Functional characterisation of genes and metabolites involved in the metabolic and neurocognitive pathophysiology of hereditary tyrosinemia type 1

Hereditary tyrosinemia type 1 (HT1), also known as hepatorenal tyrosinemia, is the most prevalent
and severe among the five tyrosine-related inherited metabolic disorders. This autosomal recessive
disorder is characterized by a deficiency or absence of the fumarylacetoacetate hydrolase (FAH)
enzyme, which is primarily active in liver and kidney cells and responsible for the final step in the
tyrosine degradation pathway. As a result, HT1 patients are unable to properly metabolize tyrosine,
leading to the accumulation of toxic tyrosine intermediates such as maleylacetoacetate (MAA), fumarylacetoacetate (FAA) and succinylacetone (SA). If left untreated, HT1 can quickly progress to acute liver failure and renal tubular defects, resulting in death within a few days to weeks after birth. Historically, tyrosine- and phenylalanine-restricted diets have been used as treatment for HT1 patients, but they have proven to be inadequate, especially in cases of acute and subacute clinical forms. Orthotopic liver transplantation is also being applied, but is only an option for those patients with acute-on-chronic liver failure or those who have developed hepatocellular carcinoma (HCC) due to delayed diagnosis from inadequate neonatal screening programs. However, since 2002, the standard therapy for HT1 patients has been the lifelong daily intake of nitisinone [2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione], a former herbicide that acts as a potent inhibitor of 4-hydroxyphenylpyruvate dioxygenase (HPD), a key enzyme in the tyrosine degradation pathway. Nitisinone causes an effective upstream metabolic block preventing the production of the aforementioned toxic metabolites. Despite rescuing HT1 patients from severe illness and early death, nitisinone therapy also leads to accumulation of tyrosine in the blood and the potential development of HCC. To control the disease and mitigate drug-related side-effects, HT1 patients must adhere to a lifelong regimen of both nitisinone treatment and a tyrosine and phenylalanine restricted diet. Unfortunately, this strict drug and dietary regime can be burdensome, leading to challenges in therapy
adherence.
In the context of therapy incompliance, we investigated which molecular mechanisms and pathways
undergo changes in the liver and blood as soon as the inhibitory effects of the drug nitisinone start to
diminish. To accomplish this, we first performed transcriptomic analysis to study gene expression patterns in liver tissue of Fah-deficient mice under continuous nitisinone therapy and after discontinuation of the therapy for seven days. We found significant modulation of molecular pathways associated with oxidative stress, glutathione metabolism, liver regeneration and liver cancer following seven days of nitisinone withdrawal. More specifically, we observed substantial alterations in the NRF2-mediated oxidative stress response and various toxicological gene classes involved in the metabolism of reactive oxygen species. The expression of several key genes related to glutathione metabolism, including Slc7a11 and Ggt1, was significantly upregulated upon short-term nitisinone deprivation. This stress response was accompanied by the transcriptional activation of several markers associated with liver progenitor cells, including Atf3, Cyr61, Ddr1, Epcam, Elovl7 and Glis3, indicating an early activation of liver regeneration upon nitisinone withdrawal.
In a subsequent set of experiments, we conducted a comparative metabolic profiling using LC-QTOFMS on mouse blood. Our aim was to investigate the relationship between the transcriptome and metabolome as it remains unclear how altered gene expression translates into metabolic changes. The metabolomics data obtained was strongly aligned with the observations in the transcriptome study, showing that depriving mice of nitisinone for seven consecutive days induces a multitude of molecular and biochemical changes associated with HT1 disease, including liver damage, HCC and depletion of glutathione. The alterations in other canonical pathways were primarily a downstream effect of the increased oxidative stress. Furthermore, we observed a general increase in amino acids and significant changes related to the aminoacyl-tRNA biosynthesis. This suggests that even shortterm deprivation of nitisinone, whether due to drug intolerance or non-compliance, may initiate the development of HCC. Moreover, we noted changes not only in numerous pathways directly and indirectly related to the metabolism of tyrosine and glutathione, but also the presence of ‘side-chain’ metabolites, such as gamma-glutamyl- and N-acetyl-coupled amino acids, suggesting that non-directly related pathways like phase II biotransformation reactions are also affected by nitisinone deprivation. Furthermore, in a pilot study, we assessed neurocognitive and behavioural profiles in response to different doses of nitisinone. We conducted the study in both Fah-deficient and healthy wildtype mice
under continues treatment, while also considering the pharmacological effects on the metabolome in
both male and female mice. Neurocognitive profiling revealed that male Fah-deficient mice treated
with low and middle doses of nitisinone performed inferior during training on the Barnes maze, suggesting a slower learning progression. However, our pilot data does not allow us to determine if this aberrancy is real genotype difference or perhaps a motivational problem. Interestingly, this effect was not observed in female mice, suggesting a potential sex difference.