Iris Garcia-Martínez

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.

Background and Objectives: To meet the long‐term demand for blood products while preserving donor health in the long run, blood establishments must recruit a sufficient number of new donors annually. To determine what will suffice, it is essential to be able to forecast future blood donation volumes as a function of new donors using estimates based on historical donation activity. Materials and Methods: Donor ( n = 11,629,873) and donation data ( n = 64,510,294) extracted from operational information systems of seven blood establishments were transformed into anonymous donation activity data (grouped by blood establishment, sex and blood group) in the form of time series of mean number of donations per donor, indexed by years since first donation. Linear models were fitted to the time series using ordinary least squares. Results: Out of the various estimated models, the best fit (mean R ² over blood establishments 99.95%) for past mean donation activity was achieved when regressing the logarithm of cumulative donation activity on the logarithm of years since first donation and an indicator variable for the year of first donation. In addition to the predictions, comparison of the estimated parameters revealed that there are significant differences between blood establishments, translating to differences in the expected number of donations accumulated over the lifetime of a donor. Conclusion: Average donation activity can be modelled using a few variables and simple models, with high precision. The estimates can be used to create forecasts of future donation volumes, which in turn can be useful in long‐term blood donation management.

Inherited primary hemostasis disorders (IPHD) comprise a clinically and genetically heterogeneous spectrum, including inherited platelet disorders (IPD), heritable disorders of connective tissue (HDCT) associated with bleeding, and bleeding disorders of unknown cause (BDUC). Their phenotypic overlap and limited access to specific functional testing make genetic analysis essential for accurate diagnosis. This study aimed to investigate the genetic basis of patients with IPHD through whole-exome sequencing (WES), providing genotype–phenotype correlations to guide clinical management. A total of 170 probands were included: 114 with IPD (67%), 28 with HDCT (16%), and 28 with BDUC (16%). Definitive genotype–phenotype correlation was achieved in 83 probands (49%), identifying 98 unique candidate variants across 48 genes. Notably, 19 patients carried variants in different genes that can contribute to the phenotype. A partial genotype–phenotype correlation was achieved in 19 probands (11%), while no variants were identified in the remaining 68 (40%), especially in the BDUC group. This multicenter study represents the first integrated analysis using a single workflow for patients with IPHD, encompassing not only IPD and BDUC but also HDCT. The high rate of genotype–phenotype correlations achieved, the identification of 68 previously undescribed variants, and the evidence of a shared and overlapped genetic and phenotypic profile among IPHD patients demonstrate the clinical value of WES. The study advocates for a paradigm shift in the clinical diagnosis of IPHD, from traditional phenotype-driven assessment to genotype-informed strategies, positioning WES as a first-line tool together with basic laboratory tests, allowing faster and more accurate diagnosis and personalized patient care.

Venous thromboembolism (VTE) is a common disease with high heritability. However, only a small portion of the genetic variance of VTE can be explained by known genetic risk factors. Neutrophil extracellular traps (NETs) have been associated with prothrombotic activity. Therefore, the genetic basis of NETs could reveal novel risk factors for VTE. A recent genome-wide association study of plasma cell-free DNA (cfDNA) levels in the Genetic Analysis of Idiopathic Thrombophilia 2 (GAIT-2) Project showed a significant associated locus near ORM1. We aimed to further explore this candidate region by next-generation sequencing, copy number variation (CNV) quantification, and expression analysis using an extreme phenotype sampling design involving 80 individuals from the GAIT-2 Project. The RETROVE study with 400 VTE cases and 400 controls was used to replicate the results. A total of 105 genetic variants and a multiallelic CNV (mCNV) spanning ORM1 were identified in GAIT-2. Of these, 17 independent common variants, a region of 22 rare variants, and the mCNV were significantly associated with cfDNA levels. In addition, eight of these common variants and the mCNV influenced ORM1 expression. The association of the mCNV and cfDNA levels was replicated in RETROVE (p-value = 1.19 × 10⁻⁶). Additional associations between the mCNV and thrombin generation parameters were identified. Our results reveal that increased mCNV dosages in ORM1 decreased gene expression and upregulated cfDNA levels. Therefore, the mCNV in ORM1 appears to be a novel marker for cfDNA levels, which could contribute to VTE risk. Received: 02 June 2022 Accepted: 20 November 2022 Article published online: 25 January 2023 © 2023. Thieme. All rights reserved. Georg Thieme Verlag KG Rüdigerstraße 14, 70469 Stuttgart, Germany

Attention-deficit/hyperactivity disorder (ADHD) is a severely impairing neurodevelopmental disorder with a prevalence of 5% in children and adolescents and of 2.5% in adults. Comorbid conditions in ADHD play a key role in symptom progression, disorder course and outcome. ADHD is associated with a significantly increased risk for substance use, abuse and dependence. ADHD and cannabis use are partly determined by genetic factors; the heritability of ADHD is estimated at 70–80% and of cannabis use initiation at 40–48%. In this study, we used summary statistics from the largest available meta-analyses of genome-wide association studies (GWAS) of ADHD (n = 53,293) and lifetime cannabis use (n = 32,330) to gain insights into the genetic overlap and causal relationship of these two traits. We estimated their genetic correlation to be r² = 0.29 (P = 1.63 × 10⁻⁵) and identified four new genome-wide significant loci in a cross-trait analysis: two in a single variant association analysis (rs145108385, P = 3.30 × 10⁻⁸ and rs4259397, P = 4.52 × 10⁻⁸) and two in a gene-based association analysis (WDPCP, P = 9.67 × 10⁻⁷ and ZNF251, P = 1.62 × 10⁻⁶). Using a two-sample Mendelian randomization approach we found support that ADHD is causal for lifetime cannabis use, with an odds ratio of 7.9 for cannabis use in individuals with ADHD in comparison to individuals without ADHD (95% CI (3.72, 15.51), P = 5.88 × 10⁻⁵). These results substantiate the temporal relationship between ADHD and future cannabis use and reinforce the need to consider substance misuse in the context of ADHD in clinical interventions.

Attention-deficit/hyperactivity disorder (ADHD) is one of the most prevalent neurodevelopmental disorders in childhood and persists into adulthood in 40–65% of cases. Given the polygenic and heterogeneous architecture of the disorder and the limited overlap between genetic studies, there is a growing interest in epigenetic mechanisms, such as microRNAs, that modulate gene expression and may contribute to the phenotype. We attempted to clarify the role of microRNAs in ADHD at a molecular level through the first genome-wide integrative study of microRNA and mRNA profiles in peripheral blood mononuclear cells of medication-naive individuals with ADHD and healthy controls. We identified 79 microRNAs showing aberrant expression levels in 56 ADHD cases and 69 controls, with three of them, miR-26b-5p, miR-185-5p, and miR-191-5p, being highly predictive for diagnostic status in an independent dataset of 44 ADHD cases and 46 controls. Investigation of downstream microRNA-mediated mechanisms underlying the disorder, which was focused on differentially expressed, experimentally validated target genes of the three highly predictive microRNAs, provided evidence for aberrant myo-inositol signaling in ADHD and indicated an enrichment of genes involved in neurological disease and psychological disorders. Our comprehensive study design reveals novel microRNA–mRNA expression profiles aberrant in ADHD, provides novel insights into microRNA-mediated mechanisms contributing to the disorder, and highlights promising candidate peripheral biomarkers.

In families at risk from monogenic diseases affected offspring, it is fundamental the development of a suitable Double Factor Preimplantation Genetic Testing (DF-PGT) method for both single-gene analysis and chromosome complement screening. Aneuploidy is not only a major issue in advanced-maternal-age patients and balanced translocation carriers, but also the aneuploidy rate is extremely high in patients undergoing in vitro fertilization (IVF), even in young donors. To adequate NGS technology to the DF-PGT strategy four different whole genome amplification systems (Sureplex, MALBAC, and two multiple displacement amplification systems-MDA) were tested using TruSight One panel on cell lines and blastocyst trophectoderm biopsies-TE. Embryo cytogenetic status was analyzed by Nexus software. Sureplex and MALBAC DNA products were considered not suitable for PGT diagnosis due to inconsistent and poor results on Trusight one (TSO) panel. Results obtained with both MDA based methods (GEH-MDA and RG-MDA) were appropriate for direct mutation detection by TSO NGS platform. Nevertheless, RG-MDA amplification products showed better coverage and lower ADO rates than GEH-MDA. The present work also demonstrates that the same TSO sequencing data is suitable not only for the direct mutation detection, but also for the indirect mutation detection by linkage analysis of informative SNPs. The present work also demonstrates that Nexus software is competent for the detection of CNV by using with TSO sequencing data from RG-MDA products, allowing for the whole cytogenetic characterization of the embryos. In conclusion, successfully development of an innovative and promising DF-PGT strategy using TSO-NGS technology in TE biopsies, performed in-house in a single laboratory experience, has been done in the present work. Additional studies should be performed before it could be used as a diagnostic alternative in order to validate this approach for the detection of chromosomal aneuploidies.

Background and aims: Cannabis is one of the most commonly used substances among adolescents and young adults. Earlier age at cannabis initiation is linked to adverse life outcomes, including multi‐substance use and dependence. This study estimated the heritability of age at first cannabis use and identified associations with genetic variants.Methods: A twin‐based heritability analysis using 8055 twins from three cohorts was performed. We then carried out a genome‐wide association meta‐analysis of age at first cannabis use in a discovery sample of 24 953 individuals from nine European, North American and Australian cohorts, and a replication sample of 3735 individuals.Results: The twin‐based heritability for age at first cannabis use was 38% [95% confidence interval (CI) = 19–60%]. Shared and unique environmental factors explained 39% (95% CI = 20–56%) and 22% (95% CI = 16–29%). The genome‐wide association meta‐analysis identified five single nucleotide polymorphisms (SNPs) on chromosome 16 within the calcium‐transporting ATPase gene (ATP2C2) at P < 5E‐08. All five SNPs are in high linkage disequilibrium (LD) (r² > 0.8), with the strongest association at the intronic variant rs1574587 (P = 4.09E‐09). Gene‐based tests of association identified the ATP2C2 gene on 16q24.1 (P = 1.33e‐06). Although the five SNPs and ATP2C2 did not replicate, ATP2C2 has been associated with cocaine dependence in a previous study. ATP2B2, which is a member of the same calcium signalling pathway, has been associated previously with opioid dependence. SNP‐based heritability for age at first cannabis use was non‐significant.Conclusion: Age at cannabis initiation appears to be moderately heritable in western countries, and individual differences in onset can be explained by separate but correlated genetic liabilities. The significant association between age of initiation and ATP2C2 is consistent with the role of calcium signalling mechanisms in substance use disorders.

Methylphenidate (MPH) is the most frequently used pharmacological treatment in children with attention-deficit/hyperactivity disorder (ADHD). However, a considerable interindividual variability exists in clinical outcome. Thus, we performed a genome-wide association study of MPH efficacy in 173 ADHD paediatric patients. Although no variant reached genome-wide significance, the set of genes containing single-nucleotide polymorphisms (SNPs) nominally associated with MPH response (P < 0.05) was significantly enriched for candidates previously studied in ADHD or treatment outcome. We prioritised the nominally significant SNPs by functional annotation and expression quantitative trait loci (eQTL) analysis in human brain, and we identified 33 SNPs tagging cis-eQTL in 32 different loci (referred to as eSNPs and eGenes, respectively). Pathway enrichment analyses revealed an over-representation of genes involved in nervous system development and function among the eGenes. Categories related to neurological diseases, psychological disorders and behaviour were also significantly enriched. We subsequently meta-analysed the association with clinical outcome for the 33 eSNPs across the discovery sample and an independent cohort of 189 ADHD adult patients (target sample) and we detected 15 suggestive signals. Following this comprehensive strategy, our results provide a better understanding of the molecular mechanisms implicated in MPH treatment effects and suggest promising candidates that may encourage future studies.