Repositório Científico do Instituto Nacional de Saúde
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Proposal of a Familial Hypercholesterolemia Pediatric Diagnostic Score (FH-PeDS)
Publication . Kafol, Jan; Miranda, Beatriz; Sikonja, Rok; Sikonja, Jaka; Wiegman, Albert; Medeiros, Ana Margarida; Alves, Ana Catarina; Freiberger, Tomas; Hutten, Barbara A.; Mlinaric, Matej; Battelino, Tadej; Humphries, Steve E.; Bourbon, Mafalda; Groselj, Urh
Background and aims: Familial hypercholesterolemia (FH) significantly increases cardiovascular risk from childhood yet remains widely underdiagnosed. This cross-sectional study aimed to evaluate existing pediatric FH diagnostic criteria in real-world cohorts and to develop two novel diagnostic tools: a semi-quantitative scoring system (FH-PeDS) and a machine learning model (ML-FH-PeDS) to enhance early FH detection.
Methods: Five established FH diagnostic criteria were assesed (Dutch Lipid Clinics Network [DLCN], Simon Broome, EAS, Simplified Canadian, and Japanese Atherosclerosis Society) in Slovenian (N=1,360) and Portuguese (N=340) pediatric hypercholesterolemia cohorts, using FH-causing variants as the reference standard. FH-PeDS was developed from the Slovenian cohort, and ML-FH-PeDS was trained and tested using a 60%/40% split before external validation in the Portuguese cohort.
Results: Only 47.4% of genetically confirmed FH cases were identified by all established criteria, while 10.9% were missed entirely. FH-PeDS outperformed DLCN in the combined cohort (AUC 0.897 vs. 0.857; p<0.01). ML-FH-PeDS showed superior predictive power (AUC 0.932 in training, 0.904 in testing vs. 0.852 for DLCN; p<0.01) and performed best as a confirmatory test in the testing subgroup (39.7% sensitivity, 87.7% PPV at 98% specificity). In the Portuguese cohort, ML-FH-PeDS maintained strong predictive performance (AUC 0.867 vs. 0.815 for DLCN; p<0.01) despite population differences.
Conclusions: Current FH diagnostic criteria perform suboptimally in children. FH-PeDS and ML-FH-PeDS provide tools to improve FH detection, particularly where genetic testing is limited. They also help guide genetic testing decisions for hypercholesterolemic children. By enabling earlier diagnosis and intervention, these tools may reduce long-term cardiovascular risk and improve outcomes.
European pilot interlaboratory comparison study on Mpox virus whole genome sequencing
Publication . Fuchs, Jonas; Bertelli, Claire; Pillonel, Trestan; Cordeiro, Rita; Izopet, Jacques; Pasquier, Christophe; Lewandowski, Kuiama; Maks, Anastasija; Michel, Janine; Rodriguez-Sanchez, Belen; Sanches-Seco, Maria Paz; Ledesma, Juan; Sobral, Daniel; Vercauteren, Koen; de Block, Tessa; Rezende, Antonio Mauro; Brinkmann, Annika; Nitsche, Andreas; Greub, Gilbert; Panning, Marcus
Objectives: Since 2022, distinct Mpox virus (MPXV) clades have been spreading across different geographic regions, causing a challenging epidemiological situation. Whole genome sequencing (WGS) proved to be instrumental for patient management and global public health. We report a pilot interlaboratory comparison study for MPXV WGS.
Methods: We distributed noninfectious DNA samples, including the main MPXV clades I and II, to eight European laboratories. We included one cowpox (CPXV) sample as a specificity control. Participants were free to choose their WGS pipeline of choice to mimic a real-world scenario and were asked to report on the sequencing pipeline used, average genome coverage, and MPXV species, clade, and subclade assignments.
Results: Seven of the eight invited laboratories reported results back. All participants largely identified the MPXV clades and reported high-quality genomes with minimal variations, specifically for MPXV clade IIb 2022 outbreak strains. However, reconstructed genomes showed high variability for nonclade IIb MPXV strains. The CPXV sample was correctly identified by three laboratories.
Conclusions: Although results for MPXV clade IIb 2022 outbreak strains are reassuring, the inclusion of MPXV clade I and IIa strains highlights pitfalls for targeted sequencing approaches and subsequent bioinformatic analyses. Our findings underscore the need for standardized external quality assessment studies.
Functional characterization of 16 variants found in the LDL receptor gene
Publication . Konečná, Kateřina; Přerovská, Tereza; Loja, Tomáš; Fajkusová, Lenka; Koutná, Jana; Kramárek, Michal; Alves, Ana Catarina; Bourbon, Mafalda; Freiberger, Tomáš; Tichý, Lukáš
Familial hypercholesterolemia (FH) is a disorder of cholesterol metabolism characterized by elevated LDL-cholesterol levels. The most common cause of FH is pathogenic variants in the LDL receptor (LDLR) gene. To shed light on the functional impact of selected LDLR variants, we functionally characterized 16 LDLR genetic variants alongside 10 control variants. We performed in vitro assays based on transient expression of WT and mutant LDLRs in LDLR-deficient Chinese hamster ovary cells. We used flow cytometry to analyze the relative amount of LDLRs expressed on the cell surface and the relative amount of internalized LDL. In addition, we analyzed the expression and maturation of LDLR protein by Western blotting. Of the 16 studied variants, two variants (p.(Asn272Thr) and p.(Arg574Leu)) did not exhibit a defect in LDLR function, one variant (p.(Ala540Thr)) exhibited a defect in LDL binding and/or internalization despite normal LDLR cell surface expression, and the remaining 13 variants had a detrimental effect on both LDLR cell surface expression and LDL internalization. The information presented in this study contributes to the clinical classification of LDLR variants and a more precise diagnosis of FH patients, highlighting the type of defect each variant produces.
The Functional Landscape Of Coding Variation In The Familial Hypercholesterolemia Gene LDLR
Publication . Tabet, Daniel R.; Coté, Atina G.; Lancaster, Megan C.; Weile, Jochen; Rayhan, Ashyad; Fotiadou, Iosifina; Kishore, Nishka; Li, Roujia; Kuang, Da; Knapp, Jennifer J.; Carrero, Carmela Serio; Taverniti, Olivia; Axakova, Anna; Castelli, Jack M. P.; Islam, Mohammad Majharul; Sowlati-Hashjin, Shahin; Gandhi, Aanshi; Maaieh, Ranim; Garton, Michael; Matreyek, Kenneth; Fowler, Douglas M.; Bourbon, Mafalda; Pfisterer, Simon G.; Glazer, Andrew M.; Kroncke, Brett M.; Parikh, Victoria N.; Ashley, Euan A.; Knowles, Joshua W.; Claussnitzer, Melina; Cirulli, Elizabeth T.; Hegele, Robert A.; Roden, Dan M.; MacRae, Calum A.; Roth, Frederick P.
Variants in the familial hypercholesterolemia gene -the most important genetic driver of cardiovascular disease-can raise circulating low-density lipoprotein (LDL) cholesterol concentrations and increase the risk of premature atherosclerosis. Definitive classifications are lacking for nearly half of clinically encountered missense variants, limiting interventions that reduce disease burden. Here, we tested the impact of ~17,000 (nearly all possible) missense coding variants on both LDLR cell-surface abundance and LDL uptake, yielding sequence-function maps that recapitulate known biochemistry, offer functional insights, and provide evidence for interpreting clinical variants. Functional scores correlated with hyperlipidemia phenotypes in prospective human cohorts and augmented polygenic scores to improve risk inference, highlighting the potential of this resource to accelerate familial hypercholesterolemia diagnosis and improve patient outcomes.
An endoribonuclease of the YicC-like family delays sporulation via sRNA degradation in Clostridioides difficile
Publication . Martins, Diogo; Salgueiro, Bruno; Sobral, Daniel; Gragera, Marcos; Hensel, Zach; Henriques, Adriano O.; Romão, Célia V.; Serrano, Mónica
Clostridioides difficile CD25890 is a YicC-like endoribonuclease involved in regulating sporulation initiation, a process critical for the host-host transmission of this anaerobic pathogen. Using comparative transcriptomics we identified a small RNA, SQ528, that accumulates at higher levels in a CD25890 deletion mutant and we show that purified CD25890 cleaves SQ528 in a metal-dependent manner. Moreover, the overexpression of SQ528 increases sporulation under certain nutritional conditions phenocopying a CD25890 deletion mutant. CD25890 is an hexamer in solution and in vivo. An N-terminal domain, which self-interacts as assessed by size exclusion chromatography and a two hybrid assay, is essential for oligomerization of CD25890. A C-terminal domain harbours residues H230, E254, and E258, conserved among orthologues, important for catalysis. AlphaFold2 modelling and cryo-EM suggest an elongated barrel-like structure with an internal cavity lined with basic residues that may aid in RNA binding. We show that CD25890 forms a complex with polynucleotide phosphorylase which combines the endoribonuclease activity of the first with the exonucleolytic activity of the latter and leads to the complete degradation of SQ528. This study identifies a native substrate for the YicC-family of ribonucleases and advances our understanding of the role of CD25890 in sporulation initiation in C. difficile.