A novel rare c. -39C>T mutation in thePROS15’UTR causing PS deficiency by creating a new upstream translation initiation codon and inhibiting the production of the natural protein

Abstract

SummaryInherited Protein S deficiency (PSD) (MIM176880) is a rare automosal dominant disorder caused by rare mutations, mainly located in the coding sequence of the structural PROS1 gene, and associated with an increased risk of venous thromboembolism. To identify the molecular defect underlying PSD observed in an extended French pedigree with 7 PSD affected members in who no candidate deleterious PROS1 mutation was detected by Sanger sequencing of PROS1 exons and their flanking intronic regions or via a MLPA approach, a whole genome sequencing strategy was adopted. This led to the identification of a never reported C to T substitution at c.-39 from the natural ATG codon of the PROS1 gene that completely segregates with PSD in the whole family. This substitution ACG->ATG creates a new start codon upstream of the main ATG. We experimentally demonstrated that the variant generates a novel overlapping ORF and inhibits the translation of the wild type protein from the main ORF in HeLa cells. This work describes the first example of 5’UTR PROS1 mutation causing PSD through the creation of an upstream ORF, a mutation that is not predicted to be deleterious by standard annotation softwares.

uORF‐introducing variants in the 5′UTR of the NIPBL gene as a cause of Cornelia de Lange syndrome

“…Interestingly, Patient 2's variant was associated with lower Kozak consensus scores by all prediction tools. These results appear to be consistent with previously published data suggesting that the amount by which the translation in reduced seems to be dependent on the uAUG match to the Kozak consensus sequence (Labrouche-Colomer et al, 2020;Wright et al, 2021). Interestingly, the phenotype of Patient 2 (and her father) described by Selicorni et al was milder than the phenotype of Patient 1, which seems consistent with the hypothesis of a milder effect of the c.-94C>T. uORFs are typically described as repressors of translation initiation at the downstream main ORF by different mechanisms (Silva et al, 2019) and we show here two variants actually acting through a similar loss-of-function mechanisms of expression regulation by reducing normal translation of the NIPBL coding sequence.…”

“…Interestingly, Patient 2's variant was associated with lower Kozak consensus scores by all prediction tools. These results appear to be consistent with previously published data suggesting that the amount by which the translation in reduced seems to be dependent on the uAUG match to the Kozak consensus sequence (Labrouche‐Colomer et al, 2020; Wright et al, 2021). Interestingly, the phenotype of Patient 2 (and her father) described by Selicorni et al was milder than the phenotype of Patient 1, which seems consistent with the hypothesis of a milder effect of the c.‐94C>T.…”

“…Despite being non conclusive on the mechanistic aspects, we show here, as previously reported for other 5′‐UTR variants (Borck et al, 2006; Hornig et al, 2016; Labrouche‐Colomer et al, 2020; Romanelli Tavares et al, 2019; Zhou et al, 2018) that a simple reporter assay, possibly associated with Patient's cells assessment when available, may be sufficient to provide evidence of a loss‐of‐function effect and thus reclassify variants of unknown significance as likely pathogenic.…”

“…Here, we assessed two variants, one of which is novel, in the 5′‐UTR of NIPBL creating uORFs and resulting in NIPBL translation repression, thus representing a novel mechanism causing CdLS. The number of diseases known to be caused by mutations that introduce or disrupt uORFs is increasing, albeit remaining scarcely reported (Barbosa, Peixeiro, et Romão 2013; Labrouche‐Colomer et al, 2020; Romanelli Tavares et al, 2019; Wright et al, 2021; Zhou et al, 2018). However, variants located in the 5′‐UTR region of NIPBL are rarely identified and require further investigation to assess putative pathogenicity (Borck et al, 2006; Selicorni et al, 2007).…”

“…Natural uORFS are considered as putative cis‐acting regulators of translation initiation, most of them acting by repressing translation of the natural protein (Lin et al, 2019). Variants creating novel upstream start codons, and variants disrupting stop codons of existing uORFs are under strong negative selection and represent a putatively important disease mechanism (Labrouche‐Colomer et al, 2020; Romanelli Tavares et al, 2019; Wright et al, 2021; Zhou et al, 2018). Several elements, such as the Kozak sequence, the distance to the 5′‐cap and the length of the uORF, make the ribosomal complex recognize the AUG translation initiation codon as such, and start the translation.…”