uAUG creating variants in the 5'UTR of ENG causing Hereditary Hemorrhagic Telangiectasia

Abstract

Hereditary Hemorrhagic Telangiectasia (HHT) is a rare, autosomal dominant, vascular disorder. About 80% of cases are caused by pathogenic variants in ACVRL1 (also known as ALK1) and ENG, with the remaining cases being unexplained. We identified two variants, c.-79C>T and c.-68G>A, in the 5'UTR of ENG in two unrelated patients. They create upstream AUGs at the origin of upstream overlapping open reading frames (uoORFs) ending at the same stop codon. To assess the pathogenicity of these variants, we performed functional assays based on the expression of wild-type and mutant constructs in human cells and evaluated their effect on ALK1 activity in a BMP-response element assay. This assay is mandatory for molecular diagnosis and has been so far only applied to coding ENG variants. These variants were associated with a decrease of protein levels in HeLa and HUVEC cells and a decreased ability to activate ALK1. We applied the same experiments on three additional uoORF-creating variants (c.-142A>T, c.-127C>T and c.-10C>T) located in the 5'UTR of ENG and previously reported in HHT patients. We found that all the analyzed variants alter protein levels and function. Additional experiments relying on an artificial deletion in our mutated constructs show that identified uAUGs could initiate the translation indicating that the associated effect is translation-dependent. Overall, we have identified two 5'UTR ENG variations in HHT patients and shed new light on the role of upstream ORFs on ENG regulation. Our findings contribute to the amelioration of molecular diagnosis in HHT.

Fig. 1. uAUG-creating variants are associated with a decrease of ENG levels in vitro.

a cDNA of the long (NM_001114753.3) isoform of ENG transcripts. Positions of the identified 5’UTR uAUG-creating variants from this project and published studies^,– as well as position of the associated uStop codon at position c.125, and of main start (c.1) and stop (c.1975) codons are indicated. 5’UTR 5’ untranslated region, CDS CoDing Sequence, WT wild type, uoORF upstream overlapping open reading frame, eCDS elongated CDS. b Schematic presentation of the pcDNA3.1-L-ENG constructs prepared and used for the evaluation of ENG steady-state levels in HeLa cells. Arrows indicate specific primers targeting the cDNA of ENG with extra sequences containing restriction sites to allow specific cloning in the expression vector. Position of the Myc-His tag is represented by the black square on the plasmid. CMV cytomegalovirus promoter, WT wild type, Var variant, 5’UTR 5’ untranslated region, L long. c, d Western blot results on total proteins extracted from transfected HeLa cells with 1 µg of pcDNA3.1-L-ENG constructs or from transduced HUVEC cells with 20 MOI of lentiviruses containing ENG, respectively. Two bands of different molecular weights are observed for endoglin likely corresponding to more glycosylated (upper band) and less/non glycosylated (lower band) ENG monomers. Anti-Myc and anti-ENG correspond to the used antibodies for the target protein from HeLa and HUVECs, respectively, and anti-β-actin corresponds to the antibody used against the reference protein. kDa kilodalton, M protein ladder, WT wild type, C- negative control corresponding to pcDNA3.1- empty vector. Shown results are representative of 5 independent experiments. Uncropped blots are shown in Supplementary Fig. 2. All blots were processed in parallel and derive from the same experiments. e Decrease of luciferase activity observed with uAUG-creating variants in ENG. Schematic presentation of the pGL3b-(ENG)-luciferase prepared and used in this assay is shown in the upper panel. Arrows indicate specific primers targeting the promoter of ENG with extra sequences containing restriction sites to allow specific cloning in the expression vector. Luc luciferase. In the lower panel, shown results with standard error of the mean correspond to Firefly/Renilla ratios normalized to the wild-type (WT) in 5 independent experiments. ***p value < 10⁻³ (two-factor ANOVA followed by Tukey’s multiple comparison test of variants versus WT).

Fig. 2. ALK1 response to BMP9 stimulation is affected by 5’UTR ENG variants.

a Schematic presentation of the co-transfected constructs in this assay. BRE BMP-response element, CMV cytomegalovirus promoter, WT wild type, Var variant, 5’UTR 5’ untranslated region, L Long. b Decrease of BRE activity observed with all the analyzed variants in co-transfected NIH3T3 cells stimulated with BMP9 (5 pg/ml). Shown results with standard error of the mean correspond to the quantification of Firefly/Renilla and normalized to the wild-type (WT) (n = 4). ***p value < 10⁻³; *p value < 5 × 10⁻², ns non-significant (two-factor ANOVA followed by Tukey’s multiple comparison test of variants versus WT).

Fig. 3. Created uAUGs in the 5’UTR of ENG seem to be able to initiate the translation.

a cDNA of the long (NM_001114753.3) isoform of ENG transcripts. Positions of the identified 5’UTR uAUG-creating variants from this project and published studies^,– as well as position of the associated uStop codon (c.125), the introduced deletion c.1_2del, and of main start (c.1) and stop (c.1975) codons are indicated. 5’UTR 5’ untranslated region, CDS coding sequence, WT wild type, uoORF overlapping upstream open reading frame. b Western blot results on total proteins extracted from transfected HeLa cells with 1 µg of pcDNA3.1-L-ENG constructs. Two bands of different molecular weights are observed for endoglin likely corresponding to more glycosylated (upper band) and less/non glycosylated (lower band) ENG monomers. Anti-Myc and anti-βactin correspond to the used antibodies for the target and the reference proteins, respectively. kDa kilodalton, M protein ladder, WT wild type, C- negative control corresponding to pcDNA3.1- empty vector. Shown results are representative of 5 independent experiments. Uncropped blots are shown in Supplementary Fig. 4. All blots were processed in parallel and derive from the same experiments. c Quantification of protein steady-state levels obtained in (b) and probably resulting from translation initiation at predicted uAUGs. For quantification, the average of each duplicate has been calculated from the quantified values and ENG levels for each sample have been normalized to the corresponding β-actin levels then to the WT (%). na, no 5’UTR variant introduced. Graphs with standard error of the mean are shown and are representative of 5 independent experiments. ***p value < 10⁻³; **p value < 10⁻²; *p value < 5 × 10⁻², ns non-significant (two-factor ANOVA followed by Tukey’s multiple comparison test of variants versus WT).