Three upstream ORFs in an alternative GRN 5′UTR influence downstream protein expression

Background Frontotemporal dementia (FTD) is the second most common subtype of dementia regarding people under 65 years. It is caused by degeneration of the frontal and temporal lobes of the brain and leads to behavioral and language impairments. Upstream open reading frames (uORFs) play an important role in controlling protein expression. Variants in these elements have been linked with certain diseases. We are investigating the role of uORFs in progranulin ( GRN )‐associated FTD. Method Patient sources are the Belgian FTD cohort (n = 366, age at onset (AAO): 63.3 ± 10.5y) and a cohort of GRN loss‐of‐function (LOF) mutation carriers (n = 112, 78 affected (AAO: 60 ± 7.1) and 34 unaffected). Targeted resequencing of uORF regions of GRN transcripts by amplicon target amplification assays ( https://www.agilent.com ). Reporter assays for characterization of GRN uORFs and identified variants. Results Sequencing of uORF regions within two different GRN 5′ untranslated regions (UTRs) revealed 2 genetic variants (rs76783532, rs75193026) located in GRN uORFs. Variant rs75193026 is found in an alternatively spliced 5′UTR of the canonical GRN transcript, which contains 3 uORFs. Reporter assays showed that this variant significantly increases the levels of the downstream protein (Kruskal‐Wallis test, p‐value = 0.0184). Abolishment of each GRN uORF in the alternative 5′UTR led to a substantial increase of the levels of the downstream protein (Kruskal‐Wallis test, p‐value <0.05). Combined mutations of all 3 uORFs initiation sites showed a 10‐fold increase in protein expression (Kruskal‐Wallis test, p‐value < 0.0001). Conclusion Differentially spliced GRN 5′UTR contains 3 uORFs that can potentially regulate expression of the downstream protein. The variant identified in the alternative 5′UTR appears to be functional based on the reporter assays, as it significantly increased protein expression. Estimation of mRNA changes in reporter assays will determine the impact of uORFs and the variant under study on transcriptional level. We are examining the possibility of uORF‐mediated translation by abolishing the canonical initiation codon of the downstream protein. We predict that our findings can contribute to novel mechanistic insights in GRN ‐associated FTD.