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

I. Torrente, F. Arturi, L. D'Aloiso, A. Colosimo, A. De Luca, E. Ferretti, D. Russo, E. Chiefari, D. Scarpelli, M. Bisceglia, B. Dallapiccola, Sebastiano Filetti

Research output: Contribution to journalArticle

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.

Original languageEnglish
Pages (from-to)111-116
Number of pages6
JournalJournal of Endocrinological Investigation
Volume27
Issue number2
Publication statusPublished - Feb 2004

Fingerprint

Germ-Line Mutation
Mutation
Codon
Exons
Proto-Oncogene Proteins c-ret
Genetic Databases
Aptitude
Genetic Testing
Reverse-Phase Chromatography
Point Mutation
Genes
Chromatography
Alleles
Medullary Thyroid cancer
Ions
Costs and Cost Analysis

Keywords

  • DHPLC
  • Medullary thyroid carcinoma
  • Multiple endocrine neoplasia type 2 (MEN 2)
  • Mutation analysis
  • RET gene

ASJC Scopus subject areas

  • Endocrinology

Cite this

Evaluation of a DHPLC-based assay for rapid detection of RET germline mutations in Italian patients with medullary thyroid carcinoma. / Torrente, I.; Arturi, F.; D'Aloiso, L.; Colosimo, A.; De Luca, A.; Ferretti, E.; Russo, D.; Chiefari, E.; Scarpelli, D.; Bisceglia, M.; Dallapiccola, B.; Filetti, Sebastiano.

In: Journal of Endocrinological Investigation, Vol. 27, No. 2, 02.2004, p. 111-116.

Research output: Contribution to journalArticle

Torrente, I, Arturi, F, D'Aloiso, L, Colosimo, A, De Luca, A, Ferretti, E, Russo, D, Chiefari, E, Scarpelli, D, Bisceglia, M, Dallapiccola, B & Filetti, S 2004, 'Evaluation of a DHPLC-based assay for rapid detection of RET germline mutations in Italian patients with medullary thyroid carcinoma', Journal of Endocrinological Investigation, vol. 27, no. 2, pp. 111-116.
Torrente, I. ; Arturi, F. ; D'Aloiso, L. ; Colosimo, A. ; De Luca, A. ; Ferretti, E. ; Russo, D. ; Chiefari, E. ; Scarpelli, D. ; Bisceglia, M. ; Dallapiccola, B. ; Filetti, Sebastiano. / Evaluation of a DHPLC-based assay for rapid detection of RET germline mutations in Italian patients with medullary thyroid carcinoma. In: Journal of Endocrinological Investigation. 2004 ; Vol. 27, No. 2. pp. 111-116.
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AU - Colosimo, A.

AU - De Luca, A.

AU - Ferretti, E.

AU - Russo, D.

AU - Chiefari, E.

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AU - Bisceglia, M.

AU - Dallapiccola, B.

AU - Filetti, Sebastiano

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