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Human Pathology (2013) 44, 1440–1446

Case study

Diagnosis of mitochondrial neurogastrointestinalencephalopathy disease in gastrointestinal biopsiesAntonio R. Perez-Atayde MD⁎

Department of Pathology, Boston Children's Hospital and Harvard Medical School, Boston, MA 02115, USA

Received 23 May 2012; revised 1 December 2012; accepted 3 December 2012

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Keywords:Leukodystrophy;Mitochondrial disease;Crohn disease;Hirschsprung disease;Myopathy;Ganglion cell;Intestinalpseudo-obstruction

Summary A 14-year-old boy with mitochondrial neurogastrointestinal encephalopathy (MNGIE)disease had a lifelong history of failure to thrive and gastrointestinal symptoms including vomiting,pain, and diarrhea, leading to progressive cachexia. At the age of 9 years, after an extensive workup, thediagnosis of Crohn disease was strongly suspected, and he underwent colonoscopy with multiplebiopsies. At 11 years of age, vision change and poor balance lead to a diagnosis of leukodystrophy bymagnetic resonance imaging. Investigations for metachromatic leukodystrophy, adrenal leukodystrophy,and globoid cell leukodystrophy were all negative. A diagnosis of MNGIE disease was suspected whenhe continued deteriorating with gastrointestinal symptoms, multiple neurologic deficits, andencephalopathy. Markedly diminished thymidine phosphorylase activity and increased thymidineplasma levels confirmed the diagnosis of MNGIE. At autopsy, megamitochondria were observed bylight microscopy in submucosal and myenteric ganglion cells and in smooth muscle cells of muscularismucosae and muscularis propria, along the entire gastrointestinal tract from the esophagus to therectum. Megamitochondria in ganglion cells were also observed in a retrospective review of theendoscopic intestinal biopsies taken at age 9 and 13 years and in the appendectomy specimen obtained1 month before his demise. This study corroborates the presence of megamitochondria ingastrointestinal ganglion cells in MNGIE disease, better illustrates their detailed morphology, anddescribes for the first time similar structures in the cytoplasm of gastrointestinal smooth muscle cells.Pathologists should be able to recognize these structures by light microscopy and be aware of theirassociation with primary mitochondriopathies.© 2013 Elsevier Inc. All rights reserved.

1. Introduction

The diagnosis of mitochondrial neurogastrointestinalencephalopathy (MNGIE) disease is usually suspected latein the clinical course when the disease is relatively fullyexpressed with progressive gastrointestinal dysmotility,progressive failure to thrive, ptosis, external ophthalmople-gia, mixed sensorimotor demyelinating neuropathy, hearing

⁎ Corresponding author.E-mail address: antonio.perezatayde@childrens.harvard.edu.

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loss, and asymptomatic leukodystrophy with increased T2signal abnormalities in the white matter by magneticresonance imaging. The order in which these manifestationsappear is unpredictable, but severe gastrointestinal dysmo-tility with weight loss, episodic abdominal pain, and diarrheaare often the earliest manifestations, and patients remainundiagnosed for many years enduring extensive workup foran etiology, in particular Crohn disease.

In 1998, we described megamitochondria seen by lightmicroscopy as brightly eosinophilic inclusions in thecytoplasm of submucosal ganglion cells in a rectal suction

1441MNGIE disease in gastrointestinal biopsies

biopsy, as a characteristic feature of MNGIE disease [1].Recently, similar structures have been observed withingastrointestinal ganglion cells in children with Alpers disease[2]. In addition, prior reports have described abnormalmitochondria by electron microscopy in intestinal smoothmuscle cells of patients with MNGIE and mitochondrialencephalomyopathy, lactic acidosis, and stroke-like episodes(MELAS) and in ganglion cells in a patient with MELAS,but megamitochondria were not observed by light micros-copy in any of these reports [3–7]. To validate our priorreport [1] and to describe additional findings, I now describea second case of MNGIE that strikingly showed by lightmicroscopy numerous characteristic megamitochondria inthe cytoplasm of gastrointestinal ganglion cells. They werepresent long before the onset of neurologic symptoms andbefore the diagnosis of MNGIE was biochemically estab-lished. In addition, this report describes for the first timeidentical inclusions within the cytoplasm of smooth musclecells of the muscularis mucosae and muscularis propria of thegastrointestinal tract.

2. Case report

This 14-year-old boy was the product of a 38 weeks'gestation born after an uncomplicated pregnancy and laborand normal spontaneous vaginal delivery. As a neonate, hedeveloped transient jaundice treated with phototherapy.Early-onset failure to thrive continued during his entire lifedespite vigorous appetite. He had intermittent episodes ofvomiting, abdominal pain, and loose stools. At age 9 years,his weight and height began to plateau, and an extensiveworkup ruled out pancreatic insufficiency, thyroid disease,celiac disease, and parasitic infection. The diagnosis ofCrohn disease was considered likely, and althoughendoscopy and colonoscopy were grossly normal andmultiple intestinal biopsies were microscopically unremark-able, the biopsy sample from the terminal ileum showedfocal active ileitis. Against this diagnosis, however, theantineutrophil cytoplasmic antibody, upper gastrointestinalseries, small bowel follow-through, hematocrit, albumin andblood sedimentation rate were all normal. At that time, theneurologic examination was unremarkable without focaldeficits. At 11 years of age because of persistent vomiting,vision change, and poor balance noted by his mother, a headmagnetic resonance imaging was done that revealedleukodystrophy with extensive T2 signal abnormality inthe periventricular white matter and generalized demyelin-ation. Workups for metachromatic leukodystrophy, adrenalleukodystrophy, and globoid cell leukodystrophy had allnegative results. He progressively developed muscle wasting,weakness, bilateral foot drop, loss of deep tendon reflexes, mildintention tremor, deterioration of gait and balance, ptosis, andabnormal behavior with anger outbursts. Nerve conduction testsshowed severe axonal demyelinating mixed sensory-motor

neuropathy. Because of these neurologic deficits, signs ofencephalopathy with behavioral and personality changes, andchronic gastrointestinal symptoms, a diagnosis of MNGIEsyndrome was suspected. The thymidine phosphorylase (TP)enzyme activity in buffy coat was 16.2 nmol/hour/mg protein(normal activity, 667 ± 212 nmol/hour/mg protein; range, 200-1340 nmol/hour/mg protein; patients with MNGIE: 0-43 nmol/hour/mg protein). A thymidine plasma level was 8.4 μmol/L(normal, b0.05 μmol/L; patients with MNGIE, 8.68 ± 5.23μmol/L) (M. Hirano, personal communication, ColumbiaUniversity). These findings are considered diagnostic ofMNGIE disease [8,9].

The patient became progressively cachectic despitevigorous oral intake, percutaneous endoscopic feeding, andparenteral nutrition. Decreased pulmonary function testswere attributed to progressive thoracic muscle weakness. Abone marrow transplant was considered, but after placementof a feeding jejunostomy tube, the patient developedruptured appendicitis, peritonitis, sepsis, acute respiratorydistress, and hemodynamic instability requiring pressors.Candida albicanswas cultured from an abdominal drain, andhe was found to have an enterocutaneous fistula drainingfrom the umbilicus. Because of progressive deterioration andafter family consent, support was withdrawn and the patientdied. An autopsy was granted.

The patient had 2 healthy siblings, and there was nofamily history of consanguinity.

3. Pathologic findings

At autopsy, there were features consistent with the clinicaldiagnosis of chronic intestinal pseudo-obstruction withdiminished diameter of small bowel and markedly dilatedstomach with hypertrophic muscular wall. The outer layer ofmuscularis propria of the small intestine was very thin withdegeneration, loss of smooth muscle cells, and replacementfibrosis (Fig. 1). Degenerating smooth muscle cells hadcytoplasmic vacuoles but not megamitochondria.

Frequent megamitochondria were identified in thesubmucosal and myenteric ganglion cells as well as insmooth muscle cells of the muscularis mucosae andmuscularis propria of the entire gastrointestinal tractincluding the esophagus, stomach, duodenum, jejunum,ileum, colon, and rectum. Megamitochondria were visible bylight microscopy as round brightly eosinophilic inclusionsthat were refractile to light by lowering the microscopecondenser lens (Figs. 2–6). Megamitochondria were presentin approximately 30% of ganglion cells in all gastrointestinalsegments examined. The number of megamitochondria percell varied from one to few to numerous and the size fromvery small, almost imperceptible to larger inclusions 3 to 5μm in diameter resembling red blood cells (Figs. 2 and 3).Although the cytoplasm of ganglion cells with few or smallmegamitochondria appeared unremarkable, those with

Fig. 1 Muscularis propria of small intestine. A, Massontrichrome stain of muscularis propria showing an atrophic externallayer (arrows). B, At higher magnification, marked interstitialfibrosis is present and residual smooth muscle cells (arrowheads)have cytoplasmic vacuoles and appear degenerated.

Fig. 2 Myenteric ganglion cells with megamitochondria andnormal-appearing cytoplasm (the photographs in the far right insetswere taken without the condenser lens revealing the refractileproperties of the megamitochondria). A, The larger ganglion cell,seen at higher magnification in the insets, contains about 3megamitochondria (arrowheads; 1 larger and 2 smaller). B, Fewclusters of medium-sized megamitochondria (arrowheads). C,Numerous smaller-sized megamitochondria within the cytoplasmand within neuritic processes (arrowheads).

1442 A. R. Perez-Atayde

numerous megamitochondria looked injured with promi-nent cytoplasmic vacuoles (Figs. 2 and 3). Smallermegamitochondria were also observed within the axonalcytoplasmic projections of ganglion cells (Fig. 2C). Theganglionic nature of these cells could be demonstrated byimmunohistochemistry using an antibody against PGP9.5, aneuron-specific protein (Leica Microsystems Inc, BuffaloGrove, IL) (Fig. 6).

Identical megamitochondria were also present by lightmicroscopy in smooth muscle cells of the muscularismucosae and muscularis propria, a feature not previouslyreported, as far as I am aware. They were frequently found inthe esophagus and small intestine (up to 20% of the smoothmuscle cells) and rarely in the colon and were better seen inlongitudinally sectioned smooth muscle cells (Fig. 4).

Retrospective review of multiple endoscopic esophagealand gastrointestinal biopsies performed during the diagnosticworkup for chronic inflammatory bowel disease, when thechild was 9 and 13 years of age, revealed megamitochondria

in the cytoplasm of submucosal ganglion cells in some of thebiopsies. There were a total of 28 endoscopic biopsies (2esophageal, 1 duodenal, 2 gastric, 1 ileal, and 5 colonic atage 9 years and 5 esophageal, 2 duodenal, 2 gastric, 1 ileal,and 7 colonic at age 13 years). Ganglion cells were observedonly in 2 duodenal biopsies (1 at age 9 years and 1 at age 13years) and in 1 colonic biopsy (at age 13 years).Megamitochondria were present in all these 3 biopsies (5/9ganglion cells [56%] had megamitochondria) (Fig. 5).Megamitochondria were also observed in approximately45% of the ganglion cells in the vermiform appendixremoved for the treatment of ruptured appendicitis 1 monthbefore his demise. Megamitochondria were observed in raresmooth muscle cells of the muscularis mucosae or

Fig. 3 Myenteric ganglion cells with megamitochondria andmarked cytoplasmic vacuolization (the photographs in the far rightinsets were taken without the condenser lens revealing the refractileproperties of the megamitochondria). A, Numerous and larger-sizedmegamitochondria within the cytoplasm of a vacuolated ganglioncell (arrows and insets). The arrowhead indicates a ganglion cellwith unremarkable cytoplasm and absence of megamitochondria. B,Numerous medium-sized megamitochondria in a vacuolatedganglion cell (arrows and insets).

ig. 4 Smooth muscle cells of muscularis propria with mega-itochondria. A, Longitudinally sectioned smooth muscle cellsontaining a variable number of megamitochondria (arrowheads).mooth muscle cell indicated by arrow is shown in the inset at higheragnification. B, Same field photograph as panel A taken without theondenser lens showing the refractile megamitochondria. C, Mega-itochondria in smooth muscle cells at higher magnification.

1443MNGIE disease in gastrointestinal biopsies

muscularis propria of the vermiform appendix and were notidentified in the very few smooth muscle cells present insome of the endoscopic intestinal biopsies.

Although there was considerable postmortem artifactultrastructurally, it was possible to see that the cytoplasmicstructures observed in the ganglion cells by lightmicroscopy were megamitochondria (Fig. 7). In addition,some ganglion cells had prominent vacuolar change ofendoplasmic reticulum membranes. Megamitochondria hadmarked reduction of cristae and showed prominentexpansion of the matrices, which appeared artifactuallyextracted. The few cristae present were displaced towardthe periphery and often showed disruption or abnormalcircular configuration. The cristae were sometimes difficultto visualize, and occasionally, the mitochondrial nature ofthe inclusions could only be discerned by their character-istic double membrane.

Electron microscopy of the smooth muscle cells alsoshowed marked postmortem artifact, but one could appreci-ate an increased number of enlarged mitochondria in somecells as well as the presence of megamitochondria withsimilar ultrastructure to those observed in ganglion cells(Fig. 8). Some enlarged mitochondria had complex convo-

FmcSmcm

luted cristae (Fig. 8A, inset). Matrical osmiophilic inclusionswere also present, some of which were probably caused bypostmortem changes.

Fig. 5 Endoscopic duodenal biopsy performed at 9 years of age.Megamitochondria in a submucosal ganglion cell are indicated byarrowheads (at higher magnification in insets; photograph on thelower inset was taken without the condenser lens).

1444 A. R. Perez-Atayde

The liver showed diffuse hepatocellular damage withcholestasis and moderate microvesicular and macrovesicularsteatosis as well as focal pericellular and marked portalfibrosis with portal-to-portal bridging. Owing to the marked

Fig. 6 Correlation of hematoxylin and eosin stain and PGP9.5immunostain. A, Two ganglion cells containing megamitochondriaare indicated by arrowheads. B, Cytoplasmic or membranousimmunoreactivity for PgP9.5 is observed in both of these ganglioncells (arrowheads).

Fig. 7 Electron microscopy of ganglion cells. A, Ganglion cellwith vacuolated endoplasmic reticulum membranes and mega-mitochondria (arrows; scale bar = 2 μm). B, At highermagnification, megamitochondria (arrows) show expanded matri-ces and marked reduction of cristae seen predominantly at theperiphery. Some cristae have a circular profile (scale bar = 500 nm).

autolysis, the presence of abnormal mitochondria could notbe assessed. Sections of the psoas muscle showedoccasional scattered single-cell myocyte damage anddegeneration, but no ragged-red fibers or megamitochon-dria. Sections of peripheral nerves did not reveal significantpathologic changes. Megamitochondria were not observedin the myocardium or in smooth muscle cells of arteries inany of the tissues and organs examined. Moreover, nomegamitochondria were identified in smooth muscle cells ofthe bronchi, urinary bladder, seminal vesicles, or skin, or inthe occasional ganglion cells that were present in sections ofthe pericardium, lung, mediastinum, pancreas, and peripan-creatic soft tissue.

Small blood vessels in the cortex and white matter of thecentral nervous system showed diffuse hyalinization withwidening of the Virchow-Robin spaces, perivascular rare-faction, and hemosiderin deposition. There were alsodemyelinating, focally destructive lesions in the basis pontiswith reactive astrocytes, foamy macrophages, and axonal

Fig. 8 Electron microscopy of smooth muscle cells. A, Smoothmuscle cells with fewmegamitochondria (arrows; scale bar = 2 μm).Inset reveals a megamitochondria with complex cristae (scale bar =500 nm). B, Smooth muscle cell with numerous megamitochondriadisplacing myofilaments to the periphery (between arrows).Matrical osmiophilic inclusions are present in most, and somemay represent postmortem artifact (scale bar = 2 μm).

1445MNGIE disease in gastrointestinal biopsies

swelling thought to be consistent with central pontinemyelinolysis. No megamitochondria were indentified in thecentral nervous system.

4. Discussion

MNGIE, first described in 1983, is a multisystem diseasewith progressive peripheral and central neurodegeneration,peripheral mixed sensory-motor neuropathy, intestinalpseudo-obstruction, progressive external ophthalmoplegia,ptosis, and leukoencephalopathy [10]. The patient's age atpresentation ranges from 5 months to 50 years, but in about60% of individuals, symptoms begin before the age of 20years. Although progressive gastrointestinal dysmotilityoccurs in virtually all individuals at some point during the

course of the illness, gastrointestinal symptoms are thepresenting complaint in 45% to 67% of individuals andtypically start during childhood [11,12]. These includenonspecific gastrointestinal signs and symptoms such asearly satiety, nausea, dysphagia, gastroesophageal reflux,postpandrial emesis, episodic abdominal pain, and/ordistention and diarrhea. Because of this, MNGIE diseasehas been misdiagnosed as anorexia nervosa and othergastrointestinal disorders, in particular inflammatory boweldisease, celiac disease, and irritable bowel syndrome [11].

MNGIE is an autosomal recessive disease that resultsfrom the mutagenic effect of TP deficiency on mitochondrialDNA (mtDNA). TP deficiency results from mutations in thenuclear gene TYMP located on chromosome 22q13.32 [8].TP activity in leukocytes from patients with MNGIE isusually less than 10% of controls, indicating that loss-of-function mutations in TP cause the disease. The pathologicconsequences of TP deficiency are thought to be theaccumulation of mtDNA defects (depletion, multiple de-letions, or duplications), in various tissues over time, becauseof aberrant thymidine metabolism that leads to impairedreplication and/or maintenance of mtDNA [8,13]. NuclearDNA damage does not appear to be a factor in thepathogenesis of MNGIE disease. Because mtDNA continuesto replicate throughout an individual's life, various tissuesthroughout the body develop abnormalities over time as aresult of progressive impairment of oxidative phosphoryla-tion. Accumulation of mtDNA mutations can be observed infibroblasts of individuals with MNGIE disease. Depletionand deletions of mtDNA are present in most individualswith MNGIE [14–16], and genetic testing of TYMP, thegene encoding TP, detects mutations in approximately 100%of them.

MNGIE is, therefore, a progressive, degenerative diseasewith poor prognosis. The mean age of death is about 38years, with a range of 26 to 58 years [13]. Although treatmentis still experimental, in one instance, allogenic stem-celltransplantation partially restored buffy coat thimidineactivity and lowered plasma nucleosides [17]. Experimentaldata suggest that mitochondrial dysfunction and clinicalsymptoms are produced after years of cumulative toxiceffects of excessive nucleosides on mtDNA, leading tosomatic mtDNA mutations in postmitotic cells and thereforeunlikely to be reversible [17]. Hence, prompt diagnosis isimportant so that treatment can be administered as soon aspossible, before irreversible damage has occurred [17].

This is the second documented patient with MNGIEdisease showing by light microscopy megamitochondria inthe cytoplasm of the gastrointestinal submucosal andmyenteric ganglion cells and the first to demonstrate similarstructures in gastrointestinal smooth muscle cells. Recently,similar structures have been observed in gastrointestinalganglion cells in children with Alpers disease [2]. Priorreports have shown abnormal mitochondria by electronmicroscopy in intestinal smooth muscle cells of patients withMNGIE and MELAS and in ganglion cells of the small

1446 A. R. Perez-Atayde

intestine in a patient with MELAS; megamitochondria,however, were not observed by light microscopy in any ofthese studies [3–7]. The first case of MNGIE reporteddescribing megamitochondria in gastrointestinal ganglioncells [1] had other features in common with the childdescribed herein, which include a long history of undiag-nosed gastrointestinal problems with a suspicion for Crohndisease, similar age at diagnosis, normal mitochondria inskeletal muscle cells by light and electron microscopy andabsence of ragged-red fibers, abnormal mitochondria ingastrointestinal smooth muscle cells, dilated hypertrophicstomach, marked atrophy and fibrosis of the intestinal outerlayer of muscularis propria, and microvesicular and macro-vesicular steatosis of the liver with focal pericellular fibrosis.This phenotype of MNGIE with an early age of presentation,a predominant clinical picture of chronic intestinal pseudo-obstruction, megamitochondria in gastrointestinal ganglioncells and smooth muscle, marked atrophy and fibrosis of theintestinal outer layer of the muscularis propria, and absenceof abnormal mitochondria in skeletal muscle may represent avariant of the disease.

The patient reported herein also had megamitochondria inthe endoscopic duodenal biopsy, performed nearly 3 yearsbefore the biochemical diagnosis of MNGIE, which wererecognized retrospectively after the patient's demise. Thepossibility of a mitochondrial disease, particularly MNGIEand Alpers disease, should be deliberately pursued inpatients with idiopathic chronic intestinal pseudo-obstruc-tion. A relatively noninvasive intestinal biopsy containingganglion cells and muscularis mucosae may be sufficient toestablish a diagnosis before more invasive types of pro-cedures are performed.

In summary, this report illustrates in close detail thesingular appearance of megamitochondria in ganglion cellsand smooth muscle cells of the gastrointestinal tract and thepresence of marked atrophy and fibrosis of the intestinalouter layer of the muscularis propria and corroborates theirimportance in the diagnosis of mitochondriopathies. Pathol-ogists should be aware of the distinctive appearance ofmegamitochondria by light microscopy and of the need toclosely evaluate the morphology of gastrointestinal ganglioncells and smooth muscle cells in patients with idiopathicchronic intestinal pseudo-obstruction.

Acknowledgments

The author thanks Michelle Hadley for the excellenttechnical assistance in the preparation of the histopathology

and immunohistochemistry slides and Howard Mulhern forhis excellent technical assistance in electron microscopy.

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