Objectives Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare auto- somal recessive disease associated with the nuclear TYMP gene mutations. As a result, the thymidine phosphorylase (TP) activity is markedly reduced / lacking leading to mitochondrial DNA point mutations, multiple deletions and depletion. MNGIE is characterized by severe gastrointestinal (GI) dysmotility (i.e. gastroparesis and intestinal pseudo-obstruction) and neurological impairment. Typical MNGIE patients have ~5% of TP residual activity with symptom onset in the second decade and a life expectancy limited to the forth decade. In contrast, MNGIE relatives (heterozygous for TYMP mutations) show ~25-35% of TP residual activity and never manifest the syndrome. Such threshold may represent the target for new treatments. So far, there are no established therapeutic options for MNGIE. In order to restore a normal TP activity, allogenic hematopoietic stem cell transplantation (AHSCT) has been used as a cellular source of TP. Data on ~20 MNGIE patients undergone AHSCT showed GI and neurological improvement, although the 5-year survival rate is ~26%. Thus, other organ transplantation may represent an alternative for MNGIE patients. Since the liver is the main organ for protein biosynthesis and the transplantation success is estimated at ~90% of cases, the aim of this study was to test whether this organ can be a reliable source of TP. Methods A number of n= 11 patients (7 males; age range: 35-55 years) undergone hepatic resection for focal disorders (i.e., uncomplicated tumors in non-cirrhotic liver) were examined. Margins of normal liver tissue were processed for a variety of methodological approaches aimed to identify, quantify and localize TP protein, i.e. western blotting (WB), ELISA, and immunohistochemistry. TYMP mRNA specific expression has been evaluated via qPCR. We used bone marrow and intestinal mucosa as positive, while skeletal muscle and MNGIE buffy coat as negative controls. Results WB showed TP protein in liver tissue with a densitometric ratio TP/GAPDH of 0.9 ± 0.5 A.U. ELISA determined a TP content of 0.5 ± 0.07 ng/ug total proteins. Notably, the liver TP concentration was six time more than that of bone marrow (P <0.05), while negative controls did not have any detectable signal. TP immunoreactivity was localized in nuclei and cytoplasm of hepatocytes as well as in reticular cells. TYMP mRNA detection provided evidence of in situ liver synthesis. Bone marrow, liver, and duodenum expressed TYMP at a comparable level, whereas no expression was identified in the skeletal muscle. Conclusions The results of this study demonstrate that the liver can be a reliable source of TP, providing a rationale for liver transplantation in MNGIE patients

Mitochondrial Neurogastrointestinal Encephalomyopathy: The Liver As a Tissue Source to Restore Thymidine Phosphorylase Activity

BOSCHETTI, ELISA;LATORRE, ROCCO;TUGNOLI, VITALIANO;CARELLI, VALERIO;BIANCO, FRANCESCA;BONORA, ELENA;BARBARA, GIOVANNI;STANGHELLINI, VINCENZO;DE GIORGIO, ROBERTO
2014

Abstract

Objectives Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a rare auto- somal recessive disease associated with the nuclear TYMP gene mutations. As a result, the thymidine phosphorylase (TP) activity is markedly reduced / lacking leading to mitochondrial DNA point mutations, multiple deletions and depletion. MNGIE is characterized by severe gastrointestinal (GI) dysmotility (i.e. gastroparesis and intestinal pseudo-obstruction) and neurological impairment. Typical MNGIE patients have ~5% of TP residual activity with symptom onset in the second decade and a life expectancy limited to the forth decade. In contrast, MNGIE relatives (heterozygous for TYMP mutations) show ~25-35% of TP residual activity and never manifest the syndrome. Such threshold may represent the target for new treatments. So far, there are no established therapeutic options for MNGIE. In order to restore a normal TP activity, allogenic hematopoietic stem cell transplantation (AHSCT) has been used as a cellular source of TP. Data on ~20 MNGIE patients undergone AHSCT showed GI and neurological improvement, although the 5-year survival rate is ~26%. Thus, other organ transplantation may represent an alternative for MNGIE patients. Since the liver is the main organ for protein biosynthesis and the transplantation success is estimated at ~90% of cases, the aim of this study was to test whether this organ can be a reliable source of TP. Methods A number of n= 11 patients (7 males; age range: 35-55 years) undergone hepatic resection for focal disorders (i.e., uncomplicated tumors in non-cirrhotic liver) were examined. Margins of normal liver tissue were processed for a variety of methodological approaches aimed to identify, quantify and localize TP protein, i.e. western blotting (WB), ELISA, and immunohistochemistry. TYMP mRNA specific expression has been evaluated via qPCR. We used bone marrow and intestinal mucosa as positive, while skeletal muscle and MNGIE buffy coat as negative controls. Results WB showed TP protein in liver tissue with a densitometric ratio TP/GAPDH of 0.9 ± 0.5 A.U. ELISA determined a TP content of 0.5 ± 0.07 ng/ug total proteins. Notably, the liver TP concentration was six time more than that of bone marrow (P <0.05), while negative controls did not have any detectable signal. TP immunoreactivity was localized in nuclei and cytoplasm of hepatocytes as well as in reticular cells. TYMP mRNA detection provided evidence of in situ liver synthesis. Bone marrow, liver, and duodenum expressed TYMP at a comparable level, whereas no expression was identified in the skeletal muscle. Conclusions The results of this study demonstrate that the liver can be a reliable source of TP, providing a rationale for liver transplantation in MNGIE patients
Gastroenterology
795
795
E. Boschetti; R. Latorre; V. Tugnoli; V. Carelli; F. Bianco; E. Bonora; G. Barbara; V. Stanghellini; R. De Giorgio
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11585/394848
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