doi:10.1093/brain/awh410Brain (2005), 128, 723–731
Infantile hepatocerebral syndromes associated
with mutations in the mitochondrial DNA
Gianfrancesco Ferrari,1Eleonora Lamantea,1Alice Donati,2Massimiliano Filosto,3Egill Briem,1
Franco Carrara,1Rossella Parini,4Alessandro Simonati,3Rene ´ Santer5and Massimo Zeviani1
Correspondence to: Massimo Zeviani, MD, PhD, Unit of
Molecular Neurogenetics, National Neurological Institute
‘Carlo Besta’, via Temolo 4, 20126 Milan, Italy
1Unit of Molecular Neurogenetics, Pierfranco and Luisa
Mariani Center for the Study of Children’s Mitochondrial
Disorders, National Institute of Neurology, Milano,2Unit of
Child Neurology, ‘Meyer’ Children’s Hospital (Florence),
3Institute of Neurology, University of Verona, Verona,
4Unit of Pediatrics, Pierfranco and Luisa Mariani Center
for the Study of Children’s Metabolic Disorders, University
Hospital, Monza, Italy and5University Children’s Hospital,
We studied nine infant patients with a combination of
progressive neurological and hepatic failure. Eight
children, including two sibling pairs and four singletons,
were affected by Alpers’ hepatopathic poliodystrophy.
A ninth baby patient suffered of a severe floppy infant
syndrome associated with liver failure. Analysis of
POLG1, the gene encoding the catalytic subunit of mito-
chondrial DNA polymerase, revealed that all the patients
carried different allelic mutations in this gene. POLG1 is
a major disease gene in mitochondrial disorders.
Mutations in this gene can be associated with multiple
deletions, depletion or point mutations of mitochondrial
DNA (mtDNA). In turn, these different molecular pheno-
types dictate an extremely heterogeneous spectrum of
clinical outcomes, ranging from adult-onset progressive
ophthalmoplegia to juvenile ataxic syndromes with
epilepsy, to rapidly fatal hepatocerebral presentations,
including Alpers’ syndrome.
Abbreviations: ad = autosomal dominant; ar = autosomal recessive; MDS = mitochondrial DNA depletion syndrome;
mtDNA = mitochondrial DNA; PCR = polymerase chain reaction; PEO = progressive external ophthalmoplegia;
POLG1 = gene encoding mitochondrial DNA polymerase-gA; pol-gA = DNA polymerase-gA; SANDO = sensory atactic
neuropathy with dysarthria and ophthalmoplegia
Received October 11, 2004. Revised December 27, 2004. Accepted January 4, 2005. Advance Access publication
February 2, 2005
Mutations in POLG1, the gene encoding the catalytic subunit
of mitochondrial DNA (mtDNA) polymerase (pol-gA) are the
prevalent cause of autosomal dominant (ad) or autosomal
recessive (ar) forms of familial progressive external ophthal-
moplegia (PEO) syndromes (Van Goethem et al., 2001;
Lamantea et al., 2002; Agostino et al., 2003). However, addi-
tional clinical presentations associated with POLG1 muta-
tions have been reported recently, including autosomal
recessive sensory atactic neuropathy with dysarthria and
ophthalmoplegia (SANDO) (Van Goethem et al., 2003a),
a juvenile-onset mixed sensory and cerebellar atactic syn-
drome complicated by epileptic seizures and myoclonus,
(Van Goethem et al., 2004; Winterthun et al., 2005) and,
very recently, Alpers’ hepatopathic poliodystrophy (Naviaux
and Nguyen, 2004). The latter is an early-onset, fatal disease,
characterized by hepatic failure, intractable seizures and
global neurological deterioration (OMIM #203700). It is
commonly believed that the main target of POLG1 mutations
is mtDNA, and that lesions in this genome may ultimately
determine the clinical phenotypes. For instance, both adPEO
#The Author (2005). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: email@example.com
and arPEO are characterized by the accumulation of multiple
mtDNA rearrangements (mainly deletions) in post-mitotic
tissues, notably skeletal muscle and brain (Zeviani et al.,
1990; Servidei et al., 1991; Suomalainen et al., 1992;
Moslemi et al., 1999). In contrast, much lesser amounts of
mtDNA rearranged molecules are detected in the muscle
tissueof patients affected
cerebellar ataxia–epilepsy syndrome, but no information is
available for these conditions on the integrity of mtDNA in
the critical tissues (i.e. cerebellum, dorsal spinal ganglia
and spinal cord). Finally, depletion of liver mtDNA, rather
than accumulation of multiple deletions, has been reported
in the case with Alpers’ syndrome associated with a homo-
zygous POLG1 stop mutation (Naviaux et al., 1999; Tesarova
et al., 2004).
We describe here nine patients (two sibling pairs and five
singleton cases) with POLG1 mutations associated with
combined infantile fatal encephalopathy and hepatopathy.
The two sibling pairs and four singletons were patients
with typical Alpers’ syndrome, while the ninth patient was
affected by a severe floppy infant syndrome with signs of
by SANDO or sensory–
Patients and methods
All patients were of Caucasian origin. The families of patients 5,
6 and 7 (see below) were from Germany, and those of the other
patients were from Italy.
Patient 1 was a boy, born at term from non-consanguineous Italian
parents. He developed normally in the first months after birth, but
head control was reached at 6 months, and autonomous deambula-
tion at 14 months. An apparently myogenic torticollis was noticed
after a few months of life; at 10 months, he had two episodes of
sudden head drop, followed by the onset of psychomotor arrest/
regression, hypotonia, ataxia and focal myoclonus of the right upper
limb. In the following weeks, the myoclonus became subcontinuous,
and was associated with rapidly progressive, severe generalized
hypotonia and weakness requiring ventilatory assistance. Seizures
were partially controlled with a combination of topiramate, clob-
azam and phenylbarbiturate. The EEG worsened in the following
weeks, with diffuse disorganization of the basal activity and multiple
foci of paroxysmal activity. A brain MRI showed symmetrical
lesions of the basal ganglia, thalami, cerebellar dentate nuclei and
left occipital cortical and subcortical regions (Fig. 1A and B). Proton
magnetic resonance spectrometry (MRS) revealed an abnormal
accumulation of lactic acid in the putamen and a reduction of the
N-acetyl aspartate (NAA) peak, an index of neuronal loss. Light
microscopy examination and the activities of the respiratory chain
complexes were both normal in a muscle biopsy, while no liver and
skin biopsies were taken. In the following months, increasingly high
levels of g-glutamyltransferase and transaminases, severe, predom-
inantly cholestatic jaundice, hypoglycaemia and hypocoagulation
were accompanied by dramatic worsening of the neurological symp-
toms, leading to the death of the child at 30 months of age. Autoptic
examination was refused by the parents. The diagnostic conclusion
was Alpers’ syndrome.
The elder sister of patient 1 had a very similar clinical course,
characterized by a disease-free interval in the first 4 months after
birth, followed by persistent vomiting, epileptic crises with loss
of eye contact, and myoclonus in the upper limbs with secondary
generalization and loss of consciousness. Similarly to her brother,
she developed a fixed flexionof the neckwith rotation toward the left
side. Persistent myoclonus in the upper limbs was associated with
profound hypotonia and loss of eyecontact. A brain MRI at 5 months
of age showed the presence of cortical atrophy and leukoencephalo-
g-glutamyltransferase and transaminases were firstly documented
after the administration of valproate for the control of myoclonic
seizures. The activities of the mitochondrial trifunctional protein
(a component of the fatty acid b-oxidation pathway) and those of
and muscle homogenate; unfortunately, liver tissue was notavailable
for biochemical studies. A rapidly progressive hepatic failure with
enlarged liver, hypoalbuminaemia and predominantly cholestatic
jaundice led to the death of the child at 8 months of age. The autopsy
showed diffuse encephalomalacia and severe liver steatosis with
lobular fibrosis and bile ductule proliferation.
Fig. 1 Brain MRI of patients 1 (A and B) and 3 (C and D).
(A) T2-weighted transversal section showing bilateral cortical
atrophy more evident in the parietal lobe and peri-silvian regions.
Bilateral lesions are present in thalamus, globus pallidus and
caudate nucleus. The internal capsule and surrounding white
matter are relatively normal. (B) Diffusion-weighted transversal
section showing two areas of lesion in the left parieto-occipital
region. (C) T1-weighted coronal section showing bilateral dilation
of lateral ventricles and cortical atrophy. (D) T1-weighted
inter-hemispheric sagittal section showing atrophy of both
cerebral cortex and cerebellar vermis.
G. Ferrari et al.
Patients 3 and 4
The clinical and neuropathological features of these two brothers,
born from non-consanguineous Italian parents, have already been
reported elsewhere (Simonati et al., 2003a). Briefly, patient 3 had a
slowly progressive history with onset at 19 months, characterized by
ataxia, spasticity and myoclonic seizures. At 9 years of age, atrophy
of the occipital cortex was shown by MRI (Fig. 1C and D), and
hepatic cirrhosis was documented on a liver biopsy. The clinical
picture worsened thereafter, leading to a vegetative state and even-
tually todeath at 15 years. The clinical history of the younger brother
(patient 4) started with myoclonic fits in the right limbs at 5 months,
followed by an atactic–dystonic syndrome with rapid global deteri-
oration. Diffuse cortical atrophy was documented by CT scan at
6 months of age, and the EEG showed multifocal spikes in the left
fronto-temporal region on a slow background activity. At 12 months,
valproate therapy was started because of refractory seizures;
however, the patient developed acute hepatic failure and died
1 month later. The neuropathological examination of the brain
showed severe atrophy of the anterior portions of the frontal lobes,
insula and parieto-occipital cortex, and necrotizing lesions with
neuronal loss, neuropil microcysts and vascular proliferation in
the brain cortex, thalamus, cerebellum and inferior olives. In both
patients, the diagnosis was Alpers’ syndrome.
This patient was a baby boy, born from non-consanguineous German
parents. He developed normally in the first 6 months. Refusal of food
and failure to thrive were noted from the 7th month of life. At 1 year
of age, increased transaminase levels in the blood were accompanied
by liver enlargement and hyperecogenicity documented by ultra-
sound examination. Motor development was delayed. His condition
gradually deteriorated, with recurrent hypoglycaemic episodes
and lactic acidosis. A liver biopsy at the age of 16 months revealed
steatosis and fibrosis. At 20 months of age, he had numerous
episodes of status epilepticus and epilepsia partialis continua. An
increase of lactate and a decrease of choline were detected by MRS
of the brain. The patient died at 2.5 years of age during an episode of
status epilepticus. Autopsy showed liver fibrosis with transition to
liver cirrhosis, spongiform changes in the cerebrum, focal infarcts in
the cerebellar cortex, and mesial temporal sclerosis. Abnormal mito-
chondria were found in liver, heart and brain, but the mtDNA was
not available for molecular studies. The diagnostic conclusion was
The clinical and neuroradiological features in this male patient,
diagnosed as typical Alpers’ syndrome, have already been reported
(Flemming et al., 2002). He was born from non-consanguineous
German parents. Mental and motor developmental delay was first
noted at 4 years of age. At 7 years, generalized tonic–clonic seizures,
myoclonus and recurrent focal motor seizures evolving into epilep-
sia partialis continua started after an operation for correction of a
squint. An otherwise intractable focal motor status was treated with
valproate, which was followed by life-threatening hepatopathy. The
boy became blind and incontinent, and lost independent ambulation.
He is now, at age 13 years, in a vegetative state.
The first child of non-consanguineous parents from Germany, this
boy was born at term after a normal pregnancy and delivery. After a
brief disease-free interval, mild motor retardation was first noted at
6 months. At the age of 16 months, he was admitted to a local
hospital for status epilepticus. An MRI performed at 18 months
of age showed mild generalized brain atrophy. He was treated
with valproate, but during the following days he developed severe
hepatopathy with an increase of transaminases in the blood, hypo-
coagulation and severe mixed jaundice. His neurological condition
evolved into epilepsia partialis continua, which was partially con-
trolled by carbamazepine, later switched to oxcarbazepine. A brain
MRI showed hyperintense signals in the brain cortex, in both thalami
and in the periventricular area of the mesencephalon. The MRS
showed a lactate peak and a decrease of the choline peak. The blood
lactate concentration was also increased. A diagnosis of Alpers’
syndrome was given to this patient. He died at 2 years of age after
severe complications from liver failure.
This male patient has already been reported as a juvenile case of
Alpers’ syndrome with central–peripheral axonopathy (Simonati
et al., 2003b). He had recurrent vomiting episodes in infancy. At
age 7 years, he developed epilepsia partialis continua, followed by
progressive ataxia. At 18 years, neurological examination revealed
sensory ataxia, absent deep tendon reflexes, cerebellar dysfunction,
lactate concentration was increased. An MRI showed mild brain
cortical and cerebellar atrophy, with focal hyperintensity signals
in the frontal and occipital cortex and in both thalami. Electro-
physiological studies revealed the presence of abnormal central
and peripheral conduction, and a sural nerve biopsy showed marked
reduction of the myelinated fibres. The following months were char-
acterized by numerous episodes of partial and generalized seizures
and myoclonic fits, which were barely responsive to therapy. The
administration of valproate to control myoclonus was followed
by the development of severe acute hepatic failure, which required
liver transplantation. However, relentless deterioration of the neuro-
logical conditions led to the patient’s death at 19 years of age.
A baby girl, the second child of non-consanguineous Italian parents,
was born at the 38th week by caesarian delivery after an otherwise
uneventful pregnancy. However, both the patient and her healthy
4-year-old brother were conceived by in vitro fertilization because of
hypofertility of the parents. The father was diagnosed as having
severe astheno-terato-zoospermia, while the mother, at age 36 years,
was found to have mild hypergonadotrophic hypogonadism with
reduced levels of dehydroepiandrosterone (75.5 mg%, normal
120–360) and levels of follicle-stimulating hormone (FSH) in the
upper part of the normal range (6.5 IU/l, normal 1.1–9.6), as typ-
ically found in pre-menopausal women. The body weight of the
patient at birth was 2760 g, body length was 49 cm and head cir-
cumference was 34 cm. After a short disease-free interval, she star-
ted having frequent vomiting with increasing feeding difficulty
leading to growth arrest at 5 months of age. From the third month
of life, the patient suffered from progressive generalized hypotonia,
with some signs of psychic regression but no loss of eye contact. The
SMN1 gene, responsible for the most common forms of hereditary
spinal muscular atrophy (SMA) syndromes, including SMA type 1,
was normal. Laboratory examination disclosed high levels of serum
transaminases and g-glutamyltransferase, high levels of lactate and
pyruvate in both serum and CSF, and normal levels of blood glucose
Infantile hepatocerebral syndromes associated with POLGI mutations725
and ammonia. High levels of lactic acid and dicarboxylic acids were
found in the urine. An ultrasound examination of the abdomen
showed a hyperecogenic liver with cholestasis and cholelithiasis,
and moderate ascites, with no alteration of the kidneys and spleen.
The ultrasound examination of the heart was normal. A brain
MRI was normal as well, but the EMG examination showed a
diffuse, marked reduction of the nerve conduction velocities
(3.4 m/s in the left median nerve; 6.0 m/s in the right common
peroneal nerve, normal values for a 5-month-child 30 m/s), suggest-
ing the presence of a severe hypomyelinating peripheral neuropathy,
accounting for the profound hypotonia, generalized weakness and
deep tendon areflexia. No other abnormality was detected on the
EMG. The visual evoked potentials were normal for the age. Activ-
ities of arylsulfatase A and cerebroside-b-galactosidase were normal
in blood leukocytes. Light microscopy examination of a muscle
biopsy did not disclose significant alterations. Biochemical invest-
igation on the homogenate from a liver biopsy revealed the presence
of multiple defects of the mtDNA-related respiratory chain activities
(?30% of age-matched controls), while no abnormality was found in
muscle homogenate and in cultured fibroblasts. The child died at
6 months of age of ventilatory insufficiency due to generalized
muscle weakness and profound hypotonia. She had persistent eleva-
tion of serum transaminases and g-glutamuyltransferase, but no
jaundice, hyperammonaemia, hypoglyacemia, abnormalities of
blood coagulation or other signs of overt hepatic failure. No autopsy
Biochemical analysis of the respiratory chain complexes was per-
formed on the 800 g supernatants obtained from muscle or liver
homogenates and on digitonin-treated cultured skin fibroblasts, as
previously described (Bugiani et al., 2004).
DNA extracted from fibroblasts, skeletal muscle, brain and
liver was used for quantification of mtDNA versus nuclear DNA
by real-time polymerase chain reaction (PCR) (He et al., 2002;
von Wurmb-Schwark et al., 2002). Specific fluorochrome-labelled
oligonucleotides (Applied Biosystems) were used as probes in real-
time PCR experiments. In some cases, DNA was extracted from
paraffin-embedded, formalin-fixed specimens of probands and con-
trols, as described (Wright and Manos, 1990). Whenever possible,
quantitative Southern blot analysis was also performed, as described
(Zeviani et al., 1989). In both real-time PCR and Southern blot
analyses, the amount of mtDNA was then compared with the amount
of the nuclear gene cluster encoding the 18S rRNA on chromosome
21, contained in the same sample. The mtDNA/18S rDNA ratio
obtained in the patients’ samples was expressed as a percentage
of the mean obtained in control samples, taken as 100%.
DNA from fibroblasts or lymphocytes was used as a template to
amplify the 23 exons of the POLG1 gene, as described (Lamantea
et al., 2002). PCR fragments were analysed by automated nucleotide
sequencing (Applied Biosystems).
Analysis of mtDNA
Southern blot and real-time quantitative PCR analyses on
muscle and fibroblast mtDNA in patients 1–3 and 9 failed
to show large-scale rearrangements or abnormalities of the
mtDNA copy number.
No liver or brain DNA was available for patients 1 and 4–7.
Total DNA was extracted from paraffin-embedded post-
mortem liver tissue from patients 2 and 3 and from two
age-matched post-mortem paraffin-embedded control liver
specimens. The results of real-time PCR showed a reduction
of the mtDNA/18S rDNA ratio ranging from 25 to 40% of the
controls’ mean in different determinations for both patients.
Likewise, a 30% reduction in mtDNA content was obtained
by real-time PCR assays on post-mortem frozen frontal cortex
of patient 8, compared with similar specimens from two age-
matched control individuals. Finally, profound depletion
(3–5% of the controls’ mean) was demonstrated repeatedly
in bioptic liver of patient 9 by both Southern blot and
real-time PCR analyses.
Analysis of the POLG1 gene
Mutations in the dGK or TK2 genes, which are known to
be responsible for some cases of liver or muscle mtDNA
depletion syndromes (MDS) (Mandel et al., 2001; Saada
et al., 2001), were first ruled out.
The recent identification of POLG1 mutations in three
children with Alpers’ hepatopathic poliodystrophy belonging
to two families (Naviaux and Nguyen, 2004) prompted us to
investigate the POLG1 gene in our patients. The results are
reported in Table 1 and Fig. 2.
Patients 1 and 2 had two identical POLG1 mutations,
including (i) a 2243G!C transversion in exon 13, predicting
a W748S change in the spacer region of the pol-gA protein;
and (ii) the insertion of a cytosine at position 3630 of the
POLG1 cDNA (3630Cins) in exon 22. The first mutation was
inherited from the father, while the second was inherited from
Table 1 POLG1 mutations
Patient no.Mutations* Gene
1 and 22243G!C
3 and 4
*The nucleotide and amino acid positions are indicated for both
alleles of each patient according to the nomenclature of the
human POLG1 cDNA and pol-gA sequences in Lamantea et al.
(2002);+Associated with the SNP 3428A!G (E1143G);
G. Ferrari et al.
the mother. Both parents were heterozygous carriers for the
(paternal) mutation 2243G!C (W748S) which was found in
patients 1 and 2, and for a (maternal) 731T!C mutation,
predicting a L244P change in the amino acid sequence. In
both sibling pairs, the 2243G!C (W748S) mutation was
syntenic to a known single-nucleotide polymorphism (SNP),
3428A!G, predicting an E1143G amino acid change. The
latter is relatively frequent (3–4%) in Caucasians and can
be found in homozygosity in normal adult individuals. The
allele carrying both 2243G!C and 3428A!G has been
reported in other POLG1 mutant patients of European origin
(Van Goethem et al., 2004; Winthertun et al., 2005).
Patient 5 was a compound heterozygote for a (paternal)
1399G!A mutation predicting an A467Tamino acid change,
and a (maternal) 2542G!A mutation predicting a G848S
amino acid change. The POLG1 gene in a healthy brother
had neither mutation.
Patient 6 was a compound heterozygote for the same
1399G!A (A467T) mutation, which was inherited from the
mother, and for a paternal 3482+2T!C splicing mutation
affecting an invariant position in the 30exon/intron donor
site of exon 21. A healthy brother had neither mutation.
Patient 7 was a compound heterozygote, again carrying the
1399G!A (A467T) mutation, which was inherited from the
father, and a new maternal 2869G!C transversion predicting
an A957P amino acid change.
Patient 8 was homozygous for the 1399G!A (A467T)
Finally, allelic mutations were also found in patient 9. The
paternal mutant allele contained the cis mutations 752C!T
in exon 3 and 1760C!T in exon 10, predicting a T251I and a
P587L amino acid change in the same pol-gA protein
molecule. The maternal mutant allele contained a 694C!G
transversion in exon 3, predicting an R232G amino acid
change. The parents were heterozygous carriers for the cor-
responding mutations; the healthy brother of the patient,
now 4 years old, was also a heterozygous carrier for the pater-
nal allele, while the maternal allele was normal.
We report here the association of POLG1 mutations with two
severe infantile syndromes, both characterized by a combina-
tion of hepatic and neurological failure. The first syndrome
meets the clinical and pathological hallmarks of Alpers’
hepatopathic poliodystrophy (Blackwood et al., 1963;
Huttenlocher et al., 1976), including (i) refractory seizures,
in particular epilepsia partialis continua; (ii) progressive
neurological deterioration with poliodystrophic changes;
and (iii) progressive hepatic failure. The association between
POLG1 mutations and Alpers’ syndrome was shown recently
in two apparently unrelated families (Naviaux and Nguyen,
2004). We found POLG1 mutations in eight out of 10 patients
Fig. 2 POLG1 mutations. Each pair of mutations is plotted against a scheme of the POLG1 gene and corresponding protein. Exons of the
gene are indicated by numbers; the ‘‘exo’’ and ‘‘pol’’ motifs indicate conserved regions in the exonucleolytic (proofreading) and
polymerase (catalytic) domains of the protein.
Infantile hepatocerebral syndromes associated with POLGI mutations727
that were diagnosed in, or referred to our Institute as affected
by typical Alpers’ syndrome over the past 10 years, suggest-
ing that POLG1 is a prevalent disease gene in this condition.
The classification of Alpers’ syndrome as a mitochondrial
disorder has long remained controversial. This uncertainty
may explain the scarcity of biochemical or molecular studies
on mitochondrial metabolism carried out in patients with
Alpers’ syndrome, including most of those reported here.
In the very few cases in which quantitative analysis of
mtDNA was performed, a decrease of mtDNA content
ranging from 10 to 30% of the normal mean was documented
in liver and brain, while results in skeletal muscle were less
consistent (Naviaux et al., 1999; Tesarova et al., 2004). Our
own results suggest the presence of a partial decrease of
mtDNA content in the only two liver specimens (from
patients 2 and 3) that could be examined, although the value
of these data is limited by the fact that they were obtained
from, and compared with, formalin-fixed autoptic specimens,
i.e. in conditions in which DNA can be damaged or degraded.
We had more reliable results by studying a frozen brain
autoptic specimen from patient 8, which showed a partial
but clearly detectable reduction in mtDNA content compared
with two control brains. Taken together, these findings sug-
gest that tissue-specific, partial mtDNA depletion is indeed a
molecular feature of Alpers’ syndrome. However, more work
is necessary to establish whether depletion alone can account
for the damage to liver and brain typical of this condition,
or if additional pathogenetic mechanisms, for example the
accumulation of mtDNA point mutations, may cooperate
with mtDNA depletion in determining the clinical phenotype.
The reported association of POLG1 mutations and hepatic
mtDNA depletion (Naviaux et al., 1999; Tesarova et al.,
2004) prompted us to extend the analysis of the gene to
patients with hepatocerebral MDS. A well-established cause
of this syndrome is mutations in the dGK gene (Mandel et al.,
2001); the clinical features are characterized by a more severe
and rapid involvement of the liver compared with Alpers’
syndrome (Filosto et al., 2004). Of the two dGK-negative
patients with early onset, rapidly progressive hepatic MDS
that were included in our study, only one patient (patient 9)
resulted as a compound heterozygote for POLG1 mutations.
Her clinical phenotype was dominated by signs of progressive
hepatic damage associated with profound depletion of
mtDNA in liver and by early-onset muscle hypotonia.
SMA1-like presentations have been reported in cases of
muscle-specific MDS (Mancuso et al., 2002), and depletion
of mtDNA associated with denervation has been documented
in the skeletal muscle of SMA patients. However, no mtDNA
depletion in either skeletal muscle or cultured fibroblasts was
found in our patient 9, possibly because of the rapidly fatal
course of her disease. On the other hand, we found markedly
decreased nerve conduction velocities in this patient,
which suggested the presence of a severe hypomyelinating
peripheral neuropathy. Peripheral neuropathy is frequent in
syndromes associated with mutations in POLG1, including
adPEO, arPEO, SANDO and the ataxia–epilepsy syndrome,
(DiFonzo et al., 2003; Van Goethem et al., 2003a, 2004;
Lamantea and Zeviani, 2004; Luoma et al., 2004; Winthertun
et al., 2005). To our knowledge, this is the first case of early-
onset hepatocerebral MDS with electrophysiological signs of
predominant myelin abnormalities in the peripheral nerves.
Unfortunately, early death and lack of autoptic examination
prevented us from verifying whether the EMG signs found in
this patient were accompanied by morphological alterations
in the motor nerves and/or in the motor areas of the spinal
cord, and in other regions of the CNS.
Interestingly, both parents of patient 9 suffered from severe
hypofertility, which required in vitro insemination. Astheno-
zoospermia has been associated with length variations of the
triplet repeat stretch encoding a polyglutamine tract located
in the N-terminal portion of the pol-gA protein (Rovio et al.,
2001). Hypofertility in males and precocious menopause in
females were reported in POLG1-positive adPEO patients
(Luoma et al., 2004), and severe hypofertility has been docu-
mented in male and female POLG1 knock-in mutant mice
(Trifunovic et al., 2004). Our observation supports the idea
that a search for heterozygous POLG1 mutations should be
included in the genetic screening for non-syndromic
hypofertility in both male and female subjects.
It is unclear why the syndromes associated with mutations
in POLG1 are tissue specific. Pol-gA is a housekeeping
protein essential for life, and appears to be the only mtDNA-
specific polymerase in mammals. Nevertheless, mtDNA
depletion was present in the liver, but not in the skeletal
muscle, or in cultured skin fibroblasts of our patients. Clinical
signs of liver damage or failure, including exquisite sensit-
ivity to valproate hepatotoxicity, was a consistent finding in
our patient series and has been reported previously by others
(Van Goethem et al., 2004). Unfortunately, in only one case
could nervous tissue be investigated. Because of this limita-
tion, we also cannot establish whether the cerebral involve-
ment in our patients was secondary to the liver failure or
rather, and more probably, could be the consequence of a
primary involvement of neural cells. This latter hypothesis is
supported by the progression of the neurological symptoms
which occurred in patient 8 after liver transplantation.
Our findings confirm that recessive POLG1 mutations can
determine hepatocerebral syndromes, possibly associated
with MDS, and further expand the spectrum of clinical
presentations associated with defects in this gene.
Nine mutant alleles were found in our patients, two carry-
ing nonsense and seven carrying missense changes (Table 1).
The first nonsense mutation, 3630Cins, predicts the synthesis
of an aberrant sequence of five amino acid residues, down-
stream from position Y1210 in the extreme C-terminus of the
pol-gA protein, followed by a premature opa stop codon.
A second nonsense mutation, 3482+2T!C, alters an invari-
ant position in the donor splicing site between exon 21 and the
adjacent intron. Each nonsense mutation was allelic to an
G. Ferrari et al.
already reported missense mutation, namely the W748S and
the A467T mutations (Table 1).
Three missense mutations, 694C!G, predicting an R232G
amino acid change, 731T!C (L244P) and 2869G!C
(A957P) were never reported before. Both the R232 and the
motifs in the N-terminal proofreading domain of the protein.
vertebrates and in Drosophila melanogaster, while L244 is
invariant in many organisms, from vertebrates to yeast
(Ropp and Copeland, 1996). Both mutations, which were
found in Italian patients, were absent in 350 DNA samples
from consecutive, ethnic-matched control individuals, for a
total of 700 alleles, indicating a frequency of <0.14% for each
mutation. The novel 2869G!C mutation (A957P) affects a
highly conserved alanine residue in the polymerase domain of
ing the same amino acid residue, was associated with a very
mild, late-onset phenotype in heterozygous Italian individuals
with adPEO (Lamantea et al., 2002). The young mother of
patient 7, who carries the A957P allele in heterozygosity, has
no neurological abnormality for the time being, but, given the
dominant, albeit very mild, behaviour of the A957S mutation,
from Northern Germany, was absent in 200 consecutive con-
the Italian controls.
Of the four already reported missense mutations, W748S
is located in the ‘linker’ region between the N-terminal
proofreading domain and the C-terminal polymerase domain
of the protein (Fig. 2). This mutation was found recently in
homozygosity in Finnish and Norwegian patients, and in a
heterozygous British patient, all affected by ataxia–epilepsy
Likewise, both the A467T and the G848S mutations affect
highly conserved positions in the spacer region of the protein.
The G848S mutation was reported previously in hetero-
zygosity in two unrelated patients: an Italian PEO patient
housing a T251I-P587L mutation in the second allele
(Lamantea et al., 2002), and a Belgian patient with PEO
that housed a heterozygous mutation in the C10orf2/Twinkle
gene, suggesting digenic inheritance (Van Goethem et al.,
2003b). The A467T mutation was found in several patients
affected by arPEO, SANDO or ataxia–epilepsy syndrome
(Van Goethem et al., 2003a, 2004; Winterthun et al., 2005),
as well as in the two Alpers’ syndrome patients originally
reported by Naviaux and Nguyen (2004) (see also the
Human Polymerase Gamma Mutation Database website
mutations, T251I-P587L, which have been reported in several
cases of arPEO. Since T251I and P587L were consistently
found as syntenic changes, it is not yet established whether
the pathogenicity of this allele is brought about by either one
mutation or by a combination of the two. The T251I-P587L
variant is a relatively frequent allele. It is indeed the most
common recessive mutation in POLG1-related phenotypes,
and we founditinheterozygosity,incombinationwith awild-
type allele, in ?1% of Italian controls.
We have reported previously the presence of the T251I-
whose second allele carried a nonsense mutation predicting
et al., 2002; Lamantea and Zeviani, 2004). A second arPEO
patient was homozygous for T251I-P587L (Lamantea and
Zeviani, 2004). Therefore, both homo- and hemizygosity
of T251I-P587L are associated with a relatively mild pheno-
type. The results of molecular analysis in our patient 9
suggest that the phenotype determined by T251I-P587L
can be substantially worsened by the R232G change, from
a relatively benign, juvenile-
myopathy with accumulation of multiple mtDNA deletions,
to a rapidly progressive infantile hepatocerebral syndrome
associated with profound depletion of mtDNA. One possib-
ility is that the R232G mutation may have a partially
dominant-negative effect over a second allele expressing an
abnormal protein, such as that carrying the T251I-P587L
mutation. Interestingly, depletion of mtDNA can be produced
by the inducible expression of a dominant-negative pol-gA
variant in human cells (Jazayeri et al., 2003), as well as by
exposing cultured cells (Janes et al., 2004) or individuals to
pol-g inhibitors (Divi et al., 2004; Walker et al., 2004).
Different functional effects on the catalytic activity, pro-
cessivity and fidelity of pol-gA are expected to occur in the
presence of mutations affecting different domains of the
and reduced processivity, with or without abnormalities in the
accuracy of nucleotide selectivity and fidelity to the template
(Ponomarev et al., 2002; Graziewicz et al., 2004). On the
other hand, mutations of the N-terminal proofreading domain
are expected to cause an increase in copying errors, while
mutations in the linker region could possibly affect the
stability and processivity of the enzyme. The multiple
functions carried out by the protein could account, at least
in part, for the bewildering heterogeneity of the mtDNA
abnormalities and clinical phenotypes identified in pol-gA
mutations. The functional characterization of the different
mutant variants of the enzyme will contribute to understand-
ingthe pathogenesis ofthe different syndromesand also make
or adult-onset skeletal
We wish to thank Ms B. Geehan for revising the manuscript.
GGP030039), Fondazione Pierfranco e Luisa Mariani
(to M.Z. and R.P.), Ricerca Finalizzata Ministero della Salute
RF-2002/158, Fondazione Cariplo and a EUMITOCOMBAT
Infantile hepatocerebral syndromes associated with POLGI mutations729
network grant from the European Union Framework
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