Department of Medical Biochemistry and Molecular Biology, University of Oulu, Oulu, Finland [Correction added after online publication Feb. 20, 2008: The affiliation: Jukka S. Moilanen, †Clinical Research Center, Oulu University Hospital, Oulu, Finland; **Department of Clinical Genetics, University of Turku, Turku, Finland has been changed to: Jukka S. Moilanen, †Clinical Research Center, Oulu University Hospital, Oulu, Finland; **Department of Clinical Genetics, University of Oulu, Oulu, Finland]
Address correspondence to Dr. Kari Majamaa, M.D., University of Turku, Department of Neurology, FIN-20014 Turku, Finland. E-mail: firstname.lastname@example.org
Purpose: Polymerase gamma (POLG) is the sole enzyme in the replication of mitochondrial DNA (mtDNA). Numerous mutations in the POLG1 gene have been detected recently in patients with various phenotypes including a classic infantile-onset Alpers-Huttenlocher syndrome (AHS). Here we studied the molecular etiology of juvenile-onset AHS manifesting with status epilepticus and liver disease in three teenagers.
Patients and Methods: We examined 14- and 17-year-old female siblings (patients 1 and 2) and an unrelated 15-year-old girl (patient 3) with juvenile-onset AHS, sequenced POLG1, and the entire mtDNA, examined mtDNA deletions by amplification of the full-length mtDNA with the long PCR method and used real-time PCR to quantify mtDNA in the tissue samples.
Results: The initial manifestations were migraine-like headache and epilepsy, and the terminal manifestations status epilepticus and hepatic failure. A homozygous W748S mutation in POLG1 was detected in the three patients. No deletions or pathogenic point mutations were found in mtDNA, but all three patients had mtDNA depletion.
Conclusions: POLG mutations should be considered in cases of teenagers and young adults with a sudden onset of intractable seizures or status epilepticus, and acute liver failure. The W748S POLG1 mutation seems to lead to tissue-specific, partial mtDNA depletion in patients with juvenile-onset Alpers syndrome. Valproic acid should be avoided in the treatment of epileptic seizures in these patients.
Polymerase gamma (POLG) is the only polymerase involved in the replication of mitochondrial DNA (mtDNA) and base excision repair in mammals (Kaguni, 2004; Longley et al., 2005). Functional POLG consists of a catalytic α-subunit containing the polymerase and exonuclease domains and an accessory β-subunit (Kaguni, 2004). The POLG1 gene (NM_002693) that encodes the POLG α-subunit is located in chromosome 15q22-26 encoding 23 exons.
The homozygous W748S mutation in the POLG1 gene has recently been found to be a common cause of spinocerebellar ataxia (Hakonen et al., 2005), while the compound heterozygous mutations W748S/L24P, W748S/G848S, and W748S/Y1210fs1216X (Davidzon et al., 2005; Ferrari et al., 2005; Nguyen et al., 2006) have been reported to lead to classic AHS with onset in infancy or early childhood. We here describe three young women with juvenile-onset AHS associated with a homozygous W748S mutation in the POLG1 gene causing the mtDNA depletion in tissues.
Patients and Methods
Patient 1 was the second child of unrelated healthy Finnish parents. She had been healthy prior to age 14 years, when she began to experience episodes of flashing bright balls with impaired vision. Within 2 weeks, attacks of left- or right-sided headache emerged with nausea and vomiting, which increased in frequency and severity. Ophthalmological examination revealed partial defects in the left visual fields. An EEG showed 2–4 Hz delta waves with spikes and sharp waves over the temporooccipital and parietooccipital regions of the right hemisphere. Brain MRI (1.5 Tesla Signa scanner, GE Medical Systems, Milwaukee, WI, U.S.A.) revealed symmetric signals in both thalami and in the cerebellum in T2-weighted images (Fig. 1A). All the other clinical examinations were normal. Apart from the visual field defects, the symptoms disappeared spontaneously, but the migraine-like headaches recurred. Liver function tests and serum and cerebrospinal fluid (CSF) lactate and pyruvate were normal on this occasion.
Eleven months after the onset of visual symptoms, she had her first tonic–clonic seizure, which rapidly progressed to a status epilepticus that responded poorly to standard medications. After 3 weeks, she continued to have occasional short jerks of her right extremities, complained of blurred vision and diplopia and was found to have horizontal nystagmus. She became seizure-free with a combination of oxcarbazepine and sodium valproate. Her plasma amylases were temporarily increased after the introduction of sodium valproate, but liver enzymes were normal.
After the status epilepticus, she had a right hemiparesis and occasional athetoid movements of the right arm. A lactate doublet peak was found in a proton magnetic resonance spectroscopic imaging (1H-MRSI). Blood and CSF lactate and pyruvate were elevated (Table 1). Three months after the status epilepticus, she developed acute pancreatitis, necrosis of the ascending colon, septic shock, and disseminated intravascular coagulopathy. She died 12 days later. Acute liver failure occurred during the last few days (Table 1).
Table 1. Clinical and laboratory features of three patients with juvenile-onset AHS and the W748S mutation in POLG1
MLH, migraine-like headache; SE, status epilepticus; VPA, valproic acid; n.i., not investigated; n.a., not applicable. The minimum and maximum are shown for all laboratory values.
bThe highest value of 18.6 mmol/l of lactic acid for patient 1 was measured during status epilepticus. Laboratory references: blood lactate 0.33–1.33 mmol/l, CSF lactate 1.1–2.2 mmol/l, plasma amylases 70–300 U/l, P-ASAT 10–35 U/l, P-ALAT 10–35 U/l, P-AFOS 70–250 U/l, P-GT 5–50 U/l, fB-NH4 <80 μmol/l, and S-bil 2–20 μmol/l.
Age at onset (yr)
Headache, visual symptoms
MLH, visual symptoms
No headache, episodic visual symptoms
MLH, visual symptoms
Initial seizure type
Focal, generalized seizures
Span between first seizures and SE
VPA before liver manifestation
Athetosis, nystagmus, hemiparesis
Age at death, 15
Current age, 21
Age at death, 20
Status praesens/cause of death
Liver failure, DIC, sepsis, pancreatitis, SE
Focal seizures, visual symptoms, nystagmus
Severe liver failure
Epileptic discharge in the right temporooccipital and parietooccipital regions
Epileptic discharge in the right temporooccipital region
Epileptic discharge in the occipital and centroparietal regions
Brain MRI (T2-weighted images)
Symmetric signal intensities in thalami and cerebellum
Symmetric signal intensities in thalami
Increased intensity in the right thalamus
Increased microvesicular fat (at autopsy)
Slight increase in fat, nonspecific changes in mitochondrial shape and size
Muscle biochemistry (OXPHOS)
Liver enzymes (U/l)
ASAT 30–97, ALAT 13–118, AFOS 58-441
ASAT 20–120, ALAT 4–35, AFOS 89–275, GT 18–105
ALAT 18-233, AFOS 263–2121, GT 27–1438
fB-NH4, S-bil (μmol/l)
NH4 188–284, bil 3–138
NH4 76, bil 4–9
NH4 12–139, bil 3–277
Autopsy findings and histology
Bleedings and blots caused by disseminated intravascular coagulation were seen in the parenchymal organs. The liver was heavy (2094 g, normal 1500 g) and yellow. Histology showed severe fat infiltration, mild fibrosis, and some bile stasis with bile thrombi. The pancreas was necrotic, as was the large intestine. The fresh weight of the brain was 1304 g. There was severe global ischemia in the brain. In addition, there was generalized spongiosis in the brain and patchy loss of neurons in the calcarine cortex and thalamus. In the cerebellum, there was a loss of Purkinje cells, which had pyknotic nuclei. The dentate nuclei were gliotic and also showed patchy neuronal loss. The cerebellar changes were consistent with the preexisting pathology.
Patient 2 was the elder sister of patient 1. She had been healthy until the age of 17 years, when she had a generalized tonic–clonic seizure. EEG showed continuous spike and slow wave discharges over the right temporooccipital region. Treatment with valproic acid was started, but she developed a status epilepticus on the following day. MRI showed symmetrical signal intensities in both thalami in T2-weighted images, similar to the initial MRI findings in the case of her sister (Fig. 1B), but a 1H-MRSI was normal. Her status epilepticus was successfully treated with propofol. Liver enzymes were mildly elevated, but they normalized during the follow-up. The liver was normal in ultrasonography and CT. Blood lactate and pyruvate were elevated (Table 1). One year after the status epilepticus, her neurological examination was normal except for nystagmus. In spite of antiepileptic medication she still has up to six monthly episodes of seeing bright balls for 1–2 min and occasional seizures with dizziness and slurred speech. Continuous delta activity and occasional spike, and slow wave complexes in the right occipital region were still seen in her EEG. An ophthalmological examination revealed nystagmus and left-eye esotrophia. Visual acuity, color vision, and visual fields were normal.
Patient 3 is the younger of two daughters of nonconsanguineous parents. Having previously had migraine without aura, she experienced a partial seizure with visual aura at age 15 years. Oxcarbazepine was started after the second seizure, which occurred 4 months later. During the next 3 years, she had three generalized seizures that were preceded by visual disturbances lasting for minutes. Liver enzymes increased during oxcarbazepine medication but normalized when oxcarbazepine was replaced with lamotrigine. Liver enzymes remained normal during this medication.
At age 18 years, she experienced a 2-week period of headache and visual disturbance. Brain MRI was normal, but EEG showed an abnormal slowing in background activity (6–7 Hz) in the occipital region without epileptiform features. At 20 years, she had partial seizures involving the left upper extremity, and these progressed to generalized tonic–clonic seizures, which could be arrested with diazepam. EEG revealed an intensive epileptic discharge in the occipital region and severe generalized disturbance. MRI pointed to increased intensity in the right thalamus. Liver enzymes were elevated. Clonazepine and topiramate were started, but she developed a therapy-resistant status epilepticus. Seizures were controlled with thiopentone, but an electrophysiological status epilepticus was seen in the right hemisphere. Other antiepileptic drugs used during the following 6 weeks were midatzolam, sodium valproate, gabapentin, and fosphenytoin. After the 6 weeks of treatment for status epilepticus the patient was drowsy, but obeyed simple commands. Bilirubin and ammonium ions increased within 3 weeks; however (Table 1), severe liver damage and hepatic coma ensued and the patient died 3 months after the initial status epilepticus.
Autopsy findings and histology
The autopsy showed a necrotic liver weighing 1980 g (normal 1500 g), and histological examination revealed a fatty liver with marked necrosis. There was increased fibrosis around the central veins (Fig. 2A) and a focal accumulation of inflammatory cells (Fig. 2B). Copper staining was negative. The lungs showed changes similar to those in adult respiratory distress syndrome. The brain weighed 1350 g, and gross sections showed mild atrophy of the cerebellar vermis but no remarkable changes otherwise. Histological examination of the brain revealed a spongiform cortex. No changes were found in basal ganglia. In the cerebellum, there was a marked loss of the Purkinje cells and gliosis. The dentate nucleus was gliotic with loss of neurons. Alzheimer type II astrocytes similar to those noted in cases of hepatic coma were found (Fig. 2C).
A skeletal muscle biopsy of 0.5–1.0 g was taken surgically from the lateral vastus muscle of the quadriceps femoris. Part of the sample was used for the isolation of DNA and for histological examinations, and the majority was taken for the isolation of mitochondria. The activities of respiratory chain enzymes in the isolated mitochondria were measured by spectrophotometric methods. The activity of complex IV was measured as described previously (Rustin et al., 1994) and those of complexes I, III, I+III, and II+III with minor modifications (Hinttala et al., 2005).
The protocol was approved by the Ethics Committee of the Medical Faculty, University of Oulu, Oulu, Finland, and informed consent for DNA analysis was obtained from the parents and patient 2.
Total genomic DNA was isolated from the blood cells using a QIAamp Blood Kit (Qiagen, Hilden, Germany) and from frozen skeletal muscle samples by the standard sodium dodecyl sulfate–proteinase K method. MtDNA was submitted to automated sequencing (ABI PRISM 377 Sequencer, Applied Biosystems, Foster City, CA, U.S.A.) using the Dye Terminator Cycle Sequencing Ready Kit (Perkin Elmer, Foster City, CA, U.S.A.) after treatment with exonuclease I and shrimp alkaline phosphatase. MtDNA deletions were analyzed by long PCR (XL-PCR) carried out using the Expand Long Template PCR System kit (Boehringer Mannheim, Mannheim, Germany), as described earlier (Remes et al., 2005).
The amount of mtDNA relative to nuclear DNA in patient and control samples from different tissues were determined by real-time quantitative PCR. MtDNA was amplified using PCR primers (forward primer, L3485-3504; reverse primer, H3532-3553) and the TAMRA labelled fluorogenic probe (Sigma Genosys, Suffolk, U.K.) spanning from nt 3506 to nt 3529 of the ND1 gene (He et al., 2002). The values were normalized using a nuclear-encoded brain natriuretic peptide (BNP) gene as a reference. DNA (10 ng) was amplified separately with the ND1 and BNP primer/probe combinations using the ABI 7300 Sequence Detection System (Applied Biosystems). The concentration of PCR primers was 3 μM and that of the fluorogenic probe 5 μM. Samples were analyzed in triplicate, and DNA isolated from rho0 cells lacking mtDNA was used as a negative control.
DNA from blood and skeletal muscle was used as a template to amplify and sequence the 23 coding exons of the POLG1 gene (NM_002693). Allele-specific amplification was used to verify the W748S mutation. The primers, containing a locked nucleic acid (LNA) nucleoside base at the 3′ end (Proligo LLC, Paris, France), were designed to anneal with either the wild type sequence or the sequence containing the mutation (Braasch & Corey, 2001).
The initial symptoms experienced by the three patients were migraine-like headache and visual symptoms or seizures leading to status epilepticus (Table 1). Brain MRI revealed signal intensities in the thalami, and EEG changes were localized mainly to the occipital regions. Patients 1 and 3 manifested with severe liver failure 3 months after their first status epilepticus. Histological examination of muscle samples from patient 2 was normal, but electron microscopy revealed markedly increased alterations in the size and shape of the mitochondria and an increased amount of fat. The activities of the mitochondrial respiratory chain of the patient 2 were normal, whereas these activities had not been measured before the death of the patients 1 and 3. Autopsy findings with regard to patients 1 and 3 supported the clinical diagnosis of juvenile-onset AHS. There were no family histories of mitochondrial disorders.
Analysis of mtDNA
XL-PCR amplification of mtDNA did not reveal any deletions in the muscle of patients 1 and 2, whereas no muscle sample was available from patient 3. The mtDNA of patients 1 and 2 was found to harbor common polymorphisms which defined mtDNA subhaplogroup V1 (72T>C, 93A>G, 263A>G, 303insC, 311insC, 485T>C, 709G>A, 750A>G, 1438A>G, 2706A>G, 4580G>A, 4639T>C, 4769A>G, 5263C>T, 7028C>T, 8860A>G, 8869A>G, 15326A>G, 15904C>T, 16153G>A, 16298T>C, and 16519T>C) Patient 3 belonged to subhaplogroup W1.
Quantification of mtDNA
MtDNA in the available tissue samples from the three patients was quantified by real-time PCR. The mtDNA content of the liver of patient 1 was 15% of that measured in the control liver, muscle mtDNA content was 33% of that in control muscle in patient 1 and 57% in patient 2, and the brain mtDNA content was 76% in patient 3, whereas that in patient 1 was normal.
Analysis of the POLG1 gene
All three patients had a homozygous c.2243G>C transversion in exon 13 of POLG1 (p.W748S), and a homozygous polymorphism c.3428A>G in exon 21 (p.E1143G). There were no other changes in the POLG1 gene. The parents of patients 1 and 2 were heterozygous for the two mutations, but no family members were available for mutation analysis in the case of patient 3.
We detected a homozygous W748S mutation in the POLG1 gene in 14- and 17-year-old female siblings and in an unrelated 15-year-old girl, all with juvenile-onset AHS. All three patients presented with subacute visual symptoms, migraine-like headaches, and refractory seizures leading to status epilepticus in their teens. Patients 1 and 3 died of hepatic failure within 3 months of the onset of the first status epilepticus. Patients 1 and 2 had both been treated with sodium valproate before the first hepatic manifestations, but liver transaminases were elevated in patient 3 even before the valproic acid treatment had been started. Acute liver failure after the administration of valproic acid is common in patients with infantile-onset AHS, but hepatic changes have also been described before its administration (Ferrari et al., 2005). Our patients 1 and 3 developed severe hepatopathy suggesting that juvenile-onset AHS patients should also avoid valproic acid. Avoidance of valproic acid has been recommended with regard to the treatment of epileptic seizures in patients with Alpers syndrome (Gordon, 2006), a diagnosis which should be considered in any child with developmental delay, cerebellar signs, and partial seizures (Schwabe et al., 1997) in addition to the juvenile-onset phenotype presented here. Acute pancreatitis was also present in patients 1 and 3, and patient 2 had temporarily elevated amylase values. Pancreatitis is rarely described in patients with AHS, but acute pancreatitis has been reported earlier in two out of seven patients with juvenile-onset AHS (Table 2).
Table 2. Alpers patients with juvenile-onset reported in the literature
Age at onset (yr)
Death after the onset of symptoms
Myoclonic and focal suddenly appearing seizures, SE
Visual illusions, right temporooccipital lesions with left hemianopsia or blindness
The symptoms and clinical and pathological findings of patients 1 and 3 were typical of AHS (MIM 203700) (Alpers, 1931; Harding, 1990; Ulmer et al., 2002; Simonati et al., 2003), the clinical features of which include intractable seizures, cortical blindness, episodic progression of neurological symptoms, liver failure and pharmacogenetic sensitivity to valproic acid toxicity, and a developmental delay during early infancy in some cases (Harding, 1990). AHS is usually seen in infancy or early childhood, and only rarely have patients presented with a delayed onset of symptoms in later childhood or early adulthood (Harding et al., 1995). To our knowledge, there have so far been only seven reported cases (in addition to those described in this paper) with juvenile-onset AHS manifesting itself in the first or the second decades of life (Klein & Dichgans, 1969; Bohnert & Noetzel, 1974; Harding et al., 1995; Montine et al., 1995; Wörle et al., 1998). The clinical presentation and history of the disease in the patients reported here (Table 1) were very similar to those described in the literature (Table 2).
Our patients harbored the homozygous W748S mutation the carrier frequency of which has been estimated to be as high as 1:125 in the Finnish population (Hakonen et al., 2005), suggesting that the frequency of people in Finland with the homozygous W748S mutation should be 1/62,500. It was somewhat surprising that we found three patients with juvenile-onset AHS within a short period, since only a few patients with a similar clinical phenotype have been reported during the last 27 years.
Variable phenotypes are associated with the homozygous W748S + E1143G mutation in Finnish patients, including adult- or juvenile-onset ataxia, peripheral neuropathy, dysarthria, mild cognitive impairment, involuntary movements, psychiatric symptoms, and epileptic seizures (Rantamäki et al., 2001; Van Goethem et al., 2004; Hakonen et al., 2005). Interestingly, hepatic involvement or status epilepticus has not been described in these patients, whereas the brain MRI findings were similar to those in our patients, including symmetrical lesions of high signal intensity in the thalamus or cerebellar white matter, or atrophy of the cerebellum or vermis (Rantamäki et al., 2001; Van Goethem et al., 2004; Hakonen et al., 2005).
Our patients did not harbor pathogenic point mutations, large-scale rearrangements or multiple deletions in their mtDNA, but instead, the mtDNA content had decreased, so that the muscle mtDNA content in patient 1 was 33% of that in the control muscle sample and that in patient 2 was 57% of the control value. Furthermore, the mtDNA content of the liver in patient 1 was 15% of that in the control liver and that of the brain in patient 3 was 76% of the control. Quantitative analyses of mtDNA have so far been performed only in a few cases of AHS with a POLG1 mutation (Naviaux et al., 1999; Naviaux & Nguyen, 2004; Tesarova et al., 2004; Ferrari et al., 2005), and a range of mtDNA content from 3 to 40% of the normal mean has been documented in the liver and brain, while the results from skeletal muscle samples have been less consistent (Naviaux et al., 1999; Naviaux & Nguyen, 2004; Tesarova et al., 2004; Ferrari et al., 2005). Our findings support the suggestion that tissue-specific, partial mtDNA depletion is a molecular feature of Alpers syndrome.
The cases reported here add fatal hepatocerebral syndrome with juvenile-onset to the clinical spectrum of recessive POLG1 mutations, and emphasize that POLG1 mutations should be considered in teenagers and young adults reporting episodic visual symptoms with migraine-like headaches and sudden-onset intractable seizures. Due to the risk of liver failure, valproic acid should be avoided when treating patients with Alpers syndrome or Alpers-like phenotypes.
The authors thank Ms. Anja Heikkinen and Ms. Pirjo Keränen for their excellent technical assistance. This study was supported by grants from the Medical Research Council, Academy of Finland (decision numbers 79843, 107174, 107490, 108953), the Sigrid Juselius Foundation, the Foundation for Pediatric Research, and the Arvo and Lea Ylppö Foundation.
Conflict of interest: The authors confirm that they have read about the journal's position on ethical issues involved in publication and affirm that the present report is consistent with these guidelines and provide full disclosure of any conflicts of interest.