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Rapid Diagnosis of MEN2B Using Unlabeled Probe Melting Analysis and the LightCycler 480 Instrument

2008; Elsevier BV; Volume: 10; Issue: 2 Linguagem: Inglês

10.2353/jmoldx.2008.070111

1943-7811

Rebecca L. Margraf, Rong Mao, Carl T. Wittwer,

Electron and X-Ray Spectroscopy Techniques

Multiple endocrine neoplasia type 2B (MEN2B) is an autosomal dominant, inherited cancer syndrome. MEN2B patients have a high risk of developing medullary thyroid carcinoma, and prophylactic thyroidectomy is recommended by 6 months of age. Genetic testing can identify MEN2B patients before cancer progression. Two RET proto-oncogene mutations, in exon 15 at codon 883 (GCT>TTT) and in exon 16 at codon 918 (ATG>ACG), account for more than 98% of MEN2B cases. An assay using unlabeled probes and the LightCycler 480 instrument was developed to genotype these two common MEN2B RET mutations. Asymmetric polymerase chain reaction was used to increase ssDNA products followed by melting analysis of the unlabeled probe/ssDNA amplicon duplex. The available samples were either patient DNA of known RET genotype or artificial templates. Analysis of the codon 883 heterozygous mutation demonstrated a ΔTm of 5.70 ± 0.11°C, while the codon 918 heterozygous mutation generated a ΔTm of −5.72 ± 0.11°C. Samples with the targeted RET mutation genotypes were accurately detected and easily distinguishable from five other reported sequence changes using these probes. Thus, MEN2B diagnosis using unlabeled probes and the LightCycler 480 is a rapid, closed-tube method that is less time consuming and less expensive than sequencing. This assay demonstrates 100% specificity and sensitivity for the identification of RET mutations causative of MEN2B. Multiple endocrine neoplasia type 2B (MEN2B) is an autosomal dominant, inherited cancer syndrome. MEN2B patients have a high risk of developing medullary thyroid carcinoma, and prophylactic thyroidectomy is recommended by 6 months of age. Genetic testing can identify MEN2B patients before cancer progression. Two RET proto-oncogene mutations, in exon 15 at codon 883 (GCT>TTT) and in exon 16 at codon 918 (ATG>ACG), account for more than 98% of MEN2B cases. An assay using unlabeled probes and the LightCycler 480 instrument was developed to genotype these two common MEN2B RET mutations. Asymmetric polymerase chain reaction was used to increase ssDNA products followed by melting analysis of the unlabeled probe/ssDNA amplicon duplex. The available samples were either patient DNA of known RET genotype or artificial templates. Analysis of the codon 883 heterozygous mutation demonstrated a ΔTm of 5.70 ± 0.11°C, while the codon 918 heterozygous mutation generated a ΔTm of −5.72 ± 0.11°C. Samples with the targeted RET mutation genotypes were accurately detected and easily distinguishable from five other reported sequence changes using these probes. Thus, MEN2B diagnosis using unlabeled probes and the LightCycler 480 is a rapid, closed-tube method that is less time consuming and less expensive than sequencing. This assay demonstrates 100% specificity and sensitivity for the identification of RET mutations causative of MEN2B. Multiple endocrine neoplasia type 2 (MEN2) is an autosomal dominant, inherited disorder consisting of three syndromes: MEN2A, MEN2B, and familial medullary thyroid carcinoma (FMTC). “Gain of function” mutations in the RET proto-oncogene cause the MEN2 syndromes. MEN2 RET mutations are almost exclusively heterozygous, with only a few reports of rare homozygous mutations.1Elisei R Cosci B Romei C Agate L Piampiani P Miccoli P Berti P Basolo F Ugolini C Ciampi R Nikiforov Y Pinchera A Identification of a novel point mutation in the RET gene (Ala883Thr), which is associated with medullary thyroid carcinoma phenotype only in homozygous condition.J Clin Endocrinol Metab. 2004; 89: 5823-5827Crossref PubMed Scopus (50) Google Scholar MEN2 syndromes result in a high lifetime risk of developing medullary thyroid carcinoma (MTC). MTC can metastasize early resulting in poor prognosis, since MTC responds poorly to chemotherapy. The only known cure is thyroidectomy before MTC metastasis, and is usually preformed between 6 months and 10 years of age, depending on the RET mutation.2Wiesner G Snow-Bailey K GeneReviews: Multiple Endocrine Neoplasia Type 2.http://www.geneclinics.org/servlet/access?db=geneclinics&site=gt&id=8888892&key=2paoNNdpDmi-r&gry=&fcn=y&fw=ZVFB&filename=/profiles/men2/index.htmlGoogle Scholar Genetic diagnostic testing can identify MEN2 patients before cancer progression, significantly improving patient survival. FMTC families present the clinical manifestation of MTC only. In addition to MTC, MEN2A families can develop pheochromocytomas and hyperparathyroidism. MEN2B patients can also develop pheochromocytomas and (rarely) hyperparathyroidism, but these patients have some additional distinguishing physical symptoms as well, such as ganglioneuromas of the digestive tract, mucosal neuromas of the lips and tongue, corneal nerve thickening, marfanoid habitus, and skeletal abnormalities.2Wiesner G Snow-Bailey K GeneReviews: Multiple Endocrine Neoplasia Type 2.http://www.geneclinics.org/servlet/access?db=geneclinics&site=gt&id=8888892&key=2paoNNdpDmi-r&gry=&fcn=y&fw=ZVFB&filename=/profiles/men2/index.htmlGoogle Scholar,3Gimm O Marsh DJ Andrew SD Frilling A Dahia PL Mulligan LM Zajac JD Robinson BG Eng C Germline dinucleotide mutation in codon 883 of the RET proto-oncogene in multiple endocrine neoplasia type 2B without codon 918 mutation.J Clin Endocrinol Metab. 1997; 82: 3902-3904Crossref PubMed Google Scholar The MEN2B patients have a very high risk of developing MTC and prophylactic thyroidectomy is recommended by 6 months of age.2Wiesner G Snow-Bailey K GeneReviews: Multiple Endocrine Neoplasia Type 2.http://www.geneclinics.org/servlet/access?db=geneclinics&site=gt&id=8888892&key=2paoNNdpDmi-r&gry=&fcn=y&fw=ZVFB&filename=/profiles/men2/index.htmlGoogle Scholar This report demonstrates a RET genotyping assay that can identify patients with MEN2B in a cost effective, rapid manner compared to sequencing or restriction digestion assays. An unlabeled probe genotyping assay uses asymmetric polymerase chain reaction (PCR) to generate ssDNA product.4Zhou L Myers AN Vandersteen JG Wang L Wittwer CT Closed-tube genotyping with unlabeled oligonucleotide probes and a saturating DNA dye.Clin Chem. 2004; 50: 1328-1335Crossref PubMed Scopus (174) Google Scholar The unlabeled probe is 3′ blocked to prevent probe extension during PCR. After PCR, the unlabeled probe and ssDNA target are annealed and subjected to high-resolution melting analysis. The detection is by a saturating dsDNA dye, LCGreenPlus+. Since a dsDNA dye is used, both the amplicon data as well as the unlabeled probe data can be analyzed, if necessary.5Margraf RL Mao R Highsmith WE Holtegaard LM Wittwer CT RET proto-oncogene genotyping using unlabeled probes, the masking technique, and amplicon high-resolution melting analysis.J Mol Diagn. 2007; 9: 184-196Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar,6Zhou L Wang L Palais R Pryor R Wittwer CT High-resolution DNA melting analysis for simultaneous mutation scanning and genotyping in solution.Clin Chem. 2005; 51: 1770-1777Crossref PubMed Scopus (182) Google Scholar The closed-tube unlabeled probe genotyping assay reduces contamination and hands-on time compared to other genotyping assays that require post-PCR processing. Unlabeled probe assays have proven to be rapid, cost effective, and accurate for genotyping.4Zhou L Myers AN Vandersteen JG Wang L Wittwer CT Closed-tube genotyping with unlabeled oligonucleotide probes and a saturating DNA dye.Clin Chem. 2004; 50: 1328-1335Crossref PubMed Scopus (174) Google Scholar5Margraf RL Mao R Highsmith WE Holtegaard LM Wittwer CT RET proto-oncogene genotyping using unlabeled probes, the masking technique, and amplicon high-resolution melting analysis.J Mol Diagn. 2007; 9: 184-196Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar6Zhou L Wang L Palais R Pryor R Wittwer CT High-resolution DNA melting analysis for simultaneous mutation scanning and genotyping in solution.Clin Chem. 2005; 51: 1770-1777Crossref PubMed Scopus (182) Google Scholar7Liew M Seipp M Durtschi J Margraf RL Dames S Erali M Voelkerding K Wittwer C Closed-tube SNP genotyping without labeled probes/a comparison between unlabeled probe and amplicon melting.Am J Clin Pathol. 2007; 127: 341-348Crossref PubMed Scopus (52) Google Scholar8Liew M Nelson L Margraf R Mitchell S Erali M Mao R Lyon E Wittwer C Genotyping of human platelet antigens 1 to 6 and 15 by high-resolution amplicon melting and conventional hybridization probes.J Mol Diagn. 2006; 8: 97-104Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar The newly available LightCycler 480 (LC480) instrument, with its 96- or 384-well format, has great potential as a platform for high-throughput and rapid unlabeled probe assays. A recent report by Herrmann et al9Herrmann MG Durtschi JD Wittwer CT Voelkerding KV Expanded instrument comparison of amplicon DNA melting analysis for mutation scanning and genotyping.Clin Chem. 2007; 53: 1544-1548Crossref PubMed Scopus (147) Google Scholar compares the LC480 and other commercially available instruments for amplicon melting analysis. RET mutations within exons 15 and 16 can cause MEN2B. The MEN2B RET mutation for exon 15 is a deletion/insertion mutation at codon 883(GCT>TTT) of genotype c.2647_2648delinsTT.3Gimm O Marsh DJ Andrew SD Frilling A Dahia PL Mulligan LM Zajac JD Robinson BG Eng C Germline dinucleotide mutation in codon 883 of the RET proto-oncogene in multiple endocrine neoplasia type 2B without codon 918 mutation.J Clin Endocrinol Metab. 1997; 82: 3902-3904Crossref PubMed Google Scholar The MEN2B RET mutation for exon 16 is a missense mutation at codon 918(ATG>ACG) of genotype c.2753T>C. Approximately 5% of MEN2B patients have the codon 883(GCT>TTT) mutation, while ∼94% of MEN2B patients have the codon 918(ATG>ACG) mutation.2Wiesner G Snow-Bailey K GeneReviews: Multiple Endocrine Neoplasia Type 2.http://www.geneclinics.org/servlet/access?db=geneclinics&site=gt&id=8888892&key=2paoNNdpDmi-r&gry=&fcn=y&fw=ZVFB&filename=/profiles/men2/index.htmlGoogle Scholar,10Hansford JR Mulligan LM Multiple endocrine neoplasia type 2 and RET: from neoplasia to neurogenesis.J Med Genet. 2000; 37: 817-827Crossref PubMed Scopus (211) Google Scholar A genotyping assay targeting these two RET mutations will identify >98% of MEN2B patients. In addition, there are other RET sequence changes within the region of the two targeted MEN2B RET mutations that instead cause FMTC or Hirschsprung disease (HSCR). HSCR can be caused by “loss of function” mutations within the RET proto-oncogene that result in the congenital absence of ganglia in portions of the digestive tract and can lead to potentially lethal intestinal blockage. The MEN2B RET genotyping assay distinguishes the two targeted MEN2B RET mutations from other reported sequence variations under the analysis probes. In this report, RET sequence variation was listed by the codon number, the wild-type codon DNA sequence followed by the codon sequence change. The nucleotide changes are in bold for the targeted RET mutations, 883(GCT>TTT) and 918(ATG>ACG), while the nontargeted RET nucleotide changes will be underlined; for example, 883(GCT>ACT). Normal samples have the RET consensus, wild-type sequence (under the probe) that is not associated with disease. De-identified normal samples and the heterozygous RET exon 16 codon 918(ATG>ACG) mutation sample were obtained from the Coriell Institute (Camden, NJ) as described previously.11Margraf RL Mao R Highsmith WE Holtegaard LM Wittwer CT Mutation scanning of the RET protooncogene using high-resolution melting analysis.Clin Chem. 2006; 52: 138-141Crossref PubMed Scopus (40) Google Scholar Coriell samples GM16658 (RET exon 15 and 16 normal controls) and GM11629 (heterozygous for the exon 16, codon 918 mutation) were used for the data in the figures and table. Coriell samples were purified using a Qiagen QIAquick kit (Qiagen, Valencia, CA); while 12 additional de-identified normal samples were purified using MagNa PURE (Roche Diagnostics Corp, Indianapolis IN). All sample genotypes were confirmed by sequence analysis (GenBank RET genomic reference NC_000010.9). The PCR amplification oligos were synthesized at Integrated DNA Technologies, Inc. (IDT, Coralville, IA). Exon 15 primer sequences were 5′-CTGGGAGCCCCGCCTCAT-3′ for the reverse and 5′-CACACACCACCCCTCTGCTG-3′ for forward. Exon 16 primer sequences were 5′-ACACATCACTTTGCGTGGTG-3′ for the reverse and 5′-TCTCCTTTACCCCTCCTTCCT-3′ for forward. The unlabeled probes were oligos that incorporated a 3′ amino modifier (3 AmM) to prevent DNA polymerase extension during PCR.4Zhou L Myers AN Vandersteen JG Wang L Wittwer CT Closed-tube genotyping with unlabeled oligonucleotide probes and a saturating DNA dye.Clin Chem. 2004; 50: 1328-1335Crossref PubMed Scopus (174) Google Scholar,12Dames S Margraf RL Pattison DC Wittwer CT Voelkerding KV Characterization of aberrant melting peaks in unlabeled probe assays.J Mol Diagn. 2007; 9: 290-296Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar The unlabeled probes were synthesized by IDT. The exon 15 probe had RET wild-type sequence 5′-CATCCTGGTAGCTGAGGGGCGGAAGATGAAGATT/3AmM/-3′ (34 nucleotides). The exon 16 probe had the codon 918(ATG>ACG) mutation-specific sequence 5′-GGTCGGATTCCAGTTAAATGGACGGCAAT/ 3AmM/-3′ (29 nucleotides). The thirteen artificial templates of the unavailable RET sequence variations used oligos that were 3′ blocked with an amino modifier (synthesized at IDT) to prevent amplification during PCR.12Dames S Margraf RL Pattison DC Wittwer CT Voelkerding KV Characterization of aberrant melting peaks in unlabeled probe assays.J Mol Diagn. 2007; 9: 290-296Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar These oligos complement the analysis probe sequences but include the mutation sequence change(s), and therefore functioned as a mutant allele template for hybridization of the analysis probe. The oligos were 34 nucleotides for exon 15 and 29 nucleotides for exon 16. The 10 μmol/L oligo stock was added at 1 to 9 μl of either water or a normal genomic DNA sample (10–50 ng/μL) to generate the artificial templates for the homozygous mutation and heterozygous samples, respectively. The heterozygous exon 15 codon 883(GCT>TTT) mutation, all homozygous sequence variation samples, and all of the heterozygous non-MEN2B RET sequence variations were artificial templates. A final PCR reaction volume of 10 μl contained either 2 μl of sample genomic DNA (20–100 ng total) or 2 μl of an artificial template. The asymmetric PCR reaction contained final concentrations of 1X FastStart master hybridization mix (LightCycler FastStart DNA Master Hybridization Probe Kit from Roche), 2 mmol/L additional of MgCl2, 0.01 U/μl AmpErase uracil N-glycosylase (Applied Biosystems, Foster City, CA) and 1X LCGreen Plus+ (Idaho Technology, Salt Lake City, UT). The two targeted MEN2B RET mutations were tested in separate reactions, with 0.5 μmol/L final concentration of either the exon 15 or exon 16 unlabeled probe. The exon 15 reaction used 0.5 μmol/L final concentration for the reverse primer and 0.1 μmol/L for the forward primer. The exon 16 reaction used 0.54 μmol/L for the reverse primer and 0.06 μmol/L for the forward primer. Reactions were overlaid with 13 μl of mineral oil to minimize a rise in background fluorescence, improving data interpretation. The plate was sealed with LC480 sealing foil (Roche) and spun at 1000 RPM for 1 minute before loading into the LC480 instrument (Roche). Thermocycling was performed on a LC480 instrument with the following parameters: initial uracil N-glycosylase step (50°C for 3 minutes) and polymerase activation (95°C for 7 minutes), followed by 55 PCR cycles (denaturation at 95°C for 10 seconds, annealing at 64°C for 10 seconds, and extension at 72°C for 10 seconds). After PCR, probe/ssDNA amplicon duplexes were generated by heating samples to 95°C for 30 seconds, then cooling to 50°C for 30 seconds. The melting data were collected between 53°C and 95°C at 30 acquisitions per °C, using the “melting curves” analysis mode. Then samples were cooled to 50°C for 10 seconds. The LC480 run was finished in approximately 1 hour and 45 minutes. All LC480 default values for rates were used: 2.2°C/s for cooling or 4.4°C/s for heating. Reaction volume was set at 10 μl (even though 13 μl of oil overlay was added). The LCGreen dye detection format used the 450 excitation filter and 500 emission filter combination. The unlabeled probe data were analyzed between 56°C and 77°C. The melting data were directly converted to a derivative plot using the LC480 software module, Tm Calling. This automatic Tm Calling function was designed for fluorescently labeled hybridization probes only and did not reliably call the melting temperatures for the unlabeled probe assay. Therefore, derivative melting peak Tms were called using the “manual Tm method” within the Tm Calling software. The Tm for the unlabeled probes was visually assigned as the temperature at the approximate (1/2) area of the derivative melting curve peak. ΔTms for heterozygous samples were defined by calculating the wild-type allele Tm minus the mutant allele Tm. The average allele Tms and ΔTm values for the normal samples and the targeted MEN2B RET mutation samples are given in Table 1, as well as the standard deviation. Note that since exon 16 uses a mutation-specific probe, the 918(ATG>ACG) mutant allele Tm is higher than the wild-type allele Tm, resulting in a negative ΔTm value.Table 1MEN2B RET Genotyping Assay PrecisionWithin run†Within run samples were in triplicate.Between run‡Between run samples were tested in five different runs.ExonGenotypeAllele*WT, wild-type allele; MUT, mutant allele; ΔTm = wild-type allele Tm − mutant allele Tm. or ΔTmAve§Ave, average (°C). For WT and MUT alleles, the average Tm is listed. The ΔTm was calculated individually per each heterozygous sample (ΔTm = wild-type allele Tm − mutant allele Tm), then average ΔTm and SD were calculated. SD, 1 standard deviation (°C).SDAveSD15Codon 883(GCT>TTT) heterozygousWT73.800.0673.710.12MUT68.050.0968.010.07ΔTm5.750.085.700.11Normal controlWT73.810.0373.620.1416Codon 918(ATG>ACG) heterozygousWT65.930.0165.850.15MUT71.710.0371.580.10ΔTm−5.790.03−5.720.11Normal controlWT65.970.0065.850.20* WT, wild-type allele; MUT, mutant allele; ΔTm = wild-type allele Tm − mutant allele Tm.† Within run samples were in triplicate.‡ Between run samples were tested in five different runs.§ Ave, average (°C). For WT and MUT alleles, the average Tm is listed. The ΔTm was calculated individually per each heterozygous sample (ΔTm = wild-type allele Tm − mutant allele Tm), then average ΔTm and SD were calculated. SD, 1 standard deviation (°C). Open table in a new tab The exon 15 reaction used an unlabeled probe of wild-type RET sequence to detect the targeted MEN2B RET mutation: 883(GCT>TTT). Fifteen normal samples were tested and had a wild-type allele Tm of 73.74 ± 0.11°C (average ± 1 SD). Since the targeted mutation sample was not available, a codon 883(GCT>TTT) artificial template was used and had a ΔTm value of 5.70 ± 0.11°C (Table 1, “between run” value). Occasionally, the exon 15 no template control had primer-dimer peak at ∼84°C which was confirmed by gel, but it did not interfere with the unlabeled probe data between 56°C and 77°C. Example data for a normal sample, the heterozygous artificial template for a codon 883(GCT>TTT) mutation sample, and a no template control is shown in Figure 1A. Precision values for within run and between runs are given in Table 1. The exon 16 reaction used a codon 918(ATG>ACG) mutation-specific unlabeled probe to identify the targeted MEN2B RET mutation: 918(ATG>ACG). Fourteen normal samples were tested and had an average wild-type allele Tm of 66.03 ± 0.1°C. A patient sample was available for the targeted MEN2B RET mutation at codon 918(ATG>ACG), and had a ΔTm value of −5.72 ± 0.11°C (Table 1, “between run” values). Example data for a normal sample, the heterozygous 918(ATG>ACG) mutation sample, and a no template control is shown in Figure 2A. Precision values for within run and between runs are given in Table 1. There are five other reported RET sequence changes under the analysis probes which can cause FMTC or HSCR.13Stenson PD Ball EV Mort M Phillips AD Shiel JA Thomas NS Abeysinghe S Krawczak M Cooper DN Human Gene Mutation Database (HGMD): 2003 update.Hum Mutat. 2003; 21: 577-581Crossref PubMed Scopus (1377) Google Scholar,14Prazeres HJ Rodrigues F Figueiredo P Naidenov P Soares P Bugalho MJ Lacerda M Campos B Martins TC Occurrence of the Cys611Tyr mutation and a novel Arg886Trp substitution in the RET proto-oncogene in multiple endocrine neoplasia type 2 families and sporadic medullary thyroid carcinoma cases originating from the central region of Portugal.Clin Endocrinol (Oxford). 2006; 64: 659-666Crossref PubMed Scopus (18) Google Scholar Although patients with these diseases have different clinical symptoms than an MEN2B patient, heterozygous artificial templates were generated for these sequence changes to determine whether the ΔTm values were distinguishable from the two targeted MEN2B RET mutations (Figure 1, Figure 2). Homozygous artificial templates were also tested for all RET sequence variations (data not shown). Exon 15 had three reported non-MEN2B RET sequence changes under the unlabeled probe (Figure 1B and data not shown). The first sequence change is a rare, single family FMTC mutation at codon 883(GCT>ACT) of the genotype c.2647G>A, which overlaps the targeted mutation position.1Elisei R Cosci B Romei C Agate L Piampiani P Miccoli P Berti P Basolo F Ugolini C Ciampi R Nikiforov Y Pinchera A Identification of a novel point mutation in the RET gene (Ala883Thr), which is associated with medullary thyroid carcinoma phenotype only in homozygous condition.J Clin Endocrinol Metab. 2004; 89: 5823-5827Crossref PubMed Scopus (50) Google Scholar This mutation is homozygous in two family members with MTC, while heterozygous family members were unaffected. The ΔTm value for this FMTC mutation was 2.32 ± 0.06°C. The second sequence change is at codon 886(CGG>TGG) of genotype c.2656C>T.14Prazeres HJ Rodrigues F Figueiredo P Naidenov P Soares P Bugalho MJ Lacerda M Campos B Martins TC Occurrence of the Cys611Tyr mutation and a novel Arg886Trp substitution in the RET proto-oncogene in multiple endocrine neoplasia type 2 families and sporadic medullary thyroid carcinoma cases originating from the central region of Portugal.Clin Endocrinol (Oxford). 2006; 64: 659-666Crossref PubMed Scopus (18) Google Scholar It is unknown at this time if this rare, single family variant is an FMTC mutation or a benign variant found in a sporadic MTC patient. The third sequence change causes HSCR and is at codon 884(GAG>GAC) of genotype c.2652G>C. The ΔTm value for 886(CGG>TGG) was 3.4°C and for the codon 884 HSCR mutation was 0.9°C. Exon 16 had two reported non-MEN2B RET sequence changes under the unlabeled probe (Figure 2B). The first sequence change is a rare, single family FMTC mutation (two family members had MTC) at codon 912(CGG>CCG) of the genotype c.2735G>C.15Jimenez C Dang GT Schultz PN El-Naggar A Shapiro S Barnes EA Evans DB Vassilopoulou-Sellin R Gagel RF Cote GJ Hoff AO A novel point mutation of the RET protooncogene involving the second intracellular tyrosine kinase domain in a family with medullary thyroid carcinoma.J Clin Endocrinol Metab. 2004; 89: 3521-3526Crossref PubMed Scopus (38) Google Scholar The ΔTm value for this mutation was 3.47 ± 0.19°C. The other sequence change is a HSCR mutation at codon 912 (CGG>CAG) of genotype c.2735G>A,16Fitze G Cramer J Ziegler A Schierz M Schreiber M Kuhlisch E Roesner D Schackert HK Association between c135G/A genotype and RET proto-oncogene germline mutations and phenotype of Hirschsprung's disease.Lancet. 2002; 359: 1200-1205Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar with a ΔTm value of 3.0°C. To test the accuracy of the artificial templates for predicting ΔTms for patient samples, the ΔTm data for a codon 918(ATG>ACG) artificial template was compared to the available codon 918(ATG>ACG) mutation sample. The artificial template mutant allele was 0.9°C higher Tm than the patient sample mutant allele, resulting in a 0.9°C increase of ΔTm to 6.6°C for the artificial template (data not shown). The MEN2B RET genotyping assay targets two mutations in exons 15 and 16 using unlabeled probes, detecting >98% of known MEN2B RET mutations. There are five other reported RET sequence variations under the analysis probes that could cause FMTC or HSCR. Although FMTC and HSCR patients will have different clinical symptoms than MEN2B patients, these non-MEN2B RET mutations were tested since they would also be detected in the MEN2B RET genotyping assay. The reported non-MEN2B RET sequence variations under the exon 16 mutation-specific probe were easily distinguished from the targeted 918(ATG>ACG) mutation by a lower Tm than the wild-type allele. The exon 15 MEN2B RET mutation is a dinucleotide sequence change and had a greater ΔTm than the three other reported RET point mutations under the analysis probe. All tested heterozygous and homozygous non-MEN2B RET sequence variation samples were distinguished from the two targeted MEN2B RET mutations. Artificial templates were used for the unavailable heterozygous and homozygous sequence variation to estimate the Tm and ΔTm values. For the available exon 16 codon 918(ATG>ACG) mutation, the artificial template mutant allele was 0.9°C higher Tm than the mutant allele from the actual patient's sample. Depending on the mutant allele Tm relative to the wild-type allele Tm, this will create either a higher or lower ΔTm than expected with a patient's sample. The difference between the artificial template and patient's sample may be due to sample salt concentration. Initial differences in sample salt concentrations (due to different DNA purification methods' elution buffers) can lead to differences in sample Tm values.17Seipp MT Durtschi JD Liew MA Williams J Damjanovich K Pont-Kingdon G Lyon E Voelkerding KV Wittwer CT Unlabeled oligonucleotides as internal temperature controls for genotyping by amplicon melting.J Mol Diagn. 2007; 9: 284-289Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar We recommend purifying control and patient DNA by the same method to avoid this potential problem. Another possible reason for the difference in Tms; the patient's sample was amplified with dUTPs (present in the Roche kit), while the artificial template oligos were made with dTTPs (complementary 3′ blocked oligo used to create the mutant allele; not amplified during PCR). The artificial templates can only estimate the expected patient ΔTm values, so patient samples should be used as positive controls when available. Exon 15 has been excluded from some MEN2 assays due to the rarity of mutations within that exon. However, the exon 15 codon 883(GCT>TTT) mutation is found in ∼5% of MEN2B patients and should be included in any MEN2B-specific diagnostic assay to identify these rare individuals that will develop MTC early in life. Although this is the first report for genotyping the codon 883(GCT>TTT) mutation using unlabeled probes, the exon 16 codon 918(ATG>ACG) mutation has been included in an unlabeled probe assay on the LightCycler and HR-1 instrument.5Margraf RL Mao R Highsmith WE Holtegaard LM Wittwer CT RET proto-oncogene genotyping using unlabeled probes, the masking technique, and amplicon high-resolution melting analysis.J Mol Diagn. 2007; 9: 184-196Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar However, in the present study, the exon 16 forward primer was moved to exclude a position of nucleotide discrepancy between primers reported in the literature and some GenBank sequences for the intronic position c.2731–70, since this may be a previously unreported polymorphism. Also, Komminoth et al reported an intronic polymorphism at position c.2731–74 found in three MTC tumor DNA samples.18Komminoth P Kunz EK Matias-Guiu X Hiort O Christiansen G Colomer A Roth J Heitz PU Analysis of RET protooncogene point mutations distinguishes heritable from nonheritable medullary thyroid carcinomas.Cancer. 1995; 76: 479-489Crossref PubMed Scopus (136) Google Scholar Furthermore, the probe was shifted off the codon 922 position, since an MEN2B family was reported to have a codon 922 sequence variant in cis with the codon 918 mutation.19Kitamura Y Scavarda N Wells Jr, SA Jackson CE Goodfellow PJ Two maternally derived missense mutations in the tyrosine kinase domain of the RET protooncogene in a patient with de novo MEN 2B.Hum Mol Genet. 1995; 4: 1987-1988Crossref PubMed Scopus (37) Google Scholar The LC480 data with the new exon 16 primer and probe is similar to the previous HR-1 data for detecting and identifying the exon 16 MEN2B RET mutation.5Margraf RL Mao R Highsmith WE Holtegaard LM Wittwer CT RET proto-oncogene genotyping using unlabeled probes, the masking technique, and amplicon high-resolution melting analysis.J Mol Diagn. 2007; 9: 184-196Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar Both assays are closed-tube and reduce possible contamination that could occur with genotyping assays that require post-PCR processing. Although the unlabeled probe assay using the LightCycler and HR-1 melting is more cost effective for testing one patient's sample, the LC480 has the cost advantage when more than one sample is tested, since the fixed cost of the 96-well plate is divided among two or more tested samples. The MEN2B RET genotyping assay, using unlabeled probes and the LC480, has less hands-on time and is more cost effective than other assays commonly used to detect MEN2B RET mutations, such as sequencing and restriction enzyme digestion. The RET exon 15 codon 883(GCT>TTT) and exon 16 codon 918(ATG>ACG) mutations were accurately genotyped. The targeted MEN2B RET mutations were easily distinguished from normal samples and other reported sequence variations under the analysis probes for 100% analytical specificity and sensitivity. Targeted mutation analysis of these two common MEN2B RET mutations has a 95% clinical sensitivity.2Wiesner G Snow-Bailey K GeneReviews: Multiple Endocrine Neoplasia Type 2.http://www.geneclinics.org/servlet/access?db=geneclinics&site=gt&id=8888892&key=2paoNNdpDmi-r&gry=&fcn=y&fw=ZVFB&filename=/profiles/men2/index.htmlGoogle Scholar Approximately 5% of MEN2B patients either have no detectable RET mutation, even upon sequencing, or have extremely rare, double mutations in exons 14–15 (<2% of known MEN2B RET mutations). Therefore, if neither of the targeted MEN2B RET mutations are found and clinical symptoms or family history still indicate the MEN2B phenotype, then further RET proto-oncogene analysis is recommended. RET exons 14–16 should be sequenced to detect the rare double mutations causative of MEN2B or full RET sequencing could be done. If no mutation is found in the entire RET gene, affected family members can be identified by linkage analysis. The MEN2B RET genotyping assay using the LC480 and unlabeled probes was 100% accurate with the advantages of a closed-tube assay, hands-off operation, and is less expensive than sequencing or other genotyping alternatives. We thank Dr. Karl Voelkerding, Shale Dames, and Maria Erali for helpful discussions and review of the manuscript.