| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Letters |
1
Unidad de Medicina Molecular (INGO), and,
2
Departamento de Fisiologia, Complejo Hospitalario, Universitario de Santiago, Universidad de Santiago de Compostela, 15706 Santiago de Compostela, Spain
aAddress correspondence to this author at: Unidad de Medicina Molecular, Hospital de Conxo, Rua Ramón Baltar, 15706 Santiago de Compostela, Spain. Fax 34-981-951679, e-mail lloidi{at}usc.es.
To the Editor:
Mutations of the RET protooncogene are associated with several disorders, including Hirschsprung disease, familial medullary thyroid carcinoma (FMTC), and multiple endocrine neoplasia type 2A (MEN 2A) and type 2B (MEN 2B). More than 85% of the causing mutations in MEN 2A families affect codon 634 in exon 11 of the RET protooncogene (1)(2)(3)(4)(5). Although all possible changes except the conservative TGC to TGT and the TGC to the termination codon TGA have been found in codon 634 in MEN 2A families, TGC (Cys) to TAC (Tyr) and TGC to CGC (Arg) are by far the most prevalent. Mutations in codon 634 are also responsible for 25–30% of patients with FMTC.
Mutation analysis of RET permits identification of MEN 2A carriers and can reduce morbidity and mortality through early intervention (6).
The methods used to detect RET mutations are time-consuming and require optimization of the PCR to avoid nonspecific PCR products that may interfere with the result. We used real-time PCR and melting curves on a LightCycler (Roche Molecular Biochemicals) to analyze the two most frequent RET mutations, C634Y and C634R, and the less frequent C634S (TGC to TCC).
All subjects had been genotyped previously by sequencing of exon 11 of the RET protooncogene. DNA was isolated from peripheral blood leukocytes by standard methods. The primers used for the amplification were RET 11 Forward (5'-CCTCTGCGGTGCCAAGCCT-3') and RET 11 Reverse (5'-GCTGACCGGGAAGGTGGG-3'), which gave a 217-bp PCR product. The wild-type probe stretched from codon 624 to codon 637. The sequence of the sensor 3' fluorescein-labeled probe was 5'-ACCGTGCGGCACAGCTC-3', and the sequence of the anchor probe, labeled 5' with LC-Red 640, was 5'-TCGCACAGTGGATCTGTGGGTGG-3'.
PCR reactions were performed in a total volume of 20 µL in the LightCycler glass capillaries. The reaction mixture contained 9.6 µL of distilled water, 2.5 µL of MgCl2 (25 mM), 1 µL of each primer (10 µM), 1 µL of each probe (4 µM), 2 µL of DNA-Master Hybridization Probes (Roche Molecular Biochemicals), and 2 µL of genomic DNA (100–500 ng). We used the master mixture for all samples (to reduce sample-to-sample differences) and a control without DNA. PCR conditions were as follows: initial denaturation at 94 °C for 30 s, followed by 35 cycles of denaturation at 94 °C for 0 s, annealing at 68 °C for 5 s, and extension at 72 °C for 10 s. After amplification, the melting analysis was performed by denaturation at 94 °C for 0 s, annealing at 50 °C for 0 s, and increasing the temperature to 90 °C with a ramp rate of 0.5 °C/s. The fluorescence emitted was measured during this process, and the melting curves (F/T) were automatically converted to melting peaks (-dF/dT).
The wild-type melting curve showed a single peak at 69.5 °C (Fig. 1
), whereas each of the mutation carriers showed two different
peaks, one for the wild-type allele at 69.5 °C and a lower one for
the mutated allele. The T-to-C transition of the C634R mutation
produced a melting peak at 61.5 °C, the G-to-A transition of the
C634Y mutation produced a melting peak at 60 °C, and the G-to-C
transversion of the C634S mutation produced a melting peak at 58 °C.
The assay variation of the melting temperatures was assessed. The
interassay variation (CV) was <1.5% and the intraassay variation was
<0.5% for the wild-type and the mutant alleles studied.
|
This method has several advantages over other methods used. The possibility of contamination is reduced because no post-PCR handling is necessary. Nonspecific PCR products will not affect the result because they will not be recognized by the probes. Finally, this method is very rapid and reduces labor and reagent costs. We think it is useful in the screening of RET for the most common mutation in MEN 2A and especially to establish the carrier status in members of families with MEN 2A and FMTC already characterized as having the 634 mutation. All family members can be analyzed simultaneously and in a very short time.
Acknowledgments
We are grateful to Dr. Josep Oriola (Hospital Clinic, Barcelona, Spain) for providing us with DNA samples from patients affected with MEN 2A and to Dr. Olfert Landt (TIB MOLBIOL, Berlin, Germany) for design of the probes.
References
The following articles in journals at HighWire Press have cited this article:
|
|
 |
|
  R. L. Margraf, R. Mao, W. E. Highsmith, L. M. Holtegaard, and C. T. Wittwer RET Proto-Oncogene Genotyping Using Unlabeled Probes, the Masking Technique, and Amplicon High-Resolution Melting Analysis J. Mol. Diagn., April 1, 2007; 9(2): 184 - 196. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. L. Margraf, R. Mao, W. E. Highsmith, L. M. Holtegaard, and C. T. Wittwer Mutation Scanning of the RET Protooncogene Using High-Resolution Melting Analysis Clin. Chem., January 1, 2006; 52(1): 138 - 141. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Gorgens, G. Fitze, D. Roesner, and H. K. Schackert One-Step Analysis of Ten Functional Haplotype Combinations of the Basic RET Promoter with a LightCycler Assay Clin. Chem., September 1, 2004; 50(9): 1693 - 1695. [Full Text] [PDF]
|
|
|
|
|
|
H. Gorgens, P. Schwarz, J. Schulze, and H. K. Schackert LightCycler Assay in the Analysis of Haplotypes of the Type 2 Diabetes Susceptibility Gene CAPN10 Clin. Chem., August 1, 2003; 49(8): 1405 - 1408. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
CGC) shows two peaks at 61.5
and 69.5 °C. (C), sample heterozygous for the C634Y
mutation (TGC