Recessive POLG mutations presenting with sensory and ataxic neuropathyin compound heterozygote patients with progressive external

ophthalmoplegia

G. Van Goethema,b,c,*, J.J. Martina,b,d, B. Dermautc, A. Lofgrenc, A. Wibailb,D. Ververkene, P. Tackf, I. Dehaeneg, M. Van Zandijckeg, M. Moonenh, C. Ceuterickd,

P. De Jonghea,b,c, C. Van Broeckhovenc

aNeuromuscular Reference Center, University Hospital of Antwerp (UZA), Antwerpen, BelgiumbDepartment of Neurology, University Hospital of Antwerp (UZA), Antwerpen, Belgium

cDepartment of Molecular Genetics, Flanders Interuniversity Institute for Biotechnology (VIB-8), Born-Bunge Foundation (BBS), University of Antwerp

(UIA), Universiteitsplein 1, B-2610 Antwerpen, BelgiumdLaboratory of Neuropathology, Born-Bunge Foundation (BBS), University of Antwerp (UA), Department of Medicine, Antwerpen, Belgium

eSint-Jozefskliniek, Izegem, BelgiumfSint-Andriesziekenhuis, Tielt, Belgium

gA.Z. Sint-Jan, Brugge, BelgiumhZiekenhuis De Bijtjes Koninklijke Instelling V.Z.W., Vlezenbeek, Belgium

Received 10 June 2002; received in revised form 18 August 2002; accepted 23 September 2002

Abstract

Autosomal recessive progressive external ophthalmoplegia is a mitochondrial disease characterized by accumulation of multiple large-

scale deletions of mitochondrial DNA. We previously reported missense mutations in POLG, the gene encoding the mitochondrial DNA

polymerase gamma in two nuclear families compatible with autosomal recessive progressive external ophthalmoplegia. Here, we report a

novel POLG missense mutation (R627W) in a sporadic patient and we provide genetic support that all these POLG mutations are actually

causal and recessive. The novel patient presented with sensory ataxic neuropathy and has the clinical triad of sensory ataxic neuropathy,

dysarthria and ophthalmoparesis (SANDO). This is the first finding of a genetic cause of Sensory Ataxic Neuropathy, Dysarthria and

Ophthalmoparesis and it implies that this disorder may actually be a variant of autosomal recessive progressive external ophthalmoplegia.

Sensory neuropathy is the initial feature in Belgian compound heterozygote autosomal recessive progressive external ophthalmoplegia

patients, all carrying the POLG A467T mutation, which occurs at a frequency of 0.6% in the Belgian population.

q 2002 Elsevier Science B.V. All rights reserved.

Keywords: Autosomal recessive inheritance; Progressive external ophthalmoplegia; Mitochondrial DNA polymerase gamma; Sensory and ataxic neuropathy;

Multiple mitochondrial DNA deletions

1. Introduction

Multiple deletions of mitochondrial DNA (mtDNA) were

first reported in families with autosomal dominant inherited

progressive external ophthalmoplegia (adPEO) [1]. Since

that time multiple mtDNA deletions were also found in

recessive forms of PEO and in many sporadic cases with

variable clinical features, often including PEO [2–7].

Recently, mutations in the gene encoding the heart/skeletal

muscle isoform of the adenine nucleotide translocator

(ANT1), in chromosome 10 open reading frame 2

(C10orf2; encoding the novel mitochondrial protein twin-

kle) and in DNA polymerase gamma (POLG) were identi-

fied in adPEO families linked to chromosomes 4q34–35,

10q23.3–q24.3 and 15q22–q26, respectively [8–10]. In

mitochondrial neurogastrointestinal encephalomyopathy

(MNGIE) patients homozygote or compound heterozygote

recessive mutations were found in the thymidine phosphor-

ylase (TP) gene [11].

We previously reported POLG mutations in two nuclear

PEO families compatible with recessive inheritance

(arPEO) [10]. The clinical phenotype of arPEO is more

heterogeneous than that of adPEO and can be more severe

Neuromuscular Disorders 13 (2003) 133–142

0960-8966/02/$ - see front matter q 2002 Elsevier Science B.V. All rights reserved.

doi:10.1016/S0960-8966(02)00216-X

www.elsevier.com/locate/nmd

* Corresponding author. Tel.: 132-3-820-2307; fax: 132-3-820-2541.

E-mail address: vgoethge@uia.ua.ac.be (G. Van Goethem).

[6]. Here, we describe the updated and detailed clinical

features of six arPEO patients with POLG missense muta-

tions. We report a novel missense mutation of POLG in a

sporadic arPEO patient with sensory ataxic neuropathy as

the presenting feature. We also report the segregation of the

mutations in all families, confirming their recessive nature.

2. Patients and methods

2.1. Case histories

2.1.1. Sporadic case

A 19-year-old man was medically rejected from compul-

sory military service due to a disturbance of balance, which

progressed slowly during the third decade and became

disabling with frequent falls. His gait unsteadiness

worsened in the dark. He presented at age 39. On examina-

tion, he had a mild asymmetric ptosis, a moderately severe

external ophthalmoparesis and mild dysarthria. There was

no other muscle weakness. There were pseudoathetoid

movements of the hands. Deep tendon reflexes were absent.

Vibratory sensation was decreased below the knee and

kinesthetic joint position sense was severely decreased at

both upper and lower limbs, whereas static joint position

sense was only mildly decreased. Romberg’s sign was

present. Two-point discrimination was decreased at the

fingertips. Touch and temperature sensations were

decreased in a stocking like pattern at the lower limbs.

Finger-to-nose testing was normal, without intention

tremor, but markedly deteriorated with eye closure. Heel-

to-shin testing was similarly abnormal with hypermetria, but

no tremor. Rapid alternating movements were normal in the

upper limbs. The gait was severely ataxic. Nerve conduction

studies showed normal motor nerve responses, but absence

of sural and sensory median and ulnar responses. Electro-

myography (EMG) was normal. Sural nerve biopsy showed

a profound loss of both large and small myelinated fibers as

well as unmyelinated fibers. Regenerative clusters were

present and there were no signs of active axonal degenera-

tion. The number of Schwann cells was increased and endo-

neurial fibrosis was severe. The myelin sheaths around the

few remaining fibers appeared normal. A quadriceps muscle

biopsy showed minor non-specific changes on light micro-

scopy without any ragged red fibers (RRF) on the modified

Gomori trichrome stain and normal histochemical activity

of cytochrome c oxidase. Electron microscopy revealed one

muscle fiber with abnormally structured mitochondria

containing crystalline inclusions. Respiratory chain enzyme

activities were normal. Muscle mtDNA analysis showed

none of the common pathogenic mtDNA point mutations

and polymerase chain reaction-single-strand conformation

polymorphism (PCR-SSCP) of mitochondrial tRNA genes

was normal. Southern blot showed no large-scale mtDNA

deletions. Cranial magnetic resonance imaging (MRI)

showed symmetric bilateral thalamic lesions with prolonged

T2-time adjacent to the posterior limb of the capsula interna

(Fig. 1). There was no atrophy of the pons, medulla oblon-

gata or cerebellum. Creatine kinase (CK) was 1.5 times

normal. Serum lactate was 1.3 meq/l (normal, ,2.2). Cere-

brospinal fluid (CSF) lactate was 2.0 meq/l (normal, ,2.8)

and CSF protein was 90 mg/dl. No pigmentary abnormal-

ities were seen at fundoscopy, but scotopic electroretinogra-

phy (ERG) showed a mild reduction in the amplitude of the

b-wave to 160 mV (normal .300 mV), compatible with rod

pathology, whereas ERG was entirely normal. Audiometry

revealed unilateral mild perceptive hearing loss and electro-

nystagmography was indicative of mild peripheral vestibu-

lar dysfunction. Electrocardiogram (ECG) Holter

monitoring was normal. On echocardiography increased fill-

ing pressures were suggestive of diastolic heart disease.

Clinical examination of the patient’s parents, both 70-

year-old, was normal. His only brother was also clinically

unaffected.

2.1.2. Patient B.II.1 (Fig. 3)

This son of non-consanguineous parents had complained

since age 16 about frequent falls followed by progressive

muscle weakness and weight loss. He had progressive diffi-

culties in lifting weights, walking, dressing and swallowing

with occasionally food entering the nose. He had also noted

progressive atrophy of the face, neck and breast muscles.

Since age 18 he received psychiatric treatment because of

depression. At age 22, mitral valve prolapse with mitral

G. Van Goethem et al. / Neuromuscular Disorders 13 (2003) 133–142134

Fig. 1. Cranial MRI of the sporadic patient showing bilateral thalamic

lesions adjacent to the posterior limb of the capsula interna appearing as

intense signals on a T2-weighted image.

insufficiency had been accidently discovered. On examina-

tion at age 28, he had ptosis, external ophthalmoplegia,

marked facial weakness and atrophy of the temporal

muscles. He had dysarthria with a nasal voice. Atrophy of

the sternocleidomastoid and dorsal neck muscles gave the

neck a thin swan neck like appearance. He had prominent

clavicles, bilateral scapula alata and a thin thorax. There was

generalized moderate to severe muscle weakness, most

pronounced in the upper limbs, particularly in the biceps

and small hand muscles (1–2/5 according to MRC scale).

At the lower limbs, paresis was most severe at the foot

dorsiflexors. The gait was waddling with bilateral steppage.

Deep tendon reflexes were absent, but sensibility appeared

normal. Coordination could not be evaluated because of the

limb paresis. Electromyography (EMG) was performed

elsewhere and allegedly showed myogenic signs in all tested

muscles and myotonic discharges in the biceps and extensor

digitorum communis muscles. Fibrillations were reported in

the abductor pollicis brevis muscle. There were also signs of

mild chronic reinnervation. An anterior tibial muscle biopsy

showed an increased variation in fiber size, necrotic muscle

fibers and excessive central nuclei. The modified Gomori

trichrome stain showed numerous RRF. Pulmonary function

tests indicated a borderline normal vital capacity. CK was

elevated at 2060 mU/ml on one occasion (normal,

,110 mU/ml). CSF protein was normal. Electroencephalo-

gram (EEG) showed atypical poly-spike and wave

complexes. Repeated examination at age 33 documented

increased blepharoptosis, pronounced dysarthria, increased

proximal muscle weakness at the lower limbs and extreme

generalized muscle atrophy. He died at age 37 because of

respiratory insufficiency complicated by aspiration pneumo-

nia with hypercapnea and severe oxygen desaturation.

Autopsy was not performed.

2.1.3. Patient B.II.2

At age 27 this patient was examined due to the presence

of ptosis and ophthalmoplegia in her younger sister who was

admitted to the hospital because of a head trauma. She had

no complaints, but ptosis and opthalmoparesis were noted,

which progressed during the following decade until she

presented at 38 years. On repeated clinical examination

she had marked bilateral ptosis and external ophthalmople-

gia. Muscle tone and power were normal. Deep tendon

reflexes were absent. She had a bilateral pes cavus. Vibra-

tory sensation was absent at the ankles. Romberg’s sign was

present. Finger-to-nose testing was normal, but heel-to shin

testing was ataxic. The gait was ataxic with a broad base.

Motor and sensory nerve conductions were slightly

decreased and the amplitudes of the sensory nerve action

potentials were markedly decreased. EMG showed fibrilla-

tions, positive sharp waves and myotonic discharges in all

examined muscles at rest. During contraction the action

potentials were polyphasic with a short duration and low

amplitude. Magnetic resonance (MR) spectroscopy of arm

muscle showed a higher increase of inorganic phosphate and

a higher decrease of phosphocreatine at a lower work load

than in controls and during the recovery phase the ratio of

inorganic phosphate/phosphocreatine remained higher than

in controls. A quadriceps muscle biopsy showed an

increased number of muscle fibers with internal nuclei and

numerous RRF. On histochemical staining cytochrome c

oxidase activity (COX) was almost entirely absent in type

2 fibers but in the RRF this activity was strongly increased

(Fig. 2A, B). Electron microscopy showed large subsarco-

lemmal accumulations of abnormally structured mitochon-

dria in the RRF (Fig. 2C). Southern blot of muscle DNA

demonstrated multiple mtDNA deletions [12].

CK was normal. Echocardiography showed mitral valve

prolapse with mild mitral insufficiency. Computed tomo-

graphic (CT) scan of the brain was normal. At age 50 she

has become wheelchair dependent because of increased

sensory ataxia, whereas remarkably muscle weakness has

not increased much. She has a normal brain MRI.

2.1.4. Patient B.II.3

This woman was examined at age 24, at the occasion of a

head trauma, and ptosis and external ophthalmoplegia were

noticed as well as global tendon areflexia and a minor

paresis of the distal upper limb muscles. By the end of the

third decade, she suffered from progressive exercise intol-

erance, weakness at the upper limbs, resulting in difficulties

to work with the hands and problems to dress. She

complained about progressive swallowing difficulties for

solid food. On repeated examination at age 34, she had

bilateral ptosis and ophthalmoparesis, facial weakness and

a generalized muscle weakness that was most severe at the

neck, the shoulder girdle, and the distal upper limb. Atrophy

was most severe in the shoulder muscles. She had bilateral

pes plano valgus. Deep tendon reflexes were absent. Heel-

to-shin test revealed a mild dysmetria. Romberg’s sign was

present. The gait was waddling and mildly ataxic. Fundo-

scopy revealed a pepper and salt retinopathy. EMG showed

fibrillations and signs of mild chronic regeneration as well

as myopathic signs and nerve conduction studies revealed a

mild axonal neuropathy. A muscle biopsy of the deltoid had

been performed during thyroid surgery and had shown an

increased variation in muscle fiber size, groups of atrophic

fibers, fiber-type grouping, an increased number of muscle

fibers with internal nuclei and numerous RRF. On electron

microscopy there were numerous abnormal mitochondria

with a proliferation of parallel or concentric cristae. On

biochemical analysis muscular carnitine was decreased to

0.41 mmol/g (normal, .3.15 mmol/g). Serum lactate was

2.8 meq/l (normal, ,2.2 meq/l). CK was minimally

elevated. CT-scan of the brain was normal. CSF protein

was elevated at 59 mg/dl. Echocardiography demonstrated

mild mitral valve prolapse and mitral insufficiency. She

suddenly died at age 38 due to respiratory insufficiency.

The following family members were clinically examined

on different occasions and considered unaffected: individual

B.I.1, a 55-year-old man who remained asymptomatic until

G. Van Goethem et al. / Neuromuscular Disorders 13 (2003) 133–142 135

he died at age 76; individual B.I.2, a 50-year-old woman

who died from cancer at age 67; individual B.III.1, a 20-

year-old man who presented for genetic counseling and had

a normal muscle biopsy and individual B.III.1, an 18-year-

old man who volunteered in the genetic study (Fig. 3).

2.1.5. Individual C.II.1

This 74-year-old woman was severely demented.

Informed consent to participate in the genetic study was

obtained from her husband. On examination, ankle jerks

were absent, but the other deep tendon reflexes were very

brisk. The plantar responses were extensor. Ocular motility

was normal.

2.1.6. Patient C.II.2 (Fig. 3)

This son of non-consanguinous parents had complained

about painful feet since age 20. Peripheral neuropathy with

areflexia was documented at age 44. From age 48 he

suffered from progressive swallowing difficulties and

progressive muscle atrophy. In photographs ptosis was not

noticed before age 52. Sensibility in the fingers was

decreased resulting in cigarette burns. At presentation at

age 61, he mainly complained about generalized fatigue,

muscle weakness and stiffness that was most severe at the

distal lower limbs with difficulties in climbing stairs. He had

a weight loss of 11 kg during the last year. On examination,

he had marked bilateral ptosis and external ophthalmople-

gia. He had dysarthria with a nasal voice. He had a general-

ized muscle atrophy and weakness, most severe at the neck

and proximal limbs. Deep tendon reflexes were absent. He

had a glove and stocking pattern of hypoesthesia for all

tested modalities. Romberg’s sign was present. The gait

was ataxic. CK was mildly elevated. Nerve conduction

studies showed absent sural and median sensory responses.

Motor nerve conduction velocities were mildly delayed.

EMG showed myopathic changes and a mild chronic rein-

nervation. Muscle biopsy showed numerous fibers with an

increased number of large nuclei in the subsarcolemmal

region, increased variation in fiber size and numerous

RRF. COX activity was strongly increased in the RRF and

very weak in the type 2 fibers. Electron microscopy showed

large subsarcolemmal accumulations of giant mitochondria

with abnormal cristae and crystalline inclusions and dense

granules (Fig. 2D). Southern blot of muscle DNA showed

multiple mtDNA deletions [12]. The patient’s condition

later further worsened with progressive dysphagia necessi-

tating enteral nutrition. His speech became hardly under-

standable and he became too weak to stand upright. After

G. Van Goethem et al. / Neuromuscular Disorders 13 (2003) 133–142136

Fig. 2. Muscle biopsy: morphologic findings. (A) Succinate dehydrogenase (SDH) histochemistry: RRF (arrows). Magnification £ 174. (B) COX histochem-

istry: RRF showing increased activity (arrow), while type 2 fibers (asterisks) are staining very weak. Magnification £ 174. (C) Electron micrograph of

subsarcolemmal accumulation of abnormal mitochondria showing circular cristae and numerous paracrystalline inclusions. Note abnormal mitochondria

and rows of lipid droplets between the myofibrils. Bar ¼ 5 mm. (D) Electron micrograph of giant mitochondrion in muscle biopsy specimen of patient C.II.2.

Bar ¼ 1 mm.

a Bilroth II operation because of continuing bleeding gastric

ulcer he developed progressive respiratory insufficiency

with CO2 retention resulting in death at the age of 70

years. Autopsy was not performed.

2.1.7. Patient C.II.3

This woman complained about painful feet since age 30.

Because of clawed toes she underwent radiotherapy compli-

cated by osteitis. Ptosis was first noticed at age 51. Then she

had progressive swallowing difficulties. On examination at

age 58, she had marked bilateral ptosis and external ophthal-

moplegia. She had mild dysarthria. Deep tendon reflexes

were absent. She was logorrheic and had emotional instabil-

ity. Nerve conduction studies showed a predominantly

sensory axonal neuropathy. There were myopathic changes

and mild chronic reinnervation signs on EMG. CK was

normal. CT-scan of the brain was normal.

2.1.8. Individual C.II.4

This asymptomatic woman died at age 44 in a car acci-

dent.

2.1.9. Individuals C.III.1–5

These family members volunteered in the genetic study.

They were 46-, 43-, 45-, 40- and 37-year olds, respectively.

They were asymptomatic and on examination, they had no

clinical signs (Fig. 3).

2.2. Enzymatic analysis

Spectrophotometric analysis of respiratory chain enzyme

complexes was performed on mitochondria isolated from

fresh skeletal muscle as described [13].

2.3. Molecular analysis

Extraction of total DNA from frozen muscle was

performed using standard procedures and mtDNA analysis

as described elsewhere [12]. Direct fluorescent cycle

sequencing of PCR amplicons of POLG, C10orf2 and

ANT1 genes was done according to standard procedures

and segregation analysis of all mutations was performed

using pyrosequencing techniques [10]. Belgian control

chromosomes numbering 612 were examined for the

presence of a c.1879 C ! T transition (R627W). Controls

were also analyzed by pyrosequencing.

In order to examine a common founder effect of the

A467T mutation haplotype analysis was performed using

a polymorphic microsatellite marker D15S127 (ABI

PRISMw Linkage Mapping Set), about 1 cM downstream

of the POLG gene, and sequencing of intragenic POLG

polymorphisms. Frequencies of intragenic POLG variations

were estimated genotyping 96 Belgian control chromo-

somes using a pyrosequencing assay.

3. Results

3.1. Clinical findings

Onset age was 20 years in case of the sporadic patient,

16–25 years in the patients of family B and 20–30 years in

family C. Disease duration appeared shortest in two patients

from family B. Their relatively early death at ages 38 and 39

is compatible with a more severe disease course in this

family. The most prominent and presenting feature in the

sporadic case was a sensory ataxic neuropathy with loss of

kinesthetic and vibratory sensation, ataxic gait, positive

Romberg’s sign and areflexia. The other clinical features

of ophthalmoplegia and dysarthria led to the clinical diag-

nosis of sensory ataxic neuropathy, dysarthria and ophthal-

moparesis (SANDO). Contrary to previously reported

SANDO cases, this patient also had thalamic lesions on

brain MRI and skeletal muscle mtDNA deletions were

absent [7]. Other potential etiologies for the peripheral

G. Van Goethem et al. / Neuromuscular Disorders 13 (2003) 133–142 137

Fig. 3. Segregation analysis showing that compound heterozygote POLG

mutations cause autosomal recessive PEO. Squares represent males, circles

represent females, filled symbols represent affected individuals and slashed

symbols represent deceased individuals. Reconstructed intragenic three

locus haplotypes are shown based on the presence or absence of the disease

causing mutations and the two insertion/deletion polymorphisms in introns

9 and 17 on each pair of chromosomes.

neuropathy including cancer, alcoholism, diabetes,

Sjogren’s syndrome or other collagen vascular diseases

were excluded.

Although signs of sensory ataxic neuropathy were also

encountered in family B, particularly in case B.II.2, the most

prominent feature in the other two affected individuals was a

severe and early onset myopathy. Although we previously

suggested that death at the age of 37 and 38 years might be

related to the mitral valve prolapse and mitral insufficiency

in these patients, a careful study of the hospital records

shows that respiratory insufficiency with CO2 retention

and complicated by respiratory tract infection caused the

death. A similar mortality cause was observed in patient

C.II.2 who recently died and on adPEO patients with the

POLG Y955C mutation [14]. In none of these deceased

patients autopsy has been performed. Psychiatric features,

reported as major depressive episodes and necessitating

specialized treatment were encountered at an early age in

the three patients from family B, but not in the other

families.

In family C the initial symptoms point to small fiber

sensory neuropathy that preceded other features by several

decades. Sensory ataxia is milder than in the sporadic

patient and patient B.II.2. Electrophysiological studies

demonstrated a sensory gangliopathy in both patients.

Muscle atrophy and severe dysphagia preceded blepharop-

tosis by several years. Life expectancy in this family is

conspicuously longer than in family B and significantly

longer than in Y955C adPEO patients. Dysphagia occurred

in families B and C and progressed until death necessitating

enteric feeding. Dysarthria or dysphonia occurred in all

patients. None of our patients had Parkinsonism.

3.2. POLG mutations

Four different missense POLG mutations were identified

in the arPEO patients. In addition, we observed two

common intronic POLG polymorphisms in patients and

control samples. In intron 9 there is a G insertion/deletion

(IVS9 1 78_79insG) with allele frequencies of 64% (Del)

and 36% (Ins), in intron 17 a GTAG insertion/deletion

(IVS17 1 38_39insGTAG) with allele frequencies of 48%

(Ins) and 52% (Del). In the sporadic patient a novel POLG

missense mutation was found. This C ! T transition at

c.1879 in exon 10 predicts a substitution of Arg with Trp

at codon 627 (R627W). The R627W mutation was absent in

612 Belgian control chromosomes. The patient also carried

the previously reported A467T mutation (exon 7), which

occurs in the Belgian population with a 0.6% allele T

frequency, and which is also present in the patients from

families B and C, carrying in addition the L304P (exon 4)

and R3P (exon 2) mutations, respectively [10]. Haplotype

analysis in all three families and all controls with the A467T

mutation did not show a common allele for the marker

D15S127. All unrelated A467T carriers share the same

intragenic polymorphisms in introns 9 and 17 (Fig. 3).

The segregation pattern of POLG mutations in the three

families indicated their causal and recessive nature in the six

compound heterozygote PEO patients (Fig. 3). Single

heterozygote family members were asymptomatic and the

examined individuals related to the sporadic patient and

individuals B.I.1, B.I.2, B.III.1, B.III.2, C.II.1, C.III.1–5

had no clinical signs. In the most recent photographs of

the deceased individuals C.I.1–2, ptosis or myopathic facial

signs were absent. Muscle biopsy of individual B.III.1

revealed no abnormalities. Direct sequencing of coding

exons of ANT1 and C10orf2 excluded the possibility that

mutations in these genes would cause disease in any patient.

Altogether these findings provide sufficient evidence to

support our earlier suggestion that mutations in POLG not

only cause dominant PEO, but also recessive PEO [10].

4. Discussion

We have previously demonstrated that dominant muta-

tions in POLG are associated with PEO and multiple

mtDNA deletions and we suggested that recessive PEO

could be caused by compound heterozygous POLG muta-

tions [10]. Here, we provide further evidence about the

causal and recessive nature of previously reported POLG

mutations and of a novel POLG missense mutation in a

nuclear family presenting as a sporadic PEO case. All

patients carry compound heterozygous missense mutations

including L304R, R3P and R627W on one allele in combi-

nation with A467T on the other allele. The shared A467T

mutation occurs with an allele T frequency of 0.6% in the

Belgian population in contrast to the other mutations, which

were not found in Belgian control chromosomes. The clin-

ical phenotype of these recessive POLG mutations shows

considerable variability, but the initial symptoms are due to

sensory neuropathy, which can precede PEO by many years.

One patient, who met the clinical criteria of SANDO had no

mtDNA deletions on Southern blot.

4.1. Compound heterozygous POLG mutations are a cause

of arPEO

The segregation analysis of the POLG mutations in each

of the three families is consistent with autosomal recessive

inheritance (Fig. 3). Heterozygote family members have

none of the clinical features encountered in the compound

heterozygote patients, even at a high age. The current find-

ing implies that both dominant and recessive mutations of

POLG can cause progressive external ophthalmoplegia with

multiple mtDNA deletions. Since the biochemical functions

of POLG are well known, our data are in contradiction with

earlier speculations that different pathogenetic mechanisms

should be involved in recessive and dominant disorders with

multiple mtDNA deletions [15]. Hence, it would be tempt-

ing to search for structural alterations in POLG and other

adPEO genes in PEO families with apparent recessive

G. Van Goethem et al. / Neuromuscular Disorders 13 (2003) 133–142138

inheritance and consanguinity, which was absent in our

three families [6,16].

4.2. Clinical variability in arPEO patients with POLG

mutations

Table 1 summarizes the major clinical and laboratory

findings in all Belgian arPEO patients with different

POLG mutations and compares them with the findings in

the previously reported PEO family with a dominant POLG

mutation. Although the number of patients is too small to

draw statistically significant conclusions about the correla-

tion between the different POLG mutations and the clinical

phenotype, some findings deserve attention.

Although onset age is on an average earlier in the

presented patients than in patients with adPEO caused by

the Y955C POLG mutation, further studies of POLG muta-

tions in other arPEO and adPEO families are necessary to

confirm this finding. Also, Belgian arPEO patients are more

severely disabled at a younger age, which leads to the spec-

ulation that the combined effect of two mutations in these

families leads to a higher probability of generation of

mtDNA deletions in postmitotic tissues, especially in the

sensory ganglia of the long peripheral nerves.

4.3. Sensory neuropathy as the presenting feature in arPEO

patients with mutated POLG

All patients developed PEO at some disease stage, but

peripheral neuropathy or limb muscle involvement preceded

ptosis in most cases, in some with several decades. This

contrasts with the Y955C patients that have ptosis as the

first symptom if one excludes the alcoholic adPEO patients

(Table 1). Also in Y955C patients, peripheral neuropathy is

asymptomatic except for the alcoholic patients who

presented with axonal sensorimotor neuropathy rather than

sensory neuropathy [12].

4.4. Recessive POLG mutations can cause SANDO and

sensory ataxia is common to all Belgian arPEO families

In the sporadic R627W patient, gait ataxia was the initial

feature and at presentation he had the clinical triad of

sensory ataxic neuropathy, dysarthria and ophthalmoplegia

(SANDO) that has been proposed as a unique mitochondrial

phenotype associated with multiple mtDNA deletions in

muscle or nerve of sporadic cases [7]. Nerve biopsy study

on our patient revealed pathology of small nerve fibers as

well as the clinically affected large myelinated sensory

nerve fibers, just as in the reported SANDO patients. We

report here for the first time a genetic cause of SANDO and

indicate the need for POLG-analysis in other cases with

SANDO or arPEO with multiple mtDNA deletions [17–20].

In previously described cases of SANDO brain MRI was

normal, whereas our patient in addition had bilateral thala-

mic lesions on MRI (Fig. 1). Similar lesions were reported

in other mitochondrial disorders, including adPEO caused

by a duplication in C2orf10 [21]. The minor abnormalities at

laboratory examinations, including ERG, audiometry, and

echocardiography need further follow-up as they might

herald the development of pigmentary retinopathy, deafness

and cardiomyopathy in this patient.

A variable degree of sensory ataxia due to peripheral

nerve involvement was also found in patients of families

B and C. In patient B.II.1 sensory ataxic neuropathy may

have been missed notwithstanding a long history of frequent

falls because of his severe limb muscle weakness at presen-

tation. EMG in one distal limb muscle, however, did show

fibrillations, and signs of mild chronic reinnervation were

present in most examined muscles. In the patients from

family C, the initial symptoms point to involvement of

small nerve fibers that preceded other features by several

decades. Moreover, muscle atrophy and severe dysphagia

preceded blepharoptosis by several years and only the

presence of axonal, predominantly sensory neuropathy,

G. Van Goethem et al. / Neuromuscular Disorders 13 (2003) 133–142 139

Table 1

Phenotypic expression of different POLG mutationsa

Patient Clinical features Onset age

(years)

Age of death

(years)

Neuroimaging Muscle biopsy POLG mutations

Sporadic Sensory ataxic neuropathy,

PEO, dysarthria

20 Thalamic lesions Normal R627W 1 A467T

B.II.1 Myopathy, PEO, depression,

dysarthria

16 37 NP RRF L304R 1 A467T

B.II.2 Sensory ataxic neuropathy,

PEO, depression, dysarthria

25 Normal RRF, multiple mtDNA deletions L304R 1 A467T

B.II.3 Myopathy, PEO, depression,

sensory ataxic neuropathy

24 38 Normal RRF NP

C.II.2 Sensory neuropathy,

dysphagia, myopathy, PEO

20 70 NP RRF, multiple mtDNA deletions R3P 1 A467T

C.II.3 Sensory neuropathy,

dysphagia, myopathy, PEO

30 NP NP R3P 1 A467T

Family A PEO, dysphagia, myopathy,

neuropathy in three alcoholics

25–39 54–65 NP RRF, multiple mtDNA deletions Y955C

a RRF, ragged red fibers; NP, not performed.

can differentiate this disorder from oculopharyngeal muscu-

lar dystrophy on clinical examination. Sensory ataxia is

milder than in the sporadic patient and patient B.II.2. The

later onset of skeletal muscle myopathy correlates with a

longer life expectancy in this family.

4.5. Towards lumping of different arPEO syndromes?

Altogether, our data indicate that there is clinical overlap

between SANDO patients and arPEO patients. The finding

of POLG mutations in both conditions suggests that

SANDO could be merely one clinical manifestation of

arPEO. It is also clear from the present study that there is

considerable variability in the phenotypic expression of

recessive POLG mutations, which might in part be due to

epigenetic or environmental factors. The clinical classifica-

tion of mitochondrial disorders has been the subject of a

vigorous debate between ‘lumpers’ and ‘splitters’ during

several decades. In our view, the currently used classifica-

tion of mitochondrial disorders associated with PEO has

become all too complicated and we would like to make a

case for clinically lumping these disorders into the ancient

nosological concept of Ophthalmoplegia-plus syndrome

[22]. The recent findings of different nuclear and mitochon-

drial genes involved in progressive external ophthalmople-

gia may eventually lead to a classification on a genetic

rather than a clinical basis given the considerable overlap-

ping of clinical syndromes and the variable phenotypic

expression of known mutations. Nevertheless, some clinical

concepts such as the Kearns–Sayre syndrome may remain

useful in the diagnostic work-up of these patients [23].

4.6. Increased cytochrome c oxidase (COX) activity in RRF

of patients with POLG mutations

The histochemical findings of increased COX activity in

RRF (Fig. 2) is puzzling and contrasts with what is usually

reported in patients with multiple mtDNA deletions

although this finding has also been reported in the Belgian

adPEO patients with the POLG Y955C mutation and in a

Sicilian adPEO family [12,24]. The meaning of this remains

unclear and future studies may reveal whether such findings

are typical for POLG mutations or not.

4.7. Reported arPEO mutations do not involve functional

POLG motifs

POLG is the only DNA polymerase identified in mito-

chondria and it mediates mtDNA replication and base exci-

sion repair [25,26]. It has an exonuclease domain with three

motifs (I, II and III) and a polymerase domain with three

motifs (A, B and C) [27]. Unlike the Y955C mutation in

adPEO, which is located in the polymerase motif B, the

recessive mutations reported here do not involve functional

POLG exonuclease or polymerase domains. Nevertheless,

the combined effect of two mutations may affect POLG

function sufficiently to lead to the multiple mtDNA dele-

tions encountered in the muscle of the probands of families

B and C. All four recessive POLG mutations change evolu-

tionary conserved residues. The R627 residue, which is

altered by the novel mutation in the sporadic case, is

conserved in mouse and Drosophila melanogaster, but not

in yeast.

4.8. The A467T mutation is common in Belgian arPEO

patients

All six arPEO patients have the A467T mutation, whereas

in the other allele three different POLG mutations were

found. The A467T mutation has an allele T frequency of

0.6% in the Belgian population [10] and one may wonder

whether it could be derived of an ancient common founder.

Although haplotype analysis did not show a common allele

for the marker D15S127, about 1 cM downstream of POLG,

which was linked to adPEO with the highest LOD score in

the 10-cM genome-wide scan in the POLG Y955C family

[10], all unrelated A467T carriers (three arPEO families and

three controls) shared the same intragenic POLG poly-

morphisms in introns 9 and 17 (Fig. 3). While this is compa-

tible with an ancient founder effect in the Belgian

population, a more thorough haplotype analysis is needed

to further validate this finding.

4.9. How do these mutations generate mtDNA deletions?

The molecular mechanism that is responsible for the

generation and accumulation of mtDNA deletions in these

patients remains unknown. One could speculate that

reported POLG mutations might increase the probability

of slipped strand mispairing of mtDNA H-strands during

replication [25], because of a lower replication rate, replica-

tion pausing or changes in POLG tertiary structure. In order

to clarify the molecular pathogenesis, biochemical charac-

terization of the mutant variants expressed in vitro [28] and

the study of in vivo models are necessary. Unfortunately,

there exists no biochemical phenotype in cultured cells of

arPEO patients and in patients it takes several decades

before the mtDNA deletions can be demonstrated in muscle

or other postmitotic tissues. Functional studies that might be

helpful in elucidating the result of the recessive POLG

mutations would imply their overexpression in mammalian

cells, which may not reflect their actual in vivo effect. It is

likely that by screening different sporadic PEO patients,

novel POLG mutations will be identified and investigating

the pathogenicity of these novel mutations may be a major

challenge.

4.10. Absence of mtDNA deletions in muscle of sporadic

arPEO case

The absence of skeletal muscle mtDNA deletions on

Southern blot of the sporadic patient with sensory ataxic

neuropathy and ophthalmoplegia is interesting and may

seem paradoxical at first sight. However, in adPEO pedi-

G. Van Goethem et al. / Neuromuscular Disorders 13 (2003) 133–142140

grees with multiple mtDNA deletions in the muscle biopsy

of several family members, a normal Southern blot of

muscle was also reported in some patients notwithstanding

the presence of PEO for several years before biopsy

[24,29]. This indicates that mtDNA deletions may be

absent in unaffected muscles, and that mutations in nuclear

genes affecting mtDNA integrity can be overlooked in PEO

patients because of normal muscle mtDNA analysis. In our

patient the absence of mtDNA deletions on Southern blot

correlates with the normal clinical, electrophysiological,

morphological and biochemical findings in the biopsied

muscle. The patients’ disease burden was apparently else-

where. Unfortunately affected nerve tissue from the Sural

nerve biopsy was not available for mtDNA analysis. Mole-

cular analysis in other SANDO patients has demonstrated

multiple mtDNA deletions in Sural nerve biopsy specimens

[7].

4.11. From mtDNA deletions to pathology

How do mtDNA deletions that are de novo being gener-

ated cause pathology in these patients? In postmitotic tissues

with a high energy demand and high mtDNA turnover, dele-

tions of mtDNA with clonal expansion probably accumulate

over decades and their proportion increases with time

[30,31]. The issue whether mtDNA deletions are function-

ally dominant over wild-type mtDNA or not remains unre-

solved since the limited data in the literature are conflicting

[32,33]. Most likely impairment of mitochondrial transla-

tion, possibly due to imbalance of tRNAs, would cause the

respiratory chain malfunction with defective ATP produc-

tion, leading to dysfunction and pathology of the cells or

muscle fiber segments involved [33].

Acknowledgements

We are grateful to the patients and family members who

volunteered in this study. We also thank Rudy Van Coster,

Department of Pediatrics, University Hospital of Gent (UZ

Gent), Belgium for the respiratory chain enzyme analysis in

the sporadic patient and Sara Seneca, Center for Medical

Genetics, University Hospital of Brussel (AZ-VUB),

Belgium for the mtDNA analysis in the sporadic patient.

This work was in part funded by the Fund for Scientific

Research-Flanders (FWO-F). Bart Dermaut is a doctoral

fellow of the FWO-F.

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