Project description:Cerebral cavernous malformations (CCM) are vascular malformations associated with abnormally dilated blood vessels and leaky capillaries that often result in hemorrhages. Despite recent advances, precise understanding of the cellular and molecular mechanism leading to the pathogenesis of CCM remains elusive. Emerging evidence indicates that small nucleolar RNAs (snoRNAs), belonging to the class of non-coding RNAs, may play a significant role as diagnostic markers in human diseases. However, there is no report till date that studied the role of snoRNAs in CCM biology. The objective of the current study was to identify snoRNAs associated with CCM pathogenesis. Using genome-wide small RNA sequencing, we identified a total of 271 snoRNAs reliably expressed in CCM. By applying additional statistical stringency, three snoRNAs (SNORD115-32, SNORD114-22 and SNORD113-3) were found to be significantly downregulated in CCM patient tissue samples (n = 3) as compared to healthy brains (n = 3). Deregulation of the selected snoRNAs was further validated by qRT-PCR. Further, cellular localization via in situ hybridization also confirmed robust reduction in the expression of SNORD115-32 and SNORD114-22 in CCM tissues as compared to the healthy controls. By applying high throughput sequencing and cellular localization analyses, we report here for the first time the genome-wide expression profile of snoRNAs in CCM tissues and a robust downregulation of candidate snoRNAs in CCM conditions. Future studies should warrant the screening in large CCM patient cohorts and will be helpful in the development of potential biomarkers and improved clinical diagnosis.
Project description:Purpose: Cerebral cavernous malformations (CCMs) are hemorrhagic neurovascular malformations that may lead to stroke, seizures and other clinical sequelae. Recent studies have shown that somatic mutations in MAP3K3 and PIK3CA also contribute to CCM pathogenesis; however, it remains unclear how these mutations contribute to sporadic versus familial cases. In our previous research, we’ve shown that co-occurring MAP3K3 and PIK3CA mutations are present within the same clonal population of cells. The overall goal of this study was to identify PIK3CA mutations in CCM-associated developmental venous anomalies (DVA). We also analyzed the plasma miRNome of patients with (1) DVA without associated CCM, as well as (2) DVA with an associated CCM) to identify circulating miRNAs that might serve as biomarkers reflecting PIK3CA activity. Methods: We collected and sequenced the plasma miRNome of 12 individuals with a sporadic CCM associated with a DVA (CCM + DVA), 6 individuals with a DVA without a CCM (DVA only), and 7 healthy controls. Results: We found that the identical PIK3CA mutation is found in endothelial cells of both the DVA and its associated CCM, but that an activating MAP3K3 mutation appears only in the CCM. The analyses miR-134-5p was downregulated in the groups of patients with only a DVA only group (when compared to healthy controls). This miRNA has been shown to target PIK3CA. In addition, miR-182-5p, was upregulated and targets MAP3K3; while let-7c-5p was downregulated and targets both PIK3CA and MAP3K3 in the group of patients with CCM and an associated DVA (when compared to DVA only). Conclusions: These results support a mechanism where DVA develop as the result of a PIK3CA mutation, creating a region of the brain vasculature that functions as a genetic primer for CCM development following acquisition of an additional somatic mutation.
Project description:Patients with familial cerebral cavernous malformation (CCM) inherit germline loss of function mutations and are susceptible to progressive development of brain lesions and neurological sequelae during their lifetime. To date, no homologous circulat- ing molecules have been identified that can reflect the presence of germ line pathogenetic CCM mutations, either in animal models or patients. We hypothesize that homologous differentially expressed (DE) plasma miRNAs can reflect the CCM germline mutation in preclinical murine models and patients. Herein, homologous DE plasma miRNAs with mechanistic putative gene targets within the transcriptome of preclinical and human CCM lesions were identified. Several of these gene targets were additionally found to be associated with CCM-enriched pathways identified using the Kyoto Encyclopedia of Genes and Genomes. DE miRNAs were also identified in familial-CCM patients who developed new brain lesions within the year following blood sample collection. The miRNome results were then validated in an independent cohort of human subjects with real-time-qPCR quantification, a technique facilitating plasma assays. Finally, a Bayesian-informed machine learning approach showed that a combination of plasma levels of miRNAs and circulating proteins improves the associa- tion with familial-CCM disease in human subjects to 95% accuracy. These findings act as an important proof of concept for the future development of translatable circulating biomarkers to be tested in preclinical studies and human trials aimed at monitoring and restoring gene function in CCM and other diseases.
Project description:Patients with familial cerebral cavernous malformation (CCM) inherit germline loss of function mutations and are susceptible to progressive development of brain lesions and neurological sequelae during their lifetime. To date, no homologous circulat- ing molecules have been identified that can reflect the presence of germ line pathogenetic CCM mutations, either in animal models or patients. We hypothesize that homologous differentially expressed (DE) plasma miRNAs can reflect the CCM germline mutation in preclinical murine models and patients. Herein, homologous DE plasma miRNAs with mechanistic putative gene targets within the transcriptome of preclinical and human CCM lesions were identified. Several of these gene targets were additionally found to be associated with CCM-enriched pathways identified using the Kyoto Encyclopedia of Genes and Genomes. DE miRNAs were also identified in familial-CCM patients who developed new brain lesions within the year following blood sample collection. The miRNome results were then validated in an independent cohort of human subjects with real-time-qPCR quantification, a technique facilitating plasma assays. Finally, a Bayesian-informed machine learning approach showed that a combination of plasma levels of miRNAs and circulating proteins improves the associa- tion with familial-CCM disease in human subjects to 95% accuracy. These findings act as an important proof of concept for the future development of translatable circulating biomarkers to be tested in preclinical studies and human trials aimed at monitoring and restoring gene function in CCM and other diseases.
Project description:Cerebral cavernous malformation (CCM) immunothrombosis is the connection between immune cells, platelet activation, coagulation cascades and astrocyte-CCM endothelium interaction, and its excessive activation may contribute to neurological disabilities in CCM disease. We characterized the spatial organization of CCM immunothrombosis observed in Pdcd10BECKObrains under normoxic conditions using the murine 10X Genomics Visium Spatial Gene Expression platform.