Javier Corral

Structural variants (SVs) in F8 and F9 are major causes of severe hemophilia, yet their mechanistic basis and clinical impact remain incompletely understood. We aimed to implement an integrated workflow to define hemophilia-associated SVs at nucleotide resolution and to explore genomic signatures and clinical correlates. We analyzed 25 unrelated families non-recurrent SVs with hemophilia A (HA;n=20) or hemophilia B (HB;n=5) using genome walking, short-read sequencing, and nanopore whole-genome long-read sequencing (WGS-LRS). In HA, we resolved 15 partial F8 deletions (from 249 bp to ~0.5 Mb), one tandem duplication, and four complex rearrangements. In HB, we characterized three large F9 deletions (including one partial and two whole-gene deletions, up to >4 Mb) and two pathogenic exonic Alu insertions. In four cases, MLPA calls were incomplete or misleading, whereas WGS-LRS uncovered breakpoints and structures missed by standard workflows. Notably, repetitive elements were present at least one breakpoint in nearly all SVs (22/25), implicating local genomic architecture in SV formation. Breakpoint-junction analysis revealed short microhomologies in most events (F8: 18/20; F9: 3/5), supporting microhomology-mediated end joining or replication-based mechanisms as predominant pathways. WGS-LRS enabled methylation-based assessment of skewed X-inactivation in a carrier with a Xq28 deletion. Inhibitors were reported in 4/16 and 3/4 evaluable HA and HB cases, respectively. Overall, this study provides the first systematic application of nanopore WGS-LRS as a transformative genomic diagnostic approach for comprehensive SV analysis in hemophilia, enabling definitive SV delineation, correcting misclassification by conventional testing, and supporting mechanism-informed interpretation to improve molecular diagnosis and genetic counseling.

Hereditary protein C (PC) deficiency, which is caused by PROC gene mutations, increases the risk of venous thromboembolism and offers limited treatment options. In this study, we developed adeno-associated virus serotype 8 (AAV8) vectors carrying either murine PC (AAV8-mPROC) or human PC (AAV8-hPROC) transgenes. These vectors were delivered through tail vein injection into PROC knockout mice. The highest dose of AAV8-mPROC (6.00E + 12 vg/kg) resulted in PC activity and antigen levels reaching 200.7% and 190.1%, respectively, which were maintained at 171.8% and 165.2%, respectively, by week 48. Similarly, the highest dose of AAV8-hPROC (8.00E + 12 vg/kg) resulted in 295.4% PC activity and 3.72 μg/ml human protein C antigen, which were maintained at 195.1% and 1.13 μg/ml, respectively, by week 48. In the vein thrombosis model, AAV8-mPROC and AAV8-hPROC significantly reduced the thrombus weight from 12.11 ± 3.39 mg to 7.19 ± 2.28 mg and 6.81 ± 2.28 mg, respectively. In the pulmonary embolism model, the proportion of embolized vessels decreased from 88.53% to approximately 60.62% in the AAV8-mPROC group and 62.33% in the AAV8-hPROC group. Our study has established a preclinical foundation for the safe and effective application of AAV vector-based gene therapy in treating inherited PC deficiency.

Objectives: Proteoforms affect biological and clinical processes and are anticipated to play a major role in establishing precision medicine. However, mass spectrometry-based proteoform tests generate complex data which needs to be processed thoroughly prior to reporting to clinical chemists and clinicians. We developed a workflow for handling (clinical) data, based on our in-house developed test for the molecular characterization of antithrombin proteoforms by LC-MRM-MS, which serves as a proof-of-concept for laboratories aiming to develop MS-based proteoform tests. Methods: Using previously collected study data investigating the diagnostic value of an MS-based antithrombin proteoform test, a workflow was developed based on three layers. Quantitative information assessed deficiencies, qualitative information assessed mutations or PTMs, and a last confirmatory step verified mutations. All steps were (semi-)automated by a custom R script. Results: The workflow comprised of quantitation through verification of a quantifying peptide and interpretation to previously established reference intervals. Qualitative information was based on the ratio between qualitative peptides and the established concentration, for which deviations >20 % indicated the presence of variant proteoforms, subsequently confirmed by variant peptide transitions. The R script analyzed and condensed the data per sample to a bi-level diagnosis report providing concentration and molecular proteoforms for easy integration in a LIS. Conclusions: MS-based proteoform analysis is a feasible and robust application for the clinical laboratory when combined with automated data analysis and we here provide guidance for establishing such analyses. MS-based proteoform tests will aid the transition to personalized medicine, placing the clinical chemist at the forefront of precision diagnostics.

Arginine residues in antithrombin are essential for heparin binding and for the activation of this key anticoagulant serpin. Mutations affecting these residues may cause antithrombin deficiency, specifically Type II heparin‐binding site (HBS) defects, and increase the risk of thrombosis. However, previous alanine‐scanning mutagenesis and crystallographic studies have yielded conflicting results regarding the specific residues involved in heparin binding. Our aim was to characterize natural variants affecting arginine residues in antithrombin. Genetic, biochemical, and functional characterization of antithrombin was done in 663 unrelated patients, most with antithrombin deficiency. Recombinant expression of the variants was performed in two different N‐glycosylation backgrounds. We identified nine SERPINC1 missense variants affecting eight arginine residues located at or near the HBS, four of them novel. Two variants, the most distant from the HBS (p.R291H and p.R177C), were found to be benign. In contrast, p.R45W, p.R56C, p.R79C, p.R79H, and p.R161Q caused Type II HBS deficiency. p.R78Q, causing a mild Type II HBS defect, was identified in a patient with compound heterozygosity with other HBS mutations. N‐glycosylation at N167 modulated heparin affinity and influenced the clinical severity of mutations affecting arginine residues involved in heparin interaction. Finally, p.R89S caused Type I deficiency by creating a novel N‐glycosylation motif, leading to hyperglycosylation and intracellular retention. Natural variants provide valuable insights into the functional contribution of arginine residues to antithrombin–heparin interaction. Our findings highlight the biochemical, functional, and clinical heterogeneity of these mutations and underscore the importance of comprehensive characterization for accurate diagnosis and prognosis in affected individuals.

Transposable elements (TEs) make up 45% of the human genome, are a source of genetic variability difficult to detect, and involved in processes related to gene regulation and disease. Nanopore sequencing is recognized as one of the best technologies for detecting TEs; however, tools for analyzing of human TE insertions and deletions with nanopore-based data can be improved. RetroInspector is an easy to use, configurable Snakemake pipeline that performs detection, annotation, enrichment, and genotyping of TEs. RetroInspector requires the FASTQ files of the samples and the reference genome to start the identification and analysis of TEs. The user can also set the threshold for the number of supporting reads for the variant filtering. RetroInspector also allows users to compare the results of two samples. Different versions of the reference genome can be used and the presence of retrotransposition features can be annotated. RetroInspector has been run on three nanopore sequencing datasets and validated experimentally using proprietary and public data with over 80% precision.