Academic literature on the topic 'Compound mutation'
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Journal articles on the topic "Compound mutation"
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Li, Jin, Zhenyu Yan, Agus Darwanto, Peng Fang, Weihua Liu, Kristy Drafahl, Julie Toplin, Cindy Spittle, and Chad Galderisi. "Phasing Analysis Of TKI Resistance Mutations In The BCR-ABL1 Kinase Domain and Neighboring Domains Using Next-Generation Sequencing." Blood 122, no. 21 (November 15, 2013): 3817. http://dx.doi.org/10.1182/blood.v122.21.3817.3817.
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Abstract Background Many CML patients treated with tyrosine kinase inhibitors (TKIs) eventually develop resistance as a result of ABL1 kinase domain (KD) mutations, and sequential treatment with different TKIs may select for multiple BCR-ABL1 mutations. Whether multiple mutations arise in distinct clones (in trans, or polyclonal mutations) or instead are present within the same BCR-ABL1 molecule (in cis, or compound mutations), has been shown to have important implications with respect to TKI sensitivities (Eide, C.A. et al., Blood 2011). Distinguishing between polyclonal and compound mutations, or mutation phasing, for the ABL1 KD has not been clinically practical with standard mutation detection methods. Here we have developed a highly sensitive next-generation sequencing (NGS) assay on the Ion Torrent PGM along with a proprietary data analysis pipeline that together enable deep sequencing of the BCR-ABL1 KD and neighboring domains with a 1% limit of detection and quantitative reporting of mutation phasing. Methods RT and long range PCR was performed to amplify BCR-ABL1 e1a2/3, e13a2/3, and e14a2/3 fusion transcripts and the PCR products were enzymatically randomly fragmented and ligated with Ion Torrent sequencing adaptors. Size-selected libraries were quantified, pooled, amplified with the OneTouch system and sequenced with the Ion Torrent PGM using 400 bp sequencing chemistry. Sequencing data were analyzed with Torrent Suite 3.4.2 with variant frequency cutoff adjusted to 1%. Variants were further annotated with a proprietary analysis pipeline and variant report was produced after manually reviewing variants by Integrative Genomics Viewer. If more than one non-synonymous variant was reported in a sample, a proprietary phasing analysis pipeline was applied to report the mutation spectrum of all of the combinations of multiple mutations in the sample. Results To validate the accuracy of the sequencing method which employs 400 bp sequencing chemistry, we compared this assay with our previously validated BCR-ABL1 NGS assay based on Ion Torrent 200 bp sequencing chemistry for a set of clinical specimens from CML patients previously treated with TKI. Results were highly concordant and similarly sensitive, with 11/11 variants (frequencies ranging from 2% to 100%) identified with comparable frequencies by both methods. To evaluate the specificity of the phasing analysis, an artificial sample was created by mixing two samples (b2_10 and b2_6) with 6 distinct variants present at ratio of 1:19. Because variants are unique in each sample, any compound mutation composed of b2_10 variant and b2_6 variant identified would be false positive. The false positive error rate (percentage of b2_6 variant as compound mutation with b2_10 variant and vice versa) ranged from 0 – 0.6%, which was consistent with sequencing error rate. We conservatively define a compound mutation as true if it is present in at least 5% of any one of the component variants in the compound mutation. Mutation detection and phasing analysis were reproducible on different chips (314 v2 and 318 v2) and different library preps from the same long range PCR product of BCR-ABL1. Table 1 shows the mutation spectrum from sample b2_6. Of the four variants detected, L248V and G250E were mutually exclusive (in trans), while T315I and M351T were present as compound mutations with each other and, separately, with either L248V or G250E. Notably, >86% of the molecules harbored single mutations, and no compound mutations containing more than 2 variants were observed. Conclusions We have developed and validated a sensitive NGS assay that enables deep sequencing of the BCR-ABL1 KD and neighboring domains along with quantitative mutational phasing. This method has been applied in evaluating >250 clinical specimens for a clinical trial of a third-generation TKI(results reported separately). The ability to easily determine the mutation phasing of a CML patients’ mutation profile using this assay will allow for investigations into compound mutation-based resistance mechanisms and may be used to better guide treatment decisions. Disclosures: Li: MolecularMD: Employment. Yan:MolecularMD: Employment. Darwanto:MolecularMD: Employment. Fang:MolecularMD: Employment. Liu:MolecularMD: Employment. Drafahl:MolecularMD: Employment. Toplin:MolecularMD: Employment. Spittle:MolecularMD: Employment. Galderisi:MolecularMD: Employment.
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Bhatwadekar, Seema S., and Parth Shah. "Mutational Phasing: Clinical Relevance in Tyrosine Kinase Domain Mutations Using Next Generation Sequencing in Chronic Myeloid Leukemia." Blood 132, Supplement 1 (November 29, 2018): 4269. http://dx.doi.org/10.1182/blood-2018-99-114130.
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Abstract Background: Tyrosine kinase mutation analysis in BCR/ABL1 gene is important for management of patients with chronic myeloid leukemia. Sanger Sequencing has been the mainstay for testing with Next Generation Sequencing (NGS) now becoming the primary technology. In this study we show a comparison between NGS versus Sanger Seqencing based ABL kinase domain mutation analysis with a likely trend of clinical relevance based on a compound versus polyclonal state of mutational distribution which may also need to be considered for patient management and therapy. Methodology: A total of 213 Imatinib-resistant patients with CML for BCR-ABL1 mutation analysis were processed on both technologies.Initial blood counts were assessed and RNA was extractedfollowed by cDNA conversion. NGS libraries were prepared with 400bp multiplexed amplicons to allow optimal phasing. Results: 179 samples were negative by both technologies. A total of only 20 samples were positive and concordant by both technologies(58.2%). Mutations in 14 other samples however were only detected in NGS(41.17%). In these 14 samples (41.17%), NGS was able to detect 23 mutations with mutation frequencies of 3-28%, which were missed by Sanger. Conclusions: Moreover 11/34 patients had 2 or >2 mutations. An inhouse script delineated mutations as compound or polyclonal from NGS data. 2/11 cases demonstrated compound mutations (Mutations in the same clone) while 7/11 cases were polyclonal per NGS. Sanger sequencing cannot differentiate between polyclonal and compound mutations. 2/11 cases appeared to have polyclonal and compound mutations. 4/11 patients presented in a blast crisis or accelerated phase CML. Interestingly, most of these patients hadat leasttwo mutations and were polyclonal(3/4). Significantly previously archived samples patients with polyclonal mutations showed polyclonality at extremely low frequency percentages in initial samples. None of the single mutation patients had presented in a blast crisis or an accelerated phase. Disclosures No relevant conflicts of interest to declare.
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Chen, Jiaqi, Hongxing Liu, Fang Wang, Yang Zhang, Xue Chen, Daijing Nie, Yu Li, Yincheng Tan, Yuanli Xu, and Xiaoli Ma. "Dynamic Evolution of Ponatinib Resistant BCR-ABL1 T315 and Compound Mutations." Blood 134, Supplement_1 (November 13, 2019): 3796. http://dx.doi.org/10.1182/blood-2019-129579.
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The third-generation tyrosine kinase inhibitor (TKI) ponatinib exhibits activity against all common BCR-ABL1 kinase domain (KD) single mutations, including the highly resistant gatekeeper T315I. However, the drug response is variable and the clinical resistance mutations may still befall with few reports to date. We performed next-generation sequencing (NGS) detection of BCR-ABL1 KD mutations in sequential samples of three BCR-ABL1-positive leukemia patients who developed clinical resistance to ponatinib, to explore the dynamic evolution of ponatinib mutations. Case 1 was diagnosed as chronic myeloid leukemia (CML) chronic phase when he was 29 years old in 2009. His maintenance therapies were imatinib and dasatinib for seven years and then replaced with ponatinib due to the blast crisis of CML and the T315I mutation with variant allele frequency (VAF) of 46%. He did not achieved molecular remission after attempting multiple combined chemotherapy and TKIs including ponatinib, with the T315I mutation persists and eventually increases to VAF of 97%. He was then was medicated with a combined chemotherapy plus dasatinib and ponatinib. But NGS KD mutation investigation showed multiple T315I compound mutations, T315M and T315I mutations six months later. The patients went through salvage allogenic hemopoietic stem cell transplantation (allo-HSCT), and the above polyclonal and compound mutations were still carried after transplantation. Finally, Q252H/T315I (VAF,50%) became the dominant clone, and ponatinib and HQP1351 (domestic TKI designed for T315I) were ineffective for this compound mutation (Figure a). A similar dynamic evolution to the BCR-ABL1 KD mutation of Case1 also occurred in case 2. The 7-year-old boy was diagnosed with BCR-ABL1-positive acute B-lymphocytic leukemia (Ph+B-ALL) in April 2014. NGS results showed that he had D276G (7%), F311I (27%) and F317L (31%) polyclonal mutations in BCR-ABL1 KD after 18 months of imatinib administration which were subsequently treated with ponatinib. After 5 months of treatment with ponatinib, F311I and F317L mutations disappeared, but G250E (5%) and D276G/T315L (4%) compound mutation appeared; subsequent progression to D276G/T315L (32%), G250W/E255V/F311I (4%) and F311I/T315I (58%) polyclonal compound mutations; long induction chemotherapy combined with ponatinib treatment remained unresolved, and finally there was only D276G/T315L compound mutation (VAF, 100%, figure b and d). Notably, a rare mutation T315L (c.943_945delinsCTC/p.T315L) appeared in the BCR-ABL1 KD. D276G is known to be sensitive to various TKIs, so we speculate that ponatinib is ineffective for the T315L mutation. Case 3 was a 46-year-old woman who diagnosed with Ph+B-ALL in July 2018. The Q252H (20%) and T315I (44%) double mutations appeared after oral administration of imatinib for 2 months, and then switched to ponatinib for remission. After 8 months, the bone marrow and peripheral blood samples showed not only the T315I/F359V compound mutation (VAF, 90% and 94%), but also the T315L mutation (VAF, 5% and 6%, c.943_944AC>CT/p.T315L). The clinician combined her BCR-ABL1 KD mutation and condition, ponatinib was discontinued. After 1 month of chemotherapy combined with dasatinib, the patient's condition improved, but the BCR-ABL1 KD mutation progressed to T315L (18%, figure c and e) and T315I/F359V compound mutation (76%). She eventually died from severe pulmonary infection and sepsis. NGS analysis identify KD mutation with sensitivity about 2%, and can also distinguish between compound and polyclonal mutations. All of the ultimately dominant ponatinib resistant mutations (Q252H/T315I, D276G/T315L, and T351I/F359V) in these three cases were T315 compound mutations derived from the T315I or other original mutation with additional mutation event. The T315L/M mutations and compound mutations collaborated by T315 and other KD mutations may confer the major component of ponatinib resistance. The dynamic resistant mutation in these three patients adds to the currently less content compendium of ponatinib clinical resistance. All of the three patients encountered ponatinib toxic side effects and had to discontinue or reduce the dose, which also confer favorable opportunity for the development of drug-resistant mutations. Figure Disclosures No relevant conflicts of interest to declare.
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Mian, Afsar Ali, Hadiqa Raees, Sujjawal Ahmad, Oliver Ottmann, and El-Nasir M. A. Lalani. "Arsenic Trioxide Suppresses Growth of BCR-ABL1 Positive Cells with "Gatekeeper" or Compound Mutation." Blood 138, Supplement 1 (November 5, 2021): 4346. http://dx.doi.org/10.1182/blood-2021-154511.
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Abstract Introduction: Chronic myeloid leukemia (CML) and 30% of adult acute lymphatic leukemia (ALL) are characterized by the Philadelphia chromosome (Ph +), having a (9;22) chromosomal translocation. The BCR-ABL1 fusion protein is the hallmark of Ph + leukemia. BCR-ABL1 is characterized by constitutively activated ABL1 tyrosine kinase activity that determines its transformation potential. Tyrosine kinase inhibitors (TKI) have greatly improved the overall prognosis of these diseases. However, unsatisfactory responses in advanced disease stages, resistance and long-term tolerability of BCR-ABL1 inhibitors represent major clinical problems. The most important resistance mechanism against TKIs is the acquisition of point mutations within the BCR-ABL1 kinase domain that impair drug binding, restoring the oncoprotein's constitutively active tyrosine kinase activity. The selection of leukemic clones driven by BCR-ABL1 harboring point mutations, such as the E255K, Y253F/H (P-loop), H396R (activation loop) or the T315I (gatekeeper). Second- and third generation TKIs such as nilotinib, dasatinib, and ponatinib effectively overcome point mutation-mediated resistance. Ponatinib is the only U.S. Food and Drug Administration approved TKI with activity against all known BCR-ABL1 point mutations, including BCR-ABL1-T315I. However, the emergence of compound mutations (two mutations within the same BCR-ABL1 allele) has been linked to resistance to all approved TKIs, including ponatinib, posing a clinical challenge with limited treatment options. The anti-cancer agent arsenic trioxide (ATO) has been used to treat patients with acute promyelocytic leukemia (APL). APL patients respond very well to ATO therapy and achieve complete remission, possibly through induction of apoptosis and differentiation. In addition, it has been demonstrated that combined treatment of ATO with interferon or nilotinib significantly suppressed cell proliferation. However, the potential effects of ATO on BCR-ABL1 mutations and especially on compound mutation is not apparent. This study aimed to investigate the role of ATO in BCR-ABL1 resistant mutations, including compound mutation in Ph + leukemias. Methods: We undertook preclinical evaluation of ATO and compared it with approved TKIs e.g. imatinib, nilotinib, dasatinib, ponatinib and ABL inhibitor asciminib, in vitro models of CML and primary patient-derived long term cultures (PD-LTC) of Ph + ALL patients with or without mutation. The effects on mutational resistance were investigated in Ba/F3 cells expressing BCR-ABL1 with T315I mutation and T315I-E255K mutation. For non-mutational resistance, we used PD-LTCs from Ph + ALL patients with different levels of non-mutational drug resistance. Cell proliferation was assessed by XTT. Results: ATO efficiently inhibited the growth of all PD-LTCs in cellular assays at dosages of 200-500nM. It also suppressed the growth of Ph + PD-LTC with non- mutational resistance (BV) and the BCR-ABL1-T315I positive PD-LTC (KO) in this dosage range. In all modelsWe treated Ba/F3 cells expressing native BCR-ABL1, BCR-ABL1-T315I mutation and BCR-ABL1-T315I-E255K (compound mutation) with increasing concentrations of imatinib (250, 500 and 1000nM), nilotinib (100, 200 and 400nM), dasatinib (10, 25 and 50nM), ponatinib (10, 50 and 100nM), asciminib) (ABL allosteric inhibitor) (5, 10 and 20nM) and ATO (0.5, 1.0 and 2.0 µM). We found that all the inhibitors significantly inhibited the proliferation of Ba/F3 cells expressing wild type BCR-ABL1 in a dose-dependent manner. In contrast, the growth of Ba/F3 cells expressing BCR-ABL1-T315I was inhibited by increasing concentration of ponatinib, asciminib and ATO. ATO potently inhibited the most challenging mutation (T315I-E255K) with a clinically relevant concentration (IC50 250nM). All approved ABL kinase inhibitors (AKIs) and allosteric inhibitors like asciminib could not inhibit the growth of Ba/F3 cells expressing BCR-ABL1 compound mutation. Conclusions: Our findings indicate that ATO significantly suppressed the proliferation of cells expressing non-mutated BCR-ABL1, single and compound mutated BCR-ABL1. These results support including ATO in treating patients with Ph + leukemias having BCR-ABL1 resistant single or compound mutati Disclosures Ottmann: Novartis: Honoraria; Amgen: Honoraria, Research Funding; Celgene/BMS: Honoraria, Research Funding; Fusion: Honoraria; Incyte: Honoraria, Research Funding.
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Kim, Soo-Hyun, Soo Young Choi, Sung-Eun Lee, Ju-Hee Bang, Ji-Young Byeun, Jin-Eok Park, Hye-Rim Jeon, Eun-Jung Jang, Saengsuree Jootar, and Dong-Wook Kim. "Dynamics and Characteristics of BCR-ABL1 Multiple Mutations in Tyrosine Kinase Inhibitor Resistant CML." Blood 120, no. 21 (November 16, 2012): 1677. http://dx.doi.org/10.1182/blood.v120.21.1677.1677.
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Abstract Abstract 1677 Background: BCR-ABL1 kinase domain (KD) point mutation causes resistance to tyrosine kinase inhibitors (TKI) in CML patients through impaired binding of TKI to the target site. One of the characteristics of patients with BCR-ABL1 kinase domain point mutations is the fact that some patients have multiple mutations. However there have not been many studies showing that data about clinical relevance or dynamics of multiple mutation during CML treatment. Methods: Since 2002, 414 CML patients were screened for mutation analysis due to sign of resistance to TKI including imatinib, nilotinib, dasatinib, bosutinib, radotinib or ponatinib at Seoul St Mary's Hospital using direct sequencing and ASO-PCR. Among them, 31 patients showed multiple mutations. We analyzed serial samples from the 31 patients using subcloning and sequencing to investigate whether the multiple mutations are on same clone (defined as compound clone), separated clones (defined as multiple clone) or co-existent clones (defined as mixed clone) and characterize its clinical relevance and dynamics. Results: Status of the patients with multiple mutations is shown in Table 1. In order to investigate whether the multiple mutations are on same clone or on separated clone, we cloned serial samples from the 31 patients. Cloning of cDNA region corresponding to BCR-ABL1 KD into plasmid was performed and followed by transformation into competent cells, colony formation, plasmid preparation of 20 colonies from each sample, and then direct sequencing. Multiple mutations of 65% patients (20 out of 31) existed compound mutation which means the individual mutant types are located on the same BCR-ABL1 molecule. In addition of major mutation types which were detectable in direct sequencing analysis, all the patients showed to have minor types of mutations which were found only through BCR-ABL1 KD cloning and subsequent colony sequencing. To make sure that this minor mutation types were not caused by sequencing error, we also analyzed of 3 patients who showed TKI resistance, but had no BCR-ABL1 mutation. In addition, samples from 3 normal persons were analyzed with the same method. The frequency of appearance of the minor types of point mutation was reduced in the patient group who showed TKI resistance, but had no BCR-ABL1 mutation, and then dramatically decreased in the normal person group, indicating that BCR-ABL1 gene in patients with point mutation are relatively unstable. Among 20 patients with compound mutation, 9 patients were available for serial timepoint samples under same TKI therapy. In all nine patients (100%), portion of compound clone was increased as treatment went on. With a median follow-up 53.3 months (range, 0–113.2 months), of 31 patients with multiple mutation, 7 patients remained alive; 4 of 11 (36%) in the multiple clone group vs 3 of 20 (15%) in the mixed clone group (P = 0.066). Conclusion: Analysis of serial samples from a same patient provided evidence of dynamic change of portion of compound mutation. In most case, portion of the clone containing compound mutation was increased as treatment went on, indicating the clone harboring compound mutation can take survival advantage over TKI treatment in comparison of the clone containing individual type of mutation. In addition, some patients showed change in individual mutation type comprising multiple mutations as treatment went on. Patients with compound clone showed poor outcomes compared with multiple clone group in our cohort, further investigation on a large patient cohort will be needed. Updated data with longer follow-up duration will be presented in the meeting. Disclosures: No relevant conflicts of interest to declare.
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Jung, Hyun Ae, Sehhoon Park, Jong-Mu Sun, Se-Hoon Lee, Jin Seok Ahn, Myung-Ju Ahn, and Keunchil Park. "Treatment and Outcomes of Metastatic Non-Small-Cell Lung Cancer Harboring Uncommon EGFR Mutations: Are They Different from Those with Common EGFR Mutations?" Biology 9, no. 10 (October 7, 2020): 326. http://dx.doi.org/10.3390/biology9100326.
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Approximately 10% of the epidermal growth factor receptor (EGFR) mutations in non-small-cell lung cancer (NSCLC) are uncommon EGFR mutations. Although the efficacy of second (2G) or third generation (3G) EGFR tyrosine kinase inhibitors (EGFR-TKIs) in the patients with uncommon EGFR mutation has been proven, further studies are warranted to define the optimal treatment approach for uncommon EGFR mutation-positive NSCLC. This study retrospectively investigated the treatment patterns and outcomes of patients with uncommon EGFR mutation-positive NSCLC from January 2011 to December 2019 at the Samsung Medical Center, Seoul, Korea. During the study, 2121 patients with EGFR mutation-positive NSCLC received first-generation (1G, gefitinib or erlotinib) or 2G EGFR-TKI (afatinib) as the first-line (1L) systemic therapy. Of this, 135 (6.4%) patients harbored uncommon EGFR mutations. Of 135, 54 (40%, 54/135) patients had overlapping mutations with major EGFR mutations. The objective response rate (ORR) for the 1L EGFR-TKI was 63.3%. The median progression-free survivals (PFSs) were 8.6 months (95% CI: 3.8–13.5), 11.7 months (95% CI: 6.6–16.7), 7.7 months (95% CI: 4.9–17.4), and 5.0 months (95% CI: 3.7–6.1) for major uncommon EGFR mutation (G719X, L861Q), compound mutation with major EGFR mutation (Del 19 or EGFR exon 21 p.L858R), other compound mutation, and other uncommon mutations, respectively. The median overall survivals (OSs) were 25.6 months (16.9–34.2), 28.8 (95% CI: 24.4–33.4), 13.5 months (95% CI: 7.4–27.8), and 9.4 months (95% CI: 3.4–10.5) for major uncommon EGFR mutation (G719X), compound mutation with major EGFR mutation (Del 19 or EGFR exon 21 p.L858R), other compound mutation, and other uncommon mutations, respectively. The response rate, median PFS, and OS were 63.3%, 16.3 months (95% CI: 15.6–16.9), and 37.5 months (95% CI: 35.4–39.6) for common EGFR mutation-positive NSCLC. After failing 1L EGFR-TKI, repeated tissue or liquid biopsy were carried out on 44.9% (35/78) of patients with T790M detected in 10/35 (28.6%) patients. With subsequent 3G EGFR-TKI after failing the first-line EGFR-TKI, the ORR and PFS for 3G EGFR-TKI were 80% and 8.9 months (95% CI: 8.0–9.8). These patients showed a median OS of 34.6 months (95% CI: 29.8–39.4). The ORR, PFS and OS were poorer in patients with uncommon (especially other compound and other uncommon mutation) than those with common EGFR mutations. T790M was detected in 28.6% of the uncommon EGFR mutation-positive patients for whom prior 1G/2G EGFR-TKIs failed and underwent repeat biopsy at the time of progression.
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Kim, Dong-Wook, Dongho Kim, Soo-Hyun Kim, Saengsuree Jootar, Hyun-Gyung Goh, Jeong Lee, Soo-Young Choi, Young-Seok Lee, and Sang-Mi Oh. "Dynamics and Characteristics of BCR-ABL Multiple Mutations In Tyrosine Kinase Inhibitor Resistant Chronic Myeloid Leukemia." Blood 116, no. 21 (November 19, 2010): 3443. http://dx.doi.org/10.1182/blood.v116.21.3443.3443.
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Abstract Abstract 3443 BCR-ABL kinase domain (KD) point mutation causes resistance to tyrosine kinase inhibitors (TKI) in CML patients through impaired binding of TKI to the target site. One of the characteristics of patients with BCR-ABL kinase domain point mutations is the fact that some patients have multiple mutations. However there have not been many studies showing that data about clinical relevance or dynamics of multiple mutation during CML treatment. From January 2002 to June 2010 at Seoul St Mary's Hospital, 277 CML patients were screened for mutation analysis due to sign of resistance to tyrosine kinase inhibitors including imatinib, nilotinib, dasatinib or bosutinib. We found that 95 patients have point mutation in BCR-ABL kinase domain through direct sequencing or ASO-PCR. Among them, 17 patients showed multiple mutation containing more than one type of point mutations in BCR-ABL KD. We investigated the patients with multiple mutations to characterize its clinical relevance and dynamics. Once mutation found, follow-up samples from the corresponding patients were collected and analyzed prospectively, or mutation status was analyzed retrospectively with cryopreserved samples if they were available. Status of the patients with multiple mutation is shown in Table 1. In order to investigate whether the multiple mutations are on same clone or on separated clone, we cloned serial samples from the 17 patients. Cloning of cDNA region corresponding to BCR-ABL KD into plasmid was performed and followed by transformation into competent cells, colony formation, plasmid preparation of 20 colonies from each sample, and then direct sequencing. Multiple mutations of 88% patients (15 out of 17) existed compound mutation which means the individual mutant types are located on the same BCR-ABL molecule. In addition of major mutation types which were detectable in direct sequencing analysis, all the patients showed to have minor types of mutations which were found only through BCR-ABL KD cloning and subsequent colony sequencing. To make sure that this minor mutation types were not caused by sequencing error, we also analyzed of 3 patients who showed TKI resistance, but had no BCR-ABL mutation. In addition, samples from 3 normal persons were analyzed with the same method. The frequency of appearance of the minor types of point mutation was reduced in the patient group who showed TKI resistance, but had no BCR-ABL mutation, and then dramatically decreased in the normal person group, indicating that BCR-ABL gene in patients with point mutation are relatively unstable. Analysis of serial samples from a same patient provided evidence of dynamic change of portion of compound mutation. In most case, portion of the clone containing compound mutation was increased as treatment went on, indicating the clone harboring compound mutation can take survival advantage over TKI treatment in comparison of the clone containing individual type of mutation. In addition, some patients showed change in individual mutation type comprising multiple mutation as treatment went on. Currently investigation of clinical relevance of compound mutation and other analyses are being carried on and more results will be provided in detail at the conference. Table 1. Patients Tx at mutation detection (mg) Compound type Compound % 1 Nilotinib400 G250E+T315I 6.7 G250E+D444G 33.3 T315I+D444G 6.7 2 Nilotinib400 M244V+T315I 95.0 3 Dasatinib100 Y253H+T315I 95.0 4 Dasatinib140 T315I+E459K 55.6 5 Dasatinib200 T315I+M351T 66.7 6 Dasatinib100 NCM Dasatinib80 NCM Dasatinib100 M244V+F359V 16.7 7 Bosutinib500 NCM 8 Dasatinib140 T315I+F359C 35.3 9 Imatinib400 E255K+T315I 5.6 10 Dasatinib80 E255V+T315I 90.0 11 Imatinib800 E255K+T315I 10.5 12 Nilotinib800 E255K+T315I 12.5 13 Dasatinib100 F311I+T315I 35.0 F311I+F317Lb 10.0 Imatinib400 F311I+T315I 10.0 F311I+F317La 15.0 F311I+F317Lb 55.0 14 Nilotinib800 Y253H+F359I 5.6 15 Bosutinib500 V299L+E459K 95.0 Nilotinib400 + Dasatinib100 V299L+F359I 5.0 V299L+E459K 55.0 V299L+F317La+E459K 15.0 V299L+F359I+E459K 15.0 V299L+F317La+F359I+E459K 5.0 16 Imatinib600 NCM 17 Imatinib400 NCM NCM: no compound mutation. Disclosures: No relevant conflicts of interest to declare.
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Finkielstain, Gabriela P., Wuyan Chen, Sneha P. Mehta, Frank K. Fujimura, Reem M. Hanna, Carol Van Ryzin, Nazli B. McDonnell, and Deborah P. Merke. "Comprehensive Genetic Analysis of 182 Unrelated Families with Congenital Adrenal Hyperplasia due to 21-Hydroxylase Deficiency." Journal of Clinical Endocrinology & Metabolism 96, no. 1 (January 1, 2011): E161—E172. http://dx.doi.org/10.1210/jc.2010-0319.
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Background: Genetic analysis is commonly performed in patients with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency. Study Objective: The objective of the study was to describe comprehensive CYP21A2 mutation analysis in a large cohort of CAH patients. Methods: Targeted CYP21A2 mutation analysis was performed in 213 patients and 232 parents from 182 unrelated families. Complete exons of CYP21A2 were sequenced in patients in whom positive mutations were not identified by targeted mutation analysis. Copy number variation and deletions were determined using Southern blot analysis and PCR methods. Genotype was correlated with phenotype. Results: In our heterogeneous U.S. cohort, targeted CYP21A2 mutation analysis did not identify mutations on one allele in 19 probands (10.4%). Sequencing identified six novel mutations (p.Gln262fs, IVS8+1G>A, IVS9-1G>A, p.R408H, p.Gly424fs, p.R426P) and nine previously reported rare mutations. The majority of patients (79%) were compound heterozygotes and 69% of nonclassic (NC) patients were compound heterozygous for a classic and a NC mutation. Duplicated CYP21A2 haplotypes, de novo mutations and uniparental disomy were present in 2.7% of probands and 1.9 and 0.9% of patients from informative families, respectively. Genotype accurately predicted phenotype in 90.5, 85.1, and 97.8% of patients with salt-wasting, simple virilizing, and NC mutations, respectively. Conclusions: Extensive genetic analysis beyond targeted CYP21A2 mutational detection is often required to accurately determine genotype in patients with CAH due to the high frequency of complex genetic variation.
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Liu, Shiguo, Shasha Zhang, Wenjie Li, Aiqing Zhang, Fengguang Qi, Guohua Zheng, Shengli Yan, and Xu Ma. "Clinical and Genetic Analysis of a Compound Heterozygous Mutation in the Thyroglobulin Gene in a Chinese Twin Family With Congenital Goiter and Hypothyroidism." Twin Research and Human Genetics 15, no. 1 (February 2012): 126–32. http://dx.doi.org/10.1375/twin.15.1.126.
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Mutations in the thyroglobulin (TG) gene, which has an estimated incidence of approximately 1 in 100,000 new-borns, cause autosomal recessive congenital hypothyroidism. The mutational spectrum of the TG gene and the phenotype–genotype correlations have not yet fully been established. We report a compound heterozygous mutation in the TG gene in a Chinese twin family with congenital goiter and hypothyroidism. We also describe the gene mutation associated with the genotype–phenotype of these children with congenital goiter and hypothyroidism. The whole coding sequence of the TG gene was analyzed by direct sequence, and the identified changes in the sequence were tested for benign polymorphism by denaturing high-performance liquid chromatography screening of the mutation and sequencing 200 chromosomes from normal controls. Analysis of the TG gene of the affected twin revealed a compound heterozygous mutation, including a novel missense mutation G2687A, which is predicted to result in a glutamine to arginine substitution at codon 877, and a known nonsense mutation C7006T, predicted to result in an arginine to stop codon at codon 2317. Analysis of 200 normal chromosomes did not identify the same change in healthy subjects. This is the first report of a TG gene mutation in the Chinese Han population. Our study provides further evidence that mutations in the TG gene cause congenital goiter and hypothyroidism, demonstrates genetic heterogeneity of the mutation, and increases our understanding of phenotype–genotype correlations in congenital hypothyroidism.
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Rao, Vamshi K., Christine J. DiDonato, and Paul D. Larsen. "Friedreich’s Ataxia: Clinical Presentation of a Compound Heterozygote Child with a Rare Nonsense Mutation and Comparison with Previously Published Cases." Case Reports in Neurological Medicine 2018 (August 9, 2018): 1–5. http://dx.doi.org/10.1155/2018/8587203.
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Friedreich’s ataxia is a neurodegenerative disorder associated with a GAA trinucleotide repeat expansion in intron 1 of the frataxin (FXN) gene. It is the most common autosomal recessive cerebellar ataxia, with a mean age of onset at 16 years. Nearly 95-98% of patients are homozygous for a 90-1300 GAA repeat expansion with only 2-5% demonstrating compound heterozygosity. Compound heterozygous individuals have a repeat expansion in one allele and a point mutation/deletion/insertion in the other. Compound heterozygosity and point mutations are very rare causes of Friedreich’s ataxia and nonsense mutations are a further rarity among point mutations. We report a rare compound heterozygous Friedrich’s ataxia patient who was found to have one expanded GAA FXN allele and a nonsense point mutation in the other. We summarize the four previously published cases of nonsense mutations and compare the phenotype to that of our patient. We compared clinical information from our patient with other nonsense FXN mutations reported in the literature. This nonsense mutation, to our knowledge, has only been described once previously; interestingly the individual was also of Cuban ancestry. A comparison with previously published cases of nonsense mutations demonstrates some common clinical characteristics.
Dissertations / Theses on the topic "Compound mutation"
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Mazur, Artur, Katrin Köhler, Markus Schülke, Mandy Skunde, Mariusz Ostański, and Angela Hübner. "Familial Glucocorticoid Deficiency Type 1 due to a Novel Compound Heterozygous MC2R Mutation." Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2014. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-134512.
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Objective: Description of the clinical, biochemical and genetic features of a Polish patient with familial glucocorticoid deficiency. Methods: Detailed clinical investigation, hormonal analysis and sequencing of the coding region of the melanocortin 2 receptor (MC2R) gene in this patient. Results: We report on a 3-month-old boy with familial glucocorticoid deficiency who presented at the age of 3 months with skin hyperpigmentation, muscle weakness, mild jaundice and constipation. Hormonal analyses revealed high ACTH and TSH serum concentrations, low serum cortisol concentration along with normal blood electrolytes. On hydrocortisone supplementation, the disease symptoms disappeared and the child recovered completely. His physical and mental development progresses normally. Genetic analysis disclosed a novel compound heterozygous MC2R mutation p.Leu46fs and p.Val49Met. Conclusion: The heterozygous p.Leu46fs mutation adds to the small number of MC2R nonsense mutations and is the first frameshift mutation within the first transmembrane domain of the receptor. According to molecular modeling the Val49Met mutation results in a structural change of the first transmembrane domain and in a potential novel interaction of the transmembrane domains I and VIIDieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich
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Mazur, Artur, Katrin Köhler, Markus Schülke, Mandy Skunde, Mariusz Ostański, and Angela Hübner. "Familial Glucocorticoid Deficiency Type 1 due to a Novel Compound Heterozygous MC2R Mutation." Karger, 2008. https://tud.qucosa.de/id/qucosa%3A27575.
Full textAbstract:
Objective: Description of the clinical, biochemical and genetic features of a Polish patient with familial glucocorticoid deficiency. Methods: Detailed clinical investigation, hormonal analysis and sequencing of the coding region of the melanocortin 2 receptor (MC2R) gene in this patient. Results: We report on a 3-month-old boy with familial glucocorticoid deficiency who presented at the age of 3 months with skin hyperpigmentation, muscle weakness, mild jaundice and constipation. Hormonal analyses revealed high ACTH and TSH serum concentrations, low serum cortisol concentration along with normal blood electrolytes. On hydrocortisone supplementation, the disease symptoms disappeared and the child recovered completely. His physical and mental development progresses normally. Genetic analysis disclosed a novel compound heterozygous MC2R mutation p.Leu46fs and p.Val49Met. Conclusion: The heterozygous p.Leu46fs mutation adds to the small number of MC2R nonsense mutations and is the first frameshift mutation within the first transmembrane domain of the receptor. According to molecular modeling the Val49Met mutation results in a structural change of the first transmembrane domain and in a potential novel interaction of the transmembrane domains I and VII.Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.
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MOROSINI, SARA. "Integrated genetic diagnosis of neurofibromatosis type 1 (NF1) and molecular characterization of one case of compound heterozygosity." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2014. http://hdl.handle.net/10281/83314.
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Genetic analysis of Neurofibromatosis type 1 (NF1) may facilitate the identification of patients in early phases of the disease. Here, we present an overview of our diagnostic research spanning the last eleven years, with a focus on the description of 225 NF1 mutations, 126 of which are novel, found in a series of 605 patients (513 unrelated) in Italy. Between 2003 and 2013, 443 unrelated patients were profiled by DHPLC analysis of 60 amplicons derived from genomic NF1 DNA and subsequent sequencing of heterozygotic PCR products. In addition, a subset of patients was studied by MLPA to identify any duplications, large deletions or microdeletions present at the locus. Over the last year, 70 unrelated patients were investigated by MLPA and sequencing of 22 amplicons spanning the entire NF1 cDNA. Mutations were found in 70% of the 293 patients studied by DHPLC, thereby fulfilling the NIH criterion for the clinical diagnosis of NF1 (detection rate: 70%); furthermore, 87% of the patients studied by RNA sequencing were genetically characterized. Mutations were also found in 36 of the 159 patients not fulfilling the NIH clinical criteria. These data support the use of RNA-based methods for genetic analysis and provide novel information for relevant for improving the management of symptoms in oligosymptomatic patients.
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MOLLA, DAVID. "INVESTIGATION ON PHARMACOLOGICAL AND AGE-INDUCED MODULATIONS OF CARDIAC PEACEMAKING AND ELECTROPHYSIOLOGICAL CHARACTERIZATION OF A COMPOUND MUTATION IN THE CARDIAC SODIUM CHANNEL." Doctoral thesis, Università degli Studi di Milano, 2021. http://hdl.handle.net/2434/816791.
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Abstract:
During my Ph.D. internship at the ‘PaceLab’, I have been involved in several scientific projects that, although involving different experimental models and with different aims, share the common background of cardiac pacemaker modulation and ion channels activity. In this sec-tion are briefly described the three main research lines in which I took part; a more complete discussion of the data will be presented later in this thesis. Notably, by the time I am writing this proposition, two of these studies have been submitted to scientific journals.
- Identification of the bradycardic molecule contained in the traditional Chinese medi-cine drug Tongmai Yangxin:
The identification of new bradycardic agents able to specifically bind HCN channels, rises great interest in the scientific community since a decreased cardiac pacemaker cur-rent would lead to an general heart rate lowering without side-effects. To date, despite a long and intensive investigation, only one pure bradycardic agent (Ivabradine)1-3 is used in the clinical setting. Ivabradine is considered virtually free of negative side-effects even though, in some rare cases, it can elicit minor optical limitations.
A few years ago, our research group started a collaboration with a traditional Chinese medicine (TMC) drug producer (Le Ren Tang Pharmaceutical Factory, Tianjin, China) with the aim of unravelling the molecular mechanism of one of their products: Tongmai Yangxin (TMYX). This drug is currently used in China for the treatment of several car-diac diseases like coronary artery disease, palpitation, heart failure and angina4 and, like other TCM compounds, it is a mixture of botanical and animal products. Remarkably, recent investigations highlighted its ability to reduce cardiac metabolic disorders, oxida-tive stress and inflammation on stable angina patients4-6.
Our previous studies explored the bradycardic action of this drug; in particular, its ef-fects were evaluated on freshly isolated rabbit SAN myocytes. TMYX displayed a dose dependent and fully reversible rate slowing of the spontaneous action potential (AP) ac-tivity, specifically acting on the pacemaker current by shifting the activation curve of HCN channels towards more negative potentials; furthermore, its efficacy appeared to be strictly correlated to the intracellular cAMP concentration. Detailed analysis demon-strate that this drug acts as a functional cAMP surmountable competitive antagonist, competing for the CNBD of f-channels according to a mode of action never observed before for the regulation of this current. Still, the nature of the bio-active molecule is un-known.
With the aim of identify this principle, TMYX was divided into 4 fractions (F1-4) by our Chinese colleagues at the Pharmaceutical Informatics Institute of Zhejiang University (Hangzhou, China) according to the solubility of its components, and the ability of each preparation to modulate spontaneous rate and the If current was evaluated by patch-clamp experiments in rabbit SAN myocytes. Data clearly demonstrate that only the most hydrophilic fraction (F1) displayed features similar to the total drug, decreasing the AP rate up to ~20% specifically acting on the pacemaker current.
To further narrow down the number of molecules to examine, F1 was subsequently di-vided into 4 sub-fractions (F1.1-1.4) and, as for the previous preparations, their effects were examined on rabbit SAN myocytes. The results pointed out the presence of the target molecule both in F1.1 and F1.2 since they were able to reduce the spontaneous AP firing by ~25 and ~20%, respectively, with a specific action on If current.
Eventually, HPLC data revealed the presence of uridine in both these sub-fraction, sug-gesting that this molecule (or one of its derivates) could be involved in the bradycardic process. Therefore, a preliminary experiment was carried out to analyze its possible car-diac rate regulation properties on rabbit SAN myocytes. Surprisingly, the perfusion of uridine (1 μM) generated a small increase in the AP rate (+3.45%) but a decrease in the pacemaker current at -65mV. Additional experiment are requested in order to shed more light on the properties of this molecule, however, given that uridine, and in partic-ular its cyclic nuclidic form (cUMP), have been reported to interact with some isoforms of the HCN channels family7-9, it appears to be a good starting point for the identifica-tion of the active principle of TMYX.
- Age-related changes in cardiac autonomic modulation and heart rate variability in mice:
The incidence of mortality caused by age-associated cardiovascular diseases is increas-ing dramatically and it will represent a serious clinical issue in the next decades10,11.
In humans, the natural process of aging is associated with progressive changes in cardi-ac autonomic nervous system (ANS) regulation that may predispose to higher cardiac risks. Animal models of aging are needed to gain insights into the relation between the aging of the ANS and cardiac pathophysiology. Specifically, the aim of this study is to verify the translational relevance of mouse models for further in-depth evaluation of the link between cardiac ANS regulation and increased arrhythmic risk with advancing age.
Therefore, heart rate and time- and frequency-domain indexes of HRV were calculated from ECG recordings in two groups of conscious C57BL6/J male mice of different ages (4- and 19-months-old) during daily undisturbed conditions following peripheral β‐adrenergic (atenolol), muscarinic (methylscopolamine), and β‐adrenergic + muscarinic blockades and β‐adrenergic (isoprenaline) stimulation. Eventually, vulnerability to ar-rhythmias was evaluated during daily, undisturbed conditions and following β‐adrenergic stimulation.
HRV analysis and heart rate responses to autonomic blockades revealed that 19-month-old mice had a lower vagal modulation of cardiac function compared with 4-month-old mice. This age-related autonomic effect did not however affect basal heart rate, since it compensated for the lower intrinsic heart rate observed in 19-month-old compared with 4-month-old mice. Both time- and frequency-domain indexes of HRV were reduced fol-lowing muscarinic, but not β‐adrenergic, blockade, suggesting that HRV is largely modulated by vagal tone in mice. Finally, 19-month-old mice showed a larger vulnera-bility to both spontaneous and isoprenaline-induced arrhythmias.
These results reveal the presence of a reduced cardiac vagal modulation and HRV asso-ciated with an increased vulnerability to cardiac arrhythmias in older mice, which is consistent with the human condition. Given their short life span, mice could be further exploited as an aged model for studying the trajectory of vagal decline with advancing age using HRV measures, and the mechanisms underlying its association with proarrhythmic remodeling of the senescent heart.
- Electrophysiological characterization of a SCN5A compound mutation (K1578N-G1866fs) discovered in a young patient affected by sinus node disfunction, atrial flut-ters and drug-induced long QT syndrome:
Given its high relevance in the generation of the cardiac AP event, alterations in the ge-netic sequence encoding for NaV1.5 channel (SCN5A) are often related to severe dis-function in the heart function12.
In this situation, a compound mutation (K1578N/G1866fs) has been reported in a child affected by severe bradycardia, atrial flutter and drug-induced QT prolongation. Nota-bly, the parents, who present a heterozygous mutation each, did not suffer of any cardi-ac problem and their ECG signals were unremarkable. Following clinical examinations, the diagnosis was sinus node disfunction and the patient was implanted with a pace-maker13.
With this study we intended to characterize the electrical properties of the Na+ current carried by the mutated NaV1.5 channels in order to better understand the impact of these alteration and explore whether the patient will benefit from a specific pharmaco-logical treatment.
According to the literature14,15, SCN5A gene can undergo a series of alternative splicing events, among which the inclusion of different exon 6 sequences that identify the neona-tal and the adult NaV1.5 isoform. Consequently, the electrical difference between these two isoforms was assessed by patch-clamp experiments on HEK-293 cells transfected with a vector containing their genetic sequence. The results displayed no differences in the current density compared to the adult isoform (HP: -120 mV) while a positive shift of both activation and inactivation curves were detected (5.1 mV and 8.8 mV, respec-tively).
The impacts of the mutations on the Na+ current were then evaluated on the same mod-el both in the patient (compound mutation K1578N/G1866fs in the neonatal isoform) and in the parents (heterozygous expression in the adult isoform) conditions. For what concerns the parents, the data collected are reasonably in agreement with the clinical in-vestigation indicating no pathologic conditions. On the other hand, the transfection of both the mutations in the neonatal SCN5A isoform caused a dramatic reduction in the current density associated to a rightward shift of the activation curve (6.5 mV), com-pared to the corresponding WT isoform.
In addition, since the time at which the neonatal to adult isoform switch occurs has still not been clearly identify, the compound mutation was also inserted in the WT adult vector. However, no changes were detected in the current density while a leftward shift of the activation curve (7.5 mV), which can be ascribed to the isoform change, suggested that the alterations have a similar effect on the activation kinetics regarding of the iso-form in which are expressed.
Eventually, no changes in the recovery from inactivation process were found between all the conditions investigated.
All in all, these data show an important loss-of-function of NaV1.5 channels in the pa-tient’s condition, suggesting that the two mutations (K1578N–G1866fs), when ex-pressed together, generate a far worst phenotype than their single heterozygous expres-sion. Furthermore, even when incorporated in the adult SCN5A isoform, the effects caused by the alterations did not change suggesting that the neonatal to adult isoform switch will probably grant no benefits for the patient.
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GEETA, GEETA. "In vitro and in vivo characterization of resistance to lorlatinib treatment in ALK mutated cancers." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2019. http://hdl.handle.net/10281/241123.
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La terapia personalizzata ha cambiato lo scenario clinico dei tumori ALK-positivi. Tuttavia, la resistenza farmacologica rimane un ostacolo importante cui far fronte. Lorlatinib è un farmaco di terza generazione che inibisce la maggior parte dei mutanti di ALK resistenti agli attuali inibitori. In questo studio sono state utilizzate in vitro e in vivo linee cellulari resistenti a lorlatinib derivanti da linfoma anaplastico a grandi cellule (ALCL), carcinoma polmonare non a piccole cellule (NSCLC) e neuroblastoma, allo scopo di studiare i meccanismi che guidano l'acquisizione della resistenza. In vitro, cellule di ALCL trattate con alte concentrazioni di lorlatinib hanno acquisito le seguenti mutazioni composte di ALK: G1202R/G1269A e C1156F/L1198F. In vivo, Le cellule di ALCL xenotrapiantate in topi immunocompromessi hanno acquisito le seguenti mutazioni ricorrenti: N1178H (5 topi su 10) e G1269A (4 topi su 10). È interessante notare che nelle cellule umane, dove l’espressione di NPM/ALK è tipicamente nucleare, la presenza della mutazione induce la localizzazione citoplasmatica di NPM/ALKN1178H, probabilmente mimandone l’overespressione. Nelle cellule resistenti si è osservata una significativa alterazione dei pathway di PI3K/AKT e RAS/MAPK, come dimostrato dall’analisi dell’espressione genica tramite RNA-seq. Il coinvolgimento di questi pathway nella resistenza a lorlatinib è stato confermato dalla validazione funzionale con inibitori. Le cellule di NSCLC trattate in vitro con lorlatinib hanno acquisito iperattivazione di EGFR. Il trattamento con un inibitore di EGFR, erlotinib, , è stato in grado di ripristinare la sensibilità a lorlatinib. In cellule di neuroblastoma resistenti a lorlatinib, il sequenziamento dell'intero esoma e l’analisi proteomica hanno rivelato una forma tronca di NF1 e l’iperattivazione di EGFR e ErbB4. Questi dati forniscono un'ampia caratterizzazione dei meccanismi di resistenza che possono insorgere in diversi tumori ALK-positivi dopo il trattamento con lorlatinib.Targeted therapy changed the standard of care in ALK-dependent tumors. However, resistance remains a major challenge. Lorlatinib is a third-generation ALK inhibitor that inhibits most ALK mutants resistant to current ALK inhibitors. In this study, we utilize lorlatinib-resistant anaplastic large cell lymphoma (ALCL), non–small cell lung cancer (NSCLC), and neuroblastoma cell lines in vitro and in vivo to investigate the acquisition of resistance and its underlying mechanisms. ALCL cells acquired compound ALK mutations G1202R/G1269A and C1156F/L1198F in vitro at high drug concentrations. ALCL xenografts selected in vivo showed recurrent N1178H (5/10 mice) and G1269A (4/10 mice) mutations. Interestingly, intracellular localization of NPM/ALKN1178H skewed toward the cytoplasm in human cells, possibly mimicking overexpression. RNA sequencing of resistant cells showed significant alteration of PI3K/AKT and RAS/MAPK pathways. Functional validation by small-molecule inhibitors confirmed the involvement of these pathways in resistance to lorlatinib. NSCLC cells exposed in vitro to lorlatinib acquired hyperactivation of EGFR, which was blocked by erlotinib to restore sensitivity to lorlatinib. In neuroblastoma, whole-exome sequencing and proteomic profiling of lorlatinib-resistant cells revealed a truncating NF1 mutation and hyperactivation of EGFR and ErbB4. These data provide an extensive characterization of resistance mechanisms that may arise in different ALK-positive cancers following lorlatinib treatment.
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Polack, Natalie Pia. "Free radical damage to DNA and related compounds." Thesis, University of York, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.261114.
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Petrie, Kirsten. "Novel mutations causing fibrodysplasia ossificans progressiva and potential therapeutic compounds." Thesis, University of Oxford, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.711611.
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Davidson, Bruce Paul, University of Western Sydney, and School of Biological Sciences. "Compound mutations in the mammalian EGFR signalling pathway affect epidermal development, growth and viability." THESIS_XXXX_SBS_Davidson_B.xml, 1997. http://handle.uws.edu.au:8081/1959.7/518.
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The widespread expression of polypeptide growth factors from the earliest stages of embryonic development through to mature issues in the adult organism suggests an involvement in a reiterated developmental process affecting the underlying cellular growth and differentiation of many tissues. The hair follicle has taken on increased significance with the observation that many genetic mutations in these peptide growth factor genes affect its development. The targeted disruption of genes encoding members of the EpidermalGrowth Factor (EGF) and Fibroblast Growth Factor (FGF) families in the mouse has revealed a functional role for these proteins in the regulation of hair follicle growth. Experimental data and other factors are examined and results given. A second experimental system was used to determine if a functional relationship between certain peptide growth factors was conserved in the Merino sheep. The induction of a catagen-like state in the wool follicle and other epidermal changes associated with EGF treatment may be related to the transciptional induction of these peptide growth factorsDoctor of Philosophy (PhD)
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Davidson, Bruce Paul. "Compound mutations in the mammalian EGFR signalling pathway affect epidermal development, growth and viability /." View thesis, 1997. http://library.uws.edu.au/adt-NUWS/public/adt-NUWS20030826.115144/index.html.
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Harnevik, Lotta. "Molecular genetic studies on cystinuria." Doctoral thesis, Linköping : Univ, 2007. http://www.bibl.liu.se/liupubl/disp/disp2007/med1034s.pdf.
Full textBooks on the topic "Compound mutation"
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Widmark, Gun. Fornvästnordiska förleder i omljudsperspektiv: With a summary--First elements of compounds in Old West Scandinavian from the viewpoint of mutation. Uppsala: [Universitetet], 1991.
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Goldberg, Ann. Women and Men: 1760–1960. Edited by Helmut Walser Smith. Oxford University Press, 2012. http://dx.doi.org/10.1093/oxfordhb/9780199237395.013.0004.
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This article is about the power of a norm and its mutation over time: the gender role division of the private nuclear family composed of a male provider and protector, and his dependent children and homemaker wife. Those roles corresponded to rigid distinctions that were made between a male public world of work, money, and politics, on the one hand, and a female private sphere of reproduction and nurturance, on the other. These were prescribed ideals of gender. However, as such, the ideals have had tremendous power, shaping personal identity and the daily lives of men and women, as well as influencing the development of the state, civil society, politics, and the economy, according to a vast and growing scholarship. This article highlights the powerful role played by the norm of separate spheres over two centuries of German history along with the development of civil society and the welfare state.
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Sebastio, Gianfranco, Manuel Schiff, and Hélène Ogier de Baulny. Lysinuric Protein Intolerance and Hartnup Disease. Oxford University Press, 2016. http://dx.doi.org/10.1093/med/9780199972135.003.0025.
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Lysinuric protein intolerance (LPI) is an inherited aminoaciduria caused by defective cationic amino acid transport at the basolateral membrane of epithelial cells in intestine and kidney. LPI is caused by mutations in the SLC7A7 gene, which encodes the y+LAT-1 protein, the catalytic light chain subunit of a complex belonging to the heterodimeric amino acid transporter family. Symptoms usually begin after weaning with refusal of feeding, vomiting, and consequent failure to thrive. Hepatosplenomegaly, hematological anomalies, and neurological involvement including hyperammonemic coma will progressively appear. Lung involvement (specifically pulmonary alveolar proteinosis), chronic renal disease that may lead to end stage renal disease, and hemophagocytic lymphohistiocytosis with macrophage activation all represent complications of LPI that may appear at any time from childhood to adulthood. The great variability of the clinical presentation frequently causes misdiagnosis or delayed diagnosis. The basic therapy of LPI consist of a low-protein diet, low-dose citrulline supplementation, nitrogen-scavenging compounds to prevent hyperammonemia, lysine, and carnitine supplements.
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Klepper, Joerg. Glut1 Deficiency and the Ketogenic Diets. Edited by Eric H. Kossoff. Oxford University Press, 2016. http://dx.doi.org/10.1093/med/9780190497996.003.0005.
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Glucose is the essential fuel for the brain. Transport into brain is exclusively mediated by the facilitative glucose transporter Glut1. Glut1 deficiency results in a “brain energy crisis,” causing global developmental delay, epilepsy, and complex movement disorders including paroxysmal nonepileptic events. Early-onset absence epilepsy, paroxysmal exertion-induced dystonia, and stomatin-deficient cryohydrocytosis have been recognized as variants. Diagnosis is based on phenotype, isolated low CSF glucose, and mutations in the SLC2A1 gene. The condition is treated effectively by classical ketogenic diets providing ketones as an alternative fuel for the brain. The modified Atkins diet in adolescents and adults improves palatability and compliance at the expense of lower ketosis. Dietary treatment is continued into adolescence to meet the energy demand of the developing brain, raising concerns about long-term adverse effects. Current fields of research include novel compounds such as ketoesters and genetic approaches in Glut1-deficient mice as potential treatment options.
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Suman, Shankar, Shivam Priya, and Akanksha Nigam, eds. Breast Cancer: Current Trends in Molecular Research. BENTHAM SCIENCE PUBLISHERS, 2022. http://dx.doi.org/10.2174/97816810895221120101.
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Breast cancer is one of the most common cancer types worldwide, and is a leading cause of cancer related deaths in women. In this book, medical experts review our current understanding of the molecular biology and characteristics of breast cancer. The topics covered in this book provide comprehensive knowledge of mechanisms underlying breast carcinogenesis, and are intended for a wide audience including scientists, teachers, and students. 11 chapters present information about several topics on breast cancer, including the role of cell growth and proliferation pathways, androgen and cytokine signaling, germline mutations in breast cancer susceptibility genes, and molecular factors causing invasive and metastatic breast cancer. In addition, the editors discuss the recent advancements in multi-omics data analysis based on inter-and intra-tumor molecular profiles. The reference highlights how the knowledge and understanding of the biological behavior of breast neoplasms have facilitated ongoing investigations into dietary polyphenolic compounds with antioxidant properties, making them function as cancer chemopreventive agents. Along with this, the current development of treatment strategies such as targeted molecular therapy, and radiation therapy is brought to the fore to update readers.
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Zhang, Marina, Mark Dodgson, and David Gann. Demystifying China's Innovation Machine. Oxford University Press, 2021. http://dx.doi.org/10.1093/oso/9780198861171.001.0001.
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China’s extraordinary economic development is explained in large part by the way it innovates. This book explains how it innovates, which has important implications not only for China but also for the rest of the world. Contrary to widely held views, China’s innovation machine is not created and controlled by an all-powerful government. Instead, it is a complex, interdependent system composed of hundreds of millions of elements, involving bottom-up innovation driven by innovators and entrepreneurs and highly pragmatic and adaptive top-down policy. Using case studies of leading firms and industries, statistics, and policy analysis, the book argues that China’s innovation machine is similar to a natural ecosystem. Innovations in technology, organization, and business model resemble genetic mutations which are random, self-serving and isolated initially, but the best fitting are selected by the market and their impacts are amplified by the innovation machine. This machine draws on China’s massive number of manufacturers, supply chains, innovation clusters, and digitally literate population, connected through supersized digital platforms. China’s innovation suffers from a lack of basic research and reliance upon certain critical technologies from overseas; its scale (size) and scope (diversity) possess attributes that make it self-correcting and stronger in the face of challenges. China’s innovation machine is most effective in a policy environment where the market prevails; policy intervention plays a significant role when market mechanisms are premature or fail. The book concludes that the future success of China’s innovation will depend on continuing policy pragmatism, mass entrepreneurship and innovation, and the development of the ‘new infrastructures’.
Book chapters on the topic "Compound mutation"
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Belachew, Dina, Traci Kazmerski, Ingrid Libman, Amy C. Goldstein, Susan T. Stevens, Stephanie DeWard, Jerry Vockley, Mark A. Sperling, and Arcangela L. Balest. "Infantile Hypophosphatasia Secondary to a Novel Compound Heterozygous Mutation Presenting with Pyridoxine-Responsive Seizures." In JIMD Reports, 17–24. Berlin, Heidelberg: Springer Berlin Heidelberg, 2013. http://dx.doi.org/10.1007/8904_2013_217.
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Ido, Masaru, Tatsuya Hayashi, Junji Nishioka, and Koji Suzuki. "Hereditary Thrombophilia Caused by Abnormality of the Anticoagulant Protein C Pathway: Prenatal Diagnosis of Compound Heterozygous Protein C Deficiency by Direct Detection of the Mutation Sites." In Pulmonary Embolism, 21–35. Tokyo: Springer Japan, 1999. http://dx.doi.org/10.1007/978-4-431-66893-0_2.
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Schröder, J., S. Rost, R. Schwaab, H. H. Brackmann, C. R. Müller, and J. Oldenburg. "Haemophilia A in a Female Due to Compound Heterozygosity of a Maternally Inherited Point Mutation and a Paternally De Novo Large Deletion Within the Factor VIII Gene." In 29. Hämophilie-Symposion, 270–73. Berlin, Heidelberg: Springer Berlin Heidelberg, 2000. http://dx.doi.org/10.1007/978-3-642-59633-9_47.
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Maher, Veronica M., M. Chia-Miao Mah, Jia-Ling Yang, Nitai P. Bhattacharyya, and J. Justin McCormick. "Mutations and Homologous Recombination Induced by N-Substituted Aryl Compounds in Mammalian Cells." In Nitroarenes, 149–56. Boston, MA: Springer US, 1990. http://dx.doi.org/10.1007/978-1-4615-3800-4_13.
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Morita, Masashi, Shun Matsumoto, Airi Sato, Kengo Inoue, Dzmitry G. Kostsin, Kozue Yamazaki, Kosuke Kawaguchi, et al. "Stability of the ABCD1 Protein with a Missense Mutation: A Novel Approach to Finding Therapeutic Compounds for X-Linked Adrenoleukodystrophy." In JIMD Reports, 23–31. Berlin, Heidelberg: Springer Berlin Heidelberg, 2018. http://dx.doi.org/10.1007/8904_2018_118.
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Varfolomeev, Sergey, Bella Grigorenko, Sofya Lushchekina, Patrick Masson, Galina Mahaeva, Dana Novichkova, and Alexander Nemuchin. "Study and modeling of mechanisms of cholinesterasis reactions in order to improve their catalytic properties in the neutralization reactions of organophosphorus compounds." In ORGANOPHOSPHORUS NEUROTOXINS, 140–80. ru: Publishing Center RIOR, 2020. http://dx.doi.org/10.29039/23_140-180.
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“Biocleaners” or “bioscavengers” are biological objects (enzymes, catalytic antibodies) that are capable of binding and/or hydrolyzing organophosphorus compounds (OPC). Their use seems to be the most effective alternative to traditional antidotes to neutralize or detoxify OPC. The introduction of bioscavengers allows neutralizing toxicant molecules in the bloodstream before they reach their biological targets, thereby providing protection against poisoning. Bioscavengers of the first-generation neutralized OPC molecules by stoichiometrically binding to them. The safety and efficacy of human butyrylcholinesterase (BChE) for protecting against OPC poisoning has been shown. However, the stoichiometric neutralization of OPC requires the introduction of a huge amount of expensive biopharmaceuticals. Catalytic bioscavengers that hydrolytically neutralize OPC were introduced at a much lower dose to achieve the same degree of effectiveness. The most effective catalytic bioscavengers are enzymes. The most promising enzymes are artificial mammalian paraoxonase mutants and bacterial phosphotriesterases. However, studies of other enzymes, such as prolidases, oxidases, artificial mutants of cholinesterases and carboxyl esterases and catalytic antibodies are actively ongoing. Since OPC are pseudosubstrates of cholinesterases (ChEs), a detailed description of the mechanisms of inhibition, dealkylation, and spontaneous reactivation of phosphorylated ChEs is critical for the development of ChEs mutants with a high rate of hydrolysis of OPC. The review presents an analysis of different views on the mechanisms of interaction of ChEs with OPC, discusses the possible directions of creating effective catalytic biological traps based on BChE and changes in their mechanism of action as compared to the native enzyme. A separate section is devoted to the effect of mutations, both polymorphic and artificial, on the stability of the protein molecule of BChE.
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Varfolomeev, Sergey, Bella Grigorenko, Sofya Lushchekina, Patrick Masson, Galina Mahaeva, Dana Novichkova, and Alexander Nemuchin. "Study and modeling of mechanisms of cholinesterasis reactions in order to improve their catalytic properties in the neutralization reactions of organophosphorous compounds." In Organophosphorous Neurotoxins, 134–74. ru: Publishing Center RIOR, 2020. http://dx.doi.org/10.29039/chapter_5e4132b603bfc4.70818543.
Full textAbstract:
“Biocleaners” or “bioscavengers” are biological objects (enzymes, catalytic antibodies) that are capable of binding and/or hydrolyzing organophosphorus compounds (OPC). Their use seems to be the most effective alternative to traditional antidotes to neutralize or detoxify OPC. The introduction of bioscavengers allows neutralizing toxicant molecules in the bloodstream before they reach their biological targets, thereby providing protection against poisoning. Bioscavengers of the first-generation neutralized OPC molecules by stoichiometrically binding to them. The safety and efficacy of human butyrylcholinesterase (BChE) for protecting against OPC poisoning has been shown. However, the stoichiometric neutralization of OPC requires the introduction of a huge amount of expensive biopharmaceuticals. Catalytic bioscavengers that hydrolytically neutralize OPC were introduced at a much lower dose to achieve the same degree of effectiveness. The most effective catalytic bioscavengers are enzymes. The most promising enzymes are artificial mammalian paraoxonase mutants and bacterial phosphotriesterases. However, studies of other enzymes, such as prolidases, oxidases, artificial mutants of cholinesterases and carboxyl esterases and catalytic antibodies are actively ongoing. Since OPC are pseudosubstrates of cholinesterases (ChEs), a detailed description of the mechanisms of inhibition, dealkylation, and spontaneous reactivation of phosphorylated ChEs is critical for the development of ChEs mutants with a high rate of hydrolysis of OPC. The review presents an analysis of different views on the mechanisms of interaction of ChEs with OPC, discusses the possible directions of creating effective catalytic biological traps based on BChE and changes in their mechanism of action as compared to the native enzyme. A separate section is devoted to the effect of mutations, both polymorphic and artificial, on the stability of the protein molecule of BChE.
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Jacobsen, Jessie C., Whitney Whitford, Brendan Swan, Juliet Taylor, Donald R. Love, Rosamund Hill, Sarah Molyneux, et al. "Compound Heterozygous Inheritance of Mutations in Coenzyme Q8A Results in Autosomal Recessive Cerebellar Ataxia and Coenzyme Q10 Deficiency in a Female Sib-Pair." In JIMD Reports, 31–36. Berlin, Heidelberg: Springer Berlin Heidelberg, 2017. http://dx.doi.org/10.1007/8904_2017_73.
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Kawabata, Takeshi, Masayuki Oda, Nobutaka Numoto, and Fusako Kawai. "Structural and Mutational Analysis of Polyethylene Terephthalate–Hydrolyzing Enzyme, Cut190, Based on Three-Dimensional Docking Structure with Model Compounds of Polyethylene Terephthalate." In Green Polymer Chemistry: New Products, Processes, and Applications, 63–75. Washington, DC: American Chemical Society, 2018. http://dx.doi.org/10.1021/bk-2018-1310.ch005.
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Riley, Susan E., and R. Colin Garner. "The Testing of four UKEMS Trial Compounds for Bacterial Mutation in the Salmonella/Microsome Assay Using (1) Aroclor-induced Rat Liver, (2) Chicken Liver S9 and (3) The York Modification of the Standard Ames Protocol." In Comparative Genetic Toxicology, 79–83. London: Palgrave Macmillan UK, 1985. http://dx.doi.org/10.1007/978-1-349-07901-8_11.
Full textConference papers on the topic "Compound mutation"
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Li, Fachao, Tingyu Zhang, and Chenxia Jin. "A Kind of Genetic Algorithm Based on Compound Mutation Strategy and Performance Study." In 2009 5th International Conference on Wireless Communications, Networking and Mobile Computing (WiCOM). IEEE, 2009. http://dx.doi.org/10.1109/wicom.2009.5300865.
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Adam, Kristine, Cornelia Köhler, Charlotte Thiels, and Thomas Lücke. "Tyrosine Hydroxylase Deficiency due to a Compound-heterozygote Mutation with One Kwon Mutation and One Previously not Described Variant on the TH-Gen." In Abstracts of the 45th Annual Meeting of the Society for Neuropediatrics. Georg Thieme Verlag KG, 2019. http://dx.doi.org/10.1055/s-0039-1698229.
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Zhang, Xin. "Notice of Retraction: Research on Improving Keratinase Vitality of Bacillus Subtilis Based on Compound Mutation." In 2011 5th International Conference on Bioinformatics and Biomedical Engineering. IEEE, 2011. http://dx.doi.org/10.1109/icbbe.2011.5780287.
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Mammadova, D. "Cortical Blindness and Epileptic Encephalopathy Due to a Previously Unknown Compound Heterozygous DIAPH1 Gene Mutation." In Abstracts of the 46th Annual Meeting of the Society for Neuropediatrics. Georg Thieme Verlag KG, 2021. http://dx.doi.org/10.1055/s-0041-1739614.
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Sinha, Ian, Chris Ritchieson, David Heaf, and David Lacy. "A Novel Compound Heterozygote Mutation Leading To Surfactant Metabolism Dysfunction Presenting As Childhood Interstitial Lung Disease." In American Thoracic Society 2010 International Conference, May 14-19, 2010 • New Orleans. American Thoracic Society, 2010. http://dx.doi.org/10.1164/ajrccm-conference.2010.181.1_meetingabstracts.a6735.
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Wang, Lili, Rutendo Gambe, Jean Fan, Angela N. Brooks, Jing Sun, Donna Neuberg, Peter Kharchenko, et al. "Abstract 669: Compound heterozygous Sf3b1-K700E mutation and Atm deletion in B cells leads to CLL in mice." In Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA. American Association for Cancer Research, 2016. http://dx.doi.org/10.1158/1538-7445.am2016-669.
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Farias, Igor Braga, Bruno de Mattos Lombardi Badia, Gustavo Carvalho Costa, Roberta Ismael Lacerda Machado, Carolina Maria Marin, Wladimir Bocca Vieira de Rezende Pinto, Paulo Victor Sgobbi de Souza, and Acary Souza Bulle Oliveira. "Clinical and genetic profile of Brazilian patients with dysferlinopathies – A retrospective study." In XIII Congresso Paulista de Neurologia. Zeppelini Editorial e Comunicação, 2021. http://dx.doi.org/10.5327/1516-3180.054.
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Introduction: Dysferlinopathies are a group of conditions that are caused by mutations in the dysferlin gene. Objectives: To characterize the clinical phenotypes and genotypic spectrum of dysferlinopathies patients and to estimate the progression of functional and motor decline. Design and setting: Retrospective analysis of the medical records of patients followed up at our institution between 1995 and 2020. Methods: Patients were selected based on the following inclusion criteria:(i) Identification of a mutation defined as pathogenic in homozygosis or compound heterozygosis in the Dysf gene;or (ii)compatible clinical manifestations and decreased expression of dysferlin in immunohistochemistry on muscle biopsy. Classification of the phenotype was based on the first symptoms. Functionality was defined by the Gardner–Medwin & Walton(GMW) scale modified for dysferlinopathy. Results: 23 patients were included in the study. 16 were classified as limb-girdle muscular dystrophy autosomal recessive 2 (LGMDR2), 4 as Miyoshi muscular dystrophy, 2 as proximo-distal onset and 1 as asymptomatic hyperCKemia. Thighs adduction was the most affected movement in the first evaluation (mean strength=3). Plantar flexion was the movement with the greatest decline in strength(mean=-0.10 points on MRC/year;pT, Arg2042Cys and c.2643+1G>A, p.?(splicing), found 3 times each. There was no statistical difference in muscle strength in the first evaluation, motor and functional decline between the phenotypes. Conclusion: While LGMDR2 was the most common phenotype at onset, with the exception of asymptomatic hyperCKemia, there were not a clear difference in the pattern of progression between them.
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Ba Sharahil, N., M. J. Rock, P. M. Farrell, H. J. Lai, D. Pfeil, P. Modaff, M. Gajapathy, B. M. Wilk, D. M. Brown, and E. A. Worthey. "Identification of Compound Heterozygous Mutation in HFE Gene in a Child with Cystic Fibrosis: A Case Report with Data on HFE Genetic Variants." In American Thoracic Society 2021 International Conference, May 14-19, 2021 - San Diego, CA. American Thoracic Society, 2021. http://dx.doi.org/10.1164/ajrccm-conference.2021.203.1_meetingabstracts.a3375.
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Pelish, Henry E., Anupong Tangpeerachaikul, Nancy E. Kohl, James R. Porter, Matthew D. Shair, and Joshua C. Horan. "Abstract 1468: NUV-655 (NVL-655) is a selective, brain-penetrant ALK inhibitor with antitumor activity against the lorlatinib-resistant G1202R/L1196M compound mutation." In Proceedings: AACR Annual Meeting 2021; April 10-15, 2021 and May 17-21, 2021; Philadelphia, PA. American Association for Cancer Research, 2021. http://dx.doi.org/10.1158/1538-7445.am2021-1468.
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van Dorland, H. A., M. M. Taleghani, K. D. Friedman, J. N. George, I. Hrachovinova, P. N. Knöbl, M. Matsumoto, et al. "More severe ADAMTS13 Deficiency in Homozygous versus Compound Heterozygous Carriers of the ADAMTS13 c.4143_4144dupA Mutation in Congenital Thrombotic Thrombocytopenic Purpura (cTTP): Impact on Disease Onset?" In 63rd Annual Meeting of the Society of Thrombosis and Haemostasis Research. Georg Thieme Verlag KG, 2019. http://dx.doi.org/10.1055/s-0039-1680105.
Full textReports on the topic "Compound mutation"
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Ori, Naomi, and Mark Estelle. Role of GOBLET and Auxin in Controlling Organ Development and Patterning. United States Department of Agriculture, January 2012. http://dx.doi.org/10.32747/2012.7697122.bard.
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The size and shape of plant leaves are extremely diverse within and among species, and are also sensitive to growth conditions. Compound leaves, such as those of tomato, maintain morphogenetic activity during early stages of their development, enabling them to elaborate lateral appendages such as leaflets. The aim of the research project was to understand the interaction between the plant hormone auxin, the putative auxin response inhibitor ENTIRE (E, SlIAA9) and the NAM/CUC transcription factor GOBLET (GOB) in compound-leaf development in tomato (Solanum lycopersicum). The specific aims of the project were: 1. Investigation of the role of GOB in compound-leaf development. 2. Characterization of E function in auxin signaling. 3. Characterization of the role of auxin in compound-leaf development. 4. Investigation of the genetic and molecular interaction between E and GOB. 5. Investigate the role of these factors in fruit development. There were no major changes in these objectives. GOB was shown to mark and promote the boundaries between the leaf and initiating leaflets. Its accurate distribution was found to be required for proper leaflet initiation and separation. E was found to interact with the TIR1 and AFB6 proteins in an auxin-dependant manner, indicating that these are functional auxin receptors that mediate E degradation in the presence of auxin. This was further supported by the stabilization of E by a mutation in domain II of the protein, which is thought to mediate its auxin-dependant degradation. Over expression of this stabilized form in tomato leaves and characterization of the e mutant phenotype and the E expression domain indicated that E acts between initiating leaflets to inhibit auxin response and lamina growth. Generation and analysis of tomato plants expressing the auxin response reporter DR5::VENUS, and analysis of the effect of auxin microapplication or overexpression of an auxin biosynthesis gene, indicated that auxin marks the sites of leaflet initiation and promotes lamina growth. Investigation of the molecular and genetic interaction between auxin, GOB and E revealed a complex network of mutual regulation that is utilized to precisely pattern the leaf margin in a manner that enables the combination of tight control and flexibility. E, auxin and GOB were shown to affect fruit development and fruit set, and in an extension of the project are currently utilized to identify new players that affect these processes. The research project yielded enhanced understanding of the mechanisms of compound leaf patterning and provided tools that will enable the manipulation of leaf shape and fruit set.
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Ori, Naomi, and Mark Estelle. Specific mediators of auxin activity during tomato leaf and fruit development. United States Department of Agriculture, January 2012. http://dx.doi.org/10.32747/2012.7597921.bard.
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The plant hormone auxin is involved in numerous developmental processes, including leaf and fruit development. The tomato (Solanumlycopersicum) gene ENTIRE (E) encodes an auxin-response inhibitor from the Aux/IAA family. While most loss-offunction mutations in Aux/IAA genes are similar to the wild type due to genetic redundancy, entire (e) mutants show specific effects on leaf and fruit development. e mutants have simple leaves, in contrast to the compound leaves of wild type tomatoes. In addition, e plants produce parthenocarpic fruits, in which fruit set occurs independently of fertilization. The aim of this research program was to utilize the e mutation to identify and characterize genes that mediate the specific effect of auxin in leaf and fruit development. The specific objectives of the project were to: 1. Characterize and map modifiers of the e leaf phenotype. 2. Characterize and map suppressors of the e fruit phenotype. 3. Dissect the developmental specificity of the E gene. 4. Examine the effect of fruit-overexpression of identified genes on fruit set and seed production. To identify mediators of auxin in leaf development, we mainly focused on one mutant, crawling elephant (crel, previously called t282), which showed substantial suppression of the e phenotype and other auxin-relatedphenotypes. We have identified the CREL gene as a homolog of the Arabidopsis VRN5 gene, involved in recruiting polycomb silencing complexes to specific targets. We showed that CREL affects auxin sensitivity in tomato. Suppressors of the e fruit phenotype have been further characterized and selected for more profound effects. Expression profiling by RNAseq was used to analyze the effect of e as well as crel on gene expression in leaves and fruits. This analysis has identified putative E and CREL targets. We have initiated studies to assess the role of some of these targets in flower and fruit development. The research has identified potential mediators of auxin response in leaf, flower and fruit development.
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Sela, Shlomo, and Michael McClelland. Investigation of a new mechanism of desiccation-stress tolerance in Salmonella. United States Department of Agriculture, January 2013. http://dx.doi.org/10.32747/2013.7598155.bard.
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Low-moisture foods (LMF) are increasingly involved in foodborne illness. While bacteria cannot grow in LMF due to the low water content, pathogens such as Salmonella can still survive in dry foods and pose health risks to consumer. We recently found that Salmonella secretes a proteinaceous compound during desiccation, which we identified as OsmY, an osmotic stress response protein of 177 amino acids. To elucidate the role of OsmY in conferring tolerance against desiccation and other stresses in Salmonella entericaserovarTyphimurium (STm), our specific objectives were: (1) Characterize the involvement of OsmY in desiccation tolerance; (2) Perform structure-function analysis of OsmY; (3) Study OsmY expression under various growth- and environmental conditions of relevance to agriculture; (4) Examine the involvement of OsmY in response to other stresses of relevance to agriculture; and (5) Elucidate regulatory pathways involved in controlling osmY expression. We demonstrated that an osmY-mutant strain is impaired in both desiccation tolerance (DT) and in long-term persistence during cold storage (LTP). Genetic complementation and addition of a recombinantOsmY (rOsmY) restored the mutant survival back to that of the wild type (wt). To analyze the function of specific domains we have generated a recombinantOsmY (rOsmY) protein. A dose-response DT study showed that rOsmY has the highest protection at a concentration of 0.5 nM. This effect was protein- specific as a comparable amount of bovine serum albumin, an unrelated protein, had a three-time lower protection level. Further characterization of OsmY revealed that the protein has a surfactant activity and is involved in swarming motility. OsmY was shown to facilitate biofilm formation during dehydration but not during bacterial growth under optimal growth conditions. This finding suggests that expression and secretion of OsmY under stress conditions was potentially associated with facilitating biofilm production. OsmY contains two conserved BON domains. To better understand the role of the BON sites in OsmY-mediated dehydration tolerance, we have generated two additional rOsmY constructs, lacking either BON1 or BON2 sites. BON1-minus (but not BON2) protein has decreased dehydration tolerance compared to intact rOsmY, suggesting that BON1 is required for maximal OsmY-mediated activity. Addition of BON1-peptide at concentration below 0.4 µM did not affect STm survival. Interestingly, a toxic effect of BON1 peptide was observed in concentration as low as 0.4 µM. Higher concentrations resulted in complete abrogation of the rOsmY effect, supporting the notion that BON-mediated interaction is essential for rOsmY activity. We performed extensive analysis of RNA expression of STm undergoing desiccation after exponential and stationary growth, identifying all categories of genes that are differentially expressed during this process. We also performed massively in-parallel screening of all genes in which mutation caused changes in fitness during drying, identifying over 400 such genes, which are now undergoing confirmation. As expected OsmY is one of these genes. In conclusion, this is the first study to identify that OsmY protein secreted during dehydration contributes to desiccation tolerance in Salmonella by facilitating dehydration- mediated biofilm formation. Expression of OsmY also enhances swarming motility, apparently through its surfactant activity. The BON1 domain is required for full OsmY activity, demonstrating a potential intervention to reduce pathogen survival in food processing. Expression and fitness screens have begun to elucidate the processes of desiccation, with the potential to uncover additional specific targets for efforts to mitigate pathogen survival in desiccation.
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Chamovitz, Daniel A., and Zhenbiao Yang. Chemical Genetics of the COP9 Signalosome: Identification of Novel Regulators of Plant Development. United States Department of Agriculture, January 2011. http://dx.doi.org/10.32747/2011.7699844.bard.
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This was an exploratory one-year study to identify chemical regulators of the COP9 signalosome. Chemical Genetics uses small molecules to modify or disrupt the function of specific genes/proteins. This is in contrast to classical genetics, in which mutations disrupt the function of genes. The underlying concept is that the functions of most proteins can be altered by the binding of a chemical, which can be found by screening large libraries for compounds that specifically affect a biological, molecular or biochemical process. In addition to screens for chemicals which inhibit specific biological processes, chemical genetics can also be employed to find inhibitors of specific protein-protein interactions. Small molecules altering protein-protein interactions are valuable tools in probing protein-protein interactions. In this project, we aimed to identify chemicals that disrupt the COP9 signalosome. The CSN is an evolutionarily conserved eight-subunit protein complex whose most studied role is regulation of E3 ubiquitinligase activity. Mutants in subunits of the CSN undergo photomorphogenesis in darkness and accumulate high levels of pigments in both dark- and light-grown seedlings, and are defective in a wide range of important developmental and environmental-response pathways. Our working hypothesis was that specific molecules will interact with the CSN7 protein such that binding to its various interacting proteins will be inhibited. Such a molecule would inhibit either CSN assembly, or binding of CSN-interacting proteins, and thus specifically inhibit CSN function. We used an advanced chemical genetic screen for small-molecule-inhibitors of CSN7 protein-protein interactions. In our pilot study, following the screening of ~1200 unique compounds, we isolated four chemicals which reproducibly interfere with CSN7 binding to either CSN8 or CSN6.
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Shai, Yechiel, Arthur Aronson, Aviah Zilberstein, and Baruch Sneh. Study of the Basis for Toxicity and Specificity of Bacillus thuringiensis d-Endotoxins. United States Department of Agriculture, January 1996. http://dx.doi.org/10.32747/1996.7573995.bard.
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The report contains three parts which summarizes the three years achievements of the three participating research groups; The Weizmann group, Tel-Aviv group and Purdue group. The firs part describes the achievements obtained by Shai's group toward the elucidation of the mechanism of membrane insertion and the structural organization of the pores formed by the Cry3A and Cry1Ac B. thuringiensis d-endotoxins. For that purpose Shai's group synthesized, fluorescently labeled and structurally and functionally characterized peptides corresponding to the seven helices that compose the pore-forming domain of Cry3A toxin, including mutants peptides and the hairpin a4G-a5 of both Cry3A and Cry 1Ac toxins composed of a4, a5 and the loop connecting a4-a5. Among the synthesized peptides were three mutated a4 helices based on site directed mutagenesis done at Aronson's group that decreased or increased Cry 1Ac toxicity. The results of these studies are consistent with a situation in which only helices a4 anda5 insert into the membrane as a helical hairpin in an antiparallel manner, while the other helices lie on the membrane surface like ribs of an umbrella (the "umbrella model"). In order to test this model Shai's group synthesized the helical hairpin a4<-->a5 of both Cry3A and Cry 1 Ac toxins, as well. Initial functional and structural studies showed direct correlation between the properties of the mutated helices and the mutated Cry1Ac. Based on Shai's findings that a4 is the second helix besides a5 that insert into the membrane, Aronson and colleagues performed extensive mutation on this helix in the CrylAc toxin, as well as in the loop connecting helices 4 and 5, and helix 3 (part two of the report). In addition, Aronson performed studies on the effect of mutations or type of insect which influence the oligomerization either the Cry 1Ab or Cry 1Ac toxins with vesicles prepared from BBMV. In the third part of the report Zilberstein's and Sneh's groups describe their studies on the three domains of Cry 1C, Cry 1E and crylAc and their interaction with the epithelial membrane of the larval midgut. In these studies they cloned all three domains and combinations of two domains, as well as cloning of the pore forming domain alone and studying its interaction with BBMV. In addition they investigated binding of Cry1E toxin and Cry1E domains to BBMV prepared from resistant (R) or sensitive larvae. Finally they initiated expression of the loop a4G<-->a5 Cry3A in E. coli to be compared with the synthetic one done by Shai's group as a basis to develop a system to express all possible pairs for structural and functional studies by Shai's group (together with Y. Shai).
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Rafaeli, Ada, and Russell Jurenka. Molecular Characterization of PBAN G-protein Coupled Receptors in Moth Pest Species: Design of Antagonists. United States Department of Agriculture, December 2012. http://dx.doi.org/10.32747/2012.7593390.bard.
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The proposed research was directed at determining the activation/binding domains and gene regulation of the PBAN-R’s thereby providing information for the design and screening of potential PBAN-R-blockers and to indicate possible ways of preventing the process from proceeding to its completion. Our specific aims included: (1) The identification of the PBAN-R binding domain by a combination of: (a) in silico modeling studies for identifying specific amino-acid side chains that are likely to be involved in binding PBAN with the receptor and; (b) bioassays to verify the modeling studies using mutant receptors, cell lines and pheromone glands (at tissue and organism levels) against selected, designed compounds to confirm if compounds are agonists or antagonists. (2) The elucidation ofthemolecular regulationmechanisms of PBAN-R by:(a) age-dependence of gene expression; (b) the effect of hormones and; (c) PBAN-R characterization in male hair-pencil complexes. Background to the topic Insects have several closely related G protein-coupled receptors (GPCRs) belonging to the pyrokinin/PBAN family, one with the ligand pheromone biosynthesis activating neuropeptide or pyrokinin-2 and another with diapause hormone or pyrokinin-1 as a ligand. We were unable to identify the diapause hormone receptor from Helicoverpa zea despite considerable effort. A third, related receptor is activated by a product of the capa gene, periviscerokinins. The pyrokinin/PBAN family of GPCRs and their ligands has been identified in various insects, such as Drosophila, several moth species, mosquitoes, Triboliumcastaneum, Apis mellifera, Nasoniavitripennis, and Acyrthosiphon pisum. Physiological functions of pyrokinin peptides include muscle contraction, whereas PBAN regulates pheromone production in moths plus other functions indicating the pleiotropic nature of these ligands. Based on the alignment of annotated genomic sequences, the primary and secondary structures of the pyrokinin/PBAN family of receptors have similarity with the corresponding structures of the capa or periviscerokinin receptors of insects and the neuromedin U receptors found in vertebrates. Major conclusions, solutions, achievements Evolutionary trace analysisof receptor extracellular domains exhibited several class-specific amino acid residues, which could indicate putative domains for activation of these receptors by ligand recognition and binding. Through site-directed point mutations, the 3rd extracellular domain of PBAN-R was shown to be critical for ligand selection. We identified three receptors that belong to the PBAN family of GPCRs and a partial sequence for the periviscerokinin receptor from the European corn borer, Ostrinianubilalis. Functional expression studies confirmed that only the C-variant of the PBAN-R is active. We identified a non-peptide agonist that will activate the PBAN-receptor from H. zea. We determined that there is transcriptional control of the PBAN-R in two moth species during the development of the pupa to adult, and we demonstrated that this transcriptional regulation is independent of juvenile hormone biosynthesis. This transcriptional control also occurs in male hair-pencil gland complexes of both moth species indicating a regulatory role for PBAN in males. Ultimate confirmation for PBAN's function in the male tissue was revealed through knockdown of the PBAN-R using RNAi-mediated gene-silencing. Implications, both scientific and agricultural The identification of a non-peptide agonist can be exploited in the future for the design of additional compounds that will activate the receptor and to elucidate the binding properties of this receptor. The increase in expression levels of the PBAN-R transcript was delineated to occur at a critical period of 5 hours post-eclosion and its regulation can now be studied. The mysterious role of PBAN in the males was elucidated by using a combination of physiological, biochemical and molecular genetics techniques.
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McClure, Michael A., Yitzhak Spiegel, David M. Bird, R. Salomon, and R. H. C. Curtis. Functional Analysis of Root-Knot Nematode Surface Coat Proteins to Develop Rational Targets for Plantibodies. United States Department of Agriculture, October 2001. http://dx.doi.org/10.32747/2001.7575284.bard.
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The goal of this research was to provide a better understanding of the interface between root-knot nematodes, Meloidogyne spp., and their host in order to develop rational targets for plantibodies and other novel methods of nematode control directed against the nematode surface coat (SC). Specific objectives were: 1. To produce additional monoclonal SC antibodies for use in Objectives 2, 3, and 4 and as candidates for development of plantibodies. 2. To determine the production and distribution of SC proteins during the infection process. 3. To use biochemical and immunological methods to perturbate the root-knot nematode SC in order to identify SC components that will serve as targets for rationally designed plantibodies. 4. To develop SC-mutant nematodes as additional tools for defining the role of the SC during infection. The external cuticular layer of nematodes is the epicuticle. In many nematodes, it is covered by a fuzzy material termed "surface coat" (SC). Since the SC is the outermost layer, it may playa role in the interaction between the nematode and its surroundings during all life stages in soil and during pathogenesis. The SC is composed mainly of proteins, carbohydrates (which can be part of glycoproteins), and lipids. SC proteins and glycoproteins have been labeled and extracted from preparasitic second-stage juveniles and adult females of Meloidogyne and specific antibodies have been raised against surface antigens. Antibodies can be used to gain more information about surface function and to isolate genes encoding for surface antigens. Characterization of surface antigens and their roles in different life-stages may be an important step towards the development of alternative control. Nevertheless, the role of the plant- parasitic nematode's surface in plant-nematode interaction is still not understood. Carbohydrates or carbohydrate-recognition domains (CROs) on the nematode surface may interact with CROs or carbohydrate molecules, on root surfaces or exudates, or be active after the nematode has penetrated into the root. Surface antigens undoubtedly play an important role in interactions with microorganisms that adhere to the nematodes. Polyclonal (PC) and monoclonal (MC) antibodies raised against Meloidogyne javanica, M. incognita and other plant-parasitic nematodes, were used to characterize the surface coat and secreted-excreted products of M. javanica and M. incognita. Some of the MC and PC antibodies raised against M. incognita showed cross-reactivity with the surface coat of M. javanica. Further characterization, in planta, of the epitopes recognized by the antibodies, showed that they were present in the parasitic juvenile stages and that the surface coat is shed during root penetration by the nematode and its migration between root cells. At the molecular level, we have followed two lines of experimentation. The first has been to identify genes encoding surface coat (SC) molecules, and we have isolated and characterized a small family of mucin genes from M. incognita. Our second approach has been to study host genes that respond to the nematode, and in particular, to the SC. Our previous work has identified a large suite of genes expressed in Lycopersicon esculentum giant cells, including the partial cDNA clone DB#131, which encodes a serine/threonine protein kinase. Isolation and predicted translation of the mature cDNA revealed a frame shift mutation in the translated region of nematode sensitive plants. By using primers homologous to conserved region of DB#131 we have identified the orthologues from three (nematode-resistant) Lycopersicon peruvianum strains and found that these plants lacked the mutation.
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Horwitz, Benjamin, and Nicole M. Donofrio. Identifying unique and overlapping roles of reactive oxygen species in rice blast and Southern corn leaf blight. United States Department of Agriculture, January 2017. http://dx.doi.org/10.32747/2017.7604290.bard.
Full textAbstract:
Plants and their fungal pathogens both produce reactive oxygen species (ROS). CytotoxicROS act both as stressors and signals in the plant-fungal interaction. In biotrophs, a compatible interaction generates little ROS, but is followed by disease. An incompatible interaction results in a strong oxidative burst by the host, limiting infection. Necrotrophs, in contrast, thrive on dead and dying cells in an oxidant-rich local environment. Rice blast, Magnaportheoryzae, a hemibiotroph, occurs worldwide on rice and related hosts and can decimate enough rice each year to feed sixty million people. Cochliobolusheterostrophus, a necrotroph, causes Southern corn leaf blight (SLB), responsible for a major epidemic in the 1970s. The objectives of our study of ROS signaling and response in these two cereal pathogens were: Confocal imaging of ROS production using genetically encoded redox sensor in two pathosystems over time. Forward genetic screening of HyPer sensor lines in two pathosystems for fungal genes involved in altered ROSphenotypes. RNA-seq for discovery of genes involved in ROS-related stress and signaling in two pathosystems. Revisions to the research plan: Library construction in SLB was limited by low transformation efficiency, compounded by a protoplasting enzyme being unavailable during most of year 3. Thus Objective 2 for SLB re-focused to construction of sensor lines carrying deletion mutations in known or candidate genes involved in ROS response. Imaging on rice proved extremely challenging, so mutant screening and imaging were done with a barley-infecting line, already from the first year. In this project, ROS imaging at unprecedented time and spatial resolution was achieved, using genetically-encoded ratio sensors in both pathogens. This technology is currently in use for a large library of rice blast mutants in the ROS sensor background, and Southern corn leaf blight mutants in final stages of construction. The imaging methods developed here to follow the redox state of plant pathogens in the host tissue should be applicable to fungal pathogens in general. Upon completion of mutant construction for SCLB we hope to achieve our goal of comparison between intracellular ROS status and response in hemibiotroph and necrotroph cereal pathogens.
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Jander, Georg, and Daniel Chamovitz. Investigation of growth regulation by maize benzoxazinoid breakdown products. United States Department of Agriculture, January 2015. http://dx.doi.org/10.32747/2015.7600031.bard.
Full textAbstract:
Introduction Previous research had suggested that benzoxazinoids, a class of defensive metabolites found in maize, wheat, rye, and wild barley, are not only direct insect deterrents, but also influence other areas of plant metabolism. In particular, the benzoxazinoid 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxa- zin-3(4H)- one (DIMBOA) was implicated in: (i) altering plant growth by interfering with auxin signaling, and (ii) leading to the induction of gene expression changes and secondary plant defense responses. The overall goal of this proposal was to identify mechanisms by which benzoxazinoids influence other aspects of plant growth and defense. Specifically, the following hypotheses were proposed to be tested as part of an approved BARD proposal: Benzoxazinoid breakdown products directly interfere with auxin perception Global changes in maize and barley gene expression are induced by benzoxazinoid activation. There is natural variation in the maize photomorphogenic response to benzoxazinoids. Although the initial proposal included experiments with both maize and barley, there were some technical difficulties with the proposed transgenic barley experiments and most of the experimental results were generated with maize. Summary of major findings Previous research by other labs, involving both maize and other plant species, had suggested that DIMBOA alters plant growth by interfering with auxin signaling. However, experiments conducted in both the Chamovitz and the Jander labs using Arabidopsis and maize, respectively, were unable to confirm previously published reports of exogenously added DIMBOA effects on auxin signaling. Nevertheless, analysis of bx1 and bx2 maize mutant lines, which have almost no detectable benzoxazinoids, showed altered responses to blue light signaling. Transcriptomic analysis of maize mutant lines, variation in inbred lines, and responses to exogenously added DIMBOA showed alteration in the transcription of a blue light receptor, which is required for plant growth responses. This finding provides a novel mechanistic explanation of the trade-off between growth and defense that is often observed in plants. Experiments by the Jander lab and others had demonstrated that DIMBOA not only has direct toxicity against insect pests and microbial pathogens, but also induces the formation of callose in both maize and wheat. In the current project, non-targeted metabolomic assays of wildtype maize and mutants with defects in benzoxazinoid biosynthesis were used to identify unrelated metabolites that are regulated in a benzoxazinoid-dependent manner. Further investigation identified a subset of these DIMBOA-responsive compounds as catechol, as well as its glycosylated and acetylated derivatives. Analysis of co-expression data identified indole-3-glycerol phosphate synthase (IGPS) as a possible regulator of benzoxazinoid biosynthesis in maize. In the current project, enzymatic activity of three predicted maize IGPS genes was confirmed by heterologous expression. Transposon knockout mutations confirmed the function of the maize genes in benzoxazinoid biosynthesis. Sub-cellular localization studies showed that the three maize IGPS proteins are co-localized in the plastids, together with BX1 and BX2, two previously known enzymes of the benzoxazinoid biosynthesis pathway. Implications Benzoxazinoids are among the most abundant and effective defensive metabolites in maize, wheat, and rye. Although there is considerable with-in species variation in benzoxazinoid content, very little is known about the regulation of this variation and the specific effects on plant growth and defense. The results of this research provide further insight into the complex functions of maize benzoxazinoids, which are not only toxic to pests and pathogens, but also regulate plant growth and other defense responses. Knowledge gained through the current project will make it possible to engineer benzoxazinoid biosynthesis in a more targeted manner to produce pest-tolerant crops without negative effects on growth and yield.