Exploring the therapeutic potential of modern and ancestral phenylalanine/tyrosine ammonia-lyases as supplementary treatment of hereditary tyrosinemia.

Phenylalanine/tyrosine ammonia-lyases (PAL/TALs) have been approved by the FDA for treatment of phenylketonuria and may harbour potential for complementary treatment of hereditary tyrosinemia Type I. Herein, we explore ancestral sequence reconstruction as an enzyme engineering tool to enhance the therapeutic potential of PAL/TALs. We reconstructed putative ancestors from fungi and compared their catalytic activity and stability to two modern fungal PAL/TALs. Surprisingly, most putative ancestors could be expressed as functional tetramers in Escherichia coli and thus retained their ability to oligomerize. All ancestral enzymes displayed increased thermostability compared to both modern enzymes, however, the increase in thermostability was accompanied by a loss in catalytic turnover. One reconstructed ancestral enzyme in particular could be interesting for further drug development, as its ratio of specific activities is more favourable towards tyrosine and it is more thermostable than both modern enzymes. Moreover, long-term stability assessment showed that this variant retained substantially more activity after prolonged incubation at 25 °C and 37 °C, as well as an increased resistance to incubation at 60 °C. Both of these factors are indicative of an extended shelf-life of biopharmaceuticals. We believe that ancestral sequence reconstruction has potential for enhancing the properties of enzyme therapeutics, especially with respect to stability. This work further illustrates that resurrection of putative ancestral oligomeric proteins is feasible and provides insight into the extent of conservation of a functional oligomerization surface area from ancestor to modern enzyme.

Diagnosis and treatment of tyrosinemia type I: a US and Canadian consensus group review and recommendations.

Tyrosinemia type I (hepatorenal tyrosinemia, HT-1) is an autosomal recessive condition resulting in hepatic failure with comorbidities involving the renal and neurologic systems and long term risks for hepatocellular carcinoma. An effective medical treatment with 2-[2-nitro-4-trifluoromethylbenzoyl]-1,3-cyclohexanedione (NTBC) exists but requires early identification of affected children for optimal long-term results. Newborn screening (NBS) utilizing blood succinylacetone as the NBS marker is superior to observing tyrosine levels as a way of identifying neonates with HT-1. If identified early and treated appropriately, the majority of affected infants can remain asymptomatic. A clinical management scheme is needed for infants with HT-1 identified by NBS or clinical symptoms. To this end, a group of 11 clinical practitioners, including eight biochemical genetics physicians, two metabolic dietitian nutritionists, and a clinical psychologist, from the United States and Canada, with experience in providing care for patients with HT-1, initiated an evidence- and consensus-based process to establish uniform recommendations for identification and treatment of HT-1. Recommendations were developed from a literature review, practitioner management survey, and nominal group process involving two face-to-face meetings. There was strong consensus in favor of NBS for HT-1, using blood succinylacetone as a marker, followed by diagnostic confirmation and early treatment with NTBC and diet. Consensus recommendations for both immediate and long-term clinical follow-up of positive diagnoses via both newborn screening and clinical symptomatic presentation are provided.

Predicting tyrosinaemia: a mathematical model of 4-hydroxyphenylpyruvate dioxygenase inhibition by nitisinone in rats.

Nitisinone or 2-(2-nitro-4-trifluoromethylbenzoyl)cyclohexane-1,3-dione is a reversible inhibitor of 4-hydroxyphenylpyruvate dioxygenase (HPPD), an enzyme important in tyrosine catabolism. Today, nitisinone is successfully used to treat Hereditary Tyrosinaemia type 1, although its original expected role was as a herbicide. In laboratory animals, treatment with nitisinone leads to the elevation of plasma tyrosine (tyrosinaemia). In rats and Beagle dogs, repeat low-dose exposure to nitisinone leads to corneal opacities whilst similar studies in the mouse and Rhesus monkey showed no comparable toxicities or other treatment related findings. The differences in toxicological sensitivities have been related to the upper limit of the concentration of tyrosine that accumulates in plasma, which is driven by the amount/activity of tyrosine aminotransferase. A physiologically based, pharmacodynamics ordinary differential equation model of HPPD inhibition to bolus exposure of nitisinone in vivo is presented. Going beyond traditional approaches, asymptotic analysis is used to separate the different timescales of events involved in HPPD inhibition and tyrosinaemia. This analysis elucidates, in terms of the model parameters, a critical inhibitor concentration (at which tyrosine concentration starts to rise) and highlights the contribution of in vitro measured parameters to events in an in vivo system. Furthermore, using parameter-fitting methods, a systematically derived reduced model is shown to fit well to rat data, making explicit how the parameters are informed by such data. This model in combination with in vitro descriptors has potential as a surrogate for animal experimentation to predict tyrosinaemia, and further development can extend its application to other related medical scenarios.

Biochemical and molecular diagnosis of tyrosinemia type I with two novel FAH mutations in a Hong Kong chinese patient: recommendation for expanded newborn screening in Hong Kong.

OBJECTIVES: Tyrosinemia type I is an autosomal recessive disorder in tyrosine metabolism. In areas without expanded newborn screening, patients present with acute hepatorenal failure in early infancy. Diagnosis can be elusive when clinical presentation is non-specific and biochemical abnormalities are masked by secondary changes. This is the first Hong Kong Chinese report. DESIGN AND METHODS: A two-month-old Chinese male infant with unremarkable antenatal and postnatal history presented with progressive abdominal distension for three days. He suffered from end-stage liver failure, hypoglycemia and hepatic encephalopathy. Diagnostic work-up was complicated starting from rule-out sepsis, intestinal obstruction, volvulus, peritonitis, septic ileus, poisoning to metabolic diseases. Clinical, biochemical and genetic data was described. RESULTS: The patient showed increases in multiple plasma amino acids including tyrosine, phenylalanine and methionine, and hyper-excretions of 4-hydroxyphenyl-acetate, -pyruvate, and -lactate, as well as N-acetyltyrosine which could be seen in liver failure due to both tyrosinemia type I and non-metabolic conditions. Because of the volatile nature, succinylacetone was almost undetectable. The diagnosis was confirmed by genetic analysis of FAH with two novel mutations, viz. NM_000137.2:c.1063-1G>A and NM_000137.2:c.1035_1037del. Living-related liver transplantation was done. However, the patient still suffered many complications after the severe metabolic insult with hypoxic ischemic encephalopathy, cerebral atrophy, global developmental delay and cortical visual impairment. CONCLUSIONS: Because of the lack of expanded newborn screening in Hong Kong, this child unfortunately presented in the most severe form of tyrosinemia type I. Expanded newborn screening can save life and reduce the burden of diagnostic complexity. This illustrates the need for expanded newborn screening in Hong Kong.

High protein diet mimics hypertyrosinemia in newborn infants.

Tyrosinemia resulting from administration of protein-dense infant diets was detected by newborn screening in two infants. Change of formula resulted in rapid resolution of the hypertyrosinemia. These cases identify nonstandard infant diets as a benign and reversible cause of tyrosinemia and a potential cause of positive newborn phenylketonuria screening.

Outcome of tyrosinaemia type III.

Tyrosinaemia type III is a rare disorder caused by a deficiency of 4-hydroxyphenylpyruvate dioxygenase, the second enzyme in the catabolic pathway of tyrosine. The majority of the nine previously reported patients have presented with neurological symptoms after the neonatal period, while others detected by neonatal screening have been asymptomatic. All have had normal liver and renal function and none has skin or eye abnormalities. A further four patients with tyrosinaemia type III are described. It is not clear whether a strict low tyrosine diet alters the natural history of tyrosinaemia type III, although there remains a suspicion that treatment may be important, at least in infancy.