Arginine does not rescue p.Q188R mutation deleterious effect in classic galactosemia

Minela Haskovic, Britt Derks, Liesbeth van der Ploeg, Jorn Trommelen, Jean Nyakayiru, Luc J C van Loon, Sabrina Mackinnon, Wyatt W Yue, Roy W A Peake, Li Zha, Didem Demirbas, Wanshu Qi, Xiaoping Huang, Gerard T Berry, Jelle Achten, Jörgen Bierau, M Estela Rubio-Gozalbo, Ana I Coelho, Minela Haskovic, Britt Derks, Liesbeth van der Ploeg, Jorn Trommelen, Jean Nyakayiru, Luc J C van Loon, Sabrina Mackinnon, Wyatt W Yue, Roy W A Peake, Li Zha, Didem Demirbas, Wanshu Qi, Xiaoping Huang, Gerard T Berry, Jelle Achten, Jörgen Bierau, M Estela Rubio-Gozalbo, Ana I Coelho

Abstract

Background: Classic galactosemia is a rare genetic metabolic disease with an unmet treatment need. Current standard of care fails to prevent chronically-debilitating brain and gonadal complications. Many mutations in the GALT gene responsible for classic galactosemia have been described to give rise to variants with conformational abnormalities. This pathogenic mechanism is highly amenable to a therapeutic strategy based on chemical/pharmacological chaperones. Arginine, a chemical chaperone, has shown beneficial effect in other inherited metabolic disorders, as well as in a prokaryotic model of classic galactosemia. The p.Q188R mutation presents a high prevalence in the Caucasian population, making it a very clinically relevant mutation. This mutation gives rise to a protein with lower conformational stability and lower catalytic activity. The aim of this study is to assess the potential therapeutic role of arginine for this mutation.

Methods: Arginine aspartate administration to four patients with the p.Q188R/p.Q188R mutation, in vitro studies with three fibroblast cell lines derived from classic galactosemia patients as well as recombinant protein experiments were used to evaluate the effect of arginine in galactose metabolism. This study has been registered at https://ichgcp.net/clinical-trials-registry/NCT03580122" title="See in ClinicalTrials.gov">NCT03580122) on 09 July 2018. Retrospectively registered.

Results: Following a month of arginine administration, patients did not show a significant improvement of whole-body galactose oxidative capacity (p = 0.22), erythrocyte GALT activity (p = 0.87), urinary galactose (p = 0.52) and urinary galactitol levels (p = 0.41). Patients' fibroblasts exposed to arginine did not show changes in GALT activity. Thermal shift analysis of recombinant p.Q188R GALT protein in the presence of arginine did not exhibit a positive effect.

Conclusions: This short pilot study in four patients homozygous for the p.Q188R/p.Q188R mutation reveals that arginine has no potential therapeutic role for galactosemia patients homozygous for the p.Q188R mutation.

Keywords: Amino acid supplementation; Arginine; Chemical chaperones; Classic galactosemia; Galactose metabolism; Inherited metabolic disorder.

Conflict of interest statement

Ethics approval and consent to participate

The Medical Ethical Committee (Medisch Ethische Toetsingscommissie, METC) of the Maastricht University Medical Center + approved the clinical study (METC reference: 172012). Written consent was obtained from all four participants before the beginning of the study.

This study was conducted in accordance with the International Conference on Harmonization Good Clinical Practice guidelines, the Principles of the Declaration of Helsinki. Fibroblast samples GM1703 AND GM727 were acquired from Coriell Institute. The other fibroblast line was generated through a Boston Children’s Hospital IRB approved protocol.

Consent for publication

Consent was obtained from all patients for publication.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Mean galactose oxidative capacity before and after arginine aspartate supplementation. Mean galactose oxidative capacity of the four patients, expressed as CUMPCD (cumulative percent of the dose) 13CO2 eliminated in air. Baseline: CUMPCD (120 min) = 0.7 ± 0.06%; CUMPCD (360 min) = 2.8 ± 1.3%. After intervention: CUMPCD (120 min) = 0.7 ± 0.02%; CUMPCD (360 min) = 2.7 ± 1.2%
Fig. 2
Fig. 2
Mean GALT activity before and after arginine aspartate supplementation. GALT activity is expressed as μmol of UDP-Gal formed per hour per mmol hemoglobin
Fig. 3
Fig. 3
Mean urinary galactitol and galactose levels before and after arginine aspartate supplementation
Fig. 4
Fig. 4
Representative thermal shift assay curves with no added ligand, arginine (10 mM) or galactose (10 mM) for A. wildtype GALT and B. p.Q188R GALT. Fluorescence values were normalized to allow comparison of Tm values. Curves for single replicate per ligand state plotted, with no added ligand, arginine (10 mM) or galactose (10 mM). Values stated are the mean values of three replicates

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