Infusion of platelets transiently reduces nucleoside overload in MNGIE

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disease caused by mutations in the gene encoding thymidine phosphorylase (ECGF1).¹ The disease is characterized by progressive external ophthalmoplegia, gastrointestinal dysmotility, cachexia, peripheral neuropathy, leukoencephalopathy, and mitochondrial dysfunction with alterations in mitochondrial DNA (mtDNA).² In patients with MNGIE, severe reduction of thymidine phosphorylase (TP) activity results in systemic accumulation of its substrates, thymidine (dThd) and deoxyuridine (dUrd).^3,4 Recently, in vitro studies have demonstrated that excess of dThd and dUrd leads to deoxynucleotide pool imbalances.⁵ The observation that several forms of mtDNA depletion syndrome are due to mutations in mitochondrial nucleoside/nucleotide metabolism enzymes² supports the notion that nucleotide imbalances can cause mtDNA instability.

Based on these findings, we proposed that reduction of circulating dThd and dUrd concentrations may be therapeutic for MNGIE.³ Here, we used platelet infusions to restore TP enzyme in MNGIE. Administration of platelets restored circulating TP activity and reduced plasma dThd and dUrd levels, suggesting that permanent restoration of TP would ameliorate the biochemical imbalances that cause this disorder.

Methods.

Results.

Platelet infusions partially restored TP catabolism of dThd and dUrd in both patients. Decline of nucleoside concentration is directly related to the amount of supplemental platelets (figure 1).

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Figure 1. Catabolism of thymidine (dThd) and deoxyuridine (dUrd) by platelets in blood in vitro. A: dThd; B: dUrd. Series represents blood of Patient 1 with different concentrations of exogenous platelets (♦ = no addition; ▪ = 10,000 platelets/μL; ▴ = 40,000 platelets/μL; = 100,000 platelets/μL) and blood from a healthy control spiked with dThd and dUrd at time zero (⋄), with no platelets added. C: calculated rates. To quantify the rates of nucleoside decay, data were adjusted through nonlinear regression to the expression C = C₀ × e^−kt, where C is the nucleoside concentration at a given time t, C₀ is the nucleoside concentration at t = 0, and k is the parameter determining the rate of nucleoside decay (elimination rate constant). R was always between 0.981 and 0.999. Half-life times were calculated from the expression as t_1/2 = (ln2)/k, using the estimated k values.

In Patient 1, circulating TP activity peaked 24 hours after each infusion, followed by a gradual decline (figure 2). Platelets transiently reduced nucleoside concentrations in both patients (figure 3, A and B). Urinary excretion of nucleosides decreased in both patients after the platelet infusions (figure 3, C and D). Patient 1 virtually stopped excreting dThd and dUrd, whereas Patient 2 excreted lower amounts of nucleosides (figure 3 and table E-1 on the Neurology Web site at www.neurology.org).

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Figure 2. Thymidine phosphorylase (TP) activity in buffy coat of Patient 1 during the period of platelet infusions. Vertical dotted lines indicate the times of the three platelet infusions.

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Figure 3. Plasma levels (A and B) and urinary excretion (C and D) of thymidine (dThd) and deoxyuridine (dUrd), over the time of platelet infusions. = dThd levels; ○ = dUrd levels; dashed horizontal lines = baseline levels of dThd and dUrd; vertical dotted lines = times of platelet infusions.

Discussion.

The identification of the causative gene for MNGIE¹ has allowed us to expand our understanding of the pathogenesis of this disease. A recent report of three patients with late-onset MNGIE demonstrated a correlation between the extent of TP dysfunction and severity of the clinical phenotype.⁷ These patients harbor less deleterious mutations in the ECGF1 gene, leading to a partial loss of TP activity (residual activity approximately 15% of normal) and only moderate increases of circulating dThd and dUrd levels (between 0.8 and 1.4 μM). Heterozygous ECGF1 mutation carriers with 26% to 35% residual TP activity are asymptomatic and have undetectable levels of nucleosides, indicating that TP activity exceeds requirements in healthy individuals.^3,7 Therefore, restoration of 26% to 35% of the total body TP activity should be sufficient to reduce nucleoside levels and halt the progression of the disease.

We previously proposed reduction of circulating nucleosides as a possible therapy for MNGIE; however, attempts to clear dThd from blood in two patients with MNGIE through hemodialysis indicated that only continuous elimination of nucleosides will lead to its permanent reduction.³ Here, we have analyzed the biochemical responses of patients with MNGIE to platelet infusions, a circulating source of TP activity. Our results demonstrate that platelets transiently provide TP activity and reduce plasma dThd and dUrd levels. The ability to deplete dThd and dUrd depends on the number of platelets, as shown by our in vitro study.

In Patient 1, peak TP activities reached levels 30% to 40% of normal activity in controls, and plasma dThd and dUrd were lowered to levels 50% to 70% of baseline. Maximum reductions coincided with highest TP activities. The reduction of nucleosides observed in Patient 2 was more pronounced and sustained. Because blood from Patient 2 was frozen and thawed before analysis, artifactual in vitro catabolism might account for the greater apparent effect of the platelet infusion. Alternatively, the difference in the circulating nucleoside levels after platelet treatment may be due to the second patient's elevated urinary nucleoside excretion, which might have synergistically enhanced the effect of platelet TP degradation of dThd and dUrd.

Daily production of dThd and dUrd in the Patient 1 can be estimated to be 68 to 107 μmol, based on the observation that she excreted these amounts over a 24-hour period before the treatment (table E-1). Pharmacokinetics studies suggest that dThd distributes in total body water volume,⁸ which can be estimated as 29 L for the patient.⁹ If total circulating TP were restored to the level found in a normal individual (k = 1.0647 hours⁻¹; figure 1), a production (K₀) of 2.83 μmol of dThd/hour (68 μmol/day) with a distribution volume (V_d) = 29 L would result in a steady-state dThd concentration (C_ss = K₀/V_d/k) of 0.09 μM. Similar calculations result in dUrd C_ss = 0.25 μM. These values could be even lower, because we did not consider urinary elimination of the nucleosides. Taken together, data from our in vitro and in vivo studies indicate that restoration of blood TP activity to patients with MNGIE could reduce nucleoside concentrations to undetectable levels, and such reduction is likely to extend to the tissues where dThd and dUrd cross membranes through nucleoside transporters.¹⁰

A more effective restoration of circulating TP could be achieved through direct administration of stabilized active TP protein or through strategies such as allogeneic stem cell transplantation or introduction of functional ECGF1 gene through viral vectors. In MNGIE, mitochondrial dysfunction and clinical symptoms are produced after years of cumulative toxic effects of imbalanced nucleosides on mtDNA. We anticipate that the process can be halted through the reduction of dThd and dUrd to normal or nearly normal levels. Unfortunately, somatic mtDNA mutations accumulated in postmitotic cells are unlikely to be reversible; therefore, treatments for MNGIE based on reduction of dThd and dUrd should be initiated early in the course of the disease, to prevent as much mitochondrial damage as possible.

Acknowledgment

The authors thank the patients and their families for their participation. The authors also thank Saba Tadesse for technical support.