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An Orthotopic Mouse Model of Anaplastic Thyroid Carcinoma
Published on: April 17, 2013
Retroposed copies of RET gene: a somatically acquired event in medullary thyroid carcinoma
Larissa V Bim1, Fábio C P Navarro2,3, Flávia O F Valente4
1Laboratório As Bases Genéticas dos Tumores da Tiroide, Universidade Federal de São Paulo, São Paulo, SP, Brazil.
Background:
Different pathogenic germline mutations in the RET oncogene are identified in MEN 2, a hereditary syndrome characterized by medullary thyroid carcinoma (MTC) and other endocrine tumors. Although genetic predisposition is recognized, not all RET mutation carriers will develop the disease during their lifetime or, likewise, RET mutation carriers belonging to the same family may present clinical heterogeneity. It has been suggested that a single germline mutation might not be sufficient for development of MEN 2-associated tumors and a somatic bi-allelic alteration might be required. Here we investigated the presence of somatic second hit mutation in the RET gene in MTC.
Methods:
We integrated Multiplex Ligation-dependent Probe Amplification (MLPA) and whole exome sequencing (WES) to search for copy number alteration (CNA) in the RET gene in MTC samples and medullary thyroid cell lines (TT and MZ-CR-1). We next found reads spanning exon-exon boundaries on RET, an indicative of retrocopy. We subsequently searched for RET retrocopies in the human reference genome (GRCh37) and in the 1000 Genomes Project data, by looking for reads reporting joined exons in the RET locus or distinct genomic regions. To determine RET retrocopy specificity and recurrence, DNA isolated from sporadic and MEN 2-associated MTC (n = 37), peripheral blood (n = 3) and papillary thyroid carcinomas with RET fusion (n = 10) samples were tested using PCR-sequencing methodology.
Results:
Through MLPA we have found evidence of CNA in the RET gene in MTC samples and MTC cell lines. WES analysis reinforced the presence of the CNA and hinted for a retroposed copy of RET not found in the human reference genome and 1.000 Genomes Project. Extended analysis confirmed the presence of a somatic MTC-related retrocopy of RET in both sporadic and hereditary tumors. We further unveiled a recurrent (28%) novel point mutation (p.G548 V) found exclusively in the retrocopy of RET. The mutation was also found in cDNA of mutated samples, suggesting it might be functional.
Conclusion:
We here report a somatic specific RET retroposed copy in MTC samples and cell lines. Our results support the idea that generation of retrocopies in somatic cells is likely to contribute to MTC genesis and progression.
Insights
A novel RET retrocopy, a second genetic hit, was discovered in medullary thyroid carcinoma (MTC) tumors. This somatic mutation in RET retrocopies may contribute to MTC development and progression.
Area of Science:
- Oncology
- Genetics
- Molecular Biology
Background:
- Germline mutations in the RET oncogene are linked to Multiple Endocrine Neoplasia type 2 (MEN 2), a hereditary syndrome often causing medullary thyroid carcinoma (MTC).
- Clinical heterogeneity and incomplete penetrance in MEN 2 suggest that germline mutations alone may not be sufficient for tumor development, implying the need for additional genetic alterations.
- The hypothesis of a somatic 'second hit' in the RET gene is proposed as a potential requirement for MTC pathogenesis in both hereditary and sporadic cases.
Purpose of the Study:
- To investigate the presence and role of somatic second hit mutations in the RET gene within medullary thyroid carcinoma (MTC) samples.
- To identify potential copy number alterations (CNAs) and other somatic mutations in the RET gene that may contribute to MTC development.
Main Methods:
- Multiplex Ligation-dependent Probe Amplification (MLPA) and whole exome sequencing (WES) were employed to detect RET gene CNAs in MTC samples and cell lines.
- Analysis of sequencing reads revealed evidence of RET retrocopies, which were then specifically searched for in patient tumor DNA using PCR-sequencing.
- Samples included sporadic MTC (n=37), hereditary MEN 2-associated MTC, peripheral blood (n=3), and papillary thyroid carcinomas with RET fusion (n=10).
Main Results:
- MLPA and WES identified CNAs in the RET gene in MTC samples and cell lines, confirming the presence of a novel, somatic RET retrocopy not present in the human reference genome.
- This RET retrocopy was found in both sporadic and hereditary MTC tumors, indicating a common mechanism in MTC genesis.
- A recurrent novel point mutation (p.G548V) was identified exclusively within the RET retrocopy in 28% of MTC samples and detected in cDNA, suggesting functional relevance.
Conclusions:
- The study reports the discovery of a somatic RET retroposed copy in medullary thyroid carcinoma (MTC) samples and cell lines.
- The findings support the hypothesis that the generation of RET retrocopies in somatic cells is a significant factor contributing to the genesis and progression of MTC.
- This somatic alteration, particularly when combined with a novel point mutation, represents a crucial step in MTC tumorigenesis.
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