Absence of NEFL in patient-specific neurons in early-onset Charcot-Marie-Tooth neuropathy

Objective: We used patient-specific neuronal cultures to characterize the molecular genetic mechanism of recessive nonsense mutations in neurofilament light (NEFL) underlying early-onset Charcot-Marie-Tooth (CMT) disease. Methods: Motor neurons were differentiated from induced pluripotent stem cells of a patient with early-onset CMT carrying a novel homozygous nonsense mutation in NEFL. Quantitative PCR, protein analytics, immunocytochemistry, electron microscopy, and single-cell transcriptomics were used to investigate patient and control neurons. Results: We show that the recessive nonsense mutation causes a nearly total loss of NEFL messenger RNA (mRNA), leading to the complete absence of NEFL protein in patient's cultured neurons. Yet the cultured neurons were able to differentiate and form neuronal networks and neurofilaments. Single-neuron gene expression fingerprinting pinpointed NEFL as the most downregulated gene in the patient neurons and provided data of intermediate filament transcript abundancy and dynamics in cultured neurons. Blocking of nonsense-mediated decay partially rescued the loss of NEFL mRNA. Conclusions: The strict neuronal specificity of neurofilament has hindered the mechanistic studies of recessive NEFL nonsense mutations. Here, we show that such mutation leads to the absence of NEFL, causing childhood-onset neuropathy through a loss-of-function mechanism. We propose that the neurofilament accumulation, a common feature of many neurodegenerative diseases, mimics the absence of NEFL seen in recessive CMT if aggregation prevents the proper localization of wild-type NEFL in neurons. Our results suggest that the removal of NEFL as a proposed treatment option is harmful in humans.Peer reviewe

Influenza pH1N1 virus accumulated H275Y mutation neuraminidase during propagation in MDCK cells

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Neurocan expression associates with better survival and viral positivity in Merkel cell carcinoma

Merkel cell carcinoma (MCC) is a rare cutaneous neuroendocrine carcinoma that is frequently divided into Merkel cell polyomavirus negative and positive tumors due their distinct genomic and transcriptomic profiles, and disease outcomes. Although some prognostic factors in MCC are known, tumorigenic pathways, which that explain outcome differences in MCC are not fully understood. We investigated transcriptomes of 110 tissue samples of a formalin-fixed, paraffin-embedded MCC series by RNA sequencing to identify genes showing a bimodal expression pattern and predicting outcome in cancer and that potentially could play a role in tumorigenesis. We discovered 19 genes among which IGHM, IGKC, NCAN, OTOF, and USH2A were associated also with overall survival (all p-values < 0.05). From these genes, NCAN (neurocan) expression was detected in all 144 MCC samples by immunohistochemistry. Increased NCAN expression was associated with presence of Merkel cell polyomavirus DNA (p = 0.001) and viral large T antigen expression in tumor tissue (p = 0.004) and with improved MCC-specific survival (p = 0.027) and overall survival (p = 0.034). We conclude that NCAN expression is common in MCC, and further studies are warranted to investigate its role in MCC tumorigenesis.Peer reviewe