Analysis of peripheral blood or tumor DNA samples from 101 patients with apparent sporadic medullary thyroid carcinoma (MTC) was performed to assess the frequency of RET proto-oncogene mutations in this patient population. Peripheral blood and/or tumor DNA was amplified by polymerase chain reaction. DNA sequence or restriction enzyme analysis was performed to detect mutations of RET proto-oncogene codons 609, 611, 618, 620, 634, 768, and 918. Six of 101 patients with apparent sporadic MTC had peripheral blood DNA mutations more commonly associated with hereditary MTC. In 4 patients, these mutations led to the identification of previously unrecognized kindreds. The remaining 2 patients were examples of de novo mutations. A codon 918 mutation was found in 14 of 57 (approximately 25%) tumor DNA samples. Mutations were not identified in the remaining patients. In this large cancer center population, approximately 6% of patients with sporadic MTC carry peripheral blood DNA mutations, either inherited or de novo, more commonly associated with MEN 2A or familial MTC. Seven additional gene carriers were identified as a direct result of these studies, a 2-fold multiplying effect. We conclude routine application of RET proto-oncogene testing should be included in all cases of apparent sporadic MTC.
Objective: Recent studies have assigned clinical signi®cance and prognostic value to the detection of thyroglobulin (Tg) mRNA in the blood of patients subjected to total thyroidectomy for a papillary or follicular thyroid carcinoma. In this study, we investigated the diagnostic speci®city of Tg mRNA detection, analysing blood samples from healthy volunteers and from patients previously subjected to total thyroidectomy for reasons other than a carcinoma of the follicular epithelium. Design and Methods: Total RNA was extracted from whole blood, reverse-transcribed and the cDNA ampli®ed for Tg and glyceraldehyde-3-phosphate dehydrogenase with speci®c primers. Expression levels were analysed by using a semi-quantitative PCR. In a few cases, Lymphoprep gradients were used to separate the mononuclear and polymorphonuclear cells prior to further analysis by reverse transcription/PCR. Results: Our data suggested that all individuals expressed Tg mRNA. Moreover, no differences in the expression levels between subjects with and without thyroid glands were documented. Documentation of Tg expression by the mononuclear and polymorphonuclear layers in patients without thyroid glands support the hypothesis that both lymphocytes and granulocytes express Tg and may justify a background expression in blood, independently of the presence of follicular cells in circulation. Conclusions: Tg mRNA expression is not limited to follicular cells of the thyroid gland, and its expression by normal blood cells should be considered in tests performed for diagnostic purposes.
Objective: Restriction analysis is a straightforward procedure for mutational analysis. It is commonly used for screening RET mutations. Incomplete digestion is a well-known cause of false results. Herein, we report another limitation of the method. Design and Methods: Screening for somatic mutations in RET exons 16, 13 and 15 was performed in a patient with a sporadic medullary thyroid carcinoma. Genetic study was carried out by both restriction analysis and direct sequencing. Results: A somatic trinucleotide change encompassing codons 882 and 883 of the RET proto-oncogene (GTA GCT to GTT TTT) was documented. Particular to this case is the silent mutation (GTA→GTT) at codon 882. Independently, both the novel silent mutation and the missense mutation at codon 883 may disrupt the same AluI restriction site. Based on the restriction pattern we were able to say that both mutations occurred in the same allele. Conclusions: Restriction analysis is an easy approach for screening RET mutations; however, it is not enough to assign a final diagnosis.
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