Evaluation Of NOTCH3 Protein And GOM In The Pathological Mechanism Of Cerebral Autosomal Dominant Arteriopathy With Subcortical Infarcts And Leukoencephalopathy (CADASIL)

Part 1 NOTCH3 gene mutationObjective:To make the gene diagnosis through analyzing the mutation on exon 2 to 23 of the NOTCH3 gene in clinical doubted cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) patients.Methods:25 index cases of clinical doubted CADASIL were detected for the mutation on exon 2 to 23 of NOTCH3 gene. The mutation-proven probands from 8 families (totally 116 members) were mutation-detecting in exons.100 normal samples of control groups were genically sequened.Results:â‘ There were 9 cases of missense mutations in the NOTCH3 gene, including R90C in exon 3 in a probands, R133C, R153C, C185Y, R182C (2 cases) and R133S in exon 4, and R544C (2 cases) in exon 11.â‘¡The mutation of R133S in exon 4 is a novel NOTCH3 gene mutation of remaining cysteine, which has not been reported at home and abroad.â‘¢It hasn't been reported in China that a sporadic patient has the mutation of R182C in exon 4.Conclusion:â‘ The mutation of R133S in exon 4 is a novel NOTCH3 gene mutation of remaining cysteine, which has not been reported at home and abroad.â‘¡It hasn't been reported in China that a sporadic patient has the mutation of R182C in exon 4. Part 2 Evaluation of diagnostic NOTCH3 protein and GOM in CADASILObjective:To probe the value of diagnose of an antibody against the amino-terminal(N) domain of the NOTCH3 receptor and the presence of specific granular osmiophilic deposits(GOM) in the reported 31 CADASIL patients who were made a definite diagnosis by genes.Methods:Applying a fragment of inhibin subunits synthetic NOTCH3 molecule(N) made by the means of KLH to resist polyclonal antibody with the amino-terminal(N) domain of the NOTCH3 receptor and by the means of purifying and as well as the specific a NOTCH3 polyclonal antibody as the result of the verifying by ELISA and Western blot. Through the contrast of the skin biopsy between the 31 CADASIL patients and 25 same-aged ones without CADASIL, observing the micro change of structure in blood vessels, evaluating the sensibility and the specificity of the immunohistochemical method with the NOTCH3-N polyclonal antibody and the deposition of ultramicrostructure GOMResults:â‘ Developing the NOTCH3-N polyclonal antibody successfully.â‘¡Demonstration of ultramicrostructure in skin blood vessels of the 31 patients:The thickening of most vessel's wall, particles of osmiophilic matter mainly depositing the surface of vascular smooth muscle cells (VSMC) in arteriole and cellvenule, occasionally locating around the skin cells in blood capillary, denaturation of smooth muscle in blood vessels and of endothelial cells, denaturation of cytoplasm with full of osmiophilic matter vesicle and mitochondria vesicle, and only one old man in the 25 contrast was identified thickening of basement membrane in blood vessels occasionally.â‘¢The sensibility of the 31 patients with ultramicrostructure GOM deposit is 54.5%, speficity 96%, whereas through the means of the NOTCH3-N polyclonal antibody, sensibility is 75.7%, speficity 98%. However if connecting the two, the sensibility can reach 91%, and speficity 97%.Conclusions:Both the sensibiliities evaluated by ultramicrostructure GOM and by the means of the NOTCH3-N polyclonal antibody being low, but the rate of diagnosis being enhanced effectively by combining the two means. Part 3 Relationship between NOTCH3 protein and GOMObjective:To discuss the relationship among GOM, the NOTCH3 protein and the degeneration of VSMC. Moreover, to illustrate the biochemical specificity and source of GOM,Methods:The two peptide fragments, amino terminus (N) and carboxy terminal(C) of the artifical composition NOTCH3, by means of KLH, were applied to respectively prepare the antibody resisting NOTCH3 N-terminal polyclonal and the antibody resisting NOTCH3 C-terminal polyclonal. What's more, by verifying, ELISA and Western blot, they were confirmed to be specific antiboies. There were 9 cases of skin biopsies from 9 different CADASIL families, and among them, the two cases were brain biopsies. Comparing the skin of the 25 patients at same age without CADASIL and the brain tissue of the two patients without CADASIL and using immune electron microscope revealed the relationship between GOM together with the amino-terminal domain of the NOTCH3 receptor and its carboxy terminal domain.Results:â‘ The the NOTCH3-C polyclonal antibody and the NOTCH3-N polyclonal antibody were prepared successfully.â‘¡GOM deposits and the specifically colloidal gold particle deposits of the NOTCH3-N polyclonal antibody exist in the surface of VSMC of the skin and the brain tissue in CADASIL patients where specifically colloidal gold particle deposits of the NOTCH3-C polyclonal antibody doesn't. These deposits of the NOTCH3-N polyclonal antibody are next to GOM, even part of them in GOM.â‘¢In control groups, there are neither GOM deposits, nor colloidal gold particle deposits of any antibody.Conclusion:The main components of GOM may be the amino-terminal(N) domain of the NOTCH3 receptor and illustrates the biological character of GOM and the relation of GOM and NOTCH3 gene. All of thses will provide a new way to studying pathological mechanism of CADASIL.