Rapid Mutation Scanning of Genes Associated with Familial Cancer Syndromes Using Denaturing High-Performance Liquid Chromatography

Marsh, Deborah J.; Theodosopoulos, George; Howell, Viive M.; Richardson, Anne; Benn, Diana E.; Proos, Anné; Eng, Charis; Robinson, Bruce G.

doi:10.1038/sj.neo.7900154

Cited by 34 publications

(15 citation statements)

References 37 publications

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“…The previously used mutation screening strategy in our laboratory for MEN1 (Crépin et al 2003) required the combination of the two techniques (MDGA and HMA) to yield 100% sensitivity and was therefore much more time consuming and expensive. Marsh et al (2001) and Roberts et al (2001) showed that DHPLC is well adapted to rapid mutation scanning. Due to frequent polymorphisms, especially in exon 9, DHPLC patterns could hide another mutation in the polymorphic DNA fragments.…”

Section: Discussionmentioning

confidence: 99%

Evaluation of denaturing high performance liquid chromatography for the mutational analysis of the MEN1 gene

Crépin¹,

Pigny²,

Escande³

et al. 2006

Journal of Molecular Endocrinology

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The identification of mutations in the MEN1 gene causing MEN1 has represented a challenge since the cloning of the gene in 1997 because of the lack of mutation hot-spots in the gene and the lack of phenotype-genotype correlations. The use of denaturing high performance liquid chromatography (DHPLC), a high throughput, reliable and automated heteroduplex-based technique, is the ideal for mutation detection in MEN1. In this work, DHPLC was optimised for the screening of the nine coding exons and splice junctions of MEN1. Thanks to collaboration between two French laboratories recognised as reference centres for genotypic MEN1 diagnosis (Lyon and Lille), a blind retrospective study conducted in a cohort of 160 unrelated MEN1 probands with (or without) known germline mutations was undertaken to evaluate the sensitivity of DHPLC. We were able to detect 101 different sequence variations by DHPLC, distributed in the 10 analysed DNA fragments and corresponding to 100% of mutation detection compared with direct sequencing. 1·2% of samples were considered as false positive, exhibiting a heterogenous profile. DHPLC did not detect five cases of deletion or duplication of complete exons, neither did direct sequencing, showing the limits of the technique. Nevertheless, the method appeared to allow automated, rapid and low-cost mutation detection with high accuracy. Direct sequencing can be then applied to identify the sequence variations on the targeted DNA fragments showing heterozygous profile by DHPLC. In conclusion, genotypic diagnosis of MEN1 can benefit from DHPLC in terms of efficacy, rapidity and cost.

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Section: Discussionmentioning

confidence: 99%

Evaluation of denaturing high performance liquid chromatography for the mutational analysis of the MEN1 gene

Crépin¹,

Pigny²,

Escande³

et al. 2006

Journal of Molecular Endocrinology

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“…35 In brief, a 5-l aliquot of heteroduplex enhanced PCR product was injected into the mobile phase in the direction of the flow path of the DNASep cartridge (Transgenomic Inc.). The conditions for each amplicon are listed in Table 3.…”

Section: Dhplcmentioning

confidence: 99%

“…34 Mutation scanning by DHPLC has been successfully applied to a number of tumor suppressor genes including PTEN, VHL, and RET. 35 A limitation of this and other mutation scanning techniques is likely interference from the detection of common polymorphisms. An anticipated problem specific to tumor suppressor genes is the possibility of false negative results attributable to LOH when testing tumor DNA.…”

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confidence: 99%

Rapid Mutation Screening for HRPT2 and MEN1 Mutations Associated with Familial and Sporadic Primary Hyperparathyroidism

Howell

Cardinal

Richardson

et al. 2006

The Journal of Molecular Diagnostics

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Familial hyperparathyroidism, a disease of the parathyroid glands, may occur in conjunction with pituitary and pancreatic tumors (multiple endocrine neoplasia type I), kidney and bone tumors (hyperparathyroidism jaw tumor syndrome) , or alone (familial isolated hyperparathyroidism). This study describes the development and validation of rapid scanning for mutations in two tumor suppressor genes linked to familial hyperparathyroidism-MEN1 and HRPT2. Denaturing high-performance liquid chromatography mutation scanning for MEN1 was performed using a set of 10 amplicons covering the nine coding exons and flanking intronic regions and for HRPT2 using a set of three amplicons for exons 1 , 2 , and 7 and flanking intronic regions , in which 80% of the mutations identified to date are located. All 52 MEN1 mutations or polymorphisms , 46 known and six unknown , were successfully detected. Mutation detection in exon 9 was not confounded by the presence of the common polymorphism D418D. In addition , all 10 HRPT2 mutations were successfully detected , and a two-step approach was able to distinguish IVS2 common polymorphisms from exon 2 mutations. The development of rapid denaturing high performance liquid chromatography mutation scanning of MEN1 and HRPT2 facilitates a molecular diagnosis of the associated familial syndromes for both clinically affected and at-risk family members.

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“…Many different prescreening methodologies have been applied to breast cancer genes, including denaturing gradient gel electrophoresis (6,7 ), single-strand conformation polymorphism analysis (8,9 ), heteroduplex analysis (10 ), conformation-sensitive gel electrophoresis (11,12 ), denaturing HPLC (13 ), and mismatch-cleavage assays (14 ). The throughput of these techniques is limited, however, and their diagnostic accuracies have generally been poorly reported in the literature (15 ).…”

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confidence: 99%

Interlaboratory Diagnostic Validation of Conformation-Sensitive Capillary Electrophoresis for Mutation Scanning

et al. 2010

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Background: Indirect alternatives to sequencing as a method for mutation scanning are of interest to diagnostic laboratories because they have the potential for considerable savings in both time and costs. Ideally, such methods should be simple, rapid, and highly sensitive, and they should be validated formally to a very high standard. Currently, most reported methods lack one or more of these characteristics. We describe the optimization and validation of conformation-sensitive capillary electrophoresis (CSCE) for diagnostic mutation scanning. Methods: We initially optimized the performance of CSCE with a systematic panel of plasmid-based controls. We then compared manual analysis by visual inspection with automated analysis by BioNumerics software (Applied Maths) in a blinded interlaboratory validation with 402 BRCA1 (breast cancer 1, early onset) and BRCA2 (breast cancer 1, early onset) variants previously characterized by Sanger sequencing. Results: With automated analysis, we demonstrated a sensitivity of >99% (95% CI), which is indistinguishable from the sensitivity for conventional sequencing by capillary electrophoresis. The 95% CI for specificity was 90%–93%; thus, CSCE greatly reduces the number of fragments that need to be sequenced to fully characterize variants. By manual analysis, the 95% CIs for sensitivity and specificity were 98.3%–99.4% and 93.1%–95.5%, respectively. Conclusions: CSCE is amenable to a high degree of automation, and analyses can be multiplexed to increase both capacity and throughput. We conclude that once it is optimized, CSCE combined with analysis with BioNumerics software is a highly sensitive and cost-effective mutation-scanning technique suitable for routine genetic diagnostic analysis of heterozygous nucleotide substitutions, small insertions, and deletions.

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Rapid Mutation Scanning of Genes Associated with Familial Cancer Syndromes Using Denaturing High-Performance Liquid Chromatography

Cited by 34 publications

References 37 publications

Evaluation of denaturing high performance liquid chromatography for the mutational analysis of the MEN1 gene

Evaluation of denaturing high performance liquid chromatography for the mutational analysis of the MEN1 gene

Rapid Mutation Screening for HRPT2 and MEN1 Mutations Associated with Familial and Sporadic Primary Hyperparathyroidism

Interlaboratory Diagnostic Validation of Conformation-Sensitive Capillary Electrophoresis for Mutation Scanning

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