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Hereditary tyrosinemia type 1 (HT1) is a genetic disorder of the tyrosine degradation pathway (TIMD) with unmet therapeutic needs. HT1 patients are unable to fully break down the amino acid tyrosine due to a deficient fumarylacetoacetate hydrolase (FAH) enzyme and, therefore, accumulate toxic tyrosine intermediates. If left untreated, they experience hepatic failure with comorbidities involving the renal and neurological system and the development of hepatocellular carcinoma (HCC). Nitisinone (NTBC), a potent inhibitor of the 4-hydroxyphenylpyruvate dioxygenase (HPD) enzyme, rescues HT1 patients from severe illness and death. However, despite its demonstrated benefits, HT1 patients under continuous NTBC therapy are at risk to develop HCC and adverse reactions in the eye, blood and lymphatic system, the mechanism of which is poorly understood. Moreover, NTBC does not restore the enzymatic defects inflicted by the disease nor does it cure HT1. Here, the changes in molecular pathways associated to the development and progression of HT1-driven liver disease that remains uncorrected under NTBC therapy were investigated using whole transcriptome analyses on the livers of Fah- and Hgd-deficient mice under continuous NTBC therapy and after seven days of NTBC therapy discontinuation. Alkaptonuria (AKU) was used as a tyrosine-inherited metabolic disorder reference disease with non-hepatic manifestations. The differentially expressed genes were enriched in toxicological gene classes related to liver disease, liver damage, liver regeneration and liver cancer, in particular HCC. Most importantly, a set of 25 genes related to liver disease and HCC development was identified that was differentially regulated in HT1 vs. AKU mouse livers under NTBC therapy. Some of those were further modulated upon NTBC therapy discontinuation in HT1 but not in AKU livers. Altogether, our data indicate that NTBC therapy does not completely resolves HT1-driven liver disease and supports the sustained risk to develop HCC over time as different HCC markers, including Moxd1, Saa, Mt, Dbp and Cxcl1, were significantly increased under NTBC.

BACKGROUND: Alkaptonuria (AKU) is caused by homogentisate 1,2-dioxygenase deficiency that leads to homogentisic acid (HGA) accumulation, ochronosis and severe osteoarthropathy. Recently, nitisinone treatment, which blocks HGA formation, has been effective in AKU patients. However, a consequence of nitisinone is elevated tyrosine that can cause keratopathy. The effect of tyrosine and phenylalanine dietary restriction was investigated in nitisinone-treated AKU mice, and in an observational study of dietary intervention in AKU patients. METHODS: Nitisinone-treated AKU mice were fed tyrosine/phenylalanine-free and phenylalanine-free diets with phenylalanine supplementation in drinking water. Tyrosine metabolites were measured pre-nitisinone, post-nitisinone, and after dietary restriction. Subsequently an observational study was undertaken in 10 patients attending the National Alkaptonuria Centre (NAC), with tyrosine >700μmol/L who had been advised to restrict dietary protein intake and where necessary, to use tyrosine/phenylalanine-free amino acid supplements. RESULTS: Elevated tyrosine (813μmol/L) was significantly reduced in nitisinone-treated AKU mice fed a tyrosine/phenylalanine-free diet in a dose responsive manner. At 3 days of restriction, tyrosine was 389.3μmol/L, 274.8μmol/L and 144.3μmol/L with decreasing phenylalanine doses. In contrast, tyrosine was not effectively reduced in mice by a phenylalanine-free diet; at 3 days tyrosine was 757.3μmol/L, 530.2μmol/L and 656.2μmol/L, with no dose response to phenylalanine supplementation. In NAC patients, tyrosine was significantly reduced (p=0.002) when restricting dietary protein alone, and when combined with tyrosine/phenylalanine-free amino acid supplementation; 4 out of 10 patients achieved tyrosine

Alkaptonuria is an ultra-rare autosomal recessive disorder of tyrosine metabolism, whereby the homogentisate 1,2-dioxygenase (HGD) enzyme is deficient, causing an elevation of its substrate homogentisic acid (HGA). Overtime, elevated HGA causes connective tissue ochronosis, leading to a severe and early onset osteoarthropathy. The use of HGD deficient mouse models in this metabolic bone disease have provided the opportunity to investigate AKU pathophysiology and potential treatments. An ENU mutagenesis AKU mouse model (BALB/c Hgd−/−) provided the means to explore the onset of pigmentation in cartilage and treatment of AKU with nitisinone, an inhibitor of the upstream enzyme forming HGA. This work provided evidence that nitisinone could not only lower circulating HGA, but could also prevent ochronosis and halt disease progression, leading to its off-label use at the National Alkaptonuria Centre (Liverpool, UK) and its subsequent testing in human clinical trials (DevelopAKUre). Recently, a new targeted AKU mouse model (Hgd tm1a−/−, C57BL/6) has been established, offering a LacZ reporter gene for localising gene expression and LoxP and FRT sites that enabled generation of an inducible and liver-specific HGD knockout model (Hgd tm1d MxCre+/−). This conditional model determined the importance of the liver as a target organ for future gene/enzyme replacement therapies in AKU. The contribution of AKU mouse models has clearly accelerated the treatment and knowledge of this rare disease, and will continue to be used.

Alkaptonuria (AKU) is characterised by increased circulating homogentisic acid and deposition of ochronotic pigment in collagen-rich connective tissues (ochronosis), stiffening the tissue. This process over many years leads to a painful and severe osteoarthropathy, particularly affecting the cartilage of the spine and large weight bearing joints. Evidence in human AKU tissue suggests that pigment binds to collagen. The exposed collagen hypothesis suggests that collagen is initially protected from ochronosis, and that ageing and mechanical loading causes loss of protective molecules, allowing pigment binding. Schmorl’s staining has previously demonstrated knee joint ochronosis in AKU mice. This study documents more comprehensively the anatomical distribution of ochronosis in two AKU mouse models (BALB/c Hgd−/−, Hgd tm1a−/−), using Schmorl’s staining. Progression of knee joint pigmentation with age in the two AKU mouse models was comparable. Within the knee, hip, shoulder, elbow and wrist joints, pigmentation was associated with chondrons of calcified cartilage. Pigmented chondrons were identified in calcified endplates of intervertebral discs and the calcified knee joint meniscus, suggesting that calcified tissues are more susceptible to pigmentation. There were significantly more pigmented chondrons in lumbar versus tail intervertebral disc endplates (p = 0.002) and clusters of pigmented chondrons were observed at the insertions of ligaments and tendons. These observations suggest that loading/strain may be associated with increased pigmentation but needs further experimental investigation. The calcified cartilage may be the first joint tissue to acquire matrix damage, most likely to collagen, through normal ageing and physiological loading, as it is the first to become susceptible to pigmentation.

Alkaptonuria (AKU) is caused by homogentisate 1,2-dioxygenase (HGD) deficiency. This study aimed to determine if HGD and other enzymes related to tyrosine metabolism are associated with the location of ochronotic pigment. Liver, kidney, skin, bone, brain, eyes, spleen, intestine, lung, heart, cartilage, and muscle were harvested from 6 AKU BALB/c Hgd -/- (3 females, 3 males) and 4 male C57BL/6 wild type (WT) mice. Hgd, 4-hydroxyphenylpyruvate dioxygenase (4-Hppd), tyrosine hydroxylase (Th), and tyrosinase (Tyr) mRNA expression was investigated using qPCR. Adrenal gland and gonads from AKU Hgd tm1a -/- mice were LacZ stained, followed by qPCR analysis of Hgd mRNA. The liver had the highest expression of Hgd, followed by the kidney, with none detected in cartilage or brain. Low-level Hgd expression was observed within developing male germ cells within the testis and epididymis in Hgd tm1a -/-. 4-Hppd was most abundant in liver, with smaller amounts in kidney and low-level expression in other tissues. Th was expressed mainly in brain and Tyr was found primarily in the eyes. The tissue distribution of both Hgd and 4-Hppd suggest that ochronotic pigment in AKU mice is a consequence of enzymes within the liver, and not from enzymatic activity within ochronotic tissues. Excessive accumulation of HGA as ochronotic pigment in joints and other connective tissues originates from the circulation and therefore the extracellular fluid. The tissue distribution of both Th and Tyr suggests that these enzymes are not involved in the formation of HGA-derived ochronotic pigment.

Background Alkaptonuria is a rare, genetic, multisystem disease characterised by the accumulation of homogentisic acid (HGA). No HGA-lowering therapy has been approved to date. The aim of SONIA 2 was to investigate the efficacy and safety of once-daily nitisinone for reducing HGA excretion in patients with alkaptonuria and to evaluate whether nitisinone has a clinical benefit. Methods SONIA 2 was a 4-year, open-label, evaluator-blind, randomised, no treatment controlled, parallel-group study done at three sites in the UK, France, and Slovakia. Patients aged 25 years or older with confirmed alkaptonuria and any clinical disease manifestations were randomly assigned (1:1) to receive either oral nitisinone 10 mg daily or no treatment. Patients could not be masked to treatment due to colour changes in the urine, but the study was evaluator-blinded as far as possible. The primary endpoint was daily urinary HGA excretion (u-HGA24) after 12 months. Clinical evaluation Alkaptonuria Severity Score Index (cAKUSSI) score was assessed at 12, 24, 36, and 48 months. Efficacy variables were analysed in all randomly assigned patients with a valid u-HGA24 measurement at baseline. Safety variables were analysed in all randomly assigned patients. The study was registered at ClinicalTrials.gov (NCT01916382). Findings Between May 7, 2014, and Feb 16, 2015, 139 patients were screened, of whom 138 were included in the study, with 69 patients randomly assigned to each group. 55 patients in the nitisinone group and 53 in the control group completed the study. u-HGA24 at 12 months was significantly decreased by 99·7% in the nitisinone group compared with the control group (adjusted geometric mean ratio of nitisinone/control 0·003 [95% CI 0·003 to 0·004], p

Alkaptonuria is an inherited disease caused by homogentisate 1,2-dioxygenase (HGD) deficiency. Circulating homogentisic acid (HGA) is elevated and deposits in connective tissues as ochronotic pigment. In this study, we aimed to define developmental and adult HGD tissue expression and determine the location and amount of gene activity required to lower circulating HGA and rescue the alkaptonuria phenotype.We generated an alkaptonuria mouse model using a knockout-first design for the disruption of the HGD gene. Hgd tm1a −/− mice showed elevated HGA and ochronosis in adulthood. LacZ staining driven by the endogenous HGD promoter was localised to only liver parenchymal cells and kidney proximal tubules in adulthood, commencing at E12.5 and E15.5 respectively. Following removal of the gene trap cassette to obtain a normal mouse with a floxed 6th HGD exon, a double transgenic was then created with Mx1-Cre which conditionally deleted HGD in liver in a dose dependent manner. 20% of HGD mRNA remaining in liver did not rescue the disease, suggesting that we need more than 20% of liver HGD to correct the disease in gene therapy.Kidney HGD activity which remained intact reduced urinary HGA, most likely by increased absorption, but did not reduce plasma HGA nor did it prevent ochronosis. In addition, downstream metabolites of exogenous 13C6-HGA, were detected in heterozygous plasma, revealing that hepatocytes take up and metabolise HGA.This novel alkaptonuria mouse model demonstrated the importance of targeting liver for therapeutic intervention, supported by our observation that hepatocytes take up and metabolise HGA.

The clinical effects of alkaptonuria (AKU) are delayed and ageing influences disease progression. Morbidity of AKU is secondary to high circulating homogentisic acid (HGA) and ochronosis. It is not known whether HGA is produced by or processed in the kidney in AKU. Data from AKU patients from four studies were merged to form a single AKU group. A control group of non-AKU subjects was generated by merging data from two non-AKU studies. Data were used to derive renal clearance and fractional excretion (FE) ratios for creatinine, HGA, phenylalanine (PHE) and tyrosine (TYR) using standard calculations, for comparison between the AKU and the control groups. There were 225 AKU patients in the AKU group and 52 in the non-AKU control group. Circulating HGA increased with age (P

The original publication of this article contained an incorrect version that did not include some final reviewers' suggestions, was inadvertently received for production and published. The original article has been corrected.

Background and Purposealkaptonuria (AKU) is an inherited disorder of tyrosine metabolism caused by lack of the enzyme homogentisate 1,2-dioxygenase (HGD). The primary biochemical consequence of HGD-deficiency is increased circulating homogentisic acid (HGA), which is central to AKU disease pathology. The aim of this study was to investigate the wider metabolic consequences of targeted Hgd disruption.Experimental Approachthe first metabolomic analysis of the Hgd−/− AKU mouse model was performed. Urinary metabolites altered in Hgd−/− were further validated by showing that the HGA-lowering drug nitisinone reversed their direction of alteration in AKUKey Resultscomparison of Hgd−/− (AKU) versus Hgd+/− (heterozygous control) urine revealed increases in HGA and a group of 8 previously unreported HGA-derived transformation products from phase I and II metabolism. HGA biotransformation products HGA-sulfate, HGA-glucuronide, HGA-hydrate and hydroxymethyl-HGA were also decreased in urine from both mice and patients with AKU on the HGA-lowering agent nitisinone. Hgd knockout also revealed a host of previously unrecognised associations between tyrosine, purine and TCA cycle metabolic pathways.Conclusion and ImplicationsAKU is rare, but our findings further what is currently understood about tyrosine metabolism more generally, and show for the first time that phase I and II detoxification is recruited to prevent accumulation of endogenously-produced metabolites in inborn errors of metabolism. The data highlight the misconception that phase I and II metabolic biotransformations are reserved solely for drug clearance; these are ancient mechanisms, which represent new potential treatment targets in inherited metabolic diseases.Abstract FigureBullet point summaryWhat is already known Increased circulating homogentisic acid is central to disease pathology in the inherited metabolic disease alkaptonuriaThe Hgd knockout mouse, created in our laboratory, accurately models human alkaptonuriaWhat this study adds Phase I and II biotransformations are recruited in alkaptonuria for detoxification of homogentisic acidThese data challenge misconceptions that phase I and II metabolism is solely for drug clearanceClinical significance Phase I and II metabolic processes represent new treatment targets in inherited metabolic diseasesThe molecular pathology of AKU extends much further than the known alteration to tyrosine metabolism

Alkaptonuria (AKU) is an inherited disorder of tyrosine metabolism caused by lack of active enzyme homogentisate 1,2-dioxygenase (HGD). The primary consequence of HGD deficiency is increased circulating homogentisic acid (HGA), the main agent in the pathology of AKU disease. Here we report the first metabolomic analysis of AKU homozygous Hgd knockout (Hgd−/−) mice to model the wider metabolic effects of Hgd deletion and the implication for AKU in humans. Untargeted metabolic profiling was performed on urine from Hgd−/− AKU (n = 15) and Hgd+/− non-AKU control (n = 14) mice by liquid chromatography high-resolution time-of-flight mass spectrometry (Experiment 1). The metabolites showing alteration in Hgd−/− were further investigated in AKU mice (n = 18) and patients from the UK National AKU Centre (n = 25) at baseline and after treatment with the HGA-lowering agent nitisinone (Experiment 2). A metabolic flux experiment was carried out after administration of 13C-labelled HGA to Hgd−/−(n = 4) and Hgd+/−(n = 4) mice (Experiment 3) to confirm direct association with HGA. Hgd−/− mice showed the expected increase in HGA, together with unexpected alterations in tyrosine, purine and TCA-cycle pathways. Metabolites with the greatest abundance increases in Hgd−/− were HGA and previously unreported sulfate and glucuronide HGA conjugates, these were decreased in mice and patients on nitisinone and shown to be products from HGA by the 13C-labelled HGA tracer. Our findings reveal that increased HGA in AKU undergoes further metabolism by mainly phase II biotransformations. The data advance our understanding of overall tyrosine metabolism, demonstrating how specific metabolic conditions can elucidate hitherto undiscovered pathways in biochemistry and metabolism.