Determination of Thymidine Phosphorylase Activity in Human Blood Cells and Fibroblasts by a Nonradiochemical Assay Using Reversed-Phase High-Performance Liquid Chromatography

In this study, we demonstrated that the highest activity of thymidine phosphorylase (TP) was found in peripheral blood mononuclear (PBM) cells followed by that of thrombocytes and granulocytes whereas no activity of TP could be detected in erythrocytes. The activity of TP in leukocytes proved to be intermediate compared to the TP activity observed in PBM cells and granulocytes. The activity of TP also was readily detectable in human fibroblasts.     

CoQ10 deficiencies and MNGIE: Two treatable mitochondrial disorders

Background

Although causative mutations have been identified for numerous mitochondrial disorders, few disease-modifying treatments are available. Two examples of treatable mitochondrial disorders are coenzyme Q₁₀ (CoQ₁₀ or ubiquinone) deficiency and mitochondrial neurogastrointestinal encephalomyopathy (MNGIE).

Scope of review

Here, we describe clinical and molecular features of CoQ₁₀ deficiencies and MNGIE and explain how understanding their pathomechanisms have led to rationale therapies. Primary CoQ₁₀ deficiencies, due to mutations in genes required for ubiquinone biosynthesis, and secondary deficiencies, caused by genetic defects not directly related to CoQ₁₀ biosynthesis, often improve with CoQ₁₀ supplementation. In vitro and in vivo studies of CoQ₁₀ deficiencies have revealed biochemical alterations that may account for phenotypic differences among patients and variable responses to therapy. In contrast to the heterogeneous CoQ₁₀ deficiencies, MNGIE is a single autosomal recessive disease due to mutations in the TYMP gene encoding thymidine phosphorylase (TP). In MNGIE, loss of TP activity causes toxic accumulations of the nucleosides thymidine and deoxyuridine that are incorporated by the mitochondrial pyrimidine salvage pathway and cause deoxynucleoside triphosphate pool imbalances, which, in turn cause mtDNA instability. Allogeneic hematopoetic stem cell transplantation to restore TP activity and eliminate toxic metabolites is a promising therapy for MNGIE.

Major conclusions

CoQ₁₀ deficiencies and MNGIE demonstrate the feasibility of treating specific mitochondrial disorders through replacement of deficient metabolites or via elimination of excessive toxic molecules.

General significance

Studies of CoQ₁₀ deficiencies and MNGIE illustrate how understanding the pathogenic mechanisms of mitochondrial diseases can lead to meaningful therapies. This article is part of a Special Issue entitled: Biochemistry of Mitochondria, Life and Intervention 2010.     

Discovery profiling and bioinformatics analysis of serum microRNA in Mitochondrial NeuroGastroIntestinal Encephalomyopathy (MNGIE)

Abstract

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare and fatal inherited metabolic disorder due to mutations in the nuclear TYMP gene and leads to a deficiency in the enzyme thymidine phosphorylase. This results in an accumulation of the deoxynucleosides, thymidine and deoxyuridine in the cellular and extracellular compartments, ultimately leading to mitochondrial failure. The understanding of the precise molecular mechanisms that underlie the disease pathology is limited, being hampered by the rarity of the disorder. Expression profiling of serum based mircoRNAs and subsequent bioinformatical analyses provide an approach to facilitate the identity of dysregulated genes and signalling pathways potentially involved in the pathogenesis of MNGIE.     

Thymidine Phosphorylase Deficiency Causes MNGIE: An Autosomal Recessive Mitochondrial Disorder

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by mutations in the gene encoding thymidine phosphorylase (TP). The disease is characterized clinically by impaired eye movements, gastrointestinal dysmotility, cachexia, peripheral neuropathy, myopathy, and leukoencephalopathy. Molecular genetic studies of MNGIE patients' tissues have revealed multiple deletions, depletion, and site‐specific point mutations of mitochondrial DNA. TP is a cytosolic enzyme required for nucleoside homeostasis. In MNGIE, TP activity is severely reduced and consequently levels of thymidine and deoxyuridine in plasma are dramatically elevated. We have hypothesized that the increased levels of intracellular thymidine and deoxyuridine cause imbalances of mitochondrial nucleotide pools that, in turn, lead to the mtDNA abnormalities. MNGIE was the first molecularly characterized genetic disorder caused by abnormal mitochondrial nucleoside/nucleotide metabolism. Future studies are likely to reveal further insight into this expanding group of diseases.     

Thymidine and deoxyuridine accumulate in tissues of patients with mitochondrial neurogastrointestinal encephalomyopathy (MNGIE)

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disease due to ECGF1 gene mutations causing thymidine phosphorylase (TP) deficiency. Analysis of post-mortem samples of five MNGIE patients and two controls, revealed TP activity in all control tissues, but not in MNGIE samples. Converse to TP activity, thymidine and deoxyuridine were absent in control samples, but present in all tissues of MNGIE patients. Concentrations of both nucleosides in the tissues were generally higher than those observed in plasma of MNGIE patients. Our observations indicate that in the absence of TP activity, tissues accumulate nucleosides, which are excreted into plasma.     

Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE): Biochemical Features and Therapeutic Approaches

Over the last 15 years, important research has expanded our knowledge of the clinical, molecular genetic, and biochemical features of mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). The characterization of mitochondrial involvement in this disorder and the seminal determination of its genetic cause, have opened new possibilities for more detailed and deeper studies on the pathomechanisms in this progressive and fatal disease. It has been established that MNGIE is caused by mutations in the gene encoding thymidine phosphorylase (TP), which lead to absolute or nearly complete loss of its catalytic activity, producing systemic accumulations of its substrates, thymidine (dThd) and deoxyuridine (dUrd). Findings obtained from in vitro and in vivo studies indicate that the biochemical imbalances specifically impair mitochondrial DNA (mtDNA) replication, repair, or both leading to mitochondrial dysfunction. We have proposed that therapy for MNGIE should be aimed at reducing the concentrations of these toxic nucleosides to normal or nearly normal levels. The first treatment, allogeneic stem-cell transplantation (alloSCT) reported in 2006, produced a nearly full biochemical correction of the dThd and dUrd imbalances in blood. Clinical follow-up of this and other patients receiving alloSCT is necessary to determine whether this and other therapies based on a permanent restoration of TP will be effective treatment for MNGIE.     

The Role of Mitochondria in Stem Cell Biology

This mini-review summarizes the current literature on the role of mitochondrial DNA mutations and mitochondrial metabolism in stem cell biology. The possible uses of stem cells as a therapeutic tool in mitochondrial disorders are also reported.     

Molecular investigations of mitochondrial deletions: Evaluating the usefulness of different genetic tests

Deletions in mitochondrial DNA are a common cause of mitochondrial disorders. The molecular diagnosis of mtDNA deletions for years was based on Southern hybridization later replaced by PCR methods such as PCR with primers specific for a particular deletion (mainly the so-called common deletion of 4977bp) and long PCR. In order to evaluate the usefulness of MLPA (Multiplex Ligation-dependent Probe Amplification) in molecular diagnosis of large scale mtDNA deletions we compare four diagnostic methods: Southern hybridization, PCR, long-PCR and MLPA in a group of 16 patients with suspected deletions. Analysis was performed on blood, muscle and in one case hepatic tissue DNA. The MLPA was not able to confirm all the deletions detected by PCR methods, but due to its relative ease of processing, minimal equipment, low costs and the additional possibility to detect frequent point mtDNA mutations in one assay it is worth considering as a screening method. We recommend to always confirm MLPA results by PCR methods.     

Elevated plasma deoxyuridine in patients with thymidine phosphorylase deficiency

Mutations in the nuclear gene encoding thymidine phosphorylase (TP) cause mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), an autosomal recessive disease with mitochondrial dysfunction and mitochondrial DNA abnormalities. We have demonstrated alterations of thymidine (dThd) metabolism in MNGIE patients. Here, we report the accumulation of another substrate of TP, deoxyuridine (dUrd), whose circulating levels ranged from 5.5 to 24.4 microM (average 14.2) in MNGIE and were undetectable (<0.05 microM) in both TP mutation carriers and controls. The dramatic accumulation of dUrd may contribute to nucleotide pool imbalances and, together with the increased levels of dThd, is likely to contribute to the pathogenesis of MNGIE.