High-Resolution Melting Analysis Is a More Effective Approach for Screening<i>TSC</i>Genes Mutations

Tsai, Tz Shiu; Huang, Mu Yi; Chang, Yu Tsui; Wang, Chuan Yu; Lin, Jainn-Jim; Hung, Po Cheng; Lin, Shuan Pei; Sun, Chien Feng; Wang, Wei Shun; Chang, Chung Ming; Chang, Shih Cheng; Chu, Da Chang

doi:10.1089/gtmb.2010.0133

Cited by 4 publications

(2 citation statements)

References 26 publications

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“…Of note, this patient had multifocal tumors (paraffin tissue from the other tumor in the same kidney was not available for testing). Four of the seven alterations detected ( TSC2 p.Y130*; TSC2 splice acceptor variant c.976-1G>A between exon 10 and 11; TSC2 p.L361Sfs; TSC1 p.Y761*) have been previously reported in patients with tuberous sclerosis complex and are thought to be pathogenic 7,8,9,10,11,12 , while the other three ( TSC2 p.L243Hfs; TSC2 p.D1677Afs; TSC1 p.L853Cfs) are considered to be likely pathogenic 6,7 .…”

Section: Resultsmentioning

confidence: 85%

Eosinophilic Solid and Cystic (ESC) Renal Cell Carcinomas Harbor TSC Mutations

Palsgrove

Pratilas

et al. 2018

American Journal of Surgical Pathology

110

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Eosinophilic solid and cystic (ESC) renal cell carcinoma (RCC) has recently been described as a potentially new subtype of RCC based upon morphologic and immunohistochemical features. These neoplasms typically demonstrate solid and cystic architecture, and the neoplastic cells contain voluminous eosinophilic cytoplasm with granular cytoplasmic stippling. There is frequently focal immunoreactivity for cytokeratin 20. Although the initial cases all occurred in adult females and had benign outcome, we recently expanded the proposed spectrum of this neoplasm to include pediatric cases, multifocal neoplasms, and a case with hematogenous metastasis. ESC has been postulated to be analogous to a subtype of RCC consistently identified in tuberous sclerosis complex patients, and while previous work has demonstrated loss of heterozygosity at the TSC1 locus and copy number gains at TSC2 in ESC RCC, these genes have not been sequenced in ESC RCC. Using capture-based and amplicon-based next-generation sequencing, we now demonstrate the consistent presence of either TSC1 or TSC2 gene mutations in pediatric ESC RCC (8/9 cases) and adult ESC RCC (6/6 cases). These included a metastatic ESC RCC which had a complete response to mTOR targeted therapy. We also found these mutations in some neoplasms with variant morphology and thus potentially expand the spectrum of ESC RCC. These include one of our adult cases which demonstrated dominant "type 2" papillary RCC morphology and 2 of 3 previously unclassified pediatric RCC with features of ESC RCC minus granular cytoplasmic stippling. We also demonstrate TSC mutations in a case of so-called "oncocytoid RCC after neuroblastoma" with identical morphology and immunoprofile, providing a molecular link between the latter and ESC RCC. In summary, ESC RCC consistently harbors actionable TSC1 or TSC2 mutations, which are infrequently seen in established subtypes of RCC. These findings support TSC1/2 mutation as a molecular marker of ESC RCC, and suggest expansion of the clinicopathologic spectrum to include neoplasms with papillary architecture, occasional cases lacking well-developed granular cytoplasmic stippling, and a subset of RCC with oncocytic features in patients who have survived neuroblastoma.

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Section: Resultsmentioning

confidence: 85%

Eosinophilic Solid and Cystic (ESC) Renal Cell Carcinomas Harbor TSC Mutations

Palsgrove

Pratilas

et al. 2018

American Journal of Surgical Pathology

110

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“…The sensitivities of HRM and DHPLC were estimated as 95% and 75% and the specificities were assessed as 91% and 98%, respectively. Based on these results, the authors concluded that their HRM protocol can be used for TSC gene mutation screening for clinical applications (32).…”

Section: Discussionmentioning

confidence: 99%

Comparison of high-resolution melting analysis to denaturing high performance liquid chromatography in the detection of point mutations in MEFV, F5, and F2 genes

Çelebi¹,

Özdağ²

2014

Turk J Med Sci

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Background/aim: Sensitive and cost-effective detection of point mutations is important in genetics research. Denaturing highperformance liquid chromatography (DHPLC) is known to be one of the most sensitive techniques for point mutation detection. A more recent technique, high-resolution melting (HRM), is based on the melting behavior of PCR products. In this study, the efficiency and sensitivity of HRM and DHPLC for the detection of MEFV, F5, and F2 gene point mutations were evaluated. Materials and methods:We studied 15 patients with MEFV mutations (E148Q, M680I, M694V, or V726A), 7 patients with the F51691G>A mutation, and 12 patients with the F220210G>A mutation. All mutations were screened by HRM and DHPLC.Results: All mutations were successfully detected by HRM. However, only 4 (MEFV E148Q and M680I, F51691G>A, and F220210G>A) of 6 mutations were successfully detected with DHPLC. Conclusion:Our study showed that HRM is more sensitive than DHPLC for detection of the studied point mutations.

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Validation of a quantitative PCR-high-resolution melting protocol for simultaneous screening ofCOL1A1andCOL1A2point mutations and large rearrangements: Application for diagnosis of osteogenesis imperfecta

et al. 2012

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Osteogenesis imperfecta (OI) is a connective tissue disorder mostly characterized by autosomal dominant inheritance. Over 1,100 causal mutations have been identified scattered along all exons of genes encoding type I collagen precursors, COL1A1 and COL1A2. Because of the absence of mutational hotspots, Sanger sequencing is considered the gold standard for molecular analysis even if the workload is very laborious and expensive. To overcome this issue, different prescreening methods have been proposed, including DHPLC and biochemical studies on cultured dermal fibroblasts; however, both approaches present different drawbacks. Moreover, in case of patients who screen negative for point mutations, an additional screening step for complex rearrangements is required; the added causative variants expected from this approach are about 1-2%. The aim of this study was to optimize and validate a new protocol that combines quantitative PCR (qPCR) and high-resolution melting (HRM) curve analysis to reduce time and costs for molecular diagnosis. Results of qPCR-HRM screening on 57 OI patients, validated by DHPLC-direct sequencing and multiplex ligation-dependent probe amplification (MLPA), indicate that all alterations identified with the mentioned methodologies are successfully detected by qPCR-HRM. Moreover, HRM was able to discriminate complex genotypes and homozygous variants. Finally, qPCR-HRM outperformed direct sequencing and DHPLC-MLPA in terms of rapidity and costs.

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High-Resolution Melting Analysis Is a More Effective Approach for ScreeningTSCGenes Mutations

Cited by 4 publications

References 26 publications

Eosinophilic Solid and Cystic (ESC) Renal Cell Carcinomas Harbor TSC Mutations

Eosinophilic Solid and Cystic (ESC) Renal Cell Carcinomas Harbor TSC Mutations

Comparison of high-resolution melting analysis to denaturing high performance liquid chromatography in the detection of point mutations in MEFV, F5, and F2 genes

Validation of a quantitative PCR-high-resolution melting protocol for simultaneous screening ofCOL1A1andCOL1A2point mutations and large rearrangements: Application for diagnosis of osteogenesis imperfecta

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