Screening of RET gene mutations in multiple endocrine neoplasia type-2 using Conformation Sensitive Gel Electrophoresis (CSGE)

Rastreamento de mutações do gene RET na neoplasia endócrina múltipla tipo 2 por Eletroforese em Gel Sensível à Conformação (CSGE)

ARTIGO ORIGINAL

Screening of RET gene mutations in multiple endocrine neoplasia type-2 using Conformation Sensitive Gel Electrophoresis (CSGE)

Rastreamento de mutações do gene RET na neoplasia endócrina múltipla tipo 2 por Eletroforese em Gel Sensível à Conformação (CSGE)

Marcelo A. C. G. dos Santos; Elisangela Pereira de S. Quedas; Rodrigo de Almeida Toledo; Delmar M. Lourenço-Júnior; Sergio Pereira de A. Toledo

Endocrine Genetics Unit (LIM-25), Endocrinology, Department of Internal Medicine, Hospital das Clínicas, and University of São Paulo School of Medicine, SP

^{Address for correspondence}

ABSTRACT

Multiple endocrine neoplasia type 2 (MEN2) is an autosomal dominant inherited tumor syndrome caused by RET proto-oncogene germline mutations (RET). Here we tested the Conformation Sensitive Gel Electrophoresis (CSGE) as a screening method for RET hot-spot mutations. Seven MEN2 families were studied by direct sequencing analysis, CSGE and Single Strand Conformational Polymorphism (SSCP). Using CSGE/SSCP, we were able to detect four out of five types of RET mutations verified by sequencing analysis: Cys620Arg, Cys634Arg, Cys634Tyr, and Met918Thr, furthermore a missense substitution at codon 648 (Val648Ile). RET polymorphisms 691 and 769 were also verified. Data obtained using CSGE/SSCP were fully concordant. We conclude that CSGE showed to be a sensitive, fast, low-cost, and simple procedure to detect RET mutations in codons which are reported as the most prevalent RET variants (~ 95%) in large MEN2 series. As to the Val804Met mutation, this method still needs to be optimized.

Keywords: Genetic screening; CSGE; SSCP; Genetic sequencing; MEN-2; RET proto-oncogene

RESUMO

A neoplasia endócrina múltipla tipo 2 (NEM2) é uma síndrome tumoral herdada por mutações germinativas no proto-oncogene RET (RET). Analisamos a aplicação do método Eletroforese em Gel Sensível à Conformação (CSGE) no rastreamento de mutações hot spots do RET. Sete famílias com NEM2 foram rastreadas pelo seqüenciamento gênico, CSGE e análise do Polimorfismo Conformacional de Cadeia Simples (SSCP). Usando ambas as metodologias de rastreamento, identificamos quatro dos cinco tipos de mutações verificadas pelo seqüenciamento: Cys620Arg, Cys634Arg, Cys634Tyr e Met918Thr, além da variação gênica Val648Ile. Das análises englobando mutações hot spots do RET, 90,6% concordaram com o seqüenciamento genético (incluindo a variação gênica Val648Ile). Polimorfismos nos códons 691 e 769 foram documentados. Os dados obtidos por CSGE/SSCP foram totalmente concordantes. Concluímos que o CSGE revelou ser metodologia sensível, rápida, de fácil execução e baixo custo no rastreamento de mutações nos códons associados à grande maioria (~ 95%) dos pacientes com NEM2.

Descritores: Rastreamento gênico; CSGE; SSCP; Seqüenciamento gênico; NEM2; Proto-oncogene RET

MULTIPLE ENDOCRINE NEOPLASIA type 2 (MEN2) is an inherited tumor syndrome characterized by the presence of medullary thyroid carcinoma (MTC), primary hyperparathyroidism (HPT), and pheochromocytoma (PHEO). MEN2 is classified as MEN2A (MTC, PHEO, and HPT); MEN2B (MTC, PHEO, and mucosal neuromas); and familial MTC (FMTC) (1-5). The genetic basis of MEN2 is associated with germline activating mutations in the RET proto-oncogene (RET). RET gene contains 21 exons, which encode a tyrosine kinase receptor protein with an extracellular domain rich in cysteine residues and an intracellular domain enriched in tyrosine residues (1,6).

Several Brazilian studies on MEN2 have been recently published, in which clinical and genetic aspects of MEN2 were extensively reviewed (2-5,7-14). All clinical variants of MEN2 have a high penetrance rate for MTC and most (90%) RET mutation carriers will exhibit evidence for MTC during their lifetime (15). Briefly, MEN2A comprises 75% of MEN2 cases and most cases (98%) harbor a RET mutation in exons 10 or 11 (codons 609, 611, 618, 620, 630, and 634). RET variants in codon 634 cause 85% of MEN2A cases, mostly with the Cys634Arg and Cys634Tyr mutations (1,7). Furthermore, FMTC cases comprise 20% of MEN2 patients and most of them (85%) have RET mutations in codons 10 or 11 (1,8,15). Less frequent mutations in codons 609, 611, 768, and 804 are usually associated with FMTC, but rarely to MEN2A (1,7). Met918Thr (exon 16) mutation has been reported in 95% of MEN2B cases, which comprise 5% of MEN2 cases (1,15). Rarely, two RET variants have been reported as co-segregating in MEN2 cases (3,8). Thus, the vast majority (95%) of RET disease-causing mutations are associated to codons 620, 634, and 918 (15).

Molecular diagnosis of RET mutations has become a crucial tool for the management of MEN2 as it may (a) identify 1–7% of inherited MTC cases among MTC patients previously considered as "sporadic" cases; (b) identify RET mutation carriers in at-risk family members and eliminate the need for annual follow-up in relatives who do not carry RET mutations; and finally (c) it is the rational basis for indicating preventive total thyroidectomy, usually under 5 years of age, in all RET mutation carriers (4-6,15). Direct DNA sequencing is the gold-standard method for genetic studies in the detection of disease-causing mutations. However, several genetic screening techniques, such as denaturating gradient gel electrophoresis (DGGE), restriction enzymes, single strand conformational polymorphism (SSCP) and denaturing high performance liquid chromatography (DHPLC) have been applied to genetic screening of at-risk relatives in families with several inherited diseases (1-8,15,16).

Conformation Sensitive Gel Electrophoresis (CSGE) has been shown to be a useful method for mutation detection. CSGE detects the differences in electrophoresis migration patterns of homo- and heteroduplex formations. These diverse bands are caused by structural alterations in DNA double helix, which are favored by mild denaturing solvents (16-18). Interestingly, this method presents important advantages when compared to restriction enzymes, SSCP, DGGE (19), and DHPLC methodologies. CSGE may present higher sensitivity for 200–800bp PCR fragment sizes, when compared to SSCP sensitivity (16,20). Comparing to DGGE, CSGE is a feasible method to standardize and does not need a 50bp GC-clamp coupled to one of the sequences (16,20). Moreover, CSGE does not require an expensive wave DNA fragment analysis system, such as in DHPLC. Although CSGE has been applied to several inherited conditions, limited information is available on CSGE to identify RET mutations (19,20). As there is an increasing demand for RET genetic diagnosis in the endocrine practice, the optimization of RET mutations is a worthwhile effort. Thus, this study aimed at validating CSGE as genetic screening approach for RET hot-spot mutations in patients with classical MEN2 phenotype presentations.

MATERIALS AND METHODS

Patients

This study was approved by local ethic committees (CAPPesq — HC-FMUSP, protocol number 265/ 04). A written informed consent was obtained from all individuals involved in this project. In this study, seven previously identified (clinically and genetically) MEN2 families were included: two with MEN2A; one with MEN2A cosegregating with congenital megacolon; three with FMTC; and one MEN2B (3-5,8). A total of 64 individuals (7 index cases and 57 at-risk family members) were screened by CSGE and a subset group of 37 patients were compared to SSCP screening. Initially, mutation analysis of all index cases was performed by directly sequencing the RET hot-spots exons 10, 11, 13, 14, 15, and 16. Other at-risk family members were also submitted to DNA sequencing, although it was restricted to the specific exon in which the RET germline mutation and/or polymorphism were identified. The PCR products of index cases and at-risk relatives found to be mutated and/or polymorphic were then screened by CSGE, and a subset of them by SSCP. Samples from 27 at-risk family members identified as healthy individuals were used as controls. All individuals have been followed at the Outpatient Service of the Division of Endocrinology, Hospital das Clínicas, University of Sao Paulo School of Medicine. Index cases were diagnosed by classical clinical, biochemical, and genetic parameters (2,3,8-12,15).

Methods

Genomic DNA extraction and PCR

Genomic DNA was extracted using the salting-out method (21). DNA concentration was measured by spectrophotometry (Pharmacia Biotech, Sweden). Exons 10, 11, 13, 14, 15, and 16 were amplified by PCR (Minicycler — MJ Research, Waterstone, MA, USA) (22), using previously reported primers (7,13,23). Other primers were designed specifically for CSGE and SSCP analyses, as shown in table 1. PCR protocols were optimized as follows: 10 mM Tris HCl pH 8.4, 50 mM KCl pH 8.4, 0.4 mM of dNTPs, 2.0U of Taq-polimerase, 5% of Dimethyl sulfoxide – DMSO. The MgCl₂ and primer concentrations were: 1.76 mM; 20 pmol for exon 10; 2.40 mM; 14 pmol for exon 11; 2.40 mM; 20 pmol for exons 13 and 15; 3 mM; 10 pmol for exon 14; and 3 mM; 20 pmol for exon 16. Genomic DNA concentration ranged from 100–200 ng. The optimized cycling programs used were: single cycle at 94ºC for 10 min. (exons 10, primer setting 11A, 11B, 15, and 16) and 3 min. (exons 13, primers settings 14A and 14B); 38 cycles at 94^ºC for 30 sec. (all exons), annealing for 30 sec. (exons 10 and 15) and 1 min. (primers settings 11A, 11B, 13, 14A, 14B, and 16), as indicated in table 1, and extension at 72^ºC for 1 min. (all exons), followed by an extension cycle at 72^ºC for 4 min. For exon 14, amplification with clamp primers (primer setting 14C) cycle temperatures were: single cycle at 95^ºC for 4 min.; 35 cycles at 94^ºC for 45 sec., annealing for 45 sec. and extension at 72^ºC for 10 min. PCR products were confirmed by electrophoresis at 70V for 1 h. (Power PAC 3000, Biorad, USA) in 2% agarose gel with TAE 1X buffer stained with ethydium bromide (0.5 mg/ml), where 5 ml of genomic amplified material were mixed to 1 ml of Loading Buffer 6 x and visualized in UV transiluminator (Foto Analyst Mini Visionary, Fotodyne, USA).

Sequencing analysis

RET direct sequencing was performed as follows: 2 ml of Big Dye terminator buffer (Applied Biosystems, Foster City, CA, USA), 6 ml of 0.25% buffer (Tris-HCl 0.5 M pH 9.0 with 25 mM of MgCl₂), 1.60 pm of primer and 5–10 ng of amplified DNA, in a total volume of 20 ml. Cycle sequencing conditions were: single cycle at 96ºC for 2 min.; 40 cycles at 96ºC for 10 sec., annealing at 60ºC for 20 sec. and extension at 60ºC for 4 min. After sequencing, reactions were purified with isopropanol/ethanol according to the manufacturer's instructions, and diluted in 2.5 ml of blue dextran buffer with deionized formamide (TSR, Applied Biosystems). Sequencing products were denaturated at 90ºC for 2 min. and immediately cooled before applied in an ABI 310 DNA sequencer. Electropherograms were analyzed by AB Navigator software (Applied Biosystems).

CSGE

The MDE gel was prepared as follows: MDE 0.5 X (Cambrex Bio Science, Rockland, ME, USA), 6% de TBE 10 X, 2.5% de glycerol, 220 ml of ammonium persulfate 10% and 22 ml of N,N,N',N'-Tetramethylethylenediamine. Gel was applied into plates (coated with repel-silane and gamma-methacryloxypropyltrimethoxysilane) and left to polymerize for 2 h. The electrophoresis system was pre-run (Sequencing System Model S2, Life Technologies, Gibco/BRL, Gaithersburg, USA) for 45 min. at 8W with TBE 0.6 X buffer. Temperature conditions for heteroduplex formation were: single cycle at 94^ºC for 5 min. (exons 10, primer setting 11A, 13, and 16) or 10 min. for exon 11 (primer setting B), followed by annealing at room temperature for 40 min. (exons 10, primers settings 11A and 11B, 13 and 16). 5 ml of sample (2:1 of amplicon and Loading Buffer 1 X) (Triple Dye Loading Buffer 6 X, Cambrex Bio Science) were loaded in gel and electrophoresed for 1 h. at 15 W (exons 11 primer setting A, and exon 13). Subsequently, exon 16 was loaded in gel, and all exons were electrophoresed at 8 W for 11 h. Amplified genomic material from exons 10 and 11 (primer setting B) were electrophoresed for 11 h. at 2 W. CSGE gel was silver stained.

SSCP

SSCP gel and electrophoresis conditions were the same as used for CSGE. After denaturation (same temperatures used in CSGE), amplicons were rested in ice before loading in gel. A loading buffer (95% formamide, 10 mM NaOH, 0.025% Bromophenol Blue, 0.025% Xylene Cyanol) was used. SSCP gel was silver stained.

RESULTS

Figure 1 and table 3 summarize RET mutation findings when CSGE, SSCP, and sequencing methods were applied.

Sequencing analysis

RET hot-spots exons (10, 11, 13, 14, 15, and 16) of all index cases (7 subjects) and in one genetically non-affected at-risk member of each family (7 subjects), used as healthy normal controls, were performed. Other 50 at-risk family members were submitted to RET sequencing of the specific exons segregating mutations and/or polymorphisms. Using direct sequencing we totally performed 186 amplicon analyses, and were able to identify mutations at RET codons Cys620Arg (TGC-CGC) in 11 MEN2 cases; Cys634Arg (TGC-CGC) in 12 MEN2 cases; Cys634Tyr (TGC-TAC) in 8 MEN2 cases; Val804Met (GTG-ATG) in 3 MEN2 cases; and Met918Thr (ATG-ACG) in one MEN2 case. We also identified 2 other at-risk members from a MEN2A family, who presented Val648Ile (GTC-ATC) missense substitution, as previously described (2,8). Some of these RET mutation data had been partially published by us elsewhere (5) (figure 1). Polymorphisms at codons Gly691Ser (GGT-AGT), Leu769Leu (CTT-CTG), and Ser904Ser (TCC-TCG) were also identified. The RET Val804Met mutation and the Ser904Ser polymorphism could only be identified by direct sequencing. Of the seven MEN2 families here studied, 35 of 64 individuals (54.7%) were found to be RET mutant carriers (disease-causing mutations): 17 of them (48.6%) were MEN2A cases; 2 cases (5.7%) presented MEN2A disease associated with congenital megacolon; 15 of them (42.9%) were FMTC and 1 patient (2.8%) had MEN2B.

CSGE

Regarding CSGE screening mutation, the PCR products of the index cases (7 subjects) and at-risk relatives (57 family members) found to be mutated or polymorphic were screened by CSGE and compared to wild-type exons from 27 at-risk family members identified as healthy individuals. For CSGE-screened mutations, we analyzed 128 amplicons (encompassing RET hot-spot mutations and codon 648 missense substitution). 116 of 128 amplicons (90.6%) were found to be concordant with DNA sequencing (gold standard). In twelve amplicons (9.4%), all representing the Val804Met mutation (exon 14), the electrophoretic migration pattern could not be standardized even with alternative primer settings (14B and 14C) and electrophoresis conditions. In relation to the polymorphism analysis, we screened 168 amplicons: 100 of them (59.5%) were concordant regarding CSGE and DNA sequencing. In 68 (40.5%) of the analyzed amplicons (all representing the Ser904Ser polymorphism), CSGE analysis could not be standardized. Briefly, patients presented the following genotype-phenotype correlations when examined by CSGE: (a) RET exon 10 Cys620Arg (TGC-CGC) in 5 MEN2A, 2 cases of congenital megacolon and 4 FMTC cases; (b) RET exon 11 Cys634Arg (TGC-CGC) in 12 MEN2A cases; (c) RET exon 11 Cys634Tyr (TGC-TAC) in 8 FMTC carriers; (d) RET exon 11 Val648Ile (GTC-ATC) in 2 so far clinically non-affected MEN2A carriers; and (e) RET exon 16 Met918Thr (ATG-ACG) in a case presenting MEN2B phenotype. Polymorphisms Gly691Ser (GGT-AGT) and Leu769Leu (CTT-CTG) were found to occur in RET mutation carriers, codon 648 missense substitution-carriers and non-carriers (13, 1 and 7 subjects, respectively).

SSCP

For RET hot-spot screening analysis using SSCP, we selected a subset of 37 subjects comprising all mutation, missense substitution and polymorphism genotypes here studied: 4 subjects presenting Cys620Arg + Gly691Ser + Ser904Ser; 3 Cys634Arg; 3 Cys634Tyr; 2 Val648Ile; 3 Val804Met (one of them carrying Leu769Leu); 1 Met918Thr; 2 Leu769Leu and 19 wild-type subjects. Considering SSCP-screened mutations, 14 of 37 subjects were RET hot-spot mutants carriers for codons 620, 634, 804, and 918; 2 of 37 subjects were codon 648 missense substitution carriers and nineteen subjects were healthy controls. As it was observed when applying CSGE technology, the same 12 amplicons (17%) representing Val804Met mutation (exon 14), could not be standardized by SSCP, either. For SSCP polymorphism screening analysis, the subset group (37 subjects) comprised four subjects presenting codon 691 + 904 polymorphisms, three 769 polymorphism carriers and twelve healthy control subjects. In 16 (35%) of the analyzed polymorphic amplicons (Ser904Ser), electrophoretic migration patterns could not be standardized. Patients presented the same genotype-phenotype correlations when screened by either SSCP or CSGE. SSCP screening method showed the same migration patterns for all exons, when examining either RET mutations or polymorphisms. Data obtained by CSGE were fully concordant (100%) with those observed when SSCP was applied.

Comparing data encompassing RET hot-spot mutations and codon 648 missense substitution obtained from SSCP/CSGE assays with those from genetic sequencing, we documented that 58 analyses (83%) were concordant. Also, comparing polymorphism data obtained by SSCP/CSGE with those obtained with genetic sequencing, 30 analyses (65%) were concordant. Using CSGE and SSCP, we were able to detect four out of the five types (80%) of RET mutations verified by direct sequencing analysis in our MEN2 patients.

DISCUSSION AND CONCLUSION

Molecular diagnosis offers a specific and highly accurate indication for prophylactic total thyroidectomy in human RET mutation carriers, which may alter MEN2 natural course of disease (15,24). In our MEN2 cases, likely due to the smallness of our sample, Val804Met RET mutation was relatively over-represented, with 3 individuals out of 35 (8.6%), compared to its less than 3% prevalence in large MEN2 series (1,15).

In the present study, we reported the CSGE use as a RET mutation screening method in typical MEN2 families. It presented high specificity for hot-spot mutations and for polymorphism analyses, as no false-positive result was detected. As for sensitivity (false negatives), Val804Met mutation could not be detected by this method. Importantly, when using the CSGE method we were able to identify RET mutations (or polymorphisms) in exons 10, 11, 13, and 16, which have been reported to cause the majority of MEN2 cases (15). Val804Met (exon 14) is a rare genetic variation, representing less than 3% of RET disease-causing mutations reported in MEN2 cases so far (1,15,25). We have occasionally successfully screened Val804Met using DGGE (3,4,8).

Direct genetic sequencing is the "gold standard" in genetic mutation analysis. However, for extended genealogies or populations, the cost of this procedure may be significantly higher than other screening methods. For instance, CSGE has been successfully used in several genetic screenings and may result in six-fold lower costs than sequencing, considering only reagents. Furthermore, it has been shown that screening methods such as DHPLC (that uses a Wave system analyzer), DGGE, SSCP, and restriction enzymes are also adequate alternatives for direct DNA sequencing (4,8,16). Each of these methods has advantages and disadvantages, as follows: DHPLC uses an automatic system for reading heteroduplex bands and is a sensitive method (90–95%), although its specific wave analyzer costs must be considered (23). Restriction enzyme sensitivity and reproduction analysis may present some drawbacks, such as: (a) inadequate recognition of restriction site and a consequently non-defined band in agarose gel electrophoresis, and (b) incomplete discrimination of amplified sequences with similar base pair sizes (4,14,16,26). Despite these limitations, a previous study reported total correlation when comparing restriction enzymes to direct sequencing (14). SSCP sensitivity usually reaches 80% for PCR fragment sizes smaller than 300 bp, meaning that non-mutant electrophoresis migration patterns do not exclude the possibility of a mutation carrier (4,14,16,26). According to other studies, SSCP presents an overall sensitivity of 95–100% for RET hot-spots mutation analysis (27,28). Also, DGGE is a highly sensitive method (90–100%) and is a very useful screening approach for RET mutations, which has been used in our laboratory (3,4,8). DGGE has several advantages, such as: (a) sensitivity and reproducibility higher than SSCP; (b) a high correlation with direct sequencing; (c) non-radioactive protocols; (d) allows little operational manipulation; and (e) does not need to recognize restriction sites. On the other hand, DGGE has some disadvantages such as: (a) requires a 50 bp GC-clamp coupled to one of the primer sequences; (b) each analyzed exon melting temperature needs to be addressed by a special computer software or hard standardization; (c) initial investment at specific electrophoresis system; (d) difficult standardization for guanine-cytosine rich sequences; and (e) polymorphism analysis may yield false-positive results (3,4,8).

In the present study, we reported CSGE application as a RET screening method in 7 typical MEN2 families with a sensitivity of 90.6% for hot-spot mutations (encompassing codon 648 missense substitution) and 59.5% for polymorphism analyses. According to the MEN Consensus, the Val804Met mutation, which was not detected in our CSGE assay, is a rare genetic variation, representing less than 3% of RET disease-causing mutations (1,15,25).

Additionally, we were interested in testing CSGE for detecting RET polymorphisms as they may act as epigenetic factors interacting with RET mutations and possibly altering the clinical presentation, course, and morbidity of MEN2 disease (29-33).

In summary, our study has shown that CSGE (and SSCP) is a useful method for screening RET hot-spot mutations, considering the following points: (a) using CSGE, we have successfully screened 4 of 5 types of RET mutations among our cases; (b) these four mutations detected by CSGE have been reported as being responsible for most (95%) of the disease-causing mutations in different MEN2 samples; (c) there was a total correlation (100%) between CSGE and SSCP data; (d) CSGE is a low-cost, fast and feasible to standardize method; (e) it does not use a radioactive protocol; and (f) it allows little operational manipulation, which decreases the possible chance of error. Moreover, CSGE dispenses with some requirements, such as: (a) the recognition of restriction sites; (b) different gel concentrations for each studied exon and 50 bp GC-clamp coupled to one of the primers sequences; and finally, (c) this method does not require the use of specific software to analyze each exon melting temperature.

In conclusion, CSGE is an effective, rapid, and low-cost screening method for the most frequently occurring RET mutations in MEN2. It is also worthwhile to note that our results reflect a retrospective study of a small number of MEN2 families (seven families) that need to be addressed to a prospective study from a greater number of families, where genetic sequencing and CSGE screening may be simultaneously applied.

ACKNOWLEDGMENTS

We thank the Papaiz group and CNPq–CAPES for supporting this research, aiming at providing a faster, safe, and accurate diagnosis of multiple endocrine neoplasia type 2. We also thank: Drs. Adriana B. Nunes, Marilza C.L. Ezabella, Neusa Abelin, Cesar Y. Hayashida, Ivone I. Mackwiack and all the staff members of the Endocrinology Genetics Unit, Endocrinology, University of Sao Paulo, School of Medicine (http://medicina.fm.usp.br/ueg). We also thank Prof. Dr. Maria Rita S.P. Bueno, Head of the Dept. of Genetics and Evolutive Biology, Institute of Bioscience IB–USP, for laboratory support.

Recebido em 22/01/07

Aceito em 20/04/07

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• 31. Baumgarter-Parzer SM, Lang R, Wagner L, Heinze G, Niederle B, Kaserer K, et al. Polymorphisms in exon 13 and intron 14 of the RET proto-oncogene: Genetic modifiers of medullary thyroid carcinoma? J Clin Endocrinol Metab 2005;90:6232-6.
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• 33. Wohllk N, Soto E, Bravo M, Becker P. Polimorfismos G691S, L769L e S836S del proto-oncogene RET no se associan a mayor riesgo de cancer medular tiroideo esporádico en pacientes chilenos. Rev Med Chile 2005;133:397-402.

Publication Dates

• Publication in this collection
  18 Jan 2008
• Date of issue
  Dec 2007

History

• Received
  22 Jan 2007
• Accepted
  20 Apr 2007