In the past few years, patients harbouring iron metabolism disorders have been undergoing high throughput sequencing. These technologies generate an important number of variants, complexifying their classification, especially when identified in non-coding sequences. 5’ and 3’ untranslated regions (UTRs) are involved in mRNA post-transcriptional regulation. UTRs are the target of different factors (proteins, ribonucleoproteins, LcRNA, mir RNA,...), that recognise specific nucleotides sequences or structures. Variants in these regions can affect transcript expression, stabilisation or degradation, which may lead to the translation of an incorrect protein expression. In this study, we propose a functional test to study the impact of 3’UTR variations. 3’UTR sequences are often associated with transcript stability or degradation. Wild type and mutated 3’UTRs are cloned in a luciferase reporter vector. The secreted luciferase depends on the 3’UTR and can be measure by a chemiluminescence assay. A SEAP gene is included in the vector and serves as a transfection normalizer in appropriate cell lines.

This model has been tested on the SLC11A2 3’UTR, using a variant identified in our patient cohort and a variant induced by site-directed mutagenesis in the IRE loop.

We thus provide proof of concept for this model to validate variants identified in 3'UTR