Late-onset MNGIE without peripheral neuropathy due to incomplete loss of thymidine phosphorylase activity.
ABSTRACT Mitochondrial NeuroGastroIntestinal Encephalomyopathy (MNGIE) is an autosomal recessive disorder characterized by severe gastrointestinal dysmotility, cachexia, peripheral neuropathy, ptosis, ophthalmoplegia, and leukoencephalopathy with early onset and severe prognosis. Mutations in the TYMP/ECGF1 gene cause a loss of thymidine phosphorylase catalytic activity, disrupting the homeostasis of intramitochondrial nucleotide pool. We report a woman with a very late onset of MNGIE, lacking peripheral neuropathy. Thymidine phosphorylase activity was markedly reduced in cultured fibroblasts, but only mildly reduced in buffy coat, where the defect is usually detected, and plasma thymidine was mildly increased compared to typical MNGIE patients. TYMP/ECGF1 analysis detected two heterozygous mutations, including a novel missense mutation. These findings indicate that a partial loss of thymidine phosphorylase activity may induce a late-onset and incomplete MNGIE phenotype.
- SourceAvailable from: Ichizo Nishino[show abstract] [hide abstract]
ABSTRACT: Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive human disease associated with multiple deletions of skeletal muscle mitochondrial DNA (mtDNA), which have been ascribed to a defect in communication between the nuclear and mitochondrial genomes. Examination of 12 MNGIE probands revealed homozygous or compound-heterozygous mutations in the gene specifying thymidine phosphorylase (TP), located on chromosome 22q13.32-qter. TP activity in leukocytes from MNGIE patients was less than 5 percent of controls, indicating that loss-of-function mutations in TP cause the disease. The pathogenic mechanism may be related to aberrant thymidine metabolism, leading to impaired replication or maintenance of mtDNA, or both.Science 02/1999; 283(5402):689-92. · 31.03 Impact Factor
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ABSTRACT: Over the last 15 years, important research has expanded our knowledge of the clinical, molecular genetic, and biochemical features of mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). The characterization of mitochondrial involvement in this disorder and the seminal determination of its genetic cause, have opened new possibilities for more detailed and deeper studies on the pathomechanisms in this progressive and fatal disease. It has been established that MNGIE is caused by mutations in the gene encoding thymidine phosphorylase (TP), which lead to absolute or nearly complete loss of its catalytic activity, producing systemic accumulations of its substrates, thymidine (dThd) and deoxyuridine (dUrd). Findings obtained from in vitro and in vivo studies indicate that the biochemical imbalances specifically impair mitochondrial DNA (mtDNA) replication, repair, or both leading to mitochondrial dysfunction. We have proposed that therapy for MNGIE should be aimed at reducing the concentrations of these toxic nucleosides to normal or nearly normal levels. The first treatment, allogeneic stem-cell transplantation (alloSCT) reported in 2006, produced a nearly full biochemical correction of the dThd and dUrd imbalances in blood. Clinical follow-up of this and other patients receiving alloSCT is necessary to determine whether this and other therapies based on a permanent restoration of TP will be effective treatment for MNGIE.Bioscience Reports 07/2007; 27(1-3):151-63. · 1.88 Impact Factor
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ABSTRACT: We studied the clinical, biochemical, and genetic features of eight patients with the autosomal recessive mitochondrial syndrome mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). MNGIE is clinically characterized by ophthalmoparesis, peripheral neuropathy, leukoencephalopathy, gastrointestinal symptoms (recurrent nausea, vomiting, or diarrhea) with intestinal dysmotility, and histologically abnormal mitochondria in muscle. Brain MRI scans were consistent with leukodystrophy in seven patients examined. Nerve conduction and EMG studies were compatible with a sensorimotor neuropathy; quantitative EMG of two patients suggested a myogenic process. Muscle mitochondrial enzyme analysis revealed a partial defect of cytochrome c oxidase activity in five patients; three had additional respiratory chain enzyme defects. Two patients had isolated complex I defects, and one had normal respiratory chain function. Southern blot analysis revealed multiple deletions of mitochondrial DNA in four of eight patients.Neurology 05/1994; 44(4):721-7. · 8.25 Impact Factor
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Late-onset MNGIE without peripheral neuropathy due to incomplete loss
of thymidine phosphorylase activity
Roberto Massaa,*, Alessandra Tessab, Maria Margolliccic, Vanna Michelid, Andrea Romigia, Giulia Tozzib,
Chiara Terraccianoa, Fiorella Piemonteb, Giorgio Bernardia, Filippo M. Santorellib
aDepartment of Neurosciences, University of Rome-Tor Vergata and IRCCS-Fondazione S. Lucia, Via Montpellier 1, I-00135 Rome, Italy
bMolecular Medicine & Neurosciences, IRCCS-Bambino Gesù Hospital, Rome, Italy
cLaboratory of Metabolic Genetics, Department of Pediatrics, University of Siena, Siena, Italy
dSection of Biochemistry, University of Siena, Siena, Italy
a r t i c l e i n f o
Received 3 July 2009
Received in revised form 10 August 2009
Accepted 27 August 2009
a b s t r a c t
Mitochondrial NeuroGastroIntestinal Encephalomyopathy (MNGIE) is an autosomal recessive disorder
characterized by severe gastrointestinal dysmotility, cachexia, peripheral neuropathy, ptosis, ophthalmo-
plegia, and leukoencephalopathy with early onset and severe prognosis. Mutations in the TYMP/ECGF1
gene cause a loss of thymidine phosphorylase catalytic activity, disrupting the homeostasis of intramito-
chondrial nucleotide pool. We report a woman with a very late onset of MNGIE, lacking peripheral neu-
ropathy. Thymidine phosphorylase activity was markedly reduced in cultured fibroblasts, but only mildly
reduced in buffy coat, where the defect is usually detected, and plasma thymidine was mildly increased
compared to typical MNGIE patients. TYMP/ECGF1 analysis detected two heterozygous mutations, includ-
ing a novel missense mutation. These findings indicate that a partial loss of thymidine phosphorylase
activity may induce a late-onset and incomplete MNGIE phenotype.
? 2009 Elsevier B.V. All rights reserved.
The syndrome of Mitochondrial NeuroGastroIntestinal Enceph-
alomyopathy (MNGIE, MIM #603041) is an autosomal recessive
disorder of oxidative phosphorylation caused by mutations in
TYMP/ECGF1, the gene encoding thymidine phosphorylase (TP), a
crucial enzyme for the homeostasis of intramitochondrial nucleo-
tide pool . Mutations in TYMP/ECGF1 lead to nearly absent TP
catalytic activity, producing systemic accumulations of its sub-
strates, thymidine (dThd) and deoxyuridine (dUrd) in body fluids.
Toxic levels of dThd and dUrd induce nucleotide pool imbalances
that in turn lead to mtDNA abnormalities (point mutations, multi-
ple deletions, and depletion) .
The typical clinical features of MNGIE consist in severe gastro-
intestinal dysmotility, cachexia, peripheral neuropathy (PN), eyelid
ptosis and ophthalmoplegia, and are associated with remarkable
leukoencephalopathy evidenced at brain MRI . Clinical onset is
usually in young adulthood, with a mean of 19 years, and the prog-
nosis is severe, with a mean age at death of 37 years. The clinical
diagnosis can be confirmed by measuring TP activity in buffy coat
or the plasma levels of dThd and dUrd , and it is subsequently
corroborated by molecular investigations.
Although MNGIE has a relatively homogeneous biochemical and
clinical phenotype, a few patients with milder biochemical altera-
tions and less severe clinical manifestations have been described in
association with mutations in TYMP/ECGF1 . Cases have also
been reported presenting highly similar clinical features but lack-
ing leukoencephalopathy, and displaying normal enzyme activity.
None of these phenocopies presented mutations in TYMP/ECGF1.
We report on a 67-year-old woman presenting, since age
53 years, the typical MNGIE phenotype but lacking PN. TP activity
was mildly reduced in buffy coat, but it was markedly reduced in
cultured skin fibroblasts. Direct gene sequencing showed patho-
genic mutations in TYMP/ECGF1.
2. Patients and methods
2.1. Case description
A 67-year-old woman, the first of two daughters born to unre-
lated parents, was healthy until the age of 53 when she underwent
a first exploratory laparotomy for profuse melena due to bleeding
from small intestine ulcers. During surgery, colic-peritoneal adhe-
sions were released and some nodules in the small bowel were
0960-8966/$ - see front matter ? 2009 Elsevier B.V. All rights reserved.
* Corresponding author. Tel.: +39 06 72596004; fax: +39 06 72596022.
E-mail address: email@example.com (R. Massa).
Neuromuscular Disorders 19 (2009) 837–840
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/nmd
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biopsied. Histological examination revealed fibro-adipose transfor-
mation with chronic inflammation. In the following years, the pa-
gastrointestinal manifestations such as early satiety, vomiting,
irregular bowel movements and borborygmi. At age 63 years, she
presented abdominal pain and distension with fever for which
she underwent a second laparotomy that disclosed a purulent peri-
tonitis due to perforation of a small bowel diverticulum. A small
bowel resection, with a termino-terminal anastomosis was per-
formed at this time. The following year the patient underwent a
new resection of the ileus, after presenting abdominal pain and
distension. The patient has always been thin, and her weight pro-
gressively declined from 48 to 39 kg. Around age 55 years she
started complaining of diplopia, associated with a progressive loss
of ocular motility and bilateral eyelid ptosis. At age 64 years, she
noticed a progressive gait unbalance, a subjective sensation of
‘‘light head” and loss of short-term memory, and a pulsating head-
ache. Moreover, she referred of a recent episode of loss of con-
sciousness without a history of epilepsy. When admitted to our
department at age 65 years, the patient was cachectic: her weight
was 39 kg and her height 161 cm. Neurological examination re-
vealed diffuse muscle atrophy, mild static and dynamic ataxia,
bilateral external ophthalmoplegia and ptosis, and a fine head tre-
mor. All other functions, including strength and sensation, were
normal. Deep tendon reflexes were uniformly brisk, and plantar re-
flexes were flexor. There were neither serological nor cerebrospinal
fluid (CSF) overt abnormalities, except for moderately increased
lactate levels, which were 27.2 mg/dl in serum (n.v. = 4.5–19.8),
and 26.7 mg/dl in the CSF (n.v. = 10–22). CSF protein level was
46.4 mg/dl (n.v. = 10–45 mg/dl). EMG studies showed a myopathic
pattern in the four limbs, whereas nerve conduction studies (NCS)
showed normal distal latencies, amplitude, and nerve conduction
velocities of the explored nerves (Table 1). Neuropsychological
testing revealed a mild impairment of visuo-spatial memory. A
brain MRI showed diffuse leukoencephalopathy of the cerebral
hemispheres and ponto-mesencephalic junction (Fig. 1A). Audio-
metric testing revealed a subclinical, bilateral, sensorineural hear-
ing loss. An EEG showed isolated spikes and/or sharp waves
localized in right temporal region and less evident in left temporal
region (Fig. 1B). An open muscle biopsy showed mildly increased
variation in fiber diameter, several ragged red and succinate dehy-
drogenase (SDH)-hyper reactive fibers and cytochrome c oxidase
(COX)-negative fibers but no neurogenic changes (Fig. 1C). Spectro-
photometric determination of respiratory chain complex activities
performed in muscle homogenate and cultured skin fibroblasts dis-
closed a slightly reduced activity of complex I (65% of normal va-
lue) but normal complex IV activity.
of melena andrecurrent
Family history was only significant for a maternal cousin who
had died at age 35 years because of an allegedly severe muscle dis-
ease. Inspection of one of her ID photographs showed a cachectic
young lady with bilateral eyelid ptosis. No additional clinical notes
were produced by her relatives.
2.2. Biochemical and molecular analyses
TP activity was measured using spectrophotometric method as
described previously . Briefly, buffy coat obtained from venous
blood and cultured fibroblasts, were homogenized in lysis buffer
(50 mM Tris–HCl, pH 7.2, containing 1% Triton X-100, 2 mM phen-
ylmethylsulfonyl fluoride and 0.02% mercaptoethanol) and then
sonicated for 30 s. Samples were centrifuged at 13,000g for
30 min at 4 ?C. Supernatants protein concentration was deter-
mined according to the bicinchoninic acid method . The reaction
mixture (final volume 100 ll), containing 100 lg of protein,
10 mM dThd in 0.1 M Tris–arsenate, pH 6.5, was incubated at
37 ?C for 1 h. The reaction was terminated by the addition of
1 ml of 0.3 N NaOH. In parallel with each sample, a blank was also
processed, to which dThd was added after the addition of NaOH.
The amount of thymine formed was measured at 300 nm wave-
length and determined based on the 3.4 ? 103l/mol/cm difference
in the molar extinction coefficient between dThd and thymine at
alkaline pH. Enzyme activity was expressed as nmol of thymine
formed per hour per milligram of protein.
Nucleoside concentration was measured in plasma by HPLC.
Freshly collected plasma (200 ll) was added to 200 ll of ice-cold
0.6 M HClO4under vortex mixing. Precipitated proteins were spun
down by centrifugation (2 min at 12,000g) and clear supernatants
were brought to pH 6–7 by adding suitable amounts of ice-cold
3.5 M K2CO3and discarding precipitated KClO4by centrifugation.
Extracts were processed by HPLC or stored at ?20 ?C. HPLC appara-
tus consisted of a Beckman System Gold Module 126, with a
mod.168 Nouveau diode array detector (Beckman San Ramon,
CA, USA). Phenomenex Luna C18 columns (3 lm particle size, 75
? 4.6 mm) equipped with guard columns (Phenomenex Security
guard 4 mm L ? 3 mm ID) were used. A gradient elution pattern
of eluant A (0.01 M potassium phosphate buffer, pH 5.5) and eluant
B (methanol) was used as follows: isocratic phase at 100% A for
4 min, then to 21% B in 4 min, then immediately to 30% B and back
to initial conditions after 3 min; initial conditions were restored in
7 min, and total run time was 18 min. HPLC elution was performed
at room temperature; the flow rate was 1 ml/min and the absor-
bance was monitored at 260 and 280 nm. Retention time, co-elu-
tion with added internal standards, absorption spectra and/or the
280/260 nm absorbance ratios confirmed peak identities; concen-
Summary of nerve conduction study findings.
Sensory nervesMotor nerves
Left radialRight ulnarWrist–ADMBe Elb–WristAb Elb–Be Elb
Right suralRight tibial Ankle–AHBKnee–Ankle
Left suralLeft peroneal Ankle–EDBBe Knee–AnkleAb Knee–Be Knee
ADM, abductor digiti minimi; Be Elb, below elbow; Ab Elb, above elbow; AHB, abductor hallucis brevis; EDB, extensor digitorum brevis; Be Knee, below knee; Ab Knee, above
R. Massa et al./Neuromuscular Disorders 19 (2009) 837–840
Author's personal copy
tration/area linear plots were developed for quantification. A mix-
ture of all standard solutions was injected daily to check the reli-
ability of separation and any modification of retention times due
to the chromatographic system.
Total DNA was extracted from peripheral blood and tissue
homogenates following standard procedures. Direct sequencing
of the TYMP/ECGF1 gene used methods reported elsewhere .
Southern blotting to determine qualitative or quantitative altera-
tions of mtDNA  and qPCR determination of the levels of mito-
chondrial genome in muscle homogenate  used described
Residual TP activity in the patient’s leukocytes was 132 nmol/h/
mg, corresponding to 18% of mean normal value (range = 250.6–
1211 nmol/h/mg of proteins; n = 50), whereas TP activity in the pa-
tient’s cultured fibroblasts was 5.7 nmol/h/mg, corresponding to
4.75% of mean normal value (range = 40–200 nmol/h/mg; n = 5).
Plasma dThd was mildly elevated (2.1 lM/l) in the patient
(n.v. = <0.05 lM/l;
n = 10),whereas
(n.v. = <0.05 lM/l; n = 10).
Direct sequencing of TYMP/ECGF1 identified the c.1160-1G>A
mutation affecting the splice acceptor site of intron 8, and already
described to lead to skipping of exon 9 , in compound heterozy-
gosity with the novel c.1135G>A (p.E379K) mutation which in-
volves a highly conserved residue in TP (Fig. 1D). The former
variant was also detected in the healthy mother but not in the sis-
ter of the proband, whereas the novel mutation was not detected in
300 control alleles. Southern blotting did not reveal gross deletions
nor severe depletion of mtDNA whereas qPCR analyses showed
partial loss of mtDNA genomes in muscle and cultured cells (43%
and 58% of normal).
The patient reported here shows a multisystem disease sugges-
tive of a defect of oxidative metabolism and, to the best of our
knowledge, she represents the oldest living MNGIE patient re-
ported and the one with the latest onset. Despite a later age at on-
set, the complex clinical setting reproduces the MNGIE syndrome
with a high degree of central nervous system symptoms possibly
related to the patient’s leukoencephalopathy, including cerebellar
ataxia, tremor and memory loss, associated with EEG focal
Contrary to patients reported so far, however, there was no evi-
dence of PN, which is regarded as a cardinal feature of MNGIE 
and, at times, mimics the neurophysiological and clinical presenta-
tion observed in CIDP or Charcot–Marie–Tooth disease [11,12].
Interestingly, rare cases with incomplete syndrome have been de-
scribed, including one late-onset patient with normal NCS after
12 years of disease progression [5,13]. Although follow-up NCS
may be necessary, it is possible that, in late-onset MNGIE, PN is dis-
pensable. A variable penetrance might be related to different
TYMP/ECGF1 mutations and their ability to promote a toxic effect
exerted by the accumulation of unprocessed nucleosides upon
the turnover of mtDNA.
Fig. 1. (A) Axial T2-FLAIR brain MRI showing diffuse hyperintensity of the cerebral white matter partially involving U-fibers. (B) The EEG recording shows isolated spikes and/
or sharp waves localized in right and left temporal region. (C) Cross-section of the muscle biopsy stained for cytochrome c oxidase (COX) showing the presence of a hyper
reactive fiber (circle) and some COX-negative fibers (asterisks). (D) Electropherogram of the sequences flanking the c.1135G>A (resulting in the p.E379K variant) (left) and the
c.1160-1G>A (right) mutations detected in the patient. Mutations are indicated by an arrow.
R. Massa et al./Neuromuscular Disorders 19 (2009) 837–840
Author's personal copy
Attempts to establish genotype–phenotype correlations in se-
vere MNGIE have been generally disappointing and the degree of
clinical severity does not appear to be fully influenced by the ex-
tent of reduced TP activity. However, there are cases in whom a
less severe TP dysfunction in buffy coat (about 15% of control val-
ues) and a moderate accumulation of plasma nucleosides appear to
correlate well with a later-onset, milder clinical form . This is in
part questioned by findings in our patient in whom the moderate
reduction of enzyme activity observed in buffy coat and the mild
plasma nucleoside accumulation might well explain both an older
age at disease manifestations and lack of a full-blown syndrome,
whereas the more marked TP deficit determined in cultured cells
would be compatible with a more severe phenotype. It remains
too speculative to hypothesize that the differential reduction of
TP activity among tissues in the same patient would be attribut-
able to the specific mutations identified, including the novel
p.E379K variant. Selection in vitro of cells with high concentrations
of nucleosides or a higher mtDNA mutation rate or other factors
might also be considered. Regardless of these hypotheses, determi-
nation of TP activity in buffy coat should be considered as the stan-
dard approach, due to the higher cost and time consumption of cell
culturing. Molecular studies remain, however, conclusive for a pre-
The editorial assistance of Mr. James Lynch is acknowledged.
We are grateful to Dr. Elena Campione for performing skin biopsy
and to Mr. Graziano Bonelli for photographic work. This study was
supported by a grant from the University of Rome ‘‘Tor Vergata”
(RSA 2008) to R.M. and the Italian Ministry of Health (to F.M.S.).
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R. Massa et al./Neuromuscular Disorders 19 (2009) 837–840