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• W2022731255 abstract "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 (118) 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 (123) 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 (192) 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 (123) 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 (192) 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 (192) 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 (102) Google ScholarCIPO 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 (1267) 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 (295) 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 (295) 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 (189) 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 (61) 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 (189) 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 (82) 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 (72) 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 (98) 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 (58) 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.Materials and MethodsPatientsA 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 (295) 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 (295) 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 (295) 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 Tissue Sample PreparationAll 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).Laser Capture MicrodissectionParaffin 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 (58) 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.Molecular AnalysisTotal 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 (50) 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 (50) 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 (82) 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 (82) 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 (82) 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 (160) Google ScholarStatistical AnalysisStatistical 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.ResultsHistological FindingsAll 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)Molecular Analysis mtDNA DeletionsAnalyses 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 (82) Google Scholar and corresponded to the “common deletion,” Δ5.0 kb, Δ7.7 kb, Δ8.1 kb, and Δ9.5 kb (Fi" @default.
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• W2022731255 title "Gastrointestinal Dysmotility in Mitochondrial Neurogastrointestinal Encephalomyopathy Is Caused by Mitochondrial DNA Depletion" @default.
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