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Additional file 1: Additional Table 1. Plasma biochemical analyses in PEX13-variants carriers. Legend: VLCFAs (very long chain fatty acids): C22:0 docosanoate, C24:0 tetracosanoate, C26:0 hexacosenoate, NA not available. Additional Figure 1. Sanger sequencing representative of the p.Arg294Trp variant. Electropherogram of individual A.II-3 (A) showing p.Arg294Trp variant inherited from his father (B). Electropherogram of individual A.II-3 (C) showing p.Y192QfsTer.14 variant inherited from his mother (D). Additional Figure 2. Electroencephalography (EEG) of individual D.II-3. EEG performed at one day of life (A) showing numerous multifocal sharp waves, especially on the temporal regions bilaterally, in the context of continuous electrical activity. EEG control performed at one month of age (B) in the same individual. A slightly hypovolted theta-delta background activity with a reduction in multifocal sharp-waves are observed. Additional Figure 3. Fundoscopy examination of individual E.II-1. Note the cherry-red spot of the macula and the diffuse white dots. Additional Figure 4. Modelling. Comparison between molecular dynamics simulation snapshots at 0ns (dark) and 250ns (light) for (A) PEX13-WT:PEX14:PEX5 tetramer; (B) PEX13 Arg294Trp:PEX14:PEX5 tetramer. Configurations were obtained by PEX13 homology modelling followed by 500ns of molecular dynamics simulations followed by blind docking to PEX14:PEX5. Color code: PEX13-WT (green), PEX13-Arg294Trp (magenta), PEX14 (white), PEX5 (blue). Arg and Trp 294 are highlighted in PEX13-WT and PEX13-Arg294Trp, respectively. Additional Figure 5. Modelling. A collection of possible homodimeric conformations for (A) PEX13-WT and (B) PEX13-Arg294Trp. Representative conformations of the first eight clusters identified by blind docking. Arg294 is highlighted in PEX13-WT (green) and Trp294 is highlighted in PEX13-Arg294Trp (magenta). Additional Figure 6. Closest contact distance distribution. The closest distance between Arg294 (green) [Trp294 (mauve)] and its binding partner in PEX13:PEX13 homodimers calculated over all the generated docking poses.

NAA20 is the catalytic subunit of the NatB complex, which is responsible for N-terminal acetylation of approximately 20% of the human proteome. Recently, pathogenic biallelic variants in NAA20 were associated with a novel neurodevelopmental disorder in five individuals with limited clinical information. We report two sisters harboring compound heterozygous variant (c.100C>T (p.Gln34Ter) and c.11T>C p.(Leu4Pro)) in the NAA20 gene, identified by exome sequencing. In vitro studies showed that the missense variant p.Leu4Pro resulted in a reduction of NAA20 catalytic activity due to weak coupling with the NatB auxiliary subunit. In addition, unpublished data of the previous families were reported, outlining the core phenotype of the NAA20-related disorder mostly characterized by cognitive impairment, microcephaly, ataxia, brain malformations, dysmorphism and variable occurrence of cardiac defect and epilepsy. Remarkably, our two patients featured epilepsy onset in adolescence suggesting this may be a part of syndrome evolution. Functional studies are needed to better understand the complexity of NAA20 variants pathogenesis as well as of other genes linked to N-terminal acetylation.

Background Pathogenic variants in PEX-genes can affect peroxisome assembly and function and cause Zellweger spectrum disorders (ZSDs), characterized by variable phenotypes in terms of disease severity, age of onset and clinical presentations. So far, defects in at least 15 PEX-genes have been implicated in Mendelian diseases, but in some of the ultra-rare ZSD subtypes genotype–phenotype correlations and disease mechanisms remain elusive. Methods We report five families carrying biallelic variants in PEX13. The identified variants were initially evaluated by using a combination of computational approaches. Immunofluorescence and complementation studies on patient-derived fibroblasts were performed in two patients to investigate the cellular impact of the identified mutations. Results Three out of five families carried a recurrent p.Arg294Trp non-synonymous variant. Individuals affected with PEX13-related ZSD presented heterogeneous clinical features, including hypotonia, developmental regression, hearing/vision impairment, progressive spasticity and brain leukodystrophy. Computational predictions highlighted the involvement of the Arg294 residue in PEX13 homodimerization, and the analysis of blind docking predicted that the p.Arg294Trp variant alters the formation of dimers, impairing the stability of the PEX13/PEX14 translocation module. Studies on muscle tissues and patient-derived fibroblasts revealed biochemical alterations of mitochondrial function and identified mislocalized mitochondria and a reduced number of peroxisomes with abnormal PEX13 concentration. Conclusions This study expands the phenotypic and mutational spectrum of PEX13-related ZSDs and also highlight a variety of disease mechanisms contributing to PEX13-related clinical phenotypes, including the emerging contribution of secondary mitochondrial dysfunction to the pathophysiology of ZSDs.