scholarly journals A Fast, Sensitive and Accurate High Resolution Melting (HRM) Technology-Based Assay to Screen for Common K-ras Mutations

2009 ◽  
Vol 31 (3) ◽  
pp. 161-167
Author(s):  
D. Kramer ◽  
F. B. Thunnissen ◽  
M. I. Gallegos-Ruiz ◽  
E. F. Smit ◽  
P. E. Postmus ◽  
...  
Keyword(s):  
High Resolution ◽  
High Resolution Melting ◽  
Mutational Analysis ◽  
Direct Sequencing ◽  
Homozygous Mutation ◽  
Analytical Sensitivity ◽  
Nucleotide Substitutions ◽  
Cycle Sequencing ◽  
Ras Mutations ◽  
Exon 1

Background: Increasing evidence points to a negative correlation between K-ras mutations and patient’s response to, or survival benefit after, treatment with EGFR-inhibitors. Therefore, rapid and reliable assays for mutational analysis of the K-ras gene are strongly needed.Methods: We designed a high resolution melting (HRM) technology-based approach followed by direct sequencing to determine K-ras exon 1 (codons 12/13) tumour genotype.Results: Reconstruction experiments demonstrated an analytical sensitivity of the K-ras exon 1 HRM assay following sequencing of 1.5–2.5% of mutated DNA in a background of wild-type DNA. Assay reproducibility and accuracy were 100%. Application of the HRM assay following sequencing onto genomic DNA isolated from formalin-fixed paraffin-embedded tumour specimens of non-small cell lung cancer (n=91) and colorectal cancer (n=7) patients revealed nucleotide substitutions at codons 12 or 13, including a homozygous mutation, in 33 (34%) and 5 (5%) cases, respectively. Comparison to conventional nested-PCR following cycle-sequencing showed an overall high agreement in genotype findings (kappa value of 0.96), with more mutations detected by the HRM assay following sequencing.Conclusion: HRM allows rapid, reliable and sensitive pre-screening of routine diagnostic specimens for subsequent genotyping of K-ras mutations, even if present at low abundance or homozygosity, and may considerably facilitate personalized therapy planning.

scholarly journals A Panel of High Resolution Melting (HRM) Technology-Based Assays with Direct Sequencing Possibility for Effective Mutation Screening of EGFR and K-ras Genes

2009 ◽  
Vol 31 (5) ◽  
pp. 329-333
Author(s):  
D. A. M. Heideman ◽  
F. B. Thunnissen ◽  
M. Doeleman ◽  
D. Kramer ◽  
H. M. Verheul ◽  
...  
Keyword(s):  
High Resolution ◽  
Targeted Therapies ◽  
Nested Pcr ◽  
High Resolution Melting ◽  
Direct Sequencing ◽  
Mutation Screening ◽  
Tumour Response ◽  
Egfr Mutations ◽  
Cycle Sequencing ◽  
Ras Genes

Background: Increasing data from clinical trials support EGFR and K-ras mutation status as predictive markers of tumour response to EGFR-targeted therapies. Consequently, rapid and reliable mutation screening assays are demanded to guide rational use of EGFR-targeted therapies.Methods: In this study, we describe the development of high resolution melting (HRM) technology-based assays with direct sequencing confirmation possibility for mutation screening of the EGFR gene (exons 19, 20 and 21) in routine diagnostic specimens, and compared assay findings to those of conventional nested-PCR following cycle-sequencing.Results: In reconstruction experiments, each HRM assay following sequencing demonstrated a sensitivity of ≤5% of mutated DNA in a background of wild-type DNA. The panel of EGFR HRM assays following sequencing applied to a series of genomic DNA samples isolated from 68 FFPE NSCLC specimens correctly identified all EGFR mutations that were previously found by nested-PCR following cycle-sequencing. The HRM approach additionally scored two mutations not detected by the conventional assay.Complementary HRM following sequencing for K-ras revealed three mutations. EGFR and K-ras mutations were mutually exclusive.Conclusions: The panel of designed HRM assays with direct reflex sequencing possibility provides an effective method for mutation screening of EGFR and K-ras genes in routine diagnostic specimens, thereby allowing the selection of the treatment of choice in clinical practice.

scholarly journals Parental mosaicism in Marfan and Ehlers–Danlos syndromes and related disorders

2021 ◽  
Author(s):  
Bertrand Chesneau ◽  
Aurélie Plancke ◽  
Guillaume Rolland ◽  
Nicolas Chassaing ◽  
Christine Coubes ◽  
...  
Keyword(s):  
High Resolution ◽  
Marfan Syndrome ◽  
High Resolution Melting ◽  
De Novo ◽  
Direct Sequencing ◽  
Causal Variant ◽  
Connective Tissue Disorder ◽  
High Resolution Melting Analysis ◽  
Aortic Diseases ◽  
Melting Analysis

AbstractMarfan syndrome (MFS) is a heritable connective tissue disorder (HCTD) caused by pathogenic variants in FBN1 that frequently occur de novo. Although individuals with somatogonadal mosaicisms have been reported with respect to MFS and other HCTD, the overall frequency of parental mosaicism in this pathology is unknown. In an attempt to estimate this frequency, we reviewed all the 333 patients with a disease-causing variant in FBN1. We then used direct sequencing, combined with High Resolution Melting Analysis, to detect mosaicism in their parents, complemented by NGS when a mosaicism was objectivized. We found that (1) the number of apparently de novo events is much higher than the classically admitted number (around 50% of patients and not 25% as expected for FBN1) and (2) around 5% of the FBN1 disease-causing variants were not actually de novo as anticipated, but inherited in a context of somatogonadal mosaicisms revealed in parents from three families. High Resolution Melting Analysis and NGS were more efficient at detecting and evaluating the level of mosaicism compared to direct Sanger sequencing. We also investigated individuals with a causal variant in another gene identified through our “aortic diseases genes” NGS panel and report, for the first time, on an individual with a somatogonadal mosaicism in COL5A1. Our study shows that parental mosaicism is not that rare in Marfan syndrome and should be investigated with appropriate methods given its implications in patient’s management.

scholarly journals Extreme PCR: Efficient and Specific DNA Amplification in 15–60 Seconds

2015 ◽  
Vol 61 (1) ◽  
pp. 145-153 ◽  
Author(s):  
Jared S Farrar ◽  
Carl T Wittwer
Keyword(s):  
High Resolution ◽  
Single Molecule ◽  
High Resolution Melting ◽  
Dna Amplification ◽  
Temperature Cycling ◽  
High Yield ◽  
Temperature Cycle ◽  
Amplification Efficiency ◽  
Analytical Sensitivity ◽  
Agarose Gels

Abstract BACKGROUND PCR is a key technology in molecular biology and diagnostics that typically amplifies and quantifies specific DNA fragments in about an hour. However, the kinetic limits of PCR are unknown. METHODS We developed prototype instruments to temperature cycle 1- to 5-μL samples in 0.4–2.0 s at annealing/extension temperatures of 62 °C–76 °C and denaturation temperatures of 85 °C–92 °C. Primer and polymerase concentrations were increased 10- to 20-fold above typical concentrations to match the kinetics of primer annealing and polymerase extension to the faster temperature cycling. We assessed analytical specificity and yield on agarose gels and by high-resolution melting analysis. Amplification efficiency and analytical sensitivity were demonstrated by real-time optical monitoring. RESULTS Using single-copy genes from human genomic DNA, we amplified 45- to 102-bp targets in 15–60 s. Agarose gels showed bright single bands at the expected size, and high-resolution melting curves revealed single products without using any “hot start” technique. Amplification efficiencies were 91.7%–95.8% by use of 0.8- to 1.9-s cycles with single-molecule sensitivity. A 60-bp genomic target was amplified in 14.7 s by use of 35 cycles. CONCLUSIONS The time required for PCR is inversely related to the concentration of critical reactants. By increasing primer and polymerase concentrations 10- to 20-fold with temperature cycles of 0.4–2.0 s, efficient (>90%), specific, high-yield PCR from human DNA is possible in <15 s. Extreme PCR demonstrates the feasibility of while-you-wait testing for infectious disease, forensics, and any application where immediate results may be critical.

Common Genetic Variants of MUTYH are not Associated with Cutaneous Malignant Melanoma: Application of Molecular Screening by Means of High-Resolution Melting Technique in a Pilot Case-Control Study

2011 ◽  
Vol 26 (1) ◽  
pp. 37-42 ◽  
Author(s):  
Concetta Santonocito ◽  
Andrea Paradisi ◽  
Rodolfo Capizzi ◽  
Eleonora Torti ◽  
Sara Lanza-Silveri ◽  
...  
Keyword(s):  
High Resolution ◽  
High Resolution Melting ◽  
Direct Sequencing ◽  
Strong Association ◽  
Laboratory Data ◽  
Gene Mutations ◽  
Case Control ◽  
Common Genetic Variants ◽  
Mutyh Gene ◽  
Embryonic Origin

MUTYH glycosylase recognizes the 8-oxoG:A mismatch and is able to excise the adenine base using proofreading mechanisms. Some papers have reported a strong association between cancer development or aggressiveness and MUTYH gene mutations. The aim of this study was to find a possible association between the most frequent MUTYH mutations and melanoma in the context of a case-control pilot study. One hundred ninety-five melanoma patients and 195 healthy controls were matched for sex and age. Clinical and laboratory data were collected in a specific database and all individuals were analyzed for MUTYH mutations by high-resolution melting and direct sequencing techniques. Men and women had significantly different distributions of tumor sites and phototypes. No significant associations were observed between the Y165C, G382D and V479F MUTYH mutations and risk of melanoma development or aggressiveness. Our preliminary findings therefore do not confirm a role for MUTYH gene mutations in the melanoma risk. Further studies are necessary for the assessment of MUTYH not only in melanoma but also other cancer types with the same embryonic origin, in the context of larger arrays studies of genes involved in DNA stability or integrity.

In-Vitro Cytotoxicity of Tipifarnib (Zarnestra TM) in Pediatric AML and ALL Samples Is Independent of RAS Mutations.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1172-1172 ◽  
Author(s):  
Bianca F. Goemans ◽  
Christian M. Zwaan ◽  
Gertjan J.L. Kaspers ◽  
Karel Hählen ◽  
Dirk Reinhardt ◽  
...  
Keyword(s):  
Clinical Studies ◽  
Direct Sequencing ◽  
Farnesyltransferase Inhibitors ◽  
Exon 2 ◽  
Ras Mutations ◽  
Lc50 Value ◽  
Significant Difference ◽  
Exon 1 ◽  
Pediatric Aml

Abstract The farnesyltransferase inhibitor tipifarnib (Zarnestra™) was originally developed to target malignancies harbouring RAS mutations. In the first clinical studies with tipifarnib, in adults with leukemia, it was found that patients who responded did not harbour any RAS mutations, suggesting a different mechanism of response. In a previous study we showed that 18% of 150 untreated pediatric AML patients harbour mutations in RAS, of which 30% were CBF-AML. We now studied 44 untreated and 13 relapsed pediatric AML, as well as 22 untreated ALL samples for mutations in RAS, using D-HPLC and direct sequencing. In vitro tipifarnib resistance was determined by a 4-day MTT assay (concentration 0.016-51μM, kindly provided by Janssen Research). The LC50 value, the concentration at which 50% of cells are killed by tipifarnib, was used as a measure of resistance. Patient characteristics were; for untreated AML: 64% boys; median age 9.3 years; median WBC 74.8x109/L; FAB 2xM0, 2xM1, 8xM2, 3xM3, 16xM4, 8xM5, 5x unclassified; for relapsed AML: 77% boys; median age 4.0 years; median WBC 41.6x109/L; FAB 2xM0, 2xM2, 3xM4, 2xM5, 2xM7, 2x unclassified; for untreated ALL: 73%boys; median age 6.0 years; median WBC 10.2x109/L; 15 B-cell precursor (BCP) ALL and 7 T-ALL. We found RAS mutations in 14 (32%) untreated AML samples (N-RAS : 8 samples exon 1, 1 sample exon 2; K-RAS: 5 samples exon 1 mutations). In relapsed AML 2 samples showed an N-RAS exon 1 mutation (15.4%). In ALL 18.2% had a RAS mutation: an N-RAS exon 1 mutation was found in 2 patients (9.1%) and a K-RAS exon 1 mutation in another 2 patients (9.1%). The distribution of tipifarnib sensitivity was similar in RAS mutated- and non-mutated untreated AML patients [median LC50 RAS mutated 7.1μM (P25-P75: 6.0-9.6μM) vs. non-mutated 4.9μM (P25-P75 2.3-8.2μM); p=0.199]. When we compared N-RAS mutated samples with K-RAS mutated samples there was no statistically significant difference in sensitivity to tipifarnib (median LC50 [p25-p75] 3.2μM [2.9-3.9μM] and 4.9μM [3.7-23.1μM], p=0.20), and comparing them separately with non-mutated AML did not show differences in sensitivity to tipifarnib (p=0.172 and p=0.463 respectively). One out of 9 (11%) N-RAS mutated and 3 out of 5 (60%) K-RAS mutated samples had an LC50 value above the 75th percentile for non-mutated AML and were considered resistant. Within relapsed AML the 2 RAS mutated samples had LC50 values of 0.83 and 6.3μM, versus a median value of 6.9μM for non-mutated relapsed AML. In ALL, we found similar results [median LC50 RAS mutated 7.8μM (P25-75: 4.1-12.8μM) vs. non-mutated 17.4μM (P25-75: 4.5-22.9μM), p=0.3], but the groups were very small. In conclusion, primary pediatric AML and ALL samples withRAS mutations show similar distributions of tipifarnib sensitivity as samples withoutRAS mutations. Hence, some RAS mutated samples may be relatively in-vitro resistant to tipifarnib, and some non-mutated samples may be relatively sensitive. Therefore, clinical studies with these compounds should not be restricted to RAS-mutated leukemia. Further studies are necessary to determine the molecular targets of farnesyltransferase inhibitors.

scholarly journals Evaluation of High-Resolution Melting Curve Analysis (HRM) assay for Detection of Pseudomonas aeruginosa PASGNDM699: A dangerous New Delhi metallo-β-lactamase (NDM) strain

2019 ◽  
Author(s):  
Sanaz Dehbashi ◽  
Hamed Tahmasebi ◽  
Mohammad Arabestani
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Melting Point ◽  
High Resolution ◽  
High Resolution Melting ◽  
Melting Curve ◽  
Curve Analysis ◽  
Melting Curve Analysis ◽  
New Delhi ◽  
Analytical Sensitivity ◽  
High Resolution Melting Curve

Abstract Background: New Delhi metallo-β-lactamase (NDM-1) is a broad spectrum β-lactamase that is able to inactivate all β-lactams except aztreonam, as is typical of metallo-β-lactamases. NDM-1 producers in Pseudomonas aeruginosa, especially PASGNDM699 strain, cause a range of infections such as urinary tract, diarrhoea and soft tissue infections. The aim of this study was to Standardization of High-Resolution Melting Curve Analysis (HRM) assay for detection of P. aeruginosa, especially PASGNDM699 strain. Methods: The HRM method was done on standard strains of P. aeruginosa strains. 9-fold Serial dilutions of known DNA concentrations, extracted from standard isolates were prepared and tested by Real Time Melting curve and HRM assay. Data analysis was performed using the StepOne Software v2.3 and HRM Software v3.0.1 (Applied Biosystems, Ltd). Results: Based on the results of the Real Time PCR assay and melt curve analysis, melting point temperatures of the N-1, N-2 and N-3 amplicon for isolates identified as NDM strains were 87.57°C, 76.92°C and 82.97°C, respectively. Furthermore, melting point temperatures of the blaVIM, blaSPM and blaSIM amplicon for isolates identified as MBL strains were 84.56°C, 85.35°C and 86.62°C, respectively. Due to the analytical specificity of the primers, all dilutions with a similar Tm and melt peaks were obtained in the melting curves. Moreover, the analytical sensitivity of NDM primer were able to detected 100CFU/mL, 103CFU/mL and 104CFU/mL of standard DAN by N-1, N-2 and N-3 primers, respectively. Also, according to analytical sensitivity of MBL primers, blaVIM was able detected of 100CFU/mL, blaSPM primer 105CFU/mL and blaSIM primer 102CFU/mL of PASGNDM699 strain. HRM results showed that N-1 primers with 55 bp and blaVIM primers with 124 bp had the highest sensitivity and specificity for P. aeruginosa PASGNDM699 strain identification. Conclusion: The data from our study indicated that the sensitivity and specificity of the HRM method linked to the primer length and the fluorescent dye. Further, we can identify antibiotic resistance in substrates such as P. aeruginosa PASGNDM699 by software analysis and melting curve analysis.

EGFR mutations detected by high-resolution melting analysis (HRMA) as a predictor of response and survival in non-small cell lung cancer (NSCLC) patients treated with gefitinib

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 7075-7075
Author(s):  
T. Takano ◽  
Y. Ohe ◽  
K. Furuta ◽  
K. Tsuta ◽  
K. Nomoto ◽  
...  
Keyword(s):  
High Resolution ◽  
High Resolution Melting ◽  
Response Rate ◽  
Direct Sequencing ◽  
False Negative ◽  
High Resolution Melting Analysis ◽  
Egfr Mutations ◽  
Wild Type ◽  
Minor Response ◽  
Melting Analysis

7075 Background: Recent studies have shown that EGFR mutations, mainly deletions in exon 19 (DEL) and L858R, are associated with gefitinib sensitivity in patients (pts) with NSCLC. We established a new easy method, using high-resolution melting analysis (HRMA), for detecting DEL and L858R mutations even from small biopsy or cytology samples, and evaluated the significance of EGFR mutations in NSCLC on a larger scale. Methods: Among 364 advanced or recurrent NSCLC pts treated with gefitinib between Jul 2002 and Dec 2004, HRMA was performed in 207 pts from whom specimens were available. DNA extracted from the archival tissue or cytology samples not subjected to microdissection was analyzed to detect DEL and L858R using HR-1 (Idaho Technology), an HRMA device. To validate this method, the results were compared with direct sequencing data obtained from microdissected tumor cells from surgical specimens in 66 pts. Results: Tissue/cytology/both samples were analyzed in 91/77/39 pts. EGFR mutations were detected in 85 (41%; DEL/L858R: 49/36) of the 207 pts. In the comparison with direct sequencing, consistent results were obtained from all of the 66 tissue samples, while false negative results were obtained in 2 of the 28 cytology samples. EGFR mutations were seen more frequently in women (54% vs. 31%; P = .001), never-smokers (53% vs. 32%; P = .002), and pts with adenocarcinoma (44% vs. 11%; P = .007). CR/PR/SD/PD was observed in 2/64/11/8 pts with EGFR mutations and in 0/10/23/89 pts with wild-type EGFR. The response rate (78% vs. 8%), time to progression (median, 9.1 vs. 1.6 months) and overall survival (median, 19.9 vs. 9.1 months) were all significantly superior in pts with EGFR mutations (P < .0001). Minor response and/or long SD (>6 months) was observed more frequently in SD pts with EGFR mutations than in those with wild-type EGFR (91% vs. 26%; P < .001). Among the pts with EGFR mutations, the response rate was significantly higher in the pts with DEL than in those with L858R (86% vs. 67%; P = .037). Conclusions: HRMA is a practical and precise method to detect DEL and L858R mutations. EGFR mutations strongly predict a better response and longer survival in NSCLC pts treated with gefitinib. No significant financial relationships to disclose.

scholarly journals A Novel Homozygous Mutation in the EC1/EC2 Interaction Domain of the Gap Junction Complex Connexon 26 Leads to Profound Hearing Impairment

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Ralf Birkenhäger ◽  
Nicola Prera ◽  
Antje Aschendorff ◽  
Roland Laszig ◽  
Susan Arndt
Keyword(s):  
Gap Junction ◽  
Inner Ear ◽  
Hearing Impairment ◽  
Mutational Analysis ◽  
Extended Family ◽  
Direct Sequencing ◽  
Ion Homeostasis ◽  
Homozygous Mutation ◽  
Interaction Domain ◽  
Function Relationship

To date, about 165 genetic loci or genes have been identified which are associated with nonsyndromal hearing impairment. In about half the cases, genetic defects in theGJB2gene (connexin 26) are the most common cause of inner-ear deafness. The genesGJB2andGJB6are localized on chromosome 13q11-12 in tandem orientation. Connexins belong to the group of “gap junction” proteins, which form connexons, each consisting of six connexin molecules. These are responsible for the exchange of ions and smaller molecules between neighboring cells. Mutational analysis in genesGJB2andGJB6was brought by direct sequencing of the coding exons including the intron transitions. Here we show in the participating extended family a homozygous mutation c.506G>A, (TGC>TAC) p.Cys169Tyr, in theGJB2gene, which could be proven for the first time and led to nonsyndromal severe hearing impairment in the afflicted patients. The mutation is located in the EC1/EC2 interaction complex of the gap junction connexon 26 complex and interrupts the K+circulation and therefore the ion homeostasis in the inner ear. The homozygous mutation p.Cys169Tyr identified here provides a novel insight into the structure-function relationship of the gap junction complex connexin/connexon 26.

scholarly journals UGT1A1 (TA)n genotyping in sickle-cell disease: High resolution melting (HRM) curve analysis or direct sequencing, what is the best way?

2013 ◽  
Vol 424 ◽  
pp. 258-260 ◽  
Author(s):  
Vincent Thomas ◽  
Blandine Mazard ◽  
Caroline Garcia ◽  
Philippe Lacan ◽  
Marie-Claude Gagnieu ◽  
...  
Keyword(s):  
High Resolution ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
High Resolution Melting ◽  
Direct Sequencing ◽  
Curve Analysis ◽  
Cell Disease

scholarly journals Rapid and Sensitive Detection of BRCA1/2 Mutations in a Diagnostic Setting: Comparison of Two High-Resolution Melting Platforms

2008 ◽  
Vol 54 (6) ◽  
pp. 982-989 ◽  
Author(s):  
Kim De Leeneer ◽  
Ilse Coene ◽  
Bruce Poppe ◽  
Anne De Paepe ◽  
Kathleen Claes
Keyword(s):  
High Resolution ◽  
High Resolution Melting ◽  
Direct Sequencing ◽  
Turnaround Time ◽  
High Resolution Melting Analysis ◽  
Coding Region ◽  
Brca1 And Brca2 ◽  
Diagnostic Laboratory ◽  
Sensitivity Setting ◽  
Melting Analysis

Abstract Background: High-resolution melting is an emerging technique for detection of nucleic acid sequence variations. Developments in instrumentation and saturating intercalating dyes have made accurate high-resolution melting analysis possible and created opportunities to use this technology in diagnostic settings. We evaluated 2 high-resolution melting instruments for screening BRCA1 and BRCA2 mutations. Methods: To cover the complete coding region and splice sites, we designed 112 PCR amplicons (136–435 bp), amplifiable with a single PCR program. LCGreen® Plus was used as the intercalating dye. High-resolution melting analysis was performed on the 96-well Lightscanner™ (Idaho Technology Inc.) and the 96-well LightCycler® 480 (Roche) instruments. We evaluated sensitivity by analyzing 212 positive controls scattered over almost all amplicons and specificity by blind screening of 22 patients for BRCA1 and BRCA2. In total, we scanned 3521 fragments. Results: All 212 known heterozygous sequence variants were detected on the Lightscanner by analysis on normal sensitivity setting. On the LightCycler 480, the standard instrument sensitivity setting of 0.3 had to be increased to 0.7 to detect all variants, decreasing the specificity to 95.9% (vs 98.7% for the Lightscanner). Conclusions: Previously, we screened BRCA1/2 by direct sequencing of the large exon 11 and denaturing gel gradient electrophoresis (DGGE) for all other coding exons. Since the introduction of high-resolution melting, our turnaround time has been one third of that with direct sequencing and DGGE, as post-PCR handling is no longer required and the software allows fast analyses. High-resolution melting is a rapid, cost-efficient, sensitive method simple enough to be readily implemented in a diagnostic laboratory.

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