Mitochondrial Neurogastrointestinal Encephalomyopathy: Evidence of Mitochondrial DNA Depletion in the Small Intestine
2006; Elsevier BV; Volume: 130; Issue: 3 Linguagem: Inglês
10.1053/j.gastro.2006.01.004
ISSN1528-0012
AutoresCarla Giordano, Mariangela Sebastiani, Giuseppe Plazzi, Claudia Travaglini, Patrizio Sale, Marcello Pinti, Andrea Tancredi, Rocco Liguori, Pasquale Montagna, Marzio Bellan, Maria Lucia Valentino, Andrea Cossarizza, Michio Hirano, Giulia d’Amati, Valério Carelli,
Tópico(s)Biochemical and Molecular Research
ResumoBackground & Aims: Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disease clinically defined by gastrointestinal dysmotility, cachexia, ptosis, ophthalmoparesis, peripheral neuropathy, white-matter changes in brain magnetic resonance imaging, and mitochondrial abnormalities. Loss-of-function mutations in thymidine phosphorylase gene induce pathologic accumulations of thymidine and deoxyuridine that in turn cause mitochondrial DNA (mtDNA) defects (depletion, multiple deletions, and point mutations). Our study is aimed to define the molecular basis of gastrointestinal dysmotility in a case of MNGIE. Methods: By using laser capture microdissection techniques, we correlated histologic features with mtDNA abnormalities in different tissue components of the gastrointestinal wall in a MNGIE patient and ten controls. Results: The patient's small intestine showed marked atrophy and mitochondrial proliferation of the external layer of muscularis propria. Genetic analysis revealed selective depletion of mtDNA in the small intestine compared with esophagus, stomach, and colon, and microdissection analysis revealed that mtDNA depletion was confined to the external layer of muscularis propria. Multiple deletions were detected in the upper esophagus and skeletal muscle. Site-specific somatic point mutations were detected only at low abundance both in the muscle and nervous tissue of the gastrointestinal tract. Analysis of the gastrointestinal tract from 10 controls revealed a non-homogeneous distribution of mtDNA content; the small intestine had the lowest levels of mtDNA. Conclusion: Atrophy, mitochondrial proliferation, and mtDNA depletion in the external layer of muscularis propria of small intestine indicate that visceral myopathy is responsible for gastrointestinal dysmotility in this MNGIE patient. Background & Aims: Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disease clinically defined by gastrointestinal dysmotility, cachexia, ptosis, ophthalmoparesis, peripheral neuropathy, white-matter changes in brain magnetic resonance imaging, and mitochondrial abnormalities. Loss-of-function mutations in thymidine phosphorylase gene induce pathologic accumulations of thymidine and deoxyuridine that in turn cause mitochondrial DNA (mtDNA) defects (depletion, multiple deletions, and point mutations). Our study is aimed to define the molecular basis of gastrointestinal dysmotility in a case of MNGIE. Methods: By using laser capture microdissection techniques, we correlated histologic features with mtDNA abnormalities in different tissue components of the gastrointestinal wall in a MNGIE patient and ten controls. Results: The patient's small intestine showed marked atrophy and mitochondrial proliferation of the external layer of muscularis propria. Genetic analysis revealed selective depletion of mtDNA in the small intestine compared with esophagus, stomach, and colon, and microdissection analysis revealed that mtDNA depletion was confined to the external layer of muscularis propria. Multiple deletions were detected in the upper esophagus and skeletal muscle. Site-specific somatic point mutations were detected only at low abundance both in the muscle and nervous tissue of the gastrointestinal tract. Analysis of the gastrointestinal tract from 10 controls revealed a non-homogeneous distribution of mtDNA content; the small intestine had the lowest levels of mtDNA. Conclusion: Atrophy, mitochondrial proliferation, and mtDNA depletion in the external layer of muscularis propria of small intestine indicate that visceral myopathy is responsible for gastrointestinal dysmotility in this MNGIE patient. Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive mitochondrial disease clinically defined by severe gastrointestinal dysmotility, cachexia, ptosis, ophthalmoparesis, peripheral neuropathy, white-matter changes in brain magnetic resonance imaging, and mitochondrial abnormalities.1Hirano M. Silvestri G. Blake D.M. Lombes A. Minetti C. Bonilla E. Hays A.P. Lovelace R.E. Butler I. Bertorini T.E. DiMauro S. Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) clinical, biochemical, and genetic features of an autosomal recessive mitochondrial disorder.Neurology. 1994; 44: 721-727Crossref PubMed Google Scholar, 2Nishino I. Spinazzola A. Papadimitriou A. Hammans S. Steiner I. Hahn C.D. Connolly A.M. Verloes A. Guimaraes J. Maillard I. Hamano H. Donati M.A. Semrad C.E. Russell J.A. Andreu A.L. Hadjigeorgiou G.M. Vu T.H. Tadesse S. Nygaard T.G. Nonaka I. Hirano I. Bonilla E. Rowland L.P. 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 Loss-of-function mutations in the thymidine phosphorylase (TP) gene3Nishino I. Spinazzola A. Hirano M. Thymidine phosphorylase gene mutations in MNGIE, a human mitochondrial disorder.Science. 1999; 283: 689-692Crossref PubMed Scopus (765) Google Scholar induce pathologic accumulation of thymidine (deoxythymidine) and uridine (deoxyuridine) and mitochondrial nucleotide pool imbalance4Spinazzola A. Marti R. Nishino I. Andreu A.L. Naini A. Tadesse S. Pela I. Zammarchi E. Donati M.A. Oliver J.A. Hirano M. Altered thymidine metabolism due to defects of thymidine phosphorylase.J Biol Chem. 2002; 277: 4128-4133Crossref PubMed Scopus (200) Google Scholar, 5Marti R. Nishigaki Y. Hirano M. Elevated plasma deoxyuridine in patients with thymidine phosphorylase deficiency.Biochem Biophys Res Commun. 2003; 303: 14-18Crossref PubMed Scopus (85) Google Scholar that in turn generate defects of mitochondrial DNA (mtDNA; depletion, multiple deletions, and point mutations) impairing mtDNA replication, maintenance, or both.6Nishigaki Y. Marti R. Copeland W.C. 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, 7Nishigaki 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 Gastrointestinal dysmotility is the most prominent and severe clinical feature in MNGIE. This is mainly characterized by dysphagia, borborygmi, gastroparesis, early satiety, abdominal cramps, nausea, vomiting, intestinal pseudo-obstruction, and diarrhea that lead to progressive weight loss and cachexia of the patient. Diverticulosis of small intestine is also frequent and is thought to be a direct consequence of dysmotility. Inflammation and perforation of diverticuli often lead to death in early adulthood. Aspiration pneumonia caused by dysphagia is another cause of death. The pathogenic mechanisms causing the gastrointestinal dysmotility in MNGIE are still unclear and to date, only one study addressed the presence of mtDNA defects in small intestine failing to detect deletions or significant depletion.7Nishigaki 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 Thus, somatic mtDNA point mutations have been proposed as a possible cause of small intestine dysfunction.6Nishigaki Y. Marti R. Copeland W.C. 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 To better understand the pathogenesis of gastrointestinal (GI) dysmotility in MNGIE patients, we performed a detailed morphologic and molecular genetic study of the entire GI tract in a well-characterized MNGIE patient. By using laser capture microdissection (LCM) techniques, we correlated histologic features with mtDNA abnormalities in different tissue components of the GI wall, providing for the first time a direct link between mtDNA depletion and myopathic changes of the external layer of muscularis propria in MNGIE. A 28-year-old man was admitted in 1993 to our institution to define the cause of his ophthalmoparesis. He reported childhood-onset bilateral ptosis, ophthalmoparesis, deafness, and epilepsy well controlled by carbamazepine. The neurologic examination was remarkable for facial diparesis, bilateral ptosis and ophthalmoplegia, bilateral hypoacusia, pes cavus, generalized muscle atrophy, and hyporeflexia. Cerebral T2-weighted MRI showed diffuse white-matter hyperintensity. Audiometry confirmed sensorineural deafness with profound defects for high frequencies. Electromyography showed a mixed picture of widespread demyelinating sensorimotor polyneuropathy and signs of myopathy. Muscle biopsy was characterized by scattered fibers with increased subsarcolemmal staining of succinic dehydrogenase (SDH). The same fibers also showed decreased cytochrome c oxidase (COX) staining. There were no ragged red fibers. The morphologic evidence of mitochondrial myopathy prompted molecular genetic studies that revealed the presence of multiple deletions of mtDNA by Southern blot analysis. A diagnosis of chronic progressive external ophthalmoplegia was initially considered. On a second admission in 2002, he was markedly cachectic and weighed 36 kg, with evidence of proximal muscle weakness. He reported an episode of gastrointestinal dysmotility with diarrhea, abdominal pain, and dysphagia 5 years earlier, followed by an acute intestinal obstruction in 1999, which was resolved by surgical ileal resection. In retrospect, he also said he suffered from important borborygmi and early satiety. On a standardized cycloergometer test, performed as previously reported,8Montagna P. Plazzi G. Cortelli P. Carelli V. Lugaresi E. Barboni P. Fiocchi M. Abnormal lactate after effort in healthy carriers of Leber's hereditary optic neuropathy.J Neurol Neurosurg Psychiatry. 1995; 58: 640-641Crossref PubMed Scopus (22) Google Scholar the patient had basal lactic acidosis worsened by muscle exercise (basal 1, 2.9; basal 2, 2.8; after exercise, 4.6; 15-minute resting recovery, 3.7; normal range 0.5–2 mmol/L). The diagnosis of MNGIE was considered. Thymidine phosphorylase activity in buffy coats was undetectable, and thymidine concentration in plasma was 7.6 μmol/L (normal, A confirming the diagnosis of MNGIE. Both parents and 2 brothers were heterozygous for the 1443G>A mutation. Within 1 year, he developed a severe episode of dysphagia and new acute abdominal symptoms leading to death. A complete autopsy was performed that revealed aspiration pneumonia and diffuse peritonitis. At autopsy, multiple tissue samples were obtained from different segments of the gastrointestinal tract (upper and lower esophagus, stomach, ileum, colon, and rectum) and from deltoid muscle and the heart. After obtaining informed consent from the relatives and approval of the University of Bologna Ethical Board for this study, we had access to samples from surgical specimen and postmortem tissues of the MNGIE patient. Tissues were fixed in 10% formalin. Postmortem samples were also frozen in liquid nitrogen–chilled isopentane. Normal tissues were obtained at autopsy from ten control subjects of comparable age, according to "La Sapienza" University Ethical Committee protocols. Histologic slides were stained with H&E, Masson Trichrome, periodic acid Schiff, and periodic acid Schiff plus diastase. COX and SDH stains were performed on frozen samples of deltoid muscle. Immunohistochemical stains for S-100, synaptophysin, neuronal-specific enolase, and glial fibrillary acidic protein (DAKO Glostrup, Denmark) were also performed. For ultrastructural analysis, autopsy 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 (Eindhoven, The Netherlands) electron microscope. Total DNA from postmortem tissues of the patient was extracted by phenol-chloroform standard procedures. A Southern blot analysis was performed to screen for the presence of mtDNA deletions. Approximately 5 μg of total DNA were linearized by digestion with the restriction enzymes PvuII and BamHI (New England Biolabs Inc., Beverly, MA), which cut mtDNA at single sites. The DNA samples were electrophoresed through a 0.8% agarose gel and transferred to nylon membrane (BioBond Nylon Membrane; SIGMA, St. Louis, MO). A mixture of partially overlapping fragments cloned in M13 covering the entire mtDNA (a kind gift of Michael King and Giuseppe Attardi) were labeled with α32P-dCTP (Random Primed DNA Labeling Kit; Roche Diagnostic Corp, Austria; BioRad, Hercules, CA). The signals were analyzed in the Molecular Imager FX System (Bio-Rad USA). Based on the results of light microscopy analysis, LCM was performed to search for the mtDNA defects within the different tissues of the GI wall. Paraffin sections from the patient and 5 controls were subjected to LCM with the MMI NIKON UV-CUT System as previously described.9Pistilli D. di Gioia C.R.T. d'Amati G. Sciacchitano S. Quaglione R. Quitadamo R. Casali C. Gallo P. Santorelli F. Detection of deleted mitochondrial DNA in Kearns-Sayre syndrome using laser capture microdissection.Hum Pathol. 2003; 34: 1058-1061Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar Histologic recognition of cell types for LCM was based on strict morphologic criteria. Smooth-muscle cells from internal and external muscular layers, as well as nerve fibers, satellite cells, and ganglia from the myenteric plexus, were separately dissected from serial tissue sections of the small intestinal wall obtained 2 cm distal to the ligament of Treitz. The dissection was performed by an ultraviolet laser, which performs circumferential dissection of selected cells, following precisely a drawn incision path. By this cold ablation technique, extracted material is never exposed directly to the laser. The microdissected tissue areas were measured, documented, and collected on an adhesive cap of nanotubes for nucleic acid extraction. A number of 100–150 cells, pooled on the same cap, were collected for each cell population. Total DNA was extracted from dissected samples with Picopure DNA extraction Kit (Arcturus; Los Altos, CA). mtDNA content was measured on both homogenate and microdissected tissues by real-time quantitative polymerase chain reaction (PCR) assays by using a previously described method.10Cossarizza A. Riva A. Pinti M. Ammannato S. Fedeli P. Mussini C. Esposito R. Galli M. Increased mitochondrial DNA content in peripheral blood lymphocytes from HIV-infected patients with lipodystrophy.Antivir Ther. 2003; 8: 51-57PubMed Google Scholar Statistical analysis was performed modeling the data by a mixed-effect model.11Pinhero J.C. Bates D.M. Mixed-effects models in S and S-PLUS. Springer, New York, NY2000Crossref Google Scholar Such a model can explain the different values of mtDNA in different tissues taking account of patient heterogeneity. Numeric estimates have been obtained by the statistical software R Foundation for Statistical Computing, Vienna, Austria with the package nlme (www.r-project.org). We performed several PCR reactions with shifted primers to detect even a small level of deletions in both homogenate and microdissected tissues. Several sets of oligonucleotide primers were used: forward primer nt 3087–3107, nt 4181–4201, nt 4370–4390, nt 5454–5474, nt 5651–5671, nt 7361–7381, nt 8123–8143, and nt 8430–8450, backward primer nt 14268–14249, nt 14840–14820, and nt 16911–16891. The PCR cycle conditions were as described.7Nishigaki 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 The PCR products were visualized by electrophoresis in a 2% agarose gel extracted by using the QIA quick gel extraction kit (Quiagen, Valencia, CA), 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 by using Wizard Plus SV Minipreps DNA Purification Systems (Promega) and then sequenced directly in an ABI Prism 310 Genetic analyzer (Applied-Biosystem, Foster City, CA) following standard procedures. To screen for the presence of mtDNA point mutation, we amplified by PCR 3 selected mtDNA regions corresponding to nt 5651–6022, nt 9917–10568, and nt 15756–16119. Within these mtDNA segments, point mutations have been identified in most MNGIE patients.6Nishigaki Y. Marti R. Copeland W.C. 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 We then directly sequenced PCR fragments. To detect very low levels of mutation, each PCR fragment was subcloned, and at least 15 clones were sequenced. Gross examination of the ileal surgical specimen revealed the presence of segmental dilation and thinning of the bowel wall, adjacent to areas of visceral stenosis because of fibrous adhesions. Diverticulitis and peritonitis were also observed. Light microscopic examination showed chronic inflammation of the mucosa with significant submucosal edema. The internal circular layer of the muscularis propria was unremarkable; in contrast, the external longitudinal layer showed marked atrophy of smooth muscle and interstitial fibrosis compared with controls (Figure 1A and B). The residual smooth-muscle cells from the atrophic external layer showed extensive cytoplasmic microvacuolation and pycnotic nuclei (Figure 1C). The myenteric and submucosal nervous plexi appeared well preserved and showed a normal distribution with immunohistochemical stains for S-100, synaptophysin, neuronal-specific enolase, and glial fibrillar acidic protein (Figure 1D). At autopsy, the entire gastrointestinal tract was carefully examined and sampled. The remarkable finding on gross inspection was the intestinal dilation and peritonitis. Histology confirmed the presence of severe atrophy and vacuolation of the smooth muscle cells, limited to the external layer of the small intestine. At electron microscopy, the vacuoles within smooth-muscle cells corresponded to cytoplasmic lipid droplets and abundant mitochondria (Figure 1E). Several myocytes showed shrunken nuclei with condensed chromatin. No obvious abnormalities were detected by light and electron microscopy in the esophagus, stomach, and large bowel (not shown). Histologic analysis of deltoid muscle confirmed a mitochondrial myopathy characterized by numerous COX negative fibers with increased SDH stain. No abnormal mtDNA bands were detected by Southern blot analyses of total DNA extracted from postmortem stomach, small intestine, colon, and rectum of the patient, whereas multiple deletions were evident in DNA from the proximal esophagus and deltoid muscle (data not shown). In our case, the 3 most abundant deletions were the same already reported in the skeletal muscle from most MNGIE patients and corresponded to the "common deletion" Δ5.0 kb, the Δ7.7 kb, and the Δ8.1 kb. Because it is well known that mtDNA deletions could be missed by Southern blot analyses, we performed several PCR reactions with shifted primers to detect additional deletions present at low levels in the stomach, small intestine colon, and rectum of the patient. However, we failed to detect any deleted mtDNA molecules by this approach (data not shown). We characterized, by PCR analyses, the Δ8.1-kb molecule in the esophagus. Because it was not possible to define the breakpoint of this deletion by direct sequencing of the PCR product, we cloned the fragment and sequenced 10 clones. By this technique, we detected a group of heterogeneous breakpoints differing a few bases length at the site of the imperfectly homologous sequence between tRNACys and ND5 genes, as already described in the skeletal muscle of MNGIE patients.7Nishigaki 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 We identified the same Δ8.1-kb deletion with similar variable breakpoints in deltoid muscle and the heart. Real-time PCR analysis revealed a nonhomogeneous distribution of mtDNA copy number in different segments of the GI tract from both the control group and the MNGIE proband. In fact, the small intestine had a consistently lower amount of mtDNAs compared with the esophagus, stomach, and colon in the 10 control subjects. The mtDNA copy number in the proband's tissues followed a similar pattern, but we observed even more striking depletion of mtDNA in the small intestine compared with the controls (Figure 2). A profound depletion was also observed in deltoid muscle with a mean of 54 ± 21 mtDNA copies per cell. Direct sequencing of the candidate regions chosen for point mutation detection identified similar heteroplasmic nucleotide changes (Table 1) in tissue homogenates from the entire GI tract (15–20 clones sequenced). Most of them consisted of T-to-C transitions preceded by at least 2 A residues, as previously described.6Nishigaki Y. Marti R. Copeland W.C. 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 Three T-to-C transitions, at nucleotide 5814, 15831, and 15956, have been already reported in different tissues of MNGIE patients.6Nishigaki Y. Marti R. Copeland W.C. 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 The amount of point mutations, evaluated as the percentage of positive clones, was consistently low (Table 1). In addition, the mtDNA base substitution T10238C, A10398G, A15954G was reported as homoplasmic polymorphic variants by a public database (www.mitomap.org), as was the T10034C change (A Achilli and A Torroni, personal communication, June 2005).Table 1mtDNA Point Mutations in Gastrointestinal Tissues of MNGIE PatientSequence T position Mutation AA change RegionAAAAAT 10158 T > C Ser > Pro ND3AAAAT 5814 T > C tRNACysAAAT 15956 T > C tRNAProAAT 15831 T > C Ile > Thr CytbAAT 15861 T > C Ile > Thr CytbTCAG 15959 G > A tRNAProTissueEsophagus551012.5n10Stomach10555n12.5Ileum5n65n16.6 Muscularis propriann16.613.5n16.6 Myenteric plexus12.5nnn1610Colon5n5nn12NOTE. Mutations were identified by sequencing. Mutation levels expressed as the percentage of positive clones within 15 to 20 sequenced. n, negative. Open table in a new tab NOTE. Mutations were identified by sequencing. Mutation levels expressed as the percentage of positive clones within 15 to 20 sequenced. n, negative. Based on the histologic findings and the results of mtDNA analysis of tissue homogenates, we performed LCM to localize the mtDNA defects within the internal and external muscle layers of the small bowel wall. Furthermore, we microdissected and analyzed nerve fibers, satellite cells, and ganglia of the myenteric plexus (Figure 3). PCR analyses failed to detect mtDNA deletions in microdissected tissues of small intestine. In addition, we performed LCM to localize mtDNA deletions within different tissues of the lower third of the esophagus. Analysis of the smooth muscle cells of the tunica muscularis and of nervous tissue failed to detect any mtDNA-deleted molecule. Histologic samples of the upper third of the esophagus, in which tunica muscularis is made up by striated muscle, were not available. The results of mtDNA analyses are shown in Figure 3B. In controls, the mtDNA copy number was lower in the external layer of muscularis propria compared with the internal one (internal/external layer ratio 2:1). However, this difference was more striking in the patient. Although the internal layer showed mtDNA levels similar to controls, the external was markedly depleted, reflecting the pathologic changes found at histology (Figure 1). No significant differences were found in mtDNA content of myenteric plexus between patient and controls. The microdissected muscle cells, nerve fibers, and ganglia of the small intestine showed the same low amount of mutated mtDNA. In particular, none of the mutations studied showed segregation to high mutant loads in specific tissue. In this study, we provide the first detailed morphologic and genetic analysis of the entire GI tract in an MNGIE patient. The correlations between histologic and ultrastructural changes with mtDNA abnormalities led us to the conclusion that the GI dysmotility in our patient is mainly because of myopathy of the small intestine wall. This is limited to the external layer of muscularis propria where marked mitochondrial proliferation and profound mtDNA depletion were found. We also show the novel finding that, in normal subjects, the small intestine has a constitutive lower amount of mtDNA copy number compared with the other segments of the GI tract. This may significantly predispose MNGIE patients to visceral myopathy and development of small intestine dysmotility. The hypothesis that mtDNA depletion is the major molecular determinant in the pathogenesis of GI dysmotility in our patient is strengthened by the results of mtDNA deletion and point-mutation analysis. In fact, we could not detect mtDNA deletions in his stomach, small intestine, or colon. In addition, we found only very low levels of site-specific point mutations in the different tissues of the GI wall, including the T-to-C substitution at nucleotide 5814 in tRNACys that has been described as pathogenic in several reports.12Manfredi G. Schon E.A. Bonilla E. Moraes C.T. Shanske S. DiMauro S. Identification of a mutation in the mitochondrial tRNA(Cys) gene associated with mitochondrial encephalopathy.Hum Mutat. 1996; 7: 158-163Crossref PubMed Google Scholar, 13Santorelli F.M. Siciliano G. Casali C. Basirico M.G. Carrozzo R. Calvosa F. Sartucci F. Bonfiglio L. Murri L. DiMauro S. Mitochondrial tRNA(Cys) gene mutation (A5814G) a second family with mitochondrial encephalopathy.Neuromuscul Disord. 1997; 7: 156-159Abstract Full Text PDF PubMed Scopus (28) Google Scholar, 14Karadimas C. Tanji K. Geremek M. Chronopoulou P. Vu T. Krishna S. Sue C.M. Shanske S. Bonilla E. DiMauro S. Lipson M. Bachman R. A5814G mutation in mitochondrial DNA can cause mitochondrial myopathy and cardiomyopathy.J Child Neurol. 2001; 16: 531-533Crossref PubMed Scopus (17) Google Scholar Moreover, a similar marked mtDNA depletion was detected in deltoid muscle that showed typical histologic features of mitochondrial myopathy. Another novel observation of our study is that multiple mtDNA deletions were found only in the proximal esophagus, in which the tunica muscularis is made up by striated muscle cells. Moreover, we found identical deletions in deltoid muscle and the heart. Deletions in the striated muscle of proximal esophagus likely contributed to dysphagia in our patient, as already described in patients with cricopharyngeal achalasia.15Kornblum C. Broicher R. Walther E. Seibel O. Reichmann H. Klockgether T. Herberhold C. Schroder R. Cricopharyngeal achalasia is a common cause of dysphagia in patients with mtDNA deletions.Neurology. 2001; 56: 1409-1412Crossref PubMed Scopus (39) Google Scholar The possible explanation for the selective localization of mtDNA deletions is that they accumulate in postmitotic skeletal muscle cells, whereas negative selection may decrease the load of deleted mtDNA molecules during turnover of smooth-muscle cells.16McShane M.A. Hammans S.R. Sweeney M. Holt I.J. Beattie T.J. Brett E.M. Harding A.E. Pearson syndrome and mitochondrial encephalopathy in a patient with a deletion of mtDNA.Am J Hum Genet. 1991; 48: 39-42PubMed Google Scholar The same mechanism does not interfere with regulation of mtDNA copy number. Thus, mtDNA depletion is the only abnormality observed in intestinal smooth-muscle cells. Our histologic findings are not novel but rather are very similar to those of most autopsy studies previously reported, which were performed before the molecular diagnosis of MNGIE was available.17Ionasescu V. Thompson S.H. Ionasescu R. Searby C. Anuras S. Christensen J. Mitros F. Hart M. Bosch P. Inherited ophthalmoplegia with intestinal pseudo-obstruction.J Neurol Sci. 1983; 59: 215-228Abstract Full Text PDF PubMed Scopus (44) Google Scholar, 18Anuras S. Mitros F.A. Nowak T.V. Ionasescu V.V. Gurll N.J. Christensen J. Green J.B. A familial visceral myopathy with external ophthalmoplegia and autosomal recessive transmission.Gastroenterology. 1983; 84: 346-353Abstract Full Text PDF PubMed Scopus (91) Google Scholar, 19Cave D. Compton C. Case records of the Massachusetts General Hospital.N Engl J Med. 1990; 322: 829-841Crossref PubMed Scopus (4) Google Scholar The external layer of muscularis propria of the small intestine resulted selectively atrophic, in particular when compared with the well-preserved internal layer and the intact myenteric and submucosal plexi. The ganglion cells did not show any obvious sign of pathology. These features suggested a primary myopathic pathogenesis of GI dismotility in this MNGIE patient. Moreover, a functional study by manometric evaluation of the GI motility in an MNGIE patient revealed features compatible with a visceral myopathy because of myocytes loss in the small intestine.20Mueller L.A. Camilleri M. Emslie-Smith A.M. Mitochondrial neurogastrointestinal encephalomyopathy manometric and diagnostic features.Gastroenterology. 1999; 116: 959-963Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar Although the relationship between TP mutations and biochemical defects in MNGIE has been thoroughly investigated,4Spinazzola A. Marti R. Nishino I. Andreu A.L. Naini A. Tadesse S. Pela I. Zammarchi E. Donati M.A. Oliver J.A. Hirano M. Altered thymidine metabolism due to defects of thymidine phosphorylase.J Biol Chem. 2002; 277: 4128-4133Crossref PubMed Scopus (200) Google Scholar, 5Marti R. Nishigaki Y. Hirano M. Elevated plasma deoxyuridine in patients with thymidine phosphorylase deficiency.Biochem Biophys Res Commun. 2003; 303: 14-18Crossref PubMed Scopus (85) Google Scholar only a limited number of studies addressed the specific molecular pathogenesis of GI dysmotility, the major clinical feature of this mitochondrial disease. A previous report failed to recognize mtDNA deletions and significant depletion in tissue homogenates of small intestine of 6 patients7Nishigaki 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; however, there was a trend toward lower mtDNA levels in the patients. The lack of established standards for mtDNA content in the normal gut and small number of samples limited the assessment of mtDNA depletion in this tissue. In addition, multiple site-specific somatic mtDNA point mutations have been identified in small intestine homogenates, but their amount seemed too low to produce a biochemical phenotype.7Nishigaki 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 By using the technique of LCM,9Pistilli D. di Gioia C.R.T. d'Amati G. Sciacchitano S. Quaglione R. Quitadamo R. Casali C. Gallo P. Santorelli F. Detection of deleted mitochondrial DNA in Kearns-Sayre syndrome using laser capture microdissection.Hum Pathol. 2003; 34: 1058-1061Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar we were able to correlate atrophy and mitochondrial proliferation in the external muscular layer of the small intestine with mtDNA depletion. The role of mtDNA depletion as a cause of small intestine morphologic and functional abnormalities is supported by previous reports on patients infected by the human immunodeficiency virus (HIV) treated with long-term zidovudine therapy. Zidovudine is a nucleoside analog that inhibits HIV replication by blocking the activity of the viral enzyme reverse transcriptase and that can also interfere with the enzymatic activity of polymerase gamma (POLG), which is responsible for replication of the mitochondrial genome. The consequent mtDNA depletion can cause several side effects, including lactic acidosis and mitochondrial myopathy.21Arnaudo E. Dalakas M. Shanske S. Moraes C.T. DiMauro S. Schon E.A. Depletion of muscle mitochondrial DNA in AIDS patients with zidovudine-induced myopathy.Lancet. 1991; 337: 508-510Abstract PubMed Scopus (482) Google Scholar A recent report showed that infants born to women infected with HIV and exposed to perinatal zidovudine developed hypoperistalsis and intestinal pseudo-obstruction.22Neuman M.I. Molle Z. Handelsman E.L. Desai N. Orentlicher R.J. Rabinowitz S.S. Neonatal hypoperistalsis associated with perinatal zidovudine administration.J Perinatol. 1998; 18: 20-23PubMed Google Scholar Moreover, the small intestine of patients with long-term zidovudine therapy shows marked atrophy and vacuolization of the external layer of muscularis propria.23Fenoglio-Preiser C.M. Motility disorders.in: Fenoglio-Preiser C.M. Gastrointestinal pathology. An atlas and text. 2nd ed. Lippincott-Raven, Philadelphia1998: 614Google Scholar A further example of a potentially similar pathogenic mechanism for GI dysmotility is the recent report of this clinical manifestation in patients with progressive external ophthalmoplegia caused by recessive mutations in the nuclear-encoded POLG.24Filosto M. Mancuso M. Nishigaki Y. Pancrudo J. Harati Y. Gooch C. Mankodi A. Bayne L. Bonilla E. Shanske S. Hirano M. DiMauro S. Clinical and genetic heterogeneity in progressive external ophthalmoplegia due to mutations in polymerase gamma.Arch Neurol. 2003; 60: 1279-1284Crossref PubMed Scopus (112) Google Scholar Analogous to TP deficiency in MNGIE, mutations in POLG may lead to various somatic defects of mtDNA ranging from multiple deletions to point mutations and depletion.25Van Goethem G. Dermaut B. Lofgren A. Martin J.J. Van Broeckhoven C. Mutation of POLG is associated with progressive external ophthalmoplegia characterized by mtDNA deletions.Nat Genet. 2001; 28: 211-212Crossref PubMed Scopus (686) Google Scholar The simplest explanation for the selective damage of the external muscular layer of the intestinal wall, both in MNGIE and in patients treated with zidovudine, could reside in the baseline low abundance of mtDNA molecules within smooth-muscle cells at this site. In fact, the analysis of normal controls shows that the amount of mtDNA within the internal muscle layer of small intestine is twice that of the external layer. The analysis of additional cases of MNGIE is necessary to confirm our findings. The authors thank our MNGIE patient and his family for their extraordinary collaboration. We gratefully acknowledge Dr Domenico Alvaro for critically reviewing the manuscript and for helpful advice and Massimo Zani for his excellent technical support. We also acknowledge Dr Gian Piero Casadei and Dr Giuseppe Baruzzi for performing the tissue sampling at autopsy and for making the surgical specimens available.
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