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Technical Briefs |
Departments of1 Surgical Research and2 Pediatric Surgery, Universitätsklinikum Carl Gustav Carus, Dresden University of Technology, Dresden, Germany;
aaddress correspondence to this author at: Department of Surgical Research, Universitätsklinikum Carl Gustav Carus, Dresden University of Technology, Fetscherstrasse 74, D-01307 Dresden, Germany; fax 49-351-458-4350, e-mail schacker{at}rcs.urz.tu-dresden.de
The RET protooncogene is expressed in human tissues of neural crest origin and has been recognized as a susceptibility gene for several autosomal inherited diseases, such as the multiple endocrine neoplasia type 2 syndromes, familial medullary thyroid carcinoma, and Hirschsprung disease (HSCR) (1). In addition to RET germline mutations, associations of several polymorphisms of the RET protooncogene with HSCR have been reported previously, and in particular, the c.135A RET variant has been shown to be strongly associated with the HSCR phenotype (2)(3). In addition to the mutation-independent effect of the c.135A allele in the etiology of HSCR, we have been reporting a HSCR-phenotype-modifying effect of the RET c.135G>A alleles (rs1800858) that result from a within-gene interaction in patients harboring RET germline mutations (4).
We have demonstrated (5) that variants of two RET promoter polymorphisms, –5G>A (rs10900296) and –1C>A (rs10900297), from the transcription start site are associated with HSCR and that the –5G>A polymorphism is in strong linkage disequilibrium with the c.135G>A polymorphism. Because the promoter haplotype –5/–1AC associated with HSCR has a significantly lower activity in an in vitro dual-luciferase expression assay than do those haplotypes identified in the majority of healthy controls, we thus provided evidence that the c.135G>A polymorphism is a marker for a functional variant in the RET promoter (5). Our findings support the concept that the involvement of both RET gene copies occurs in a dose-dependent fashion in RET-associated diseases and confirm the notion of a within-gene interaction between mutation and polymorphisms, with the consecutive modulation of RET-associated phenotypes (5).
Unambiguous haplotype reconstruction from two genotypes at –5 and –1 from the transcription start site of the RET promoter is possible only if one or both genotypes are homozygous by PCR and Sanger sequencing. Otherwise, PCR products of the basic RET promoter have to be cloned, replicated after transformation into competent Escherichia coli, and sequenced. We therefore sought to develop a LightCyclerTM assay that enables a one-step analysis of 10 functional haplotype combinations of the basic RET promoter to rapidly and cost-effectively screen various patient populations for the involvement of certain haplotypes in the development of neural-crest-associated diseases.
PCR was performed on a LightCycler (Roche Diagnostics) using hybridization probes in combination with the LightCycler-FastStart DNA Master Hybridization Probes Kit (Roche Diagnostics). PCR primers and hybridization probes were synthesized commercially by TIB MOLBIOL. We used primers HU RET-PROM Forward (5'-CGCAGCCAGAGCAAGCACT-3') and HU RET-PROM Reverse (5'-CACGTTCCGGGGCACTCA-3') for PCR amplification, and hybridization anchor probe 5'-CTACCCGCTCCTCCGGCGCAGC X-3', labeled with fluorescein, and hybridization sensor probe 5'-CGCTTGCCTCGCTTCAGTCCp-3', labeled with LC-Red 640, for melting curve analysis. PCR for promoter sequences was performed in a total volume of 20 µL in glass capillaries. The reaction mixture used in each PCR consisted of 50 ng of genomic DNA, 2 µL of each primer (5 µM), 2 µL of the LightCycler-FastStart DNA Master Hybridization Probes Kit, 0.4 µL of each hybridization probe (20 µM), and 1.8 µL of MgCl2 (25 mM). Because the fourth haplotype, –5/–1AA, could not be detected in 400 chromosomes sequenced, it was generated by site-directed mutagenesis with the megaprimer method, as described previously (5).
The PCR products were cloned into the pGL3-Basic plasmid (Promega), 0.1 pg of which was used for PCR. Plasmid containing the AA haplotype was mixed with plasmids containing all other haplotypes to generate positive controls. After an initial polymerase activation step for 10 min at 94 °C and initial denaturation at 98 °C for 5 s and at 96 °C for 5 s, 45 PCR cycles were performed with denaturation at 98 °C for 4 s and 96 °C for 6 s, 15 s of annealing at 63 °C, and extension at 72 °C for 12 s. After completion, the reaction was heated to 94 °C for 1 min, followed by cooling to 75 °C at 20 °C/s and to 35 °C at 1 °C/s. After the temperature was maintained at 35 °C for 2 min, a melting curve was recorded during slow heating at 0.05 °C/s to 90 °C. Both PCR and the melting procedure were detected online with the LightCycler instrument. The fluorescence signal (F) was plotted in real time against the temperature (T) to generate melting curves for every sample (F vs T), which were then converted to melting peaks by plotting the negative derivative of F with respect to T against T (–dF/dT against T) (6)(7)(8)(9). All samples analyzed on the LightCycler were verified independently by DNA sequence analysis using Sanger sequencing on an A.L.F. Express sequencer (Amersham Pharmacia Biotech) as described elsewhere (5).
The sensor probe perfectly matched promoter haplotype –5/–1GC. In addition, haplotypes –5/–1AC, –5/–1GA, and –5/–1AA have been reported (10). Fig. 1
shows the melting curves of 10 possible haplotype combinations.
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We analyzed genomic DNA from 130 control individuals for haplotype combinations, using Sanger sequencing and cloning and sequencing of compound heterozygous genotypes at positions –5 and –1. LightCycler analysis with this assay confirmed all haplotypes and haplotype combinations without exception (Table 1
). We could not identify the –5/–1AA haplotype in 260 chromosomes tested. Frequencies of haplotypes containing the –5G>A variant were very similar to the allele frequencies of the exon 2 codon 45 c.135G>A variant (data not shown), suggesting a strong linkage disequilibrium (5).
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Haplotype reconstruction from two genotypes at –5 and –1 from the transcription start site of the RET promoter presents a technical challenge because PCR products of the basic RET promoter have to be cloned and replicated after transformation into competent E. coli when using PCR and Sanger sequencing, and several clones have to be sequenced to determine both haplotypes. Furthermore, GC-rich gene sequences, such as the RET basic promoter, are difficult to resolve because of the formation of stable secondary structures and therefore require repeated sequencing in both directions to verify the DNA sequence.
For the solution of these technical obstacles, we present a LightCycler assay for a one-step analysis of 10 functional RET basic promoter haplotype combinations. This is the first LightCycler assay for haplotyping of two single-nucleotide polymorphisms located four bases apart with use of only one hybridization probe.
In conclusion, this approach enables rapid, cost-effective, and highly accurate determination of haplotypes and their combinations, especially when compared with the conventional approach, which uses PCR, Sanger sequencing, and in the case of heterozygous genotypes, additional subcloning and resequencing.
Acknowledgments
We are grateful to Dr. Olfert Landt (TIB MOLBIOL, Berlin, Germany) for design of the probes.
Footnotes
1 Heike Görgens and Guido Fitze contributed equally to this project; 
References
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