Results

Case Report

The patient is a 32‐year‐old German man, born at term to non‐consanguineous parents after an uneventful pregnancy. Early psychomotor development was largely normal, except for a speech delay during childhood that resolved with logopedic therapy. Mild short‐term memory difficulties during school age were also reported. No relevant family history of metabolic, neurological, or gastrointestinal disorders was known.

From adolescence (approximately age 15), the patient developed pronounced gastrointestinal symptoms including painful intestinal spasms, loud bowel sounds, diarrhea, and marked stress sensitivity, significantly impairing quality of life. Routine laboratory investigations by general practitioners found no organic cause, and irritable bowel syndrome was assumed. No metabolic investigations were performed. At age 20, the patient independently began supplementation with a multivitamin preparation and magnesium (250 mg/day), reporting improvement in fatigue, sleep quality, and abdominal symptoms.

At age 25, the patient independently underwent urinary testing for vitamin B12 deficiency. While serum B12 was normal, urinary MMA was markedly elevated (70.32 mg/g creatinine; reference < 2.4 mg/g creatinine), leading to referral to a specialized metabolic center. Repeated investigations consistently showed elevated MMA with normal B12 and homocysteine. In October 2021, serum MMA was 8007 nmol/L and urinary MMA 95 mg/g creatinine. MMA levels were subsequently monitored annually (Table 1).

 | MMA serum | MMA urin | MA urin | Homocystein | Coenzyme Q10
Dec 2017 |  | > 70 mg/g crea (< 2.4) |  |  | 
Jan 2018 |  |  |  | 9.6 μmol/l (< 12 μmol/l) | 
June 2021 |  | 81.1 nmol/mol Hb (< 3.7) |  |  | 
Oct 2021 | 8007 nmol/L (50–300 nmol/L) | 95 mg/g crea (< 4mg/g) |  |  | 
Sept 2022 |  | 105 mmol/mol crea (0–18) | 6 mmol/mol crea (0–28) |  | 
Sept 2023 |  | 104 mmol/mol crea (0–18) | 5 mmol/mol crea (0–28) |  | 
Jan 2024 |  |  |  |  | 0.42 mg/l (0.64–2.16 mg/l)
Sept 2024 |  | 86 mmol/mol crea (0–18) | 9 mmol/mol crea (0–28) |  | 

Molecular genetic testing at age 29 identified a homozygous pathogenic variant in ACSF3 (c.1672C>T; p.Arg558Trp), confirming CMAMMA. No additional pathogenic variants were detected. Despite repeated investigations, malonic acid was never detected in urine.

Following diagnosis, a moderately protein‐restricted, higher‐carbohydrate diet was recommended; however, this worsened gastrointestinal symptoms and anxiety. Increased protein intake led to rapid symptom improvement (Table 2). In an attempt to alleviate symptoms, the patient (height: 188 cm; body weight: 81.8 kg) systematically evaluated: (i) a low‐protein diet (60 g/day), (ii) a high‐protein diet (224 g/day), and (iii) a moderate‐protein diet (73 g/day) with glutamine supplementation (20 g/day). Dietary interventions were independently initiated by the patient and part of a controlled clinical protocol. Each dietary intervention was performed over 2 months. The greatest improvement in intestinal spasms, stress sensitivity, and anxiety occurred during the third intervention. Across all regimens, amino acid profiling showed consistently elevated plasma glutamine and histidine. No adverse events were reported by the patient.

Amino acids | Reference values | Unit | 60 g prot/day | 223 g prot/day | 73 g prot/day +20 g glutamine/day
Essential amino acids
Leucine | 43–180 | nmol/mL | 130.6 | 142.9 | 116.3
Isoleucine | 24–98 | nmol/mL | 632 | 75.3 | 57.9
Threonine | 62–170 | nmol/mL | 162 | 164.3 | 112.5
Valine | 90–285 | nmol/mL | 241.7 | 282.9 | 210.7
Lysine | 90–320 | nmol/mL | 188.1 | 186.3 | 149
Methionine | 8–26 | nmol/mL | 25.3 | 23.4 | 19.6
Phenylalanine | 30–73 | nmol/mL | 62.4 | 63 | 58.5
Triptophan | 21–48 | nmol/mL | 74.1 | 49 | 49.8
Histidine | 38–86 | nmol/mL | 89.7 | 93.5 | 74.4
Nonessential amino acids
Glycine | 135–375 | nmol/mL | 410.8 | 307.1 | 304.2
Alanine | 159–464 | nmol/mL | 435.9 | 333.5 | 331.7
Serine | 90–210 | nmol/mL | 220.7 | 170.2 | 159.3
Arginine | 5–55 | nmol/ml | 33.3 | 44.8 | 18.3
Tyrosine | 25–95 | nmol/mL | 64.2 | 59 | 58.2
Proline | 76–300 | nmol/mL | 289.9 | 288.9 | 224.8
Glutamate | 95–205 | nmol/mL | 150.5 | 147 | 180.3
Glutamine | 180–440 | nmol/mL | 675.6 | 549.3 | 641.6
Aspartate | 35–180 | nmol/mL | 146.1 | 137 | 122.4
Asparagine | 47–97 | nmol/mL | 63.4 | 175.7 | 72.6
Non‐proteinogenic amino acids
Citrulline | 11–38 | nmol/mL | 35.1 | 48.4 | 43.6
Taurine | 115–275 | nmol/mL | 131.6 | 266.8 | 111.8
Ornithine | 50–160 | nmol/mL | 163.1 | 45.3 | 120.3

The patient reports that gastrointestinal symptoms profoundly affected his quality of life from adolescence through his 20s (Figure S1B). Self‐initiated multivitamin and magnesium supplementation at age 20 provided partial relief. Following diagnosis, he found that protein restriction worsened symptoms, while higher protein intake combined with glutamine (20 g/day), riboflavin (200–400 mg/day), acetyl‐l‐carnitine, and magnesium substantially improved abdominal pain, anxiety, and overall well‐being. He highlights riboflavin as particularly effective for reducing stress sensitivity. The patient values the diagnosis for enabling targeted symptom management but expresses concern about the lack of disease‐specific treatment guidelines and long‐term prognosis.

Deficiency of ACSF3 Protein Severely Affects Lipoylation and Pyruvate Oxidation in Patient Fibroblasts

Global malonylation levels were analyzed in fibroblast lysates using a pan‐antibody recognizing malonylated lysine residues. Although a quantification of the malonylation degree was not performed, results seemed to show a selective increase in specific protein subsets (Figure S1C), suggesting redistribution rather than uniform upregulation. ACSF3 protein expression was only moderately reduced (45.1% ± 6.73%; p = 0.082) (Figure 1B).

FIGURE 1: Effects of ASCF3 deficiency on mitochondrial proteins and pyruvate oxidation. (A) ACSF3 protein and lipoylation degree measured on the DLAT and DLST subunits of the PDC and αKGDH, respectively. (B) Quantification of the protein expression. (C) Pyruvate dehydrogenase enzyme activity. (D) OXPHOS; CI: Complex I—NDUFB8; CII: Complex II—SDHB; CIII: Complex III—MTCO1; CIV: Complex IV—UQCRC2; CV: Complex V—ATP5A. Western blots are representative of n = 3 biological replicates for healthy control and one patient's cell line. The values are mean ± SEM of four independent experiments (four replicates). Values denoted by * and *** were considered significant if p < 0.05 or p < 0.0005, respectively (Student's t‐test).

OXPHOS complex analysis revealed a pronounced reduction of Complexes I, II, III, and IV, with complex V unaffected (Figure 1D). Lipoylation of PDC and αKGDH subunits was severely reduced, to 24.97% ± 3.25% (p = 0.000361) and 1.15% ± 0.12% (p = 0.000416), respectively (Figure 1A,B). Consistent with reduced lipoylation, PDH activity was significantly lower in fibroblasts of this patient (12.1 ± 2.05 mU/mg) compared to controls (23.3 ± 1.32 mU/mg; p = 0.0246) (Figure 1C). These cellular findings are particularly noteworthy in light of recent evolutionary evidence demonstrating that elevated ACSF3 expression, driven by the human‐specific variant rs34590044‐A, enhances mitochondrial respiratory activity and reduces MMA accumulation in liver cells [14]. Our data in this patient fibroblasts represent, in essence, the pathological mirror image of that evolutionary gain‐of‐function: where the ancestral ACSF3 variant supported metabolic homeostasis across human evolution, loss‐of‐function mutations collapse the same mitochondrial machinery, leading to respiratory chain deficiency and impaired energy substrate oxidation [9].