Results

Genetic Testing

Targeted genetic testing was performed as part of routine clinical care after appropriate patient consent. DNA was isolated from EDTA blood samples and sequencing of the following genes was performed: MMUT, MMAA, MMAB, MCEE, MMADHC, ALDH6A1, and ACSF3. The diagnosis was established using Alamut Visual 2.0 and comparison with ClinVar and HGMD databases.

Cell Culture, Protein Homogenates, Western Blot Analysis, and PDH Enzyme Testing

Experiments were performed with fibroblasts from three healthy controls (Coriell Institute: GM23964, GM23976, and GM08399) and the CMAMMA patient. Patient fibroblasts were obtained after signed informed consent. Cells were grown and homogenized in RIPA buffer with protease and phosphatase inhibitors. Total protein concentration was measured by BCA assay. Protein homogenates (25 and 50 μg) were separated by SDS‐PAGE and transferred to nitrocellulose. Antibodies used: anti‐malonylated lysine (Cell Signaling Technology, 1:1000), anti‐lipoic acid (Abcam, 1:2000), anti‐ACSF3 (Invitrogen, 1:1000), anti‐OXPHOS (Abcam, 1:250), and anti‐β‐actin as loading control. HRP‐conjugated secondary antibodies were used at 1:5000. Signals were detected and quantified with a Fusion Fx Analyzer and ImageJ. PDH enzyme activity was measured using the PDH enzyme assay Kit (MAK567‐1KT, Sigma‐Aldrich) per manufacturer's instructions using 30 mg protein.

Statistical Analysis and Use of AI Tools

In designing and reporting our experiments, we followed the current recommendations for distinguishing biological from technical replication in cell‐based studies [15, 16, 17, 18]. All experiments were performed using primary fibroblasts derived from a single CMAMMA patient and the corresponding control cell line. Biological replicates (n = 4) were generated by independently culturing the cells on three separate occasions. For each biological replicate, cells were cultured independently, lysed separately, and processed independently for the respective biochemical analyses. When technical replicates were performed, the corresponding measurements were averaged and used to obtain a single value for each biological replicate. Statistical analyses were therefore conducted using the independent biological replicates as the experimental unit, in accordance with current recommendations distinguishing biological from technical replication in cell‐based experiments.

Data are presented as means ± SEM. Significance was assessed by unpaired Student's t‐tests using GraphPad Prism 6.0 (GraphPad Software, San Diego California USA); p < 0.05 was considered significant. The AIHub tool of University of Freiburg was used for the grammatical correction of the text.