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Technical Briefs |
(Department of Molecular Pathology, Canterbury Health Laboratories, Christchurch, New Zealand;
aaddress correspondence to this author at: P.O. Box 151, Christchurch, New Zealand; fax 64-3-3640545, e-mail andrew.laurie{at}cdhb.govt.nz)
Abstract
Background: Single base-pair substitution mutations in the gene for coagulation factor VIII, procoagulant component (hemophilia A) (F8) account for approximately 50% of severe cases of hemophilia A (HA), and almost all moderate or mild cases. Because F8 is a large gene, mutation screening using denaturing HPLC or DNA sequencing is time-consuming and expensive.
Methods: We evaluated high-resolution melting analysis as an option for screening for F8 gene mutations. The melting curves of amplicons heterozygous for known F8 gene mutations were compared with melting curves of the corresponding normal amplicons to assess whether melting analysis could detect these variants. We examined 2 platforms, the Roche LightCycler 480 (LC480) and the Idaho Technology LightScanner.
Results: On both instruments, 18 (90%) of the 20 F8 gene variants we examined were resolved by melting analysis. For the other 2 mutations, the melting curves of the heterozygous amplicons were similar to the corresponding normal amplicons, suggesting these variants may not be detected by this approach in a mutation-scanning screen.
Conclusion: High-resolution melting analysis is an appealing technology for F8 gene screening. It is rapid and quickly identifies mutations in the majority of HA patients; samples in which no mutation is detected require further testing by DNA sequencing. The LC480 and LightScanner platforms performed similarly.
Hemophilia A (HA) is a coagulation disorder caused by a lack of normal coagulation factor VIII (FVIII) activity(1). This disease is caused by mutations in the coagulation factor VIII, procoagulant component (hemophilia A) (F8) gene, which encodes FVIII and is located at Xq28. F8 is a large gene, comprising 26 exons across 186 kb, and produces a 9-kb mRNA transcript. All exons are small (69–262 bp) except exon 14, which is 3.1 kb.
Approximately 45% of severe HA cases are the result of a large inversion that disrupts the F8 gene in intron 22, and a further 1%–5% are caused by an inversion affecting intron 1. In the remaining severe cases, and in cases of mild or moderate HA, a single-base substitution, small insertion, or deletion is usually the causative mutation. Such mutations create either missense, nonsense, or frameshift mutations, or affect consensus splice sites [reviewed in(2)]. Many such variants have been reported in HA patients, and more than 900 different mutations have been described(3).
Many hematology services now offer genetic screening to identify the F8 gene mutation in their HA patients. This information allows identification of carrier females, prenatal testing for affected pregnancies, preimplantation genetic diagnosis, and in some cases prediction of the likelihood of FVIII inhibitor production in HA patients receiving prophylactic FVIII treatment(2).
Genetic screening of HA patients who do not have either the intron 1 or intron 22 inversions is problematic because of the size of the F8 gene and the variety of mutations that can occur. Before capillary-based DNA sequencing platforms were widely available it was not practical to sequence all 26 exons, so mutation-scanning strategies such as single-strand conformational polymorphism analysis were used(4). A more sensitive technique, denaturing HPLC (dHPLC), has also been used successfully for F8 mutation scanning(5). In our laboratory we have used dHPLC combined with DNA sequencing to identify 20 different F8 gene mutations in 28 HA patients we have analyzed(6). Although we have found dHPLC to be a sensitive scanning method, it is a low-throughput technology and has many ongoing costs. Likewise, DNA sequencing of the F8 gene is time-consuming and expensive.
High-resolution melting analysis represents the next generation of mutation scanning technology and offers considerable time and cost savings over both dHPLC and sequencing. This closed-tube assay is performed on amplicons post-PCR. In the presence of a saturating double-stranded DNA-binding dye, amplicons are slowly heated until fully denatured while the fluorescence is monitored(7). Amplicons heterozygous for a sequence variant yield altered melting curves compared with normal control samples. High-resolution melting analysis has recently been tested in a variety of clinical mutation-scanning applications and shown to be a sensitive and cost-effective technique(8)(9)(10)(11)(12)(13)(14).
In this study we assessed whether 20 different F8 gene mutations, located across 13 exons, could be detected by high-resolution melting analysis. These are mutations we have previously identified by dHPLC and DNA sequencing during our F8 gene-screening program(6). For each mutation we assessed whether the melting curve of the heterozygous amplicon differed from that of the corresponding normal amplicon to evaluate whether a melting analysis screen of the F8 gene would detect the variant. We tested 2 different platforms—the Roche LightCycler 480 (LC480) and the Idaho Technology LightScanner.
Genomic DNA from 20 male HA patients, each with a different F8 gene mutation, was mixed with an equal amount of DNA from a healthy male control. Because males are hemizygous for the F8 gene, this mixing is necessary to ensure the amplified exon is heterozygous for the F8 mutation, which is a requirement for detection by melting analysis. Each mixed sample, and a mixed normal control, was amplified using primers targeting only the F8 exon where the mutation is located. We used primer sets previously optimized for dHPLC by Oldenburg et al.(5), because the requirements of amplicons for melting analysis are likely to be similar to those for dHPLC. Samples were amplified in duplicate 15-µL PCRs using the LightCycler 480 Genotyping Master PCR mixture (Roche), in the presence of 1x LCGreen PLUS (Idaho Technology), 0.5 µmol/L forward and reverse primers, and 50 ng DNA template. The thermal cycling protocol was as follows: polymerase activation (95 °C for 10 min); touchdown cycling step [5 cycles of: denaturation at 95 °C for 20 s, annealing at 61 °C for 20 s (decreased by 1 °C per cycle), extension at 72 °C for 20 s]; amplification (40 cycles of: denaturation at 95 °C for 20 s, annealing at 56 °C for 20 s, extension at 72 °C for 20 s); and a final extension (72 °C for 5 min). The touchdown cycling step was included to improve specificity of the PCR because LCGreen PLUS is known to increase the melting temperature of primers by 2–4 °C(15). For samples analyzed on the LC480, the melting step was appended to the amplification step; samples melted on the LightScanner were amplified on the LC480 as above, then transferred to the LightScanner for the melting analysis.
To analyze melting data on both the LC480 and the LightScanner software, melting curves were normalized by defining regions in the pre- and postdenaturation parts of the curve, and the value was set for the melting temperature shift adjustment. Output plots are in the form of normalized temperature-shifted melting curves that show the decrease in fluorescence (Fl) against increasing temperature, and difference curves that show the difference in fluorescence (
Fl) between the melting curves of the mutation sample and the normal control, against temperature. The difference curves provided the best resolution to differentiate mutation and normal samples, with the amplitude of the peak (Flmax) recorded as a measure of the resolution. On the basis of the observed variation in 4 normal controls (see Figs. 1–3 in the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol53/issue12), we considered that a Flmax value of at least 5 on the LC480, which is equivalent to 0.05 on the LightScanner, was sufficient resolution to distinguish the mutant sample from the normal control, although the shape of the curve is also an important consideration and may indicate the presence of a variant even if Flmax 
5/0.05.
Analysis of the data indicated that 18 of 20 F8 gene mutations included in this study were detected by melting analysis on both LC480 and LightScanner platforms, with Flmax values of at least 5/0.05 (Table 1
). The variants F1775L (c.5380T>C, exon 16) and c.6901–2A>G [intron 25 (exon 26 amplicon)] had Flmax of <5/0.05, indicating a lower confidence in the ability of the melting analysis to resolve the mutant sample from the normal controls (see Figs. 1 and 3 in the online Data Supplement). Even when PCR products from the mutant samples were mixed with products from normal DNA post-PCR, the resolution of these samples was still poor (see Figs. 4 and 5 in the online Data Supplement).
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Data for the melting analysis of F8 exon 23 variants on the LightScanner is shown in Fig. 1
. These curves are representative of the other samples in the study listed in Table 1
and show the difference curve for each variant (in duplicate) derived using the normal sample melting curve as a reference, which appears on the difference plot as the baseline. Each exon 23 variant has a distinct curve profile, indicating this technique can distinguish different variants from each other, as well as from the normal sample. Melting curves for these exon 23 variants on the LC480 are shown in Fig. 2 of the online Data Supplement, along with 4 controls to indicate the variability in normal samples.
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The LC480 and LightScanner platforms performed similarly overall, although each has strengths and weaknesses. The LC480 is a thermal cycler, so samples for melting analysis can be amplified on this machine, with the melting step appended to the PCR protocol. The amplification of samples can be monitored in real time, because LCGreen PLUS fluorescence is proportional to the amount of double-stranded DNA in the PCR, and any samples in which amplification failed can be removed from the melting analysis. The LightScanner performs only the melting step, but in our hands gave greater consistency between replicate samples. Both machines use a 96-well plate format (with the option for a 384-well format), enabling rapid throughput of samples.
In summary, high-resolution melting analysis is a powerful and cost-effective option for mutation scanning of the F8 gene. The sensitivity of this technique is comparable to dHPLC, and it offers advantages in the speed and cost of analysis. To support the F8 mutation-scanning screen in the clinical setting, DNA sequencing of amplicons in which a putative mutation (or polymorphism) was detected can be used to confirm and identify the variant and to rescreen any samples in which no mutations were detected across all F8 exons. In this context, scanning by high-resolution melting analysis is an ideal technology because it is rapid, economical, and capable of detecting most mutations (90% in this study). Mutations not detected by the melting analysis screen would be identified by subsequent sequencing of all F8 exons for that sample.
Acknowledgments
Grant/funding support: We acknowledge Roche Diagnostics (New Zealand) for the use of the Roche LC480, and John Morris Scientific for the use of the Idaho Technology LightScanner.
Financial disclosures: None declared.
Acknowledgments: We are grateful to Scott Mead and Campbell Sheen for helpful comments.
References
The following articles in journals at HighWire Press have cited this article:
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  M.-P. Audrezet, A. Dabricot, C. Le Marechal, and C. Ferec Validation of High-Resolution DNA Melting Analysis for Mutation Scanning of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Gene J. Mol. Diagn., September 1, 2008; 10(5): 424 - 434. [Abstract] [Full Text] [PDF]
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 K. De Leeneer, I. Coene, B. Poppe, A. De Paepe, and K. Claes Rapid and Sensitive Detection of BRCA1/2 Mutations in a Diagnostic Setting: Comparison of Two High-Resolution Melting Platforms Clin. Chem., June 1, 2008; 54(6): 982 - 989. [Abstract] [Full Text] [PDF]
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