Fig 1: TWIST1 5' UTR variants and effect on translation. A: Genomic sequence showing the locations of the variants identified in Families 1 and 2 within the 5' UTR of TWIST1 (NM_000474.3). The TATAA box and transcription start site (TSS) are denoted by blue shading. The reading frames from the uAUGs at -263 (Family 1) and -255 (Family 2) are indicated by red and blue dotted lines, respectively. Note that the ORF from c.-263 terminates at a stop codon (TGA; yellow text with red shading) that is 62 bp upstream of the reference start codon of TWIST1 (denoted by red highlighting and turquoise arrow). The uAUG in Family 2 is in-frame with the TWIST1 start codon. B: Kozak consensus sequence (Kozak, 1986) aligned to the uAUGs of Families 1 and 2, and to the TWIST1 start codon (sAUG). The relative strengths of these possible translation initiation sequences were assessed by three online tools, DNA functional site miner (DNAFSMiner), NetStart, and ATGpr with scores shown on the right. C: Luciferase analysis to determine the effect of the Family 1, 5' UTR variants on translation. Luciferase reporter DNA constructs are shown on the left and normalized luciferase activity generated from each is shown on the right. (i) WT construct. (ii) The Dual construct contains both c.-263 and c.-281 variants, while the c.-281 (iii) and c.-263 (iv) constructs contain each variant in isolation. The c.-263C > A variant is in-frame with a TGA stop codon at c.-62_-60 generating a large uORF of 204 bp (grey shading; 68 codons). (v) The c.-263, c.-252Stop construct incorporates a new stop codon at -254_-252, shortening the uORF to four codons and extending the distance from the uORF to the mORF from 59 bp to 251 bp. D: Luciferase analysis of the Family 2 variant c.-255G > A. (i) WT construct. (ii) The ATG created by c.-255 is in-frame with the luciferase ORF adding a further 85 codons. (iii) The c.-255, c.-246Stop construct incorporates a new stop codon at -246_-244 to create a short four codon uORF, while the c-255, c.-75Stop construct (iv) contains a longer uORF of 61 codons. Plots are shown as mean±standard error based on three separate experiments carried out in triplicate. E: The top panel shows RT-qPCR (blue) and dual luciferase reporter (red) assays in HEK 293T cells comparing Renilla luciferase expression and activity using WT, Dual, and c.-255 constructs (plots are shown as mean±SD). The y-axis shows relative expression or activity of the Renilla reporter gene (normalized against firefly and to WT). mRNA levels and luciferase activity are indicated in blue and red, respectively. Bottom panel: western blot analysis of transfected HEK293 cell lysates showing expression of Renilla luciferase produced from WT (lanes 1 and 2) and c.-255 constructs (lanes 3 and 4), from separate experiments. The N-terminal extension produced by translation from c.-255 uAUG is predicted to increase the molecular weight of Renilla by ~9 kDa, but a larger product was not detected. Anti-Renilla luciferase antibody (Abcam ab185925) and a-tubulin (Santa Cruz, sc-32293) at 1/1000 dilutions were used against 10 µg of protein lysate (BCA protein assay kit, Thermo)