Melting temperature of molecular beacons as an indicator of the ligase detection reaction for multiplex detection of point mutations

A novel strategy based on the ligase detection reaction (LDR) using the melting temperature of molecular beacons as the indicator is presented for the multiplex detection of gene mutations.

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Molecular Basis of In Vivo Resistance to Sulfadoxine-Pyrimethamine in African Adult Patients Infected withPlasmodium falciparum Malaria Parasites

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.42.7.1811 ◽

1998 ◽

Vol 42 (7) ◽

pp. 1811-1814 ◽

Cited By ~ 45

Author(s):

Leonardo K. Basco ◽

Rachida Tahar ◽

Pascal Ringwald

Keyword(s):

Dihydrofolate Reductase ◽

Point Mutations ◽

Gene Mutations ◽

Adult Patients ◽

Wild Type ◽

Dihydropteroate Synthase ◽

Reductase Gene ◽

Parasite Populations

ABSTRACT In vitro sulfadoxine and pyrimethamine resistance has been associated with point mutations in the dihydropteroate synthase and dihydrofolate reductase domains, respectively, but the in vivo relevance of these point mutations has not been well established. To analyze the correlation between genotype and phenotype, 10 Cameroonian adult patients were treated with sulfadoxine-pyrimethamine and followed up for 28 days. After losses to follow-up (n = 1) or elimination of DNA samples due to mixed parasite populations with pyrimethamine-sensitive and pyrimethamine-resistant profiles (n = 3), parasite genomic DNA from day 0 blood samples of six patients were analyzed by DNA sequencing. Three patients who were cured had isolates characterized by a wild-type or mutant dihydrofolate reductase gene (with one or two mutations) and a wild-type dihydropteroate synthase gene. Three other patients who failed to respond to sulfadoxine-pyrimethamine treatment carried isolates with triple dihydrofolate reductase gene mutations and either a wild-type or a mutant dihydropteroate synthase gene. Three dihydrofolate reductase gene codons (51, 59, and 108) may be reliable genetic markers that can accurately predict the clinical outcome of sulfadoxine-pyrimethamine treatment in Africa.

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The Wiskott-Aldrich syndrome and X-linked congenital thrombocytopenia are caused by mutations of the same gene

Blood ◽

10.1182/blood.v86.10.3797.bloodjournal86103797 ◽

1995 ◽

Vol 86 (10) ◽

pp. 3797-3804 ◽

Cited By ~ 150

Author(s):

Q Zhu ◽

M Zhang ◽

RM Blaese ◽

JM Derry ◽

A Junker ◽

...

Keyword(s):

Clinical Symptoms ◽

Point Mutations ◽

Gene Mutations ◽

Exon Skipping ◽

Splice Site Mutation ◽

Missense Mutations ◽

Site Mutation ◽

Exon 2 ◽

Wiskott Aldrich Syndrome ◽

Was Gene

The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized by thrombocytopenia, small platelets, eczema, recurrent infections, and immunodeficiency. Besides the classic WAS phenotype, there is a group of patients with congenital X-linked thrombocytopenia (XLT) who have small platelets but only transient eczema, if any, and minimal immune deficiency. Because the gene responsible for WAS has been sequenced, it was possible to correlate the WAS phenotypes with WAS gene mutations. Using a fingerprinting screening technique, we determined the approximate location of the mutation in 13 unrelated WAS patients with mild to severe clinical symptoms. Direct sequence analysis of cDNA and genomic DNA obtained from patient-derived cell lines showed 12 unique mutations distributed throughout the WAS gene, including insertions, deletions, and point mutations resulting in amino acid substitutions, termination, exon skipping, or splicing defects. Of 4 unrelated patients with the XLT phenotype, 3 had missense mutations affecting exon 2 and 1 had a splice-site mutation affecting exon 9. Patients with classic WAS had more complex mutations, resulting in termination codons, frameshift, and early termination. These findings provide direct evidence that XLT and WAS are caused by mutations of the same gene and suggest that severe clinical phenotypes are associated with complex mutations.

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Genetic diseases of the Kennedy pathways for membrane synthesis

Journal of Biological Chemistry ◽

10.1074/jbc.rev120.013529 ◽

2020 ◽

Vol 295 (51) ◽

pp. 17877-17886

Author(s):

Mahtab Tavasoli ◽

Sarah Lahire ◽

Taryn Reid ◽

Maren Brodovsky ◽

Christopher R. McMaster

Keyword(s):

Single Gene ◽

Genetic Diseases ◽

Point Mutations ◽

Gene Mutations ◽

Hereditary Diseases ◽

Biological Functions ◽

Phospholipid Synthesis ◽

Spastic Paraplegia ◽

Kennedy Pathway ◽

Single Gene Disorders

The two branches of the Kennedy pathways (CDP-choline and CDP-ethanolamine) are the predominant pathways responsible for the synthesis of the most abundant phospholipids, phosphatidylcholine and phosphatidylethanolamine, respectively, in mammalian membranes. Recently, hereditary diseases associated with single gene mutations in the Kennedy pathways have been identified. Interestingly, genetic diseases within the same pathway vary greatly, ranging from muscular dystrophy to spastic paraplegia to a childhood blinding disorder to bone deformations. Indeed, different point mutations in the same gene (PCYT1; CCTα) result in at least three distinct diseases. In this review, we will summarize and review the genetic diseases associated with mutations in genes of the Kennedy pathway for phospholipid synthesis. These single-gene disorders provide insight, indeed direct genotype-phenotype relationships, into the biological functions of specific enzymes of the Kennedy pathway. We discuss potential mechanisms of how mutations within the same pathway can cause disparate disease.

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Solid-Phase Amplification for Detection of C282Y and H63D Hemochromatosis (HFE) Gene Mutations

Clinical Chemistry ◽

10.1093/clinchem/47.8.1384 ◽

2001 ◽

Vol 47 (8) ◽

pp. 1384-1389 ◽

Cited By ~ 11

Author(s):

Mark S Turner ◽

Sarah Penning ◽

Angela Sharp ◽

Valentine J Hyland ◽

Ray Harris ◽

...

Keyword(s):

Solid Phase ◽

Point Mutations ◽

Gene Mutations ◽

Pcr Primers ◽

Clinical Samples ◽

Hfe Gene ◽

Target Sequence ◽

Nucleotide Polymorphisms ◽

Nitrophenyl Phosphate ◽

Hemochromatosis Gene

Abstract Background: There is a need for simple, rapid, and inexpensive methods for the detection of single-nucleotide polymorphisms. Our aim was to develop a single-tube ELISA-like PCR assay and evaluate it by detecting the common C282Y and H63D mutations found in the hemochromatosis gene (HFE) by use of clinical samples. Methods: The method, termed solid-phase amplification (SPA), involves dual liquid- and solid-phase amplification of a target sequence by the use of two PCR primers, one of which is in two forms: the first is covalently immobilized to the wall of a microwell, and the second is free in solution. During allele-specific amplification, both the free and solid-phase amplicons are labeled by incorporation of digoxigenin (DIG)-dUTP. The amount of surface-bound amplicon is determined colorimetrically by the use of an alkaline phosphatase-anti-DIG-Fab conjugate and p-nitrophenyl phosphate. Results: Two different amplicon-labeling methods were evaluated. Analysis of 173 clinical samples for the C282Y and H63D HFE point mutations with SPA revealed that only one sample was incorrectly diagnosed, apparently because of operator error, when compared with conventional restriction fragment length polymorphism assay results. Conclusions: The SPA assay has potential for medium-scale mutation detection, having the advantage of being manipulatively simple and immediately adaptable for use in clinical laboratories with existing ELISA instrumentation.

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Point mutation bias in SARS-CoV-2 variants results in increased ability to stimulate inflammatory responses

Scientific Reports ◽

10.1038/s41598-020-74843-x ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Masato Kosuge ◽

Emi Furusawa-Nishii ◽

Koyu Ito ◽

Yoshiro Saito ◽

Kouetsu Ogasawara

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Rna Editing ◽

Inflammatory Responses ◽

Rna Viruses ◽

Point Mutations ◽

Gene Mutations ◽

Severe Pneumonia ◽

Mutation Bias ◽

Enhanced Production ◽

Tnf Α

Abstract Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection induces severe pneumonia and is the cause of a worldwide pandemic. Coronaviruses, including SARS-CoV-2, have RNA proofreading enzymes in their genomes, resulting in fewer gene mutations than other RNA viruses. Nevertheless, variants of SARS-CoV-2 exist and may induce different symptoms; however, the factors and the impacts of these mutations are not well understood. We found that there is a bias to the mutations occurring in SARS-CoV-2 variants, with disproportionate mutation to uracil (U). These point mutations to U are mainly derived from cytosine (C), which is consistent with the substrate specificity of host RNA editing enzymes, APOBECs. We also found the point mutations which are consistent with other RNA editing enzymes, ADARs. For the C-to-U mutations, the context of the upstream uracil and downstream guanine from mutated position was found to be most prevalent. Further, the degree of increase of U in SARS-CoV-2 variants correlates with enhanced production of cytokines, such as TNF-α and IL-6, in cell lines when compared with stimulation by the ssRNA sequence of the isolated virus in Wuhan. Therefore, RNA editing is a factor for mutation bias in SARS-CoV-2 variants, which affects host inflammatory cytokines production.

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DPY19L2 gene mutations are a major cause of globozoospermia: identification of three novel point mutations

MHR Basic science of reproductive medicine ◽

10.1093/molehr/gat018 ◽

2013 ◽

Vol 19 (6) ◽

pp. 395-404 ◽

Cited By ~ 30

Author(s):

F. Zhu ◽

F. Gong ◽

G. Lin ◽

G. Lu

Keyword(s):

Point Mutations ◽

Gene Mutations

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Mutations in the AML1 Gene Define an Unfavorable Subgroup in Acute Myeloid Leukemia with FAB M0.

Blood ◽

10.1182/blood.v104.11.4340.4340 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4340-4340

Author(s):

Frank Dicker ◽

Mirjam Klaus ◽

Torsten Haferlach ◽

Wolfgang Kern ◽

Wolfgang Hiddemann ◽

...

Keyword(s):

High Performance ◽

De Novo ◽

Mutation Screening ◽

Point Mutations ◽

Gene Mutations ◽

Normal Karyotype ◽

Runt Domain ◽

Screening Efficiency ◽

Genetic Aberration ◽

Aml1 Gene

Abstract The AML1/RUNX1 gene is the most frequent target for chromosomal translocations in leukemia. Recently point mutations in the AML1 gene have been demonstrated as another mode of genetic aberration. AML1 mutations have been reported in de novo MDS and AML, as well as in therapy related MDS and AML. The AML M0 subtype has been found to be most frequently affected by sporadic AML1 gene mutations. We analysed AML1 gene mutations in a cohort of 49 M0 patients. Mutation screening was performed either with SSCP (n=21) and/or denaturating High Performance Liquid Chromatography (dHPLC) (n=33), 5 cases were analyzed by both methods. SSCP screening of exons 3–5 of the AML1 gene was carried out at the genomic level. These exons cover the socalled Runt domain, which is most frequently mutated. Fragments with aberrant mobility were sequenced. With this method 5 cases were found to be mutated. Subsequently, to improve the screening efficiency an assay using dHPLC was established. Hereby, we screened the cDNA of patient samples for mutations in amino acid codons 1–277 of the AML1b transcript, where the Runt domain is located between codons 49 and 178. All 5 cases detected by SSCP were confirmed by dHPLC. Nine mutations were detected in the cohort of 28 cases (32%) which had not been analyzed by SSCP. In total, 14 of the 49 samples (29%) tested were identified to be mutated, which is a slightly higher frequency than previously reported. In the cohort of 35 AML1 non-mutated cases 20 (57%) had a normal karyotype and 15 (43%) an aberrant karyotypes, whereas only 6 of the 14 AML1 mutated cases (43%) had a normal karyotype (p=0.001). Three of the AML1 mutated cases (21%) also had FLT3 mutations. One had an FLT3-LM, one an FLT3-TKD mutation, and one case both LM and TKD mutations. Clinical follow up data were available for 33 patients (22 AML1 non- mutated, 11 AML1 mutated). The median OS and EFS of the AML1 non-mutated versus the mutated group was 276 days versus 63 days (p = 0.0679) and 276 vs. 63 days (p=0.0630) respectively. Thus the AML1 mutated cases tend to have a worse clinical outcome. When other AML subtypes were screened for AML1 mutations, i.e. M1 (n=26), M2 (n=21) and M4 (n=3), only 1 additional AML1 mutation was detected, confirming the highest prevalence of AML1 mutations in M0. In conclusion, 1) we established a new assay to screen for AML1 mutations. 2) We confirmed the high incidence of AML1 gene mutations in AML M0, both in cases with normal and aberrant karyotype. 3) For the first time we demonstrated that AML1 mutations define an unfavorable subentity in AML M0.

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Recurrent Mutations Found In Acute Monocytic Leukemia by Exomes Sequencing

Blood ◽

10.1182/blood.v116.21.2490.2490 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2490-2490

Author(s):

Xiao-Jing Yan ◽

Jie Xu ◽

Zhao-Hui Gu ◽

Chun-Ming Pan ◽

Gang Lv ◽

...

Keyword(s):

Conflicts Of Interest ◽

Prognostic Significance ◽

Point Mutations ◽

Gene Mutations ◽

Distinct Group ◽

Acute Monocytic Leukemia ◽

Monocytic Leukemia ◽

Genetic Changes ◽

Recurrent Mutations ◽

Acute Myelomonocytic Leukemia

Abstract Abstract 2490 Acute monocytic leukemia, the M5 subtype of acute myeloid leukemia (AML-M5), is a distinct group with characteristic clinical features and has been reported to have a poor prognosis. A subset of AML-M5 is associated with various chromosomal translocations involving the MLL locus at 11q23, while mutations are also reported in genes such as NPM1, FLT3, NRAS at different frequencies in this disease. However, these genetic changes occur only in a part of AML-M5 patients and some of them lack specificity due to the presence in other AML subtypes. To address the important genetic and molecular factors for the pathogenesis of M5 in a comprehensive manner, we sequenced exomes from nine AML-M5 initial bone marrow (BM) samples and matched control samples. We identified 64 somatic mutations within the coding sequences of 61 genes including 57 point mutations and 7 insertions or deletions (indels), among which 17 genes had at least 2 cases of mutations in 100 validated AML-M5 BM samples. We tested a part of these gene mutations in different subtypes of AML and found that some mutations are restricted to AML-M5 and AML-M4 (acute myelomonocytic leukemia) with more than 15% and 10% frequency, respectively. Thus, by systemic sequencing of exomes from a group of AML-M5 cases, we discovered recurring mutations that may play an essential role in the pathogenesis of AML with monocytic features.The prognostic significance of the mutations will be present in details. Disclosures: No relevant conflicts of interest to declare.

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Selection, adaptation, and bacterial operons

Genome ◽

10.1139/g89-044 ◽

1989 ◽

Vol 31 (1) ◽

pp. 265-271 ◽

Cited By ~ 22

Author(s):

Barry G. Hall

Keyword(s):

Structural Gene ◽

Point Mutations ◽

Gene Mutations ◽

Adaptive Mutation ◽

Evolutionary Potential ◽

Adaptive Mutations ◽

Metabolic Functions ◽

Metabolic Capability ◽

Regulatory Mutations ◽

Selection For

Bacteria are especially useful as systems to study the molecular basis of adaptive evolution. Selection for novel metabolic capabilities has allowed us to study the evolutionary potential of organisms and has shown that there are three major "strategies" for the evolution of new metabolic functions. (i) Regulatory mutations may allow a gene to be expressed under unusual conditions. If the product ofthat gene is already active toward a novel resource, then a regulatory mutation alone may confer a new metabolic capability. (ii) Structural gene mutations may alter the catalytic properties of enzymes so that they can act on novel substrates. These structural gene mutations may dramatically improve catalytic capabilities, and in some cases they can confer entirely new capabilities upon enzymes. In most cases both regulatory and structural gene mutations are required for the effective evolution of new metabolic functions. (iii) Operons that are normally silent, or cryptic, may be activated by either point mutations or by the action of mobile genetic elements. When activated, these operons can provide entirely new pathways for the metabolism of novel resources. Selection can also play a role in modulating the probability that a particular adaptive mutation will occur. In this paper I present evidence that a specific adaptive mutation, reversion of the metB1 mutation, occurs 60 to 80 times more frequently during prolonged selection on plates under conditions where the members of the population are not growing than it does in growing cells under nonselective conditions. This selective condition, methionine starvation, does not increase the frequency of other mutations unrelated to methionine biosynthesis. Thus, contrary to our present notions, selection can act not only to reveal preexisting mutations but to modulate the frequency with which adaptive mutations occur.Key words: mutation rates, molecular evolution, adaptive mutations, cryptic genes.

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