Fig. 1

(A) Schematic representation of human 4-hydroxyphenylpyruvate dioxygenase-like protein (HPDL) with the mitochondrial targeting sequence (MTS), the two vicinal oxygen chelate (VOC) domains, and the three Fe binding sites. All the HPDL variants described in the literature are reported. The variants identified in our study are shown in bold black type, and the novel ones in bold red type. (B) Immunoblot analysis of HPDL in skin fibroblasts from patients and control (CTRL) individuals. Determination of β-tub content was used as a normalization control. The variants identified in each patient were the following: Gly50Asp/Gly50Asp in F-A-ID1, F-A-ID2, F-E-ID6, F-M-ID12; Phe31Leu/Gly278Ser in F-B-ID3; Ser49Arg/Ser49Arg in F-C-ID4; Gly50Asp/Ser173Cys in F-D-ID5; Trp358Gly/Trp358Gly in F-G-ID7, F-G-ID8, F-G-ID9; Ala86ArgfsTer45/Ile266Thr in F-H-ID10; Leu234GlyfsTer94/Ser172Asn in F-L-ID11. (C) Oxygen consumption rate (OCR) measured using a Seahorse XF analyzer. HPDL patients presented altered OCR with reduced maximal respiration and reduced spare respiratory capacity (SRC). (D) Increased reactive oxygen species (ROS) production in HPDL patients' skin fibroblasts versus controls after TBHP (tert-butyl hydroperoxide, an inducer of ROS production in cells) administration (RM = regular medium). (E) (left) Plasma glial fibrillary acidic protein (GFAP) levels in HPDL patients and controls. Boxplots within violin plots show median (thick horizontal line), interquartile range (thin horizontal lines), and whiskers extending to 1.5 × IQR. Statistical analyses demonstrated significantly higher GFAP levels in HPDL patients compared with age-matched controls. Correlation plots between GFAP levels and (center) the localization variants within the putative domains of HPDL protein, (right) patients' phenotypes (mild/intermediate/severe). (F) Dysregulated metabolites identified in patients' serum through variable importance in projection (VIP) score analysis. Pink bars represent metabolites found to be decreased in HPDL patients, while green bars represent metabolites found to be increased. In all graphs, asterisks denote statistically significant differences: *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ***P < 0.001, whereas “ns” indicates non-significant results.