Identifying Common Genetic Variants by High-Resolution Melting

Abstract Background: Heteroduplex scanning techniques usually detect all heterozygotes, including common variants not of clinical interest. Methods: We conducted high-resolution melting analysis on the 24 exons of the ACVRL1 and ENG genes implicated in hereditary hemorrhagic telangiectasia (HHT). DNA in samples from 13 controls and 19 patients was PCR amplified in the presence of LCGreen® I, and all 768 exons melted in an HR-1® instrument. We used 10 wild-type controls to identify common variants, and the remaining samples were blinded, amplified, and analyzed by melting curve normalization and overlay. Unlabeled probes characterized the sequence of common variants. Results: Eleven common variants were associated with 8 of the 24 HHT exons, and 96% of normal samples contained at least 1 variant. As a result, the positive predictive value (PPV) of a heterozygous exon was low (31%), even in a population of predominantly HHT patients. However, all common variants produced unique amplicon melting curves that, when considered and eliminated, resulted in a PPV of 100%. In our blinded study, 3 of 19 heterozygous disease-causing variants were missed; however, 2 were clerical errors, and the remaining false negative would have been identified by difference analysis. Conclusions: High-resolution melting analysis is a highly accurate heteroduplex scanning technique. With many exons, however, use of single-sample instruments may lead to clerical errors, and routine use of difference analysis is recommended. Common variants can be identified by their melting curve profiles and genotyped with unlabeled probes, greatly reducing the false-positive results common with scanning techniques.

Download Full-text

Amplicon Melting Analysis with Labeled Primers: A Closed-Tube Method for Differentiating Homozygotes and Heterozygotes

Clinical Chemistry ◽

10.1373/49.3.396 ◽

2003 ◽

Vol 49 (3) ◽

pp. 396-406 ◽

Cited By ~ 235

Author(s):

Cameron N Gundry ◽

Joshua G Vandersteen ◽

Gudrun H Reed ◽

Robert J Pryor ◽

Jian Chen ◽

...

Keyword(s):

High Resolution ◽

High Resolution Melting ◽

Rapid Heating ◽

Melting Curve ◽

Pcr Primers ◽

High Resolution Melting Analysis ◽

Melting Transition ◽

Sequence Variant ◽

Sequence Variants ◽

Melting Analysis

Abstract Background: Common methods for identification of DNA sequence variants use gel electrophoresis or column separation after PCR. Methods: We developed a method for sequence variant analysis requiring only PCR and amplicon melting analysis. One of the PCR primers was fluorescently labeled. After PCR, the melting transition of the amplicon was monitored by high-resolution melting analysis. Different homozygotes were distinguished by amplicon melting temperature (Tm). Heterozygotes were identified by low-temperature melting of heteroduplexes, which broadened the overall melting transition. In both cases, melting analysis required ∼1 min and no sample processing was needed after PCR. Results: Polymorphisms in the HTR2A (T102C), β-globin [hemoglobin (Hb) S, C, and E], and cystic fibrosis (F508del, F508C, I507del, I506V) genes were analyzed. Heteroduplexes produced by amplification of heterozygous DNA were best detected by rapid cooling (>2 °C/s) of denatured products, followed by rapid heating during melting analysis (0.2–0.4 °C/s). Heterozygotes were distinguished from homozygotes by a broader melting transition, and each heterozygote had a uniquely shaped fluorescent melting curve. All homozygotes tested were distinguished from each other, including Hb AA and Hb SS, which differed in Tm by <0.2 °C. The amplicons varied in length from 44 to 304 bp. In place of one labeled and one unlabeled primer, a generic fluorescent oligonucleotide could be used if a 5′ tail of identical sequence was added to one of the two unlabeled primers. Conclusion: High-resolution melting analysis of PCR products amplified with labeled primers can identify both heterozygous and homozygous sequence variants.

Download Full-text

High resolution melting analysis to genotype the most common variants in the HFE gene

Clinical Chemistry and Laboratory Medicine (CCLM) ◽

10.1515/cclm.2011.237 ◽

2011 ◽

Vol 49 (9) ◽

Cited By ~ 3

Author(s):

Roberta V. Marotta ◽

Olivia Turri ◽

Antonella Morandi ◽

Manuela Murano ◽

Gianlodovico Melzi d'Eril ◽

...

Keyword(s):

High Resolution ◽

High Resolution Melting ◽

High Resolution Melting Analysis ◽

Hfe Gene ◽

Common Variants ◽

Melting Analysis

Download Full-text

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

Journal of Clinical Oncology ◽

10.1200/jco.2006.24.18_suppl.7075 ◽

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.

Download Full-text

Rapid Detection of Human Torque Teno Viruses Using High-Resolution Melting Analysis

Balkan Journal of Medical Genetics ◽

10.2478/bjmg-2013-0018 ◽

2013 ◽

Vol 16 (1) ◽

pp. 55-62 ◽

Cited By ~ 4

Author(s):

S Spandole ◽

D Cimponeriu ◽

M Toma ◽

I Radu ◽

D.A. Ion

Keyword(s):

High Resolution ◽

High Resolution Melting ◽

Fluorescent Dyes ◽

Melting Curve ◽

High Resolution Melting Analysis ◽

Dna Viruses ◽

Mutation Scanning ◽

Method Optimization ◽

Melting Analysis ◽

Curve Patterns

Abstract Torque teno viruses (TTVs) are recently discovered DNA viruses, with heterogeneous genomes, highly prevalent in populations worldwide. The species that infect humans are Torque teno virus (TTV), Torque teno midi virus (TTMDV) and Torque teno mini virus (TTMV). High-resolution melting analysis (HRMA) is a sensitive and effective method for genotyping and mutation scanning. Up to now, HRMA has not been utilized for detection of TTVs. The aim of this study was to asses if HRMA is suitable for detecting TTVs variants. DNA was extracted from the blood and saliva of 13 healthy subjects for method optimization. Additionally, saliva samples from 100 healthy individuals were collected for estimating the TTVs’ prevalence. Viral DNA was amplified by heminested polymerase chain reaction (PCR). Second round amplicons were used for the HRMA. The samples were analyzed using two fluorescent dyes, SYBR® Green I and EvaGreen®. The prevalence values for TTV, TTMDV and TTMV were 71.0, 31.0 and 54.0%, respectively. The three major melting curve patterns corresponding to TTV, TTMDV and TTMV on HRMA can be easily distinguished regardless of kit used. Our results showed that HRMA is a rapid and efficient method of detecting human TTVs.

Download Full-text

Duplex high resolution melting analysis (dHRMA) to detect two hot spot CYP24A1 pathogenic variants (PVs) associated to idiopathic infantile hypercalcemia (IIH)

Molecular Biology Reports ◽

10.1007/s11033-021-06324-x ◽

2021 ◽

Author(s):

Maria De Bonis ◽

Elisa De Paolis ◽

Maria Elisabetta Onori ◽

Giorgia Mazzuccato ◽

Antonio Gatto ◽

...

Keyword(s):

High Resolution ◽

High Resolution Melting ◽

Hot Spot ◽

Melting Curve ◽

High Resolution Melting Analysis ◽

Pathogenic Variants ◽

Melting Analysis ◽

Idiopathic Infantile Hypercalcemia ◽

Set Up ◽

Peculiar Case

AbstractPathogenic variants (PVs) in CYP24A1 gene are associated with Idiopathic Infantile Hypercalcemia disease (IIH). The identification of CYP24A1 PVs can be a useful tool for the improvement of target therapeutic strategies. Aim of this study is to set up a rapid and inexpensive High Resolution Melting Analysis (HRMA)-based method for the simultaneous genotyping of two hot spot PVs in CYP24A1 gene, involved in IIH. A duplex-HRMA (dHRMA) was designed in order to detect simultaneously CYP24A1 c.428_430delAAG, p.(Glu143del) (rs777676129) and c.1186C > T, p.(Arg396Trp) (rs114368325), in peculiar cases addressed to our Laboratory. dHRMA was able to identify clearly and simultaneously both hot spot CYP24A1 PVs evaluating melting curve shape and melting temperature (Tm). This is the first dHRMA approach to rapidly screen the two most frequent CYP24A1 PVs in peculiar case, providing useful information for diagnosis and patient management in IIH disease.

Download Full-text

DNA-based Identification of Peucedanum ostruthium by High-Resolution-Melting Analysis (HRM)

Planta Medica ◽

10.1055/s-0032-1307632 ◽

2012 ◽

Vol 78 (05) ◽

Author(s):

C Schmiderer ◽

C Franz ◽

J Novak

Keyword(s):

High Resolution ◽

High Resolution Melting ◽

High Resolution Melting Analysis ◽

Melting Analysis

Download Full-text

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

European Journal of Human Genetics ◽

10.1038/s41431-020-00797-3 ◽

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.

Download Full-text