Molecular genetic evaluation of pediatric renovascular hypertension due to renal artery stenosis and abdominal aortic coarctation in neurofibromatosis type 1.

The etiology of renal artery stenosis (RAS) and abdominal aortic coarctation (AAC) causing the midaortic syndrome (MAS), often resulting in renovascular hypertension (RVH), remains ill-defined. Neurofibromatosis type 1 (NF-1) is frequently observed in children with RVH. Consecutive pediatric patients (N = 102) presenting with RVH secondary to RAS with and without concurrent AAC were prospectively enrolled in a clinical data base, and blood, saliva and operative tissue, when available, were collected. Among the 102 children, 13 were having a concurrent clinical diagnosis of NF-1 (12.5%). Whole exome sequencing was performed for germline variant detection, and RNA-Seq analysis of NF1, MAPK pathway genes and MCP1 levels were undertaken in five NF-1 stenotic renal arteries, as well as control renal and mesenteric arteries from children with no known vasculopathy or NF-1. In 11 unrelated children with sequencing data, 11 NF1 genetic variants were identified, of which 10 had not been reported in gnomAD. Histologic analysis of NF-1 RAS specimens consistently revealed intimal thickening, disruption of the internal elastic lamina and medial thinning. Analysis of transcript expression in arterial lesions documented an approximately 5-fold reduction in NF1 expression, confirming heterozygosity, MAPK pathway activation and increased MCP1 expression. In summary, NF-1-related RVH in children is rare but often severe and progressive and, as such, important to recognize. It is associated with histologic and molecular features consistent with an aggressive adverse vascular remodeling process. Further research is necessary to define the mechanisms underlying these findings.

Lysyl oxidase interactions with transforming growth factor-ß during angiogenesis are mediated by endothelin 1.

Crosstalk between multiple components underlies the formation of mature vessels. Although the players involved in angiogenesis have been identified, mechanisms underlying the crosstalk between them are still unclear. Using the ex vivo aortic ring assay, we set out to dissect the interactions between two key angiogenic signaling pathways, vascular endothelial growth factor (VEGF) and transforming growth factor ß (TGFß), with members of the lysyl oxidase (LOX) family of matrix modifying enzymes. We find an interplay between VEGF, TGFß, and the LOXs is essential for the formation of mature vascular smooth muscle cells (vSMC)-coated vessels. RNA sequencing analysis further identified an interaction with the endothelin-1 pathway. Our work implicates endothelin-1 downstream of TGFß in vascular maturation and demonstrate the complexity of processes involved in generating vSMC-coated vessels.

The in vivo endothelial cell translatome is highly heterogeneous across vascular beds.

Endothelial cells (ECs) are highly specialized across vascular beds. However, given their interspersed anatomic distribution, comprehensive characterization of the molecular basis for this heterogeneity in vivo has been limited. By applying endothelial-specific translating ribosome affinity purification (EC-TRAP) combined with high-throughput RNA sequencing analysis, we identified pan EC-enriched genes and tissue-specific EC transcripts, which include both established markers and genes previously unappreciated for their presence in ECs. In addition, EC-TRAP limits changes in gene expression after EC isolation and in vitro expansion, as well as rapid vascular bed-specific shifts in EC gene expression profiles as a result of the enzymatic tissue dissociation required to generate single-cell suspensions for fluorescence-activated cell sorting or single-cell RNA sequencing analysis. Comparison of our EC-TRAP with published single-cell RNA sequencing data further demonstrates considerably greater sensitivity of EC-TRAP for the detection of low abundant transcripts. Application of EC-TRAP to examine the in vivo host response to lipopolysaccharide (LPS) revealed the induction of gene expression programs associated with a native defense response, with marked differences across vascular beds. Furthermore, comparative analysis of whole-tissue and TRAP-selected mRNAs identified LPS-induced differences that would not have been detected by whole-tissue analysis alone. Together, these data provide a resource for the analysis of EC-specific gene expression programs across heterogeneous vascular beds under both physiologic and pathologic conditions.

A Novel Recurrent COL5A1 Genetic Variant Is Associated With a Dysplasia-Associated Arterial Disease Exhibiting Dissections and Fibromuscular Dysplasia.

OBJECTIVE: While rare variants in the COL5A1 gene have been associated with classical Ehlers-Danlos syndrome and rarely with arterial dissections, recurrent variants in COL5A1 underlying a systemic arteriopathy have not been described. Monogenic forms of multifocal fibromuscular dysplasia (mFMD) have not been previously defined. Approach and Results: We studied 4 independent probands with the COL5A1 pathogenic variant c.1540G>A, p.(Gly514Ser) who presented with arterial aneurysms, dissections, tortuosity, and mFMD affecting multiple arteries. Arterial medial fibroplasia and smooth muscle cell disorganization were confirmed histologically. The COL5A1 c.1540G>A variant is predicted to be pathogenic in silico and absent in gnomAD. The c.1540G>A variant is on a shared 160.1 kb haplotype with 0.4% frequency in Europeans. Furthermore, exome sequencing data from a cohort of 264 individuals with mFMD were examined for COL5A1 variants. In this mFMD cohort, COL5A1 c.1540G>A and 6 additional relatively rare COL5A1 variants predicted to be deleterious in silico were identified and were associated with arterial dissections (P=0.005). CONCLUSIONS: COL5A1 c.1540G>A is the first recurring variant recognized to be associated with arterial dissections and mFMD. This variant presents with a phenotype reminiscent of vascular Ehlers-Danlos syndrome. A shared haplotype among probands supports the existence of a common founder. Relatively rare COL5A1 genetic variants predicted to be deleterious by in silico analysis were identified in ≈2.7% of mFMD cases, and as they were enriched in patients with arterial dissections, may act as disease modifiers. Molecular testing for COL5A1 should be considered in patients with a phenotype overlapping with vascular Ehlers-Danlos syndrome and mFMD.

PHACTR1 Is a Genetic Susceptibility Locus for Fibromuscular Dysplasia Supporting Its Complex Genetic Pattern of Inheritance.

Fibromuscular dysplasia (FMD) is a nonatherosclerotic vascular disease leading to stenosis, dissection and aneurysm affecting mainly the renal and cerebrovascular arteries. FMD is often an underdiagnosed cause of hypertension and stroke, has higher prevalence in females (~80%) but its pathophysiology is unclear. We analyzed ~26K common variants (MAF>0.05) generated by exome-chip arrays in 249 FMD patients and 689 controls. We replicated 13 loci (P<10-4) in 402 cases and 2,537 controls and confirmed an association between FMD and a variant in the phosphatase and actin regulator 1 gene (PHACTR1). Three additional case control cohorts including 512 cases and 669 replicated this result and overall reached the genomic level of significance (OR = 1.39, P = 7.4×10-10, 1,154 cases and 3,895 controls). The top variant, rs9349379, is intronic to PHACTR1, a risk locus for coronary artery disease, migraine, and cervical artery dissection. The analyses of geometrical parameters of carotids from ~2,500 healthy volunteers indicate higher intima media thickness (P = 1.97×10-4) and wall to lumen ratio (P = 0.002) in rs9349379-A carriers, suggesting indices of carotid hypertrophy previously described in carotids of FMD patients. Immunohistochemistry detected PHACTR1 in endothelium and smooth muscle cells of FMD and normal human carotids. The expression of PHACTR1 by genotypes in primary human fibroblasts showed higher expression in rs9349379-A carriers (N = 86, P = 0.003). Phactr1 knockdown in zebrafish resulted in dilated vessels indicating subtle impaired vascular development. We report the first susceptibility locus for FMD and provide evidence for a complex genetic pattern of inheritance and indices of shared pathophysiology between FMD and other cardiovascular and neurovascular diseases.

Clinical and biochemical profiles suggest fibromuscular dysplasia is a systemic disease with altered TGF-ß expression and connective tissue features.

Fibromuscular dysplasia (FMD) is a rare, nonatherosclerotic arterial disease for which the molecular basis is unknown. We comprehensively studied 47 subjects with FMD, including physical examination, spine magnetic resonance imaging, bone densitometry, and brain magnetic resonance angiography. Inflammatory biomarkers in plasma and transforming growth factor ß (TGF-ß) cytokines in patient-derived dermal fibroblasts were measured by ELISA. Arterial pathology other than medial fibrodysplasia with multifocal stenosis included cerebral aneurysm, found in 12.8% of subjects. Extra-arterial pathology included low bone density (P<0.001); early onset degenerative spine disease (95.7%); increased incidence of Chiari I malformation (6.4%) and dural ectasia (42.6%); and physical examination findings of a mild connective tissue dysplasia (95.7%). Screening for mutations causing known genetically mediated arteriopathies was unrevealing. We found elevated plasma TGF-ß1 (P=0.009), TGF-ß2 (P=0.004) and additional inflammatory markers, and increased TGF-ß1 (P=0.0009) and TGF-ß2 (P=0.0001) secretion in dermal fibroblast cell lines from subjects with FMD compared to age- and gender-matched controls. Detailed phenotyping of patients with FMD allowed us to demonstrate that FMD is a systemic disease with alterations in common with the spectrum of genetic syndromes that involve altered TGF-ß signaling and offers TGF-ß as a marker of FMD.

Mutations in Grxcr1 are the basis for inner ear dysfunction in the pirouette mouse.

Recessive mutations at the mouse pirouette (pi) locus result in hearing loss and vestibular dysfunction due to neuroepithelial defects in the inner ear. Using a positional cloning strategy, we have identified mutations in the gene Grxcr1 (glutaredoxin cysteine-rich 1) in five independent allelic strains of pirouette mice. We also provide sequence data of GRXCR1 from humans with profound hearing loss suggesting that pirouette is a model for studying the mechanism of nonsyndromic deafness DFNB25. Grxcr1 encodes a 290 amino acid protein that contains a region of similarity to glutaredoxin proteins and a cysteine-rich region at its C terminus. Grxcr1 is expressed in sensory epithelia of the inner ear, and its encoded protein is localized along the length of stereocilia, the actin-filament-rich mechanosensory structures at the apical surface of auditory and vestibular hair cells. The precise architecture of hair cell stereocilia is essential for normal hearing. Loss of function of Grxcr1 in homozygous pirouette mice results in abnormally thin and slightly shortened stereocilia. When overexpressed in transfected cells, GRXCR1 localizes along the length of actin-filament-rich structures at the dorsal-apical surface and induces structures with greater actin filament content and/or increased lengths in a subset of cells. Our results suggest that deafness in pirouette mutants is associated with loss of GRXCR1 function in modulating actin cytoskeletal architecture in the developing stereocilia of sensory hair cells.

Burden of Rare Genetic Variants in Spontaneous Coronary Artery Dissection With High-risk Features.

Importance: The emerging genetic basis of spontaneous coronary artery dissection (SCAD) has been defined as both partially complex and monogenic in some patients, involving variants predominantly in genes known to underlie vascular connective tissue diseases (CTDs). The effect of these genetic influences has not been defined in high-risk SCAD phenotypes, and the identification of a high-risk subgroup of individuals may help to guide clinical genetic evaluations of SCAD. Objective: To identify and quantify the burden of rare genetic variation in individuals with SCAD with high-risk clinical features. Design, Setting, and Participants: Whole-exome sequencing (WES) was performed for subsequent case-control association analyses and individual variant annotation among individuals with high-risk SCAD. Genetic variants were annotated for pathogenicity by in-silico analysis of genes previously defined by sequencing for vascular CTDs and/or SCAD, as well as genes prioritized by genome-wide association study (GWAS) and colocalization of arterial expression quantitative trait loci. Unbiased genome-wide association analysis of the WES data was performed by comparing aggregated variants in individuals with SCAD to healthy matched controls or the Genome Aggregation Database (gnomAD). This study was conducted at a tertiary care center. Individuals in the Canadian SCAD Registry genetics study with a high-risk SCAD phenotype were selected and defined as peripartum SCAD, recurrent SCAD, or SCAD in an individual with family history of arteriopathy. Main Outcomes and Measures: Burden of genetic variants defined by DNA sequencing in individuals with high-risk SCAD. Results: This study included a total of 336 participants (mean [SD] age, 53.0 [9.5] years; 301 female participants [90%]). Variants in vascular CTD genes were identified in 17.0% of individuals (16 of 94) with high-risk SCAD and were enriched (OR, 2.6; 95% CI, 1.6-4.2; P = 7.8 × 10-4) as compared with gnomAD, with leading significant signals in COL3A1 (OR, 13.4; 95% CI, 4.9-36.2; P = 2.8 × 10-4) and Loeys-Dietz syndrome genes (OR, 7.9; 95% CI, 2.9-21.2; P = 2.0 × 10-3). Variants in GWAS-prioritized genes, observed in 6.4% of individuals (6 of 94) with high-risk SCAD, were also enriched (OR, 3.6; 95% CI, 1.6-8.2; P = 7.4 × 10-3). Variants annotated as likely pathogenic or pathogenic occurred in 4 individuals, in the COL3A1, TGFBR2, and ADAMTSL4 genes. Genome-wide aggregated variant testing identified novel associations with peripartum SCAD. Conclusions and Relevance: In this genetic study, approximately 1 in 5 individuals with a high-risk SCAD phenotype harbored a rare genetic variant in genes currently implicated for SCAD. Genetic screening in this subgroup of individuals presenting with SCAD may be considered.

Fibromuscular Dysplasia and Abdominal Aortic Aneurysms Are Dimorphic Sex-Specific Diseases With Shared Complex Genetic Architecture.

BACKGROUND: The risk of arterial diseases may be elevated among family members of individuals having multifocal fibromuscular dysplasia (FMD). We sought to investigate the risk of arterial diseases in families of individuals with FMD. METHODS: Family histories for 73 probands with FMD were obtained, which included an analysis of 463 total first-degree relatives focusing on FMD and related arterial disorders. A polygenic risk score for FMD (PRSFMD) was constructed from prior genome-wide association findings of 584 FMD cases and 7139 controls and evaluated for association with an abdominal aortic aneurysm (AAA) in a cohort of 9693 AAA cases and 294 049 controls. A previously published PRSAAA was also assessed among the FMD cases and controls. RESULTS: Of all first degree relatives of probands, 9.3% were diagnosed with FMD, aneurysms, and dissections. Aneurysmal disease occurred in 60.5% of affected relatives and 5.6% of all relatives. Among 227 female first-degree relatives of probands, 4.8% (11) had FMD, representing a relative risk (RR)FMD of 1.5 ([95% CI, 0.75-2.8]; P=0.19) compared with the estimated population prevalence of 3.3%, though not of statistical significance. Of all fathers of FMD probands, 11% had AAAs resulting in a RRAAA of 2.3 ([95% CI, 1.12-4.6]; P=0.014) compared with population estimates. The PRSFMD was found to be associated with an AAA (odds ratio, 1.03 [95% CI, 1.01-1.05]; P=2.6×10-3), and the PRSAAA was found to be associated with FMD (odds ratio, 1.53 [95% CI, 1.2-1.9]; P=9.0×10-5) as well. CONCLUSIONS: FMD and AAAs seem to be sex-dimorphic manifestations of a heritable arterial disease with a partially shared complex genetic architecture. Excess risk of having an AAA according to a family history of FMD may justify screening in family members of individuals having FMD.

SARS-CoV-2 Spike Protein S1-Mediated Endothelial Injury and Pro-Inflammatory State Is Amplified by Dihydrotestosterone and Prevented by Mineralocorticoid Antagonism.

Men are disproportionately affected by the coronavirus disease-2019 (COVID-19), and face higher odds of severe illness and death compared to women. The vascular effects of androgen signaling and inflammatory cytokines in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-mediated endothelial injury are not defined. We determined the effects of SARS-CoV-2 spike protein-mediated endothelial injury under conditions of exposure to androgen dihydrotestosterone (DHT) and tumor necrosis factor-a (TNF-α) and tested potentially therapeutic effects of mineralocorticoid receptor antagonism by spironolactone. Circulating endothelial injury markers VCAM-1 and E-selectin were measured in men and women diagnosed with COVID-19. Exposure of endothelial cells (ECs) in vitro to DHT exacerbated spike protein S1-mediated endothelial injury transcripts for the cell adhesion molecules E-selectin, VCAM-1 and ICAM-1 and anti-fibrinolytic PAI-1 (p < 0.05), and increased THP-1 monocyte adhesion to ECs (p = 0.032). Spironolactone dramatically reduced DHT+S1-induced endothelial activation. TNF-α exacerbated S1-induced EC activation, which was abrogated by pretreatment with spironolactone. Analysis from patients hospitalized with COVID-19 showed concordant higher circulating VCAM-1 and E-Selectin levels in men, compared to women. A beneficial effect of the FDA-approved drug spironolactone was observed on endothelial cells in vitro, supporting a rationale for further evaluation of mineralocorticoid antagonism as an adjunct treatment in COVID-19.

Epidemiologic and Genetic Associations of Erythropoietin With Blood Pressure, Hypertension, and Coronary Artery Disease.

[Figure: see text].

Rare loss-of-function mutations of PTGIR are enriched in fibromuscular dysplasia.

AIMS: Fibromuscular dysplasia (FMD) and spontaneous coronary artery dissection (SCAD) are related, non-atherosclerotic arterial diseases mainly affecting middle-aged women. Little is known about their physiopathological mechanisms. We aimed to identify rare genetic causes to elucidate molecular mechanisms implicated in FMD and SCAD. METHODS AND RESULTS: We analysed 29 exomes that included familial and sporadic FMD. We identified one rare loss-of-function variant (LoF) (frequencygnomAD = 0.000075) shared by two FMD sisters in the prostaglandin I2 receptor gene (PTGIR), a key player in vascular remodelling. Follow-up was conducted by targeted or Sanger sequencing (1071 FMD and 363 SCAD patients) or lookups in exome (264 FMD) or genome sequences (480 SCAD), all independent and unrelated. It revealed four additional LoF allele carriers, in addition to several rare missense variants, among FMD patients, and two LoF allele carriers among SCAD patients, including one carrying a rare splicing mutation (c.768 + 1C>G). We used burden test to test for enrichment in patients compared to gnomAD controls, which detected a putative enrichment in FMD (PTRAPD = 8 × 10-4), but not a significant enrichment (PTRAPD = 0.12) in SCAD. The biological effects of variants on human prostaclycin receptor (hIP) signalling and protein expression were characterized using transient overexpression in human cells. We confirmed the LoFs (Q163X and P17RfsX6) and one missense (L67P), identified in one FMD and one SCAD patient, to severely impair hIP function in vitro. CONCLUSIONS: Our study shows that rare genetic mutations in PTGIR are enriched among FMD patients and found in SCAD patients, suggesting a role for prostacyclin signalling in non-atherosclerotic stenosis and dissection.

Genetic investigation of fibromuscular dysplasia identifies risk loci and shared genetics with common cardiovascular diseases.

Fibromuscular dysplasia (FMD) is an arteriopathy associated with hypertension, stroke and myocardial infarction, affecting mostly women. We report results from the first genome-wide association meta-analysis of six studies including 1556 FMD cases and 7100 controls. We find an estimate of SNP-based heritability compatible with FMD having a polygenic basis, and report four robustly associated loci (PHACTR1, LRP1, ATP2B1, and LIMA1). Transcriptome-wide association analysis in arteries identifies one additional locus (SLC24A3). We characterize open chromatin in arterial primary cells and find that FMD associated variants are located in arterial-specific regulatory elements. Target genes are broadly involved in mechanisms related to actin cytoskeleton and intracellular calcium homeostasis, central to vascular contraction. We find significant genetic overlap between FMD and more common cardiovascular diseases and traits including blood pressure, migraine, intracranial aneurysm, and coronary artery disease.

Chromosome 1q21.2 and additional loci influence risk of spontaneous coronary artery dissection and myocardial infarction.

Spontaneous coronary artery dissection (SCAD) is a non-atherosclerotic cause of myocardial infarction (MI), typically in young women. We undertook a genome-wide association study of SCAD (Ncases = 270/Ncontrols = 5,263) and identified and replicated an association of rs12740679 at chromosome 1q21.2 (Pdiscovery+replication = 2.19 × 10-12, OR = 1.8) influencing ADAMTSL4 expression. Meta-analysis of discovery and replication samples identified associations with P < 5 × 10-8 at chromosome 6p24.1 in PHACTR1, chromosome 12q13.3 in LRP1, and in females-only, at chromosome 21q22.11 near LINC00310. A polygenic risk score for SCAD was associated with (1) higher risk of SCAD in individuals with fibromuscular dysplasia (P = 0.021, OR = 1.82 [95% CI: 1.09-3.02]) and (2) lower risk of atherosclerotic coronary artery disease and MI in the UK Biobank (P = 1.28 × 10-17, HR = 0.91 [95% CI :0.89-0.93], for MI) and Million Veteran Program (P = 9.33 × 10-36, OR = 0.95 [95% CI: 0.94-0.96], for CAD; P = 3.35 × 10-6, OR = 0.96 [95% CI: 0.95-0.98] for MI). Here we report that SCAD-related MI and atherosclerotic MI exist at opposite ends of a genetic risk spectrum, inciting MI with disparate underlying vascular biology.

Grxcr2 is required for stereocilia morphogenesis in the cochlea.

Hearing and balance depend upon the precise morphogenesis and mechanosensory function of stereocilia, the specialized structures on the apical surface of sensory hair cells in the inner ear. Previous studies of Grxcr1 mutant mice indicated a critical role for this gene in control of stereocilia dimensions during development. In this study, we analyzed expression of the paralog Grxcr2 in the mouse and evaluated auditory and vestibular function of strains carrying targeted mutations of the gene. Peak expression of Grxcr2 occurs during early postnatal development of the inner ear and GRXCR2 is localized to stereocilia in both the cochlea and in vestibular organs. Homozygous Grxcr2 deletion mutants exhibit significant hearing loss by 3 weeks of age that is associated with developmental defects in stereocilia bundle orientation and organization. Despite these bundle defects, the mechanotransduction apparatus assembles in relatively normal fashion as determined by whole cell electrophysiological evaluation and FM1-43 uptake. Although Grxcr2 mutants do not exhibit overt vestibular dysfunction, evaluation of vestibular evoked potentials revealed subtle defects of the mutants in response to linear accelerations. In addition, reduced Grxcr2 expression in a hypomorphic mutant strain is associated with progressive hearing loss and bundle defects. The stereocilia localization of GRXCR2, together with the bundle pathologies observed in the mutants, indicate that GRXCR2 plays an intrinsic role in bundle orientation, organization, and sensory function in the inner ear during development and at maturity.

The transmembrane inner ear (tmie) gene contributes to vestibular and lateral line development and function in the zebrafish (Danio rerio).

The inner ear is a complex organ containing sensory tissue, including hair cells, the development of which is not well understood. Our long-term goal is to discover genes critical for the correct formation and function of the inner ear and its sensory tissue. A novel gene, transmembrane inner ear (Tmie), was found to cause hearing-related disorders when defective in mice and humans. A homologous tmie gene in zebrafish was cloned and its expression characterized between 24 and 51 hours post-fertilization. Embryos injected with morpholinos (MO) directed against tmie exhibited circling swimming behavior (approximately 37%), phenocopying mice with Tmie mutations; semicircular canal formation was disrupted, hair cell numbers were reduced, and maturation of electrically active lateral line neuromasts was delayed. As in the mouse, tmie appears to be required for inner ear development and function in the zebrafish and for hair cell maturation in the vestibular and lateral line systems as well.