Liver as a source for thymidine phosphorylase replacement in mitochondrial neurogastrointestinal encephalomyopathy.

Elisa Boschetti,Giovanna Cenacchi,Carlo Casali,Francesca Bianco,Vitaliano Tugnoli,Rita Rinaldi,Antonio D Pinna,Kerry Rhoden,Loris Pironi,Vincenzo Stanghellini,Massimo Del Gaudio,Valerio Carelli,Roberto D’Alessandro,Roberto De Giorgio,Paul A Cobine

doi:10.1371/journal.pone.0096692

Abstract

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare autosomal recessive mitochondrial disease associated with mutations in the nuclear TYMP gene. As a result, the thymidine phosphorylase (TP) enzyme activity is markedly reduced leading to toxic accumulation of thymidine and therefore altered mitochondrial DNA. MNGIE is characterized by severe gastrointestinal dysmotility, neurological impairment, reduced life expectancy and poor quality of life. There are limited therapeutic options for MNGIE. In the attempt to restore TP activity, allogenic hematopoietic stem cell transplantation has been used as cellular source of TP. The results of this approach on ∼20 MNGIE patients showed gastrointestinal and neurological improvement, although the 5-year mortality rate is about 70%. In this study we tested whether the liver may serve as an alternative source of TP. We investigated 11 patients (7M; 35–55 years) who underwent hepatic resection for focal disorders. Margins of normal liver tissue were processed to identify, quantify and localize the TP protein by Western Blot, ELISA, and immunohistochemistry, and to evaluate TYMP mRNA expression by qPCR. Western Blot identified TP in liver with a TP/GAPDH ratio of 0.9±0.5. ELISA estimated TP content as 0.5±0.07 ng/μg of total protein. TP was identified in both nuclei and cytoplasm of hepatocytes and sinusoidal lining cells. Finally, TYMP mRNA was expressed in the liver. Overall, our study demonstrates that the liver is an important source of TP. Orthotopic liver transplantation may be considered as a therapeutic alternative for MNGIE patients.

Highlights

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare autosomal recessive mitochondrial disease due to mutations in the nuclear TYMP gene encoding thymidine phosphorylase (TP)
TYMP mutations lead to a marked reduction of TP activity [2] resulting in a toxic accumulation of nucleosides in plasma of MNGIE patients
TP analysis and quantification The presence and the amount of TP in healthy human liver tissues were assessed by Western blot (WB) and ELISA (Figure 1)

Summary

Introduction

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare autosomal recessive mitochondrial disease due to mutations in the nuclear TYMP gene encoding thymidine phosphorylase (TP). This enzyme converts the nucleosides thymidine (dThd) and deoxyuridine into thymine and uracil, respectively [1]. TYMP mutations lead to a marked reduction (or virtual absence) of TP activity [2] resulting in a toxic accumulation of nucleosides in plasma of MNGIE patients This biochemical imbalance leads to secondary mitochondrial DNA (mtDNA) point mutations, multiple deletions and, more importantly, mtDNA depletion [3,4]. Since nuclear and mitochondrial nucleotide pools originate from different pathways, TYMP mutations do not influence nuclear DNA turnover [5]

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Results

Conclusion