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Technical Briefs |
1 Northcott Neuroscience Laboratory, ANZAC Research Institute, Concord NSW, Australia; 2 Molecular Medicine Laboratory, Concord Hospital, Concord NSW, Australia; 3 John Morris Scientific, Victoria, Australia; 4 Neurogenetics Group and 6 Peripheral Neuropathy Group, Department of Molecular Genetics, Flanders Interuniversity Institute for Biotechnology, Institute Born-Bunge, University of Antwerp, Antwerpen, Belgium; 5 Department of Pathology, School of Medical Sciences, University of New South Wales, Kensington, NSW, Australia;
aaddress correspondence to this author at: Northcott Neuroscience Laboratory, ANZAC Research Institute, Concord, NSW, Australia 2139; fax 61-2-9767-9101, e-mail marinak{at}anzac.edu.au
Abstract
Background: X-linked Charcot-Marie-Tooth type 1 disease has been associated with 280 mutations in the GJB1 [gap junction protein, beta 1, 32kDa (connexin 32, Charcot-Marie-Tooth neuropathy, X-linked)] gene. High-resolution melting analysis with an automated instrument can be used to scan DNA for alterations, but its use in X-linked disorders has not been described.
Methods: A 96-well LightScanner for high resolution melting analysis was used to scan amplicons of the GJB1 gene. All mutations reported in this study had been confirmed previously by sequence analysis. DNA samples were amplified with the double-stranded DNA-binding dye LC Green Plus. Melting curves were analyzed as fluorescence difference plots. The shift and curve shapes of melting profiles were used to distinguish controls from patient samples.
Results: The method detected each of the 23 mutations used in this study. Eighteen known mutations provided validation of the high-resolution melting method and a further 5 mutations were identified in a blind study. Altered fluorescence difference curves for all the mutations were easily distinguished from the wild-type melting profile.
Conclusion: High-resolution melting analysis is a simple, sensitive, and cost-efficient alternative method to scan for gene mutations in the GJB1 gene. The technology has the potential to reduce sequencing burden and would be suitable for mutation screening of exons of large multiexon genes that have been discovered to be associated with Charcot Marie Tooth neuropathy.
Charcot-Marie-Tooth (CMT) neuropathy is the most common group of hereditary disorders presenting to genetic clinics and affects 
1 in 2500 individuals (1). The CMT syndrome includes many hereditary disorders of peripheral nerves and affects both motor and sensory neurons. Patients suffering from CMT show progressive distal wasting and weakness, pes cavus or foot drop, and loss of deep tendon reflexes. X-linked CMT (CMTX) is the 2nd most common form of demyelinating CMT after Charcot-Marie-Tooth disease type 1A and accounts for 10%–15% of all CMT cases(2). CMTX1 (MIM 302800) is an X-linked dominant trait caused by mutations in the GJB1 [gap junction protein, beta 1, 32kDa (connexin 32, Charcot-Marie-Tooth neuropathy, X-linked)] gene(3). Over 280 mutations have been reported in this gene according to the Inherited Peripheral Neuropathies Mutation Database (www.molgen.ua.ac.be/CMTMutations).
Our laboratory currently uses sequencing to identify gene mutations in the GJB1 gene. High-resolution melting (HRM) curve analysis is a powerful tool for scanning entire amplicons and detecting sequence variations, made possible through the discovery of the saturating double-stranded (ds) DNA dye, LC Green Plus (4), and by advances in instrumentation that enable acquisition of high-resolution fluorescent data(5). LC Green Plus (Idaho Technology) can be used at concentrations that do not inhibit PCR amplification but efficiently saturate PCR products. HRM analysis of PCR products amplified in the presence of LC Green Plus can detect heterozygous and most homozygous sequence variations by the difference in shape and position of the melting curve when compared with a wild-type melt profile(4)(6). The method would therefore be amenable to screening an X-linked dominant disorder with heterozygous females and hemizygous male patients.
Informed consent for DNA studies was obtained from all patients according to protocols approved by the Concord Hospital and the University of Antwerp Ethics Review Committees. To validate the HRM method, we selected 18 known patient samples that were positive for GJB1 mutations (10 males and 8 females) and 4 control individuals (3 females and 1 male). In addition, 10 deidentified DNA samples (6 males, 4 females) with and without GJB1 mutations were selected for blind analysis from the Molecular Genetics Department, University of Antwerp, Belgium. The control and patient samples used in both the validation and blind study were previously confirmed by sequence analysis with primers described by Bergoffen et al. (3). The GenBank sequence NM_000166 was used as the reference sequence for the cDNA. Nucleotide numbering of the A in the ATG translation initiation site was designated +1. There is 1 reported single-nucleotide polymorphism (SNP; rs11551260) in the coding sequence of the GJB1 gene; however, none of the individuals used in this study contained the SNP (c.287 C>G).
Primers to amplify 4 overlapping amplicons were designed with the Oligo Program Version 6 (Molecular Biology Insights) to provide comprehensive coverage of the GJB1 single-exon 852-bp open reading frame. Primer information, fragment size, fragment coverage, and pathogenic codons are shown in Table 1
. Fragments 1, 2, and 4 provided adequate coverage for gene scanning, and fragment 3 was designed to test for mutations close to the primer. To facilitate heteroduplex formation, samples from hemizygous males were mixed with samples of male wild-type DNA (1:1 w/w) before PCR amplification. Amplifications were performed in 10-µL reactions containing 50 ng DNA, 200 µmol/L dNTP, 1 U Bio-X-Act Taq Polymerase (Bioline), 3 mmol/L MgCl2, 2X PCR Enhancer (Invitrogen) 0.6X LC Green Plus (Idaho Technology), and 0.25 µmol/L primers. PCR was performed on either a 9700 thermocycler (Applied Biosystems) or Mastercycler (Eppendorf) with an initial denaturation of 95 °C for 5 min, followed by 35–40 cycles of 95 °C for 30 s, 55 °C for 30 s, and 68 °C for 40 s, with a final extension of 68 °C for 5 min. We used LC Green Plus at 0.6X in our reactions and found no difference in the fluorescence detection sensitivity at this concentration compared with the 1X concentration. To facilitate heteroduplex formation, we subjected the samples to a 2-temperature hold profile (95 °C for 5 s followed by 50 °C for 5 min).
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We performed melting acquisition on a 96-well LightScanner (Idaho Technology). The plate was heated from 80 °C to 98 °C at 0.1 °C/s with a 300-ms frame interval, 15-ms exposure, and 100% LED power, giving 14 points/°C (5)(7). Melting-curve analysis was performed by use of previously described methods(7) with LightScanner Software (version 1.0.1.524). Melting curves were normalized by selecting linear regions before and after the melting transition. These regions were defined for each curve, with an upper (100%) fluorescence and lower (0%) baseline being common for all curves. To eliminate slight temperature errors between samples, the normalized melting curves were temperature shifted by moving the curves along the X-axis to bring them through a common temperature that facilitates clustering into groups. To avoid false negatives, we performed this procedure at a temperature at which the entire mixture of duplexes had melted. Fluorescent difference curves were generated from normalized temperature-shifted data by selecting a control for comparison and subtracting the fluorescence of the control from all other melting curves. The fluorescence difference between all other curves and the comparison curve was then plotted against temperature.
A total of 18 known GJB1 mutations (mutations 1–18, Fig. 1A–1F
.) were used to validate the HRM method. Mutation detection sensitivity was 100% for all the known mutations. The control (wild-type) sample melting curves grouped tightly for all fragments, and altered difference curves were easily distinguished for the 18 mutations. HRM analysis also demonstrated detection sensitivity for mutations close to the primer. The mutations located closest to either end of fragment 3 were present in patient 7 (located 3-bp in from the 5' end of the forward primer) and in patient 16 (located 2-bp from the 3' end of the reverse primer). In both instances, the mutations gave altered melting curves compared with the control group (Fig. 1C
and 1D
). The 2 deletion mutations in patients 5 and 7 clearly showed an altered fluorescence curve for the 1-bp (Fig. 1A
) and the 18-bp deletion (Fig. 1B
and 1C
) compared with the wild-type profile. For the blind analysis, the same controls used in the validation experiments were amplified in addition to the 10 deidentified samples. Mutation detection sensitivity was 100%, with 5 of 5 mutations (mutations 19–23, Fig. 1G–1I
) being identified. Altered melting curves were observed on fragments 1, 3, and 4 and samples negative for mutations grouped tightly with the known control melting profiles. The melting curves for mutation 23 (509T>A) confirmed localization of the base change to an overlapping region on fragments 3 and 4. Mutations 1 (Fig. 1A
), 8, 10 (Fig. 1B
and 1C
), 18 (Fig. 1F
), and 21 (Fig. 1G
) are previously unreported novel mutations that further demonstrate the allelic heterogeneity of CMTX1.
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We have demonstrated a rapid and sensitive method for mutation scanning the GJB1 gene by use of the ds DNA-binding dye LC Green Plus and a 96-well format dedicated melting and detection instrument (LightScanner). GJB1 provided an excellent gene model, enabling us to analyze many different mutations spanning the GJB1 gene. The HRM results for the 18 known mutations and the detection of the 5 mutations in the blind study were 100% concordant with the results obtained by sequencing. We recognize that HRM analysis will not replace sequencing for confirmation of altered fluorescence melt curves; however, in a gene scanning scenario, samples in the blind study producing a melt curve consistent with known normal controls would not be sequenced, a situation that demonstrates the potential of this technology to reduce sequencing burden. Although the GJB1 gene contained a reported SNP (rs11551260), retrospective sequence analysis of all individuals confirmed the absence of this neutral variant in our study, as evidenced by the tight grouping of control samples in fragment 1 of both the validation (Fig. 1A
) and blind (Fig. 1I
) study. Accounting for reported SNPs in an amplicon is important in HRM analysis, as is eliminating the amplification of neutral intronic DNA variants through primer design. These neutral variants will produce altered fluorescence difference curves and will require sequence analysis because the specificity of scanning methods is not 100%.
We have shown that HRM analysis requires simple PCR protocols to prepare samples for melting curve analysis, and that the method has the sensitivity required for the detection of deletion gene rearrangements. Other methods relying on heteroduplex formation to detect sequence variation [e.g., dHPLC and temperature gradient capillary electrophoresis (TGCE)] often fail to detect small homozygous insertions and deletions unless sequential analyses are performed and manual processing is undertaken (8). Our findings complement the report for detecting internal tandem duplications (ranging from 6 bp to 102 bp) by this method(9).
Because HRM can scan an entire amplicon for sequence variation, this method is ideal for screening the complete open reading frame of a gene. Although the coding region of the GJB1 gene is only a single-exon gene, it allowed us to test and validate HRM as a gene-scanning method for an X-linked disorder, in which all heterozygous female and hemizygous male mutations were detected. In contrast, many of the genes reported for CMT are large (>15 exons) e.g., DNM2 (dynamin 2) and MFN2 (mitofusin 2), and would be suited to this method of mutation scanning. We have estimated the cost of reagents to be $0.60 per sample per amplicon, which is one tenth of our current sequencing cost. HRM analysis of single exons of large multiexon genes is a simple, sensitive, and cost-efficient gene-scanning method that would clearly reduce the sequencing burden.
References
The following articles in journals at HighWire Press have cited this article:
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M. Pichler, M. Balic, E. Stadelmeyer, C. Ausch, M. Wild, C. Guelly, T. Bauernhofer, H. Samonigg, G. Hoefler, and N. Dandachi Evaluation of High-Resolution Melting Analysis as a Diagnostic Tool to Detect the BRAF V600E Mutation in Colorectal Tumors J. Mol. Diagn., March 1, 2009; 11(2): 140 - 147. [Abstract] [Full Text] [PDF]
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J. G. Vandersteen, P. Bayrak-Toydemir, R. A. Palais, and C. T. Wittwer Identifying Common Genetic Variants by High-Resolution Melting Clin. Chem., July 1, 2007; 53(7): 1191 - 1198. [Abstract] [Full Text] [PDF]
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