Recessive POLG mutations presenting with sensory and ataxic neuropathy in compound heterozygote patients with progressive external ophthalmoplegia

Neuromuscular Disorders 13 (2003) 133–142 www.elsevier.com/locate/nmd

Recessive POLG mutations presenting with sensory and ataxic neuropathy in compound heterozygote patients with progressive external ophthalmoplegia G. Van Goethem a,b,c,*, J.J. Martin a,b,d, B. Dermaut c, A. Lo¨fgren c, A. Wibail b, D. Ververken e, P. Tack f, I. Dehaene g, M. Van Zandijcke g, M. Moonen h, C. Ceuterick d, P. De Jonghe a,b,c, C. Van Broeckhoven c a

Neuromuscular Reference Center, University Hospital of Antwerp (UZA), Antwerpen, Belgium b Department of Neurology, University Hospital of Antwerp (UZA), Antwerpen, Belgium c Department of Molecular Genetics, Flanders Interuniversity Institute for Biotechnology (VIB-8), Born-Bunge Foundation (BBS), University of Antwerp (UIA), Universiteitsplein 1, B-2610 Antwerpen, Belgium d Laboratory of Neuropathology, Born-Bunge Foundation (BBS), University of Antwerp (UA), Department of Medicine, Antwerpen, Belgium e Sint-Jozefskliniek, Izegem, Belgium f Sint-Andriesziekenhuis, Tielt, Belgium g A.Z. Sint-Jan, Brugge, Belgium h Ziekenhuis 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 largescale 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 * Corresponding author. Tel.: 132-3-820-2307; fax: 132-3-820-2541. E-mail address: [email protected] (G. Van Goethem).

(ANT1), in chromosome 10 open reading frame 2 (C10orf2; encoding the novel mitochondrial protein twinkle) and in DNA polymerase gamma (POLG) were identified 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 phosphorylase (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

0960-8966/02/$ - see front matter q 2002 Elsevier Science B.V. All rights reserved. doi:10.1016/S0 960-8966(02)00 216-X

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[6]. Here, we describe the updated and detailed clinical features of six arPEO patients with POLG missense mutations. 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 compulsory 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 examination, 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. Heelto-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. Electromyography (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 degeneration. The number of Schwann cells was increased and endoneurial fibrosis was severe. The myelin sheaths around the few remaining fibers appeared normal. A quadriceps muscle biopsy showed minor non-specific changes on light microscopy 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

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.

T2-time adjacent to the posterior limb of the capsula interna (Fig. 1). There was no atrophy of the pons, medulla oblongata or cerebellum. Creatine kinase (CK) was 1.5 times normal. Serum lactate was 1.3 meq/l (normal, ,2.2). Cerebrospinal fluid (CSF) lactate was 2.0 meq/l (normal, ,2.8) and CSF protein was 90 mg/dl. No pigmentary abnormalities were seen at fundoscopy, but scotopic electroretinography (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 electronystagmography was indicative of mild peripheral vestibular dysfunction. Electrocardiogram (ECG) Holter monitoring was normal. On echocardiography increased filling pressures were suggestive of diastolic heart disease. Clinical examination of the patient’s parents, both 70year-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 difficulties 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

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insufficiency had been accidently discovered. On examination 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. Electroencephalogram (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 pneumonia 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 ophthalmoplegia. Muscle tone and power were normal. Deep tendon reflexes were absent. She had a bilateral pes cavus. Vibratory 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 fibrillations, 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

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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 subsarcolemmal accumulations of abnormally structured mitochondria 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 tomographic (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 intolerance, 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. Heelto-shin test revealed a mild dysmetria. Romberg’s sign was present. The gait was waddling and mildly ataxic. Fundoscopy 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

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

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 20year-old man who presented for genetic counseling and had a normal muscle biopsy and individual B.III.1, an 18-yearold 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 ophthalmoplegia. He had dysarthria with a nasal voice. He had a generalized 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 reinnervation. 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 necessitating enteral nutrition. His speech became hardly understandable and he became too weak to stand upright. After

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2.1.8. Individual C.II.4 This asymptomatic woman died at age 44 in a car accident. 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

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.

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 complicated 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 ophthalmoplegia. She had mild dysarthria. Deep tendon reflexes were absent. She was logorrheic and had emotional instability. 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.

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 PRISM w 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 chromosomes 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 diagnosis of sensory ataxic neuropathy, dysarthria and ophthalmoparesis (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

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neuropathy including cancer, alcoholism, diabetes, Sjo¨ gren’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 blepharoptosis 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 mutations in POLG are associated with PEO and multiple mtDNA deletions and we suggested that recessive PEO could be caused by compound heterozygous POLG mutations [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 combination 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 clinical 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 finding 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 tempting to search for structural alterations in POLG and other adPEO genes in PEO families with apparent recessive

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Table 1 Phenotypic expression of different POLG mutations a Patient

Clinical features

Onset age (years)

Sporadic

Sensory ataxic neuropathy, PEO, dysarthria Myopathy, PEO, depression, dysarthria Sensory ataxic neuropathy, PEO, depression, dysarthria Myopathy, PEO, depression, sensory ataxic neuropathy Sensory neuropathy, dysphagia, myopathy, PEO Sensory neuropathy, dysphagia, myopathy, PEO PEO, dysphagia, myopathy, neuropathy in three alcoholics

20

B.II.1 B.II.2 B.II.3 C.II.2 C.II.3 Family A a

16

Age of death (years)

37

25

Neuroimaging

Muscle biopsy

POLG mutations

Thalamic lesions

Normal

R627W 1 A467T

NP

RRF

L304R 1 A467T

Normal

RRF, multiple mtDNA deletions

L304R 1 A467T

24

38

Normal

RRF

NP

20

70

NP

RRF, multiple mtDNA deletions

R3P 1 A467T

NP

NP

R3P 1 A467T

NP

RRF, multiple mtDNA deletions

Y955C

30 25–39

54–65

RRF, ragged red fibers; NP, not performed.

inheritance and consanguinity, which was absent in our three families [6,16].

4.4. Recessive POLG mutations can cause SANDO and sensory ataxia is common to all Belgian arPEO families

4.2. Clinical variability in arPEO patients with POLG mutations

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 thalamic 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 presentation. 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,

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 correlation 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 mutations 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 speculation 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].

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can differentiate this disorder from oculopharyngeal muscular 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.

tions encountered in the muscle of the probands of families B and C. All four recessive POLG mutations change evolutionary 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.

4.5. 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 classification of mitochondrial disorders has been the subject of a vigorous debate between ‘lumpers’ and ‘splitters’ during several decades. In our view, the currently used classification 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 mitochondrial genes involved in progressive external ophthalmoplegia may eventually lead to a classification on a genetic rather than a clinical basis given the considerable overlapping 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 mitochondria and it mediates mtDNA replication and base excision 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-

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 polymorphisms in introns 9 and 17 (Fig. 3). While this is compatible 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, replication pausing or changes in POLG tertiary structure. In order to clarify the molecular pathogenesis, biochemical characterization 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-

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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 elsewhere. Unfortunately affected nerve tissue from the Sural nerve biopsy was not available for mtDNA analysis. Molecular 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 generated cause pathology in these patients? In postmitotic tissues with a high energy demand and high mtDNA turnover, deletions of mtDNA with clonal expansion probably accumulate over decades and their proportion increases with time [30,31]. The issue whether mtDNA deletions are functionally dominant over wild-type mtDNA or not remains unresolved since the limited data in the literature are conflicting [32,33]. Most likely impairment of mitochondrial translation, possibly due to imbalance of tRNAs, would cause the respiratory chain malfunction with defective ATP production, 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. References [1] Zeviani M, Servidei S, Gellera C, Bertini E, DiMauro S, DiDonato S. An autosomal dominant disorder with multiple deletions of mitochondrial DNA starting at the D-loop region. Nature 1989;339:309–311. [2] Yuzaki M, Ohkoshi N, Kanazawa I, Kagawa Y, Ohta S. Multiple deletions in mitochondrial DNA at direct repeats of non-D-loop regions in cases of familial mitochondrial myopathy. Biochem Biophys Res Commun 1989;164:1352–1357.

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