Programmable endonuclease combined with isothermal polymerase amplification to selectively enrich for rare mutant allele fractions

2021 ◽  
Author(s):  
Junman Chen ◽  
Tian Qiu ◽  
Michael G. Mauk ◽  
Zheng Su ◽  
Yaguang Fan ◽  
...  
Keyword(s):  
Mutant Allele

INTERRELATION OF ALLELIC VARIANTS OF HEMOSTASIS SYSTEM GENES ON THE DEVELOPMENT AND COURSE OF LUPUS NEPHRITIS

2019 ◽  
Vol 23 (2) ◽  
pp. 77-81
Author(s):  
E. N. Borisov ◽  
L. V. Ivanitsky ◽  
L. M. Samokhodskaya ◽  
T. N. Krasnova ◽  
E. P. Pavlikova ◽  
...  
Keyword(s):  
Lupus Nephritis ◽  
Mutant Allele ◽  
Impaired Renal Function ◽  
Mthfr C677t ◽  
Mthfr Gene ◽  
Renal Survival ◽  
Mutant Genotype ◽  
Hemostatic System ◽  
Allelic Variations ◽  
Pai 1

THE AIM: to evaluate the effect of allelic variations in the hemostatic system genes on the development and course of lupus nephritis. PATIENTS AND METHODS. The study analyzed 100 patients with SLE Caucasians. 80 women and 20 men aged 16 to 73 years (mean age 37, ± 14 years). The duration of observation was for 73 patients over 5 years, for 18 – from 1 year to 5 years and for 9 – less than 1 year A rise in the level of creatinine in the blood above or equal to 2 mg / dl was considered a significant sign of impaired renal function. RESULTS. Among the patients included in the study, kidney damage was detected in 61 people (61%). In 33 of them (54.1%), a variant of renal pathology was observed according to the type of rapidly progressive lupus nephritis (BPVN). In patients with BH, mutations in the MTHFR (C677T) gene were statistically significantly more frequent (p = 0.033). The OR for the mutant genotype is 6.146 with 95% CI from 1.692 to 22.326. In patients with PWHD, mutations in the MTHFR (C677T) gene were statistically significantly more frequent (p = 0.031). The OR for the mutant genotype is 1.625 with 95% CI from 1.034 to 4.771. The five-year renal survival in carriers of the mutant allele of the MTHFR gene (C677T) is statistically significantly lower (72.8%) than in patients without this mutation (81.9%) (p = 0.027). Ten-year renal survival in carriers of the mutant allele of the MTHFR gene (C677T) is statistically significantly less (55.6%) than in patients without this mutation (70.5%) (p = 0.016). In patients with BH, mutations in the PAI-1 gene (4G / 5G 675) were statistically significantly more frequent (p = 0.046). OR for mutant genotype – 1.766 with 95% CI from 1.061 to 4.758. CONCLUSION. The mutant alleles of the MTHFR (C677T) and PAI-1 (4G / 5G 675) genes are likely to be associated with the development of BH. Polymorphism of the MTHFR gene (C677T) is associated with an unfavorable course of HH.

scholarly journals MULTIPLE ALLELE APPROACH TO THE STUDY OF GENES IN DROSOPHILA MELANOGASTER THAT ARE INVOLVED IN IMAGINAL DISC DEVELOPMENT

Genetics ◽  
1978 ◽  
Vol 89 (2) ◽  
pp. 355-370 ◽  
Author(s):  
Allen Shearn ◽  
Grafton Hersperger ◽  
Evelyn Hersperger ◽  
Ellen Steward Pentz ◽  
Paul Denker
Keyword(s):  
Drosophila Melanogaster ◽  
Phenotypic Variation ◽  
Mutant Allele ◽  
Imaginal Disc ◽  
Reference Allele ◽  
Multiple Allele ◽  
Significant Difference ◽  
Chromosome Mutations ◽  
Cold Sensitive

ABSTRACT The phenotypes of five different lethal mutants of Drosophila melanogaster that have small imaginal discs were analyzed in detail. From these results, we inferred whether or not the observed imaginal disc phenotype resulted exclusively from a primary imaginal disc defect in each mutant. To examine the validity of these inferences, we employed a multiple-allele method. Lethal alleles of the five third-chromosome mutations were identified by screening EMS-treated chromosomes for those which fail to complement with a chromosome containing all five reference mutations. Twenty-four mutants were isolated from 13,197 treated chromosomes. Each of the 24 was then tested for complementation with each of the five reference mutants. There was no significant difference in the mutation frequencies at these five loci. The stage of lethality and the imaginal disc morphology of each mutant allele were compared to those of its reference allele in order to examine the range of defects to be found among lethal alleles of each locus. In addition, hybrids of the alleles were examined for intracistronic complementation. For two of the five loci, we detected no significant phenotypic variation among lethal alleles. We infer that each of the mutant alleles at these two loci cause expression of the null activity phenotype. However, for the three other loci, we did detect significant phenotypic variation among lethal alleles. In fact, one of the mutant alleles at each of these three loci causes no detectable imaginal disc defect. This demonstrates that attempting to assess the developmental role of a gene by studying a single mutant allele may lead to erroneous conclusions. As a byproduct of the mutagenesis procedure, we have isolated two dominant, cold-sensitive mutants.

scholarly journals Assessing Genetic Variants in Matched Biocompartments From Patients With Serous Ovarian Cancer

2021 ◽  
Vol 20 ◽  
pp. 153303382110279
Author(s):  
Brooke E. Sanders ◽  
Lisa Ku ◽  
Paul Walker ◽  
Benjamin G. Bitler
Keyword(s):  
Ovarian Cancer ◽  
Genetic Variants ◽  
Mutant Allele ◽  
Serous Ovarian Cancer ◽  
Brca Testing ◽  
Panel Testing ◽  
University Of Colorado ◽  
Gene Panel Testing ◽  
The University ◽  
Tumor Profiling

The clinical use of molecular tumor profiling (MTP) is expanding and there is an increasing use of MTP data to manage patient care. At the University of Colorado, 18 patients were diagnosed with primary serous ovarian cancer between 9/2015 and 6/2019 and consented for banking and analysis of tumor, ascites and plasma. All 18 patients had tumor and plasma samples that were sent for MTP, and 13 of 18 patients additionally had ascites collected and sent for MTP. 50-gene panel testing and BRCA testing were performed on primary tumor. BRCA genetic variants were more likely to be identified in plasma as compared to ascites or tumor, though not statistically significant ( P = 0.17). Co-occurring genetic variants between plasma and ascites were less common in comparison to co-occurring variants between tumor and plasma or tumor and ascites, though not statistically significant ( P = 0.68). Variants in KDR (VEGFR2) and TP53 were most likely to be conserved across all 3 biocompartments. Mutant allele frequencies (MAF) of individual genetic variants varied across biocompartments, though tended to be highest in the tumor, followed by ascites.

scholarly journals Mutator-Suppressible Alleles of rough sheath1 and liguleless3 in Maize Reveal Multiple Mechanisms for Suppression

Genetics ◽  
2000 ◽  
Vol 154 (1) ◽  
pp. 437-446 ◽  
Author(s):  
Lisa Girard ◽  
Michael Freeling
Keyword(s):  
Untranslated Region ◽  
Mutant Allele ◽  
Negative Regulation ◽  
Wild Type ◽  
Knox Gene ◽  
Transcript Accumulation ◽  
Mu Suppression ◽  
Different Types ◽  
Rna Blot Analysis

Abstract Insertions of Mutator transposons into maize genes can generate suppressible alleles. Mu suppression is when, in the absence of Mu activity, the phenotype of a mutant allele reverts to that of its progenitor. Here we present the characterization of five dominant Mu-suppressible alleles of the knox (knotted1-like homeobox) genes liguleless3 and rough sheath1, which exhibit neomorphic phenotypes in the leaves. RNA blot analysis suggests that Mu suppression affects only the neomorphic aspect of the allele, not the wild-type aspect. Additionally, Mu suppression appears to be exerting its effects at the level of transcription or transcript accumulation. We show that truncated transcripts are produced by three alleles, implying a mechanism for Mu suppression of 5′ untranslated region insertion alleles distinct from that which has been described previously. Additionally, it is found that Mu suppression can be caused by at least three different types of Mutator elements. Evidence presented here suggests that whether an allele is suppressible or not may depend upon the site of insertion. We cite previous work on the knox gene kn1, and discuss our results in the context of interactions between Mu-encoded products and the inherently negative regulation of neomorphic liguleless3 and rough sheath1 transcription.

scholarly journals Likelihoods and Simulation Methods for a Class of Nonneutral Population Genetics Models

Genetics ◽  
2001 ◽  
Vol 159 (2) ◽  
pp. 853-867 ◽  
Author(s):  
Peter Donnelly ◽  
Magnus Nordborg ◽  
Paul Joyce
Keyword(s):  
Population Genetics ◽  
Population Size ◽  
Mutant Allele ◽  
Mutation Rates ◽  
Simulation Methods ◽  
Large Populations ◽  
Selection Intensities ◽  
Infinite Alleles Model

Abstract Methods for simulating samples and sample statistics, under mutation-selection-drift equilibrium for a class of nonneutral population genetics models, and for evaluating the likelihood surface, in selection and mutation parameters, are developed and applied for observed data. The methods apply to large populations in settings in which selection is weak, in the sense that selection intensities, like mutation rates, are of the order of the inverse of the population size. General diploid selection is allowed, but the approach is currently restricted to models, such as the infinite alleles model and certain K-models, in which the type of a mutant allele does not depend on the type of its progenitor allele. The simulation methods have considerable advantages over available alternatives. No other methods currently seem practicable for approximating likelihood surfaces.

scholarly journals PCR-based genotyping assays to detect germline APC variant associated with hereditary gastrointestinal polyposis in Jack Russell terriers

2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Kyoko Yoshizaki ◽  
Akihiro Hirata ◽  
Hiroyuki Matsushita ◽  
Naohito Nishii ◽  
Mifumi Kawabe ◽  
...  
Keyword(s):  
Mutant Allele ◽  
False Negative ◽  
Disease Onset ◽  
Pcr Amplification ◽  
Buccal Swab ◽  
Wild Type ◽  
Gastrointestinal Polyposis ◽  
Pcr Rflp ◽  
Formalin Fixed Paraffin Embedded ◽  
Rflp Assay

Abstract Background The prevalence of gastrointestinal (GI) neoplastic polyps in Jack Russell terriers (JRTs) has increased in Japan since the late 2000s. Recently, we demonstrated that JRTs with GI polyps harbor identical germline variant in the APC gene (c.[462_463delinsTT]) in the heterozygous state. Thus, this disease is an autosomal dominant hereditary disorder. Although the affected JRTs have distinct features, such as the development of multiple GI polyps and an early age of disease onset, genetic testing is indispensable for a definitive diagnosis. Here, polymerase chain reaction (PCR)-based assays capable of detecting germline APC variant were designed and validated using synthetic wild-type and mutant DNAs and genomic DNAs from carrier and non-carrier dogs. Result First, the PCR-restriction fragment length polymorphism (PCR-RFLP) assay was developed by taking advantage of the germline APC variant creating a new restriction site for MseI. In the PCR-RFLP assay, the 156-bp region containing the variant site was amplified by PCR and subsequently digested with MseI, yielding diagnostic 51 and 58 bp fragments from the mutant allele and allowing determination of the APC genotypes. It was possible to determine the genotypes using genomic DNA extracted from the peripheral blood, buccal swab, or formalin-fixed paraffin-embedded tissue. Next, a TaqMan duplex real-time PCR assay was developed, where a 78-bp region flanking the variant was amplified in the presence of wild-type allele- and mutant allele-specific fluorescent probes. Using blood-derived DNA, altogether 40 cycles of PCR amplification determined the APC genotypes of all examined samples by measuring the fluorescence intensities. Importantly, false-positive and false-negative errors were never detected in both assays. Conclusion In this study, we developed highly reliable genetic tests for hereditary GI polyposis in JRTs, providing accurate assessment of the presence of the causative germline APC variant. The genotyping assays could contribute to the diagnosis and prevention of hereditary GI polyposis in dogs.

The clp1 Gene of the Mushroom Coprinus cinereus Is Essential for A-Regulated Sexual Development

Genetics ◽  
2001 ◽  
Vol 157 (1) ◽  
pp. 133-140
Author(s):  
Kazumi Inada ◽  
Yoshinori Morimoto ◽  
Toshihide Arima ◽  
Yukio Murata ◽  
Takashi Kamada
Keyword(s):  
Sexual Development ◽  
Genomic Dna ◽  
Mutant Allele ◽  
Coprinus Cinereus ◽  
Mating Partner ◽  
Essential Step ◽  
Genes Encoding ◽  
Dna Fragment ◽  
Necessary And Sufficient ◽  
Novel Protein

Abstract Sexual development in the mushroom Coprinus cinereus is under the control of the A and B mating-type loci, both of which must be different for a compatible, dikaryotic mycelium to form between two parents. The A genes, encoding proteins with homeodomain motifs, regulate conjugate division of the two nuclei from each mating partner and promote the formation of clamp connections. The latter are hyphal configurations required for the maintenance of the nuclear status in the dikaryotic phase of basidiomycetes. The B genes encode pheromones and pheromone receptors. They regulate the cellular fusions that complete clamp connections during growth, as well as the nuclear migration required for dikaryosis. The AmutBmut strain (326) of C. cinereus, in which both A- and B-regulated pathways are constitutively activated by mutations, produces, without mating, dikaryon-like, fertile hyphae with clamp connections. In this study we isolated and characterized clampless1-1 (clp1-1), a mutation that blocks clamp formation, an essential step in A-regulated sexual development, in the AmutBmut background. A genomic DNA fragment that rescues the clp1-1 mutation was identified by transformations. Sequencing of the genomic DNA, together with RACE experiments, identified an ORF interrupted by one intron, encoding a novel protein of 365 amino acids. The clp1-1 mutant allele carries a deletion of four nucleotides, which is predicted to cause elimination of codon 128 and frameshifts thereafter. The clp1 transcript was normally detected only in the presence of the A protein heterodimer formed when homokaryons with compatible A genes were mated. Forced expression of clp1 by promoter replacements induced clamp development without the need for a compatible A gene combination. These results indicate that expression of clp1 is necessary and sufficient for induction of the A-regulated pathway that leads to clamp development.

Effectiveness of personalized selection of antihypertensive therapy based on genetic polymorphism

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
S.V Shlyk ◽  
N.V Drobotya ◽  
L.A Khaisheva ◽  
L.V Arytyunyan ◽  
A.A Pirozhenko
Keyword(s):  
Blood Pressure ◽  
Genetic Polymorphism ◽  
Antihypertensive Therapy ◽  
Mutant Allele ◽  
Polymorphic Marker ◽  
Left Ventricular ◽  
Wall Stiffness ◽  
Echocardiographic Parameters ◽  
Functional Features ◽  
Agt Gene

Abstract   Recent data indicate that it is important to develop the problem of genetic polymorphism in patients with arterial hypertension (AH) for the long-term possibility of using it as a justification for choosing the optimal treatment strategy. Currently, despite the availability of effective antihypertensive agents, the percentage of patients who have reached the target blood pressure level remains still not enough. A rational basis for choosing a particular class of drugs and / or their combinations in a concrete patient with AH can be the detection of genetic markers that determine the degree of sensitivity to therapy, since the relationship of its effectiveness with the genetic features is not in doubt today. Material and methods The work is based on the results of a clinical, instrumental, laboratory and genetic examination of 41 patients with AH with insufficient effectiveness of previous antihypertensive therapy. The median age and duration of the disease were 54 (32; 70) years and 7 (1; 20) years, respectively. Taking into account the identified gene polymorphism, a fixed combination of an ACE inhibitor (perindopril 10 mg) and a thiazide-like diuretic (indapamide 2.5 mg) was assigned. The comparative dynamics of blood pressure daily monitoring, left ventricle echocardiographic parameters, as well as indicators of vascular wall stiffness were analyzed before therapy and 3 months later. Results The study established a relationship between the clinical and morpho-functional features of AH in the examined patients with polymorphism of the AGT, AGTR2, CYP11B2, GNB3, NOS3 genes:-786 of their heterozygotes and “mutant” homozygotes, of which 3 polymorphic genes (AGT, AGTR2, CYP11B2) encode the activity of ACE. The obtained results allowed to establish that positive dynamics of the studied indicators was revealed in all patients. Though patients, carriers of the mutant allele C of polymorphic marker T704C AGT gene, had statistically significant more expressed benefit changes in blood pressure daily profile, echocardiographic parameters (such as left ventricular mass index, indicators of left ventricular diastolic function) and all parameters of arterial wall stiffness compared with patients who do not carry the “mutant” allele. Conclusion Thus, the selected treatment regimen demonstrated maximum antihypertensive, cardio - and vasoprotective effectiveness in the group of AH patients with the presence of the allele 704C of the polymorphic marker T704C of the AGT gene, which indicates the perspectivity of using genetic approaches to develop personalized tactics of AH patients drug treatment. Funding Acknowledgement Type of funding source: None

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