Functional Studies of Tyrosine Hydroxylase Missense Variants Reveal Distinct Patterns of Molecular Defects in Dopa ‐Responsive Dystonia

Abstract

Congenital tyrosine hydroxylase deficiency (THD) is found in autosomal-recessive Dopa-responsive dystonia and related neurological syndromes. The clinical manifestations of THD are variable, ranging from early-onset lethal disease to mild Parkinson disease-like symptoms appearing in adolescence. Until 2014, approximately 70 THD patients with a total of 40 different disease-related missense mutations, five nonsense mutations, and three mutations in the promoter region of the tyrosine hydroxylase (TH) gene have been reported. We collected clinical and biochemical data in the literature for all variants, and also generated mutant forms of TH variants previously not studied (N = 23). We compared the in vitro solubility, thermal stability, and kinetic properties of the TH variants to determine the cause(s) of their impaired enzyme activity, and found great heterogeneity in all these properties among the mutated forms. Some TH variants had specific kinetic anomalies and phenylalanine hydroxylase, and Dopa oxidase activities were measured for variants that showed signs of altered substrate binding. p.Arg233His, p.Gly247Ser, and p.Phe375Leu had shifted substrate specificity from tyrosine to phenylalanine and Dopa, whereas p.Cys359Phe had an impaired activity toward these substrates. The new data about pathogenic mechanisms presented are expected to contribute to develop individualized therapy for THD patients.

Discovery of compounds that protect tyrosine hydroxylase activity through different mechanisms

“…In order to do so PC12Adh (ATCC CRL-1721.1) cells expressing these hTH1 forms were grown for 24 or 48 h in the presence of 0, 40, and 100 μM of compound IV [28]. All three mutants were well expressed and presented b25% immunoreactive TH protein and 5-15% TH activity compared with wt, in the cell extracts of controls in the absence of compounds, which reflects the associated folding and catalytic defects, as expected from previous expression analyses [16]. Previous studies with wt-mice treated with compound IV (5 mg/kg/day given orally for 12 days) have shown a stimulating effect on the steady state level of TH activity in brain extracts, without increasing the amount of TH protein, which is the customary effect of pharmacological chaperones such as compound III [28].…”

“…THD is a very rare genetic disease, with about 70 patients reported worldwide [7,16,51], thus making it an ultra-orphan disease [52]. Research on drug development for these uncommon disorders is rather limited both in academia and in the pharmaceutical industry.…”

“…The different responses of each mutant, e.g. p.R202H and p.L205P, to each compound at either 24 or 48 h, appear to be associated with the low stabilizing effect of compound IV as well as the instability and misfolding degree of the mutation, which is highest for p.L205P [16], which may explain the lower activating effect at longer times for this mutant. We then studied the effect of compounds 2, 4, and 5.…”

“…THD-associated mutations correlate with loss-of-function-misfolding and instability of TH [14][15][16], and the mutants are rapidly degraded by intracellular proteases, leading to decreased TH and dopamine levels in THD patients. Increasing evidence points to pharmacological chaperones as a promising therapeutic strategy for loss-offunction misfolding diseases [17][18][19][20].…”

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“…Until now, approximately 70 THD patients with a total of 40 different disease-related missense mutations of the TH gene have been reported [14] . Genetic analysis revealed a compound heterozygote for novel mutations on TH gene: c.1451G>A (p.R484H) harbored by the father and a gene mutation c.1559T>A (p.L520H) of maternal origin.…”