutation can be detected in the majority of myeloproliferative neoplasm (MPN) patients. The JAK2 inhibitor Ruxolitinib is the first FDA-approved treatment for MPNs. However, its use is limited by various dose related toxicities. Here, we studied the metabolic state and glutamine metabolism of BaF3-hEPOR-JAK2V617F and BaF3-hEPOR-JAK2WT cells. We found that the JAK2V617F-mutant cells were associated with increased oxygen consumption rate and extracellular acidification rate than the JAK2WT cells and there was an increased glutamine metabolism in JAK2V617F-mutant cells compared to wild-type cells. Glutaminase (GLS), the key enzyme in gluta-mine metabolism, was upregulated in the JAK2V617F-mutant BaF3 cells compared to the JAK2WT BaF3 cells. In MPN patient peripheral blood CD34+ cells, GLS expression was increased in JAK2V617F-mutant progenitor cells compared to JAK2 wild-type progenitor cells from the same patients and GLS levels were increased at the time of disease progression compared to at earlier time points. Moreover, GLS inhibitor increased the growth inhibitory effect of Ruxolitinib in both JAK2V617F-mutant cell lines and peripheral blood CD34+ cells from MPN patients. Therefore, GLS inhibitor should be further explored to enhance the therapeutic effectiveness of JAK2 inhibitor and allow the administration of lower doses of the drug to avoid its toxicity. PMID:26227854

Loss of Ezh2 synergizes with JAK2-V617F in initiating myeloproliferative neoplasms and promoting myelofibrosis

PubMed Central

Nienhold, Ronny; Zmajkovic, Jakub; Hao-Shen, Hui; Geier, Florian; Dirnhofer, Stephan; Feenstra, Jelena D. Milosevic

2016-01-01

Myeloproliferative neoplasm (MPN) patients frequently show co-occurrence of JAK2-V617F and mutations in epigenetic regulator genes, including EZH2. In this study, we show that JAK2-V617F and loss of Ezh2 in hematopoietic cells contribute synergistically to the development of MPN. The MPN phenotype induced by JAK2-V617F was accentuated in JAK2-V617F;Ezh2−/− mice, resulting in very high platelet and neutrophil counts, more advanced myelofibrosis, and reduced survival. These mice also displayed expansion of the stem cell and progenitor cell compartments and a shift of differentiation toward megakaryopoiesis at the expense of erythropoiesis. Single cell limiting dilution transplantation with bone marrow from JAK2-V617F;Ezh2+/− mice showed increased reconstitution and MPN disease initiation potential compared with JAK2-V617F alone. RNA sequencing in Ezh2-deficient hematopoietic stem cells (HSCs) and megakaryocytic erythroid progenitors identified highly up-regulated genes, including Lin28b and Hmga2, and chromatin immunoprecipitation (ChIP)–quantitative PCR (qPCR) analysis of their promoters revealed decreased H3K27me3 deposition. Forced expression of Hmga2 resulted in increased chimerism and platelet counts in recipients of retrovirally transduced HSCs. JAK2-V617F–expressing mice treated with an Ezh2 inhibitor showed higher platelet counts than vehicle controls. Our data support the proposed tumor suppressor function of EZH2 in patients with MPN and call for caution when considering using Ezh2 inhibitors in MPN. PMID:27401344

JAK2 inhibitor therapy in myeloproliferative disorders: rationale, preclinical studies and ongoing clinical trials.

PubMed

Pardanani, A

2008-01-01

The recent identification of somatic mutations such as JAK2V617F that deregulate Janus kinase (JAK)-signal transducer and activator of transcription signaling has spurred development of orally bioavailable small-molecule inhibitors that selectively target JAK2 kinase as an approach to pathogenesis-directed therapy of myeloproliferative disorders (MPD). In pre-clinical studies, these compounds inhibit JAK2V617F-mediated cell growth at nanomolar concentrations, and in vivo therapeutic efficacy has been demonstrated in mouse models of JAK2V617F-induced disease. In addition, ex vivo growth of progenitor cells from MPD patients harboring JAK2V617F or MPLW515L/K mutations is also potently inhibited. JAK2 inhibitors currently in clinical trials can be grouped into those designed to primarily target JAK2 kinase (JAK2-selective) and those originally developed for non-MPD indications, but that nevertheless have significant JAK2-inhibitory activity (non-JAK2 selective). This article discusses the rationale for using JAK2 inhibitors for the treatment of MPD, as well as relevant aspects of clinical trial development for these patients. For instance, which group of MPD patients is appropriate for initial Phase I studies? Should JAK2V617F-negative MPD patients be included in the initial studies? What are the likely consequences of 'off-target' JAK3 and wild-type JAK2 inhibition? How should treatment responses be monitored?

Co-targeting the PI3K/mTOR and JAK2 signalling pathways produces synergistic activity against myeloproliferative neoplasms

PubMed Central

Bartalucci, Niccolò; Tozzi, Lorenzo; Bogani, Costanza; Martinelli, Serena; Rotunno, Giada; Villeval, Jean-Luc; Vannucchi, Alessandro M

2013-01-01

Aberrant JAK2 signalling plays a central role in myeloproliferative neoplasms (MPN). JAK2 inhibitors have proven to be clinically efficacious, however, they are not mutation-specific and competent enough to suppress neoplastic clonal haematopoiesis. We hypothesized that, by simultaneously targeting multiple activated signalling pathways, MPN could be more effectively treated. To this end we investigated the efficacy of BEZ235, a dual PI3K/mTOR inhibitor, alone and in combination with the JAK1/JAK2 inhibitor ruxolitinib, in different preclinical models of MPN. Single-agent BEZ235 inhibited the proliferation and induced cell cycle arrest and apoptosis of mouse and human JAK2V617F mutated cell lines at concentrations significantly lower than those required to inhibit the wild-type counterpart, and preferentially prevented colony formation from JAK2V617F knock-in mice and patients' progenitor cells compared with normal ones. Co-treatment of BEZ235 and ruxolitinib produced significant synergism in all these in-vitro models. Co-treatment was also more effective than single drugs in reducing the extent of disease and prolonging survival of immunodeficient mice injected with JAK2V617F-mutated Ba/F3-EPOR cells and in reducing spleen size, decreasing reticulocyte count and improving spleen histopathology in conditional JAK2V617F knock-in mice. In conclusion, combined inhibition of PI3K/mTOR and JAK2 signalling may represent a novel therapeutic strategy in MPN. PMID:24237791

An MPL W515L mutation in refractory anemia with ringed sideroblasts associated with marked thrombocytosis: A case report.

PubMed

Hao, Lin; Sen, Sandeep; Sugumar, Dhivya

2015-02-01

The current study presents the case of a 63-year-old patient exhibiting refractory anemia with ringed sideroblasts associated with marked thrombocytosis (RARS-T), who was positive for the MPL W515L mutation, but negative for the JAK2 V617F mutation. Following diagnosis, the patient remained asymptomatic for over three years, however, in August 2012, the patient relapsed and was administered with supportive treatment in the form of subcutaneous darbepoetin α at a dose of 300 μg/week, which resulted in an increased hemoglobin concentration, allowing the patient to remain transfusion-independent. The MPL W515L mutation has been reported in two previous cases of myelodysplastic/myeloproliferative neoplasms (MDS/MPN) with ringed sideroblasts, however, to the best of our knowledge, the current report is the first to present a case of RARS-T with an MPL W515L mutation. A clinical trial designed to evaluate the efficacy of a targeted agent against the JAK2 V617F mutation is currently ongoing, with the aim of providing a novel therapeutic strategy for treating MDS/MPN patients. As MPL is located upstream of the JAK-STAT signaling pathway, it is a possible therapeutic target in MDS/MPN patients positive for an MPL W515L mutation, but negative for a JAK2 V617F mutation.

SOCS3 tyrosine phosphorylation as a potential bio-marker for myeloproliferative neoplasms associated with mutant JAK2 kinases

PubMed Central

Elliott, Joanne; Suessmuth, Yvonne; Scott, Linda M.; Nahlik, Krystyna; McMullin, Mary Frances; Constantinescu, Stefan N.; Green, Anthony R.; Johnston, James A.

2009-01-01

JAK2 V617F, identified in the majority of patients with myeloproliferative neoplasms, tyrosine phosphorylates SOCS3 and escapes its inhibition. Here, we demonstrate that the JAK2 exon 12 mutants described in a subset of V617F-negative MPN cases, also stabilize tyrosine phosphorylated SOCS3. SOCS3 tyrosine phosphorylation was also observed in peripheral blood mononuclear cells and granulocytes isolated from patients with JAK2 H538QK539L or JAK2 F537-K539delinsL mutations. JAK kinase inhibitors, which effectively inhibited the proliferation of cells expressing V617F or K539L, also caused a dose-dependent reduction in both mutant JAK2 and SOCS3 tyrosine phosphorylation. We propose, therefore, that SOCS3 tyrosine phosphorylation may be a novel bio-marker of myeloproliferative neoplasms resulting from a JAK2 mutation and a potential reporter of effective JAK2 inhibitor therapy currently in clinical development. PMID:19229050

Preclinical characterization of the selective JAK1/2 inhibitor INCB018424: therapeutic implications for the treatment of myeloproliferative neoplasms

PubMed Central

Quintás-Cardama, Alfonso; Vaddi, Kris; Liu, Phillip; Manshouri, Taghi; Li, Jun; Scherle, Peggy A.; Caulder, Eian; Wen, Xiaoming; Li, Yanlong; Waeltz, Paul; Rupar, Mark; Burn, Timothy; Lo, Yvonne; Kelley, Jennifer; Covington, Maryanne; Shepard, Stacey; Rodgers, James D.; Haley, Patrick; Kantarjian, Hagop

2010-01-01

Constitutive JAK2 activation in hematopoietic cells by the JAK2V617F mutation recapitulates myeloproliferative neoplasm (MPN) phenotypes in mice, establishing JAK2 inhibition as a potential therapeutic strategy. Although most polycythemia vera patients carry the JAK2V617F mutation, half of those with essential thrombocythemia or primary myelofibrosis do not, suggesting alternative mechanisms for constitutive JAK-STAT signaling in MPNs. Most patients with primary myelofibrosis have elevated levels of JAK-dependent proinflammatory cytokines (eg, interleukin-6) consistent with our observation of JAK1 hyperactivation. Accordingly, we evaluated the effectiveness of selective JAK1/2 inhibition in experimental models relevant to MPNs and report on the effects of INCB018424, the first potent, selective, oral JAK1/JAK2 inhibitor to enter the clinic. INCB018424 inhibited interleukin-6 signaling (50% inhibitory concentration [IC50] = 281nM), and proliferation of JAK2V617F+ Ba/F3 cells (IC50 = 127nM). In primary cultures, INCB018424 preferentially suppressed erythroid progenitor colony formation from JAK2V617F+ polycythemia vera patients (IC50 = 67nM) versus healthy donors (IC50 > 400nM). In a mouse model of JAK2V617F+ MPN, oral INCB018424 markedly reduced splenomegaly and circulating levels of inflammatory cytokines, and preferentially eliminated neoplastic cells, resulting in significantly prolonged survival without myelosuppressive or immunosuppressive effects. Preliminary clinical results support these preclinical data and establish INCB018424 as a promising oral agent for the treatment of MPNs. PMID:20130243

MPLW515L Is a Novel Somatic Activating Mutation in Myelofibrosis with Myeloid Metaplasia

PubMed Central

Pikman, Yana; Lee, Benjamin H; Mercher, Thomas; McDowell, Elizabeth; Ebert, Benjamin L; Gozo, Maricel; Cuker, Adam; Wernig, Gerlinde; Moore, Sandra; Galinsky, Ilene; DeAngelo, Daniel J; Clark, Jennifer J; Lee, Stephanie J; Golub, Todd R; Wadleigh, Martha; Gilliland, D. Gary; Levine, Ross L

2006-01-01

Background The JAK2V617F allele has recently been identified in patients with polycythemia vera (PV), essential thrombocytosis (ET), and myelofibrosis with myeloid metaplasia (MF). Subsequent analysis has shown that constitutive activation of the JAK-STAT signal transduction pathway is an important pathogenetic event in these patients, and that enzymatic inhibition of JAK2V617F may be of therapeutic benefit in this context. However, a significant proportion of patients with ET or MF are JAK2V617F-negative. We hypothesized that activation of the JAK-STAT pathway might also occur as a consequence of activating mutations in certain hematopoietic-specific cytokine receptors, including the erythropoietin receptor (EPOR), the thrombopoietin receptor (MPL), or the granulocyte-colony stimulating factor receptor (GCSFR). Methods and Findings DNA sequence analysis of the exons encoding the transmembrane and juxtamembrane domains of EPOR, MPL, and GCSFR, and comparison with germline DNA derived from buccal swabs, identified a somatic activating mutation in the transmembrane domain of MPL (W515L) in 9% (4/45) of JAKV617F-negative MF. Expression of MPLW515L in 32D, UT7, or Ba/F3 cells conferred cytokine-independent growth and thrombopoietin hypersensitivity, and resulted in constitutive phosphorylation of JAK2, STAT3, STAT5, AKT, and ERK. Furthermore, a small molecule JAK kinase inhibitor inhibited MPLW515L-mediated proliferation and JAK-STAT signaling in vitro. In a murine bone marrow transplant assay, expression of MPLW515L, but not wild-type MPL, resulted in a fully penetrant myeloproliferative disorder characterized by marked thrombocytosis (Plt count 1.9–4.0 × 10 12/L), marked splenomegaly due to extramedullary hematopoiesis, and increased reticulin fibrosis. Conclusions Activation of JAK-STAT signaling via MPLW515L is an important pathogenetic event in patients with JAK2V617F-negative MF. The bone marrow transplant model of MPLW515L-mediated myeloproliferative disorders (MPD) exhibits certain features of human MF, including extramedullary hematopoiesis, splenomegaly, and megakaryocytic proliferation. Further analysis of positive and negative regulators of the JAK-STAT pathway is warranted in JAK2V617F-negative MPD. PMID:16834459

MPLW515L is a novel somatic activating mutation in myelofibrosis with myeloid metaplasia.

PubMed

Pikman, Yana; Lee, Benjamin H; Mercher, Thomas; McDowell, Elizabeth; Ebert, Benjamin L; Gozo, Maricel; Cuker, Adam; Wernig, Gerlinde; Moore, Sandra; Galinsky, Ilene; DeAngelo, Daniel J; Clark, Jennifer J; Lee, Stephanie J; Golub, Todd R; Wadleigh, Martha; Gilliland, D Gary; Levine, Ross L

2006-07-01

The JAK2V617F allele has recently been identified in patients with polycythemia vera (PV), essential thrombocytosis (ET), and myelofibrosis with myeloid metaplasia (MF). Subsequent analysis has shown that constitutive activation of the JAK-STAT signal transduction pathway is an important pathogenetic event in these patients, and that enzymatic inhibition of JAK2V617F may be of therapeutic benefit in this context. However, a significant proportion of patients with ET or MF are JAK2V617F-negative. We hypothesized that activation of the JAK-STAT pathway might also occur as a consequence of activating mutations in certain hematopoietic-specific cytokine receptors, including the erythropoietin receptor (EPOR), the thrombopoietin receptor (MPL), or the granulocyte-colony stimulating factor receptor (GCSFR). DNA sequence analysis of the exons encoding the transmembrane and juxtamembrane domains of EPOR, MPL, and GCSFR, and comparison with germline DNA derived from buccal swabs, identified a somatic activating mutation in the transmembrane domain of MPL (W515L) in 9% (4/45) of JAKV617F-negative MF. Expression of MPLW515L in 32D, UT7, or Ba/F3 cells conferred cytokine-independent growth and thrombopoietin hypersensitivity, and resulted in constitutive phosphorylation of JAK2, STAT3, STAT5, AKT, and ERK. Furthermore, a small molecule JAK kinase inhibitor inhibited MPLW515L-mediated proliferation and JAK-STAT signaling in vitro. In a murine bone marrow transplant assay, expression of MPLW515L, but not wild-type MPL, resulted in a fully penetrant myeloproliferative disorder characterized by marked thrombocytosis (Plt count 1.9-4.0 x 10(12)/L), marked splenomegaly due to extramedullary hematopoiesis, and increased reticulin fibrosis. Activation of JAK-STAT signaling via MPLW515L is an important pathogenetic event in patients with JAK2V617F-negative MF. The bone marrow transplant model of MPLW515L-mediated myeloproliferative disorders (MPD) exhibits certain features of human MF, including extramedullary hematopoiesis, splenomegaly, and megakaryocytic proliferation. Further analysis of positive and negative regulators of the JAK-STAT pathway is warranted in JAK2V617F-negative MPD.

Characteristics and clinical correlates of MPL 515W>L/K mutation in essential thrombocythemia.

PubMed

Vannucchi, Alessandro M; Antonioli, Elisabetta; Guglielmelli, Paola; Pancrazzi, Alessandro; Guerini, Vittoria; Barosi, Giovanni; Ruggeri, Marco; Specchia, Giorgina; Lo-Coco, Francesco; Delaini, Federica; Villani, Laura; Finotto, Silvia; Ammatuna, Emanuele; Alterini, Renato; Carrai, Valentina; Capaccioli, Gloria; Di Lollo, Simonetta; Liso, Vincenzo; Rambaldi, Alessandro; Bosi, Alberto; Barbui, Tiziano

2008-08-01

Among 994 patients with essential thrombocythemia (ET) who were genotyped for the MPLW515L/K mutation, 30 patients carrying the mutation were identified (3.0%), 8 of whom also displayed the JAK2V671F mutation. MPLW515L/K patients presented lower hemoglobin levels and higher platelet counts than did wild type (wt) MPL; these differences were highly significant compared with MPLwt/JAK2V617F-positive patients. Reduced hemoglobin and increased platelet levels were preferentially associated with the W515L and W515K alleles, respectively. MPL mutation was a significant risk factor for microvessel disturbances, suggesting platelet hyperreactivity associated with constitutively active MPL; arterial thromboses were increased only in comparison to MPLwt/JAK2wt patients. MPLW515L/K patients presented reduced total and erythroid bone marrow cellularity, whereas the numbers of megakaryocytes, megakaryocytic clusters, and small-sized megakaryocytes were all significantly increased. These data indicate that MPLW515L/K mutations do not define a distinct phenotype in ET, although some differences depended on the JAK2V617F mutational status of the counterpart.

Genetic variation at MECOM, TERT, JAK2 and HBS1L-MYB predisposes to myeloproliferative neoplasms

PubMed Central

Tapper, William; Jones, Amy V.; Kralovics, Robert; Harutyunyan, Ashot S.; Zoi, Katerina; Leung, William; Godfrey, Anna L.; Guglielmelli, Paola; Callaway, Alison; Ward, Daniel; Aranaz, Paula; White, Helen E.; Waghorn, Katherine; Lin, Feng; Chase, Andrew; Joanna Baxter, E.; Maclean, Cathy; Nangalia, Jyoti; Chen, Edwin; Evans, Paul; Short, Michael; Jack, Andrew; Wallis, Louise; Oscier, David; Duncombe, Andrew S.; Schuh, Anna; Mead, Adam J.; Griffiths, Michael; Ewing, Joanne; Gale, Rosemary E.; Schnittger, Susanne; Haferlach, Torsten; Stegelmann, Frank; Döhner, Konstanze; Grallert, Harald; Strauch, Konstantin; Tanaka, Toshiko; Bandinelli, Stefania; Giannopoulos, Andreas; Pieri, Lisa; Mannarelli, Carmela; Gisslinger, Heinz; Barosi, Giovanni; Cazzola, Mario; Reiter, Andreas; Harrison, Claire; Campbell, Peter; Green, Anthony R.; Vannucchi, Alessandro; Cross, Nicholas C.P.

2015-01-01

Clonal proliferation in myeloproliferative neoplasms (MPN) is driven by somatic mutations in JAK2, CALR or MPL, but the contribution of inherited factors is poorly characterized. Using a three-stage genome-wide association study of 3,437 MPN cases and 10,083 controls, we identify two SNPs with genome-wide significance in JAK2V617F-negative MPN: rs12339666 (JAK2; meta-analysis P=1.27 × 10−10) and rs2201862 (MECOM; meta-analysis P=1.96 × 10−9). Two additional SNPs, rs2736100 (TERT) and rs9376092 (HBS1L/MYB), achieve genome-wide significance when including JAK2V617F-positive cases. rs9376092 has a stronger effect in JAK2V617F-negative cases with CALR and/or MPL mutations (Breslow–Day P=4.5 × 10−7), whereas in JAK2V617F-positive cases rs9376092 associates with essential thrombocythemia (ET) rather than polycythemia vera (allelic χ2 P=7.3 × 10−7). Reduced MYB expression, previously linked to development of an ET-like disease in model systems, associates with rs9376092 in normal myeloid cells. These findings demonstrate that multiple germline variants predispose to MPN and link constitutional differences in MYB expression to disease phenotype. PMID:25849990

Spectrum of mutations in RARS-T patients includes TET2 and ASXL1 mutations.

PubMed

Szpurka, Hadrian; Jankowska, Anna M; Makishima, Hideki; Bodo, Juraj; Bejanyan, Nelli; Hsi, Eric D; Sekeres, Mikkael A; Maciejewski, Jaroslaw P

2010-08-01

While a majority of patients with refractory anemia with ring sideroblasts and thrombocytosis harbor JAK2V617F and rarely MPLW515L, JAK2/MPL-negative cases constitute a diagnostic problem. 23 RARS-T cases were investigated applying immunohistochemical phospho-STAT5, sequencing and SNP-A-based karyotyping. Based on the association of TET2/ASXL1 mutations with MDS/MPN we studied molecular pattern of these genes. Two patients harbored ASXL1 and another 2 TET2 mutations. Phospho-STAT5 activation was present in one mutated TET2 and ASXL1 case. JAK2V617F/MPLW515L mutations were absent in TET2/ASXL1 mutants, indicating that similar clinical phenotype can be produced by various MPN-associated mutations and that additional unifying lesions may be present in RARS-T. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Peptide nucleic acid probe-based fluorescence melting curve analysis for rapid screening of common JAK2, MPL, and CALR mutations.

PubMed

Park, Joonhong; Song, Minsik; Jang, Woori; Chae, Hyojin; Lee, Gun Dong; Kim, KyungTak; Park, Heekyung; Kim, Myungshin; Kim, Yonggoo

2017-02-01

We developed and evaluated the feasibility of peptide nucleic acid (PNA)-based fluorescence melting curve analysis (FMCA) to detect common mutations in myeloproliferative neoplasms (MPNs). We have set up two separate reactions of PNA-based FMCA: JAK2 V617F &CALR p.Leu367fs*46 (set A) and MPL W515L/K &CALR p.Lys385fs*47 (set B). Clinical usefulness was validated with allele-specific real-time PCR, fragment analysis, Sanger sequencing in 57 BCR-ABL1-negative MPNs. The limit of detection (LOD) of PNA-based FMCA was approximately 10% for each mutation and interference reactions using mixtures of different mutations were not observed. Non-specific amplification was not observed in normal control. PNA-based FMCA was able to detect all JAK2 V617F (n=20), CALR p.Leu367fs*46 (n=10) and p.Lys385fs*47 (n=8). Three of six MPL mutations were detected except three samples with low mutant concentration in out of LOD. JAK2 exon 12 mutations (n=7) were negative without influencing V617F results. Among six variant CALR exon 9 mutations, two were detected by this method owing to invading of probe binding site. PNA-based FMCA for detecting common JAK2, MPL, and CALR mutations is a rapid, simple, and sensitive technique in BCR-ABL1-negative MPNs with >10% mutant allele at the time of initial diagnosis. Copyright © 2016 Elsevier B.V. All rights reserved.

Germline and somatic JAK2 mutations and susceptibility to chronic myeloproliferative neoplasms

PubMed Central

2009-01-01

Myeloproliferative neoplasms (MPNs) are a group of closely related stem-cell-derived clonal proliferative diseases. Most cases are sporadic but first-degree relatives of MPN patients have a five- to seven-fold increased risk for developing an MPN. The tumors of most patients carry a mutation in the Janus kinase 2 gene (JAK2V617F). Recently, three groups have described a strong association of JAK2 germline polymorphisms with MPN in patients positive for JAK2V617F. The somatic mutation occurs primarily on one particular germline JAK2 haplotype, which may account for as much as 50% of the risk to first-degree relatives. This finding provides new directions for unraveling the pathogenesis of MPN. PMID:19490586

CYT387, a selective JAK1/JAK2 inhibitor: in vitro assessment of kinase selectivity and preclinical studies using cell lines and primary cells from polycythemia vera patients.

PubMed

Pardanani, A; Lasho, T; Smith, G; Burns, C J; Fantino, E; Tefferi, A

2009-08-01

Somatic mutations in Janus kinase 2 (JAK2), including JAK2V617F, result in dysregulated JAK-signal transducer and activator transcription (STAT) signaling, which is implicated in myeloproliferative neoplasm (MPN) pathogenesis. CYT387 is an ATP-competitive small molecule that potently inhibits JAK1/JAK2 kinases (IC(50)=11 and 18 nM, respectively), with significantly less activity against other kinases, including JAK3 (IC(50)=155 nM). CYT387 inhibits growth of Ba/F3-JAK2V617F and human erythroleukemia (HEL) cells (IC(50) approximately 1500 nM) or Ba/F3-MPLW515L cells (IC(50)=200 nM), but has considerably less activity against BCR-ABL harboring K562 cells (IC=58 000 nM). Cell lines harboring mutated JAK2 alleles (CHRF-288-11 or Ba/F3-TEL-JAK2) were inhibited more potently than the corresponding pair harboring mutated JAK3 alleles (CMK or Ba/F3-TEL-JAK3), and STAT-5 phosphorylation was inhibited in HEL cells with an IC(50)=400 nM. Furthermore, CYT387 selectively suppressed the in vitro growth of erythroid colonies harboring JAK2V617F from polycythemia vera (PV) patients, an effect that was attenuated by exogenous erythropoietin. Overall, our data indicate that the JAK1/JAK2 selective inhibitor CYT387 has potential for efficacious treatment of MPN harboring mutated JAK2 and MPL alleles.

The JH2 domain and SH2-JH2 linker regulate JAK2 activity: A detailed kinetic analysis of wild type and V617F mutant kinase domains.

PubMed

Sanz Sanz, Arturo; Niranjan, Yashavanthi; Hammarén, Henrik; Ungureanu, Daniela; Ruijtenbeek, Rob; Touw, Ivo P; Silvennoinen, Olli; Hilhorst, Riet

2014-10-01

JAK2 tyrosine kinase regulates many cellular functions. Its activity is controlled by the pseudokinase (JH2) domain by still poorly understood mechanisms. The V617F mutation in the pseudokinase domain activates JAK2 and causes myeloproliferative neoplasms. We conducted a detailed kinetic analysis of recombinant JAK2 tyrosine kinase domain (JH1) and wild-type and V617F tandem kinase (JH1JH2) domains using peptide microarrays to define the functions of the kinase domains. The results show that i) JAK2 follows a random Bi-Bi reaction mechanism ii) JH2 domain restrains the activity of the JH1 domain by reducing the affinity for ATP and ATP competitive inhibitors iii) V617F decreases affinity for ATP but increases catalytic activity compared to wild-type and iv) the SH2-JH2 linker region participates in controlling activity by reducing the affinity for ATP. Copyright © 2014 Elsevier B.V. All rights reserved.

AML1 is overexpressed in patients with myeloproliferative neoplasms and mediates JAK2V617F-independent overexpression of NF-E2

PubMed Central

Wang, Wei; Schwemmers, Sven; Hexner, Elizabeth O.

2010-01-01

The transcription factor NF-E2 is overexpressed in the majority of patients with polycythemia vera (PV). Concomitantly, 95% of these patients carry the JAK2V617F mutation. Although NF-E2 levels correlate with JAK2V671F allele burden in some PV cohorts, the molecular mechanism causing aberrant NF-E2 expression has not been described. Here we show that NF-E2 expression is also increased in patients with essential thrombocythemia and primary myelofibrosis independent of the presence of the JAK2V617F mutation. Characterization of the NF-E2 promoter revealed multiple functional binding sites for AML1/RUNX-1. Chromatin immunoprecipitation demonstrated AML1 binding to the NF-E2 promoter in vivo. Moreover, AML1 binding to the NF-E2 promoter was significantly increased in granulocytes from PV patients compared with healthy controls. AML1 mRNA expression was elevated in patients with PV, essential thrombocythemia, and primary myelofibrosis both in the presence and absence of JAK2V617F. In addition, AML1 and NF-E2 expression were highly correlated. RNAi-mediated suppression of either AML1 or of its binding partner CBF-β significantly decreased NF-E2 expression. Moreover, expression of the leukemic fusion protein AML/ETO drastically decreased NF-E2 protein levels. Our data identify NF-E2 as a novel AML1 target gene and delineate a role for aberrant AML1 expression in mediating elevated NF-E2 expression in MPN patients. PMID:20339092

A germline JAK2 SNP is associated with predisposition to the development of JAK2V617F-positive myeloproliferative neoplasms

PubMed Central

Kilpivaara, Outi; Mukherjee, Semanti; Schram, Alison M; Wadleigh, Martha; Mullally, Ann; Ebert, Benjamin L; Bass, Adam; Marubayashi, Sachie; Heguy, Adriana; Garcia-Manero, Guillermo; Kantarjian, Hagop; Offit, Kenneth; Stone, Richard M; Gilliland, D Gary; Klein, Robert J; Levine, Ross L

2013-01-01

Polycythemia vera, essential thrombocythemia and primary myelofibrosis are myeloproliferative neoplasms (MPN) characterized by multilineage clonal hematopoiesis1–5. Given that the identical somatic activating mutation in the JAK2 tyrosine kinase gene (JAK2V617F) is observed in most individuals with polycythemia vera, essential thrombocythemia and primary myelofibrosis6–10, there likely are additional genetic events that contribute to the pathogenesis of these phenotypically distinct disorders. Moreover, family members of individuals with MPN are at higher risk for the development of MPN, consistent with the existence of MPN predisposition loci11. We hypothesized that germline variation contributes to MPN predisposition and phenotypic pleiotropy. Genome-wide analysis identified an allele in the JAK2 locus (rs10974944) that predisposes to the development of JAK2V617F-positive MPN, as well as three previously unknown MPN modifier loci. We found that JAK2V617F is preferentially acquired in cis with the predisposition allele. These data suggest that germline variation is an important contributor to MPN phenotype and predisposition. PMID:19287384

JAK2 mutations and clinical practice in myeloproliferative neoplasms.

PubMed

Tefferi, Ayalew

2007-01-01

With the discovery in the last 3 years of novel Janus kinase 2 (JAK2) and thrombopoietin receptor (MPL) mutations, the pathogenetic understanding of and clinical practice for myeloproliferative neoplasms (MPNs) have entered a new era. Each one of these newly discovered mutations, including JAK2V617F, MPLW515L, and a JAK2 exon 12 mutation, has been shown to result in constitutive activation of JAK-STAT signaling and also induce a MPN phenotype in mice. Thus, JAK2 is now considered to be a legitimate target for drug development in MPNs, and small molecule JAK2 inhibitors have already gone through successful preclinical testing, and early-phase human trials in primary myelofibrosis have already begun. Furthermore, JAK2 mutation screening has now become a front-line diagnostic test in the evaluation of both "erythrocytosis" and thrombocytosis and the 2001 World Health Organization diagnostic criteria for polycythemia vera, essential thrombocythemia, and primary myelofibrosis have now been revised to incorporate JAK2V617F mutation screening.

JAK2V617F-mutant megakaryocytes contribute to hematopoietic stem/progenitor cell expansion in a model of murine myeloproliferation

PubMed Central

Zhan, H; Ma, Y; Lin, CHS; Kaushansky, K

2016-01-01

The myeloproliferative neoplasms (MPNs) are characterized by hematopoietic stem/progenitor cell (HSPC) expansion and overproduction of mature blood cells. The JAK2V617F mutation is present in hematopoietic cells in a majority of patients with MPNs, but the mechanism(s) responsible for MPN stem cell expansion remain incomplete. One hallmark feature of the marrow in patients with MPNs is megakaryocyte (MK) hyperplasia. We report here that mice bearing a human JAK2V617F gene restricted exclusively to the MK lineage develop many of the features of a MPN. Specifically, these mice exhibit thrombocytosis, splenomegaly, increased numbers of marrow and splenic hematopoietic progenitors and a substantial expansion of HSPCs. In addition, wild-type mice transplanted with cells from JAK2V617F-bearing MK marrow develop a myeloproliferative syndrome with thrombocytosis and erythrocytosis as well as pan-hematopoietic progenitor and stem cell expansion. As marrow histology in this murine model of myeloproliferation reveals a preferentially perivascular localization of JAK2V617F-mutant MKs and an increased marrow sinusoid vascular density, it adds to accumulating data that MKs are an important component of the marrow HSPC niche, and that MK expansion might indirectly contribute to the critical role of the thrombopoietin/c-Mpl signaling pathway in HSPC maintenance and expansion. PMID:27133820

Efficacy of NS-018, a potent and selective JAK2/Src inhibitor, in primary cells and mouse models of myeloproliferative neoplasms.

PubMed

Nakaya, Y; Shide, K; Niwa, T; Homan, J; Sugahara, S; Horio, T; Kuramoto, K; Kotera, T; Shibayama, H; Hori, K; Naito, H; Shimoda, K

2011-07-01

Aberrant activation of Janus kinase 2 (JAK2) caused by somatic mutation of JAK2 (JAK2V617F) or the thrombopoietin receptor (MPLW515L) plays an essential role in the pathogenesis of myeloproliferative neoplasms (MPNs), suggesting that inhibition of aberrant JAK2 activation would have a therapeutic benefit. Our novel JAK2 inhibitor, NS-018, was highly active against JAK2 with a 50% inhibition (IC(50)) of <1 n, and had 30-50-fold greater selectivity for JAK2 over other JAK-family kinases, such as JAK1, JAK3 and tyrosine kinase 2. In addition to JAK2, NS-018 inhibited Src-family kinases. NS-018 showed potent antiproliferative activity against cell lines expressing a constitutively activated JAK2 (the JAK2V617F or MPLW515L mutations or the TEL-JAK2 fusion gene; IC(50)=11-120 n), but showed only minimal cytotoxicity against most other hematopoietic cell lines without a constitutively activated JAK2. Furthermore, NS-018 preferentially suppressed in vitro erythropoietin-independent endogenous colony formation from polycythemia vera patients. NS-018 also markedly reduced splenomegaly and prolonged the survival of mice inoculated with Ba/F3 cells harboring JAK2V617F. In addition, NS-018 significantly reduced leukocytosis, hepatosplenomegaly and extramedullary hematopoiesis, improved nutritional status, and prolonged survival in JAK2V617F transgenic mice. These results suggest that NS-018 will be a promising candidate for the treatment of MPNs.

Efficacy of NS-018, a potent and selective JAK2/Src inhibitor, in primary cells and mouse models of myeloproliferative neoplasms

PubMed Central

Nakaya, Y; Shide, K; Niwa, T; Homan, J; Sugahara, S; Horio, T; Kuramoto, K; Kotera, T; Shibayama, H; Hori, K; Naito, H; Shimoda, K

2011-01-01

Aberrant activation of Janus kinase 2 (JAK2) caused by somatic mutation of JAK2 (JAK2V617F) or the thrombopoietin receptor (MPLW515L) plays an essential role in the pathogenesis of myeloproliferative neoplasms (MPNs), suggesting that inhibition of aberrant JAK2 activation would have a therapeutic benefit. Our novel JAK2 inhibitor, NS-018, was highly active against JAK2 with a 50% inhibition (IC50) of <1 n, and had 30–50-fold greater selectivity for JAK2 over other JAK-family kinases, such as JAK1, JAK3 and tyrosine kinase 2. In addition to JAK2, NS-018 inhibited Src-family kinases. NS-018 showed potent antiproliferative activity against cell lines expressing a constitutively activated JAK2 (the JAK2V617F or MPLW515L mutations or the TEL–JAK2 fusion gene; IC50=11–120 n), but showed only minimal cytotoxicity against most other hematopoietic cell lines without a constitutively activated JAK2. Furthermore, NS-018 preferentially suppressed in vitro erythropoietin-independent endogenous colony formation from polycythemia vera patients. NS-018 also markedly reduced splenomegaly and prolonged the survival of mice inoculated with Ba/F3 cells harboring JAK2V617F. In addition, NS-018 significantly reduced leukocytosis, hepatosplenomegaly and extramedullary hematopoiesis, improved nutritional status, and prolonged survival in JAK2V617F transgenic mice. These results suggest that NS-018 will be a promising candidate for the treatment of MPNs. PMID:22829185

Aberrant let7a/HMGA2 signaling activity with unique clinical phenotype in JAK2-mutated myeloproliferative neoplasms

PubMed Central

Chen, Chih-Cheng; You, Jie-Yu; Lung, Jrhau; Huang, Cih-En; Chen, Yi-Yang; Leu, Yu-Wei; Ho, Hsing-Ying; Li, Chian-Pei; Lu, Chang-Hsien; Lee, Kuan-Der; Hsu, Chia-Chen; Gau, Jyh-Pyng

2017-01-01

High mobility group AT-hook 2 (HMGA2) is an architectural transcription factor that is negatively regulated by let-7 microRNA through binding to it’s 3′-untranslated region. Transgenic mice expressing Hmga2 with a truncation of its 3′-untranslated region has been shown to exhibit a myeloproliferative phenotype. To decipher the let-7-HMGA2 axis in myeloproliferative neoplasms, we employed an in vitro model supplemented with clinical correlation. Ba/F3 cells with inducible JAK2V617F expression (Ton.JAK2.V617F cells) showed upregulation of HMGA2 with concurrent let-7a repression. Ton.JAK2.V617F cells treated with a let-7a inhibitor exhibited further escalation of Hmga2 expression, while a let-7a mimic diminished the Hmga2 transcript level. Hmga2 overexpression conferred JAK2-mutated cells with a survival advantage through inhibited apoptosis. A pan-JAK inhibitor, INC424, increased the expression of let-7a, downregulated the level of Hmga2, and led to increased apoptosis in Ton.JAK2.V617F cells in a dose-dependent manner. In samples from 151 patients with myeloproliferative neoplasms, there was a modest inverse correlation between the expression levels of let-7a and HMGA2. Overexpression of HMGA2 was detected in 29 (19.2%) of the cases, and it was more commonly seen in patients with essential thrombocythemia than in those with polycythemia vera (26.9% vs. 12.7%, P=0.044). Patients with upregulated HMGA2 showed an increased propensity for developing major thrombotic events, and they were more likely to harbor one of the 3 driver myeloproliferative neoplasm mutations in JAK2, MPL and CALR. Our findings suggest that, in a subset of myeloproliferative neoplasm patients, the let-7-HMGA2 axis plays a prominent role in the pathogenesis of the disease that leads to unique clinical phenotypes. PMID:28057739

Mutant calreticulin-expressing cells induce monocyte hyperreactivity through a paracrine mechanism

PubMed Central

Garbati, Michael R.; Welgan, Catherine A.; Landefeld, Sally H.; Newell, Laura F.; Agarwal, Anupriya; Dunlap, Jennifer B.; Chourasia, Tapan K.; Lee, Hyunjung; Elferich, Johannes; Traer, Elie; Rattray, Rogan; Cascio, Michael J.; Press, Richard D.; Bagby, Grover C.; Tyner, Jeffrey W.; Druker, Brian J.; Dao, Kim-Hien T.

2016-01-01

Mutations in the calreticulin gene (CALR) were recently identified in approximately 70–80% of patients with JAK2-V617F-negative essential thrombocytosis and primary myelofibrosis. All frameshift mutations generate a recurring novel C-terminus. Here we provide evidence that mutant calreticulin does not accumulate efficiently in cells and is abnormally enriched in the nucleus and extracellular space compared to wildtype calreticulin. The main determinant of these findings is the loss of the calcium-binding and KDEL domains. Expression of type I mutant CALR in Ba/F3 cells confers minimal IL-3-independent growth. Interestingly, expression of type I and type II mutant CALR in a non-hematopoietic cell line does not directly activate JAK/STAT signaling compared to JAK2-V617F expression. These results led us to investigate paracrine mechanisms of JAK/STAT activation. Here we show that conditioned media from cells expressing type I mutant CALR exaggerate cytokine production from normal monocytes with or without treatment with a toll-like receptor agonist. These effects are not dependent on the novel C-terminus. These studies offer novel insights into the mechanism of JAK/STAT activation in patients with JAK2-V617F-negative essential thrombocytosis and primary myelofibrosis. PMID:26573090

Two Clinical Phenotypes in Polycythemia Vera

PubMed Central

Spivak, Jerry L.; Considine, Michael; Williams, Donna M.; Talbot, Conover C.; Rogers, Ophelia; Moliterno, Alison R.; Jie, Chunfa; Ochs, Michael F.

2014-01-01

BACKGROUND Polycythemia vera is the ultimate phenotypic consequence of the V617F mutation in Janus kinase 2 (encoded by JAK2), but the extent to which this mutation influences the behavior of the involved CD34+ hematopoietic stem cells is unknown. METHODS We analyzed gene expression in CD34+ peripheral-blood cells from 19 patients with polycythemia vera, using oligonucleotide microarray technology after correcting for potential confounding by sex, since the phenotypic features of the disease differ between men and women. RESULTS Men with polycythemia vera had twice as many up-regulated or down-regulated genes as women with polycythemia vera, in a comparison of gene expression in the patients and in healthy persons of the same sex, but there were 102 genes with differential regulation that was concordant in men and women. When these genes were used for class discovery by means of unsupervised hierarchical clustering, the 19 patients could be divided into two groups that did not differ significantly with respect to age, neutrophil JAK2 V617F allele burden, white-cell count, platelet count, or clonal dominance. However, they did differ significantly with respect to disease duration; hemoglobin level; frequency of thromboembolic events, palpable splenomegaly, and splenectomy; chemotherapy exposure; leukemic transformation; and survival. The unsupervised clustering was confirmed by a supervised approach with the use of a top-scoring-pair classifier that segregated the 19 patients into the same two phenotypic groups with 100% accuracy. CONCLUSIONS Removing sex as a potential confounder, we identified an accurate molecular method for classifying patients with polycythemia vera according to disease behavior, independently of their JAK2 V617F allele burden, and identified previously unrecognized molecular pathways in polycythemia vera outside the canonical JAK2 pathway that may be amenable to targeted therapy. PMID:25162887

JAK2, MPL, and CALR mutations in Chinese Han patients with essential thrombocythemia.

PubMed

Wang, Jing; Zhang, Biao; Chen, Bing; Zhou, Rong-Fu; Zhang, Qi-Guo; Li, Juan; Yang, Yong-Gong; Zhou, Min; Shao, Xiao-Yan; Xu, Yong; Xu, Xi-Hui; Ouyang, Jian; Xu, Jingyan; Ye, Qing

2017-04-01

Mutations in Janus kinase 2 (JAK2), myeloproliferative leukemia (MPL), and CALR are highly relevant to Philadelphia chromosome (Ph)-negative myeloproliferative neoplasms. Assessing the prevalence of molecular mutations in Chinese Han patients with essential thrombocythemia (ET), and correlating their mutational profile with disease characteristics/phenotype. Of the 110 subjects studied, 62 carried the JAK2 V617F mutation, 21 had CALR mutations, one carried an MPL (W515) mutation, and 28 had non-mutated JAK2, CALR, and MPL (so-called triple-negative ET). Mutations in JAK2 exon 12 were not detected in any patient. Two ET patients had both CALR and JAK2 V617F mutations. Comparing the hematological parameters of the patients with JAK2 mutations with those of the patients with CALR mutations showed that the ET patients with CALR mutations were younger (p = 0.045) and had higher platelet counts (p = 0.043). Genotyping for CALR could be a useful diagnostic tool for JAK2/MPL-negative ET, since the data suggest that CALR is much more prevalent than MPL, therefore testing for CALR should be considered in patients who are JAK2 negative as its frequency is almost 20 times that of MPL mutation.

Myeloproliferative neoplasms with concurrent BCR-ABL1 translocation and JAK2 V617F mutation: a multi-institutional study from the bone marrow pathology group.

PubMed

Soderquist, Craig R; Ewalt, Mark D; Czuchlewski, David R; Geyer, Julia T; Rogers, Heesun J; Hsi, Eric D; Wang, Sa A; Bueso-Ramos, Carlos E; Orazi, Attilio; Arber, Daniel A; Hexner, Elizabeth O; Babushok, Daria V; Bagg, Adam

2018-05-01

Myeloproliferative neoplasms arise from hematopoietic stem cells with somatically altered tyrosine kinase signaling. Classification of myeloproliferative neoplasms is based on hematologic, histopathologic and molecular characteristics including the presence of the BCR-ABL1 and JAK2 V617F. Although thought to be mutually exclusive, a number of cases with co-occurring BCR-ABL1 and JAK2 V617F have been identified. To characterize the clinicopathologic features of myeloproliferative neoplasms with concomitant BCR-ABL1 and JAK2 V617F, and define the frequency of co-occurrence, we conducted a retrospective multi-institutional study. Cases were identified using a search of electronic databases over a decade at six major institutions. Of 1570 patients who were tested for both BCR-ABL1 and JAK2 V617F, six were positive for both. An additional five patients were identified via clinical records providing a total of 11 cases for detailed evaluation. For each case, clinical variables, hematologic and genetic data, and bone marrow histomorphologic features were analyzed. The sequence of identification of the genetic abnormalities varied: five patients were initially diagnosed with a JAK2 V617F+ myeloproliferative neoplasm, one patient initially had BCR-ABL1+ chronic myeloid leukemia, while both alterations were identified simultaneously in five patients. Classification of the BCR-ABL1-negative myeloproliferative neoplasms varied, and in some cases, features only became apparent following tyrosine kinase inhibitor therapy. Seven of the 11 patients showed myelofibrosis, in some cases before identification of the second genetic alteration. Our data, reflecting the largest reported study comprehensively detailing clinicopathologic features and response to therapy, show that the co-occurrence of BCR-ABL1 and JAK2 V617F is rare, with an estimated frequency of 0.4%, and most often reflects two distinct ('composite') myeloproliferative neoplasms. Although uncommon, it is important to be aware of this potentially confounding genetic combination, lest these features be misinterpreted to reflect resistance to therapy or disease progression, considerations that could lead to inappropriate management.

Single Nucleotide Polymorphism (SNP)-Based Loss of Heterozygosity (LOH) Testing by Real Time PCR in Patients Suspect of Myeloproliferative Disease

PubMed Central

Huijsmans, Cornelis J. J.; Poodt, Jeroen; Damen, Jan; van der Linden, Johannes C.; Savelkoul, Paul H. M.; Pruijt, Johannes F. M.; Hilbink, Mirrian; Hermans, Mirjam H. A.

2012-01-01

During tumor development, loss of heterozygosity (LOH) often occurs. When LOH is preceded by an oncogene activating mutation, the mutant allele may be further potentiated if the wild-type allele is lost or inactivated. In myeloproliferative neoplasms (MPN) somatic acquisition of JAK2V617F may be followed by LOH resulting in loss of the wild type allele. The occurrence of LOH in MPN and other proliferative diseases may lead to a further potentiating the mutant allele and thereby increasing morbidity. A real time PCR based SNP profiling assay was developed and validated for LOH detection of the JAK2 region (JAK2LOH). Blood of a cohort of 12 JAK2V617F-positive patients (n = 6 25–50% and n = 6>50% JAK2V617F) and a cohort of 81 patients suspected of MPN was stored with EDTA and subsequently used for validation. To generate germ-line profiles, non-neoplastic formalin-fixed paraffin-embedded tissue from each patient was analyzed. Results of the SNP assay were compared to those of an established Short Tandem Repeat (STR) assay. Both assays revealed JAK2LOH in 1/6 patients with 25–50% JAK2V617F. In patients with >50% JAK2V617F, JAK2LOH was detected in 6/6 by the SNP assay and 5/6 patients by the STR assay. Of the 81 patients suspected of MPN, 18 patients carried JAK2V617F. Both the SNP and STR assay demonstrated the occurrence of JAK2LOH in 5 of them. In the 63 JAK2V617F-negative patients, no JAK2LOH was observed by SNP and STR analyses. The presented SNP assay reliably detects JAK2LOH and is a fast and easy to perform alternative for STR analyses. We therefore anticipate the SNP approach as a proof of principle for the development of LOH SNP-assays for other clinically relevant LOH loci. PMID:22768290

A Multiplex Snapback Primer System for the Enrichment and Detection of JAK2 V617F and MPL W515L/K Mutations in Philadelphia-Negative Myeloproliferative Neoplasms

PubMed Central

Zhang, Yunqing; Zhang, Xinju; Xu, Xiao; Kang, Zhihua; Li, Shibao; Zhang, Chen; Su, Bing

2014-01-01

A multiplex snapback primer system was developed for the simultaneous detection of JAK2 V617F and MPL W515L/K mutations in Philadelphia chromosome- (Ph-) negative myeloproliferative neoplasms (MPNs). The multiplex system comprises two snapback versus limiting primer sets for JAK2 and MPL mutation enrichment and detection, respectively. Linear-After exponential (LATE) PCR strategy was employed for the primer design to maximize the amplification efficiency of the system. Low ionic strength buffer and rapid PCR protocol allowed for selective amplification of the mutant alleles. Amplification products were analyzed by melting curve analysis for mutation identification. The multiplex system archived 0.1% mutation load sensitivity and <5% coefficient of variation inter-/intra-assay reproducibility. 120 clinical samples were tested by the multiplex snapback primer assay, and verified with amplification refractory system (ARMS), quantitative PCR (qPCR) and Sanger sequencing method. The multiplex system, with a favored versatility, provided the molecular diagnosis of Ph-negative MPNs with a suitable implement and simplified the genetic test process. PMID:24729973

Structure-Function Correlation of G6, a Novel Small Molecule Inhibitor of Jak2

PubMed Central

Majumder, Anurima; Govindasamy, Lakshmanan; Magis, Andrew; Kiss, Róbert; Polgár, Tímea; Baskin, Rebekah; Allan, Robert W.; Agbandje-McKenna, Mavis; Reuther, Gary W.; Keserű, György M.; Bisht, Kirpal S.; Sayeski, Peter P.

2010-01-01

Somatic mutations in the Jak2 protein, such as V617F, cause aberrant Jak/STAT signaling and can lead to the development of myeloproliferative neoplasms. This discovery has led to the search for small molecule inhibitors that target Jak2. Using structure-based virtual screening, our group recently identified a novel small molecule inhibitor of Jak2 named G6. Here, we identified a structure-function correlation of this compound. Specifically, five derivative compounds of G6 having structural similarity to the original lead compound were obtained and analyzed for their ability to (i) inhibit Jak2-V617F-mediated cell growth, (ii) inhibit the levels of phospho-Jak2, phospho-STAT3, and phospho-STAT5; (iii) induce apoptosis in human erythroleukemia cells; and (iv) suppress pathologic cell growth of Jak2-V617F-expressing human bone marrow cells ex vivo. Additionally, we computationally examined the interactions of these compounds with the ATP-binding pocket of the Jak2 kinase domain. We found that the stilbenoid core-containing derivatives of G6 significantly inhibited Jak2-V617F-mediated cell proliferation in a time- and dose-dependent manner. They also inhibited phosphorylation of Jak2, STAT3, and STAT5 proteins within cells, resulting in higher levels of apoptosis via the intrinsic apoptotic pathway. Finally, the stilbenoid derivatives inhibited the pathologic growth of Jak2-V617F-expressing human bone marrow cells ex vivo. Collectively, our data demonstrate that G6 has a stilbenoid core that is indispensable for maintaining its Jak2 inhibitory potential. PMID:20667821

TLR4 and RAGE conversely mediate pro-inflammatory S100A8/9-mediated inhibition of proliferation-linked signaling in myeloproliferative neoplasms.

PubMed

Kovačić, Marijana; Mitrović-Ajtić, Olivera; Beleslin-Čokić, Bojana; Djikić, Dragoslava; Subotički, Tijana; Diklić, Miloš; Leković, Danijela; Gotić, Mirjana; Mossuz, Pascal; Čokić, Vladan P

2018-06-26

Previously, the family of S100A proteins has been found to be associated with inflammation and myelopoiesis and to be able to induce or support myeloproliferation during chronic inflammation. Here, we studied the inflammatory myeloid-related proteins S100A4, S100A8, S100A9 and S100A12 in myeloproliferative neoplasms (MPNs) in order to assess the involvement of chronic inflammation in the pathogenesis of MPN. We analyzed the S100A4, S100A8, S100A9 and S100A12 mRNA and protein levels in the bone marrow and circulation of 140 patients with MPN and 15 healthy controls using Western blotting, microarray-based mRNA expression profiling and ELISA assays, respectively. In addition we performed functional studies on the proliferation-related AKT and ERK1/2 signaling pathways in MPN-derived granulocytes using Western blotting and proteomic analyses. We found that the S100A mRNA levels were increased in MPN patient-derived circulatory CD34 + cells, and that their protein expression levels were also augmented in their granulocytes and bone marrow stroma cells, depending on the JAK2V617F mutation allele burden. We also found that calreticulin (CALR) mutations were related to reduced S100A8 plasma levels in primary myelofibrosis (PMF). The S100A8 plasma levels were found to be increased in MPN, the S100A9 plasma levels in PMF and essential thrombocythemia (ET), and the S100A12 plasma levels in polycythemia vera (PV). These S100A plasma levels showed a positive correlation with the systemic inflammation marker IL-8, as well as with the numbers of leukocytes and thrombocytes, depending on the JAK2V617F mutation status. Additionally, we found that heterodimeric S100A8/9 can inhibit the AKT pathway in MPN-derived granulocytes mediated by the Toll-like receptor 4 (TLR4), depending on the CALR mutation status. Conversely, we found that blocking of the receptor for advanced glycation end products (RAGE) increased the S100A8/9-mediated inhibition of AKT signaling in the MPN-derived granulocytes. Moreover, we found that heterodimeric S100A8/9 generally induced TLR4-mediated ERK1/2 dephosphorylation proportionally to the JAK2V617F mutation allele burden. TLR4/RAGE blocking prevented the S100A8/9-mediated inhibition of ERK1/2 phosphorylation in PV. From our data we conclude that the S100A8 and S100A9 granulocyte and plasma levels are increased in MPN patients, along with inflammation markers, depending on their JAK2V617F mutation allele burden. We also found that S100A8/9-mediated inhibition of the proliferation-related AKT and ERK1/2 signaling pathways can be decreased by CALR mutation-dependent TLR4 blocking and increased by RAGE inhibition in MPN.

[The quantitative testing of V617F mutation in gen JAK2 using pyrosequencing technique].

PubMed

Dunaeva, E A; Mironov, K O; Dribnokhodova, T E; Subbotina, E E; Bashmakova; Ol'hovskiĭ, I A; Shipulin, G A

2014-11-01

The somatic mutation V617F in gen JAK2 is a frequent cause of chronic myeloprolific diseases not conditioned by BCR/ABL mutation. The quantitative testing of relative percentage of mutant allele can be used in establishing severity of disease and its prognosis and in prescription of remedy inhibiting activity of JAK2. To quantitatively test mutation the pyrosequencing technique was applied. The developed technique permits detecting and quantitatively, testing percentage of mutation fraction since 7%. The "gray zone" is presented by samples with percentage of mutant allele from 4% to 7%. The dependence of expected percentage of mutant fraction in analyzed sample from observed value of signal is described by equation of line with regression coefficients y = - 0.97, x = -1.32 and at that measurement uncertainty consists ± 0.7. The developed technique is approved officially on clinical material from 192 patients with main forms of myeloprolific diseases not conditioned by BCR/ABL mutation. It was detected 64 samples with mautant fraction percentage from 13% to 91%. The developed technique permits implementing monitoring of therapy of myeloprolific diseases and facilitates to optimize tactics of treatment.

Analysis of genomic aberrations and gene expression profiling identifies novel lesions and pathways in myeloproliferative neoplasms

PubMed Central

Rice, K L; Lin, X; Wolniak, K; Ebert, B L; Berkofsky-Fessler, W; Buzzai, M; Sun, Y; Xi, C; Elkin, P; Levine, R; Golub, T; Gilliland, D G; Crispino, J D; Licht, J D; Zhang, W

2011-01-01

Polycythemia vera (PV), essential thrombocythemia and primary myelofibrosis, are myeloproliferative neoplasms (MPNs) with distinct clinical features and are associated with the JAK2V617F mutation. To identify genomic anomalies involved in the pathogenesis of these disorders, we profiled 87 MPN patients using Affymetrix 250K single-nucleotide polymorphism (SNP) arrays. Aberrations affecting chr9 were the most frequently observed and included 9pLOH (n=16), trisomy 9 (n=6) and amplifications of 9p13.3–23.3 (n=1), 9q33.1–34.13 (n=1) and 9q34.13 (n=6). Patients with trisomy 9 were associated with elevated JAK2V617F mutant allele burden, suggesting that gain of chr9 represents an alternative mechanism for increasing JAK2V617F dosage. Gene expression profiling of patients with and without chr9 abnormalities (+9, 9pLOH), identified genes potentially involved in disease pathogenesis including JAK2, STAT5B and MAPK14. We also observed recurrent gains of 1p36.31–36.33 (n=6), 17q21.2–q21.31 (n=5) and 17q25.1–25.3 (n=5) and deletions affecting 18p11.31–11.32 (n=8). Combined SNP and gene expression analysis identified aberrations affecting components of a non-canonical PRC2 complex (EZH1, SUZ12 and JARID2) and genes comprising a ‘HSC signature' (MLLT3, SMARCA2 and PBX1). We show that NFIB, which is amplified in 7/87 MPN patients and upregulated in PV CD34+ cells, protects cells from apoptosis induced by cytokine withdrawal. PMID:22829077

Clinical effect of driver mutations of JAK2, CALR, or MPL in primary myelofibrosis.

PubMed

Rumi, Elisa; Pietra, Daniela; Pascutto, Cristiana; Guglielmelli, Paola; Martínez-Trillos, Alejandra; Casetti, Ilaria; Colomer, Dolors; Pieri, Lisa; Pratcorona, Marta; Rotunno, Giada; Sant'Antonio, Emanuela; Bellini, Marta; Cavalloni, Chiara; Mannarelli, Carmela; Milanesi, Chiara; Boveri, Emanuela; Ferretti, Virginia; Astori, Cesare; Rosti, Vittorio; Cervantes, Francisco; Barosi, Giovanni; Vannucchi, Alessandro M; Cazzola, Mario

2014-08-14

We studied the impact of driver mutations of JAK2, CALR, (calreticulin gene) or MPL on clinical course, leukemic transformation, and survival of patients with primary myelofibrosis (PMF). Of the 617 subjects studied, 399 (64.7%) carried JAK2 (V617F), 140 (22.7%) had a CALR exon 9 indel, 25 (4.0%) carried an MPL (W515) mutation, and 53 (8.6%) had nonmutated JAK2, CALR, and MPL (so-called triple-negative PMF). Patients with CALR mutation had a lower risk of developing anemia, thrombocytopenia, and marked leukocytosis compared with other subtypes. They also had a lower risk of thrombosis compared with patients carrying JAK2 (V617F). At the opposite, triple-negative patients had higher incidence of leukemic transformation compared with either CALR-mutant or JAK2-mutant patients. Median overall survival was 17.7 years in CALR-mutant, 9.2 years in JAK2-mutant, 9.1 years in MPL-mutant, and 3.2 years in triple-negative patients. In multivariate analysis corrected for age, CALR-mutant patients had better overall survival than either JAK2-mutant or triple-negative patients. The impact of genetic lesions on survival was independent of current prognostic scoring systems. These observations indicate that driver mutations define distinct disease entities within PMF. Accounting for them is not only relevant to clinical decision-making, but should also be considered in designing clinical trials. © 2014 by The American Society of Hematology.

Clinical effect of driver mutations of JAK2, CALR, or MPL in primary myelofibrosis

PubMed Central

Rumi, Elisa; Pietra, Daniela; Pascutto, Cristiana; Guglielmelli, Paola; Martínez-Trillos, Alejandra; Casetti, Ilaria; Colomer, Dolors; Pieri, Lisa; Pratcorona, Marta; Rotunno, Giada; Sant’Antonio, Emanuela; Bellini, Marta; Cavalloni, Chiara; Mannarelli, Carmela; Milanesi, Chiara; Boveri, Emanuela; Ferretti, Virginia; Astori, Cesare; Rosti, Vittorio; Cervantes, Francisco; Barosi, Giovanni; Vannucchi, Alessandro M.

2014-01-01

We studied the impact of driver mutations of JAK2, CALR, (calreticulin gene) or MPL on clinical course, leukemic transformation, and survival of patients with primary myelofibrosis (PMF). Of the 617 subjects studied, 399 (64.7%) carried JAK2 (V617F), 140 (22.7%) had a CALR exon 9 indel, 25 (4.0%) carried an MPL (W515) mutation, and 53 (8.6%) had nonmutated JAK2, CALR, and MPL (so-called triple-negative PMF). Patients with CALR mutation had a lower risk of developing anemia, thrombocytopenia, and marked leukocytosis compared with other subtypes. They also had a lower risk of thrombosis compared with patients carrying JAK2 (V617F). At the opposite, triple-negative patients had higher incidence of leukemic transformation compared with either CALR-mutant or JAK2-mutant patients. Median overall survival was 17.7 years in CALR-mutant, 9.2 years in JAK2-mutant, 9.1 years in MPL-mutant, and 3.2 years in triple-negative patients. In multivariate analysis corrected for age, CALR-mutant patients had better overall survival than either JAK2-mutant or triple-negative patients. The impact of genetic lesions on survival was independent of current prognostic scoring systems. These observations indicate that driver mutations define distinct disease entities within PMF. Accounting for them is not only relevant to clinical decision-making, but should also be considered in designing clinical trials. PMID:24986690

Presence of atypical thrombopoietin receptor (MPL) mutations in triple-negative essential thrombocythemia patients.

PubMed

Cabagnols, Xénia; Favale, Fabrizia; Pasquier, Florence; Messaoudi, Kahia; Defour, Jean Philippe; Ianotto, Jean Christophe; Marzac, Christophe; Le Couédic, Jean Pierre; Droin, Nathalie; Chachoua, Ilyas; Favier, Remi; Diop, M'boyba Khadija; Ugo, Valérie; Casadevall, Nicole; Debili, Najet; Raslova, Hana; Bellanné-Chantelot, Christine; Constantinescu, Stefan N; Bluteau, Olivier; Plo, Isabelle; Vainchenker, William

2016-01-21

Mutations in signaling molecules of the cytokine receptor axis play a central role in myeloproliferative neoplasm (MPN) pathogenesis. Polycythemia vera is mainly related to JAK2 mutations, whereas a wider mutational spectrum is detected in essential thrombocythemia (ET) with mutations in JAK2, the thrombopoietin (TPO) receptor (MPL), and the calreticulin (CALR) genes. Here, we studied the mutational profile of 17 ET patients negative for JAK2V617F, MPLW515K/L, and CALR mutations, using whole-exome sequencing and next-generation sequencing (NGS) targeted on JAK2 and MPL. We found several signaling mutations including JAK2V617F at very low allele frequency, 1 homozygous SH2B3 mutation, 1 MPLS505N, 1 MPLW515R, and 2 MPLS204P mutations. In the remaining patients, 4 presented a clonal and 7 a polyclonal hematopoiesis, suggesting that certain triple-negative ETs are not MPNs. NGS on 26 additional triple-negative ETs detected only 1 MPLY591N mutation. Functional studies on MPLS204P and MPLY591N revealed that they are weak gain-of-function mutants increasing MPL signaling and conferring either TPO hypersensitivity or independence to expressing cells, but with a low efficiency. Further studies should be performed to precisely determine the frequency of MPLS204 and MPLY591 mutants in a bigger cohort of MPN. © 2016 by The American Society of Hematology.

LNK mutations and myeloproliferative disorders.

PubMed

McMullin, Mary Frances; Cario, Holger

2016-02-01

The lymphocyte adaptor protein (LNK) is one of a family of adaptor proteins involved cell signaling and control of B cell populations. It has a critical role in regulation of signaling in hematopoiesis. Lnk negatively regulates cytokine initiated cell signaling and it functions as a negative regulator of the mutant protein in myeloproliferative neoplasms JAK2V617F. A number of mutations in LNK have been described in a variety of myeloproliferative neoplasms some of which have been demonstrated to cause increased cellular proliferation. The majority of mutations occur in exon 2. In a small number of cases idiopathic erythrocytosis with subnormal erythropoietin levels LNK mutations have been found which may account for the clinical phenotype. Thus investigation for LNK mutations should be considered in the investigation of idiopathic erythrocytosis and perhaps other myeloproliferative neoplasms. © 2015 Wiley Periodicals, Inc.

A risk of essential thrombocythemia in carriers of constitutional CHEK2 gene mutations.

PubMed

Janiszewska, Hanna; Bak, Aneta; Pilarska, Maria; Heise, Marta; Junkiert-Czarnecka, Anna; Kuliszkiewicz-Janus, Małgorzata; Całbecka, Małgorzata; Jaźwiec, Bozena; Wołowiec, Dariusz; Kuliczkowski, Kazimierz; Haus, Olga

2012-03-01

Germline mutations of the CHEK2 gene have been reported in some myeloid and lymphoid malignancies, but their impact on development of essential thrombocythemia has not been studied. In 16 out of 106 (15.1%) consecutive patients, newly diagnosed with essential thrombocythemia, we found one of four analyzed CHEK2 mutations: I157T, 1100delC, IVS2+1G>A or del5395. They were associated with the increased risk of disease (OR=3.8; P=0.002). The median age at ET diagnosis among CHEK2+/JAK2V617F+ patients was seven years lower than that among CHEK2-/JAK2V617F+ (52 vs. 59 years; P=0.04), whereas there was no difference in the medians of hematologic parameters between these groups. The results obtained suggest that CHEK2 mutations could potentially contribute to the susceptibility to essential thrombocythemia. The germline inactivation of CHEK2, as it seems, has no direct impact on the development of disease, but it could cause disruption of cell cycle checkpoints and initiate or support the cancerogenic process of essential thrombocythemia at a younger age.

A risk of essential thrombocythemia in carriers of constitutional CHEK2 gene mutations

PubMed Central

Janiszewska, Hanna; Bąk, Aneta; Pilarska, Maria; Heise, Marta; Junkiert-Czarnecka, Anna; Kuliszkiewicz-Janus, Małgorzata; Całbecka, Małgorzata; JaŸwiec, Bożena; Wołowiec, Dariusz; Kuliczkowski, Kazimierz; Haus, Olga

2012-01-01

Germline mutations of the CHEK2 gene have been reported in some myeloid and lymphoid malignancies, but their impact on development of essential thrombocythemia has not been studied. In 16 out of 106 (15.1%) consecutive patients, newly diagnosed with essential thrombocythemia, we found one of four analyzed CHEK2 mutations: I157T, 1100delC, IVS2+1G>A or del5395. They were associated with the increased risk of disease (OR=3.8; P=0.002). The median age at ET diagnosis among CHEK2+/JAK2V617F+ patients was seven years lower than that among CHEK2−/JAK2V617F+ (52 vs. 59 years; P=0.04), whereas there was no difference in the medians of hematologic parameters between these groups. The results obtained suggest that CHEK2 mutations could potentially contribute to the susceptibility to essential thrombocythemia. The germline inactivation of CHEK2, as it seems, has no direct impact on the development of disease, but it could cause disruption of cell cycle checkpoints and initiate or support the cancerogenic process of essential thrombocythemia at a younger age. PMID:22058216

Essential role for Stat5a/b in myeloproliferative neoplasms induced by BCR-ABL1 and JAK2V617F in mice

PubMed Central

Walz, Christoph; Ahmed, Wesam; Lazarides, Katherine; Betancur, Monica; Patel, Nihal; Hennighausen, Lothar; Zaleskas, Virginia M.

2012-01-01

STAT5 proteins are constitutively activated in malignant cells from many patients with leukemia, including the myeloproliferative neoplasms (MPNs) chronic myeloid leukemia (CML) and polycythemia vera (PV), but whether STAT5 is essential for the pathogenesis of these diseases is not known. In the present study, we used mice with a conditional null mutation in the Stat5a/b gene locus to determine the requirement for STAT5 in MPNs induced by BCR-ABL1 and JAK2V617F in retroviral transplantation models of CML and PV. Loss of one Stat5a/b allele resulted in a decrease in BCR-ABL1–induced CML-like MPN and the appearance of B-cell acute lymphoblastic leukemia, whereas complete deletion of Stat5a/b prevented the development of leukemia in primary recipients. However, BCR-ABL1 was expressed and active in Stat5-null leukemic stem cells, and Stat5 deletion did not prevent progression to lymphoid blast crisis or abolish established B-cell acute lymphoblastic leukemia. JAK2V617F failed to induce polycythemia in recipients after deletion of Stat5a/b, although the loss of STAT5 did not prevent the development of myelofibrosis. These results demonstrate that STAT5a/b is essential for the induction of CML-like leukemia by BCR-ABL1 and of polycythemia by JAK2V617F, and validate STAT5a/b and the genes they regulate as targets for therapy in these MPNs. PMID:22234689

How I treat polycythemia vera.

PubMed

Passamonti, Francesco

2012-07-12

Polycythemia vera (PV) is a clonal disorder characterized by unwarranted production of red blood cells. In the majority of cases, PV is driven by oncogenic mutations that constitutively activate the JAK-STAT signal transduction pathway, such as JAK2 V617F, or exon 12 mutations or LNK mutations. Diagnosis of PV is based on the WHO criteria. Diagnosis of post-PV myelofibrosis is established according to the International Working Group for Myeloproliferative Neoplasms Research and Treatment criteria. Different clinical presentations of PV are discussed. Prognostication of PV is tailored to the most frequent complication during follow-up, namely, thrombosis. Age older than 60 years and prior history of thrombosis are the 2 main risk factors for disease stratification. Correlations are emerging between leukocytosis, JAK2(V617F) mutation, BM fibrosis, and different outcomes of PV, which need to be confirmed in prospective studies. In my practice, hydroxyurea is still the "gold standard" when cytoreduction is needed, even though pegylated IFN-alfa-2a and ruxolitinib might be useful in particular settings. Results of phase 1 or 2 studies concerning these latter agents should however be confirmed by the ongoing randomized phase 3 clinical trials. In this paper, I discuss the main problems encountered in daily clinical practice with PV patients regarding diagnosis, prognostication, and therapy.

Thrombocytosis and thrombosis.

PubMed

Vannucchi, Alessandro M; Barbui, Tiziano

2007-01-01

The aim of this review is to discuss current diagnostic approaches to, and classification of, patients presenting with thrombocytosis, in light of novel information derived from the discovery of specific molecular abnormalities in chronic myeloproliferative disorders (CMPD), which represent the most common cause of primary thrombocytosis. The JAK2V617F and the MPLW515L/K mutations have been found in patients with essential thrombocythemia, polycythemia vera, and primary myelofibrosis, and less frequently in other myeloproliferative disorders complicated by thrombocytosis. However, neither mutation is disease specific nor is it universally present in patients with elevated platelet counts due to a CMPD; therefore, distinguishing between reactive and primary forms of thrombocytosis, as well as among the different clinical entities that constitute the CMPD, still requires a multifaceted diagnostic approach that includes as a key step the accurate evaluation of bone marrow histology. The role of elevated platelet counts in thrombosis, which represent the predominant complication of CMPD,significantly affecting prognosis and quality of life as well as, paradoxically, in the pathogenesis of the hemorrhagic manifestations, will be discussed. Established and novel potential risk factors for thrombosis, including the clinical relevance of the JAK2V617F mutation, and current management strategies for thrombocytosis are also briefly discussed.

Clonal heterogeneity as a driver of disease variability in the evolution of myeloproliferative neoplasms.

PubMed

Prick, Janine; de Haan, Gerald; Green, Anthony R; Kent, David G

2014-10-01

Myeloproliferative neoplasms (MPNs) are clonal hematological diseases in which cells of the myelo-erythroid lineage are overproduced and patients are predisposed to leukemic transformation. Hematopoietic stem cells are the suspected disease-initiating cells, and these cells must acquire a clonal advantage relative to nonmutant hematopoietic stem cells to perpetuate disease. In 2005, several groups identified a single gain-of-function point mutation in JAK2 that associated with the majority of MPNs, and subsequent studies have led to a comprehensive understanding of the mutational landscape in MPNs. However, confusion still exists as to how a single genetic aberration can be associated with multiple distinct disease entities. Many explanations have been proposed, including JAK2V617F homozygosity, individual patient heterogeneity, and the differential regulation of downstream JAK2 signaling pathways. Several groups have made knock-in mouse models expressing JAK2V617F and have observed divergent phenotypes, each recapitulating some aspects of disease. Intriguingly, most of these models do not observe a strong hematopoietic stem cell self-renewal advantage compared with wild-type littermate controls, raising the question of how a clonal advantage is established in patients with MPNs. This review summarizes the current molecular understanding of MPNs and the diversity of disease phenotypes and proposes that the increased proliferation induced by JAK2V617F applies a selection pressure on the mutant clone that results in highly diverse clonal evolution in individuals. Copyright © 2014 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

JAK and MPL mutations in myeloid malignancies.

PubMed

Tefferi, Ayalew

2008-03-01

The Janus family of non-receptor tyrosine kinases (JAK1, JAK2, JAK3 and tyrosine kinase 2) transduces signals downstream of type I and II cytokine receptors via signal transducers and activators of transcription (STATs). JAK3 is important in lymphoid and JAK2 in myeloid cell proliferation and differentiation. The thrombopoietin receptor MPL is one of several JAK2 cognate receptors and is essential for myelopoiesis in general and megakaryopoiesis in particular. Germline loss-of-function (LOF) JAK3 and MPL mutations cause severe combined immunodeficiency and congenital amegakaryocytic thrombocytopenia, respectively. Germline gain-of-function (GOF) MPL mutation (MPLS505N) causes familial thrombocytosis. Somatic JAK3 (e.g. JAK3A572V, JAK3V722I, JAK3P132T) and fusion JAK2 (e.g. ETV6-JAK2, PCM1-JAK2, BCR-JAK2) mutations have respectively been described in acute megakaryocytic leukemia and acute leukemia/chronic myeloid malignancies. However, current attention is focused on JAK2 (e.g. JAK2V617F, JAK2 exon 12 mutations) and MPL (e.g. MPLW515L/K/S, MPLS505N) mutations associated with myeloproliferative neoplasms (MPNs). A JAK2 mutation, primarily JAK2V617F, is invariably associated with polycythemia vera (PV). The latter mutation also occurs in the majority of patients with essential thrombocythemia (ET) or primary myelofibrosis (PMF). MPL mutational frequency in MPNs is substantially less (<10%). In general, despite a certain degree of genotype - phenotype correlations, the prognostic relevance of harbouring one of these mutations, or their allele burden when present, remains dubious. Regardless, based on the logical assumption that amplified JAK-STAT signalling is central to the pathogenesis of PV, ET and PMF, several anti-JAK2 tyrosine kinase inhibitors have been developed and are currently being tested in humans with these disorders.

Regenerative nodular hyperplasia, portal vein thrombosis and primary myelofibrosis: an unusual triple association.

PubMed

Sández Montagut, Víctor Manuel; Giráldez Gallego, Álvaro; Ontanilla Clavijo, Guilermo

2018-03-01

We report a case of a regenerative nodular hyperplasia with a portal vein cavernomatosis with a subsequent progression to symptomatic, occlusive thrombosis of the superior mesenteric vein. A thorough investigation resulted in a final diagnosis of primary myelofibrosis associated with the V617F mutation in the JAK2 gene.

Antagonistic activities of the immunomodulator and PP2A-activating drug FTY720 (Fingolimod, Gilenya) in Jak2-driven hematologic malignancies

PubMed Central

Oaks, Joshua J.; Santhanam, Ramasamy; Walker, Christopher J.; Roof, Steve; Harb, Jason G.; Ferenchak, Greg; Eisfeld, Ann-Kathrin; Van Brocklyn, James R.; Briesewitz, Roger; Saddoughi, Sahar A.; Nagata, Kyosuke; Bittman, Robert; Caligiuri, Michael A.; Abdel-Wahab, Omar; Levine, Ross; Arlinghaus, Ralph B.; Quintas-Cardama, Alfonso; Goldman, John M.; Apperley, Jane; Reid, Alistair; Milojkovic, Dragana; Ziolo, Mark T.; Marcucci, Guido; Ogretmen, Besim; Neviani, Paolo

2013-01-01

FTY720 (Fingolimod, Gilenya) is a sphingosine analog used as an immunosuppressant in multiple sclerosis patients. FTY720 is also a potent protein phosphatase 2A (PP2A)–activating drug (PAD). PP2A is a tumor suppressor found inactivated in different types of cancer. We show here that PP2A is inactive in polycythemia vera (PV) and other myeloproliferative neoplasms characterized by the expression of the transforming Jak2V617F oncogene. PP2A inactivation occurs in a Jak2V617F dose/kinase-dependent manner through the PI-3Kγ-PKC–induced phosphorylation of the PP2A inhibitor SET. Genetic or PAD-mediated PP2A reactivation induces Jak2V617F inactivation/downregulation and impairs clonogenic potential of Jak2V617F cell lines and PV but not normal CD34+ progenitors. Likewise, FTY720 decreases leukemic allelic burden, reduces splenomegaly, and significantly increases survival of Jak2V617F leukemic mice without adverse effects. Mechanistically, we show that in Jak2V617F cells, FTY720 antileukemic activity requires neither FTY720 phosphorylation (FTY720-P) nor SET dimerization or ceramide induction but depends on interaction with SET K209. Moreover, we show that Jak2V617F also utilizes an alternative sphingosine kinase-1–mediated pathway to inhibit PP2A and that FTY720-P, acting as a sphingosine-1-phosphate-receptor-1 agonist, elicits signals leading to the Jak2-PI-3Kγ-PKC-SET–mediated PP2A inhibition. Thus, PADs (eg, FTY720) represent suitable therapeutic alternatives for Jak2V617F MPNs. PMID:23926298

Stimulation of the amino acid transporter SLC6A19 by JAK2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bhavsar, Shefalee K.; Hosseinzadeh, Zohreh; Merches, Katja

Highlights: Black-Right-Pointing-Pointer The amino acid transporter SLC6A19 is upregulated by Janus kinase-2 JAK2. Black-Right-Pointing-Pointer The {sup V617F}JAK2 mutant, causing myeloproliferative disease, is more effective. Black-Right-Pointing-Pointer JAK2 inhibitor AG490 reverses stimulation of SLC6A19 by {sup V617F}JAK2. Black-Right-Pointing-Pointer JAK2 enhances SLC6A19 protein insertion into the cell membrane. Black-Right-Pointing-Pointer SLC6A19 may contribute to amino acid uptake into {sup V617F}JAK2 expressing tumor cells. -- Abstract: JAK2 (Janus kinase-2) is expressed in a wide variety of cells including tumor cells and contributes to the proliferation and survival of those cells. The gain of function mutation {sup V617F}JAK2 mutant is found in the majority of myeloproliferativemore » diseases. Cell proliferation depends on the availability of amino acids. Concentrative cellular amino acid uptake is in part accomplished by Na{sup +} coupled amino acid transport through SLC6A19 (B(0)AT). The present study thus explored whether JAK2 activates SLC6A19. To this end, SLC6A19 was expressed in Xenopus oocytes with or without wild type JAK2, {sup V617F}JAK2 or inactive {sup K882E}JAK2 and electrogenic amino acid transport determined by dual electrode voltage clamp. In SLC6A19-expressing oocytes but not in oocytes injected with water or JAK2 alone, the addition of leucine (2 mM) to the bath generated a current (I{sub le}), which was significantly increased following coexpression of JAK2 or {sup V617F}JAK2, but not by coexpression of {sup K882E}JAK2. Coexpression of JAK2 enhanced the maximal transport rate without significantly modifying the affinity of the carrier. Exposure of the oocytes to the JAK2 inhibitor AG490 (40 {mu}M) resulted in a gradual decline of I{sub le}. According to chemiluminescence JAK2 enhanced the carrier protein abundance in the cell membrane. The decline of I{sub le} following inhibition of carrier insertion by brefeldin A (5 {mu}M) was similar in the absence and presence of JAK2 indicating that JAK2 stimulates carrier insertion into rather than inhibiting carrier retrival from the cell membrane. In conclusion, JAK2 up-regulates SLC6A19 activity which may foster amino acid uptake into JAK2 expressing cells.« less

A rapid, highly accurate method for quantifying CALR mutant allele burden in persons with myeloproliferative neoplasms.

PubMed

Yao, Qiu-Mei; Zhou, Jiao; Gale, Robert Peter; Li, Jin-Lan; Li, Ling-Di; Li, Ning; Chen, Shan-Shan; Ruan, Guo-Rui

2015-10-01

Calreticulin (CALR) mutations were recently identified in a substantial proportion of persons with essential thrombocythemia (ET) and with primary myelofibrosis (PMF) without JAK2(V617F). Consequently rapid, sensitive, and specific methods to detect and quantify these mutations are needed. We studied samples from 1088 persons with myeloproliferative neoplasms (MPNs) including 421 JAK2(V617F) negative subjects with ET, PMF, polycythemia vera (PV), chronic myeloid leukemia (CML) and hyper-eosinophilic syndrome (HES). Detection of CALR exon 9 mutations was done by PCR amplification followed by fragment length analysis and direct sequencing. Dilution assays were used to determine CALR mutant allele burden. We detected CALR mutations in blood and bone marrow samples from 152 subjects with ET and with PMF but not in samples from normal or persons with PV, CML, or HES. CALR mutant peaks were distinct from wild-type peaks and dilution experiments indicated a sensitivity level of 0.5-5% for a CALR mutant allele in a wild-type background. Diverse types of mutations were detected including deletions, insertions, and complex indels. All mutations were confirmed by direct sequencing. We also used dilution experiments to quantify mutant allele burden. We were able to reproducibly detect mutant allele levels as low 5% (0.5-5%) in a wild-type background. PCR amplification followed by fragment length analysis is a rapid, sensitive, and specific method for screening persons with MPNs for CALR mutations, especially those with ET and PMF and for estimating mutant allele burden.

Evaluation of Inflammation Parameters in Philadelphia Negative Chronic Myeloproliferative Neoplasia Patients.

PubMed

Hacibekiroglu, Tuba; Akinci, Sema; Basturk, Abdulkadir; inal, Besime; Guney, Tekin; Bakanay, Sule Mine; Dilek, Imdat

2015-01-01

Chronic myeloproliferative diseases are clonal stem cell diseases which occur as a result of uncontrollable growth and reproduction of hematopoietic stem cells, which are the myeloid series source in bone marrow. Recent studies have suggested that chronic inflammation can be a triggering factor in the clonal change in chronic myeloproliferative neoplasia (CMPN). In our study, we evaluated the existence of a chronic inflammation process in our Philadelphia negative (Ph-)CMPN patients using inflammation parameters in combination with demographic, laboratory and clinical characteristics of the patients. Demographic characteristics, clinical and laboratorial data, and thrombosis histories of 99 Ph-CMPN patients, who were diagnosed at our outpatient clinic of hematology in accordance with WHO 2008 criteria, were analyzed retrospectively,with 80 healthy individuals of matching gender and age included as controls. Complete blood counts, sedimentation, C reactive protein (CRP), JAK V617F gene mutations, abdomen ultrasound images and previous thrombosis histories of these patients were retrospectively analyzed. Ph-CMPN and healthy control groups included 99 and 80 cases, respectively. PV, ET and MF diagnoses of patients were 43 (%43.4), 44 (44.4%) and 12 (12.1%), respectively. JAK V617F gene mutation was found to be positive in 64 (71.1%) of all cases and in 27(65.8%), 32 (82%), 5 (50%) of the cases in PV, ET and PMF groups, respectively. Thrombosis was determined as 12 (12%) in the entire group, 12.5% in the JAK V617F negative and 15.3% in the positive patients, with no statistical significance (p=0.758). No significant difference was observed between patients with and without previous thrombosis history in respect to hemogram parameters, sedimentation and CRP (p>0.05), neutrophil to lymphocyte ratio (NLR), erythrocyte distribution width (RDW), mean platelet volume (MPV) and sedimentation levels of the patient.

Jak2 FERM Domain Interaction with the Erythropoietin Receptor Regulates Jak2 Kinase Activity▿

PubMed Central

Funakoshi-Tago, Megumi; Pelletier, Stéphane; Moritake, Hiroshi; Parganas, Evan; Ihle, James N.

2008-01-01

Janus kinases are essential for signal transduction by a variety of cytokine receptors and when inappropriately activated can cause hematopoietic disorders and oncogenesis. Consequently, it can be predicted that the interaction of the kinases with receptors and the events required for activation are highly controlled. In a screen to identify phosphorylation events regulating Jak2 activity in EpoR signaling, we identified a mutant (Jak2-Y613E) which has the property of being constitutively activated, as well as an inactivating mutation (Y766E). Although no evidence was obtained to indicate that either site is phosphorylated in signaling, the consequences of the Y613E mutation are similar to those observed with recently described activating mutations in Jak2 (Jak2-V617F and Jak2-L611S). However, unlike the V617F or L611S mutant, the Y613E mutant requires the presence of the receptor but not Epo stimulation for activation and downstream signaling. The properties of the Jak2-Y613E mutant suggest that under normal conditions, Jak2 that is not associated with a receptor is locked into an inactive state and receptor binding through the FERM domain relieves steric constraints, allowing the potential to be activated with receptor engagement. PMID:18160720

JAK2 V617F positive essential thrombocythemia developing in a patient with CD5⁻ chronic lymphocytic leukemia.

PubMed

Wei, Ju; Wang, Chun; Qin, You-Wen; Zhu, Jun; Gao, Yang-Rong; Cai, Qi; Yan, Shi-Ke

2012-06-01

Coexistence of chronic lymphocytic leukemia (CLL) and essential thrombocythemia (ET) in a patient is extremely rare, with only 10 cases reported thus far in literature. This paper describes a 94-year-old male having atypical B-CLL with CD5⁻ (CD5⁻) phenotype and ET. In this patient, we performed interphase fluorescence in situ hybridization (FISH) analysis which revealed 13q14.3 deletion in 31% of B-lymphocyte nuclei and RB1 deletion in 27% of B-lymphocyte nuclei, but not in neutrophils and T-lymphocytes. Furthermore, we identified JAK2 V617F mutation in the peripheral blood nucleated cells and neutrophils, but not in the B- and T-lymphocyte populations. Therefore, it was concluded that the occurrence of CD5− B-CLL and ET in this patient was pathogenically independent.

Emergence of MPLW515 mutation in a patient with CALR deletion: Evidence of secondary acquisition of MPL mutation in the CALR clone.

PubMed

Partouche, Nicolas; Conejero, Carole; Barathon, Quentin; Moroch, Julien; Tulliez, Michel; Cordonnier, Catherine; Giraudier, Stephane

2018-02-01

Myeloproliferative neoplasms are characterized by transduction pathway recognized as mutually exclusive molecular abnormalities such as BCR-ABL translocation, JAK2V617F or JAK2 exon 12 mutations, MPL w515, and CALR mutations. However, in some rare cases, associations of such mutations are found in 1 patient. This can be related to 2 pathologies (at least 2 different clones harboring 2 mutations) or associated mutations in 1 clone. We describe here such an association of CALR and MPL mutations in a patient harboring the second mutation in a subclone during the phenotypic evolution of the myeloproliferative neoplasms. Copyright © 2017 John Wiley & Sons, Ltd.

Acquired copy-neutral loss of heterozygosity of chromosome 1p as a molecular event associated with marrow fibrosis in MPL-mutated myeloproliferative neoplasms.

PubMed

Rumi, Elisa; Pietra, Daniela; Guglielmelli, Paola; Bordoni, Roberta; Casetti, Ilaria; Milanesi, Chiara; Sant'Antonio, Emanuela; Ferretti, Virginia; Pancrazzi, Alessandro; Rotunno, Giada; Severgnini, Marco; Pietrelli, Alessandro; Astori, Cesare; Fugazza, Elena; Pascutto, Cristiana; Boveri, Emanuela; Passamonti, Francesco; De Bellis, Gianluca; Vannucchi, Alessandro; Cazzola, Mario

2013-05-23

We studied mutations of MPL exon 10 in patients with essential thrombocythemia (ET) or primary myelofibrosis (PMF), first investigating a cohort of 892 consecutive patients. MPL mutation scanning was performed on granulocyte genomic DNA by using a high-resolution melt assay, and the mutant allele burden was evaluated by using deep sequencing. Somatic mutations of MPL, all but one involving codon W515, were detected in 26/661 (4%) patients with ET, 10/187 (5%) with PMF, and 7/44 (16%) patients with post-ET myelofibrosis. Comparison of JAK2 (V617F)-mutated and MPL-mutated patients showed only minor phenotypic differences. In an extended group of 62 MPL-mutated patients, the granulocyte mutant allele burden ranged from 1% to 95% and was significantly higher in patients with PMF or post-ET myelofibrosis compared with those with ET. Patients with higher mutation burdens had evidence of acquired copy-neutral loss of heterozygosity (CN-LOH) of chromosome 1p in granulocytes, consistent with a transition from heterozygosity to homozygosity for the MPL mutation in clonal cells. A significant association was found between MPL-mutant allele burden greater than 50% and marrow fibrosis. These observations suggest that acquired CN-LOH of chromosome 1p involving the MPL location may represent a molecular mechanism of fibrotic transformation in MPL-mutated myeloproliferative neoplasms.

Acquired copy-neutral loss of heterozygosity of chromosome 1p as a molecular event associated with marrow fibrosis in MPL-mutated myeloproliferative neoplasms

PubMed Central

Pietra, Daniela; Guglielmelli, Paola; Bordoni, Roberta; Casetti, Ilaria; Milanesi, Chiara; Sant’Antonio, Emanuela; Ferretti, Virginia; Pancrazzi, Alessandro; Rotunno, Giada; Severgnini, Marco; Pietrelli, Alessandro; Astori, Cesare; Fugazza, Elena; Pascutto, Cristiana; Boveri, Emanuela; Passamonti, Francesco; De Bellis, Gianluca; Vannucchi, Alessandro; Cazzola, Mario

2013-01-01

We studied mutations of MPL exon 10 in patients with essential thrombocythemia (ET) or primary myelofibrosis (PMF), first investigating a cohort of 892 consecutive patients. MPL mutation scanning was performed on granulocyte genomic DNA by using a high-resolution melt assay, and the mutant allele burden was evaluated by using deep sequencing. Somatic mutations of MPL, all but one involving codon W515, were detected in 26/661 (4%) patients with ET, 10/187 (5%) with PMF, and 7/44 (16%) patients with post-ET myelofibrosis. Comparison of JAK2 (V617F)–mutated and MPL-mutated patients showed only minor phenotypic differences. In an extended group of 62 MPL-mutated patients, the granulocyte mutant allele burden ranged from 1% to 95% and was significantly higher in patients with PMF or post-ET myelofibrosis compared with those with ET. Patients with higher mutation burdens had evidence of acquired copy-neutral loss of heterozygosity (CN-LOH) of chromosome 1p in granulocytes, consistent with a transition from heterozygosity to homozygosity for the MPL mutation in clonal cells. A significant association was found between MPL-mutant allele burden greater than 50% and marrow fibrosis. These observations suggest that acquired CN-LOH of chromosome 1p involving the MPL location may represent a molecular mechanism of fibrotic transformation in MPL-mutated myeloproliferative neoplasms. PMID:23575445

Janus kinase 2 inhibitors in myeloproliferative disorders.

PubMed

Lucia, Eugenio; Recchia, Anna Grazia; Gentile, Massimo; Bossio, Sabrina; Vigna, Ernesto; Mazzone, Carla; Madeo, Antonio; Morabito, Lucio; Gigliotti, Vincenzo; De Stefano, Laura; Caruso, Nadia; Servillo, Pasquale; Franzese, Stefania; Bisconte, Maria Grazia; Gentile, Carlo; Morabito, Fortunato

2011-01-01

JAK2 is an obligatory kinase for the proliferation and differentiation of erythroid cells and megakaryocytes thus representing a relevant therapeutic target for agents that specifically inhibit its activity particularly in myeloproliferative disorders (MPD) harboring JAK2(V617F) mutations. We discuss the physiopathology of the JAK2 signaling pathway and review clinical trials of JAK2 inhibitors for the treatment of MPD using papers and meeting abstracts published up to September 2010. This review helps in understanding the potential role of JAK2 inhibitors in MPD clinical trials and provides a comprehensive review regarding their efficacy and safety in these disorders. JAK2 inhibitors may prove to be useful only for suppressing disease manifestations. However, unlike drugs such as IFN which are capable of eliminating the malignant clone, JAK2 inhibitors are unable to eradicate the disease. In fact, results to date indicate that although these inhibitors reduce splenomegaly and alleviate constitutional symptoms irrespective of JAK2 mutational status, most have only a modest impact on the JAK2(V617F) allele burden. Considering the relevant risk of serious complications in patients undergoing splenectomy, these drugs could find a suitable indication in patients with myelofibrosis awaiting bone marrow transplantation.

The mutation profile of JAK2 and CALR in Chinese Han patients with Philadelphia chromosome-negative myeloproliferative neoplasms

PubMed Central

2014-01-01

Mutations in JAK2, MPL and CALR are highly relevant to the Philadelphia chromosome (Ph)-negative myeloproliferative neoplasms (MPNs). We performed high resolution melting analysis and Sanger sequencing together with T-A cloning to elucidate the unique mutation profile of these genes, in Chinese patients with MPNs. Peripheral blood DNA samples were obtained from 80 patients with polycythemia vera (PV), 80 patients with essential thrombocytosis (ET) and 50 patients with primary myelofibrosis (PMF). Ten PV patients were identified with diverse JAK2 exon 12 mutations. Five novel JAK2 Exon 12 mutation patterns (M532V/E543G, N533D, M535I/H538Y/K549I, E543G and D544N) were described. JAK2 V617F was detected in 140 samples (66 PV, 45 ET and 29 PMF). JAK2 Exon 12 mutations were prevalent (13%) and variable in the Chinese patients. Compared with PV patients with JAK2 V617F mutations, PV patients with JAK2 exon 12 mutations had an earlier median onset of disease (P = 0.0013). MPL W515L/K mutations were discerned in 4 ET and 3 PMF patients. Two kinds of CALR mutation, c. 1179_1230del and c. 1234_1235insTTGTC were detected in 20 ET and 16 PMF patients. A novel CALR mutation pattern (c. 1173_1223del/c. 1179_1230del) was identified in 2 PMF samples. In addition, 17 scattered point mutations in CALR c.1153 to c.1255 were also detected in 13 cases with CALR frame-shifting variations and 2 cases without CALR frame-shifting variations. Female patients showed a predisposition to CALR mutations (P = 0.0035). Chinese Ph-negative MPN patients have a unique mutation landscape in the common molecular markers of MPN diagnosis. Validation of the molecular diagnostic pipeline should be emphasized since there is a considerable ethnical diversity in the molecular profiles of Ph-negative MPNs. PMID:25023898

FLT3 and JAK2 Mutations in Acute Myeloid Leukemia Promote Interchromosomal Homologous Recombination and the Potential for Copy Neutral Loss of Heterozygosity.

PubMed

Gaymes, Terry J; Mohamedali, Azim; Eiliazadeh, Anthony L; Darling, David; Mufti, Ghulam J

2017-04-01

Acquired copy neutral LOH (CN-LOH) is a frequent occurrence in myeloid malignancies and is often associated with resistance to standard therapeutic modalities and poor survival. Here, we show that constitutive signaling driven by mutated FLT3 and JAK2 confers interchromosomal homologous recombination (iHR), a precedent for CN-LOH. Using a targeted recombination assay, we determined significant iHR activity in internal tandem duplication FLT3 (FLT3-ITD) and JAK2V617F-mutated cells. Sister chromatid exchanges, a surrogate measure of iHR, was significantly elevated in primary FLT3-ITD normal karyotype acute myeloid leukemia (NK-AML) compared with wild-type FLT3 NK-AML. HR was harmonized to S phase of the cell cycle to repair broken chromatids and prevent iHR. Increased HR activity in G 0 arrested primary FLT3-ITD NK-AML in contrast to wild-type FLT3 NK-AML. Cells expressing mutated FLT3-ITD demonstrated a relative increase in mutation frequency as detected by thymidine kinase (TK) gene mutation assay. Moreover, resistance was associated with CN-LOH at the TK locus. Treatment of FLT3-ITD- and JAK2V617F-mutant cells with the antioxidant N -acetylcysteine diminished reactive oxygen species (ROS), restoring iHR and HR levels. Our findings show that mutated FLT3-ITD and JAK2 augment ROS production and HR, shifting the cellular milieu toward illegitimate recombination events such as iHR and CN-LOH. Therapeutic reduction of ROS may thus prevent leukemic progression and relapse in myeloid malignancies. Cancer Res; 77(7); 1697-708. ©2017 AACR . ©2017 American Association for Cancer Research.

Use of CBL exon 8 and 9 mutations in diagnosis of myeloproliferative neoplasms and myelodysplastic/myeloproliferative disorders: an analysis of 636 cases

PubMed Central

Schnittger, Susanne; Bacher, Ulrike; Alpermann, Tamara; Reiter, Andreas; Ulke, Madlen; Dicker, Frank; Eder, Christiane; Kohlmann, Alexander; Grossmann, Vera; Kowarsch, Andreas; Kern, Wolfgang; Haferlach, Claudia; Haferlach, Torsten

2012-01-01

We analyzed 636 patients with diverse myeloproliferative neoplasms or myelodysplastic/myeloproliferative neoplasms for mutations of the Casitas B-cell lymphoma gene (CBLmut) in exons 8 and 9 and performed correlations to other genetic alterations. CBLmut were detected in 63 of 636 (9.9%) of these selected patients. CBLmut were more frequent in myelodysplastic/myeloproliferative neoplasms than myeloproliferative neoplasms (51 of 328, 15.5% vs. 12 of 291, 4.1%; P<0.001). Frequency was 48 of 278 (17.3%) in chronic myelomonocytic leukemia and 3 of 33 (9.1%) in unclassifiable myelodysplastic/myeloproliferative neoplasms. CBLmut was not detected in polycythemia vera, primary myelofibrosis, essential thrombocythemia, or refractory anemia with ring sideroblasts and marked thrombocytosis. CBLmut were underrepresented in JAK2V617F mutated as compared to JAK2V617wt cases (P<0.001), and mutually exclusive of JAK2exon12mut and MPLW515mut. CBLmut were associated with monosomy 7 (P=0.008) and TET2mut (P=0.003). In chronic myelomonocytic leukemia, CBLmut had no significant impact on survival outcomes. Therefore, CBLmut are frequent in chronic myelomonocytic leukemia, absent in classical myeloproliferative neoplasms, and are only exceptionally found in coincidence with JAK-STAT pathway activating mutations. PMID:22733026

CBL, CBLB, TET2, ASXL1, and IDH1/2 mutations and additional chromosomal aberrations constitute molecular events in chronic myelogenous leukemia

PubMed Central

Makishima, Hideki; Jankowska, Anna M.; McDevitt, Michael A.; O'Keefe, Christine; Dujardin, Simon; Cazzolli, Heather; Przychodzen, Bartlomiej; Prince, Courtney; Nicoll, John; Siddaiah, Harish; Shaik, Mohammed; Szpurka, Hadrian; Hsi, Eric; Advani, Anjali; Paquette, Ronald

2011-01-01

Progression of chronic myelogenous leukemia (CML) to accelerated (AP) and blast phase (BP) is because of secondary molecular events, as well as additional cytogenetic abnormalities. On the basis of the detection of JAK2, CBL, CBLB, TET2, ASXL1, and IDH1/2 mutations in myelodysplastic/myeloproliferative neoplasms, we hypothesized that they may also contribute to progression in CML. We screened these genes for mutations in 54 cases with CML (14 with chronic phase, 14 with AP, 20 with myeloid, and 6 with nonmyeloid BP). We identified 1 CBLB and 2 TET2 mutations in AP, and 1 CBL, 1 CBLB, 4 TET2, 2 ASXL1, and 2 IDH family mutations in myeloid BP. However, none of these mutations were found in chronic phase. No cases with JAK2V617F mutations were found. In 2 cases, TET2 mutations were found concomitant with CBLB mutations. By single nucleotide polymorphism arrays, uniparental disomy on chromosome 5q, 8q, 11p, and 17p was found in AP and BP but not involving 4q24 (TET2) or 11q23 (CBL). Microdeletions on chromosomes 17q11.2 and 21q22.12 involved tumor associated genes NF1 and RUNX1, respectively. Our results indicate that CBL family, TET2, ASXL1, and IDH family mutations and additional cryptic karyotypic abnormalities can occur in advanced phase CML. PMID:21346257

MECOM, HBS1L-MYB, THRB-RARB, JAK2, and TERT polymorphisms defining the genetic predisposition to myeloproliferative neoplasms: A study on 939 patients.

PubMed

Trifa, Adrian P; Bănescu, Claudia; Bojan, Anca S; Voina, Cristian M; Popa, Ștefana; Vișan, Simona; Ciubean, Alina D; Tripon, Florin; Dima, Delia; Popov, Viola M; Vesa, Ștefan C; Andreescu, Mihaela; Török-Vistai, Tünde; Mihăilă, Romeo G; Berbec, Nicoleta; Macarie, Ioan; Coliţă, Andrei; Iordache, Maria; Cătană, Alina C; Farcaș, Marius F; Tomuleasa, Ciprian; Vasile, Kinga; Truică, Cristina; Todincă, Adriana; Pop-Muntean, Lavinia; Manolache, Raluca; Bumbea, Horia; Vlădăreanu, Ana-Maria; Gaman, Mihaela; Ciufu, Cristina M; Popp, Radu A

2018-01-01

Polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) are classical myeloproliferative neoplasms (MPN), characterized by specific somatic mutations in JAK2, CALR or MPL genes. JAK2 46/1 and TERT rs2736100 polymorphisms are known to significantly predispose to MPN. This study aimed to establish the additional contribution of the recently described MECOM rs2201862, HBS1L-MYB rs9376092 and THRB-RARB rs4858647 polymorphisms to the occurrence of MPN. These three polymorphisms, along with JAK2 46/1 and TERT rs2736100 were genotyped in 939 MPN patients (454 with ET, 337 with PV and 148 with PMF) and 483 controls. MECOM rs2201862 associated significantly with each MPN entity, except for ET, and with all major molecular sub-types, especially those CALR-mutated (OR = 1.4; 95% CI = 1.1-1.8; P-value = .005). HBS1L-MYB rs9376092 associated only with JAK2 V617F-mutated ET (OR = 1.4; 95% CI = 1.1-1.7; P-value = .003). THRB-RARB rs4858647 had a weak association with PMF only (OR = 1.5; 95% CI = 1-2.1; P-value = .04). Surprisingly, JAK2 46/1 haplotype was associated significantly not only with JAK2 V617F-mutated MPN, but also with CALR-mutated MPN (OR = 1.4; 95% CI = 1.1-1.8; P-value = .01). TERT rs2736100 was associated equally strong with all MPN, regardless of phenotype or molecular sub-type. In conclusion, JAK2 46/1, TERT rs2736100 and MECOM rs2201862 are the chief predisposing polymorphisms to MPN. © 2017 Wiley Periodicals, Inc.

Whole-exome sequencing identifies novel MPL and JAK2 mutations in triple-negative myeloproliferative neoplasms

PubMed Central

Milosevic Feenstra, Jelena D.; Nivarthi, Harini; Gisslinger, Heinz; Leroy, Emilie; Rumi, Elisa; Chachoua, Ilyas; Bagienski, Klaudia; Kubesova, Blanka; Pietra, Daniela; Gisslinger, Bettina; Milanesi, Chiara; Jäger, Roland; Chen, Doris; Berg, Tiina; Schalling, Martin; Schuster, Michael; Bock, Christoph; Constantinescu, Stefan N.; Cazzola, Mario

2016-01-01

Essential thrombocythemia (ET) and primary myelofibrosis (PMF) are chronic diseases characterized by clonal hematopoiesis and hyperproliferation of terminally differentiated myeloid cells. The disease is driven by somatic mutations in exon 9 of CALR or exon 10 of MPL or JAK2-V617F in >90% of the cases, whereas the remaining cases are termed “triple negative.” We aimed to identify the disease-causing mutations in the triple-negative cases of ET and PMF by applying whole-exome sequencing (WES) on paired tumor and control samples from 8 patients. We found evidence of clonal hematopoiesis in 5 of 8 studied cases based on clonality analysis and presence of somatic genetic aberrations. WES identified somatic mutations in 3 of 8 cases. We did not detect any novel recurrent somatic mutations. In 3 patients with clonal hematopoiesis analyzed by WES, we identified a somatic MPL-S204P, a germline MPL-V285E mutation, and a germline JAK2-G571S variant. We performed Sanger sequencing of the entire coding region of MPL in 62, and of JAK2 in 49 additional triple-negative cases of ET or PMF. New somatic (T119I, S204F, E230G, Y591D) and 1 germline (R321W) MPL mutation were detected. All of the identified MPL mutations were gain-of-function when analyzed in functional assays. JAK2 variants were identified in 5 of 57 triple-negative cases analyzed by WES and Sanger sequencing combined. We could demonstrate that JAK2-V625F and JAK2-F556V are gain-of-function mutations. Our results suggest that triple-negative cases of ET and PMF do not represent a homogenous disease entity. Cases with polyclonal hematopoiesis might represent hereditary disorders. PMID:26423830

Whole-exome sequencing identifies novel MPL and JAK2 mutations in triple-negative myeloproliferative neoplasms.

PubMed

Milosevic Feenstra, Jelena D; Nivarthi, Harini; Gisslinger, Heinz; Leroy, Emilie; Rumi, Elisa; Chachoua, Ilyas; Bagienski, Klaudia; Kubesova, Blanka; Pietra, Daniela; Gisslinger, Bettina; Milanesi, Chiara; Jäger, Roland; Chen, Doris; Berg, Tiina; Schalling, Martin; Schuster, Michael; Bock, Christoph; Constantinescu, Stefan N; Cazzola, Mario; Kralovics, Robert

2016-01-21

Essential thrombocythemia (ET) and primary myelofibrosis (PMF) are chronic diseases characterized by clonal hematopoiesis and hyperproliferation of terminally differentiated myeloid cells. The disease is driven by somatic mutations in exon 9 of CALR or exon 10 of MPL or JAK2-V617F in >90% of the cases, whereas the remaining cases are termed "triple negative." We aimed to identify the disease-causing mutations in the triple-negative cases of ET and PMF by applying whole-exome sequencing (WES) on paired tumor and control samples from 8 patients. We found evidence of clonal hematopoiesis in 5 of 8 studied cases based on clonality analysis and presence of somatic genetic aberrations. WES identified somatic mutations in 3 of 8 cases. We did not detect any novel recurrent somatic mutations. In 3 patients with clonal hematopoiesis analyzed by WES, we identified a somatic MPL-S204P, a germline MPL-V285E mutation, and a germline JAK2-G571S variant. We performed Sanger sequencing of the entire coding region of MPL in 62, and of JAK2 in 49 additional triple-negative cases of ET or PMF. New somatic (T119I, S204F, E230G, Y591D) and 1 germline (R321W) MPL mutation were detected. All of the identified MPL mutations were gain-of-function when analyzed in functional assays. JAK2 variants were identified in 5 of 57 triple-negative cases analyzed by WES and Sanger sequencing combined. We could demonstrate that JAK2-V625F and JAK2-F556V are gain-of-function mutations. Our results suggest that triple-negative cases of ET and PMF do not represent a homogenous disease entity. Cases with polyclonal hematopoiesis might represent hereditary disorders. © 2016 by The American Society of Hematology.

Sequential treatment of CD34+ cells from patients with primary myelofibrosis with chromatin-modifying agents eliminate JAK2V617F-positive NOD/SCID marrow repopulating cells

PubMed Central

Wang, Xiaoli; Zhang, Wei; Tripodi, Joseph; Lu, Min; Xu, Mingjiang; Najfeld, Vesna; Li, Yan

2010-01-01

Because primary myelofibrosis (PMF) originates at the level of the pluripotent hematopoietic stem cell (HSC), we examined the effects of various therapeutic agents on the in vitro and in vivo behavior of PMF CD34+ cells. Treatment of PMF CD34+ cells with chromatin-modifying agents (CMAs) but not hydroxyurea, Janus kinase 2 (JAK2) inhibitors, or low doses of interferon-α led to the generation of greater numbers of CD34+ chemokine (C-X-C motif) receptor (CXCR)4+ cells, which were capable of migrating in response to chemokine (C-X-C motif) ligand (CXCL)12 and resulted in a reduction in the proportion of hematopoietic progenitor cells (HPCs) that were JAK2V617F+. Furthermore, sequential treatment of PMF CD34+ cells but not normal CD34+ cells with decitabine (5-aza-2′-deoxycytidine [5azaD]), followed by suberoylanilide hydroxamic acid (SAHA; 5azaD/SAHA), or trichostatin A (5azaD/TSA) resulted in a higher degree of apoptosis. Two to 6 months after the transplantation of CMAs treated JAK2V617F+ PMF CD34+ cells into nonobese diabetic/severe combined immunodeficient (SCID)/IL-2Rγnull mice, the percentage of JAK2V617F/JAK2total in human CD45+ marrow cells was dramatically reduced. These findings suggest that both PMF HPCs, short-term and long-term SCID repopulating cells (SRCs), are JAK2V617F+ and that JAK2V617F+ HPCs and SRCs can be eliminated by sequential treatment with CMAs. Sequential treatment with CMAs, therefore, represents a possible effective means of treating PMF at the level of the malignant SRC. PMID:20858855

Ruxolitinib reduces JAK2 p.V617F allele burden in patients with polycythemia vera enrolled in the RESPONSE study.

PubMed

Vannucchi, Alessandro Maria; Verstovsek, Srdan; Guglielmelli, Paola; Griesshammer, Martin; Burn, Timothy C; Naim, Ahmad; Paranagama, Dilan; Marker, Mahtab; Gadbaw, Brian; Kiladjian, Jean-Jacques

2017-07-01

In patients with polycythemia vera (PV), an elevated JAK2 p.V617F allele burden is associated with indicators of more severe disease (e.g., leukocytosis, splenomegaly, and increased thrombosis risk); however, correlations between allele burden reductions and clinical benefit in patients with PV have not been extensively evaluated in a randomized trial. This exploratory analysis from the multicenter, open-label, phase 3 Randomized Study of Efficacy and Safety in Polycythemia Vera With JAK Inhibitor INCB018424 Versus Best Supportive Care trial evaluated the long-term effect of ruxolitinib treatment on JAK2 p.V617F allele burden in patients with PV. Evaluable JAK2 p.V617F-positive patients randomized to ruxolitinib (n = 107) or best available therapy (BAT) who crossed over to ruxolitinib at week 32 (n = 97) had consistent JAK2 p.V617F allele burden reductions throughout the study. At all time points measured (up to weeks 208 [ruxolitinib-randomized] and 176 [ruxolitinib crossover]), mean changes from baseline over time in JAK2 p.V617F allele burden ranged from -12.2 to -40.0% (ruxolitinib-randomized) and -6.3 to -17.8% (ruxolitinib crossover). Complete or partial molecular response was observed in 3 patients (ruxolitinib-randomized, n = 2; ruxolitinib crossover, n = 1) and 54 patients (ruxolitinib-randomized, n = 33; ruxolitinib crossover, n = 20; BAT, n = 1), respectively. Among patients treated with interferon as BAT (n = 13), the mean maximal reduction in allele burden from baseline was 25.6% after crossover to ruxolitinib versus 6.6% before crossover. Collectively, the data from this exploratory analysis suggest that ruxolitinib treatment for up to 4 years provides progressive reductions in JAK2 p.V617F allele burden in patients with PV who are resistant to or intolerant of hydroxyurea. The relationship between allele burden changes and clinical outcomes in patients with PV remains unclear.

Pathogenesis of Myeloproliferative Neoplasms: Role and Mechanisms of Chronic Inflammation.

PubMed

Hermouet, Sylvie; Bigot-Corbel, Edith; Gardie, Betty

2015-01-01

Myeloproliferative neoplasms (MPNs) are a heterogeneous group of clonal diseases characterized by the excessive and chronic production of mature cells from one or several of the myeloid lineages. Recent advances in the biology of MPNs have greatly facilitated their molecular diagnosis since most patients present with mutation(s) in the JAK2, MPL, or CALR genes. Yet the roles played by these mutations in the pathogenesis and main complications of the different subtypes of MPNs are not fully elucidated. Importantly, chronic inflammation has long been associated with MPN disease and some of the symptoms and complications can be linked to inflammation. Moreover, the JAK inhibitor clinical trials showed that the reduction of symptoms linked to inflammation was beneficial to patients even in the absence of significant decrease in the JAK2-V617F mutant load. These observations suggested that part of the inflammation observed in patients with JAK2-mutated MPNs may not be the consequence of JAK2 mutation. The aim of this paper is to review the different aspects of inflammation in MPNs, the molecular mechanisms involved, the role of specific genetic defects, and the evidence that increased production of certain cytokines depends or not on MPN-associated mutations, and to discuss possible nongenetic causes of inflammation.

Pathogenesis of Myeloproliferative Neoplasms: Role and Mechanisms of Chronic Inflammation

PubMed Central

Bigot-Corbel, Edith; Gardie, Betty

2015-01-01

Myeloproliferative neoplasms (MPNs) are a heterogeneous group of clonal diseases characterized by the excessive and chronic production of mature cells from one or several of the myeloid lineages. Recent advances in the biology of MPNs have greatly facilitated their molecular diagnosis since most patients present with mutation(s) in the JAK2, MPL, or CALR genes. Yet the roles played by these mutations in the pathogenesis and main complications of the different subtypes of MPNs are not fully elucidated. Importantly, chronic inflammation has long been associated with MPN disease and some of the symptoms and complications can be linked to inflammation. Moreover, the JAK inhibitor clinical trials showed that the reduction of symptoms linked to inflammation was beneficial to patients even in the absence of significant decrease in the JAK2-V617F mutant load. These observations suggested that part of the inflammation observed in patients with JAK2-mutated MPNs may not be the consequence of JAK2 mutation. The aim of this paper is to review the different aspects of inflammation in MPNs, the molecular mechanisms involved, the role of specific genetic defects, and the evidence that increased production of certain cytokines depends or not on MPN-associated mutations, and to discuss possible nongenetic causes of inflammation. PMID:26538820

The Stilbenoid Tyrosine Kinase Inhibitor, G6, Suppresses Jak2-V617F-mediated Human Pathological Cell Growth in Vitro and in Vivo*

PubMed Central

Kirabo, Annet; Embury, Jennifer; Kiss, Róbert; Polgár, Tímea; Gali, Meghanath; Majumder, Anurima; Bisht, Kirpal S.; Cogle, Christopher R.; Keserű, György M.; Sayeski, Peter P.

2011-01-01

Using structure-based virtual screening, we previously identified a novel stilbenoid inhibitor of Jak2 tyrosine kinase named G6. Here, we hypothesized that G6 suppresses Jak2-V617F-mediated human pathological cell growth in vitro and in vivo. We found that G6 inhibited proliferation of the Jak2-V617F expressing human erythroleukemia (HEL) cell line by promoting marked cell cycle arrest and inducing apoptosis. The G6-dependent increase in apoptosis levels was concomitant with increased caspase 3/7 activity and cleavage of PARP. G6 also selectively inhibited phosphorylation of STAT5, a downstream signaling target of Jak2. Using a mouse model of Jak2-V617F-mediated hyperplasia, we found that G6 significantly decreased the percentage of blast cells in the peripheral blood, reduced splenomegaly, and corrected a pathologically low myeloid to erythroid ratio in the bone marrow by eliminating HEL cell engraftment in this tissue. In addition, drug efficacy correlated with the presence of G6 in the plasma, marrow, and spleen. Collectively, these data demonstrate that the stilbenoid compound, G6, suppresses Jak2-V617F-mediated aberrant cell growth. As such, G6 may be a potential therapeutic lead candidate against Jak2-mediated, human disease. PMID:21127060

Efficacy of the JAK2 inhibitor INCB16562 in a murine model of MPLW515L-induced thrombocytosis and myelofibrosis.

PubMed

Koppikar, Priya; Abdel-Wahab, Omar; Hedvat, Cyrus; Marubayashi, Sachie; Patel, Jay; Goel, Aviva; Kucine, Nicole; Gardner, Jeffrey R; Combs, Andrew P; Vaddi, Kris; Haley, Patrick J; Burn, Timothy C; Rupar, Mark; Bromberg, Jacqueline F; Heaney, Mark L; de Stanchina, Elisa; Fridman, Jordan S; Levine, Ross L

2010-04-08

The discovery of JAK2 and MPL mutations in patients with myeloproliferative neoplasms (MPNs) provided important insight into the genetic basis of these disorders and led to the development of JAK2 kinase inhibitors for MPN therapy. Although recent studies have shown that JAK2 kinase inhibitors demonstrate efficacy in a JAK2V617F murine bone marrow transplantation model, the effects of JAK2 inhibitors on MPLW515L-mediated myeloproliferation have not been investigated. In this report, we describe the in vitro and in vivo effects of INCB16562, a small-molecule JAK2 inhibitor. INCB16562 inhibited proliferation and signaling in cell lines transformed by JAK2 and MPL mutations. Compared with vehicle treatment, INCB16562 treatment improved survival, normalized white blood cell counts and platelet counts, and markedly reduced extramedullary hematopoeisis and bone marrow fibrosis. We observed inhibition of STAT3 and STAT5 phosphorylation in vivo consistent with potent inhibition of JAK-STAT signaling. These data suggest JAK2 inhibitor therapy may be of value in the treatment of JAK2V617F-negative MPNs. However, we did not observe a decrease in the size of the malignant clone in the bone marrow of treated mice at the end of therapy, which suggests that JAK2 inhibitor therapy, by itself, was not curative in this MPN model.

Efficacy of the JAK2 inhibitor INCB16562 in a murine model of MPLW515L-induced thrombocytosis and myelofibrosis

PubMed Central

Koppikar, Priya; Abdel-Wahab, Omar; Hedvat, Cyrus; Marubayashi, Sachie; Patel, Jay; Goel, Aviva; Kucine, Nicole; Gardner, Jeffrey R.; Combs, Andrew P.; Vaddi, Kris; Haley, Patrick J.; Burn, Timothy C.; Rupar, Mark; Bromberg, Jacqueline F.; Heaney, Mark L.; de Stanchina, Elisa; Fridman, Jordan S.

2010-01-01

The discovery of JAK2 and MPL mutations in patients with myeloproliferative neoplasms (MPNs) provided important insight into the genetic basis of these disorders and led to the development of JAK2 kinase inhibitors for MPN therapy. Although recent studies have shown that JAK2 kinase inhibitors demonstrate efficacy in a JAK2V617F murine bone marrow transplantation model, the effects of JAK2 inhibitors on MPLW515L-mediated myeloproliferation have not been investigated. In this report, we describe the in vitro and in vivo effects of INCB16562, a small-molecule JAK2 inhibitor. INCB16562 inhibited proliferation and signaling in cell lines transformed by JAK2 and MPL mutations. Compared with vehicle treatment, INCB16562 treatment improved survival, normalized white blood cell counts and platelet counts, and markedly reduced extramedullary hematopoeisis and bone marrow fibrosis. We observed inhibition of STAT3 and STAT5 phosphorylation in vivo consistent with potent inhibition of JAK-STAT signaling. These data suggest JAK2 inhibitor therapy may be of value in the treatment of JAK2V617F-negative MPNs. However, we did not observe a decrease in the size of the malignant clone in the bone marrow of treated mice at the end of therapy, which suggests that JAK2 inhibitor therapy, by itself, was not curative in this MPN model. PMID:20154217

The oral HDAC inhibitor pracinostat (SB939) is efficacious and synergistic with the JAK2 inhibitor pacritinib (SB1518) in preclinical models of AML

PubMed Central

Novotny-Diermayr, V; Hart, S; Goh, K C; Cheong, A; Ong, L-C; Hentze, H; Pasha, M K; Jayaraman, R; Ethirajulu, K; Wood, J M

2012-01-01

Acute myeloid leukemia (AML) is currently treated with aggressive chemotherapy that is not well tolerated in many elderly patients, hence the unmet medical need for effective therapies with less toxicity and better tolerability. Inhibitors of FMS-like tyrosine kinase 3 (FLT3), JAK2 and histone deacetylase inhibitors (HDACi) have been tested in clinical studies, but showed only moderate single-agent activity. High efficacy of the HDACi pracinostat treating AML and synergy with the JAK2/FLT3 inhibitor pacritinib is demonstrated. Both compounds inhibit JAK-signal transducer and activator of transcription (STAT) signaling in AML cells with JAK2V617F mutations, but also diminish FLT3 signaling, particularly in FLT3-ITD (internal tandem duplication) cell lines. In vitro, this combination led to decreased cell proliferation and increased apoptosis. The synergy translated in vivo in two different AML models, the SET-2 megakaryoblastic AML mouse model carrying a JAK2V617F mutation, and the MOLM-13 model of FLT3-ITD-driven AML. Pracinostat and pacritinib in combination showed synergy on tumor growth, reduction of metastases and synergistically decreased JAK2 or FLT signaling, depending on the cellular context. In addition, several plasma cytokines/growth factors/chemokines triggered by the tumor growth were normalized, providing a rationale for combination therapy with an HDACi and a JAK2/FLT3 inhibitor for the treatment of AML patients, particularly those with FLT3 or JAK2 mutations. PMID:22829971

Clinical and molecular characterisation of a prospectively collected cohort of children and adolescents with polycythemia vera.

PubMed

Cario, Holger; Schwarz, Klaus; Herter, Jan M; Komrska, Vladimir; McMullin, Mary F; Minkov, Milen; Niemeyer, Charlotte; Pospisilova, Dagmar; Reinhard, Harald; Debatin, Klaus-Michael; Pahl, Heike L

2008-08-01

The clinical, haematological, molecular and treatment data of eight paediatric patients with polycythemia vera (PV) were collected prospectively. One patient developed PV after treatment for large-cell anaplastic lymphoma. Budd-Chiari syndrome was diagnosed in two patients, necessitating orthotopic liver transplantation in one and transjugular portosystemic shunting in the other. The remaining patients presented with non-specific symptoms. Endogenous erythroid colonies were detected in all cases examined. The JAK2(V617F) mutation was found in six patients; two patients displayed JAK2 exon 12 mutations, including one novel mutation (JAK2(H538-K539delinsI)). CD177 (PRV-1) mRNA expression was increased in three of five patients tested.

Clinical and molecular characterisation of a prospectively collected cohort of children and adolescents with polycythemia vera

PubMed Central

Cario, Holger; Schwarz, Klaus; Herter, Jan M.; Komrska, Vladimir; McMullin, Mary F.; Minkov, Milen; Niemeyer, Charlotte; Pospisilova, Dagmar; Reinhard, Harald; Debatin, Klaus-Michael; Pahl, Heike L.

2014-01-01

Summary The clinical, haematological, molecular and treatment data of eight paediatric patients with polycythemia vera (PV) were collected prospectively. One patient developed PV after treatment for large-cell anaplastic lymphoma. Budd-Chiari syndrome was diagnosed in two patients, necessitating orthotopic liver transplantation in one and transjugular portosystemic shunting in the other. The remaining patients presented with non-specific symptoms. Endogenous erythroid colonies were detected in all cases examined. The JAK2V617F mutation was found in six patients; two patients displayed JAK2 exon 12 mutations, including one novel mutation (JAK2H538-K539delinsI). CD177 (PRV-1) mRNA expression was increased in three of five patients tested. PMID:18557746

Investigation of T-cell immunoglobulin- and mucin-domain-containing molecule-3 (TIM-3) polymorphisms in essential thrombocythaemia (ET).

PubMed

Han, Fuyan; Wang, Guanghai; Li, Yuantang; Tian, Wenjun; Dong, Zhenfang; Cheng, Shiqing; Liu, Yiqing; Qu, Teng; Wang, Xiaoying; Wang, Yong; Zhang, Bingchang; Ju, Ying

2017-07-01

T-cell immunoglobulin- and mucin-domain-containing molecule-3 (TIM-3) is preferentially expressed on terminally differentiated Th1 cells and inhibits their IFN-γ production. It has been reported that chronic inflammation may be an important driving force for myeloproliferative neoplasms (MPNs). Therefore, we hypothesized that as an important inflammation regulator, TIM-3 may be involved in essential thrombocythaemia (ET). The goal of this study was to investigate whether the -1516G > T, -574G > T and +4259T > G single-nucleotide polymorphisms (SNPs) within the TIM-3 gene contribute to the genetic susceptibility of individuals to ET. Genotyping of the TIM-3 -1516G > T, -574G > T and + 4259T > G SNPs was performed in 175 patients with ET and in 151 controls via a polymerase chain reaction-restriction fragment length polymorphism assay. We also investigated the relationships between the genotypes of each SNP and the risk factors of ET such as routine blood indexes, age and JAK2 V617F mutation. The genotype and allele frequencies of the -1516G > T SNP (p = 0.016 and 0.019, respectively), the -574G > T SNP (p = 0.035 and 0.038, respectively) and the +4259T > G SNP (p = 0.036 and 0.038, respectively) of the ET patients and the controls were significantly different. A haplotype analysis found that the GGT and TGT haplotypes had significantly different distributions between ET and controls (p = 0.041 and 0.041, respectively). However, no significant differences were detected between the genotypes of all SNPs and routine blood indexes, age and JAK2V617F mutation. The -1516G > T, -574G > T and +4259T > G SNPs within TIM-3 gene might play an important role as a genetic risk factor in the pathogenesis of ET.

Use of molecular testing to identify a cluster of patients with polycythemia vera in eastern Pennsylvania.

PubMed

Seaman, Vincent; Jumaan, Aisha; Yanni, Emad; Lewis, Brian; Neyer, Jonathan; Roda, Paul; Xu, Mingjiang; Hoffman, Ronald

2009-02-01

The role of the environment in the origin of polycythemia vera has not been well documented. Recently, molecular diagnostic tools have been developed to facilitate the diagnosis of polycythemia vera. A cluster of patients with polycythemia vera was suspected in three countries in eastern Pennsylvania where there have long been a concern about environment hazards. Rigorous clinical criteria and JAK2 617V>F testing were used to confirm the diagnosis of polycythemia vera in patients in this area. Participants included cases of polycythemia vera from the 2001 to 2005 state cancer registry as well as self- and physician-referred cases. A diagnosis of polycythemia vera was confirmed in 53% of 62 participants using WHO criteria, which includes JAK2 617V>F testing. A statistically significant cluster of cases (P < 0.001) was identified where the incidence of polycythemia vera was 4.3 times that of the rest of the study area. The area of the cluster contained numerous sources of hazardous material including waste-coal power plants and U.S. Environmental Protection Agency Superfund sites. The diagnosis of polycythemia vera based solely on clinical criteria is frequently erroneous, suggesting that our prior knowledge of the epidemiology of this disease might be inaccurate. The JAK2 617V>F mutational analysis provides diagnostic clarity and permitted the confirmation of a cluster of polycythemia vera cases not identified by traditional clinical and pathologic diagnostic criteria. The close proximity of this cluster to known areas of hazardous material exposure raises concern that such environmental factors might play a role in the origin of polycythemia vera.

The Amelioration of Myelofibrosis with Thrombocytopenia by a JAK1/2 Inhibitor, Ruxolitinib, in a Post-polycythemia Vera Myelofibrosis Patient with a JAK2 Exon 12 Mutation.

PubMed

Ikeda, Kazuhiko; Ueda, Koki; Sano, Takahiro; Ogawa, Kazuei; Ikezoe, Takayuki; Hashimoto, Yuko; Morishita, Soji; Komatsu, Norio; Ohto, Hitoshi; Takeishi, Yasuchika

2017-01-01

Less than 5% of patients with polycythemia vera (PV) show JAK2 exon 12 mutations. Although PV patients with JAK2 exon 12 mutations are known to develop post-PV myelofibrosis (MF) as well as PV with JAK2V617F, the role of JAK inhibitors in post-PV MF patients with JAK2 exon 12 mutations remains unknown. We describe how treatment with a JAK1/2 inhibitor, ruxolitinib, led to the rapid amelioration of marrow fibrosis, erythrocytosis and thrombocytopenia in a 77-year-old man with post-PV MF who carried a JAK2 exon 12 mutation (JAK2H538QK539L). This case suggests that ruxolitinib is a treatment option for post-PV MF in patients with thrombocytopenia or JAK2 exon 12 mutations.

The Jak2 Inhibitor, G6, Alleviates Jak2-V617F-Mediated Myeloproliferative Neoplasia by Providing Significant Therapeutic Efficacy to the Bone Marrow1

PubMed Central

Kirabo, Annet; Park, Sung O; Majumder, Anurima; Gali, Meghanath; Reinhard, Mary K; Wamsley, Heather L; Zhao, Zhizhuang Joe; Cogle, Christopher R; Bisht, Kirpal S; Keserü, György M; Sayeski, Peter P

2011-01-01

We recently developed a Janus kinase 2 (Jak2) small-molecule inhibitor called G6 and found that it inhibits Jak2-V617F-mediated pathologic cell growth in vitro, ex vivo, and in vivo. However, its ability to inhibit Jak2-V617F-mediated myeloproliferative neoplasia, with particular emphasis in the bone marrow, has not previously been examined. Here, we investigated the efficacy of G6 in a transgenic mouse model of Jak2-V617F-mediated myeloproliferative neoplasia. We found that G6 provided therapeutic benefit to the peripheral blood as determined by elimination of leukocytosis, thrombocytosis, and erythrocytosis. G6 normalized the pathologically high plasma concentrations of interleukin 6 (IL-6). In the liver, G6 eliminated Jak2-V617F-driven extramedullary hematopoiesis. With respect to the spleen, G6 significantly reduced both the splenomegaly and megakaryocytic hyperplasia. In the critically important bone marrow, G6 normalized the pathologically high levels of phospho-Jak2 and phospho-signal transducer and activator of transcription 5 (STAT5). It significantly reduced the megakaryocytic hyperplasia in the marrow and completely normalized the M/E ratio. Most importantly, G6 selectively reduced the mutant Jak2 burden by 67%on average, with virtual elimination of mutant Jak2 cells in one third of all treated mice. Lastly, clonogenic assays using marrow stem cells from the myeloproliferative neoplasm mice revealed a time-dependent elimination of the clonogenic growth potential of these cells by G6. Collectively, these data indicate that G6 exhibits exceptional efficacy in the peripheral blood, liver, spleen, and, most importantly, in the bone marrow, thereby raising the possibility that this compound may alter the natural history of Jak2-V617F-mediated myeloproliferative neoplasia. PMID:22131881

Refractory Anemia with Ring Sideroblasts (RARS) and RARS with Thrombocytosis (RARS-T) – “2017 Update on Diagnosis, Risk-stratification, and Management”

PubMed Central

Patnaik, Mrinal M.; Tefferi, Ayalew

2017-01-01

Disease Overview Ring sideroblasts (RS) are erythroid precursors with abnormal perinuclear mitochondrial iron accumulation. Two myeloid neoplasms defined by the presence of RS, include refractory anemia with ring sideroblasts (RARS), now classified under myelodysplastic syndromes with RS (MDS-RS) and RARS with thrombocytosis (RARS-T); now called myelodysplastic/myeloproliferative neoplasm with RS and thrombocytosis (MDS/MPN-RS-T). Diagnosis MDS-RS is a lower risk MDS, with single or multilineage dysplasia (SLD/MLD), <5% bone marrow (BM) blasts and ≥15% BM RS (≥5% in the presence of SF3B1 mutations). MDS/MPN-RS-T, now a formal entity in the MDS/MPN overlap syndromes, has diagnostic features of MDS-RS-SLD, along with a platelet count ≥ 450 × 10(9)/L and large atypical megakaryocytes (similar to BCR-ABL1 negative MPN). Mutations and Karyotype Mutations in SF3B1 are seen in ≥80% of patients with MDS-RS-SLD and MDS/MPN-RS-T, and strongly correlate with the presence of BM RS; MDS/MPN-RS-T patients also demonstrate JAK2V617F, ASXL1, DNMT3A, SETBP1, and TET2 mutations; with ASXL1/SETBP1 mutations adversely impacting survival. Cytogenetic abnormalities are uncommon in both diseases. Risk stratification Most patients with MDS-RS-SLD are stratified into lower risk groups by the revised-International Prognostic Scoring System (R-IPSS). Disease outcome in MDS/MPN-RS-T is better than that of MDS-RS-SLD, but worse than that of essential thrombocythemia. Both diseases have a low risk of leukemic transformation. Treatment Anemia and iron overload are complications seen in both and are managed similar to lower risk MDS and MPN. Aspirin therapy is reasonable in MDS/MPN-RS-T, especially in the presence of JAK2V617F, but the value of platelet-lowering drugs is uncertain. PMID:28188970

Prospect of JAK2 inhibitor therapy in myeloproliferative neoplasms

PubMed Central

Atallah, Ehab; Verstovsek, Srdan

2016-01-01

The discovery of the Janus kinase (JAK)2 V617F mutation in patients with myeloproliferative neoplasms was a major milestone in understanding the biology of those disorders. Several groups simultaneously reported on the high incidence of this mutation in patients with myeloproliferative neoplasms: almost all patients with polycythemia vera harbor the mutation and about 50% of patients with essential thrombocythemia and primary myelofibrosis have the mutation, making the development of JAK2 tyrosine kinase inhibitors an attractive therapeutic goal. In addition, inhibition of JAK2 kinase may have a therapeutic role in other hematologic malignancies, such as chronic myeloid leukemia or lymphoma. A number of molecules that inhibit JAK2 kinase have been described in the literature, and several are being evaluated in a clinical setting. Here, we summarize current clinical experience with JAK2 inhibitors. PMID:19445582

Increased neutrophil extracellular trap formation promotes thrombosis in myeloproliferative neoplasms.

PubMed

Wolach, Ofir; Sellar, Rob S; Martinod, Kimberly; Cherpokova, Deya; McConkey, Marie; Chappell, Ryan J; Silver, Alexander J; Adams, Dylan; Castellano, Cecilia A; Schneider, Rebekka K; Padera, Robert F; DeAngelo, Daniel J; Wadleigh, Martha; Steensma, David P; Galinsky, Ilene; Stone, Richard M; Genovese, Giulio; McCarroll, Steven A; Iliadou, Bozenna; Hultman, Christina; Neuberg, Donna; Mullally, Ann; Wagner, Denisa D; Ebert, Benjamin L

2018-04-11

Thrombosis is a major cause of morbidity and mortality in Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs), clonal disorders of hematopoiesis characterized by activated Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling. Neutrophil extracellular trap (NET) formation, a component of innate immunity, has been linked to thrombosis. We demonstrate that neutrophils from patients with MPNs are primed for NET formation, an effect blunted by pharmacological inhibition of JAK signaling. Mice with conditional knock-in of Jak2 V617F , the most common molecular driver of MPN, have an increased propensity for NET formation and thrombosis. Inhibition of JAK-STAT signaling with the clinically available JAK2 inhibitor ruxolitinib abrogated NET formation and reduced thrombosis in a deep vein stenosis murine model. We further show that expression of PAD4, a protein required for NET formation, is increased in JAK2 V617F -expressing neutrophils and that PAD4 is required for Jak2 V617F -driven NET formation and thrombosis in vivo. Finally, in a population study of more than 10,000 individuals without a known myeloid disorder, JAK2 V617F -positive clonal hematopoiesis was associated with an increased incidence of thrombosis. In aggregate, our results link JAK2 V617F expression to NET formation and thrombosis and suggest that JAK2 inhibition may reduce thrombosis in MPNs through cell-intrinsic effects on neutrophil function. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

The role of JAK1/2 inhibitors in the treatment of chronic myeloproliferative neoplasms.

PubMed

Keohane, Clodagh; Mesa, Ruben; Harrison, Claire

2013-01-01

In 2005, the description of the JAK2V617F mutation for the first time provided a molecular key to enable more rapid diagnosis and target for novel therapeutics in the myeloproliferative neoplasms. In 2007, the first-in-class agent INC18424, ruxolitinib, JAKafi, or JAKAVI was first tested in patients with intermediate-risk 2 or high-risk myelofibrosis regardless of whether they possessed the JAK2V617F mutation. Patients treated with this agent had major reduction in splenomegaly as well as impressive reduction, and in some cases resolution, of symptoms. This study was followed by the two Controlled Myelofibrosis Study with Oral JAK Inhibitor Therapy (COMFORT) trials (the first-ever phase III trials in myelofibrosis), which confirmed results in these aspects were superior to either placebo or standard care, and updated results show a survival advantage with this therapy. This paper discusses these results and data from other JAK inhibitors while speculating on the future of these therapies. It also reflects on the fact that the true targets and agents' mode of action are uncertain. Unlike targeted therapy for chronic myeloid leukemia (CML), these agents do not deliver molecular remission, and it is not clear whether their predominant benefit is mediated via JAK2, JAK1, or both. Nonetheless, the advent of the JAK inhibitor is a welcome advance and has made a dramatic improvement to the therapeutic landscape of these conditions.

Presence of calreticulin mutations in JAK2-negative polycythemia vera.

PubMed

Broséus, Julien; Park, Ji-Hye; Carillo, Serge; Hermouet, Sylvie; Girodon, François

2014-12-18

Calreticulin (CALR) mutations have been reported in Janus kinase 2 (JAK2)- and myeloproliferative leukemia (MPL)-negative essential thrombocythemia and primary myelofibrosis. In contrast, no CALR mutations have ever been reported in the context of polycythemia vera (PV). Here, we describe 2 JAK2(V617F)-JAK2(exon12)-negative PV patients who presented with a CALR mutation in peripheral granulocytes at the time of diagnosis. In both cases, the CALR mutation was a 52-bp deletion. Single burst-forming units-erythroid (BFU-E) from 1 patient were grown in vitro and genotyped: the same CALR del 52-bp mutation was noted in 31 of the 37 colonies examined; 30 of 31 BFU-E were heterozygous for CALR del 52 bp, and 1 of 31 BFU-E was homozygous for CALR del 52 bp. In summary, although unknown mutations leading to PV cannot be ruled out, our results suggest that CALR mutations can be associated with JAK2-negative PV. © 2014 by The American Society of Hematology.

The Hepatocyte Growth Factor (HGF)/Met Axis: A Neglected Target in the Treatment of Chronic Myeloproliferative Neoplasms?

PubMed Central

Boissinot, Marjorie; Vilaine, Mathias; Hermouet, Sylvie

2014-01-01

Met is the receptor of hepatocyte growth factor (HGF), a cytoprotective cytokine. Disturbing the equilibrium between Met and its ligand may lead to inappropriate cell survival, accumulation of genetic abnormalities and eventually, malignancy. Abnormal activation of the HGF/Met axis is established in solid tumours and in chronic haematological malignancies, including myeloma, acute myeloid leukaemia, chronic myelogenous leukaemia (CML), and myeloproliferative neoplasms (MPNs). The molecular mechanisms potentially responsible for the abnormal activation of HGF/Met pathways are described and discussed. Importantly, inCML and in MPNs, the production of HGF is independent of Bcr-Abl and JAK2V617F, the main molecular markers of these diseases. In vitro studies showed that blocking HGF/Met function with neutralizing antibodies or Met inhibitors significantly impairs the growth of JAK2V617F-mutated cells. With personalised medicine and curative treatment in view, blocking activation of HGF/Met could be a useful addition in the treatment of CML and MPNs for those patients with high HGF/MET expression not controlled by current treatments (Bcr-Abl inhibitors in CML; phlebotomy, hydroxurea, JAK inhibitors in MPNs). PMID:25119536

AKT is a therapeutic target in myeloproliferative neoplasms

PubMed Central

Khan, Irum; Huang, Zan; Wen, Qiang; Stankiewicz, Monika J.; Gilles, Laure; Goldenson, Benjamin; Schultz, Rachael; Diebold, Lauren; Gurbuxani, Sandeep; Finke, Christy M.; Lasho, Terra L.; Koppikar, Priya; Pardanani, Animesh; Stein, Brady; Altman, Jessica K.; Levine, Ross L.; Tefferi, Ayalew; Crispino, John D.

2014-01-01

The majority of patients with BCR-ABL1-negative myeloproliferative neoplasms (MPN) harbor mutations in JAK2 or MPL, which lead to constitutive activation of the JAK/STAT, PI3K, and ERK signaling pathways. JAK inhibitors by themselves are inadequate in producing selective clonal suppression in MPN and are associated with hematopoietic toxicities. MK-2206 is a potent allosteric AKT inhibitor that was well tolerated, including no evidence of myelosuppression, in a phase I study of solid tumors. Herein, we show that inhibition of PI3K/AKT signaling by MK-2206 affected the growth of both JAK2V617F or MPLW515L-expressing cells via reduced phosphorylation of AKT and inhibition of its downstream signaling molecules. Moreover, we demonstrate that MK-2206 synergizes with Ruxolitinib in suppressing the growth of JAK2V617F mutant SET2 cells. Importantly MK-2206 suppressed colony formation from hematopoietic progenitor cells in patients with primary myelofibrosis (PMF) and alleviated hepatosplenomegaly and reduced megakaryocyte burden in the bone marrows, livers and spleens of mice with MPLW515L-induced MPN. Together, these findings establish AKT as a rational therapeutic target in the MPNs. PMID:23748344

AKT is a therapeutic target in myeloproliferative neoplasms.

PubMed

Khan, I; Huang, Z; Wen, Q; Stankiewicz, M J; Gilles, L; Goldenson, B; Schultz, R; Diebold, L; Gurbuxani, S; Finke, C M; Lasho, T L; Koppikar, P; Pardanani, A; Stein, B; Altman, J K; Levine, R L; Tefferi, A; Crispino, J D

2013-09-01

The majority of patients with BCR-ABL1-negative myeloproliferative neoplasms (MPN) harbor mutations in JAK2 or MPL, which lead to constitutive activation of the JAK/STAT, PI3K and ERK signaling pathways. JAK inhibitors by themselves are inadequate in producing selective clonal suppression in MPN and are associated with hematopoietic toxicities. MK-2206 is a potent allosteric AKT inhibitor that was well tolerated, including no evidence of myelosuppression, in a phase I study of solid tumors. Herein, we show that inhibition of PI3K/AKT signaling by MK-2206 affected the growth of both JAK2V617F- or MPLW515L-expressing cells via reduced phosphorylation of AKT and inhibition of its downstream signaling molecules. Moreover, we demonstrate that MK-2206 synergizes with ruxolitinib in suppressing the growth of JAK2V617F-mutant SET2 cells. Importantly, MK-2206 suppressed colony formation from hematopoietic progenitor cells in patients with primary myelofibrosis and alleviated hepatosplenomegaly and reduced megakaryocyte burden in the bone marrows, livers and spleens of mice with MPLW515L-induced MPN. Together, these findings establish AKT as a rational therapeutic target in the MPNs.

A novel splicing site IRP1 somatic mutation in a patient with pheochromocytoma and JAK2V617F positive polycythemia vera: a case report.

PubMed

Pang, Ying; Gupta, Garima; Yang, Chunzhang; Wang, Herui; Huynh, Thanh-Truc; Abdullaev, Ziedulla; Pack, Svetlana D; Percy, Melanie J; Lappin, Terence R J; Zhuang, Zhengping; Pacak, Karel

2018-03-13

The role of the hypoxia signaling pathway in the pathogenesis of pheochromocytoma/paraganglioma (PPGL)-polycythemia syndrome has been elucidated. Novel somatic mutations in hypoxia-inducible factor type 2A (HIF2A) and germline mutations in prolyl hydroxylase type 1 and type 2 (PHD1 and PHD2) have been identified to cause upregulation of the hypoxia signaling pathway and its target genes including erythropoietin (EPO) and its receptor (EPOR). However, in a minority of patients presenting with this syndrome, the genetics and molecular pathogenesis remain unexplained. The aim of the present study was to uncover novel genetic causes of PPGL-polycythemia syndrome. A female presented with a history of JAK2 V617F positive PV, diagnosed in 2007, and right adrenal pheochromocytoma diagnosed and resected in 2011. Her polycythemia symptoms and hematocrit levels continued to worsen from 2007 to 2011, with an increased frequency of phlebotomies. Postoperatively, until early 2013, her hematocrit levels remained normalized. Following this, the hematocrit levels ranged between 46.4 and 48.9% [35-45%]. Tumor tissue from the patient was further tested for mutations in genes related to upregulation of the hypoxia signaling pathway including iron regulatory protein 1 (IRP1), which is a known regulator of HIF-2α mRNA translation. Functional studies were performed to investigate the consequences of these mutations, especially their effect on the HIF signaling pathway and EPO. Indel mutations (c.267-1_267delGGinsTA) were discovered at the exon 3 splicing site of IRP1. Minigene construct and splicing site analysis showed that the mutation led to a new splicing site and a frameshift mutation of IRP1, which caused a truncated protein. Fluorescence in situ hybridization analysis demonstrated heterozygous IRP1 deletions in tumor cells. Immunohistochemistry results confirmed the truncated IRP1 and overexpressed HIF-2α, EPO and EPOR in tumor cells. This is the first report which provides direct molecular genetic evidence of association between a somatic IRP1 loss-of-function mutation and PHEO and secondary polycythemia. In patients diagnosed with PHEO/PGL and polycythemia with negative genetic testing for mutations in HIF2A, PHD1/2, and VHL, IRP1 should be considered as a candidate gene.

Physiological Jak2V617F expression causes a lethal myeloproliferative neoplasm with differential effects on hematopoietic stem and progenitor cells.

PubMed

Mullally, Ann; Lane, Steven W; Ball, Brian; Megerdichian, Christine; Okabe, Rachel; Al-Shahrour, Fatima; Paktinat, Mahnaz; Haydu, J Erika; Housman, Elizabeth; Lord, Allegra M; Wernig, Gerlinde; Kharas, Michael G; Mercher, Thomas; Kutok, Jeffery L; Gilliland, D Gary; Ebert, Benjamin L

2010-06-15

We report a Jak2V617F knockin mouse myeloproliferative neoplasm (MPN) model resembling human polycythemia vera (PV). The MPN is serially transplantable and we demonstrate that the hematopoietic stem cell (HSC) compartment has the unique capacity for disease initiation but does not have a significant selective competitive advantage over wild-type HSCs. In contrast, myeloid progenitor populations are expanded and skewed toward the erythroid lineage, but cannot transplant the disease. Treatment with a JAK2 kinase inhibitor ameliorated the MPN phenotype, but did not eliminate the disease-initiating population. These findings provide insights into the consequences of JAK2 activation on HSC differentiation and function and have the potential to inform therapeutic approaches to JAK2V617F-positive MPN. Copyright 2010 Elsevier Inc. All rights reserved.

Stem and Progenitor Cell Subsets Are Affected by JAK2 Signaling and Can Be Monitored by Flow Cytometry

PubMed Central

Iida, Ryuji; Welner, Robert S.; Zhao, Wanke; Alberola-lla, José; Medina, Kay L.; Zhao, Zhizhuang Joe; Kincade, Paul W.

2014-01-01

Although extremely rare, hematopoietic stem cells (HSCs) are divisible into subsets that differ with respect to differentiation potential and cell surface marker expression. For example, we recently found that CD86− CD150+ CD48− HSCs have limited potential for lymphocyte production. This could be an important new tool for studying hematological abnormalities. Here, we analyzed HSC subsets with a series of stem cell markers in JAK2V617F transgenic (Tg) mice, where the mutation is sufficient to cause myeloproliferative neoplasia with lymphocyte deficiency. Total numbers of HSC were elevated 3 to 20 fold in bone marrow of JAK2V617F mice. Careful analysis suggested the accumulation involved multiple HSC subsets, but particularly those characterized as CD150HI CD86− CD18L°CD41+ and excluding Hoechst dye. Real-Time PCR analysis of their HSC revealed that the erythropoiesis associated gene transcripts Gata1, Klf1 and Epor were particularly high. Flow cytometry analyses based on two differentiation schemes for multipotent progenitors (MPP) also suggested alteration by JAK2 signals. The low CD86 on HSC and multipotent progenitors paralleled the large reductions we found in lymphoid progenitors, but the few that were produced functioned normally when sorted and placed in culture. Either of two HSC subsets conferred disease when transplanted. Thus, flow cytometry can be used to observe the influence of abnormal JAK2 signaling on stem and progenitor subsets. Markers that similarly distinguish categories of human HSCs might be very valuable for monitoring such conditions. They could also serve as indicators of HSC fitness and suitability for transplantation. PMID:24699465

Roles of germline JAK2 activation mutation JAK2 V625F in the pathology of myeloproliferative neoplasms.

PubMed

Wu, Qing-Yun; Ma, Meng-Meng; Fu, Lin; Zhu, Yuan-Yuan; Liu, Yang; Cao, Jiang; Zhou, Ping; Li, Zhen-Yu; Zeng, Ling-Yu; Li, Feng; Wang, Xiao-Yun; Xu, Kai-Lin

2018-05-18

Janus tyrosine kinase 2 (JAK2) mediates downstream signaling of cytokine receptors in all hematological lineages, constitutively active somatic JAK2 mutations play key roles in the pathology of myeloproliferative neoplasms (MPNs). Recently, germline JAK2 mutations are also associated with triple-negative MPNs. A novel germline mutation JAK2 V625F is reported to be involved in a subset of MPNs patients. However, the pathogenesis of this mutation caused MPN is still unclear. In this study, the homology models of JAK2 V625F showed that the newly formed interaction between F625 and Y613 disrupted the JAK2 JH1-JH2 domain interactions was responsible for its activation, when F625 and Y613 interaction was disrupted, its activity significantly decreased. While, when this interaction was repaired whether by forming hydrogen bond or salt bond, it would cause JAK2 activation. Biochemical studies also demonstrated that JAK2 V625F mutation led to JAK2-STAT5 pathway activation and promoted the proliferation of BaF3 cells. Thus, our results herein provide clues to understand the mechanism JAK2 V625F mutation caused MPNs and give information for the development of JAK2 mutation specific inhibitors. Copyright © 2018 Elsevier B.V. All rights reserved.

CYT387, a novel JAK2 inhibitor, induces hematologic responses and normalizes inflammatory cytokines in murine myeloproliferative neoplasms

PubMed Central

Tyner, Jeffrey W.; Bumm, Thomas G.; Deininger, Jutta; Wood, Lisa; Aichberger, Karl J.; Loriaux, Marc M.; Druker, Brian J.; Burns, Christopher J.; Fantino, Emmanuelle

2010-01-01

Activating alleles of Janus kinase 2 (JAK2) such as JAK2V617F are central to the pathogenesis of myeloproliferative neoplasms (MPN), suggesting that small molecule inhibitors targeting JAK2 may be therapeutically useful. We have identified an aminopyrimidine derivative (CYT387), which inhibits JAK1, JAK2, and tyrosine kinase 2 (TYK2) at low nanomolar concentrations, with few additional targets. Between 0.5 and 1.5μM CYT387 caused growth suppression and apoptosis in JAK2-dependent hematopoietic cell lines, while nonhematopoietic cell lines were unaffected. In a murine MPN model, CYT387 normalized white cell counts, hematocrit, spleen size, and restored physiologic levels of inflammatory cytokines. Despite the hematologic responses and reduction of the JAK2V617F allele burden, JAK2V617F cells persisted and MPN recurred upon cessation of treatment, suggesting that JAK2 inhibitors may be unable to eliminate JAK2V617F cells, consistent with preliminary results from clinical trials of JAK2 inhibitors in myelofibrosis. While the clinical benefit of JAK2 inhibitors may be substantial, not the least due to reduction of inflammatory cytokines and symptomatic improvement, our data add to increasing evidence that kinase inhibitor monotherapy of malignant disease is not curative, suggesting a need for drug combinations to optimally target the malignant cells. PMID:20385788

Blast transformation and fibrotic progression in polycythemia vera and essential thrombocythemia: a literature review of incidence and risk factors

PubMed Central

Cerquozzi, S; Tefferi, A

2015-01-01

Polycythemia vera (PV) and essential thrombocythemia (ET) constitute two of the three BCR-ABL1-negative myeloproliferative neoplasms and are characterized by relatively long median survivals (approximately 14 and 20 years, respectively). Potentially fatal disease complications in PV and ET include disease transformation into myelofibrosis (MF) or acute myeloid leukemia (AML). The range of reported frequencies for post-PV MF were 4.9–6% at 10 years and 6–14% at 15 years and for post-ET MF were 0.8–4.9% at 10 years and 4–11% at 15 years. The corresponding figures for post-PV AML were 2.3–14.4% at 10 years and 5.5–18.7% at 15 years and for post-ET AML were 0.7–3% at 10 years and 2.1–5.3% at 15 years. Risk factors cited for post-PV MF include advanced age, leukocytosis, reticulin fibrosis, splenomegaly and JAK2V617F allele burden and for post-ET MF include advanced age, leukocytosis, anemia, reticulin fibrosis, absence of JAK2V617F, use of anagrelide and presence of ASXL1 mutation. Risk factors for post-PV AML include advanced age, leukocytosis, reticulin fibrosis, splenomegaly, abnormal karyotype, TP53 or RUNX1 mutations as well as use of pipobroman, radiophosphorus (P32) and busulfan and for post-ET AML include advanced age, leukocytosis, anemia, extreme thrombocytosis, thrombosis, reticulin fibrosis, TP53 or RUNX1 mutations. It is important to note that some of the aforementioned incidence figures and risk factor determinations are probably inaccurate and at times conflicting because of the retrospective nature of studies and the inadvertent labeling, in some studies, of patients with prefibrotic primary MF or ‘masked' PV, as ET. Ultimately, transformation of MPN leads to poor outcomes and management remains challenging. Further understanding of the molecular events leading to disease transformation is being investigated. PMID:26565403

Oncogenes in myeloproliferative disorders.

PubMed

Tefferi, Ayalew; Gilliland, D Gary

2007-03-01

Myeloproliferative disorders (MPDs) constitute a group of hematopoietic malignancies that feature enhanced proliferation and survival of one or more myeloid lineage cells. William Dameshek is credited for introducing the term "MPDs" in 1951 when he used it to group chronic myeloid leukemia (CML), polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF) under one clinicopathologic category. Since then, other myeloid neoplasms have been added to the MPD member list: chronic neutrophilic (CNL), eosinophilic (CEL) and myelomonocytic (CMML) leukemias; juvenile myelomonocytic leukemia (JMML); hypereosinophilic syndrome (HES); systemic mastocytosis (SM); and others. Collectively, MPDs are stem cell-derived clonal proliferative diseases whose shared and diverse phenotypic characteristics can be attributed to dysregulated signal transduction--a consequence of acquired somatic mutations. The most recognized among the latter is BCR-ABL, the disease-causing mutation in CML. Other mutations of putative pathogenetic relevance in MPDs include: JAK2V617F in PV, ET, and PMF; JAK2 exon 12 mutations in PV; MPLW515L/K in PMF and ET; KITD816V in SM; FIP1L1-PDGFRA in CEL-SM; rearrangements of PDGFRB in CEL-CMML and FGFR1 in stem cell leukemia-lymphoma syndrome; and RAS/PTPN11/NF1 mutations in JMML. This increasing repertoire of mutant molecules has streamlined translational research and molecularly targeted drug development in MPDs.

Splanchnic vein thrombosis as a first manifestation of Primary myelofibrosis

PubMed

Campos-Cabrera, Gregorio; Campos-Cabrera, Virginia; Campos-Cabrera, Salvador; Campos-Villagómez, José-Luis; Romero-González, Alejandra

2017-01-01

Myeloproliferative neoplasms are chronic disorders of clonal hematopoietic stem cells, characterized by an overproduction of functional granulocytes, red blood cells and / or platelets, and one of the major complications is the occurrence of venous and arterial thrombotic problems caused by increased platelet aggregation and thrombin generation. In this study 11 cases of primary myelofibrosis (PM) were evaluated and 2 debuted with splanchnic venous thrombosis (SVT); so after seeing the results of this study and of world literature, it is suggested that in patients with SVT, diagnostic methods for PM like the JAK2V617F mutation should be included. Copyright: © 2017 SecretarÍa de Salud

Novel mutations in the inhibitory adaptor protein LNK drive JAK-STAT signaling in patients with myeloproliferative neoplasms

PubMed Central

Oh, Stephen T.; Simonds, Erin F.; Jones, Carol; Hale, Matthew B.; Goltsev, Yury; Gibbs, Kenneth D.; Merker, Jason D.; Zehnder, James L.; Nolan, Garry P.

2010-01-01

Dysregulated Janus kinase–signal transducer and activator of transcription (JAK-STAT) signaling due to activation of tyrosine kinases is a common feature of myeloid malignancies. Here we report the first human disease-related mutations in the adaptor protein LNK, a negative regulator of JAK-STAT signaling, in 2 patients with JAK2 V617F–negative myeloproliferative neoplasms (MPNs). One patient exhibited a 5 base-pair deletion and missense mutation leading to a premature stop codon and loss of the pleckstrin homology (PH) and Src homology 2 (SH2) domains. A second patient had a missense mutation (E208Q) in the PH domain. BaF3-MPL cells transduced with these LNK mutants displayed augmented and sustained thrombopoietin-dependent growth and signaling. Primary samples from MPN patients bearing LNK mutations exhibited aberrant JAK-STAT activation, and cytokine-responsive CD34+ early progenitors were abnormally abundant in both patients. These findings indicate that JAK-STAT activation due to loss of LNK negative feedback regulation is a novel mechanism of MPN pathogenesis. PMID:20404132

Molecular diagnostics of myeloproliferative neoplasms.

PubMed

Langabeer, Stephen E; Andrikovics, Hajnalka; Asp, Julia; Bellosillo, Beatriz; Carillo, Serge; Haslam, Karl; Kjaer, Lasse; Lippert, Eric; Mansier, Olivier; Oppliger Leibundgut, Elisabeth; Percy, Melanie J; Porret, Naomi; Palmqvist, Lars; Schwarz, Jiri; McMullin, Mary F; Schnittger, Susanne; Pallisgaard, Niels; Hermouet, Sylvie

2015-10-01

Since the discovery of the JAK2 V617F mutation in the majority of the myeloproliferative neoplasms (MPN) of polycythemia vera, essential thrombocythemia and primary myelofibrosis ten years ago, further MPN-specific mutational events, notably in JAK2 exon 12, MPL exon 10 and CALR exon 9 have been identified. These discoveries have been rapidly incorporated into evolving molecular diagnostic algorithms. Whilst many of these mutations appear to have prognostic implications, establishing MPN diagnosis is of immediate clinical importance with selection, implementation and the continual evaluation of the appropriate laboratory methodology to achieve this diagnosis similarly vital. The advantages and limitations of these approaches in identifying and quantitating the common MPN-associated mutations are considered herein with particular regard to their clinical utility. The evolution of molecular diagnostic applications and platforms has occurred in parallel with the discovery of MPN-associated mutations, and it therefore appears likely that emerging technologies such as next-generation sequencing and digital PCR will in the future play an increasing role in the molecular diagnosis of MPN. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Recombinant interferon-α in myelofibrosis reduces bone marrow fibrosis, improves its morphology and is associated with clinical response.

PubMed

Pizzi, Marco; Silver, Richard T; Barel, Ariella; Orazi, Attilio

2015-10-01

Recombinant interferon-α represents a well-established therapeutic option for the treatment of polycythemia vera and essential thrombocythemia. Recent studies also suggest a role for recombinant interferon-α in the treatment of 'early stage' primary myelofibrosis, but few studies have reported the bone marrow changes after clinically successful interferon therapy. The aim of the present study is to detail the histological responses to recombinant interferon-α in primary myelofibrosis and post-polycythemia vera/post-essential thrombocythemia myelofibrosis and to correlate these with clinical findings. We retrospectively studied 12 patients with primary myelofibrosis or post-polycythemia vera/post-essential thrombocythemia myelofibrosis, who had been treated with recombinant interferon-α. Six patients had received other prior cytoreductive therapies. Bone marrow biopsy was assessed for the following histological parameters: (i) cellularity; (ii) myeloid-to-erythroid ratio; (iii) megakaryocyte tight clusters; (iv) megakaryocyte and naked nuclei density; (v) megakaryocytic atypia; (vi) fibrosis; and (vii) the percentage of blasts. Clinical and laboratory data were included: (i) constitutional symptoms; (ii) splenomegaly, if present; and (iii) complete cell blood count. The clinical response to therapy was evaluated using the International Working Group for Myelofibrosis Research and Treatment/European LeukemiaNet response criteria. The Dynamic International Prognostic Scoring System (DIPSS) score was calculated before and after recombinant interferon-α administration. Successful interferon therapy for myelofibrosis was associated with a significant reduction of marrow fibrosis, cellularity, megakaryocyte density and naked nuclei density. The presence of JAK2(V617F) mutation correlated with improved DIPSS score. JAK2(V617F)-negative cases showed worsening of such score or evolution to acute myeloid leukemia. Cytogenetic analysis documented a normal karyotype in all cases. In conclusion, successful clinical response to interferon-α correlates well with an improvement of bone marrow morphology. The prognostic effect of such therapy may be influenced by the JAK2 mutational status. Additional studies are needed to confirm these preliminary data.

Splanchnic vein thrombosis in myeloproliferative neoplasms: pathophysiology and molecular mechanisms of disease

PubMed Central

How, Joan; Zhou, Amy; Oh, Stephen T.

2016-01-01

Myeloproliferative neoplasms (MPNs) are the most common underlying prothrombotic disorder found in patients with splanchnic vein thrombosis (SVT). Clinical risk factors for MPN-associated SVTs include younger age, female sex, concomitant hypercoagulable disorders, and the JAK2 V617F mutation. These risk factors are distinct from those associated with arterial or deep venous thrombosis (DVT) in MPN patients, suggesting disparate disease mechanisms. The pathophysiology of SVT is thought to derive from local interactions between activated blood cells and the unique splanchnic endothelial environment. Other mutations commonly found in MPNs, including CALR and MPL, are rare in MPN-associated SVT. The purpose of this article is to review the clinical and molecular risk factors for MPN-associated SVT, with particular focus on the possible mechanisms of SVT formation in MPN patients. PMID:28246554

Mutations close to a hub residue affect the distant active site of a GH1 β-glucosidase.

PubMed

Souza, Valquiria P; Ikegami, Cecília M; Arantes, Guilherme M; Marana, Sandro R

2018-01-01

The tertiary structure of proteins has been represented as a network, in which residues are nodes and their contacts are edges. Protein structure networks contain residues, called hubs or central, which are essential to form short connection pathways between any pair of nodes. Hence hub residues may effectively spread structural perturbations through the protein. To test whether modifications nearby to hub residues could affect the enzyme active site, mutations were introduced in the β-glycosidase Sfβgly (PDB-ID: 5CG0) directed to residues that form an α-helix (260-265) and a β-strand (335-337) close to one of its main hub residues, F251, which is approximately 14 Å from the Sfβgly active site. Replacement of residues A263 and A264, which side-chains project from the α-helix towards F251, decreased the rate of substrate hydrolysis. Mutation A263F was shown to weaken noncovalent interactions involved in transition state stabilization within the Sfβgly active site. Mutations placed on the opposite side of the same α-helix did not show these effects. Consistently, replacement of V336, which side-chain protrudes from a β-strand face towards F251, inactivated Sfβgly. Next to V336, mutation S337F also caused a decrease in noncovalent interactions involved in transition state stabilization. Therefore, we suggest that mutations A263F, A264F, V336F and S337F may directly perturb the position of the hub F251, which could propagate these perturbations into the Sfβgly active site through short connection pathways along the protein network.

ASXL1/EZH2 mutations promote clonal expansion of neoplastic HSC and impair erythropoiesis in PMF.

PubMed

Triviai, Ioanna; Zeschke, Silke; Rentel, Jan; Spanakis, Marios; Scherer, Theo; Gabdoulline, Razif; Panagiota, Victoria; Thol, Felicitas; Heuser, Michael; Stocking, Carol; Kröger, Nicolaus

2018-06-15

Primary myelofibrosis (PMF) is a hematopoietic stem cell (HSC) disease, characterized by aberrant differentiation of all myeloid lineages and profound disruption of the bone marrow niche. PMF samples carry several mutations, but their cell origin and hierarchy in regulating the different waves of clonal and aberrant myeloproliferation from the prime HSC compartment is poorly understood. Genotyping of >2000 colonies from CD133+HSC and progenitors from PMF patients confirmed the complex genetic heterogeneity within the neoplastic population. Notably, mutations in chromatin regulators ASXL1 and/or EZH2 were identified as the first genetic lesions, preceding both JAK2-V617F and CALR mutations, and are thus drivers of clonal myelopoiesis in a PMF subset. HSC from PMF patients with double ASXL1/EZH2 mutations exhibited significantly higher engraftment in immunodeficient mice than those from patients without histone modifier mutations. EZH2 mutations correlate with aberrant erythropoiesis in PMF patients, exemplified by impaired maturation and cell cycle arrest of erythroid progenitors. These data underscore the importance of post-transcriptional modifiers of histones in neoplastic stem cells, whose clonal growth sustains aberrant myelopoiesis and expansion of pre-leukemic clones in PMF.

[Diagnostic molecular pathology of lymphatic and myeloid neoplasms].

PubMed

Klapper, W; Kreipe, H

2015-03-01

Molecular pathology has been an integral part of the diagnostics of tumors of the hematopoietic system substantially longer than for solid neoplasms. In contrast to solid tumors, the primary objective of molecular pathology in hematopoietic neoplasms is not the prediction of drug efficacy but the diagnosis itself by excluding reactive proliferation and by using molecular features for tumor classification. In the case of malignant lymphomas, the most commonly applied molecular tests are those for gene rearrangements for immunoglobulin heavy chains and T-cell receptors. However, this article puts the focus on new and diagnostically relevant assays in hematopathology. Among these are mutations of MYD88 codon 265 in lymphoplasmacytic lymphomas, B-raf V600E in hairy cell leukemia and Stat3 exon 21 in indolent T-cell lymphomas. In myeloproliferative neoplasms, MPL W515, calreticulin exon 9 and the BCR-ABL and JAK2 V617F junctions are the most frequently analyzed differentiation series. In myelodysplastic and myeloproliferative neoplasms, SRSF2, SETBP1 and CSF3R mutations provide important differential diagnostic information. Genes mutated in myelodysplastic syndromes (MDS) are particularly diverse but their analysis significantly improves the differential diagnostics between reactive conditions and MDS. The most frequent changes in MDS include mutations of TET2 and various genes encoding splicing factors.

Thrombosis in Philadelphia negative classical myeloproliferative neoplasms: a narrative review on epidemiology, risk assessment, and pathophysiologic mechanisms.

PubMed

Ball, Somedeb; Thein, Kyaw Zin; Maiti, Abhishek; Nugent, Kenneth

2018-05-01

Thrombosis is common in cancer patients and is associated with increased morbidity and mortality. Myeloproliferative neoplasms (MPN) are common malignancies in elderly individuals and are known for a high incidence of thrombotic complications. Different risk factors have been identified in studies, and risk models have been developed to identify patients with MPN at higher risk for thrombosis. Several pathophysiological mechanisms help explain the increased likelihood of thrombosis in these patients. Factors, such as leukocyte and platelet activation leading to the formation of leukocyte-platelet aggregates, activation of the coagulation cascade by microparticles, high levels of inflammatory cytokines, and endothelial dysfunction have a crucial role in thrombosis in MPN patients. Recent studies have demonstrated a significant association between the allele burden of specific genetic mutations (mainly JAK2V617F) associated with MPN and the incidence of thrombotic events, thus suggesting a possible role for these mutations in thrombogenesis.

Recent advances in diagnosis and treatment of chronic myeloproliferative neoplasms

PubMed Central

Guglielmelli, Paola

2010-01-01

The Philadelphia chromosome-negative chronic myeloproliferative neoplasms (MPNs) have recently been the focus of tremendous advances in basic knowledge of disease pathophysiology following the recognition of mutations in JAK2 and MPL. These discoveries also led to refinement of the criteria employed for diagnosis. The prognostic roles of the JAK2 V617F mutation and of leukocytosis as independent risk factors for thrombosis, which represents the leading cause of death in patients with polycythemia vera and essential thrombocythemia, are supported by retrospective studies. A new risk stratification approach to the patient with primary myelofibrosis allows clinicians to distinguish categories of patients with significantly different expected survival. Finally, new drugs are currently being tested for MPNs, and molecular discoveries could ultimately lead to the development of a specific targeted therapy. Overall, significant advances in diagnosis, prognostication, and treatment have taken place in the last couple of years in the field of MPNs. PMID:20948870

Frequent POLE1 p.S297F mutation in Chinese patients with ovarian endometrioid carcinoma.

PubMed

Zou, Yang; Liu, Fa-Ying; Liu, Huai; Wang, Feng; Li, Wei; Huang, Mei-Zhen; Huang, Yan; Yuan, Xiao-Qun; Xu, Xiao-Yun; Huang, Ou-Ping; He, Ming

2014-03-01

The catalytic subunit of DNA polymerase epsilon (POLE1) functions primarily in nuclear DNA replication and repair. Recently, POLE1 mutations were detected frequently in colorectal and endometrial carcinomas while with lower frequency in several other types of cancer, and the p.P286R and p.V411L mutations were the potential mutation hotspots in human cancers. Nevertheless, the mutation frequency of POLE1 in ovarian cancer still remains largely unknown. Here, we screened a total of 251 Chinese samples with distinct subtypes of ovarian carcinoma for the presence of POLE1 hotspot mutations by direct sequencing. A heterozygous somatic POLE1 mutation, p.S297F (c.890C>T), but not p.P286R and p.V411L hotspot mutations observed in other cancer types, was identified in 3 out of 37 (8.1%) patients with ovarian endometrioid carcinoma; this mutation was evolutionarily highly conserved from Homo sapiens to Schizosaccharomyces. Of note, the POLE1 mutation coexisted with mutation in the ovarian cancer-associated PPP2R1A (protein phosphatase 2, regulatory subunit A, α) gene in a 46-year-old patient, who was also diagnosed with ectopic endometriosis in the benign ovary. In addition, a 45-year-old POLE1-mutated ovarian endometrioid carcinoma patient was also diagnosed with uterine leiomyoma while the remaining 52-year-old POLE1-mutated patient showed no additional distinctive clinical manifestation. In contrast to high frequency of POLE1 mutations in ovarian endometrioid carcinoma, no POLE1 mutations were identified in patients with other subtypes of ovarian carcinoma. Our results showed for the first time that the POLE1 p.S297F mutation, but not p.P286R and p.V411L hotspot mutations observed in other cancer types, was frequent in Chinese ovarian endometrioid carcinoma, but absent in other subtypes of ovarian carcinoma. These results implicated that POLE1 p.S297F mutation might be actively involved in the pathogenesis of ovarian endometrioid carcinoma, but might not be actively involved in other subtypes of ovarian carcinoma. Copyright © 2014 Elsevier B.V. All rights reserved.

JAK inhibitors for the treatment of myeloproliferative neoplasms and other disorders

PubMed Central

Vainchenker, William; Leroy, Emilie; Gilles, Laure; Marty, Caroline; Plo, Isabelle; Constantinescu, Stefan N.

2018-01-01

JAK inhibitors have been developed following the discovery of the JAK2V617F in 2005 as the driver mutation of the majority of non- BCR-ABL1 myeloproliferative neoplasms (MPNs). Subsequently, the search for JAK2 inhibitors continued with the discovery that the other driver mutations ( CALR and MPL) also exhibited persistent JAK2 activation. Several type I ATP-competitive JAK inhibitors with different specificities were assessed in clinical trials and exhibited minimal hematologic toxicity. Interestingly, these JAK inhibitors display potent anti-inflammatory activity. Thus, JAK inhibitors targeting preferentially JAK1 and JAK3 have been developed to treat inflammation, autoimmune diseases, and graft-versus-host disease. Ten years after the beginning of clinical trials, only two drugs have been approved by the US Food and Drug Administration: one JAK2/JAK1 inhibitor (ruxolitinib) in intermediate-2 and high-risk myelofibrosis and hydroxyurea-resistant or -intolerant polycythemia vera and one JAK1/JAK3 inhibitor (tofacitinib) in methotrexate-resistant rheumatoid arthritis. The non-approved compounds exhibited many off-target effects leading to neurological and gastrointestinal toxicities, as seen in clinical trials for MPNs. Ruxolitinib is a well-tolerated drug with mostly anti-inflammatory properties. Despite a weak effect on the cause of the disease itself in MPNs, it improves the clinical state of patients and increases survival in myelofibrosis. This limited effect is related to the fact that ruxolitinib, like the other type I JAK2 inhibitors, inhibits equally mutated and wild-type JAK2 (JAK2WT) and also the JAK2 oncogenic activation. Thus, other approaches need to be developed and could be based on either (1) the development of new inhibitors specifically targeting JAK2V617F or (2) the combination of the actual JAK2 inhibitors with other therapies, in particular with molecules targeting pathways downstream of JAK2 activation or the stability of JAK2 molecule. In contrast, the strong anti-inflammatory effects of the JAK inhibitors appear as a very promising therapeutic approach for many inflammatory and auto-immune diseases. PMID:29399328

Identification and functional analysis of CBLB mutations in type 1 diabetes.

PubMed

Yokoi, Norihide; Fujiwara, Yuuka; Wang, He-Yao; Kitao, Mai; Hayashi, Chihiro; Someya, Tomohiro; Kanamori, Masao; Oiso, Yutaka; Tajima, Naoko; Yamada, Yuichiro; Seino, Yutaka; Ikegami, Hiroshi; Seino, Susumu

2008-03-28

Casitas B-lineage lymphoma b (Cblb) is a negative regulator of T-cell activation and dysfunction of Cblb in rats and mice results in autoimmunity. In particular, a nonsense mutation in Cblb has been identified in a rat model of autoimmune type 1 diabetes. To clarify the possible involvement of CBLB mutation in type 1 diabetes in humans, we performed mutation screening of CBLB and characterized functional properties of the mutations in Japanese subjects. Six missense mutations (A155V, F328L, N466D, K837R, T882A, and R968L) were identified in one diabetic subject each, excepting N466D. Of these mutations, F328L showed impaired suppression of T-cell activation and was a loss-of-function mutation. These data suggest that the F328L mutation is involved in the development of autoimmune diseases including type 1 diabetes, and also provide insight into the structure-function relationship of CBLB protein.

Myeloproliferative neoplasms and inflammation: whether to target the malignant clone or the inflammatory process or both.

PubMed

Koschmieder, S; Mughal, T I; Hasselbalch, H C; Barosi, G; Valent, P; Kiladjian, J-J; Jeryczynski, G; Gisslinger, H; Jutzi, J S; Pahl, H L; Hehlmann, R; Maria Vannucchi, A; Cervantes, F; Silver, R T; Barbui, T

2016-05-01

The Philadelphia-negative myeloproliferative neoplasms (MPNs) are clonal disorders involving hematopoietic stem and progenitor cells and are associated with myeloproliferation, splenomegaly and constitutional symptoms. Similar signs and symptoms can also be found in patients with chronic inflammatory diseases, and inflammatory processes have been found to play an important role in the pathogenesis and progression of MPNs. Signal transduction pathways involving JAK1, JAK2, STAT3 and STAT5 are causally involved in driving both the malignant cells and the inflammatory process. Moreover, anti-inflammatory and immune-modulating drugs have been used successfully in the treatment of MPNs. However, to date, many unresoved issues remain. These include the role of somatic mutations that are present in addition to JAK2V617F, CALR and MPL W515 mutations, the interdependency of malignant and nonmalignant cells and the means to eradicate MPN-initiating and -maintaining cells. It is imperative for successful therapeutic approaches to define whether the malignant clone or the inflammatory cells or both should be targeted. The present review will cover three aspects of the role of inflammation in MPNs: inflammatory states as important differential diagnoses in cases of suspected MPN (that is, in the absence of a clonal marker), the role of inflammation in MPN pathogenesis and progression and the use of anti-inflammatory drugs for MPNs. The findings emphasize the need to separate the inflammatory processes from the malignancy in order to improve our understanding of the pathogenesis, diagnosis and treatment of patients with Philadelphia-negative MPNs.

Causes of Adult Splanchnic Vein Thrombosis in the Mediterranean Area

PubMed Central

De Stefano, Valerio; Za, Tommaso; Ciminello, Angela; Betti, Silvia; Rossi, Elena

2011-01-01

The term splanchnic vein thrombosis encompasses Budd-Chiari syndrome (BCS), extrahepatic portal vein obstruction (EHPVO), and mesenteric vein thrombosis. Risk factors can be local or systemic. A local precipitating factor is rare in BCS, while it is common in patients with portal vein thrombosis. Chronic myeloproliferative neoplasms (MPN) are the leading systemic cause of splanchnic vein thrombosis, and are diagnosed in half BCS patients and one-third of EHPVO patients; the somatic mutation JAK2 V617F is detectable in a large majority of patients with overt MPN, and up to 40% of patients without overt MPN. Inherited thrombophilia is present in at least one-third of patients, and the factor V Leiden or the prothrombin G20210A mutations are the most common mutations found in BCS or EHPVO patients, respectively. Multiple factors are present in approximately one-third of patients with BCS and two- thirds of patients with portal vein thrombosis. In a few patient series from the Southern Mediterranean area the high prevalence of MPN and thrombophilia as underlying cause of BCS is confirmed, although the data should be considered preliminary. Peculiar risk factors present in the area are Behçet’s disease and hydatidosis; moreover, membraneous webs, typically found in Asian patients, are present in a significant portion of cases. PMID:22220260

Quantification of the Mutant CALR Allelic Burden by Digital PCR: Application to Minimal Residual Disease Evaluation after Bone Marrow Transplantation.

PubMed

Mansier, Olivier; Migeon, Marina; Saint-Lézer, Arnaud; James, Chloé; Verger, Emmanuelle; Robin, Marie; Socié, Gérard; Bidet, Audrey; Mahon, François-Xavier; Cassinat, Bruno; Lippert, Eric

2016-01-01

With the recent discovery of CALR mutations, >80% of patients with myeloproliferative neoplasms carry a phenotype-driving mutation. For JAK2 V617F, the most frequent mutation in myeloproliferative neoplasms, accurate determination of mutational loads is of interest at diagnosis, for phenotypic and prognostic purposes, and during follow-up for minimal residual disease assessment. We developed a digital PCR technique that allowed the accurate determination of CALR allelic burdens for the main mutations (types 1 and 2). Compared with the commonly used fluorescent PCR product analysis, digital PCR is more precise, reproducible, and accurate. Furthermore, this method reached a very high sensitivity. We detected at least 0.025% CALR mutants. It can thus be used for patient characterization at diagnosis and for minimal residual disease monitoring. When applied to patients with primary myelofibrosis who underwent hematopoietic stem cell transplant, the digital PCR detected low levels of minimal residual disease. After negativation of the mutational load in all patients, the disease reappeared at a low level in one patient, preceding hematologic relapse. In conclusion, digital PCR adapted to type 1 and 2 CALR mutations is an inexpensive, highly precise, and sensitive technique suitable for evaluation of myeloproliferative neoplasm patients during follow-up. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

CMML AND aCML: NOVEL PATHOGENETIC LESIONS

PubMed Central

Muramatsu, Hideki; Makishima, Hideki; Maciejewski, Jaroslaw P.

2012-01-01

Summary Chronic myelomonocytic leukemia (CMML) and atypical chronic myeloid leukemia (aCML) are distinct, yet related, entities of myelodysplastic/myeloproliferative neoplasms (MDS/MPN) characterized by morphologic dysplasia with accumulation of monocytes or neutrophils, respectively. Our understanding of the molecular pathogenesis of CMML and aCML has advanced, mainly due to the application of novel technologies such as array-based karyotyping or next generation sequencing. In addition to previously known recurrent aberrations, somatic uniparental disomy affecting chromosomes 3, 4, 7, and 11 frequently occurs in CMML. Novel somatic mutations of genes, including those associated with proliferation signaling (CBL, RAS, RUNX1, JAK2 (V617F)) and with modification of epigenetic status (TET2, ASXL1, UTX, EZH2) have been found. Various combinations of mutations suggest a multistep pathogenesis and may account for clinical heterogeneity. The prognostic and diagnostic significance of these molecular lesions, in particular their value as biomarkers of response or resistance to specific therapies, while uncertain now is likely to be clarified as large systematic studies come to completion. PMID:22289493

Secondary Hypertension, Erythrocytosis, and Unilateral Renal Cystic Disease in a Submariner: A Case Report.

PubMed

Forbes, Angela S; Yeo, Fred E

Erythrocytosis, or increased red blood cell mass, may be primary as in the case of polycythemia vera (PV), or secondary due to a variety of causes related to erythropoietin (EPO) secretion and hypoxia. Chronic pulmonary disease and certain EPO-secreting tumors should be addressed and excluded early during the course of evaluation for a patient presenting with increased red blood cell mass. Inclusion of the JAK2 V617F gene mutation in the recent World Health Organization criteria for the diagnosis of PV allows for facilitated diagnosis and guides therapy. EPO levels can be helpful in diagnosis and guiding therapy, but in the case of cystic renal diseases, EPO levels are often not elevated, creating diagnostic uncertainty. This report describes a case of symptoms directly attributable to erythrocytosis in the setting of negative JAK2 mutation and normal EPO levels. The subsequent discovery of a large cystic renal kidney and PV were the leading diagnostic considerations. 2016.

Historical views, conventional approaches, and evolving management strategies for myeloproliferative neoplasms.

PubMed

Stein, Brady L; Gotlib, Jason; Arcasoy, Murat; Nguyen, Marie Huong; Shah, Neil; Moliterno, Alison; Jamieson, Catriona; Pollyea, Daniel A; Scott, Bart; Wadleigh, Martha; Levine, Ross; Komrokji, Rami; Klisovic, Rebecca; Gundabolu, Krishna; Kropf, Patricia; Wetzler, Meir; Oh, Stephen T; Ribeiro, Raul; Paschal, Rita; Mohan, Sanjay; Podoltsev, Nikolai; Prchal, Josef; Talpaz, Moshe; Snyder, David; Verstovsek, Srdan; Mesa, Ruben A

2015-04-01

The classical Philadelphia chromosome-negative myeloproliferative neoplasms (MPN), which include essential thrombocythemia, polycythemia vera, and myelofibrosis (MF), are in a new era of molecular diagnosis, ushered in by the identification of the JAK2(V617F) and cMPL mutations in 2005 and 2006, respectively, and the CALR mutations in 2013. Coupled with increased knowledge of disease pathogenesis and refined diagnostic criteria and prognostic scoring systems, a more nuanced appreciation has emerged of the burden of MPN in the United States, including the prevalence, symptom burden, and impact on quality of life. Biological advances in MPN have translated into the rapid development of novel therapeutics, culminating in the approval of the first treatment for MF, the JAK1/JAK2 inhibitor ruxolitinib. However, certain practical aspects of care, such as those regarding diagnosis, prevention of vascular events, choice of cytoreductive agent, and planning for therapies, present challenges for hematologists/oncologists, and are discussed in this article. Copyright © 2015 by the National Comprehensive Cancer Network.

Single molecule molecular inversion probes for targeted, high-accuracy detection of low-frequency variation.

PubMed

Hiatt, Joseph B; Pritchard, Colin C; Salipante, Stephen J; O'Roak, Brian J; Shendure, Jay

2013-05-01

The detection and quantification of genetic heterogeneity in populations of cells is fundamentally important to diverse fields, ranging from microbial evolution to human cancer genetics. However, despite the cost and throughput advances associated with massively parallel sequencing, it remains challenging to reliably detect mutations that are present at a low relative abundance in a given DNA sample. Here we describe smMIP, an assay that combines single molecule tagging with multiplex targeted capture to enable practical and highly sensitive detection of low-frequency or subclonal variation. To demonstrate the potential of the method, we simultaneously resequenced 33 clinically informative cancer genes in eight cell line and 45 clinical cancer samples. Single molecule tagging facilitated extremely accurate consensus calling, with an estimated per-base error rate of 8.4 × 10(-6) in cell lines and 2.6 × 10(-5) in clinical specimens. False-positive mutations in the single molecule consensus base-calls exhibited patterns predominantly consistent with DNA damage, including 8-oxo-guanine and spontaneous deamination of cytosine. Based on mixing experiments with cell line samples, sensitivity for mutations above 1% frequency was 83% with no false positives. At clinically informative sites, we identified seven low-frequency point mutations (0.2%-4.7%), including BRAF p.V600E (melanoma, 0.2% alternate allele frequency), KRAS p.G12V (lung, 0.6%), JAK2 p.V617F (melanoma, colon, two lung, 0.3%-1.4%), and NRAS p.Q61R (colon, 4.7%). We anticipate that smMIP will be broadly adoptable as a practical and effective method for accurately detecting low-frequency mutations in both research and clinical settings.

Single molecule molecular inversion probes for targeted, high-accuracy detection of low-frequency variation

PubMed Central

Hiatt, Joseph B.; Pritchard, Colin C.; Salipante, Stephen J.; O'Roak, Brian J.; Shendure, Jay

2013-01-01

The detection and quantification of genetic heterogeneity in populations of cells is fundamentally important to diverse fields, ranging from microbial evolution to human cancer genetics. However, despite the cost and throughput advances associated with massively parallel sequencing, it remains challenging to reliably detect mutations that are present at a low relative abundance in a given DNA sample. Here we describe smMIP, an assay that combines single molecule tagging with multiplex targeted capture to enable practical and highly sensitive detection of low-frequency or subclonal variation. To demonstrate the potential of the method, we simultaneously resequenced 33 clinically informative cancer genes in eight cell line and 45 clinical cancer samples. Single molecule tagging facilitated extremely accurate consensus calling, with an estimated per-base error rate of 8.4 × 10−6 in cell lines and 2.6 × 10−5 in clinical specimens. False-positive mutations in the single molecule consensus base-calls exhibited patterns predominantly consistent with DNA damage, including 8-oxo-guanine and spontaneous deamination of cytosine. Based on mixing experiments with cell line samples, sensitivity for mutations above 1% frequency was 83% with no false positives. At clinically informative sites, we identified seven low-frequency point mutations (0.2%–4.7%), including BRAF p.V600E (melanoma, 0.2% alternate allele frequency), KRAS p.G12V (lung, 0.6%), JAK2 p.V617F (melanoma, colon, two lung, 0.3%–1.4%), and NRAS p.Q61R (colon, 4.7%). We anticipate that smMIP will be broadly adoptable as a practical and effective method for accurately detecting low-frequency mutations in both research and clinical settings. PMID:23382536

Differential effects of hydroxyurea and INC424 on mutant allele burden and myeloproliferative phenotype in a JAK2-V617F polycythemia vera mouse model.

PubMed

Kubovcakova, Lucia; Lundberg, Pontus; Grisouard, Jean; Hao-Shen, Hui; Romanet, Vincent; Andraos, Rita; Murakami, Masato; Dirnhofer, Stephan; Wagner, Kay-Uwe; Radimerski, Thomas; Skoda, Radek C

2013-02-14

To establish a preclinical animal model for testing drugs with potential effects on myeloproliferative neoplasms (MPNs), we first performed a detailed phenotypic characterization of Cre-inducible transgenic JAK2-V617F mice. Deleting the conditional mouse Jak2-knockout alleles increased erythropoiesis and accentuated the polycythemia vera phenotype, but did not alter platelet or granulocyte levels. In a transplantation assay, JAK2-V617F(+) BM cells had an advantage over wild-type competitor cells. Using this competitive repopulation assay, we compared the effects of INC424 (ruxolitinib), a dual Jak1/Jak2 inhibitor, and hydroxyurea (HU). HU led to weight loss, but did not reduce spleen weight. The hematologic parameters were lowered and a slight decrease of the mutant allele burden was noted. INC424 had little effect on body weight, but strongly decreased spleen size and rapidly normalized RBC and neutrophil parameters. No significant decrease in the mutant allele burden was observed. INC424 reduced the phospho-Stat5 levels, whereas HU strongly increased phospho-Stat5, most likely because of the elevated erythropoietin levels in response to the HU-induced anemia. This compensatory increase in JAK/STAT signaling may counteract the beneficial effects of cytoreduction at higher doses of HU and represents an adverse effect that should be avoided.

The novel complex allele [A238V;F508del] of the CFTR gene: clinical phenotype and possible implications for cystic fibrosis etiological therapies.

PubMed

Diana, Anna; Polizzi, Angela Maria; Santostasi, Teresa; Ratclif, Luigi; Pantaleo, Maria Giuseppina; Leonetti, Giuseppina; Iusco, Danila Rosa; Gallo, Crescenzio; Conese, Massimo; Manca, Antonio

2016-06-01

Few mutations in cis have been annotated for F508del homozygous patients. Southern Italy patients who at a first analysis appeared homozygous for the F508del mutation (n=63) or compound heterozygous for the F508del and another mutation in the cystic fibrosis transmembrane conductance regulator gene (n=155) were searched for the A238V mutation in exon 6. The allelic frequency of the complex allele [A238V;F508del] was 0.04. When the whole data set was used (comprised also of 56 F508del/F508del and 34 F508del/other mutation controls), no differences reached the statistical significance in the clinical parameters, except chloride concentrations which were lower in [A238V;F508del]/other mutation compared with F508del/other mutation (P=0.03). The two study groups presented less complications than the control groups. Within the minimal data set (34 F508del/F508del, 27 F508del/other mutation, 4 [A238V;F508del]/F508del cases and 5 [A238V;F508del]/other mutation cases); that is, presenting all the variables in each patient, forced expiratory volume in 1 s and forced vital capacity presented a trend to lower levels in the study groups in comparison with the F508del/F508del group, and C-reactive protein approximated statistically significant higher levels in the [A238V;F508del]/other mutation as compared with F508del/F508del patients (P=0.09). The analysis of statistical dependence among the variables showed a significant anticorrelation between chloride and body mass index in the [A238V;F508del]/other mutation group. In conclusion, the complex allele [A238V;F508del] seems to be associated with less general complications than in the control groups, on the other hand possibly giving a worse pulmonary phenotype and higher systemic/local inflammatory response. These findings have implications for the correct recruitment and clinical response of F508del patients in the clinical trials testing the new etiological drugs for cystic fibrosis.

Sodium channel dysfunction in intractable childhood epilepsy with generalized tonic–clonic seizures

PubMed Central

Rhodes, Thomas H; Vanoye, Carlos G; Ohmori, Iori; Ogiwara, Ikuo; Yamakawa, Kazuhiro; George, Alfred L

2005-01-01

Mutations in SCN1A, the gene encoding the brain voltage-gated sodium channel α1 subunit (NaV1.1), are associated with genetic forms of epilepsy, including generalized epilepsy with febrile seizures plus (GEFS+ type 2), severe myoclonic epilepsy of infancy (SMEI) and related conditions. Several missense SCN1A mutations have been identified in probands affected by the syndrome of intractable childhood epilepsy with generalized tonic–clonic seizures (ICEGTC), which bears similarity to SMEI. To test whether ICEGTC arises from molecular mechanisms similar to those involved in SMEI, we characterized eight ICEGTC missense mutations by whole-cell patch clamp recording of recombinant human SCN1A heterologously expressed in cultured mammalian cells. Two mutations (G979R and T1709I) were non-functional. The remaining alleles (T808S, V983A, N1011I, V1611F, P1632S and F1808L) exhibited measurable sodium current, but had heterogeneous biophysical phenotypes. Mutant channels exhibited lower (V983A, N1011I and F1808L), greater (T808S) or similar (V1611F and P1632S) peak sodium current densities compared with wild-type (WT) SCN1A. Three mutations (V1611F, P1632S and F1808L) displayed hyperpolarized conductance–voltage relationships, while V983A exhibited a strong depolarizing shift in the voltage dependence of activation. All mutants except T808S had hyperpolarized shifts in the voltage dependence of steady-state channel availability. Three mutants (V1611F, P1632S and F1808L) exhibited persistent sodium current ranging from ∼1–3% of peak current amplitude that was significantly greater than WT-SCN1A. Several mutants had impaired slow inactivation, with V983A showing the most prominent effect. Finally, all of the functional alleles exhibited reduced use-dependent channel inhibition. In summary, SCN1A mutations associated with ICEGTC result in a wide spectrum of biophysical defects, including mild-to-moderate gating impairments, shifted voltage dependence and reduced use dependence. The constellation of biophysical abnormalities for some mutants is distinct from those previously observed for GEFS+ and SMEI, suggesting possible, but complex, genotype–phenotype correlations. PMID:16210358

Replication restart in UV-irradiated Escherichia coli involving pols II, III, V, PriA, RecA and RecFOR proteins.

PubMed

Rangarajan, Savithri; Woodgate, Roger; Goodman, Myron F

2002-02-01

In Escherichia coli, UV-irradiated cells resume DNA synthesis after a transient inhibition by a process called replication restart. To elucidate the role of several key proteins involved in this process, we have analysed the time dependence of replication restart in strains carrying a combination of mutations in lexA, recA, polB (pol II), umuDC (pol V), priA, dnaC, recF, recO or recR. We find that both pol II and the origin-independent primosome-assembling function of PriA are essential for the immediate recovery of DNA synthesis after UV irradiation. In their absence, translesion replication or 'replication readthrough' occurs approximately 50 min after UV and is pol V-dependent. In a wild-type, lexA+ background, mutations in recF, recO or recR block both pathways. Similar results were obtained with a lexA(Def) recF strain. However, lexA(Def) recO or lexA(Def) recR strains, although unable to facilitate PriA-pol II-dependent restart, were able to perform pol V-dependent readthrough. The defects in restart attributed to mutations in recF, recO or recR were suppressed in a recA730 lexA(Def) strain expressing constitutively activated RecA (RecA*). Our data suggest that in a wild-type background, RecF, O and R are important for the induction of the SOS response and the formation of RecA*-dependent recombination intermediates necessary for PriA/Pol II-dependent replication restart. In con-trast, only RecF is required for the activation of RecA that leads to the formation of pol V (UmuD'2C) and facilitates replication readthrough.

F1174V mutation alters the ALK active conformation in response to Crizotinib in NSCLC: Insight from molecular simulations.

PubMed

Dehghanian, Fariba; Kay, Maryam; Vallian, Sadeq

2017-08-01

Crizotinib is an efficient antineoplastic drug for treatment of non-small cell lung carcinoma (NSCLC), which is identified as an anaplastic lymphoma kinase (ALK) inhibitor. F1174V is a recently identified acquired point mutation relating to the Crizotinib resistance in NSCLC patients. The mechanism of Crizotinib resistance relating to F1174V mutation as a non-active site mutation remains unclear. In this study, the molecular dynamic simulation was used to investigate the possible mechanisms by which F1174V mutation may affect the structure and activity of ALK kinase domain. The results suggested that F1174V mutation could cause two important secondary structure alterations, which led to the local conformational change in ALK kinase domain. This causes more positive free energy in the mutant complex in comparison with the wild-type one. In addition, our structural analyses illustrated that F1174V mutation could result in some important interactions, which represent the key characteristics of the ALK active conformation. This study provided a molecular mechanism for ALK Crizotinib resistance caused by F1174V mutation,which could facilitate designing more efficient drugs. Copyright © 2017 Elsevier Inc. All rights reserved.

[Diagnostic algorithm in chronic myeloproliferative diseases (CMPD)].

PubMed

Haferlach, Torsten; Bacher, Ulrike; Kern, Wolfgang; Schnittger, Susanne; Haferlach, Claudia

2007-09-15

The Philadelphia-negative chronic myeloproliferative diseases (CMPD) are very complex and heterogeneous disorders. They are represented by polycythemia vera (PV), chronic idiopathic myelofibrosis (CIMF), essential thrombocythemia (ET), CMPD/unclassifiable (CMPD-U), chronic neutrophilic leukemia (CNL), and chronic eosinophilic leukemia/hypereosinophilic syndrome (CEL/HES) according to the WHO classification. Before, diagnostics were mainly focused on clinical and morphological aspects, but in recent years cytogenetics and fluorescence in situ hybridization (FISH) found entrance in routine schedules as chromosomal abnormalities are relevant for prognosis and classification. Recently, there is rapid progress in the field of molecular characterization: the JAK2V617F mutation which shows a high incidence in PV, CIMF, and ET already plays a central role and will probably soon be included in follow-up procedures. Due to the detection of mutations in exon 12 of the JAK2 gene or mutations in the MPL gene the variety of activating mutations in the CMPD is still increasing. In CEL/HES the detection of the FIP1L1-PDGFRA fusion gene and overexpression of PDGFRA and PDGFRB led to targeted therapy with tyrosine kinase inhibitors. Thus, diagnostics in the CMPD transform toward a multimodal diagnostic concept based on a combination of methods - cyto-/histomorphology, cytogenetics, and individual molecular methods which can be included in a diagnostic algorithm.

Detection of MPL mutations by a novel allele-specific PCR-based strategy.

PubMed

Furtado, Larissa V; Weigelin, Helmut C; Elenitoba-Johnson, Kojo S J; Betz, Bryan L

2013-11-01

MPL mutation testing is recommended in patients with suspected primary myelofibrosis or essential thrombocythemia who lack the JAK2 V617F mutation. MPL mutations can occur at allelic levels below 15%, which may escape detection by commonly used mutation screening methods such as Sanger sequencing. We developed a novel multiplexed allele-specific PCR assay capable of detecting most recurrent MPL exon 10 mutations associated with primary myelofibrosis and essential thrombocythemia (W515L, W515K, W515A, and S505N) down to a sensitivity of 2.5% mutant allele. Test results were reviewed from 15 reference cases and 1380 consecutive specimens referred to our laboratory for testing. Assay performance was compared to Sanger sequencing across a series of 58 specimens with MPL mutations. Positive cases consisted of 45 with W515L, 6 with S505N, 5 with W515K, 1 with W515A, and 1 with both W515L and S505N. Seven cases had mutations below 5% that were undetected by Sanger sequencing. Ten additional cases had mutation levels between 5% and 15% that were not consistently detected by sequencing. All results were easily interpreted in the allele-specific test. This assay offers a sensitive and reliable solution for MPL mutation testing. Sanger sequencing appears insufficiently sensitive for robust MPL mutation detection. Our data also suggest the relative frequency of S505N mutations may be underestimated, highlighting the necessity for inclusion of this mutation in MPL test platforms. Copyright © 2013 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Investigating the effects of tropomyosin mutations on its flexibility and interactions with filamentous actin using molecular dynamics simulation.

PubMed

Zheng, Wenjun; Hitchcock-DeGregori, Sarah E; Barua, Bipasha

2016-10-01

Tropomyosin (Tpm) is a two-chained α-helical coiled-coil protein that binds to filamentous actin (F-actin), and regulates its interactions with myosin by occupying three average positions on F-actin (blocked, closed, and open). Mutations in the Tpm are linked to heart diseases including hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM). To elucidate the molecular mechanisms of Tpm mutations (including DCM mutation E54K, HCM mutations E62Q, A63V, K70T, V95A, D175N, E180G, L185R, E192K, and a designed synthetic mutation D137L) in terms of their effects on Tpm flexibility and its interactions with F-actin, we conducted extensive molecular dynamics simulations for the wild-type and mutant Tpm in complex with F-actin (total simulation time 160 ns per mutant). The mutants exhibited distinct changes (i.e., increase or decrease) in the overall and local flexibility of the Tpm coiled-coil, with each mutation causing both local and long-range modifications of the Tpm flexibility. In addition, our binding calculations revealed weakened Tpm-F-actin interactions (except for L185R, D137L and A63V) involving five periods of Tpm, which correlate with elevated fluctuation of Tpm relative to the blocked position on F-actin that may lead to easier activation and increased Ca 2+ -sensitivity. We also simulated the αβ/βα-Tpm heterodimer in comparison with the αα-Tpm homodimer, which revealed greater flexibility and weaker actin binding in the heterodimer. Our findings are consistent with a complex mechanism underlying how different Tpm mutations perturb the Tpm function in distinct ways (e.g., by affecting specific sites of Tpm), which bear no simple links to the disease phenotypes (e.g., HCM vs. DCM).

Is it time to revisit contraindications to organ donation from donors with a JAK-2 mutation? Safe use of a liver allograft from a donor with essential thrombocythaemia.

PubMed

Haldar, Debashis; Chen, Frederick; Byron, Jane; Elsharkawy, Ahmed Mohamed; Perera, M Thamara Pr

2015-07-01

Transplantation can cure end-stage liver disease and hepatocellular carcinoma. However, the balance of organ demand and provision is heavily tipped to the detriment of patients. Patients awaiting transplantation rely on the greater use of marginal donors that may carry a risk to the recipient. UK authorities have decreed donor haematological malignancy an absolute contraindication. The authors describe the first report of a patient being safely transplanted with a liver from a donor who suffered from JAK2 V617F mutation-driven essential thrombocythaemia to a patient with a critical burden of hepatocellular carcinoma. A year after transplantation, the patient has neither evidence of acquisition of the donor's pathology, nor evidence of carcinoma recurrence. The case highlights the responsibility of the recipient team to maximize the use of organs by expert risk assessment. Dissemination of experience should inform future decisions, benefit patients and bolster utility in an era of growing waiting-list mortality. © 2015 Steunstichting ESOT.

A Point Mutation V419L in the Sodium Channel Gene from Natural Populations of Aedes aegypti Is Involved in Resistance to λ-Cyhalothrin in Colombia

PubMed Central

Granada, Yurany; Mejía-Jaramillo, Ana María; Strode, Clare

2018-01-01

Resistance to pyrethroids in mosquitoes is mainly caused by target site insensitivity known as knockdown resistance (kdr). In this work, we examined the point mutations present in portions of domains I, II, III, and IV of the sodium channel gene in Aedes aegypti mosquitoes from three Colombian municipalities. A partial region coding for the sodium channel gene from resistant mosquitoes was sequenced, and a simple allele-specific PCR-based assay (AS-PCR) was used to analyze mutations at the population level. The previously reported mutations, V1016I and F1534C, were found with frequencies ranging from 0.04 to 0.41, and 0.56 to 0.71, respectively, in the three cities. Moreover, a novel mutation, at 419 codon (V419L), was found in Ae. aegypti populations from Bello, Riohacha and Villavicencio cities with allelic frequencies of 0.06, 0.36, and 0.46, respectively. Interestingly, the insecticide susceptibility assays showed that mosquitoes from Bello were susceptible to λ-cyhalothrin pyrethroid whilst those from Riohacha and Villavicencio were resistant. A positive association between V419L and V1016I mutations with λ-cyhalothrin resistance was established in Riohacha and Villavicencio. The frequency of the F1534C was high in the three populations, suggesting that this mutation could be conferring resistance to insecticides other than λ-cyhalothrin, particularly type I pyrethroids. Further studies are required to confirm this hypothesis. PMID:29443870

A Point Mutation V419L in the Sodium Channel Gene from Natural Populations of Aedes aegypti Is Involved in Resistance to λ-Cyhalothrin in Colombia.

PubMed

Granada, Yurany; Mejía-Jaramillo, Ana María; Strode, Clare; Triana-Chavez, Omar

2018-02-14

Resistance to pyrethroids in mosquitoes is mainly caused by target site insensitivity known as knockdown resistance ( kdr ). In this work, we examined the point mutations present in portions of domains I, II, III, and IV of the sodium channel gene in Aedes aegypti mosquitoes from three Colombian municipalities. A partial region coding for the sodium channel gene from resistant mosquitoes was sequenced, and a simple allele-specific PCR-based assay (AS-PCR) was used to analyze mutations at the population level. The previously reported mutations, V1016I and F1534C, were found with frequencies ranging from 0.04 to 0.41, and 0.56 to 0.71, respectively, in the three cities. Moreover, a novel mutation, at 419 codon (V419L), was found in Ae. aegypti populations from Bello, Riohacha and Villavicencio cities with allelic frequencies of 0.06, 0.36, and 0.46, respectively. Interestingly, the insecticide susceptibility assays showed that mosquitoes from Bello were susceptible to λ-cyhalothrin pyrethroid whilst those from Riohacha and Villavicencio were resistant. A positive association between V419L and V1016I mutations with λ-cyhalothrin resistance was established in Riohacha and Villavicencio. The frequency of the F1534C was high in the three populations, suggesting that this mutation could be conferring resistance to insecticides other than λ-cyhalothrin, particularly type I pyrethroids. Further studies are required to confirm this hypothesis.

The Small Molecule Inhibitor G6 Significantly Reduces Bone Marrow Fibrosis and the Mutant Burden in a Mouse Model of Jak2-Mediated Myelofibrosis

PubMed Central

Kirabo, Annet; Park, Sung O.; Wamsley, Heather L.; Gali, Meghanath; Baskin, Rebekah; Reinhard, Mary K.; Zhao, Zhizhuang J.; Bisht, Kirpal S.; Keserű, György M.; Cogle, Christopher R.; Sayeski, Peter P.

2013-01-01

Philadelphia chromosome–negative myeloproliferative neoplasms, including polycythemia vera, essential thrombocytosis, and myelofibrosis, are disorders characterized by abnormal hematopoiesis. Among these myeloproliferative neoplasms, myelofibrosis has the most unfavorable prognosis. Furthermore, currently available therapies for myelofibrosis have little to no efficacy in the bone marrow and hence, are palliative. We recently developed a Janus kinase 2 (Jak2) small molecule inhibitor called G6 and found that it exhibits marked efficacy in a xenograft model of Jak2-V617F–mediated hyperplasia and a transgenic mouse model of Jak2-V617F–mediated polycythemia vera/essential thrombocytosis. However, its efficacy in Jak2-mediated myelofibrosis has not previously been examined. Here, we hypothesized that G6 would be efficacious in Jak2-V617F–mediated myelofibrosis. To test this, mice expressing the human Jak2-V617F cDNA under the control of the vav promoter were administered G6 or vehicle control solution, and efficacy was determined by measuring parameters within the peripheral blood, liver, spleen, and bone marrow. We found that G6 significantly reduced extramedullary hematopoiesis in the liver and splenomegaly. In the bone marrow, G6 significantly reduced pathogenic Jak/STAT signaling by 53%, megakaryocytic hyperplasia by 70%, and the Jak2 mutant burden by 68%. Furthermore, G6 significantly improved the myeloid to erythroid ratio and significantly reversed the myelofibrosis. Collectively, these results indicate that G6 is efficacious in Jak2-V617F–mediated myelofibrosis, and given its bone marrow efficacy, it may alter the natural history of this disease. PMID:22796437

Comparison Between 64Cu-PSMA-617 PET/CT and 18F-Choline PET/CT Imaging in Early Diagnosis of Prostate Cancer Biochemical Recurrence.

PubMed

Cantiello, Francesco; Crocerossa, Fabio; Russo, Giorgio Ivan; Gangemi, Vincenzo; Ferro, Matteo; Vartolomei, Mihai Dorin; Lucarelli, Giuseppe; Mirabelli, Maria; Scafuro, Chiara; Ucciero, Giuseppe; De Cobelli, Ottavio; Morgia, Giuseppe; Damiano, Rocco; Cascini, Giuseppe Lucio

2018-06-04

To evaluate the diagnostic performance of 64 Cu-PSMA-617 positron emission tomography (PET) with computed tomography (CT) for restaging prostate cancer after biochemical recurrence (BCR) and to compare it with 18 F-choline PET/CT in a per-patient analysis. An observational study was performed of 43 patients with BCR after laparoscopic radical prostatectomy who underwent 64 Cu-PSMA-617 PET/CT and subsequently 18 F-choline PET/CT for restaging. The detection rates (DR) of 64 Cu-PSMA-617 PET/CT and of 18 F-choline PET/CT were calculated by standardized maximum uptake value (SUV max ) at 4 hours and SUV max at 1 hour as reference, respectively. Furthermore, univariate logistic regression analysis was carried out to identify independent predictive factors of positivity with 64 Cu-PSMA-617 PET/CT. An overall positivity with 64 Cu-PSMA-617 PET/CT was found in 32 patients (74.4%) versus 19 (44.2%) with 18 F-choline PET/CT. Specifically, after stratifying for prostate-specific antigen (PSA) values, we found a good performance of 64 Cu-PSMA-617 PET/CT at low PSA levels compared to 18 F-choline PET/CT, with a DR of 57.1% versus 14.3% for PSA 0.2-0.5 ng/mL (P = .031), and of 60% versus 30% with PSA 0.5-1 ng/mL. At univariate binary logistic regression analysis, PSA level was the only independent predictor of 64 Cu-PSMA-617 PET/CT positivity. No significant difference in terms of DR for both 64 Cu-PSMA-617 PET/CT and 18 F-choline PET/CT was found according to different Gleason score subgroups. In our study cohort, a better performance was observed for 64 Cu-PSMA-617 PET/CT compared to 18 F-choline PET/CT in restaging after BCR, especially in patients with low PSA values. Copyright © 2018 Elsevier Inc. All rights reserved.

Bleeding complications in BCR-ABL negative myeloproliferative neoplasms: prevalence, type, and risk factors in a single-center cohort.

PubMed

Kander, Elizabeth M; Raza, Sania; Zhou, Zheng; Gao, Juehua; Zakarija, Anaadriana; McMahon, Brandon J; Stein, Brady L

2015-11-01

The BCR-ABL1-negative myeloproliferative neoplasms (MPN) share an increased risk of thrombotic and hemorrhagic complications. Risk factors for hemorrhage are less well defined than those for thrombosis. Because patients with CALR mutations have higher platelet counts compared to JAK2 V617F-mutated patients, bleeding rates may be increased in this group. Our aim was to retrospectively evaluate whether acquired von Willebrand disease (AvWD), thrombocytosis, mutational status, or treatment history are associated with bleeding in a cohort of MPN patients. Using an electronic database, MPN patients seen between 2005 and 2013 were retrospectively identified using ICD-9 codes and billing records. A bleeding event was defined as one that was identified in the medical record and graded based on the Common Terminology Criteria for Adverse Event (CTCAE) version 4.0. Among 351 MPN patients, 15.6 % experienced 64 bleeding event types. There was no association of bleeding with mutational status, gender, MPN subtype, aspirin use, prior thrombosis, or platelet count at presentation. There was an association between bleeding and older age at diagnosis. aVWD was identified in six patients. In this single-center retrospective study, bleeding events were identified in 15 % of patients, and associated with older age at diagnosis. aVWD was rarely tested for in this cohort.

Protein kinase Cɛ inhibition restores megakaryocytic differentiation of hematopoietic progenitors from primary myelofibrosis patients.

PubMed

Masselli, E; Carubbi, C; Gobbi, G; Mirandola, P; Galli, D; Martini, S; Bonomini, S; Crugnola, M; Craviotto, L; Aversa, F; Vitale, M

2015-11-01

Among the three classic Philadelphia chromosome-negative myeloproliferative neoplasms, primary myelofibrosis (PMF) is the most severe in terms of disease biology, survival and quality of life. Abnormalities in the process of differentiation of PMF megakaryocytes (MKs) are a hallmark of the disease. Nevertheless, the molecular events that lead to aberrant megakaryocytopoiesis have yet to be clarified. Protein kinase Cɛ (PKCɛ) is a novel serine/threonine kinase that is overexpressed in a variety of cancers, promoting aggressive phenotype, invasiveness and drug resistance. Our previous findings on the role of PKCɛ in normal (erythroid and megakaryocytic commitment) and malignant (acute myeloid leukemia) hematopoiesis prompted us to investigate whether it could be involved in the pathogenesis of PMF MK-impaired differentiation. We demonstrate that PMF megakaryocytic cultures express higher levels of PKCɛ than healthy donors, which correlate with higher disease burden but not with JAK2V617F mutation. Inhibition of PKCɛ function (by a negative regulator of PKCɛ translocation) or translation (by target small hairpin RNA) leads to reduction in PMF cell growth, restoration of PMF MK differentiation and inhibition of PKCɛ-related anti-apoptotic signaling (Bcl-xL). Our data suggest that targeting PKCɛ directly affects the PMF neoplastic clone and represent a proof-of-concept for PKCɛ inhibition as a novel therapeutic strategy in PMF.

High Resolution Melting Analysis for JAK2 Exon 14 and Exon 12 Mutations

PubMed Central

Rapado, Inmaculada; Grande, Silvia; Albizua, Enriqueta; Ayala, Rosa; Hernández, José-Angel; Gallardo, Miguel; Gilsanz, Florinda; Martinez-Lopez, Joaquin

2009-01-01

JAK2 mutations are important criteria for the diagnosis of Philadelphia chromosome-negative myeloproliferative neoplasms. We aimed to assess JAK2 exon 14 and exon 12 mutations by high-resolution melting (HRM) analysis, which allows variation screening. The exon 14 analysis included 163 patients with polycythemia vera, secondary erythrocytoses, essential thrombocythemia, or secondary thrombocytoses, and 126 healthy subjects. The study of exon 12 included 40 JAK2 V617F-negative patients (nine of which had polycythemia vera, and 31 with splanchnic vein thrombosis) and 30 healthy subjects. HRM analyses of JAK2 exons 14 and 12 gave analytical sensitivities near 1% and both intra- and interday coefficients of variation of less than 1%. For HRM analysis of JAK2 exon 14 in polycythemia vera and essential thrombocythemia, clinical sensitivities were 93.5% and 67.9%, clinical specificities were 98.8% and 97.0%, positive predictive values were 93.5% and 79.2%, and negative predictive values were 98.8% and 94.6, respectively. Correlations were observed between the results from HRM and three commonly used analytical methods. The JAK2 exon 12 HRM results agreed completely with those from sequencing analysis, and the three mutations in exon 12 were detected by both methods. Hence, HRM analysis of exons 14 and 12 in JAK2 shows better diagnostic values than three other routinely used methods against which it was compared. In addition, HRM analysis has the advantage of detecting unknown mutations. PMID:19225136

Novel mutations in DNAJB6 gene cause a very severe early-onset limb-girdle muscular dystrophy 1D disease.

PubMed

Palmio, Johanna; Jonson, Per Harald; Evilä, Anni; Auranen, Mari; Straub, Volker; Bushby, Kate; Sarkozy, Anna; Kiuru-Enari, Sari; Sandell, Satu; Pihko, Helena; Hackman, Peter; Udd, Bjarne

2015-11-01

DNAJB6 is the causative gene for limb-girdle muscular dystrophy 1D (LGMD1D). Four different coding missense mutations, p.F89I, p.F93I, p.F93L, and p.P96R, have been reported in families from Europe, North America and Asia. The previously known mutations cause mainly adult-onset proximal muscle weakness with moderate progression and without respiratory involvement. A Finnish family and a British patient have been studied extensively due to a severe muscular dystrophy. The patients had childhood-onset LGMD, loss of ambulation in early adulthood and respiratory involvement; one patient died of respiratory failure aged 32. Two novel mutations, c.271T > A (p.F91I) and c.271T > C (p.F91L), in DNAJB6 were identified by whole exome sequencing as a cause of this severe form of LGMD1D. The results were confirmed by Sanger sequencing. The anti-aggregation effect of the mutant DNAJB6 was investigated in a filter-trap based system using transient transfection of mammalian cell lines and polyQ-huntingtin as a model for an aggregation-prone protein. Both novel mutant proteins show a significant loss of ability to prevent aggregation. Copyright © 2015 Elsevier B.V. All rights reserved.

Backbone dynamics and global effects of an activating mutation in minimized Mtu RecA inteins.

PubMed

Du, Zhenming; Liu, Yangzhong; Ban, David; Lopez, Maria M; Belfort, Marlene; Wang, Chunyu

2010-07-23

Inteins mediate protein splicing, which has found many applications in biotechnology and protein engineering. A single valine-to-leucine mutation (V67L) can globally enhance splicing and related cleavage reactions in minimized Mycobacterium tuberculosis RecA inteins. However, V67L mutation causes little change in crystal structures. To test whether protein dynamics contribute to activity enhancement in the V67L mutation, we have studied the conformations and dynamics of the minimized and engineered intein DeltaDeltaIhh-V67CM and a single V67L mutant, DeltaDeltaIhh-L67CM, by solution NMR. Chemical shift perturbations established that the V67L mutation causes global changes, including changes at the N-terminus and C-terminus of the intein, which are active sites for protein splicing. The single V67L mutation significantly slows hydrogen-exchange rates globally, indicating a shift to more stable conformations and reduction in ensemble distribution. Whereas the V67L mutation causes little change for motions on the picosecond-to-nanosecond timescale, motions on the microsecond-to-millisecond timescale affect a region involving the conserved F-block histidine and C-terminal asparagine, which are residues important for C-terminal cleavage. The V67L mutation is proposed to activate splicing by reducing the ensemble distribution of the intein structure and by modifying the active sites. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Computational design of variants for cephalosporin C acylase from Pseudomonas strain N176 with improved stability and activity.

PubMed

Tian, Ye; Huang, Xiaoqiang; Li, Qing; Zhu, Yushan

2017-01-01

In this report, redesigning cephalosporin C acylase from the Pseudomonas strain N176 revealed that the loss of stability owing to the introduced mutations at the active site can be recovered by repacking the nearby hydrophobic core regions. Starting from a quadruple mutant M31βF/H57βS/V68βA/H70βS, whose decrease in stability is largely owing to the mutation V68βA at the active site, we employed a computational enzyme design strategy that integrated design both at hydrophobic core regions for stability enhancement and at the active site for activity improvement. Single-point mutations L154βF, Y167βF, L180βF and their combinations L154βF/L180βF and L154βF/Y167βF/L180βF were found to display improved stability and activity. The two-point mutant L154βF/L180βF increased the protein melting temperature (T m ) by 11.7 °C and the catalytic efficiency V max /K m by 57 % compared with the values of the starting quadruple mutant. The catalytic efficiency of the resulting sixfold mutant M31βF/H57βS/V68βA/H70βS/L154βF/L180βF is recovered to become comparable to that of the triple mutant M31βF/H57βS/H70βS, but with a higher T m . Further experiments showed that single-point mutations L154βF, L180βF, and their combination contribute no stability enhancement to the triple mutant M31βF/H57βS/H70βS. These results verify that the lost stability because of mutation V68βA at the active site was recovered by introducing mutations L154βF and L180βF at hydrophobic core regions. Importantly, mutation V68βA in the six-residue mutant provides more space to accommodate the bulky side chain of cephalosporin C, which could help in designing cephalosporin C acylase mutants with higher activities and the practical one-step enzymatic route to prepare 7-aminocephalosporanic acid at industrial-scale levels.

WHO-defined 'myelodysplastic syndrome with isolated del(5q)' in 88 consecutive patients: survival data, leukemic transformation rates and prevalence of JAK2, MPL and IDH mutations.

PubMed

Patnaik, M M; Lasho, T L; Finke, C M; Gangat, N; Caramazza, D; Holtan, S G; Pardanani, A; Knudson, R A; Ketterling, R P; Chen, D; Hoyer, J D; Hanson, C A; Tefferi, A

2010-07-01

The 2008 World Health Organization (WHO) criteria were used to identify 88 consecutive Mayo Clinic patients with 'myelodysplastic syndrome with isolated del(5q)' (median age 74 years; 60 females). In all, 60 (68%) patients were followed up to the time of their death. Overall median survival was 66 months; leukemic transformation was documented in five (5.7%) cases. Multivariable analysis identified age >or=70 years (P=0.01), transfusion need at diagnosis (P=0.04) and dysgranulopoiesis (P=0.02) as independent predictors of shortened survival; the presence of zero (low risk), one (intermediate risk) or >or=2 (high risk) risk factors corresponded to median survivals of 102, 52 and 27 months, respectively. Janus kinase 2 (JAK2), thrombopoietin receptor (MPL), isocitrate dehydrogenase 1 (IDH1) and IDH2 mutational analysis was performed on archived bone marrows in 78 patients; JAK2V617F and MPLW515L mutations were shown in five (6.4%) and three (3.8%) patients, respectively, and did not seem to affect phenotype or prognosis. IDH mutations were not detected. Survival was not affected by serum ferritin and there were no instances of death directly related to iron overload. The current study is unique in its strict adherence to WHO criteria for selecting study patients and providing information on long-term survival, practical prognostic factors, baseline risk of leukemic transformation and the prevalence of JAK2, MPL and IDH mutations.

WHO-defined ‘myelodysplastic syndrome with isolated del(5q)' in 88 consecutive patients: survival data, leukemic transformation rates and prevalence of JAK2, MPL and IDH mutations

PubMed Central

Patnaik, M M; Lasho, T L; Finke, C M; Gangat, N; Caramazza, D; Holtan, S G; Pardanani, A; Knudson, R A; Ketterling, R P; Chen, D; Hoyer, J D; Hanson, C A; Tefferi, A

2010-01-01

The 2008 World Health Organization (WHO) criteria were used to identify 88 consecutive Mayo Clinic patients with ‘myelodysplastic syndrome with isolated del(5q)' (median age 74 years; 60 females). In all, 60 (68%) patients were followed up to the time of their death. Overall median survival was 66 months; leukemic transformation was documented in five (5.7%) cases. Multivariable analysis identified age ⩾70 years (P=0.01), transfusion need at diagnosis (P=0.04) and dysgranulopoiesis (P=0.02) as independent predictors of shortened survival; the presence of zero (low risk), one (intermediate risk) or ⩾2 (high risk) risk factors corresponded to median survivals of 102, 52 and 27 months, respectively. Janus kinase 2 (JAK2), thrombopoietin receptor (MPL), isocitrate dehydrogenase 1 (IDH1) and IDH2 mutational analysis was performed on archived bone marrows in 78 patients; JAK2V617F and MPLW515L mutations were shown in five (6.4%) and three (3.8%) patients, respectively, and did not seem to affect phenotype or prognosis. IDH mutations were not detected. Survival was not affected by serum ferritin and there were no instances of death directly related to iron overload. The current study is unique in its strict adherence to WHO criteria for selecting study patients and providing information on long-term survival, practical prognostic factors, baseline risk of leukemic transformation and the prevalence of JAK2, MPL and IDH mutations. PMID:20485371

Critical Problems in Very Large Scale Computer Systems

DTIC Science & Technology

1989-03-31

253-6043 Srinivas Devadas (617) 253-0454 Thomas F. Knight, Jr. (617) 253-7807 F. Thomson Leighton (617) 253-3662 Charles E. Leiserson (617) 253-5833...VLSI Memo No. 88-477, October 1988. S. Devadas , "General Decomposition of Sequential Machines: Relationships to State Assignment," to appear in...Perspective, C. Hewitt and G. Agha editors, MIT Press, 1989. Also MIT VLSI Memo No. 88-491, December 1988. * T. Leighton, B . Maggs, and S. Rao, "Universal

Critical Problems in Very Large Scale Computer Systems

DTIC Science & Technology

1990-03-31

Srinivas Devadas (617) 253-0454 Thomas F. Knight, Jr. (617) 253-7807 F. Thomson Leighton (617) 253-3662 Charles E. Leiserson (617) 253-5833 Jacob K...Aided Design of Integrated Circuits and Systems, pages 19-29, January 1990. [5] S. Arora, T. Leighton, and B . Maggs. On-line algorithms for path...selection in a non-blocking network. In Proceedings of the 21st Annual ACM Symposium on Theory of Computing, May 1990. To appear. [6] P. Ashar, S. Devadas

A KCNQ1 mutation causes age-dependant bradycardia and persistent atrial fibrillation.

PubMed

Ki, Chang-Seok; Jung, Chae Lim; Kim, Hyun-ji; Baek, Kwan-Hyuck; Park, Seung Jung; On, Young Keun; Kim, Ki-Suk; Noh, Su Jin; Youm, Jae Boum; Kim, June Soo; Cho, Hana

2014-03-01

Atrial fibrillation (AF) is the most common arrhythmia. Gain-of-function mutations in KCNQ1, the pore-forming α-subunit of the slow delayed rectifier K current (IKs) channel, have been associated with AF. The purpose of this study was functional assessment of a mutation in KCNQ1 identified in a family with persistent AF and sinus bradycardia. We investigated whether this KCNQ1 missense mutation could form the genetic basis for AF and bradycardia simultaneously in this family. Sanger sequencing in a family with hereditary persistent AF identified a novel KCNQ1 variant (V241F) in a highly conserved region of S4 domain. The proband and her son developed bradycardia and persistent AF in an age-dependent fashion. The other son was a mutation carrier but he showed sinus bradycardia and not AF. Whole-cell patch clamp electrophysiology showed that V241F mutation in KCNQ1 shifted the activation curve to the left and dramatically slowed deactivation, leading to a constitutively open-like phenotype. Computer modeling showed that V241F would slow pacemaker activity. Also, simulations of atrial excitation predicted that V241F results in extreme shortening of action potential duration, possibly resulting in AF. Our study indicates that V241F might cause sinus bradycardia by increasing IKs. Additionally, V241F likely shortens atrial refractoriness to promote a substrate for reentry. KCNQ1 mutations have previously been described in AF, yet this is the first time a mutation in KCNQ1 is associated with age-dependent bradycardia and persistent AF. This finding further supports the hypothesis that sinus node dysfunction contributes to the development of AF.

Risk Factors for and Management of MPN-Associated Bleeding and Thrombosis.

PubMed

Martin, Karlyn

2017-10-01

The Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) are characterized by both thrombotic and bleeding complications. The purpose of this review is to describe the risk factors associated with bleeding and thrombosis in MPN, as well as to review prevention strategies and management of these complications. Well-described risk factors for thrombotic complications include older age and history of prior thrombosis, along with traditional cardiovascular and venous thromboembolic risk factors. More recently, JAK2 V617F mutation has been found to carry an increased risk of thrombotic complications, whereas CALR has a lower risk than JAK2 mutation. Factors associated with an increased risk of bleeding in MPN include a prior history of bleeding, acquired von Willebrand syndrome, and primary myelofibrosis. Recent findings suggest that thrombocytosis carries a higher risk of bleeding than thrombosis in MPN, and aspirin may exacerbate this risk of bleeding, particularly in CALR-mutated ET. Much of the management of MPN focuses on predicting risk of bleeding and thrombosis and initiating prophylaxis to prevent complications in those at high risk of thrombosis. Emerging evidence suggests that sub-populations may have bleeding risk that outweighs thrombotic risk, particularly in setting of antiplatelet therapy. Future work is needed to better characterize this balance. At present, a thorough assessment of the risks of bleeding and thrombosis should be undertaken for each patient, and herein, we review risk factors for and management of these complications.

Impact of Molecular Genetics on Outcome in Myelofibrosis Patients after Allogeneic Stem Cell Transplantation.

PubMed

Kröger, Nicolaus; Panagiota, Victoria; Badbaran, Anita; Zabelina, Tatjana; Triviai, Ioanna; Araujo Cruz, Michelle Maria; Shahswar, Rabia; Ayuk, Francis; Gehlhaar, Marten; Wolschke, Christine; Bollin, Robin; Walter, Carolin; Dugas, Martin; Wiehlmann, Lutz; Lehmann, Ulrich; Koenecke, Christian; Chaturvedi, Anuhar; Alchalby, Haefaa; Stadler, Michael; Eder, Matthias; Christopeit, Max; Göhring, Gudrun; Koenigsmann, Michael; Schlegelberger, Brigitte; Kreipe, Hans-Heinrich; Ganser, Arnold; Stocking, Carol; Fehse, Boris; Thol, Felicitas; Heuser, Michael

2017-07-01

Molecular genetics may influence outcome for patients with myelofibrosis. To determine the impact of molecular genetics on outcome after allogeneic stem cell transplantation, we screened 169 patients with primary myelofibrosis (n = 110), post-essential thrombocythemia/polycythemia vera myelofibrosis (n = 46), and myelofibrosis in transformation (n = 13) for mutations in 16 frequently mutated genes. The most frequent mutation was JAK2V617F (n = 101), followed by ASXL1 (n = 49), calreticulin (n = 34), SRSF2 (n = 16), TET2 (n = 10), U2AF1 (n = 11), EZH2 (n = 7), MPL (n = 6), IDH2 (n = 5), IDH1 (n = 4), and CBL (n = 1). The cumulative incidence of nonrelapse mortality (NRM) at 1 year was 21% and of relapse at 5 years 25%. The 5-year rates progression-free (PFS) and overall survival (OS) were and 56%, respectively. In a multivariate analysis CALR mutation was an independent factor for lower NRM (HR, .415; P = .05), improved PFS (HR, .393; P = .01), and OS (HR, .448; P = .03). ASXL1 and IDH2 mutations were independent risk factors for lower PFS (HR, 1.53 [P = .008], and HR, 5.451 [P = .002], respectively), whereas no impact was observed for "triple negative" patients. Molecular genetics, especially CALR, IDH2, and ASXL1 mutations, may thus be useful to predict outcome independently from known clinical risk factors after allogeneic stem cell transplantation for myelofibrosis. Copyright © 2017 The American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

Prognostication in Philadelphia Chromosome Negative Myeloproliferative Neoplasms: a Review of the Recent Literature.

PubMed

Zhou, Amy; Afzal, Amber; Oh, Stephen T

2017-10-01

The prognosis for patients with Philadelphia chromosome (Ph)-negative myeloproliferative neoplasms (MPNs) is highly variable. All Ph-negative MPNs carry an increased risk for thrombotic complications, bleeding, and leukemic transformation. Several clinical, biological, and molecular prognostic factors have been identified in recent years, which provide important information in guiding management of patients with Ph-negative MPNs. In this review, we critically evaluate the recent published literature and discuss important new developments in clinical and molecular factors that impact survival, disease transformation, and thrombosis in patients with polycythemia vera, essential thrombocythemia, and primary myelofibrosis. Recent studies have identified several clinical factors and non-driver mutations to have prognostic impact on Ph-negative MPNs independent of conventional risk stratification and prognostic models. In polycythemia vera (PV), leukocytosis, abnormal karyotype, phlebotomy requirement on hydroxyurea, increased bone marrow fibrosis, and mutations in ASXL1, SRSF2, and IDH2 were identified as additional adverse prognostic factors. In essential thrombocythemia (ET), JAK2 V617F mutation, splenomegaly, and mutations in SH2B3, SF3B1, U2AF1, TP53, IDH2, and EZH2 were found to be additional negative prognostic factors. Bone marrow fibrosis and mutations in ASXL1, SRSF2, EZH2, and IDH1/2 have been found to be additional prognostic factors in primary myelofibrosis (PMF). CALR mutations appear to be a favorable prognostic factor in PMF, which has not been clearly demonstrated in ET. The prognosis for patients with PV, ET, and PMF is dependent upon the presence or absence of several clinical, biological, and molecular risk factors. The significance of additional risk factors identified in these recent studies will need further validation in prospective studies to determine how they may be best utilized in the management of these disorders.

Critical Problems in Very Large Scale Computer Systems

DTIC Science & Technology

1988-09-30

253-6043 Srinivas Devadas (617) 253-0454 Thomas F. Knight, Jr. (617) 253-7807 F. Thomson Leighton (617) 253-3662 Charles E. Leiserson (617) 253-5833...J. Keen, P. Nuth, J. Larivee, and B . Totty, "Message-Driven Processor Architecture," MIT VLSI Memo No. 88-468, August 1988. *W. J. Dally and A. A...losses and gains) which are the first polynomial-time combinatorial algorithms for this problem. One algorithm runs in O(n2m2 lg 2 n Ig B ) time and the

A Genetic Screen Reveals an Unexpected Role for Yorkie Signaling in JAK/STAT-Dependent Hematopoietic Malignancies in Drosophila melanogaster

PubMed Central

Anderson, Abigail M.; Bailetti, Alessandro A.; Rodkin, Elizabeth; De, Atish; Bach, Erika A.

2017-01-01

A gain-of-function mutation in the tyrosine kinase JAK2 (JAK2V617F) causes human myeloproliferative neoplasms (MPNs). These patients present with high numbers of myeloid lineage cells and have numerous complications. Since current MPN therapies are not curative, there is a need to find new regulators and targets of Janus kinase/Signal transducer and activator of transcription (JAK/STAT) signaling that may represent additional clinical interventions . Drosophila melanogaster offers a low complexity model to study MPNs as JAK/STAT signaling is simplified with only one JAK [Hopscotch (Hop)] and one STAT (Stat92E). hopTumorous-lethal (Tum-l) is a gain-of-function mutation that causes dramatic expansion of myeloid cells, which then form lethal melanotic tumors. Through an F1 deficiency (Df) screen, we identified 11 suppressors and 35 enhancers of melanotic tumors in hopTum-l animals. Dfs that uncover the Hippo (Hpo) pathway genes expanded (ex) and warts (wts) strongly enhanced the hopTum-l tumor burden, as did mutations in ex, wts, and other Hpo pathway genes. Target genes of the Hpo pathway effector Yorkie (Yki) were significantly upregulated in hopTum-l blood cells, indicating that Yki signaling was increased. Ectopic hematopoietic activation of Yki in otherwise wild-type animals increased hemocyte proliferation but did not induce melanotic tumors. However, hematopoietic depletion of Yki significantly reduced the hopTum-l tumor burden, demonstrating that Yki is required for melanotic tumors in this background. These results support a model in which elevated Yki signaling increases the number of hemocytes, which become melanotic tumors as a result of elevated JAK/STAT signaling. PMID:28620086

From leeches to personalized medicine: evolving concepts in the management of polycythemia vera.

PubMed

Vannucchi, Alessandro M

2017-01-01

Polycythemia vera is a clonal disorder of hematopoietic stem/progenitor cells. It manifests as an expansion of red cell mass. It is the most common chronic myeloproliferative neoplasm. In virtually all cases, it is characterized by a V617F point mutation in JAK2 exon 14 or less common mutations in exon 12. The landmark discovery of the autonomously activated JAK/STAT signaling pathway paved the way for the clinical development of the first target drug, the JAK1 and JAK2 inhibitor ruxolitinib. This is now approved for patients with resistance or intolerance to hydroxyurea. Phlebotomies and hydroxyurea are still the cornerstone of treatment, and aim to prevent the first appearance or recurrence of cardiovascular events that, together with progression to post-polycythemia vera myelofibrosis and leukemia, represent the main causes of death. Interferon-α is an alternative drug and has been shown to induce molecular remissions. It is currently undergoing phase III trials that might eventually lead to its approval for clinical use. The last few years have witnessed important advances towards an accurate early diagnosis of polycythemia vera, greater understanding of its pathogenesis, and improved patient management. This review will focus on the most recent achievements and will aim to unify the different concepts involved in a personalized approach to the patient with polycythemia vera. In spite of many recent advances in the understanding of its pathogenesis and improved disease management, polycythemia vera remains a life-threatening myeloproliferative neoplasm for which there is no cure. This review will present a critical overview of evolving concepts in diagnosis and treatment of this disease. Copyright© Ferrata Storti Foundation.

From leeches to personalized medicine: evolving concepts in the management of polycythemia vera

PubMed Central

Vannucchi, Alessandro M.

2017-01-01

Polycythemia vera is a clonal disorder of hematopoietic stem/progenitor cells. It manifests as an expansion of red cell mass. It is the most common chronic myeloproliferative neoplasm. In virtually all cases, it is characterized by a V617F point mutation in JAK2 exon 14 or less common mutations in exon 12. The landmark discovery of the autonomously activated JAK/STAT signaling pathway paved the way for the clinical development of the first target drug, the JAK1 and JAK2 inhibitor ruxolitinib. This is now approved for patients with resistance or intolerance to hydroxyurea. Phlebotomies and hydroxyurea are still the cornerstone of treatment, and aim to prevent the first appearance or recurrence of cardiovascular events that, together with progression to post-polycythemia vera myelofibrosis and leukemia, represent the main causes of death. Interferon-α is an alternative drug and has been shown to induce molecular remissions. It is currently undergoing phase III trials that might eventually lead to its approval for clinical use. The last few years have witnessed important advances towards an accurate early diagnosis of polycythemia vera, greater understanding of its pathogenesis, and improved patient management. This review will focus on the most recent achievements and will aim to unify the different concepts involved in a personalized approach to the patient with polycythemia vera. In spite of many recent advances in the understanding of its pathogenesis and improved disease management, polycythemia vera remains a life-threatening myeloproliferative neoplasm for which there is no cure. This review will present a critical overview of evolving concepts in diagnosis and treatment of this disease. PMID:27884974

Stability enhancement of cytochrome c through heme deprotonation and mutations.

PubMed

Sonoyama, Takafumi; Hasegawa, Jun; Uchiyama, Susumu; Nakamura, Shota; Kobayashi, Yuji; Sambongi, Yoshihiro

2009-01-01

The chemical denaturation of Pseudomonas aeruginosa cytochrome c(551) variants was examined at pH 5.0 and 3.6. All variants were stabilized at both pHs compared with the wild-type. Remarkably, the variants carrying the F34Y and/or E43Y mutations were more stabilized than those having the F7A/V13M or V78I ones at pH 5.0 compared with at pH 3.6 by ~3.0-4.6 kJ/mol. Structural analyses predicted that the side chains of introduced Tyr-34 and Tyr-43 become hydrogen donors for the hydrogen bond formation with heme 17-propionate at pH 5.0, but less efficiently at pH 3.6, because the propionate is deprotonated at the higher pH. Our results provide an insight into a stabilization strategy for heme proteins involving variation of the heme electronic state and introduction of appropriate mutations.

Extramedullary haematopoiesis presenting with cardiac tamponade in a patient with polycythaemia vera.

PubMed

Haroun, Faysal; Elkis, Viktoria; Chen, Anne; Lee, Elsie

2017-08-10

A 71-year-old man with a history of polycythaemia vera, diagnosed 4 years ago, presented to the emergency room with shortness of breath. A bedside echocardiogram revealed a large pericardial effusion with features concerning for pericardial tamponade. A left anterior thoracotomy and a pericardial window were emergently performed in the operating room and relieved the patient's symptoms. Histology evaluation of the pericardial fragments and pericardial fluid revealed the presence of trilineage haematopoietic elements without any increase in the blasts. A bone marrow core biopsy revealed an increase in reticulin fibre and increase in the number of blasts of 5%-10%, whereas peripheral blood testing was positive for JAK2 V617F mutation. This case report reviews the literature for cases of extramedullary haematopoiesis associated with myeloproliferative neoplasms. © BMJ Publishing Group Ltd (unless otherwise stated in the text of the article) 2017. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Elevated serum erythropoietin in a patient with polycythaemia vera presenting with Budd-Chiari syndrome

PubMed Central

Jones, Catherine; Levy, Yair; Tong, Alex W

2014-01-01

Polycythaemia vera (PV) is a clonal disorder of bone marrow stem cells characterised by erythrocytosis. Diagnosis of PV requires exclusion of secondary causes of polycythaemia. It has been held that an elevated erythropoietin (Epo) level strongly indicates secondary erythrocytosis and excludes PV diagnosis, to the extent that the reduced serum Epo level is currently listed as a minor criterion in the WHO classification scheme for PV. However, patients with PV who co-present with Budd-Chiari syndrome have been documented with elevated serum Epo levels. For these patients, identification of the Janus kinase 2 (JAK2) V617F point mutation along with the transient nature of the Epo elevation provides certainty of PV diagnosis, as illustrated by the proband. In this case report, the patient's positive response to cytoreductive therapy (hydroxyurea 500 mg daily) and phlebotomy (750 mL over three phlebotomies) further supports validity of PV diagnosis with elevated Epo. The patient remains on rivaroxaban (Xarelto) for treatment of her portal vein thrombosis. PMID:25452296

Novel C617Y mutation in the 7th transmembrane segment of luteinizing hormone/choriogonadotropin receptor in a Japanese boy with peripheral precocious puberty.

PubMed

Nagasaki, Keisuke; Katsumata, Noriyuki; Ogawa, Yohei; Kikuchi, Toru; Uchiyama, Makoto

2010-01-01

Testotoxicosis, also known as familial male-limited precocious puberty, is an autosomal dominant form of gonadotropin-independent precocious puberty caused by heterozygous constitutively activating mutations of the LHCGR gene encoding the luteinizing hormone/choriogonadotropin receptor (LH/CGR). The patient is an 8-year-old boy who started to develop pubic hair and penile enlargement at 6 years of age. The patient had elevated serum testosterone levels, but initially exhibited a prepubertal response of gonadotropins to GnRH, which was followed by central activation of the hypothalamo-pituitary-gonadal axis. The father reported having experienced precocious puberty, and is 158 cm tall. There is no history of short stature and precocious puberty in the family except for the father. The LHCGR gene was analyzed by direct DNA sequencing of amplified PCR products from the patient and his parents. The wild-type and mutant LH/CGRs were transiently expressed in COS-1 cells and cAMP levels in the cells were determined with or without hCG stimulation. Genetic analysis revealed a novel C617Y mutation of the LHCGR gene in the patient and his mother, while his father had no mutations. Functional expression study demonstrated around 15% increase in the basal intracellular cAMP level in cells expressing the mutant LH/CGR compared with that in cells expressing the wild-type receptor. We have reported the first missense C617Y mutation located in the 7th transmembrane segment of LH/CGR causing testotoxicosis. The modest phenotype of our patient may be explained, at least in part, by the modest increase in the intracellular cAMP level caused by the C617Y mutation.

Estrogen signaling selectively induces apoptosis of hematopoietic progenitors and myeloid neoplasms without harming steady-state hematopoiesis.

PubMed

Sánchez-Aguilera, Abel; Arranz, Lorena; Martín-Pérez, Daniel; García-García, Andrés; Stavropoulou, Vaia; Kubovcakova, Lucia; Isern, Joan; Martín-Salamanca, Sandra; Langa, Xavier; Skoda, Radek C; Schwaller, Jürg; Méndez-Ferrer, Simón

2014-12-04

Estrogens are potent regulators of mature hematopoietic cells; however, their effects on primitive and malignant hematopoietic cells remain unclear. Using genetic and pharmacological approaches, we observed differential expression and function of estrogen receptors (ERs) in hematopoietic stem cell (HSC) and progenitor subsets. ERα activation with the selective ER modulator (SERM) tamoxifen induced apoptosis in short-term HSCs and multipotent progenitors. In contrast, tamoxifen induced proliferation of quiescent long-term HSCs, altered the expression of self-renewal genes, and compromised hematopoietic reconstitution after myelotoxic stress, which was reversible. In mice, tamoxifen treatment blocked development of JAK2(V617F)-induced myeloproliferative neoplasm in vivo, induced apoptosis of human JAK2(V617F+) HSPCs in a xenograft model, and sensitized MLL-AF9(+) leukemias to chemotherapy. Apoptosis was selectively observed in mutant cells, and tamoxifen treatment only had a minor impact on steady-state hematopoiesis in disease-free animals. Together, these results uncover specific regulation of hematopoietic progenitors by estrogens and potential antileukemic properties of SERMs. Copyright © 2014 Elsevier Inc. All rights reserved.

Highly preferential association of NonF508del CF mutations with the M470 allele.

PubMed

Ciminelli, B M; Bonizzato, A; Bombieri, C; Pompei, F; Gabaldo, M; Ciccacci, C; Begnini, A; Holubova, A; Zorzi, P; Piskackova, T; Macek, M; Castellani, C; Modiano, G; Pignatti, P F

2007-01-01

On the basis of previous findings on random individuals, we hypothesized a preferential association of CF causing mutations with the M allele of the M470V polymorphic site of the CFTR gene. We have determined the M/V-CF mutation haplotype in a series of 201 North East Italian and 73 Czech CF patients who were not F508del homozygotes, as F508del was already known to be fully associated with the M allele. Out of 358 not F508del CF genes, 84 carried the V allele and 274 the less common M allele. In the N-E Italian population, MM subjects have a risk of carrying a CF causing mutation 6.9x greater than VV subjects when F508del is excluded and 15.4x when F508del is included. In the Czech population a similar, although less pronounced, association is observed. Besides the possible biological significance of this association, the possibility of exploiting it for a pilot screening program has been explored in a local North East Italian population for which CF patients were characterized for their CF mutation. General M470V genotyping followed by common CF mutation screening limited to couples in which each partner carries at least one M allele would need testing only 39% of the couples, which contribute 89% of the total risk, with a cost benefit.

Polycythemia is associated with bone loss and reduced osteoblast activity in mice.

PubMed

Oikonomidou, P R; Casu, C; Yang, Z; Crielaard, B; Shim, J H; Rivella, S; Vogiatzi, M G

2016-04-01

Increased fragility has been described in humans with polycythemia vera (PV). Herein, we describe an osteoporotic phenotype associated with decreased osteoblast activity in a mouse model of PV and another mouse of polycythemia and elevated circulating erythropoietin (EPO). Our results are important for patients with PV or those treated with recombinant EPO (rEPO). PV and other myeloproliferative syndromes have been recently associated with an increased risk for fractures. However, the presence of osteoporosis in these patients has not been well documented. EPO, a hormone primarily known to stimulate erythropoiesis, has been shown recently to regulate bone homeostasis in mice. The aim of this study was to examine the bone phenotype of a mouse model of PV and compare it to that of animals with polycythemia caused by elevated circulating EPO. Bone mass and remodeling were evaluated by micro-computed tomography and histomorphometry. The JAK2(V617F) knock-in mouse, a model of human PV, manifests polycythemia and low circulating EPO levels. Results from this mouse were compared to wild type (wt) controls and the tg6 transgenic mouse that shows polycythemia caused by increased constitutive expression of EPO. Compared to wt, both JAK2(V617F) and tg6 mice had a decrease in trabecular bone mass. Tg6 mice showed an additional modest decrease in cortical thickness and cortical bone volume per tissue volume (P < 0.01) suggesting a more severe bone phenotype than JAK2(V617F). Decreased osteoblast numbers and bone formation along with normal osteoclast numbers and activity were found in both mice. This study indicates that PV is associated with low bone mass and decreased osteoblast activity in mice. Our results support future studies of osteoporosis in affected humans. Polycythemia caused by chronically elevated circulating EPO also results in bone loss, and implications on patients treated with rEPO should be evaluated.

Prevalence and prognostic impact of allelic imbalances associated with leukemic transformation of Philadelphia chromosome–negative myeloproliferative neoplasms

PubMed Central

Krug, Utz O.; Lee, Dhong Hyun Tony; Kawamata, Norihiko; Iwanski, Gabriela B.; Lasho, Terra; Weiss, Tamara; Nowak, Daniel; Koren-Michowitz, Maya; Kato, Motohiro; Sanada, Masashi; Shih, Lee-Yung; Nagler, Arnon; Raynaud, Sophie D.; Müller-Tidow, Carsten; Mesa, Ruben; Haferlach, Torsten; Gilliland, D. Gary; Tefferi, Ayalew; Ogawa, Seishi; Koeffler, H. Phillip

2010-01-01

Philadelphia chromosome–negative myeloproliferative neoplasms (MPNs) including polycythemia vera, essential thrombocythemia, and primary myelofibrosis show an inherent tendency for transformation into leukemia (MPN-blast phase), which is hypothesized to be accompanied by acquisition of additional genomic lesions. We, therefore, examined chromosomal abnormalities by high-resolution single nucleotide polymorphism (SNP) array in 88 MPN patients, as well as 71 cases with MPN-blast phase, and correlated these findings with their clinical parameters. Frequent genomic alterations were found in MPN after leukemic transformation with up to 3-fold more genomic changes per sample compared with samples in chronic phase (P < .001). We identified commonly altered regions involved in disease progression including not only established targets (ETV6, TP53, and RUNX1) but also new candidate genes on 7q, 16q, 19p, and 21q. Moreover, trisomy 8 or amplification of 8q24 (MYC) was almost exclusively detected in JAK2V617F− cases with MPN-blast phase. Remarkably, copy number–neutral loss of heterozygosity (CNN-LOH) on either 7q or 9p including homozygous JAK2V617F was related to decreased survival after leukemic transformation (P = .01 and P = .016, respectively). Our high-density SNP-array analysis of MPN genomes in the chronic compared with leukemic stage identified novel target genes and provided prognostic insights associated with the evolution to leukemia. PMID:20068225

Proteomic analysis reveals heat shock protein 70 has a key role in polycythemia Vera.

PubMed

Gallardo, Miguel; Barrio, Santiago; Fernandez, Marisol; Paradela, Alberto; Arenas, Alicia; Toldos, Oscar; Ayala, Rosa; Albizua, Enriqueta; Jimenez, Ana; Redondo, Santiago; Garcia-Martin, Rosa Maria; Gilsanz, Florinda; Albar, Juan Pablo; Martinez-Lopez, Joaquin

2013-11-19

JAK-STAT signaling through the JAK2V617F mutation is central to the pathogenesis of myeloproliferative neoplasms (MPN). However, other events could precede the JAK2 mutation. The aim of this study is to analyze the phenotypic divergence between polycytemia vera (PV) and essential thrombocytemia (ET) to find novel therapeutics targets by a proteomic and functional approach to identify alternative routes to JAK2 activation. Through 2D-DIGE and mass spectrometry of granulocyte protein from 20 MPN samples, showed differential expression of HSP70 in PV and ET besides other 60 proteins. Immunohistochemistry of 46 MPN bone marrow samples confirmed HSP70 expression. The median of positive granulocytes was 80% in PV (SD 35%) vs. 23% in ET (SD 34.25%). In an ex vivo model KNK437 was used as an inhibition model assay of HSP70, showed dose-dependent inhibition of cell growth and burst formation unit erythroid (BFU-E) in PV and ET, increased apoptosis in the erythroid lineage, and decreased pJAK2 signaling, as well as a specific siRNA for HSP70. These data suggest a key role for HSP70 in proliferation and survival of the erythroid lineage in PV, and may represent a potential therapeutic target in MPN, especially in PV.

High Frequency of Copy-Neutral Loss of Heterozygosity in Patients with Myelofibrosis.

PubMed

Rego de Paula Junior, Milton; Nonino, Alexandre; Minuncio Nascimento, Juliana; Bonadio, Raphael S; Pic-Taylor, Aline; de Oliveira, Silviene F; Wellerson Pereira, Rinaldo; do Couto Mascarenhas, Cintia; Forte Mazzeu, Juliana

2018-01-01

Myelofibrosis is the rarest and most severe type of Philadelphia-negative classical myeloproliferative neoplasms. Although mutually exclusive driver mutations in JAK2, MPL, or CALR that activate JAK-STAT pathway have been related to the pathogenesis of the disease, chromosome abnormalities have also been associated with the phenotype and prognosis of the disease. Here, we report the use of a chromosomal microarray platform consisting of both oligo and SNP probes to improve the detection of chromosome abnormalities in patients with myelofibrosis. Sixteen patients with myelofibrosis were tested, and the results were compared to karyotype analysis. Driver mutations in JAK2, MPL, or CALR were investigated by PCR and MLPA. Conventional cytogenetics revealed chromosome abnormalities in 3 out of 16 cases (18.7%), while chromosomal microarray analysis detected copy-number variations (CNV) or copy-neutral loss of heterozygosity (CN-LOH) alterations in 11 out of 16 (68.7%) patients. These included 43 CN-LOH, 14 deletions, 1 trisomy, and 1 duplication. Ten patients showed multiple chromosomal abnormalities, varying from 2 to 13 CNVs or CN-LOHs. Mutational status for JAK2, CALR, and MPL by MLPA revealed a total of 3/16 (18.7%) patients positive for the JAK2 V617F mutation, 9 with CALR deletion or insertion and 1 positive for MPL mutation. Considering that most of the CNVs identified were smaller than the karyotype resolution and the high frequency of CN-LOHs in our study, we propose that chromosomal microarray platforms that combine oligos and SNP should be used as a first-tier genetic test in patients with myelofibrosis. © 2018 S. Karger AG, Basel.

A novel mutation in the alpha-helix 1 of the C subunit of the F(1)/F(0) ATPase responsible for optochin resistance of a Streptococcus pneumoniae clinical isolate.

PubMed

Cogné, N; Claverys, J; Denis, F; Martin, C

2000-10-01

Previously reported mutations involved in optochin resistance of Streptococcus pneumoniae clinical isolates changed residues 48, 49 or 50, in the transmembrane alpha-helix 2 of the F(1)/F(0) ATPase subunit. We report here an unusual mutation which changes the sequence of the transmembrane alpha-helix 1 of the AtpC subunit. This mutation involves a Gly to Ser substitution resulting from a G to A transition at codon 14 of the atpC gene.

Autosomal dominant deficiency of the interleukin-17F in recurrent aphthous stomatitis: Possible novel mutation in a new entity.

PubMed

Zare Bidoki, Alireza; Massoud, Ahmad; Najafi, Shamsolmoulouk; Mohammadzadeh, Mahsa; Rezaei, Nima

2018-05-15

Recurrent Aphthous Stomatitis (RAS) is a common oral inflammatory disease with unknown pathogenesis. Although the immune system alterations could be involved in predisposition of individuals to oral candidiasis, precise etiologies of RAS have not been understood yet. A recent study showed that autosomal dominant IL17F deficiency could cause chronic mucocutaneous candidiasis. Considering the inflammatory nature of interleukin (IL)-17F and RAS, this study was performed to check any disease-associated mutation in a number of patients with RAS. Sixty-two Iranian individuals with RAS were investigated in this study. After DNA extraction using a phenol-chloroform method from the whole blood, amplification was accomplished by polymerase chain reaction and the products were sequenced using a 3730 ABI sequencer. The results of sequencing revealed a missense, heterozygous mutation of IL17F, converting a threonine to proline in a patient with RAS (T79P). The Poly-phen software suggested a damaging probability predicting this substitution to have a harmful effect on IL-17F protein function. This mutation was checked in fifty healthy individuals, and was not detected in any of them. This is the first study showing that a mutation in IL-17F is associated with susceptibility to RAS. However, functional studies and further studies on more patients with RAS are required to confirm such association. Copyright © 2018 Elsevier B.V. All rights reserved.

Improving olefin tolerance and production in E. coli using native and evolved AcrB

DOE PAGES

Mingardon, Florence; Clement, Camille; Hirano, Kathleen; ...

2015-01-20

Microorganisms can be engineered for the production of chemicals utilized in the polymer industry. However many such target compounds inhibit microbial growth and might correspondingly limit production levels. Here, we focus on compounds that are precursors to bioplastics, specifically styrene and representative alpha-olefins; 1-hexene, 1-octene, and 1-nonene. We evaluated the role of the Escherichia coli efflux pump, AcrAB-TolC, in enhancing tolerance towards these olefin compounds. AcrAB-TolC is involved in the tolerance towards all four compounds in E. coli. Both styrene and 1-hexene are highly toxic to E. coli. Styrene is a model plastics precursor with an established route for productionmore » in E. coli (McKenna and Nielsen, 2011). Though our data indicates that AcrAB-TolC is important for its optimal production, we observed a strong negative selection against the production of styrene in E. coli. Thus we used 1-hexene as a model compound to implement a directed evolution strategy to further improve the tolerance phenotype towards this alpha-olefin. We focused on optimization of AcrB, the inner membrane domain known to be responsible for substrate binding, and found several mutations (A279T, Q584R, F617L, L822P, F927S, and F1033Y) that resulted in improved tolerance. Several of these mutations could also be combined in a synergistic manner. Our study shows efflux pumps to be an important mechanism in host engineering for olefins, and one that can be further improved using strategies such as directed evolution, to increase tolerance and potentially production.« less

Germline variations at JAK2, TERT, HBS1L-MYB and MECOM and the risk of myeloproliferative neoplasms in Taiwanese population

PubMed Central

Chiang, Yi-Hao; Chang, Yu-Cheng; Lin, Huan-Chau; Huang, Ling; Cheng, Chun-Chia; Wang, Wei-Ting; Cheng, Hung-I; Su, Nai-Wen; Chen, Caleb Gon-Shen; Lin, Johnson; Chang, Yi-Fang; Chang, Ming-Chih; Hsieh, Ruey-Kuen; Chou, Wen-Chien; Lim, Ken-Hong; Kuo, Yuan-Yeh

2017-01-01

Germline variations at JAK2, TERT, HBS1L-MYB and MECOM have been found to associate with myeloproliferative neoplasms (MPNs) in European populations. Whether these germline variations are associated with MPNs in Taiwanese population is obscure. Here we aimed to evaluate the association of five germline variations (JAK2 46/1 haplotype tagged by rs12343867, JAK2 intron 8 rs12339666, TERT rs2736100, HBS1L-MYB rs9376092 and MECOM rs2201862) and the risk of MPNs in Taiwanese population. A total of 178 MPN patients (109 essential thrombocythemia, 54 polycythemia vera and 15 primary myelofibrosis) were enrolled into this study. The information of 17033 control subjects was obtained from Taiwan Biobank database. The JAK2 46/1 haplotype, JAK2 rs12339666 and TERT rs2736100 were significantly associated with Taiwanese MPNs (P = 3.6×10-19, 1.9×10-19 and 3.1×10-6, respectively), and JAK2V617F-positive MPNs (n=121) (P = 5.6×10-21, 4.4×10-21 and 8.6×10-7, respectively). In JAK2V617F-negative cases (n=55), only the JAK2 46/1 haplotype and JAK2 rs12339666 remained statistically significant (P= 0.009 and 0.007, respectively). When stratified by disease subtypes, the JAK2 46/1 haplotype and JAK2 rs12339666 were significantly associated with all three MPN subtypes, but TERT rs2736100 was only associated with essential thrombocythemia and polycythemia vera. We did not find any association of these five SNPs with CALR mutations in our cohort. Furthermore, the risk alleles of MECOM rs2201862 and HBS1L-MYB rs9376092 were demonstrated to be negatively associated with the risk of developing polycythemia vera. In conclusion, germline variations at JAK2 (both the 46/1 haplotype and rs12339666) and TERT rs2736100 were associated with MPNs in Taiwanese population. PMID:29100304

Co-occurrence of Point Mutations in the Voltage-Gated Sodium Channel of Pyrethroid-Resistant Aedes aegypti Populations in Myanmar

PubMed Central

Kawada, Hitoshi; Oo, Sai Zaw Min; Thaung, Sein; Kawashima, Emiko; Maung, Yan Naung Maung; Thu, Hlaing Myat; Thant, Kyaw Zin; Minakawa, Noboru

2014-01-01

Background Single amino acid substitutions in the voltage-gated sodium channel associated with pyrethroid resistance constitute one of the main causative factors of knockdown resistance in insects. The kdr gene has been observed in several mosquito species; however, point mutations in the para gene of Aedes aegypti populations in Myanmar have not been fully characterized. The aim of the present study was to determine the types and frequencies of mutations in the para gene of Aedes aegypti collected from used tires in Yangon City, Myanmar. Methodology/Principal Findings We determined high pyrethroid resistance in Aedes aegypti larvae at all collection sites in Yangon City, by using a simplified knockdown bioassay. We showed that V1016G and S989P mutations were widely distributed, with high frequencies (84.4% and 78.8%, respectively). By contrast, we were unable to detect I1011M (or I1011V) or L1014F mutations. F1534C mutations were also widely distributed, but with a lower frequency than the V1016G mutation (21.2%). High percentage of co-occurrence of the homozygous V1016G/S989P mutations was detected (65.7%). Additionally, co-occurrence of homozygous V1016G/F1534C mutations (2.9%) and homozygous V1016G/F1534C/S989P mutations (0.98%) were detected in the present study. Conclusions/Significance Pyrethroid insecticides were first used for malaria control in 1992, and have since been constantly used in Myanmar. This intensive use may explain the strong selection pressure toward Aedes aegypti, because this mosquito is generally a domestic and endophagic species with a preference for indoor breeding. Extensive use of DDT for malaria control before the use of this chemical was banned may also explain the development of pyrethroid resistance in Aedes aegypti. PMID:25077956

Co-occurrence of point mutations in the voltage-gated sodium channel of pyrethroid-resistant Aedes aegypti populations in Myanmar.

PubMed

Kawada, Hitoshi; Oo, Sai Zaw Min; Thaung, Sein; Kawashima, Emiko; Maung, Yan Naung Maung; Thu, Hlaing Myat; Thant, Kyaw Zin; Minakawa, Noboru

2014-01-01

Single amino acid substitutions in the voltage-gated sodium channel associated with pyrethroid resistance constitute one of the main causative factors of knockdown resistance in insects. The kdr gene has been observed in several mosquito species; however, point mutations in the para gene of Aedes aegypti populations in Myanmar have not been fully characterized. The aim of the present study was to determine the types and frequencies of mutations in the para gene of Aedes aegypti collected from used tires in Yangon City, Myanmar. We determined high pyrethroid resistance in Aedes aegypti larvae at all collection sites in Yangon City, by using a simplified knockdown bioassay. We showed that V1016G and S989P mutations were widely distributed, with high frequencies (84.4% and 78.8%, respectively). By contrast, we were unable to detect I1011M (or I1011V) or L1014F mutations. F1534C mutations were also widely distributed, but with a lower frequency than the V1016G mutation (21.2%). High percentage of co-occurrence of the homozygous V1016G/S989P mutations was detected (65.7%). Additionally, co-occurrence of homozygous V1016G/F1534C mutations (2.9%) and homozygous V1016G/F1534C/S989P mutations (0.98%) were detected in the present study. Pyrethroid insecticides were first used for malaria control in 1992, and have since been constantly used in Myanmar. This intensive use may explain the strong selection pressure toward Aedes aegypti, because this mosquito is generally a domestic and endophagic species with a preference for indoor breeding. Extensive use of DDT for malaria control before the use of this chemical was banned may also explain the development of pyrethroid resistance in Aedes aegypti.

The natural history and treatment outcome of blast phase BCR-ABL− myeloproliferative neoplasms

PubMed Central

Tam, Constantine S.; Nussenzveig, Roberto M.; Popat, Uday; Bueso-Ramos, Carlos E.; Thomas, Deborah A.; Cortes, Jorge A.; Champlin, Richard E.; Ciurea, Stefan E.; Manshouri, Taghi; Pierce, Sherry M.; Kantarjian, Hagop M.

2008-01-01

We analyzed the outcomes of 74 patients diagnosed with BCR-ABL− myeloproliferative neoplasms in blast phase receiving induction chemotherapy (55%), low-intensity therapy (16%), stem cell transplantation (SCT; 3%), or supportive care (26%). Median survival from the date of blastic transformation was 5 months. Patients receiving supportive therapy had a median survival of 6 weeks. Complete remission with or without blood recovery was achieved in 46% of patients receiving induction chemotherapy, but remissions were not durable with a median progression-free survival of only 5 months. Eight patients received SCT either as first therapy or after responding to antileukemia therapy. These patients had a markedly superior survival, with 73% alive at a median follow-up of 31 months. JAK2V617F kinetics were assessed in 16 patients: 0 of 4 negative patients became positive at transformation, and among 12 positive patients, 1 had an increase in JAK2V617F% at transformation, 7 had a substantial decrease, and 4 had stable levels. Myeloproliferative neoplasm blast phase is associated with a dismal prognosis. Responses to chemotherapy can be achieved but are not durable. Long-term survivors had all received SCT either as first therapy or in first remission. PMID:18566326

Selected missense mutations impair frataxin processing in Friedreich ataxia.

PubMed

Clark, Elisia; Butler, Jill S; Isaacs, Charles J; Napierala, Marek; Lynch, David R

2017-08-01

Frataxin (FXN) is a highly conserved mitochondrial protein. Reduced FXN levels cause Friedreich ataxia, a recessive neurodegenerative disease. Typical patients carry GAA repeat expansions on both alleles, while a subgroup of patients carry a missense mutation on one allele and a GAA repeat expansion on the other. Here, we report that selected disease-related FXN missense mutations impair FXN localization, interaction with mitochondria processing peptidase, and processing. Immunocytochemical studies and subcellular fractionation were performed to study FXN import into the mitochondria and examine the mechanism by which mutations impair FXN processing. Coimmunoprecipitation was performed to study the interaction between FXN and mitochondrial processing peptidase. A proteasome inhibitor was used to model traditional therapeutic strategies. In addition, clinical profiles of subjects with and without point mutations were compared in a large natural history study. FXN I 154F and FXN G 130V missense mutations decrease FXN 81-210 levels compared with FXN WT , FXN R 165C , and FXN W 155R , but do not block its association with mitochondria. FXN I 154F and FXN G 130V also impair FXN maturation and enhance the binding between FXN 42-210 and mitochondria processing peptidase. Furthermore, blocking proteosomal degradation does not increase FXN 81-210 levels. Additionally, impaired FXN processing also occurs in fibroblasts from patients with FXN G 130V . Finally, clinical data from patients with FXN G 130V and FXN I 154F mutations demonstrates a lower severity compared with other individuals with Friedreich ataxia. These data suggest that the effects on processing associated with FXN G 130V and FXN I 154F mutations lead to higher levels of partially processed FXN, which may contribute to the milder clinical phenotypes in these patients.

Comprehensive screening of the USH2A gene in Usher syndrome type II and non-syndromic recessive retinitis pigmentosa.

PubMed

Seyedahmadi, Babak Jian; Rivolta, Carlo; Keene, Julia A; Berson, Eliot L; Dryja, Thaddeus P

2004-08-01

A screen of the entire coding region of the USH2A gene in 129 unrelated patients with Usher syndrome type II (USH2) and in 146 unrelated patients with non-syndromic autosomal recessive retinitis pigmentosa (ARRP) uncovered 54 different sequence variations, including 18 likely pathogenic mutations (13 frameshift, three nonsense, and two missense), 12 changes of uncertain pathogenicity (11 missense changes and one in-frame deletion), and 24 non-pathogenic rare variants or polymorphisms. Of the 18 likely pathogenic mutations, nine were novel. Among the USH2 patients, 50 (39%) had one or two likely pathogenic mutations. The most common mutant allele in USH2 patients was E767fs, which was found in 29 patients, including one homozygote. Among the ARRP patients, we found 17 (12%) with one or two likely pathogenic mutations. The most common mutant allele in ARRP patients was C759F and it was found in 10 patients. The C759F allele was also found in two USH2 patients; in neither of them was a change in the other allele found. The second most common mutant allele in both patient groups was L1447fs (found in 6/50 USH2 patients and 6/17 ARRP patients). Of the 50+17=67 patients with identified USH2A mutations, only one mutation in one allele was found in 41+12=53 (79%); the reason for the high proportion of patients with only one identified mutation is obscure. Our results indicate that USH2A mutations are found in about 7% of all cases of RP in North America, a frequency similar to the RPGR gene (8%) and the rhodopsin gene (10%).

[Prognostic value of JAK2, MPL and CALR mutations in Chinese patients with primary myelofibrosis].

PubMed

Xu, Z F; Li, B; Liu, J Q; Li, Y; Ai, X F; Zhang, P H; Qin, T J; Zhang, Y; Wang, J Y; Xu, J Q; Zhang, H L; Fang, L W; Pan, L J; Hu, N B; Qu, S Q; Xiao, Z J

2016-07-01

To evaluate the prognostic value of JAK2, MPL and CALR mutations in Chinese patients with primary myelofibrosis (PMF). Four hundred and two Chinese patients with PMF were retrospectively analyzed. The Kaplan-Meier method, the Log-rank test, the likelihood ratio test and the Cox proportional hazards regression model were used to evaluate the prognostic scoring system. This cohort of patients included 209 males and 193 females with a median age of 55 years (range: 15- 89). JAK2V617F mutations were detected in 189 subjects (47.0% ), MPLW515 mutations in 13 (3.2%) and CALR mutations in 81 (20.1%) [There were 30 (37.0%) type-1, 48 (59.3%) type-2 and 3 (3.7%) less common CALR mutations], respectively. 119 subjects (29.6%) had no detectable mutation in JAK2, MPL or CALR. Univariate analysis indicated that patients with CALR type-2 mutations or no detectable mutations had inferior survival compared to those with JAK2, MPL or CALR type- 1 or other less common CALR mutations (the median survival was 74vs 168 months, respectively [HR 2.990 (95% CI 1.935-4.619),P<0.001]. Therefore, patients were categorized into the high-risk with CALR type- 2 mutations or no detectable driver mutations and the low- risk without aforementioned mutations status. The DIPSS-Chinese molecular prognostic model was proposed by adopting mutation categories and DIPSS-Chinese risk group. The median survival of patients classified in low risk (132 subjects, 32.8% ), intermediate- 1 risk (143 subjects, 35.6%), intermediate- 2 risk (106 subjects, 26.4%) and high risk (21 subjects, 5.2%) were not reached, 156 (95% CI 117- 194), 60 (95% CI 28- 91) and 22 (95% CI 10- 33) months, respectively, and there was a statistically significant difference in overall survival among the four risk groups (P<0.001). There was significantly higher predictive power for survival according to the DIPSS-Chinese molecular prognostic model compared with the DIPSS-Chinese model (P=0.005, -2 log-likelihood ratios of 855.6 and 869.7, respectively). The impact of the CALR type- 2 mutations or no detectable driver mutation on survival was independent of current prognostic scoring systems. The DIPSS- Chinese molecular prognostic model based on the molecular features of Chinese patients was proposed and worked well for prognostic indication.

Clinical, histopathologic, and genetic features of pediatric primary myelofibrosis--an entity different from adults.

PubMed

DeLario, Melissa R; Sheehan, Andrea M; Ataya, Ramona; Bertuch, Alison A; Vega, Carlos; Webb, C Renee; Lopez-Terrada, Dolores; Venkateswaran, Lakshmi

2012-05-01

Primary myelofibrosis is a chronic myeloproliferative neoplasm characterized by cytopenias, leukoerythroblastosis, extramedullary hematopoiesis, hepatosplenomegaly and bone marrow fibrosis. Primary myelofibrosis is a rare disorder in adults; children are even less commonly affected by this entity, with the largest pediatric case series reporting on three patients. Most literature suggests spontaneous resolution of myelofibrosis without long term complications in the majority of affected children. We describe the clinical, pathologic, and molecular characteristics and outcomes of nineteen children with primary myelofibrosis treated in our center from 1984 to 2011. Most patients had cytopenia significant enough to require supportive therapy. No child developed malignant transformation and only five of the 19 children (26%) had spontaneous resolution of disease. Sequence analyses for JAK2V617F and MPLW515L mutations were performed on bone marrow samples from 17 and six patients, respectively, and the results were negative. In conclusion, analysis of this large series of pediatric patients with primary myelofibrosis demonstrates distinct clinical, hematologic, bone marrow, and molecular features from adult patients. Copyright © 2012 Wiley Periodicals, Inc.

The real-world outcomes of treating Polycythemia Vera: Physician adherence to treatment guidelines.

PubMed

Jackson Chornenki, Nicholas L; Chai-Adisaksopha, Chatree; Leong, Darryl P; Siegal, Deborah M; Hillis, Christopher M

2018-06-04

Therapy in Polycythemia Vera (PV), a myeloproliferative neoplasm, focuses on reducing cardiovascular (CV) risk without increasing bleeding or hematological progression. However, the real-world practice of treating PV in North America is understudied. We performed a retrospective cohort study of newly diagnosed PV (JAK2V617F mutation positive) patients in Hamilton, Canada to fill this knowledge gap. Out of 108 patients included, (n = 45, 41.7%) patients did not receive therapy consistent with contemporary treatment guidelines. Multivariable analysis showed increased white blood cell count at diagnosis (HR, 1.09; 95% CI, 1.04-1.14; p < 0.001), older age (HR, 1.15; 95% CI, 1.07-1.23; p < 0.001) and diabetic history (HR, 3.71; 95% CI, 1.27-10.78; p = 0.012) associated with greater mortality. Not receiving pharmacological treatment according to guidelines was also independently associated with increased mortality (HR, 3.12; 95% CI, 1.13-8.65; p = 0.029). Copyright © 2018 Elsevier Ltd. All rights reserved.

Management of polycythaemia vera: a critical review of current data.

PubMed

McMullin, Mary F; Wilkins, Bridget S; Harrison, Claire N

2016-02-01

Polycythaemia vera (PV) is a chronic blood cancer; its clinical features are dominated by myeloproliferation (erythrocytosis, often leucocytosis and/or thrombocytosis) and a tendency for thrombosis and transformation to myelofibrosis or acute myeloid leukaemia. In the past 10 years the pathophysiology of this condition has been defined as JAK/STAT pathway activation, almost always due to mutations in JAK2 exons 12 or 14 (JAK2 V617F). In the same time period our understanding of the optimal management of PV has expanded, most recently culminating in the approval of JAK inhibitors for the treatment of PV patients who are resistant or intolerant to therapy with hydroxycarbamide. It has also been demonstrated that life expectancy for many patients with PV is not normal, nor is their quality of life. We critically explore these findings and discuss their impact. In addition, we highlight persisting gaps in our current management strategy; for example, what is the optimal first line cytoreductive therapy and, indeed, which patients need cytoreductive drugs. © 2015 John Wiley & Sons Ltd.

Polycythemia Vera: An Appraisal of the Biology and Management 10 Years After the Discovery of JAK2 V617F

PubMed Central

Stein, Brady L.; Oh, Stephen T.; Berenzon, Dmitriy; Hobbs, Gabriela S.; Kremyanskaya, Marina; Rampal, Raajit K.; Abboud, Camille N.; Adler, Kenneth; Heaney, Mark L.; Jabbour, Elias J.; Komrokji, Rami S.; Moliterno, Alison R.; Ritchie, Ellen K.; Rice, Lawrence; Mascarenhas, John; Hoffman, Ronald

2015-01-01

Polycythemia vera (PV) is a chronic myeloproliferative neoplasm that is associated with a substantial symptom burden, thrombohemorrhagic complications, and impaired survival. A decade after the seminal discovery of an activating mutation in the tyrosine kinase JAK2 in nearly all patients with PV, new treatment options are finally beginning to emerge, necessitating a critical reappraisal of the underlying pathogenesis and therapeutic modalities available for PV. Herein, we comprehensively review clinical aspects of PV including diagnostic considerations, natural history, and risk factors for thrombosis. We summarize recent studies delineating the genetic basis of PV, including their implications for evolution to myelofibrosis and secondary acute myeloid leukemia. We assess the quality of evidence to support the use of currently available therapies, including aspirin, phlebotomy, hydroxyurea, and interferon. We analyze recent studies evaluating the safety and efficacy of JAK inhibitors, such as ruxolitinib, and evaluate their role in the context of other available therapies for PV. This review provides a framework for practicing hematologists and oncologists to make rational treatment decisions for patients with PV. PMID:26324368

Leber's hereditary optic neuropathy is associated with mitochondrial ND6 T14502C mutation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao, Fuxin; Zhejiang Provincial Key Laboratory of Medical Genetics, School of Life Sciences, Wenzhou Medical College, Wenzhou, Zhejiang 325003; Guan, Minqiang

2009-11-20

We report here the clinical, genetic, and molecular characterization of three Chinese families with Leber's hereditary optic neuropathy (LHON). There were variable severity and age of onset in visual impairment among these families. Strikingly, there were extremely low penetrances of visual impairment in these Chinese families. Sequence analysis of complete mitochondrial genomes in these pedigrees showed the homoplasmic T14502C (I58V) mutation, which localized at a highly conserved isoleucine at position 58 of ND6, and distinct sets of mtDNA polymorphisms belonging to haplogroups M10a, F1a1, and H2. The occurrence of T14502C mutation in these several genetically unrelated subjects affected by visualmore » impairment strongly indicates that this mutation is involved in the pathogenesis of visual impairment. Here, mtDNA variants I187T in the ND1, A122V in CO1, S99A in the A6, and V254I in CO3 exhibited an evolutionary conservation, indicating a potential modifying role in the development of visual impairment associated with T14502C mutation in those families. Furthermore, nuclear modifier gene(s) or environmental factor(s) may play a role in the phenotypic manifestation of the LHON-associated T14502C mutation in these Chinese families.« less

77 FR 51573 - Certain Automated Media Library Devices; Determination To Review in Part a Final Initial...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-08-24

... convincing evidence that the IBM 3570, 3575, 7331, 7336 and 3494 documents qualify as printed publications.... To the extent the IBM 3570, 7331, 7336 and 3494 documents qualify as printed publications under 35 U... Elecs., Inc., 553 U.S. 617 (2008); LG Elecs., Inc. v. Bizcom Elecs., Inc., 453 F.3d 1364 (Fed. Cir. 2006...

Essential thrombocythemia in young individuals: frequency and risk factors for vascular events and evolution to myelofibrosis in 126 patients.

PubMed

Alvarez-Larrán, A; Cervantes, F; Bellosillo, B; Giralt, M; Juliá, A; Hernández-Boluda, J C; Bosch, A; Hernández-Nieto, L; Clapés, V; Burgaleta, C; Salvador, C; Arellano-Rodrigo, E; Colomer, D; Besses, C

2007-06-01

The frequency of vascular events and evolution to myelofibrosis (MF) in young individuals with essential thrombocythemia (ET) is not well known. The incidence and predisposing factors to such complications was studied in 126 subjects diagnosed with ET at a median age of 31 years (range: 5-40). Overall survival and probability of survival free of thrombosis, bleeding and MF were analyzed by the Kaplan-Meier method and the presence of the Janus Kinase 2 (JAK2) V617F mutation correlated with the appearance of such complications. The JAK2 mutation (present in 43% of patients) was associated with higher hemoglobin (Hb) (P<0.001) and lower platelets at diagnosis. With a median follow-up of 10 years (range: 4-25), 31 thrombotic events were registered (incidence rate: 2.2 thromboses/100 patients/year). When compared with the general population, young ET patients showed a significant increase in stroke (odds ratio 50, 95% CI: 21.5-115) and venous thromboses (odds ratio 5.3, 95% CI: 3.9-10.6). Thrombosis-free survival was 84% at 10 years, with tobacco use being associated with higher risk of thrombosis. Actuarial freedom from evolution to MF was 97% at 10 years. In conclusion, young ET patients have thrombotic events, especially stroke and venous thrombosis, more frequently than generally considered, whereas they rarely transform to MF.

Myeloproliferative Neoplasms in Children.

PubMed

Hofmann, Inga

2015-09-01

Myeloproliferative neoplasms (MPN) are a group of clonal hematopoietic stem cell disorders characterized by aberrant proliferation of one or more myeloid lineages often with increased immature cells in the peripheral blood. The three classical BCR-ABL-negative MPNs are: 1) polycythemia vera (PV), 2) essential thrombocythemia (ET), and 3) primary myelofibrosis (PMF), which are typically disorders of older adults and are exceedingly rare in children. The diagnostic criteria for MPNs remain largely defined by clinical, laboratory and histopathology assessments in adults, but they have been applied to the pediatric population. The discovery of the JAK2 V617F mutation, and more recently, MPL and CALR mutations, are major landmarks in the understanding of MPNs. Nevertheless, they rarely occur in children, posing a significant diagnostic challenge given the lack of an objective, clonal marker. Therefore, in pediatric patients, the diagnosis must rely heavily on clinical and laboratory factors, and exclusion of secondary disorders to make an accurate diagnosis of MPN. This review focuses on the clinical presentation, diagnostic work up, differential diagnosis, treatment and prognosis of the classical BCR-ABL-negative MPNs (PV, ET and PMF) in children and highlights key differences to the adult diseases. Particular attention will be given to pediatric PMF, as it is the only disorder of this group that is observed in infants and young children, and in many ways appears to be a unique entity compared to adult PMF.

EXEL-8232, a small-molecule JAK2 inhibitor, effectively treats thrombocytosis and extramedullary hematopoiesis in a murine model of myeloproliferative neoplasm induced by MPLW515L.

PubMed

Wernig, G; Kharas, M G; Mullally, A; Leeman, D S; Okabe, R; George, T; Clary, D O; Gilliland, D G

2012-04-01

About 10% of patients with essential thrombocythemia (ET) or myelofibrosis (MF) that lack mutations in JAK2 harbor an activating mutation in the thrombopoietin receptor, MPLW515L. Distinct from the JAK2V617F retroviral transplant model, the MPLW515L model recapitulates many features of ET and MF, including severe fibrosis and thrombocytosis. We have tested EXEL-8232, an experimental potent JAK2 inhibitor, for efficacy in suppression of thrombocytosis in vivo and for its ability to attenuate MPLW515L myeloproliferative disease. EXEL-8232 was administered for 28 days q12 h by oral gavage at doses of 30 or 100 mg/kg, prospectively. Animals treated with EXEL-8232 at 100 mg/kg had normalized high platelet counts, eliminated extramedullary hematopoiesis in the spleen and eliminated bone marrow fibrosis, whereas the wild-type controls did not develop thrombocytopenia. Consistent with a clinical response in this model, we validated surrogate end points for response to treatment, including a reduction of endogenous colony growth and signaling inhibition in immature erythroid and myeloid primary cells both in vitro and upon treatment in vivo. We conclude that EXEL-8232 has efficacy in treatment of thrombocytosis in vivo in a murine model of ET and MF, and may be of therapeutic benefit for patients with MPL-mutant MPN.

Comprehensive review of JAK inhibitors in myeloproliferative neoplasms

PubMed Central

Sonbol, Mohamad Bassam; Firwana, Belal; Zarzour, Ahmad; Morad, Mohammad; Rana, Vishal

2013-01-01

Myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem-cell disorders, characterized phenotypically by the abnormal accumulation of mature-appearing myeloid cells. Polycythemia vera, essential thrombocythemia, primary myelofibrosis (also known as ‘BCR-ABL1-negative’ MPNs), and chronic myeloid leukemia (CML) are the primary types of MPNs. After the discovery of the BCR-ABL1 fusion protein in CML, several oncogenic tyrosine kinases have been identified in ‘BCR-ABL1-negative’ MPNs, most importantly, JAK2V617F mutation. The similarity in the clinical characteristics of the BCR-ABL1-negative MPN patients along with the prevalence of the Janus kinase mutation in this patient population provided a strong rationale for the development of a new class of pharmacologic inhibitors that target this pathway. The first of its class, ruxolitinib, has now been approved by the food and drug administration (FDA) for the management of patients with intermediate- to high-risk myelofibrosis. Ruxolitinib provides significant and sustained improvements in spleen related and constitutional symptoms secondary to the disease. Although noncurative, ruxolitinib represents a milestone in the treatment of myelofibrosis patients. Other types of JAK2 inhibitors are being tested in various clinical trials at this point and may provide better efficacy data and safety profile than its predecessor. In this article, we comprehensively reviewed and summarized the available preclinical and clinical trials pertaining to JAK inhibitors. PMID:23610611

The myeloproliferative neoplasms, unclassifiable: clinical and pathological considerations.

PubMed

Gianelli, Umberto; Cattaneo, Daniele; Bossi, Anna; Cortinovis, Ivan; Boiocchi, Leonardo; Liu, Yen-Chun; Augello, Claudia; Bonometti, Arturo; Fiori, Stefano; Orofino, Nicola; Guidotti, Francesca; Orazi, Attilio; Iurlo, Alessandra

2017-02-01

In this study, we investigate in detail the morphological, clinical and molecular features of 71 consecutive patients with a diagnosis of myeloproliferative neoplasms, unclassifiable. We performed a meticulous morphological analysis and found that most of the cases displayed a hypercellular bone marrow (70%) with normal erythropoiesis without left-shifting (59%), increased granulopoiesis with left-shifting (73%) and increased megakaryocytes with loose clustering (96%). Megakaryocytes displayed frequent giant forms with hyperlobulated or bulbous nuclei and/or other maturation defects. Interestingly, more than half of the cases displayed severe bone marrow fibrosis (59%). Median values of hemoglobin level and white blood cells count were all within the normal range; in contrast, median platelets count and lactate dehydrogenase were increased. Little less than half of the patients (44%) showed splenomegaly. JAK2V617F mutation was detected in 72% of all patients. Among the JAK2-negative cases, MPLW515L mutation was found in 17% and CALR mutations in 67% of the investigated cases, respectively. Finally, by multiple correspondence analysis of the morphological profiles, we found that all but four of the cases could be grouped in three morphological clusters with some features similar to those of the classic BCR-ABL1-negative myeloproliferative neoplasms. Analysis of the clinical parameters in these three clusters revealed discrepancies with the morphological profile in about 55% of the patients. In conclusion, we found that the category of myeloproliferative neoplasm, unclassifiable is heterogeneous but identification of different subgroups is possible and should be recommended for a better management of these patients.

Whole exome sequencing for the identification of a new mutation in TGFB2 involved in a familial case of non-syndromic aortic disease.

PubMed

Gago-Díaz, Marina; Blanco-Verea, Alejandro; Teixidó-Turà, Gisela; Valenzuela, Irene; Del Campo, Miguel; Borregan, Mar; Sobrino, Beatriz; Amigo, Jorge; García-Dorado, David; Evangelista, Artur; Carracedo, Angel; Brion, María

2014-11-01

Non-syndromic aortic disease (NSAD) is a frequently asymptomatic but potentially lethal disease characterised by familial cases of thoracic aortic aneurysms and dissections. This monogenic but genetically heterogeneous condition is primarily inherited as an autosomal dominant disorder with low penetrance and variable expression. Mutations in ACTA2, TGFBR1, TGFBR2, MYH11, SMAD3, MYLK, and FBN1 genes have been described but still, there are many unresolved familial cases. The whole exome of two distantly related and affected members of a Spanish family with multiple cases of NSAD was analysed through 5500 SOLiD(™) System for the identification of shared and putative pathogenic variants. A new mutation termed c.C1042T:p.R348C (NM_001135599.2) was identified in TGFB2, a gene located in an evolutionary highly conserved region (Chr1: 218,519,577-218,617,961) that has been recently connected to this disease. The analysis of other family members using capillary sequencing confirmed cosegregation of the mutation with the disease and its incomplete penetrance. The repeated implication of TGFB2 in the development of thoracic aortic aneurysms and dissections suggests that this gene should be considered during genetic diagnosis of this disease. An accurate diagnosis of affected individuals and additional family members at risk allows for a personalised and more efficient gene-based follow-up and treatment. Finally, the reiterative presence of common musculoskeletal and craniofacial additional features in patients with TGFB2 mutations suggests the existence of a new yet undefined connective tissue syndrome responsible for not only aortic dilation, but also for the other extracardiac alterations present in the affected patients. Copyright © 2014 Elsevier B.V. All rights reserved.

U.S. Civil Airmen Statistics: 1983.

DTIC Science & Technology

1984-05-31

PA 15222, TS 722-2721. Commercial Street, Boston, MA 0120.’ M1 W2-6071. Conmmercial 4124 -2.721. 617-223.407. PIel Bookstore, 720 North Miain Street... CDb 0 go r% 0 .0 .0 0 I 0 a .4 . 44 45 !.~~~ N 0 a4 0a 44 0 a4 a SO . .0 -4. A. adV 0 0 61WaI 0 V4 is S IL4 U j .01-6 ~~i A. a.4~ F A.. 16 4 0 * 1

Widespread Distribution of a Newly Found Point Mutation in Voltage-Gated Sodium Channel in Pyrethroid-Resistant Aedes aegypti Populations in Vietnam

PubMed Central

Kawada, Hitoshi; Higa, Yukiko; Komagata, Osamu; Kasai, Shinji; Tomita, Takashi; Thi Yen, Nguyen; Loan, Luu Lee; Sánchez, Rodrigo A. P.; Takagi, Masahiro

2009-01-01

Background Resistance of Aedes aegypti to photostable pyrethroid insecticides is a major problem for disease-vector control programs. Pyrethroids target the voltage-gated sodium channel on the insects' neurons. Single amino acid substitutions in this channel associated with pyrethroid resistance are one of the main factors that cause knockdown resistance in insects. Although kdr has been observed in several mosquito species, point mutations in the para gene have not been fully characterized in Ae. aegypti populations in Vietnam. The aim of this study was to determine the types and frequencies of mutations in the para gene in Ae. aegypti collected from used tires in Vietnam. Methods and Findings Several point mutations were examined that cause insensitivity of the voltage-gated sodium channel in the insect nervous system due to the replacement of the amino acids L1014F, the most commonly found point mutation in several mosquitoes; I1011M (or V) and V1016G (or I), which have been reported to be associated to knockdown resistance in Ae. aegypti located in segment 6, domain II; and a recently found amino acid replacement in F1269 in Ae. aegypti, located in segment 6, domain III. Among 756 larvae from 70 locations, no I1011M or I1011V nor L1014F mutations were found, and only two heterozygous V1016G mosquitoes were detected. However, F1269C mutations on domain III were distributed widely and with high frequency in 269 individuals among 757 larvae (53 collection sites among 70 locations surveyed). F1269C frequencies were low in the middle to north part of Vietnam but were high in the areas neighboring big cities and in the south of Vietnam, with the exception of the southern mountainous areas located at an elevation of 500–1000 m. Conclusions The overall percentage of homozygous F1269C seems to remain low (7.4%) in the present situation. However, extensive and uncontrolled frequent use of photostable pyrethroids might be a strong selection pressure for this mutation to cause serious problems in the control of dengue fever in Vietnam. PMID:19806205

[A rare case of myeloproliferative disease with t(8;13)(p11;q12) associated with eosinophilia and lymphadenopathy].

PubMed

Tsyba, N N; Turkina, A G; Chelysheva, E Yu; Nemchenko, I S; Kovrigina, A M; Obukhova, T N; Urnova, E S; Kuzmina, L A; Savchenko, V G

Myeloproliferative disease associated with FGFR1 rearrangement (8p11), which is included in the 2008 WHO Classification of Myeloid Neoplasms, is a rare and extremely aggressive abnormality. The paper describes a clinical case of a 39-year-old female patient who was detected to have leukocytosis (as high as 47.2·109/l), absolute eosinophilia (as high as 3.1·109/l), and enlarged peripheral lymph nodes during her visit to a doctor. The bone marrow (BM) showed the changes typically encountered in myeloproliferative disease with eosinophilia. The patient was found to have t(8;13)(p11;q12) translocation associated with the rearrangement of the FGFR1 gene located at the 8p11 locus. Molecular and cytogenetic examinations failed to reveal BCR-ABL chimeric transcript, Jak2 V617F mutation, and deletions and translocations involving PDGFRA (4q12) and PDGFRB (5q32-33). The similar changes in the karyotype were also found in the lymph node cells. The undertaken treatment with hydroxyurea and the tyrosine kinase inhibitor dasatinib turned out to be ineffective. The patient underwent allogeneic BM transplantation from a HLA-identical sibling. Graft rejection occurred 6 months later. Allogeneic BM transplantation from the same donor (100% donor chimerism; FGFR1/8р11 translocation was not detected), which was complicated by the development of chronic graft-versus-host reaction, was performed again in March 2015. The patient is being followed up and continues to receive immunosuppressive therapy.

Implications of fALS Mutations on Sod1 Function and Oligomerization in Cell Models.

PubMed

Brasil, Aline A; Magalhães, Rayne S S; De Carvalho, Mariana D C; Paiva, Isabel; Gerhardt, Ellen; Pereira, Marcos D; Outeiro, Tiago F; Eleutherio, Elis C A

2018-06-01

Among the familial forms of amyotrophic lateral sclerosis (fALS), 20% are associated with the Cu,Zn-superoxide dismutase (Sod1). fALS is characterized by the accumulation of aggregated proteins and the increase in oxidative stress markers. Here, we used the non-invasive bimolecular fluorescence complementation (BiFC) assay in human H4 cells to investigate the kinetics of aggregation and subcellular localization of Sod1 mutants. We also studied the effect of the different Sod1 mutants to respond against oxidative stress by following the levels of reactive oxygen species (ROS) after treatment with hydrogen peroxide. Our results showed that only 30% of cells transfected with A4VSod1 showed no inclusions while for the other Sod1 mutants tested (L38V, G93A and G93C), this percentage was at least 70%. In addition, we found that 10% of cells transfected with A4VSod1 displayed more than five inclusions per cell and that A4V and G93A Sod1 formed inclusions more rapidly than L38V and G93C Sod1. Expression of WTSod1 significantly decreased the intracellular oxidation levels in comparison with expression of fALS Sod1 mutants, suggesting the mutations induce a functional impairment. All fALS mutations impaired nuclear localization of Sod1, which is important for maintaining genomic stability. Consistently, expression of WTSod1, but not of fALS Sod1 mutants, reduced DNA damage, as measured by the comet assay. Altogether, our study sheds light into the effects of fALS Sod1 mutations on inclusion formation, dynamics, and localization as well as on antioxidant response, opening novel avenues for investigating the role of fALS Sod1 mutations in pathogenesis.

IDENTIFICATION OF NOVEL FIBROBLAST GROWTH FACTOR RECEPTOR 3 GENE MUTATIONS IN ACTINIC CHEILITIS

PubMed Central

Chou, Annie; Dekker, Nusi; Jordan, Richard C.K.

2009-01-01

Objective Activating mutations in the fibroblast growth factor receptor 3 (FGFR3) gene are responsible for several craniosynostosis and chondrodysplasia syndromes as well as some human cancers including bladder and cervical carcinoma. Despite a high frequency in some benign skin disorders, FGFR3 mutations have not been reported in cutaneous malignancies. Actinic cheilitis (AC) is a sun-induced premalignancy affecting the lower lip that frequently progresses to squamous cell carcinoma (SCC). The objective of this study was to determine if FGFR3 gene mutations are present in AC and SCC of the lip. Study Design DNA was extracted and purified from micro-dissected, formalin-fixed, paraffin-embedded tissue sections of 20 cases of AC and SCC arising in AC. Exons 7, 15, and 17 were PCR amplified and direct sequenced. Results Four novel somatic mutations in the FGFR3 gene were identified: exon 7 mutation 742C→T (amino acid change R248C), exon 15 mutations 1850A→G (D617G) and 1888G→A (V630M), and exon 17 mutation 2056G→A (E686K). Grade of dysplasia did not correlate with presence of mutations. Conclusion The frequency of FGFR3 receptor mutations suggests a functional role for the FGFR3 receptor in the development of epithelial disorders and perhaps a change may contribute to the pathogenesis of some AC and SCC. PMID:19327639

Irritable bowel syndrome patients have SCN5A channelopathies that lead to decreased NaV1.5 current and mechanosensitivity.

PubMed

Strege, Peter R; Mazzone, Amelia; Bernard, Cheryl E; Neshatian, Leila; Gibbons, Simon J; Saito, Yuri A; Tester, David J; Calvert, Melissa L; Mayer, Emeran A; Chang, Lin; Ackerman, Michael J; Beyder, Arthur; Farrugia, Gianrico

2018-04-01

The SCN5A-encoded voltage-gated mechanosensitive Na + channel Na V 1.5 is expressed in human gastrointestinal smooth muscle cells and interstitial cells of Cajal. Na V 1.5 contributes to smooth muscle electrical slow waves and mechanical sensitivity. In predominantly Caucasian irritable bowel syndrome (IBS) patient cohorts, 2-3% of patients have SCN5A missense mutations that alter Na V 1.5 function and may contribute to IBS pathophysiology. In this study we examined a racially and ethnically diverse cohort of IBS patients for SCN5A missense mutations, compared them with IBS-negative controls, and determined the resulting Na V 1.5 voltage-dependent and mechanosensitive properties. All SCN5A exons were sequenced from somatic DNA of 252 Rome III IBS patients with diverse ethnic and racial backgrounds. Missense mutations were introduced into wild-type SCN5A by site-directed mutagenesis and cotransfected with green fluorescent protein into HEK-293 cells. Na V 1.5 voltage-dependent and mechanosensitive functions were studied by whole cell electrophysiology with and without shear force. Five of 252 (2.0%) IBS patients had six rare SCN5A mutations that were absent in 377 IBS-negative controls. Six of six (100%) IBS-associated Na V 1.5 mutations had voltage-dependent gating abnormalities [current density reduction (R225W, R433C, R986Q, and F1293S) and altered voltage dependence (R225W, R433C, R986Q, G1037V, and F1293S)], and at least one kinetic parameter was altered in all mutations. Four of six (67%) IBS-associated SCN5A mutations (R225W, R433C, R986Q, and F1293S) resulted in altered Na V 1.5 mechanosensitivity. In this racially and ethnically diverse cohort of IBS patients, we show that 2% of IBS patients harbor SCN5A mutations that are absent in IBS-negative controls and result in Na V 1.5 channels with abnormal voltage-dependent and mechanosensitive function. NEW & NOTEWORTHY The voltage-gated Na + channel Na V 1.5 contributes to smooth muscle physiology and electrical slow waves. In a racially and ethnically mixed irritable bowel syndrome cohort, 2% had mutations in the Na V 1.5 gene SCN5A. These mutations were absent in irritable bowel syndrome-negative controls. Most mutant Na V 1.5 channels were loss of function in voltage dependence or mechanosensitivity.

ALK F1174V mutation confers sensitivity while ALK I1171 mutation confers resistance to alectinib. The importance of serial biopsy post progression.

PubMed

Ou, Sai-Hong; Milliken, Jeffrey C; Azada, Michele C; Miller, Vincent A; Ali, Siraj M; Klempner, Samuel J

2016-01-01

Many acquired resistant mutations to the anaplastic lymphoma kinase (ALK) gene have been identified during treatment of ALK-rearranged non-small cell lung cancer (NSCLC) patients with crizotinib, ceritinib, and alectinib. These various acquired resistant ALK mutations confer differential sensitivities to various ALK inhibitors and may provide guidance on how to sequence the use of many of the second generation ALK inhibitors. We described a patient who developed an acquired ALK F1174V resistant mutation on progression from crizotinib that responded to alectinib for 18 months but then developed an acquired ALK I1171S mutation to alectinib. Both tumor samples had essentially the same genomic profile by comprehensive genomic profiling otherwise. This is the first patient report that demonstrates ALK F1174V mutation is sensitive to alectinib and further confirms missense acquired ALK I1171 mutation is resistant to alectinib. Sequential tumor re-biopsy for comprehensive genomic profiling (CGP) is important to appreciate the selective pressure during treatment with various ALK inhibitors underpinning the evolution of the disease course of ALK+NSCLC patients while on treatment with the various ALK inhibitors. This approach will likely help inform the optimal sequencing strategy as more ALK inhibitors become available. This case report also validates the importance of developing structurally distinct ALK inhibitors for clinical use to overcome non-cross resistant ALK mutations. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Dimerization deficiency of enigmatic retinitis pigmentosa-linked rhodopsin mutants

NASA Astrophysics Data System (ADS)

Ploier, Birgit; Caro, Lydia N.; Morizumi, Takefumi; Pandey, Kalpana; Pearring, Jillian N.; Goren, Michael A.; Finnemann, Silvia C.; Graumann, Johannes; Arshavsky, Vadim Y.; Dittman, Jeremy S.; Ernst, Oliver P.; Menon, Anant K.

2016-10-01

Retinitis pigmentosa (RP) is a blinding disease often associated with mutations in rhodopsin, a light-sensing G protein-coupled receptor and phospholipid scramblase. Most RP-associated mutations affect rhodopsin's activity or transport to disc membranes. Intriguingly, some mutations produce apparently normal rhodopsins that nevertheless cause disease. Here we show that three such enigmatic mutations--F45L, V209M and F220C--yield fully functional visual pigments that bind the 11-cis retinal chromophore, activate the G protein transducin, traffic to the light-sensitive photoreceptor compartment and scramble phospholipids. However, tests of scramblase activity show that unlike wild-type rhodopsin that functionally reconstitutes into liposomes as dimers or multimers, F45L, V209M and F220C rhodopsins behave as monomers. This result was confirmed in pull-down experiments. Our data suggest that the photoreceptor pathology associated with expression of these enigmatic RP-associated pigments arises from their unexpected inability to dimerize via transmembrane helices 1 and 5.

Transjugular local thrombolysis with/without TIPS in patients with acute non-cirrhotic, non-malignant portal vein thrombosis.

PubMed

Klinger, Christoph; Riecken, Bettina; Schmidt, Arthur; De Gottardi, Andrea; Meier, Benjamin; Bosch, Jaime; Caca, Karel

2017-12-01

Therapeutic anticoagulation is the standard treatment in patients with acute non-cirrhotic portal vein thrombosis (PVT). In critically ill patients, anticoagulation only may not suffice to achive rapid and stable recanalization. This study evaluates efficacy and safety of transjugular interventional therapy in acute non-cirrhotic PVT. This retrospective study includes 17 consecutive patients with acute noncirrhotic, non-malignant PVT. Main indication for interventional therapy was imminent intestinal infarction (n=10). Treatment consisted of a combination of transjugular thrombectomy, local fibrinolysis and - depending on thrombus resolution - transjugular intrahepatic portosystemic shunt. Recanalization was successful in 94.1%. One- and two-year secondary PV patency rates were 88.2%. Major complications (n=3) resolved spontaneously in all but one patient (heparin induced thrombocytopenia type 2 with intestinal infarction). Symptoms improved in all patients. However, segmental bowel resection had to be performed in two (11.8%). During a median follow-up of 28.6 months, no patient experienced portal hypertensive complications. Presence of JAK2 V617F mutation predicted both short-term and long-term technical success. Transjugular recanalization is safe and effective in patients with acute non-cirrhotic, non-malignant PVT. It should be considered especially in patients with imminent bowel infarction and low likelihood of recanalization following therapeutic anticoagulation. Patients with JAK2 mutation ought to be followed meticulously. Copyright © 2017 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.

Navigating the evolving paradigms in the diagnosis and treatment of myeloproliferative disorders.

PubMed

Mesa, Ruben A

2007-01-01

The diagnosis and management of the BCR-ABL-negative myeloproliferative disorders (MPDs) of polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) are at an explosive crossroads of scientific investigation and evolving paradigms since the discovery of the tyrosine kinase-activating JAK2V617F mutation in 2005. Additional discovery of relevant molecular lesions (JAK2 exon 12 mutations and c-MplW515L/K) have only further enriched our understanding of MPD pathogenesis. The improved diagnostic certainty these molecular markers provide have resulted in the modification, and simplification, of the World Health Organization (WHO) diagnostic algorithms for MPDs. Despite these scientific advances, however, the initial management of MPDs continues to rely upon a risk-based strategy to minimize the risk of vascular events with control of erythrocytosis, targeted antiplatelet therapy, and risk-based myelosuppressive therapy. No current medical therapy has altered the natural trend of the MPDs to lead to overt severe myelofibrosis or acute leukemia. Investigations into targeted therapies for MPDs are proceeding at a brisk pace with agents aimed at immunomodulation, decreasing marrow stromal reaction to the aberrant clone, DNA hypomethylation, or the inhibition of tyrosine kinases. Specific inhibition of JAK2 itself appears promising by in vitro investigations, and clinical trials with multiple agents are planned to commence enrollment in 2007. The potential impact of JAK2 inhibitors on the manifestations of the MPDs is unclear, but is awaited with great interest.

Quantitative Sequencing for the Determination of Kdr-type Resistance Allele (V419L, L925I, I936F) Frequencies in Common Bed Bug (Hemiptera: Cimicidae) Populations Collected from Israel.

PubMed

Palenchar, Daniel J; Gellatly, Kyle J; Yoon, Kyong Sup; Mumcuoglu, Kosta Y; Shalom, Uri; Clark, J Marshall

2015-09-01

Human bed bug infestations have dramatically increased worldwide since the mid-1990s. A similar phenomenon was also observed in Israel since 2005, when infestations were reported from all over the country. Two single nucleotide polymorphisms (V419L and L925I) in the bed bug voltage-sensitive sodium channel confer kdr-type resistance to pyrethroids. Using quantitative sequencing (QS), the resistance allele frequencies of Israeli bed bug populations from across the country were determined. Genomic DNA was extracted from samples of 12 populations of bed bugs collected from Israel and DNA fragments containing the V419L or L925I and I936F mutations sites were PCR amplified. The PCR products were analyzed by QS and the nucleotide signal ratios calculated and used to predict the resistance allele frequencies of the unknown populations. Results of the genetic analysis show that resistant nucleotide signals are highly correlated to resistance allele frequencies for both mutations. Ten of the 12 tested populations had 100% of the L925I mutation and 0% of the V419L mutation. One population was heterogeneous for the L925I mutation and had 0% of the V419L mutation and another population was heterogeneous for the V419L mutation and had 100% of the L925I mutation. I936F occurred only at low levels. These results indicate that bed bugs in Israel are genetically resistant to pyrethroids. Thus, pyrethroids should only be used for bed bug management with caution using effective application and careful monitoring procedures. Additionally, new and novel-acting insecticides and nonchemical means of controlling bed bugs should be explored. © The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Detection of the KIT D816V mutation in peripheral blood of systemic mastocytosis: diagnostic implications.

PubMed

Jara-Acevedo, Maria; Teodosio, Cristina; Sanchez-Muñoz, Laura; Álvarez-Twose, Ivan; Mayado, Andrea; Caldas, Carolina; Matito, Almudena; Morgado, José M; Muñoz-González, Javier I; Escribano, Luis; Garcia-Montero, Andrés C; Orfao, Alberto

2015-08-01

Recent studies have found the KIT D816V mutation in peripheral blood of virtually all adult systemic mastocytosis patients once highly sensitive PCR techniques were used; thus, detection of the KIT D816V mutation in peripheral blood has been proposed to be included in the diagnostic work-up of systemic mastocytosis algorithms. However, the precise frequency of the mutation, the biological significance of peripheral blood-mutated cells and their potential association with involvement of bone marrow hematopoietic cells other than mast cells still remain to be investigated. Here, we determined the frequency of peripheral blood involvement by the KIT D816V mutation, as assessed by two highly sensitive PCR methods, and investigated its relationship with multilineage involvement of bone marrow hematopoiesis. Overall, our results confirmed the presence of the KIT D816V mutation in peripheral blood of most systemic mastocytosis cases (161/190; 85%)--with an increasing frequency from indolent systemic mastocytosis without skin lesions (29/44; 66%) to indolent systemic mastocytosis with skin involvement (124/135; 92%), and more aggressive disease subtypes (11/11; 100%)--as assessed by the allele-specific oligonucleotide-qPCR method, which was more sensitive (P<.0001) than the peptide nucleic acid-mediated PCR approach (84/190; 44%). Although the presence of the KIT mutation in peripheral blood, as assessed by the allele-specific oligonucleotide-qPCR technique, did not accurately predict for multilineage bone marrow involvement of hematopoiesis, the allele-specific oligonucleotide-qPCR allele burden and the peptide nucleic acid-mediated-PCR approach did. These results suggest that both methods provide clinically useful and complementary information through the identification and/or quantification of the KIT D816V mutation in peripheral blood of patients suspected of systemic mastocytosis.

Confirmation and revision on the orbital period change of the possible type Ia supernova progenitor V617 Sagittarii

NASA Astrophysics Data System (ADS)

Shi, Guang; Qian, Sheng-Bang; Fernández Lajús, Eduardo

2014-02-01

This work reports new photometric results of eclipsing cataclysmic variable V617 Sagittarii (V617 Sgr). We analyzed the orbital period change of V617 Sgr by employing three new (since 2010) CCD eclipse timings along with all the available data from the literature. It was found that the orbital period of V617 Sgr undergoes an obvious long-term increase, which confirms the result revealed by Steiner et al. (2006). The rate of orbital period increase was calculated to be {dot{P}} = +2.14(0.05) × 10-7 d yr-1. This suggests the lifetime of the secondary star will end in a timescale of 0.97 × 106 yr faster than that predicted previously. In particular, a cyclic variation with a period of 4.5 yr and an amplitude of 2.3 min may appear in the O - C diagram. Dominated by the wind-accretion mechanism, high mass transfer from the low mass secondary to the white dwarf is expected to continue in the V Sge-type star V617 Sgr during its long-term evolution. The mass transfer rate |skew4dot{M}_{ tr}| was estimated to be in the range of about 2.2 × 10-7 to 5.2 × 10-7 M⊙ yr-1. Accordingly, the already massive (≥ 1.2 M⊙) white dwarf primary will process stable nuclear burning, accrete a fraction of the mass from its companion to reach the standard Chandrasekhar mass limit (≃ 1.38 M⊙), and ultimately produce a type Ia supernova (SN Ia) within about 4-8 × 105 yr or earlier.

Antibody production of wild-type and enzyme V279F variants of PAF-AH as a risk factor for Cardiovascular disease

NASA Astrophysics Data System (ADS)

Ramadhani, Anggia N.; Puspitarini, Sapti; Sari, Anissa N.; Widodo

2017-11-01

Coronary artery disease (CAD) has emerged as a leading cause of death in Indonesia nowadays. WHO data in 2012 revealed that 37% of the Indonesian population died from this disease. CAD occurs because of endothelial dysfunction in the arteries. Lipoprotein-associated phospholipase A2 (Lp-PLA2), also known as platelet-activating factor acetylhydrolase (PAF-AH), is a phospholipase A2 enzyme, encoded by the PLA2G7 gene. This protein is predicted to be involved in inflammatory phospholipid metabolism so it can be used as a biomarker of CAD in the early phase. Thus, the purpose of this research is to discover the difference in antibody production between wild-type and mutant V279F. The PAF-AH enzyme was isolated from mice lymphocyte cells in order to develop this enzyme as a biomarker of cardiovascular disease. PAF-AH migrates at 55kDa according to SDS-PAGE analysis. Flow cytometry analysis showed that mutant PAF-AH (V279F) is more antigenic than wild-type PAF-AH. The missense mutation of V279F PAF-AH means this enzyme cannot catabolize the acetyl group at the sn-2 position of PAF.

Myeloproliferative Neoplasm Animal Models

PubMed Central

Mullally, Ann; Lane, Steven W.; Brumme, Kristina; Ebert, Benjamin L.

2012-01-01

Synopsis Myeloproliferative neoplasm (MPN) animal models accurately re-capitulate human disease in mice and have been an important tool for the study of MPN biology and therapy. Transplantation of BCR-ABL transduced bone marrow cells into irradiated syngeneic mice established the field of MPN animal modeling and the retroviral bone marrow transplantation (BMT) assay has been used extensively since. Genetically engineered MPN animal models have enabled detailed characterization of the effects of specific MPN associated genetic abnormalities on the hematopoietic stem and progenitor cell (HSPC) compartment and xenograft models have allowed the study of primary human MPN-propagating cells in vivo. All models have facilitated the pre-clinical development of MPN therapies. JAK2V617F, the most common molecular abnormality in BCR-ABL negative MPN, has been extensively studied using retroviral, transgenic, knock-in and xenograft models. MPN animal models have also been used to investigate additional genetic lesions found in human MPN and to evaluate the bone marrow microenvironment in these diseases. Finally, several genetic lesions, although not common, somatically mutated drivers of MPN in humans induce a MPN phenotype in mice. Future uses for MPN animal models will include modeling compound genetic lesions in MPN and studying myelofibrotic transformation. PMID:23009938

[Hungarian Philadelphia negative chronic myeloproliferative neoplasia registry. Evaluation of the Polycythemia vera patients].

PubMed

Dombi, Péter; Illés, Árpád; Demeter, Judit; Homor, Lajos; Simon, Zsófia; Kellner, Ádám; Karádi, Éva; Valasinyószki, Erika; Udvardy, Miklós; Egyed, Miklós

2017-06-01

Intruduction and aim: The Hungarian National Registry for Philadelphia chromosome negative myeloproliferative neoplasms has been developed. The aim of the recent study is to assess the clinical characteristics of Hungarian patients with polycythemia vera. Data of 351 JAK2 V617F and exon 12 mutation positive polycythemia vera patients were collected online from 15 haematology centres reporting epidemiologic, clinical characteristics, diagnostic tools, therapeutic interventions, thromboembolic complications, disease transformations. Vascular events prior to and after diagnosis were evaluated upon the Landolfi risk assessment scale. 116 thromboembolic events were reported in 106 PV patients prior to diagnosis and 152 occasions in 102 patients during follow-up. The frequency of major arterial events were significantly reduced (p<0.0001) and the minor venous events were significantly elevated (p<0.0001) after the diagnosis. Major hemorrhagic complications were found in 25 and transformation in 26 cases. Our registry allows to collect and evaluate the features of patients with polycythemia vera. The Landolfi risk stratification was proven to be useful. Based on evaluated data, accuracy of diagnostic criteria and compliance to risk-adapted therapeutic guidelines are needed. Orv Hetil. 2017; 158(23): 901-909.

Development of Improved Seals and Closures for Dry Diving Suits.

DTIC Science & Technology

1979-01-31

7 AA SIS 17 2 BATTELLE COL4 4 St LABS 0O 4 F/6 6/17DEVLOPMENT OF IMPROVED SEALS AND CLOSURES FOR DRY DIVAlM SUITS--ETC (U) ULS JAN 79 M61331-76-C...A I Columbus Laboratories Report DTI ELIT V~ omn mbIIap" pww =dt 0lu FINAL REPORT on DEVELOPMENT OF IMPROVED SEALS AND CLOSURES FOR DRY DIVING SUITS...6 Multiple Lip Wrist Seal ..................................... 6 Closures

Resolution of bone marrow fibrosis in a patient receiving JAK1/JAK2 inhibitor treatment with ruxolitinib.

PubMed

Wilkins, Bridget S; Radia, Deepti; Woodley, Claire; Farhi, Sarah El; Keohane, Clodagh; Harrison, Claire N

2013-12-01

Ruxolitinib, a JAK1/JAK2 inhibitor, is currently the only pharmacological agent approved for the treatment of myelofibrosis. Approval was based on findings from two phase 3 trials comparing ruxolitinib with placebo (COMFORT-I) and with best available therapy (COMFORT-II) for the treatment of primary or secondary myelofibrosis. In those pivotal trials, ruxolitinib rapidly improved splenomegaly, disease-related symptoms, and quality of life and prolonged survival compared with both placebo and conventional treatments. However, for reasons that are currently unclear, there were only modest histomorphological changes in the bone marrow, and only a subset of patients had significant reductions in JAK2 V617F clonal burden. Here we describe a patient with post-polycythemia vera myelofibrosis who received ruxolitinib at our institution (Guy's and St. Thomas' NHS Foundation Trust, London, United Kingdom) as part of the COMFORT-II study. While on treatment, the patient had dramatic improvements in splenomegaly and symptoms shortly after starting ruxolitinib. With longer treatment, the patient had marked reductions in JAK2 V617F allele burden, and fibrosis of the bone marrow resolved after approximately 3 years of ruxolitinib treatment. To our knowledge, this is the first detailed case report of resolution of fibrosis with a JAK1/JAK2 inhibitor. ClinicalTrials.gov Identifier: NCT00934544.

Autoxidation and Oxygen Binding Properties of Recombinant Hemoglobins with Substitutions at the αVal-62 or βVal-67 Position of the Distal Heme Pocket*

PubMed Central

Tam, Ming F.; Rice, Natalie W.; Maillett, David H.; Simplaceanu, Virgil; Ho, Nancy T.; Tam, Tsuey Chyi S.; Shen, Tong-Jian; Ho, Chien

2013-01-01

The E11 valine in the distal heme pocket of either the α- or β-subunit of human adult hemoglobin (Hb A) was replaced by leucine, isoleucine, or phenylalanine. Recombinant proteins were expressed in Escherichia coli and purified for structural and functional studies. 1H NMR spectra were obtained for the CO and deoxy forms of Hb A and the mutants. The mutations did not disturb the α1β2 interface in either form, whereas the H-bond between αHis-103 and βGln-131 in the α1β1 interfaces of the deoxy α-subunit mutants was weakened. Localized structural changes in the mutated heme pocket were detected for the CO form of recombinant Hb (rHb) (αV62F), rHb (βV67I), and rHb (βV67F) compared with Hb A. In the deoxy form the proximal histidyl residue in the β-subunit of rHb (βV67F) has been altered. Furthermore, the interactions between the porphyrin ring and heme pocket residues have been perturbed in rHb (αV62I), rHb (αV62F), and rHb (βV67F). Functionally, the oxygen binding affinity (P50), cooperativity (n50), and the alkaline Bohr Effect of the three α-subunit mutants and rHb (βV67L) are similar to those of Hb A. rHb (βV67I) and rHb (βV67F) exhibit low and high oxygen affinity, respectively. rHb (βV67F) has P50 values lower that those reported for rHb (αL29F), a B10 mutant studied previously in our laboratory (Wiltrout, M. E., Giovannelli, J. L., Simplaceanu, V., Lukin, J. A., Ho, N. T., and Ho, C. (2005) Biochemistry 44, 7207–7217). These E11 mutations do not slow down the autoxidation and azide-induced oxidation rates of the recombinant proteins. Results from this study provide new insights into the roles of E11 mutants in the structure-function relationship in hemoglobin. PMID:23867463

HIV type-1 genotypic resistance profiles in vertically infected patients from Argentina reveal an association between K103N+L100I and L74V mutations.

PubMed

Aulicino, Paula C; Rocco, Carlos A; Mecikovsky, Debora; Bologna, Rosa; Mangano, Andrea; Sen, Luisa

2010-01-01

Patterns and pathways of HIV type-1 (HIV-1) antiretroviral (ARV) drug resistance-associated mutations in clinical isolates are conditioned by ARV history and factors such as viral subtype and fitness. Our aim was to analyse the frequency and association of ARV drug resistance mutations in a group of long-term vertically infected patients from Argentina. Plasma samples from 71 patients (38 children and 33 adolescents) were collected for genotypic HIV-1 ARV resistance testing during the period between February 2006 and October 2008. Statistically significant pairwise associations between ARV resistance mutations in pol, as well as associations between mutations and drug exposure, were identified using Fisher's exact tests with Bonferroni and false discovery rate corrections. Phylogenetic analyses were performed for subtype assignment. In protease (PR), resistance-associated mutations M46I/L, I54M/L/V/A/S and V82A/F/T/S/M/I were associated with each other and with minor mutations at codons 10, 24 and 71. Mutations V82A/F/T/S/M/I were primarily selected by the administration of ritonavir (RTV) in an historical ARV regimen. In reverse transcriptase, thymidine analogue mutation (TAM)1 profile was more common than TAM2. The non-nucleoside K103N+L100I mutations were observed at high frequency (15.5%) and were significantly associated with the nucleoside mutation L74V in BF recombinants. Associations of mutations at PR sites reflect the frequent use of RTV at an early time in this group of patients and convergent resistance mechanisms driven by the high exposure to protease inhibitors, as well as local HIV-1 diversity. The results provide clinical evidence of a molecular interaction between K103N+L100I and L74V mutations at the reverse transcriptase gene in vivo, limiting the future use of second-generation non-nucleoside reverse transcriptase inhibitors such as etravirine.

Gradual Loss of ACTH Due to a Novel Mutation in LHX4: Comprehensive Mutation Screening in Japanese Patients with Congenital Hypopituitarism

PubMed Central

Takagi, Masaki; Ishii, Tomohiro; Inokuchi, Mikako; Amano, Naoko; Narumi, Satoshi; Asakura, Yumi; Muroya, Koji; Hasegawa, Yukihiro; Adachi, Masanori; Hasegawa, Tomonobu

2012-01-01

Mutations in transcription factors genes, which are well regulated spatially and temporally in the pituitary gland, result in congenital hypopituitarism (CH) in humans. The prevalence of CH attributable to transcription factor mutations appears to be rare and varies among populations. This study aimed to define the prevalence of CH in terms of nine CH-associated genes among Japanese patients. We enrolled 91 Japanese CH patients for DNA sequencing of POU1F1, PROP1, HESX1, LHX3, LHX4, SOX2, SOX3, OTX2, and GLI2. Additionally, gene copy numbers for POU1F1, PROP1, HESX1, LHX3, and LHX4 were examined by multiplex ligation-dependent probe amplification. The gene regulatory properties of mutant LHX4 proteins were characterized in vitro. We identified two novel heterozygous LHX4 mutations, namely c.249-1G>A, p.V75I, and one common POU1F1 mutation, p.R271W. The patient harboring the c.249-1G>A mutation exhibited isolated growth hormone deficiency at diagnosis and a gradual loss of ACTH, whereas the patient with the p.V75I mutation exhibited multiple pituitary hormone deficiency. In vitro experiments showed that both LHX4 mutations were associated with an impairment of the transactivation capacities of POU1F1 andαGSU, without any dominant-negative effects. The total mutation prevalence in Japanese CH patients was 3.3%. This study is the first to describe, a gradual loss of ACTH in a patient carrying an LHX4 mutation. Careful monitoring of hypothalamic–pituitary -adrenal function is recommended for CH patients with LHX4 mutations. PMID:23029363

The combinatorial PP1-binding consensus Motif (R/K)x( (0,1))V/IxFxx(R/K)x(R/K) is a new apoptotic signature.

PubMed

Godet, Angélique N; Guergnon, Julien; Maire, Virginie; Croset, Amélie; Garcia, Alphonse

2010-04-01

Previous studies established that PP1 is a target for Bcl-2 proteins and an important regulator of apoptosis. The two distinct functional PP1 consensus docking motifs, R/Kx((0,1))V/IxF and FxxR/KxR/K, involved in PP1 binding and cell death were previously characterized in the BH1 and BH3 domains of some Bcl-2 proteins. In this study, we demonstrate that DPT-AIF(1), a peptide containing the AIF(562-571) sequence located in a c-terminal domain of AIF, is a new PP1 interacting and cell penetrating molecule. We also showed that DPT-AIF(1) provoked apoptosis in several human cell lines. Furthermore, DPT-APAF(1) a bi-partite cell penetrating peptide containing APAF-1(122-131), a non penetrating sequence from APAF-1 protein, linked to our previously described DPT-sh1 peptide shuttle, is also a PP1-interacting death molecule. Both AIF(562-571) and APAF-1(122-131) sequences contain a common R/Kx((0,1))V/IxFxxR/KxR/K motif, shared by several proteins involved in control of cell survival pathways. This motif combines the two distinct PP1c consensus docking motifs initially identified in some Bcl-2 proteins. Interestingly DPT-AIF(2) and DPT-APAF(2) that carry a F to A mutation within this combinatorial motif, no longer exhibited any PP1c binding or apoptotic effects. Moreover the F to A mutation in DPT-AIF(2) also suppressed cell penetration. These results indicate that the combinatorial PP1c docking motif R/Kx((0,1))V/IxFxxR/KxR/K, deduced from AIF(562-571) and APAF-1(122-131) sequences, is a new PP1c-dependent Apoptotic Signature. This motif is also a new tool for drug design that could be used to characterize potential anti-tumour molecules.

Direct engagement of the PI3K pathway by mutant KIT dominates oncogenic signaling in gastrointestinal stromal tumor.

PubMed

Bosbach, Benedikt; Rossi, Ferdinand; Yozgat, Yasemin; Loo, Jennifer; Zhang, Jennifer Q; Berrozpe, Georgina; Warpinski, Katherine; Ehlers, Imke; Veach, Darren; Kwok, Andrew; Manova, Katia; Antonescu, Cristina R; DeMatteo, Ronald P; Besmer, Peter

2017-10-03

Gastrointestinal stromal tumors (GISTs) predominantly harbor activating mutations in the receptor tyrosine kinase KIT. To genetically dissect in vivo the requirement of different signal transduction pathways emanating from KIT for tumorigenesis, the oncogenic Kit V558Δ mutation was combined with point mutations abrogating specific phosphorylation sites on KIT. Compared with single-mutant Kit V558Δ/+ mice, double-mutant Kit V558Δ;Y567F/Y567F knock-in mice lacking the SRC family kinase-binding site on KIT (pY567) exhibited attenuated MAPK signaling and tumor growth. Surprisingly, abrogation of the PI3K-binding site (pY719) in Kit V558Δ;Y719F/Y719F mice prevented GIST development, although the interstitial cells of Cajal (ICC), the cells of origin of GIST, were normal. Pharmacologic inhibition of the PI3K pathway in tumor-bearing Kit V558Δ/+ mice with the dual PI3K/mTOR inhibitor voxtalisib, the pan-PI3K inhibitor pilaralisib, and the PI3K-alpha-restricted inhibitor alpelisib each diminished tumor proliferation. The addition of the MEK inhibitor PD-325901 or binimetinib further decreased downstream KIT signaling. Moreover, combining PI3K and MEK inhibition was effective against imatinib-resistant Kit V558Δ;T669I/+ tumors.

Direct engagement of the PI3K pathway by mutant KIT dominates oncogenic signaling in gastrointestinal stromal tumor

PubMed Central

Bosbach, Benedikt; Rossi, Ferdinand; Yozgat, Yasemin; Loo, Jennifer; Zhang, Jennifer Q.; Berrozpe, Georgina; Warpinski, Katherine; Ehlers, Imke; Kwok, Andrew; Manova, Katia; Antonescu, Cristina R.; DeMatteo, Ronald P.; Besmer, Peter

2017-01-01

Gastrointestinal stromal tumors (GISTs) predominantly harbor activating mutations in the receptor tyrosine kinase KIT. To genetically dissect in vivo the requirement of different signal transduction pathways emanating from KIT for tumorigenesis, the oncogenic KitV558Δ mutation was combined with point mutations abrogating specific phosphorylation sites on KIT. Compared with single-mutant KitV558Δ/+ mice, double-mutant KitV558Δ;Y567F/Y567F knock-in mice lacking the SRC family kinase-binding site on KIT (pY567) exhibited attenuated MAPK signaling and tumor growth. Surprisingly, abrogation of the PI3K-binding site (pY719) in KitV558Δ;Y719F/Y719F mice prevented GIST development, although the interstitial cells of Cajal (ICC), the cells of origin of GIST, were normal. Pharmacologic inhibition of the PI3K pathway in tumor-bearing KitV558Δ/+ mice with the dual PI3K/mTOR inhibitor voxtalisib, the pan-PI3K inhibitor pilaralisib, and the PI3K-alpha–restricted inhibitor alpelisib each diminished tumor proliferation. The addition of the MEK inhibitor PD-325901 or binimetinib further decreased downstream KIT signaling. Moreover, combining PI3K and MEK inhibition was effective against imatinib-resistant KitV558Δ;T669I/+ tumors. PMID:28923937

Detection of the V1016G mutation in the voltage-gated sodium channel gene of Aedes aegypti (Diptera: Culicidae) by allele-specific PCR assay, and its distribution and effect on deltamethrin resistance in Thailand.

PubMed

Stenhouse, Steven A; Plernsub, Suriya; Yanola, Jintana; Lumjuan, Nongkran; Dantrakool, Anchalee; Choochote, Wej; Somboon, Pradya

2013-08-30

Resistance to pyrethroid insecticides is widespread among populations of Aedes aegypti, the main vector for the dengue virus. Several different point mutations within the voltage-gated sodium channel (VGSC) gene contribute to such resistance. A mutation at position 1016 in domain II, segment 6 of the VGSC gene in Ae. aegypti leads to a valine to glycine substitution (V1016G) that confers resistance to deltamethrin. This study developed and utilized an allele-specific PCR (AS-PCR) assay that could be used to detect the V1016G mutation. The assay was validated against a number of sequenced DNA samples of known genotype and was determined to be in complete agreement. Larvae and pupae were collected from various localities throughout Thailand. Samples were reared to adulthood and their resistance status against deltamethrin was determined by standard WHO susceptibility bioassays. Deltamethrin-resistant and susceptible insects were then genotyped for the V1016G mutation. Additionally, some samples were genotyped for a second mutation at position 1534 in domain III (F1534C) which is also known to confer pyrethroid resistance. The bioassay results revealed an overall mortality of 77.6%. Homozygous 1016G individuals survived at higher rates than either heterozygous or wild-type (1016 V) mosquitoes. The 1016G mutation was significantly and positively associated with deltamethrin resistance and was widely distributed throughout Thailand. Interestingly, wild-type 1016 V mosquitoes tested were homozygous for the 1534C mutation, and all heterozygous mosquitoes were also heterozygous for 1534C. Mutant homozygous (G/G) mosquitoes expressed the wild-type (F/F) at position 1534. However, the presence of the 1534C mutation was not associated with deltamethrin resistance. Our bioassay results indicate that all populations sampled display some degree of resistance to deltamethrin. Homozygous 1016G mosquitoes were far likelier to survive such exposure. However, resistance in some populations cannot be explained due to kdr mutations and indicates that other resistance mechanisms are operating. The presence of this mutation alone does not fully explain the resistance phenotype we see among Thai Ae. aegypti populations.

DNA Methylation as an Epigenetic Factor in the Development and Progression of Polycythemia Vera

DTIC Science & Technology

2009-06-01

Table 2). ID Diagnosis at analysis Original Dx Transformation JAK2 V617F MPD01 PV PV 41% MPD02 PV PV 48% MPD03 PV PV 86% MPD04 MF PV 47...Pathol Biol ( Paris ). 2001;49:164-166. 2. Spivak JL. Diagnosis of the myeloproliferative disorders: resolving phenotypic mimicry. Semin Hematol...Less than 10% of a single isolated BFU-E colony, originating from a single progenitor, is sufficient for determination of allele frequency. The

Epilepsy in hemiplegic migraine: Genetic mutations and clinical implications.

PubMed

Prontera, P; Sarchielli, P; Caproni, S; Bedetti, C; Cupini, L M; Calabresi, P; Costa, C

2018-02-01

Objective We performed a systematic review on the comorbidities of familial/sporadic hemiplegic migraine (F/SHM) with seizure/epilepsy in patients with CACNA1A, ATP1A2 or SCN1A mutations, to identify the genotypes associated and investigate for the presence of mutational hot spots. Methods We performed a search in MEDLINE and in the Human Gene Mutation and Leiden Open Variation Databases for mutations in the CACNA1A, ATP1A2 and SCN1A genes. After having examined the clinical characteristics of the patients, we selected those having HM and seizures, febrile seizures or epilepsy. For each gene, we determined both the frequency and the positions at protein levels of these mutations, as well as the penetrance of epilepsy within families. Results Concerning F/SHM-Epilepsy1 (F/SHME1) and F/SHME2 endophenotypes, we observed a prevalent involvement of the transmembrane domains, and a strong correlation in F/SHME1 when the positively charged amino acids were involved. The penetrance of epilepsy within the families was highest for patients carrying mutation in the CACNA1A gene (60%), and lower in those having SCN1A (33.3%) and ATP1A2 (30.9%) mutations. Conclusion Among the HM cases with seizure/epilepsy, we observed mutational hot spots in the transmembrane domains of CACNA1A and ATP1A2 proteins. These findings could lead to a better understanding of the pathological mechanisms underlying migraine and epilepsy, therein guaranteeing the most appropriate therapeutic approach.

Familial Mediterranean fever with a single MEFV mutation: comparison of rare and common mutations in a Turkish paediatric cohort.

PubMed

Soylemezoglu, Oguz; Kandur, Yasar; Duzova, Ali; Ozkaya, Ozan; Kasapcopur, Ozgür; Baskin, Esra; Fidan, Kibriya; Yalcinkaya, Fatos

2015-01-01

Presence of common MEFV gene mutations strengthened the diagnosis of FMF in addition to the typical clinical characteristics of FMF. However, there are also rare mutations. P369S, A744S, R761H, K695R, F479L are the main rare mutations in Turkish population. We aimed to evaluate FMF patients with a single allele MEFV mutation and to compare patients with common and rare mutations. We retrospectively reviewed the medical records of FMF patients with a single allele mutation who were followed up between 2008 and 2013 in six centres. We compared the patients with rare and common mutations for disease severity score, frequent exacerbations ( >1 attack per month), long attack period (>3 day), symptoms, age at the onset of symptoms, gender, consanguinity, and family history. Two hundred and seventeen patients (M/F=101/116) with the diagnosis of FMF and single mutation were included. Heterozygote mutations were defined as common (M694V, V726A, M68OI) and rare mutations (A744S, P369S, K695R, R761H, F479L). Sixty-seven patients (27 males, 40 females) had one single rare mutation and 150 (74 males, 76 females) had one single common mutation. No difference was found between the rare and common mutations with respect to the disease severity score. There was no significant difference between common and rare heterozygote form of mutations in terms of disease severity. Patients with typical characteristics of FMF, with some rare mutations (A744S, P369S) should be treated in the same manner as patients with a common mutation.

Critical Problems in Very Large Scale Computer Systems

DTIC Science & Technology

1990-09-30

Massachusetts Institute of Technology Cambridge, Massachusetts 02139 Anant Agarwal (617) 253-1448 William J. Dally (617) 253-6043 Srinivas Devadas ...Contrac t No. N00014-87-K-0825. Srinivs Devada (67 5305 F.pproved f r Public rgt (1 236 Ditribution Unlimited (7 5 8 Contents 1 Research Overview 2...d/or Dist Special I 1 Research Overview • The research vehicle for this contract i/the largest possible computerthat ’ can b conceived for the mid to

A Single Crossing-Over Event in Voltage-Sensitive Na+ Channel Genes May Cause Critical Failure of Dengue Mosquito Control by Insecticides

PubMed Central

Hirata, Koichi; Komagata, Osamu; Itokawa, Kentaro; Yamamoto, Atsushi; Tomita, Takashi; Kasai, Shinji

2014-01-01

The voltage-sensitive sodium (Na+) channel (Vssc) is the target site of pyrethroid insecticides. Pest insects develop resistance to this class of insecticide by acquisition of one or multiple amino acid substitution(s) in this channel. In Southeast Asia, two major Vssc types confer pyrethroid resistance in the dengue mosquito vector Aedes aegypti, namely, S989P+V1016G and F1534C. We expressed several types of Vssc in Xenopus oocytes and examined the effect of amino acid substitutions in Vssc on pyrethroid susceptibilities. S989P+V1016G and F1534C haplotypes reduced the channel sensitivity to permethrin by 100- and 25-fold, respectively, while S989P+V1016G+F1534C triple mutations reduced the channel sensitivity to permethrin by 1100-fold. S989P+V1016G and F1534C haplotypes reduced the channel sensitivity to deltamethrin by 10- and 1-fold (no reduction), respectively, but S989P+V1016G+F1534C triple mutations reduced the channel sensitivity to deltamethrin by 90-fold. These results imply that pyrethroid insecticides are highly likely to lose their effectiveness against A. aegypti if such a Vssc haplotype emerges as the result of a single crossing-over event; thus, this may cause failure to control this key mosquito vector. Here, we strongly emphasize the importance of monitoring the occurrence of triple mutations in Vssc in the field population of A. aegypti. PMID:25166902

Mutations of the central tyrosines of putative cholesterol recognition amino acid consensus (CRAC) sequences modify folding, activity, and sterol-sensing of the human ABCG2 multidrug transporter.

PubMed

Gál, Zita; Hegedüs, Csilla; Szakács, Gergely; Váradi, András; Sarkadi, Balázs; Özvegy-Laczka, Csilla

2015-02-01

Human ABCG2 is a plasma membrane glycoprotein causing multidrug resistance in cancer. Membrane cholesterol and bile acids are efficient regulators of ABCG2 function, while the molecular nature of the sterol-sensing sites has not been elucidated. The cholesterol recognition amino acid consensus (CRAC, L/V-(X)(1-5)-Y-(X)(1-5)-R/K) sequence is one of the conserved motifs involved in cholesterol binding in several proteins. We have identified five potential CRAC motifs in the transmembrane domain of the human ABCG2 protein. In order to define their roles in sterol-sensing, the central tyrosines of these CRACs (Y413, 459, 469, 570 and 645) were mutated to S or F and the mutants were expressed both in insect and mammalian cells. We found that mutation in Y459 prevented protein expression; the Y469S and Y645S mutants lost their activity; while the Y570S, Y469F, and Y645F mutants retained function as well as cholesterol and bile acid sensitivity. We found that in the case of the Y413S mutant, drug transport was efficient, while modulation of the ATPase activity by cholesterol and bile acids was significantly altered. We suggest that the Y413 residue within a putative CRAC motif has a role in sterol-sensing and the ATPase/drug transport coupling in the ABCG2 multidrug transporter. Copyright © 2014. Published by Elsevier B.V.

Safety and efficacy of ruxolitinib in splanchnic vein thrombosis associated with myeloproliferative neoplasms.

PubMed

Pieri, Lisa; Paoli, Chiara; Arena, Umberto; Marra, Fabio; Mori, Fabio; Zucchini, Mery; Colagrande, Stefano; Castellani, Alessandro; Masciulli, Arianna; Rosti, Vittorio; De Stefano, Valerio; Betti, Silvia; Finazzi, Guido; Ferrari, Maria Luisa; Rumi, Elisa; Ruggeri, Marco; Nichele, Ilaria; Guglielmelli, Paola; Fjerza, Rajmonda; Mannarelli, Carmela; Fanelli, Tiziana; Merli, Lucia; Corbizi Fattori, Giuditta; Massa, Margherita; Cimino, Giuseppe; Rambaldi, Alessandro; Barosi, Giovanni; Cazzola, Mario; Barbui, Tiziano; Vannucchi, Alessandro M

2017-02-01

Splanchnic vein thrombosis (SVT) is one of the vascular complications of myeloproliferative neoplasms (MPN). We designed a phase 2 clinical trial to evaluate safety and efficacy of ruxolitinib in reducing splenomegaly and improving disease-related symptoms in patients with MPN-associated SVT. Patients diagnosed with myelofibrosis (12 cases), polycythemia vera (5 cases) and essential thrombocythemia (4 cases) received ruxolitinib for 24 weeks in the core study period. Spleen volume was assessed by magnetic resonance imaging (MRI) and splanchnic vein circulation by echo-Doppler analysis. Nineteen patients carried JAK2V617F, one had MPLW515L, and one CALRL367fs*46 mutation. Eighteen patients had spleno-portal-mesenteric thrombosis, two had Budd-Chiari syndrome, and one had both sites involved; 16 patients had esophageal varices. Ruxolitinib was well tolerated with hematological toxicities consistent with those of patients without SVT and no hemorrhagic adverse events were recorded. After 24 weeks of treatment, spleen volume reduction ≥35% by MRI was achieved by 6/21 (29%) patients, and a ≥50% spleen length reduction by palpation at any time up to week 24 was obtained by 13/21 (62%) patients. At week 72, 8 of the 13 (62%) patients maintained the spleen response by palpation. No significant effect of treatment on esophageal varices or in splanchnic circulation was observed. MPN-related symptoms, evaluated by MPN-symptom assessment form (SAF) TSS questionnaire, improved significantly during the first 4 weeks and remained stable up to week 24. In conclusion, this trial shows that ruxolitinib is safe in patients with MPN-associated SVT, and effective in reducing spleen size and disease-related symptoms. © 2016 Wiley Periodicals, Inc.

Low-concentration methylene blue maintains energy production and strongly improves survival of Leigh syndrome French Canadian skin fibroblasts.

PubMed

Legault, Jean; Larouche, Pierre-Luc; Côté, Isabelle; Bouchard, Line; Pichette, André; Robinson, Brian H; Morin, Charles

2011-01-01

Leigh syndrome French Canadian (LSFC) is a recessive disease caused by mutations in the LRPPRC gene (leucine-rich pentatricopeptide repeat containing protein). These mutations induce a cytochrome c oxidase (COX) deficiency resulting in episodes of acute acidotic crisis that will often lead to death. There is no effective treatment. Methylene blue (MB) is a redox dye that increases COX content and activity in vitro and in vivo suggesting that MB could prevent and treat LSFC. In this study, the protective effect of low-concentration MB was tested on two LSFC cell lines, including LSFC-F1, homozygous for the mutation A354V, and LSFC-F2 a compound heterozygous for the mutations A354V and C12775STOP. MB effect on metabolic activity was assessed on both LSFC cells in stable and acidotic conditions. For LSFC-F1, results showed that metabolic activity drastically decline after 96 hours in both conditions but not LSFC-F2 and normal cells. MB completely prevents the decrease of metabolic activity in LSFC-F1. Intracellular ATP content was also measured in both culture media. After 96 hours in acidotic medium, ATP content was almost completely depleted for both LSFC cells. Interestingly, MB completely restores ATP content in LSFC-F1 and LSFC-F2 cells. Finally, MB strongly improves the survival of both LSFC cells.

Two novel sodium channel mutations associated with resistance to indoxacarb and metaflumizone in the diamondback moth, Plutella xylostella.

PubMed

Wang, Xing-Liang; Su, Wen; Zhang, Jian-Heng; Yang, Yi-Hua; Dong, Ke; Wu, Yi-Dong

2016-02-01

Indoxacarb and metaflumizone belong to a relatively new class of sodium channel blocker insecticides (SCBIs). Due to intensive use of indoxacarb, field-evolved indoxacarb resistance has been reported in several lepidopteran pests, including the diamondback moth Plutella xylostella, a serious pest of cruciferous crops. In particular, the BY12 population of P. xylostella, collected from Baiyun, Guangdong Province of China in 2012, was 750-fold more resistant to indoxacarb and 70-fold more resistant to metaflumizone compared with the susceptible Roth strain. Comparison of complementary DNA sequences encoding the sodium channel genes of Roth and BY12 revealed two point mutations (F1845Y and V1848I) in the sixth segment of domain IV of the PxNav protein in the BY population. Both mutations are located within a highly conserved sequence region that is predicted to be involved in the binding sites of local anesthetics and SCBIs based on mammalian sodium channels. A significant correlation was observed among 10 field-collected populations between the mutant allele (Y1845 or I1848) frequencies (1.7% to 52.5%) and resistance levels to both indoxacarb (34- to 870-fold) and metaflumizone (1- to 70-fold). The two mutations were never found to co-exist in the same allele of PxNav , suggesting that they arose independently. This is the first time that sodium channel mutations have been associated with high levels of resistance to SCBIs. F1845Y and V1848I are molecular markers for resistance monitoring in the diamondback moth and possibly other insect pest species. © 2015 Institute of Zoology, Chinese Academy of Sciences.

BRAF Mutation Correlates With High-Risk Langerhans Cell Histiocytosis and Increased Resistance to First-Line Therapy.

PubMed

Héritier, Sébastien; Emile, Jean-François; Barkaoui, Mohamed-Aziz; Thomas, Caroline; Fraitag, Sylvie; Boudjemaa, Sabah; Renaud, Florence; Moreau, Anne; Peuchmaur, Michel; Chassagne-Clément, Catherine; Dijoud, Frédérique; Rigau, Valérie; Moshous, Despina; Lambilliotte, Anne; Mazingue, Françoise; Kebaili, Kamila; Miron, Jean; Jeziorski, Eric; Plat, Geneviève; Aladjidi, Nathalie; Ferster, Alina; Pacquement, Hélène; Galambrun, Claire; Brugières, Laurence; Leverger, Guy; Mansuy, Ludovic; Paillard, Catherine; Deville, Anne; Armari-Alla, Corinne; Lutun, Anne; Gillibert-Yvert, Marion; Stephan, Jean-Louis; Cohen-Aubart, Fleur; Haroche, Julien; Pellier, Isabelle; Millot, Frédéric; Lescoeur, Brigitte; Gandemer, Virginie; Bodemer, Christine; Lacave, Roger; Hélias-Rodzewicz, Zofia; Taly, Valérie; Geissmann, Frédéric; Donadieu, Jean

2016-09-01

Langerhans cell histiocytosis (LCH) is an inflammatory myeloid neoplasia with a broad spectrum of clinical manifestations and outcomes in children. The somatic BRAF(V600E) mutation occurs frequently, but clinical significance remains to be determined. BRAF(V600E) mutation was investigated in a French LCH cohort. We analyzed associations between mutation status and clinical presentation, extent of disease, reactivation rate, response to therapy, and long-term permanent sequelae. Among 315 patients with successfully determined BRAF status, 173 (54.6%) carried a BRAF(V600E) mutation. Patients with BRAF(V600E) manifested more severe disease than did those with wild-type BRAF. Patients with BRAF(V600E) comprised 87.8% of patients (43 of 49) with multisystem LCH with risk organ involvement (liver, spleen, hematology), 68.6% of patients (35 of 51) with multisystem LCH without risk organ involvement, 43.9% of patients (86 of 196) with single-system LCH, and 42.1% of patients (8 of 19) with lung-involved LCH (P < .001). BRAF(V600E) mutation was also associated with organ involvement that could lead to permanent, irreversible damage, such as neurologic (75%) and pituitary (72.9%) injuries. Compared with patients with wild-type BRAF, patients with BRAF(V600E) more commonly displayed resistance to combined vinblastine and corticosteroid therapy (21.9% v 3.3%; P = .001), showed a higher reactivation rate (5-year reactivation rate, 42.8% v 28.1%; P = .006), and had more permanent, long-term consequences from disease or treatment (27.9% v 12.6%; P = .001). In children with LCH, BRAF(V600E) mutation was associated with high-risk features, permanent injury, and poor short-term response to chemotherapy. Further population-based studies should be undertaken to confirm our observations and to assess the impact of BRAF inhibitors for this subgroup of patients who may benefit from targeted therapy. © 2016 by American Society of Clinical Oncology.

In silico identification of genetic variants in glucocerebrosidase (GBA) gene involved in Gaucher's disease using multiple software tools.

PubMed

Manickam, Madhumathi; Ravanan, Palaniyandi; Singh, Pratibha; Talwar, Priti

2014-01-01

Gaucher's disease (GD) is an autosomal recessive disorder caused by the deficiency of glucocerebrosidase, a lysosomal enzyme that catalyses the hydrolysis of the glycolipid glucocerebroside to ceramide and glucose. Polymorphisms in GBA gene have been associated with the development of Gaucher disease. We hypothesize that prediction of SNPs using multiple state of the art software tools will help in increasing the confidence in identification of SNPs involved in GD. Enzyme replacement therapy is the only option for GD. Our goal is to use several state of art SNP algorithms to predict/address harmful SNPs using comparative studies. In this study seven different algorithms (SIFT, MutPred, nsSNP Analyzer, PANTHER, PMUT, PROVEAN, and SNPs&GO) were used to predict the harmful polymorphisms. Among the seven programs, SIFT found 47 nsSNPs as deleterious, MutPred found 46 nsSNPs as harmful. nsSNP Analyzer program found 43 out of 47 nsSNPs are disease causing SNPs whereas PANTHER found 32 out of 47 as highly deleterious, 22 out of 47 are classified as pathological mutations by PMUT, 44 out of 47 were predicted to be deleterious by PROVEAN server, all 47 shows the disease related mutations by SNPs&GO. Twenty two nsSNPs were commonly predicted by all the seven different algorithms. The common 22 targeted mutations are F251L, C342G, W312C, P415R, R463C, D127V, A309V, G46E, G202E, P391L, Y363C, Y205C, W378C, I402T, S366R, F397S, Y418C, P401L, G195E, W184R, R48W, and T43R.

Detection of knockdown resistance mutations in the common bed bug, Cimex lectularius (Hemiptera: Cimicidae), in Australia.

PubMed

Dang, Kai; Toi, Cheryl S; Lilly, David G; Bu, Wenjun; Doggett, Stephen L

2015-07-01

Pyrethroid resistance in the common bed bug, Cimex lectularius L., has been reported worldwide. An important resistance mechanism is via knockdown resistance (kdr) mutations, notably V419L and L925I. Information regarding this kdr-type resistance mechanism is unknown in Australia. This study aims to examine the status of kdr mutations in Australian C. lectularius strains. Several modern field-collected strains and museum-preserved reference collections of Australian C. lectularius were examined. Of the field strains (2007-2013), 96% had the known kdr mutations (L925I or both V419L/L925I). The 'Adelaide' strain (2013) and samples from the preserved reference collections (1994-2002) revealed no known kdr mutations. A novel mutation I936F was apparent in the insecticide-resistant 'Adelaide' strain, one strain from Perth (with L925I) and the majority of the reference collection specimens. The laboratory insecticide-resistant 'Sydney' strain showed a mixture of no kdr mutations (20%) and L925I (80%). The novel mutation I936F may be a kdr mutation but appeared to contribute less resistance to the pyrethroids than the V419L and L925I mutations. The detection of high frequencies of kdr mutations indicates that kdr-type resistance is widespread across Australia. Hence, there should be a reduced reliance on pyrethroid insecticides and an integrated management approach for the control of C. lectularius infestations. © 2014 Society of Chemical Industry.

Dimerization deficiency of enigmatic retinitis pigmentosa-linked rhodopsin mutants

PubMed Central

Ploier, Birgit; Caro, Lydia N.; Morizumi, Takefumi; Pandey, Kalpana; Pearring, Jillian N.; Goren, Michael A.; Finnemann, Silvia C.; Graumann, Johannes; Arshavsky, Vadim Y.; Dittman, Jeremy S.; Ernst, Oliver P.; Menon, Anant K.

2016-01-01

Retinitis pigmentosa (RP) is a blinding disease often associated with mutations in rhodopsin, a light-sensing G protein-coupled receptor and phospholipid scramblase. Most RP-associated mutations affect rhodopsin's activity or transport to disc membranes. Intriguingly, some mutations produce apparently normal rhodopsins that nevertheless cause disease. Here we show that three such enigmatic mutations—F45L, V209M and F220C—yield fully functional visual pigments that bind the 11-cis retinal chromophore, activate the G protein transducin, traffic to the light-sensitive photoreceptor compartment and scramble phospholipids. However, tests of scramblase activity show that unlike wild-type rhodopsin that functionally reconstitutes into liposomes as dimers or multimers, F45L, V209M and F220C rhodopsins behave as monomers. This result was confirmed in pull-down experiments. Our data suggest that the photoreceptor pathology associated with expression of these enigmatic RP-associated pigments arises from their unexpected inability to dimerize via transmembrane helices 1 and 5. PMID:27694816

Resistance profile of darunavir: combined 24-week results from the POWER trials.

PubMed

de Meyer, Sandra; Vangeneugden, Tony; van Baelen, Ben; de Paepe, Els; van Marck, Herwig; Picchio, Gaston; Lefebvre, Eric; de Béthune, Marie-Pierre

2008-03-01

The resistance profile of darunavir (TMC114) in treatment-experienced patients was explored using pooled week 24 data from POWER 1, 2, and 3 at the recommended dose of darunavir with low-dose ritonavir (darunavir/r, 600/100 mg bid, N = 458). Baseline darunavir fold change in EC(50) was a strong predictor of virological response at week 24. Preliminary phenotypic clinical cut-offs of 10 and 40 were established. Virological response to darunavir/r was maintained in the presence at baseline of a high number of IAS-USA PI resistance-associated mutations (IAS-USA PI RAMS); a diminished response occurred with >or=14. Eleven protease mutations associated with diminished darunavir/r virological response were identified (V11I, V32I, L33F, I47V, I50V, I54L/M, G73S, L76V, I84V, and L89V). These darunavir resistance-associated mutations (DRV RAMS) occurred in the presence of a high number of IAS-USA PI RAMS. Virological response was diminished with three or more DRV RAMS in the background of a high number of IAS-USA PI RAMS. Incremental numbers of DRV RAMS were more predictive of outcome than were IAS-USA PI RAMS. Mutations developing during darunavir/r virological failure (V32I, L33F, I47V, I54L, and L89V) were also featured in the DRV RAMS list. Site-directed mutants carrying these five mutations, or any one of these mutations either alone or together with one or two IAS-USA PI RAMS, showed no reduced darunavir susceptibility, suggesting that a high number of additional background mutations is required for darunavir resistance. In this population of treatment-experienced patients, darunavir/r demonstrated significantly greater efficacy than investigator-selected control PIs of trials POWER 1 and 2, regardless of baseline viral genotype or phenotype, while exhibiting a high genetic barrier to the development of resistance.

Predicting the impact of mutations on the specific activity of Bacillus thermocatenulatus lipase using a combined approach of docking and molecular dynamics.

PubMed

Yukselen, Onur; Timucin, Emel; Sezerman, Ugur

2016-10-01

Lipases are important biocatalysts owing to their ability to catalyze diverse reactions with exceptional substrate specificities. A combined docking and molecular dynamics (MD) approach was applied to study the chain-length selectivity of Bacillus thermocatenulatus lipase (BTL2) towards its natural substrates (triacylglycerols). A scoring function including electrostatic, van der Waals (vdW) and desolvation energies along with conformational entropy was developed to predict the impact of mutation. The native BTL2 and its 6 mutants (F17A, V175A, V175F, D176F, T178V and I320F) were experimentally analyzed to determine their specific activities towards tributyrin (C4) or tricaprylin (C8), which were used to test our approach. Our scoring methodology predicted the chain-length selectivity of BTL2 with 85.7% (6/7) accuracy with a positive correlation between the calculated scores and the experimental activity values (r = 0.82, p = 0.0004). Additionally, the impact of mutation on activity was predicted with 75% (9/12) accuracy. The described study represents a fast and reliable approach to accurately predict the effect of mutations on the activity and selectivity of lipases and also of other enzymes. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Results of a phase 2 study of pacritinib (SB1518), a JAK2/JAK2(V617F) inhibitor, in patients with myelofibrosis

PubMed Central

Seymour, John F.; Roberts, Andrew W.; Wadleigh, Martha; To, L. Bik; Scherber, Robyn; Turba, Elyce; Dorr, Andrew; Zhu, Joy; Wang, Lixia; Granston, Tanya; Campbell, Mary S.; Mesa, Ruben A.

2015-01-01

Pacritinib (SB1518) is a Janus kinase 2 (JAK2), JAK2(V617F), and Fms-like tyrosine kinase 3 inhibitor that does not inhibit JAK1. It demonstrated a favorable safety profile with promising efficacy in phase 1 studies in patients with primary and secondary myelofibrosis (MF). This multicenter phase 2 study further characterized the safety and efficacy of pacritinib in the treatment of patients with MF. Eligible patients had clinical splenomegaly poorly controlled with standard therapies or were newly diagnosed with intermediate- or high-risk Lille score. Patients with any degree of cytopenia were eligible. Thirty-five patients were enrolled. At entry, 40% had hemoglobin <10 g/dL and 43% had platelets <100 000× 109/L. Up to week 24, 8 of 26 evaluable patients (31%) achieved a ≥35% decrease in spleen volume determined by magnetic resonance imaging and 14 of 33 (42%) attained a ≥50% reduction in spleen size by physical examination. Median MF symptom improvement was ≥50% for all symptoms except fatigue. Grade 1 or 2 diarrhea (69%) and nausea (49%) were the most common treatment-emergent adverse events. The study drug was discontinued in 9 patients (26%) due to adverse events (4 severe). Pacritinib is an active agent in patients with MF, offering a potential treatment option for patients with preexisting anemia and thrombocytopenia. This trial was registered at www.clinicaltrials.gov as #NCT00745550. PMID:25762180

Crystallographic Analysis Reveals a Novel Second Binding Site for Trimethoprim in Active Site Double Mutants of Human Dihydrofolate Reductase†,‡

PubMed Central

Cody, Vivian; Pace, Jim; Piraino, Jennifer; Queener, Sherry F.

2011-01-01

In order to produce a more potent replacement for trimethoprim (TMP) used as a therapy for Pneumocystis pneumonia and targets dihydrofolate reductase from Pneumocystis jirovecii (pjDHFR), it is necessary to understand the determinants of potency and selectivity against DHFR from the mammalian host and fungal pathogen cells. To this end, active site residues in human (h)DHFR were replaced with those from pjDHFR. Structural data are reported for two complexes of TMP with the double mutants Gln35Ser/Asn64Phe (Q35S/N64F) and Gln35Lys/Asn64Phe (Q35K/N64F) of hDHFR that unexpectedly show evidence for the binding of two molecules of TMP: one molecule that binds in the normal folate binding site and the second molecule that binds in a novel subpocket site such that the mutated residue Phe64 is involved in van der Waals contacts to the trimethoxyphenyl ring of the second TMP molecule. Kinetic data for the binding of TMP to hDHFR and pjDHFR reveal an 84-fold selectivity of TMP against pjDHFR (Ki 49 nM) compared to hDHFR (Ki 4093 nM). Two mutants that contain one substitution from pj- and one from the closely related Pneumocystis carinii DHFR (pcDHFR) (Q35K/N64F and Q35S/N64F) show Ki values of 593 and 617 nM, respectively; these Ki values are well above both the Ki for pjDHFR and are similar to pcDHFR (Q35K/N64F) and Q35S/N64F) (305 nM). These results suggest that active site residues 35 and 64 play key roles in determining selectivity for pneumocystis DHFR, but that other residues contribute to the unique binding of inhibitors to these enzymes. PMID:21684339

Identification of a novel large deletion in a patient with severe factor V deficiency using an in-house F5 MLPA assay.

PubMed

Nuzzo, F; Paraboschi, E M; Straniero, L; Pavlova, A; Duga, S; Castoldi, E

2015-01-01

Factor V (FV) deficiency is a rare autosomal recessive bleeding disorder caused by mutations in the F5 gene. FV-deficient patients in whom no mutation or only one mutation is found may harbour large gene rearrangements, which are not detected by conventional mutation screening strategies. The aim of this study was to develop and validate a multiplex ligation-dependent probe amplification (MLPA) assay for the detection of large deletions and duplications in the F5 gene. Twenty-two MLPA probes targeting 19 of the 25 exons and the upstream and downstream regions of the F5 gene were designed and tested in 10 normal controls, a patient with a known heterozygous deletion of F5 exons 1-7 (positive control) and 14 genetically unexplained FV-deficient patients. MLPA results were confirmed by digital PCR on a QuantStudio(™) 3D Digital PCR System. The F5-specific probes yielded a reproducible peak profile in normal controls, correctly detected the known deletion in the positive control and suggested the presence of a novel deletion of exons 9-10 in a patient with undetectable FV levels and only one identified mutation. Follow-up by chip-based digital PCR, long-range PCR and direct sequencing confirmed that this patient carried a heterozygous F5 deletion of 1823 bp extending from intron 8 to intron 10. Bioinformatics sequence analysis pinpointed repetitive elements that might have originated the deletion. In conclusion, we have developed and validated an MLPA assay for the detection of gross F5 gene rearrangements. This assay may represent a valuable tool for the molecular diagnosis of FV deficiency. © 2014 John Wiley & Sons Ltd.

Trimethoprim Resistance of Dihydrofolate Reductase Variants from Clinical Isolates of Pneumocystis jirovecii

PubMed Central

Cody, V.; Pace, J.; Torkelson, P.; Gangjee, A.

2013-01-01

Pneumocystis jirovecii is an opportunistic pathogen that causes serious pneumonia in immunosuppressed patients. Standard therapy and prophylaxis include trimethoprim (TMP)-sulfamethoxazole; trimethoprim in this combination targets dihydrofolate reductase (DHFR). Fourteen clinically observed variants of P. jirovecii DHFR were produced recombinantly to allow exploration of the causes of clinically observed failure of therapy and prophylaxis that includes trimethoprim. Six DHFR variants (S31F, F36C, L65P, A67V, V79I, and I158V) showed resistance to inhibition by trimethoprim, with Ki values for trimethoprim 4-fold to 100-fold higher than those for the wild-type P. jirovecii DHFR. An experimental antifolate with more conformational flexibility than trimethoprim showed strong activity against one trimethoprim-resistant variant. The two variants that were most resistant to trimethoprim (F36C and L65P) also had increased Km values for dihydrofolic acid (DHFA). The catalytic rate constant (kcat) was unchanged for most variant forms of P. jirovecii DHFR but was significantly lowered in F36C protein; one naturally occurring variant with two amino acid substitutions (S106P and E127G) showed a doubling of kcat, as well as a Km for NADPH half that of the wild type. The strongest resistance to trimethoprim occurred with amino acid changes in the binding pocket for DHFA or trimethoprim, and the strongest effect on binding of NADPH was linked to a mutation involved in binding the phosphate group of the cofactor. This study marks the first confirmation that naturally occurring mutations in the gene for DHFR from P. jirovecii produce variant forms of DHFR that are resistant to trimethoprim and may contribute to clinically observed failures of standard therapy or prophylaxis. PMID:23896474

Conserved Glycine Residues in the Fusion Peptide of the Paramyxovirus Fusion Protein Regulate Activation of the Native State

PubMed Central

Russell, Charles J.; Jardetzky, Theodore S.; Lamb, Robert A.

2004-01-01

Hydrophobic fusion peptides (FPs) are the most highly conserved regions of class I viral fusion-mediating glycoproteins (vFGPs). FPs often contain conserved glycine residues thought to be critical for forming structures that destabilize target membranes. Unexpectedly, a mutation of glycine residues in the FP of the fusion (F) protein from the paramyxovirus simian parainfluenza virus 5 (SV5) resulted in mutant F proteins with hyperactive fusion phenotypes (C. M. Horvath and R. A. Lamb, J. Virol. 66:2443-2455, 1992). Here, we constructed G3A and G7A mutations into the F proteins of SV5 (W3A and WR isolates), Newcastle disease virus (NDV), and human parainfluenza virus type 3 (HPIV3). All of the mutant F proteins, except NDV G7A, caused increased cell-cell fusion despite having slight to moderate reductions in cell surface expression compared to those of wild-type F proteins. The G3A and G7A mutations cause SV5 WR F, but not NDV F or HPIV3 F, to be triggered to cause fusion in the absence of coexpression of its homotypic receptor-binding protein hemagglutinin-neuraminidase (HN), suggesting that NDV and HPIV3 F have stricter requirements for homotypic HN for fusion activation. Dye transfer assays show that the G3A and G7A mutations decrease the energy required to activate F at a step in the fusion cascade preceding prehairpin intermediate formation and hemifusion. Conserved glycine residues in the FP of paramyxovirus F appear to have a primary role in regulating the activation of the metastable native form of F. Glycine residues in the FPs of other class I vFGPs may also regulate fusion activation. PMID:15564482

Structures of oncogenic, suppressor and rescued p53 core-domain variants: mechanisms of mutant p53 rescue

PubMed Central

Wallentine, Brad D.; Wang, Ying; Tretyachenko-Ladokhina, Vira; Tan, Martha; Senear, Donald F.; Luecke, Hartmut

2013-01-01

To gain insights into the mechanisms by which certain second-site suppressor mutations rescue the function of a significant number of cancer mutations of the tumor suppressor protein p53, X-ray crystallographic structures of four p53 core-domain variants were determined. These include an oncogenic mutant, V157F, two single-site suppressor mutants, N235K and N239Y, and the rescued cancer mutant V157F/N235K/N239Y. The V157F mutation substitutes a smaller hydrophobic valine with a larger hydrophobic phenylalanine within strand S4 of the hydrophobic core. The structure of this cancer mutant shows no gross structural changes in the overall fold of the p53 core domain, only minor rearrangements of side chains within the hydrophobic core of the protein. Based on biochemical analysis, these small local perturbations induce instability in the protein, increasing the free energy by 3.6 kcal mol−1 (15.1 kJ mol−1). Further biochemical evidence shows that each suppressor mutation, N235K or N239Y, acts individually to restore thermodynamic stability to V157F and that both together are more effective than either alone. All rescued mutants were found to have wild-type DNA-binding activity when assessed at a permissive temperature, thus pointing to thermodynamic stability as the critical underlying variable. Interestingly, thermodynamic analysis shows that while N239Y demonstrates stabilization of the wild-type p53 core domain, N235K does not. These observations suggest distinct structural mechanisms of rescue. A new salt bridge between Lys235 and Glu198, found in both the N235K and rescued cancer mutant structures, suggests a rescue mechanism that relies on stabilizing the β-sandwich scaffold. On the other hand, the substitution N239Y creates an advantageous hydrophobic contact between the aromatic ring of this tyrosine and the adjacent Leu137. Surprisingly, the rescued cancer mutant shows much larger structural deviations than the cancer mutant alone when compared with wild-type p53. These suppressor mutations appear to rescue p53 function by creating novel intradomain interactions that stabilize the core domain, allowing compensation for the destabilizing V157F mutation. PMID:24100332

Mutations increasing exposure of a receptor binding site epitope in the soluble and oligomeric forms of the caprine arthritis-encephalitis lentivirus envelope glycoprotein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hoetzel, Isidro; Cheevers, William P.

2005-09-01

The caprine arthritis-encephalitis (CAEV) and ovine maedi-visna (MVV) viruses are resistant to antibody neutralization, a feature shared with all other lentiviruses. Whether the CAEV gp135 receptor binding site(s) (RBS) in the functional surface envelope glycoprotein (Env) is protected from antibody binding, allowing the virus to resist neutralization, is not known. Two CAEV gp135 regions were identified by extrapolating a gp135 structural model that could affect binding of antibodies to the RBS: the V1 region and a short sequence analogous in position to the human immunodeficiency virus type 1 gp120 loop B postulated to be located between two major domains ofmore » CAEV gp135. Mutation of isoleucine-166 to alanine in the putative loop B of gp135 increased the affinity of soluble gp135 for the CAEV receptor(s) and goat monoclonal antibody (Mab) F7-299 which recognizes an epitope overlapping the gp135 RBS. The I166A mutation also stabilized or exposed the F7-299 epitope in anionic detergent buffers, indicating that the I166A mutation induces conformational changes and stabilizes the RBS of soluble gp135 and enhances Mab F7-299 binding. In contrast, the affinity of a V1 deletion mutant of gp135 for the receptor and Mab F7-299 and its structural stability did not differ from that of the wild-type gp135. However, both the I166A mutation and the V1 deletion of gp135 increased cell-to-cell fusion activity and binding of Mab F7-299 to the oligomeric Env. Therefore, the CAEV gp135 RBS is protected from antibody binding by mechanisms both dependent and independent of Env oligomerization which are disrupted by the V1 deletion and the I166A mutation, respectively. In addition, we found a correlation between side-chain {beta}-branching at amino acid position 166 and binding of Mab F7-299 to oligomeric Env and cell-to-cell fusion, suggesting local secondary structure constraints in the region around isoleucine-166 as one determinant of gp135 RBS exposure and antibody binding.« less

Altered heme catabolism by heme oxygenase-1 caused by mutations in human NADPH cytochrome P450 reductase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pandey, Amit V., E-mail: amit@pandeylab.org; Flueck, Christa E.; Mullis, Primus E.

2010-09-24

Research highlights: {yields} Mutations in POR identified from patients lead to reduced HO-1 activities. {yields} POR mutation Y181D affecting FMN binding results in total loss of HO-1 activity. {yields} POR mutations A287P, C569Y and V608F, lost 50-70% activity. {yields} Mutations in FAD binding domain, R457H, Y459H and V492E lost all HO-1 activity. {yields} POR polymorphisms P228L, R316W, G413S, A503V and G504R have normal activity. -- Abstract: Human heme oxygenase-1 (HO-1) carries out heme catabolism supported by electrons supplied from the NADPH through NADPH P450 reductase (POR, CPR). Previously we have shown that mutations in human POR cause a rare formmore » of congenital adrenal hyperplasia. In this study, we have evaluated the effects of mutations in POR on HO-1 activity. We used purified preparations of wild type and mutant human POR and in vitro reconstitution with purified HO-1 to measure heme degradation in a coupled assay using biliverdin reductase. Here we show that mutations in POR found in patients may reduce HO-1 activity, potentially influencing heme catabolism in individuals carrying mutant POR alleles. POR mutants Y181D, A457H, Y459H, V492E and R616X had total loss of HO-1 activity, while POR mutations A287P, C569Y and V608F lost 50-70% activity. The POR variants P228L, R316W and G413S, A503V and G504R identified as polymorphs had close to WT activity. Loss of HO-1 activity may result in increased oxidative neurotoxicity, anemia, growth retardation and iron deposition. Further examination of patients affected with POR deficiency will be required to assess the metabolic effects of reduced HO-1 activity in affected individuals.« less

Assisted stellar suicide in V617 Sagittarii

NASA Astrophysics Data System (ADS)

Steiner, J. E.; Oliveira, A. S.; Cieslinski, D.; Ricci, T. V.

2006-02-01

Context: .V617 Sgr is a V Sagittae star - a group of binaries thought to be the galactic counterparts of the Compact Binary Supersoft X-ray Sources - CBSS. Aims: .To check this hypothesis, we measured the time derivative of its orbital period. Methods: .Observed timings of eclipse minima spanning over 30 000 orbital cycles are presented. Results: .We found that the orbital period evolves quite rapidly: P/dot{P} = 1.1×106 years. This is consistent with the idea that V617 Sgr is a wind driven accretion supersoft source. As the binary system evolves with a time-scale of about one million years, which is extremely short for a low mass evolved binary, it is likely that the system will soon end either by having its secondary completely evaporated or by the primary exploding as a supernova of type Ia. Conclusions: .

Temporal frequency of knockdown resistance mutations, F1534C and V1016G, in Aedes aegypti in Chiang Mai city, Thailand and the impact of the mutations on the efficiency of thermal fogging spray with pyrethroids.

PubMed

Plernsub, Suriya; Saingamsook, Jassada; Yanola, Jintana; Lumjuan, Nongkran; Tippawangkosol, Pongsri; Walton, Catherine; Somboon, Pradya

2016-10-01

In Thailand, control of dengue outbreaks is currently attained by the use of space sprays, particularly thermal fogging using pyrethroids, with the aim of killing infected Aedes mosquito vectors in epidemic areas. However, the principal dengue vector, Aedes aegypti, is resistant to pyrethroids conferred mainly by mutations in the voltage-gated sodium channel gene, F1534C and V1016G, termed knockdown resistance (kdr). The objectives of this study were to determine the temporal frequencies of F1534C and V1016G in Ae. aegypti populations in relation to pyrethroid resistance in Chiang Mai city, and to evaluate the impact of the mutations on the efficacy of thermal fogging with the pyrethroid deltamethrin. Larvae and pupae were collected from several areas around Chiang Mai city during 2011-2015 and reared to adulthood for bioassays for deltamethrin susceptibility. These revealed no trend of increasing deltamethrin resistance during the study period (mortality 58.0-69.5%, average 62.8%). This corresponded to no overall change in the frequencies of the C1534 allele (0.55-0.66, average 0.62) and G1016 allele (0.34-0.45, average 0.38), determined using allele specific amplification. Only three genotypes of kdr mutations were detected: C1534 homozygous (VV/CC); G1016/C1534 double heterozygous (VG/FC); and G1016 homozygous (GG/FF) indicating that the F1534C and V1016G mutations occurred on separate haplotypic backgrounds and a lack of recombination between them to date. The F1 progeny females were used to evaluate the efficacy of thermal fogging spray with Damthrin-SP(®) (deltamethrin+S-bioallethrin+piperonyl butoxide) using a caged mosquito bioassay. The thermal fogging spray killed 100% and 61.3% of caged mosquito bioassay placed indoors and outdoors, respectively. The outdoor spray had greater killing effect on C1534 homozygous and had partially effect on double heterozygous mosquitoes, but did not kill any G1016 homozygous mutants living outdoors. As this selection pressure would be expected to have led to an increase in frequency of the G1016 allele, it is likely that the relatively stable kdr mutation allele frequencies observed here result from balancing selection, in the form of overdominance for VG/FC genotypes and/or the effects of fluctuating environments that vary in insecticide exposure. Copyright © 2016 Elsevier B.V. All rights reserved.

Structures of oncogenic, suppressor and rescued p53 core-domain variants: mechanisms of mutant p53 rescue

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wallentine, Brad D.; Wang, Ying; Tretyachenko-Ladokhina, Vira

2013-10-01

X-ray crystallographic structures of four p53 core-domain variants were determined in order to gain insights into the mechanisms by which certain second-site suppressor mutations rescue the function of a significant number of cancer mutations of the tumor suppressor protein p53. To gain insights into the mechanisms by which certain second-site suppressor mutations rescue the function of a significant number of cancer mutations of the tumor suppressor protein p53, X-ray crystallographic structures of four p53 core-domain variants were determined. These include an oncogenic mutant, V157F, two single-site suppressor mutants, N235K and N239Y, and the rescued cancer mutant V157F/N235K/N239Y. The V157F mutationmore » substitutes a smaller hydrophobic valine with a larger hydrophobic phenylalanine within strand S4 of the hydrophobic core. The structure of this cancer mutant shows no gross structural changes in the overall fold of the p53 core domain, only minor rearrangements of side chains within the hydrophobic core of the protein. Based on biochemical analysis, these small local perturbations induce instability in the protein, increasing the free energy by 3.6 kcal mol{sup −1} (15.1 kJ mol{sup −1}). Further biochemical evidence shows that each suppressor mutation, N235K or N239Y, acts individually to restore thermodynamic stability to V157F and that both together are more effective than either alone. All rescued mutants were found to have wild-type DNA-binding activity when assessed at a permissive temperature, thus pointing to thermodynamic stability as the critical underlying variable. Interestingly, thermodynamic analysis shows that while N239Y demonstrates stabilization of the wild-type p53 core domain, N235K does not. These observations suggest distinct structural mechanisms of rescue. A new salt bridge between Lys235 and Glu198, found in both the N235K and rescued cancer mutant structures, suggests a rescue mechanism that relies on stabilizing the β-sandwich scaffold. On the other hand, the substitution N239Y creates an advantageous hydrophobic contact between the aromatic ring of this tyrosine and the adjacent Leu137. Surprisingly, the rescued cancer mutant shows much larger structural deviations than the cancer mutant alone when compared with wild-type p53. These suppressor mutations appear to rescue p53 function by creating novel intradomain interactions that stabilize the core domain, allowing compensation for the destabilizing V157F mutation.« less

Mpl traffics to the cell surface through conventional and unconventional routes

PubMed Central

Cleyrat, Cédric; Darehshouri, Anza; Steinkamp, Mara P.; Vilaine, Mathias; Boassa, Daniela; Ellisman, Mark H.; Hermouet, Sylvie; Wilson, Bridget S.

2014-01-01

Myeloproliferative neoplasms (MPNs) are often characterized by JAK2 or calreticulin mutations, indicating aberrant trafficking in pathogenesis. This study focuses on Mpl trafficking and Jak2 association using two model systems: Human Erythroleukemia cells (HEL; JAK2V617F) and K562 myeloid leukemia cells (JAK2WT). Consistent with a putative chaperone role for Jak2, Mpl and Jak2 associate on both intracellular and plasma membranes (shown by proximity ligation assay) and siRNA-mediated knockdown of Jak2 led to Mpl trapping in the ER. Even in Jak2 sufficient cells, Mpl accumulates in punctate structures that partially co-localize with ER-Tracker, the ER exit site marker (ERES) Sec31a, the autophagy marker LC3 and LAMP1. Mpl was fused to miniSOG, a genetically-encoded tag for correlated light and electron microscopy. Results suggest that a fraction of Mpl is taken up into autophagic structures from the ER and routed to autolyososomes. Surface biotinylation shows that both immature and mature Mpl reach the cell surface; in K562 cells Mpl is also released in exosomes. Both forms rapidly internalize upon ligand addition, while recovery is primarily attributed to immature Mpl. Mpl appears to reach the plasma membrane via both conventional ER-Golgi and autolysosome secretory pathways, as well as recycling. PMID:24931576

Efficacy of ALK5 inhibition in myelofibrosis

PubMed Central

Zhao, Wanke; Ho, Wanting Tina; Han, Ying; Murdun, Cem; Mailloux, Adam W.; Zhang, Ling; Wang, Xuefeng; Budhathoki, Anjali; Pradhan, Kith; Rapaport, Franck; Wang, Huaquan; Shao, Zonghong; Ren, Xiubao; Steidl, Ulrich; Levine, Ross L.; Zhao, Zhizhuang Joe; Verma, Amit; Epling-Burnette, Pearlie K.

2017-01-01

Myelofibrosis (MF) is a bone marrow disorder characterized by clonal myeloproliferation, aberrant cytokine production, extramedullary hematopoiesis, and bone marrow fibrosis. Although somatic mutations in JAK2, MPL, and CALR have been identified in the pathogenesis of these diseases, inhibitors of the Jak2 pathway have not demonstrated efficacy in ameliorating MF in patients. TGF-β family members are profibrotic cytokines and we observed significant TGF-β1 isoform overexpression in a large cohort of primary MF patient samples. Significant overexpression of TGF-β1 was also observed in murine clonal MPLW515L megakaryocytic cells. TGF-β1 stimulated the deposition of excessive collagen by mesenchymal stromal cells (MSCs) by activating the TGF-β receptor I kinase (ALK5)/Smad3 pathway. MSCs derived from MPLW515L mice demonstrated sustained overproduction of both collagen I and collagen III, effects that were abrogated by ALK5 inhibition in vitro and in vivo. Importantly, use of galunisertib, a clinically active ALK5 inhibitor, significantly improved MF in both MPLW515L and JAK2V617F mouse models. These data demonstrate the role of malignant hematopoietic stem cell (HSC)/TGF-β/MSC axis in the pathogenesis of MF, and provide a preclinical rationale for ALK5 blockade as a therapeutic strategy in MF. PMID:28405618

A single mutation in the PBC loop of VP2 is involved in the in vitro replication of infectious bursal disease virus.

PubMed

Qi, Xiaole; Gao, Xiang; Lu, Zhen; Zhang, Lizhou; Wang, Yongqiang; Gao, Li; Gao, Yulong; Li, Kai; Gao, Honglei; Liu, Changjun; Cui, Hongyu; Zhang, Yanping; Wang, Xiaomei

2016-07-01

To test whether amino acid mutations in the PBC and PHI loops of VP2 are involved in the replication and virulence of infectious bursal disease virus (IBDV), a pair of viruses, namely the moderately virulent IBDV (rGx-F9VP2) and the attenuated strain (rGt), were used. Residue mutations A222P (PBC) and S330R (PHI), selected by sequence comparison, were introduced individually into rGx-F9VP2 by using a reverse genetics system. In addition, the reverse mutation of either P222A or R330S was introduced into rGt. The four modified viruses were then rescued and evaluated in vitro (CEF cells) and in vivo (SPF chickens). Results showed that A222P elevated the replication efficiency of rGx-F9VP2 while P222A reduced that of rGt in CEF cells. A mutation at residue 330 did not alter IBDV replication. In addition, animal experiments showed that a single mutation at either residue 222 or 330 did not significantly influence the virulence of IBDV. In conclusion, residue 222 in PBC of VP2 is involved in the replication efficiency of IBDV in vitro but does not affect its virulence in vivo, further facilitating our understanding of the gene-function of IBDV.

Insecticides resistance in the Culex quinquefasciatus populations from northern Thailand and possible resistance mechanisms.

PubMed

Yanola, Jintana; Chamnanya, Saowanee; Lumjuan, Nongkran; Somboon, Pradya

2015-09-01

The mosquito vector Culex quinquefasciatus is known to be resistant to insecticides worldwide, including Thailand. This study was the first investigation of the insecticide resistance mechanisms, involving metabolic detoxification and target site insensitivity in C. quinquefasciatus from Thailand. Adult females reared from field-caught larvae from six provinces of northern Thailand were determined for resistant status by exposing to 0.05% deltamethrin, 0.75% permethrin and 5% malathion papers using the standard WHO susceptibility test. The overall mortality rates were 45.8%, 11.4% and 80.2%, respectively. A fragment of voltage-gated sodium channel gene was amplified and sequenced to identify the knock down resistance (kdr) mutation. The ace-1 gene mutation was determined by using PCR-RFLP. The L1014F kdr mutation was observed in all populations, but the homozygous mutant F/F1014 genotype was found only in two of the six provinces where the kdr mutation was significantly correlated with deltamethrin resistance. However, none of mosquitoes had the G119S mutation in the ace-1 gene. A laboratory deltamethrin resistant strain, Cq_CM_R, has been established showing a highly resistant level after selection for a few generations. The mutant F1014 allele frequency was significantly increased after one generation of selection. A synergist assay was performed to assess the metabolic detoxifying enzymes. Addition of bis(4-nitrophenyl)-phosphate (BNPP) and diethyl maleate (DEM), inhibitors of esterases and glutathione S-transferases (GST), respectively, into the larval bioassay of the Cq_CM strain with deltamethrin showed no significant reduction. By contrast, addition of piperonyl butoxide (PBO), an inhibitor of cytochrome P450 monooxygenases, showed a 9-fold reduction of resistance. Resistance to pyrethroids in C. quinquefasciatus is widely distributed in northern Thailand. This study reports for the first time for the detection of the L1014F kdr mutation in wild populations of C. quinquefasciatus in Thailand. At least two major mechanisms, kdr and cytochrome P450 monooxygenases, confer resistance to deltamethrin in Thai C. quinquefasciatus populations. Copyright © 2015 Elsevier B.V. All rights reserved.

Diagnostic contribution of molecular analysis of the cystic fibrosis transmembrane conductance regulator gene in patients suspected of having mild or atypical cystic fibrosis *

PubMed Central

Dal'Maso, Vinícius Buaes; Mallmann, Lucas; Siebert, Marina; Simon, Laura; Saraiva-Pereira, Maria Luiza; Dalcin, Paulo de Tarso Roth

2013-01-01

OBJECTIVE: To evaluate the diagnostic contribution of molecular analysis of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in patients suspected of having mild or atypical cystic fibrosis (CF). METHODS: This was a cross-sectional study involving adolescents and adults aged ≥ 14 years. Volunteers underwent clinical, laboratory, and radiological evaluation, as well as spirometry, sputum microbiology, liver ultrasound, sweat tests, and molecular analysis of the CFTR gene. We then divided the patients into three groups by the number of mutations identified (none, one, and two or more) and compared those groups in terms of their characteristics. RESULTS: We evaluated 37 patients with phenotypic findings of CF, with or without sweat test confirmation. The mean age of the patients was 32.5 ± 13.6 years, and females predominated (75.7%). The molecular analysis contributed to the definitive diagnosis of CF in 3 patients (8.1%), all of whom had at least two mutations. There were 7 patients (18.9%) with only one mutation and 26 patients (70.3%) with no mutations. None of the clinical characteristics evaluated was found to be associated with the genetic diagnosis. The most common mutation was p.F508del, which was found in 5 patients. The combination of p.V232D and p.F508del was found in 2 patients. Other mutations identified were p.A559T, p.D1152H, p.T1057A, p.I148T, p.V754M, p.P1290P, p.R1066H, and p.T351S. CONCLUSIONS: The molecular analysis of the CFTR gene coding region showed a limited contribution to the diagnostic investigation of patients suspected of having mild or atypical CF. In addition, there were no associations between the clinical characteristics and the genetic diagnosis. PMID:23670503

Diagnostic contribution of molecular analysis of the cystic fibrosis transmembrane conductance regulator gene in patients suspected of having mild or atypical cystic fibrosis.

PubMed

Dal'Maso, Vinícius Buaes; Mallmann, Lucas; Siebert, Marina; Simon, Laura; Saraiva-Pereira, Maria Luiza; Dalcin, Paulo de Tarso Roth

2013-01-01

To evaluate the diagnostic contribution of molecular analysis of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in patients suspected of having mild or atypical cystic fibrosis (CF). This was a cross-sectional study involving adolescents and adults aged > 14 years. Volunteers underwent clinical, laboratory, and radiological evaluation, as well as spirometry, sputum microbiology, liver ultrasound, sweat tests, and molecular analysis of the CFTR gene. We then divided the patients into three groups by the number of mutations identified (none, one, and two or more) and compared those groups in terms of their characteristics. We evaluated 37 patients with phenotypic findings of CF, with or without sweat test confirmation. The mean age of the patients was 32.5 ± 13.6 years, and females predominated (75.7%). The molecular analysis contributed to the definitive diagnosis of CF in 3 patients (8.1%), all of whom had at least two mutations. There were 7 patients (18.9%) with only one mutation and 26 patients (70.3%) with no mutations. None of the clinical characteristics evaluated was found to be associated with the genetic diagnosis. The most common mutation was p.F508del, which was found in 5 patients. The combination of p.V232D and p.F508del was found in 2 patients. Other mutations identified were p.A559T, p.D1152H, p.T1057A, p.I148T, p.V754M, p.P1290P, p.R1066H, and p.T351S. The molecular analysis of the CFTR gene coding region showed a limited contribution to the diagnostic investigation of patients suspected of having mild or atypical CF. In addition, there were no associations between the clinical characteristics and the genetic diagnosis.

A mutation of RNA polymerase β' subunit (RpoC) converts heterogeneously vancomycin-intermediate Staphylococcus aureus (hVISA) into "slow VISA".

PubMed

Matsuo, Miki; Hishinuma, Tomomi; Katayama, Yuki; Hiramatsu, Keiichi

2015-07-01

Various mutations in the rpoB gene, which encodes the RNA polymerase β subunit, are associated with increased vancomycin (VAN) resistance in vancomycin-intermediate Staphylococcus aureus (VISA) and heterogeneously VISA (hVISA) strains. We reported that rpoB mutations are also linked to the expression of the recently found "slow VISA" (sVISA) phenotype (M. Saito, Y. Katayama, T. Hishinuma, A. Iwamoto, Y. Aiba, K Kuwahara-Arai, L. Cui, M. Matsuo, N. Aritaka, and K. Hiramatsu, Antimicrob Agents Chemother 58:5024-5035, 2014, http://dx.doi.org/10.1128/AAC.02470-13). Because RpoC and RpoB are components of RNA polymerase, we examined the effect of the rpoC(P440L) mutation on the expression of the sVISA phenotype in the Mu3fdh2*V6-5 strain (V6-5), which was derived from a previously reported hVISA strain with the VISA phenotype. V6-5 had an extremely prolonged doubling time (DT) (72 min) and high vancomycin MIC (16 mg/liter). However, the phenotype of V6-5 was unstable, and the strain frequently reverted to hVISA with concomitant loss of low growth rate, cell wall thickness, and reduced autolysis. Whole-genome sequencing of phenotypic revertant strain V6-5-L1 and comparison with V6-5 revealed a second mutation, F562L, in rpoC. Introduction of the wild-type (WT) rpoC gene using a multicopy plasmid resolved the sVISA phenotype of V6-5, indicating that the rpoC(P440L) mutant expressed the sVISA phenotype in hVISA. To investigate the mechanisms of resistance in the sVISA strain, we independently isolated an additional 10 revertants to hVISA and VISA. In subsequent whole-genome analysis, we identified compensatory mutations in the genes of three distinct functional categories: the rpoC gene itself as regulatory mutations, peptidoglycan biosynthesis genes, and relQ, which is involved in the stringent response. It appears that the rpoC(P440L) mutation causes the sVISA phenotype by augmenting cell wall peptidoglycan synthesis and through the control of the stringent response. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Examination of in vivo mutagenicity of sodium arsenite and dimethylarsinic acid in gpt delta rats.

PubMed

Fujioka, Masaki; Gi, Min; Kawachi, Satoko; Tatsumi, Kumiko; Ishii, Naomi; Doi, Kenichiro; Kakehashi, Anna; Wanibuchi, Hideki

2016-11-01

Arsenic is a well-known human bladder and liver carcinogen, but its exact mechanism of carcinogenicity is not fully understood. Dimethylarsinic acid (DMA V ) is a major urinary metabolite of sodium arsenite (iAs III ) and induces urinary bladder cancers in rats. DMA V and iAs III are negative in in vitro mutagenicity tests. However, their in vivo mutagenicities have not been determined. The purpose of present study is to evaluate the in vivo mutagenicities of DMA V and iAs III in rat urinary bladder epithelium and liver using gpt delta F344 rats. Ten-week old male gpt delta F344 rats were randomized into 3 groups and administered 0, 92mg/L DMA V , or 87mg/L iAs III (each 50mg/L As) for 13weeks in the drinking water. In the mutation assay, point mutations are detected in the gpt gene by 6-thioguanine selection (gpt assay) and deletion mutations are identified in the red/gam genes by Spi - selection (Spi - assay). Results of the gpt and Spi - assays showed that DMA V and iAs III had no effects on the mutant frequencies or mutation spectrum in urinary bladder epithelium or liver. These findings indicate that DMA V and iAs III are not mutagenic in urinary bladder epithelium or liver in rats. Copyright © 2016. Published by Elsevier B.V.

Novel KCNQ3 Mutation in a Large Family with Benign Familial Neonatal Epilepsy: A Rare Cause of Neonatal Seizures

PubMed Central

Maljevic, Snezana; Vejzovic, Sabina; Bernhard, Matthias K.; Bertsche, Astrid; Weise, Sebastian; Döcker, Miriam; Lerche, Holger; Lemke, Johannes R.; Merkenschlager, Andreas; Syrbe, Steffen

2016-01-01

Benign familial neonatal seizures (BFNS) present a rare familial epilepsy syndrome caused by genetic alterations in the voltage-gated potassium channels Kv7.2 and Kv7.3, encoded by KCNQ2 and KCNQ3. While most BFNS families carry alterations in KCNQ2, mutations in KCNQ3 appear to be less common. Here, we describe a family with 6 individuals presenting with neonatal focal and generalized seizures. Genetic testing revealed a novel KCNQ3 variant, c.835G>T, cosegregating with seizures in 4 tested individuals. This variant results in a substitution of the highly conserved amino acid valine localized within the pore-forming transmembrane segment S5 (p.V279F). Functional investigations in Xenopus laevis oocytes revealed a loss of function, which supports p.V279F as a pathogenic mutation. When p.V279F was coexpressed with the wild-type (WT) Kv7.2 subunits, the resulting potassium currents were about 10-fold reduced compared to the WT Kv7.3 and Kv7.2 coexpression. Genotype-phenotype correlation shows an incomplete penetrance of p.V279F. Response to antiepileptic treatment was variable, but evaluation of treatment response remained challenging due to the self-limiting character of the disease. The identification of the pathogenic variant helped to avoid unnecessary investigations in affected family members and allowed guided therapy. PMID:27781029

Novel KCNQ3 Mutation in a Large Family with Benign Familial Neonatal Epilepsy: A Rare Cause of Neonatal Seizures.

PubMed

Maljevic, Snezana; Vejzovic, Sabina; Bernhard, Matthias K; Bertsche, Astrid; Weise, Sebastian; Döcker, Miriam; Lerche, Holger; Lemke, Johannes R; Merkenschlager, Andreas; Syrbe, Steffen

2016-09-01

Benign familial neonatal seizures (BFNS) present a rare familial epilepsy syndrome caused by genetic alterations in the voltage-gated potassium channels Kv7.2 and Kv7.3, encoded by KCNQ2 and KCNQ3. While most BFNS families carry alterations in KCNQ2 , mutations in KCNQ3 appear to be less common. Here, we describe a family with 6 individuals presenting with neonatal focal and generalized seizures. Genetic testing revealed a novel KCNQ3 variant, c.835G>T, cosegregating with seizures in 4 tested individuals. This variant results in a substitution of the highly conserved amino acid valine localized within the pore-forming transmembrane segment S5 (p.V279F). Functional investigations in Xenopus laevis oocytes revealed a loss of function, which supports p.V279F as a pathogenic mutation. When p.V279F was coexpressed with the wild-type (WT) Kv7.2 subunits, the resulting potassium currents were about 10-fold reduced compared to the WT Kv7.3 and Kv7.2 coexpression. Genotype-phenotype correlation shows an incomplete penetrance of p.V279F. Response to antiepileptic treatment was variable, but evaluation of treatment response remained challenging due to the self-limiting character of the disease. The identification of the pathogenic variant helped to avoid unnecessary investigations in affected family members and allowed guided therapy.

Mutated and Bacteriophage T4 Nanoparticle Arrayed F1-V Immunogens from Yersinia pestis as Next Generation Plague Vaccines

PubMed Central

Tao, Pan; Mahalingam, Marthandan; Kirtley, Michelle L.; van Lier, Christina J.; Sha, Jian; Yeager, Linsey A.; Chopra, Ashok K.; Rao, Venigalla B.

2013-01-01

Pneumonic plague is a highly virulent infectious disease with 100% mortality rate, and its causative organism Yersinia pestis poses a serious threat for deliberate use as a bioterror agent. Currently, there is no FDA approved vaccine against plague. The polymeric bacterial capsular protein F1, a key component of the currently tested bivalent subunit vaccine consisting, in addition, of low calcium response V antigen, has high propensity to aggregate, thus affecting its purification and vaccine efficacy. We used two basic approaches, structure-based immunogen design and phage T4 nanoparticle delivery, to construct new plague vaccines that provided complete protection against pneumonic plague. The NH2-terminal β-strand of F1 was transplanted to the COOH-terminus and the sequence flanking the β-strand was duplicated to eliminate polymerization but to retain the T cell epitopes. The mutated F1 was fused to the V antigen, a key virulence factor that forms the tip of the type three secretion system (T3SS). The F1mut-V protein showed a dramatic switch in solubility, producing a completely soluble monomer. The F1mut-V was then arrayed on phage T4 nanoparticle via the small outer capsid protein, Soc. The F1mut-V monomer was robustly immunogenic and the T4-decorated F1mut-V without any adjuvant induced balanced TH1 and TH2 responses in mice. Inclusion of an oligomerization-deficient YscF, another component of the T3SS, showed a slight enhancement in the potency of F1-V vaccine, while deletion of the putative immunomodulatory sequence of the V antigen did not improve the vaccine efficacy. Both the soluble (purified F1mut-V mixed with alhydrogel) and T4 decorated F1mut-V (no adjuvant) provided 100% protection to mice and rats against pneumonic plague evoked by high doses of Y. pestis CO92. These novel platforms might lead to efficacious and easily manufacturable next generation plague vaccines. PMID:23853602

Association between Three Mutations, F1565C, V1023G and S996P, in the Voltage-Sensitive Sodium Channel Gene and Knockdown Resistance in Aedes aegypti from Yogyakarta, Indonesia

PubMed Central

Rochmijati Wuliandari, Juli; Lee, Siu Fai; White, Vanessa Linley; Tantowijoyo, Warsito; Hoffmann, Ary Anthony; Endersby-Harshman, Nancy Margaret

2015-01-01

Mutations in the voltage-sensitive sodium channel gene (Vssc) have been identified in Aedes aegypti and some have been associated with pyrethroid insecticide resistance. Whether these mutations cause resistance, alone or in combination with other alleles, remains unclear, but must be understood if mutations are to become markers for resistance monitoring. We describe High Resolution Melt (HRM) genotyping assays for assessing mutations found in Ae. aegypti in Indonesia (F1565C, V1023G, S996P) and use them to test for associations with pyrethroid resistance in mosquitoes from Yogyakarta, a city where insecticide use is widespread. Such knowledge is important because Yogyakarta is a target area for releases of Wolbachia-infected mosquitoes with virus-blocking traits for dengue suppression. We identify three alleles across Yogyakarta putatively linked to resistance in previous research. By comparing resistant and susceptible mosquitoes from bioassays, we show that the 1023G allele is associated with resistance to type I and type II pyrethroids. In contrast, F1565C homozygotes were rare and there was only a weak association between individuals heterozygous for the mutation and resistance to a type I pyrethroid. As the heterozygote is expected to be incompletely recessive, it is likely that this association was due to a different resistance mechanism being present. A resistance advantage conferred to V1023G homozygotes through addition of the S996P allele in the homozygous form was suggested for the Type II pyrethroid, deltamethrin. Screening of V1023G and S996P should assist resistance monitoring in Ae. aegypti from Yogyakarta, and these mutations should be maintained in Wolbachia strains destined for release in this city to ensure that these virus-blocking strains of mosquitoes are not disadvantaged, relative to resident populations. PMID:26463408

Association between Three Mutations, F1565C, V1023G and S996P, in the Voltage-Sensitive Sodium Channel Gene and Knockdown Resistance in Aedes aegypti from Yogyakarta, Indonesia.

PubMed

Wuliandari, Juli Rochmijati; Lee, Siu Fai; White, Vanessa Linley; Tantowijoyo, Warsito; Hoffmann, Ary Anthony; Endersby-Harshman, Nancy Margaret

2015-07-23

Mutations in the voltage-sensitive sodium channel gene (Vssc) have been identified in Aedes aegypti and some have been associated with pyrethroid insecticide resistance. Whether these mutations cause resistance, alone or in combination with other alleles, remains unclear, but must be understood if mutations are to become markers for resistance monitoring. We describe High Resolution Melt (HRM) genotyping assays for assessing mutations found in Ae. aegypti in Indonesia (F1565C, V1023G, S996P) and use them to test for associations with pyrethroid resistance in mosquitoes from Yogyakarta, a city where insecticide use is widespread. Such knowledge is important because Yogyakarta is a target area for releases of Wolbachia-infected mosquitoes with virus-blocking traits for dengue suppression. We identify three alleles across Yogyakarta putatively linked to resistance in previous research. By comparing resistant and susceptible mosquitoes from bioassays, we show that the 1023G allele is associated with resistance to type I and type II pyrethroids. In contrast, F1565C homozygotes were rare and there was only a weak association between individuals heterozygous for the mutation and resistance to a type I pyrethroid. As the heterozygote is expected to be incompletely recessive, it is likely that this association was due to a different resistance mechanism being present. A resistance advantage conferred to V1023G homozygotes through addition of the S996P allele in the homozygous form was suggested for the Type II pyrethroid, deltamethrin. Screening of V1023G and S996P should assist resistance monitoring in Ae. aegypti from Yogyakarta, and these mutations should be maintained in Wolbachia strains destined for release in this city to ensure that these virus-blocking strains of mosquitoes are not disadvantaged, relative to resident populations.

Congenital hypopituitarism due to POU1F1 gene mutation.

PubMed

Lee, Ni-Chung; Tsai, Wen-Yu; Peng, Shinn-Forng; Tung, Yi-Ching; Chien, Yin-Hsiu; Hwu, Wuh-Liang

2011-01-01

POU1F1 (Pit-1; Gene ID 5449) is an anterior pituitary transcriptional factor, and POU1F1 mutation is known to cause anterior pituitary hypoplasia, growth hormone and prolactin deficiency and various degree of hypothyroidism. We report here a patient who presented with growth failure and central hypothyroidism since early infancy. However, treatment with thyroxine gave no effect and he subsequently developed calf muscle pseudohypertrophy (Kocher-Debre-Semelaigne syndrome), elevation of creatinine kinase, dilated cardiomyopathy and pericardial effusion. Final diagnosis was made by combined pituitary function test and sequencing analysis that revealed POU1F1 gene C.698T > C (p.F233S) mutation. The rarity of the disease can result in delayed diagnosis and treatment. Copyright © 2011 Formosan Medical Association & Elsevier. Published by Elsevier B.V. All rights reserved.

Experimentally Dissecting the Origins of Peroxiredoxin Catalysis.

PubMed

Nelson, Kimberly J; Perkins, Arden; Van Swearingen, Amanda E D; Hartman, Steven; Brereton, Andrew E; Parsonage, Derek; Salsbury, Freddie R; Karplus, P Andrew; Poole, Leslie B

2018-03-01

Peroxiredoxins (Prxs) are ubiquitous cysteine-based peroxidases involved in oxidant defense and signal transduction. Despite much study, the precise roles of conserved residues remain poorly defined. In this study, we carried out extensive functional and structural characterization of 10 variants of such residues in a model decameric bacterial Prx. Three active site proximal mutations of Salmonella typhimurium AhpC, T43V, R119A, and E49Q, lowered catalytic efficiency with hydrogen peroxide by 4-5 orders of magnitude, but did not affect reactivity toward their reductant, AhpF. pK a values of the peroxidatic cysteine were also shifted up by 1-1.3 pH units for these and a decamer disruption mutant, T77I. Except for the decamer-stabilizing T77V, all mutations destabilized decamers in the reduced form. In the oxidized form, three mutants-T77V, T43A, and T43S-exhibited stabilized decamers and were more efficiently reduced by AhpF than wild-type AhpC. Crystal structures of most mutants were solved and many showed alterations in stability of the fully folded active site loop. This is the first study of Prx mutants to comprehensively assess the effects of mutations on catalytic activities, the active site cysteine pK a , and the protein structure and oligomeric status. The Arg119 side chain must be properly situated for efficient catalysis, but for other debilitating variants, the functional defects could be explained by structural perturbations and/or associated decamer destabilization rather than direct effects. This underscores the importance of our comprehensive approach. A remarkable new finding was the preference of the reductant for decamers. Antioxid. Redox Signal. 28, 521-536.

Predicting Chromophoric Dissolved Organic Matter Distributions in Coastal Waters

DTIC Science & Technology

2006-01-01

Predicting Chromophoric Dissolved Organic Matter Distributions in Coastal Waters Robert F. Chen Environmental , Earth and Ocean Sciences...G. Bernard Gardner Environmental , Coastal and Ocean Sciences University of Massachusetts Boston 100 Morrissey Boulevard Boston, MA 02125-3393...phone: (617)287-7451 fax: (617)287-7474 email: bernie.gardner@umb.edu Yong Tian Environmental , Coastal and Ocean Sciences University of

Predicting Chromophoric Dissolved Organic Matter Distributions in Coastal Waters

DTIC Science & Technology

2007-09-30

Predicting Chromophoric Dissolved Organic Matter Distributions in Coastal Waters Robert F. Chen Environmental , Earth and Ocean Sciences...G. Bernard Gardner Environmental , Coastal and Ocean Sciences University of Massachusetts Boston 100 Morrissey Boulevard Boston, MA 02125-3393...phone: (617)287-7451 fax: (617)287-7474 email: bernie.gardner@umb.edu Yong Tian Environmental , Coastal and Ocean Sciences University of

Predicting Chromophoric Dissolved Organic Matter Distributions in Coastal Waters

DTIC Science & Technology

2008-09-30

Predicting Chromophoric Dissolved Organic Matter Distributions in Coastal Waters Robert F. Chen Environmental , Earth and Ocean...umb.edu G. Bernard Gardner Environmental , Coastal and Ocean Sciences University of Massachusetts Boston 100 Morrissey Boulevard Boston, MA...02125-3393 phone: (617) 287-7451 fax: (617) 287-7474 email: bernie.gardner@umb.edu Yong Tian Environmental , Coastal and Ocean Sciences

KIT D816V-mutated bone marrow mesenchymal stem cells in indolent systemic mastocytosis are associated with disease progression.

PubMed

Garcia-Montero, Andres C; Jara-Acevedo, Maria; Alvarez-Twose, Ivan; Teodosio, Cristina; Sanchez-Muñoz, Laura; Muñiz, Carmen; Muñoz-Gonzalez, Javier I; Mayado, Andrea; Matito, Almudena; Caldas, Carolina; Morgado, Jose M; Escribano, Luis; Orfao, Alberto

2016-02-11

Multilineage involvement of bone marrow (BM) hematopoiesis by the somatic KIT D816V mutation is present in a subset of adult indolent systemic mastocytosis (ISM) patients in association with a poorer prognosis. Here, we investigated the potential involvement of BM mesenchymal stem cells (MSCs) from ISM patients by the KIT D816V mutation and its potential impact on disease progression and outcome. This mutation was investigated in highly purified BM MSCs and other BM cell populations from 83 ISM patients followed for a median of 116 months. KIT D816V-mutated MSCs were detected in 22 of 83 cases. All MSC-mutated patients had multilineage KIT mutation (100% vs 30%, P = .0001) and they more frequently showed involvement of lymphoid plus myeloid BM cells (59% vs 22%; P = .03) and a polyclonal pattern of inactivation of the X-chromosome of KIT-mutated BM mast cells (64% vs 0%; P = .01) vs other multilineage ISM cases. Moreover, presence of KIT-mutated MSCs was associated with more advanced disease features, a greater rate of disease progression (50% vs 17%; P = .04), and a shorter progression-free survival (P ≤ .003). Overall, these results support the notion that ISM patients with mutated MSCs may have acquired the KIT mutation in a common pluripotent progenitor cell, prior to differentiation into MSCs and hematopoietic precursor cells, before the X-chromosome inactivation process occurs. From a clinical point of view, acquisition of the KIT mutation in an earlier BM precursor cell confers a significantly greater risk for disease progression and a poorer outcome. © 2016 by The American Society of Hematology.

Two novel sodium channel mutations associated with resistance to indoxacarb and metaflumizone in the diamondback moth, Plutella xylostella

PubMed Central

Wang, Xing-Liang; Su, Wen; Zhang, Jian-Heng; Yang, Yi-Hua; Dong, Ke; Wu, Yi-Dong

2015-01-01

Indoxacarb and metaflumizone belong to a relatively new class of sodium channel blocker insecticides (SCBIs). Due to intensive use of indoxacarb, field-evolved indoxacarb resistance has been reported in several lepidopteran pests including the diamondback moth, Plutella xylostella, a serious pest of cruciferous crops. In particular, the BY12 population of P. xylostella, collected from Baiyun, Guangdong province of China in 2012, was 750-fold more resistant to indoxacarb and 70-fold more resistant to metaflumizone compared with the susceptible Roth strain. Comparison of cDNA sequences encoding the sodium channel genes of Roth and BY12 revealed two point mutations (F1845Y and V1848I) in the 6th segment of domain IV of the PxNav protein in the BY population. Both mutations are located within a highly conserved sequence region that is predicted to be involved in the binding sites of local anesthetics and SCBIs based on mammalian sodium channels. A significant correlation was observed among ten field-collected populations between the mutant allele (Y1845 or I1848) frequencies (1.7% to 52.5%) and resistance levels to both indoxacarb (34- to 870-fold) and metaflumizone (1- to 70-fold). The two mutant alleles were never found to co-exist in the same allele of PxNav, suggesting that they arose independently. This is the first time that sodium channel mutations have been associated with high levels of resistance to SCBIs. F1845Y and V1848I are molecular markers for resistance monitoring in the diamondback moth and possibly other insect pest species. PMID:25850422

Analysis of trafficking, stability and function of human connexin 26 gap junction channels with deafness-causing mutations in the fourth transmembrane helix.

PubMed

Ambrosi, Cinzia; Walker, Amy E; Depriest, Adam D; Cone, Angela C; Lu, Connie; Badger, John; Skerrett, I Martha; Sosinsky, Gina E

2013-01-01

Human Connexin26 gene mutations cause hearing loss. These hereditary mutations are the leading cause of childhood deafness worldwide. Mutations in gap junction proteins (connexins) can impair intercellular communication by eliminating protein synthesis, mis-trafficking, or inducing channels that fail to dock or have aberrant function. We previously identified a new class of mutants that form non-functional gap junction channels and hemichannels (connexons) by disrupting packing and inter-helix interactions. Here we analyzed fourteen point mutations in the fourth transmembrane helix of connexin26 (Cx26) that cause non-syndromic hearing loss. Eight mutations caused mis-trafficking (K188R, F191L, V198M, S199F, G200R, I203K, L205P, T208P). Of the remaining six that formed gap junctions in mammalian cells, M195T and A197S formed stable hemichannels after isolation with a baculovirus/Sf9 protein purification system, while C202F, I203T, L205V and N206S formed hemichannels with varying degrees of instability. The function of all six gap junction-forming mutants was further assessed through measurement of dye coupling in mammalian cells and junctional conductance in paired Xenopus oocytes. Dye coupling between cell pairs was reduced by varying degrees for all six mutants. In homotypic oocyte pairings, only A197S induced measurable conductance. In heterotypic pairings with wild-type Cx26, five of the six mutants formed functional gap junction channels, albeit with reduced efficiency. None of the mutants displayed significant alterations in sensitivity to transjunctional voltage or induced conductive hemichannels in single oocytes. Intra-hemichannel interactions between mutant and wild-type proteins were assessed in rescue experiments using baculovirus expression in Sf9 insect cells. Of the four unstable mutations (C202F, I203T, L205V, N206S) only C202F and N206S formed stable hemichannels when co-expressed with wild-type Cx26. Stable M195T hemichannels displayed an increased tendency to aggregate. Thus, mutations in TM4 cause a range of phenotypes of dysfunctional gap junction channels that are discussed within the context of the X-ray crystallographic structure.

BCR-ABL1- positive chronic myeloid leukemia with erythrocytosis presenting as polycythemia vera: a case report.

PubMed

Cornea, Mihaela I Precup; Levrat, Emmanuel; Pugin, Paul; Betticher, Daniel C

2015-04-08

The World Health Organization classification of chronic myeloproliferative disease encompasses eight entities of bone marrow neoplasms, among them Breakpoint cluster region-Abelson murine leukemia viral oncogene homolog 1-positive chronic myeloid leukemia and polycythemia vera. Polycythemia vera requires, in the majority of cases (95%), the negativity of Breakpoint cluster region-Abelson murine leukemia viral oncogene homolog 1 rearrangement and the presence of the Janus kinase 2 mutation. We report a case of erythrocytosis as the primary manifestation of a chronic myeloid leukemia, with the presence of the Philadelphia chromosome and the Breakpoint cluster region-Abelson murine leukemia viral oncogene homolog 1 fusion gene, and in the absence of any Janus kinase 2 mutation. A 68-year-old Caucasian woman, with a history of cigarette consumption and obstructive sleep apnoea syndrome (undergoing continuous positive airway pressure treatment) had presented to our institution with fatigue and a hemoglobin level of 18.6g/L, with slight leukocytosis at 16G/L, and no other anomalies on her complete blood cell count. Examination of her arterial blood gases found only a slight hypoxemia; erythropoietin and ferritin levels were very low and could not explain a secondary erythrocytosis. Further analyses revealed the absence of any Janus kinase 2 mutation, thus excluding polycythemia vera. Taken together with a high vitamin B12 level, we conducted a Breakpoint cluster region-Abelson murine leukemia viral oncogene homolog 1 gene analysis and bone marrow cytogenetic analysis, both of which returned positive, leading to the diagnosis of chronic myeloid leukemia. To date, this case is the first description of a Breakpoint cluster region-Abelson murine leukemia viral oncogene homolog 1-positive chronic myeloid leukemia, presenting with erythrocytosis as the initial manifestation, and mimicking a Janus kinase 2 V617F-negative polycythemia vera. Her impressive response to imatinib therapy underscores the importance of not missing this diagnosis.

Selectivity in the Use of Gi/o Proteins Is Determined by the DRF Motif in CXCR6 and Is Cell-Type Specific.

PubMed

Singh, Satya P; Foley, John F; Zhang, Hongwei H; Hurt, Darrell E; Richards, Jennifer L; Smith, Craig S; Liao, Fang; Farber, Joshua M

2015-11-01

CXCR6, the receptor for CXCL16, is expressed on multiple cell types and can be a coreceptor for human immunodeficiency virus 1. Except for CXCR6, all human chemokine receptors contain the D(3.49)R(3.50)Y(3.51) sequence, and all but two contain A(3.53) at the cytoplasmic terminus of the third transmembrane helix (H3C), a region within class A G protein-coupled receptors that contacts G proteins. In CXCR6, H3C contains D(3.49)R(3.50)F(3.51)I(3.52)V(3.53) at positions 126-130. We investigated the importance and interdependence of the canonical D126 and the noncanonical F128 and V130 in CXCR6 by mutating D126 to Y, F128 to Y, and V130 to A singly and in combination. For comparison, we mutated the analogous positions D142, Y144, and A146 to Y, F, and V, respectively, in CCR6, a related receptor containing the canonical sequences. Mutants were analyzed in both human embryonic kidney 293T and Jurkat E6-1 cells. Our data show that for CXCR6 and/or CCR6, mutations in H3C can affect both receptor signaling and chemokine binding; noncanonical H3C sequences are functionally linked, with dual changes mitigating the effects of single mutations; mutations in H3C that compromise receptor activity show selective defects in the use of individual Gi/o proteins; and the effects of mutations in H3C on receptor function and selectivity in Gi/o protein use can be cell-type specific. Our findings indicate that the ability of CXCR6 to make promiscuous use of the available Gi/o proteins is exquisitely dependent on sequences within the H3C and suggest that the native sequence allows for preservation of this function across different cellular environments. U.S. Government work not protected by U.S. copyright.

Selectivity in the Use of Gi/o Proteins Is Determined by the DRF Motif in CXCR6 and Is Cell-Type Specific

PubMed Central

Singh, Satya P.; Foley, John F.; Zhang, Hongwei H.; Hurt, Darrell E.; Richards, Jennifer L.; Smith, Craig S.; Liao, Fang

2015-01-01

CXCR6, the receptor for CXCL16, is expressed on multiple cell types and can be a coreceptor for human immunodeficiency virus 1. Except for CXCR6, all human chemokine receptors contain the D3.49R3.50Y3.51 sequence, and all but two contain A3.53 at the cytoplasmic terminus of the third transmembrane helix (H3C), a region within class A G protein–coupled receptors that contacts G proteins. In CXCR6, H3C contains D3.49R3.50F3.51I3.52V3.53 at positions 126–130. We investigated the importance and interdependence of the canonical D126 and the noncanonical F128 and V130 in CXCR6 by mutating D126 to Y, F128 to Y, and V130 to A singly and in combination. For comparison, we mutated the analogous positions D142, Y144, and A146 to Y, F, and V, respectively, in CCR6, a related receptor containing the canonical sequences. Mutants were analyzed in both human embryonic kidney 293T and Jurkat E6-1 cells. Our data show that for CXCR6 and/or CCR6, mutations in H3C can affect both receptor signaling and chemokine binding; noncanonical H3C sequences are functionally linked, with dual changes mitigating the effects of single mutations; mutations in H3C that compromise receptor activity show selective defects in the use of individual Gi/o proteins; and the effects of mutations in H3C on receptor function and selectivity in Gi/o protein use can be cell-type specific. Our findings indicate that the ability of CXCR6 to make promiscuous use of the available Gi/o proteins is exquisitely dependent on sequences within the H3C and suggest that the native sequence allows for preservation of this function across different cellular environments. PMID:26316539

Succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency: two pathogenic mutations, V133E and C456F, in Japanese siblings.

PubMed

Song, X Q; Fukao, T; Watanabe, H; Shintaku, H; Hirayama, K; Kassovska-Bratinova, S; Kondo, N; Mitchell, G A

1998-01-01

Succinyl-CoA:3-ketoacid CoA transferase (SCOT; EC 2.8.3.5; locus symbol OXCT) is the key enzyme of ketone body utilization. Hereditary SCOT deficiency (MIM 245050) causes episodes of severe ketoacidosis. We developed a transient expression system for mutant SCOT cDNAs, using immortalized SCOT-deficient fibroblasts. This paper describes and characterizes three missense mutations in two SCOT-deficient siblings from Japan. They are genetic compounds who inherited the mutation C456F (c1367 G-->T) from their mother. Their paternal allele contains two mutations in cis, T58M (c173 C-->T) and V133E (c398T-->A). Expression of SCOT cDNAs containing either V133E or C456F produces no detectable SCOT activity, whereas T58M is functionally neutral. T58M is a rare sequence variant not detected in 100 control Japanese alleles. In fibroblasts from the proband (GS02), in whom immunoblot demonstrated no detectable SCOT peptide, we measured an apparent residual SCOT activity of 20-35%. We hypothesize that the high residual SCOT activity in homogenates may be an artifact caused by use of the substrate, acetoacetyl-CoA by other enzymes. Expression of mutant SCOT cDNAs more accurately reflects the residual activity of SCOT than do currently available assays in cell or tissue homogenates.

Tensile properties of haynes alloy 230 and inconel 617 after long exposures to LiF-22CaF2 and vacuum at 1093 K

NASA Astrophysics Data System (ADS)

Whittenberger, J. D.

1994-12-01

As a part of a study of a space-based thermal energy storage system utilizing the latent heat of fusion of the eutectic salt LiF-20CaF2 (mole%), the two wrought Ni-base superalloys Haynes alloy 230 and Inconel 617 were subjected to molten salt, its vapor, and vacuum for periods as long as 10,000 h at 1093 K. Following exposure, the microstructures were characterized, and samples from each superalloy were tensile tested between 77 and 1200 K. Neither the structure nor mechanical properties revealed evidence for additional degradation due to exposures to the salt. Although some loss in tensile properties was noted, particularly at 77 K, this reduction could be ascribed to the influence of simple aging at 1093 K.

HBV core promoter mutations promote cellular proliferation through E2F1-mediated upregulation of S-phase kinase-associated protein 2 transcription.

PubMed

Huang, Yuehua; Tai, Andrew W; Tong, Shuping; Lok, Anna S F

2013-06-01

Hepatitis B virus (HBV) core promoter (CP) mutations have been associated with an increased risk of hepatocellular carcinoma (HCC) in clinical studies. We previously reported that a combination of CP mutations seen in HCC patients, expressed in HBx gene, increased SKP2 (S-phase kinase-associated protein 2) expression, thereby promoting cellular proliferation. Here, we investigate the possible mechanisms by which CP mutations upregulate SKP2. We used immunoblotting and ATPlite assay to validate the effect of CP mutations in full-length HBV genome on cell cycle regulator levels and cell proliferation. Activation of SKP2 mRNA was assessed by quantitative real-time PCR in primary human hepatocytes (PHH) and HCC cell lines. Effect of CP mutations on SKP2 promoter activity was determined by luciferase assay. Target regulation of E2F1 on SKP2 was analyzed by siRNAs. CP mutations in full-length HBV genome upregulated SKP2 expression, thereby downregulating cell cycle inhibitors and accelerating cellular proliferation. CP mutations enhanced SKP2 promoter activity but had no effect on SKP2 protein stability. Mapping of the SKP2 promoter identified a region necessary for activation by CP mutations that contains an E2F1 response element. Knocking down E2F1 reduced the effects of CP mutations on SKP2 and cellular proliferation. The effect of CP mutations on E2F1 might be mediated through hyperphosphorylation of RB. HBV CP mutations enhance SKP2 transcription by activating the E2F1 transcription factor and in turn downregulate cell cycle inhibitors, thus providing a potential mechanism for an association between CP mutations and HCC. Copyright © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Unraveling the spectrum of KIT mutations in gastrointestinal stromal tumors: An Indian Tertiary Cancer Center Experience.

PubMed

Pai, Trupti; Bal, Munita; Shetty, Omshree; Gurav, Mamta; Ostwal, Vikas; Ramaswamy, Anant; Ramadwar, Mukta; Desai, Sangeeta

2017-01-01

Primary mutations in the KIT gene are the driving force for gastrointestinal stromal tumors (GIST) tumorigenesis. Predictive role of KIT mutation status aids oncologists in patient management. There is a paucity of comprehensive data on the frequency of mutations in the KIT gene in GIST affecting Indian patients. The aims of this study were to determine the frequency and spectrum of molecular alterations affecting the KIT gene and assess their association with clinicopathologic features in a cohort of patients of GIST. Morphological and immunohistochemically confirmed GIST cases ( n = 114) accessioned from August 2014-June 2015 were analyzed for mutations in KIT exons 9, 11, 13, and 17 and subjected to Sanger sequencing onto the ABI 3500 Genetic Analyzer. The sequences were analyzed using sequence analysis software: SeqScape ® and Chromas Lite. KIT mutations were seen in 70% of cases and the majority of KIT mutations involved exon 11 (57%), followed by exon 9 (10%), exon 13 (3%), and exon 17 (1%). Most common exon 11 mutations were in-frame deletions (61.4%) followed by substitution mutations (19.3%). Exon 9 mutations showed identical duplication of Ala-Tyr at codons 502-503. Simultaneous mutations affecting exon 11 and 13 were discovered. Novel variations, namely, p.Q556E (c.1666C>G), p.Q556dup (c.1666_1668dupCAG), p.K558_V559delinsS (c.1672_1677delAAGGTTinsAGT), p.Y503_F504insTY (c.1509_1510insACCTAT), and p.K642R (c.1925A>G) involving exons 11, 9, and 13, respectively, were observed. First study with complete analysis of all 4 exons of KIT (exons 9, 11, 13, and 17) in Indian GIST patients. Along with well-described KIT mutations, several rare double mutations as well as novel alterations were reported in this series.

Synergistic effect of factor VII gene polymorphisms causing mild factor VII deficiency in a case of severe factor X deficiency.

PubMed

Deshpande, Rutuja; Ghosh, Kanjaksha; Shetty, Shrimati

2017-01-01

Congenital combined deficiency of coagulation factors VII and X are mainly attributed to large deletions involving both the genes in chromosome 13 or occasionally due to the coincidental occurrence of independently occurring mutations. We report the molecular basis of congenital combined deficiency of factors VII and X in a 6-year-old female child. Direct DNA sequencing of both factor VII (F7) and factor X (F10) genes showed a novel homozygous missense mutation p.Cys90Tyr (c.307G>A) in exon 4 of F10. No mutations were detected in F7; however, the patient was homozygous for three polymorphic alleles known to be associated with reduced factor VII levels. The present case illustrates the synergistic effect of multiple polymorphisms resulting in phenotypic factor VII deficiency in the absence of a pathogenic mutation.

Emergence of novel and dominant acquired EGFR solvent-front mutations at Gly796 (G796S/R) together with C797S/R and L792F/H mutations in one EGFR (L858R/T790M) NSCLC patient who progressed on osimertinib.

PubMed

Ou, Sai-Hong Ignatius; Cui, Jean; Schrock, Alexa B; Goldberg, Michael E; Zhu, Viola W; Albacker, Lee; Stephens, Philip J; Miller, Vincent A; Ali, Siraj M

2017-06-01

Acquired epidermal growth factor receptor (EGFR) resistance mutations to osimertinib are common, including the EGFR C797S that abolishes the covalent binding of osimertinib to EGFR. Here we report the emergence of novel EGFR solvent front mutations at Gly796 (G796S/R) in addition to a hinge pocket L792F/H mutations, and C797S/G all in cis with T790M in a single patient on progression on osimertinib as detected by plasma circulating tumor DNA (ctDNA) assay in the course of clinical care. A 69-year-old Caucasian female former light-smoker presented with stage IV EGFR L858R positive adenocarcinoma who developed EGFR T790M mutation after 8 month treatment of erlotinib. The patient was initiated on osimertinib with disease shrinkage after 2 months, but tumor regrowth was observed after 5 months of osimertinib treatment. Assay of plasma ctDNA at this time revealed these different secondary resistance mutations all in trans with each other including distinct mutations at the same codon producing different amino acid changes: G796S/R (mutant allele frequency [MAF]; 14.4%), C797S/G (MAF: 2.26%), L792F/H (MAF: 0.36%), and V802F (MAF: 0.40%), in addition to the pre-existing L858R (MAF:17.9%) and T790M (MAF:18.2%) but all in cis with T790M. The G796S/R mutations are homologous with known reported solvent front mutations in ALK G1202R, ROS1 G2032R, TrkA G595R and TrkC G623R, all of which are associated with acquired resistance to type I TKIs. In silico modeling revealed mutation at G796 interferes with osimertinib binding to the EGFR kinase domain at the phenyl aromatic ring position as this residue forms a narrow "hydrophobic sandwich" with L718, while L792F/H mutation interferes with osimertinib binding at the methoxyl group on the phenyl ring. Multiple resistance mutations at differing allele frequencies including novel EGFR solvent front mutations can emerge in a single patient with progression on osimertinib potentially due to tumor hetereogeneity and definitely present a significant therapeutic and drug development challenge. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

Cystic fibrosis transmembrane conductance regulator mutations at a referral center for cystic fibrosis.

PubMed

Coutinho, Cyntia Arivabeni de Araújo Correia; Marson, Fernando Augusto de Lima; Ribeiro, Antônio Fernando; Ribeiro, José Dirceu; Bertuzzo, Carmen Silvia

2013-01-01

To determine the frequency of six mutations (F508del, G542X, G551D, R553X, R1162X, and N1303K) in patients with cystic fibrosis (CF) diagnosed, at a referral center, on the basis of abnormal results in two determinations of sweat sodium and chloride concentrations. This was a cross-sectional study involving 70 patients with CF. The mean age of the patients was 12.38 ± 9.00 years, 51.43% were female, and 94.29% were White. Mutation screening was performed with polymerase chain reaction (for F508del), followed by enzymatic digestion (for other mutations). Clinical analysis was performed on the basis of gender, age, ethnicity, pulmonary/gastrointestinal symptoms, and Shwachman-Kulczycki (SK) score. All of the patients showed pulmonary symptoms, and 8 had no gastrointestinal symptoms. On the basis of the SK scores, CF was determined to be mild, moderate, and severe in 22 (42.3%), 17 (32.7%), and 13 (25.0%) of the patients, respectively. There was no association between F508del mutation and disease severity by SK score. Of the 140 alleles analyzed, F508del mutation was identified in 70 (50%). Other mutations (G542X, G551D, R553X, R1162X, and N1303K) were identified in 12 (7.93%) of the alleles studied. In F508del homozygous patients with severe disease, the OR was 0.124 (95% CI: 0.005-0.826). In 50% of the alleles studied, the molecular diagnosis of CF was confirmed by identifying a single mutation (F508del). If we consider the analysis of the six most common mutations in the Brazilian population (including F508del), the molecular diagnosis was confirmed in 58.57% of the alleles studied.

Multifunctional imaging signature for V-KI-RAS2 Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations in colorectal cancer.

PubMed

Miles, Kenneth A; Ganeshan, Balaji; Rodriguez-Justo, Manuel; Goh, Vicky J; Ziauddin, Zia; Engledow, Alec; Meagher, Marie; Endozo, Raymondo; Taylor, Stuart A; Halligan, Stephen; Ell, Peter J; Groves, Ashley M

2014-03-01

This study explores the potential for multifunctional imaging to provide a signature for V-KI-RAS2 Kirsten rat sarcoma viral oncogene homolog (KRAS) gene mutations in colorectal cancer. This prospective study approved by the institutional review board comprised 33 patients undergoing PET/CT before surgery for proven primary colorectal cancer. Tumor tissue was examined histologically for presence of the KRAS mutations and for expression of hypoxia-inducible factor-1 (HIF-1) and minichromosome maintenance protein 2 (mcm2). The following imaging parameters were derived for each tumor: (18)F-FDG uptake ((18)F-FDG maximum standardized uptake value [SUVmax]), CT texture (expressed as mean of positive pixels [MPP]), and blood flow measured by dynamic contrast-enhanced CT. A recursive decision tree was developed in which the imaging investigations were applied sequentially to identify tumors with KRAS mutations. Monte Carlo analysis provided mean values and 95% confidence intervals for sensitivity, specificity, and accuracy. The final decision tree comprised 4 decision nodes and 5 terminal nodes, 2 of which identified KRAS mutants. The true-positive rate, false-positive rate, and accuracy (95% confidence intervals) of the decision tree were 82.4% (63.9%-93.9%), 0% (0%-10.4%), and 90.1% (79.2%-96.0%), respectively. KRAS mutants with high (18)F-FDG SUVmax and low MPP showed greater frequency of HIF-1 expression (P = 0.032). KRAS mutants with low (18)F-FDG SUV(max), high MPP, and high blood flow expressed mcm2 (P = 0.036). Multifunctional imaging with PET/CT and recursive decision-tree analysis to combine measurements of tumor (18)F-FDG uptake, CT texture, and perfusion has the potential to identify imaging signatures for colorectal cancers with KRAS mutations exhibiting hypoxic or proliferative phenotypes.

Characterization of an apparently synonymous F5 mutation causing aberrant splicing and factor V deficiency.

PubMed

Nuzzo, F; Bulato, C; Nielsen, B I; Lee, K; Wielders, S J; Simioni, P; Key, N S; Castoldi, E

2015-03-01

Coagulation factor V (FV) deficiency is a rare autosomal recessive bleeding disorder. We investigated a patient with severe FV deficiency (FV:C < 3%) and moderate bleeding symptoms. Thrombin generation experiments showed residual FV expression in the patient's plasma, which was quantified as 0.7 ± 0.3% by a sensitive prothrombinase-based assay. F5 gene sequencing identified a novel missense mutation in exon 4 (c.578G>C, p.Cys193Ser), predicting the abolition of a conserved disulphide bridge, and an apparently synonymous variant in exon 8 (c.1281C>G). The observation that half of the patient's F5 mRNA lacked the last 18 nucleotides of exon 8 prompted us to re-evaluate the c.1281C>G variant for its possible effects on splicing. Bioinformatics sequence analysis predicted that this transversion would activate a cryptic donor splice site and abolish an exonic splicing enhancer. Characterization in a F5 minigene model confirmed that the c.1281C>G variant was responsible for the patient's splicing defect, which could be partially corrected by a mutation-specific morpholino antisense oligonucleotide. The aberrantly spliced F5 mRNA, whose stability was similar to that of the normal mRNA, encoded a putative FV mutant lacking amino acids 427-432. Expression in COS-1 cells indicated that the mutant protein is poorly secreted and not functional. In conclusion, the c.1281C>G mutation, which was predicted to be translationally silent and hence neutral, causes FV deficiency by impairing pre-mRNA splicing. This finding underscores the importance of cDNA analysis for the correct assessment of exonic mutations. © 2014 John Wiley & Sons Ltd.

Quantification of mutant SPOP proteins in prostate cancer using mass spectrometry-based targeted proteomics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Hui; Barbieri, Christopher E.; He, Jintang

Speckle-type POZ protein (SPOP) is an E3 ubiquitin ligase adaptor protein that functions as a potential tumor suppressor, and SPOP mutations have been identified in ~10% of human prostate cancers. However, it remains unclear if mutant SPOP proteins can be utilized as biomarkers for early detection, diagnosis, prognosis or targeted therapy of prostate cancer. Moreover, the SPOP mutation sites are distributed in a relatively short region where multiple lysine residues, posing significant challenges for bottom-up proteomics analysis of the SPOP mutations. To address this issue, PRISM (high-pressure, high-resolution separations coupled with intelligent selection and multiplexing)-SRM (selected reaction monitoring) mass spectrometrymore » assays have been developed for quantifying wild-type SPOP protein and 11 prostate cancer-derived SPOP mutations. Despite inherent limitations due to amino acid sequence constraints, all the PRISM-SRM assays developed using Arg-C digestion showed a linear dynamic range of at least two orders of magnitude, with limits of quantification range from 0.1 to 1 fmol/μg of total protein in the cell lysate. Applying these SRM assays to analyze HEK293T cells with and without expression of the three most frequent SPOP mutations in prostate cancer (Y87N, F102C or F133V) led to confident detection of all three SPOP mutations in corresponding positive cell lines but not in the negative cell lines. Expression of the F133V mutation and wild-type SPOP was at much lower levels compared to that of F102C and Y87N mutations; however, at present it is unknown if this also affects the activity of the SPOP protein. In summary, PRISM-SRM enables multiplexed, isoform-specific detection of mutant SPOP proteins in cell lysates, which holds great potential in biomarker development for prostate cancer.« less

Clinical impact of factor V Leiden, prothrombin G20210A, and MTHFR C677T mutations among sickle cell disease patients of Central India.

PubMed

Nishank, Sudhansu Sekhar; Singh, Mendi Prema Shyam Sunder; Yadav, Rajiv

2013-11-01

It is known that patients with sickle cell disease (SCD) present activation of the blood coagulation and fibrinolytic systems, especially during vaso-occlusive crises and also during the steady state of the disease. We determined whether the presence of the factor prothrombin gene G20210A variant, factor V gene G1691A mutation (factor V Leiden), and methylenetetrahydrofolate reductase (MTHFR) C677T polymorphisms may be risk factors for vascular complications in individuals with SCD. The study involved 150 patients with sickle cell anemia and 150 healthy controls of Central India. Genotyping of three thrombophilic mutations was carried out by PCR-RFLP methods using MnlI, Hind III, and Hinf I, respectively, for factor V Leiden, prothrombin, and MTHFR mutations. Patients with SCD had significantly higher prevalence of mutant variants of MTHFR gene (28.0% heterozygotes and 14.6% homozygotes) and FVL gene (14.6% heterozygotes) as compared to normal/control individuals, but complete absence of mutant variants of prothrombin gene. The patients with SCD having mutant variants of MTHFR and FVL genes showed higher incidence of pain in chest, abdomen, and bone joints along with early age of onset of clinical manifestations as well as frequent dependence on blood transfusion than those patients with SCD having wild variants of these thrombotic genes. As compared to control subjects, SCD individuals having mutant variants of FVL and MTHFR genes had significant association with higher levels of prothrombin fragment (F1+2), D-dimer, thrombin-antithrombin (TAT), and lower level of protein C. MTHFR C677T and FVL G1691A polymorphisms may be risk factors for increased vascular complications in patient with SCD. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Incomplete APOBEC3G/F Neutralization by HIV-1 Vif Mutants Facilitates the Genetic Evolution from CCR5 to CXCR4 Usage

PubMed Central

Alteri, Claudia; Surdo, Matteo; Bellocchi, Maria Concetta; Saccomandi, Patrizia; Continenza, Fabio; Armenia, Daniele; Parrotta, Lucia; Carioti, Luca; Costa, Giosuè; Fourati, Slim; Di Santo, Fabiola; Scutari, Rossana; Barbaliscia, Silvia; Fedele, Valentina; Carta, Stefania; Balestra, Emanuela; Alcaro, Stefano; Marcelin, Anne Genevieve; Calvez, Vincent; Ceccherini-Silberstein, Francesca; Artese, Anna

2015-01-01

Incomplete APOBEC3G/F neutralization by a defective HIV-1Vif protein can promote genetic diversification by inducing G-to-A mutations in the HIV-1 genome. The HIV-1 Env V3 loop, critical for coreceptor usage, contains several putative APOBEC3G/F target sites. Here, we determined if APOBEC3G/F, in the presence of Vif-defective HIV-1 virus, can induce G-to-A mutations at V3 positions critical to modulation of CXCR4 usage. Peripheral blood mononuclear cells (PBMC) and monocyte-derived macrophages (MDM) from 2 HIV-1-negative donors were infected with CCR5-using 81.A-VifWT virus (i.e., with wild-type [WT] Vif protein), 81.A-VifE45G, or 81.A-VifK22E (known to incompletely/partially neutralize APOBEC3G/F). The rate of G-toA mutations was zero or extremely low in 81.A-VifWT- and 81.A-VifE45G-infected PBMC from both donors. Conversely, G-to-A enrichment was detected in 81.A-VifK22E-infected PBMC (prevalence ranging from 2.18% at 7 days postinfection [dpi] to 3.07% at 21 dpi in donor 1 and from 10.49% at 7 dpi to 8.69% at 21 dpi in donor 2). A similar scenario was found in MDM. G-to-A mutations occurred at 8 V3 positions, resulting in nonsynonymous amino acid substitutions. Of them, G24E and E25K strongly correlated with phenotypically/genotypically defined CXCR4-using viruses (P = 0.04 and 5.5e−7, respectively) and increased the CXCR4 N-terminal binding affinity for V3 (WT, −40.1 kcal/mol; G24E, −510 kcal/mol; E25K, −522 kcal/mol). The analysis of paired V3 and Vif DNA sequences from 84 HIV-1-infected patients showed that the presence of a Vif-defective virus correlated with CXCR4 usage in proviral DNA (P = 0.04). In conclusion, incomplete APOBEC3G/F neutralization by a single Vif amino acid substitution seeds a CXCR4-using proviral reservoir. This can have implications for the success of CCR5 antagonist-based therapy, as well as for the risk of disease progression. PMID:26055363

V67L Mutation Fills an Internal Cavity To Stabilize RecA Mtu Intein

DOE PAGES

Zwarycz, Allison S.; Fossat, Martin; Akanyeti, Otar; ...

2017-05-10

Inteins mediate protein splicing, which has found extensive applications in protein science and biotechnology. In the Mycobacterium tuberculosis RecA mini–mini intein (ΔΔIhh), a single valine to leucine substitution at position 67 (V67L) dramatically increases intein stability and activity. However, crystal structures show that the V67L mutation causes minimal structural rearrangements, with a root-mean-square deviation of 0.2 Å between ΔΔIhh-V67 and ΔΔIhh-L67. Thus, the structural mechanisms for V67L stabilization and activation remain poorly understood. In this paper, we used intrinsic tryptophan fluorescence, high-pressure nuclear magnetic resonance (NMR), and molecular dynamics (MD) simulations to probe the structural basis of V67L stabilization ofmore » the intein fold. Guanidine hydrochloride denaturation monitored by fluorescence yielded free energy changes (ΔG f°) of -4.4 and -6.9 kcal mol –1 for ΔΔIhh-V67 and ΔΔIhh-L67, respectively. High-pressure NMR showed that ΔΔIhh-L67 is more resistant to pressure-induced unfolding than ΔΔIhh-V67 is. The change in the volume of folding (ΔV f) was significantly larger for V67 (71 ± 2 mL mol –1) than for L67 (58 ± 3 mL mol –1) inteins. The measured difference in ΔV f (13 ± 3 mL mol –1) roughly corresponds to the volume of the additional methylene group for Leu, supporting the notion that the V67L mutation fills a nearby cavity to enhance intein stability. In addition, we performed MD simulations to show that V67L decreases side chain dynamics and conformational entropy at the active site. Finally, it is plausible that changes in cavities in V67L can also mediate allosteric effects to change active site dynamics and enhance intein activity.« less

V67L Mutation Fills an Internal Cavity To Stabilize RecA Mtu Intein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zwarycz, Allison S.; Fossat, Martin; Akanyeti, Otar

Inteins mediate protein splicing, which has found extensive applications in protein science and biotechnology. In the Mycobacterium tuberculosis RecA mini–mini intein (ΔΔIhh), a single valine to leucine substitution at position 67 (V67L) dramatically increases intein stability and activity. However, crystal structures show that the V67L mutation causes minimal structural rearrangements, with a root-mean-square deviation of 0.2 Å between ΔΔIhh-V67 and ΔΔIhh-L67. Thus, the structural mechanisms for V67L stabilization and activation remain poorly understood. In this paper, we used intrinsic tryptophan fluorescence, high-pressure nuclear magnetic resonance (NMR), and molecular dynamics (MD) simulations to probe the structural basis of V67L stabilization ofmore » the intein fold. Guanidine hydrochloride denaturation monitored by fluorescence yielded free energy changes (ΔG f°) of -4.4 and -6.9 kcal mol –1 for ΔΔIhh-V67 and ΔΔIhh-L67, respectively. High-pressure NMR showed that ΔΔIhh-L67 is more resistant to pressure-induced unfolding than ΔΔIhh-V67 is. The change in the volume of folding (ΔV f) was significantly larger for V67 (71 ± 2 mL mol –1) than for L67 (58 ± 3 mL mol –1) inteins. The measured difference in ΔV f (13 ± 3 mL mol –1) roughly corresponds to the volume of the additional methylene group for Leu, supporting the notion that the V67L mutation fills a nearby cavity to enhance intein stability. In addition, we performed MD simulations to show that V67L decreases side chain dynamics and conformational entropy at the active site. Finally, it is plausible that changes in cavities in V67L can also mediate allosteric effects to change active site dynamics and enhance intein activity.« less

Prevalence of etravirine-associated mutations in clinical samples with resistance to nevirapine and efavirenz.

PubMed

Llibre, J M; Santos, J R; Puig, T; Moltó, J; Ruiz, L; Paredes, R; Clotet, B

2008-11-01

To evaluate the expected activity of etravirine in clinical samples, according to mutational patterns associated with decreased virological response (VR). We identified 1586 routine clinical samples with resistance-associated mutations (RAMs) to nevirapine and efavirenz (K103N 60%, Y181C 37%, G190A 27%, V108I 13%). Concerning in vitro identified etravirine mutations, samples with F227C, Y181I, M230L or L100I plus K103N plus Y181C were considered highly resistant. Samples with two RAMs plus Y181C or V179D or K101E or Y188L were considered intermediate. The prevalence of 13 RAMs recently associated with decreased VR to etravirine in the DUET clinical trials was also investigated. Most samples (69%) harboured more than one IAS-USA RAM to first-generation non-nucleoside reverse transcriptase inhibitors (NNRTIs): 42% harboured two RAMs, 21% three RAMs and 6% four or more RAMs. The prevalence of 13 specific etravirine RAMs was V179F 0.12%, G190S 3.9%, Y181V 0.1%, V106I 2.6%, V179D 1.6%, K101P 2.0%, K101E 10.1%, Y181C 36.9%, A98G 5.9%, V90I 6.9%, Y181I 3.6%, G190A 27% and L100I 9.1%. The five RAMs with the most impact on VR (V179F/D, G190S, Y181V and V106I) occurred less often. Overall, 8.2% of the samples had three or more etravirine RAMs and only 1.1% had four or more. In addition, patterns of RAMs previously associated with intermediate etravirine resistance were present in 26.2% of the samples, whereas 4.85% displayed patterns of high-degree resistance. For RAMs associated with decreased VR, etravirine resistance in routine clinical samples was lower than previously reported. High-degree resistance was uncommon, even in patients with resistance to first-generation NNRTIs, whereas low-to-intermediate etravirine resistance was more common.

Effects of a mutation on the folding mechanism of a beta-hairpin.

PubMed

Juraszek, Jarek; Bolhuis, Peter G

2009-12-17

The folding mechanism of a protein is determined by its primary sequence. Yet, how the mechanism is changed by a mutation is still poorly understood, even for basic secondary structures such as beta-hairpins. We perform an extensive simulation study of the effects of mutating the GB1 beta-hairpin into Trpzip4 (Y5W, F12W, V14W) on the folding mechanism. While Trpzip4 has a much more stable native state due to very strong hydrophobic interactions of the side chains, its folding rate does not differ significantly from the wild type beta-hairpin. We sample the free-energy landscapes of both hairpins with Replica Exchange Molecular Dynamics (REMD) and identify the four (meta)stable states (U, H, F, and N). Using Transition Path Sampling (TPS), we then harvest ensembles of unbiased pathways between the H and F states and between the F and N states to investigate the unbiased folding mechanisms. In both hairpins, the hydrophobic collapse (U-H) is followed by the middle hydrogen bond formation (H-F), and finally a closing of the strands in a zipper-like fashion (F-N). For the Trpzip4, the path ensembles indicate that the final F-N step is much more difficult than for GB1 and involves partial unfolding, rezipping of hydrogen bonds, and rearrangement of the Trp-14 side chain. For the rate-limiting (H-F) step, the path ensembles show that in GB1 desolvation and strand closure go hand in hand, while in Trpzip4 desolvation is decoupled from strand closure. Nevertheless, likelihood maximization shows that the reaction coordinate for both hairpins remains the interstrand distance. We conclude that the folding mechanism of both hairpins is a combination of hydrophobic collapse and zipping of hydrogen bonds but that the zipper mechanism is more visible in Trpzip4. A major difference between the two hairpins is that in the transition state of the rate-limiting step for Trpzip4 one tryptophan is exposed to the solvent due to steric hindrance, making the folding mechanism more complex and leading to an increased F-N barrier. Thus, our results show in atomistic detail how a mutation leads to a different folding mechanism and results in a more frustrated folding free-energy landscape.

MIL-STD-1760 Application Guidelines

DTIC Science & Technology

1991-09-01

64 6.9 Video Switch Functional Diagram ......................................................... 67 6.10 F-16... Video Control .............................................................................. 68 6.11 F-16 Video System...76 6.17 Second Video Line to STAs 4, 5, and 6 1760 ASIs ................................ 77 6.18 Functional

Expanding the substrate scope of phenylalanine ammonia-lyase from Petroselinum crispum towards styrylalanines.

PubMed

Bencze, László Csaba; Filip, Alina; Bánóczi, Gergely; Toşa, Monica Ioana; Irimie, Florin Dan; Gellért, Ákos; Poppe, László; Paizs, Csaba

2017-05-03

This study focuses on the expansion of the substrate scope of phenylalanine ammonia-lyase from Petroselinum crispum (PcPAL) towards the l-enantiomers of racemic styrylalanines rac-1a-d - which are less studied and synthetically challenging unnatural amino acids - by reshaping the aromatic binding pocket of the active site of PcPAL by point mutations. Ammonia elimination from l-styrylalanine (l-1a) catalyzed by non-mutated PcPAL (wt-PcPAL) took place with a 777-fold lower k cat /K M value than the deamination of the natural substrate, l-Phe. Computer modeling of the reactions catalyzed by wt-PcPAL indicated an unproductive and two major catalytically active conformations and detrimental interactions between the aromatic moiety of l-styrylalanine, l-1a, and the phenyl ring of the residue F137 in the aromatic binding region of the active site. Replacing the residue F137 by smaller hydrophobic residues resulted in a small mutant library (F137X-PcPAL, X being V, A, and G), from which F137V-PcPAL could transform l-styrylalanine with comparable activity to that of the wt-PcPAL with l-Phe. Furthermore, F137V-PcPAL showed superior catalytic efficiency in the ammonia elimination reaction of several racemic styrylalanine derivatives (rac-1a-d) providing access to d-1a-d by kinetic resolution, even though the d-enantiomers proved to be reversible inhibitors. The enhanced catalytic efficiency of F137V-PcPAL towards racemic styrylalanines rac-1a-d could be rationalized by molecular modeling, indicating the more relaxed enzyme-substrate complexes and the promotion of conformations with higher catalytic activities as the main reasons. Unfortunately, ammonia addition onto the corresponding styrylacrylates 2a-d failed with both wt-PcPAL and F137V-PcPAL. The low equilibrium constant of the ammonia addition, the poor ligand binding affinities of 2a-d, and the non-productive binding states of the unsaturated ligands 2a-d within the active sites of either wt-PcPAL or F137V-PcPAL - as indicated by molecular modeling - might be responsible for the inactivity of the PcPAL variants in the reverse reaction. Modeling predicted that the F137V mutation is beneficial for the KRs of 4-fluoro-, 4-cyano- and 4-bromostyrylalanines, but non-effective for the KR process of 4-trifluoromethylstyrylalanine.

Mpl traffics to the cell surface through conventional and unconventional routes.

PubMed

Cleyrat, Cédric; Darehshouri, Anza; Steinkamp, Mara P; Vilaine, Mathias; Boassa, Daniela; Ellisman, Mark H; Hermouet, Sylvie; Wilson, Bridget S

2014-09-01

Myeloproliferative neoplasms (MPNs) are often characterized by JAK2 or calreticulin (CALR) mutations, indicating aberrant trafficking in pathogenesis. This study focuses on Mpl trafficking and Jak2 association using two model systems: human erythroleukemia cells (HEL; JAK2V617F) and K562 myeloid leukemia cells (JAK2WT). Consistent with a putative chaperone role for Jak2, Mpl and Jak2 associate on both intracellular and plasma membranes (shown by proximity ligation assay) and siRNA-mediated knockdown of Jak2 led to Mpl trapping in the endoplasmic reticulum (ER). Even in Jak2 sufficient cells, Mpl accumulates in punctate structures that partially colocalize with ER-tracker, the ER exit site marker (ERES) Sec31a, the autophagy marker LC3 and LAMP1. Mpl was fused to miniSOG, a genetically encoded tag for correlated light and electron microscopy. Results suggest that a fraction of Mpl is taken up into autophagic structures from the ER and routed to autolyososomes. Surface biotinylation shows that both immature and mature Mpl reach the cell surface; in K562 cells Mpl is also released in exosomes. Both forms rapidly internalize upon ligand addition, while recovery is primarily attributed to immature Mpl. Mpl appears to reach the plasma membrane via both conventional ER-Golgi and autolysosome secretory pathways, as well as recycling. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Multifaceted Intervention by the Hsp90 Inhibitor Ganetespib (STA-9090) in Cancer Cells with Activated JAK/STAT Signaling

PubMed Central

Proia, David A.; Foley, Kevin P.; Korbut, Tim; Sang, Jim; Smith, Don; Bates, Richard C.; Liu, Yuan; Rosenberg, Alex F.; Zhou, Dan; Koya, Keizo; Barsoum, James; Blackman, Ronald K.

2011-01-01

There is accumulating evidence that dysregulated JAK signaling occurs in a wide variety of cancer types. In particular, mutations in JAK2 can result in the constitutive activation of STAT transcription factors and lead to oncogenic growth. JAK kinases are established Hsp90 client proteins and here we show that the novel small molecule Hsp90 inhibitor ganetespib (formerly STA-9090) exhibits potent in vitro and in vivo activity in a range of solid and hematological tumor cells that are dependent on JAK2 activity for growth and survival. Of note, ganetespib treatment results in sustained depletion of JAK2, including the constitutively active JAK2V617F mutant, with subsequent loss of STAT activity and reduced STAT-target gene expression. In contrast, treatment with the pan-JAK inhibitor P6 results in only transient effects on these processes. Further differentiating these modes of intervention, RNA and protein expression studies show that ganetespib additionally modulates cell cycle regulatory proteins, while P6 does not. The concomitant impact of ganetespib on both cell growth and cell division signaling translates to potent antitumor efficacy in mouse models of xenografts and disseminated JAK/STAT-driven leukemia. Overall, our findings support Hsp90 inhibition as a novel therapeutic approach for combating diseases dependent on JAK/STAT signaling, with the multimodal action of ganetespib demonstrating advantages over JAK-specific inhibitors. PMID:21533169

Evaluation of the association between the JAK2 46/1 haplotype and chronic myeloproliferative neoplasms in a Brazilian population

PubMed Central

Silva, Sarah Pagliarini- e-; Santos, Bruna Cunha; de Figueiredo Pereira, Elizangela Mendes; Ferreira, Mari Ellen; Baraldi, Elaine Cristina; Sell, Ana Maria; Visentainer, Jeane Eliete Laguila

2013-01-01

OBJECTIVE: The JAK2 46/1 haplotype has recently been described as a major contributing factor to the development of myeloproliferative neoplasm, whether positive or negative for the JAK2 V617F mutation. The G allele, identified by a single-nucleotide polymorphism known as JAK2 rs10974944, is part of the JAK2 46/1 haplotype. The aim of this study was to verify the association between the presence of the G allele and the development of BCR-ABL-negative chronic myeloproliferative neoplasms in our population. METHODS: Blood and oral mucosa swab samples were obtained from 56 patients of two local Brazilian hospitals who had previously been diagnosed with BCR-ABL-negative chronic myeloproliferative neoplasms. Blood samples from 90 local blood donors were used as controls. The presence of the G allele was assessed using a PCR-RFLP assay after extracting DNA from the samples. RESULTS: The presence of the G allele was strongly associated with the presence of BCR-ABL-negative chronic myeloproliferative neoplasms (p = 0.0001; OR = 2.674; 95% CI = 1.630−4.385) in the studied population. CONCLUSION: In agreement with previous reports, the JAK2 46/1 haplotype, represented in this study by the presence of the G allele, is an important predisposing factor in the oncogenetic development of these neoplasms in our population. PMID:23420150

First report of HGD mutations in a Chinese with alkaptonuria.

PubMed

Yang, Yong-jia; Guo, Ji-hong; Chen, Wei-jian; Zhao, Rui; Tang, Jin-song; Meng, Xiao-hua; Zhao, Liu; Tu, Ming; He, Xin-yu; Wu, Ling-qian; Zhu, Yi-min

2013-04-15

Alkaptonuria (AKU) is one of the first prototypic inborn errors in metabolism and the first human disease found to be transmitted via Mendelian autosomal recessive inheritance. It is caused by HGD mutations, which leads to a deficiency in homogentisate 1,2-dioxygenase (HGD) activity. To date, several HGD mutations have been identified as the cause of the prototypic disease across different ethnic populations worldwide. However, in Asia, the HGD mutation is very rarely reported. For the Chinese population, no literature on HGD mutation screening is available to date. In this paper, we describe two novel HGD mutations in a Chinese AKU family, the splicing mutation of IVS7+1G>C, a donor splice site of exon 7, and a missense mutation of F329C in exon 12. The predicted new splicing site of the mutated exon 7 sequence demonstrated a 303bp extension after the mutation site. The F329C mutation most probably disturbed the stability of the conformation of the two loops critical to the Fe(2+) active site of the HGD enzyme. Copyright © 2013 Elsevier B.V. All rights reserved.

Serbo-Croatian. SC-15A. Part 1. Basic Structure,

DTIC Science & Technology

1983-09-30

regularly replace certain others in specific linguistic conditions. There are two main kinds of such mutations . One set is associated with sonority (voicing...or non-voicing), the other with palatalization. (a) Mutations caused by voicing or devoicing concern the plosive and sibilant consonants, since no... mutations exist of consonants f, h, c, J, r, v. The plosives and sibilants mutate as follows: p alternates with b p/b s alternates with z s/z t alternates

Functional interaction of CCAAT/enhancer-binding-protein-α basic region mutants with E2F transcription factors and DNA.

PubMed

Kowenz-Leutz, Elisabeth; Schuetz, Anja; Liu, Qingbin; Knoblich, Maria; Heinemann, Udo; Leutz, Achim

2016-07-01

The transcription factor CCAAT/enhancer-binding protein α (C/EBPα) regulates cell cycle arrest and terminal differentiation of neutrophils and adipocytes. Mutations in the basic leucine zipper domain (bZip) of C/EBPα are associated with acute myeloid leukemia. A widely used murine transforming C/EBPα basic region mutant (BRM2) entails two bZip point mutations (I294A/R297A). BRM2 has been discordantly described as defective for DNA binding or defective for interaction with E2F. We have separated the two BRM2 mutations to shed light on the intertwined reciprocity between C/EBPα-E2F-DNA interactions. Both, C/EBPα I294A and R297A retain transactivation capacity and interaction with E2F-DP. The C/EBPα R297A mutation destabilized DNA binding, whereas the C/EBPα I294A mutation enhanced binding to DNA. The C/EBPα R297A mutant, like BRM2, displayed enhanced interaction with E2F-DP but failed to repress E2F-dependent transactivation although both mutants were readily suppressed by E2F1 for transcription through C/EBP cis-regulatory sites. In contrast, the DNA binding enhanced C/EBPα I294A mutant displayed increased repression of E2F-DP mediated transactivation and resisted E2F-DP mediated repression. Thus, the efficient repression of E2F dependent S-phase genes and the activation of differentiation genes reside in the balanced DNA binding capacity of C/EBPα. Copyright © 2016 Elsevier B.V. All rights reserved.

A multiplex PCR for detection of knockdown resistance mutations, V1016G and F1534C, in pyrethroid-resistant Aedes aegypti.

PubMed

Saingamsook, Jassada; Saeung, Atiporn; Yanola, Jintana; Lumjuan, Nongkran; Walton, Catherine; Somboon, Pradya

2017-10-10

Mutation of the voltage-gated sodium channel (VGSC) gene, or knockdown resistance (kdr) gene, is an important resistance mechanism of the dengue vector Aedes aegypti mosquitoes against pyrethroids. In many countries in Asia, a valine to glycine substitution (V1016G) and a phenylalanine to cysteine substitution (F1534C) are common in Ae. aegypti populations. The G1016 and C1534 allele frequencies have been increasing in recent years, and hence there is a need to have a simple and inexpensive tool to monitor the alleles in large scale. A multiplex PCR to detect V1016G and F1534C mutations has been developed in the current study. This study utilized primers from previous studies for detecting the mutation at position 1016 and newly designed primers to detect variants at position 1534. The PCR conditions were validated and compared with DNA sequencing using known kdr mutant laboratory strains and field collected mosquitoes. The efficacy of this method was also compared with allele-specific PCR (AS-PCR). The results of our multiplex PCR were in complete agreement with sequencing data and better than the AS-PCR. In addition, the efficiency of two non-toxic DNA staining dyes, Ultrapower™ and RedSafe™, were evaluated by comparing with ethidium bromide (EtBr) and the results were satisfactory. Our multiplex PCR method is highly reliable and useful for implementing vector surveillance in locations where the two alleles co-occur.

Correction of the Abnormal Trafficking of Primary Myelofibrosis CD34+ Cells by Treatment with Chromatin Modifying Agents

PubMed Central

Wang, Xiaoli; Zhang, Wei; Ishii, Takefumi; Sozer, Selcuk; Wang, Jiapeng; Xu, Mingjiang; Hoffman, Ronald

2011-01-01

The abnormal trafficking of CD34+ cells is a unique characteristic of primary myelofibrosis (PMF). We have further studied the behavior of PMF CD34+ cells by examining their homing to the marrow and the spleens of NOD/SCID mice. Following the infusion of PMF and normal G-CSF mobilized peripheral blood (mPB) CD34+ cells into NOD/SCID mice, reduced numbers of PMF CD34+ cells and CFU-GM as compared to mPB were detected in the marrow of these mice while similar numbers of PMF and mPB CD34+ cells and CFU-GM homed to their spleens. The abnormal homing of PMF CD34+ cells was associated with reduced expression of CXCR4, but was not related to the presence of JAK2V617F. The sequential treatment of PMF CD34+ cell with the chromatin modifying agents, 5-aza-2'-deoxycytidine (5azaD) and trichostatin A (TSA) but not treatment with small molecule inhibitors of JAK2 resulted in the generation of increased numbers of CD34+CXCR4+ cells which was accompanied by enhanced homing of PMF CD34+ cells to the marrow but not the spleens of NOD/SCID mice. Following 5azaD/TSA treatment JAK2V617F negative PMF hematopoietic progenitor cells preferentially homed to the marrow but not the spleens of recipient mice. Our data suggest that PMF CD34+ cells are characterized by a reduced ability to home to the marrow but not the spleens of NOD/SCID mice and that this homing defect can be corrected by sequential treatment with chromatin modifying agents. PMID:19752087

The occurrence and the type of germline mutations in the RET gene in patients with medullary thyroid carcinoma and their unaffected kindred's from Central Poland.

PubMed

Paszko, Z; Sromek, M; Czetwertynska, M; Skasko, E; Czapczak, D; Wisniewska, A; Prokurat, A; Chrupek, M; Jagielska, A; Kozlowicz-Gudzinska, I

2007-12-01

We aimed to investigate the occurrence and types of pathogenic mutations in the RET gene in patients with MTC of the Central Poland population and in their relatives. DNA was extracted from the peripheral blood lymphocytes of a total of 330 persons, including 235 MTC patients and 95 of their unaffected kindred's. Exons 10, 11, 13, 14, 15 and 16 of the RET gene were amplified by PCR and sequenced. Sixty-seven people were found to carry pathogenic, germline mutations in the RET gene. In exon 10, C609F, C609R and C609Y (3 families), C618G, C618F (2 families), and C620G (4 families) mutations were identified. In exon 11, C634R (8 families) and C649L mutations (1 patient) were found. Five families carried Y791F mutation in exon 13. One patient with PTC revealed the presence of a Y791F mutation. In 3 families, exon 14 of the RET gene harbored the following mutations: V804L (1 patient), E819K (1 patient) and R844Q (1 patient). In 1 family, the S891A mutation was identified in exon 15, 3 families were found to carry mutations in exon16, R912P in 1 family and M918T in 2 families. In summary, of the 235 patients affected by MTC, 46 (19.6%) carried pathogenic RET gene mutations, 1 patient with RET mutation had kidney carcinoma, and 1 had PTC. The results show the occurrence of a variety of mutations prevalent in patients with MTC in the population of Central Poland. These results may contribute to a better diagnosis of medullary thyroid carcinoma.

Structural characterization of B and non-B subtypes of HIV-protease: insights into the natural susceptibility to drug resistance development.

PubMed

Sanches, Mario; Krauchenco, Sandra; Martins, Nadia H; Gustchina, Alla; Wlodawer, Alexander; Polikarpov, Igor

2007-06-15

Although a majority of HIV-1 infections in Brazil are caused by the subtype B virus (also prevalent in the United States and Western Europe), viral subtypes F and C are also found very frequently. Genomic differences between the subtypes give rise to sequence variations in the encoded proteins, including the HIV-1 protease. The current anti-HIV drugs have been developed primarily against subtype B and the effects arising from the combination of drug-resistance mutations with the naturally existing polymorphisms in non-B HIV-1 subtypes are only beginning to be elucidated. To gain more insights into the structure and function of different variants of HIV proteases, we have determined a 2.1 A structure of the native subtype F HIV-1 protease (PR) in complex with the protease inhibitor TL-3. We have also solved crystal structures of two multi-drug resistant mutant HIV PRs in complex with TL-3, from subtype B (Bmut) carrying the primary mutations V82A and L90M, and from subtype F (Fmut) carrying the primary mutation V82A plus the secondary mutation M36I, at 1.75 A and 2.8 A resolution, respectively. The proteases Bmut, Fwt and Fmut exhibit sevenfold, threefold, and 54-fold resistance to TL-3, respectively. In addition, the structure of subtype B wild type HIV-PR in complex with TL-3 has been redetermined in space group P6(1), consistent with the other three structures. Our results show that the primary mutation V82A causes the known effect of collapsing the S1/S1' pockets that ultimately lead to the reduced inhibitory effect of TL-3. Our results further indicate that two naturally occurring polymorphic substitutions in subtype F and other non-B HIV proteases, M36I and L89M, may lead to early development of drug resistance in patients infected with non-B HIV subtypes.

Impact of Mutations on the Midpoint Potential of the [4Fe-4S]+1,+2 Cluster and on Catalytic Activity in Electron Transfer Flavoprotein-ubiquinone Oxidoreductase (ETF-QO)†

PubMed Central

Usselman, Robert J.; Fielding, Alistair J.; Frerman, Frank E.; Watmough, Nicholas J.; Eaton, Gareth R.; Eaton, Sandra S.

2011-01-01

Electron transfer flavoprotein - ubiquinone oxidoreductase (ETF-QO) is an iron-sulfur flavoprotein that accepts electrons from electron-transfer flavoprotein (ETF) and reduces ubiquinone from the Q-pool. ETF-QO contains a single [4Fe-4S]2+,1+ cluster and one equivalent of FAD, which are diamagnetic in the isolated oxidized enzyme and can be reduced to paramagnetic forms by enzymatic donors or dithionite. Mutations were introduced by site-directed mutagenesis of amino acids in the vicinity of the iron-sulfur cluster of Rhodobacter sphaeroides ETF-QO. Y501 and T525 are equivalent to Y533 and T558 in the porcine ETF-QO. In the porcine protein, these residues are within hydrogen bonding distance of the Sγ of the cysteine ligands to the iron-sulfur cluster. Y501F, T525A, and Y501F/T525A substitutions were made to determine the effects on midpoint potential, activity, and EPR spectral properties of the cluster. The integrity of the mutated proteins was confirmed by optical spectra, EPR g-values, and spin-lattice relaxation rates, and the cluster to flavin point-dipole distance was determined by relaxation enhancement. Potentiometric titrations were monitored by changes in the CW EPR signals of the cluster and semiquinone. Single mutations decreased the mid-point potentials of the iron-sulfur cluster from +37 mV for wild type to −60 mV for Y501F and T525A and to −128 mV for Y501F/T525A. Lowering the midpoint potential resulted in a decrease in steady-state ubiquinone reductase activity and in ETF semiquinone disproportionation. The decrease in activity demonstrates that reduction of the iron-sulfur cluster is required for activity. There was no detectable effect of the mutations on the flavin midpoint potentials. PMID:18069858

Impact of mutations on the midpoint potential of the [4Fe-4S]+1,+2 cluster and on catalytic activity in electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO).

PubMed

Usselman, Robert J; Fielding, Alistair J; Frerman, Frank E; Watmough, Nicholas J; Eaton, Gareth R; Eaton, Sandra S

2008-01-08

Electron-transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO) is an iron-sulfur flavoprotein that accepts electrons from electron-transfer flavoprotein (ETF) and reduces ubiquinone from the Q-pool. ETF-QO contains a single [4Fe-4S]2+,1+ cluster and one equivalent of FAD, which are diamagnetic in the isolated oxidized enzyme and can be reduced to paramagnetic forms by enzymatic donors or dithionite. Mutations were introduced by site-directed mutagenesis of amino acids in the vicinity of the iron-sulfur cluster of Rhodobacter sphaeroides ETF-QO. Y501 and T525 are equivalent to Y533 and T558 in the porcine ETF-QO. In the porcine protein, these residues are within hydrogen-bonding distance of the Sgamma of the cysteine ligands to the iron-sulfur cluster. Y501F, T525A, and Y501F/T525A substitutions were made to determine the effects on midpoint potential, activity, and EPR spectral properties of the cluster. The integrity of the mutated proteins was confirmed by optical spectra, EPR g-values, and spin-lattice relaxation rates, and the cluster to flavin point-dipole distance was determined by relaxation enhancement. Potentiometric titrations were monitored by changes in the CW EPR signals of the cluster and semiquinone. Single mutations decreased the midpoint potentials of the iron-sulfur cluster from +37 mV for wild type to -60 mV for Y501F and T525A and to -128 mV for Y501F/T525A. Lowering the midpoint potential resulted in a decrease in steady-state ubiquinone reductase activity and in ETF semiquinone disproportionation. The decrease in activity demonstrates that reduction of the iron-sulfur cluster is required for activity. There was no detectable effect of the mutations on the flavin midpoint potentials.

Efficacy of ponatinib against ABL tyrosine kinase inhibitor-resistant leukemia cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Okabe, Seiichi, E-mail: okabe@tokyo-med.ac.jp; Tauchi, Tetsuzo; Tanaka, Yuko

2013-06-07

Highlights: •Efficacy of ponatinib against ABL tyrosine kinase inhibitor-resistant leukemia cells okabe et al. •Imatinib or nilotinib resistance was involved Src family kinase. •The BCR-ABL point mutation (E334V) was highly resistant to imatinib or nilotinib. •Ponatinib was a powerful strategy against imatinib or nilotinib resistant Ph-positive cells. -- Abstract: Because a substantial number of patients with chronic myeloid leukemia acquire resistance to ABL tyrosine kinase inhibitors (TKIs), their management remains a challenge. Ponatinib, also known as AP24534, is an oral multi-targeted TKI. Ponatinib is currently being investigated in a pivotal phase 2 clinical trial. In the present study, we analyzedmore » the molecular and functional consequences of ponatinib against imatinib- or nilotinib-resistant (R) K562 and Ba/F3 cells. The proliferation of imatinib- or nilotinib-resistant K562 cells did not decrease after treatment with imatinib or nilotinib. Src family kinase Lyn was activated. Point mutation Ba/F3 cells (E334 V) were also highly resistant to imatinib and nilotinib. Treatment with ponatinib for 72 h inhibited the growth of imatinib- and nilotinib-resistant cells. The phosphorylation of BCR-ABL, Lyn, and Crk-L was reduced. This study demonstrates that ponatinib has an anti-leukemia effect by reducing ABL and Lyn kinase activity and this information may be of therapeutic relevance.« less

SHOCKS Impulse-Jerk(I-J) Plasticity/Fracture Burst Acoustic-Emission(BAE) NON:``1''/ ω -``Noise'' Power-Law; Universality Power-Spectrum is I-J Time-Series Fourier-Transform: 1687 < < < 1988: VERY-LONG PRE-``Bak''!!!

NASA Astrophysics Data System (ADS)

Chavira, Aldo; Gregson, Victor, Jr.; Green, Sidney; Siegel, Edward

2011-06-01

SHOCKS impulse-jerk(I-J) [apply strain/impulse to get stress/jerk ],{VS. NON-shocks[apply stress to get strain]}, plasticity/fracture BAE[E. S.: MSE 8.,310(71); PSS: (a) 5, 601/607(71); Xl..-Latt. Defects 5, 277(74); Scripta Met.: 6, 785(72); 8, 587/617(74); 3rd Tokyo A.-E. Symp. (76);Acta Met.25,383(77); JMMM 7, 312(78)] NON: ``1''/ ω -``Noise'' Zipf(NON-Pareto); power-law ; universality power-spectrum is manifestly-demonstrated in ONLY ``PURE''-MATHS way to be nothing but d[F(t)=m(t)a(t)=Newton's (3rd) Law of Motion=(I-J)]/dt I-Jderivative d(I-J)/dt=dF(t)/dt=[m(t)da(t)/dt+a(t)dm(t)/dt] REdiscovery!!! A/Siegel NON-shock PHYSICS derivation fails!!!; ''PURE''-MATHS: dF(t)/dt=d2p(t)/dt2=[m(t)da(t)/dt+a(t)dm(t)/dt] TRIPLE-integral [VS. NON -shocks F = ma time-series DOUBLE-integral] Dichotomy: s(t) = [v0+(1/2)a(t)t2+EXTRA-TERM(S)], {VS. s(t) = [v0t+(1/2) at2]}, integral-transform formally defines power-spectrum Dichotomy:

Next-generation sequencing reveals a Novel NSCLC ALK F1174V mutation and confirms ALK G1202R mutation confers high-level resistance to alectinib (CH5424802/RO5424802) in ALK-rearranged NSCLC patients who progressed on crizotinib.

PubMed

Ignatius Ou, Sai-Hong; Azada, Michele; Hsiang, David J; Herman, June M; Kain, Tatiana S; Siwak-Tapp, Christina; Casey, Cameron; He, Jie; Ali, Siraj M; Klempner, Samuel J; Miller, Vincent A

2014-04-01

Acquired secondary mutations in the anaplastic lymphoma kinase (ALK) gene have been identified in ALK-rearranged (ALK+) non-small-cell lung cancer (NSCLC) patients who developed disease progression while on crizotinib treatment. Here, we identified a novel secondary acquired NSCLC ALK F1174V mutation by comprehensive next-generation sequencing in one ALK+ NSCLC patient who progressed on crizotinib after a prolonged partial response to crizotinib. In a second case, we identified a secondary acquired ALK G1202R, which also confers resistance to alectinib (CH5424802/RO5424802), a second-generation ALK inhibitor that can inhibit ALK gatekeeper L1196M mutation in vitro. ALK G1202R is located at the solvent front of the ALK kinase domain and exhibits a high level of resistance to all other ALK inhibitors currently in clinical development in vitro. Comprehensive genomic profiling of resistant tumor is increasingly important in tailoring treatment decisions after disease progression on crizotinib in ALK+ NSCLC given the promise of second-generation ALK inhibitors and other therapeutic strategies.

Essential thrombocythemia

PubMed Central

Brière, Jean B

2007-01-01

Essential thrombocythemia (ET) is an acquired myeloproliferative disorder (MPD) characterized by a sustained elevation of platelet number with a tendency for thrombosis and hemorrhage. The prevalence in the general population is approximately 30/100,000. The median age at diagnosis is 65 to 70 years, but the disease may occur at any age. The female to male ratio is about 2:1. The clinical picture is dominated by a predisposition to vascular occlusive events (involving the cerebrovascular, coronary and peripheral circulation) and hemorrhages. Some patients with ET are asymptomatic, others may experience vasomotor (headaches, visual disturbances, lightheadedness, atypical chest pain, distal paresthesias, erythromelalgia), thrombotic, or hemorrhagic disturbances. Arterial and venous thromboses, as well as platelet-mediated transient occlusions of the microcirculation and bleeding, represent the main risks for ET patients. Thromboses of large arteries represent a major cause of mortality associated with ET or can induce severe neurological, cardiac or peripheral artery manifestations. Acute leukemia or myelodysplasia represent only rare and frequently later-onset events. The molecular pathogenesis of ET, which leads to the overproduction of mature blood cells, is similar to that found in other clonal MPDs such as chronic myeloid leukemia, polycythemia vera and myelofibrosis with myeloid metaplasia of the spleen. Polycythemia vera, myelofibrosis with myeloid metaplasia of the spleen and ET are generally associated under the common denomination of Philadelphia (Ph)-negative MPDs. Despite the recent identification of the JAK2 V617F mutation in a subset of patients with Ph-negative MPDs, the detailed pathogenetic mechanism is still a matter of discussion. Therapeutic interventions in ET are limited to decisions concerning the introduction of anti-aggregation therapy and/or starting platelet cytoreduction. The therapeutic value of hydroxycarbamide and aspirin in high risk patients has been supported by controlled studies. Avoiding thromboreduction or opting for anagrelide to postpone the long-term side effects of hydrocarbamide in young or low risk patients represent alternative options. Life expectancy is almost normal and similar to that of a healthy population matched by age and sex. PMID:17210076

Molecular analysis of PROP1, POU1F1, LHX3, and HESX1 in Turkish patients with combined pituitary hormone deficiency: a multicenter study.

PubMed

Baş, Firdevs; Uyguner, Z Oya; Darendeliler, Feyza; Aycan, Zehra; Çetinkaya, Ergun; Berberoğlu, Merih; Şiklar, Zeynep; Öcal, Gönül; Darcan, Şükran; Gökşen, Damla; Topaloğlu, Ali Kemal; Yüksel, Bilgin; Özbek, Mehmet Nuri; Ercan, Oya; Evliyaoğlu, Olcay; Çetinkaya, Semra; Şen, Yaşar; Atabek, Emre; Toksoy, Güven; Aydin, Banu Küçükemre; Bundak, Rüveyde

2015-06-01

To investigate the specific mutations in PROP1, POU1F1, LHX3, and HESX1 genes in patients with combined pituitary hormone deficiency (CPHD) in Turkey. Seventy-six patients with CPHD were included in this study. Based on clinical, hormonal, and neuro-radiological data, relevant transcription factor genes were evaluated by Sanger sequencing and multiplex ligation-dependent probe amplification. Total frequency of mutations was 30.9 % in patients with CPHD. Frequency was significantly higher in familial patients (p = 0.001). Three different types of mutations in PROP1 gene (complete gene deletion, c.301-302delAG, a novel mutation; IVS1+2T>G) were found in 12 unrelated patients (21.8 %). Mutations in PROP1 gene were markedly higher in familial than in sporadic cases (58.8 vs. 5.3 %, p < 0.001). Homozygous complete gene deletion was the most common mutation in PROP1 gene (8/12) and was identified in six familial patients. Four different homozygous mutations [p.Q4X, novel mutations; exons 1-2 deletion, p.V153F, p.I244S] were detected in POU1F1 gene. Central precocious puberty was firstly observed in a sporadic-male patient with homozygous POU1F1 (p.I244S) mutation. A homozygous mutation in HESX1 gene (p.R160H) was detected in one patient. This study is the first to investigate specific mutations in CPHD patients in Turkey. Complete deletion in PROP1 gene was the most common mutation encountered in patients with CPHD. We believe that the results of this study will contribute to the establishment of genetic screening strategies in Turkey, as well as to the studies on phenotype-genotype correlations and early diagnosis of CPHD patients.

Mutations in the KIAA0196 Gene at the SPG8 Locus Cause Hereditary Spastic Paraplegia

PubMed Central

Valdmanis, Paul N.; Meijer, Inge A.; Reynolds, Annie; Lei, Adrienne; MacLeod, Patrick; Schlesinger, David; Zatz, Mayana; Reid, Evan; Dion, Patrick A.; Drapeau, Pierre; Rouleau, Guy A.

2007-01-01

Hereditary spastic paraplegia (HSP) is a progressive upper-motor neurodegenerative disease. The eighth HSP locus, SPG8, is on chromosome 8p24.13. The three families previously linked to the SPG8 locus present with relatively severe, pure spastic paraplegia. We have identified three mutations in the KIAA0196 gene in six families that map to the SPG8 locus. One mutation, V626F, segregated in three large North American families with European ancestry and in one British family. An L619F mutation was found in a Brazilian family. The third mutation, N471D, was identified in a smaller family of European origin and lies in a spectrin domain. None of these mutations were identified in 500 control individuals. Both the L619 and V626 residues are strictly conserved across species and likely have a notable effect on the structure of the protein product strumpellin. Rescue studies with human mRNA injected in zebrafish treated with morpholino oligonucleotides to knock down the endogenous protein showed that mutations at these two residues impaired the normal function of the KIAA0196 gene. However, the function of the 1,159-aa strumpellin protein is relatively unknown. The identification and characterization of the KIAA0196 gene will enable further insight into the pathogenesis of HSP. PMID:17160902

Allele-Specific Polymerase Chain Reaction for the Imatinib-Resistant KIT D816V and D816F Mutations in Mastocytosis and Acute Myelogenous Leukemia

PubMed Central

Corless, Christopher L.; Harrell, Patina; Lacouture, Mario; Bainbridge, Troy; Le, Claudia; Gatter, Ken; White, Clifton; Granter, Scott; Heinrich, Michael C.

2006-01-01

Oncogenic mutations of the receptor tyrosine kinase KIT contribute to the pathogenesis of gastrointestinal stromal tumors, systemic mastocytosis (SM), and some cases of acute myelogenous leukemia (AML). The D816V substitution in the activation loop of KIT results in relative resistance to the kinase inhibitor imatinib (Gleevec). Because this mutation occurs in 80 to 95% of adult SM, its detection has diagnostic and predictive significance. Unfortunately, the fraction of mutation-positive cells in clinical SM samples is often below the 20 to 30% threshold needed for detection by direct DNA sequencing. We have developed an allele-specific polymerase chain reaction assay using a mutation-specific primer combined with a wild-type blocking oligonucleotide that amplifies D816V at the level of 1% mutant allele in DNA extracted from formalin-fixed, paraffin-embedded tissue. There were no amplifications among 64 KIT wild-type tumors and cell lines, whereas all D816V-mutant samples (eight AML and 11 mast cell disease) were positive. Other D816 substitutions associated with resistance to imatinib in vitro are rare in SM. Among these D816F was detectable with the assay whereas D816H, D816Y, and D816G did not amplify. Nine biopsies (bone marrow, skin, or colon) with suspected SM were negative by denaturing high performance liquid chromatography and/or DNA sequencing but positive by allele-specific polymerase chain reaction. Thus, the assay may be useful in confirming the diagnosis of SM. PMID:17065430

Crystallization of a non-B and a B mutant HIV protease.

PubMed

Sanches, Mario; Martins, Nádia Helena; Calazans, Alexandre; Brindeiro, Rodrigo de Moraes; Tanuri, Amilcar; Antunes, Octavio Augusto Ceva; Polikarpov, Igor

2004-09-01

HIV polymorphism is responsible for the selection of variant viruses resistant to inhibitors used in AIDS treatment. Knowledge of the mechanism of resistance of those viruses is determinant to the development of new inhibitors able to stop, or at least slow down, the disease's progress caused by new mutations. In this paper, the crystallization and preliminary crystallographic structure solution for two multi-resistant 99 amino acid HIV proteases, both isolated from Brazilian patients failing intensive anti-AIDS therapy are presented, viz. the subtype B mutant, with mutations Q7K, S37N, R41K, K45R, I54V, L63P, A71V, V82A and L90M, and the subtype F (wild type), naturally carrying mutations Q7K, I15V, E35D, M36I, S37N, R41K, R57K, D60E, Q61N, I62V, L63S, I64L and L89M, with respect to the B consensus sequence. Both proteins crystallized as a complex with the inhibitor TL-3 in space group P6(1)22. X-ray diffraction data were collected from these crystals to resolutions of 2.1 and 2.6 A for the subtype B mutant and subtype F wild type, respectively, and the enzyme structures were solved by molecular replacement. The crystals of subtype F HIV protease are, to the best of the authors' knowledge, the first protein crystals obtained for a non-B HIV protease.

Discovery of Point Mutations in the Voltage-Gated Sodium Channel from African Aedes aegypti Populations: Potential Phylogenetic Reasons for Gene Introgression.

PubMed

Kawada, Hitoshi; Higa, Yukiko; Futami, Kyoko; Muranami, Yuto; Kawashima, Emiko; Osei, Joseph H N; Sakyi, Kojo Yirenkyi; Dadzie, Samuel; de Souza, Dziedzom K; Appawu, Maxwell; Ohta, Nobuo; Suzuki, Takashi; Minakawa, Noboru

2016-06-01

Yellow fever is endemic in some countries in Africa, and Aedes aegpyti is one of the most important vectors implicated in the outbreak. The mapping of the nation-wide distribution and the detection of insecticide resistance of vector mosquitoes will provide the beneficial information for forecasting of dengue and yellow fever outbreaks and effective control measures. High resistance to DDT was observed in all mosquito colonies collected in Ghana. The resistance and the possible existence of resistance or tolerance to permethrin were suspected in some colonies. High frequencies of point mutations at the voltage-gated sodium channel (F1534C) and one heterozygote of the other mutation (V1016I) were detected, and this is the first detection on the African continent. The frequency of F1534C allele and the ratio of F1534C homozygotes in Ae. aegypti aegypti (Aaa) were significantly higher than those in Ae. aegypti formosus (Aaf). We could detect the two types of introns between exon 20 and 21, and the F1534C mutations were strongly linked with one type of intron, which was commonly found in South East Asian and South and Central American countries, suggesting the possibility that this mutation was introduced from other continents or convergently selected after the introgression of Aaa genes from the above area. The worldwide eradication programs in 1940s and 1950s might have caused high selection pressure on the mosquito populations and expanded the distribution of insecticide-resistant Ae. aegypti populations. Selection of the F1534C point mutation could be hypothesized to have taken place during this period. The selection of the resistant population of Ae. aegypti with the point mutation of F1534C, and the worldwide transportation of vector mosquitoes in accordance with human activity such as trading of used tires, might result in the widespread distribution of F1534C point mutation in tropical countries.

The JAK2 GGCC (46/1) Haplotype in Myeloproliferative Neoplasms: Causal or Random?

PubMed Central

Anelli, Luisa; Zagaria, Antonella; Specchia, Giorgina

2018-01-01

The germline JAK2 haplotype known as “GGCC or 46/1 haplotype” (haplotypeGGCC_46/1) consists of a combination of single nucleotide polymorphisms (SNPs) mapping in a region of about 250 kb, extending from the JAK2 intron 10 to the Insulin-like 4 (INLS4) gene. Four main SNPs (rs3780367, rs10974944, rs12343867, and rs1159782) generating a “GGCC” combination are more frequently indicated to represent the JAK2 haplotype. These SNPs are inherited together and are frequently associated with the onset of myeloproliferative neoplasms (MPN) positive for both JAK2 V617 and exon 12 mutations. The association between the JAK2 haplotypeGGCC_46/1 and mutations in other genes, such as thrombopoietin receptor (MPL) and calreticulin (CALR), or the association with triple negative MPN, is still controversial. This review provides an overview of the frequency and the role of the JAK2 haplotypeGGCC_46/1 in the pathogenesis of different myeloid neoplasms and describes the hypothetical mechanisms at the basis of the association with JAK2 gene mutations. Moreover, possible clinical implications are discussed, as different papers reported contrasting data about the correlation between the JAK2 haplotypeGGCC_46/1 and blood cell count, survival, or disease progression. PMID:29641446

The JAK2 GGCC (46/1) Haplotype in Myeloproliferative Neoplasms: Causal or Random?

PubMed

Anelli, Luisa; Zagaria, Antonella; Specchia, Giorgina; Albano, Francesco

2018-04-11

The germline JAK2 haplotype known as "GGCC or 46/1 haplotype" (haplotype GGCC_46/1 ) consists of a combination of single nucleotide polymorphisms (SNPs) mapping in a region of about 250 kb, extending from the JAK2 intron 10 to the Insulin-like 4 ( INLS4 ) gene. Four main SNPs (rs3780367, rs10974944, rs12343867, and rs1159782) generating a "GGCC" combination are more frequently indicated to represent the JAK2 haplotype. These SNPs are inherited together and are frequently associated with the onset of myeloproliferative neoplasms (MPN) positive for both JAK2 V617 and exon 12 mutations. The association between the JAK2 haplotype GGCC_46/1 and mutations in other genes, such as thrombopoietin receptor ( MPL ) and calreticulin ( CALR ), or the association with triple negative MPN, is still controversial. This review provides an overview of the frequency and the role of the JAK2 haplotype GGCC_46/1 in the pathogenesis of different myeloid neoplasms and describes the hypothetical mechanisms at the basis of the association with JAK2 gene mutations. Moreover, possible clinical implications are discussed, as different papers reported contrasting data about the correlation between the JAK2 haplotype GGCC_46/1 and blood cell count, survival, or disease progression.

The impact of active site mutations of South African HIV PR on drug resistance: Insight from molecular dynamics simulations, binding free energy and per-residue footprints.

PubMed

Ahmed, Shaimaa M; Maguire, Glenn E M; Kruger, Hendrik G; Govender, Thirumala

2014-04-01

Molecular dynamics simulations and binding free energy calculations were used to provide an understanding of the impact of active site drug-resistant mutations of the South African HIV protease subtype C (C-SA HIV PR), V82A and V82F/I84V on drug resistance. Unique per-residue interaction energy 'footprints' were developed to map the overall drug-binding profiles for the wild type and mutants. Results confirmed that these mutations altered the overall binding landscape of the amino acid residues not only in the active site region but also in the flaps as well. Four FDA-approved drugs were investigated in this study; these include ritonavir (RTV), saquinavir (SQV), indinavir (IDV), and nelfinavir (NFV). Computational results compared against experimental findings were found to be complementary. Against the V82F/I84V variant, saquinavir, indinavir, and nelfinavir lose remarkable entropic contributions relative to both wild-type and V82A C-SA HIV PRs. The per-residue energy 'footprints' and the analysis of ligand-receptor interactions for the drug complexes with the wild type and mutants have also highlighted the nature of drug interactions. The data presented in this study will prove useful in the design of more potent inhibitors effective against drug-resistant HIV strains. © 2013 John Wiley & Sons A/S.

Year to year change in FEV1 in patients with cystic fibrosis and different mutation classes.

PubMed

De Boeck, K; Zolin, A

2017-03-01

In patients with cystic fibrosis, most treatments addressing the underlying basic defect are mutation or mutation class specific. These treatments are disease modifying if they lower the year to year change in lung function. We therefore calculated the current loss of lung function, measured by year to year change in forced expired volume in 1s in 11,417 patients included in the European Cystic Fibrosis Society Patient Registry. Whereas patients with at least one mutation of class IV or V have on average a lower year to year change, we did not find a difference between patients with a stop codon mutation, homozygous for F508del or at least one class III mutation. These data are useful background information to discuss the impact of different disease modifying treatments. Copyright © 2016 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

Identification, characterization, immobilization, and mutational analysis of a novel acetylesterase with industrial potential (LaAcE) from Lactobacillus acidophilus.

PubMed

Wang, Ying; Ryu, Bum Han; Yoo, Wanki; Lee, Chang Woo; Kim, Kyeong Kyu; Lee, Jun Hyuck; Kim, T Doohun

2018-01-01

Lactic acid bacteria, which are involved in the fermentation of vegetables, meats, and dairy products, are widely used for the productions of small organic molecules and bioactive peptides. Here, a novel acetylesterase (LaAcE) from Lactobacillus acidophilus NCFM was identified, functionally characterized, immobilized, and subjected to site-directed mutagenesis for biotechnological applications. The enzymatic properties of LaAcE were investigated using biochemical and biophysical methods including native polyacrylamide gel electrophoresis, acetic acid release, biochemical assays, enzyme kinetics, and spectroscopic methods. Interestingly, LaAcE exhibited the ability to act on a broad range of substrates including glucose pentaacetate, glyceryl tributyrate, fish oil, and fermentation-related compounds. Furthermore, immobilization of LaAcE showed good recycling ability and high thermal stability compared with free LaAcE. A structural model of LaAcE was used to guide mutational analysis of hydrophobic substrate-binding region, which was composed of Leu 156 , Phe 164 , and Val 204 . Five mutants (L156A, F164A, V204A, L156A/F164A, and L156A/V204A) were generated and investigated to elucidate the roles of these hydrophobic residues in substrate specificity. This work provided valuable insights into the properties of LaAcE, and demonstrated that LaAcE could be used as a model enzyme of acetylesterase in lactic acid bacteria, making LaAcE a great candidate for industrial applications. Copyright © 2017 Elsevier B.V. All rights reserved.

Antisense-based RNA therapy of factor V deficiency: in vitro and ex vivo rescue of a F5 deep-intronic splicing mutation.

PubMed

Nuzzo, Francesca; Radu, Claudia; Baralle, Marco; Spiezia, Luca; Hackeng, Tilman M; Simioni, Paolo; Castoldi, Elisabetta

2013-11-28

Antisense molecules are emerging as a powerful tool to correct splicing defects. Recently, we identified a homozygous deep-intronic mutation (F5 c.1296+268A>G) activating a cryptic donor splice site in a patient with severe coagulation factor V (FV) deficiency and life-threatening bleeding episodes. Here, we assessed the ability of 2 mutation-specific antisense molecules (a morpholino oligonucleotide [MO] and an engineered U7 small nuclear RNA [snRNA]) to correct this splicing defect. COS-1 and HepG2 cells transfected with a F5 minigene construct containing the patient's mutation expressed aberrant messenger RNA (mRNA) in excess of normal mRNA. Treatment with mutation-specific antisense MO (1-5 µM) or a construct expressing antisense U7snRNA (0.25-2 µg) dose-dependently increased the relative amount of correctly spliced mRNA by 1 to 2 orders of magnitude, whereas control MO and U7snRNA were ineffective. Patient-derived megakaryocytes obtained by differentiation of circulating progenitor cells did not express FV, but became positive for FV at immunofluorescence staining after administration of antisense MO or U7snRNA. However, treatment adversely affected cell viability, mainly because of the transfection reagents used to deliver the antisense molecules. Our data provide in vitro and ex vivo proof of principle for the efficacy of RNA therapy in severe FV deficiency, but additional cytotoxicity studies are warranted.

High prevalence of natural polymorphisms in Gag (CA-SP1) associated with reduced response to Bevirimat, an HIV-1 maturation inhibitor.

PubMed

Seclén, Eduardo; González, María Del Mar; Corral, Angélica; de Mendoza, Carmen; Soriano, Vincent; Poveda, Eva

2010-01-28

Mutations H358Y, L363F/M, A364V and A366T/V confer in-vitro resistance to bevirimat. Moreover, polymorphisms at the Glutamine-Valine-Threonine (QVT) motif (369-371) have been associated with reduced bevirimat activity in vivo. The rate of these changes was assessed in 389 HIV+ patients naïve for bevirimat. QVT polymorphisms were frequent (47%), especially in non-B subtypes (93%). Conversely, only four patients (1%) harbored major bevirimat resistance mutations. Finally, specific gag changes were associated with protease inhibitor resistance mutations in subtype B viruses.

Primary genotypic resistance of HIV-1 to the maturation inhibitor PA-457 in protease inhibitor-experienced patients.

PubMed

Malet, Isabelle; Wirden, Marc; Derache, Anne; Simon, Anne; Katlama, Christine; Calvez, Vincent; Marcelin, Anne-Geneviève

2007-04-23

Sequences from 82 protease inhibitors (PI)-experienced patients were analysed for the presence of previously described in-vitro resistance mutations to PA-457 located in the C-terminal capside (H226Y, L231F, L231M) and in the N-terminal SP1 (A1V, A3T, A3V) within the CA-SP1 boundary domain. Overall, the CA-SP1 cleavage site was highly conserved in PI pre-treated patients and only one patient showed an L231M mutation. The impact of this mutation should be further addressed in vivo.

Lack of in vitro constitutive activity for four previously reported TSH receptor mutations identified in patients with nonautoimmune hyperthyroidism and hot thyroid carcinomas.

PubMed

Jaeschke, Holger; Mueller, Sandra; Eszlinger, Markus; Paschke, Ralf

2010-12-01

Constitutively activating mutations (CAMs) of the TSHR are the major cause for nonautoimmune hyperthyroidism. Re-examination of constitutive activity previously determined in CHO cell lines recently demonstrated the caveats for the in vitro determination of constitutive TSHR activity, which leads to false positive conclusions regarding the molecular origin of hyperthyroidism or hot thyroid carcinomas. Mutations L677V and T620I identified in hot thyroid carcinomas were previously characterized in CHO and in 3T3-Vill cell lines, respectively, stably expressing the mutant without determination of TSHR expression. F666L identified in a patient with hot thyroid nodules, I691F in a family with nonautoimmune hyperthyroidism and F631I identified in a hot thyroid carcinoma were not characterized for their in vitro function. Therefore, we decided to (re)evaluate the in vitro function of these five TSHR variants by determination of cell surface expression, and intracellular cAMP and inositol phosphate levels and performed additionally linear regression analyses to determine basal activity independently from the mutant's cell surface expression in COS-7 and HEK(GT) cells. Only one (F631I) of the five investigated TSHR variants displayed constitutive activity for G(α) s signalling and showed correlation with the clinical phenotype. The previous false classification of T620I and L677V as CAMs is most likely related to the fact that both mutations were characterized in cell lines stably expressing the mutated receptor construct without assessing the respective receptor number per cell. Other molecular aetiologies for the nonautoimmune hyperthyroidism and/or hot thyroid carcinomas in these three patients and one family should be elucidated. © 2010 Blackwell Publishing Ltd.

Cystic fibrosis transmembrane conductance regulator mutations at a referral center for cystic fibrosis*

PubMed Central

Coutinho, Cyntia Arivabeni de Araújo Correia; Marson, Fernando Augusto de Lima; Ribeiro, Antônio Fernando; Ribeiro, José Dirceu; Bertuzzo, Carmen Silvia

2013-01-01

OBJECTIVE: To determine the frequency of six mutations (F508del, G542X, G551D, R553X, R1162X, and N1303K) in patients with cystic fibrosis (CF) diagnosed, at a referral center, on the basis of abnormal results in two determinations of sweat sodium and chloride concentrations. METHODS: This was a cross-sectional study involving 70 patients with CF. The mean age of the patients was 12.38 ± 9.00 years, 51.43% were female, and 94.29% were White. Mutation screening was performed with polymerase chain reaction (for F508del), followed by enzymatic digestion (for other mutations). Clinical analysis was performed on the basis of gender, age, ethnicity, pulmonary/gastrointestinal symptoms, and Shwachman-Kulczycki (SK) score. RESULTS: All of the patients showed pulmonary symptoms, and 8 had no gastrointestinal symptoms. On the basis of the SK scores, CF was determined to be mild, moderate, and severe in 22 (42.3%), 17 (32.7%), and 13 (25.0%) of the patients, respectively. There was no association between F508del mutation and disease severity by SK score. Of the 140 alleles analyzed, F508del mutation was identified in 70 (50%). Other mutations (G542X, G551D, R553X, R1162X, and N1303K) were identified in 12 (7.93%) of the alleles studied. In F508del homozygous patients with severe disease, the OR was 0.124 (95% CI: 0.005-0.826). CONCLUSIONS: In 50% of the alleles studied, the molecular diagnosis of CF was confirmed by identifying a single mutation (F508del). If we consider the analysis of the six most common mutations in the Brazilian population (including F508del), the molecular diagnosis was confirmed in 58.57% of the alleles studied. PMID:24310628

Modeling cystic fibrosis disease progression in patients with the rare CFTR mutation P67L.

PubMed

MacKenzie, Isobel E R; Paquette, Valerie; Gosse, Frances; George, Sheenagh; Chappe, Frederic; Chappe, Valerie

2017-05-01

The progression of cystic fibrosis (CF) in patients with the rare mutation P67L was examined to determine if it induced a milder form of CF compared to the common severe ΔF508 mutation. Parameters of lung function, level of bacterial infection, nutritional status and hospitalization were used to represent CF progression. Age at diagnosis and pancreatic status were used to assess CF presentation. Analysis of data from the CF Canada Registry collected over a 15-year period included 266 ΔF508/ΔF508 homozygote patients from CF clinics in Atlantic Canada and 26 compound heterozygote patients with the rare P67L mutation from clinics across Canada. Late age at diagnosis, high incidence of pancreatic sufficiency, maintained Body Mass Index (BMI) with age, delayed life-threatening bacterial infection, and fewer days in hospital were observed for P67L heterozygote patients included in this study. Although the decline of lung function did not differ from ΔF508 homozygotes, the fact that a greater proportion of P67L heterozygotes live to an older age suggests that lung function is not the primary factor determining CF progression for P67L heterozygote patients. The P67L mutation is associated with a mild disease, even when combined with the severe ΔF508 mutation. Copyright © 2017 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

[Genetic analysis of the mutations in HIV-1 infected population in Ecuador].

PubMed

González-González, Manuel; Correa-Sierra, Consuelo; Hermida-Álava, Katherine; Machado-Díaz, Ana; Gómez-Andrade, L Fernando; Castillo-Segovia, Martha; Pérez-Santos, C Lissette; Kourí-Cardellá, Vivian

Background The international recommendations of antiretroviral treatment include resistance tests to guide the treatment regimen in each patient, which is not available on a regular basis in Ecuador. Aim To describe mutations that confer resistance to antiretrovirals in a population of Ecuadorian patients. Methods Plasma samples from 101 HIV-1 patients with failure to antiretroviral therapy, divided into 15 children and 86 adults, were studied with the GS Junior (Roche) and the sequences were analyzed with the DeepChek program. Results The most frequent mutations were M184V/I, K101E/P/H, K103N/S, D30N, M46L/I, I54L/M, V82T/F/A/S/L and L90M in adults and F77L, K103N/S, M46L/I, V82T/F/A/S/L and L90M in children. High resistance to non-nucleoside reverse transcriptase (RT) inhibitors in minority viral populations of adults and children (34.9% and 70%) was detected; in children both viral populations (majority and minority viral populations) (> 45%) were protease inhibitor resistant. Patients who had a greater number of therapeutic regimens had higher levels of resistance to antiretrovirals. Most of the samples were subtype B in the TR and protease region, and CRF25_cpx in integrase. Conclusions Mutations and resistance to antiretrovirals are shown in a population of Ecuadorian patients with HIV-1. These results will make it possible to issue a warning to health authorities about the need for resistance studies.

Low frequency of genotypic resistance in HIV-1-infected patients failing an atazanavir-containing regimen: a clinical cohort study.

PubMed

Dolling, David I; Dunn, David T; Sutherland, Katherine A; Pillay, Deenan; Mbisa, Jean L; Parry, Chris M; Post, Frank A; Sabin, Caroline A; Cane, Patricia A

2013-10-01

To determine protease mutations that develop at viral failure for protease inhibitor (PI)-naive patients on a regimen containing the PI atazanavir. Resistance tests on patients failing atazanavir, conducted as part of routine clinical care in a multicentre observational study, were randomly matched by subtype to resistance tests from PI-naive controls to account for natural polymorphisms. Mutations from the consensus B sequence across the protease region were analysed for association and defined using the IAS-USA 2011 classification list. Four hundred and five of 2528 (16%) patients failed therapy containing atazanavir as a first PI over a median (IQR) follow-up of 1.76 (0.84-3.15) years and 322 resistance tests were available for analysis. Recognized major atazanavir mutations were found in six atazanavir-experienced patients (P < 0.001), including I50L and N88S. The minor mutations most strongly associated with atazanavir experience were M36I, M46I, F53L, A71V, V82T and I85V (P < 0.05). Multiple novel mutations, I15S, L19T, K43T, L63P/V, K70Q, V77I and L89I/T/V, were also associated with atazanavir experience. Viral failure on atazanavir-containing regimens was not common and major resistance mutations were rare, suggesting that adherence may be a major contributor to viral failure. Novel mutations were described that have not been previously documented.

NON-Shock-Plasticity/Fracture Burst Acoustic-Emission(BAE) ``1''/f -``Noise'' Power-Spectrum Power-Law UNIVERSALITY is Merely F=ma Time-Series Integral-Transform, aka ``Bak'' -``SOC'' REdiscovery'' PRE(1687)-``Bak''(1988)

NASA Astrophysics Data System (ADS)

Siegel, Edward; Nabarro, Frank; Brailsford, Alan; Tatro, Clement

2011-06-01

NON-shock-plasticity/fracture BAE[E.S.:MSE 8,310(71);PSS:(a)5,601/607(71);Xl.-Latt. Defects 5,277(74);Scripta Met.:6,785(72); 8,587/617(74);3rd Tokyo AE Symp.(76);Acta Met. 25,383(77);JMMM 7,312(78)] ``1''/ ω-``noise'' power-spectrum ``pink''-Zipf-(NOT ``red''-Pareto) power-law UNIVERSALITY is manifestly-demonstrated in two distinct ways to be nothing but Newton Law of Motion F = ma REdiscovery!!!(aka ``Bak''(1988)-``SOC'':1687 < < < 1988: 1988-1687=301-years!!! PHYSICS:(1687) cross-multiplied F=ma rewritten as 1/m=a/F=OUTPUT/IN-PUT=EFFECT/CAUSE=inverse-mass mechanical-susceptibility=X(`` ω'') X(`` ω '') ~(F.-D. thm.) ~P(`` ω'') ``noise'' power-spectrum; (``Max & Al show''): E ~ ω , & E ~(or any/all media with upper-limiting-speeds) ~m. Thus: ω ~ E ~m inverting: 1/ ω ~ 1/E ~1/m ~a/F= X(`` ω'') ~ P(`` ω'') thus: F=ma integral-transform(I-T) is ```SOC'''s'' P(ω) ~ 1/ ω !!!; ``PURE''-MATHS: F=ma DOUBLE-integral time-series(T-S) s(t)=[v0t+(1/2)at2] I-T formally defines power-spectrum:

Incomplete APOBEC3G/F Neutralization by HIV-1 Vif Mutants Facilitates the Genetic Evolution from CCR5 to CXCR4 Usage.

PubMed

Alteri, Claudia; Surdo, Matteo; Bellocchi, Maria Concetta; Saccomandi, Patrizia; Continenza, Fabio; Armenia, Daniele; Parrotta, Lucia; Carioti, Luca; Costa, Giosuè; Fourati, Slim; Di Santo, Fabiola; Scutari, Rossana; Barbaliscia, Silvia; Fedele, Valentina; Carta, Stefania; Balestra, Emanuela; Alcaro, Stefano; Marcelin, Anne Genevieve; Calvez, Vincent; Ceccherini-Silberstein, Francesca; Artese, Anna; Perno, Carlo Federico; Svicher, Valentina

2015-08-01

Incomplete APOBEC3G/F neutralization by a defective HIV-1Vif protein can promote genetic diversification by inducing G-to-A mutations in the HIV-1 genome. The HIV-1 Env V3 loop, critical for coreceptor usage, contains several putative APOBEC3G/F target sites. Here, we determined if APOBEC3G/F, in the presence of Vif-defective HIV-1 virus, can induce G-to-A mutations at V3 positions critical to modulation of CXCR4 usage. Peripheral blood mononuclear cells (PBMC) and monocyte-derived macrophages (MDM) from 2 HIV-1-negative donors were infected with CCR5-using 81.A-VifWT virus (i.e., with wild-type [WT] Vif protein), 81.A-VifE45G, or 81.A-VifK22E (known to incompletely/partially neutralize APOBEC3G/F). The rate of G-toA mutations was zero or extremely low in 81.A-VifWT- and 81.A-VifE45G-infected PBMC from both donors. Conversely, G-to-A enrichment was detected in 81.A-VifK22E-infected PBMC (prevalence ranging from 2.18% at 7 days postinfection [dpi] to 3.07% at 21 dpi in donor 1 and from 10.49% at 7 dpi to 8.69% at 21 dpi in donor 2). A similar scenario was found in MDM. G-to-A mutations occurred at 8 V3 positions, resulting in nonsynonymous amino acid substitutions. Of them, G24E and E25K strongly correlated with phenotypically/genotypically defined CXCR4-using viruses (P = 0.04 and 5.5e-7, respectively) and increased the CXCR4 N-terminal binding affinity for V3 (WT, -40.1 kcal/mol; G24E, -510 kcal/mol; E25K, -522 kcal/mol). The analysis of paired V3 and Vif DNA sequences from 84 HIV-1-infected patients showed that the presence of a Vif-defective virus correlated with CXCR4 usage in proviral DNA (P = 0.04). In conclusion, incomplete APOBEC3G/F neutralization by a single Vif amino acid substitution seeds a CXCR4-using proviral reservoir. This can have implications for the success of CCR5 antagonist-based therapy, as well as for the risk of disease progression. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Reduction in hepatic drug metabolizing CYP3A4 activities caused by P450 oxidoreductase mutations identified in patients with disordered steroid metabolism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Flueck, Christa E.; Mullis, Primus E.; Pandey, Amit V., E-mail: amit@pandeylab.org

2010-10-08

Research highlights: {yields} Cytochrome P450 3A4 (CYP3A4), metabolizes 50% of drugs in clinical use and requires NADPH-P450 reductase (POR). {yields} Mutations in human POR cause congenital adrenal hyperplasia from diminished activities of steroid metabolizing P450s. {yields} We are reporting that mutations in POR may reduce CYP3A4 activity. {yields} POR mutants Y181D, A457H, Y459H, V492E and R616X lost 99%, while A287P, C569Y and V608F lost 60-85% CYP3A4 activity. {yields} Reduction of CYP3A4 activity may cause increased risk of drug toxicities/adverse drug reactions in patients with POR mutations. -- Abstract: Cytochrome P450 3A4 (CYP3A4), the major P450 present in human liver metabolizesmore » approximately half the drugs in clinical use and requires electrons supplied from NADPH through NADPH-P450 reductase (POR, CPR). Mutations in human POR cause a rare form of congenital adrenal hyperplasia from diminished activities of steroid metabolizing P450s. In this study we examined the effect of mutations in POR on CYP3A4 activity. We used purified preparations of wild type and mutant human POR and in vitro reconstitution with purified CYP3A4 to perform kinetic studies. We are reporting that mutations in POR identified in patients with disordered steroidogenesis/Antley-Bixler syndrome (ABS) may reduce CYP3A4 activity, potentially affecting drug metabolism in individuals carrying mutant POR alleles. POR mutants Y181D, A457H, Y459H, V492E and R616X had more than 99% loss of CYP3A4 activity, while POR mutations A287P, C569Y and V608F lost 60-85% activity. Loss of CYP3A4 activity may result in increased risk of drug toxicities and adverse drug reactions in patients with POR mutations.« less

Efficient infectious cell culture systems of the hepatitis C virus (HCV) prototype strains HCV-1 and H77.

PubMed

Li, Yi-Ping; Ramirez, Santseharay; Mikkelsen, Lotte; Bukh, Jens

2015-01-01

The first discovered and sequenced hepatitis C virus (HCV) genome and the first in vivo infectious HCV clones originated from the HCV prototype strains HCV-1 and H77, respectively, both widely used in research of this important human pathogen. In the present study, we developed efficient infectious cell culture systems for these genotype 1a strains by using the HCV-1/SF9_A and H77C in vivo infectious clones. We initially adapted a genome with the HCV-1 5'UTR-NS5A (where UTR stands for untranslated region) and the JFH1 NS5B-3'UTR (5-5A recombinant), including the genotype 2a-derived mutations F1464L/A1672S/D2979G (LSG), to grow efficiently in Huh7.5 cells, thus identifying the E2 mutation S399F. The combination of LSG/S399F and reported TNcc(1a)-adaptive mutations A1226G/Q1773H/N1927T/Y2981F/F2994S promoted adaptation of the full-length HCV-1 clone. An HCV-1 recombinant with 17 mutations (HCV1cc) replicated efficiently in Huh7.5 cells and produced supernatant infectivity titers of 10(4.0) focus-forming units (FFU)/ml. Eight of these mutations were identified from passaged HCV-1 viruses, and the A970T/I1312V/C2419R/A2919T mutations were essential for infectious particle production. Using CD81-deficient Huh7 cells, we further demonstrated the importance of A970T/I1312V/A2919T or A970T/C2419R/A2919T for virus assembly and that the I1312V/C2419R combination played a major role in virus release. Using a similar approach, we found that NS5B mutation F2994R, identified here from culture-adapted full-length TN viruses and a common NS3 helicase mutation (S1368P) derived from viable H77C and HCV-1 5-5A recombinants, initiated replication and culture adaptation of H77C containing LSG and TNcc(1a)-adaptive mutations. An H77C recombinant harboring 19 mutations (H77Ccc) replicated and spread efficiently after transfection and subsequent infection of naive Huh7.5 cells, reaching titers of 10(3.5) and 10(4.4) FFU/ml, respectively. Hepatitis C virus (HCV) was discovered in 1989 with the cloning of the prototype strain HCV-1 genome. In 1997, two molecular clones of H77, the other HCV prototype strain, were shown to be infectious in chimpanzees, but not in vitro. HCV research was hampered by a lack of infectious cell culture systems, which became available only in 2005 with the discovery of JFH1 (genotype 2a), a genome that could establish infection in Huh7.5 cells. Recently, we developed in vitro infectious clones for genotype 1a (TN), 2a (J6), and 2b (J8, DH8, and DH10) strains by identifying key adaptive mutations. Globally, genotype 1 is the most prevalent. Studies using HCV-1 and H77 prototype sequences have generated important knowledge on HCV. Thus, the in vitro infectious clones developed here for these 1a strains will be of particular value in advancing HCV research. Moreover, our findings open new avenues for the culture adaptation of HCV isolates of different genotypes. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Promoter-dependent activity on androgen receptor N-terminal domain mutations in androgen insensitivity syndrome.

PubMed

Tadokoro-Cuccaro, Rieko; Davies, John; Mongan, Nigel P; Bunch, Trevor; Brown, Rosalind S; Audi, Laura; Watt, Kate; McEwan, Iain J; Hughes, Ieuan A

2014-01-01

Androgen receptor (AR) mutations are associated with androgen insensitivity syndrome (AIS). Missense mutations identified in the AR-N-terminal domain (AR-NTD) are rare, and clinical phenotypes are typically mild. We investigated 7 missense mutations and 2 insertion/deletions located in the AR-NTD. This study aimed to elucidate the pathogenic role of AR-NTD mutants in AIS and to use this knowledge to further define AR-NTD function. AR-NTD mutations (Q120E, A159T, G216R, N235K, G248V, L272F, and P380R) were introduced into AR-expression plasmids. Stably expressing cell lines were established for del57L and ins58L. Transactivation was measured using luciferase reporter constructs under the control of GRE and Pem promoters. Intrinsic fluorescence spectroscopy and partial proteolysis studies were performed for mutations which showed reduced activities by using a purified AR-AF1 protein. Pem-luciferase reporter activation was reduced for A159T, N235K, and G248V but not the GRE-luciferase reporter. Protein structure analysis detected no significant change in the AR-AF1 region for these mutations. Reduced cellular expression and transactivation activity were observed for ins58L. The mutations Q120E, G216R, L272F, P380R, and del57L showed small or no detectable changes in function. Thus, clinical and experimental analyses have identified novel AR-signalling defects associated with mutations in the structurally disordered AR-NTD domain in patients with AIS. © 2014 S. Karger AG, Basel.

Structure-function analysis of the extracellular domains of the Duffy antigen/receptor for chemokines: characterization of antibody and chemokine binding sites.

PubMed

Tournamille, Christophe; Filipe, Anne; Wasniowska, Kazimiera; Gane, Pierre; Lisowska, Elwira; Cartron, Jean-Pierre; Colin, Yves; Le Van Kim, Caroline

2003-09-01

The Duffy antigen/receptor for chemokines (DARC), a seven-transmembrane glycoprotein carrying the Duffy (Fy) blood group, acts as a widely expressed promiscuous chemokine receptor. In a structure-function study, we analysed the binding of chemokines and anti-Fy monoclonal antibodies (mAbs) to K562 cells expressing 39 mutant forms of DARC with alanine substitutions spread out on the four extracellular domains (ECDs). Using synthetic peptides, we defined previously the Fy6 epitope (22-FEDVW-26), and we characterized the Fya epitope as the linear sequence 41-YGANLE-46. In agreement with these results, mutations of F22-E23, V25 and Y41, G42, N44, L45 on ECD1 abolished the binding of anti-Fy6 and anti-Fya mAbs to K562 cells respectively, Anti-Fy3 binding was abolished by D58-D59 (ECD1), R124 (ECD2), D263 and D283 (ECD4) substitutions. Mutations of C51 (ECD1), C129 (ECD2), C195 (ECD3) and C276 (ECD4 severely reduced anti-Fy3 and CXC-chemokine ligand 8 (CXCL-8) binding. CXCL-8 binding was also abrogated by mutations of F22-E23, P50 (ECD1) and D263, R267, D283 (ECD4). These results defined the Fya epitope and suggested that (1) two disulphide bridges are involved in the creation of an active chemokine binding pocket; (2) a limited number of amino acids in ECDs 1-4 participate in CXCL-8 binding; and (3) Fy3 is a conformation-dependent epitope involving all ECDs. We also showed that N-glycosylation of DARC occurred on N16SS and did not influence antibody and chemokine binding.

The Involvement of Ser1898 of the Human L-Type Calcium Channel in Evoked Secretion

PubMed Central

Bachnoff, Niv; Cohen-Kutner, Moshe; Atlas, Daphne

2011-01-01

A PKA consensus phosphorylation site S1928 at the α 11.2 subunit of the rabbit cardiac L-type channel, CaV1.2, is involved in the regulation of CaV1.2 kinetics and affects catecholamine secretion. This mutation does not alter basal CaV1.2 current properties or regulation of CaV1.2 current by PKA and the beta-adrenergic receptor, but abolishes CaV1.2 phosphorylation by PKA. Here, we test the contribution of the corresponding PKA phosphorylation site of the human α 11.2 subunit S1898, to the regulation of catecholamine secretion in bovine chromaffin cells. Chromaffin cells were infected with a Semliki-Forest viral vector containing either the human wt or a mutated S1898A α 11.2 subunit. Both subunits harbor a T1036Y mutation conferring nifedipine insensitivity. Secretion evoked by depolarization in the presence of nifedipine was monitored by amperometry. Depolarization-triggered secretion in cells infected with either the wt α 11.2 or α 11.2/S1898A mutated subunit was elevated to a similar extent by forskolin. Forskolin, known to directly activate adenylyl-cyclase, increased the rate of secretion in a manner that is largely independent of the presence of S1898. Our results are consistent with the involvement of additional PKA regulatory site(s) at the C-tail of α 11.2, the pore forming subunit of CaV1.2. PMID:22216029

Mutations in a signal sequence for the thylakoid membrane identify multiple protein transport pathways and nuclear suppressors

PubMed Central

1994-01-01

The apparatus that permits protein translocation across the internal thylakoid membranes of chloroplasts is completely unknown, even though these membranes have been the subject of extensive biochemical analysis. We have used a genetic approach to characterize the translocation of Chlamydomonas cytochrome f, a chloroplast-encoded protein that spans the thylakoid once. Mutations in the hydrophobic core of the cytochrome f signal sequence inhibit the accumulation of cytochrome f, lead to an accumulation of precursor, and impair the ability of Chlamydomonas cells to grow photosynthetically. One hydrophobic core mutant also reduces the accumulation of other thylakoid membrane proteins, but not those that translocate completely across the membrane. These results suggest that the signal sequence of cytochrome f is required and is involved in one of multiple insertion pathways. Suppressors of two signal peptide mutations describe at least two nuclear genes whose products likely describe the translocation apparatus, and selected second-site chloroplast suppressors further define regions of the cytochrome f signal peptide. PMID:8034740

Severe hypertriglyceridemia due to two novel loss-of-function lipoprotein lipase gene mutations (C310R/E396V) in a Chinese family associated with recurrent acute pancreatitis.

PubMed

Lun, Yu; Sun, Xiaofang; Wang, Ping; Chi, Jingwei; Hou, Xu; Wang, Yangang

2017-07-18

Lipoprotein lipase (LPL) is widely expressed in skeletal muscles, cardiac muscles as well as adipose tissue and involved in the catabolism of triglyceride. Herein we have systematically characterized two novel loss-of-function mutations in LPL from a Chinese family in which afflicted members were manifested by severe hypertriglyceridemia and recurrent pancreatitis. DNA sequencing revealed that the proband was a heterozygote carrying a novel c.T928C (p.C310R) mutation in exon 6 of the LPL gene. Another member of the family was detected to be a compound heterozygote who along with the c.T928C mutation also carried a novel missense mutation c.A1187T (p.E396V) in exon 8 of the LPL gene. Furthermore, COS-1 cells were transfected with lentiviruses containing the mutant LPL genes. While C310R markedly reduced the overall LPL protein level, COS-1 cells carrying E396V or double mutations contained similar overall LPL protein levels to the wild-type. The specific activity of the LPL mutants remained at comparable magnitude to the wild-type. However, few LPL were detected in the culture medium for the mutants, suggesting that both mutations caused aberrant triglyceride catabolism. More specifically, E396V and double mutations dampened the transport of LPL to the cell surface, while for the C310R mutation, reducing LPL protein level might be involved. By characterizing these two novel LPL mutations, this study has expanded our understanding on the pathogenesis of familial hypertriglyceridemia (FHTG).

Profound, prelingual nonsyndromic deafness maps to chromosome 10q21 and is caused by a novel missense mutation in the Usher syndrome type IF gene PCDH15.

PubMed

Doucette, Lance; Merner, Nancy D; Cooke, Sandra; Ives, Elizabeth; Galutira, Dante; Walsh, Vanessa; Walsh, Tom; MacLaren, Linda; Cater, Tracey; Fernandez, Bridget; Green, Jane S; Wilcox, Edward R; Shotland, Lawrence I; Shotland, Larry; Li, Xiaoyan Cindy; Li, X C; Lee, Ming; King, Mary-Claire; Young, Terry-Lynn

2009-05-01

We studied a consanguineous family (Family A) from the island of Newfoundland with an autosomal recessive form of prelingual, profound, nonsyndromic sensorineural hearing loss. A genome-wide scan mapped the deafness trait to 10q21-22 (max LOD score of 4.0; D10S196) and fine mapping revealed a 16 Mb ancestral haplotype in deaf relatives. The PCDH15 gene was mapped within the critical region and was an interesting candidate because truncating mutations cause Usher syndrome type IF (USH1F) and two missense mutations have been previously associated with isolated deafness (DFNB23). Sequencing of the PCDH15 gene revealed 33 sequencing variants. Three of these variants were homozygous exclusively in deaf siblings but only one of them was not seen in ethnically matched controls. This novel c.1583 T>A transversion predicts an amino-acid substitution of a valine with an aspartic acid at codon 528 (V528D). Like the two DFNB23 mutations, the V528D mutation in Family A occurs in a highly conserved extracellular cadherin (EC) domain of PCDH15 and is predicted to be more deleterious than the previously identified DFNB23 missense mutations (R134G and G262D). Physical assessment, vestibular and visual function testing in deaf adults ruled out syndromic deafness because of Usher syndrome. This study validates the DFNB23 designation and supports the hypothesis that missense mutations in conserved motifs of PCDH15 cause nonsyndromic hearing loss. This emerging genotype-phenotype correlation in USH1F is similar to that in several other USH1 genes and cautions against a prognosis of a dual sensory loss in deaf children found to be homozygous for hypomorphic mutations at the USH1F locus.

Caspase-6 Activation in Familial Alzheimer Disease Brains Carrying Amyloid Precursor Protein, Presenilin I or Presenilin II Mutations

PubMed Central

Albrecht, Steffen; Bogdanovic, Nenad; Ghetti, Bernardino; Winblad, Bengt; LeBlanc, Andréa C.

2010-01-01

We previously demonstrated the activation of Caspase-6 in the hippocampus and cortex in cases of mild, moderate, severe and very severe Alzheimer disease (AD). To determine whether Caspase-6 is also activated in familial AD, we performed an immunohistochemical analysis of active Caspase-6 and Tau cleaved by Caspase-6 in temporal cortex and hippocampal tissue sections from cases of familial AD. The cases included 5 carrying the amyloid precursor protein K670N, M671L Swedish mutation, 1 carrying the amyloid precursor protein E693G Arctic mutation, 2 each carrying the Presenilin I M146V, F105L, A431E, V261F, Y115C mutations, and 1 with the Presenilin II N141I mutation. Active Caspase-6 immunoreactivity was found in all cases. Caspase-6 immunoreactivity was observed in neuritic plaques or cotton wool plaques in some cases, neuropil threads and neurofibrillary tangles. These results indicate that Caspase-6 is activated in familial forms of AD, as previously observed in sporadic forms. Since sporadic and familial AD cases have similar pathological features, these results support a fundamental role of Caspase-6 in the pathophysiology of both familial and sporadic AD. PMID:19915487

Further Insights into the Allan-Herndon-Dudley Syndrome: Clinical and Functional Characterization of a Novel MCT8 Mutation.

PubMed

Armour, Christine M; Kersseboom, Simone; Yoon, Grace; Visser, Theo J

2015-01-01

Mutations in the thyroid hormone (TH) transporter MCT8 have been identified as the cause for Allan-Herndon-Dudley Syndrome (AHDS), characterized by severe psychomotor retardation and altered TH serum levels. Here we report a novel MCT8 mutation identified in 4 generations of one family, and its functional characterization. Proband and family members were screened for 60 genes involved in X-linked cognitive impairment and the MCT8 mutation was confirmed. Functional consequences of MCT8 mutations were studied by analysis of [125I]TH transport in fibroblasts and transiently transfected JEG3 and COS1 cells, and by subcellular localization of the transporter. The proband and a male cousin demonstrated clinical findings characteristic of AHDS. Serum analysis showed high T3, low rT3, and normal T4 and TSH levels in the proband. A MCT8 mutation (c.869C>T; p.S290F) was identified in the proband, his cousin, and several female carriers. Functional analysis of the S290F mutant showed decreased TH transport, metabolism and protein expression in the three cell types, whereas the S290A mutation had no effect. Interestingly, both uptake and efflux of T3 and T4 was impaired in fibroblasts of the proband, compared to his healthy brother. However, no effect of the S290F mutation was observed on TH efflux from COS1 and JEG3 cells. Immunocytochemistry showed plasma membrane localization of wild-type MCT8 and the S290A and S290F mutants in JEG3 cells. We describe a novel MCT8 mutation (S290F) in 4 generations of a family with Allan-Herndon-Dudley Syndrome. Functional analysis demonstrates loss-of-function of the MCT8 transporter. Furthermore, our results indicate that the function of the S290F mutant is dependent on cell context. Comparison of the S290F and S290A mutants indicates that it is not the loss of Ser but its substitution with Phe, which leads to S290F dysfunction.

Further Insights into the Allan-Herndon-Dudley Syndrome: Clinical and Functional Characterization of a Novel MCT8 Mutation

PubMed Central

Yoon, Grace; Visser, Theo J.

2015-01-01

Background Mutations in the thyroid hormone (TH) transporter MCT8 have been identified as the cause for Allan-Herndon-Dudley Syndrome (AHDS), characterized by severe psychomotor retardation and altered TH serum levels. Here we report a novel MCT8 mutation identified in 4 generations of one family, and its functional characterization. Methods Proband and family members were screened for 60 genes involved in X-linked cognitive impairment and the MCT8 mutation was confirmed. Functional consequences of MCT8 mutations were studied by analysis of [125I]TH transport in fibroblasts and transiently transfected JEG3 and COS1 cells, and by subcellular localization of the transporter. Results The proband and a male cousin demonstrated clinical findings characteristic of AHDS. Serum analysis showed high T3, low rT3, and normal T4 and TSH levels in the proband. A MCT8 mutation (c.869C>T; p.S290F) was identified in the proband, his cousin, and several female carriers. Functional analysis of the S290F mutant showed decreased TH transport, metabolism and protein expression in the three cell types, whereas the S290A mutation had no effect. Interestingly, both uptake and efflux of T3 and T4 was impaired in fibroblasts of the proband, compared to his healthy brother. However, no effect of the S290F mutation was observed on TH efflux from COS1 and JEG3 cells. Immunocytochemistry showed plasma membrane localization of wild-type MCT8 and the S290A and S290F mutants in JEG3 cells. Conclusions We describe a novel MCT8 mutation (S290F) in 4 generations of a family with Allan-Herndon-Dudley Syndrome. Functional analysis demonstrates loss-of-function of the MCT8 transporter. Furthermore, our results indicate that the function of the S290F mutant is dependent on cell context. Comparison of the S290F and S290A mutants indicates that it is not the loss of Ser but its substitution with Phe, which leads to S290F dysfunction. PMID:26426690

Mucopolysaccharidosis type I in 21 Czech and Slovak patients: Mutation analysis suggests a functional importance of C-terminus of the IDUA protein

PubMed Central

Vazna, Alzbeta; Beesley, Clare; Berna, Linda; Stolnaja, Larisa; Myskova, Helena; Bouckova, Michaela; Vlaskova, Hana; Poupetova, Helena; Zeman, Jiri; Magner, Martin; Hlavata, Anna; Winchester, Bryan; Hrebicek, Martin; Dvorakova, Lenka

2009-01-01

Abstract Mucopolysaccharidosis type I (MPS I) is an autosomal recessive lysosomal storage disorder that is caused by a deficiency of the enzyme α-l-iduronidase (IDUA). Of the 21 Czech and Slovak patients who have been diagnosed with MPS I in the last 30 years, 16 have a severe clinical presentation (Hurler syndrome), 2 less severe manifestations (Scheie syndrome), and 3 an intermediate severity (Hurler/Scheie phenotype). Mutation analysis was performed in 20 MPS I patients and 39 mutant alleles were identified. There was a high prevalence of the null mutations p.W402X (12 alleles) and p.Q70X (7 alleles) in this cohort. Four of the 13 different mutations were novel: p.V620F (3 alleles), p.W626X (1 allele), c.1727 + 2T > G (1 allele) and c.1918_1927del (2 alleles). The pathogenicity of the novel mutations was verified by transient expression studies in Chinese hamster ovary cells. Seven haplotypes were observed in the patient alleles using 13 intragenic polymorphisms. One of the two haplotypes associated with the mutation p.Q70X was not found in any of the controls. Haplotype analysis showed, that mutations p.Q70X, p.V620F, and p.D315Y probably have more than one ancestor. Missense mutations localized predominantly in the hydrophobic core of the enzyme are associated with the severe phenotype, whereas missense mutations localized to the surface of the enzyme are usually associated with the attenuated phenotypes. Mutations in the 130 C-terminal amino acids lead to clinical manifestations, which indicates a functional importance of the C-terminus of the IDUA protein. © 2009 Wiley-Liss, Inc. PMID:19396826

Analysis of IgV gene mutations in B cell chronic lymphocytic leukaemia according to antigen-driven selection identifies subgroups with different prognosis and usage of the canonical somatic hypermutation machinery.

PubMed

Degan, Massimo; Bomben, Riccardo; Bo, Michele Dal; Zucchetto, Antonella; Nanni, Paola; Rupolo, Maurizio; Steffan, Agostino; Attadia, Vincenza; Ballerini, Pier Ferruccio; Damiani, Daniela; Pucillo, Carlo; Poeta, Giovanni Del; Colombatti, Alfonso; Gattei, Valter

2004-07-01

Cases of B-cell chronic lymphocytic leukaemia (B-CLL) with mutated (M) IgV(H) genes have a better prognosis than unmutated (UM) cases. We analysed the IgV(H) mutational status of B-CLL according to the features of a canonical somatic hypermutation (SHM) process, correlating this data with survival. In a series of 141 B-CLLs, 124 cases were examined for IgV(H) gene per cent mutations and skewing of replacement/silent mutations in the framework/complementarity-determining regions as evidence of antigen-driven selection; this identified three B-CLL subsets: significantly mutated (sM), with evidence of antigen-driven selection, not significantly mutated (nsM) and UM, without such evidence and IgV(H) gene per cent mutations above or below the 2% cut-off. sM B-CLL patients had longer survival within the good prognosis subgroup that had more than 2% mutations of IgV(H) genes. sM, nsM and UM B-CLL were also characterized for the biased usage of IgV(H) families, intraclonal IgV(H) gene diversification, preference of mutations to target-specific nucleotides or hotspots, and for the expression of enzymes involved in SHM (translesion DNA polymerase zeta and eta and activation-induced cytidine deaminase). These findings indicate the activation of a canonical SHM process in nsM and sM B-CLLs and underscore the role of the antigen in defining the specific clinical and biological features of B-CLL.

A novel flux-switching permanent magnet machine with v-shaped magnets

NASA Astrophysics Data System (ADS)

Zhao, Guishu; Hua, Wei

2017-05-01

In this paper, firstly a novel 6-stator-coil/17-rotor-pole (6/17) flux-switching permanent magnet (FSPM) machine with V-shaped magnets, deduced from conventional 12/17 FSPM machines is proposed to achieve more symmetrical phase back-electromotive force (back-EMF), and smaller torque ripple by comparing with an existing 6/10 V-shaped FSPM machine. Then, to obtain larger electromagnetic torque, less torque ripple, and easier mechanical processing, two improved variants based on the original 6/17 V-shaped topology are proposed. For the first variant, the separate stator-core segments located on the stator yoke are connected into a united stator yoke, while for the second variant the stator core is a whole entity by adding magnetic bridges at the ends of permanent magnets (PMs). Consequently, the performances of the three 6/17 V-shaped FSPM machines, namely, the original one and the two variants, are conducted by finite element analysis (FEA). The results reveal that the first variant exhibits significantly larger torque and considerably improved torque per magnet volume, i.e., the magnet utilization ratio than the original one, and the second variant exhibits the smallest torque ripple, least total harmonic distribution (THD) of phase back-EMF, and easiest mechanical processing for manufacturing.

In vitro resistance development for RO-0335, a novel diphenylether nonnucleoside reverse transcriptase inhibitor.

PubMed

Javanbakht, H; Ptak, R G; Chow, E; Yan, J M; Russell, J D; Mankowski, M K; Hogan, P A; Hogg, J H; Vora, H; Hang, J Q; Li, Y; Su, G; Paul, A; Cammack, N; Klumpp, K; Heilek, G

2010-05-01

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are important components of current combination therapies for the treatment of human immunodeficiency virus type 1 (HIV-1) infection. However, their low genetic barriers against resistance development, cross-resistance and serious side effects can compromise the benefits of the first generation compounds in this class (efavirenz and nevirapine). To study potential pathways leading to resistance against the novel diphenylether NNRTI, RO-0335, sequential passage experiments at low multiplicity of infection (MOI) were performed to solicit a stepwise selection of resistance mutations. Two pathways to loss of susceptibility to RO-0335 were observed, containing patterns of amino acid changes at either V106I/A plus F227C (with additional contributions from A98G, V108I, E138K, M230L and P236L) or V106I/Y188L (with a potential contribution from L100I, E138K and Y181C). Characterization of the observed mutations by site-directed mutagenesis in the isogenic HXB2D background demonstrated that a minimum of two or more mutations were required for significant loss of susceptibility, with the exception of Y188L, which requires a two-nucleotide change. Patterns containing F227C or quadruple mutations selected by RO-0335 showed a low relative fitness value when compared to wild-type HXB2D.

Frequent germline deleterious mutations in DNA repair genes in familial prostate cancer cases are associated with advanced disease.

PubMed

Leongamornlert, D; Saunders, E; Dadaev, T; Tymrakiewicz, M; Goh, C; Jugurnauth-Little, S; Kozarewa, I; Fenwick, K; Assiotis, I; Barrowdale, D; Govindasami, K; Guy, M; Sawyer, E; Wilkinson, R; Antoniou, A C; Eeles, R; Kote-Jarai, Z

2014-03-18

Prostate cancer (PrCa) is one of the most common diseases to affect men worldwide and among the leading causes of cancer-related death. The purpose of this study was to use second-generation sequencing technology to assess the frequency of deleterious mutations in 22 tumour suppressor genes in familial PrCa and estimate the relative risk of PrCa if these genes are mutated. Germline DNA samples from 191 men with 3 or more cases of PrCa in their family were sequenced for 22 tumour suppressor genes using Agilent target enrichment and Illumina technology. Analysis for genetic variation was carried out by using a pipeline consisting of BWA, Genome Analysis Toolkit (GATK) and ANNOVAR. Clinical features were correlated with mutation status using standard statistical tests. Modified segregation analysis was used to determine the relative risk of PrCa conferred by the putative loss-of-function (LoF) mutations identified. We discovered 14 putative LoF mutations in 191 samples (7.3%) and these mutations were more frequently associated with nodal involvement, metastasis or T4 tumour stage (P=0.00164). Segregation analysis of probands with European ancestry estimated that LoF mutations in any of the studied genes confer a relative risk of PrCa of 1.94 (95% CI: 1.56-2.42). These findings show that LoF mutations in DNA repair pathway genes predispose to familial PrCa and advanced disease and therefore warrants further investigation. The clinical utility of these findings will become increasingly important as targeted screening and therapies become more widespread.

Structural Studies of a Rationally Selected Multi-Drug Resistant HIV-1 Protease Reveal Synergistic Effect of Distal Mutations on Flap Dynamics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Agniswamy, Johnson; Louis, John M.; Roche, Julien

We report structural analysis of HIV protease variant PRS17 which was rationally selected by machine learning to represent wide classes of highly drug-resistant variants. Crystal structures were solved of PRS17 in the inhibitor-free form and in complex with antiviral inhibitor, darunavir. Despite its 17 mutations, PRS17 has only one mutation (V82S) in the inhibitor/substrate binding cavity, yet exhibits high resistance to all clinical inhibitors. PRS17 has none of the major mutations (I47V, I50V, I54ML, L76V and I84V) associated with darunavir resistance, but has 10,000-fold weaker binding affinity relative to the wild type PR. Comparable binding affinity of 8000-fold weaker thanmore » PR is seen for drug resistant mutant PR20, which bears 3 mutations associated with major resistance to darunavir (I47V, I54L and I84V). Inhibitor-free PRS17 shows an open flap conformation with a curled tip correlating with G48V flap mutation. NMR studies on inactive PRS17 D25N unambiguously confirm that the flaps adopt mainly an open conformation in solution very similar to that in the inhibitor-free crystal structure. In PRS17, the hinge loop cluster of mutations, E35D, M36I and S37D, contributes to the altered flap dynamics by a mechanism similar to that of PR20. An additional K20R mutation anchors an altered conformation of the hinge loop. Flap mutations M46L and G48V in PRS17/DRV complex alter the Phe53 conformation by steric hindrance between the side chains. Unlike the L10F mutation in PR20, L10I in PRS17 does not break the inter-subunit ion pair or diminish the dimer stability, consistent with a very low dimer dissociation constant comparable to that of wild type PR. Distal mutations A71V, L90M and I93L propagate alterations to the catalytic site of PRS17. PRS17 exhibits a molecular mechanism whereby mutations act synergistically to alter the flap dynamics resulting in significantly weaker binding yet maintaining active site contacts with darunavir.« less

Stabilization of luciferase from Renilla reniformis using random mutations.

PubMed

Shigehisa, Megumi; Amaba, Norie; Arai, Shigeki; Higashi, Chisato; Kawanabe, Ryo; Matsunaga, Ayano; Laksmi, Fina Amreta; Tokunaga, Masao; Ishibashi, Matsujiro

2017-01-01

We expressed luciferase (RLuc) from Renilla reniformis in Escherichia coli RLuc was purified using a Ni-NTA column and subsequently characterized. It was unstable in acidic solutions and at 30°C. To increase the stability of RLuc, the Rluc gene was randomly mutated using error-prone polymerase chain reaction. E. coli harboring the mutated gene was screened by detecting luminescence on a plate containing the substrate coelenterazine at 34°C. Three mutants, i.e. N264SS287P, N178D and F116LI137V, were obtained. The solubilities and specific activities of these mutants were higher than those of the wild type. Furthermore, the N264SS287P mutant maintained stability at a temperature approximately 5°C higher than that of the wild type, while denaturation of the F116LI137V mutant started at a temperature that was 5°C lower than the wild type, and ended at a temperature that was 7°C higher. We examined the obtained mutations using thermal shift assays and a computer program Coot in this study. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Polymorphisms and resistance mutations in the protease and reverse transcriptase genes of HIV-1 F subtype Romanian strains.

PubMed

Paraschiv, Simona; Otelea, Dan; Dinu, Magdalena; Maxim, Daniela; Tinischi, Mihaela

2007-03-01

To evaluate the prevalence of resistance mutations in the genome of HIV-1 F subtype strains isolated from Romanian antiretroviral (ARV) treatment-naïve patients and to assess the phylogenetic relatedness of these strains with other HIV-1 strains. Twenty-nine HIV-1 strains isolated from treatment-naïve adolescents (n=15) and adults (n=14) were included in this study. Resistance genotyping was performed by using Big Dye Terminator chemistry provided by the ViroSeq Genotyping System. The sequences of the protease and reverse transcriptase genes were aligned (ClustalW) and a phylogenetic tree was built (MEGA 3 software). For subtyping purposes, all the nucleotide sequences were submitted to the Stanford database. All the studied strains were found to harbor accessory mutations in the protease gene. The most frequent mutation was M36I (29 of 29 strains), followed by L63T, K20R, and L10V. The number of polymorphisms associated with protease inhibitor resistance was different for the two age groups. Intraphylogenetic divergence was greater for adults than for adolescents infected in childhood. All the strains were found to belong to the F1 subtype. The phylogenetic analysis revealed that Romanian strains clustered together, but distinctly from F1 HIV-1 strains isolated in other parts of the world (Brazil, Finland, and Belgium). Protease secondary mutations are present with high frequency in the HIV-1 F subtype strains isolated from Romanian ARV treatment-naïve patients, but no major resistance mutations were found.

Comparison of kinetics, toxicity, oligomers formation and membrane binding capacity of α-synuclein familial mutations at A53 site including newly discovered A53V mutation.

PubMed

Mohite, Ganesh M; Kumar, Rakesh; Panigrahi, Rajlaxmi; Navalkar, Ambuja; Singh, Nitu; Datta, Debalina; Mehra, Surabhi; Ray, Soumik; Gadhe, Laxmikant G; Das, Subhadeep; Singh, Namrata; Chatterjee, Debdeep; Kumar, Ashutosh; Maji, Samir K

2018-05-17

The involvement of α-synuclein (α-Syn) amyloid formation in Parkinson's disease (PD) pathogenesis is supported by the discovery of α-Syn gene (SNCA) mutations linked with familial PD, which are known to modulate the oligomerization and aggregation of α-Syn. Recently, the A53V mutation has been discovered, which leads to the late-onset PD. In the present study, we characterized for the first time the biophysical properties including the aggregation propensities, toxicity of aggregated species and membrane binding capability of A53V along with all familial mutations at A53 position. Present data suggest that A53V accelerate fibrillation of α-Syn without affecting the overall morphology and cytotoxicity of fibrils compared to wild-type protein. The aggregation propensity for A53 mutants is found to be; A53T>A53V>WT>A53E. Further, time course aggregation study reveals that A53V mutant promotes early oligomerization similar to A53T mutation. It promotes the highest amount of oligomer formation immediate after dissolution, which are cytotoxic. Although in the presence of membrane-mimicking environments, A53V mutation showed similar extent of helix-induction capacity as of WT protein, however, it exhibited lesser binding to lipid vesicle. The NMR study revealed unique chemical shift perturbation by A53V mutation com-pared to other mutations at A53 site. The present study might help to establish the disease-causing mechanism of A53V in PD pathology.

Molecular basis of cystic fibrosis in Lithuania: incomplete CFTR mutation detection by PCR-based screening protocols.

PubMed

Giannattasio, S; Bobba, A; Jurgelevicius, V; Vacca, R A; Lattanzio, P; Merafina, R S; Utkus, A; Kucinskas, V; Marra, E

2006-01-01

Mutational analysis of the cystic fibrosis transmembrane regulator (CFTR) gene was performed in 98 unrelated CF chromosomes from 49 Lithuanian CF patients through a combined approach in which the p.F508del mutation was first screened by allele-specific PCR while CFTR mutations in nonp.F508del chromosomes have been screened for by denaturing gradient gel electrophoresis analysis. A CFTR mutation was characterized in 62.2% of CF chromosomes, two of which (2.0%) have been previously shown to carry a large gene deletion CFTRdele2,3(21 kb). The most frequent Lithuanian CF mutation is p.F508del (52.0%). Seven CFTR mutations, p.N1303K (2.0%), p.R75Q (1.0%), p.G314R (1.0%), p.R553X (4.2%), p.W1282X (1.0%), and g.3944delGT (1.0%), accounted for 10.1% of Lithuanian CF chromosomes. It was not possible to characterize 35.8% of the CF Lithuanian chromosomes. Analysis of intron 8 (TG)mTn and M470V polymorphic loci did not permit the characterization of the CFTR dysfunction underlying the CF phenotype in the patients for which no CFTR mutation was identified. Thus, screening of the eight CFTR mutations identified in this study and of the large deletion CFTRdele2,3(21 kb) allows the implementation of an early molecular or confirmatory CF diagnosis for 65% of Lithuanian CF chromosomes.

Low frequency of genotypic resistance in HIV-1-infected patients failing an atazanavir-containing regimen: a clinical cohort study

PubMed Central

Dolling, David I.; Dunn, David T.; Sutherland, Katherine A.; Pillay, Deenan; Mbisa, Jean L.; Parry, Chris M.; Post, Frank A.; Sabin, Caroline A.; Cane, Patricia A.; Aitken, Celia; Asboe, David; Webster, Daniel; Cane, Patricia; Castro, Hannah; Dunn, David; Dolling, David; Chadwick, David; Churchill, Duncan; Clark, Duncan; Collins, Simon; Delpech, Valerie; Geretti, Anna Maria; Goldberg, David; Hale, Antony; Hué, Stéphane; Kaye, Steve; Kellam, Paul; Lazarus, Linda; Leigh-Brown, Andrew; Mackie, Nicola; Orkin, Chloe; Rice, Philip; Pillay, Deenan; Phillips, Andrew; Sabin, Caroline; Smit, Erasmus; Templeton, Kate; Tilston, Peter; Tong, William; Williams, Ian; Zhang, Hongyi; Zuckerman, Mark; Greatorex, Jane; Wildfire, Adrian; O'Shea, Siobhan; Mullen, Jane; Mbisa, Tamyo; Cox, Alison; Tandy, Richard; Hale, Tony; Fawcett, Tracy; Hopkins, Mark; Ashton, Lynn; Booth, Claire; Garcia-Diaz, Ana; Shepherd, Jill; Schmid, Matthias L.; Payne, Brendan; Hay, Phillip; Rice, Phillip; Paynter, Mary; Bibby, David; Kirk, Stuart; MacLean, Alasdair; Gunson, Rory; Coughlin, Kate; Fearnhill, Esther; Fradette, Lorraine; Porter, Kholoud; Ainsworth, Jonathan; Anderson, Jane; Babiker, Abdel; Fisher, Martin; Gazzard, Brian; Gilson, Richard; Gompels, Mark; Hill, Teresa; Johnson, Margaret; Kegg, Stephen; Leen, Clifford; Nelson, Mark; Palfreeman, Adrian; Post, Frank; Sachikonye, Memory; Schwenk, Achim; Walsh, John; Huntington, Susie; Jose, Sophie; Thornton, Alicia; Glabay, Adam; Orkin, C.; Garrett, N.; Lynch, J.; Hand, J.; de Souza, C.; Fisher, M.; Perry, N.; Tilbury, S.; Gazzard, B.; Nelson, M.; Waxman, M.; Asboe, D.; Mandalia, S.; Delpech, V.; Anderson, J.; Munshi, S.; Korat, H.; Welch, J.; Poulton, M.; MacDonald, C.; Gleisner, Z.; Campbell, L.; Gilson, R.; Brima, N.; Williams, I.; Schwenk, A.; Ainsworth, J.; Wood, C.; Miller, S.; Johnson, M.; Youle, M.; Lampe, F.; Smith, C.; Grabowska, H.; Chaloner, C.; Puradiredja, D.; Walsh, J.; Weber, J.; Ramzan, F.; Mackie, N.; Winston, A.; Leen, C.; Wilson, A.; Allan, S.; Palfreeman, A.; Moore, A.; Wakeman, K.

2013-01-01

Objectives To determine protease mutations that develop at viral failure for protease inhibitor (PI)-naive patients on a regimen containing the PI atazanavir. Methods Resistance tests on patients failing atazanavir, conducted as part of routine clinical care in a multicentre observational study, were randomly matched by subtype to resistance tests from PI-naive controls to account for natural polymorphisms. Mutations from the consensus B sequence across the protease region were analysed for association and defined using the IAS-USA 2011 classification list. Results Four hundred and five of 2528 (16%) patients failed therapy containing atazanavir as a first PI over a median (IQR) follow-up of 1.76 (0.84–3.15) years and 322 resistance tests were available for analysis. Recognized major atazanavir mutations were found in six atazanavir-experienced patients (P < 0.001), including I50L and N88S. The minor mutations most strongly associated with atazanavir experience were M36I, M46I, F53L, A71V, V82T and I85V (P < 0.05). Multiple novel mutations, I15S, L19T, K43T, L63P/V, K70Q, V77I and L89I/T/V, were also associated with atazanavir experience. Conclusions Viral failure on atazanavir-containing regimens was not common and major resistance mutations were rare, suggesting that adherence may be a major contributor to viral failure. Novel mutations were described that have not been previously documented. PMID:23711895

Nonsensing residues in S3-S4 linker's C terminus affect the voltage sensor set point in K+ channels.

PubMed

Carvalho-de-Souza, Joao L; Bezanilla, Francisco

2018-02-05

Voltage sensitivity in ion channels is a function of highly conserved arginine residues in their voltage-sensing domains (VSDs), but this conservation does not explain the diversity in voltage dependence among different K + channels. Here we study the non-voltage-sensing residues 353 to 361 in Shaker K + channels and find that residues 358 and 361 strongly modulate the voltage dependence of the channel. We mutate these two residues into all possible remaining amino acids (AAs) and obtain Q-V and G-V curves. We introduced the nonconducting W434F mutation to record sensing currents in all mutants except L361R, which requires K + depletion because it is affected by W434F. By fitting Q-Vs with a sequential three-state model for two voltage dependence-related parameters ( V 0 , the voltage-dependent transition from the resting to intermediate state and V 1 , from the latter to the active state) and G-Vs with a two-state model for the voltage dependence of the pore domain parameter ( V 1/2 ), Spearman's coefficients denoting variable relationships with hydrophobicity, available area, length, width, and volume of the AAs in 358 and 361 positions could be calculated. We find that mutations in residue 358 shift Q-Vs and G-Vs along the voltage axis by affecting V 0 , V 1 , and V 1/2 according to the hydrophobicity of the AA. Mutations in residue 361 also shift both curves, but V 0 is affected by the hydrophobicity of the AA in position 361, whereas V 1 and V 1/2 are affected by size-related AA indices. Small-to-tiny AAs have opposite effects on V 1 and V 1/2 in position 358 compared with 361. We hypothesize possible coordination points in the protein that residues 358 and 361 would temporarily and differently interact with in an intermediate state of VSD activation. Our data contribute to the accumulating knowledge of voltage-dependent ion channel activation by adding functional information about the effects of so-called non-voltage-sensing residues on VSD dynamics. © 2018 Carvalho-de-Souza and Bezanilla.

HER2 activating mutations are targets for colorectal cancer treatment

PubMed Central

Kavuri, Shyam M.; Jain, Naveen; Galimi, Francesco; Cottino, Francesca; Leto, Simonetta M.; Migliardi, Giorgia; Searleman, Adam C.; Shen, Wei; Monsey, John; Trusolino, Livio; Jacobs, Samuel A.; Bertotti, Andrea; Bose, Ron

2015-01-01

The Cancer Genome Atlas project identified HER2 somatic mutations and gene amplification in 7% of colorectal cancer patients. Introduction of the HER2 mutations, S310F, L755S, V777L, V842I, and L866M, into colon epithelial cells increased signaling pathways and anchorage-independent cell growth, indicating that they are activating mutations. Introduction of these HER2 activating mutations into colorectal cancer cell lines produced resistance to cetuximab and panitumumab by sustaining MAPK phosphorylation. HER2 mutations are potently inhibited by low nanomolar doses of the irreversible tyrosine kinase inhibitors, neratinib and afatinib. HER2 gene sequencing of 48 cetuximab resistant, quadruple (KRAS, NRAS, BRAF, and PIK3CA) WT colorectal cancer patient-derived xenografts (PDX’s) identified 4 PDX’s with HER2 mutations. HER2 targeted therapies were tested on two PDX’s. Treatment with a single HER2 targeted drug (trastuzumab, neratinib, or lapatinib) delayed tumor growth, but dual HER2 targeted therapy with trastuzumab plus tyrosine kinase inhibitors produced regression of these HER2 mutated PDX’s. PMID:26243863

Physical results from 2+1 flavor domain wall QCD and SU(2) chiral perturbation theory

DOE Office of Scientific and Technical Information (OSTI.GOV)

Allton, C.; Antonio, D. J.; Boyle, P. A.

2008-12-01

We have simulated QCD using 2+1 flavors of domain wall quarks and the Iwasaki gauge action on a (2.74 fm){sup 3} volume with an inverse lattice scale of a{sup -1}=1.729(28) GeV. The up and down (light) quarks are degenerate in our calculations and we have used four values for the ratio of light quark masses to the strange (heavy) quark mass in our simulations: 0.217, 0.350, 0.617, and 0.884. We have measured pseudoscalar meson masses and decay constants, the kaon bag parameter B{sub K}, and vector meson couplings. We have used SU(2) chiral perturbation theory, which assumes only the upmore » and down quark masses are small, and SU(3) chiral perturbation theory to extrapolate to the physical values for the light quark masses. While next-to-leading order formulas from both approaches fit our data for light quarks, we find the higher-order corrections for SU(3) very large, making such fits unreliable. We also find that SU(3) does not fit our data when the quark masses are near the physical strange quark mass. Thus, we rely on SU(2) chiral perturbation theory for accurate results. We use the masses of the {omega} baryon, and the {pi} and K mesons to set the lattice scale and determine the quark masses. We then find f{sub {pi}}=124.1(3.6){sub stat}(6.9){sub syst} MeV, f{sub K}=149.6(3.6){sub stat}(6.3){sub syst} MeV, and f{sub K}/f{sub {pi}}=1.205(0.018){sub stat}(0.062){sub syst}. Using nonperturbative renormalization to relate lattice regularized quark masses to regularization independent momentum scheme masses, and perturbation theory to relate these to MS, we find m{sub ud}{sup MS}(2 GeV)=3.72(0.16){sub stat}(0.33){sub ren}(0.18){sub syst} MeV, m{sub s}{sup MS}(2 GeV)=107.3(4.4){sub stat}(9.7){sub ren}(4.9){sub syst} MeV, and m-tilde{sub ud} ratio m-tilde{sub s}=1 ratio 28.8(0.4){sub stat}(1.6){sub syst}. For the kaon bag parameter, we find B{sub K}{sup MS}(2 GeV)=0.524(0.010){sub stat}(0.013){sub ren}(0.025){sub syst}. Finally, for the ratios of the couplings of the vector mesons to the vector and tensor currents (f{sub V} and f{sub V}{sup T}, respectively) in the MS scheme at 2 GeV we obtain f{sub {rho}}{sup T}/f{sub {rho}}=0.687(27); f{sub K*}{sup T}/f{sub K*}=0.712(12), and f{sub {phi}}{sup T}/f{sub {phi}}=0.750(8)« less

Measurement of the Absolute Branching Fraction of Ds+→τ+ντ Decay

NASA Astrophysics Data System (ADS)

Ecklund, K. M.; Love, W.; Savinov, V.; Lopez, A.; Mendez, H.; Ramirez, J.; Ge, J. Y.; Miller, D. H.; Shipsey, I. P. J.; Xin, B.; Adams, G. S.; Anderson, M.; Cummings, J. P.; Danko, I.; Hu, D.; Moziak, B.; Napolitano, J.; He, Q.; Insler, J.; Muramatsu, H.; Park, C. S.; Thorndike, E. H.; Yang, F.; Artuso, M.; Blusk, S.; Khalil, S.; Li, J.; Mountain, R.; Nisar, S.; Randrianarivony, K.; Sultana, N.; Skwarnicki, T.; Stone, S.; Wang, J. C.; Zhang, L. M.; Bonvicini, G.; Cinabro, D.; Dubrovin, M.; Lincoln, A.; Rademacker, J.; Asner, D. M.; Edwards, K. W.; Naik, P.; Reed, J.; Briere, R. A.; Ferguson, T.; Tatishvili, G.; Vogel, H.; Watkins, M. E.; Rosner, J. L.; Alexander, J. P.; Cassel, D. G.; Duboscq, J. E.; Ehrlich, R.; Fields, L.; Gibbons, L.; Gray, R.; Gray, S. W.; Hartill, D. L.; Heltsley, B. K.; Hertz, D.; Jones, C. D.; Kandaswamy, J.; Kreinick, D. L.; Kuznetsov, V. E.; Mahlke-Krüger, H.; Mohapatra, D.; Onyisi, P. U. E.; Patterson, J. R.; Peterson, D.; Riley, D.; Ryd, A.; Sadoff, A. J.; Shi, X.; Stroiney, S.; Sun, W. M.; Wilksen, T.; Athar, S. B.; Patel, R.; Yelton, J.; Rubin, P.; Eisenstein, B. I.; Karliner, I.; Mehrabyan, S.; Lowrey, N.; Selen, M.; White, E. J.; Wiss, J.; Mitchell, R. E.; Shepherd, M. R.; Besson, D.; Pedlar, T. K.; Cronin-Hennessy, D.; Gao, K. Y.; Hietala, J.; Kubota, Y.; Klein, T.; Lang, B. W.; Poling, R.; Scott, A. W.; Zweber, P.; Dobbs, S.; Metreveli, Z.; Seth, K. K.; Tomaradze, A.; Libby, J.; Powell, A.; Wilkinson, G.

2008-04-01

Using a sample of tagged Ds+ decays collected near the Ds*±Ds∓ peak production energy in e+e- collisions with the CLEO-c detector, we study the leptonic decay Ds+→τ+ντ via the decay channel τ+→e+νeν¯τ. We measure B(Ds+→τ+ντ)=(6.17±0.71±0.34)%, where the first error is statistical and the second systematic. Combining this result with our measurements of Ds+→μ+νμ and Ds+→τ+ντ (via τ+→π+ν¯τ), we determine fDs=(274±10±5)MeV.

New Carbon Dioxide Line Parameters for Atmospheric and High Temperature Applications

DTIC Science & Technology

1989-10-16

10 LO, z W *0 iZ w 0 0 D V) 0c W +~ 2:.’ 0t -XJ N- O0 0 0 z U- WE 0 2: 0 2: w r-= cc wcccc 0 LOI r 0 3 0w C4o,) 00 0~ U) w cw > ZEC L 4 JZ c0 cc OW...02173 Mr. Charles Randall The Aerospace Corp. (213)366-5977 P.O. Box 92957 Los Angeles, CA 90009 Mr. Peter J. Rayer Dept. of Atmospheric, Oceanic and...National Laboratory (505)667-7672 ESS-5 MS F665 Los Alamos, NM 87545 Dr. Peter Wintersteiner ARCON Corp. (617)890-3330 260 Bear Hill Road Waltham, MA 02154

Discovery of Point Mutations in the Voltage-Gated Sodium Channel from African Aedes aegypti Populations: Potential Phylogenetic Reasons for Gene Introgression

PubMed Central

Muranami, Yuto; Kawashima, Emiko; Osei, Joseph H. N.; Sakyi, Kojo Yirenkyi; Dadzie, Samuel; de Souza, Dziedzom K.; Appawu, Maxwell; Ohta, Nobuo; Minakawa, Noboru

2016-01-01

Background Yellow fever is endemic in some countries in Africa, and Aedes aegpyti is one of the most important vectors implicated in the outbreak. The mapping of the nation-wide distribution and the detection of insecticide resistance of vector mosquitoes will provide the beneficial information for forecasting of dengue and yellow fever outbreaks and effective control measures. Methodology/Principal Findings High resistance to DDT was observed in all mosquito colonies collected in Ghana. The resistance and the possible existence of resistance or tolerance to permethrin were suspected in some colonies. High frequencies of point mutations at the voltage-gated sodium channel (F1534C) and one heterozygote of the other mutation (V1016I) were detected, and this is the first detection on the African continent. The frequency of F1534C allele and the ratio of F1534C homozygotes in Ae. aegypti aegypti (Aaa) were significantly higher than those in Ae. aegypti formosus (Aaf). We could detect the two types of introns between exon 20 and 21, and the F1534C mutations were strongly linked with one type of intron, which was commonly found in South East Asian and South and Central American countries, suggesting the possibility that this mutation was introduced from other continents or convergently selected after the introgression of Aaa genes from the above area. Conclusions/Significance The worldwide eradication programs in 1940s and 1950s might have caused high selection pressure on the mosquito populations and expanded the distribution of insecticide-resistant Ae. aegypti populations. Selection of the F1534C point mutation could be hypothesized to have taken place during this period. The selection of the resistant population of Ae. aegypti with the point mutation of F1534C, and the worldwide transportation of vector mosquitoes in accordance with human activity such as trading of used tires, might result in the widespread distribution of F1534C point mutation in tropical countries. PMID:27304430

Mutations in the Transmembrane Domain and Cytoplasmic Tail of Hendra Virus Fusion Protein Disrupt Virus-Like-Particle Assembly.

PubMed

Cifuentes-Muñoz, Nicolás; Sun, Weina; Ray, Greeshma; Schmitt, Phuong Tieu; Webb, Stacy; Gibson, Kathleen; Dutch, Rebecca Ellis; Schmitt, Anthony P

2017-07-15

Hendra virus (HeV) is a zoonotic paramyxovirus that causes deadly illness in horses and humans. An intriguing feature of HeV is the utilization of endosomal protease for activation of the viral fusion protein (F). Here we investigated how endosomal F trafficking affects HeV assembly. We found that the HeV matrix (M) and F proteins each induced particle release when they were expressed alone but that their coexpression led to coordinated assembly of virus-like particles (VLPs) that were morphologically and physically distinct from M-only or F-only VLPs. Mutations to the F protein transmembrane domain or cytoplasmic tail that disrupted endocytic trafficking led to failure of F to function with M for VLP assembly. Wild-type F functioned normally for VLP assembly even when its cleavage was prevented with a cathepsin inhibitor, indicating that it is endocytic F trafficking that is important for VLP assembly, not proteolytic F cleavage. Under specific conditions of reduced M expression, we found that M could no longer induce significant VLP release but retained the ability to be incorporated as a passenger into F-driven VLPs, provided that the F protein was competent for endocytic trafficking. The F and M proteins were both found to traffic through Rab11-positive recycling endosomes (REs), suggesting a model in which F and M trafficking pathways converge at REs, enabling these proteins to preassemble before arriving at plasma membrane budding sites. IMPORTANCE Hendra virus and Nipah virus are zoonotic paramyxoviruses that cause lethal infections in humans. Unlike that for most paramyxoviruses, activation of the henipavirus fusion protein occurs in recycling endosomal compartments. In this study, we demonstrate that the unique endocytic trafficking pathway of Hendra virus F protein is required for proper viral assembly and particle release. These results advance our basic understanding of the henipavirus assembly process and provide a novel model for the interplay between glycoprotein trafficking and paramyxovirus assembly. Copyright © 2017 American Society for Microbiology.

Rare emergence of drug resistance in HIV-1 treatment-naïve patients receiving elvitegravir/cobicistat/emtricitabine/tenofovir alafenamide for 144 weeks.

PubMed

Margot, Nicolas; Cox, Stephanie; Das, Moupali; McCallister, Scott; Miller, Michael D; Callebaut, Christian

2018-06-01

The single tablet regimen (STR) composed of elvitegravir (E), cobicistat (C), emtricitabine (F), and tenofovir alafenamide (TAF) (E/C/F/TAF) was compared to the STR composed of E, C, F, and tenofovir disoproxil fumarate (TDF) (E/C/F/TDF) in 2 phase 3 studies in 1733 HIV-1 infected treatment-naïve adults. Superior efficacy of E/C/F/TAF compared to E/C/F/TDF was demonstrated at Week 144 with 84% treatment success compared to 80%, respectively, along with significantly better outcomes of bone and renal safety. Analyze the emergence of HIV-1 resistance in treatment-naïve adults receiving E/C/F/TAF for 144 weeks. We conducted an integrated resistance analysis of the 2 Phase 3 studies, comprising pretreatment HIV-1 sequencing for all participants (N = 1733) and post-baseline HIV-1 resistance analysis for participants with virologic failure (HIV-1 RNA ≥400 copies/mL). Primary resistance-associated mutations (RAMs) were observed pre-treatment in 7.4% (NRTI-RAMs), 18.1% (NNRTI-RAMs), and 3.3% (PI-RAMs) of enrolled subjects. Baseline HIV-1 subtype or pre-existing RAMs did not affect E/C/F/TAF treatment response at week 144. Virologic failure resistance analyses were conducted for 28/866 (3.2%) and 30/867 (3.5%) patients in the E/C/F/TAF and E/C/F/TDF arms, respectively. Over the 3-year study, the rate of resistance emergence remained low at 1.4% in each group (12/866 in E/C/F/TAF; 12/867 in E/C/F/TDF). Resistant virus emerged in 24 patients who developed resistance to antiretrovirals in the regimens (E/C/F/TAF: M184V/I [1.3%], INSTI-RAMs [0.9%], K65R/N [0.2%]; E/C/F/TDF: M184V/I [1.0%], INSTI-RAMs [0.9%], K65R/N [0.5%]). Resistance emergence was rare (1.4%) with similar patterns of emergent mutations in both groups. M184V/I was the most prevalent RAM (1.2% overall). Copyright © 2018 Elsevier B.V. All rights reserved.

A dominant TRPV4 variant underlies osteochondrodysplasia in Scottish fold cats.

PubMed

Gandolfi, B; Alamri, S; Darby, W G; Adhikari, B; Lattimer, J C; Malik, R; Wade, C M; Lyons, L A; Cheng, J; Bateman, J F; McIntyre, P; Lamandé, S R; Haase, B

2016-08-01

Scottish fold cats, named for their unique ear shape, have a dominantly inherited osteochondrodysplasia involving malformation in the distal forelimbs, distal hindlimbs and tail, and progressive joint destruction. This study aimed to identify the gene and the underlying variant responsible for the osteochondrodysplasia. DNA samples from 44 Scottish fold and 54 control cats were genotyped using a feline DNA array and a case-control genome-wide association analysis conducted. The gene encoding a calcium permeable ion channel, transient receptor potential cation channel, subfamily V, member 4 (TRPV4) was identified as a candidate within the associated region and sequenced. Stably transfected HEK293 cells were used to compare wild-type and mutant TRPV4 expression, cell surface localisation and responses to activation with a synthetic agonist GSK1016709A, hypo-osmolarity, and protease-activated receptor 2 stimulation. The dominantly inherited folded ear and osteochondrodysplasia in Scottish fold cats is associated with a p.V342F substitution (c.1024G>T) in TRPV4. The change was not found in 648 unaffected cats. Functional analysis in HEK293 cells showed V342F mutant TRPV4 was poorly expressed at the cell surface compared to wild-type TRPV4 and as a consequence the maximum response to a synthetic agonist was reduced. Mutant TRPV4 channels had a higher basal activity and an increased response to hypotonic conditions. Access to a naturally-occurring TRPV4 mutation in the Scottish fold cat will allow further functional studies to identify how and why the mutations affect cartilage and bone development. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

In vitro resistance to the human immunodeficiency virus type 1 maturation inhibitor PA-457 (Bevirimat).

PubMed

Adamson, Catherine S; Ablan, Sherimay D; Boeras, Ioana; Goila-Gaur, Ritu; Soheilian, Ferri; Nagashima, Kunio; Li, Feng; Salzwedel, Karl; Sakalian, Michael; Wild, Carl T; Freed, Eric O

2006-11-01

3-O-(3',3'-dimethylsuccinyl)betulinic acid (PA-457 or bevirimat) potently inhibits human immunodeficiency virus type 1 (HIV-1) maturation by blocking a late step in the Gag processing pathway, specifically the cleavage of SP1 from the C terminus of capsid (CA). To gain insights into the mechanism(s) by which HIV-1 could evolve resistance to PA-457 and to evaluate the likelihood of such resistance arising in PA-457-treated patients, we sought to identify and characterize a broad spectrum of HIV-1 variants capable of conferring resistance to this compound. Numerous independent rounds of selection repeatedly identified six single-amino-acid substitutions that independently confer PA-457 resistance: three at or near the C terminus of CA (CA-H226Y, -L231F, and -L231M) and three at the first and third residues of SP1 (SP1-A1V, -A3T, and -A3V). We determined that mutations CA-H226Y, CA-L231F, CA-L231M, and SP1-A1V do not impose a significant replication defect on HIV-1 in culture. In contrast, mutations SP1-A3V and -A3T severely impaired virus replication and inhibited virion core condensation. The replication defect imposed by SP1-A3V was reversed by a second-site compensatory mutation in CA (CA-G225S). Intriguingly, high concentrations of PA-457 enhanced the maturation of SP1 residue 3 mutants. The different phenotypes associated with mutations that confer PA-457 resistance suggest the existence of multiple mechanisms by which HIV-1 can evolve resistance to this maturation inhibitor. These findings have implications for the ongoing development of PA-457 to treat HIV-1 infection in vivo.

SLFL Genes Participate in the Ubiquitination and Degradation Reaction of S-RNase in Self-compatible Peach.

PubMed

Chen, Qiuju; Meng, Dong; Gu, Zhaoyu; Li, Wei; Yuan, Hui; Duan, Xuwei; Yang, Qing; Li, Yang; Li, Tianzhong

2018-01-01

It has been proved that the gametophytic self-incompatibility (GSI), mainly exists in Rosaceae and Solanaceae, is controlled by S genes, which are two tightly linked genes located at highly polymorphic S -locus: the S-RNase for pistil specificity and the F-box gene ( SFB/SLF ) for pollen specificity, respectively. However, the roles of those genes in SI of peach are still a subject of extensive debate. In our study, we selected 37 representative varieties according to the evolution route of peach and identified their S genotypes. We cloned pollen determinant genes mutated PperSFB1m, PperSFB2m, PperSFB4m , and normal PperSFB2 , and style determinant genes PperS1-RNase, PperS2-RNase, PperS2m-RNase , and PperS4-RNase . The mutated PperSFBs encode truncated SFB proteins due to a fragment insertion. The truncated PperSFBs and normal PperSFB2 interacted with PperS-RNases demonstrated by Y2H. Normal PperSFB2 was divided into four parts: box, box-V1, V1-V2, and HVa-HVb. The box domain of PperSFB2 did not interact with PperS-RNases, both of the box-V1 and V1-V2 had interactions with PperS-RNases, while the hypervariable region of PperSFB2 HVa-HVb only interacted with PperS2-RNase showed by Y2H and BiFC assay. Bioinformatics analysis of peach genome revealed that there were other F-box genes located at S-locus, and of which three F-box genes were specifically expressed in pollen, named as PperSLFL1, PperSLFL2 , and PperSLFL3 , respectively. In phylogenetic analysis PperSLFLs clustered with Maloideae SFBB genes, and PperSFB genes were clustered into the other group with other SFB genes of Prunus . Protein interaction analysis revealed that the three PperSLFLs interacted with PperSSK1 and PperS-RNases with no allelic specificity. In vitro ubiquitination assay showed that PperSLFLs could tag ubiquitin molecules onto PperS-RNases. The above results suggest that three PperSLFLs are the appropriate candidates for the "general inhibitor," which would inactivate the S-RNases in pollen tubes, involved in the self-incompatibility of peach.

SLFL Genes Participate in the Ubiquitination and Degradation Reaction of S-RNase in Self-compatible Peach

PubMed Central

Chen, Qiuju; Meng, Dong; Gu, Zhaoyu; Li, Wei; Yuan, Hui; Duan, Xuwei; Yang, Qing; Li, Yang; Li, Tianzhong

2018-01-01

It has been proved that the gametophytic self-incompatibility (GSI), mainly exists in Rosaceae and Solanaceae, is controlled by S genes, which are two tightly linked genes located at highly polymorphic S-locus: the S-RNase for pistil specificity and the F-box gene (SFB/SLF) for pollen specificity, respectively. However, the roles of those genes in SI of peach are still a subject of extensive debate. In our study, we selected 37 representative varieties according to the evolution route of peach and identified their S genotypes. We cloned pollen determinant genes mutated PperSFB1m, PperSFB2m, PperSFB4m, and normal PperSFB2, and style determinant genes PperS1-RNase, PperS2-RNase, PperS2m-RNase, and PperS4-RNase. The mutated PperSFBs encode truncated SFB proteins due to a fragment insertion. The truncated PperSFBs and normal PperSFB2 interacted with PperS-RNases demonstrated by Y2H. Normal PperSFB2 was divided into four parts: box, box-V1, V1-V2, and HVa-HVb. The box domain of PperSFB2 did not interact with PperS-RNases, both of the box-V1 and V1-V2 had interactions with PperS-RNases, while the hypervariable region of PperSFB2 HVa-HVb only interacted with PperS2-RNase showed by Y2H and BiFC assay. Bioinformatics analysis of peach genome revealed that there were other F-box genes located at S-locus, and of which three F-box genes were specifically expressed in pollen, named as PperSLFL1, PperSLFL2, and PperSLFL3, respectively. In phylogenetic analysis PperSLFLs clustered with Maloideae SFBB genes, and PperSFB genes were clustered into the other group with other SFB genes of Prunus. Protein interaction analysis revealed that the three PperSLFLs interacted with PperSSK1 and PperS-RNases with no allelic specificity. In vitro ubiquitination assay showed that PperSLFLs could tag ubiquitin molecules onto PperS-RNases. The above results suggest that three PperSLFLs are the appropriate candidates for the “general inhibitor,” which would inactivate the S-RNases in pollen tubes, involved in the self-incompatibility of peach. PMID:29520292

Resin/graphite fiber composites

NASA Technical Reports Server (NTRS)

Cavano, P. J.

1974-01-01

Techniques were developed that provided thermo-oxidatively stable A-type polyimide/graphite fiber composites using the approach of in situ polymerization of monomeric reactants directly on reinforcing fibers, rather than employing separately prepared prepolymer varnish. This was accomplished by simply mixing methylene dianiline and two ester-acids and applying this solution to the fibers for subsequent molding. Five different formulated molecular weight resins were examined, and an optimized die molding procedure established for the 1500 formulated molecular weight system. Extensive ultrasonic inspection of composites was successfully utilized as a technique for monitoring laminate quality. Composite mechanical property studies were conducted with this polyimide resin at room temperature and after various time exposures in a thermo-oxidative environment at 561 K (550 F), 589 K (600 F) and 617 K (650 F). It was determined that such composites have a long term life in the temperature range of 561 K to 589 K. The final phase involved the fabrication and evaluation of a series of demonstration airfoil specimens.

Drug-resistant molecular mechanism of CRF01_AE HIV-1 protease due to V82F mutation

NASA Astrophysics Data System (ADS)

Liu, Xiaoqing; Xiu, Zhilong; Hao, Ce

2009-05-01

Human immunodeficiency virus type 1 protease (HIV-1 PR) is one of the major targets of anti-AIDS drug discovery. The circulating recombinant form 01 A/E (CRF01_AE, abbreviated AE) subtype is one of the most common HIV-1 subtypes, which is infecting more humans and is expanding rapidly throughout the world. It is, therefore, necessary to develop inhibitors against subtype AE HIV-1 PR. In this work, we have performed computer simulation of subtype AE HIV-1 PR with the drugs lopinavir (LPV) and nelfinavir (NFV), and examined the mechanism of resistance of the V82F mutation of this protease against LPV both structurally and energetically. The V82F mutation at the active site results in a conformational change of 79's loop region and displacement of LPV from its proper binding site, and these changes lead to rotation of the side-chains of residues D25 and I50'. Consequently, the conformation of the binding cavity is deformed asymmetrically and some interactions between PR and LPV are destroyed. Additionally, by comparing the interactive mechanisms of LPV and NFV with HIV-1 PR we discovered that the presence of a dodecahydroisoquinoline ring at the P1' subsite, a [2-(2,6-dimethylphenoxy)acetyl]amino group at the P2' subsite, and an N2 atom at the P2 subsite could improve the binding affinity of the drug with AE HIV-1 PR. These findings are helpful for promising drug design.

Cystic fibrosis transmembrane regulator haplotypes in households of patients with cystic fibrosis.

PubMed

Furgeri, Daniela Tenório; Marson, Fernando Augusto Lima; Correia, Cyntia Arivabeni Araújo; Ribeiro, José Dirceu; Bertuzzo, Carmen Sílvia

2018-01-30

Nearly 2000 mutations in the cystic fibrosis transmembrane regulator (CFTR) gene have been reported. The F508del mutation occurs in approximately 50-65% of patients with cystic fibrosis (CF). However, molecular diagnosis is not always possible. Therefore, silent polymorphisms can be used to label the mutant allele in households of patients with CF. To verify the haplotypes of four polymorphisms at the CFTR locus in households of patients with CF for pre-fertilization, pre-implantation, and prenatal indirect mutation diagnosis to provide better genetic counseling for families and patients with CF and to associate the genotypes/haplotypes with the F508del mutation screening. GATT polymorphism analysis was performed using direct polymerase chain reaction amplification, and the MP6-D9, TUB09 and TUB18 polymorphism analyses were performed using restriction fragment length polymorphism. Nine haplotypes were found in 37 CFTR alleles, and of those, 24 were linked with the F508del mutation and 13 with other CFTR mutations. The 6 (GATT), C (MP6-D9), G (TUB09), and C (TUB18) haplotypes showed the highest prevalence (48%) of the mutant CFTR allele and were linked to the F508del mutation (64%). In 43% of households analyzed, at least one informative polymorphism can be used for the indirect diagnostic test. CFTR polymorphisms are genetic markers that are useful for identifying the mutant CFTR alleles in households of patients with CF when it is not possible to establish the complete CFTR genotype. Moreover, the polymorphisms can be used for indirect CFTR mutation identification in cases of pre-fertilization, pre-implantation and prenatal analysis. Copyright © 2017 Elsevier B.V. All rights reserved.

Novel somatic mutations in the catalytic subunit of the protein kinase A as a cause of adrenal Cushing's syndrome: a European multicentric study.

PubMed

Di Dalmazi, Guido; Kisker, Caroline; Calebiro, Davide; Mannelli, Massimo; Canu, Letizia; Arnaldi, Giorgio; Quinkler, Marcus; Rayes, Nada; Tabarin, Antoine; Laure Jullié, Marie; Mantero, Franco; Rubin, Beatrice; Waldmann, Jens; Bartsch, Detlef K; Pasquali, Renato; Lohse, Martin; Allolio, Bruno; Fassnacht, Martin; Beuschlein, Felix; Reincke, Martin

2014-10-01

Somatic mutations in PRKACA gene, encoding the catalytic subunit of protein kinase A (PKA), have been recently found in a high proportion of sporadic adenomas associated with Cushing's syndrome. The aim was to analyze the PRKACA mutation in a large cohort of patients with adrenocortical masses. Samples from nine European centers were included (Germany, n = 4; Italy, n = 4; France, n = 1). Samples were drawn from 149 patients with nonsecreting adenomas (n = 32 + 2 peritumoral), subclinical hypercortisolism (n = 36), Cushing's syndrome (n = 64 + 2 peritumoral), androgen-producing tumors (n = 4), adrenocortical carcinomas (n = 5 + 2 peritumoral), and primary bilateral macronodular adrenal hyperplasias (n = 8). Blood samples were available from patients with nonsecreting adenomas (n = 15), subclinical hypercortisolism (n = 10), and Cushing's syndrome (n = 35). Clinical and hormonal data were collected. DNA amplification by PCR of exons 6 and 7 of the PRKACA gene and direct sequencing were performed. PRKACA heterozygous mutations were found in 22/64 samples of Cushing's syndrome patients (34%). No mutations were found in peritumoral tissue and blood samples or in other tumors examined. The c.617A>C (p.Leu206Arg) occurred in 18/22 patients. Furthermore, two novel mutations were identified: c.600_601insGTG/p.Cys200_Gly201insVal in three patients and c.639C>G+c.638_640insATTATCCTGAGG/p.Ser213Arg+p.Leu212_Lys214insIle-Ile-Leu-Arg) in one. All the mutations involved a region implicated in interaction between PKA regulatory and catalytic subunits. Patients with somatic PRKACA mutations showed higher levels of cortisol after dexamethasone test and a smaller adenoma size, compared with nonmutated subjects. These data confirm and extend previous observations that somatic PRKACA mutations are specific for adrenocortical adenomas causing Cushing's syndrome.

[SH and HN Protein Genetic Characterization Analysis of Mumps Virus Isolated in Liaoning Province from 2008 to 2014].

PubMed

Wang, Yan; Ma, Yan; Hao, Shuang; Xu, Xiaoting; Han, Yue; Yao, Wenqing; Zhao, Zhuo

2016-03-01

To analyze the genetic characterization of epidemic mumps virus strains in Liaoning Province and provide the basis for mumps control. A total of 32 mumps viruses strains were isolated during 2008-2104. The fragment of SH genes and HN genes were amplified by RT-PCR, the PCR products were sequenced and analyzed. Basing on the 316 nucleotides of SH gene, The phylogenetic analyses were processed with the data of WHO mumps reference strains downloaded from GenBank and 32 mumps viruses strains. It showed that the 31 mumps virus strains belong to F genotype except MuVi/Liaoning. CHN/16.11 which was G genotype . Comparing to the A reference strains (Jeryl-Lynn and S-79), F genotype MuV were mutated on 12 amino acids sites and 27 amino acids siteson on HN gene. F genotype MuV added one N-glycosylation site in 464th-466th amino acids. The antigenic sites on HN were mutated on 121th, 123th, 279th, 287th, 336th, 356th and 442th. Maybe, it will influence the MuV antigenic.

Epilepsy-causing mutations in Kv7.2 C-terminus affect binding and functional modulation by calmodulin.

PubMed

Ambrosino, Paolo; Alaimo, Alessandro; Bartollino, Silvia; Manocchio, Laura; De Maria, Michela; Mosca, Ilaria; Gomis-Perez, Carolina; Alberdi, Araitz; Scambia, Giovanni; Lesca, Gaetan; Villarroel, Alvaro; Taglialatela, Maurizio; Soldovieri, Maria Virginia

2015-09-01

Mutations in the KCNQ2 gene, encoding for voltage-gated Kv7.2K(+) channel subunits, are responsible for early-onset epileptic diseases with widely-diverging phenotypic presentation, ranging from Benign Familial Neonatal Seizures (BFNS) to epileptic encephalopathy. In the present study, Kv7.2 BFNS-causing mutations (W344R, L351F, L351V, Y362C, and R553Q) have been investigated for their ability to interfere with calmodulin (CaM) binding and CaM-induced channel regulation. To this aim, semi-quantitative (Far-Western blotting) and quantitative (Surface Plasmon Resonance and dansylated CaM fluorescence) biochemical assays have been performed to investigate the interaction of CaM with wild-type or mutant Kv7.2 C-terminal fragments encompassing the CaM-binding domain; in parallel, mutation-induced changes in CaM-dependent Kv7.2 or Kv7.2/Kv7.3 current regulation were investigated by patch-clamp recordings in Chinese Hamster Ovary (CHO) cells co-expressing Kv7.2 or Kv7.2/Kv7.3 channels and CaM or CaM1234 (a CaM isoform unable to bind Ca(2+)). The results obtained suggest that each BFNS-causing mutation prompts specific biochemical and/or functional consequences; these range from slight alterations in CaM affinity which did not translate into functional changes (L351V), to a significant reduction in the affinity and functional modulation by CaM (L351F, Y362C or R553Q), to a complete functional loss without significant alteration in CaM affinity (W344R). CaM overexpression increased Kv7.2 and Kv7.2/Kv7.3 current levels, and partially (R553Q) or fully (L351F) restored normal channel function, providing a rationale pathogenetic mechanism for mutation-induced channel dysfunction in BFNS, and highlighting the potentiation of CaM-dependent Kv7.2 modulation as a potential therapeutic approach for Kv7.2-related epilepsies. Copyright © 2015 Elsevier B.V. All rights reserved.

Suppression of the UV-sensitive phenotype of Escherichia coli recF mutants by recA(Srf) and recA(Tif) mutations requires recJ+.

PubMed Central

Thoms, B; Wackernagel, W

1988-01-01

Mutations in recA, such as recA801(Srf) (suppressor of RecF) or recA441(Tif) (temperature-induced filamentation) partially suppress the deficiency in postreplication repair of UV damage conferred by recF mutations. We observed that spontaneous recA(Srf) mutants accumulated in cultures of recB recC sbcB sulA::Mu dX(Ap lac) lexA51 recF cells because they grew faster than the parental strain. We show that in a uvrA recB+ recC+ genetic background there are two prerequisites for the suppression by recA(Srf) of the UV-sensitive phenotype of recF mutants. (i) The recA(Srf) protein must be provided in increased amounts either by SOS derepression or by a recA operator-constitutive mutation in a lexA(Ind) (no induction of SOS functions) genetic background. (ii) The gene recJ, which has been shown previously to be involved in the recF pathway of recombination and repair, must be functional. The level of expression of recJ in a lexA(Ind) strain suffices for full suppression. Suppression by recA441 at 30 degrees C also depends on recJ+. The hampered induction by UV of the SOS gene uvrA seen in a recF mutant was improved by a recA(Srf) mutation. This improvement did not require recJ+. We suggest that recA(Srf) and recA(Tif) mutant proteins can operate in postreplication repair independent of recF by using the recJ+ function. PMID:2841294

Novel SLC34A3 mutation causing hereditary hypophosphataemic rickets with hypercalciuria in a Gambian family.

PubMed

Braithwaite, Vickie; Pettifor, John M; Prentice, Ann

2013-03-01

Three siblings, aged 12, 4 and 2 years, presented at a Gambian clinic with bone deformities. Radiographs of knees and wrists confirmed the presence of florid rickets. The family (including 2 unaffected siblings and the mother) were investigated for hereditary rickets. The three affected siblings had biochemical features of hereditary hypophosphataemic rickets with hypercalciuria (HHRH) with normal plasma calcium and 25-hydroxyvitamin D concentrations, elevated 1,25-dihydroxyvitamin D, hypophosphataemia, hyperphosphaturia and hypercalciuria. At presentation, two of the three affected siblings had an elevated fibroblast growth factor-23 (FGF23) concentration. The mother and clinically unaffected siblings had largely normal biochemistry. Genetic analysis of the SLC34A3 gene, encoding the type IIc sodium-phosphate cotransporter, in DNA samples from the siblings and their mother was conducted. Three single nucleotide polymorphisms (SNPs) S168F, E513V and L599L were identified. E513V and L599L had been previously identified as benign polymorphisms. S168F however, is a previously unreported variant. In silico mutation evaluation predicted that the S168F mutation causes changes in the protein product which are damaging to its function. In addition, the three clinically affected siblings were homozygous in the S168F variant whereas the unaffected family members were carriers. This study describes a biochemical profile and complementary gene data consistent with a rare genetic hypophosphataemic rickets disease in a family from rural Gambia. To our knowledge, this study reports the first cases of HHRH in Africa and describes a novel causal mutation within the SLC34A3 gene. Copyright © 2012 Elsevier Inc. All rights reserved.

Functional Studies and In Silico Analyses to Evaluate Non-Coding Variants in Inherited Cardiomyopathies.

PubMed

Frisso, Giulia; Detta, Nicola; Coppola, Pamela; Mazzaccara, Cristina; Pricolo, Maria Rosaria; D'Onofrio, Antonio; Limongelli, Giuseppe; Calabrò, Raffaele; Salvatore, Francesco

2016-11-10

Point mutations are the most common cause of inherited diseases. Bioinformatics tools can help to predict the pathogenicity of mutations found during genetic screening, but they may work less well in determining the effect of point mutations in non-coding regions. In silico analysis of intronic variants can reveal their impact on the splicing process, but the consequence of a given substitution is generally not predictable. The aim of this study was to functionally test five intronic variants ( MYBPC3 -c.506-2A>C, MYBPC3 -c.906-7G>T, MYBPC3 -c.2308+3G>C, SCN5A -c.393-5C>A, and ACTC1 -c.617-7T>C) found in five patients affected by inherited cardiomyopathies in the attempt to verify their pathogenic role. Analysis of the MYBPC3 -c.506-2A>C mutation in mRNA from the peripheral blood of one of the patients affected by hypertrophic cardiac myopathy revealed the loss of the canonical splice site and the use of an alternative splicing site, which caused the loss of the first seven nucleotides of exon 5 ( MYBPC3 -G169AfsX14). In the other four patients, we generated minigene constructs and transfected them in HEK-293 cells. This minigene approach showed that MYBPC3 -c.2308+3G>C and SCN5A -c.393-5C>A altered pre-mRNA processing, thus resulting in the skipping of one exon. No alterations were found in either MYBPC3 -c.906-7G>T or ACTC1 -c.617-7T>C. In conclusion, functional in vitro analysis of the effects of potential splicing mutations can confirm or otherwise the putative pathogenicity of non-coding mutations, and thus help to guide the patient's clinical management and improve genetic counseling in affected families.

SEMA3A, a Gene Involved in Axonal Pathfinding, Is Mutated in Patients with Kallmann Syndrome

PubMed Central

Parkash, Jyoti; Espy, Cécile; Fouveaut, Corinne; Leroy, Chrystel; Baron, Stéphanie; Campagne, Céline; Vanacker, Charlotte; Collier, Francis; Cruaud, Corinne; Meyer, Vincent; García-Piñero, Alfons; Dewailly, Didier; Cortet-Rudelli, Christine; Gersak, Ksenija; Metz, Chantal; Chabrier, Gérard; Pugeat, Michel; Young, Jacques; Hardelin, Jean-Pierre; Prevot, Vincent; Dodé, Catherine

2012-01-01

Kallmann syndrome (KS) associates congenital hypogonadism due to gonadotropin-releasing hormone (GnRH) deficiency and anosmia. The genetics of KS involves various modes of transmission, including oligogenic inheritance. Here, we report that Nrp1 sema/sema mutant mice that lack a functional semaphorin-binding domain in neuropilin-1, an obligatory coreceptor of semaphorin-3A, have a KS–like phenotype. Pathohistological analysis of these mice indeed showed abnormal development of the peripheral olfactory system and defective embryonic migration of the neuroendocrine GnRH cells to the basal forebrain, which results in increased mortality of newborn mice and reduced fertility in adults. We thus screened 386 KS patients for the presence of mutations in SEMA3A (by Sanger sequencing of all 17 coding exons and flanking splice sites) and identified nonsynonymous mutations in 24 patients, specifically, a frameshifting small deletion (D538fsX31) and seven different missense mutations (R66W, N153S, I400V, V435I, T688A, R730Q, R733H). All the mutations were found in heterozygous state. Seven mutations resulted in impaired secretion of semaphorin-3A by transfected COS-7 cells (D538fsX31, R66W, V435I) or reduced signaling activity of the secreted protein in the GN11 cell line derived from embryonic GnRH cells (N153S, I400V, T688A, R733H), which strongly suggests that these mutations have a pathogenic effect. Notably, mutations in other KS genes had already been identified, in heterozygous state, in five of these patients. Our findings indicate that semaphorin-3A signaling insufficiency contributes to the pathogenesis of KS and further substantiate the oligogenic pattern of inheritance in this developmental disorder. PMID:22927827

SEMA3A, a gene involved in axonal pathfinding, is mutated in patients with Kallmann syndrome.

PubMed

Hanchate, Naresh Kumar; Giacobini, Paolo; Lhuillier, Pierre; Parkash, Jyoti; Espy, Cécile; Fouveaut, Corinne; Leroy, Chrystel; Baron, Stéphanie; Campagne, Céline; Vanacker, Charlotte; Collier, Francis; Cruaud, Corinne; Meyer, Vincent; García-Piñero, Alfons; Dewailly, Didier; Cortet-Rudelli, Christine; Gersak, Ksenija; Metz, Chantal; Chabrier, Gérard; Pugeat, Michel; Young, Jacques; Hardelin, Jean-Pierre; Prevot, Vincent; Dodé, Catherine

2012-08-01

Kallmann syndrome (KS) associates congenital hypogonadism due to gonadotropin-releasing hormone (GnRH) deficiency and anosmia. The genetics of KS involves various modes of transmission, including oligogenic inheritance. Here, we report that Nrp1(sema/sema) mutant mice that lack a functional semaphorin-binding domain in neuropilin-1, an obligatory coreceptor of semaphorin-3A, have a KS-like phenotype. Pathohistological analysis of these mice indeed showed abnormal development of the peripheral olfactory system and defective embryonic migration of the neuroendocrine GnRH cells to the basal forebrain, which results in increased mortality of newborn mice and reduced fertility in adults. We thus screened 386 KS patients for the presence of mutations in SEMA3A (by Sanger sequencing of all 17 coding exons and flanking splice sites) and identified nonsynonymous mutations in 24 patients, specifically, a frameshifting small deletion (D538fsX31) and seven different missense mutations (R66W, N153S, I400V, V435I, T688A, R730Q, R733H). All the mutations were found in heterozygous state. Seven mutations resulted in impaired secretion of semaphorin-3A by transfected COS-7 cells (D538fsX31, R66W, V435I) or reduced signaling activity of the secreted protein in the GN11 cell line derived from embryonic GnRH cells (N153S, I400V, T688A, R733H), which strongly suggests that these mutations have a pathogenic effect. Notably, mutations in other KS genes had already been identified, in heterozygous state, in five of these patients. Our findings indicate that semaphorin-3A signaling insufficiency contributes to the pathogenesis of KS and further substantiate the oligogenic pattern of inheritance in this developmental disorder.

Diabetes Insipidus in Mice with a Mutation in Aquaporin-2

PubMed Central

Lloyd, David J; Hall, Frank Wesley; Tarantino, Lisa M; Gekakis, Nicholas

2005-01-01

Congenital nephrogenic diabetes insipidus (NDI) is a disease characterized by failure of the kidney to concentrate urine in response to vasopressin. Human kindreds with nephrogenic diabetes insipidus have been found to harbor mutations in the vasopressin receptor 2 (Avpr2) gene or the vasopressin-sensitive water channel aquaporin-2 (Aqp2) gene. Development of a treatment is rendered difficult due to the lack of a viable animal model. Through forward genetic screening of ethylnitrosourea-mutagenized mice, we report the identification and characterization of a mouse model of NDI, with an F204V mutation in the Aqp2 gene. Unlike previously attempted murine models of NDI, our mice survive to adulthood and more exactly recapitulate the human disorder. Previous in vitro experiments using renal cell lines suggest recessive Aqp2 mutations result in improper trafficking of the mutant water pore. Using these animals, we have directly proven this hypothesis of improper AQP2 translocation as the molecular defect in nephrogenic diabetes insipidus in the intact organism. Additionally, using a renal cell line we show that the mutated protein, AQP2-F204V, is retained in the endoplasmic reticulum and that this abnormal localization can be rescued by wild-type protein. This novel mouse model allows for further mechanistic studies as well as testing of pharmacological and gene therapies for NDI. PMID:16121255

Mutational analysis of the major soybean UreF paralogue involved in urease activation

USDA-ARS?s Scientific Manuscript database

In soybean, mutation at Eu2 or Eu3 eliminates the urease activities of both the embryo-specific and the tissue-ubiquitous (assimilatory) isozymes, encoded by Eu1 and Eu4, respectively. Eu3 encodes UreG, a GTP’ase necessary for proper emplacement of Ni and carbon dioxide in the urease active site. ...

Characterization of CD34+ hematopoietic cells in systemic mastocytosis: Potential role in disease dissemination.

PubMed

Mayado, A; Teodosio, C; Dasilva-Freire, N; Jara-Acevedo, M; Garcia-Montero, A C; Álvarez-Twose, I; Sánchez-Muñoz, L; Matito, A; Caldas, C; Muñoz-González, J I; Henriques, A; Sánchez-Gallego, J I; Escribano, L; Orfao, A

2018-01-13

Recent studies show that most systemic mastocytosis (SM) patients, including indolent SM (ISM) with (ISMs+) and without skin lesions (ISMs-), carry the KIT D816V mutation in PB leukocytes. We investigated the potential association between the degree of involvement of BM hematopoiesis by the KIT D816V mutation and the distribution of different maturation-associated compartments of bone marrow (BM) and peripheral blood (PB) CD34 + hematopoietic precursors (HPC) in ISM and identified the specific PB cell compartments that carry this mutation. The distribution of different maturation-associated subsets of BM and PB CD34 + HPC from 64 newly diagnosed (KIT-mutated) ISM patients and 14 healthy controls was analyzed by flow cytometry. In 18 patients, distinct FACS-purified PB cell compartments were also investigated for the KIT mutation. ISM patients showed higher percentages of both BM and PB MC-committed CD34 + HPC vs controls, particularly among ISM cases with MC-restricted KIT mutation (ISM MC ); this was associated with progressive blockade of maturation of CD34 + HPC to the neutrophil lineage from ISM MC to multilineage KIT-mutated cases (ISM ML ). Regarding the frequency of KIT-mutated cases and cell populations in PB, variable patterns were observed, the percentage of KIT-mutated PB CD34 + HPC, eosinophils, neutrophils, monocytes and T cells increasing from ISMs- MC and ISMs+ MC to ISM ML patients. The presence of the KIT D816V mutation in PB of ISM patients is associated with (early) involvement of circulating CD34 + HPC and multiple myeloid cell subpopulations, KIT-mutated PB CD34 + HPC potentially contributing to early dissemination of the disease. © 2018 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.

Deletion mutation analysis on C-terminal domain of plant vacuolar H(+)-pyrophosphatase.

PubMed

Lin, Hsin Hung; Pan, Yih Jiuan; Hsu, Shen Hsing; Van, Ru Chuan; Hsiao, Yi Yuong; Chen, Jiun Hsien; Pan, Rong Long

2005-10-15

Vacuolar H(+)-translocating inorganic pyrophosphatase (V-PPase; EC 3.6.1.1) is a homodimeric proton-translocase; it contains a single type of polypeptide of approximately 81kDa. A line of evidence demonstrated that the carboxyl terminus of V-PPase is relatively conserved in various plant V-PPases and presumably locates in the vicinity of the catalytic site. In this study, we attempt to identify the roles of the C-terminus of V-PPase by generating a series of C-terminal deletion mutants over-expressed in Saccharomyces cerevisiae, and determining their enzymatic and proton translocating reactions. Our results showed that the deletion mutation at last 5 amino acids in the C-terminus (DeltaC5) induced a dramatic decline in enzymatic activity, proton translocation, and coupling efficiency of V-PPase; but the mutant lacking last 10 amino acids (DeltaC10) retained about 60-70% of the enzymatic activity of wild-type. Truncation of the C-terminus by more than 10 amino acids completely abolished the enzymatic activity and proton translocation of V-PPase. Furthermore, the DeltaC10 mutant displayed a shift in T(1/2) (pretreatment temperature at which half enzymatic activity is observed) but not the optimal pH for PP(i) hydrolytic activity. The deletion of the C-terminus substantially modified apparent K(+) binding constant, but exert no significant changes in the Na(+)-, F(-)-, and Ca(2+)-inhibition of the enzymatic activity of V-PPase. Taken together, we speculate that the C-terminus of V-PPase may play a crucial role in sustaining enzymatic activity and is likely involved in the K(+)-regulation of the enzyme in an indirect manner.

A novel mutation m.8561C>G in MT-ATP6/8 causing a mitochondrial syndrome with ataxia, peripheral neuropathy, diabetes mellitus, and hypergonadotropic hypogonadism.

PubMed

Kytövuori, Laura; Lipponen, Joonas; Rusanen, Harri; Komulainen, Tuomas; Martikainen, Mika H; Majamaa, Kari

2016-11-01

Defects in the respiratory chain or mitochondrial ATP synthase (complex V) result in mitochondrial dysfunction that is an important cause of inherited neurological disease. Two of the subunits of complex V are encoded by MT-ATP6 and MT-ATP8 in the mitochondrial genome. Pathogenic mutations in MT-ATP6 are associated with the Leigh syndrome, the syndrome of neuropathy, ataxia, and retinitis pigmentosa (NARP), as well as with non-classical phenotypes, while MT-ATP8 is less frequently mutated in patients with mitochondrial disease. We investigated two adult siblings presenting with features of cerebellar ataxia, peripheral neuropathy, diabetes mellitus, sensorineural hearing impairment, and hypergonadotropic hypogonadism. As the phenotype was suggestive of mitochondrial disease, mitochondrial DNA was sequenced and a novel heteroplasmic mutation m.8561C>G in the overlapping region of the MT-ATP6 and MT-ATP8 was found. The mutation changed amino acids in both subunits. Mutation heteroplasmy correlated with the disease phenotype in five family members. An additional assembly intermediate of complex V and increased amount of subcomplex F 1 were observed in myoblasts of the two patients, but the total amount of complex V was unaffected. Furthermore, intracellular ATP concentration was lower in patient myoblasts indicating defective energy production. We suggest that the m.8561C>G mutation in MT-ATP6/8 is pathogenic, leads biochemically to impaired assembly and decreased ATP production of complex V, and results clinically in a phenotype with the core features of cerebellar ataxia, peripheral neuropathy, diabetes mellitus, and hypergonadotropic hypogonadism.

Genetics Home Reference: generalized pustular psoriasis

MedlinePlus

... C, Valeyrie-Allanore L, Vicente MA, Trembath RC, Smith CH, Barker JN, Capon F. IL36RN mutations define ... A, Simpson MA, Pink AE, Di Meglio P, Smith CH, Pullabhatla V, Knight J, Spain SL, Nestle ...

CFTR Genotype and Maximal Exercise Capacity in Cystic Fibrosis: A Cross-sectional Study.

PubMed

Radtke, Thomas; Hebestreit, Helge; Gallati, Sabina; Schneiderman, Jane E; Braun, Julia; Stevens, Daniel; Hulzebos, Erik Hj; Takken, Tim; Boas, Steven R; Urquhart, Don S; Lands, Larry C; Tejero, Sergio; Sovtic, Aleksandar; Dwyer, Tiffany; Petrovic, Milos; Harris, Ryan A; Karila, Chantal; Savi, Daniela; Usemann, Jakob; Mei-Zahav, Meir; Hatziagorou, Elpis; Ratjen, Felix; Kriemler, Susi

2018-02-01

Cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in human skeletal muscle cells. Variations of CFTR dysfunction among patients with cystic fibrosis may be an important determinant of maximal exercise capacity in cystic fibrosis. Previous studies on the relationship between CFTR genotype and maximal exercise capacity are scarce and contradictory. This study was designed to explore factors influencing maximal exercise capacity, expressed as peak oxygen uptake (V.O2peak), with a specific focus on CFTR genotype in children and adults with cystic fibrosis. In an international, multicenter, cross-sectional study, we collected data on CFTR genotype and cardiopulmonary exercise tests in patients with cystic fibrosis who were ages 8 years and older. CFTR mutations were classified into functional classes I–V. The final analysis included 726 patients (45% females; age range, 8–61 yr; forced expiratory volume in 1 s, 16 to 123% predicted) from 17 cystic fibrosis centers in North America, Europe, Australia, and Asia, all of whom had both valid maximal cardiopulmonary exercise tests and complete CFTR genotype data. Overall, patients exhibited exercise intolerance (V.O2peak, 77.3 ± 19.1% predicted), but values were comparable among different CFTR classes. We did not detect an association between CFTR genotype functional classes I–III and either V.O2peak (percent predicted) (adjusted β = −0.95; 95% CI, −4.18 to 2.29; P = 0.57) or maximum work rate (Wattmax) (adjusted β = −1.38; 95% CI, −5.04 to 2.27; P = 0.46) compared with classes IV–V. Those with at least one copy of a F508del-CFTR mutation and one copy of a class V mutation had a significantly lower V.O2peak (β = −8.24%; 95% CI, −14.53 to −2.99; P = 0.003) and lower Wattmax (adjusted β = −7.59%; 95% CI, −14.21 to −0.95; P = 0.025) than those with two copies of a class II mutation. On the basis of linear regression analysis adjusted for relevant confounders, lung function and body mass index were associated with V.O2peak. CFTR functional genotype class was not associated with maximal exercise capacity in patients with cystic fibrosis overall, but those with at least one copy of a F508del-CFTR mutation and a single class V mutation had lower maximal exercise capacity.

Mutation and polymorphism analysis of the human homogentisate 1, 2-dioxygenase gene in alkaptonuria patients.

PubMed Central

Beltrán-Valero de Bernabé, D; Granadino, B; Chiarelli, I; Porfirio, B; Mayatepek, E; Aquaron, R; Moore, M M; Festen, J J; Sanmartí, R; Peñalva, M A; de Córdoba, S R

1998-01-01

Alkaptonuria (AKU), a rare hereditary disorder of phenylalanine and tyrosine catabolism, was the first disease to be interpreted as an inborn error of metabolism. AKU patients are deficient for homogentisate 1,2 dioxygenase (HGO); this deficiency causes homogentisic aciduria, ochronosis, and arthritis. We cloned the human HGO gene and characterized two loss-of-function mutations, P230S and V300G, in the HGO gene in AKU patients. Here we report haplotype and mutational analysis of the HGO gene in 29 novel AKU chromosomes. We identified 12 novel mutations: 8 (E42A, W97G, D153G, S189I, I216T, R225H, F227S, and M368V) missense mutations that result in amino acid substitutions at positions conserved in HGO in different species, 1 (F10fs) frameshift mutation, 2 intronic mutations (IVS9-56G-->A, IVS9-17G-->A), and 1 splice-site mutation (IVS5+1G-->T). We also report characterization of five polymorphic sites in HGO and describe the haplotypic associations of alleles at these sites in normal and AKU chromosomes. One of these sites, HGO-3, is a variable dinucleotide repeat; IVS2+35T/A, IVS5+25T/C, and IVS6+46C/A are intronic sites at which single nucleotide substitutions (dimorphisms) have been detected; and c407T/A is a relatively frequent nucleotide substitution in the coding sequence, exon 4, resulting in an amino acid change (H80Q). These data provide insight into the origin and evolution of the various AKU alleles. PMID:9529363

Genetic study of the BRAF gene reveals new variants and high frequency of the V600E mutation among Iranian ameloblastoma patients.

PubMed

Soltani, Maryam; Tabatabaiefar, Mohammad Amin; Mohsenifar, Zhaleh; Pourreza, Mohammad Reza; Moridnia, Abbas; Shariati, Laleh; Razavi, Seyyed Mohammad

2018-01-01

Ameloblastoma is a benign, slow-growing and locally invasive tumor. It is one of the most prevalent odontogenic tumors, with an incidence rate of 1% of all oral tumors and approximately 18% of odontogenic tumors. A group of genes have been investigated in patients with ameloblastoma. The BRAF V600E mutation has been implicated as the most common mutation in ameloblastoma. The presence or absence of this mutation has been associated with several clinicopathological properties, including location, age at diagnosis, histology, and prognosis. Although some populations have been investigated so far, little data are available on the Iranian population. The current research was launched to study the BRAF V600E mutation among a cohort of Iranian patients with ameloblastoma. In this clinicopathological and molecular biology study, a total of 19 formalin-fixed, paraffin-embedded tissues were studied. DNA extraction was performed, followed by PCR-sequencing of exons 10 and 15 of the BRAF gene to identify mutations. In silico analysis was performed for the identified variants. Results were analyzed by T test, Chi-square, and Fisher's exact test. Totally, 12 of 19 samples (63%) harbored the p. V600E hotspot mutation. In addition, we identified several variants, two of which were novel. The c.1769T>G (p. V590G) and c.1751C>T (p.L584F) as the novel variants showed a possible damaging effect by in silico analysis. No variant was found within exon 10. Our study confirms the role of BRAF mutations in ameloblastoma in the Iranian patients studied. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

L1198F Mutation Resensitizes Crizotinib to ALK by Altering the Conformation of Inhibitor and ATP Binding Sites

PubMed Central

Li, Jian; Sun, Rong; Wu, Yuehong; Song, Mingzhu; Li, Jia; Yang, Qianye; Chen, Xiaoyi; Bao, Jinku; Zhao, Qi

2017-01-01

The efficacy of anaplastic lymphoma kinase (ALK) positive non-small-cell lung cancer (NSCLC) treatment with small molecule inhibitors is greatly challenged by acquired resistance. A recent study reported the newest generation inhibitor resistant mutation L1198F led to the resensitization to crizotinib, which is the first Food and Drug Administration (FDA) approved drug for the treatment of ALK-positive NSCLC. It is of great importance to understand how this extremely rare event occurred for the purpose of overcoming the acquired resistance of such inhibitors. In this study, we exploited molecular dynamics (MD) simulation to dissect the molecular mechanisms. Our MD results revealed that L1198F mutation of ALK resulted in the conformational change at the inhibitor site and altered the binding affinity of ALK to crizotinib and lorlatinib. L1198F mutation also affected the autoactivation of ALK as supported by the identification of His1124 and Tyr1278 as critical amino acids involved in ATP binding and phosphorylation. Our findings are valuable for designing more specific and potent inhibitors for the treatment of ALK-positive NSCLC and other types of cancer. PMID:28245558

Expansion of the genotypic and phenotypic spectrum of xeroderma pigmentosum in Chinese population.

PubMed

Zhang, Jia; Cheng, Ruhong; Yu, Xia; Sun, Zhonghui; Li, Ming; Yao, Zhirong

2017-01-01

Xeroderma pigmentosum (XP) is a rare genodermatosis characterized by exaggerated sunburn reactions, freckle-like pigmentation, and a high possibility of developing cutaneous tumors. XP comprised seven complementation groups (from XP-A to XP-G) and a variant form XP-V. This study was based on five unrelated Chinese families with six patients clinically suspected to be XP. Mutation screening was performed by direct sequencing of the entire coding region of eight XP genes. All of the pathogenic mutations were identified by mutational analysis, including four novel mutations. Our study successfully identified the pathogenic mutations in six XP patients (three XP-A, one XP-G, one XP-V, and a rare XP-D group in Chinese population). We reviewed the reported XP cases with mutations in the Chinese population and concluded that four complementation groups (XP-A, XP-C, XP-G, and XP-V) that occupy the major proportion should be considered as a first step in genetic detection (especially, XPA is the most common group, and unlike in other populations, XP-G is not rare in the Chinese population). Moreover, XP-D and XP-F, two rare subgroups, should also be added for further mutational analysis. Further, we provide some information for Chinese dermatologists that, when an early diagnosis is made, XP-C and XP-V patients can have relatively good prognoses. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Altered epidermal lipid processing and calcium distribution in the KID syndrome mouse model Cx26S17F.

PubMed

Bosen, Felicitas; Celli, Anna; Crumrine, Debra; vom Dorp, Katharina; Ebel, Philipp; Jastrow, Holger; Dörmann, Peter; Winterhager, Elke; Mauro, Theodora; Willecke, Klaus

2015-07-08

The keratitis-ichthyosis-deafness (KID) syndrome is caused by mutations in the gap junctional channel protein connexin 26 (Cx26), among them the mutation Cx26S17F. Heterozygous Cx26S17F mice resemble the human KID syndrome, i.e. exhibiting epidermal hyperplasia and hearing impairments. Newborn Cx26S17F mice show a defective epidermal water barrier as well as altered epidermal lipid secretion and location. Linoleoyl ω-esterified ceramides are strongly decreased on the skin surface of Cx26S17F mice. Moreover, the epidermal calcium gradient is altered in the mutant mice. These alterations may be caused by an abnormal Cx26S17F channel function that leads to a defective epidermal water barrier, which in turn may trigger the hyperproliferation seen in the KID syndrome. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Structural Insights into HIV Reverse Transcriptase Mutations Q151M and Q151M Complex That Confer Multinucleoside Drug Resistance

DOE Office of Scientific and Technical Information (OSTI.GOV)

Das, Kalyan; Martinez, Sergio E.; Arnold, Eddy

HIV-1 reverse transcriptase (RT) is targeted by multiple drugs. RT mutations that confer resistance to nucleoside RT inhibitors (NRTIs) emerge during clinical use. Q151M and four associated mutations, A62V, V75I, F77L, and F116Y, were detected in patients failing therapies with dideoxynucleosides (didanosine [ddI], zalcitabine [ddC]) and/or zidovudine (AZT). The cluster of the five mutations is referred to as the Q151M complex (Q151Mc), and an RT or virus containing Q151Mc exhibits resistance to multiple NRTIs. To understand the structural basis for Q151M and Q151Mc resistance, we systematically determined the crystal structures of the wild-type RT/double-stranded DNA (dsDNA)/dATP (complex I), wild-type RT/dsDNA/ddATPmore » (complex II), Q151M RT/dsDNA/dATP (complex III), Q151Mc RT/dsDNA/dATP (complex IV), and Q151Mc RT/dsDNA/ddATP (complex V) ternary complexes. The structures revealed that the deoxyribose rings of dATP and ddATP have 3'-endo and 3'-exo conformations, respectively. The single mutation Q151M introduces conformational perturbation at the deoxynucleoside triphosphate (dNTP)-binding pocket, and the mutated pocket may exist in multiple conformations. The compensatory set of mutations in Q151Mc, particularly F116Y, restricts the side chain flexibility of M151 and helps restore the DNA polymerization efficiency of the enzyme. The altered dNTP-binding pocket in Q151Mc RT has the Q151-R72 hydrogen bond removed and has a switched conformation for the key conserved residue R72 compared to that in wild-type RT. On the basis of a modeled structure of hepatitis B virus (HBV) polymerase, the residues R72, Y116, M151, and M184 in Q151Mc HIV-1 RT are conserved in wild-type HBV polymerase as residues R41, Y89, M171, and M204, respectively; functionally, both Q151Mc HIV-1 and wild-type HBV are resistant to dideoxynucleoside analogs.« less

Prevalence of drug-resistant mutation among drug-treated HIV/AIDS inpatient in Airlangga University teaching hospital, Surabaya, Indonesia

NASA Astrophysics Data System (ADS)

Rachman, B. E.; Khairunisa, S. Q.; Witaningrum, A. M.; Yunifiar, M. Q.; Widiyanti, P.; Nasronudin

2018-03-01

Increased use of antiretroviral therapy did not completely reduce the incidence of HIV/AIDShospitalization. Various factors can be involved. The aim of this study is to examine HIV-1 drug resistance mutations profile in drug-treated HIV/AIDS patients who underwent hospitalization. HIV/AIDS patients who are admitted to hospital who had received ART are included in the study and then examined for the presence of drug resistance-associated mutations. A total of 17 samples were included in the study, but only 11 samples that could be sequence analyzed. On the mutation examination of drug resistance in reverse transcriptase gene, it werefound a major mutation in K103N (9%) and G190A (9%). Most minor mutations were found in A98S (18.1%), followed by M41L, M184V, L210W, T215Y, V108l, Y181C and H221Y at 9% each. Whereas, on examination of drug resistance mutations in protease genes, there is a major mutation in I84V of 9%. Most minor mutations on M36I (45.4%), followed by L10I (36.3%), H69K (36.3%), I93L (27.2%), G16E, L89M, K20R 18.1%, L64V and V771I 9% respectively.A large number of mutated samples pose a challenge in long-term antiretroviral treatment, so a breakthrough policy is needed to minimize the impact.

An active-site phenylalanine directs substrate binding and C-H cleavage in the alpha-ketoglutarate-dependent dioxygenase TauD.

PubMed

McCusker, Kevin P; Klinman, Judith P

2010-04-14

Enzymes that cleave C-H bonds are often found to depend on well-packed hydrophobic cores that influence the distance between the hydrogen donor and acceptor. Residue F159 in taurine alpha-ketoglutarate dioxygenase (TauD) is demonstrated to play an important role in the binding and orientation of its substrate, which undergoes a hydrogen atom transfer to the active site Fe(IV)=O. Mutation of F159 to smaller hydrophobic side chains (L, V, A) leads to substantially reduced rates for substrate binding and for C-H bond cleavage, as well as increased contribution of the chemical step to k(cat) under steady-state turnover conditions. The greater sensitivity of these substrate-dependent processes to mutation at position 159 than observed for the oxygen activation process supports a previous conclusion of modularity of function within the active site of TauD (McCusker, K. P.; Klinman, J. P. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 19791-19795). Extraction of intrinsic deuterium kinetic isotope effects (KIEs) using single turnover transients shows 2- to 4-fold increase in the size of the KIE for F159V in relation to wild-type and F159L. It appears that there is a break in behavior following removal of a single methylene from the side chain of F159L to generate F159V, whereby the protein active site loses its ability to restore the internuclear distance between substrate and Fe(IV)=O that supports optimal hydrogenic wave function overlap.

Role of the Simian Virus 5 Fusion Protein N-Terminal Coiled-Coil Domain in Folding and Promotion of Membrane Fusion

PubMed Central

West, Dava S.; Sheehan, Michael S.; Segeleon, Patrick K.; Dutch, Rebecca Ellis

2005-01-01

Formation of a six-helix bundle comprised of three C-terminal heptad repeat regions in antiparallel orientation in the grooves of an N-terminal coiled-coil is critical for promotion of membrane fusion by paramyxovirus fusion (F) proteins. We have examined the effect of mutations in four residues of the N-terminal heptad repeat in the simian virus 5 (SV5) F protein on protein folding, transport, and fusogenic activity. The residues chosen have previously been shown from study of isolated peptides to have differing effects on stability of the N-terminal coiled-coil and six-helix bundle (R. E. Dutch, G. P. Leser, and R. A. Lamb, Virology 254:147-159, 1999). The mutant V154M showed reduced proteolytic cleavage and surface expression, indicating a defect in intracellular transport, though this mutation had no effect when studied in isolated peptides. The mutation I137M, previously shown to lower thermostability of the six-helix bundle, resulted in an F protein which was properly processed and transported to the cell surface but which had reduced fusogenic activity. Finally, mutations at L140M and L161M, previously shown to disrupt α-helix formation of isolated N-1 peptides but not to affect six-helix bundle formation, resulted in F proteins that were properly processed. Interestingly, the L161M mutant showed increased syncytium formation and promoted fusion at lower temperatures than the wild-type F protein. These results indicate that interactions separate from formation of an N-terminal coiled-coil or six-helix bundle are important in the initial folding and transport of the SV5 F protein and that mutations that destabilize the N-terminal coiled-coil can result in stimulation of membrane fusion. PMID:15650180

Somatic mutations of GUCY2F, EPHA3, and NTRK3 in human cancers.

PubMed

Wood, Laura D; Calhoun, Eric S; Silliman, Natalie; Ptak, Janine; Szabo, Steve; Powell, Steve M; Riggins, Gregory J; Wang, Tian-Li; Yan, Hai; Gazdar, Adi; Kern, Scott E; Pennacchio, Len; Kinzler, Kenneth W; Vogelstein, Bert; Velculescu, Victor E

2006-10-01

Tyrosine kinases are major regulators of signal transduction cascades involved in cellular proliferation and have important roles in tumorigenesis. We have recently analyzed the tyrosine kinase gene family for alterations in human colorectal cancers and identified somatic mutations in seven members of this gene family. In this study we have used high-throughput sequencing approaches to further evaluate this subset of genes for genetic alterations in other human tumors. We identified somatic mutations in GUCY2F, EPHA3, and NTRK3 in breast, lung, and pancreatic cancers. Our results implicate these tyrosine kinase genes in the pathogenesis of other tumor types and suggest that they may be useful targets for diagnostic and therapeutic intervention in selected patients.

Flavobacterium johnsoniae sprB Is Part of an Operon Spanning the Additional Gliding Motility Genes sprC, sprD, and sprF ▿ †

PubMed Central

Rhodes, Ryan G.; Nelson, Shawn S.; Pochiraju, Soumya; McBride, Mark J.

2011-01-01

Cells of Flavobacterium johnsoniae move rapidly over surfaces by a process known as gliding motility. Gld proteins are thought to comprise the gliding motor that propels cell surface adhesins, such as the 669-kDa SprB. A novel protein secretion apparatus called the Por secretion system (PorSS) is required for assembly of SprB on the cell surface. Genetic and molecular analyses revealed that sprB is part of a seven-gene operon spanning 29.3 kbp of DNA. In addition to sprB, three other genes of this operon (sprC, sprD, and sprF) are involved in gliding. Mutations in sprB, sprC, sprD, and sprF resulted in cells that failed to form spreading colonies on agar but that exhibited some motility on glass in wet mounts. SprF exhibits some similarity to Porphyromonas gingivalis PorP, which is required for secretion of gingipain protease virulence factors via the P. gingivalis PorSS. F. johnsoniae sprF mutants produced SprB protein but were defective in localization of SprB to the cell surface, suggesting a role for SprF in secretion of SprB. The F. johnsoniae PorSS is involved in secretion of extracellular chitinase in addition to its role in secretion of SprB. SprF was not needed for chitinase secretion and may be specifically required for SprB secretion by the PorSS. Cells with nonpolar mutations in sprC or sprD produced and secreted SprB and propelled it rapidly along the cell surface. Multiple paralogs of sprB, sprC, sprD, and sprF are present in the genome, which may explain why mutations in sprB, sprC, sprD, and sprF do not result in complete loss of motility and suggests the possibility that semiredundant SprB-like adhesins may allow movement of cells over different surfaces. PMID:21131497

Frequency of V1016I and F1534C mutations in the voltage-gated sodium channel gene in Aedes aegypti in Venezuela.

PubMed

Alvarez, Leslie C; Ponce, Gustavo; Saavedra-Rodriguez, Karla; Lopez, Beatriz; Flores, Adriana E

2015-06-01

The V1016I and F1534C mutations in the voltage-gated sodium channel gene have been associated with resistance to pyrethroids and DDT in Aedes aegypti mosquitoes. A study was carried out to determine the frequency of I1016 and C1534 by real-time PCR in five natural populations of Ae. aegypti in Venezuela during 2008, 2010 and 2012, as well as in a strain selected with 0.14 µg of deltamethrin for 15 generations. In natural populations, frequencies of I1016 varied between 0.01 and 0.37, and frequencies of C1534 between 0.35 and 1.0. In the Pampanito strain, the frequency of I1016 increased from 0.02 in F1 up to 0.5 in F15 and from 0.35 up to fixation for C1534 after selection with deltamethrin. The results showed that C1534 frequencies are higher than I1016 frequencies in natural populations of Ae. aegypti in Venezuela, and that deltamethrin selected the C1534 more rapidly than I1016. © 2014 Society of Chemical Industry.

Single-tube tetradecaplex panel of highly polymorphic microsatellite markers < 1 Mb from F8 for simplified preimplantation genetic diagnosis of hemophilia A.

PubMed

Zhao, M; Chen, M; Tan, A S C; Cheah, F S H; Mathew, J; Wong, P C; Chong, S S

2017-07-01

Essentials Preimplantation genetic diagnosis (PGD) of severe hemophilia A relies on linkage analysis. Simultaneous multi-marker screening can simplify selection of informative markers in a couple. We developed a single-tube tetradecaplex panel of polymorphic markers for hemophilia A PGD use. Informative markers can be used for linkage analysis alone or combined with mutation detection. Background It is currently not possible to perform single-cell preimplantation genetic diagnosis (PGD) to directly detect the common inversion mutations of the factor VIII (F8) gene responsible for severe hemophilia A (HEMA). As such, PGD for such inversion carriers relies on indirect analysis of linked polymorphic markers. Objectives To simplify linkage-based PGD of HEMA, we aimed to develop a panel of highly polymorphic microsatellite markers located near the F8 gene that could be simultaneously genotyped in a multiplex-PCR reaction. Methods We assessed the polymorphism of various microsatellite markers located ≤ 1 Mb from F8 in 177 female subjects. Highly polymorphic markers were selected for co-amplification with the AMELX/Y indel dimorphism in a single-tube reaction. Results Thirteen microsatellite markers located within 0.6 Mb of F8 were successfully co-amplified with AMELX/Y in a single-tube reaction. Observed heterozygosities of component markers ranged from 0.43 to 0.84, and ∼70-80% of individuals were heterozygous for ≥ 5 markers. The tetradecaplex panel successfully identified fully informative markers in a couple interested in PGD for HEMA because of an intragenic F8 point mutation, with haplotype phasing established through a carrier daughter. In-vitro fertilization (IVF)-PGD involved single-tube co-amplification of fully informative markers with AMELX/Y and the mutation-containing F8 amplicon, followed by microsatellite analysis and amplicon mutation-site minisequencing analysis. Conclusions The single-tube multiplex-PCR format of this highly polymorphic microsatellite marker panel simplifies identification and selection of informative markers for linkage-based PGD of HEMA. Informative markers can also be easily co-amplified with mutation-containing F8 amplicons for combined mutation detection and linkage analysis. © 2017 International Society on Thrombosis and Haemostasis.

IFITM5 mutations and osteogenesis imperfecta.

PubMed

Hanagata, Nobutaka

2016-03-01

Interferon-induced transmembrane protein 5 (IFITM5) is an osteoblast-specific membrane protein that has been shown to be a positive regulatory factor for mineralization in vitro. However, Ifitm5 knockout mice do not exhibit serious bone abnormalities, and thus the function of IFITM5 in vivo remains unclear. Recently, a single point mutation (c.-14C>T) in the 5' untranslated region of IFITM5 was identified in patients with osteogenesis imperfecta type V (OI-V). Furthermore, a single point mutation (c.119C>T) in the coding region of IFITM5 was identified in OI patients with more severe symptoms than patients with OI-V. Although IFITM5 is not directly involved in the formation of bone in vivo, the reason why IFITM5 mutations cause OI remains a major mystery. In this review, the current state of knowledge of OI pathological mechanisms due to IFITM5 mutations will be reviewed.

Mutations in ATP6V1B1 and ATP6V0A4 genes cause recessive distal renal tubular acidosis in Mexican families.

PubMed

Escobar, Laura I; Simian, Christopher; Treard, Cyrielle; Hayek, Donia; Salvador, Carolina; Guerra, Norma; Matos, Mario; Medeiros, Mara; Enciso, Sandra; Camargo, María Dolores; Vargas-Poussou, Rosa

2016-05-01

Autosomal recessive distal renal tubular acidosis (dRTA) is a rare disease characterized by a hyperchloremic metabolic acidosis with normal anion gap, hypokalemia, hypercalciuria, hypocitraturia, nephrocalcinosis, and conserved glomerular filtration rate. In some cases, neurosensorial deafness is associated. dRTA is developed during the first months of life and the main manifestations are failure to thrive, vomiting, dehydration, and anorexia. Nine unrelated families were studied: seven children, a teenager, and an adult with dRTA. Hearing was preserved in four children. Coding regions of the genes responsible for recessive dRTA were analysed by Sanger sequencing. Molecular defects were found in the genes ATP6V1B1 and ATP6V0A4. We identified three homozygous variants in ATP6V1B: a frameshift mutation (p.Ile386Hisfs*56), a nucleotide substitution in exon 10 (p.Pro346Arg), and a new splicing mutation in intron 5. Three patients were homozygous for one novel (p.Arg743Trp) and one known (p.Asp411Tyr) missense mutations in the ATP6V0A4 gene. Three patients were compound heterozygous: one proband displayed two novel mutations, the frameshift mutation p.Val52Metfs*25, and a large deletion of exons 18-21; two probands showed the missense mutation p.Asp411Tyr and as a second mutation, p.Arg194Ter and c.1691+2dup, respectively. ATP6V0A4 and ATP6V1B1 genes were involved in recessive dRTA of Mexican families. All ATP6V1B1 mutations detected were homozygous and all patients developed sensorineural hearing loss (SNHL) early in infancy. ATP6V0A4 mutations were found in one infant and three children without SNHL, and in one teenager and one adult with SNHL confirming the phenotypic variability in this trait. The mutation p.Asp411Tyr detected in four Mexican families was due to a founder effect. Screening of these mutations could provide a rapid and valuable tool for diagnosis of dRTA in this population.

Human knockouts and phenotypic analysis in a cohort with a high rate of consanguinity.

PubMed

Saleheen, Danish; Natarajan, Pradeep; Armean, Irina M; Zhao, Wei; Rasheed, Asif; Khetarpal, Sumeet A; Won, Hong-Hee; Karczewski, Konrad J; O'Donnell-Luria, Anne H; Samocha, Kaitlin E; Weisburd, Benjamin; Gupta, Namrata; Zaidi, Mozzam; Samuel, Maria; Imran, Atif; Abbas, Shahid; Majeed, Faisal; Ishaq, Madiha; Akhtar, Saba; Trindade, Kevin; Mucksavage, Megan; Qamar, Nadeem; Zaman, Khan Shah; Yaqoob, Zia; Saghir, Tahir; Rizvi, Syed Nadeem Hasan; Memon, Anis; Hayyat Mallick, Nadeem; Ishaq, Mohammad; Rasheed, Syed Zahed; Memon, Fazal-Ur-Rehman; Mahmood, Khalid; Ahmed, Naveeduddin; Do, Ron; Krauss, Ronald M; MacArthur, Daniel G; Gabriel, Stacey; Lander, Eric S; Daly, Mark J; Frossard, Philippe; Danesh, John; Rader, Daniel J; Kathiresan, Sekar

2017-04-12

A major goal of biomedicine is to understand the function of every gene in the human genome. Loss-of-function mutations can disrupt both copies of a given gene in humans and phenotypic analysis of such 'human knockouts' can provide insight into gene function. Consanguineous unions are more likely to result in offspring carrying homozygous loss-of-function mutations. In Pakistan, consanguinity rates are notably high. Here we sequence the protein-coding regions of 10,503 adult participants in the Pakistan Risk of Myocardial Infarction Study (PROMIS), designed to understand the determinants of cardiometabolic diseases in individuals from South Asia. We identified individuals carrying homozygous predicted loss-of-function (pLoF) mutations, and performed phenotypic analysis involving more than 200 biochemical and disease traits. We enumerated 49,138 rare (<1% minor allele frequency) pLoF mutations. These pLoF mutations are estimated to knock out 1,317 genes, each in at least one participant. Homozygosity for pLoF mutations at PLA2G7 was associated with absent enzymatic activity of soluble lipoprotein-associated phospholipase A2; at CYP2F1, with higher plasma interleukin-8 concentrations; at TREH, with lower concentrations of apoB-containing lipoprotein subfractions; at either A3GALT2 or NRG4, with markedly reduced plasma insulin C-peptide concentrations; and at SLC9A3R1, with mediators of calcium and phosphate signalling. Heterozygous deficiency of APOC3 has been shown to protect against coronary heart disease; we identified APOC3 homozygous pLoF carriers in our cohort. We recruited these human knockouts and challenged them with an oral fat load. Compared with family members lacking the mutation, individuals with APOC3 knocked out displayed marked blunting of the usual post-prandial rise in plasma triglycerides. Overall, these observations provide a roadmap for a 'human knockout project', a systematic effort to understand the phenotypic consequences of complete disruption of genes in humans.

CAV3 mutations causing exercise intolerance, myalgia and rhabdomyolysis: Expanding the phenotypic spectrum of caveolinopathies.

PubMed

Scalco, Renata Siciliani; Gardiner, Alice R; Pitceathly, Robert D S; Hilton-Jones, David; Schapira, Anthony H; Turner, Chris; Parton, Matt; Desikan, Mahalekshmi; Barresi, Rita; Marsh, Julie; Manzur, Adnan Y; Childs, Anne-Marie; Feng, Lucy; Murphy, Elaine; Lamont, Phillipa J; Ravenscroft, Gianina; Wallefeld, William; Davis, Mark R; Laing, Nigel G; Holton, Janice L; Fialho, Doreen; Bushby, Kate; Hanna, Michael G; Phadke, Rahul; Jungbluth, Heinz; Houlden, Henry; Quinlivan, Ros

2016-08-01

Rhabdomyolysis is often due to a combination of environmental trigger(s) and genetic predisposition; however, the underlying genetic cause remains elusive in many cases. Mutations in CAV3 lead to various neuromuscular phenotypes with partial overlap, including limb girdle muscular dystrophy type 1C (LGMD1C), rippling muscle disease, distal myopathy and isolated hyperCKemia. Here we present a series of eight patients from seven families presenting with exercise intolerance and rhabdomyolysis caused by mutations in CAV3 diagnosed by next generation sequencing (NGS) (n = 6). Symptoms included myalgia (n = 7), exercise intolerance (n = 7) and episodes of rhabdomyolysis (n = 2). Percussion-induced rapid muscle contractions (PIRCs) were seen in five out of six patients examined. A previously reported heterozygous mutation in CAV3 (p.T78M) and three novel variants (p.V14I, p.F41S, p.F54V) were identified. Caveolin-3 immunolabeling in muscle was normal in 3/4 patients; however, immunoblotting showed more than 50% reduction of caveolin-3 in five patients compared with controls. This case series demonstrates that exercise intolerance, myalgia and rhabdomyolysis may be caused by CAV3 mutations and broadens the phenotypic spectrum of caveolinopathies. In our series, immunoblotting was a more sensitive method to detect reduced caveolin-3 levels than immunohistochemistry in skeletal muscle. Patients presenting with muscle pain, exercise intolerance and rhabdomyolysis should be routinely tested for PIRCs as this may be an important clinical clue for caveolinopathies, even in the absence of other "typical" features. The use of NGS may expand current knowledge concerning inherited diseases, and unexpected/atypical phenotypes may be attributed to well-known human disease genes. Copyright © 2016 Elsevier B.V. All rights reserved.

Inflammation as a Driver of Clonal Evolution in Myeloproliferative Neoplasm

DTIC Science & Technology

2017-10-01

ligation due to a defect in the negative regulatory feedback loop which normally serves to dampen TNF production . We have localized this defect to a...excessive inflammatory cytokine production in MPN. 15. SUBJECT TERMS Myeloproliferative neoplasm, JAK2V617F, IL-10, TNF, inflammation 16. SECURITY...4 4. Impact…………………………...……………………………………9 5. Changes/Problems...….………………………………………………11 6. Products …………………………………….……….….…………….12 7

AB071. Mutations of AR gene in Vietnamese patients: genotype and phenotype

PubMed Central

Dung, Vu Chi; Fukami, Maki; Ngoc, Can Thi Bich; Thao, Bui Phuong; Khanh, Nguyen Ngoc; Nga, Pham Thu; Dat, Nguyen Phu; Ogata, Tsutomu

2015-01-01

Androgen insensitivity syndrome (AIS) is the most common specific cause of 46,XY disorder in sex development. The androgen signaling pathway is complex but so far, the only gene linked with AIS is the androgen receptor (AR). Mutations in the AR are found in most subjects with complete AIS but in partial AIS, the rate has varied 28-73%, depending on the case selection. More than over 800 entries of mutations causing AIS, representing over 500 different AR mutations from more than 850 patients with AIS have been reported. We aim to describe clinical manifestations and to identify mutation of AR in Vietnamese patients with AIS. This case series study included 12 patients from 9 unrelated families with AIS. The gonadal position and external genitalia were evaluated clinically and using ultrasound. The mutation analysis of AR was performed using PCR and direct sequencing. The age of diagnosis was 1 to 83 years old. 8/12 cases were complete androgen insensitivity syndrome (CAIS) (female external genitalia) and 4 cases were predominantly female partial AIS phenotype. Four cases had two labial testes, six cases had inguinal testes and two cases had abdominal testes. Five different mutations of AR were identified from seven cases of three unrelated families including three novel ones. The novel missense mutation p.L701F (c.2103G > T) was identified in a patient of 83 years of age. The novel missense mutation p.L705F (c.2113C > T) was identified in two sibs. The novel mutation p. W752S (c.2256G > T) was identified in a child with CAIS phenotype and had family history. The reported missense mutation p.V747M was identified in two sibs. The reported mutation p.V867M (c.2599G > A) was identified in a child with female phenotype. Our study identified three novel and two reported mutation in the AR gene that may provide us new insights into the molecular mechanisms of AIS. The expanded database of these mutations should benefit patients in the diagnosis and treatment of this syndrome.

Distribution and Frequency of Pyrethroid Resistance-Associated Mutations in Host Lineages of the Bed Bug (Hemiptera: Cimicidae) Across Europe.

PubMed

Balvín, Ondrej; Booth, Warren

2018-03-15

For over two decades, the bed bug, Cimex lectularius L. (Hemiptera: Cimicidae) has been undergoing a dramatic global resurgence, likely in part to the evolution of mechanisms conferring resistance to insecticides. One such mechanism is knock-down resistance (kdr), resulting from nonsynonymous mutations within the voltage-gated sodium channel (VGSC) gene. To date, three mutations have been identified in C. lectularius, V419L, L925I, and I936F. Using Sanger sequencing, the frequency and distribution of these VGSC mutations across 131 populations collected from the bat-associated and human-associated lineages of C. lectularius found in Europe are documented. All populations from the bat-associated lineage lacked mutations at the three sites. In contrast, the majority of populations associated with humans (93.5%) possessed the mutation at the L925I site. The I936F mutation, previously only reported in Israel and Australia, was found in nine populations spread across several European countries, including the Czech Republic and Switzerland. The high frequency of kdr-associated resistance already reported in C. lectularius and the occurrence and broad geographic distribution of this additional VGSC mutation, questions the continued use of pyrethroids in the treatment of infestations.

Naturally occurring hepatitis C virus protease inhibitors resistance-associated mutations among chronic hepatitis C genotype 1b patients with or without HIV co-infection.

PubMed

Cao, Ying; Zhang, Yu; Bao, Yi; Zhang, Renwen; Zhang, Xiaxia; Xia, Wei; Wu, Hao; Xu, Xiaoyuan

2016-05-01

The aim of this study was to measure the frequency of natural mutations in hepatitis C virus (HCV) mono-infected and HIV/HCV co-infected protease inhibitor (PI)-naive patients. Population sequence of the non-structural (NS)3 protease gene was evaluated in 90 HCV mono-infected and 96 HIV/HCV co-infected PI treatment-naive patients. The natural prevalence of PI resistance mutations in both groups was compared. Complete HCV genotype 1b NS3 sequence information was obtained for 152 (81.72%) samples. Seven sequences (8.33%) of the 84 HCV mono-infected patients and 21 sequences (30.88%) of the 68 HIV/HCV co-infected patients showed amino acid substitutions associated with HCV PI resistance. There was a significant difference in the natural prevalence of PI resistance mutations between these two groups (P = 0.000). The mutations T54S, R117H and N174F were observed in 1.19%, 5.95% and 1.19% of HCV mono-infected patients. The mutations F43S, T54S, Q80K/R, R155K, A156G/V, D168A/E/G and V170A were found in 1.47%, 4.41%, 1.47%/1.47%, 2.94%, 23.53%/1.47%, 1.47%/1.47%/1.47% and 1.47% of HIV/HCV co-infected patients, respectively. In addition, the combination mutations in the NS3 region were detected only in HIV/HCV genotype 1b co-infected patients. Naturally occurring HCV PI resistance mutations existed in HCV mono-infected and HIV/HCV co-infected genotype 1b PI-naive patients. HIV co-infection was associated with a greater frequency of PI resistance mutations. The impact of HIV infection on baseline HCV PI resistance mutations and treatment outcome in chronic hepatitis C (CHC) patients should be further analyzed. © 2015 The Japan Society of Hepatology.

Manipulating proteostasis to repair the F508del-CFTR defect in cystic fibrosis.

PubMed

Esposito, Speranza; Tosco, Antonella; Villella, Valeria R; Raia, Valeria; Kroemer, Guido; Maiuri, Luigi

2016-12-01

Cystic fibrosis (CF) is a lethal monogenic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that entails the (diagnostic) increase in sweat electrolyte concentrations, progressive lung disease with chronic inflammation and recurrent bacterial infections, pancreatic insufficiency, and male infertility. Therapies aimed at restoring the CFTR defect have emerged. Thus, a small molecule which facilitates chloride channel opening, the potentiator Ivacaftor, has been approved for the treatment of CF patients bearing a particular class of rare CFTR mutations. However, small molecules that directly target the most common misfolded CFTR mutant, F508del, and improve its intracellular trafficking in vitro, have been less effective than expected when tested in CF patients, even in combination with Ivacaftor. Thus, new strategies are required to circumvent the F508del-CFTR defect. Airway and intestinal epithelial cells from CF patients bearing the F508del-CFTR mutation exhibit an impressive derangement of cellular proteostasis, with oxidative stress, overactivation of the tissue transglutaminase (TG2), and disabled autophagy. Proteostasis regulators such as cysteamine can rescue and stabilize a functional F508del-CFTR protein through suppressing TG2 activation and restoring autophagy in vivo in F508del-CFTR homozygous mice, in vitro in CF patient-derived cell lines, ex vivo in freshly collected primary patient's nasal cells, as well as in a pilot clinical trial involving homozygous F508del-CFTR patients. Here, we discuss how the therapeutic normalization of defective proteostasis can be harnessed for the treatment of CF patients with the F508del-CFTR mutation.

An interactive mutation database for human coagulation factor IX provides novel insights into the phenotypes and genetics of hemophilia B.

PubMed

Rallapalli, P M; Kemball-Cook, G; Tuddenham, E G; Gomez, K; Perkins, S J

2013-07-01

Factor IX (FIX) is important in the coagulation cascade, being activated to FIXa on cleavage. Defects in the human F9 gene frequently lead to hemophilia B. To assess 1113 unique F9 mutations corresponding to 3721 patient entries in a new and up-to-date interactive web database alongside the FIXa protein structure. The mutations database was built using MySQL and structural analyses were based on a homology model for the human FIXa structure based on closely-related crystal structures. Mutations have been found in 336 (73%) out of 461 residues in FIX. There were 812 unique point mutations, 182 deletions, 54 polymorphisms, 39 insertions and 26 others that together comprise a total of 1113 unique variants. The 64 unique mild severity mutations in the mature protein with known circulating protein phenotypes include 15 (23%) quantitative type I mutations and 41 (64%) predominantly qualitative type II mutations. Inhibitors were described in 59 reports (1.6%) corresponding to 25 unique mutations. The interactive database provides insights into mechanisms of hemophilia B. Type II mutations are deduced to disrupt predominantly those structural regions involved with functional interactions. The interactive features of the database will assist in making judgments about patient management. © 2013 International Society on Thrombosis and Haemostasis.

Analysis of HFE and non-HFE gene mutations in Brazilian patients with hemochromatosis.

PubMed

Bittencourt, Paulo Lisboa; Marin, Maria Lúcia Carnevale; Couto, Cláudia Alves; Cançado, Eduardo Luiz Rachid; Carrilho, Flair José; Goldberg, Anna Carla

2009-01-01

Approximately one-half of Brazilian patients with hereditary hemochromatosis (HH) are neither homozygous for the C282Y mutation nor compound heterozygous for the H63D and C282Y mutations that are associated with HH in Caucasians. Other mutations have been described in the HFE gene as well as in genes involved in iron metabolism, such as transferrin receptor 2 (TfR2) and ferroportin 1 (SCL40A1). To evaluate the role of HFE, TfR2 and SCL40A1 mutations in Brazilian subjects with HH. Nineteen male subjects (median age 42 [range: 20-72] years) with HH were evaluated using the Haemochromatosis StripAssay A. This assay is capable of detecting twelve HFE mutations, which are V53M, V59M, H63D, H63H, S65C, Q127H, P160delC, E168Q, E168X, W169X, C282Y and Q283, four TfR2 mutations, which are E60X, M172K, Y250X, AVAQ594-597del, and two SCL40A1 mutations, which are N144H and V162del. In our cohort, nine (47%) patients were homozygous for the C282Y mutation, two (11%) were heterozygous for the H63D mutation, and one each (5%) was either heterozygous for C282Y or compound heterozygous for C282Y and H63D. No other mutations in the HFE, TfR2 or SCL40A1 genes were observed in the studied patients. One-third of Brazilian subjects with the classical phenotype of HH do not carry HFE or other mutations that are currently associated with the disease in Caucasians. This observation suggests a role for other yet unknown mutations in the aforementioned genes or in other genes involved in iron homeostasis in the pathogenesis of HH in Brazil.

Interallelic Complementation at the Suppressor of Forked Locus of Drosophila Reveals Complementation between Suppressor of Forked Proteins Mutated in Different Regions

PubMed Central

Simonelig, M.; Elliott, K.; Mitchelson, A.; O'Hare, K.

1996-01-01

The Su(f) protein of Drosophila melanogaster shares extensive homologies with proteins from yeast (RNA14) and man (77 kD subunit of cleavage stimulation factor) that are required for 3' end processing of mRNA. These homologies suggest that su(f) is involved in mRNA 3' end formation and that some aspects of this process are conserved throughout eukaryotes. We have investigated the genetic and molecular complexity of the su(f) locus. The su(f) gene is transcribed to produce three RNAs and could encode two proteins. Using constructs that contain different parts of the locus, we show that only the larger predicted gene product of 84 kD is required for the wild-type function of su(f). Some lethal alleles of su(f) complement to produce viable combinations. The structures of complementing and noncomplementing su(f) alleles indicate that 84-kD Su(f) proteins mutated in different domains can act in combination for partial su(f) function. Our results suggest protein-protein interaction between or within wild-type Su(f) molecules. PMID:8846900

Proton-coupled electron transfer in the catalytic cycle of Alcaligenes xylosoxidans copper-dependent nitrite reductase.

PubMed

Leferink, Nicole G H; Han, Cong; Antonyuk, Svetlana V; Heyes, Derren J; Rigby, Stephen E J; Hough, Michael A; Eady, Robert R; Scrutton, Nigel S; Hasnain, S Samar

2011-05-17

We demonstrated recently that two protons are involved in reduction of nitrite to nitric oxide through a proton-coupled electron transfer (ET) reaction catalyzed by the blue Cu-dependent nitrite reductase (Cu NiR) of Alcaligenes xylosoxidans (AxNiR). Here, the functionality of two putative proton channels, one involving Asn90 and the other His254, is studied using single (N90S, H254F) and double (N90S--H254F) mutants. All mutants studied are active, indicating that protons are still able to reach the active site. The H254F mutation has no effect on the catalytic activity, while the N90S mutation results in ~70% decrease in activity. Laser flash-photolysis experiments show that in H254F and wild-type enzyme electrons enter at the level of the T1Cu and then redistribute between the two Cu sites. Complete ET from T1Cu to T2Cu occurs only when nitrite binds at the T2Cu site. This indicates that substrate binding to T2Cu promotes ET from T1Cu, suggesting that the enzyme operates an ordered mechanism. In fact, in the N90S and N90S--H254F variants, where the T1Cu site redox potential is elevated by ∼60 mV, inter-Cu ET is only observed in the presence of nitrite. From these results it is evident that the Asn90 channel is the main proton channel in AxNiR, though protons can still reach the active site if this channel is disrupted. Crystallographic structures provide a clear structural rationale for these observations, including restoration of the proton delivery via a significant movement of the loop connecting the T1Cu ligands Cys130 and His139 that occurs on binding of nitrite. Notably, a role for this loop in facilitating interaction of cytochrome c(551) with Cu NiR has been suggested previously based on a crystal structure of the binary complex.

Restoration of R117H CFTR folding and function in human airway cells through combination treatment with VX-809 and VX-770

PubMed Central

Gentzsch, Martina; Ren, Hong Y.; Houck, Scott A.; Quinney, Nancy L.; Cholon, Deborah M.; Sopha, Pattarawut; Chaudhry, Imron G.; Das, Jhuma; Dokholyan, Nikolay V.; Randell, Scott H.

2016-01-01

Cystic fibrosis (CF) is a lethal recessive genetic disease caused primarily by the F508del mutation in the CF transmembrane conductance regulator (CFTR). The potentiator VX-770 was the first CFTR modulator approved by the FDA for treatment of CF patients with the gating mutation G551D. Orkambi is a drug containing VX-770 and corrector VX809 and is approved for treatment of CF patients homozygous for F508del, which has folding and gating defects. At least 30% of CF patients are heterozygous for the F508del mutation with the other allele encoding for one of many different rare CFTR mutations. Treatment of heterozygous F508del patients with VX-809 and VX-770 has had limited success, so it is important to identify heterozygous patients that respond to CFTR modulator therapy. R117H is a more prevalent rare mutation found in over 2,000 CF patients. In this study we investigated the effectiveness of VX-809/VX-770 therapy on restoring CFTR function in human bronchial epithelial (HBE) cells from R117H/F508del CF patients. We found that VX-809 stimulated more CFTR activity in R117H/F508del HBEs than in F508del/F508del HBEs. R117H expressed exclusively in immortalized HBEs exhibited a folding defect, was retained in the ER, and degraded prematurely. VX-809 corrected the R117H folding defect and restored channel function. Because R117 is involved in ion conductance, VX-770 acted additively with VX-809 to restore CFTR function in chronically treated R117H/F508del cells. Although treatment of R117H patients with VX-770 has been approved, our studies indicate that Orkambi may be more beneficial for rescue of CFTR function in these patients. PMID:27402691

Restoration of R117H CFTR folding and function in human airway cells through combination treatment with VX-809 and VX-770.

PubMed

Gentzsch, Martina; Ren, Hong Y; Houck, Scott A; Quinney, Nancy L; Cholon, Deborah M; Sopha, Pattarawut; Chaudhry, Imron G; Das, Jhuma; Dokholyan, Nikolay V; Randell, Scott H; Cyr, Douglas M

2016-09-01

Cystic fibrosis (CF) is a lethal recessive genetic disease caused primarily by the F508del mutation in the CF transmembrane conductance regulator (CFTR). The potentiator VX-770 was the first CFTR modulator approved by the FDA for treatment of CF patients with the gating mutation G551D. Orkambi is a drug containing VX-770 and corrector VX809 and is approved for treatment of CF patients homozygous for F508del, which has folding and gating defects. At least 30% of CF patients are heterozygous for the F508del mutation with the other allele encoding for one of many different rare CFTR mutations. Treatment of heterozygous F508del patients with VX-809 and VX-770 has had limited success, so it is important to identify heterozygous patients that respond to CFTR modulator therapy. R117H is a more prevalent rare mutation found in over 2,000 CF patients. In this study we investigated the effectiveness of VX-809/VX-770 therapy on restoring CFTR function in human bronchial epithelial (HBE) cells from R117H/F508del CF patients. We found that VX-809 stimulated more CFTR activity in R117H/F508del HBEs than in F508del/F508del HBEs. R117H expressed exclusively in immortalized HBEs exhibited a folding defect, was retained in the ER, and degraded prematurely. VX-809 corrected the R117H folding defect and restored channel function. Because R117 is involved in ion conductance, VX-770 acted additively with VX-809 to restore CFTR function in chronically treated R117H/F508del cells. Although treatment of R117H patients with VX-770 has been approved, our studies indicate that Orkambi may be more beneficial for rescue of CFTR function in these patients. Copyright © 2016 the American Physiological Society.

Circadian-relevant genes are highly polymorphic in autism spectrum disorder patients.

PubMed

Yang, Zhiliang; Matsumoto, Ayumi; Nakayama, Kazuhiro; Jimbo, Eriko F; Kojima, Karin; Nagata, Koh-ichi; Iwamoto, Sadahiko; Yamagata, Takanori

2016-01-01

The genetic background of autism spectrum disorder (ASD) is considered a multi-genetic disorder with high heritability. Autistic children present with a higher prevalence of sleep disorders than has been observed in children with normal development. Some circadian-relevant genes have been associated with ASD (e.g., PER1, PER2, NPAS2, MTNR1A, and MTNR1B). We analyzed 28 ASD patients (14 with sleep disorders and 14 without) and 23 control subjects of Japanese descent. The coding regions of 18 canonical clock genes and clock-controlled genes were sequenced. Detected mutations were verified by direct sequencing analysis, and additional control individuals were screened. Thirty-six base changes with amino acid changes were detected in 11 genes. Six missense changes were detected only in individuals with ASD with sleep disturbance: p.F498S in TIMELESS, p.S20R in NR1D1, p.R493C in PER3, p.H542R in CLOCK, p.L473S in ARNTL2, and p.A325V in MTNR1B. Six missense changes were detected only in individuals with ASD without sleep disturbance: p.S1241N in PER1, p.A325T in TIMELESS, p.S13T in ARNTL, p.G24E in MTNR1B, p.G24E in PER2, and p.T1177A in PER3. The p.R493C mutation in PER3 was detected in both groups. One missense change, p.P932L in PER2, was detected only in the control group. Mutations in NR1D1, CLOCK, and ARNTL2 were detected only in individuals with ASD with sleep disorder. The prevalence of the mutations detected only single time differed significantly among all ASD patients and controls (p=0.003). Two kinds of mutations detected only in individuals with ASD with sleep disorder, p.F498S in TIMELESS and p.R366Q in PER3, were considered to affect gene function by three different methods: PolyPhen-2, scale-invariant feature transform (SIFT) prediction, and Mutation Taster (www.mutationtaster.org). The mutations p.S20R in NR1D1, p.H542R in CLOCK, p.L473S in ARNTL2, p.A325T in TIMELESS, p.S13T in ARNTL, and p.G24E in PER2 were diagnosed to negatively affect gene function by more than one of these methods. Mutations in circadian-relevant genes affecting gene function are more frequent in patients with ASD than in controls. Circadian-relevant genes may be involved in the psychopathology of ASD. Copyright © 2015 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.

Human knockouts and phenotypic analysis in a cohort with a high rate of consanguinity

PubMed Central

Saleheen, Danish; Natarajan, Pradeep; Armean, Irina M.; Zhao, Wei; Rasheed, Asif; Khetarpal, Sumeet; Won, Hong-Hee; Karczewski, Konrad J.; O’Donnell-Luria, Anne H.; Samocha, Kaitlin E.; Weisburd, Benjamin; Gupta, Namrata; Zaidi, Mozzam; Samuel, Maria; Imran, Atif; Abbas, Shahid; Majeed, Faisal; Ishaq, Madiha; Akhtar, Saba; Trindade, Kevin; Mucksavage, Megan; Qamar, Nadeem; Zaman, Khan Shah; Yaqoob, Zia; Saghir, Tahir; Rizvi, Syed Nadeem Hasan; Memon, Anis; Mallick, Nadeem Hayyat; Ishaq, Mohammad; Rasheed, Syed Zahed; Memon, Fazal-ur-Rehman; Mahmood, Khalid; Ahmed, Naveeduddin; Do, Ron; Krauss, Ronald M.; MacArthur, Daniel G.; Gabriel, Stacey; Lander, Eric S.; Daly, Mark J.; Frossard, Philippe; Danesh, John; Rader, Daniel J.; Kathiresan, Sekar

2017-01-01

A major goal of biomedicine is to understand the function of every gene in the human genome.1 Loss-of-function (LoF) mutations can disrupt both copies of a given gene in humans and phenotypic analysis of such ‘human knockouts’ can provide insight into gene function. Consanguineous unions are more likely to result in offspring who carry LoF mutations in a homozygous state. In Pakistan, consanguinity rates are notably high.2 Here, we sequenced the protein-coding regions of 10,503 adult participants in the Pakistan Risk of Myocardial Infarction Study (PROMIS) designed to understand the determinants of cardiometabolic diseases in South Asians.3 We identified individuals carrying predicted LoF (pLoF) mutations in the homozygous state, and performed phenotypic analysis involving >200 biochemical and disease traits. We enumerated 49,138 rare (<1 % minor allele frequency) pLoF mutations. These pLoF mutations are predicted to knock out 1,317 genes in at least one participant. Homozygosity for pLoF mutations at PLAG27 was associated with absent enzymatic activity of soluble lipoprotein-associated phospholipase A2; at CYP2F1, with higher plasma interleukin-8 concentrations; at TREH, with lower concentrations of apoB-containing lipoprotein subfractions; at either A3GALT2 or NRG4, with markedly reduced plasma insulin C-peptide concentrations; and at SLC9A3R1, with mediators of calcium and phosphate signaling. Finally, APOC3 is a gene which retards clearance of plasma triglyceride-rich lipoproteins and where heterozygous deficiency confers protection against coronary heart disease.4,5 In Pakistan, we now observe APOC3 homozygous pLoF carriers; we recalled these knockout humans and challenged with an oral fat load. Compared with wild-type family members, APOC3 knockouts displayed marked blunting of the usual post-prandial rise in plasma triglycerides. Overall, these observations provide a roadmap for a ‘human knockout project’, a systematic effort to understand the phenotypic consequences of complete disruption of genes in humans. PMID:28406212

The Arrhythmogenic Calmodulin p.Phe142Leu Mutation Impairs C-domain Ca2+ Binding but Not Calmodulin-dependent Inhibition of the Cardiac Ryanodine Receptor*

PubMed Central

Liu, Yingjie; Larsen, Kamilla Taunsig; Nani, Alma; Tian, Xixi; Holt, Christian; Wang, Ruiwu; Fill, Michael

2017-01-01

A number of point mutations in the intracellular Ca2+-sensing protein calmodulin (CaM) are arrhythmogenic, yet their underlying mechanisms are not clear. These mutations generally decrease Ca2+ binding to CaM and impair inhibition of CaM-regulated Ca2+ channels like the cardiac Ca2+ release channel (ryanodine receptor, RyR2), and it appears that attenuated CaM Ca2+ binding correlates with impaired CaM-dependent RyR2 inhibition. Here, we investigated the RyR2 inhibitory action of the CaM p.Phe142Leu mutation (F142L; numbered including the start-Met), which markedly reduces CaM Ca2+ binding. Surprisingly, CaM-F142L had little to no aberrant effect on RyR2-mediated store overload-induced Ca2+ release in HEK293 cells compared with CaM-WT. Furthermore, CaM-F142L enhanced CaM-dependent RyR2 inhibition at the single channel level compared with CaM-WT. This is in stark contrast to the actions of arrhythmogenic CaM mutations N54I, D96V, N98S, and D130G, which all diminish CaM-dependent RyR2 inhibition. Thermodynamic analysis showed that apoCaM-F142L converts an endothermal interaction between CaM and the CaM-binding domain (CaMBD) of RyR2 into an exothermal one. Moreover, NMR spectra revealed that the CaM-F142L-CaMBD interaction is structurally different from that of CaM-WT at low Ca2+. These data indicate a distinct interaction between CaM-F142L and the RyR2 CaMBD, which may explain the stronger CaM-dependent RyR2 inhibition by CaM-F142L, despite its reduced Ca2+ binding. Collectively, these results add to our understanding of CaM-dependent regulation of RyR2 as well as the mechanistic effects of arrhythmogenic CaM mutations. The unique properties of the CaM-F142L mutation may provide novel clues on how to suppress excessive RyR2 Ca2+ release by manipulating the CaM-RyR2 interaction. PMID:27927985

Impact of human immunodeficiency virus type 1 resistance to protease inhibitors on evolution of resistance to the maturation inhibitor bevirimat (PA-457).

PubMed

Adamson, Catherine S; Waki, Kayoko; Ablan, Sherimay D; Salzwedel, Karl; Freed, Eric O

2009-05-01

The maturation inhibitor bevirimat [3-O-(3',3'dimethysuccinyl)betulinic acid; BVM; also known as PA-457 or DSB] potently inhibits human immunodeficiency virus type 1 (HIV-1) replication by blocking protease (PR)-mediated cleavage at the junction between capsid (CA) and spacer peptide 1 (SP1) in Gag. We previously isolated a panel of single-amino-acid substitutions that confer resistance to BVM in vitro (C. S. Adamson, S. D. Ablan, I. Boeras, R. Goila-Gaur, F. Soheilian, K. Nagashima, F. Li, K. Salzwedel, M. Sakalian, C. T. Wild, and E. O. Freed, J. Virol. 80:10957-10971, 2006). The BVM resistance mutations cluster at or near the CA-SP1 cleavage site. Because BVM likely will be used clinically in patients harboring viruses resistant to PR inhibitors (PIs), in this study we evaluated the interplay between a PI-resistant (PIR) PR and the BVM resistance mutations in Gag. As expected, the PIR mutations had no effect on inhibition by BVM; however, we observed general processing defects and a slight delay in viral replication in Jurkat T cells associated with the PIR mutations, even in the absence of compound. When combined, most BVM resistance and PIR mutations acted additively to impair viral replication, particularly in the presence of BVM. The BVM-resistant mutant SP1-A1V was an exception, as it supported robust replication in the context of either wild-type (WT) or PIR PR, even at high BVM concentrations. Significantly, the emergence of BVM resistance was delayed in the context of the PIR PR, and the SP1-A1V mutation was acquired most frequently with either WT or PIR PR. These results suggest that resistance to BVM is less likely to emerge in patients who have failed PIs than in patients who are PI naive. We predict that the SP1-A1V substitution is the most likely to emerge in vivo, as this mutant replicates robustly independently of PR mutations or BVM. These findings offer insights into the effect of PIR mutations on the evolution of BVM resistance in PI-experienced patients.

Kinase Regulation by Hydrophobic Spine Assembly in Cancer

PubMed Central

Ahuja, Lalima G.; Meharena, Hiruy S.; Kannan, Natarajan; Kornev, Alexandr P.

2014-01-01

A new model of kinase regulation based on the assembly of hydrophobic spines has been proposed. Changes in their positions can explain the mechanism of kinase activation. Here, we examined mutations in human cancer for clues about the regulation of the hydrophobic spines by focusing initially on mutations to Phe. We identified a selected number of Phe mutations in a small group of kinases that included BRAF, ABL1, and the epidermal growth factor receptor. Testing some of these mutations in BRAF, we found that one of the mutations impaired ATP binding and catalytic activity but promoted noncatalytic allosteric functions. Other Phe mutations functioned to promote constitutive catalytic activity. One of these mutations revealed a previously underappreciated hydrophobic surface that functions to position the dynamic regulatory αC-helix. This supports the key role of the C-helix as a signal integration motif for coordinating multiple elements of the kinase to create an active conformation. The importance of the hydrophobic space around the αC-helix was further tested by studying a V600F mutant, which was constitutively active in the absence of the negative charge that is associated with the common V600E mutation. Many hydrophobic mutations strategically localized along the C-helix can thus drive kinase activation. PMID:25348715

Germline BRCA mutations are associated with higher risk of nodal involvement, distant metastasis, and poor survival outcomes in prostate cancer.

PubMed

Castro, Elena; Goh, Chee; Olmos, David; Saunders, Ed; Leongamornlert, Daniel; Tymrakiewicz, Malgorzata; Mahmud, Nadiya; Dadaev, Tokhir; Govindasami, Koveela; Guy, Michelle; Sawyer, Emma; Wilkinson, Rosemary; Ardern-Jones, Audrey; Ellis, Steve; Frost, Debra; Peock, Susan; Evans, D Gareth; Tischkowitz, Marc; Cole, Trevor; Davidson, Rosemarie; Eccles, Diana; Brewer, Carole; Douglas, Fiona; Porteous, Mary E; Donaldson, Alan; Dorkins, Huw; Izatt, Louise; Cook, Jackie; Hodgson, Shirley; Kennedy, M John; Side, Lucy E; Eason, Jacqueline; Murray, Alex; Antoniou, Antonis C; Easton, Douglas F; Kote-Jarai, Zsofia; Eeles, Rosalind

2013-05-10

To analyze the baseline clinicopathologic characteristics of prostate tumors with germline BRCA1 and BRCA2 (BRCA1/2) mutations and the prognostic value of those mutations on prostate cancer (PCa) outcomes. This study analyzed the tumor features and outcomes of 2,019 patients with PCa (18 BRCA1 carriers, 61 BRCA2 carriers, and 1,940 noncarriers). The Kaplan-Meier method and Cox regression analysis were used to evaluate the associations between BRCA1/2 status and other PCa prognostic factors with overall survival (OS), cause-specific OS (CSS), CSS in localized PCa (CSS_M0), metastasis-free survival (MFS), and CSS from metastasis (CSS_M1). PCa with germline BRCA1/2 mutations were more frequently associated with Gleason ≥ 8 (P = .00003), T3/T4 stage (P = .003), nodal involvement (P = .00005), and metastases at diagnosis (P = .005) than PCa in noncarriers. CSS was significantly longer in noncarriers than in carriers (15.7 v 8.6 years, multivariable analyses [MVA] P = .015; hazard ratio [HR] = 1.8). For localized PCa, 5-year CSS and MFS were significantly higher in noncarriers (96% v 82%; MVA P = .01; HR = 2.6%; and 93% v 77%; MVA P = .009; HR = 2.7, respectively). Subgroup analyses confirmed the poor outcomes in BRCA2 patients, whereas the role of BRCA1 was not well defined due to the limited size and follow-up in this subgroup. Our results confirm that BRCA1/2 mutations confer a more aggressive PCa phenotype with a higher probability of nodal involvement and distant metastasis. BRCA mutations are associated with poor survival outcomes and this should be considered for tailoring clinical management of these patients.

Germline BRCA Mutations Are Associated With Higher Risk of Nodal Involvement, Distant Metastasis, and Poor Survival Outcomes in Prostate Cancer

PubMed Central

Castro, Elena; Goh, Chee; Olmos, David; Saunders, Ed; Leongamornlert, Daniel; Tymrakiewicz, Malgorzata; Mahmud, Nadiya; Dadaev, Tokhir; Govindasami, Koveela; Guy, Michelle; Sawyer, Emma; Wilkinson, Rosemary; Ardern-Jones, Audrey; Ellis, Steve; Frost, Debra; Peock, Susan; Evans, D. Gareth; Tischkowitz, Marc; Cole, Trevor; Davidson, Rosemarie; Eccles, Diana; Brewer, Carole; Douglas, Fiona; Porteous, Mary E.; Donaldson, Alan; Dorkins, Huw; Izatt, Louise; Cook, Jackie; Hodgson, Shirley; Kennedy, M. John; Side, Lucy E.; Eason, Jacqueline; Murray, Alex; Antoniou, Antonis C.; Easton, Douglas F.; Kote-Jarai, Zsofia; Eeles, Rosalind

2013-01-01

Purpose To analyze the baseline clinicopathologic characteristics of prostate tumors with germline BRCA1 and BRCA2 (BRCA1/2) mutations and the prognostic value of those mutations on prostate cancer (PCa) outcomes. Patients and Methods This study analyzed the tumor features and outcomes of 2,019 patients with PCa (18 BRCA1 carriers, 61 BRCA2 carriers, and 1,940 noncarriers). The Kaplan-Meier method and Cox regression analysis were used to evaluate the associations between BRCA1/2 status and other PCa prognostic factors with overall survival (OS), cause-specific OS (CSS), CSS in localized PCa (CSS_M0), metastasis-free survival (MFS), and CSS from metastasis (CSS_M1). Results PCa with germline BRCA1/2 mutations were more frequently associated with Gleason ≥ 8 (P = .00003), T3/T4 stage (P = .003), nodal involvement (P = .00005), and metastases at diagnosis (P = .005) than PCa in noncarriers. CSS was significantly longer in noncarriers than in carriers (15.7 v 8.6 years, multivariable analyses [MVA] P = .015; hazard ratio [HR] = 1.8). For localized PCa, 5-year CSS and MFS were significantly higher in noncarriers (96% v 82%; MVA P = .01; HR = 2.6%; and 93% v 77%; MVA P = .009; HR = 2.7, respectively). Subgroup analyses confirmed the poor outcomes in BRCA2 patients, whereas the role of BRCA1 was not well defined due to the limited size and follow-up in this subgroup. Conclusion Our results confirm that BRCA1/2 mutations confer a more aggressive PCa phenotype with a higher probability of nodal involvement and distant metastasis. BRCA mutations are associated with poor survival outcomes and this should be considered for tailoring clinical management of these patients. PMID:23569316

Molecular mechanistic explanation for the spectrum of cholestatic disease caused by the S320F variant of ABCB4.

PubMed

Andress, Edward J; Nicolaou, Michael; Romero, Marta R; Naik, Sandhia; Dixon, Peter H; Williamson, Catherine; Linton, Kenneth J

2014-05-01

ABCB4 flops phosphatidylcholine into the bile canaliculus to protect the biliary tree from the detergent activity of bile salts. Homozygous-null ABCB4 mutations cause the childhood liver disease, progressive familial intrahepatic cholestasis, but cause and effect is less clear, with many missense mutations linked to less severe cholestatic diseases. ABCB4(S320F), in particular, is described in 13 patients, including in heterozygosity with ABCB4(A286V), ABCB4(A953D), and null mutants, whose symptoms cover the spectrum of cholestatic disease. We sought to define the impact of these mutations on the floppase, explain the link with multiple conditions at the molecular level, and investigate the potential for reversal. ABCB4(S320F), ABCB4(A286V), and ABCB4(A953D) expression was engineered in naïve cultured cells. Floppase expression, localization, and activity were measured by western blot, confocal microscopy, and lipid transport assays, respectively. ABCB4(S320F) was fully active for floppase activity but expression at the plasma membrane was reduced to 50%. ABCB4(A286V) expressed and trafficked efficiently but could not flop lipid, and ABCB4(A953D) expressed poorly and was impaired in floppase activity. Proteasome inhibition stabilized nascent ABCB4(S320F) and ABCB4(A953D) but did not improve plasma membrane localization. Cyclosporin-A improved plasma membrane localization of both ABCB4(S320F) and ABCB4(A953D), but inhibited floppase activity. The level of ABCB4 functionality correlates with, and is the primary determinant of, cholestatic disease severity in these patients. ABCB4(S320F) homozygosity, with half the normal level of ABCB4, is the tipping point between more benign and potentially fatal cholestasis and makes these patients more acutely sensitive to environmental effects. Cyclosporin-A increased expression of ABCB4(S320F) and ABCB4(A953D), suggesting that chemical chaperones could be exploited for therapeutic benefit to usher in a new era of personalized medicine for patients with ABCB4-dependent cholestatic disease. © 2014 by the American Association for the Study of Liver Diseases.

The Dual Regulatory Role of Amino Acids Leu480 and Gln481 of Prothrombin*

PubMed Central

Wiencek, Joesph R.; Hirbawi, Jamila; Yee, Vivien C.; Kalafatis, Michael

2016-01-01

Prothrombin (FII) is activated to α-thrombin (IIa) by prothrombinase. Prothrombinase is composed of a catalytic subunit, factor Xa (fXa), and a regulatory subunit, factor Va (fVa), assembled on a membrane surface in the presence of divalent metal ions. We constructed, expressed, and purified several mutated recombinant FII (rFII) molecules within the previously determined fVa-dependent binding site for fXa (amino acid region 473–487 of FII). rFII molecules bearing overlapping deletions within this significant region first established the minimal stretch of amino acids required for the fVa-dependent recognition exosite for fXa in prothrombinase within the amino acid sequence Ser478–Val479–Leu480–Gln481–Val482. Single, double, and triple point mutations within this stretch of rFII allowed for the identification of Leu480 and Gln481 as the two essential amino acids responsible for the enhanced activation of FII by prothrombinase. Unanticipated results demonstrated that although recombinant wild type α-thrombin and rIIaS478A were able to induce clotting and activate factor V and factor VIII with rates similar to the plasma-derived molecule, rIIaSLQ→AAA with mutations S478A/L480A/Q481A was deficient in clotting activity and unable to efficiently activate the pro-cofactors. This molecule was also impaired in protein C activation. Similar results were obtained with rIIaΔSLQ (where rIIaΔSLQ is recombinant human α-thrombin with amino acids Ser478/Leu480/Gln481 deleted). These data provide new evidence demonstrating that amino acid sequence Leu480–Gln481: 1) is crucial for proper recognition of the fVa-dependent site(s) for fXa within prothrombinase on FII, required for efficient initial cleavage of FII at Arg320; and 2) is compulsory for appropriate tethering of fV, fVIII, and protein C required for their timely activation by IIa. PMID:26601957

Occurrence of etravirine/rilpivirine-specific resistance mutations selected by efavirenz and nevirapine in Kenyan patients with non-B HIV-1 subtypes failing antiretroviral therapy.

PubMed

Crawford, Keith W; Njeru, Dorothy; Maswai, Jonah; Omondi, Milton; Apollo, Duncan; Kimetto, Jane; Gitonga, Lawrence; Munyao, James; Langat, Raphael; Aoko, Appolonia; Tarus, Jemutai; Khamadi, Samoel; Hamm, Tiffany E

2014-01-28

Resistance to efavirenz and nevirapine has not been associated with mutations at position 138 of reverse transcriptase. In an evaluation of virologic suppression rates in PEPFAR (President's Emergency Plan For AIDS Relief) clinics in Kenya among patients on first-line therapy (RV288), 63% (617/975) of randomly selected patients on antiretroviral therapy were suppressed (HIV RNA<400 copies/ml). Among those with non-nucleoside reverse transcriptase inhibitor resistance (n = 101), 14 (13.8%) had substitutions at 138 (A, G, K or Q), mutations selected only by etravirine and rilpivirine in subtype B viruses. All 14 patients received efavirenz or nevirapine, not etravirine or rilpivirine, and were predominantly subtype A1. This may be the first report of efavirenz and nevirapine selecting these mutations in these subtypes.

Analysis of some polymorphic markers of the CFTR gene in cystic fibrosis patients and healthy donors from the Moscow region

DOE Office of Scientific and Technical Information (OSTI.GOV)

Amosenko, F.A.; Sazonova, M.A.; Kapranov, N.I.

1995-04-01

Allelic frequencies of three polymorphic markers in the CFTR gene were estimated on chromosomes derived from cystic fibrosis (CF) patients and healthy donors from Moscow and the Moscow region. These polymorphic markers are tetranucleotide tandem repeats GATT in intron 6B, M470V in exon 10, and T854T in exon 14 (fragment A). Frequencies at allele 1 of the M470V marker, along with allele 2 of GATT and T854T, are two times higher for CF patients without {Delta}F508 mutation than for healthy donors, and there is linkage disequilibrium of these alleles of the polymorphic markers analyzed with the CF gene. Allele 1more » of M470V and T854T markers, as well as allele 2 of the GATT marker (six repeats), are absolutely linked to mutation F508 of the CFTR gene. Using the polymorphic markers studied, family analysis of CF was carried out in two families. 10 refs., 1 fig., 1 tab.« less

Missense variants in plakophilin-2 in arrhythmogenic right ventricular cardiomyopathy patients--disease-causing or innocent bystanders?

PubMed

Christensen, Alex Hørby; Benn, Marianne; Tybjaerg-Hansen, Anne; Haunso, Stig; Svendsen, Jesper Hastrup

2010-01-01

Mutations in genes encoding desmosomal proteins have been linked to arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D). We hypothesized that a Scandinavian ARVC/D population would have a different spectrum of plakophilin-2 (PKP2) mutations and that some of the reported missense mutations may not be pathogenic. We screened 53 unrelated patients fulfilling Task Force criteria for ARVC/D for mutations in PKP2 by direct sequencing. Seven different mutations were identified: two insertion/deletions (E329fsX352, P401fsX406), 1 splice site (2146-2A>T), 1 non-sense (R79X) and 4 missense mutations (Q62K in 2 patients, G489R, G673V) of undeterminable pathogeneity. None of these mutations was present in 650 controls. Five of the mutations were novel. Seven patients carried reported missense mutations (D26N, S140F, V587I); however, these mutations were identified in our healthy controls, although at a lower frequency. Evaluation of all reported missense mutations in PKP2 showed unclear pathogeneity of several reported mutations. Fifteen percent of Danish ARVC/D patients carried PKP2 mutations. Our finding of reported disease-causing mutations at a low frequency among healthy controls suggests that these variants are disease modifying but not directly disease causing. We recommend conservative interpretation of missense variants in PKP2, functional characterization and large-scale sequencing to clarify normal variation in the gene.

Financial consequences of a payment-by-results scheme in Catalonia: gefitinib in advanced EGFR-mutation positive non-small-cell lung cancer.

PubMed

Clopes, Ana; Gasol, Montse; Cajal, Rosana; Segú, Luis; Crespo, Ricard; Mora, Ramón; Simon, Susana; Cordero, Luis A; Calle, Candela; Gilabert, Antoni; Germà, Josep R

2017-01-01

In 2011 the first payment-by-results (PbR) scheme in Catalonia was signed between the Catalan Institute of Oncology (ICO), the Catalan Health Service, and AstraZeneca (AZ) for the introduction of gefitinib in the treatment of advanced EGFR-mutation positive non-small-cell lung cancer. The PbR scheme includes two evaluation points: at week 8, responses, stabilization and progression were evaluated, and at week 16 stabilization was confirmed. AZ was to reimburse the total treatment cost of patients that failed treatment, defined as progression at weeks 8 or 16. To estimate the financial consequences of this PbR reimbursement model and determine the perception of the stakeholders involved in the agreement. Differential drug costs between two scenarios, with and without the PbR, were calculated. A qualitative investigation of the organizational elements was performed by interviewing the parties involved in the agreement. Forty-one patients were included from June 2011 to October 2013 and assessed at two evaluation points. Clinical results were comparable to those observed in the pivotal studies of gefitinib. The difference in the cost of gefitinib using the PbR compared to the traditional purchasing scenario was 6.17% less at 8 weeks, 11.18% at 16 weeks and 4.15% less for the overall treatment. The PbR resulted in total savings of around €36,000 (€880 per patient). From an operational and organizational perspective, the availability of adequate data systems to measure outcomes and monitor accountability and the involvement of healthcare professionals were acknowledged as crucial. Tangible and intangible benefits were identified with respect to the interests of the parties involved. This has led to the incorporation of innovation for patients under acceptable conditions.

How We Identify and Manage Patients with Inadequately Controlled Polycythemia Vera.

PubMed

Reiter, Andreas; Harrison, Claire

2016-10-01

Polycythemia vera (PV) is a chronic myeloproliferative neoplasm (MPN) characterized by an overactive Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway through mutations in JAK2 exons 12 or 14 (JAK2 V617F). The dominant clinical characteristics include erythrocytosis (with or without leukocytosis/thrombocytosis), thrombotic events, and symptoms. Increased risk of mortality is mainly caused by thrombotic events and progression to post-polycythemia vera myelofibrosis (PPV-MF) or secondary acute myeloid leukemia (sAML). The most important prognostic factors include age and a history of thrombotic events, although recent evidence has indicated that leukocytosis and additional cytogenetic aberrations may also be of significant prognostic value. First-line therapies include aspirin and phlebotomies, which significantly reduce the incidence of thrombotic events and prolong survival. Cytoreductive treatment with hydroxyurea (approved) and conventional or pegylated interferon-α (effective, but not approved in many countries) is initiated for high-risk or inadequately controlled disease, e.g., uncontrolled hematocrit, leukocytosis, thrombocytosis, thrombotic events, splenomegaly, or symptoms. However, some patients may not receive initial benefit from first-line therapy or may become resistant or intolerant in due course. Although second-line treatment options are limited, clinical trials have shown the efficacy of ruxolitinib toward improving blood counts, enlarged spleen, and symptoms and potentially reducing thrombotic events. Identification of patients with uncontrolled PV is important for clinical care, as such patients have a high risk of complications, and future studies with JAK inhibitors or other agents alone or in combination are needed to test their potential to reduce rates of thrombotic events and transformation to PPV-MF or sAML.