Identification of additional risk loci for stroke and small vessel disease: a meta-analysis of genome-wide association studies

BACKGROUND: Genetic determinants of stroke, the leading neurological cause of death and disability, are poorly understood and have seldom been explored in the general population. Our aim was to identify additional loci for stroke by doing a meta-analysis of genome-wide association studies. METHODS: For the discovery sample, we did a genome-wide analysis of common genetic variants associated with incident stroke risk in 18 population-based cohorts comprising 84 961 participants, of whom 4348 had stroke. Stroke diagnosis was ascertained and validated by the study investigators. Mean age at stroke ranged from 45·8 years to 76·4 years, and data collection in the studies took place between 1948 and 2013. We did validation analyses for variants yielding a significant association (at p<5 × 10(-6)) with all-stroke, ischaemic stroke, cardioembolic ischaemic stroke, or non-cardioembolic ischaemic stroke in the largest available cross-sectional studies (70 804 participants, of whom 19 816 had stroke). Summary-level results of discovery and follow-up stages were combined using inverse-variance weighted fixed-effects meta-analysis, and in-silico lookups were done in stroke subtypes. For genome-wide significant findings (at p<5 × 10(-8)), we explored associations with additional cerebrovascular phenotypes and did functional experiments using conditional (inducible) deletion of the probable causal gene in mice. We also studied the expression of orthologs of this probable causal gene and its effects on cerebral vasculature in zebrafish mutants. FINDINGS: We replicated seven of eight known loci associated with risk for ischaemic stroke, and identified a novel locus at chromosome 6p25 (rs12204590, near FOXF2) associated with risk of all-stroke (odds ratio [OR] 1·08, 95% CI 1·05-1·12, p=1·48 × 10(-8); minor allele frequency 21%). The rs12204590 stroke risk allele was also associated with increased MRI-defined burden of white matter hyperintensity-a marker of cerebral small vessel disease-in stroke-free adults (n=21 079; p=0·0025). Consistently, young patients (aged 2-32 years) with segmental deletions of FOXF2 showed an extensive burden of white matter hyperintensity. Deletion of Foxf2 in adult mice resulted in cerebral infarction, reactive gliosis, and microhaemorrhage. The orthologs of FOXF2 in zebrafish (foxf2b and foxf2a) are expressed in brain pericytes and mutant foxf2b(-/-) cerebral vessels show decreased smooth muscle cell and pericyte coverage. INTERPRETATION: We identified common variants near FOXF2 that are associated with increased stroke susceptibility. Epidemiological and experimental data suggest that FOXF2 mediates this association, potentially via differentiation defects of cerebral vascular mural cells. Further expression studies in appropriate human tissues, and further functional experiments with long follow-up periods are needed to fully understand the underlying mechanisms

The Foxf2 gene in development and disease

The Foxf2 gene in development and disease Azadeh Reyahi Department of Chemistry and Molecular Biology, University of Gothenburg, Box 462, SE 405 30, Göteborg, Sweden Abstract In this thesis I present our recent data on the involvement and the mechanism of action of the forkhead transcription factor Foxf2 in development of the brain microvasculature, formation of the blood-brain barrier, control of the intestinal stem cell niche, and fusion of the secondary palate. The potential clinical signifi- cance of these findings is strengthened by a correlation between Foxf2 expres- sion and intestinal adenoma formation, and by association between genetic vari- ants in human FOXF2 and incident stroke. We showed that Foxf2 is expressed in brain pericytes, but not in mural cells of other organs. Foxf2 null mutants have a defective brain vasculature and suffer from intracranial hemorrhage and a leaky blood-brain barrier with increased endothelial vesicular trans-cytosis. Foxf2-/- brain pericytes have diminished Pdg- frβ expression, and the cerebral vasculature a reduced activity of the Tgfβ –Alk5– Smad2/3 signaling pathway, associated with decreased expression of integrins, Tgfb2, Tgfbr2, Alk5 and other pathway components. In a large GWAS performed by an international consortium, we identified a genome-wide significant association of common variants near FOXF2 with risk of stroke. Conditional knockout mice, in which Foxf2 was deleted in healthy adults, developed clinical signs of stroke and exhibited cerebral ischemia, reactive glio- sis and microhemorrhage. The animal model results thus corroborate the human genetic association and identifies FOXF2 as a novel risk locus for stroke. In the intestine we showed that Foxf2 is expressed by subepithelial fibroblasts and restricts the size of the stem cell niche, and thereby the number and prolif- eration of Lgr5+ stem cells. Foxf2 is a target of epithelial hedegehog signaling and inhibits the Wnt pathway by increasing the expression of the extracellular Wnt inhibitor Sfrp1. As a consequence, reduced Foxf2 expression significantly in- creases both initiation and growth of intestinal tumors. Reduced proliferation and decreased extracellular matrix production in the neural crest-derived mesenchyme of the palatal shelves was found to be respon- sible for the cleft palate phenotype in Foxf2 null mutants. Mechanistically, the defect is associated with reduced canonical Tgfβ signaling and integrin expres- sion. The Tgfb2 mRNA level was not affected, but the amount of Tgfβ2 protein was significantly decreased in mutant palatal shelf mesenchyme

Lipid Nanoparticles Deliver the Therapeutic VEGFA mRNA In Vitro and In Vivo and Transform Extracellular Vesicles for Their Functional Extensions

Lipid nanoparticles (LNPs) are currently used to transport functional mRNAs, such as COVID-19 mRNA vaccines. The delivery of angiogenic molecules, such as therapeutic VEGF-A mRNA, to ischemic tissues for producing new blood vessels is an emerging strategy for the treatment of cardiovascular diseases. Here, the authors deliver VEGF-A mRNA via LNPs and study stoichiometric quantification of their uptake kinetics and how the transport of exogenous LNP-mRNAs between cells is functionally extended by cells’ own vehicles called extracellular vesicles (EVs). The results show that cellular uptake of LNPs and their mRNA molecules occurs quickly, and that the translation of exogenously delivered mRNA begins immediately. Following the VEGF-A mRNA delivery to cells via LNPs, a fraction of internalized VEGF-A mRNA is secreted via EVs. The overexpressed VEGF-A mRNA is detected in EVs secreted from three different cell types. Additionally, RNA-Seq analysis reveals that as cells’ response to LNP-VEGF-A mRNA treatment, several overexpressed proangiogenic transcripts are packaged into EVs. EVs are further deployed to deliver VEGF-A mRNA in vitro and in vivo. Upon equal amount of VEGF-A mRNA delivery via three EV types or LNPs in vitro, EVs from cardiac progenitor cells are the most efficient in promoting angiogenesis per amount of VEGF-A protein produced. Intravenous administration of luciferase mRNA shows that EVs could distribute translatable mRNA to different organs with the highest amounts of luciferase detected in the liver. Direct injections of VEGF-A mRNA (via EVs or LNPs) into mice heart result in locally produced VEGF-A protein without spillover to liver and circulation. In addition, EVs from cardiac progenitor cells cause minimal production of inflammatory cytokines in cardiac tissue compared with all other treatment types. Collectively, the data demonstrate that LNPs transform EVs as functional extensions to distribute therapeutic mRNA between cells, where EVs deliver this mRNA differently than LNPs. CC BY 4.0© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.E-mail: [email protected] authors acknowledge the support from the National Genomics Infrastructure in Stockholm funded by Science for Life (SciLife) Laboratory, the Knut and Alice Wallenberg Foundation and the Swedish Research Council, and SNIC/Uppsala Multidisciplinary Center for Advanced Computational Science for assistance with massively parallel sequencing and access to the UPPMAX computational infrastructure. Moreover, the authors acknowledge Mr. Mario Soriano Navarro at the Responsable Servicio Microscopía Electrónica, Valencia Spain, for technical assistance. This work was supported by grants from the Swedish Foundation of Strategic Research (Stiftelsen för strategisk forskning: SSF) in the Industrial Research Centre, FoRmulaEx - Nucleotide Functional Drug Delivery (Grant ID: IRC15-0065), the Swedish research council (VR, Grant ID: 2020-01316), and the Swedish governmental agency for innovation systems (VINNOVA, Grant ID: 2017-02960). This research was also funded by the Systems Biology Research Centre at the University of Skövde under grants from the Knowledge Foundation (Grant ID: 20160330).</p

Identification of additional risk loci for stroke and small vessel disease: a meta-analysis of genome-wide association studies

Genetic determinants of stroke, the leading neurological cause of death and disability, are poorly understood and have seldom been explored in the general population. Our aim was to identify additional loci for stroke by doing a meta-analysis of genome-wide association studies.For the discovery sample, we did a genome-wide analysis of common genetic variants associated with incident stroke risk in 18 population-based cohorts comprising 84 961 participants, of whom 4348 had stroke. Stroke diagnosis was ascertained and validated by the study investigators. Mean age at stroke ranged from 45·8 years to 76·4 years, and data collection in the studies took place between 1948 and 2013. We did validation analyses for variants yielding a significant association (at p<5 × 10(-6)) with all-stroke, ischaemic stroke, cardioembolic ischaemic stroke, or non-cardioembolic ischaemic stroke in the largest available cross-sectional studies (70 804 participants, of whom 19 816 had stroke). Summary-level results of discovery and follow-up stages were combined using inverse-variance weighted fixed-effects meta-analysis, and in-silico lookups were done in stroke subtypes. For genome-wide significant findings (at p<5 × 10(-8)), we explored associations with additional cerebrovascular phenotypes and did functional experiments using conditional (inducible) deletion of the probable causal gene in mice. We also studied the expression of orthologs of this probable causal gene and its effects on cerebral vasculature in zebrafish mutants.We replicated seven of eight known loci associated with risk for ischaemic stroke, and identified a novel locus at chromosome 6p25 (rs12204590, near FOXF2) associated with risk of all-stroke (odds ratio [OR] 1·08, 95% CI 1·05-1·12, p=1·48 × 10(-8); minor allele frequency 21%). The rs12204590 stroke risk allele was also associated with increased MRI-defined burden of white matter hyperintensity-a marker of cerebral small vessel disease-in stroke-free adults (n=21 079; p=0·0025). Consistently, young patients (aged 2-32 years) with segmental deletions of FOXF2 showed an extensive burden of white matter hyperintensity. Deletion of Foxf2 in adult mice resulted in cerebral infarction, reactive gliosis, and microhaemorrhage. The orthologs of FOXF2 in zebrafish (foxf2b and foxf2a) are expressed in brain pericytes and mutant foxf2b(-/-) cerebral vessels show decreased smooth muscle cell and pericyte coverage.We identified common variants near FOXF2 that are associated with increased stroke susceptibility. Epidemiological and experimental data suggest that FOXF2 mediates this association, potentially via differentiation defects of cerebral vascular mural cells. Further expression studies in appropriate human tissues, and further functional experiments with long follow-up periods are needed to fully understand the underlying mechanisms.NIH, NINDS, NHMRC, CIHR, European national research institutions, Fondation Leducq

FOXF2 is required for cochlear development in humans and mice

Molecular mechanisms governing the development of the human cochlea remain largely unknown. Through genome sequencing, we identified a homozygous FOXF2 variant c.325A>T (p.I109F) in a child with profound sensorineural hearing loss (SNHL) associated with incomplete partition type I anomaly of the cochlea. This variant is not found in public databases or in over 1000 ethnicity-matched control individuals. I109 is a highly conserved residue in the forkhead box (Fox) domain of FOXF2, a member of the Fox protein family of transcription factors that regulate the expression of genes involved in embryogenic development as well as adult life. Our in vitro studies show that the half-life of mutant FOXF2 is reduced compared to that of wild type. Foxf2 is expressed in the cochlea of developing and adult mice. The mouse knockout of Foxf2 shows shortened and malformed cochleae, in addition to altered shape of hair cells with innervation and planar cell polarity defects. Expressions of Eya1 and Pax3, genes essential for cochlear development, are reduced in the cochleae of Foxf2 knockout mice. We conclude that FOXF2 plays a major role in cochlear development and its dysfunction leads to SNHL and developmental anomalies of the cochlea in humans and mice

Abnormal Hair Development and Apparent Follicular Transformation to Mammary Gland in the Absence of Hedgehog Signaling

We show that removing the Shh signal tranducer Smoothened from skin epithelium secondarily results in excess Shh levels in the mesenchyme. Moreover, the phenotypes we observe reflect decreased epithelial Shh signaling, yet increased mesenchymal Shh signaling. For example, the latter contributes to exuberant hair follicle (HF) induction, while the former depletes the resulting follicular stem cell niches. This disruption of the niche apparently also allows the remaining stem cells to initiate hair formation at inappropriate times. Thus, the temporal structure of the hair cycle may depend on the physical structure of the niche. Finally, we find that the ablation of epithelial Shh signaling results in unexpected transformations: the follicular outer root sheath takes on an epidermal character, and certain HFs disappear altogether, having adopted a strikingly mammary gland-like fate. Overall, our study uncovers a multifaceted function for Shh in sculpting and maintaining the integrity and identity of the developing HF

Identification of additional risk loci for stroke and small vessel disease : a meta-analysis of genome-wide association studies

Background Genetic determinants of stroke, the leading neurological cause of death and disability, are poorly understood and have seldom been explored in the general population. Our aim was to identify additional loci for stroke by doing a meta-analysis of genome-wide association studies. Methods For the discovery sample, we did a genome-wide analysis of common genetic variants associated with incident stroke risk in 18 population-based cohorts comprising 84 961 participants, of whom 4348 had stroke. Stroke diagnosis was ascertained and validated by the study investigators. Mean age at stroke ranged from 45.8 years to 76.4 years, and data collection in the studies took place between 1948 and 2013. We did validation analyses for variants yielding a significant association (at p<5 x10(-6)) with all-stroke, ischaemic stroke, cardioembolic ischaemic stroke, or non-cardioembolic ischaemic stroke in the largest available cross-sectional studies (70 804 participants, of whom 19 816 had stroke). Summary-level results of discovery and follow-up stages were combined using inverse-variance weighted fixed effects meta-analysis, and in-silico lookups were done in stroke subtypes. For genome-wide significant findings (at p<5 x10(-8)), we explored associations with additional cerebrovascular phenotypes and did functional experiments using conditional (inducible) deletion of the probable causal gene in mice. We also studied the expression of orthologs of this probable causal gene and its effects on cerebral vasculature in zebrafish mutants. Findings We replicated seven of eight known loci associated with risk for ischaemic stroke, and identified a novel locus at chromosome 6p25 (rs12204590, near FOXF2) associated with risk of all-stroke (odds ratio [OR] 1.08, 95% CI 1.05-1-12, p=1.48 x10(-8); minor allele frequency 21%). The rs12204590 stroke risk allele was also associated with increased MRI-defined burden of white matter hyperintensity a marker of cerebral small vessel disease in stroke-free adults (n=21079; p=0.0025). Consistently, young patients (aged 2-32 years) with segmental deletions of FOXF2 showed an extensive burden of white matter hyperintensity. Deletion of Foxf2 in adult mice resulted in cerebral infarction, reactive gliosis, and microhaemorrhage. The orthologs of FOXF2 in zebrafish (fox2b and foxf2a) are expressed in brain pericytes and mutant foxf2b(-/-) cerebral vessels show decreased smooth muscle cell and pericyte coverage. Interpretation We identified common variants near FOXF2 that are associated with increased stroke susceptibility. Epidemiological and experimental data suggest that FOXF2 mediates this association, potentially via differentiation defects of cerebral vascular mural cells. Further expression studies in appropriate human tissues, and further functional experiments with long follow-up periods are needed to fully understand the underlying mechanisms