Treatment of ARS deficiencies with specific amino acids

Abstract

Purpose: Recessive cytosolic aminoacyl-tRNA synthetase (ARS) deficiencies are severe multiorgan diseases, with limited treatment options. By loading transfer RNAs (tRNAs) with their cognate amino acids, ARS are essential for protein translation. However, it remains unknown why ARS deficiencies lead to specific symptoms, especially early life and during infections. We set out to increase pathophysiological insight and improve therapeutic possibilities.

Methods: In fibroblasts from patients with isoleucyl-RS (IARS), leucyl-RS (LARS), phenylalanyl-RS-beta-subunit (FARSB), and seryl-RS (SARS) deficiencies, we investigated aminoacylation activity, thermostability, and sensitivity to ARS-specific amino acid concentrations, and developed personalized treatments.

Results: Aminoacylation activity was reduced in all patients, and further diminished at 38.5/40 °C (P^LARS and P^FARSB), consistent with infectious deteriorations. With lower cognate amino acid concentrations, patient fibroblast growth was severely affected. To prevent local and/or temporal deficiencies, we treated patients with corresponding amino acids (follow-up: 1/2-2 2/3rd years), and intensified treatment during infections. All patients showed beneficial treatment effects, most strikingly in growth (without tube feeding), head circumference, development, coping with infections, and oxygen dependency.

Conclusion: For these four ARS deficiencies, we observed a common disease mechanism of episodic insufficient aminoacylation to meet translational demands and illustrate the power of amino acid supplementation for the expanding ARS patient group. Moreover, we provide a strategy for personalized preclinical functional evaluation.

Fig. 1. Enzyme activity is decreased but not absent in all patients, and IARS, LARS, and FARSB patient fibroblasts are increased sensitive to isoleucine, leucine and phenylalanine deprivation, respectively.

(a) Enzyme activity is decreased but not absent in all patients. Aminoacylation activity in fibroblasts of P^IARS, P^IARS-2, P^LARS, P^FARSB, and two siblings with the same homozygous variant as P^SARS, presented as percentage of age-matched controls. GARS activity was measured as an internal control. All measurements were performed in triplicate (n = 3), except IARS controls (n = 6) and GARS controls (n = 12). Error bars show standard deviation. Unpaired t-test: ns p ≥ 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (b–e) IARS, LARS, and FARSB patient fibroblasts are sensitive to isoleucine, leucine, and phenylalanine deprivation, respectively. Seventy-two hours proliferation of fibroblasts of P^IARS, P^IARS-2, P^LARS, P^FARSB, and two siblings with the same homozygous variant as P^SARS, compared to age-matched control fibroblasts, exposed to decreasing concentrations of isoleucine (b: P^IARS/P^IARS-2), leucine (c: P^LARS), phenylalanine (d: P^FARSB), or serine (e: two siblings of P^SARS), shown as normalized impedance, measured with an xCELLigence MP. Amino acid concentrations were compared to average plasma concentrations. Every donor was measured once (a; n = 1 each) or twice (b–d; n = 2 each). Two (a–c) or three (d) controls were measured once (n = 1 each). Error bars show standard deviation. Unpaired t-test: ns p ≥ 0.05, *p < 0.05, **p < 0.01, ***p < 0.001. (f–i) LARS and FARS activity deteriorate in LARS and FARSB patient fibroblasts, respectively. Aminoacylation activity in fibroblasts of P^IARS and P^IARS-2 (f), P^LARS (g), P^FARSB (h), and two siblings with the same homozygous variant as P^SARS (i) at 37  °C, 38.5  °C, and 40 °C. Data are presented compared to the enzymatic activity at 37 °C. All measurements were performed in triplicate (n = 3). Error bars show standard deviation. Unpaired t-test: ns p ≥ 0.05, *p < 0.05, **p < 0.01.

Fig. 2. Summary of symptoms with treatment effects, and growth charts for all patients.

(a–d) Summary of symptoms and treatment effects. Summary of the symptoms of P^IARS (top left), P^LARS (top right), P^FARSB (bottom left), and P^SARS (bottom right). Colors indicate the generalized treatment effect on the symptom (green: improved; white: stable or stabilized; red: progressed). Standard deviation scores (Z) for height, weight, and head circumference were calculated using Netherlands (NL) reference charts (P^IARS; head circumference of P^LARS), World Health Organization (WHO) reference charts (P^FARSB), Centers for Disease Control and Prevention (CDC) reference charts (height and weight of P^LARS), and French (FR) reference charts (P^SARS). CRP C-reactive protein, FSIQ full-scale intelligence quotient, G-tube gastrostomy-tube, ILD interstitial lung disease, FRI fluid reasoning index, INR international normalized ratio, LI language index, PAP pulmonary alveolar proteinosis, PEG percutaneous endoscopic gastrostomy, PIQ performance intelligence quotient, VIQ verbal intelligence quotient, VCI verbal comprehension index, VSI visual–spatial index, WISC-V Wechsler Intelligence Scale for Children, fifth edition, WPPSI-III/IV Wechsler Preschool and Primary Scale of Intelligence, third/fourth edition. (e–h) Growth charts: Z-scores of height, weight and head circumference for age. Growth charts of height, weight, and head circumference in standard deviation score (Z) of (a) P^IARS (NL reference charts); (b) P^LARS (CDC reference charts for height and weight, NL reference charts for head circumference); (c) P^FARSB (WHO reference charts); and (d) P^SARS (FR reference charts). T in months from start of treatment.