Internal ribosome entry site-mediated translation of UPF1 contributes to its oncogenic role in colorectal cancer

Colorectal cancer (CRC) is the third cause of death worldwide and projections point towards an increase for the next two decades. Many genes are misregulated in CRC, contributing to the development of the disease. Up-frameshift 1 (UPF1) is involved in many cellular mechanisms such as nonsense-mediated mRNA decay, cell cycle progression, or telomere maintenance and homeostasis. It also works as a tumour suppressor protein in most cancers but not in CRC, in which UPF1 plays an oncogenic role. We used the Xena platform to perform in silico analyses that revealed UPF1 is overexpressed in CRC contrary to several other analysed cancers. Besides, UPF1 expression levels are increased in CRC compared to the counterpart normal tissues. Experimentally, we confirmed these data and observed that endogenous UPF1 expression is maintained in different CRC cell lines under endoplasmic reticulum (ER) stress. To understand the mechanism underlying such maintenance, we used a bicistronic reporter construct to test whether UPF1 5’UTR can mediate alternative translation initiation and we concluded that such sequence contains an internal ribosome entry site (IRES) that maintains UPF1 expression in both normal and stress conditions in a 5’ cap-independent way. Deletional and mutational analysis of UPF1 5’UTR showed that nucleotides 1–100 (stem loop (SL) I) and 151–275 (SL III) — out of 275 nucleotides — are the minimal required sequences for the IRES to work properly. Using RNA antisense oligonucleotides (ASOs) targeting UPF1 IRES SL I and III, we observed a reduced UPF1 expression in HCT116 (CRC) cells. Altogether, these results suggest that UPF1 expression levels are maintained by the IRES-mediated translation mechanisms under conditions impairing canonical translation, such as those that mimic the tumour microenvironment. Thus, ASOs may be an upcoming therapy to target such alternative mechanism of translation initiation and prevent CRC development.Work supported by INSA and UIDB/04046/2020 (DOI: 10.54499/UIDB/04046/2020 ) and UIDP/04046/2020 (DOI: 10.54499/UIDP/04046/2020) Centre grants from FCT, Portugal (to BioISI).N/

Regulation of Δ160p53: a p53 short isoform with oncogenic functions

p53 is a transcription factor that activates tumor suppressor genes in response to various cellular stresses.Work supported by INSA and UIDB/04046/2020 (DOI: 10.54499/UIDB/04046/2020 ) and UIDP/04046/2020 (DOI: 10.54499/UIDP/04046/2020) Centre grants from FCT, Portugal (to BioISI).N/

Reclassification of BRCA1/2 variants previously classified as VUS (ACMG-AMP guidelines) with gene-specific guidelines from ClinGen ENIGMA and CanVIG-UK

In recent years, the number of BRCA1/2 germline variants associated with hereditary breast/ovarian cancer syndrome (HBOC), classified as variants of uncertain significance (VUS) according to ACMG-AMP guidelines (ACMGg) has been increasing. Reclassification of VUS as (likely) benign or (likely) pathogenic is crucial for maximizing diagnostic yield and appropriately managing HBOC patients. Recently, specific guidelines to improve classification of BRCA1/2 variants were independently developed by ClinGen ENIGMA1 (CG-Eg) and CanVIG-UK2 (CV-UKg). Main goals: i) independently reclassify BRCA1/2 variants previously classified as VUS (ACMGg) with the new guidelines (CG-Eg and CV-UKg); ii) compare the results between the different guidelines iii) evaluate the potential clinical impact of this reclassification. BRCA1/2 germline variants identified in patients with suspected HBOC and previously classified as VUS (8 missense, 5 intronic) were independently reclassified according to CG-Eg and CV-UKg. Variant assessment included: query of clinical/population databases and use of VEP, Alamut, VarSome and Franklin-Genoox. VUS reclassification (using CG-Eg versus CV-UKg) was in agreement for 10 variants (2 VUS, 6 likely benign (LB) and 2 benign (B)). The remaining 3 VUS were reclassified as LB with CG-Eg and kept as VUS with CV-UK. Application of specific guidelines reduced the number of VUS from 10 to 2 (CG-Eg) or to 5 (CV-UKg). The main difference between CG-Eg and CV-UKg is related with the downgrading strength of PM2 and the upgrading strength of BP1 criteria (in CG-Eg) for missense variants present outside clinically important functional domains and without splicing impact. The difference in BP1 strength has a major impact, making CG-Eg more stringent and reducing the number of VUS. The use of different guidelines, even if gene-specific, can lead to dissimilar classifications, a general consensus leading to a unique international guideline will be useful.FCT/MCTES, ToxOmics and Human Health (UIDB/00009/2020). GenomePT(POCI-01-0145-FEDER-022184).info:eu-repo/semantics/publishedVersio

Relevance of Multigene Panels in the Molecular diagnosis of patients with disorders of sexual development

Introduction: Next generation sequencing (NGS) is increasingly used in the molecular diagnosis of rare diseases, such as disorders of sexual development (DSD), allowing the analysis of a greater number of genes in a single assay, providing faster results with reduced costs. DSD patients may present high phenotypic overlap (genital ambiguity, sex reversal, delayed/absent puberty, infertility) posing challenges to clinical diagnosis. NGS technology using multigene panels has higher hypothesis to identify the genetic cause and novel genetic variants in a large number of cases. Methodology: 33 genomic DNA samples from DSD patients were sequenced on MiSeq using the Ampliseq technology (Illumina). A customized gene panel covering 40 genes was used to prepare the libraries of the target sequences. The 40 genes were subdivided into 5 subpanels: primary sex determination, sex differentiation, hypogonadotropic hypogonadism/infertility, steroidogenesis and premature ovarian insufficiency. Variant classification, according to ACMG-AMP, was based on bioinformatics tools (ex. VEP, HSF, VarSome, Alamut) and databases (gnomAD, HGMD, ClinVar, dbSNP). Pathogenic, Likely pathogenic and VUS variants were confirmed by Sanger sequencing. Results: 16 of the 33 patients were previously studied in our laboratory with negative results for the main genes associated with DSD (SRY, AR, ANOS1, GNRHR, CYP21A2). We identified 9 causative variants (8P/1LP) in 7 patients (21.2%) in AR, HSD17B3, LHCGR, FGFR1 and NR5A1. One patient was a compound heterozygous for HSD17B3 and another simultaneously homozygous for LHCGR and hemizygous for AR. The remaining 5 were homozigous, heterozigous or hemizygous for HSD17B3, LHCGR, NR5A1, FGFR1 or AR. One VUS, FGFR1:c.566G>A p.Arg189His, requires familial studies to revaluate its pathogenicity. Discussion: Multigene NGS studies allows to increase the rate of variant detection, mainly in genes not included in a first molecular approach. It also contributes to establishing or confirming the clinical diagnosis, assisting in decisions regarding the treatment and reproductive management of patients and families, as well as in genetic counselling.FCT/MCTES, Projects - ToxOmics and Human Health (UIDB/00009/2020) and GenomePT (POCI-01-0145-FEDER-022184)info:eu-repo/semantics/publishedVersio

Human TP53 is a tumour suppressor that acquires oncogenic functions during integrated stress response (ISR) due to a translational switch

Eukaryotic cells have developed different mechanisms and adaptive pathways that allow them to cope with external stress stimuli. Under stress conditions, global protein synthesis is shut down, and some alternative mechanisms of mRNA translation initiation are induced. Although the tumour suppressor protein p53 — the most mutated gene in cancer — has been considered the guardian of the genome and a master regulator of several cellular functions, the truth is it is not just one isoform, the full-length (FLp53), but also many other p53 isoforms that have been described so far. Based on our previous results, some functions of the shorter isoforms are different from and complement FLp53 activity. Here we show the specific induction of Δ160p53 isoform during integrated stress response (ISR). We confirmed the presence of an Internal Ribosome Entry Site (IRES) in p53 mRNA that controls Δ160p53 isoform translation, using a bicistronic reporter construct. When subjecting cells to endoplasmic reticulum stress, we showed that eIF2α phosphorylation is a key event leading to cap-independent expression of Δ160p53 during ISR. Also, some cancer-specific mutations in the DNA-binding domain of p53 enhance cap-independent translation of Δ160p53 via Δ160p53IRES. Using an antisense morpholino oligo targeting Δ160IRES significantly reduces Δ160p53 protein levels and impaired its oncogenic functions. Additionally, we found the 5’untranslated region of Δ160p53 inhibits the IRES activity. Our data support a model in which an IRES structure in the coding region of p53 is activated under stress conditions, leading to the expression of the oncogenic shorter Δ160p53 isoform, whose structure is affected by cancer-specific mutations in the p53 gene. A better understanding of Δ160p53IRES structure and function may be advantageous for a more efficient therapeutic targeting of p53.FCTinfo:eu-repo/semantics/publishedVersio