Laboratório As Bases Genéticas dos Tumores da Tiroide, Universidade Federal de São Paulo, São Paulo, SP, Brazil.
Centro de Oncologia Molecular, Hospital Sírio-libanês, São Paulo, SP, Brazil.
BMC Med Genomics. 2019 Jul 9;12(1):104. doi: 10.1186/s12920-019-0552-1.
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.
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.
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.
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.
在遗传性综合征 MEN 2 中，不同的 RET 致癌基因突变被确定为病因，该综合征的特征是甲状腺髓样癌（MTC）和其他内分泌肿瘤。尽管已经认识到遗传易感性，但并非所有 RET 突变携带者在其一生中都会患上这种疾病，同样，属于同一家庭的 RET 突变携带者也可能表现出临床异质性。有人提出，单个种系突变可能不足以导致 MEN 2 相关肿瘤的发生，可能需要体细胞双等位基因改变。在这里，我们研究了 MTC 中 RET 基因的体细胞二次打击突变的存在。
我们整合了多重连接依赖性探针扩增（MLPA）和全外显子组测序（WES），以搜索 MTC 样本和甲状腺髓样癌细胞系（TT 和 MZ-CR-1）中 RET 基因的拷贝数改变（CNA）。接下来，我们在 RET 上找到了跨越外显子-外显子边界的reads，这表明存在 retrocopy。随后，我们通过寻找报告在 RET 基因座或不同基因组区域中连接外显子的 reads，在人类参考基因组（GRCh37）和 1000 基因组计划数据中寻找 RET retrocopies。为了确定 RET retrocopy 的特异性和重现性，我们使用 PCR-测序方法测试了来自散发性和 MEN 2 相关 MTC（n=37）、外周血（n=3）和具有 RET 融合的甲状腺乳头状癌（n=10）样本的 DNA。
通过 MLPA，我们在 MTC 样本和 MTC 细胞系中发现了 RET 基因的 CNA 证据。WES 分析进一步证实了 CNA 的存在，并暗示了在人类参考基因组和 1000 基因组计划中未发现的 RET 反转录副本。扩展分析证实了在散发性和遗传性肿瘤中均存在与 MTC 相关的 RET 体细胞 retrocopy。我们进一步揭示了一种新的（28%）突变（p.G548V），仅在 RET 的 retrocopy 中发现。该突变也存在于突变样本的 cDNA 中，提示其可能具有功能。
我们在此报告了 MTC 样本和细胞系中存在体细胞特异性的 RET 反转录副本。我们的结果支持这样一种观点，即在体细胞中产生 retrocopies 可能有助于 MTC 的发生和进展。
