Computational protocol: Effect of 3′UTR RET Variants on RET mRNA Secondary Structure and Disease Presentation in Medullary Thyroid Carcinoma

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Protocol publication

[…] The bioinformatics analyses were realized based on haplotypes inferred by Phase program. Additionally, we also used the haplotype carrying only the S836S variant (TTCGT). No patient harbored only S836S polymorphism was observed in our study population, this variant occurs only in combination with L769L and 3’UTR variants (GTCAC haplotype).The preliminary analysis was realized in both, wild type (TCCGT_WT haplotype) and polymorphic sequences (GCCGT, TCGGT and GTCAC haplotypes), with the RNAfold algorithm provided by the Vienna Package 2.1.5. This algorithm provides the optimal RNA secondary structure and its associated minimal free energy (MFE) for each input sequence [].The Vienna Package [] program RNAsubopt with default parameters was used to generate suboptimal secondary structures of all haplotypes. Since the number of possible suboptimal structures for each sequence is exceedingly large we randomly selected a subset of those using the -p option. In this analysis we used subsets of 2900 structures for each sequence. The resulting dot-bracket files were evaluated as follows: 1) by counting the number of continuous base-pairs in double helices of at least 6 bp (number of double helices sections—NDH); 2) by calculating the resulting Gibbs free energy (kcal/mol) with RNAeval from the Vienna Package [].First we performed these calculations for the wild type sequence and obtained average numbers of base-pairs, total melting index and Gibbs free energies. Then we compared each suboptimal structure of the polymorphic haplotype to those averages and counted how many times they present higher thermodynamic stability (lower Gibbis free energy). This gives us a measure that represents the probability which polymorphic haplotype may result into a more stable structure than the wild-type sequence.The NCBI Reference Sequence accession number for the RET gene was NM_020975.4 (mRNA). […]

Pipeline specifications

Software tools RNAfold, RNAsubopt, RNAeval
Application RNA structure analysis
Organisms Homo sapiens
Diseases Hirschsprung Disease, Thyroid Neoplasms