Causal relationship between gut microbiota and intracranial hemorrhage: A two-sample Mendelian randomization study.

Patients with intracranial hemorrhage (ICH) usually have an imbalance in the gut microbiota (GM); however, whether this is a causal correlation remains unclear. This study used summary data from an open genome-wide association study to conduct double-sample Mendelian randomization (MR) to test the causal correlation between GM and ICH. First, we used a cutoff value of P < 10E-5 to select single nucleotide polymorphisms critical for each GM. Inverse variance weighted, weighted median, and MR-PRESSO methods were used to evaluate the strength of this causal association. Finally, functional maps and annotations from genome-wide association studies were used to determine the biological functions of the genes. MR analysis revealed that Rikenellaceae RC9 gut group was significantly positively correlated with ICH risk. For every unit increase in Rikenellaceae RC9 gut group, the relative risk of ICH increased by 34.4%(P = 4.62E-04). Rhodospirillales, Terrisporobacter, Veillonellaceae, Coprococcus 3, unknown genus, Alphaproteobacteria, and Allisonella groups were negatively correlated with the risk of ICH, while Anaerofilum, Eubacteriumbrachy group, Clostridia, Howardella, and Romboutsia were negatively correlated with the risk of ICH. Nonetheless, the specific role of single nucleotide polymorphisms gene enrichment requires further investigation. This study suggests the causal effect on ICH. The discovery of >10 GMs associated with ICH could be used to prevent and treat ICH.

Pathologic features of brain hemorrhage after radiation treatment: case series with somatic mutation analysis.

BACKGROUND: Radiation treatment for diseases of the brain can result in hemorrhagic adverse radiation effects. The underlying pathologic substrate of brain bleeding after irradiation has not been elucidated, nor potential associations with induced somatic mutations. METHODS: We retrospectively reviewed our department's pathology database over 5 years and identified 5 biopsy specimens (4 patients) for hemorrhagic lesions after brain irradiation. Tissues with active malignancy were excluded. Samples were characterized using H&E, Perl's Prussian Blue, and Masson's Trichrome; immunostaining for B-cells (anti-CD20), T-cells (anti-CD3), endothelium (anti-CD31), macrophages (anti-CD163), α-smooth muscle actin, and TUNEL. DNA analysis was done by two panels of next-generation sequencing for somatic mutations associated with known cerebrovascular anomalies. RESULTS: One lesion involved hemorrhagic expansion among multifocal microbleeds that had developed after craniospinal irradiation for distant medulloblastoma treatment. Three bleeds arose in the bed of focally irradiated arteriovenous malformations (AVM) after confirmed obliteration. A fifth specimen involved the radiation field distinct from an irradiated AVM bed. From these, 2 patterns of hemorrhagic vascular pathology were identified: encapsulated hematomas and cavernous-like malformations. All lesions included telangiectasias with dysmorphic endothelium, consistent with primordial cavernous malformations with an associated inflammatory response. DNA analysis demonstrated genetic variants in PIK3CA and/or PTEN genes but excluded mutations in CCM genes. CONCLUSIONS: Despite pathologic heterogeneity, brain bleeding after irradiation is uniformly associated with primordial cavernous-like telangiectasias and disruption of genes implicated in dysangiogenesis but not genes implicated as causative of cerebral cavernous malformations. This may implicate a novel signaling axis as an area for future study.

Postmortem diagnosis of severe factor X deficiency in a fetus with intracranial hemorrhage resulting in intrauterine death.

In patients with severe congenital factor X deficiency, spontaneous intracranial hemorrhage (ICH) is particularly frequent in early childhood. We describe a case of fetal death at 26 weeks due to massive ICH. Gene panel analysis of postmortem samples revealed homozygosity for a pathologic F10 gene variant (c.1210T>C, p.Cys404Arg), which impedes correct folding of the catalytic serine protease domain and, therefore, causes a significant reduction in FX levels. The parents, not consanguineous but of the same ethnic community, were found to be heterozygous for this variant and did not have any personal or family history of abnormal bleeding. To the best of our knowledge, this is the first reported case of severe FX deficiency resulting in ICH diagnosed through postmortem genetic analysis. It illustrates the importance of exploring the etiology of fetal or neonatal ICH, which may impact future pregnancies, and the treatment of a potential coagulopathy in the child.

Spectrum of Fetal Intraparenchymal Hemorrhage in COL4A1/A2-Related Disorders.

BACKGROUND: COL4A1/A2 variants affecting the alpha 1 and 2 chains of type IV collagen are increasingly recognized as a cause of fetal and neonatal intracranial hemorrhage, porencephaly, and schizencephaly. Fetal magnetic resonance imaging (MRI) findings in COL4A1/A2-related disorders are not well characterized. METHODS: This is a retrospective case series of fetal MRI findings in eight patients with intraparenchymal hemorrhage (IPH) and COL4A1/A2 variants, five of whom have postnatal imaging and clinical follow-up. RESULTS: IPH was multifocal and bilateral in four of eight patients. IPH involved the frontal lobes in all cases and basal ganglia in six of eight. The median maximum diameter of IPH was 16 mm (range 6 to 65 mm). All patients had ventriculomegaly, and four of eight had intraventricular hemorrhage. Prenatal IPH size correlated clinically with motor outcomes, and none had clinically symptomatic recurrent hemorrhage. CONCLUSION: COL4A1/A2 variants can present with a spectrum of IPH prenatally, including small and/or unifocal IPH, as well as multifocal and bilateral IPH, involving the frontal lobes and basal ganglia. Given the wide spectrum of IPH severity seen on fetal brain MRI, genetic testing for COL4A1/A2 variants should be considered in all cases of fetal IPH.

Bi-allelic variants in the ESAM tight-junction gene cause a neurodevelopmental disorder associated with fetal intracranial hemorrhage.

The blood-brain barrier (BBB) is an essential gatekeeper for the central nervous system and incidence of neurodevelopmental disorders (NDDs) is higher in infants with a history of intracerebral hemorrhage (ICH). We discovered a rare disease trait in thirteen individuals, including four fetuses, from eight unrelated families associated with homozygous loss-of-function variant alleles of ESAM which encodes an endothelial cell adhesion molecule. The c.115del (p.Arg39Glyfs∗33) variant, identified in six individuals from four independent families of Southeastern Anatolia, severely impaired the in vitro tubulogenic process of endothelial colony-forming cells, recapitulating previous evidence in null mice, and caused lack of ESAM expression in the capillary endothelial cells of damaged brain. Affected individuals with bi-allelic ESAM variants showed profound global developmental delay/unspecified intellectual disability, epilepsy, absent or severely delayed speech, varying degrees of spasticity, ventriculomegaly, and ICH/cerebral calcifications, the latter being also observed in the fetuses. Phenotypic traits observed in individuals with bi-allelic ESAM variants overlap very closely with other known conditions characterized by endothelial dysfunction due to mutation of genes encoding tight junction molecules. Our findings emphasize the role of brain endothelial dysfunction in NDDs and contribute to the expansion of an emerging group of diseases that we propose to rename as "tightjunctionopathies."

Intracranial Hemorrhage Rate and Lesion Burden in Patients With Familial Cerebral Cavernous Malformation.

Background Familial cerebral cavernous alformation (CCM) is an autosomal dominant disease caused by mutations in KRIT1, CCM2, or PDCD10. Cases typically present with multiple lesions, strong family history, and neurological symptoms, including seizures, headaches, or other deficits. Intracranial hemorrhage (ICH) is a severe manifestation of CCM, which can lead to death or long-term neurological deficits. Few studies have reported ICH rates and risk factors in familial CCM. We report ICH rates and assess whether CCM lesion burden, a disease severity marker, is associated with risk of symptomatic ICH during follow-up in a well-characterized cohort of familial CCM cases. Methods and Results We studied 386 patients with familial CCM with follow-up data enrolled in the Brain Vascular Malformation Consortium CCM Project. We estimated symptomatic ICH rates overall and stratified by history of ICH before enrollment. CCM lesion burden (total lesion count and large lesion size) assessed at baseline enrollment was tested for association with increased risk of subsequent ICH during follow-up using Cox regression models adjusted for history of ICH before enrollment, age, sex, and family structure and stratified on recruitment site. The symptomatic ICH rate for familial CCM cases was 2.8 per 100 patient-years (95% CI, 1.9-4.1). Those with ICH before enrollment had a follow-up ICH rate of 4.5 per 100 patient-years (95% CI, 2.6-8.1) compared with 2.0 per 100 patient-years (95% CI, 1.3-3.5) in those without (P=0.042). Total lesion count was associated with increased risk of ICH during follow-up (hazard ratio [HR], 1.37 per doubling of total lesion count [95% CI, 1.10-1.71], P=0.006). The symptomatic ICH rate for familial CCM cases was 2.8 per 100 patient-years (95% CI, 1.9-4.1). Those with ICH before enrollment had a follow-up ICH rate of 4.5 per 100 patient-years (95% CI, 2.6-8.1) compared with 2.0 per 100 patient-years (95% CI, 1.3-3.5) in those without (P=0.042). Total lesion count was associated with increased risk of ICH during follow-up (hazard ratio [HR], 1.37 per doubling of total lesion count [95% CI, 1.10-1.71], P=0.006). Conclusions Patients with familial CCM with prior history of an ICH event are at higher risk for rehemorrhage during follow-up. In addition, total CCM lesion burden is significantly associated with increased risk of subsequent symptomatic ICH; hence lesion burden may be an important predictor of patient outcome and aid patient risk stratification.

Congenital coagulation factor V deficiency with intracranial hemorrhage.

BACKGROUND: Congenital coagulation factor V (FV) deficiency is a very rare hemorrhagic disease with an incidence of approximately one in a million. The common clinical manifestations of FV deficiency include ecchymosis and mucosal bleeding. Life-threatening intracranial bleeding is rare. It has been reported in several cases. However, the molecular basis has been established in only a few cases. METHODS: We reported a 2-month-old girl with congenital FV deficiency and intracranial hemorrhage. Coagulation screening combined with clinical manifestations was performed to diagnose congenital FV deficiency. Genetic testing was performed to identify the pathogenic genes. A literature review was included to emphasize the clinical manifestation, diagnosis, and treatment for congenital FV deficiency with intracranial bleeding. RESULTS: The coagulation tests revealed a significantly prolonged prothrombin time (PT) of 51 s and an activated partial thromboplastin time (APTT) of 73.7 s. The patient had a plasma FV activity of 0.9%. Genetic testing showed compound heterozygous mutations of the patient's FV gene. A literature review showed that patients with homozygous or compound heterozygous variants of the FV gene were often associated with a severe bleeding phenotype. CONCLUSION: Our study provides a direction for the rapid and accurate diagnosis and treatment for FV deficiency to avoid life-threatening bleeding. Infants with spontaneous cranial hematoma and intracranial hemorrhage should be investigated for underlying hemostatic defects. Congenital coagulation factor deficiency should be considered. Once congenital FV deficiency is diagnosed, fresh frozen plasma (FFP) should be given on a regular basis. Liver transplantation may be performed in severe cases.

COL4A1/COL4A2 and inherited platelet disorder gene variants in fetuses showing intracranial hemorrhage.

BACKGROUND: Variants of COL4A1/COL4A2 genes have been reported in fetal intracranial hemorrhage (ICH) cases but their prevalence and characteristics have not been established in a large series of fetuses. Fetal neonatal alloimmune thrombocytopenia is a major acquired ICH factor but the prevalence and characteristics of inherited platelet disorder (IPD) gene variants leading to thrombocytopenia are unknown. Herein, we screened COL4A1/COL4A2 and IPD genes in a large series of ICH fetuses. METHODS: A cohort of 194 consecutive ICH fetuses were first screened for COL4A1/COL4A2 variants. We manually curated a list of 64 genes involved in IPD and investigated them in COL4A1/COL4A2 negative fetuses, using exome sequencing data from 101 of these fetuses. RESULT: Pathogenic variants of COL4A1/COL4A2 genes were identified in 36 fetuses (19%). They occurred de novo in 70% of the 32 fetuses for whom parental DNA was available. Pathogenic variants in two megakaryopoiesis genes (MPL and MECOM genes) were identified in two families with recurrent and severe fetal ICH, with variable extraneurological pathological features. CONCLUSION: Our study emphasizes the genetic heterogeneity of fetal ICH and the need to screen both COL4A1/COL4A2 and IPD genes in the etiological investigation of fetal ICH to allow proper genetic counseling.

Somatic mosaicism in the MAPK pathway in sporadic brain arteriovenous malformation and association with phenotype.

OBJECTIVE: Sporadic brain arteriovenous malformation (BAVM) is a tangled vascular lesion characterized by direct artery-to-vein connections that can cause life-threatening intracerebral hemorrhage (ICH). Recently, somatic mutations in KRAS have been reported in sporadic BAVM, and mutations in other mitogen-activated protein kinase (MAPK) signaling pathway genes have been identified in other vascular malformations. The objectives of this study were to systematically evaluate somatic mutations in MAPK pathway genes in patients with sporadic BAVM lesions and to evaluate the association of somatic mutations with phenotypes of sporadic BAVM severity. METHODS: The authors performed whole-exome sequencing on paired lesion and blood DNA samples from 14 patients with sporadic BAVM, and 295 genes in the MAPK signaling pathway were evaluated to identify genes with somatic mutations in multiple patients with BAVM. Digital droplet polymerase chain reaction was used to validate KRAS G12V and G12D mutations and to assay an additional 56 BAVM samples. RESULTS: The authors identified a total of 24 candidate BAVM-associated somatic variants in 11 MAPK pathway genes. The previously identified KRAS G12V and G12D mutations were the only recurrent mutations. Overall, somatic KRAS G12V was present in 14.5% of BAVM lesions and G12D was present in 31.9%. The authors did not detect a significant association between the presence or allelic burden of KRAS mutation and three BAVM phenotypes: lesion size (maximum diameter), age at diagnosis, and age at ICH. CONCLUSIONS: The authors confirmed the high prevalence of somatic KRAS mutations in sporadic BAVM lesions and identified several candidate somatic variants in other MAPK pathway genes. These somatic variants may contribute to understanding of the etiology of sporadic BAVM and the clinical characteristics of patients with this condition.

Deciphering the Irregular Risk of Stroke Increased by Obesity Classes: A Stratified Mendelian Randomization Study.

Background: To investigate the relationship between different classes of obesity and stroke, we conducted a stratified Mendelian randomization (MR) study. Methods: The body mass index (BMI) data of 263,407 Europeans with three classes of obesity (obesity class I, 30 kg/m2 ≤ BMI < 35 kg/m2; obesity class II, 35 kg/m2 ≤ BMI < 40 kg/m2; obesity class III, 40 kg/m2 ≤ BMI) were extracted from the Genetic Investigation of ANthropometric Traits (GIANT) consortium. Summary-level data of stroke and its subtypes [ischemic stroke (IS) and intracerebral hemorrhage (ICH)] were obtained from the genome-wide association study (GWAS) meta-analysis, which was performed by the MEGASTROKE consortium. MR methods were used to identify the causal relationships. Results: The MR analysis revealed that both obesity class I [odds ratio (OR) = 1.08, 95% CI: 1.05-1.12, p = 1.0 × 10-5] and obesity class II (OR = 1.06, 95% CI: 1.03-1.09, p = 1 × 10-4) were significantly positively related to IS, while obesity class III was not (OR = 1.01, 95% CI: 0.96-1.06, p = 0.65). In contrast to IS, there was no class of obesity associated with ICH risk. Further examination of the relationship between obesity classification and IS subtypes revealed that certain degrees of obesity were related to large artery stroke (LAS) (OR = 1.14, 95% CI: 1.04-1.24, p = 2.8 × 10-3 for class I; OR = 1.08, 95% CI: 1.01-1.16, p = 0.002 for class II) and cardioembolic stroke (CES) (OR = 1.11, 95% CI: 1.02-1.20, p = 0.02 for class I; OR = 1.08, 95% CI: 1.02-1.15, p = 0.007 for class II). Conclusions: A higher risk of IS, but not ICH, could be linked to obesity classes I and II. A strong association between LAS and CES and obesity was observed among all IS subtypes in the obese population.

Clinical RNA sequencing confirms compound heterozygous intronic variants in RYR1 in a patient with congenital myopathy, respiratory failure, neonatal brain hemorrhage, and d-transposition of the great arteries.

BACKGROUND: Defects in the RYR1 (OMIM#180901) gene lead to Ryanodine receptor type 1-related myopathies (RYR1-RM); the most common subgroup of congenital myopathies. METHODS: Congenital myopathy presents a diagnostic challenge due to the need for multiple testing modalities to identify the many different genetic etiologies. In this case, the patient remained undiagnosed after whole-exome sequencing (WES), chromosomal microarray, methylation analysis, targeted deletion and duplication studies, and targeted repeat expansion studies. Clinical whole-genome sequencing (WGS) was then pursued as part of a research study to identify a diagnosis. RESULTS: WGS identified compound heterozygous RYR1 intronic variants, RNA sequencing confirmed both variants to be pathogenic causing RYR1-RM in a phenotype of severe congenital hypotonia with respiratory failure from birth, neonatal brain hemorrhage, and congenital heart disease involving transposition of the great arteries. CONCLUSION: While there is an ongoing debate about the clinical superiority of WGS versus WES for patients with a suspected genetic condition, this scenario highlights a weakness of WES as well as the added cost and delay in diagnosis timing with having WGS follow WES or even ending further genetic testing with a negative WES. While knowledge gaps still exist for many intronic variants, transcriptome analysis provides a way of validating the resulting dysfunction caused by these variants and thus allowing for appropriate pathogenicity classification. This is the second published case report of a patient with pathogenic intronic variants in RYR1-RM, with clinical RNA testing confirming variant pathogenicity and therefore the diagnosis suggesting that for some patients careful analysis of a patient's genome and transcriptome are required for a complete genetic evaluation. The diagnostic odyssey experienced by this patient highlights the importance of early, rapid WGS.

Proteomic Profiling of Exosomes From Hemorrhagic Moyamoya Disease and Dysfunction of Mitochondria in Endothelial Cells.

Background and Purpose: Moyamoya disease (MMD) is a rare steno-occlusive and slowly progressing cerebrovascular disorder. The detailed mechanism of the underlying pathogenesis is still blurry. Methods: Tandem Mass Tag-labeled quantitative proteomics was performed on serum-derived exosomes (SDEs) extracted from adult patients diagnosed with pure ischemic MMD or hemorrhagic MMD and healthy controls. Then mouse brain vascular endothelial cell (EC), human umbilical vein EC, neuroblastoma cell, and human hepatocyte cell were treated with exosomes, and changes of the protein expression in mouse brain vascular EC cells were identified. Results: Proteomics analysis results showed that 859 shared proteins were detected in SDEs from ischemic and hemorrhagic MMD patients with 231 differently expressed compared with healthy controls. Bioinformatic analysis revealed dysregulated cell growth and maintenance and indicated disturbed actin dynamics in MMD, with CFL1 (Cofilin-1) and ACTR2/3 (actin-related protein 2/3; also known as ARP2/3) downregulated in ischemic and hemorrhagic patients' SDEs. We also found immunity dysfunction in hemorrhagic MMD. Following treatment with MMD SDEs, mouse brain vascular EC cells showed significantly higher levels of proliferation and more ethynyl-2-deoxyuridine-positive cells compared with the healthy control group, while there were no obvious changes in the human umbilical vein EC and human hepatocyte cell. Interestingly, we also found that SDEs from ischemic MMD promoted neuroblastoma cell proliferation. Proteomic analysis of mouse brain vascular EC cells suggested that SDEs from hemorrhagic MMD patients induced dysfunction of the mitochondria in cerebrovascular ECs. Conclusions: This study highlighted potential molecular mechanisms underlying the pathogenesis of MMD patients, thereby providing new therapeutic strategies for MMD.

Expression and methylation status of vascular endothelial growth factor and thrombospondin-1 genes in congenital factor XIII-deficient patients with intracranial hemorrhage.

Congenital factor XIII (FXIII) deficiency is one of the rarest bleeding disorders, with an incidence of one per 2 million persons. Intracranial hemorrhage (ICH), a major cause of mortality in FXIII deficiency, is reported to be associated with vascular endothelial growth factor (VEGF) and thrombospondin-1 (TSP-1). Therefore, we investigated the association of VEGF and TSP-1 expression and methylation patterns with ICH in congenital FXIII deficiency patients. This study was conducted on 40 participants with FXIII, 20 of whom experienced ICH (cases), and 20 who did not (controls). Methylation pattern, gene expression, and plasma protein level were assessed using bisulfite sequencing PCR, quantitative real-time PCR, and ELISA. We found a partially methylated pattern for both VEGF and TSP-1 (P > 0.05). VEGF mRNA levels of the case group were significantly higher than those of the control group (P < 0.05), whereas TSP-1 mRNA levels did not show significant upregulation (P > 0.05). Plasma VEGF and TSP-1 concentrations in the case group were higher, but not statistically significant (P > 0.05). Our findings showed no obvious correlation between VEGF or TSP-1 methylation patterns and expression, suggesting that their expression in FXIII deficiency may not solely be controlled by gene methylation.

MiR-18a Aggravates Intracranial Hemorrhage by Regulating RUNX1-Occludin/ZO-1 Axis to Increase BBB Permeability.

OBJECTIVES: To study the molecular mechanisms of miR-18a aggravating intracranial hemorrhage (ICH) by increasing the blood-brain barrier (BBB) permeability. METHODS: Brain microvascular endothelial cells (BMVECs) and astrocytes were isolated, identified, and co-cultured to establish in vitro BBB model. BMVECs co-cultured with astrocytes were stimulated with or without thrombase and then transfected with miR-18a mimic and/or si-RUNX1. The trans-endothelial electric resistance (TEER) and FlNa flux were measured, respectively. The potential interaction between RUNX1 and miR-18a was also detected. Additionally, SD rats were injected with fresh autologous non-anticoagulant blood into the brain basal ganglia to establish ICH model. After administration with miR-18a, sh-miR-18a, miR-18a+RUNX1, sh-miR-18a+sh-RUNX1, respectively, BBB permeability was assessed. RESULTS: After overexpressing miR-18a, the expression levels of RUNX1, Occludin and ZO-1 were decreased, but the Evan's blue contents and brain water contents were significantly increased in ICH rats. Additionally, rat neurological function was impaired, accompanying with an increase of TEER and fluorescein sodium flux. MiR-18a was a direct target of RUNX1 and it could bind to the promoters of RUNX1 to inhibit the expression of Occuldin and ZO-1. Consistently, these phenomena could also be observed in the corresponding cell model. Conversely, miR-18a knockdown or RUNX1 overexpression just presented an improvement effect on ICH. CONCLUSIONS: MiR-18a plays a critical role during ICH because it targets to RUNX1 to inhibit the expression of tight junction proteins (Occludin and ZO-1) and then disrupt BBB permeability. MiR-18a might be a probable therapeutic target for ICH diseases.

Review of treatment and therapeutic targets in brain arteriovenous malformation.

Brain arteriovenous malformations (bAVM) are an important cause of intracranial hemorrhage (ICH), especially in younger patients. The pathogenesis of bAVM are largely unknown. Current understanding of bAVM etiology is based on studying genetic syndromes, animal models, and surgically resected specimens from patients. The identification of activating somatic mutations in the Kirsten rat sarcoma viral oncogene homologue (KRAS) gene and other mitogen-activated protein kinase (MAPK) pathway genes has opened up new avenues for bAVM study, leading to a paradigm shift to search for somatic, de novo mutations in sporadic bAVMs instead of focusing on inherited genetic mutations. Through the development of new models and understanding of pathways involved in maintaining normal vascular structure and functions, promising therapeutic targets have been identified and safety and efficacy studies are underway in animal models and in patients. The goal of this paper is to provide a thorough review or current diagnostic and treatment tools, known genes and key pathways involved in bAVM pathogenesis to summarize current treatment options and potential therapeutic targets uncovered by recent discoveries.

TT genotype of the MMP-9-1562C/T polymorphism may be a risk factor for thrombolytic therapy-induced hemorrhagic complications after acute ischemic stroke.

INTRODUCTION: Levels of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) influence recombinant tissue plasminogen activator (rtPA) therapy response in patients with acute ischemic stroke (AIS). Serum levels of MMPs and TIMPs along with the expression of genes coding these proteins are related to the recovery and appearance of adverse effects (AE) after AIS. Consequently, it is important to explore whether polymorphisms in regulatory sequences of MMPs and TIMPs are associated with rtPA response in AIS patients. OBJECTIVES: To determine whether selected polymorphic variants within MMP-2, MMP-9, and TIMP-2 genes may influence rtPA therapy response with regard to outcomes in patients with AIS and the occurrence of AE. METHODS: Our study included 166 patients suffering AIS, treated with rtPA. Patients' recovery was estimated using the Modified Rankin Scale (mRS) 3 months after the AIS occurred. Favorable outcome was defined with scores 0-1 and poor outcome with scores 2-6. Genotyping was performed using real-time PCR (rs243866, rs243865, rs243864, rs2277698, and rs8179090) and PCR-RFLP (rs2285053, rs3918242) methods. Additionally, rtPA AE were followed during the hospitalization. RESULTS: There was no significant association between genotypes and alleles of selected polymorphisms and rtPA therapy response measured through the decrease of the mRS score in patients with AIS. Intracranial hemorrhage, as well as parenchymal hematoma type 2, was significantly more frequent in patients with TT genotype of the MMP-9-1562C/T polymorphism (p = 0.047, p = 0.011, respectively). Patients with intracranial hemorrhages after rtPA were significantly more likely to have the TT genotype of TIMP-2-303C/T polymorphism and the TT genotype of MMP-9-1562C/T polymorphism (p < 0.001). CONCLUSION: TT genotype of the MMP-9-1562C/T polymorphism may be a risk factor for rtPA-induced hemorrhagic complications after AIS.

Genetic Profiling of Idiopathic Antenatal Intracranial Haemorrhage: What We Know?

Intracranial hemorrhage (ICH) is reported in premature infants and rarely, in prenatal life. Fetal ICH can be accurately identified in utero and categorized by antenatal sonography and/or MRI. Infectious disease, maternal drug exposure, alloimmune thrombocytopenia, maternal trauma, coagulation disorders and twin-to-twin transfusion syndrome can cause fetal ICH. However, in many cases, the cause is not identified and a genetic disorder should be taken into consideration. We conducted a review of the literature to investigate what we know about genetic origins of fetal ICH. We conducted targeted research on the databases PubMed and EMBASE, ranging from 1980 to 2020. We found 311 studies and 290 articles were excluded because they did not meet the inclusion criteria, and finally, 21 articles were considered relevant for this review. Hemostatic, protrombotic, collagen and X-linked GATA 1 genes were reported in the literature as causes of fetal ICH. In cases of ICH classified as idiopathic, possible underlying genetic causes should be accounted for and investigated. The identification of ICH genetic causes can guide the counselling process with respect to the recurrence risk, in addition to producing relevant clinical data to the neonatologist for the optimal management and prompt treatment of the newborn.

Molecular Characterization of Two Homozygous Factor VII Variants Associated with Intracranial Bleeding.

Clinical parameters have been extensively studied in factor (F) VII deficiency, but the knowledge of molecular mechanisms of this disease is scarce. We report on three probands with intracranial bleeds at an early age, one of which had concomitant high titer of FVII inhibitor. The aim of the present study was to identify the causative mutations and to elucidate the underlying molecular mechanisms. All nine F7 exons were sequenced in the probands and the closest family members. A homozygous deletion in exon 1, leading to a frame shift and generation of a premature stop codon (p.C10Pfs*16), was found in proband 1. Probands 2 and 3 (siblings) were homozygous for a missense mutation in exon 8, resulting in a glycine (G) to arginine (R) substitution at amino acid 240 (p.G240R). All probands had severely reduced FVII activity (FVII:C < 1 IU/dL). Treatment consisted of recombinant FVIIa and/or plasma concentrate, and proband 1 developed a FVII inhibitor shortly after initiation of treatment. The FVII variants were overexpressed in mammalian cell lines. No FVII protein was produced in cells expressing the p.C10Pfs*16 variant, and the inhibitor development in proband 1 was likely linked to the complete absence of circulating FVII. Structural analysis suggested that the G to R substitution in FVII found in probands 2 and 3 would destabilize the protein structure, and cell studies demonstrated a defective intracellular transport and increased endoplasmic reticulum stress. The molecular mechanism underlying the p.G240R variant could be reduced secretion caused by protein destabilization and misfolding.

Rs41291957 polymorphism in the promoter region of microRNA­143 serves as a prognostic biomarker for patients with intracranial hemorrhage.

The present study aimed to investigate the function of the single nucleotide polymorphism (SNP) rs41291957 in the prognosis of intracerebral hemorrhage (ICH). In addition, the molecular mechanisms underlying the role of microRNA (miR)­143, Toll­like receptor 2 (TLR2) and interleukin­16 (IL­16) were studied in patients with ICH that carried different alleles in the locus of the rs41291957 SNP. Kaplan­Meier survival curves were calculated for 182 patients with ICH, genotyped as CC, presenting a cytosine in both chromosome, CT, presenting both variants, and TT, presents a thymine in both chromosomes. In addition, the possible regulatory relationships between miR­143 and TLR2/IL­16 were studied using computational analysis, luciferase assays and western blot assay. In addition, the inflammatory profiles of cerebrospinal fluid (CSF) and serum samples collected from the subjects were compared. The patients genotyped as TT presented the lowest survival rate, while patients genotyped as CC presented the highest survival rate. TLR2 mRNA was identified as a potential target of miR­143, while IL­16 showed no direct interaction with miR­143. The above regulatory relationships were further investigated using cells transfected with miR­143 precursor or TLR2 small interfering RNA. In addition, the expression levels of inflammatory factors, such as tumor necrosis factor α, interferon, IL­6, IL­10 and NF­L­6, were highest in the CSF/serum samples collected from patients genotyped as TT and lowest in patients genotyped as CC. By contrast, the expression levels of miR­143 showed an opposite trend in the expression of the above inflammatory factors. The rs41291957 SNP, located in the promoter region of miR­143, reduced the expression of miR­143 and upregulated the expression of the pro­inflammatory factor TLR2, eventually leading to a poorer prognosis in patients with ICH.

Selective Endothelial Hyperactivation of Oncogenic KRAS Induces Brain Arteriovenous Malformations in Mice.

OBJECTIVE: Brain arteriovenous malformations (bAVMs) are a leading cause of hemorrhagic stroke and neurological deficits in children and young adults, however, no pharmacological intervention is available to treat these patients. Although more than 95% of bAVMs are sporadic without family history, the pathogenesis of sporadic bAVMs is largely unknown, which may account for the lack of therapeutic options. KRAS mutations are frequently observed in cancer, and a recent unprecedented finding of these mutations in human sporadic bAVMs offers a new direction in the bAVM research. Using a novel adeno-associated virus targeting brain endothelium (AAV-BR1), the current study tested if endothelial KRASG12V mutation induces sporadic bAVMs in mice. METHODS: Five-week-old mice were systemically injected with either AAV-BR1-GFP or -KRASG12V . At 8 weeks after the AAV injection, bAVM formation and characteristics were addressed by histological and molecular analyses. The effect of MEK/ERK inhibition on KRASG12V -induced bAVMs was determined by treatment of trametinib, a US Food and Drug Administration (FDA)-approved MEK/ERK inhibitor. RESULTS: The viral-mediated KRASG12V overexpression induced bAVMs, which were composed of a tangled nidus mirroring the distinctive morphology of human bAVMs. The bAVMs were accompanied by focal angiogenesis, intracerebral hemorrhages, altered vascular constituents, neuroinflammation, and impaired sensory/cognitive/motor functions. Finally, we confirmed that bAVM growth was inhibited by trametinib treatment. INTERPRETATION: Our innovative approach using AAV-BR1 confirms that KRAS mutations promote bAVM development via the MEK/ERK pathway, and provides a novel preclinical mouse model of bAVMs which will be useful to develop a therapeutic strategy for patients with bAVM. ANN NEUROL 2021;89:926-941.