Mutation detection in lung tumors: Comparing high-resolution melting with direct sequencing

2008 ◽  
Vol 26 (15_suppl) ◽  
pp. 19021-19021
Author(s):  
E. H. Lim ◽  
C. Hough ◽  
J. McKinney ◽  
S. Zhang ◽  
R. Martin ◽  
...  
Keyword(s):  
High Resolution ◽  
Mutation Detection ◽  
High Resolution Melting ◽  
Direct Sequencing ◽  
Lung Tumors

High Resolution Melting Analysis for Rapid JAK2 Exon 14 and Exon12 Mutation Detection In Formalin Fixed Paraffin-Embeded Trephine Bone Marrow Biopsies of Patients with Polycythemia Vera. Detection of Novel JAK2 mutations

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1984-1984
Author(s):  
Tatiana Burjanivova ◽  
Roberto H Nussenzveig ◽  
Mohamed E Salama ◽  
Naser Aboud ◽  
Juraj Marcinek ◽  
...  
Keyword(s):  
Bone Marrow ◽  
High Resolution ◽  
Polycythemia Vera ◽  
Mutation Detection ◽  
High Resolution Melting ◽  
Direct Sequencing ◽  
High Resolution Melting Analysis ◽  
Jak2v617f Mutation ◽  
Specific Pcr ◽  
Melting Analysis

Abstract Abstract 1984 Polycythemia vera (PV) belongs to the group of Ph1 negative myeloproliferative neoplasms and is characterized by the presence of JAK2V617F somatic mutation in >95% of PV patients. In some patients lacking this mutation, alternative mutations in exon 12 of the JAK2 gene have been reported. Identification of JAK2 mutations is a major WHO criterion for diagnosis of PV. Therefore, reliable and sensitive methods are needed for detection of these mutations. High resolution melting analysis (HRM) for mutation detection in JAK2 exons 12 and 14 have been previously described using DNA isolated from fresh patient samples. We report here the development of an HRM screening method for both JAK2 exons 12 and 14. We have applied these methods for retrospective screening of archived paraffin-embedded BM biopsies from PV patients. A cohort of 101 PV patients was screened for JAK2 exons 12 and 14 mutations in DNA isolated from paraffin-embedded tissues. All included patients had bone marrow trephine biopsies exhibiting features consistent with or suspicious for PV. In addition, 6 samples with previously confirmed exon12 mutation by allele-specific PCR were tested by this HRM method. DNA was isolated from paraffin-embedded tissues after deparaffinization process in graded alcohols and xylene. Briefly, deparaffinized samples were digested with proteinase K for 3days and DNA was isolated using the DNeasy Micro Kit (Qiagen, Valencia, CA). Following DNA isolation, samples were analyzed by HRM for JAK2 exons 12 and 14. Short exon-specific PCR amplicons (90-100 bp) were generated, which allowed the detection of the mutation in degradated material. All abnormal samples were bi-directionally sequenced. All 6 known exon 12 mutated samples were identified by HRM. Moreover, we repeatedly identified an abnormal exon 12 melting curve in 1/101 paraffin embedded samples. Sequencing results for this sample indicate the presence of a large (~32 bp) duplication. All 7 positive samples were confirmed by direct sequencing (see table 1). Interestingly, all three delH538K539 insL samples identified used a distinct codon for L and also had reproducibly different melting profiles. Ninety-two of 101 patients were positive for JAK2V617F mutation. One patient sample exhibited an abnormal HRM profile from wild-type and V617F positive control analysis. Two mutations were found in this patient by direct sequencing including JAK2V617F mutation and a previously unreported mutation in exon 14 (L611S) and we are now determining if these two separate JAK2 mutations are in cis or trans. In summary, we developed an HRM assay suitable for detection JAK2 exons 12 and exon14 mutations in archival material. This method is suitable for routine laboratory detection of these mutations as well as screening of archived biological material for additional mutations. We report one case of JAK2V617F-positive PV presenting with an additional JAK2L611S. This work has been supported by the MPD-RC consortium (J.P.) and a “UICC International Cancer Technology Transfer Fellowship“ awarded to Dr. Burjanivova. TB, RN and MES contributed equally to this work. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Snapback Primer Mediated Clamping PCR for Detection ofEGFRandKRASMutations in NSCLC Patients by High Resolution Melting Analysis

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Haiyan Sun ◽  
Yang Yang ◽  
Lixin Yang ◽  
Bo Su ◽  
Gening Jiang ◽  
...  
Keyword(s):  
High Resolution ◽  
Somatic Mutations ◽  
Mutation Detection ◽  
High Resolution Melting ◽  
Direct Sequencing ◽  
Detection Sensitivity ◽  
Double Blind ◽  
Direct Dna Sequencing ◽  
Detection Of Mutations ◽  
Nsclc Patients

Assays for detecting somatic mutations are requested with higher sensitivity and more convenience. Here, we describe snapback primer mediated allele clamping enrichment polymerase chain reaction (SPACE-PCR), a novel form of PCR that amplifies minority alleles selectively from mixtures. We replaced regular PCR with SPACE-PCR before sequencing or genotyping assays to improve mutation detection sensitivity by up to 100-fold in detectingEGFRandKRASsomatic mutations. Combined SPACE-PCR with analysis of snapback primer by high resolution melting (SPACE-HRM), the high sensitive system that enables a closed-tube detection of mutations after isolating mutants has been established, as low as 1/105–1/1000 mutant samples can be diagnosed. And finally, in a double-blind experiment of 150 cases of non-small-cell lung cancer (NSCLC) patients, compared with direct DNA sequencing and ADX-EGFR/KRASmutation detection kit, up to 25% of the PCR-direct sequencing negative cases turned out to be positive in SPACE-HRM mutation tests; the specificity is 100%. Results demonstrated that the SPACE-HRM system we set up is a high sensitive assay that can be used forEGFRandKRASallele enrichment and reliable detection. We anticipate that the method will be employed in multiple applications in the clinic, including diagnosis, scanner recurrence monitoring, and treatment management.

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 Comparative analysis of pyrosequencing and QMC-PCR in conjunction with high resolution melting for KRAS/BRAF mutation detection

2010 ◽  
Vol 91 (6) ◽  
pp. 500-505 ◽  
Author(s):  
Salih Ibrahem ◽  
Rashmi Seth ◽  
Brendan O’Sullivan ◽  
Wakkas Fadhil ◽  
Philippe Taniere ◽  
...  
Keyword(s):  
Comparative Analysis ◽  
High Resolution ◽  
Braf Mutation ◽  
Mutation Detection ◽  
High Resolution Melting

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.

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.

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