Atypical Melting Curve Resulting from Genetic Variation in the 3′ Untranslated Region at Position 20218 in the Prothrombin Gene Analyzed with the LightCycler Factor II (Prothrombin) G20210A Assay

Tag, Carmen G.; Schifflers, Marie-Claire; Mohnen, Monika; Gressner, A. M.; Weiskirchen, Ralf

doi:10.1373/clinchem.2005.050021

Cited by 9 publications

(15 citation statements)

References 9 publications

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“…The other point mutation is a transition (A ! G) at nucleotide 20218, which has been described in a single individual; a 60-year-old woman of Slavic descent with a family history of thrombosis [8].…”

Section: Discussionmentioning

confidence: 99%

Abnormal melt curve profile during prothrombin 20210G → A analysis due to the 20209C → T variant

Dunn

Allen

Bates

et al. 2006

Blood Coagulation &Amp; Fibrinolysis

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The common factor II 20210G --> A mutation, located in the 3'-untranslated region, is an important risk factor for the development of thromboembolic disorders, especially in Caucasians. A number of methods are employed for clinical laboratory diagnosis of this mutation, some of which are capable of detecting adjacent 3'-end sequence variations. We present results from an African deep vein thrombosis patient tested for the 20210G --> A mutation by real-time polymerase chain reaction and melt-curve analysis using hybridization probes that incidentally detected an adjacent 3'-untranslated region variant. The patient sample was tested using the Factor II (Prothromobin) G20210A Kit (Roche Diagnostics, Indianapolis, Indiana, USA), in conjunction with the Roche LightCycler. A polymerase chain reaction fragment from the 3'-end of the F2 gene was subsequently sequenced for identification of the variant. Melt-curve analysis revealed a normal 20210*G peak and an unknown aberrant allelic peak. Following sequence analysis, the patient was determined to be heterozygous for 20209C --> T. The presence of the 20209C --> T variant in the current patient and in eight other reported individuals of African descent, most with thrombosis-associated complaints, suggests that this rare variant poses a potential increased risk for thromboembolic disease in this ethnic group.

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Section: Discussionmentioning

confidence: 99%

Abnormal melt curve profile during prothrombin 20210G → A analysis due to the 20209C → T variant

Dunn

Allen

Bates

et al. 2006

Blood Coagulation &Amp; Fibrinolysis

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“…Some of these well-established diagnostic assays have reported atypical melting peaks due to the presence of new nucleotide substitutions in the hybridization probe area beside those the assays were originally designed for. These additional nucleotide substitutions have shown varying clinical effects [23][24][25][26]. Atypical melting peaks are usually detected by T m shifts of 5-6…”

Section: Discussionmentioning

confidence: 99%

Rapid Genotyping of the Human Renin (REN) Gene by the LightCycler® Instrument: Identification of Unexpected Nucleotide Substitutions within the Selected Hybridization Probe Area

et al. 2010

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Abstract. Preeclampsia is a serious disorder affecting nearly 3% of all pregnancies in the Western world. It is associated with hypertension and proteinuria, and several lines of evidence suggest that the renin-angiotensin system (RAS) may be involved in the development of hypertension at different stages of a preeclamptic pregnancy. In this study, we developed rapid genotyping assays on the LightCycler instrument to allow the detection of genetic variants in the renin gene (REN) that may predispose to preeclampsia. The method is based on real-time PCR and allele-specific hybridization probes, followed by fluorescent melting curve analysis to expose a change in melting temperature (Tm). Ninety-two mother-father-child triads (n = 276) from preeclamptic pregnancies were genotyped for three haplotype-tagging single nucleotide polymorphisms (htSNPs) in REN. All three htSNPs (rs5705, rs1464816 and rs3795575) were successfully genotyped. Furthermore, two unexpected nucleotide substitutions (rs11571084 and rs61757041) were identified within the selected hybridization probe area of rs1464816 and rs3795575 due to aberrant melting peaks. In conclusion, genotyping on the LightCycler instrument proved to be rapid and highly reproducible. The ability to uncover additional nucleotide substitutions is particularly important in that it allows the identification of potentially etiological variants that might otherwise be overlooked by other genotyping methods.

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“…22 Similarly, the A20218G mutation was identified. 23 Because different samples vary slightly in absolute Tm, relying on the Tm difference between normal and variant alleles (⌬Tm) is preferable for identifying new variants. 24,25 An unexpected F5 1689GϾA heterozygote was initially interpreted as a F5 Leiden heterozygote by absolute Tm, but was later correctly identified by ⌬Tm.…”

Section: Identifying Novel Variantsmentioning

confidence: 99%

LightCycler Technology in Molecular Diagnostics

Lyon

Wittwer

2009

The Journal of Molecular Diagnostics

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From the first report of the LightCycler as a real-time polymerase chain reaction (PCR) instrument 1 clinical laboratories quickly realized its potential as a diagnostic tool. Quantitative applications important for infectious disease and oncology applications were followed by genotyping assays that took advantage of accurate temperature control to melt DNA amplicons or probe/amplicon duplexes. The first Food and Drug Administration-cleared molecular genetic assays were developed for this platform, specifically genotyping single base variants in F5 and F2. According to a College of American Pathologists survey, 2 ϳ30% of clinical laboratories report using the LightCycler for these two assays. This article will review the history of LightCycler technology, focusing on those applications widely incorporated into molecular diagnostics. In addition we will review recent developments that may impact future clinical testing.Real-time PCR instruments simultaneously amplify and detect, eliminating the need to open tubes containing PCR amplicon and therefore reducing the risk of future contamination. Fluorescent dyes or probes allow continuous monitoring as template DNA is amplified.3 By monitoring fluorescence every cycle, the amount of original target can be calculated. By monitoring fluorescence as the temperature changes, genotyping and heterozygote scanning can be performed by melting analysis, often removing the need for downstream analysis.The first two platforms developed for real-time PCR were the LightCycler (Roche, Indianapolis, IN) and the ABI 7700 (Applied Biosystems, Foster City, CA). The instruments were as different as the groups that created them. At the time, ABI was the undisputed corporate leader of PCR technology and the 7700 was a large 96-well laser-based instrument focused on throughput.

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Atypical Melting Curve Resulting from Genetic Variation in the 3′ Untranslated Region at Position 20218 in the Prothrombin Gene Analyzed with the LightCycler Factor II (Prothrombin) G20210A Assay

Cited by 9 publications

References 9 publications

Abnormal melt curve profile during prothrombin 20210G → A analysis due to the 20209C → T variant

Abnormal melt curve profile during prothrombin 20210G → A analysis due to the 20209C → T variant

Rapid Genotyping of the Human Renin (REN) Gene by the LightCycler® Instrument: Identification of Unexpected Nucleotide Substitutions within the Selected Hybridization Probe Area

LightCycler Technology in Molecular Diagnostics

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