High-Resolution Melting Analysis (HRMA)-More than just sequence variant screening

Vossen, Rolf H. A. M.; Aten, Emmelien; Roos, Anja; Dunnen, Johan T. den

doi:10.1002/humu.21019

Cited by 436 publications

(345 citation statements)

References 29 publications

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“…Compared with other methods, such as denaturing high-performance liquid chromatography, HRM offers at least two additional benefits: (1) the temperature gradient covers all potential melting domains of amplicons, which provides superior detection sensitivity; and (2) the closed-tube method reduces post-PCR manipulations and the risk of pipetting errors and contaminations. 17,18 Moreover, our identification of the rs3804769 SNP represents a positive control and argues for the general validity of our approach.…”

Section: Methodsmentioning

confidence: 85%

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A total of 220 patients with autosomal dominant spastic paraplegia do not display mutations in the SLC33A1 gene (SPG42)

et al. 2010

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The most frequent causes of autosomal dominant (AD) hereditary spastic paraplegias (HSP) (ADHSP) are mutations in the SPAST gene (SPG4 locus). However, roughly 60% of patients are negative for SPAST mutations, despite their family history being compatible with AD inheritance. A mutation in the gene for an acetyl-CoA transporter (SLC33A1) has recently been reported in one Chinese family to cause ADHSP-type SPG42. In this study, we screened 220 independent SPAST mutationnegative ADHSP samples for mutations in the SLC33A1 gene by high-resolution melting curve analysis. Conspicuous samples were validated by direct sequencing. Moreover, copy number variations affecting SLC33A1 were screened by multiplex ligation-dependent probe amplification assay. We could not identify potentially disease-causing mutations in our patients either by mutation scanning or by gene dosage analysis, as for the latter specific positive controls are not available to date. As our sample represents ADHSP patients for whom SPAST mutations and almost in all cases ATL1 and REEP1 mutations had been excluded, we consider SLC33A1 gene mutations as being very rare in a European ADHSP cohort, if present at all. To date, as SPG42 has still not been identified in a second, unrelated family, systematic genetic testing for SLC33A1 mutations is not recommended.

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Section: Methodsmentioning

confidence: 85%

“…16 No other sequence alterations were detected. As HRM analysis is a screening method with B99% sensitivity, 17,18 we handled the results with care. Along with this high sensitivity, HRM is a simple, rapid and low-cost method to screen for unknown sequence variants in a high-throughput modality.…”

Section: Methodsmentioning

confidence: 99%

A total of 220 patients with autosomal dominant spastic paraplegia do not display mutations in the SLC33A1 gene (SPG42)

et al. 2010

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“…The small amplicon genotyping method in this study using the high-resolution melting curve analysis is a one-step, single-tube method for detection of specific mutations and faster, simpler and less expensive than the approaches requiring separations or labeled probes. 19 The screening for c.1559delT in ALPL may be useful for diagnosis of pl-HPP in Japanese to provide optimum genetic counseling for fetal skeletal dysplasia. pl-HPP occasionally could not be diagnosed with sonographic examination in the first trimester because incomplete ossification is an usual finding at this stage of development.…”

Section: Discussionmentioning

confidence: 99%

Prevalence of c.1559delT in ALPL, a common mutation resulting in the perinatal (lethal) form of hypophosphatasia in Japanese and effects of the mutation on heterozygous carriers

et al. 2010

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Hypophosphatasia (HPP) is an inherited disorder caused by mutations in ALPL that encodes an isozyme of alkaline phosphatase (ALP), TNSALP. One of the most frequent ALPL mutations is c.1559delT, which causes the most severe HPP, the perinatal (lethal) form (pl-HPP). c.1559delT has been found only in Japanese and its prevalence is suspected to be high; however, the allele frequency of c.1559delT in Japanese remains unknown. We designed a screening system for the mutation based on high-resolution melting curve analysis, and examined the frequency of c.1559delT. We found that the c.1559delT carrier frequency is 1/480 (95% confidence interval, 1/1562-1/284). This indicates that B1 in 900 000 individuals to have pl-HPP caused by a homozygous c.1559delT mutation. In our analysis, the majority of c.1559delT carriers had normal values of HPP biochemical markers, such as serum ALP and urine phosphoethanolamine. Our results indicate that the only way to reliably detect whether individuals are pl-HPP carriers is to perform the ALPL mutation analysis.

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“…The RAS family genes, originally identified as oncogenes in acutely transforming retroviruses (Vossen et al, 2009) have three highly homologous RAS proteins encoded by KRAS, HRAS and NRAS genes. A high frequency of RAS mutations has been found in many types of tumor; approximately 30% of all human cancers develop a mutation in a RAS gene with mutations most frequently occurring in KRAS.…”

Section: Discussionmentioning

confidence: 99%

Co-amplification at Lower Denaturation-temperature PCR Combined with Unlabled-probe High-resolution Melting to Detect KRAS Codon 12 and 13 Mutations in Plasma-circulating DNA of Pancreatic Adenocarcinoma Cases

Zhou²,

Song³

et al. 2015

Asian Pacific Journal of Cancer Prevention

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Background: The aim of our study was to establish COLD-PCR combined with an unlabeled-probe HRM approach for detecting KRAS codon 12 and 13 mutations in plasma-circulating DNA of pancreatic adenocarcinoma (PA) cases as a novel and effective diagnostic technique. Materials and Methods: We tested the sensitivity and specificity of this approach with dilutions of known mutated cell lines. We screened 36 plasma-circulating DNA samples, 24 from the disease control group and 25 of a healthy group, to be subsequently sequenced to confirm mutations. Simultaneously, we tested the specimens using conventional PCR followed by HRM and then used target-DNA cloning and sequencing for verification. The ROC and respective AUC were calculated for KRAS mutations and/or serum CA 19-9. Results: It was found that the sensitivity of Sanger reached 0.5% with COLD-PCR, whereas that obtained after conventional PCR did 20%; that of COLD-PCR based on unlabeled-probe HRM, 0.1%. KRAS mutations were identified in 26 of 36 PA cases (72.2%), while none were detected in the disease control and/or healthy group. KRAS mutations were identified both in 26 PA tissues and plasma samples. The AUC of COLD-PCR based unlabeled probe HRM turned out to be 0.861, which when combined with CA 19-9 increased to 0.934. Conclusions: It was concluded that COLD-PCR with unlabeled-probe HRM can be a sensitive and accurate screening technique to detect KRAS codon 12 and 13 mutations in plasma-circulating DNA for diagnosing and treating PA.

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High-Resolution Melting Analysis (HRMA)-More than just sequence variant screening

Cited by 436 publications

References 29 publications

A total of 220 patients with autosomal dominant spastic paraplegia do not display mutations in the SLC33A1 gene (SPG42)

A total of 220 patients with autosomal dominant spastic paraplegia do not display mutations in the SLC33A1 gene (SPG42)

Prevalence of c.1559delT in ALPL, a common mutation resulting in the perinatal (lethal) form of hypophosphatasia in Japanese and effects of the mutation on heterozygous carriers

Co-amplification at Lower Denaturation-temperature PCR Combined with Unlabled-probe High-resolution Melting to Detect KRAS Codon 12 and 13 Mutations in Plasma-circulating DNA of Pancreatic Adenocarcinoma Cases

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