Genetic Risk Factors for Neurological Disorders in Children with Adverse Events Following Immunization: A Descriptive Study of a Polish Case Series

Studies conducted on large populations show a lack of connection between vaccination and serious neurological symptoms. However, there are isolated cases that indicate such a relationship. These reports on adverse effects following immunization (AEFI) reduce social confidence in vaccination; however, their background may be rare genetic defects. The aim of the presented study was to examine if neurological AEFI in children may be associated with variants in genes related to neurodevelopment. To identify such possible associations, a descriptive study of the Polish case series was conducted. We performed next-generation sequencing in patients who, up to 4 weeks of injection of any vaccine, manifested neurological AEFI. We included 23 previously normally developing children with first seizures that occurred after vaccination. We identified pathogenic/likely pathogenic variants in genes engaged in neurodevelopment in nine patients and variants of uncertain significance in another nine patients. The mutated genes belonged to the group of genes related to epilepsy syndromes/epileptic encephalopathy. We showed that AEFI might have a genetic background. We hypothesized that in some AEFI patients, the vaccine might only trigger neurological symptoms that would have been manifested anyway as a result of a pathogenic variant in a gene engaged in neurodevelopment

The PINK1 p.I368N mutation affects protein stability and ubiquitin kinase activity

Abstract Background Mutations in PINK1 and PARKIN are the most common causes of recessive early-onset Parkinson’s disease (EOPD). Together, the mitochondrial ubiquitin (Ub) kinase PINK1 and the cytosolic E3 Ub ligase PARKIN direct a complex regulated, sequential mitochondrial quality control. Thereby, damaged mitochondria are identified and targeted to degradation in order to prevent their accumulation and eventually cell death. Homozygous or compound heterozygous loss of either gene function disrupts this protective pathway, though at different steps and by distinct mechanisms. While structure and function of PARKIN variants have been well studied, PINK1 mutations remain poorly characterized, in particular under endogenous conditions. A better understanding of the exact molecular pathogenic mechanisms underlying the pathogenicity is crucial for rational drug design in the future. Methods Here, we characterized the pathogenicity of the PINK1 p.I368N mutation on the clinical and genetic as well as on the structural and functional level in patients’ fibroblasts and in cell-based, biochemical assays. Results Under endogenous conditions, PINK1 p.I368N is expressed, imported, and N-terminally processed in healthy mitochondria similar to PINK1 wild type (WT). Upon mitochondrial damage, however, full-length PINK1 p.I368N is not sufficiently stabilized on the outer mitochondrial membrane (OMM) resulting in loss of mitochondrial quality control. We found that binding of PINK1 p.I368N to the co-chaperone complex HSP90/CDC37 is reduced and stress-induced interaction with TOM40 of the mitochondrial protein import machinery is abolished. Analysis of a structural PINK1 p.I368N model additionally suggested impairments of Ub kinase activity as the ATP-binding pocket was found deformed and the substrate Ub was slightly misaligned within the active site of the kinase. Functional assays confirmed the lack of Ub kinase activity. Conclusions Here we demonstrated that mutant PINK1 p.I368N can not be stabilized on the OMM upon mitochondrial stress and due to conformational changes in the active site does not exert kinase activity towards Ub. In patients’ fibroblasts, biochemical assays and by structural analyses, we unraveled two pathomechanisms that lead to loss of function upon mutation of p.I368N and highlight potential strategies for future drug development

Additional file 4: Table S2. of The PINK1 p.I368N mutation affects protein stability and ubiquitin kinase activity

FoldX measurement for mutational energy effect on stability of binding pocket. Using the FoldX algorithm, which compares the WT structure with the mutant, calculations on WT and mutant at t = 0 and after 100 ns were performed. While WT is nearly zero over that time interval, the N368 mutant increases energy (∆∆G) by 1.5–2 kcal/mol*Å2. This modest increase supports that the mutation distorts the local region via an increase of Gibb’s free energy. (DOCX 16 kb

Additional file 5: Figure S1. of The PINK1 p.I368N mutation affects protein stability and ubiquitin kinase activity

Reduced full-length PINK1 levels in p.I368N mutant fibroblasts upon treatment with valinomycin. (A) Representative confocal IF images of control and two PINK1 p.I368N fibroblasts showing mitochondrial localization but greatly reduced levels of the mutant protein. Cells were left untreated (-) or treated with 10 μM CCCP for 6 days (+) and stained with antibodies against PINK1 (green) and TOM20 (mitochondria, red). Nuclei were counterstained with Hoechst (blue). Scale bars represent 10 μm. (B) Subcellular fractionation of WT control and PINK1 p.I368N fibroblasts treated with or without 1 μM valinomycin for 24 h. Despite different protein levels, both WT and p.I368N mutant full-length PINK1 localized to the mitochondrial fraction. A shift of PARKIN into higher molecular weights species indicative of PINK1-dependent activation was not observed in lysates from PINK1 p.I368N mutant cells. Purity of the mitochondrial and cytosolic fractions was determined using antibodies recognizing TOM20 and p38 MAPK, respectively. PNS denotes post-nuclear supernatant. (C) Control and two PINK1 p.I368N fibroblasts were treated with 1 μM valinomycin for 0, 2, 4 or 8 h and total lysates were analyzed by WB with indicated antibodies. GAPDH served as a loading control. Similar to CCCP treatment (Fig. 3b), rapid stabilization of full-length PINK1 along with an increase of p-Ser65-Ub levels was observed in control cells, but not in PINK1 p.I368N mutant fibroblasts. (D) Representative Images obtained with a 20x magnification on the BD pathway 855 system. Control fibroblasts and PINK1 I368N cells were seeded in 96-well imaging plates and treated with valinomycin as indicated. Cells were fixed and stained with p-S65-Ub antibodies (green) and Hoechst (blue). Scale bars indicate 10 μm. (TIF 13839 kb