GGC repeat expansions in NOTCH2NLC cause uN2CpolyG cerebral amyloid angiopathy

Abstract The expansion of GGC repeats within NOTCH2NLC leads to translation of the uN2CpolyG protein, the primary pathogenic factor in neuronal intranuclear inclusion disease (NIID). The aim of this study was to explore the deposition of uN2CpolyG as an amyloid in the vessel wall, leading to uN2CpolyG cerebral amyloid angiopathy-related cerebral microbleeds (CMBs). A total of 97 patients with genetically confirmed NIID were enrolled in this study. We analysed the presence of CMBs using susceptibility-weighted imaging sequences and compared general clinical information, cerebrovascular risk factors, stroke history, antiplatelet medication use and MRI features between NIID patients with and without CMBs. We also performed haematoxylin and eosin, Perl’s, Congo red and Thioflavin S staining, ubiquitin, p62 and uN2CpolyG immunostaining on brain tissue obtained from four NIID patients. A total of 354 CMBs were detected among 41 patients with NIID, with nearly half located in the deep brain, one-third in the lobes and ∼20% in the infratentorial area. No significant differences in cerebrovascular disease risk factors or history of antiplatelet drug use were observed between patients with and without CMBs. However, patients with CMBs had suffered a higher incidence of previous ischaemic and haemorrhagic stroke events. This group also had a higher incidence of recent subcortical infarcts and a higher proportion of white matter lesions in the external capsule and temporal pole. Conversely, patients without CMBs showed higher detection of high signals at the corticomedullary junction on diffusion-weighted imaging and more pronounced brain atrophy. Haematoxylin and eosin staining showed blood vessel leakage and haemosiderin-laden macrophage clusters, and Prussian blue staining revealed iron deposition in brain tissue. CMBs occurred more frequently in small vessels lacking intranuclear inclusions, and extensive degeneration of endothelial cells and smooth muscle fibres was observed mainly in vessels lacking inclusions. Congo red-positive amyloid deposition was observed in the cerebral vessels of NIID patients, with disordered filamentous fibres appearing under an electron microscope. Additionally, the co-localization of Thioflavin S-labelled amyloid and uN2CpolyG protein in the cerebral vascular walls of NIID patients further suggested that uN2CpolyG is the main pathogenic protein in this form of amyloid angiopathy. In conclusion, we reviewed patients with GGC repeat expansion of NOTCH2NLC from a new perspective, providing initial clinical, neuroimaging and pathological evidence suggesting that uN2CpolyG might contribute to a distinct type of cerebral amyloid angiopathy.