Modulation of Ca2+ Channels by Heterologously Expressed Wild-Type and Mutant Human μ-Opioid Receptors (hMORs) Containing the A118G Single-Nucleotide Polymorphism

2007 ◽  
Vol 97 (2) ◽  
pp. 1058-1067 ◽  
Author(s):  
Wojciech Margas ◽  
Ira Zubkoff ◽  
H. Gregg Schuler ◽  
Piotr K. Janicki ◽  
Victor Ruiz-Velasco
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Rank Order ◽  
Sympathetic Neurons ◽  
Signal Transduction Pathway ◽  
Wild Type ◽  
Nucleotide Polymorphism ◽  
Patch Clamp Technique ◽  
Single Nucleotide ◽  
Cervical Ganglion ◽  
Ganglion Neurons

The most common single-nucleotide polymorphism (SNP) of the human μ-opioid receptor (hMOR) gene occurs at position 118 (A118G) and results in substitution of asparagine to aspartate at the N-terminus. The purpose of the present study was to compare the pharmacological profile of several opioid agonists to heterologously expressed hMOR and N-type Ca2+ channels in sympathetic neurons. cDNA constructs coding for wild-type and mutant hMOR were microinjected in rat superior cervical ganglion neurons and N-type Ca2+ channel modulation was investigated using the whole cell variant of the patch-clamp technique. Concentration–response relationships were generated with the following selective MOR agonists: DAMGO, morphine, morphine-6-glucuronide (M-6-G), and endomorphin I. The estimated maximal inhibition for the agonists ranged from 52 to 64% for neurons expressing either hMOR subtype. The rank order of potencies for estimated EC50 values (nM) in cells expressing wild-type hMOR was: DAMGO (31) ≫ morphine (76) ≅ M-6-G (77) ≅ endomorphin I (86). On the other hand, the rank order in mutant-expressing neurons was: DAMGO (14) ≫ morphine (39) ≫ endomorphin I (74) ≅ M-6-G (82), with a twofold leftward shift for both DAMGO and morphine. The DAMGO-mediated Ca2+ current inhibition was abolished by the selective MOR blocker, CTAP, and by pertussis toxin pretreatment of neurons expressing either hMOR subtype. These results suggest that the A118G variant MOR exhibits an altered signal transduction pathway and may help explain the variability of responses to opiates observed with carriers of the mutant allele.

scholarly journals Serotonin 5-HT2C Receptor Cys23Ser Single Nucleotide Polymorphism Associates with Receptor Function and Localization In Vitro

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Michelle A. Land ◽  
Holly L. Chapman ◽  
Brionna D. Davis-Reyes ◽  
Daniel E. Felsing ◽  
John A. Allen ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Subcellular Localization ◽  
Cell Lines ◽  
Intracellular Signaling ◽  
Calcium Release ◽  
Wild Type ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Recycling Pathway

Abstract A non-synonymous single nucleotide polymorphism of the human serotonin 5-HT2C receptor (5-HT2CR) gene that converts a cysteine to a serine at amino acid codon 23 (Cys23Ser) appears to impact 5-HT2CR pharmacology at a cellular and systems level. We hypothesized that the Cys23Ser alters 5-HT2CR intracellular signaling via changes in subcellular localization in vitro. Using cell lines stably expressing the wild-type Cys23 or the Ser23 variant, we show that 5-HT evokes intracellular calcium release with decreased potency and peak response in the Ser23 versus the Cys23 cell lines. Biochemical analyses demonstrated lower Ser23 5-HT2CR plasma membrane localization versus the Cys23 5-HT2CR. Subcellular localization studies demonstrated O-linked glycosylation of the Ser23 variant, but not the wild-type Cys23, may be a post-translational mechanism which alters its localization within the Golgi apparatus. Further, both the Cys23 and Ser23 5-HT2CR are present in the recycling pathway with the Ser23 variant having decreased colocalization with the early endosome versus the Cys23 allele. Agonism of the 5-HT2CR causes the Ser23 variant to exit the recycling pathway with no effect on the Cys23 allele. Taken together, the Ser23 variant exhibits a distinct pharmacological and subcellular localization profile versus the wild-type Cys23 allele, which could impact aspects of receptor pharmacology in individuals expressing the Cys23Ser SNP.

scholarly journals Novel Genotyping and Quantitative Analysis of Neuraminidase Inhibitor Resistance-Associated Mutations in Influenza A Viruses by Single-Nucleotide Polymorphism Analysis

2011 ◽  
Vol 55 (10) ◽  
pp. 4718-4727 ◽  
Author(s):  
Susu Duan ◽  
David A. Boltz ◽  
Jiang Li ◽  
Christine M. Oshansky ◽  
Henju Marjuki ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Influenza Viruses ◽  
Polymorphism Analysis ◽  
Snp Analysis ◽  
Pandemic H1n1 ◽  
Wild Type ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Inhibitor Resistance ◽  
Public Health Epidemiology

ABSTRACTNeuraminidase (NA) inhibitors are among the first line of defense against influenza virus infection. With the increased worldwide use of the drugs, antiviral susceptibility surveillance is increasingly important for effective clinical management and for public health epidemiology. Effective monitoring requires effective resistance detection methods. We have developed and validated a novel genotyping method for rapid detection of established NA inhibitor resistance markers in influenza viruses by single nucleotide polymorphism (SNP) analysis. The multi- or monoplex SNP analysis based on single nucleotide extension assays was developed to detect NA mutations H275Y and I223R/V in pandemic H1N1 viruses, H275Y in seasonal H1N1 viruses, E119V and R292K in seasonal H3N2 viruses, and H275Y and N295S in H5N1 viruses. The SNP analysis demonstrated high sensitivity for low-content NA amplicons (0.1 to 1 ng/μl) and showed 100% accordant results against a panel of defined clinical isolates. The monoplex assays for the H275Y NA mutation allowed precise and accurate quantification of the proportions of wild-type and mutant genotypes in virus mixtures (5% to 10% discrimination), with results comparable to those of pyrosequencing. The SNP analysis revealed the lower growth fitness of an H275Y mutant compared to the wild-type pandemic H1N1 virus by quantitatively genotyping progeny viruses grown in normal human bronchial epithelial cells. This novel method offers high-throughput screening capacity, relatively low costs, and the wide availability of the necessary equipment, and thus it could provide a much-needed approach for genotypic screening of NA inhibitor resistance in influenza viruses.

The role of a single nucleotide polymorphism of MDM2 in glioblastoma multiforme

2008 ◽  
Vol 109 (5) ◽  
pp. 842-848 ◽  
Author(s):  
Rina G. Khatri ◽  
Kapila Navaratne ◽  
Robert J. Weil
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Glioblastoma Multiforme ◽  
Age Of Onset ◽  
Polymerase Chain Reaction Analysis ◽  
Genetic Alterations ◽  
Primary Brain Tumor ◽  
Healthy Controls ◽  
Wild Type ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide

Object Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults, with a 5-year survival rate of < 5%. Aberrant function of TP53 is common in GBM. Although mutational inactivation of p53 is found in many cases, there remain tumors in which genetic alterations of p53 are absent. Negative regulators of the TP53 pathway such as MDM2, which directly inhibits TP53 expression and activity, may influence the pathogenesis of GBM. To understand its potential function in gliomagenesis, the authors analyzed a novel single nucleotide polymorphism (SNP) in the MDM2 promoter that enhances MDM2 expression. Methods The investigators isolated DNA from 98 patients with GBM and 102 healthy, cancer-free controls. A polymerase chain reaction analysis was performed to determine the MDM2 SNP309 genotype by using distinct primer pairs for the wild-type (T) and mutant (G) alleles. Results The frequency of the mutant MDM2 polymorphism was found to be higher (p = 0.0092) in patients with GBM (54.6%) compared with healthy controls (41.2%); the TT and GG genotypes were more common in healthy controls and patients with GBM (p = 0.0004 and p = 0.02, respectively). Although there was no association between the MDM2 SNP309 and overall survival, the GG genotype was associated with development of GBM at a younger age in patients with tumors harboring wild-type p53, which may mitigate the effect of the MDM2 SNP. Conclusions Although the MDM2 SNP309 does not portend decreased survival, the increased incidence of the mutant G allele in patients with GBM and its influence on age of onset suggest a potential role in the molecular pathogenesis of GBM, and may be a therapeutic target.

scholarly journals A Caenorhabditis elegans Wild Type Defies the Temperature–Size Rule Owing to a Single Nucleotide Polymorphism in tra-3

PLoS Genetics ◽  
2007 ◽  
Vol 3 (3) ◽  
pp. e34 ◽  
Author(s):  
Jan E Kammenga ◽  
Agnieszka Doroszuk ◽  
Joost A. G Riksen ◽  
Esther Hazendonk ◽  
Laurentiu Spiridon ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Caenorhabditis Elegans ◽  
Wild Type ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide

A C. elegans Wild-type Defies the Temperature-size Rule due to a Single Nucleotide Polymorphism in tra-3

PLoS Genetics ◽  
2005 ◽  
Vol preprint (2007) ◽  
pp. e34
Author(s):  
Jan E Kammenga ◽  
Agnieszka Doroszuk ◽  
Joost A. G. Riksen ◽  
Esther Hazendonk ◽  
Laurentio Spiridon ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Wild Type ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
C Elegans

scholarly journals Sensitivity and Specificity of Single-Nucleotide Polymorphism Scanning by High-Resolution Melting Analysis

2004 ◽  
Vol 50 (10) ◽  
pp. 1748-1754 ◽  
Author(s):  
Gudrun H Reed ◽  
Carl T Wittwer
Keyword(s):  
Single Nucleotide Polymorphism ◽  
High Resolution ◽  
Sensitivity And Specificity ◽  
High Resolution Melting ◽  
Wild Type ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Product Size ◽  
Pcr Products ◽  
Melting Analysis

Abstract Background: Screening for heterozygous sequence changes in PCR products, also known as “mutation scanning”, is an important tool for genetic research and clinical applications. Conventional methods require a separation step. Methods: We evaluated the sensitivity and specificity of homogeneous scanning, using a saturating DNA dye and high-resolution melting. Heterozygous single-nucleotide polymorphism (SNP) detection was studied in three different sequence backgrounds of 40%, 50%, and 60% GC content. PCR products of 50–1000 bp were generated in the presence of LCGreen™ I. After fluorescence normalization and temperature overlay, melting curve shape was used to judge the presence or absence of heterozygotes among 1632 cases. Results: For PCR products of 300 bp or less, all 280 heterozygous and 296 wild-type cases were correctly called without error. In 672 cases between 400 and 1000 bp with the mutation centered, the sensitivity and specificity were 96.1% and 99.4%, respectively. When the sequence background and product size with the greatest error rate were used, the sensitivity of off-center SNPs (384 cases) was 95.6% with a specificity of 99.4%. Most false negatives occurred with SNPs that were compared with an A or T wild type sequence. Conclusions: High-resolution melting analysis with the dye LCGreen I identifies heterozygous single-base changes in PCR products with a sensitivity and specificity comparable or superior to nonhomogeneous techniques. The error rate of scanning depends on the PCR product size and the type of base change, but not on the position of the SNP. The technique requires only PCR reagents, the dye LCGreen I, and 1–2 min of closed-tube, post-PCR analysis.

A 3′ UTR Single Nucleotide Polymorphism 829C→T in Dihydrofolate Reductase Gene Results in an Increase in DHFR Protein Level and MTX Resistance.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2084-2084
Author(s):  
Prasunkumar J. Mishra ◽  
Giuseppe S.A. Longo ◽  
Lata G. Menon ◽  
Emine Abali ◽  
Debabrata Banerjee ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Cell Lines ◽  
Dihydrofolate Reductase ◽  
Protein Level ◽  
Gene Copy ◽  
Mrna Levels ◽  
Wild Type ◽  
Nucleotide Polymorphism ◽  
Dhfr Gene ◽  
Single Nucleotide

Abstract Dihydrofolate reductase (DHFR) catalyzes the reduction of dihydrofolate to tetrahydrofolate (THF) required for the synthesis of thymidylate and purines. Methotrexate (MTX) acts as a tight-binding inhibitor of DHFR and remains an important chemotherapeutic agent for treatment of leukemias and lymphomas. Increased DHFR confers resistance to antifolates in target cells. A previously reported single nucleotide polymorphism (SNP) 829C/C→829T/T (829C→T) found in the 3′- untranslated region of DHFR gene transcript (between the first and second polyadenylation site) was associated with higher expression of the DHFR transcript. The SNP was identified in 5.4% of the cases and 6.0% in the controls of Japanese patients with childhood leukemia/lymphomas (Goto et al. 2001, Clinical Cancer Research, Vol. 7, 1952-1956). The objective of the present study was to determine the role of the 3′ UTR SNP 829C→T in DHFR gene expression, DHFR protein level and resistance to MTX. The mutation 829C→T in the 3′ UTR of wild type DHFR was introduced by site directed mutagenesis and the mutant cDNA expressed in DHFR deficient CHO cells (DG-44), wild type DHFR and vector alone constructs were also transfected into DG44 as controls. After two weeks of selection in G418 containing media, several well-isolated surviving colonies were picked and expanded as cell lines in media containing G418. Real-time quantitative PCR was used to compare mRNA and genomic DNA level of the clones while Western blotting was used to compare the protein levels. MTX cytotoxicity assay was carried out in media lacking thymidine. Clones expressing the mutant 829C→T showed greater than two fold enhanced expression of DHFR transcripts as compared to wild type clones. Corresponding to the high mRNA levels, an increase in DHFR protein level was observed in the mutant clones without an increase in DHFR gene copy number. Cytotoxicity studies showed that cell lines with increased levels of DHFR were significantly more resistant to MTX than cells with wild type 3′ UTR. Of interest clonogenic efficiency of the mutants in medium lacking thymidine was greater than wild type and was directly proportional to the level of DHFR expressed in the clones. This study demonstrates that when SNP 829C→T is introduced in the 3′ UTR of wild type DHFR, the expression of the DHFR mRNA is enhanced with a corresponding increase in the protein level. The presence of a SNP 829C→T in patients with ALL may contribute to treatment failure, as MTX is a key drug in curative regimen for this disease. Future studies are directed toward determining the abundance of this SNP in other populations, and the correlation between this SNP and clinical methotrexate resistance and or decreased MTX toxicity.

scholarly journals Single-Nucleotide Polymorphism Leading to False Allelic Fraction by Droplet Digital PCR

2017 ◽  
Vol 63 (8) ◽  
pp. 1370-1376 ◽  
Author(s):  
Eric S Christenson ◽  
W Brian Dalton ◽  
David Chu ◽  
Ian Waters ◽  
Karen Cravero ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Careful Analysis ◽  
Digital Pcr ◽  
Droplet Digital Pcr ◽  
Clinical Samples ◽  
Unexpected Finding ◽  
Wild Type ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Patients With Cancer

Abstract BACKGROUND Molecular-based diagnostics have great utility for cancer detection. We have used droplet digital PCR (ddPCR) as a platform for identifying mutations in circulating plasma tumor DNA (ptDNA). We present the unexpected finding of a spurious mutant allele fraction that was discovered to be artifactual because of the presence of a single-nucleotide polymorphism (SNP) in a patient sample. DESIGN AND METHODS Probe and primer combinations for the K700 and V701 loci of the SF3B1 spliceosome gene were designed for ddPCR to identify the percentage of mutant and wild-type alleles. Clinical samples from patients with cancer with known SF3B1 mutations were collected and tested to evaluate the assays' ability to detect SF3B1 mutations in ptDNA. RESULTS Patient samples showed SF3B1 K700E mutations within the ptDNA of 4 patients with acute leukemia and 3 with myelodysplastic syndrome who were known to harbor this mutation. A blood sample from a patient with lung cancer with a known SF3B1 V701F mutation was also analyzed and this mutation was successfully identified in ptDNA. However, 1 of the patients with a K700E mutation was found to have a mutational burden of 98%. After careful analysis of this locus by Sanger sequencing and ddPCR, this patient was found to have an SNP (R702R), which prevented binding of the ddPCR wild-type probe to its cognate allele. CONCLUSIONS These results further support that ddPCR-based assays may be valuable companion diagnostics for the identification and monitoring of patients with cancer, but the results also emphasize the need to identify SNPs at loci that are being analyzed.

Sign in / Sign up

Export Citation Format

Share Document