Mutational analysis of RET proto‐oncogene among patients with medullary thyroid carcinoma and ‘at risk’ carriers from India

Mutations associated with the RET proto-oncogene have been extensively investigated in patients with medullary thyroid carcinoma (MTC) since its first description in 1993. Several germline activating point mutations on exons 10, 11, 13, 14, 15 and 16 of this gene, are known to be associated with multiple endocrine neoplasia type 2 (MEN 2) and familial MTC (FMTC). These mutations are inherited in an autosomal dominant manner necessitating screening of index cases and their families to identify carriers who could benefit from early prophylactic thyroidectomy. In addition to these mutations, several single nucleotide polymorphisms (SNPs) have also been described though it is debatable whether these polymorphisms have either a predisposing or a modifying role in the pathogenesis of MTC. MTC is a malignancy with a good prognosis where patients have reasonable longevity even in the presence of metastases. Our previous experience over a 20-year period has shown that survival rates are favorable even in cases with metastases (43%) at the time of diagnosis. Understanding the mutational profile of MTC in the Indian population is therefore crucial to establishing a diagnosis and performing early prophylactic thyroidectomy before development of disease. However, a detailed report of the mutational profile among Indian patients is lacking. We report RET mutational analysis of the six ‘risk exons’ along with characterization of the four known exonic RET polymorphisms among patients with MTC and among ‘at risk’ carriers. Thirty-six patients with MTC who attended the outpatient clinic between January 2008 to December 2009 were included in the study. The diagnosis of MTC was based on the histopathology of the tumor after thyroidectomy. Peripheral venous blood was collected from all these patients and 11 ‘at risk’ carriers after obtaining consent. Genomic DNA extracted from 200 ll of blood was amplified with primers for exons 10, 11, 13, 14, 15 and 16. PCR products were sequenced using the ABI PRISM 310 genetic analyzer (Applied Biosystems, Foster City, CA, USA). The association between mutational status and various demographic, clinical factors were evaluated by Chi-square test or Fisher’s exact test using stata 10.0 (StataCorp, College Station, TX, USA). The 36 index cases (24 males and 13 females), included cases of MEN IIA (n = 3), MEN IIB (n = 2), FMTC (n = 2) and apparently sporadic MTC (sMTC, n = 29). The mean age of patients included was 47 years (range 19–74 years) and median serum calcitonin was 4650 pg/ml (range 133–97, 8000 pg/ml). Metastases were observed in 72% of patients (n = 26). Mutational analysis showed that 28% (10/36) of patients in this study carried a germline mutation which included all cases of MEN II and FMTCs and three patients (10%; 3/36) who were apparently sporadic cases (Table 1). Mutations at codon 634/exon 11 was most common (n = 5), while two patients had a mutation at codon 618 and one each at codon 804, 891 and 918. Similar sequence analysis was performed for all the 11 ‘at risk’ carriers and six harbored the same mutations as in the index case. Four of them underwent prophylactic thyroidectomy. While the percentage of mutations among the familial and sporadic cases of MTC in this study are comparable to the previous reported studies from other geographic regions of the world, it differs from the only other Indian study which reported a larger number (18/24) of sMTC. Irrespective of these differences, the presence of mutations in sporadic cases and among ‘at risk’ carriers, demonstrates the benefit of routine screening of all patients with MTC and their first degree relatives even in a resource limited setting. Sequences were also analyzed for the four known exonic polymorphisms (G691S/exon 11, L769L/exon 13, S836S/ exon 14 and S904S/exon 15). SNPs at 691 and 769 were more common (47% each) than polymorphisms at 904 (35%) and 836 (20%). Interestingly, nine sporadic cases with no mutations also had the G691S polymorphism, which is the only SNP associated with an amino acid change. Further, all cases that had G691S polymorphism also had S904S polymorphism. Although G691S is not considered an oncogenic mutation, a functional role for this SNP has not been entirely ruled out. In fact Elsei et al. reported a significantly higher allelic frequency of G691S among sMTC when compared to normal controls suggesting a putative role for this SNP as a genetic modifier. However, our study has the drawback of characterizing these polymorphisms only among patients’ with MTCs without