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Gastrointestinal Dysmotility in Mitochondrial Neurogastrointestinal Encephalomyopathy Is Caused by Mitochondrial DNA Depletion

2008; Elsevier BV; Volume: 173; Issue: 4 Linguagem: Inglês

10.2353/ajpath.2008.080252

1525-2191

Carla Giordano, Mariangela Sebastiani, Roberto De Giorgio, Claudia Travaglini, Andrea Tancredi, Maria Lucia Valentino, Marzio Bellan, Andrea Cossarizza, Michio Hirano, Giulia d’Amati, Valério Carelli,

ATP Synthase and ATPases Research

Chronic intestinal pseudo-obstruction is a life-threatening condition of unknown pathogenic mechanisms. Chronic intestinal pseudo-obstruction can be a feature of mitochondrial disorders, such as mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), a rare autosomal-recessive syndrome, resulting from mutations in the thymidine phosphorylase gene. MNGIE patients show elevated circulating levels of thymidine and deoxyuridine, and accumulate somatic mitochondrial DNA (mtDNA) defects. The present study aimed to clarify the molecular basis of chronic intestinal pseudo-obstruction in MNGIE. Using laser capture microdissection, we correlated the histopathological features with mtDNA defects in different tissues from the gastrointestinal wall of five MNGIE and ten control patients. We found mtDNA depletion, mitochondrial proliferation, and smooth cell atrophy in the external layer of the muscularis propria, in the stomach and in the small intestine of MNGIE patients. In controls, the lowest amounts of mtDNA were present at the same sites, as compared with other layers of the gastrointestinal wall. We also observed mitochondrial proliferation and mtDNA depletion in small vessel endothelial and smooth muscle cells. Thus, visceral mitochondrial myopathy likely causes gastrointestinal dysmotility in MNGIE patients. The low baseline abundance of mtDNA molecules may predispose smooth muscle cells of the muscularis propria external layer to the toxic effects of thymidine and deoxyuridine, and exposure to high circulating levels of nucleosides may account for the mtDNA depletion observed in the small vessel wall. Chronic intestinal pseudo-obstruction is a life-threatening condition of unknown pathogenic mechanisms. Chronic intestinal pseudo-obstruction can be a feature of mitochondrial disorders, such as mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), a rare autosomal-recessive syndrome, resulting from mutations in the thymidine phosphorylase gene. MNGIE patients show elevated circulating levels of thymidine and deoxyuridine, and accumulate somatic mitochondrial DNA (mtDNA) defects. The present study aimed to clarify the molecular basis of chronic intestinal pseudo-obstruction in MNGIE. Using laser capture microdissection, we correlated the histopathological features with mtDNA defects in different tissues from the gastrointestinal wall of five MNGIE and ten control patients. We found mtDNA depletion, mitochondrial proliferation, and smooth cell atrophy in the external layer of the muscularis propria, in the stomach and in the small intestine of MNGIE patients. In controls, the lowest amounts of mtDNA were present at the same sites, as compared with other layers of the gastrointestinal wall. We also observed mitochondrial proliferation and mtDNA depletion in small vessel endothelial and smooth muscle cells. Thus, visceral mitochondrial myopathy likely causes gastrointestinal dysmotility in MNGIE patients. The low baseline abundance of mtDNA molecules may predispose smooth muscle cells of the muscularis propria external layer to the toxic effects of thymidine and deoxyuridine, and exposure to high circulating levels of nucleosides may account for the mtDNA depletion observed in the small vessel wall. Chronic intestinal pseudo-obstruction (CIPO) is a highly morbid and often life-threatening condition characterized by marked dysfunction of gut propulsive motility, which results in a clinical picture mimicking mechanical obstruction.1Connor FL Di Lorenzo C Chronic intestinal pseudo-obstruction: assessment and management.Gastroenterology. 2006; 30: S29-S36Abstract Full Text Full Text PDF Scopus (127) Google Scholar, 2Stanghellini V Cogliandro RF De Giorgio R Barbara G Salvioli B Corinaldesi R Chronic intestinal pseudo-obstruction: manifestations, natural history and management.Neurogastroenterol Motil. 2007; 19: 440-452Crossref PubMed Scopus (136) Google Scholar, 3De Giorgio R Sarnelli G Corinaldesi R Stanghellini V Advances in our understanding of the pathology of chronic intestinal pseudo-obstruction.Gut. 2004; 53: 1549-1552Crossref PubMed Scopus (208) Google Scholar Patients with CIPO usually complain of severe symptoms including abdominal pain and distension, early satiety, bloating, and vomiting, as well as constipation and/or diarrhea. CIPO is an important cause of chronic intestinal failure, because affected individuals become unable to maintain normal nutrition and body weight. Concerning etiologies, CIPO may be primary or secondary to a variety of systemic diseases.2Stanghellini V Cogliandro RF De Giorgio R Barbara G Salvioli B Corinaldesi R Chronic intestinal pseudo-obstruction: manifestations, natural history and management.Neurogastroenterol Motil. 2007; 19: 440-452Crossref PubMed Scopus (136) Google Scholar, 3De Giorgio R Sarnelli G Corinaldesi R Stanghellini V Advances in our understanding of the pathology of chronic intestinal pseudo-obstruction.Gut. 2004; 53: 1549-1552Crossref PubMed Scopus (208) Google Scholar Primary CIPO may be due to abnormalities of smooth muscle cells of muscularis propria (ie, visceral myopathy) and/or enteric neuronal supplies of gastrointestinal (GI) wall (ie, visceral neuropathy).3De Giorgio R Sarnelli G Corinaldesi R Stanghellini V Advances in our understanding of the pathology of chronic intestinal pseudo-obstruction.Gut. 2004; 53: 1549-1552Crossref PubMed Scopus (208) Google Scholar In addition, abnormalities of the GI pacemaker cells, the interstitial cells of Cajal have been reported.4Jain D Moussa K Tandon M Culpepper-Morgan J Proctor DD Role of interstitial cells of Cajal in motility disorders of the bowel.Am J Gastroenterol. 2003; 98: 618-624Crossref PubMed Scopus (112) Google Scholar CIPO is an increasingly recognized clinical feature of mitochondrial encephalomyopathies.5Bindoff L Mitochondrial gastroenterology.in: Di Mauro S Hirano M Schon EA Mitochondrial Medicine. Informa Healthcare, Abington, UK2006: 143-159Crossref Google Scholar This heterogeneous group of genetic disorders is caused by dysfunction of the mitochondrial respiratory chain that usually affects highly energy dependent tissues such as brain and muscle.6DiMauro S Schon EA Mitochondrial respiratory-chain diseases.N Engl J Med. 2003; 348: 2656-2668Crossref PubMed Scopus (1343) Google Scholar Among mitochondrial encephalomyopathies, one most frequently associated with GI dysmotility and CIPO is mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), an autosomal recessive syndrome due to mutations in the thymidine phosphorylase gene TYMP.7Nishino I Spinazzola A Papadimitriou A Hammans S Steiner I Hahn CD Connolly AM Verloes A Guimarães J Maillard I Hamano H Donati MA Semrad CE Russell JA Andreu AL Hadjigeorgiou GM Vu TH Tadesse S Nygaard TG Nonaka I Hirano I Bonilla E Rowland LP DiMauro S Hirano M Mitochondrial neurogastrointestinal encephalomyopathy: an autosomal recessive disorder due to thymidine phosphorylase mutations.Ann Neurol. 2000; 47: 792-800Crossref PubMed Scopus (309) Google Scholar MNGIE is defined clinically by severe GI dysmotility, cachexia, ptosis, ophthalmoparesis, peripheral neuropathy, white matter changes in brain magnetic resonance imaging, and mitochondrial abnormalities.7Nishino I Spinazzola A Papadimitriou A Hammans S Steiner I Hahn CD Connolly AM Verloes A Guimarães J Maillard I Hamano H Donati MA Semrad CE Russell JA Andreu AL Hadjigeorgiou GM Vu TH Tadesse S Nygaard TG Nonaka I Hirano I Bonilla E Rowland LP DiMauro S Hirano M Mitochondrial neurogastrointestinal encephalomyopathy: an autosomal recessive disorder due to thymidine phosphorylase mutations.Ann Neurol. 2000; 47: 792-800Crossref PubMed Scopus (309) Google Scholar GI dysmotility leads to progressive weight loss and cachexia of MNGIE patients, and diverticulosis of small intestine complicated by inflammation and perforation often causes their death in early adulthood. Biochemical abnormalities in MNGIE include drastically reduced thymidine phosphorylase activity leading to accumulation of thymidine (dThd) and deoxyuridine (dUrd) in blood and tissues.8Spinazzola A Marti R Nishino I Andreu AL Naini A Tadesse S Pela I Zammarchi E Donati MA Oliver JA Hirano M Altered thymidine metabolism due to defects of thymidine phosphorylase.J Biol Chem. 2002; 277: 4128-4133Crossref PubMed Scopus (198) Google Scholar, 9Valentino ML Martí R Tadesse S López LC Manes JL Lyzak J Hahn A Carelli V Hirano M Thymidine and deoxyuridine accumulate in tissues of patients with mitochondrial neurogastrointestinal encephalomyopathy (MNGIE).FEBS Lett. 2007; 581: 3410-3414Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar Toxic levels of dThd and dUrd induce nucleotide pool imbalances that in turn lead to mtDNA abnormalities (point mutations, multiple deletions, and depletion).8Spinazzola A Marti R Nishino I Andreu AL Naini A Tadesse S Pela I Zammarchi E Donati MA Oliver JA Hirano M Altered thymidine metabolism due to defects of thymidine phosphorylase.J Biol Chem. 2002; 277: 4128-4133Crossref PubMed Scopus (198) Google Scholar, 10Nishigaki Y Marti R Hirano M ND5 is a hot-spot for multiple atypical mitochondrial DNA deletions in mitochondrial neurogastrointestinal encephalomyopathy.Hum Mol Genet. 2004; 13: 91-101Crossref PubMed Scopus (84) Google Scholar, 11Ferraro P Pontarin G Crocco L Fabris S Reichard P Bianchi V Mitochondrial deoxynucleotide pools in quiescent fibroblasts: a possible model for mitochondrial neurogastrointestinal encephalomyopathy (MNGIE).J Biol Chem. 2005; 280: 24472-24480Crossref PubMed Scopus (74) Google Scholar, 12Song S Wheeler LJ Mathews CK Deoxyribonucleotide pool imbalance stimulates deletions in HeLa cell mitochondrial DNA.J Biol Chem. 2003; 278: 43893-43896Crossref PubMed Scopus (103) Google Scholar The pathogenic mechanisms causing GI dysmotility in MNGIE are still unclear. We recently showed atrophy, mitochondrial proliferation, and mtDNA depletion in muscularis propria of small intestine in one patient.13Giordano C Sebastiani M. Plazzi G. Travaglini C. Sale P. Pinti M. Tancredi A. Liguori R. Montagna P. Bellan M. Valentino M.L. Cossarizza A. Hirano M. Carelli V. Mitochondrial neurogastrointestinal encephalomyopathy: evidence of mitochondrial DNA depletion in the small intestine.Gastroenterology. 2006; 130: 893-901Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar In the present study, we provide a detailed morphological and molecular investigation of the entire GI tract in five MNGIE patients, hence establishing a link between marked mtDNA depletion and myopathic changes of the external layer of muscularis propria in this syndrome. A summary of clinical features of the five MNGIE patients investigated is reported in Table 1.Table 1Summary of the Clinical Data from MNGIE PatientsPatient (reference no.)GenderOnsetAge at diagnosisAge at deathTYMP mutationExon/intronBlood thymidine levels1 (13)M5 years epilepsy and childhood-onset bilateral ptosis, 25 years onset GI symptoms35 years38 yearsc.457 G > A homozygousExon 47.6 μmol/L2MChildhood-onset GI symptoms, 37 years onset bilateral ptosis38 years39 yearsc.522T > A homozygousExon 57.0 μmol/L3 (patient 3 in reference 7Nishino I Spinazzola A Papadimitriou A Hammans S Steiner I Hahn CD Connolly AM Verloes A Guimarães J Maillard I Hamano H Donati MA Semrad CE Russell JA Andreu AL Hadjigeorgiou GM Vu TH Tadesse S Nygaard TG Nonaka I Hirano I Bonilla E Rowland LP DiMauro S Hirano M Mitochondrial neurogastrointestinal encephalomyopathy: an autosomal recessive disorder due to thymidine phosphorylase mutations.Ann Neurol. 2000; 47: 792-800Crossref PubMed Scopus (309) Google Scholar)M18 years foot numbness (neuropathy)34 years37 yearsc.433 G > A homozygousExon 4Not determined4 (patient 4 in reference 7Nishino I Spinazzola A Papadimitriou A Hammans S Steiner I Hahn CD Connolly AM Verloes A Guimarães J Maillard I Hamano H Donati MA Semrad CE Russell JA Andreu AL Hadjigeorgiou GM Vu TH Tadesse S Nygaard TG Nonaka I Hirano I Bonilla E Rowland LP DiMauro S Hirano M Mitochondrial neurogastrointestinal encephalomyopathy: an autosomal recessive disorder due to thymidine phosphorylase mutations.Ann Neurol. 2000; 47: 792-800Crossref PubMed Scopus (309) Google Scholar)F15 years borborgymi, abdominal pain, diarrhea26 years28 yearsc.665 A > G c.1406 insCExon 6 exon 10Not determined5 (patient 7 in reference 7Nishino I Spinazzola A Papadimitriou A Hammans S Steiner I Hahn CD Connolly AM Verloes A Guimarães J Maillard I Hamano H Donati MA Semrad CE Russell JA Andreu AL Hadjigeorgiou GM Vu TH Tadesse S Nygaard TG Nonaka I Hirano I Bonilla E Rowland LP DiMauro S Hirano M Mitochondrial neurogastrointestinal encephalomyopathy: an autosomal recessive disorder due to thymidine phosphorylase mutations.Ann Neurol. 2000; 47: 792-800Crossref PubMed Scopus (309) Google Scholar)F26 PEO36 years39 yearsc.866 A > C IVS9-1 G > CExon 7 intron 85.7 μmol/LGI symptomsNeuromuscularCNSOtherBorborygmi, diarrhea, abdominal pain, dysphagia, diverticulosis, acute intestinal pseudo-obstructionPEO, demyelinating sensorimotor polyneuropathy and myopathy (EMG), COX-negative fibers at muscle biopsyEpilepsy, sensorineural deafness, diffuse white matter hyperintensity (MRI)Markedly cachectic BMI = 12.9Borborygmi, diarrhea, abdominal pain, dysphagiaPEO, demyelinating sensorimotor polyneuropathy and signs of myopathy (EMG), COX-negative fibers at muscle biopsySensorineural deafness, diffuse white matter hyperintensity (MRI)Markedly cachectic BMI = 15.7Borborygmi, diarrhea, abdominal cramps and pain, intestinal pseudo-obstruction, diverticulosisPEO, demyelinating sensorimotor polyneuropathy and signs of myopathy (EMG), COX-negative fibers and RRF at muscle biopsyDiffuse white matter hyperintensity (MRI)Markedly cachectic BMI = 13.8Borborygmi, abdominal pain, diarrhea, intestinal pseudo-obstruction, diverticulosisPEO, demyelinating sensorimotor polyneuropathy, epilepsy, COX-negative fibers and RRF at muscle biopsyDiffuse white matter hyper intensity (MRI)Markedly cachectic BMI = 13.8Borborygmi, early satiety, abdominal cramps and painPEO, demyelinating sensorimotor polyneuropathy, COX-negative fibers and RRF at muscle biopsyDiffuse white matter hyperintensity (MRI)Markedly cachectic BMI = 14.8BMI, body mass index; COX, cytochrome c oxidase; EMG, electromyography; GI, gastrointestinal; MRI, magnetic resonance imaging; PEO, progressive external ophthalmoplegia; RRF, ragged red fibers. Open table in a new tab BMI, body mass index; COX, cytochrome c oxidase; EMG, electromyography; GI, gastrointestinal; MRI, magnetic resonance imaging; PEO, progressive external ophthalmoplegia; RRF, ragged red fibers. All studies conformed to Sapienza, University of Rome, Ethical Committee protocols. At autopsy, after informed consent was provided from relatives, multiple tissue samples were obtained from esophagus, stomach, small intestine, and colon from five MNGIE patients. We used as controls, ten age-matched sudden cardiac death cases whose autopsies were performed in the Department of Pathology, Sapienza, University of Rome. Autopsies from both MNGIE patients and controls were performed after 24 to 30 hours after death. For molecular analysis of whole tissue homogenate, samples were snap-frozen in liquid nitrogen-chilled isopentane. For histological analysis, tissue sections obtained from formalin-fixed, paraffin-embedded samples, were stained with H&E and Masson's trichrome. Immunohistochemistry for S-100, synaptophysin, neuronal-specific enolase, glial fibrillary acidic protein (DAKO Glostrup, Denmark), and mitochondrial antigens (Clone MTC, UCS Diagnostic, Morlupo, Italy) was also performed. Combined cytochrome c oxidase/succinate dehydrogenase (COX/SDH) stain was performed on frozen sections of proximal esophagus and small intestine obtained from patients 3 and 4 (see Table 1). Combining the histoenzymatic mitochondrial activities of COX (orange) and SDH (blue) results in a brown stain. Single cells bearing mtDNA defects commonly lose their COX activity; however, leaving intact the nuclear-encoded SDH activity, thus becoming highlighted in blue on the brown background. For ultrastructural analysis, samples were fixed in 4% paraformaldehyde-phosphate buffered saline and postfixed in osmium tetroxide. Thin sections were stained with uracyl acetate and lead citrate and examined with a CM10 Philips electron microscope (Eindhoven, the Netherlands). Paraffin sections from patients and controls were subjected to laser capture microdissection with the MMI NIKON UV-CUT System (Molecular Machines & Industries, Glattbrug, Switzerland) as previously described.13Giordano C Sebastiani M. Plazzi G. Travaglini C. Sale P. Pinti M. Tancredi A. Liguori R. Montagna P. Bellan M. Valentino M.L. Cossarizza A. Hirano M. Carelli V. Mitochondrial neurogastrointestinal encephalomyopathy: evidence of mitochondrial DNA depletion in the small intestine.Gastroenterology. 2006; 130: 893-901Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar Briefly, serial 5-μm-thick cut sections were mounted on a polyethylene foil slide and stained with H&E. Sections were observed under light microscope with a ×40 objective. Selected tissue areas were microdissected by an UV laser, which performs circumferential dissection, following precisely a drawn incision path. The microdissected tissue areas were measured, documented, and collected on an adhesive cap of nanotubes for nucleic acid extraction. All microdissection experiments were performed in triplicate. The following cell types were separately microdissected: 1) smooth muscle cells from tunica muscularis of esophagus; 2) skeletal fibers of cricopharyngeal muscle; 3) smooth muscle cells from internal and external layers of muscularis propria; 4) myenteric ganglion cells from stomach, small intestine, and colon; and 5) smooth muscle and endothelial cells from the wall of small arteries and arterioles from submucosal layer of the GI tract and from liver, kidney, and pancreas parenchyma. Histological recognition of cell types for laser capture microdissection was based on strict morphological criteria. In selected cases, to confirm the detection of smooth muscle cells within fibrous tissue, slides were stained with Masson's trichrome (not shown). Between 50 and 100 cells were collected for each cell population and pooled for analyses. In addition, frozen sections of proximal esophagus stained with combined COX/SDH were subjected to laser capture microdissection to isolate single COX-positive and COX-negative skeletal muscle cells from cricopharyngeal muscle. Total DNA was extracted from whole tissue homogenates by phenol-chloroform standard procedures and from dissected samples with Picopure DNA extraction Kit (Arcturus, Los Altos, CA). Amounts of mtDNA were measured in both homogenate and microdissected tissues by real-time quantitative PCR (RT-PCR) assays using a previously described method.14Mussini C Pinti M Bugarini R Borghi V Nasi M Nemes E Troiano L Guaraldi G Bedini A Sabin C Esposito R Cossarizza A Effect of CD4-monitored treatment interruption on mitochondrial DNA content in HIV-infected patients: a prospective study.AIDS. 2005; 19: 1627-1633Crossref PubMed Scopus (51) Google Scholar Briefly, a mtDNA fragment (nt 4625 to 4714) and a nuclear DNA fragment corresponding to FasL gene were co-amplified by multiplex polymerase chain reaction using TaqMan probe system and Platinum Quantitative PCR SuperMix-UDG (Invitrogen, Life Technologies, Parsley, UK). PCR conditions, primers, and probes were as previously detailed.14Mussini C Pinti M Bugarini R Borghi V Nasi M Nemes E Troiano L Guaraldi G Bedini A Sabin C Esposito R Cossarizza A Effect of CD4-monitored treatment interruption on mitochondrial DNA content in HIV-infected patients: a prospective study.AIDS. 2005; 19: 1627-1633Crossref PubMed Scopus (51) Google Scholar With each assay, a standard curve for mtDNA and nDNA was generated using serial known dilutions of a vector (provided by Genemore, Modena, Italy) in which the regions used as template for the two amplifications were cloned tail to tail, to have a ratio of 1:1 of the reference molecules. The absolute mtDNA copy number per cell was obtained by the ratio of mtDNA to nDNA values multiplied by 2 (as two copies of the nuclear gene are present in a cell). PCR was performed in an “iCycler” Thermal cycler (BioRad, Hercules, CA) and at least three measurements were obtained for each sample. Evaluation of mtDNA deletions (ΔmtDNA) on tissue homogenates was performed by Southern blot analysis.10Nishigaki Y Marti R Hirano M ND5 is a hot-spot for multiple atypical mitochondrial DNA deletions in mitochondrial neurogastrointestinal encephalomyopathy.Hum Mol Genet. 2004; 13: 91-101Crossref PubMed Scopus (84) Google Scholar Several PCR reactions with shifted primers were performed to detect the mtDNA deletions on microdissected tissues, as described.10Nishigaki Y Marti R Hirano M ND5 is a hot-spot for multiple atypical mitochondrial DNA deletions in mitochondrial neurogastrointestinal encephalomyopathy.Hum Mol Genet. 2004; 13: 91-101Crossref PubMed Scopus (84) Google Scholar To evaluate the deletion junctions of mtDNA molecules, a series of PCR experiments, with the following set of oligonucleotide primers, were performed as described10Nishigaki Y Marti R Hirano M ND5 is a hot-spot for multiple atypical mitochondrial DNA deletions in mitochondrial neurogastrointestinal encephalomyopathy.Hum Mol Genet. 2004; 13: 91-101Crossref PubMed Scopus (84) Google Scholar: forward primer, nt 8287 to 8306 and reverse primer, nt 13590 to 13571, for the Δ5 kb fragment; forward primer, nt 6229 to 6249 and reverse primer, nt 14268 to 14249, for the Δ7.7 Kb fragment; forward primer, nt 5651 to 5671 and reverse primer, nt 14268 to 14249 for the Δ8.1 Kb fragment; and forward primer, nt 4370 to 4390 and reverse primer, nt 14268 to 14249 for the Δ9.5 Kb fragment. PCR-amplified fragments were visualized by electrophoresis in a 2% agarose gel, extracted using the QIA quick gel extraction kit (Quiagen, Valencia, CA), and sequenced in an ABI Prism 310 Genetic analyzer (Applied-Biosystem, Foster City, CA) following standard procedures. Since it was not possible to define the breakpoint of the Δ8.1-Kb and of the Δ9.5-Kb deletions by direct sequencing of the PCR product, PCR-amplified fragments were ligated into pGEM-T Easy Vector and subcloned using pGEM-T Easy Vector System (Promega, Madison, WI). Approximately 10 cloned plasmids of each PCR product were purified using Wizard Plus SV Minipreps DNA Purification Systems (Promega, Madison, WI) and then sequenced. To screen for the presence of mtDNA point mutation in microdissected tissues, we amplified by PCR and sequenced three selected mtDNA regions corresponding to nt 5651 to 6022, nt 9917 to 10568, and nt 15756 to 16119. Within these mtDNA segments, point mutations have been identified in most MNGIE patients.15Nishigaki Y Marti' R Copeland WC Hirano M Site-specific somatic mitochondrial DNA point mutations in patients with thymidine phosphorylase deficiency.J Clin Invest. 2003; 111: 1913-1921Crossref PubMed Scopus (167) Google Scholar Statistical analysis was performed on the data with a mixed effect model. This model can explain the various values of mtDNA in different tissues taking account of patient heterogeneity.16Pinhero JC Bates DM Linear mixed effects models, basics concepts and examples.in: Pinhero JC Bates DM Mixed-Effects Models in S and S-PLUS. Springer, New York2000: 3-52Crossref Google Scholar Numerical estimate have been obtained by the statistical software R Foundation for Statistical Computing, Vienna, Austria with the package nlme (www.r-project.org last accessed November 19, 2007). Simple linear regression was performed with mtDNA copy number/cells means in different tissue components of GI in MNGIE patients and controls. All five MNGIE patients showed similar morphological features in the GI wall. The most remarkable abnormalities were observed in the external longitudinal layer of muscularis propria of stomach and small intestine, which showed atrophy and vacuolization of smooth muscle cells, and interstitial fibrosis. These features were more prominent in the small intestine, which showed patchy areas with only a few residual muscle cells within the fibrous tissue (Figure 1, A and B). In contrast, the external layer of muscularis propria from the large bowel (not shown) and the internal layer of muscularis propria of the entire GI tract appeared normal. The myenteric and submucosal nervous plexi appeared well preserved and showed a normal distribution with immunostains for S-100, synaptophysin, neuronal-specific enolase, and glial fibrillar acidic protein (not shown). Immunostain with anti-mitochondria antibodies showed marked mitochondrial proliferation at the level of muscularis propria of GI tract in all patients, as compared with controls (Figure 1, C and D). Electron microscopy confirmed this finding (Figure 1E). The combined COX/SDH stain revealed clusters of COX-negative smooth muscle cells in the muscularis propria of small intestine and focally, COX deficient ganglion cells in the myenteric plexus in two patients (patients 3 and 4) (Figure 1, F and H). These abnormalities were absent in control tissues (Figure 1G). A marked mitochondrial proliferation was observed also in smooth muscle and endothelial cells from the wall of small arteries and arterioles in the GI tract and other visceral organs (liver, kidney, heart, and pancreas, not shown) of MNGIE patients (Figure 2A), as compared with controls (Figure 2B). These vessels appeared COX-negative with combined COX/SDH stain (Figure 2C). Electron microscopy confirmed the presence of abundant mitochondria in endothelial cells from small vessels (Figure 2D). Tunica muscularis of proximal esophagus did not show morphological alterations (Figure 3A). However, combined COX/SDH stain revealed numerous COX-negative fibers with increased SDH intensity in the cricopharyngeal muscle (Figure 3B).Figure 2Mitochondrial proliferation in smooth muscle and endothelial cells of small vessels in MNGIE. A: Immunostain for mitochondrial antigens shows mitochondrial proliferation in the tunica media of a small artery from a MNGIE patient (patient 1, anti-mitochondrial antigens antibody, clone MTC, UCS Diagnostic, original magnification ×10). B: Immunostain for mitochondrial antigens in a small artery from a control subject (Anti-mitochondrial antigens antibody, clone MTC, UCS Diagnostic, original magnification ×10). C: The combined COX/SDH stain shows a blue COX-negative vessel from the small bowel surrounded by brown COX-positive smooth muscle cells of muscularis propria (patient 3, COX/SDH, original magnification ×40). D: Ultrastructural features of endothelial cells from a small vessel of MNGIE patient 2. Note the numerous mitochondria (arrows).with postmortem artifactual swelling. A red blood cell is marked by the asterisk.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Histopathology and molecular analysis of esophagus in MNGIE. A: The wall of proximal esophagus has a normal appearance. The arrow indicates smooth muscle cells of tunica muscularis and the asterisk marks the striated fibers of the cricopharyngeal muscle (patient 2, H&E, original magnification ×4). B: The combined COX/SDH stain shows frequent COX-negative fibers in blue (patient 3, COX/SDH, original magnification ×40). C: Southern blot analysis of DNA from tissue homogenates of patient 1 shows multiple deletions only in the esophagus and skeletal muscle. In addition to the signal from the 16.6-kb mtDNA molecule (WT), signals of lower molecular weight, corresponding to mtDNA deleted molecules (arrows), are evident. C, colon; S, stomach; I, ileum; E, esophagus; and M, skeletal muscle. M+ is a positive control with multiple deletion. M− is a control subject. U, uncut sample. D: The Δ5-Kb, Δ7.7-Kb, Δ8.1-Kb, and Δ9.5-Kb deletions were investigated by PCR assays using oligonucleotide primers flanking the regions upstream and downstream breakpoints in the parental molecule (see Materials and Methods). Representative 2% agarose gels from patient 3. The bands corresponding to the mtDNA deletions were detected in esophagus homogenate (E) and in the microdissected striated fibers from cricopharyngeal muscle (Sk). No deletions were detected in smooth muscle cells from tunica muscularis (Sm). (Negative control, C−; the arrow indicate 500 bp). E: MtDNA content was evaluated in single COX-positive (blue bar) and COX-negative (green bar) skeletal muscle fibers from cricopharyngeal muscle from MNGIE patients 3 and 4 versus three controls (red bar). In MNGIE patients, mtDNA/nuclear DNA ratio is less than normal controls, in both COX-positive and COX-negative fibers. Severely COX-deficient fibers have lower mtDNA amount than fibers with residual COX activity.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Analyses of tissue homogenates from GI tract of MNGIE patients demonstrated mtDNA deletions only in the upper esophagus. The most abundant deletions were the same as those reported in skeletal muscle of MNGIE patients,10Nishigaki Y Marti R Hirano M ND5 is a hot-spot for multiple atypical mitochondrial DNA deletions in mitochondrial neurogastrointestinal encephalomyopathy.Hum Mol Genet. 2004; 13: 91-101Crossref PubMed Scopus (84) Google Scholar and corresponded to the “common deletion,” Δ5.0 kb, Δ7.7 kb, Δ8.1 kb, and Δ9.5 kb (Figure 3C). The deletion junctions were as described.10Nishigaki Y Marti R Hirano M ND5 is a hot-spot for multiple atypical mitochondrial DNA deletions in mitochondrial neurogastrointestinal encephalomyopathy.Hum Mol Genet. 2004; 13: 91-101Crossref PubMed Scopus (84) Google Scholar, 13Giordano C Sebastiani M. Plazzi G. Travaglini C. Sale P. Pinti M. Tancredi A. Liguori R. Montagna P. Bellan M. Valentino M.L. Cossarizza A. Hirano M. Carelli V. Mitochondrial neurogastrointestinal encephalomyopathy: evidence of mitochondrial DNA depletion in the small intestine.Gastroenterology. 2006; 130: 893-901Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar Analysis of microdissected smooth muscle cells from tunica muscularis and striated muscle cells from cricopharyngeal muscle revealed detectable levels of deleted mtDNA molecules only in striated muscle fibers (Figure 3D). Attempts to detect mtDNA deletions in single COX-positive and COX-negative cricopharyngeal fibers failed, due to the inability to amplify mtDNA from COX-negative fibers. The likely explanation for this phenomenon is the marked mtDNA depletion at this site, as described below. However, we confirmed the presence of mtDNA deletions in COX-positive fibers as reported17Krishnan KJ Bender A Taylor RW Turnbull DM A multiplex real-time PCR method to detect and quantify mitochondrial DNA deletions in individual cells.Anal Biochem. 2007; 370: 127-129Crossref PubMed Scopus (76) Google Scholar (not shown). Analyses of microdissected smooth muscle cells from muscularis propria and ganglion cells from myenteric plexi of stomach, small intestine, and colon, and from smooth muscle and endothelial cells of small arteries and arterioles did not show mtDNA deletions in MNGIE patients (not shown). Analyses of tissue homogenates revealed, in all MNGIE patients, a marked mtDNA depletion confined to the small intestine (91% decreased compared to controls). A milder reduction in mtDNA amount was observed also in the stomach (43% decrease), whereas esophagus and colon did not show differences between patients and controls (Figure 4A). Noteworthy, even in the control group, we observed a non-homogeneous distribution of mtDNA within the different segments of GI tract; the relative amount of mtDNA in the small intestine was 55% of the level in esophagus, 49% of stomach, and 61% of colon (Figure 4A), confirming our previous report.13Giordano C Sebastiani M. Plazzi G. Travaglini C. Sale P. Pinti M. Tancredi A. Liguori R. Montagna P. Bellan M. Valentino M.L. Cossarizza A. Hirano M. Carelli V. Mitochondrial neurogastrointestinal encephalomyopathy: evidence of mitochondrial DNA depletion in the small intestine.Gastroenterology. 2006; 130: 893-901Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar Microdissected tissues from the esophageal wall revealed marked mtDNA depletion limited to the skeletal muscle fibers from cricopharyngeal muscle. In fact, in MNGIE patients, the mtDNA/nuclear DNA ratio was less than normal controls, both in single COX-positive (decreased by 67% as compared to controls) and COX-negative fibers (decreased by 96% as compared to controls) (Figure 3E). In contrast, mtDNA amount in smooth muscle cells from tunica muscularis was comparable in patients and controls (not shown). A marked mtDNA depletion (93% decrease) was detected in the external layer of muscularis propria of small intestine in patients as compared with controls, whereas a milder reduction was observed in its internal layer (48% decrease) and in the ganglion cells of the myenteric plexus (79% decrease). Significant reductions of mtDNA were also observed in the external layer of muscularis propria of stomach (65% decrease) and colon (76% decrease), whereas their internal layer and the ganglion cells of the myenteric plexus showed only mild decreases of mtDNA (less than 45%) (Figure 4B). The control group also demonstrated a non-homogeneous distribution of mtDNA within different tissues of GI wall, with the external layer of muscularis propria showing the lowest amount (internal/external layer ratio 2:1) (Figure 4B). Intriguingly, regression analysis showed a significant linear relationship between the residual mtDNA level in different segments and tissue types of the GI tract of MNGIE patients and the mtDNA levels in the corresponding sections of control subjects (Figure 4C). A marked reduction of mtDNA (87% decrease) was observed also in microdissected vascular smooth muscle and endothelial cells from MNGIE patients (Figure 4C), despite the high mtDNA content in controls at this site. Direct sequencing of the candidate regions screened for point mutation detection, based on the previously reported analysis,15Nishigaki Y Marti' R Copeland WC Hirano M Site-specific somatic mitochondrial DNA point mutations in patients with thymidine phosphorylase deficiency.J Clin Invest. 2003; 111: 1913-1921Crossref PubMed Scopus (167) Google Scholar identified similar heteroplasmic nucleotide changes in all microdissected tissues from the entire GI tract. None of the mutations studied showed segregation to high mutant loads in any of the tissues analyzed (data not shown). To determine the pathogenic mechanism of CIPO in MNGIE, we performed a systematic correlation of morphological and molecular features of the entire GI tract, in autopsy samples of patients and controls. We demonstrated, in five MNGIE patients, that visceral mitochondrial myopathy is likely to cause GI dysmotility. This is due to mtDNA depletion and is characterized by patchy reactivity of smooth muscle cells to cytochrome c oxidase. As usually occurs in mitochondrial disorders, the mtDNA depletion is associated with marked mitochondrial proliferation, a well known compensatory strategy for the reduced respiratory chain function.18Heddi A Stepien G Benke PJ Wallace DC Coordinate induction of energy gene expression in tissue of miyochondrial disease patients.J Biol Chem. 1999; 274: 22968-22976Crossref PubMed Scopus (152) Google Scholar The mtDNA depletion involves the muscularis propria of the entire GI tract and is most dramatically prominent in its external layer. In stomach and small intestine, the profound mtDNA depletion at this site correlates well with the observed atrophy and interstitial fibrosis. In addition, only in the small intestine, we reported significant mtDNA depletion (79% reduction of mtDNA in patients as compared with controls) with evidence of COX-deficient ganglion cells in the myenteric plexus. Notably, we observed a linear relationship between the residual mtDNA content in different segments and tissues of the GI tract in MNGIE patients and the mtDNA amount in the corresponding sections in normal controls. Thus, the baseline low abundance of mtDNA molecules may predispose smooth muscle cells of the external layer of muscularis propria to the toxic effects of circulating dThd and dUrd, with resultant mtDNA depletion. Based on our findings, the physiological abundance of mtDNA copies in different tissues may be one of the factors accounting for the selective threshold effect and the phenotypic expression of mtDNA abnormalities. A novel interesting observation of our study is the ubiquitous finding of mtDNA depletion, COX deficiency, and marked mitochondrial proliferation, within small vessels in all MNGIE patients. In contrast to the external layer of muscularis propria, vascular endothelial and smooth muscle cells of vessels wall normally contain high numbers of mtDNAs to support their high energy requirements.19Gerlach E Nees S Becker BF The vascular endothelium: a survey of some newly evolving biochemical and physiological features.Basic Res Cardiol. 1985; 80: 459-474Crossref PubMed Scopus (63) Google Scholar A possible explanation for our findings is that small vessels, because of their anatomical position at the blood-tissue interface, play a key role in regulation of extracellular and vascular nucleoside levels. In fact, they possess high-affinity nucleoside transporters20Löffler M Morote-Garcia JC Eltzschig SA Coe IR Eltzschig HK Physiological roles of vascular nucleoside transporters.Arterioscler Thromb Vasc Biol. 2007; 27: 1004-1013Crossref PubMed Scopus (137) Google Scholar and have a high capacity for nucleoside accumulation.21Archer RGE Pitelka V Hammond JR Nucleoside transporter subtype expression and function in rat skeletal muscle microvascular endothelial cells.Br J Pharmacol. 2004; 143: 202-214Crossref PubMed Scopus (35) Google Scholar Thus, we hypothesize that in MNGIE, deoxynucleotides accumulate in small vessels wall, where they exert their toxic effect. The observation of a marked mtDNA depletion in small vessel wall supports the hypothesis that the edematous features shown at cerebral magnetic resonance imaging in MNGIE patients could be due to breakdown of the blood-brain barrier.22Szigeti K Sule N Adesina AM Armstrong DL Saifi GM Bonilla E Hirano M Lupski JR Increased blood-brain barrier permeability with thymidine phosphorylase deficiency.Ann Neurol. 2004; 56: 881-886Crossref PubMed Scopus (26) Google Scholar Regarding the other types of mtDNA abnormalities described in MNGIE (ie, mtDNA deletion and point mutations), our results confirm the finding of very low levels of site-specific mtDNA point mutations in the different tissues of the GI wall. In addition, we show that mtDNA deletions are limited to the skeletal muscle component of the upper esophagus, in combination with partial mtDNA depletion, which is virtually complete in the COX-negative fibers as demonstrated in our single-fiber analysis. The morphological and molecular features of cricopharyngeal muscle account for the oropharyngeal dysfunction (dysphagia) observed in MNGIE patients. The selective localization of mtDNA deletions confirms our previous findings in a single MNGIE patient13Giordano C Sebastiani M. Plazzi G. Travaglini C. Sale P. Pinti M. Tancredi A. Liguori R. Montagna P. Bellan M. Valentino M.L. Cossarizza A. Hirano M. Carelli V. Mitochondrial neurogastrointestinal encephalomyopathy: evidence of mitochondrial DNA depletion in the small intestine.Gastroenterology. 2006; 130: 893-901Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar and strengthens the hypothesis that deleted mtDNA molecules accumulate over the years only in postmitotic tissues such as skeletal muscle.10Nishigaki Y Marti R Hirano M ND5 is a hot-spot for multiple atypical mitochondrial DNA deletions in mitochondrial neurogastrointestinal encephalomyopathy.Hum Mol Genet. 2004; 13: 91-101Crossref PubMed Scopus (84) Google Scholar In contrast, the load of deleted mtDNA molecules may be reduced in replicating cells because of negative selection.23Pontarin G Ferraro P Valentino ML Hirano M Reichard P Bianchi V Mitochondrial DNA depletion and thymidine phosphate pool dynamics in a cellular model of mitochondrial neurogastrointestinal encephalomyopathy.J Biol Chem. 2006; 11;281: 22720-22728Crossref Scopus (68) Google Scholar In summary, our study demonstrates that severe depletion of mtDNA is the most striking molecular defect in smooth muscle of the GI tract and vascular wall of MNGIE patients. The depletion of mtDNA correlates with histopathological abnormalities and is likely due to toxic levels of dThd and dUrd disrupting mitochondrial nucleotide pool, as confirmed by in vitro studies.12Song S Wheeler LJ Mathews CK Deoxyribonucleotide pool imbalance stimulates deletions in HeLa cell mitochondrial DNA.J Biol Chem. 2003; 278: 43893-43896Crossref PubMed Scopus (103) Google Scholar, 23Pontarin G Ferraro P Valentino ML Hirano M Reichard P Bianchi V Mitochondrial DNA depletion and thymidine phosphate pool dynamics in a cellular model of mitochondrial neurogastrointestinal encephalomyopathy.J Biol Chem. 2006; 11;281: 22720-22728Crossref Scopus (68) Google Scholar The mechanism for this effect remains unknown. The constitutive low abundance of mtDNA within smooth muscle cells of the external layer of muscularis propria may account for the marked mtDNA depletion at this site. Exposure of vascular smooth muscle to the high circulating levels of nucleosides may contribute to the selective vulnerability of these cells. Because mtDNA depletion is more apt to correction than point mutations or deletions, the severe CIPO in MNGIE may be amenable to therapies that reduce circulating dThd and dUrd as recently reported.24Hirano M Martí R Casali C Tadesse S Uldrick T Fine B Escolar DM Valentino ML Nishino I Hesdorffer C Schwartz J Hawks RG Martone DL Cairo MS DiMauro S Stanzani M Garvin Jr, JH Savage DG Allogenic stem cell transplantation corrects biochemical derangements in MNGIE.Neurology. 2006; 67: 1458-1460Crossref PubMed Scopus (153) Google Scholar We are extremely grateful to Dr Patrizio Sale for his advice on LCM technique. We are deeply indebted to the patients’ families.