Effect of 3'UTR RET Variants on RET mRNA Secondary Structure and Disease Presentation in Medullary Thyroid Carcinoma

Abstract

Background: The RET S836S variant has been associated with early onset and increased risk for metastatic disease in medullary thyroid carcinoma (MTC). However, the mechanism by which this variant modulates MTC pathogenesis is still open to discuss. Of interest, strong linkage disequilibrium (LD) between RET S836S and 3'UTR variants has been reported in Hirschsprung's disease patients.

Objective: To evaluate the frequency of the RET 3'UTR variants (rs76759170 and rs3026785) in MTC patients and to determine whether these variants are in LD with S836S polymorphism.

Methods: Our sample comprised 152 patients with sporadic MTC. The RET S836S and 3'UTR (rs76759170 and rs3026785) variants were genotyped using Custom TaqMan Genotyping Assays. Haplotypes were inferred using the phase 2.1 program. RET mRNA structure was assessed by Vienna Package.

Results: The mean age of MTC diagnosis was 48.5±15.5 years and 57.9% were women. The minor allele frequencies of RET polymorphisms were as follows: S836S, 5.6%; rs76759170, 5.6%; rs3026785, 6.2%. We observed a strong LD among S836S and 3'UTR variants (|D'| = -1, r2 = 1 and |D'| = -1, r2 = 0,967). Patients harboring the S836S/3'UTR variants presented a higher percentage of lymph node and distant metastasis (P = 0.013 and P<0.001, respectively). Accordingly, RNA folding analyses demonstrated different RNA secondary structure predictions for WT(TCCGT), S836S(TTCGT) or 3'UTR(GTCAC) haplotypes. The S836S/3'UTR haplotype presented a greater number of double helices sections and lower levels of minimal free energy when compared to the wild-type haplotype, suggesting that these variants provides the most thermodynamically stable mRNA structure, which may have functional consequences on the rate of mRNA degradation.

Conclusion: The RET S836S polymorphism is in LD with 3'UTR variants. In silico analysis indicate that the 3'UTR variants may affect the secondary structure of RET mRNA, suggesting that these variants might play a role in posttranscriptional control of the RET transcripts.

Fig 1. Kaplan–Meier estimates the proportion of sporadic MTC patients with lymph node (A; n = 131; P = 0.011) or distant metastasis (B; n = 116; P<0.001).

The log rank test was used to compare curves.

Fig 2. The figure shows the optimal mRNA secondary structure of the RET haplotypes.

A) Total mRNA secondary structure of the RET wild-type (WT) haplotype. B) Part of mRNA secondary structure of the RET WT haplotype. C) Part of mRNA secondary structure of the RET S836S haplotype (TTCGT). D) Part of mRNA secondary structure of the RET 3’UTR haplotype (GTCAC). Haplotypes generated by RNAfold program (Vienna Package).

Fig 3. The minimal free energy (MFE, kcal/mol) and number of double helices (N_DH) were available in both, optimal and suboptimal structures.

For suboptimal structures MFE and NDH are averages over 2900 samples. The variant fragment carrying the S836S and 3’UTR variants (GTCAC haplotype) presented greater N_DH (B,D) and lower levels of MFE (A,C) when compared to wild-type haplotype (WT, TCCGT), this fact happens in both, optimal and suboptimal structures. *These analysis included only synonymous polymorphisms.

Fig 4. Immunostaining of the RET proto-oncogene in GTCAC haplotype carriers & non-carriers.

A) Two representative slices of RET Immunostaining in a sample carrier S836S/3’UTR (GTCAC haplotype) (left) and non-carrier of this haplotype (right). B) Intensity of RET Immunostaining in samples with or without S836S/3’UTR (GTCAC haplotype), P = 0.054.