High prevalence of the C634Y mutation in theRET proto-oncogene in MEN 2A families inSpain

Beatriz Sánchez, Mercedes Robledo, Josefina Biarnes, María-Eugenia Sáez, Victor Volpini,Javier Benítez, Elena Navarro, Agustín Ruiz, Guillermo Antiñolo, Salud Borrego

AbstractThe RET proto-oncogene encodes a re-ceptor tyrosine kinase expressed in neuralcrest derived tissues. Germline mutationsin the RET proto-oncogene are responsi-ble for three diVerent dominantly inher-ited cancer syndromes: multipleendocrine neoplasia type 2A (MEN 2A),type 2B (MEN 2B), and familial medullarythyroid carcinoma (FMTC). MTC canalso occur sporadically. Molecular char-acterisation of the RET proto-oncogenehas been performed by PCR-SSCP analy-sis, direct DNA sequencing, and restric-tion enzyme analysis in 49 unrelated,Spanish, MEN 2 families: 30 MEN 2Afamilies, six FMTC families, and 13 fami-lies classified as “other”. Germline mis-sense mutations in one of six cysteinecodons (609, 611, 618, and 620 in exon 10,and codons 630 and 634 in exon 11), whichencode part of the extracellular cysteinerich domain of RET, have been detected inthe majority of these families: 100% ofMEN 2A families, 67% of FMTC families,and 54% of families classified as “other”.No RET mutations in exons 10, 11, 13, 14,15, or 16 were detected in the remainingfamilies. The most frequent RET muta-tion in MEN 2A Spanish families is C634Y,occurring in 73% of cases. Haplotypeanalysis does not exclude the possibility offounder eVects in Spanish MEN 2A fami-lies with the C634Y mutation.(J Med Genet 1999;36:68–70)

Keywords: medullary thyroid carcinoma; RET proto-oncogene; molecular analysis

Medullary thyroid carcinoma (MTC) is atumour of the thyroid C cells which may occursporadically or as part of the autosomal domi-nantly inherited cancer syndrome multipleendocrine neoplasia type 2 (MEN 2). Depend-ing on the tissues involved, MEN 2 is dividedinto MEN 2A, MEN 2B, and familial medul-lary thyroid carcinoma (FMTC). MEN 2A ischaracterised by hyperplasia of the parafollicu-lar C cells of the thyroid, with subsequent neo-plastic progression to MTC in 95% of cases,phaeochromocytoma in 50% of cases, and para-thyroid hyperplasia (HPT) in 15-30% of cases.The MEN 2B syndrome consists of MTC,phaeochromocytoma, and a variety of develop-mental abnormalities including mucosal neu-romas, marfanoid habitus, and ganglioneuro-

matosis of the intestinal tract. In FMTC, thepresence of MTC is the only disease pheno-type. Germline mutations in the RET proto-oncogene are responsible for the MEN 2syndromes.1–3 The RET proto-oncogene en-codes a member of the receptor tyrosine kinase(RTK) family. Unlike most members of theRTK super family, RET activation requires theformation of a multimeric receptor complexthat includes glial cell line derived neuro-trophic factor (GDNF) as ligand, and a cellsurface associated accessory protein designatedGFRá-1 (GDNFR-á, RETL1, or TrnR1).4–6

Recently, a related ligand, neurturin (NTN),and an adaptor molecule GFRá-2 (GDNFR-â,RETL2, TrnR2, NTNR-á) have beendescribed.7–11 The majority of MEN 2A andFMTC families have germline missense muta-tions in one of six highly conserved cysteinecodons (609, 611, 618, and 620 in exon 10,and codons 630 and 634 in exon 11), whichencode part of the extracellular cysteine richdomain of RET.1–3 12–14 In FMTC families,other less frequent missense RET mutations incodons 768 (exon 13) and 804 (exon 14),within the intracellular tyrosine kinase domain,have been detected.3 15–18

Recently, the results of a study carried out bythe International RET Mutation Consortiumshowed that 85% of MEN 2A families had amutation at codon 634 and the most frequentmutation at this codon was C634R (52%) fol-lowed by C634Y (26%). On the other hand, inFMTC families, the most frequent mutation atcodon 634 was C634Y and there was noC634R mutation among these families.3

In this study, we analysed the presence,nature, and position of germline RET muta-tions in 49 Spanish MEN 2 families. Of thesefamilies, 30 were diagnosed with MEN 2A, sixwith FMTC, and 13 families were classified as“other”, according to the criteria of theInternational RET Mutation Consortium.3

Germline DNA was extracted from blood leu-cocytes by standard procedures.19 DNA from arepresentative member of each family was PCRamplified for RET exons 10, 11, 13, 14, 15, and16 using primers previously described.20–23 Ini-tially, we screened for the RET mutations morefrequently identified in MEN 2 by restrictionenzyme analysis. When a RET mutation wasdetected, the result was confirmed by directsequencing of the corresponding exon in bothsense and antisense directions by the dideoxy-nucleotide terminator cycle sequencingmethod (fmolTM DNA Sequencing System,

J Med Genet 1999;36:68–7068

Unidad de GenéticaMédica y DiagnósticoPrenatal, HospitalUniversitario “Virgendel Rocío”, AvdaManuel Siurot s/n,41013 Sevilla, SpainB SánchezM-E SáezA RuizG AntiñoloS Borrego

Departamento deGenética, FundaciónJiménez Diaz, Madrid,SpainM RobledoJ Benítez

Departament deGenètica Molecular,Institut de RecercaOncológica,L’Hospitalet deLlobregat, Barcelona,SpainJ BiarnesV Volpini

Servicio deEndocrinología,Hospital Universitario“Virgen del Rocío”,Sevilla, SpainE Navarro

Correspondence to:Dr Borrego.

Received 6 March 1998Revised version accepted forpublication 5 June 1998

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Promega) using fluorescently labelled primers(synthetised 5' labelled with Cy5 Amidite)described elsewhere.20–23 Gel electrophoresisand analysis were carried out on an ALFExpressTM Automatic DNA sequencer (Phar-macia Biotech). When no RET mutation wasdetected by restriction enzyme analysis, exons10, 11, 13, 14, 15, and 16 were screened formutations by single strand conformationalpolymorphism analysis (SSCP) using fluores-cently labelled primers describedelsewhere.20–23 Electrophoresis was carried outin an automated DNA sequencer (ALFExpressTM Automatic DNA sequencer). Whenan aberrant SSCP pattern was observed, wesearched for the mutation responsible by directsequencing, as we have described previously.Haplotype analyses at the RET locus were per-formed using two extragenic flanking microsat-ellite polymorphisms, D10S141 in the centro-meric position and ZNF22 in the telomericposition, and the highly polymorphic RET-INT5 intragenic CA repeat.24–26 Electrophore-sis and analysis of these polymorphic loci werecarried out in an ALF ExpressTM AutomaticDNA sequencer.

Mutations of the RET proto-oncogene havebeen identified in the majority of SpanishMEN 2 families. The identification of thesemutations has an important predictive value,allowing earlier diagnosis of at risk subjects andthe exclusion of mutation negative subjectsfrom further biochemical tests and prophylac-tic surgery. We have identified germline mis-sense mutations in the extracellular domain ofthe RET proto-oncogene in all 30 MEN 2Afamilies (100%). The most frequent mutationsin MEN 2A families are at codon 634 in exon11, occurring in 97% of cases (29/30 families).In 3% (1/30) of families, a mutation at codon620 in exon 10 has been identified. In SpanishFMTC and “other” families, we have identified

RET mutations in 67% (4/6) and 54% (7/13)of the cases, respectively. As described in previ-ous studies, the position and nature of thesemutations were more heterogeneous and theyare distributed among the cysteine codons inexons 10 and 11. Mutations at codon 634 werefound in 50% (3/6) of our FMTC families, in17% (1/6) a mutation in exon 10 wasidentified, and no mutations in exons 10, 11,13, 14, 15, or 16 were detected in 33% (2/6). Infamilies classified as “other”, we have detecteda mutation at codon 634 in exon 11 in 31%(4/13) of families, in 23% (3/13) a mutation inexon 10 was identified, and in 46% (6/13) offamilies, no mutation in exons 10, 11, 13, 14,15, or 16 was detected. The distribution ofamino acid changes in Spanish MEN 2 familiesare showed in table 1.

We have studied the relationship between thenature and position of the RET germlinemutation and the disease phenotype. Ourresults also show the association of mutationsat codon 634 and the presence of phaeochro-mocytoma or parathyroid disease observed inthe Consortium series.3 An interesting result ofour study is that of 19 families with MTC andphaeochromocytoma, 15 have the specificmutation C634Y. The other three MEN 2Afamilies with the C634Y mutation have MTCand parathyroid disease only.

The results of the mutation analysis in Span-ish MEN 2 families are consistent with the dataof the large series studied by the InternationalRET Mutation Consortium. An exception isthe mutation C634R detected in one SpanishFMTC family, which was not found among theFMTC families included in the Consortiumstudy. This FMTC family consists of sevenaVected family members over two generationswith MTC confirmed by postsurgical histo-logical study. The aVected subjects range in agefrom 12 to 44 years. All aVected subjects andsix of their first degree relatives were screenedfor phaeochromocytoma and HPT and foundto be negative. It is possible that this FMTCfamily is one where the other features of MEN2A have yet to develop. Another interestingfinding is the high frequency of the C634Ymutation in Spanish MEN 2A families (73%),in contrast to the large excess of C634R (52%)observed in the MEN 2A families in the Con-sortium study. Our finding is similar to that ofOriola et al,27 in a smaller Spanish MEN 2Aseries, in which they found the C634Ymutation in five of seven families (71%). Ourdata, together with those reported by Oriola etal,27 show the relative prevalence of the C634Ymutation in MEN 2A families in Spain.

To investigate whether the high frequency ofthis mutation in Spain might be the result of acommon ancestor, we have performed haplo-type analysis, using polymorphic markerswithin and flanking the RET gene, in MEN 2Afamilies with the C634Y mutation.28 ThediVerent haplotypes observed associated withmutated chromosomes (table 2) seem toexclude a founder eVect. However, table 2shows that although we have detected sevenhaplotypes among 15 patients with this muta-tion, 14 subjects share one of two alleles at the

Table 1 Distribution of amino acid substitutions in Spanish MEN 2 families

Mutation Nucleotide change MEN 2A (n=30) FMTC (n=6) Other (n=13)

Codon 634C634Y TGC→TAC 22 0 2C634R TGC→CGC 3 1 1C634W TGC→TGG 3 2 1C634G TGC→GGC 1 0 0

Codon 620C620S TGC→TCC 1 0 0

Codon 618C618R TGC→CGC 0 1 0C618F TGC→TTC 0 0 2

Codon 611C611F TGC→TTT 0 0 1None 0 2 6

Table 2 Haplotypes observed in chromosomes withC634Y RET mutation in Spanish MEN 2A families

Haplotype*DiseasechromosomesZNF22 RET-INT5 D10S141

9 1 4 69 2 13 49 2 8 19 2 12 19 2 11 14 2 7 17 4 4 1

*Alleles are designated according to references 24, 25, and 26.

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RET-INT5. Without having information onhaplotype frequencies in the Spanish popula-tion, we cannot rule out two mutational eventscombined with founder eVects and recombina-tion. This hypothesis may explain the discrep-ancy between our findings in the Spanishpopulation and the RET Mutation Consor-tium data with regard to the relative prevalenceof the C634Y and C634R mutations.

We thank all the families involved in this study for theircooperation. We are very grateful to Professor Charis Eng whoprovided invaluable help and comments on this paper. Thiswork was supported by grants 95/1667, 97/0339, and 98/0898from the Fondo de Investigaciones Sanitarias (Spain).

1 Mulligan LM, Kwok JBJ, Healey CS, et al. Germline muta-tions of the RET proto-oncogene in multiple endocrineneoplasia type 2A. Nature 1993;363:458-60.

2 Donis-Keller H, Dou S, Chi D, et al. Mutations in the RETproto-oncogene are associated with MEN 2A and FMTC.Hum Mol Genet 1993;2:851-6.

3 Eng C, Clayton D, SchuVenecker I, et al. The relationshipbetween specific RET proto-oncogene mutations anddisease phenotype in multiple endocrine neoplasia type 2:International RET Mutation Consortium analysis. JAMA1996;276:1575-9.

4 Davies AM, Dixon JE, Fox GM, et al. Nomenclature ofGPI-linked receptors for the GDNF ligand family. Neuron1997;19:485.

5 Durbec P, Marcos-Gutierrez CV, Kilkenny C, et al. GDNFsignalling through the Ret receptor tyrosine kinase. Nature1996;381:789-93.

6 Vega QC, Worby CA, Lechner MS, Dixon JE, Dressler GR.Glial cell line-derived neurotrophic factor activates thereceptor tyrosine kinase RET and promotes kidneymorphogenesis. Proc Natl Acad Sci USA 1996;93:10657-61.

7 Sanicola M, Hession C, Worley D, et al. Glial cellline-derived neurotrophic factor-dependent RET activa-tion can be mediated by two diVerent cell-surface accessoryproteins. Proc Natl Acad Sci USA 1997;94:6238-43.

8 Kotzbauer PT, Lampe PA, Heuckeroth RO, et al. Neur-turin, a relative of glial-cell-line-derived neurotrophicfactor. Nature 1996;384:467-70.

9 Baloh RH, Tansey MG, Golden JP, et al. TrnR2, a novelreceptor that mediates neurturin and GDNF signalingthrough Ret. Neuron 1997;18:793-802.

10 Buj-Bello A, Adu J, Pinon LG, et al. Neurturin responsive-ness requires a GPI-linked receptor and the Ret receptortyrosine kinase. Nature 1997;387:721-4.

11 Klein RD, Sherman D, Ho WH, et al. A GPI-linked proteinthat interacts with Ret to form a candidate neurturinreceptor. Nature 1997;387:717-21.

12 Mulligan LM, Eng C, Healey CS, et al. Specific mutationsof the RET proto-oncogene are related to diseasephenotype in MEN 2A and FMTC. Nat Genet 1994;6:70-4.

13 Komminoth P, Kunz EK, Matais-Guiu X, et al. Analysis ofRET protooncogene point mutations distinguishes heredit-able from nonhereditable medullary thyroid carcinomas.Cancer 1995;76:479-89.

14 Kitamura Y, Goodfellow PJ, Shimizu K, et al. Novelgermline RET proto-oncogene mutations associated withmedullary thyroid carcinoma (MTC): mutation analysis inJapanese patients with MTC. Oncogene 1997;14:3103-6.

15 Eng C, Smith DP, Mulligan LM, et al. A novel point muta-tion in the tyrosine kinase domain of the RET proto-oncogene in sporadic medullary thyroid carcinoma and in afamily with FMTC. Oncogene 1995;10:509-13.

16 Bolino A, SchuVenecker I, Yin L, et al. RET mutations inexons 13 and 14 of FMTC patients. Oncogene 1995;10:2415-19.

17 Fink M, Weinhusel A, Niederle B, Haas OA. Distinctionbetween sporadic and hereditary medullary thyroid carci-noma (MTC) by mutation analysis of the RET proto-oncogene. “Study Group Multiple Endocrine NeoplasiaAustria (SMENA)”. Int J Cancer 1996;69:312-16.

18 Fattoruso O, Quadro L, Libroia A, et al. A GTG to ATGnovel point mutation at codon 804 in exon 14 of the RETproto-oncogene in two families aVected by familial medul-lary thyroid carcinoma. Hum Mutat 1998;suppl 1:S167-71.

19 Dracopoli NC, Haines JL, Korf BR, et al, eds. Current proto-cols in human genetics. New York: John Wiley, 1994.

20 Zedenius J, Wallin G, Hamberger B, et al. Somatic andMEN 2A de novo mutations identified in the RETproto-oncogene by screening of sporadic MTCs. Hum MolGenet 1994;3:1259-62.

21 McMahon R, Mulligan LM, Healey CS, et al. Direct, non-radioactive detection of mutations in multiple endocrineneoplasia type 2A families. Hum Mol Genet 1994;3:643-6.

22 Mulligan LM, Eng C, Attié T, et al. Diverse phenotypesassociated with exon 10 mutations of the RET proto-oncogene. Hum Mol Genet 1994;3:2163-7.

23 Marsh DJ, Learoyd DL, Andrew SD, et al. Somaticmutations in the RET proto-oncogene in sporadic medul-lary thyroid carcinoma. Clin Endocrinol 1996;44:249-57.

24 Love DR, Gardner E, Ponder BAJ. A polymorphic dinucle-otide repeat at the D10S141 locus. Hum Mol Genet 1993;2:491.

25 Love DR, Gardner E, Ponder BAJ. A polymorphic dinucle-otide repeat at the ZNF22 locus. Hum Mol Genet1993;2:491.

26 Pasini B, Hofstra RMW, Yin L, et al. The physical map ofthe RET proto-oncogene. Oncogene 1995;11:1737-43.

27 Oriola J, Hernandez C, Simo R, et al. Genetic analysis ofseven Mediterranean families with multiple endocrine neo-plasia type 2A. Clin Endocrinol 1996;44:207-12.

28 Gardner E, Mulligan LM, Eng C, et al. Haplotype analysisof MEN 2 mutations. Hum Mol Genet 1994;3:1771-4.

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