# Overlapping upstream ORFs ending at c.125 lead to reduced Endoglin, contributing to Hereditary Hemorrhagic Telangiectasia

Omar Soukarieh, Carole Proust, Clémence Deiber, Caroline Meguerditchian, Alexandre Guilhem, Shirine Mohamed, Aurelie Goyenvalle, Beatrice Jaspard-Vinassa, Sophie Dupuis-Girod, David-Alexandre Trégouët

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Omar Soukarieh, Carole Proust, Clémence Deiber, Caroline Meguerditchian, Alexandre Guilhem, et al.. Overlapping upstream ORFs ending at c.125 lead to reduced Endoglin, contributing to Hereditary Hemorrhagic Telangiectasia. 2025. hal-05059184

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# Overlapping upstream ORFs ending at c.125 lead to reduced Endoglin, contributing to Hereditary Hemorrhagic Telangiectasia

Omar Soukarieh

omar.soukarieh@inserm.fr

Inserm U1034 https://orcid.org/0000-0003-4923-4353

Carole Proust

University of Bordeaux, INSERM

Clémence Deiber

University of Bordeaux, INSERM

Caroline Meguerditchian

University of Bordeaux, INSERM

Alexandre Guilhem

Hospices Civils de Lyon,

Shirine Mohamed

CHRU BRABOIS

Aurelie Goyenvalle

Université de Versailles Saint Quentin https://orcid.org/0000-0003-3938-1165

Beatrice Jaspard-vinassa

Délégation Aquitaine, Poitou-Charentes

Sophie Dupuis-Girod

Hospices Civils de Lyon

David-Alexandre Trégouët

INSERM https://orcid.org/0000-0001-9084-7800

## Article

Keywords:

Posted Date: May 5th, 2025

DOI: https://doi.org/10.21203/rs.3.rs-4808835/v1
**License:** © Ⓟ This work is licensed under a Creative Commons Attribution 4.0 International License.

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**Additional Declarations:** There is NO Competing Interest.
**Abstract**

Hereditary Hemorrhagic Telangiectasia (HHT) is a rare vascular disease mainly caused by pathogenic mutations in *ACVRL1* and *ENG* genes. Despite advances in HHT diagnosis, the molecular origin of some cases remains unclear. Recently, we observed a high prevalence of HHT-causing 5'UTR variants in *ENG*. These variants commonly introduce upstream AUG codons (uAUGs) at the origin of upstream open reading frames (upORFs) overlapping the coding sequence, all terminating at the same stop codon located at position c.125 (uAUG-c.125). Here, we analyzed all 5'UTR ENG single nucleotide variants that could alter upORFs in silico. Moreover, we found that 85% of uAUG-c.125 variants alter the protein levels. Furthermore, we identified 2 variants creating uAUG and uCUG in HHT patients and experimentally demonstrated their association with reduced endoglin levels This study provides new elements for the interpretation of upORF-altering variants in the 5'UTR of ENG with new insights for the molecular diagnosis of HHT.

**Introduction**

Hereditary Hemorrhagic Telangiectasia (HHT) is a multiorgan rare vascular disease¹ caused by pathogenic mutations in *ACVRL1* and *ENG* genes in more than 90% of cases². The clinical diagnosis of HHT is based on Curaçao criteria which include family history, epistaxis, multiple telangiectasias, and visceral vascular lesions such as gastrointestinal telangiectasia and/or arteriovenous malformations (AVMs)³. Depending on the identified criteria in patients, the clinical diagnosis of HHT can be classified as definite, possible, or unlikely. Tremendous efforts have been made in order to identify new genetic drivers² and molecular explanations at the origin of HHT⁴,⁵. However, around 10% of cases are still with unclear molecular origin¹,². Unresolved cases can be attributed to rare variants in poorly explored and complex regions of the genome that have yet to be identified and characterized⁶,⁷. In this study, we aim to contribute to the optimization of HHT molecular diagnosis related to the involvement of rare variants in HHT.

For a long time, the search for rare pathogenic mutations in HHT patients was mainly restricted to exonic/flanking intronic regions¹,⁵. However, we and others have recently reported 5 rare pathogenic variations in the 5'UTR of *ENG* identified in HHT patients with definite diagnosis⁸⁻¹³. Acting as Loss-of-function (LoF) mutations, these variations create upstream AUGs (uAUGs) in frame with the same stop codon in position c.125 (uAUG-c.125) and resulting in overlapping upstream Open Reading Frames (uoORFs). In addition, these variants were associated with a decrease of the protein levels of Endoglin, encoded by the *ENG* gene. Endoglin is a membrane glycoprotein that plays a major role in endothelial cell biology and is highly involved in angiogenesis¹⁴,¹⁵. It has mainly been known as co-receptor in response to bone morphogenetic proteins belonging to the transforming growth factor β (TGF-β) superfamily¹⁶⁻¹⁸.
Our previous data suggested that *ENG* could be enriched in HHT-causing uAUG-creating variants carrying the common characteristic to create overlapping upORFs ending with the same stop codon (c.125).

Furthermore, given the growing evidence that non-canonical translation initiation sites (TIS) differing by one nucleotide from the AUG codon can also initiate translation<sup>19,20</sup>, and despite such evidence in the context of HHT, we hypothesize that upstream ORFs (upORFs) starting with non-canonical TIS could also contribute to regulate the translation of the Endoglin coding sequence (CDS). Interestingly, upstream CUGs have particularly been described to be associated with human diseases<sup>21,22</sup>. To improve the molecular diagnosis of HHT, we here extracted all possible single nucleotide variants (SNVs) in the 5'UTR of *ENG* that could create or alter upORFs from the MORFEEdb database<sup>23</sup> resulting from the latest version of the bioinformatics tool MORFEE. In addition, we completed the functional analysis of all uAUG-c.125 variants and found that 85% of them are associated with a decrease of Endoglin levels *in vitro*. Finally, we identified a uAUG- and a uCUG- creating variants in French HHT patients and found that they also decrease protein levels in our assays. This study will facilitate the detection and interpretation of non-coding variants in HHT, contributing to improve the molecular diagnosis for patients and their families.

## Results

Identification of all possible upORF-altering variations in the 5'UTR of *ENG*

A total of 909 SNVs resulted from the *in silico* mutational saturation of the 5'UTR of *ENG* (Supplementary Table 1). MORFEE annotated 328 of them as creating uTIS, uStop, and/or deleting existing uStop. The annotated variations can be at the origin of 360 upORFs (Fig. 1a, Supplementary Table 2). More precisely, 255 variants are predicted to create new uTIS, 30 to create new uStop and 12 to delete existing uStop (Fig. 1a). The remaining 31 SNVs show multiple consequences (Fig. 1a; Supplementary Table 3). While the majority of annotated variants do not exist in databases and correspond to artificial ones, 29 of them are reported in ClinVar as candidates for HHT. Of the 29 ClinVar *ENG* upORF variants, 27 are predicted to create uTIS (Supplementary Table 4), among which 21 are classified as variants of unknown significance (VUS) or with conflicting interpretations. Moreover, at least 14 *ENG* uTIS-creating variants have been reported in GnomAD V4 0.0 with minor allele frequency lower than 0.01% but without any evidence of association with HHT (Supplementary Table 5).

In more detail, 286 SNVs creating canonical or non-canonical uTIS could be at the origin of 294 upORFs, with the understanding that a given SNV may create different uTIS (Fig. 1b; Supplementary Table 6). Among these upORFs, 86 are fully located in the 5'UTR (uORFs) ending at 2 different stop codons (c.-166 and c.-34), 122 are overlapping with the CDS (uoORFs) ending with stop codons at position c.90 or c.125, and 86 correspond to elongated CDS (eCDS) ending at the main stop codon (i.e. natural stop codon of the CDS) (Fig. 1c).

Interestingly, 8 upORFs have been reported in public databases (www.sORFs.org<sup>24</sup>;
https://metamorf.hb.univ-amu.fr/<sup>25</sup>; https://vutr.rarediseaseagenomics.org/; and
https://smorfs.ddnetbio.com<sup>26)</sup> as naturally existing in the 5'UTR of *ENG*. Five of these upORFs end with the stop codon located at c.-166, 2 with the stop codon at position c.-33 and 1 is overlapping the CDS and ends at the uStop-c.125 (Supplementary Fig. 1a). MORFEE annotated 8 variations as deleting the uStop codon located at position c.-166, and 7 that delete the uStop codon at position c.-34, thus elongating these existing upORFs into either longer fully upstream ORFs (uORFs) ending at positions c.-34 or into uoORFs ending at new stop codon c.90, respectively (Supplementary Fig. 1b). By contrast, MORFEE identified 17 variations that could create new stop codons then shortening existing upORFs reported in databases and 42 that could create new stop codons at the origin of new upORFs (Supplementary Fig. 1c). Of note, two variants creating new stop codons (c.-200C > T and c.-133C > T) have been reported as variants of unknown significance in ClinVar and 2 others (c.-253C > A and c.-87C > T) have been reported as rare in GnomAD V4 0.0 database with the c.-87C > T showing multiple consequences on upORFs (Supplementary Tables 3&5).

Most ENG SNVs creating uAUGs in frame with the uStop-c.125 drastically alter the protein levels

Recently, we have demonstrated that 5 *ENG* 5'UTR variants identified in HHT patients and creating uAUG-initiated uoORFs all ending at the uStop-c.125, were responsible of decreased protein levels¹³, revealing their pathogenicity (Table 1). To extrapolate whether these deleterious effects also hold for any other uAUG-creating SNVs at the origin of uoORFs ending at the uStop-c.125, we conducted the same experimental work¹³ on the remaining 8 variations predicted by the MORFEE *in silico* analysis (Fig. 2a; Table 1). No decrease of protein levels was observed for the c.-287C > A and c.-37G > T variations (Fig. 2b-c; Supplementary Fig. 2; Supplementary Table 7). While the c.-287C > A variant showed similar protein levels comparing to the wild-type (WT), the c.-37G > T tended to associate with an increase of *ENG* levels in our assay (Fig. 2b-c; Supplementary Fig. 3a). Interestingly, the c.-37G > T variant is predicted to simultaneously create a uAUG-initiated uoORF and to shorten an existing upORF (Supplementary Table 3). The 6 remaining variations were associated with a mean protein level lower than 40% compared to the wild-type construct. After correcting for multiple testing, the association was statistically significant (p < 0.05) for 4 of these variants (c.-271G > T, c.-249C > G, c.-128C > A, c.-33A > G). Despite similar trends, the other two (c.-167C > A and c.-31G > T) did not reach significance (p ~ 0.10). No significant difference was observed in our RT-qPCR experiments between mutants and WT (Supplementary Fig. 3b).
<table><caption>Table 1
Characteristics of the 13 uAUGs and the studied uCUG created by variants in the 5'UTR of ENG.</caption><thead><tr><th>Variants</th><th>uTIS-SNV</th><th>Protein levels in vitro (%)<sup>‡</sup></th><th>uoORF size (nt)</th><th>Kozak sequence</th><th>TIS-Predictor (KSS score)</th><th>PreTIS score</th></tr></thead><tbody><tr><td rowspan="4">Previously studied<sup>13</sup></td><td>c.-142A > T<sup>$</sup></td><td>[0-14]</td><td>270</td><td>CAGATGG</td><td><strong>0.66</strong></td><td><strong>0.84</strong></td></tr><tr><td>c.-127C > T<sup>#, $</sup></td><td>[0-5]</td><td>255</td><td>GGGATGC</td><td><strong>0.69</strong></td><td><strong>0.84</strong></td></tr><tr><td>c.-79C > T<sup>#, $</sup></td><td>[8-27]</td><td>207</td><td>CCCATGC</td><td><strong>0.65</strong></td><td><strong>0.67</strong></td></tr><tr><td>c.-68G > A</td><td>[30-46]</td><td>195</td><td>CCGATGC</td><td>0.61</td><td><strong>0.89</strong></td></tr><tr><td></td><td>c.-10C > T<sup>#, $</sup></td><td>[21-49]</td><td>138</td><td>CCCATGT</td><td>0.61</td><td><strong>0.85</strong></td></tr><tr><td rowspan="10">New</td><td>c.-287C > A<sup>*</sup></td><td>[61-115]</td><td>414</td><td>CATATGC</td><td>0.42</td><td>na</td></tr><tr><td>c.-271G > T<sup>*</sup></td><td>[4-19]</td><td>399</td><td>GCCATGA</td><td><strong>0.78</strong></td><td>na</td></tr><tr><td>c.-249C > G</td><td>[6-12]</td><td>378</td><td>GCCATGC</td><td><strong>0.67</strong></td><td>na</td></tr><tr><td>c.-182C > A</td><td>[4-17]</td><td>309</td><td>TCCATGT</td><td>0.57</td><td><strong>0.91</strong></td></tr><tr><td>c.-167C > A</td><td>[7-77]</td><td>294</td><td>CTCATGA</td><td>0.55</td><td><strong>0.87</strong></td></tr><tr><td>c.-37G > T<sup>*</sup></td><td>[94-251]</td><td>165</td><td>CACATGA</td><td>0.57</td><td><strong>1</strong></td></tr><tr><td>c.-33A > G</td><td>[0-25]</td><td>162</td><td>AGGATGA</td><td><strong>0.73</strong></td><td><strong>1</strong></td></tr><tr><td>c.-31G > T</td><td>[12-41]</td><td>159</td><td>ATAATGC</td><td>0.63</td><td><strong>0.96</strong></td></tr><tr><td>c.-76C > T<sup>#, $</sup></td><td>[50-182]</td><td>204</td><td>ACGCTGG</td><td><strong>0.87</strong></td><td><strong>0.93</strong></td></tr></tbody></table>
<table><thead><tr><th>Variants</th><th>uTIS-SNV</th><th>Protein levels in vitro (%)<sup>‡</sup></th><th>uoORF size (nt)</th><th>Kozak sequence</th><th>TIS-Predictor (KSS score)</th><th>PreTIS score</th></tr></thead><tbody><tr><td colspan="7">uTIS (upstream Translation Initiation Start site) position in the L-ENG (Long isoform); NM_001114753.3 transcript (c.1 corresponds to the A of the main AUG) is indicated. Nucleotides at positions -3 and +4 relative to the predicted uTIS are in bold when corresponding to the most conserved nucleotide. The uTIS are underlined. Two additional scores have also been extracted from TIS-predictor<sup>30</sup> and PreTIS<sup>31</sup> tools, to predict the strength of Kozak sequence and the translation efficiency of the created uTIS, respectively. Bolded scores are those higher than the predefined thresholds.</td></tr><tr><td colspan="7">§, the c.-142A > T, c.-127C > T, c.-79C > T, c.-10C > T, and c.-76C > T variants are reported in ClinVar database. The c.-142A > T, c.-127C > T are classified as pathogenic/likely pathogenic, the c.-79C > T and c.-76C > T as variants of unknown significance, and the c.-10C > T has conflicting interpretations, according to ClinVar. #, the c.-127C > T, c.-79C > T, c.-10C > T, and c.-76C > T variants are reported in dbSNP database as rs1060501408, rs1564466502, rs756994701, and rs943786398, respectively. *, in addition of the uAUG-creation, c.-287C > A and c.-37G > T are predicted to create new stop codons shortening existing upORFs, and c.-271G > T is predicted to simultaneously create a uAUA at the origin of a fully upstream upORF. nt, nucleotide; ‡ Range of Endoglin protein levels observed across 5 independent experiments from Soukarieh et al.,<sup>13</sup>, and this study; na, non-applicable.</td></tr></tbody></table>

In total, 85% (11/13) of these variants (current and previous works<sup>13</sup>) decrease the Endoglin protein levels *in vitro*.

Among the 13 5'UTR variants we evaluated *in vitro*, 6 are characterized by a moderate Kozak sequence (Table 1). These 6 variants (c.-271G > T, c.-249C > G, c.-142A > T, c.-127C > T, c.-33A > G and c.-31G > T) were all associated with decreased protein levels in our assay with 5/6 variants showing the most drastic effects. The 7 remaining variants were associated with a weak Kozak sequence and 5 of them (c.-182C > A, c.-167C > A, c.-79C > T, c.-68G > A and c.-10C > T) were associated with a decrease of the protein levels. Finally, the 2 variants with no decrease of Endoglin protein levels (c.-287C > A and c.-37G > T) are associated with weak Kozak sequences. Our analysis suggests that the sole information on the Kozak sequence of uTIS cannot be used to predict the potential effect of uAUG-creating variants in *ENG*.

We extended the predictions of the Kozak sequence by applying predictions from TIS-predictor, which takes into account the 10 nucleotides surrounding a given canonical or non-canonical TIS to generate KSS scores reflecting the strength of Kozak sequences. The authors defined a threshold of 0.64 for translation initiation by uAUGs. We extracted KSS scores for our 13 uAUG-creating variants of interest (Table 1; Supplementary Table 2) and found that 6/13 uAUGs have scores higher than 0.64. These 6 variants (c.-271G > T, c.-249C > G, c.-142A > T, c.-127C > T, c.-79C > T and c.-33A > G) were observed to decrease the protein levels of Endoglin in our experiments. Interestingly, these variants are those showing the most drastic effects in our assays. However, 5/11 variants decreasing the protein levels (c.-182C > A, c.-167C > A, c.-68G > A, c.-31G > T and c.-10C > T) presented with KSS scores below the recommended threshold of 0.64 as the two variants with no decrease of protein levels.

Furthermore, we evaluated the efficiency of PreTIS scores to predict uTIS that can alter the protein levels. These scores predict the efficiency of a given uTIS to initiate the translation. We hypothesized
that the highest PreTIS scores predicting the most efficient uTIS would be associated with the most drastic effect on the protein levels. We collected PreTIS scores for the 10/13 uAUGs created by 5'UTR variants in ENG. The remaining 3 variants being located within the first 99 nucleotides of the 5'UTR (c.-287C > A, c.-271G > T and c.-249C > G), they cannot be predicted with PreTIS. The totality of the predicted uAUGs (10/10) has PreTIS scores higher than the predefined threshold for translation efficiency (0.54) (Table 1). Importantly, all but the c.-37G > T are associated with a decrease of the protein levels.

# New uoORF-creating variants identified in HHT patients

We used the generated catalog of variants annotated with MORFEE to retrospectively analyze ENG variants discovered in patients from the French National reference center for HHT with unresolved molecular diagnosis. We thus identified 2 uTIS-creating variants, the aforementioned c.-33A > G and the c.-76C > T variants. The first one, creating a uAUG predicted to generate a uoORF ending at the c.125 codon and never reported in public databases, was identified in a patient with definite HHT according to Curaçao criteria. Our experimental study (Fig. 2b-c) provided strong argument for its pathogenicity. The second variant, creating a non-canonical TIS (uCUG), is also predicted to generate a uoORF ending at the c.125 codon (Fig. 3a). This variant (rs943786398) was detected in 2 unrelated patients referred for genetic screening for suspicious HHT, although they only fulfilled one of the Curaçao criteria (Supplementary Table 8). This variant has been classified as variant of unknown significance (VUS) associated to HHT in ClinVar. The proband in the first family had only few telangiectasias and recurrent epistaxis during childhood with atypical presence of stroke and deep vein thrombosis. In the second family, the proband presented with pulmonary AVM, one epistaxis per month and not more than 2 telangiectasias on her face. In this family, the father was an asymptomatic carrier of the variant. Following the same experimental workflow as above, we observed that the c.-76C > T variant was associated with decreased Endoglin levels of more than 25% in comparison with the WT (Fig. 3b-c; Supplementary Fig. 4). Significant difference of ENG transcript amounts was observed between c.-76C > T and WT by RT-qPCR (Supplementary Fig. 3c), suggesting that an additional potential effect of this variant on RNA should not be excluded. Very interestingly, the uCUG created by ENG c.-76C > T is encompassed by a strong Kozak sequence and carries very high scores with TIS-predictor and PreTIS (Table 1). Five additional variants creating non-canonical uTIS in frame with the stop codon at position c.125 have been reported in ClinVar (Supplementary Table 4).

# Discussion

The diagnosis wandering in HHT is one of the main challenges for the molecular diagnosis and management of HHT patients and their families. This could be explained by the lack of information about non-coding or structural variants in HHT genes. Recent studies have however shown the association of this kind of variants in HHT-patients, suggesting that they are still underexplored⁶,⁷. Curiously, at least 5 non-coding variants carrying very similar characteristics in the 5'UTR of ENG have
been associated with HHT<sup>8-13</sup>. They all create overlapping upORFs ending with the same stop codon at position c.125 and are associated with severe HHT phenotypes<sup>13</sup>, being a very particular characteristic for *ENG*.

To tackle the challenge of diagnostic wandering, we conducted an exhaustive characterization of 5'UTR variants altering upORFs in *ENG*. We started by bioinformatically characterizing all 5'UTR *ENG* variants that can create or modify upORFs, aiming to facilitate the identification of pathogenic variants for HHT. This was achieved by cataloguing all possible SNVs (already reported or yet unreported artificial ones) that can create canonical or non-canonical TIS, create new stop codons or delete existing stop codon in the *ENG*. From this catalog, we were able to identify, in patients followed by the French National Reference Center for HHT, two extremely rare variants including one, c.-33A > G, that had never been reported and another, c.-76C > T, classified as VUS in ClinVar. Both of these variants create uoORFs ending with stop codon located at c.125 position and are associated with a decrease of Endoglin levels in our experimental assays. While the c.-33A > G is predicted to create a uAUG, the c.-76C > T creates a uCUG and is the first example of 5'UTR creating a non-canonical TIS in HHT. This variant was identified in 2 patients with unlikely HHT. These findings suggest that rare *ENG* variants predicted to create non-canonical uTIS in frame with the uStop-c.125 should also be considered as candidates for causing HHT or explaining clinical heterogeneity. Moreover, we demonstrated that most uAUG creating variants at the origin of uoORFs ending with the uStop-c.125 are associated with decreased Endoglin levels *in vitro*. It would be interesting to conduct similar experimental investigations for variants creating uTIS in frame with the uStop c.90 (Fig. 2c), thus generating similar type of upORFs (uoORFs), and to better explore the context of these stop codons that could influence the effects of the associated uoORFs.

Beyond the 2 identified variants in French HHT patients, the generated bioinformatics catalog (Supplementary Table 2), together with our experimental findings will serve to identify candidate rare 5'UTR *ENG* variants in HHT, contributing to resolve molecular origins of HHT. For instance, among *ENG* variants reported in ClinVar, 56 are located in the 5'UTR with 18 (~ 42% of 5'UTR variants) annotated with MORFEE as creating uTIS. Very importantly, the recent availability of a comprehensive catalog of all 5'UTR variants with the potential to alter upORFs across all human genes<sup>23</sup> will greatly facilitate the identification of novel 5'UTR pathogenic variants in other HHT genes and beyond. Of note, we also showed that prediction metrics dedicated to upORF-altering variants (Kozak strength, KSS and PreTIS scores) deserve to be evaluated with a larger dataset of variants with different impacts (increase, decrease or null effect) on protein levels, in particular for variants with predicted multiple consequences on upORF alteration (see Supplementary Table 3 on c-37C > T).

In conclusion, our work provides new insights into the interpretation of *ENG* non-coding variants in HHT patients.

# Methods

## Nomenclature
DNA sequence variant nomenclature follows current recommendations of the HGVS²⁷.

Search for all upORF-altering variants in the 5'UTR of ENG

We *in silico* mutated each position in the 5'UTR of the main transcript of ENG (MANE select, ENST00000373203.9) reported in the latest version of Ensembl database (GRCh38.p14) with the 3 alternative nucleotides to generate a vcf file containing all possible SNVs between positions c.-303 (i.e., first nucleotide of the 5'UTR) and c.-1 (Supplementary Table 1). The generated vcf file was then annotated using an updated version of the MORFEE bioinformatics tool¹³,²⁸ now available on https://doi.org/10.5281/zenodo.14864790. This version can annotate variations predicted to (i) create canonical and non-canonical TIS; (ii) create new stop codons (TAA, TAG and TGA); and/or (iii) delete existing stop codons along a given transcript. The resulting list of SNVs is provided in Supplementary Table 2.

Additionally, we extracted known upORFs in the 5'UTR of *ENG* from public databases reporting small ORFs that have been identified through ribosome profiling and/or mass spectrometry in human cells: sORFs repository (www.sORFs.org)²⁴; metamORF database (https://metamorf.hb.univ-amu.fr/)²⁵; vUTR interface (https://vutr.rarediseaseagenomics.org/); and smoRFs browser (https://smorfs.ddnetbio.com)²⁶.

Selection of ENG variants for experimental validation

Five rare variants creating uAUGs in frame with the stop codon at position c.125 have been previously identified in HHT patients and have shown drastic effects on ENG protein levels¹³. We here selected all additional 8 variations identified by MORFEE to create uAUGs in frame with the stop codon at position c.125 for experimental validation (Supplementary Table 2).

We also selected the c.-76C > T variation, creating a non-canonical uTIS in frame with the c.125 stop codon. This variation was further identified in a collection of HHT patients with unresolved molecular diagnosis (Supplementary Table 2).

## Plasmid constructs

Preparation of pcDNA3.1-L-ENG (L, Long, NM_001114753.3) constructions was performed by directed mutagenesis on pcDNA3.1-L-ENG-WT construct¹³ using the 2-step overlap extension PCR method²⁹ and primers listed in Supplementary Table 9. BamH*I* and *Sac*II or BamH*I* and Blp*I* were used as cloning sites, depending on the inserted variant (Supplementary Table 9). Only pcDNA3.1-L-ENG-c.-287C > A construction was prepared by a simple PCR reaction with a forward primer carrying the variant downstream of BamH*I* cloning site and a reverse primer overlapping Sac*II* (Supplementary Table 9). All new constructs were verified by Sanger sequencing of the insert and cloning sites (Azenta/Genewiz).

Functional analysis of ENG 5'UTR variants
Transfection of HeLa cells, RNA and protein extractions as well as western blot and RT-qPCR analyses were carried out as described in Soukarieh *et al.*, 2023¹³. Reverse transcription was performed by using oligo-dT and random hexamers. Primers are listed in Supplementary Table 9.

## Kozak sequence interpretation and bioinformatics predictions

We here used the obtained results in our functional assays on *ENG* variants (Table 1) to evaluate the predictive power of the strength of the Kozak sequence and of 2 predictive scores, TIS-predictor³⁰ (KSS scores) and PreTIS³¹ tools.

The Kozak sequence is defined as the genomic sequence surrounding a TIS. The optimal Kozak sequence is [A/G]CCATGG, underlined nucleotides corresponding to the TIS and bolded nucleotides to the most conserved positions. We have arbitrarily considered a given Kozak sequence as (i) strong when it contains a purine at position -3 and a guanine at position +4; (ii) moderate when it contains a purine at position -3 or a guanine at position +4, and; (iii) weak when it does not contain a purine at position -3 nor a guanine at position +4.

### Statistical analysis of in vitro data

Differential protein and RNA levels were assessed using Kruskal-Wallis test followed by Dunn test for handling multiple comparison when appropriate. A threshold of p < 0.05 was used to declare statistical significance.

## Declarations

### Competing interests

The authors declare that they have no known competing financial or non-financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Author contributions

OS and DAT conceived the project. OS, CP, and BJV designed the experiments. OS, CP, CD, and BJV performed the experiments. OS, CP, CD, and BJV analyzed the data. BJV and AG provided technical support and suggestions on the project and the experiments. SDG and MT were in charge of clinical management of HHT patients. CM performed the mutational saturation and variant annotation with MORFEE. OS and DAT drafted the paper that was further shared to co-authors who read/corrected/ and approved the final manuscript.
# Acknowledgments

This project was carried out in the framework of the French National Research Agency (ANR) ANR-23-CE17-0042-01 program as part of the ENDOMORF project, of the INSERM GOLD Cross-Cutting program (D-A.T.) and of the Precision and global vascular brain health institute funded by the France 2030 investment plan as part of the IHU3 initiative under grant agreement ANR-23-IAHU-0001. O.S was financially supported by a grant of the Lefoulon-Delalande Foundation.

# Data availability

ENG constructs generated during this study are available upon request by email from the corresponding author (omar.soukarieh@inserm.fr).

All raw experimental data analysed in this work are provided in Supplementary Table 7.

## Code Availability

The used version of MORFEE tool is available at https://doi.org/10.5281/zenodo.14864790.

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## Figures
<figure><img src="image_3.png" /><figcaption>(b)</figcaption></figure>

<figure><img src="image_4.png" /></figure>

<figure><img src="image_5.png" /><figcaption>Figure 1</figcaption></figure>

Identification of all possible single nucleotide variants (SNVs) modifying or creating upstream open reading frames (upORFs) in the 5'UTR of ENG. (a) MORFEE annotated SNVs that modify existing upORFs or create new ones (New_STOP-creating, uStop-deleting SNVs and uTIS-creating SNVs). (b) Three types of upORFs can result from the creation of upstream translation initiation sites (uTIS) in the 5'UTR of ENG. uoORF-SNVs, variants at the origin of upORFs overlapping the coding sequence (CDS); eCDS-SNVs, variants creating elongated CDS; uORF-SNVs, variants creating fully upstream upORFs. (c) Detailed illustration of upORFs generated by uTIS-creating SNVs. The type of uTIS and position of stop codons associated with the generated upORFs, as well as the number of each type of upORF and of uTIS are indicated. TSS, translation start site.
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /><figcaption>Figure 2</figcaption></figure>

**ENG** variants at the origin of uAUGs in frame with the same stop codon at position c.125. (a) Eight variants in the 5'UTR create uAUG-initiated upstream overlapping open reading frames (uoORFs) with different sizes but ending at the same position. Position and identity of these variants and of created uAUG, size of the uoORFs, associated stop codon located at position c.125 and first and last positions of the CoDing Sequence (CDS) (c.1 and c.1977, respectively) are indicated. TSS, translation start site; CDS, CoDing Sequence. (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-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. All blots were processed in parallel and derive from the same experiments. (c) Quantification of ENG steady-state levels in HeLa cells from (b). 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 (%). The two bands obtained for the Endoglin, corresponding to the more glycosylated (upper band) and less/non glycosylated (lower band) ENG monomers 1, were taken together for the quantification. Graphs with standard error of the mean are representative of 5 independent experiments.
**, p < 10⁻²; *, p < 0.05, ns, non-significant (Kruskal-Wallis followed by Dunn comparison test of variants versus WT).

<figure><img src="image_12.png" /><figcaption>Figure 3</figcaption></figure>

**uoORF-creating variants identified in French HHT patient.** (a) ENG c.-76C>T creating an upstream CUG in frame with the stop codon at position c.125 at the origin of an upstream Overlapping Open Reading Frame (uoORF) of 204 nucleotides (nt). Position and identity of the variant and of created uCUG, and first and last positions of the CoDing Sequence (CDS) (c.1 and c.1977, respectively) are indicated. (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-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. All blots were processed
in parallel and derive from the same experiments. (c) Quantification of ENG steady-state levels in HeLa
cells from (b). For quantification, the average of each duplicate has been calculated from the quantified
values and ENG levels for c.-76C>T sample have been normalized to the corresponding β-actin levels
then to the WT (%). The two bands obtained for the Endoglin, corresponding to the more glycosylated
(upper band) and less/non glycosylated (lower band) ENG monomers 1, were taken together for the
quantification. Graphs with standard error of the mean are representative of 12 independent
experiments. **, p < 10⁻² (Kruskal-Wallis test of the c.-76C>T versus WT).

**Supplementary Files**

This is a list of supplementary files associated with this preprint. Click to download.

* Proustetal.SuppTables.xlsx

• Proustet.al2025SupplementaryData.pdf