Abstract
CADASIL is a small vessel disease caused by mutations in NOTCH3 that lead to an odd number of cysteines in the EGF-like repeat domain, causing protein misfolding and aggregation. The main symptoms are migraine, psychiatric disturbances, recurrent strokes and dementia, being executive function characteristically impaired. The molecular pathways altered by this receptor aggregation need to be studied further. A genome-wide transcriptome study (four cases paired with three healthy siblings) was carried out, in addition to a qRT-PCR for validation purposes (ten new cases and eight new controls). To study the expression profile by cell type of the significant mRNAs found, we performed an in situ hybridization (ISH) (nine cases and eight controls) and a research in the Single-nuclei Brain RNA-seq expression browser (SNBREB). Pathway analysis enrichment was carried out with Gene Ontology and Reactome. Neuropsychological tests were performed in five of the qRT-PCR cases. The two most significant differentially expressed mRNAs (BANP, p-value = 7.23 × 10–4 and PDCD6IP, p-value = 8.36 × 10–4) were selected for the validation study by qRT-PCR. Additionally, we selected two more mRNAs (CAMK2G, p-value = 4.52 × 10–3 and E2F4, p-value = 4.77 × 10–3) due to their association with ischemic neuronal death. E2F4 showed differential expression in the genome-wide transcriptome study and in the qRT-PCR (p = 1.23 × 10–3), and it was upregulated in CADASIL cases. Furthermore, higher E2F4 expression was associated with worse executive function (p = 2.04 × 10–2) and attention and information processing speed (IPS) (p = 8.73 × 10–2). In situ hibridization showed E2F4 expression in endothelial and vascular smooth vessel cells. In silico studies indicated that E2F4 is also expressed in brain endothelial cells. Among the most significant pathways analyzed, there was an enrichment of vascular development, cell adhesion and vesicular machinery terms and autophagy process. E2F4 is more highly expressed in the skin biopsy of CADASIL patients compared to controls, and its expression is present in endothelial cells and VSMCs. Further studies are needed to understand whether E2F4 could be useful as a biomarker, to monitor the disease or be used as a therapeutic target.
Highlights
CADASIL is a small vessel disease caused by mutations in NOTCH3 that lead to an odd number of cysteines in the EGF-like repeat domain, causing protein misfolding and aggregation
For the genome-wide transcriptome study (GWTS) study, we had four CADASIL patients corresponding to three different families, and three sibling controls without cysteine-affecting NOTCH3 missense mutation (CNMM), one per family, in the CADAGENIA registry
No statistically significant differences between cases and controls were observed in age, sex, smoking habits, hypertension, diabetes mellitus, dyslipidemia, migraine, psychiatric disease, stroke or dementia (Table 1)
Summary
CADASIL is a small vessel disease caused by mutations in NOTCH3 that lead to an odd number of cysteines in the EGF-like repeat domain, causing protein misfolding and aggregation. Abbreviations CADASIL Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy SVD Small vessel disease ECD Extracellular domain EGFr Epidermal growth factor-like repeats VTN Vitronectin qRT-PCR Real-time quantitative reverse transcription polymerase chain reaction GOMs Granular osmiophilic materials EF Executive function GWTS Genome-wide transcriptome study ISH In situ hybridization CNMM Cysteine-affecting NOTCH3 missense mutation MOCA Montreal Cognitive Assessment WMS-III Wechsler Memory Scale-III WAIS-III Wechsler Adult Intelligence Scale TMT-B Trail Making Test part B TMT-A Trail Making Test part A RQ Relative quantification RMA Robust multi-array average algorithm SD Standard deviation IPS Information processing speed αSMA Alpha smooth muscle actin ROI Region of interest GSEA Gene set enrichment analysis GO Gene Ontology BP Biological process CC Cellular component MF Molecular function TGFβ Transforming growth factor-β IH Intimal hyperplasia. The hallmark of the disease, granular osmiophilic materials (GOMs) constituted partly by Notch3 ECD15, has a sensitivity of 45–96% and a specificity of 100% for CADASIL diagnosis[16]
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