Validation of High-Resolution DNA Melting Analysis for Mutation Scanning of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene

From the first report of the LightCycler as a real-time polymerase chain reaction (PCR) instrument 1 clinical laboratories quickly realized its potential as a diagnostic tool. Quantitative applications important for infectious disease and oncology applications were followed by genotyping assays that took advantage of accurate temperature control to melt DNA amplicons or probe/amplicon duplexes. The first Food and Drug Administration-cleared molecular genetic assays were developed for this platform, specifically genotyping single base variants in F5 and F2. According to a College of American Pathologists survey, 2 ϳ30% of clinical laboratories report using the LightCycler for these two assays. This article will review the history of LightCycler technology, focusing on those applications widely incorporated into molecular diagnostics. In addition we will review recent developments that may impact future clinical testing.Real-time PCR instruments simultaneously amplify and detect, eliminating the need to open tubes containing PCR amplicon and therefore reducing the risk of future contamination. Fluorescent dyes or probes allow continuous monitoring as template DNA is amplified.3 By monitoring fluorescence every cycle, the amount of original target can be calculated. By monitoring fluorescence as the temperature changes, genotyping and heterozygote scanning can be performed by melting analysis, often removing the need for downstream analysis.The first two platforms developed for real-time PCR were the LightCycler (Roche, Indianapolis, IN) and the ABI 7700 (Applied Biosystems, Foster City, CA). The instruments were as different as the groups that created them. At the time, ABI was the undisputed corporate leader of PCR technology and the 7700 was a large 96-well laser-based instrument focused on throughput.

Section: Mutation Scanning By Meltingmentioning

confidence: 99%

LightCycler Technology in Molecular Diagnostics

Lyon

Wittwer

2009

“…Because abnormal normalized curves were based on the nucleotide composition of an amplicon and the type of sequence changes in the amplicon, it is possible that certain variants might not be detected by HRM. 20,21 In many cases, shorter amplicons improve the sensitivity of variant detection by HRM (see Supplemental Figure S2 at http://jmd.amjpathol.org). Adding an unlabeled probe to target a specific variant in the HRM reaction, which would generate a new melting curve at a lower melting temperature in addition to that from the full-length amplicon, uses the same principle as using shorter amplicons and could resolve this type of issue.…”

Section: Discussionmentioning

confidence: 99%

“…20,48 Furthermore, mixing the PCR products from test samples with wild-type reference samples could improve the sensitivity of detecting homozygous variants. 21 However, caution has to be taken in mixing samples or PCR products for clinical diagnosis. Because the "temperature shift" setting would overlay melting curves with the same shape but different melting temperatures, 17 disabling the temperature shift step in HRM data analysis would also help identify homozygous variants in case these generated melting curves that shifted only the melting temperature but had the same general shape as the references.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Mutation Analysis of SLC26A4 for Pendred Syndrome and Nonsyndromic Hearing Loss by High-Resolution Melting

Chen

Tranebjærg

Rendtorff

et al. 2011

Pendred syndrome and DFNB4 (autosomal recessive nonsyndromic congenital deafness, locus 4) are associated with autosomal recessive congenital sensorineural hearing loss and mutations in the SLC26A4 gene. Extensive allelic heterogeneity, however, necessitates analysis of all exons and splice sites to identify mutations for individual patients. Although Sanger sequencing is the gold standard for mutation detection, screening methods supplemented with targeted sequencing can provide a cost-effective alternative. One such method, denaturing high-performance liquid chromatography, was developed for clinical mutation detection in SLC26A4. However, this method inherently cannot distinguish homozygous changes from wild-type sequences. High-resolution melting (HRM), on the other hand, can detect heterozygous and homozygous changes cost-effectively, without any post-PCR modifications. We developed a closed-tube HRM mutation detection method specific for SLC26A4 that can be used in the clinical diagnostic setting. Twenty-eight primer pairs were designed to cover all 21 SLC26A4 exons and splice junction sequences. Using the resulting amplicons, initial HRM analysis detected all 45 variants previously identified by sequencing. Subsequently, a 384-well plate format was designed for up to three patient samples per run. Blinded HRM testing on these plates of patient samples collected over 1 year in a clinical diagnostic laboratory accurately detected all variants identified by sequencing. In conclusion, HRM with targeted sequencing is a reliable, simple, and cost-effective method for SLC26A4 mutation screening and detection. Pendred syndrome and DFNB4 (autosomal recessive nonsyndromic congenital deafness, locus 4) are associated with autosomal recessive congenital sensorineural hearing loss. Whereas Pendred syndrome is thought to be the most common cause of syndromic hearing loss, 1,2 DFNB4 is a nonsyndromic form of hearing loss caused by mutations in the same gene. 3 Clinically, both can be associated with temporal bone abnormalities. 4 In addition, an abnormal perchlorate discharge test result and/or euthyroid goiter can be present in patients with Pendred syndrome but not in those with DFNB4. 3,4 This may be due to allelic heterogeneity because differences in residual activity of the protein products render different phenotypes. 5

“…High-resolution melting (HRM) is a simple closed-tube PCR assay that precisely monitors changes in fluorescence on the basis of various sizes and nucleotide components of the amplicons during melting of the DNA duplex. This procedure can provide highly sensitive, high-throughput, cost-effective genotyping and detection of genetic mutations 15 and single-nucleotide polymorphisms and DNA methylation, 16 -18 in particular in imprinting disorders. 19,20 Some HRM assays based on methylation-dependent or methylation-independent PCR have been used to evaluate aberrant DNA hypermethylation in malignant tumors 12,21 ; however, these methods require generation of a standard curve of various methylation levels and are unsuitable in cases with heterogeneous methylation patterns frequently observed in malignant lesions.…”

mentioning

confidence: 99%

Rapid Assessment of the Heterogeneous Methylation Status of CEBPA in Patients with Acute Myeloid Leukemia by Using High-Resolution Melting Profile

Lin

Jiang

Chou

et al. 2011

Epigenetic inactivation of tumor-suppressor genes, often in association with aberrant DNA methylation of CpG islands in the promoter region of these genes, is a key factor in tumorigenesis. CCAAT/enhancer binding protein alpha (CEBPA) methylation is a favorable prognostic biomarker for acute myeloid leukemia; however, rather than the complete methylation observed in inherited disorders, CEBPA methylation is heterogeneous. In this study, we established an algorithm called the "methylation index," deduced from high-resolution melting profiles, which includes Tm shifting (⌬Tm) and Tm width ratio (fold of width), to evaluate the heterogeneous methylation status. The methylation index was highly correlated with the exact methylation levels detected by using the MassARRAY method (R 2 ‫؍‬ 0.80; P < 0.001). Within-run reproducibility for the methylation index was 0.9% as the coefficient of variation, and between-run reproducibility was 2.6%. It was determined that with a cutoff methylation index of 1.412, the best measures of sensitivity and specificity could be obtained (97.14% and 95.89%, respectively) to discern low or high CEBPA methylation status. This novel algorithm for calculation of the methylation index from high-resolution melting profiles for CEBPA methylation is compatible with measurement of the methylation level as assayed using MassARRAY and could be a simple and efficient screening method for determination of CEBPA methylation status in acute myeloid leukemia.