Implications On Drug Resistance and Survival of ABCB1 Single Nucleotide Polymorphisms in Normal Karyotype De Novo AML.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2648-2648
Author(s):  
Christer Paul ◽  
Henrik Green ◽  
Ingrid Jakobsen Falk ◽  
Kourosh Lotfi ◽  
Esbjorn Paul ◽  
...  
Keyword(s):  
Drug Sensitivity ◽  
De Novo ◽  
Normal Karyotype ◽  
University Hospital ◽  
Leukemic Cells ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
The Mean ◽  
De Novo Aml

Abstract Abstract 2648 Poster Board II-624 Background: Multidrug resistance and expression of the ATP-dependent drug transporting protein ABCB1 is a clinically relevant problem in the treatment of acute myeloid leukaemia. Several single nucleotide polymorphisms (SNPs) in the ABCB1 have been associated with altered P-glycoprotein expression and phenotype. These SNPs might influence the clinical outcome in AML and predict individual differences in response to therapy with ABCB1 substrates. Aims: To investigate the impact of the ABCB1 SNPs in exon 11, 12, 21 and 26 on treatment response, survival and in vitro drug sensitivity in AML patients. Methods: PCR and Pyrosequencing were used to determine the genotype of the SNPs G1199T/A, C1236T, A1308G, G2677T/A and C3435T in 100 de novo AML patients with normal karyotype treated at Linköping University Hospital or Karolinska University Hospital. Almost all patients were treated with one anthracycline and Ara-C during the induction regime. The affect of the genetic variants in ABCB1 on survival were analysed by Kaplan-Meier Log-rank tests and multivariant analysis by Cox regression. Patients receiving transplantation were censored at that point in the analysis. A Nordic reference material of 400 healthy volunteers of equal age and sex distribution was also included. NPM1 and FLT3-ITD mutations were determined by PCR. Leukemic cells were isolated and drug sensitivity was measured after 4 days culturing by a bioluminescence ATP-assay. Results: The survival of the AML patients was significantly correlated to the ABCB1 genotypes C1236T (P=0.02) and G2677T (P=0.02), with a borderline significance for G1199A (p=0.06). For the C1236T SNP the mean survival was 0.7, 1.3 and 1.8 years for the wild type, heterozygous and homozygous variants, respectively. The mean survival for patients with G/G, G/T and T/T genotype of SNP G2677T/A was 0.7, 1.2 and 1.7 years, respectively. Only the wild type of A1308T was found in the material and C3435T did not correlate to survival. Multivariate analysis showed that 1236T/T and 2677T/T were independent factors for survival (hazard ratio 0.24 and 0.22). Comparison of allele frequencies between AML patients and healthy volunteers showed no significant difference. In vitro testing showed that leukemic cells from patients carrying 1236T/T or 2677T/T were significantly more susceptible for mitoxantrone and borderline susceptible to daunorubicine and etoposide, substrates for Pgp but not to Ara-C. Conclusions: Our findings suggest that ABCB1 SNPs do not affect the development of the disease but the survival after chemotherapy possibly by impact on drug sensitivity. The correlation between ABCB1 genotype and the overall survival of AML patients might provide useful information for treatment strategies and individualized chemotherapy. Disclosures: Paul: Aprea AB: Consultancy, Research Funding.

Expression of p14ARF in De Novo AML with Normal Karyotype. Implication on Drug Resistance and Survival.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4261-4261
Author(s):  
Esbjorn O. Paul ◽  
Stefan Deneberg ◽  
Soren Lehmann ◽  
Sofia Bengtzén ◽  
Hareth Nahi
Keyword(s):  
Drug Resistance ◽  
De Novo ◽  
P53 Protein ◽  
Normal Karyotype ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Low Levels ◽  
De Novo Aml ◽  
Vitro Effect

Abstract The p53 protein exerts a significant role in growth control of cells and impaired function of p53 by mutation or otherwise is regarded to be important for development of many human cancers. The p53 protein is strongly regulated by the E3-ligase HDM-2 that specifically binds to p53 and causes proteosomal degradation. P14ARF encoded from the INK 4a/ARF gene locus of chromosome 9p, activates the p53 pathway by binding to and inhibiting HDM-2. The objective was to study if the level of mRNA for p14ARF in leukemic cells from patients with AML was related to in vitro drug resistance and clinical outcome. Leukemic cells (>90% pure) isolated from 46 adult patients with normal karyotype de novo AML were incubated with antileukemic drugs (daunorubicin, mitoxantrone, etoposide and Ara-C) cultured for 4 days. The effect was determined by bioluminiscence measuring of intracellular ATP compared to an untreated control. We also tested the activity against PRIMA (p53-dependent reactivation and induction of massive apoptosis), a novel, small molecule shown to activate down regulated p53. mRNA for p14ARF was determined by real time PCR. Results: Patients whose leukemic cells expressed high level of p14ARF mRNA in the leukemic cells (≥0.2 compared to the housekeeping gene) had a significantly better survival compared to those with low level (<0.02). Median survival not obtained compared to 9 months. The mean activity of all tested conventional antileukemic drugs was higher on leukemic cell samples expressing p14ARF mRNA ≥0.2 compared to those with low levels. In contrast, PRIMA exerted significantly higher in vitro effect on leukemic cell samples with low levels of p14ARF mRNA. We conclude that low levels of p14ARF in leukemic cells from patients with normal karyotype AML is a marker for poor prognosis, which may depend on impaired p53 activity causing resistance against conventional antileukemic drugs. Treatment with p53 targeting drugs as PRIMA may be a future possibility to improve the outcome for these patients.

Low Expression of p14ARF in De Novo AML with Normal Karyotype Is Associated with Short Survival but Increased in Vitro Sensitivity to p53 Modulating Drugs

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2937-2937
Author(s):  
Esbjörn Paul ◽  
Bertil Uggla ◽  
Kals G Wiman ◽  
Christer Paul ◽  
Soren Lehmann ◽  
...  
Keyword(s):  
De Novo ◽  
P53 Protein ◽  
Statistical Significance ◽  
Normal Karyotype ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Prognostic Impact ◽  
Future Possibility ◽  
De Novo Aml

Abstract Background: The p53 protein is strongly regulated by the E3 ligase HDM-2, which specifically binds to p53 and causes proteosomal degradation. The p14ARF protein activates p53 by binding to and inhibiting HDM-2. Design and methods: To further study the prognostic impact of p14ARF in AML, leukemic cells from 57 adult patients with normal karyotype de novo AML were analysed for p14ARF mRNA expression level using real-time PCR. We also measured the cytotoxicity against conventional cytostatics and PRIMA-1, a novel small molecule shown to activate mutated p53 (Nature Medicine2002; 8: 282–288) using an ATP-assay. Intracellular p53 protein was measured by FACS after incubation with PRIMA-1 in combination with the HDM-2 blocking agent RITA (Nature Medicine2004; 10: 1321–1328) Results: Patients whose cells expressed more p14ARF mRNA than the 75th percentile (0.26) showed significantly better survival compared with those with lower levels, 61% vs. 30% 3-year survival (p=0.033). The difference remained significant also when NPM1/FLT3 status was considered. The mean effects of all tested conventional antileukemic drugs were greater in leukemic cell samples expressing p14ARF mRNA ≥0.26, but the differences did not reach statistical significance. In contrast, PRIMA-1 had a significantly greater effect on leukemic cell samples with low levels of p14ARF mRNA and PRIMA-1 and RITA significantly increased intracellular p53 levels. Conclusions: Low level of p14ARF mRNA in leukemic cells from patients with normal karyotype AML is a strong marker for poor prognosis and decreased sensitivity to conventional cytostatics. Treatment with drugs targeting p53 can be a future possibility to improve the outcome for these patients.

scholarly journals Genomic Epidemiology of a Protracted Hospital Outbreak Caused by a Toxin A-Negative Clostridium difficile Sublineage PCR Ribotype 017 Strain in London, England

2015 ◽  
Vol 53 (10) ◽  
pp. 3141-3147 ◽  
Author(s):  
M. D. Cairns ◽  
M. D. Preston ◽  
T. D. Lawley ◽  
T. G. Clark ◽  
R. A. Stabler ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
De Novo ◽  
University Hospital ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Conjugative Transposon ◽  
Content Type ◽  
Genomic Epidemiology ◽  
Hospital Outbreak ◽  
Nosocomial Diarrhea

Clostridium difficileremains the leading cause of nosocomial diarrhea worldwide, which is largely considered to be due to the production of two potent toxins: TcdA and TcdB. However, PCR ribotype (RT) 017, one of five clonal lineages of human virulentC. difficile, lacks TcdA expression but causes widespread disease. Whole-genome sequencing was applied to 35 isolates from hospitalized patients withC. difficileinfection (CDI) and two environmental ward isolates in London, England. The phylogenetic analysis of single nucleotide polymorphisms (SNPs) revealed a clonal cluster of temporally variable isolates from a single hospital ward at University Hospital Lewisham (UHL) that were distinct from other London hospital isolates.De novoassembled genomes revealed a 49-kbp putative conjugative transposon exclusive to this hospital clonal cluster which would not be revealed by current typing methodologies. This study identified three sublineages ofC. difficileRT017 that are circulating in London. Similar to the notorious RT027 lineage, which has caused global outbreaks of CDI since 2001, the lineage of toxin-defective RT017 strains appears to be continually evolving. By utilization of WGS technologies to identify SNPs and the evolution of clonal strains, the transmission of outbreaks caused by near-identical isolates can be retraced and identified.

Identification and Effects of Novel Promoter Region Haplotypes in the Human Equilibrative Nucleoside Transporter, hENT1.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2083-2083
Author(s):  
Scott N. Myers ◽  
Rakesh K. Goyal ◽  
Jennifer D. Roy ◽  
Robert E. Ferrell
Keyword(s):  
De Novo ◽  
Remission Rate ◽  
Leukemic Cells ◽  
Luciferase Reporter ◽  
Luciferase Expression ◽  
Nucleoside Transporter ◽  
Nucleotide Polymorphisms ◽  
Flanking Sequence ◽  
Equilibrative Nucleoside Transporter

Abstract Front-line induction chemotherapy regimens containing cytosine arabinoside (Ara-C) and anthracyclines result in 80% complete remission rate in childhood acute myeloid leukemia (AML) but their cure rate is about 35 – 50%, one of the lowest of all childhood cancers. Understanding the factors that contribute to emergence of chemoresistant leukemic cells is crucial to improving treatment outcome in children with AML. We are interested in studying the role of variation in Ara-C transport and biotransformation pathway genes in the efficacy and toxicity of treatment of childhood AML. To permeate the cell membrane, Ara-C is mainly dependent on human equilibrative nucleoside transporter 1 (hENT1; SLC29A1; gene localized to 6p21.1). Several studies have suggested an important role for altered levels of hENT1 in the chemosensitivity of AML blasts to Ara-C (Galmarini et al. Leukemia2001; 15(6):87; Gati et al. Leuk Lymphoma1998; 32(1–2):45). Osato and colleagues identified two single nucleotide polymorphisms (SNPs) in the hENT1 coding sequence that led to missense changes, but their in vitro analysis did not detect differences in the activity of variant alleles in a yeast transfection system (Osato et al. Pharmacogenetics2003;13(5):297). To identify variation in hENT1 that might influence its expression, we sequenced 1.6Kb of the proximal 5′-flanking sequence of the gene in 42 unrelated individuals and identified three SNPs at positions C-1345G, G-1050A, and G-706C. TRANSFAC analysis (www.genomatix.de) predicted that two of these (C-1345G & G-706C) would alter consensus transcription factor binding site sequences. We cloned four naturally occurring haplotypes (CGG, CAG, CGC, and GAG) using the TOPO-TA cloning kit, then transfected Cos-1 cells using the Lipofectamine 2000 protocol. Gene expression was assayed using the Promega Dual-Luciferase Reporter Assay System and read on a Molecular Devices HT Analyzer. Luciferase activity was measured at 24 and 48 hours after transfection for six replicates of every condition during three separate transfections. To correct for differences in transfection efficiencies, experimental (Photinus pyralis) luciferase activities were normalized by co-transfection with control (Renilla reniformis) luciferase plasmid. Compared to the wild type CGG haplotype, variant haplotypes CAG, CGC, and GAG drive luciferase expression at approximately 2x (p <0.0001), 1.4x (p <0.001) and 1.2x (p =0.08), respectively. This leads to the hypothesis that individuals carrying CAG or CGC haplotypes (17% of the population) exhibit higher levels of hENT1 expression and are more sensitive to Ara-C exposure. Experiments are underway to quantify gene transcripts in people of known hENT1 haplotypes. We also plan to genotype a large cohort of children with de novo AML for these three SNPs in hENT1 and correlate clinical outcomes in individuals carrying the low- versus the high-expressing haplotypes.

Prognostic Significance Of TP53 mutations and Single Nucleotide Polymorphisms In Acute Myeloid Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4971-4971
Author(s):  
Simon B. Zeichner ◽  
Sarah Alghamdi ◽  
Gina Elhammady ◽  
Robert Poppiti ◽  
Amilcar Castellano-Sanchez
Keyword(s):  
Myeloid Leukemia ◽  
Tp53 Mutation ◽  
De Novo ◽  
Performance Status ◽  
Nucleotide Polymorphisms ◽  
Complex Karyotype ◽  
Single Nucleotide ◽  
Tp53 Mutations ◽  
Significant Difference ◽  
De Novo Aml

Abstract Background The response to treatment and overall survival (OS) of patients with acute myeloid leukemia (AML) is variable, with a median OS ranging from several months to more than 10 years. Age at diagnosis, performance status (PS), and karyotype expression have long been established in prognostication. Loss of TP53, a tumor suppressor gene located on the short arm of chromosome 17, is one of the most frequent genetic abnormalities in human cancer and is one of the more promising prognostic markers for AML. Studies have shown that TP53 mutations are present in 5-25% of all AML patients, in 70% of those with complex karyotypes, and are associated with old age, chemotherapy resistance, and worse OS. Single nucleotide polymorphisms (SNPs), changes in DNA seen in an appreciable amount of the population, have been examined in AML and studies have suggested a possible correlation with worse outcomes. Using genetic sequencing, we set out to look at our own experience with AML, and hypothesized TP53 mutations and SNPs would mimic the literature, occurring in a minority of patients, and conferring a worse OS. Methods We performed a pilot study of randomly selected, newly diagnosed AML patients at Mount Sinai Medical Center, diagnosed from 2005-2008 (n =10). Immunohistochemical (IHC) analysis of bone marrows and peripheral blood smears was assessed via DO-1 antibody on paraffin embedded tissue. Conventional cytogenetic analyses were performed on short-term cultured bone marrow and peripheral blood cells with the use of the GTG-banding technique. TP53 PCR sequencing was performed using DNA from bone marrow smears using the Sanger sequencing platform and resolved by capillary electrophoresis. Analysis was performed using Mutation Surveyor software with confirmation of the variants using the COSMIC and dbSNP databases. Descriptive frequencies and median survivals were calculated for demographic information, prognostic factors, and treatment variables. A univariate analysis was performed. Results The majority of patients in our pilot study were older than age 60 (80%), male (60%), Hispanic (60%), and had a poor PS (ECOG 2-3: 60%). Most patients had de-novo AML (50%) with an intermediate (50%) non-complex (70%) karyotype and a TP53 P72R SNP (50%). Fewer than half of these patients harbored TP53 mutations (40%). There was no significant difference in OS based on sex, AML history, risk-stratified karyotype, or TP53 mutation. There was a trend toward improved survival among patients younger than age 60 (11, 4 mo, p = 0.09), of Hispanic ethnicity (8, 1 mo, p = 0.11), and those not harboring P72R (8, 2, p = 0.10). There was a significant improvement in survival among patients with a better PS (28, 4 mo, p = 0.01) and those who did not have a complex karyotype (8, 1 mo, p = 0.03). Among patients with a TP53-mutation, there were a larger number of individuals who were younger than age 60 (25.0, 16.7%), who were male (75.0, 50.0%), had a good performance status (ECOG 0-1: 50.0, 16.7%), had de-novo AML (50.0, 66.7%), and who had an adverse karyotype (50.0, 33%). Patients with a P72R SNP were more often male (80, 40%) and had a worse PS (ECOG 2-3: 80, 40%) with AML secondary to MDS (60, 20%) and a complex karyotype (40, 0%). The most commonly observed TP53 mutation was a missense N310K (40%) and the most commonly observed SNP was P72R (100.0%). Patients with more than one TP53 mutation had a worse clinical course than those with only a single mutation. Conclusion Our study demonstrated that poor PS and the presence of a complex karyotype were associated with a decreased OS. TP53 mutations were relatively uncommon, occurring more frequently in male patients with an adverse karyotype. Although there was no significant difference in survival between TP53 mutated and un-mutated patients, there was a trend toward worse OS among patients with a specific SNP. These results suggest that different TP53 mutations and SNPs should not be treated the same, and that some may confer a worse prognosis than others. Larger studies are needed to validate these findings. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Somatic variants for seed and fruit set in grapevine

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Laura Costantini ◽  
Paula Moreno-Sanz ◽  
Chinedu Charles Nwafor ◽  
Silvia Lorenzi ◽  
Annarita Marrano ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphisms ◽  
Fruit Set ◽  
Climatic Factors ◽  
Embryo Sac ◽  
Wine Industry ◽  
Nucleotide Polymorphisms ◽  
Wine Quality ◽  
Single Nucleotide ◽  
Germplasm Collections

Abstract Background Grapevine reproductive development has direct implications on yield. It also impacts on berry and wine quality by affecting traits like seedlessness, berry and bunch size, cluster compactness and berry skin to pulp ratio. Seasonal fluctuations in yield, fruit composition and wine attributes, which are largely driven by climatic factors, are major challenges for worldwide table grape and wine industry. Accordingly, a better understanding of reproductive processes such as gamete development, fertilization, seed and fruit set is of paramount relevance for managing yield and quality. With the aim of providing new insights into this field, we searched for clones with contrasting seed content in two germplasm collections. Results We identified eight variant pairs that seemingly differ only in seed-related characteristics while showing identical genotype when tested with the GrapeReSeq_Illumina_20K_SNP_chip and several microsatellites. We performed multi-year observations on seed and fruit set deriving from different pollination treatments, with special emphasis on the pair composed by Sangiovese and its seedless variant locally named Corinto Nero. The pollen of Corinto Nero failed to germinate in vitro and gave poor berry set when used to pollinate other varieties. Most berries from both open- and cross-pollinated Corinto Nero inflorescences did not contain seeds. The genetic analysis of seedlings derived from occasional Corinto Nero normal seeds revealed that the few Corinto Nero functional gametes are mostly unreduced. Moreover, three genotypes, including Sangiovese and Corinto Nero, were unexpectedly found to develop fruits without pollen contribution and occasionally showed normal-like seeds. Five missense single nucleotide polymorphisms were identified between Corinto Nero and Sangiovese from transcriptomic data. Conclusions Our observations allowed us to attribute a seedlessness type to some variants for which it was not documented in the literature. Interestingly, the VvAGL11 mutation responsible for Sultanina stenospermocarpy was also discovered in a seedless mutant of Gouais Blanc. We suggest that Corinto Nero parthenocarpy is driven by pollen and/or embryo sac defects, and both events likely arise from meiotic anomalies. The single nucleotide polymorphisms identified between Sangiovese and Corinto Nero are suitable for testing as traceability markers for propagated material and as functional candidates for the seedless phenotype.

Assessing single nucleotide polymorphism selection methods for the development of a low-density panel optimized for imputation in South African Drakensberger beef cattle

2021 ◽  
Author(s):  
Simon F Lashmar ◽  
Donagh P Berry ◽  
Rian Pierneef ◽  
Farai C Muchadeyi ◽  
Carina Visser
Keyword(s):  
South African ◽  
Clustering Algorithm ◽  
Imputation Accuracy ◽  
Developed Countries ◽  
Genotype Imputation ◽  
Selection Strategy ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Single Nucleotide Polymorphism Selection ◽  
The Mean

Abstract A major obstacle in applying genomic selection (GS) to uniquely adapted local breeds in less-developed countries has been the cost of genotyping at high densities of single nucleotide polymorphisms (SNP). Cost reduction can be achieved by imputing genotypes from lower to higher densities. Locally adapted breeds tend to be admixed and exhibit a high degree of genomic heterogeneity thus necessitating the optimization of SNP selection for downstream imputation. The aim of this study was to quantify the achievable imputation accuracy for a sample of 1,135 South African (SA) Drakensberger using several custom-derived lower-density panels varying in both SNP density and how the SNP were selected. From a pool of 120,608 genotyped SNP, subsets of SNP were chosen 1) at random, 2) with even genomic dispersion, 3) by maximizing the mean minor allele frequency (MAF), 4) using a combined score of MAF and linkage disequilibrium (LD), 5) using a partitioning-around-medoids (PAM) algorithm, and finally 6) using a hierarchical LD-based clustering algorithm. Imputation accuracy to higher density improved as SNP density increased; animal-wise imputation accuracy defined as the within-animal correlation between the imputed and actual alleles ranged from 0.625 to 0.990 when 2,500 randomly selected SNP were chosen versus a range of 0.918 to 0.999 when 50,000 randomly selected SNP were used. At a panel density of 10,000 SNP, the mean (standard deviation) animal-wise allele concordance rate was 0.976 (0.018) versus 0.982 (0.014) when the worst (i.e., random) as opposed to the best (i.e., combination of MAF and LD) SNP selection strategy was employed. A difference of 0.071 units was observed between the mean correlation-based accuracy of imputed SNP categorized as low (0.01&lt;MAF≤0.1) versus high MAF (0.4&lt;MAF≤0.5). Greater mean imputation accuracy was achieved for SNP located on autosomal extremes when these regions were populated with more SNP. The presented results suggested that genotype imputation can be a practical cost-saving strategy for indigenous breeds such as the South African Drakensberger. Based on the results, a genotyping panel consisting of approximately 10,000 SNP selected based on a combination of MAF and LD would suffice in achieving a less than 3% imputation error rate for a breed characterized by genomic admixture on the condition that these SNP are selected based on breed-specific selection criteria.

In-Silico and In-vitro Analysis of Human SOS1 Protein Causing Noonan Syndrome – A Novel Approach to Explore the Molecular Pathways

2021 ◽  
Vol 22 ◽  
Author(s):  
Vinoth Sigamani ◽  
Sheeja Rajasingh ◽  
Narasimman Gurusamy ◽  
Arunima Panda ◽  
Johnson Rajasingh
Keyword(s):  
Noonan Syndrome ◽  
In Silico ◽  
Genetic Disorder ◽  
In Vitro Study ◽  
Human Skin Fibroblast ◽  
Nucleotide Polymorphisms ◽  
Gene Expressions ◽  
Single Nucleotide ◽  
Nucleotide Mutation

Aims: Noonan syndrome (NS) is an autosomal dominant genetic disorder caused by single nucleotide mutation in PTPN11, SOS1, RAF1, and KRAS genes. Background: We hypothesize that in-silico analysis of human SOS1 mutations would be a promising predictor in identifying the pathogenic effect of NS. Methods: Here, we computationally analyzed the SOS1 gene to identify the pathogenic non-synonymous single nucleotide polymorphisms (nsSNPs) to cause NS. The variant information of SOS1 was collected from the SNP database (dbSNP). The variants were further analyzed by in-silico tools I-Mutant, iPTREE-STAB, and MutPred to elucidate their structural and functional characteristics. Results: We found that 11 nsSNPs of SOS1 were more pathogenic to cause NS. The 3D modeling of the wild-type and the 11 nsSNPs were performed using I-TASSER and validated via ERRAT and RAMPAGE. SOS1 interacting proteins were analysed through STRING, which showed that SOS1 interacted with cardiac proteins GATA4, TNNT2, and ACTN2. During these interactions, GRB2 and HRAS act as an intermediate molecules between SOS1 and cardiac proteins. These in-silico analyses were validated using induced cardiomyocytes (iCMCs) derived from NS patients carrying SOS1 gene variant c.1654A>G (NS-iCMCs) and compared with control human skin fibroblast-derived iCMCs (C-iCMCs). Our in vitro data further confirmed that the SOS1, GRB2 and HRAS gene expressions as well as the activated ERK protein, were significantly decreased in NS-iCMCs compared to C-iCMCs. Conclusion: This is the first in-silico and in vitro study demonstrating that 11 nsSNPs of SOS1 were playing a deleterious pathogenic role in causing NS.

Contribution of boars to reproductive performance and paternity after homospermic and heterospermic artificial insemination

2015 ◽  
Vol 27 (7) ◽  
pp. 1012 ◽  
Author(s):  
C. E. R. Ferreira ◽  
D. B. Sávio ◽  
A. C. Guarise ◽  
M. J. Flach ◽  
G. D. A. Gastal ◽  
...  
Keyword(s):  
Reproductive Performance ◽  
Artificial Insemination ◽  
Penetration Rate ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Blood Samples ◽  
Precise Evaluation ◽  
Umbilical Cords ◽  
Farrowing Rate

Heterospermic AI is commonly used in swine despite preventing precise evaluation of individual boar fertility. The present study compared the contribution of four boars (A, B, C and D) for reproductive performance and for paternity using homospermic and heterospermic (AB, AC, AD, BC, BD and CD) AI (n = 204 for homospermic AI; n = 307 for heterospermic AI). Blood samples from the four boars, from all sows inseminated with heterospermic doses and from the umbilical cords of their piglets, as well as tissue smears from mummified fetuses, were genotyped using single nucleotide polymorphisms (SNPs). Differences among boars were detected for the in vitro oocyte penetration rate and for the number of spermatozoa per oocyte (P < 0.05), but not for sperm motility, mitochondrial functionality and integrity of the membrane, acrosome and DNA (P > 0.05). Homospermic and heterospermic AI resulted in similar (P > 0.05) farrowing rates (90.5% and 89.9%, respectively) and total litter size (12.4 ± 0.4 and 12.7 ± 0.7, respectively). Farrowing rate was lower for Boar B than for Boar C (P < 0.05), but no other differences in reproductive performance among boars were observed with homospermic AI. The SNPs determined the paternity of 94.2% of the piglets sired by heterospermic AI. In the AC pool, paternity contribution per boar was similar (P > 0.05), but differences between boars occurred in all other pools (P < 0.05). Boar D achieved the greatest paternity contribution in all pools and parity categories (nearly 60%), whereas Boar B sired the fewest piglets (at most 40%). Reproductive performance was similar with homospermic and heterospermic AI, but differences in performance among boars undetected with homospermic AI were only evident after genotyping the piglets sired through heterospermic AI.

scholarly journals The Molecular Basis of Long First Remissions in Normal Karyotype AML Patients

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3827-3827
Author(s):  
Francesca Ferraro ◽  
Christopher A Miller ◽  
Amy Abdalla ◽  
Nichole Helton ◽  
Nathan Salomonis ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Somatic Mutations ◽  
Conflicts Of Interest ◽  
Normal Karyotype ◽  
High Dose ◽  
Intermediate Risk ◽  
The Mean ◽  
Cebpa Mutations

Currently, it is not clear why some patients with acute myeloid leukemia (AML) can be "cured" with chemotherapy alone; are they living with small amounts of disease that is held in check by immunologic (or other) mechanisms, or is their disease really eradicated? The percentage of cytogenetically normal AML patients who have long (>5 years) first remissions (LFRs) after chemotherapy alone is low (about 9.1% in patients <60 years and 1.6% in >60 years1). For this reason, most intermediate risk patients are offered allogeneic transplantation to decrease their risk for relapse. To better understand mechanisms of chemotherapy sensitivity in AML, we performed an analysis of the mutation landscape and persistence, using samples from 8 normal karyotype LFR patients (without CEBPA mutations) who received standard "7+3" induction and high dose cytarabine consolidation as their only therapy. The mean age at diagnosis was 43.5 years, and the mean follow up in first remission is 7.6 years; none of these patients has relapsed to date. For each case, we performed enhanced exome sequencing at diagnosis (235x coverage of the entire exome, and ~1008x coverage of recurrently mutated AML genes). Each case had at least one documented AML driver mutation, with a median of 29 somatic mutations in the exome space. We created probes for 225 mutations (mean 28 per case), and performed error-corrected sequencing (Haloplex) for all available remission samples. The mean depth of Haloplex coverage was 1607x, and each sample had at least one AML-specific mutation assayed, with a sensitivity of 1 cell in 1,750 (0.06%). 7/8 patients demonstrated complete clearance of all mutations in all remission samples tested, which was confirmed with digital droplet PCR for 5 cases, with a sensitivity of detection of 1 cell in 100,000. In one case, we detected a persistent ancestral clone harboring DNMT3AR882H, which can be associated with long first remissions for some patients2. Strikingly, the founding clone in all 8 cases had one or more somatic mutations in genes known to drive cell proliferation (e.g. MYC, FLT3, NRAS, PTPN11, Figure 1 top panel). These are usually subclonal mutations that occur late during leukemic progression, suggesting that the presence of a "proliferative hit" in the founding clone might be important for chemotherapy clearance of all the AML cells in a given patient. To support this hypothesis, we analyzed the mutational clearance of 82 AML cases with paired diagnosis and day 30 post-chemotherapy bone marrow samples. We observed that, whether present in the founding clone or in subclones, mutations in MYC, CEBPA, FLT3, NRAS, and PTPN11 cleared after induction chemotherapy in all samples, while other mutations were often persistent at day 30 (e.g. DNMT3A, IDH1, IDH2, NPM1, TET2; Figure 1 bottom panel). Compared to other published sequencing studies of AML, MYC and NRAS mutations were significantly enriched in this small cohort (MYC p= 0.002, and NRAS p= 0.034), with MYC enrichment being particularly striking (37.5% versus 1.8%). All MYC mutations were canonical single base substitutions occurring in the highly conserved MYC Box 2 domain at the N-terminus of MYC (p.P74Q or p.T73N). Overexpression of MYCP74Q in murine hematopoietic progenitors prolonged MYC half life (89 min vs. 44 min for wild type), and enhanced cytarabine sensitivity at all concentrations tested (range 10-1000 nM, p=0.0003), both in vitro and in a MYC-driven leukemia model in vivo. MYC expression measured with flow cytometry in the blasts of the LFR samples was significantly higher (p=0.045) compared to unfavorable risk (complex karyotype) or other intermediate risk categories, but similar to good risk AML (biallelic CEBPA mutations, core binding factor fusion-associated AML, and AML with isolated NPMc), suggesting that activation of the MYC pathway may represent a shared feature of chemosensitive patients. Taken together, these data suggest that some intermediate patients who are effectively "cured" with chemotherapy alone may not have persistent subclinical disease, nor retained ancestral clones that could potentially contribute to relapse. Importantly, these patients often have mutations driving cell proliferation in the founding clone, indicating that the presence of specific mutations in all malignant cells may be critical for complete AML cell clearance with chemotherapy. 1. Blood Adv. 2018 Jul 10; 2(13): 1645-1650 2. N Engl J Med 2018; 378:1189-1199 Disclosures No relevant conflicts of interest to declare.

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