Detection of RET proto-oncogene point mutations in paraffin-embedded pheochromocytoma specimens by nonradioactive single-strand conformation polymorphism analysis and direct sequencing

Komminoth, P.; Kunz, E.; Hiort, O.; Schröder, S.; Matias-Guiu, X.; Christiansen, G.; Roth, J.; Heitz, P.U.

American Journal of Pathology 145(4): 922-929


ISSN/ISBN: 0002-9440
PMID: 7943181
Accession: 008437858

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The suitability of formalin-fixed and paraffin-embedded tumor material was evaluated for molecular analysis of the RET proto-oncogene. We analyzed exons 10, 11, and 16 for point mutations in seven sporadic and six multiple endocrine neoplasia (MEN) 2A-associated pheochromocytomas by a nonradioactive single-strand conformation polymorphism assay followed by nonradioactive direct sequencing of PCR-amplified DNA using an automated DNA sequencer. AM MEN 2A-associated pheochromocytomas contained a heterozygous missense germline mutation within cysteine codons of the cysteine-rich extracellular domain encoded by exons 10 and 11. Mutations were located in codon 619 (TCG fwdarw TCC; Cys fwdarw Ser) in one, in codon 635 (TGC fwdarw CGC; Cys fwdarw Arg) in three, and in codon 635 (TGC fwdarw TAC; Cys fwdarw Tyr) in two pheochromocytomas. No tumor-specific (somatic) mutations were detected in exons 10, 11, and 16 of the sporadic pheocbromocytomas. These data support recent findings that germline point mutations that are clustered in distinct cysteine codons of the RET proto-oncogene are involved in the neoplastic Phenotype of the MEN 2A syndrome. Our results demonstrate that both nonradioactive single-strand conformation polymorphism and direct sequencing are suitable metbods to detect single base substitutions in DNA extracted from archival material.