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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - TIME (Time of damage in classic galactosemia: is prenatal toxicity a determinant factor?)

Teaser

Summary

One of the long-standing questions in the galactosemia field is whether there is prenatal damage. With the TIME project, we provided evidence of early postnatal damage in classic galactosemia.
Classic galactosemia (CG) is an inherited metabolic disorder in galactose metabolism. Galactose is a sugar extremely important as energy source for infants and for the glycosylation of complex molecules. The main pathway of galactose metabolism is the Leloir pathway.
The Leloir pathway metabolizes galactose through the sequential action of three enzymes: galactokinase (GALK1), which converts galactose into galactose-1-phosphate (Gal-1-P); galactose-1-phosphate uridylyltransferase (GALT), which transfers a uridine monophosphate group from uridine diphosphate-glucose (UDP-Glc) to Gal-1-P, thereby releasing Glc-1-P and forming UDP-galactose (UDP-Gal); and UDP-Gal 4-epimerase (GALE), which interconverts UDP-Gal and UDP-Glc. In CG, there is an accumulation of galactose metabolites, namely of Gal-1-P.
CG is caused by deficient activity of GALT. It affects between 1:16,000 and 1:60,000 live births and presents in the neonatal period as a life-threatening disease. Exclusion of galactose from the diet results in complete resolution of the acute complications. This treatment shows, however, limited efficacy in the prevention of long-term complications, with patients suffering from cognitive, fertility and social impairments. There is an urgent need to address the clinical and social impairments observed in CG patients.
CG is an autosomal recessive disorder caused by mutations in the GALT gene. As with many other inherited metabolic diseases, CG mutational spectrum is dominated by missense mutations (>60%). Previous studies described that GALT impairment results from misfolding and aggregation, thus establishing CG as a conformational disorder. Conformational disorders are highly amenable to a pharmacological chaperones- and/or proteostasis modulators-based therapy. The recent description of the crystal structure of human GALT will greatly facilitate the design of GALT-targeted therapeutic compounds. These findings are extremely important towards the development of a truly effective therapy for CG. Accordingly, there is an urgent need to clarify the time of damage onset, as it will define the time at which therapy should be initiated in order to prevent the development of the chronic complications.
The overall objective of this study is to provide new insights on time of damage onset in CG, particularly if it begins prenatally.

Work performed

With the TIME project, using our recently developed CG zebrafish model, we provided evidence of early postnatal damage in CG, but not prenatal.
We have performed a biochemical characterization of galactose metabolism in CG zebrafish and compared it with the healthy fish throughout development, from embryonal stage to adulthood, with a particular focus of the CG target-organs of damage (brain and ovary).
Enzymes of the Leloir pathway are ubiquitously expressed in zebrafish throughout development, which is in agreement with the human phenotype. Brain analysis of adult fish revealed no significant differences in galactose metabolism both at the gene expression and at the catalytic activity levels, whereas the ovary revealed higher galk1 and gale expression in CG fish, suggestive of gonadal-specific needs of these enzymes in adulthood. Notably, there was a significant inter-tissue difference on the catalytic activity of galk1, gale, and ugp (for both healthy and CG fish), strengthening the notion of organ-specific needs.
Regarding the clinical phenotype, we studied brain and gonadal damage throughout development. Brain impairments were evaluated by histological, motor activity and myelin structure analysis. Whereas histological analysis revealed no major anatomic abnormalities in adult CG fish, motor activity was decreased from juvenile stage onwards. Myelin analysis revealed a decreased length in a specific tract in the early postnatal period, whose clinical significance is being further investigated. Ovarian impairments were evaluated by macroscopic, histological and fertility analysis, which revealed ovarian impairments and reduced reproductive capacity in CG fish. Additionally, primordial germ cells (PGCs) were investigated and revealed that migration pattern is more frequently abnormal in CG fish than in healthy fish.
Overall, the TIME findings suggest that there is early postnatal damage in CG. No evidence of prenatal damage was found. The progress made within this project will be crucial in the development of new therapeutic strategies that prevent the development of CG long-term complications. Within our international galactosemia network (GalNet, www.galactosemianetwork.org), various research groups are collaborating towards the common goal of developing an effective therapy for CG. The insights provided by the TIME project are of utmost importance for this international cooperative project and will be used by this consortium in the development of a safe and effective treatment for CG.
These findings have been presented at both national and international meetings and symposia, and will continue to be further disseminated beyond the period of the TIME project.

Final results

CG is a challenging disorder of galactose metabolism, with very disappointing outcomes, representing a heavy burden on the patients and families. Its current standard of care, a galactose-restricted diet, fails to prevent chronically-debilitating complications.
Even though the first description of galactosemia dates back to 1908, nowadays its exact pathophysiology is not yet fully understood and there is still no effective treatment.
Recent advances in the molecular bases of CG took research a step further towards therapy development. CG has been demonstrated to be a conformational disorder, and thus amenable by a pharmacological chaperones-/proteostasis modulators-based therapy. The recent description of the crystal structure of human GALT will greatly facilitate the design of GALT-targeted therapeutic compounds. Additionally, a therapy based on GALK1 inhibitors aiming to mitigate Gal-1-P accumulation (widely pointed out as a key pathogenic agent) has, thus far, shown very promising results.
At present, within our international galactosemia network (GalNet), various galactosemia research groups are working in unison to develop an effective therapy for CG. The TIME findings suggest early postnatal damage, whereas no prenatal damage was observed. From the therapeutic viewpoint, this observation entails that therapy can be effectively implemented after birth, although very early in life. This notion is crucial for the GalNet international cooperative project, and will be taken into account by this consortium in the development of an effective and safe therapy for CG.
An effective treatment for CG will have a positive socio-economic impact: i) on the health care systems by preventing chronic complications and decreasing the medical assistance needed; and ii) on the social impairments, allowing CG patients to fully engage in society.