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Evaluation of a DHPLC-based assay for rapid detection of RET germline mutations in Italian patients with medullary thyroid carcinoma

Journal of Endocrinological Investigation volume 27, pages 111–116 (2004)Cite this article

Abstract

Causative gain-of-function mutations of the RET tyrosine-kinase receptor gene have been reported in more than 95% of inherited cases of medullary thyroid carcinoma (MTC; OMIM# 155240). Most RET activating mutations are clustered in mutational “hot spots” in exons 10, 11, 13, 14, 15 and 16 and are usually detected by single- strand conformation polymorphism (SSCP) followed by direct sequencing. To improve sensitivity, time and costs of mutational screening we have developed a denaturing high performance chromatography (DHPLC) protocol, based on the detection of heteroduplex molecules by ion-pair reverse-phase liquid chromatography under partially denaturing conditions. The mutational screening of RET exons 10, 11, 13–16 was performed in a total of 111 subjects, including 45 MTC patients and 49 relatives with known RET mutations and 17 individuals, being at risk of hereditary MTC and carrying unknown RET alleles. Heteroduplex peaks with a distinct and reproducible DHPLC elution profile allowed the detection of both rare and common RET mutations. Overall, the DHPLC-based methodology showed a high level of sensitivity and accuracy, nearing 100%. Furthermore, our protocol showed the ability to identify: 1) all the mutated codons of RET located in the “hot spots” domain; 2) the different point mutations occurring in the same codon of RET gene; 3) less frequent or rare mutations; 4) polymorphisms. As such, it can be proposed as a relatively simple and highly accurate method for a rapid genetic testing for members of MTC families.

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References

  1. Brandi ML, Gagel RF, Angeli A, et al. Guidelines for diagnosis and therapy of MEN type 1 and type 2. J Clin Endocrinol Metab 2001, 86: 5658–71.

    PubMed  Article  CAS  Google Scholar 

  2. Farndon JR, Leight GS, Dilley WG, et al. Familial medullary thyroid carcinoma without associated endocrinopathies: a distinct clinical entity. Br J Surg 1986, 73: 278–81.

    PubMed  Article  CAS  Google Scholar 

  3. Mulligan LM, Kwok JBJ, Healey CS, et al. Germline mutations of the RET proto-oncogene in multiple endocrine neoplasia type 2A. Nature 1993, 363: 458–60.

    PubMed  Article  CAS  Google Scholar 

  4. Donis-Keller H, Dou S, Chi D, et al. Mutations in the RET proto-oncogene are associated with MEN IIA and FMTC. Hum Mol Genet 1993, 2: 851–6.

    PubMed  Article  CAS  Google Scholar 

  5. Eng C, Smith DP, Mulligan LM, et al. Point mutation within the tyrosine kinase domain of the RET proto-oncogene in multiple endocrine neoplasia type 2B and related sporadic tumours. Hum Mol Genet 1994, 3: 237–41.

    PubMed  Article  CAS  Google Scholar 

  6. Takahashi M, Buma Y, Iwamoto T, Inaguma Y, Ikeda H, Hiai H. Cloning and expression of the RET Proto-oncogene encoding a tyrosine kinase with two potential transmembrane domains. Oncogene 1988, 3: 571–8.

    PubMed  CAS  Google Scholar 

  7. Takahashi M. The GDNF/RET signaling pathway and human diseases. Cytokine Growth Factor Rev 2001, 12: 361–73.

    PubMed  Article  CAS  Google Scholar 

  8. Manie S, Santoro M, Fusco A, Billaud M. The RET receptor: function in development and dysfunction in congenital malformation. Trends Genet 2001, 17: 580–9.

    PubMed  Article  CAS  Google Scholar 

  9. Eng C, Clayton D, Schuffenecker I, et al. The relationship between specific ret proto-oncogene mutation and disease phenotype in multiple endocrine neoplasia type 2: International Ret Mutation Consortium Analysis. JAMA 1996, 276: 1575–9.

    PubMed  Article  CAS  Google Scholar 

  10. Mulligan LM, Ponder BA. Genetic basis of endocrine disease: multiple endocrine neoplasia type 2. J Clin Endocrinol Metab 1995, 80: 1989–95.

    PubMed  CAS  Google Scholar 

  11. Lombardo F, Baudin E, Chiefari E, et al. Familial medullary thyroid carcinoma: clinical variability and low aggressiveness associated with RET mutation at codon 804. J Clin Endocrinol Metab 2002, 87: 1674–80.

    PubMed  Article  CAS  Google Scholar 

  12. Lecube A, Hernandez C, Oriola J, et al. V804M RET mutation and familial medullary thyroid carcinoma: report of a large family with expression of the disease only in the homozygous gene carriers. Surgery 2002, 131: 509–14.

    PubMed  Article  Google Scholar 

  13. Underhill PA, Jin L, Lin AA, et al. Detection of numerous Y chromosome biallelic polymorphisms by denaturing high-performance liquid chromatography. Genome Res 1997, 7: 996–1005.

    PubMed Central  PubMed  CAS  Google Scholar 

  14. Xiao L, Li L, Visvesvara GS, Moura H, Didier ES, Lal AA. Genotyping Encephalitozoon cuniculi by multilocus analyses of genes with repetitive sequences. J Clin Microbiol 2001, 6: 2248–53.

    Article  Google Scholar 

  15. Colosimo A, Guida V, De Luca A, et al. Reliability of DHPLC in mutational screening of beta-globin (HBB) alleles. Hum Mutat 2002, 19: 287–95.

    PubMed  Article  CAS  Google Scholar 

  16. Flex E, De Luca A, D’Apice MR, Buccino A, Dallapiccola B, Novelli G. Rapid scanning of myotubularin (MTM1) gene by denaturing high-performance liquid chromatography (DHPLC). Neuromuscul Disord 2002, 12: 501–5.

    PubMed  Article  Google Scholar 

  17. De Luca A, Buccino A, Gianni D, et al. NF1 gene analysis based on DHPLC. Hum Mutat 2003, 21: 171–2.

    PubMed  Article  CAS  Google Scholar 

  18. Chiefari E, Russo D, Giuffrida D, et al. Analysis of RET proto- oncogene abnormalities in patients with MEN 2A, MEN 2B, familial or sporadic medullary thyroid carcinoma. J Endocrinol Invest 1998, 21: 358–64.

    PubMed  CAS  Article  Google Scholar 

  19. Ceccherini I, Hofstra RM, Luo Y, et al. DNA polymorphisms and conditions for SSCP analysis of the 20 exons of the RET proto-oncogene [published erratum appears in Oncogene 1995, 10: 1257]. Oncogene 1994, 10: 3025–9.

    Google Scholar 

  20. Nilsson O, Tisell LE, Jansson S, Ahlman H, Gimm O, Eng C. Adrenal and extra-adrenal pheochromocytomas in a family with germline RET V804L mutation. JAMA 1999, 281: 1587–8.

    PubMed  Article  CAS  Google Scholar 

  21. Nakao A, Naomoto Y, Kataoka M, et al. A family of multiple endocrine neoplasia type 2A with the RET proto-oncogene mutation in codon 618 (Cys?Arg). Jpn J Clin Oncol 2001, 31: 157–61.

    PubMed  Article  CAS  Google Scholar 

  22. Berndt I, Reuter M, Saller B, et al. A new hot spot for mutations in the ret protooncogene causing familial medullary thyroid carcinoma and multiple endocrine neoplasia type 2A. J Clin Endocrinol Metab 1998, 83: 770–4.

    PubMed  CAS  Google Scholar 

  23. Gimm O, Neuberg DS, Marsh DJ, et al. Over-representation of a germline RET sequence variant in patients with sporadic medullary thyroid carcinoma and somatic RET codon 918 mutation. Oncogene 1999, 18: 1369–73.

    PubMed  Article  CAS  Google Scholar 

  24. Ruiz A, Antinolo G, Fernandez RM, Eng C, Marcos I, Borrego S. Germline sequence variant S836S in the RET proto-oncogene is associated with low level predisposition to sporadic medullary thyroid carcinoma in the Spanish population. Clin Endocrinol (Oxf) 2001, 55: 399–402.

    Article  CAS  Google Scholar 

  25. Bounacer A, Du Villard JA, Wicker R, et al. Association of RET codon 691 polymorfism in radiation-induced human thyroid tumors with C- cell hyperplasia in peritumoral tissue. Br J Cancer 2002, 86: 1929–36.

    PubMed Central  PubMed  Article  CAS  Google Scholar 

  26. Robledo M, Gil L, Pollan M, et al. Polymorfisms G691S/S904S of RET as genetic modifiers of MEN 2A. Cancer Res 2003, 63: 1814–7.

    PubMed  CAS  Google Scholar 

  27. Liu W, Smith DI, Rechtzigel KJ, Thibodeau SN, James CD. Denaturing high performance liquid chromatography (DHPLC) used in the detection of germline and somatic mutations. Nucleic Ac Res 1998, 26: 1396–400.

    Article  CAS  Google Scholar 

  28. Marsh DJ, Theodosopoulos G, Howell V, et al. Rapid mutation scanning of genes associated with familial cancer syndromes using denaturing high-performance liquid chromatography. Neoplasia 2001, 3: 236–44.

    PubMed Central  PubMed  Article  CAS  Google Scholar 

  29. Peacock ML, Borst MJ, Sweet JD, Decker RA. Detection of RET mutations in multiple endocrine neoplasia type 2a and familial medullary thyroid carcinoma by Denaturing gradient gel electrophoresis. Hum Mutat 1996, 7: 100–4.

    PubMed  Article  CAS  Google Scholar 

  30. Kim IJ, Kang HC, Park JH, et al. RET oligonucleotide microarray for the detection of RET mutations in multiple endocrine neoplasia type 2 syndromes. Clin Cancer Res 2002, 8: 457–63.

    PubMed  CAS  Google Scholar 

  31. Sanchez B, Robledo M, Biarnes J, et al. High prevalence of the C634Y mutation in the RET proto-oncogene in MEN 2A families in Spain. J Med Genet 1999, 36: 68–70.

    PubMed Central  PubMed  CAS  Google Scholar 

  32. Liu WO, Oefner PJ, Qian C, Odom RS, Francke U. Denaturing HPLC-identified novel FBN1 mutations, polymorphisms, and sequence variants in Marfan syndrome and related connective tissue disorders. Genet Test 1997–98, 1: 237–42.

    PubMed  Article  CAS  Google Scholar 

  33. Bunn CF, Lintott CJ, Scott RS, George PM. Comparison of SSCP and DHPLC for the detection of LDLR mutations in a New Zealand cohort. Hum Mutat 2002, 19: 311.

    PubMed  Article  Google Scholar 

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Affiliations

  1. IRCCS-CSS, San Giovanni Rotondo and CSS-Mendel Institute, Rome, Italy

    I. Torrente, L. D’Aloiso, A. Colosimo, A. De Luca, M. Bisceglia & B. Dallapiccola

  2. Department of Clinical and Experimental Medicine ”G. Salvatore” and Department of Pharmacobiologic Sciences, University of Catanzaro, Catanzaro, Italy

    F. Arturi, D. Russo, E. Chiefari & D. Scarpelli

  3. Department of Biomedical Sciences, University “G. D’Annunzio”, Chieti, Italy

    A. Colosimo

  4. Department of Experimental Medicine and Pathology, University of Rome “La Sapienza”, Rome, Italy

    E. Ferretti & B. Dallapiccola

  5. Department of Clinical Sciences, University of Rome “La Sapienza”, Rome, Italy

    S. Filetti MD

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  1. I. Torrente
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  2. F. Arturi
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  3. L. D’Aloiso
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  4. A. Colosimo
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  5. A. De Luca
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  6. E. Ferretti
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  7. D. Russo
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  8. E. Chiefari
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  10. M. Bisceglia
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  11. B. Dallapiccola
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  12. S. Filetti MD
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Correspondence to S. Filetti MD.

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Torrente, I., Arturi, F., D’Aloiso, L. et al. Evaluation of a DHPLC-based assay for rapid detection of RET germline mutations in Italian patients with medullary thyroid carcinoma. J Endocrinol Invest 27, 111–116 (2004). https://doi.org/10.1007/BF03346254

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  • DOI: https://doi.org/10.1007/BF03346254

Key-words

  • Medullary thyroid carcinoma
  • multiple endocrine neoplasia type 2 (MEN 2)
  • DHPLC
  • mutation analysis
  • RET gene