®
LightCycler® 480 Real-Time PCR System in Human
Genetics
Featured project: High-Resolution Melting for CFTR Mutation
Scanning
Stefanie Beck-Wödl and Peter Bauer
Molecular Pathology Research and Development Laboratory, Department of Pathology, University
of Tübingen, Germany
Read in this article:
Introduction
Materials and Methods
Results and Discussion
Conclusions
References
Introduction
Cystic fibrosis (CF) is the most common autosomal recessive monogenic disease (1:2500, with a
carrier frequency of 1 in 25). More than 1,000 mutations are described in the CFTR (cystic fibrosis
transmembrane conductance regulator) gene. In Germany, 86% of all CFTR alleles are defined by
19 different mutations, with F508del being the most prevalent (73%). These alleles are located in
nine different PCR fragments. Complete gene analysis by scanning and/or sequencing is seldom
performed because of the cost, time, and labor involved. High-resolution melting analysis (HRM) is
a rapid, closed-tube alternative.
To assess the feasibility of using HRM in CFTR mutation screening for research purposes, we set
up PCR protocols for CFTR exons 4, 7, 10, 11, 14b, 19, 20, and 21, enabling the analysis of the
most prevalent CFTR mutations in the German population. In parallel, unlabeled probes were
tested to directly discern known CFTR mutations in these exons.
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Materials and Methods
Several CFTR-mutation-positive samples were selected for this study, including samples for
F508del, I507del, R117H, R347P, G542X, G551D, R553X, 1717-1G>A, 2789+5G>A, S1255X,
W1272X, and N1303K. CFTR genotypes had been previously confirmed for all samples, either by
direct sequencing or RFLP-analysis. Unlabeled probes were designed complementary to the wildtype sequences. PCR fragments usually had amplicon lengths of less than 400 bp. Amplification
was carried out for 45 cycles followed by a melting curve from +50 to +85°C. Ten picomoles of
each primer and 3 pmol of each unlabeled probe were used for detection.
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Results and Discussion
So far, we have been able to establish an HRM analysis panel with 18 CFTR mutations amplified
in one PCR assay. This panel covers approximately 85% of the German population. All mutations
in this panel were determined to be heterozygous, as indicated by aberrant melting curves.
F508del and compound heterozygote alleles in exon 11 (G551D/R553X) where clearly discernible
by analysis of the melting curves of the unlabeled probes.
Figure 1: Genetic variation in the human CFTR gene analyzed by high-resolution melting. A
198 bp fragment of the human CFTR gene was amplified using the LightCycler® 480 High
Resolution Melting Master and subjected to amplicon melting at high resolution. Difference plot
analysis revealed three different groups of heterozygotes (blue, red, and green) in addition to the
homozygous samples (black).
Figure 2: Genotype differentiation using high-resolution melting analysis in the presence of
unlabeled probes. Samples not differentiated by amplicon melting alone (green in Figure 1) could
be separated based on the Tm of an unlabeled probe covering both mutated sites (see insert).
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Conclusions
High-resolution melting analysis with unlabeled probes enables accurate genotyping and mutation
screening in the CFTR gene. The sensitivity obtained in this experiment was almost 100%, and
specificity was guaranteed by mutation-specific melting peaks.
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References
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[1] Riordan JR, Rommens JM, Kerem B, Alon N, Rozmahel R, Grzelczak Z, et al. Identification
of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science
1989;245:1066-1073.
[2] Bobadilla JL, Macek M, Jr, Fine JP, Farrell PM. Cystic fibrosis: a worldwide analysis of
CFTR mutations: correlation with incidence data and application to screening. Hum Mutat
2002;19:575-606.
[3] Dork T, Mekus F, Schmidt K, Bosshammer J, Fislage R, Heuer T, et al. Detection of more
than 50 different CFTR mutations in a large group of German cystic fibrosis patients. Hum
Genet 1994;94:533-542.
[4] Chou LS, Lyon E, Wittwer CT. A comparison of high-resolution melting analysis with
denaturing high-performance liquid chromatography for mutation scanning: cystic fibrosis
transmembrane conductance regulator gene as a model. Am J Clin Pathol 2005;124:330-338.
[5] Wittwer CT, Marshall BC, Reed GH, Cherry JL. Rapid cycle allele-specific amplification:
studies with the cystic fibrosis delta F508 locus. Clin Chem 1993;39:804-809.
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