Patient homozygous for a recessive POLG mutation presents with features of MERRF G. Van Goethem, MD, PhD; R. Mercelis, MD, PhD; A. Löfgren, MSc; S. Seneca, PhD; C. Ceuterick, PhD; J.J. Martin, MD, PhD; and C. Van Broeckhoven, PhD, DSc
Abstract—Both dominant and recessive missense mutations were recently reported in the gene encoding the mitochondrial DNA polymerase gamma (POLG) in patients with progressive external ophthalmoplegia (PEO). The authors report on a patient homozygous for a recessive missense mutation in POLG who presented with a multisystem disorder without PEO. The most prominent features were myoclonus, seizure, and sensory ataxic neuropathy, so the clinical picture overlapped with the syndrome of myoclonus, epilepsy, and ragged red fibers (MERRF). NEUROLOGY 2003;61:1811–1813
Mutations in different nuclear genes cause dominant and recessive progressive external ophthalmoplegia (PEO).1 These forms of PEO are characterized by secondary accumulation of multiple deletions in mitochondrial DNA (mtDNA). In different families, primary dominant and recessive mutations were observed in the POLG gene.2-4 Patients with reported recessive PEO are compound heterozygotes of two different mutations in POLG.2-4 Here, we report the clinical phenotype of a patient homozygous for a recessive missense mutation (A476T) in POLG. Case history. The 18-year-old patient is the elder son of nonconsanguineous parents, aged 45 and 41 years. Clinical examination was normal. His 11-yearold brother was unaffected. The patient had a mild disturbance of balance since age 15 years. At age 17 years, he twice was examined for sudden episodes of vigorous left-sided myoclonic jerks at the arm, face, and upper trunk. On the second occasion, these were followed by a secondary generalized epileptic seizure. Myoclonus and seizures did not recur after treatment with clonazepam and sodium valproate, but the latter was replaced by lamotrigine because of toxic hepatitis, which next abated. Tremor and imbalance notably increased after the seizure but then steadily improved. He experienced numbness at the right leg. Cognitive functioning decreased mildly. On examination, he had myoclonic jerks, gait ataxia (which worsened in the dark), and a discrete head tremor. Deep tendon reflexes were absent. He had no muscle weakness, dysphonia, or blepharoptosis. Saccadic and pursuit ocular movements were repeatedly normal. There was transient hypoesthesia below the
knees. Results of funduscopy were normal. Nerve conduction studies demonstrated axonal sensory neuropathy and normal motor nerve responses. EMG results were normal. Somatosensory evoked potentials (SEPs) were repeatedly suggestive of proximal peripheral nerve pathology or radiculopathy. Visual evoked potentials (VEPs) were repeatedly prolonged. Repeated EEG and cranial MRI results were normal. Cervical spine MRI results were also normal. Serum lactate was within the normal range. Creatine kinase (CK) was 366 U/L (normal ⬍170). During sodium valproate therapy, total serum bilirubin was elevated at 3.8 mg/dL (normal ⬍1.3) with elevated liver enzymes. CSF lactate was 2.0 mEq/L (normal ⬍2.8), and CSF protein was 90 mg/dL. Videooculography results were normal. EKG Holter and echocardiography results were normal. Gated blood pool scan suggested incipient cardiomyopathy. Quadriceps muscle biopsy (at age 17 years) was normal on light microscopy without ragged red fibers and without cytochrome c oxidase negative fibers on histochemical staining. Muscle and skin appeared normal on electron microscopy. Respiratory chain enzyme activities on freshly isolated muscle mitochondrial fractions were normal. Methods. Isolation of genomic DNA and mutation analyses were performed as described.2 For allele and haplotype sharing analysis, we used 10 microsatellite markers located in a 1.6-Mb region surrounding POLG on chromosome 15q25. Besides D15S979, D15S1045, and D15S202, we used seven novel microsatellite markers (markers 1 to 7) identified in the genomic sequence. The number of alleles of microsatellite markers varied from 5 to 12, and allele frequencies were calculated in 96 Belgian control subjects. Detailed information on the novel microsatellite sequences is available on request. Microsatellite markers were ana-
From the Division of Neurology and the Neuromuscular Reference Center (Drs. Van Goethem, Mercelis, and Martin), University Hospital Antwerp, Belgium; Department of Molecular Genetics (Drs. Van Goethem and Van Broeckhoven, A. Löfgren), Flanders Interuniversity Institute for Biotechnology (VIB8); Born-Bunge Foundation (Drs. Van Goethem, Ceuterick, Martin, and Van Broeckhoven, A. Löfgren), University of Antwerp, Belgium; and Department of Medical Genetics (Dr. Seneca), University of Brussels (VUB), Belgium. Received March 19, 2003. Accepted in final form August 20, 2003. Address correspondence and reprint requests to Dr. Christine Van Broeckhoven, Department of Molecular Genetics VIB8, University of Antwerp UA/UIA, Universiteitsplein 1, B-2610 Antwerpen, Belgium; e-mail:
[email protected] Copyright © 2003 by AAN Enterprises, Inc.
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Figure. Allele and haplotype analysis using 10 microsatellite markers surrounding POLG in the patient and three unrelated families with recessive progressive external ophthalmoplegia (PEO).2,4 Marker alleles are ordered from the chromosome centromere (top) to the telomere. *Indicates the homozygous A476T patient described in this article. In the three families with PEO, the patients with PEO are compound heterozygotes for A476T and another missense mutation in POLG, i.e., L304R, R3P, and R627W.2,4 Patients are indicated by filled symbols, and the common haplotype is boxed.
lyzed by PCR amplification using fluorescently labeled flanking primers, analyzed on an ABI 3700 automated sequencer (Applied Biosystems, Weiterstadt, Germany), and PCR fragment lengths were determined using the software program GENESCAN (Applied Biosystems). Mitochondrial DNA was isolated from skeletal muscle using standard procedures. Selection of PCR fragments amplified from mutant mtDNA was obtained using two different pairs of oligonucleotides that primed on widely separated regions of mtDNA. Such “long-range PCR” selectively amplifies the smaller-sized deletioncontaining fragments.
Results. Clinical findings. The most prominent and presenting clinical features in this patient were myoclonus, seizure, and sensory ataxia with demonstration of axonal sensory neuropathy on electrophysiologic investiga1812
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tions. Appropriate laboratory investigations in this patient excluded Lafora body disease, storage disorders, and congenital disorders of glycosylation (CDG). Cancer, diabetes, and collagen vascular diseases were ruled out as potential causes of peripheral neuropathy. The clinical features of myoclonus, seizure, axonal sensory ataxic neuropathy, and hepatotoxicity induced by valproate and mild cognitive decline and cardiomyopathy were indicative of a multisystem disorder and suggestive of mitochondrial disease, particularly MERRF.5 However, ragged red fibers, a hallmark of MERRF, were absent on muscle biopsy. Genetic findings. mtDNA analysis. Pathogenic point mutations in any of the 22 transfer RNA (tRNA) genes were excluded.5,6
Diagnostic Southern blot analysis showed no large-scale mtDNA rearrangements (deletions or duplications). However, long-range PCR revealed multiple small DNA fragments in co-occurrence with the expected normal-length PCR product derived from wild-type mtDNA (data available on request). This indicates the presence of low proportions of mtDNA deletions in the patient’s muscle. POLG analysis. Sequencing of coding exons of POLG revealed that the patient was homozygous for the recessive A476T mutation, which was previously demonstrated in compound heterozygote recessive PEO patients and in 0.6% of Belgian control chromosomes.2,4 Both parents were heterozygote A467T carriers. Mutations in the genes ANT1 and C10orf2/Twinkle were excluded. To examine a possible founder effect for A476T in the Belgian population, allele and haplotype sharing was examined in the patient and in three unrelated PEO families segregating A476T (figure).2,4 The patient homozygous for A476T was also homozygous for 8 of 10 microsatellite markers (see figure). Data indicated that all A476T carriers shared a common haplotype of seven microsatellite markers (marker 1 to D15S202). The frequencies of shared alleles in control subjects varied from 9 to 35%, predicting a frequency of 0.003% for the shared haplotype in a region of approximately 350 kb around POLG.
Discussion. Our patient’s disorder resembled MERRF; the patient was homozygous for the recessive A467T mutation in POLG. The sensory ataxic neuropathy in this teenager prompted us to sequence POLG because neuropathy can precede PEO in patients with recessive POLG mutations.4 Reported patients with POLG mutations have PEO,2-4 which our patient may contract in the future. Here, the presentation with myoclonus and seizure is unique, expanding the clinical phenotype of recessive POLG mutations. Our patient also resembles previously reported patients without identified genetic cause, who may be candidates for sequencing POLG.7,8 Why the phenotype in this case is different from PEO caused by recessive POLG mutations remains unclear. The absence of skeletal muscle mtDNA deletions on Southern blot analysis is notable. However, we detected low levels of multiple mtDNA deletions on the more sensitive long-range PCR. Previously, mtDNA
deletions were not always demonstrated in patients with PEO and recessive or dominant POLG mutations.4,9 This implies that mutations in nuclear genes affecting mtDNA integrity can be overlooked because of normal Southern blot analysis of muscle mtDNA. One may expect a higher proportion of mtDNA deletions in the clinically affected sural nerve.10 Nerve biopsy was not obtained in this case. Acknowledgment The authors thank Rudy Van Coster, MD, PhD, Department of Pediatrics, University Hospital of Ghent (UZ Gent), Belgium, for assistance with respiratory chain enzyme analysis, and Marc Cruts, PhD, Department of Molecular Genetics, for assistance with haplotype analysis.
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